( 12 ) Patent Application Publication ( 10 ) Pub . No . : US 2019 / 0262399 A1

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US 20190262399A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2019 /0262399 A1 Wang et al . ( 43) Pub . Date: Aug. 29 , 2019

(54 ) COMPOSITIONS AND METHODS FOR A61P 35/ 00 ( 2006 . 01 ) EVALUATING AND MODULATING IMMUNE G16H 50 / 70 (2006 .01 ) RESPONSES G16H 50 / 30 ( 2006 . 01 ) G16B 25 / 10 ( 2006 .01 ) (71 ) Applicants : The Broad Institute , Inc. , Cambridge , C12Q 1/ 6809 (2006 . 01) MA (US ); Massachusetts Institute of C12Q 1/ 6811 ( 2006 .01 ) Technology , Cambridge , MA (US ) ; The C120 1/ 6851 ( 2006 . 01 ) Brigham and Women ' s Hospital , Inc ., C12Q 1/ 6853 ( 2006 . 01 ) Boston ,MA (US ) ( 52 ) U . S . CI. (72 ) Inventors : Chao Wang , Boston , MA (US ) ; CPC ...... A61K 35 / 17 ( 2013 .01 ) ; GOIN 33 /5023 Meromit Singer , Cambridge , MA (US ); ( 2013 .01 ) ; A61P 35 /00 ( 2018 . 01 ) ; G16H Aviv Regev, Cambridge , MA (US ) ; 50 / 70 ( 2018 .01 ) ; G16H 50 /30 (2018 .01 ) ; Vijay K . Kuchroo, Boston , MA (US ) GOIN 2800 / 24 (2013 .01 ); C120 1 /6809 (2013 .01 ) ; C12Q 1 /6811 (2013 .01 ) ; C12Q ( 21 ) Appl. No. : 16 /331 , 410 1 /6851 ( 2013 .01 ) ; C120 1 /6853 ( 2013 . 01 ) ; GOIN 2800 / 52 (2013 .01 ) ; G16B 25 / 10 (22 ) PCT Filed : Sep . 7 , 2017 ( 2019 .02 ) (86 ) PCT No .: PCT/ US2017 / 050469 $ 371 (c )( 1 ), ( 57) ABSTRACT ( 2 ) Date : Mar. 7, 2019 Related U . S . Application Data The present invention provides markers , marker signatures (60 ) Provisional application No . 62/ 384 ,557 , filed on Sep . and molecular targets that correlate with dysfunction or 7 , 2016 . activation of immune cells . The present markers , marker signatures and molecular targets provide for new ways to Publication Classification evaluate and modulate immune responses. Therapeutic (51 ) Int . Cl. methods are also provided to treat a patient in need thereof A61K 35 / 17 ( 2006 .01 ) who would benefit from a modulated immune response . GOIN 33 /50 ( 2006 .01 ) Specification includes a Sequence Listing.

Tim3Tims - PD1PDI to Tim3- Pont+ Function Tim3 + PD1+ + Tim3 PD1- 090 MTY Similar 0.00 Timp PD1+ 90 function MTT - Tim3 + PD1 + we Implant Wait CD8 + tumor 2 – 3 wks T - cells Sequencing Patent Application Publication Aug. 29, 2019 Sheet 1 of 66 US 2019 / 0262399 A1

FIG . 1A

Implant carcinoma WIKI

11 2 – 3 weeks

htyy

Tumor dissociation

CD8 +

Tim3 PD1- Tim3- - PD1+ Tim3* PD1+

Function Patent Application Publication Aug. 29, 2019 Sheet 2 of 66 US 2019 / 0262399 A1

FIG . 1B

Tim3 Tim3 Tim3+ Naïve EffMem PD1- PD1+ PD1 +

MY 3031 genes

WAN * * * :343

WXXXXXXX

:..

. . . .

1 2 3 Z - score Patent Application Publication Aug. 29 , 2019 Sheet 3 of 66 US 2019 /0262399 A1

VYNYVYYNYN 10dEWIL C5(n*273) C10 (n=157) Od - EWL wwwwwwwww EftMen

VYN PD11 -Tim3 C4)356-n( arengarenesancongannaman C9 (n=164) VYNYNY . LOC . WIL EffMem Naive

* PD1* Tim3 WYNNYANNN PD1 Tim3 FIG.1C C3(n-420) C8(n=191) "PD1 -Tim3 EffMem WANANAW Naive

wy *PD1 * Tim3 442) * * * 2014 -Tim3 C2 ontwerowaniewannowe. (n=215) - lad - ul in EffMem wwwwwwwwww Naive

*PD1 Tim3 Tim3mPD1 )577-1( C6(n=236) PD1 - Tim3 EffMem . WWW Naive com . thermomindannmann continue in humeurteilunarheiminutione humminne tuomannuitium bewonerinen bewurkunde teminarne whenuateman wanawwwwwwwwwwwwwww or oen score - Z score - Z Patent Application Publication Aug. 29, 2019 Sheet 4 of 66 US 2019 / 0262399 A1

FIG . 1D

666666666 Sinza 23K H444444wr2NR

KLUBI/12

E Maine Men: DN DF Patent Application Publication Aug. 29, 2019 Sheet 5 of 66 US 2019 / 0262399 A1

FIG . 1E

+-log10(pvalue)

" " " " " " " " " " " » » » * * * * * * " - - 30 = = = = = Cust ASE4 stusta iste Chust10

memelulog10(p-value)

02 SUAL Qus10 Patent Application Publication Aug. 29 , 2019 Sheet 6 of 66 US 2019 /0262399 A1

FIG . 1F LCMV CD8 in vivo exhaustion activation

oolxoco - - - SignificanceWW threshold

w - Log (p -value ) Patent Application Publication Aug. 29, 2019 Sheet 7 of 66 US 2019 / 0262399 A1 FIG . 1G Wherry2012 day 15 Acute vs. naïve

-log10(pvalue) * * * * * * * * * * * * * M * * * * -* * * * *- * * * * * * - * * * -* * * * * * * * * * * * * * *

& dar4 dlar5 dier_7 6 ODNOT ZIPIP Pip dlar PIP DIERP Wherry2012 day 8 Acute vs. day 30 acute

Oandnowto -log10(pvalue) 17 4 " A " " - . ann. * * * * * * * * * * 7 N - TTTT ------NE LE AT . . + 1 LV - N 1 m . " N N * MR + ith - - - - - XNY LE * * . . - - - - * . . . - - - dari dar2 der_+ dar_ duur gar_4| Sarkar2008 KLRG -eff vs . naive

-log10(pvalue) - * * * * * * * * * * . + + + + 2 1 1 * 7 * - * -* * * 717 -- 747 * 27 * 1 - A

597 Chice I1912 chut9 01Fres Sarkar2008 KLRG - eff vs . [naïve OR memory ]

-log10(pvalue) OhnowTI .

with . . . 1 . N . XI V ...... NY LE - T - - * * ------* th X L . . . . . * * L

. T - = - - = - - ? ? ? ?? ?? ?? ?? ? ??? ? ? ?? ? ? ?? ? ?? ??? ? ?? ?? ? ?? ?? ?? ??? * " ?? ? ? ???? ?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? - - - ? ?? ?? ?? ? ? ???? ? - ?? - ? ? ? ? ? ? ?? ?? ??? upp ?_??? du du dau BD BIRD Dip Patent Application Publication Aug. 29, 2019 Sheet 8 of 66 US 2019 / 0262399 A1

FIG . 1H

1 Wherry2012 day (D15C vs. D15A ] UNION1 [D30C vs. D30A ]

- + + + + + + hi ------+ N ------

8* ckey_10

Wherry2012Y " day (D15C vs . D15A) INTERSECT [D30C vs. D30A ]

. EU LIELU L . . . - - V A U L . . VAL H L EI LELE LE L . . . . . 2 I 2 2 LH - . - . U LE LI L LU ......

ckm_9 Patent Application Publication Aug. 29, 2019 Sheet 9 of 66 US 2019 / 0262399 A1

FIG . 11 Tim3 Tim3 - Tim3 + Naïve EffMemm PD1 - PD1 + PD17 Rank SERPINE2 GZMO DUSP4 OSBPL3 NRNT COKN3 CSFT PLSCR1 CDKN2B TOX ARSB TNFRSF9 CCRL2 C1QTNF6 SERPINA3G CD81 CD244 . LO0877008 / TGAT FASL CREB3L2 D630039A03RIK GEM TBX21 DENNA 4 - 2 0 2 4 Centered intensity Patent Application Publication Aug. 29, 2019 Sheet 10 of 66 US 2019 /0262399 A1

ODOLG C0276 TNPSF14 CD icos TNFRSF18 TNFSR4 TNFRSP9 TNF9F11 TNERSF4 0

DP -2

DN Co-Stimulatoryreceptors EffM Naive

FIG.1J

KLRA3.KLI CTLA4 LAGS CD244 0020 CD160 HAVCP2 PILRA PDCD1 KLRD1 KLRAZ PDPN LAR1 CEACAM1 BTLA DP

SP Co-Inhibitoryreceptors DN EffM Naive Patent Application Publication Aug. 29, 2019 Sheet 11 of 66 US 2019 /0262399 A1

EffMem 2Tim3-PD1 Tim3-PD11 PD1+Tim3O

MT1:B16 MT2,B16 . wwwwwwwwww WWW ww 2500m FIG.2A 8000 6000 4000 2000 2000 1500 1000 RE RE

! TERROR

CT26MT1: CT26:MT2,6000

- - - 5000- 4000- 2000+ 20,000 10,000 RE Patent Application Publication Aug. 29, 2019 Sheet 12 of 66 US 2019 / 0262399 A1 FIG . 2B

Up in De Zinc associated Down I DE ZINE associated

140121*** 1

Not significant { p -value = 0 .79 )

RERE

T :+ 8 + FIG . 2C labile Zn2+ 100000

??????? labileZn2+MFI M

. . . 3 * " .. . *** 20000 DP SP DN OP SP DN OP SP ON CD8 * TIL CD4 " TIL CD8 * DLN Patent Application Publication Aug. 29, 2019 Sheet 13 of 66 US 2019 /0262399 A1

FIG . 2D

Free Zn in CD8 + TILS * * 100 ,000 - NS WT 80 , 000 O MT- | Zinpyr-1MFI 60 ,000 000 -

20 ,000 TIL .DN TIL .SP TIL .DP Patent Application Publication Aug. 29, 2019 Sheet 14 of 66 US 2019 /0262399 A1

FIG . 3A OMT- - 400 WT

* * * Tumorsize(mm2)

do 5 10 15 20 Days Patent Application Publication Aug. 29, 2019 Sheet 15 of 66 US 2019 /0262399 A1

FIG . 3B dLN MT- - 20 ,000 WT 15 ,000 10, 000 5000 10 5 2. 5 1. 250 O MT- A 105 TIL WT 104 1034 udo 102 101_ gp102 100 peptide ( UM.5 ) Patent Application Publication Aug. 29, 2019 Sheet 16 of 66 US 2019 /0262399 A1

FIG . 3C comprehens WT Mel (n = 14 ) 300m p = 0 .011 @ MT- - pMel (n = 14 )

Tumorsize(mm*2)

cusssssssy - SSSSSSSSSSSSS S SSSS Success . . . 0 5 10 15 20 days Patent Application Publication Aug. 29, 2019 Sheet 17 of 66 US 2019 /0262399 A1

FIG . 3D O No cell 2007 OMTOE– Control OT1OT1

Tumorsize(mm)

+ +

5 10 15 20 25 Days Patent Application Publication Aug. 29, 2019 Sheet 18 of 66 US 2019 /0262399 A1

FIG . 3E WT MT - - 10943. 62 3030 . 8 - 33 .. 78 78 38 .6 104

wymianaik 5 0 . 2 5 . 21 133 6 24 0 103 104 105 103 104 105 PD - 1

100 , p = 0 . 078

Tim-3PD1+ofCD8+TILs(%)

WT MT- - Patent Application Publication Aug. 29, 2019 Sheet 19 of 66 US 2019 /0262399 A1

FIG . 3F WT MT - - 10 % 377 M 17. 7 ) 4312A 33 .8 * 160 TNFat of DO 40 Tim - 3 + CD8 + 20 TILS ( % ) -TNF[ 17. 6 orgenrense 27 19: 16 APAARA 10918 .51 2 . 09 10. 8 6 .08 1041 15 IL - 2 of 1 103 110 Tim - 3 + CD8+ with* 15 TILS ( % ) LinhT + 44 . 3 45 . 1 38 .2 105 32 .6 23 . 6 32 . 4 35 ,31 104 60 IFNyt of 103 40 Tim - 3 + CD8 + 20 TILS (% ) 21. 8 13 . 41 IFNY 0 103 104 105 103 104 105 wwwwwwwwwwwwwww LTim - 3 WT MT - - Patent Application Publication Aug. 29, 2019 Sheet 20 of 66 US 2019 /0262399 A1

FIG . 3G Polyfunctionality 40 * NS O MT- 1 • WT

IFNy*TNFaIL-2ofCD8(%) .Hi Tim - 3 + Tim - 3

FIG . 3H Free Zn in - TILS 100 ,0007 * O MT - / 80 ,000 IWT Zinpyr-1MEI 60, 000 40, 000 + I 20 , 000 OL TIL DP TIL SP TIL DN Patent Application Publication Aug. 29, 2019 Sheet 21 of 66 US 2019 /0262399 A1

-MTWT GrzmB •H TILS + CD8 + B Granzyme %

? 29.2

. SHAL . FIG.31 A | 21 .

MT-I:TILCD8 SSENSACAAAAAAAA

? WT:TILCD8

|800- - B Granzyme Patent Application Publication Aug. 29, 2019 Sheet 22 of 66 US 2019 /0262399 A1 Sequencing Function Similar function hastitatu

PD1 PD1 Tim3+PD120 PD1+Tim3ons Tim3+PD1 Theport Tim Timonalainen 3 FIG.4A CD8+ 90 T-cells

-mu

::: :

Wait 2-3wks

Implanttumor

MT+ WWT Patent Application Publication Aug. 29, 2019 Sheet 23 of 66 US 2019 /0262399 A1

Tim3+PD1 PD1+-3Tim• PD1+3Tim Tim3PD1 OTim3PD1 oTim3PD1+ Wildtype (mean*) Knockout (mean)

20 FIG.4B ó PC1

-20

-40

PC2 Patent Application Publication Aug. 29, 2019 Sheet 24 of 66 US 2019 /0262399 A1

7+ PD7 *Tim3 I )*PD1 -Tim3 2 7-PD1 -Tim3 MT +PD1 * Tim3 PD17“Tim3 *PD1 -Tim3 Wildtype -420 -OZ meanPC2 FIG.4C

PD147*Tim3 *PD1 - Tim3 ,MT-2 PD1-Tim3 PD11*Tim3 JE *PD1 " Tim3 typeWild )PDT -Tim3

-25 meanPC1 Patent Application Publication Aug. 29, 2019 Sheet 25 of 66 US 2019 /0262399 A1

FIG . 4D Tim3 Tim3+ Tim3+ PD1 PD1+ PD1+ WT WT MT

Geneexpressiongroups Patent Application Publication Aug. 29, 2019 Sheet 26 of 66 US 2019 /0262399 A1

FIG . 4E

Naïve CD8 CD8 in vitro act Mean WT( ) * Tim3 - PD1 PC2PC2 * Tim3 - PD1+ Tm3 + PD1+ Mean (KO ) Tim3- PD1 Tim3 - PD1 + Tim3 + PD1 + -40 - 20 o 20 40 PC1 FIG . 4F

1 CD8 in vivo activation stik i 2 LOMV exhaustion - - ...... mon i 3 Cluster 2 signature toring on the international 4 Memory CD Pearsoncoefficient nimentiinftitifolentinenfutterfuffufafanulifelinininfinitat 5 Naive CD8 wiwili

Dus To Pearson coefficient Patent Application Publication Aug. 29, 2019 Sheet 27 of 66 US 2019 /0262399 A1

FIG . 5A PPT "Dysfunctionscore (CorrelationofWTsampleswithPC2) utv

??????.?????????????????

WWW HORA HTH

Activation score (Correlation of KO samples with Pc1}

Modules Dysfunctionscore(DYS) ODYS - specific ACT- specific DYS / ACT

11!: : : : .. . . " Neither ryyyy

????????????????????????????????????????????????????????????????????????????

. .. Activation score (ACT ) Each gene score is projected onto both diagonals , resulting in two gene rankings : DYS - specific aparte state 20: 39 angle. ACT- specific DYS / ACT kwa Neither Patent Application Publication Aug. 29, 2019 Sheet 28 of 66 US 2019 /0262399 A1

FIG . 5B Co - inhibitory receptors Co - stimulatory receptors OOOOOOO ????????????

TheRosta TNFRSFO SLAMF6 C D 160 .

* C027 : TNES# 14 ©BILA ACSI Dysfunctionscore ATOER4

- 10 - 0. 5 0. 5 10TrrrrrrrrrrrrrFFFFFFFFFFRFORIFFIFFINTEFIAPRTREFPREFINERIPARITERFAFFFFFAFFFFFFFFAIR WWWWWWWWW Activation score Patent Application Publication Aug. 29, 2019 Sheet 29 of 66 US 2019 /0262399 A1

WWW.

20 DownregulatedinCD8activation ac?vationCD8UpregulatedinU WWW.

thesentere thewonder thewonder netwondewhether FIG.5C (CorrelationofKOsampleswithPC1) - Activationscore

. .. i

0 .1 5. 0

) PC2 with samples WTof Correlation ( score " Dysfunction" Patent Application Publication Aug. 29, 2019 Sheet 30 of 66 US 2019 /0262399 A1 ???????????????????????????????????????????????? activationCD8Upregulatedin DownregulatedinCD8activation ????????

90 FIG.5D 10 (CorrelationofKOsampleswithPC1)

. BAN ! ! 00 Activationscore *

* *

0.5

natemat score " Dysfunction " Patent Application Publication Aug. 29, 2019 Sheet 31 of 66 US 2019 /0262399 A1

FIG . 5E

ACT / DYS ACTDYS Neither

LCMV exhaustion w ?????????????????????????????????????????????????????????? Cluster 2 signature CD8 in vivo activation

CD8 in vitro activation org CD8 Treg (Ly49 + ) Memory CDS WWW MU Naive CD8

D - -- SignificanceM threshold - Log ( p - value ) Patent Application Publication Aug. 29, 2019 Sheet 32 of 66 US 2019 /0262399 A1

FIG . 5F

Exhaustion - specific set (human Activation set (human ) ???????? TS Dysfunctionscore WWWWWWWWWW . wwwwwwww NWZERIA* . 0 . 0

Activation score

Wilcox . test KS .test DYS- specific VS . ACT -specific 0 .036 0 . 027 w Exhaustion - specific set (human ) www Activation set (human )

Fn(x)

down DYS pumunta ACT projection score Patent Application Publication Aug. 29, 2019 Sheet 33 of 66 US 2019 /0262399 A1

FIG . 5G MDFIC

varrewerytrawwerwyller DTXI •KLF2 i Dysfunctionscore *ŽEB2 verervar

. 3 Vyvrtulevarulevyarverview SPB TSC2207 the withWhat twitchineswww. www. AAAAAAAAA

* * * * WWWWWWUNASE Top -ranking TFS ACTAceptank DYSDÝSIACT / ACT NeitherNeither

D2 GATA3 IKZF2 SUDS3 DTX1 KLF2 YEATS2 FHL2 PRDM1 ZEB2 BHLHE40 RBPJTRPSI MDFIC HIFIA TSC22D1 POU6F1 TCF4 Ranking TY MALU ???????????????? DYS CE DYS - ACT

+ ???????????????

+ + ????????????????????? WW ACT Neither

Log ( p -value ) Patent Application Publication Aug. 29, 2019 Sheet 34 of 66 US 2019 /0262399 A1

FIG . 5H 2 - 1 - 3 P - FY3 P - 1 * 3

? 14 . 449 ????? ?????? . ???? ?????? ??? ??????????? ?????????

? ????????????????? ????????????????????

? ????????? ?? ? . 5 ???? . . . . ! . . . ??? ??????????????????? ??????? -

? ???? ????????????????????????????????????????? ???????????????????????????????????????????????????? Patent Application Publication Aug. 29, 2019 Sheet 35 of 66 US 2019 /0262399 A1

FIG . 6A

Gata3 6 . 99

Khi

inter - Tim3

FIG . 6B Foxp3 WT uths wat .ini TTTTTTTT CD8 TY .

. uluh yeye Wederom . 14 .4 ?????????????????????????????? TIT CD4 Tim3 Patent Application Publication Aug. 29, 2019 Sheet 36 of 66 US 2019 /0262399 A1

11.9 37.0 1051041030 GATA+GATA-

105.687 1044 108t Gata3 ) % ( cells- T 2 - IL CD8 of 21 - IL

6.01 5.89 1051041030 WWW GATA- FIG.6C 1053.93 of Gata34 10 - IL ) % ( cells- T CD8 of + 10 - IL

8.22 4.05 222222V77227 105104103Ö VI21 GATA- 77 +44.9

105428 104 103 1027 40,000 Gata3 20,000 10,000 IFNY 000,30 IFNY of MFI Patent Application Publication Aug. 29, 2019 Sheet 37 of 66 US 2019 /0262399 A1

14days Monitortumorgrowth

Transferof KO/controlcells .

i SuccessfulCRISPRinclusion days FIG.6D Injecttumor toWTmouse Drugselection

naïveCD8 CRISPRKO *Control PMelWT KE Patent Application Publication Aug. 29, 2019 Sheet 38 of 66 US 2019 /0262399 A1

VIIEIIE Rerelt Vre CtrlGata3-4 PMEL?WT

( % ) 57 +CD8 ) %of ( TILS TNFat + CD8 of + 10 - IL 4Gata3-Ctrl PMEL-WT

FIG.6E ) %( TILS ) % ( TILs + CD8 of IFNyt +CD8 of + 2 - IL

2.09 0.84 6.35 913.77 0103104105

9109415. 10°30D 59. IL-2 104 1941 OF IFNY L W-Ctrl Gata3+/-?W Patent Application Publication Aug. 29, 2019 Sheet 39 of 66 US 2019 /0262399 A1

pMel(CD8+)

. rrirurrrr PD-1 R

FIG.6F WTPMel->Gata3/pMel IFTTTT TIT

wwwww * * * * * * * * * * * * * * . ' . ' . ' . .

RAY *

rrrrrrrrrrrrr 2

. Y 13.8 TTTTTTTTTTTTTTTTTTT T

low Foxp3 Patent Application Publication Aug. 29, 2019 Sheet 40 of 66 US 2019 /0262399 A1

FIG . 6G

+ Ctrl PMEL1 ? WT O Gata3 del. PMEL ? WT 2000 - No cell transferN

oppo Tumorsize(mm2)

ö 5 10 15 20 25 Days Patent Application Publication Aug. 29, 2019 Sheet 41 of 66 US 2019 /0262399 A1

FIG . 6H Gata3 0 . 0047 * * * 0 . 003 I 0. 002 0 .001 -

0 .000 Control GATA3 LV LV Patent Application Publication Aug. 29, 2019 Sheet 42 of 66 US 2019 /0262399 A1

PMEL?WT 4-Gata3Ctrl

) % ( TILs + CD8 of + 1 - PD + 3 - Tim

3.10 ZTTTT7777777 159. FIG.61 COXED.75 0103104105 Gata3-WT 2

2.16 78.4

6.11| Ctrl—WT . 0103104105 10%+1.75928 82.9 PD-14 3 - Tim Patent Application Publication Aug. 29, 2019 Sheet 43 of 66 US 2019 /0262399 A1

FIG . 7A FIG . 7B No TIL (gray ) MT- - DP WTDP No TIL (gray ) ON SP DP

wwwwwwwwww NINISFREE ARTIA

RMS TTTTTTTTTTTTTTTTTT W WNU*

xxxxxxx CFSE with CFSE

was en WT TIL 015 (TIMO PO1" ) WT TIL J15 ( Tim PON } i nto consideren RAMAw KWELIER %CFSETcells CFSETcells

* * * . * * Teff :co : ratio Teffico & ratio Patent Application Publication Aug. 29, 2019 Sheet 44 of 66 US 2019 /0262399 A1

FIG . 7C

MT- / TIL : day 11 0 . 17 341 Foxp3 ?????? ++ %Foxp3+ofCD3CD8Tcells

??????????????????? 31 LULLLLL . . . Fa TTTTTTT " ! ! ! ! ! ! ! fummum Od- T M1- FIG . 7D % FOXP3 * Edxoy 31. 8

at %FOXP3+ofGata36CD8Tcells MENEM HTTTTTTTTTTTTTTTTTTTTTTTTT uuluu N t - seribog Tim3 FIG . 7E

WYT#Zn " Patent Application Publication Aug. 29, 2019 Sheet 45 of 66 US 2019 /0262399 A1

FIG . 8A Exp 1 : total Pou?afi KO (Mixed SNP profile of B6 and 12956 /SVEVTac ) : 73 .47 % B6 ; the WT used in shitthis experiment is 100 % B6

WT (n = 9 ) Pou2af1- 4 ( n = 5 )

. B16Tumorsize(cm^2)

???????????PI 0 5 10 15 20 days Patent Application Publication Aug. 29, 2019 Sheet 46 of 66 US 2019 /0262399 A1

FIG . 8B Exp 2 - WT CD8 - > WT (n = 5 ) wyth WT CD4 - > WT (n = 5 ) Pou2af1 -/ - CD8 > WT (n = 5 ) wyty Pou2af1 -/ - CD4 - > WT (n = 5 )

B16Tumorsize(cm^2)

itis

D * * ???1221222222222222222222222222222222222211222222222222222222222222222222222221 - 0 5 10 15 20 † days

Transferredw splenic CD8 or CD4 T cells from individualmice from9 exp 1 Patent Application Publication Aug. 29, 2019 Sheet 47 of 66 US 2019 /0262399 A1

FIG . 9 CD8 + TIL Activation V

. WWW Upregulation of Co - stimulatory Co - inhibitory ( Tim3, PD1 , Lag3 , TIGIT etc) VUUKKIIIIIIIIIIIIIIIIIIIIIIIII Metallothioneins

Gata3

Dysfunction / Exhaustion Patent Application Publication Aug. 29, 2019 Sheet 48 of 66 US 2019 /0262399 A1

FIG . 10

EVE PMEL 1 transfer - > WT 500 Ctrl PMEL 222122222 LV31 PMEL Tumorsize(mm^2) 222

. T o 12 18 24 days Patent Application Publication Aug. 29, 2019 Sheet 49 of 66 US 2019 /0262399 A1

15 71

litti:

-5010 CD8invivoactivation .113

MWI

ullit

FIG.11A

•Knockout

-20 Activationmodule

** 11 tit. •Wildtype ill 27

module Dysfunction Patent Application Publication Aug. 29, 2019 Sheet 50 of 66 US 2019 /0262399 A1

ó5to15

11 11 eHullut : CD8invivoactivation

ETHit tits 1.itsitTulliist With"

-10121 signature 2 Cluster

-5010 CD8invivoactivation

exhaustion LCMV

15 FIG.11B wwwwwwwwwwww

510 1.Ali www wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww CD8invivoactivation module DYSI ACT

!111 Knockout 11 blir# !Yii -501015 CD8invivoactivation Wildtype

module Activation Patent Application Publication Aug. 29, 2019 Sheet 51 of 66 US 2019 /0262399 A1

FIG . 11C • 1 2 3 4 5 6 7

. .X

USNE2 W 423

WL91

- - 40 - 20 ó 20 40 ISNE 1 Patent Application Publication Aug. 29, 2019 Sheet 52 of 66 US 2019 /0262399 A1

Top20% *2040% *60-80% *00-100%

Naive/memory-likemodule WWW ISNE1

Dysfunctionmodule ISNE;

FIG.11D gevoegwrongorol ACT/DYSinodule Werewewyowegoor ISNE1

Activationmodule 7ISNE

-30 2 SNE Patent Application Publication Aug. 29, 2019 Sheet 53 of 66 US 2019 /0262399 A1

#Wildtype u Knockout 27 Cluster

Activationmodule ACT!DYSmodule Dysfunctionmodule Naive/Memorymodule

FIG.11E-11G Cluster

mL0910co-value) 07 21

31 wi, uw Knockout SNE1 ??????????????????????????????????????? in Wildtype 1966

2 ISNE Patent Application Publication Aug. 29, 2019 Sheet 54 of 66 US 2019 /0262399 A1

**GZMR **GZMK GZMA *GZME GZMO GZMM IFNG Centerednormalizedexpression

Cluster7 *

*pvalue<0.05 *p-value<0.001

FIG.12

Cluster5 Patent Application Publication Aug. 29, 2019 Sheet 55 of 66 US 2019 /0262399 A1

FIG . 13A CD83 50

25 rrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrr

. CD83 12 . 5 10 . 0 tSNE_2 crrrrrrrrrrrrrrrr

- 25 ......

. rrrrrrrrrrrrrrrrrrcrrrrrrrrrrrrrrrrrrrrrre

- 50 * * * * * * * * * * * * * * * * * * * * rrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrr - 50 - 25 50 ISNEYtSNE _ 1 Patent Application Publication Aug. 29, 2019 Sheet 56 of 66 US 2019 /0262399 A1

FIG . 13B CD83

ha ',.;:

WE W :,'.;si gene 12 .5 10 . 0 YOU WY WW 7 . 5 M 5 . 0 25

- .1

. WA 00

W W . .. . . : :

. - 50 . - 50 - 25 o 25 5077777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777, ISNE _ 1 Patent Application Publication Aug. 29, 2019 Sheet 57 of 66 US 2019 /0262399 A1

FIG . 13C CCR8

......

HA WWW 32 YA 118/14+4144111481111111111°714441111111111111111111111111111111111111 gene

B ITS : V W . . . . . : 717

Allalily t.'

. 7797227222222V/2A122277W772727272727272727277122122772782717F102727272727272717F112112717127227W8112722728112727177277777777777777771mm.

112 Tiisi

11/414118871718811811111111111111111 Hill : : : : : : ...... : : :: :: : : : :: : : :: : : :: : : : :: : : : : : :: : : : : : : M : : : : : : : :: : : : : :: : : : :: : : : : : :: : : : : :: : W HI! - 50 - 25 25 50 tSNE _ 1

Patent Application Publication Aug. 29, 2019 Sheet 59 of 66 US 2019 /0262399 A1

FIG . 13E TNFSF11

VVVY YYYYYYYYYY /

VAA / 215 : : W :: : : : : :: ...... /

tot / gene

Hill / SU 1. Hill 2 att /

. tSNE_2 1 Wema MW NN 14211UIDATA /

/ VU

/ ...... 111111111111111 WW / M

01 /

-50 7/ - 50 - 25? 25 50 11 IN Patent Application Publication Aug. 29, 2019 Sheet 60 of 66 US 2019 /0262399 A1

FIG . 13F CD811

...... gene

WWW

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COMPOSITIONS AND METHODS FOR development of improved vaccines and novel immune EVALUATING AND MODULATING IMMUNE based therapies for various cancers . It is believed that the RESPONSES breadth of the functional potential of CD8 + T cells is far from understood , and that gaining a deeper understanding RELATED APPLICATIONS AND will lead to further advancements. INCORPORATION BY REFERENCE [0007 ] During persistent immune activation , such as [0001 ] This application claims the benefit of U .S . Provi uncontrolled tumor growth or chronic viral infections , the sional Patent Application No . 62 /384 ,557 , filed Sep . 7 , 2016 . ability of CD8 + lymphocytes to secrete pro - inflammatory [0002 ] Reference is made to PCT Publication No . cytokines and elaborate cytotoxic function becomes com WO / 2014 / 134351 published on Feb . 27 , 2014 , PCT Publi promised to different extents ( Anderson et al . , 2016 , Immu cation No. WO /2014 / 145631 published on Sep . 18 , 2014 , nity 44 , 989 - 1004 ; Baitsch et al. , 2012 , Trends Immunol 33 , PCT Publication No. WO /2014 / 172606 published on Oct. 364 - 372 ; Kim and Ahmed , 2010 , Curr Opin Immunol 22 , 23 , 2014 and PCT Publication No. WO /2015 / 130968 pub 223 -230 ; Wherry and Kurachi, 2015 , Nature reviews Immu lished on Feb . 26 , 2015 . Reference is also made to Interna nology 15 , 486 -499 ; Zuniga et al. , 2015 , Annu Rev Virol 2 , tional application serial number PCT/ US2016 /040015 filed 573 - 597 ) . These CD8 + populations are frequently referred to on Jun . 29 , 2016 , International application serial numbers as “ dysfunctional” or “ exhausted ” , and are believed to PCT /US2016 /059507 , PCT /US2016 / 059487 and PCT / constitute a barrier to successful anti - tumor and anti - viral US2016 / 059463 filed on Oct . 28 , 2016 , and U . S . provisional immunity . Such dysfunctional or exhausted CD8+ lympho application Ser. Nos . 62 / 247 ,432 , 62 / 247 ,446 and 62 / 247 , cytes are typically compromised for their ex vivo cytokine 430 , filed on Oct. 28 , 2015 . secretion capabilities and are present in an environment in 10003] The foregoing applications, and all documents which there is persistent antigen . Gaining a clear molecular cited therein or during their prosecution (" appln cited docu understanding of the dysfunctional T cell state can thus help ments " ) and all documents cited or referenced in the appln develop successful therapeutic interventions. cited documents , and all documents cited or referenced [0008 ] Dysfunctional CD8+ T cells can be both protective herein (“ herein cited documents ” ) , and all documents cited and detrimental against disease control. Attempts to manipu or referenced in herein cited documents , together with any late pathways associated with T cell dysfunction have manufacturer ' s instructions, descriptions, product specifica resulted in different biological consequences to the host . On tions , and product sheets for any products mentioned herein one hand , blocking co - inhibitory pathways such as PD - 1 and or in any document incorporated by reference herein , are Tim3 that frequently coincide with T cell dysfunction has hereby incorporated herein by reference , and may be promoted T cell function and is particularly effective in employed in the practice of the invention . More specifically , promoting tumor regression in several types of cancer . On all referenced documents are incorporated by reference to the other hand , the targeted deletion of PD - 1 at disease onset the same extent as if each individual document was specifi causes immune pathology and death of the host in a chronic cally and individually indicated to be incorporated by ref viral infection model , suggesting that T cell dysfunction may erence . have evolved to prevent immunopathology (Barber et al. , 2006 , Nature , vol. 439 , 682 - 687 ) . Also , current therapies STATEMENT REGARDING FEDERALLY targeting co - inhibitory or immune checkpoint receptors such SPONSORED RESEACH as CTLA - 4 and PD - 1 that are highly expressed on dysfunc tional T cells are showing promise in the clinic . However , [0004 ] This invention was made with government support not all patients respond and some cancers remain largely under Grant Nos . MH105960 , NS045937 , A1073748 and refractory to these therapies. Furthermore , depletion of CA187975 awarded by the National Institutes of Health . The exhausted T cells by targeting known regulators such as Tbet government has certain rights in the invention . and Eomes resulted in high viral load suggesting that FIELD OF THE INVENTION dysfunctional T cells provide partially effective immune control (Paley et al. , 2012 , Science, vol. 338 , 1220 - 1225 ). [0005 ] The invention relates to substances , compositions These findings highlight the complex roles of dysfunctional and methods useful in evaluating and modulating immune CD8 + T cells during unsuccessful antigen clearance . The responses. ability to delineate the diverse roles of dysfunctional CD8 + T cells at the molecular level can help with more specific BACKGROUND OF THE INVENTION targeting of truly exhausted T cells while sparing those [0006 ] T cell fitness is closely linked to health and disease . dysfunctional T cells that may be still protective . Activated CD8 + T lymphocytes have been reported to exist [ 0009 ] CD8 + T cell function is associated with their in various functional states characterized by different cytokine profiles . It has been reported that effector CD8 + T cytokine secretion potentials , proliferation capabilities and cells with the ability to simultaneously produce multiple the ability and potential to become long - term memory cells . cytokines (polyfunctional CD8 + T cells ) are associated with The types of CD8 + subpopulations and their functional protective immunity in patients with controlled chronic viral states can vary kinetically in response to different pathogens infections as well as cancer patients responsive to immune and are dependent on the status of pathogen clearance . therapy (Spranger et al. , 2014 , J . Immunother . Cancer , vol. Characterizing the different CD8 + T cell subpopulations and 2 , 3 ) . In the presence ofpersistent antigen CD8 + T cells were their underlying driving mechanisms is an active field of found to have lost cytolytic activity completely over time research contributing to our understanding of protective (Moskophidis et al. , 1993 , Nature , vol . 362 , 758 - 761) . It was immunity in successful pathogen clearance , and of T cell subsequently found that dysfunctional T cells can differen regulation during uncontrolled tumor growth and chronic tially produce IL - 2 , TNFa and IFNg in a hierarchical order infections. Recent advances on this front have enabled the (Wherry et al. , 2003 , J . Virol. , vol. 77 , 4911 - 4927 ) . Recent US 2019 / 0262399 A1 Aug . 29 , 2019 studies also suggest that Tbet and Eomes regulate early and provide additional and preferably improved molecular tar terminally exhausted T cells with distinct cytokine profiles gets involved in immune responses, as well as therapeuti ( Paley et al. , 2012 , supra ) and that these subpopulations cally useful substances and compositions impinging on such coexist in both murine models and in humans with chronic molecular targets to modulate immune responses. infections over a long period of time (Buggert et al. , 2014 , [0014 ] Citation or identification of any document in this PLoS Pathog ., vol. 10 , e1004251 ). application is not an admission that such document is [0010 ] These findings show that even dysfunctional T available as prior art to the present invention . cells can exist in various functional states . It is therefore likely that a plethora of different CD8+ T cell dysfunctional SUMMARY OF THE INVENTION states , regulated by multiple molecular pathways , is present [0015 ] The various aspects of the invention as disclosed in across different diseases and during different stages of this specification are based , at least in part , on the novel disease progression , distinctively contributing to immune discovery of useful markers , marker signatures and molecu control. Deciphering molecular pathways associated with lar targets associated with immune cell dysfunction and / or the various cytokine profiles of dysfunctional CD8 + T cells activation . More particularly , certain of the present markers, is crucial to gain a better understanding of the heterogeneity marker signatures and molecular targets correlate with the and function of the CD8 + response during unsuccessful loss of effector function of the immune cells and are advan antigen clearance, and can greatly contribute to the design of tageously distinct, separate or uncoupled from , or indepen targeted therapies. dent of the immune cell activation status . Certain other of [0011 ] Dysfunctional CD8 + T cells from LCMV infected the present markers , marker signatures and molecular targets mice ( Blackburn et al. , 2009, Nature immunology 10 , 29 - 37 ; correlate with immune cell activation and are advanta Wherry et al. , 2007 , Immunity 27, 670 -684 ) and cancer geously distinct, separate or uncoupled from , or independent ( Baitsch et al. , 2011, J Clin Invest 121 , 2350 -2360 ; Fourcade of the immune cell dysfunction status . et al ., 2010 , The Journal of experimental medicine 207 , [0016 ] Previously , obtaining molecular signatures for T 2175 - 2186 ; Matsuzaki et al. , 2010 , Proceedings of the cell dysfunction has been complicated by the fact that T cell National Academy of Sciences of the United States of dysfunction arises from chronic T cell activation , whereby America 107 , 7875 - 7880 ; Sakuishi et al. , 2010 , The Journal molecular signatures of T cell dysfunction and activation are of experimentalmedicine 207, 2187 -2194 ) differ profoundly closely intertwined . Hence , co - inhibitory receptors that from memory CD8 + T cells, and co - express multiple co mark dysfunctional T cells are also up -regulated during T inhibitory receptors such as PD - 1 , Lag -3 , and Tim - 3 . Indeed , cell activation , where they function to contract the effector therapeutic targeting of co - inhibitory receptors with block T cell population and restore immune homeostasis . Further ing antibodies has achieved great success in cancer patients . more , dysfunctional CD8 + T cells and activated CD8 + T However , many patients still fail to respond and some cells both up - regulate genes that regulate activation of the cancers are refractory to these therapies (Restifo et al . , 2016 , cell cycle , T cell homing and migration and effector mol Nat Rev Cancer 16 , 121- 126 ) . Thus, to identify novel ecules such as granzymes , and both down - regulate memory therapeutic targets and stratify patients , it is important to cell gene signatures (Wherry et al. 2007 , supra ; Doering et better understand the dysfunctional T cell state . al. 2012 , supra ) . Indeed , T cell “ dysfunction ” may have [0012 ] A major challenge to developing therapies that likely evolved as a physiological process to carefully bal specifically target the dysfunctional CD8 + T cell state is that ance T cell activation and self- regulation in the face of current markers and transcriptional signatures of dysfunc chronic antigen persistence, thereby limiting immunopathol tion are closely intertwined with the activated CD8 + T cell ogy and minimizing collateral damage to the host . state (Doering et al ., 2012 , Immunity 37, 1130 -1144 ; Fuertes [0017 ] The present inventors devised an integrated experi Marraco et al. , 2015 , Frontiers in immunology 6 , 310 ; Tirosh mental and computational approach to systematically dissect et al. , 2016 , Science 352 , 189 - 196 ) . This is not surprising the molecular pathways associated with activation and “ dys given that T cell dysfunction arises in the face of chronic T function ” within CD8 + tumor - infiltrating lymphocytes cell activation . Thus, both dysfunctional CD8 + T cells and ( TILs) , allowing to uncouple molecular signatures for T cell activated CD8 + T cells up - regulate genes involved in acti dysfunction and activation . The present analysis identifies vation of the cell cycle , T cell homing and migration , as well gene modules that are uniquely associated with the dysfunc as effector molecules, such as granzymes and co - stimulatory tional T cell state and activated T cell state , and key and co - inhibitory receptors that mark T cells for subsequent molecular nodes that control them . The present markers , regulation (Giordano et al. , 2015 , EMBO J 34 , 2042 -2058 ; marker signatures and molecular targets thus provide for Wherry et al. , 2007 , supra ) . Moreover, both cell types new ways to evaluate and modulate immune responses , such down -regulate memory cell gene signatures (Doering et al. , as to specifically evaluate and target the dysfunctional T cell 2012 , supra ; Wherry et al ., 2007 , supra ). Indeed , T cell state while leaving T cell activation programs intact . dysfunction likely evolved as a physiological process to 0018 ] The present inventors further realised that the acti balance T cell activation with self- regulation in the face of vation and dysfunction module scores were negatively cor chronic antigen persistence , thereby limiting immunopathol related across T cells , such that a higher expression of one ogy. As a result , it has been challenging to identify markers module ' s genes by a cell predicts lower expression of the and approaches that would specifically target the dysfunc other module ' s genes in the same cell . Hence , the dysfunc tional T cell state , while preserving the activated T cell state , tion and activation transcriptional signatures are enriched in aswell as to identify bona fide dysfunctional T cells in vivo . different cells , and the activation versus dysfunction mod [0013 ] Consequently , there exists a continuous need to ules can be distinguished in individual T cells in vivo . The provide additional and preferably improved markers, prod inventors identified genes and gene products differentially ucts and methods allowing to determine the functional state upregulated in T cells expressing the dysfunction module , of immune cells. Likewise , there exists a continuous need to which thus provide useful markers , marker signatures and US 2019 /0262399 A1 Aug . 29 , 2019 molecular targets specifically for dysfunction in T cells . The [ 0023 ] d ) a signature comprising or consisting of one or inventors also identified genes and gene products differen more markers selected from the group consisting of CD83 , tially upregulated in T cells expressing the activation mod TNFRSF4 , CD74 , CCR7, CD81 , TNFSF4 , KIT , ITGB1, ule , which thus provide useful markers , marker signatures CD200 , TNFSF8 , CD160 , CD82 , ITGB3 , CD44 , ALCAM , and molecular targets specifically for activation in T cells . GYPC , IL2RA , CDON , LAG3 , CD9 , CSF2RA , FAS , CD97 , [0019 ] Accordingly, an aspect of the invention provides a TNFSF9, BSG , IFNAR1, TRAF3 , CD96 , and TNFRSF9; method of detecting dysfunctional immune cells comprising 10024 ] e ) a signature comprising or consisting of one or detection of a gene expression signature of dysfunction more markers selected from the group consisting of CD83 , selected from the group consisting of: CD81, TNFRSF4 , CXCL16 , IL21R , and IL18R1; [0025 ) f ) a signature comprising or consisting of one or [ 0020 ] a ) a signature comprising or consisting of one or more markers selected from the group consisting of REL , more markers selected from the group consisting of CD83 , BCL6 , MNDA , BHLHE40 , NFKB2 , ZHX2, KLF4 , CCRS, TNFRSF4 , CD74 , CCR7, TNFSF11 , CD81 , NFKB1, NFKBIA , RARG , FOSB , HIVEP1, ZFP36L1, TBC1D4 , REL , PLK2 , XCL1 , TNFSF4 , SLC2A6 , NFAT5 , ELK3, JUNB , LIMK1, TGIF1, KDM2B , IRF5, A1836003 , LAD1, 1700019D03RIK , BCL6 , MNDA , RELB , HSF2 , HIF1A , NR4A3 , PHTF2, STAT5A , DTX3 , RAMP3 , GPM6B , BHLHE40 , AXL , ECE1, FILIPIL , KIT , NRIP1, IRF8 , STAT3 , NCOA3 , CALCOCO1, PCGF5 , ITGB1 , CCL1, NFKB2, PLXDC2, ARC , DUSP4 , CD200 , NFKBIE , ETV6 , RNF19A , STAT4 , NR4A2, NFKBIB , TRAF1 , ZHX2, NCF1, CCDC28B , PTPRS , PERI, GTF2A1, SPRY1, TFE3 , TGIF2 , RORA , RPL6 , STOGALNAC3 , TUSC3 , PDCDILG2, SDHAF1 , ARAP2, EGR2 , FOXP4 , TBLIX , KDM4A , COPS2 , FOS , DEDD , KLF4 , E130308A19RIK , FAM46A , TNFRSF18 , SYNJ2 , SOSTMI, NT5C , PIAS4 , ZMYM2, DMTF1 , AEBP2 , CYTH3, TNFSF8 , CD160 , RPL10 , CRTAM , RAB6B , TRPSI , SP3, HBP1, NR4A1, TLE3 , RPL7 , MED21, PTGER2, NFKB1, ANKRD46 , STOGALNAC6 , ITPR1, DRAP1, TCF7 , CREB3L2, ZFHX2, KDM5B , and NR3C1; ITM2C , BTLA , TSPAN32 , CD82 , NFKBIA , MS4A4C , or RARG , NRGN , TRIB1, ZC3H12D , BMYC , IFI27L2A , 10026 ] g ) a signature comprising or consisting of two or GADD45B , NAPSA , KLRB1F, RASGEF1A , FOSB , MAP3K8, HIVEP1, SSH1, RABGAPIL , ZFP36L1, more markers each independently selected from any one of ARL4D , CACNAIS , NFAT5 , DNAJC12 , SOWAHC , SDF4 , the groups as defined in any one of a ) to f ) . TMEM120B , DUSP1, ELK3, JUNB , GRAMD1B , LIMK1, [0027 ] In certain embodiments , the signature comprises, ZFC3H1, OSTF1, LTA , DNMT3A , BCL7C , TSPAN13 , consists essentially of, or consists of one or more markers ASNSD1, TGIF1, NRN1, SYNGR2 , MSI2 , UAP1, selected from the group consisting of CD83 , CD81 , and UNC93B1, JAK2, KDM2B , ANXA5, PRDX2, TMEM173 , TNFRSF4 . PHACTR2, CCDC104 , CEP85L , IRF5 , INF2 , ITGB3 , 10028 ] In certain embodiments , the signature further com MPC1, BCL2A1D , PARP3 , ASAP1, MRPS6 , RELB , prises one or more additional markers of dysfunction . In an FAM110A , GPR68 , NRP1, CAPG , SCYL2 , SAMD3 , embodiment , the one or more additional markers of dys H2- AB1, HSF2 , CD44 , STX6 , POLG2, TESPA1, ALCAM , function is a co - inhibitory receptor selected from the group NSMF, LRRC8D , HIF1A , PACSIN1, PKP4, ASS1, NR4A3 , consisting of PD1, CTLA4 , TIGIT , TIM3 , LAG3, KLRC1, ENO3, GYPC , KIF3B , IL2RA , RAB37 , SGMS1 , HLCS , BTLA , NRP1, CD160 , CD274 , IDO , CD200 , CD244 , SEMA6D , NMRK1, SLC17A6 , SLC39A1, RPS4X , CDON , KLRD1, LAIRI, CEACAMI, KLRAT , FAS, GPR132 , ZFP445 , LAG3, RPS26 , PHTF2 , CST3 , CD9, STAT5A , CD74 , SLAMF6 , CD5 , GPR35 , CD28 , CD44 , and ABCA3 , CSF2RA , DTX3, RSPH3A , NRIP1, SDHA , PTGER4 . In an embodiment, the one or more additional PNKD , FLNB , MGRN1, SLC26A2 , HMOX2 , PEX16 , markers ofdysfunction is selected from the group consisting INPP4A , TNFRSF25 , IRF8 , RGCC , IFITM2, TNFSF14 , of PD1 , CTLA4 , TIGIT , TIM3, LAG3 , and KLRC1. NSUNO , STAT3 , PFKFB3 , TYROBP, HTRA2 , KLRI2 , [ 0029 ] In certain embodiments , the signature comprises , CTSS , ARL5C , KLHL24 , SESN3, GM5424 , FAS , NCOA3 , consists essentially of, or consists of at least two markers , or FAM53B , CALCOCO1, ERGIC3 , 4930523CO7RIK , at least three markers , or at least four markers , or at least five PCGF5 , ANXA4 , and HERPUD1; markers , or six or more markers; or the signature comprises , consists essentially of, or consists of at least one , at least [ 0021] b ) a signature comprising or consisting of one or two, or at least three markers each independently selected more markers selected from the group consisting of CD74 , from any one of the groups as defined in c ) or d ) ; or the CCR7, TBC1D4 , SLC2A6 , BCL6 , JAK2, PARP3 , ASAP1, signature comprises, consists essentially of, or consists of at RELB , H2- AB1 , CD44 , ABCA3 , PFKFB3 , SESN3, FAS , least one, at least two, or at least three markers selected from 4930523CO7RIK , PCGF5 , TNIP1, SPRY1, NCOA7, the group as defined in f ) ; or — the signature comprises or RPLPO , SMIM8, ANTXR2, NSMCE1, DEDD , B3GNT2 , consists of at least one , at least two , or at least three markers CABLES1, SLAMF6 , UBL3 , NR4A1 , ATG7, and KDM5B ; each independently selected from any one of the groups as [ 0022 ] c ) a signature comprising or consisting of one or defined in c ) or d ) and at least one, at least two , or at least more markers selected from the group consisting of CD83 , three markers selected from the group as defined in f ) CCRS , TNFRSF4 , CD74, CCR7 , TNFSF11 , CD81 , XCL1, [0030 ] In certain embodiments , the signature comprises , TNFSF4 , AXL , ECE1, KIT , ITGB1, CCL1 , CD200 , or consists essentially of, or consists of at least one , at least TNFRSF18 , TNFSF8, CD160 , PTGER2 , BTLA , TSPAN32 , two, or at least three markers selected from the group CD82 , KLRB1F , LTA , ANXA5 , ITGB3 , NRP1, H2- AB1, consisting of CD83 , CD81 , TNFRSF4 , CCR8 , TNFSF11 , CD44 , ALCAM , GYPC , IL2RA , CDON , LAG3, CD9, CCL1 and AXL ; or the signature comprises , consists essen TNFSF14 , FAS , GD12 , TNIP1, IL21R , IL18R1, H2- AA , tially of, or consists of CD83 ; or CD83 and CD81 , optionally NR4A2, IL18RAP, CD97 , TNFSF9 , IRAK1BP1, GABA with one or more of TNFRSF4 , CCR8 , TNFSF11 , CCL1 RAPL1, TRPV2, EBAGY, GRN , RAMP1 , AIMP1, BSG , and /or AXL ; or CD83 and TNFRSF4 , optionally with one or IFNAR1, PRKCA , TRAF3 , CD96 , TNFRSF9 , and NR3C1; more of CD81, CCR8, TNFSF11 , CCL1 and / or AXL ; or US 2019 /0262399 A1 Aug . 29 , 2019

CD83 and CCR8 , optionally with one or more CD81, CDCA3 , ESPLI , CASC5 , PBK , KIF2C , SGOLI, CDK1, TNFRSF4 , TNFSF11 , CCL1 and / or AXL ; or CD83 and SHCBP1, ASPM , FBXO5, MIS18BP1 , SPAG5 , KIF4 , TNFSF11, optionally with one or more of CD81 , TNFRSF4 , ASF1B , BUB1B , AURKB , NCAPG , DEPDC1A , ESCO2 , CCR8, TNFSF11 , CCL1 and /or AXL ; or CD83 and CCL1, CDCA2 , BC030867 , KIF20A , HIST1H2AK , SMC2 , ECT2 , optionally with one or more of CD81, TNFRSF4 , CCRS , RRM2, MK167, 2810417H13RIK , CIT , GTSE1, NCAPG2 , TNFSF11, and /or AXL ; or CD83 and AXL , optionally with NCAPH , CDCA8, SAPCD2, NEK2, CEP55 , CDCA5 , one or more of CD81 , TNFRSF4 , CCR8, TNFSF11 , and / or TOP2A , CCNB2 , MASTL , ARHGAP19 , AURKA , KIF23 , CCL1. CCNB1, RAD51, TACC3, MELK , STMN1, HIST1H2AE , [0031 ] Another aspect of the invention provides a method HIST1H4D , CENPH , HIST1H1B , CDC25C , CCDC34 , for determining whether or not an immune cell has a CDC20 , KIF11 , ARHGAP11A , 4930427A07RIK , dysfunctional immune phenotype , said method comprising FAM83D , INCENP, MAD2L1 , HIST1H2AO , CKAP2 , determining in said immune cell the expression of the NDC80 , RAD51AP1, NUF2 , E2F7 , CKSIB , NCAPD2 , signature of dysfunction as defined above , whereby expres GEN1, ANLN , TICRR , POLO , PRC1, TTK , RAD54B , sion of the signature indicates that the immune cell has a E2F8 , STIL , KIF18A , PARPBP, CDC45 , BIRC5 , KIF15 , dysfunctional immune phenotype . SKA2 . KIF20B , TK1, PLK4 . FANCD2 , CENPM , [ 0032 ] A further aspect of the invention relates to a C330027C09RIK , HIST2H4, SKA3 , RRM1, TROAP, method for determining whether or not a patient would RAD54L , KNTC1, ZWILCH , CLSPN , TPX2, CMC2, benefit from a therapy aimed at reducing dysfunction of TCF19 , MCM10 , HMGB2, HIST1H3C , HIST1H1E , immune cells or a therapy aimed at upregulating of an UBE2T, CHTF18 , TUBAIB , TUBBS , H2AFX , GPSM2, immune response , the method comprising determining , in SPC24 , UHRF1 , TRIP13 , PMF1 , ZFP367, RACGAP1, immune cells from said patient the expression of the signa CDC25B , UBE2C , CDKN3, CENPI, HIST1H3B , KPNA2, ture of dysfunction as defined above , whereby expression of HJURP, BRCA1, WDR62 , CENPN , GMNN , POC1A , the signature indicates the patient will benefit from the TMPO , KNSTRN , FANCI, CENPF , 6430706D22RIK , therapy ; or for determining whether or not a patient would CKS2 , DIAP3 , WDR67 , FIGNL1, BRCA2, HMGB3 , benefit from a therapy aimed at increasing dysfunction of MYBL2, PKMYT1 , TRAIP , RFC5 , CEP128 , POLAI, immune cells or a therapy aimed at downregulating of an ANKLEI, HMGB1, FEN1, H2AFZ , TUBB4B , CTC1, immune response , the method comprising determining, in CKAP5 , CDC6 , LIG1, POLE , MCMI, RFWD3 , HMGN2 , immune cells from said patient the expression of the signa TYMS, CENPP , NCAPD3 , SUV39H1, A730008H23RIK , ture of dysfunction as defined above , whereby expression of TUBA1C , EMEI, EXO1, PTMA, BLM , ULBP1, the signature indicates the patient will likely not benefit from 1190002F15RIK , CDC7 , DLGAP5 , TUBG1, PRIM2, the therapy. TMEM48 , NRM , CENPA , BARD1, HAUS4 , RCC1 , [0033 ] Another aspect of the invention provides a method LMNB1, and HIST1H2AG ; for determining the efficacy of a treatment of a patient with [0038 ] b ) a signature comprising or consisting of one or a therapy, particularly immune therapy, said method com more markers selected from the group consisting of prising determining in immune cells from said patient the HIST1H1E , HMGB1, HAUS4 , RCC1, HAUS5 , REEP4 , expression of the signature of dysfunction as defined above SLBP, FKBP2, ARSB , HIST1H3E , RAD18 , RAD50 , TAF6 , before and after said treatment and determining the efficacy ANAPC5, FANCG , CTCF , TONSL , LMNB2 , SEPHS1, of said therapy based thereon . HNRNPA2B1 , ANAPC15 , STARD3NL , 2700029MOORIK , [0034 ] A further aspect of the invention relates to a DPY30 , SDF2L1, RDM1, CDCA7L , MEAF6 , MYEF2 . method for determining the suitability of a compound for DNAAF2 , POLR3K , IPP, TARDBP, GPAA1, KPNB1, modulating a dysfunctional immune phenotype and / or FTSJD2 , RPRD1B , HISTIHIC , DPYSL2 , TAF12 , modulating an immune response , said method comprising ARPP19 , TMCO1, EXOC4 , ASRGL1, CPSF6 , EIF2D . contacting an immune cell expressing the signature of CCNH , MYG1, VMA21 , TFIP11 , NDUFAB1, NUP35 , dysfunction as defined above with said compound and GRPEL1, C1D , GBP3 , CYB5B , PPM1G , SRSF10 , PIGF , determining whether or not said compound can affect the KLRED , COG4 , PDIA4 , CDT1 , DUSP19 , ACAT2 , expression of the signature by said cell . COMMD3, HCFC2 , LMAN1, ABHD10 , TIMM50 , CALU , 10035 ] Further aspects of the invention relate to an isolated AP1M1, GGH , GCDH , MRPS33 , TAX1BP3 , DYNLT1C , immune cell characterised in that the immune cell comprises ERP44 , TMEM129 , COG2, TMEM167 , RBX1, the signature of dysfunction as defined above ; to a popula 0610009020RIK , PSMB9, PUF60 , LSM4 , PEX19 , NTANI, tion of said immune cells ; to a composition or pharmaceu ELP2 , AKR1B3 , PHF11B , POLC3 , ZFP142 , PRADC1, tical composition comprising said immune cell or said TMEM209 , DYNCILII , FARSB , MTHFSD , immune cell population , and to a method for eliciting 1700052N19RIK , SARS2 , LAMTORI, ALDH3A2. immune tolerance in a subject comprising administering to EHBP1L1, D16ERTD472E , CCDC127 , COMMD10 , the subject said immune cell or said immune cell population DNAJC14 , MRPL16 , SSBP1 , EXOSC4, UPF1, DOHH , or said pharmaceutical composition . PTPN23 , ZADH2 , ARFIP1, STX18 , VMP1, TCEAI, [ 0036 ] Another aspect of the invention provides a method ERGICI, PIAS3 , RAD17 , EXOSC3 , ACOTS , MINA , of detecting activated immune cells comprising detection of LRRK1, MOGS , METTL10 , CERS4 , ATPBD4 , ZDHHC6 , a gene expression signature of activation selected from the MAVS , PARL , GNG2, CD200R4 , USF1 , TYK2, SNAPC1, group consisting of: RBM18 , VPS53, ACTR10 , and DPCD ; [0037 ] a ) a signature comprising or consisting of one or [ 0039 ] c ) a signature comprising , consisting essentially of, more markers selected from the group consisting of or consisting of one or more markers selected from the group NUSAP1 , CCNA2, NEIL3 , SPC25 , HIST1H2AB , consisting of HMGB1, ULBP1 , REEP4 , HMMR , CMTM7, CKAP2L , PLK1, HIST2H3C2 , MXD3, FAM64A , BUB1, SIVAI, PAQR4 , ATPIF1 , NUP85 , HSPA2 , ENTPD1, FOXM1, HIST2H3B , KIF22 , CENPE , SKAI, CCNF, HNRNPU , CLIC4 , FLOT2 , ENOX2, ENPP1, TFRC , US 2019 /0262399 A1 Aug . 29 , 2019

HAVCR2, CD2BP2 , LGALSI , PGRMC1, USP14 , SNW1, RASSF7 , KEAP1, CAMTAI, MED15 , MED8 , ENTPD5 , ATP6AP2 , CCL3 , ILLORA , ARNT2, KLREI , ING3 , CREBZF , TMF1, BOLA2 , IKZF2 , ARID5B , SND1, CLPTM1, ITGAV , KLRC1, PDIA4, SP1, CCR5 , ADAM17 , PHF20 , ZBTB24 , SMARCE1, ARID1A , MTA2, KLF10 , CXCL10 , IGSF8 , ADAM10 , IFNG , TNFRSF9 , CD244 , CCNC , IRF8 , ASH2L , MIER3 , UIMC1, ELK3 , AEBP2 , CTLA4 , ERP44 , PSENI, LY6A , CCRL2 , NCOR2 , BRF1, SPRY2, RLF, IRF3 , NFYA , FLII , BCLAF1 , FIZI, HSP90AA1, IGF2R , PGP, KLRC3, CKLF, TNFRSF1A , SP3 , PARD6A ,MTA1 , CCDC71 , PBX4 , PHF5A , NACC1, TMX3 , KLRC2 , PDE4D , SMPD1, IDE , SERPINE2 , ATF7IP , HDAC2, GATAD2B , MGA, TRIM27 , TCF20 , LRPAP1, CSF1, CYSLTR2, LSM1, GRN , IL1F9, LDLR , RUFY2, GOLGB1, PYGO2 , ZFPL1, HIF1AN , BUD31, CD80 , GPR174 , MIF , MYO9B , ROCK1, GPR56 , GPR160 , TERF2 , NOTCH2, UBN1, DNM2, PPIE , HIVEP2 , RAC1, PTPN11 , CMTM6 , ADA , NOTCH2, GPI1 , GDI2 , TBC1D2B , COPS2 , TAF2 , PNRC2 , ESRRA , IRF9 , SAP30 , P4HB , NRP1, F2R , AGTRAP, PGLYRP1, STX4A , MIER1, EYA3, NCOA3, LMO4 , PREB , NMI, ZBTB40 , ADAMS, LYST, ITGAI, XPOT, KLRK1, CX3CR1, PCGF1 , HMGA1, SARNP, HSBP1, CREM , PTTG1, SEPT2 , CCL4 , CAST, RPS6KB1, H2- M3 , LAG3 , CD99L2 , AGGF1, BMI1 , NCOA2, CBX4, TFEB , XAB2 , ERF , PDCD1 , ECE1 , EZR, NR4A2 , SEMA4D, NAMPT, PEAR1 , SAP18 , RYBP, MED17 , GTF2H3, ZMYND19 , SKIL , IL12RB1, CD200R4 , CD48 , LAMP2 , IRAK2, CXCR6 , LZTR1, FOXK2, HTATSF1, TBL1X , PLEKHF2 , CIC , GPR65 , and GCNT1 ; TCEA1, CIZI, TAF9B , TULP4 , HMG20B , PIAS3, MED21 , [ 0040] d ) a signature comprising , consisting essentially of, TBL1XR1, UTP6 , KDM3A , TBX21 , ZBTB5 , EGRI , or consisting of one ormore markers selected from the group ZFYVE27 , PHTF1, THOC2, TRAK1, GTF2H1, RNF14 , consisting of HMMR , SIVAL, ENTPD1, ENPP1, TFRC , LCORL , IKZF3 , HEXIMI, RNF19A , FLII , MED27 , CD2BP2 , ENTPD5, ITGAV , KLRC1, CCR5 , ADAM17 , THAP7 , MAFF , GATA3 , PNN , CBX8, ADNP, NAB2, TNFRSF9 , CD244 , CTLA4 , IL3RA , IGF2R , TNFRSF1A , MED13 , NR4A2, PHRF1, SREBF1 , STAT2 , CHD2, CD80 , ADAM8 , LAGU , MGL2 , CD99L2 , SEMA4D , MEF2D , TRIP12 , MLX , RLIM , ABT1, KAT5 , ETS2 , IL12RB1, CD200R4, CD48 , and LAMP2 ; SCAP, RELA , BATF , MED26 , KDM2B , KDM6A , [ 0041 ] e ) a signature comprising , consisting essentially of, CBFA2T2 , PIAS4 , USF1, ZBTB7A , RNF25, ZBTB1 , or consisting of one or more markers selected from the group MED23 , ZBTB41 , PHF2, KAT2B , KDM2A , CIR1 , consisting of MXD3, FOXM1, E2F7 , RAD54B , E2F8 , ZC3H15 , ZEB2 , REXO4, ZSCAN21, BLZF1, and CENPB ; TCF19 , HMGB2, UHRF1 , TRIP13 , PMF1, BRCA1, or BRCA2 , HMGB3, MYBL2 , HMGB1, PTMA , WHSC1, [0042 ] f) a signature comprising, consisting essentially of, CHAF1A , RBL1, DNMT1, CCNE1, DEK , E2F2 , CHAF1B , or consisting of two or more markers each independently EZH2, G2E3 , WDHD1, SUZ12 , TFDP1 , RBBP4 , selected from any one of the groups as defined in any one of CASP8AP2 , RFC1, CDCA4 , RBBP8, SSRP1, ANAPC11 , a ) to e ) . TERF1, POLE3, CBFB , CBX3, CTCF , MED30 , PBRM1, [0043 ] In certain embodiments , the signature further com TFDP2, ITGB3BP, CBX6 , NRF1, BAZIB , E2F3 , PIASI, prises one or more additional markers of activation . In ILF2 , HDAC6 , TIMELESS , SMARCA5, MYEF2 , TAR certain embodiments , the one or more additional markers of DBP, EED , HMGXB4, METTL14 , E2F4 , LINO, MTF2 , activation is a co -stimulatory receptor selected from the MAZ , ATF1 , TAF1, TOX , NFYB , HNRNPD , SMARCB1 , group consisting of TNFRSF9 , TNFRSF4 , TNFSF4 , UHRF2 , ASXL1, MED14 , NAB1, BRD , ILF3 , MED7, TNFRSF18 , TNFSF11 , TNFRSF13C , CD27 , CD28 , CD86 , RB1, HDAC3, ERH , TSG101 , RNPS1 , CCNH , NONO , ICOS, and TNFSF14 . DEAF1, ZFP91 , PKNOX1, L3MBTL2 , CDC5L , SP4 , [0044 ] In certain embodiments . the signature comprises , KLF11, SMARCC1, HDAC1, GTF2H5 , SMARCC2, consists essentially of, or consists of at least two markers, or RUVBL2 , ZBTB45 , NMRALI, WIZ , ING1 , FOSB , CID , at least three markers , or at least four markers , or at least five DICER1, E2F1 , THRAP3 , RNF4 , TSHZ1, SF1 , GABPA , markers, or six or more markers ; or the signature comprises , GABPB1, SMYD4, CBY1 , ARNT2 , GABPB2, RFX1, consists essentially of , or consists of at least one , at least MORF4L2, ZFYVE19 , SUB1, HCFC1, TARBP2, GTF3C2 , two , or at least three markers each independently selected POU2F1 , ZNHIT3 , TBP, CAND1, PCM1, BAZIA , ATF6 , from any one of the groups as defined in c ) or d ) ; or the SREBF2 , YAF2 , TCERG1, BRPF1, LITAF , SMYD3, signature comprises, consists essentially of, or consists of at RNF5 , DPF2, SMARCD2, E2F5 , PML , GMEB1, SP1, least one, at least two , or at least three markers selected from PSIP1, SP2, CNOT6 , OVCA2 , PFDN1, COMMD3 , ING2, the group as defined in e ) ; or the signature comprises, MYNN , HCFC2, AES , LRRFIP2 , GTF2E2 , YEATS4 , consists essentially of, or consists of at least one , at least CNOT2 , MYCBP, PA2G4, TOE1, SMARCD1, NFYC , two, or at least three markers each independently selected GMEB2, CEBPB , IKZF5 , TFAM , NFIL3 , CNOT4 , COPS5 , from any one of the groups as defined in c ) or d ) and at least GTF2A2, CNOT1 , SIN3A , GTF2F1, TSC22D2, ZBTB2 , one , at least two , or at least three markers selected from the MED4 , RUVBL1, AIP, TRIM28 , GTF2B , DEDD , CREB1, PRDM1, CTBP1, GTF3C5, TAF10 , PSMC3, MED31 , group as defined in e ). RBX1, FUS , POBP1, ELF2 , ATF2 , CNOT8 , NCOR2 , [0045 ] In certain embodiments , the signature comprises, SMARCA4, GNPTAB , TAF6L , CRAMPIL , TCF3 , consists essentially of, or consists of at least one , at least CEBPG ,GTF3C3 , TAF3 , ID2, YBX1, TAF11, YY2, MEN1, two , or at least three markers selected from the group PHF6 , PHF17 , SMARCAD1, RBL2 , HTT , HDAC5 , ING5 , consisting of ULBP1 , HMMRhigh and REEP4 ; or the sig CXXC1, NFKBIL1, CSDA , GTF3A , PRDM10 , MECP2 , nature comprises , consists essentially of, or consists of SUDS3, CUX1, ZBTB22 , PLRG1, MED24 , ETV5 , ULBP1 and one or both of HMMRhigh or REEP4 . SFMBT1, HLTF , MEF2A , JAZF1, GATADI, ZBTB11 , [0046 ] A further aspect of the invention provides a method ZNRD1, RBPJ, XRCC6 , GTF2A1 , CTNNB1, PURB , for determining whether or not an immune cell has an CNOT7 , ZZEF1, TAF15 , TSC22D4 , HIRA , ELF4 , activated immune phenotype , said method comprising deter MED13L ,MMS19 , MBD1, VHL , VPS72, FOXJ3 , UBE2K , mining in said immune cell the expression of the signature US 2019 / 0262399 A1 Aug . 29 , 2019 of activation as defined above , whereby expression of the TRAF1, ZHX2, NCF1 , CCDC28B , PTPRS, signature indicates that the immune cell has an activated STOGALNAC3 , TUSC3, PDCDILG2, SDHAF1, ARAP2 , immune phenotype . KLF4 , E130308A19RIK , FAM46A , TNFRSF18 , SYNJ2 , [0047 ] Another aspect of the invention relates to a method CYTH3 , TNFSF8, CD160 , RPL10 , CRTAM , RAB6B , for determining whether or not a patient would benefit from PTGER2 , NFKB1, ANKRD46 , STOGALNAC6, ITPR1, a therapy aimed at reducing activation of immune cells or a ITM2C , BTLA , TSPAN32 , CD82 , NFKBIA , MS4A4C , therapy aimed at downregulating of an immune response , RARG , NRGN , TRIB1, ZC3H12D , BMYC , IF127L2A , the method comprising determining, in immune cells from GADD45B , NAPSA , KLRB1F , RASGEF1A , FOSB , said patient the expression of the signature of activation as MAP3K8, HIVEP1, SSH1, RABGAPIL , ZFP36L1, defined above , whereby expression of the signature indicates ARL4D , CACNAIS , NFAT5 , DNAJC12 , SOWAHC , SDF4 , the patient will benefit from the therapy ; or for determining TMEM120B , DUSP1, ELK3, JUNB , GRAMD1B , LIMK1, whether or not a patient would benefit from a therapy aimed ZFC3H1, OSTF1, LTA , DNMT3A , BCL7C , TSPAN13 , at increasing activation of immune cells or a therapy aimed ASNSD1, TGIF1 , NRN1, SYNGR2, MSI2 , UAP1, at upregulating of an immune response , the method com UNC93B1, JAK2, KDM2B , ANXA5, PRDX2 , TMEM173 , prising determining, in immune cells from said patient the PHACTR2, CCDC104 , CEP85L , IRF5 , INF2, ITGB3, expression of the signature of activation as defined above , MPC1 , BCL2A1D , PARP3 , ASAP1, MRPS6 , RELB , whereby expression of the signature indicates the patient FAM110A , GPR68 , NRP1 , CAPG , SCYL2 , SAMD3. will likely not benefit from the therapy. H2 -AB1 , HSF2 , CD44 , STX6 , POLG2, TESPA1, ALCAM , [ 0048 ] A further aspect of the invention relates to a NSMF, LRRC8D , HIF1A , PACSIN1, PKP4 , ASS1, NR4A3, method for determining the efficacy of a treatment of a ENO3 , GYPC , KIF3B , IL2RA , RAB37 , SGMS1, HLCS , patient with a therapy, particularly immune therapy , said SEMA6D , NMRK1, SLC17A6 , SLC39A1, RPS4X , CDON , method comprising determining in immune cells from said ZFP445 , LAGU , RPS26 , PHTF2 , CST3 , CD9 , STAT5A , patient the expression of the signature of activation as ABCA3 , CSF2RA , DTX3, RSPH3A , NRIP1, SDHA , defined above before and after said treatment and determin PNKD , FLNB , MGRN1, SLC26A2 , HMOX2, PEX16 , ing the efficacy of said therapy based thereon . INPP4A , TNFRSF25 , IRF8 , RGCC , IFITM2, TNFSF14 , 10049 ] Another aspect of the invention relates to a method NSUN , STAT3 , PFKFB3, TYROBP, HTRA2, KLRI2 , for determining the suitability of a compound for modulat CTSS , ARL5C , KLHL24 , SESN3, GM5424 , FAS , NCOA3, ing an activated immune phenotype and /or modulating an FAM53B , CALCOCO1, ERGIC3 , 4930523COORIK , immune response , said method comprising contacting an PCGF5 , ANXA4 , and HERPUD1; immune cell expressing the signature of activation as [0055 ] b ) the group consisting of CD74 , CCR7, TBC1D4 , defined above with said compound and determining whether SLC2A6 , BCL6 , JAK2 , PARP3 , ASAP1, RELB , H2- AB1, or not said compound can affect the expression of the CD44 , ABCA3 , PFKFB3 , SESN3, FAS , 4930523C07RIK , signature by said cell . PCGF5 , TNIP1, SPRY1, NCOA7, RPLPO , SMIM8, 10050 ] Further aspects of the invention provide an isolated ANTXR2, NSMCE1, DEDD , B3GNT2, CABLES1 , immune cell characterised in that the immune cell comprises SLAMF6 , UBL3 , NR4A1, ATG7, and KDM5B ; the signature of activation as defined above ; to a population [0056 ] c) the group consisting of CD83 , CCR8, of said immune cells ; to a composition or pharmaceutical TNFRSF4 , CD74 , CCR7 , TNFSF11, CD81, XCL1 , composition comprising said immune cell or said immune TNFSF4 , AXL , ECE1, KIT, ITGB1 , CCL1, CD200 , cell population , and to a method for eliciting an immune TNFRSF18 , TNFSF8, CD160 , PTGER2, BTLA , TSPAN32 , response in a subject comprising administering to the subject CD82 , KLRB1F, LTA , ANXA5 , ITGB3 , NRP1, H2 -AB1 , said immune cell or said immune cell population or said CD44 , ALCAM , GYPC , IL2RA , CDON , LAG3 , CD9, pharmaceutical composition . TNFSF14 , FAS, GD12 , TNIP1, IL21R , IL18R1, H2 -AA , [0051 ] Another aspect of the invention relates to a method NR4A2 , IL18RAP, CD97 , TNFSF9, IRAK1BP1, GABA of isolating an immune cell as defined in any one of the RAPL1, TRPV2 , EBAG9, GRN , RAMP1, AIMP1, BSG , preceding aspects comprising binding of an affinity ligand to IFNAR1, PRKCA , TRAF3 , CD96 , TNFRSF9 , and NR3C1; a signature gene expressed on the surface of the immune [0057 ] d ) the group consisting of CD83 , TNFRSF4, cell. CD74 , CCR7, CD81 , TNFSF4, KIT , ITGB1, CD200 , [0052 ] A further aspect of the invention provides a method TNFSF8 , CD160 , CD82 , ITGB3, CD44 , ALCAM , GYPC , of treating a subject in need thereof, comprising adminis IL2RA , CDON , LAG3 , CD9 , CSF2RA , FAS , CD97 , tering to said subject an agent capable of modulating the TNFSF9, BSG , IFNAR1, TRAF3 , CD96 , and TNFRSF9 ; immune cell as defined in any one of the preceding aspects . 10058 ] e ) the group consisting of CD83 , CD81 , TNFRSF4 , [0053 ] A further aspect of the invention provides a kit of CXCL16 , IL21R , and IL18R1 ; parts comprising means for detection of the signature as 0059 ] f ) the group consisting of REL , BCL6 , MNDA , defined in any one of the preceding aspects . BHLHE40 , NFKB2 , ZHX2, KLF4 , NFKB1, NFKBIA , [0054 ] Another aspect of the invention relates to an iso RARG , FOSB , HIVEP1, ZFP36L1, NFAT5 , ELK3, JUNB , lated immune cell modified to comprise an altered expres LIMK1, TGIF1, KDM2B , IRF5 , RELB , HSF2, HIF1A , sion or activity of , or modified to comprise an agent capable NR4A3 , PHTF2 , STAT5A , DTX3, NRIP1 , IRF8 , STAT3 , of inducibly altering expression or activity of, one or more NCOA3 , CALCOCO1, PCGF5 , NFKBIE , ETV6 , RNF19A , genes or gene products selected from a ) the group consisting STAT4 , NR4A2 , NFKBIB , PERI, GTF2A1, SPRY1, TFE3 , of CD83 , CCR8, TNFRSF4 , CD74 , CCR7, TNFSF11 , TGIF2 , RORA , RPL6 , EGR2, FOXP4 , TBLIX , KDM4A , CD81, TBC1D4 , REL , PLK2, XCL1, TNFSF4 , SLC2A6 , COPS2 , FOS, DEDD , SQSTM1, NT5C , PIAS4 , ZMYM2, A1836003 , LAD1, 1700019D03RIK , BCL6 , MNDA , DMTF1 , AEBP2 , TRPS1, SP3 , HBP1, NR4A1, TLE3 , RAMP3, GPM6B , BHLHE40 , AXL , ECE1, FILIPIL , KIT , RPL7 , MED21, DRAP1, TCF7 , CREB3L2 , ZFHX2 , ITGB1, CCL1, NFKB2 , PLXDC2 , ARC , DUSP4 , CD200 , KDM5B , and NR3C1 ; or US 2019 / 0262399 A1 Aug . 29 , 2019

[ 0060 ] g ) the group consisting of two or more genes or STX18 , VMP1, TCEA1, ERGIC1 , PIAS3 , RAD17 , gene products each independently selected from any one of EXOSC3, ACOTS , MINA, LRRK1, MOGS , METTL10 , the groups as defined in any one of a ) to f ). CERS4 , ATPBD4 , ZDHHC6, MAVS , PARL , GNG2, [ 0061] Another aspect of the invention provides an iso CD200R4 , USF1, TYK2, SNAPC1, RBM18, VPS53, lated immune cell modified to comprise an altered expres ACTR10 , and DPCD ; sion or activity of, or modified to comprise an agent capable [0063 ] c ) the group consisting of HMGB1, ULBP1, of inducibly altering expression or activity of, one or more REEP4 , HMMR , CMTM7, SIVAI, PAQR4 , ATPIF1 , genes or gene products selected from a ) the group consisting NUP85 , HSPA2, ENTPD1, HNRNPU , CLIC4, FLOT2 , of NUSAP1, CCNA2 , NEIL3 , SPC25 , HIST1H2AB , ENOX2, ENPP1, TFRC , HAVCR2, CD2BP2 , LGALS1, CKAP2L , PLK1, HIST2H3C2 , MXD3 , FAM64A , BUB1, PGRMC1, USP14 , ENTPD5 , ATP6AP2 , CCL3 , ILBORA , FOXM1, HIST2H3B , KIF22 , CENPE , SKA1, CCNF , ARNT2 , KLRE1, CLPTM1, ITGAV , KLRC1, PDIA4, SP1, CDCA3, ESPL1, CASC5 , PBK , KIF2C , SGOL1, CDK1, CCR5 , ADAM17 , CXCL10 , IGSF8 , ADAM10 , IFNG , SHCBP1, ASPM , FBXO5, MIS18BP1, SPAG5, KIF4 , TNFRSF9 , CD244 , CTLA4, ERP44 , PSEN1, LY6A , ASF1B , BUB1B , AURKB , NCAPG , DEPDC1A , ESCO2 , CCRL2 , NCOR2, HSP90AAI, IGF2R , PGP, KLRC3, CDCA2 , BC030867 , KIF20A , HIST1H2AK , SMC2 , ECT2 , CKLF , TNFRSF1A , TMX3 , KLRC2 , PDE4D , SMPD1 , RRM2, MK167 , 2810417H13RIK , CIT , GTSE1, NCAPG2, IDE , SERPINE2, LRPAP1, CSF1 , CYSLTR2, LSM1, GRN , NCAPH , CDCA8 , SAPCD2, NEK2, CEP55 , CDCA5 , IL1F9 , LDLR , CD80 , GPR174 , MIF , MYO9B , ROCK1, TOP2A , CCNB2 , MASTL , ARHGAP19 , AURKA , KIF23 , GPR56 , GPR160 , RAC1, PTPN11 , CMTM6 , ADA , CCNB1, RAD51, TACC3 , MELK , STMN1, HIST1H2AE , NOTCH2, GPI1 , GDI2 , P4HB , NRP1, F2R , AGTRAP , HIST1H4D , CENPH , HIST1H1B , CDC25C , CCDC34 , PGLYRP1 , STX4A , ADAMS, LYST , ITGA1, XPOT, CDC20 , KIF11 , ARHGAP11A , 4930427A07RIK , KLRK1, CX3CR1, SEPT2 , CCL4 , CAST, RPS6KB1, FAM83D , INCENP, MAD2L1, HIST1H2AO , CKAP2 , H2 -M3 , LAG3 , CD99L2 , PDCD1, ECE1 , EZR , NR4A2 , NDC80 , RAD51API, NUF2, E2F7, CKSIB , NCAPD2 , SEMA4D, NAMPT, PEAR1 , IL12RB1 , CD200R4 , CD48 , GEN1, ANLN , TICRR , POLQ , PRC1, TTK , RAD54B , LAMP2 , IRAK2, CXCR6 , GPR65 , and GCNT1; E2F8 , STIL , KIF18A , PARPBP, CDC45 , BIRC5 , KIF15 , [0064 ] d ) the group consisting of HMMR , SIVAI , SKA2, KIF20B , TK1, PLK4, FANCD2, CENPM , ENTPD1, ENPP1, TFRC , CD2BP2 , ENTPD5, ITGAV , C330027C09RIK , HIST2H4, SKA3, RRM1, TROAP , KLRC1, CCR5, ADAM17 , TNFRSF9 , CD244 , CTLA4, RAD54L , KNTC1, ZWILCH , CLSPN , TPX2 , CMC2, IL3RA , IGF2R , TNFRSF1A , CD80 , ADAM8, LAG3, TCF19 , MCM10 , HMGB2, HIST1H3C , HIST1H1E , MGL2 , CD99L2 , SEMA4D , IL12RB1, CD200R4 , CD48 , UBE2T , CHTF18 , TUBA1B , TUBBS , H2AFX , GPSM2, and LAMP2 ; SPC24 , UHRF1, TRIP13, PMF1, ZFP367 , RACGAP1, [0065 ] e ) the group consisting of MXD3, FOXM1, E2F7 , CDC25B , UBE2C , CDKN3 , CENPI, HIST1H3B , KPNA2 , RAD54B , E2F8 , TCF19 , HMGB2, UHRF1 , TRIP13 , HJURP, BRCA1, WDR62 , CENPN , GMNN , POC1A , PMF1, BRCA1, BRCA2 , HMGB3 , MYBL2, HMGB1, TMPO , KNSTRN , FANCI, CENPF , 6430706D22RIK , PTMA , WHSC1 , CHAF1A , RBLI , DNMT1 , CCNE1 . CKS2 , DIAP3 , WDR67 , FIGNL1, BRCA2 , HMGB3, DEK , E2F2 , CHAF1B , EZH2 , G2E3, WDHD1, SUZ12 , MYBL2, PKMYT1, TRAIP , RFC5 , CEP128 , POLAI, TFDP1, RBBP4 , CASP / AP2 , RFC1 , CDCA4 , RBBP8 , ANKLE1, HMGB1, FEN1, H2AFZ , TUBB4B , CTC1 , SSRP1, ANAPC11 , TERF1, POLE3 , CBFB , CBX3 , CTCF, CKAP5 , CDC6 , LIG1, POLE , MCMI, RFWD3, HMGN2, MED30 , PBRM1, TFDP2 , ITGB3BP, CBX6 , NRF1 , TYMS, CENPP , NCAPD3 , SUV39H1, A730008H23RIK , BAZIB , E2F3 , PIASI , ILF2 , HDAC6 , TIMELESS , TUBAIC , EMEI, EXO1, PTMA , BLM , ULBP1 , SMARCA5 , MYEF2 , TARDBP, EED , HMGXB4 , 1190002F15RIK , CDC7 , DLGAP5 , TUBG1, PRIM2, METTL14 , E2F4 , LIN9, MTF2 , MAZ , ATF1 , TAF1 , TOX . TMEM48 , NRM , CENPA , BARDI, HAUS4 , RCC1, NFYB , HNRNPD , SMARCB1, UHRF2, ASXL1, MED14 , LMNB1, and HIST1H2AG ; NAB1, BRDS , ILF3 , MED7, RB1, HDAC3 , ERH , TSG101, [0062 ] b ) the group consisting of HIST1H1E , HMGB1, RNPS1 , CCNH , NONO , DEAF1 , ZFP91 , PKNOX1. HAUS4 , RCC1, HAUS5 , REEP4, SLBP, FKBP2 , ARSB , L3MBTL2 , CDC5L , SP4 , KLF11 , SMARCC1, HDAC1, HIST1H3E , RAD18 , RAD50 , TAF6 , ANAPC5 , FANCG , GTF2H5 , SMARCC2, RUVBL2, ZBTB45 , NMRALI, CTCF, TONSL , LMNB2, SEPHS1, HNRNPA2B1, WIZ , ING1, FOSB , CID , DICER1, E2F1, THRAP3 , RNF4 , ANAPC15 , STARD3NL , 2700029MOORIK , DPY30 , TSHZ1, SF1, GABPA ,GABPB1 , SMYD4 , CBY1 , ARNT2 , SDF2L1, RDM1, CDCA7L , MEAF6 , MYEF2 , DNAAF2 , GABPB2 , RFX1, MORF4L2 , ZFYVE19 , SUB1, HCFC1, POLR3K , IPP , TARDBP, GPAA1, KPNB1, FTSJD2, TARBP2, GTF3C2 , POU2F1 , ZNHIT3 , TBP , CAND1, RPRD1B , HIST1H1C , DPYSL2, TAF12 , ARPP19 , PCM1, BAZ1A , ATF6 , SREBF2 , YAF2, TCERG1, BRPF1 , TMCO1, EXOC4 , ASRGL1 , CPSF6 , EIF2D , CCNH , LITAF , SMYD3, RNF5 , DPF2 , SMARCD2, E2F5 , PML , MYG1, VMA21 , TFIP11, NDUFAB1, NUP35 , GRPEL1, GMEB1, SP1 , PSIP1 , SP2 , CNOT6 , OVCA2 , PFDN1, CID , GBP3 , CYB5B , PPM1G , SRSF10 , PIGF, KLREI , COMMD3, ING2, MYNN , HCFC2, AES , LRRFIP2 , COG4, PDIA4, CDT1, DUSP19 , ACAT2 , COMMD3 , GTF2E2 , YEATS4 , CNOT2 , MYCBP, PA2G4, TOE1, HCFC2, LMAN1, ABHD10 , TIMM50 , CALU , AP1M1, SMARCD1, NFYC , GMEB2 , CEBPB , IKZF5 , TFAM , GGH , GCDH , MRPS33 , TAX1BP3 , DYNLTIC , ERP44 , NFIL3 , CNOT4 , COPS5 , GTF2A2, CNOT1 , SIN3A , TMEM129 , COG2 , TMEM167 , RBX1, 0610009020RIK , GTF2F1, TSC22D2, ZBTB2, MED4 , RUVBL1, AIP , PSMB9, PUF60 , LSM4 , PEX19 , NTAN1, ELP2 , AKR1B3 , TRIM28 , GTF2B , DEDD , CREB1, PRDM1, CTBP1, PHF11B , POLC3 , ZFP142 , PRADC1, TMEM209, GTF3C5 , TAF10 , PSMC3, MED31, RBX1, FUS , PQBP1, DYNC1LI1, FARSB ,MTHFSD , 1700052N19RIK , SARS2 , ELF2 , ATF2 , CNOT8 , NCOR2, SMARCA4 , GNPTAB , LAMTORI, ALDH3A2 , EHBP1L1, D16ERTD472E , TAF6L , CRAMPIL , TCF3 , CEBPG , GTF3C3, TAF3 , ID2 , CCDC127 , COMMD10 , DNAJC14 , MRPL16 , SSBP1, YBX1, TAF11, YY2, MEN1, PHF6 , PHF17 , SMARCADI, EXOSC4 , UPF1 , DOHH , PTPN23, ZADH2 , ARFIP1, RBL2, HTT , HDAC5 , ING5 , CXXC1, NFKBIL1, CSDA , US 2019 / 0262399 A1 Aug . 29 , 2019

GTF3A , PRDM10 , MECP2 , SUDS3, CUX1, ZBTB22, defined in any one of the above aspects , or modifying said PLRG1, MED24 , ETV5, SFMBT1 , HLTF, MEF2A , JAZF1 , isolated immune cell such as to comprise an agent capable GATADI, ZBTB11 , ZNRD1, RBPJ, XRCC6 , GTF2A1, of inducibly altering expression or activity of one or more CTNNB1, PURE , CNOT7 , ZZEF1, TAF15 , TSC22D4 , genes or gene products as defined in any one of the above HIRA , ELF4 , MED13L , MMS19 , MBD1, VHL , VPS72 , aspects ; and ( c) reintroducing themodified isolated immune FOXJ3 , UBE2K , SNW1, RASSF7 , KEAP1, CAMTAI , cell to the subject. MED15 , MED8, ING3, CREBZF , TMF1, BOLA2 , IKZF2, [0070 ] Accordingly , it is an object of the invention not to ARID5B , SND1, PHF20 , ZBTB24 , SMARCE1 , ARIDIA , encompass within the invention any previously known prod MTA2, KLF10 , CCNC , IRF8 , ASH2L , MIER3 , UIMC1, uct, process ofmaking the product, or method of using the ELK3 , AEBP2 , BRF1 , SPRY2, RLF , IRF3 , NFYA , FLII , product such that Applicants reserve the right and hereby BCLAF1, FIZI, SP3 , PARD6A , MTA1, CCDC71, PBX4 , disclose a disclaimer of any previously known product, PHF5A , NACCI, ATF7IP , HDAC2 , GATAD2B , MGA , process, or method . It is further noted that the invention does TRIM27 , TCF20 , RUFY2, GOLGB1, PYGO2 , ZFPL1, not intend to encompass within the scope of the invention HIF1AN , BUD31 , TERF2 , NOTCH2, UBN1, DNM2, PPIE , any product, process , or making of the product or method of HIVEP2 , TBC1D2B , COPS2 , TAF2 , PNRC2 , ESRRA , using the product, which does not meet the written descrip IRF9 , SAP30 , MIER1, EYA3 , NCOA3, LM04, PREB , tion and enablement requirements of the USPTO ( 35 U . S . C . NMI, ZBTB40 , PCGF1 , HMGA1, SARNP, HSBP1 , CREM , $ 112 , first paragraph ) or the EPO ( Article 83 of the EPC ) , PTTG1, AGGF1, BMI1 , NCOA2 , CBX4 , TFEB , XAB2, such that Applicants reserve the right and hereby disclose a ERF, SAP18 , RYBP , MED17 , GTF2H3, ZMYND19 , SKIL , disclaimer of any previously described product, process of LZTRI, FOXK2, HTATSF1 , TBLIX , PLEKHF2 , CIC , making the product, or method of using the product . It may TCEA1, CIZI , TAF9B , TULP4, HMG20B , PIAS3 , MED21 , be advantageous in the practice of the invention to be in TBLIXR1, UTP6 , KDM3A , TBX21, ZBTB5 , EGR1, compliance with Art . 53 ( c ) EPC and Rule 28 ( b ) and (c ) EPC . ZFYVE27 , PHTF1, THOC2 , TRAK1, GTF2H1, RNF14 , All rights to explicitly disclaim any embodiments that are LCORL , IKZF3 , HEXIM1, RNF19A , FLI1, MED27 , the subject of any granted patent( s ) of applicant in the THAP7 , MAFF, GATA3 , PNN , CBX8 , ADNP, NAB2, lineage of this application or in any other lineage or in any MED13 , NR4A2, PHRF1, SREBF1, STAT2 , CHD2 , prior filed application of any third party is explicitly MEF2D , TRIP12 , MLX , RLIM , ABT1 , KAT5 , ETS2, reserved Nothing herein is to be construed as a promise . SCAP, RELA , BATF , MED26 , KDM2B , KDM6A , [0071 ] It is noted that in this disclosure and particularly in CBFA2T2 , PIAS4 , USF1, ZBTB7A , RNF25 , ZBTB1 , the claims and / or paragraphs , terms such as " comprises” , MED23 , ZBTB41 , PHF2 , KAT2B , KDM2A , CIR1, “ comprised ” , “ comprising ” and the like can have the mean ZC3H15 , ZEB2, REXO4, ZSCAN21, BLZF1, and CENPB ; ing attributed to it in U . S . Patent law ; e . g. , they can mean or " includes” , “ included ” , “ including” , and the like; and that [0066 ] f ) the group consisting of two or more genes or terms such as " consisting essentially of” and “ consists gene products each independently selected from any one of essentially of” have the meaning ascribed to them in U . S . the groups as defined in any one of a ) to e ) . Patent law , e . g . , they allow for elements not explicitly [0067 ] Further aspects of the invention provide a cell recited , but exclude elements that are found in the prior art population of said modified immune cells ; methods for or that affect a basic or novel characteristic of the invention . generating said modified immune cell ; a composition or 10072 ] These and other embodiments are disclosed or are pharmaceutical composition comprising said isolated obvious from and encompassed by, the following Detailed immune cell or said immune cell population ; said isolated Description . immune cell or said immune cell population for use in therapy ; and said isolated immune cell or said immune cell BRIEF DESCRIPTION OF THE DRAWINGS population for use in immunotherapy or adoptive immuno [0073 ] The patent or application file contains at least one therapy, preferably immunotherapy or adoptive immuno drawing executed in color. Copies of this patent or patent therapy of a proliferative disease , such as a tumor or cancer , application publication with color drawing ( s ) will be pro or a chronic infection , such as a chronic viral infection . vided by the Office upon request and payment of the [0068 ] A further aspect of the invention provides a method necessary fee . of treating a subject in need thereof, preferably a subject in [0074 ] The following detailed description , given by way need of immunotherapy or adoptive immunotherapy, more of example , but not intended to limit the invention solely to preferably immunotherapy or adoptive immunotherapy of a the specific embodiments described , may best be understood proliferative disease , such as a tumor or cancer, or a chronic in conjunction with the accompanying drawings . infection , such as a chronic viral infection , comprising f00751. FIG . 1A - 1J illustrate experimental results related to administering to said subject said isolated immune cell or determination of a signature of CD8 + T cell exhaustion in said cell population . cancer . FIG . 1A outlines the experimental setup for obtain [ 0069 ] A further aspect of the invention provides a method ing Tim3 + PD -1 + (DP ), Tim3- PD -1 + (SP ) , and Tim3- PD - 1 of treating a subject in need thereof, preferably a subject in (DN ) TIL cell populations. FIG . 1B illustrates that differ need of immunotherapy or adoptive immunotherapy, more entially expressed genes across the Tim - 3 /PD - 1 defined preferably immunotherapy or adoptive immunotherapy of a subpopulations define a dysfunctional signature in CD8 + proliferative disease , such as a tumor or cancer , or a chronic TILs, and presents a heatmap of the 3031 genes determined infection , such as a chronic viral infection , comprising : ( a ) as differentially expressed across the TILs subpopulations providing an isolated immune cell from the subject , or ( Naïve: CD8 + CD62L " iCD44low cells from spleens of non isolating an immune cell from a subject ; ( b ) modifying said tumor- bearing Balb / c mice , EffMem : Effector memory isolated immune cell such as to comprise an altered expres CD8 + CD62L lowCD44hi cells extracted from non - tumor sion or activity of one or more genes or gene products as bearing Balb / c mice , DN : CD8 + Tim3- PD -1 , SP : CD8 + US 2019 / 0262399 A1 Aug . 29 , 2019

Tim3- PD - 1 *, DP : CD8 + Tim3+ PD - 1 + TILs) . FIG . 1C empty retrovirus ( ctl OT1 ) or MT1 retrovirus (MTOE OT1 ) illustrates clustering of the genes differentially expressed prior to transfer into WT mice that were subsequently across the TILs subpopulations , and in conjunction with implanted with MC38 -OVA tumor. Mean tumor growth is Table 1 demonstrates that dysfunctional CD8 + TILs are shown . FIGS . 3E - G and 31, TIL were isolated and stimulated enriched for CD8 effector -like and cell cycle features. FIG . with PMA / ionomyicin in the presence of brefeldin A for 4 1D provides an enlarged representation of cluster C2 of FIG . hours prior to extracellular and intracellular staining and 1C , which in the present experiments appeared to best analysis by flow cytometry. FIG . 311 , reduced zinc level in represent a CD8 + dysfunctional signature . FIG . 1E illus DP TILs in MT- / - as compared to WT. FIG . 31 , Granzyme trates that TILs' cluster C2 is associated with both activation expression in WT and MT- / - CD8 + TILS. and exhaustion signatures. y -axis is - log 10 ( p - value enrich [ 0078 ] FIG . 4A - 4F show that MT- / - transcriptome ment ) for genes upregulated in signature ( values plotted in enables decoupling of activation and dysfunction in CD8 + red ) and + log 10 ( p - value enrichment ) for genes downregu TILS. FIG . 4A outlines the experimental setup for transcrip lated in signature (values plotted in blue ) . Cluster C2 is tional profiling of MT -/ - CD8 + TILS. FIG . 4B illustrates significant for enrichment with genes upregulated in both unbiased PCA analysis of WT and MT- / - DN , SP, and DP CD8 + activation and viral exhaustion , and cluster C3 is TILs populations . FIG . 4C illustrates bar plots showing the enriched for genes downregulated during CD8 + activation . mean of values of each of DN , SP, and DP subpopulation Upper panel : enrichments for CD8 + activation ( data from from WT and MT- / - for PC1 (first panel) and PC2 (second Doering et al. , 2012 , Immunity , vol. 37 (6 ), 1130 -44 2012 ; panel) separately . Error bars are the standard error of the day 15 acute vs. naïve ) ; lower panel: enrichments for CD8 + mean estimator. P -values for significance are computed LCMV exhaustion ( data from Doering et al. , 2012 , supra ; using standard t- test. ( * ) p - value < 0 .05 (* * ) p - value < 0 .01 . day 15 chronic vs. day 15 acute ) . FIG . 1F illustrates that FIG . 4D illustrates that genes differentially expressed Cluster 2 is significantly enriched with genes up - regulated in between the WT DN and DP populations and between the a CD8 + viral exhaustion signature (Doering et al ., 2012 , WT DP and MT -/ - DP populations were split into four supra ) as well as an in vivo CD8 + activation signature groups based on their expression trend . Groups I and II show (Sarkar et al. , 2008, supra ). p - values determined by hyper a trend of further increased or decreased expression , respec geometric test . Dashed line marks p = 0 .05 significance tively . Groups III and IV show reversal of expression . FIG . threshold . FIG . 16 compares with previous activation sig 4E illustrates overlay of naïve and in vitro activated CD8 + natures . FIG . 1H compares with previous exhaustion signa T cell transcriptomes on PC1 and PC2, which supports tures . FIG . 11 illustrates heatmap of the top ranking genes association of PC1, but not PC2, with CD8 + activation . FIG . from cluster C2 . FIG . 1J illustrates expression of co - inhibi 4F illustrates that correlations of PC1 and PC2 values with tory and co -stimulatory receptors in CD8 + TILs populations. various signatures show a strong association of PC1 with Shown are genes from pre -determined co - inhibitory and activation signatures , previously annotated signatures of co - stimulatory lists , that were upregulated in the DP sub exhaustion , and our cluster 2 gene signature and of PC2 with population . genes showing a reversal of expression in WT and MT- / [0076 ] FIG . 2A - 2D illustrates experimental results related TILS (Groups III and IV from D ) . to metallothionein and zinc metabolism . FIG . 2A illustrates [0079 ] FIG . 5A -511 illustrate identification of gene mod the expression of MT1 and MT2 as determined by qPCR in ules associated with T cell activation and dysfunction , sorted CD8 TILs isolated from mice bearing CT26 colon leading up to a novel signature which decouples dysfunction carcinoma and B16 melanoma tumors . FIG . 2B illustrates from activation of CD8 TILs. FIG . 5A illustrates the distri that 158 zinc related genes were in the present DE ( differ bution of genes by their dysfunction and activation scores , entially expressed ) set , and a significant proportion of the which reveals genes associated to different extents with the downregulated genes were zinc associated . FIG . 2C illus dysfunction and /or activation transcriptional programs, i . e . , trates that dysregulation of Zinc metabolism was reflected each gene ' s placement in the " Activation -Dysfunction also at the level of increasing of free Zn in Tim3 + PD - 1 * of space ” . Each gene is projected onto both diagonal axes to CD8 + but not CD4 + TIL or DLN . FIG . 2D shows availability determine a score of its association with the two modules of intracellular zinc in CD8 + TILs populations . WT and each axis represents ( lower panel ) . FIG . 5B illustrates place MT- / - TILs were stained with Zinpyr- 1 for measuring free ment of known co - inhibitory and co -stimulatory receptors in Zn followed by cell surface staining and analyzed by flow the “ Activation - Dysfunction space ”. The majority of co cytometry. inhibitory receptors (blue ) and co - stimulatory receptors [0077 ] FIG . 3A -31 illustrate that metallothionein defi ( red ) are associated with both activation and dysfunction , as ciency improves tumor control and reverses CD8 + T cell previously reported in the literature . FIG . 5C illustrates dysfunction . FIG . 3A , mice deficient in both MT1 and MT2 placement of CD8 activation signature genes in the " Acti ( i. e ., MT- / - mice ) and wild type (WT ) littermate controls vation - Dysfunction space ” . FIG . 5D illustrates the place were implanted subcutaneously with B16F10 melanoma, ment of genes reported as constituting the viral LCMV plot shows mean tumor growth . FIG . 3B , tumor draining exhaustion signature (Doering et al. 2012 , supra ) in the lymph node (dLN , upper panel) and tumor - infiltrating lym “ Activation -Dysfunction space ” . FIG . 5E illustrates enrich phocytes ( TIL , lower panel) were isolated from WT and ments of different signatures for the differentmodules of the MT- / - mice 15 days post tumor inoculation and stimulated dysfunction / activation plot ( Sarkar 2008 : Sarkar et al. 2008 , with tumor antigen gp100 or irrelevant peptide in vitro . On J Exp Med , vol. 205 ( 3 ) , 625 - 40 ) . Dashed line marks p = 0 . 05 day 3 , tumor antigen - specific proliferation was measured by significance threshold . FIG . 5F illustrates genes from an 3H incorporation . FIG . 3C , recipients ofMT - / - pmel CD8 + exhaustion and activation signature defined in a human T cells show slower tumor growth compared to those melanoma study ( Tirosh et al. , 2016 , Science 352 , 189 - 196 ) transferred with wild type pmel CD8 + T cells . FIG . 3D , separate on the Dysfunction < - > Activation axis Applicants naïve OT- 1 cells were sorted , activated , and infected with have defined (as shown in A ). Shown is the distribution of US 2019 /0262399 A1 Aug . 29 , 2019 genes on the Dysfunction / Activation plot ( top ) and the different gene signatures with the single -cell clusters ( XL Kolmogorov - Smirnov plot of the values of the human mHG test , threshold at top 30 % of list) . Dashed line marks signatures on the Dysfunction < - > Activation axis (Axis 1 - 2 p = 0 .05 significance threshold . FIG . 116 , Counts of cells in ( A ) ) (Kolmogorov - Smirnoff test p - value = 0 .027 ) . ( ) . FIG . from WT/MT - / - in the different clusters . Clusters signifi 5G illustrates the distribution of genes by their dysfunction cantly enriched for presence of WT (blue ) or MT- / - cells and activation scores , highlighting the position of Gata3 , a (red ) are marked . * p -value < 0 .05 , * * p - value < 0 .01 , * * * p zinc -binding TF , and Top 5 transcription factors ( TFs) for value < 0 .001 (hypergeometric test ) . each module from the set of differentially expressed TFs in [ 0086 ] FIG . 12 , related to FIG . 11. Cytokine and effector the original dysfunctional signature (upper panel ) ; a heat molecule expression in singlecell clusters 7 and 5 . Centered map of the ranking for the marked TFs in each of the four and normalized RNA levels are shown for different cytok modules ( lower panel ) . FIG . 5H illustrates that NRP1 recep ines or effector molecules ( rows) for each of the cells tor was highly expressed in PD - 1 * Tim3+ CD8 TILs. ( columns ) in clusters 5 and 7 from the single - cell analysis of [0080 ] FIG . 6A -61 illustrate further experimental corrobo FIG . 5 . To correct for differences in library complexity ration of the involvement of Gata3 in regulating CD8 T cell between cells and allow a comparison at the single - gene dysfunction in cancer. FIG . 6A - C , WT mice were implanted level, expression levels for all genes and cells analyzed subcutaneously with B16F10 melanoma cells . TILs were ( FIG . 5 ) were normalized by partitioning cells into 10 bins isolated on day 15 post tumor cell injection and analyzed for by their library complexity and conducting a median -nor Gata3 expression and T cell function . A , representative flow malization procedure for each gene , as previously described cytometry data showing Gata3 expression gated on CD8 + (Gaublomme et al. , 2015 ). IL2 , GZMD and GZME are not TIL , B , Foxp3 expression by Gata3 + CD8 + T cells , C , included in this analysis because they did not pass the cytokine expression of Gata3 + and Gata3 - CD8 + TIL . FIG . required expression thresholds to be included in the overall 6D , schematics of experimental setup . FIG . 6E , F , I , TIL single - cell analysis (Methods and Resources ) . * p - value were isolated on day 21 after tumor cell injection and < 0 . 05 , * * * p - value < 0 . 001. Overall p - value for cytokine/ analyzed for surface molecule expression and function by effector molecule signature was p < 10 -8 , (Wilcoxon rank flow cytometry . FIG . 6G , 1x106 CRISPR /Cas9 - targeted sum test) . cells (Gata3 - ' - ) were transferred to WT mice ( n = 5 / group ) [0087 ] FIG . 13A - 13H provides plots of expression of bearing B16F10melanoma tumors (day 5 post tumor graft CD83 , CD81, TNFRSF4 , CCR8, TNFSF11, CCL1 and AXL ing ) . Mean tumor growth is shown . Data are representative on the tSNE plot as shown in FIG . 11C . of 3 independent experiments. Statistical analysis was per - [0088 ] FIG . 14A - 14D provides plots of expression of formed using linear regression . * * p - value < 0 .01 . FIG . 6H , ULBP1, HMMR and REEP4 on the tSNE plot as shown in targeted deletion of Gata3 using crispr / cas9 genome editing . FIG . 11C . Naïve CD8 + T cells were sorted from PMEL transgenic mice , infected with control or Gata3 LV and activated with DETAILED DESCRIPTION OF THE plate -bound anti- CD3 and anti -CD28 antibodies in the pres INVENTION ence of IL - 2 (Experimental procedures) . Representative [0089 ] All documents cited or referenced herein (“ herein qPCR results showing Gata3 mRNA level in control versus cited documents ” ), and all documents cited or referenced in Gata3 LV targeted CD8 T cells . herein cited documents, together with any manufacturer' s [0081 ] FIG . 7A -7E illustrate that MT and Gata3 coopera instructions , descriptions , product specifications, and prod tively promote a suppressive phenotype of dysfunctional uct sheets for any products mentioned herein or in any CD8 + T cells in tumor. document incorporated by reference herein , are hereby [0082 ] FIG . 8A -8B illustrate experiments corroborating incorporated herein by reference , and may be employed in involvement of Pou2afl in dysfunction of CD8 + TILS . the practice of the invention . More specifically, all refer [0083 ] FIG . 9 illustrates a putative molecular model of T enced documents are incorporated by reference to the same cell activation / dysfunction . extent as if each individual document was specifically and [0084 ] FIG . 10 , CRISPR targeted pmel CD8 + T cells individually indicated to be incorporated by reference . ( Foxo1 - / - ; denoted LV31 in the figure ) were transferred to 100901 Unless otherwise defined , all terms used in disclos WT mice bearing B16F10 melanoma tumors on the indi ing the invention , including technical and scientific terms, cated day . Mean tumor growth is shown . have the meaning as commonly understood by one of [0085 ] FIG . 11A - 116 , The dysfunction and activation ordinary skill in the art to which this invention belongs . By transcriptional programs are anti - correlated at the single -cell means of further guidance , term definitions are included to level . FIG . 11A , Expression of the dysfunction module at the better appreciate the teaching of the invention . When spe single -cell level is negatively correlated with expression of cific terms are defined in connection with a particular aspect the activation module (left , r = - 0 .42 ) and of an in vivo CD8 + of the invention or a particular embodiment of the invention , activation signature (Sarkar et al ., 2008 , supra ) ( right, r = - 0 . such connotation is meant to apply throughout this specifi 47 ) . FIG . 11B , Expression of an in vivo CD8 + activation cation , i . e ., also in the context of other aspects or embodi signature at the single - cell level is positively correlated with ments of the invention , unless otherwise defined . expression of the activation module ( r = 0 .57 ) , the activation / [ 0091 ] As used herein , the singular forms “ a ” , “ an ” , and dysfunction module ( r = 0 .79 ) , a viral LCMV exhaustion “ the ” include both singular and plural referents unless the signature ( r = 0 . 85 ) and the cluster 2 genes ( FIG . 1B ) ( r = 0 . context clearly dictates otherwise . 68 ) . FIG . 11C , D , E , A USNE visualization ( van der Maaten 0092 ] The recitation of numerical ranges by endpoints and Hinton , 2008 ) of the 1061 single - cells analyzed , colored includes all numbers and fractions subsumed within the by ( C ) the partitioning into 7 clusters ( infomap ) , ( D ) gene respective ranges , as well as the recited endpoints . signatures of the four gene modules defined (by quantile ), [0093 ] The terms " about” or “ approximately ” as used and ( E ) mouse type (WT or MT" ) FIG . 11F , Association of herein when referring to a measurable value such as a US 2019 / 0262399 A1 Aug . 29 , 2019

parameter, an amount, a temporal duration , and the like, are STOGALNAC3, TUSC3, PDCDILG2, SDHAF1, ARAP2 , meant to encompass variations of and from the specified KLF4, E130308A19RIK , FAM46A , TNFRSF18 , SYNJ2 , value , such as variations of + / - 10 % or less , preferably CYTH3 , TNFSF8 , CD160 , RPL10 , CRTAM , RAB6B , + / - 5 % or less , more preferably + / - 1 % or less , and still more PTGER2, NFKB1, ANKRD46 , STOGALNAC6 , ITPR1, preferably + / - 0 . 1 % or less of and from the specified value, ITM2C , BTLA , TSPAN32 , CD82 , NFKBIA , MS4A4C , insofar such variations are appropriate to perform in the RARG , NRGN , TRIB1, ZC3H12D , BMYC , IF127L2A , disclosed invention . It is to be understood that the value to GADD45B , NAPSA , KLRB1F, RASGEF1A , FOSB , which the modifier “ about” or “ approximately ” refers is MAP3K8 , HIVEP1, SSH1, RABGAPIL , ZFP36L1, itself also specifically , and preferably , disclosed . ARL4D , CACNAIS , NFAT5 , DNAJC12 , SOWAHC , SDF4, 10094 ] Whereas the terms “ one or more " or " at least one” , TMEM120B , DUSP1, ELK3, JUNB , GRAMD1B , LIMK1, such as one or more members or at least one member of a ZFC3H1, OSTF1, LTA , DNMT3A , BCL7C , TSPAN13 , group of members , is clear per se , by means of further ASNSD1, TGIF1, NRN1, SYNGR2 , MSIZ , UAP1. exemplification , the term encompasses inter alia a reference UNC93B1, JAK2 , KDM2B , ANXA5 , PRDX2 , TMEM173 , to any one of said members , or to any two or more of said PHACTR2 , CCDC104 , CEP85L , IRF5 , INF2 , ITGB3 , members , such as, e . g . , any 23 , 24 , 25 , 26 or 7 etc . of said MPC1, BCL2A1D , PARP3 , ASAP1 , MRPS6 , RELB , members , and up to all said members. In another example , FAM110A , GPR68, NRP1, CAPG , SCYL2, SAMD3, " one or more ” or “ at least one ” may refer to 1 , 2 , 3 , 4 , 5 , 6 , H2 - AB1, HSF2 , CD44 , STX6 , POLG2, TESPA1, ALCAM , 7 or more . NSMF, LRRC8D , HIF1A , PACSIN1, PKP4 , ASSI , NR4A3 , [ 0095 ] The term " optional” or “ optionally ” means that the ENO3 , GYPC , KIF3B , IL2RA , RAB37 , SGMS1, HLCS , subsequent described event, circumstance or substituent SEMA6D , NMRK1, SLC17A6 , SLC39A1, RPS4X , CDON , may or may not occur , and that the description includes ZFP445 , LAG3, RPS26 , PHTF2, CST3 , CD9, STAT5A , instances where the event or circumstance occurs and ABCA3 , CSF2RA , DTX3 , RSPH3A , NRIP1, SDHA , instances where it does not . PNKD , FLNB , MGRN1, SLC26A2, HMOX2 , PEX16 , [0096 ] In the following passages , different aspects or INPP4A , TNFRSF25 , IRF8 , RGCC , IFITM2, TNFSF14 , embodiments of the invention are defined in more detail . NSUNO , STAT3 , PFKFB3, TYROBP, HTRA2, KLRI2 , Each aspect or embodiment so defined may be combined CTSS , ARL5C , KLHL24 , SESN3 , GM5424 , FAS , NCOA3, with any other aspect( s ) or embodiment( s ) unless clearly FAM53B , CALCOCOI, ERGIC3, 4930523COORIK , indicated to the contrary . In particular, any feature indicated PCGF5 , ANXA4 , and HERPUD1; as being preferred or advantageous may be combined with [0101 ] b ) a signature comprising or consisting of one or any other feature or features indicated as being preferred or more markers selected from the group consisting of CD74 , advantageous. CCR7, TBC1D4 , SLC2A6 , BCL6 , JAK2, PARP3 , ASAP1, [0097 ] Reference throughout this specification to " one RELB , H2 - AB1, CD44 , ABCA3, PFKFB3 , SESN3, FAS , embodiment” , “ an embodiment” means that a particular 4930523C07RIK , PCGF5 , TNIP1, SPRY1, NCOA7 , feature , structure or characteristic described in connection RPLPO , SMIM8, ANTXR2 , NSMCE1, DEDD , B3GNT2 , with the embodiment is included in at least one embodiment CABLES1 , SLAMF6 , UBL3 , NR4A1, ATG7 , and KDM5B ; of the present invention . Thus, appearances of the phrases [0102 ] c ) a signature comprising or consisting of one or " in one embodiment” or “ in an embodiment” in various more markers selected from the group consisting of CD83 , places throughout this specification are not necessarily all CCR8, TNFRSF4 , CD74 , CCR7, TNFSF11, CD81 , XCL1, referring to the same embodiment, but may . Furthermore , TNFSF4 , AXL , ECEV, KIT , ITGB1, CCL1, CD200 , the particular features , structures or characteristics may be TNFRSF18 , TNFSF8 , CD160 , PTGER2, BTLA , TSPAN32 , combined in any suitable manner, as would be apparent to a CD82 , KLRB1F , LTA , ANXA5 , ITGB3 , NRP1 , H2- AB1, person skilled in the art from this disclosure, in one or more CD44 , ALCAM , GYPC , IL2RA , CDON , LAGU , CD9, embodiments . Furthermore , while some embodiments TNFSF14 , FAS , GDI2 , TNIP1, IL21R , IL18R1, H2 - AA , described herein include some but not other features NR4A2 , IL18RAP , CD97 , TNFSF9, IRAK1BP1 , GABA included in other embodiments , combinations of features of RAPL1, TRPV2, EBAGY, GRN , RAMP1, AIMP1 , BSG , different embodiments are meant to be within the scope of IFNAR1, PRKCA , TRAF3 , CD96 , TNFRSF9, and NR3C1; the invention , and form different embodiments , as would be [0103 ] d ) a signature comprising or consisting of one or understood by those in the art . For example , in the appended more markers selected from the group consisting of CD83 , claims, any of the claimed embodiments can be used in any TNFRSF4 , CD74 , CCR7, CD81, TNFSF4 , KIT , ITGB1, combination . CD200 , TNFSF8 , CD160 , CD82 , ITGB3 , CD44, ALCAM , [0098 ] The present invention relates generally to novel GYPC , IL2RA , CDON , LAG3, CD9, CSF2RA , FAS, CD97 , markers , marker signatures and molecular targets useful for TNFSF9, BSG , IFNAR1 , TRAF3 , CD96 , and TNFRSF9 ; evaluating and modulating immune responses. [0104 ] e ) a signature comprising or consisting of one or [ 0099] In an aspect the present invention teaches a gene more markers selected from the group consisting of CD83 , expression signature of immune cell dysfunction , wherein CD81, TNFRSF4 , CXCL16 , IL21R , and IL18R1; the signature is selected from the group consisting of: [0105 ] f ) a signature comprising or consisting of one or [0100 ] a ) a signature comprising or consisting of one or more markers selected from the group consisting of REL , more markers selected from the group consisting of CD83 , BCL6 , MNDA , BHLHE40 , NFKB2, ZHX2, KLF4 , CCRS , TNFRSF4 , CD74 , CCR7 , TNFSF11, CD81, NFKB1, NFKBIA , RARG , FOSB , HIVEP1, ZFP36L1, TBC1D4 , REL , PLK2, XCL1, TNFSF4 , SLC2A6 , NFAT5 , ELK3 , JUNB , LIMK1, TGIF1, KDM2B , IRF5 , A1836003 , LAD1, 1700019D03RIK , BCL6 , MNDA , RELB , HSF2, HIF1A , NR4A3, PHTF2, STAT5A , DTX3, RAMP3 , GPM6B , BHLHE40 , AXL , ECE1 , FILIPIL , KIT, NRIP1, IRF8 , STAT3 , NCOA3 , CALCOCO1, PCGF5 , ITGB1 , CCL1, NFKB2, PLXDC2, ARC , DUSP4 , CD200 , NFKBIE , ETV6 , RNF19A , STAT4 , NR4A2, NFKBIB , TRAF1, ZHX2, NCF1, CCDC28B , PTPRS , PERI, GTF2A1, SPRY1, TFE3 , TGIF2 , RORA , RPL6 , US 2019 /0262399 A1 Aug . 29 , 2019

EGR2, FOXP4, TBL1X , KDM4A , COPS2 , FOS , DEDD , TNFRSF4 , CXCL16 , IL21R , IL18R1, REL , BCL6 , MNDA , SOSTMI, NT5C , PIAS4 , ZMYM2, DMTF1 , AEBP2 , BHLHE40 , NFKB2, ZHX2, KLF4 , NFKB1, NFKBIA , TRPS1, SP3, HBP1 , NR4A1, TLE3 , RPL7 , MED21, RARG , FOSB , HIVEP1 , ZFP36L1 , NFAT5 , ELK3, JUNB , DRAP1, TCF7 , CREB3L2, ZFHX2, KDM5B , and NR3C1; LIMK1, TGIF1 , KDM2B , IRF5 , RELB , HSF2, HIF1A , or NR4A3 , PHTF2, STAT5A , DTX3 , NRIP1 , IRF8 , STAT3 , [0106 ] g ) a signature comprising or consisting of two or NCOA3, CALCOCO1, PCGF5 , NFKBIE , ETV6 , RNF19A , more markers each independently selected from any one of STAT4 , NR4A2 , NFKBIB , PERI, GTF2A1, SPRY1, TFE3 . the groups as defined in any one of a ) to f ). TGIF2 , RORA , RPL6 , EGR2 , FOXP4 , TBLIX , KDM4A , 10107 ] In a related aspect the present invention teaches a COPS2, FOS, DEDD , SOSTM1, NT5C , PIAS4 , ZMYM2, gene expression signature of immune cell dysfunction , the DMTF1 , AEBP2 , TRPS1, SP3 , HBP1, NR4A1, TLE3 , signature comprising or consisting of one or more markers RPL7 , MED21, DRAP1, TCF7 , CREB3L2 , ZFHX2 , selected from the group consisting of CD83 , CCR8, KDM5B , and NR3C1. TNFRSF4 , CD74 , CCR7 , TNFSF11, CD81, TBC1D4 , REL , 10108 ] In certain embodiments , the signature of immune PLK2, XCL1 , TNFSF4 , SLC2A6 , A1836003 , LADI, cell dysfunction comprises or consists of one or more 1700019D03RIK , BCL6 , MNDA , RAMP3 , GPM6B , markers selected from the group consisting of CD83 , CD81, BHLHE40 , AXL , ECE1, FILIPIL , KIT, ITGB1 , CCL1 , and TNFRSF4 . NFKB2 , PLXDC2 , ARC , DUSP4, CD200 , TRAF1 , ZHX2 , f0109 . In certain embodiments , the signature of immune NCF1 , CCDC28B , PTPRS , STOGALNAC3 , TUSC3 , cell dysfunction further comprises one or more additional PDCD1LG2, SDHAF1 , ARAP2 , KLF4 , E130308A19RIK , markers of dysfunction . By means of example and not FAM46A , TNFRSF18 , SYNJ2 , CYTH3 , TNFSF8 , CD160 , limitation , the one or more additional markers of dysfunc RPL10 , CRTAM , RAB6B , PTGER2, NFKB1, ANKRD46 , tion may be a co - inhibitory receptor selected from the group STOGALNAC6 , ITPR1, ITM2C , BTLA , TSPAN32 , CD82 , consisting of PD1, CTLA4 , TIGIT , TIM3, LAGU , KLRC1, NFKBIA , MS4A4C , RARG , NRGN , TRIB1, ZC3H12D , BTLA , NRP1, CD160 , CD274 , IDO , CD200 , CD 244 , BMYC , IF127L2A , GADD45B , NAPSA , KLRB1F , KLRD1, LAIR1, CEACAM1, KLRA7 , FAS, GPR132 , RASGEF1A , FOSB , MAP3K8, HIVEP1 , SSH1, CD74 , SLAMF6 , CD5, GPR35 , CD28 , CD44 , and RABGAPIL , ZFP36L1, ARL4D , CACNAIS , NFAT5 , PTGER4 . By means of another example and not limitation , DNAJC12 , SOWAHC , SDF4 , TMEM120B , DUSP1 , ELK3 , the one or more additional markers of dysfunction may be JUNB , GRAMD1B , LIMK1, ZFC3H1, OSTF1, LTA , selected from the group consisting of PD1, CTLA4 , TIGIT , DNMT3A , BCL7C , TSPAN13 , ASNSD1, TGIF1, NRNI , TIM3, LAG3 , and KLRC1. SYNGR2 , MSI2 , UAP1, UNC93B1, JAK2 , KDM2B , [0110 ] In certain embodiments , the signature of immune ANXA5 , PRDX2 , TMEM173, PHACTR2 , CCDC104 , cell dysfunction comprises or consists of at least two mark CEP85L , IRF5 , INF2 , ITGB3, MPC1, BCL2A1D , PARP3, ers , or at least three markers , or at least four markers , or at ASAP1, MRPS6 , RELB , FAM110A , GPR68, NRP1, least five markers, or six or more markers . In certain CAPG , SCYL2 , SAMD3, H2- AB1, HSF2 , CD44 , STX6 , embodiments , the signature of immune cell dysfunction POLG2, TESPAI, ALCAM , NSMF, LRRC8D , HIF1A , comprises or consists of at least one, at least two, or at least PACSINI, PKP4 , ASS1 , NR4A3, ENO3 , GYPC , KIF3B , three markers each independently selected from any one of IL2RA , RAB37 , SGMS1 , HLCS , SEMA6D , NMRK1, the groups as defined in c ) or d ) . The group as defined in c ) SLC17A6 , SLC39A1, RPS4X , CDON , ZFP445 , LAG3 , comprises cell surface molecules and cytokines; the group as RPS26 , PHTF2, CST3 , CD9 , STAT5A , ABCA3 , CSF2RA , defined in d ) comprises CD molecules . In certain embodi DTX3, RSPH3A , NRIP1 , SDHA , PNKD , FLNB , MGRN1, ments , the signature of immune cell dysfunction comprises SLC26A2, HMOX2, PEX16 , INPP4A , TNFRSF25 , IRF8 , or consists of at least one , at least two , or at least three RGCC , IFITM2, TNFSF14 , NSUN6 , STAT3 , PFKFB3 , markers selected from the group as defined in f ) . The group TYROBP , HTRA2, KLRI2 , CTSS , ARL5C , KLHL24 , as defined in f ) comprises transcription factors and intrac SESN3 , GM5424 , FAS , NCOA3 , FAM53B , CALCOCO1, ellular molecules. In certain embodiments , the signature of ERGIC3 , 4930523CO7RIK , PCGF5 , ANXA4 , HERPUDI, immune cell dysfunction comprises or consists of at least CD74 , CCR7, TBC1D4 , SLC2A6 , BCL6 , JAK2, PARP3 , one, at least two , or at least three markers each indepen ASAP1 , RELB , H2 - AB1, CD44 , ABCA3 , PFKFB3 , dently selected from any one of the groups as defined in c ) SESN3 , FAS , 4930523C07RIK , PCGF5 , TNIP1 , SPRY1, or d ) and at least one , at least two , or at least three markers NCOA7, RPLPO , SMIMS, ANTXR2, NSMCE1, DEDD , selected from the group as defined in f ) . B3GNT2 , CABLES1, SLAMF6 , UBL3 , NR4A1, ATG7, [0111 ] The gene expression signature of immune cell KDM5B , CD83 , CCRS , TNFRSF4 , CD74 , CCR7, dysfunction can be useful inter alia in cell analysis , diag TNFSF11, CD81 , XCL1, TNFSF4 , AXL , ECE1, KIT , nostic , therapeutic and candidate compound screening appli ITGB1 , CCL1, CD200 , TNFRSF18 , TNFSF8 , CD160 , cations . PTGER2 , BTLA , TSPAN32 , CD82 , KLRB1F , LTA , [0112 ] Hence , an aspect provides a method of detecting ANXA5 , ITGB3 , NRP1, H2- AB1, CD44 , ALCAM ,GYPC , dysfunctional immune cells comprising detection of the IL2RA , CDON , LAG3 , CD9, TNFSF14 , FAS , GDI2 , gene expression signature of immune cell dysfunction as TNIP1, IL21R , IL18R1, H2- AA , NR4A2, IL18RAP , CD97 , taught herein . TNFSF9, IRAK1BP1 , GABARAPL1 , TRPV2, EBAG9, [0113 ] Another aspect provides a method for determining GRN , RAMP1 , AIMP1 , BSG , IFNARL PRKCA , TRAF3 , whether or not an immune cell has a dysfunctional immune CD96 , TNFRSF9 , NR3C1, CD83 , TNFRSF4 , CD74 , CCR7, phenotype, said method comprising determining in said CD81, TNFSF4 , KIT, ITGB1 , CD200 , TNFSF8 , CD160 , immune cell the expression of the signature of immune cell CD82 , ITGB3 , CD44 , ALCAM , GYPC , IL2RA , CDON , dysfunction as taught herein , whereby expression of the LAG3, CD9, CSF2RA , FAS , CD97 , TNFSF9, BSG , signature indicates that the immune cell has a dysfunctional IFNAR1 , TRAF3 , CD96 , TNFRSF9, CD83 , CD81, immune phenotype . US 2019 /0262399 A1 Aug . 29 , 2019

[0114 ] A further aspect relates to a method for determining CDCA3 , ESPLI , CASC5 , PBK , KIF2C , SGOLI, CDK1, whether or not a patient would benefit from a therapy aimed SHCBP1, ASPM , FBXO5 , MIS18BP1, SPAG5 , KIF4, at reducing dysfunction of immune cells or a therapy aimed ASF1B , BUB1B , AURKB , NCAPG , DEPDC1A , ESCO2, at upregulating of an immune response , the method com CDCA2 , BC030867 , KIF20A , HIST1H2AK , SMC2 , ECT2 , prising determining , in immune cells from said patient the RRM2, MK167 , 2810417H13RIK , CIT , GTSE1, NCAPG2 , expression of the signature of immune cell dysfunction as NCAPH , CDCAS , SAPCD2, NEK2, CEP55 , CDCA5 , taught herein , whereby expression of the signature indicates TOP2A , CCNB2 , MASTL , ARHGAP19 , AURKA , KIF23 , the patient will benefit from the therapy. A further aspect CCNB1, RAD51 , TACC3, MELK , STMN1, HIST1H2AE , relates to a method for determining whether or not a patient HIST1H4D , CENPH , HIST1H1B , CDC25C , CCDC34 , would benefit from a therapy aimed at increasing dysfunc CDC20 , KIF11 , ARHGAP11A , 4930427A07RIK , tion of immune cells or a therapy aimed at downregulating FAM83D , INCENP, MAD2L1 , HIST1H2AO , CKAP2 , of an immune response, the method comprising determining , NDC80 , RAD51AP1, NUF2 , E2F7 , CKS1B , NCAPD2, in immune cells from said patient the expression of the GEN1, ANLN , TICRR , POLO , PRC1, TTK , RAD54B , signature of immune cell dysfunction as taught herein , E2F8 , STIL , KIF18A , PARPBP, CDC45 , BIRC5 , KIF15 , whereby expression of the signature indicates the patient SKA2 . KIF20B , TK1, PLK4 . FANCD2 , CENPM , will likely not benefit from the therapy . C330027C09RIK , HIST2H4, SKA3 , RRM1, TROAP, [ 0115 ] Another aspect provides a method for determining RAD54L , KNTC1, ZWILCH , CLSPN , TPX2 , CMC2 , the efficacy of a treatment of a patient with a therapy, TCF19 , MCM10 , HMGB2, HIST1H3C , HIST1H1E , particularly immune therapy, said method comprising deter UBE2T , CHTF18 , TUBAIB , TUBBS , H2AFX , GPSM2, mining in immune cells from said patient the expression of SPC24 , UHRF1 , TRIP13 , PMF1 , ZFP367, RACGAP1, the signature of immune cell dysfunction as taught herein CDC25B , UBE2C , CDKN3, CENPI, HIST1H3B , KPNA2, before and after said treatment and determining the efficacy HJURP, BRCA1, WDR62 , CENPN , GMNN , POC1A , of said therapy based thereon . In certain embodiments, the TMPO , KNSTRN , FANCI, CENPF , 6430706D22RIK , method may be employed for determining the efficacy of CKS2 , DIAP3 , WDR67 , FIGNL1, BRCA2, HMGB3 , therapy or immune therapy aimed at reducing dysfunction of MYBL2, PKMYT1 , TRAIP , RFC5 , CEP128 , POLAI, immune cells or aimed at upregulating of an immune ANKLEI, HMGB1, FEN1, H2AFZ , TUBB4B , CTC1, response , whereby unchanged or increased expression of the CKAP5 , CDC6 , LIGI, POLE , MCMI, RFWD3 , HMGN2 , signature indicates that the treatment should be adjusted . In TYMS, CENPP , NCAPD3 , SUV39H1, A730008H23RIK , certain other embodiments , the method may be employed TUBA1C , EMEI, EXO1, PTMA, BLM , ULBP1, for determining the efficacy of therapy or immune therapy 1190002F15RIK , CDC7 , DLGAP5 , TUBG1, PRIM2, aimed at increasing dysfunction of immune cells or aimed at TMEM48, NRM , CENPA , BARDI, HAUS4 , RCC1, downregulating of an immune response , whereby LMNB1, and HIST1H2AG ; unchanged or reduced expression of the signature indicates [0119 ] b ) a signature comprising or consisting of one or that the treatment should be adjusted . more markers selected from the group consisting of [ 0116 ] A further aspect relates to a method for determining HIST1H1E , HMGB1, HAUS4 , RCC1, HAUS5, REEP4 , the suitability of a compound for modulating a dysfunctional SLBP, FKBP2 , ARSB , HIST1H3E , RAD18 , RAD50 , TAF6 , immune phenotype and /or modulating an immune response , ANAPC5 , FANCG , CTCF, TONSL , LMNB2, SEPHS1, said method comprising contacting an immune cell express HNRNPA2B1 , ANAPC15 , STARD3NL , 2700029MOORIK , ing the signature of immune cell dysfunction as taught DPY30 , SDF2L1, RDM1, CDCA7L , MEAF6 , MYEF2 . herein with said compound and determining whether or not DNAAF2 , POLR3K , IPP, TARDBP, GPAAI, KPNB1, said compound can affect the expression of the signature by FTSJD2 , RPRD1B , HISTIHIC , DPYSL2 , TAF12 , said cell. In certain embodiments , the method may be ARPP19 , TMCO1, EXOC4 , ASRGL1, CPSF6 , EIF2D . employed for determining the suitability of the compound CCNH , MYG1, VMA21, TFIP11, NDUFAB1, NUP35 , for reducing a dysfunctional immune phenotype and /or GRPEL1, C1D , GBP3 , CYB5B , PPM1G , SRSF10 , PIGF , upregulating of an immune response , whereby decreased KLRED , COG4 , PDIA4 , CDT1 , DUSP19 , ACAT2 , expression of the signature indicates that the compound is COMMD3, HCFC2 , LMAN1, ABHD10 , TIMM50 , CALU , suitable for reducing dysfunctional immune phenotype and AP1M1, GGH , GCDH , MRPS33 , TAX1BP3 , DYNLTIC , or upregulating of an immune response . In certain other ERP44 , TMEM129 , COG2, TMEM167 , RBX1, embodiments , the method may be employed for determining 0610009020RIK , PSMB9, PUF60 , LSM4 , PEX19 , NTANI, the suitability of the compound for increasing a dysfunc ELP2 , AKR1B3 , PHF11B , POLC3 , ZFP142 , PRADC1 , tional immune phenotype and /or downregulating of an TMEM209 , DYNCILII , FARSB , MTHFSD , immune response , whereby increased expression of the 1700052N19RIK , SARS2 , LAMTORI, ALDH3A2. signature indicates that the compound is suitable for increas EHBP1L1, D16ERTD472E , CCDC127 , COMMD10 , ing dysfunctional immune phenotype and / or downregulating DNAJC14 , MRPL16 , SSBP1 , EXOSC4, UPF1, DOHH , of an immune response . PTPN23 , ZADH2 , ARFIP1, STX18 , VMP1, TCEAI, [0117 ] In another aspect the present invention teaches a ERGICI, PIAS3 , RAD17 , EXOSC3, ACOT8 , MINA , gene expression signature of immune cell activation , LRRK1, MOGS , METTL10 , CERS4 , ATPBD4 , ZDHHC6 , wherein the signature is selected from the group consisting MAVS , PARL , GNG2, CD200R4 , USF1 , TYK2, SNAPC1, of: RBM18 , VPS53 , ACTR10 , and DPCD ; [ 0118 ] a ) a signature comprising or consisting of one or [0120 ] c ) a signature comprising or consisting of one or more markers selected from the group consisting of more markers selected from the group consisting of NUSAP1 , CCNA2, NEIL3 , SPC25 . HIST1H2AB , HMGB1, ULBP1 , REEP4 , HMMR , CMTM7, SIVAI, CKAP2L , PLK1, HIST2H3C2 , MXD3, FAM64A , BUB1, PAQR4, ATPIF1 , NUP85 , HSPA2 , ENTPD1, HNRNPU , FOXM1, HIST2H3B , KIF22 , CENPE , SKAI, CCNF, CLIC4 , FLOT2 , ENOX2 , ENPP1, TFRC , HAVCR2, US 2019 / 0262399 A1 Aug . 29 , 2019 14

CD2BP2 , LGALS1 , PGRMC1, USP14 , ENTPD5, ARID5B , SND1, PHF20 , ZBTB24 , SMARCE1, ARIDIA , ATP6AP2 , CCL3, ILIORA , ARNT2 , KLRE1 , CLPTM1, MTA2, KLF10 , CCNC , IRF8 , ASH2L , MIER3, UIMC1, ITGAV , KLRC1, PDIA4, SP1 , CCR5 , ADAM17 , CXCL10 , ELK3, AEBP2 , BRF1 , SPRY2, RLF, IRF3 , NFYA , FLII , IGSF8 , ADAM10 , IFNG , TNFRSF9 , CD244 , CTLA4 , BCLAF1, FIZI, SP3 , PARD6A , MTA1, CCDC71 , PBX4 , ERP44 , PSENI, LY6A , CCRL2 , NCOR2, HSP90AA1, PHF5A , NACC1, ATF7IP , HDAC2 , GATAD2B , MGA . IGF2R , PGP, KLRC3 , CKLF, TNFRSF1A , TMX3, KLRC2, TRIM27 , TCF20 , RUFY2 , GOLGB1, PYGO2, ZFPL1 , PDE4D , SMPD1, IDE , SERPINE2 , LRPAP1 , CSF1, CYS HIF1AN , BUD31 , TERF2 , NOTCH2 , UBN1, DNM2, PPIE . LTR2, LSMI, GRN , IL1F9 , LDLR , CD80 , GPR174 , MIF , HIVEP2 , TBC1D2B , COPS2 , TAF2 , PNRC2, ESRRA , MYO9B , ROCK1, GPR56 , GPR160 , RAC1 , PTPN11 , IRF9 , SAP30 , MIER1, EYA3, NCOA3, LMO4 , PREB , CMTM6 , ADA , NOTCH2 , GPI1 , GDI2 , P4HB , NRP1, NMI, ZBTB40 , PCGF1 , HMGA1, SARNP, HSBP1, CREM . F2R , AGTRAP , PGLYRP1 , STX4A , ADAMS, LYST, PTTG1, AGGF1, BMI1 , NCOA2 , CBX4 , TFEB , XAB2 , ITGA1, XPOT, KLRK1, CX3CR1, SEPT2 , CCL4 , CAST , ERF, SAP18 , RYBP, MED17 , GTF2H3, ZMYND19 , SKIL , RPS6KB1, H2 -M3 , LAG3 , CD99L2 , PDCD1, ECE1, EZR , LZTRI, FOXK2 , HTATSF1 , TBLIX , PLEKHF2 , CIC , NR4A2, SEMA4D, NAMPT, PEAR1 , IL12RB1 , CD200R4, TCEA1, CIZI , TAF9B , TULP4 , HMG20B , PIAS3 , MED21, CD48 , LAMP2 , IRAK2, CXCR6 , GPR65, and GCNTI ; TBL1XR1, UTP6 , KDM3A , TBX21 , ZBTB5, EGR1, [0121 ] d ) a signature comprising or consisting of one or ZFYVE27 , PHTF1, THOC2, TRAK1, GTF2H1, RNF14 , more markers selected from the group consisting ofHMMR , LCORL , IKZF3, HEXIMI, RNF19A , FLII , MED27 , SIVAI , ENTPD1, ENPP1, TFRC , CD2BP2 , ENTPD5 , THAP7 , MAFF , GATA3 , PNN , CBX8 , ADNP, NAB2 , ITGAV , KLRC1, CCR5, ADAM17 , TNFRSF9 , CD244 , MED13 , NR4A2 , PHRF1, SREBF1 , STAT2 , CHD2, CTLA4 , IL3RA , IGF2R , TNFRSF1A , CD80 , ADAM8, MEF2D , TRIP12 , MLX , RLIM , ABT1 , KAT5 , ETS2 , LAG3, MGL2 , CD99L2 , SEMA4D , IL12RB1, CD200R4 , SCAP, RELA , BATF , MED26 , KDM2B , KDM6A , CD48 , and LAMP2 ; CBFA2T2 , PIAS4 , USF1 , ZBTB7A , RNF25 , ZBTB1, [0122 ] e ) a signature comprising or consisting of one or MED23 , ZBTB41 , PHF2 , KAT2B , KDM2A , CIR1 . more markers selected from the group consisting of MXD3, ZC3H15 , ZEB2, REXO4 , ZSCAN21, BLZF1 , and CENPB ; FOXM1, E2F7 , RAD54B , E2F8 , TCF19 , HMGB2 , UHRF1, or TRIP13 , PMF1, BRCA1, BRCA2, HMGB3 , MYBL2 , (0123 ] f ) a signature comprising or consisting of two or HMGB1, PTMA , WHSC1, CHAF1A , RBLI, DNMT1 , more markers each independently selected from any one of CCNE1 , DEK , E2F2, CHAF1B , EZH2, G2E3 , WDHD1, the groups as defined in any one of a ) to e) . SUZ12 , TFDP1, RBBP4 , CASP8AP2 , RFC1, CDCA4 , [0124 ] In a related aspect the present invention teaches a RBBP8 , SSRP1, ANAPC11, TERF1 , POLE3 , CBFB , gene expression signature of immune cell activation , the CBX3 , CTCF , MED30 , PBRM1, TFDP2 , ITGB3BP, CBX6 , signature comprising or consisting of one or more markers NRF1 , BAZIB , E2F3 , PIAS1, ILF2 , HDAC6 , TIMELESS , selected from the group consisting of NUSAP1, CCNA2 , SMARCA5 , MYEF2 , TARDBP, EED , HMGXB4, NEIL3 , SPC25 , HIST1H2AB , CKAP2L , PLK1, METTL14 , E2F4 , LIN9, MTF2 , MAZ , ATF1 , TAF1 , TOX , HIST2H3C2 , MXD3 , FAM64A , BUB1, FOXM1, NFYB , HNRNPD , SMARCB1, UHRF2 , ASXL1, MED14 , HIST2H3B , KIF22 , CENPE , SKA1, CCNF, CDCA3, NAB1, BRDS, ILF3 ,MEDT , RB1, HDAC3 , ERH , TSG101 , ESPL1, CASC5 , PBK , KIF2C , SGOL1, CDK1, SHCBP1, RNPS1, CCNH , NONO , DEAF1, ZFP91 , PKNOX1, ASPM , FBX05, MIS18BP1 , SPAG5 , KIF4 , ASF1B , L3MBTL2 , CDC5L , SP4 , KLF11, SMARCC1, HDACI, BUB1B , AURKB , NCAPG , DEPDC1A , ESCO2, CDCA2, GTF2H5, SMARCC2 , RUVBL2 , ZBTB45 , NMRALI , BC030867 , KIF20A , HIST1H2AK , SMC2 , ECT2 , RRM2, WIZ , ING1, FOSB , CID , DICER1, E2F1, THRAP3, RNF4 , MKI67, 2810417H13RIK , CIT, GTSEL , NCAPG2, TSHZ1, SF1 , GABPA , GABPB1, SMYD4 , CBY1, ARNT2 , NCAPH , CDCAS , SAPCD2 , NEK2 , CEP55 , CDCA5 . GABPB2, RFX1, MORF4L2 , ZFYVE19 , SUB1, HCFC1, TOP2A , CCNB2 , MASTL , ARHGAP19 , AURKA , KIF23 , TARBP2, GTF3C2 , POU2F1, ZNHIT3 , TBP, CANDI, CCNB1, RAD51 , TACC3 , MELK , STMN1, HIST1H2AE , PCM1, BAZ1A , ATF6 , SREBF2, YAF2, TCERG1, BRPF1, HIST1H4D , CENPH , HIST1H1B , CDC25C , CCDC34 , LITAF, SMYD3, RNF5 , DPF2, SMARCD2, E2F5 , PML , CDC20 , KIF11 , ARHGAP11A , 4930427A07RIK , GMEB1, SP1 , PSIP1 , SP2 , CNOT6 , OVCA2 , PFDN1, FAM83D , INCENP, MAD2L1, HIST1H2AO , CKAP2 , COMMD3 , ING2, MYNN , HCFC2, AES , LRRFIP2 , NDC80 , RAD51 AP1, NUF2 , E2F7 , CKS1B , NCAPD2 , GTF2E2 , YEATS4 , CNOT2, MYCBP , PA2G4, TOEI, GEN1, ANLN , TICRR , POLQ , PRC1 , TTK , RAD54B , SMARCD1, NFYC , GMEB2 , CEBPB , IKZF5 , TFAM , E2F8 , STIL , KIF18A , PARPBP, CDC45 , BIRC5 , KIF15 , NFIL3 , CNOT4 , COPS5 , GTF2A2 , CNOTI , SIN3A , SKA2 , KIF20B , TKI, PLK4, FANCD2, CENPM , GTF2F1, TSC22D2, ZBTB2 , MED4, RUVBL1, AIP , C330027C09RIK , HIST2H4, SKA3 , RRM1, TROAP , TRIM28 , GTF2B , DEDD , CREB1, PRDM1, CTBP1, RAD54L , KNTC1 , ZWILCH , CLSPN , TPX2 , CMC2, GTF3C5, TAF10 , PSMC3, MED31, RBX1, FUS, PQBP1 , TCF19 , MCM10 , HMGB2 , HIST1H3C , HIST1HIE , ELF2 , ATF2, CNOT8 , NCOR2, SMARCA4, GNPTAB , UBE2T , CHTF18 , TUBA1B , TUBBS, H2AFX , GPSM2, TAF6L , CRAMPIL , TCF3 , CEBPG , GTF3C3, TAF3 , ID2, SPC24 , UHRF1, TRIP13 , PMF1, ZFP367, RACGAP1, YBX1, TAF11, YY2, MEN1, PHF6 , PHF17 , SMARCADI, CDC25B , UBE2C , CDKN3, CENPI, HIST1H3B , KPNA2, RBL2, HTT , HDAC5, ING5, CXXC1, NFKBIL1, CSDA , HJURP, BRCA1, WDR62 , CENPN , GMNN , POC1A , GTF3A , PRDM10 , MECP2 , SUDS3 , CUX1, ZBTB22 , TMPO , KNSTRN , FANCI, CENPF , 6430706D22RIK , PLRG1, MED24 , ETV5 , SFMBT1, HLTF , MEF2A , JAZF1, CKS2 , DIAP3 , WDR67 , FIGNL1, BRCA2 , HMGB3, GATADI, ZBTB11 , ZNRD1, RBPJ, XRCC6 , GTF2A1 , MYBL2 , PKMYT1 , TRAIP , RFC5 , CEP128 , POLA1, CTNNB1 , PURE , CNOT7 , ZZEF1, TAF15 , TSC22D4 , ANKLEI , HMGB1, FEN1, H2AFZ , TUBB4B , CTC1, HIRA , ELF4 , MED13L , MMS19 , MBDI, VHL , VPS72 , CKAP5 , CDC6 , LIG1, POLE , MCMI, RFWD3 , HMGN2 . FOXJ3 , UBE2K , SNW1, RASSF7 , KEAP1, CAMTA1, TYMS, CENPP , NCAPD3, SUV39H1, A730008H23RIK , MED15 , MED8, ING3, CREBZF, TMF1, BOLA2 , IKZF2 , TUBAIC , EMEI, EXO1, PTMA , BLM , ULBP1, US 2019 / 0262399 A1 Aug . 29 , 2019 15

1190002F15RIK , CDC7 , DLGAP5 , TUBG1, PRIM2, HDAC1, GTF2H5, SMARCC2, RUVBL2 , ZBTB45 , TMEM48 , NRM , CENPA , BARDI, HAUS4 , RCC1 , NMRALI, WIZ , ING1, FOSB , CID , DICER1, E2F1, LMNB1, HIST1H2AG , HIST1H1E , HMGB1, HAUS4 , THRAP3 , RNF4, TSHZ1, SF1 , GABPA , GABPB1, RCC1, HAUS5 , REEP4 , SLBP, FKBP2, ARSB , SMYD4, CBY1, ARNT2 , GABPB2 , RFX1, MORF4L2 , HIST1H3E , RAD18, RAD50 , TAF6 , ANAPC5 , FANCG , ZFYVE19 , SUB1, HCFC1, TARBP2 , GTF3C2, POU2F1 , CTCF , TONSL , LMNB2 , SEPHS1, HNRNPA2B1, ZNHIT3 , TBP , CAND1, PCM1, BAZ1A , ATF6 , SREBF2, ANAPC15 , STARD3NL , 2700029MOORIK , DPY30 , YAF2 , TCERG1, BRPF1 , LITAF , SMYD3 , RNF5 , DPF2 . SDF2L1, RDM1, CDCA7L , MEAF6 , MYEF2 , DNAAF2 , SMARCD2, E2F5 , PML , GMEB1, SP1 , PSIP1 , SP2 , CNOT6 , OVCA2, PFDN1, COMMD3, ING2, MYNN , POLR3K , IPP , TARDBP, GPAAI, KPNB1, FTSJD2, HCFC2, AES , LRRFIP2 , GTF2E2 , YEATS4 , CNOT2 . RPRD1B , HIST1H1C , DPYSL2, TAF12 , ARPP19 , MYCBP, PA2G4, TOE1, SMARCD1, NFYC , GMEB2, TMCO1, EXOC4, ASRGL1, CPSF6 , EIF2D , CCNH , CEBPB , IKZF5 , TFAM , NFIL3 , CNOT4 , COPS5 , MYG1, VMA21 , TFIP11, NDUFAB1 , NUP35 , GRPEL1, GTF2A2 , CNOT1, SIN3A , GTF2F1, TSC22D2 , ZBTB2 , CID , GBP3 , CYB5B , PPM1G , SRSF10 , PIGF, KLRE1, MED4 , RUVBL1, AIP , TRIM28 , GTF2B , DEDD , CREB1, COG4, PDIA4, CDT1, DUSP19 , ACAT2 , COMMD3 , PRDM1, CTBP1, GTF3C5 , TAF10 , PSMC3, MED31, HCFC2, LMAN1, ABHD10 , TIMM50 , CALU , AP1M1, RBX1, FUS, PQBP1, ELF2 , ATF2 , CNOT8, NCOR2, GGH , GCDH , MRPS33 , TAX1BP3, DYNLTIC , ERP44 , SMARCA4 , GNPTAB , TAF6L , CRAMPIL , TCF3 , TMEM129 , COG2 , TMEM167 , RBX1, 0610009020RIK , CEBPG ,GTF3C3 , TAF3 , ID2, YBX1, TAF11 , YY2, MENI, PSMB9, PUF60 , LSM4, PEX19 , NTAN1, ELP2 , AKR1B3 , PHF6 , PHF17 , SMARCAD1, RBL2 , HTT, HDAC5 , ING5, PHF11B , PQLC3 , ZFP142 , PRADC1, TMEM209 , CXXC1, NFKBILI, CSDA , GTF3A , PRDM10 , MECP2 , DYNC1LI1 , FARSB , MTHFSD , 1700052N19RIK , SARS2 , SUDS3 , CUX1, ZBTB22 , PLRG1, MED24 , ETV5 , LAMTOR1, ALDH3A2, EHBP1L1, D16ERTD472E , SFMBT1 , HLTF , MEF2A , JAZF1, GATADI, ZBTB11 , CCDC127 , COMMD10 , DNAJC14 , MRPL16 , SSBP1, ZNRD1 , RBPJ , XRCC6 , GTF2A1, CTNNB1, PURB , EXOSC4 , UPF1, DOHH , PTPN23 , ZADH2, ARFIP1 , CNOT7 , ZZEF1, TAF15 , TSC22D4 , HIRA , ELF4 , STX18 , VMP1, TCEAN, ERGICI, PIAS3 , RAD17 , MED13L , MMS19 , MBD1, VHL , VPS72 , FOXJ3 , UBE2K , EXOSC3 , ACOTS , MINA , LRRK1, MOGS , METTL10 , SNW1, RASSF7 , KEAP1, CAMTA1, MED15 , MED8, CERS4 , ATPBD4, ZDHHC6 , MAVS , PARL , GNG2, ING3 , CREBZF, TMF1, BOLA2 , IKZF2 , ARID5B , SND1, CD200R4 , USF1 , TYK2 , SNAPC1, RBM18 , VPS53 , PHF20 , ZBTB24 , SMARCE1, ARIDIA , MTA2 , KLF10 , ACTR10 , DPCD , HMGB1, ULBP1, REEP4 , HMMR , CCNC , IRF8 , ASH2L , MIER3 , UIMC1, ELK3 , AEBP2 , CMTM7, SIVAI , PAQR4 , ATPIF1, NUP85 , HSPA2 , BRF1, SPRY2 , RLF , IRF3 , NFYA , FLII, BCLAF1, FIZI , ENTPD1, HNRNPU , CLIC4 , FLOT2 , ENOX2, ENPP1 , SP3 , PARD6A , MTA1, CCDC71, PBX4 , PHF5A , NACCI, TFRC , HAVCR2 , CD2BP2 , LGALS1, PGRMC1 , USP14 , ATF7IP , HDAC2, GATAD2B , MGA, TRIM27 , TCF20 , ENTPD5 , ATP6AP2 , CCL3 , ILIORA , ARNT2 , KLREI , RUFY2 , GOLGB1, PYGO2 , ZFPLI, HIF1AN , BUD31, CLPTM1, ITGAV ,KLRC1 , PDIA4, SP1, CCR5 , ADAM17 , TERF2 , NOTCH2, UBN1, DNM2, PPIE , HIVEP2 , CXCL10 , IGSF8 , ADAM10 , IFNG , TNFRSF9 , CD244 , TBC1D2B , COPS2, TAF2 , PNRC2, ESRRA , IRF9 , SAP30 , CTLA4, ERP44 , PSEN1, LY6A , CCRL2 , NCOR2, MIER1, EYAZ, NCOA3, LMO4 , PREB , NMI, ZBTB40 , HSP90AA1, IGF2R , PGP, KLRC3 , CKLF, TNFRSF1A , PCGF1 , HMGAI, SARNP, HSBP1 , CREM , PTTG1. TMX3 , KLRC2, PDE4D , SMPD1, IDE , SERPINE2 , AGGF1, BMI1 , NCOA2 , CBX4 , TFEB , XAB2, ERF, LRPAP1, CSF1, CYSLTR2, LSM1, GRN , IL1F9 , LDLR , SAP18 , RYBP , MED17 , GTF2H3, ZMYND19 , SKIL , CD80 , GPR174 , MIF , MYO9B , ROCK1, GPR56 , GPR160 , LZTR1, FOXK2, HTATSF1 , TBLIX , PLEKHF2 , CIC . RAC1 , PTPN11, CMTM6 , ADA , NOTCH2, GPII , GDI2 , TCEA1, CIZI , TAF9B , TULP4, HMG20B , PIAS3 ,MED21 , P4HB , NRP1, F2R , AGTRAP , PGLYRP1, STX4A , TBL1XR1, UTP6 , KDM3A , TBX21 , ZBTB5 , EGR1, ADAMS, LYST, ITGA1, XPOT , KLRK1, CX3CR1, ZFYVE27 , PHTF1, THOC2 , TRAK1, GTF2H1, RNF14 , SEPT2 , CCL4 , CAST, RPS6KB1, H2- M3 , LAG3 , CD99L2 , LCORL , IKZF3 , HEXIMI, RNF19A , FLI1, MED27 , PDCD1 , ECE1 , EZR , NR4A2 , SEMA4D, NAMPT, PEAR1 , THAP7 , MAFF, GATA3 , PNN , CBX8 , ADNP, NAB2. IL12RB1, CD200R4 , CD48 , LAMP2 , IRAK2, CXCR6 , GPR65 , GCNT1 , HMMR , SIVA1, ENTPD1, ENPP1 , MED13 , NR4A2 , PHRF1, SREBF1, STAT2 , CHD2, TFRC , CD2BP2 , ENTPD5 , ITGAV , KLRC1, CCR5 , MEF2D , TRIP12 , MLX , RLIM , ABT1 , KAT5 , ETS2 , ADAM17 , TNFRSF9 , CD244 , CTLA4, IL3RA , IGF2R , SCAP, RELA , BATF , MED26 , KDM2B , KDM6A , TNFRSF1A , CD80 , ADAM8, LAGU , MGL2 , CD99L2 , CBFA2T2 , PIAS4 , USF1, ZBTB7A , RNF25 , ZBTB1, SEMA4D , IL12RB1, CD200R4, CD48 , LAMP2 , MXD3 , MED23 , ZBTB41 , PHF2 , KAT2B , KDM2A , ORE ZC3H15 , FOXM1, E2F7 , RAD54B , E2F8 , TCF19 , HMGB2, UHRF1 , ZEB2, REXO4, ZSCAN21 , BLZF1 , and CENPB . TRIP13 , PMF1, BRCA1, BRCA2, HMGB3, MYBL2 , [0125 ] In certain embodiments , the signature of immune HMGB1, PTMA , WHSC1, CHAF1A , RBL1 , DNMT1 , cell activation further comprises one or more additional CCNE1, DEK , E2F2 , CHAF1B , EZH2, G2E3 , WDHD1, markers of activation . By means of example , the one or more SUZ12 , TFDP1 , RBBP4 , CASP8AP2 , RFC1, CDCA4, additional markers of activation may be a co - stimulatory RBBP8 , SSRP1 , ANAPC11 , TERF1 , POLE3 , CBFB , receptor selected from the group consisting of TNFRSF9 , CBX3 , CTCF, MED30 , PBRM1, TFDP2, ITGB3BP, CBX6 , TNFRSF4 , TNFSF4 , TNFRSF18 , TNFSF11 , TNFRSF13C , NRF1 , BAZIB , E2F3 , PIAS1, ILF2 , HDAC6 , TIMELESS , CD27 , CD28 , CD86 , ICOS, and TNFSF14 . SMARCA5 , MYEF2 , TARDBP, EED , HMGXB4 , 101261. In certain embodiments , the signature of immune METTL14 , E2F4 , LIN9, MTF2 , MAZ , ATF1, TAF1, TOX , cell activation comprises or consists of at least two markers , NFYB , HNRNPD , SMARCB1, UHRF2 , ASXL1, MED14 , or at least three markers , or at least four markers , or at least NAB1, BRD8, ILF3 , MED7, R131 , HDAC3 , ERH , five markers, or six or more markers . In certain embodi TSG101 , RNPS1 , CCNH , NONO , DEAF1 , ZFP91, ments, the signature of immune cell activation comprises or PKNOX1, L3MBTL2 , CDC5L , SP4 , KLF11 , SMARCC1, consists of at least one , at least two , or at least three markers US 2019 /0262399 A1 Aug . 29 , 2019 16 each independently selected from any one of the groups as immune phenotype and /or modulating an immune response , defined in c ) or d ). The group as defined in c ) comprises cell said method comprising contacting an immune cell express surface molecules and cytokines ; the group as defined in d ) ing the signature of immune cell activation as taught herein comprises CD molecules. In certain embodiments, the sig with said compound and determining whether or not said nature of immune cell activation comprises or consists of at compound can affect the expression of the signature by said least one , at least two , or at least three markers selected from cell . In certain embodiments , the method is employed for the group as defined in e ) . The group as defined in e ) determining the suitability of the compound for reducing an comprises transcription factors and intracellular molecules. activated immune phenotype and /or downregulating of an In certain embodiments , the signature of immune cell acti immune response , whereby decreased expression of the vation comprises or consists of at least one , at least two , or signature indicates that the compound is suitable for reduc at least three markers each independently selected from any ing activated immune phenotype and/ or downregulating of one of the groups as defined in c ) or d ) and at least one, at an immune response . In certain other embodiments , the least two , or at least three markers selected from the group method is employed for determining the suitability of the as defined in e ) . compound for increasing an activated immune phenotype [0127 ] The gene expression signature of immune cell and / or upregulating of an immune response , whereby activation can be useful inter alia in cell analysis , diagnostic , increased expression of the signature indicates that the therapeutic and candidate compound screening applications . compound is suitable for increasing activated immune phe [0128 ] Hence , an aspect provides a method of detecting notype and / or upregulating of an immune response . activated immune cells comprising detection of the gene [0133 ] The term “ gene expression signature” refers to one expression signature of immune cell activation as taught or more genes, such as a panel of genes , whose expression herein . correlates with a specific phenotype . According to certain [ 0129 ] A further aspect provides a method for determining aspects of the present invention , " high " expression of one or whether or not an immune cell has an activated immune more markers comprised by the gene expression of dysfunc phenotype, said method comprising determining in said tion correlates with an immune cell that has a dysfunctional immune cell the expression of the signature of immune cell phenotype . The gene expression signature of dysfunction activation as taught herein , whereby expression of the sig may also be used to determine cells with a phenotype that nature indicates that the immune cell has an activated does not correlate with an immune cell that has a dysfunc immune phenotype . tional phenotype . According to certain other aspects of the [0130 ] Another aspect relates to a method for determining present invention , " high " expression of one or more markers whether or not a patient would benefit from a therapy aimed comprised by the gene expression of activation correlates at reducing activation of immune cells or a therapy aimed at with an immune cell that has an activated phenotype . The downregulating of an immune response , the method com gene expression signature of activation may also be used to prising determining , in immune cells from said patient the determine cells with a phenotype that does not correlate with expression of the signature of immune cell activation as an immune cell that has an activated phenotype . taught herein , whereby expression of the signature indicates [0134 ] A gene expression signature may be determined by the patient will benefit from the therapy. Another aspect any method known in the art. Gene expression can be relates to a method for determining whether or not a patient determined by sequencing , preferably RNA -seq , quantita would benefit from a therapy aimed at increasing activation tive reverse transcription PCR , western blot, ELISA , immu of immune cells or a therapy aimed at upregulating of an nofluorescence , FACS, CYTOF, or microarray . Preferably, immune response , the method comprising determining , in the expression signature may be determined by a method immune cells from said patient the expression of the signa allowing for single - cell resolution of the measurement, i . e . , ture of immune cell activation as taught herein , whereby on a single - cell level, such as by single cell RNA sequenc expression of the signature indicates the patient will likely ing , FACS , or CYTOF. In certain embodiments , determining not benefit from the therapy. whether or not an immune cell expresses a signature may [0131 ] A further aspect relates to a method for determining comprise cell sorting . In certain embodiments , a sample the efficacy of a treatment of a patient with a therapy, from a human subject may be obtained prior to detecting the particularly immune therapy, said method comprising deter one or more markers therein . mining in immune cells from said patient the expression of [0135 ] The term “ high ” as used herein generally means a the signature of immune cell activation as taught herein higher by a statically significant amount relative to a refer before and after said treatment and determining the efficacy ence ; for the avoidance of doubt, “ high ” means a statistically of said therapy based thereon . In certain embodiments , the significant value at least 10 % higher than a reference level , method is employed for determining the efficacy of therapy for example at least 20 % higher, at least 30 % higher , at least or immune therapy aimed at reducing activation of immune 40 % higher , at least 50 % higher , at least 60 % higher, at least cells or aimed at downregulating of an immune response , 70 % higher , at least 80 % higher, at least 90 % higher, at least whereby unchanged or increased expression of the signature 100 % higher , at least 2 - fold higher, at least 3 - fold higher, at indicates that the treatment should be adjusted . In certain least 4 - fold higher, at least 5 - fold higher, at least 10 - fold other embodiments , themethod is employed for determining higher or more, as compared to a reference level. the efficacy of therapy or immune therapy aimed at increas - 0136 ] The term " low ” as used herein generally means ing activation of immune cells or aimed at upregulating of lower by a statically significant amount; for the avoidance of an immune response , whereby unchanged or reduced doubt, " low ” means a statistically significant value at least expression of the signature indicates that the treatment 10 % lower than a reference level, for example a value at should be adjusted . least 20 % lower than a reference level, at least 30 % lower [ 0132 ] Another aspect relates to a method for determining than a reference level , at least 40 % lower than a reference the suitability of a compound for modulating an activated level, at least 50 % lower than a reference level, at least 60 % US 2019 /0262399 A1 Aug . 29 , 2019 17 lower than a reference level , at least 70 % lower than a f0140 ] The signature according to certain embodiments of reference level, at least 80 % lower than a reference level , at the present invention may comprise or consist of one or least 90 % lower than a reference level, up to and including more genes and/ or proteins , such as for instance 1 , 2 , 3 , 4 , 100 % lower than a reference level (i . e ., absent level as 5 , 6 , 7, 8 , 9 , 10 or more . In certain embodiments , the compared to a reference sample ) . signature may comprise or consist of two or more genes [0137 ] The term “ statistically significant” or “ signifi and /or proteins , such as for instance 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 cantly ” refers to statistical significance and generally means or more. In certain embodiments , the signature may com a two standard deviation below normal, or lower, concen prise or consist of three or more genes and / or proteins, such tration of the marker. The term refers to statistical evidence as for instance 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 or more . In certain that there is a difference . It is defined as the probability of embodiments, the signature may comprise or consist of four making a decision to reject the null hypothesis when the null or more genes and/ or proteins , such as for instance 4 , 5 , 6 , hypothesis is actually true . The decision is often made using 7 , 8 , 9 , 10 or more . In certain embodiments, the signature the p -value . may comprise or consist of five or more genes and / or [ 0138 ] As used herein a signature may encompass any proteins , such as for instance 5 , 6 , 7 , 8 , 9 , 10 or more . In gene or genes , or protein or proteins, whose expression certain embodiments , the signature may comprise or consist profile or whose occurrence is associated with a specific cell of six or more genes and / or proteins, such as for instance 6 , type , subtype , or cell state of a specific cell type or subtype 7 , 8 , 9 , 10 or more . In certain embodiments, the signature within a population of cells . Increased or decreased expres may comprise or consist of seven or more genes and / or sion or activity or prevalence may be compared between proteins, such as for instance 7 , 8 , 9 , 10 or more . In certain different cells in order to characterize or identify for instance embodiments , the signature may comprise or consist of eight specific cell (sub ) populations . A gene signature as used or more genes and /or proteins , such as for instance 8 , 9 , 10 herein , may thus refer to any set of up - and down -regulated or more . In certain embodiments, the signature may com genes between different cells or cell ( sub populations prise or consist of nine or more genes and / or proteins , such derived from a gene -expression profile . For example , a gene as for instance 9 , 10 or more . In certain embodiments, the signature may comprise a list of genes differentially signature may comprise or consist of ten or more genes expressed in a distinction of interest. It is to be understood and /or proteins, such as for instance 10 , 11 , 12 , 13 , 14 , 15 , that also when referring to proteins ( e . g . differentially or more . It is to be understood that a signature according to expressed proteins ) , such may fall within the definition of the invention may for instance also include a combination of " gene ” signature . genes or proteins . [0139 ] The signatures as defined herein ( being it a gene [0141 ] It is to be understood that “ differentially expressed ” signature , protein signature or other genetic signature ) can genes/ proteins include genes /proteins which are up - or be used to indicate the presence of a cell type , a subtype of down - regulated as well as genes /proteins which are turned the cell type, the state of the microenvironment of a popu on or off . When referring to up - or down - regulation , in lation of cells , a particular cell type population or subpopu certain embodiments , such up - or down -regulation is pref lation , and / or the overall status of the entire cell ( sub ) erably at least two - fold , such as two -fold , three - fold , four population . Furthermore , the signature may be indicative of fold , five - fold , or more , such as for instance at least ten - fold , cells within a population of cells in vivo . The signature may at least 20 - fold , at least 30 - fold , at least 40 - fold , at least also be used to suggest for instance particular therapies , or 50 - fold , or more . Alternatively , or in addition , differential to follow up treatment, or to suggest ways to modulate expression may be determined based on common statistical immune systems. The signatures of the present invention tests , as is known in the art . may be discovered by analysis of expression profiles of [0142 ] As discussed herein , differentially expressed single - cells within a population of cells from isolated genes /proteins may be differentially expressed on a single samples ( e . g . blood samples ) , thus allowing the discovery of cell level, or may be differentially expressed on a cell novel cell subtypes or cell states that were previously population level. Preferably , the differentially expressed invisible or unrecognized . The presence of subtypes or cell genes/ proteins as discussed herein , such as constituting the states may be determined by subtype specific or cell state gene signatures as discussed herein , when as to the cell specific signatures. The presence of these specific cell ( sub ) population level, refer to genes that are differentially types or cell states may be determined by applying the expressed in all or substantially all cells of the population signature genes to bulk sequencing data in a sample . Not (such as at least 80 % , preferably at least 90 % , such as at least being bound by a theory, a combination of cell subtypes 95 % of the individual cells ) . This allows one to define a having a particular signature may indicate an outcome. Not particular subpopulation of cells . As referred to herein , a being bound by a theory , the signatures can be used to " subpopulation ” of cells preferably refers to a particular deconvolute the network of cells present in a particular subset of cells of a particular cell type which can be pathological condition . Not being bound by a theory the distinguished or are uniquely identifiable and set apart from presence of specific cells and cell subtypes are indicative of other cells of this cell type . The cell subpopulation may be a particular response to treatment, such as including phenotypically characterized , and is preferably character increased or decreased susceptibility to treatment . The sig ized by the signature as discussed herein . A cell ( sub ) nature may indicate the presence of one particular cell type . population as referred to herein may constitute of a (sub ) In one embodiment, the novel signatures are used to detect population of cells of a particular cell type characterized by multiple cell states or hierarchies that occur in subpopula a specific cell state . tions of immune cells that are linked to particular patho - [0143 ] When referring to induction , or alternatively sup logical condition ( e . g . cancer ) , or linked to a particular pression of a particular signature, preferable is meant induc outcome or progression of the disease , or linked to a tion or alternatively suppression (or upregulation or down particular response to treatment of the disease . regulation ) of at least one gene/ protein of the signature, such US 2019 /0262399 A1 Aug . 29 , 2019 as for instance at least two , at least three , at least four, at least detect multiple cell states that occur in a subpopulation of five, at least six , or all genes/ proteins of the signature . tumor cells that are linked to resistance to targeted therapies [0144 ] Signatures may be functionally validated as being and progressive tumor growth . In preferred embodiments , uniquely associated with a particular immune phenotype . immune cell states of tumor infiltrating lymphocytes are Induction or suppression of a particular signature may detected consequentially be associated with or causally drive a par [0149 ] In one embodiment, the signature genes are ticular immune phenotype . detected by immunofluorescence, mass cytometry (CyTOF ) , [0145 ] Various aspects and embodiments of the invention FACS , drop -seq , RNA - seq , single cell qPCR , MERFISH may involve analyzing gene signatures , protein signature , (multiplex ( in situ ) RNA FISH ) , microarray and / or by in situ and / or other genetic signature based on single cell analyses hybridization . Other methods including absorbance assays ( e . g . single cell RNA sequencing ) or alternatively based on and colorimetric assays are known in the art and may be cell population analyses , as is defined herein elsewhere . used herein . In some aspects, measuring expression of [ 0146 ] In further aspects , the invention relates to gene signature genes comprises measuring protein expression signatures , protein signature , and / or other genetic signature levels. Protein expression levels may be measured , for of particular immune cell subpopulations, as defined herein . example , by performing a Western blot, an ELISA or bind The invention hereto also further relates to particular ing to an antibody array . In another aspect, measuring immune cell subpopulations, which may be identified based expression of said genes comprises measuring RNA expres on the methods according to the invention as discussed sion levels . RNA expression levels may be measured by herein ; as well as methods to obtain such cell ( sub ) popula tions and screening methods to identify agents capable of performing RT- PCR , Northern blot, an array hybridization , inducing or suppressing particular immune cell ( sub )popu or RNA sequencing methods . lations. [0150 ] An enzyme- linked immunosorbent assay, or 101471. The invention further relates to various uses of the ELISA , may be used to measure the differential expression gene signatures , protein signature , and / or other genetic of a plurality of signature genes . There are many variations signature as defined herein , as well as various uses of the of an ELBA assay. All are based on the immobilization of an immune cells or immune cell ( sub )populations as defined antigen or antibody on a solid surface , generally a microtiter herein . Particular advantageous uses include methods for plate . The original ELISA method comprises preparing a identifying agents capable of inducing or suppressing par sample containing the biomarker proteins of interest , coating ticular immune cell ( sub populations based on the gene the wells of a microtiter plate with the sample , incubating signatures , protein signature, and / or other genetic as defined each well with a primary antibody that recognizes a specific herein . The invention further relates to agents capable of antigen , washing away the unbound antibody, and then inducing or suppressing particular immune cell ( sub )popu detecting the antibody -antigen complexes . The antibody lations based on the gene signatures , protein signature , antibody complexes may be detected directly . For this , the and / or other genetic signature as defined herein , as well as primary antibodies are conjugated to a detection system , their use for modulating, such as inducing or repressing , a such as an enzyme that produces a detectable product . The particular gene signature , protein signature , and / or other antibody -antibody complexes may be detected indirectly . genetic signature . In related aspects , modulating , such as For this , the primary antibody is detected by a secondary inducing or repressing , a particular gene signature , protein antibody that is conjugated to a detection system , as signature , and / or other genetic signature may modify overall described above . The microtiter plate is then scanned and the immune cells composition , such as activated or dysfunc raw intensity data may be converted into expression values tional immune cell composition , or distribution , or function using means known in the art . ality . [0151 ] Detection of signature genes may be by FACS . The [0148 ] As used herein the term " signature gene” means term “ fluorescent activated cell sorting ” or “ FACS ” , as used any gene or genes whose expression profile is associated herein , refers to a technique for counting , examining , and with a specific cell type , subtype , or cell state of a specific sorting microscopic particles suspended in a stream of fluid . cell type or subtype within a population of cells . The It allows simultaneous multiparametric analysis of the signature gene can be used to indicate the presence of a cell physical and / or chemical characteristics of single cells flow type , a subtype of the cell type , the state of the microenvi ing through an optical and /or electronic detection apparatus . ronment of a population of cells , and /or the overall status of Generally , a beam of light ( usually laser light ) of a single the entire cell population . Furthermore , the signature genes wavelength is directed onto a hydro - dynamically focused may be indicative of cells within a population of cells in stream of fluid . A number of detectors are aimed at the point vivo . Not being bound by a theory , the signature genes can where the stream passes through the light beam ; one in line be used to deconvolute the cells present in a tumor based on with the light beam ( Forward Scatter, correlates to cell comparing them to data from bulk analysis of a tumor volume) and several perpendicular to the beam , (Side Scat sample . The signature gene may indicate the presence of one ter , correlates to the inner complexity of the particle and /or particular cell type . In one embodiment, the signature genes surface roughness ) and one or more fluorescent detectors . may indicate that dysfunctional or activated tumor infiltrat Each suspended particle passing through the beam scatters ing T -cells are present. The presence of cell types within a the light in some way , and fluorescent chemicals found in the tumor may indicate that the tumor will be resistant to a particle or attached to the particle may be excited into treatment. In one embodiment the signature genes of the emitting light at a lower frequency than the light source . By present invention are applied to bulk sequencing data from analyzing the combinations of scattered and fluorescent light a tumor sample to transform the data into information picked up by the detectors it is then possible to derive relating to disease outcome and personalized treatments . In information about the physical and chemical structure of one embodiment, the novel signature genes are used to each individual particle . US 2019 / 0262399 A1 Aug . 29 , 2019

[0152 ] Detection of signature genes may involve a cell a population of said immune cells ; b ) in vitro expanding the sorting step to enrich for cells of interest and thus facilitate immune cell or immune cell population of a ) ; and c ) or enhance their sensitive and specific detection . Cell sorting administering the in vitro expanded immune cell or immune techniques are commonly based on tagging the cell with cell population of b ) to the subject . antibody against the cell membrane antigen specific to the [0156 ] A related aspect thus relates to a method of isolat target subpopulation of cells . The antibody is conjugated to ing an immune cell comprising the signature of immune cell a magnetic bead and /or fluorophore or other label to enable dysfunction as taught herein . Such method may typically cell sorting and detection . Such methods may include affin comprise binding of an affinity ligand to a signature gene ity chromatography , particle magnetic separation , centrifu expressed on the surface of the immune cell . By means of an gation , or filtration , and flow cytometry ( including fluores example and not limitation , one or more, two or more, three cence activated cell sorting ; FACS) . Approaches based on or more , four or more , five or more , or six or more signature antibody -coated microbeads can use magnetic fields (Racila gene products may be bound by corresponding affinity et at, 1998 ) , column chromatography, centrifugation , filtra ligands to effect the separation and isolation of the cells . tion or FACS to achieve separation . [0157 ] A further aspect provides an isolated immune cell [ 0153 ] Cells may be sequenced by any method known in characterised in that the immune cell comprises (displays , the art for determining a gene signature . Methods of pre expresses ) the signature of immune cell activation as taught paring cDNA is known in the art . Single cells may be herein . Preferably , the immune cell is a T cell , more pref sequenced for detection of a gene signature . Single cells of e rably a CD8 + T cell . Another aspect relates to a population the present invention may be divided into single droplets of said immune cells . A further aspect relates to a compo using a microfluidic device . The single cells in such droplets sition or pharmaceutical composition comprising said may be further labeled with a barcode . In this regard immune cell or said immune cell population . A further reference is made to Macosko et al ., 2015 , “ Highly Parallel aspect relates to a method for eliciting an immune response Genome -wide Expression Profiling of Individual Cells in a subject comprising administering to the subject said Using Nanoliter Droplets ” Cell 161 , 1202 - 1214 ; Interna immune cell or said immune cell population or said phar tional patent application number PCT/ US2015 /049178 , pub maceutical composition . In certain embodiments , the lished as WO2016 /040476 on Mar. 17 , 2016 ; Klein et al. , method may comprise the steps of: a ) isolating from a 2015 , “ Droplet Barcoding for Single - Cell Transcriptomics biological sample of the subject an immune cell comprising Applied to Embryonic Stem Cells ” Cell 161 , 1187 - 1201; the signature of immune cell activation as taught herein or Zheng , et al. , 2016 , “Haplotyping germline and cancer a population of said immune cells ; b ) in vitro expanding the genomes with high - throughput linked - read sequencing ” immune cell or immune cell population of a ); and c) Nature Biotechnology 34 , 303 - 311 ; and International patent administering the in vitro expanded immune cell or immune publication number WO 2014210353 A2, all the contents cell population of b ) to the subject. and disclosure of each of which are herein incorporated by [0158 ] A related aspect thus relates to a method of isolat reference in their entirety . ing an immune cell comprising the signature of immune cell 10154 ] The gene expression signatures as taught herein activation as taught herein . Such method may typically also allow to isolate and characterise previously unknown comprise binding of an affinity ligand to a signature gene types or subtypes of immune cells . The novel immune cell expressed on the surface of the immune cell . By means of an types or subtypes , can be useful inter alia in diagnostic , example and not limitation , one or more , two or more , three therapeutic and candidate compound screening applications . or more , four or more , five or more , or six ormore signature Optionally , the novel immune cell types may be modified to gene products may be bound by corresponding affinity introduce genetic , epigenetic, gene expression and/ or phe ligands to effect the separation and isolation of the cells . notypic alterations thereto , e . g . , may be genetically engi [0159 ] Immune cells may be obtained using any method neered to express a heterologous gene or protein therein , or known in the art. In one embodiment T cells that have may be genetically engineered to downregulate or abolish or infiltrated a tumor are isolated . T cells may be removed upregulate the expression of an endogenous gene or protein during surgery . T cells may be isolated after removal of therein . By means of an example and not limitation , the tumor tissue by biopsy . T cells may be isolated by any means novel immune cell types or subtypes may be engineered to known in the art . In one embodiment the method may comprise an antigen - specific chimeric antigen receptor comprise obtaining a bulk population of T cells from a tumor (CAR ), such as a tumor antigen - specific CAR . sample by any suitable method known in the art . For [0155 ] Hence , an aspect relates to an isolated immune cell example , a bulk population of T cells can be obtained from characterised in that the immune cell comprises ( displays , a tumor sample by dissociating the tumor sample into a cell expresses ) the signature of immune cell dysfunction as suspension from which specific cell populations can be taught herein . Preferably , the immune cell is a T cell , more selected . Suitable methods of obtaining a bulk population of preferably a CD8 + T cell. Another aspect relates to a T cells may include , but are not limited to , any one or more population of said immune cells . A further aspect relates to of mechanically dissociating ( e . g ., mincing ) the tumor, a composition or pharmaceutical composition comprising enzymatically dissociating ( e . g ., digesting ) the tumor, and said immune cell or said immune cell population . A further aspiration ( e . g . , as with a needle ) . aspect relates to a method for eliciting immune tolerance in (0160 ] The bulk population of T cells obtained from a a subject comprising administering to the subject said tumor sample may comprise any suitable type of T cell . immune cell or said immune cell population or said phar Preferably , the bulk population of T cells obtained from a maceutical composition . In certain embodiments , the tumor sample comprises tumor infiltrating lymphocytes method may comprise the steps of: a ) isolating from a ( TILS ) . biological sample of the subject an immune cell comprising [0161 ] The tumor sample may be obtained from any the signature of immune cell dysfunction as taught herein or mammal. Unless stated otherwise , as used herein , the term US 2019 / 0262399 A1 Aug . 29 , 2019 20

“ mammal ” refers to any mammal including , but not limited as compared to other cell types , such in isolating tumor to , mammals of the order Logomorpha , such as rabbits; the infiltrating lymphocytes ( TIL ) from tumor tissue or from order Carnivora , including Felines ( cats ) and Canines immunocompromised individuals . Further, use of longer ( dogs ) ; the order Artiodactyla , including Bovines ( cows) and incubation times can increase the efficiency of capture of Swines (pigs ) ; or of the order Perssodactyla , including CD8 + T cells . Equines (horses ) . The mammals may be non -human pri [0164 ] Enrichment of a T cell population by negative mates, e . g ., of the order Primates , Ceboids, or Simoids selection can be accomplished with a combination of anti (monkeys ) or of the order Anthropoids ( humans and apes ) . bodies directed to surface markers unique to the negatively In some embodiments , the mammal may be a mammal of the selected cells , A preferred method is cell sorting and / or order Rodentia , such as mice and hamsters . Preferably , the selection via negative magnetic immunoadherence or flow mammal is a non -human primate or a human . An especially cytometry that uses a cocktail of monoclonal antibodies preferred mammal is the human . directed to cell surface markers present on the cells nega [0162 ] T cells can be obtained from a number of sources , tively selected . For example , to enrich for CD4 + cells by including peripheral blood mononuclear cells , bone marrow , negative selection , a monoclonal antibody cocktail typically lymph node tissue , spleen tissue , and tumors . In certain includes antibodies to CD14 , CD20 , CD11b , CD16 , HLA embodiments of the present invention , T cells can be DR , and CD8 . obtained from a unit of blood collected from a subject using [ 0165 ] Further, monocyte populations ( i. e ., CD14 + cells ) any number of techniques known to the skilled artisan , such may be depleted from blood preparations by a variety of as Ficoll separation . In one preferred embodiment, cells methodologies, including anti- CD14 coated beads or col from the circulating blood of an individual are obtained by umns, or utilization of the phagocytotic activity of these apheresis or leukapheresis . The apheresis product typically cells to facilitate removal. Accordingly , in one embodiment, contains lymphocytes, including T cells , monocytes , granu the invention uses paramagnetic particles of a size sufficient locytes , B cells , other nucleated white blood cells , red blood to be engulfed by phagocytotic monocytes. In certain cells , and platelets. In one embodiment , the cells collected embodiments , the paramagnetic particles are commercially by apheresis may be washed to remove the plasma fraction available beads , for example , those produced by Life Tech and to place the cells in an appropriate buffer or media for nologies under the trade name DynabeadsTM . In one embodi subsequent processing steps . In one embodiment of the ment , other non - specific cells are removed by coating the invention , the cells are washed with phosphate buffered paramagnetic particles with “ irrelevant” proteins ( e . g . , saline (PBS ) . In an alternative embodiment, the wash solu serum proteins or antibodies ). Irrelevant proteins and anti tion lacks calcium and may lack magnesium or may lack bodies include those proteins and antibodies or fragments many if not all divalent cations. Initial activation steps in the thereof that do not specifically target the T cells to be absence of calcium lead to magnified activation . As those of isolated . In certain embodiments the irrelevant beads include ordinary skill in the art would readily appreciate a washing beads coated with sheep anti -mouse antibodies , goat anti step may be accomplished by methods known to those in the mouse antibodies , and human serum albumin . art , such as by using a semi- automated “ flow - through ” . [0166 ] In brief, such depletion of monocytes is performed centrifuge ( for example , the Cobe 2991 cell processor) by preincubating T cells isolated from whole blood , according to the manufacturer ' s instructions. After washing , apheresed peripheral blood , or tumors with one or more the cells may be resuspended in a variety of biocompatible varieties of irrelevant or non -antibody coupled paramagnetic buffers, such as , for example , Ca - free, Mg- free PBS . Alter particles at any amount that allows for removal of mono natively , the undesirable components of the apheresis cytes (approximately a 20 : 1 bead : cell ratio ) for about 30 sample may be removed and the cells directly resuspended minutes to 2 hours at 22 to 37 degrees C ., followed by in culture media . magnetic removal of cells which have attached to or [0163 ] In another embodiment, T cells are isolated from engulfed the paramagnetic particles. Such separation can be peripheral blood lymphocytes by lysing the red blood cells performed using standard methods available in the art. For and depleting the monocytes, for example, by centrifugation example , any magnetic separation methodology may be through a PERCOLLTM gradient. A specific subpopulation used including a variety of which are commercially avail of T cells , such as CD28 + , CD4 + , CDC , CD45RAT, and able , ( e . g . , DYNAL® Magnetic Particle Concentrator CD45RO + T cells , can be further isolated by positive or (DYNAL MPC? )) . Assurance of requisite depletion can be negative selection techniques . For example , in one preferred monitored by a variety ofmethodologies known to those of embodiment, T cells are isolated by incubation with anti ordinary skill in the art, including flow cytometric analysis CD3/ anti -CD28 ( i. e ., 3x28 ) - conjugated beads , such as of CD14 positive cells , before and after depletion . DYNABEADS® M - 450 CD3 /CD28 T , or XCYTE [0167 ] For isolation of a desired population of cells by DYNABFADSTM for a time period sufficient for positive positive or negative selection , the concentration of cells and selection of the desired T cells . In one embodiment, the time surface ( e. g. , particles such as beads) can be varied . In period is about 30 minutes . In a further embodiment, the certain embodiments , it may be desirable to significantly time period ranges from 30 minutes to 36 hours or longer decrease the volume in which beads and cells are mixed and all integer values there between . In a further embodi together ( i . e . , increase the concentration of cells ) , to ensure ment, the time period is at least 1 , 2 , 3 , 4 , 5 , or 6 hours. In maximum contact of cells and beads . For example , in one yet another preferred embodiment, the time period is 10 to embodiment, a concentration of 2 billion cells /ml is used . In 24 hours . In one preferred embodiment, the incubation time one embodiment, a concentration of 1 billion cells /ml is period is 24 hours . For isolation of T cells from patients with used . In a further embodiment, greater than 100 million leukemia , use of longer incubation times, such as 24 hours , cells /ml is used . In a further embodiment, a concentration of can increase cell yield . Longer incubation times may be used cells of 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 , or 50 million cells /ml to isolate T cells in any situation where there are few T cells is used . In yet another embodiment, a concentration of cells US 2019 / 0262399 A1 Aug . 29 , 2019 from 75 , 80 , 85 , 90 , 95, or 100 million cells / nil is used . In 902 ). Tetramers are limited by the need to utilize predicted further embodiments , concentrations of 125 or 150 million binding peptides based on prior hypotheses, and the restric cells /ml can be used . Using high concentrations can result in tion to specific HLAs . Peptide -MHC tetramers can be gen increased cell yield , cell activation , and cell expansion . erated using techniques known in the art and can be made Further, use of high cell concentrations allowsmore efficient with any MHC molecule of interest and any antigen of capture of cells that may weakly express target antigens of iniinterest as described herein . Specific epitopes to be used in interest, such as CD28 -negative T cells , or from samples this context can be identified using numerous assays known where there are many tumor cells present ( i. e ., leukemic in the art . For example , the ability of a polypeptide to bind blood , tumor tissue , etc ) . Such populations of cells may have to MHC class I may be evaluated indirectly by monitoring therapeutic value and would be desirable to obtain . For the ability to promote incorporation of 1251 labeled B2- mi example , using high concentration of cells allows more croglobulin (B2m ) into MHC class 1/ 32m / peptide heterotri efficient selection of CD8 + T cells that normally have weaker meric complexes ( see Parker et al. , J . Immunol. 152 : 163 , CD28 expression . 1994 ). 10168 ] In a related embodiment, it may be desirable to use [0172 ] In one embodiment cells are directly labeled with lower concentrations of cells . By significantly diluting the an epitope - specific reagent for isolation by flow cytometry mixture of l ' cells and surface ( e . g . , particles such as beads ) , followed by characterization of phenotype and TCRs. In one interactions between the particles and cells is minimized . T cells are isolated by contacting the T cell specific anti This selects for cells that express high amounts of desired bodies. Sorting of antigen - specific T cells , or generally any antigens to be bound to the particles . For example , CD4 + T cells of the present invention , can be carried out using any cells express higher levels of CD28 and are more efficiently of a variety of commercially available cell sorters , including , captured than CD8 + T cells in dilute concentrations. In one but not limited to , MoFlo sorter (DakoCytomation , Fort embodiment, the concentration of cells used is 5x10?/ ml . In Collins, Colo . ), FACSAriaTM , FACSArrayTM , FACSVan other embodiments , the concentration used can be from about 1x10° /ml to 1x10°/ ml , and any integer value in tageTM , BDTM LSI II, and FACSCaliburTM (BD Biosciences, between . San Jose , Calif . ). [ 0169 ] T cells can also be frozen . Wishing not to be bound [0173 ] In a preferred embodiment, the method comprises by theory , the freeze and subsequent thaw step provides a selecting cells that also express CD3 . The method may more uniform product by removing granulocytes and to comprise specifically selecting the cells in any suitable some extent monocytes in the cell population . After a manner . Preferably , the selecting is carried out using flow washing step to remove plasma and platelets , the cells may cytometry . The flow cytometry may be carried out using any be suspended in a freezing solution . While many freezing suitable method known in the art. The flow cytometry may solutions and parameters are known in the art and will be employ any suitable antibodies and stains. Preferably , the useful in this context, one method involves using PBS antibody is chosen such that it specifically recognizes and containing 20 % DMSO and 8 % human serum albumin , or binds to the particular biomarker being selected . For other suitable cell freezing media , the cells then are frozen example , the specific selection of CD3 , CD8, TIM - 3 , LAG to - 80 " C at a rate of 1° per minute and stored in the vapor 3 , 4 - 1BB , or PD - 1 may be carried out using anti -CD3 . phase of a liquid nitrogen storage tank . Other methods of anti- CD8 , anti - TIM -3 , anti -LAG -3 , anti -4 - IBB , or anti controlled freezing may be used as well as uncontrolled PD - 1 antibodies, respectively . The antibody or antibodies freezing immediately at - 20° C . or in liquid nitrogen . may be conjugated to a bead ( e . g . , a magnetic bead ) or to a [0170 ] T cells for use in the present invention may also be fluorochrome. Preferably, the flow cytometry is fluores antigen - specific T cells . For example , tumor - specific T cells cence -activated cell sorting (FACS ) . TCRs expressed on T can be used . In certain embodiments , antigen -specific T cells cells can be selected based on reactivity to autologous can be isolated from a patient of interest , such as a patient tumors . Additionally, T cells that are reactive to tumors can afflicted with a cancer or an infectious disease . In one be selected for based on markers using the methods embodiment neoepitopes are determined for a subject and T described in patent publication Nos . WO2014133567 and cells specific to these antigens are isolated . Antigen -specific WO2014133568, herein incorporated by reference in their cells for use in expansion may also be generated in vitro entirety . Additionally , activated T cells can be selected for using any number ofmethods known in the art, for example , based on surface expression of CD107a . as described in U . S . Patent Publication No . US [0174 ] In one embodiment of the invention , the method 20040224402 entitled , Generation And Isolation of Antigen further comprises expanding the numbers of T cells in the Specific Cells , or in U . S . Pat. No . 6 ,040 ,177 . Antigen enriched cell population . Such methods are described in specific cells for use in the present invention may also be U . S . Pat . No. 8 ,637 , 307 and is herein incorporated by generated using any number of methods known in the art , for reference in its entirety . The numbers of T cells may be example, as described in Current Protocols in immunology , increased at least about 3 - fold (or 4 - , 5 - , 6 - , 7 - , 8 - , or 9 - fold ) , or Current Protocols in Cell Biology , both published by John more preferably at least about 10 - fold (or 20 - , 30 - , 40 -, 50 - , Wiley & Sons . Inc. , Boston , Mass . 60 -, 70 -, 80 -, or 90 - fold ) , more preferably at least about [0171 ] In a related embodiment, it may be desirable to sort 100 - fold , more preferably at least about 1 ,000 fold , or most or otherwise positively select ( e . g . via magnetic selection ) preferably at least about 100 ,000 - fold . The numbers of T the antigen specific cells prior to or following one or two cells may be expanded using any suitable method known in rounds of expansion . Sorting or positively selecting antigen the art . Exemplary methods of expanding the numbers of specific cells can be carried out using peptide -MHC tetram cells are described in patent publication No . WO ers ( Altman , et al. , Science. 1996 Oct. 4 ; 274 (5284 ) : 94 - 6 ) . In 2003057171 , U . S . Pat. No . 8 , 034 , 334 , and U . S . Patent another embodiment the adaptable tetramer technology Application Publication No . 2012 /0244133 , each of which is approach is used ( Andersen et al. , 2012 Nat Protoc . 7: 891 incorporated herein by reference . US 2019 / 0262399 A1 Aug . 29 , 2019

[0175 ] In one embodiment, ex vivo T cell expansion can example modulate the proliferation , differentiation , matura be performed by isolation of T cells and subsequent stimu tion , migration , cytokine expression , antigen presentation , lation or activation followed by further expansion . In one and /or viability of the immune cell . embodiment of the invention , the T cells may be stimulated [0180 ] In certain embodiments , the agent is capable of or activated by a single agent. In another embodiment, T reducing the dysfunction of the immune cell ( e .g . , to treat a cells are stimulated or activated with two agents , one that proliferative disease , such as a tumor or cancer, or a chronic induces a primary signal and a second that is a co - stimula infection , such as a chronic viral infection ), or is capable of tory signal. Ligands useful for stimulating a single signal or increasing the dysfunction of the immune cell ( e . g . , to treat stimulating a primary signal and an accessory molecule that an autoimmune condition or disease ), or is capable of stimulates a second signal may be used in soluble form . reducing the activation of the immune cell ( e . g ., to treat an Ligands may be attached to the surface of a cell, to an autoimmune condition or disease ) , or is capable of increas Engineered Multivalent Signaling Platform (EMSP ) , or ing the activation of the immune cell (e .g ., to treat a immobilized on a surface . In a preferred embodiment both proliferative disease , such as a tumor or cancer, or a chronic primary and secondary agents are co - immobilized on a infection , such as a chronic viral infection ) . surface , for example a bead or a cell . In one embodiment, the [0181 ] In certain embodiments , the agent is capable of molecule providing the primary activation signal may be a modulating expression or activity of one or more genes or CD3 ligand, and the co - stimulatory molecule may be a gene products comprised by the signature of immune cell CD28 ligand or 4 - 1BB ligand . dysfunction or immune cell activation taught herein . For [0176 ] In accordance with the present invention , model example , in certain embodiments , (a ) the agent is capable of cellular systems using cell lines, primary cells , or tissue downregulating or abolishing expression or activity of one samples may be maintained in growth medium and may be or more genes or gene products comprised by the signature treated with compounds that may be at a single concentra of dysfunction as taught herein , thereby reducing dysfunc tion or at a range of concentrations . At specific times after tion of the immune cell ( e . g ., to treat a proliferative disease , treatment, cellular RNAs may be isolated from the treated such as a tumor or cancer, or a chronic infection , such as a cells , primary cells or tumors, which RNAs are indicative of chronic viral infection ) ; ( b ) the agent is capable of upregu expression of selected genes from a signature described lating expression or activity of one or more genes or gene herein . The cellular RNA is analyzed for the presence and /or products comprised by the signature of dysfunction as taught quantity of specific RNA transcripts . Transcripts may be herein , thereby increasing dysfunction of the immune cell amplified for detection purposes using standard methodolo ( e . g . , to treat an autoimmune condition or disease ) ; (c ) the gies , such as, for example, reverse transcriptase polymerase agent is capable ofupregulating expression or activity of one chain reaction (RT -PCR ), etc . The presence or absence , or or more genes or gene products comprised by the signature levels , of specific RNA transcripts are determined from these of activation as taught herein , thereby increasing the acti measurements and a metric derived for the type and degree vation of the immune cell ( e . g . , to treat a proliferative of response of the sample to the treated compound compared disease , such as a tumor or cancer, or a chronic infection , to control samples . Also in accordance with the present such as a chronic viral infection ) ; or ( d ) the agent is capable invention , there are disclosed herein characteristic , or sig of downregulating or abolishing expression or activity of nature , sets of genes and gene sequences whose expression one or more genes or gene products comprised by the is , or can be , as a result of the methods of the present signature of activation as taught herein , thereby reducing invention , linked to , or used to characterize , the dysfunction , activation of the immune cell ( e . g ., to treat an autoimmune activation or immune state of the immune cells of the present condition or disease ) . invention . Thus , the methods of the present invention iden [0182 ] In certain embodiments , any of the immune cells tify novel immunotherapeutic agents based on their altera may be a T cell or said immune cells comprise T cells , tion of expression of small sets of characteristic , or indicator, preferably CD8 + T cells . or signature genes in specific model systems. The methods [ 0183 ] In certain embodiments , any of the immune cells of the invention may therefore be used with a variety of cell may display tumor specificity . Such immune cells may be lines or with primary samples from tumors maintained in particularly useful for treating proliferative diseases , such as vitro under suitable culture conditions for varying periods of a tumor or cancer. By means of an example, said immune time, or in situ in suitable animal models . In preferred cell may have been isolated from a tumor of a subject, embodiments , tumor infiltrating lymphocytes ( TILs ) are preferably the immune cell may be a tumor infiltrating screened . lymphocyte . By means of another example , said immune [0177 ] The immune cell types or subtypes newly defined cell may comprise a tumor -specific chimeric antigen recep herein , can be also constitute valuable targets for use in tor (CAR ). therapeutic methods, such as for use in immunotherapy . [ 0184 ] Any methods of treatment as contemplated [ 0178 ] Accordingly , an aspect provides a method of treat throughout the present specification may be for a condition , ing a subject in need thereof , comprising administering to disease or disorder where an enhanced immune response is said subject an agent capable ofmodulating the immune cell required , such as but not limited to a cancer, or a condition , comprising the signature of immune cell dysfunction as disease or disorder where a decreased immune response is taught herein . required , such as but not limited to an autoimmune disease . [ 0179 ] Another aspect provides a method of treating a The immune cell may be modified , such that expression of subject in need thereof, comprising administering to said a gene signature is altered . The immune cell may be modi subject an agent capable of modulating the immune cell fied by treatment with an agent specific for downregulating comprising the signature of immune cell activation as taught expression or activity of at least one gene of one gene herein . By means of example , the agent may modulate one signature. The immune cell may be modified by treatment or more phenotypic aspects of the immune , such as for with an agent specific for upregulating expression or activity US 2019 / 0262399 A1 Aug . 29 , 2019 23 in at least one gene of an opposing gene signature . A gene CEP85L , IRF5 , INF2 , ITGB3 , MPC1, BCL2A1D , PARP3 , in the dysfunctional gene signature and a gene in the ASAP1, MRPS6 , RELB , FAM110A , GPR68 , NRP1, activation signature may be such modified . Not being bound CAPG , SCYL2, SAMD3, H2- AB1, HSF2 , CD44 , STX6 , by a theory , cancer may be treated by obtaining a dysfunc POLG2, TESPA1, ALCAM , NSMF, LRRC8D , HIF1A , tional T cell and treating with an agent that activates the cell. PACSIN1, PKP4 , ASS1 , NR4A3 , ENO3 , GYPC , KIF3B . Not being bound by a theory , introducing dysfunctional cells IL2RA , RAB37 , SGMS1, HLCS , SEMA6D , NMRK1, to a subject with an autoimmune disease may be performed . SLC17A6 , SLC39A1, RPS4X , CDON , ZFP445 , LAG3. Dysfunctional cells secrete suppressive cytokines that may RPS26 , PHTF2 , CST3 , CD9, STAT5A , ABCA3 , CSF2RA , suppress immune cells causing the autoimmunity . A gene , DTX3, RSPH3A , NRIP1 , SDHA , PNKD , FLNB , MGRN1, gene signature or immune cell may be modified ex vivo . A SLC26A2, HMOX2 , PEX16 , INPP4A , TNFRSF25 , IRF8 , gene, gene signature or immune cell may be modified ex RGCC , IFITM2, TNFSF14 , NSUNÁ , STAT3 , PFKFB3 , vivo . A gene, gene signature or immune cell may be modi TYROBP, HTRA2 , KLRI2 , CTSS , ARL5C , KLHL24 . fied in vivo . Not being bound by a theory, modifying SESN3 , GM5424 , FAS , NCOA3, FAM53B , CALCOCO1, immune cells in vivo , such that dysfunctional immune cells ERGIC3, 4930523COORIK , PCGF5 , ANXA4 , and HER are decreased can provide a therapeutic effect by enhancing PUD1; an immune response in a subject . A gene , gene signature or [0189 ] b ) the group consisting of CD74 , CCR7, TBC1D4, immune cell may be modified by a small molecule, a DNA SLC2A6 , BCL6 , JAK2 , PARP3 , ASAP1, RELB , H2 - AB1, targeting agent, or a therapeutic antibody or antibody frag CD44 , ABCA3 , PFKFB3, SESN3, FAS , 4930523C07RIK , ment thereof. As described herein , a DNA targeting agent PCGF5 , TNIP1, SPRY1, NCOA7, RPLPO , SMIM8, may be a CRISPR system . ANTXR2, NSMCE1, DEDD , B3GNT2, CABLES1 , [ 0185 ] A further aspect provides a kit of parts comprising SLAMF6 , UBL3 , NR4A1, ATG7 , and KDM5B ; means for detection of the signature of dysfunction and /or [ 0190 ] c ) the group consisting of CD83 , CCR8 , the signature of activation as taught herein . By means of TNFRSF4 , CD74 , CCR7, TNFSF11 , CD81, XCL1, example , such means for detection may comprise primers , TNFSF4 , AXL , ECE1, KIT, ITGB1 , CCL1, CD200 , probes , or antibodies. TNFRSF18 , TNFSF8 , CD160 , PTGER2 , BTLA , TSPAN32 , [0186 ] The inventors further realised that modulation of CD82 , KLRB1F , LTA , ANXA5 , ITGB3 , NRP1 , H2 - AB1, genes or gene products comprised by the gene signatures as CD44 , ALCAM , GYPC , IL2RA , CDON , LAG3 , CD9, taught herein in isolated immune cells can modulate the TNFSF14 , FAS , GDI2 , TNIP1 , IL21R , IL18R1, H2 - AA , properties of the cells and thereby provide for advantageous NR4A2 , IL18RAP, CD97 , TNFSF9, IRAK1BP1, GABA effects , such as increasing or decreasing dysfunctional phe RAPL1, TRPV2, EBAGO, GRN , RAMP1, AIMP1, BSG , notype of the immune cells , or rendering the immune cells IFNAR1, PRKCA , TRAF3 , CD96 , TNFRSF9 , and NR3C1; more resistant or more sensitive to becoming dysfunctional, [0191 ] d ) the group consisting of CD83 , TNFRSF4 , or increasing or decreasing activated phenotype of the CD74 , CCR7 , CD81 , TNFSF4 , KIT , ITGB1, CD200 , immune cells , or rendering the immune cells more resistant TNFSF8 , CD160 , CD82 , ITGB3, CD44 , ALCAM , GYPC , or more sensitive to becoming activated . Such modulation IL2RA , CDON , LAG3, CD9, CSF2RA , FAS , CD97 , can be of value inter alia in therapeutic applications , such as TNFSF9, BSG , IFNAR1, TRAF3 , CD96 , and TNFRSF9 ; for example but without limitation in ex vivo or allogeneic [0192 ] e ) the group consisting of CD83 , CD81, TNFRSF4 , therapies involving immune cells , such as T cells , such as CXCL16 , IL21R , and IL18R1; CD8 + T cells , e. g ., CAR - T therapies. [0193 ] f) the group consisting of REL , BCL6 , MNDA , [0187 ]. Accordingly , an aspect relates to an isolated BHLHE40 , NFKB2, ZHX2, KLF4 , NFKB1, NFKBIA , immune cell modified to comprise an altered expression or RARG , FOSB , HIVEP1, ZFP36L1, NFAT5 , ELK3, JUNB , activity of, or modified to comprise an agent capable of LIMK1, TGIF1, KDM2B , IRF5 , RELB , HSF2, HIF1A , inducibly altering expression or activity of, one or more NR4A3, PHTF2 , STAT5A , DTX3 , NRIP1, IRF8 , STAT3 , genes or gene products selected from . NCOA3, CALCOCO1, PCGF5 , NFKBIE , ETV6 , RNF19A , [0188 ] a ) the group consisting of CD83 , CCR8 , STAT4 , NR4A2 , NFKBIB , PERI, GTF2A1, SPRY1, TFE3 , TNFRSF4 , CD74 , CCR7, TNFSF11 , CD81, TBC1D4, REL , TGIF2 , RORA , RPL6 , EGR2 , FOXP4 , TBL1X , KDM4A , PLK2, XCL1, TNFSF4 , SLC2A6 , A1836003 , LAD1, COPS2, FOS , DEDD , SOSTM1, NT5C , PIAS4 , ZMYM2 , 1700019D03RIK , BCL6 , MNDA , RAMP3 , GPM6B , DMTF1, AEBP2, TRPS1 , SP3 , HBP1, NR4A1, TLE3 , BHLHE40 , AXL , ECE1, FILIPIL , KIT, ITGB1, CCL1 , RPL7 , MED21, DRAP1, TCF7 , CREB3L2 , ZFHX2, NFKB2 , PLXDC2, ARC , DUSP4, CD200 , TRAF1 , ZHX2, KDM5B , and NR3C1 ; or NCF1, CCDC28B , PTPRS , ST6GALNAC3 , TUSC3, [0194 ] g ) the group consisting of two or more genes or PDCD1LG2, SDHAF1 , ARAP2, KLF4 , E130308A19RIK , gene products each independently selected from any one of FAM46A , TNFRSF18 , SYNJ2 , CYTH3, TNFSF8 , CD160 , the groups as defined in any one of a ) to f ) . RPL10 , CRTAM , RAB6B , PTGER2, NFKB1, ANKRD46 , [0195 ] A related aspect provides an isolated immune cell STOGALNAC6 , ITPR1, ITM2C , BTLA , TSPAN32, CD82, modified to comprise an altered expression or activity of, or NFKBIA , MS4A4C , RARG , NRGN , TRIB1 , ZC3H12D , modified to comprise an agent capable of inducibly altering BMYC , IF127L2A , GADD45B , NAPSA , KLRB1F , expression or activity of, one or more genes or gene products RASGEF1A , FOSB , MAP3K8 , HIVEP1 , SSHI, selected from CD83 , CCR8 , TNFRSF4 , CD74 , CCR7, RABGAPIL , ZFP36L1, ARL4D , CACNAIS , NFAT5 , TNFSF11 , CD81, TBC1D4, REL , PLK2, XCL1 , TNFSF4 , DNAJC12 , SOWAHC , SDF4 , TMEM120B , DUSP1 , ELK3, SLC2A6 , A836003 , LAD1 , 1700019D03RIK , BCL6 , JUNB , GRAMDIB , LIMK1, ZFC3H1, OSTF1 , LTA , MNDA , RAMP3, GPM6B , BHLHE40 , AXL , ECE1, DNMT3A , BCL7C , TSPAN13 , ASNSD1, TGIF1, NRN1, FILIPIL , KIT , ITGB1, CCL1, NFKB2 , PLXDC2 , ARC , SYNGR2, MSI2 , UAP1, UNC93B1, JAK2, KDM2B , DUSP4 , CD200 , TRAF1, ZHX2, NCF1 , CCDC28B , ANXA5, PRDX2, TMEM173 , PHACTR2 , CCDC104 , PTPRS, STOGALNAC3 , TUSC3, PDCDILG2, SDHAF1 , US 2019 / 0262399 A1 Aug . 29 , 2019 24

ARAP2 , KLF4 , E130308A19RIK , FAM46A , TNFRSF18 , [0197 ] Another aspect provides an isolated immune cell SYNJ2 , CYTH3, TNFSF8 , CD160 , RPL10 , CRTAM , modified to comprise an altered expression or activity of, or RAB6B , PTGER2, NFKB1, ANKRD46 , STOGALNAC6 , modified to comprise an agent capable of inducibly altering ITPR1, ITM2C , BTLA , TSPAN32 , CD82 , NFKBIA , expression or activity of, one or more genes or gene products MS4A4C , RARG , NRGN , TRIB1, ZC3H12D , BMYC , selected from IFI27L2A , GADD45B , NAPSA , KLRB1F , RASGEF1A , [0198 ] a ) the group consisting of NUSAP1, CCNA2, FOSB , MAP3K8, HIVEP1, SSH1, RABGAPIL , ZFP36L1, NEIL3, SPC25 . HIST1H2AB , CKAP2L . PLK1. ARL4D , CACNAIS , NFAT5 , DNAJC12 , SOWAHC , SDF4 , HIST2H3C2 , MXD3, FAM64A , BUB1, FOXM1, TMEM120B , DUSP1, ELK3, JUNB , GRAMD1B , LIMK1, HIST2H3B , KIF22 , CENPE , SKAI, CCNF , CDCA3 , ZFC3H1, OSTF1 , LTA , DNMT3A , BCL7C , TSPAN13 , ESPLI, CASC5 , PBK , KIF2C , SGOLI, CDK1, ASNSD1, TGIF1, NRN1, SYNGR2 , MSI2 , UAP1 , SHCBP1, ASPM , FBXO5 , MIS18BP1, SPAG5 , KIF4 , UNC93B1, JAK2, KDM2B , ANXA5, PRDX2 , TMEM173 , ASF1B , BUB1B , AURKB , NCAPG , DEPDC1A . PHACTR2, CCDC104 , CEP85L , IRF5, INF2 , ITGB3 , ESCO2 , CDCA2 , BC030867, KIF20A , HIST1H2AK , MPC1, BCL2A1D , PARP3 , ASAP1, MRPS6 , RELB , SMC2, ECT2, RRM2, MK167 , 2810417H13RIK , CIT , GTSE1, NCAPG2, NCAPH , CDCAS, SAPCD2 , FAM110A , GPR68 , NRP1, CAPG , SCYL2, SAMD3, NEK2, CEP55 , CDCA5 , TOP2A , CCNB2 , MASTL , H2 -AB1 , HSF2 , CD44 , STX6 , POLG2 , TESPA1, ALCAM , ARHGAP19 , AURKA , KIF23 , CCNB1, RAD51 , NSMF, LRRC8D , HIF1A , PACSIN1, PKP4 , ASS1 , NR4A3 , TACC3, MELK , STMN1, HIST1H2AE , HIST1H4D , ENO3, GYPC , KIF3B , IL2RA , RAB37 , SGMS1 , HLCS , CENPH , HIST1H1B , CDC25C , CCDC34 , CDC20 , SEMA6D , NMRK1, SLC17A6 , SLC39A1, RPS4X , CDON , KIF11 , ARHGAP11A , 4930427A07RIK , FAM83D , ZFP445 , LAG3, RPS26 , PHTF2 , CST3 , CD9, STAT5A , INCENP, MAD2L1, HIST1H2AO , CKAP2 , NDC80 , ABCA3, CSF2RA , DTX3 , RSPH3A , NRIP1, SDHA , RAD51AP1 , NUF2, E2F7, CKSIB , NCAPD2, GEN1, PNKD , FLNB , MGRN1, SLC26A2, HMOX2, PEX16 , ANLN , TICRR , POLQ , PRC1, TTK , RAD54B , E2F8, INPP4A , TNFRSF25 , IRF8 , RGCC , IFITM2, TNFSF14 , STIL , KIF18A , PARPBP, CDC45 , BIRC5, KIF15 , NSUNG , STAT3 , PFKFB3 , TYROBP, HTRA2 , KLRI2 , SKA2 , KIF20B , TKI, PLK4 , FANCD2, CENPM , CTSS , ARL5C , KLHL24 , SESN3, GM5424 , FAS , NCOA3 , C330027C09RIK , HIST2H4 , SKA3 , RRM1, TROAP , FAM53B , CALCOCO1, ERGIC3, 4930523C07RIK , RAD54L , KNTC1, ZWILCH , CLSPN , TPX2 , CMC2, PCGF5 , ANXA4, HERPUD1, CD74 , CCR7 , TBC1D4, TCF19 , MCM10 , HMGB2 , HIST1H3C , HIST1H1E , SLC2A6 , BCL6 , JAK2, PARP3 , ASAP1 , RELB , H2- AB1, UBET, CHTF18 , TUBA1B , TUBBS , H2AFX , CD44, ABCA3 , PFKFB3 , SESN3 , FAS , 4930523C07RIK , GPSM2, SPC24 , UHRF1, TRIP13 , PMF1, ZFP367 , PCGF5 , TNIP1, SPRY1, NCOA7, RPLPO , SMIM8 , RACGAP1, CDC25B , UBE2C , CDKN3, CENPI, ANTXR2, NSMCE1, DEDD , B3GNT2 , CABLES1 , HIST1H3B , KPNA2 , HJURP, BRCA1, WDR62 , SLAMF6 , UBL3 , NR4A1, ATG7, KDM5B , CD83, CCR8, CENPN , GMNN , POC1A , TMPO , KNSTRN , FANCI, TNFRSF4 , CD74 , CCR7, TNFSF11 , CD81 , XCL1, CENPF , 6430706D22RIK , CKS2 , DIAP3 , WDR67 , TNFSF4 , AXL , ECEI, KIT , ITGB1, CCL1 , CD200 , FIGNL1, BRCA2, HMGB3 , MYBL2 , PKMYT1 , TNFRSF18 , TNFSF8 , CD160 , PTGER2, BTLA , TSPAN32 , TRAIP , RFC5 , CEP128 , POLA1, ANKLE1, HMGB1, CD82 , KLRB1F , LTA , ANXA5 , ITGB3, NRP1, H2 - AB1, FEN1, H2AFZ , TUBB4B , CTC1, CKAP5 , CDC6 , CD44 , ALCAM , GYPC , IL2RA , CDON , LAG3, CD9 , LIGI, POLE , MCMI, RFWD3 , HMGN2 , TYMS, TNFSF14 , FAS , GDI2 , TNIP1, IL21R , IL18R1, H2 -AA , CENPP , NCAPD3 , SUV39H1, A730008H23RIK , NR4A2 , IL18RAP, CD97 , TNFSF9 , IRAK1BP1, GABA TUBAIC , EME1, EXO1, PTMA , BLM , ULBP1, RAPL1, TRPV2 , EBAGY, GRN , RAMP1 , AIMP1, BSG , 1190002F15RIK , CDC7 , DLGAP5 , TUBG1, PRIM2, IFNAR1, PRKCA , TRAF3 , CD96 , TNFRSF9 , NR3C1, TMEM48 , NRM , CENPA , BARD1, HAUS4 , RCC1, CD83, TNFRSF4, CD74 , CCR7 , CD81, TNFSF4 , KIT , LMNB1, and HIST1H2AG ; ITGB1 , CD200 , TNFSF8 , CD160 , CD82 , ITGB3 , CD44 , [0199 ] b ) the group consisting of HIST1H1E , HMGB1, ALCAM , GYPC , IL2RA , CDON , LAG3 , CD9, CSF2RA , HAUS4 , RCC1, HAUS5 , REEP4 , SLBP, FKBP2 , ARSB , FAS , CD97 , TNFSF9 , BSG , IFNAR1, TRAF3 , CD96 , HIST1H3E , RAD18 , RAD50 , TAF6 , ANAPC5 , FANCG , TNFRSF9, CD83 , CD81, TNFRSF4 , CXCL16 , IL21R , CTCF, TONSL , LMNB2 , SEPHSI, HNRNPA2B1, IL18R1, REL , BCL6 , MNDA , BHLHE40 , NFKB2 , ZHX2, ANAPC15 , STARD3NL , 2700029MOORIK , DPY30 , KLF4 , NFKB1, NFKBIA , RARG , FOSB , HIVEP1 , SDF2L1, RDM1, CDCA7L , MEAF6 , MYEF2 , DNAAF2 , ZFP36L1, NFAT5 , ELK3 , JUNB, LIMK1, TGIF1 , KDM2B , POLR3K , IPP , TARDBP, GPAA1, KPNB1, FTSJD2, IRF5 , RELB , HSF2 , HIF1A , NR4A3 , PHTF2 , STAT5A , RPRD1B , HIST1H1C , DPYSL2 , TAF12 , ARPP19 , DTX3, NRIP1, IRF8 , STAT3 , NCOA3, CALCOCO1, TMCO1, EXOC4 , ASRGL1, CPSF6 , EIF2D , CCNH , PCGF5 , NFKBIE , ETV6 , RNF19A , STAT4 , NR4A2, NFK MYG1, VMA21 , TFIP11 , NDUFAB1, NUP35 , GRPEL1, BIB , PERI, GTF2A1, SPRY1, TFE3 , TGIF2 , RORA , RPL6 , C1D , GBP3 , CYB5B , PPM1G , SRSF10 , PIGF, KLREI , EGR2, FOXP4 , TBLIX , KDM4A , COPS2 , FOS, DEDD , COG4, PDIA4 , CDT1 , DUSP19 , ACAT2 , COMMD3 , SOSTMI, NT5C , PIAS4 , ZMYM2 , DMTF1 , AEBP2, HCFC2, LMAN1, ABHD10 , TIMM50 , CALU , AP1M1, TRPS1, SP3 , HBP1 , NR4A1 , TLE3 , RPL7 , MED21, GGH , GCDH , MRPS33 , TAX1BP3 , DYNLTIC , ERP44, DRAP1 , TCF7 , CREB3L2 , ZFHX2, KDM5B , and NR3C1 . TMEM129 , COG2 , TMEM167 , RBX1, 0610009020RIK , 0196 ] In certain embodiments , the isolated immune cell is PSMB9, PUF60 , LSM4, PEX19 , NTAN1, ELP2 , AKR1B3 , modified to comprise an altered expression or activity of, or PHF11B , POLC3, ZFP142 , PRADC1, TMEM209 , modified to comprise an agent capable of inducibly altering DYNC1LI1 , FARSB ,MTHFSD , 1700052N19RIK , SARS2 , expression or activity of , one or more genes or gene products LAMTORI, ALDH3A2 , EHBP1L1, D16ERTD472E , selected from the group consisting of CD83 , CD81, and CCDC127 , COMMD10 , DNAJC14 , MRPL16 , SSBP1, TNFRSF4 . EXOSC4 , UPF1, DOHH , PTPN23 , ZADH2 , ARFIP1 , US 2019 / 0262399 A1 Aug . 29 , 2019 25

STX18 , VMP1, TCEA1, ERGICI, PIAS3 , RAD17 , GTF3A , PRDM10 , MECP2 , SUDS3 , CUX1, ZBTB22 , EXOSC3, ACOT8 , MINA , LRRK1, MOGS , METTL10 , PLRG1, MED24 , ETV5 , SFMBT1, HLTF , MEF2A , JAZF1 , CERS4 , ATPBD4 , ZDHHC6 , MAVS , PARL , GNG2, GATADI, ZBTB11 , ZNRD1, RBPJ, XRCC6 , GTF2A1, CD200R4 , USF1, TYK2, SNAPC1, RBM18 , VPS53 , CTNNB1, PURE , CNOT7 , ZZEF1, TAF15 , TSC22D4 , ACTR10 , and DPCD ; HIRA , ELF4 , MED13L , MMS19 , MBDI, VHL , VPS72, [0200 ] c ) the group consisting of HMGB1, ULBP1, FOXJ3 , UBE2K , SNW1, RASSF7 , KEAP1, CAMTA1 , REEP4, HMMR , CMTM7, SIVAI , PAQR4, ATPIF1 , MED15 , MED8, ING3 , CREBZF , TMF1 , BOLA2 , IKZF2 . NUP85 , HSPA2 , ENTPD1, HNRNPU , CLIC4, FLOT2 , ARID5B , SND1, PHF20 , ZBTB24 , SMARCE1, ARIDIA , ENOX2, ENPP1 , TFRC , HAVCR2 , CD2BP2, LGALSI , MTA2, KLF10 , CCNC , IRF8 , ASH2L , MIER3, UIMC1, PGRMC1, USP14 , ENTPD5, ATP6AP2 , CCL3 , ILLORA , ELK3, AEBP2 , BRF1 , SPRY2, RLF, IRF3 , NFYA , FLIT , ARNT2 , KLRE1, CLPTM1, ITGAV , KLRC1, PDIA4 , SP1, BCLAF1 , FIZ1, SP3 , PARD6A , MTA1, CCDC71, PBX4 , CCR5 , ADAM17 , CXCL10 , IGSF8 , ADAM10 , IFNG , PHF5A , NACCI , ATF7IP , HDAC2 , GATAD2B , MGA , TNFRSF9 , CD244 , CTLA4, ERP44 , PSEN1, LY6A , TRIM27 , TCF20 , RUFY2 , GOLGB1, PYGO2 , ZFPL1, CCRL2 , NCOR2 , HSP90AA1 , IGF2R , PGP, KLRC3, HIF1AN , BUD31, TERF2, NOTCH2, UBN1, DNM2, PPIE , CKLF, TNFRSF1A , TMX3, KLRC2, PDE4D , SMPD1, HIVEP2 , TBC1D2B , COPS2 , TAF2 , PNRC2, ESRRA , IDE, SERPINE2 , LRPAP1, CSF1 , CYSLTR2 , LSM1, GRN , IRF9 , SAP30 , MIER1, EYA3, NCOA3 , LMO4 , PREB , IL1F9 , LDLR , CD80 , GPR174 , MIF , MYO9B , ROCK1, NMI, ZBTB40 , PCGF1 , HMGA1, SARNP , HSBP1, CREM , GPR56 , GPR160 , RAC1, PTPN11 , CMTM6 , ADA , PTTG1, AGGF1, BMI1 , NCOA2, CBX4 , TFEB , XAB2, NOTCH2, GPII , GDI2 , P4HB , NRP1 , F2R , AGTRAP , ERF, SAP18 , RYBP, MED17 , GTF2H3 , ZMYND19 , SKIL , PGLYRP1, STX4A , ADAMS, LYST, ITGA1, XPOT, LZTR1, FOXK2 , HTATSF1, TBL1X , PLEKHF2 , CIC , KLRK1 , CX3CR1, SEPT2 , CCL4 , CAST, RPS6KB1 , TCEA1, CIZI , TAF9B , TULP4, HMG20B , PIAS3 ,MED21 , H2- M3 , LAG3, CD99L2 , PDCD1, ECE1, EZR , NR4A2, TBLIXR1, UTP6 , KDM3A , TBX21 , ZBTB5 , EGRI, SEMA4D, NAMPT, PEAR1, IL12RB1 , CD200R4 , CD48, ZFYVE27 , PHTF1, THOC2, TRAK1, GTF2H1, RNF14 , LAMP2, IRAK2, CXCR6 , GPR65 , and GCNTI ; LCORL , IKZF3 , HEXIMI, RNF19A , FLI1, MED27 , [ 0201 ] d ) the group consisting of HMMR , SIVAI , THAP7 , MAFF, GATA3 , PNN , CBX8 , ADNP, NAB2, ENTPD1, ENPP1 , TFRC , CD2BP2, ENTPD5 , ITGAV, MED13 , NR4A2, PHRF1, SREBF1 , STAT2 , CHD2, KLRC1, CCR5 , ADAM17 , TNFRSF9 , CD244 , CTLA4 , MEF2D , TRIP12 , MLX , RLIM , ABT1 , KAT5 , ETS2 , IL3RA , IGF2R , TNFRSF1A , CD80 , ADAMS, LAG3, SCAP, RELA , BATF , MED26 , KDM2B , KDM6A , MGL2 , CD99L2 , SEMA4D , IL12RB1 , CD200R4 , CD48 , CBFA2T2 , PIAS4 , USF1 , ZBTB7A , RNF25 , ZBTB1, and LAMP2 ; MED23 , ZBTB41 , PHF2 , KAT2B , KDM2A , CIR1 . [ 0202 ] e ) the group consisting of MXD3, FOXM1, E2F7, ZC3H15 , ZEB2, REXO4 , ZSCAN21, BLZF1, and CENPB ; RAD54B , E2F8 , TCF19 , HMGB2, UHRF1, TRIP13 , or PMF1, BRCA1, BRCA2, HMGB3 , MYBL2 , HMGB1, [0203 ] f ) the group consisting of two or more genes or PTMA , WHSC1, CHAF1A , RBL1, DNMT1 , CCNE1, gene products each independently selected from any one of DEK , E2F2, CHAF1B , EZH2, G2E3 , WDHD1, SUZ12 , the groups as defined in any one of a ) to e ) . TFDP1 , RBBP4, CASP8AP2 , RFC1, CDCA4 , RBBP8 , [0204 ] A related aspect provides an isolated immune cell SSRP1, ANAPC11 , TERF1 , POLE3, CBFB , CBX3, CTCF , modified to comprise an altered expression or activity of, or MED30 , PBRMI, TFDP2 , ITGB3BP, CBX6 , NRF1 , modified to comprise an agent capable of inducibly altering BAZIB , E2F3 , PIASI , ILF2, HDAC6 , TIMELESS , expression or activity of, one or more genes or gene products SMARCA5 , MYEF2, TARDBP , EED , HMGXB4 , selected from : NUSAP1, CCNA2 , NEIL3, SPC25 , METTL14 , E2F4 , LIN9, MTF2 , MAZ , ATF1 , TAF1 , TOX , HIST1H2AB , CKAP2L , PLK1, HIST2H3C2 , MXD3 , NFYB , HNRNPD , SMARCB1, UHRF2 , ASXL1, MED14 , FAM64A , BUB1, FOXM1, HIST2H3B , KIF22 , CENPE , NAB1, BRD , ILF3 , MED7 , RB1, HDAC3, ERH , TSG101, SKAI , CCNF, CDCA3 , ESPL1, CASC5 , PBK , KIF2C , RNPS1, CCNH , NONO , DEAF1 , ZFP91 , PKNOX1, SGOLI , CDK1, SHCBP1, ASPM , FBX05 , MIS18BP1 , L3MBTL2, CDC5L , SP4 , KLF11 , SMARCC1, HDACI, SPAG5, KIF4 , ASF1B , BUB1B , AURKB , NCAPG , GTF2H5, SMARCC2, RUVBL2, ZBTB45 , NMRALI , DEPDC1A , ESCO2, CDCA2 , BC030867 , KIF20A , WIZ , ING1, FOSB , CID , DICER1, E2F1, THRAP3 , RNF4 , HIST1H2AK , SMC2, ECT2 , RRM2, MKI67 , TSHZ1, SF1, GABPA ,GABPB1 , SMYD4 , CBY1, ARNT2 , 2810417H13RIK , CIT, GTSE1, NCAPG2, NCAPH , GABPB2 , RFX1 , MORF4L2 , ZFYVE19, SUB1, HCFC1, CDCAS, SAPCD2 , NEK2, CEP55 , CDCA5 , TOP2A , TARBP2 , GTF3C2, POU2F1, ZNHIT3 , TBP, CANDI, CCNB2, MASTL , ARHGAP19 , AURKA , KIF23 , CCNB1, PCM1, BAZ1A , ATF6 , SREBF2 , YAF2, TCERG1, BRPF1 , RAD51, TACC3, MELK , STMN1, HIST1H2AE , LITAF, SMYD3, RNF5 , DPF2, SMARCD2, E2F5, PML , HIST1H4D , CENPH , HIST1H1B , CDC25C , CCDC34 , GMEB1, SP1 , PSIP1, SP2 , CNOT , OVCA2 , PFDN1, CDC20 , KIF11 , ARHGAP11A , 4930427A07RIK , COMMD3 , ING2, MYNN , HCFC2 , AES, LRRFIP2 , FAM83D , INCENP, MAD2L1, HIST1H2AO , CKAP2 , GTF2E2, YEATS4 , CNOT2 , MYCBP, PA2G4, TOEI , NDC80 , RAD51AP1, NUF2 , E2F7 , CKS1B , NCAPD2, SMARCD1, NFYC , GMEB2, CEBPB , IKZF5 , TFAM , GEN1, ANLN , TICRR , POLQ , PRC1 , TTK , RAD54B , NFIL3 , CNOT4 , COPS5 , GTF2A2 , CNOT1 , SIN3A , E2F8 , STIL , KIF18A , PARPBP , CDC45 , BIRC5 , KIF15 , GTF2F1 , TSC22D2, ZBTB2 , MED4 , RUVBL1, AIP , SKA2, KIF20B , TK1, PLK4, FANCD2 , CENPM , TRIM28 , GTF2B , DEDD , CREB1, PRDM1, CTBP1, C330027COORIK , HIST2H4, SKA3 , RRMI, TROAP , GTF3C5 , TAF10 , PSMC3 , MED31, RBX1, FUS , POBP1 , RAD54L , KNTC1, ZWILCH , CLSPN , TPX2, CMC2, ELF2, ATF2 , CNOT8 , NCOR2, SMARCA4 , GNPTAB , TCF19 , MCM10 , HMGB2 , HIST1H3C , HISTIHIE , TAF6L , CRAMPIL , TCF3 , CEBPG , GTF3C3 , TAF3 , ID2, UBE2T , CHTF18 , TUBA1B , TUBBS , H2AFX , GPSM2, YBX1, TAF11, YY2 , MEN1, PHF6 , PHF17 , SMARCAD1, SPC24 , UHRF1 , TRIP13 , PMF1 , ZFP367 , RACGAP1, RBL2, HTT , HDAC5 , ING5 , CXXC1, NFKBIL1 , CSDA , CDC25B , UBE2C , CDKN3, CENPI, HIST1H3B , KPNA2 , US 2019 /0262399 A1 Aug . 29 , 2019

HJURP, BRCA1, WDR62 , CENPN , GMNN , POC1A CBX3 , CTCF ,MED30 , PBRM1, TFDP2, ITGB3BP, CBX6 , TMPO , KNSTRN , FANCI, CENPF , 6430706D22RIK , NRF1, BAZIB , E2F3 , PIASI, ILF2 , HDAC6 , TIMELESS , CKS2, DIAP3 , WDR67, FIGNL1, BRCA2 , HMGB3, SMARCA5 , MYEF2 , TARDBP, EED , HMGXB4 , MYBL2 , PKMYT1, TRAIP, RFC5, CEP128 , POLAI, METTL14 , E2F4 , LIN9, MTF2 , MAZ , ATF1 , TAF1 , TOX , ANKLE1, HMGB1, FEN1, H2AFZ , TUBB4B , CTC1, NFYB , HNRNPD , SMARCB1, UHRF2 , ASXL1, MED14 , CKAP5 , CDC6 , LIG1, POLE , MCMI, RFWD3 , HMGN2 , NAB1, BRDS, ILF3 , MED7, R131 , HDAC3, ERH , TYMS , CENPP , NCAPD3, SUV39H1, A730008H23RIK , TSG101, RNPS1, CCNH , NONO , DEAF1, ZFP91 , TUBAIC , EMEI, EXO1, PTMA, BLM , ULBP1, PKNOX1, L3MBTL2 , CDC5L , SP4 , KLF11 , SMARCC1, 1190002F15RIK , CDC7 , DLGAP5 , TUBG1, PRIM2, HDACI, GTF2H5, SMARCC2 , RUVBL2 , ZBTB45 , TMEM48 , NRM , CENPA , BARD1, HAUS4 , RCC1, NMRALI, WIZ , ING1, FOSB , CID , DICER1, E2F1, LMNB1, HIST1H2AG , HIST1HIE , HMGB1, HAUS4 , THRAP3 , RNF4 , TSHZI , SF1 , GABPA , GABPB1, RCC1 , HAUS5 , REEP4 , SLBP, FKBP2 , ARSB , SMYD4, CBY1 , ARNT2, GABPB2, RFX1, MORF4L2 , HIST1H3E , RAD18 , RAD50 , TAF6 , ANAPC5, FANCG , ZFYVE19 , SUB1, HCFC1 , TARBP2 , GTF3C2, POU2F1, CTCF, TONSL , LMNB2 , SEPHS1, HNRNPA2B1, ZNHIT3 , TBP, CAND1, PCM1, BAZIA , ATF6 , SREBF2 , ANAPC15 , STARD3NL , 2700029MOORIK , DPY30 , YAF2, TCERGI, BRPF1, LITAF , SMYD3, RNF5 , DPF2 , SDF2L1, RDM1, CDCA7L , MEAF6 , MYEF2 , DNAAF2 , SMARCD2 , E2F5, PML , GMEB1, SP1 , PSIP1 , SP2 , POLR3K , IPP , TARDBP , GPAA1 KPNB1, FTSJD2 , CNOT6 , OVCA2, PFDN1, COMMD3, ING2, MYNN , RPRD1B , HISTIHIC , DPYSL2 , TAF12 , ARPP19 , HCFC2, AES , LRRFIP2 , GTF2E2 , YEATS4 , CNOT2 , TMCO1, EXOC4 , ASRGLI, CPSF6 , EIF2D , CCNH , MYCBP , PA2G4, TOEI, SMARCD1, NFYC , GMEB2 , MYG1, VMA21 , TFIP11, NDUFAB1, NUP35 , GRPELI , CEBPB , IKZF5 , TFAM , NFIL3 , CNOT4 , COPS5 . C1D , GBP3 , CYB5B , PPM1G , SRSF10 , PIGF, KLRE1, GTF2A2 , CNOT1 , SIN3A , GTF2F1, TSC22D2 , ZBTB2, COG4 , PDIA4 , CDT1, DUSP19 , ACAT2 , COMMD3 , MED4 , RUVBL1 , AIP, TRIM28 ,GTF2B , DEDD , CREB1, HCFC2, LMANI, ABHD10 , TIMM50 , CALU , AP1M1, PRDM1, CTBP1 , GTF3C5 , TAF10 , PSMC3 , MED31. GGH , GCDH , MRPS33 , TAX1BP3, DYNLTIC , ERP44 , RBX1, FUS, PQBP1 , ELF2 , ATF2 , CNOT8 , NCOR2, TMEM129 , COG2 , TMEM167 , RBX1, 0610009020RIK , SMARCA4 , GNPTAB , TAF6L , CRAMPIL , TCF3 , PSMB9, PUF60 , LSM4, PEX19 , NTAN1, ELP2 , AKR1B3 , CEBPG ,GTF3C3 , TAF3 , ID2, YBX1, TAF11 , YY2 ,MEN1 , PHF11B , POLC3 , ZFP142 , PRADC1, TMEM209 , PHF6 , PHF17 , SMARCAD1, RBL2 , HTT, HDAC5 , ING5 , DYNC1LI1 , FARSB , MTHFSD , 1700052N19RIK , SARS2 , CXXC1, NFKBIL1, CSDA , GTF3A , PRDM10 , MECP2 , LAMTOR1, ALDH3A2 , EHBP1L1, D16ERTD472E , SUDS3 , CUX1, ZBTB22 , PLRGI, MED24 , ETV5 , CCDC127 , COMMD10 , DNAJC14 , MRPL16 , SSBP1, SFMBT1 , HLTF , MEF2A , JAZF1 , GATADI, ZBTB11 , EXOSC4 , UPF1, DOHH , PTPN23 , ZADH2 , ARFIP1, ZNRD1, RBPJ, XRCC6 , GTF2A1, CTNNB1, PURB , STX18 , VMP1, TCEAI, ERGIC1, PIAS3 , RAD17 , CNOT7 , ZZEF1 , TAF15 , TSC22D4 , HIRA , ELF4 , EXOSC3 , ACOT , MINA , LRRK1, MOGS , METTL10 , MED13L , MMS19 , MBD1, VHL , VPS72 , FOXJ3 , UBE2K , CERS4 , ATPBD4, ZDHHC6 , MAVS , PARL , GNG2, SNW1, RASSF7 , KEAP1 , CAMTAI, MED15 , MED , CD200R4 , USF1, TYK2, SNAPC1, RBM18 , VPS53 , ING3 , CREBZF , TMF1, BOLA2 , IKZF2, ARID5B , SND1 , ACTR10 , DPCD , HMGB1, ULBP1, REEP4, HMMR , PHF20 , ZBTB24 , SMARCE1, ARID1A , MTA2, KLF10 , CMTM7, SIVAI, PAQR4 , ATPIF1 , NUP85 , HSPA2 , CCNC , IRF8 , ASH2L , MIER3, UIMCI , ELK3, AEBP2 , ENTPD1, HNRNPU , CLIC4, FLOT2 , ENOX2 , ENPP1, BRF1, SPRY2 , RLF, IRF3, NFYA , FLII , BCLAF1 , FIZI , TFRC , HAVCR2, CD2BP2 , LGALS1 , PGRMC1, USP14 , SP3 , PARD6A , MTA1, CCDC71, PBX4 , PHF5A , NACC1, ENTPD5 , ATP6AP2, CCL3 , IL10RA, ARNT2 , KLREI , ATF7IP , HDAC2, GATAD2B , MGA, TRIM27 , TCF20 , CLPTM1, ITGAV , KLRC1, PDIA4 , SP1, CCR5 , ADAM17 , RUFY2 , GOLGB1, PYGO2, ZFPL1 , HIF1AN , BUD31, CXCL10 , IGSF8 , ADAM10 , IFNG , TNFRSF9 , CD244 , TERF2 , NOTCH2, UBN1, DNM2, PPIE , HIVEP2 , CTLA4, ERP44, PSEN1, LY6A , CCRL2, NCOR2 , TBC1D2B , COPS2 , TAF2 , PNRC2, ESRRA , IRF9 , SAP30 , HSP90AAI, IGF2R , PGP, KLRC3, CKLF, TNFRSF1A , MIER1, EYAZ, NCOA3, LMO4 , PREB , NMI, ZBTB40 , TMX3 , KLRC2, PDE4D , SMPD1, IDE , SERPINE2 , PCGF1 , HMGAI, SARNP, HSBP1 , CREM , PTTG1, LRPAP1, CSF1, CYSLTR2, LSM1, GRN , IL1F9, LDLR , AGGF1, BMI1 , NCOA2 , CBX4 , TFEB , XAB2 , ERF, CD80 , GPR174 , MIF , MYO9B , ROCK1, GPR56 , GPR160 , SAP18 , RYBP, MED17 , GTF2H3 , ZMYND19 , SKIL , RAC1, PTPN11, CMTM6, ADA , NOTCH2 , GPI1, GDI2 , LZTR1, FOXK2, HTATSF1, TBLIX , PLEKHF2 , CIC , P4HB , NRP1, F2R , AGTRAP , PGLYRP1 , STX4A , TCEA1, CIZI , TAF9B , TULP4, HMG20B , PIAS3 ,MED21 , ADAMS , LYST , ITGAI, XPOT, KLRK1, CX3CR1, TBL1XR1, UTP6 , KDM3A , TBX21 , ZBTB5 , EGRI, SEPT2 , CCL4, CAST, RPS6KB1, H2 -M3 , LAG3, CD99L2 , ZFYVE27 , PHTF1, THOC2, TRAK1, GTF2H1, RNF14 , PDCD1 , ECE1 , EZR, NR4A2, SEMA4D , NAMPT , PEAR1 , LCORL , IKZF3 , HEXIMI, RNF19A , FLI1 , MED27 , IL12RB1, CD200R4 , CD48 , LAMP2 , IRAK2, CXCR6 , THAP7 , MAFF , GATA3 , PNN , CBX8, ADNP, NAB2 , GPR65 , GCNTI, HMMR , SIVAI, ENTPD1, ENPP1, MED13 , NR4A2 , PHRF1, SREBF1, STAT2 , CHD2 , TFRC , CD2BP2 , ENTPD5 , ITGAV , KLRC1, CCR5 , MEF2D , TRIP12 , MLX , RLIM , ABT1, KAT5 , ETS2 , ADAM17 , TNFRSF9 , CD244 , CTLA4 , IL3RA , IGF2R , SCAP, RELA , BATF , MED26 , KDM2B , KDM6A , TNFRSF1A , CD80 , ADAMS, LAG3, MGL2, CD99L2 , CBFA2T2 , PIAS4 , USF1, ZBTB7A , RNF25 , ZBTB1, SEMA4D , IL12RB1, CD200R4 , CD48 , LAMP2, MXD3 , MED23 , ZBTB41 , PHF2 , KAT2B , KDM2A , CIR1 , FOXM1, E2F7 , RAD54B , E2F8 , TCF19 , HMGB2 , UHRF1, ZC3H15 , ZEB2 , REX04 , ZSCAN21, BLZF1, and CENPB . TRIP13 , PMF1 , BRCA1, BRCA2 , HMGB3, MYBL2, [0205 ] As used herein , the term “ modulation of at least HMGB1, PTMA, WHSC1, CHAF1A , RBLI, DNMT1 , one function of the immune cell ” includes the modulation of CCNE1, DEK , E2F2 , CHAF1B , EZH2, G2E3 , WDHD1, any of a variety of T cell- related functions and /or activities , SUZ12 , TFDP1 , RBBP4, CASP8AP2 , RFC1, CDCA4, including by way of non - limiting example , controlling or RBBP8 , SSRP1, ANAPC11 , TERF1, POLE3 , CBFB , otherwise influencing the networks that regulate T cell US 2019 / 0262399 A1 Aug . 29 , 2019 differentiation ; controlling or otherwise influencing the net - manipulated by a man -made process, such as a man -made works that regulate T cell maintenance , for example , over molecular - or cell biology process , resulting in the modifi the lifespan of a T cell; controlling or otherwise influencing cation of at least one characteristic of the immune cell. Such the networks that regulate T cell function ; controlling or man -made process may for example be performed in vitro or otherwise influencing the networks that regulate helper T ex vivo . cell ( Th cell ) differentiation ; controlling or otherwise influ [0210 ] The term “ altered expression " denotes that the encing the networks that regulate Th cell maintenance , for modification of the immune cell alters , i. e . , changes or example , over the lifespan of a Th cell , controlling or modulates, the expression of the recited gene ( s ) or polypep otherwise influencing the networks that regulate Th cell tides( s ) . The term " altered expression " encompasses any function ; controlling or otherwise influencing the networks direction and any extent of said alteration . Hence , “ altered that regulate Th17 cell differentiation ; controlling or other expression ” may reflect qualitative and/ or quantitative wise influencing the networks that regulate Th17 cell main change( s ) of expression , and specifically encompasses both tenance , for example , over the lifespan of a Th17 cell ; increase ( e . g . , activation or stimulation ) or decrease ( e . g . , controlling or otherwise influencing the networks that regu inhibition ) of expression . late Th17 cell function ; controlling or otherwise influencing [0211 ] The terms “ increased ” or “ increase” or “ upregu the networks that regulate regulatory T cell ( Treg ) differen lated ” or “ upregulate ” as used herein generally mean an tiation ; controlling or otherwise influencing the networks increase by a statically significant amount. For avoidance of that regulate Treg cell maintenance , for example , over the doubt, " increased ” means a statistically significant increase lifespan of a Treg cell ; controlling or otherwise influencing of at least 10 % as compared to a reference level, including the networks that regulate Treg cell function ; controlling or an increase of at least 20 % , at least 30 % , at least 40 % , at otherwise influencing the networks that regulate other CD4 + least 50 % , at least 60 % , at least 70 % , at least 80 % , at least T cell differentiation ; controlling or otherwise influencing 90 % , at least 100 % or more , including, for example at least the networks that regulate other CD4 + T cell maintenance ; 2 - fold , at least 3 - fold , at least 4 -fold , at least 5 -fold , at least controlling or otherwise influencing the networks that regu 10 - fold increase or greater as compared to a reference level , late other CD4 + T cell function ; controlling or otherwise as that term is defined herein . influencing the networks that regulate other CD8 + T cell [0212 ] The term “ reduced ” or “ reduce” or “ decrease ” or differentiation , controlling or otherwise influencing the net " decreased ” or “ downregulate ” or “ downregulated ” as used works that regulate other CD8 + T cell maintenance ; or herein generally means a decrease by a statistically signifi controlling or otherwise influencing the networks that regu cant amount relative to a reference . For avoidance of doubt, late other CD8 + T cell function . “ reduced ” means statistically significant decrease of at least [ 0206 ] The term “ isolated ” with reference to a particular 10 % as compared to a reference level, for example a component generally denotes that such component exists in decrease by at least 20 % , at least 30 % , at least 40 % , at least separation from — for example , has been separated from or t 50 % , or least 60 % , or least 70 % , or least 80 % , at least 90 % prepared and / or maintained in separation from one or or more , up to and including a 100 % decrease ( i. e ., absent more other components of its natural environment. More level as compared to a reference sample ) , or any decrease particularly , the term “ isolated ” as used herein in relation to between 10 - 100 % as compared to a reference level, as that a cell or cell population denotes that such cell or cell term is defined herein . The term " abolish ” or “ abolished " population does not form part of an animal or human body . may in particular refer to a decrease by 100 % , i . e . , absent 02071. The term “ immune cell ” as used herein generally level as compared to a reference sample . encompasses any cell derived from a hematopoietic stem [0213 ] The modification may produce an immune cell cell that plays a role in the immune response . Immune cells comprising altered expression or activity of the one or more include , without limitation , lymphocytes, such as T cells and genes or gene products as taught herein ; or themodification B cells , antigen -presenting cells (APC ), dendritic cells , may produce an immune cell which does not comprise monocytes, macrophages, natural killer (NK ) cells , mast altered expression or activity of the one or more genes or cells, basophils , eosinophils , or neutrophils , as well as any gene products as taught herein , but which has acquired the progenitors of such cells . In certain preferred embodiments , ability to exhibit altered expression or activity of the one or the immune cell may be a T cell. As used herein , the term more genes or gene products as taught herein in response to “ T cell” ( i. e ., T lymphocyte ) is intended to include all cells an external signal. The latter cell has thus been modified to within the T cell lineage, including thymocytes , immature T comprise an agent capable of inducibly ( i . e . , in response to cells , mature T cells and the like. The term “ T cell” may a signal, more particularly to an external signal, such as to include CD4 + and /or CD8 + T cells , T helper ( T ) ) cells , e . g . , an external chemical, biological and /or physical signal ) Th1, T12 and T , 17 cells , and T regulatory ( Treg ) cells . altering expression or activity of the one or more genes or [ 0208 ] In certain more preferred embodiments , the gene products as taught herein . immune is a T cell , preferably a CD8 + T cell, also known as [0214 ] Hence , in certain embodiments , the modification cytotoxic T cell or T A CD8 + T cell is a T cell expressing may comprise exposing the immune cell to an agent or the CD8 cell surface marker , and recognizes antigens in the contacting the immune cell with an agent or introducing into context of MHC class I presentation . CD8 + T cells have the immune cell an agent capable of altering the expression cytotoxic activity and proliferate in response to IFN - gamma or activity of the one or more genes or gene products as and other cytokines . Engagement of CD8 + T -cell to the TCR taught herein , whereby the expression or activity of the one receptor of a CD8 + T - cell antigen presented by Class I MHC or more genes or gene products as taught herein in the molecules and co -stimulating molecules lead to cytotoxic immune cell is altered . In certain embodiments , the agent or activity , proliferation and / or cytokine production . one or more elements thereof may be under inducible [ 0209 ] The term " modified ” as used herein broadly control . For example , the expression of the agent or one or denotes that an immune cell has been subjected to or more elements thereof by the immune cell and /or the activity US 2019 / 0262399 A1 Aug . 29 , 2019 28 of the agent or one or more elements thereof in the cell may [ 0218 ] Aswill be clear to the skilled person , " modulating” be under inducible control. The immune cell thereby can also involve effecting a change ( which can either be an acquires the ability to exhibit altered expression or activity increase or a decrease in affinity , avidity , specificity and / or of the one or more genes or gene products as taught herein selectivity of a target or antigen , e . g ., the one or more genes in response to an external signal configured to modulate the or gene products as taught herein , for one or more of its agent or one or more elements thereof , such as the expres targets compared to the same conditions but without the sion and /or the activity of the agent or one or more elements presence of a modulating agent. Again , this can be deter thereof. mined in any suitable manner and / or using any suitable assay known per se , depending on the target. In particular, [ 0215 ]. Any one or more of the several successive molecu an action as an inhibitor / antagonist or activator / agonist can lar mechanisms involved in the expression of a given gene be such that an intended biological or physiological activity or polypeptide may be targeted by the immune cell modi is increased or decreased , respectively , by at least 5 % , at fication as intended herein . Without limitation , these may include targeting the gene sequence ( e . g . , targeting the least 10 % , at least 25 % , at least 50 % , at least 60 % , at least polypeptide -encoding , non -coding and /or regulatory por 70 % , at least 80 % , or 90 % or more , compared to the tions of the gene sequence ) , the transcription of the gene into biological or physiological activity in the same assay under RNA , the polyadenylation and where applicable splicing the same conditions but without the presence of the inhibi and / or other post - transcriptional modifications of the RNA tor/ antagonist agent or activator/ agonist agent. Modulating into mRNA , the localisation of the mRNA into cell cyto can also involve activating the target or antigen or the plasm , where applicable other posttranscriptional modifi mechanism or pathway in which it is involved . cations of the mRNA , the translation of the mRNA into a 102191. The term “ agent” as used herein generally refers to polypeptide chain , where applicable post- translational any substance or composition , such as a chemical entity or modifications of the polypeptide , and / or folding of the biological product , or combination of chemical entities or polypeptide chain into the mature conformation of the biological products , capable of achieving a desired effect in polypeptide . For compartmentalised polypeptides , such as a system , more particularly in a biological system , e . g . , in a secreted polypeptides and transmembrane polypeptides, this cell , tissue, organ , or an organism . In the present context, an may further include targeting trafficking of the polypeptides, agent may be exposed to , contacted with or introduced into i . e ., the cellular mechanism by which polypeptides are an immune cell to modify at least one characteristic of the transported to the appropriate sub - cellular compartment or immune cell, such as to ( inducibly ) alter the expression or organelle , membrane, e. g . the plasmamembrane , or outside activity of the one or more genes or gene products as taught the cell . herein by the immune cell . Further in the present context, an agent may be administered to a subject to treat or prevent or [0216 ] Hence , “ altered expression ” may particularly control a disease or condition , for example by ( inducibly ) denote altered production of the recited gene products by the altering the expression or activity of the one or more genes modified immune cell . As used herein , the term " gene or gene products as taught herein by immune cells of the product ( s )” includes RNA transcribed from a gene ( e .g ., subject mRNA ), or a polypeptide encoded by a gene or translated [0220 ] The chemical entity or biological product is pref from RNA . erably , but not necessarily a low molecular weight com [ 0217 ] Also , “ altered expression ” as intended herein may pound, but may also be a larger compound , or any organic encompass modulating the activity of the one or more genes or inorganic molecule effective in the given situation , or gene products as taught herein . Accordingly , “ altered including modified and unmodified nucleic acids such as expression ” , “ altering expression ” , “ modulating expres antisense nucleic acids , RNAi, such as siRNA or shRNA , sion " , or " detecting expression ” or similar may be used CRISPR -Cas systems, peptides, peptidomimetics , receptors , interchangeably with respectively “ altered expression or ligands, and antibodies, aptamers , polypeptides, nucleic acid activity ” , “ altering expression or activity ” , “ modulating analogues or variants thereof . Examples include an oligomer expression or activity " , or " detecting expression or activity ” of nucleic acids , amino acids , or carbohydrates including or similar. As used herein , " modulating ” or “ to modulate ” without limitation proteins , oligonucleotides , ribozymes , generally means either reducing or inhibiting the activity of DNAzymes, glycoproteins , siRNAs, lipoproteins, aptamers , a target or antigen , e .g ., the one or more genes or gene and modifications and combinations thereof. Agents can be products as taught herein , or alternatively increasing the selected from a group comprising : chemicals ; small mol activity of the target or antigen , e . g . , the one or more genes ecules ; nucleic acid sequences ; nucleic acid analogues ; or gene products as taught herein , as measured using a proteins ; peptides; aptamers ; antibodies ; or fragments suitable in vitro , cellular or in vivo assay . In particular , thereof. A nucleic acid sequence can be RNA or DNA , and " modulating ” or “ to modulate ” can mean either reducing or can be single or double stranded , and can be selected from inhibiting the ( relevant or intended ) activity of, or alterna a group comprising ; nucleic acid encoding a protein of tively increasing the ( relevant or intended ) biological activ interest, oligonucleotides, nucleic acid analogues , for ity of the target or antigen , e. g ., the one or more genes or example peptide- nucleic acid (PNA ) , pseudo - complemen gene products as taught herein , as measured using a suitable tary PNA (PC - PNA ) , locked nucleic acid (LNA ), modified in vitro , cellular or in vivo assay (which will usually depend RNA (mod -RNA ) , single guide RNA etc . Such nucleic acid on the target or antigen involved ), by at least 5 % , at least sequences include , for example , but are not limited to , 10 % , at least 25 % , at least 50 % , at least 60 % , at least 70 % , nucleic acid sequence encoding proteins, for example that at least 80 % , or 90 % or more , compared to activity of the act as transcriptional repressors, antisense molecules , target or antigen in the sameassay under the same conditions ribozymes , small inhibitory nucleic acid sequences , for but without the presence ofthe inhibitor/ antagonist agents or example but are not limited to RNAi, shRNAi, siRNA , activator/ agonist agents described herein . micro RNAi (mRNAi ) , antisense oligonucleotides , CRISPR US 2019 /0262399 A1 Aug . 29 , 2019 guide RNA , for example that target a CRISPR enzyme to a length , preferably about 19 -30 base nucleotides, preferably specific DNA target sequence etc . A protein and /or peptide about 20 - 25 nucleotides in length , e . g . , 20 , 21, 22 , 23 , 24 , or fragment thereof can be any protein of interest, for 25 , 26 , 27 , 28 , 29 , or 30 nucleotides in length ). example, but are not limited to : mutated proteins ; therapeu tic proteins and truncated proteins , wherein the protein is [0224 ] As used herein “ shRNA ” or “ small hairpin RNA ” normally absent or expressed at lower levels in the cell. ( also called stem loop ) is a type of siRNA . In one embodi Proteins can also be selected from a group comprising ; ment, these shRNAs are composed of a short , e . g . about 19 mutated proteins, genetically engineered proteins, peptides , to about 25 nucleotide , antisense strand , followed by a synthetic peptides, recombinant proteins , chimeric proteins, nucleotide loop of about 5 to about 9 nucleotides , and the antibodies , midibodies, minibodies, triabodies, humanized analogous sense strand . Alternatively, the sense strand can proteins , humanized antibodies, chimeric antibodies, modi precede the nucleotide loop structure and the antisense fied proteins and fragments thereof. Alternatively, the agent strand can follow . can be intracellular within the cell as a result of introduction [0225 ] The terms “ microRNA ” or “ miRNA ” are used of a nucleic acid sequence into the cell and its transcription interchangeably herein are endogenous RNAs , some of resulting in the production of the nucleic acid and / or protein which are known to regulate the expression of protein modulator of a gene within the cell . In some embodiments, coding genes at the posttranscriptional level. Endogenous the agent is any chemical, entity or moiety , including with microRNAs are small RNAs naturally present in the genome out limitation synthetic and naturally - occurring non -pro that are capable of modulating the productive utilization of teinaceous entities . In certain embodiments the agent is a mRNA . The term artificial microRNA includes any type of small molecule having a chemical moiety . Agents can be RNA sequence , other than endogenous microRNA , which is known to have a desired activity and /or property, or can be capable of modulating the productive utilization of mRNA . selected from a library of diverse compounds. MicroRNA sequences have been described in publications [ 0221] As used herein , " gene silencing ” or “ gene such as Lim , et al ., Genes & Development, 17 , p . 991 - 1008 silenced ” in reference to an activity of an RNAi molecule , ( 2003 ) , Lim et al Science 299 , 1540 ( 2003 ) , Lee and Ambros for example a siRNA or miRNA refers to a decrease in the Science , 294 , 862 (2001 ) , Lau et al . , Science 294 , 858 - 861 mRNA level in a cell for a target gene by at least about 5 % , ( 2001 ) , Lagos -Quintana et al, Current Biology , 12 , 735 -739 about 10 % , about 20 % , about 30 % , about 40 % , about 50 % , ( 2002 ) , Lagos Quintana et al, Science 294 , 853 - 857 (2001 ) , about 60 % , about 70 % , about 80 % , about 90 % , about 95 % , and Lagos- Quintana et al , RNA , 9, 175 - 179 ( 2003 ), which about 99 % , about 100 % of themRNA level found in the cell are incorporated by reference . Multiple microRNAs can also without the presence of the miRNA or RNA interference be incorporated into a precursor molecule . Furthermore , molecule . In one preferred embodiment , the mRNA levels miRNA - like stem - loops can be expressed in cells as a are decreased by at least about 70 % , about 80 % , about 90 % , vehicle to deliver artificial miRNAs and short interfering about 95 % , about 99 % , about 100 % . RNAs ( siRNAs) for the purpose of modulating the expres [0222 ] As used herein , the term “ RNAi” refers to any type sion of endogenous genes through the miRNA and or RNAi of interfering RNA , including but not limited to , siRNAi, pathways . shRNAi, endogenous microRNA and artificial microRNA . For instance , it includes sequences previously identified as [0226 ] As used herein , “ double stranded RNA ” or siRNA , regardless of the mechanism of downstream pro “ dsRNA ” refers to RNA molecules that are comprised of cessing of the RNA ( i. e . although siRNAs are believed to two strands . Double - stranded molecules include those com have a specific method of in vivo processing resulting in the prised of a single RNA molecule that doubles back on itself cleavage ofmRNA , such sequences can be incorporated into to form a two - stranded structure . For example , the stem loop the vectors in the context of the flanking sequences structure of the progenitor molecules from which the single described herein ) . The term “ RNAi” can include both gene stranded miRNA is derived , called the pre -miRNA ( Bartel et silencing RNAi molecules , and also RNAi effector mol al. 2004 . Cell 1 16 : 281 - 297 ) , comprises a dsRNA molecule . ecules which activate the expression of a gene . By way of an [0227 ] The term “ nucleic acid ” is well known in the art . A example only, in some embodiments RNAi agents which “ nucleic acid ” as used herein will generally refer to a serve to inhibit or gene silence are useful in themethods , kits molecule ( i . e . , strand ) of DNA , RNA or a derivative or and compositions disclosed herein to alter the expression of , analog thereof, comprising a nucleobase . A nucleobase such as in particular inhibit the expression of a GATA3 includes , for example , a naturally occurring purine or and / or FOXO1 gene and / or of the one or more genes as pyrimidine base found in DNA ( e . g . , an adenine , “ A ” , a taught herein . guanine , “ G ” , a thymine “ T ” , or a cytosine , “ C ” ) or RNA 10223 ] As used herein , a “ siRNA” refers to a nucleic acid ( e . g ., an A , a G , an uracil, “ U ” , or a C ) . The term “ nucleic that forms a double stranded RNA , which double stranded acid ” encompasses the terms " oligonucleotide” and “ poly RNA has the ability to reduce or inhibit expression of a gene nucleotide ” " each as a subgenus of the term “ nucleic acid ” . or target gene when the siRNA is present or expressed in the The term “ oligonucleotide” refers to a molecule of between same cell as the target gene . The double stranded RNA about 3 and about 100 nucleobases in length . The term siRNA can be formed by the complementary strands . In one “ polynucleotide ” refers to at least one molecule of greater embodiment, a siRNA refers to a nucleic acid that can form than about 100 nucleobases in length . The term “ nucleic a double stranded siRNA . The sequence of the siRNA can acid ” also refers to polynucleotides such as deoxyribo correspond to the full - length target gene , or a subsequence nucleic acid (DNA ) , and , where appropriate , ribonucleic thereof. Typically , the siRNA is at least about 15 - 50 nucleo acid (RNA ) . The term should also be understood to include , tides in length ( e . g ., each complementary sequence of the as equivalents, analogs of either RNA or DNA made from double stranded siRNA is about 15 - 50 nucleotides in length , nucleotide analogs, and , as applicable to the embodiment and the double stranded siRNA is about 15 -50 base pairs in being described , single ( sense or antisense) and double US 2019 /0262399 A1 Aug . 29 , 2019 30 stranded polynucleotides. The terms “ polynucleotide peutic peptides. In some embodiments , a modulator or a sequence ” and “ nucleotide sequence” are also used inter modulator of any one of the one or more gene products as changeably herein . taught herein comprises a protein or fragment thereof, or [ 0228 ] The terms " polypeptide” and “ protein ” are used comprises the gene product or fragment thereof, respec interchangeably to refer to a polymer of amino acid residues, tively , fused to a Fc fragment, which is comprised of D - or and are not limited to a minimum length . Peptides , oligo L -amino acid residues , as use of naturally occurring peptides, dimers , multimers, and the like, are also composed L - amino acid residues has the advantage that any break of linearly arranged amino acids linked by peptide bonds , down products should be relatively non - toxic to the cell or and whether produced biologically , recombinantly , or syn organism . thetically and whether composed of naturally occurring or [0230 ] In yet a further embodiment, a modulator of the one non - naturally occurring amino acids , are included within or more gene products as taught herein , which is a peptide this definition . Both full -length proteins and fragments or fragments or derivatives thereof can be a retro - inverso thereof are encompassed by the definition . The terms also peptide . A “ retro - inverso peptide ” refers to a peptide with a include co - translational and post - translational modifications reversal of the direction of the peptide bond on at least one of the polypeptide , such as , for example , disulfide -bond position , i . e . , a reversal of the amino - and carboxy - termini formation , glycosylation , acetylation , phosphorylation , pro with respect to the side chain of the amino acid . Thus, a teolytic cleavage ( e . g . , cleavage by furins or metallopro retro - inverso analogue has reversed termini and reversed teases and prohormone convertases (PCs ) ), and the like . direction of peptide bonds while approximately maintaining Furthermore, for purposes of the present invention , a “ poly the topology of the side chains as in the native peptide peptide” encompasses a protein that includes modifications, sequence . The retro - inverso peptide can contain L - amino such as deletions , additions , and substitutions ( generally acids or D - amino acids, or a mixture of L - amino acids and conservative in nature as would be known to a person in the D - amino acids , up to all of the amino acids being the art ) , to the native sequence , as long as the protein maintains D - isomer . Partial retro - inverso peptide analogues are poly the desired activity . These modifications can be deliberate , peptides in which only part of the sequence is reversed and as through site -directed mutagenesis , or can be accidental, replaced with enantiomeric amino acid residues . Since the such as through mutations of hosts that produce the proteins, retro - inverted portion of such an analogue has reversed or errors due to PCR amplification or other recombinant amino and carboxyl termini, the amino acid residues flank DNA methods . Polypeptides or proteins are composed of ing the retro - inverted portion are replaced by side -chain linearly arranged amino acids linked by peptide bonds, but analogous a -substituted geminal- diaminomethanes and in contrast to peptides, have a well - defined conformation . malonates , respectively . Retro - inverso forms of cell pen Proteins , as opposed to peptides , generally consist of chains etrating peptides have been found to work as efficiently in of 50 or more amino acids . For the purposes of the present translocating across a membrane as the natural forms. Syn invention , the term “ peptide ” as used herein typically refers thesis of retro - inverso peptide analogues are described in to a sequence of amino acids of made up of a single chain Bonelli, F . et al ., Int J Pept Protein Res . 24 ( 6 ) : 553 - 6 ( 1984 ) ; of D - or L - amino acids or a mixture of D - and L - amino acids Verdini, A and Viscomi, G . C , J . Chem . Soc . Perkin Trans . joined by peptide bonds. Generally , peptides contain at least 1 :697 -701 ( 1985 ); and U . S . Pat . No . 6 , 261, 569 , which are two amino acid residues and are less than about 50 amino incorporated herein in their entirety by reference. Processes acids in length . for the solid -phase synthesis of partial retro - inverso peptide [ 0229 ] The incorporation of non -natural amino acids , analogues have been described (EP 97994 - B ) which is also including synthetic non - native amino acids, substituted incorporated herein in its entirety by reference . amino acids, or one ormore D - amino acids into the peptides [0231 ] The term “ antibody ” is meant to be an immuno ( or other components of the composition , with exception for globulin protein that is capable of binding an antigen . protease recognition sequences ) is desirable in certain situ Antibody as used herein is meant to include antibody ations. D - amino acid -containing peptides can exhibit fragments , e . g . F (ab ') 2 , Fab ', Fab , capable of binding the increased stability in vitro or in vivo compared to L -amino antigen or antigenic fragment of interest. Exemplary frag acid - containing forms. Thus, the construction of peptides ments include Fab , Fab ' , F ( ab ' ) 2 , Fabc, Fd , dAb , VHH and incorporating D - amino acids can be particularly useful when scFv and/ or Fv fragments . As used herein , the term “ anti greater in vivo or intracellular stability is desired or required . body ” is used in its broadest sense and generally refers to More specifically , D - peptides are resistant to endogenous any immunologic binding agent, such as a whole antibody , peptidases and proteases , thereby providing better oral trans including without limitation a chimeric , humanized , human , epithelial and transdermal delivery of linked drugs and recombinant, transgenic , grafted and single chain antibody, conjugates, improved bioavailability of membrane -perma and the like, or any fusion proteins , conjugates, fragments , nent complexes (see below for further discussion ) , and or derivatives thereof that contain one or more domains that prolonged intravascular and interstitial lifetimes when such selectively bind to an antigen of interest. The term antibody properties are desirable . The use of D - isomer peptides can thereby includes a whole immunoglobulin molecule , a also enhance transdermal and oral trans -epithelial delivery monoclonal antibody , a chimeric antibody , a humanized of linked drugs and other cargo molecules. Additionally , antibody , a human antibody , or an immunologically effective D - peptides cannot be processed efficiently for major histo fragment of any of these . The term thus specifically encom compatibility complex class II -restricted presentation to T passes intact monoclonal antibodies, polyclonal antibodies , helper cells , and are therefore less likely to induce humoral multivalent ( e . g . , 2 - , 3 - or more -valent ) and / or multi -specific immune responses in the whole organism . Peptide conju antibodies ( e . g . , bi- or more -specific antibodies ) formed gates can therefore be constructed using, for example , from at least two intact antibodies, and antibody fragments D - isomer forms of cell penetrating peptide sequences , L - iso insofar they exhibit the desired biological activity (particu mer forms of cleavage sites, and D - isomer forms of thera larly, ability to specifically bind an antigen of interest ) , as US 2019 /0262399 A1 Aug . 29 , 2019 31 well as multivalent and /or multi -specific composites of such cells normally proliferate and produce cell killing enzymes, fragments . The term “ antibody ” is not only inclusive of e .g ., they can release the cytotoxins perforin , granzymes, antibodies generated by methods comprising immunisation , and granulysin . However , exhausted / dysfunctional T cells but also includes any polypeptide , e .g . , a recombinantly do not respond adequately to TCR stimulation , and display expressed polypeptide , which is made to encompass at least poor effector function , sustained expression of inhibitory one complementarity - determining region (CDR ) capable of receptors and a transcriptional state distinct from that of specifically binding to an epitope on an antigen of interest . functional effector or memory T cells. Dysfunction / exhaus Hence , the term applies to such molecules regardless tion of T cells thus prevents optimal control of infection and whether they are produced in vitro , in cell culture, or in vivo . tumors . Exhausted / dysfunctional immune cells , such as T [0232 ] The term “ humanized antibody " is used herein to cells , such as CD8 + T cells , may produce reduced amounts describe complete antibody molecules, i. e . composed of two of IFN - gamma , TNF - alpha and /or one or more immunos complete light chains and two complete heavy chains, as timulatory cytokines, such as IL - 2 , compared to functional well as antibodies consisting only of antibody fragments , immune cells . Exhausted /dysfunctional immune cells , such e . g . Fab , Fab ' , F ( ab ) 2 , and Fv, wherein the CDRs are as T cells , such as CD8+ T cells , may further produce derived from a non -human source and the remaining portion ( increased amounts of) one or more immunosuppressive of the Ig molecule or fragment thereof is derived from a transcription factors or cytokines , such as IL - 10 and /or human antibody, preferably produced from a nucleic acid Foxp3 , compared to functional immune cells , thereby con sequence encoding a human antibody . tributing to local immunosuppression . [0233 ] The terms “ human antibody ” and “ humanized anti [0236 ] As used herein , the term “ unresponsiveness ” also body ” are used herein to describe an antibody of which all includes refractivity to activating receptor -mediated stimu portions of the antibody molecule are derived from a nucleic lation . Such refractivity is generally antigen -specific and acid sequence encoding a human antibody . Such human persists after exposure to the antigen has ceased . Unrespon antibodies are most desirable for use in antibody therapies , sive immune cells can have a reduction of at least 10 % , 20 % , as such antibodies would elicit little or no immune response 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 95 % , or even 100 % in the human subject. in cytotoxic activity , cytokine production , proliferation ( e. g ., [ 0234 ] All gene name symbols refer to the gene as com in response to a cytokine , such as IFN - gamma) , migration monly known in the art. Gene symbols may be those referred and trafficking , phagocytotic activity , or any combination to by the HUGO Gene Nomenclature Committee (HGNC ) . thereof, relative to a corresponding control immune cell of Any reference to the gene symbol is a reference made to the the same type . entire gene or variants of the gene . The HUGO Gene [0237 ] The Applicants have demonstrated that altering , Nomenclature Committee is responsible for providing and more particularly downregulating or abolishing, the human gene naming guidelines and approving new , unique expression of genes in the dysfunction module , in dysfunc human gene names and symbols . All human gene names and tional immune cells may at least partly counter the observed symbols can be searched at www .genenames . org , the HGNC dysfunction , whereby the immune cell may display an website , and the guidelines for their formation are available improved tumor - or infection - clearing ability . Without limi there (www . genenames . org / guidelines ) . tation , altering , and more particularly downregulating or [ 0235 ] During persistent immune activation , such as dur abolishing , the expression or activity of the expression of ing uncontrolled tumor growth or chronic infections, sub genes in the dysfunction module , in dysfunctional immune populations of immune cells , particularly of CD8 + T cells , cells can improve one or more aspects of the immune cell become compromised to different extents with respect to function , such as the immune cell ' s proliferation ( e . g ., in their cytokine and /or cytolytic capabilities . Such immune response to a cytokine , such as IFN - gamma ) or cell division , cells , particularly CD8 + T cell , are commonly referred to as entrance into the cell cycle , differentiation , cytokine pro " dysfunctional” or as " functionally exhausted ” or duction , cytotoxicity , migration and trafficking , phagocy " exhausted ” . As used herein , the term " dysfunctional” or totic activity , or any combination thereof, preferably at least “ functional exhaustion ” refer to a state of a cell where the the immune cell ' s cytokine production or cytotoxicity or cell does not perform its usual function or activity in both . Similarly , without limitation , altering , and more par response to normal input signals , and includes refractivity of ticularly downregulating or abolishing , the expression or immune cells to stimulation , such as stimulation via an activity of the expression of genes in the dysfunction mod activating receptor or a cytokine . Such a function or activity ule , in immune cells, preferably immune cells for adoptive includes, but is not limited to , proliferation ( e . g ., in response cell transfer, can prevent the immune cell from becoming to a cytokine , such as IFN - gamma ) or cell division , entrance dysfunctional or exhausted . Thus, downregulating or abol into the cell cycle , cytokine production , cytotoxicity , migra ishing, the expression or activity of the expression of genes tion and trafficking , phagocytotic activity , or any combina in the dysfunction module can inhibit induction of dysfunc tion thereof. Normal input signals can include, but are not tion or exhaustion . Similarly, without limitation , altering , limited to , stimulation via a receptor ( e . g . , T cell receptor, B and more particularly downregulating or abolishing, the cell receptor , co - stimulatory receptor ) . Unresponsive expression or activity of one or more genes or gene products immune cells can have a reduction of at least 10 % , 20 % , selected from the group consisting of the genes or gene 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 95 % , or even 100 % products listed in Table 3 , part “ Dysfunction _ module ” , in cytotoxic activity , cytokine production , proliferation , traf Table 5A or Table 5B , in dysfunctional immune cells can ficking , phagocytotic activity , or any combination thereof, improve one or more aspects of the immune cell function , relative to a corresponding control immune cell of the same such as the immune cell ' s proliferation ( e . g ., in response to type. In some particular embodiments of the aspects a cytokine , such as IFN - gamma ) or cell division , entrance described herein , a cell that is dysfunctional is a CD8 + T cell into the cell cycle , differentiation , cytokine production , that expresses the CD8 + cell surface marker. Such CD8+ cytotoxicity ,migration and trafficking, phagocytotic activity , US 2019 / 0262399 A1 Aug . 29 , 2019 32 or any combination thereof, preferably at least the immune [0238 ] In certain embodiments , the immune cell displays cell' s cytokine production or cytotoxicity or both . Further, tumor specificity . The immune cell may be isolated from a without limitation , altering , and more particularly upregu tumor of a subject, preferably wherein the immune cell is a lating , the expression or activity of one or more genes or tumor infiltrating lymphocyte . Generally , “ tumor infiltrating gene products selected from the group consisting of the lymphocytes” or “ TILs” refer to white blood cells that have left the bloodstream and migrated into a tumor. Such T cells genes or gene products listed in Table 3 , part “ Activation _ typically endogenously express a T cell receptor having module ” , in immune cells (e . g ., dysfunctional or non -dys specificity to an antigen expressed by the tumor cells ( tumor functional ) can improve one or more aspects of the immune antigen specificity ). In alternative embodiments , an immune cell function , such as the immune cell' s proliferation ( e . g ., cell , such as a T cell, preferably a CD8 + T cell, may be in response to a cytokine, such as IFN - gamma ) or cell engineered to express a T cell receptor having specificity to division , entrance into the cell cycle , differentiation , a desired antigen , such as a tumor cell antigen . For example , cytokine production , cytotoxicity, migration and trafficking , the immune cell, such as a T cell, preferably a CD8 + T cell , phagocytotic activity , or any combination thereof, prefer may comprise a chimeric antigen receptor (CAR ) having ably at least the immune cell ' s cytokine production or specificity to a desired antigen , such as a tumor - specific cytotoxicity or both . As used herein , a " cytokine ” is a chimeric antigen receptor (CAR ) . generic term for proteins released by any of the lymph cells [0239 ] Hence , in certain embodiments , the immune cell as that act on other cells as intercellular mediators and affect intended herein , such as a T cell , preferably a CD8 + T cell , cellular activity and control inflammation . Cytokines are may be modified to comprise downregulated or abolished typically soluble proteins or peptides which are naturally expression or activity of, or modified to comprise an agent produced by mammalian cells and which act in vivo as capable of inducibly downregulating or abolishing expres humoral regulators at micro - to picomolar concentrations . sion or activity of one or more genes or gene products Cytokines can , either under normal or pathological condi described herein . tions , modulate the functional activities of individual cells and tissues . A proinflammatory cytokine is a cytokine that is [0240 ] In further embodiments , the immune cell as intended herein , such as a T cell , preferably a CD8 + T cell, capable of causing any of the following physiological reac may be modified to comprise downregulated or abolished tions associated with inflammation : vasodialation , hyper expression or activity of the one or more genes or gene emia , increased permeability of vessels with associated products selected from the group consisting of the genes or edema , accumulation of granulocytes and mononuclear gene products listed in Table 3 , part “ Dysfunction _module ” , phagocytes, or deposition of fibrin . In some cases, the Table 5A or Table 5B , or to comprise an agent capable of pro - inflammatory cytokine can also cause apoptosis , such as inducibly downregulating or abolishing expression or activ in chronic heart failure , where TNF has been shown to ity of the one or more genes or gene products selected from stimulate cardiomyocyte apoptosis . Non - limiting examples the group consisting of the genes or gene products listed in of pro - inflammatory cytokines are tumor necrosis factor Table 3 , part “ Dysfunction _module ”, Table 5A or Table 5B . ( TNF ) , interleukin ( IL ) - 1 . alpha , IL - 1 .beta , IL - 6 , IL - 8 , IL - 18 , interferon - gamma ( INFy ) , HMG - 1 , platelet -activating fac [ 0241] In further embodiments , the immune cell as tor ( PAF ) , and macrophage migration inhibitory factor intended herein , such as a T cell, preferably a CD8 + T cell, (MIF ) . Additionally examples of cytokines include , lym may be modified to comprise upregulated expression or phokines , monokines , and traditionalpolypeptide hormones . activity of the one or more genes or gene products selected Included among the cytokines are growth hormones such as from the group consisting of the genes or gene products human growth hormone , N -methionyl human growth hor listed in Table 3 , part “ Activation _ module ” , or to comprise mone, and bovine growth hormone ; parathyroid hormone ; an agent capable of inducibly upregulating expression or thyroxine ; insulin ; proinsulin ; relaxin ; prorelaxin ; glycopro activity of the one or more genes or gene products selected tein hormones such as follicle stimulating hormone ( FSH ), from the group consisting of the genes or gene products thyroid stimulating hormone ( TSH ), and luteinizing hor listed in Table 3, part “ Activation _ module ” . mone (LH ) ; hepatic growth factor ; fibroblast growth factor; [0242 ] In further embodiments , the immune cell as prolactin ; placental lactogen ; tumor necrosis factor - a and - B ; intended herein , such as a T cell , preferably a CD8 + T cell , mullerian - inhibiting substance (MIS ) ; mouse gonadotropin may be modified to comprise downregulated or abolished associated peptide ; inhibin ; activin ; vascular endothelial expression or activity of the one or more genes or gene growth factor (VEGF ) ; integrin ; thrombopoietin ( TPO ) ; products selected from the group consisting of the genes or nerve growth factors such as NGF - B ; platelet - growth factor; gene products listed in Table 3 , part “ Dysfunction _module ” , transforming growth factors ( TGFs ) such as TGF- a and Table 5A or Table 5B , or to comprise an agent capable of TGF- B ; insulin - like growth factor - I and — II ; erythropoietin inducibly downregulating or abolishing expression or activ ( EPO ) ; osteoinductive factors ; interferons such as inter ity of the one or more genes or gene products selected from feron - a , - B , and - y ; colony stimulating factors (CSFs ) such the group consisting of the genes or gene products listed in as macrophage -CSF ( M -CSF ) ; granulocyte -macrophage Table 3 , part “ Dysfunction _module ” , Table 5A or Table 5B ; CSF (GM -CSF ) ; and granulocyte -CSF ( G -CSF ) ; interleu and further modified to comprise upregulated expression or kins ( ILs) such as , for example and not for limitation , IL - 1, activity of the one or more genes or gene products selected IL - 1 . a , IL - 1. beta , IL - 2 , IL - 3 , IL - 4 , IL - 5 , IL - 6 , IL - 7 , IL - 8 , from the group consisting of the genes or gene products IL - 9 , IL - 10 , IL - 11 , IL - 12 ; a tumor necrosis factor such as listed in Table 3 , part “ Activation _module ” , or to comprise TNF - a or TNF -B ; and other polypeptide factors including an agent capable of inducibly upregulating expression or leukemia inhibitory factor (LIF ) and kit ligand ( KL ) . As activity of the one or more genes or gene products selected used herein , when referring to a patient the term " cytokine ” from the group consisting of the genes or gene products refers to on one or more of those produced by the patient . listed in Table 3 , part " Activation _ module ” . US 2019 /0262399 A1 Aug . 29 , 2019 33

[0243 ] In yet further embodiments , the immune cell as The adoptive transfer of autologous tumor infiltrating lym intended herein , such as a T cell , preferably a CD8 + T cell, phocytes ( TIL ) (Besser et al. , (2010 ) Clin . Cancer Res 16 ( 9 ) may be modified to comprise upregulated expression or 2646 -55 ; Dudley et al. , (2002 ) Science 298 (5594 ) : 850 - 4 ; activity of the one or more genes or gene products selected and Dudley et al. , ( 2005 ) Journal of Clinical Oncology 23 from the group consisting of the genes or gene products ( 10 ): 2346 -57 .) or genetically re -directed peripheral blood listed in Table 3 , part “ Dysfunction _ module” , Table 5A or mononuclear cells ( Johnson et al. , ( 2009 ) Blood 114 ( 3 ) : Table 5B , or to comprise an agent capable of inducibly 535 - 46 ; and Morgan et al. , (2006 ) Science 314 (5796 ) 126 - 9 ) upregulating expression or activity of the one or more genes has been used to successfully treat patients with advanced or gene products selected from the group consisting of the solid tumors, including melanoma and colorectal carcinoma, genes or gene products listed in Table 3 , part “ Dysfunction as well as patients with CD19 - expressing hematologic module” , Table 5A or Table 5B . malignancies (Kalos et al. , ( 2011 ) Science Translational [ 0244 ] In further embodiments , the immune cell as MedicineTY 3 ( 95 ) : 95ra73 ). intended herein , such as a T cell, preferably a CD8 + T cell, [ 0248 ] Aspects of the invention involve the adoptive trans may be modified to comprise downregulated or abolished fer of immune system cells, such as T cells , specific for expression or activity of the one or more genes or gene selected antigens , such as tumor associated antigens or products selected from the group consisting of the genes or tumor specific neoantigens (see Maus et al. , 2014 , Adoptive gene products listed in Table 3 , part “ Activation _ module” , Immunotherapy for Cancer or Viruses, Annual Review of or to comprise an agent capable of inducibly downregulating Immunology, Vol. 32 : 189 - 225 ; Rosenberg and Restifo , or abolishing expression or activity of the one or more genes 2015 , Adoptive cell transfer as personalized immunotherapy or gene products selected from the group consisting of the for human cancer , Science Vol. 348 no . 6230 pp . 62 - 68 ; genes or gene products listed in Table 3 , part “ Activation _ Restifo et al ., 2015 , Adoptive immunotherapy for cancer : module ” . harnessing the T cell response. Nat . Rev . Immunol . 12 ( 4 ) : [ 0245 ] In further embodiments , the immune cell as 269 - 281 ; and Jenson and Riddell, 2014 , Design and imple intended herein , such as a T cell, preferably a CD8 + T cell, mentation of adoptive therapy with chimeric antigen recep may be modified to comprise upregulated expression or tor -modified T cells. Immunol Rev . 257 ( 1 ) : 127 - 144 ; and activity of the one or more genes or gene products selected Rajasagi et al. , 2014 , Systematic identification of personal from the group consisting of the genes or gene products tumor -specific neoantigens in chronic lymphocytic leuke listed in Table 3 , part “ Dysfunction _module ” , Table 5A or mia . Blood . 2014 Jul. 17 ; 124 ( 3 ) :453 -62 ) . Table 5B , or to comprise an agent capable of inducibly [0249 ] In certain embodiments , an antigen ( such as a upregulating expression or activity of the one or more genes tumor antigen ) to be targeted in adoptive cell therapy ( such or gene products selected from the group consisting of the as particularly CAR or TCR T - cell therapy ) of a disease genes or gene products listed in Table 3 , part “ Dysfunction ( such as particularly of tumor or cancer ) may be selected module ” , Table 5A or Table 5B ; and further modified to from a group consisting of: B cell maturation antigen comprise downregulated or abolished expression or activity (BCMA ) ; PSA ( prostate - specific antigen ); prostate - specific of the one or more genes or gene products selected from the membrane antigen (PSMA ) ; PSCA (Prostate stem cell anti group consisting of the genes or gene products listed in gen ) ; Tyrosine -protein kinase transmembrane receptor Table 3 , part “ Activation _module ” , or to comprise an agent ROR1; fibroblast activation protein (FAP ) ; Tumor -associ capable of inducibly downregulating or abolishing expres ated glycoprotein 72 ( TAG72 ) ; Carcinoembryonic antigen sion or activity of the one or more genes or gene products (CEA ) ; Epithelial cell adhesion molecule ( EPCAM ) ; Meso selected from the group consisting of the genes or gene thelin ; Human Epidermal growth factorReceptor 2 (ERBB2 products listed in Table 3 , part “ Activation _module ” . (Her2 /neu ) ) ; Prostate ; Prostatic acid phosphatase (PAP ) ; [0246 ] In certain embodiments , cells as described herein elongation factor 2 mutant ( ELF2M ) ; Insulin - like growth and below may be used for adoptive cell therapy (ACT ) . The factor 1 receptor ( IGF- 1R ) ; gp100 ; BCR -ABL ( breakpoint transferred cells may include and be modulated by immune cluster region - Abelson ) ; tyrosinase ; New York esophageal cells or immune cell populations as taught herein . In certain squamous cell carcinoma 1 (NY - ESO - 1 ) ; K - light chain , embodiments , the dysfunctional T cells of the present inven LAGE ( L antigen ); MAGE (melanoma antigen ); Melanoma tion are depleted from cells used in ACT and the depleted associated antigen 1 (MAGE - A1 ); MAGE A3 ; MAGE A6 ; cells may be transferred to a subject suffering from a disease legumain ; Human papillomavirus (HPV ) E6 ; HPV E7; pros ( e .g ., cancer) . In certain embodiments , the dysfunctional T tein ; survivin ; PCTA1 (Galectin 8 ) ; Melan - A /MART - 1 ; Ras cells of the present invention may be modulated , such as to mutant ; TRP - 1 (tyrosinase related protein 1 , or gp75 ) ; be activated as described herein , and may be transferred to Tyrosinase -related Protein 2 ( TRP2 ) ; TRP - 2 / INT2 ( TRP - 2 / a subject suffering from a disease ( e . g . , cancer ) . In certain intron 2 ) ; RAGE (renal antigen ) ; receptor for advanced embodiments , the activated T cells of the present may be glycation end products 1 (RAGE1 ) ; Renal ubiquitous 1 , 2 transferred to a subject suffering from a disease ( e . g ., ( RU1, RU2 ) ; intestinal carboxyl esterase ( iCE ) ; Heat shock cancer ). protein 70 - 2 (HSP70 - 2 ) mutant; thyroid stimulating hor [ 0247 ] The immune cells or immune cell populations as mone receptor ( TSHR ) ; CD123 ; CD171; CD19 ; CD20 ; taught herein may be used for adoptive cell transfer (ACT ) . CD22 ; CD26 ; CD30 ; CD33 ; CD44v7 / 8 ( cluster of differen In certain embodiments , the present invention comprises tiation 44 , exons 7 / 8 ) ; CD53 ; CD92 ; CD100 ; CD148 ; adoptive cell therapy . Adoptive cell therapy can refer to the CD150 ; CD200 ; CD261 ; CD262 ; CD362 ; CS - 1 (CD2 sub transfer of cells , most commonly immune- derived cells , set 1 , CRACC , SLAMF7 , CD319 , and 19A24 ) ; C -type back into the same patient or into a new recipient host with lectin - like molecule - 1 ( CLL - 1 ) ; ganglioside GD3 ( aNeu5Ac the goal of transferring the immunologic functionality and ( 2 - 8 )aNeu5Ac ( 2 - 3) bDGalp ( 1- 4 )bDG1cp ( 1- 1 )Cer ); Tn anti characteristics into the new host. If possible , use of autolo gen ( Tn Ag) ; Fms- Like Tyrosine Kinase 3 ( FLT3 ) ; CD38 ; gous cells helps the recipient by minimizing GVHD issues . CD138 ; CD44v6 ; B7H3 (CD276 ); KIT (CD117 ) ; Interleu