A

Vehicle IL-15cx treated Events CD71 CD98 CD44 GZMB

B

60 P = 0.03 300 P = 0.005 100 P = 0.02 4000 P < 0.0001 80 3000 FI ) FI ) FI ) 40 200 (%) 60 high 2000 40 20 100 CD 44 (gM CD 98 (gM CD 71 MB (gM GZ MB 1000 20

0 0 0 0 Vehicle IL-15cx Vehicle IL-15cx Vehicle IL-15cx Vehicle IL-15cx

Supplemental Figure 1: Five day IL-15cx treatment results in splenic CD8+ T cell activation and up-regulation of cytotoxic molecule expression in PyMT tumor-bearing mice. (A,B) Vehicle or IL-15cx was injected for five days (days 0 - 4) into PyMT tumor-bearing mice, as depicted in Fig.1C, and splenocytes were assayed on day 5. (A) Phenotype of splenic CD8+ T cells in vehicle versus IL-15cx treated PyMT tumor bearing mice. CD71 (transferrin ) and CD98 (large neutral trans- porter, or LAT1) are up-regulated on T cells upon activation and growth (ref. 28 and not shown). As indicated, filled histograms are CD8+ splenocytes from mice treated with vehicle, while line trace is with IL-15cx. (B) As in A, but for multiple samples. These data are representative of two independent experiments. Error bars indicate S.D., and P-values are from student’s unpaired t-test. A Unsorted tumor Splenic CD8+CD44high + IL-15 CD8+CD44high + IL-15 Events CD71 B C Splenic only Tumor:Splenic 512 71% 1:10 High IL-2 stimulation/ 59% no tumor suspension 256

128 1:1 36% 64 CD71 gMFI 32

10:1 22% 16

Low IL-2 stimulation/ 0 no tumor suspension 1:10 1:1 10:1 Events Ratio tumor suspension: Splenocyte CD71 splenocyte suspension

Supplemental Figure 2: PyMT tumor single-cell suspensions induce IL-2 / IL-15 resistance in CD8+ T cells. (A) High-dose IL-15 (1μg/mL) added to PyMT tumor single-cell suspension does not induce CD8+ T cell activation/proliferation as measured by CD71 up-regulation (solid trace). Unstimulated CD8+ T cell CD71 fluorescence is shown in filled grey histogram, IL-15 (1μg/mL) treated splenocytes are shown in dotted trace. (B) CD71 abundance on total CD8+ congenically-distinct splenocytes (WT, non-tumor T cells) after 72h incubation with 1μg/mL IL-2 in vitro with or without increasing ratios of a PyMT single-cell suspension; ratio of tumor suspension to splenocyte suspension indicated in upper histogram inset, lower inset is % of splenocytes postive for CD71. Left, 1μg/mL IL-2 up-regulates CD71 on the majority of spleen-derived CD8+ T cells after 72 hours in vitro. Right, increasing ratios of a PyMT single-cell suspension dampens in vitro CD8+ splenocyte IL-2 responsive- ness. For all histograms, filled, grey trace depicts CD71 fluorescence on CD8+ splenocytes treated with 20ng/mL IL-2, sufficient to maintain survival in vitro, but not to activate T cells; black trace is CD71 expression gated on splenocytes. (C) Graphical representation of CD71 data from B, again gating on CD8+CD44high splenocytes. Each line represents a separate PyMT tumor single-cell suspension. The gMFI of CD71 on splenocytes incubated with 20ng/mL or 1μg/mL IL-2 are indicated with horizontal dashed lines for reference of unstimulated and near-maximal stimulated conditions. This experiment was performed twice. A B C CD44low/ CD44high/ Spleen Tumor naive 6000 memory phenotype phenotype

4000 ress io n exp 2000 ea n M Events PD1 0 CD122 CD25 CD122 CD132 Il2ra Il2rb Il2rg Il15ra Total splenic CD8+ PyMT tumor CD8+

D E 1000 Il2ra (CD25) Splenic103- Brain103- Brain103+ 800 8000 600 400 LCMV-Arm 6000 200 LCMV-cl13

0 ress io n 4000 exp 1500 Il2rb (CD122) ea n 2000 M 1000 0 Il2ra Il2rb Il2rg Il15ra 500

0

4000 Il2rg (CD132) 3000

2000

1000

0 250 Il15ra (CD215) 200 150 100 50 Mean expression 0 0 10 20 30 Time post-infection (days)

Supplemental Figure 3: Abundance of IL-2 / IL-15 cytokine receptor chains in PyMT tumor, acute/persistent infection, and + + TRM CD8 T cells. (A) Mean expression of IL-2 / IL-15 cytokine receptors; derived from the PyMT CD8 T cell microarray data in Fig.3-5. The splenic population was CD8+CD44high; error bars indicate S.E.M. (B) Additional flow cytometry of CD122 expression on PyMT tumor CD8+ T cells versus spleen; these data derived from a different experiment from that in Fig.3F. Note the bimodal CD122 expresion in splenic CD8+ T cells, which corresponds to naive CD44lowCD122low and antigen-experienced CD44high- CD122high; error bars indicate S.E.M. (C) PD-1 versus cytokine receptor expression in PyMT MEC model. (D) Mean expression of indicated cytokine receptors on virus-specific cells in acute and persistent infection; data derived from GSE41870. (E) Expres- - - + sion of indicated cytokine receptors on subsets of splenic CD103 , brain CD103 , or brain 103 (TRM) virus-specific cells 20 days post VSV-OVA infection; data derived from GSE39152. Error bars indicate S.D. VSV day8 VSV day8 PyMT tumor PyMT tumor Replicate: 1 2 3 1 2 3 symbol Gene name Replicate: 1 2 3 1 2 3 Gene symbol Gene name Tnfrsf9 receptor superfamily, member 9 Nanp n-acetylneuraminic acid phosphatase Litaf lipopolysaccharide-induced tnf factor Kifap3 kinesin-associated 3 Cxcl10 chemokine (c-x-c motif) ligand 10 Ubash3b ubiquitin associated and SH3 domain containing, B Inpp4b inositol polyphosphate-4-phosphatase, type ii, 105kda Gldc dehydrogenase (decarboxylating) Dusp4 dual specificity phosphatase 4 Hmgn3 high mobility group nucleosomal binding domain 3 Ctla4 cytotoxic t-lymphocyte-associated protein 4 Nrgn neurogranin (protein kinase c substrate, rc3) Pdcd1 programmed cell death 1 Cpt1a carnitine palmitoyltransferase 1a (liver) Tfrc (p90, cd71) Cd83 molecule Coro2a coronin, actin binding protein, 2a Serpine2 serpin peptidase inhibitor, clade e (nexin, plasminogen activator inhibitor type 1), m.2 Myo1e myosin ie Stau2 staufen, rna binding protein, homolog 2 (drosophila) Plod2 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 Art3 adp-ribosyltransferase 3 Cdk6 cyclin-dependent kinase 6 Kif22 kinesin family member 22 Ifitm3 interferon induced transmembrane protein 3 (1-8u) Il21 interleukin 21 Dtl denticleless homolog (drosophila) Fam81a family with sequence similarity 81, member a Rtp4 receptor transporter protein 4 Pex11a peroxisomal biogenesis factor 11a Gcnt1 glucosaminyl (n-acetyl) transferase 1, core 2 (beta-1,6-n-acetylglucosaminyltransferase) Id3 inhibitor of dna binding 3, dominant negative helix-loop-helix protein Ncf1 neutrophil cytosolic factor 1, (chronic granulomatous disease, autosomal 1) Synpo synaptopodin Smyd2 set and mynd domain containing 2 Cd109 molecule Ncapg2 non-smc ii complex, subunit g2 Adk adenosine kinase Xcl1 chemokine (c motif) ligand 1 Psmc3ip psmc3 interacting protein Crmp1 collapsin response mediator protein 1 Oxsr1 oxidative-stress responsive 1 Irf8 interferon regulatory factor 8 Rcsd1 rcsd domain containing 1 Mad2l1 mad2 mitotic arrest deficient-like 1 (yeast) Flnb filamin b, beta (actin binding protein 278) Sema4c sema domain, (ig), (semaphorin) 4c Ccr9 chemokine (c-c motif) receptor 9 Nop58 nop58 ribonucleoprotein Cd81 molecule Ehd4 eh-domain containing 4 Vamp5 vesicle-associated 5 (myobrevin) Bst2 bone marrow stromal cell antigen 2 Slc6a13 6 (neurotransmitter transporter, gaba), member 13 Psat1 phosphoserine aminotransferase 1 Zan zonadhesin Rsad2 radical s-adenosyl methionine domain containing 2 Fkbp5 fk506 binding protein 5 Ccna2 cyclin a2 Myb v-myb myeloblastosis viral oncogene homolog (avian) Pcgf5 polycomb group ring finger 5 Dynlt3 dynein, chain, tctex-type 3 Bub1 bub1 budding uninhibited by benzimidazoles 1 homolog (yeast) Aim2 absent in 2 Cdc14a cdc14 cell division cycle 14 homolog a (s. cerevisiae) Blm bloom syndrome Uhrf1 ubiquitin-like, containing phd and ring finger domains, 1 Hat1 histone acetyltransferase 1 Cdk2ap1 cdk2-associated protein 1 Ranbp1 binding protein 1 Chek1 chk1 checkpoint homolog (s. pombe) Rgs10 regulator of g-protein signalling 10 Lap3 leucine aminopeptidase 3 Cd160 molecule Tfdp2 transcription factor dp-2 (e2f dimerization partner 2) Tox thymocyte selection-associated high mobility group box St6gal1 st6 beta-galactosamide alpha-2,6-sialyltranferase 1 Gnl3 guanine nucleotide binding protein-like 3 (nucleolar) Naa50 N(alpha)-acetyltransferase 50, NatE catalytic subunit Bcap29 b-cell receptor-associated protein 29 Spats2 spermatogenesis associated, serine-rich 2 C1qbp complement component 1, q subcomponent binding protein Eif1ax eukaryotic translation initiation factor 1a, x-linked Hells helicase, lymphoid-specific Atad5 ATPase family, AAA domain containing 1 Phlpp1 PH domain and leucine rich repeat protein phosphatase 1 Umps uridine monophosphate synthetase St14 suppression of tumorigenicity 14 (colon carcinoma) Ctps ctp synthase Itm2a integral membrane protein 2a Ccdc58 coiled-coil domain containing 58 Myc v-myc myelocytomatosis viral oncogene homolog (avian) Rad51 rad51 homolog (reca homolog, e. coli) (s. cerevisiae) Fcer1g fc fragment of ige, high affinity i, receptor for; gamma polypeptide Skap2 src kinase associated phosphoprotein 2 Ccnb2 cyclin b2 Arhgap11a rho gtpase activating protein 11a Hook1 hook homolog 1 (drosophila) Spag5 sperm associated antigen 5 Mmd monocyte to macrophage differentiation-associated Hmgb3 high-mobility group box 3 Casp3 caspase 3, apoptosis-related cysteine peptidase Mthfd2 methylenetetrahydrofolate dehydrogenase (nadp+ dependent) 2 Fignl1 fidgetin-like 1 Chst2 carbohydrate (n-acetylglucosamine-6-o) sulfotransferase 2 Gcsh glycine cleavage system protein h (aminomethyl carrier) Napsa napsin a aspartic peptidase Hspa9 heat shock protein 9 Plk4 polo-like kinase 4 (drosophila) Uchl5 ubiquitin carboxyl-terminal hydrolase l5 Pwp1 pwp1 homolog (s. cerevisiae) Txndc12 thioredoxin domain containing 12 (endoplasmic reticulum) Pctp phosphatidylcholine transfer protein Ddit4 dna-damage-inducible transcript 4 Exo1 exonuclease 1 Lag3 lymphocyte-activation gene 3 Shcbp1 shc sh2-domain binding protein 1 Tpd52 tumor protein d52 Stard3nl stard3 n-terminal like Dut dutp pyrophosphatase Ndrg1 n-myc downstream regulated gene 1 Wdr67 wd repeat domain 67 Spock2 sparc/, cwcv and kazal-like domains (testican) 2 Melk maternal embryonic leucine zipper kinase Optn optineurin Snx9 sorting nexin 9 Srfbp1 serum response factor binding protein 1 Frmd4a ferm domain containing 4a Fancd2 fanconi anemia, complementation group d2 Rad54l rad54-like (s. cerevisiae) Xrcc2 x-ray repair complementing defective repair in chinese hamster cells 2 Cdc6 cdc6 cell division cycle 6 homolog (s. cerevisiae) Trip13 thyroid hormone receptor interactor 13 Psmd12 proteasome (prosome, macropain) 26s subunit, non-atpase, 12 Gca grancalcin, ef-hand calcium binding protein Tfdp1 transcription factor dp-1 Jazf1 jazf zinc finger 1 Gmps guanine monphosphate synthetase Slc37a2 solute carrier family 37 (glycerol-3-phosphate transporter), member 2 Ckap2 cytoskeleton associated protein 2 Egr2 early growth response 2 (krox-20 homolog, drosophila) Spc25 SPC25, NDC80 kinetochore complex component, homolog (S. cerevisiae) Mpp6 membrane protein, palmitoylated 6 (maguk p55 subfamily member 6) Utp6 utp6, small subunit (ssu) processome component, homolog (yeast) Rnaseh2b ribonuclease h2, subunit b Rnf128 ring finger protein 128 Kpna2 alpha 2 (rag cohort 1, alpha 1) Celf2 CUGBP, Elav-like family member 2 Hsh2d hematopoietic sh2 domain containing Slc25a13 solute carrier family 25, member 13 () Rad18 rad18 homolog (s. cerevisiae) Mboat7 membrane bound O-acyltransferase domain containing 7 Tacc2 transforming, acidic coiled-coil containing protein 2 Gas2 growth arrest-specific 2 Bcl3 b-cell cll/lymphoma 3 Cblb cas-br-m (murine) ecotropic retroviral transforming sequence b Ptger2 prostaglandin e receptor 2 (subtype ep2), 53kda Lpgat1 lysophosphatidylglycerol acyltransferase 1 Suv39h1 suppressor of variegation 3-9 homolog 1 (drosophila) Etv5 ets variant gene 5 (ets-related molecule) Nek2 nima (never in mitosis gene a)-related kinase 2 Filip1 filamin a interacting protein 1 Cdc27 cell division cycle 27 Mef2c mads box transcription enhancer factor 2, polypeptide c (myocyte enhancer factor 2c) Sgol1 shugoshin-like 1 (s. pombe) Bmi1 b lymphoma mo-mlv insertion region (mouse) Cenpp centromere protein p Tox2 TOX high mobility group box family member 2 Set set translocation (myeloid leukemia-associated) Bcat1 branched chain aminotransferase 1, cytosolic Supplemental Figure 4: in tumor versus acute infection CD8+ T cells. GSEA heatmaps of expression in PyMT tumor CD8+ T cells versus OT-I responding to VSV-OVA infection, day 8. GSEA/Geneset is LCMV acute versus chronic infection, from Fig.3C. At left are up-regulated in PyMT tumor vs. acute infection (VSV-OVA), at right are unchanged or down-regulated. day 106 postVSV-OVA infection andday 100postLM-OVA infection(GSM605904-6 andGSM538401-2). day 1LM-OVA OT-I (GSM920637-920639), day5VSV-OVA OT-I (GSM538387-8, GSM605897),andmemoryare pooledOT-I from expression filter of>50ineithertheImmgen orPyMT datasetwasapplied. Immgendataderived from:naiveOT-I (GSM605909-11), down-regulated inthecomparisonsabove areplotted. cells (y-axis);color-codedfoldchange barsharedfornaive,day1andpooledmemory. Below , the50transcriptsmostup- and Supplemental Figure5:Differentially-regulatedtranscripts inPyMTtumorCD8 effector and memory T cells. Above, 10

-3 Up in tumor Tumor 10 10 10 10 1 2 3 4 Naive LOC100504914 10 Gzmb 10 LOC641050 1 -1 Cd200r1 Gm8721 Sema6d Bhlhe40 Tcrg-V2 Tcrg-V3 S100a6 Entpd1 Osbpl3 Fcer1g Gp49a Myo1e Tcrg-C Cd244 Dapk2 Ctla2a Dusp1 Pdcd1 Socs2 Nr4a2 Nr4a3 Gzmb Gzma Gzmk Lilrb4 Fosl2 Chn2 Cdh1 Rgs1 Klra5 Ctla4 Klra6 Rgs2 Klrc1 Klrc2 Itga1 Lag3 Gem Ccr5 Ccr8 Ptprj Ccl5 Ccl3 Fgl2 Plek Litaf Fasl Tigit 10 1600012F09Rik Usp33 Vipr1 Tet1 Amigo2 Gm16489 Gm6683 Fam101b Cnga1 Pecam1 Als2cl Chst15 Pik3ip1 Il6st Ggt1 Ccr7 Tet1 Nsg2 Actn1 Aff3 Tcf7 Treml2 Slc16a5 Usp28 Ifngr2 2610019F03Rik A930002I21Rik St6gal1 Gm14085 Qser1 St6gal1 Atp1b1 Tlr1 H2-Ob Cd55 Cnr2 Gm16591 Lef1 A630038E17Rik Xkrx Il6ra Cpm Cmah Sell Rapgef4 Slc6a19 Art2b Igfbp4 Dapl1 Ccr9 2 10 10 1 3 Ccr9 Tcf7

Up in naive 10 10 4 3 -128 -16 1 +16 +128 10 -3 Up in tumor Day 1effector mean class expression of indicated Immgen datasets (x-axis) versus PyMT tumor CD8 CD200r1 10 A130082M07Rik LOC100504914 1 10 Serpina3f Gm5486 Cd200r1 Cd200r4 Gm8721 Sema6d -1 Tcra-V8 Tcrg-V2 Tcrg-V3 S100a6 Vps37b B3galt5 Entpd1 Osbpl3 Fcer1g Mir27b Myo1e Tcrg-C Cd244 Dapk2 Klra10 Dusp1 Pdcd1 Klrb1c Socs2 Nr4a2 Cxcr6 Gzmk Capg Cd38 Cdh1 Chn2 Klra9 Rgs2 Klra5 Klra6 Rgs1 Klrk1 Klrc1 Itgae Itga1 Car2 10 Nt5e Ccr5 Ctse Ccl5 Fgl2 Fasl 2 Sell Rpl7a Dctd Umps Ppa1 Rrp9 Gemin6 Rangrf Nip7 Npm1 Ifng Rps25 Marcksl1 Eef1e1 Adk Nop16 Pno1 Sssca1 Timm8a1 Shmt1 Hspd1 Car12 Cd200 Myc Tspan6 Hk2 Gemin6 Timm8a1 Rps13 Wdr12 Irf4 Rpp40 Impdh2 Utf1 Wdr12 Chchd4 Cnn3 Bcat1 Ccr9 Lad1 Srm Rpl15 Dapl1 E430024C06Rik Sema7a Il2ra Lif Il2 Il3 Mela Pdcd1 10 10 3 1 Il2 Il2ra The complete listsareincludedassupplementary tables;ameanclass

Up in effector day 1 10 Fold change 4 10 3 10 -3 Day 5effector Lilrb4

Up in tumor 10 A130082M07Rik 1 LOC100504914 Dusp1 10 LOC641050 LOC641050 LOC641050 BC005685 Gm10008 -1 Cd200r1 Gm8721 Pmepa1 Sema6d Cables1 Tcra-V8 Tcrg-V2 Tcrg-V3 10 Vps37b Tnfaip3 Gpr114 Bcl2l11 Fcer1g Cxcl10 Gp49a Tcrg-C Cd244 Neurl3 Klra10 Dusp1 Socs2 Tnfsf8 Nr4a3 Nr4a1 Nr4a2 Rpl36 Fosl2 Lilrb4 Cdh1 Chn2 Klra9 Rgs1 Klra6 Klra5 Rgs2 Itgae Ccr8 Cish Sik1 Bcl2 Fgl2 Fasl Tigit 2 Prr11 Cmah Kif23 Hist1h2bk Kif14 Anxa1 Sgol2 Hist1h1a Ect2 F630043A04Rik Ncapg2 Fignl1 Ckap2l Depdc1a Ncapg Tpx2 Arhgap19 4930547N16Rik Hba-a1 Cenpe Cep55 Kif18b Esco2 Dlgap5 Rrm2 Casc5 Birc5 E2f8 Ccnb1 Kif15 Shcbp1 Mki67 Hist1h1b Rpl13 Cdca8 Hist1h3h Nek2 Kif4 Ccnb1 Nuf2 Rps23 Bub1 Plk1 Hmmr Kif20a Ccnb2 2810417H13Rik Kif2c Ccna2 Itgam 10 + 10 3 Tcellsversusnaive,early effector, peak 1 10 Up in effector day 5 4 10 -16 1 +16 +256 -256 3 10 -3 Day 100+memory 10 1 Tigit 1810011H11Rik 10 Up in tumor LOC641050 -1 Hist1h2bk 10 Gm8721 Cd200r1 Gm5486 Sema6d Pmepa1 Bhlhe40 Cables1 Tcrg-V2 Tcrg-V3 Tcra-V8 Vps37b Tnfaip3 Bcl2l11 Fcer1g Tnfrsf9 Cxcl10 Myo1e Tcrg-C Cd244 Dusp1 Rgs16 Dusp4 Pdcd1 Nr4a2 Nr4a1 Nr4a3 Gzma Gzmb Isg20 Fosl2 2 Chn2 Klra5 Rgs1 Klra6 Ctla4 Rgs2 Itgae Lag3 Gem Car2 Ikzf2 Ccr8 Fgl2 Sik1 Litaf Tigit Ahr Gramd4 Rnf144a Fgf13 Rpl13 Dtx1 Arhgef18 AI467606 Aff3 Itgax Gab3 Ccr2 Usp33 Wfikkn2 Il6ra Arhgap26 Ccl9 Cx3cr1 Prss12 Gm10785 Lef1 Arl4c Aqp9 Pde2a I830127L07Rik Kcnj8 Mir342 Slc9a7 Elovl7 Gm11435 Zeb2 Dusp7 Dapl1 Tnfrsf22 Gm6683 S1pr1 Gm13927 As3mt Il18rap S1pr5 Sell AY036118 Anxa1 Trio Tcf7 Il18r1 Klf3 Il7r Cmah Fcgr2b A630038E17Rik 10 Cmah 3 10 Il18r1 1 10 Il7r 4 Up in memory 10 3 + T post-operative human memory and effector memory breast CD8+ TIL isotype naive PBL(CD8+CD45RAhighCCR7high) PBL (CD8+CD45RA+/-/CCR7low) (CD45RA+/-/CCR7low)

Healthy donor PBL PBL and I II III 1 2 3

CD69

PD-1

CD244

PTPRJ/ CD148 Established and putative activation/exhaustion markers

CD200R

TIM3

CD25

CD122

Selected IL-2/-15 receptors CD132

Supplemental Figure 6: Human breast cancer CD8+ T cell infiltrate. Relative to matched patient effector/memory CD8+ T cells in the PBL, breast cancer CD8+ tumor T cells are enriched for CD69highPD1highCD132med cells with a trend towards brighter CD200R expression. One healthy donor PBL and one breast cancer sample and matched PBL sample were collected for each experiment, and this was performed three times as indicated by arabic or roman numerals above. A

100 pos CD103 brain TRM vs. spln PyMT tumor CD8+ T cells vs. spln LCMV cl13 vs. Arm 10

1 Litaf* /// Ly6c2 Ly6c1 Usp33 Fgf13 Elovl7 Eomes Fam65b S1pr5 Tlr1 Rasgrp2 Sidt1 Cmah Slamf6 S1pr1 Skil Icos Litaf Sik1 Xcl1 Fold change Qpct Itgae Itga1 Rgs2 Ctla4 Rgs1 Klre1 Chn2 Nr4a2 Nr4a1 Hpgds Inpp4b Vps37b Hspa1a Tmem123

-10 /// Ly6c2 Ly6c1 Hspa1b /// Hspa1a Cd244 /// LOC677008

-100 There were two probesets for Litaf, hence one is marked with a * to avoid confusion.

B Sort +IL-15 17 14 23 20 CD8+ 60h in vitro

CD103low

PyMT Tumor tumor- 8 45 15 9 bearing CD103high

0.3 2 3 1

Spleen CD44high PD-1 IKZF2 TOX PD-1 CD71 SSC

Supplemental Figure 7: TRM “core signature” gene expression and transcriptional regulator TOX correlation with IL-15

resistance. (A) Fold change of core signature in indicated datasets. TRM control was splenic OT-I cells day 20 post-in- fection with VSV-OVA; PyMT tumor CD8+CD44high control was splenic CD8+CD44high T cells; the LCMV-cl13 day 15 control

was LCMV-Arm day 15 post-infection. The TRM data was derived from GSE39152, the “core signature” of TRM was reported in Mackay et al., NI, 2013, and the LCMV data is from GSE41867. As these datasets were normalized independently with differ- ent controls, interpretation should be limited to overall expression patterns with consideration of control populations. (B) CD8+CD103high or CD8+CD103low cells were sorted from single-cell suspensions of PyMT tumors. CD71 and SSC vs. indicated marker after 60h in 1μg/mL IL-15 in vitro is plotted. This experiment was performed once. A B 80 P < 0.0001 5

Day 0 21 23 25 27 28 (%) + 4 60 3 Infect of CD8

40 + LCMV-Arm P = 0.0004 or -cl13 2 IL-15cx IL-15cx IL-15cx IL-15cx abundance (%) gp33 + 20 b 1

Assess CD8 + CD8 T cell 0 H-2D 0 populations Untx +IL-15cx Untx Untx +IL-15cx Untx clone13 Arm clone13 Arm

C +IL-15: 4 days 1μg/mL in vitro

Uninfected LCMV-Arm d24 LCMV-cl13 d24

Uninfected LCMV-Arm

high Events

PD-1 LCMV-cl13, PD-1 CD71 CD71

Uninfected LCMV-Arm

high PD-1 Events LCMV-cl13, PD-1 CD25 CD25

Uninfected LCMV-Arm

PD-1 high

Events LCMV-cl13, PD-1 SSC SSC

Supplemental Figure 8: IL-15cx treatment of persistent virus-infected mice results in decreased relative abundance of exhausted antigen-specific CD8+ T cells. (A) Experimental design for testing virally-exhausted CD8+ T cell responsive- ness to IL-15cx. Mice were infected with LCMV-Arm (acute infection) or LCMV-cl13 (persistent infection) on day 0. One group of mice was then dosed with IL-15cx every other day for one week as depicted. (B) Relative abundance (%) of total CD8+ and of virus-specific CD8+ T cells (H-2Db gp33-41 tetramer+) in splenic single-cell suspensions of uninfected, LCMV-Arm or LCMV-cl13 infected mice with or without treatment with IL-15cx, as depicted in A. H-2Db gp33-41 tetramer+PD-1+ virus-specif- ic cells are under-represented in the IL-15cx expanded CD8+ T cell pool. P-value is from Student’s unpaired t-test, error bars indicate S.D. This experiment performed once. (C) Splenocytes from uninfected, LCMV-Arm or LCMV-cl13 infected mice were incubated with 1μg/mL IL-15 in vitro for 4 days. CD71, CD25 and SSC versus PD-1 on CD8+CD4--gated cells. Histo- grams at right depict CD71, CD25 and SSC on total CD8+CD4- from uninfected, LCMV-Arm, and LCMV-cl13 PD-1high cells; n=3 for all groups. This experiment was performed twice. A B

103 15 ) 3 (%) + P = 0.02 10 102

cells GZMB 5 + Tumor volume (mm Tumor CD8 101 0 Before After Untreated αPD- IL-15cx IL-15cx+ only only αPD-L1Ab C D IL-15 1μg/ml + Unstim 30 P = 0.009 αPD-L1/ αTGF-β (%) + 20 Sorted CD8+CD44high splenocytes cells IFN γ

+ 10

CD8 Sorted PyMT 0 tumor Untreated αPD-L1 IL-15cx IL-15cx+ CD8+CD44high only only αPD-L1Ab

Supplemental Figure 9: Combined in vivo IL-15cx and αPD-L1 antibody treatment does not rescue PyMT CD8+ T cell cytokine non-responsiveness. (A) Tumor volumes before and after five days IL-15cx + αPD-L1 antibody on day 1 (150 ug), day 2 (100ug) and day 3 (100ug; clone 10F.9G2, BioXcell). (B) From the experiment in A, tumor single-cell suspensions were generated and the % of tumor-infiltrating CD8+ T cells that were granzymeB positive was determined for IL-15cx/αPD-L1 treated animals and additonal controls depicted; error bars indicate S.D. (C) As in B, but single-cell PyMT tumor suspensions were stimulated with αCD3/αCD28 antibodies for 5-8 h in vitro in the presence of golgi transport inhibitor golgistop (BD Biosci- ences). The % of PyMT CD8+ T cells that produced IFNγ was determined by intracellular staining and flow cytometry. This experiment was performed once with n indicated in plot; error bars indicate S.E.M. (D) Sorted CD8+ T cells from the spleen or PyMT tumors were plated with or without IL-15 as indicated in figure with αPD-L1 (10F.9G2) and αTGF-β (1D11.16.8) antibod- ies (both BioXcell, 20ng/mL); note unstimulated condition is the same as in Fig.2C, but was part of the same experiment and is included here for reference. This experiment was performed twice. P-values in B and C are from student’s unpaired t test. LCMV-cl13 d23 PyMT MEC tum.

Control (FMO)

LCMV-cl13, day 23, PD-1high FMO CD80 PD-1 high

Events PyMT MEC tumor, PD-1 CD80

No Ab LCMV-cl13 d23 PyMT MEC tum. PD-1

Control (FMO)

LCMV-cl13, day 23, PD-1high CD200R

PD-1 high

Events PyMT MEC tumor, PD-1 CD200R

Supplemental Figure 10: Expression of CD80 and CD200R on CD8+ T cells from PyMT tumor and persistent viral infec- tion. A CD8+CD4- gate was applied in all cases. Left, a no antibody/fluorescence minus one (FMO) control in the same chan- nel as the CD80 and CD200R fluorochrome-conjugated antibodies. Center, the cell-surface abundance of CD80 and CD200R versus PD-1 is plotted; histograms at right depict expression of indicated antibody on CD8+PD-1high T cells from day 23 post- infection with LCMV-clone13 or from a PyMT MEC tumor. This experiment was performed twice. A αCD3/αCD28 activate CD45.1+ OT-I splenocytes 24-48 h Expand in IL-2 72h

~2 weeks Inject 1 x 107 OT-I/CD45.1 Isolate spleen and tumor at Inject 1x106 B16-OVA cells iv into tumor-bearing mice day 2,5,12 post-transfer

B C D Spleen Tumor Spleen Tumor Spleen Tumor 30 200 30

150 20 20 100

10 PD-1 MFI 10 50 CD244 MFI Abundance (%)

0 0 0 0 5 10 15 0 5 10 15 0 5 10 15 Days post-transfer Days post-transfer Days post-transfer

Supplemental Figure 11: Activated and expanded tumor-specific CD8+ T cells adoptively transferred to tumor-bearing mice localize to tumor, but rapidly up-regulate PD-1 and CD244. (A) Experimental procedure, (B) localization, abun- dance, (C) PD-1 MFI and (D) CD244 MFI of adoptively transferred tumor-specific OT-I cells over time. This experiment was performed once with a n=3; error bars indicate S.E.M.