A Vehicle IL-15cx treated Events CD71 CD98 CD44 GZMB B 60 P = 0.03 300 P = 0.005 100 4000 P = 0.02 P < 0.0001 ) ) ) 80 3000 FI FI FI 40 200 (%) 60 high (gM (gM 2000 44 98 71 40 (gM MB 20 100 CD CD CD GZ 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 receptor) and CD98 (large neutral amino acid 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 n io 4000 ress exp n 2000 ea 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 n 6000 200 io LCMV-cl13 0 ress 4000 exp 1500 n Il2rb (CD122) ea 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 Gene symbol Gene name Replicate: 1 2 3 1 2 3 Gene symbol Gene name Tnfrsf9 tumor necrosis factor receptor superfamily, member 9 Nanp n-acetylneuraminic acid phosphatase Litaf lipopolysaccharide-induced tnf factor Kifap3 kinesin-associated protein 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 glycine 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 transferrin receptor (p90, cd71) Cd83 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 cd109 molecule Ncapg2 non-smc condensin 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, immunoglobulin domain (ig), (semaphorin) 4c Ccr9 chemokine (c-c motif) receptor 9 Nop58 nop58 ribonucleoprotein Cd81 cd81 molecule Ehd4 eh-domain containing 4 Vamp5 vesicle-associated membrane protein 5 (myobrevin) Bst2 bone marrow stromal cell antigen 2 Slc6a13 solute carrier family 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, light chain, tctex-type 3 Bub1 bub1 budding uninhibited by benzimidazoles 1 homolog (yeast) Aim2 absent in melanoma 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 ran binding protein 1 Chek1 chk1 checkpoint homolog (s. pombe) Rgs10 regulator of g-protein signalling 10 Lap3 leucine aminopeptidase 3 Cd160 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