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Background Aging is the biggest risk factor for cancer, yet little is known about cancer immunotherapy effects. ?PD-1 can block PD-L1 and PD-L2 while ?PD-L1 blocks PD-1 and CD80.1 A recent key finding in young hosts including humans is that melanoma response to aPD-1/aPD-L1 correlates with CD8+TCF-1+ T cell stem cell (TCSC) generation.2 Methods We tested aPD-1 (100 or 200 µg/mouse), aPD-L1 (100 µg/mouse) or aPD-L2 (200 µg/mouse) in aged (18-33 months) and young (3-8 months) mice challenged orthotopically with B16 (WT or PD-L1ko) melanoma (SQ) or ID8agg ovarian cancer (IP). Tumors were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF. Results We reported that aPD-1 treats young and aged with B16 and aPD-L1 treats young not aged.3 aPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibiting this property [figure 1]. Efficacy in young (aPD-1, aPD-L1) and aged (aPD-L2) correlated with increased tumor TCSC [figure 3], but TCSC differed by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3) [figure 2]. aPD-L2 efficacy against B16 in aged mice required host IFN-g and IL-17 [figure 4]. IP ID8agg ovarian cancer did not respond to aPD-L2 in aged or young mice. Aged expressed up to 40-fold more PD-L2 versus young on various immune cells suggesting high PD-L2 helps aPD-L2 response [figure 6]. Host IFN-g contributed to aged PD-L2 expression, which did not appear cell-autonomous [figure 6]. PD-L1KO aged but not young mice challenged with PD-L1KO B16 responded to aPD-1 [figure 5], consistent with PD-L2 block as a mechanism. Conclusions Treatment differences in aged versus young could depend on immune checkpoint or TCSC differences, which could be related to CD8+ T-cell infiltration, including TCSC. aPD-1 efficacy in aged PD-L1KO mice challenged with PD-L1KO B16 suggests that aPD-1 efficacy is through PD-L2 block in aged. PD-L2 expression differences and anatomical compartment differences in tumor microenvironment may also contribute to treatment efficacy differences. We are now identifying mechanisms for increased PD-L2 and other mechanisms for aPD-L2 efficacy in aged, and testing TCSC effects. Our work can improve cancer immunotherapy in aged hosts and provides insights in treatment failures, including in young hosts. Acknowledgements South Texas MSTP training grant (NIH T32GM113896), TL1TR002647, Graduate Research in Immunology Program training grant (NIH T32 AI138944), R01 CA231325, Samuel Waxman Cancer Research Foundation Grant Ethics Approval The study was approved by UTHSA IACUC, approval number 20180021. References Schildberg FA, Klein SR, Freeman GJ, Sharpe AH. Coinhibitory pathways in the B7-CD28 ligand-receptor family. Immunity 2016;44(5):955-72. Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537(7620):417-21. Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol 2018;105:146-54.

Background Ovarian cancer (OC) responds poorly to immunotherapies. Regulatory T cells (Treg) engage IL-2 by high-affinity CD25 for differentiation and function,1 and anti-tumor effector T cells (Teff) use intermediate affinity CD122. We studied IL-2 complexes (IL-2c) that selectively activate CD122 (Teff) over CD25 (Tregs). Methods Orthotopic ID8agg-luc mouse OC burden was measured by in vivo imaging. Tumor, ascites and draining lymph nodes (TDLN) were analyzed by flow and tSNE. IL-2c was complexed using 1.5 µg/mouse IL-2 and 7.5 µg/mouse aIL-2 (clone JES6-5H4) before i.p. injection every other day x 4 starting at day 7. antiPD-L1 was given at 100ug/mouse every 3 days x 4 starting from Day 11. FIR mice2 were used to sort live Tregs. Results IL-2c but not antiPD-L1 potently inhibits ID8agg (figure 1). IL-2c decreased ascites Treg functional markers (e.g., CD25, granzymeB) while upregulating the same markers on Teffs (figure 2). IL-2c inhibited Treg suppression in ascites while TDLN Tregs were unaffected (figure 3). tSNE showed great similarity of TDLN Tregs treated with isotype and IL-2c while ascites Tregs after IL-2c showed a fragile phenotype (e.g., increased PD-1, T-bet, and IFNgamma with maintained FoxP3 expression [figure 4]) which is known to contribute to better response to cancer immunotherapy.3 4 We observed a complete reduction of tumor bioluminescence with IL-2c and antiPD-L1 combo treatment in nearly all subjects significantly exceeding effect of IL-2c alone (figure 5). A CD8+CXCR5+TCF-1+ T cell stem cell (TCSC) population reportedly improves immune checkpoint blockade efficacy.5 6 Since CD122 is regulated by TCF-1,7 we explored the effect of IL-2c on these TCSC. IL-2c significantly induced a CD8+TCF-1+ TCSC population in ID8agg tumors (figure 6), possibly through a positive feedback loop by further enhancing CD122 expression on TCF-1+, but not TCF-1- cells (figure 7). tSNE analysis of detailed immune phenotype of IL-2c induced TCSC revealed that these TCSC differed from those induced by antiPD-L1. In ID8agg, antiPD-L1-induced TCSC are mostly CXCR5+ and PD1+, consistent with previous reports in other cancers3 4 while IL-2c-induced TCSC were PD1- (figure 8), expressed CCR2 and CXCR3, and produced TNFalpha (figure 9). Conclusions We define two novel IL-2c effects: inducing Treg fragility therefore reducing immunosuppression while promoting TCSC that could enhance effective anti-tumor immunity. Current work tests if effects are related and help efficacy, and mechanisms for IL-2c Treg effects. We also show that elicited TCSC differ by treatment and tumor, requiring additional investigations. Acknowledgements This work is supported by CPRIT Research Training Award (RP 170345), Ovarian Cancer Research Alliance Ann and Sol Schreiber Mentored Investigator Award to YD and R01 CA205965to TC. Ethics Approval All mice studies were approved by UT Health San Antonio Institutional Animal Care and Use Committee (IACUC). Approval number 20150093AR, 20140001AR, 20170035AR, 20140039AR, 20140027AR, 20090128AR, 20120071AR, 20180021AR. References Malek TR: The biology of interleukin-2. Annu Rev Immunol 2008, 26:453–479. Fantini MC, Dominitzki S, Rizzo A, Neurath MF, Becker C: In vitro generation of CD4+ CD25+ regulatory cells from murine naive T cells. Nat Protoc 2007, 2(7):1789–1794. Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, Horne W, Moskovitz JM, Kolls JK, Sander C, et al: Interferon-gamma Drives Treg Fragility to Promote Anti-tumor Immunity. Cell 2017, 169(6):1130–1141e1111. Overacre-Delgoffe AE, Vignali DAA: Treg Fragility: A Prerequisite for Effective Antitumor Immunity? Cancer Immunol Res 2018, 6(8):882–887. Brummelman J, Mazza EMC, Alvisi G, Colombo FS, Grilli A, Mikulak J, Mavilio D, Alloisio M, Ferrari F, Lopci E, et al: High-dimensional single cell analysis identifies stem-like cytotoxic CD8(+) T cells infiltrating human tumors. J Exp Med 2018. Sade-Feldman M, Yizhak K, Bjorgaard SL, Ray JP, de Boer CG, Jenkins RW, Lieb DJ, Chen JH, Frederick DT, Barzily-Rokni M, et al: Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell 2018, 175(4):998–1013 e1020. Jeevan-Raj B, Gehrig J, Charmoy M, Chennupati V, Grandclement C, Angelino P, Delorenzi M, Held W: The Transcription Factor Tcf1 Contributes to Normal NK Cell Development and Function by Limiting the Expression of Granzymes. Cell Rep 2017, 20(3):613–626.

Background αPD-L1 bladder cancer (BC) immunotherapy is effective in Methods We used PD-L1+ mouse BC cell lines MB49 and MBT-2, for orthotopic, intravesical (i.e., in bladder) and intravenous challenge studies of local versus lung metastatic BC. Results αPD-L1 or IL-2c alone reduced tumor burden and extended survival in local MB49 and MBT-2. Using in vivo cell depletions, we found that γδ T cells and NK cells, but strikingly not CD8+ T cells, were necessary for IL-2c efficacy in bladder. We confirmed γδ T cell requirements for IL-2c, but not αPD-L1 efficacy in γδ T cell-null TCRδKO mice. TCRβKO conventional T cell-null mice exhibited IL-2c, but not αPD-L1 responsiveness for orthotopic BC treatment. Neither agent alone treated lung metastatic MB49 or MBT-2 but the drug combination improved survival in both tumor models. Combination treatment effects in lungs were distinct from bladder, requiring CD8+ T and NK cells, but not γδ T cells. Conclusions BC immunotherapy effects differ by anatomic compartment and use distinct mechanisms to treat primary and metastatic BC. CD122-directed IL-2 is a promising BC immunotherapy strategy, and IL-2c is a candidate mediator through innate immune effects. αPD-L1 could improve IL-2c efficacy by engagement of adaptive immune responses including to improve metastatic disease treatment efficacy. Ethics Approval All procedures involving animals in this study were approved by the UT Health San Antonio Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with UT Health San Antonio Department of Laboratory Animal Resources standards. References Shah AY, Gao J, Siefker-Radtke AO. Five new therapies or just one new treatment? A critical look at immune checkpoint inhibition in urothelial cancer: Future Medicine, 2017. Arenas-Ramirez N, Zou C, Popp S, et al. Improved cancer immunotherapy by a CD25-mimobody conferring selectivity to human interleukin-2. Science translational medicine 2016;8(367):367ra166-367ra166.

BackgroundAnti-programmed death-ligand 1 (αPD-L1) immunotherapy is approved to treat bladder cancer (BC) but is effective in + antitumor T cells known to improve αPD-L1 efficacy. We hypothesized that the tumor microenvironment, including local immune cells in primary versus metastatic BC, differentially affects immunotherapy responses and that IL-2c effects could differ from, and thus complement αPD-L1.MethodsWe studied mechanisms of IL-2c and αPD-L1 efficacy using PD-L1+ mouse BC cell lines MB49 and MBT-2 in orthotopic (bladder) and metastatic (lung) sites.ResultsIL-2c reduced orthotopic tumor burden and extended survival in MB49 and MBT-2 BC models, similar to αPD-L1. Using antibody-mediated cell depletions and genetically T cell-deficient mice, we unexpectedly found that CD8+ T cells were not necessary for IL-2c efficacy against tumors in bladder, whereas γδ T cells, not reported to contribute to αPD-L1 efficacy, were indispensable for IL-2c efficacy there. αPD-L1 responsiveness in bladder required conventional T cells as expected, but not γδ T cells, altogether defining distinct mechanisms for IL-2c and αPD-L1 efficacy. γδ T cells did not improve IL-2c treatment of subcutaneously challenged BC or orthotopic (peritoneal) ovarian cancer, consistent with tissue-specific and/or tumor-specific γδ T cell contributions to IL-2c efficacy. IL-2c significantly altered bladder intratumoral γδ T cell content, activation status, and specific γδ T cell subsets with antitumor or protumor effector functions. Neither IL-2c nor αPD-L1 alone treated lung metastatic MB49 or MBT-2 BC, but their combination improved survival in both models. Combination treatment efficacy in lungs required CD8+ T cells but not γδ T cells.ConclusionsMechanistic insights into differential IL-2c and αPD-L1 treatment and tissue-dependent effects could help develop rational combination treatment strategies to improve treatment efficacy in distinct cancers. These studies also provide insights into γδ T cell contributions to immunotherapy in bladder and engagement of adaptive immunity by IL-2c plus αPD-L1 to treat refractory lung metastases.

OBJECTIVES/GOALS: Aging is the biggest risk factor for cancer, yet little is known about cancer immunotherapy effects. Here we investigate melanoma response to αPD-1, αPD-L1 and αPD-L2 in young vs. aged hosts. We look at different immune outcomes as possible mechanism. METHODS/STUDY POPULATION: We tested αPD-1 (100 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18-24 months) and young (3-8 months) mice challenged orthotopically with B16. Tumors and draining lymph nodes (TDLN) were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF. RESULTS/ANTICIPATED RESULTS: We reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young. aPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibiting this property. Efficacy in young (aPD-1, aPD-L1) and aged (aPD-1, aPD-L2) correlated with increased T cell stem cells (TCSC) and total tumor-infiltrating lymphocytes (TIL), but TCSC differed by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3, PD-1 and TIM- expression). Aged expressed significantly more T-cell PD-1 and up to 40-fold more PD-L2 versus young in myeloid and NK cells, and TCSC. Bone marrow-derived DC experiments suggest aged DC are destined for high PD-L2 versus young. DISCUSSION/SIGNIFICANCE OF IMPACT: Treatment differences in aged vs. young could depend on immune checkpoint or TCSC differences. We are now identifying mechanisms for increased PD-L2 and contributions to aPD-L2 efficacy in aged, and testing TCSC effects. Our work can improve cancer immunotherapy in aged hosts and further provide important insights even in young hosts.