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Tumor cell‐intrinsic programmed death‐ligand 1 (PD‐L1) signals mediate immunopathologic effects in breast, colon, and ovarian cancers and in melanomas, but bladder cancer (BC) effects are unreported. We show here that BC cell‐intrinsic PD‐L1 signals in mouse MB49 and human RT4, UM‐UC3, and UM‐UC‐14 BC cells regulate important pathologic pathways and processes, including effects not reported in other cancers. α‐PD‐L1 antibodies reduced BC cell proliferation in vitro, demonstrating direct signaling effects. BC cell‐intrinsic PD‐L1 promoted mammalian target of rapamycin complex 1 (mTORC1) signals in vitro and augmented in vivo immune‐independent cell growth and metastatic cancer spread, similar to effects we reported in melanoma and ovarian cancer. BC cell‐intrinsic PD‐L1 signals also promoted basal and stress‐induced autophagy, whereas these signals inhibited autophagy in melanoma and ovarian cancer cells. BC cell‐intrinsic PD‐L1 also mediated chemotherapy resistance to the commonly used BC chemotherapy agents cis‐platinum and gemcitabine and to the mTORC1 inhibitor, rapamycin. Thus, BC cell‐intrinsic PD‐L1 signals regulate important virulence and treatment resistance pathways that suggest novel, actionable treatment targets meriting additional studies. As a proof‐of‐concept, we showed that the autophagy inhibitor chloroquine improved cis‐platinum treatment efficacy in vivo, with greater efficacy in PD‐L1 null versus PD‐L1‐replete BC., We provide evidence that tumor‐intrinsic PD‐L1 mediates pathologic signals in human and mouse bladder cancers, independent of molecular subtype. Signals include control of proliferation, mTORC1, autophagy, and chemotherapy resistance. We show that countering the pathologic cell‐intrinsic PD‐L1 signals improves chemotherapy response in vivo of mice bearing bladder cancer tumors.

Our laboratory has investigated the role of an evolutionarily conserved RNA species called microRNAs (miRs) in regulation of anti-chlamydial protective immunity. MiRs including miR-155 expressed in specific immune effector cells are critical for antigen specific protective immunity and IFN-γ production. Using miR-155 deficient mice, and a murine pulmonary model for chlamydial infection, we report here 1) the effect of host miR-155 on bacterial burden, and 2) identify probable immune genes regulated by miR-155.

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.

Aging is the biggest risk factor for cancer, yet little is known about how it affects cancer immunotherapy. Melanoma response to αPD-1/αPD-L1 correlates with CD8+ TCF-1+ T cell stem cell (TCSC) generation. 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 melanoma (SQ) or ID8agg ovarian cancer (IP). Tumors and draining lymph nodes (TDLN) were analyzed by flow. We previously reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young. αPD-L2 treated B16 in aged but not young, the first single agent cancer immunotherapy exhibiting this property. Preliminary data indicate survival benefit of αPD-L2 against ID8agg in aged but not young mice. Aged mice tolerated all treatments comparable to young. B16 efficacy in young (αPD-1, αPD-L1) and aged (αPD-1, αPD-L2) correlated with increased TCSC and total TIL, but TCSC differed by age and treatment. PD-1 interacts with PD-L1 and PD-L2 while PD-L1 interacts with PD-1 and CD80. Aged mice expressed significantly more T-cell PD-1 and PD-L2 and up to 40-fold more PD-L2, PD-L1 and CD80 vs. young on myeloid cells and NK cells. We generated bone marrow-derived DC with GM-CSF. Aged DC expressed much high PD-L2 versus young, suggesting a cell-intrinsic PD-L2 age bias. Treatment differences in aged versus young could depend on these immune checkpoint or TCSC differences. We are now identifying mechanisms for increased PD-L2 and contributions to αPD-L2 efficacy in aged, and testing TCSC effects on treatments. Our work can improve cancer immunotherapy in aged hosts and provide models for treatment resistance in young hosts.

Tumor-intrinsic PD-L1 signals contribute to tumor virulence and pathology, but mechanisms are poorly understood. We previously showed that tumor-intrinsic PD-L1 increases mTORC1 and tumor initiating cell (TIC) generation and function in ovarian cancer and melanoma. We used CRISPR/Cas9 (KO) and shRNA (lo) to reduce PD-L1 in mouse melanoma (B16) and ovarian cancer (ID8agg), and human ovarian cancer (ES2). PD-L1lo reduced mTORC1 and TIC as expected, but unexpectedly increased actin polymerization and filopodia, and altered cell morphology. The actin depolymerizing agents Latrunculin-A or Cytochalasin-D reversed actin polymerization in PD-L1lo ID8agg cells and increased mTORC1 (p-rpS6) without affecting mTORC2 (pAktS473). Latrunculin-A and Cytochalasin-D increased TIC numbers and stemness gene expression (Oct4) in PD-L1lo ID8agg cells with no effect on control ID8agg cells. Similar actin effects were seen in PD-L1KO B16 and PD-L1KO ES2. To confirm TIC function, Latrunculin-A increased tumorospheres (TIC self-renewal function) in PD-L1lo, but not control ID8agg cells. The mTORC1 inhibitor rapamycin reduced TIC numbers and functions with no effect on actin polymerization, suggesting mTORC1 is downstream of actin polymerization. Rptorlo ID8agg cells (with low mTORC1 signaling) did not exhibit the PD-L1lo actin polymerization, nor did Latrunculin-A increase Rptorlo ID8agg TIC numbers, tumorospheres, or stemness genes, confirming the flow of signals is PD-L1 to actin to mTORC1 to TIC (stemness). Our data define a highly novel tumor virulence control mechanism of cell-intrinsic PD-L1 through inhibiting actin polymerization suggesting new drug discovery targets. Preliminary data show some similar effects of tumor PD-1.