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Figure S6 - Figure.S6 shows combination of rapamycin and BCG increase percentage of effector memory γδ T cells and production of cytokine such as IFNγ within γδ T cells.

Reagent information for flow cytometry and ELISPOT assay.

Legends for all 7 supplementary figures

Bacillus Calmette-Guérin (BCG) is the most effective intravesical agent at reducing recurrence for patients with high-grade, non–muscle-invasive bladder cancer. Nevertheless, response to BCG is variable and strategies to boost BCG efficacy have not materialized. Prior work demonstrated a requirement for either conventional αβ or nonconventional γδ T cells in mediating BCG treatment efficacy, yet the importance of T-cell antigen specificity for BCG's treatment effect is unclear. Here, we provide direct evidence to show that BCG increases the number of tumor antigen–specific αβ T cells in patients with bladder cancer and protects mice from subsequent same-tumor challenge, supporting BCG induction of tumor-specific memory and protection. Adoptive T-cell transfers of antigen-specific αβ T cells into immunodeficient mice challenged with syngeneic MB49 bladder tumors showed that both tumor and BCG antigen–specific αβ T cells contributed to BCG efficacy. BCG-specific antitumor immunity, however, also required nonconventional γδ T cells. Prior work shows that the mTOR inhibitor rapamycin induces the proliferation and effector function of γδ T cells. Here, rapamycin increased BCG efficacy against both mouse and human bladder cancer in vivo in a γδ T cell–dependent manner. Thus, γδ T cells augment antitumor adaptive immune effects of BCG and support rapamycin as a promising approach to boost BCG efficacy in the treatment of non–muscle-invasive bladder cancer.

Supplementary figures 1-10. Figure S1. IL-2c increases conventional CD4 T cell in ascites three weeks after final IL-2c, but does not affect ascites Treg and effector T cell numbers at one week after final IL-2c administration. Figure S2. IL-2c treatment increases CD8+ central and effector memory T cells in the ascites, and central memory T cells in TDLN. Fig S3. Additional Treg phenotype in ascites and TDLN after IL-2c treatment. Fig S4. IL-2c directly inhibits Treg suppression specifically in the tumor microenvironment. Fig S5. IL-2 signaling is not impaired in TDLN after IL-2c treatment. Figure S6. Specific Treg depletion is highly effective against ID8agg. Figure S7. αCD25 attenuates IL-2c-induced tumor rejection and T cell function in ID8agg OC. Figure S8. IL-2c promotes intratumoral T cell infiltration. Figure S9. IL-2c reduces intratumoral Treg activation and functional molecules in B16 melanoma. Figure S10. IL-2c increases TIGIT+LAG-3+PD-1+ CD8+ T cells in B16 tumors.

Regulatory T cells (Treg) engage IL-2 by high affinity CD25, 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. We found IL-2c but not aPD-L1 potently inhibits ID8agg, an aggressive mouse ovarian cancer (OC) model. IL-2c decreased ascites Treg functional markers (CD25, GranzymeB) while upregulating them on Teffs. IL-2c inhibited Treg suppressive function in ascites but not TDLN. Ascites Tregs after IL-2c showed a fragile phenotype (increased PD-1, T-bet, and IFNg with maintained FoxP3) known to contribute to better immunotherapy response. These data suggest IL-2c inhibits Treg in the ascites through inducing fragile Tregs. Neither CD8+ T cells nor gd T cells is required for IL-2c induced Treg fragility. Studies testing other populations are ongoing. CD8+TCF-1+T cell stem cells (TCSC) improve immune checkpoint blockade in many cancer types. In ID8agg tumors, IL-2c induces CD8+TCF-1+ TCSC with tumor inhibitory capacity when transferred into Rag KO mice. tSNE analysis revealed IL-2c-induced TCSC are CXCR5+PD-1− express CCR2 CXCR3 and produce TNFa, while aPD-L1 induced TCSC are CXCR5+ and PD-1+, consistent with previous reports. These data suggest IL-2c induces beneficial CD8+TCSC distinct from aPD-L1. Although aPD-L1 is not efficacious alone in ID8agg, it significantly promoted IL-2c efficacy. Mice treated with IL-2c+αPD-L1 are resistant to tumor re-challenge even 6 weeks after final dose, suggesting durable immune memory. In sum, IL-2c is a novel OC immunotherapy that targets distinct immune pathways simultaneously, including inhibiting Tregs, boosting TCSCs, and inducing immune memory when combined with aPD-L1.

Tumor PD-L1 mediates diverse cell-intrinsic signals that increase cancer pathogenesis including the DNA damage response (DDR). Although immune consequences of tumor-extrinsic PD-L1 blockade are known, novel therapeutic vulnerabilities from inhibiting tumor-intrinsic PD-L1 signals are little studied. To assess tumor-intrinsic PD-L1 DDR control, we depleted PD-L1 by CRISPR/Cas9 or shRNA (PD-L1KO) in mouse B16 (melanoma), 4T1 (breast), ID8agg (ovarian), and human bladder RT4 cancer lines. We identified specific DDR signaling defects in PD-L1KO versus control (con) cells after exposure to distinct DNA damaging agents. 24h gemcitabine (or 2 Gy X-ray) in vitro in PD-L1KO vs. con RT4 cells elevated γH2AX (DNA damage marker) and reduced Chk2 DDR gene expression but not ATM, ATR, Chk1, or BRCA1 (immunoblots), and decreased BRCA1/p-RPA32 nuclear foci formation (confocal) altogether suggesting tumor-intrinsic PD-L1 promotes specific DDR/DNA repair pathways. PD-L1 loss could thus predict synthetic lethality to DDR inhibitors (DDRi) Chk1i or PARPi. PD-L1KORT4, ID8, and 4T1 cells were more sensitive to small molecule Chk1i in vitro (>15 fold vs. con, MTT) and in vivo in NSG mice. Chk1i and PARPi led to γH2AX increase in PD-L1KO B16, RT4, and 4T1 vs. sham treated tumor cells as predicted by DDR defects. Genetic cellular PD-L1 depletion, but not anti-PD-L1 (extrinsic), strongly sensitized B16 and 4T1 tumors to PARPi in vivo. Strikingly, PARPi had no effect on PD-L1KO B16 in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi treatment efficacy. Thus, DDRi plus inhibiting tumor intrinsic PD-L1 signals could improve immunotherapy in immunotherapy-sensitive or resistant tumors.

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.

PD-L1, expressed on many tumors, inhibits anti-tumor T cells by programmed death (PD)-1 engagement. Tumor PD-L1 predicts anti-PD-L1 treatment outcomes through incompletely understood mechanisms. We used naturally PD-L1+ murine B16 melanoma (ctrl) and made PD-L1KO B16 by CRISPR/Cas9. PD-L1KO B16 grew faster when ctrl B16 was on the opposite flank (trans). By contrast, ctrl B16 grew slower when trans to PD-L1KO B16. Anti-PD-L1 slowed ctrl but not PD-L1KO B16 tumors as expected. However, when PD-L1KO was trans to ctrl αPD-L1 now also slowed PD-L1KO growth. Anti-PD-L1 increased CD3+ T cell infiltration into ctrl and PD-L1KO tumors similarly in vivo, but increased natural killer (NK) cell numbers and functions (e.g., CD107a, IFN-g) in PD-L1KO tumors > ctrl. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly > ctrl, but NK cells migrated 2-fold more to PD-L1KO vs ctrl. PD-L1KO tumors produced more CCL2 than ctrl in vitro and in vivo, and this CCL2 induced CCR2+ NK chemotaxis in vitro. Both NK cells and T cells were required for optimal trans responses to anti-PD-L1 treatment in vivo. In wild type mice, anti-CCL2 reduced growth of trans PD-L1KO tumors > ctrl, and reduced anti-PD-L1 efficacy against PD-L1KO> ctrl tumors. In CCR2KO mice, anti-PD-L1 efficacy was retained against trans ctrl B16, but abolished against PD-L1KO trans tumors. PD-L1 also regulated other chemokines recruiting receptor+ cells, with similar effects seen in MB49 bladder cancer and 4T1 breast cancer cells. Thus, tumor PD-L1 alters immune infiltrates and anti-PD-L1 efficacy through novel chemokine effects. Our models help understand anti-PD-L1 (and likely anti-PD-1) responses based on tumor PD-L1 expression and can define ways to improve immunotherapy for PD-L1 null tumors.

PD-1/PD-L1 provides a mechanism of immune escape, the blockage of which has reinvigorated interest in the treatment of urothelial cancer. We recently reported on cell-intrinsic PD-L1 effects in melanoma and ovarian cancer, and considered that effects in bladder cancer cells could differ based on distinct mutational landscapes. Using CRISPER/Cas9 methodology, we knockout PD-L1 in the murine bladder cancer cell line MB49 and the human bladder caner cell line RT. We show here that cell-intrinsic MB49 and RT4 cells express PD-L1 and PD-1 that each mediate cell-intrinsic signals. PD-L1 knockout has little effect on cells proliferation in vitro, but significant difference could be found in tumor size when we used MB49 Ctrl or MB49 PD-L1KO cells to challenge C57B16 mice. αPD-L1 and αPD-1 antibodies reduced bladder cancer cell proliferation in vitro demonstrating direct signaling effects. Bladder cancer cell-intrinsic PD-L1 promoted mTORC1 and tumor initiating cell generation similar to melanoma and ovarian cancer cells. By contrast to melanoma and ovarian cancer cells, bladder cancer cell PD-L1 promoted autophagy and had little effect on in vivo immune-independent growth. Base one the functional role, PD-L1 knockout increased proliferation inhibition induced by Rapamycin in both mouse and human bladder cancer cells. Furthermore, we found intrinsic PD-L1 suppress cytokine production, including CXCL10. Corresponding with increased CXCL10 secretion, more CXCR3+ NK cells could be attracted by PD-L1KO cancer cells. Overall, our findings further illustrated the intrinsic role of PD-L1 in bladder cancer development and found PD-1 expression on human bladder cancer cells for the first time.