Immune checkpoint expression and relationships to anti-PD-L1 immune checkpoint blockade cancer immunotherapy efficacy in aged versus young mice.

Introduction: Aging is the biggest cancer risk, and immune checkpoint (IC) inhibition (ICI) is a revolutionary cancer immunotherapy approach. Nonetheless, there are limited preclinical/clinical data regarding aging effects on ICI outcomes or age effects on IC expression in different organs or tumors. Methods: Flow cytometry assessed IC on immune and non-immune cells in various organs in young and aged BL6 mice. Comparisons: aged versus young naïve WT versus interferon-γ KO mice and WT challenged with B16F10 melanoma and treated with αPD-1 or αPD-L1 ICI. We co-cultured young and aged T cells and myeloid cells in vitro and used OMIQ analyses to test cell-cell interactions. Results: αPD-1 ICI treated melanoma in young and aged hosts, whereas αPD-L1 ICI was only effective in young. We found considerable, previously undescribed age effects on expression of various IC molecules participating in the ICI treatment, including PD-1, PD-L1, PD-L2, and CD80, in distinct organs and in the tumor. These data help explain differential ICI efficacy in young and aged hosts. Host interferon-γ influenced age effects on IC expression in both directions depending on specific IC molecule and tissue. IC expression was further affected by tumor challenge on immune, non-immune, and tumor cells in tumor and other organs. In in vitro co-culture, αPD-1 versus αPD-L1 distinctly influenced polyclonal T cells in young versus aged, suggesting mechanisms for distinct age-related ICI outcomes. Conclusion: Age affects IC expression on specific immune cells in an organ- and tissue-specific manner. ICs were generally higher on aged immune cells. High immune-cell PD-1 could help explain αPD-1 efficacy in aged. High co-expression of CD80 with PD-L1 on dendritic cells could help explain lack of αPD-L1 efficacy in aged hosts. Factors other than myeloid cells and interferon-γ also affect age-related IC expression and T cell function, meriting additional studies.

CD122-targeted interleukin-2 and αPD-L1 treat bladder cancer and melanoma via distinct mechanisms, including CD122-driven natural killer cell maturation.

Bladder cancer (BC) and melanoma are amenable to immune checkpoint blockade (ICB) therapy, yet most patients with advanced/metastatic disease do not respond. CD122-targeted interleukin (IL)-2 can improve ICB efficacy, but mechanisms are unclear. We tested αPD-L1 and CD122-directed immunotherapy with IL-2/αIL-2 complexes (IL-2c) in primary and metastatic bladder and melanoma tumors. IL-2c treatment of orthotopic MB49 and MBT-2 BC generated NK cell antitumor immunity through enhanced activation, reduced exhaustion, and promotion of a mature, effector NK cell phenotype. By comparison, subcutaneous B16-F10 melanoma, which is IL-2c sensitive, requires CD8+ T and not NK cells, yet we found αPD-L1 efficacy requires both CD8+ T and NK cells. We then explored αPD-L1 and IL-2c mechanisms at distinct metastatic sites and found intraperitoneal B16-F10 metastases were sensitive to αPD-L1 and IL-2c, with IL-2c but not αPD-L1, increasing CD122+ mature NK cell function, confirming conserved IL-2c effects in distinct cancer types and anatomic compartments. αPD-L1 failed to control tumor growth and prolong survival in B16-F10 lung metastases, yet IL-2c treated B16-F10 lung metastases effectively even in T cell and adaptive immunity deficient mice, which was abrogated by NK cell depletion in wild-type mice. Flow cytometric analyses of NK cells in B16-F10 lung metastases suggest that IL-2c directly boosts NK cell activation and effector function. Thus, αPD-L1 and IL-2c mediate nonredundant, immune microenvironment-specific treatment mechanisms involving CD8+ T and NK cells in primary and metastatic BC and melanoma. Mechanistic differences suggest effective treatment combinations including in other tumors or sites, warranting further studies.

γδ T Cells Support Antigen-Specific αß T cell-Mediated Antitumor Responses during BCG Treatment for Bladder Cancer.

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 αß 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 αß 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 αß T cells into immunodeficient mice challenged with syngeneic MB49 bladder tumors showed that both tumor and BCG antigen-specific αß 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.

A Randomized Feasibility Trial Comparing Surveillance Regimens for Patients with Low and Low-Intermediate Risk Non-Muscle Invasive Bladder Cancer.

BACKGROUND: Surveillance regimens for non-muscle invasive bladder cancer (NMIBC) are disparate and controlled trials could inform guidelines. The feasibility of randomizing patients to variable frequency surveillance is unknown. OBJECTIVES: To determine patient willingness to randomization to high frequency (HF) versus low frequency (LF) surveillance regimen for NMIBC and compare patient comfort and healthcare costs across regimens. METHODS: A non-blinded, two-arm, randomized-controlled study of patients with low or low-intermediate risk NMIBC was conducted at two institutions where patients were offered randomization to HF vs. LF surveillance following initial tumor resection. The HF group underwent cystoscopy every three months for 2 years, then every 6 months for 2 years, then annually. The LF group underwent cystoscopy at 9 months following the 3-month cystoscopy, then annually. Assuming 75% of patients approached would agree to enrollment, a sample size of n = 35 patients per arm provided a one-sided 95% exact Clopper-Pearson confidence lower-limit of 60%. RESULTS: Of 70 patients approached, 45 (64.3%) agreed to participate and 25 (35.7%) declined enrollment due to preference for HF. Twelve biopsies were performed, including 4 (19%) of 21 patients in the HF group and 8 (33.3%) of 24 patients in the LF group. Disease recurrence (low grade Ta) was observed in 3 (14.3%) and 5 (20.8%) patients in the HF and LF groups, respectively. No patients experienced high grade recurrence or progression. Both groups had similar patient-reported procedure-related discomfort and quality of life measures over time. Patient out-of-pocket cost and healthcare systems costs were $383.80 more per patient annually in the HF group. CONCLUSIONS: Randomization to variable frequency surveillance is challenging as over a third of patients declined participation. However, these data provide important preliminary insights into the potential effects of surveillance frequency on oncologic and economic outcomes in patients with low and low-intermediate risk bladder cancer.

Rapamycin enhances BCG-specific γδ T cells during intravesical BCG therapy for non-muscle invasive bladder cancer: a randomized, double-blind study.

BACKGROUND: Although intravesical BCG is the standard treatment of high-grade non-muscle invasive bladder cancer (NMIBC), response rates remain unsatisfactory. In preclinical models, rapamycin enhances BCG vaccine efficacy against tuberculosis and the killing capacity of γδ T cells, which are critical for BCG's antitumor effects. Here, we monitored immunity, safety, and tolerability of rapamycin combined with BCG in patients with NMIBC. METHODS: A randomized double-blind trial of oral rapamycin (0.5 or 2.0 mg daily) versus placebo for 1 month was conducted in patients with NMIBC concurrently receiving 3 weekly BCG instillations (NCT02753309). The primary outcome was induction of BCG-specific γδ T cells, measured as a percentage change from baseline. Post-BCG urinary cytokines and immune cells were examined as surrogates for local immune response in the bladder. Secondary outcomes measured were adverse events (AEs) and tolerability using validated patient-reported questionnaires. RESULTS: Thirty-one patients were randomized (11 placebo, 8 rapamycin 2.0 mg, and 12 rapamycin 0.5 mg). AEs were similar across groups and most were grade 1-2. One (12.5%) patient randomized to 2.0 mg rapamycin was taken off treatment due to stomatitis. No significant differences in urinary symptoms, bowel function, or bother were observed between groups. The median (IQR) percentage change in BCG-specific γδ T cells from baseline per group was as follows: -26% (-51% to 24%) for placebo, 9.6% (-59% to 117%) for rapamycin 0.5 mg (versus placebo, p=0.18), and 78.8% (-31% to 115%) for rapamycin 2.0 mg (versus placebo, p=0.03). BCG-induced cytokines showed a progressive increase in IL-8 (p=0.02) and TNF-α (p=0.04) over time for patients on rapamycin 2.0 mg, whereas patients receiving placebo had no significant change in urinary cytokines. Compared with placebo, patients receiving 2.0 mg rapamycin had increased urinary γδ T cells at the first week of BCG (p=0.02). CONCLUSIONS: Four weeks of 0.5 and 2.0 mg oral rapamycin daily is safe and tolerable in combination with BCG for patients with NMIBC. Rapamycin enhances BCG-specific γδ T cell immunity and boosts urinary cytokines during BCG treatment. Further study is needed to determine long-term rapamycin safety, tolerability and effects on BCG efficacy.

Bladder cancer cell-intrinsic PD-L1 signals promote mTOR and autophagy activation that can be inhibited to improve cytotoxic chemotherapy.

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.

CD122-Selective IL2 Complexes Reduce Immunosuppression, Promote Treg Fragility, and Sensitize Tumor Response to PD-L1 Blockade.

The IL2 receptor (IL2R) is an attractive cancer immunotherapy target that controls immunosuppressive T regulatory cells (Treg) and antitumor T cells. Here we used IL2Rß-selective IL2/anti-IL2 complexes (IL2c) to stimulate effector T cells preferentially in the orthotopic mouse ID8agg ovarian cancer model. Despite strong tumor rejection, IL2c unexpectedly lowered the tumor microenvironmental CD8+/Treg ratio. IL2c reduced tumor microenvironmental Treg suppression and induced a fragile Treg phenotype, helping explain improved efficacy despite numerically increased Tregs without affecting Treg in draining lymph nodes. IL2c also reduced Treg-mediated, high-affinity IL2R signaling needed for optimal Treg functions, a likely mechanism for reduced Treg suppression. Effector T-cell IL2R signaling was simultaneously improved, suggesting that IL2c inhibits Treg functions without hindering effector T cells, a limitation of most Treg depletion agents. Anti-PD-L1 antibody did not treat ID8agg, but adding IL2c generated complete tumor regressions and protective immune memory not achieved by either monotherapy. Similar anti-PD-L1 augmentation of IL2c and degradation of Treg functions were seen in subcutaneous B16 melanoma. Thus, IL2c is a multifunctional immunotherapy agent that stimulates immunity, reduces immunosuppression in a site-specific manner, and combines with other immunotherapies to treat distinct tumors in distinct anatomic compartments. SIGNIFICANCE: These findings present CD122-targeted IL2 complexes as an advancement in cancer immunotherapy, as they reduce Treg immunosuppression, improve anticancer immunity, and boost PD-L1 immune checkpoint blockade efficacy in distinct tumors and anatomic locations.

Urinary Diversion Disparity Following Radical Cystectomy for Bladder Cancer in the Hispanic Population.

OBJECTIVE: To determine if disparities in quality of surgical care exist between Hispanics and non-Hispanics undergoing radical cystectomy for bladder cancer. MATERIALS AND METHODS: An observational cohort study was conducted retrospectively on patients who underwent radical cystectomy for urothelial carcinoma of the bladder at our institution between January 2005 and July 2018. Data was collected on demographic, clinical, and pathological characteristics of patients, including self-reported ethnicity. Univariable and multivariable logistic or linear regression analyses were used to evaluate the association of ethnicity with receipt of neoadjuvant chemotherapy, utilization of laparoscopic surgery, number of lymph nodes removed, and continent urinary diversion. RESULTS: We identified 507 patients in our database out of which, 136 (27%) were Hispanic and 371 (73%) were non-Hispanic. Compared to non-Hispanics, Hispanics had a higher body mass index (26.9 kg/m2 vs 28.2 kg/m2, P = .006) and lived further away from site of surgery (34 vs 96 miles, P = .02). No significant differences were observed in receipt of neoadjuvant chemotherapy, laparoscopic surgery, or number of lymph nodes removed during cystectomy between ethnicity groups. However, Hispanics were less likely than non-Hispanics to receive a continent urinary diversion on multivariable analysis (odds ratio 0.30, 95% confidence interval 0.10 - 0.92, P = .03). CONCLUSION: Disparity exists in the delivery of continent urinary diversions for Hispanic patients undergoing radical cystectomy for bladder cancer. Further investigation is needed to determine the potential causes for this disparity in care delivered.