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Background: Bladder tumor-infiltrating CD56 bright NK cells are more tumor cytotoxic than their CD56 dim counterparts. Identification of NK cell subsets is labor-intensive and has limited utility in the clinical setting. Here, we sought to identify a surrogate marker of bladder CD56 bright NK cells and to test its prognostic significance., Methods: CD56 bright and CD56 dim NK cells were characterized with the multiparametric flow (n = 20) and mass cytometry (n = 21) in human bladder tumors. Transcriptome data from bladder tumors (n = 351) profiled by The Cancer Genome Atlas (TCGA) were analyzed. The expression levels of individual markers in intratumoral CD56 bright and CD56 dim NK cells were visualized in tSNE plots. Expressions of activation markers were also compared between Killer Cell Lectin-Like Receptor Subfamily F Member 1 (KLRF1) + and KLRF1 - NK cells., Results: Intratumoral CD56 bright NK cells displayed a more activated phenotype compared to the CD56 dim subset. Multiple intratumoral cell types expressed CD56, including bladder tumor cells and nonspecific intratumoral CD56 expression was associated with worse patient survival. Thus, an alternative to CD56 as a marker of CD56 bright NK cells was sought. The activation receptor KLRF1 was significantly increased on CD56 bright but not on CD56 dim NK cells. Intratumoral KLRF1 + NK cells were more activated and expressed higher levels of activation molecules compared with KLRF1 - NK cells, analogous to the distinct effector function of NK cells across CD56 expression. High intratumoral KLRF1 was associated with improved recurrence-free survival (hazard ratio [HR] 0.53, p = 0.01), cancer-specific survival (HR 0.47, p = 0.02), and overall survival (HR 0.54, p = 0.02) on multivariable analyses that adjusted for clinical and pathologic variables., Conclusions: KLRF1 is a promising prognostic marker in bladder cancer and may guide treatment decisions upon validation., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Purpose: Repeated instillations of bacillus Calmette et Guérin (BCG) are the gold standard immunotherapeutic treatment for reducing recurrence for patients with high-grade papillary non-muscle invasive bladder cancer (NMIBC) and for eradicating bladder carcinoma-in situ. Unfortunately, some patients are unable to tolerate BCG due to treatment-associated toxicity and bladder removal is sometimes performed for BCG-intolerance. Prior studies suggest that selectively delipidated BCG (dBCG) improves tolerability of intrapulmonary delivery reducing tissue damage and increasing efficacy in preventing Mycobacterium tuberculosis infection in mice. To address the lack of treatment options for NMIBC with BCG-intolerance, we examined if selective delipidation would compromise BCG's antitumor efficacy and at the same time increase tolerability to the treatment., Materials and Methods: Murine syngeneic MB49 bladder cancer models and in vitro human innate effector cell cytotoxicity assays were used to evaluate efficacy and immune impact of selective delipidation in Tokyo and TICE BCG strains., Results: Both dBCG-Tokyo and dBCG-TICE effectively treated subcutaneous MB49 tumors in mice and enhanced tumor-infiltrating CD8 + T and natural killer cells, similar to conventional BCG. However, when compared to conventional BCG, only dBCG-Tokyo retained a significant effect on intratumoral tumor-specific CD8 + and γδ T cells by increasing their frequencies in tumor tissue and their production of antitumoral function-related cytokines, i.e., IFN-γ and granzyme B. Further, dBCG-Tokyo but not dBCG-TICE enhanced the function and cytotoxicity of innate effector cells against human bladder cancer T24 in vitro., Conclusions: These data support clinical investigation of dBCG-Tokyo as a treatment for patients with BCG-intolerant NMIBC., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (©2021 American Association for Cancer Research.)

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., Competing Interests: Ethics approval and consent to participate: All animal studies were approved by the UT Health San Antonio Institutional Animal Care and Use Committee and each experiment was conducted in accordance with the standards required by the UT Health San Antonio Department of Laboratory Animal Resources., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Purpose: Human innate lymphoid cells (hILCs) are lineage-negative immune cells that do not express rearranged adaptive antigen receptors. Natural killer (NK) cells are hILCs that contribute to cancer defense. The role of non-NK hILCs in cancer is unclear. Our study aimed to characterize non-NK hILCs in bladder cancer., Experimental Design: Mass cytometry was used to characterize intratumoral non-NK hILCs based on 35 parameters, including receptors, cytokines, and transcription factors from 21 muscle-invasive bladder tumors. Model-based clustering was performed on t-distributed stochastic neighbor embedding (t-SNE) coordinates of hILCs, and the association of hILCs with tumor stage was analyzed., Results: Most frequent among intratumoral non-NK hILCs were hILC1s, which were increased in higher compared with lower stage tumors. Intratumoral hILC1s were marked by Th17-like phenotype with high RORγt, IL-17, and IL-22 compared to Th1 differentiation markers, including Tbet, perforin, and IFN-γ. Compared with intratumoral hILC2s and hILC3s, hILC1s also had lower expression of activation markers (NKp30, NKp46, and CD69) and increased expression of exhaustion molecules (PD-1 and Tim3). Unsupervised clustering identified nine clusters of bladder hILCs, which were not defined by the primary hILC subtypes 1-3. hILC1s featured in all the nine clusters indicating that intratumoral hILC1s displayed the highest phenotypic heterogeneity among all hILCs., Conclusions: hILC1s are increased in higher stage tumors among patients with muscle-invasive bladder cancer. These intratumoral hILC1s exhibit an exhausted phenotype and Th17-like differentiation, identifying them as potential targets for immunotherapy., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Anti-programmed death-ligand 1 (αPD-L1) immunotherapy is approved to treat bladder cancer (BC) but is effective in <30% of patients. Interleukin (IL)-2/αIL-2 complexes (IL-2c) that preferentially target IL-2 receptor β (CD122) augment CD8 + 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., Methods: We 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., Results: IL-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., Conclusions: Mechanistic 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., Competing Interests: Competing interests: OB holds a patent on IL-2c., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: Competing interests: The studies related to this manuscript were conducted abided by research ethics and approved by IRB guidelines for clinical sample use. All authors have consent for publishing the data from the studies. RS discloses other roles as Consultant for FerGene and Clinical Research Support for JBL(SWOG), FKD and Decipher Biosciences. All other authors disclose no conflict of interest., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The mechanistic target of rapamycin (mTOR) integrates environmental inputs to regulate cellular growth and metabolism in tumors. However, mTOR also regulates T-cell differentiation and activation, rendering applications of mTOR inhibitors toward treating cancer complex. Preclinical data support distinct biphasic effects of rapamycin, with higher doses directly suppressing tumor cell growth and lower doses enhancing T-cell immunity. To address the translational relevance of these findings, the effects of the mTOR complex 1 (mTORC1) inhibitor, rapamycin, on tumor and T cells were monitored in patients undergoing cystectomy for bladder cancer. MB49 syngeneic murine bladder cancer models were tested to gain mechanistic insights. Surgery-induced T-cell exhaustion in humans and mice and was associated with increased pulmonary metastasis and decreased PD-L1 antibody efficacy in mouse bladder cancer. At 3 mg orally daily, rapamycin concentrations were 2-fold higher in bladder tissues than in blood. Rapamycin significantly inhibited tumor mTORC1, shown by decreased rpS6 phosphorylation in treated versus control patients ( P = 0.008). Rapamycin reduced surgery-induced T-cell exhaustion in patients, evidenced by a significant decrease in the prevalence of dysfunctional programmed death-1 (PD-1)-expressing T cells. Grade 3 to 4 adverse event rates were similar between groups, but rapamycin-treated patients had a higher rate of wound complications versus controls. In conclusion, surgery promoted bladder cancer metastasis and decreased the efficacy of postoperative bladder cancer immunotherapy. Low-dose (3 mg daily) oral rapamycin has favorable pharmacodynamic and immune modulating activity in surgical patients and has the potential to decrease surgery-induced immune dysfunction., (©2018 American Association for Cancer Research.)