Myrna Garcia

Abstract 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.

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.

The interaction between tumor surface-expressed PDL1 and immune cell PD1 for the evasion of antitumor immunity is well established and is targeted by FDA-approved anti-PDL1 and anti-PD1 antibodies. Nonetheless, recent studies highlight the immunopathogenicity of tumor-intrinsic PDL1 signals that can contribute to the resistance to targeted small molecules, cytotoxic chemotherapy, and αPD1 immunotherapy. As genetic PDL1 depletion is not currently clinically tractable, we screened FDA-approved drugs to identify those that significantly deplete tumor PDL1. Among the candidates, we identified the β-lactam cephalosporin antibiotic cefepime as a tumor PDL1-depleting drug (PDD) that increases tumor DNA damage and sensitivity to DNA-damaging agents in vitro in distinct aggressive mouse and human cancer lines, including glioblastoma multiforme, ovarian cancer, bladder cancer, and melanoma. Cefepime reduced tumor PDL1 post-translationally through ubiquitination, improved DNA-damaging-agent treatment efficacy in vivo in immune-deficient and -proficient mice, activated immunogenic tumor STING signals, and phenocopied specific genetic PDL1 depletion effects. The β-lactam ring and its antibiotic properties did not appear contributory to PDL1 depletion or to these treatment effects, and the related cephalosporin ceftazidime produced similar effects. Our findings highlight the rapidly translated potential for PDDs to inhibit tumor-intrinsic PDL1 signals and improve DNA-damaging agents and immunotherapy efficacy.