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BackgroundAnti-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.MethodsWe 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.ResultsIL-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.ConclusionsMechanistic 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.

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 influencedThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

BRAF mutation was noted in 38 tumors (76%). Systemic treatment was administered in 23 patients (51%), including targeted therapy in 13 (56%) and immunotherapy in 3 (13%). Patients with BRAF-mutated tumors received systemic treatment in 22 cases (58%), including targeted therapy in 13 patients (59%). Intrathecal treatment was given in 23 patients (45%). Whole brain radiotherapy was used in 16 patients (31%), 33 patients (65%) had no brain radiotherapy. The combination of systemic and intrathecal treatment (n¼13, 25%) was the most commonly used combination. No specific treatment was given to 11 patients (22%). Median OS for the whole cohort was 1.7 months (IQR: 0.9-5.2). Median OS was 1.5 months (0.8-4.3) in type I LM versus 2.2 months (1.5-8.1) in type II LM; 1.9 months (IQR: 0.9-4.5 months) in BRAF mutated tumors versus 4.6 (IQR:1.4-8.6) in the BRAF non-mutated tumors; and 1.4 (IQR: 0.5-1.6) without versus 2.9 months (IQR: 1-6) with treatment. Conclusions:The prognosis of LM in melanoma patients remains poor despite novel systemic treatment options. New therapeutic approaches are urgently needed in this population.

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Abstract28-day-old weanling rats were fed a diet containing 3% casein as the only source of protein for eight weeks to induce protein deficiency. When compared to control animals (fed a diet containing 25% casein), these rats had significantly lowered body (5.2-fold reduction) and liver (2.5-fold reduction) weights. The circulatory level of retinol (nmoi per ml plasma) as well as retinol (nmol per g tissue) in the liver of these protein-deficient animals were also reduced significantly, although their liver concentration of retinyl palmitate (nmol per g tissue) was comparable to that of the control group. Assay of liver tissue for retinyl palmitate hydrolase activity revealed a 4-fold reduction (compared to that of control animals) of specific enzyme activity (nmol retinol formed per g protein per h). These findings suggest that severe protein deficiency results in a decreased hydrolysis of retinyl esters in the liver, which may be in part responsible for the reduced level of metabolically 'active' retinoids available for normal physiological functions.

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.

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