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PD-1/PD-L1 blockade can promote robust tumor regression yet secondary resistance often occurs as immune selective pressure drives outgrowth of resistant tumor clones. Here using a genome-wide CRISPR screen in B16.SIY melanoma cells, we confirm Ifngr2 and Jak1 as important genes conferring sensitivity to T cell-mediated killing in vitro. However, when implanted into mice, these Ifngr2- and Jak1- deficient tumors paradoxically are better controlled immunologically. This phenotype maps to defective PD-L1 upregulation on mutant tumor cells, which improves anti-tumor efficacy of CD8 + T cells. To reconcile these observations with clinical reports of anti-PD-1 resistance linked to emergence of IFN-γ signaling mutants, we show that when mixed with wild-type tumor cells, IFN-γ-insensitive tumor cells indeed grow out, which depends upon PD-L1 expression by wild-type cells. Our results illustrate the complexity of functions for IFN-γ in anti-tumor immunity and demonstrate that intratumor heterogeneity and clonal cooperation can contribute to immunotherapy resistance. Melanoma is a cancer that responds relatively well to immunotherapy but resistance does ensue. Here, the authors show that loss of IFNGR2 or JAK1 in a melanoma cell line enhances T cell mediated lysis, however these cells are paradoxically more sensitive to immune-mediated tumor control in vivo due to the loss of PD-L1.

BackgroundPatients with cancer were excluded from phase 3 COVID-19 vaccine trials, and the immunogenicity and side effect profiles of these vaccines in this population is not well understood. Patients with cancer can be immunocompromised from chemotherapy, corticosteroids, or the cancer itself, which may affect cellular and/or humoral responses to vaccination. PD-1 is expressed on T effector cells, T follicular helper cells and B cells, leading us to hypothesize that anti-PD-1 immunotherapies may augment antibody or T cell generation after vaccination.MethodsAntibodies to the SARS-CoV-2 receptor binding domain (RBD) and spike protein were assessed in patients with cancer (n=118) and healthy donors (HD, n=22) after 1, 2 or 3 mRNA vaccine doses. CD4+ and CD8+ T cell reactivity to wild-type (WT) or B.1.617.2 (delta) spike peptides was measured by intracellular cytokine staining.ResultsOncology patients without prior COVID-19 infections receiving immunotherapy (n=36), chemotherapy (n=15), chemoimmunotherapy (n=6), endocrine or targeted therapies (n=6) and those not on active treatment (n=26) had similar RBD and Spike IgG antibody titers to HDs after two vaccinations. Contrary to our hypothesis, PD-1 blockade did not augment antibody titers or T cell responses. Patients receiving B-cell directed therapies (n=14) including anti-CD20 antibodies and multiple myeloma therapies had decreased antibody titers, and 9/14 of these patients were seronegative for RBD antibodies. No differences were observed in WT spike-reactive CD4+ and CD8+ T cell generation between treatment groups. 11/13 evaluable patients seronegative for RBD had a detectable WT spike-reactive CD4+ T cell response. T cells cross-reactive against the B.1.617.2 variant spike peptides were detected in 31/59 participants. Two patients with prior immune checkpoint inhibitor-related adrenal insufficiency had symptomatic hypoadrenalism after vaccination.ConclusionsCOVID-19 vaccinations are safe and immunogenic in patients with solid tumors, who developed similar antibody and T cell responses compared with HDs. Patients on B-cell directed therapies may fail to generate RBD antibodies after vaccination and should be considered for prophylactic antibody treatments. Many seronegative patients do develop a T cell response, which may have an anti-viral effect. Patients with pre-existing adrenal insufficiency may need to take stress dose steroids during vaccination to avoid adrenal crisis.

Checkpoint blockade immunotherapy (CBI) is a cornerstone of modern cancer treatment, but efficacy is not universal, partly due to a wide variation in baseline immune cell infiltration in the tumor microenvironment (TME). One mechanism contributing to this variability is tumor cell-intrinsic activation of the β-catenin signaling pathway, which has been shown to drive a non-T cell-inflamed TME leading to a loss of therapeutic efficacy of a range of immunotherapies. There is great interest in drugging the Wnt/β-catenin pathway in an effort to restore immune interactions in β-catenin-active tumors. As a proof-of-concept, we reasoned that a genetic experiment eliminating β-catenin expression after establishment of a non-T cell-inflamed TME would illustrate the maximal biologic effect that could be expected with total blockade of the pathway. We developed a genetically engineered mouse (GEM) model that allows for dynamic regulation of β-catenin on and off via doxycycline. We observed that administration of doxycycline resulted in robust nuclear accumulation of melanocyte-specific β-catenin which was accompanied by a reduction in CD3+ T cell infiltration. Discontinuing doxycycline treatment led to loss of nuclear β-catenin expression, associated with a substantial return of CD3+ T cells into the TME. However, despite the re-infiltration of CD3+ T cells, the tumors previously expressing β-catenin did not regain therapeutic responsiveness to anti-CTLA-4 plus anti-PD-L1 therapy. Single cell RNA sequencing of the ex-β-catenin tumors indicated the enrichment of an immunosuppressive-like macrophage population, characterized by the expression of Ccl8, Gas6, Cd163 and Cd209g Moreover, spatial transcriptomic analysis demonstrated that CD3+ T cells are in close contact with these M2-like macrophages, likely resulting in their inhibition. This finding suggests that the prior presence of β-catenin may induce long-lasting changes in the TME that continue to suppress the immune response even after β-catenin levels are reduced, having important therapeutic implications.

Inclusion of a tag in CARs facilitates cell manipulation and downstream research studies. Adoptive immunotherapy with genetically engineered T cells has the potential to treat cancer and other diseases. The introduction of Strep -tag II sequences into specific sites in synthetic chimeric antigen receptors or natural T-cell receptors of diverse specificities provides engineered T cells with a marker for identification and rapid purification, a method for tailoring spacer length of chimeric receptors for optimal function, and a functional element for selective antibody-coated, microbead-driven, large-scale expansion. These receptor designs facilitate cGMP manufacturing of pure populations of engineered T cells for adoptive T-cell therapies and enable in vivo tracking and retrieval of transferred cells for downstream research applications.