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Advances in the understanding of the tumor microenvironment have led to development of immunotherapeutic strategies, such as chimeric antigen receptor T cells (CAR-Ts). However, despite success in blood malignancies, CAR-T therapies in solid tumors have been hampered by their restricted infiltration. Here, we used our understanding of early cytotoxic lymphocyte infiltration of human lymphocytes in solid tumors in vivo to investigate the receptors in normal, adjacent, and tumor tissues of primary non-small-cell lung cancer specimens. We found that CX3CL1-CX3CR1 reduction restricts cytotoxic cells from the solid-tumor bed, contributing to tumor escape. Based on this, we designed a CAR-T construct using the well-established natural killer group 2, member D (NKG2D) CAR-T expression together with overexpression of CX3CR1 to promote their infiltration. These CAR-Ts infiltrate tumors at higher rates than control-activated T cells or IL-15-overexpressing NKG2D CAR-Ts. This construct also had similar functionality in a liver-cancer model, demonstrating potential efficacy in other solid malignancies.

We performed multiplex immunofluorescence (mIF) using an array of myeloid and lymphoid markers on primary tumor samples from 122 patients with RCC. Regions of interest (ROIs) were selected from three distinct tumor zones: the tumor-core, stroma, and tumor-stroma interface. Digital pathologic imaging analysis was leveraged to quantify the geospatial location and distribution of immune cells within the TIME, and these findings were correlated with a variety of tumor molecular profiles. For patients with ccRCC, as clinical stage increased, immunosuppressive M2-like CD163+ TAMs migrated from the tumor compartment and into the stroma at the tumor-stroma interface and became increasingly co-localized with CD8+ T-cells. Furthermore, high CD163+ TAM clustering into the stromal compartment and high CD8+/CD163+ stromal co-localization were independently associated with worse OS and cancer-specific survival (CSS), while accounting for clinical stage. Overall, this data suggests that the pro-tumor effect of M2-like TAMs and their effect on CD8+ T-cells may be dependent on their specific geospatial location and distribution within the TIME -- namely, that M2-like TAMs exert their pro-tumor effect and interact with CD8+ T-cells most effectively from the stromal compartment at the tumor-stroma interface.

Background Clear cell renal cell carcinoma (ccRCC) tumors have low frequencies of genetic alterations compared with other malignancies, but very high levels of immune cell infiltration and favorable response rates to immunotherapy. Currently, the interplay between specific ccRCC somatic mutations and immune infiltration pattern is unclear. Objective To analyze the associations between common ccRCC somatic mutations and immune cell infiltration patterns within the tumor immune microenvironment (TIME). Design, setting, and participants The study included tumor samples (24 primary and 24 metastatic) from 48 patients with stage IV ccRCC. Targeted sequencing was performed for well-characterized recurrent somatic mutations in ccRCC, with the analysis focusing on the six most common ones: VHL, BAP1, PBRM1, SETD2, TP53, and KDM5C. For each sample, multiplex immunofluorescence (IF) was performed in lymphoid and myeloid panels, for seven regions of interest in three zones (tumor core, stroma, and tumor-stroma interface). IF-derived cellular densities were compared across patients, stratified by their somatic mutation status, using a linear mixed-model analysis. External validation was pursued using RNA-seq enrichment scoring from three large external data sources. Results and limitations Tumors with SETD2 mutations demonstrated significantly decreased levels of FOXP3+ T cells in the tumor core, stroma, and tumor-stroma interface. PBRM1 mutations were associated with decreased FOXP3+ T cells in the tumor core. Primary KDM5C mutations were associated with significantly increased CD206+ macrophage tumor infiltration in the tumor core. A computational method estimating immune cell types in the TIME using bulk RNA-seq data, xCell scoring, failed to validate associations from the IF analysis in large external data sets. A major limitation of the study is the relatively small patient population studied. Conclusions This study provides evidence that common somatic mutations in ccRCC, such as SETD2, PBRM1, and KDM5C, are associated with distinct immune infiltration patterns within the TIME. Patient summary In this study, we analyzed tumor samples from patients with metastatic kidney cancer to determine whether common genetic mutations that arise from the cancer cells are associated with the density of immune cells found within those tumors. We found several distinct immune cell patterns that were associated with specific genetic mutations. These findings provide insight into the interaction between cancer genetics and the immune system in kidney cancer.

Immune infiltration is typically quantified using cellular density, not accounting for cellular clustering. Tumor-associated macrophages (TAM) activate oncogenic signaling through paracrine interactions with tumor cells, which may be better reflected by local cellular clustering than global density metrics. Using multiplex immunohistochemistry and digital pathologic analysis we quantified cellular density and cellular clustering for myeloid cell markers in 129 regions of interest from 55 samples from 35 patients with metastatic ccRCC. CD68+ cells were found to be clustered with tumor cells and dispersed from stromal cells, while CD163+ and CD206+ cells were found to be clustered with stromal cells and dispersed from tumor cells. CD68+ density was not associated with OS, while high tumor/CD68+ cell clustering was associated with significantly worse OS. These novel findings would not have been identified if immune infiltrate was assessed using cellular density alone, highlighting the importance of including spatial analysis in studies of immune cell infiltration of tumors.SIGNIFICANCEIncreased clustering of CD68+ TAMs and tumor cells was associated with worse overall survival for patients with metastatic ccRCC. This effect would not have been identified if immune infiltrate was assessed using cell density alone, highlighting the importance of including spatial analysis in studies of immune cell infiltration of tumors.