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Background: Primary and secondary resistance is a major hurdle in cancer immunotherapy. Therefore, a better understanding of the underlying mechanisms involved in immunotherapy resistance is of pivotal importance to improve therapy outcome., Method: Here, two mouse models with resistance against therapeutic vaccine-induced tumor regression were studied. Exploration of the tumor microenvironment by high dimensional flow cytometry in combination with therapeutic in vivo settings allowed for the identification of immunological factors driving immunotherapy resistance., Results: Comparison of the tumor immune infiltrate during early and late regression revealed a change from tumor-rejecting toward tumor-promoting macrophages. In concert, a rapid exhaustion of tumor-infiltrating T cells was observed. Perturbation studies identified a small but discernible CD163 hi macrophage population, with high expression of several tumor-promoting macrophage markers and a functional anti-inflammatory transcriptome profile, but not other macrophages, to be responsible. In-depth analyses revealed that they localize at the tumor invasive margins and are more resistant to Csf1r inhibition when compared with other macrophages. In vivo studies validated the activity of heme oxygenase-1 as an underlying mechanism of immunotherapy resistance. The transcriptomic profile of CD163 hi macrophages is highly similar to a human monocyte/macrophage population, indicating that they represent a target to improve immunotherapy efficacy., Conclusions: In this study, a small population of CD163 hi tissue-resident macrophages is identified to be responsible for primary and secondary resistance against T-cell-based immunotherapies. While these CD163 hi M2 macrophages are resistant to Csf1r-targeted therapies, in-depth characterization and identification of the underlying mechanisms driving immunotherapy resistance allows the specific targeting of this subset of macrophages, thereby creating new opportunities for therapeutic intervention with the aim to overcome immunotherapy resistance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre-existing tumor-specific T cells. Here, the blueprint of the TIME associated with such an ongoing tumor-specific T-cell response was dissected in a unique prospective oropharyngeal squamous cell carcinoma (OPSCC) cohort, in which tumor-specific tumor-infiltrating T cells were detected (immune responsiveness (IR + )) or not (lack of immune responsiveness (IR - ))., Methods: A comprehensive multimodal, high-dimensional strategy was applied to dissect the TIME of treatment-naive IR + and IR - OPSCC tissue, including bulk RNA sequencing (NanoString), imaging mass cytometry (Hyperion) for phenotyping and spatial interaction analyses of immune cells, and combined single-cell gene expression profiling and T-cell receptor (TCR) sequencing (single-cell RNA sequencing (scRNAseq)) to characterize the transcriptional states of clonally expanded tumor-infiltrating T cells., Results: IR + patients had an excellent survival during >10 years follow-up. The tumors of IR + patients expressed higher levels of genes strongly related to interferon gamma signaling, T-cell activation, TCR signaling, and mononuclear cell differentiation, as well as genes involved in several immune signaling pathways, than IR - patients. The top differently overexpressed genes included CXCL12 and LTB, involved in ectopic lymphoid structure development. Moreover, scRNAseq not only revealed that CD4 + T cells were the main producers of LTB but also identified a subset of clonally expanded CD8 + T cells, dominantly present in IR + tumors, which secreted the T cell and dendritic cell (DC) attracting chemokine CCL4. Indeed, immune cell infiltration in IR + tumors is stronger, highly coordinated, and has a distinct spatial phenotypical signature characterized by intratumoral microaggregates of CD8 + CD103 + and CD4 + T cells with DCs. In contrast, the IR - TIME comprised spatial interactions between lymphocytes and various immunosuppressive myeloid cell populations. The impact of these chemokines on local immunity and clinical outcome was confirmed in an independent The Cancer Genome Atlas OPSCC cohort., Conclusion: The production of lymphoid cell attracting and organizing chemokines by tumor-specific T cells in IR + tumors constitutes a positive feedback loop to sustain the formation of the DC-T-cell microaggregates and identifies patients with excellent survival after standard therapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: A profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint molecule expression was performed in early-stage and late-stage VSCC., Methods: The type, location, and infiltration pattern of T cells were studied in 109 patients with primary VSCC FIGO stage I-III. RNA expression of genes involved in immune oncology and oncogenic signaling pathways was analyzed in 40 VSCC, matched for prognostic clinicopathological variables, analyzed for HPV and p53 status, and selected based on T cell infiltration., Results: High intraepithelial infiltration with CD4 or CD8 T cells was associated with longer overall and recurrence-free survival and formed an independent prognostic factor, outperforming molecular subtype and stage of the disease. Strong T cell infiltrated VSCC displayed a coordinated immune response reflected by a positive association between T cells and different lymphocyte and myeloid cell subsets. The expression of genes involved in the migration of T cells and myeloid cells, T cell activation and costimulation, interferon (IFN)-γ signaling, cytotoxicity and apoptosis was higher than in low infiltrated tumors. An active immune signaling profile was observed in all inflamed, part of the altered-excluded and not in altered-immunosuppressed or deserted VSCC. While several checkpoint molecules were overexpressed, only PD-L1 expression displayed discriminatory ability and clinical usefulness. High PD-L1 expression was detected in all inflamed and ~60% of the altered-excluded VSCC., Conclusion: An active immune signaling profile is present in 35% of primary FIGO I-III VSCCs, suggesting potential responsiveness to neoadjuvant PD-1/PD-L1 immunotherapy., Competing Interests: Competing interests: The authors (KEK, SJS, MIEvP and SHvdB) have filed a patent relating to the treatment of vulvar squamous cell carcinoma (VSCC) and methods for identifying subjects with VSCC who are likely to benefit from such therapy., (© 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.)

Purpose: Human papillomavirus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) has a much better prognosis than HPV-negative OPSCC, and this is linked to dense tumor immune infiltration. As the viral antigens may trigger potent immunity, we studied the relationship between the presence of intratumoral HPV-specific T-cell responses, the immune contexture in the tumor microenvironment, and clinical outcome. Experimental Design: To this purpose, an in-depth analysis of tumor-infiltrating immune cells in a prospective cohort of 97 patients with HPV16-positive and HPV16-negative OPSCC was performed using functional T-cell assays, mass cytometry (CyTOF), flow cytometry, and fluorescent immunostaining of tumor tissues. Key findings were validated in a cohort of 75 patients with HPV16-positive OPSCC present in the publicly available The Cancer Genome Atlas database. Results: In 64% of the HPV16-positive tumors, type I HPV16-specific T cells were present. Their presence was not only strongly related to a better overall survival, a smaller tumor size, and less lymph node metastases but also to a type I-oriented tumor microenvironment, including high numbers of activated CD161 + T cells, CD103 + tissue-resident T cells, dendritic cells (DC), and DC-like macrophages. Conclusions: The viral antigens trigger a tumor-specific T-cell response that shapes a favorable immune contexture for the response to standard therapy. Hence, reinforcement of HPV16-specific T-cell reactivity is expected to boost this process. Clin Cancer Res; 24(3); 634-47. ©2017 AACR See related commentary by Laban and Hoffmann, p. 505 ., (©2017 American Association for Cancer Research.)

Background & Aims: Colorectal cancer is a complex disease involving immune defense mechanisms within the tumor. Herein, we used data integration and biomolecular network reconstruction to generate hypotheses about the mechanisms underlying immune responses in colorectal cancer that are relevant to tumor recurrence., Methods: Mechanistic hypotheses were formulated on the basis of data from 108 patients and tested using different assays (gene expression, phenome mapping, tissue-microarrays, T-cell receptor [TCR] repertoire)., Results: This integrative approach revealed that chemoattraction and adhesion play important roles in determining the density of intratumoral immune cells. The presence of specific chemokines (CX3CL1, CXCL10, CXCL9) and adhesion molecules (ICAM1, VCAM1, MADCAM1) correlated with different subsets of immune cells and with high densities of T-cell subpopulations within specific tumor regions. High expression of these molecules correlated with prolonged disease-free survival. Moreover, the expression of certain chemokines associated with particular TCR repertoire and specific TCR use predicted patient survival., Conclusions: Data integration and biomolecular network reconstruction is a powerful approach to uncover molecular mechanisms. This study shows the utility of this approach for the investigation of malignant tumors and other diseases. In colorectal cancer, the expression of specific chemokines and adhesion molecules were found as being critical for high densities of T-cell subsets within the tumor and associated with particular TCR repertoire. Intratumoral-specific TCR use correlated with the prognosis of the patients., (2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)