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Although elevated plasma interleukin-8 (pIL-8) has been associated with poor outcome to immune checkpoint blockade 1, this has not been comprehensively evaluated in large randomized studies. Here we analyzed circulating pIL-8 and IL8 gene expression in peripheral blood mononuclear cells and tumors of patients treated with atezolizumab (anti-PD-L1 monoclonal antibody) from multiple randomized trials representing 1,445 patients with metastatic urothelial carcinoma (mUC) and metastatic renal cell carcinoma. High levels of IL-8 in plasma, peripheral blood mononuclear cells and tumors were associated with decreased efficacy of atezolizumab in patients with mUC and metastatic renal cell carcinoma, even in tumors that were classically CD8+ T cell inflamed. Low baseline pIL-8 in patients with mUC was associated with increased response to atezolizumab and chemotherapy. Patients with mUC who experienced on-treatment decreases in pIL-8 exhibited improved overall survival when treated with atezolizumab but not with chemotherapy. Single-cell RNA sequencing of the immune compartment showed that IL8 is primarily expressed in circulating and intratumoral myeloid cells and that high IL8 expression is associated with downregulation of the antigen-presentation machinery. Therapies that can reverse the impacts of IL-8-mediated myeloid inflammation will be essential for improving outcomes of patients treated with immune checkpoint inhibitors.

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFβ and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15+ CAFs in human patients was confirmed in >80,000 single cells from 22 patients with PDAC as well as by using IHC on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising more than 600 patients across six cancer types revealed elevated levels of the LRRC15+ CAF signature correlated with poor response to anti–PD-L1 therapy. This work has important implications for targeting nonimmune elements of the tumor microenvironment to boost responses of patients with cancer to immune checkpoint blockade therapy. Significance: This study describes the single-cell landscape of CAFs in pancreatic cancer during in vivo tumor evolution. A TGFβ-driven, LRRC15+ CAF lineage is associated with poor outcome in immunotherapy trial data comprising multiple solid-tumor entities and represents a target for combinatorial therapy. This article is highlighted in the In This Issue feature, p. 161

Close proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what controls the spatial distribution of T cells in the tumour microenvironment is not well understood. Here we couple digital pathology and transcriptome analysis on a large ovarian tumour cohort and develop a machine learning approach to molecularly classify and characterize tumour-immune phenotypes. Our study identifies two important hallmarks characterizing T cell excluded tumours: 1) loss of antigen presentation on tumour cells and 2) upregulation of TGFβ and activated stroma. Furthermore, we identify TGFβ as an important mediator of T cell exclusion. TGFβ reduces MHC-I expression in ovarian cancer cells in vitro. TGFβ also activates fibroblasts and induces extracellular matrix production as a potential physical barrier to hinder T cell infiltration. Our findings indicate that targeting TGFβ might be a promising strategy to overcome T cell exclusion and improve clinical benefits of cancer immunotherapy., The exclusion of T cells from solid tumours is a potentially important mechanism that regulates whether or not cancer patients respond well to checkpoint blocking immunotherapies. Here the authors identify immune phenotypes and mediators of T cell exclusion among ovarian cancer patient samples from the ICON7 phase III trial.

Upon encountering their cognate antigens, T cells can undergo clonal expansion to produce multiple copies of a cell with a shared T cell receptor (TCR). Despite the fundamental role of clonal expansion in cancer immunity, little is known about its relationship with T cell subpopulations or antitumor responses in cancer patients. Here we have performed single-cell RNA sequencing (scRNA-seq) and deep analysis of TCR clonotypes (scTCR-seq) in cancer patients across several indications, assessing the profiles of TCRs in the various populations of T cells in tumors, normal adjacent tissue (NAT), and peripheral blood. We found that, although most clonotypes were represented by a single cell, the remaining clonal lineages showed expansion in either NAT or tumor exclusively, or dual-residence with expansion in both compartments. In a subset of patients, we find clear evidence of clonotypic expansion of T effector- and effector memory-like cells not only within the tumor but also in NAT. Importantly, expanded clonotypes found in the tumor and NAT can also typically be detected in peripheral blood, suggesting a continuously replenishment from sites outside of the tumor with fresh, non-exhausted replacement cells. Our data further suggests a continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response. Citation Format: Shravan Madireddi, Thomas D. Wu, Patricia E. de Almeida, Romain Banchereau, Ying-Jiun J. Chen, Avantika S. Chitre, Hina Iftikhar, William E. O'Gorman, Amelia Au-Yeung, Chikara Takahashi, Leonard D. Goldstein, Chungkee Poon, Shilpa Keerthivasan, Sanjeev Mariathasan, Meghna Das Thakur, Mahrukh A. Huseni, Marcus Ballinger, Ivette Estay, Patrick Caplazi, Zora Modrusan, Lélia Delamarre, Ira Mellman, Richard Bourgon, Jane L. Grogan. Patterns of T cell clonal expansion in cancer patients associate with response to immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO084.

Elevated plasma interleukin-8 (IL-8) is a poor prognostic factor for many cancers. However, the association of IL-8 with clinical outcomes to checkpoint inhibition has not been comprehensively evaluated in randomized studies. Moreover, the source of IL-8 and the underlying biology that influences resistance to immune checkpoint inhibitors remain unknown. Here we analyzed circulating IL-8 protein in plasma, and IL8 gene expression in peripheral blood mononuclear cells (PBMC) and tumors of patients treated with atezolizumab (anti-PD-L1 mAb), from multiple randomized trials in metastatic urothelial carcinoma (mUC) and metastatic renal cell carcinoma (mRCC). High levels of IL-8 in plasma, PBMCs and tumors, were associated with decreased efficacy in mUC and mRCC patients treated with atezolizumab, even in tumors that were classically CD8+ T cell inflamed. mUC patients treated with atezolizumab, but not with chemotherapy, who experienced an on-treatment decrease in plasma IL-8, exhibited improved overall survival. IL-8 is primarily expressed in circulating and intratumoral myeloid cells, and high IL8 expression was associated with the downregulation of the antigen presentation machinery in myeloid cells. A better understanding of IL-8-mediated myeloid inflammation in curtailing responses to checkpoint inhibitors is essential for developing new therapies for patients. Citation Format: Kobe Yuen, Lifen Liu, Congfen Li, Deepali Rishipathak, Patrick Williams, Edward Kadel, Hartmut Koeppen, Shravan Madireddi, Shilpa Keerthivasan, Ying-Jun Chen, Zora Modrusen, Romain Banchereau, Ning Leng, Priti Hegde, Mahrukh Huseni, Sanjeev Mariathasan. Systemic and tumor associated IL-8 correlates with resistance to PD-L1 blockade [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2000.

Background Close proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what determines the spatial distribution of T cells in the tumour microenvironment is not well understood. Coupling digital pathology and transcriptome analysis on large ovarian tumour cohorts, here we report classification and functionally dissection of tumour-immune contexture in human ovarian cancer. Methods CD8 IHC and RNAseq analysis were performed on 370 ovarian tumours from the ICON7 phase III clinical trial. Coupling digital pathology with transcriptome analysis, a random forest machine learning algorithm was developed and independently validated for classifying tumour-immune phenotypes in ovarian cancer. Anti-tumour activity of TGFβ blockade in combination with anti-PD-L1 was evaluated in an ovarian cancer mouse model. Results We show the identified tumour-immune phenotypes are of biological and clinical importance with interconnection to molecular subtypes and association with clinical outcome in ovarian cancer. Two important hallmarks of T cell exclusion were identified: 1) loss of antigen presentation on tumour cells and 2) upregulation of TGFβ and activated stroma. We identified TGFβ as a key mediator of T cell exclusion. TGFβ reduced MHC class I expression in ovarian cancer cells and induced extracellular matrix (ECM) production and immunosuppressive molecules in human primary fibroblasts. Finally, we demonstrated that combination of anti-TGFβ and anti-PD-L1 in a mouse ovarian cancer model significantly improved the anti-tumour efficacy and survival. Conclusions This study provided the first systematic and in-depth characterization of the molecular features and mechanisms underlying the tumour-immune phenotypes in ovarian cancer. We illuminated a multi-faceted role of TGFβ in mediating consequential crosstalk between tumour cells and cancer associated fibroblasts to shape the tumour-immune contexture. Our findings support that targeting the TGFβ pathway represents a promising therapeutic strategy to overcome T cell exclusion and optimize response to cancer immunotherapy. Legal entity responsible for the study The authors. Funding Genentech/Roche. Disclosure All authors have declared no conflicts of interest.

Th17 cells are known to play a critical role in adaptive immune responses to several important extracellular pathogens. Additionally, Th17 cells are implicated in the pathogenesis of several autoimmune and inflammatory disorders as well as in cancer. Therefore, it is essential to understand the mechanisms that regulate Th17 differentiation. Notch signaling is known to be important at several stages of T cell development and differentiation. In this study, we report that Notch1 is activated in both mouse and human in vitro-polarized Th17 cells and that blockade of Notch signaling significantly downregulates the production of Th17-associated cytokines, suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies, as well as chromatin immunoprecipitation, that IL-17 and retinoic acid-related orphan receptor γt are direct transcriptional targets of Notch signaling in Th17 cells. Finally, in vivo inhibition of Notch signaling reduced IL-17 production and Th17-mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Thus, this study highlights the importance of Notch signaling in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.

The density and type of lymphocytes that infiltrate colon tumors are predictive of the clinical outcome of colon cancer. High densities of T helper 17 (T H 17) cells and inflammation predict poor outcome, whereas infiltration by T regulatory cells (T regs ) that naturally suppress inflammation is associated with longer patient survival. However, the role of T regs in cancer remains controversial. We recently reported that T regs in colon cancer patients can become proinflammatory and tumor-promoting. These properties were directly linked with their expression of RORγt (retinoic acid–related orphan receptor-γt), the signature transcription factor of T H 17 cells. We report that Wnt/β-catenin signaling in T cells promotes expression of RORγt. Expression of β-catenin was elevated in T cells, including T regs , of patients with colon cancer. Genetically engineered activation of β-catenin in mouse T cells resulted in enhanced chromatin accessibility in the proximity of T cell factor-1 (Tcf-1) binding sites genome-wide, induced expression of T H 17 signature genes including RORγt, and promoted T H 17-mediated inflammation. Strikingly, the mice had inflammation of small intestine and colon and developed lesions indistinguishable from colitis-induced cancer. Activation of β-catenin only in T regs was sufficient to produce inflammation and initiate cancer. On the basis of these findings, we conclude that activation of Wnt/β-catenin signaling in effector T cells and/or T regs is causatively linked with the imprinting of proinflammatory properties and the promotion of colon cancer.

Graft versus Host disease (GVHD) is a major lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). As skin is one of the major organs involved and the most accessible, it serves as an ideal organ site to further understand the pathogenesis and progression of acute GVHD and could serve as a window into pathophysiology of other organ sites. Cytokine network aberrations are a central feature of the pathogenesis of acute cutaneous GVHD, terminating in the hallmark of premature apoptosis of keratinocytes. The purpose of this study is to delineate the cytokine network aberrations at the molecular and cellular level in affected skin from patients with acute GVHD with the goal of developing novel therapeutic approaches to defined targets with the goal of preventing or reducing acute GVHD. We performed immuno-phenotypic profiling of acute cutaneous GVHD biopsies determining relative numbers of various types of immunocytes including T cells, dendritic cells, and macrophages. Cytokine expression (IFNγ and TNFα) was determined by RT-PCR on RNA extracted from eight different lesional skin and two non-lesional biopsies. We analyzed for TH-1 (IFN γ and TNFα), TH-2 (IL-4) and TH-17 (IL-17A, IL-22) cytokine mRNAs. We observed that TH-1 cytokine mRNAs were detected in 7 out of 8 different lesional skin biopsies but none of two nonlesional skin biopsies. Interestingly TH-2 and TH-17 cytokine mRNAs were not detectable in acute cutaneous GVHD lesions or nonlesional skin biopsies. RNA extracted from a 2-way mixed lymphocyte reaction (MLR) served as a positive control for these RT-PCR results. Given recent data that suggested that Notch-1 regulated TH-1 polarization by transcriptionally controlling the Tbx gene (MinterLM et al 2005), we hypothesized that inhibiting Notch signaling might be of potential therapeutic importance given these results of TH-1 polarization in acute skin GVHD. To block Notch signaling we utilized several pharmacological gamma secretase inhibitors (which may have therapeutic applications), and supplemented this pan-Notch receptor inhibition approach by use of a Notch-1 specific siRNA. Preliminary studies suggest that both approaches reduce IFN γ levels in allogeneic T cell responses in-vitro. Further experiments are in progress to confirm these findings and to determine the molecular mechanism. This study begins to define the cytokine network operative within acute cutaneous GVHD lesions highlighting the finding that TH-1 rather than TH-17 cytokines may be the more important target.