Cancer Cell-Intrinsic Expression of MHC Class II Regulates the Immune Microenvironment and Response to Anti-PD-1 Therapy in Lung Adenocarcinoma.

MHC class II (MHCII) expression is usually restricted to APC but can be expressed by cancer cells. We examined the effect of cancer cell-specific MHCII (csMHCII) expression in lung adenocarcinoma on T cell recruitment to tumors and response to anti-PD-1 therapy using two orthotopic immunocompetent murine models of non-small cell lung cancer: CMT167 (CMT) and Lewis lung carcinoma (LLC). We previously showed that CMT167 tumors are eradicated by anti-PD1 therapy, whereas LLC tumors are resistant. RNA sequencing analysis of cancer cells recovered from tumors revealed that csMHCII correlated with response to anti-PD1 therapy, with immunotherapy-sensitive CMT167 cells being csMHCII positive, whereas resistant LLC cells were csMHCII negative. To test the functional effects of csMHCII, MHCII expression was altered on the cancer cells through loss- and gain-of-function of CIITA, a master regulator of the MHCII pathway. Loss of CIITA in CMT167 decreased csMHCII and converted tumors from anti-PD-1 sensitive to anti-PD-1 resistant. This was associated with lower levels of Th1 cytokines, decreased T cell infiltration, increased B cell numbers, and decreased macrophage recruitment. Conversely, overexpression of CIITA in LLC cells resulted in csMHCII in vitro and in vivo. Enforced expression of CIITA increased T cell infiltration and sensitized tumors to anti-PD-1 therapy. csMHCII expression was also examined in a subset of surgically resected human lung adenocarcinomas by multispectral imaging, which provided a survival benefit and positively correlated with T cell infiltration. These studies demonstrate a functional role for csMHCII in regulating T cell infiltration and sensitivity to anti-PD-1.

A Beginner's Guide to Analyzing and Visualizing Mass Cytometry Data.

Mass cytometry has revolutionized the study of cellular and phenotypic diversity, significantly expanding the number of phenotypic and functional characteristics that can be measured at the single-cell level. This high-dimensional analysis platform has necessitated the development of new data analysis approaches. Many of these algorithms circumvent traditional approaches used in flow cytometric analysis, fundamentally changing the way these data are analyzed and interpreted. For the beginner, however, the large number of algorithms that have been developed, as well as the lack of consensus on best practices for analyzing these data, raise multiple questions: Which algorithm is the best for analyzing a dataset? How do different algorithms compare? How can one move beyond data visualization to gain new biological insights? In this article, we describe our experiences as recent adopters of mass cytometry. By analyzing a single dataset using five cytometry by time-of-flight analysis platforms (viSNE, SPADE, X-shift, PhenoGraph, and Citrus), we identify important considerations and challenges that users should be aware of when using these different methods and common and unique insights that can be revealed by these different methods. By providing annotated workflow and figures, these analyses present a practical guide for investigators analyzing high-dimensional datasets. In total, these analyses emphasize the benefits of integrating multiple cytometry by time-of-flight analysis algorithms to gain complementary insights into these high-dimensional datasets.

CD8+ T cells modulate autosomal dominant polycystic kidney disease progression.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent inherited nephropathy. To date, therapies alleviating the disease have largely focused on targeting abnormalities in renal epithelial cell signaling. ADPKD has many hallmarks of cancer, where targeting T cells has brought novel therapeutic interventions. However, little is known about the role and therapeutic potential of T cells in ADPKD. Here, we used an orthologous ADPKD model, Pkd1 p.R3277C (RC), to begin to define the role of T cells in disease progression. Using flow cytometry, we found progressive increases in renal CD8+ and CD4+ T cells, correlative with disease severity, but with selective activation of CD8+ T cells. By immunofluorescence, T cells specifically localized to cystic lesions and increased levels of T-cell recruiting chemokines (CXCL9/CXCL10) were detected by qPCR/in situ hybridization in the kidneys of mice, patients, and ADPKD epithelial cell lines. Importantly, immunodepletion of CD8+ T cells from one to three months in C57Bl/6 Pkd1RC/RC mice resulted in worsening of ADPKD pathology, decreased apoptosis, and increased proliferation compared to IgG-control, consistent with a reno-protective role of CD8+ T cells. Thus, our studies suggest a functional role for T cells, specifically CD8+ T cells, in ADPKD progression. Hence, targeting this pathway using immune-oncology agents may represent a novel therapeutic approach for ADPKD.

Cancer cell-intrinsic expression of MHC II in lung cancer cell lines is actively restricted by MEK/ERK signaling and epigenetic mechanisms.

BACKGROUND: Programmed death 1/programmed death ligand 1 (PD-1/PD-L1) targeted immunotherapy affords clinical benefit in ~20% of unselected patients with lung cancer. The factor(s) that determine whether a tumor responds or fails to respond to immunotherapy remains an active area of investigation. We have previously defined divergent responsiveness of two KRAS-mutant cell lines to PD-1/PD-L1 blockade using an orthotopic, immunocompetent mouse model. Responsiveness to PD-1/PD-L1 checkpoint blockade correlates with an interferon gamma (IFNγ)-inducible gene signature and major histocompatibility complex class II (MHC II) expression by cancer cells. In the current study, we aim to identify therapeutic targets that can be manipulated in order to enhance cancer-cell-specific MHC II expression. METHODS: Responsiveness to IFNγ and induction of MHC II expression was assessed after various treatment conditions in mouse and human non-small cell lung cancer (NSCLC) cell lines using mass cytometric and flow cytometric analysis. RESULTS: Single-cell analysis using mass and flow cytometry demonstrated that IFNγ consistently induced PD-L1 and MHC class I (MHC I) across multiple murine and human NSCLC cell lines. In contrast, MHC II showed highly variable induction following IFNγ treatment both between lines and within lines. In mouse models of NSCLC, MHC II induction was inversely correlated with basal levels of phosphorylated extracellular signal-regulated kinase (ERK) 1/2, suggesting potential mitogen-activated protein (MAP) kinase-dependent antagonism of MHC II expression. To test this, cell lines were subjected to varying levels of stimulation with IFNγ, and assessed for MHC II expression in the presence or absence of mitogen-activated protein kinase kinase (MEK) inhibitors. IFNγ treatment in the presence of MEK inhibitors significantly enhanced MHC II induction across multiple lung cancer lines, with minimal impact on expression of either PD-L1 or MHC I. Inhibition of histone deacetylases (HDACs) also enhanced MHC II expression to a more modest extent. Combined MEK and HDAC inhibition led to greater MHC II expression than either treatment alone. CONCLUSIONS: These studies emphasize the active inhibitory role that epigenetic and ERK signaling cascades have in restricting cancer cell-intrinsic MHC II expression in NSCLC, and suggest that combinatorial blockade of these pathways may engender new responsiveness to checkpoint therapies.

Activation of PPARγ in Myeloid Cells Promotes Progression of Epithelial Lung Tumors through TGFß1.

Lung cancer is a heterogeneous disease in which patient-specific treatments are desirable and the development of targeted therapies has been effective. Although mutations in KRAS are frequent in lung adenocarcinoma, there are currently no targeted agents against KRAS. Using a mouse lung adenocarcinoma cell line with a Kras mutation (CMT167), we previously showed that PPARγ activation in lung cancer cells inhibits cell growth in vitro yet promotes tumor progression when activated in myeloid cells of the tumor microenvironment. Here, we report that PPARγ activation in myeloid cells promotes the production of TGFß1, which, in turn, acts on CMT167 cancer cells to increase migration and induce an epithelial-mesenchymal transition (EMT). Targeting TGFß1 signaling in CMT167 cells prevented their growth and metastasis in vivo. Similarly, another mouse lung adenocarcinoma cell line with a Kras mutation, LLC, induced TGFß1 in myeloid cells through PPARγ activation. However, LLC cells are more mesenchymal and did not undergo EMT in response to TGFß1, nor did LLC require TGFß1 signaling for metastasis in vivo. Converting CMT167 cells to a mesenchymal phenotype through overexpression of ZEB1 made them unresponsive to TGFß1 receptor inhibition. The ability of TGFß1 to induce EMT in lung tumors may represent a critical process in cancer progression. We propose that TGFß receptor inhibition could provide an additional treatment option for KRAS-mutant epithelial lung tumors.Implications: This study suggests that TGFß receptor inhibitors may be an effective therapy in a subset of KRAS-mutant patients with non-small cell lung cancer, which show an epithelial phenotype.

Tumor-intrinsic response to IFNγ shapes the tumor microenvironment and anti-PD-1 response in NSCLC.

Targeting PD-1/PD-L1 is only effective in ∼20% of lung cancer patients, but determinants of this response are poorly defined. We previously observed differential responses of two murine K-Ras-mutant lung cancer cell lines to anti-PD-1 therapy: CMT167 tumors were eliminated, whereas Lewis Lung Carcinoma (LLC) tumors were resistant. The goal of this study was to define mechanism(s) mediating this difference. RNA sequencing analysis of cancer cells recovered from lung tumors revealed that CMT167 cells induced an IFNγ signature that was blunted in LLC cells. Silencing Ifngr1 in CMT167 resulted in tumors resistant to IFNγ and anti-PD-1 therapy. Conversely, LLC cells had high basal expression of SOCS1, an inhibitor of IFNγ. Silencing Socs1 increased response to IFNγ in vitro and sensitized tumors to anti-PD-1. This was associated with a reshaped tumor microenvironment, characterized by enhanced T cell infiltration and enrichment of PD-L1hi myeloid cells. These studies demonstrate that targeted enhancement of tumor-intrinsic IFNγ signaling can induce a cascade of changes associated with increased therapeutic vulnerability.

Complement Activation via a C3a Receptor Pathway Alters CD4+ T Lymphocytes and Mediates Lung Cancer Progression.

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3-/- mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5-dependent pathway.Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143-56. ©2017 AACR.