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Purpose: This study characterizes intratumoral macrophage populations within baseline melanoma biopsies from patients with advanced melanoma who received either anti-PD-1 monotherapy or a combination with anti-CTLA-4. Particularly, FcγRIIIa (CD16)-expressing macrophage densities were investigated for associations with response and progression-free survival. Experimental Design: Patients with advanced melanoma who received either anti-PD-1 monotherapy or combination anti-PD-1 and anti-CTLA-4 were retrospectively identified. Macrophage populations were analyzed within baseline melanoma biopsies via multiplex IHC in relation to treatment outcomes. Results: Patients who responded to combination immune checkpoint inhibitor contained higher CD16+ macrophage densities than those who did not respond (196 vs. 7 cells/mm2; P = 0.0041). There was no diffidence in CD16+ macrophage densities in the PD-1 monotherapy-treated patients based on response (118 vs. 89 cells/mm2; P = 0.29). A significantly longer 3-year progression-free survival was observed in combination-treated patients with high intratumoral densities of CD16+ macrophages compared with those with low densities (87% vs. 42%, P = 0.0056, n = 40). No association was observed in anti-PD-1 monotherapy-treated patients (50% vs. 47%, P = 0.4636, n = 50). Melanoma biopsies with high densities of CD16+ macrophages contained upregulated gene expression of critical T-cell recruiting chemokines (CXCL9, CXCL10, and CXCL11). Conclusions: Our data demonstrate that tumor microenvironments enriched with CD16+ macrophages are favorable for response to combination anti-PD-1 and anti-CTLA-4 therapy but not anti-PD-1 monotherapy. These data provides a potential biomarker of response for combination immunotherapies in patients with metastatic melanoma.

Cohort and dataset overview

Differential expression analysis gene list of FACS sorted CD16+ compared to CD16- macrophages from human melanoma dissociates.

Purpose:This study characterizes intratumoral macrophage populations within baseline melanoma biopsies from patients with advanced melanoma who received either anti-PD-1 monotherapy or a combination with anti-CTLA-4. Particularly, FcγRIIIa (CD16)-expressing macrophage densities were investigated for associations with response and progression-free survival.Experimental Design:Patients with advanced melanoma who received either anti-PD-1 monotherapy or combination anti-PD-1 and anti-CTLA-4 were retrospectively identified. Macrophage populations were analyzed within baseline melanoma biopsies via multiplex IHC in relation to treatment outcomes.Results:Patients who responded to combination immune checkpoint inhibitor contained higher CD16+ macrophage densities than those who did not respond (196 vs. 7 cells/mm2; P = 0.0041). There was no diffidence in CD16+ macrophage densities in the PD-1 monotherapy-treated patients based on response (118 vs. 89 cells/mm2; P = 0.29). A significantly longer 3-year progression-free survival was observed in combination-treated patients with high intratumoral densities of CD16+ macrophages compared with those with low densities (87% vs. 42%, P = 0.0056, n = 40). No association was observed in anti-PD-1 monotherapy-treated patients (50% vs. 47%, P = 0.4636, n = 50). Melanoma biopsies with high densities of CD16+ macrophages contained upregulated gene expression of critical T-cell recruiting chemokines (CXCL9, CXCL10, and CXCL11).Conclusions:Our data demonstrate that tumor microenvironments enriched with CD16+ macrophages are favorable for response to combination anti-PD-1 and anti-CTLA-4 therapy but not anti-PD-1 monotherapy. These data provides a potential biomarker of response for combination immunotherapies in patients with metastatic melanoma.

Background: Guidelines for pathological evaluation of neoadjuvant specimens and pathological response categories have been developed by the International Neoadjuvant Melanoma Consortium (INMC). As part of the Optimal Neo-adjuvant Combination Scheme of Ipilimumab and Nivolumab (OpACIN-neo) clinical trial of neoadjuvant combination anti-programmed cell death protein 1/anti-cytotoxic T-Iymphocyte-associated protein 4 immunotherapy for stage III melanoma, we sought to determine interobserver reproducibility of INMC histopathological assessment principles, identify specific tumour bed histopathological features of immunotherapeutic response that correlated with recurrence and relapse-free survival (RFS) and evaluate proposed INMC pathological response categories for predicting recurrence and RFS.Patients and methods: Clinicopathological characteristics of lymph node dissection specimens of 83 patients enrolled in the OpACIN-neo clinical trial were evaluated. Two methods of assessing histological features of immunotherapeutic response were evaluated: the previously described immune-related pathologic response (irPR) score and our novel immunotherapeutic response score (ITRS). For a subset of cases (n = 29), cellular composition of the tumour bed was analysed by flow cytometry.Results: There was strong interobserver reproducibility in assessment of pathological response (kappa = 0.879) and percentage residual viable melanoma (intraclass correlation coefficient = 0.965). The immunotherapeutic response subtype with high fibrosis had the strongest association with lack of recurrence (P = 0.008) and prolonged RFS (P = 0.019). Amongst patients with criteria for pathological non-response (pNR, >50% viable tumour), all who recurred had >= 70% viable melanoma. Higher ITRS and irPR scores correlated with lack of recurrence in the entire cohort (P = 0.002 and P = 70% viable melanoma and incorporating additional criteria of

Effective treatment or prevention of immune side effects associated with checkpoint inhibitor therapy of cancer is an important goal in this new era of immunotherapy. Hepatitis due to immunotherapy with antibodies against PD-1 is uncommon and generally of low severity. We present an unusually severe case arising in a melanoma patient after more than 6 months uncomplicated treatment with anti-PD-1 in an adjuvant setting. The hepatitis rapidly developed resistance to high-dose steroids, requiring anti-thymocyte globulin (ATG) to achieve control. Mass cytometry allowed comprehensive phenotyping of circulating lymphocytes and revealed that CD4+ T cells were profoundly depleted by ATG, while CD8+ T cells, B cells, NK cells and monocytes were relatively spared. Multiple abnormalities in CD4+ T cell phenotype were stably present in the patient before disease onset. These included a population of CCR4−CCR6− effector/memory CD4+ T cells expressing intermediate levels of the Th1-related chemokine receptor CXCR3 and abnormally high multi-drug resistance type 1 transporter (MDR1) activity as assessed by a rhodamine 123 excretion assay. Expression of MDR1 has been implicated in steroid resistance and may have contributed to the severity and lack of a sustained steroid response in this patient. The number of CD4+ rhodamine 123-excreting cells was reduced > 3.5-fold after steroid and ATG treatment. This case illustrates the need to consider this form of steroid resistance in patients failing treatment with corticosteroids. It also highlights the need for both better identification of patients at risk and the development of treatments that involve more specific immune suppression. Electronic supplementary material The online version of this article (10.1007/s00262-017-2107-7) contains supplementary material, which is available to authorized users.

The success of immune checkpoint inhibitors in cancer immunotherapy has been widely heralded. However many cancer patients do not respond to immune checkpoint therapy and some relapse due to acquired tumor resistance. Epigenetic targeting may be beneficial in cancer immunotherapy by reversing immune avoidance and escape mechanisms employed by cancer cells, as well as by modulating immune cell differentiation and function. In this manuscript we review recent findings suggesting how epigenetics may be used to improve cancer immunotherapy. We focus on the inhibitors of the CTLA4 and PD1 immune checkpoints and epigenetic modifiers of histone acetylation and methylation and DNA methylation.

Transcriptomic signatures designed to predict melanoma patient responses to PD-1 blockade have been reported but rarely validated. We now show that intra-patient heterogeneity of tumor responses to PD-1 inhibition limit the predictive performance of these signatures. We reasoned that resistance mechanisms will reflect the tumor microenvironment, and thus we examined PD-1 inhibitor resistance relative to T-cell activity in 94 melanoma tumors collected at baseline and at time of PD-1 inhibitor progression. Tumors were analyzed using RNA sequencing and flow cytometry, and validated functionally. These analyses confirm that major histocompatibility complex (MHC) class I downregulation is a hallmark of resistance to PD-1 inhibitors and is associated with the MITFlow/AXLhigh de-differentiated phenotype and cancer-associated fibroblast signatures. We demonstrate that TGFß drives the treatment resistant phenotype (MITFlow/AXLhigh) and contributes to MHC class I downregulation in melanoma. Combinations of anti-PD-1 with drugs that target the TGFß signaling pathway and/or which reverse melanoma de-differentiation may be effective future therapeutic strategies., A significant proportion of patients develop innate or acquired resistance to immune checkpoint inhibitors. Here, the authors show that resistance to anti-PD-1 blockade is associated with TGF-beta driven major histocompatibility complex I (MHCI) down-regulation and a de-differentiated phenotype in melanoma patients.

It is now well accepted that the immune system can control cancer growth. However, tumors escape immune-mediated control through multiple mechanisms and the downregulation or loss of major histocompatibility class (MHC)-I molecules is a common immune escape mechanism in many cancers. MHC-I molecules present antigenic peptides to cytotoxic T cells, and MHC-I loss can render tumor cells invisible to the immune system. In this review, we examine the dysregulation of MHC-I expression in cancer, explore the nature of MHC-I-bound antigenic peptides recognized by immune cells, and discuss therapeutic strategies that can be used to overcome MHC-I deficiency in solid tumors, with a focus on the role of natural killer (NK) cells and CD4 T cells.

The primary immune role of B cells is to produce antibodies, but they can also influence T cell function via antigen presentation and, in some contexts, immune regulation. Whether their roles in tumour immunity are similar to those in other chronic immune responses such as autoimmunity and chronic infection, where both pro- and anti-inflammatory roles have been described, remains controversial. Many studies have aimed to define the role of B cells in antitumor immune responses, but despite this considerable body of work, it is not yet possible to predict how they will affect immunity to any given tumour. In many human cancers, the presence of tumour-infiltrating B cells and tumour-reactive antibodies correlates with extended patient survival, and this clinical observation is supported by data from some animal models. On the other hand, T cell responses can be adversely affected by B cell production of immunoregulatory cytokines, a phenomenon that has been demonstrated in humans and in animal models. The isotype and concentration of tumour-reactive antibodies may also influence tumour progression. Recruitment of B cells into tumours may directly reflect the subtype and strength of the anti-tumour T cell response. As the response becomes chronic, B cells may attenuate T cell responses in an attempt to decrease host damage, similar to their described role in chronic infection and autoimmunity. Understanding how B cell responses in cancer are related to the effectiveness of the overall anti-tumour response is likely to aid in the development of new therapeutic interventions against cancer.

Simple Summary Immunotherapy leads to durable responses in a proportion of patients with advanced melanoma. Combination immunotherapy is more efficacious than single-agent immunotherapy, yet it is associated with significant toxicity. Currently there are no robust biomarkers to guide first-line immunotherapy selection. We have developed a flow cytometry-based score, to quantify the expression of antigen-presenting molecules MHC-I and MHC-II on melanoma cells, that incorporates both the fraction of tumor cells expressing MHC molecules and the level of expression. We demonstrate that the evaluation of tumor cell surface MHC-I expression aids in treatment selection, with combination immunotherapy providing clinical benefit over single-agent immunotherapy in MHC-I low melanoma with poor immune cell infiltration. Abstract Immunotherapy targeting T-cell inhibitory receptors, namely programmed cell death-1 (PD-1) and/or cytotoxic T-lymphocyte associated protein-4 (CTLA-4), leads to durable responses in a proportion of patients with advanced metastatic melanoma. Combination immunotherapy results in higher rates of response compared to anti-PD-1 monotherapy, at the expense of higher toxicity. Currently, there are no robust molecular biomarkers for the selection of first-line immunotherapy. We used flow cytometry to profile pretreatment tumor biopsies from 36 melanoma patients treated with anti-PD-1 or combination (anti-PD-1 plus anti-CTLA-4) immunotherapy. A novel quantitative score was developed to determine the tumor cell expression of antigen-presenting MHC class I (MHC-I) molecules, and to correlate expression data with treatment response. Melanoma MHC-I expression was intact in all tumors derived from patients who demonstrated durable response to anti-PD-1 monotherapy. In contrast, melanoma MHC-I expression was low in 67% of tumors derived from patients with durable response to combination immunotherapy. Compared to MHC-I high tumors, MHC-I low tumors displayed reduced T-cell infiltration and a myeloid cell-enriched microenvironment. Our data emphasize the importance of robust MHC-I expression for anti-PD-1 monotherapy response and provide a rationale for the selection of combination immunotherapy as the first-line treatment in MHC-I low melanoma.

Immunotherapies blocking immune inhibitory receptors programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) on T-cells have dramatically improved patient outcomes in a range of advanced cancers. However, the lack of response, and the development of resistance remain major obstacles to long-term improvements in patient outcomes. There is significant interest in the clinical use of biomarkers to improve patient selection, and the expression of PD-1 ligand 1 (PD-L1) is often reported as a potential biomarker of response. However, accumulating evidence suggests that the predictive value of PD-L1 expression in tumor biopsies is relatively low due, in part, to its complex biology. In this review, we discuss the biological consequences of PD-L1 expression by various cell types within the tumor microenvironment, and the complex mechanisms that regulate PD-L1 expression at the genomic, transcriptomic and proteomic levels.

Summary Constitutive expression of the immune checkpoint, PD-L1, inhibits anti-tumor immune responses in cancer, although the factors involved in PD-L1 regulation are poorly understood. Here we show that loss of global DNA methylation, particularly in intergenic regions and repeat elements, is associated with constitutive (PD-L1CON), versus inducible (PD-L1IND), PD-L1 expression in melanoma cell lines. We further show this is accompanied by transcriptomic up-regulation. De novo epigenetic regulators (e.g., DNMT3A) are strongly correlated with PD-L1 expression and methylome status. Accordingly, decitabine-mediated inhibition of global methylation in melanoma cells leads to increased PD-L1 expression. Moreover, viral mimicry and immune response genes are highly expressed in lymphocyte-negative plus PD-L1-positive melanomas, versus PD-L1-negative melanomas in The Cancer Genome Atlas (TCGA). In summary, using integrated genomic analysis we identified that global DNA methylation influences PD-L1 expression in melanoma, and hence melanoma's ability to evade anti-tumor immune responses. These results have implications for combining epigenetic therapy with immunotherapy., Graphical Abstract, Highlights • Global DNA hypomethylation in melanoma promotes constitutive PD-L1 expression • Transcriptomic up-regulation accompanies constitutive PD-L1 expression in melanoma • Constitutive PD-L1 is associated with reduced DNMT3A expression and viral mimicry • DNA methylation inhibitor treatment of melanoma cells increases PD-L1 expression, Genetics; Genomics; Cancer; Transcriptomics

Introduction The cytokines IFNγ and TNFα are abundantly expressed by cytotoxic T cells. These cytokines have distinct and overlapping downstream effects. In this study, we assessed the effects of IFNγ and TNFα on the expression of immune regulatory factors. Material and methods We analysed a large panel of melanoma cell lines, including BRAF mutant (n=11), NRAS mutant (n=10), BRAF and NRAS wild type (n=10) cutaneous melanomas, and GNAQ/11-mutant uveal melanomas (n=8). These cells were treated with TNFα or IFNγ for 48 hour and the expression of PD-L1, PD-L2, HLA-ABC and HLA-DR was analysed by flow cytometry. Results and discussions IFNγ showed consistently stronger induction of PD-L1, PD-L2, HLA-ABC and HLA-DR compared to TNFα. For instance, 35/39 cells (90%) showed IFNγ-mediated up regulation of PD-L1, PD-L2, HLA-ABC and HLA-DR, whereas TNFα induced expression of all immune markers in only 23/39 cells (59%). We also noted variation in the activity of IFNγ and TNFα that reflected melanoma subtypes. For instance, in the 31 cutaneous melanoma cell lines, the relative expression of HLA-ABC and HLA-DR was similar after IFNγ stimulation. In contrast, TNFα preferentially induced HLA-ABC, rather than HLA-DR, in the cutaneous cell lines and HLA-ABC was also preferentially stimulated by both cytokines in the 8 uveal melanoma cell lines. Response of the four immune markers to IFNγ and TNFα was often correlated. This was most evident in the case of PD-L2. We noted strong correlation between baseline, IFNγ- and TNFα-induced PD-L2 levels (Spearman’s rank correlation coefficient: p Conclusion These findings have important implications for immunotherapy, confirming that most melanomas are responsive to IFNγ and/or TNFα.

The programmed death-ligand 1 (PD-L1) receptor is an important immune checkpoint and is often upregulated in cancer cells to allow immune evasion. In melanoma, the patients with PD-L1 expression and absense of tumour infiltrating lymphocytes (TILs) (i.e. “constitutive PD-L1 or PD-L1CON”) show worse response rates and prognosis than patients with PD-L1 expression and the presence of TILs (i.e. “inducible PD-L1 or PD-L1IND”). However, how PD-L1 expression is regulated in melanoma cells remains elusive. Understanding the mechanisms of how PD-L1 is regulated is important for predicting responses for anti-PD-L1 treatment and for developing new combinatorial therapies. We hypothesised that epigenetic state regulates constitutive and inducible PD-L1 expression in melanoma. To address the hypothesis, we have generated whole-genome scale DNA methylomes (using reduced representation bisulfite sequencing) and transcriptomes (RNA-Seq) for patient derived melanoma cell lines (in PD-L1IND and PD-L1CON groups). We discovered extensive global hypomethylation in the constitutive lines, particularly pronounced in intergenic repeat regions and gene bodies. A high proportion of hypomethylated regions exhibited dichotomous methylation patterns indicating a common regulatory mechanism between the PD-L1IND and PD-L1CON lines. RNA-Sequening data indicated that the hypomethylated state of the PD-L1CON cells was correlated with higher upregulation of the differentially expressed genes at a global-scale and the upregulated genes were associated with cancer hallmark properties. The upregulated genes exhibited signatures of viral mimicry and cytosolic sensing of dsRNA similar to what has been observed after DNA methyltransferase inhibitor (DNMTi) treatment in cancers. DNMTi treatment increased PD-L1 transcription in the PD-L1IND cell lines. PD-L1CON samples showed greater resistance to DNMTi compared to PD-L1 negative samples. This was observed at protein-coding genes, transposable elements and long non-coding RNAs. Moreover, genes involved in the innate immune pathway was strongly increased upon DNMTi mediated hypomethylation in the PD-L1IND cell lines (FDR-adjusted P-value = 0.02), however there was no expression change in the PD-L1 positive cells (Adjusted P-value = 1.0). Furthermore, we found that the IRF1 transcription factor, which plays a critical role in the innate immune pathway and also binds to the PD-L1 promoter, is significantly upregulated upon DNMTi in the PD-L1 negative cell lines (Adjusted P-value = 0.01) but not in the PD-L1 positive cells (Adjusted P-value = 0.9). These results show that global hypomethylation levels regulate PD-L1 expression in melanoma. We believe these results are the first to show that DNA methylation levels play a role in regulating PD-L1 on melanoma and suggest they may have important implications for combined treatments targeting methylation (DNMTi) and PD1/PD-L1 (anti-PD1 antibodies). Citation Format: Aniruddha Chatterjee, Antonio Ahn, Euan J. Rodger, Peter A. Stockwell, Matthew Parry, Jyoti Motwani, Stuart J. Gallagher, Elena Shklovskaya, Jessamy Tiffen, Peter Hersey, Michael R. Eccles. Global DNA methylation levels regulate PD-L1 expression in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 826.

Summary The dendritic cell (DC) lineage is remarkably heterogeneous. It has been postulated that specialized DC subsets have evolved in order to select and support the multitude of possible T cell differentiation pathways. However, defining the function of individual DC subsets has proven remarkably difficult, and DC subset control of key T cell fates such as tolerance, T helper cell commitment and regulatory T cell induction is still not well understood. While the difficulty in assigning unique functions to particular DC subsets may be due to sharing of functions, it may also reflect a lack of appropriate physiological in-vivo models for studying DC function. In this paper we review the limitations associated with many of the current DC models and highlight some of the underlying difficulties involved in studying the function of murine DC subsets.

Antigen-dependent interactions between T lymphocytes and dendritic cells (DCs) can produce two distinct outcomes: tolerance and immunity. It is generally considered that all DC subsets are capable of supporting both tolerogenic and immunogenic responses, depending on their exposure to activating signals. Here, we tested whether epidermal Langerhans cells (LCs) can support immunogenic responses in vivo in the absence of antigen presentation by other DC subsets. CD4 T cells responding to antigen presentation by activated LCs initially proliferated but then failed to differentiate into effector/memory cells or to survive long term. The tolerogenic function of LCs was maintained after exposure to potent adjuvants and occurred despite up-regulation of the costimulatory molecules CD80, CD86, and IL-12, but was consistent with their failure to translocate the NF-κB family member RelB from the cytoplasm to the nucleus. Commitment of LCs to tolerogenic function may explain why commensal microorganisms expressing Toll-like receptor (TLR) ligands but confined to the skin epithelium are tolerated, whereas invading pathogens that breach the epithelial basement membrane and activate dermal DCs stimulate a strong immune response.

Antibodies directed against the programmed cell death protein 1 (PD-1) immune checkpoint have significantly improved survival of patients with advanced metastatic melanoma. Innate and acquired resistance to anti-PD-1 treatment represents a significant treatment obstacle, yet the mechanisms of resistance are poorly understood and remain difficult to model at the cellular level. Here we used multiparameter flow cytometry to examine the immune profiles of 39 tumor biopsies obtained from 31 patients with stage III-IV metastatic melanoma prior to (baseline, n = 21) or during (n = 18) single-agent anti-PD1 immunotherapy; twelve patients developed resistance to treatment. Samples were enzymatically dissociated and cryopreserved until thawed and stained with fluorescently labeled antibodies to enable a comprehensive analysis of melanoma cells and tumor infiltrating lymphocytes (TILs). Mean melanoma cell content was 57+4.7% (mean+s.e.m.) while mean immune infiltrate (CD45 positive cells) was 31.5+4.5%. Analysis of melanoma cell expression of antigen presenting molecules and PD-1 ligands revealed frequent downregulation of HLA-ABC expression in both baseline (9/21, 43%) and on-treatment tumors (10/18, 56%), including a complete HLA-ABC loss in 2/39 samples. HLA-ABC up-regulation was observed in 9/39 (23%) tumors. Expression of HLA-DR and PD-L1 strongly correlated with that of HLA-ABC (Spearman r=0.68 and =0.89, respectively), indicative of local interferon exposure. Unlike PD-L1, melanoma cell expression of PD-L2 was low in the biopsied tissue. In contrast, PD-L2 expression was readily induced by gamma interferon and correlated with HLA-ABC expression in matching melanoma cell lines established from the dissociated biopsies (9/39). We found multiple correlations between parameters characterizing the immune profiles of tumor cells and TILs in both baseline and on-treatment samples. Among significant treatment-associated changes, conventional T-cells bearing the αβ T-cell receptor were 1.4-fold higher in the on-treatment group compared to baseline (65+2.7% versus 46.2+4.1%, P Citation Format: Elena Shklovskaya, Jenny Lee, Su Yin Lim, Sara Alavi, John Thompson, Robyn Saw, Matteo Carlino, Richard Scolyer, Alexander Menzies, Georgina Long, Richard Kefford, Helen Rizos. Flow cytometric analysis of immune responses in the melanoma tissue biopsies before or during anti-PD1 immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A154.

The dermis contains a novel population of γδT cells that are distinct from epidermal γδT cells and produce IL-17 in response to mycobacterial infection., The presence of γδ T cell receptor (TCR)–expressing cells in the epidermis of mice, termed dendritic epidermal T cells (DETCs), is well established. Because of their strict epidermal localization, it is likely that DETCs primarily respond to epithelial stress, such as infections or the presence of transformed cells, whereas they may not participate directly in dermal immune responses. In this study, we describe a prominent population of resident dermal γδ T cells, which differ from DETCs in TCR usage, phenotype, and migratory behavior. Dermal γδ T cells are radioresistant, cycle in situ, and are partially depend on interleukin (IL)-7, but not IL-15, for their development and survival. During mycobacterial infection, dermal γδ T cells are the predominant dermal cells that produce IL-17. Absence of dermal γδ T cells is associated with decreased expansion in skin draining lymph nodes of CD4+ T cells specific for an immunodominant Mycobacterium tuberculosis epitope. Decreased CD4+ T cell expansion is related to a reduction in neutrophil recruitment to the skin and decreased BCG shuttling to draining lymph nodes. Thus, dermal γδ T cells are an important part of the resident cutaneous immunosurveillance program. Our data demonstrate functional specialization of T cells in distinct microcompartments of the skin.

Despite major advances in immuno- and targeted therapies for metastatic melanoma, not all patients respond to such treatments and the development of therapy resistance remains a key obstacle. Novel targets are required and the epigenetic modifier EZH2 may represent such a solution. EZH2 methylates histones causing chromatin compaction and silencing of gene expression. Aberrant EZH2 in cancer results in the silencing of hundreds of tumor-suppressor genes that would normally constrain cancer growth, and 26% of patients in the Australian Melanoma Genome Project (AMGP) display such abnormal EZH2. Using ChIP-seq and microarrays to reveal downstream EZH2 target genes, we identified many tumor-suppressor genes including some that play a role in the antitumor immune response. Interestingly, xenografts in immune-deficient NOD SCID mice showed no significant inhibition in tumor growth when treated with an EZH2 inhibitor. However, growth inhibition was achieved when the same experiment was performed in nude mice, which retain some mediators of innate immunity. The effect of EZH2 overexpression in melanoma on immune-mediated rejection was studied in an inducible in vivo model. Subcutaneous B16 tumors expressing model antigen were initially rejected but relapsed in the majority of immunodeficient mice transferred with CD4 T cells. Long-term (120-day) survival was reduced from 40% to 22% if the tumors also overexpressed EZH2. Our studies suggest that aberrant EZH2 in melanoma drives aggressive disease via suppression of genes that would normally trigger the antitumor immune response. The progression of several preclinical, small-molecule inhibitors of EZH2 remains promising. Citation Format: Jessamy Tiffen, Elena Shklovskaya, Stuart Gallagher, Dilini Gunatilake, Peter Hersey. The epigenetic modifier EZH2 represses antitumor immunity in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1384.

Modulating the host-immune response by the use of recombinant vaccines is a potential strategy to improve protection against microbial pathogens. In this study, we sought to determine whether secretion of murine GM-CSF by the bacillus Calmette-Guérin (BCG) vaccine influenced protective immunity against Mycobacterium tuberculosis. BCG-derived GM-CSF stimulated the in vitro generation of functional APCs from murine bone marrow precursors, as demonstrated by the infection-induced secretion of IL-12 by differentiated APCs, and the ability of these cells to present Ag to mycobacterium-specific T cells. Mice vaccinated with BCG-secreting murine GM-CSF (BCG:GM-CSF) showed increased numbers of CD11c+MHCII+ and CD11c−CD11b+F480+ cells compared with those vaccinated with control BCG, and this effect was most apparent in the draining lymph nodes at 7 and 14 days postvaccination. Vaccination with BCG:GM-CSF also resulted in enhanced expression of costimulatory molecules on migratory dendritic cells in the draining lymph nodes. The increased APC number was associated with an increase in the frequency of anti-mycobacterial IFN-γ-secreting T cells generated after BCG:GM-CSF vaccination compared with vaccination with control BCG, and this effect was sustained up to 17 wk in the spleens of immunized mice. Vaccination with BCG:GM-CSF resulted in an ∼10-fold increase in protection against disseminated M. tuberculosis infection compared with control BCG. This study demonstrates the potential of BCG-secreting immunostimulatory molecules as vaccines to protect against tuberculosis and suggests BCG:GM-CSF merits further appraisal as a candidate to control M. tuberculosis infection in humans.

The importance of CD4 T cells in tumour immunity has been increasingly recognised, with recent reports describing robust CD4 T cell-dependent tumour control in mice whose immune-regulatory mechanisms have been disturbed by irradiation, chemotherapy, immunomodulatory therapy and/or constitutive immunodeficiency. Tumour control in such models has been attributed in large part to direct Major Histocompatibility Complex (MHC) class II-dependent CD4 T cell killing of tumour cells. To test whether CD4 T cells can eradicate tumours without directly killing tumour cells, we developed an animal model in which tumour-derived antigen could be presented to T-cell receptor (TCR)-transgenic CD4 T cells by host but not tumour MHC class II molecules. In I-E(+) mice bearing I-E(null) tumours, naive I-E-restricted CD4 T cells proliferated locally in tumour-draining lymph nodes after recognising tumour-derived antigen on migratory dendritic cells. In lymphopaenic but not immunosufficient hosts, CD4 T cells differentiated into polarised T helper type 1 (Th1) cells expressing interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα) and interleukin (IL)-2 but little IL-17, and cleared established tumours. Tumour clearance was enhanced by higher TCR affinity for tumour antigen-MHC class II and was critically dependent on IFNγ, as demonstrated by early tumour escape in animals treated with an IFNγ blocking antibody. Thus, CD4 T cells and IFNγ can control tumour growth without direct T-cell killing of tumour cells, and without requiring additional adaptive immune cells such as CD8 T cells and B cells. Our results support a role for effective CD4 T cell-dependent tumour immunity against MHC class II-negative tumours.

Regulatory T cells (Tregs) have been shown to enhance immune reconstitution and prevent graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation; however, it is unclear how Tregs mediate these effects. Here, we developed a model to examine the mechanism of Treg-dependent regulation of immune reconstitution. Lymphopenic mice were selectively reconstituted with Tregs prior to transfer of conventional CD4+ T cells. Full Treg reconstitution prevented the rapid oligoclonal proliferation that gives rise to pathogenic CD4 effector T cells, while preserving the slow homeostatic form of lymphopenia-induced peripheral expansion that repopulates a diverse peripheral T cell pool. Treg-mediated CTLA-4-dependent downregulation of CD80/CD86 on DCs was critical for inhibition of rapid proliferation and was a function of the Treg/DC ratio achieved by reconstitution. In an allogeneic BM transplant model, selective Treg reconstitution before T cell transfer also normalized DC costimulation and provided complete protection against GVHD. In contrast, cotransfer of Tregs was not protective. Our results indicate that achieving optimal recovery from lymphopenia should aim to improve early Treg reconstitution in order to increase the relative number of Tregs to DCs and thereby inhibit spontaneous oligoclonal T cell proliferation.

EZH2 contains the catalytic subunit of the PRC2 complex, which plays a role in maintenance of stem cell- like state of cancer cells and in the progression of melanoma. Our previous studies have shown that hyperactivation of EZH2 by somatic mutations or copy number gain occurs in 20% of melanoma patients and impacts overall survival. Elevated activity of EZH2 in cancer can be targeted by small molecule drugs (e.g. GSK126) and is associated with sensitivity to apoptotic cell death. Transcriptomic profiling showed that several well-known tumor suppressors such as BOK, NDRG1 and RASSF5 are down-regulated in cells with abnormalities in EZH2, which can be reversed by inhibitor treatment. By using our in vitro EZH2 drug targeting assay we validated genes associated with immune recognition and MHC class II antigen presentation such as HLA A and B,DR and CD74 (HLA DR invariant chain). In addition, ChIP-Seq studies identified adhesion molecules, chemokine ligands, and receptors involved in traffic of lymphocytes to tumors as well as the function of NK cells. Treatment of melanoma with EZH2 inhibitors showed increased killing by NK cells suggesting that an immunosuppressive effect of EZH2 involving both innate and adaptive immunity. In a mouse model of subcutaneous B16 melanoma, a course of daily treatment with an EZH2 inhibitor did not change tumour growth or immune responses in immunosufficient mice but caused a transient 9-fold increase in circulating Ly6G+ [granulocytic] myeloid-derived suppressor cells [MDSCs] suggesting the effect of EZH2 modifiers on the myeloid compartment. In summary, patients with somatic activation of EZH2 may benefit from treatment combinations that include inhibitors of EZH2. Supported by NHMRC program grant 633004, Sydney medical school foundation and Cancer Institute NSW. F.V.F. is grateful for the support of grants CA154887 from the National Institutes of Health, National Cancer Institute and CRCC CRN-17-427258. Citation Format: Peter Hersey, Stuart Gallagher, Jessamy Tiffen, Elena Shklovskaya, Stephen Wilson, Fabian Filipp. EZH2 inhibitors in immunotherapy of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1676. doi:10.1158/1538-7445.AM2017-1676

The cytokine thymic stromal lymphopoietin (TSLP) is produced by epithelia exposed to the contact sensitizer dibutyl phthalate (DBP), and it is critical for the induction of Th2 immune responses by DBP-FITC. TSLP is thought to act on dendritic cells (DC), but the precise DC subsets involved in the response to TSLP remain to be fully characterized. In this study we show that a subset of CD326loCD103loCD11blo dermal DC, which we termed “triple-negative (TN) DC,” is highly responsive to TSLP. In DBP-FITC–treated mice, TN DC upregulated expression of CD86 and rapidly migrated to the draining lymph node to become the most abundant skin-derived DC subset at 24 and 48 h after sensitization. None of these responses was observed in TSLPR-deficient mice. In contrast, TN DC numbers were not increased after treatment with the allergen house dust mite or the bacteria Escherichia coli and bacillus Calmette–Guérin, which increased other DC subsets. In vivo, treatment with rTSLP preferentially increased the numbers of TN DC in lymph nodes. In vitro, TN DC responded to rTSLP treatment with a higher level of STAT5 phosphorylation compared with other skin-derived DC subsets. The TN DC subset shared the morphology, phenotype, and developmental requirements of conventional DC, depending on FLT3 expression for their optimal development from bone marrow precursors, and CCR7 for migration to the draining lymph node. Thus, TN DC represent a dermal DC subset that should be considered in future studies of TSLP-dependent contact sensitization and skin immune responses.

Dendritic cells (DCs) are critical for regulating CD4 and CD8 T cell immunity, controlling Th1, Th2, and Th17 commitment, generating inducible Tregs, and mediating tolerance. It is believed that distinct DC subsets have evolved to control these different immune outcomes. However, how DC subsets mount different responses to inflammatory and/or tolerogenic signals in order to accomplish their divergent functions remains unclear. Lipopolysaccharide (LPS) provides an excellent model for investigating responses in closely related splenic DC subsets, as all subsets express the LPS receptor TLR4 and respond to LPS in vitro. However, previous studies of the LPS-induced DC transcriptome have been performed only on mixed DC populations. Moreover, comparisons of the in vivo response of two closely related DC subsets to LPS stimulation have not been reported in the literature to date. We compared the transcriptomes of murine splenic CD8 and CD11b DC subsets after in vivo LPS stimulation, using RNA-Seq and systems biology approaches. We identified subset-specific gene signatures, which included multiple functional immune mediators unique to each subset. To explain the observed subset-specific differences, we used a network analysis approach. While both DC subsets used a conserved set of transcription factors and major signalling pathways, the subsets showed differential regulation of sets of genes that ‘fine-tune’ the network Hubs expressed in common. We propose a model in which signalling through common pathway components is ‘fine-tuned’ by transcriptional control of subset-specific modulators, thus allowing for distinct functional outcomes in closely related DC subsets. We extend this analysis to comparable datasets from the literature and confirm that our model can account for cell subset-specific responses to LPS stimulation in multiple subpopulations in mouse and man.

A factor inhibiting the proliferation of splenic colony-forming units, injected bothin vivo and after preincubation of mouse bone marrow cellsin vitro, had a dose-dependent effect on the increased proliferative activity of splenic colony-forming units from the bone marrow of mice treated with testosterone propionate. This was associated with a reduction in the number of early hemopoietic precursors of mouse bone marrow. The counts of clonogenic granulocytic-macrophagal and macrophagal colony-forming units decreased and that of burst-forming units in murine bone marrow increased after exposure to the hormone. Testosterone propionate promoted a decrease of the repopulating potential of bone marrow cells, which recovered after their preincubation with the factor inhibiting the proliferation of splenic colony-forming units.

One reason proposed for the failure of Mycobacterium bovis bacille Calmette Guérin (BCG) vaccination to adequately control the spread of tuberculosis is a limited ability of the vaccine to induce effective CD8 T cell responses. However, the relative capacity of the BCG vaccine and virulent Mycobacterium tuberculosis to induce activation of CD8 T cells, and the factors that govern the initial priming of these cells after mycobacterial infection, are poorly characterized. Using a TCR transgenic CD8 T cell transfer model, we demonstrate significant activation of Ag-specific CD8 T cells by BCG, but responses were delayed and of reduced magnitude compared with those following infection with M. tuberculosis. The degree of CD8 T cell activation was critically dependent on the level of antigenic stimulation, as modifying the infectious dose to achieve comparable numbers of BCG or M. tuberculosis in draining lymph nodes led to the same pattern of CD8 T cell responses to both strains. Factors specific to M. tuberculosis infection did not influence the priming of CD8 T cells, as codelivery of M. tuberculosis with BCG did not alter the magnitude of BCG-induced T cell activation. Following transfer to RAG-1−/− recipients, BCG and M. tuberculosis-induced CD8 T cells conferred equivalent levels of protection against M. tuberculosis infection. These findings demonstrate that BCG is able to prime functional CD8 T cells, and suggest that effective delivery of Ag to sites of T cell activation by vaccines may be a key requirement for optimal CD8 T cell responses to control mycobacterial infection.

The interaction between dendritic cells and T cells is crucial for the regulation of immunological tolerance and immunity. Although our understanding of the mechanisms responsible for these phenomena has advanced significantly in recent years, we are still lacking a fully integrated model of how dendritic cell phenotype correlates with function, and how complex interactions with multiple dendritic and T cell subpopulations shape the course of the immune response in vivo. In this review, we summarize the current state of knowledge in the field, highlighting the areas where further investigation is likely to advance our understanding of this fundamental immunological interaction.

The interaction between dendritic cells and T cells is crucial for the regulation of immunological tolerance and immunity. Although our understanding of the mechanisms responsible for these phenomena has advanced significantly in recent years, we are still lacking a fully integrated model of how dendritic cell phenotype correlates with function, and how complex interactions with multiple dendritic and T cell subpopulations shape the course of the immune response in vivo. In this review, we summarize the current state of knowledge in the field, highlighting the areas where further investigation is likely to advance our understanding of this fundamental immunological interaction.

Background Group 2 innate lymphoid cells (ILC2) have been implicated in the pathogenesis of allergic lung diseases. However, the upstream signals that regulate ILC2 function during pulmonary inflammation remain poorly understood. ILC2s have been shown to respond to exogenous IL-2, but the importance of endogenous IL-2 in ILC2 function in vivo remains unclear. Objective We sought to understand the role of IL-2 in the regulation of ILC2 function in the lung. Methods We used histology, flow cytometry, immunohistochemistry, ELISA, and quantitative PCR with knockout and reporter mice to dissect pulmonary ILC2 function in vivo . We examined the role of ILC2s in eosinophilic crystalline pneumonia, an idiopathic type 2 inflammatory lung condition of mice, and the effect of IL-2 deficiency on this disease. We determined the effect of IL-2 administration on pulmonary ILC2 numbers and function in mice in the steady state and after challenge with IL-33. Results We discovered an unexpected role for innate cell–derived IL-2 as a major cofactor of ILC2 function during pulmonary inflammation. Specifically, we found that IL-2 was essential for the development of eosinophilic crystalline pneumonia, a type 2 disease characterized by increased numbers of activated ILC2s. We show that IL-2 signaling serves 2 distinct functions in lung ILC2s, namely promoting cell survival/proliferation and serving as a cofactor for the production of type 2 cytokines. We further demonstrate that group 3 innate lymphoid cells are an innate immune source of IL-2 in the lung. Conclusion Innate cell–derived IL-2 is a critical cofactor in regulating ILC2 function in pulmonary type 2 pathology.

Systemic administration of high doses of soluble Ag induces peripheral CD4+ T cell tolerance in unmanipulated hosts. To test whether tolerance is modified under conditions of transient lymphopenia, we tracked the response of 5C.C7 TCR-transgenic CD4+ T cells to i.v. moth cytochrome c peptide in mice that received low-dose gamma irradiation 10 days previously. This model was chosen because it does not support spontaneous lymphopenia-induced proliferation of 5C.C7 cells, allowing the study of Ag-specific responses without interference from simultaneous spontaneous proliferation. Clonal expansion in response to i.v. peptide was increased in irradiated mice, while clonal deletion was severely impaired in comparison with untreated animals. Amplified TCR triggering was observed in irradiated hosts, consistent with dendritic cell activation leading to enhanced Ag presentation. Failure of deletion was accompanied by persistent T cell activation and accumulation of Th1 effector cells. Up-regulated expression of IL-7R and the prosurvival protein Bcl-xL was associated with clonal persistence. Cells with memory and naive phenotypes were both represented within persistent clones, but no Th1 function could be demonstrated within the long-term memory population. Failure of clonal deletion in irradiated hosts represents a novel mechanism limiting TCR diversity in a lymphopenic environment and may contribute to subsequent autoimmunity.

Migrated Langerhans cells (m-LCs) have recently been shown to comprise only a minority of skin-derived dendritic cells (DCs) expressing Langerin in cutaneous lymph nodes. We have used BM chimeric mice that differ in CD45 and MHC class II alleles to unequivocally distinguish between radioresistant m-LCs and radiosensitive migrated dermal DCs (m-dDCs), to determine their phenotype, response to contact sensitization, and ability to activate naive CD4+ T cells in vivo. We have also characterized three subsets of dDCs and their migratory counterparts, as distinguished by expression of CD11b and Langerin. Each of the four subsets of skin DCs showed differential migration to draining LN in response to contact sensitizing agents. Migration of Langerin−CD11b+ and Langerin+CD11blow dDCs peaked after 1 day, followed by Langerin−CD11blow dDCs at 2 days and Langerin+ LCs at 4 days. Moreover, while m-LCs and m-dDCs had similar surface phenotypes in the steady state, they displayed unexpectedly different activation responses to contact sensitization: m-dDCs markedly up-regulated CD80 and CD86 at day 1, whereas only m-LCs up-regulated CD40, with delayed kinetics. Thus, m-dDCs are likely to be responsible for the initial response to skin immunization. However, when expression of cognate MHC class II was restricted to LCs and m-LCs, they were also capable of processing and presenting protein Ag to drive naive CD4 T cell proliferation in vivo. Thus, m-dDCs and m-LCs display distinct behavior in cutaneous lymph nodes while sharing the ability to interact specifically with T cells to control the immune response.