Anti-melanoma Immune Responses Research Articles

Interfering with aggregated α-synuclein in advanced melanoma leads to a major upregulation of MHC class II proteins.

Melanoma is the most serious and deadly form of skin cancer and with progression to advanced melanoma, the intrinsically disordered protein α-synuclein is upregulated to high levels. While toxic to dopaminergic neurons in Parkinson's disease, α-synuclein is highly beneficial for primary and metastatic melanoma cells. To gain detailed insights into this exact opposite role of α-synuclein in advanced melanoma, we performed proteomic studies of high-level α-synuclein-expressing human melanoma cell lines that were treated with the diphenyl-pyrazole small-molecule compound anle138b, which binds to and interferes with the oligomeric structure of α-synuclein. We also performed proteomic and transcriptomic studies of human melanoma xenografts that were treated systemically with the anle138b compound. The results reveal that interfering with oligomerized α-synuclein in the melanoma cells in these tumor xenografts led to a substantial upregulation and expression of major histocompatibility complex proteins, which are pertinent to enhancing anti-melanoma immune responses.

Enhancing Immunogenicity in Metastatic Melanoma: Adjuvant Therapies to Promote the Anti-Tumor Immune Response.

Advanced melanoma is an aggressive form of skin cancer characterized by low survival rates. Less than 50% of advanced melanoma patients respond to current therapies, and of those patients that do respond, many present with tumor recurrence due to resistance. The immunosuppressive tumor-immune microenvironment (TIME) remains a major obstacle in melanoma therapy. Adjuvant treatment modalities that enhance anti-tumor immune cell function are associated with improved patient response. One potential mechanism to stimulate the anti-tumor immune response is by inducing immunogenic cell death (ICD) in tumors. ICD leads to the release of damage-associated molecular patterns within the TIME, subsequently promoting antigen presentation and anti-tumor immunity. This review summarizes relevant concepts and mechanisms underlying ICD and introduces the potential of non-ablative low-intensity focused ultrasound (LOFU) as an immune-priming therapy that can be combined with ICD-inducing focal ablative therapies to promote an anti-melanoma immune response.

CD16 CAR-T cells enhance antitumor activity of CpG ODN-loaded nanoparticle-adjuvanted tumor antigen-derived vaccinevia ADCC approach

BackgroundCombinatorial immunotherapy strategies for enhancing the responsiveness of immune system have shown great promise for cancer therapy. Engineered nanoformulation incorporated toll-like receptor (TLR) 9 agonist CpG ODN has shown more positive results in suppressing tumor growth and can significantly enhance other immunotherapy activity with combinatorial effects due to the innate and adaptive immunostimulatory effects of CpG.ResultsIn the present work, protamine sulfate (PS) and carboxymethyl β-glucan (CMG) were used as nanomaterials to form nanoparticles through a self-assembly approach for CpG ODN encapsulation to generate CpG ODN-loaded nano-adjuvant (CNPs), which was subsequently mixed with the mixture of mouse melanoma-derived antigens of tumor cell lysates (TCL) and neoantigens to develop vaccine for anti-tumor immunotherapy. The obtained results showed that CNPs was able to effectively deliver CpG ODN into murine bone marrow-derived dendritic cells (DC) in vitro, and remarkably stimulate the maturation of DC cells with proinflammatory cytokine secretion. In addition, in vivo analysis showed that CNPs enhanced anti-tumor activity of PD1 antibody and CNPs-adjuvanted vaccine based on the mixture antigens of melanoma TCL and melanoma-specific neoantigen could not only induce anti-melanoma cellular immune responses, but also elicit melanoma specific humoral immune responses, which significantly inhibited xenograft tumor growth. Furthermore, CD16 CAR-T cells were generated by expressing CD16-CAR in CD3+CD8+ murine T cells.ConclusionOur results eventually showed that anti-melanoma antibodies induced by CNPs-adjuvanted TCL vaccines were able to collaborate with CD16-CAR-T cells to generate an enhanced targeted anti-tumor effects through ADCC (antibody dependent cell cytotoxicity) approach. CD16 CAR-T cells has thus a great potential to be an universal promising strategy targeting on solid tumor synergistic immunotherapy via co-operation with TCL-based vaccine.

Sensory Nerves inhibit the development of protective anti-melanoma immune response and formation of tertiary lymphoid structures

Abstract Peripheral nervous system (PNS) is an important component of the tumor microenvironment (TME). It has been established that autonomic innervation plays an important role in cancer. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Precisely, sensory ablation enhanced leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Furthermore, Depletion of CD8+ lymphocytes prevented denervation-restricted tumor growth. Importantly, denervated tumors contained higher densities of mature high endothelial venules (HEV) associated with the formation of classic tertiary lymphoid structures (TLS), which associate with a more favorable melanoma prognosis. Transcriptional profiling of human melanomas confirmed our findings in murine melanoma models. Specifically, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival.. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby providing a new therapeutic target for melanoma treatment.

453 The heat shock protein 70 (Hsp70) induces effective anti-melanoma immune defense

Immune checkpoint inhibitors have revolutionized the treatment of patients with advanced and metastatic melanoma and led to prolonged overall survival. However, long-term treatment responses are still limited to few patients, where tumor-specific effector T cells at the tumor sites are considered predictors for treatment success. This raises the question of new treatment strategies. We were recently able to show in preclinical models, that targeted activation of melanoma-associated mast cells (MC) with lipopolysaccharide (LPS) induces an effective anti-melanoma immune response in a TLR4-dependent manner.

660 Epigenetic regulation of Slamf6 expression in the immune response to melanoma

Many patients with advanced melanoma do not mount a successful anti-melanoma immune response, become refractory to checkpoint inhibitors, and unfortunately succumb to their disease. There is a great unmet need for improved mechanistic understanding of the anti-melanoma immune response, as well as novel strategies to improve long term survival. Failure of the anti-melanoma immune response has been linked to the establishment of a hypofunctional/exhausted T-cell state, with at least two distinct T-cell populations.

Sensory Nerves Impede the Formation of Tertiary Lymphoid Structures and Development of Protective Antimelanoma Immune Responses.

Peripheral neurons comprise a critical component of the tumor microenvironment (TME). The role of the autonomic innervation in cancer has been firmly established. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Specifically, sensory ablation led to improved leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Cutaneous sensory nerves hindered the maturation of intratumoral high endothelial venules and limited the formation of mature tertiary lymphoid-like structures containing organized clusters of CD4+ T cells and B cells. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Importantly, CD8a depletion prevented denervation-dependent antitumor effects. Finally, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby defining a novel target for therapeutic intervention in the melanoma setting.

A protocol for phase II study investigating the safety and efficacy of TM5614 in combination with nivolumab in the treatment of unresectable malignant melanoma

Abstract Introduction: Plasminogen activating inhibitor-1 (PAI-1) plays a significant role in the development of various cancers, including melanomas, and modulation of tumor-associated inflammation is one of the key anti-tumor functions of PAI-1. A recent report suggested that PAI-1 inhibitors suppress the expression of programmed cell death 1 ligand 1 (PD-L1) on tumor-associated macrophages (TAMs), suppressing the induction of regulatory T cells and promoting anti-melanoma immune responses. Methods and analysis: This is a single-arm, open-label, multi-institutional, phase 2 clinical trial to assess the efficacy and safety of nivolumab in combination with TM5614 (pPAI-1 inhibitor) in patients with advanced malignant melanoma. Nivolumab will be administered every 4 weeks, and TM5614 will be administered daily for 8 weeks. For the first-line cohort, assuming the null hypothesis of a response rate of 22.2% and an alternative hypothesis of 40%, a minimum of 22 patients are required to achieve a two-sided, type I error of 5% and power of 70% based on the exact binomial distribution. For the second-line and beyond cohorts, assuming the null hypothesis of a response rate of 5% and the alternative hypothesis of 22.2%, a minimum of 18 patients are required to achieve a two-sided, type I error of 5%, and power of 70% based on the exact binomial distribution. Discussion: This study will contribute to the development of novel combination therapy for anti- programmed cell death 1 antibodies-resistant advanced melanoma patients, which remains an unmet clinical need. Trial registration: Trial registration number: jRCT2021210029

196 Effective anti-melanoma immune response by peritumoral delivery of the heat shock protein 70 (Hsp70)

Immune checkpoint inhibitors increased overall survival for patients with advanced and metastatic melanoma, but long-term treatment responses are still limited to few patients with tumor specific effector T-cells at the tumor sites as predictors for treatment success. Thus, new treatment strategies are required. In preclinical models we could recently show, that targeted activation of melanoma- associated mast cells (MC) with lipopolysaccharide (LPS) induces an effective anti-melanoma immune response in a Toll-like receptor (TLR) 4-dependent manner.

Variable GILT protein expression in melanoma cells of metastatic tumor specimens

Melanoma is the sixth most common cancer in the US. Immunotherapies, such as anti-CTLA-4 and anti-PD-1, have transformed the treatment of advanced melanoma; however, a subset of patients fail to respond to immunotherapy. Thus, improved predictors of the anti-melanoma immune response and response to immunotherapy are needed. We have previously shown that 1) gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of a melanoma antigen, 2) there is variable GILT protein expression in melanoma cells from primary and cutaneous metastatic melanoma specimens, and 3) high GILT mRNA expression in melanoma specimens is associated with improved overall survival. Here, we created a database of n = 43 metastatic melanoma patients (stage III or IV) annotated with clinical outcome. Formalin-fixed, paraffin-embedded sections of pre-treatment metastatic melanoma specimens were tested for GILT protein expression using immunohistochemistry and scored by a dermatopathologist and dermatologist. GILT expression in melanoma cells was detected in 65% of lymph node and distant organ metastases, consistent with our prior finding of GILT expression in 58% of cutaneous metastases. The frequency of GILT-staining melanoma cells was determined by the ratio of the area of tumor cells with positive GILT staining to the total area of tumor cells. The frequency of GILT-staining melanoma cells among specimens with positive GILT staining was 15.2% ± 24.7% (mean ± SD). The median frequency of GILT staining was used to divide the patients into no/low and high GILT expression groups. The median overall survival was 52.8 months in the no/low GILT expression group, while the median overall survival was not reached in the high GILT expression group (log-rank, p = 0.67). For the n = 34 patients who were treated with immunotherapy targeting CTLA-4 and/or PD-1, the median overall survival was 38.2 months in the no/low GILT expression group, while the median overall survival was not reached in the high GILT expression group (log-rank, p = 0.83). These data suggest that high GILT expression in melanoma cells may be associated with improved overall survival, although a statistically significant association of GILT with survival was not observed in the whole group or the subset of patients treated with immunotherapy. A limitation of the current analysis is that 65% of patients have incomplete clinical outcome data. This study demonstrates variable GILT protein expression in tumor cells in metastatic melanoma specimens. Updated clinical outcome data will allow us to better evaluate the association of GILT protein expression in melanoma cells with overall survival and response to immunotherapy in metastatic melanoma patients.

MITF induces escape from innate immunity in melanoma

BackgroundThe application of immune-based therapies has revolutionized cancer treatment. Yet how the immune system responds to phenotypically heterogeneous populations within tumors is poorly understood. In melanoma, one of the major determinants of phenotypic identity is the lineage survival oncogene MITF that integrates diverse microenvironmental cues to coordinate melanoma survival, senescence bypass, differentiation, proliferation, invasion, metabolism and DNA damage repair. Whether MITF also controls the immune response is unknown.MethodsBy using several mouse melanoma models, we examine the potential role of MITF to modulate the anti-melanoma immune response. ChIP-seq data analysis, ChIP-qPCR, CRISPR-Cas9 genome editing, and luciferase reporter assays were utilized to identify ADAM10 as a direct MITF target gene. Western blotting, confocal microscopy, flow cytometry, and natural killer (NK) cytotoxicity assays were used to determine the underlying mechanisms by which MITF-driven phenotypic plasticity modulates melanoma NK cell-mediated killing.ResultsHere we show that MITF regulates expression of ADAM10, a key sheddase that cleaves the MICA/B family of ligands for NK cells. By controlling melanoma recognition by NK-cells MITF thereby controls the melanoma response to the innate immune system. Consequently, while melanoma MITFLow cells can be effectively suppressed by NK-mediated killing, MITF-expressing cells escape NK cell surveillance.ConclusionOur results reveal how modulation of MITF activity can impact the anti-melanoma immune response with implications for the application of anti-melanoma immunotherapies.

Specific Patterns of Blood ILCs in Metastatic Melanoma Patients and Their Modulations in Response to Immunotherapy.

Simple SummaryAnti-CTLA-4 and anti-PD-1 immune checkpoints inhibitors (ICI) have revolutionized the treatment of metastatic melanoma patients, leading to durable responses. However, some patients still not respond to this clinically used immunotherapies and there is a lack of biomarkers leading to the choice of first-line therapies. Innate lymphoid cells (ILC) express immune checkpoint receptors and are involved in anti-melanoma immune response. The aim of this article is to study ILCs from peripheral blood of melanoma patients receiving Ipilimumab, an anti-CTLA-4 treatment, and their association with clinical responses to this therapy. Our results show an impact of Ipilimumab on ILCs proportions and phenotype in blood. Moreover, the presence of anergic CD56dimCD16−DNAM-1− NK cells were associated with progression of the disease. These findings demonstrate the important role of ILC in the response to ICI.Immunotherapy targeting immune checkpoint receptors brought a breakthrough in the treatment of metastatic melanoma patients. However, a number of patients still resist these immunotherapies. Present on CD8+T cells, immune checkpoint receptors are expressed by innate lymphoid cells (ILCs), which may contribute to the clinical response. ILCs are composed of natural killer (NK) cells, which are cytotoxic effectors involved in tumor immunosurveillance. NK cell activation is regulated by a balance between activating receptors that detect stress molecules on tumor cells and HLA-I-specific inhibitory receptors. Helper ILCs (h-ILCs) are newly characterized ILCs that secrete cytokines and regulate the immune homeostasis of tissue. We investigated the modulation of blood ILCs in melanoma patients treated with ipilimumab. Circulating ILCs from metastatic stage IV melanoma patients and healthy donors were studied for their complete phenotypic status. Patients were studied before and at 3, 6, and 12 weeks of ipilimumab treatment. A comparison of blood ILC populations from donors and melanoma patients before treatment showed changes in proportions of ILC subsets, and a significant inverse correlation of CD56dim NK cells and h-ILC subsets was identified in patients. During treatment with ipilimumab, percentages of all ILC subsets were reduced. Ipilimumab also impacted the expression of the CD96/TIGIT/DNAM-1 pathway in all ILCs and increased CD161 and CTLA-4 expression by h-ILCs. When considering the response to the treatment, patients without disease control were characterized by higher percentages of CD56bright NK cells and ILC1. Patients with disease control displayed larger populations of activated CD56dimCD16+ DNAM-1+ NK cells, while anergic CD56dimCD16−DNAM-1− NK cells were prominent in patients without disease control. These results provide original findings on the distribution of ILC subsets in advanced melanoma patients and their modulation through immunotherapy. The effects of ipilimumab on these ILC subsets may critically influence therapeutic outcomes. These data indicate the importance of considering these innate cell subsets in immunotherapeutic strategies for melanoma patients.

Neutral Sphingomyelinase 2 Heightens Anti-Melanoma Immune Responses and Anti-PD-1 Therapy Efficacy.

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell-dependent immune responses and overcome resistance to anti-PD-1.

Novel Oncolytic Herpes Simplex Virus 1 VC2 Promotes Long-Lasting, Systemic Anti-melanoma Tumor Immune Responses and Increased Survival in an Immunocompetent B16F10-Derived Mouse Melanoma Model.

Oncolytic virotherapy (OVT) is now understood to be an immunotherapy that uses viral infection to liberate tumor antigens in an immunogenic context to promote the development of antitumor immune responses. The only currently FDA-approved oncolytic virotherapy, T-Vec, is a modified type 1 herpes simplex virus (HSV-1). While T-Vec is associated with limited response rates, its modest efficacy supports the continued development of novel OVT viruses. Herein, we test the efficacy of a recombinant HSV-1, VC2, as an OVT in a syngeneic B16F10-derived mouse model of melanoma. VC2 possesses mutations that block its ability to enter neurons via axonal termini. This greatly enhances its safety profile by precluding the ability of the virus to establish latent infection. VC2 has been shown to be a safe, effective vaccine against both HSV-1 and HSV-2 infection in mice, guinea pigs, and nonhuman primates. We found that VC2 slows tumor growth rates and that VC2 treatment significantly enhances survival of tumor-engrafted, VC2-treated mice over control treatments. VC2-treated mice that survived initial tumor engraftment were resistant to a second engraftment as well as colonization of lungs by intravenous introduction of tumor cells. We found that VC2 treatment induced substantial increases in intratumoral T cells and a decrease in immunosuppressive regulatory T cells. This immunity was critically dependent on CD8+ T cells and less dependent on CD4+ T cells. Our data provide significant support for the continued development of VC2 as an OVT for the treatment of human and animal cancers.IMPORTANCE Current oncolytic virotherapies possess limited response rates. However, when certain patient selection criteria are used, oncolytic virotherapy response rates have been shown to increase. This, in addition to the increased response rates of oncolytic virotherapy in combination with other immunotherapies, suggests that oncolytic viruses possess significant therapeutic potential for the treatment of cancer. As such, it is important to continue to develop novel oncolytic viruses as well as support basic research into their mechanisms of efficacy. Our data demonstrate significant clinical potential for VC2, a novel type 1 oncolytic herpes simplex virus. Additionally, due to the high rates of survival and the dependence on CD8+ T cells for efficacy, our model will enable study of the immunological correlates of protection for VC2 oncolytic virotherapy and oncolytic virotherapy in general. Understanding the mechanisms of efficacious oncolytic virotherapy will inform the rational design of improved oncolytic virotherapies.

Cascade of reactive oxygen species generation by polyprodrug for combinational photodynamic therapy

The redox status of cancer cells is well regulated by the balance between the reactive oxygen species (ROS) generation and elimination. Thus, the overall elevation of ROS level above the cellular tolerability threshold would lead to apoptotic or necrotic cell death. Herein, cinnamaldehyde (CA), a kind of oxidative stress amplified agent, was combined with photosensitizer pheophorbide A (PA) to promote the generation of ROS though synergistically endogenous and exogenous pathways. Firstly, acid-responsive polygalactose-co-polycinnamaldehyde polyprodrug (termed as PGCA) was synthesized, which could self-assemble into stable nanoparticles for the delivery of PA (termed as PGCA@PA NPs). The abundant expression of galactose receptor on tumor cells facilitated the positive targeting and cellular uptake efficiency of PGCA@PA NPs, after which PA could be synchronously released in company with the intracellular disassembly of PGCA NPs, due to the detaching of CA moieties under acidic microenvironment in endo/lysosomal compartment. Significantly increased ROS level was induced by the combined action of CA and PA with light irradiation, resulting in dramatically enhanced apoptosis of cancer cells. Importantly, intravenous injection of PGCA@PA NPs potently inhibited the tumor growth in hepatocellular carcinoma with negligible adverse effects. Moreover, combined with anti-programmed cell death protein 1 (anti-PD-1) therapy, PGCA@PA NPs treatment elicited anti-melanoma T-cell immune response and significantly promoted T cells infiltration in tumors. Hence, this novel polyprodrug nano delivery system was able to target and modulate the unique redox regulatory mechanisms of cancer cells through endogenous and exogenous pathways, providing a feasible approach to achieve synergetic therapeutic activity and selectivity.

Association of pathogenic germline variant KDR Q472H with angiogenesis and resistance to treatment in melanoma.

10060 Background: Preclinical data suggest that melanoma angiogenesis promotes resistance to MAPK-pathway (MAPKi) and immune checkpoint inhibitors (ICIs). However, phase II clinical trials of anti-angiogenic therapy in melanoma were disappointing. We previously identified a pathogenic germline variant Q472H in the kinase insert domain receptor [KDR Q472H; vascular endothelial growth factor receptor-2 (VEGFR-2)] in 35% of primary melanoma patients. We hypothesize that KDR Q472H promotes resistance to MAPKi or ICIs, and that combined MAPKi or ICI and VEGF pathway inhibition may improve outcomes in patients harboring the variant. Methods: Metastatic melanoma (MM) patient clinical data and biospecimens enrolled in the NYU Langone Medical Center Melanoma program were studied. KDR status was determined by TaqMan assays. Tumor microvessel density (MVD) was assessed by CD34 immunohistochemistry. The impact of KDR Q472H on the tumor microenvironment was determined by RNA-seq and Nanostring. Synergy between BRAF (dabrafenib) and VEGFR-2 (lenvatinib) inhibitors in KDR-genotyped MM cell lines was assessed using cell proliferation assays and the Chou-Talalay method. Synergy between ICIs and anti-VEGFR-2 was evaluated in vivo using a B16 melanoma model. Results: We studied 221 MM patients (38% KDR Q472H variants). KDR Q472H variant was significantly associated with higher tumor MVD (P = 0.002). Among the MAPKi-treated patients, KDR Q472H homozygotes had shorter median progression-free survival (PFS, 3.3 vs 9.7 months, P = 0.009) than KDR wild type (WT). In patients treated with anti-PD-1-based therapies, response rates were lower in KDR Q472H variant patients compared to WT (P = 0.012), with shorter median PFS (8.4 months vs not reached, P = 0.0443). Transcriptomic analyses identified an immunosuppressive phenotype in KDR Q472H tumors, with reduced expression of genes associated with chemotaxis, inflammation, T cell activity, and antigen presentation. Consistent with this finding, VEGFR-2 blockade in a KDR Q472H B16 mouse melanoma model augmented the anti-melanoma immune response. KDR Q472H cell lines displayed synergistic cytotoxicity with dabrafenib and lenvatinib, compared to KDR WT cells. Conclusions: Our data demonstrate that melanoma patients with pathogenic germline variant KDR Q472H may be more resistant to both ICIs and MAPKi. Anti-angiogenic therapy should be reconsidered within this specific subset of patients in prospective clinical trials.

Identifying a uniform T cell signature associated with protective anti-tumor immunity and autoimmune systemic sclerosis

Abstract Precise tug-of-war between immunosuppressive regulatory T (Treg) cells and inflammatory IL-17-producing (Th17) cells ensures appropriate reactivity to pathogenic challenge. While an imbalance towards Tregs leads to suboptimal responses as observed in cancer, unchecked Th17 activity leads to autoimmunity. Though numerous mechanisms have been identified for how Tregs suppress effector cells, productive effector T cell responses are believed to result from passive escape of Treg suppression. Here we report a novel active role for Th17 cells in dampening Tregs to promote effector activity in the context of melanoma. We hypothesize that Th17 cells can physically impede Treg suppression resulting in enrichment of CD8 T cell prevalence and effector function. Using a melanoma mouse model to characterize Th17 effectors responsible for inhibiting Tregs, we found elimination of IL-17-producing cells prevents vaccine-induced anti-melanoma immune responses. This suggests Th17 cells are important for induction of CD8-mediated anti-tumor immunity. Using RNA-Seq analysis, we generated a gene expression (GE) signature to characterize Th17 effectors responsible for inhibiting Tregs in the context of tumor vaccination. Interestingly, increased ratio of Th17 to Treg cells may contribute to pathogenesis of localized and systemic scleroderma. Diminished Treg- and enhanced Th17-associated genes are consistently found in scleroderma patient skin, leading us to hypothesize that scleroderma pathogenesis has a similar Th17 GE signature to that associated with enhanced tumor clearance. Using the tumor-associated Th17 GE signature to examine scleroderma patient samples we determined transcriptional overlap between the Th17 cell subsets.

Targeting tumor-resident mast cells for effective anti-melanoma immune responses.

Immune checkpoint blockade has revolutionized cancer treatment. Patients developing immune mediated adverse events, such as colitis, appear to particularly benefit from immune checkpoint inhibition. Yet, the contributing mechanisms are largely unknown. We identified a systemic LPS signature in melanoma patients with colitis following anti-cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) checkpoint inhibitor treatment and hypothesized that intestinal microbiota-derived LPS contributes to therapeutic efficacy. Because activation of immune cells within the tumor microenvironment is considered most promising to effectively control cancer, we analyzed human and murine melanoma for known sentinels of LPS. We identified mast cells (MCs) accumulating in and around melanomas and showed that effective melanoma immune control was dependent on LPS-activated MCs recruiting tumor-infiltrating effector T cells by secretion of CXCL10. Importantly, CXCL10 was also upregulated in human melanomas with immune regression and in patients with colitis induced by anti-CTLA-4 antibody. Furthermore, we demonstrate that CXCL10 upregulation and an MC signature at the site of melanomas are biomarkers for better patient survival. These findings provide conclusive evidence for a "Trojan horse treatment strategy" in which the plasticity of cancer-resident immune cells, such as MCs, is used as a target to boost tumor immune defense.

508 Melanogenic property of melanoma is associated with aryl hydrocarbon receptor-induced suppression of anti-melanoma immune response

Melanoma is the solid tumor with high mutational burden, which leads the generation of neo-antigens and the infiltration of cytotoxic T cells (CTLs) recognizing these antigens. Recent improvement of prognosis of melanoma resulting from anti PD-1 antibody elucidated the importance of the expression of PD-1 on tumor-infiltrating CTLs for melanoma cells to evade the cytotoxic activity of CTLs. Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that responds to a wide range of chemicals and induces the battery of genes associated with detoxification, including CYP1A1 and CYP1B1. Recently Liu and colleagues reported the upregulation of PD-1 on CTLs is induced via AHR activation by binding of AHR ligands released from melanoma cells. Among various solid tumors, melanoma exerts the relatively stronger suppression to anti-tumor immunity. Notably, characteristic biology in melanoma compared to other solid tumors is the ability of melanogenesis, though little is known about the association between melanogenesis and anti-tumor immune response. Therefore, we investigated the correlation among melanogenesis, AHR activation and PD-1 expression in the lesion of melanoma by using published RNA-seq data and immunohistochemistry of biopsy samples. Gene set variation analysis of RNA-seq data from microdissected lesions of melanoma revealed the correlation between the gene set of AHR system and that of melanogenesis in melanoma. Moreover, immunohistochemical analysis revealed CYP1A1 is expressed in Tyrosinase+ melanoma cells and their surrounding CTLs, which express PD-1. Furthermore, given the clinical data, the level of CYP1A1 expression in melanoma lesion tends to correlate with the efficacy of immunotherapies including anti PD-1 antibody. These results indicate the melanogenic property of melanoma cells enables them to activate AHR system, which suppresses anti-tumor immunity via the upregulation of PD-1 on CTLs in some way related to melanogenesis.

Abstract 941: B cells are required to generate optimal antitumor immunity in response to PD-1 blockade treatment

Abstract Background: Melanoma is the most deadly form of skin cancer. Although, the immunogenic nature of melanoma makes this disease susceptible to immunotherapy, metastatic melanoma remains highly resistant to established immunotherapies like cancer vaccines. Anti-PD-1 antibodies alone showed great clinical activity but were still ineffective in many melanoma patients. These reports highlight the need to understand the molecular mechanism of immunotherapy induced anti-melanoma immune responses to develop new approaches for effective therapy against melanoma. B cells augment CD8+ T cell-mediated immunity during autoimmunity and allograft rejection, but the role of B cells in anti-tumor CD8+ T cell immunity and in cancer patient survival is controversial. Recently, we reported that intratumoral stimulation of TLR-7/8 in a mouse model of melanoma induced tumor regression and activation of B cells. B cells were required for tumor suppression in an antibody-independent manner. Also, we found that melanoma patients who responded to anti-PD-1 blockade therapy had significantly more B cells and CD8+ T cells in tumors than non-responders. Based on these evidences, we hypothesized that activated B cells induce and/or enhance anti-melanoma CD8+ T cell immunity in response to immunotherapy. Methods: B16 melanoma mouse model was used. Wild type and B cell Knockout mice were used to determine treatment efficacy and generation of tumor-specific CD8+T cells in response to PD-L1 blockade and/or TLR-7/8 activation. TLR-7/8 agonist activated B cells were isolated and adoptively transferred into tumor-bearing B cell KO mice, which were treated with anti-PD-L1.Therapeutic efficacy and melanoma-specific CD8+ T cell immunity were determined. Results: We found that absence of B cells not only decreases the efficacy of TLR-7/8 agonist or anti-PDL-1 abs monotherapy but also TLR-7/8 agonist and anti-PD-L1 abs combination therapy. In addition, mice were unable to develop potent anti- melanoma CD8+T cell immunity in the absence of B cells in response to either mono or combination therapy. Interestingly, we reported prolonged survival of tumor bearing B cell KO mice which received TLR-7/8 agonist activated B cells compared to inactivated B cells recipients in response to anti-PD-L1 antibody therapy. Conclusions: These results suggest that activated B cells enhance anti-melanoma CD8+ T cell immunity in response to immunotherapy. We expect our findings to lead the field closer to development of biomarkers based on B cell activation that predict response to immunotherapy. Our findings may also facilitate design of more effective anti-melanoma immunotherapy. Citation Format: Shubhra Singh, Karishma Bavisi, Jason Roszik, Adi Diab, Richard E. Davis, Patrick Hwu, Willem W. Overwijk, Manisha Singh. B cells are required to generate optimal antitumor immunity in response to PD-1 blockade treatment [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 941.