Adoptive T Cell Therapy Targeting Different Gene Products Reveals Diverse and Context-Dependent Immune Evasion in Melanoma.

Tumor immune escape limits durable responses to T cell therapy. Here, we examined how regulation and function of gene products that provide the target epitopes for CD8+ T cell anti-tumor immunity influence therapeutic efficacy and resistance. We used a CRISPR-Cas9-based method (CRISPitope) in syngeneic melanoma models to fuse the same model CD8+ T cell epitope to the C-termini of different endogenous gene products. Targeting melanosomal proteins or oncogenic CDK4R24C (Cyclin-dependent kinase 4) by adoptive cell transfer (ACT) of the same epitope-specific CD8+ T cells revealed diverse genetic and non-genetic immune escape mechanisms. ACT directed against melanosomal proteins, but not CDK4R24C, promoted melanoma dedifferentiation, and increased myeloid cell infiltration. CDK4R24C antigen persistence was associated with an interferon-high and T-cell-rich tumor microenvironment, allowing for immune checkpoint inhibition as salvage therapy. Thus, the choice of target antigen determines the phenotype and immune contexture of recurrent melanomas, with implications to the design of cancer immunotherapies.

Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.

Amplification of N-Myc is associated with a T-cell-poor microenvironment in metastatic neuroblastoma restraining interferon pathway activity and chemokine expression.

Immune checkpoint inhibitors have significantly improved the treatment of several cancers. T-cell infiltration and the number of neoantigens caused by tumor-specific mutations are correlated to favorable responses in cancers with a high mutation load. Accordingly, checkpoint immunotherapy is thought to be less effective in tumors with low mutation frequencies such as neuroblastoma, a neuroendocrine tumor of early childhood with poor outcome of the high-risk disease group. However, spontaneous regressions and paraneoplastic syndromes seen in neuroblastoma patients suggest substantial immunogenicity. Using an integrative transcriptomic approach, we investigated the molecular characteristics of T-cell infiltration in primary neuroblastomas as an indicator of pre-existing immune responses and potential responsiveness to checkpoint inhibition. Here, we report that a T-cell-poor microenvironment in primary metastatic neuroblastomas is associated with genomic amplification of the MYCN (N-Myc) proto-oncogene. These tumors exhibited lower interferon pathway activity and chemokine expression in line with reduced immune cell infiltration. Importantly, we identified a global role for N-Myc in the suppression of interferon and pro-inflammatory pathways in human and murine neuroblastoma cell lines. N-Myc depletion potently enhanced targeted interferon pathway activation by a small molecule agonist of the cGAS-STING innate immune pathway. This promoted chemokine expression including Cxcl10 and T-cell recruitment in microfluidics migration assays. Hence, our data suggest N-Myc inhibition plus targeted IFN activation as adjuvant strategy to enforce cytotoxic T-cell recruitment in MYCN-amplified neuroblastomas.

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma.

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

T-cell immune function in tumor, skin, and peripheral blood of advanced stage melanoma patients: implications for immunotherapy.

PURPOSE: To predict the potential antitumor effect of antigen-specific T cells in melanoma patients, we investigated T-cell effector function in relation to tumor-escape mechanisms. EXPERIMENTAL DESIGN: CD8(+) T cells isolated from tumor, adjacent normal skin, and peripheral blood of 17 HLA-A2(+) patients with advanced-stage melanoma were analyzed for their antigen specificity and effector function against melanocyte differentiation antigens MART-1, gp100, and tyrosinase by using HLA-A2/peptide tetramers and functional assays. In addition, the presence of tumor-escape mechanisms PD-L1/PD-1 pathway, FoxP3 and loss of HLA or melanocyte differentiation antigens, both required for tumor cell recognition and killing, were studied. RESULTS: Higher percentages of melanocyte antigen-specific CD8(+) T cells were found in the melanoma tissues as compared with adjacent normal skin and peripheral blood. Functional analysis revealed 2 important findings: (i) in 5 of 17 patients, we found cytokine production after specific peptide stimulation by tumor-infiltrating lymphocytes (TIL), not by autologous peripheral blood lymphocytes (PBL); (ii) CD8(+) T cells from 7 of 17 patients did not produce cytokines after specific stimulation, which corresponded with significant loss of tumor HLA-A2 expression. The presence of other tumor-escape mechanisms did not correlate to T-cell function. CONCLUSIONS: Our data show that functional T-cell responses could be missed when only PBL and not TIL are evaluated, emphasizing the importance of TIL analysis for immunomonitoring. Furthermore, loss of tumor HLA-A2 may explain the lack of T-cell functionality. These findings have important implications for selecting melanoma patients who may benefit from immunotherapy.

Skin-depigmenting agent monobenzone induces potent T-cell autoimmunity toward pigmented cells by tyrosinase haptenation and melanosome autophagy.

In this study, we report the previously unknown mechanism of inducing robust anti-melanoma immunity by the vitiligo-inducing compound monobenzone. We show monobenzone to increase melanocyte and melanoma cell immunogenicity by forming quinone-haptens to the tyrosinase protein and by inducing the release of tyrosinase- and melanoma antigen recognized by T cells-1 (MART-1)-containing CD63+ exosomes following melanosome oxidative stress induction. Monobenzone further augments the processing and shedding of melanocyte-differentiation antigens by inducing melanosome autophagy and enhanced tyrosinase ubiquitination, ultimately activating dendritic cells, which induced cytotoxic human melanoma-reactive T cells. These T cells effectively eradicate melanoma in vivo, as we have reported previously. Monobenzone thereby represents a promising and readily applicable compound for immunotherapy in melanoma patients.

Effective melanoma immunotherapy in mice by the skin-depigmenting agent monobenzone and the adjuvants imiquimod and CpG.

BACKGROUND: Presently melanoma still lacks adequate treatment options for metastatic disease. While melanoma is exceptionally challenging to standard regimens, it is suited for treatment with immunotherapy based on its immunogenicity. Since treatment-related skin depigmentation is considered a favourable prognostic sign during melanoma intervention, we here aimed at the reverse approach of directly inducing vitiligo as a shortcut to effective anti-melanoma immunity. METHODOLOGY AND PRINCIPAL FINDINGS: We developed an effective and simple to use form of immunotherapy by combining the topical skin-bleaching agent monobenzone with immune-stimulatory imiquimod cream and cytosine-guanine oligodeoxynucleotides (CpG) injections (MIC therapy). This powerful new approach promptly induced a melanoma antigen-specific immune response, which abolished subcutaneous B16.F10 melanoma growth in up to 85% of C57BL/6 mice. Importantly, this regimen induced over 100 days of tumor-free survival in up to 60% of the mice, and forcefully suppressed tumor growth upon re-challenge either 65- or 165 days after MIC treatment cessation. CONCLUSIONS: MIC therapy is effective in eradicating melanoma, by vigilantly incorporating NK-, B- and T cells in its therapeutic effect. Based on these results, the MIC regimen presents a high-yield, low-cost and simple therapy, readily applicable in the clinic.

Autoimmune destruction of skin melanocytes by perilesional T cells from vitiligo patients.

In vitiligo, cytotoxic T cells infiltrating the perilesional margin are suspected to be involved in the pathogenesis of the disease. However, it remains to be elucidated whether these T cells are a cause or a consequence of the depigmentation process. T cells we obtained from perilesional skin biopsies, were significantly enriched for melanocyte antigen recognition, compared with healthy skin-infiltrating T cells, and were reactive to melanocyte antigen-specific stimulation. Using a skin explant model, we were able to dissect the in situ activities of perilesional T cells in the effector phase of depigmentation. We show that these T cells could infiltrate autologous normally pigmented skin explants and efficiently kill melanocytes within this microenvironment. Interestingly, melanocyte apoptosis was accompanied by suprabasal keratinocyte apoptosis. Perilesional T cells did, however, not induce apoptosis in lesional skin, which is devoid of melanocytes, indicating the melanocyte-specific cytotoxic activity of these cells. Melanocyte killing correlated to local infiltration of perilesional T cells. Our data show that perilesional cytotoxic T cells eradicate pigment cells, the characteristic hallmark of vitiligo, thereby providing evidence of T cells being able to mediate targeted autoimmune tissue destruction.