Acral Melanoma Is Infiltrated with cDC1s and Functional Exhausted CD8 T Cells Similar to the Cutaneous Melanoma of Sun-Exposed Skin.

Acral melanoma (AM) is the most common melanoma in non-Caucasian populations, yet it remains largely understudied. As AM lacks the UV-radiation mutational signatures that characterize other cutaneous melanomas, it is considered devoid of immunogenicity and is rarely included in clinical trials assessing novel immunotherapeutic regimes aiming to recover the antitumor function of immune cells. We studied a Mexican cohort of melanoma patients from the Mexican Institute of Social Security (IMSS) (n = 38) and found an overrepresentation of AM (73.9%). We developed a multiparametric immunofluorescence technique coupled with a machine learning image analysis to evaluate the presence of conventional type 1 dendritic cells (cDC1) and CD8 T cells in the stroma of melanoma, two of the most relevant immune cell types for antitumor responses. We observed that both cell types infiltrate AM at similar and even higher levels than other cutaneous melanomas. Both melanoma types harbored programmed cell death protein 1 (PD-1+) CD8 T cells and PD-1 ligand (PD-L1+) cDC1s. Despite this, CD8 T cells appeared to preserve their effector function and expanding capacity as they expressed interferon-γ (IFN-γ) and KI-67. The density of cDC1s and CD8 T cells significantly decreased in advanced stage III and IV melanomas, supporting these cells' capacity to control tumor progression. These data also argue that AM could respond to anti-PD-1-PD-L1 immunotherapy.

The cholera toxin B subunit induces trained immunity in dendritic cells and promotes CD8 T cell antitumor immunity.

Introduction: Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches hematopoietic precursors in the bone marrow to sustain a memory-like phenotype. Dendritic cells (DCs) can exhibit memory-like responses, enhanced upon subsequent exposure to a pathogen; however, whether this imprinting is lineage and stimulus-restricted is still being determined. Nevertheless, the functional consequences of DCs training on the adaptive and protective immune response against non-infectious diseases remain unresolved. Methods: We evaluated the effect of the nontoxic cholera B subunit (CTB), LPS and LTA in the induction of trained immunity in murine DCs revealed by TNFa and LDH expression, through confocal microscopy. Additionally, we obtained bone marrow DCs (BMDCs) from mice treated with CTB, LPS, and LTA and evaluated training features in DCs and their antigen-presenting cell capability using multiparametric cytometry. Finally, we design an experimental melanoma mouse model to demonstrate protection induced by CTB-trained DCs in vivo. Results: CTB-trained DCs exhibit increased expression of TNFa, and metabolic reprogramming indicated by LDH expression. Moreover, CTB training has an imprint on DC precursors, increasing the number and antigen-presenting function in BMDCs. We found that training by CTB stimulates the recruitment of DC precursors and DCs infiltration at the skin and lymph nodes. Interestingly, training-induced by CTB promotes a highly co-stimulatory phenotype in tumor-infiltrating DCs (CD86+) and a heightened functionality of exhausted CD8 T cells (Ki67+, GZMB+), which were associated with a protective response against melanoma challenge in vivo. Conclusion: Our work indicates that CTB can induce innate immune training on DCs, which turns into an efficient adaptive immune response in the melanoma model and might be a potential immunotherapeutic approach for tumor growth control.

TCF1-positive and TCF1-negative TRM CD8 T cell subsets and cDC1s orchestrate melanoma protection and immunotherapy response.

BACKGROUND: Melanoma, the most lethal form of skin cancer, has undergone a transformative treatment shift with the advent of checkpoint blockade immunotherapy (CBI). Understanding the intricate network of immune cells infiltrating the tumor and orchestrating the control of melanoma cells and the response to CBI is currently of utmost importance. There is evidence underscoring the significance of tissue-resident memory (TRM) CD8 T cells and classic dendritic cell type 1 (cDC1) in cancer protection. Transcriptomic studies also support the existence of a TCF7+ (encoding TCF1) T cell as the most important for immunotherapy response, although uncertainty exists about whether there is a TCF1+TRM T cell due to evidence indicating TCF1 downregulation for tissue residency activation. METHODS: We used multiplexed immunofluorescence and spectral flow cytometry to evaluate TRM CD8 T cells and cDC1 in two melanoma patient cohorts: one immunotherapy-naive and the other receiving immunotherapy. The first cohort was divided between patients free of disease or with metastasis 2 years postdiagnosis while the second between CBI responders and non-responders. RESULTS: Our study identifies two CD8+TRM subsets, TCF1+ and TCF1-, correlating with melanoma protection. TCF1+TRM cells show heightened expression of IFN-γ and Ki67 while TCF1- TRM cells exhibit increased expression of cytotoxic molecules. In metastatic patients, TRM subsets undergo a shift in marker expression, with the TCF1- subset displaying increased expression of exhaustion markers. We observed a close spatial correlation between cDC1s and TRMs, with TCF1+TRM/cDC1 pairs enriched in the stroma and TCF1- TRM/cDC1 pairs in tumor areas. Notably, these TCF1- TRMs express cytotoxic molecules and are associated with apoptotic melanoma cells. Both TCF1+ and TCF1- TRM subsets, alongside cDC1, prove relevant to CBI response. CONCLUSIONS: Our study supports the importance of TRM CD8 T cells and cDC1 in melanoma protection while also highlighting the existence of functionally distinctive TCF1+ and TCF1- TRM subsets, both crucial for melanoma control and CBI response.

CXCR5 and TIM-3 expressions define distinct exhausted T cell subsets in experimental cutaneous infection with Leishmania mexicana.

T-cell exhaustion is a key stage in chronic infections since it limits immunopathology, but also hinders the elimination of pathogens. Exhausted T (Tex) cells encompass dynamic subsets, including progenitor cells that sustain long-term immunity through their memory/stem like properties, and terminally-differentiated cells, resembling the so-called Tex cells. The presence of Tex cells in chronic leishmaniasis has been reported in humans and murine models, yet their heterogeneity remains unexplored. Using flow cytometry, we identified Tex cells subtypes based on PD-1, CXCR5 and TIM-3 expressions in draining lymph nodes (dLNs) and lesion sites of C57BL/6 mice infected with L. mexicana at 30-, 60- and 90-days post-infection. We showed that infected mice developed a chronic infection characterized by non-healing lesions with a high parasite load and impaired Th1/Th2 cytokine production. Throughout the infection, PD-1+ cells were observed in dLNs, in addition to an enhanced expression of PD-1 in both CD4+ and CD8+ T lymphocytes. We demonstrated that CD4+ and CD8+ T cells were subdivided into PD-1+CXCR5+TIM-3- (CXCR5+), PD-1+CXCR5+TIM-3+ (CXCR5+TIM-3+), and PD-1+CXCR5-TIM-3+ (TIM-3+) subsets. CXCR5+ Tex cells were detected in dLNs during the whole course of the infection, whereas TIM-3+ cells were predominantly localized in the infection sites at day 90. CXCR5+TIM-3+ cells only increased at 30 and 60 days of infection in dLNs, whereas no increase was observed in the lesions. Phenotypic analysis revealed that CXCR5+ cells expressed significantly higher levels of CCR7 and lower levels of CX3CR1, PD-1, TIM-3, and CD39 compared to the TIM-3+ subset. CXCR5+TIM-3+ cells expressed the highest levels of all exhaustion-associated markers and of CX3CR1. In agreement with a less exhausted phenotype, the frequency of proliferating Ki-67 and IFN-γ expressing cells was significantly higher in the CXCR5+ subset within both CD4+ and CD8+ T cells compared to their respective TIM-3+ subsets, whereas CD8+CXCR5+TIM-3+ and CD8+TIM-3+ subsets showed an enhanced frequency of degranulating CD107a+ cells. In summary, we identified a novel, less-differentiated CXCR5+ Tex subset in experimental cutaneous leishmaniasis caused by L. mexicana. Targeting these cells through immune checkpoint inhibitors such as anti-PD-1 or anti PD-L1 might improve the current treatment for patients with the chronic forms of leishmaniasis.

Immunotherapy Options for Acral Melanoma, A fast-growing but Neglected Malignancy.

Melanoma is the deadliest form of skin cancer. It is classified as cutaneous and non-cutaneous, with the former characterized by developing in sun-exposed areas of the skin, UV-light radiation being its most important risk factor and ordinarily affecting fair skin populations. In recent years, the incidence of melanoma has been increasing in populations with darker complexion, for example, Hispanics, in which acral melanoma is highly prevalent. The WHO estimates that the incidence and mortality of melanoma will increase by more than 60% by 2040, particularly in low/medium income countries. Acral melanoma appears in the palms, soles and nails, and because of these occult locations, it is often considered different from other cutaneous melanomas even though it also originates in the skin. Acral melanoma is very rare in Caucasian populations and is often not included from genetic analysis and clinical trials. In this review, we present the worldwide epidemiology of acral melanoma; we summarize its genetic characterization and point out important signaling pathways for targeted therapy. We also discuss how genetic analyses have shown that acral melanoma carries a sufficient mutational load and neoantigen formation to be targeted by the immune system, arguing for a potential benefit with novel immunotherapeutic strategies, alone or combined with targeted therapy. This is important because chemotherapy remains the first-line treatment in non-developed nations despite a disheartening response. In summary, the increased incidence and mortality of acral melanoma in low/medium income countries calls for increasing our knowledge about its nature and therapeutic options and leveling off the asymmetric research conducted primarily on Caucasian populations.