Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19.

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19.

Immunodeficiency syndromes differentially impact the functional profile of SARS-CoV-2-specific T cells elicited by mRNA vaccination.

Many immunocompromised patients mount suboptimal humoral immunity after SARS-CoV-2 mRNA vaccination. Here, we assessed the single-cell profile of SARS-CoV-2-specific T cells post-mRNA vaccination in healthy individuals and patients with various forms of immunodeficiencies. Impaired vaccine-induced cell-mediated immunity was observed in many immunocompromised patients, particularly in solid-organ transplant and chronic lymphocytic leukemia patients. Notably, individuals with an inherited lack of mature B cells, i.e., X-linked agammaglobulinemia (XLA) displayed highly functional spike-specific T cell responses. Single-cell RNA-sequencing further revealed that mRNA vaccination induced a broad functional spectrum of spike-specific CD4+ and CD8+ T cells in healthy individuals and patients with XLA. These responses were founded on polyclonal repertoires of CD4+ T cells and robust expansions of oligoclonal effector-memory CD45RA+ CD8+ T cells with stem-like characteristics. Collectively, our data provide the functional continuum of SARS-CoV-2-specific T cell responses post-mRNA vaccination, highlighting that cell-mediated immunity is of variable functional quality across immunodeficiency syndromes.

Dynamic MAIT Cell Recovery after Severe COVID-19 Is Transient with Signs of Heterogeneous Functional Anomalies.

Mucosal-associated invariant T (MAIT) cells are an abundant population of unconventional T cells in humans and play important roles in immune defense against microbial infections. Severe COVID-19 is associated with strong activation of MAIT cells and loss of these cells from circulation. In the present study, we investigated the capacity of MAIT cells to recover after severe COVID-19. In longitudinal paired analysis, MAIT cells initially rebounded numerically and phenotypically in most patients at 4 mo postrelease from the hospital. However, the rebounding MAIT cells displayed signs of persistent activation with elevated expression of CD69, CD38, and HLA-DR. Although MAIT cell function was restored in many patients, a subgroup displayed a predominantly PD-1high functionally impaired MAIT cell pool. This profile was associated with poor expression of IFN-γ and granzyme B in response to IL-12 + L-18 and low levels of polyfunctionality. Unexpectedly, although the overall T cell counts recovered, normalization of the MAIT cell pool failed at 9-mo follow-up, with a clear decline in MAIT cell numbers and a further increase in PD-1 levels. Together, these results indicate an initial transient period of inconsistent recovery of MAIT cells that is not sustained and eventually fails. Persisting MAIT cell impairment in previously hospitalized patients with COVID-19 may have consequences for antimicrobial immunity and inflammation and could potentially contribute to post-COVID-19 health problems.

COVID-19-specific metabolic imprint yields insights into multiorgan system perturbations.

Corona disease 2019 (COVID-19) affects multiple organ systems. Recent studies have indicated perturbations in the circulating metabolome linked to COVID-19 severity. However, several questions pertain with respect to the metabolome in COVID-19. We performed an in-depth assessment of 1129 unique metabolites in 27 hospitalized COVID-19 patients and integrated results with large-scale proteomic and immunology data to capture multiorgan system perturbations. More than half of the detected metabolic alterations in COVID-19 were driven by patient-specific confounding factors ranging from comorbidities to xenobiotic substances. Systematically adjusting for this, a COVID-19-specific metabolic imprint was defined which, over time, underwent a switch in response to severe acute respiratory syndrome coronavirus-2 seroconversion. Integration of the COVID-19 metabolome with clinical, cellular, molecular, and immunological severity scales further revealed a network of metabolic trajectories aligned with multiple pathways for immune activation, and organ damage including neurological inflammation and damage. Altogether, this resource refines our understanding of the multiorgan system perturbations in severe COVID-19 patients.

MAIT cell compartment characteristics are associated with the immune response magnitude to the BNT162b2 mRNA anti-SARS-CoV-2 vaccine.

Mucosa-associated invariant T (MAIT) cells are unconventional T cells with innate-like capacity to rapidly respond to microbial infection via MR1-restricted antigen recognition. Emerging evidence indicate that they can also act as rapid sensors of viral infection via innate cytokine activation. However, their possible role in the immune response to mRNA vaccination is unknown. Here, we evaluated the involvement of MAIT cells in individuals vaccinated with the BNT162b2 mRNA SARS-CoV-2 vaccine. MAIT cell levels, phenotype and function in circulation were preserved and unperturbed through day 35 post-vaccination in healthy donor (HD) vaccinees, as well as people living with HIV (PLWH) or with primary immunodeficiency (PID). Unexpectedly, pre-vaccination and post-vaccination levels of MAIT cells correlated positively with the magnitude of the SARS-CoV-2 spike protein-specific CD4 T cell and antibody responses in the HD vaccinees. This pattern was largely preserved in the PID group, but less so in the PLWH group. Furthermore, in the HD vaccinees levels of MAIT cell activation and cytolytic potential correlated negatively to the adaptive antigen-specific immune responses. These findings indicate an unexpected association between MAIT cell compartment characteristics and the immune response magnitude to the BNT162b2 mRNA vaccine.

NK cell frequencies, function and correlates to vaccine outcome in BNT162b2 mRNA anti-SARS-CoV-2 vaccinated healthy and immunocompromised individuals.

Adaptive immune responses have been studied extensively in the course of mRNA vaccination against COVID-19. Considerably fewer studies have assessed the effects on innate immune cells. Here, we characterized NK cells in healthy individuals and immunocompromised patients in the course of an anti-SARS-CoV-2 BNT162b2 mRNA prospective, open-label clinical vaccine trial. See trial registration description in notes. Results revealed preserved NK cell numbers, frequencies, subsets, phenotypes, and function as assessed through consecutive peripheral blood samplings at 0, 10, 21, and 35 days following vaccination. A positive correlation was observed between the frequency of NKG2C+ NK cells at baseline (Day 0) and anti-SARS-CoV-2 Ab titers following BNT162b2 mRNA vaccination at Day 35. The present results provide basic insights in regards to NK cells in the context of mRNA vaccination, and have relevance for future mRNA-based vaccinations against COVID-19, other viral infections, and cancer.Trial registration: The current study is based on clinical material from the COVAXID open-label, non-randomized prospective clinical trial registered at EudraCT and clinicaltrials.gov (no. 2021-000175-37). Description: https://clinicaltrials.gov/ct2/show/NCT04780659?term=2021-000175-37&draw=2&rank=1 .

The Karolinska KI/K COVID-19 Immune Atlas: An open resource for immunological research and educational purposes.

The Karolinska KI/K COVID-19 Immune Atlas project was conceptualized in March 2020 as a part of the academic research response to the developing SARS-CoV-2 pandemic. The aim was to rapidly provide a curated dataset covering the acute immune response towards SARS-CoV-2 infection in humans, as it occurred during the first wave. The Immune Atlas was built as an open resource for broad research and educational purposes. It contains a presentation of the response evoked by different immune and inflammatory cells in defined naïve patient-groups as they presented with moderate and severe COVID-19 disease. The present Resource Article describes how the Karolinska KI/K COVID-19 Immune Atlas allow scientists, students, and other interested parties to freely explore the nature of the immune response towards human SARS-CoV-2 infection in an online setting.

MAIT cell counts are associated with the risk of hospitalization in COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation associated with chronic inflammation in the airways. Mucosal-associated invariant T (MAIT) cells are unconventional, innate-like T cells highly abundant in mucosal tissues including the lung. We hypothesized that the characteristics of MAIT cells in circulation may be prospectively associated with COPD morbidity. METHODS: COPD subjects (n = 61) from the Tools for Identifying Exacerbations (TIE) study were recruited when in stable condition. At study entry, forced expiratory volume in 1 s (FEV1) was measured and peripheral blood mononuclear cells were cryopreserved for later analysis by flow cytometry. Patients were followed for 3 years to record clinically meaningful outcomes. RESULTS: Patients who required hospitalization at one or more occasions during the 3-year follow-up (n = 21) had lower MAIT cell counts in peripheral blood at study inclusion, compared with patients who did not get hospitalized (p = 0.036). In contrast, hospitalized and never hospitalized patients did not differ in CD8 or CD4 T cell counts (p = 0.482 and p = 0.221, respectively). Moreover, MAIT cells in hospitalized subjects showed a more activated phenotype with higher CD38 expression (p = 0.014), and there was a trend towards higher LAG-3 expression (p = 0.052). Conventional CD4 and CD8 T cells were similar between the groups. Next we performed multi-variable logistic regression analysis with hospitalizations as dependent variable, and FEV1, GOLD 2017 group, and quantity or activation of MAIT and conventional T cells as independent variables. MAIT cell count, CD38 expression on MAIT cells, and LAG-3 expression on both MAIT and CD8 T cells were all independently associated with the risk of hospitalization. CONCLUSIONS: These findings suggest that MAIT cells might reflect a novel, FEV1-independent immunological dimension in the complexity of COPD. The potential implication of MAIT cells in COPD pathogenesis and MAIT cells' prognostic potential deserve further investigation.

HCMV triggers frequent and persistent UL40-specific unconventional HLA-E-restricted CD8 T-cell responses with potential autologous and allogeneic peptide recognition.

Immune response against human cytomegalovirus (HCMV) includes a set of persistent cytotoxic NK and CD8 T cells devoted to eliminate infected cells and to prevent reactivation. CD8 T cells against HCMV antigens (pp65, IE1) presented by HLA class-I molecules are well characterized and they associate with efficient virus control. HLA-E-restricted CD8 T cells targeting HCMV UL40 signal peptides (HLA-EUL40) have recently emerged as a non-conventional T-cell response also observed in some hosts. The occurrence, specificity and features of HLA-EUL40 CD8 T-cell responses remain mostly unknown. Here, we detected and quantified these responses in blood samples from healthy blood donors (n = 25) and kidney transplant recipients (n = 121) and we investigated the biological determinants involved in their occurrence. Longitudinal and phenotype ex vivo analyses were performed in comparison to HLA-A*02/pp65-specific CD8 T cells. Using a set of 11 HLA-E/UL40 peptide tetramers we demonstrated the presence of HLA-EUL40 CD8 αßT cells in up to 32% of seropositive HCMV+ hosts that may represent up to 38% of total circulating CD8 T-cells at a time point suggesting a strong expansion post-infection. Host's HLA-A*02 allele, HLA-E *01:01/*01:03 genotype and sequence of the UL40 peptide from the infecting strain are major factors affecting the incidence of HLA-EUL40 CD8 T cells. These cells are effector memory CD8 (CD45RAhighROlow, CCR7-, CD27-, CD28-) characterized by a low level of PD-1 expression. HLA-EUL40 responses appear early post-infection and display a broad, unbiased, Vß repertoire. Although induced in HCMV strain-dependent, UL4015-23-specific manner, HLA-EUL40 CD8 T cells are reactive toward a broader set of nonapeptides varying in 1-3 residues including most HLA-I signal peptides. Thus, HCMV induces strong and life-long lasting HLA-EUL40 CD8 T cells with potential allogeneic or/and autologous reactivity that take place selectively in at least a third of infections according to virus strain and host HLA concordance.

Chronic hepatitis delta virus infection leads to functional impairment and severe loss of MAIT cells.

BACKGROUND & AIMS: Hepatitis delta virus (HDV) infection is the most severe form of viral hepatitis. Although HDV-associated liver disease is considered immune-mediated, adaptive immune responses against HDV are weak. Thus, the role of several other cell-mediated mechanisms such as those driven by mucosa-associated invariant T (MAIT) cells, a group of innate-like T cells highly enriched in the human liver, has not been extensively studied in clinical HDV infection. METHODS: MAIT cells from a sizeable cohort of patients with chronic HDV were analyzed ex vivo and in vitro after stimulation. Results were compared with MAIT cells from hepatitis B virus (HBV) monoinfected patients and healthy controls. RESULTS: Circulating MAIT cells were dramatically decreased in the peripheral blood of HDV-infected patients. Signs of decline were also observed in the liver. In contrast, only a modest decrease of circulating MAIT cells was noted in HBV monoinfection. Unsupervised high-dimensional analysis of residual circulating MAIT cells in chronic HDV infection revealed the appearance of a compound phenotype of CD38hiPD-1hiCD28loCD127loPLZFloEomesloHelioslo cells indicative of activation. Corroborating these results, MAIT cells exhibited a functionally impaired responsiveness. In parallel to MAIT cell loss, HDV-infected patients exhibited signs of monocyte activation and increased levels of proinflammatory cytokines IL-12 and IL-18. In vitro, IL-12 and IL-18 induced an activated MAIT cell phenotype similar to the one observed ex vivo in HDV-infected patients. These cytokines also promoted MAIT cell death, suggesting that they may contribute to MAIT cell activation and subsequent loss during HDV infection. CONCLUSIONS: These results suggest that chronic HDV infection engages the MAIT cell compartment causing activation, functional impairment, and subsequent progressive loss of MAIT cells as the HDV-associated liver disease progresses. LAY SUMMARY: Hepatitis delta virus (HDV) infection is the most severe form of viral hepatitis. We found that in patients with HDV, a subset of innate-like T cells called mucosa-associated invariant T cells (or MAIT cells), which are normally abundant in peripheral blood and the liver, are activated, functionally impaired and severely depleted.

IL-9 promotes the survival and function of human melanoma-infiltrating CD4(+) CD8(+) double-positive T cells.

We previously demonstrated an accumulation of tumor-reactive CD4(+) CD8(+) double positive (DP) T cells within melanoma-infiltrating lymphocytes, supporting their role in the regulation of anti-tumor immune responses. Similarly to their CD8(+) counterparts, intra-tumor DP T cells are MHC class-I restricted but differed by a limited lytic activity against autologous melanoma cells. Based on these observations and to further characterize DP T cells, both populations were compared at the transcriptional level. Our results revealed the overexpression of the IL-9 receptor (IL-9R) by DP T cells and prompted us to investigate the impact of IL-9 on their biology. We show that IL-9 favors DP T-cell survival by protecting them from apoptosis and by promoting their proliferation. In addition, IL-9 enhances their ability to produce cytokines and increased their levels of granzyme B/perforin as well as degranulation capacity, leading to a strengthened cytotoxic activity against melanoma cells. Taken together, the IL-9R(high) DP T-cell population could be a new preferential target for IL-9, which could take part in their retention within the melanoma infiltrate while also favoring their anti-tumor activity. More generally, our results extend the pleiotropic effects of IL-9 to IL-9R-expressing intra-tumor T cells, which could further potentiate anti-tumor immune responses.

Expansion of donor-unrestricted MAIT cells with enhanced cytolytic function suitable for TCR redirection.

Progress in our understanding of MR1-restricted mucosa-associated invariant T (MAIT) cells has raised interest in harnessing these cells for immunotherapy. The innate-like response characteristics, abundance in the blood, donor-unrestricted nature, and tropism for tissues make MAIT cells suitable candidates for adoptive cell transfer therapies. However, reliable methods and tools to utilize MAIT cells in such approaches are lacking. Here, we established methodology for efficient expansion of human MAIT cells in culture with high purity and yield, while preserving their functional response toward their natural ligand and increasing their cytotoxic potential. The cultured MAIT cells retained their effector memory characteristics without signs of terminal differentiation and expressed a more diverse set of chemokine receptors, potentially widening their already broad tissue tropism. To investigate the potential of MAIT cells in a context outside their main role in controlling bacterial infection, we engineered cultured MAIT cells with a new TCR specificity to mediate effective antiviral HLA class I-restricted effector function. In summary, we developed robust and effective methodology for the expansion of human MAIT cells with enhanced cytolytic capacity and for their engineering with a new specificity. These findings form a basis for the development of MAIT cells as a platform for adoptive immunotherapy.

Human MAIT cells endowed with HBV specificity are cytotoxic and migrate towards HBV-HCC while retaining antimicrobial functions.

BACKGROUND & AIMS: Virus-specific T cell dysfunction is a common feature of HBV-related hepatocellular carcinoma (HBV-HCC). Conventional T (ConT) cells can be redirected towards viral antigens in HBV-HCC when they express an HBV-specific receptor; however, their efficacy can be impaired by liver-specific physical and metabolic features. Mucosal-associated invariant T (MAIT) cells are the most abundant innate-like T cells in the liver and can elicit potent intrahepatic effector functions. Here, we engineered ConT and MAIT cells to kill HBV expressing hepatoma cells and compared their functional properties. METHODS: Donor-matched ConT and MAIT cells were engineered to express an HBV-specific T cell receptor (TCR). Cytotoxicity and hepatocyte homing potential were investigated using flow cytometry, real-time killing assays, and confocal microscopy in 2D and 3D HBV-HCC cell models. Major histocompatibility complex (MHC) class I-related molecule (MR1)-dependent and MR1-independent activation was evaluated in an Escherichia coli THP-1 cell model and by IL-12/IL-18 stimulation, respectively. RESULTS: HBV TCR-MAIT cells demonstrated polyfunctional properties (CD107a, interferon [IFN] γ, tumour necrosis factor [TNF], and IL-17A) with strong HBV target sensitivity and liver-homing chemokine receptor expression when compared with HBV TCR-ConT cells. TCR-mediated lysis of hepatoma cells was comparable between the cell types and augmented in the presence of inflammation. Coculturing with HBV+ target cells in a 3D microdevice mimicking aspects of the liver microenvironment demonstrated that TCR-MAIT cells migrate readily towards hepatoma targets. Expression of an ectopic TCR did not affect the ability of the MAIT cells to be activated via MR1-presented bacterial antigens or IL-12/IL-18 stimulation. CONCLUSIONS: HBV TCR-MAIT cells demonstrate anti-HBV functions without losing their endogenous antimicrobial mechanisms or hepatotropic features. Our results support future exploitations of MAIT cells for liver-directed immunotherapies. LAY SUMMARY: Chronic HBV infection is a leading cause of liver cancer. T cell receptor (TCR)-engineered T cells are patients' immune cells that have been modified to recognise virus-infected and/or cancer cells. Herein, we evaluated whether mucosal-associated invariant T cells, a large population of unconventional T cells in the liver, could recognise and kill HBV infected hepatocytes when engineered with an HBV-specific TCR. We show that their effector functions may exceed those of conventional T cells currently used in the clinic, including antimicrobial properties and chemokine receptor profiles better suited for targeting liver tumours.

Transcriptomic features of tumour-infiltrating CD4lowCD8high double positive αß T cells in melanoma.

Peripheral CD4+CD8+ double positive (DP) T cells are a phenotypically and functionally heterogeneous population depending on their origin and pathologic context. We previously identified among tumour infiltrating lymphocytes in melanoma, a tumour-reactive MHC class-I restricted CD4lowCD8high DP αß T-cell subpopulation with CD4-like function. In this study, we used an in-depth comparative transriptomic analysis of intra-melanoma DP T cells and CD4 and CD8 single positive (SP) T cells, to better comprehend the origin of this DP phenotype, and define the transcriptomic signature of activated DP T cells. We observed that intra-melanoma DP T cells were transcriptome-wise closer to their CD8 SP T-cell counterparts in terms of number of genes differentially expressed (97 in common with CD8 SP T cells and 15 with CD4 SP T cells) but presented hallmarks of a transition to a CD4-like functional profile (CD40LG) with a decreased cytotoxic signature (KLRC1) in favour of an increased cytokine-receptor interaction signature (IL4, IL24, IL17A…). This unleashed CD4-like program could be the results of the observed unbalanced expression of the THPOK/Runx3 transcription factors in DP T cells. Overall, this study allow us to speculate that intra-melanoma DP T cells arise from CD8 SP T cells being reprogrammed to a helper function.

Innate lymphoid cell composition associates with COVID-19 disease severity.

OBJECTIVES: The role of innate lymphoid cells (ILCs) in coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is unknown. Understanding the immune response in COVID-19 could contribute to unravel the pathogenesis and identification of treatment targets. Here, we describe the phenotypic landscape of circulating ILCs in COVID-19 patients and identified ILC phenotypes correlated to serum biomarkers, clinical markers and laboratory parameters relevant in COVID-19. METHODS: Blood samples collected from moderately (n = 11) and severely ill (n = 12) COVID-19 patients, as well as healthy control donors (n = 16), were analysed with 18-parameter flow cytometry. Using supervised and unsupervised approaches, we examined the ILC activation status and homing profile. Clinical and laboratory parameters were obtained from all COVID-19 patients, and serum biomarkers were analysed with multiplex immunoassays. RESULTS: Innate lymphoid cells were largely depleted from the circulation of COVID-19 patients compared with healthy controls. Remaining circulating ILCs revealed decreased frequencies of ILC2 in severe COVID-19, with a concomitant decrease of ILC precursors (ILCp) in all patients, compared with controls. ILC2 and ILCp showed an activated phenotype with increased CD69 expression, whereas expression levels of the chemokine receptors CXCR3 and CCR4 were significantly altered in ILC2 and ILCp, and ILC1, respectively. The activated ILC profile of COVID-19 patients was associated with soluble inflammatory markers, while frequencies of ILC subsets were correlated with laboratory parameters that reflect the disease severity. CONCLUSION: This study provides insights into the potential role of ILCs in immune responses against SARS-CoV-2, particularly linked to the severity of COVID-19.

MAIT cell activation and dynamics associated with COVID-19 disease severity.

Severe COVID-19 is characterized by excessive inflammation of the lower airways. The balance of protective versus pathological immune responses in COVID-19 is incompletely understood. Mucosa-associated invariant T (MAIT) cells are antimicrobial T cells that recognize bacterial metabolites, and can also function as innate-like sensors and mediators of antiviral responses. Here, we investigated the MAIT cell compartment in COVID-19 patients with moderate and severe disease, as well as in convalescence. We show profound and preferential decline in MAIT cells in the circulation of patients with active disease paired with strong activation. Furthermore, transcriptomic analyses indicated significant MAIT cell enrichment and pro-inflammatory IL-17A bias in the airways. Unsupervised analysis identified MAIT cell CD69high and CXCR3low immunotypes associated with poor clinical outcome. MAIT cell levels normalized in the convalescent phase, consistent with dynamic recruitment to the tissues and later release back into the circulation when disease is resolved. These findings indicate that MAIT cells are engaged in the immune response against SARS-CoV-2 and suggest their possible involvement in COVID-19 immunopathogenesis.

Factors Influencing Functional Heterogeneity in Human Mucosa-Associated Invariant T Cells.

Mucosa-associated invariant T (MAIT) cells are unconventional innate-like T cells that recognize microbial riboflavin metabolites presented by the monomorphic MHC class I-related (MR1) molecule. Despite the high level of evolutionary conservation of MR1 and the limited diversity of known antigens, human MAIT cells and their responses may not be as homogeneous as previously thought. Here, we review recent findings indicating that MAIT cells display microbe-specific response patterns with multiple layers of heterogeneity. The natural killer cell receptor CD56 marks a MAIT cell subset with distinct response profile, and the T cell receptor ß-chain diversity influences responsiveness at the single cell level. The MAIT cell tissue localization also influences their response profiles with higher IL-17 in tissue-resident MAIT cells. Furthermore, there is emerging evidence that the type of antigen-presenting cells, and innate cytokines produced by such cells, influence the quality of the ensuing MAIT cell response. On the microbial side, the expression patterns of MR1-presented antigenic and non-antigenic compounds, expression of other bioactive microbial products, and of innate pattern recognition ligands all influence downstream MAIT cell responses. These recent findings deepen our understanding of MAIT cell functional diversity and adaptation to the type and location of microbial challenge.

Emergence of High-Avidity Melan-A-Specific Clonotypes as a Reflection of Anti-PD-1 Clinical Efficacy.

Therapeutic strategies using anti-PD-1-blocking antibodies reported unparalleled effectiveness for melanoma immunotherapy, but deciphering immune responses modulated by anti-PD-1 treatment remains a crucial issue. Here, we analyzed the composition and functions of the large Melan-A-specific T-cell repertoire in the peripheral blood of 9 melanoma patients before and after 2 months of treatment with anti-PD-1. We observed amplification of Melan-A-specific Vß subfamilies undetectable before therapy (thereafter called emerging Vß subfamilies) in responding patients, with a predominant expansion in patients with a complete response. These emerging Vß subfamilies displayed a higher functional avidity for their cognate antigen than Vß subfamilies not amplified upon anti-PD-1 therapy and could be identified by a sustained coexpression of PD-1 and TIGIT receptors. Thus, in addition to the emergence of neoantigen-specific T cells previously documented upon anti-PD-1 therapy, our work describes the emergence of high-avidity Melan-A-specific clonotypes as a surrogate marker of treatment efficacy. Cancer Res; 77(24); 7083-93. ©2017 AACR.

CD40L confers helper functions to human intra-melanoma class-I-restricted CD4+CD8+ double positive T cells.

Although CD4+CD8+ double positive (DP) T cells represent a small fraction of peripheral T lymphocytes in healthy human donors, their frequency is often increased under pathological conditions (in blood and targeted tissues). In solid cancers such as melanoma, we previously demonstrated an enrichment of tumor reactive CD4lowCD8highαß DP T cells among tumor-infiltrating lymphocytes of unknown function. Similarly to their single positive (SP) CD8+ counterparts, intra-melanoma DP T cells recognized melanoma cell lines in an HLA-class-I restricted context. However, they presented a poor cytotoxic activity but a strong production of diverse Th1 and Th2 cytokines. The aim of this study was to clearly define the role of intra-melanoma CD4lowCD8highαß DP T cells in the antitumor immune response. Based on a comparative transcriptome analysis between intra-melanoma SP CD4+, SP CD8+ and DP autologous melanoma-infiltrating T-cell compartments, we evidenced an overexpression of the CD40L co-stimulatory molecule on activated DP T cells. We showed that, like SP CD4+ T cells, and through CD40L involvement, DP T cells are able to induce both proliferation and differentiation of B lymphocytes and maturation of functional DCs able to efficiently prime cytotoxic melanoma-specific CD8 T-cell responses. Taken together, these results highlight the helper potential of atypical DP T cells and their role in potentiating antitumor response.