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.

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.

Mucosa-Associated Invariant T Cell Hypersensitivity to Staphylococcus aureus Leukocidin ED and Its Modulation by Activation.

Mucosa-associated invariant T (MAIT) cells recognize bacterial riboflavin metabolite Ags presented by MHC class Ib-related protein (MR1) and play important roles in immune control of microbes that synthesize riboflavin. This includes the pathobiont Staphylococcus aureus, which can also express a range of virulence factors, including the secreted toxin leukocidin ED (LukED). In this study, we found that human MAIT cells are hypersensitive to LukED-mediated lysis and lost on exposure to the toxin, leaving a T cell population devoid of MAIT cells. The cytolytic effect of LukED on MAIT cells was rapid and occurred at toxin concentrations lower than those required for toxicity against conventional T cells. Furthermore, this coincided with high MAIT cell expression of CCR5, and loss of these cells was efficiently inhibited by the CCR5 inhibitor maraviroc. Interestingly, exposure and preactivation of MAIT cells with IL-12 and IL-18, or activation via TCR triggering, partially protected from LukED toxicity. Furthermore, analysis of NK cells indicated that LukED targeted the mature cytotoxic CD57+ NK cell subset in a CCR5-independent manner. Overall, these results indicate that LukED efficiently eliminates immune cells that can respond rapidly to S. aureus in an innate fashion without the need for clonal expansion, and that MAIT cells are exceptionally vulnerable to this toxin. Thus, the findings support a model where LukED secretion may allow S. aureus to avoid recognition by the rapid cell-mediated responses mediated by MAIT cells and NK cells.

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 .

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.

Emerging Role for MAIT Cells in Control of Antimicrobial Resistance.

Antimicrobial resistance is a serious threat to global public health as antibiotics are losing effectiveness due to rapid development of resistance. The human immune system facilitates control and clearance of resistant bacterial populations during the course of antimicrobial therapy. Here we review current knowledge of mucosa-associated invariant T (MAIT) cells, an arm of the immune system on the border between innate and adaptive, and their critical place in human antibacterial immunity. We propose that MAIT cells play important roles against antimicrobial-resistant infections through their capacity to directly clear multidrug-resistant bacteria and overcome mechanisms of antimicrobial resistance. Finally, we discuss outstanding questions pertinent to the possible advancement of host-directed therapy as an alternative intervention strategy for antimicrobial-resistant bacterial infections.

MR1-Restricted T Cells with MAIT-like Characteristics Are Functionally Conserved in the Pteropid Bat Pteropus alecto.

Bats are reservoirs for a large number of viruses which have potential to cause major human disease outbreaks, including the current coronavirus disease 2019 (COVID-19) pandemic. Major efforts are underway to understand bat immune response to viruses, whereas much less is known about their immune responses to bacteria. In this study, MR1-restricted T (MR1T) cells were detected through the use of MR1 tetramers in circulation and tissues of Pteropus alecto (Pa) bats. Pa MR1T cells exhibited weak responses to MR1-presented microbial metabolites at resting state. However, following priming with MR1-presented agonist they proliferated, upregulated critical transcription factors and cytolytic proteins, and gained transient expression of Th1/17-related cytokines and antibacterial cytotoxicity. Collectively, these findings show that the Pa bat immune system encompasses an abundant and functionally conserved population of MR1T cells with mucosal-associated invariant T-like characteristics, suggesting that MR1 and MR1T cells also play a significant role in bat immune defense.

Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance in Escherichia coli.

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli.

Opsonization-Enhanced Antigen Presentation by MR1 Activates Rapid Polyfunctional MAIT Cell Responses Acting as an Effector Arm of Humoral Antibacterial Immunity.

Mucosa-associated invariant T (MAIT) cells are innate-like antimicrobial T cells recognizing a breadth of important pathogens via presentation of microbial riboflavin metabolite Ags by MHC class Ib-related (MR1) molecules. However, the interaction of human MAIT cells with adaptive immune responses and the role they may play in settings of vaccinology remain relatively little explored. In this study we investigated the interplay between MAIT cell-mediated antibacterial effector functions and the humoral immune response. IgG opsonization of the model microbe Escherichia coli with pooled human sera markedly enhanced the capacity of monocytic APC to stimulate MAIT cells. This effect included greater sensitivity of recognition and faster response kinetics, as well as a markedly higher polyfunctionality and magnitude of MAIT cell responses involving a range of effector functions. The boost of MAIT cell responses was dependent on strongly enhanced MR1-mediated Ag presentation via increased FcγR-mediated uptake and signaling primarily mediated by FcγRI. To investigate possible translation of this effect to a vaccine setting, sera from human subjects before and after vaccination with the 13-valent-conjugated Streptococcus pneumoniae vaccine were assessed in a MAIT cell activation assay. Interestingly, vaccine-induced Abs enhanced Ag presentation to MAIT cells, resulting in more potent effector responses. These findings indicate that enhancement of Ag presentation by IgG opsonization allows innate-like MAIT cells to mount a faster, stronger, and qualitatively more complex response and to function as an effector arm of vaccine-induced humoral adaptive antibacterial immunity.

Quantification of Human MAIT Cell-Mediated Cellular Cytotoxicity and Antimicrobial Activity.

The mucosa-associated invariant T (MAIT) cells represent the most abundant population of antimicrobial T cells in humans. When encountering cells infected with riboflavin-producing bacteria, this innate-like T cell population rapidly release a plethora of pro-inflammatory cytokines, mediates antimicrobial activity, and kill infected cells. Here, we describe methodological approaches and protocols to measure their cytotoxicity and antimicrobial effector function using multi-color flow cytometry-based and standard microbiological techniques. We provide specific guidance on protocols and describe potential pitfalls for each of the presented methodologies. Finally, we discuss potential applications and current limitations of our approaches to the study of human MAIT cell antimicrobial properties.

The CD4-CD8- MAIT cell subpopulation is a functionally distinct subset developmentally related to the main CD8+ MAIT cell pool.

Mucosa-associated invariant T (MAIT) cells are unconventional innate-like T cells that recognize microbial riboflavin metabolites presented by the MHC class I-like protein MR1. Human MAIT cells predominantly express the CD8α coreceptor (CD8+), with a smaller subset lacking both CD4 and CD8 (double-negative, DN). However, it is unclear if these two MAIT cell subpopulations distinguished by CD8α represent functionally distinct subsets. Here, we show that the two MAIT cell subsets express divergent transcriptional programs and distinct patterns of classic T cell transcription factors. Furthermore, CD8+ MAIT cells have higher levels of receptors for IL-12 and IL-18, as well as of the activating receptors CD2, CD9, and NKG2D, and display superior functionality following stimulation with riboflavin-autotrophic as well as riboflavin-auxotrophic bacterial strains. DN MAIT cells display higher RORγt/T-bet ratio, and express less IFN-γ and more IL-17. Furthermore, the DN subset displays enrichment of an apoptosis gene signature and higher propensity for activation-induced apoptosis. During development in human fetal tissues, DN MAIT cells are more mature and accumulate over gestational time with reciprocal contraction of the CD8+ subset. Analysis of the T cell receptor repertoire reveals higher diversity in CD8+ MAIT cells than in DN MAIT cells. Finally, chronic T cell receptor stimulation of CD8+ MAIT cells in an in vitro culture system supports the accumulation and maintenance of the DN subpopulation. These findings define human CD8+ and DN MAIT cells as functionally distinct subsets and indicate a derivative developmental relationship.

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.

Reversing HIV latency via sphingosine-1-phosphate receptor 1 signaling.

OBJECTIVE: In this study, we looked for a new family of latency reversing agents. DESIGN: We searched for G-protein-coupled receptors (GPCR) coexpressed with the C-C chemokine receptor type 5 (CCR5) in primary CD4 T cells that activate infected cells and boost HIV production. METHODS: GPCR coexpression was unveiled by reverse transcriptase-PCR. We used fluorescence resonance energy transfer to analyze the dimerization with CCR5 of the expressed GPCR. Viral entry was measured by flow cytometry, reverse transcription by quantitative PCR, nuclear factor-kappa B translocation by immunofluorescence, long terminal repeat activation using a gene reporter assay and viral production by p24 quantification. RESULTS: Gαi-coupled sphingosine-1-phophate receptor 1 (S1P1) is highly coexpressed with CCR5 on primary CD4 T cells and dimerizes with it. The presence of S1P1 had major effects neither on viral entry nor on reverse transcription. Yet, S1P1 signaling induced NFκB activation, boosting the expression of the HIV LTR. Consequently, in culture medium containing sphingosine-1-phophate, the presence of S1P1 enhanced the replication of a CCR5-, but also of a CXCR4-using HIV-1 strain. The S1P1 ligand FTY720, a drug used in multiple sclerosis treatment, inhibited HIV-1 productive infection of monocyte-derived dendritic cells and of severe combined immunodeficiency mice engrafted with human peripheral blood mononuclear cells. Conversely, S1P1 agonists were able to force latently infected peripheral blood mononuclear cells and lymph node cells to produce virions in vitro. CONCLUSION: Altogether these data indicate that the presence of S1P1 facilitates HIV-1 replicative cycle by boosting viral genome transcription, S1P1 antagonists have anti-HIV effects and S1P1 agonists are HIV latency reversing agents.