Altered differentiation is central to HIV-specific CD4+ T cell dysfunction in progressive disease.

Dysfunction of virus-specific CD4+ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4+ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cell)-like profile characterized HIV-specific CD4+ T cells in viremic infection. HIV-specific CD4+ T cells from people spontaneously controlling the virus (elite controllers) robustly expressed genes associated with the TH1, TH17 and TH22 subsets of helper T cells. Viral suppression by ART resulted in a distinct transcriptional landscape, with a reduction in the expression of genes associated with TFH cells, but persistently low expression of genes associated with TH1, TH17 and TH22 cells compared to the elite controller profile. Thus, altered differentiation is central to the impairment of HIV-specific CD4+ T cells and involves both gain of function and loss of function.

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.

Functional SARS-CoV-2 cross-reactive CD4+ T cells established in early childhood decline with age.

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (ß-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein-Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination.

T cell immunity to SARS-CoV-2.

Exceptional efforts have been undertaken to shed light into the biology of adaptive immune responses to SARS-CoV-2. T cells occupy a central role in adaptive immunity to mediate helper functions to different arms of the immune system and are fundamental to mediate protection, control, and clearance of most viral infections. Even though many questions remain unsolved, there is a growing literature linking specific T cell characteristics to differential COVID-19 severity and vaccine outcome. In this review, we summarize our current understanding of CD4+ and CD8+ T cell responses in acute and convalescent COVID-19. Further, we discuss the T cell literature coupled to pre-existing immunity and vaccines and highlight the need to look beyond blood to fully understand how T cells function in the tissue space.

Tools for Visualizing HIV in Cure Research.

PURPOSE OF REVIEW: The long-lived HIV reservoir remains a major obstacle for an HIV cure. Current techniques to analyze this reservoir are generally population-based. We highlight recent developments in methods visualizing HIV, which offer a different, complementary view, and provide indispensable information for cure strategy development. RECENT FINDINGS: Recent advances in fluorescence in situ hybridization techniques enabled key developments in reservoir visualization. Flow cytometric detection of HIV mRNAs, concurrently with proteins, provides a high-throughput approach to study the reservoir on a single-cell level. On a tissue level, key spatial information can be obtained detecting viral RNA and DNA in situ by fluorescence microscopy. At total-body level, advancements in non-invasive immuno-positron emission tomography (PET) detection of HIV proteins may allow an encompassing view of HIV reservoir sites. HIV imaging approaches provide important, complementary information regarding the size, phenotype, and localization of the HIV reservoir. Visualizing the reservoir may contribute to the design, assessment, and monitoring of HIV cure strategies in vitro and in vivo.

RNA flow cytometric FISH for investigations into HIV immunology, vaccination and cure strategies.

Despite the tremendous success of anti-retroviral therapy (ART) no current treatment can eradicate latent HIV reservoirs from HIV-infected individuals or generate, effective HIV-specific immunity. Technological limitations have hampered the identification and characterization of both HIV-infected cells and HIV-specific responses in clinical samples directly ex vivo. RNA-flow cytometric fluorescence in situ hybridisation (RNA Flow-FISH) is a powerful technique, which enables detection of mRNAs in conjunction with proteins at a single-cell level. Here, we describe how we are using this technology to address some of the major questions remaining in the HIV field in the era of ART. We discuss how CD4 T cell responses to HIV antigens, both following vaccination and HIV infection, can be characterized by measurement of cytokine mRNAs. We describe how our development of a dual HIV mRNA/protein assay (HIVRNA/Gag assay) enables high sensitivity detection of very rare HIV-infected cells and aids investigations into the translation-competent latent reservoir in the context of HIV cure.

Impaired Th17 polarization of phenotypically naive CD4(+) T-cells during chronic HIV-1 infection and potential restoration with early ART.

BACKGROUND: Depletion of mucosal Th17 cells during HIV/SIV infections is a major cause for microbial translocation, chronic immune activation, and disease progression. Mechanisms contributing to Th17 deficit are not fully elucidated. Here we investigated alterations in the Th17 polarization potential of naive-like CD4(+) T-cells, depletion of Th17-commited subsets during HIV pathogenesis, and Th17 restoration in response to antiretroviral therapy (ART). RESULTS: Peripheral blood CD4(+) T-cells expressing a naive-like phenotype (CD45RA(+)CCR7(+)) from chronically HIV-infected subjects receiving ART (CI on ART; median CD4 counts 592 cells/µl; viral load: <50 HIV-RNA copies/ml; time since infection: 156 months) compared to uninfected controls (HIV-) were impaired in their survival and Th17 polarization potential in vitro. In HIV- controls, IL-17A-producing cells mainly originated from naive-like T-cells with a regulatory phenotype (nTregs: CD25(high)CD127(-)FoxP3(+)) and from CD25(+)CD127(+)FoxP3(-) cells (DP, double positive). Th17-polarized conventional naive CD4(+) T-cells (nT: CD25(-)CD127(+)FoxP3(-)) also produced IL17A, but at lower frequency compared to nTregs and DP. In CI on ART subjects, the frequency/counts of nTreg and DP were significantly diminished compared to HIV- controls, and this paucity was further associated with decreased proportions of memory T-cells producing IL-17A and expressing Th17 markers (CCR6(+)CD26(+)CD161(+), mTh17). nTregs and DP compared to nT cells harbored superior levels of integrated/non-integrated HIV-DNA in CI on ART subjects, suggesting that permissiveness to integrative/abortive infection contributes to impaired survival and Th17 polarization of lineage-committed cells. A cross-sectional study in CI on ART subjects revealed that nTregs, DP and mTh17 counts were negatively correlated with the time post-infection ART was initiated and positively correlated with nadir CD4 counts. Finally, a longitudinal analysis in a HIV primary infection cohort demonstrated a tendency for increased nTreg, DP, and mTh17 counts with ART initiation during the first year of infection. CONCLUSIONS: These results support a model in which the paucity of phenotypically naive nTregs and DP cells, caused by integrative/abortive HIV infection and/or other mechanisms, contributes to Th17 deficiency in HIV-infected subjects. Early ART initiation, treatment intensification with integrase inhibitors, and/or other alternative interventions aimed at preserving/restoring the pool of cells prone to acquire Th17 functions may significantly improve mucosal immunity in HIV-infected subjects.

MAIT cell heterogeneity across paired human tissues reveals specialization of distinct regulatory and enhanced effector profiles.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize microbial riboflavin pathway metabolites presented by evolutionarily conserved MR1 molecules. We explored the human MAIT cell compartment across organ donor-matched blood, barrier, and lymphoid tissues. MAIT cell population size was donor dependent with distinct tissue compartmentalization patterns and adaptations: Intestinal CD103+ resident MAIT cells presented an immunoregulatory CD39highCD27low profile, whereas MAIT cells expressing NCAM1/CD56 dominated in the liver and exhibited enhanced effector capacity with elevated response magnitude and polyfunctionality. Both intestinal CD39high and hepatic CD56+ adaptations accumulated with donor age. CD56+ MAIT cells displayed limited T cell receptor-repertoire breadth, elevated MR1 binding, and a transcriptional profile skewed toward innate activation pathways. Furthermore, CD56 was dynamically up-regulated to a persistent steady-state equilibrium after exposure to antigen or IL-7. In summary, we demonstrate functional heterogeneity and tissue site adaptation in resident MAIT cells across human barrier tissues with distinct regulatory and effector signatures.

Sustained IFN signaling is associated with delayed development of SARS-CoV-2-specific immunity.

Plasma RNAemia, delayed antibody responses and inflammation predict COVID-19 outcomes, but the mechanisms underlying these immunovirological patterns are poorly understood. We profile 782 longitudinal plasma samples from 318 hospitalized patients with COVID-19. Integrated analysis using k-means reveals four patient clusters in a discovery cohort: mechanically ventilated critically-ill cases are subdivided into good prognosis and high-fatality clusters (reproduced in a validation cohort), while non-critical survivors segregate into high and low early antibody responders. Only the high-fatality cluster is enriched for transcriptomic signatures associated with COVID-19 severity, and each cluster has distinct RBD-specific antibody elicitation kinetics. Both critical and non-critical clusters with delayed antibody responses exhibit sustained IFN signatures, which negatively correlate with contemporaneous RBD-specific IgG levels and absolute SARS-CoV-2-specific B and CD4+ T cell frequencies. These data suggest that the "Interferon paradox" previously described in murine LCMV models is operative in COVID-19, with excessive IFN signaling delaying development of adaptive virus-specific immunity.

Additive effects of booster mRNA vaccination and SARS-CoV-2 Omicron infection on T cell immunity across immunocompromised states.

Suboptimal immunity to SARS-CoV-2 mRNA vaccination has frequently been observed in individuals with various immunodeficiencies. Given the increased antibody evasion properties of emerging SARS-CoV-2 subvariants, it is necessary to assess whether other components of adaptive immunity generate resilient and protective responses against infection. We assessed T cell responses in 279 individuals, covering five different immunodeficiencies and healthy controls, before and after booster mRNA vaccination, as well as after Omicron infection in a subset of patients. We observed robust and persistent Omicron-reactive T cell responses that increased markedly upon booster vaccination and correlated directly with antibody titers across all patient groups. Poor vaccination responsiveness in immunocompromised or elderly individuals was effectively counteracted by the administration of additional vaccine doses. Functionally, Omicron-reactive T cell responses exhibited a pronounced cytotoxic profile and signs of longevity, characterized by CD45RA+ effector memory subpopulations with stem cell-like properties and increased proliferative capacity. Regardless of underlying immunodeficiency, booster-vaccinated and Omicron-infected individuals appeared protected against severe disease and exhibited enhanced and diversified T cell responses against conserved and Omicron-specific epitopes. Our findings indicate that T cells retain the ability to generate highly functional responses against newly emerging variants, even after repeated antigen exposure and a robust immunological imprint from ancestral SARS-CoV-2 mRNA vaccination.