TLR signaling in human antigen-presenting cells regulates MR1-dependent activation of MAIT cells.

Mucosal-associated invariant T (MAIT) cells are an abundant innate-like T lymphocyte population that are enriched in liver and mucosal tissues. They are restricted by MR1, which presents antigens derived from a metabolic precursor of riboflavin synthesis, a pathway present in many microbial species, including commensals. Therefore, MR1-mediated MAIT cell activation must be tightly regulated to prevent inappropriate activation and immunopathology. Using an in vitro model of MR1-mediated activation of primary human MAIT cells, we investigated the mechanisms by which it is regulated. Uptake of intact bacteria by antigen presenting cells (APCs) into acidified endolysosomal compartments was required for efficient MR1-mediated MAIT cell activation, while stimulation with soluble ligand was inefficient. Consistent with this, little MR1 was seen at the surface of human monocytic (THP1) and B-cell lines. Activation with a TLR ligand increased the amount of MR1 at the surface of THP1 but not B-cell lines, suggesting differential regulation in different cell types. APC activation and NF-κB signaling were critical for MR1-mediated MAIT cell activation. In primary cells, however, prolonged TLR signaling led to downregulation of MR1-mediated MAIT cell activation. Overall, MR1-mediated MAIT cell activation is a tightly regulated process, dependent on integration of innate signals by APCs.

Human APOBEC3 induced mutation of human immunodeficiency virus type-1 contributes to adaptation and evolution in natural infection.

Human APOBEC3 proteins are cytidine deaminases that contribute broadly to innate immunity through the control of exogenous retrovirus replication and endogenous retroelement retrotransposition. As an intrinsic antiretroviral defense mechanism, APOBEC3 proteins induce extensive guanosine-to-adenosine (G-to-A) mutagenesis and inhibit synthesis of nascent human immunodeficiency virus-type 1 (HIV-1) cDNA. Human APOBEC3 proteins have additionally been proposed to induce infrequent, potentially non-lethal G-to-A mutations that make subtle contributions to sequence diversification of the viral genome and adaptation though acquisition of beneficial mutations. Using single-cycle HIV-1 infections in culture and highly parallel DNA sequencing, we defined trinucleotide contexts of the edited sites for APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H. We then compared these APOBEC3 editing contexts with the patterns of G-to-A mutations in HIV-1 DNA in cells obtained sequentially from ten patients with primary HIV-1 infection. Viral substitutions were highest in the preferred trinucleotide contexts of the edited sites for the APOBEC3 deaminases. Consistent with the effects of immune selection, amino acid changes accumulated at the APOBEC3 editing contexts located within human leukocyte antigen (HLA)-appropriate epitopes that are known or predicted to enable peptide binding. Thus, APOBEC3 activity may induce mutations that influence the genetic diversity and adaptation of the HIV-1 population in natural infection.

Suppression of HIV-1 infection by APOBEC3 proteins in primary human CD4(+) T cells is associated with inhibition of processive reverse transcription as well as excessive cytidine deamination.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4(+) T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4(+) T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3' termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4(+) T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself.

HIV-1 replication and APOBEC3 antiviral activity are not regulated by P bodies.

The APOBEC3 cytidine deaminases play a critical role in host-mediated defense against exogenous viruses, most notably, human immunodeficiency virus type-1 (HIV-1) and endogenous transposable elements. APOBEC3G and APOBEC3F interact with numerous proteins that regulate cellular RNA metabolism, including components of the RNA-induced silencing complex (RISC), and colocalize with a subset of these proteins to mRNA processing bodies (P bodies), which are sites of mRNA translational repression and decay. We sought to determine the role of P bodies and associated proteins in HIV-1 replication and APOBEC3 antiviral activity. While we established a positive correlation between APOBEC3 protein incorporation into virions and localization to P bodies, depletion of the P-body components DDX6 or Lsm1 did not affect HIV-1 replication, APOBEC3 packaging into virions or APOBEC3 protein mediated inhibition of HIV-1 infectivity. In addition, neither HIV-1 genomic RNA nor Gag colocalized with P-body proteins. However, simultaneous depletion of multiple Argonaute family members, the effector proteins of RISC, could modestly increase viral infectivity. Because some APOBEC3 proteins interact with several Argonaute proteins, we also tested whether they could modulate microRNA (miRNA) activity. We found no evidence for the specific regulation of miRNA function by the APOBEC3 proteins, though more general effects on transfected gene expression were observed. In sum, our results indicate that P bodies and certain associated proteins do not regulate HIV-1 replication or APOBEC3 protein antiviral activity. Localization to P bodies may therefore provide a means of sequestering APOBEC3 enzymatic activity away from cellular DNA or may be linked to as yet unidentified cellular functions.

MR1: An unconventional twist in the tail.

MR1 is a conserved molecule that binds microbial vitamin B metabolites and presents them to unconventional T cells. Lim and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202110125) uncover the role of AP2 in ensuring MR1 surface presentation, which relies on an atypical motif within the MR1 cytoplasmic tail.

Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses.

The role of immune responses to previously seen endemic coronavirus epitopes in severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and disease progression has not yet been determined. Here, we show that a key characteristic of fatal outcomes with coronavirus disease 2019 (COVID-19) is that the immune response to the SARS-CoV-2 spike protein is enriched for antibodies directed against epitopes shared with endemic beta-coronaviruses and has a lower proportion of antibodies targeting the more protective variable regions of the spike. The magnitude of antibody responses to the SARS-CoV-2 full-length spike protein, its domains and subunits, and the SARS-CoV-2 nucleocapsid also correlated strongly with responses to the endemic beta-coronavirus spike proteins in individuals admitted to an intensive care unit (ICU) with fatal COVID-19 outcomes, but not in individuals with nonfatal outcomes. This correlation was found to be due to the antibody response directed at the S2 subunit of the SARS-CoV-2 spike protein, which has the highest degree of conservation between the beta-coronavirus spike proteins. Intriguingly, antibody responses to the less cross-reactive SARS-CoV-2 nucleocapsid were not significantly different in individuals who were admitted to an ICU with fatal and nonfatal outcomes, suggesting an antibody profile in individuals with fatal outcomes consistent with an "original antigenic sin" type response.

Human MAIT cells respond to and suppress HIV-1.

Human MAIT cells sit at the interface between innate and adaptive immunity, are polyfunctional and are capable of killing pathogen infected cells via recognition of the Class IB molecule MR1. MAIT cells have recently been shown to possess an antiviral protective role in vivo and we therefore sought to explore this in relation to HIV-1 infection. There was marked activation of MAIT cells in vivo in HIV-1-infected individuals, which decreased following ART. Stimulation of THP1 monocytes with R5 tropic HIVBAL potently activated MAIT cells in vitro. This activation was dependent on IL-12 and IL-18 but was independent of the TCR. Upon activation, MAIT cells were able to upregulate granzyme B, IFNγ and HIV-1 restriction factors CCL3, 4, and 5. Restriction factors produced by MAIT cells inhibited HIV-1 infection of primary PBMCs and immortalized target cells in vitro. These data reveal MAIT cells to be an additional T cell population responding to HIV-1, with a potentially important role in controlling viral replication at mucosal sites.

Human MAIT Cell Activation In Vitro.

Mucosal-associated invariant T (MAIT) cells are an abundant innate-like T cell subset in humans, enriched in mucosal tissues and the liver. MAIT cells express a semi-invariant T cell receptor (TCR) and recognize microbial-derived riboflavin metabolites presented on the MHC Class I-like molecule MR1. In addition to activation via the TCR, MAIT cells can also be activated in response to cytokines such as IL-12 and IL-18, in contrast to conventional T cells. Here we describe TCR-dependent and -independent methods for MAIT cell activation. The TCR-dependent approaches include stimulation with microbead- or plate-bound anti-CD3/anti-CD28 antibodies, and with 5-OP-RU or paraformaldehyde (PFA)-fixed E. coli in the presence of antigen-presenting cells (APCs). The latter method includes a combination of TCR- and cytokine-mediated stimulation. The TCR-independent methods include direct stimulation with the recombinant cytokines IL-12 and IL-18, and indirect stimulation with TLR-4/TLR-8 agonists or influenza A virus in the presence of APCs. Finally, we outline a protocol to analyze activated MAIT cells using flow cytometry.

Short Communication: Lack of Effect of Maraviroc Intensification on Blood and Gut Reservoir.

We show that intensification of treatment with maraviroc in patients chronically infected with HIV-1 receiving successful long-term antiretroviral therapy was not associated with improvements in HIV-related morbidity, HIV reservoir, microbial translocation, immune activation, or immune exhaustion in either gut or peripheral blood. The measurement of reservoir in both gut and blood longitudinally contributes to a paucity of data in the area.

Molecular Analyses Define Vα7.2-Jα33+ MAIT Cell Depletion in HIV Infection: A Case-Control Study.

Mucosal-associated invariant T (MAIT) cells are an abundant antibacterial innate-like lymphocyte population. There are conflicting reports as to their fate in HIV infection. The objective of this study was to determine whether MAIT cells are truly depleted in HIV infection. In this case-control study of HIV-positive patients and healthy controls, quantitative real-time polymerase chain reaction was used to assess the abundance of messenger RNA (mRNA) and genomic DNA (gDNA) encoding the canonical MAIT cell T cell receptor (Vα7.2-Jα33). Comparison was made with flow cytometry. Significant depletion of both Vα7.2-Jα33 mRNA and gDNA was seen in HIV infection. Depletion of Vα7.2+CD161++ T cells was confirmed by flow cytometry. In HIV infection, the abundance of Vα7.2-Jα33 mRNA correlated most strongly with the frequency of Vα7.2+CD161++ cells. No increase was observed in the frequency of Vα7.2+CD161- cells among CD3+CD4- lymphocytes. MAIT cells are depleted from blood in HIV infection as confirmed by independent assays. Significant accumulation of a CD161- MAIT cell population is unlikely. Molecular approaches represent a suitable alternative to flow cytometry-based assays for tracking of MAIT cells in HIV and other settings.

Direct ex vivo evaluation of long-lived protective antiviral memory B cell responses against hepatitis B virus.

BACKGROUND: The frequency of protective antiviral memory B cells after hepatitis B virus (HBV) vaccination is unknown. METHODS: A novel 2-step immunomagnetic protocol to assess the ex vivo frequency of protective HBV surface antigen (HBsAg)-specific memory B cells was used. RESULTS: HBsAg-specific memory B cells were detected in vaccinated individuals, although at very low frequency (median, 0.2% of CD19(+) cells [range, 0%-4% of CD19(+) cells]). No correlation existed between the frequency of HBsAg-specific memory B cells and the corresponding serum antibody titer or B cell enzyme-linked immunosorbent spot findings. CONCLUSION: Our results indicate sustained B cell-mediated protection against HBV despite waning antibody titers, which is consistent with clinical observations.