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Mucosa-associated invariant T (MAIT) cells are an evolutionarily conserved population of T cells serving a sentinel function at mucosal barrier sites to mediate immune protection. MAIT cells utilize their semi-invariant T cell receptor (TCR) to react to non-peptidic antigens derived from microbial riboflavin metabolites, presented on monomorphic MHC-class I-like MR1 molecules. In addition to TCR-dependent recognition of vitamin B2metabolites, MAIT cells sense and respond to local inflammatory cytokines in an innate-like manner. Surface expression of CD56 was previously shown to be associated with enhanced MAIT cell sensitivity to TCR-independent activation. However, the regulation, frequency, or relevance of CD56 expression on human MAIT cells is currently not well understood. Flow cytometry revealed heterogeneity of CD56 expression across donor-matched tissues obtained from human organ donors. Strikingly, the majority of liver MAIT cells expressed CD56, whereas expression was consistently lower in other tissues. We evaluated de novo CD56 expression on blood-derived sorted CD56-negative MAIT cells following IL-7 or antigen exposure which is likely to occur in the hepatic acute phase response. CD56-negative MAIT cells strongly upregulated CD56 expression after IL-7 stimulation and maintained CD56 expression for up to 19 days. Exposure to MR1-presented antigen induced CD56 expression similarly but required prior cell proliferation. In summary, we identify heterogeneity between MAIT cell populations of different tissue origin and determine the dynamic regulation of the CD56-associated innate-like characteristics to shed light on unknown aspects of MAIT cell diversity and immunobiology.

Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell–mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms.

NK cells play an important role in the defense against viral infections. However, little is known about the regulation of NK cell responses during the first days of acute viral infections in humans. In this study, we used the live attenuated yellow fever virus (YFV) vaccine 17D as a human in vivo model to study the temporal dynamics and regulation of NK cell responses in an acute viral infection. YFV induced a robust NK cell response in vivo, with an early activation and peak in NK cell function at day 6, followed by a delayed peak in Ki67 expression, which was indicative of proliferation, at day 10. The in vivo NK cell response correlated positively with plasma type I/III IFN levels at day 6, as well as with the viral load. YFV induced an increased functional responsiveness to IL-12 and IL-18, as well as to K562 cells, indicating that the NK cells were primed in vivo. The NK cell responses were associated primarily with the stage of differentiation, because the magnitude of induced Ki67 and CD69 expression was distinctly higher in CD57− NK cells. In contrast, NK cells expressing self- and nonself-HLA class I–binding inhibitory killer cell Ig-like receptors contributed, to a similar degree, to the response. Taken together, our results indicate that NK cells are primed by type I/III IFN in vivo early after YFV infection and that their response is governed primarily by the differentiation stage, independently of killer cell Ig-like receptor/HLA class I–mediated inhibition or education.

The live attenuated yellow fever virus (YFV) 17D vaccine provides a good model to study immune responses to an acute viral infection in humans. We studied the temporal dynamics, composition, and character of the primary human T cell response to YFV. The acute YFV-specific effector CD8 T cell response was broad and complex; it was composed of dominant responses that persisted into the memory population, as well as of transient subdominant responses that were not detected at the memory stage. Furthermore, HLA-A2– and HLA-B7–restricted YFV epitope–specific effector cells predominantly displayed a CD45RA−CCR7−PD-1+CD27high phenotype, which transitioned into a CD45RA+CCR7−PD-1−CD27low memory population phenotype. The functional profile of the YFV-specific CD8 T cell response changed in composition as it matured from an effector- to a memory-type response, and it tended to become less polyfunctional during the course of this transition. Interestingly, activation of CD4 T cells, as well as FOXP3+ T regulatory cells, in response to YFV vaccination preceded the kinetics of the CD8 T cell response. The present results contribute to our understanding of how immunodominance patterns develop, as well as the phenotypic and functional characteristics of the primary human T cell response to a viral infection as it evolves and matures into memory.

Invariant CD1d-restricted NKT (iNKT) cells play important roles in generating protective immune responses against infections. In this study, we have investigated the role of human iNKT cells in HSV-1 infection and their interaction with epidermal keratinocytes. These cells express CD1d and are the primary target of the virus. Keratinocytes loaded with α-galactosyl ceramide (α-GalCer) could stimulate IFN-γ production and CD25 upregulation by iNKT cells. However, both α-GalCer–dependent and cytokine-dependent activation of iNKT cells was impaired after coculture with HSV-1–infected cells. Notably, CD1d downregulation was not observed on infected keratinocytes, which were also found to inhibit TCR-independent iNKT cell activation. Further examination of the cytokine profile of iNKT–keratinocyte cocultures showed inhibition of IFN-γ, IL-5, IL-10, IL-13, and IL-17 secretion but upregulation of IL-4 and TNF-α after the infection. Moreover, cell-to-cell contact between infected keratinocytes and iNKT cells was required for the inhibition of activation, as the cell-free supernatants containing virus did not affect activation. Productive infection of iNKT cells was however not required for the inhibitory effect. After coculture with infected cells, iNKT cells were no longer responsive to further stimulation with α-GalCer–loaded CD1d-expressing cells. We found that exposure to HSV-1–infected cells resulted in impaired TCR signaling downstream of ZAP70. Additionally, infected cells upregulated the expression of the negative T cell regulator, galectin-9; however, blocking experiments indicated that the impairment of iNKT cell responses was independent of galectin-9. Thus, interference with activation of human iNKT cells by HSV-1 may represent a novel immunoevasive strategy used by the virus to avoid immune clearance.

NK cells are important innate immune effector cells, normally characterized as CD56+CD3− lymphocytes. In this study, we report that CD56−CD16+ NK cells expand in many patients with chronic hepatitis C virus infection. These CD56− NK cells were functionally impaired with respect to cytokine production upon target cell recognition, in comparison to CD56dim and CD56bright NK cell subsets. In particular, CD56− NK cells were strikingly defective in their polyfunctional response as measured by the coexpression of MIP-1β, IFN-γ, TNF-α, and CD107a degranulation. The ability of these cells to mediate three or four of these functions was poor; expression of MIP-1β alone dominated their response. CD56− NK cells retained expression of receptors such as the natural cytotoxicity receptors and NKG2D, whereas the expression of CD57 and perforin was lower when compared with CD56dim NK cells. Interestingly, pretreatment levels of CD56− NK cells correlated with the outcome of pegylated IFN-α and ribavirin treatment. In patients with CD56− NK cells in the range of healthy subjects, 80% reached a sustained virological response to treatment, whereas only 25% of patients with levels clearly above those in healthy subjects experienced a sustained virological response. Thus, chronic hepatitis C virus infection is associated with an expansion of CD56− NK cells functionally skewed toward MIP-1β production only. Furthermore, high levels of these cells reveal a disturbance in innate cellular immunity that is associated with an impaired ability to respond to antiviral treatment with IFN-α and ribavirin.

Chronic viral infection and persistent antigenic stimulation is associated with a rise of late-stage differentiated CD8 T cells including a unique subset of CD8 T cells expressing FCg receptor IIIa (CD16). Expansion of CD16+ CD8 T cells is observed in other chronic diseases like HCV, but not HIV-1 infection. CD16 mediates antibody dependent cellular cytotoxicity (ADCC), most notably on NK cells, and is associated with protection from HIV-1 acquisition. Using cryopreserved PBMC from healthy and HIV-1 chronically infected donors from Uganda, we compared phenotypic, transcriptional, and functional changes in CD16+ CD8 T cells. We found that chronic, untreated HIV-1 infection is associated with elevated numbers of CD45RA+CD57+ terminal effector CD16+ CD8 T cells compared to healthy individuals, and display a distinct transcriptional profile in between conventional CD8 T cells and NK cells, characterized by high IKZF2 and KLRF1, and low expression of IL7R. This transcriptional profile translates into a NKp80+ IL-7Rα-surface phenotype and high expression of the Helios transcription factor. Additionally, CD16+ CD8 T cells are capable of mediating ADCC at levels similar to that of NK cells. These CD16+ CD8 T cells are highly activated and correlate positively with expansion of the CD8 T cell compartment and plasma levels of soluble factors of antiviral immunity and inflammation, such as IP-10, TNF, IL-6, and TNFRII. These data indicate that terminally differentiated, effector CD8 T cells acquire innate, NK cell-like characteristics during chronic HIV-1 infection, and suggest that HIV-specific ADCC is part of the repertoire of functions CD8 T cells use to combat the ability of HIV-1 to escape the TCR-specific, anti-viral effects of CD8 T cells.

Mucosa-associated invariant T (MAIT) cells are a class of non-classical T lymphocytes defined by their invariant Vα7.2-containing MR1-restricted T cell receptor, as well as high expression of CD161. At present, the characteristics of MAIT cells in the oral mucosa are poorly defined. In this study, we have analyzed buccal biopsies and matched blood samples from a cohort of healthy volunteers. MAIT cells were present in the buccal mucosa, with a tendency to cluster around the basal layer, a region also found to contain a higher frequency of MR1-expressing antigen-presenting cells. The frequency of MAIT cells was similar in the buccal mucosa compared to matched peripheral blood, in contrast to other major T cell subsets. However, the buccal mucosal MAIT cells displayed a tissue resident activated profile, with high CD69, HLA-DR and PD-1 expression, as well as a skewed subset distribution with higher representation of CD4/CD8 double-negative cells and of CD8αα+ cells within the CD8+ subset. The Jα chain usage in the MAIT cell TCR was more variable in oral mucosal MAIT cells than in peripheral blood MAIT cells. Functionally, the oral mucosal MAIT cells had lower perforin levels, indicative of a lower cytolytic potential. Interestingly, aspects of the oral MAIT cell population were associated with life-style factors such as consumption of bacterially fermented milk products and alcohol intake. Together, these data show that MAIT cells form a part of the oral mucosal T cell compartment, where they exhibit a tissue-resident activated profile. Thus, MAIT cells are positioned in the oral cavity and equipped to handle resident microbiota as well as invading pathogens.

Mucosa-associated invariant T (MAIT) cells are a large innate-like T-cell subset in humans defined by invariant TCR Vα7.2 use and expression of CD161. MAIT cells recognize microbial riboflavin metabolites of bacterial or fungal origin presented by the monomorphic MR1 molecule. The extraordinary level of evolutionary conservation of MR1 and the limited known diversity of riboflavin metabolite antigens have suggested that MAIT cells are relatively homogeneous and uniform in responses against diverse microbes carrying the riboflavin biosynthesis pathway. The ability of MAIT cells to exhibit microbe-specific functional specialization has not been thoroughly investigated. Here, we found that MAIT cell responses against Escherichia coli and Candida albicans displayed microbe-specific polyfunctional response profiles, antigen sensitivity, and response magnitudes. MAIT cell effector responses against E. coli and C. albicans displayed differential MR1 dependency and TCR β-chain bias, consistent with possible divergent antigen subspecificities between these bacterial and fungal organisms. Finally, although the MAIT cell immunoproteome was overall relatively homogenous and consistent with an effector memory-like profile, it still revealed diversity in a set of natural killer cell-associated receptors. Among these, CD56, CD84, and CD94 defined a subset with higher expression of the transcription factors promyelocytic leukemia zinc finger (PLZF), eomesodermin, and T-bet and enhanced capacity to respond to IL-12 and IL-18 stimulation. Thus, the conserved and innate-like MAIT cells harbor multiple layers of functional heterogeneity as they respond to bacterial or fungal organisms or innate cytokines and adapt their antimicrobial response patterns in a stimulus-specific manner.

The activated PD-1+ CD4 T cell population is associated with disease progression in untreated HIV-1 infection, and with residual viral replication in individuals on ART. Few studies have examined cell associated viral load (CAVL) in phenotypically different activated CD4 T cell populations to define the HIV reservoir. We followed 32 HIV-1 chronically infected individuals, from Kampala, Uganda, and determined their CD4 T cell counts, and viral load at baseline, 6, and 12 months after the initiation of ART. Peripheral blood T cell populations were sorted based on activation profiles and gag DNA was measured to determine CAVL within these populations. Soluble inflammatory factors were measured in plasma using a multiplexed platform. The median HIV-1 viral load declined from 95,315 copies/ml at baseline to below detection for all patients after 12 months of ART, whereas CD4 T cells counts averaged 204 cells/μl at baseline and increased to 373 cells/μl by month 12. Median levels of the activated PD-1+ CD4 T cell population contracted from 4.5% of CD4 T cells at baseline to 2.3% of CD4 T cells after 12 months of ART. Baseline levels of activated PD-1+ CD4 T cells positively correlated with plasma levels of IP-10 (R2=0.362, P

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1α (EF1α). EF1α occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1α peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1α peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1α isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.

To examine possible interference patterns between immunodominant CTL Ags, we analyzed the response to mixtures of five well-characterized H-2Kb-restricted epitopes, each of which had earlier been described as immunodominant within its antigenic system. Clear patterns of dominance were observed between peptides in the mixture, with the CTL response focusing on the Sendai virus nucleoprotein 324–332 and vesicular stomatitis virus nucleoprotein 52–59 epitopes. The dominance of these epitopes correlated with high CTL availability. Subdominance of the OVA257–264 and the MCF1233 murine leukemia virus envelope 574–581 peptides could not be explained by inferior ability to bind and stabilize MHC class I molecules. Interestingly, immunodominance was broken if the peptide mixture was pulsed on bone marrow-derived dendritic cells, a mode of immunization allowing efficient recognition of a broader set of specificities. Our results show that immunodominance is neither an absolute feature of a given epitope nor does it apply only in relation to other epitopes within the same protein, micro-organism, or cell. Novel “superdominant” hierarchies emerge in the response against multiple “dominant” epitopes. A T cell competition model to explain the data in terms of a balance influenced by CTL frequencies and available APC capacity is discussed.

Chronic immune activation is strongly associated with Human Immunodeficiency virus 1 (HIV-1) disease progression. Here, we have investigated the state of T cell immune activation, as well as T cell responses to hepatitis C virus (HCV), in patients with HCV/HIV-1 co-infection on effective anti-retroviral therapy (ART). Co-infected subjects displayed elevated immune activation as determined by CD38 expression in both CD4 and CD8 T cells, as compared to mono-infected control groups. HCV-specific T cells in response to HCV antigens tended to be stronger in the co-infected patients with a more pronounced IFNγ profile by CD8 T cells and this group also had higher HCV loads at baseline. Patients who reached a sustained virological response to peg-interferon-α and ribavirin treatment displayed significantly lower pre-treatment levels of chronic T cell activation and a trend towards stronger HCV-specific T cell responses, as compared to patients who failed to clear. Data suggest a link between high levels of chronic immune activation, poor adaptive immune responses and HCV treatment failure in HCV/HIV-1 co-infected subjects. We suggest that the high level of chronic immune activation may provide a clue as to why patients co-infected with these viruses in general display faster viral disease progression. This work was supported by the Swedish Research Council, SIDA and the Swedish National Board of Health and Welfare.