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Mucosal-associated invariant T (MAIT) cells are an innate-like population of T cells that display a TCR Vα7.2+ CD161+ phenotype and are restricted by the nonclassical MHC-related molecule 1 (MR1). Although B cells control MAIT cell development and function, little is known about the mechanisms underlying their interaction(s). Here, we report, for the first time, that during Salmonella enterica serovar Typhi (S. Typhi) infection, HLA-G expression on B cells downregulates IFN-γ production by MAIT cells. In contrast, blocking HLA-G expression on S. Typhi-infected B cells increases IFN-γ production by MAIT cells. After interacting with MAIT cells, kinetic studies show that B cells upregulate HLA-G expression and downregulate the inhibitory HLA-G receptor CD85j on MAIT cells resulting in their loss. These results provide a new role for HLA-G as a negative feedback loop by which B cells control MAIT cell responses to antigens.

Salmonella enterica serovar Typhi (S. Typhi) causes substantial morbidity and mortality worldwide, particularly among young children. Humans develop an array of mucosal immune responses following S. Typhi infection. Whereas the cellular mechanisms involved in S. Typhi infection have been intensively studied, very little is known about the early chromatin modifications occurring in the human gut microenvironment that influence downstream immune responses. To address this gap in knowledge, cells isolated from human terminal ileum exposed ex vivo to the wild-type S. Typhi strain were stained with a 33-metal-labeled antibody panel for mass cytometry analyses of the early chromatin modifications modulated by S. Typhi. We measured the cellular levels of 6 classes of histone modifications, and 1 histone variant in 11 major cell subsets (i.e., B, CD3 + T, CD4 + T, CD8 + T, NK, TCR-γδ, Mucosal associated invariant (MAIT), and NKT cells as well as monocytes, macrophages, and epithelial cells). We found that arginine methylation might regulate the early-differentiation of effector-memory CD4+ T-cells following exposure to S. Typhi. We also found S. Typhi-induced post-translational modifications in histone methylation and acetylation associated with epithelial cells, NKT, MAIT, TCR-γδ, Monocytes, and CD8 + T-cells that are related to both gene activation and silencing.

It is widely accepted that CD4(+) and CD8(+) T-cells play a significant role in protection against Salmonella enterica serovar Typhi (S. Typhi), the causative agent of the typhoid fever. However, the antigen specificity of these T-cells remains largely unknown. Previously, we demonstrated the feasibility of using a recombinant Escherichia coli (E. coli) expression system to uncover the antigen specificity of CD4(+) and CD8(+) T cells. Here, we expanded these studies to include the evaluation of 12 additional S. Typhi proteins: 4 outer membrane proteins (OmpH, OmpL, OmpR, OmpX), 3 Vi-polysaccharide biosynthesis proteins (TviA, TviB, TviE), 3 cold shock proteins (CspA, CspB, CspC), and 2 conserved hypothetical proteins (Chp 1 and Chp2), all selected based on the bioinformatic analyses of the content of putative T-cell epitopes. CD4(+) and CD8(+) T cells from 15 adult volunteers, obtained before and 42 days after immunization with oral live attenuated Ty21a vaccine, were assessed for their functionality (i.e., production of cytokines and cytotoxic expression markers in response to stimulation with selected antigens) as measured by flow cytometry. Although volunteers differed on their T-cell antigen specificity, we observed T-cell immune responses against all S. Typhi proteins evaluated. These responses included 9 proteins, OmpH, OmpR, TviA, TviE, CspA, CspB, CspC, Chp 1 and Chp 2, which have not been previously reported to elicit T-cell responses. Interestingly, we also observed that, regardless of the protein, the functional patterns of the memory T-cells were different between CD4(+) and CD8(+) T cells. In sum, these studies demonstrated the feasibility of using bioinformatic analysis and the E. coli expressing system described here to uncover novel immunogenic T-cell proteins that could serve as potential targets for the production of protein-based vaccines.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Although immunity induced by typhoid fever is moderated and short-lived, typhoid vaccination with the attenuated Ty21a oral vaccine generates long-lasting protection rates reaching up to 92%. Thus, there are important differences on how wild-type Salmonella and typhoid vaccine strains stimulate host immunity. We hypothesize that vaccine strains with different mutations might affect gut inflammation and intestinal permeability by different mechanisms. To test this hypothesis, we used an in vitro organotypic model of the human intestinal mucosa composed of human intestinal epithelial cells, lymphocytes/monocytes, endothelial cells, and fibroblasts. We also used six Salmonella enterica serovar Typhi (S. Typhi) strains: the licensed Ty21a oral vaccine, four typhoid vaccine candidates (i.e., CVD 908, CVD 909, CVD 910, and CVD 915) and the wild-type Ty2 strain. We found that genetically engineered S. Typhi vaccine strains elicit differential host changes not only in the intestinal permeability and secretion of inflammatory cytokines, but also in the phenotype and activation pathways of innate cells. These changes were distinct from those elicited by the parent wild-type S. Typhi and depended on the genetic manipulation. In sum, these results emphasize the importance of carefully selecting specific manipulations of the Salmonella genome in the development of typhoid vaccines.

Gastrointestinal infections by Salmonella enterica serovar Typhi (S. Typhi) are rare in industrialized countries. However, they remain a major public health problem in the developing world with an estimated 26.9 million new cases annually and significant mortality when untreated. Recently, we provided the first direct evidence that CD8(+) MAIT cells are activated and have the potential to kill cells exposed to S. Typhi, and that these responses are dependent on bacterial load. However, MAIT cell kinetics and function during bacterial infections in humans remain largely unknown. In this study, we characterize the human CD8(+) MAIT cell immune response to S. Typhi infection in subjects participating in a challenge clinical trial who received a low- or high dose of wild-type S. Typhi. We define the kinetics of CD8(+) MAIT cells as well as their levels of activation, proliferation, exhaustion/apoptosis, and homing potential. Regardless of the dose, in volunteers resistant to infection (NoTD), the levels of CD8(+) MAIT cells after S. Typhi challenge fluctuated around their baseline values (day 0). In contrast, volunteers susceptible to the development of typhoid disease (TD) exhibited a sharp decline in circulating MAIT cells during the development of typhoid fever. Interestingly, MAIT cells from low-dose TD volunteers had higher levels of CD38 coexpressing CCR9, CCR6, and Ki67 during the development of typhoid fever than high-dose TD volunteers. No substantial perturbations on the levels of these markers were observed in NoTD volunteers irrespective of the dose. In sum, we describe, for the first time, that exposure to an enteric bacterium, in this case S. Typhi, results in changes in MAIT cell activation, proliferation, and homing characteristics, suggesting that MAIT cells are an important component of the human host response to bacterial infection.

Salmonella enterica serovar Typhi, the causative agent of typhoid fever, is a facultative intracellular bacterial pathogen (5, 7, 37). In industrialized countries, Salmonella infection is rare, with most infections occurring in military personnel and in individuals traveling to areas where typhoid fever is endemic. However, it remains an important public health priority in undeveloped parts of the world. Overall, it is estimated that 16 million new cases and 600,000 deaths occur annually (5, 12, 18). Our laboratory has demonstrated that oral immunization of volunteers with attenuated typhoid vaccine strain Ty21a triggers the generation of CD8+ T cells that kill S. Typhi-infected cells displaying human leukocyte antigen E (HLA-E) on the cell surface (27). T cells are likely to play an important role in the host defense against S. Typhi. They might contribute to Salmonella control by secreting cytokines such as gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) and by directly killing bacterium-infected cells (27, 28, 30-32, 38-40). HLA-E is a protein that is part of a family of molecules known as class Ib or nonclassical HLA molecules (26). HLA-E is expressed ubiquitously on the peripheral blood mononuclear cell (PBMC; e.g., B cells, T cells, natural killer [NK] cells, and macrophages) surface at various levels, depending on the cell type (15, 34). Because HLA-E molecules are essentially monomorphic, they are likely to present a more conserved set of bacterial peptides that could be universally recognized by CD8+ T cells from most, if not all, individuals. Given the potential significance of HLA-E-restricted CD8+ responses in resistance to Salmonella infection, it is of great importance to study these responses in depth, including their kinetics of appearance and persistence, which currently remains largely unknown. These responses might provide key insights into the open question concerning the dual role of the HLA-E molecules in short-lived innate and long-lived adaptive immunity (35). In fact, HLA-E molecules were primarily described as ligands for CD94/NKG2A, -B, and -C receptors present on the innate NK cell surface (3). However, HLA-E-restricted T cells have recently been implicated in adaptive immunity not only to Salmonella but also to cytomegalovirus and Mycobacterium (10, 22). Oral immunization with the licensed Ty21a typhoid vaccine is a particularly relevant model of Salmonella infection, which is transmitted by the ingestion of virulent S. Typhi. In the present studies, volunteers were immunized with the standard regimen of four spaced doses of Ty21a given every other day (8, 28). This vaccination regimen results in levels of protection ranging from 60 to 80% (9). Using cells from Ty21a vaccinees, we investigated the kinetics of appearance and longevity of HLA-E-restricted CD8+ T-cell responses for up to 2 years after immunization with the Ty21a typhoid vaccine and defined in detail the various memory T-cell subsets and effector functions mediating these responses.

Induction of effective memory T cells is likely to be critical to the level and duration of protection elicited by novel live oral typhoid vaccines. Using cells from volunteers who ingested Salmonella Typhi vaccine strain CVD 909, we characterized the induction of interferon (IFN)-gamma-secreting central (T(CM), CD45RO(+)CD62L(+)) and effector (T(EM), CD45RO(+)CD62L(-)) memory T populations, and their gut-homing potential based on integrin alpha4/beta7 expression. Both CD4(+) T(EM) and T(CM) populations secreted IFN-gamma. However, although CD4(+) T(EM) expressed, or not, integrin alpha(4)/beta(7), CD4(+) T(CM) cells were predominantly integrin alpha(4)/beta(7)(+). In contrast, IFN-gamma-secreting CD8(+) cells were predominantly classical T(EM) and CD45RA(+) T(EM) (T(EMRA), CD45RO(-)CD62L(-)) subsets. However, although CD8(+) T(EM) expressed, or not, integrin alpha(4)/beta(7), CD8(+) T(EMRA) were predominantly integrin alpha(4)/beta(7)(+). This is the first demonstration that oral immunization of humans with S. Typhi elicits diverse IFN-gamma-secreting CD4(+) and CD8(+) T(CM) and T(EM) subsets able to migrate to the gut and other lymphoid tissues.

Our previous studies in volunteers immunized with Salmonella enterica serovar Typhi (S. Typhi) have suggested an important role for CD8+ T cells in host defense. In this study we describe a novel subset of nonclassical human HLA-E-restricted S. Typhi-specific CD8+ T cells derived from PBMC of Ty21a typhoid vaccinees. CD3+CD8+CD4−CD56− T cells effectively killed S. Typhi-infected targets regardless of whether they share classical HLA class I molecules with them, by a FAS-independent, granule-dependent mechanism, as evidenced by induction of granzyme B release and the blocking effects of concanamycin and strontium ions. The expression of HLA-E Ags, but not CD1-a, -b, or -c, on the membrane of S. Typhi-infected targets rendered them susceptible to lysis. Moreover, anti-HLA-E Abs partially blocked these responses. We also demonstrated that presentation of S. Typhi Ags via HLA-E could stimulate IFN-γ production. Increases in the net frequency of IFN-γ spot-forming cells were observed in the presence of targets coated with peptides that contain S. Typhi GroEL HLA-E binding motifs. These results demonstrate that HLA-E binds nonamer peptides derived from bacterial proteins and trigger CD8+-mediated lysis and IFN-γ production when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections.

Type 1 cell-mediated immunity might play an important role in protection from typhoid fever. We evaluated whether immunization with Salmonella enterica serovar Typhi (S. Typhi) strain CVD 908-htrA (a ΔaroC ΔaroD ΔhtrA mutant), a leading live oral typhoid vaccine candidate, elicits specific CD4+ and CD8+ S. Typhi immune responses. Potent CTL responses and IFN-γ secretion by CD8+ T cells were detected following immunization with CVD 908-htrA in high (4.5 × 108 CFU) and low (5 × 107 CFU) dosages. S. Typhi-specific CTL were observed in six of eight vaccinees (four high and two low dose) after immunization. Mean increases in the frequency of IFN-γ spot-forming cells (SFC) in the presence of S. Typhi-infected targets were 221 ± 41 SFC/106 PBMC and 233 ± 87 SFC/106 PBMC, in the high and low dose groups, respectively. Strong CD4+ T cell responses were also observed. Increases in the IFN-γ production to soluble S. Typhi flagella (STF) occurred in 82 and 38% of the volunteers who received the high and low doses, respectively. Robust correlations were observed between volunteers that responded with IFN-γ SFC to stimulation with S. Typhi-infected cells and IFN-γ released in response to stimulation with STF Ags (r = 0.822, p < 0.001) and between CTL and IFN-γ production to STF (r = 0.818, p = 0.013). These data demonstrating the concomitant induction of both CD4- and CD8-mediated CMI are consistent with a significant role for type 1 immunity in controlling typhoid infection and support the continuing evaluation of CVD 908-htrA as a typhoid vaccine candidate.

We have measured in 22 asymptomatic human immunodeficiency virus type 1-infected patients (10 rapid progressors and 12 slow progressors) the proviral load of CD4+T cells homogeneously superinfected by the same dose of a non-syncytium-inducing virus in the presence or in the absence of autologous CD8+T cells. We demonstrated that the antiviral activity of CD8+T cells was highly predictive of the rate of peripheral CD4+T-cell decline.

BACKGROUND:The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8(+) T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-gamma secretion. However, how S. Typhi regulates the development of specific CD8(+) responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8(+) T cells. METHODOLOGY/PRINCIPAL FINDINGS:We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-alpha, but low levels of IL-12 p70 and IFN-gamma. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells. CONCLUSIONS/SIGNIFICANCE:This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8(+) cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.

CD8+T cells are likely to play an important role in host defense againstSalmonella entericaserovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-γ) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8+T cells in humans. To this end, we determined the TCR beta chain (Vβ) usage of CD8+T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vβ usage varied among volunteers, we identified oligoclonal Vβ subset expansions in individual volunteers (Vβ 2, 5.1, 8, 17, and 22 in volunteer 1; Vβ 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vβ 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-γ secretion assays on Vβ sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7−CD27−CD45RO+CD62L−) and coexpressed gut homing molecules (e.g., high levels of integrin α4β7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vβ families. Moreover, these serovar Typhi-specific CD8+T cells bearing defined Vβ specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4 + T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV + asymptomatic patient (PT) as effector cells and the CD4 + lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3 + , CD4 − , CD8 + , TCR α β + , TCR γ δ − , CD56 − ) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4 + T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.