Challenge of Humans with Wild-type Salmonella enterica Serovar Typhi Elicits Changes in the Activation and Homing Characteristics of Mucosal-Associated Invariant T Cells

Abstract

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.

Keywords: MAIT cells; Salmonella; T cells; bacteria; gut; human.

Figure 1

Kinetics of MAIT cells over a 28-day post-challenge follow-up period. Ex vivo peripheral blood mononuclear cells were stained with YEVID, followed by surface staining with monoclonal antibodies to CD3, CD4, CD8, CD14, CD19, CD161, and TCRα 7.2 and analyzed by multichromatic flow cytometry. For the analysis, following the elimination of doublets and other debris, the cells were gated on lymphocytes, and then a “dump” channel was used to eliminate dead cells (YEVID⁺) as well as macrophages (CD14⁺), and B cells (CD19⁺) from the analyses. This was followed by additional gating on CD3, CD4, and CD8, as well as CD161 vs. TCRα 7.2 to analyze MAIT cells. (A) A representative gating strategy for MAIT cells. (B) Individual volunteers and (C) combined data of MAIT cell kinetics. Bar graphs extend from the 25th to 75th percentiles, and the line in the middle represents the median of the pooled data from all the volunteers. The whiskers delineate the smallest to the largest value. Numbers in the “X” axis represent days after the challenge, except for the numbers inside of the green box that represent 48 and 96 h after diagnosis of typhoid disease. NoTD, volunteers who did not develop typhoid disease; TD, volunteers who developed typhoid disease.

Figure 2

MAIT cell activation following Salmonella enterica serovar Typhi challenge. Ex vivo peripheral blood mononuclear cells were analyzed as described in Figure 1. Expression of CD38 and HLA-DR was evaluated to identify activated MAIT cells. Activated MAIT cells in individual volunteers expressing (A) CD38 or coexpressing (B) CD38 and HLA-DR surface markers. (C) Combined data of the activated MAIT cells coexpressing CD38 and HLA-DR surface markers in all volunteers. (D) Representative data of MAIT cells coexpressing CD38 and HLA-DR surface markers within the CD3⁺CD4⁻CD8⁺ T cell population. Bar graphs extend from the 25th to 75th percentiles, and the line in the middle represents the median of the pooled data from the five different subjects. The whiskers delineate the smallest to the largest value. Numbers in the “X” axis represent days after the challenge, except for the numbers inside of the green box that represent 48 and 96 h after diagnosis of typhoid disease. NoTD, volunteers who did not develop typhoid disease; TD, volunteers who developed typhoid disease.

Figure 3

Identification of exhausted and apoptotic MAIT cells following Salmonella enterica serovar Typhi challenge. Ex vivo peripheral blood mononuclear cells were analyzed as described in Figure 1. Expression of CD57 and caspase-3 were performed to identify exhausted and apoptotic MAIT cells, respectively. Combined data of MAIT cells expressing (A) CD57 or coexpressing (B) CD57 and CD38 surface markers. Combined data of MAIT cells expressing (C) caspase-3 or coexpressing (D) caspase-3 and CD38 surface markers. Bar graphs extend from the 25th to 75th percentiles, and the line in the middle represents the median of the pooled data from the five different subjects. The whiskers delineate the smallest to the largest value. Numbers in the “X” axis represent days after the challenge, except for the numbers inside of the green box that represent 48 and 96 h after diagnosis of typhoid disease. NoTD, volunteers who did not develop typhoid disease; TD, volunteers who developed typhoid disease.

Figure 4

MAIT cell proliferation following Salmonella enterica serovar Typhi challenge. Ex vivo peripheral blood mononuclear cells were analyzed as described in Figure 1. Ki67 expression was used to identify proliferating MAIT cells. (A) Representative data of intracellular expression of Ki67⁺ MAIT cells over a 28-day post-challenge follow-up period in NoTD and typhoid disease (TD) volunteers who received a low- or high-dose challenge. (B) Individual and (C) combined data of proliferating MAIT cells from volunteers receiving a low-dose challenge. (D) Individual and (E) combined data of proliferating MAIT cells from volunteers receiving a high-dose challenge. (F) Individual and (G) combined data of CD38-positive cells gated on proliferating (Ki67⁺) MAIT cells. Bar graphs extend from the 25th to 75th percentiles, and the line in the middle represents the median of the pooled data from the five different subjects. The whiskers delineate the smallest to the largest value. Numbers in the “X” axis represent days after the challenge, except for the numbers inside of the green box that represent 48 and 96 h after diagnosis of typhoid disease. NoTD, volunteers who did not develop typhoid disease; TD, volunteers who developed typhoid disease.

Figure 5

Evaluation of exhaustion and apoptosis in proliferating MAIT cells. Ex vivo peripheral blood mononuclear cells were analyzed as described in Figure 1. Expression of CD57 and caspase-3 were performed to identify exhausted and apoptotic in proliferating MAIT cells, respectively. Individual and combined data of Ki67⁺ MAIT cells expressing (A) capsase-3 or (B) CD57 surface markers are shown. MAIT cells were sequentially gated on Ki67⁺ and then on capsase-3 or CD57 surface markers. Bar graphs extend from the 25th to 75th percentiles, and the line in the middle represents the median of the pooled data from the five different subjects. The whiskers delineate the smallest to the largest value. Numbers in the “X” axis represent days after the challenge, except for the numbers inside of the green box that represent 48 and 96 h after diagnosis of typhoid disease. NoTD, volunteers who did not develop typhoid disease; TD, volunteers who developed typhoid disease.

Figure 6

Evaluation of the homing potential of MAIT cells following Salmonella enterica serovar Typhi challenge. Ex vivo peripheral blood mononuclear cells were analyzed as described in Figure 1. Additional gating on CCR6 and CCR9 were performed to identify MAIT cells with the potential to home to inflamed and gut tissues, respectively. Combined data of MAIT cells coexpressing (A) CCR6 and CD38, (B) CCR9 and CD38, (C) CCR6 and CCR9, (D) CCR6 and caspase-3, and (E) CCR9 and caspase-3 surface markers. Bar graphs extend from the 25th to 75th percentiles, and the line in the middle represents the median of the pooled data from the five different subjects. The whiskers delineate the smallest to the largest value. Numbers in the “X” axis represent days after the challenge, except for the numbers inside of the green box that represent 48 and 96 h after diagnosis of typhoid disease. NoTD, volunteers who did not develop typhoid disease; TD, volunteers who developed typhoid disease.