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

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.


Supplementary Figure 2. Comparison of the kinetics and activation markers on MAIT cells among volunteers receiving a low-dose inoculum.
Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) total MAIT cells or (B) MAIT cells co-expressing CD38 and/or HLA-DR surface markers were evaluated and compared among NoTD and TD volunteers who received a lowdose inoculum. Data was grouped by time frames as follows: day 0, days 1-4, days 7-9 (for NoTD, or 48-96 hours for TD volunteers) and days 14-28. NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.

Supplementary Figure 3. Comparison of the kinetics and activation markers on MAIT cells among volunteers receiving a high-dose inoculum.
Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) total MAIT cells or (B) MAIT cells co-expressing CD38 and/or HLA-DR surface markers were evaluated and compared among NoTD and TD volunteers who received a high-dose inoculum. Data was group by time frame as follows: day 0, days 1-4, days 7-9 (for NoTD, or 48-96 hours for TD volunteers) and days 14-28. NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant. Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) total MAIT cells or (B) MAIT cells coexpressing CD38 and/or HLA-DR surface markers were evaluated and compared among TD volunteers receiving a high or low-dose inoculum. Data was grouped by time frame as follows: before (days 1-4) and after development of disease (48-96 hours after diagnosis. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.

Supplementary Figure 8. Comparison of exhaustion and apoptosis markers on MAIT cells among volunteers receiving a low-dose inoculum.
Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) exhaustion (CD57) or (B) apoptosis (Caspase-3) on total or activated MAIT cells were evaluated and compared among NoTD and TD volunteers who received a low-dose inoculum. Data was grouped by time frame as follows: day 0, days 1-4, days 7-9 (for NoTD, or 48-96 hours for TD volunteers) and days 14-28. NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.

Supplementary Figure 9. Comparison of exhaustion and apoptosis markers on MAIT cells among volunteers receiving a high-dose inoculum.
Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) exhaustion (CD57) or (B) apoptosis (Caspase-3) on total or activated MAIT cells were evaluated and compared among NoTD and TD volunteers who received a high-dose inoculum. Data was grouped by time frame as follows: day 0, days 1-4, days 7-9 (for NoTD, or 48-96 hours for TD volunteers) and days 14-28. NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.

Supplementary Figure 10. Comparison of the exhaustion and apoptosis markers on MAIT cells among TD volunteers receiving a high or low-dose inoculum.
Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) exhaustion (CD57) or (B) apoptosis (Caspase-3) on total or activated MAIT cells were evaluated and compared among TD volunteers receiving a high or low-dose inoculum. Data was grouped by time frame as follows: before (days 1-4) and after development of disease (48-96 hours after diagnosis). TD, Individuals who developed typhoid disease.

Supplementary Figure 12. Comparison of the exhaustion and apoptosis markers on proliferating MAIT cells among volunteers receiving a low-dose inoculum.
Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) total proliferating MAIT cells or (B) proliferating MAIT cells coexpressing CD38, CD57 or Caspase-3 surface markers were evaluated and compared among NoTD and TD volunteers who received a low-dose inoculum. Data was grouped by time frame as follows: day 0, days 1-4, days 7-9 (for NoTD, or 48-96 hours for TD volunteers) and days 14-28. NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.

Days after challenge
High-dose -Gated on ki67 + MAIT cells Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) total proliferating MAIT cells or (B) proliferating MAIT cells co-expressing CD38, CD57 or Caspase-3 surface markers were evaluated and compared among NoTD and TD volunteers who received a high-dose inoculum. Data was group by time frame as follows: day 0, days 1-4, days 7-9 (for NoTD, or 48-96 hours for TD volunteers) and days 14-28. NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.  Figure 14. Comparison of the exhaustion and apoptosis markers on proliferating MAIT cells among TD volunteers receiving a low or high-dose inoculum. Ex vivo PBMC isolated before (Day 0, baseline) and after challenge were analyzed as described in Figure 1. Levels of (A) total proliferating MAIT cells or (B) proliferating MAIT cells co-expressing CD38, CD57 or Caspase-3 surface markers were evaluated and compared among TD volunteers receiving high and low-dose inoculums. Data was grouped by time frame as follows: before (days 1-4) and after development of disease (48-96 hours after diagnosis). NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant. Ex vivo PBMC isolated before (Day 0, baseline) and after challenge 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 (CCR6) and gut (CCR9) tissues, respectively. Combined data of MAIT cells single expressing CCR6 or CCR9, or co-expressing CCR6 and CD38, CCR9 and CD38, CCR6 and CCR9 were compared among NoTD and TD volunteers who received a low-dose inoculum. Data was group by time frame as follows: day 0, days 1-4, days 7-9 (for NoTD, or 48-96 hours for TD volunteers) and days 14-28. NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant. Ex vivo PBMC isolated before (Day 0, baseline) and after challenge 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 (CCR6) and gut (CCR9) tissues, respectively. Combined data of MAIT cells single expressing CCR6 or CCR9, or co-expressing CCR6 and CD38, CCR9 and CD38, CCR6 and CCR9 were compared among NoTD and TD volunteers who received a high-dose inoculum. Data was grouped by time frame as follows: day 0, days 1-4, days 7-9 (for NoTD, or 48-96 hours for TD volunteers) and days 14-28. NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.  Figure 17. Comparison of the homing markers on MAIT cells among volunteers receiving a high-or low-dose inoculum who developed TD. Ex vivo PBMC isolated before (Day 0, baseline) and after challenge 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 (CCR6) and gut (CCR9) tissues, respectively. Combined data of MAIT cells single expressing CCR6 or CCR9, or co-expressing CCR6 and CD38, CCR9 and CD38, CCR6 and CCR9, CCR6 and Caspase-3, or CCR9 and Caspase-3 were evaluated and compared among TD volunteers receiving a low or high-dose inoculum. Data was grouped by time frame as follows: before (days 1-4) and after development of disease (48-96 hours after diagnosis). TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.  Figure 18. Comparison of the homing markers on MAIT cells among volunteers receiving a low or high-dose inoculum. Ex vivo PBMC isolated before (Day 0, baseline) and after challenge 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 (CCR6) and gut (CCR9) tissues, respectively. Combined data of MAIT cells co-expressing CCR6 and Caspase-3, or CCR9 and Caspase-3 were evaluated and compared among NoTD and TD volunteers receiving a low-(A) or high-dose (B) inoculum. Data was grouped by time frame as follows: before (days 1-4) and after development of disease (48-96 hours after diagnosis). NoTD, individuals who did not develop typhoid disease. TD, Individuals who developed typhoid disease. P values of <0.05 were considered statistically significant.