Participants’ text message responses to the nine questions.

<p>Nine of the 36 text messages sent to the study participants asked direct questions. Counting the number of responses provided a measure of engagement and retention in the study. Thirty participants (88%) responded to these questions. More than half (18 men; 53%) replied to seven or more of the nine questions with two replying to all nine questions.</p

Studies of the effects of Opa proteins on CD4⁺ T cell proliferation.

<p>Studies of the effects of Opa proteins on CD4⁺ T cell proliferation.</p

Serum bactericidal antibody titres of pooled murine sera against 4 target strains, following immunisation with wild-type and mutant recombinant OpaA and OpaD proteins.

<p>Serum bactericidal antibody titres of pooled murine sera against 4 target strains, following immunisation with wild-type and mutant recombinant OpaA and OpaD proteins.</p

CD4⁺ T cell proliferation following incubation of PBMCs ex vivo with different antigens.

<p>Proliferation of each antigen relative to media only control. Each coloured dot represents the value for a single individual (control antigens—blue; recombinant proteins—purple, liposomal proteins—green, outer membrane vesicles—yellow, inactivated bacteria—red). Black dots represent mean value for each antigen, with 95% confidence intervals shown. Number in parentheses after each antigen represents number on individuals in that group. CEACAM = carcinoembryonic antigen-related cell adhesion molecule; SEB = Staphylococcal enterotoxin B; PBS = phosphate-buffered saline; LDAO = llauryldimethylamine-oxide; wt = wild-type. PHA (phytohaemagglutinin) result not shown for clarity, to confine y-axis to relevant range for other antigens.</p

CD4⁺ T cell proliferation following incubation of PBMCs with different antigens, plus IL-2, anti-CD3 and anti-CD28.

<p>Proliferation of each antigen relative to media only control. Each coloured dot represents the value for a single individual (control antigens—blue; recombinant proteins—purple, liposomal proteins—green, outer membrane vesicles—yellow, inactivated bacteria—red). Black dots represent mean value for each antigen, with 95% confidence intervals shown. Number in parentheses after each antigen represents number on individuals in that group. PHA = phytohaemagglutinin; CEACAM = carcinoembryonic antigen-related cell adhesion molecule; SEB = Staphylococcal enterotoxin B; PBS = phosphate-buffered saline; LDAO = llauryldimethylamine-oxide; wt = wild-type.</p

Antigens used for T cell proliferation assays.

<p>Antigens used for T cell proliferation assays.</p

CD4⁺ T cell proliferation following incubation of PBMCs with different antigens, plus IL-2.

<p>CD4⁺ T cell proliferation following incubation of PBMCs with different antigens, plus IL-2.</p

MOESM1 of Salmonella Typhi-specific multifunctional CD8+ T cells play a dominant role in protection from typhoid fever in humans

Additional file 1: Table S1. Participant Demographic Characteristics. Figure S1. Baseline hematological parameters and S. typhi-specific responses. (a) Total white cell count (WCC) and absolute numbers of lymphocytes were assessed by routine blood hematology before challenge. Absolute numbers of CD3+, CD3+CD8+, CD3+CD4+ cells, and of CD3+CD8+ memory subsets (TEM, TEMRA and TCM) were calculated using the percentages of positive cells obtained by flow cytometry analysis. Statistical analyses were performed using the Mann–Whitney test. (b) Individual representation of baseline CD8+ TEM immune responses following stimulation with S. typhi-infected cells. PBMC isolated at baseline from each participant (TD n = 7, blue; NoTD n = 9, red) were stimulated for 18 h with S. typhi-infected B-EBV cells (circles) or S. typhi-infected blasts (triangles). After co-culture, cells were immunostained with a 14-color panel of mAbs and analyzed by follow cytometry as described in Methods. Each symbol represents the net percentage of positive CD8+ TEM cells measured for CD107a, IFN-γ, TNF-α, MIP-1β, as indicated. Horizontal lines represent the median for each group. Statistical analyses were performed using the Mann–Whitney test. *p < 0.05; **p < 0.01. Figure S2. Absolute numbers of T cell subsets in circulation after challenge. Shown are the kinetics of various T cell subsets in representative participants from the TD and NoTD groups. One participant who did not meet diagnosis definition (NoTD) but showed the presence of S. typhi in blood by PCR assay is also represented. (a) Absolute numbers of CD3+ and CD3+CD8+cells following challenge were calculated using the percentages of positive cells obtained by flow cytometry analysis. (b) Absolute numbers of IFN-γ+ and CD107a expressing S. Typhi-specific CD8+ TEM following challenge. Figure S3. Kinetics and amplitude of S. Typhi-specific CD8+ T cell responses after challenge for each participant. (a) Kinetics of IFN-γ production by CD8+ TEM following stimulation with S. Typhi-infected B-EBV cells are presented for each participant. The day at which each of the TD participants was diagnosed is indicated after their id number. (b) Areas under the curve were measured around the time of diagnosis for each biomarker in CD8+ TEM, TEMRA and TCM subsets. Each bar represents mean ± SEM of area under the curve obtained after stimulation with S. Typhi-infected AEH cells, B-EBV cells and blasts. Figure S4. Homing potential of the dominant S. Typhi-specific CD8+ TEM multifunctional populations at baseline. Flow cytometry data were analyzed using the FCOM function of Winlist to determine the proportion of all possible combinations of the six measured biomarkers to identify MF cells (i.e., positive for multiple biomarkers concomitantly). Each symbol represents the percentage of the different populations measured after stimulation with S. Typhi-infected cells (AEH cells [squares], B-EBV [circles] cells or blasts [triangles]) for each participant. (a) The percentages of single positive cells (1+) or of total MF cells (i.e., the sum of all cells concomitantly positive for 2 or more biomarkers) are represented for CD8+ TEM integrin α4β7− and integrin α4β7+ cells. (b) Total MF cells were divided into four groups on the basis of the number of biomarkers they expressed. (C) Shown are the six major individual populations of MF in integrin α4β7− and integrin α4β7+ S. Typhi-specific CD8+ TEM at baseline in all NoTD participants. Horizontal lines represent the median for each group. Statistical analyses were performed using mixed effects models to account for multiple observations per person. *p < 0.05, **p < 0.01, ***p < 0.001. Figure S5. Dominant populations and gut homing capabilities of MF S. Typhi-specific CD8+ TEM responses in TD and NoTD participants after challenge. Flow cytometry data were analyzed using the FCOM function of Winlist to determine the proportion of all possible combinations of the six measured biomarkers to identify MF cells (i.e., positive for several biomarkers concomitantly). Percentages were measured at day 7 for NoTD participants and at 48 h after typhoid diagnosis for TD participants. Each symbol represents the percentage of the different populations measured after stimulation with S. typhi-infected cells (AEH cells [squares], B-EBV [circles] cells or blasts [triangles]) for each participant. (a) The percentages of single positive cells (1+) or of total MF cells (i.e., the sum of all cells concomitantly positive for two or more biomarkers) are represented for CD8+ TEM integrin α4β7− and integrin α4β7 + cells. (b) Total MF cells were divided into four groups on the basis of the number of biomarkers they expressed. (c) The six major individual populations of MF in CD8+ TEM are represented separately for S. typhi-specific integrin α4β7− (green) and integrin α4β7+ (purple) CD8+ TEM in NoTD participants at day 7 post-challenge. Horizontal lines represent the median for each group. Statistical analyses were performed using mixed effects models to account for multiple observations per person. *p < 0.05, **p < 0.01, ***p < 0.001

Opposite associations of hepcidin with serum iron and transferrin saturation in the presence and absence of inflammation.

<p>Associations between (A) hepcidin and serum iron, (B) hepcidin and transferrin saturation (Tsat), (C) ferritin and serum iron, and (D) ferritin and Tsat, when acute inflammation (defined as CRP>5 mg/L, open circles) or when no inflammation (CRP<5 mg/L, closed circles) was evident. The analysis considered all data obtained between challenge day and Day 14 post-challenge for each study participant, whether diagnosed with typhoid or not. To normalize data, each parameter was log-transformed prior to analysis. The analysis accounts for individuals contributing more than single data points by using regression with clustered errors (Serum Iron analyses (A) and (C): CRP <5 mg/L: 118 observations, 47 clusters; CRP >5 mg/L: 69 observations, 36 clusters. Tsat analyses (B) and (D): CRP <5 mg/L: 117 observations, 46 clusters; CRP >5 mg/L: 69 observations, 36 clusters). Regression with clustered errors adjusts the confidence intervals of the regression coefficients to account for intra-cluster correlation, as is likely when multiple observations from the same individuals are included. Pearson correlation coefficients and p-values are stated.</p

Changes in hepcidin, iron and inflammatory indices between baseline–the day of typhoid challenge–and the day of typhoid diagnosis.

<p>Serum samples were available from both day of typhoid challenge and day of typhoid diagnosis from 19 individuals from Study A (placebo arm of vaccine/typhoid challenge study). (A) temperature, (B) hepcidin, (C) serum iron, (D) transferrin saturation, (E) CRP, (F) ferritin and (G) hemoglobin were measured on the day of challenge and day of typhoid diagnosis. P-values represent results of paired t tests based on geometric means for hepcidin (baseline, 10.4 ng/mL [95% CI: 7.1–15.3]; diagnosis, 98.2 ng/mL [75.9–126.9]), ferritin (baseline, 46.3 μg/L [30.8–69.8]; diagnosis, 86.4μg/L [56.9–131.0]) and CRP (baseline: 1.46 mg/L [0.84–2.54]; diagnosis, 34.1 mg/L [24.2–48.2]), and arithmetic means for temperature (baseline, 36.3°C [36.1–36.5]; diagnosis, 37.6°C [37.3–37.9]), serum iron (baseline, 12.6 μmol/L [9.9–15.3]; diagnosis, 4.4 μmol/L [2.7–6.1]), transferrin saturation (baseline, 22.3% [17.2–27.4]; diagnosis, 7.4% [4.7–10.2]) and hemoglobin (baseline, 14.3 g/dL [13.6–15.1]; diagnosis 13.9 g/dL [13.1–14.8]).</p