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1 % Lymphocyte in CBC A Lymphocyte count /µl B. x C. 50 D % CD3+CD8+ Cells Calculated CD8+ count/µl #61 #63 #64 #65 #68 #71 #72 #75 Figure S1. Blood lymphocyte counts and absolute numbers of CD3+CD8+ cells before and following Ty21a vaccination. Percentages (panel A) and absolute lymphocyte counts/µl (panel B) were obtained from available Complete Blood Cell counts (CBC) performed in blood specimens collected at Days 0 (n=14), 42 (n=13) and 84 (n=10) from Ty21a vaccinated volunteers. The % of CD3+CD8+ cells (panel C) were determined by flow cytometry. Absolute numbers of CD8+ cells/µl (panel D) were calculated as: [(Absolute counts of lymphocyte/µl) X (% of CD3+CD8+ cells; by flow cytometry)/100]] Horizontal bars represent the mean values for each group. A one way ANOVA with Bonferroni's Multiple Comparison Test showed no significant differences (p>0.15) in any of the parameters shown for D0, D42 or D84 samples. #76 #77 #80 #92 #95 #97

2 Number of cells S. Typhi (70%) S. Para A (53%) Gated on live EBV-B cells S. Para B (62%) No infection (1.5%) CSA-FITC Figure S2. Preparation of EBV-infected targets. EBV transformed B Cell lines were infected with S. Typhi, S. Paratyphi A and S. Paratyphi A (MOI: 10:1, Bacteria:Cell) and stained 18 hours post-infection. Percentages of Salmonella antigen expressing cells (CSA-1 + : solid bar) are shown in parenthesis.

3 R96: % R97: % R98: % R99: % R96 A. Gating protocol A1. Lymphocyte gate SSC-A R2: 39.46% FSC-H FSC-A R3: 95.17% FSC-H YEVID + CD14/19/45 BV570-A R4: 54.70% CD3 BV650-A CD8 PerCP-Cy5-5-A A2. Doublets exclusion A3. Live CD3 + A4. Live CD3 + CD4 - CD8 + T CM A5. CD8 + T cell memory R5: 27.31% CD4 PE-Cy5-A CD62L APC-EF780-A T EM T N T EMRA CD45RA Qdot 800-A B. CD8 + C. CD D. CD IFN- + E. CD IFN- + a + CD62L-APC-E780 CD62L APC-EF780-A T CM T EM T EMRA R100: 10.22% R99: 31.48% R98: 24.59% R97: 32.20% CD45RA Qdot 800-A T N CD45RA Qdot-800 Figure S3. Gating protocol and characteristics of Ty21a-induced effector CD8 + T cells. A sequential gating protocol (Panels A1-A5) was used to define memory subsets of CD8+ T cells, i.e., T central memory (T CM ; CD62L+CD45RA ), T naive (T N ; CD62L+CD45RA+), T effector memory (T EM ; CD62L CD45RA ), and CD45RA positive T effector (T EMRA ; CD62L CD45RA+). PBMC collected from a representative volunteer 42 days following immunization with Ty21a was stimulated with S. Typhi-infected targets. Representative histograms show memory T subpopulations (gated on live CD14- CD3+CD4-CD8- depicted in gray dots; panel B) on which back-gated cells were superimposed as follows: panel C (CD69+ activated cells in red), panel D (CD69+ IFN- + cells in blue) and panel E (CD69+IFN- +a+ cells in purple). The numbers shown in the boxes within histograms (panels C, D, E) are the % of the superimposed gated populations into the different T effector and memory subsets (as shown in panel B).

4 A. Gated on CD8+ T EM Gated on CD69+ IFN + CD8+ T EM B. C. D. 3.8% 3.74% IFN CD69 Gated on CD69+ IFN + CD8+ T EMRA F. G. H IFN- E. Gated on CD8+ T EMRA CD69 CD69 Figure S4. Representative histograms of multifunctional cells. PBMC collected from a representative volunteer (#92) 42 days following immunization with Ty21a were stimulated with S. Typhi-infected targets. Shown are two parameter histograms of activated (CD69+) IFN- + cells corresponding to T EM (panel A) and T EMRA (panel E) subsets of CD8+ cells (gating protocol is shown in Fig. S3). CD8+CD69+IFN + cells in T EM (panels B,C,D) and T EMRA subsets (panels F,G,H) were further analyzed regarding their co-production of,, or. The numbers shown within histograms represent the % of gated subsets in the corresponding quadrant.

5 A. MF IFN- + CD8+T EM B. MF IFN- + CD8+T EMRA * % of CD8+ T EM C. MF + CD8+T EM % of CD8+ T EMRA D. MF + CD8+T EMRA * 0.1 #61 #64 #68 #72 #75 #77 #80 #95 #63 #65 #71 #74 #76 #78 #92 #97 Figure S5. Post-vaccination peak increases in Salmonella-specific cross-reactive multifunctional cells in individual Ty21a vaccinees: Shown are the peak post-vaccination increases of S. Typhi (ST)-, S. Paratyphi A (PA)- or S. Paratyphi B (PB)- specific IFN- + (Panels A,B) and + (Panels C,D) of total multifunctional (MF, the sum of all multifunctional subsets) cells in CD8+T EM (n=16, panels A, C) and CD8+T EMRA (n=15, panels B,D) subsets. Post-vaccination peaks: Peak responses at days 42 or 84 minus pre-vaccination [day 0] levels Horizontal bars represent Mean p<1. *p<5 compared to the corresponding T EM (in panels A and C) by Wilcoxon signed rank test, 2-tail. ST specific increases in CD8+ T EMRA subsets in volunteer #74 were outliers (above mean+3sd) and thus were excluded for this comparative analysis

6 CD8+T EM MF cells A B C * # S. Typhi S. Para A S. Para B CD8+T EMRA MF cells D E F S. Typhi S. Para A S. Para B Figure S6. Characterization of post-vaccination increases in multifunctional responses by CD8+ T EM and T EMRA subsets. Post-vaccination peak increases (peak level at days 42 or 84 post-vaccination minus the corresponding pre-vaccination levels) in IFN-, TNF- and producing and/or a expressing MF subpopulations were determined by FCOM analysis in 16 subjects. CD8+ T EM (panels A,B,C) and CD8+T EMRA (panels D,E,F) cells concomitantly producing double (2+), triple (3+) or all (quadruple, 4+) cytokines or expressing cells are shown as percentages of the corresponding total Salmonella-specific MF cells. Data were analyzed by Mann-Whitney tests. Comparisons represent those between CD8+ T EM and T EMRA subsets. *p<5 p<1. #p=6

7 % of CD8+ T EM A. MF CD8+T EM B. MF CD8+T EMRA % of CD8+ T EM # # 65U 71U 72U 74U 80U 92U 95U 97U Figure S7. Post-vaccination increases in MIP-1 producing CD8 + cells in individual Ty21a vaccinees. Shown are the post-vaccination peak increases in total multifunctional (MF) MIP-1 + cells in CD8+T EM (panel A) and CD8+T EMRA (panel B) subsets following stimulation with S. Typhi (ST)-, S. Paratyphi A (PA)- and S. Paratyphi B (PB)-infected targets. Horizontal Bars indicate the means of each group (n=8). #p=0.12 by Wilcoxon signed rank test, 2-tail.

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