To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell–receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4+ T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single–amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell–vaccination strategies against Salmonella.
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This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.), Cancer Research UK (CRUK) (C399/A2291) (V.C.), the NIHR Clinical Research Network Thames Valley (A.J.P.), the Oxford Biomedical Research Centre (A.J.P., V.C.), the Bill & Melinda Gates Foundation (OPP1084259) (A.J.P.), the European Vaccine Initiative (PIM) (A.J.P.), and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.). G.D. is supported by The Wellcome Trust. We thank C. Waugh and the WIMM FACS facility for assistance with cell sorting, and T. Rostron for assistance with next-generation sequencing. The DRB4 tetramers were provided by B. Kwok and I.-T. Chow, from the Tetramer Core Laboratory at the Benaroya Research Institute at Virginia Mason.
The authors declare no competing interests.
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Integrated supplementary information
(a)Gating strategy to identify CD4+ and CD4+Ki67+ cells in Mass Cytometry experiments. Frequency of Cluster 19 cells within Ki67+ (b) and total CD4+ T cells (c) at different time points after infection (n = 6). (b) Friedman Test p = 0.0147, Dunn’s Multiple Comparison Test D0 vs D4, D28, D90 p = ns, D0 vs EF p = 0.027. (c)Friedman Test p = 0.0159, Dunn’s Multiple Comparison Test D0 vs D4, D28, D90 p = ns, D0 vs EF p = 0.0247. (d) Total Ki67+ events (grey) and Cluster 19 events (red) across all samples were concatenated and expression of 20 markers against CD38 in the distinct populations is shown.
Supplementary Figure 2 CCR7–CD38+CD4+ cells are enriched in Ki67+ accumulating during enteric fever.
(a) Expression of CCR7 and CD38 in the 20 distinct clusters identified by PhenoGraph analysis. (b) Proportion of PhenoGraph clusters within Ki67+ CD38+CCR7- cells at the peak of Ki67+ cell accumulation. (c) Frequency of Ki67+ CD38+CCR7- cells (Friedman Test p = 0.0130, Dunn’s Multiple Comparison Test D0 vs D4, D28, D90p = ns, D0 vs EF p = 0.0139) and of KI67 + cells non comprised within CD38+CCR7- cells over time in challenged volunteers (n = 6) (Friedman Test p = 0.23, Dunn’s Multiple Comparison Test D0 vs D4, EF, D28, D90 p = ns). Box extends from the 25th to the 75th percentile and whiskers from the minimum to the maximum value; line indicates median.
Supplementary Figure 3 Accumulation of CD4+CD38+CCR7– in whole blood of volunteers with enteric fever
(a) Identification of CD4+CD38+CCR7- cells during enteric fever by Multicolour Flow Cytometry in whole blood from a representative volunteer challenged with S. Typhi, and one challenged with S. Paratyphi A. (b) Accumulation of CD38+CCR7- cells after diagnosis in volunteers challenged with S. Typhi and Paratyphi A who developed (Diagnosed) but not in those who did not develop enteric fever (Not Diagnosed). In challenged volunteers not diagnosed blood was collected 14 days after challenge (D14). Friedman Multi comparison Test p = 0.006 (S. Typhi), p < 0.0001 (S. Paratyphi). Dunn’s Multiple Comparison Test ED vs D0, p = 0.0065 (Typhi, n = 9); p = 0.0002 (S. Paratyphi A, n = 9). Box extends from the 25th to the 75th percentile and whiskers from the minimum to the maximum value; line indicates median.
Shown is an example with four live fluorescent dyes. Cell were labelled with 9 combinations of three distinct dilution of Violet and Far red dyes. Cells with each of the nine cluster were labelled with 6 combinations of 4 distinct dilutions of CFSE and and 3 of CMTMR orange dyes. Dilutions used for adjacent clusters across the 640-670 axis were labelled with alternate dilutions of CFSE and CMTMR Orange (as shown in the overlaid plots) in order to reduce spill-over in case of suboptimal separation between clusters across the channel-dye combination with the lowest stain index.
Supplementary Figure 5 Increased frequency of HlyE- and CdtB- specific T cells in volunteers diagnosed with enteric fever
(a) Frequency of HlyE, CdtB and Flu specific T cell responses 28 days after challenge measured by IFN-g ELISPOT in volunteers challenged with S. Typhi or S. Paratyphi A who developed (Diagnosed, D, n = 12) or did not develop (Not Diagnosed, ND, n = 9)) enteric fever compared to uninfected controls (HC. n = 10). Freshly isolated PBMC were rested overnight and incubated with 5μg/ml of HlyE or CdtB peptide pools, or with inactivated seasonal influenza vaccine (Flu). Mann Witney two tailed t-test. * = p < 0.05; ** = p < 0.005. Box extends from the 25th to the 75th percentile and whiskers from the minimum to the maximum value; line indicates median.
(a) Monocyte derived macrophages (MF) were differentiated for 5 days in the lower chamber of 24 well transwell plates. At day 5 a pool of 3 CdtB specific clones (T) was added to the upper well in the presence or absence of CdtB peptide pool (CdtB). After O.N. incubation, upper well was removed, macrophages were washed, infected with S. Typhimurium (LT2 strain) for 30 m, washed again and incubated with Gentamicin (30 mg/ml) to limit bacterial overgrowth. 2 h after infection cell culture supernatant was plated in agar plates and colonies counted after over night culture. Indicated is the percentage of bacterial growth compared to macrophages alone (n = 6, Friedman test (p = 0.0008) with Dunn’s multiple comparisons test (control vs T + CdtB p = 0.0169). Box extends from the 25th to the 75th percentile and whiskers from the minimum to the maximum value; line indicates median. (b) CFU after O.N. culture of macrophage supernatant in agar plate in one representative experiment.
Supplementary Figure 7 Increased frequency of CdtB, HlyE, and PhoN T cell responses after diagnosis with enteric fever
(a) Frequency of HlyE, CdtB, and Flu specific T cell responses at baseline and 28 days after challenge measured by IFN-g ELISPOT in frozen PBMC from volunteers challenged with S. Typhi or S. Paratyphi A who developed (n = 19) enteric fever (Wilcoxon matched-pairs signed rank test). (b) Frequency of HlyE, CdtB, PhoN and Flu specific T cell responses in HLADRB4 + and HLADRB4- volunteers 28 days after challenge (Mann Whitney test two-tailed p value). (c) Frequency of PhoN specific T cell responses at baseline and 28 days after challenge measured by IFN-g ELISPOT in frozen PBMC from volunteers challenged with S. Typhi or S. Paratyphi A who developed (n = 19) enteric fever and in HLADRB4+ and HLADRB4- individuals. ELISPOT analysis done on the same frozen samples as in panel a and b.
(a) Rarefaction curve describing the estimated diversity of the CDR3β repertoire of CD38+CCR7- and CD38-CCR7- subsets in volunteers P1 and T4. Dots between continuous and dashed curves indicate the number of TCRs identified within each sample. Number of cells probed: P1 CD38+CCR7- = 5087 cells, CD38-CCR7- = 1.15e105 cells; T4 CD38+CCR7- = 9130 cells, CD38-CCR7- = 1.3e105 cells. (b) Pathogen selectivity, antigen specificity, restriction, CDR3α and CDR3β sequence of clones isolated from volunteer P1. Starred(*) sequences were identified also in the polyclonal analysis. (c) Pie Chart depicting the frequency of CDR3β sequences within the polyclonal repertoire of CD38+CCR7- cells (5087 cells probed) from T4, highlighted are the CDR3β sequences identified in the isolated clones. (d) Frequency of CDR3β within the polyclonal repertoire of CD38+CCR7- cells and CD38-CCR7- cells in volunteer P1 (1.15e105 cells probed). Red circles represent CDR3β sequences identified in the isolated clones.
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Napolitani, G., Kurupati, P., Teng, K.W.W. et al. Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses. Nat Immunol 19, 742–754 (2018). https://doi.org/10.1038/s41590-018-0133-z
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