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. 2024 Sep 12:15:1384642.
doi: 10.3389/fimmu.2024.1384642. eCollection 2024.

Role of circulating T follicular helper subsets following Ty21a immunization and oral challenge with wild type S. Typhi in humans

Jayaum S Booth  1   2 , Rekha R Rapaka  1   3 , Monica A McArthur  1   2   4 , Stephanie Fresnay  1   2   5 , Thomas C Darton  6   7 , Christoph J Blohmke  6   8 , Claire Jones  6 , Claire S Waddington  6   9   10 , Myron M Levine  1   2   3 , Andrew J Pollard  6 , Marcelo B Sztein  1   2   3   11
Affiliations

Role of circulating T follicular helper subsets following Ty21a immunization and oral challenge with wild type S. Typhi in humans

Jayaum S Booth et al. Front Immunol. .

Abstract

Despite decades of intense research, our understanding of the correlates of protection against Salmonella Typhi (S. Typhi) infection and disease remains incomplete. T follicular helper cells (TFH), an important link between cellular and humoral immunity, play an important role in the development and production of high affinity antibodies. While traditional TFH cells reside in germinal centers, circulating TFH (cTFH) (a memory subset of TFH) are present in blood. We used specimens from a typhoid controlled human infection model whereby participants were immunized with Ty21a live attenuated S. Typhi vaccine and then challenged with virulent S. Typhi. Some participants developed typhoid disease (TD) and some did not (NoTD), which allowed us to assess the association of cTFH subsets in the development and prevention of typhoid disease. Of note, the frequencies of cTFH were higher in NoTD than in TD participants, particularly 7 days after challenge. Furthermore, the frequencies of cTFH2 and cTFH17, but not cTFH1 subsets were higher in NoTD than TD participants. However, we observed that ex-vivo expression of activation and homing markers were higher in TD than in NoTD participants, particularly after challenge. Moreover, cTFH subsets produced higher levels of S. Typhi-specific responses (cytokines/chemokines) in both the immunization and challenge phases. Interestingly, unsupervised analysis revealed unique clusters with distinct signatures for each cTFH subset that may play a role in either the development or prevention of typhoid disease. Importantly, we observed associations between frequencies of defined cTFH subsets and anti-S. Typhi antibodies. Taken together, our results suggest that circulating TFH2 and TFH17 subsets might play an important role in the development or prevention of typhoid disease. The contribution of these clusters was found to be distinct in the immunization and/or challenge phases. These results have important implications for vaccines aimed at inducing long-lived protective T cell and antibody responses.

Keywords: CHIM; S. Typhi; cTfh; circulating follicular helper T cells; typhoid fever.

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Conflict of interest statement

ML: Co-inventor of a live attenuated S. Typhi vaccine strain CVD 909 and a S. Paratyphi A vaccine strain CVD 1902 that have been licensed to Bharat Biotech International BBI, Hyderabad, India for clinical development. ML is also a co-inventor of a Trivalent Salmonella Conjugate vaccine that includes S. Enteritidis, S. Typhimurium conjugates core plus O-polysaccharide covalently linked to FliC flagellin subunits of the homologous serovars in combination with BBI’s Vi conjugate Typbar TCV. AP: is chair of the UK department of Health and Social Cares Joint Committee on vaccination and immunisation. He has research grants on typhoid/paratyphoid vaccines from Serum Institute of India, Medical Research Council, Wellcome Trust and Bill & Melinda gates Foundation. Author MM was employed by company Sanofi. Authors SF and CB were employed by company GlaxsoSmithKline. Author RR is presently employed at Moderna Therapeutics and owns shares/options. Her contributions to this project were made prior to her employment at Moderna. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Control human infection model. Schematic of a typhoid control human infection model (CHIM). Participants were recruited and immunized with three doses of the live oral attenuated typhoid vaccine, Ty21a, at days minus 28 (D-28), D-26 and D-24. Participants were then challenged on day 0 (D0) with wt S. Typhi (Quailes Strain) at a dose of 1-5 X 104 CFU. At around day 7, some of the participants developed typhoid disease (TD) (blue) while others did not (NoTD) (red). TD48 and TD96 denote PBMC collected 48 or 96 hours after TD diagnosis. On day 14, all participants (TD and NoTD) received antibiotics (Abx). PBMC were collected from multiple time points (shown in red) from baseline (D-28) up to 28 days after challenge. The number of participants studied for each time point in the TD and NoTD groups are shown (n=x).
Figure 2
Figure 2
Gating strategy and frequencies of circulating T follicular helper cells (cTFH) and their subsets following Ty21a immunization and wt S. Typhi challenge. (A) Gating strategy showing cTFH (CD4+CD45RA-CXCR5+) in PBMC CD4+ T cells in a representative participant based on expression of CXCR5 and lack of expression of CD45RA. cTFH subsets (cTFH1, cTFH2, cTFH17 and cTFHDP) were characterized based on the expression of CXCR3 and/or CCR6 molecules. The frequencies of (B) total cTFH, (C) cTFH1, (D) cTFH2 and (E) cTFH17 subsets were measured in the immunization and challenge phases and compared between TD (blue lines) and NoTD (red lines) participants. *Represents significant (p<0.05) differences in frequencies between TD and NoTD at the indicated time points.
Figure 3
Figure 3
Homing and activation of cTFH subsets following Ty21a immunization and wt S. Typhi challenge. Ex-vivo expression of homing markers (A) integrin α4β7 and (B) CCR7 were measured and compared between cTFH subsets (cTFH1, cTFH2 and cTFH17) in TD (Blue lines) and NoTD (red lines) participants following immunization and wt S. Typhi challenge. Similarly, the ex-vivo expression of activation markers, (C) CD69, (D) CD154 (CD40L), (E) ICOS and (F) PD1 were assessed and compared between cTFH subsets in TD and NoTD participants following immunization and wt S. Typhi challenge. Significant differences between TD and NoTD participants for each subset are represented by *p<0.05. symbols indicate trends (p ≤ 0.1) to show differential responses between TD and NoTD groups for each cTFH subsets.
Figure 4
Figure 4
cTFH subsets have increased expression of gut homing and activation markers in TD participants. The areas under the curve (AUC) for each participant was calculated for the immunization phase (D-28 to D0) and for the challenge phase (D0 to D28) for integrin α4β7 expression for (A) cTFH1, (B) cTFH2 and (C) cTFH17. Similarly, AUC were calculated for the ICOS expression for the immunization phase (D-28 to D0) and for the challenge phase (D0 to D28) in (D) cTFH1, (E) cTFH2 and (F) cTFH17. Significant differences between TD and NoTD are represented by *p<0.05 and **p<0.005 respectively. Trends to show significant differences (p ≤ 0.1) between TD and NoTD groups.
Figure 5
Figure 5
S. Typhi-specific responses induced in cTFH subsets following Ty21a immunization and wt S. Typhi challenge. S. Typhi responses were determined by stimulation of cTFH with (i) S. Typhi-infected (ST) or (ii) non-infected (NI) autologous EBV-B. Net S. Typhi responses were calculated as the difference of ST minus NI in the immunization and challenge phases in participants in the TD and NoTD groups. (A) Net IL-21 and IFNγ S. Typhi responses were measured in cTFH1. (B) Net IL-21 and IL-2 S. Typhi responses were measured in cTFH2. (C) net IL-21 and IL-17A S. Typhi responses were measured in cTFH17. Significant differences between TD and NoTD groups are represented by *p<0.05. Trends to show significant differences (p ≤ 0.1) between TD and NoTD groups.
Figure 6
Figure 6
Effect of wt S. Typhi challenge on net S. Typhi-specific responses elicited by cTFH subsets. Net S. Typhi responses of cTFH subsets following wt S. Typhi challenge was determined by stimulation of cTFH with (i) S. Typhi-infected (ST) or (ii) non-infected (NI) autologous EBV-B. Net S. Typhi responses were calculated by the difference of ST minus NI in PBMC samples from participants in both TD and NoTD groups at D0 (before challenge) and D7 (7 days following challenge). Symbols are individual participants. Net S. Typhi responses in (A) IL-21, (B) IFNγ, (C) IL-2, (D) IL-17A, (E) TNFα and (F) granzyme B were determined and compared between days 0 and 7 after challenge and between TD and NoTD groups as indicated by the horizontal bars. Significant differences between days 0 and 7 or between TD and NoTD participants for each subset are represented by *p<0.05. Trends to show significant differences (p ≤ 0.1) between days 0 and 7 and between TD and NoTD groups for each cTFH subset.
Figure 7
Figure 7
TD and NoTD cTFH grouped into 11 clusters following unsupervised analysis. Concatenated TD and NoTD cTFH at the various time points (D-28 to D28) (same number of events per participant per time point) were found to segregate into 11 clusters with varying levels of activation, homing and cytokines markers. (A) Uniform Manifold Approximation and Projection (UMAP) was used to perform dimensionality reductions and plots were generated as described previously (44). Unsupervised clustering was performed using PhenoGraph (57). PhenoGraph clusters were then visualized on UMAP to create a reference map of all automatically detected cTFH subsets. The analyses showed 11 cTFH clusters with different number of events as shown in the table. Based on the UMAP plots, the expression of (B) Activation markers (CD69, CD27, PD-1, ICOS, CD154), (C) Cytokines and Chemokines (IL-17A, CD107a, IL-2, GranzymeB (GrzB), IFNγ, TNFα, IL-21 and MIP1β), and (D) homing markers (CCR4, CXCR3, CD62L, integrin α4β7, CCR7 and CCR6) were evaluated in the 11 cTFH clusters.
Figure 8
Figure 8
cTFH clusters are present differentially in TD and NoTD participants and during Ty21a vaccination and S. Typhi challenge. (A) Clusters expressing various markers at different levels of expression as shown by a Red-Blue color scheme (red-maximum expression; blue-low/no expression). The frequencies of the clusters are compared between TD and NoTD participants and their intensity shown by a Yellow-Black/Dark Blue color scheme (Yellow-maximum frequency; black/dark blue-low/no frequency). (B) Comparison of the frequencies of the 11 clusters of cTFH across the various time points (D-28, D-14, D0, D7, TD48, TD96, D14 and D28) as shown by a Yellow-Black/Dark Blue color scheme (Yellow-maximum frequency; black/dark blue-low/no frequency).
Figure 9
Figure 9
Distinct clusters are associated with the development of typhoid disease (TD) or lack of development of typhoid disease (NoTD). Each single cluster (A-K) (1–11) frequencies (% events) were evaluated and compared between TD (Blue line) and NoTD (red line) at all time points (D-28, D-14, D0, D7, TD48, TD96, D14 and D28). Arrows indicate the kinetics of which of the 11 cTFH clusters is being evaluated in each panel.
Figure 10
Figure 10
Clusters are differentially expressed, and in some cases present exclusively in individual cTFH subsets. (A) Phenotypic markers in the individual clusters showed varying levels of expression of the various homing and activation markers as shown by a Red-Blue color scheme (red-maximum expression; blue-low/no expression). The frequencies of the clusters are compared between cTFH1, cTFH2, cTFH17 and cTFH-double positive (DP) subsets as shown by a Yellow-Black/Dark Blue color scheme (Yellow-maximum frequency; black/dark blue-low/no frequency). (B) Frequencies of the 11 clusters of cTFH in each of the four cTFH subsets, as defined by CXCR3 vs CCR6, were compared on UMAP plots. (C) Frequencies (% events) of the 11 clusters for each cTFH subset are shown in a representative volunteer (Ox 2001).
Figure 11
Figure 11
Each cTFH subset has distinct clusters that are associated with TD or NoTD in the vaccination and/or challenge phases. For each cTFH subset, the kinetics of cluster (1-11) frequencies (% events) were evaluated and compared between TD (blue lines) and NoTD (red lines) at all time points (D-28, D-14, D0, D7, TD48, TD96, D14 and D28). For cTFH1 subsets, (A-D) clusters 3, 4, 7, 8; TFH2 (E-G) clusters 4, 7, 11; TFH17 (H-K) clusters 5, 7, 8, 9; and TFH-DP (L-O) clusters 4, 5, 7 and 10 were evaluated for their frequencies in TD (blue) and NoTD (red) at all time points. Significant differences between TD and NoTD are indicated by *p<0.05. Trends to show significant differences (p ≤ 0.1) between TD and NoTD groups.
Figure 12
Figure 12
Clusters 4 and 7 are associated with the development or protection from typhoid disease and are present in all subsets. (A) Cluster 4 is present in TFH1, TFH2, TFH17 and TFH-DP and their phenotypic, homing, and functional markers are expressed in each cTFH subsets at different levels as shown by the heatmap Red-Blue color scheme (Maximum level-red and minimum level-blue) in TD and NoTD. (B) Cluster 7 is present in TFH1, TFH2, TFH17 and TFH-DP and their phenotypic, homing, and functional markers are expressed in each cTFH subset at various levels as shown by the Red-Blue color scheme (Maximum level-red and minimum level-blue) in TD and NoTD.
Figure 13
Figure 13
Unique clusters defined functional cTFH subsets that are associated with the development of typhoid disease. (A) cluster 5 is mostly present in TFH-DP but with marked differences in the expression of phenotypic, homing, and functional markers. (B) Cluster 10 is unique to cTFH-DP subsets while cluster 11 (C) is observed in TFH2 and TFH17 subsets. The phenotypic, homing, and functional markers are expressed in each cTFH subset at different levels as shown by the Red-Blue color scheme (Maximum level-red and minimum level-blue) in TD and NoTD.
Figure 14
Figure 14
Defined clusters of each cTFH subsets are associated with the development of typhoid disease. Clusters showing significant differences between the participants who developed, or not, typhoid disease were evaluated at each phase of the study, baseline (D-28), immunization (Immun) phase (D-14) and challenge phase (D7) for each cTFH subset: (A) cTFH1, (B) cTFH2, (C) cTFH17 and (D) CTFH-DP. Differences are shown between the TD and NoTD groups. Symbols are individual participants. *Significant differences (p<0.05). Trends to show significant differences (p ≤ 0.1) between TD and NoTD groups.
Figure 15
Figure 15
Association between the frequencies of cTFH subsets and S. Typhi-specific anti-LPS antibodies production and functional serum bactericidal antibodies (SBA) in TD and NoTD. ELISAs and SBA assays were performed in a set of serum samples obtained at multiple time points (pre-vaccination -D-28-, pre-challenge -D0-, and post-challenge day 28 -D28-) corresponding to the participants (TD n = 8, NoTD n = 8) in whom the cTFH subsets frequencies and responses were evaluated. Correlation between the frequencies of cTFH subsets (cTFH1, cTFH2, cTFH17, cTFHDP) and S. Typhi-specific anti-LPS (A) IgG, (B) IgM and (C) IgA were determined using Spearman’s correlation analysis. (D) Correlation between bactericidal SBA titers and the frequencies of cTFH subsets (cTFH1, cTFH2, cTFH17, cTFHDP) were evaluated. * Strong significant correlation (r>0.7) (p<0.05) (Red).
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