Clonal composition and persistence of antigen-specific circulating T follicular helper cells

Abstract

T follicular helper (T_FH ) cells play an essential role in promoting B cell responses and antibody affinity maturation in germinal centers (GC). A subset of memory CD4⁺ T cells expressing the chemokine receptor CXCR5 has been described in human blood as phenotypically and clonally related to GC T_FH cells. However, the antigen specificity and relationship of these circulating T_FH (cT_FH ) cells with other memory CD4⁺ T cells remain poorly defined. Combining antigenic stimulation and T cell receptor (TCR) Vβ sequencing, we found T cells specific to tetanus toxoid (TT), influenza vaccine (Flu), or Candida albicans (C.alb) in both cT_FH and non-cT_FH subsets, although with different frequencies and effector functions. Interestingly, cT_FH and non-cT_FH cells specific for C.alb or TT had a largely overlapping TCR Vβ repertoire while the repertoire of Flu-specific cT_FH and non-cT_FH cells was distinct. Furthermore, Flu-specific but not C.alb-specific PD-1⁺ cT_FH cells had a "GC T_FH -like" phenotype, with overexpression of IL21, CXCL13, and BCL6. Longitudinal analysis of serial blood donations showed that Flu-specific cT_FH and non-cT_FH cells persisted as stable repertoires for years. Collectively, our study provides insights on the relationship of cT_FH with non-cT_FH cells and on the heterogeneity and persistence of antigen-specific human cT_FH cells.

Keywords: Candida albicans; T follicular helper cells; TCR Vβ sequencing; influenza vaccine; tetanus toxoid.

Figure 1

Variable TCR Vβ repertoire overlap between cT_FH and non‐cT_FH cells specific for different microbial antigens. (A) Flow cytometry analysis on a representative donor showing the expression of CXCR5 and CCR6 on CD4⁺ memory T cells. (B) Four memory T cell subsets were FACS‐sorted according to CXCR5 and CCR6 differential expression, labeled with CFSE, and stimulated with influenza vaccine (Flu), tetanus toxoid (TT) or C. albicans (C.alb) in the presence of autologous monocytes. Shown are the percentages of proliferating CFSE^low cells at 6–7 days post stimulation. In all conditions, 85%‐98% of CFSE^low cells expressed the activation markers CD25 and/or ICOS. Each symbol indicates a different healthy donor (n = 5 donors for Flu, n = 3 for TT, and n = 4 for C.alb). * p‐value < 0.05, ** p‐value < 0.01, *** p‐value < 0.001, **** p‐value < 0.0001, as determined by two‐tailed paired t test. (C) Flu‐, TT‐ and C.alb‐specific CFSE^low T cells from each subset were FACS‐sorted and their repertoire was analyzed by next generation TCR Vβ sequencing. Shown are the cytoscape clonal networks of TCR Vβ repertoire of antigen‐specific T cells isolated from CXCR5⁺CCR6⁺ (purple), CXCR5^–CCR6⁺ (blue), CXCR5⁺CCR6^– (pink), and CXCR5^–CCR6^– (green) T cell subsets. Numbers of total (colored) and shared (black) TCR Vβ clonotypes are indicated. Clonotypes shared among two or three subsets are shown as black dots and clonotypes shared among all four subsets are shown as red dots. Data are from three representative donors (one for each antigen condition, indicated in panel B with blue symbols) out of six donors analyzed. (D) Shown is the fraction of unique and shared TCR Vβ clonotypes in each sample reported in panel C. Only the major responder T cell subsets for each type of antigen are reported. (E) Dendrogram plots of repertoire similarity among the four subsets based on the Morisita‐Horn similarity index between pairs of TCR Vβ repertoires. (F) Pairwise comparisons of TCR Vβ clonotype frequency distribution in antigen‐specific CFSE^low T cell lines isolated from cT_FH and non‐cT_FH cells reported in panel C. Frequencies are reported as a percentage of productive templates. The total number of clonotypes is indicated in the x‐ and y‐axes. The number of clonotypes shared between two samples are reported in the upper right corner. (G) Shown is the cumulative frequency of unique and shared TCR Vβ clonotypes in each sample reported in panel C. Only the major responder T cell subsets for each type of antigen are reported.

Figure 2

Flu‐specific cells are enriched in PD‐1⁺ cT_FH and have a distinct repertoire. (A) Percentages of proliferating CFSE^low cells measured at day 6–7 post stimulation of T cell subsets with Flu or C. al C.alb) in the presence of autologous monocytes. Each symbol indicates a different healthy donor (n = 7 for Flu, and n = 7 for C.alb). * p‐value < 0.05, ** p‐value < 0.01, as determined by two‐tailed paired t test. (B) The TCR Vβ repertoire of Flu‐ and C.alb‐specific CFSE^low T cells from each subset was determined by next generation sequencing. The numbers of TCR Vβ clonotypes detected from each antigen‐specific PD‐1⁺ cT_FH, PD‐1^– cT_FH and CXCR5^– subsets and technical replicates are reported in the black cells of the table. The numbers of clonotypes shared between technical replicates are shown in the grey cells. The numbers of clonotypes shared between any pair of two different subsets are reported in the white cells. Data are from two representative donors (one for each antigen condition, indicated in panel A with blue symbols) out of three donors analyzed. (C) Percentages of unique and shared clonotypes between technical replicates and between different subsets, determined by the Jaccard index calculated on the samples reported in panel B. (D) Cumulative frequencies of unique and shared clonotypes between technical replicates and between different subsets, determined as the average of the cumulative frequencies of the shared clonotypes in each of the two subsets calculated on the samples reported in panel B. (E) Normalized TCR overlap score between pairs of TCR Vβ repertoires of antigen‐specific PD‐1⁺ cT_FH, PD‐1^– cT_FH and non‐cT_FH cells, calculated by Chao‐Jaccard similarity index normalized on technical replicates. Data are multiple pairwise comparisons of the samples reported in panel B. * p‐value < 0.05, ** p‐value < 0.01, *** p‐value < 0.001, **** p‐value < 0.0001, as determined by one‐way ANOVA with Tukey multiple comparisons test. (F) Heat map reporting the frequencies of the top 5% most expanded TCR Vβ clonotypes in the samples reported in panel B (433 Flu‐specific and 201 C.alb‐specific clonotypes, respectively) ranked by the sum frequency of each clone. Dendrograms are based on the Morisita‐Horn similarity index between pairs of TCR Vβ repertoires.

Figure 3

Flu‐specific PD‐1⁺ cT_FH cells phenotypically resemble GC T_FH cells. (A) Heat map of the mRNA expression level of selected genes from antigen‐specific PD‐1⁺ cT_FH, PD‐1^– cT_FH, and non‐cT_FH cells. T_FH‐related and Th1‐related genes are shown for Flu‐specific cells (upper panels). T_FH and Th17‐related genes are shown for C. albicans‐specific cells (lower panels). Gene expression is normalized based on the average mRNA counts of each gene from the three T cell subsets. Data from n = 4 healthy donors (HD1‐4) are shown. (B) Shortlist of GC T_FH‐related genes ranked based on the gene expression ratio of PD‐1⁺ cT_FH to PD‐1^– cT_FH specific for influenza (left panel) or C. albicans (right panel). Ratios falling into the range of 0.67‐1.5 were considered not significant. (C) Concentrations of CXCL13 in the day 6–7 culture supernatants of T cell subsets stimulated with Flu or C.alb, as measured by ELISA. For each antigen condition, shown are the experimental replicates (n = 4‐10) from one representative donor out of n = 3 donors analysed. Background cytokine production measured in T cells cultured with monocytes in the absence of the antigen is shown as dashed lines. * p‐value < 0.05, ** p‐value < 0.01, **** p‐value < 0.0001, as determined by two‐tailed unpaired t test. (D) Concentrations of IL‐21 in the day 6–7 culture supernatants of T cell subsets stimulated with Flu or C.alb, as measured by ELISA. For each antigen condition, shown are the experimental replicates (n = 6‐20) from one representative donor out of n = 3 donors analyzed. Background cytokine production measured in T cells cultured with monocytes in the absence of the antigen is shown as dashed lines. ** p‐value < 0.01, as determined by two‐tailed unpaired t test.

Figure 4

Flu‐specific cT_FH and T_EM cells induced by infection or vaccination are persistent and phenotypically stable. (A) Serial blood donations from a Flu vaccinated individual were collected and next generation TCR Vβ sequencing was performed on CFSE^low proliferating T cells isolated from Flu‐stimulated cT_FH, T_CM, and T_EM subsets (initial input 5×10⁵ cells per subset) at each time‐point. Shown is the pairwise comparison of TCR Vβ clonotype frequency distribution in Flu‐specific cT_FH, T_CM, and T_EM subsets at the initial time‐point of analysis (t1: May 2014). Frequencies are shown as percentage of productive templates. The total number of clonotypes is indicated in the x‐ and y‐axes. Values in the upper right corner represent the number of clonotypes shared between two samples. The Venn diagrams show the number of unique and shared Flu‐specific TCR Vβ clonotypes between cT_FH, T_CM and T_EM subsets. (B) Sample overlap between pairs of Flu‐specific TCR Vβ repertoires was calculated using the Chao–Jaccard similarity index. The bar histograms show the Chao‐Jaccard index between pairs of Flu‐specific cT_FH, T_CM, and T_EM TCR Vβ repertoires obtained from serial blood donations of a healthy donor (t1: May 2014, t2: Feb 2015, t3: Sep 2015, t4: Jan 2016, t5: Nov 2017). *p < 0.05 as determined by two‐tailed paired t test. (C) Number of productive TCR Vβ clonotypes detected by next generation sequencing of Flu‐specific cT_FH (red circles), T_CM (green squares), and T_EM (blue triangles) cells at each time‐point of the longitudinal analysis. (D and E) The Flu‐specific TCR Vβ clonotypes found at the initial time‐point (t1) in CFSE^low cultures from cT_FH (3529 clonotypes), T_CM (2900 clonotypes), and T_EM (3090 clonotypes) were followed in a longitudinal analysis of serial blood donations. The fraction of TCR Vβ clonotypes detected in CFSE^low cultures at each subsequent time‐point is reported in (D) as percentage of start for cT_FH (red circles), T_CM (green squares), and T_EM (blue triangles). Dates of seasonal Flu vaccination are reported as dashed lines. The stacked barplots in (E) show a longitudinal analysis of the phenotype distribution of Influenza‐specific clonotypes originally detected at the initial time‐point (t1) in CFSE^low cultures from T cell subsets (cT_FH plotted on the left, T_CM in the center, T_EM on the right). Each sector reports the percentage of TCR Vβ clonotypes that are found within the original subset (cT_FH, T_CM and T_EM for the plots on the left, center and right, respectively) or transitioned to other subsets at each time‐point. Data are expressed as percentage of start, and subsets are color‐coded (cT_FH in red, T_CM in green, and T_EM in blue). (F) Within the Flu‐specific TCR Vβ clonotypes found at the initial time‐point (t1) only in CFSE^low cultures from cT_FH (cT_FH‐only: 2541 clonotypes), T_CM (T_CM‐only: 1743 clonotypes), or T_EM (T_EM‐only: 1819 clonotypes), the top 5% clonotypes were shortlisted (top 5% cT_FH‐only: 127 clonotypes, top 5% T_CM‐only: 87 clonotypes, top 5% T_EM‐only: 91 clonotypes) and followed in CFSE^low cultures from subsequent time‐points. The stacked barplots report a longitudinal analysis of the phenotype distribution of the shortlisted TCR Vβ clonotypes detected at each subsequent time‐point (top 5% cT_FH‐only plotted on the left, top 5% T_CM‐only in the center, top 5% T_EM‐only on the right). Each sector reports the percentage of TCR Vβ clonotypes that are found within the original subset (cT_FH, T_CM and T_EM for the plots on the left, center and right, respectively) or transitioned to other subsets at each time‐point. Data are expressed as percentage of start, and subsets are color‐coded (cT_FH in red, T_CM in green, and T_EM in blue). (G and H). The Flu‐specific TCR Vβ clonotypes found at the initial time‐point (t1) shared among CFSE^low cultures from cT_FH, T_CM and T_EM were shortlisted (318 clonotypes) and followed in subsequent time‐points. The fraction of TCR Vβ clonotypes detected at each subsequent time‐point is reported in (G) as percentage of start. Dates of seasonal Flu vaccination are reported as dashed lines. Panel (H) shows the frequency distribution of TCR Vβ clonotypes from total cT_FH, T_CM, and T_EM subsets sequenced directly after ex vivo isolation from the same individual at t1 (May 2014, plots on the left) and t5 (Nov 2017, plots on the right). Frequencies are shown as percentage of productive templates. Colored circles mark the TCR Vβ clonotypes belonging to the shortlist of 318 shared clonotypes. The total number of TCR Vβ clonotypes and the number of retrieved clonotypes from the group of 318 shared clonotypes are reported on top of each graph in black and orange, respectively. Dotted lines in the graphs indicate the frequency threshold of the top 5% expanded clonotypes.