Fig 2: ICOS and CD38 mark cTfh cells following seasonal influenza vaccination. (A and B) Flow cytometric contour plots (A) and quantification (B) of the frequency of CD38+ICOS+CXCR5+PD-1+ cells among CD45RA-CD4+CD3+ cells in the peripheral blood of healthy UK donors at days 0 and 7 relative to seasonal influenza vaccination; n = 41. (B) Each symbol represents a volunteer; an individual donor is connected by a line at the two time points; n = 41. P < 0.0001; the P value was generated with a Wilcoxon signed-rank test. (C and D) Correlation of the frequency of CD38+ICOS+CXCR5+PD-1+ cTfh cells 7 d after vaccination with the change in antibody titer of anti-Cal09 IgG (C, an influenza A HA, P = 5.3 × 10-7, Rho = 0.75) and anti-Bris08 IgG (D, an influenza B HA, P = 5.5 × 10-7, Rho = 0.79) 7 d after vaccination. Statistical analysis by Spearman’s correlation (Rho = coefficient); n = 41. (E) Scatterplot of whole transcriptome RNA-sequencing data comparing the expression of all genes expressed in CD38+ICOS+CXCR5+PD-1+ cells before and 7 d following seasonal influenza vaccination; n = 4. Differentially expressed genes (DESeq2) are indicated in blue. (F) Flow cytometric contour plots of CXCR3 and CCR6 expression on ICOS+CXCR5+PD-1+ Tfh cells at the indicated time points relative to vaccination, a representative example of 36 individuals. (G) The number of unique TCRß CDR3 amino acid sequences identified in RNA-sequencing libraries from CD38+ICOS+CXCR5+PD-1+ cTfh cells from four volunteers were analyzed at days 0 and 7 relative to vaccination; n = 4. P = 0.0017; the P value was generated with a paired Student’s t test. (H) Quantification of the percentage of total TCRß CDR3 reads per unique clone: each symbol represents a unique CDR3 amino acid sequence. The dotted line indicates a frequency of 2%. In H, each letter on the x axis corresponds to a different volunteer. Data are from one seasonal influenza vaccination cohort.

Fig 3: Transcriptional analysis identifies circulating counterparts of GC-Tfh cells. (A and B) Flow cytometric plots showing the gating strategy of the four populations of CD4+CD3+CD45RA- cells sorted from iliac LNs (left) and peripheral blood (right) for RNA-sequencing and the expression of BCL6 protein in these populations, with representative plots shown; n = 6 individuals. (C and D) Hierarchical clustering (C) and PC analysis (D) of the most highly variable transcripts (standard deviation, >0.7) from RNA-sequencing data from four populations sorted in A from blood and LN samples. Three peripheral blood samples from the blood population no. 4 were excluded from the analysis as they did not pass quality control. In D, each symbol represents one individual. (E) Supervised analysis of the RNA-sequencing data from the four populations isolated from the blood using a gene signature for the LN GC-Tfh cells (LN 4); n = 254 genes. (F) Expression of IL7R, ASCL2, IL21, ICOS, and S1PR2 mRNA from RNA-sequencing data of the populations sorted in A; cell types are indicated by the colors shown in A. RNA-sequencing data were standardized relative to a library size of 1 million reads, and then read counts were log2 transformed; a value of -5 represents the limit of detection. P values were determined using ANOVA and the Tukey multiple-comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (G) Quantification of the number of TCRß CDR3 amino acid sequence clonotypes found in each blood cell population that are shared with the different LN populations (for each cell type n = 6 donors, except blood population no. 4 where n = 3; shared numbers of clonotypes were converted to normalized pseudocounts as described in Statistical analyses and Materials and methods). PBMC1, P = 0.055; PBMC2, P = 0.59; PBMC3, P = 0.0062; PBMC4, P = 0.029. The P values were determined using Kruskal-Willis tests. Data are from six individuals in one experiment.

Fig 4: A GLA-SE adjuvanted vaccine promotes cTfh expansion. (A and B) Flow cytometric contour plots of (A) PD-1 and CXCR5 on total CD45RA-CD4+CD3+ cells and (B) ICOS and CD38 on the population gated in A on peripheral blood cells from individuals 7 d after the third P27A vaccination. Alum group, n = 7; GLA-SE group, n = 8. (C) Fold change of CD38+ICOS+CXCR5+PD-1+ cTfh cells 7 d after the third P27A vaccination (frequency of cTfh cells at day 63/baseline). Alum group, n = 7; GLA-SE, group n = 8. P = 0.027. The P value is from an unpaired Student’s t test. (D) Correlation of anti-P27A IgG antibody titer 28 d after the third vaccination against fold change in CD38+ICOS+CXCR5+PD-1+ cTfh cells 7 d after the third vaccination. Rho = 0.73; P = 0.003 by Spearman’s correlation (Rho = coefficient); n = 15. (E) Scatterplot of RNA-sequencing data comparing all genes expressed in CD38+ICOS+CXCR5+PD-1+ cTfh cells 7 d after the third P27A vaccination in either Alum- (x axis) or GLA-SE– (y axis) vaccinated groups. Alum group, n = 7; GLA-SE group, n = 8. (F–I) Bar plots showing log-normalized reads from RNA-sequencing data of CD40LG. P = 0.593 (F), IL21 P = 0.846 (G), CXCR3 P = 0.606 (H), and CCR6 P = 0.977 (I) for transcripts in ICOS in CD38+ICOS+CXCR5+PD-1+ cTfh cells 7 d after the third P27A vaccination; Alum group, n = 7; GLA-SE group, n = 8. P values were calculated using DESeq2 with a false discovery rate correction. Each symbol represents one individual; those who received Alum are shown in black, and those who received GLA-SE are in white. Data are from one clinical trial.

Fig 5: Expression of surface markers of pTFH and pTFR cells. (A–D) Quantification of the percentage of cells expressing CCR5, TIGIT, PD1, or ICOS on pTFR and pTFH cells, respectively. HIV- (n = 8, mean ± SEM). HIV+ (in red, n = 6, mean ± SEM) (Mann–Whitney U test). (A,B) In both pTFH and pTFR cells, the percentage of TIGIT-expressing cells was similar in HIV-infected and non-infected individuals. In HIV-infected and non-infected individuals, the expression of TIGIT was higher in pTFR than in pTFH cells. (C,D). In both pTFH and pTFR cells, the percentage of cells expressing PD1/ICOS was similar in HIV-infected and non-infected participants of the MACS. (ns: p > 0.05, *: p = 0.05, **: p = 0.01).