Diversification of the antigen-specific T cell receptor repertoire after varicella zoster vaccination

Abstract

Diversity and size of the antigen-specific T cell receptor (TCR) repertoire are two critical determinants for successful control of chronic infection. Varicella zoster virus (VZV) that establishes latency during childhood can escape control mechanisms, in particular with increasing age. We examined the TCR diversity of VZV-reactive CD4 T cells in individuals older than 50 years by studying three identical twin pairs and three unrelated individuals before and after vaccination with live attenuated VZV. Although all individuals had a small number of dominant T cell clones, the breadth of the VZV-specific repertoire differed markedly. A genetic influence was seen for the sharing of individual TCR sequences from antigen-reactive cells but not for repertoire richness or the selection of dominant clones. VZV vaccination favored the expansion of infrequent VZV antigen-reactive TCRs, including those from naïve T cells with lesser boosting of dominant T cell clones. Thus, vaccination does not reinforce the in vivo selection that occurred during chronic infection but leads to a diversification of the VZV-reactive T cell repertoire. However, a single-booster immunization seems insufficient to establish new clonal dominance. Our results suggest that repertoire analysis of antigen-specific TCRs can be an important readout to assess whether a vaccination was able to generate memory cells in clonal sizes that are necessary for immune protection.

Copyright © 2016, American Association for the Advancement of Science.

Figures

Fig. 1. The T cell receptor repertoire of VZV antigen-reactive CD4 T cells

(A) CFSE-labeled PBMCs were stimulated with VZV lysate or mock lysate for 8 days. Scatter plots are gated on CD4 T cells and are representative of four experiments. (B) 8-day VZV lysate activated PBMCs were either restimulated with PMA and ionomycin or VZV lysate together with fresh T cell-depleted CTV color-coded PBMCs. The frequencies of cytokine-producing cells stimulated by VZV lysate in CFSElow and CFSEhigh CTVnegativeCD4 T cells were normalized for the number of cytokine-producing cells after stimulation with PMA and ionomycin. (C) The number of distinct TCRβ chains in VZV antigen-reactive CD4 T cells from three twin pairs (A, B and C) and three unrelated individuals (D, E and F) was determined. Identical twins are indicated by the same color. (D) Frequencies of VZV antigen-reactive T cells were correlated with the richness of their TCRβ chain repertoires (r=−0.5, p=0.18). (E) Total CD4 T cells were analyzed for the presence and frequencies of VZV-reactive TCR sequences. Sequences that were too infrequent to be re-identified were set at a minimum frequency of 1 in 106. Clonal size distributions are shown by plotting normalized clonal size bins vs. the log number of TCR sequences found at that particular frequency. Identical twins are shown in the same color, but different symbols. (F) The stacked bar plot shows the proportion of the VZV antigen-reactive CD4 TCRβ repertoire that the most abundant clones occupy. (G) The number of distinct VZV-reactive CD4 TCRβ chains inversely correlated with the space taken up by the most abundant clones (r=−0.8, p=0.01). Results are from two-sided non-parametric Spearman correlation.

Fig. 2. Genetic impact on naïve, memory and VZV antigen-reactive CD4 TCRβ repertoires

(A) Heatmaps show hierarchical clustering of TRBV-TRBJ combination frequencies in naïve (left panel), memory (middle panel), and VZV antigen-reactive CD4 T cells (right panel) for three twin pairs (A-C) and three unrelated individuals (D-F). The color represents pairwise Pearson correlation coefficients ranging from 0.85 to 1 for naïve and memory cells and 0.2 to 1 for VZV-reactive T cells. (B) TRBV (left) and TRBJ (right) genes that were significantly more similar in twin pairs than unrelated individuals were identified using a nonparametric permutation test (p<0.05). Similarities are expressed as the ratio of differences between unrelated individuals and differences within twins. Naïve (orange) and memory cells (green) are compared. (C) The repertoires in twins and non-twins were analyzed for the presence of TCRs with identical amino acid sequences. Frequencies of shared TCRβ chain sequences were normalized by dividing the number of shared TCRβ chain sequences within a pair by the product of the total number of distinct TCRβ chain sequences from these two individuals. Similarities were compared using nonparametric permutation test.

Fig. 3. Influence of vaccination on the CD4 T cell receptor repertoire of VZV-reactive cells

VZV antigen-reactive TCRβ chain sequences were identified as described in Fig. 1 on days 0, 8, 14 and 28 and their individual frequencies in total CD4 T cells were determined. (A) Line graphs show the sum of all frequencies of detected TCRβ-chains in one individual. Unrelated individuals are indicated by different colors, twins by different symbols. (B) The percent of VZV-reactive TCRβ chains that had a significant change in clonal size between day 0 and day 8 after vaccination is shown. Changes between two time points from a non-vaccinated control individual are shown for comparison. (C) The number of unique VZV-specific TCRβ chains before and on days 8, 14, and 28 after vaccination is shown. Paired Wilcoxon-Mann-Whitney tests were performed for comparison in A and C, n=9.

Fig. 4. VZV vaccination recruits antigen-specific CD4 T cells from the naïve compartment in addition to expanding memory T cells

(A) Stacked bars show the number of distinct VZV-specific CD4 TCRβ chains that could be recovered only in the naïve or in the memory compartment and those present in both compartments before vaccination. (B) CFSE-labeled purified naïve T cells and CTV-labeled PBMCs were cultured together in the presence of VZV lysate for 8 days. The scatter plot of gated CD4 T cells identifies proliferating naïve CD4 T cells and total CD4 T cells. Data are representative of two experiments. (C) Clonal size distributions of VZV-specific TCRβ chains that were originally derived from either naïve or memory CD4 T cells are shown as box plots of median frequencies of the nine individuals. Compared to day 0, clonal sizes were significantly increased for both naïve and memory population at all time-points after vaccination (p<0.01, paired Wilcoxon-Mann-Whitney test, n=9).

Fig. 5. VZV vaccination diversifies the VZV-reactive TCR repertoire

(A) The numbers of distinct VZV antigen-reactive TCRβ chains ordered by decreasing clonal size are plotted versus the cumulative space they occupy. Line graphs with different colors illustrate the clonal size distributions for different time points after vaccination. Data from one individual is shown. (B) The number of the largest distinct T cell clones that take up 80% of VZV-reactive CD4 TCR repertoire was determined as illustrated by the intersection with the indicated line in Fig. 5A. Results for the nine individuals before and 28 days after vaccination are shown as box plots. (C) Results are expressed as the percentage of clones that take up 80% of the antigen-specific repertoire space by dividing the number of different clones shown in Fig. 5B by the total number of clones for each time point and individual. (D) Distribution of clonal frequencies in VZV-reactive CD4 TCRβ repertoire at day 0 pre-vaccination is plotted against those at day 28 after vaccination. Each smoothed line represents the fitting curve that summarizes the distribution of clonal sizes for one individual. For comparison, lack of changes in clonal sizes is included as a dotted line. Paired Wilcoxon-Mann-Whitney tests were performed for comparison in B and C, n=9.