Diversity and clonal selection in the human T-cell repertoire

Abstract

T-cell receptor (TCR) diversity, a prerequisite for immune system recognition of the universe of foreign antigens, is generated in the first two decades of life in the thymus and then persists to an unknown extent through life via homeostatic proliferation of naïve T cells. We have used next-generation sequencing and nonparametric statistical analysis to estimate a lower bound for the total number of different TCR beta (TCRB) sequences in human repertoires. We arrived at surprisingly high minimal estimates of 100 million unique TCRB sequences in naïve CD4 and CD8 T-cell repertoires of young adults. Naïve repertoire richness modestly declined two- to fivefold in healthy elderly. Repertoire richness contraction with age was even less pronounced for memory CD4 and CD8 T cells. In contrast, age had a major impact on the inequality of clonal sizes, as estimated by a modified Gini-Simpson index clonality score. In particular, large naïve T-cell clones that were distinct from memory clones were found in the repertoires of elderly individuals, indicating uneven homeostatic proliferation without development of a memory cell phenotype. Our results suggest that a highly diverse repertoire is maintained despite thymic involution; however, peripheral fitness selection of T cells leads to repertoire perturbations that can influence the immune response in the elderly.

Keywords: T-cell homeostasis; adaptive immune responses; aging; immunosenescence.

Fig. 1.

Age is associated with a modest decrease in diversity of the TCRB repertoire. TCRB sequences were obtained from replicate samples of naïve (A and B) and memory (C and D) CD4 and CD8 T cells. A lower bound of TCRB richness was estimated by applying nonparametric statistics using the Chao2 estimator. Results are shown for nucleotide (A and C) and derived amino acid sequences (B and D). Estimates were compared by Wilcoxon–Mann–Whitney test. Increase in age is associated with a decline in richness of naïve CD4 and CD8 T cells; however, the repertoire in the elderly remains highly diverse. Richness in CD4 and CD8 memory T cells markedly differed, whereas the impact of age was negligibly small.

Fig. 2.

Increase in clonal expansions within naïve CD4 and CD8 T-cell compartments with age. (A) The mean number of replicate TCRB sequences was used as an estimate of approximate clonal sizes. The frequency distribution of clonal size bins is shown as (log mean) ± SD of the four young and the five elderly adults. Nondetectable clonal sizes were set at a frequency of 1 in 10⁷. (B) Replicate samples of naïve CD4 or naïve CD8 T cells were analyzed for the shared occurrence of TCRB sequences to estimate a clonality score, defined as the probability of two independently identified sequences originating from the same clone. Clonality scores were compared by Wilcoxon–Mann–Whitney test. The observed increase indicate inequality in clonal sizes in the elderly naïve T-cell repertoire with age with increasing number of large clones.

Fig. 3.

Greater clonality in the repertoire of CD8 than CD4 memory T cells. Frequency distributions of categorized clonal size bins (A) and clonality scores (B) for memory CD4 and CD8 T cells were determined as described in Fig. 2 for naïve T cells. Nondetectable clonal sizes were set at the minimal frequency of 1 in 10⁶ (A). Clonality scores were compared by Wilcoxon–Mann–Whitney test. Large clonal expansions were more frequent in CD8 than in CD4 T cells with only minor influence of age on clonality in both compartments.

Fig. 4.

Characterization of clonally expanded naive T cells. TCRB sequences that had frequencies of ≥0.01% and were found in the naïve as well as the memory compartment were analyzed for their frequencies in five independent naïve and memory replicate samples (samples A–E). Representative results of two elderly individuals (one in A and the other in B) are shown as heat maps using the color scheme from infrequent (blue) to frequent (red). The data indicate that T cells expanded in the naïve and memory compartments are different.

Fig. 5.

Increased responsiveness of in vivo expanded naïve CD8 T cells to cytokine-induced proliferation. Naïve CD8 T cells were cultured with IL-7 and IL-15. TCRB sequences from naïve CD8 T cells that had divided equal to or more than once or twice were compared with sequences present in the peripheral blood repertoire of the individual. Results from two individuals are shown. The proportions of sequences from the cultured cells that were members of clones detected in four (purple), three (blue), two (green), or one (red) replicates of the original noncultured T-cell libraries from the peripheral blood are represented as cumulative bar graphs. The fastest-proliferating cultured T cells (right column) show enrichment of large clones found in four replicate libraries from the blood (P < 0.001).