Next-Generation Sequencing Analysis of the Human TCRγδ+ T-Cell Repertoire Reveals Shifts in Vγ- and Vδ-Usage in Memory Populations upon Aging

Abstract

Immunological aging remodels the immune system at several levels. This has been documented in particular for the T-cell receptor (TCR)αβ+ T-cell compartment, showing reduced naive T-cell outputs and an accumulation of terminally differentiated clonally expanding effector T-cells, leading to increased proneness to autoimmunity and cancer development at older age. Even though TCRαβ+ and TCRγδ+ T-cells follow similar paths of development involving V(D)J-recombination of TCR genes in the thymus, TCRγδ+ T-cells tend to be more subjected to peripheral rather than central selection. However, the impact of aging in shaping of the peripheral TRG/TRD repertoire remains largely elusive. Next-generation sequencing analysis methods were optimized based on a spike-in method using plasmid vector DNA-samples for accurate TRG/TRD receptor diversity quantification, resulting in optimally defined primer concentrations, annealing temperatures and cycle numbers. Next, TRG/TRD repertoire diversity was evaluated during TCRγδ+ T-cell ontogeny, showing a broad, diverse repertoire in thymic and cord blood samples with Gaussian CDR3-length distributions, in contrast to the more skewed repertoire in mature circulating TCRγδ+ T-cells in adult peripheral blood. During aging the naive repertoire maintained its diversity with Gaussian CDR3-length distributions, while in the central and effector memory populations a clear shift from young (Vγ9/Vδ2 dominance) to elderly (Vγ2/Vδ1 dominance) was observed. Together with less clear Gaussian CDR3-length distributions, this would be highly suggestive of differentially heavily selected repertoires. Despite the apparent age-related shift from Vγ9/Vδ2 to Vγ2/Vδ1, no clear aging effect was observed on the Vδ2 invariant T nucleotide and canonical Vγ9-Jγ1.2 selection determinants. A more detailed look into the healthy TRG/TRD repertoire revealed known cytomegalovirus-specific TRG/TRD clonotypes in a few donors, albeit without a significant aging-effect, while Mycobacterium tuberculosis-specific clonotypes were absent. Notably, in effector subsets of elderly individuals, we could identify reported TRG and TRD receptor chains from TCRγδ+ T-cell large granular lymphocyte leukemia proliferations, which typically present in the elderly population. Collectively, our results point to relatively subtle age-related changes in the human TRG/TRD repertoire, with a clear shift in Vγ/Vδ usage in memory cells upon aging.

Keywords: TCRγδ+; aging; development; next-generation sequencing; repertoire.

Figure 1

Technical optimization of next-generation sequencing assays for TRG/TRD loci. Multiplex PCR assays were optimized with balanced primer concentrations, annealing temperatures, and cycle numbers as summarized in Table S3 in Supplementary Material. Plasmid pools were used as spike-in samples to determine the percentage expected sequences per V and J gene vs. the observed percentage after sequencing (Table S2 in Supplementary Material). Expected percentages are indicated in black bars, observed percentages are indicated in colored bars. TRG assays showed high overlap between frequencies of expected and observed sequences for Vγ and Jγ (A) genes, with some variation due to single primers covering multiple genes (Vγ1F covering Vγ2-8, Jγ1.1/2.1 covering Jγ1.1 and Jγ2.1, and Jγ1.3/2.3 covering Jγ1.3 and Jγ2.3). TRD assays showed nearly similar percentages of expected and observed sequences for Vδ and Jδ (B) genes. Error bars represent SD of PCR replicates (N = 4).

Figure 2

Circoletto visualization of the TRG/TRD repertoire during ontogeny. Optimized multiplex PCR next-generation sequencing assays were applied on total TCRγδ+ T-cells sorted from thymus (Thy), neonatal cord blood (CB), and adult peripheral blood (PB). TRG assays showed high repertoire diversity in both Thy and CB samples, with low interindividual variation, while adult PB samples showed individual-specific repertoire patterns with less receptor diversity (A). TRD assays showed high dominance of Vδ1 (light blue bars), which was also observed in CB samples, both with low intersample variation. Adult PB samples showed donor-specific patterns with sometimes skewing toward Vδ2 and even Vδ3 (B). Three representative samples of each samples type are visualized: Thy04-10, Thy05-13, Thy10-03, CB2, CB3, CB4, PB30, PB31, and PB50. Plots were made using the Circoletto online software tool [http://www.circos.ca (35)]. Each band represents a V–J rearrangement, with colors based on V-gene usage.

Figure 3

V gene diversity in the combinatorial TRG/TRD repertoire of different TCRγδ+ T-cell subsets from young and elderly individuals. V-gene usage per subset visualized in bar graphs indicating a diverse repertoire in naive and effector subsets of both young and elderly, with skewing toward Vγ2 and Vγ9 in memory populations (A). Naive populations showed diversity in Vδ usage with Vδ1 dominance in young and elderly. Young individuals showed relatively higher Vδ2 usage in especially the memory and effector subsets, whereas elderly individuals showed clear Vδ1 dominance in all subsets, except for effector memory cells (B). Median V-gene frequencies of productive sequences with SD bars were indicated in bar graphs for young (N = 11) and elderly (N = 12) individuals. Color legends are indicated in the figure. Statistical significance was tested using the Mann–Whitney U-test. Level of significance is indicated in the plots: *p < 0.05; **p < 0.01; ***p < 0.0001.

Figure 4

CDR3-length distributions of TCRγδ+ T-cell subsets from young and elderly individuals. Frequencies of CDR3-lengths from different TCRγδ+ T-cell subsets are summarized per T-cell receptor chain and age group. Naive subsets (red lines) showed distributions resembling Gaussian profiles, while memory subsets showed dominant peaks suggestive of selection and receptor skewing (green and purple lines). TRG CDR3-lengths showed similar distributions between young and elderly (A), TRD CDR3-lengths in young individuals showed a Gaussian profile in the naive subset and more skewing in memory and effector subsets, whereas in elderly individuals all subsets showed dominant peaks (B). Mean frequencies per subset were indicated for young (N = 11) and elderly (N = 12) individuals. Data normality was tested using the diptest package in R.

Figure 5

Canonical Vγ9–Jγ1.2 sequences and Vδ2–Jδ1 invariant T selection determinant in young and elderly healthy individuals. GCA sequence in bold mediates preferential recombination of Vγ9 with Jγ1.2, resulting in specific CDR3 length and amino acid composition, adapted from Ref. (45) (A). Frequencies of all productive Vγ9–Jγ1.2 sequences containing the canonical sequence per age group and subset marked in dark gray. In light gray Vγ9–Jγ1.2 rearrangements with other, non-canonical sequences are indicated. Numbers of total Vγ9–Jγ1.2 sequences are indicated in center of plots (B). The invariant T selection determinant is located at the relative second position of the first codon in the junction of Vδ2–Jδ1 rearrangements, adapted from Ref. (13) (C). Frequencies of invariant T selection determinant in young and elderly subsets (dark gray part outer ring), leading to L, V, or I amino acid residues (inner blue pie chart, dark blue part). Absolute number of unique productive Vδ2–Jδ1 sequences indicated next to plots (D).