Discovery of invariant T cells by next-generation sequencing of the human TCR α-chain repertoire

Abstract

During infection and autoimmune disease, activation and expansion of T cells take place. Consequently, the TCR repertoire contains information about ongoing and past diseases. Analysis and interpretation of the human TCR repertoire are hampered by its size and stochastic variation and by the diversity of Ags and Ag-presenting molecules encoded by the MHC, but are highly desirable and would greatly impact fundamental and clinical immunology. A subset of the TCR repertoire is formed by invariant T cells. Invariant T cells express interdonor-conserved TCRs and recognize a limited set of Ags, presented by nonpolymorphic Ag-presenting molecules. Discovery of the three known invariant T cell populations has been a tedious and slow process, identifying them one by one. Because conservation of the TCR α-chain of invariant T cells is much higher than the β-chain, and because the TCR α-chain V gene segment TRAV1-2 is used by two of the three known invariant TCRs, we employed next-generation sequencing of TCR α-chains that contain the TRAV1-2 gene segment to identify 16 invariant TCRs shared among many blood donors. Frequency analysis of individual clones indicates these T cells are expanded in many donors, implying an important role in human immunity. This approach extends the number of known interdonor-conserved TCRs and suggests that many more exist and that these TCR patterns can be used to systematically evaluate human Ag exposure.

Figure 1. Identification of invariant TRAV1-2⁺α chains

A TRAV1-2⁺CD4⁺ and TRAV1-2⁺CD4⁻ cells were sorted from asymptomatic tuberculin positive patients (designated with C), or random blood bank donors (designated with B). B TCR α chain sequences were derived from the sorted populations, as well as from external sources of non-sorted T cells. C A method of filtering for characteristics that define invariant TCRα chains was applied. Confirmation of the obtained candidates was sought in external datasets. D For each of the resulting 16 candidates, motifs were determined. ^aClones are defined as a group of nucleotide sequences using the same V and J segment and the same CDR3 sequence. To obtain the number of clones in a dataset, duplicates of the same rearrangement are removed, and the result is a number of unique rearrangements. ^bThe publicly available dataset described by Wang (3) was analyzed using our pipeline. ^cThe most abundant variant that passed the filters was selected as the consensus invariant α chain, and the less abundant variant was considered to be a variant covered by the motif. ^dX denotes the location of a variable amino acid position in a motif.

Figure 2. Convergent recombination in invariant α chains

A Proposed mechanisms of convergent recombination for two representative new invariant α chains from Figure 1d. All nucleotide sequences that are shown are present in the TRAV1-2 dataset. Nucleotides that can be attributed to the germline TRAV1-2 segment are shown in cyan, to TRAJ30 in pink, and to TRAJ4 in yellow. Amino acids that occupy the variable position in the motif are shown in red. B The number of unique clones that encode each peptide in the TRAV1-2 dataset was determined. The black line represents the mean of the number of clones per α chain for GEM, MAIT, new invariant, and all other α chains. The box plot shows the upper and lower quartiles, the whiskers indicate the 2^nd and the 98^th percentile, and the blue dots represent the outliers.

Figure 3. Clonal size of invariant α chains as a measure of T cell expansion

A-B The clonal size of each clone that is present in each sample of the TRAV1-2 dataset is expressed as the percentage of the total number of reads. A MAIT clones are shown in green, the two types of GEM clones in red and blue, and other clones in light grey. B Only the clones that represent a new invariant α chain are shown. Each color represents one of the 16 new invariant α chains. All dots with the same color represent nucleotide sequences that encode the same amino acid sequence. C The quantitative contribution by the 16 newly identified invariant α chains to the total TRAV1-2⁺ population is shown, as well as the contribution by MAIT cells, GEM cells, and other TCR α chains.