Discovery of invariant T cells by next generation sequencing of the human T cell receptor α chain repertoire

Patients

For the TRAV1-2 dataset, blood was obtained from asymptomatic tuberculin-positive donors clinically assessed to have latent tuberculosis but with no clinical or radiographic evidence of active tuberculosis (samples starting with “C”), and from blood-bank donors (samples starting with “B”), after informed consent was obtained, as approved by the institutional review boards of the Lemuel Shattuck Hospital and Partners Healthcare, Boston, USA. For the AMC dataset, blood was obtained from five different random blood bank donors at Sanquin, Amsterdam, Netherlands. High resolution HLA genotyping was performed by next generation sequencing using the 454 Life Sciences GS FLX system and Titanium chemistry as previously described (4).

Cell sorting and sequencing

Monoclonal antibodies against TRAV1-2 and CD4 (3C10 from Biolegend and RPA-T4 APC from BD Bioscience) were incubated for 30 min on ice. Cells were were pre-gated for lymphocytes based on forward and side scatter and sorted on an 11-color FACSAria (Becton Dickinson). RNA was isolated with an RN easy kit (Qiagen), and first strand cDNA was synthesized with a Quantitect reverse-transcription kit (Qiagen), including a genomic DNA–removal step. The generation of amplicons for next generation sequencing was previously described (5, 6). The cDNA was amplified using a full repertoire approach, using multiple primers to cover all known V genes in a linear amplification protocol to prevent bias between primers. For this paper only the TRAV1-2 primer is relevant as either the cells used were FACS sorted using a TRAV1-2-specific antibody (TRAV1-2 dataset) or only the TRAV1-2 sequences were used (AMC dataset). The TRAV1-2-specific primer is 5′-GGACAAARCMTTGASCAGCC-3′ (7). The Vα primers are tailed with the primer B sequence of the LibA system (Titanium protocol) (5′-CTATGCGCCTTGCCAGCCCGCTCAG-3′) (Roche/454). In the first step of the linear amplification the cDNA samples are amplified in a volume of 20μl in the presence of 1x Buffer B (Solis BioDyne), 0.1 mM dNTPs (each), 1 mM MgCl₂, 0.25 U/μl HotFire polymerase (Solis BioDyne), 0.2 pmol/μl of each Vα primer. The amplification was run on a T1 thermocycler (Biometra) using the cycling conditions: 1×96°C for 15′, 40× (96°C for 30″, 60°C for 60″, 72°C for 30″), 1×72°C for 10′. After amplification, the amplicons were purified using AMPURE SPRI beads (Agencourt) according to the manufacturers instructions using equal amounts of amplicon volume and bead volume. In the second step, a PCR is performed using primer B sequence of the Roche 454 LibA system as forward primer and a Cα specific primer 5′-TCTCAGCTGGTACACGGCAG-3′ (7) tailed with both a genetic barcode and the primer A sequence of the LibA system (Titanium protocol) (5′-CGTATCGCCTCCCTCGCGCCATCAG-3′) (Roche/454). Amplification conditions are equal to the linear amplification step. The cycling conditions were: 1×96°C for 15′, 35× (96°C for 30″, 60°C for 60″, 72°C for 30”), 1×72°C for 10′. After amplification the amplicons purified again using AMPURE SPRI beads (Agencourt).

Sequencing was performed on the Roche/454 Genome sequencer using the Titanium platform. Preparations and quality controls were performed according to the manufacturers instructions.

Bioinformatics

TCR α chain sequences were analyzed using a pipeline described previously (5). Briefly, sequences were sorted per sample using genetic barcodes. Subsequently V and J segments were identified by comparison against the IMGT database (8) using the BLAST-like alignment tool (BLAT) (9) with default settings. V and J segments with the highest score and percent identity were assigned to the sequences. Sequences that were out of frame or too short to unequivocally assign V and J segments were discarded. The CDR3 was defined as the region from the Cysteine at amino acid position 104 to the Phenylalanine-Glycine motif at amino acid position 118–119) (10). N and P nucleotides were reported as the region between V and J alignments, and are together treated as N nucleotides in the filtering strategy. Sequences were denoted as duplicates if they had an identical Vα, Jα and CDR3α at nucleotide level. Data were analysed and filtered as described in the results section using in-house developed R scripts. The amount of expansion was calculated as the percentage of sequences per clone compared to the total amount of valid sequences per sample. Clones with a percentage above 1% are considered highly expanded, between 0.1 and 1% medium expanded, and below 0.1% low or not expanded.

TRAV1-2⁺ T cell receptor α chain datasets

To generate sequences of TCR α chains that utilize TRAV1-2, we sorted T cells from six human subjects based on binding to antibodies against TRAV1-2 and CD4 (Table I and Fig. 1A). We chose to split the TRAV1-2⁺ population in TRAV1-2⁺CD4⁺ and TRAV1-2⁺CD4⁻ and treat them as separate samples because MAIT and GEM cells both express TRAV1-2 but have been described to exist mostly as CD4⁻ and CD4⁺, respectively (11, 12). Unlike classical MHC-restricted T cells, the expression of CD4 or CD8 or the lack thereof has no known effects on the functionality of T cells that recognize non-polymorphic antigen presenting molecules (13, 14). We did not separate based on CD8 expression to avoid effects of cell activation as most anti-CD8 antibodies detect CD8αα, which is regulated according to the activation status of the cell. The separation based on CD4 appears more robust, since the presence or absence of CD4 is a stable feature of a T cell. Thus, the TRAV1-2⁺CD4⁻ population contains CD8αβ⁺ T cells and double negative T cells, regardless of their activation status, and the CD4⁺ population contains CD4⁺ CD8αβ⁻ cells, regardless of their activation status. Using 454 sequencing technology, approximately 10,000 TCR α chain sequences were obtained from each of the 12 samples (TRAV1-2⁺CD4⁺ and TRAV1-2⁺CD4⁻ populations from six subjects). Sequence read length was between 379–403 nucleotides and averaged 385 nucleotides. Non-productive α chains, which consisted of out of frame sequences and sequences with internal stop codons, were removed, as well as sequences that were too short to assign V and J segment usage (Fig. 1B). Subsequently, we removed all duplicates. Duplicates were defined as sequence reads with identical V and J segment usage and identical joints and thus identical CDR3s and are most likely derived from one T cell clone. The resulting 10823 sequences (called “clones”) are thought to represent different, unique T cell clones.

Identification of new invariant α chains

The known invariant α chains expressed by NKT cells, MAIT cells, and GEM T cells fulfill two criteria: they are present in most, if not all, individuals, and they contain very few N nucleotides (12, 15,16). These invariant α chains consist of one predominant consensus amino acid sequence, and occasionally diverges from the consensus sequence by up to two amino acids, while keeping chain length identical. Also, invariant consensus sequences can be encoded by slightly different nucleotide sequences. Therefore, to identify invariant α chain candidates, we only considered α chains with 0, 1, 2, or 3 N nucleotides (Fig. 1C) (15). We then translated these α chains into protein sequences and generated a list of the unique protein sequences. Of these protein sequences, we only considered sequences that were present in at least half of the samples, as one feature of invariant T-cells is their presence in multiple subjects. Among the 33 α chains that passed that filter (Supplementary Fig. 1A), 14 were removed because they were identified as a known type of invariant T cell, as they expressed MAIT α chains that utilize TRAJ33 to form a CDR3 sequence CAXXDSNYQLIWGAG, or TRAJ12 to form a CDR3 sequence CAVXDSSYKLIFG, where X can be any amino acid. The 19 consensus sequences that were left were grouped into 16 invariant α chain motifs with identical TRAJ segment and CDR3 length (Fig. 1D). Of the 16 invariant α chain motifs 1 is found in 4 out of 6 subjects, 7 are found in 5 out of 6 subjects and 8 are found in all 6 subjects. Subsequently, we indicated the presence or absence of the new invariant α chain consensus sequences in independent external samples that were not sorted for TRAV1-2 (3). Together, these external datasets contained approximately 1600 TRAV1-2 α chains. Finally, because variants of MAIT, NKT, and GEM T cells exist that differ at one or sometimes two positions of the amino acid consensus sequence, we looked for variants of our 21 new consensus CDR3α sequences that differ at one amino acid position from the consensus sequences and that were present in at least 3 out of 12 samples. The location of the variable amino acids is indicated with an X in the CDR3 sequence (Fig. 1D). Thus we were able to assign 16 different new invariant α chain motifs within the human TRAV1-2 repertoire.

Of note, T cells have been described that recognize MR1 and were classified as MAIT cells based on their specificity, but that do not express the typical MAIT TCR α chain sequence that uses TRAJ33 (11, 17–18). Instead these cells express a particular TRAV1-2 and TRAJ20-utilizing α chain with a CDR3 length of 13 amino acids, or a particular TRAV1-2 and TRAJ12-utilizing α chain with a CDR3 length of 13 amino acids. The latter type fits a motif that we identified independently here. Thus, using a sequence of heuristic filters, we re-identified canonical and atypical MAIT TCR α chains, and found 16 new invariant α chains.

Alternative explanations for interdonor-conserved sequences are cross-contamination of samples and public TCRs. The simple V-J joint of invariant TCR α chains is a feature that enables their interdonor-conserved nature (19). Sharing among donors is not predicted to occur among TCRs with complex joints that incorporate multiple N nucleotides. As a control for cross contamination of samples, we generated a list of interdonor-conserved TCR α chains that were filtered for >3 N nucleotides (Supplementary Fig. 2). With one exception, all sequences on that list are MAIT sequences. MAIT cells, regardless of the number of N nucleotides they use, are expanded in vivo due to antigen exposure. Because we found only one non-MAIT sequence among the sequences with N>3 that was shared among donors (as opposed to 16 non MAIT with N<4), we conclude that cross contamination is not the driving force behing the identification of our 16 new invariant TCR α chains.

Public TCRs are defined as MHC-restricted TCRs that are shared among donors that share at least one MHC allele and relevant antigen exposure. To get an indication whether there are public TCRs among the16 new invariant α chains, we HLA-typed 5 of the 6 human subjects that were used for the generation of the TRAV1-2 dataset and asked whether the occurrence of any of the new invariant α chains was limited to subjects that express the same MHC proteins. For HLA-A, HLA-B, and HLA-DRB1, the maximum number of donors that shared a protein was two. For HLA-C, HLA-DQ, HLA-DRB3, and HLA-DRB4, the maximum number of donors that shared a protein was three (Table I and Supplementary Fig. 3). Because four of the 16 α chains were present in 4 out of 5 subjects with available HLA typing results, and the other 12 α chains were present in all 5 subjects (Supplementary Fig. 3), we conclude that none of the16 new invariant α chains is limited to subjects that share MHC alleles, and thus none fulfills the criteria for public TCRs.

Convergent recombination in invariant TCR α chains

The two main factors that contribute to MAIT and NKT α chain prevalence are convergent recombination and in vivo T cell expansion. Convergent recombination causes high precursor frequencies of certain TCR α chains because many independent ways of joining a V and J segment lead to the same amino acid sequence (19–21), whereas T cell expansion increases the number of daughter cells of individual T cell clones (22). Illustrating known mechanisms of convergent recombination in our dataset, we identified examples of identical nucleotide sequences where nucleotides could have been contributed by either the V or J gene without addition of N nucleotides, allowing the formation of identical nucleotide sequences from germline-encoded gene segments (Fig. 2A). In addition, different nucleotide sequences that encode the same TCR α chain amino acid sequence were identified, as well as amino acid variants of the consensus sequence. The analysis of two examples out of 16 CDR3 motifs of newly identified invariant α chains are shown in Fig. 2A. To get an overall indication of the extent of convergent recombination in the newly identified invariant α chains, we assessed how many different nucleotide sequences (clones) encode each MAIT, GEM, and new invariant TCR α chain, and compared this with other TRAV1-2 α chains in our dataset (Supplementary Fig. 1 and Fig. 2B). The data show that MAIT and GEM cells are more often subject to convergent recombination than the newly identified invariant α chains and the TRAV1-2 α chains in our dataset.

Expansion of invariant T cell clones in vivo

Homeostatic or cytokine-driven T cell expansion causes all the T cells that express certain cytokine receptors or other stimulatory receptors other than the TCR to expand, while antigen-driven T cell expansion causes T cell clones with a certain antigen-specific TCR to expand upon specific stimulation of that TCR. NKT and MAIT cells are subject to both mechanisms (22–24) while GEM T cells seem to be at least subject to antigen-driven expansion (12). Insight in these mechanisms is relevant for the question whether a T cell recognizes a specific exogenous antigen and expands upon encounter of that antigen in vivo, or whether the T cell has a function in homeostasis or amplification of other immune cell signals. To assess expansion in our dataset, we plotted the frequency of reads that represent clones encoding GEM, MAIT, or other α chains (Fig. 3A), or new invariant α chains (Fig. 3B) to see whether they can be classified a low, medium, or highly expanded clones (Fig. 3A). As expected, clones with MAIT α chains are among the highly expanded clones (clones that form >1% of the TRAV1-2 repertoire). Clones with GEM α chains include one highly expanded clone, but mostly represent medium expanded clones (clones that form between 0.1 and 1% of the TRAV1-2 repertoire).

GEM TCR α chains are defined as using TRAV1-2 and TRAJ9 to form the CDR3 sequence CAVRXTGGFKTIF or CAVLXTGGFKTIF (12). These individual variants do not make it through the filter of being detectable in > 6 out of 12 samples, but together, they exist in 8 samples (Supplementary Fig. 1B).

Of note, subjects C28, C34, C52, and C58 are latent tuberculosis patients, while subjects B36 and B38 are random blood bank donors. Because Mycobacterium tuberculosis produces glucose monomycolate and mycolic acid, the antigens for GEM T cells, we hypothesized that GEM T cell clones in tuberculosis patients have undergone antigen-driven expansion, while this is not the case in random blood donors, which are less likely to be exposed to these antigens. However, we observed expanded clones with GEM TCR α chains in all subjects (Fig. 3A). Nevertheless, the reads of all GEM TCR α clones together form a bigger fraction of the total TRAV1-2 repertoire in the tuberculosis patients (0.22%, 13.6%, 0.49%, and 0.33%) than in the random blood bank donors (0.09% and 0.47%). A carefully designed study with more subjects and confirmed tuberculosis-unexposed control subjects is needed to confirm this preliminary observation.

Among the clones that represent the 16 new invariant α chain motifs, there are many that fall in the category of medium and highly expanded clones (Fig. 3B). In addition to the frequency of individual T cell clones with invariant α chains (Fig. 3B), we determined the fraction of the TRAV1-2 population formed by all clones combined that fit one motif (Fig. 3C). Because TRAV1-2⁺ T cells typically form 1 – 2% of the total T cell population, these data give insight in the prevalence of these T cells in blood. As expected, the frequency of MAIT α chains among CD4⁺ cells is lower than among CD4⁻ cells in all donors (11, 23). Consistent with findings by Gold et al. (17), MAIT populations in our dataset are smaller in tuberculosis patients than in healthy blood bank donors (Fig. 3C). Migration into infected tissues has been suggested to account for this effect. Together, our quantitative data on MAIT and GEM cells are consistent with published findings, and the data on the newly described invariant α chains show a big clonal size of some of these clones, indicating antigen-driven expansion in vivo.