Preferential Use of Public TCR during Autoimmune Encephalomyelitis

The MHC is the principal genetic locus conveying susceptibility for tissue-specific autoimmunity. For example, homozygosity for HLA-DRB1*15:01 increases the probability of developing multiple sclerosis ∼7-fold (1, 2). In contrast, few non-MHC polymorphisms impart a >1.2-fold increase in risk (3). Potential mechanisms for these allele-specific effects include differential binding of self-antigen–derived epitopes to different MHC specificities and modulation of the selection and activation of pathologic effector and protective regulatory T (Treg) cells (4, 5).

These risk-associated MHC engage a TCR repertoire that has the potential to generate 10¹⁵–10¹⁸ distinct receptors. A much smaller number of unique T cell clones is present in the circulation and represents only a small fraction of this potential diversity (6, 7). Nevertheless, due to the TCR’s capacity for diversification, each individual’s TCR repertoire is essentially unique. Considering the extensive variability of TCR repertoires and the large number of tissue-specific Ags capable of binding MHC molecules that TCR may engage, it may be considered surprising that single MHC alleles so effectively impose population-wide disease risk.

Despite the theoretical diversity of the TCR repertoire, a small fraction of α and β monomers are shared by most individuals or public (8–10). Public TCR α or β-chains can pair with a vast array of independently rearranged and distinct β or α-chains that contribute roughly equally to ligand recognition (11) and therefore would not be expected to bias TCR recognition. However, it has also been shown that certain TRAV and TRBV are preferentially employed in specific responses (12–15), and in one case, structural data identified a binding hot spot between a single TRBV and Ag–MHC ligand that explained this preference (16). Considering this, it is possible that public α or β-chains, which are fixed not only for the V region, but also for J and CDR3 sequences, are capable of modulating a TCRαβ heterodimer’s likelihood of productively engaging autoantigen.

Public TCR largely result from recombinatorial biases in TCR α and β-chain formation and, due to their preferential formation, are often present at high frequency (17). Public TCR have been found to be prevalent in some infectious, autoimmune, and other responses (18, 19). However, clarifying the role of public TCR in disease has been previously limited by several factors. These include prior assessments of responses with low levels of heterogeneity such as that in MBP-induced experimental autoimmune encephalomyelitis (EAE) in H-2^u mice, low-resolution sequencing, or spectratyping analyses that are unable to robustly quantify individual public sequences, absence of data defining differential publicity in the repertoires used by different T cell lineages, and, importantly, the general inability to establish whether public sequences are overrepresented compared with expectations from preimmune TCR frequencies.

In this study, we use saturation sequencing to assess in depth the use of public TCR in a model autoimmune disease, myelin oligodendroglial glycoprotein (MOG)_35–55-induced EAE. Analyses of >18 million TCRβ from Foxp3⁺ Treg and Foxp3⁻ conventional T (Tconv) cells from different organs and time points and from diseased and preimmune mice identified both a high level of TCR diversity as well as extensive use of public TCR within the autoimmune response. Comparisons of disease-associated and unassociated repertoires from preimmune and immune mice further demonstrated that public TCR were preferentially deployed from the preimmune repertoire. Assessment of developing thymocytes identified a markedly increased formation of disease-associated public TCR in preselection thymocytes, and comparison of early and late disease repertoires indicated a focusing and enrichment of the public TCR response over time. These results indicate that public TCR are preferentially incorporated into the responding repertoire during MOG-EAE. They further support the hypothesis that recognition biases imposed by the public TCR repertoire directly bear on the autoimmune response.

C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred with GFP-Foxp3 knockin mice on a B6 background obtained from Dr. A. Rudensky (Memorial Sloan Kettering Cancer Center) (20). Mice were bred under specific pathogen-free conditions, and animal experiments were carried out in compliance with Institutional Animal Care and Use Committee guidelines.

RBCs were lysed prior to staining. FcRs were blocked with FcR blocking reagent (Miltenyi Biotec, San Diego, CA). Cell-surface staining was performed for 20 min at 4°C in PBS containing 0.1% sodium azide and 2% (v/v) FBS. mAbs specific for CD4 (clone RM4-5), CD8 (clone 53-6.7), TCRβ (clone H57-597), CD44 (clone 1M7), CD45RB (clone C363.16A), CD69 (clone H1-2F3), and TRBV13-2, 3 (KJ-16) were from BD Biosciences (San Jose, CA). Flow cytometric analysis was performed on an LSRFortessa (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).

MOG_35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized by the St. Jude Hartwell Center for Biotechnology and HPLC purified prior to use. EAE was induced and scored as described (21). B6 mice were immunized s.c. with 100 μg MOG_35–55 in CFA containing 0.4 mg Mycobacterium tuberculosis H37RA (Difco, Lawrence, KS). Two hundred nanograms pertussis toxin (List Biological Laboratories, Campbell, CA) was administered i.v. on days 0 and 2. Clinical scoring was: 1, limp tail; 2, hind limb paresis or partial paralysis; 3, total hind limb paralysis; 4, hind limb paralysis and body or front limb paresis or paralysis; and 5, moribund.

T cells were isolated from spleen, CNS, and thymus as described (22). Splenic and CNS cells were stained with CD4 Ab and flow cytometrically sorted into CD4⁺GFP-Foxp3⁺ (Treg cell) and CD4⁺GFP-Foxp3⁻ (Tconv cell) populations. Some splenic Tconv cells were further sorted into CD4⁺CD44^hiCD45RB^lo (memory/effector) and CD4⁺CD44^loCD45RB^hi (naive) populations. Thymic T cells were stained with CD4, CD8, and TCR Abs and sorted into CD4⁺CD8⁺TCR^lo double-positive, CD4⁺CD8⁻Foxp3⁻, and CD4⁺CD8⁻Foxp3⁺ single-positive T cell populations. Flow cytometric sorting used a Reflection (Sony Biotechnology, Champaign, IL) sorter.

DNA was prepared and high-throughput sequencing performed as previously described (21). Sequencing was performed on an Illumina Genome Analyzer IIx sequencer (Illumina, San Diego, CA), and 125-bp reads covering the entire CDR3β region were obtained. Raw sequencing data were subsequently trimmed, and the CDR3β sequences were filtered for quality using criteria we have previously described (23). Raw unprocessed data are accessible at European Nucleotide Archive (http://www.ebi.ac.uk/ena). Study accession number is PRJEB13451.

T cell hybridomas were generated using isolated CNS-infiltrating T cells derived from 50 B6 mice with EAE. Cells were stimulated for 3 d with anti-CD3/anti-CD28 prior to fusion with partner cells as previously described and single cells cloned by limiting dilution (22). Resulting hybridomas were screened for KJ-16 (TRBV13-2, 13-3) positivity prior to cDNA production and TCRβ amplification using TRBV13 and TCRα specific primers and capillary sequencing. Responsiveness to MOG_35–55 was measured by coculture of the hybridomas with irradiated splenic APCs with or without 100 μg/ml MOG_35–55 or with control anti-CD3. Sequences were considered shared if they were identified in the CNS repertoires of any of the 12 mice subjected to high-throughput sequencing.

Culture supernatants from hybridomas were collected 24 h after culture with irradiated splenic APCs in the absence of stimulation, in the presence of 100 μg/ml MOG_35–55, or in the presence of anti-CD3 and analyzed for IL-2 using the Milliplex MAP mouse cytokine/chemokine immunoassay kit (Millipore, Billerica, MA) on a Luminex (Bio-Rad) instrument.

Foxp3-GFP reporter mice were immunized with MOG_35–55 to induce EAE as described above. CNS tissue was harvested 17 d postinjection and pooled from nine mice, and CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ TRBV13-2⁺ populations were isolated by flow cytometric sorting. 5′ Rapid amplification of cDNA ends of TCR α-chains was performed as previously reported (24). Briefly, mRNA was extracted using Oligotex Direct mRNA mini kit (Qiagen) and used as a template for cDNA amplification with the SMARTer PCR cDNA synthesis kit (Clontech). Amplified cDNA was cleaned using the PCR purification kit (Qiagen), and rearranged TCR products were amplified using a primer specific for the TCRα constant domain (5′-TCAACTGGACCACAGCCTCAGCGTCA-3′). Products were run on a 1% agarose gel, and fragments between 500 and 700 bp were extracted using a Qiagen gel purification kit. Extracted fragments were cloned into the PCR2.1-TOPO vector for miniprep and sequence analysis.

Except as indicated below, means and SDs were calculated using Excel (Microsoft) or Prism (GraphPad). Plots demonstrate mean ± 1 SD. Two-tailed Student t tests were applied to compare any two groups and ANOVA for three or more groups. For multiple comparisons, significance is only shown for indicated groups. Bayesian analysis was conducted using the Fisher exact test using GraphPad Prism. A p value <0.05 was considered statistically significant. For analysis of CDR3β length, the weighted linear regression method was applied to assess the correlation between CDR3β length and degree of publicity. The total number of available sequences identified as private or public occurring in the specified number of mice was used as weight to determine the significance of this correlation. To assess preferential amino acid usage at specified positions of the CDR3β sequence, sequences were segregated into three groups: private, moderately public (occurring in two to four mice), and highly public (occurring in five to nine mice). The χ² tests were applied to compare the frequency of each amino acid at the indicated position across these groups. To offset the problem of multiple comparisons made across the same data set, the raw p values were adjusted using the Holm-Bonferroni method. Adjusted p values <0.05 were considered significant. Significant differences in amino acid frequency were plotted as a heat map showing preferential usage in public or private TCRs and the magnitude of the difference in the frequency.

TRBV13-2 is the dominant TCRβ in MOG-specific T cells during EAE in C57BL/6 mice (25). To understand the composition and dynamics of autoimmune effector and regulatory repertoires, we flow cytometrically analyzed sorted TRBV13-2⁺ T cell populations from mice with MOG-induced EAE and healthy controls. Cells were isolated from the CNS and spleens of mice with early (day 15–18) and late (day 26) EAE and the spleens and thymi of healthy controls. CNS-infiltrating TRBV13-2⁺ T cell percentages were similar at the early and late time points. CD4⁺Foxp3⁻ (Tconv) and Foxp3⁺ (Treg) populations were acquired for most analyses. From some mice, the CD4⁺Foxp3⁻ cells were further sorted into naive (CD45Rb^hiCD44^lo) and memory/effector (CD45Rb^loCD44^hi) subsets. Saturation sequencing was performed using high-throughput techniques, with >18 × 10⁶ TCR studied (Supplemental Table I) (8, 21, 23, 26, 27).

TRBJ use, an indicator of the heterogeneity of the sequenced populations, was overall similar in Tconv and Treg from the CNS and spleens of mice with EAE and spleens of controls (Fig. 1). This was true whether tallying unique amino acid sequences or total sequences acquired. Small perturbations, particularly an increased TRBJ2.1 representation in the CNS, were seen for both Foxp3⁺ and Foxp3⁻ cells. However, the CNS TRBV13-2 response in MOG-EAE proved overall to be highly diverse, and TRBJ were incorporated roughly in proportion to their frequency in the preimmune population. Not surprisingly, T cells infiltrating the autoimmune CNS were virtually exclusively memory/activated (not shown). Splenic TCR sequences that were also identified in the CNS were also heavily biased toward the memory/activated subset (Fig. 2A, 2B; 77.7 ± 9.0% of unique and 99.0 ± 0.7% of total TCRβ sequences acquired). Therefore a highly diverse, activated T cell population infiltrates the CNS in MOG-EAE, and this population shows concordant signs of activation in the spleen.

Due to the limited size of the CNS infiltrates, substantially fewer unique sequences were identified in the CNS compared with splenic repertoires (Supplemental Table I). Of these, 9.2% of the unique Foxp3⁻ CNS sequences present in mice with early EAE (day 15–18) were shared by two or more of nine mice (Fig. 3A). However, these shared sequences comprised 50.8% of total CNS sequences acquired, indicating a high prevalence of the shared TCRβ. Similarly, in three mice analyzed with late disease (day 26), 8.1% of unique sequences were shared, but these comprised 49.6% of total CNS Foxp3⁻ sequences (Fig. 3D). Progressively smaller numbers of unique Foxp3⁻ TCRβ were shared in the CNS by increasing numbers of mice (Fig. 3B, 3E), but these shared TCRβ were heavily used (Fig. 3C, 3F). For example, 0.05% of unique CD4⁺Foxp3⁻–derived TCRβ sequences were identified in seven or more of the nine early EAE CNS, but comprised >1.5% of total sequences acquired, a 30-fold enrichment. A similar pattern of highly shared TCR representation was evident with CNS-infiltrating Treg cells (Fig. 3G–L).

To further characterize private and shared TCR CDR3β sequences, we compared their average lengths. A slight but highly significant decrease in the average length of the CDR3β loops of public TCRs was observed, and this was proportional to the degree of publicity (Fig. 4A). In addition, differences were observed in amino acid use at positions 1–12 of private and public TCR CDR3βs (Fig. 4B). Public TCRs had a preference for a glycine at position 3, aspartic acid at position 4, alanine at position 5, and glycine at position 6. The magnitude of the increased frequency of these amino acids at these positions also increased with the degree of publicity (not shown). The selection of the glycine at P3 and the aspartic acid at P4 indicated that public TCRs preferentially used the germline-encoded C-terminal TRBV13-2 sequence (P1–P4 = ASGD), whereas private TCRs were more likely to have non–germline-encoded amino acids at these positions. Increased use of a tyrosine residue toward the end of public CDR3 sequence was also seen. This likely resulted from the slightly shorter average length of public TCRs and thus an increased probability of the CDR3 terminal tyrosine residue locating to positions 10–12. The trends in public TCR CDR3β sequence length and amino acid use were observed in both Foxp3⁻ and Foxp3⁺ sequences, as well as in both spleen and CNS samples, suggesting that they are not related to regulatory properties or the presence of EAE, but are biases more generally associated with public TCR.

Sabatino et al. (28), using two-dimensional–based affinity analyses, previously estimated that ∼60% of CNS- infiltrating T cells in MOG-EAE recognize the MOG_35–55 autoantigen. To assess fractional response in this study and determine if there is a differential responsiveness to autoantigen among T cells expressing CNS shared or nonshared TCR, we produced T cell hybridomas from the pooled CNSs of 50 mice with EAE. TCRβ cDNA from KJ-16 Ab (TRBV13-2, 13-3 specific)–positive hybridomas were sequenced, and results were correlated with the reactivity of the hybridomas to MOG_35–55 Ag. Concordant with Fig. 3A and 3D demonstrating that approximately one-half of CNS sequences are shared, 42.5% of 73 TRBV13-2⁺ hybridoma TCRβ sequences identified were also found in the CNS-infiltrating repertoire data sets described above (Fig. 5A, 5B). An identical value, 42.5%, of the 73 hybridomas responded to MOG_35–55. A modest and nonsignificant increase was seen in the proportion of hybridomas incorporating shared versus nonshared TCRβ sequences that demonstrated MOG_35–55 responsiveness (45.2% shared versus 40.5% nonshared; p > 0.05, Fisher exact test). This indicates that a large proportion of CNS-infiltrating T cells are specific for cognate autoantigen and that publicity does not demonstrably alter this. As it is possible that not all hybridomas derived from T cells responding to autoantigen showed detectable reactivity in the in vitro assay in this study, these results set only a minimum threshold for the extent of MOG responsiveness among CNS-infiltrating T cells.

The high prevalence of shared TCRβ in the CNS repertoire (Fig. 3) did not necessarily indicate that shared sequences were preferentially deployed from the peripheral immune repertoire. Public sequences are often present at high frequency. The frequency of public sequences incorporated into the CNS repertoire may therefore have been proportionate to their representation in the larger repertoire and to that of all public sequences, whether involved or uninvolved in the autoimmune response (29). To assess this and establish the extent to which CNS-infiltrating T cells are disproportionately public, we analyzed the more abundant TCRβ sequences present in the spleen, where TCR from cells involved and uninvolved in the CNS response could be directly compared. If TCR engaged in the CNS response were randomly and equivalently deployed from the public and private peripheral repertoires, the proportions of splenic sequences that are identified and not identified in the CNS would be expected to be similarly public (Supplemental Fig. 1). In contrast, if public TCRβ were preferentially incorporated into the CNS response, the splenic sequences also seen in the CNS should be enriched in public sequences.

Consistent with prior reports in other systems, few Foxp3⁺ and Foxp3⁻ TCRβ from the spleens of mice with EAE were shared (Fig. 6). However, TCRβ sequences also seen in the CNS were markedly more likely to be shared than sequences not observed in the CNS (Fig. 7A). For example, 28.5% of the unique CNS Foxp3⁻ sequences from mice with early disease were identified in all spleens analyzed compared with 2.7% of non-CNS sequences, indicating that many CNS sequences were derived from the public repertoire. Pairwise analysis of the sharing of CNS and non-CNS sequences between individual mice verified this finding (Fig. 7C). Results were similar in mice with late disease (Fig. 7B, 7C) and for Foxp3⁺ T cells (Fig. 7D–F). This indicates that the high prevalence of shared sequences within the CNS reflects a selectively elevated use of public TCR among CNS-infiltrating T cells compared with that anticipated if public and private sequences were randomly used.

The increased incorporation of unique public TCRβ sequences in the CNS may have resulted from a greater TCRα heterogeneity among T cell clones bearing public versus private TCRβ. This would lead to greater clonal diversity of cells expressing public compared with private TCRs. Individual public TCRβ sequences would then be associated with a larger number of specificities and therefore an increased probability of being incorporated into the autoimmune response. To exclude this potential confounding factor and determine whether public TCRβ were more generally overrepresented among CNS-infiltrating T cell clones, the analyses above enumerating unique TCRβ sequences was replicated tabulating total splenic TCRβ sequences acquired. This analysis, however, also showed a dramatically enhanced identification of CNS compared with non-CNS sequences among the shared sequences (Fig. 8). Therefore, measurements of both unique and total TCR sequences indicate that public TCRβ are present at a markedly elevated frequency within the CNS repertoire compared with that anticipated if public and private sequences were randomly selected from the repertoire.

If public T cells were preferentially recruited and expanded in the CNS, CNS-infiltrating TCRβ should be disproportionately public in preimmune mice too. To test this, we mapped CNS-infiltrating and uninvolved splenic TCRβ sequences from mice with EAE onto preimmune repertoires from four unimmunized mice. For all samples analyzed, either Foxp3⁻ or Foxp3⁺ and early or late disease, substantially more CNS-infiltrating than noninfiltrating TCRβ sequences were identified as public in the preimmune repertoire (Fig. 9). This, again, was true both for unique and total TCRβ sequences analyzed. Together, these data support a heavily biased public TCRβ usage in the EAE repertoire, with this preference traceable to the preimmune repertoire.

To determine whether the extent of sharing of autoimmune repertoires differed in early and late EAE, we next compared CNS repertoires from individual mice. Overlap of CNS sequences was lowest when pairs of day 15–18 mice were compared with each other. Repertoire overlap increased when day 15–18 mice were compared with day 26 mice and was greatest when day 26 mice were compared (Fig. 10A, 10B). Splenic TCR overlap, unlike that of CNS TCR, did not change with disease stage (Fig. 10C, 10D). Therefore, the extent of sharing increases with time in the disease-associated repertoire. That mice with early disease shared more of their CNS repertoire with late disease mice than with each other further indicates a temporal focusing of the shared CNS repertoire.

Considering the relatedness of the days 15–18 and day 26 repertoires, we combined the 12 CNS data sets we acquired to identify common TCRβ sequences. Forty-eight TCRβ were identified in ≥10 of the 12 CNS repertoires (Supplemental Table II). These receptors were oligoclonal with multiple nucleotide sequences used for each CNS amino acid sequence cumulatively (range 2–59, median = 13) and within single mice (mean 2.8 ± 1.3). Consistent with their oligoclonal presence, in individual CNS, 46.7 ± 19.9% of the 48 public TCRβ were identified in both the Foxp3⁺ and Foxp3⁻ lineages compared with 2.9 ± 1.9% of private sequences (p < 10⁻⁵).

It is hypothesized that public TCRs have a high probability of forming in the thymus. Biases in V(D)J recombination selects for TCRs with fewer nucleotide additions at the junctions, and public TCRs often have CDR3 sequences that more closely resemble germline V, D, and J sequences relative to private TCRs. Consistently, we observed a trend toward slightly shorter CDR3 lengths in autoimmune public TCRs, as well as preferential use of the germline TRBV sequence relative to private TCRs (Fig. 4). Additionally, public TCRs with a single amino acid sequence are often found to be encoded by multiple nucleotide sequences, indicating that they are formed repeatedly in the thymus through multiple independent rearrangements, also providing an explanation for their commonality (9, 17, 30).

To test for a preferential thymic formation of public TRBV13-2⁺ TCR sequences in MOG-EAE, we assessed their nucleotide use in preselection CD4⁺CD8⁺TCR^lo (double-positive) thymocytes from preimmune mice (5). These cells were further identified as CD5 and CD69 negative, verifying their preselection status. Nucleotide sequence diversity in preselection thymocytes for each of the 48 public TCRβ was compared with that of paired control private TCRβ from the same mouse. These paired private sequences were selected based on having identical Vβ, Jβ, and CDR3 length and similar identification frequencies (>0.25- to <4-fold). Average nucleotide sequence numbers across 4 mice for each of the 48 sequences and their controls were calculated. If individual public sequences had a high probability of forming due to independent preselection recombination events, multiple nucleotide sequences encoding a single public amino acid sequence would be expected within an individual thymus. The public sequences indeed displayed markedly greater mean preselection nucleotide variability than control private sequences (Fig. 11; 6.2 ± 2.9 versus 1.1 ± 0.1 nt sequences), indicating that public sequences have an increased probability of forming and that preselection recombinatorial biases foster disease-associated public repertoire formation.

In preliminary studies, we assessed TCRα-chain use in TRBV13-2⁺ CNS-infiltrating TCR. We used 5′ rapid amplification of cDNA ends of rearranged TCRs in sorted Foxp3⁺ and Foxp3⁻ TRBV13-2⁺ CD4⁺ T cells followed by template-switch anchored RT-PCR to isolate and then sequence recombined TCRα products. TRAV7 was preferentially used among Foxp3⁻ clones, both in total sequences and unique sequences (Supplemental Fig. 2). A more moderate use of TRAV7 and an increase in TRAV12 sequences was observed among Foxp3⁺ clones. The most common TRAJ gene detected, TRAJ23, exhibited relatively consistent use between the Foxp3⁻ and Foxp3⁺ clones examined in this study. This difference in TRAV use between Foxp3⁻ and Foxp3⁺ clones suggests that TRAV selection may be skewed depending on the regulatory status of the cells. However, these results are based on a small data set, and more extensive analyses are needed to define the role of TCRα in this model system.

Using saturation sequencing of disease-associated and unassociated TRBV13-2 TCR repertoires, we demonstrate a selective and prominent role of public TCR in the CNS autoimmune response. TRBV13-2⁺ TCR were analyzed because of this Vβ-chain’s documented prominence in the MOG-specific T cell response. We find that the frequencies at which unique and total CNS-infiltrating TCRβ sequences were shared among mice was markedly elevated when compared with TCRβ unengaged in autoimmunity both from mice with EAE and from preimmune mice. The extent of use of public TCR did not substantially differ in Foxp3⁺ and Foxp3⁻ T cells, indicating that publicity is intrinsic to the response regardless of lineage assignment.

Recombinatorial biases in preselection thymocytes serve as the primary source for the generation of the broad public repertoire (10, 31, 32). Predispositions in VDJ use and activity levels of TdT and nucleases modifying junctional sequences increase the probability these sequences will form. We implicate similar preselection biases also in generating the public autoimmune repertoire and identify substantial ongoing oligoclonal production of public autoimmune-associated TCR within the thymus. Indeed, many public TCRβ were identified in both Treg and effector T cells in the CNS of individual mice, consistent with their increased formation in preselection thymocytes. As previously documented by Furmanski et al. (33) with the larger public repertoire, analyses in this study of the frequency of individual receptors in pre- and postselection thymocytes and splenic T cells in preimmune mice further failed to indicate that, once formed, disease-associated public TCR are preferentially selected (data not shown).

Most prior studies exploring public TCR representation have used low-resolution techniques that identify small numbers of response-enriched public TCR. Our results with high-resolution sequencing indicate that public TCR-produced repertoire distortions need not result from idiosyncratic effects due to the presence of rare high-frequency dominant clonotypes. Rather, the TCR used by public CNS-infiltrating T cells proved to be highly heterogeneous. Indeed, the most common public TCR identified was found in a median of only 0.2% of TRBV13-2⁺ TCR sequenced.

Our results also do not imply that public TCR possess unique structural properties that distinguish them from private TCR. Indeed, despite comprising a small fraction of the total repertoire, public sequences remain diverse. It would therefore seem unlikely that their preferential recruitment into the autoimmune response when compared with private sequences is due to a distinct biochemistry. Rather, one possibility is that because public TCR are pervasive across a population, specificity distortions they introduce within the repertoire will be introduced into all individuals bearing relevant MHC alleles. Thus, we can speculate that MOG_35–55 serves as the dominant autoantigen in C57BL/6 EAE precisely because the public repertoire in conjunction with the restricting MHC, IA^b, augments repertoire reactivity toward this autoantigen. Other neuroantigens transparent to the public repertoire and preferentially engaged by private TCR would only be capable of mediating autoimmunity in the small number of individuals that stochastically possess adequate numbers and subsets of Ag-specific private T cells.

An alternative explanation for the preferential incorporation of public sequences into the autoimmune repertoire is that these sequences more generally predispose TCRαβ toward self-reactivity. Other repertoire studies, though more limited in scope and definition compared with these, have also identified public sequences among autoreactive T cells (18, 34, 35). However, it would seem more likely that overrepresentation of public TCR is not specific to self-reactive populations, but rather reflects a generically increased responsiveness of public TCR to Ag. Public TCR use has also been found in the responses to several pathogens and other Ags (10, 17). Although the extent to which public TCR are actually overrepresented rather than just prevalent in immune responses requires further study, a number of analyses demonstrate a strong presence of public TCRs, implying a central role in specific epitope responses (36, 37). It is reasonable to expect that the recombinase machinery is tuned to preferentially generate sequences well adapted to support MHC engagement. If so, public sequences, which have a high likelihood of forming, may be particularly well suited to support TCR associations with MHC–Ag. The majority of the TCR interface with Ag–MHC binds the MHC rather than antigenic peptide, and MHC-specific associations are critical to orienting the TCR and effective recognition (11, 38). An overall enhanced compatibility with MHC may thereby lead to the increased use of public TCR in diverse responses. An element of enhanced self-reactivity would be expected to accompany any generically increased binding fitness of TCR for MHC, and indeed, this has been observed in mutational analyses increasing TCR affinity (39, 40).

Although this latter model remains speculative, in potential support, we find that though the representation of public TCR among the autoimmune response in EAE is elevated, the fraction of CNS-infiltrating cells that functionally respond to MOG_35–55 is similar among shared and nonshared sequences. This suggests that publicity in and of itself did not increase the proportion of activated CNS-infiltrating T cells with threshold reactivity to cognate MOG_35–55 autoantigen. However, the increased presence of public TCR in the CNS implicates these cells as having an increased likelihood of more generally responding to the immunizing stimulus. Ultimately, further functional assessments of the public and private immune response in this study as well as to additional immunizing events will be necessary to establish patterns of response distinguishing the public and private repertoires and the generality of these findings.

The capacity to interrogate the repertoire continues to increase with improving sequencing technologies (26). Data sets are being collected in several disease models, and these may be linked to functional analyses of individual TCR, similar to those we describe in this study, to identify the relevance of specific sequences. Indeed, the identification of preferential TRBV and TRAV usage in several diseases may imply that public TCRα or TCRβ sequences bearing predefined V, J, and CDR3 sequences will strongly skew response characteristics as in this study. If so, specific public sequences may prove useful for the longitudinal monitoring of immune responses during autoimmune diseases. Likewise, if public TCR ultimately prove not only to be overrepresented, but also drivers of the autoimmune response as originally hypothesized by Wilson et al. (41), it may be possible to modulate the autoimmune response by targeting the selection or activity of T cells bearing public TCR sequences.

We thank Richard Cross, Greig Lennon, and Parker Ingle for assistance with flow cytometric sorting and Jackie Wright and Bofeng Li for assistance with hybridoma production and DNA sequencing.

The authors have no financial conflicts of interest.