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. Author manuscript; available in PMC: 2015 Aug 15.
Published in final edited form as: J Immunol. 2014 Jul 11;193(4):1778–1786. doi: 10.4049/jimmunol.1400532

The Ability to Rearrange Dual TCRs Enhances Positive Selection, Leading to Increased Allo- and Autoreactive T Cell Repertoires

Peggy P Ni *, Benjamin Solomon †, Chyi-Song Hsieh †, Paul M Allen *, Gerald P Morris ‡
PMCID: PMC4119549  NIHMSID: NIHMS607501  PMID: 25015825

Abstract

Thymic selection is designed to ensure T cell receptor (TCR) reactivity to foreign antigens presented by self-MHC while minimizing reactivity to self-antigens. We hypothesized that the repertoire of T cells with unwanted specificities such as alloreactivity or autoreactivity are a consequence of simultaneous rearrangement of both TCRα loci. We hypothesized that this process helps maximize production of thymocytes capable of successfully completing thymic selection, but results in secondary TCRs that escape stringent selection. In T cells expressing 2 TCRs, 1 TCR can mediate positive selection and mask secondary TCR from negative selection. Examination of mice heterozygous for TRAC (TCRα+/−), capable of only one functional TCRα rearrangement, demonstrated a defect in generating mature T cells attributable to decreased positive selection. Elimination of secondary TCRs did not broadly alter the peripheral T cell compartment, though deep sequencing of TCRα repertoires of dual TCR T cells and TCRα+/− T cells demonstrated unique TCRs in the presence of secondary rearrangements. The functional impact of secondary TCRs on the naive peripheral repertoire was evidenced by reduced frequencies of T cells responding to autoantigen and alloantigen pMHC tetramers in TCRα+/− mice. T cell populations with secondary TCRs had significantly increased ability to respond to altered peptide ligands related to their allogeneic ligand as compared to TCRα+/− cells, suggesting increased breadth in peptide recognition may be a mechanism for their reactivity. Our results imply that the role of secondary TCRs in forming the T cell repertoire is perhaps more significant than what has been assumed.

Introduction

The T cell receptor is comprised of TCRα and TCRβ chains generated by gene segment recombination during thymocyte development. Generation of operational TCRs is critical for development of a functional T cell repertoire, as TCRs must specifically and sensitively recognize self and foreign peptide-MHC (pMHC) ligands to appropriately navigate development and mediate immune responses (1). The TCRβ chain rearranges in double-negative (DN) thymocytes under tight allelic exclusion and ceases when an in-frame product is made and expressed (2–4). CD4 and CD8 co-receptors are then upregulated, and in these double-positive (DP) cells TCRα chain recombination occurs until halted by positively selecting signals (5–8). Positive selection requires specific recognition of self-pMHC ligands (9–13). This strict requirement results in a majority of thymocytes dying from an inability to undergo positive selection (14,15). However, presumably in a measure to maximize generation of TCRs capable of mediating positive selection, TCRα gene recombination occurs in DP thymocytes in a simultaneous and iterative fashion on both loci (7, 16). Iterative revision of TCRα, sequential recombination of TRAV and TRAJ segments on the same chromosome, has been demonstrated to be important for efficient positive selection of T cells by enabling multiple opportunities for formation of a successful in-frame TCRα rearrangement (17). However, the impact of simultaneous rearrangement of TCRα loci on both chromosomes on thymocyte selection has not been defined.

Simultaneous rearrangement of both TCRα loci results in a lack of allelic exclusion for TCRα, evidenced by thymocytes and peripheral T cells with 2 in-frame rearrangements of TCRα (3, 18), and mature T cells with dual TCR expression on the surface (8, 19, 20). In these cells, each TCRα chain pairs with the same β chain, giving the cell 2 distinct pMHC ligand specificities (21, 22). The expression of dual TCRs presents a unique change to the requirements of a thymocyte for successful selection. One TCR can successfully mediate positive selection, enabling the presence of a secondary TCR that does not participate in positive selection (19, 21, 22). Expression of secondary TCRs can also factor importantly during negative selection, masking autoreactive TCRs from deletion (23–25). This masking effect is likely mediated through decreased surface expression of the pathogenic TCR due to TCRα chain competition for the single TCRβ chain (26, 27). Thus, the presence of dual TCRs in developing thymocytes provides an unusual lessening of the stringent requirements for thymic selection, which could significantly impact the naive T cell repertoire.

This potential prompted us to examine dual TCR T cell alloreactivity as a model of naive T cell responses. Examination of alloreactive responses in mice genetically lacking dual TCR T cells (TCRα+/−, heterozygous for a mutation in TRAC disrupting formation of a functional TCRα chain), revealed that secondary TCRs, which comprise approximately 10% of the peripheral TCR repertoire in mice, constitute over 40% of the response to allogeneic stimuli (22). The impact of secondary TCRs in pathologic alloreactivity is demonstrated in patients developing acute graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation. In these patients, dual TCR T cells were expanded, activated, and responded preferentially to mismatched alloantigens (28). These data indicate that dual TCR T cells contribute significantly to alloreactive T cell responses. A functional contribution of naturally-arising dual TCR T cells to the autoreactive T cell repertoire has been suggested by studies of diabetes in NOD mice (29, 30).

We hypothesized that the potential for unwanted TCR specificities harbored by dual TCR T cells must be balanced by a significant benefit of simultaneous TCRα rearrangement during thymocyte development. We theorized that this benefit likely occurs though improved efficiency of DP thymocyte positive selection. Examination of thymocyte development in TCRα+/− mice demonstrated decreased positive selection, confirming the importance of simultaneous TCRα rearrangements for effective thymocyte development. While this did not broadly impact peripheral T cell numbers or TCRVα and TCRJα gene segment use, deep sequencing of peripheral TCRs demonstrated the presence of unique TCR sequences among dual TCR T cells that were notably absent from TCRα+/− T cell populations. Functional examination of naive peripheral T cell repertoires demonstrated that TCRα+/− mice had diminished binding to autopeptide-MHC and allopeptide-MHC tetramers but not to foreign antigen. Population-level analysis of binding to altered peptide ligands (APLs) of an allostimulatory peptide showed that the presence of secondary TCRs enabled recognition of related pMHC ligands, providing a possible mechanism for dual TCR T cell alloreactivity.

Materials and Methods

Mice

B6, B6.Ly5.1, B6.Thy1.1 and B6.K mice were originally purchased from The Jackson Laboratory. TCRα+/− mice, incapable of expressing two TCRα chains due to a targeted disruption at the 5’ end of one copy of the TRAC gene, were derived by crossing TRAC−/− B6 mice (6) with B6 or B6.Thy.1.1 mice. Mice were bred and housed in specific pathogen-free conditions at Washington University Medical Center (St. Louis, MO). All use of laboratory animals was approved and performed in accordance with the Washington University Division of Comparative Medicine guidelines.

Flow cytometry

Thymocyte and T cell analyses were performed using anti-CD3 (145-2C11)-PE-Cy7, anti-CD45.2 (104)-PerCP-Cy5.5, anti-CD90.1 (HIS51)-eFluor 450 (eBioscience), anti-CD69 (H1.2F3)-PE-Cy7, anti-CD4 (RM4-5)-AF700, anti-CD8α (53-6.7)-APC-Cy7 (Biolegend), and anti-CD45.1 (A20)-APC (BD Biosciences). Non-T cells were excluded by labeling with Pacific Blue-labeled anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-CD11c (N418), and anti-F4/80 (BM8) (Biolegend). TCRVα2+ and dual TCR T cells were identified among CD3+B220−CD11b−CD11c−F4/80− splenocytes using anti-TCRVα2 (B20.1)-PE and anti-TCRVα3 (RR3-16)-FITC, anti-TCRVα8 (KT50)-FITC, and anti-TCRVα11 (RR8-1)-FITC (BD Biosciences). Samples were analyzed using LSR II or LSR Fortessa cytometers (BD Biosciences) with calculated compensation, and data were analyzed with FlowJo software (Tree Star). FACSAria II (BD Biosciences) was used for sorting.

Thymocyte culture

Survival was assessed by culturing 107 thymocytes/mL in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum (PAA), 2 mM GlutaMAX, and 50 µg/ml gentamycin for 6 days. Cultures were labeled for CD4, CD8, Annexin V, and 7-AAD (Biolegend), and analyzed by flow cytometry.

Thymocyte transfers

B6.Ly5.1 and TCRα+/−.Thy1.1 pre-selection thymocyte populations were bead-enriched for CD53− cells using anti-CD53 mAb (OX-79, Biolegend) and anti-IgM paramagnetic beads (Miltenyi Biotec). Enriched cell populations were mixed at a 1:1 ratio and injected intrathymically into sublethally irradiated (5 Gy) B6 recipient mice. Thymi from recipient mice were analyzed 7 days post-injection by flow cyomtetry.

BrdU labeling

Mice were injected intraperitoneally with 1.2 mg BrdU (Sigma) and thymi were harvested at 24, 48, and 96 h timepoints. Thymocytes were labeled for surface markers, fixed with BD CytoFix (BD Biosciences), permeabilized with Permeabilization Wash Buffer (Biolegend), treated with 1 mL DNase I (Sigma) at 50 U/mL, intracellularly labeled with anti-BrdU (BU20A)-FITC (eBioscience), and analyzed by flow cytometry.

TCR repertoire analysis

TCRVα2+ T cells and dual TCR T cells were sorted from TCRα+/− and B6 splenocytes by flow cytometry. TCRα cDNA libraries were generated by PCR (31, 32) and sequenced by 250 cycle paired-end sequencing using an Illumina MiSeq at the Washington University Genome Sequencing Center. TRAV and TRAJ gene segment use was determined by sequence analysis using the International ImMunoGeneTics Information System nucleotide sequence database (33).

Tetramer enrichment

Lymphocytic choriomeningitis virus glycoprotein (LCMV)66–77 (DIYKGVYQFKSV) / I-Ab (LCMV-Ab tetramer)-APC, mouse MHC class II antigen Eα52–68 (ASFEAQGALANIAVDKA) / I-Ab (Eα-Ab tetramer)-APC, and myelin oligodendrocyte glycoprotein (MOG)38–49 (GWYRSPFSRVVH) / I-Ab (MOG-Ab tetramer)-PE were obtained from the NIH Tetramer Core Facility at Emory University (Atlanta, GA). Murine CD22654–666 (SGQDLHSSGQKLR) / I-Ek (CD22-Ek tetramer)-PE and murine TFR231–244 (SGKLVHANFGTKKD) / I-Ek (TFR-Ek tetramer)-PE tetramers were generated using soluble I-Ek produced in E. coli inclusion bodies and refolded with peptide (34). Tetramer enrichment was performed according to published protocols (35). Briefly, cells were incubated with tetramer for 1 h at room temperature, washed, incubated with anti-PE and/or anti-APC conjugated microbeads for 30 min at 4°C, and passed over magnetized LS columns for positive selection (Miltenyi Biotec). Enriched populations were labeled for surface markers for 20 min. at 4°C and analyzed or sorted by flow cytometry.

Intracellular cytokine staining

T cell antigen-specific responses were assessed by 24 h culture with 105 Chinese hamster ovary (CHO) cells stably expressing I-Ek and stimulated with 20 ng/ml PMA and 1 µM ionomycin or 10–50 µM peptide in round-bottom 96-well plates at 37°C. During the last 4 h of culture, 10 µg/mL Brefeldin A (Sigma-Aldrich) was added. Live cells were stained using LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Molecular Probes), labeled for surface markers, and intracellularly labeled with anti-IFNγ (XMG1.2)-FITC or isotype control (Biolegend).

In vivo alloreactive response

T cells were enriched from B6.Ly5.1 and TCRα+/−.Thy1.1 spleens using anti-CD4 and anti-CD8 paramagnetic beads and LS columns (Miltenyi Biotec), mixed 1:1, pulsed with 5 mM CFSE (Sigma), and 5–10 × 106 cells were injected i.v. into lethally irradiated (10 Gy) B6.K mice. Splenocytes were harvested at 8 h and 24 h after injection, labeled for surface markers, and analyzed by flow cytometry for CFSE dilution.

Statistical analyses

Data were analyzed using Prism 6 software (Graph Pad). Mean values of mouse replicate data were analyzed using Student’s t test. Paired data were analyzed using paired ratio t-test. Multi-categorical data were analyzed using 2-way ANOVA. TCR sequence data were analyzed by linear regression analysis with computation of 99% confidence and predictive intervals and tabular analysis of outliers.

Results

Secondary TCRα rearrangements enable efficient positive selection

The effects of simultaneous TCRα rearrangement on thymocyte development were evaluated by analyzing thymus cellularity and populations in TCRα+/− mice incapable of forming functional secondary TCRα chains (6). Thymi from B6 and TCRα+/− mice had similar total number of cells (Fig. 1A). There were comparable percentages of immature DN (8.8±1.5% vs. 7.6±2.0%), DP (70.1±3.0% vs. 76.4±3.6%), and SP cells (14.7±2.2% vs. 19.7±2.5%) in TCRα+/− mice compared to B6 (Figs. 1B and 1C). However, closer examination revealed that TCRα+/− mice had significantly reduced percentages of post-selection CD3high thymocytes (8.7±0.6%) compared to B6 (13.3±0.7%) (Figs. 1D and 1E). This specific decrease of post-positive selection thymocytes implies that secondary TCRα rearrangements are important for efficient thymic development.

FIGURE 1.

FIGURE 1

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Thymic generation of mature T cells is deficient in the absence of secondary TCRα rearrangements. (A) Comparison of numbers of thymocytes in 6 week old B6 and TCRα+/− mice. Each point represents a single mouse, n = 17, 4 independent experiments, mean ± sem, Student’s t test. (B–E) The effect of secondary TCRα rearrangements on T cell development examined by comparing thymi of B6 and TCRα+/− mice by flow cytometry, n = 10, 3 independent experiments. (B) Representative plots of CD4 and CD8 labeling of thymocytes from individual mice. (C) Percentages of mature CD4+ or CD8+ single positive (SP), CD4+CD8+ double positive (DP), or CD4−CD8− double negative (DN) thymocytes. Each point represents a single thymus, mean ± sem, Student’s t test. (D) Representative plots of CD3 and CD69 labeling of thymocytes from individual mice. (E) Percentages of post-positive selection CD3high thymocytes. Each point represents a single thymus, mean ± sem, Student’s t test.

We hypothesized that positive selection would be the most critical window for the influence of secondary TCRα chains, as it is estimated that pre-selection DP thymocytes have an average lifespan of only approximately 60 h during which they must form a functional TCR to mediate positive selection and continue development (14, 15). To examine positive selection kinetics, we injected B6 and TCRα+/− mice with a pulse dose of BrdU and evaluated subsequent development of labeled thymocytes. There was no difference in the percentage of thymocytes (predominantly DN thymocytes) that incorporated BrdU (Fig. 2A). However, over the following 96 h, pulsed TCRα+/− thymocytes demonstrated a deficient progression to a post-selection CD3high phenotype (18.7±2.2%) compared to B6 (27.1±1.8%) (Fig. 2B).

FIGURE 2.

FIGURE 2

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Deficiency of secondary TCRα rearrangements impairs positive selection. (A–B) BrdU (1.6 mg) was administered i.p. on d 0. (A) Thymocyte BrdU incorporation was evaluated at 24 h post-injection (n = 6, 3 independent experiments, mean ± sem, Student’s t test). (B) Thymocyte development in Brd U-pulsed B6 and B6.TCRα+/− mice compared by analyzing development of CD3high post-positive selection thymocytyes by flow cytometry at 24 h, 48 h, and 96 h post-BrdU injection. Mean ± sem of 6 mice per group in 3 independent experiments, Student’s t test. (C–E) Pre-selection thymocytes from congenically-marked B6.Ly5.1 and TCRα+/−.Thy1.1 mice were enriched by magnetic bead selection, combined at a 1:1 ratio, and 107 cells transferred by intrathymic injection into B6 mice to examine development (n = 8, 3 independent experiments). (C) Transferred cells were detected 7 d after injection by flow cytometric analysis of thymocytes. Representative plot with congenically-marked transferred cells expressed as percentage of total thymocytes. (D) Development of post-selection CD3high thymocytes was compared between B6.Ly5.1 and TCRα+/−.Thy1.1 thymocytes in individual mice. Transferred populations in a single mouse are linked by the line between points, paired ratio t test. (E) Ratio of B6.Ly5.1 to B6.Cα+/−.Thy1.1 cells among total thymocyes and post-selection CD3high thymocytes. Mean ± sem, paired ratio t test. (F) Flow cytometry analysis of thymocytes from 6 wk old B6 and TCRα+/− mice to assess thymocyte apoptosis. Thymocytes were labeled with CD4, CD8, Annexin V, and stained with 7-AAD. Each point represents a single mouse (n = 7) from 3 independent experiments, mean ± sem, Student’s t test. (G) Intrinsic thymocyte survival was compared between thymocytes from 6 wk old B6 and TCRα+/− mice by in vitro culture of 107 thymocytes/ml. Cultures were sampled at indicated time points and labeled for CD4, CD8, Annexin V, and stained with 7-AAD. Points are mean ± sem of 6 thymic cultures from 3 independent experiments, data analyzed by ANOVA.

Co-transfer studies of congenically marked B6 and TCRα+/− cells were performed to attribute the deficiency of generating mature SP thymocytes to a cell-intrinsic defect. Pre-selection CD53− cells from B6.Ly5.1 and TCRα+/−.Thy1.1 thymocytes were mixed at a 1:1 ratio and intra-thymically injected into recipient B6 mice. Transferred cells were analyzed 7 days post-injection by flow cytometry. The ratio of B6.Ly5.1/TCRα+/−.Thy1.1 among total thymocytes was 1.36±0.11, similar to the pre-injection ratio. However, examination of post-selection CD3high thymocytes revealed a significant skewing toward B6.Ly5.1 cells (1.86±0.15 ratio), suggesting that the inability to perform simultaneous TCRα rearrangements impaired the ability of thymocytes to mature. Comparison of B6.Ly5.1 and TCRα+/−.Thy1.1 thymocyte development within individual thymi underscores this observation (Figs. 2C–E).

To rule out a survival defect as the mechanism for this deficiency, we examined viability in B6 and TCRα+/− thymocytes. Direct ex vivo analysis of Annexin V and 7-AAD labeling of thymocytes did not reveal any differences between B6 and B6.TCRα+/− mice (Fig. 2F). Likewise, B6 and B6.TCRα+/− thymocytes demonstrated similar viability during 6 days of in vitro culture (Fig. 2G) indicating that the competitive advantage of secondary TCR-sufficient thymocytes was not due to a difference in viability.

Absence of secondary TCRα rearrangements eliminates certain TCR specificities

The contribution of secondary TCRα chains in mediating thymocyte development suggests that their absence could significantly affect the peripheral T cell repertoire. The lack of secondary TCRα rearrangements did not affect peripheral T cell numbers in the spleen (Fig. 3A) or CD4 or CD8 subsets (Fig. 3B). To examine potential skewing of the TCR repertoire, TRAV and TRAJ gene segment use was analyzed by TCRα cDNA sequence analysis of peripheral T cells from 3 B6 and 3 TCRα+/− mice. Analysis of 123,655 B6 and 104,434 TCRα+/− TCRα transcripts revealed similar TRAV (Fig. 3C) and TRAJ (Fig. 3D) gene segment use, indicating that the absence of secondary TCRs does not broadly affect the T cell repertoire.

FIGURE 3.

FIGURE 3

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Elimination of secondary TCRα rearrangements does not broadly alter the peripheral T cell compartment. (A) Comparison of numbers of T cells in the spleens of 6 week old B6 and TCRα+/− mice. Each point represents a single mouse (n = 6) from 3 independent experiments, mean ± sem, Student’s t test. (B) Comparison of CD4+ and CD8+ T cell composition, measured by flow cytometry of splenocytes. Mean ± sem of 6 mice from 3 independent experiments, Student’s t test. Comparison of (C) TRAV and (D) TRAJ gene segment use by DNA sequence analysis of splenic T cells from 6 week old B6 and TCRα+/− mice (3 mice each, 23,877 – 34,995 TCRα sequence reads/mouse). Mean ± sd for each group, data analyzed by ANOVA.

For a more focused examination, we compared TCRα sequences from TCRVα2+ (TRAV14) T cells from TCRα+/− mice with TRAV14+ TCRα transcripts from dual TCR T cells (TCRVα2+ and TCRVα3/8/11+) from B6 mice. This enabled direct comparison of TCRα repertoire composition in the presence or absence of secondary TCRs. From 3 independent TRAV14+ TCRα transcript libraries for each mouse strain, we analyzed 141,353 B6 TRAV14+ TCRα sequences and 148,228 TCRα+/− TRAV14+ TCRα sequences, yielding 13,646 and 14,002 unique CDR3α sequences respectively. Of the unique TRAV14+ CDR3α sequences, 4609 (33.8% of the B6 repertoire) were shared between the 2 groups (Fig. 4A), indicating a potential for significant differences between the 2 repertoires. There was no difference between TCRα+/− and dual TCR B6 TRAV14+ TCRα sequences in CDR3 length (Fig. 4B), amino acid composition (Fig. 4C), and TRAJ gene segment use (Fig. 4D). However, our hypothesis proposes that the impact of secondary TCRα rearrangements would be more subtle and reflected in the presence or absence of specific TCRα sequences that might normally be negatively selected against if present as the only TCR. Comparison of the most abundant transcripts revealed considerable overlap (3/10 top-ranked dual TCR and 4/10 TCRα+/− transcripts were shared) (Fig. 4E). Comparison of all TRAV14 transcript abundance demonstrated a significant correlation between the 2 populations (r = 0.788) (Fig. 4F). The slope of the correlation suggests that the TCRα repertoire is less diverse in TCRα+/− T cells than B6.

FIGURE 4.

FIGURE 4

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Elimination of secondary TCRα rearrangements results in elimination of specific TCRs from the naive T cell repertoire. TRAV14 T cell receptor libraries were generated from TCRα+/− TCRVα2+ T cells and B6 TCRVα2+ dual TCR T cells (identified by co-expression of TCRVα3, TCRVα8, or TCRVα11) sorted by flow cytometry. (A) Three independent libraries per group were sequenced and generated 141,353 B6 TRAV14+ TCRα sequences and 148,228 TCRα+/− TRAV14+ TCRα sequences yielding 23,039 total unique sequences divided among the groups as indicated. Analysis of (B) CDR3 length, (C) CDR3 amino acid composition, and (D) TRAJ gene segment use was compared between the 2 groups. (E) CDR3 sequence abundance was determined as a proportion of total TRAV14 transcripts for each population. The 10 most abundant CDR3 sequences for both populations are shown with corresponding frequencies. (F) Total TRAV14 CDR3 sequence frequencies for both populations, with correlation and 99% confidence interval determined by linear regression (Pearson correlation analysis). (G) The 5 most abundant TRAV14 CDR3 sequences unique to each population are shown with corresponding frequencies.

The limits on TCRα diversity implied by regression analysis are more clearly illustrated by examining CDR3 sequences unique to each repertoire. If the absence of a TCRα sequence in one repertoire were due to sampling error or random effect, it would be expected that the frequency of the sequence would fall within the confidence intervals determined by regression analysis. This is true for the majority of TRAV14+TCRα sequences unique to the dual TCR (8430/9037, 93.3%) and TCR±+/− (8884/9393, 94.6%) repertoires, which fall within the 99% confidence intervals. However, the increased frequency of TCRα sequences unique to dual TCR T cells that are outside of expected random sampling error as compared to secondary TCRα-insufficient T cells (P < 0.001, Fisher’s exact test) suggests that the repertoire lacking secondary TCRs is not a stochastic process, but the result of actively excluding some sequences. Indeed, many TCRα sequences unique to the B6 dual TCR T cells were of relatively high abundance (> 0.1% of the TRAV14+ repertoire) as compared to TCRα+/− cells (Fig. 4G). These results illustrate the effect of secondary TCRα rearrangements on the diversity of the peripheral T cell repertoire.

Secondary TCRs contribute to naive autoreactive and alloreactive repertoires

Findings from TCRα repertoire analysis suggest that exclusion of distinct sequences in TCRα+/− mice could manifest as a differential ability of the naive T cell repertoire to respond to antigens. We measured the reactivity of naive B6 and TCRα+/− T cells using class II peptide-MHC tetramers to estimate the frequency of antigen-specific T cells (35). We generated I-Ek tetramers loaded with allogeneic peptides CD22654–666 and transferrin receptor (TFR)231–244, previously identified by mass spectrometry of endogenous peptides eluted from I-Ek molecules and known to stimulate alloreactive T cells (36). T cells were labeled with alloantigen pMHC tetramers, enriched by magnetic bead selection, and identified by flow cytometry (Fig. 5A). CD22/I-Ek and TFR/I-Ek tetramer positive and negative cells from B6 mice were sorted by flow cytometry and assessed for response to CD22 and TFR peptides. Tetramer positive populations responded to their peptide, whereas the tetramer negative cells did not (Fig. 5B), demonstrating the efficiency of our pMHC tetramer labeling. TCRα+/− mice had a consistently decreased frequency of response to individual allogeneic pMHC complexes (Fig. 5C). The average frequencies of TCRα+/− T cells recognizing the TFR/I-Ek and CD22/I-Ek tetramers (8.6±0.9 and 61.0±8.1 cells/105 T cells, respectively) were significantly less those of B6 T cells (19.9±4.7 and 99.9±14.8 cells/105 T cells). There was no significant difference between B6 and TCRα+/− T cell response to a minor histocompatibility antigen, Eα52–68 presented by self-MHC I-Ab (4.3±1.0 vs. 3.1±0.9 cells/105 T cells, respectively).

FIGURE 5.

FIGURE 5

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Secondary TCRα rearrangements specifically increase the alloreactive and autoreactive T cell repertoire. Splenocytes, axillary, inguinal, and popliteal lymph nodes were collected from B6 and TCRα+/− mice, incubated with APC- or PE-labeled allogeneic pMHC tetramers, enriched by magnetic selection using anti-APC or anti-PE beads, and singlet T cells were analyzed by flow cytometry. (A) Representative B6 mouse sample labeled with TFR/I-Ek is shown. (B) Reactivity of pMHC tetramer-labeled cells was assessed by sorting tetramer+ and tetramer− T cells by flow cytometry and measuring IFN-γ production following 18 h culture with CHO-Ek cells and 10 µM CD22 or 50 µM TFR peptide. IFN-γ production was measured by intracellular cytokine staining, with percent of IFN-γ+ cells indicated. Data are representative examples of 3 independent experiments. (C) The frequency of alloantigen-reactive T cells was calculated as the number of tetramer+ cells/105 T cells collected from each mouse. Data points are individual mice (n = 8–11) from 5 independent experiments, mean ± sem, Student’s t test. (D) The frequency of autoantigen- and cognate peptide-reactive T cells was calculated as the number of tetramer+ cells/105 T cells collected from each mouse. Data points are individual mice (n = 5–6) from 3 independent experiments, mean ± sem, Student’s t test.

Consistent with our hypothesis proposing that secondary TCRα rearrangements contribute specifically to the recognition of atypical ligands, TCRα+/− mice also had reduced frequencies of naive CD4+ T cells recognizing the autoantigen MOG38–49 presented by I-Ab (8.6±2.5 cells/105 T cells) as compared to B6 (28.3±9.3 cells/105 T cells) (Fig. 5D). Conversely, recognition of I-Ab tetramers presenting a foreign antigen, LCMV66–77 was comparable between B6 and TCRα+/− CD4+ T cells (4.7±2.3 vs. 5.8±2.5 cells/105 T cells, respectively). These results demonstrate specific contribution of secondary TCRα rearrangements to the alloreactive and autoreactive T cell repertoires.

Secondary TCRs increase early during in vivo alloreactivity

To evaluate the contribution of secondary TCRs during in vivo alloreactivity, we used a MHC-mismatched model of GVHD. B6.Ly5.1 and TCRα+/−.Thy1.1 T cells were mixed 1:1, pulsed with CFSE, and injected into lethally irradiated B6.K recipient mice (Fig. 6A). Expansion of transferred T cells was assessed by flow cytometry 24 h after transfer. While the 2 cell populations were transferred at equal numbers, recovery of TCRα+/− cells was decreased (ratio TCRα+/−/B6 T cells 0.6±0.1%) (Fig. 6B). Examination of CFSE dilution in the recovered cells demonstrated that TCRα+/−.Thy1.1 T cells were less likely to divide in the first 24 h post-transfer as compared to the secondary TCR-sufficient B6.Ly5.1 cells (Figs. 6C and D). The diminished response of TCRα+/− T cells early after allogeneic stimulation in vivo illustrates a functional consequence of the decreased frequency of alloreactive T cells in the absence of secondary TCRα rearrangements and underscores the potential importance of dual TCR T cells in driving early pathologic alloreactive responses.

FIGURE 6.

FIGURE 6

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Elimination of secondary TCRα chains reduces T cell in vivo alloresponse. Congenically-marked B6.Ly5.1 and TCRα+/−.Thy1.1 T cells (both H-2b) were mixed at a 1:1 ratio, pulsed with CFSE, and injected into lethally-irradiated MHC mismatched B6.K (H-2k) recipients. (A) Pre-injection cells were labeled for congenic markers to confirm ratio of cells. Representative example from 3 independent experiments. Transferred cells were analyzed at 24 h by flow cytometry of splenocytes. (B) Cells were labeled for congenic markers to examine ratio of TCRα+/− and B6 T cells pre-transplant and 24 h post-transfer. Results shown are mean ± sem of 12 recipient mice from 3 independent experiments, Student’s t test. (C) CFSE dilution was analyzed in congenically marked cells recovered 24 hours post-transfer. Representative example from 1 recipient mouse. (D) In vivo proliferative response to allogeneic stimulation was assessed in recovered cells by comparing the percentage of cells having undergone at least 1 cell division after 24 h. Data are composite of 12 recipient mice from 3 independent experiments, mean + sem, Student’s t test.

Elimination of secondary TCRs inhibits recognition of altered allogeneic peptide ligands

The contribution of secondary TCRs to the alloreactive and autoreactive T cell repertoire has multiple possible mechanistic explanations. One is that elimination of secondary TCRα rearrangements results in a decreased number of TCRs, which reduces the probability of recognizing a specific antigen. However, although secondary TCRs comprise less than 10% of the T cell repertoire, our data indicate that they encompass significantly more of the alloreactive and autoreactive repertoires (Figs. 5C and 5D, Refs. 22, 30) but do not affect recognition of conventional foreign antigens. This specific and disproportionate effect suggests that it is not simple stochastic addition of antigen specificities to the repertoire by secondary TCRs, but rather a unique property of secondary TCRs.

To investigate this, we compared functional responses of CD22/I-Ek tetramer binding T cells from B6 and TCRα+/− mice to altered peptide ligands (APLs). Tetramer positive cells from naive mice were sorted by flow cytometry and stimulated with the wild-type CD22 peptide or CD22 APLs containing single amino acid substitutions at TCR contact sites (P2 and P5). Responses were assessed by measurement of IFNγ production after 24 h culture. IFNγ production to either non-specific stimulation or wild-type CD22 peptide was similar between the 2 cell types (Fig. 7A). However, I-Ek/CD22-specific B6 T cell populations responded to APLs with mutations at either the P2 or P5 positions while I-Ek/CD22-specific TCRα+/− cells did not (Figs. 7A and 7B). These data suggest that an increased breadth in ligand recognition may underlie the specific atypical ligand recognition by dual TCR T cells at a population level.

FIGURE 7.

FIGURE 7

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Secondary TCRs enable reaction to closely-related allogeneic peptide antigens. CD22/I-Ek alloantigen-reactive T cells were collected from B6 and TCRα+/− mice by tetramer-labeling magnetic enrichment and flow cytometry sorting strategy and cultured with CHO-Ek cells for 24 h, either in the presence of the CD22 peptide, or CD22 peptide APLs P2A (H to A change at TCR contact P2) and P5S (G to S change at TCR contact P5). PMA (20 ng/ml) and ionomycin (1 µM) was used as a positive control. (A) T cell responses were measured by flow cytometry with intracellular labeling for IFN-γ production. Representative experiment shown. (B) Aggregate data from 3 independent experiments with APL response normalized to the response against wild-type CD22 peptide, mean ± sem, ANOVA.

Discussion

The existence of a minority of T cells expressing 2 TCRs in mice and humans has been recognized for some time (19), though the biological significance of these cells has not been well understood. Studies of transgenic TCR systems demonstrated that secondary TCRα recombination may enable TCRs with unwanted reactivities to escape thymic selection and emigrate to the periphery (24, 25). Thus, the potential benefit of simultaneous TCRα chain development may detrimentally alter the T cell repertoire. Here, we used mice genetically deficient for secondary TCRα rearrangements to define their role in thymocyte development and generation of the T cell repertoire.

Analysis of thymocyte development in TCRα+/− mice demonstrated diminished production of mature post-positive selection CD3high SP cells (Figs. 1 and 2), supporting the notion that simultaneous TCRα rearrangements occur to maximize the efficiency of mature T cell production from thymocytes successfully mediating β-selection. Our results demonstrate that secondary TCRα rearrangements are quantitatively important for thymopoiesis, though we cannot ascribe the relative importance of their effects on positive versus negative selection. This will require further investigation in systems where these questions can be separated.

The elimination of secondary TCRα rearrangements did not broadly affect the peripheral T cell repertoire, either in the number of T cells, or in the general use of TCRVα and TCRJα gene segments (Fig. 3). A focused comparison of TRAV14 TCR transcripts between T cells lacking secondary TCRα rearrangements and T cells with dual TCR expression similarly revealed no differences in the use of TCRJα gene segments or TCR properties such as CDR3 length and amino acid composition. However, dual TCR T cells had numerous high abundance CDR3 sequences that were absent among cells lacking secondary TCRα chains (Fig. 4). Statistical analysis indicated that the nonappearance of these sequences from TCRα+/− T cells was not simply sampling error but represented a specific absence from their repertoire. The reciprocal was not true, indicating that the presence of these unique TCRα sequences depended on secondary TCRα rearrangements. While it is not possible to exclude a stochastic process that excluded these TCR sequences from TCRα+/− T cells, we propose that this supports our hypothesis that secondary TCRα rearrangements contribute specificities to the peripheral repertoire that would otherwise be negatively selected in cells expressing a single TCR.

We propose that secondary TCRα rearrangements increase the T cell repertoire for atypical self and allogeneic pMHC ligands due to the effects of dual TCR expression on the stringency of thymic selection. Indeed, secondary TCRs contribute significantly and specifically in responses to peptides involved in autoimmunity and alloreactivity, but not cognate ligands (Fig. 5). The decreased response of CD4+ T cells from TCRα+/− mice to MOG/I-Ab tetramers was somewhat unexpected given a previous study reporting no impact on EAE disease outcome in mice lacking secondary TCRs (29). In that study, the effect of secondary TCRs was measured following immunization with antigen and adjuvant as compared to our interrogation of the naive T cell repertoire. Given that the NOD diabetes model, which does not rely on adjuvants, also indicated a role for dual TCR T cells (29, 30), it seems that secondary TCRs may be important contributors to autoimmunity under physiological conditions. Interestingly, a report indicating that a majority of regulatory T cells (Tregs) in humans express 2 TCRs (37) underscores the potential for dual TCR T cells to recognize self-pMHC with high affinity.

The potential for secondary TCRs to contribute to pathogenic responses has been demonstrated by our previous studies examining dual TCR T cells in mouse models and patients with GVHD (22, 28). We hypothesized that the disproportionate alloreactive responses we observed among dual TCR T cells resulted from increased frequencies of alloantigen-specific T cells among naive dual TCR T cells. This hypothesis is now supported by our data here demonstrating decreased numbers of alloreactive precursors and proliferative potential during the early phase of GVHD in the absence of secondary TCRs (Fig. 6). These data highlight the potentially important role for dual TCR T cells in GVHD and autoimmunity during the earliest phases of disease.

We hypothesize that secondary TCRs are important in alloreactivity and autoimmunity due to their uniquely relaxed constraints of thymic selection which may enable T cell crossreactivity. It has been demonstrated that negative selection is important for eliminating crossreactive TCRs (38), and thus secondary TCRs masked from negative selection may be more crossreactive. A potential contribution for secondary TCRs to crossreactivity is supported by the increased breadth of recognition of allopeptide APLs in wild-type T cells as compared to T lacking secondary TCRs (Fig. 7). Numerous studies have demonstrated that TCR interaction with the peptide is critical for response to allogeneic ligands (36, 39). However, there is uncertainty regarding how much flexibility the TCR has in recognizing multiple ligands and how this relates to alloreactivity (40, 41). While our data do not question the importance of peptide recognition in alloreactivity, it does indicate a potential mechanism for the increased propensity for dual TCR T cells to respond to specific pMHC ligands. The mechanistic basis for the unusual reactivity of dual TCR T cells warrants further investigation in a system where the parameters of thymic selection and ligand specificity can be correlated for individual TCRs.

In summary, we show that efficient thymocyte development requires simultaneous rearrangement of both TCRα loci. However, this comes at a cost, with resulting secondary TCRs having an increased ability to respond to self and allogeneic pMHC. Our results highlight a hitherto underappreciated role of dual TCR-expressing T cells in the development of the T cell repertoire and suggest that therapeutic strategies for combatting autoimmune diseases or transplant rejection and GVHD take into account the significant contribution of these uniquely powerful T cells.

Acknowledgements

The authors would like to thank the NIH Tetramer Core Facility for I-Ab tetramer production, S. Horvath for production of peptides and I-Ek tetramers, D. Kreamalmeyer for maintaining the mouse colony, and J.D. Bui for comments.

Abbreviations used in this article

APL

altered peptide ligand

B6

C57BL/6

CHO

Chinese hamster ovary

DN

double-negative

DP

double-positive

GVHD

graft versus host disease

LCMV

lymphocytic choriomeningitis virus

MOG

myelin oligodendrocyte glycoprotein

pMHC

peptide-MHC

Footnotes

Disclosures

The authors declare no financial conflicts of interests.

1

This work was supported by National Institutes of Health grants K08AI085039 (G.P.M.) and R01AI061173 (P.M.A.) and by the American Heart Association grant 10PRE3050039 (P.P.N.).

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