Development and function of autospecific dual TCR+ T lymphocytes

Robin K. Paterson1, Horst Bluethmann2, Pi-ou Tseng3, Anne Dunlap3 and Terri H. Finkel1,3,4

1 Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262, USA
2 Central Research Units, F. Hoffmann-LaRoche, 4002 Basel, Switzerland
3 Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA
4 Departments of Pediatrics and Biochemistry & Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO 80262, USA

Correspondence to: R. K. Paterson


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Recent studies have challenged the long held concept that each T lymphocyte expresses on its surface only a single, unique {alpha}ßTCR. Dual TCR+ T cells have been recognized, however, their origin and potential to escape screening for self-reactivity remain obscure. We now report the thymic generation of dual {alpha}ßTCR+ T cells in the H-2Db/H-Y-specific TCR transgenic (Tg) mouse. Dual TCR+ thymocytes were positively selected less efficiently than single TCR+ thymocytes, although a subset attained maturity. Importantly, when TCR Tg mice were bred onto a negatively selecting background, auto-specific cells survived central deletion and matured as CD4+ dual TCR+ cells. These cells were autoreactive when CD8 expression was restored. The existence of autospecific, dual TCR+ T cells may have implications for the maintenance of self tolerance.

Keywords: autoreactivity, CD4, CD8, IL-4, TCR, thymic development, thymic selection, thymocytes


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Early studies of T cell clones suggested that rearrangement of the TCR{alpha} gene was not restricted by allelic exclusion (1), as was thought to be the case for the TCRß gene (2,3). However, a phenomenon termed `phenotypic exclusion' was postulated to prevent dual TCR{alpha} surface expression, maintaining the one cell–one TCR status quo (1). More recently, cells bearing two surface TCR{alpha} have been cloned from human blood or murine lymph node and are estimated to comprise up to ~30% of mature T cells (4,5). Dual TCR{alpha}+ or TCRß+ T cells are also observed among mature T cells from TCR transgenic (Tg) mice (69). Recently, ~1% of human T cells have been recognized to co-express two surface TCRß (10,11).

Although documented to exist, the origin and function of the dual TCR+ cell as well as its potential for autoreactivity remain the subject of speculation (47,12). The present findings document the existence of a novel population of immature dual TCR+ thymocytes which are amenable to positive selection and export. These cells are subject to escape from both central and peripheral tolerance.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Mice and reagents
H-Y TCR Tg mice were bred for 10 generations on the C57Bl/6 (H-2Db) or DBA/2 (H-2Dd) backgrounds in our facility and studied at 1–4 months of age. Phycoerythrin (PE)-conjugated heat shock antigen (HSA), CD69, CD4, V{alpha}2, CD8{alpha} and CD8ß antibodies, and FITC-conjugated V{alpha}8 and V{alpha}2 mAb were obtained from PharMingen (San Diego, CA). In some experiments, V{alpha}2 mAb was purified from the B20.1 hybridoma (provided by B. Malissen) and conjugated to FITC. Biotinylated T3.70 (provided by H.-S. Teh) is specific for the V{alpha} region of the H-Y TCR.

Flow cytometry
Cells were suspended in BSS containing 2% FCS, 0.1% sodium azide and NMS or Fc block (PharMingen). Biotinylated antibodies were visualized with streptavidin-conjugated PE (Tago, Burlingame, CA) or streptavidin–perCP (Becton Dickinson, Mountain View, CA) for two- or three-color FACS respectively. Cells were analyzed using a Coulter Elite, Epics Profile or XL flow cytometer. Instrument alignments were checked with Coulter DNA-check beads. The calibration points were set by eye. Standard Coulter software was used to generate plots. Frequency data is expressed as a ratio of total viable lymphocytes after gating on forward and side scatter.

Cell culture
T cells were cultured with 500,000 antigen-presenting cells (APC) (prepared from spleens of male or female C57Bl/6 mice by treatment with mitomycin C) at a.3:1 ratio in complete IMDM (containing 10% FCS, 0.04 M sodium bicarbonate, 0.1 M HEPES buffer, 0.35% ß-mercaptoethanol, 10 mM sodium pyruvate, 0.02% L-glutamine, 0.01% penicillin, 0.01% streptomycin and 0.01% gentamycin) supplemented with IL-2 (plasmacytoma-conditioned media; provided by P. Marrack). CD8 cells were enriched by cell sorting of lymph node populations stained with a CD8{alpha}-specific mAb. For proliferation experiments, wells were pulsed with 1 µCi of [3H]thymidine (ICN, Irvine, CA) for 18 h before scintillation counting.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Development of dual TCR+ thymocytes
Most studies of dual TCR+ expression have been limited to mature populations of cells. That the TCR{alpha} chain can somatically mutate (13) and that RAG is expressed extrathymically (14,15) raise the possibility that TCR duality might arise within peripheral lymphoid tissues. Indeed, it was recently proposed that the dual TCR+ phenotype is excluded from the mature thymic compartment via post-translational mechanisms (16). Thus, the origin of the dual TCR+ T cell remains to be clarified.

We used the anti-H-Y TCR Tg mouse (17) to study the development of dual TCR+ thymocytes of defined specificity. When bred onto a non-selecting (NS; H-2Dd) background, thymocytes from anti-H-Y mice are forced to undergo extensive rearrangement of their endogenous TCR{alpha} genes to produce a self-compatible TCR. Mature thymocytes often lack the Tg TCR{alpha} from their surface (18), which might be the result of competition with an endogenous TCR{alpha} for pairing with the stably expressed Tg TCRß. This predicts the occurrence, at least transiently, of immature thymocytes co-expressing two distinct TCR{alpha}. Indeed, dual TCR{alpha}+ cells were identified in NS thymus as V{alpha}Tg+V{alpha}2+ (Fig. 1aGo) or V{alpha}Tg+V{alpha}8+ cells (not shown). A subpopulation of dual TCR+ thymocytes appeared to be mature based on their relatively high density of V{alpha}2. Maturity was confirmed by assessment of HSA, which is dull on mature cells (19), and CD69, which increases following positive selection (20). Both HSAdull (Fig. 1bGo) and CD69+ (Fig. 1cGo) cells were observed among V{alpha}2+V{alpha}Tg+ thymocytes, consistent with their intrathymic maturation. We have reported similar findings for dual TCR{alpha}+ thymocytes in normal mice (Paterson et al., manuscript submitted). Thus, dual TCR+ T cells may arise as the consequence of normal T cell development.




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Fig. 1. Thymic differentiation of dual TCR+ cells. Thymus and lymph node T cells from non-selecting (H-2Dd) anti-H-Y mice were analyzed flow cytometrically for TCR expression. (a) Dual TCR+ thymocytes were co-stained with V{alpha}Tg and V{alpha}2 mAb (top). Irrelevant IgG2a mAb (middle) and staining of DBA/2 cells (bottom) served as non-specific controls. (b and c) V{alpha}Tg+V{alpha}2+ thymocytes were analyzed for HSA (b) and CD69 (c) expression. (d) Dual TCR+ lymph node cells were identified by co-staining with V{alpha}Tg and V{alpha}2 mAb (top). Irrelevant IgG2a mAb (middle) and staining of DBA/2 cells (bottom) served as controls. Data are representative of at least five experiments.

 
Although maturation of dual TCR+ thymocytes could be demonstrated, the process indeed appeared to be inefficient. V{alpha}2high cells represented 38.0 ± 1.1% of single TCR+ (V{alpha}TgV{alpha}2+), and 18.3 ± 3.0% of dual TCR+ (V{alpha}Tg+V{alpha}2+) thymocytes (P < 0.001). Using HSA as the criterion for maturity, 26.7 ± 4.3% of dual TCR+, compared with 42.4 ± 4.3% of single TCR+, thymocytes were HSAdull (P < 0.02). CD69 positivity was also lower among dual TCR+ versus single TCR+ thymocytes in two experiments (30.8 ± 7.3 versus 43.2 ± 0.8%).

These findings are consistent with an avidity model of thymic selection (2122). In NS mice, the Tg TCR cannot signal and therefore its presence limits the signaling capacity of a co-expressed, endogenous TCR, which must compete for signaling molecules. Among dual TCR+ thymocytes, only those with high V{alpha}2/V{alpha}Tg surface ratios (i.e. relatively high occupancy of the TCRß–CD3 complex by the V{alpha}2+ receptor) can achieve positive selection. Inefficient signaling in dual TCR+ cells might represent a physiologic mechanism limiting their development or function. However, a subset of developing dual TCR+ thymocytes is exported and appears in the periphery as mature cells (Fig. 1dGo).

Autospecificity and autoreactivity among dual TCR+ T cells
On theoretical grounds, the dual TCR+ thymocyte can be envisioned to escape central tolerance. Figure 2Go illustrates two models which predict the maturation of autospecific dual TCR+ cells. The antagonist-TCR model (Fig. 2AGo) predicts that an autospecific thymocyte might be effectively `rescued' by the acquisition of a second TCR which disrupts negative signaling through competition for signaling molecules. In the present study, we focus mainly on the utilization of the so-called stochastic pathway of T cell development as a mechanism by which potentially autoreactive dual TCR+ T cells might be generated (Fig. 2BGo).



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Fig. 2. Intrathymic development of auto-specific dual TCR+ T cells. (A) In the antagonist-TCR model, expression of a self-incompatible or an autospecific (as depicted in the figure) antigen receptor might put pressure on a developing thymocyte to produce a new TCR{alpha}. The new TCR{alpha} competes for TCRß and for downstream signaling molecules, and thus compromises the negative signal mediated by the first {alpha}ßTCR. Clonal deletion is prevented (or aborted; see ref. 35, pertaining to the transiently reversible nature of clonal deletion among B cells) and the immature thymocyte may then become positively selected. The new {alpha}ßTCR would have to have high affinity for the self-MHC complex to mediate positive selection under the compromised signaling conditions. Theoretically, an increase in the relative amount of either of the two co-expressed {alpha}ßTCRs could qualitatively alter the outcome of an antigen-specific signaling event in the mature T cell, possibly leading to autoreactivity. (B) Stochastic lineage commitment allows the generation of auto-specific dual TCR+ T cells with an unscreened TCR. If the auto-specific TCR is MHC class I restricted, as depicted, an unselected thymocyte could escape deletion by committing to the CD4 lineage. Since unselected thymocytes stably express RAG (3) even after lineage commitment (27), a new class II-restricted TCR could be generated and could mediate positive selection in conjunction with CD4. The mature T cell would carry the class I-restricted, auto-specific TCR as a `bystander'.

 
It was predicted that dual TCR+ thymocytes might escape central tolerance by mechanisms involving loss of the appropriate co-receptor required for TCR signaling (see Fig. 2BGo). Were autospecific T cells to be exported from the thymus, they might jeopardize peripheral tolerance when a response to foreign antigen releases an inappropriate anti-self response (6,10). In support of this theory, recent studies have emphasized the reduced threshold requirements for self-antigen recognition by T cells that become activated inappropriately through immunization or molecular mimicry (reviewed in 23). On the other hand, the potential for autoreactivity among dual TCR+ cells remains controversial, since it has been argued that TCR duality occurs only when one of the TCR is self-MHC incompatible and thus unable to be triggered under physiologic circumstances (7).

The anti-H-Y male mouse [deletor (D)] provides an in vivo model for studying the fate of autospecific TCR (24). In the D thymus, dual TCR+ cells might have a survival advantage over single Tg TCR+ cells, which are clonally deleted. As predicted by models of stochastic thymic differentiation (25–29 and see Fig. 2BGo), a population of mature CD4+ dual TCR+ cells was found to exist in D mice. Figure 3Go(A) illustrates the enrichment of an endogenous V{alpha}, V{alpha}2, among Tg TCR+ T cells of the CD4 lineage, as compared with the CD8 lineage. V{alpha}2 expression among CD4+ Tg TCR+ cells from D mice averaged 11.1 ± .6% (n = 3) and averaged 2.0 ± .2% (n = 4) among CD8+TgTCR+ cells.





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Fig. 3. Autoreactivity of dual TCR+ T cells. (A) Lymph node T cells from a 3-month-old male (D; H-2Db) anti-H-Y mice (D mouse) were analyzed by FACS for expression of the Tg TCR{alpha}, the CD8 or CD4 co-receptor and for endogenous V{alpha}2. CD8dullTg TCR+ cells (top left) may avoid central deletion (24). CD8dullTg TCR+ cells contained very few dual TCR+ cells (middle left). Irrelevant IgG2a mAb was used as a negative control (bottom left). CD4+V{alpha}Tg+ cells (top right) were enriched for dual TCR+ cells (middle right). The data are representative of at least five similar experiments measuring V{alpha}2 and/or V{alpha}8 (not shown) expression among Tg TCR+ cells of the CD4 and CD8 lineages. (B) T cells from D mice were depleted of CD8+ cells by cell sorting. CD8-depleted cells were >95% V{alpha}Tg+ and contaminating CD8+ cells averaged 0.11 ± 0.05% (mean ± SEM). CD8-depleted cells were cultured in the presence or absence of IL-4 (400 U/ml) and either female (H-Y) or male (H-Y+) H-2Db APC, and assessed for [3H]thymidine uptake after 3 days in culture. The data are expressed as mean ± SEM c.p.m. from triplicate wells (P < 0.01) and are representative of two separate experiments. As a control, CD8+-enriched T cells from an over-selecting anti-H-Y mouse (female H-2Db) were examined in parallel. [3H]Thymidine incorporation was 47,382 ± 3941 c.p.m. in the presence of male APC (H-Y+), and 36,586 ± 1673 c.p.m. in the presence of male APC and IL-4. (C) T cells from D mice were depleted of CD8+ cells, and analyzed for antigen receptor and co-receptor expression following exposure to H-Y antigen in culture. (A) A representative analysis of the post-sort purity of the CD8-depleted population is shown. Residual CD8+ cells were 0.005% and are plotted as forward scatter versus CD8. (B) CD8-depleted cells were cultured with either female (H-Y) (left panels) or male (H-Y+) (right panels) APC (H-2Db). Cultures were treated with IL-4 (400 U/ml) or left untreated (Ø), as indicated. Three-color FACS analysis was performed to determine the proportion of CD8+ cells within the CD4+V{alpha}Tg+ population. An irrelevant isotype-matched mAb was studied in parallel as a control for the measurement of CD8 (bottom right). The observed increase in CD8 was specific since the expression of CD4 among V{alpha}Tg+ cells did not increase: CD4 expression among V{alpha}Tg+ cells was 5.6 ± 0.8% in untreated cultures and 3.8 ± 0.9% in IL-4-treated cultures. Data are representative of five experiments performed (see Table 1Go).

 
CD4+ dual TCR+ cells might be predisposed to autoreactivity if CD8 were reinduced post-thymically. De novo expression of CD8 can be induced among CD4+ mature cells by IL-4 (30). Thus, the dual TCR+-enriched population of CD4+V{alpha}Tg+ cells that we observed might become autoreactive if CD8 were restored by IL-4. D T cells were depleted of CD8+ cells and cultured for 3.5 days with syngeneic spleen cells from female mice (H-Y) in the presence or absence of IL-4 (see Table 1Go). Although CD8 was undetectable among CD4+V{alpha}Tg+ cells post-depletion, some spontaneous reappearance of dull levels of CD8 (~1/10 the surface density of CD8 on cells from S or non-Tg mice) occurred after 3.5 days in culture. IL-4 exposure led to a further increase in the percentage and density of CD8; the percentage of CD8+ cells within the CD4+V{alpha}Tg+ population increased from 12.9 ± 2.1 to 23.5 ± 4.2% (paired Student's t-test; P = 0.0002) and the fluorescence intensity increased from 2.2 ± 0.13 to 3.1 ± 0.21 (P = 0.0001). These data are consistent with other demonstrations that IL-4 induces CD8 on CD4+ cells (30). We cannot, however, formally rule out the possibility of a preferential expansion of the CD8+CD4+V{alpha}Tg+ cells contaminating the 3.5 day cultures.


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Table 1. IL-4 induction of CD8 on autospecific CD4+V{alpha}Tg+ T cells
 
The autoreactive potential of the IL-4-induced population of CD8+CD4+V{alpha}Tg+ cells was assessed by measuring [3H]thymidine uptake in cultures exposed to the male H-Y antigen. As shown in Fig. 3Go(B), CD8+CD4+V{alpha}Tg+ cells did not proliferate in response to female APC, even when IL-4 was present. In the absence of IL-4, male APC did not drive the proliferation of CD8+CD4+V{alpha}Tg+ cells, presumably due to the low levels at which they expressed CD8. This is consistent with other reports that CD8dullV{alpha}Tg+ cells are antigen unresponsive (31). However, [3H]thymidine incorporation occurred in the presence of combined IL-4 and male APC, suggesting that the induced CD8+CD4+V{alpha}Tg+ population was responsible for mediating the auto-antigen-specific response. Indeed, we observed a relative expansion of CD8+CD4+V{alpha}Tg+ cells in IL-4-treated cultures that were exposed to the male antigen (Fig. 3cGo and Table 1Go). This IL-4-induced responsiveness to H-Y probably reflects both the increased percentage of CD8+CD4+V{alpha}Tg+ responding cells as well as their increased surface density of CD8. In fact, others have demonstrated the potential for autoreactivity among H-Y-specific D T cells that were rendered CD8high through the expression of a transgene (32).

In summary, this work demonstrates that dual TCR+ cells arise intrathymically and can circumvent central tolerance, perhaps by utilizing a stochastic pathway of differentiation. Among such cells, autoreactivity may be regulated by cytokine exposure in the periphery. Stochastic differentiation might be a minor pathway of thymic development in the normal host (29). However, it could assume importance under physiologic or pathologic conditions that promote or mimic thymocyte lineage commitment. Pathogens that down-regulate CD4, and which are linked to autoimmunity, include HIV (33) and Epstein–Barr virus (34).


    Acknowledgments
 
The authors would like to thank H.-S. Teh for providing T3.70 antibody, S. Sobus and W. Townend for assistance with flow cytometry, and D. Nemazee for critical review of the manuscript. This work was supported by National Institutes of Health grants R01 AI30575, P01 AI22295 (T. H. F.), the Arthritis Foundation (R. P. and T. H. F.), the Juvenile Diabetes Foundation (R. P.), the University of Colorado Health Sciences Center Cancer (T. H. F.), the Bender Foundation, and the Eleanor and Michael Stobin Trust (T. H. F.).


    Abbreviations
 
APCantigen-presenting cell
Ddeletor
HSAheat stable antigen
NSnon-selecting
PEphycoerythrin
Sover-selecting
Tgtransgenic

    Notes
 
Transmitting editor: L. Glimcher

Received 27 April 1998, accepted 6 October 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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