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
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Abstract
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Recent studies have challenged the long held concept that each T lymphocyte expresses on its surface only a single, unique
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
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
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Introduction
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Early studies of T cell clones suggested that rearrangement of the TCR
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
surface expression, maintaining the one cellone TCR status quo (1). More recently, cells bearing two surface TCR
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
+ 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.
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Methods
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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 14 months of age. Phycoerythrin (PE)-conjugated heat shock antigen (HSA), CD69, CD4, V
2, CD8
and CD8ß antibodies, and FITC-conjugated V
8 and V
2 mAb were obtained from PharMingen (San Diego, CA). In some experiments, V
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
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 streptavidinperCP (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
-specific mAb. For proliferation experiments, wells were pulsed with 1 µCi of [3H]thymidine (ICN, Irvine, CA) for 18 h before scintillation counting.
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Results and discussion
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Development of dual TCR+ thymocytes
Most studies of dual TCR+ expression have been limited to mature populations of cells. That the TCR
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
genes to produce a self-compatible TCR. Mature thymocytes often lack the Tg TCR
from their surface (18), which might be the result of competition with an endogenous TCR
for pairing with the stably expressed Tg TCRß. This predicts the occurrence, at least transiently, of immature thymocytes co-expressing two distinct TCR
. Indeed, dual TCR
+ cells were identified in NS thymus as V
Tg+V
2+ (Fig. 1a
) or V
Tg+V
8+ cells (not shown). A subpopulation of dual TCR+ thymocytes appeared to be mature based on their relatively high density of V
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. 1b
) and CD69+ (Fig. 1c
) cells were observed among V
2+V
Tg+ thymocytes, consistent with their intrathymic maturation. We have reported similar findings for dual TCR
+ thymocytes in normal mice (Paterson et al., manuscript submitted). Thus, dual TCR+ T cells may arise as the consequence of normal T cell development.
Although maturation of dual TCR+ thymocytes could be demonstrated, the process indeed appeared to be inefficient. V
2high cells represented 38.0 ± 1.1% of single TCR+ (V
TgV
2+), and 18.3 ± 3.0% of dual TCR+ (V
Tg+V
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
2/V
Tg surface ratios (i.e. relatively high occupancy of the TCRßCD3 complex by the V
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. 1d
).
Autospecificity and autoreactivity among dual TCR+ T cells
On theoretical grounds, the dual TCR+ thymocyte can be envisioned to escape central tolerance. Figure 2
illustrates two models which predict the maturation of autospecific dual TCR+ cells. The antagonist-TCR model (Fig. 2A
) 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. 2B
).
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. 2B
). 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 (2529 and see Fig. 2B
), a population of mature CD4+ dual TCR+ cells was found to exist in D mice. Figure 3
(A) illustrates the enrichment of an endogenous V
, V
2, among Tg TCR+ T cells of the CD4 lineage, as compared with the CD8 lineage. V
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.
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
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 1
). Although CD8 was undetectable among CD4+V
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
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
Tg+ cells contaminating the 3.5 day cultures.
The autoreactive potential of the IL-4-induced population of CD8+CD4+V
Tg+ cells was assessed by measuring [3H]thymidine uptake in cultures exposed to the male H-Y antigen. As shown in Fig. 3
(B), CD8+CD4+V
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
Tg+ cells, presumably due to the low levels at which they expressed CD8. This is consistent with other reports that CD8dullV
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
Tg+ population was responsible for mediating the auto-antigen-specific response. Indeed, we observed a relative expansion of CD8+CD4+V
Tg+ cells in IL-4-treated cultures that were exposed to the male antigen (Fig. 3c
and Table 1
). This IL-4-induced responsiveness to H-Y probably reflects both the increased percentage of CD8+CD4+V
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 EpsteinBarr virus (34).
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Acknowledgments
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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.).
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Abbreviations
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APC | antigen-presenting cell |
D | deletor |
HSA | heat stable antigen |
NS | non-selecting |
PE | phycoerythrin |
S | over-selecting |
Tg | transgenic |
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Notes
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Transmitting editor: L. Glimcher
Received 27 April 1998,
accepted 6 October 1998.
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References
|
---|
-
Malissen, M., Trucy, J., Jouvin-Marche, E., Cazenave, P.-A., Scollary, R. and Malissen, B. 1992. Regulation of TCR
and ß gene allelic exclusion during T-cell development. Immunol. Today 13:315.[ISI][Medline]
-
Uematsu, Y., Ryser, S., Dembic, Z., Borgulya, P., Krimpenfort, P., Berns, A., von Boehmer, H. and Steinmetz, M. 1988. In transgenic mice the introduced functional T cell receptor ß chain prevents expression of endogenous ß-genes. Cell 52:831.[ISI][Medline]
-
Borgulya, P., Kishi, H., Uematsu, Y. and von Boehmer, H. 1992. Exclusion and inclusion of
and ß T cell receptor alleles. Cell 69:529.[ISI][Medline]
-
Padovan, E., Casorati, G., Dellabona, P., Meyer, S., Brockhaus, M. and Lanzavecchia, A. 1993. Expression of two T cell receptor chains: dual receptor T cells. Science 262:422.[ISI][Medline]
-
Heath, W. R., Carbone, F. R., Bertolino, P., Kelly, J. Cose, S. and Miller, J. F. A. P. 1995. Expression of two T cell receptor
chains on the surface of normal murine T cells. Eur. J. Immunol. 25:1617.[ISI][Medline]
-
Heath, W. R. and Miller, J. F. A. P. 1993. Expression of two alpha chains on the surface of T cells in T cell receptor transgenic mice. J. Exp. Med. 178:1807.[Abstract]
-
Hardardottir, F., Baron, J. L. and Janeway, C. 1995. T cells with two functional antigen-specific receptors. Proc. Natl Acad. Sci. USA 92:354.[Abstract]
-
Munthe, L. A., Sollien, A., Dembic, Z. and Bogen, B. 1995. Preferential positive selection of T lymphocytes which express two different TCR
chains, an endogenous and a transgenic. Scand. J. Immunol. 42:651.[ISI][Medline]
-
Munthe, L. A., Blichfeldt, E., Sollien, A., Dembic, Z. and Bogen, B. 1996. T cells with two TCR ß chains and reactivity to both MHC/idiotypic peptide and superantigen. Cell. Immunol. 170:283.[ISI][Medline]
-
Davodeau, F., Peyrat, M.-A., Romagne, F., Necker, A., Hallet, M.-M., Vie, H. and Bonneville, M. 1995. Dual T cell receptor ß chain expression on human T lymphocytes. J. Exp. Med. 181:1391.[Abstract]
-
Padovan, E., Giachino, C., Cella, M., Valitutti, S., Acuto, O. and Lanzavecchia, A. 1995. Normal T lymphocytes can express two different T cell receptor ß chains: implications for the mechanism of allelic exclusion. J. Exp. Med. 181:1587.[Abstract]
-
Mason, D. 1994. Allelic exclusion of
chains in TCRs. Int. Immunol. 6:881.[Abstract]
-
Zheng, B., Xue, W. and Kelsoe, G. 1995. Locus-specific somatic hypermutation in germinal centre T cells. Nature 372:556.[ISI]
-
Guy Grand, D., Vanden Broecke, C., Briottet, C., Malassis-Seris, M., Selz, F. and Vassalli, P. 1992. Different expression of the recombination activity gene RAG-1 in various populations of thymocytes, peripheral T cells and gut thymus-independent intraepithelial lymphocytes suggests two pathways of T cell receptor rearrangement. Eur. J. Immunol. 22:505.[ISI][Medline]
-
Collins, C., Norris, S., McEntee, G., Traynor, O., Bruno, L., von Boehmer, H., Hegarty, J. and O'Farrelly, C. 1996. RAG1, RAG2 and pre-T cell receptor
chain expression by adult human hepatic T cells: evidence for extrathymic T cell maturation. Eur. J. Immunol. 26:3114.[ISI][Medline]
-
Alam, S. M., Crispe, I. N. and Gascoigne, N. R. 1995. Allelic exclusion of mouse T cell receptor
chains occurs at the time of thymocyte TCR up-regulation. Immunity 3:449.[ISI][Medline]
-
Blüthmann, H., Kisielow, P., Uematsu, Y., Malissen, M., Krimpenfort, P., Berns, A., von Boehmer, H. and Steinmetz, M. 1988. T-cell-specific deletion of T-cell transgenes allows functional rearrangement of endogenous
- and ß-genes. Nature 334:156.[ISI][Medline]
-
Teh, H.-S., Kisielow, P., Scott, B., Kishi, H., Uematsu, Y., Blüthmann, H. and von Boehmer, H. 1988. Thymic major histocompatibility complex antigens and the
ß T-cell receptor determine the CD4/CD8 phenotype of T cells. Nature 335:293.[ISI]
-
Wilson, A., Day, L. M., Scollay, R. and Shortman, K. 1988. Subpopulations of mature murine thymocytes: properties of CD4CD8+ and CD4+CD8 thymocytes lacking the heat-stable antigen. Cell. Immunol. 117:312.[ISI][Medline]
-
Swat, W., Dessing, M., von Boehmer, H. and Kisielow, P. 1993. CD69 expression during selection and maturation of CD4+8+ thymocytes. Eur. J. Immunol. 23:739.[ISI][Medline]
-
Ashton-Rickardt, P. G. and Tonegawa, S. 1994. A differential-avidity model for T-cell selection. Immunol Today 15:362.[ISI][Medline]
-
Hogquist, K. A., Jameson, S. C., Heath, W. R., Howard, J. L., Bevan, M. J. and Carbone, F. R. 1994. T cell receptor antagonist peptides induce positive selection. Cell 76:17.[ISI][Medline]
-
Kruisbeek, A. M. and Amsen, D. 1996. Mechanisms underlying T-cell tolerance. Curr. Opin. Immunol. 8: 233.[ISI][Medline]
-
Kisielow, P., Blüthmann H., Staerz, U. D., Steinmetz, M. and von Boehmer, H. 1988. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature 333:742.[ISI][Medline]
-
Davis, C. B., Killeen, N., Casey Crooks, M. E., Raulet, C. and Littman, D. R. 1993. Evidence for a stochastic mechanisms in the differentiation of mature subsets of T lymphocytes. Cell 73:237.[ISI][Medline]
-
van Meerwijk, J. P. M. and Germain, R. 1993. Development of mature CD8+ thymocytes: selection rather than instruction? Science 261:911.[ISI][Medline]
-
Chan, S. H., Cosgrove, C., Waltzinger, C., Benoist, C. and Mathis, D. 1993. Another view of the selective model of thymocyte selection. Cell 73:225.[ISI][Medline]
-
Kaufman Paterson, R., Burkly, L. C., Kurahara, D. K., Dunlap, A., Flavell, R. A. and Finkel, T. H. 1994. Thymic development in human CD4 transgenic mice. Positive selection occurs after commitment to the CD8 lineage. J. Immunol. 153:3491.[Abstract/Free Full Text]
-
Robey, E., Itano, A., Fanslow, W. C. and Fowlkes, B. J. 1994. Constitutive CD8 expression allows inefficient maturation of CD4+ helper T cells in class II major histocompatibility complex mutant mice. J. Exp. Med. 179:1997.[Abstract]
-
Paliard, X., de Waal Malefij, R., de Vries, J. E. and Spits, H. 1988. Interleukin-4 mediates CD8 induction on human CD4+ T cell clones. Nature 335:642.[ISI][Medline]
-
von Boehmer, H., Kirberg, J. and Rocha, B. 1991. An unusual lineage of
/ß T cells that contains autoreactive cells. J. Exp. Med. 174:1001.[Abstract]
-
Robey, E. A., Ramsdell, F., Gordon, J. W., Mamalaki, C., Kioussis, D., Youn, H. J., Gottlieb, P. D., Axel, R. and Folkes, B. J. 1992. A self-reactive T cell population that is not subject to negative selection. Int. Immunol. 4:969.[Abstract]
-
Levy, J. A. 1993. Pathogenesis of human immunodeficiency virus infection. Microbiol. Rev. 57:183.[Abstract]
-
Kaufman Paterson, R. L., Kelleher, C., Amenkonah, T., Streib, J. E., Xu, J. W., Jones, J. F. and Gelfand, E. W. 1995. Model of EpsteinBarr virus infection of human thymocytes: Expression of viral genome and impact on cellular receptor expression in the T-lymphoblastoid cell line, HPB-ALL. Blood 85:456.[Abstract/Free Full Text]
-
Hartley, S. B., Cook, M. P., Fulcher, D. A., Harris, A. W., Cory, S., Basten, A. and Goodnow, C. C. 1993. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell 72:325.[ISI][Medline]