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CD8⁺ T Lymphocytes in Double TCR Transgenic Mice. I. TCR Expression and Thymus Selection in the Absence or in the Presence of Self-Antigen¹

Lymphocyte Population Biology Unit, Unité de Recherche Associée, Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France

	Abstract

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We derived Rag2-deficient mice bearing two rearranged TCR transgenes, one specific for the HY male Ag and the second specific for the gp33-41 peptide of lymphocytic choriomeningitis virus, both restricted to the MHC H-2D^b class I molecule. We found that, in female double transgenic (DTg) mice, most CD8 T cells express only the TCR chain from the aHY transgene. By comparing the mRNA species for both -chains, we observed that in T cells from DTg mice the aHY TCR chain transcripts are abundant, whereas the anti-lymphocytic choriomeningitis virus TCR chain transcripts are rare. In contrast to TCR chain expression, most of the T cells from DTg mice express two TCR chains. We examined the thymus selection of the dual-receptor CD8 T cells in the presence of self-Ag. We found that the presence of a second TCR chain allows a significant number of CD8 T cells expressing a self-reactive receptor to escape central deletion and migrate to the peripheral pools of male mice. Differences in TCR and coreceptor expression between female and male MoaHY and DTg mice suggest that peripheral T cell survival requires an optimal level of signaling, which implies a process of "adaptation" of lymphocyte populations to the host environment.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antigen receptors of B and T lymphocytes are subjected to allelic exclusion (1). In general, for T cells the TCR chain gene segments start to rearrange on one chromosome and continue on the second chromosome only when the first attempt resulted in a nonproductive gene (2). Productive rearrangement results in the expression of a TCR chain, which alone (3) or in association with the pre-TCR (pT)³ chain (4, 5) during the early phases of T cell development is essential for TCR chain allelic exclusion. Allelic exclusion of the TCR, however, is never fail proof. Different studies indicate that 1% of the mature T cells contain two productive TCR alleles (6, 7). Differential kinetics of TCR chain expression, however, may still contribute to inhibition of the expression of one of the two productive rearrangements. Thus, TCR chains may compete for limited amounts of pT (5) and/or the CD3 complex, and in this case it is possible that the first -chain expressed preempts surface expression of a second -chain. At the protein level, earlier expression of one TCR chain may also change the efficiency of the association and expression of distinct TCR heterodimers (8). In contrast to the TCR locus, TCR chain rearrangements proceed simultaneously in both chromosomes (9) and 30% of the human T cells express two TCR chains (10, 11, 12). In normal mice, 30% of T cells show two rearranged -chains (10) and a variable fraction express two -chains, suggesting that the likelihood of generation of T cells with dual specificity is elevated.

Potentially the allelically included T cells could play an important role in autoimmunity. In dual-receptor cells, a second self-reactive TCR could bypass negative selection in the thymus, by virtue of its lower expression. If a non-self Ag stimulates the naive dual-receptor T cells, these T cells once activated would acquire a lower threshold of activation, respond to self-peptide/MHC, and cause disease. Several in vivo experimental models have examined this possibility. Studies on the susceptibility to spontaneous autoimmune diseases of nonobese diabetic mice hemizygous for the TCR locus did not support the role of dual T cells in autoimmune diabetes (13). However, in different mouse models, co-expression of two TCR transgenes rescues self-reactive T cells from tolerance induction and allows their exit into the periphery (14, 15). These cells could be stimulated in vitro to anti-self effector functions via the second receptor (14), and their in vivo presence was correlated to the induction of autoimmune diabetes when the relevant Ag was expressed by the pancreatic tissue (15).

The role of dual-receptor T cells in autoimmune diseases, however, is strictly dependent in their ability to maintain surface expression of two functional receptors and to be positively selected in the thymus. Because thymus positive selection requires receptor engagement by MHC molecules (16), it is likely that, in the presence of two receptors bearing a different specificity, each may impose some constraints to the selection of dual-receptor T cells. Indeed, it was recently shown that reduced specific receptor density results in reduced thymus positive selection of dual-receptor T cells (17). To examine these questions, we studied thymus T cell selection and TCR expression in Rag2-deficient animals bearing two complete rearranged TCR transgenes. One receptor is specific for the HY male Ag (18, 19) and the second is specific for the gp33-41 peptide of lymphocytic choriomeningitis virus (LCMV) (20), both restricted to the same MHC H-2D^b class I molecule.

	Materials and Methods

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Mice

C57BL/6 mice transgenic (Tg) for the anti-HY TCR (VT3.70;V8.2) (18, 19), or the P14 TCR (V2;V8.1) (20), were crossed into a Rag2-deficient background (21). The mice obtained (MoaHY and MoP14) were intercrossed to give rise to MoaHY.MoP14 double-transgenic (DTg) mice. All these strains were maintained in specific pathogen-free isolators at the Centre de Développement des Techniques Avancées pour l’Expérimentation Animale-Centre National de la Recherche Scientifique (Orléans, France). B6.CD3^-/- mice (22) were from the Centre de Développement des Techniques Avancées pour l’Expérimentation Animale-Centre National de la Recherche Scientifique. Double CD3^-/-H-2D^b-/- mice (23) were maintained specific pathogen-free in our animal facilities at the Pasteur Institute and at the Centre de Développement des Techniques Avancées pour l’Expérimentation Animale.

Flow cytometry

The following mAbs were used: anti-CD8 (53-6.7), anti-V2 (B20.1), pan-TCR chain (H57-597), anti-V8.1/2 (MR5-2), anti-CD3 (145-2C11), anti-CD4 (L3T4/RM4-5), anti-CD69 (H1.2F3), anti-CD25 (PC61), and anti-CD24/HSA (M1/69) from BD PharMingen (San Diego, CA) and anti-CD44 (IM781) and anti-CD62L (MEL14) from Caltag Laboratories (San Francisco, CA). The anti-VT3.70 and the F23.2 anti-V8.2 were from B. Rocha (Institut National de la Santé et de la Recherche Médicale U345, Institut Necker, Paris, France). Cell-surface staining was performed with the appropriate combinations of FITC, PE, TRI-Color (Caltag Laboratories), PerCP (BD Biosciences, San Jose, CA), Biotin, and APC-coupled Abs. Biotin-coupled Abs were revealed by APC-, TRI-Color-, or PerCP-coupled streptavidin. Dead cells were excluded by light-scattering gating. All analyses were performed with a FACScalibur (BD Biosciences) interfaced to Macintosh CellQuest software (Apple Computer, Cupertino, CA). For TCR co-internalization analysis, spleen cells from Tg mice were incubated (37°C, 5% CO₂) in NaN₃-free balanced salt solution for different periods of time in the presence of Abs specific for either one of the Tg TCR or TCR chains. At the end of the incubation periods, cells were washed in 0.2% NaN₃ and kept in the dark at 4°C. Next, the cells were stained with fluorescent Abs directed against TCR chains and were analyzed. Results are expressed as a percentage of the geometric mean of fluorescence intensity (MFI), compared with control cells incubated in absence of the first capping Ab.

Immunoscope

We followed the Immunoscope analysis previously described (24). In brief, total RNA extracted from lymph node (LN) CD8 T cells of the different Tg mice by TRIzol (Life Technologies, Mannheim, Germany) was treated with DNase I (Boehringer Mannheim, Mannheim, Germany). From each RNA sample, cDNA was prepared using (dT)_12–18 and Superscript II reverse transcriptase (Life Technologies). The cDNAs for the different TCR chains were amplified by PCR (40 cycles) using primers specific for the TCRV8 family (5'-TCC CTG ATG GRT ACA AGG CC-3') and TCR-C2 (5'-GCC AGA AGG TAG CAG AGA CCC-3'). The resulting PCR products were used for a run-off reaction (two cycles) using a nested fluorescent primer specific for the TCR-C2 segment (5'-CCT GGG TGG AGT CAC ATT TCT C-3'). Finally, the run-off products were resolved on an automatic 373A DNA sequencer (Applied Biosystems, Foster City, CA), and the results were analyzed using the Immunoscope software (24).

Bone marrow (BM) chimeras

Host mice were lethally irradiated (900 rad) in a ¹³⁷Ce source and injected i.v. with 2–4 x 10⁶ BM cells. Thymus, spleen, inguinal, and mesenteric LN cell suspensions were prepared, and the number and phenotype of the cells were evaluated.

In vitro proliferation assays

Spleen cells from Tg mice were incubated in 96-well plates (10⁵ cells/well) at 37°C, 5% CO₂, in a final volume of 200 µl in complete RPMI 1640 medium supplemented with 10% FCS (Boehringer Mannheim). Cells were stimulated with Con A (Sigma-Aldrich, St. Louis, MO), anti-CD3 (BD PharMingen), or the TCR Tg specific peptides gp33-41 (25) (KAVYNFATM) and Smcy-3 peptides (26) (KCSRNRQYL) purchased from Neosystem (Strasbourg, France) and used with a >95% purity. After 1–4 days of culture, cells were pulsed overnight with 0.5 µCi of [³H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD8⁺ T cells in female TCR DTg Rag2-deficient mice

Mice expressing Tg TCRs specific for the male HY Ag (aHY) and for the gp33-41 peptide of the LCMV (P14) were crossed with B6.Rag2^-/- mice to obtain two lines of mice harboring homogeneous populations of CD8⁺ T cells all expressing the same TCR specificity. Monoclonal MoaHY (V8.2⁺VT3.70⁺) and MoP14 (V8.1⁺V2⁺) mice were intercrossed to obtain MoaHY.MoP14 (DTg) mice bearing two MHC H-2D^b class I-restricted TCR transgenes.

In the DTg female mice, dual TCR chain-expressing (V2⁺T3.70⁺) cells represent the majority (80%) of the peripheral CD8 T cells (Fig. 1A). About 20% of the CD8 T cells express a single V chain; i.e., they are either V2⁺T3.70^- (5–15%) or V2^-T3.70⁺ (5–15%) (Fig. 1A). Expression of VT3.70 was lower in DTg than in MoaHY mice, but varied as in aHY.Rag⁺ TCR Tg mice, where endogenous TCR chains can be co-expressed (18). The vast majority (99%) of the peripheral CD8 T cells express the aHY TCR V8.2 chain. Only <1% of the total CD8 T cells are V8⁺8.2^-V2⁺T3.70^-, corresponding to the P14 TCR (data not shown). Although detection of T cells expressing each of the -chains from the V8 family can be done using the F23.1, KJ16, and F23.2 Abs (27), we found that direct analysis of dual TCR expression was not conclusive due to the lack of a V8.1 monospecific Ab. The assembly of the TCR transgenes at the cell surface of the DTg T cells was studied by following the kinetics of co-internalization of the different TCR Tg chains (Fig. 1B). Pre-incubation of DTg cells with anti-V8.2 promotes the complete internalization of both VT3.70⁺ and V2⁺ chains, suggesting that the V8.2 aHY TCR Tg chain associates with both the aHY and the P14 TCR Tg chains. These results also indicate that, in the DTg cells, equivalent levels of surface expression of two TCR chains must be a rare event. Studies on the in vitro response of DTg CD8 T cells to specific peptides provided further evidence of the poor expression of the V8.1 P14 TCR transgene. Thus, whereas the dual-receptor T cells proliferated in response to the Scmy-3 HY peptide (Fig. 2, A and B), the P14-specific gp33-41 LCMV peptide failed to induce proliferation of DTg cells (Fig. 2C). However, it induced the early CD69 expression in 50% of V2⁺T3.70⁺ cells (Fig. 2D), indicating that those cells were still capable of peptide recognition and of a partial agonistic response (28).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Peripheral T cells in MoP14, MoaHY and MoaHY.MoP14DTg female mice. A, Expression of CD8 (histograms) and of V2 and VT3.70 TCR transgenes by gated peripheral LN CD8 T cells (dot plots). B, Spleen cells from DTg females were incubated for 6 h in the presence of a first anti-V8.2 capping Ab. The expression of the TCR at different times after incubation was compared with the TCR expression in absence of the first Ab. The left graph shows the kinetics of V2 internalization (expressed as a percentage of MFI of control cells) among the V2+VT3.70- () and V2+VT3.70+ () T cells. The right graph shows VT3.70 down-regulation in V2-VT3.70+ () and V2+VT3.70+ () T cells. We must mention that, in CD8 T cells from monoclonal mice, down-regulation of a TCR chain correlates with the TCR chain disappearance and vice versa. Thus, pre-incubation with T3.70 does not internalize the TCR of T cells from MoP14 origin, and pre-incubation with the P14- chain-specific B20 Ab does not internalize the aHY TCR in T cells from MoaHY mice. Similarly, the V8.2 Ab specific for the aHY TCR did not internalize the P14 TCR (data not shown).

View larger version (32K): [in this window] [in a new window]   FIGURE 2. In vitro stimulation of T cells from MoP14, MoaHY, and MoaHY.MoP14 DTg mice. A, Spleen T cells from MoaHY female () and DTg female () mice were stimulated in cultures with the Scmy3 aHY TCR-specific peptide. Results show the [3H]thymidine incorporation at day 2 of culture obtained in one typical experiment (mean of triplicate ± SD). B, Peptide activated (solid line) and nonactivated (dotted line) cells were stained at 24 h for the surface expression of the early activation marker CD69. The percentage of CD69+ cells is indicated inside each histogram. C and D, The same analysis was performed for T cells from MoP14 mice () and DTg female () using the gp33-41 P14 TCR-specific peptide.

Thymus T cell development in TCR (DTg) Rag2^-/- female mice

To understand the cellular mechanisms that could lead to the exclusion of one TCR chain transgene, we compared T cell development in the thymus of the DTg females, MoaHY females, and MoP14 mice (Fig. 3). The total number of thymocytes (75 x 10⁶ cells) and the double negative (DN) (10%), double positive (DP) (80%), and single positive (SP) CD8⁺ (10%) distributions were similar in all mice (Fig. 3A). The composition of the DN populations as well as their levels of TCR expression (Fig. 3B) varied. In both MoaHY and DTg mice, CD44^-CD25^high populations were absent, and all DN T cells, CD44^-CD25^int and CD44^-CD25^-, expressed high levels of the aHY TCR Tg V8.2. In the MoP14 mice, CD44^-CD25^high cells were present and they did not express TCR. Expression of the P14 TCR transgene was first detected among CD44^-CD25^int cells, but the frequency of these cells with TCR^high expression was much lower than that found at similar stages of differentiation in the thymus of both MoaHY and DTg mice. Therefore, the aHY TCR transgene was expressed earlier than the P14 TCR chain. This earlier expression, probably before the CD44^-CD25^high stage, induces the rapid transition of immature thymocytes into CD44^-CD25^int DN cells and the disappearance of the CD44^-CD25^high DN population. The kinetics of expression of the TCR chain transgenes also differed. In MoaHY females, all CD44^-CD25^int DN cells expressed high levels of VT3.70, whereas in the MoP14 thymus, V2 expression was low in CD44^-CD25^int DN cells and high levels of expression could only be detected among CD44^-CD25^- DN cells.

View larger version (54K): [in this window] [in a new window]   FIGURE 3. T cell development in the thymus of single- and double-TCR Tg Rag2-deficient female mice. A, Thymus cells from MoaHY.MoP14DTg, MoaHY, and MoP14 mice were stained for the expression of CD4 and CD8 (left panels). The relative proportions of the different DN, DP, and SP populations are shown in the quadrants inside each dot plot. The right panels show the pattern of expression of CD25 and CD44 among the gated DN thymocytes. B, The histograms show the expression of the V and V TCR Tg chains among the different subpopulations of DN thymocytes defined according to intensity of expression of CD25 and CD44.

Decreased P14 TCR chain mRNA levels in dual TCR Tg CD8 T cells

The exclusion of the P14 TCR transgene could occur at the transcription and/or at a posttranscription (protein) level. The high sequence homology between the two Tg TCR chains prevented RT-PCR analysis of the specific mRNA transcripts. However, the size of the hypervariable CDR3-like region of the two chains differs. Therefore, it was possible to evaluate the frequency of the TCR chain transcripts using a method of PCR amplification and primer extension with fluorescent oligonucleotides (24). We must mention that we used the same amplification and extension primers for both TCR chains. By measuring the area corresponding to the transcripts of each -chain from a 1:1 mixture of both transgene T cells, we found that the level of the P14 TCR Tg mRNA was 3- to 4-fold lower than that of the aHY TCR transgene (Fig. 4). Correspondingly, the surface expression of TCR by mature CD8 T cells is 20–30% lower in MoP14 mice than in MoaHY mice. The lower level of transcription of the P14 transgene may be due to the types of promoters and transgene vectors used. Indeed, the aHY vector includes both the TCR chain promoter and the TCR chain enhancer, whereas the P14 vector uses an MHC class I promoter and the Ig H chain enhancer, without regulatory elements specific for TCR expression (19, 20).

View larger version (32K): [in this window] [in a new window]   FIGURE 4. TCR chain expression in single- and double-TCR Tg Rag2-deficient female mice. A, The aHY and P14 TCR chain mRNA expression was studied by Immunoscope, using lymph node cells from MoaHY, MoP14, and MoaHY.MoP14DTg female and male mice. A 1:1 mixture of LN CD8 T cells from MoaHY and MoP14 mice was used to compare the relative transcription of both TCR chain transgenes. B, CD8+ T cells from MoaHY, MoP14, and MoaHY.MoP14DTg mice were stained for total TCR chain surface expression using a pan Ab (H57). The MFI obtained is indicated in each histogram.

DTg T cells are H-2D^b restricted

Do new combinations of TCR chains due to the random association between the different TCR Tg chains confer a new MHC restriction specificity to the MoaHY.MoP14 DTg cells? Lymphocyte development in the thymus of the DTg females only gives rise to CD4^-CD8⁺ SP cells (Fig. 3A). The lack of CD4 SP cells indicates that any potential association between - and -chains from the two TCR transgenes does not permit positive selection by MHC class II. Selection of DTg cells in the absence of the aHY and P14 TCR MHC class I H-2D^b restricting element was also investigated. In female CD3^-/-H-2D^b-/- chimeras reconstituted with BM cells from female DTg donors (Fig. 5), the number of thymocytes is reduced. The few CD8⁺ SP cells present in the thymus are immature because TCR^highCD8⁺ SP cells are almost absent (Fig. 5) and the number of peripheral CD8 T cells is 20-fold lower (i.e., <0.5 x 10⁶ cells) than in CD3^-/-H-2D^b+/+ chimeras. The few peripheral CD8 SP cells present express the aHY V8.2 and the P14 V2 chains (data not shown). From these results, we conclude that, in DTg mice, dual-receptor expression does not hinder thymus T cell positive selection and that SP CD8 cells are restricted to MHC H-2D^b class I molecules.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Development of MoaHY.MoP14DTg T cells in BM chimeras. Irradiated CD3-/- (top) or CD3-/-H-2Db-/- mice (bottom) were reconstituted with BM cells from DTg donors. Two months later, T cell development was examined. The panels show the expression of CD4 and CD8 in the thymus of both BM chimeras and the expression of V2 and VT3.70 among the SP CD8+ cells (R1 gate). Note the rarity of SP TCR+ cells in the CD3-/-H-2Db-/- hosts. The relative proportions of the DN, DP, and SP populations are shown in the quadrants inside each dot plot. The total numbers of thymocytes and SP CD8 thymus cells are indicated above each dot plot.

We next investigated whether the presence of two TCR transgenes could rescue cells from deletion in the presence of male HY self-Ag. We compared T cell development in the thymus of MoaHY and DTg male mice. In MoaHY male mice, the presence of the HY Ag blocks thymus T cell differentiation at the DN to DP transition. The number of thymocytes is reduced to 5 x 10⁶, of which 92% are DN (18). In the thymus of DTg males, T cell development is also impaired, but in contrast to MoaHY male mice, the presence of the second V2 TCR transgene rescues a significant fraction of cells into the DP compartment (Fig. 6, A and B). This allows the selection of cells co-expressing both P14-specific V2 and low levels of HY-specific VT3.70 into the SP CD8 T cell compartment (Fig. 6B). At the periphery of the MoaHY males, 70% of the peripheral TCR⁺ cells are CD8^-CD4^- DN (Fig. 7A), and the few TCR⁺ CD8⁺ T cells present (4 x 10⁶) express low levels of TCR (18). In the DTg males, TCR⁺ DN cells are practically absent and we recovered 3-fold more peripheral CD8⁺ T cells (12 x 10⁶), of which most (80%) express two TCR chains (Fig. 7A) and diminished levels of CD8 (data not shown). In contrast with DTg females (Fig. 1A), single V2⁺T3.70^- cells are more abundant (20%) and V2^-T3.70⁺ cells are rare (2%). We examined the density of expression and the assembly of the different TCR chains in DTg cells from male mice. Compared with dual TCR cells from DTg females, the expression of the VT3.70 TCR chain, though variable, was consistently reduced (compare Figs. 1A and 7A). Pre-incubation of dual receptor cells from DTg males with anti-V8.2 Abs fails to internalize 50% of the VT3.70 chains (Fig. 7B). This observation contrasts with the results obtained with dual-receptor cells from DTg females and suggests an increased expression of the P14 V8.1, which associates with the VT3.70 chains at the surface (Figs. 1B and 7B). Indeed, the level of the P14 TCR Tg mRNA in cells from DTg male mice is higher than in cells from DTg female mice (Fig. 4A). Thus, deletion of dual-receptor T cells bearing the aHY TCR is abrogated, allowing the selection of the cells expressing low levels of the self-reactive TCR and higher levels of the second non-self-reactive P14 TCR (15). These findings show that most peripheral CD8⁺ T cells in DTg males are V8.1^+/low8.2⁺V2⁺T3.70^low. We conclude that in male DTg mice, the presence of a second TCR transgene prevents deletion of cells expressing a self-reactive receptor. The types and the levels of receptor expression differ from those observed in female mice, indicating a process of adaptation of the population of CD8 T cells determined by their antigenic microenvironment.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. T cell development in the presence of the HY male Ag. A, Thymocytes from MoaHY and DTg male mice were stained for CD4 and CD8 surface expression. The relative proportions of the different DN, DP, and SP populations are shown in the quadrants inside each dot plot. B, The dot plots show the expression of V2 and VT3.70 chains by SP CD8 thymus cells in DTg male mice.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Peripheral T cells in MoaHY and MoaHY.MoP14DTg male mice. A, Expression of CD8 (histograms) by gated TCR+ LN cells and of V2 and VT3.70 TCR transgenes by gated peripheral LN CD8 T cells (dot plots). B, Spleen cells from DTg male mice were incubated for 5 h in the presence of a first anti-V8.2 capping Ab. The expression of the TCR at different times after incubation was compared with the TCR expression in absence of the first Ab. The left graph shows the kinetics of V2 internalization (expressed as a percentage of MFI of control cells) among the V2+VT3.70- () and V2+VT3.70+ () T cells. The right graph shows VT3.70 down-regulation in V2-VT3.70+ () and V2+VT3.70+ () T cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the MoaHY.MoP14 DTg mice, thymus T cell development only gives rise to CD8 T cells, indicating that the association between the different -chains of the two TCR transgenes was not able to generate MHC class II-restricted CD4 T cells. The lack of significant T cell development in MHC class I H-2D^b-/- chimeras further demonstrates that the all TCR chain combinations that can be generated from the two transgenes are restricted only to the H-2D^b molecule. These results are not due to the inability of the different TCR chains to associate (29). Indeed, the study of the co-internalization of the TCR chains in CD8 cells from female and male DTg mice shows that the two -chains and the two -chains of both transgenes can form heterodimers in all possible combinations. These observations contrast with previous reports, in which DTg mice using TCR transgenes restricted to MHC class I and MHC class II or to two different MHC class I molecules were shown to express a new MHC specificity (14, 17, 30). The fidelity of the dual MoaHY.MoP14 TCR transgene cells to the H-2D^b restricting element allows comparing the selection of dual- and single-receptor T cells, without the biases that could be introduced by the generation of a new MHC restriction specificity.

In female DTg mice, whereas 80% of the peripheral CD8 T cells express two TCR chains, the vast majority of the CD8 T cells only express one TCR chain; i.e., they functionally "excluded" one of the TCR chain transgenes. Indeed, several findings suggest that, although 99% of the CD8 T cells from DTg mice are V8.2⁺, most cells either do not express or poorly express the V8.1 chain of the P14 TCR. First, internalization of the V8.2 chain of the aHY TCR transgene leads to complete co-internalization of both V chains. Secondly, in vitro stimulation of CD8 T cells from DTg mice with the LCMV gp33-41 peptide failed to induce cell proliferation. Because the peripheral pools of MoP14 mice contain a high number of CD8 T cells, the absence of the P14 -chain in DTg mice is not due to its poor expression or the inability of cells expressing it to be positively selected. Moreover, in DTg mice a minority (1%) of CD8 T cells are V8⁺8.2^-V2⁺T3.70^-; i.e., they express only the P14 V8.1 chain. By comparing the kinetics of expression of the Tg TCR chains during T cell development, we found that, in MoaHY mice, 96% of the CD44^-CD25^int DN cells already express the TCR chain, whereas, in MoP14 mice, expression of the TCR chain occurs at a later CD44^-CD25^- stage. The different kinetics of TCR expression may relate to the type of vectors used. The aHY transgene contains both the TCR chain promoter and enhancer, whereas the P14 transgene uses an MHC class I promoter and an IgH enhancer (19, 20). The presence of the correct TCR chain enhancer justifies both the earlier and the higher transcription of the aHY TCR chain. By comparing the mRNA species of both -chains, we observed that the TCR chain transcripts in the CD8 T cells from the MoP14 mice are less abundant than in the CD8 T cells from the MoaHY mice. Accordingly, TCR chain surface expression was found to be lower in MoP14 CD8 T cells than in MoaHY CD8 T cells. More importantly, in the dual-receptor T cells, the P14 -chain transcripts are very poorly represented (10% of total). These results suggest that, in dual-receptor T cells, silencing of the P14 -chain occurs at the mRNA level. The exact mechanisms that regulate the levels of the P14 -chain transcripts remain unknown. Double-TCR transgenic mice have been previously shown to produce cells with mutual exclusion of the TCR transgenes posttranscriptionally (31) or which express both receptors equally (32, 33). The effect on P14 TCR chain allelic exclusion could be due to the earlier and stronger expression of the aHY -chain that may affect expression of the second P14 -chain transgene at the posttranscriptional level. It may reduce the stability and the half-life of the P14 -chain transcripts. It may compete for limited amounts of pT or for the CD3 complex required for surface receptor expression. It may also inhibit transcription of the second P14 -chain transgene. In the latter case, any possible regulatory mechanisms involved in suppressing transcription of the P14 -chain might act through the IgH enhancer used in the P14 vector. In the rare V8⁺8.2^-V2⁺T3.70^- CD8 T cells, the reverse must occur because these cells express only the P14 V8.1 chain and exclude the aHY -chain. These effects may simply reflect vagaries of the TCR constructs. However, all these mechanisms are selective for the -chain transgene, in that they do not affect expression of the P14 TCR transgene under the control of the same promoter and enhancer (20), suggesting that they may have some physiological relevance. Examples of silencing of one transgene have also been reported in mice that bear two complete rearranged Ig transgenes (34, 35). Overall, these findings indicate that the presence of two productive -chain rearrangements (6) does not necessarily correlate with double -chain expression, as shown for the TCR locus (29). This represents yet another mechanism for functional TCR chain exclusion. As a result of this process, most of the CD8 T cells from the DTg mice express one TCR associated with two TCR chains, like 30% of the peripheral T cells from a normal mouse.

What happens to the development of dual-receptor-expressing T cells in the presence of the male HY self-Ag? Thymus T cell development has been shown to be particularly sensitive to changes in TCR expression levels (17, 36). In male DTg mice, the presence of a second TCR transgene reduces the expression of the aHY TCR, allowing the appearance of significant numbers of DP thymocytes and TCR^highCD8⁺ SP T cells. During the DP to SP transition, only the cells expressing lower levels of CD8 and of the aHY TCR are positively selected. SP cells in the thymus and CD8⁺ cells at the periphery of DTg males mostly express two V chains, as in DTg females, but in contrast to the female mice, single T3.70⁺ cells are rare and single V2⁺ cells are more abundant. The lack of complete internalization of the TCR chains after anti-V8.2 treatment indicates that the dual-receptor cells express increased levels of the P14 V8.1 chain. In agreement, P14 -chain transcripts were more represented in the cells from DTg male mice than in those from DTg females. Overall, the presence of the HY Ag leads to the counterselection of cells bearing high levels of the aHY-specific receptors both in DTg and in MoaHY mice. Alternatively, cells may modulate expression of specific receptors as a function of the levels of Ag encountered in the developing environment.

In contrast with MoaHY males, in which TCR⁺CD8^- (DN) cells represent 60% of the peripheral TCR⁺ cell pool (18), in DTg male mice most peripheral TCR⁺ cells are CD8⁺. It has been claimed that, in aHY Tg mice, the peripheral DN cells belong to T cell lineage (37, 38) and that expression of the TCR receptor could ensure maturation and survival of the cells. Our current observations in Rag2^-/- MoaHY mice demonstrate that expression of TCRs is not required for the survival of the TCR⁺ DN cells. Overall, these findings suggest that successful lymphocyte survival at the periphery requires optimal levels of signaling. Thus, in male MoaHY the presence of an excess of self-Ag counter selects CD8⁺ cells and allows the peripheral accumulation and survival of aHY TCR⁺ T cells, which do not express the CD8 or CD4 coreceptors. In DTg males, in the presence of the same levels of self-Ag, T cells expressing CD8 can accumulate due to the dilution of the aHY TCR. In fact, the few peripheral TCR⁺ DN cells that survive in DTg male mice express higher levels of the aHY-specific TCR (data not shown).

We show here, by using double-TCR Tg mice, that TCR chain usage is tightly controlled and that mechanisms other than classical allelic exclusion can affect expression of a second TCR chain transgene. In contrast, control of TCR chain is loose, allowing the co-expression of two TCR chain transgenes. As a consequence, the majority of CD8 T cells from the MoaHY.MoP14 DTg female and male mice, like 30% of the peripheral T cells from a normal mouse (11, 12), express one TCR chain associated with two different TCR chains. Therefore, these DTg mice provide a unique model for studying the in vivo functional behavior of the dual-TCR CD8 T cells. We examined the thymus selection of the dual-receptor CD8 T cells in the presence of self-Ag. We found that the presence of a second TCR chain allows a significant number of CD8 T cells expressing a self-reactive receptor to escape central deletion and migrate to the peripheral pools of male mice. Differences in TCR and coreceptor expression between female and male MoaHY and DTg mice suggest that peripheral T cell survival requires an optimal level of signaling, which implies a process of "adaptation" of lymphocyte populations to the host environment.

	Acknowledgments

 
We thank Benedita Rocha for criticism, Laurent Ferradini for suggestions, James Di Santo for reviewing the manuscript, and Annick Lim for help with the immunoscope analysis.

	Footnotes

 
¹ This work was supported by the Institute Pasteur, Centre National de la Recherche Scientifique, Agence Nationale de Recherches sur le SIDA, Association pour la Recherche sur le Cancer, Sidaction, and Ministère de la Recherche et de l’Technologie, France.

² Address correspondence and reprint requests to Dr. Antonio A. Freitas, Lymphocyte Population Biology Unit, Unité de Recherche Associée, Centre National de la Recherche Scientifique, 1961 Institut Pasteur, 25 Rue du Dr. Roux, 75015 Paris, France. E-mail address: afreitas{at}pasteur.fr

³ Abbreviations used in this paper: pT, pre-TCR; LCMV, lymphocytic choriomeningitis virus; Tg, transgenic; DTg, double transgenic; MFI, mean fluorescence intensity; LN, lymph node; SP, single positive; BM, bone marrow; DN, double negative; DP, double positive.

Received for publication July 13, 2001. Accepted for publication September 21, 2001.

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