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Quantitative and Qualitative Adjustment of Thymic T Cell Production by Clonal Expansion of Premigrant Thymocytes¹

Armelle Le Campion^2,*, Bruno Lucas^*, Nicole Dautigny^*, Sandrine Léaument, Florence Vasseur^* and Claude Pénit^*

^* Institut National de la Santé et de la Recherche Médicale Unité 345 and Laboratoire d’Expérimentation Animale et de Transgénèse, Institut Necker, Paris, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In normal mice, single-positive thymocytes proliferate before being exported into the peripheral T cell pool. We measured the in vivo proliferation rates of mature thymocytes in several TCR transgenic mice. Different monoclonal TCR transgenic single-positive thymocytes proliferated at different rates in a given MHC context. Conversely, mature thymocytes expressing a given TCR, generated in mice of different MHC haplotypes, also showed different rates of proliferation. In p59^fyn-deficient mice, the proliferation rate of mature thymocytes was diminished. Thus, premigrant thymocyte expansion is TCR mediated and depends on TCR affinity for self peptide/MHC ligands. In addition, we show that mature thymocyte expansion is clonotypic, increases the daily thymic T cell output, and modifies the TCR repertoire of newly produced T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The thymus is the site of production of new naive T cells expressing a highly diverse TCR repertoire. During T cell development, the TCR repertoire expressed by maturing T cells is influenced by interactions with endogenous peptide/MHC molecules. The T cell repertoire enables T cells to respond to a broad spectrum of foreign antigenic peptides while tolerating self peptides presented by self MHC molecules. Starting from a given number of immature CD4⁺CD8⁺ double-positive (DP)³ cells, mature thymocyte production is primarily determined by the yield of positive vs negative selection. Positive selection biases the repertoire of TCRs on thymocytes toward cells that can bind self peptide/MHC complexes with low affinity, while negative selection eliminates thymocytes with TCRs that bind self peptide/MHC complexes with high affinity (1). Thymocytes with TCRs unable to perceive self peptide/MHC complexes die by neglect. Thus, postrearrangement-selective mechanisms allow death by neglect of the useless, active elimination of the dangerous, and survival of the useful (2). Self peptide/MHC molecules are crucial in the generation of a T cell repertoire with a wide spectrum of specificities. Furthermore, naive T cells continue to require contacts with self peptide/MHC molecules for their survival in the periphery (3, 4, 5, 6, 7, 8, 9).

We have recently described a late intrathymic expansion phase of mature single-positive (SP) thymocytes (10, 11). Indeed, some mature thymocytes that have successfully passed both selection processes proliferate before leaving the thymus. In the steady state normal murine adult thymus, 1.7% of the TCR^high CD4⁺CD8^- SP (CD4SP) and 5.2% of the TCR^high CD4^-CD8⁺ SP (CD8SP) incorporate the DNA precursor bromodeoxyuridine (BrdU) within 4 h (10). In previous studies, we found that SP thymocytes that are submitted to such proliferation (also suggested in monkeys (12)) are totally mature (CD24^-, Qa-2^high), thus being more similar to recent thymic emigrants (RTEs) and peripheral T cells than to the bulk of thymic medullary SP TCR^high cells. This proliferation concerns cells that recently matured in the thymus, and therefore excludes mature T cells reentering the thymus (10). However, on the basis of activation and adhesion surface molecule expression, cycling SP thymocytes are phenotypically different from activated peripheral T cells. Compared with the bulk of mature (CD24^-) SP thymocytes, they did not change expression of CD44 and CD62L like in vivo activated T cells (11). While the generation of mature CD4⁺ and CD8⁺ T cells depends on effective TCR signaling induced by recognition of self peptide/MHC molecules in the thymus, it remains to be determined whether mature SP thymocyte proliferation also depends on similar signals. If so, would such proliferation be dependent on TCR affinity for self peptide/MHC ligands? Furthermore, as mature SP thymocytes proliferate before leaving the thymus (10), what is the quantitative and qualitative impact of this late expansion on thymic T cell production?

To address these issues, we examined several rag-2-deficient mouse strains expressing a transgenic (Tg) TCR, to compare the proliferation rates of distinct individual monoclonal T cell clones at the mature thymocyte stage. We studied the proliferation of mature thymocytes when the affinity of the TCR for the peptide/self MHC complexes was varied by changing the MHC context for the same TCR transgene, and evaluated the contribution of SP cell expansion to thymic T cell production.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Tg rag-2-deficient mice expressing the AND TCR transgene in the H-2^b/b, H-2^b/k, or H-2^k/k MHC haplotype, and H-2^k/k CD3-deficient mice were maintained in our animal facilities (13, 14). C57BL/Ba mice were bred and maintained in our animal facilities. C57BL/6 mice were purchased from Charles Rivers Breeders (Elbeuf, France). C57BL/6 H-2^b/b CD3-deficient mice (15) and C57BL/6 rag-2-deficient mice (16) were obtained from the Center for Development of Advanced Experimentation Techniques (Orléans, France), and C57BL/6 p59^fyn-deficient mice (17) were from The Jackson Laboratory (Bar Harbor, ME). Female C57BL/6 H-Y TCR Tg rag-2^-/- mice (18) were generously provided by B. Rocha (Necker Institut, Institut National de la Santé et de la Recherche Médicale Unité 345, Paris, France) and C57BL/6 P14 TCR Tg rag-2^-/- mice (19) by A. Freitas (Pasteur Institut). All these mice were studied between 6 and 8 wk of age.

Adoptive transfer

Bone marrow (BM) chimeras were generated by sublethal gamma-irradiation (4 Gy) of host mice before i.v. injection of CD4-depleted and CD8-depleted BM cells. BM cells were obtained from femurs and tibias of H-Y TCR Tg rag-2^-/-, H-2^b/b, or H-2^k/k AND TCR Tg rag-2^-/- mice or C57BL/Ba mice. A 1:1 mixture (4 x 10⁶ total cells) of AND TCR Tg or H-Y TCR Tg (Thy-1.1^-) and C57BL/Ba (Thy-1.1⁺) CD4-depleted and CD8-depleted BM cells was injected i.v. into irradiated C57BL/6 rag-2^-/- hosts. Irradiated H-2^b/b or H-2^k/k CD3-deficient mice were injected i.v. with 6 x 10⁶ CD4-depleted and CD8-depleted BM cells from AND TCR Tg rag-2^-/- mice of the same haplotype. Sixteen or eighteen days later, the chimera were pulsed with BrdU, and thymuses were recovered and analyzed for thymocyte proliferation. BM cell suspensions were depleted of CD4 or CD8 T cells with a mixture of mAbs and magnetic sorting with coated Dynabeads (Dynal, Oslo, Norway).

BrdU labeling

One milligram of BrdU (Sigma, St. Louis, MO) was injected twice i.p. at a half-hour interval, except for the mixed chimera experiment (4-h interval; Fig. 1). In all experiments in which DNA-synthesizing cells were detected and characterized, the thymus was harvested 30 min after the second injection. To study thymus emigrants, BrdU labeling was prolonged to 24 h by injecting BrdU three times at 8-h intervals before injecting the mice with FITC.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Proliferation rates of mature monoclonal and polyclonal CD4SP or CD8SP thymocytes differ in a given thymic environment. A 1:1 mixture of BM cells from AND TCR Tg rag-2 -/- (a) or H-Y TCR Tg rag-2-/- (b) and from C57BL/Ba (Thy-1.1) mice was injected i.v. into irradiated B6 rag-2-/- mice. On day 16 (a) or 18 (b) after BM transfer, mouse chimeras received a double BrdU pulse (4-h interval) and were tested for CD4SP or CD8SP proliferation according to the type of chimera. Data are presented as BrdU/Thy-1.1 fluorescence dot plots of gated CD24-/int CD4+ or CD24-/int CD8+ thymocytes. Monoclonal AND TCR Tg or H-Y TCR Tg SP cells are Thy-1.1-. Polyclonal CD4SP or CD8SP cells are Thy-1.1+. Numbers indicate the percentage of BrdU+ cells in the corresponding Thy-1.1- or Thy-1.1+ cells.

BrdU-injected mice were anesthetized and, after opening the thorax cavity, 10 µl of FITC (5 mg/ml in PBS) was injected into each thymic lobe. The thymus, spleen, and peripheral lymph nodes (LNs; pooled axillary, inguinal, cervical, and mesenteric nodes) were recovered 16 h later.

Abs and immunofluorescence analysis

Cell surface staining. Four-color immunofluorescence analysis was performed using a FACSCalibur system (Becton Dickinson, San Jose, CA). List-mode data files were analyzed using CellQuest software (Becton Dickinson).

Cell suspensions were prepared in PBS plus 4% FBS (Life Technologies, Grand Island, NY) and 0.2% sodium azide (Sigma). Cells were distributed in round-bottom 96-well plates, and surface molecules were stained with PE-conjugated anti-CD8 Ab (CT-CD8; Caltag, San Francisco, CA), PerCP-conjugated anti-CD4 Ab (RM4-5), biotinylated anti-Thy-1.1 (CD90.1, HO22.1), anti-CD44 (1M7), anti-CD69 (H1.2F3), anti-CD5 (53-7.3), or anti-V3 (KJ25) revealed with streptavidin-allophycocyanin. Abs were purchased from PharMingen (San Diego, CA), unless otherwise indicated.

BrdU detection. Surface-stained cells were fixed and permeabilized in PBS containing 1% paraformaldehyde plus 0.01% Tween 20 for 48 h at 4°C, and then submitted to the BrdU DNase detection technique, as previously described (20), using FITC-conjugated anti-BrdU Ab (3D4; Becton Dickinson). For BrdU detection in FITC-labeled thymic emigrants, we used acid DNA denaturation (21) and PE-conjugated anti-BrdU (3D4; Becton Dickinson).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TCR/MHC-dependent interactions induce the proliferation of mature SP thymocytes

We have shown that normal mature SP thymocytes proliferate before emigrating to the periphery in normal adult mice (10). These cycling mature SP thymocytes are phenotypically different from activated peripheral T cells, and their proliferation rate is dependent on the peripheral pool size (11). One possible origin of the SP cell proliferation in the thymus might be signals mediated through the TCR. In normal mice, the measured proliferation rate of SP thymocytes is likely to be the sum of the proliferation of diverse T cell clones. In this case, different T cell clones should have different mitotic activities in a given peptide/self MHC environment.

In this study, we used TCR Tg mice to compare the proliferation rates of distinct isolated monoclonal T cell clones. We first studied mature CD8SP thymocytes from two different TCR Tg mice expressing distinct clonotypic H-2D^b-restricted TCRs, namely P14 TCR Tg mice expressing an anti-lymphocytic choriomeningitis virus TCR (19), and female H-Y TCR Tg mice expressing a TCR directed against the male Ag (18). We also studied mature CD4SP thymocytes expressing the AND TCR Tg directed against pigeon cytochrome c. These cells are positively selected in both H-2^k/k and H-2^b/b haplotypes (13). All Tg strains were crossed with rag-2-deficient mice to rule out endogenous TCR and gene rearrangements, and will be simply referred to as TCR Tg mice.

We first compared these three H-Y, P14, AND TCR Tg strains in the same haplotype and in the same genetic background (H-2^b/b, C57BL/6 (B6)). Contrary to CD4SP thymocytes, CD8SP thymocytes contain a high proportion of immature cells, intermediate between double-negative precursors and DP cells. These immature SP cells proliferate extensively (22), and cannot be distinguished from mature SP cells on the basis of TCR expression, which is already high at this stage in TCR Tg mice. We thus restricted our analysis to CD24^-/int cells, as most mature CD8SP cells have down-regulated CD24, at least partially (23). We compared the proliferation rate of SP subsets by measuring BrdU incorporation after a short pulse (2 injections at a 30-min interval). A small, but significant proportion of P14 TCR Tg CD8SP thymocytes was BrdU labeled (0.46 ± 0.19%; n = 4), compared with only 0.04 ± 0.01% (n = 3) in H-Y TCR Tg mice (Table I). Furthermore, CD8SP cells from these two different B6 Tg rag-2^-/- mice exhibited a lower proliferation rate than the polyclonal CD8SP subset from their normal B6 counterparts. Likewise, few, if any CD4SP thymocytes proliferated in B6 AND TCR Tg mice (0.05 ± 0.03%; n = 6), contrary to polyclonal CD4SP thymocytes from B6 normal mice. Thus, in a given MHC context (H-2^b/b), the proliferation rate of clones expressing different TCRs varies. These results suggest that proliferation of mature SP cells in the thymus is induced by TCR/MHC interactions.

To exclude the possibility that the different proliferation rates observed above might be simply due to microenvironmental defects in TCR Tg mice, we developed an in vivo model in which monoclonal and polyclonal SP cells coexist in the same environment. We have previously shown that, in chimeras made by normal BM transfer to irradiated rag-2^-/- mice, SP thymocytes show significantly higher proliferative activity than the baseline value found in the adult steady state. Peaks of BrdU incorporation are detected on day 16 and on day 18 after BM transfer in CD4 and CD8 SP thymocytes, respectively (11).

We therefore used this transfer model to evaluate the proliferation rate of two distinct CD4SP populations, namely monoclonal AND TCR Tg cells and polyclonal C57BL/Ba cells. A similar comparison was made with monoclonal H-Y TCR Tg CD8SP cells and polyclonal C57BL/Ba CD8SP cells. Irradiated rag-2^-/- mice were reconstituted with equal numbers of C57BL/Ba (Thy-1.1⁺) BM cells and either AND TCR Tg (Thy-1.1^-) BM cells or H-Y TCR Tg (Thy-1.1^-) BM cells. On day 16 or 18 posttransfers, the chimeras received a double BrdU injection at a 4-h interval. CD4SP thymocytes from AND TCR Tg mice exhibited a very low proliferation rate (1.4% of BrdU⁺ cells) relative to the polyclonal CD4SP subset from C57BL/Ba mice (21.2% of BrdU⁺ cells) (Fig. 1a). Similarly, only 3.2% of H-Y TCR Tg CD8SP thymocytes proliferated, compared with 14.3% of CD8SP cells from C57BL/Ba mice (Fig. 1b). These data show that, in a given thymic environment, CD4SP proliferation is 20-fold lower in the monoclonal AND TCR Tg population and that CD8SP proliferation is 5-fold lower in the monoclonal H-Y TCR Tg population than in the CD4SP and CD8SP polyclonal populations, respectively. Thus, the observed differences in proliferation between various monoclonal and polyclonal thymocyte populations are not due to microenvironmental defects.

Mature SP thymocyte proliferation is altered in p59^fyn-deficient mice

If SP thymocyte proliferation is induced by TCR signals, it must be affected in mice with defective TCR signal transduction. The protein kinase p59^fyn is associated with the TCR/CD3 complex and plays an important role in thymic selection (24), even if thymus size and the distribution of CD4 and CD8 thymocyte subsets are unaffected by the fyn mutation (17). p59^fyn-deficient mice received a double BrdU pulse injection. Gated mature TCR^high CD4SP and CD8SP thymocytes from p59^fyn-deficient mice exhibited 1.6-fold-lower proliferation compared with their normal B6 CD4SP and CD8SP counterparts (Table II). Mixed chimeras were made by transfer of a 1:1 mixture of p59^fyn-/- and B6 BM cells. On day 28 posttransfer, BrdU incorporation by mature SP thymocytes was measured. The results showed that, when generated in the same thymus, p59^fyn-/- SP cells proliferated 2- to 3-fold less than B6 SP cells (data not shown). This proliferative defect in fyn^-/- mature SP thymocytes is probably due to blockade of TCR signal transduction. The residual proliferation might result from cells capable of propagating TCR signals in fyn-independent fashion, the most likely candidate being p56^lck (25). However, the role of p56^lck cannot be evaluated because its absence or inactivation disturbs T cell differentiation at early stages (26). These results confirm that TCR/MHC interactions are involved in mature thymocyte proliferation. Nevertheless, we cannot formally exclude that some proliferation might be TCR independent.

The intensity of TCR signaling depends on the affinity of the TCR-peptide/self MHC interaction. We postulated that TCR affinity for stimulatory peptide/self MHC ligands might modulate the proliferation of mature SP thymocytes. To investigate this possibility, we studied AND TCR Tg mice expressing H-2^b/b, H-2^b/k, or H-2^k/k MHC haplotypes. The distribution of the different thymocyte subpopulations was assessed in these mice on the basis of CD4/CD8 fluorescence intensities (Fig. 2a). H-2^k/k AND TCR Tg mice showed a reduced thymus size and contained a lower percentage of DP cells than did H-2^b/k and H-2^b/b AND TCR Tg mice. Although no significant differences in the percentages of CD4SP Tg thymocytes were observed among the three haplotypes, the absolute number of CD4SP thymocytes was 5-fold lower in H-2^k/k mice than in H-2^b/b mice. The small thymus size and the reduction in DP Tg thymocyte numbers observed in the H-2^k/k haplotype are consistent with the induction of a clonal deletion (thymic negative selection) as a result of higher AND TCR affinity for I-E^k than for I-A^b MHC molecules (27, 28, 29). It has been shown that CD5 surface levels on CD4SP thymocytes from Tg mice increase with TCR affinity for the selecting ligands (30). In agreement with these data, the level of CD5 expression by AND TCR Tg CD4SP thymocytes gradually increased from the H-2^b/b to the H-2^b/k and the H-2^k/k haplotype, whereas V3 transgene levels in CD4SP Tg thymocytes were similar (Fig. 2b).

View larger version (48K): [in this window] [in a new window]   FIGURE 2. Thymocyte subsets in H-2b/b, H-2b/k, and H-2k/k AND TCR Tg rag-2-/- mice. Thymocytes derived from AND TCR Tg rag-2-/- mice expressing various MHC haplotypes were labeled with PE anti-CD8, PerCP anti-CD4, and biotinylated anti-V3 or anti-CD5, with streptavidin-allophycocyanin revelation. CD4+CD8+ DP and CD4+CD8- SP thymocyte subsets were defined on the basis of CD4/CD8 fluorescence intensities. The proportions of each subset are shown as percentages (a). Comparison of V3 and CD5 expression levels by CD4SP thymocytes from H-2b/b (solid line), H-2b/k (broken line), and H-2k/k (dotted line) AND TCR Tg rag-2-/- mice (b).

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Proliferation of mature monoclonal AND TCR Tg CD4SP thymocytes in mice with different MHC haplotypes. BrdU was injected twice at a 30-min interval, and thymocytes from H-2b/b, H-2b/k, and H-2k/k AND TCR Tg rag-2-/- mice were stained with PE anti-CD8, PerCP anti-CD4, and biotinylated anti-CD69 or anti-CD44, with streptavidin-allophycocyanin revelation. BrdU/CD69 fluorescence dot plots for CD4SP thymocytes (gated as indicated in Fig. 2a) from H-2b/b, H-2b/k, and H-2k/k AND TCR Tg rag-2-/- mice (a). Irradiated H-2b/b or H-2k/k CD3-deficient hosts were injected with either H-2b/b or H-2k/k (syngeneic) AND TCR Tg rag-2-/- T cell-depleted BM cells, respectively (b). Results are representative of six mice and show BrdU/CD69 or BrdU/CD44 fluorescence dot plots from AND TCR Tg CD4SP thymocytes 16 days after transfer. Percentages of BrdU+ cells are shown in each dot plot.

Daily T cell emigration in H-2^b/b, H-2^b/k, and H-2^k/k AND TCR Tg rag-2^-/- mice

Mature cycling T cells leave the thymus within 24 h after cycling (10, 11). We examined whether the different proliferation rates observed in the AND TCR Tg mouse system influenced thymic output in these mice.

We first determined the thymus export rate in H-2^b/b, H-2^b/k, and H-2^k/k AND TCR Tg mice injected intrathymically with FITC 16 h previously. Spleen and LNs (pooled axillary, cervical, inguinal, and mesenteric nodes) were assayed for the presence of FITC⁺ cells. Thymus, LNs, and spleen cells were stained with Abs against CD4, CD8, and V3; RTEs were defined as V3⁺ CD4⁺ FITC⁺ cells outside the thymus. No differences in RTE absolute numbers were observed among mice of the three haplotypes (Fig. 4a, left). However, when the number of V3⁺ CD4⁺ CD8^- RTEs was compared with that of V3⁺ CD4SP thymocytes (Fig. 4a, middle), the RTE/CD4SP ratio in H-2^k/k mice was 2.5-fold higher than in H-2^b/b mice (Fig. 4a, right). This indicated that the thymic export rate was higher in H-2^k/k than in H-2^b/k mice; the latter value was higher than that observed in H-2^b/b AND TCR Tg mice.

View larger version (46K): [in this window] [in a new window]   FIGURE 4. Daily thymic T cell emigration in H-2b/b, H-2b/k, and H-2k/k AND TCR Tg rag-2-/- mice. H-2b/b, H-2b/k, and H-2k/k AND TCR Tg rag-2-/- mice were injected intrathymically with FITC. Sixteen hours later, thymus, LN, and spleen cells were harvested. Absolute number of RTEs per 24 h (left), absolute number of CD4SP thymocytes (middle), and ratio absolute number of RTEs/absolute number of CD4SP thymocytes (right) in H-2b/b, H-2b/k, and H-2k/k AND TCR Tg rag-2-/- mice (a). Total RTEs were detected as FITC+ V3+ CD4+ cells and include all those found in spleen plus those in pooled axillary, cervical, inguinal, and mesenteric LNs. No difference in the absolute number of RTEs was observed among mice of the three MHC haplotypes. Each value represents the mean of three independent experiments (at least two mice per experiment). All values are means ± SDs of values determined for individual mice. BrdU histograms of gated FITC+ cells from LNs and spleen (b). Mice were injected i.p. with BrdU three times at 8-h intervals and were then injected intrathymically with FITC. RTEs were detected in LNs and spleen (SP) 16 h after intrathymic FITC labeling. Results show percentages of BrdU+ cells among gated FITC+ cells. RTE cells from the spleen of H-2k/k AND TCR Tg rag-2-/- mice were highly enriched in BrdU+ cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interactions between thymocyte TCRs and self peptide/MHC complexes presented in the thymus are crucial for the generation of a functional T cell repertoire with a wide spectrum of specificities. Negative selection induces the death of thymocytes with TCRs whose interactions with self peptide/MHC molecule ligands might result in full activation of mature T cells and autoimmune responses (2, 31). Thymocytes also require TCR-mediated signals for survival, maturation, and the choice between the CD4⁺ and CD8⁺ lineages (32), all these processes being part of positive selection. Positive and negative selection processes lead to fine shaping of the T cell TCR repertoire: only a few percent of DP thymocytes generated during pre-TCR-driven expansion reach the fully mature ( TCR^high, CD24^low, Qa-2^high) SP stage. Neglected cells never leave the thymus cortex. By contrast, negative and positive selection do not appear to be strictly restricted to the cortex or medulla (24, 33), even if negative selection-related apoptotic cells have been exclusively observed in the medulla (34). Furthermore, TCR repertoire shaping by positive selection, while mainly detected in the cortex (35), seems to continue in the medulla (36).

We have shown that the last physiological event occurring in the thymic medulla is the proliferation of mature premigrant SP thymocytes (10, 11). Proliferation of CD4⁺CD8^- and CD4^-CD8⁺ thymocytes has previously been studied in vitro, and it has been suggested that normal B6 and TCR Tg SP cells can proliferate in an MHC^-/- environment (37). By contrast, another report suggested that SP proliferation is TCR mediated in vivo (38). In this study, we directly show the involvement of active TCR signaling in this late expansion, as proliferation of both CD4SP and CD8SP thymocytes was significantly diminished in p59^fyn-deficient mice. In the normal thymus, mature thymocytes expressing different clonotypic TCRs exhibit a large range of affinities for peptide/self MHC molecule complexes, and there is no way of detecting possible differences in their mitotic activities. To measure the proliferation rate of individual T cell clones, we compared BrdU incorporation by a given TCR Tg clone in different MHC contexts, and by SP thymocytes with different clonotypic Tg TCRs in a given MHC context. Several reports have suggested that the affinity of the AND TCR is higher for I-E^k than for I-A^b MHC molecules (27, 28, 29). Indeed, this is consistent with the ability of I-E^k bound to self peptides to negatively select many Tg thymocytes. Moreover, CD5 expression on AND Tg CD4SP thymocytes increased gradually from H-2^b/b to H-2^k/k haplotypes, reflecting a stepwise progression of AND TCR Tg affinity for self peptide/MHC molecule complexes (30). The percentages of BrdU⁺ cells among AND TCR Tg CD4SP cells followed a similar pattern (0.05% and 0.65% in H-2^b/b and H-2^k/k adult mice, respectively). This difference was even clearer in chimeras studied on day 16 after BM transfer (0.4% in H-2^b/b and 7% in H-2^k/k hosts). It thus appears that the proliferation rate of AND TCR Tg CD4SP cells correlated with the affinity of this TCR for self peptide/MHC complexes. A similar conclusion can be drawn for TCR Tg CD8SP cells. A higher degree of phosphorylation of the TCR -chain (39) and higher CD5 expression (data not shown) (30) in P14 TCR Tg CD8SP cells suggest a higher affinity of the P14 TCR for the selecting ligands relative to the H-Y TCR. Again, we found that mature CD8SP thymocytes proliferated more actively in P14 than in H-Y TCR Tg mice. Thus, we provide clear evidence that the proliferation of mature SP thymocytes in vivo is driven by clone-specific TCR interactions with self MHC molecules, loaded with self peptides. Furthermore, the rate of this proliferation correlates with the affinity of the interaction. In the normal B6 mouse thymus, the global proliferation rate can be considered as resulting from the various proliferation rates of diverse T cell clones. This would imply that some individual natural clones display very high mitotic activity, particularly as the polyclonal SP cell population from normal mice proliferates more actively than all the TCR Tg cells that we have tested.

TCR stimulation leading to proliferation of SP thymocytes could involve environmental Ag-derived peptides. Indeed, cycling mature thymocytes are almost ready to migrate, and are thus located in the close vicinity of efferent blood vessels (our preliminary data). We can exclude this possibility in Tg mice, as it seems very unlikely that generated T cells in these mice encounter their nominal Ag (e.g., pigeon cytochrome c, lymphocytic choriomeningitis virus, or male Ag H-Y). However, in normal B6 mice, some T cell clones may proliferate in response to Ag-derived peptides. We have previously shown that proliferating mature SP thymocytes in the medulla resemble naive T cells phenotypically, but display an activation phenotype when they specifically respond to exogenous Ag (11). Thus, if Ag-derived peptides are indeed involved in TCR-mediated proliferation of definite mature thymocytes, they would not induce an activated phenotype. Most likely, in B6 normal mice, as observed in TCR Tg mice, low affinity TCR self peptide/MHC interactions induce the proliferation of mature SP thymocytes.

The mechanisms regulating thymic export are still far from clear. We have previously shown that mature T cells rapidly leave the thymus after cycling (10, 11). In the AND TCR Tg system, H-2^k/k mice contain far fewer CD4SP thymocytes than do H-2^b/k and H-2^b/b mice, but we found that similar numbers of RTEs were produced per 24 h in the three mouse strains. Interestingly, this equal T cell emigration was matched by an equal number of CD4SP thymocytes incorporating BrdU. Concomitantly, H-2^k/k mouse RTEs were highly enriched in BrdU⁺ CD4⁺ T cells relative to H-2^b/b mouse RTEs. As shown in Fig. 4b, a 4-fold increase in BrdU⁺ cells among CD4SP cells was found in H-2^k/k mouse RTEs. These data suggest that proliferation of T cell clones leads to an increase in their daily thymic export.

This TCR dependence implies that SP thymocyte expansion is not a random phenomenon. In normal mice, SP thymocyte clones that proliferate belong to the subset that has been positively selected and has escaped negative selection at the semimature stage; among these, proliferation is restricted to cells with a very narrow window of affinity for presented self peptide/MHC ligands. Indeed, thymocytes escaping negative selection are known as low affinity clones, but only those with affinities in the upper range of these low affinity values will respond to the signal by expanding. Proliferation of SP thymocytes therefore appears to be a last-step selection event, resulting in fine tuning of the repertoire of T cells leaving the thymus, skewing this repertoire toward T cell clones in the higher range of the low affinity window. In this way, T cells reaching the peripheral pool might be more suited to perceiving survival signals transmitted by TCR-MHC interactions, this low self-reactivity serving to maintain the survival and homeostasis of naive T cells (3, 4, 5, 6, 7, 8, 9).

Why does proliferation occur at the precise SP mature (CD24^-/low) stage, and not earlier (at the semimature stage) or later (in RTEs)? It has been shown that immature DP thymocytes are more sensitive to low affinity ligands than are their SP progeny (40, 41). SP thymocytes are able to respond to self peptide stimuli with a sensitivity intermediate between that of DP and mature peripheral T cells. More precisely, in H-2^b/b AND TCR Tg rag-2^-/- mice, T cell sensitivity to a partial agonist peptide decreases with T cell maturation from DP to thymic CD4SP, and peripheral CD4⁺ T cells appear unresponsive to the ligand formed by this peptide (41). These differences could explain why fully mature CD4SP thymocytes display higher proliferative activity than mature peripheral T cells (personal data). Kishimoto and Sprent (42) have shown that negative selection of SP thymocytes is restricted to the semimature CD24^high population. Thus, for a given T cell clone, owing to maturation-related differences in sensitivity, signaling by self peptides might lead to cell death of DP and semimature SP thymocytes, and to proliferation of fully mature thymocytes, with no effect on peripheral T cells.

We have previously shown that the first polyclonal SP thymocytes generated in BM chimeras proliferate more strongly than normal adult SP cells, and that a similar proliferation peak is found in mouse neonates (11). In the present study, we show that the first AND TCR Tg CD4SP thymocytes appearing in the medulla, 16 days after BM transfer to CD3-deficient hosts, display a 10-fold increase in proliferation when compared with baseline values found in adult mice. Moreover, differences between MHC haplotypes are amplified. Similar results were obtained in AND TCR Tg H-2^b/b and H-2^k/k neonates (data not shown). Sixteen days after transfer, T cells are still absent from the periphery, suggesting that the degree of peripheral pool filling could influence the SP thymocyte proliferation rate. Furthermore, when LN T cells are coinjected with BM cells to rag-deficient hosts, this increase in proliferation is abolished, values remaining similar to those in normal adult mice at all times after transfer (11). This greater proliferation of mature SP thymocytes in a lymphopenic environment is reminiscent of the proliferation of peripheral naive T cells observed by several authors in lymphopenic situations. Indeed, in normal mice, peripheral naive T cells are essentially resting, in the absence of Ag stimulation (43, 44, 45, 46, 47). However, naive T cells have a great potential for division when transferred to lymphopenic hosts (14, 47, 48, 49, 50, 51, 52). In particular, we have shown that CD4⁺ T cells from AND TCR Tg rag-2^-/- mice transferred to syngeneic hosts depleted of T cells divide in the absence of direct antigenic stimulation, and that this proliferation requires interactions with self MHC molecules (14). In this latter setting, peripheral AND TCR Tg CD4⁺ T cells divided more actively in the H-2^k/k haplotype than in the H-2^b/b haplotype. It has also been suggested that such peripheral T cell expansion would be dependent on self peptide-specific low affinity interactions similar to those required for positive selection in the thymus (48, 49, 52). However, in most cases, proliferating naive T cells acquire a memory-like phenotype, whereas proliferating mature thymocytes continue to exhibit a naive phenotype. We cannot exclude the possibility that a single low affinity TCR self peptide interaction step mediates both mature SP thymocyte and peripheral naive T cell proliferation in the lymphopenic environment. Experiments are underway to determine whether this is the case.

The expansion resulting from recognition of self peptide/MHC molecules by developing thymocytes allows preferential selection of T cells to proceed on the basis of their ability to detect self ligands with an affinity situated in the upper part of the positive selection window. In this sense, this last intrathymic step of T cell development is not conceptually different from positive selection. This interaction of mature SP thymocytes with self peptide/MHC complexes would result in fine tuning of the repertoire of T cells leaving the thymus, skewing this repertoire toward T cell clones in the higher range of this window. In this way, recent thymicemigrants might be more suited to perceiving self peptide-specific recognition to survive and contribute to the long-lived pool of peripheral T cells.

	Acknowledgments

 
We thank B. Rocha and A. Freitas for their kind gift of mice, andJ. P. Guezet and I. Guezet for providing products.

	Footnotes

 
¹ This work was supported by Institut National de la Santé et de la Recherche Médicale and Université René Descartes Paris V, and by grants from the Association de la Recherche sur le Cancer. A.L. was supported by fellowships from Ensemble contre le SIDA and Fondation pour la Recherche Médicale (France).

² Address correspondence and reprint requests to Dr. Armelle Le Campion, Institut National de la Santé et de la Recherche Médicale Unité 345, Institut Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France. E-mail address: lecampio{at}necker.fr

³ Abbreviations used in this paper: DP, double-positive; BM, bone marrow; BrdU, bromodeoxyuridine; LN, lymph node; RTE, recent thymic emigrant; SP, single positive; Tg, transgenic.

Received for publication October 18, 2001. Accepted for publication December 5, 2001.

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