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The Journal of Immunology, 2002, 169: 4347-4353.
Copyright © 2002 by The American Association of Immunologists

Human CD4 Expression at the Late Single-Positive Stage of Thymic Development Supports T Cell Maturation and Peripheral Export in CD4-Deficient Mice1

Olivier Boyer*, Gilles Marodon*, José L. Cohen*, Laurence Lejeune*, Théano Irinopoulou{ddagger}, Roland Liblau{dagger}, Patrick Bruneval{ddagger} and David Klatzmann2,*

* Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires and Centre National de la Recherche Scientifique Unité Mixte de Recherche 7087, {dagger} Laboratoire de Neuro-immunologie, Centre de Recherche en Virologie et Immunologie, Hôpital Pitié-Salpêtrière, Paris, France; and {ddagger} Institut National de la Santé et de la Recherche Médicale Unité 430, Hôpital Broussais, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Positive selection of developing thymocytes is initiated at the double-positive (DP) CD4+CD8+ stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient mice. However, it is unclear whether later onset CD4 expression would still allow such a restoration. To investigate this issue, we used transgenic mice in which a hCD4 transgene is not expressed on DP, but only on single-positive cells. By crossing these animals with CD4-deficient mice, we show that late hCD4 expression supports the maturation of T cell precursors and the peripheral export of mature TCR{alpha}{beta}+ CD8- T cells. These results were confirmed in two different MHC class II-restricted TCR transgenic mice. T cells arising by this process were functional in the periphery because they responded to agonist peptide in vivo. Interestingly, thymocytes of these mice appeared refractory to peptide-induced negative selection. Together, these results indicate that the effect of CD4 on positive selection of class II-restricted T cells extends surprisingly late into the maturation process by a previously unrecognized pathway of differentiation, which might contribute to the generation of autoreactive T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the thymus, positive and negative selection shape the T cell repertoire (1, 2). Phenotypically and functionally mature CD4+CD8- and CD4-CD8+ single-positive (SP)3 cells are generated from immature CD4+CD8+ double-positive (DP) precursors by a complex process of positive selection, which results in the restriction of TCR recognition by MHC molecules. In addition, negative selection of thymocytes with high affinity/avidity for self Ags contributes to the limitation of self reactivity by deleting overtly autoreactive T cells (3) or positively selecting regulatory CD4+CD25+ T cells (4). Different cell types have been implicated in these processes: thymic cortical epithelium sustains positive selection, whereas medullary epithelium and/or bone marrow-derived cells support negative selection (5). Nevertheless, it is now apparent that both positive and negative selections are closely interrelated. Indeed, positive selection seems to require sustained signaling from continuous or multiple MHC interactions over several stages of differentiation (6).

Positive selection of developing thymocytes is thought to be initiated at the DP stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient (CD40/0) mice (7, 8). However, it is unclear whether later onset CD4 expression would still allow such a restoration. We investigated in this study whether CD4 expression at the SP stage would restore T cell development in CD40/0 mice. For this, we used our previously described EpCD4 mice (referred to as Ep mice in this work) in which a hCD4 transgene is not expressed on DP, but only on SP cells in the thymus (9). In the present study, we show that late CD4 expression still allows the maturation of T cell precursors and the peripheral export of Ag-responsive T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Ep (line 10) transgenic mice were described previously (9). They express a hCD4 cDNA under the transcriptional control of hCD4 gene promoter and murine CD4 (mCD4) gene enhancer sequences. CD40/0 mice have a targeted disruption of the CD4 gene (7). AND transgenic mice possess a V{alpha}11 V{beta}3 TCR that recognizes a pigeon cytochrome c peptide on the I-Ek molecule (10, 11). Because homozygous expression of I-Ek induces some clonal deletion in this model, experiments with AND mice were performed in the H-2k/b background in which positive selection is optimal (12). HNT transgenic mice possess a V{alpha}15 V{beta}8.3 TCR that recognizes an influenza hemagglutinin peptide on the I-Ad molecule (13). Experiments with HNT mice were performed in the H-2b/d background. Recombinase-activating gene (Rag)0/0 mice have a targeted disruption of the Rag-2 gene (14). Mice were bred in the animal facility of the Faculté de Médecine Pitié-Salpêtrière (Paris, France) or in the Centre de Distribution, Typage et Archivage Animal, Centre National de la Recherche Scientifique (Orléans, France). Combinations of mutant mice were obtained by appropriate breedings. Screening of progeny was performed by flow cytometry analysis of a blood sample, and included a determination of the H-2 phenotype. Mice were manipulated according to the European Union guidelines.

Flow cytometry analysis

Thymus, spleen, and lymph nodes (brachial, axillary, and inguinal) were cut into small fragments and incubated in RPMI 1640 supplemented with 1.6 mg/ml type IV collagenase (Sigma-Aldrich, Saint-Quentin Fallavier, France) and 200 µg/ml DNase I (Boehringer Mannheim, Mannheim, Germany) at 37°C for 30 min. Cells were dissociated by repeated pipetting, reincubated at 37°C for 10 min, and washed. Cell suspensions were then incubated with 200 µg/ml DNase I for 15 min at room temperature and resuspended in staining buffer (3% FCS, 0.02% azide PBS). Spleen cell suspensions were additionally mixed with 2 vol 0.8% ammonium chloride for RBC lysis, immediately centrifuged, and resuspended in staining buffer.

Two-, three-, or four-color stainings were performed by incubating 1–2 x 106 cells for 30 min at +4°C with different combinations of the following mAbs (quantum red, tricolor (TC), allophycocyanin, streptavidin): anti-mCD4 (FITC, PE, TC, or allophycocyanin labeled), anti-CD8 (FITC, PE, or TC labeled), anti-CD90, anti-CD3, anti-TCR{alpha}{beta} (FITC labeled) from Caltag Laboratories (San Francisco, CA); anti-heat-stable Ag (HSA; FITC or PE labeled), anti-CD3 (PE labeled), anti-CD62L, anti-Qa-2, anti-CD69, anti-V{beta}8.3 (PE labeled) from BD PharMingen (San Diego, CA); anti-hCD4 or IgG1 negative control (quantum red labeled) from Sigma-Aldrich; and anti-V{alpha}11 (biotinylated) from BD PharMingen revealed by streptavidin-TC from Caltag Laboratories. After a final wash, cells were fixed with 1% paraformaldehyde PBS. Events were acquired and analyzed with CellQuest software on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Confocal microscopy

The thymuses were snap frozen. For single immunofluorescence, acetone-fixed frozen sections were incubated for 60 min with either anti-hCD4 mAb (biotinylated) from BD Biosciences diluted at 1/50 in PBS buffer (pH 7.4, 0.1 M), or anti-CD3 (PE labeled) from BD PharMingen diluted at 1/200. After a rinse in PBS buffer, the hCD4 sections were incubated with cyanin 2 (Cy2)-labeled streptavidin (Amersham, Les Ulis, France) diluted at 1/400. After a rinse, the sections were mounted with Immunmount (DAKO, Trappes, France). For double immunofluorescence, the sections were incubated first with anti-hCD4 mAb, followed by Cy2-streptavidin, and second with anti-CD3.

Immunofluorescence images were acquired with a Leica TCS SP confocal scanning laser microscope (Leica Microsystems, Manheim, Germany), equipped with an ArKr laser, and mounted on an inverted microscope. Slides were observed with a x16 or a x63 (1.32 NA) oil immersion objective. Anti-hCD4 mAb coupled with Cy2 was excited at 488 nm and detected at 500–600 nm, and anti-CD3 mAb coupled with PE was excited at 568 nm and detected at 580–650 nm. Acquisition was done in a sequential way to avoid cross talk between the emission spectra of the fluorochromes, with all acquisition settings kept constant for all images.

Peptide experiments

The pigeon cytochrome c88–104 peptide (KAERADLIAYLKQATAK) was previously described to induce clonal deletion of immature thymocytes bearing the AND TCR (15). This peptide (Cybergene, Saint-Malo, France) was adjusted to a concentration of 10 µg/µl in sterile PBS. A quantity of 1 mg was injected i.v., while control mice received PBS only. Flow cytometry analysis was performed on lymph node (LN) cells, and thymocytes were recovered 40 h after injection.

Statistical analyses

Statistical analyses were performed using StatView software (SAS Institute, Cary, NC). The Mann-Whitney test was used to compare data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Timing of hCD4 expression in Ep mice

Ep transgenic mice were described previously (9). They harbor a hCD4 transgene placed under the transcriptional control of CD4 gene-derived regulatory sequences that govern expression in both CD4+ and CD8+ mature T cells. As shown in Fig. 1GoA, hCD4 is not detectable at the surface of DP thymocytes, while expressed at the surface of all mature CD4+ peripheral T cells. Further analysis revealed that maturing thymocytes start to express hCD4 at the CD4SP stage slightly before the down-regulation of HSA and the up-regulation of Qa-2, two markers of thymocyte maturation with an antiparallel expression pattern (16). Thus, transgene expression in Ep mice occurs at a late stage of T cell development that we will refer to as the late CD4SP stage.



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  		FIGURE 1. Expression profile of the hCD4 transgene during T cell development in Ep mice. Ep mice express a hCD4 transgene placed under the transcriptional control of CD4 gene-derived regulatory sequences. Flow cytometry analysis was performed after gating on A, CD4+CD8+ (DP) thymocytes or CD4+ PBMC, or on B, CD4+CD8- (CD4SP) thymocytes. On single-parameter histograms, the isotype-matched negative control is overlaid (dotted line).

 
Development of TCR{alpha}{beta}+ CD8- T cells in CD40/0 mice by expression of hCD4 at the late CD4SP stage

To test whether CD4 expression at the late CD4SP stage would still support T cell maturation, we crossed CD40/0 mice with Ep mice, generating CD40/0 Ep mice. In CD40/0 mice, T cells of the CD4 lineage can be identified by their TCR/CD3+CD8- phenotype (17). In the thymus of CD40/0 Ep mice, the frequency of CD8- cells among HSAlow thymocytes was significantly increased as compared with CD40/0 mice (Fig. 2GoA). The absolute number of CD8- thymocytes was also significantly augmented after gating on HSAlow CD62L+ thymocytes (Fig. 2GoB), indicating that this increase cannot be ascribed to the re-entry of activated CD62L- peripheral T cells to the thymus (18). In the periphery, the frequency of CD3+CD8- T cells and that of TCR{alpha}{beta}+ CD8- T cells was significantly increased in CD40/0 Ep mice as compared with CD40/0 mice (Fig. 2Go, C and D), and >90% of these cells expressed hCD4. Moreover, a similar increase was also found in naive CD44low T cells (Fig. 2GoE), and in CD45RBhigh and CD62Lhigh T cells (not shown). Finally, the TCR-V{beta}3, 4, 6, 8, 10, 11, and 14 usage in CD3+CD8- T cells was similar in CD40/0 Ep and CD40/0 mice, and no increase in NK1.1+ CD3+CD8- T cells was observed in CD40/0 Ep mice (not shown). Altogether, these results reveal an increased production of polyclonal naive TCR{alpha}{beta}+ CD8- T cells when hCD4 is expressed at the late CD4SP stage.



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  		FIGURE 2. Expression of hCD4 at the late CD4SP stage increases the generation of TCR+ CD8- T cells in CD40/0 mice. Flow cytometry analysis of thymus, spleen, and LN cells from CD40/0 and CD40/0 Ep mice was performed. Cell frequencies are given as mean ± SEM (number of mice is indicated in parentheses). Total numbers of splenocytes and thymocytes were not statistically different between CD40/0 and CD40/0 Ep mice. A, Frequency of CD8- cells after gating on HSAlow thymocytes. Absolute numbers of HSAlow CD8- thymocytes in CD40/0, CD40/0 Ep, and CD4wt mice were (x106): 8.8 ± 1.4, 12.9 ± 1.8, and 21.2 ± 3.5, respectively. B, Total numbers of HSAlow CD62Lhigh thymocytes. C, Frequency of CD3+CD8- T cells in spleen and LN. Absolute numbers of CD3+CD8- splenocytes in CD40/0 vs CD40/0 Ep mice were (x106): 7.8 ± 0.8 vs 9.4 ± 0.7 (p < 0.05). In CD40/0 Ep mice, 94.4% ± 0.3 of LN CD3+ CD8- express hCD4. D, Frequency of TCR{alpha}{beta}+ CD8- T cells in spleen and LN. E, Frequency of CD3+CD8- T cells in spleen and LN after gating on CD44low cells. Absolute numbers of CD3+CD44lowCD8- splenocytes in CD40/0 vs CD40/0 Ep mice were (x106): 1.5 ± 0.2 vs 2.5 ± 0.3 (p < 0.05).

 
Development of CD8- T cells in MHC class II-restricted TCR transgenic CD40/0 mice by hCD4 expression at the late CD4SP stage

Because TCR{alpha}{beta}+ hCD4+CD8- T cells generated at the late CD4SP are presumably MHC class II restricted, we further investigated whether a similar phenomenon would occur in the context of a known MHC class II restriction. For this, we turned to the AND TCR transgenic mice (10, 11). Thymocyte development in the CD4 lineage in this model is highly dependent on the presence of a CD4 molecule, i.e., although class II restricted, nearly all AND TCR transgenic T cells develop in the CD8 lineage in CD40/0 mice (Fig. 3GoA) (12).



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  		FIGURE 3. Expression of hCD4 at the late CD4SP stage increases the generation of TCR+ CD8- T cells in CD40/0 mice expressing the MHC class II-restricted AND (V{alpha}11) TCR. Flow cytometry analysis of thymus and LN cells from AND CD40/0 and AND CD40/0 Ep (Ragwt or Rag0/0 background) was performed. Cell frequencies are given as mean ± SEM (number of mice is indicated in parentheses). Total numbers of thymocytes were not statistically different between AND Ragwt CD40/0 and AND Ragwt CD40/0 Ep mice, nor between AND Rag0/0 CD40/0 and AND Rag0/0 CD40/0 Ep mice. A, Frequency of V{alpha}11+ CD8- thymocytes among HSAlow thymocytes and CD90+ LN cells (Ragwt background). Absolute cell number of HSAlow V{alpha}11+ CD8- thymocytes in AND Ragwt CD40/0 vs AND Ragwt CD40/0 Ep mice were (x106): 0.9 ± 0.2 vs 7.0 ± 1.4 (p < 0.0005). B, Frequency of V{alpha}11+ CD8- thymocytes among HSAlow thymocytes and CD3+ LN cells (Rag0/0 background). Absolute cell numbers of HSAlow V{alpha}11+ CD8- thymocytes in AND Rag0/0 CD40/0 vs AND Rag0/0 CD40/0 Ep mice were (x106): 0.8 ± 0.4 vs 6.6 ± 2.4 (p < 0.01). C, Frequency of CD8- cells among V{alpha}11+ and V{alpha}11+ CD62Lhigh thymocytes. D, Frequency of CD69+ cells among V{alpha}11+ CD8- thymocytes (Rag0/0 background).

 
In AND CD40/0 Ep mice, V{alpha}11+ CD8- thymocytes were significantly more abundant among HSAlow thymocytes as compared with AND CD40/0 mice (Fig. 3GoA). This was manifested by an average 5-fold increase in frequency and 8-fold increase in absolute cell numbers. In the periphery, the frequency of V{alpha}11+ CD8- LN T cells also significantly increased (Fig. 3GoA). To avoid the generation of T cells that would express endogenously rearranged TCR chains, we performed the same experiments in recombinase-deficient Rag0/0 mice. A dramatic increase in the frequency and in the absolute numbers of V{alpha}11+ CD8- HSAlow thymocytes, and of V{alpha}11+ CD8- T cells in the LN was also found (Fig. 3GoB). CD8- T cells were significantly more abundant among V{alpha}11+ CD62L+ thymocytes in both Rag wild-type (Ragwt) and Rag0/0 mice (Fig. 3GoC). In addition, the generation of V{alpha}11+ CD8- thymocytes was accompanied by the up-regulation of CD69 (Fig. 3GoD), a transiently expressed marker characteristic of thymocytes undergoing positive selection (19, 20).

To exclude that this developmental pattern would be unique to the AND model, we analyzed mice transgenic for another MHC class II-restricted TCR, i.e., V{alpha}15 V{beta}8.3 HNT TCR (13). The frequency of V{beta}8.3+ CD8- cells in the thymus and in the periphery was also significantly increased in HNT CD40/0 Ep mice as compared with HNT CD40/0 mice (Fig. 4Go). Importantly, there was an absolute MHC requirement in this process because there was no increased generation of V{beta}8.3+ CD8- cells upon hCD4 expression in a nonselecting MHC background such as H-2b. Indeed, V{beta}8.3+ CD8- cells represented 35% of HSAlow thymocytes in H-2b/b HNT CD40/0 Ep mice (n = 8) vs 39% in H-2b/b HNT CD40/0 mice (n = 6), and there was no statistically significant difference in V{beta}8.3+ CD8- cell frequencies among CD3+ LN T cells (not shown). Besides, we occasionally observed a slight reduction in the level of CD8 expression in CD40/0 Ep mice as compared with CD40/0 controls (Figs. 2GoC, 3A, and 4). Because CD8+ T cells express hCD4 in the Ep model, this may suggest that the presence of transgenic hCD4 somehow down-regulates CD8 in this lineage. Together, these results reveal an increased production of MHC class II-restricted T cells upon hCD4 expression at the late CD4SP stage.



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  		FIGURE 4. Expression of hCD4 at the late CD4SP stage increases the generation of TCR CD8- T cells in CD40/0 mice expressing the MHC class II-restricted HNT (V{beta}8.3) TCR. Flow cytometry analysis of LN cells and thymocytes from HNT CD40/0, HNT CD40/0 Ep mice was performed. Cell frequencies are given as mean ± SEM (number of mice is indicated in parentheses).

 
Localization of the late CD4SP selection process in the thymus

To investigate the localization of late CD4SP selection, we performed confocal microscopy analysis of hCD4 expression in the thymus and found that hCD4+ thymocytes were located exclusively in the medulla (Fig. 5GoA). In addition, virtually all medullary thymocytes were CD3+, whereas hCD4 expression was limited to patches of contiguous cells (Fig. 5GoB). Colocalization analysis revealed the existence of two populations of medullar thymocytes, i.e., CD3+hCD4- and CD3+hCD4+, in agreement with the observation that only the more mature CD4SP thymocytes are hCD4+ (Fig. 1Go). Consequently, thymic maturation induced by the expression of hCD4 at the late CD4SP stage takes place in the medulla.



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  		FIGURE 5. Expression of hCD4 in CD40/0 Ep mice is restricted to the thymic medulla. Confocal microscopy was performed on frozen thymic sections after incubation with anti-hCD4 (green fluorescence) and anti-CD3 (red fluorescence) mAb. A, Lower magnification, objective x16. B, Higher magnification, objective x63, centered on the medulla. Results for hCD4 (upper) and CD3 (middle) are shown separately and overlaid (lower).

 
Activation in the periphery, but resistance to deletion in the thymus after Ag injection in AND CD40/0 Ep mice

The functionality of the MHC class II-restricted hCD4+CD8- T cells that developed in AND CD40/0 Ep mice was evaluated by their ability to respond to agonist peptide injection. Similar to AND CD4wt mice, a dramatic up-regulation of CD69 in V{alpha}11+ CD8- LN T cells from AND CD40/0 Ep mice was observed 40 h after peptide injection, demonstrating that T cells that matured upon late hCD4 expression were able to respond in the periphery (Fig. 6GoA).



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  		FIGURE 6. Agonist peptide activates peripheral T cells, but fails to induce deletion of HSAhigh V{alpha}11+ CD8- thymocytes in AND CD40/0 Ep mice. The agonist peptide was injected i.v. to AND CD4wt and AND CD40/0 Ep mice. Flow cytometry analysis was performed on LN cells, and thymocytes recovered 40 h after peptide or PBS (control) injection. Cell frequencies are given as mean ± SEM (number of mice is indicated in parentheses). A, Frequency of CD69+ cells among V{alpha}11+ CD8- LN cells. B, Frequency of HSAhigh cells among V{alpha}11+ CD8- thymocytes. C, Absolute numbers of HSAhigh (left panel) and HSAlow (right panel) cells among V{alpha}11+ CD8- thymocytes.

 
In the thymus of AND CD4wt mice, peptide injection resulted in the deletion of HSAhigh V{alpha}11+ CD8- thymocytes both in frequency and absolute numbers (Fig. 6Go, B and C). In marked contrast, in AND CD40/0 Ep mice, peptide injection did not result in the deletion of HSAhigh thymocytes within the V{alpha}11+ CD8- compartment (Fig. 6Go, B and C), suggesting that CD8- thymocytes that matured upon late hCD4 expression are resistant to negative selection. After peptide injection, the number of HSAlow thymocytes within the V{alpha}11+ CD8- subset augmented from 1.5 x 106 to 4.4 x 106 cells in AND CD40/0 Ep mice (Fig. 6GoC), suggesting that Ag recognition promotes final maturation rather than negative selection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study demonstrate that CD4 expression at the late SP stage of thymic development is capable of supporting thymocyte maturation and peripheral export of Ag-responsive MHC class II-restricted T cells. Ep transgenic mice provided an appropriate model to investigate this issue because hCD4 is not expressed in DP thymocytes, and strong experimental evidence has now accumulated to indicate that hCD4 is fully functional in mice. First, the extracellular domain of hCD4 can functionally interact with mouse MHC class II molecules (21, 22, 23). Second, there is substantial homology between the cytoplasmic regions of mCD4 and hCD4 (24), which results in an efficient biochemical coupling of the intracellular domain of hCD4 to the signaling pathways that lead to mouse T cell activation (25). Third, expression of a hCD4 transgene at the DP and subsequent stages has been shown to restore normal T cell development and function in CD40/0 mice (7, 8). This included the restoration of: 1) positive and negative selection of developing thymocytes; 2) MHC class II-restricted alloreactive and Ag-specific T cell responses; and 3) primary and secondary Ag-specific IgG humoral responses.

In agreement with our previous observation (9), there was no detectable expression of hCD4 in DP thymocytes of Ep mice (Fig. 1Go). It could be argued that low numbers of transgenic hCD4 molecules might still be expressed at the DP stage and, thus, be responsible for the increased production of CD3+CD8- T cells in mice expressing hCD4. The strong CD4 requirement for positive selection of thymocytes bearing the AND TCR renders this hypothesis very unlikely. Indeed, it has been reported that a reduction of only ~50% in surface level expression of CD4 severely impairs the capacity of AND TCR+ thymocytes to develop in the CD4 lineage (26). Hence, a very low CD4 surface level on DP thymocytes, below the level of detection of flow cytometry, would be unable to positively select T cell precursors bearing the AND TCR.

Whereas a population of bona fide MHC class II-dependent Th cells arises in CD40/0 mice, not all CD8- T cells in such animals are generated by MHC class II-driven positive selection. For instance, CD8- mature T cells may include CD1d-restricted V{alpha}14 NK T cells (27, 28) or {gamma}{delta} T cells (29, 30, 31, 32). In our model, the TCR-V{beta} usage in CD3+CD8- T cells was similar in CD40/0 Ep and CD40/0 mice, and no increase in NK1.1+ CD3+CD8- T cells was observed. This renders a role for NK T cells very unlikely because these cells are NK1.1+ and show a bias toward V{beta}8 usage. Furthermore, there was no increased generation of V{beta}8.3+ CD8- cells upon hCD4 expression in a nonselecting MHC background. Therefore, the increased production of CD3+CD8- thymocytes upon hCD4 expression at the late CD4SP stage can be attributed to an MHC class II-dependent process.

Two nonmutually exclusive mechanisms can account for the increased production of CD3+CD8- thymocytes upon hCD4 expression at the late CD4SP stage: positive selection of MHC class II-restricted thymocytes and/or postselection expansion (33, 34). Some MHC class II-restricted cortical thymocytes would have survived in the absence of a CD4 signal at the DP stage and would have proceeded to the medulla to receive the positively selecting signal provided by the hCD4 transgene. These cells may have further been activated to proliferate because of the coreceptor function provided by hCD4. Whatever the relative contribution of positive selection sensu stricto, i.e., not associated with proliferation, and postselection expansion, our results challenge the view that positive selection is exclusively an early developmental process supported solely by the cortical epithelium (35). The nature of the cells that support late positive selection in the medulla remains unknown. They may belong to a particular subset of medullar stromal cells because previous studies have ruled out the capacity of either medullary epithelium (36, 37, 38) or bone marrow-derived cells (39, 40) to support positive selection of CD4 T cells.

In CD40/0 mice, it is considered that the CD8- T cells that develop despite the absence of CD4 bear TCRs with high affinity for MHC class II (17). In this study, CD4 expression at the late CD4SP stage allowed only a partial restoration of the CD4 lineage. This raised the possibility that the process described in this work only rescues thymocytes bearing TCRs with rather high affinity for MHC class II. In contrast, those thymocytes bearing TCRs with low affinity for MHC class II would die by neglect before they could reach the medulla. This hypothesis is difficult to reconcile with the results obtained with TCR transgenic mice. One would expect that such mice would either fully restore their CD4 lineage if the affinity of the transgenic TCR were sufficient or, alternatively, would not restore this lineage at all if it were insufficient. The partial CD4 lineage restoration observed in the TCR transgenic experiments instead suggests that late positive selection is not dictated primarily by TCR affinity. Nevertheless, thymic selection can be partial, affecting some thymocytes, but not others, as a function of the level of TCR and/or other relevant receptors. Finally, it cannot be formally excluded that insufficient availability of developmental niches for positive selection in the medulla may be a limiting factor for a complete restoration of the CD4 lineage in this system.

Late positive selection was less marked with the HNT TCR than with the AND TCR. This presumably reflects the weak positive selection of thymocytes in HNT mice, as attested by an absence of skewing toward the CD4 lineage and the maintenance of a normal thymic architecture in this model of transgenic mice (41, 42). Despite the absence of CD4 bias in the HNT model (CD4/CD8 ratio = 0.7 in CD4wt HNT mice as compared with 21 in CD4wt AND mice), late expression of hCD4 was still able to significantly increase the production of CD3+CD8- T cells.

Intravenous injection of a peptide Ag in TCR transgenic animals is an efficient method to activate peripheral T cells while inducing clonal deletion of thymocytes (43). This central deletion may occur as the result of specific recognition of the antigenic peptide by the thymocyte (44) and also as a consequence of the activation of cytokine-releasing mature T cells (45). In this study, TCR transgenic thymocytes are resistant to both mechanisms of deletion upon encounter with agonist peptide. A likely explanation is that the positively selecting signal provided by hCD4 expression has been delivered at a developmental stage at which clonal deletion can no longer occur. Rather, Ag recognition promoted final maturation, as suggested by the increased production of HSAlow thymocytes in peptide-injected AND CD40/0 Ep mice. An alternative hypothesis is that the outcome of thymic selection results from the integration of positive and negative selecting signals. In our experimental conditions, the positive selecting signal would have predominated upon the peptide-dependent negative signal. Finally, it cannot be formally excluded that the presence of CD4 might be altering the sensitivity of thymocytes to cytokine-induced cell death, instead of, or in addition to effects on clonal deletion. Whatever the mechanism, some thymocytes that have escaped negative selection might leave the thymus for the periphery. Under physiological conditions, this may be of importance for T cells bearing two TCR{alpha} chains (46, 47). Should one of these TCRs find an appropriate ligand for late positive selection, a positive signal could be generated that would counteract a negatively selecting signal transmitted via the other TCR on the same thymocyte. Because mature T cells generated by late positive selection are functional in the periphery as shown by their capacity to respond to agonist peptide, this would result in the export of a self-reactive mature T cell. Altogether, these results reveal a previously unrecognized pathway of thymic maturation and raise the possibility that late positive selection contributes to the generation of autoreactive T cells.


   Acknowledgments
 
We thank B. Rocha for helpful suggestions; B. Salomon, M. Seman, and R. Cibotti for critical reading of the manuscript; J. P. Regnault and P. Delis for animal care; S. Bruel for excellent technical assistance; and P. Kitmacher for artwork.


   Footnotes
 
1 This work was supported in part by the Université Pierre et Marie Curie and the Center National de la Recherche Scientifique. G.M. was supported by Agence Nationale de Recherche sur le SIDA. Back

2 Address correspondence and reprint requests to Dr. David Klatzmann, Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, Centre de Recherche en Virologie et Immunologie, Hôpital Pitié-Salpêtrière, 83 bd de l’hôpital, F-75013 Paris, France. E-mail address: david.klatzmann{at}chups.jussieu.fr Back

3 Abbreviations used in this paper: SP, single positive; 0/0, knockout; Cy2, cyanin 2; DP, double positive; h, human; HSA, heat-stable Ag; LN, lymph node; m, murine; Rag, recombinase-activating gene; TC, tricolor; wt, wild type. Back

Received for publication May 22, 2002. Accepted for publication August 13, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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