The Common Cytokine Receptor gamma  Chain and the Pre-T Cell Receptor Provide Independent but Critically Overlapping Signals in Early alpha /beta T Cell Development

By James P. Di Santo,* Iannis Aifantis,§ Eleftheria Rosmaraki,* Corinne Garcia,§ Jacqueline Feinberg,§ Hans Jörg Fehling,parallel Alain Fischer,* Harald von Boehmer,§ and Benedita RochaDagger

From the * Institut National de la Santé et de la Recherche Médicale (INSERM) U429, Hôpital Necker-Enfants Malades, F-75743 Paris, France; Dagger  INSERM U345 and § U373, CHU Necker, F-75730 Paris, France; and the parallel  Basel Institute for Immunology, CH-4005 Basel, Switzerland

    Abstract
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Intracellular signals emanating from cytokine and antigen receptors are integrated during the process of intrathymic development. Still, the relative contributions of cytokine receptor signaling to pre-T cell receptor (TCR) and TCR-mediated differentiation remain undefined. Interleukin (IL)-7 interactions with its cognate receptor complex (IL-7Ralpha coupled to the common cytokine receptor gamma  chain, gamma c) play a dominant role in early thymopoiesis. However, alpha /beta T cell development in IL-7-, IL-7Ralpha -, and gamma c-deficient mice is only partially compromised, suggesting that additional pathways can rescue alpha /beta T lineage cells in these mice. We have investigated the potential interdependence of gamma c- and pre-TCR-dependent pathways during intrathymic alpha /beta T cell differentiation. We demonstrate that gamma c-dependent cytokines do not appear to be required for normal pre-TCR function, and that the rate-limiting step in alpha /beta T cell development in gamma c- mice does not involve TCR-beta chain rearrangements, but rather results from poor maintenance of early thymocytes. Moreover, mice double mutant for both gamma c and pre-Talpha show vastly reduced thymic cellularity and a complete arrest of thymocyte differentiation at the CD44+CD25+ cell stage. These observations demonstrate that the pre-TCR provides the gamma c-independent signal which allows alpha /beta T cell development in gamma c- mice. Thus, a series of overlapping signals derived from cytokine and T cell receptors guide the process of alpha /beta thymocyte development.

Key words: thymus;  interleukin;  lymphocyte;  development;  knockout
    Introduction
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The common cytokine receptor gamma  chain (gamma c)1 forms a critical functional component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (for a review, see reference 1). Naturally occurring mutations in gamma c are responsible for X-linked severe combined immunodeficiency disease (SCIDX1) in humans, characterized by a complete absence of T and NK cells, while B cells are present (for reviews, see references 2 and 3). Targeted deletion of gamma c in mice also provokes a wide variety of defects in lymphoid development, including a complete absence of NK cells, gamma /delta T cells, and gut-associated lymphoid tissue (4). Like SCIDX1 patients, gamma c- mice have some mature peripheral B cells, but these mice also show a remarkable degree of alpha /beta T cell development (4). These data demonstrate the important role of gamma c-dependent signals in lymphopoiesis, but also suggest that fundamental differences exist between the mechanisms that permit alpha /beta T cell development in humans and mice.

Analysis of single cytokine- or cytokine receptor-deficient mice has identified which gamma c-dependent signals are responsible for some of the observed developmental defects seen in gamma c- mice. NK cell differentiation is critically linked to the expression of the IL-2Rbeta chain (9), thereby implicating IL-2- and/or IL-15-mediated signaling pathways in the development of these cells. Since IL-2 mutant mice develop NK cells (10), this suggests that IL-15 (or another IL-2Rbeta -binding ligand) is required for the differentiation of this subset (for a review, see reference 11). In contrast, the defect in gamma /delta T cell development in gamma c- mice appears strictly IL-7 dependent (12). Moreover, since IL-7 was initially identified as an important growth factor for T and B cell precursors (13, 14), this would explain the severely reduced thymic cellularity and defects in bone marrow B cell and intrathymic precursors found in IL-7 and IL-7Ralpha mutant mice (15).

The biological consequences of gamma c-dependent receptor engagement for alpha /beta T cell development include signals which can potentially promote cell survival, proliferation, and/or differentiation. Experimentally, however, it has been difficult to conclusively define which of these processes are adversely affected in the absence of gamma c. In theory, the absence of IL-7 signaling in gamma c- mice could potentially limit thymocyte development by affecting the survival and/ or expansion of intrathymic precursors, or by reducing the efficiency of the recombination process. Evidence for the latter has been suggested by reports that the expression of functionally rearranged TCR-alpha /beta transgenes in gamma c- or IL-7Ralpha -deficient mice augmented total thymocyte numbers (18, 19). However, enforced expression of the antiapoptotic factor Bcl-2 could also rescue alpha /beta T cell development in these mice (20), supporting a major role for IL-7/gamma c signaling in promoting a survival program. In all of these cases, thymic reconstitution was not complete, suggesting either that IL-7/gamma c influences both survival and recombination, or that intrathymic development involves additional mechanisms which are critically dependent on this receptor complex, such as the efficiency of pre-TCR assembly or function.

In this report, we have analyzed the potential interplay between gamma c-dependent cytokine pathways and signaling through the pre-TCR for alpha /beta T cell development.

    Materials and Methods
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals and Cell Preparation.

gamma c- mice (5), TCR-alpha -/- mice (23), pTalpha -/- mice (24), recombination activating gene (RAG)- 2-/- mice (25), and their control littermates were maintained in a specific pathogen-free animal facility (CNRS/CDTA, Orleans, France). gamma c-/pTalpha -/- mice were generated by crossing gamma c+/- female mice to pTalpha -/- male mice, and female offspring carrying the gamma c mutation were identified by PCR (26) and backcrossed to pTalpha -/- males. gamma c-/pTalpha -/- male mice were then identified by PCR. gamma c-/TCR-alpha -/- mice were generated in a similar fashion using TCR-alpha -/- male mice. All mice were on a mixed (129Ola × C57Bl/6) background and were used between 3 and 6 wk of age. Cells isolated from thymus and spleen were prepared as described previously (5).

Antibodies and Immunofluorescence Analysis.

The following mAbs were used as conjugates to fluorescein (FITC), phycoerythrin (PE), Tricolor (TRI), or biotin: CD3 (145-2C11), CD4 (GK1.5), CD8beta (35-5.8), TCR-alpha /beta (H57-597), TCR-gamma /delta (GL3), HSA (J11d), CD44 (Pgp-1), CD25 (PC-61), IL-7Ralpha (A7R34), c-kit (2B8), NK-specific (DX5), and B220 (RA3-6B2). Biotinylated mAbs were revealed with either streptavidin-TRI or streptavidin-allophycocyanin (APC; Caltag). Cell suspensions were lysed of erythrocytes and depleted of B cells using sheep anti-mouse Ig-coated magnetic beads (Dynal). Cells were stained in microtiter plates (2 × 106 cells/well in 50 µl), using combinations of directly conjugated mAbs. Simultaneous four-color cell sorting and analysis were performed on a FACSVantage® flow cytometer (Becton Dickinson). Dead cells were excluded by gating based on forward and side scatter characteristics. Sorted populations were routinely 97% pure upon reanalysis.

Cell Cycle Analysis.

In vivo labeling of S phase cells with bromo-deoxyuridine (BrdU; Sigma) was performed by a single intraperitoneal BrdU injection at a dose of 50 mg/kg body wt 15 min before killing. Thymocyte subsets were sorted using a FACSVantage®, and cells incorporating BrdU were identified as described previously (27) using an FITC-coupled anti-BrdU mAb (Becton Dickinson) and a FACScan® flow cytometer.

Intracellular Staining for Bcl-2, pTalpha , and TCR-beta Chains.

Bcl-2 staining was performed as described previously (28). For intracellular staining of pTalpha or TCR-beta chains, surface antigens were stained as above and cells were fixed with 0.1% paraformaldehyde. Cells were subsequently permeabilized in 0.1% saponin before incubation with biotinylated anti-TCR-beta (29) or anti-pTalpha (30) mAbs and finally with streptavidin-APC before analysis on a FACSCalibur® flow cytometer.

TCR-beta Rearrangements.

TCR-beta V(D)J rearrangements were studied as described (31). In brief, genomic DNA was amplified using a combination of two 5' primers specific for Vbeta 6 and Vbeta 8 together with a 3' primer hybridizing to the 3' region of Jbeta 2.5. PCR products were fractionated on 2% agarose gels, transferred to nylon membranes, and probed by Southern hybridization using a 33P-labeled oligonucleotide specific for the 5' region of Jbeta 2.5. Five distinct PCR products ranging in size from 850 to 150 bp are expected depending on the rearrangements of the Vbeta segment with either Jbeta 2.1, Jbeta 2.2, Jbeta 2.3, Jbeta 2.4, or Jbeta 2.5.

    Results
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
Abnormal Development of Early Thymocyte Progenitors in gamma c- Mice.

We analyzed intrathymic development in gamma c- mice to better define the nature of any developmental blocks resulting from the absence of gamma c. When comparing absolute numbers of CD4-CD8- double negative (DN) cells, CD4+CD8+ double positive (DP) cells, and CD4+CD8- and CD4-CD8+ single positive (SP) mature T cells, the DN and CD8 SP populations were more affected by the absence of gamma c (Fig. 1 A). While both DP and CD4 SP cells were reduced by 15-20-fold (similar to the overall decrease in thymic cellularity), the DN and CD8 SP subsets were reduced almost 40-fold. The DN compartment contains both early thymocyte precursors (CD3-) as well as mature CD3+ TCR-gamma /delta and TCR-alpha /beta cells. While gamma /delta T cells do not develop in gamma c- mice (4, 28), DN alpha /beta T cells are present (32). To specifically evaluate gamma c- TCR- thymocyte progenitors, we studied CD44 and CD25 expression on CD3-CD4-CD8- (TN) thymocytes by four-color immunofluorescence analysis. Previous studies have demonstrated that these cells differentiate through the following stages: CD44+CD25- right-arrow CD44+CD25+ right-arrow CD44-CD25+ right-arrow CD44-CD25- (33; for a review, see reference 34).


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Fig. 1.   Early thymocyte development in control and gamma c- mice. (A) Thymocytes from 3-4-wk-old gamma c-deficient mice and their littermate controls were stained using CD4-PE and CD8beta -FITC, and absolute numbers of DN, DP, CD4 SP, and CD8 SP cells were calculated (mean ± SD from 10 mice of each genotype). (B) Thymocytes were stained with a combination of FITC-conjugated antibodies (CD3, CD4, CD8beta , TCR-alpha /beta , TCR-gamma /delta , B220, DX5, and Gr-1), CD44-PE, and CD25-biotin followed by streptavidin-TRI. CD44 versus CD25 expression is shown on electronically gated FITC- TN thymocytes. Absolute numbers were calculated (mean ± SD) from six mice of each genotype.

Compared with controls, immature thymocytes from gamma c-deficient mice showed altered patterns of CD44 and CD25 expression (Fig. 1 B). The most immature CD44+CD25- TN cells, which can also give rise to B cells, NK cells, and thymic dendritic cells (35; for a review, see reference 36), were found in normal frequency, but were clearly reduced in absolute numbers. More striking was the relative accumulation of cells at the CD44+CD25+ stage, which were almost twofold increased in frequency compared with controls (Fig. 1 B). Moreover, absence of gamma c was associated with a clear defect in the development of cells beyond the CD44+CD25+ stage. Percentages of CD44-CD25+ and CD44-CD25- cells were reduced by a factor of 2 and were markedly reduced (>100-fold) in absolute numbers (Fig. 1 B).

Altered Cell Proliferation in gamma c- Thymocyte Precursors.

The block in thymocyte maturation seen in gamma c- thymi could result from abnormal differentiation, reduced cell survival, and/or proliferative defects. The ability of gamma c- thymocyte precursors to incorporate the analogue BrdU was used as a measure of intrathymic proliferation. The number of cells in S phase of the cell cycle was analyzed after a single injection of BrdU (Fig. 2 A). Only a fraction of the immature thymocytes are labeled under these conditions, and it is clear that T cell precursors in gamma c- mice show defects in proliferation, as both CD44+CD25- and CD44+CD25+ cells have markedly reduced BrdU incorporation relative to their gamma c+ counterparts (the CD44-CD25+ and CD44-CD25- subsets were not analyzed in gamma c- thymi due to their extremely small absolute numbers; these subsets were normally labeled in gamma c+ controls [data not shown]). DP cells from gamma c- mice were similarly affected.


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Fig. 2.   Altered survival and proliferation of early thymocyte precursors in the absence of gamma c. (A) Abnormal BrdU incorporation in gamma c- thymocytes. Mice received a single pulse of BrdU before killing. Indicated thymocyte subsets were sorted, and percentages of cells incorporating BrdU were analyzed. (B) Annexin V staining of gamma c+ or gamma c- thymocyte subsets. Cells were stained for TN cells (see Fig. 1), CD25, and Annexin V. Propidium iodide-negative CD25- and CD25+ TN cells were electronically gated, and percentages of Annexin V-positive cells were calculated (mean ± SD) from four mice of each genotype. (C) Intracellular Bcl-2 staining of DN thymocyte precursors was performed; gamma c- thymocytes demonstrated severely reduced Bcl-2 levels (thick lines). Thin lines, isotype control staining.

Decreased BrdU incorporation in gamma c- thymocyte precursors could result from decreased survival of these cells. gamma c-dependent cytokines have been shown to promote lymphocyte survival by maintaining levels of antiapoptotic factors such as Bcl-2 and Bcl-XL (37, 38). Therefore, we examined intracellular levels of Bcl-2 in immature thymocytes from gamma c- mice and their control gamma c+ littermates. gamma c- precursors had markedly reduced levels of Bcl-2 compared with their gamma c+ counterparts (Fig. 2 B). Moreover, gamma c- cells showed elevated cell surface staining with Annexin V (Fig. 2 C), indicating commitment to the apoptotic process (39). Taken together, these results are consistent with a critical role for gamma c cytokines in the survival and expansion of thymocyte precursors, the absence of which could account in part for their reduction in absolute cell numbers.

Pre-TCR Signaling in the Absence of gamma c.

Having demonstrated a major role for gamma c in the most immature T cell precursors, we next addressed the impact of the gamma c mutation on pre-TCR signaling. The activity of the pre-TCR is presumed to begin at the CD44-CD25+ cell stage when functional TCR-beta chain rearrangements are achieved (24, 40). The role of gamma c-dependent cytokines in pre-TCR function has not been previously examined, although it has been suggested that cytokines (like IL-7) may play a role in the expansion of pre-T cells as they differentiate towards the DP stage. This hypothesis gains support from the fact that CD25+ intrathymic precursors express IL-7Ralpha (41). Moreover, the defect in intrathymic maturation in gamma c- mice and the marked depletion in CD44-CD25+ and CD44-CD25- cells could be the consequence of abnormal pre-TCR assembly or function.

Although the pre-TCR has its main role in the transit of early alpha /beta T cell precursors to the DP stage (for a review, see reference 42), additional mechanisms independent of the pre-TCR have been described which can permit the generation of DP cells in vivo (43, 44). These include effects mediated by gamma /delta TCRs and via alpha /beta TCRs due to early rearrangements at the TCR-alpha locus. To focus on the pre-TCR-mediated pathway and to exclude these alternative pathways of DP cell generation, we crossed gamma c- mice (which lack gamma /delta T cells) with TCR-alpha -/- mice (23; to eliminate TCR-alpha expression). Thymocyte differentiation was examined in these double mutants. Introduction of the TCR-alpha mutation did not further alter the pattern of differentiation of thymocytes to the DP stage compared with gamma c- mice or further diminish their total thymic cellularity (Fig. 3 A). These results demonstrate that early TCR-alpha rearrangements do not play a major role in the differentiation of DP cells in gamma c- mice.


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Fig. 3.   Pre-TCR function in the absence of the gamma c chain. (A) CD4/CD8 profiles of thymocytes from 3-4-wk-old TCR-alpha -deficient and TCR-alpha /gamma c double- deficient mice. Total thymocyte numbers were calculated (mean ± SD) from six mice of each genotype. (B) Intracellular TCR-beta chain expression in thymocytes from gamma c-deficient, pTalpha -deficient, or wild-type mice. (C) Thymocyte CD4/CD8 profiles from mice bearing a functional TCR Vbeta 8.2 Tg on gamma c-deficient or wild-type backgrounds. Absolute thymocyte cell numbers (mean ± SD) were derived from four to six mice of each genotype.

Pre-TCR signaling results in beta  selection, i.e., the preferential expansion of early thymocytes expressing a single, functionally rearranged TCR-beta chain (for a review, see reference 45). beta  selection was assessed in control, gamma c-, and pTalpha -/- thymocytes by intracellular staining of TCR-beta expression (Fig. 3 B). As reported previously (44), pTalpha -/- mice show clear defects in beta  selection as demonstrated by decreased intracellular levels of TCR-beta chains in DP thymocytes compared with control mice. In contrast, gamma c- DP thymocytes demonstrated intracellular TCR-beta levels comparable to controls (Fig. 3 B, and see below).

We also considered that pre-TCR function might be affected due to reduced synthesis of TCR-beta chains, thereby limiting the assembly of a pre-TCR complex. Since TCR-beta V(D)J rearrangements are detected at the CD44-CD25+ cell stages (40, 46), and previous studies have suggested that IL-7/CD127/gamma c interactions may be important for RAG expression during the TCR recombination process (for a review, see reference 47), we tested whether defects in TCR-beta rearrangements were responsible for the developmental block in gamma c- thymi. gamma c- mice were bred with mice bearing a functionally rearranged TCR Vbeta 8.2 transgene (Tg) (48), and thymocyte development was analyzed. As shown in Fig. 3 C, gamma c-/TCR-beta Tg+ mice demonstrated no change in the distribution or absolute numbers of thymocytes compared with non-Tg gamma c- littermates. The lack of discernible effect of the TCR-beta Tg in gamma c- mice was not due to an inability to express this TCR, as surface levels of Vbeta 8.2 were equivalent in gamma c+ and gamma c- total thymocytes and CD25+ thymocyte precursors (data not shown). These results suggest that defects in the TCR-beta chain rearrangement process are not rate limiting in the absence of gamma c, and are consistent with a role for gamma c in promoting survival and proliferation of early thymocytes.

gamma c and Pre-TCR Signaling Pathways Compensate for Each Other in alpha /beta T Cell Development.

To address the relative interdependence of gamma c and pre-TCR signals, we intercrossed the gamma c and pTalpha null strains to generate mice lacking both these molecules. Thymocyte development was analyzed in 3-4-wk-old double mutant mice (gamma c-/pTalpha -/-) as well as single mutants for gamma c or pTalpha and wild-type mice.

Consistent with previous reports (5, 24), the thymi from either gamma c or pTalpha mutant mice demonstrated a reduction in the size (data not shown) and cellularity (20-30-fold; Fig. 4). In contrast, gamma c-/pTalpha -/- thymi were severely hypoplastic and showed a drastic reduction (~4,000-fold) in absolute cell numbers (Fig. 4). Thymocyte cell surface phenotype was further characterized in these mice (Fig. 4, A and B). pTalpha -/- thymi showed an incomplete block in thymocyte development with an accumulation of cells at the DN stage; however, pTalpha -deficient thymocytes are capable of further maturation to DP and SP mature cells. In marked contrast, thymi from double mutant gamma c-/pTalpha -/- mice contained only immature DN cells (Fig. 4 A). Using CD44 and CD25 markers, gamma c- thymi demonstrated the characteristic incomplete block at the CD44+CD25+ to CD44-CD25+ transition (Fig. 4 B), whereas the accumulation of cells at the CD44-CD25+ stage in pTalpha -deficient mice coincides with pre-TCR-mediated cellular expansion and differentiation to the DP stage (for a review, see reference 45). Strikingly, residual thymocytes from gamma c-/pTalpha -/- mutant mice showed a developmental block with a complete arrest of differentiation at the CD44+CD25+ stage (Fig. 4 B). To our knowledge, this is the first description of mutant mice harboring this particular intrathymic defect. In terms of absolute cell numbers, gamma c-/pTalpha -/- thymi contained ~4 × 104 TN precursors, all of which were CD44+, and therefore similar to the number of CD44+ TN precursors found in gamma c- thymi (Fig. 1 B). However, unlike gamma c- or pTalpha -/- single mutant mice, the arrest in thymic development in gamma c-/pTalpha -/- double mutant thymi was complete, as no mature T cells were found intrathymically or in the peripheral lymphoid organs (data not shown, and see below). These results (a) define a critical period of intrathymic development (the CD44+CD25+ to CD44-CD25+ transition) in which signals delivered by gamma c and the pre-TCR pathways appear to overlap, and (b) suggest that pre-TCR signals are responsible for rescue of alpha /beta T cell development in gamma c- mice.


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Fig. 4.   Intrathymic development in gamma c/pTalpha -/- double mutant mice. (A) CD4/CD8 profiles of thymocytes from 3-wk-old mice. (B) CD44/CD25 expression on gated TN thymocytes (see Fig. 1). For these experiments, thymi from four to six gamma c/pTalpha -/- double mutant mice were pooled. Absolute thymocyte cell numbers (mean ± SD) were calculated for the indicated mice (n = 6-10 for each genotype).

It would follow from these results that a pre-TCR can form at the CD44+CD25+ stage. Although several studies have reported the rearrangement status of early thymocyte subsets (40, 46, 49), no studies to date have examined pre-TCR protein expression in these cells. Using reagents specific for the TCR-beta (29) and a newly developed antibody against the pTalpha chain (30), we characterized intracellular pre-TCR components in early thymocytes from gamma c+ and gamma c- mice (Fig. 5). At the CD44+CD25+ stage, thymocytes demonstrate uniform intracellular staining for pTalpha chains, whereas the level of pTalpha expression increases slightly as the cells mature to become CD44-CD25+. A small fraction of CD44+CD25+ cells (3.0 ± 1%; n = 4) also stain intracellularly for TCR-beta protein; this fraction increases to ~20% as these cells downregulate CD44 expression (Fig. 5). TCR-beta and pTalpha protein expression on a per cell basis was not qualitatively or quantitatively altered in gamma c- thymocytes (Fig. 5). These data conclusively demonstrate that a pre-TCR can form during the CD44+CD25+ to CD44-CD25+ transition, a point at which intrathymic precursors express IL-7Ralpha /gamma c (41). These results suggest that gamma c and pre-TCR signals are independent and overlapping for intrathymic development.


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Fig. 5.   Intracellular expression of pTalpha and TCR-beta chain in CD25+ thymocyte precursors. Thymocytes from gamma c+ or gamma c- mice were surface stained for TN cells (see Fig. 1), CD44, and CD25, fixed, and permeabilized with saponin before detection of intracellular (IC) TCR-beta or pTalpha chains. Gated CD44+CD25+ and CD44-CD25+ thymocyte subsets are boxed. Negative controls (dotted lines) are staining of thymocytes from RAG-2-deficient (for TCR-beta ) or pTalpha -deficient (for pTalpha ) mice.

Characterization of the gamma c-/pTalpha -/- Thymic Rudiment.

Due to the severe reduction in thymocyte cell numbers in gamma c-/pTalpha -/- mice, a PCR-based strategy (31) was used to identify any TCR-beta rearrangements present in these mutant thymi. DNA from control, gamma c-, or pTalpha -/- thymi contained abundant TCR-beta V(D)J rearrangements, which were diverse with respect to junctional sequences present in the CDR3 region (Fig. 6; and data not shown). In contrast, rearrangements from gamma c-/pTalpha -/- mutant thymi were reduced in overall amounts, although samples derived from independent thymi contained multiple and different bands, indicating rearrangements to different Jbeta segments (Fig. 6). Sequence analysis of these PCR products revealed unique Vbeta CDR3 sequences, suggesting that the observed reduction in rearrangements was related to the paucity of absolute cell numbers and not to a restricted rearrangement potential (data not shown). Finally, TCR-beta rearrangements were absent from the spleens of gamma c-/pTalpha -/- double mutant mice, demonstrating that the intrathymic block in alpha /beta T cell development was complete and that no mature alpha /beta T cells were produced that could seed the peripheral lymphoid organs (Fig. 6).


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Fig. 6.   TCR-beta rearrangements in thymus and spleen. Genomic DNA from the indicated mice were amplified by PCR using a combination of primers specific for TCR Vbeta 6 and Vbeta 8 (sense) and a primer specific for the 3' region of TCR Jbeta 2.5 (antisense). Amplification products were detected by blot hybridization using a probe specific for the 5' region of TCR Jbeta 2.5.

    Discussion
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

alpha /beta T cells are generated intrathymically through a series of developmental steps involving survival, expansion, and differentiation of immature precursor cells. The gamma c chain plays a critical role in this process, primarily by relaying signals from stromal cell-derived IL-7 to developing thymocytes. The essential role of IL-7 in early thymocyte differentiation has been difficult to define because this cytokine has been postulated both to act as a trophic factor and to influence the TCR rearrangement process (for a review, see reference 47). Moreover, deficiencies in IL-7/ IL-7Ralpha /gamma c abrogate gamma /delta T cell development, whereas alpha /beta T cell development is permissive, suggesting either a differential role for the IL-7 receptor complex in the generation of these two T cell subsets or the existence of compensatory pathways that rescue alpha /beta T cells in the absence of IL-7/IL-7Ralpha /gamma c. In this report, we identify the pre-TCR as a rescue mechanism for alpha /beta T cell development in gamma c- mice. This result suggests that a model of intrathymic differentiation involves an overlapping series of signals derived from growth factors and TCRs that guide the maturation process (Fig. 7). This model is consistent with the permissive nature of thymocyte development in c-kit-, IL-7Ralpha -, gamma c-, and pTalpha -deficient mice (4, 15, 24, 50), and sheds light on the compensatory signaling pathways that exist to insure alpha /beta T cell differentiation in these mutant mice.


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Fig. 7.   Cytokine and TCR signaling in intrathymic development. Hematopoietic stem cells (HSC) give rise to thymocytes via common lymphoid progenitor cells (CLP). Within the thymus, TN precursor cells can be further subdivided based on expression of CD44 and CD25. Immature SP cells (ISP) are the immediate precursors of DP thymocytes, which after tolerance induction via alpha /beta TCR selection develop into CD4+ or CD8+ SP cells which exit the thymus to seed peripheral lymphoid organs. gamma /delta T cells can derive from the various subsets of TN cells. Concerning the molecules required to generate and maintain alpha /beta T cells, growth factor receptors including c-kit and the common gamma  chain (gamma c) in concert with the pre-TCR and TCR-alpha /beta appear to provide overlapping signals which guide the developmental process. In the absence of either IL-7Ralpha , gamma c, or the pre-TCR, signals provided by adjacent pathway(s) permit a limited degree of alpha /beta thymocyte development. In contrast, when two adjacent signals are absent (for example, in c-kit × gamma c or gamma c × pTalpha double mutants), alpha /beta T cell development is abrogated. Moreover, alpha /beta T cells require continual TCR triggering in the periphery. In contrast, gamma /delta T cells appear strictly cytokine dependent, both intrathymically and in the periphery. Hatched circles, cycling cells.

Before the expression of a rearranged TCR chain, immature thymocytes are maintained and proliferate in response to factors provided within the thymic milieu (for a review, see reference 51). Although several cytokines have been shown to act on thymic precursors, stem cell factor (SCF) and IL-7 remain the two dominant factors that can promote their survival and/or expansion (14, 50, 52). The receptors for stem cell factor (c-kit) and for IL-7 (the IL-7Ralpha /gamma c complex) are coexpressed on early thymocytes (41, 53, 54), and proliferation of these cells is reduced in the absence of c-kit or IL-7Ralpha /gamma c (50, 55; and this report). The permissive nature of thymocyte development in c-kit or IL-7Ralpha /gamma c mutants implies redundancy in the pathways that maintain early precursors. The hypothesis that c-kit and IL-7Ralpha /gamma c signals could compensate for each other at this stage is strongly supported by the complete abrogation of thymocyte development (before the CD44+CD25- cell stage) in mice deficient in both c-kit and gamma c (26). Thus, for cells up to the CD44+CD25- stage, c-kit and gamma c act synergistically to maintain cells before TCR rearrangements. The essential nature of c-kit and gamma c signals cannot be replaced by other growth factors at this stage of development (26).

At the transition from the CD44+CD25+ stage to the CD44-CD25+ stage, rearrangements of the TCR-beta chain begin (40, 46, 49). Since IL-7- and gamma c-deficient thymocyte precursors are most severely affected at this stage (55; Fig. 1 B), the failure to signal through gamma c could have an adverse effect on the TCR rearrangement process. Consistent with this hypothesis, previous studies have demonstrated that transgenic expression of a functionally rearranged TCR-alpha /beta (against the male-specific [HY] antigen in association with H-2Db) could partially restore total thymocyte numbers in gamma c- and IL-7Ralpha -deficient mice (18, 19). However, these experiments failed to rule out potential effects associated with TCR signaling under conditions of positive selection, since increases in thymic cellularity were only observed in H-2Db female mice. Here we show that the same TCR-beta alone has no effect on thymocyte development in gamma c- mice. This observation strongly suggests that defective TCR-beta rearrangements alone cannot account for the abnormal alpha /beta T cell development in the absence of gamma c, and supports the idea that poor survival and reduced proliferation of the CD44+CD25+ and subsequent thymocyte subsets are the major limiting factors for alpha /beta T cell development in these mice.

The results presented here identify the pre-TCR as an independent and essential signal which acts in concert with gamma c during alpha /beta thymopoiesis. Although in principle the failure to signal through gamma c could have an adverse effect on pre-TCR assembly or function, we find that beta  selection and pre-TCR-mediated expansion appear gamma c independent. However, the essential role of the pre-TCR in gamma c- mice is clearly illustrated by the gamma c/pTalpha double mutants. In these mice, thymocyte development proceeds only to the CD44+CD25+ stage (thereby delimiting the role of the c-kit pathway), and implies that further development requires either gamma c or pre-TCR signals. As indicated above, gamma c can act via IL-7 to maintain early thymocytes at this stage (20, 55) and might also serve to drive differentiation to the CD44-CD25+ stage where TCR rearrangements are ongoing (40, 46, 49). How, then, could the pre-TCR rescue gamma c-deficient cells at such an early stage?

To address this issue, we examined expression of TCR-beta and pTalpha proteins in CD44+CD25+ and CD44-CD25+ thymocytes from gamma c+ and gamma c- mice. Our results clearly demonstrate that a pre-TCR complex can potentially form in a small subset of CD44+CD25+ cells. The expression of a pre-TCR at this stage could thereby provide a compensatory mechanism in gamma c- mice to enable alpha /beta T cell development. Moreover, the "window" of pre-TCR expression was similar in gamma c+ and gamma c- mice. In this respect, gamma c and pre-TCR signals are independent and overlapping at this stage of intrathymic development (Fig. 7).

The signaling cascades initiated from gamma c and pre-TCRs appear distinct. gamma c receptors activate the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway (for a review, see reference 56), whereas the pre-TCR uses immunoreceptor tyrosine-based activation motif (ITAM)-containing CD3 components which couple to the src family and ZAP-70/syk family tyrosine kinases (for reviews, see references 42 and 57). How the pre-TCR mediates proliferation of late thymocyte precursors is unknown, but our results indicate that this process does not require gamma c-dependent cytokines. Further work will be required to identify the mechanism by which triggering the pre-TCR engages the cell cycle.

Our observations provide insights into the difference between alpha /beta and gamma /delta T cell development in IL-7/IL-7Ralpha / gamma c-deficient mice (12, 17, 58). Although the pre-TCR is capable of rescuing TCR-alpha /beta cells in gamma c- mice, gamma /delta T cells lack an equivalent mechanism and therefore must rely on other signals for their final intrathymic differentiation. We have previously shown that transgenic expression of rearranged TCR-gamma or TCR-gamma /delta chains failed to rescue gamma /delta T cell development in gamma c- mice, suggesting that gamma c-dependent cytokines provide the dominant signals for gamma /delta T cell survival and/or proliferation both intrathymically and in the periphery (28). On the other hand, peripheral maintenance of alpha /beta T cells requires continual TCR stimulation (for reviews, see references 59 and 60) but appears less gamma c dependent in their development.

Finally, our model suggests that differences in T cell development between human and murine gamma c deficiency might be related to species-specific differences in the function of c-kit, IL-7/IL-7Ralpha /gamma c, or the pre-TCR. Little is known about the specific patterns of expression of these molecules with regard to the various stages of intrathymic development in humans. Moreover, the documented differences between human and mouse pTalpha cytoplasmic sequences (61) could result in differential signaling properties of the pre-TCR between species. Support for this model will require further studies focusing on these pathways in human thymocyte development.

    Footnotes

Address correspondence to James P. Di Santo, INSERM U429, Pavillon Kirmisson, Hôpital Necker- Enfants Malades, 149, rue de Sèvres, F-75743 Paris, France. Phone: 33-1-44-49-50-51; Fax: 33-1-42-73-06-40; E-mail: disanto{at}necker.fr

Received for publication 9 September 1998 and in revised form 17 November 1998.

1   Abbreviations used in this paper: BrdU, bromo-deoxyuridine; DN, double negative; DP, double positive; gamma c, common cytokine receptor gamma  chain; RAG, recombination activating gene; SP, single positive; TN, triple negative; Tg, transgene; TRI, Tricolor.

We thank D. Guy-Grand (Paris), M. Malissen, B. Malissen (Marseille), and H.-R. Rodewald (Basel) for stimulating discussions.

Supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Association pour le Recherche sur le Cancer, and the Ligue Nationale Contre le Cancer. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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