The Common Cytokine Receptor gamma  Chain Controls Survival of gamma /delta T Cells

By Marie Malissen,* Pablo Pereira,Dagger David J. Gerber,§ Bernard Malissen,* and James P. DiSantopar

From the * Centre d'Immunologie Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Marseille Luminy, Case 906, 13288 Marseille Cedex 9, France; Dagger  Unité d'Immunologie, Centre National de la Recherche Scientifique Unité Recherche Associée 1961, Institut Pasteur, 75724 Paris, France; § Center for Cancer Research, Massachusetts Institute of Technology, 02139 Cambridge, Massachusetts; and par  INSERM U429, Hôpital Necker-Enfants Malades, 75743 Paris, France

Summary
Materials and Methods
Results and Discussion
Footnotes
Acknowledgements
References


Summary

We have investigated the role of common gamma  chain (gamma c)-signaling pathways for the development of T cell receptor for antigen (TCR)-gamma /delta T cells. TCR-gamma /delta -bearing cells were absent from the adult thymus, spleen, and skin of gamma c-deficient (gamma c-) mice, whereas small numbers of thymocytes expressing low levels of TCR-gamma /delta were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-gamma /delta development through induction of V-J (variable-joining) rearrangements at the TCR-gamma locus. In contrast, we detected clearly TCR-gamma rearrangements in fetal thymi from gamma c- mice (which fail to signal in response to IL-7) and reduced TCR-gamma rearrangements in adult gamma c thymi. No gross defects in TCR-delta or TCR-beta rearrangements were observed in gamma c- mice of any age. Introduction of productively rearranged TCR Vgamma 1 or TCR Vgamma 1/Vdelta 6 transgenes onto mice bearing the gamma c mutation did not restore TCR-gamma /delta development to normal levels suggesting that gamma c-dependent pathways provide additional signals to developing gamma /delta T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from gamma c- mice were dramatically reduced compared to gamma c+ transgenic littermates. We favor the concept that gamma c-dependent receptors are required for the maintenance of TCR-gamma /delta cells and contribute to the completion of TCR-gamma rearrangements primarily by promoting survival of cells committed to the TCR-gamma /delta lineage.


Tcells can be divided into two populations based on the structure of their TCRs. Most T cells express TCR heterodimers consisting of alpha  and beta  chains, whereas a smaller population expresses an alternative TCR made of gamma  and delta  chains. These two T cell populations share a number of features, including rearranging antigen receptor chains, the products of which associate with a set of invariant CD3 polypeptides responsible for signaling to the cell that the TCR heterodimer has been engaged (for review see reference 1). In contrast, gamma /delta T cells differ from alpha /beta T cells in their ontogeny, variable (V)1 gene repertoires, and ultimate anatomical locations (for reviews see references 2 and 3). gamma /delta T cells are the first TCR-expressing cells detected in the early fetal thymus, and persist in the adult thymus in small numbers. The first two waves of gamma /delta T cells to appear during a fetal development express Vgamma 5-joining gamma 1 (Jgamma 1) and Vgamma 6-Jgamma 1 genes, both of which pair with delta  chains composed of Vdelta 1-Ddelta 1-Jdelta 2 segment combinations. The most striking feature of these TCR-gamma /delta genes is their lack of junctional diversity (2, 3). The Vgamma 5-expressing cells migrate to the skin to become the Thy1+ dendritic epidermal T cell (DETC) population, whereas Vgamma 6-expressing cells migrate to mucosal surfaces lining the reproductive tract and tongue (4). The production of Vgamma 5/Vdelta 1 and Vgamma 6/ Vdelta 1 cells slows at the end of fetal life and another wave of TCR-gamma /delta cells develops from this time onwards, which uses mainly Vgamma 4 and Vgamma 1 gene segments paired with a variety of Vdelta genes and which displays extensive junctional diversity. In the adult, these gamma /delta T cells constitute ~0.2% of the thymus and following export, seed the spleen and lymph nodes. The mechanisms that control the sequential appearance of gamma /delta T cell subsets with distinct V gene segment usage, V(D)J junctional diversity, and unique homing properties are unknown (5, 6). Recent evidence suggests that the observed TCR-gamma and TCR-delta gene rearrangements are temporally programmed and do not rely on selection of a particular subset of receptors among a diverse TCR-gamma /delta combinatorial repertoire (7).

Deciphering the role played by cell-cell interactions and soluble cytokines provided by the thymic microenvironment constitutes a central question in the development of fetal versus adult gamma /delta T cells. A variety of interleukins have been demonstrated to affect the growth and differentiation of TCR-gamma /delta cells. For instance, freshly isolated gamma /delta T cells from fetal thymus, skin, spleen, or peritoneal cavity can proliferate in vitro to IL-2, IL-7, or IL-15 (10), and in utero administration of antibodies to the IL-2Rbeta chain block development of DETC (14). Gene ablation experiments in vivo have confirmed some of these findings. Mice deficient in IL-2Rbeta (shared by IL-2 and IL-15), IL-7/IL-7Ralpha , or the common gamma  chain (gamma c; shared by IL-2, IL-4, IL-7, IL-9, and IL-15) each have defects in gamma /delta T cell development (15). Still, the mechanism by which cytokine depletion affects the differentiation program of these cells is not completely understood. Cytokines could play a role in survival or proliferation of developing thymocytes, or alternatively might directly influence the TCR rearrangement process. Along these lines, experiments using mouse fetal liver cultures supplemented with IL-7 suggested that this cytokine could specifically induce the rearrangement of TCR-gamma chain genes (21), and recently IL-7Ralpha -deficient mice were shown to have a selective block in TCR-gamma gene recombination (22). In this report, we have analyzed mice deficient in gamma c to clarify the role of the gamma c in the development of fetal and adult gamma /delta T cells. Our results demonstrate that gamma c-containing receptor complexes play a role for TCR-gamma chain rearrangements in the adult, but not the fetal thymus, and more importantly, signaling through the gamma c provides essential survival signals for gamma /delta T cells.


Materials and Methods

Mice. Mice harboring a null mutation of the gamma c have been described (19), gamma c-deficient mice, IL-7-deficient mice (kindly provided by R. Murray, DNAX Corp, Palo Alto, CA; reference 23), recombinase-activating gene 1-deficient mice (RAG1-/-; ref. 24), and their littermate controls were maintained in specific pathogen-free conditions and used between 4 and 8 wk of age. Fetal tissues were obtained from timed-pregnant mice. Day 0 of embryonic development was considered to be the day a vaginal plug was observed.

Mice transgenic (Tg) for TCR Vgamma 1 and double Tg for TCR Vgamma 1 and TCR Vdelta 6 were constructed as follows. Genomic DNA fragments containing the rearranged TCR Vgamma 1 and Vdelta 6 genes were isolated from a cosmid library prepared in pWE15 using partially digested Sau 3A I DNA from the T3.13.1 TCR-gamma /delta hybridoma (25). The Vgamma 1-Jgamma 4 clone (45 kb) contained 10 kb of upstream sequence and extended 26 kb downstream of the Cgamma 4 exon. The Vdelta 6-D-Jdelta 1 clone (34 kb) contained 5 kb of upstream DNA and 14 kb downstream of the Cdelta exon. Tg constructs were mixed and the DNA microinjected into the pronuclei of fertilized embryos. Mice carrying the Tg TCRs were identified by PCR and backcrossed onto the C57Bl/6 background. Mice were screened using tail DNA and primers specific for the Tg Vgamma 1 TCR: forward, 5'-CCGGCAAAAAGCAAAAAAGTT-3'; and reverse, 5'-CCCATGATGTGCCTGACCAG-3'. PCR conditions were as follows: denaturation at 94°C for 20 s, annealing at 59°C for 25 s, and extension at 74°C for 25 s for 33 cycles.

Antibodies. The following Abs were used as conjugates to either FITC, PE, or biotin: anti-TCR-beta (H57-597), anti-TCR-gamma /delta (GL3), anti-TCR Vgamma 5 (536), anti-CD3 (2C11), anti-CD4, anti-CD8alpha , anti-CD24 (J11d), anti-Mac-1 (M1/70), and anti-CD32 (2.4G2). Streptavidin Tricolor (CALTAG Labs., South San Francisco, CA) was used to detect biotinylated antibodies. The antibody specific for TCR Vgamma 1 (2.11) has been described (25). A clonotypic antibody recognizing Vgamma 1/Vdelta 6 TCR heterodimer (1.9) was obtained in the same fusion and its specificity verified using a panel of gamma /delta T cell hybridomas as described (25).

FACS® Analysis and Cell Sorting. Single cell suspensions obtained from thymus or spleen were lysed of red cells using hypotonic NH4Cl solution. DETCs were isolated from ear skin using trypsinization and mechanical disaggregation as described (26). Nonspecific binding of mAbs to FcRs was reduced by preincubation with anti-CD32 mAb for 15 min. For surface staining, cells were incubated with saturating amounts of directly conjugated mAbs for 20 min, washed twice, and incubated with streptavidin Tricolor. For Bcl-2 staining, surface antigens were stained as above, and cells were washed in PBS and fixed in PBS containing 1% paraformaldehyde/0.01% Tween-20 for 90 min on ice. Cells were subsequently incubated with hamster anti-mouse Bcl-2 (clone 3F11; PharMingen, San Diego, CA) or purified hamster Ig. Cells were washed, incubated with biotinylated goat anti- hamster Ig, and finally with streptavidin Tricolor. Cells were analyzed on a FACScan® flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA). For isolation of early thymocyte precursors, cells were stained with CD4-FITC and CD8-PE, and double negative (DN) cells sorted using a FACStar Plus® cell sorter (Becton Dickinson).

PCR Analysis. DNA samples were extracted from total or fractionated populations of fetal or adult thymocytes using the salting-out technique (27). PCR reactions were done in a final volume of 20 µl and included a maximum of 100 ng of template DNA, 1 mM of each primer, 200 µM of each deoxynucleotide triphosphate, and 0.2 U of Taq DNA polymerase. Whole reaction mixtures were run on a 1.5% agarose gel, blotted to nylon membrane (Gene Screen Plus, New England Nuclear, Boston, MA), and hybridized with 32P-labeled oligonucleotide probes.

The oligonucleotides and PCR conditions used for the analysis of TCR-beta rearrangements were as described (28). For the TCR-gamma and TCR-delta rearrangements, PCR were performed essentially as described (9) except that each cycle was shortened as follows: incubation at 94°C for 30 s, annealing at 50-60°C for 30 s, and extension at 72°C for 40 s. At least two sets of independent experiments were performed for each sample. To show that there is a linear relationship between product yield and the number of input target sequences, serial dilutions have been analyzed for each DNA sample. Hybridizing bands were quantitated using a phosphorimager (BAS1000; Raytest, Courbevoie, France). Before the analysis of the relative levels of TCR gene rearrangements, the quality and quantity of DNA present in each sample were checked by amplifying the nonrearranging trithorax gene (MTrx; reference 29) using primers MTrx1: 5'-AGGGTAAGCTGTGCTATGG-3' and MTrx2: 5'-AGTAGTGTTTCCTCAGTCCCC-3'.


Results and Discussion

Absence of TCR-gamma /delta Cells in gamma c-deficient Mice.

In vitro data have suggested an important role for IL-2, IL-7, and IL-15 in the survival, proliferation, and differentiation of cells belonging to the gamma /delta T cell lineage (10). Because the gamma c receptor plays an integral role in the function of these cytokine receptors, we anticipated that gamma c- mice would exhibit a defect in gamma /delta T cell development. As most adult gamma / delta  T cells fail to express the CD4 and CD8 coreceptors and are found in the DN subset of T cells, we examined wild-type (wt) and gamma c- DN thymocytes for the presence of cells bearing TCR-gamma /delta receptors. Compared to control mice, adult thymi from gamma c- mice showed a complete absence of TCR-gamma /delta + cells (Fig. 1). Further analysis of the peripheral lymphoid organs, skin, and small intestine of adult gamma c-deficient mice failed to demonstrate any TCR-gamma /delta + cells in the animal (Fig. 1 and data not shown). These results confirm and extend previous observations demonstrating the strict dependence on gamma c-containing receptors for the development of all types of gamma /delta T cells present in adult mice (15).


Fig. 1. Flow cytometric analysis of gamma /delta T cells from adult gamma c+ and gamma c- mice. (A) Thymocytes were stained with FITC-anti-pan-TCR-gamma /delta (GL3), PE-anti-CD3, biotin-anti-CD4, and biotin-anti-CD8alpha . CD4- CD8- (DN) cells were electronically gated. (B) Skin-resident DETCs were isolated as indicated in Materials and Methods, and stained with biotin-anti-TCR Vgamma 5 and FITC-anti-CD3. (C) Splenocytes were monocyte depleted by adherence and stained with FITC-anti-pan-TCR-gamma /delta , PE-anti-CD3, biotin-anti-CD4, and biotin-anti-CD8alpha . CD3+ DN cells were electronically gated. Biotinylated antibodies were revealed with streptavidin-Tricolor.
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Considering that gamma /delta T cells constitute a minor cell population in the adult thymus, we next analyzed fetal thymi from gamma c- mice, since they contain a higher frequency of gamma / delta  T cells (9). Total thymocyte cell numbers were clearly reduced in gamma c- mice relative to control mice at all stages of fetal development examined (Table 1) although CD4 and CD8 expression was unaltered (data not shown). An ~10-fold reduction in cell number is apparent in gamma c- fetal thymi, yet with age, thymocyte cell numbers increase in parallel with gamma c+ mice. This suggests that alternative gamma c-independent signaling pathways (including that of the receptor tyrosine kinase c-kit; reference 30) support continuous thymic seeding and permit progressive thymocyte accumulation. Analysis of fetal (days 16-18) gamma c-deficient thymocytes revealed a reduced percentage of cells expressing TCR-gamma /delta heterodimers (Fig. 2 and data not shown) and suggested that gamma /delta T cells might have some capacity to develop in that context, but might subsequently be lost, perhaps due to poor survival or failure to mature. Consistent with this hypothesis, the few fetal TCR-gamma /delta thymocytes found in gamma c- mice have an immature phenotype characterized by low levels of TCR-gamma /delta heterodimers and high levels of heat-stable antigen (HSAhi; Fig. 2). Similar observations have been made in IL-7-deficient mice (16), pointing to IL-7 as the gamma c-dependent cytokine responsible for the defect observed in gamma c- thymi.

Table 1. Cellularity of Fetal and Neonatal Thymi from gamma c- Mice


Total cell number (× 105)
Age  gamma c+  gamma c-

FD15.5 2.46 ± 1.5 (6)*    0.3 ± 0.2 (6)
FD17.5 33.6 ± 9.1 (9)  3.8 ± 2.1 (10)
FD18.5 56.1 ± 20.3 (7)  7.0 ± 3.3 (3)
Neonate D1 96.8 ± 30 (6)  9.2 ± 2.7 (6)
Neonate D6    500 ± 15 (3) 22.6 ± 9.6 (3)

*  Number of mice analyzed. Numbers are mean ± SEM.


Fig. 2. Flow cytometric analysis of fetal thymocytes from gamma c+ and gamma c- mice. (A) Thymocytes were stained with FITC-anti-pan-TCR-gamma /delta and PE-anti-CD3. (B) Thymocytes were stained with FITC-anti-pan-TCR-gamma /delta and PE-anti-HSA. In all experiments, nonspecific binding was blocked by the addition of anti-CD16/32.
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Taken together, these observations confirm the major role played by the IL-7/gamma c-signaling pathway in gamma /delta T cell development (16), but also suggest that factors independent of gamma c and IL-7 are present in the fetal, but not the adult, thymus and can support the appearance of the most immature gamma /delta T cells. Recently, a novel cytokine, thymic stromal cell-derived lymphopoeitin (TSLP; reference 31), has been identified which shares biological activities with IL-7 and uses the IL-7Ralpha chain for signaling (32). We would hypothesize that TSLP could partially replace IL-7 during fetal development in gamma c- mice. Two additional observations support this view. First, thymocytes from gamma c- mice respond to TSLP (33) and second, mice with a deletion of the IL-7Ralpha chain have neither fetal nor adult TCR-gamma /delta cells detectable by FACS® analysis (17). Therefore, the spectrum of cytokine receptors expressed by fetal and adult immature thymocytes may be identical, but fetal versus adult thymic stroma may differ in their abilities to produce cytokines, like TSLP. Along this line, it will be interesting to determine the relative expression of TSLP in fetal and adult thymi.

TCR Rearrangements in gamma c- Thymocytes.

We considered a number of nonexclusive hypotheses to explain the pronounced negative effect of gamma c deficiency on gamma /delta T cell development: (a) gamma c may be required for committment to the TCR-gamma /delta lineage (defect in alpha /beta -gamma /delta lineage branching), (b) gamma c may be required for initiation or completion of the site-specific DNA recombination process affecting the TCR-gamma and/or TCR-delta loci (defect in TCR rearrangements), or (c) signals via gamma c may be essential for the survival of cells during the process of gamma /delta T cell differentiation.

To explore these different possibilities, we analyzed the status of TCR gene rearrangements in fetal and adult thymocytes from wt and gamma c-deficient mice using a polymerase chain reaction technique that can specifically assess the presence of rearranged DNA from the various TCR gene loci (9, 28). The results shown in Fig. 3 were generated using fetal (day 17) thymocytes and PCR primer pairs specific for the Vgamma 5-Jgamma 1, Vgamma 1-Jgamma 4, Vdelta 1-Jdelta 2, and Vdelta 4-Jdelta 1 rearrangements. Although Vdelta 4-Jdelta 1 and Vgamma 1-Jgamma 4 rearrangements were nearly unaffected in gamma c- fetal thymi, the canonical Vdelta 1-Jdelta 2 and Vgamma 5-Jgamma 1 rearrangements found in thymocytes destined to seed the skin epithelium (4) were clearly reduced (Fig. 3). As expected from the fact that the gamma c mutation does not block the development of alpha /beta T cells (19, 20), DNA samples from gamma c thymi contained Dbeta -Jbeta and Vbeta -Dbeta -Jbeta rearrangements that were as diverse as those found in wt samples (data not shown).



Fig. 3. Quantitation of TCR-delta and TCR-gamma rearrangements in fetal gamma c+ and gamma c- thymocytes. (A) Indicated rearrangements were amplified from DNA isolated from unfractionated D17 fetal gamma c+ or gamma c- thymi. Serial dilutions of DNA template were analyzed (1×, 2×, 4×, and 8×). (B) Quantification of A. Hybridizing bands were scanned using a phosphorimager and the relative levels of rearrangements compared to wild-type (WT) gamma c+ thymi after normalizing for input DNA using MTrx primers. See Materials and Methods for details.
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Similar studies were performed on adult gamma c- thymi. As shown in Fig. 4, the Vgamma 1-Jgamma 4 rearrangements detected in 4-wk-old gamma c- thymi represent 23% of those observed in wt thymi, respectively. Only limited Vgamma 4-Jgamma 1 rearrangements are observed in adult gamma c- thymi, representing ~1% of the levels observed in adult wt thymi. In contrast, the extent of TCR-delta rearrangements found in adult thymi from gamma c-mutant mice resembled those present in wt adult thymi (Fig. 4 B, top). Thus, these results suggest that the gamma c mutation has little effect on adult TCR-delta rearrangements, but appears to selectively reduce adult TCR-gamma rearrangements. Parallel studies using IL-7-deficient mice demonstrated a similar defect in TCR-gamma rearrangements (Table 2).



Fig. 4. Quantitation of TCR-delta and TCR-gamma rearrangements in adult gamma c+ and gamma c- thymocytes. (A) Rearrangements of Vdelta 4-Jdelta 1 and Vgamma 1-Jgamma 4 were amplified from DNA isolated from unfractionated gamma c+ or gamma c- thymi. Serial dilutions of DNA template were analyzed (1×, 2×, and 4×). (B) Quantification of A. Relative levels of rearrangements are compared to wild-type (WT) gamma c+ thymi.
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Table 2. TCR-gamma and TCR-delta Rearrangements in gamma c- and IL-7-/- Thymi


DNA source Vdelta 4-Jdelta 1 Vgamma 4-Jgamma 1

 gamma c+ 100.00* 100.00
RAG 1-/-   2.1   0.4
 gamma c- 108.4   2.66
IL-7-/- 114.5   3.79

*  Rearrangements were quantitated and expressed as percentage of rearrangements found in DNA isolated from gamma c+ total thymocyte preparations from 4-wk-old mice (see Materials and Methods). Results are representative of two independent experiments.

Most developing alpha /beta T cells contain TCR-delta gene segments that have rearranged during the DN stages of development (34, 35). As a result, TCR-alpha /beta + thymocytes retain TCR-delta locus sequences (36), which could account for the split phenotype observed for the TCR-gamma and TCR-delta loci in unfractionated adult gamma c- thymocytes (Fig. 4). We therefore examined TCR-delta rearrangements in CD4-CD8- precursors from gamma c mutant mice. As summarized in Table 3, the extent of TCR-delta rearrangement present in gamma c- DN thymocytes was found to be similar to that observed in total gamma c- thymocytes and in CD3epsilon Delta 5/Delta 5 thymocytes, which are an enriched source of gamma c+ DN cells with normal levels of TCR-delta gene rearrangements (34). Therefore, these findings demonstrate that TCR-delta genes do rearrange in the CD4-CD8- precursors isolated from adult gamma c- mice. TCR-gamma rearrangements in gamma c- DN thymocytes were also reduced (data not shown).

Table 3. TCR-delta Rearrangements from gamma c- Thymocyte Precursors


DNA source Vdelta 4-Jdelta 1 Vdelta 5-Jdelta 1

 gamma c+ total thymus 100.00* 100.00
RAG 1-/- thymus   1.61   1.56
 gamma c- total thymus  94.36  75.40
 gamma c- DN sortDagger 111.92  55.58
CD3epsilon Delta 5/Delta 5 thymus  73.24 101.43

*  Indicated TCR-delta rearrangements were quantitated and expressed as percentage of rearrangements found in DNA isolated from gamma c+ total thymocyte preparations (see Materials and Methods). Results are representative of two independent experiments.
Dagger  Double negative thymocytes (CD4-CD8-) were sorted from gamma c- thymi as described in Materials and Methods, and DNA was isolated for analysis.

In conclusion, our analysis of TCR-gamma and TCR-delta rearrangements in fetal and adult gamma c- thymi distinguishes TCR-gamma /delta cell development during these two stages. Although noncanonical TCR-gamma and TCR-delta rearrangements were both present during the fetal period, TCR-gamma , but not TCR-delta , rearrangements were severely reduced in the adult gamma c- thymus. The implications of these observations are the following. First, during fetal life, the absence of gamma c signaling pathways does not impair the ability to rearrange the TCR-gamma or TCR-delta loci. This suggests that gamma c-independent factors may compensate for the lack of IL-7/gamma c-mediated signals. Second, the fact that fetal and adult thymi from gamma c- mice do contain TCR-gamma and TCR-delta rearrangements, but fail to give rise to mature gamma /delta T cells strongly suggests that other defects (e.g., survival of already committed or successfully rearranged gamma /delta T cells) likely accounts for the defective gamma /delta T cell development observed in gamma c thymi.

Lack of TCR-gamma /delta Cell Survival in gamma c-deficient Mice Tg for Rearranged TCR Vgamma 1 or TCR Vgamma 1/Vdelta 6.

To further address potential defects in alpha /beta -gamma /delta lineage branching and/ or gamma /delta T cell survival, we crossed gamma c-deficient mice with mice Tg for a functionally rearranged TCR Vgamma 1 gene or with double Tg mice harboring the same TCR Vgamma 1 and a productively rearranged TCR Vdelta 6 gene. The T3.13.3 hybridoma from which these rearranged TCR chains were isolated corresponds to the subset of adult TCR-gamma /delta cells and the TCR Vgamma 1/Vdelta 6 heterodimer demonstrates extensive junctional diversity (25). In addition, the Vgamma 1 Tg construct contains the necessary flanking DNA sequences to ensure proper expression in TCR-gamma /delta precursors, as well as the silencer element required to prevent its adventitious expression in TCR-alpha /beta lineage cells (39). Founder mice expressing the Vgamma 1 or Vgamma 1/Vdelta 6 constructs were identified and crossed onto the gamma c-deficient background.

Expression of the Vgamma 1 Tg alone in mice with or without the gamma c mutation did not alter absolute thymocyte cell numbers or the expression of mature CD4 or CD8 single positive thymocytes (Table 4; Fig. 5). Total thymocyte preparations from nontransgenic littermates contained only a very small percentage of cells marked with the anti-TCR Vgamma 1 antibody, whereas gamma c+ Vgamma 1 Tg animals demonstrate an increase in both the frequency (Fig. 5) and absolute numbers of Vgamma 1 gamma /delta T cells (Table 4). Importantly, the Vgamma 1+ cells were negative for TCR-beta chains, demonstrating that the Tg was correctly expressed in TCR-gamma /delta cells and that the flanking silencer element was operative in the alpha /beta T cells found in these Tg mice (Fig. 5). gamma c- TCR-gamma Tg animals showed a population of Vgamma 1+ cells in total thymus preparations; these Tg+ thymocytes were more clearly demonstrated in the DN compartment (Fig. 5). Despite expression of the Tg Vgamma 1 chain, gamma /delta T cells in gamma c- mice were still severely reduced. Compared with gamma c+ Tg animals, there was a 120-fold reduction in absolute numbers of Vgamma 1+ cells (Table 4). Considering that the gamma c chain plays a role in the generation of the earliest noncommitted thymic precursors (CD44+CD25- cells; reference 40) which are 15-fold reduced in gamma c- mice (data not shown), part of the dramatic reduction in Vgamma 1+ thymocytes in gamma c- Tg mice stems from the limited number of thymocyte precursors available to express the Tg Vgamma 1 receptor. Taking this into account, gamma /delta T cells are still eightfold reduced (120-fold/15-fold) in gamma c- Tg mice relative to gamma c+ Tg controls, suggesting that additional mechanisms are responsible for the defective gamma /delta T cell development. Similar results were obtained using mice expressing the same TCR Vgamma 1 chain and a rearranged TCR Vdelta 6 (Fig. 6 and data not shown). We conclude that a rearranged TCR-gamma /delta Tg does not restore normal gamma /delta T cell development in the absence of gamma c. Moreover, since gamma c- mice can express the TCR-gamma /delta transgenes, a defect in TCR-alpha / beta -gamma /delta lineage branching can be effectively ruled out.

Table 4. Transgenic gamma /delta T Cell Development in gamma c- Mice


Mouse n* Total thymocyte cell No. (× 106) Thymic TCR-gamma /delta cell No. (× 105) Splenic lymphoid cell No. (× 106) Splenic TCR-gamma /delta cell No. (× 105)

 gamma c+ Non-Tg >10    247 ± 34           2.5± 0.3 (0.1%Dagger ) 66.5 ± 10.9       3.3 ± 0.6 (0.6%Dagger )
 gamma c- Non-Tg >10 11.7 ± 6.6 ND  8.2 ± 3.2 <0.05 (<0.05%)
 gamma c+ TCR Vgamma 1 Tg 5    198 ± 23.1     29 ± 3.4 (1.4%) 45.2 ± 4.6     20 ± 6.5 (4.0%)
 gamma c- TCR Vgamma 1 Tg 7 8.22 ± 4.6 0.24 ± 0.1 (0.3%)  4.7 ± 1.1 0.14 ± 0.07 (0.4%)

*  Number of mice analyzed.
Dagger  Percentage of cells positive for TCR Vgamma 1 determined by FACS® analysis. ND: see Fig. 1.


Fig. 5. Flow cytometric analysis of thymocytes from gamma c+ or gamma c- mice transgenic for a rearranged TCR Vgamma 1 receptor. (A) Cells were stained with FITC-anti-CD8alpha and PE-anti-CD4. (B) Cells were stained with FITC-anti-TCR Vgamma 1 and biotin-anti-TCR-beta . (C) Cells were stained with FITC-anti-TCR Vgamma 1, PE-anti-CD4, and PE-anti-CD8alpha . CD4-CD8- (DN) cells were electronically gated.
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Fig. 6. Flow cytometric analysis of thymocytes and splenocytes of gamma c+ and gamma c- mice transgenic for TCR Vgamma 1 or double transgenic for TCR Vgamma 1/Vdelta 6. Cells were stained with combinations of FITC-anti-TCR Vgamma 1, FITC-anti-TCRgamma /delta clonotype, PE-anti-CD3, and PE-anti-Mac1.
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Considering that thymically derived gamma /delta T cells seed the spleen and lymph nodes of postnatal mice, we further examined the peripheral lymphoid compartments for transgenic gamma /delta T cells. As shown in Fig. 6, a large population of cells expressing the transgenic TCR-gamma /delta receptor are present in the spleen of gamma c+ Tg animals; these cells coexpress CD3, but are TCR-beta negative (data not shown) and accumulate to levels which are approximately six-fold higher in absolute numbers than gamma c+ nontransgenic animals (Table 4). In contrast, the periphery of gamma c- Tgs contain only a few cells expressing the Tg TCR-gamma /delta receptor, and these cells fail to accumulate in the spleen. In terms of absolute numbers, gamma c- Tg gamma /delta cells are reduced 142-fold compared with gamma c+ Tg littermates (Table 4). Taken together, our results suggest a major role for gamma c-dependent signals in the survival of gamma /delta T cells.

To investigate whether gamma c- Tg gamma /delta T cells had a defect in survival, we examined Tg thymocytes for the expression of the antiapoptotic factor, Bcl-2. Engagement of gamma c-dependent receptors has been shown to maintain high levels of Bcl-2, which appear to protect lymphoid cells from cell death (41). Although Vgamma 1+ thymocytes from gamma c+ mice expressed Bcl-2, Tg+ gamma /delta T cells from gamma c- mice were essentially negative for Bcl-2 (Fig. 7). These results are consistent with a defect in gamma /delta T cell survival in the absence of gamma c, although the relative contribution of Bcl-2 in supporting gamma /delta T cell development remains to be determined.


Fig. 7. Intracellular levels of Bcl-2 are diminished in gamma c- gamma /delta T cells. Cells were surface stained with FITC-anti-TCR Vgamma 1, fixed, and permeabilized. Bcl-2 staining was performed as described in Materials and Methods. Dotted lines indicate staining with hamster Ig and solid lines with hamster anti-mouse Bcl-2-specific Ig.
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Concluding Remarks.

Most gamma /delta T cells start their development within the thymus where they rearrange their TCR-gamma and TCR-delta genes via site-specific DNA recombination reactions triggered by the specialized stromal microenvironment found in the thymus. IL-2, IL-7, and IL-15 bind to specific receptors that share the gamma c and these cytokines have been postulated to play an important role in the survival, growth, and differentiation of gamma /delta cells (10). In a recent study using mice deficient in IL-7Ralpha chains, TCR-gamma gene rearrangements were found to be selectively abolished, and as a consequence, these mice lacked both fetal and adult TCR-gamma /delta cells (22). The authors concluded that ligands binding to the IL-7Ralpha chain (IL-7 and TSLP) are likely to be mandatory for the process of TCR-gamma rearrangements within intrathymic TCR-gamma /delta cell precursors (22). Although we do not refute this conclusion, our data reveal an additional function of gamma c-containing receptors (likely due to IL-7), that is independent of the TCR-gamma rearrangement process. Thymocytes from adult gamma c-deficient mice do not contain detectable TCR-gamma /delta cells and showed only low levels of TCR-gamma rearrangements, thereby limiting the potential synthesis of TCR-gamma polypeptides. Complementation of such gamma c-deficient mice with TCR-gamma and TCR-gamma /delta transgenes only partially rescued thymic gamma /delta T cell development and did not permit accumulation of peripheral gamma /delta T cells. Therefore, the developmental blockade affecting the adult gamma /delta T cell lineage in gamma c-deficient mice results not only from the limited amounts of rearranged TCR-gamma genes but also from the fact that IL-7 promotes the survival of gamma /delta T cell precursors containing TCR-gamma and TCR-gamma /delta polypeptides. The survival role played by IL-7 in gamma /delta T cell development is supported by our observation that fetal thymocytes from gamma c-deficient mice do contain TCR-gamma and TCR-delta rearrangements, but fail to generate appreciable numbers of gamma /delta T cells intrathymically or to export them to the periphery. Further evidence is provided by the fact that gamma c- gamma /delta T cells contain dramatically reduced levels of the antiapoptotic factor Bcl-2. Previous reports have shown that engagement of gamma c-containing receptors maintains cellular Bcl-2 protein levels (41), thereby promoting lymphoid cell survival. Whether the near-absent Bcl-2 levels are directly responsible for the survival defect in gamma c- gamma /delta T cells or simply an epiphenomenon related to decreased cell survival remains to be determined.

Thus, we would like to propose that for gamma /delta T cells, the primary function of gamma c-containing receptors is to promote survival. The ability of the gamma c- fetal thymus to support the survival of gamma /delta T cells (possibly via TSLP) would explain the presence of TCR-gamma rearrangements in IL-7-deficient mice and gamma c-deficient mice, and their mere absence in IL-7Ralpha -deficient mice. After successful expression of a functional TCR-gamma /delta complex, gamma /delta T cells would still require signals via gamma c to survive, mature, and seed the periphery. This idea gains support from the analysis of IL-7-deficient mice, where Vgamma 5loHSAhi fetal thymocytes are readily detectable, but fail to mature into Vgamma 5hiHSAlo cells (16). As a result, skin DETCs are not detectable in IL-7-deficient mice (data not shown).

Based on these results, one is led to ask why development of alpha /beta T cells is less severely impaired in gamma c-deficient mice than that of gamma /delta T cells. In the alpha /beta lineage, it has been recently documented that TCR-beta rearrangements are accompanied by a selective process allowing only those cells displaying a productively rearranged Vbeta gene to reach the next stage of differentiation (beta  selection). At a later time point, a second phase of selection, denoted TCR-alpha /beta selection, occurs to ensure MHC restriction and self tolerance. For gamma /delta T cells, TCR-gamma and TCR-delta rearrangements are probably achieved concurrently and not subjected to pre-TCR-based epigenetic control mechanisms operating during alpha /beta T cell development (34). In this model of gamma /delta T development, gamma /delta T cell precursors might not receive any pre-TCR or TCR signals, and engagement of gamma c-containing cytokine receptors may constitute the only means to support the survival of these cells. In marked contrast, in alpha /beta T cell precursors, the gamma c-dependent survival signals and the pre-TCR dependent survival signals may partially overlap, explaining how, in the absence of survival signals dependent on gamma c-containing receptors, signals emanating at a latter time point from the pre-TCR may rescue the development of a few alpha /beta T cell precursors.


Footnotes

Address correspondence to James P. DiSanto, INSERM U429, Pavillon Kirmisson, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, Paris F-75743 France. Phone: 33-1-44-49-50-51; FAX: 33-1-45-75-13-02; E-mail: disanto{at}necker.fr

Received for publication 22 April 1997 and in revised form 4 August 1997.

1   Abbreviations used in this paper: gamma c, common gamma  chain; DETC, dendritic epidermal T cell; DN, double negative; HSA, heat-stable antigen; J, joining; RAG, recombinase-activating gene; Tg, transgenic; TSLP, thymic stromal cell-derived lymphopoeitin; V, variable; wt, wild type.

This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the Association pour le Recherche sur le Cancer, Ligue Nationale Contre le Cancer, and the Commission of the European Communities.

We thank R. Murray (DNAX Corp.) for kindly providing IL-7-deficient mice, S.Y. Huang for help in generating the TCR Tg mice, and A. Wilson, D. Guy-Grand, and F. Rieux-Laucat for helpful discussions.


References

1. Kisielow, P., and H. von Boehmer. 1995. Development and selection of T cells: facts and puzzles. Adv. Immunol. 58: 87-209 [Medline].
2. Allison, J.P.. 1993. gamma delta T-cell development. Curr. Opin. Immunol. 5: 241-246 [Medline].
3. Hass, W., P. Pereira, and S. Tonegawa. 1993. Gamma/delta cells. Annu. Rev. Immunol. 11: 637-685 [Medline].
4. Lafaille, J.J., A. DeCloux, M. Bonneville, Y. Takagaki, and S. Tonegawa. 1989. Junctional sequences of T cell receptor gamma delta genes: implications for gamma delta T cell lineages and for a novel intermediate of V-(D)-J joining. Cell. 59: 859-870 [Medline].
5. Bogue, M., H. Mossman, U. Stauffer, C. Benoist, and D. Mathis. 1993. The level of N-region diversity in T cell receptors is not pre-ordained in the stem cell. Eur. J. Immunol. 23: 1185-1188 [Medline].
6. Ikuta, K., T. Kina, I. MacNeil, N. Uchida, B. Peault, Y.-H. Chien, and I.L. Weissman. 1990. A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell. 62: 863-874 [Medline].
7. Asarnow, D.M., D. Cado, and D.H. Raulet. 1993. Selection is not required to produce invariant T-cell receptor-gene junctional sequences. Nature (Lond.). 362: 158-160 [Medline].
8. Gerstein, R.M., and M.R. Lieber. 1993. Extent to which homology can constrain coding exon junctional diversity in V(D)J recombination. Nature (Lond.). 363: 625-627 [Medline].
9. Itohara, S., P. Monbaerts, J. Iacomini, J.J. Lafaille, A. Nelson, A.R. Clarke, M.L. Hooper, A. Farr, and S. Tonegawa. 1993. T-cell receptor delta  gene mutant mice: independent generation of alpha beta T cells and programmed rearrangement of gamma delta TCR genes. Cell. 72: 337-345 [Medline].
10. LeClercq, G., M. De Smedt, B. Tison, and J. Plum. 1990. Preferential proliferation of T cell Vgamma 3-positive cells in IL-2-stimulated fetal thymocytes. J. Immunol. 145: 3992-3999 [Abstract/Free Full Text].
11. Watanabe, Y., T. Sudo, N. Minato, A. Ohnishi, and Y. Katsura. 1991. Interleukin 7 preferentially supports the growth of gamma delta T cell receptor bearing T cells from fetal thymocytes in vitro. Int. Immunol. 3: 1067-1075 [Abstract].
12. Matsue, H., P.R. Bergstresser, and A. Takashima. 1993. Keratinocyte-derived IL-7 serves as a growth factor for dendritic epidermal T cells in mice. J. Immunol. 151: 6012-6019 [Abstract/Free Full Text].
13. Nishimura, H., K. Hiromatsu, N. Kobayashi, K.H. Grabstein, R. Paxton, K. Sugamura, J.A. Bluestone, and Y. Yoshikai. 1996. IL-15 is a novel growth factor for murine gamma delta T cells induced by Salmonella infection. J. Immunol. 156: 663-669 [Abstract].
14. Tanaka, T., F. Kitamura, Y. Nagasaka, K. Kuida, H. Suwa, and M. Miyasaka. 1993. Selective long-term elimination of natural killer cells in vivo by an anti-interleukin 2 receptor beta  chain monoclonal antibody in mice. J. Exp. Med. 178: 1103-1107 [Abstract].
15. Suzuki, H., G.S. Duncan, H. Takimoto, and T.W. Mak. 1997. Abnormal development of intestinal lymphocytes and peripheral natural killer cells in mice lacking the IL-2 receptor beta  chain. J. Exp. Med. 185: 499-505 [Abstract/Free Full Text].
16. Moore, T.A., U. von Freeden-Jeffry, R. Murray, and A. Zlotnik. 1996. Inhibition of gamma delta T cell development and early thymocyte maturation in IL-7-/- mice. J. Immunol. 157: 2366-2373 [Abstract].
17. Maki, S., S. Sunaga, Y. Komagata, Y. Kodaira, A. Mabuchi, H. Karasuyama, K. Yokomuro, J.-I. Miyazaki, and K. Ikuta. 1996. Interleukin 7 receptor-deficient mice lack gamma delta T cells. Proc. Natl. Acad. Sci. USA. 93: 7172-7177 [Abstract/Free Full Text].
18. He, Y.-W., and T.R. Malek. 1996. Interleukin-7 receptor alpha  is essential for the development of gamma delta + T cells, but not natural killer cells. J. Exp. Med. 184: 289-293 [Abstract].
19. DiSanto, J.P., W. Müller, D. Guy-Grand, A. Fischer, and K. Rajewsky. 1995. Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma  chain. Proc. Natl. Acad. Sci. USA. 92: 377-381 [Abstract].
20. Cao, X., E.W. Shores, J. Hu-Li, M.R. Anver, B.L. Kelsell, S.M. Russel, J. Drago, M. Noguchi, A. Grinberg, E.T. Bloom, et al . 1995. Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma  chain. Immunity 2: 223-238 [Medline].
21. Appasamy, P.M., T.J. Kenniston, Y. Weng, E.C. Holt, J. Kost, and W.H. Chambers. 1993. Interleukin-7-induced expression of specific T cell receptor gamma  variable region genes in murine fetal liver cultures. J. Exp. Med. 178: 2201-2206 [Abstract].
22. Maki, K., S. Sunaga, and K. Ikuta. 1996. The V-J recombination of T cell receptor-gamma genes is blocked in interleukin-7 receptor-deficient mice. J. Exp. Med. 184: 2423-2427 [Abstract/Free Full Text].
23. von Freeden-Jeffry, U., P. Vieira, L.A. Lucian, T. McNeil, S.E.G. Burdach, and R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519-1526 [Abstract].
24. Spanopoulou, E., C.A.J. Roman, L.M. Cororan, M.S. Schlissel, D.P. Silver, D. Nemazee, M.C. Nussenzweig, S.A. Shinton, R.R. Hardy, and D. Baltimore. 1994. Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice. Genes Dev. 8: 1030-1042 [Abstract].
25. Pereira, P., D.J. Gerber, S.Y. Huang, and S. Tonegawa. 1995. Ontogenic development and tissue distribution of Vgamma 1-expressing gamma /delta T lymphocytes in normal mice. J. Exp. Med. 182: 1921-1930 [Abstract].
26. Stingl, G., L.A. Gazze-Stingl, W. Aberer, and K. Wolff. 1981. Antigen presentation by murine epidermal langerhans cells and its alteration by ultraviolet B light. J. Immunol. 127: 1707-1713 [Free Full Text].
27. Miller, S.A., D.D. Dykes, and H.F. Polesky. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16: 1015 .
28. Anderson, S.J., S.D. Levin, and R.M. Perlmutter. 1993. Protein tyrosine kinase p56lck controls allelic exclusion of T-cell receptor beta -chain genes. Nature (Lond.). 365: 552-554 [Medline].
29. Ma, Q., H. Alder, K.K. Nelson, D. Chatterjee, Y. Gu, R. Nakamura, E. Canaani, C.M. Croce, L.D. Siracusa, and A.M. Buchberg. 1993. Analysis of the murine ALL-1 gene reveals conserved domains with human ALL-1 and identifies a motif shared with DNA methyltransferases. Proc. Natl. Acad. Sci. USA. 90: 6350-6354 [Abstract].
30. Rodewald, H.R., M. Ogawa, C. Haller, W. Waskow, and J.P. DiSanto. 1997. Pro-thymocyte expansion by c-kit and the common cytokine receptor gamma  chain is essential for repertoire formation. Immunity. 6: 265-272 [Medline].
31. Friend, S.L., S. Hosier, A. Nelson, D. Foxworthe, D.E. Williams, and A. Farr. 1994. A thymic stromal cell line supports in vitro development of surface IgM+ B cells and produces a novel growth factor affecting B and T lineage cells. Exp. Hematol (NY). 22: 321-328 .
32. Peschon, J.J., P.J. Morrissey, K.H. Grabstein, F.J. Ramsdell, E. Maraskovsky, B.C. Gliniak, L.S. Park, S.F. Ziegler, D.E. William, C.B. Ware, et al . 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180: 1955-1960 [Abstract].
33. Lantz, O., L.I. Sharara, F. Tilloy, A. Andersson, and J.P. DiSanto. 1997. Lineage relationships and differentiation of natural killer (NK) T cells: intrathymic selection and IL-4 production in the absence of NKR-P1 and Ly49 molecules. J. Exp. Med. 185: 1395-1401 [Abstract/Free Full Text].
34. Malissen, M., A. Gillet, L. Ardouin, G. Bouvier, J. Trucy, P. Ferrier, E. Vivier, and B. Malissen. 1995. Altered T cell development in mice with a targeted mutation of the CD3epsilon gene. EMBO (Eur. Mol. Biol. Organ.) J. 14: 4641-4653 [Abstract].
35. Wilson, A., J.P. de Villartay, and H.R. MacDonald. 1996. T cell receptor delta  gene rearrangement and T early alpha  (TEA) expression in immature alpha beta lineage thymocytes: implications for alpha beta /gamma delta lineage committment. Immunity 4: 37-45 [Medline].
36. Livak, F., H.T. Petrie, N. Crispe, and D.G. Schatz. 1995. In-frame TCR gamma  gene rearrangements play a critical role in the alpha beta /gamma delta T cell lineage decision. Immunity. 2: 617-627 [Medline].
37. Nakajima, P.B., J.P. Menetski, D.B. Roth, M. Gellert, and M.J. Bosma. 1995. V-D-J rearrangements at the T cell receptor delta  locus in mouse thymocytes of the alpha beta lineage. Immunity. 3: 609-621 [Medline].
38. Dudley, E.C., M. Girardi, M.J. Owen, and A.C. Hayday. 1995. alpha beta and gamma delta T cells can share a late common precursor. Curr. Biol. 5: 659-669 [Medline].
39. Ishida, I., S. Verbeck, M. Bonneville, S. Itohara, A. Berns, and S. Tonegawa. 1990. T-cell receptor gamma delta and gamma  transgenic mice suggest a role of gamma  gene silencer in the generation of alpha beta T-cells. Proc. Natl. Acad. Sci. USA. 87: 3067-3071 [Abstract].
40. Godfrey, D.I., and A. Zlotnik. 1993. Control points in early T-cell development. Immunol. Today. 14: 547-551 [Medline].
41. Boise, L.H., and C.B. Thompson. 1996. Hierarchical control of lymphocyte survival. Science (Wash. DC). 274: 67-68 [Medline].
42. Boise, L.H., A.J. Minn, C.H. June, T. Lindsten, and C.B. Thompson. 1995. Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division. Proc. Natl. Acad. Sci. USA. 92: 5491-5495 [Abstract].
43. Akbar, A.N., N.J. Borthwick, R.G. Wickremasinghe, P. Panayiotidis, D. Pilling, M. Bfill, S. Krajewski, J.C. Reed, and M. Salmon. 1996. Interleukin-2 receptor common gamma  chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawl: selective induction of anti-apoptotic (bcl-2, bcl-XL) but not pro-apoptotic (bax, bcl-XS) gene expression. Eur. J. Immunol. 26: 294-299 [Medline].

Copyright © 1997 by The Rockefeller University Press.