The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nakajima, H.
Right arrow Articles by Leonard, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nakajima, H.
Right arrow Articles by Leonard, W. J.
The Journal of Immunology, 1999, 162: 782-790.
Copyright © 1999 by The American Association of Immunologists

Role of Bcl-2 in {alpha}ß T Cell Development in Mice Deficient in the Common Cytokine Receptor {gamma}-Chain: The Requirement for Bcl-2 Differs Depending on the TCR/MHC Affinity

Hiroshi Nakajima1 and Warren J. Leonard2

Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice lacking the common cytokine receptor {gamma}-chain ({gamma}c) exhibit severely compromised T cell development, with diminished Bcl-2 expression in mature (CD4+ or CD8+) thymocytes and peripheral T cells. Enforced expression of Bcl-2 in these mice partially rescued {alpha}ß T cell development but not {gamma}{delta} T cell development. Transgenic expression of the OVA-specific DO11.10 (DO10) TCR also could modestly increase thymocyte numbers, and T cells expressing the transgenic TCR (KJ1-26+ T cells) were found in the periphery. Interestingly, the presence of KJ1-26+ T cells was dependent on the MHC background and was seen in the moderate affinity H-2d/d background but not in the higher affinity H-2d/b background in {gamma}c-deficient mice. In contrast, KJ1-26+ T cells exist in the periphery in both the H-2d/d and H-2d/b backgrounds in DO10 transgenic {gamma}c wild-type mice. These results suggest that the importance of {gamma}c-dependent signals for T cell development differs depending on the affinity of TCR for MHC. Moreover, enforced expression of Bcl-2 had a much greater effect on the development of {gamma}c-deficient T cells expressing the DO10 TCR in the high affinity H-2d/b background than in the H-2d/d background, suggesting that {gamma}c-dependent Bcl-2 expression influences T cell development in a TCR/MHC-dependent manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Failure to express a functional common cytokine receptor {gamma}-chain ({gamma}c)3 is the genetic defect in X-linked SCID (XSCID) (1). The profound immunological defects in this disease are explained by the observation that {gamma}c is a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (2, 3, 4). Like humans with XSCID, mice in which the {gamma}c gene has been disrupted by homologous recombination exhibit greatly diminished thymic cellularity (approximately 1–5% of normal) (5, 6, 7), underscoring the essential role of {gamma}c in thymic development. As mice lacking IL-7 (8) or IL-7R{alpha} (9) exhibit T cell defects similar to those found in {gamma}c-deficient mice, at least for mainstream {alpha}ß T cell development, a loss of IL-7 signaling is assumed to be responsible for, or to at least substantially contribute to, the diminished thymic development seen in {gamma}c-deficient mice. However, the maturation of CD4-CD8- (double negative (DN)) thymocytes is blocked at different stages in IL-7R{alpha}-deficient mice than in {gamma}c-deficient mice: there is a partial differentiation arrest at the CD44+CD25- stage in IL-7R{alpha}-deficient mice but this arrest is seen instead at the CD44+CD25+ stage in {gamma}c-deficient mice (10), suggesting that factors in addition to IL-7 may also be important for thymocyte maturation.

IL-7 can function as a cofactor for TCR rearrangement (11, 12, 13). Interestingly, expression of the rearranged HY TCR transgene can partially rescue thymic development in IL-7R{alpha}-deficient (14) and {gamma}c-deficient female mice (15), suggesting that TCR rearrangement is one of the {gamma}c-dependent signals controlling thymic development. In both IL-7R{alpha}-deficient and {gamma}c-deficient mice, however, thymic cellularity increases only fourfold (up to 10–20% of wild-type levels), indicating that {gamma}c plays a role in thymic development beyond TCR rearrangement (14, 15). Another possible role for {gamma}c in thymic development is to allow the induction of Bcl-2. Indeed, Bcl-2 is diminished in IL-7-deficient DN thymocytes, and IL-7 up-regulates Bcl-2 expression in this population (16). Moreover, it has been reported that the enforced expression of Bcl-2 rescues thymic development in IL-7R{alpha}-deficient mice (17, 18) and in {gamma}c-deficient mice (19); however, Bcl-2 transgenic {gamma}c-deficient mice have only about 10–20% of normal thymic cellularity (19). We have now performed studies to further characterize the role of {gamma}c in T cell development and found that enforced Bcl-2 expression can partially rescue {alpha}ß T cell but not {gamma}{delta} T cell development. When coexpressed, TCR and Bcl-2 transgenes synergistically rescue T cell development but this effect depends on the affinity between TCR and MHC. The implications of these findings are discussed.


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

{gamma}c-deficient mice (6) were back-crossed to BALB/c mice (H-2d/d) or C57BL/6 mice (H-2b/b) (The Jackson Laboratory, Bar Harbor, ME) for at least eight generations. Bcl-2 transgenic male (originally C3H background) mice in which Bcl-2 expression is under control of the Lck proximal promoter (20) were back-crossed to BALB/c mice or C57BL/6 mice for at least two generations. OVA-specific DO11.10 (DO10) TCR transgenic mice (H-2d/d) (21) were crossed to C57BL/6 {gamma}c+/- heterozygous females (H-2b/b) to obtain DO10 TCR transgenic {gamma}c+/- heterozygous females in the H-2d/b background. As the {gamma}c gene is located on chromosome X, mating of Bcl-2 transgenic {gamma}c-deficient male mice (H-2d/d) to DO10 TCR transgenic {gamma}c+/- heterozygous females (H-2d/b) yielded 16 genotypes resulted: DO10+ or DO10-, {gamma}c+ or {gamma}c-, Bcl-2+ or Bcl-2-, and H-2d/d or H-2d/b. Genotyping of mice was performed as described elsewhere (20, 22, 23) and was confirmed by flow cytometric analysis. Mice were housed in microisolator cages under specific pathogen-free conditions.

Flow cytometric analysis

Cells from the thymus and spleen were stained and analyzed on a FACSort (Becton Dickinson, San Jose, CA) using CELLQuest software as previously described (22). For direct staining, the following conjugated Abs were from PharMingen (San Diego, CA): anti-CD4 FITC, phycoerythrin (PE), Cy-Chrome, APC (H129.19), anti-CD8 FITC, PE, Cy-Chrome, APC (53-6.7), anti-H-2Kb PE (AF6-88.5), anti-H-2Kd FITC (SF1-1.1), anti-H-2Kk FITC (36-7-5), anti-I-Ab FITC (AF6-120.1), anti-I-Ad PE (AMS-32.1), anti-I-Ak FITC (10-3.6), anti-CD3 Cy-Chrome (145-2C11), anti-CD25 PE (3C7), anti-CD44 FITC, Cy-Chrome (IM7), anti-CD62L PE (Mel-14), anti-TCR ß FITC, PE (H57-597), and anti-TCR {gamma}{delta} FITC, PE (GL3). Anti-CD3 biotin (CD3-H5) was from Accurate Scientific (Westbury, NY). Biotinylated Ab was visualized by Streptavidin APC or Streptavidin Cy-Chrome (PharMingen). KJ1-26, an anti-Id mAb for the DO10 TCR (24), was purified from supernatants of hybridoma cells using protein G columns (Pharmacia, Uppsala, Sweden) and conjugated to FITC. Prior to staining, FcRS were blocked with anti-CD16/32 Ab (2.4G2; PharMingen).

Cell cycle analysis of thymocytes

Analysis of two-color surface staining coupled with DNA content by flow cytometry was carried out by modifying the method of Schmid et al. (25). In brief, thymocytes were stained with anti-CD4 FITC and anti-CD8 PE, washed twice with PBS, and DNA was stained with 10 µg/ml of 7-amino-actinomycin D (Sigma, St. Louis, MO) in permeabilizing buffer (PBS containing 0.04% saponin (Sigma) and 0.5% BSA) for 2 h at 4°C. DNA content was analyzed using flow cytometry. Cells containing more DNA than the G0/G1 peak were defined as cycling cells (cells in the S, G2, and M stages of the cell cycle).

Intracellular staining of murine and human Bcl-2

Bcl-2 levels in cells were analyzed as previously described (22, 26) using anti-murine Bcl-2 mAb (clone 3F11, PharMingen), anti-human Bcl-2 mAb (clone 6C8, PharMingen), or purified hamster IgG (anti-TNP, PharMingen) as a negative control.

In vivo bromodeoxyuridine (BrdU) uptake and BrdU staining

Mice from 3.5–4 wk old were injected i.p. with 1.5 mg of BrdU (Sigma) in 500 µl of PBS. Control mice were injected with PBS. Ten hours after the injection, thymocytes were fixed with 70% ethanol for 30 min on ice, denatured with 2 M HCl containing 0.5% Triton X-100 for 30 min to produce ssDNA, and neutralized with 0.1 M sodium tetraborate, pH 9.0 (Sigma). Cells were resuspended in PBS/1% BSA containing 0.5% Tween-20, stained with anti-BrdU FITC (Becton Dickinson) for 30 min at 25°C, washed once with PBS/1% BSA containing 0.5% Tween-20, resuspended in PBS/1% BSA, and analyzed on a FACSort.

Statistical analysis

Statistical determination of the difference between means of experimental groups was determined using an unpaired, Student’s two-tailed t test using the Stat View program (BrainPower, Calabasas, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Diminished cell expansion and cycling cells in thymuses in {gamma}c-deficient mice

Thymic cellularity is regulated by a number of mechanisms, including the number of migrating prothymocytes entering the organ, the efficiency of successful TCR rearrangement, the number of cell divisions, the amount of cell death resulting from the death of "neglected" cell or cells undergoing negative selection, and the rate of cell migration from thymus to periphery (reviewed in Refs. 27, 28, 29, 30, 31). As previously described (5, 6, 7), thymuses from {gamma}c-deficient mice have only approximately 3% of the normal cellularity. To address the role of {gamma}c in thymic development, we first analyzed the cell cycle status of {gamma}c-deficient thymocytes. Mice were injected i.p. with BrdU, and BrdU incorporation by thymocytes was analyzed 10 h later. Whereas approximately 23% of wild-type thymocytes incorporated BrdU, only 9% of {gamma}c-deficient thymocytes did so under the same conditions (Fig. 1A). Consistent with this diminished BrdU uptake, {gamma}c-deficient thymocytes contained fewer cycling cells (cells in the S/G2/M phases) (Fig. 1B). Cell division of thymocytes predominantly occurs in the CD4-CD8- (DN) and CD4+CD8+ double positive (DP) stages of thymic maturation (27, 32). However, as shown in Fig. 1C, cycling cells were diminished in all maturation stages (DN, DP, and CD4+ single positive (SP)) in {gamma}c-deficient mice (CD8+ SP cells were not analyzed given their near absence in {gamma}c-deficient mice). Correspondingly, {gamma}c-deficient mice had fewer large blastic DN and DP thymocytes (as determined by forward light scatter, Fig. 1D), populations which contain many cells in the cell cycle (27, 32).

Bcl-2 is diminished in the CD25+ DN and in CD4+ and CD8+ SP populations, but not in the CD3low DP population of thymocytes in {gamma}c-deficient mice

{gamma}c-dependent signals are known to induce Bcl-2 expression (33, 34) and Bcl-2 expression is diminished in {gamma}c-deficient peripheral T cells (22). Enforced expression of Bcl-2 can rescue {gamma}c-deficient peripheral T cells from increased apoptosis (23), suggesting that {gamma}c-dependent signals are physiologically important for maintaining Bcl-2 expression and preventing apoptosis of peripheral T cells. Moreover, enforced expression of Bcl-2 has been shown to increase the thymic cellularity in {gamma}c-deficient mice (19). We therefore analyzed expression levels of Bcl-2 in wild-type and {gamma}c-deficient thymcoytes (Fig. 2Go, note that the numbers in the upper right corner of each panel represent mean fluorescent intensities of Bcl-2 expression for wild-type and {gamma}c-deficient mice). In wild-type mice (thin line histograms), immature DN cKit+CD44+ thymocytes (Fig. 2B) express moderate levels of Bcl-2, whereas Bcl-2 expression has a biphasic pattern in CD44+CD25- cells (Fig. 2C) and Bcl-2 levels are uniformly high in DN CD44+CD25+ (Fig. 2D) and DN CD44-CD25+ (Fig. 2E) stages. In agreement with previous reports (26, 35, 36), after Bcl-2 is down-regulated in DN CD44-CD25- (Fig. 2F) and DP CD3low (Fig. 2G) stages, Bcl-2 is up-regulated again in DP CD3high (Fig. 2H) stages and highly expressed in CD4+CD3high (Fig. 2I) or CD8+CD3high (Fig. 2J) mature thymocytes. Interestingly, in wild-type mice, {gamma}c expression is high in DN thymocytes and SP mature thymocytes but low in DP thymocytes (ref. 37 , and our unpublished observation), correlating with the relative expression of Bcl-2 in these different populations. Therefore, it was surprising that levels of Bcl-2 in unfractionated thymocytes in {gamma}c-deficient mice were not diminished (Fig. 2A, bold histogram). However, when analyzed in more detail, Bcl-2 expression was not properly up-regulated in DN CD44+CD25+ (Fig. 2D) and DN CD44-CD25+ (Fig. 2E) thymocytes from the levels seen in DN cKit+CD44+ thymocytes (Fig. 2B) in {gamma}c-deficient mice. Whereas Bcl-2 levels were essentially normal in DP CD3low thymocytes (Fig. 2G), the up-regulation of Bcl-2 from DP CD3low (Fig. 2G) to mature thymocytes is also impaired in {gamma}c-deficient mice (Fig. 2I for CD4+CD3high thymocytes and Fig. 2J for CD8+CD3high thymocytes). It is interesting that the expression level of Bcl-2 is much lower in CD8+CD3high thymocytes than in CD4+CD3high thymocytes in {gamma}c-deficient mice; this may at least in part explain the diminished number of CD8+CD3high thymocytes and CD8+ splenic T cells in {gamma}c-deficient mice (6).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 2. {gamma}c-deficient thymocytes express diminished levels of Bcl-2 in CD8+ mature thymocytes and in CD4-CD8-CD25+ immature thymocytes, but not in CD4+CD8+ thymocytes. A to H, Shown are representative flow-cytometric profiles of Bcl-2 staining in total (A), cKit+CD44+ (B), CD44+CD25- (C), CD44+CD25+ (D), CD44-CD25+ (E), CD44-CD25- (F), CD4+CD8+CD3low (G), CD4+CD8+CD3high (H), CD4+CD8-CD3high (I), and CD4-CD8+CD3high (J) thymocytes in wild-type mice (thin line) and {gamma}c-deficient mice (bold line). Control staining for wild-type mice is shown by the dotted line. For B to F, cells that are positive for either CD3, CD4, CD8, or B220 were gated out by staining with a combination of anti-CD8 APC, anti-CD4 APC, anti-B220 APC, and anti-CD3 biotin + Streptavidin-APC. cKit+CD44+ (B), CD44+CD25- (C), CD44+CD25+ (D), CD44-CD25+ (E), CD44-CD25- and (F) subpopulations of CD3-CD4-CD8-B220- thymocytes were identified using anti-CD44 Cy-Chrome, and either anti-CD25 PE or anti-cKit PE and those cells were analyzed for Bcl-2 expression as described in Materials and Methods. For G to J, after thymocytes were stained with anti-CD8 Cy-Chrome, CD4 PE, and anti-CD3 biotin + Streptavidin APC, Bcl-2 expression was evaluated by intracellular staining. As expected, control staining for {gamma}c-deficient thymocytes was similar to that for wild-type thymocytes (data not shown).

 
Enforced expression of Bcl-2 partially increases the numbers of thymocytes in {gamma}c-deficient mice

To investigate the role of diminished levels of Bcl-2 in {gamma}c-deficient thymocytes, we generated Bcl-2 transgenic {gamma}c-deficient mice using Bcl-2 transgenic mice in which human Bcl-2 expression is under control of the Lck proximal promoter (20). We first analyzed the levels of the transgenic human Bcl-2 in each stage of thymocyte maturation (Fig. 3A). As shown in Fig. 3A-i, over 99% of thymocytes expressed transgenic Bcl-2. When analyzed in more detail, expression of the transgenic Bcl-2 was detected in under 10% of most immature DN cKit+CD44+ thymocytes (Fig. 3A-ii) and 20% of DN CD44+CD25- thymocytes (Fig. 3A-iii); however, transgenic Bcl-2 was detected in over 90% of the DN CD44+CD25+ cells (Fig. 3A-iv) and is highly expressed throughout thymocyte maturation after this stage, including DN CD44-CD25+ (Fig. 3A-v), DP, CD4+, and CD8+ thymocytes (data not shown). As expected, levels of expression of the Bcl-2 transgene were similar in Bcl-2 transgenic {gamma}c-wild-type and Bcl-2 transgenic {gamma}c-deficient thymocytes (data not shown). In Bcl-2 transgenic {gamma}c-deficient mice, the number of thymocytes was approximately 4-fold higher than in nontransgenic {gamma}c-deficient mice (p < 0.01, Fig. 3B); however, the thymic cellularity was still only 12% of that found in wild-type mice and the increase in thymocyte numbers in Bcl-2 transgenic {gamma}c-deficient mice is less significant in 8- to 9-wk-old adult mice even though the Bcl-2 transgene was still strongly expressed (data not shown).

As previously described, {gamma}c-deficient thymocytes exhibit an increased CD4+ to CD8+ ratio (6) (Fig. 3C, compare upper two dot plots). In Bcl-2 transgenic {gamma}c-deficient mice, the percentage of CD8+ thymocytes increased (Fig. 3C, lower right panel) and the ratio of CD4+ to CD8+ was normalized. This increase in CD8+ thymocytes was mainly due to an increase in mature CD8+CD3high thymocytes rather than an increase in immature CD8+CD3low thymocytes (data not shown). We next analyzed the cell-cycle status in Bcl-2 transgenic {gamma}c-deficient mice (Fig. 3D). Expression of the Bcl-2 transgene lowered the percentage of proliferating thymocytes in {gamma}c wild-type mice as assessed by BrdU uptake and cell cycle analysis (Fig. 3D), consistent with a previous report (38). Although Bcl-2 expression partially rescued thymic cellularity in {gamma}c-deficient mice (Fig. 3B), it did not correct the defect in the percentage of cycling cells and, in fact, may even inhibit cell cycling (Fig. 3D, note that the absolute number of thymocytes that incorporate BrdU is slightly increased in Bcl-2 transgenic {gamma}c-deficient mice because the absolute number of thymocytes is increased approximately 4-fold (Fig. 3B). This is consistent with the possibilities that either another {gamma}c-dependent signal besides Bcl-2 is required for thymic development or that Bcl-2 selectively rescues only certain populations of thymocytes.

Enforced expression of Bcl-2 can increase {alpha}ß T cells but not {gamma}{delta} T cells in {gamma}c-deficient mice

Because T cells express either {alpha}ß or {gamma}{delta} TCRs, we next analyzed whether the rescue of T cell development by Bcl-2 in {gamma}c-deficient mice is lineage-specific or not. Neither {gamma}c-deficient mice nor IL-7R{alpha}-deficient mice have {gamma}{delta} T cells either in the thymus or in the periphery (5, 6, 39, 40). As shown in Fig. 4Go, Bcl-2 did not promote the development of {gamma}{delta} T cells either in the thymus or in the periphery in {gamma}c-deficient mice (Fig. 4, D vs C, H vs G, and L vs K), so that the increase in thymocytes seen in Fig. 3B was primarily due to an increase in {alpha}ß lineage. These results suggest that Bcl-2 expression is not sufficient for {gamma}{delta} T cell development and that other {gamma}c-dependent signals are essential for development of this lineage. Although the human Bcl-2 transgene was equally expressed in {alpha}ß and {gamma}{delta} lineages (Fig. 4, N, P, and R) in Lckprox Bcl-2 transgenic mice, transgenic expression of Bcl-2 actually resulted in a relative increase in {alpha}ß lineage and decrease in {gamma}{delta} lineage (Fig. 4, B vs A, F vs E, and J vs I) in {gamma}c-wild-type mice. Thus, there is a marked difference in the effect of Bcl-2 on {alpha}ß and {gamma}{delta} T cell development. Interestingly, expression of the human Bcl-2 transgene (Fig. 4, N, P, and R, open histograms) diminished expression of the endogenous murine Bcl-2 (solid histograms, compare Fig. 4, N, P, and R with Fig. 4, M, O, and Q), suggesting that Bcl-2 may negatively regulate its own expression.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 4. Enforced expression of Bcl-2 did not rescue the development of {gamma}{delta} T cells. A to L, Shown are representative dot plots of TCR{alpha}ß vs TCR{gamma}{delta} staining in splenic CD3+ T cells (A to D); thymocytes expressing high levels of CD3 (E to H); and CD4-CD8- thymocytes (I to L) for {gamma}c+ (A, E, and I); Bcl-2+{gamma}c+ (B, F, and J); {gamma}c- (C, G, and K), and Bcl-2+{gamma}c- (D, H, and L) mice. M–R, Lckprox Bcl-2 is expressed in {gamma}{delta} T cells. Shown are representative histograms for endogenous (mouse) and transgenic (human) Bcl-2 staining in CD3+ {alpha}ß splenic T cells (M and N); CD3+ {gamma}{delta} splenic T cells (O and P); and {gamma}{delta} DN thymocytes (Q and R) for wild-type (M, O, and Q) and Bcl-2 transgenic mice (N, P, and R). Absolute number of thymocytes (mean ± SD) x 10-6, {gamma}c+ (155.3 ± 35.7), Bcl-2+ (136.0 ± 29.8), {gamma}c- (7.3 ± 6.3), Bcl-2+{gamma}c- (23.6 ± 11.8). Absolute number of CD3+ splenic T cells (mean ± SD) x 10-4: {gamma}c+ (1005.6 ± 62.8), Bcl-2+ (1283.0 ± 101.1), {gamma}c- (235.4 ± 18.8), Bcl-2+{gamma}c- (790.7 ± 87.5).

 
The increase in thymic cellularity in {gamma}c-deficient mice resulting from expression of a TCR transgene depends on the MHC background

IL-7 has been shown to work as cofactor for TCR rearrangement (11, 12, 13). To bypass this maturation signal, we generated TCR transgenic {gamma}c-deficient mice. In this experiment, we used young mice (3.5–4 wk of age) to minimize the degree of peripheral CD4+ T cell expansion found in {gamma}c-deficient mice (22). In Fig. 5Go, A–L are representative dot plots showing CD4 vs CD8 profile of thymocytes; above each dot plot is the total number of thymocytes. In Fig. 5M, bars A–L correspond to panels A–L and indicate the total number of thymocytes and the number of CD4+ thymocytes. Analogous to Fig. 5Go, A–L of Fig. 6Go are representative dot plots showing CD4 vs KJ1-26 (anti-idiotypic mAb for DO10 TCR) profiles for splenocytes; above each dot plot is the total number of splenocytes. In Fig. 6M, bars A–L indicate the total number of CD4+ T cells and CD4+ T cells that express DO10 TCR (CD4+KJ1-26+). As shown in Fig. 5Go, introduction of the OVA-specific DO10 TCR transgene increased the number of thymocytes in {gamma}c-deficient mice approximately sixfold, from 3.8 ± 0.6 x 106 for {gamma}c-deficient mice (mean ± SEM for 20 mice) to 21.7 ± 1.6 x 106 for DO10+{gamma}c-deficient mice (mean ± SEM for seven mice) in the H-2d/d background (compare Fig. 5, G vs H and the inset in M), but had little effect in {gamma}c wild-type mice (Fig. 5, A vs B). In contrast, the number of CD4+ splenic T cells was diminished in DO10+{gamma}c-H-2d/d mice as compared with nontransgenic {gamma}c-deficient mice (Fig. 6, G, H, and M (inset)) (21); nevertheless, DO10+{gamma}c-H-2d/d mice still have substantial numbers of CD4+KJ1-26+ splenocytes (Fig. 6, H and M (inset)). The DO10 TCR has been shown to exhibit higher affinity for I–Ab than I–Ad, and more thymocytes were therefore observed in the H-2d/d background than in the H-2d/b background (partial negative selection in the H-2d/b background) (41). Consistent with previous findings (41), the number of thymocytes and CD4+KJ1-26+ splenocytes was diminished in DO10+ {gamma}c+ mice in the H-2d/b background as compared with DO10+ {gamma}c+ mice in the H-2d/d background (compare Fig. 5, C vs B and Fig. 6, C vs B). Similarly, the number of thymocytes was diminished in DO10+ {gamma}c- mice in the H-2d/b background as compared with the number in DO10+ {gamma}c- mice in the H-2d/d background (compare Fig. 5, I vs H). Thus, the increase in thymic cellularity seen in {gamma}c-deficient mice expressing the DO10 transgene in the H-2d/d background results from bypassing the defect in TCR rearrangement in {gamma}c-deficient mice and is influenced by the MHC background. Interestingly, whereas DO10+ {gamma}c+ H-2d/b mice have substantial numbers of CD4+KJ1-26+ splenocytes, DO10+ {gamma}c- H-2d/b mice have no CD4+KJ1-26+ splenocytes (compare Fig. 6, I vs C). These results suggest that {gamma}c-dependent signals are required for development and/or survival of T cells expressing a TCR with a relatively high affinity to the self-MHC/peptide complex. Since DO10+ {gamma}c- H-2d/b mice have substantial numbers of CD4+ thymocytes (Fig. 5I) and over 90% of CD4+ thymocytes express transgenic TCR (KJ1-26+), the defect of CD4+KJ1-26+ splenocytes probably is not due to the defect in positive selection per se but rather may be due to a defect in survival and/or expansion of CD4+KJ1-26+ cells after the CD4+ SP thymocyte stage.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 5. Increased thymic cellularity by the TCR transgene differs depending on the affinity of the TCR for MHC in {gamma}c-deficient mice. A–L, Shown are the cellularity of thymus (mean ± SEM, x106) and representative flow cytometric analysis of CD4 vs CD8 staining of thymocytes from 3.5- to 4-wk-old {gamma}c+ (A), DO10+{gamma}c+ (H-2d/d) (B), DO10+{gamma}c+ (H-2d/b) (C), Bcl-2+ {gamma}c+ (D), DO10+ Bcl-2+ {gamma}c+ (H-2d/d) (E), DO10+ Bcl-2+ {gamma}c+ (H-2d/b) (F), {gamma}c- (G), DO10+ {gamma}c- (H-2d/d) (H), DO10+ {gamma}c- (H-2d/b) (I), Bcl-2+ {gamma}c- (J), DO10+ Bcl-2+ {gamma}c- (H-2d/d) (K), and DO10+ Bcl-2+ {gamma}c- (H-2d/b) (L) mice. Because the cellularity of thymuses in non-TCR transgenic mice ({gamma}c+, Bcl-2+ {gamma}c+, {gamma}c-, and Bcl-2+ {gamma}c-) is not affected by MHC, data for the mice shown in this figure include both H-2d/d and H-2d/b mice. M, The number of total thymocytes and CD4+ thymocytes (mean ± SEM, x106) is shown for the mice in A–L. In the inset, the number of total thymocytes and CD4+ thymocytes are shown for the mice in G to I using a different scale.

 


View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 6. The importance of {gamma}c-dependent signals for T cell development depends on the affinity of the TCR for MHC. A to L, Shown are the number of splenocytes (mean ± SEM, x106) and representative flow cytometric analysis of CD4 vs KJ1-26 staining of splenocytes from 3.5- to 4-wk-old {gamma}c+ (A), DO10+ {gamma}c+ (H-2d/d) (B), DO10+ {gamma}c+ (H-2d/b) (C), Bcl-2+ {gamma}c+ (D), DO10+ Bcl-2+ {gamma}c+ (H-2d/d) (E), DO10+ Bcl-2+ {gamma}c+ (H-2d/b) (F), {gamma}c- (G), DO10+ {gamma}c- (H-2d/d) (H), DO10+ {gamma}c- (H-2d/b) (I), Bcl-2+ {gamma}c- (J), DO10+ Bcl-2+ {gamma}c- (H-2d/d) (K), and DO10+ Bcl-2+ {gamma}c- (H-2d/b) (L) mice. Analogous to Fig. 5Go, data for non-TCR transgenic mice ({gamma}c+, Bcl-2+ {gamma}c+, {gamma}c-, and Bcl-2+ {gamma}c-) shown in this figure include both H-2d/d and H-2d/b mice. M, The number of total CD4+ T cells and CD4+KJ1-26+ T cells (mean ± SEM, x106) for mice is shown in A–L. In the inset, the number of total CD4+ T cells and CD4+KJ1-26+ T cells for the mice are shown in G–I, using a different scale.

 
Bcl-2 can rescue development of CD4+ KJ1-26+ T cells in {gamma}c-deficient mice in the H-2d/b background

Because CD4+ SP thymocytes in DO10+ {gamma}c- mice also exhibit slightly diminished Bcl-2 expression (data not shown) similar to those found in nontransgenic {gamma}c-deficient thymocytes (Fig. 2Go), we next investigated whether Bcl-2 could rescue development of CD4+KJ1-26+ cells in {gamma}c-deficient mice in the H-2d/b background. Whereas enforced expression of Bcl-2 at most only modestly increased the number of total thymocytes and CD4+ SP thymocytes in the H-2d/d background (Fig. 5, E vs B for DO10+ {gamma}c+ mice and K vs H for DO10+ {gamma}c- mice), in the H-2d/b background, enforced expression of Bcl-2 strongly increased the percentage of CD4+ thymocytes from 12.5% to 38% in DO10+ {gamma}c+ mice and from 31.2% to 63.8% in DO10+ {gamma}c- mice (compare Fig. 5, C and F for DO10+ {gamma}c+ mice, and I and L for DO10+ {gamma}c- mice). In addition, enforced expression of Bcl-2 strongly increased the number of thymocytes in DO10+ {gamma}c- mice in the H-2d/b background from 9.3 ± 2.2 x 106 to 49.8 ± 6.4 x 106 (mean ± SEM, Fig. 5, I and L). Furthermore, although enforced expression of Bcl-2 increased the percentage and the absolute number of CD4+KJ1-26+ splenocytes in DO10+ {gamma}c+ mice in the H-2d/b background (compare Fig. 6, C vs F), Bcl-2 expression dramatically increased the CD4+KJ1-26+ splenocytes (over 250-fold) in DO10+ {gamma}c- mice (Fig. 6, I vs L, 3.8 ± 1.3 x 104/spleen for DO10+ {gamma}c- H-2d/b mice (n = 8) and 1014.0 ± 122.1 x 104/spleen for DO10+ Bcl-2+ {gamma}c- H-2d/b mice (n = 15), see hatched bars I vs L in Fig. 6M). These data indicate that {gamma}c is required for T cell development in situations in which TCRs have relatively high affinities for self-MHC and that the importance of Bcl-2 on T cell development also differs substantially for different TCR/MHC affinities. Although thymic cellularity is higher in DO10+Bcl-2+ {gamma}c- mice than in nontransgenic {gamma}c- mice, it is still diminished as compared with wild-type mice. Thus, {gamma}c appears to have additional functions beyond TCR rearrangement and Bcl-2 induction for promoting thymic development (see Discussion).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The function of the thymus is to produce T cells in which TCRs have a sufficient affinity for non-self-peptide/MHC complexes and where potentially harmful self-reactive T cells have been eliminated (28, 29, 31, 42). {gamma}c-dependent signals are believed to be important for the T cell compartment, not only by inducing expansion and preventing apoptosis of peripheral T cells, but also by supporting thymic development (5, 6, 7, 22, 43). IL-7 is one {gamma}c-dependent cytokine clearly implicated in this process. {gamma}c-deficient mice not only have very small thymuses, but the percentage of thymocytes that incorporate BrdU and are in cell cycle are severely diminished. As previously described, cell division of thymocytes occurs predominantly in DN thymocytes and partly in DP thymocytes (27, 32). The rate of cell division is diminished in both DN and DP stages in {gamma}c-deficient mice, suggesting that {gamma}c-dependent signals are required for normal division of DN and DP thymocytes.

In addition to the importance of {gamma}c-dependent signals for expansion of thymocytes, {gamma}c is also vital for the induction of Bcl-2 and anti-apoptotic signals. We have shown here that Bcl-2 expression is diminished in immature DN thymocytes and mature SP thymocytes but not in DP thymocytes in {gamma}c-deficient mice. Recently, a similar defect in Bcl-2 expression in DN thymocytes was found in IL-7-deficient mice, and exogenous IL-7 could up-regulate Bcl-2 expression in these cells (16). In our studies, although enforced expression of Bcl-2 could increase the cellularity of {gamma}c-deficient thymuses four- to fivefold, thymic cellularity in Bcl-2 transgenic {gamma}c-deficient mice is still only 12–15% of normal levels. Because transgenic Bcl-2 in Lckprox Bcl-2 transgenic mice is only partially expressed in immature DN CD44+CD25- thymocytes, we cannot eliminate the possibility that {gamma}c-induced expression of Bcl-2 in this stage of thymocytes is vital for normal T cell development. If this is the case, it provides a possible explanation for diminished thymic development in Lckprox Bcl-2 transgenic {gamma}c-deficient mice. However, since a similar partial increase in cellularity was reported when Eµ-Bcl-2 and H-2K Bcl-2 were mated with {gamma}c-deficient mice (13 and 20% of wild-type thymus, respectively) (19), and since expression of a {gamma}c transgene under control of the same Lck proximal promoter construct normalized cellularity of {gamma}c-deficient thymocytes and allowed the development of {gamma}{delta} T cells (our unpublished data), we hypothesize that {gamma}c plays important roles in thymic development beyond the induction of Bcl-2. This is consistent with observations that IL-7 signaling is not limited to Bcl-2 induction. Indeed, IL-7 potently activates STAT proteins (44); moreover, the activation of phosphatidylinositol 3-kinase by IL-7 is known to be vital for cell cycle entry and proliferation (45). One of the other possibilities is that {gamma}c expression on a nonthymocyte might be required for thymic differentiation. However, given the ability of an Lck promoter-driven {gamma}c transgene to reconstitute thymic cellularity and a normal CD4:CD8 ratio, this possibility is diminished.

It was still striking that the effect of enforced expression of Bcl-2 differs between the {alpha}ß and {gamma}{delta} T cell lineages. Whereas enforced expression of Bcl-2 increased {alpha}ß T cells in the thymus and periphery, it did not increase the number of {gamma}{delta} T cells. Recently, an analysis of {gamma}{delta} T cell development in {gamma}c-deficient mice (46) revealed that there are some {gamma}{delta} T cells in fetal {gamma}c-deficient mice and these cells express diminished levels of Bcl-2. Our results demonstrate that the enforced expression of Bcl-2 is not sufficient for {gamma}{delta} T cell development in {gamma}c-deficient mice, suggesting that other {gamma}c-dependent functions are required for their development and highlighting a potentially important difference in {alpha}ß vs {gamma}{delta} T cell development.

In addition to its anti-apoptotic effect and cell-proliferative effects, it has been shown that IL-7 is a cofactor for TCR rearrangement (11, 12, 13). Indeed, TCR transgenic {gamma}c-deficient mice have larger thymuses than nontransgenic {gamma}c-deficient mice (ref. 15 for the MHC class I system; Fig. 5Go for the MHC class II system). We have now observed that this effect appears to be dependent on TCR/MHC affinity, with a strong increase in cellularity in a moderately high TCR/MHC affinity situation (DO10 TCR in the H-2d/d background) and a weak increase in cellularity in a high TCR/MHC affinity situation (DO10 TCR in H-2d/b background). These results suggest that the effect of a rearranged TCR on increasing thymic cellularity of {gamma}c-deficient mice is not solely due to bypassing the TCR rearrangement but also is due to the efficiency of thymic selection. Interestingly, substantial numbers of CD4+ T cells expressing the transgenic DO10 TCR (CD4+KJ1-26+) exist in the periphery in DO10+ {gamma}c+ H-2d/d, DO10+ {gamma}c+ H-2d/b, and DO10+ {gamma}c- H-2d/d mice; in contrast, DO10+ {gamma}c- H-2d/b mice have few CD4+KJ1-26+ cells in the periphery even though DO10+ {gamma}c- H-2d/b mice have substantial numbers of CD4+SP thymocytes expressing DO10 TCR. These results indicate that the greatly diminished number of CD4+ KJ1-26+ splenic T cells in these mice results from defective survival of mature thymocytes and/or peripheral T cells. This decrease is consistent with the diminished Bcl-2 expression in mature CD4+ thymocytes (Fig. 2Go) and CD4+ splenic T cells (22). In addition, the importance of {gamma}c-dependent signals for T cells expressing the same TCR differs depending on the MHC background, with {gamma}c being of great importance in a high affinity TCR/MHC environment and relatively low importance in a moderate affinity TCR/MHC environment. Although the precise mechanisms of this finding remains unclear, anti-apoptotic signals from {gamma}c such as Bcl-2 seem to be important for the survival of T cells in which the affinity of TCR and MHC is high. Moreover, this finding may be important in view of a recent observation showing the importance of the elimination of Ag-specific thymocytes at the semimature CD4+CD8- stage (47). However, the enforced expression of Bcl-2 cannot or can only partially overcome Mtv-induced deletion (20, 48, 49) and the HY-induced deletion (50, 51), suggesting that the rescue of T cells by Bcl-2 occurs only within a limited range of TCR/MHC affinities. The fact that the number of thymocytes in {gamma}c-deficient mice expressing both Bcl-2 and TCR transgenes is still diminished indicates that other functions of {gamma}c beyond Bcl-2 induction and TCR rearrangement, such as proliferation, are also important for T cell development. Because the restoration of thymocytes differs in different MHC backgrounds, it is possible, however, that such additional {gamma}c functions are not necessarily essential, depending on the TCR/MHC affinity. Nevertheless, restoration of {gamma}c-deficient thymocytes by Bcl-2 is also partial in another TCR transgenic system (the cytochrome c-specific AND transgene in the H-2b/b background; our unpublished observation), suggesting that additional functions of {gamma}c beyond Bcl-2 induction and TCR rearrangement are likely to be important for the majority of T cells.

In conclusion, {gamma}c-dependent signals play multiple roles in T cell development. First, {gamma}c-dependent signals are important for the expansion of thymocytes. Second, {gamma}c-dependent signals play a role for induction of Bcl-2 in CD25+ DN thymocytes and mature thymocytes, thereby supporting the viability of these cells. Third, {gamma}c-dependent signals play a role for both {alpha}ß and {gamma}{delta} T cell development, but only {alpha}ß T cell numbers increased following constitutive expression of a Bcl-2 transgene, indicating that other {gamma}c-dependent signals are vital for {gamma}{delta} T cell development. Fourth, the requirement of {gamma}c-dependent signals for T cell development depends on the affinity of the TCR for MHC, with {gamma}c-dependent signals being more important in the high affinity TCR/MHC environment than in the moderate affinity TCR/MHC environment. Finally, the efficiency of rescue of {gamma}c-deficient T cells by the Bcl-2 transgene also differs depending on the affinity of TCR for MHC. Given that the constitutive expression of Bcl-2 does not fully compensate for the absence of {gamma}c, it will be important to define how other signals downstream of {gamma}c together integrate with Bcl-2 to explain the multiple contributions of {gamma}c to lymphoid development.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 1. {gamma}c-deficient thymocytes exhibit diminished BrdU incorporation and decreased cycling cells. A, {gamma}c-deficient thymocytes exhibit diminished BrdU incorporation. Mice (4 wk old) were injected i.p. with 1.5 mg of BrdU. Ten hours later, mice were sacrificed and thymocytes were analyzed for BrdU incorporation. Shown are representative histograms for BrdU staining and mean ± SD of BrdU incorporation (p < 0.002, n = 3). B, {gamma}c-deficient thymocytes exhibit diminished cycling cells. Shown are representative histograms for DNA content of unfractionated thymocytes and mean ± SD of cells in the S/G2/M phases of the cell cycle (p < 0.001, n = 8). C, Thymocytes were analyzed for DNA content within CD4-CD8-, CD4+CD8+, and CD4+ thymocyte populations. Shown are mean ± SD of cells in the S/G2/M phases of the cell cycle (n = 8). D, {gamma}c-deficient thymuses exhibit diminished number of large cells. Shown are representative histograms for forward light scatter (FSC) of CD4-CD8- cells and CD4+CD8+ cells.

 


View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 3. Enforced expression of Bcl-2 partially restores thymic development in {gamma}c-deficient mice. A, A human Bcl-2 transgene under control of the Lckprox promoter is expressed in DN CD25+ immature thymocytes. The expression of human Bcl-2 (transgenic Bcl-2) was analyzed in LckproxBcl-2 transgenic mice by using a species-specific mAb for human Bcl-2. Shown are representative histograms for human Bcl-2 staining in total thymocytes (i), or in the subpopulations of thymocytes, cKit+CD44+ (ii), CD44+CD25- (iii), CD44+CD25+ (iv), and CD44-CD25+ (v). The data were generated in a fashion similar to that described for Fig. 2Go. As expected, expression of transgenic Bcl-2 in Bcl-2 transgenic {gamma}c-deficient mice was similar to that found in Bcl-2 transgenic {gamma}c wild-type mice (data not shown). B, Increased thymic cellularity in Bcl-2 transgenic {gamma}c-deficient mice. Shown is the thymic cellularity of wild-type ({gamma}c+), Bcl-2 transgenic (Bcl-2 {gamma}c+), {gamma}c-deficient ({gamma}c-), and Bcl-2 transgenic {gamma}c-deficient (Bcl-2 {gamma}c-) mice. All mice were analyzed at 3–4 wk of age. C, Enforced expression of Bcl-2 increased the number of CD8+ thymocytes in {gamma}c-deficient mice. Shown are representative CD4 vs CD8 staining of thymocytes for {gamma}c+, Bcl-2 {gamma}c+, {gamma}c-, and Bcl-2 {gamma}c- mice. D, Enforced expression of Bcl-2 did not increase the percentage of cycling cells in the thymuses of {gamma}c-deficient mice. Shown are the mean ± SD of the percentage of thymocytes incorporating BrdU of thymocytes (solid bars) and the percentage of thymocytes in the S/G2/M phases of the cell cycle (open bars) in {gamma}c+, Bcl-2 {gamma}c+, {gamma}c-, and Bcl-2 {gamma}c- mice (n = 5).

 

   Acknowledgments
 
We thank D. Y. Loh for DO11.10 mice and the sequences of the primers for detecting the DO11.10 transgene; S. J. Korsmeyer (Washington University) for Bcl-2 transgenic mice; B. J. Fowlkes for KJ1-26 cells; T. Tran for assistance with purification and labeling of KJ1-26 mAb; Y. Nakajima for secretarial help; T. Diers and J. Bollenbacher for help with animal care; and R. Germain, B. J. Fowlkes, T. Miyazaki, M. Noguchi, and E. W. Shores for critical comments.


   Footnotes
 
1 H.N. was supported in part by the Japan Society for the Promotion of Science and the Naito Foundation. Back

2 Address correspondence to Dr. Warren J. Leonard, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, Bldg. 10, Rm. 7N252, 10 Center Drive, Bethesda, MD 20892-1674. E-mail address: Back

3 Abbreviations used in this paper: {gamma}c, common cytokine receptor {gamma} chain; XSCID, X-linked severe combined immunodeficiency; DO10, DO11.10; DP, double positive; DN, double negative; SP, single positive; PE, phycoerythrin, BrdU, bromodeoxyuridine. Back

Received for publication July 2, 1998. Accepted for publication October 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Noguchi, M., H. Yi, H. M. Rosenblatt, A. H. Filipovich, S. Adelstein, W. S. Modi, O. W. McBride, W. J. Leonard. 1993. Interleukin-2 receptor {gamma} chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 73:147.[Medline]
  2. Sugamura, K., H. Asao, M. Kondo, N. Tanaka, N. Ishii, K. Ohbo, M. Nakamura, T. Takeshita. 1996. The interleukin-2 receptor {gamma} chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. Annu. Rev. Immunol. 14:179.[Medline]
  3. Leonard, W. J., E. W. Shores, P. E. Love. 1996. Role of the common cytokine receptor {gamma} chain in cytokine signaling and lymphoid development. Immunol. Rev. 148:97.
  4. Lin, J.-X., W. J. Leonard. 1997. Signaling from the IL-2 receptor to the nucleus. Cytokine Growth Factor Rev. 8:313.[Medline]
  5. DiSanto, J. P., W. Müller, D. Guy-Grand, A. Fischer, 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.[Abstract/Free Full Text]
  6. Cao, X., E. W. Shores, J. Hu-Li, M. R. Anver, B. L. Kelsall, S. M. Russell, 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.[Medline]
  7. Ohbo, K., T. Suda, M. Hashiyama, A. Mantani, M. Ikebe, K. Miyakawa, M. Moriyama, M. Nakamura, M. Katsuki, K. Takahashi, et al 1996. Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor {gamma} chain. Blood 87:956.[Abstract/Free Full Text]
  8. von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519.[Abstract/Free Full Text]
  9. 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. Williams, C. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180:1955.[Abstract/Free Full Text]
  10. He, Y. W., H. Nakajima, W. J. Leonard, B. Adkins, T. R. Malek. 1997. The common {gamma}-chain of cytokine receptors regulates intrathymic T cell development at multiple stages. J. Immunol. 158:2592.[Abstract]
  11. Muegge, K., M. P. Vila, S. K. Durum. 1993. Interleukin-7: a cofactor for V(D)J rearrangement of the T cell receptor ß gene. Science 261:93.[Abstract/Free Full Text]
  12. Oosterwegel, M. A., M. C. Haks, U. Jeffry, R. Murray, A. M. Kruisbeek. 1997. Induction of TCR gene rearrangements in uncommitted stem cells by a subset of IL-7 producing, MHC class-II-expressing thymic stromal cells. Immunity 6:351.[Medline]
  13. Candeias, S., K. Muegge, S. K. Durum. 1997. IL-7 receptor and VDJ recombination: trophic versus mechanistic actions. Immunity 6:501.[Medline]
  14. Crompton, T., S. V. Outram, J. Buckland, M. J. Owen. 1997. A transgenic T cell receptor restores thymocyte differentiation in interleukin-7 receptor {alpha} chain-deficient mice. Eur. J. Immunol. 27:100.[Medline]
  15. DiSanto, J. P., D. Guy-Grand, A. Fischer, A. Tarakhovsky. 1996. Critical role for the common cytokine receptor {gamma} chain in intrathymic and peripheral T cell selection. J. Exp. Med. 183:1111.[Abstract/Free Full Text]
  16. von Freeden-Jeffry, U., N. Solvason, M. Howard, R. Murray. 1997. The earliest T lineage-committed cells depend on IL-7 for Bcl-2 expression and normal cell cycle progression. Immunity 7:147.[Medline]
  17. Akashi, K., M. Kondo, U. von Freeden-Jeffry, R. Murray, I. L. Weissman. 1997. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. Cell 88:1033.
  18. Maraskovsky, E., L. A. O’Reilly, M. Teepe, L. M. Corcoran, J. J. Peschon, A. Strasser. 1997. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant Rag-1-/- mice. Cell 89:1011.[Medline]
  19. Kondo, M., K. Akashi, J. Domen, K. Sugamura, I. L. Weissman. 1997. Bcl-2 rescues T lymphopoiesis, but not B or NK cell development, in common {gamma} chain-deficient mice. Immunity 7:155.[Medline]
  20. Sentman, C. L., J. R. Shutter, D. Hockenbery, O. Kanagawa, S. J. Korsmeyer. 1991. Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 67:879.[Medline]
  21. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250:1720.[Abstract/Free Full Text]
  22. Nakajima, H., E. W. Shores, M. Noguchi, W. J. Leonard. 1997. The common cytokine receptor {gamma} chain plays an essential role in regulating lymphoid homeostasis. J. Exp. Med. 185:189.[Abstract/Free Full Text]
  23. Nakajima, H., W. J. Leonard. 1997. Impaired peripheral deletion of activated T cells in mice lacking the common cytokine receptor {gamma}-chain: defective Fas ligand expression in {gamma}-chain-deficient mice. J. Immunol. 159:4737.[Abstract]
  24. Marrack, P., R. Shimonkevitz, C. Hannum, K. Haskins, J. Kappler. 1983. The major histocompatibility complex-restricted antigen receptor on T cells. IV. An antiidiotypic antibody predicts both antigen and I-specificity. J. Exp. Med. 158:1635.[Abstract/Free Full Text]
  25. Schmid, I., C. H. Uittenbogaart, B. Keld, J. V. Giorgi. 1994. A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. J. Immunol. Methods 170:145.[Medline]
  26. Veis, D. J., C. L. Sentman, E. A. Bach, S. J. Korsmeyer. 1993. Expression of the Bcl-2 protein in murine and human thymocytes and in peripheral T lymphocytes. J. Immunol. 151:2546.[Abstract]
  27. Shortman, K., M. Egerton, G. J. Spangrude, R. Scollay. 1990. The generation and fate of thymocytes. Semin. Immunol. 2:3.[Medline]
  28. Robey, E., B. L. Fowlkes. 1994. Selective events in T cell development. Annu. Rev. Immunol. 12:675.[Medline]
  29. Kisielow, P., H. von Boehmer. 1995. Development and selection of T cells: facts and puzzles. Adv. Immunol. 58:87.[Medline]
  30. Shortman, K., L. Wu. 1996. Early T lymphocyte progenitors. Annu. Rev. Immunol. 14:29.[Medline]
  31. Marrack, P., J. Kappler. 1997. Positive selection of thymocytes bearing {alpha}ß T cell receptors. Curr. Opin. Immunol. 9:250.[Medline]
  32. Fehling, H. J., H. von Boehmer. 1997. Early {alpha}ß T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9:263.[Medline]
  33. Otani, H., M. Erdos, W. J. Leonard. 1993. Tyrosine kinase(s) regulate apoptosis and bcl-2 expression in a growth factor-dependent cell line. J. Biol. Chem. 268:22733.[Abstract/Free Full Text]
  34. Deng, G., E. R. Podack. 1993. Suppression of apoptosis in a cytotoxic T-cell line by interleukin 2-mediated gene transcription and deregulated expression of the protooncogene bcl-2. Proc. Natl. Acad. Sci. USA 90:2189.[Abstract/Free Full Text]
  35. Gratiot-Deans, J., R. Merino, G. Nunez, L. A. Turka. 1994. Bcl-2 expression during T-cell development: early loss and late return occur at specific stages of commitment to differentiation and survival. Proc. Natl. Acad. Sci. USA 91:10685.[Abstract/Free Full Text]
  36. Linette, G. P., M. J. Grusby, S. M. Hedrick, T. H. Hansen, L. H. Glimcher, S. J. Korsmeyer. 1994. Bcl-2 is upregulated at the CD4+CD8+ stage during positive selection and promotes thymocyte differentiation at several control points. Immunity 1:197.[Medline]
  37. Kondo, M., Y. Ohashi, K. Tada, M. Nakamura, K. Sugamura. 1994. Expression of the mouse interleukin-2 receptor {gamma} chain in various cell populations of the thymus and spleen. Eur. J. Immunol. 24:2026.[Medline]
  38. Linette, G. P., Y. Li, K. Roth, S. J. Korsmeyer. 1996. Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation. Proc. Natl. Acad. Sci. USA 93:9545.[Abstract/Free Full Text]
  39. He, Y. W., 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.[Abstract/Free Full Text]
  40. Maki, K., S. Sunaga, Y. Komagata, Y. Kodaira, A. Mabuchi, H. Karasuyama, K. Yokomuro, J-i. Miyazaki, K. Ikuta. 1996. Interleukin 7 receptor-deficient mice lack {gamma}{delta} T cells. Proc. Natl. Acad. Sci. USA 93:7172.[Abstract/Free Full Text]
  41. Liu, C.-P., J. W. Kappler, P. Marrack. 1996. Thymocytes can become mature T cells without passing through the CD4+CD8+, double positive stage. J. Exp. Med. 184:1619.[Abstract/Free Full Text]
  42. Jr Janeway, C. A.. 1994. Thymic selection: two pathways to life and two to death. Immunity 1:3.[Medline]
  43. Vella, A., T. K. Teague, J. Ihle, J. Kappler, P. Marrack. 1997. Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: stat6 is probably not required for the effect of IL-4. J. Exp. Med. 186:325.[Abstract/Free Full Text]
  44. Lin, J.-X., T.-S. Migone, M. Tsang, M. Friedmann, J. A. Weatherbee, L. Zhou, A. Yamauchi, E. T. Bloom, J. Mietz, S. John, et al 1995. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 2:331.[Medline]
  45. Corcoran, A. E., F. M. Smart, R. J. Cowling, T. Crompton, M. J. Owen, A. R. Venkitaraman. 1996. The interleukin-7 receptor {alpha} chain transmit distinct signals for proliferation and differentiation during B lymphopoiesis. EMBO J. 15:1924.[Medline]
  46. Malissen, M., P. Pereira, D. J. Gerber, B. Malissen, J. P. DiSanto. 1997. The common cytokine receptor {gamma} chain controls survival of {gamma}{delta} T cells. J. Exp. Med. 186:1277.[Abstract/Free Full Text]
  47. Kishimoto, H., C. D. Surh, J. Sprent. 1998. A role for Fas in negative selection of thymocytes in vivo. J. Exp. Med. 187:1427.[Abstract/Free Full Text]
  48. Strasser, A., A. W. Harris, S. Cory. 1991. bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell 67:889.[Medline]
  49. Siegel, R. M., M. Katsumata, T. Miyashita, D. C. Louie, M. I. Greene, J. C. Reed. 1992. Inhibition of thymocyte apoptosis and negative antigenic selection in bcl-2 transgenic mice. Proc. Natl. Acad. Sci. USA 89:7003.[Abstract/Free Full Text]
  50. Strasser, A., A. W. Harris, H. von Boehmer, S. Cory. 1994. Positive and negative selection of T cells in T-cell receptor transgenic mice expressing a bcl-2 transgene. Proc. Natl. Acad. Sci. USA 91:1376.[Abstract/Free Full Text]
  51. Tao, W., S. J. Teh, I. Melhado, F. Jirik, S. J. Korsmeyer, H. S. Teh. 1994. The T cell receptor repertorire of CD4-CD8+ thymocytes is altered by overexpression of the BCL-2 protooncogene in the thymus. J. Exp. Med. 179:145.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
S. Uehara, S. M. Hayes, L. Li, D. El-Khoury, M. Canelles, B. J. Fowlkes, and P. E. Love
Premature Expression of Chemokine Receptor CCR9 Impairs T Cell Development
J. Immunol.,