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Gads^–/– Mice Reveal Functionally Distinct Subsets of TCR⁺ CD4^–CD8^– Double-Negative Thymocytes¹

Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, KS 66160

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
TCR expression in CD4^–CD8^– double-negative (DN) thymocytes induces signaling pathways that promote survival and proliferation, as well as differentiation into CD4⁺CD8⁺ double-positive thymocytes. The signaling pathways that regulate survival, proliferation, and differentiation remain unclear. We used Gads-deficient mice to investigate the signaling pathways that regulate these cell fates. During this investigation, we focused on TCR⁺ DN thymocytes and found that there are at least three functionally distinct subsets of TCR⁺ DN thymocytes: TCR⁺ DN3E, TCR⁺ DN3L, and TCR⁺ DN4. Survival and proliferation of TCR⁺ DN3E were independent of Gads, but survival and proliferation of TCR⁺ DN3L cells were Gads dependent. Likewise, expression of Bcl-2 in TCR⁺ DN3E cells was Gads independent, but Gads was necessary for Bcl-2 expression in TCR⁺ DN3L cells. Bcl-2 expression was not dependent on Gads in TCR⁺ DN4 cells, but proliferation of TCR⁺ DN4 cells was Gads dependent. Gads was not required for the differentiation of DN thymocytes into DP thymocytes. In fact, Gads^–/– DN3E cells differentiated into DP thymocytes more readily than wild-type cells. We conclude that signaling pathways required to initiate TCR-induced survival and proliferation are distinct from the pathways that maintain survival and proliferation. Furthermore, signaling pathways that promote survival and proliferation may slow differentiation.

	Introduction

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Development of T cells is characterized by an ordered series of events that culminates in the production of peripheral CD4⁺ or CD8⁺ T cells. The earliest precursors committed to the T cell lineage are found within a population of thymocytes that lack CD4 or CD8 expression and are called CD4^–CD8^– double-negative (DN)³ thymocytes. DN thymocytes can be subdivided into at least four subpopulations, based on CD44 and CD25 expression. DN1 cells (CD44⁺CD25^–) differentiate into DN2 cells (CD44⁺CD25⁺), followed by DN3 cells (CD44^–CD25⁺). DN3 cells lose CD25 expression and become DN4 cells. DN4 cells up-regulate CD4 and CD8 to become CD4⁺CD8⁺ double-positive (DP) thymocytes before differentiating into either single-positive (SP) CD4⁺ or SP CD8⁺ thymocytes.

The first major checkpoint during early T cell development is coincident with the expression of either TCR or TCR. TCR and TCR can be first detected by intracellular staining and flow cytometry in the DN3 population. When expressed at the cell surface, TCR couples with pre-T, CD3 molecules, and -chain to form a pre-TCR (1). At this point, cells that express TCR proceed through a series of events that are collectively known as selection. selection includes signals that promote survival and proliferation (1, 2). In addition, cells receive signals to down-regulate CD25 and then express CD4 and CD8 to become DP thymocytes.

The signaling mechanisms that regulate survival, proliferation, and differentiation of TCR⁺ DN thymocytes remain unclear, but one pathway likely to regulate these events is calcium signaling. Surface expression of the pre-TCR triggers activation of Src-family protein tyrosine kinases (PTKs) and Syk/Zap70 family PTKs. Mice lacking Src-family or Syk/Zap70-family PTKs had a complete block in T cell development at the DN3 stage (3, 4, 5). This defect illustrated the importance of these kinases in the signaling pathways that regulate selection. Syk/Zap70-family PTKs phosphorylate the membrane-bound adaptor protein linker for activation of T cells (LAT) on sites that become the binding sites for several Src homology 2 (SH2) domain-containing proteins including the Gads adaptor protein. Gads constitutively binds the SH2 domain-containing leukocyte protein of 76-kDa (SLP-76) adaptor protein. Thus, the recruitment of Gads to LAT brings SLP-76 into the signaling complex and ultimately leads to calcium mobilization (6, 7, 8, 9, 10, 11). LAT^–/– mice and SLP-76^–/– mice had a complete block in T cell development at the DN3 stage (12, 13, 14), further supporting a role for pre-TCR-mediated signaling in regulating selection.

Unexpectedly, Gads^–/– mice were able to generate mature T cells, although the number of peripheral T cells in Gads^–/– mice was dramatically lower than in wild-type mice (15, 16). In addition, Gads^–/– thymi were much smaller than wild-type thymi. These data indicated that T cell development could proceed in the absence of Gads, but development was not efficient.

Among the defects in Gads^–/– mice was an increased frequency of DN thymocytes and a dramatic reduction in the absolute number of DP thymocytes (16). This observation suggested that Gads was required for the normal transition from DN thymocytes to DP thymocytes. Within the DN population, Gads^–/– mice had an increase in the percentage of DN cells that were DN3, as compared with Gads^+/+ mice. A defect at this stage of development is highly suggestive of a defect in one or more components of selection.

In this study, we extended our previous findings by examining facets of selection that might be regulated by Gads. Specifically, we investigated whether Gads regulates survival, proliferation, or differentiation of TCR⁺ DN thymocytes. We discovered that there are at least three functionally distinct subsets of TCR⁺ DN thymocytes and Gads differentially regulates survival and proliferation in these subsets. In addition, we found that Gads slows the differentiation of TCR⁺ DN thymocytes into DP thymocytes.

	Materials and Methods

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Mice

The generation of Gads^–/– mice was previously described (16). Mice were housed under specific pathogen-free conditions and all experiments were performed in compliance with the University of Kansas Medical Center Institutional Care and Use Committee. Mice were used between the ages of 3 and 5 wk.

Antibodies

Anti-CD8-Alexa 647, anti-CD25-FITC, anti-CD25-allophycocyanin-Cy7, anti-TCR-PE, anti-TCR-PE-Cy5.5, anti-TCR-allophycocyanin, anti-TCR-FITC, anti-CD28-PE, anti-CD127-PE, anti-CD4-allophycocyanin-Alexa 750, anti-TCR-PE-Cy5.5, and anti-Bcl-2-PE were purchased from BD Biosciences. Anti-CD4-Pacific Blue was purchased from Invitrogen Life Technologies. Anti-CD44-PE-Cy7 was purchased from eBioscience.

Surface and intracellular staining

Surface staining was performed in PBS containing 2% FCS. For intracellular staining, surface-labeled cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT). Then, cells were washed twice in PBS containing 2% FCS and 3 mM sodium azide before permeabilizing for 15 min at RT in PBS containing 0.1% saponin and 10% FCS. Cells were stained and washed in saponin buffer and fixed in 1% paraformaldehyde before analysis.

For staining with 4',6'-diamidino-2-phenylindole (DAPI), stained and fixed cells were washed twice with 2% FCS in PBS and resuspended in 1 µg/ml DAPI in 0.2% Tween 20 in PBS. Cells were incubated in the dark for 30 min at RT before analysis.

For annexin V-binding assays, thymocytes were surface labeled with anti-CD4-Pacific Blue, anti-CD8-Alexa 647, anti-CD44-PE-Cy7, anti-CD25-FITC, and annexin V-PE in annexin V binding buffer (0.01 M HEPES (pH 7.4), 0.14 M NaCl, and 2.5 mM CaCl₂).

Flow cytometry

Flow cytometry studies were performed using a BD LSR II (BD Immunocytometry Systems). Data were analyzed using BD FACSDiva software (BD Biosciences).

In vitro differentiation

Total thymocytes from Gads^+/+ and Gads^–/– mice were harvested and depleted of CD4⁺ and CD8⁺ cells using anti-CD4-magnetic particles DM and anti-CD8-magnetic particles DM (BD Biosciences). Remaining cells were surface labeled with anti-CD4-FITC, anti-CD8-FITC, anti-CD44-PE-Cy7, and anti-CD25-PE. DN3E, DN3L, and DN4 thymocytes were FACS purified using a BD FACSAria (BD Immunocytometry Systems). A total of 5 x 10⁴ cells were cocultured with OP9-DL1 cells, as previously described (17). Cells were cultured in DMEM or calcium-free DMEM containing 20% FCS, penicillin, streptomycin, sodium pyruvate, and nonessential amino acids. A total of 5 ng/ml IL-7 and 5 ng/ml Flt3L (PeproTech) were added to the culture, except in some experiments in which the IL-7 was omitted. At the indicated time points, cells were harvested, surface labeled with anti-CD4-Pacific Blue and anti-CD8-Alexa 647. Cells were also labeled with ethidium monoazide bromide (EMA; Invitrogen Life Technologies) and EMA was bound to DNA by exposure to a 60 W light bulb for 10 min. Also, CountBrite absolute counting beads (Invitrogen Life Technologies) were added to each sample. Cells were analyzed by flow cytometry.

Statistics

Statistics were performed using the Student t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
DN3 and DN4 cells can be subdivided into 12 subsets

To determine the mechanism by which Gads regulates early T cell development, we analyzed DN subpopulations in Gads^+/+ and Gads^–/– mice and found that the populations previously known as DN3 and DN4 can be subdivided into at least 12 subsets. First, DN3 and DN4 cells can be divided into DN3E (CD44^–/lowCD25^high), DN3L (CD44^–/lowCD25^low), and DN4 (CD44^–/lowCD25^–) thymocytes. Consistent with previous data (15, 16), there was an increase in the percentage of DN cells that were DN3E with a concurrent loss of DN4 thymocytes in Gads^–/– mice (Fig. 1A). The percentage of DN cells that were in the DN3L population was similar in Gads^+/+ and Gads^–/– mice, suggesting that this population is either a transition between DN3E and DN4 cells or a developmentally unrelated population.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. DN3E, DN3L, and DN4 thymocytes are distinct populations. Gads+/+ and Gads–/– thymocytes were harvested, counted, and surface labeled with anti-CD4, anti-CD8, anti-CD44, and anti-CD25. Then, cells were intracellularly labeled with anti-TCR and anti-TCR. A, Upper panels, CD4 and CD8 expression were analyzed on total thymocytes. Lower panels, Cells were gated on DN thymocytes and CD44 and CD25 expression were analyzed. DN3E, DN3L, and DN4 cells are defined as shown. The numbers indicate the percentage of DN cells that were DN3E, DN3L, and DN4. B, DN3E, DN3L, and DN4 cells from A were gated and analyzed for TCR and TCR expression. Data shown are representative of at least 10 mice.

The model of subdividing DN3 and DN4 thymocytes into three subsets became more compelling after analyzing Gads^–/– thymocytes. As in Gads^+/+ mice, the percentage of TCR⁺ cells in the Gads^–/– DN3L population was substantially greater than in the Gads^–/– DN3E population (Fig. 1B). However, the percentage of Gads^–/– DN4 cells that expressed TCR was substantially lower than in Gads^–/– DN3L cells and lower than in Gads^+/+ DN4 cells. Furthermore, the percentage of Gads^–/– DN4 thymocytes that expressed TCR was dramatically higher than the percentage of Gads^–/– DN3L cells or Gads^+/+ DN4 cells that expressed TCR. Even further, between 6 and 10% of Gads^–/– DN4 thymocytes expressed both TCR and TCR, a population that has been largely ignored in wild-type mice because it represents between 1 and 2% of Gads^+/+ DN4 cells.

These data indicated that the populations called DN3 and DN4 could be subdivided into at least 12 subsets. These subsets include DN3E, DN3L, and DN4 thymocytes, each of which can be further divided into TCR^–TCR^–, TCR⁺, TCR⁺, and TCR⁺TCR⁺.

Gads is not required for / T cell lineage commitment

Next, we calculated the absolute number of Gads^+/+ and Gads^–/– DN thymocytes within the T or T cell lineages. As shown in Fig. 2, the absolute number of TCR⁺ DN3E thymocytes and TCR⁺ DN3E thymocytes were identical in Gads^+/+ and Gads^–/– mice. Based on these data, we can conclude that Gads was not required for TCR, TCR, or TCR gene rearrangement. Further, Gads was not required for / T cell lineage commitment.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Gads–/– mice have a more dramatic defect in TCR+ T cell development than TCR+ T cell development. Gads+/+ and Gads–/– mice were harvested, counted, and surface labeled as described in the legend to Fig. 1. A, The absolute number of TCR+ cells in each thymic subset was calculated. Shown is mean ± SE; *, p < 0.005, n = 16 Gads+/+ and 15 Gads–/– mice. B, The absolute number of TCR+ cells in each thymic subset was calculated. Shown is mean ± SE. *, p < 0.04, n = 10 Gads+/+ and 11 Gads–/– mice.

In contrast to TCR⁺ cells, the number of TCR⁺ DN thymocytes was only mildly decreased in Gads^–/– mice, as compared with Gads^+/+ mice; the average number of TCR⁺ DN thymocytes in Gads^–/– mice was greater than half the average number of TCR⁺ DN thymocytes in Gads^+/+ mice (Fig. 2B). Also, the number of DN4 cells that expressed both TCR and TCR was nearly identical in Gads^+/+ and Gads^–/– mice, suggesting that TCR⁺TCR⁺ DN thymocytes are a normally occurring population that is independent of Gads.

In summary, the cell counts indicated that Gads is not required for / T cell lineage commitment and Gads is more critical for T cell development than T cell development.

Gads is required for proliferation of TCR⁺ DN thymocyte subsets, but not TCR⁺ DN thymocytes

We measured DNA content as an indicator of cell cycle status in thymocyte subsets (Fig. 3). In Gads^+/+ mice, 41 ± 6.6% (n = 12) of TCR⁺ DN3E thymocytes were in the S, G₂, or M phase of the cell cycle (Fig. 3A). A comparable percentage of Gads^–/– TCR⁺ DN3E thymocytes 36 ± 6.7% (n = 13) were in cycle (p = 0.07), indicating that Gads is not required for TCR-induced proliferation. However, the percentage of TCR⁺ DN3L and DN4 cells in the S, G₂, or M phase of the cell cycle was lower in Gads^–/– mice than Gads^+/+ cells. For TCR⁺ DN3L cells, 47 ± 4.0% of Gads^+/+ cells and 30 ± 6.5% of Gads^–/– cells were in the S, G₂, or M phase of the cell cycle (p < 0.0001). The percentages of TCR⁺ DN4 cells in the S, G₂, or M phase of the cell cycle were 22 ± 8.5% in Gads^+/+ mice and 12 ± 5.0% in Gads^–/– mice (p = 0.0037).

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Gads is not required for proliferation of TCR+ DN3E thymocytes, but is required for proliferation of TCR+ DN3L and DN4 cells. Cells were stained with anti-CD4, anti-CD8, anti-CD44, anti-CD25, anti-TCR, anti-TCR, and DAPI. A, The percentage of cells in the indicated populations that were in S, G2, or M phase of the cell cycle is shown (mean ± SD). *, p < 0.0001; **, p = 0.0037, n = 13 Gads+/+ and 12 Gads–/– mice for TCR+ cells. B, The percentage of TCR+ DN3E, TCR+ DN3L, and TCR+ DN4 thymocytes that were in the S, G2, or M phase of the cell cycle is shown (mean ± SD); n = at least 5 for each subset.

The differences in proliferation seen in Gads^+/+ and Gads^–/– TCR⁺ DN thymocytes may reflect a defect in survival in Gads^–/– DN3L and DN4 cells. Using annexin V staining, we found that survival of Gads^–/– DN3E thymocytes was comparable to that of Gads^+/+ cells, but survival of Gads^–/– DN3L thymocytes was impaired (Fig. 4A). A total of 2.6 ± 1.8% of Gads^+/+ DN3E thymocytes were annexin V positive, as compared with 3.6 ± 2.6% of Gads^–/– cells. In contrast, 7.8 ± 1.5% of Gads^+/+ DN3L thymocytes were annexin V positive, as compared with 17 ± 4.7% of Gads^–/– cells (p = 0.0030, n = 6). Because expression of TCR and TCR was markedly different in Gads^+/+ and Gads^–/– DN4 populations (Fig. 1B), analysis of annexin V staining on this population was not interpretable. These data are consistent with a model in which survival of DN3E cells and DN3L cells are regulated by different signaling pathways.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. Gads is not required for survival of TCR+ DN3E thymocytes, but is required for survival of TCR+ DN3L cells. A, Gads+/+ and Gads–/– thymocytes were harvested and stained with anti-CD4, anti-CD8, anti-CD44, anti-CD25, and annexin V. The percentages of DN3E and DN3L cells that were annexin V+ are shown (mean ± SD). *, p = 0.003, n = 6. B, Gads+/+ and Gads–/– thymocytes were harvested and stained with anti-CD4, anti-CD8, anti-CD44, and anti-CD25. Then, cells were permeabilized and stained with anti-TCR and anti-Bcl-2 or isotype control Ig. The percentage of TCR+ cells in each subset that expressed Bcl-2 is shown. Data shown are representative of six mice.

Collectively, these data suggested that survival and proliferation of TCR⁺ DN3E, DN3L, and DN4 thymocytes are regulated by different signaling pathways. Gads was not required for survival and proliferation TCR⁺ DN3E thymocytes, but Gads regulated survival and proliferation of TCR⁺ DN3L thymocytes. In DN4 cells, Gads was required for proliferation, but not Bcl-2 expression. If DN3E, DN3L, and DN4 cells represent consecutive stages of T cell development, then the signaling pathways regulating survival and proliferation of TCR⁺ DN thymocytes may change as cells transition from DN3E to DN3L to DN4. Alternatively, TCR⁺ DN3E and TCR⁺ DN3L cells could represent distinct lineages that are regulated differently.

Gads is not required for differentiation of DN thymocytes into DP thymocytes

To test whether Gads might regulate T cell differentiation, we used the in vitro T cell development system developed by Schmitt et al. (17). FACS-purified DN3E, DN3L and DN4 cells from Gads^+/+ and Gads^–/– mice were cocultured with OP9-DL1 cells, a bone marrow stromal cell line transfected with the Notch ligand DL-1. Immature SP CD8⁺ and DP thymocytes could be found within 1 day of culture in samples seeded with Gads^+/+ DN3E, DN3L, and DN4 cells (Fig. 5B), indicating that each population contained precursors of DP thymocytes. Fewer DP thymocytes were found in cultures seeded with DN3E thymocytes than DN3L or DN4. This likely reflected the smaller percentage of TCR-expressing cells in the DN3E population, as compared with DN3L and DN4, and supports the model in which DN3E thymocytes must first differentiate into DN3L and DN4 cells before giving rise to DP thymocytes.

View larger version (71K): [in this window] [in a new window]   FIGURE 5. Gads expression can slow DN thymocyte differentiation, but extracellular calcium is required for differentiation. Gads+/+ and Gads–/– DN3E, DN3L, and DN4 cells were FACS purified and cocultured with OP9-DL1 cells, as described in Materials and Methods. The purity of the sort is shown in A. B, At the indicated time points, cells were harvested and analyzed for CD4 and CD8 expression. The number in each dot plot represents the percentage of viable cells that expressed CD4 and CD8. Representative of six independent experiments. C, Some samples in the in vitro differentiation assay were cultured in calcium-free medium. Data shown are representative of six independent experiments.

Although cultures seeded with Gads^–/– cells had a greater percentage of DP thymocytes than cultures seeded with Gads^+/+ cells, fewer live lymphocytes were harvested from cultures seeded with Gads^–/– cells. The most striking difference was in the DN3L population. On day 4, cultures seeded with Gads^+/+ DN3L thymocytes contained an average of 391,087 ± 54,877 cells, as compared with 102,085 ± 15,237 Gads^–/– cells (p = 0.0011, n = 4). This trend was also observed in DN3E cells (126,658 ± 48,268 vs 75,798 ± 20,373) and DN4 cells (66,473 ± 42,842 vs 12,000 ± 4124), although these differences did not reach the level of statistical significance.

As another test to determine whether Gads regulates survival of DN thymocytes, cells cultured in the in vitro differentiation assay were stained with EMA, a marker for dead or dying cells. On average, 38 ± 6.0% of Gads^–/– DN3E cells in the live lymphocyte gate were EMA⁺, as compared with 24 ± 4.0% of Gads^+/+ cells (p = 0.035, n = 3). Likewise, 6.6 ± 0.92% of Gads^–/– DN3L cells were EMA⁺, as compared with 2.8 ± 2.0% of Gads^+/+ DN3L thymocytes (p = 0.024).

Extracellular calcium is required for normal survival and proliferation of DN thymocytes

Because the most understood function of Gads is to regulate calcium mobilization in T cells, we tested whether calcium was required for the differentiation of DN thymocytes into DP cells. The in vitro differentiation experiment described above was repeated using calcium-free medium. Although a smaller percentage of cells recovered from cultures seeded in calcium-free medium were DP thymocytes than cells recovered from calcium-containing medium (Fig. 5C), cells could readily differentiate in the absence of extracellular calcium. Similar results were observed when using Gads^+/+ or Gads^–/– cells. The number of cells recovered from cultures seeded in calcium-free medium was consistently and significantly lower than cultures seeded in calcium-containing medium. The average number of cells harvested from calcium-free medium seeded with Gads^+/+ DN3E thymocytes was 40% the number harvested from calcium-containing medium (p = 0.010, n = 4). The average numbers of cells recovered from cultures seeded with Gads^+/+ DN3L and DN4 in calcium-free medium was 32% (p < 0.0001) and 24% (p = 0.023) the number of cells harvested from calcium-containing medium. Similar trends were observed with Gads^–/– cells; the average numbers of thymocytes harvested from cultures seeded with Gads^–/– DN3E, DN3L, and DN4 thymocytes in calcium-free medium were 50%, (p = 0.0092), 28% (p = 0.0010), and 51% (p = 0.079) of the number of cells recovered from calcium-containing medium, respectively.

These data suggested that extracellular calcium was most required for proliferation and survival of TCR⁺ DN thymocytes, but differentiation was only mildly impaired without extracellular calcium.

Gads regulates cytokine receptor expression

We then sought to uncover the mechanism by which Gads might regulate survival, proliferation, and differentiation of TCR⁺ DN thymocytes. CD28 is more highly expressed in DN4 cells than DN3, and CD28^–/– DN thymocytes have impaired proliferation of DN4 cells (19). Based on these data, we tested whether Gads might regulate CD28 expression. In Gads^+/+ and Gads^–/– DN thymocytes, CD28 expression strongly correlated with TCR expression, but CD28 expression was slightly reduced in all Gads^–/– TCR⁺ DN subsets, as compared with Gads^+/+ cells (Fig. 6A). These data indicated that Gads was not necessary for CD28 expression.

View larger version (51K): [in this window] [in a new window]   FIGURE 6. Gads regulates CD127 expression on TCR+ DN subsets. Gads+/+ and Gads–/– thymocytes were labeled with anti-CD4, anti-CD8, anti-CD44, anti-CD25, anti-TCR, and either anti-CD28 (A), anti-CD127 (B), anti-132 (C), or anti-CD122 (D). A–C, The MFI of the indicated surface protein is shown on TCR+ and TCR– cells. D, CD122 expression is shown. The percentages represent the percentage of TCR+ cells in each subset that expressed CD122.

The high-affinity IL-2R consists of CD25 (IL-2R), CD122 (IL-2R), and CD132. CD25 was used to define the populations, so CD25 expression on Gads^+/+ and Gads^–/– cells was, by definition, comparable. CD122 was expressed only on a subset of TCR⁺ DN thymocytes (Fig. 6D). Some TCR⁺ DN3E cells expressed CD122 in a Gads-dependent fashion, but CD122 was expressed by a greater percentage of Gads^–/– TCR⁺ DN4 cells than Gads^+/+ TCR⁺ DN4 cells. These data suggested that TCR⁺ CD122⁺ DN thymocytes represent a distinct population of TCR⁺ DN thymocytes that may be regulated in a Gads-dependent manner.

IL-7 slows the differentiation of DN thymocytes into DP thymocytes

Because Gads may regulate CD127 expression on some TCR⁺ DN thymocyte subsets, we used the in vitro differentiation assay described earlier to test the role of IL-7 on the differentiation of DN thymocyte subsets. Gads^+/+ DN3E, DN3L, and DN4 thymocytes were cocultured with OP9-DL1 cells in the presence or absence of IL-7. Omission of IL-7 from the culture resulted in a greater percentage of harvested cells expressing CD4 and CD8 (Fig. 7). The results were most dramatic among wells seeded with DN3E and DN3L cells, where CD127 expression was highest. Similar data were obtained when Gads^–/– cells were used (data not shown), indicating that the lower level of CD127 expression seen in Gads^–/– cells was sufficient to transmit IL-7-dependent signals that slow differentiation in vitro.

View larger version (46K): [in this window] [in a new window]   FIGURE 7. IL-7 inhibits differentiation of DN thymocytes into DP thymocytes. Wild-type DN3E, DN3L, and DN4 cells were FACS purified and cocultured with OP9-DL1 cells, as described in the legend to Fig. 5, except IL-7 was omitted from some samples (-IL-7). Cells were harvested at 24 or 48 h and stained with anti-CD4 and anti-CD8. Shown is the percentage of viable cells that expressed CD4 and CD8. Representative of four independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

While studying Gads^–/– thymocytes, we found at least three functionally distinct populations of TCR⁺ DN thymocytes. When CD44 and CD25 were first used to described subsets of DN thymocytes, DN thymocytes were divided into four major subsets: DN1, DN2, DN3, and DN4 (21). As flow cytometers became more sensitive, the existence of intermediate populations have been proposed. For example, DN3 and DN4 cells can be divided into DN3E, DN3L, and DN4, but clear distinctions among these populations have not been found. TCR and TCR can be first detected as intracellular proteins in the DN3 stage of development. As shown in Fig. 1, the pattern of TCR and TCR expression is markedly different in DN3E, DN3L, and DN4 cells. The differences were much more evident in Gads^–/– mice than in Gads^+/+ mice. In Gads^+/+ mice, the percentage of DN thymocytes that expressed TCR was similar in DN3L and DN4 cells and much smaller in DN3E cells. This finding might suggest that DN3L and DN4 cells are similar. However, in Gads^–/– mice, the percentage of DN thymocytes that expressed TCR was dramatically higher in DN3L cells than in DN3E or DN4 cells, supporting a model in which DN3L and DN4 cells are distinct populations.

Although it is likely that the DN3E, DN3L, and DN4 subsets represent consecutive developmental stages, we cannot exclude the possibility that DN3L cells represent a separate lineage of cells. The in vitro differentiation assay revealed that all three subsets contained DP thymocyte precursors (Fig. 5). A greater percentage of DP cells were recovered from cultures seeded with DN3L and DN4 cells than with DN3E cells, consistent with the model that DN3E cells differentiate into DN3L and DN4 cells before expressing CD4 and CD8. However, the percentage of DP cells recovered from cultures seeded with DN3L cells and DN4 thymocytes were often comparable, suggesting that DN3L cells might not need to differentiate into DN4 cells before expressing CD4 and CD8. Alternatively, thymocytes may traverse the DN4 stage so rapidly that we are unable to detect the correct order of development in this assay.

There are two major considerations when using the in vitro differentiation assay to determine whether DN3L cells differentiate into DN4 cells before expressing CD4 and CD8. The first consideration is that more TCR⁺ DN3L cells were in the S, G₂, or M phases of the cell cycle than TCR⁺ DN4 cells (Fig. 3), which is consistent with reports that total DN3L cells proliferate more robustly than total DN4 cells (22, 23). This proliferation likely results in more DP precursors being generated in DN3L cultures than DN4 cultures.

The other consideration when evaluating the in vitro differentiation assay is the complexity of the TCR⁺ DN4 population. Analysis of Bcl-2 expression revealed two separate populations of TCR⁺ DN4 cells; approximately half the TCR⁺ DN4 cells expressed Bcl-2 (Fig. 4B). In addition, a subset of TCR⁺ DN4 cells expressed CD122 (Fig. 6D). The physiologic differences between Bcl-2⁺, Bcl-2^–, CD122⁺, and CD122^– TCR⁺ DN4 cells are unknown, but it is possible that not all these subpopulations will differentiate into DP cells.

Signaling pathways that regulate survival and proliferation of TCR⁺ DN thymocytes varied among the TCR⁺ DN thymocyte subsets. Gads was not required for survival and proliferation of TCR⁺ DN3E thymocytes (Fig. 3), but Gads was required for optimal survival and proliferation of TCR⁺ DN3L and DN4 cells. This may reflect a role for Gads in sustaining the signals required for survival and proliferation, but not in initiating these signals. Alternatively, the role for Gads in regulating survival and proliferation may only be evident at later stages of development where more subtle defects could become detectable over a number of cell divisions.

Multiple assays were used to show that Gads was not required for survival of DN thymocyte subsets. First, we used annexin V staining, which has the limitation of prohibiting the concurrent use of intracellular staining to differentiate TCR⁺ and TCR^– DN thymocytes. However, because the percentage of TCR⁺ DN thymocytes was comparable in Gads^+/+ and Gads^–/– DN3L population, it is likely that Gads regulates survival of TCR⁺ DN3L cells. We also measured cleaved caspase-3 in TCR⁺ and TCR^– DN thymocytes subsets, but caspase-3 cleavage appeared to more accurately reflect proliferation than apoptosis (data not shown). A correlation between caspase-3 cleavage and proliferation would be consistent with the requirement for caspases in lymphocyte proliferation (24, 25). Further, caspase-8 deficiency leads to a complete block in development at the DN3 stage (26), suggesting that caspases are required for survival, proliferation, or differentiation of TCR⁺ DN thymocytes.

For another indicator of cell survival, we measured Bcl-2 expression in TCR⁺ DN thymocyte subsets. Like proliferation, Bcl-2 expression was regulated differently in the various TCR⁺ DN thymocyte subsets. In wild-type mice, Bcl-2 protein levels were slightly higher in TCR⁺ DN3E cells than TCR⁺ DN3L cells (Fig. 4B). This is consistent with the Bcl-2 mRNA analyses performed by Mandal et al. (27) and Verschelde et al. (28) who showed that Bcl-2 mRNA declined as cells progressed from the DN3 stage to DN4 stage. Bcl-2 protein expression was comparable in Gads^+/+ and Gads^–/– TCR⁺ DN3E cells, consistent with the model that Bcl-2 expression is IL-7 dependent (29). However, Gads^–/– TCR⁺ DN3L cells had lower levels of Bcl-2 protein than Gads^+/+ cells (Fig. 4B), suggesting that Bcl-2 expression is regulated by Gads in this subset. Gads-dependent signaling pathways may either promote Bcl-2 transcription in TCR⁺ DN3L cells, stabilize Bcl-2 mRNA, or stabilize Bcl-2 protein.

Unlike DN3L cells, Bcl-2 expression among DN4 thymocytes was Gads independent (Fig. 4B). Gads-independent Bcl-2 expression was also evident in a population of TCR^– DN4 cells. Based on the percentages of TCR^– cells that expressed TCR in Gads^+/+ and Gads^–/– mice, it is likely that the TCR^– Bcl-2⁺ DN4 cells were TCR⁺. Thus, Bcl-2 expression in TCR⁺ DN cells was likely Gads independent. In addition, there was no difference in the percentage of Gads^+/+ and Gads^–/– TCR⁺ DN thymocytes in the S, G₂, or M phase of the cell cycle (Fig. 3). The different signaling requirements for survival and proliferation of TCR⁺ DN thymocytes and TCR⁺ DN thymocytes likely explain why Gads was more critical for T cell development than T cell development.

Lastly, we examined thymocyte survival in the in vitro differentiation assay and found that fewer Gads^–/– DN thymocytes survived in the experimental period than Gads^+/+ cells. This result was consistent with the annexin V staining and the Bcl-2 data and support a model in which Gads regulates survival of TCR⁺ DN thymocytes.

Whereas Gads was required for survival and proliferation of some TCR⁺ DN thymocyte subsets, Gads was not required for differentiation of TCR⁺ DN thymocytes into DP thymocytes (Fig. 5B). In fact, there was consistently a higher percentage of DP thymocytes recovered from cultures seeded with Gads^–/– DN3E cells than Gads^+/+ DN3E cells, suggesting that Gads may retard differentiation. These data support a model in which proliferation and differentiation are linked. In wild-type thymi, DN thymocytes may proliferate to expand the pool of DP precursors. CD4 and CD8 expression may not be possible until the population has sufficiently expanded. In Gads^–/– mice where proliferation of DP precursors is defective, cells may differentiate prematurely. This could explain why there are so few DP thymocytes in Gads^–/– mice, despite relatively minor differences in the absolute number of DN thymocytes (16). Accelerated differentiation might also explain why the number of TCR⁺ DN4 cells was so strikingly diminished in Gads^–/– mice, whereas the number of TCR⁺ DN3L cells was less deficient and the number of TCR⁺ DN3E cells was normal (Fig. 2A).

Because the most characterized function of Gads is to regulate TCR-mediated calcium mobilization, we tested the effects of calcium deprivation on thymocyte proliferation and differentiation. Fewer Gads^+/+ thymocytes were recovered from cultures containing calcium-free medium than calcium-containing medium, suggesting that extracellular calcium is required for survival, proliferation, or both. This result is consistent with the lower cell yields obtained from Gads^–/– thymocytes and supports a role for Gads-mediated calcium mobilization regulating survival, proliferation, or both. Whereas Gads^–/– DN thymocytes differentiated faster than Gads^+/+ cells (Fig. 5B), DN thymocytes cultured in calcium-free medium had impaired differentiation (Fig. 5C). These data suggest that Gads^–/– TCR⁺ DN thymocytes can generate a sufficient calcium signal to promote differentiation.

Finally, we sought to examine the mechanism by which Gads might regulate cell fate decisions in TCR⁺ DN thymocytes. Because CD127 expression can be regulated by the pre-TCR (30), we questioned whether Gads might regulate CD127 expression. In wild-type mice, CD127 expression decreased as TCR⁺ cells progressed from DN3E to DN3L to DN4, but CD127 was expressed at a low level in DN4 cells (Fig. 6B and Ref. 31). CD127 expression was lower on Gads^–/– TCR⁺ DN3E and DN3L cells than on Gads^+/+ cells, but Gads^–/– and Gads^+/+ TCR⁺ DN4 cells and TCR^– DN thymocytes had comparable levels of CD127 surface expression. These data suggested that Gads could regulate CD127 expression specifically on TCR⁺ DN3E and DN3L cells and Gads might regulate T cell development by enhancing CD127 expression.

When evaluating the physiologic relevance of Gads-regulated CD127 expression, it is important to consider the IL-7 concentration in the tissue housing TCR⁺ DN thymocytes. TCR⁺ DN cells are most likely found in the subcapsular zone of the thymus (32, 33, 34, 35). Because IL-7-producing cells are concentrated at the corticomedullary junction and the medulla (36), it is likely that the concentration of IL-7 is low in the subcapsular zone of the thymus. By promoting CD127 expression in TCR⁺ DN3E and TCR⁺ DN3L thymocytes, Gads may enhance the sensitivity of the DN thymocytes to low concentrations of IL-7.

We used the in vitro differentiation assay to determine whether IL-7 can regulate DN thymocyte development. Consistent with data reported by Balciunaite et al. (37), omission of IL-7 from the culture resulted in faster differentiation of DN thymocytes into DP thymocytes (Fig. 7). The results were more dramatic on DN3E and DN3L cells, where CD127 expression was higher. Similar results were obtained using Gads^–/– cells (data not shown), suggesting that, in the presence of excess IL-7, reduced CD127 expression did not impair IL-7 signaling. Alternatively, IL-7 may act on the OP9 stromal cells in a manner that enhances differentiation of the thymocytes into DP cells.

In summary, we conclude that there are at least three functionally distinct subsets of TCR⁺ DN thymocytes. The signaling pathways that regulate survival and proliferation of TCR⁺ DN thymocytes were different in each thymocyte subset.

	Acknowledgments

 
We thank Drs. Steve Benedict and Michael Parmely for critical review of this manuscript and members of the Benedict laboratory for helpful discussions. We also thank Dr. Joyce Slusser and the Flow Cytometry Core for assistance with flow cytometry and Dr. Martha Montello for assistance in preparing the manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant P20 RR016443 from the Centers of Biomedical Research Excellence Program of the National Center for Research Resources.

² Address correspondence and reprint requests to Dr. Thomas M. Yankee, Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, 3901 Rainbow Boulevard, 3025 Wahl Hall West, Mail Stop 3029 Kansas City, KS 66160. E-mail address: tyankee{at}kumc.edu

³ Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; PTK, protein tyrosine kinase; LAT, linker for activation of T cells; SH, Src homology; SLP-76, SH2 domain-containing leukocyte protein of 76-kDa; RT, room temperature; DAPI, 4',6-diamidino-2-phenylindole; EMA, ethidium monoazide bromide; MFI, mean fluorescence intensity.

Received for publication November 22, 2006. Accepted for publication May 3, 2007.

	References

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