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The Journal of Immunology, 2004, 173: 2253-2261.
Copyright © 2004 by The American Association of Immunologists

B7-CD28 Interaction Promotes Proliferation and Survival but Suppresses Differentiation of CD4–CD8– T Cells in the Thymus1

Xincheng Zheng2,*, Jian-Xin Gao2,*, Xing Chang*, Yin Wang*, Yan Liu*, Jing Wen*, Huiming Zhang*, Jian Zhang{dagger}, Yang Liu3,* and Pan Zheng*

* Division of Cancer Immunology, Department of Pathology, Ohio State University Medical Center and Comprehensive Cancer Center, Columbus, OH 43210; and {dagger} Department of Orthopedic Surgery, Rush University Medical Center, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25+CD4+ regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4–CD8– (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25+CD44– (DN3) to CD25–CD44– (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cells are educated in the thymus to gain immune competence. Mature T cells migrate into secondary lymphoid organs where they encounter Ags, expand, and differentiate into effector cells. The activated T cells are dispatched to target tissues to mediate effector function. As the major costimulators in T cell activation, B7-1 and B7-2 were first demonstrated to play a major role in the activation and differentiation of T cells in the secondary lymphoid tissues (1, 2, 3). Subsequently, the notion of T cell costimulation has been extended to T cell effector function in target tissues, including tumors (4, 5, 6, 7) and normal tissues during autoimmune destruction (8, 9). Accumulating data from several groups, including that of ours (10), have shown that B7/CD28 enhances clonal deletion of autoreactive T cells (11, 12, 13, 14). In addition, anti-CD28 Ab promotes differentiation of CD4+CD8+ double-positive (DP)4 thymocytes into single-positive (SP) T cells (15, 16). Moreover, it has been shown that costimulation is critical for the generation and homeostasis of the CD25+CD4+ regulatory T cells (17, 18, 19, 20). Because all of these stages involve interaction of Ag-specific receptors with MHC:peptide complex, it can be suggested that T cell costimulation is an important parameter whenever TCR is engaged by MHC:peptide complex. An interesting issue that has not been addressed is whether T cell costimulation participates in the {beta}-selection, in which the rearranged TCR{beta} are paired with pT{alpha} to mediate proliferation and differentiation of immature thymocytes.

The earliest T cell progenitor expresses neither TCR nor coreceptor CD4 or CD8, and is usually referred to as double-negative (DN) T cells for the lack of CD4 and CD8. They are subdivided according to the expression of surface markers CD44 and CD25. DN1, which is CD44+CD25–, contains cells that are committed to lymphoid lineage, but maintains the potential to develop into T cells, B cells, or NK cells (21). With the increased CD25 expression, DN1 becomes CD44+CD25+, which are called DN2 cells. Cells at this stage are committed to T cell lineage, and therefore are also called pro-T cells. Growth factors, such as IL-7 and stem cell factor (c-kit ligand), play important roles in this developmental step (22, 23, 24, 25, 26). DN3, or early pre-T population, down-regulates CD44 and is characterized by CD44–CD25+. At this stage, the TCR{beta} locus is rearranged by a RAG-dependent mechanism. This leads to the assembly of the pre-TCR complex consisting of CD3, pT{alpha}, and TCR{beta} chains. Disruption of the complex causes a complete arrest at DN3, as shown in RAG (27, 28, 29)-, TCR{beta} (30)-, and pT{alpha} (31)-deficient mice. As further maturation occurs, cells lose expression of CD25 to become CD44–CD25– or DN4. DN4 progresses to the CD4+CD8+ DP via an immature SP stage and then goes through positive and negative selection to become CD4+ or CD8+ SP T cells.

Although it is clear that the survival of DN4 requires rearrangement of TCR{beta} and expression of pT{alpha}, very little is known about other cell surface interactions during the early phase of T cell maturation, which is generally coupled with rapid T cell proliferation. In the process of studying the effect of B7 blockade on the development of Ag-specific T cells in the thymus, we observed that anti-B7 Abs have significant effect on the development of early T cell progenitors. To substantiate this observation, we systematically analyzed the maturation and proliferation of the T cell progenitors in mice with targeted mutation of B7-1 and B7-2, CD28, and CTLA-4. Our results demonstrate targeted mutations of B7-1 and B7-2 or CD28 diminish the proliferation and survival of DN4 T cells and accelerate DN3 to DN4 transition, most likely by increasing accumulation of the RAG-2 protein and enhancing TCR rearrangement.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals

Wild-type (WT), B7-1–/–B7-2–/– (32), CD28–/– (33) C57BL6/j mice were purchased from The Jackson Laboratory (Bar Harbor, ME). CTLA-4+/– mice in B6 background have been described (34). All animals were maintained in the University Laboratory Animal Research Facility at Ohio State University under specific pathogen-free conditions.

Abs and flow cytometry

Both cell surface markers and intracellular staining were analyzed by flow cytometry. The fluorescence-conjugated Abs anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD44 (IM7), anti-CD25 (PC61), anti-CD28 (37.57), anti-CTLA4 (UC10-4F10-11), and anti-TCR {beta}-chain (H57-597) were purchased from BD Pharmingen (San Diego, CA). The fixation and permeabilization solution kit (cytofix/cytoperm; BD Pharmingen) was used for intracellular staining, according to manufacturer’s protocol.

To measure the proliferation of thymocytes in vivo, mice were injected i.p. with BrdU (1 mg/mouse in 100 µl of PBS). Four hours later, the mice were sacrificed and thymocytes were prepared. BrdU incorporation was detected by flow cytometry with a BrdU Flow Kit, as described by manufacturer (BD Pharmingen).

The apoptotic thymocytes were determined by their binding to annexin V. After cell surface staining, the cells were resuspended in staining buffer with annexin V (BD Pharmingen) and were stained at room temperature for 15 min. The samples were analyzed by flow cytometry within 1 h.

Western blot and real-time RT-PCR

The total thymocytes were depleted twice with anti-CD4 (GK1.5), anti-CD8 (2.4.3), anti-TCR{gamma}{delta} (GL-3), anti-I-Ab (AF6-120.1), and magnetic Dynabeads (Dynal Biotech, Oslo, Norway), according to manufacturer’s manual. The RAG-2 protein level was detected by Western blot. Briefly, cells were lysed in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1 mM DTT,1 mM Na3O4, 2 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF. Total protein extracts (100 µg) were fractionated on a 12% SDS-PAGE and transferred to Hybond-P membrane (Amersham Biosciences, Buckinghamshire, U.K.). The transferred membrane was blotted by a goat polyclonal Ab against RAG-2 (Santa Cruz Biotechnology, Santa Cruz, CA). Mouse {beta}-actin mAb (Sigma-Aldrich, St. Louis, MO) was used for internal blotting control.

Total RNA was extracted from DN thymocytes and treated with RNase-free DNase I (Invitrogen Life Technologies, Carlsbad, CA). cDNA was synthesized with Superscriptase II and oligo(dT) (Invitrogen Life Technologies). The real-time PCR was conducted in ABI PRISM 7700 Cycler (Applied Biosystems, Foster City, CA) using a QuantiTect SYBR green PCR kit (Qiagen, Valencia, CA), according to manufacturers’ protocols and real-time PCR conditions. The relative expression levels in B7 knockout (B7KO) thymocytes were compared with those from WT thymocytes after normalization with internal control (ribosome L-19), as follows: comparative expression level = 2–({Delta}CTB7KO–{Delta}CTB6). {Delta}CT = CT(target gene) – CT(L-19) and represents the difference between the two threshold cycle (CT) values of two PCRs for the same initial template amount. The oligonucleotide primers were: the RAG-1 forward primer, 5'-TGCAGACATTCTAGCACTCTGG-3', and reverse primer, 5'-ACATCTGCCTTCACGTCGAT-3'; the RAG-2 forward primer, 5'-CACATCCACAAGCAGGAAGTACAC-3', and reverse primer, 5'-TCCCTCGACTATACACCACGTCAA-3'; the pre-TCR{alpha} forward primer, 5'-AGCTTCTGGCTGCAACTGGGTCAT-3', and reverse primer, 5'-TACCTGCCGCTGTGTCCCCCCGAG; TCR V{alpha}2 forward primer, 5'-CAATAAAAGGGAGAAAAAGC-3', and reverse primer, 5'-AAGTCGGTGAACAGGCAGAG3'; TCR V{alpha}4 forward primer, 5'-AGCAGCAGAGGKTTTGAAGC-3', and reverse primer, 5'-GGCACATTGATTTGGGAGTC-3' (35); CD8 {beta}-chain forward primer, 5'-CTGCTTTGAACTGCTGCAAG-3', and reverse primer, 5'-GGAAGAGTACATGGTGCGT-3'; the ribosome L-19 forward primer, 5'-CTGAAGGTCAAAGGGAATGTG-3', and reverse primer, 5'-GGACAGAGTCTTGTGATCTC-3' (36), was used as internal control.

Antimitotic drug treatment

Eight-week-old C57BL/6 mice were given three injections of either demecolcine (Sigma-Aldrich) at 200 µg/mouse/injection or PBS at 4-h intervals. This was followed by two daily treatments with the same amount of drug. The BrdU was injected in conjunction with the last treatment. The mice were sacrificed at 4 h after the last injection to harvest thymocytes for analyses.

Immunohistochemistry with anti-B7-1 or anti-B7-2 mAb

Frozen sections of the thymus fixed with acetone and were incubated with anti-B7-1 mAb 3A12 (37) or anti-B7-2 mAb GL-1 (38) hybridoma supernatants. The anti-B7-1 mAb was detected by biotinylated goat anti-hamster Abs, while anti-B7-2 Abs were detected by biotinylated goat anti-rat Abs, each followed by HRP-conjugated streptavidin.

Statistical analysis

Data were statistically analyzed with two-tailed Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Impact of defective B7-1, B7-2, and their receptors on the DN subsets

In the process of studying the effect of anti-B7-1/2 Abs for T cell development in vivo, we observed that anti-B7-1/2, but not control Ig, caused a substantial reduction of DN3 and a major increase of DN4 in thymus of adult C57BL/6 mice and perinatal BALB/c mice. A statistically significant reduction was also observed in the DN2 subset (data not shown). To substantiate this observation, we compared mice deficient for B7-1 and B7-2 with their WT controls for DN subsets. As shown in Fig. 1, in comparison with WT, B7-deficient mice had an increased DN4, but decreased DN2 and DN3.



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  		FIGURE 1. The distribution of DN subsets in the thymi of WT, B7KO, and CD28KO mice. B7KO, CD28KO, and C57BL/6 mice were sacrificed at 8 wk old, and thymocytes were harvested and stained with anti-CD4, anti-CD8, anti-CD44, and anti-CD25 Abs. Profiles of total viable cells (upper panels) or gated CD4–CD8– DN populations (lower panels) are shown. Contour graphs depict thymocyte subsets of one mouse in each group. Means and SD are given in the panels. The experiments have been done twice, using a total of nine mice per group.

 
B7-1 and B7-2 interact with two known receptors, CD28 and CTLA-4, on T cells. A putative third receptor has been suggested (39, 40), although its identity remains elusive. As a first step to determining the receptors that may be involved in the alteration of DN3 and DN4, we first analyzed cell surface expression of CD28 and intracellular accumulation of the CTLA-4 among DN subsets of WT thymus. As shown in Fig. 2, in comparison with isotype control, CD28 is expressed on ~13% of DN3 and 70–80% of DN4. CTLA-4, in contrast, is expressed on ~80% of DN3 cells and 40% of DN4. To determine the role for CD28 and CTLA-4 in the development of DN, we compared WT, B7KO, CD28KO, and CTLA-4KO for the DN subsets. As shown in Fig. 1, targeted mutation of CD28 had significant effects on the distribution of DN. Qualitatively, reductions in DN2 and DN3 and an increase in DN4 were parallel to what was found in B7–/–. However, targeted mutation of CD28 was less effective than those of the B7-1 and B7-2. To determine whether CTLA4 function explains the differences between CD28–/– and B7–/–, we compared thymi harvested from 15-day-old CTLA-4+/– and CTLA-4–/– mice for DN maturation and proliferation. At this point, the overall subset distribution of SP, DN, and DP subsets is grossly normal (41) and no autoimmune disease was observed. Fig. 3 shows the profiles of DN thymocytes in 2-wk-old CD28–/–, B7–/–, CTLA-4+/–, and CTLA-4–/– mice. The distribution of DN subsets in the 2-wk-old CTLA4+/– mice was similar to that of adult mice (Fig. 1). No statistically significant difference was detected in the relative amounts of DN subsets between CTLA4+/– and CTLA4–/– mice. Mutation of B7 and CD28, in contrast, significantly reduced DN3 and increased DN4. In contrast to what was observed in adult mice (Fig. 1), the effects of B7 mutation in the young mice were comparable to those of CD28. Thus, CTLA-4 alone is not responsible for the costimulation by B7-1 and B7-2 in the early stage of thymocyte development.



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  		FIGURE 2. The expression of CD28 and CTLA-4 on different DN subsets. WT C57BL/6 mice were sacrificed at 8 wk old, and thymocytes were harvested. Cells were surface stained with anti-CD4/CD8, anti-CD44, anti-CD25, and anti-CD28, and intracellularly stained with anti-CTLA-4 Abs. The DN were identified as the negative group in a staining using a mixture of anti-CD4 and anti-CD8 mAbs with the same fluorochrome. The number indicates the percentage of cells that bind Abs specific for CD28 and CTLA-4 or isotype control Ig (hamster IgG1). Data shown are contour graphs of one representative mouse from each group of three mice, and the numbers in the squares are the means and SD of the percentage of the subsets. This experiment has been repeated twice.

 


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  		FIGURE 3. CTLA-4 alone is not responsible for the effect of B7-1/2 in DN thymocyte development. Thymi from 2-wk-old CTLA-4+/– (n = 8), CTLA-4–/– (n = 7), B7–/– (n = 9), and CD28–/– (n = 8) mice were harvested and stained with anti-CD4, anti-CD8, anti-CD44, and anti-CD25 Abs. Percentages shown in the quadrants are summaries of data from two independent experiments. No significant difference in DN subsets was observed between CTLA4+/– and CTLA4–/– mice. The differences between B7–/– and CTLA4+/– or CD28–/– and CTLA4+/– are highly significant (p < 0.001).

 
Role for B7-1/2 and CD28 in proliferation and programmed cell death of DN

An important feature of the DN thymocytes is their high rate of proliferation (42, 43). To test whether B7 deficiency affects itsproliferation, we pulsed WT, CD28-, and B7-deficient mice with BrdU and measured the DNA synthesis of the ex vivo DN by flow cytometry 4 h later. As shown in Fig. 4, in agreement with previous publications (44, 45), two major waves of proliferation were observed in DN2 (~20%) and DN4 (~40%) in the WT mice, and somewhat less pronounced, but still substantial proliferation was found in DN3 (~10%). Interestingly, <8% of the DN4 from B7-deficient mice incorporated BrdU. As expected, this reduction corresponds to a reduction of the proportion of DN4 with large forward scatters. Thus, targeted mutations of B7-1 and B7-2 suppress both enlargement and DNA synthesis of the DN4 cells. Although less pronounced than what was found in the B7-deficient mice, DN4 from CD28-deficient mice also showed a ~2-fold reduction in the BrdU incorporation.



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  		FIGURE 4. Target mutations of B7-1 and B7-2 or CD28 decrease the proliferation among DN4 thymocytes. B7KO, CD28KO, and C57BL/6 mice were injected with BrdU i.v. The thymi were harvested at 4 h after BrdU injection and stained with anti-CD4/CD8, anti-CD44, and anti-CD25, and intracellularly stained with anti-BrdU Abs. The numbers in the panels indicate means and SD of the percentage of thymocytes incorporated with BrdU from individual subsets. The p values of Student’s t tests are also provided. Data shown are representative of two independent experiments involving a total of nine mice in each group.

 
An interesting issue is whether the CD28 and B7 deficiencies affect the survival of DN thymocytes. In this study, we investigated the number of cells underlying programmed cell death in the WT, B7-1/2-, and CD28-deficient mice by staining with annexin V. As shown in Fig. 5, B7-1/2- and CD28-deficient DN4 had significantly more annexin V+ cells than the WT counterpart. These results revealed that knocking out B7 and CD28 had similar effects in causing the death of DN4.



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  		FIGURE 5. Target mutation of B7 and CD28 promotes programmed cell death among DN4. B7KO, CD28KO, and C57BL/6 mice were sacrificed at 8–9 wk old, and thymocytes were harvested and stained with anti-CD4/CD8, anti-CD44, and anti-CD25 Abs and annexin V. Data shown are representative of three mice in each group. This experiment has been repeated twice involving seven mice in each group.

 
Targeted mutation of B7 and CD28 induces TCR expression among DN4

An important feature of the small DN4 T cells is the expression of cell surface pre-TCR complex. To test the effect of costimulation on the up-regulation of TCR{beta}, we compared DN1–4 from WT, B7-1/2-, and CD28-deficient mice for the expression of TCR. Although a significant portion of CD44highCD25– (DN1) cells expresses TCR{beta} on the cell surface, these cells belong to the NKT lineages, as has been reported by others (46). In addition, no TCR{beta}high thymocytes were found with the DN2 populations. A small number of DN3 and ~20% of DN4 thymocytes express TCR{beta} at significant levels. Interestingly, in the B7KO mice, the overwhelming majority (nearly 80%) of DN4 express TCR{beta}, while >60% of DN4 in the CD28-deficient mice are TCR{beta}high (Fig. 6). Thus, costimulation by B7-1/2 and CD28 genes inhibits the expression of the TCR{beta} on DN4.



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  		FIGURE 6. Target mutation of B7 and CD28 increases TCR expression among DN4. Thymocytes from 8- to 9-wk-old B7KO, CD28KO, and C57BL/6 mice were stained with anti-CD4/CD8, anti-CD44, anti-CD25, anti-TCR {beta}-chain (lower panels), or isotype control Ig (upper panels) Abs. Data shown are representative of three mice in each group. This experiment has been repeated twice.

 
Elimination of cycling thymocytes recapitulates the basic features of DN subsets in mice with targeted mutations of B7-1/2 or CD28

Given the overall role of B7-CD28 interaction in promoting T cell proliferation and survival (47), it is of great interest to determine whether the altered DN subset distribution and enhanced expression of TCR are a consequence of reduced TCR proliferation and survival. To test this hypothesis, we treated WT C57BL/6 mice with demecolcine, which kills cells undergoing mitosis. As shown in Fig. 7A, three consecutive treatments with demecolcine over a 3-day period removed the overwhelming majority of dividing cells within the DN thymocytes. When CD25 and CD44 expression was analyzed, it became clear that deletion of mitotic thymocytes caused a drastic increase in DN4 and reduction in DN3 (Fig. 7B). At the same time, the expression of TCR is significantly increased among DN3 and DN4 in mice that received antimitotic treatments (Fig. 7C). Thus, antimitotic treatment recapitulates the two main effects of B7 blockade, although the cellular mechanisms may differ in these two conditions.



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  		FIGURE 7. Antimitotic treatment recapitulates the effect of defective T cell costimulation on DN development. A, The efficiency of antimitotic treatment as revealed by reduction in BrdU incorporation. Data shown are means and SD (n = 4) of the percentage of BrdU+ T cells within the DN subsets. B, Antimitotic treatment increases DN4 while reducing DN2 and DN3. C, Antimitotic treatment increases TCR expression on DN3 and DN4 thymocytes. Data shown in B and C are representative contour graphs from one mouse in groups of four mice. The numbers in the quadrants are means and SD of the percentage of cells in the subset. This experiment has been repeated three times. *, p < 0.05; **, p < 0.01.

 
Accumulation of RAG-2 and increased expression of rearranged TCR{alpha} in mice with targeted mutation of B7-1/2

It is well established that expression of both RAG-1 and RAG-2 is critical for transition from DN3 to DN4, as thymocyte development in mice lacking RAG-1 or RAG-2 is blocked at DN3 (25, 26, 27). Because both TCR overexpression and increased DN4 can be explained by increased RAG activity, we analyzed expression and accumulation of RAG-1/2 mRNA by RT-PCR and RAG-2 protein by Western blot. As shown in Fig. 8, A and B, purified DN cells from B7KO mice expressed significantly more RAG-2 protein than WT mice. This increase in B7KO DN cells is most likely due to posttranscriptional mechanisms, as the RAG-1/2 mRNA was not increased (Fig. 8C). Corresponding to increased RAG-2 protein, real-time RT-PCR revealed a 2- to 3-fold increase in V{alpha}2 and V{alpha}4 expression among the DN from B7-deficient mice in comparison with WT mice (Fig. 8C).



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  		FIGURE 8. Increased accumulation of RAG-2 protein and rearranged TCR{alpha} in DN population of B7-1/2-deficient mice. Total thymocytes from 8- to 9-wk-old B7KO and C57BL/6 mice were collected, and DN thymocytes were purified by removing cells expressing CD4, CD8, TCR{gamma}{delta}, and I-Ab. In each experiment, three thymi per group were combined to isolate DN cells for protein and total RNA extraction. The experiments were repeated twice. A, Thymocyte subsets before and after purification. B, The RAG-2 protein was detected with Western blot. C, The expressions of RAG-1, RAG-2, pre-TCR{alpha}, TCR V{alpha}2 and V{alpha}4, and CD8 {beta} genes were detected with real-time RT-PCR. **, p < 0.01; ***, p < 0.001. Data shown in this figure have been repeated twice.

 
Expression of B7-1 and B7-2 in the thymus

A previous report (48) suggested that expression of B7, as revealed by immunohistochemistry with fusion protein CTLA4Ig, is restricted to the medulla. However, the development of DN takes place in the cortex. To reconcile this apparent inconsistency, we revisited the expression of B7-1 and B7-2 by immunohistochemistry using anti-B7-1 and anti-B7-2 Abs. As shown in Fig. 9, B7-1 expression was observed only in the medulla. Significant levels of B7-2, however, were detected in both cortex and medulla, although the intensity of B7-2 was also higher in the medulla. The difference between current data and the previous work most likely reflects the higher affinity of mAbs than the fusion protein, CTLA4Ig.



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  		FIGURE 9. Expression of B7-1 and B7-2 in the thymus. Frozen thymic sections were stained with either anti-B7-1 (A) and anti-B7-2 (B), followed by second-step reagents, biotinylated goat anti-hamster (A and C), or goat anti-rat (B and D) IgG. The cortex (c) and medulla (m) are marked.

 
Subset distribution of thymocytes among age-matched WT, CD28–/–, and B7–/– mice

Because the DN4 cells are the immediate precursor for the DP cells, it is possible that abnormal DN development may be associated with altered thymocyte subsets. As shown in Table I, mutations in B7 and CD28 genes resulted in 10–20% increase in CD4–CD8– thymocyte and a small, but statistically highly significant decrease in CD4+CD8+ T cells. The percentage of SP CD4 and CD8 T cells was substantially increased.


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  		Table I. Impact of targeted mutations of B7-1/2 and CD28 on the subsets of T cells in the thymusa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The T cell progenitors undergo rapid proliferation and phenotypic transitions before they emerge as the CD4+CD8+ T cell precursors for positive and negative selection (49). However, the cell surface interactions that guide the first phase of T cell development are still poorly understood. The potential involvement of costimulatory molecules in this phase has not been investigated. In this study, we provide several lines of evidence that demonstrate a critical contribution of costimulatory molecule B7-1/2 and their receptor CD28.

The most clear-cut demonstration of the impact of costimulation on DN development is the alteration of the relative amounts of CD44–CD25+ (DN3) and CD44–CD25– (DN4) cells in mice with inactivated B7-1/2 and CD28. In the WT mice, there are 2–3 times more DN3 than DN4 cells, while B7-deficient mice have 3-fold more DN4 than DN3 cells. Theoretically, there are at least four potential mechanisms that can account for the reduction of DN3 in B7-1/2- and CD28-deficient mice, namely, the proliferation and survival of DN3, a decreased transition from DN2 to DN3, an increased transition from DN3 to DN4, and a reduced rate of transition from DN to DP. Our data effectively ruled out the first possibility, as the proliferation and survival of DN3 were not inhibited by the targeted mutations. A CD28-mediated decrease in DN2 to DN3 transition is not probable, as the DN2 lacks cell surface CD28 expression, while a very small proportion of DN3 expresses CD28.

More rapid transition from DN3 to DN4 is likely to contribute to both a decrease in DN3 and increase in DN4, as these changes cannot be accounted for by death or proliferation in DN3 and DN4. This interpretation is also supported by increased accumulation of RAG-2, which mediates a critical checkpoint between DN3 and DN4. Paradoxically, despite the overall trend of decreased proliferation and survival, the number of total DN and especially DN4 is significantly increased. This can be explained if one assumes that the progression from DN4 to DP is kinetically slower in mice with mutations of B7-1, B7-2, and CD28. This interpretation is consistent with the fact that the DP subset is decreased in the mutant mice. The decrease is statistically highly significant, although not large numerically. However, the decrease caused by delayed DN to DP transition can be more substantial if one considers the decrease in the context of previous works, which shows that costimulation blockade decreases clonal deletion (10, 14). As such, the decrease in DP, resulted from delayed DN to DP progression, may have been counterbalanced by the increase of DP caused by defective clonal deletion. The increase in CD4+CD8– and CD4–CD8+ cells can be attributed directly to abnormal clonal deletion, although altered differentiated DN may have an indirect effect.

Our analysis of proliferation and programmed cell death of the T cell progenitors in the thymus reveals two more points.

First, in parallel to what was described for activation and effector function of mature T cells, the costimulatory molecules B7-1 and B7-2 promote proliferation and survival of immature T cells. Thus, in comparison with WT thymocytes, an increased proportion of DN4 in B7- and CD28-deficient mice was undergoing programmed cell death. Conversely, a substantially reduced percentage of DN4 cells in B7- or CD28-deficient mice incorporated BrdU. Interestingly, in mice with mutations of either B7 or CD28, the proportion of dividing and apoptotic cells appears to have increased among DN2 and DN3 subsets. However, because these two subsets are diminished in the mutant mice, the changes in proportions do not translate into an increased number of cells undergoing division or apoptosis. Differences in apoptosis and proliferation were also noted among the DN1 subset that are equally represented in WT and mutant mice. However, because our study does not differentiate between true DN1 and those that express TCR and CD44 (mostly NKT cells), the significance of the difference in DN1 is unclear at this point.

Second, the function of B7-CD28 interaction in promoting division of DN thymocytes may explain the more rapid DN3 to DN4 transition in mice with mutations of B7 or CD28. This is due to the fact that this transition requires RAG activity restricted to the G0/G1 phase of the cell cycle (50, 51). Indeed, RAG-2 protein is accumulated at G0/G1, and its expression level decreases rapidly at the G1-S transition of the cell cycle by cytoplasmic sequestration and ubiquitin-dependent degradation (52). Our analysis of RAG-2 protein and TCR gene expression clearly demonstrated that DN from B7-deficient mice have increased accumulation of RAG-2 protein and increased expression of rearranged TCR genes. The link between cell division and DN3-DN4 transition is supported by our data that antimitotic treatment results in changes of DN thymocytes that are not unlike those observed in B7- and CD28-deficient mice, including the ratio of DN3/DN4 and cell surface TCR. Because more cells in DN4 incorporate BrdU than those in DN3, the increased cell number in DN4 during antimitotic treatment is most likely due to a combination of two defects: an increased transition from DN3 and a delayed transition out of DN4.

The expression of B7-2 in the cortex is consistent with the idea that B7-CD28 interaction is directly involved in differentiation in DN. This is further strengthened by our observation that most of the effects in DN division and survival are seen in DN4 that express high levels of CD28. However, due to the genetic nature of the current study, it is theoretically possible that the observed effect is not an indirect consequence of such interaction. Another interesting issue is the identity of B7 receptors that may be involved in the early stage of T cell development. Although mutations of B7 and CD28 have qualitatively similar phenotypes, mutations of B7-1 and B7-2 have significantly more severe effect than those of CD28. A natural question is whether this is attributable to CTLA4, the other known B7 receptor. Although CTLA4 protein is expressed early among DN2 cells, targeted mutation of CTLA4 does not affect DN subset composition. It is therefore unlikely that CTLA4 is involved in the differentiation of DN. It is possible that another unidentified B7 receptor (40) may participate in this process. Regardless of what the additional receptor may be, our results extended the functional spectrum of T cells that are modulated by the costimulatory pathway.


   Acknowledgments
 
We thank Lynde Shaw for editorial assistance and Lijie Yin for mouse breeding.


   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 is supported by National Institutes of Health Grants AI51342 and CA58033 (to Y.L.), a grant from Department of Defense Prostate Cancer Program (DAMD 17-03-1-0013 to P.Z.), and National Institutes of Health Grants P01 AR045652 and K02 AR049047 (to J.Z.). Back

2 X.Z. and J.-X.G. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Yang Liu, Division of Cancer Immunology, Department of Pathology, Ohio State University Medical Center, 129 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. E-mail address: liu-3{at}medctr.osu.edu Back

4 Abbreviations used in this paper: DP, double positive; CT, threshold cycle; DN, double negative; KO, knockout; SP, single positive; WT, wild type. Back

Received for publication March 1, 2004. Accepted for publication June 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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