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The Journal of Immunology, 2001, 166: 2394-2403.
Copyright © 2001 by The American Association of Immunologists

A Critical Role for CD2 in Both Thymic Selection Events and Mature T Cell Function1

Tetsuro Sasada and Ellis L. Reinherz2

Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2-/- H-2b mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2Kb molecule were compared on a wild-type or CD2-/- background. In N15tg RAG-2-/- CD2-/- mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4-CD8- double-negative thymocytes at the CD25+CD44- stage. Moreover, mature N15tg RAG-2-/- CD2-/- T cells are ~100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-{gamma} production. Repertoire analysis shows substantial differences in V{alpha} usage between non-tg C57BL/6 (B6) and B6 CD2-/- mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The human CD2 molecule is expressed on virtually all T cells and thymocytes as well as NK cells. CD2 binds to the ubiquitous CD58 (LFA-3) cell surface glycoprotein, and through this counterreceptor interaction, promotes the initial stages of T cell contact with APCs or target cells (reviewed in Ref. 1). Moreover, unlike with integrin-based adhesion, CD2 binding is independent of TCR triggering (2, 3). While the affinity of the monomeric CD2-CD58 interaction is relatively low (Kd ~1 µM), the very rapid Koff (>=4 s-1) and Kon (>=400,000 M-1.s-1) rates suggest a dynamic set of binding events (4, 5, 6, 7). The recently reported crystal structure of the heterophilic adhesion complex between the amino-terminal domains of human CD2 and CD58 reveals an asymmetric, orthogonal, face-to-face interaction involving the major {beta}-sheets of the respective Ig-like domains. Poor shape, but good charge complementarity is observed (8). In the virtual absence of hydrophobic forces, interdigitating charged amino side chains form hydrogen bonds and salt links at the interface, imparting a high degree of specificity with but modest affinity.

The coligation of CD2 and CD58 on opposing cell surfaces creates an intercellular membrane distance (~135Å) suitable for TCR-peptide/MHC (pMHC)3 or NK receptor-MHC interactions (9, 10, 11, 12), thus fostering such immune recognition processes. Large molecules such as CD45 phosphatase are excluded from this conjugation area, further enhancing T cell activation (8). Furthermore, the transient nature of CD2-CD58 binding does not interfere with diffusion of TCR and pMHC complexes into the appropriate physical contact space. Indeed, in the presence of the human CD2/human CD58 interaction, T cells recognize the relevant pMHC complexes on APCs with a 50- to 100-fold greater efficiency than in cell conjugates that lack the counterreceptor interaction (13). Human CD2 signaling serves to optimize T cell activation (14, 15), enhance IL-12 responsiveness (16, 17), and reverse T cell anergy (18).

Murine CD2 is structurally homologous to human CD2. However, in rodents, the CD2 ligand is not CD58, but rather CD48 (19). No rodent CD58 gene has been identified. In this regard, it has long been recognized that CD2, CD58, and CD48 constitute an adhesion subfamily within the Ig superfamily (20). Genetic and sequence analysis suggests that a primordial gene duplicated to produce a CD2-CD48 recognition pair before divergence of humans and rodents. Subsequently, a further duplication of the CD48 gene gave rise to CD58 in the human. The CD2 gene diverged in parallel, evolving to recognize CD58 and CD48 ligands in human and rodent species, respectively. The affinity of rodent CD2 for rodent CD48 is higher than that between the human homologues, but not as great as that between human CD2/human CD58 (7). Thus, the rodent CD2-CD48 interaction has been replaced by the human CD2-CD58 interaction during evolution. Given that the rodent CD48 cell surface distribution is more restricted than that of human CD58, these adhesion pairs may subserve somewhat different functions in these species. Perhaps consistent with this view, the analysis of CD2-/- mice first suggested that CD2 was dispensable for the development and function of T cells (21). More recent studies have suggested that murine CD2 may set quantitative thresholds for T cell activation in the mouse as well as the human (22).

In this study, we have addressed the question of CD2 function in a well-defined murine system with regard to both thymic differentiation and mature T cell activation. Using normal B6 or B6 CD2-/- mice, we show that the presence or absence of CD2 dramatically affects V{alpha} repertoire selection in both CD4 as well as CD8 T cells. Moreover, we have assessed the function of CD2 in mice bearing an N15 TCR transgene derived from a representative CD8 T cell clone with a vesicular stomatitis virus nucleoprotein octapeptide N52–59 (VSV8)/Kb specificity (23). In these studies, a RAG-2-/- mouse background was used to obviate any confounding effects of endogenous TCRs. These results demonstrate an important role for CD2 not only in mature T cell function, but also in thymic pre-TCR function and selection processes.


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

N15 TCR transgenic (tg) RAG-2-/- H-2b (N15tg CD2+/+) mice were generated and maintained, as previously described (23). To create the N15 TCRtg RAG-2-/- CD2-/- H-2b (N15tg CD2-/-) mice, the N15 TCRtg RAG-2-/- H-2b (N15tg CD2+/+) mice were bred with CD2-/- H-2b mice that had been backcrossed for five generations onto C57BL/6 (B6, H-2b) mice by N. Killeen (University of California, San Francisco, CA). The heterozygous CD2+/- F1 mice were intercrossed, and offspring that were N15tg+/+ RAG-2-/- CD2-/- H-2b were used. The expression of the N15 TCR as well as lack of RAG-2 or CD2 gene expression in knockout animals were confirmed based on the FACS analysis of peripheral blood cells. The homozygosity of the N15 TCR transgenes was proven by subsequent breeding analysis. B6 mice and CD2-/- H-2b mice that were backcrossed for five generations onto B6 were purchased from Taconic (Germantown, NY). All lines were maintained and bred under sterile barrier conditions at the animal facility of Dana-Farber Cancer Institute (Boston, MA).

Peptide synthesis

VSV8 (RGYVYQGL) and its variant peptides, L4 and Norvaline4, which were substituted by leucine and norvaline (Bachem Bioscience, King of Prussia, PA) at the fourth position of VSV8, respectively, were synthesized by standard solid-phase methods on an Applied Biosystems 430A synthesizer (Foster City, CA) at the Biopolymers Laboratory of Massachusetts Institute of Technology (Cambridge, MA). All peptides were purified by reverse-phase HPLC (Hewlett Packard HPLC 1100, Palo Alto, CA) with a 2-mm C4 column. Peptides were analyzed for purity and correct molecular composition by electrospray mass spectrometry, amino acid analysis, and HPLC. VSV8 is a cognate antigenic peptide agonist that triggers negative selection of N15tg thymocytes, whereas L4 and Norvaline4 peptides are weak agonists of the N15 TCR and induce positive selection of N15tg thymocytes (24, 25).

Abs, flow cytometric analysis, and cell sorting

The following mAbs were used: FITC- or R-PE-conjugated anti-CD2 (RM2-5); FITC- or PE-conjugated anti-CD4 (RM4-5); PE- or CyChrome-conjugated anti-CD8{alpha} (53-6.7); PE-conjugated anti-CD25 (PC61); FITC-conjugated anti-CD44 (IM7); biotin-conjugated anti-V{alpha}2 (B20.1), anti-V{alpha}3.2 (RR3-16), anti-V{alpha}8 (B21.14), anti-V{alpha}11.1, 11.2 (RR8-1), anti-V{beta}2 (B20.6), anti-V{beta}4 (KT-4), anti-V{beta}5.1, 5.2 (MR9.4), anti-V{beta}6 (RR4-7), anti-V{beta}7 (TR310), anti-V{beta}8.1, 8.2 (MR5-2), anti-V{beta}9 (MR10-2), anti-V{beta}10 (B21.5), anti-V{beta}11 (RR3-15), anti-V{beta}12 (MR11-1), anti-V{beta}14 (14-2); PE-conjugated anti-V{beta}3 (KJ25), anti-V{beta}8.3 (1B3.3), anti-V{beta}13 (MR12-3) (PharMingen, San Diego, CA); anti-N15{beta}-chain clonotype (R53) (26) plus FITC-labeled goat anti-rat IgG (Caltag Laboratories, Burlingame, CA). For flow cytometry, single-cell suspensions of thymocytes, splenocytes, or lymph node cells were double or triple stained at 5 x 106 cells/ml in PBS-2% FCS containing the Abs at saturating concentrations according to standard procedures. Phenotypes and proportions of each subpopulation were analyzed on a FACScan (Becton Dickinson, San Jose, CA) using the CellQuest program. Dead cells were excluded from the analysis by forward and side scatter gating.

For CD4-CD8- double-negative (DN) cell sorting of thymocytes from the N15tg CD2+/+ and N15tg CD2-/- mice, thymocytes were stained with PE anti-CD4 and PE anti-CD8{alpha}, and CD4-CD8- DN cells were sorted on a FACSVantage cell sorter (Becton Dickinson). Immediately after sorting, sorted cells were double-stained with FITC anti-CD4 and CyChrome anti-CD8{alpha}, and reanalyzed on a FACScan to confirm their purity. The sorted DN subpopulation was found to be at least 97% pure.

Proliferation assay

Splenocytes from N15tg CD2+/+ or N15tg CD2-/- mice (1 x 105/well) were incubated at 37°C with 2 x 104 irradiated EL-4 cells, which had been preloaded for 2 h with the indicated doses of VSV8 variant peptides in AIM-V medium (Life Technologies, Grand Island, NY) containing 50 µM 2-ME. After 48-h incubation, 0.4 µCi/well of [3H]thymidine (ICN Biomedicals, Aurora, OH) was added, and after an additional 18 h of culture at 37°C, the cells were harvested and the incorporated radioactivity was measured.

Measurement of IFN-{gamma}

IFN-{gamma} production was induced under the same culture conditions used for proliferation assays (see above). After 48 h of incubation, supernatants were collected and assayed for IFN-{gamma} using a mouse IFN-{gamma} ELISA kit (Mouse IFN-{gamma} OptEIA Set; PharMingen). The sensitivity of the assay was 31.3–2000 pg/ml, and results were calculated as the mean of duplicate wells. The ability of Con A-activated mouse lymphoblasts to secrete IFN-{gamma} in response to IL-12 was assessed as follows. Splenocytes from N15tg CD2+/+ or N15tg CD2-/- mice (1 x 106/ml) were cultured in AIM-V medium containing 50 µM 2-ME, 100 U/ml rIL-2, and 2.5 µg/ml Con A. After 3 days, the Con A-activated splenocytes were harvested, washed, and resuspended in AIM-V with 50 µM 2-ME and the indicated doses of rIL-12 (PharMingen) at 5 x 104 cells/well. The cells were incubated at 37°C with or without 1 x 105 irradiated EL-4 cells for 48 h, and supernatants were collected and assayed for IFN-{gamma} production.

Cytotoxicity assay

To generate cytotoxic T cells (CTL), splenocytes from N15tg CD2+/+ or N15tg CD2-/- mice (5 x 106/well) were stimulated for 5–6 days at 37°C with 5 x 105/well irradiated N1 cells (EL-4 cells transfected with a vesicular stomatitis virus mini-gene cassette) in the presence of 10% supernatant of Con A-activated rat splenocytes. For the cytotoxicity assay, EL-4 cells were labeled with sodium 51Cr (0.1 mCi/106 cells) for 1 h at 37°C and washed three times, and 5000 cells/well in RPMI 1640 with 10% FCS were transferred to a 96-well V-bottom plate. VSV8 peptide (10-6-10-11 M) was added, and 1 h later, 5 x 104 CTL was transferred per well in a final volume of 200 µl and incubated for 4 h at 37°C. After centrifugation, 100 µl of the supernatant was removed from each well, and radioactivity was determined using a Packard gamma counter.

For NK cytotoxic assays, YAC-1 target cells were labeled with sodium 51Cr (0.1 mCi/106 cells) for 1 h at 37°C and washed three times, and 5000 cells/well in RPMI 1640 with 10% FCS were transferred to a 96-well V-bottom plate. Splenocytes from N15tg CD2+/+ or N15tg CD2-/- mice were added at the indicated ratios to a final volume of 200 µl, and the plates were incubated for 4 h at 37°C. After centrifugation, 100 µl of the supernatant was removed from each well, and radioactivity was determined using a Packard gamma counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reduced number and abnormal phenotype of thymocytes in N15tg CD2-deficient mice

To analyze the role of CD2 in thymocyte development and mature T cell function, we crossed CD2-deficient mice (H-2b) with tg mice expressing the N15 MHC class I-restricted (H-2Kb) TCR specific for VSV8, and subsequently established N15tg RAG-2-/- CD2-/- H-2b (hereafter for simplicity referred to as N15tg CD2-/-) mice. T lineage cells from the thymus and lymph nodes of these animals were then directly compared. As expected, CD8+ single-positive (SP) cells from the N15tg CD2-/- lymph nodes lack surface CD2 expression (Fig. 1GoA). However, as defined by the anti-N15{beta}-chain clonotypic mAb R53, these lymph node T cells express the same N15 TCR level as lymph node T cells derived from N15tg RAG-2-/- CD2+/+ H-2b (N15tg CD2+/+) mice. Furthermore, the surface density of CD8 coreceptor as enumerated by the anti-CD8{alpha} mAb 53-6.7 is comparable.



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  		FIGURE 1. Quantitative and qualitative T lineage abnormalities in N15tg RAG-2-/- CD2-deficient mice. A, CD2 and TCR expression on lymph node T cells of N15tg RAG-2-/- CD2+/+ and N15tg RAG-2-/- CD2-/- mice. Lymph node cells from 6-wk-old N15tg RAG-2-/- CD2+/+ and N15tg RAG-2-/- CD2-/- mice were triple stained with PE anti-CD2, CyChrome anti-CD8{alpha}, and mAb R53 (anti-N15 TCR{beta}-chain clonotype), followed by FITC anti-rat IgG. The dot-plot profiles of CD2 (y-axis) vs CD8{alpha} (x-axis) expression in 10,000 live cells and histograms of N15 TCR on CD8+ lymph node cells are shown. The percentage of cells in each subset quadrant is indicated. B, Thymocyte numbers are reduced in N15tg RAG-2-/- CD2-deficient mice. The total thymocyte numbers from 3- to 6-wk-old N15tg RAG-2-/- CD2+/+ and N15tg RAG-2-/- CD2-/- mice are enumerated. Open circles (N15tg RAG-2-/- CD2+/+ mice at 3 wk (n = 4), 4 wk (n = 10), 5 wk (n = 8), and 6 wk (n = 7) of age) and closed circles (N15tg RAG-2-/- CD2-/- mice at 3 wk (n = 6), 4 wk (n = 7), 5 wk (n = 6), and 6 wk (n = 11) of age) represent values of individual mice, and bars represent average values for a given group. The thymocyte numbers of N15tg RAG-2-/- CD2-/- mice are significantly lower than those of N15tg RAG-2-/- CD2+/+ mice at each time point (3 wk, p < 0.001; 4 wk, p < 0.00005; 5 wk, p < 0.001; 6 wk, p < 0.005). C, The number of CD8+ splenic T cells is not affected by CD2 deficiency. CD8+ splenic T cell numbers were calculated by multiplying the total numbers of splenocytes from 6- to 8-wk-old N15tg RAG-2-/- CD2+/+ and N15tg RAG-2-/- CD2-/- mice by the percentages of CD8+ SP subset, as determined by FACS analysis. Open circles (N15tg RAG-2-/- CD2+/+: n = 15) and closed circles (N15tg RAG-2-/- CD2-/-: n = 15) represent values from individual mice, and bars represent average values. The difference in the numbers of CD8+ splenic T cells between N15tg RAG-2-/- CD2+/+ and N15tg RAG-2-/- CD2-/- animals is not significant (p = 0.29). D, Thymocyte development is progressively impaired by the lack of CD2. The thymocytes from N15tg RAG-2-/- CD2+/+ (top row) and N15tg RAG-2-/- CD2-/- (bottom row) mice at 3 and 6 wk of age were double stained with PE anti-CD4 and CyChrome anti-CD8{alpha}. The expression of CD4 (y-axis) and CD8{alpha} (x-axis) on thymocytes was detected by flow cytometry after gating on 10,000 live cells. The percentages of each subset are indicated. Results are representative of 4–11 experiments.

 
Comparison of the thymocyte cell number and subset distribution between the N15tg CD2+/+ and N15tg CD2-/- mice reveals striking differences. For example, at 3 wk of age, N15tg CD2-/- mice have a markedly smaller thymus with a ~5-fold reduction in the number of thymocytes (Fig. 1GoB). Furthermore, although both the N15tg CD2+/+ and N15tg CD2-/- mice exhibit a gradual loss of thymocytes with increasing age, N15tg CD2-/- mice consistently possess fewer total thymocytes than control N15tg CD2+/+ mice at each age tested (3 wk, p < 0.001; 4 wk, p < 0.00005; 5 wk, p < 0.001; 6 wk, p < 0.005) (Fig. 1GoB). Nevertheless, the number of peripheral CD8+ SP splenic T cells is comparable in both strains (Fig. 1GoC), possibly as a result of homeostatic mechanisms involving peripheral lymphoid system components (27, 28). Perhaps more importantly, as shown by the CD4/CD8{alpha} double-staining profiles in Fig. 1GoD, there is a dramatic loss of double-positive (DP) thymocytes in N15tg CD2-/- mice as they mature from 3 to 6 wk of age. Thymocytes from 3-wk-old N15tg CD2+/+ mice are predominantly DP (87%) with 7.2% DN and 5.2% CD8+ SP cells. In contrast, in N15tg CD2-/- mice of equivalent age, less than half of the thymocytes (44%) are DP, and the percentages of DN and CD8+ SP thymocytes are increased to 22% and 33%, respectively. As the total number of N15tg CD2-/- thymocytes is about 5 times less than control N15tg CD2+/+ thymocytes at 3 wk of age, N15tg CD2-/- mice show no significant differences in the absolute numbers of DN and CD8+ SP thymocytes, but ~10-fold less DP thymocytes than N15tg CD2+/+ mice (N15tg CD2-/-, 12.7 x 106 DP cells vs N15tg CD2+/+, 100.7 x 106 DP cells). At 6 wk of age, the percentages of DN and CD8+ SP thymocytes in N15tg CD2+/+ mice are increased to 43% and 28%, respectively, and the percentage of DP thymocytes (28%) is reduced. In contrast, 6-wk-old N15tg CD2-/- mice show more dramatic changes in thymocyte phenotype; 95% of the thymocytes in 6-wk-old N15tg CD2-/- thymocytes are DN, with only small percentages of DP (0.5%) and CD8+ SP (4.1%) cells. These results indicate that the absence of CD2 influences intrathymic T cell development in N15tg mice.

Inhibition of early T cell development in N15tg CD2-deficient mice

Fig. 2GoA graphs the absolute numbers of individual DP, DN, and CD8+ SP thymocytes from N15tg CD2-/- mice as a function of age. DP and CD8+ SP thymocyte numbers decline continuously between weeks 3 and 5, but the number of DN thymocytes increases from week 3–4 and then remains relatively constant. Hence, CD2 deficiency results in a striking reduction of DP and CD8+ SP thymocytes, but not of DN thymocytes, suggesting that the defect in the intrathymic differentiation pathway occurs before the generation of DP subsets in N15tg CD2-/- mice. To further define the stage at which thymocyte development is blocked in N15tg CD2-/- mice, we next examined DN thymocytes. DN thymocytes can be subdivided into four discrete subsets according to their surface expression of the CD25 and CD44 molecules (29). The phenotype of the most immature stage is CD44+CD25-, with maturation proceeding through a CD44+CD25+ stage, then to a CD44-CD25+ stage, and finally to a CD44-CD25- stage. We sorted the DN thymocytes from N15tg CD2+/+ and N15tg CD2-/- mice and examined their CD25 and CD44 expression. As shown in Fig. 2GoB, in 4-wk-old N15tg CD2+/+ mice, 78% of DN thymocytes are CD44-CD25+ and 21% are CD44-CD25-. However, in 4-wk-old N15tg CD2-/- mice, the percentage of CD44-CD25+ cells is increased to 92%. Because the number of DN thymocytes in 4-wk-old N15tg CD2-/- mice is ~2.3-fold more than that of 4-wk-old N15tg CD2+/+ mice, this results in a 2.7-fold increase in the CD44-CD25+ thymocytes (N15tg CD2+/+, 1.6 x 106; N15tg CD2-/-, 4.3 x 106). The percentage of CD44-CD25- cells in 4-wk-old N15tg CD2-/- mice is decreased to 6.6%; however, the absolute number is only slightly decreased (N15tg CD2+/+, 4.5 x 105; N15tg CD2-/-, 3.5 x 105). Note that the percentages of CD44+CD25- and CD44+CD25+ thymocytes are comparable in both mice. Although not shown, we also observed in N15tg CD2-/- mice a striking reduction of CD44-CD25+ large DN thymocytes, a phenotype noted previously in pre-TCR-deficient animals (30). Collectively, these results suggest that N15tg CD2-/- mice exhibit a defect at the transition from CD44-CD25+ to CD44-CD25- DN thymocytes. It would appear that this partial developmental block is associated with a significant decrease in the relative proportion and absolute number of DP and CD8+ SP thymocytes and a reduction in total thymic cellularity. This result is not a consequence of perturbation of TCR expression in the tg mice: <5% of DN CD44-CD25+ thymocytes are positive for TCR expression, as judged by anti-TCR{beta} mAb H57 and anti-CD3{epsilon} mAb 2C11 reactivity in N15tg Rag-2-/- or B6 mice, whether of CD2-/- or CD2+/+ genotypes. Moreover, DN CD44-CD25- thymocytes are equivalently positive in parallel comparison of CD2-/- and CD2+/+ strains (data not shown).



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  		FIGURE 2. A defect in early T cell development in N15tg RAG-2-/- CD2-deficient mice. A, Time-dependent reduction in the numbers of DP and CD8+ SP thymocytes in N15tg RAG-2-/- CD2-deficient mice. The numbers of DP, CD8+ SP, and DN thymocytes were calculated by quantifying the total numbers of thymocytes from 3- to 6-wk-old N15tg RAG-2-/- CD2-/- mice and the percentages of each subset, as determined by FACS analysis. Each point represents average values from 6 to 11 animals (3 wk, n = 6; 4 wk, n = 7; 5 wk, n = 6; 6 wk, n = 11). B, The transition from CD44-CD25+ to CD44-CD25- DN thymocytes is blocked in N15tg RAG-2-/- CD2-deficient mice. DN thymocytes were double stained with FITC anti-CD44 and PE anti-CD25. The profile of CD44 (y-axis) vs CD25 (x-axis) expression in 10,000 live cells is shown. The percentage of each subset is indicated. The result is representative of three independent experiments.

 
Defect in pre-TCR signaling in N15tg CD2-deficient mice

Productive rearrangement of the TCR{beta} gene locus begins in CD44+CD25+ thymocytes (31). Subsequently, the TCR{beta} subunit protein, in association with the pre-TCR{alpha} and CD3 subunits, is expressed as a surface pre-TCR complex at the CD44-CD25+ DN stage (32). Signaling through the pre-TCR complex on CD44-CD25+ thymocytes then drives the differentiation of these cells to the CD44-CD25- stage (32). This developmental checkpoint is termed {beta} selection and insures that DN precursors have a functionally rearranged {beta}-chain gene to survive beyond the CD44-CD25+ stage. Mice that lack an essential component of the pre-TCR complex due to targeted gene disruption (see Discussion) exhibit severe defects in T cell differentiation at the CD44-CD25+ stage. By treatment of these mice with anti-CD3{epsilon}-specific mAb, however, pre-TCR signaling can be bypassed, leading to strong proliferation of DN thymocytes and efficient generation of DP cells (33, 34, 35). N15tg CD2-/- mice exhibit a severe block at the CD44-CD25+ developmental stage similar to that seen in pre-TCR complex-deficient animals, implying that CD2 deficiency affects pre-TCR function. Therefore, we next examined whether treatment with the anti-CD3{epsilon}-specific mAb 2C11 could also bypass the developmental arrest we observed in N15tg CD2-/- mice. Six-week-old N15tg CD2-/- mice were injected i.p. with 100 µg of purified anti-CD3{epsilon}-specific mAb 2C11. Seven days later, thymocytes were prepared, counted, and stained for CD4 and CD8{alpha} expression. As shown in Fig. 3Go, treatment of N15tg CD2-/- mice with anti-CD3{epsilon}-specific mAb 2C11 resulted in the efficient generation (60-fold increase) of DP thymocytes with a ~4-fold increase in total thymocyte number from 8.8 x 106 to 33 x 106. This result is consistent with the notion that the early developmental arrest of N15tg CD2-/- thymocytes results from impaired pre-TCR-mediated signaling.



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  		FIGURE 3. Efficient induction of DP thymocytes in N15tg RAG-2-/- CD2-deficient mice by i.p. injection of anti-CD3{epsilon} Ab. Six-week-old N15tg RAG-2-/- CD2-/- mice were injected i.p. with 100 µg of the affinity-purified CD3{epsilon}-specific Ab 145-2C11 in 0.2 ml PBS. Control animals were injected with PBS alone. Seven days later, thymocytes were counted and analyzed by flow cytometry for expression of CD4 (y-axis) and CD8{alpha} (x-axis). The percentages of each subset are indicated. The result is representative of three independent experiments. The PBS-injected control N15tg RAG-2-/- CD2-/- mouse showed no significant changes in the absolute number (8.8 x 106 cells) of thymocytes or their subset distribution. By contrast, treatment of N15tg RAG-2-/- CD2-/- mice with anti-CD3{epsilon} mAb resulted in an efficient generation of DP thymocytes and a ~4-fold increase in total thymocyte number (33 x 106 cells).

 
Altered T cell repertoire in CD2-deficient mice

The T cell repertoire is shaped by both negative and positive thymic selection processes acting primarily at the level of the immature DP thymocytes (36, 37, 38, 39). Thymic selection is affected by monomeric TCR-pMHC affinity, surface TCR and pMHC ligand densities, and CD4 and CD8 coreceptor expression. To examine whether CD2 influences thymic repertoire selection, we assessed the TCR V{alpha} and V{beta} gene usage of CD4+ and CD8+ SP lymph node T cells in C57BL/6 (CD2+/+) and non-tg CD2-/- H-2b (CD2-/-) mice by flow cytometry employing a panel of anti-V{alpha} or anti-V{beta} mAbs. As shown in Fig. 4Go, there is a striking difference in the TCR V{alpha} gene usage in peripheral lymph node T cells between CD2+/+ and CD2-/- mice. CD4+ lymph node T cells from CD2-/- mice have a reduced frequency of TCRs incorporating V{alpha}2 (mean = 9.75%, SD = 0.64 vs mean = 14.78%, SD = 0.92, p < 1 x 10-9), V{alpha}3.2 (mean = 0.32%, SD = 0.17 vs mean = 1.54%, SD = 0.37, p < 5 x 10-7), V{alpha}8 (mean = 1.94%, SD = 0.24 vs mean = 3.21%, SD = 0.30, p < 1 x 10-8), and V{alpha}11 (mean = 0.18%, SD = 0.09 vs mean = 5.47%, SD = 0.35, p < 1 x 10-11) gene segments compared with CD2+/+ mice (Fig. 4GoA). CD8+ lymph node T cells from CD2-/- mice also bear fewer TCRs incorporating V{alpha}2 (mean = 7.42%, SD = 0.93 vs mean = 8.91%, SD = 0.43, p < 0.001), V{alpha}3.2 (mean = 0.86%, SD = 0.45 vs mean = 3.77%, SD = 0.46, p < 5 x 10-11), V{alpha}8 (mean = 3.44%, SD = 0.59 vs mean = 6.19%, SD = 0.41, p < 5 x 10-9), and V{alpha}11 (mean = 0.28%, SD = 0.17 vs mean = 1.95%, SD = 0.38, p < 5 x 10-8) than those from CD2+/+ mice. As revealed by Southern blot analysis, the TCR V{alpha} locus haplotype of the CD2-/- mice is the same as that of the B6 mice, excluding differences in the TCR V{alpha} locus as the basis of the distinct V{alpha} usage observed (data not shown). In contrast, with the exception of V{beta}12 usage in CD4+ lymph node T cells (mean = 3.03%, SD = 0.41 in CD2-/- mice vs mean = 4.04%, SD = 0.42 in CD2+/+ mice, p < 0.01) and V{beta}5 usage in CD8+ lymph node T cells (mean = 13.17%, SD = 1.30 in CD2-/- mice vs mean = 15.09%, SD = 1.13 in CD2+/+ mice, p < 0.005), V{beta} gene segment expression shows no significant differences between CD2+/+ and CD2-/- mice. Taken together, these data suggest that lack of CD2 expression exerts an influence on selection of the TCR repertoire by primarily affecting usage of V{alpha} gene segments.



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  		FIGURE 4. Altered T cell repertoire development in CD2-deficient mice. Lymph node cells from 4- to 5-wk-old control C57BL/6 (CD2+/+) and CD2-/- H-2b (CD2-/-) mice were stained with FITC anti-CD4, CyChrome anti-CD8{alpha}, and PE anti-V{beta} mAb or biotin-anti-V{alpha} or anti-V{beta} mAb, followed by PE-conjugated streptavidin. Values represent the mean and SD of percentages in CD4+ (A) and CD8+ (B) SP lymph node T cell subsets, as determined by FACS analysis (CD2+/+ and CD2-/-, n = 10) (*, p < 0.01).

 
Decreased IFN-{gamma} production and proliferative responses upon Ag-triggered stimulation of splenic T cells in N15tg CD2-deficient mice

To examine the role of CD2 in mature murine T cell function, we next tested the Ag responsiveness of CD2-deficient mice. The splenic T cells from N15tg CD2+/+ and N15tg CD2-/- mice were stimulated in vitro with varying molar concentrations of the VSV8 cognate peptide or two altered peptide ligand (APL) variants, L4 and Norvaline4, using irradiated EL-4 cells as H-2Kb-bearing APC, and then assayed for IFN-{gamma} production and proliferation. L4 and Norvaline4 peptides are identical in sequence to the VSV8 octapeptide, except for a single p4 substitution of valine with leucine or norvaline, respectively. Both APLs induce positive selection of N15tg thymocytes (24, 25). As shown in Fig. 5GoA, when incubated for 48 h with VSV8-prepulsed EL-4 cells, both the N15tg CD2+/+ and N15tg CD2-/- splenocytes secrete IFN-{gamma} into the culture supernatant at each peptide concentration tested (10-5–10-11 M). However, the values of IFN-{gamma} secreted from N15tg CD2+/+ splenocytes are significantly higher than those from N15tg CD2-/- splenocytes at 10-5–10-9 M VSV8. In fact, compared with N15tg CD2+/+ splenocytes, the dose-response curve from N15tg CD2-/- splenocytes is shifted to the right by a factor of 100-1000. Furthermore, when incubated with EL-4 cells prepulsed with the weak agonists L4 or Norvaline4, the N15tg CD2+/+ splenocytes can produce readily detectable amount of IFN-{gamma} at peptide concentrations of 10-4 and 10-5 M. In contrast, N15tg CD2-/- splenocytes cannot induce IFN-{gamma} production after either L4 or Norvaline4 stimulation at any peptide concentration tested (Fig. 5Go, B and C). Similarly, as shown in Fig. 5, D–F, significant differences are also observed in the IL-2-dependent proliferative responses of the N15tg CD2+/+ vs N15tg CD2-/- splenic T cells after stimulation with antigenic peptides, as assessed by [3H]thymidine incorporation. Although both N15tg CD2+/+ and N15tg CD2-/- splenocytes show a similar dose-response curve after coculture with irradiated EL-4 cells prepulsed with VSV8 for 48 h, the amounts of incorporated [3H]thymidine in N15tg CD2-/- splenocytes are clearly less than those in N15tg CD2+/+ splenocytes at peptide concentrations from 10-5 to 10-11 M (Fig. 5GoD). In addition, compared with N15tg CD2+/+ splenocytes, N15tg CD2-/- splenocytes show significantly decreased proliferative responses to L4 or Norvaline4 stimulation at peptide concentrations from 10-4 to 10-7 M, with the dose-response curves from N15tg CD2-/- splenocytes shifted to the right by a factor of ~10 (Fig. 5Go, E and F). Collectively, these results demonstrate that responsiveness to antigenic peptides is impaired in the N15tg CD2-/- splenic T cells, as determined by peptide Ag-specific induction of proliferation and cytokine production. Hence, in the mature lymphoid compartment, CD2 enables lower concentrations of Ags to induce T cell responses.



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  		FIGURE 5. Splenic T cells of N15tg RAG-2-/- CD2-deficient mice show diminished IFN-{gamma} production and proliferation upon Ag-triggered stimulation. Splenocytes (1 x 105 cells) from 6- to 8-wk-old N15tg RAG-2-/- CD2+/+ and N15tg RAG-2-/- CD2-/- mice were incubated with the indicated concentrations of VSV8-related peptides (VSV8, L4, and Norvaline4) and irradiated Kb-bearing EL-4 cells (2 x 104 cells). Forty-eight hours later, supernatants were assayed for IFN-{gamma} production (A, B, and C) and parallel T cell proliferation, as judged by [3H]TdR incorporation (D, E, and F). Mean ± SD of results from three to five different mice is shown (*, p < 0.01; **, p < 0.05).

 
Impaired splenic T cell responsiveness to IL-12 in N15tg CD2-deficient mice

The interaction between CD2 and CD58 on opposing cells enhances T cell responsiveness to IL-12, explaining the ability of monocytes to augment human T cell activation by IL-12 (16, 17, 40). To examine whether CD2 molecules can also regulate the responsiveness of T cells to IL-12 in the murine system, we cultured Con A-activated lymphoblasts from N15tg CD2+/+ and N15tg CD2-/- mice with rIL-12 in the presence or absence of EL-4 as the CD48+ APC for 48 h, and then quantitated IFN-{gamma} secretion. We used Con A-activated T cells because treatment of murine splenocytes with Con A enhances expression of the murine IL-12R, thereby increasing the sensitivity to IL-12 (41). As shown in Fig. 6Go, in the absence of APC, Con A-activated lymphoblasts from N15tg CD2+/+ and N15tg CD2-/- mice produce equivalent amounts of IFN-{gamma} after rIL-12 stimulation. In contrast, when Con A-activated lymphoblasts are stimulated with rIL-12 in the presence of EL-4 cells, the production of IFN-{gamma} from N15tg CD2+/+ lymphoblasts is 2- to 3-fold more than that from N15tg CD2-/- lymphoblasts at equivalent concentrations of rIL-12. Moreover, equivalent concentrations of IFN-{gamma} were produced by N15tg CD2+/+ T cells at a 10-fold lower concentration of rIL-12 than required by N15tg CD2-/- T cells. While coculture with EL-4 cells also enhances IFN-{gamma} production from N15tg CD2-/- lymphoblasts at the highest concentration of rIL-12 tested (100 ng/ml), this result clearly demonstrates the critical role of CD2 in IL-12-mediated IFN-{gamma} production in the murine system. Given that CD48 is the only known ligand of CD2 in the murine system, we interpret this result as evidence for regulation of IL-12 responsiveness by the CD2-CD48 interaction.



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  		FIGURE 6. Impaired IL-12 responsiveness of activated splenic T cells from N15tg RAG-2-/- CD2-deficient mice. Con A-activated T lymphoblasts (5 x 105 cells) from 6-wk-old N15tg RAG-2-/- CD2+/+ or N15tg RAG-2-/- CD2-/- mice were incubated with indicated concentrations of rIL-12 in the presence (filled symbols) or absence (open symbols) of 1 x 105 irradiated EL-4 cells for 48 h at 37°C. Subsequently, supernatants were collected and assayed for IFN-{gamma} production. Mean ± SD of results from three different mice is shown at each concentration of rIL-12. IL-12-induced IFN-{gamma} production in the presence of APC is significantly impaired by CD2 deficiency (**, p < 0.05).

 
Normal CTL and NK cytolytic activity in N15tg CD2-deficient mice

In mature CTL, TCR occupancy can elicit proliferation, cytokine production, and cytotoxic responses. However, it has been reported that cytotoxicity is the most sensitive measure of TCR ligation events, being triggered under conditions in which no other biological sequelae are detected (42). To examine the role of CD2 in Ag-specific cytotoxic activity, CTL were generated from N15tg CD2+/+ and N15tg CD2-/- splenocytes by stimulation in vitro for 5–6 days with Kb-bearing N1 cells (which carry a VSV8 minigene) and conditioned media, and then tested for their ability to lyse 51Cr-labeled EL-4 target cells (Kb) pulsed with varying concentrations of VSV8 peptide. Interestingly, as shown in Fig. 7GoA, CTL generated from N15tg CD2-/- splenocytes killed VSV8-loaded EL-4 cells as efficiently as CTL from N15tg CD2+/+ splenocytes at each peptide concentration tested (10-6–10-11 M). This result demonstrates that CD2 deficiency does not affect CTL effector function in N15tg mice.



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  		FIGURE 7. CD2 deficiency does not alter CTL or NK effector function. A, CTL activation in N15tg RAG-2-/- CD2+/+ and N15tg RAG-2-/- CD2-/- mice is equivalent. CTL from 6- to 8-wk-old N15tg RAG-2-/- CD2+/+ or N15tg RAG-2-/- CD2-/- mice (5 x 104 cells) were incubated at 37°C with labeled EL-4 cells (5 x 103 cells) at an E:T ratio of 10:1 for 4 h, and radioactivity of the supernatant was determined. Mean ± SD of results from four different mice is shown. B, NK function is unaffected by lack of CD2. Splenocytes from N15tg RAG-2-/- CD2+/+ or N15tg RAG-2-/- CD2-/- mice were incubated with labeled YAC-1 target cells (5 x 103 cells) at the indicated ratios for 4 h at 37°C, and radioactivity of the supernatant was determined. Mean ± SD of results from four different mice is shown.

 
As CD2 is also expressed on the surface of NK cells (43), we assessed the role of CD2 in NK cytolytic effector function. To this end, freshly isolated N15tg CD2+/+ and N15tg CD2-/- splenocytes were cultured with 51Cr-labeled NK-sensitive YAC-1 mouse lymphoma cells as targets, and cytolytic activity was assessed. As shown in Fig. 7GoB, no significant difference was observed in the ability of the N15tg CD2+/+ and N15tg CD2-/- NK cells to lyse the YAC-1 cells across a wide range of E:T ratios tested. These results suggest that CD2 deficiency does not affect the cytotoxic effector activity of CTL or NK cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Earlier studies have suggested a key role for human CD2 in T cell Ag recognition (13, 44). Consequently, it was surprising that CD2-deficient mice expressed no abnormalities in T cell development or mature T cell function upon initial evaluation (21). To reexplore the role of CD2 in the mouse, we analyzed thymic development and Ag-dependent mature T cell function in MHC class I-restricted N15 TCRtg mice on a RAG-2-/- background in the presence or absence of functional CD2 gene expression (CD2+/+ vs CD2-/- strains). Our current results define a critical role for CD2 not only in mature T cell cytokine production, but also in thymic pre-TCR function in N15tg RAG-2-/- mice. Non-tg CD2-deficient mice show an altered peripheral T cell repertoire relative to B6 mice, demonstrating that one consequence of a loss of CD2 is altered thymic selection; this alteration may have been concealed in previous analyses through compensatory adaptations during T cell development in the non-tg CD2-deficient mice. Collectively, our findings clearly demonstrate an important contribution of CD2 to the murine immune system in vivo.

CD2 and mature T cell function

Human CD2-CD58-driven conjugate formation between T lymphocytes and APCs is known to facilitate TCR-mediated Ag recognition of pMHC ligands and, subsequently, the attendant downstream cellular activation events (13, 44). In addition to augmenting Ag recognition via its role in adhesion, CD2 has a very highly conserved cytoplasmic domain, which recruits several cytosolic molecules including CD2AP (45), CD2BP1 (46), and CD2BP2 (47), and facilitates Ag-triggered T cell responses via signal transduction. Coligation of CD2 and CD58 molecules on opposing cells redistributes CD2 to the region of cell-cell contact, in which the high local CD2 concentration offers a marked avidity enhancement to further adhesive forces (13, 45, 48). Thus, CD2 contributes to the formation of a specialized junction between a T cell and an APC, providing a mechanism for sustained TCR engagement and signaling. This intercellular region has been referred to by some as an immunologic synapse, and is linked to cellular polarization as well (49, 50).

Although many studies have demonstrated the importance of CD2/CD58 interaction in human T cell Ag recognition, no CD58 homologues have been identified in rodents; instead, another GPI-anchored protein, CD48, has been identified in both mice (19) and rats (51). The affinity of rodent CD2 for rodent CD48 is ~10-fold weaker than that between human CD2-CD58 (7). Nevertheless, the importance of the murine CD2-CD48 interaction in T cell activation has been recently demonstrated using murine T cell lines (52, 53). In the current study, we tested whether CD2 deficiency affects cytokine production and proliferative responses of peripheral naive T cells from N15tg mice. As expected, peripheral N15 T cells required 100- to 1000-fold more VSV8 cognate peptide to produce the same amount of IFN-{gamma} in the absence of CD2 in vitro, suggesting that CD2 can quantitatively facilitate TCR-mediated Ag recognition, thereby enabling lower concentrations of pMHC to induce a T cell inflammatory response. More importantly, in the absence of CD2, two VSV8 peptide APLs, L4 and Norvaline4, did not induce IFN-{gamma} at any concentration, indicating that the CD2 molecule is essential for immune responses against weak agonistic ligands and consistent with the data of Bachmann et al. (22).

In contrast to T cell cytokine production and proliferative responses, CD2 deficiency does not affect cytotoxic activity of alloreactive effectors (21) or peptide Ag-specific effectors (this study). In this regard, it is of interest that T cell cytotoxicity, which requires local secretion of prestored mediators, can be rapidly induced by interaction of only a few target cell pMHC complexes with the relevant TCR on a given effector T cell. Moreover, this ligation occurs in the absence of measurable TCR down-regulation (42, 54). In contrast, both cytokine production and T cell proliferation require gene transcription and necessitate higher thresholds of TCR ligation and cross-linking (55). In addition to augmenting TCR-triggered activation, the human CD2-CD58 interaction has been reported to control T cell responsiveness to IL-12, thus playing a key role in the IL-12/IFN-{gamma}-positive feedback loop between T cells and APCs (16, 17). In the current study, we show that the CD2-CD48 interaction subserves an analogous function in mice to the CD2-CD58 interaction in human beings.

CD2 and early T cell development

Early thymic differentiation is impaired in N15tg CD2-/- mice: the DP as well as SP thymocyte subpopulations are significantly reduced in size, whereas the total number of DN thymocytes is not diminished. Hence, T cell development is blocked at an early developmental stage before generation of DP thymocytes in the N15tg CD2-/- mice. Further analysis of the DN thymocytes shows a significant increase in the CD44-CD25+ subset and concurrent decrease in the CD44-CD25- subset. This block in thymic differentiation during the transition from the CD44-CD25+ to CD44-CD25- population in N15tg CD2-/- mice is similar to that observed in thymocytes from genetically targeted mice lacking an essential component of the pre-TCR complex, such as pT{alpha}-/- (56), RAG-1-/-, -2-/- (57, 58) TCR{beta}-/- (59), CD3{epsilon}-/- (60), CD3{zeta}-/- (61, 62), and CD3{gamma}-/- (63) mice. That parenteral injection with anti-CD3{epsilon} mAb rapidly restores DP thymocyte generation suggests a critical role for CD2 in early thymic development, especially in the pre-TCR-mediated transition from CD44-CD25+ to CD44-CD25- stages. Note that this thymic differentiation block is not a consequence of diminished {beta}-chain expression. TCR{beta} expression as judged by H57 mAb reactivity is equivalent on CD2+/+ and CD2-/- non-tg and N15tg Rag-2-/- CD44-CD25+ DN thymocytes (data not shown).

Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymic subpopulations derived from B6 (Ly-5.1) mice into normal, nonirradiated Ly-5.2 congenic recipient mice identifies one early differentiation sequence (Fc{gamma}RII/III+CD2- -> Fc{gamma}RII/III+CD2+ -> Fc{gamma}RII/III-CD2+) that precedes the entry of DN thymocytes into the DP stage (64). {beta}-chain V(D)J rearrangement is essentially restricted to the Fc{gamma}RII/III-CD2+ subset of DN thymocytes. Hence, CD2 is present on the pre-TCR-bearing DN thymocytes and maintained on all thymocytes at subsequent developmental stages (64). Although it is not certain whether the pre-TCR on the surface of DN thymocytes must interact with a ligand (for example, MHC) to exert its in vivo biological functions (35, 65), the primary role of CD2 in pre-TCR function may be to enhance the relatively weak affinity between the pre-TCR and its putative ligand as well as to effect intracellular signaling influencing maturation and/or expansion of DN thymocytes. Consistent with this notion, studies of human CD2 tg mice suggested that the cytoplasmic domain of CD2 is likely to be involved in early thymic development (66).

Previous reports involving other MHC class I-restricted TCR (HY and 2C) tg CD2-deficient mice on a Rag+/+ background also showed reduction in the numbers of DP thymocytes, albeit to different extents (67). Unlike many TCRtg mice (68, 69), the N15 TCR is not on the surface of the vast majority of DN thymocytes, including the pre-TCR-expressing CD44-CD25+ subset (data not shown). Hence, N15tg animals may more closely approximate the physiologic situation. However, in contrast to the TCRtg CD2-deficient mice, non-tg CD2-deficient mice showed no obvious abnormalities in development of DP thymocytes (21). One likely possibility is that CD2 can affect pre-TCR-mediated signaling to a variable degree, depending on the specific sequences of rearranged TCR{beta}-chains, including their CDR3 loops. Although more detailed analysis of individual TCRs is necessary to test this hypothesis, we suggest that only those pre-TCRs bearing rearranged TCR{beta}-chains that can activate the necessary intracellular signaling without CD2 may be selected at the CD44-CD25+ stage in non-tg CD2-deficient mice. This hypothesis can also explain why the V{beta} gene usage is not altered in the CD2-/- vs CD2+/+ non-tg animals.

CD2 and thymic repertoire generation

The T cell repertoire is shaped by negative and positive thymic selection processes at the level of immature DP thymocytes (36, 37, 38, 39). Strong interactions are negatively selecting, while weak interactions are positively selecting. Furthermore, the lack of interaction results in death by neglect. In addition to the influence of the monomeric TCR-pMHC affinity and thymocyte surface TCR and stromal pMHC ligand densities on thymocyte fate, it is also evident from studies of CD4 and CD8 coreceptor tg or knockout mice that the expression of other cell surface components can regulate selection outcome (70, 71, 72, 73). For example, as the expression of the CD4 and CD8 coreceptor increases, selection changes from being positive to negative (74, 75). Similar to the roles of CD4 and CD8 coreceptors in thymic development, our current study demonstrates that CD2 expression exerts a strong influence on TCR repertoire selection in non-tg mice. Given that CD2 is critical for Ag recognition in mature T cells, this profound effect of CD2 on the TCR repertoire selection is not unexpected. In CD2-deficient mice, it is likely that the TCR/pMHC affinity required for both positive and negative selection processes is altered so that immature thymocytes bearing TCRs with relatively high affinities for pMHCs can escape negative selection, thereby influencing the T cell repertoire generation. Because SP thymocytes show alterations similar to those of SP peripheral T cells in CD2-/- mice (data not shown), we infer that such selection variation is occurring primarily in the thymus. While it has been reported that the absence of CD2 enhances positive selection in HY TCR tg mice (67), this is not the case for the 2C TCR (67) or the N15 TCR (this study). Thus, alterations in selection are also predicated on the specific selecting peptides or their surrogates.

It is of particular interest that, despite the close similarity of TCR V{beta} gene usage between non-tg CD2+/+ and CD2-/- mice, the TCR V{alpha} gene usage shows striking differences in both CD4 and CD8 SP peripheral T cells. This differential change in TCR V{alpha}, but not in V{beta} usage, may result from the dominant role of the V{alpha} domain in pMHC Ag recognition and the fact that V{beta} usage has already been determined before the time CD2 expression influences pre-TCR function. Thus, on one hand, it is not surprising that usage of V{alpha} gene segments would have to be substantially altered in both CD4 and CD8 SP subsets to accommodate changes in thymic selection influenced by CD2 deficiency. On the other hand, however, repertoire analysis of wild-type human CD4 tg B6 mice and mice harboring nonfunctional mutant human CD4 (F43I) shows alterations in both V{alpha} and V{beta} gene usage in CD4 SP T cells (76). As pre-TCR signal components trigger transcriptional activation of TCR{alpha} genes and concurrent silencing of the pre-TCR in the pT{alpha} locus (77), it is possible that the CD2 deficiency may alter the initiation of TCR{alpha} gene transcription in a differential manner. Hence, the alteration in V{alpha} usage may be as much a manifestation of modified pre-TCR triggering at the DN thymocyte stage as it is a consequence of selection at the DP thymocyte stage. Certainly, these two possibilities are not mutually exclusive.


   Acknowledgments
 
We thank Drs. Harald von Boehmer, Linda K. Clayton, and Shigeo Koyasu for helpful comments. We acknowledge the early contributions of Dr. Yosi Ghendler in establishing the various N15tg mouse strains, and Drs. Dan Littman and Nigel Killeen for originally providing the CD2-/- B6 mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI21226. Back

2 Address correspondence and reprint requests to Dr. Ellis L. Reinherz, Laboratory of Immunobiology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Back

3 Abbreviations used in this paper: pMHC, peptide-MHC; APL, altered peptide ligand; DN, double negative; DP, double positive; SP, single positive; tg, transgenic; VSV8, vesicular stomatitis virus nucleoprotein octapeptide. Back

Received for publication July 14, 2000. Accepted for publication December 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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