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T Lymphocyte Development in the Absence of CD3 or CD3¹

Baoping Wang^2,*, Ninghai Wang^*, Charles E. Whitehurst, Jian She^*, Jianzhu Chen and Cox Terhorst^*

^* Division of Immunology, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, MA 02215; and Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD3, , , and proteins together with the pre-TCR -chain (pT) and a rearranged TCR ß-chain assemble to form the pre-TCR that controls the double negative (DN) to double positive (DP) stages of thymopoiesis. The CD3 proteins are expressed before pT and TCR ß-chains in prothymocytes and are expressed intracellularly in precursor NK cells, suggesting that the CD3 complex may function independent of pT and TCRß. In this report, both the role of CD3 exclusively, and the role of CD3 proteins collectively, in thymocyte and NK cell development were examined. In a mouse strain termed ^P, a neomycin cassette inserted within the CD3 promoter abolishes CD3 and expression and also abolishes CD3 expression in all but a small minority (1%) of prothymocytes. These prothymocytes became deficient in CD3 alone upon reconstitution of CD3 expression and were severely, but not completely, arrested at the DN stage, as small numbers of double positive thymocytes were detected. In de facto CD3^null mice generated by crossing the ^P mice with CD3^-/- mice, thymopoiesis were arrested at the CD44^-CD25⁺ DN stage as observed in RAG^-/- mice, DJ and VDJ recombination at the TCRß locus was functional, and normal numbers of NK cells were detected. Together, the findings demonstrate that during thymocyte development, the CD3 complex collectively is not essential until the critical CD44^-CD25⁺ DN stage in which pre-TCR begins to function, whereas CD3 is critical for the assembly of pre-TCR. Moreover, CD3 proteins are dispensable for NK cell development.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intrathymic T lymphocyte development is defined by different expression patterns of a series of cell surface Ag, including CD4, CD8, CD25, and CD44 (1, 2, 3). The most immature thymocytes are CD4^-CD8^- double negative (DN)³ cells that comprise 4% of thymic cellularity, and can be further divided into four subpopulations that differentiate in this order: CD44⁺CD25^- CD44⁺CD25⁺ CD44^-CD25⁺ CD44^-CD25^- (2, 4). The DN CD44^-CD25^- cells subsequently develop into immature CD4⁺CD8⁺ double positive (DP) CD44^-CD25^- thymocytes that are subjected to either positive or negative selection (5, 6, 7). The positively selected thymocytes become mature CD4⁺ or CD8⁺ single positive (SP) cells and are exported to the periphery (1, 6, 8). During this process, the genes coding for TCRs, pre-TCR (pT), and the associated CD3 proteins are also expressed in a temporal order and promote T cell development (9, 10, 11, 12).

Extensive biochemical studies have demonstrated that CD3 proteins are required for the assembly and surface expression of the TCR (13, 14, 15). In each TCR/CD3 complex, there are two copies of CD3 and CD3, but only one copy of CD3 and CD3 (16, 17, 18, 19, 20, 21). CD3 forms heterodimers with CD3 and CD3, whereas CD3 exists as a homodimer (18, 20, 22, 23). In prothymocytes, CD3 proteins associate with pT/TCRß to form the pre-TCR/CD3 complex (24, 25, 26, 27). The pre-TCR/CD3 complex plays an essential role in thymocyte differentiation from DN cells to DP cells, termed ß-selection, as targeted mutations in pT (28), TCR-ß (29), or RAG (29) genes all result in an arrest of T cell development at the DN CD44^-CD25⁺ stage (10, 11).

We, and others, have used gene knockout and transgenic approaches to study the role of CD3 proteins in T cell development (12). In CD3^-/- mice, T cell development is arrested at the immature DP stage (30, 31, 32, 33). In CD3^-/- mice, T cell development is arrested during the transition from the DP to the SP stage (34). In CD3^-/- mice, T cell development is severely arrested during the transition from the DN to DP stage (35). However, in mice deficient in both CD3 and CD3 genes, early thymocyte development was arrested at the DN CD44^-CD25⁺ stage (36). There are two independent strains of CD3 mutant mice. One is called the CD3^5/5 mice, in which exon 5 of the CD3 gene was replaced by a PGK neo^r cassette (37). We recently generated another strain of CD3 mutant mice termed the CD3^P/P (^P) mice, in which the promoter and the first two exons of the CD3 gene were replaced by a PGK-neo^r cassette (38). In both types of CD3 mutant mice, early thymocyte development was arrested at the DN CD44^-CD25⁺ stage (37, 38).

However, a specific role for CD3 in early T cell development is still unclear because CD3 and CD3 expression were also severely inhibited in both strains of CD3 mutant mice (37, 38). By reconstitution of CD3 expression in the ^P mice with a CD3 transgene, we recently revealed that CD3 does not regulate CD3 expression, and that the inhibition of CD3 and CD3 expression was caused by the PGK-neo^r cassettes inserted in the neighboring CD3 gene (38). Moreover, the study also showed that >99% of prothymocytes in the ^P mice are deficient in CD3, whereas 1% of prothymocytes express CD3 and <<1% of prothymocytes express CD3 (38). Thus, the blockade in thymopoiesis in the ^P mice represents the phenotype of mice with a triple deficiency in the CD3-chains. Therefore, we reasoned that if CD3 expression is restored in the ^P mice, we should be able to follow the development of the 1% of CD3^{+ +-} prothymocytes. We report here that prothymocytes deficient in CD3 alone can very inefficiently develop to the DP stage.

In addition to the essential role of CD3 proteins for the assembly and signal transduction of the pre-TCR and TCR, CD3 proteins are also expressed on the cell surface as a part of a clonotype-independent CD3 (CIC) complex in prothymocytes before the expression of pT and TCRß (39, 40), and are expressed intracellularly in NK cell precursors and NK cells (41, 42, 43). Because in vivo anti-CD3 stimulation of CIC in pT^-/-, TCRß^-/-, or RAG^-/- mice all result in thymocyte differentiation from DN cells to DP cells, the CIC may play a role in transducing a signal for early thymocyte development, including the onset of VDJ recombination. We have previously reported that overexpression of CD3 in transgenic mice results in an arrest of prothymocyte development at the DN CD44^-CD25^- stages before the stage in RAG^-/- mice, in addition to a complete block in NK cell development (44, 45). Thus, a remaining question is whether mice deficient in all four CD3 chains would have a block in very early thymopoiesis, VDJ recombination, and NK cell development. To answer these questions, we made use of the fact that >99% of ^P thymocytes are deficient in CD3, and hence obtained a de facto CD3^-/- mouse by breeding the ^P mice with CD3^-/- mice. We demonstrate here that: 1) prothymocytes deficient in all CD3 proteins still arrest at the same DN CD44^-CD25⁺ stage as that of RAG^-/- mice, 2) TCRß rearrangements take place in the absence of all CD3 proteins, and 3) CD3 proteins play no essential role in NK cell development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

The ^P mice and the ^Pxtg mice were generated as described (38). ^Pxtg mice were obtained by breeding the ^P mice with a strain of transgenic mice, termed tg4, that carries three copies of a human CD3 transgene (44). Double mutant mice were identified by Southern blot analysis.

Flow cytometric analysis

Single cell suspensions of thymocytes, LN cells, and spleen cells were prepared as described (44, 45). Three color staining of the cells was performed as previously reported (44, 45). Briefly, cells were incubated with prestaining buffer (0.5–1 x 10⁶ cells/50 µl) (PBS, 4% BSA, 0.5% sodium azide (SA), 15% of a mixture of normal hamster, normal rat, and normal mouse sera, 0.5 µg anti-FcR Ab) for 5 min. The cells were then stained with a biotinylated Ab (0.5 µg/50 µl per tube) for 30 min. The cells were washed once with 3 ml PBS, followed by staining with 50 µl/tube of a mixture of streptavidin-RED670 (Life Technologies, Grand Island, NY) (0.4 µl/sample), plus phycoerythrin- and FITC-conjugated Abs (0.5 µg each/sample), for 30 min. The cells were washed once with 3 ml PBS, resuspended, and fixed in 200 µl of PBS/SA and 1% formaldehyde until analyzed with a FACScan using CellQuest software (Becton Dickinson, Franklin Lakes, NJ). To exclude contamination of dead cells as CD4⁺CD8⁺ cells in thymocyte samples, some samples were stained with 200 µl of PBS/SA containing 1 µg/ml of propidium iodide after the final wash, and analyzed the same day. All procedures were performed on ice or at 4°C until analysis. For each sample, at least 10,000 cells were collected.

RNA analysis

Northern blot analysis was performed as described (36, 46).

NK cytotoxicity assay

NK-mediated cytotoxic lysis was determined by a standard ⁵¹Cr-release assay as described previously (44). Briefly, mice were injected i.p with 100 µg of poly(I:C) 16–24 h before sacrifice. Splenocytes derived from the testing mice were mixed with 5 x 10^{3 51}Cr-labeled YAC-1 cells in triplicate, at E:T ratios of 200, 100, 33, and 11, in a final volume of 200 µl in round-bottom microtiter plate, and incubated for 4 h at 37°C.

PCR assay for TCRß DJ and VDJ rearrangements

Rearrangement of Dß1. to Jß1.1 through Jß1.5 (DJ recombination) and Vß12 and Vß14 to Dß1Jß1.1 through Dß1Jß1.5 (VDJ recombination) was detected by a PCR-based assay (47) using the oligonucleotide primers Db15A, 5'-CCCCAGAGGAGCAGCTTATCTG-3'; Dß1–5', 5'-GGTAGACCTATGGGAGGGTC-3'; Jb15X, 5'-AAGACTCCTAGACTGCAGACTCAG-3'; Jb15Y, 5'-CCAGTTTGGTCCCATAGTTTACCT-3'; Vß12PS, 5'-GCTGGAGTTACCCAGACACCC-3'; and Vß14PS, 5'-GCCCTAACCTCTACTGGTACTGGCAGGC-3'. For DJ rearrangement, a primary PCR was performed using 100 ng of purified thymocyte DNA from the various mouse strains and primers Db15A and Jb15X for 15 cycles, and then 5 µl was removed and used as the template in a secondary PCR with the nested primers Dß1–5' and Jb15Y for 20 cycles. For VDJ rearrangement, a primary PCR was performed using 100 ng of purified thymocyte DNA and primers Vß12PS or Vß14PS and Jb15X for 15 cycles, and then 5 µl was removed and used as the template in a secondary seminested PCR with primers Vß12PS or Vß14PS and Jb15Y for 20 cycles. All reactions were in 50 µl and consisted of 10 mM Tris-HCl, (pH 8.8), 50 mM KCl, 2 mM MgCl², 0.2 mM dNTPs, 0.1% Triton X-100, 0.5 U Taq polymerase, and 125 ng of each primer. For all reactions, cycle conditions were as follows: 1 cycle of 95°C for 5 min, 62°C for 2 min, and 72°C for 2 min, followed by 15 or 20 cycles of 95°C for 1 min, 62°C for 1.5 min, and 72°C for 2 min. Secondary reactions were electrophoresed on 1.5% agarose gels, transferred to Immobilon nylon membrane (Bio-Rad, Richmond, CA), and all DNA products were detected by Southern blot hybridization with an end-labeled oligonucleotide probe specific for the 3' coding region of the Jß1.5 gene segment (5'-GAACAGAGAGTCGAGTC-3') (48). The m.w. of the resultant products in these assays accurately corresponded to the expected m.w. for rearrangements to Jß1.1 through Jß1.5 based on the known TCRß gene sequence.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A small number of prothymocytes in ^P mice express CD3

In addition to the absence of CD3 expression in ^P mice, the expression of the CD3 and CD3 genes was also suppressed by the neo^r cassette inserted in the neighboring CD3 gene (38). The suppression of CD3 expression was more severe than that of CD3 possibly because the neo^r cassette is closer to the CD3 gene than to the CD3 gene (38). As shown in Fig. 1A, CD3 and CD3 mRNA were apparently absent in ^P thymocytes (38). However, a minor CD3 mRNA band could be visualized in ^P thymocytes upon longer exposure, whereas CD3 mRNA was undetectable even when overexposed (Fig. 1B). Nevertheless, a minute level of CD3 transcription could be detected by a more sensitive RT-PCR method (38). We have shown recently that the detected CD3 and CD3 expression is restricted to a very small fraction of ^P thymocytes, whereas the majority of ^P thymocytes are deficient in both CD3 and CD3. Taken together, a very small fraction of the ^P thymocytes (1%) do express CD3, and a much smaller fraction of the ^P thymocytes may express both CD3 and CD3.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. CD3 deficiency in the P mice. A, Northern blot analysis of thymocytes from wt, P, and RAG-/- mice for the expression of CD3 and . The blotting of ß-actin was used as an RNA quantity control. The respective probes are indicated on the left, and the sizes of the RNA transcripts are indicated on the right. B, Longer exposure of the Northern blots shown in (A). C, Northern blot analysis of thymocytes from wt, P, and Pxtg mice for the expression of murine CD3, , and the transgenic human CD3. Of note, the introduction of the human CD3 transgene did not restore the expression of the endogenous murine CD3 and expression.

Developmental block of prothymocytes deficient in CD3 per se

Thymocytes that express both CD3 and CD3 in ^P mice should represent the phenotype of thymocytes deficient in CD3 per se. However, the extremely small number of such cells precluded a conclusive examination of this issue in the ^P mice. We reasoned that because there are more CD3-expressing cells than CD3-expressing cells in the ^P thymus, we should be able to obtain a detectable number of prothymocytes deficient in CD3 only if CD3 expression is restored in the ^P mice (Figs. 1C and 2). Thus, we bred the ^P mice with a strain of transgenic mice termed tg4^+/- (tg) that express a human CD3 transgene (45) (Fig. 1C). Indeed, a small but genuine population, averaging 0.4% (up to 2%), of DP cells was consistently detected in the ^Pxtg mice (Fig. 3). These DP cells were CD44^-CD25^-, similar to the immature DP cells in wild type (wt) mice (data not shown). The majority (>98%) of thymocytes in the ^Pxtg mice (which are CD3^-tg^+- cells) were still DN cells, with most of them being CD44^-CD25⁺ cells, the same as those in RAG^-/- or ^P mice (Figs. 2 and 3) (38, 49). However, thymic cellularity remained the same in ^Pxtg mice as compared with the ^P mice (Fig. 4), and no mature T cells (TCR-ß⁺ SP cells) were detected in the thymus or in the periphery of these mice (data not shown). Therefore, these data demonstrated that prothymocytes deficient in CD3 per se can become DP cells, whereas prothymocytes deficient in both CD3 and CD3 are arrested at the DN CD44^-CD25⁺ stage.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. Thymocyte development in the Pxtg, Pxtg and PxCD3-/- mice. Left panel, Profile of CD4 and CD8 expression. Right panel, Expression of CD44 and CD25 in the software-gated DN thymocytes.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Schematic diagram of the rationale and outcome of the experiments. Top panel, The rationale. The relative numbers of different subpopulations of cells were estimated as described (38). The patterns of CD3 expression are indicated (for reasons of simplicity, positively expressed CD3 subunit(s) is omitted in some cases). The horizontal bars at the tip of arrows represent blockades in T cell development. Bottom panel, The outcome. Blockades in T cell development from prothymocytes deficient in the expression of CD3 genes. See text for details. Of note, the number of 16.7 or 0.4% represents the average frequency of DP thymocytes in the whole thymus from Pxtg or Pxtg mice, respectively.

View larger version (51K): [in this window] [in a new window]   FIGURE 4. Thymic cellularity of the mutant mice. Each symbol represents the total number of thymocytes from a mouse. The average number of thymocytes from mutant mice was compared with that from wt (including CD3P/+) littermates or age-matched wt mice. For comparison, data from tg and Pxtg mice are also shown.

P

P

P

P

P

P

P

T cell and NK cell development in ^PxCD3^-/- mice

To investigate the collective role of CD3 proteins in very early thymopoiesis, we bred the ^P mice with CD3^-/- mice. The resulting ^PxCD3^-/- mice represent de facto CD3^-/- mice, because prothymocytes expressing CD3 or CD3 would be too few to give a detectable phenotype (without reconstitution of some CD3 expression) (Fig. 2). Nonetheless, early T cell development was arrested at the very same DN CD44^-CD25⁺ point in the ^PxCD3^-/- mice as in the ^P mice (Figs. 3 and 4). Flow cytometric analysis of thymocytes for the expression of c-kit, Sca-1, and Thy-1 revealed the same Thy-1⁺Sca-1⁺c-kit^- phenotype in the ^PxCD3^-/- mice as in the RAG-2^-/- mice (data not shown).

To assess the role of CD3 proteins in NK cell development, we analyzed the NK cell compartment in the spleen of the ^P and ^PxCD3^-/- mice. NK cells from certain strains of mice such as C57BL/6, but not other strains including 129/sv (in which embryonic stem cells were originated), express the NK1.1 marker (50). Nonetheless, NK cells from all strains express DX5 Ag (51). As the colony of knockout mice was maintained by sib-breeding, NK cells could express both NK1.1 and DX5, or DX5 only. As shown in Fig. 5, splenocytes derived from the ^P mice contained normal percentages of NK1.1⁺DX5⁺ cells and possessed normal level of cytotoxicity against NK cell-sensitive YAC-1 target cells. Although splenocytes derived from six of six ^PxCD3^-/- mice were negative for NK1.1 expression, which was in part due to the fact that NK cells in the CD3^-/- mice used in this study were mostly negative for NK1.1 expression (data not shown), these spleen cells contained normal percentages of DX5⁺ cells (Fig. 5A), and importantly, these splenocytes killed YAC-1 target cells efficiently (Fig. 5B). We conclude that NK cell development was normal in both the ^P and ^PxCD3^-/- mice. Taken together, CD3 proteins do not play an essential role in early thymocyte development before the DN CD44^-CD25⁺ stage or in NK cell development.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. NK cell development in the P and PxCD3-/- mice. A, Flow cytometric analysis of splenocytes for surface expression of NK1.1 and DX5. B, Cytotoxicity of spleen cells against NK-sensitive YAC-1 target cells. The percentages of NK cells in these splenocytes are represented in A.

Lastly, we examined the effect of a deficiency in CD3 proteins on TCR ß-chain rearrangements using a DNA-PCR assay (47). To this end, genomic DNA was isolated from the thymocytes derived from the ^P and ^PxCD3^-/- mice, as well as from mice deficient in CD3 (36). Thymocyte DNA isolated from wt mice and RAG-2^-/- mice were included in these experiments as controls. TCRß DJ rearrangements were assessed by nested PCR using 5' primers complementary to upstream of Dß1 and two 3' primers immediately downstream of Jß1.5, allowing the detection of Dß1 to Jß1.1 through Jß1.5 rearrangements. The VDJ rearrangements were assessed by nested PCR using two 5' primers complementary to Vß12 and Vß14, respectively, and the same 3' primers downstream of Jß1.5. The amplified PCR products were visualized with an oligonucleotide that specifically hybridizes to the Jß1.5 gene segment. As illustrated in Fig. 6, TCRß gene rearrangements in the mutant thymocytes were as efficient as those in wt counterparts. Thus, CD3 proteins are not required to signal initiation of TCR-ß gene rearrangements.

View larger version (49K): [in this window] [in a new window]   FIGURE 6. DNA-PCR assay for TCR ß-chain rearrangements. Left panel, Dß1 to Jß1.1 through Jß1.5 rearrangements. Middle and right panels, Vß12 and Vß14 to Jß1.1 through Jß1.5 rearrangements. P/P = P.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

P

P

No mature TCRß⁺ SP cells were detected in the thymus or the periphery of more than 10 ^Pxtg mice analyzed. One might argue that because only a very small number of CD3-deficient prothymocytes were present in these mice, the SP cells derived from the CD3^-/- prothymocytes were too few to be detected. This argument is unlikely because the same number of CD3^-/- prothymocytes, upon restoration of CD3 expression with a CD3 transgene in ^Pxtg mice, were able to give rise to up to 40% of the wt level of TCRß⁺ SP cells in the periphery (Fig. 2) (38). For instance, among more than 20 ^Pxtg mice examined, in 1 mouse only 0.5% of the 3 x 10⁶ thymocytes were DP cells, whereas 15% of its spleen cells were TCRß⁺ SP cells (data not shown). Moreover, in recently reported CD3^-/- mice, 5% of thymocytes in the small thymus were DP cells, whereas the absolute number of SP cells accounted for 20% of the T cell number in wt mice in the spleen (35). Therefore, we conclude that CD3^-/- DP thymocytes could not differentiate into the SP stage.

Based on the numbers of DP cells observed in the ^Pxtg mice vs those observed in ^Pxtg mice, we can estimate the thymic cellularity of a CD3 deficient mouse as follows: because every CD3⁺tg⁺ prothymocyte would give rise to more than 40-fold (16.7% vs 0.4%) DP cells as compared with a CD3^- (i.e., CD3⁺tg^+-) prothymocyte, thymic cellularity of potential CD3^-/- mice would be >40-fold less than the cellularity of tg mice (which is 43% of the cellularity in wt mice (38). Thus, the total number of thymocytes in potential CD3^-/- mice would be 1% of wt mice (i.e., 1 to 5 x 10⁶ cells/thymus). In other words, thymic cellularity of potential CD3^-/- mice would be similar to that of a number of mutant mice in which pre-TCR function is severely impaired, such as TCRß^-/- (29), pT^-/- (28), or CD3^-/- (35) mice. Therefore, we conclude that differentiation from DN to DP stages would be severely, but not completely, blocked in CD3^-/- mice, similar to the block in pT^-/- (28) or CD3^-/- (35) mice. This conclusion could be examined further by generating another CD3^-/- mouse with targeted embryonic stem cells in which the neo^r cassette is deleted by cre-loxP-mediated recombination (52, 53).

The conclusion of a severe block of pre-TCR-mediated early T cell development in CD3^-/- mice is consistent with the large body of biochemical evidence for a critical role of CD3 in the assembly of pre-TCR/CD3 complexes. It is known that CD3 associates with CD3 and CD3 to form the CD3 and CD3 cores in the pre-TCR/CD3 complexes (18, 22, 23, 39, 54). Thus, absence of CD3 would severely impair the formation of the pre-TCR/CD3 complexes. On the other hand, because all other components of pre-TCR/CD3 complexes are expressed (probably at very low levels for TCRß), it is likely that a partial pre-TCR can be assembled and expressed on cell surface, albeit very poorly, and this partial pre-TCR could mediate a very weak signal that induces inefficient transition of DN cells to DP cells.

The appearance of DP cells in the ^Pxtg mice differs from the DN phenotype of CD3-^5/5 and ^P mice, suggesting that the arrest in the latter CD3 mutant mice was caused by an accumulative deficiency of CD3 and CD3. Because significant expression of CD3 was observed in the CD3^5/5 mice (37), these mice may be considered CD3-deficient mice (CD3^-/-). The overwhelming majority of the ^Pxtg thymocytes were CD3^-/-. Moreover, because the frequency of the potential CD3/-expressing DN cells was very low in the ^P mice, these mice can be considered CD3-deficient mice (CD3^-/-). Likewise, ^PxCD3^-/- mice can be viewed as animals deficient in all CD3 proteins (CD3^-/-) (Fig. 2). In all of these cases, thymocyte development is completely arrested at the DN CD44^-CD25⁺ checkpoint (Fig. 2). Combining the data from this study and those from mice deficient in CD3 (35), CD3 (34), CD3 (30, 31, 32, 33), and CD3, and (36), we conclude that during T cell development, CD3 proteins are dispensable up to the DN CD44^-CD25⁺ stage in which pre-TCR begins to function, but they play an essential, yet partially overlapping, role in further T cell development (Fig. 7). Because it has been shown that under artificial circumstances, either CD3 or CD3 cytoplasmic domains alone can independently generate signals for thymocyte development to DP stage (55), the primary role of CD3 proteins in early stages of T cell development is likely their structural contribution for the assembly and surface expression of pre-TCR/CD3, whereas individual CD3 chains may execute distinctive functions in later stages of T cell ontogeny.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Schematic diagram of the block points in T cell development in various TCR/CD3-deficient mice. The timing of expression of TCR/CD3 genes is indicated on the left side. The block points in T cell development in the TCR/CD3 mutant mice are indicated on the right side, and are derived from data reported in this study and those observed from mice deficient in RAG (49, 59), TCRß (29), pT (28), TCR (60), CD3 (35), CD3 (34), and CD3 (30–33), and from CD35/5 (37) and CD3-/- (36) mice. The solid line indicates a complete blockade, whereas the dashed line indicates a severe but incomplete blockade.

P

P

We demonstrated here that TCRß rearrangements take place in the absence of all CD3 proteins. Of note, the phenotype of thymocytes and TCR ß-chain rearrangements observed in the ^PxCD3^-/- mice are consistent with those observed in the CD3^5/5xCD3^-/- mice described recently (56). However, since a significant CD3 expression and minute amounts of a truncated CD3 polypeptide were detected in the CD3^5/5 mice (37), the ^PxCD3^-/- mice may better represent animals deficient in all CD3 proteins.

Whereas earlier work using blotting techniques suggested that CD3 genes were not expressed in murine splenic NK cells (57), recent studies using more sensitive PCR-based approach demonstrated that CD3 genes were expressed in some mouse fetal liver or fetal thymus-derived immature NK cell lines (43). Moreover, human fetal NK cells also express CD3, , and (41, 42, 58). Thus, it was plausible that CD3 proteins may be involved in NK cell development. However, normal numbers of functional NK cells were detected in mice deficient in CD3 (36), CD3 (^P) and CD3 (^PxCD3^-/-). Therefore, CD3 proteins play no essential role in NK cell development.

	Acknowledgments

 
We thank Dr. C. A. Biron and D. Allen for a critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants CA 74233 (to B.W.), AI 35714 (to C.T.), AI 40146 (to J.C.), and by a Cancer Research Institute Fellowship (to C.E.W.).

² Address correspondance and reprint requests to Dr. Baoping Wang, Division of Immunology, Re-204, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail:

³ Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; pT, pre-TCR -chain; CIC, clonotype-independent CD3; SA, sodium azide; tg, transgenic mice termed tg4^+/-, wt, wild type.

Received for publication June 3, 1998. Accepted for publication September 2, 1998.

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