Essential and Partially Overlapping Role of CD3gamma and CD3delta for Development of alpha beta and gamma delta T Lymphocytes

By Baoping Wang,* Ninghai Wang,* Mariolina Salio,* Arlene Sharpe, Deborah Allen,* Jian She,* and Cox Terhorst*

From the * Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; and the Dagger  Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CD3gamma and CD3delta are two highly related components of the T cell receptor (TCR)-CD3 complex which is essential for the assembly and signal transduction of the T cell receptor on mature T cells. In gene knockout mice deficient in either CD3delta or CD3gamma , early thymic development mediated by pre-TCR was either undisturbed or severely blocked, respectively, and small numbers of TCR-alpha beta + T cells were detected in the periphery of both mice. gamma delta T cell development was either normal in CD3delta -/- mice or partially blocked in CD3gamma -/- mice. To examine the collective role of CD3gamma and CD3delta in the assembly and function of pre-TCR and in the development of gamma delta T cells, we generated a mouse strain with a disruption in both CD3gamma and CD3delta genes (CD3gamma delta -/-). In contrast to mice deficient in either CD3gamma or CD3delta chains, early thymic development mediated by pre-TCR is completely blocked, and TCR-alpha beta + or TCR-gamma delta + T cells were absent in the CD3gamma delta -/- mice. Taken together, these studies demonstrated that CD3gamma and CD3delta play an essential, yet partially overlapping, role in the development of both alpha beta and gamma delta T cell lineages.

Key words: CD3gamma CD3delta T cell receptor-CD3 complexT cell developmentknockout mouse
    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

During thymocyte development, the genes coding for TCR-alpha and -beta , pre-TCR-alpha (pTalpha ), and the associated CD3 proteins (CD3gamma , delta , epsilon , and zeta ) are expressed in a temporal order (1). The pre-TCR-CD3 complex, consisting of pTalpha , TCR-beta , and CD3 proteins, plays a major role in early thymocyte development and in the transition from CD4-CD8- (double negative, DN) to CD4+CD8+ (double positive, DP) cells, as targeted mutations in pTalpha , TCR-beta , RAG, and CD3 genes all result in an arrest of T cell development at the DN CD44-CD25+ check point (2, 3). Subsequently, TCR-alpha replaces pTalpha and the resulting TCR-CD3 complex mediates signal transduction cascades leading to further T cell development (2). Compared with alpha beta T cell development, gamma delta T cell development is less defined (4, 5). The majority of thymic gamma delta T cells do not express CD4 or CD8 antigens (6), and pTalpha and TCR-beta are not involved the development of gamma delta T cells (7, 8). However, CD3 proteins are required for the development of this lineage (2).

Ample biochemical studies have shown that the CD3 proteins are important for assembly and efficient surface expression of TCR (9). In each TCR-CD3 complex, there are two copies of CD3epsilon and CD3zeta , yet only one copy of the highly homologous CD3gamma and CD3delta (10). CD3epsilon forms heterodimers with CD3gamma and CD3delta , and can also exist as a CD3epsilon epsilon homodimer, whereas CD3zeta exists as a CD3zeta zeta homodimer (11, 13, 14). TCRs lacking CD3gamma , delta , epsilon , or zeta  can reach the cell surface, albeit 10-100-fold less efficiently than wild-type receptors, because of a certain degree of redundancy in their assembly potential (15, 16). In immature thymocytes, the CD3 proteins are expressed (17- 19), before the expression of pTalpha and TCR-beta (1). Thus, CD3 proteins can be a part of the pre-TCR-CD3 complex or part of a clonotype-independent CD3 (CIC) complex (20). In these complexes, CD3gamma epsilon dimers are consistently detected (20, 21), and some studies indicated the presence of a small quantity of CD3delta epsilon dimers (18). This led to the notion that CD3gamma may be preferentially required over CD3delta in the assembly of pre-TCR complexes (22).

Recent studies on mutant mice deficient in either the CD3gamma or CD3delta gene in part support this notion. Whereas transition from DN to DP alpha beta thymocytes appears to be normal in CD3delta -/- mice (23), alpha beta T cell development in CD3gamma -/- mice is blocked at the DN CD44-CD25+ check point (24). However, the blockade in T cell development in CD3gamma -/- mice is incomplete, as small numbers of DP thymocytes were found and TCR-alpha beta + T cells were detected in the periphery (24). Moreover, in either mutant a considerable number of gamma delta T cells is present (23, 24). Therefore, it is likely that CD3gamma and CD3delta play an essential, yet to some extent redundant, role in early development of T cells.

To examine the issue of partial overlap in function between CD3gamma and CD3delta , a mouse strain with a disruption in both the CD3gamma and CD3delta genes (CD3gamma delta -/-) would be useful. A CD3gamma delta -/- mouse, however, could not be generated by breeding the CD3delta -/- and CD3gamma -/- mice, because the genes coding for CD3gamma , delta , and epsilon  are located in a single gene cluster and a mere 1.4-kb intergenic sequence separates the first exons of CD3gamma and CD3delta genes (25). Therefore, we generated CD3gamma delta -/- mice by deleting the promoters and exons 1 of both genes.

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

Generation of CD3gamma delta -/- Mice.

The targeting construct was generated by standard methods. In brief, a genomic DNA clone containing a 15.5-kb fragment of CD3gamma delta genes was isolated from a 129/sv mouse genomic DNA library (provided by Dr. Manley Huang, GenPharm Int., Mountain View, CA) and subcloned into pBluescriptSK+ (Stratagene, La Jolla, CA). A 2.8-kb SalI-XhoI DNA fragment containing the PGK-TKr gene was isolated from pPGK-TK (provided by Dr. Manley Huang), and ligated to the XhoI site of pPGK-hygromyciner (hygr) (a gift of Dr. Richard Mortensen). A 1.9-kb XbaI-XbaI intronic fragment between exon 1 and 2 of CD3delta was obtained by XbaI digestion of the 15.5-kb CD3gamma delta genomic DNA fragment. And a 3-kb intronic fragment between exon 1 and 2 of CD3gamma was obtained by first subcloning a 5-kb EcoRI-XbaI fragment into SK+ followed by a HindIII cut, so that a HindIII site from the polylinker region of the plasmid was transferred to one end of the 3-kb fragment. The 1.9-kb XbaI-XbaI fragment and the 3-kb HindIII-HindIII fragment were inserted into the 5' and 3' sites of the PGK-Hygr gene. In the resulting construct, a 3.1-kb DNA fragment containing the 1.4-kb intergenic DNA fragment between the CD3gamma and CD3delta genes and exons 1 of both genes were replaced by the 2.8-kb PGK-Hygr cassette. 10 µg of purified targeting molecules were electroporated into 107 J-1 ES cells. ES cells were positively selected by hygromycin-B at 200 µg/ml and negatively selected by FIAU at 0.2 µM. 355 clones were selected and examined by Southern blots for homologous recombination using a 0.8-kb (StuI-XbaI) 5' probe located outside of the construct. Eight clones were identified as targeted clones, which were confirmed by another Southern analysis with a hygr probe. Four of the targeted clones were injected into the blastocysts of either C57BL/6 or BALB/C origin, and 90- 100% fur color chimerism was observed in 45 founder mice. Test breeding of the chimeras indicated that all of the males (n = 28 from 3 embryonic stem [ES] clones) transmitted the ES cell genome. Four males were mated to C57BL/6 females to generate heterozygous mice, and homozygous CD3gamma delta -/- lines were obtained by sibling breeding. Identical results were obtained from homozygous CD3gamma delta -/- lines of different ES clones.

Flow Cytometric Analysis.

Single cell suspensions of thymocytes, LN cells, spleen cells, PBL, and small intestine intraepithelial lymphocytes (iIEL) were prepared as described (26, 27). Three-color staining of the cells was performed as previously reported elsewhere (28).

RNA Analysis.

Northern blot analysis was performed as described (29).

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Generation of CD3gamma delta -/- Mice.

To generate mice deficient in both CD3gamma and CD3delta gene expression, a 3.1-kb DNA fragment containing the promoters (25) and exons 1 of the CD3gamma and CD3delta genes was replaced by a PGK-Hygr cassette (Fig. 1 A). The PGK-hygr cassette was chosen here over the PGK-neor cassette to prevent a possible suppressive effect of the PGK-neor on neighboring gene expression (30, 31). Homozygous mice carrying this mutation in the CD3gamma and delta  genes were generated (Fig. 1 B). Northern blot analysis demonstrated that the expression of both CD3gamma and CD3delta mRNA was absent in the CD3gamma delta -/- thymocytes (Fig. 2). Moreover, no aberrant expression of the truncated CD3gamma or delta  mRNAs were ever detected in Northern blotting of thymocytes from more than 20 CD3gamma delta -/- mice. However, the expression of the neighboring CD3epsilon gene and the nonlinked CD3zeta was normal (Fig. 2), and pTalpha expression was detected (data not shown).


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Fig. 1.   Disruption of CD3gamma delta genes. (A) Diagram of the CD3gamma delta targeting vector for homologous recombination. Exons 1-5 of the CD3delta gene and exon 1 of the CD3gamma gene are numbered. Arrows indicate the transcriptional orientations of the CD3gamma delta genes. The 0.8-kb probe was used for screening the ES cell clones and for Southern analysis of tail DNA. (B) Southern blot analysis of tail DNA.


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Fig. 2.   TCR-CD3 expression in the CD3gamma delta -/- mice. Northern blotting of thymocytes from wt, RAG-/- and CD3gamma delta -/- mice for the expression of CD3gamma , delta , epsilon , zeta , and TCR-alpha and -beta . The respective probes are indicated on the left, and the sizes on the right. Two mice of each type were analyzed in this blot.

alpha beta T Cell Development in the CD3gamma delta -/- Mice.

Total cellularity of the thymi of CD3gamma delta -/- mice was 2-5% of that in wild-type or heterozygous littermates (Fig. 3 A). Flow cytometric analysis of the thymocytes showed that these cells are DN, with the majority of them being CD44-CD25+c-Kit-Sca-1+, identical to the thymocytes found in RAG-/- mice (Fig. 3 B). Northern blot analyses of the thymocytes of CD3gamma delta -/- mice did not detect the mRNA for rearranged TCR-alpha and TCR-beta genes, whereas only the 1.0-kb germline Cbeta mRNA was detectable (Fig. 2). Consistent with these analyses, no mature alpha beta + T cells were detected in the LN, the spleen, or the gut of the CD3gamma delta -/- mice (Figs. 3 C and 4 C, Table 1). B cell development appeared unaffected (Table 1). Taken together, alpha beta T cell development in CD3gamma delta -/- mice is blocked at the same DN CD44-CD25+ check point as RAG-/- mice (32, 33).


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Fig. 3.   T cell deficiency in the CD3gamma delta -/- mice. (A) Thymic cellularity of the mutant mice. Each symbol represents the total number of thymocytes from a mouse. The ages of the mice are indicated. For each age group, the average number of thymocytes from mutant mice was compared with that of wild-type (including CD3gamma delta +/-) littermates or age-matched, wild-type mice (28). (B) Flow cytometric analysis of thymocytes from CD3gamma delta -/-, RAG-2-/- and wild-type mice for surface expression of CD4, CD8, CD44, CD25, Sca-1, and c-Kit. (C) Flow cytometric analysis of peripheral lymph node cells for surface expression of CD4 and CD8.


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Fig. 4.   Flow cytometric analysis of the gamma delta T cell compartment in CD3gamma delta -/- mice. (A) Thymocytes were stained with anti-TCR-gamma delta - biotinylated (detected with RED-670), anti-CD3-PE, and anti-CD4/ CD8-FITC. (Left) Profile of CD4/CD8 expression. (Right) Profile of TCR-gamma delta and CD3 expression in the analytically gated DN cells. (B) Lymph node cells were similarly analyzed as in A, except that a mixture of FITC-conjugated antibodies, i.e., anti-CD4, -CD8, -TCR-alpha beta , -B220, -Mac-3, and Gr-1 (collectively termed Lin), was used. (C) Expression of TCR-alpha beta and TCR-gamma delta in iIEL. (D) iIEL were stained with anti-CD8alpha , anti-CD8beta , and anti-CD32. (Left) Profile of CD8alpha /CD8beta expression. (Right) Profile of CD32 expression in the analytically gated CD8alpha alpha + cells. The CD8alpha + cells were all CD8alpha alpha + and were predominantly CD32+. (E) iIEL were stained with anti-CD8alpha , anti-B220, and anti-CD32, and the profile of CD8alpha /B220 expression in the analytically gated CD32+ cells is shown.

                              
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Table 1
T Cell and B Cell Compositions in CD3gamma delta -/- Mice

gamma delta T Cell Development in CD3gamma delta -/- Mice.

Next, gamma delta T cell development in CD3gamma delta -/- mice was examined. As shown in Fig. 4, A and B, gamma delta T cells were absent in the thymus and periphery of CD3gamma delta -/- mice. Since gamma delta T cells normally account for only a very small fraction of thymocytes and peripheral T cells, we assessed gamma delta T cell development in the small intestine, where gamma delta T cells represent a major population of the iIEL in wild-type mice. In CD3gamma delta -/- mice, gamma delta T cells were again nondetectable in the intestine (Fig. 4 C). However, normal number of CD8alpha alpha +B220+CD32+NK1.1- cells, representing T cell progenitors in the gut (27) could be detected in the gut of CD3gamma delta -/- mice (Fig. 4, C-E, Table 1, and data not shown). Therefore, these analyses indicate that deficiency in CD3gamma and delta  completely blocked gamma delta T cell development beyond the CD8alpha alpha + stage.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We report here that in the CD3gamma delta -/- double mutant mice, intrathymic development is completely arrested at the DN CD44-CD25+ prothymocyte stage, a central check point at which pre-TCR begins to mediate further thymocyte differentiation into the DP stage. This observation indicates that the function of pre-TCR is completely abrogated in CD3gamma delta -/- mice. In contrast, in recently reported CD3delta -/- mice, thymic development is undisturbed up to the DP stage (23), whereas the transition from DN to DP stages was severely but not completely blocked in CD3gamma -/- mice (24). The phenotypes of CD3delta -/- and CD3gamma -/- mice are consistent with the biochemical evidence that CD3gamma is preferentially required over CD3delta in prothymocytes for the assembly of the pre-TCR-CD3 complex (22). However, the present data revealed that CD3delta also participated in vivo in the assembly and function of the pre-TCR-CD3 complex. Moreover, small numbers of TCR-alpha beta + T cells were detected in the periphery of CD3delta -/- and CD3gamma -/- mice, but were absent in CD3gamma delta -/- mice. These observations are consistent with the biological evidence that in mature T cells, the TCR- CD3 complex lacking either CD3gamma or delta  could sometimes be detected on the cell surface at reduced levels. However, no surface expression of the TCR-CD3 complex could be detected in cells lacking both CD3gamma and delta  (15, 16). Taken together, CD3gamma and CD3delta collectively play an essential, yet partially overlapping, role in the assembly and function of the pre-TCR. It is most likely that in the absence of CD3gamma and CD3delta , pre-TCR cannot be expressed on the surface of prothymocytes.

In addition to the structural requirement, CD3gamma and CD3delta may regulate pre-TCR function through the signaling capacity of the immunoreceptor tyrosine-based activation motifs (ITAMs) presented in their cytoplasmic domains (34). It is known that not every ITAM plays a distinct role in pre-TCR function. For instance, pre-TCR function is competent in mutant mice deficient in the CD3zeta cytoplasmic domain (35). Moreover, the defect in pre-TCR function in CD3gamma -/- (24), CD3zeta -/- (36), or RAG-/- (19, 27, 37) mice can be overcome by anti-CD3epsilon -mediated cross-linking. However, the same anti-CD3epsilon treatment in vivo in CD3gamma delta -/- mice failed to relieve the block at the DN check point (data not shown). Since the anti-CD3epsilon antibody used in all of these studies, namely 2C11 (or 500A2), binds CD3epsilon efficiently when either CD3gamma or CD3delta is presented but poorly when both CD3gamma and CD3delta are missing (38; data not shown), the lack of thymocyte differentiation upon 2C11 treatment of CD3gamma delta -/- mice might be explained by the following nonexclusive possibilities: (a) pre-TCR could not be expressed on the surface of CD3gamma delta -/- prothymocytes; (b) the inefficient binding of 2C11 to CD3epsilon on the surface of CD3gamma delta -/- prothymocytes results in a weak signal that is below the threshold level for further thymic development; and (c) the cytoplasmic domains of CD3gamma and CD3delta collectively play an essential role in pre-TCR function. The last possibility, nevertheless, is less likely because it has been shown that under artificial circumstances, either CD3epsilon or CD3zeta cytoplasmic domain alone can independently generate signals for thymocyte development to the DP stage (39). Thus, the ultimate assessment of the physiological role of the cytoplasmic domains of CD3gamma and CD3delta awaits the generation of mutant mice in which the cytoplasmic domains of CD3gamma and CD3delta are deleted.

An important observation of this study was that gamma delta T cell development was completely blocked in the CD3gamma delta -/- mice. In comparison, gamma delta T cell development was partially blocked in the CD3gamma -/- mice and was undisturbed in CD3delta -/- mice (23, 24). Thus, this study demonstrated that CD3delta also plays a role in regulating the development of the gamma delta T cell lineage, and CD3gamma and CD3delta collectively are essential for gamma delta T cell development. Like their regulation of alpha beta T cell development, CD3gamma and CD3delta may regulate gamma delta T cell development by their structural contribution and/or signaling capacity. Nevertheless, the function of CD3gamma or CD3delta for gamma delta T cells may not be a duplication of their respective roles for alpha beta T cells. For instance, although surface expression of TCR-alpha beta is severely reduced (8-10-fold) in CD3delta -/- mice, their TCR-gamma delta expression is only mildly (less than twofold) reduced (23). On the other hand, severe reduction of both TCR-alpha beta and TCR-gamma delta expression in CD3gamma -/- mice indicated a pivotal role of CD3gamma in the assembly of TCR-alpha beta -CD3 and TCR-gamma delta -CD3 complexes (24). Taken together, it is likely that the complete block in gamma delta T cell development in CD3gamma delta -/- mice was a result of the incomplete TCR-gamma delta -CD3 complex not being expressed on cell surface in the absence of CD3gamma and CD3delta . It remains to be investigated whether the cytoplasmic domains of CD3gamma and CD3delta also have distinct functions in the development of gamma delta T cells.

In conclusion, in the CD3gamma delta -/- mice, early thymic development mediated by pre-TCR was completely blocked, and TCR-alpha beta + and TCR-gamma delta + T cells were absent. These observations are different from those made on either CD3delta -/- or CD3gamma -/- mice, in which pre-TCR function was either undisturbed or incompletely blocked, as TCR-alpha beta + and TCR-gamma delta + T cells were detected in the periphery. Taken together, these studies demonstrated that CD3gamma and CD3delta play an essential, yet partially overlapping, role in the development of both alpha beta and gamma delta T cell lineages.

    Footnotes

Address correspondence to Cox Terhorst, Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215. Phone: 617-667-7147; Fax: 617-667-7140; E-mail: cterhors @

Received for publication 26 May 1998 and in revised form 22 July 1998.

   Dr. Salio's current address is Basel Institute of Immunology, Basel, Switzerland.
   The first two authors contributed equally to this work.

This work was supported by National Institutes of Health grants CA 74233 (to B. Wang) and AI35714 and R37-17651 (to C. Terhorst).

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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