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The CD3γ chain is essential for development of both the TCRαβ and TCRγδ lineages

1998; Springer Nature; Volume: 17; Issue: 7 Linguagem: Inglês

10.1093/emboj/17.7.1871

1460-2075

Mariëlle C. Haks, Paul Krimpenfort, Jannie Borst, Ada M. Kruisbeek,

CAR-T cell therapy research

Article1 April 1998free access The CD3γ chain is essential for development of both the TCRαβ and TCRγδ lineages Mariëlle C. Haks Mariëlle C. Haks Division of Immunology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Paul Krimpenfort Paul Krimpenfort Division of Molecular Genetics The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Jannie Borst Jannie Borst Division of Cellular Biochemistry, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Ada M. Kruisbeek Corresponding Author Ada M. Kruisbeek Division of Immunology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Mariëlle C. Haks Mariëlle C. Haks Division of Immunology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Paul Krimpenfort Paul Krimpenfort Division of Molecular Genetics The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Jannie Borst Jannie Borst Division of Cellular Biochemistry, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Ada M. Kruisbeek Corresponding Author Ada M. Kruisbeek Division of Immunology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands Search for more papers by this author Author Information Mariëlle C. Haks1, Paul Krimpenfort2, Jannie Borst3 and Ada M. Kruisbeek 1 1Division of Immunology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 2Division of Molecular Genetics The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 3Division of Cellular Biochemistry, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Huis, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:1871-1882https://doi.org/10.1093/emboj/17.7.1871 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info CD3γ and CD3δ are the most closely related CD3 components, both of which participate in the TCRαβ–CD3 complex expressed on mature T cells. Interestingly, however, CD3δ does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3δ gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3δ, CD3γ is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3γ, in which expression of CD3δ, CD3ϵ, CD3ζ, pTα and TCRβ remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3γ. Nevertheless, T-cell development is severely inhibited in CD3γ-deficient mice. The number of cells in the thymus is reduced to 97% of the thymocytes resided in the CD4−CD8− DN compartment. Analysis of CD44 and CD25 expression in DN cells of newborn mice revealed that these cells, like those from adult mice, are arrested at the CD44−CD25+ DN stage, frequently with increased numbers of thymocytes within the CD44+CD25− compartment as well (Figure 3B, bottom panel). Surface expression of CD3ϵ was investigated in the thymocyte subsets that are characterized based on the differential expression of CD4 and CD8 coreceptors. CD3ϵ surface-expression can be observed within all thymocyte subsets in CD3γ−/− mice, albeit at greatly reduced levels (Figure 4A). The hierarchy of the expression levels, however, is comparable with that in the normal thymus, with SP cells expressing the highest levels, followed by CD4+CD8+ cells and finally CD4−CD8− cells, in which CD3ϵ expression is practically undetectable. The reduced expression levels are likely to reflect a role for CD3γ in (pre-)TCR complex formation and/or transport to the cell surface since mRNA expression levels for CD3ϵ are comparable between WT- and CD3γ-homozygous mutant mice (Figure 2A). Analysis of CD25 expression revealed that downregulation of CD25 at the DN stage is severely impaired in the absence of CD3γ. In CD3γ-deficient mice ∼55–70% of the DP thymocytes still express CD25, whereas in WT littermate controls this population comprises only 1–2% (Figure 4B). It has been suggested that this failure to downregulate CD25 is due to the generation of these DP cells in the absence of β-selection (Fehling and Von Boehmer, 1997), in which differentiation to the DP stage is induced without the concomitant cell expansion (Crompton et al., 1994). Figure 4.Cell-surface expression of CD3ϵ is drastically reduced in thymocytes from CD3γ−/− mice, and downregulation of CD25 at the DN stage is also severely impaired. Total thymocytes of 6- to 8-week-old WT and CD3γ homozygous mutant mice were analyzed by three-parameter flow cytometry for the expression of (A) CD3ϵ-FITC (grey) or an irrelevant mAb (white) or (B) CD25-Biotin plus SA-Tricolor in the several cell populations that are characterized, based on the differential expression of CD4-PE versus CD8-Biotin plus SA-Tricolor (A) or CD8-FITC (B). Download figure Download PowerPoint Taken together, the observations in the adult and newborn CD3γ−/− mice demonstrate that absence of the CD3γ chain results in a block in thymocyte development at a similar developmental stage as in RAG-1, CD3ϵ, TCRβ and pTα knock-out mice. These findings therefore document a crucial role for CD3γ in the pre-TCR complex. One of the ways in which CD3γ may function in the pre-TCR is by contributing to complex assembly, which is a prerequisite for transport from the ER to the cell surface. It is clear that CD3ϵ-expression levels on thymocytes from CD3γ−/− mice (Figure 4A) are severely compromised, and incomplete complex formation alone could be sufficient for reduced pre-TCR function. In a non-mutually exclusive hypothesis, the lack of unique CD3γ-dependent signaling events may further contribute to a reduction in pre-TCR function; the current production of mice lacking only the CD3γ-immunoreceptor tyrosine-based activation motif (ITAM) will resolve this issue. T cells in peripheral lymphoid organs of CD3γ−/− mice As some SP thymocytes are generated in CD3γ−/− mice (Figure 3A), we addressed whether SP T cells can be detected in the peripheral T-cell repertoire. Indeed, small numbers of B220− cells that express either CD3, CD4 and/or CD8 can be detected in the spleen and lymph nodes of these mice (Figure 5A). This population contains both CD4 SP and CD8 SP T cells, in a ratio comparable with those observed in heterozygous and WT littermate controls (Figure 5B). Nevertheless, the absolute number of peripheral T cells is significantly decreased, representing only 2–5% of the number of T cells in normal mice for lymph nodes, and only 20% of the T-cell number in normal mice for the spleen. In contrast, B-cell development proceeds unaltered in CD3γ−/− mice. Although the relative number of peripheral B220+ cells that do not express CD3, CD4 or CD8 is increased (Figure 5A), the absolute number of mature B220+IgM+ B cells in the spleen and lymph nodes of CD3γ-deficient mice was unchanged (data not shown). Figure 5.Small numbers of peripheral T cells can be detected in the spleen and lymph nodes of CD3γ−/− mice, while B-cell development is unaffected. Flow cytometry analysis of spleen and lymph node (LN) cells of 6- to 8-week-old CD3γ+/+, CD3γ+/− and CD3γ−/− mice. Total cell populations were monitored for the expression of (A) a cocktail of CD3, CD4, CD8–FITC versus B220-Biotin plus SA-Tricolor or (B) CD4-PE versus CD8-Biotin plus SA-Tricolor. The percentage of cells within each quadrant is indicated. The absolute number of spleen and lymph node cells detected in the WT, heterozygous mutant and homozygous mutant genotypes is depicted above the corresponding dot display. Download figure Download PowerPoint Since the level of TCR expression on thymocytes from CD3γ−/− mice is significantly reduced (Figure 4A), we determined whether TCR expression on peripheral T cells is also affected by absence of the CD3γ chain. To what extent the different CD3 components contribute to efficient surface expression of mature TCRαβ complexes has not been fully resolved (Sussman et al., 1988; Hall et al., 1991; Kappes and Tonegawa, 1991; Buferne et al., 1992; Geisler, 1992; Dave et al., 1997), but a dependency on CD3γ has been reported (Buferne et al., 1992; Geisler, 1992). The present findings clearly support the latter observations; in mice lacking CD3γ, CD3ϵ expression on peripheral T cells is severely reduced (Figure 6), reaching only 5–10% of the levels observed on WT peripheral T cells. Together with the findings on reduced CD3ϵ expression on the different thymocyte subsets in CD3γ-deficient mice (Figure 4A), it can be concluded that CD3γ represents an essential structural component that contributes to assembly and/or transport of the TCR–CD3 complex. Figure 6.Absence of CD3γ is associated with strongly reduced levels of CD3ϵ on peripheral T cells of CD3γ−/− mice. Lymph node cells of 6- to 8-week-old WT and CD3γ homozygous mutant mice were analyzed by three-parameter flow cytometry for the expression of CD3ϵ–FITC (grey) or an irrelevant mAb (white) in the several cell populations that are characterized based on the differential expression of CD4-PE versus CD8-Biotin plus SA-Tricolor. Download figure Download PowerPoint Development of γδ T cells in CD3γ−/− mice The above findings suggesting a block in development at the CD44−CD25+ DN stage indicate a defect in αβ T-cell development. Indeed, as illustrated in Figure 7A and B through TCRβ versus CD3ϵ staining (top panels), αβ T-cell development is severely affected by lack of CD3γ. Next we investigated to what extent the defect in pre-T–cell development in CD3γ−/− mice affected development of the TCRγδ lineage as well. A severe defect in γδ T-cell development can be observed in mice lacking CD3ϵ (Malissen et al., 1995) which suggests that γδ T cells, like αβ T cells, require signaling either through a pre-TCR-type structure or a mature γδ TCR complex to expand and/or differentiate. However, γδ T-cell development proceeds undisturbed in mice lacking TCRβ, pTα or CD3δ, and the γδ T cells of such mice express normal levels of the TCRγδ heterodimer (Mombaerts et al., 1992a; Fehling et al., 1995; Dave et al., 1997). If γδ T cells, analogous to αβ T cells, are subject to a pre-TCR-like selection step as recently suggested (Passoni et al., 1997), such a pre-TCR-type structure does not involve pTα, TCRβ or CD3δ. In addition, CD3ζ-chain-deficient mice exhibit normal γδ T-cell development (Malissen et al., 1993; Ohno et al., 1993), while their αβ T-cell development is compromised. Figure 7.Development of both αβ T cells and γδ T cells is affected in CD3γ-deficient mice. (A) Thymocytes and (B) lymph node cells of 6- to 8-week-old CD3γ+/+, CD3γ+/− and CD3γ−/− mice were analyzed by flow cytometry for the expression of TCRαβ–FITC (top panels) or TCRγδ–FITC (bottom panels) versus CD3ϵ-PE. The percentage of cells within the depicted gates is indicated in each dot display. Download figure Download PowerPoint Our results on the generation of the TCRγδ lineage in CD3γ−/− mice support a requirement for CD3γ in the development of this lineage as well (Figure 7). First, and most importantly, although the relative number of TCRγδ positive T cells in both the thymus and lymph nodes of CD3γ−/− mice was comparable, or at best slightly increased, the absolute number of γδ T cells in CD3γ−/− mice was drastically reduced (Figures 7A and B, bottom panels). In CD3γ-deficient mice, 4×104 thymic γδ T cells were detected compared with 3–4×106 thymic γδ T cells in WT and heterozygous mice. In the lymph nodes of CD3γ-deficient mice, a 4- to 5-fold reduction in the absolute number of peripheral γδ T cells was obser