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Role of the multiple T cell receptor (TCR)-zeta chain signaling motifs in selection of the T cell repertoire.

Shores EW, Tran T, Grinberg A, Sommers CL, Shen H, Love PE - J. Exp. Med. (1997)

Bottom Line: The TCR is composed of four distinct signal transducing subunits (CD3-gamma, -delta, -epsilon, and zeta) that contain either one (CD3-gamma, -delta, -epsilon) or three (-zeta) signaling motifs (ITAMs) within their intracytoplasmic domains.A possible function for multiple TCR ITAMs could be to amplify signals generated by the TCR during selection.A direct relationship was observed between the number of zeta chain ITAMs within the TCR complex and the efficiency of both positive and negative selection.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematologic Products, Food and Drug Administration, Bethesda, Maryland 20892, USA.

ABSTRACT
Immature thymocytes undergo a selection process within the thymus based on their T cell antigen receptor (TCR) specificity that results either in their maturation into functionally competent, self-MHC-restricted T cells (positive selection) or their deletion (negative selection). The outcome of thymocyte selection is thought to be controlled by signals transduced by the TCR that vary in relation to the avidity of the TCR-ligand interaction. The TCR is composed of four distinct signal transducing subunits (CD3-gamma, -delta, -epsilon, and zeta) that contain either one (CD3-gamma, -delta, -epsilon) or three (-zeta) signaling motifs (ITAMs) within their intracytoplasmic domains. A possible function for multiple TCR ITAMs could be to amplify signals generated by the TCR during selection. To determine the importance of the multiple TCR-zeta chain ITAMs in thymocyte selection, transgenes encoding alpha/beta TCRs with known specificity were bred into mice in which zeta chains lacking one or more ITAMs had been genetically substituted for endogenous zeta. A direct relationship was observed between the number of zeta chain ITAMs within the TCR complex and the efficiency of both positive and negative selection. These results reveal a role for multiple TCR ITAMs in thymocyte selection and identify a function for TCR signal amplification in formation of the T cell repertoire.

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Analysis of LN T cells from H-Y+/ζ Tg mice. Immunofluorescence and multicolor FCM analysis of LN cells from adult female and  male H-Y+/ ζ-3 ITAM Tg or H-Y+/ζ-0 ITAM Tg mice. Also shown  are LN cells from a nontransgenic C57BL/6 (H-Y−/ζ+/+) mouse. Threecolor FCM was performed on total LN cells after staining with FITC– anti–H-Y clonotypic antibody (T3.70), anti-CD8–PE and anti-CD4– biotin, followed by streptavidin–red 670. CD4 versus CD8 two-color  profiles are displayed on total (left) or gated T3.70+ (right) cells. Numbers  in the regions reflect the percentage of total cells found in that region.
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Figure 4: Analysis of LN T cells from H-Y+/ζ Tg mice. Immunofluorescence and multicolor FCM analysis of LN cells from adult female and male H-Y+/ ζ-3 ITAM Tg or H-Y+/ζ-0 ITAM Tg mice. Also shown are LN cells from a nontransgenic C57BL/6 (H-Y−/ζ+/+) mouse. Threecolor FCM was performed on total LN cells after staining with FITC– anti–H-Y clonotypic antibody (T3.70), anti-CD8–PE and anti-CD4– biotin, followed by streptavidin–red 670. CD4 versus CD8 two-color profiles are displayed on total (left) or gated T3.70+ (right) cells. Numbers in the regions reflect the percentage of total cells found in that region.

Mentions: In female H-2Db, H-Y+/ζ+/+ mice, interaction of the H-Y TCR on immature thymocytes with an unidentified ligand results in positive selection and generation of CD8 SP thymocytes that express high surface levels of the H-Y TCR (detected by reactivity with the clonotype-specific antibody, T3.70; reference 24; Fig. 3 A, Table 1 A). Importantly, a phenotype similar to that of H-Y+/ζ+/+ mice was observed in female H-Y+/ζ−/− mice reconstituted with the full-length ζ transgene (H-Y+/ζ-3 ITAM Tg; Fig. 3 A, Table 1 A). These mice also had large thymi that contained a high percentage of T3.70+, CD8 SP cells indicating that positive selection was effectively restored in ζ−/− mice by expression of a transgene-encoded full-length (3 ITAM) ζ chain (Fig. 3). However, in female H-Y+/ζ−/− mice reconstituted with a transgene encoding the signaling-deficient ζ chain (H-Y+/ζ-0 ITAM Tg), positive selection of T3.70+ thymocytes was markedly impaired as evidenced by the extremely low percentage of T3.70+, CD8 SP thymocytes (Fig. 3 A, Table 1 A). Interestingly, H-Y+/ζ−/− females reconstituted with transgenic ζ chains that contain a single ITAM (either the first [membrane proximal] or third [membrane distal]) exhibited an intermediate phenotype (Fig. 3 A). Although there were subtle differences in the two H-Y+/ζ-1 ITAM Tg lines, the percentage of T3.70+, CD8 SP thymocytes in both lines consistently fell between those observed in mice reconstituted with either the ζ-3 ITAM or ζ-0 ITAM transgenes. The direct relationship between the number of ζ chain ITAMs and the generation of clonotypic (T3.70+) CD8 SP thymocytes was even more evident when absolute numbers of T3.70+, CD8+ thymocytes in the various transgenic lines were compared (Table 1 A). Examination of LNs from H-Y+/ζ-0 ITAM Tg females also revealed a lower percentage of T3.70+, CD8+ T cells relative to H-Y+/ζ-3 ITAM Tg females (Fig. 4). A similar relationship between the number of TCR-ζ ITAMs and the efficiency of positive selection was also observed with a class II–restricted TCR-αβ Tg (AND; reference 22; Fig. 5). Together, these results demonstrate that TCR ligand interactions that can generate signals that promote positive selection in the presence of full-length ζ chains are unable to generate these signals in the absence of ζ chain ITAMs.


Role of the multiple T cell receptor (TCR)-zeta chain signaling motifs in selection of the T cell repertoire.

Shores EW, Tran T, Grinberg A, Sommers CL, Shen H, Love PE - J. Exp. Med. (1997)

Analysis of LN T cells from H-Y+/ζ Tg mice. Immunofluorescence and multicolor FCM analysis of LN cells from adult female and  male H-Y+/ ζ-3 ITAM Tg or H-Y+/ζ-0 ITAM Tg mice. Also shown  are LN cells from a nontransgenic C57BL/6 (H-Y−/ζ+/+) mouse. Threecolor FCM was performed on total LN cells after staining with FITC– anti–H-Y clonotypic antibody (T3.70), anti-CD8–PE and anti-CD4– biotin, followed by streptavidin–red 670. CD4 versus CD8 two-color  profiles are displayed on total (left) or gated T3.70+ (right) cells. Numbers  in the regions reflect the percentage of total cells found in that region.
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Figure 4: Analysis of LN T cells from H-Y+/ζ Tg mice. Immunofluorescence and multicolor FCM analysis of LN cells from adult female and male H-Y+/ ζ-3 ITAM Tg or H-Y+/ζ-0 ITAM Tg mice. Also shown are LN cells from a nontransgenic C57BL/6 (H-Y−/ζ+/+) mouse. Threecolor FCM was performed on total LN cells after staining with FITC– anti–H-Y clonotypic antibody (T3.70), anti-CD8–PE and anti-CD4– biotin, followed by streptavidin–red 670. CD4 versus CD8 two-color profiles are displayed on total (left) or gated T3.70+ (right) cells. Numbers in the regions reflect the percentage of total cells found in that region.
Mentions: In female H-2Db, H-Y+/ζ+/+ mice, interaction of the H-Y TCR on immature thymocytes with an unidentified ligand results in positive selection and generation of CD8 SP thymocytes that express high surface levels of the H-Y TCR (detected by reactivity with the clonotype-specific antibody, T3.70; reference 24; Fig. 3 A, Table 1 A). Importantly, a phenotype similar to that of H-Y+/ζ+/+ mice was observed in female H-Y+/ζ−/− mice reconstituted with the full-length ζ transgene (H-Y+/ζ-3 ITAM Tg; Fig. 3 A, Table 1 A). These mice also had large thymi that contained a high percentage of T3.70+, CD8 SP cells indicating that positive selection was effectively restored in ζ−/− mice by expression of a transgene-encoded full-length (3 ITAM) ζ chain (Fig. 3). However, in female H-Y+/ζ−/− mice reconstituted with a transgene encoding the signaling-deficient ζ chain (H-Y+/ζ-0 ITAM Tg), positive selection of T3.70+ thymocytes was markedly impaired as evidenced by the extremely low percentage of T3.70+, CD8 SP thymocytes (Fig. 3 A, Table 1 A). Interestingly, H-Y+/ζ−/− females reconstituted with transgenic ζ chains that contain a single ITAM (either the first [membrane proximal] or third [membrane distal]) exhibited an intermediate phenotype (Fig. 3 A). Although there were subtle differences in the two H-Y+/ζ-1 ITAM Tg lines, the percentage of T3.70+, CD8 SP thymocytes in both lines consistently fell between those observed in mice reconstituted with either the ζ-3 ITAM or ζ-0 ITAM transgenes. The direct relationship between the number of ζ chain ITAMs and the generation of clonotypic (T3.70+) CD8 SP thymocytes was even more evident when absolute numbers of T3.70+, CD8+ thymocytes in the various transgenic lines were compared (Table 1 A). Examination of LNs from H-Y+/ζ-0 ITAM Tg females also revealed a lower percentage of T3.70+, CD8+ T cells relative to H-Y+/ζ-3 ITAM Tg females (Fig. 4). A similar relationship between the number of TCR-ζ ITAMs and the efficiency of positive selection was also observed with a class II–restricted TCR-αβ Tg (AND; reference 22; Fig. 5). Together, these results demonstrate that TCR ligand interactions that can generate signals that promote positive selection in the presence of full-length ζ chains are unable to generate these signals in the absence of ζ chain ITAMs.

Bottom Line: The TCR is composed of four distinct signal transducing subunits (CD3-gamma, -delta, -epsilon, and zeta) that contain either one (CD3-gamma, -delta, -epsilon) or three (-zeta) signaling motifs (ITAMs) within their intracytoplasmic domains.A possible function for multiple TCR ITAMs could be to amplify signals generated by the TCR during selection.A direct relationship was observed between the number of zeta chain ITAMs within the TCR complex and the efficiency of both positive and negative selection.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematologic Products, Food and Drug Administration, Bethesda, Maryland 20892, USA.

ABSTRACT
Immature thymocytes undergo a selection process within the thymus based on their T cell antigen receptor (TCR) specificity that results either in their maturation into functionally competent, self-MHC-restricted T cells (positive selection) or their deletion (negative selection). The outcome of thymocyte selection is thought to be controlled by signals transduced by the TCR that vary in relation to the avidity of the TCR-ligand interaction. The TCR is composed of four distinct signal transducing subunits (CD3-gamma, -delta, -epsilon, and zeta) that contain either one (CD3-gamma, -delta, -epsilon) or three (-zeta) signaling motifs (ITAMs) within their intracytoplasmic domains. A possible function for multiple TCR ITAMs could be to amplify signals generated by the TCR during selection. To determine the importance of the multiple TCR-zeta chain ITAMs in thymocyte selection, transgenes encoding alpha/beta TCRs with known specificity were bred into mice in which zeta chains lacking one or more ITAMs had been genetically substituted for endogenous zeta. A direct relationship was observed between the number of zeta chain ITAMs within the TCR complex and the efficiency of both positive and negative selection. These results reveal a role for multiple TCR ITAMs in thymocyte selection and identify a function for TCR signal amplification in formation of the T cell repertoire.

Show MeSH