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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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Role of TCRζ–mediated signals in thymocyte  positive and negative selection.  (A) Phenotype of thymocytes  from H-Y+/ζ−/−; ζ Tg female  mice. (B) Phenotype of thymocytes from H-Y+/ζ−/−; ζ Tg  male mice. H-Y+/ζ−/− mice  were reconstituted with transgenes encoding either full-length  ζ chains (ζ-3 ITAM Tg), ζ  chains that contain a single  ITAM (either the first or third,  ζ-1 ITAM Tg) or ζ chains that  lack ITAMs (ζ-0 ITAM Tg).  Data show immunofluorescence  and multicolor FCM analysis of  thymocytes from adult H-2Db  mice. Three-color FCM was  performed on cells stained with  anti–H-Y clonotypic antibody  (T3.70) conjugated to FITC,  anti-CD8–PE, and anti-CD4– biotin, followed by streptavidin– red 670 (16). Two-color plots  show total thymocytes or software-gated T3.70+ thymocytes.  Numbers in the quadrants reflect  the percentage of total thymocytes in that quadrant. Singlecolor profiles (solid lines) depict  T3.70 staining on either  CD4+CD8+ thymocytes (A) or  total thymocytes (B). Dotted  lines reflect staining with negative control antibody.
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Figure 3: Role of TCRζ–mediated signals in thymocyte positive and negative selection. (A) Phenotype of thymocytes from H-Y+/ζ−/−; ζ Tg female mice. (B) Phenotype of thymocytes from H-Y+/ζ−/−; ζ Tg male mice. H-Y+/ζ−/− mice were reconstituted with transgenes encoding either full-length ζ chains (ζ-3 ITAM Tg), ζ chains that contain a single ITAM (either the first or third, ζ-1 ITAM Tg) or ζ chains that lack ITAMs (ζ-0 ITAM Tg). Data show immunofluorescence and multicolor FCM analysis of thymocytes from adult H-2Db mice. Three-color FCM was performed on cells stained with anti–H-Y clonotypic antibody (T3.70) conjugated to FITC, anti-CD8–PE, and anti-CD4– biotin, followed by streptavidin– red 670 (16). Two-color plots show total thymocytes or software-gated T3.70+ thymocytes. Numbers in the quadrants reflect the percentage of total thymocytes in that quadrant. Singlecolor profiles (solid lines) depict T3.70 staining on either CD4+CD8+ thymocytes (A) or total thymocytes (B). Dotted lines reflect staining with negative control antibody.

Mentions: Examination of ζ−/− mice that express the H-Y transgene revealed that the developmental impairment observed previously in ζ−/− mice was also observed in both male and female H-Y+/ζ−/− mice (Fig. 2). These results were expected because of the extremely low level of TCR surface expression in the absence of ζ chain (11–14) and are consistent with a requirement for the TCR for normal thymocyte development and selection. To address the importance of the ζ chain ITAMs in thymocyte selection, we next examined H-Y+/ζ−/− mice reconstituted with transgenes encoding either full-length ζ chains (H-Y+/ζ-3 ITAM Tg mice), ζ chains containing a single ITAM (H-Y+/ζ-1 ITAM Tg mice), or ζ chains lacking all three ITAMs (H-Y+/ζ- 0 ITAM Tg mice) (15). Each of the ζ transgenes was capable of restoring TCR surface expression in mice lacking endogenous ζ chain (Fig. 3; 15). For these experiments transgenic founder lines were chosen that gave comparable levels of TCR surface expression that were equal to or greater than that found on thymocytes from ζ+/+ mice (Fig. 3; 15).


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)

Role of TCRζ–mediated signals in thymocyte  positive and negative selection.  (A) Phenotype of thymocytes  from H-Y+/ζ−/−; ζ Tg female  mice. (B) Phenotype of thymocytes from H-Y+/ζ−/−; ζ Tg  male mice. H-Y+/ζ−/− mice  were reconstituted with transgenes encoding either full-length  ζ chains (ζ-3 ITAM Tg), ζ  chains that contain a single  ITAM (either the first or third,  ζ-1 ITAM Tg) or ζ chains that  lack ITAMs (ζ-0 ITAM Tg).  Data show immunofluorescence  and multicolor FCM analysis of  thymocytes from adult H-2Db  mice. Three-color FCM was  performed on cells stained with  anti–H-Y clonotypic antibody  (T3.70) conjugated to FITC,  anti-CD8–PE, and anti-CD4– biotin, followed by streptavidin– red 670 (16). Two-color plots  show total thymocytes or software-gated T3.70+ thymocytes.  Numbers in the quadrants reflect  the percentage of total thymocytes in that quadrant. Singlecolor profiles (solid lines) depict  T3.70 staining on either  CD4+CD8+ thymocytes (A) or  total thymocytes (B). Dotted  lines reflect staining with negative control antibody.
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Figure 3: Role of TCRζ–mediated signals in thymocyte positive and negative selection. (A) Phenotype of thymocytes from H-Y+/ζ−/−; ζ Tg female mice. (B) Phenotype of thymocytes from H-Y+/ζ−/−; ζ Tg male mice. H-Y+/ζ−/− mice were reconstituted with transgenes encoding either full-length ζ chains (ζ-3 ITAM Tg), ζ chains that contain a single ITAM (either the first or third, ζ-1 ITAM Tg) or ζ chains that lack ITAMs (ζ-0 ITAM Tg). Data show immunofluorescence and multicolor FCM analysis of thymocytes from adult H-2Db mice. Three-color FCM was performed on cells stained with anti–H-Y clonotypic antibody (T3.70) conjugated to FITC, anti-CD8–PE, and anti-CD4– biotin, followed by streptavidin– red 670 (16). Two-color plots show total thymocytes or software-gated T3.70+ thymocytes. Numbers in the quadrants reflect the percentage of total thymocytes in that quadrant. Singlecolor profiles (solid lines) depict T3.70 staining on either CD4+CD8+ thymocytes (A) or total thymocytes (B). Dotted lines reflect staining with negative control antibody.
Mentions: Examination of ζ−/− mice that express the H-Y transgene revealed that the developmental impairment observed previously in ζ−/− mice was also observed in both male and female H-Y+/ζ−/− mice (Fig. 2). These results were expected because of the extremely low level of TCR surface expression in the absence of ζ chain (11–14) and are consistent with a requirement for the TCR for normal thymocyte development and selection. To address the importance of the ζ chain ITAMs in thymocyte selection, we next examined H-Y+/ζ−/− mice reconstituted with transgenes encoding either full-length ζ chains (H-Y+/ζ-3 ITAM Tg mice), ζ chains containing a single ITAM (H-Y+/ζ-1 ITAM Tg mice), or ζ chains lacking all three ITAMs (H-Y+/ζ- 0 ITAM Tg mice) (15). Each of the ζ transgenes was capable of restoring TCR surface expression in mice lacking endogenous ζ chain (Fig. 3; 15). For these experiments transgenic founder lines were chosen that gave comparable levels of TCR surface expression that were equal to or greater than that found on thymocytes from ζ+/+ mice (Fig. 3; 15).

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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