Limits...
Function of CD3 epsilon-mediated signals in T cell development.

Sommers CL, Dejarnette JB, Huang K, Lee J, El-Khoury D, Shores EW, Love PE - J. Exp. Med. (2000)

Bottom Line: The T cell antigen receptor (TCR) and pre-TCR complexes are composed of multiple signal-transducing subunits (CD3 gamma, CD3 delta, CD3 epsilon, and zeta) that each contain one or more copies of a semiconserved functional motif, the immunoreceptor tyrosine-based activation motif (ITAM).Although biochemical studies indicate that individual TCR-ITAMs may bind selectively or with different affinity to various effector molecules, data from other experiments suggest that at least some ITAMs are functionally equivalent.The results demonstrate that signals transduced by CD3 epsilon are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling in a manner similar to that previously observed for zeta chain.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The T cell antigen receptor (TCR) and pre-TCR complexes are composed of multiple signal-transducing subunits (CD3 gamma, CD3 delta, CD3 epsilon, and zeta) that each contain one or more copies of a semiconserved functional motif, the immunoreceptor tyrosine-based activation motif (ITAM). Although biochemical studies indicate that individual TCR-ITAMs may bind selectively or with different affinity to various effector molecules, data from other experiments suggest that at least some ITAMs are functionally equivalent. In this study, we examined the role of CD3straightepsilon ITAM-mediated signals in T cell development by genetically reconstituting CD3 epsilon-deficient mice with transgenes encoding either wild-type or ITAM-mutant (signaling defective) forms of the protein. The results demonstrate that signals transduced by CD3 epsilon are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling in a manner similar to that previously observed for zeta chain. Unexpectedly, analysis of TCR-transgenic/CD3 epsilon-mutant mice reveals a potential role for CD3 epsilon signals in T cell survival.

Show MeSH

Related in: MedlinePlus

(A) Analysis of CD3ε and CD3εM protein tyrosine phosphorylation after stimulation of thymocytes with pervanadate. Thymocytes (108) from nontransgenic B6 mice (N.Thy), CD3εΔ/Δ; ε-tg (ε-tg), and CD3εΔ/Δ; εM-tg (εM-tg) mice were incubated for 5 min in medium lacking (−) or containing (+) pervanadate. Cell lysates were immunoprecipitated with anti-CD3ε (mAb 145-2C11). Immunoprecipitated proteins were resolved by 12% SDS-PAGE, followed by antiphosphotyrosine (mAb 4G10) Western blotting (top blot). Blots were subsequently stripped and reprobed with anti-CD3ε (mAb HMT3.1; bottom blot). (B) Genetic reconstitution of mice deficient for ζ and CD3ε (ζ−/− × εΔ/Δ) with transgenes encoding WT or mutant CD3ε proteins. Two-color plots show CD4/CD8 staining profiles on total thymocytes or total lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show CD3 surface staining on total thymocytes or total lymph node cells. Total thymocyte numbers are provided in the histograms. Results are shown for two independently generated εM-tg founder lines with similar transgene copy number.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193290&req=5

Figure 2: (A) Analysis of CD3ε and CD3εM protein tyrosine phosphorylation after stimulation of thymocytes with pervanadate. Thymocytes (108) from nontransgenic B6 mice (N.Thy), CD3εΔ/Δ; ε-tg (ε-tg), and CD3εΔ/Δ; εM-tg (εM-tg) mice were incubated for 5 min in medium lacking (−) or containing (+) pervanadate. Cell lysates were immunoprecipitated with anti-CD3ε (mAb 145-2C11). Immunoprecipitated proteins were resolved by 12% SDS-PAGE, followed by antiphosphotyrosine (mAb 4G10) Western blotting (top blot). Blots were subsequently stripped and reprobed with anti-CD3ε (mAb HMT3.1; bottom blot). (B) Genetic reconstitution of mice deficient for ζ and CD3ε (ζ−/− × εΔ/Δ) with transgenes encoding WT or mutant CD3ε proteins. Two-color plots show CD4/CD8 staining profiles on total thymocytes or total lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show CD3 surface staining on total thymocytes or total lymph node cells. Total thymocyte numbers are provided in the histograms. Results are shown for two independently generated εM-tg founder lines with similar transgene copy number.

Mentions: Based on the results of in vitro studies, the εM transgene is predicted to encode a protein that does not contribute to the TCR signaling response 452021. However, in view of the normal phenotype observed in CD3εΔ/Δ; εM-tg mice, we wished to examine this issue in greater detail in our transgenic system. A hallmark of ITAM function is its ability to become tyrosine phosphorylated upon T cell activation, thereby creating a site for stable association with and activation of ZAP-70 2. After activation with pervanadate, tyrosine phosphorylation of both CD3ε and TCR-ζ is observed in thymocytes from control (CD3ε+/+) and CD3εΔ/Δ; ε-tg mice (Fig. 2 A). In contrast, whereas pervanadate stimulation of thymocytes from CD3εΔ/Δ; εM-tg mice induced tyrosine phosphorylation of TCR ζ chain, tyrosine phosphorylation of the mutated CD3ε chain was nearly undetectable (Fig. 2 A). Similar results were obtained after stimulation of thymocytes by TCR cross-linking (data not shown). Reblotting with anti-CD3ε mAb demonstrated the presence of the mutant CD3ε protein in the immunoprecipitated CD3 complexes (Fig. 2 A). As the proximal ITAM tyrosine is retained in the εM protein, the extremely low level of tyrosine phosphorylation observed after stimulation was unexpected. Interestingly, a TCR ζ chain transgene containing Y→F mutations of the NH2- (but not the COOH-) terminal tyrosine residues in each of its three ITAMs also failed to become phosphorylated in vivo upon T cell activation 9. Recent data suggest that tyrosine phosphorylation of the TCR ζ chain follows a hierarchical progression in which the phosphorylation of specific ITAM tyrosines proceeds and perhaps facilitates phosphorylation of other ITAM tyrosines 22. Our findings may reflect a similar hierarchy for phosphorylation of the CD3ε-ITAM tyrosines. In any event, these results indicate that the εM protein is weakly (if at all) tyrosine phosphorylated upon TCR engagement.


Function of CD3 epsilon-mediated signals in T cell development.

Sommers CL, Dejarnette JB, Huang K, Lee J, El-Khoury D, Shores EW, Love PE - J. Exp. Med. (2000)

(A) Analysis of CD3ε and CD3εM protein tyrosine phosphorylation after stimulation of thymocytes with pervanadate. Thymocytes (108) from nontransgenic B6 mice (N.Thy), CD3εΔ/Δ; ε-tg (ε-tg), and CD3εΔ/Δ; εM-tg (εM-tg) mice were incubated for 5 min in medium lacking (−) or containing (+) pervanadate. Cell lysates were immunoprecipitated with anti-CD3ε (mAb 145-2C11). Immunoprecipitated proteins were resolved by 12% SDS-PAGE, followed by antiphosphotyrosine (mAb 4G10) Western blotting (top blot). Blots were subsequently stripped and reprobed with anti-CD3ε (mAb HMT3.1; bottom blot). (B) Genetic reconstitution of mice deficient for ζ and CD3ε (ζ−/− × εΔ/Δ) with transgenes encoding WT or mutant CD3ε proteins. Two-color plots show CD4/CD8 staining profiles on total thymocytes or total lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show CD3 surface staining on total thymocytes or total lymph node cells. Total thymocyte numbers are provided in the histograms. Results are shown for two independently generated εM-tg founder lines with similar transgene copy number.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193290&req=5

Figure 2: (A) Analysis of CD3ε and CD3εM protein tyrosine phosphorylation after stimulation of thymocytes with pervanadate. Thymocytes (108) from nontransgenic B6 mice (N.Thy), CD3εΔ/Δ; ε-tg (ε-tg), and CD3εΔ/Δ; εM-tg (εM-tg) mice were incubated for 5 min in medium lacking (−) or containing (+) pervanadate. Cell lysates were immunoprecipitated with anti-CD3ε (mAb 145-2C11). Immunoprecipitated proteins were resolved by 12% SDS-PAGE, followed by antiphosphotyrosine (mAb 4G10) Western blotting (top blot). Blots were subsequently stripped and reprobed with anti-CD3ε (mAb HMT3.1; bottom blot). (B) Genetic reconstitution of mice deficient for ζ and CD3ε (ζ−/− × εΔ/Δ) with transgenes encoding WT or mutant CD3ε proteins. Two-color plots show CD4/CD8 staining profiles on total thymocytes or total lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show CD3 surface staining on total thymocytes or total lymph node cells. Total thymocyte numbers are provided in the histograms. Results are shown for two independently generated εM-tg founder lines with similar transgene copy number.
Mentions: Based on the results of in vitro studies, the εM transgene is predicted to encode a protein that does not contribute to the TCR signaling response 452021. However, in view of the normal phenotype observed in CD3εΔ/Δ; εM-tg mice, we wished to examine this issue in greater detail in our transgenic system. A hallmark of ITAM function is its ability to become tyrosine phosphorylated upon T cell activation, thereby creating a site for stable association with and activation of ZAP-70 2. After activation with pervanadate, tyrosine phosphorylation of both CD3ε and TCR-ζ is observed in thymocytes from control (CD3ε+/+) and CD3εΔ/Δ; ε-tg mice (Fig. 2 A). In contrast, whereas pervanadate stimulation of thymocytes from CD3εΔ/Δ; εM-tg mice induced tyrosine phosphorylation of TCR ζ chain, tyrosine phosphorylation of the mutated CD3ε chain was nearly undetectable (Fig. 2 A). Similar results were obtained after stimulation of thymocytes by TCR cross-linking (data not shown). Reblotting with anti-CD3ε mAb demonstrated the presence of the mutant CD3ε protein in the immunoprecipitated CD3 complexes (Fig. 2 A). As the proximal ITAM tyrosine is retained in the εM protein, the extremely low level of tyrosine phosphorylation observed after stimulation was unexpected. Interestingly, a TCR ζ chain transgene containing Y→F mutations of the NH2- (but not the COOH-) terminal tyrosine residues in each of its three ITAMs also failed to become phosphorylated in vivo upon T cell activation 9. Recent data suggest that tyrosine phosphorylation of the TCR ζ chain follows a hierarchical progression in which the phosphorylation of specific ITAM tyrosines proceeds and perhaps facilitates phosphorylation of other ITAM tyrosines 22. Our findings may reflect a similar hierarchy for phosphorylation of the CD3ε-ITAM tyrosines. In any event, these results indicate that the εM protein is weakly (if at all) tyrosine phosphorylated upon TCR engagement.

Bottom Line: The T cell antigen receptor (TCR) and pre-TCR complexes are composed of multiple signal-transducing subunits (CD3 gamma, CD3 delta, CD3 epsilon, and zeta) that each contain one or more copies of a semiconserved functional motif, the immunoreceptor tyrosine-based activation motif (ITAM).Although biochemical studies indicate that individual TCR-ITAMs may bind selectively or with different affinity to various effector molecules, data from other experiments suggest that at least some ITAMs are functionally equivalent.The results demonstrate that signals transduced by CD3 epsilon are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling in a manner similar to that previously observed for zeta chain.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
The T cell antigen receptor (TCR) and pre-TCR complexes are composed of multiple signal-transducing subunits (CD3 gamma, CD3 delta, CD3 epsilon, and zeta) that each contain one or more copies of a semiconserved functional motif, the immunoreceptor tyrosine-based activation motif (ITAM). Although biochemical studies indicate that individual TCR-ITAMs may bind selectively or with different affinity to various effector molecules, data from other experiments suggest that at least some ITAMs are functionally equivalent. In this study, we examined the role of CD3straightepsilon ITAM-mediated signals in T cell development by genetically reconstituting CD3 epsilon-deficient mice with transgenes encoding either wild-type or ITAM-mutant (signaling defective) forms of the protein. The results demonstrate that signals transduced by CD3 epsilon are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling in a manner similar to that previously observed for zeta chain. Unexpectedly, analysis of TCR-transgenic/CD3 epsilon-mutant mice reveals a potential role for CD3 epsilon signals in T cell survival.

Show MeSH
Related in: MedlinePlus