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Major histocompatibility complex class I molecules modulate activation threshold and early signaling of T cell antigen receptor-gamma/delta stimulated by nonpeptidic ligands.

Carena I, Shamshiev A, Donda A, Colonna M, Libero GD - J. Exp. Med. (1997)

Bottom Line: CD94-mediated inhibition is more effective at low than at high doses of TCR ligand, which may focus T cell responses towards antigen-presenting cells presenting high amounts of antigen.CD94 engagement has major effects on TCR signaling cascade.These events may cause abortion of proximal TCR-mediated signaling and set a higher TCR activation threshold.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology, Department of Research, University Hospital, Basel, Switzerland.

ABSTRACT
Killer cell inhibitory receptors and CD94-NKG2-A/B heterodimers are major histocompatibility complex class I-specific inhibitory receptors expressed by natural killer cells, T cell antigen receptor (TCR)-gamma/delta cells, and a subset of TCR-alpha/beta cells. We studied the functional interaction between TCR-gamma/delta and CD94, this inhibitory receptor being expressed on the majority of gamma/delta T cells. When engaged by human histocompatibility leukocyte antigen class I molecules, CD94 downmodulates activation of human TCR-gamma/delta by phosphorylated ligands. CD94-mediated inhibition is more effective at low than at high doses of TCR ligand, which may focus T cell responses towards antigen-presenting cells presenting high amounts of antigen. CD94 engagement has major effects on TCR signaling cascade. It facilitates recruitment of SHP-1 phosphatase to TCR-CD3 complex and affects phosphorylation of Lck and ZAP-70 kinase, but not of CD3 zeta chain upon TCR triggering. These events may cause abortion of proximal TCR-mediated signaling and set a higher TCR activation threshold.

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Tyrosine phosphorylation is affected in CD94+ γ/δ T cells  stimulated by IPP in the presence of HLA class I+ APCs. D1C55 cells  were stimulated with IPP in the presence of normal or Daudi-β2 cells. As  control, D1C55 or Daudi cells alone were incubated with IPP. Immunoprecipitations were carried out with anti–ZAP-70 (A and B), or anti-CD3  ζ chain mAbs (C). Cells were lysed with 1% NP-40 and precipitated proteins were resolved by SDS-PAGE and immunoblotted with HRP-conjugated antiphosphotyrosine 4G10 mAbs. The blot of anti–ZAP-70 immunoprecipitation was stripped and reblotted with anti–ZAP-70 mAbs  (B). Arrows indicate ZAP-70 (A and B) and p21 CD3 ζ chain (C). H,  Heavy chain of immunoprecipitating Abs. The amount of proteins in A  and B were estimated by scanning densitometry analysis.
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Figure 5: Tyrosine phosphorylation is affected in CD94+ γ/δ T cells stimulated by IPP in the presence of HLA class I+ APCs. D1C55 cells were stimulated with IPP in the presence of normal or Daudi-β2 cells. As control, D1C55 or Daudi cells alone were incubated with IPP. Immunoprecipitations were carried out with anti–ZAP-70 (A and B), or anti-CD3 ζ chain mAbs (C). Cells were lysed with 1% NP-40 and precipitated proteins were resolved by SDS-PAGE and immunoblotted with HRP-conjugated antiphosphotyrosine 4G10 mAbs. The blot of anti–ZAP-70 immunoprecipitation was stripped and reblotted with anti–ZAP-70 mAbs (B). Arrows indicate ZAP-70 (A and B) and p21 CD3 ζ chain (C). H, Heavy chain of immunoprecipitating Abs. The amount of proteins in A and B were estimated by scanning densitometry analysis.

Mentions: To better characterize which components of TCR signaling are affected by CD94 engagement with HLA class I molecules, γ/δ T cells were stimulated with IPP in the presence of Daudi or Daudi-β2 APCs, and then ZAP-70 and the CD3 ζ chain were individually precipitated and revealed using antiphosphotyrosine mAbs (Fig. 5). In additional experiments, the CD3–TCR complex was precipitated with anti-CD3 ζ chain mAb and the coprecipitated proteins revealed with specific antibodies (Fig. 6). These studies showed that when both the TCR and CD94 are engaged, ZAP-70 is hypophosphorylated (Fig. 5, A and B), although similar amounts are associated with the CD3–TCR complex (Fig. 6 B). In the same conditions, the lck form migrating at 60 kD is less abundant (Fig. 6 A), whereas the CD3 ζ chain is normally phosphorylated (Fig. 5 C). Thus, CD94 interaction with HLA class I molecules causes hypophosphorylation of ZAP-70, but not of the CD3 ζ chain. This finding is different from what has been observed with NK cell clones stimulated with anti-FcγRIII mAbs. In this case inhibition by anti-p70 KIR mAbs leads to CD3 ζ dephosphorylation (28). This discrepancy might be due to the used stimuli or to the type of receptors analyzed in the two studies (FcγR and p70 KIR versus TCR-γ/δ and CD94).


Major histocompatibility complex class I molecules modulate activation threshold and early signaling of T cell antigen receptor-gamma/delta stimulated by nonpeptidic ligands.

Carena I, Shamshiev A, Donda A, Colonna M, Libero GD - J. Exp. Med. (1997)

Tyrosine phosphorylation is affected in CD94+ γ/δ T cells  stimulated by IPP in the presence of HLA class I+ APCs. D1C55 cells  were stimulated with IPP in the presence of normal or Daudi-β2 cells. As  control, D1C55 or Daudi cells alone were incubated with IPP. Immunoprecipitations were carried out with anti–ZAP-70 (A and B), or anti-CD3  ζ chain mAbs (C). Cells were lysed with 1% NP-40 and precipitated proteins were resolved by SDS-PAGE and immunoblotted with HRP-conjugated antiphosphotyrosine 4G10 mAbs. The blot of anti–ZAP-70 immunoprecipitation was stripped and reblotted with anti–ZAP-70 mAbs  (B). Arrows indicate ZAP-70 (A and B) and p21 CD3 ζ chain (C). H,  Heavy chain of immunoprecipitating Abs. The amount of proteins in A  and B were estimated by scanning densitometry analysis.
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Related In: Results  -  Collection

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Figure 5: Tyrosine phosphorylation is affected in CD94+ γ/δ T cells stimulated by IPP in the presence of HLA class I+ APCs. D1C55 cells were stimulated with IPP in the presence of normal or Daudi-β2 cells. As control, D1C55 or Daudi cells alone were incubated with IPP. Immunoprecipitations were carried out with anti–ZAP-70 (A and B), or anti-CD3 ζ chain mAbs (C). Cells were lysed with 1% NP-40 and precipitated proteins were resolved by SDS-PAGE and immunoblotted with HRP-conjugated antiphosphotyrosine 4G10 mAbs. The blot of anti–ZAP-70 immunoprecipitation was stripped and reblotted with anti–ZAP-70 mAbs (B). Arrows indicate ZAP-70 (A and B) and p21 CD3 ζ chain (C). H, Heavy chain of immunoprecipitating Abs. The amount of proteins in A and B were estimated by scanning densitometry analysis.
Mentions: To better characterize which components of TCR signaling are affected by CD94 engagement with HLA class I molecules, γ/δ T cells were stimulated with IPP in the presence of Daudi or Daudi-β2 APCs, and then ZAP-70 and the CD3 ζ chain were individually precipitated and revealed using antiphosphotyrosine mAbs (Fig. 5). In additional experiments, the CD3–TCR complex was precipitated with anti-CD3 ζ chain mAb and the coprecipitated proteins revealed with specific antibodies (Fig. 6). These studies showed that when both the TCR and CD94 are engaged, ZAP-70 is hypophosphorylated (Fig. 5, A and B), although similar amounts are associated with the CD3–TCR complex (Fig. 6 B). In the same conditions, the lck form migrating at 60 kD is less abundant (Fig. 6 A), whereas the CD3 ζ chain is normally phosphorylated (Fig. 5 C). Thus, CD94 interaction with HLA class I molecules causes hypophosphorylation of ZAP-70, but not of the CD3 ζ chain. This finding is different from what has been observed with NK cell clones stimulated with anti-FcγRIII mAbs. In this case inhibition by anti-p70 KIR mAbs leads to CD3 ζ dephosphorylation (28). This discrepancy might be due to the used stimuli or to the type of receptors analyzed in the two studies (FcγR and p70 KIR versus TCR-γ/δ and CD94).

Bottom Line: CD94-mediated inhibition is more effective at low than at high doses of TCR ligand, which may focus T cell responses towards antigen-presenting cells presenting high amounts of antigen.CD94 engagement has major effects on TCR signaling cascade.These events may cause abortion of proximal TCR-mediated signaling and set a higher TCR activation threshold.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology, Department of Research, University Hospital, Basel, Switzerland.

ABSTRACT
Killer cell inhibitory receptors and CD94-NKG2-A/B heterodimers are major histocompatibility complex class I-specific inhibitory receptors expressed by natural killer cells, T cell antigen receptor (TCR)-gamma/delta cells, and a subset of TCR-alpha/beta cells. We studied the functional interaction between TCR-gamma/delta and CD94, this inhibitory receptor being expressed on the majority of gamma/delta T cells. When engaged by human histocompatibility leukocyte antigen class I molecules, CD94 downmodulates activation of human TCR-gamma/delta by phosphorylated ligands. CD94-mediated inhibition is more effective at low than at high doses of TCR ligand, which may focus T cell responses towards antigen-presenting cells presenting high amounts of antigen. CD94 engagement has major effects on TCR signaling cascade. It facilitates recruitment of SHP-1 phosphatase to TCR-CD3 complex and affects phosphorylation of Lck and ZAP-70 kinase, but not of CD3 zeta chain upon TCR triggering. These events may cause abortion of proximal TCR-mediated signaling and set a higher TCR activation threshold.

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