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. 1997 Nov 17;186(10):1769-74.
doi: 10.1084/jem.186.10.1769.

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

I Carena  1 , A Shamshiev, A Donda, M Colonna, G D Libero
Affiliations

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

I Carena et al. J Exp Med. .

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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Figures

Figure 1
Figure 1
Distribution of inhibitory receptors on γ/δ clones derived from PBMC, thymus, and gut. Immunofluorescence analyses were performed on 40 clones from PBMC, 7 from thymus, and 12 from intestine using anti-CD94, and anti-p58.1, p58.2, p70, and p140 KIR mAbs (see Materials and Methods). The area of each pie is proportional to the number of clones reactive with CD94 or KIR-specific antibodies. Clones reactive with at least one KIR Ab were grouped and indicated as KIR+.
Figure 2
Figure 2
Surface expression of IR molecules on freshly isolated γ/δ T cells. PBMCs from nine donors were stained with anti–pan-γ/δ mAbs together with two anti-IR mAbs, and three-color analyses were performed. The figure presents the percentage of γ/δ T cells reactive with Q66 (anti-p140) and HP3B1 (anti-CD94, A) or with HP3E4 (anti-p58.1) and HP3B1 (anti-CD94, B). Bars indicate medians and ranges of percentage of total γ/δ T cells.
Figure 3
Figure 3
TNF-α release is highly influenced by HLA class I+ APCs when TCR-γ/δ is stimulated with low doses, but not high doses, of IPP. Two CD94+ clones, G2B2 (A and C) and D1C55 (B and D) were stimulated with increasing amounts of IPP in the presence of normal (□) or Daudi-β2 cells (○). Anti-CD94 (▵), but not an isotype-matched irrelevant mAb (○), partially restores TNF-α release in the presence of Daudi-β2 cells (C and D). Similar results were obtained in 14 independent experiments using these two clones and four other CD94+ γ/δ clones, derived from different donors. The amount of TNF-α released by a given clone in any given experiment was different and correlated with its activation state. Daudi cells do not release detectable TNF-α when incubated with IPP (not shown). Bars indicate SD.
Figure 4
Figure 4
TCR-γ/δ stimulation with IPP in the presence of HLA class I+ APC recruits increased amounts of SHP-1 phosphatase to CD94 and to TCR–CD3 complex and causes tyrosine hypophosphorylation. (A) Western blot performed with anti–SHP-1 Abs after immunoprecipitations carried out with anti-CD94 (lanes 2 and 3) or an isotype-matched irrelevant mAb (lane 1) from γ/δ T cells stimulated with IPP in the presence of Daudi (lanes 1 and 2) or Daudi-β2 APCs (lane 3). (B) Anti–SHP-1 Western blot after immunoprecipitation with anti-CD3 ζ chain mAbs. (C) Protein tyrosine phosphorylation of total cell lysates is visualized by immunoblotting with antiphosphotyrosine 4G10 mAb. Cells were lysed with 1% digitonin (A and B) or with 1% NP-40 (C). Molecular mass markers are indicated on the right in kilodaltons. Arrows indicate SHP-1 in A and B, and two hypophosphorylated proteins migrating at ∼70 and 130 kD in C. H, Ig heavy chain of immunoprecipitating Abs.
Figure 5
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.
Figure 6
Figure 6
Association of Lck but not of ZAP-70 with CD3 ζ chain is diminished by engagement of CD94. γ/δ T cells were stimulated as described in Fig. 5, lysed in 1% digitonin, and immunoprecipitated with anti-CD3 ζ chain mAbs. Precipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-Lck mAb (A). Arrows indicate the position of p56 and p60 Lck. The blot was then stripped and reblotted with anti–ZAP-70 mAbs (B). The arrow indicates ZAP-70. Molecular mass markers are shown at the right side of the figure in kilodaltons. H, Heavy chain of immunoprecipitating Abs.
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