Document Detail

Major histocompatibility complex class I molecules modulate activation threshold and early signaling of T cell antigen receptor-gamma/delta stimulated by nonpeptidic ligands.
Jump to Full Text
MedLine Citation:
PMID:  9362537     Owner:  NLM     Status:  MEDLINE    
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.
I Carena; A Shamshiev; A Donda; M Colonna; G D Libero
Related Documents :
20038637 - Development of promyelocytic zinc finger and thpok-expressing innate gamma delta t cell...
16203967 - {gamma}{delta} t cell homeostasis is established in competition with {alpha}{beta} t ce...
10415017 - Functional similarity and differences between selection-independent cd4-cd8- alphabeta ...
12517927 - Cutting edge: homeostatic proliferation of peripheral t lymphocytes is regulated by clo...
16751137 - Cellular imaging and surface marker labeling of hematopoietic cells using quantum dot b...
10214867 - Expressions of four major protein ser/thr phosphatases in human primary leukemic cells.
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of experimental medicine     Volume:  186     ISSN:  0022-1007     ISO Abbreviation:  J. Exp. Med.     Publication Date:  1997 Nov 
Date Detail:
Created Date:  1997-12-03     Completed Date:  1997-12-03     Revised Date:  2012-06-05    
Medline Journal Info:
Nlm Unique ID:  2985109R     Medline TA:  J Exp Med     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1769-74     Citation Subset:  IM    
Experimental Immunology, Department of Research, University Hospital, Basel, Switzerland.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Antigens, CD / physiology
Cell Line
Clone Cells
HLA Antigens / physiology*
Histocompatibility Antigens Class I / physiology*
Intracellular Signaling Peptides and Proteins
Lectins, C-Type*
Lymphocyte Activation*
Macromolecular Substances
Membrane Glycoproteins / physiology
NK Cell Lectin-Like Receptor Subfamily D
Peptides / immunology,  physiology*
Protein Tyrosine Phosphatase, Non-Receptor Type 11
Protein Tyrosine Phosphatase, Non-Receptor Type 6
Protein Tyrosine Phosphatases / metabolism
Protein-Tyrosine Kinases / metabolism
Receptor-CD3 Complex, Antigen, T-Cell / metabolism
Receptors, Antigen, T-Cell, gamma-delta / drug effects,  metabolism,  physiology*
Receptors, Immunologic / biosynthesis
Receptors, KIR
Signal Transduction / immunology*
T-Lymphocyte Subsets / metabolism
Tyrosine / metabolism
ZAP-70 Protein-Tyrosine Kinase
Reg. No./Substance:
0/Antigens, CD; 0/HLA Antigens; 0/Histocompatibility Antigens Class I; 0/Intracellular Signaling Peptides and Proteins; 0/KLRD1 protein, human; 0/Lectins, C-Type; 0/Ligands; 0/Macromolecular Substances; 0/Membrane Glycoproteins; 0/NK Cell Lectin-Like Receptor Subfamily D; 0/Peptides; 0/Receptor-CD3 Complex, Antigen, T-Cell; 0/Receptors, Antigen, T-Cell, gamma-delta; 0/Receptors, Immunologic; 0/Receptors, KIR; 55520-40-6/Tyrosine; EC Kinases; EC Protein-Tyrosine Kinase; EC protein, human; EC protein, human; EC protein, human; EC Tyrosine Phosphatase, Non-Receptor Type 11; EC Tyrosine Phosphatase, Non-Receptor Type 6; EC Tyrosine Phosphatases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): J Exp Med
ISSN: 0022-1007
ISSN: 1540-9538
Publisher: The Rockefeller University Press
Article Information
Download PDF
Copyright: 1997
Received Day: 13 Month: 5 Year: 1997
Revision Received Day: 8 Month: 9 Year: 1997
Print publication date: Day: 17 Month: 11 Year: 1997
Volume: 186 Issue: 10
First Page: 1769 Last Page: 1774
ID: 2199143
PubMed Id: 9362537

Major Histocompatibility Complex Class I Molecules Modulate Activation Threshold and Early Signaling of T Cell Antigen Receptor–γ/δ Stimulated by Nonpeptidic Ligands
Ilaria Carena*
Abdijapar Shamshiev*
Alena Donda*
Marco Colonna
Gennaro De Libero*
From *Experimental Immunology, Department of Research, University Hospital, CH-4031 Basel, Switzerland; and Basel Institute for Immunology, CH-4005 Basel, Switzerland
Address correspondence to Dr. Gennaro De Libero, Experimental Immunology, Department of Research, University Hospital, Hebelstrasse 20, CH-4031 Basel, Switzerland. Phone: 41-61-265-2327; FAX: 41-61-265-2350.

Activation of NK cells is modulated by inhibitory receptors that interact with MHC class I molecules. The MHC class I–binding receptors currently identified have been assigned to two distinct groups that belong to the Ig (killer inhibitory receptors; KIR) and C-type lectin superfamilies (CD94), which here are collectively referred to as inhibitory receptors (IR). The p58.1, p58.2, p70, and the p70/140 KIRs recognize different supertypic epitopes of HLA-C, HLA-B, and HLA-A isoforms (for reviews see references 14). The second type of IR includes heterodimers of CD94 covalently associated with NKG2-A or NKG2-B molecules (5, 6). Both CD94 and NKG2 molecules are type II membrane glycoproteins that belong to the superfamily of C-type lectins. These receptors have a broader specificity in that they recognize different HLA-A, -B, and -C alleles (for review see reference 2).

KIRs inhibit cell triggering through recruitment and activation of intracellular phosphatases (for reviews see references 7 and 8), via a mechanism similar to that regulating B cells (9). The natural substrates of these phosphatases are not known and it is believed that they inhibit proximal tyrosine kinase activation (7, 8).

Surface expression of KIRs is not limited to NK cells, since T cells with TCR-α/β or -γ/δ are also KIR+. Only a limited number of TCR-α/β cells express p58.1, p58.2, and p70 KIR (1012), and preferential expression of KIRs has been reported on CD45RO+ CD29+ memory cells (13). Based on these findings, it was proposed that KIRs are expressed by T cells after chronic cell activation and after generation of long-lived memory cells (2). It has also recently been reported that p70 KIR may inhibit activation of TCR-α/β by bacterial superantigens (11, 14) and by melanoma antigen (15).

Little is known about the functional role of IR on TCR-γ/δ T cells. Phenotypic studies showed that ∼60% of circulating γ/δ T cells are CD94+ (16). In contrast, expression of the p58 and p70 KIRs on γ/δ T cells is rare (12, 14, 17, 18). This study shows that CD94 finely downmodulates TCR-γ/δ activation induced by phosphorylated metabolites (1921), as γ/δ T cell response is severely impaired in the presence of low, but not high, doses of ligand. This regulation correlates with increased recruitment of SHP-1 to CD94 and to the TCR–CD3 complex, with reduced Lck–TCR–CD3 complex association, and with ZAP-70 kinase hypophosphorylation.

Materials and Methods
T Cell Clones, Lines, and Phosphorylated Ligands.

T cell lines and clones were established and maintained as previously described (22). Isopentenylpyrophosphate (IPP) was purchased from [companySigma Chemical Co.] (Buchs, Switzerland). Daudi cells were purchased from the American Type Culture Collection (Rockville, MD), and β2-microglobulin gene–transfected Daudi cells (Daudi-β2) were provided by Dr. J. Parnes (Stanford University, Stanford, CA; reference 23).

Immunofluorescence Analysis.

The following mAbs were used: B1 (IgG1) anti-pan TCR-γ/δ; B3 (IgG1) anti-Vγ9; 4A11 (IgG1) anti-Vγ4; 4G6 (IgG1) anti-Vδ2 (all characterized in our laboratory); δTCS1 (IgG1) anti-Vδ1-Jδ1/2 (Endogen, Boston, MA); P11.5B (IgG1) anti-Vδ3 (provided by Dr. M. Bonneville, INSERM Unité 211, Nantes, France); Q66 (IgM) anti-p140 (provided by Dr. L. Moretta, Laboratorio di Immunopatologia, Genova, Italy); GL183 (IgG1) anti-p58.2; HP3B1 (IgG2a) anti-CD94 (Immunotech, Marseilles, France); 5.133 (IgG1) anti-p140 and p70 (24); and HP3E4 (IgM) anti-p58.1 (provided by Dr. M. López-Botet, Hospital de la Princesa, Madrid, Spain). Three-color immunofluorescence analysis was performed using anti-TCR mAbs together with two anti-IR mAbs, when the isotype of the antibodies could be combined. First antibodies were revealed with mouse Ig isotype-specific FITC-, PE-, or biotin-labeled goat antisera (Southern Biotechnology Associates, Birmingham, AL) and streptavidin-3 color (Caltag, San Francisco, CA). A FACScan® flow cytometer with the Lysis II program was used for acquisition and analysis of data.

Cytokine Release Assays.

T cells (5 × 104/well) were stimulated by phosphorylated ligands in duplicate cultures in flat-bottomed 96-well plates in the presence of different types of APC (5 × 104/well). After 18 h of incubation, 100 μl of supernatant was removed and used to test the content of TNF-α by ELISA using commercial kits (Endotell, Bottmingen, Switzerland).

Killing Assays.

Cytotoxic assays were performed as previously described (25).

Immunoprecipitation and Western Blot Analyses.

The following mAbs were used for immunoprecipitation and immunoblotting analysis: anti-CD3 ζ chain; anti-Lck and anti–SHP-1 antibodies ([companySanta Cruz Biotechnology], Santa Cruz, CA); anti–ZAP-70 and antiphosphotyrosine 4G10 (Upstate Biotechnology, Lake Placid, NY); and H146-968 hamster mAbs which recognize human CD3 ζ chain (a gift of Dr. R. Kubo, Cytel Corp., San Diego, CA). Immunoprecipitation was performed using γ/δ T cells (5 × 106/ml) incubated with Daudi cells (8 × 106/ml) in the presence of IPP at 37°C for 30 min. To better detect the inhibitory effects of CD94 on γ/δ TCR, a suboptimal dose of IPP (10 μM) was used. Cells were washed twice in cold PBS at 4°C and then maintained for 40 min at 4°C in lysis buffer (1% digitonin, or 1% NP-40, 150 mM NaCl, 20 mM Tris-HCl, pH 7.2, containing 1 mM sodium orthovanadate, 2 mM EDTA, 1 mM NaF, 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). After centrifugation at 10,000 g for 15 min, supernatants were precleared with protein G–Sepharose beads ([companyPharmacia Biotech] Europe, Dubendorf, Germany), and then with mouse Ig coupled to protein G–Sepharose, and then supernatants were incubated with indicated mAb–protein G–Sepharose at 4°C for 3 h. Immunoprecipitates were washed three times with 1 ml of 0.3% digitonin or NP-40 buffer and solubilized in Laemmli's sample buffer. Immunoblot analysis was performed as previously described (25). The presence of NKG2A was confirmed by PCR amplification and by detection of an ∼44-kD band with an anti-NKG2A antiserum in anti-CD94 immunoprecipitates (data not shown).

Results and Discussion
The Majority of Human TCR-γ/δ Cells Express at least One IR.

Using five different IR-specific mAbs, analysis of IR expression was performed on a panel of γ/δ T cell clones isolated from different donors and tissues. As shown in Fig. 1, most of the γ/δ clones derived from peripheral blood were positive for at least one mAb, thus demonstrating that, in contrast to what has been reported for αβ T cells (13, 16), IR are often expressed on the surface of γ/δ T cells. CD94 was the most frequently expressed IR, with 27 γ/δ clones positive out of the 40 tested. A smaller number of clones (15 out of 40) were p58.1, p58.2, p70, or p140 KIRs positive. Since all clones were activated under the same conditions and CD94 clones maintained a stable phenotype, it is unlikely that CD94 expression was induced during culture. Phenotypic comparison of clones bearing different types of Vγ/Vδ pairs revealed the tendency for Vγ9 (TCRVG2S1)/Vδ2 (TCRDV102S1) cells to be CD94+ more frequently than cells using other Vγ and Vδ chains, without reaching a statistically significant difference. The six CD4+ γ/δ clones tested were negative for all the anti-KIR mAbs (data not shown). They might represent a distinct population of γ/δ T cells with different TCR regulatory mechanisms. The same analysis performed on seven γ/δ clones derived from the thymus revealed only two positive clones. Conversely, all 12 γ/δ clones from the intestine were CD94+, and 6 of them coexpressed KIRs. Although we analyzed a limited number of clones, these results suggest that the expression of IR varies in different organs.

Coexpression of p140 and CD94, or p58.1 and CD94, was studied on total γ/δ T cells freshly isolated from nine donors (Fig. 2) or on subpopulations bearing different Vδ chains (data not shown). These analyses showed that CD94 is the most frequently expressed IR and that only a minority of γ/δ T cells express p140 and p58.1 KIRs, thereby confirming the results obtained with γ/δ clones. The expression of KIRs seemed to be biased because in some donors all the p140+ γ/δ T cells were CD94+, while in others p140+ γ/δ T cells were all CD94. These findings could result from an oligoclonal expansion, as described for KIRs+ αβ T cells (13).

Taken together, our phenotypic analysis indicates that expression of CD94 and KIRs by γ/δ T cells is not random, and it suggests that the microenvironment in which T cells are activated influences expression of IR.

CD94 Modulates TCR-γ/δ Activation.

As most of Vγ9/ Vδ2 cells express at least the CD94 molecule, we investigated the inhibitory capacity of this IR on TCR-γ/δ activation. Daudi Burkitt's lymphoma cell line, which does not express the β2-microglobulin gene and is therefore HLA class I, is killed by γ/δ T cells bearing the Vγ9/Vδ2 TCR (26). We first studied whether killing of Daudi cells by CD94+ Vγ9/Vδ2 cells could be inhibited by CD94 engagement with HLA class I molecules. When Vγ9/Vδ2 cells were tested against β2-microglobulin gene–transfected Daudi cells (Daudi-β2; reference 23), which express HLA class I molecules on the surface, killing was reduced but could be partially restored by addition of anti-CD94 mAbs (data not shown). Thus, like KIRs (11, 12, 14, 17, 18), CD94 also inhibits TCR activation.

Human γ/δ T cells also react to a variety of nonpeptidic ligands, some of which are phosphorylated metabolites (1921). We studied whether TCR-γ/δ activation by IPP, a strong agonist ligand (20, 21), could be inhibited by CD94. γ/δ clones were stimulated with different doses of IPP in the presence of Daudi or Daudi-β2 cells as APCs and TNF-α release was measured. The results confirmed that HLA class I+ APCs greatly reduce TCR-γ/δ–mediated cell activation (Fig. 3, A and B). TNF-α release was partially restored by addition of anti-CD94 mAbs, but not by isotype-matched irrelevant mAb (Fig. 3, C and D), thus further supporting the involvement of CD94. More importantly, CD94 engagement shifted the dose–response curve towards higher amounts of ligand. In the presence of Daudi-β2 APCs, 5–20 times higher doses of IPP were required than in the presence of Daudi APCs for an equivalent TNF-α release. This effect was consistently observed with four additional γ/δ clones (data not shown). These findings suggest that CD94 might serve the function of setting a higher cell activation threshold, as shown for other lymphocyte surface molecules (27).

Engagement of CD94 by HLA Class I Molecules Increases Recruitment of SHP-1 Phosphatase to CD94 and to the TCR– CD3 Complex and Causes Tyrosine Hypophosphorylation.

The inhibitory activities of KIRs have been ascribed to membrane recruitment of phosphatases and subsequent dephosphorylation of Lck, ZAP-70, and phospholipase C-γ (28). We tested whether SHP-1 phosphatase, which has been coprecipitated with the NKG2-A/NKR-P1C chimeric receptor (29), associates with CD94 expressed by γ/δ T cells. Indeed, SHP-1 was associated with CD94 in γ/δ T cells stimulated with IPP and the amount of coprecipitated SHP-1 was increased in the presence of Daudi-β2 APCs (Fig. 4A).

SHP-1 phosphatase might affect TCR signaling by associating with and dephosphorylating components of the TCR–CD3 complex. In support of this hypothesis, SHP-1 was coprecipitated with the TCR–CD3 complex when γ/δ T cells were cultured in the presence of Daudi-β2, but not of Daudi APCs (Fig. 4B). Thus, expression of MHC class I molecules on APCs is mandatory to detect this association, at least under the given experimental conditions. Moreover, increased amounts of SHP-1 were coprecipitated with the TCR–CD3 complex after stimulation with IPP (Fig. 4B). TCR triggering might facilitate the association of SHP-1 with components of the TCR–CD3 complex or might initiate a cascade that facilitates the phosphorylation of CD94, the association of SHP-1 with CD94, and thus the recruitment of this phosphatase to the membrane (28). Once on the membrane, SHP-1 might be recruited to the TCR–CD3 complex more readily. This hypothesis implies that TCR and CD94 form a functional multimolecular complex where SHP-1 is brought into physical proximity with its substrates, as shown in another model (30).

The increased SHP-1–TCR association may be responsible for the alterations observed with some proteins involved in TCR signaling. In agreement with this possibility, we found that CD94 stimulation causes a reduced tyrosine phosphorylation of bands migrating at ∼70 and 130 kD (Fig. 4C).

CD94 Engagement Affects ZAP-70 Phosphorylation, but not ZAP-70–CD3–TCR Complex Association.

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. 6B). In the same conditions, the lck form migrating at 60 kD is less abundant (Fig. 6A), whereas the CD3 ζ chain is normally phosphorylated (Fig. 5C). 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).

In conclusion, in γ/δ T cells stimulated with nonpeptidic ligands, CD94 engagement by HLA class I molecules leads to increased recruitment of SHP-1 to the TCR–CD3 complex, to reduced coprecipitation of Lck with the TCR–CD3 complex and to hypophosphorylation of ZAP-70. These findings may suggest that dephosphorylation of ZAP-70 is the key event blocking downstream TCR signaling.

Our data also show that the inhibitory mechanism of CD94 is different from that of antagonist ligands. In the latter case, the CD3 immunoglobulin receptor family tyrosine-based activatory motifs are partially tyrosine-phosphorylated and ZAP-70 does not associate with the CD3 ζ chain (31, 32).

An important issue concerns the implications of IR expression on the majority of γ/δ T cells, in particular the Vγ9/Vδ2 population, which is the most abundant circulating γ/δ population. Vγ9/Vδ2 cells recognize phosphorylated metabolites which are commonly found in eukaryotic and prokaryotic cells (for review see reference 33). Perhaps the function of IR is to prevent inappropriate and potentially dangerous γ/δ T cell activation by small quantities of these ubiquitous compounds.


The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd. This work was supported by Swiss National Fund grant 31-045518.95 to G. De Libero.

We thank L. Mori and T.J. Resink for critically reading the manuscript. We also thank M. Bonneville and P. Fish for helpful discussions, M. Bonneville, R. Kubo, M. López-Botet, and L. Moretta for providing mAbs, and J. Parnes for providing Daudi-β2 cells.

1. Moretta A,Bottino C,Vitale M,Pende D,Biassoni R,Mingari MC,Moretta L. Receptors for HLA class-I molecules in human natural killer cellsAnnu Rev Immunol 1996;14:619–648. [pmid: 8717527]
2. Lanier LL,Phillips JH. Inhibitory MHC class I receptors on NK cells and T cellsImmunol Today 1996;17:86–91. [pmid: 8808056]
3. Colonna M. Natural killer cell receptors specific for MHC class I moleculesCurr Opin Immunol 1996;8:101–107. [pmid: 8729453]
4. Long EO,Burshtyn DN,Clark WP,Peruzzi M,Rajagopalan S,Rojo S,Wagtmann N,Winter CC. Killer cell inhibitory receptors: diversity, specificity and functionImmunol Rev 1997;155:135–144. [pmid: 9059889]
5. Lazetic S,Chang C,Houchins JP,Lanier LL,Phillips JP. Human natural killer cell receptors involved in MHC class I recognition are disulfide-linked heterodimers of CD94 and NKG2 subunitsJ Immunol 1996;157:4741–4745. [pmid: 8943374]
6. Carretero M,Cantoni C,Bellon T,Bottino C,Biassoni R,Rodriguez A,Perez-Villar JJ,Moretta L,Moretta A,López-Botet M. The CD94 and NKG2-A C-type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I moleculesEur J Immunol 1997;27:563–567. [pmid: 9045931]
7. Vivier E,Daeron M. Immunoreceptor tyrosine-based inhibition motifsImmunol Today 1997;18:286–291. [pmid: 9190115]
8. Leibson PJ. Signal transduction during natural killer cell activation: inside the mind of a killerImmunity 1997;6:655–661. [pmid: 9208838]
9. Satterthwaite A,Witte O. Genetic analysis of tyrosine kinase function in B cell developmentAnnu Rev Immunol 1996;14:131–154. [pmid: 8717510]
10. Falk CS,Steinle A,Schendel DJ. Expression of HLA-C molecules confers target cell resistance to some non– major histocompatibility complex–restricted T cells in a manner analogous to allospecific natural killer cellsJ Exp Med 1995;182:1005–1018. [pmid: 7561674]
11. D'Andrea A,Chang C,Phillips JH,Lanier LL. Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I allelesJ Exp Med 1996;184:789–794. [pmid: 8760835]
12. Mingari MC,Vitale C,Cambiaggi A,Schiavetti F,Melioli G,Ferrini S,Poggi A. Cytolytic T lymphocytes displaying natural killer (NK)-like activity: expression of NK-related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TCR-mediated target cell lysis or lymphokine productionInt Immunol 1995;7:697–703. [pmid: 7547697]
13. Mingari MC,Schiavetti F,Ponte M,Vitale C,Maggi E,Romagnani S,Demarest J,Pantaleo G,Fauci AS,Moretta L. Human CD8+T lymphocyte subsets that express HLA class I–specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populationsProc Natl Acad Sci USA 1996;93:12433–12438. [pmid: 8901599]
14. Phillips JH,Gumperz JE,Parham P,Lanier LL. Superantigen-dependent, cell-mediated cytotoxicity inhibited by MHC class I receptors on T lymphocytesScience (Wash DC) 1995;268:403–405. [pmid: 7716542]
15. Ikeda H,Lethé B,Lehmann F,Van Baren N,Baurain J-F,De Smet C,Chambost H,Vitale M,Moretta A,Boon T,Coulie PG. Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptorImmunity 1997;6:199–208. [pmid: 9047241]
16. Aramburu J,Balboa MA,Ramirez A,Silva A,Acevedo A,Sanchez-Madrid F,De Landazuri MO,López-Botet M. A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor-γ/δ+T lymphocytes. I. Inhibition of the IL-2 dependent proliferation by anti-Kp43 monoclonal antibodyJ Immunol 1990;144:3238–3247. [pmid: 1691231]
17. Ferrini S,Cambiaggi A,Meazza R,Sforzini S,Marciano S,Mingari MC,Moretta L. T cell clones expressing the natural killer cell–related p58 receptor molecule display heterogeneity in phenotypic properties and p58 functionEur J Immunol 1994;24:2294–2298. [pmid: 7925558]
18. Nakajima H,Tomiyama H,Takiguchi M. Inhibition of γ/δ T cell recognition by receptors for MHC class I moleculesJ Immunol 1995;155:4139–4142. [pmid: 7594567]
19. Constant P,Davodeau F,Peyrat MA,Poquet Y,Puzo G,Bonneville M,Fournie JJ. Stimulation of human gamma delta T cells by nonpeptidic mycobacterial ligandsScience (Wash DC) 1994;264:267–270. [pmid: 8146660]
20. Tanaka Y,Morita CT,Tanaka Y,Nieves E,Brenner MB,Bloom BR. Natural and synthetic non-peptide antigens recognized by human gamma delta T cellsNature (Lond) 1995;375:155–158. [pmid: 7753173]
21. Bürk MR,Mori L,De Libero G. Human Vγ9/ Vδ2 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolitesEur J Immunol 1995;25:2052–2058. [pmid: 7621879]
22. De Libero G,Casorati G,Giachino C,Carbonara C,Migone N,Matzinger P,Lanzavecchia A. Selection by two powerful antigens may account for the presence of the major population of human peripheral γ/δ T cellsJ Exp Med 1991;173:1311–1322. [pmid: 1827824]
23. Seong RH,Clayberger C,Krensky A,Parnes J. Rescue of Daudi cell HLA expression by transfection of the mouse β2-microglobulin geneJ Exp Med 1988;167:288–299. [pmid: 3279151]
24. Dohring C,Scheidegger D,Samaridis J,Cella M,Colonna M. A human killer inhibitory receptor specific for HLA-A1,2J Immunol 1996;156:3098–3101. [pmid: 8617928]
25. Bürk M,Carena I,Donda A,Mariani F,Mori L,De Libero G. Functional inactivation in the whole population of human Vγ9/Vδ2 T lymphocytes induced by a nonpeptidic antagonistJ Exp Med 1997;185:91–97. [pmid: 8996245]
26. Fisch P,Malkovsky M,Braakman E,Sturm E,Bolhuis RL,Prieve A,Sosman JA,Lam VA,Sondel PM. γ/δ T cell clones and natural killer cell clones mediate distinct patterns of non–major histocompatibility complex– restricted cytolysisJ Exp Med 1990;171:1567–1579. [pmid: 2185329]
27. Tedder TF,Tuscano J,Sato S,Kehrl JH. CD22, a B lymphocyte–specific adhesion molecule that regulates antigen receptor signalingAnnu Rev Immunol 1997;15:481–504. [pmid: 9143697]
28. Binstadt BA,Brumbaugh KM,Dick CJ,Scharenberg AM,Williams BL,Colonna M,Lanier LL,Kinet J-P,Abraham RT,Leibson PJ. Sequential involvement of lck and SHP-1 with MHC-recognizing receptors on NK cells inhibits FcR-initiated tyrosine kinase activationImmunity 1996;5:629–638. [pmid: 8986721]
29. Houchins JP,Lanier LL,Niemi EC,Phillips JH,Ryan JC. Natural killer cell cytolytic activity is inhibited by NKG2-A and activated by NKG2-CJ Immunol 1997;158:3603–3609. [pmid: 9103421]
30. Bléry M,Delon J,Trautmann A,Cambiaggi A,Olcese L,Biassoni R,Moretta L,Chavrier P,Moretta A,Daeron M,Vivier E. Reconstituted killer cell inhibitory receptors for major histocompatibility complex class I molecules control mast cell activation induced via immunoreceptor tyrosine-based activation motifsJ Biol Chem 1997;272:8989–8996. [pmid: 9083022]
31. Sloan-Lancaster J,Shaw AS,Rothbard JB,Allen PM. Partial T cell signaling: altered phospho-zeta and lack of Zap-70 recruitment in APL-induced T cell anergyCell 1994;79:913–922. [pmid: 8001128]
32. Madrenas J,Wange RL,Wang JL,Isakov N,Samelson LE,Germain RN. Zeta phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonistsScience (Wash DC) 1995;267:515–518. [pmid: 7824949]
33. De Libero G. Sentinel function of broadly reactive human γ/δ T cellsImmunol Today 1997;18:22–26. [pmid: 9018970]


[Figure ID: F1]
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 ID: F2]
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 ID: F3]
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 ID: F4]
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 ID: F5]
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 ID: F6]
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.

Article Categories:
  • Brief Definitive Report
    • Brief Definitive Reports

Previous Document:  Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of ultravi...
Next Document:  Quantitative contribution of CD4 and CD8 to T cell antigen receptor serial triggering.