The non-classical MHC class I molecule Qa-1b inhibits classical MHC class I-restricted cytotoxicity of cytotoxic T lymphocytes

Stefan Lohwasser1, Akira Kubota1, Margarita Salcedo2, Rebecca H. Lian1 and Fumio Takei11,3

1 Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3 Canada
2 Unité de Biologie Moléculaire du Gène, INSERM U277, Institute Pasteur, 75015 Paris, France
3 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada

Correspondence to: F. Takei, Terry Fox Laboratory, British Columbia Cancer Research Centre, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
The CD94/NKG2A heterodimer is an inhibitory receptor expressed on a subset of mouse NK cells. CD94/NKG2A recognizes the non-classical MHC class I (class Ib) molecule Qa-1b and inhibits NK cytotoxicity. Qa-1b presents a peptide derived from the leader sequence of classical MHC class I molecules. Here, we examined the role of CD94/NKG2A in T cell-mediated cytotoxicity. Soluble tetrameric Qa-1b bound to almost all CD8+, but not CD4+, T cells. This binding seems to be mediated by CD8, because COS cells transfected with CD8 also bound Qa-1b tetramer. Therefore, the expression of CD94/NKG2 in T cells was further examined by single-cell RT-PCR. Most murine CD8+ T cells constitutively expressed CD94 and NKG2A transcripts, whereas they were not detected in CD4+ T cells. Co-expression of Qa-1b and Dk on target cells significantly inhibited cytotoxicity of Dk-specific cytotoxic T lymphocytes generated by mixed lymphocyte reaction, indicating that Qa-1b on antigen-presenting cells interacts with CD94/NKG2A on CD8 T cells and regulates classical MHC class I-restricted cytotoxic T cells. These results suggest a significant role of CD94/NKG2A as an inhibitory receptor on CD8+ T cells.

Keywords: cell-cell interactions, cytotoxic T lymphocyte, MHC rodent


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
The cytotoxicity of NK cells is inhibited by inhibitory receptors specific for MHC class I antigens. Human NK cells express killer cell-inhibitory receptors (KIR) that belong to the Ig superfamily and the CD94/NKG2 heterodimers that belong to the C-type lectin superfamily (1,2). KIR recognize specific MHC class I molecules, whereas CD94/NKG2 receptors interact with the non-classical MHC class I molecule HLA-E that presents peptides derived from the leader sequences of the classical MHC class I proteins (reviewed in 3). Similar to human NK cells, mouse NK cells also express two different families of receptors for MHC class I, both belonging to the C-type lectin superfamily. Like KIR on human NK cells, Ly-49 molecules recognize classical MHC class I on target cells (4). Recently, murine CD94 and NKG2 have been cloned (58). In analogy to the human system, the murine CD94/NKG2A receptor recognizes the non-classical MHC class I molecule Qa-1b and seems to function as an inhibitory NK receptor (9). Although Qa-1b, like the classical MHC class I molecules, presents peptides to T cells, it predominantly binds a single species of peptide termed Qdm (for Qa-1 determinant modifier), which is derived from amino acids 3–11 of the leader sequence of the class I MHC D-region-encoded molecules including H-2Dd and H-2Db (10,11). Murine NKG2A, like its human counterpart, contains one consensus immunoreceptor tyrosine-based inhibition motif (ITIM) and one ITIM-like sequence in its cytoplasmic domain (12). The ITIM in the cytoplasmic domain of human NKG2A associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP-1 and SHP-2. It is thought that murine NKG2A uses similar inhibition pathways (13). Both human and murine NKG2A have the ITIM in the cytoplasmic domains and function as inhibitory receptors. In humans, NKG2C associates with DAP-12 and transduces activation signals (14). Although the function of mouse NKG2C and NKG2E is still unknown, the lack of ITIM in its cytoplasmic domain suggests that its function is very different from that of NKG2A.

In humans, expression of CD94/NKG2A is not restricted to NK cells but is also found on a minor subset of T cells (15). In general, <10% of human CD8+ T cells express CD94/NKG2A (16). Both IL-15 and transforming growth factor-ß effectively induce expression of CD94/NKG2A in superantigen or alloantigen-activated CD8+ T cells (17,18). Cross-linking of CD94/NKG2A with mAb leads to partial inhibition of T cell functions including TCR-mediated triggering of cytolytic activity and lymphokine production (13,1921).

In the present study we have examined the expression of CD94/NKG2A on murine T cells. Most murine CD8+ T cells were found to express transcripts for CD94 and NKG2A. Furthermore, overexpression of Qa-1b on target cells leads to inhibition of classical MHC class I-specific T cell cytotoxicity, suggesting that CD94/NKG2 may play a significant role in the regulation of murine CD8+ T cells functions.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
Mice
C57BL/6 (H-2b, Qa-1b) mice, C3H/HeJ (H-2k, Qa-1b) mice and CDF rats were purchased from the Jackson Laboratory (Bar Harbor, ME).

Expression vectors
The gene coding for H-2Dk was kindly provided by Dr W. Jefferies (Biotechnology Laboratories, Vancouver). The coding region of the Qa-1b gene was obtained from B6 mouse spleen cells by RT-PCR, cloned into pBluescript and sequenced. To generate a green fluorescence protein (GFP) fusion construct, PCR primers were designed to amplify the cDNA fragment encoding the extracellular and transmembrane domains of Qa-1b. The PCR product was cloned into pBluescript, sequenced and subcloned into pEGFP-N1 (Clonetech, Palo Alto, CA), thus replacing the cytoplasmic domain of Qa-1b by GFP. The cDNAs for mouse CD94, NKG2A and NKG2C have been previously described (5,6). The cDNA for CD8{alpha} was kindly provided by Dr Herzenberg (Stanford University, CA).

Cells, cell lines and transfection
Single-cell suspensions were prepared from freshly isolated spleens and lymph nodes of C57BL/6 and C3H/HeJ mice (1–12 weeks old), and used for flow cytometric analysis and mixed lymphocyte reaction (MLR). CD94, NKG2A, NKG2C and CD8 cDNAs that had been cloned into the pCDM8 expression vector were transfected into COS cells using Lipofectamine (Canadian Life Technologies, Burlington, Ontario, Canada) according to the manufacturer's protocols. The HLA class I-deficient human lymphoblastoid line 721.221 was kindly provided by Dr P. Parham (Stanford University, CA) and cultured in RPMI supplemented with 10% FCS. The H-2Dk gene was transfected by electroporation either alone or together with the Qa-1b/GFP fusion construct into 721.221 cells at 0.45 kV and 125 µF, and stable transfectants resistant to G418 (Canadian Life Technologies) were subsequently selected for high expression by cell sorting.

Qa-1b tetramers, mAb and flow cytometry
The Qa-1b tetramer was described previously (22). Briefly, tetrameric complexes of biotinylated Qa-1b, human ß2-microglobulin and peptide (AMAPRTLLL) were formed using streptavidin–Red670 (SAvRED; Life Technologies) at a 4:1 molar ratio. For cell staining, the Qa-1b tetramer was used at a total protein concentration of 7 µg/ml in PBS. The hybridoma 3-83P (anti-Dk/Kk) and OKT9 were obtained from ATCC (Manassas, VA). The Qa-1b-specific mAb 6A8.6F10.1A6 (6F10) was a generous gift from Dr Soloski (Johns Hopkins University, Baltimore, MD). The allophycocyanin (APC)-conjugated anti-CD3 mAb (145-2C11; PharMingen, San Diego, CA), FITC-conjugated anti-CD8a (Boehringer, Mannheim, Germany) and phycoerythrin (PE)-conjugated anti-CD4 (Becton Dickinson, San Jose, CA) were diluted in PBS with 1% FCS, and used at a concentration of 2–20 µg/ml. All incubations were done for 30 min on ice. After the final washing, labeled cells were analyzed on a FACSCalibur (Becton Dickinson) using CellQuest software.

Single-cell RT-PCR
Expression of CD94 and NKG2s in individual T cells and NK cells was examined by single-cell RT-PCR as described (27,28). In brief, cells were sorted and individually deposited into 96-well plates containing guanidinium isothiocyanate solution. RNA was isolated from each well and reverse transcribed. The cDNA thus generated was subjected to poly(A) tailing and PCR amplified using the special oligo(dT) primer for 40 cycles. The amplified total cDNA derived from each well was subjected to a second round of PCR amplification for 35 cycles using the following primers: CD94-specific 5'-primer; 5'-TTTCTTGATGGTTACTTTGGGAGTT-3'; CD94-specific 3'-primer, 5'-AAACGCTTTTGCTTGGACTGTA-3'; NKG2A-specific 5'-primer, 5'-CGAAGCAAAGGCACAGA-3'; NKG2A-specific 3'-primer, 5'-ATGGCACAGTTACATTCATCAT-3'; NKG2C-specific 5'-primer, 5'-GCTGAACTGAAGAAGCAGATCC-3'; NKG2C-specific 3'-primer, 5'-TGGGGAATTTACACTTACAAAG-3'.

PCR products were separated by agarose gel electrophoresis and transferred to nylon membranes. The following oligonucleotides were used for hybridization: CD94, 5'-AACAATTGCACTGATGCCCAA-3'; NKG2A/C/E, 5'-TCCTTCGGAAGGGCAGAGGTCA-3'; and ß-actin, 5'-CAAGTGCTTCTAGGCGGACTGT-3'. The oligonucleotides were 32P-end-labeled using terminal transferase and hybridized at 58°C to Southern blots. After hybridization the blots were washed in 3xSSC/1% SDS. The filters were exposed to X-ray films at –70°C.

MLR and cytotoxicity assay
Nylon wool non-adherent C57BL/6 mouse splenocytes and CDF rat splenocytes (8x106) were co-cultured with mitomycin C-treated C3H/HeJ splenocytes for 5 days in RPMI containing 5% FCS and 10 U/ml IL-2 using 24-well plates at a responder:stimulator ratio of 2:1. The cytolytic activity of T cells from the MLR was tested against 51Cr-labeled target cells in standard 51Cr-release assays. Data are expressed as percent specific lysis at the indicated E:T ratios.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
Binding of Qa-1b tetramers to CD8+ T cells and NK cells
A soluble tetrameric form of Qa-1b has previously been shown to bind to ~50% of NK1.1+ B6 splenocytes (22). We also found that a subset of CD3+ T cells was recognized by the Qa-1b tetramer. To further characterize the Qa-1b binding T cell subpopulations, we stained lymphocytes from spleen, lymph nodes and peripheral blood of B6 mice with anti-CD8, anti-CD4 and anti-CD3 mAb together with Qa-1b tetramer folded in the presence of the Qdm peptide (AMAPRTLLL). Four-color flow cytometric analysis showed that the Qa-1b tetramer bound to most, if not all, CD8+ T cells, whereas no significant binding to CD4+ T cells was detected (Fig. 1AGo). The level and pattern of Qa-1b binding was the same for lymphocytes from the spleen, lymph nodes or peripheral blood (data not shown). As these findings are in sharp contrast to previously published results, we speculated that the high percentage of Qa-1b tetramer-positive cells might be due to altered binding specificity of the tetrameric complexes used in the present experiments. However, in accordance with previously published results we found that ~50% of adult NK cells bound to the Qa-1b tetramer with a higher percentage (85%) of neonatal NK cells binding to the tetrameric complexes (Fig. 1BGo). As reported by Vance et al., the Qa-1b tetramer used in our experiments also recognized the CD94/NKG2A and CD94/NKG2C heterodimers which were co-expressed on COS cells (Fig 2AGo). Interestingly, the expression of the receptor for Qa-1b in the T cell lineage largely overlaps with the expression of CD8. To test whether CD8 is also involved in the binding of Qa-1b tetramers to CD8+ T cells we transiently transfected COS cells with an expression construct for CD8. Surprisingly, the Qa-1b tetramer stained CD8-transfected COS cells and the staining of the transfected COS cells with Qa-1b tetramer was very similar to that with anti-CD8 antibody (Fig. 2BGo). Pre-incubation of CD8-transfected COS or splenic T cells with anti-CD8 antibody did not inhibit the staining with the tetramer (data not shown). This is consistent with a recent report which showed that some anti-CD8 mAb does not inhibit the binding of MHC class I tetramers to CD8 T cells (23). Therefore, the contribution of CD8 to the binding of Qa-1b tetramers to CD8+ T cells is difficult to assess. Although Jameson et al. have recently shown that CD8 plays a critical role in the antigen-specific TCR binding of multimeric MHC class I complexes, it is unclear why different preparations of the same class I molecule show different binding specificities (23).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. Binding of Qa-1b tetramer to CD8+ T cells and NK cells. (A) B6 splenocytes (upper panel) and lymph node cells (lower panel) were stained with anti-CD8–FITC, anti-CD4–PE, anti-CD3–APC and tetrameric Qa-1b–SAvRED. CD3+CD8+ (left) and CD3+CD4+ cells (right) were electronically gated and analyzed for the binding of Qa-1b tetramer. The histograms of Qa-1b tetramer binding were overlapped with those of control staining with SAvRED alone. (B) B6 splenocytes of 2-day-old (left panel) and 12-week-old (right panel) mice were stained with anti-CD3–FITC, anti-NK1.1–Cy5 and tetrameric Qa-1b–SAvRED. NK1.1+CD3 NK cells were gated and analyzed for Qa-1b binding. The results are representative of five independent experiments.

 


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2. Binding of Qa-1b tetramer to CD94/NKG2A, CD94/NKG2C and CD8a. COS cells were transfected with CD94, NKG2A, NKG2C and CD8a as indicated. The histograms of Qa-1b binding and CD8–FITC staining overlapped with the controls are shown. The results are representative of two independent experiments.

 
Expression of CD94, NKG2A, NKG2C and NKG2E in CD8+ T cells and NK cells
To assess the expression of CD94/NKG2 receptors on T cells and to determine which CD94/NKG2 heterodimers are predominantly expressed on mouse T cell subsets, we examined the expression of CD94, NKG2A, NKG2C and NKG2E mRNA in individual T cells by single-cell RT-PCR as described in Methods. CD94 mRNA was detected in 14 out of 18 (77%) CD8+ splenic T cells. Similarly, NKG2A mRNA was detected in 16 out of 18 (89%) CD8+ splenic T cells (Fig. 3Go). In contrast to CD8+ T cells only three out of 18 (16%) CD4+ T cells expressed CD94 mRNA and none of them expressed NKG2A, NKG2C or NKG2E mRNA. Although we were able to detect NKG2C transcripts in NK cells [two out of 18 (11%)], we could not detect NKG2C mRNA in any of the T cell subsets in this study. In two out of 18 CD8+ T cells we could detect NKG2E mRNA. Transcripts for CD94 were found in nine out of 18 (50%) NK cells and eight out of 18 (44%) were positive for NKG2A mRNA. The percentage of CD94/NKG2 receptor-positive NK cells is very similar to the percentage of Qa-1b tetramer-positive NK cells. This indicates that the single-cell RT-PCR data properly reflects the receptor expression pattern. Although the expression analysis by single-cell RT-PCR suggests that the majority of CD8+ T cells express CD94 and NKG2A messages, this apparently does not lead to the expression of the respective proteins on the cell surface. Using a mAb against mouse CD94 and NKG2, Vance et al. have shown that CD94 and NKG2 proteins are expressed on ~50% of NK cells, but only on a minority of T cells (<1%) (8). This is in accordance with previous studies which have shown that human CD94/NKG2A is expressed in a minor subset of T cells (16,24). They are mostly CD8+, typically lack CD28 and belong to a subset of activated lymphocytes (25). IL-15 and TGF-ß dramatically increase expression of CD94/NKG2A on human T cells (17,18). This pattern of human CD94/NKG2A expression in T cell subsets is significantly different from that of murine CD94/NKG2A as determined by the single-cell RT-PCR. At the mRNA level, murine CD94/NKG2A appears to be expressed in the majority of murine CD8+ T cells but not CD4+ T cells.



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 3. Single-cell RT-PCR analysis of CD94 and NKG2 mRNA in T cells. CD4+, CD8+ splenic T cells and NK1.1+CD3 NK cells (A, B and C respectively) were individually sorted into 96-well plates. The expression of CD94, NKG2A and NKG2C/E mRNA in each cell was determined by single-cell RT-PCR as described in Methods. Aliquots of the PCR products were separated by agarose gel electrophoresis, blotted and hybridized to specific oligonucleotide probes. Each lane represents a single cell. Wells containing only guanidinium isothiocyanate solution (H2O) were used as a negative control. ß-Actin was used as a positive control.

 
Inhibition of cytotoxic T lymphocytes (CTL) by Qa-1b
To examine the functional significance of CD94/NKG2A expression in CD8+ T cells, we tested whether Qa-1b on target cells has any effects on CTL. Classical class I MHC-restricted CTL were generated by co-culturing nylon wool non-adherent B6 splenocytes (H-2b, Qa-1b) with mitomycin C-treated C3H/HeJ splenocytes (H-2k, Qa-1b). For the target cells, the human classical MHC class I-deficient B cell line 721.221 transfected with the murine classical class I MHC molecule Dk alone or in combination with the Qa-1b/GFP fusion construct (see Methods) were generated. Because 721.221 cells are deficient for human classical MHC class I genes, they do not express the dominant leader peptides that bind to Qa-1b. Flow cytometric analysis showed that Dk-transfected 721.221 cells and those co-transfected with Dk and Qa-1b/GFP expressed comparable levels of Dk (Fig. 4AGo). Qa-1b/GFP expression was also detected by the green fluorescence of GFP in 721.221 cells co-transfected with Dk and Qa-1b/GFP (Fig. 4AGo), but not those transfected with Qa-1b/GFP alone (data not shown). Therefore, the leader sequence of Dk seems to be able to provide peptides that bind and stabilize Qa-1b.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. Inhibition of CTL by recognition of Qa-1b. (A) Flow cytometric analysis of the surface expression of H-2Dk and Qa-1b/GFP on 721.221 cells. Cells were incubated with the anti-Dk mAb 3-83P followed by secondary PE-labeled goat anti-mouse antibody. The histograms of anti-Dk staining overlapped with those of control staining with secondary antibody alone are shown. The lower panel shows the histogram of expression Qa-1b/GFP detected by the green fluorescence of GFP overlapped with that of control untransfected 721.221 cells. In (B) and (C), 721.221 cells were labeled with 51Cr and used as targets for the cytotoxicity of mouse (B) and rat (C) MLR-derived CTL as described in Methods. In (B), anti-Qa-1b (6F10) and isotype-matched control (OKT9) were added at a concentration of 20 µg/ml where indicated. The results are representative of four independent experiments, each done in triplicate.

 
CTL generated by the B6 anti-C3H MLR readily killed Dk-transfected 721.221 but not control 721.221 transfected with vector alone. The cytotoxicity against 721.221 cells co-transfected with Dk and Qa-1b/GFP was significantly lower than that against the Dk-transfectants (Fig. 4BGo), indicating that expression of the non-classical MHC class I Qa-1b on the target cells inhibits the classical MHC class I Dk-restricted CTL. By adding the Qa-1b-specific mAb 6F10 the partial inhibition could be reversed, indicating that the observed inhibition is indeed due to Qa-1b expression on target cells. The isotype control antibody OKT9 that also bound to 721.221 cells had no effect on the cytotoxicity. It should be noted that all the transfected 721.221 cells were equally sensitive to rat anti-C3H MLR-derived CTL (Fig. 4CGo). Therefore, the observed effect of Qa-1b was not due to an intrinsic resistance of the Qa-1b-transfected cells to cell-mediated lysis in general.

These results suggest that Qa-1b on target cells interacts with an inhibitory receptor on CD8+ T cells and inhibits classical class I MHC-restricted CTL. As mouse CD94/NKG2A is the only known inhibitory receptor recognizing Qa-1b, it is most likely that the observed inhibition is indeed due to CD94/NKG2A. This is consistent with the previous finding that up-regulation of CD94/NKG2A on human T cells by IL-15 and TGF treatment leads to an impairment of allo-specific cytolytic activity by MLR-derived T cell populations or clones (17,18). It is noteworthy that in our experimental system we see the reduction of the cytolytic ability of mouse MLR-derived T cells without adding any cytokines or growth factors. Although mouse and human CD8+ T cells can both express CD94/NKG2A the expression of this inhibitory receptor appears to be differently regulated between the two species.


    Conclusions
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 
It is of interest that the expression of CD94/NKG2A generally overlaps with CD8 expression in the murine T cell lineage. The former recognizes the non-classical MHC class I Qa-1b, whereas the latter interacts with the classical MHC class I molecules. Furthermore, Qa-1b primarily presents peptides derived from the leader sequence of the classical MHC class I D-region gene products. Thus, Qa-1b expression is dependent on the classical class I MHC, resulting in ubiquitous co-expression of classical MHC class I and Qa-1b. Since CD8+ T cells are MHC class I restricted, it is conceivable that the activation of CD8+ T cells in general is regulated by the balance between pro-stimulatory signals generated by the co-receptor CD8 and inhibitory signals from CD94/ NKG2A. How then do CTL exert their cytotoxic functions in the presence of inhibitory signals from CD94/NKG2A? Allogeneic C3H blasts are readily killed by MLR-derived CTL although they are expressing Qa-1b (Fig. 5BGo). Flow cytometric analysis of the C3H blasts and the Qa-1b/GFP transfected 721.221 line shows that the expression of the alloantigen (H2-Dk, H2-Kk) was ~10-fold higher on the C3H blasts than on the transfected cell line, whereas the expression of Qa-1b was higher on the transfectants (Fig. 5AGo). In the case of the C3H blasts, the activation signals from the TCR and CD8 engaged by the highly expressed alloantigen are presumably dominant over the inhibitory signals from CD94/NKG2A engaged by Qa-1b, which is expressed at a lower level on the C3H blasts. In the case of the transfected 721.221 cells, the surface expression of Qa-1b may be higher than that of Dk, resulting in the inhibition of the cytotoxicity. The inhibition of cytotoxic T cells by CD94/NKG2A may be important for the prevention of self-reactivities of CD8 T cells expressing low-affinity TCR for self-peptide–MHC. Recently it has been shown that some melanoma patients bear tumor-specific CTL expressing CD94/NKG2A and that these CTL are at least partially inhibited by CD94/NKG2A engagement (26). Thus, although expression of inhibitory receptors on CD8 T cells may be, on the one hand, necessary to prevent autoimmunity it might, on the other hand, be detrimental for tumor-specific activity of CTL. Further studies on CD94/NKG2A and Qa-1b will provide insight into the mechanisms by which the delicate balance between activation and inhibition of the immune system is maintained.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. Concanavalin A blasts derived from C3H mice are readily killed by anti-Dk CTL. (A) Flow cytometric analysis of Qa-1b (6F10) and Dk (3-83P) expression of 721.221-Dk/Qa-1b and C3H concanavalin A blasts (shaded). Histograms are overlapped with control staining. (B) C3H concanavalin A blasts were used as targets for the cytotoxicity of MLR-derived CTL. Where indicated, mAb were added at a concentration of 20 µg/ml. The results are representative of three independent experiments, each done in triplicate.

 


    Acknowledgments
 
This work was supported by a grant from the National Cancer Institute of Canada with core support provided by the British Columbia Cancer Agency. S. L. is a recipient of a Deutsche Forschungsgemeinschaft fellowship and A. K. is a recipient of a Leukemia Research Fund of Canada fellowship. We thank Dr W. Jefferies for the Dk gene and Dr D. Mager for critical reading of the manuscript.


    Abbreviations
 
APC allophycocyanin
CTL cytotoxic T lymphocyte
GFP green fluorescence protein
ITIM immunoreceptor tyrosine-based inhibitory motif
KIR killer cell-inhibitory receptors
MLR mixed lymphocyte reaction
PE phycoerythrin
SAvRED streptavidin–Red670
TGF transforming growth factor

    Notes
 
Transmitting editor: J. Schrader

Received 14 April 2000, accepted 4 December 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Conclusions
 References
 

  1. Long, E. O., Burshtyn, D. N., Clark, W. P., Peruzzi, M., Rajagopalan, S., Rojo, S., Wagtmann, N. and Winter, C. C. 1997. Killer cell inhibitory receptors: diversity, specificity, and function. Immunol. Rev. 155:135.[ISI][Medline]
  2. Lopez-Botet, M., Perez-Villar, J. J., Carretero, M., Rodriguez, A., Melero, I., Bellon, T., Llano, M. and Navarro, F. 1997. Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules. Immunol. Rev. 155:165.[ISI][Medline]
  3. Braud, V. M. and McMichael, A. J. 1999. Regulation of NK cell functions through interaction of the CD94/NKG2 receptors with the nonclassical class I molecule HLA-E. Curr. Top. Microbiol. Immunol. 244:85.[ISI][Medline]
  4. Takei, F., Brennan, J. and Mager, D. L. 1997. The Ly-49 family: genes, proteins and recognition of class I MHC. Immunol. Rev. 155:67.[ISI][Medline]
  5. Vance, R. E., Tanamachi, D. M., Hanke, T. and Raulet, D. H. 1997. Cloning of a mouse homolog of CD94 extends the family of C-type lectins on murine natural killer cells. Eur. J. Immunol. 27:3236.[ISI][Medline]
  6. Lohwasser, S., Hande, P., Mager, D. L. and Takei, F. 1999. Cloning of murine NKG2A, B and C: second family of C-type lectin receptors on murine NK cells. Eur. J. Immunol. 29:755.[ISI][Medline]
  7. Silver, E. T., Lau, J. C. Y. and Kane, K. P. 1999. Molecular cloning of mouse NKG2A and C. Immunogenetics 49:727.[ISI][Medline]
  8. Vance, R. E., Jamieson, A. M. and Raulet, D. H. 1999. Recognition of the class Ib molecule Qa-1b by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells. J. Exp. Med. 190:1801.[Abstract/Free Full Text]
  9. Vance, R. E., Kraft, J. R., Altman, J. D., Jensen, P. E. and Raulet, D. H. 1998. Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). J. Exp. Med. 188:1841.[Abstract/Free Full Text]
  10. Aldrich, C. J., DeCloux, A., Woods, A. S., Cotter, R. J., Soloski, M. J. and Forman, J. 1994. Identification of a TAP-dependent leader peptide recognized by alloreactive T cells specific for a class Ib antigen. Cell 79:649.[ISI][Medline]
  11. DeCloux, A., Woods, A. S., Cotter, R. J., Soloski, M. J. and Forman, J. 1997. Dominance of a single peptide bound to the class I(B) molecule, Qa-1b. J. Immunol. 158:2183.[Abstract]
  12. Vivier, E. and Daeron, M. 1997. Immunoreceptor tyrosine-based inhibition motifs (ITIMs). Immunol. Today 18:286.[ISI][Medline]
  13. Le Drean, E., Vely, F., Olcese, L., Cambiaggi, A., Guia, S., Krystal, G., Gervois, A., Moretta, A., Jotereau, F. and Vivier, E. 1998. Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A: association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases. Eur. J. Immunol. 28:264.[ISI][Medline]
  14. Lanier, L. L., Corliss, B., Wu, J. and Phillips, J. H. 1998. Association of DAP12 with activating CD94/NKG2C NK cell receptors. Immunity 8:693.[ISI][Medline]
  15. Ferrini, S., Cambiaggi, A., Meazza, R., Sforzini, S., Marciano, S., Mingari, M. C. and Moretta, L. 1994. T cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function. Eur. J. Immunol. 24:2294.[ISI][Medline]
  16. Mingari, M. C., Schiavetti, F., Ponte, M., Vitale, C., Maggi, E., Romagnani, S., Demarest, J., Pantaleo, G., Fauci, A. and Moretta, L. 1996. Human CD8+ T lymphocytes subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations. Proc. Natl Acad. Sci. USA 93:12433.[Abstract/Free Full Text]
  17. Mingari, M. C., Ponte, M., Bertone, S., Schiavetti, F., Vitale, C., Bellomo, R., Moretta, A. and Moretta, L. 1998. HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin 15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+ T cells. Proc. Natl Acad. Sci. USA 95:1172.[Abstract/Free Full Text]
  18. Bertone, S., Schiavetti, F., Bellomo, R., Vitale, C., Ponte, M., Moretta, L. and Mingari, M. C. 1999. Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur. J. Immunol. 29:23.[ISI][Medline]
  19. D'Andrea, A., Chang, C., Phillips, J. H. and Lanier, L. L. 1996. Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles. J. Exp. Med. 184:789.[Abstract]
  20. Carena, I., Shamshiev, A., Donda, A., Colonna, M. and Libero, G. D. 1997. Major histocompatibility complex class I molecules modulate activation threshold and early signaling of T cell antigen receptor-gamma/delta stimulated by nonpeptidic ligands. J. Exp. Med. 186:1769.[Abstract/Free Full Text]
  21. Bakker, A. B., Phillips, J. H., Figdor, C. G. and Lanier, L. L. 1998. Killer cell inhibitory receptors for MHC class I molecules regulate lysis of melanoma cells mediated by NK cells, gamma delta T cells, and antigen-specific CTL. J. Immunol. 160:5239.[Abstract/Free Full Text]
  22. Salcedo, M., Bousso, P., Ljunggren, H. G., Kourilsky, P. and Abastado, J. P. 1998. The Qa-1b molecule binds to a large subpopulation of murine NK cells. Eur. J. Immunol. 28: 4356.[ISI][Medline]
  23. Daniels, M. A. and Jameson, S. C. 2000. Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers. J. Exp. Med. 191:335.[Abstract/Free Full Text]
  24. Noppen, C., Schaefer, C., Zajac, P., Schutz, A., Kocher, T., Kloth, J., Heberer, M., Colonna, M., De Libero, G. and Spagnoli, G. C. 1998. C-type lectin-like receptors in peptide-specific HLA class I-restricted cytotoxic T lymphocytes: differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope specificity. Eur. J. Immunol. 28:1134.[ISI][Medline]
  25. Speiser, D. E., Valmori, D., Rimoldi, D., Pittet, M. J., Lienard, D., Cerundolo, V., MacDonald, H. R., Cerottini, J. C. and Romero, P. 1999. CD28-negative cytolytic effector T cells frequently express NK receptors and are present at variable proportions in circulating lymphocytes from healthy donors and melanoma patients. Eur. J. Immunol. 29:1990.[ISI][Medline]
  26. Speiser, D. E., Pittet, M. J., Valmori, D., Dunbar, R., Rimoldi, D., Lienard, D., MacDonald, H. R., Cerottini, J. C., Cerundolo, V. and Romero, P. 1999. In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes. J. Exp. Med. 190:775.[Abstract/Free Full Text]
  27. Sauvageau, G., Lansdorp, P. M., Eaves, C. J., Hogge, D. E., Dragowska, W. H., Reid, D. S., Largman, C., Lawrence, H. J. and Humphries, R. K. 1994. Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc. Natl Acad. Sci. USA 91:12223.[Abstract/Free Full Text]
  28. Kubota, A., Kubota, S., Lohwasser, S., Mager, D. and Takei, F. 1999. Diversity of NK cell receptor repertoire in adult and neonatal mice. J. Immunol. 163:212.[Abstract/Free Full Text]