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(Journal of Leukocyte Biology. 2001;70:219-224.)
© 2001 by Society for Leukocyte Biology

Expression of CD94 and NKG2 molecules on human CD4+ T cells in response to CD3-mediated stimulation

Pilar Romero*, Consuelo Ortega*, Agustín Palma*, Ignacio J. Molina{dagger}, José Peña* and Manuel Santamaría*

* Departamento de Inmunología, Facultad de Medicina, Hospital Universitario "Reina Sofía," Universidad de Córdoba, Córdoba, Spain, and
{dagger} Unidad de Inmunología, Facultad de Medicina, Universidad de Granada, Granada, Spain

Correspondence: Dr. Manuel Santamaría, Servicio de Inmunología, Hospital Universitario Reina Sofía, Avenida Menendez Pidal s/n, E-14004 Córdoba, Spain. E-mail: msantamaria{at}uco.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the ability of human peripheral CD4+ cells to express CD94 and NKG2 molecules as a consequence of CD3-mediated activation. Using highly purified peripheral CD4+ T cells, we found expression of both CD94 and NKG2A 15 days after CD3-mediated stimulation of cells. We also determined by reverse transcriptase-PCR that all gene members of NKG2 family—namely, NKG2A, -C, -D, and -E—are sequentially expressed on CD4+ cells. We found that this expression is tightly regulated by cytokines, and we identified transforming growth factor-ß1 and interleukin-10 as the main factors that, on CD3-dependent stimulation, positively contribute to the expression of CD94 and NKG2A on CD4+ cells. We also investigated the functional role of NKG2A and found that coligation of CD3 and NKG2A by specific monoclonal antibodies results in significant inhibition of interferon {gamma} and tumor necrosis factor {alpha} production by stimulated CD4+ cells. The presence and function of these receptors on CD4+ lymphocytes support a more general role for NKG2 molecules, whose functions were originally thought to be confined to cytotoxic cells, in the immune system.

Key Words: C-type lectin receptors • cytokine regulation • TNF-{alpha} • IFN-{gamma}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD94/NKG2 receptors are C-type lectin heterodimers composed of CD94 covalently associated with one of the NKG2 molecules [reviewed in references 1–3]. The invariant component of the receptors, CD94, is a type II integral membrane protein with a very short, nonsignaling, intracytoplasmic tail [4 ]. To generate functional receptors, CD94 is disulfide linked with a member of the NKG2 family, namely NKG2A, -B, -C, or -E molecules [5 , 6 ]. NKG2D does not associate with CD94 [7 ] and shares little homology with the other members of the NKG2 family [8 ]. The natural ligand for the CD94/NKG2 complex is the nonclassical class I human leukocyte antigen (HLA) E [9 ]. NKG2A and -B contain immunoreceptor tyrosine-based inhibition motifs within their intracellular domains [10 ] that transduce inhibitory signals. Conversely, all other NKG2 members transduce activation signals because they lack immunoreceptor tyrosine-based inhibition motif sequences and are linked with transmembrane proteins such as DAP10 and DAP12 that contain immunoreceptor tyrosine-based activating motifs [11 12 13 ]. CD94/NKG2 receptors are expressed on natural killer (NK) cells and some subset of CD8+ (either {alpha}ß+ or {gamma}{delta}+) T-peripheral lymphocytes [14 , 15 ]. Expression of these receptors on T-cell populations is dependent on cell activation and is positively regulated by certain cytokines. In this context, we have recently shown that interleukin (IL)-10 contributes to the expression of the CD94/NKG2 receptor on long-term-activated human CD8+ T lymphocytes [16 ]. Other cytokines able to induce membrane expression of CD94/NKG2A on CD8+ T lymphocytes are IL-15 [17 ] and transforming growth factor ß (TGF-ß) [18 ]. Functional studies have revealed the relationship of these receptors to cytotoxicity processes mediated by NK cells and CD8+ T cells, leading to inhibition or enhancement of cell lysis [19 20 21 ].

However, expression of CD94/NKG2 molecules on CD4+ cells has barely been studied, and therefore its function in this T-cell subgroup remains unknown. Because CD4+ lymphocytes play a central role in the regulation of the immune response, we explored whether CD94/NKG2 receptors are functionally expressed on this subpopulation of T cells and, if so, the function(s) in which they are involved. We found that CD3-stimulated CD4+ cells sequentially express all NKG2 gene family members during the culture period. We also found that IL-10 and TGF-ß induced CD94/NKG2 expression. Remarkably, cross-linking of CD94/NKG2A inhibited interferon (IFN) {gamma} and tumor necrosis factor {alpha} (TNF-{alpha}) production by CD4+ human T lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
The following phycoerythrin- or fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (mAbs) were purchased from Becton Dickinson (Mountain View, CA): Leu-4-SK7 (anti-CD3), Leu-3a-SK3 (anti-CD4), 25723.11 (anti-IFN{gamma}), 6401.1111 (anti-TNF-{alpha}), and goat anti-mouse immunoglobulin (Ig) G (GAM) secondary antibody. FITC-labeled HP-3D9 (anti-CD94) and JES3-19F1 (anti-IL-10) were obtained from PharMingen (San Diego, CA). OKT3 (anti-CD3)-producing hybridoma cells were purchased from the American Type Culture Collection (Manassas, VA). The culture supernatants were ammonium sulfate cut and purified over Affi-Gel-protein A columns (Bio-Rad Laboratories, Hercules, CA). FITC-labeled C15 [anti-T-cell receptor (TCR) V{alpha}24] was obtained from Immunotech (Marseilles, France). The following unconjugated, purified mAbs were used: Z199 (anti-NKG2A), purchased from Immunotech; 9016.2 (anti-TGF-ß1), from R&D (Minneapolis, MN); 9D7 (anti-IL-10), a generous gift from M. G. Roncarolo (Hospital San Raffaele, Milan, Italy); and HP3D1 (anti-CD94), kindly donated by M. López-Botet (Universitat Pompeu Fabra, Barcelona, Spain). The culture medium used was RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 100 U/mL of penicillin plus 100 µg/mL of streptomycin (Bio-Whittaker, Verviers, Belgium). Human recombinant IL-2 was a gift from Hoffman-LaRoche (Nutley, NJ). Lymphocyte density gradient isolation medium (Lymphoprep) was purchased from Nycomed Pharma (Oslo, Norway).

Flow cytometry
For direct immunofluorescence assays, 105 cells were incubated with the mAbs on ice for 30 min, washed with phosphate-buffered saline, and analyzed on a FACScalibur flow cytometer (Becton Dickinson). For indirect immunofluorescence, a second incubation with the isotype-specific, FITC-labeled GAM antibody was carried out as described above. Live cells were selectively gated on forward- and side-scatter parameters, and for double-fluorescence experiments, the flow cytometer was appropriately compensated with Calli-Brite beads, and the data were analyzed with FACS Comp 4.1 software (Becton Dickinson).

Isolation and culture of T lymphocytes
Peripheral blood lymphocytes were obtained by density gradient centrifugation. Adherent cells were removed by incubation of the cells in 100-mm-diameter plastic petri dishes (Nunc, Roskilde, Denmark) at 37°C for 60 min. The cells were then passed over a nylon-wool column to remove residual B cells and macrophages. This resulted in a population of T cells that was typically >90% CD3+, <2% CD56+, <1% CD19+, and <1% CD14+, as determined by flow cytometry. Further isolation of the major T-cell subpopulation and removal of CD94+ cells were achieved by negative selection using relevant antibodies followed by a second incubation with GAM-coupled magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells that bound to the immunobeads were removed with a magnetic particle concentrator (Miltenyi Biotec). The T lymphocytes were resuspended in complete medium supplemented with 25 U/mL of recombinant (r)IL-2 and cultured at a concentration of 106/mL in 24-well plates (Nunc), which had been previously coated in phosphate-buffered saline (pH 8.0) with 2 µg/well of OKT3 mAb in the presence or absence of 2 ng/mL of IL-10 and incubated overnight at 37°C. Every 5 days, the cells were restimulated in plates freshly coated with OKT3, and the medium and cytokines were replaced. For functional blocking experiments, 2.5 µg/mL of 9D7 mAb (anti-IL-10) or 1 µg/mL of 9016.2 mAb (anti-TGF-ß1) were present from the initiation of cultures and freshly added at each restimulation of the cells. The surface expression of CD94/NKG2A was determined at day 0 and every 5 days thereafter.

Detection of intracellular cytokines
CD4+ cells that had been stimulated as indicated above were harvested on day 20 of culture, washed, and resuspended in complete medium supplemented with 10 µg/mL of Brefeldin A (Sigma) but lacking IL-2. The cells were stimulated for 6 h in 24-well plates coated with either 1.5 µg/well of OKT3 mAb alone or a combination of OKT3 plus 2 µg/well of either mAb Z199 (anti-NKG2A) or mAb HP-3D9 (anti-CD94). After stimulation, the cells were lysed with fluorescence-activated cell sorter permeabilizing solution (Becton Dickinson) and incubated for 10 min at room temperature in the dark with labeled anticytokine antibodies (0.15 µg/106 cells). The cells were finally analyzed with the flow cytometer. Profiles of the stimulated cells were overlaid with profiles of the cells cultured in the presence of isotype-matched irrelevant antibodies, permeabilized, and stained as indicated in the flow cytometry methods.

RT-PCR of NKG2 genes
Total RNA from activated CD4+ T cells was extracted at the indicated time points by the LiCl/urea method. Briefly, 4 µg of total RNA were reverse transcribed using the avian myeloblastosis virus reverse transcription system (Promega, Madison, WI), and equal amounts of the resulting cDNA templates were amplified by PCR using the following specific oligonucleotides previously deduced [22 ] from the NKG2 gene sequences: NKG2A, forward primer 5'-CCAGAGAAGCTCATTGTTGG (spanning nucleotides 202–222) and reverse primer 5'-CCAATCCATGAGGATGGTG (spanning nucleotides 654–675); NKG2C, forward primer 5'-GGAAATATTCCAAGTAGAATTAAAT (spanning nucleotides 108–133) and reverse primer 5'-CTGATGCACTGTAAACGCAAAT (spanning nucleotides 677–700); NKG2D, forward primer 5'-CTGGGAGATGAGTGAATTTCATA (nucleotides 35 to 57), and reverse primer 5–-GACTTCACCAGTTTAAGTAAATC (nucleotides 451 to 429); and NKG2E, forward primer 5–-CTGTGCTTCAAAGAACTCTTCT (spanning nucleotides 432–454) and reverse 5'-CTGGTCTGATATAAGTCCACGT. After an initial denaturation step (2 min at 94°C), PCR amplification was carried out in a Gene-Amp 2400 thermal cycler (Perkin-Elmer, Norwalk, CT) for 30 cycles with the following conditions: annealing, 60°C for 15 s; extension, 72°C for 75 s; and denaturing, 94°C for 20 s. There was a final extension of 10 min at 72°C. Aliquots of PCR products were loaded onto 1.5% agarose gels, and the DNA was stained with ethidium bromide.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We have investigated the ability of human peripheral CD4+ cells to express of CD94/NKG2 molecules as a consequence of CD3-mediated activation. Thus, CD4 cells were obtained by immunomagnetic selection yielding consistently >95% of the CD3+ CD4+ populations, as detected by double immunofluorescence flow cytometry (Fig. 1 , left insert). These CD4+ resting lymphocytes did not express surface CD94 (Fig. 1 , right insert). The cells were stimulated with anti-CD3 mAb in the presence of exogenous IL-2 and restimulated as indicated in Materials and Methods. We detected a time-dependent expression of CD94 on activated CD4+ cells, which reached maximum levels at days 20–25 of culture (Fig. 1 , open squares). Because it is known that CD94 may be expressed by fresh peripheral CD8+ lymphocytes as well as NK cells, we monitored the presence of these populations throughout the culture. We found 1–3% of residual CD8+ contaminant cells on days of maximum CD94 expression, whereas the presence of NK cells was negligible (data not shown).



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  		Figure 1. Peripheral human CD4 lymphocytes express CD94/NKG2A molecules after CD3-mediated stimulation. CD4 T lymphocytes obtained from fresh peripheral blood mononuclear cells were purified as indicated in Materials and Methods, typically yielding populations that were 100% CD3+ CD4+ and CD4+ CD94-, as determined by double-immunofluorescence flow cytometry (top inserts). Cells were activated with immobilized anti-CD3 mAbs, and expression of CD94 ({square}) or CD94/NKG2A ({circ}) on CD4+ cells was determined every 5 days by flow cytometry. Data are the means and SD of three experiments.

 
The expression of different members of the NKG2 family on CD4+ cells was assessed with both serological and molecular probes. Thus, the presence of NKG2A on CD4+ cells was studied with the mAb Z199, and the peak of NKG2A surface expression paralleled that of CD94 on days 20–25 (Fig. 1 , open circles). At these time points, 10–15% of the cells present in the culture expressed NKG2A, whereas CD94 was detected on nearly 30% of the cells. Therefore, we investigated the expression of other molecules of the NKG2 family by RT-PCR because of the lack of mAbs that properly define other members of the NKG2 family at the protein level. We found amplification bands of expected sizes that indicated a sequential expression of NKG2 genes on activated CD4+ cells. Thus, NKG2D and -E gene products were detected as early as day 10 of culture, whereas NKG2A was not amplified by RT-PCR until day 15 of culture (Fig. 2 ). The remaining NKG2 member studied, NKG2C, appeared only after 20 days of culture.



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  		Figure 2. Expression of NKG2 gene family members on CD3-activated human peripheral CD4+ cells. Total RNA from activated CD4+ cells was extracted at the indicated time points after initiation of culture, reverse transcribed into cDNA, and amplified by PCR with specific oligonucleotide primers that discriminate gene products from NKG2 family members. Amplification of NKG2A yielded a 325-bp specific band, NKG2C yielded a 619-bp band, NKG2D yielded a 416-bp band, and NKG2E yielded a 225-bp band.

 
We studied next whether signals delivered by the cross-linking of the NKG2A receptor influence the profile of cytokine production by CD4 cells. We found that occupancy of the NKG2A by a specific mAb resulted in strong inhibition of IFN-{gamma} as well as TNF-{alpha} production, as detected by intracytoplasmic staining of cells (Fig. 3 , middle row). The occurrence of extensive cellular crosstalk mediated by the NKG2A receptor is noteworthy because most of the 34% of cells producing IFN{gamma} or of the 25% of those CD4 cells producing TNF-{alpha} did not express the CD94/NKG2A heterodimer, as indicated in Figure 1 . Cross-linking of the NKG2A molecule did not influence the production of IL-10 (Fig. 3) or other cytokines such as IL-5, -7, -13, and lymphotoxin (data not shown). Stimulation of the cells with a combination of anti-CD3 plus anti-CD94 mAbs did not modify the pattern of cytokine production observed in CD4 cells stimulated with OKT3 alone (Fig. 3 , bottom row). As expected, incubation of cells with anti-CD94 or anti-NKG2A mAb alone did not induce any cytokine production or inhibition (data not shown).



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  		Figure 3. Cross-linking of the NKG2A surface receptor inhibits production of IFN{gamma} and TNF-{alpha} by activated human CD4 cells. Activated cells were harvested at day 20 of culture, and cytokine production was assessed by flow cytometry after intracytoplasmic staining of IFN{gamma}, TNF-{alpha}, and IL-10. Cells were stimulated for 6 h with either OKT3 mAb alone (top row), a combination of OKT3 and anti-NKG2A (middle row) mAbs, or anti-CD94 mAb alone (bottom row). A representative experiment out of three is shown.

 
Because it is known that IL-10 [16 ] and TGF-ß [18 ] induce CD94 expression on CD8 cells, we examined whether some of these cytokines were involved in the surface induction of this molecule on CD4 cells. Thus, we performed blocking experiments with saturating amounts of neutralizing mAbs directed against those cytokines and found that the blockage of endogenous IL-10 resulted in inhibition of the number of CD4+ cells expressing CD94 (Fig. 4 ) as well as a complete absence of NKG2A surface expression. Neutralization of TGF-ß1 modestly decreased CD94 expression, affecting significantly the percentage of cells expressing NKG2A (Fig. 4) . The binding of specific mAbs to other cytokines, such as IL-15, did not influence the surface expression of the proteins. To address the functional significance of IL-10 in the process of CD94 induction on activated CD4 cells, cell cultures were carried out in the continuous presence of exogenous IL-10. Under these conditions, we observed a significantly higher level of expression of CD94 surface molecules on cells cultured in the presence of IL-10 (Fig. 5 , closed squares) than that for the control group of cells grown in the absence of this cytokine (Fig. 5 , open squares). Thus, >50% of the CD4 cells cultured with IL-10 expressed CD94, representing a twofold increase over the maximum percentages of stimulated cells expressing these molecules in the absence of IL-10. In the absence of CD3-mediated activation, IL-10 showed no effect on the expression of CD94/NKG2 on CD4+ T cells (data not shown).



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  		Figure 4. The surface expression of CD94/NKG2A on activated CD4+ cells is diminished by the continuous presence in the culture of anti-TGF-ß1 or anti-IL-10 mAbs. Human peripheral blood CD4 cells were stimulated via CD3/TCR in the continuous presence of saturating amounts of indicated anti-cytokine mAbs. Expression of CD94 or CD94/NKG2A on cells was determined at day 20 of culture. Data are percentages of inhibition versus cells expressing CD94 or CD94/NKG2A cultured in the presence of isotype-matched irrelevant antibodies (means ± SD of three experiments). Statistical analysis (Student’s t-test) indicated significant differences between cells cultured with irrelevant antibodies and those cultured with anti-TGF-ß1 (P <0.01) or anti-IL-10 (P <0.01 for CD94 and P <0.001 for NKG2A).

 


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  		Figure 5. Exogenous IL-10 enhances surface expression of CD94/NKG2A. Cells activated via CD3 were cultured in the presence (closed symbols) or absence (open symbols) of exogenous IL-10. Expression of CD94 (squares) and NKG2A (circles) was assessed by flow cytometry every 5 days of culture. Data are means ± SD of three experiments. Statistical analysis (Student’s t-test) revealed statistical differences between the groups of cells expressing CD94 in the presence or absence of IL-10 (P <0.05 for cells at day 15 and P <0.01 at day 20).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides evidence that CD3/TCR-mediated cell activation enabled CD4+ lymphocytes to express de novo CD94/NKG2 gene products. We observed a strong surface expression of CD94/NKG2A on CD4 cells, noting that the latter molecule was the only NKG2 member that could be defined by mAbs. A significant expression of NKG2A is detected only after a prolonged period of culture (15–20 days). These results are consistent with recent reports noting low-level expression of CD94/NKG2A on CD4+ cells 10 days after stimulation [18 ]. However, the higher expression of NKG2A molecules on CD4 cells found in this study compared with others might be explained by differences in the stimuli used, the composition of the cell population, the length of culture, or the cytokines added [17 , 18 ]. The percentage of cells that acquired surface expression of CD94 exceeded that of cells expressing NKG2A. The gap was filled by the coupling of the CD94 molecule to other members of the NKG2 gene family (NKG2C and -E), as revealed by RT-PCR, thereby representing the full array of activating and inhibitory receptors in CD4+ cells. This observation might be of particular relevance in view of our preliminary data indicating that no other killer Ig-like receptors are expressed on CD4+ T lymphocytes [P. Romero, C. Ortega, A. Palma, I. J. Molina, and M. Santamaria, unpublished observations]. However, the possibility that some cells express CD94 as homodimers cannot be excluded, although no function has been ascribed to such a form of CD94 when found on NK cells and CD8+ lymphocytes [23 ]. Remarkably, we have detected expression of NKG2D, which does not associate with CD94 [7 ] but does associate with DAP10 [11 ], forming an activating receptor that triggers cell-mediated cytotoxicity and that might also augment production of cytokines initiated by signals delivered by other receptors. NKG2D does not recognize the HLA-E antigen as other NKG2 members do; instead, it binds several ligands structurally related to the major histocompatibility complex class I, such as MICA, MICB [24 ], ULBP1, ULBP2, and retinoic acid-inducible genes, whose products are frequently found on tumor cells [25 ]. The existence of a number of inducible ligands for NKG2D and the ability of ULBP1 and ULBP2 to bind cytomegalovirus (CMV)-encoded UL16 proteins support an active participation of NKG2D receptors in immune surveillance against tumors and pathogen-infected cells [3 ]. Expression of NKG2D on NK cells, {gamma}{delta}TCR+ cells, and CD8+ {alpha}ßTCR+ cells has been described [26 ]; however, to the best of our knowledge, this is the first time that expression of NKG2D molecules has been detected on human CD4+ {alpha}ßTCR+ lymphocytes. It is also noteworthy that, among the NKG2 gene family members, NKG2D mRNA is the first to appear strongly and that its expression is well sustained throughout the culture period. However, NKG2D expression at the protein level in polyclonal CD4+ populations cannot be demonstrated until specific serological probes become available.

NKG2 molecules appear sequentially in a time-dependent fashion, suggesting that each type of receptor might be required at different stages of the response. Indeed, we showed that coligation of NKG2A and CD3 resulted in a significant down-modulation of functions mediated by the CD3/TCR complex, as demonstrated by the inhibition of IFN-{gamma} and TNF-{alpha} production. This is consistent with recently reported results supporting the functional involvement of CD94/NKG2 receptors in either the up- or down-regulation of IFN{gamma} and TNF-{alpha} production in both CD8+ T cells and NK cells [27 ]. It is noteworthy that the production of all the T helper (Th) 2 cytokines studied was not affected by coupling of the NKG2A receptor, suggesting that NKG2A is mainly involved with the modulation of Th1 cytokines. The involvement of CD94/NKG2A receptors with the control of cytokine production in CD4+ cells is plausible because inhibitory heterodimers act by preventing tyrosine phosphorylation of activation receptors such as 2B4, whose engagement triggers IFN{gamma} secretion by NK cells [28 , 29 ]. The fact that occupancy of the NKG2A receptor, which was expressed on 15–20% of cells, results in selective and complete inhibition of IFN{gamma} and TNF-{alpha} , which are produced by a higher proportion of cells, suggests that there is extensive intercellular communication. However, because Brefeldin A was used during the assays and protein secretion was therefore prevented, the signal(s) to down-regulate the cytokine secretion could not be delivered by a secreted protein. Thus, the prime candidates for mediating this effect are membrane-expressed receptor-ligand pair molecules. The ability to produce large amounts of IFN{gamma} and to express CD94/NKG2A might suggest that the CD4+ cells used in this study belong to the NK T-cell population, which is defined by expression of V{alpha}24+ TCR and IFN{gamma} and IL-4 production [30 ]; however, this possibility is unlikely because these cells do not express the V{alpha}24+ TCR (data not shown).

The contribution of these receptors to the immunobiology of CD4+ lymphocytes and the functional characterization of these populations (i.e., whether they are naïve or memory cells or polarized, regulatory, effector, or anergic T lymphocytes) need to be examined. In this context, it could be speculated that the expression of CD94/NKG2 receptor(s) on human CD4 cells during late stages of cell activation might contribute to the regulation of chronic immune responses (i.e., certain viral infections or chronic allograft rejection). Given the fact that CD94/NKG2 receptors are actively involved in the regulation of cytotoxic responses, their roles in CD4+ cells would be related to control of cytotoxicity by modulating cytokine production. In light of our results, it is conceivable that inhibitory and activating receptors of the CD94/NKG2 family might help to adjust the overall Th1 cytokine levels on interaction with its ligand, the HLA-E molecule, thereby leading to either inhibition or enhancement of cytotoxic function by CD8+ and NK cells. It is interesting that HLA-E antigen, which may be expressed on all nucleated cells, is strongly up-regulated in human CMV infection [31 ]. This model also accommodates very recent data showing that NKG2D, which does not associate with CD94, acts as a surrogate costimulatory molecule for CMV-specific CD8+{alpha}ß+ CD28- effector T cells. Remarkably, the ligand for NKG2D, MIC, is also strongly up-regulated during CMV infection, and it has been suggested that cytokine modulation also plays a role in this costimulatory process [32 ]. The data reported here showing the expression of CD94/NKG2 receptors as well as NKG2D molecules on CD4+ cells, together with the ability of NKG2D to function as either an inhibitory or an activating receptor, provide new insights regarding the resources available to CD4+ cells to control and drive immune responses.


   ACKNOWLEDGEMENTS
 
This work was supported by CICYT grants SAF98-0165-CO2-02 and FIS99-862 (to M. S.), SAF98-0165-CO2-01 (to I. J. M.), and FIS98-1051 (to J. P.). P. R. is supported by a fellowship grant from Pharmacia Diagnostic (Spain).

We are grateful to Miguel López-Botet and Maria Grazia Roncarolo for their generous gifts of antibodies. We are also indebted to Hoffman-LaRoche for their continuous supply of rIL-2.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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B. T. Wilhelm, J.-R. Landry, F. Takei, and D. L. Mager
Transcriptional Control of Murine CD94 Gene: Differential Usage of Dual Promoters by Lymphoid Cell Types
J. Immunol., October 15, 2003; 171(8): 4219 - 4226.
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