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The Journal of Immunology, 2002, 168: 3295-3302.
Copyright © 2002 by The American Association of Immunologists

Expression of Inhibitory Receptors Ly49E and CD94/NKG2 on Fetal Thymic and Adult Epidermal TCR V{gamma}3 Lymphocytes1

Katrien Van Beneden, An De Creus, Frederik Stevenaert, Veronique Debacker, Jean Plum and Georges Leclercq2

Department of Clinical Chemistry, Microbiology, and Immunology, University of Ghent, University Hospital, Ghent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ly49 and CD94/NKG2 inhibitory receptors are predominantly expressed on murine NK cells, but they are also expressed on a subpopulation of peripheral CD8 memory TCR {alpha}{beta} lymphocytes. In this study we demonstrate that Ly49E and CD94/NKG2 receptors are expressed on mature TCR V{gamma}3+ cells in the fetal thymus. Expression correlated with a memory phenotype, such as expression of CD44, 2B4, and IL-2R{beta} (CD122), and absence of IL-2R{alpha} (CD25) expression. No expression of Ly49A, C, D, G2, or I receptors was observed. This phenotype is similar to that of fetal thymic NK cells. Skin-located V{gamma}3 T cells, the progeny of fetal thymic V{gamma}3 cells, also expressed CD94/NKG2 and Ly49E but not the other members of the Ly49 family. The development and survival of Ly49E+ or CD94/NKG2+ V{gamma}3 T lymphocytes was not dependent upon expression of MHC class I molecules. The cytotoxicity of TCR V{gamma}3 cells was inhibited when Qdm, the ligand for CD94/NKG2, was presented by Qa1b-transfected target cells. Also, upon cross-linking of CD94/NKG2 with mAb 3S9, TCR V{gamma}3 thymocytes were prevented from killing Fc{gamma}R+ P815 target cells. These effects were most pronounced in the CD94/NKG2high subpopulation as compared with the CD94/NKG2low subpopulation of V{gamma}3 cells. Our data demonstrate that V{gamma}3 T cells expressing inhibitory Ly49E and CD94/NKG2 receptors are mature and display a memory phenotype, and that CD94/NKG2 functions as an inhibitory receptor on these T lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells express inhibitory receptors specific for MHC class I molecules. Interaction of these receptors with their ligands results in inhibition of NK cell-mediated cytotoxicity. This mechanism enables NK cells to discriminate between MHC class I+ and MHC class I- target cells and to lyse virus-infected and transformed cells that lack self-MHC class I Ags (1, 2). NK cell receptors can be divided in three families. One family contains the killer cell inhibitory receptors (KIRs)3 expressed on human NK cells (3). The second family has been identified in mouse and contains the Ly49 receptors. In C57BL/6 mice, the Ly49 family consists of 14 highly related genes (Ly49A–N) encoding lectin-like glycoproteins expressed as disulfide-linked homodimers (4, 5, 6, 7, 8). Similar to human KIRs, murine Ly49 receptors have been shown to recognize different classical MHC class I molecules (3, 9, 10, 11). The third family consists of lectin-like CD94/NKG2A–F heterodimeric receptors that are expressed in human and rodents (3, 12, 13, 14, 15). The human and murine CD94/NKG2 heterodimers recognize the nonclassical MHC class I molecules HLA-E and Qa-1b, respectively. Like HLA-E, the murine homolog Qa-1b binds TAP-dependent peptides derived from MHC class I signal sequences (15, 16, 17, 18, 19).

NK receptors were initially described on NK cells but are also detected on NKT cells and on TCR {alpha}{beta} and {gamma}{delta} T cells. In humans, KIR and CD94/NKG2 receptors are expressed on a minor subset of peripheral CD8+ {alpha}{beta} T lymphocytes and are rare on CD4+ {alpha}{beta} T cells (20, 21). The distribution of CD94/NKG2 includes the majority of circulating human cytotoxic {gamma}{delta} T cells, while a minor subpopulation of these {gamma}{delta} T cells expresses KIRs (22, 23, 24, 25, 26). Circulating human {gamma}{delta} T cells predominantly express the V{gamma}9V{delta}2 TCR and recognize phosphoantigens in a MHC-unrestricted manner (27). KIRs and CD94/NKG2 are infrequently distributed on the V{delta}1 subset (23, 24, 25, 26). KIR+ or CD94/NKG2+ TCR {alpha}{beta} and {gamma}{delta} cells bear surface markers characteristic of a memory phenotype, and analysis of T lymphocytes from cord blood, fetal, or adult thymus failed to detect KIR or CD94/NKG2 expression on these cells, suggesting that inhibitory NK receptors are expressed on peripheral T lymphocytes following activation (20, 22, 23, 28). Functional studies have demonstrated that activation of KIRs and CD94/NKG2 receptors on human {alpha}{beta} and {gamma}{delta} T cells leads to inhibition of cytotoxicity and cytokine production (29).

In mouse, Ly49 and CD94/NKG2 molecules are preferentially expressed on CD8+ TCR {alpha}{beta} cells and are infrequent on conventional CD4+ {alpha}{beta} T cells (29, 30, 31, 32, 33). NK receptor expressing CD8+ TCR {alpha}{beta} cells are memory T cells phenotypically defined as CD8+CD44+ (31). Additionally, Ly49 and CD94/NKG2 expression has been observed on gut intraepithelial TCR {alpha}{beta}+ T cells, which differentiate extrathymically (32, 34), and on a considerable part of NKT cells (29, 32, 33, 35, 36). Expression of Ly49 inhibitory receptors on {alpha}{beta} T cells has been shown to confer inhibition on T cell activation and effector functions (9, 30, 31, 37, 38).

Little information is available regarding the NK cell receptor distribution on murine TCR {gamma}{delta} cells. One report shows CD94 expression on intraepithelial {gamma}{delta} T cells in adult mice (32). In addition, NK1.1, Ly49A, Ly49C/I, and Ly49G2 receptors have been shown to be expressed on a fraction of adult thymic {gamma}{delta} T cells and are also distributed on peripheral {gamma}{delta} T cells isolated from spleen and liver. The surface expression of Ly49 receptors on these {gamma}{delta} T cells is correlated with NK1.1 expression (30, 39, 40, 41). Evidence for an inhibitory role of these NK receptors in {gamma}{delta} T cells is missing.

In humans, KIR expressing memory CD8+ TCR {alpha}{beta} cells express both 2B4 and IL-2R{beta} molecules and are negative for CD25 expression. These cells are referred to as T memory type 1 (Tm1) cells (42). Similar to human Tm1 cells, murine Ly49 expressing CD8+ memory {alpha}{beta} T cells have a CD25-IL-2R{beta}+ phenotype (31). It remains to be defined whether murine {gamma}{delta} T cells also contain Tm1 phenotype cells. There is evidence that one subpopulation of murine TCR {gamma}{delta} cells, namely TCR V{gamma}3 T cells, might have a Tm1 phenotype. V{gamma}3 T cells express the canonical V{gamma}3/V{delta}1 TCR and appear around fetal days (FD)14–15 as the first wave of T lymphocytes during fetal thymic development (43, 44). TCR V{gamma}3 T lymphocytes are the precursors of V{gamma}3/V{delta}1 T cells in the epidermis of adult mice (45). V{gamma}3+ T cells express IL-2R{beta} (46) and are CD25- (47), and the expression of 2B4 has been described on skin-located V{gamma}3 T cells (48). Because these data indicate that V{gamma}3+ T cells might have a Tm1 phenotype, we investigated whether these cells also express inhibitory NK receptors. In this report, we demonstrate that Ly49 and CD94/NKG2 receptors are expressed on fetal thymic and skin-located V{gamma}3 T cells. V{gamma}3 T cells express Ly49E but not the other members of the Ly49 family. The development of Ly49E+ or CD94/NKG2+ V{gamma}3 T lymphocytes is not dependent upon expression of MHC class I molecules. Finally, functional analysis demonstrated that the CD94/NKG2 receptor inhibits the cytolytic activity of V{gamma}3 T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

C57BL/6J (B6) mice were originally purchased from Harlan (Zeist, The Netherlands). {beta}2-microglobulin-deficient ({beta}2m-/-) mice backcrossed five generations onto the B6 strain from the original 129 x B6 chimera were obtained from Taconic Farms (Germantown, NY). Mice were bred in our breeding facility. To obtain dated pregnant mice, mice were mated for 15 h and the fetuses were removed from FD15 to FD18 (plug date = day 0). Mice were treated and used in agreement with the institutional guidelines.

Media and reagents

RPMI 1640 or DMEM media were supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.03% glutamine, and 5 x 10-5 M 2-ME (all from Life Technologies, Paisley, U.K.). These media will be further referred to as complete RPMI 1640 or complete DMEM, respectively. Purified human rIL-2 and rIL-7 were kindly provided by Dr. M. Gately (Hoffmann-LaRoche, Nutley, NJ) and Dr. S. Gillis (Immunex, Seattle, WA), respectively.

Preparation of cell suspensions

Thymuses were removed and disrupted using a small potter homogenizer. Spleens from 8- to 12-wk-old mice were removed and teased apart. Erythrocytes from spleens were lysed with 0.17 M NH4Cl, and the remaining lymphocytes were washed three times. Cells were counted with trypan blue to exclude dead cells and were suspended in complete RPMI 1640 medium. Skin was removed from killed adult mice and was freed of fat tissue followed by flotation, dermal side down, on 0.3% trypsin solution (Sigma-Aldrich, St. Louis, MO) at 4°C for 18 h. Skin samples were pooled in complete DMEM containing 0.25% DNase (Boehringer Mannheim, Mannheim, Germany). Single cell suspensions were prepared by mechanical agitation and separated from dead cells by lympholyte-M (Cedarlane Laboratories, Hornby, Canada) density gradient centrifugation. Interface cells were collected and cultured overnight in complete RPMI 1640 medium supplemented with 250 U/ml IL-7 to allow recovery of surface expression of membrane proteins.

IL-2 stimulation

Thymus cell suspensions were cultured in 24-well plates (Falcon; BD Biosciences, Mountain View, CA) at 2 x 106 cells per well in 2 ml complete RPMI 1640 medium with a final concentration of 1000 U/ml IL-2. After culture for 4 days at 37°C, cells were harvested, washed twice, and counted with trypan blue.

Antibodies

mAbs used were FITC-, biotin-, and PE-conjugated anti-TCR V{gamma}3 (hamster IgG, clone F536 (44); BD PharMingen, San Diego, CA), biotin-conjugated anti-{gamma}{delta} TCR (hamster IgG, clone 13D5), anti-Fc{gamma}RII/III (unconjugated, rat IgG2b, clone 2.4G2; kindly provided by Dr. J. Unkeless, Mount Sinai School of Medicine, New York, NY), biotin-conjugated anti-HSA (rat IgG2b, clone M1/69; BD PharMingen), PE-conjugated anti-CD44 (rat IgG2b, clone IM7; BD PharMingen), biotin-conjugated anti-CD25 (rat IgG1, clone PC61; kindly provided by Dr. M. Nabholz, Epalinges, Switzerland) (49), PE-conjugated and unconjugated anti-2B4 (mouse IgG2b, clone 2B4; BD PharMingen), FITC-conjugated anti-IL-2R{beta} (rat IgG2b, clone TM-{beta}1; kindly provided by Dr. T. Tanaka, Tokyo, Japan) (50), FITC-conjugated anti-Ly49E/C (rat IgG2a, clone 4D12) (33), FITC-conjugated and unconjugated anti-NKG2A/C/E (rat IgG2b, clone 3S9) (33), biotin-conjugated anti-Ly49A/D (rat IgG2a, clone 12A8; ascites kindly provided by Dr. J. Ortaldo, National Cancer Institute, Frederick, MD), PE-conjugated anti-Ly49C/I (mouse IgG2a, clone 5E6; BD PharMingen), FITC-conjugated anti-Ly49G2 (rat IgG2a, clone 4D11; American Type Culture Collection, Manassas, VA), unconjugated rat IgG2b isotype control (clone A95-1; BD PharMingen), and unconjugated anti-Qa1b (clone 6F10; BD PharMingen).

FCA and sorting

Where indicated, freshly isolated thymocytes and adult splenocytes were depleted of CD4 T cells using unconjugated L3T4 mAb and sheep anti-mouse IgG Dynabeads (Dynal Biotech, Hamburg, Germany). To avoid aspecific binding, Fc{gamma}R was blocked by preincubation of cells with saturating amounts of anti-Fc{gamma}RII/III mAb. Cells were incubated with the indicated mAbs for 45 min at 4°C. After washing, biotin-conjugated mAbs were revealed with second-step streptavidin-allophycocyanin or streptavidin-PE (BD Biosciences). Cells were analyzed for fluorescence using a FACSCalibur flow cytometer (BD Immunocytometry Systems, Mountain View, CA) equipped with an argon laser (488 nm) and a helium-neon laser (633 nm) with the CellQuest software program (BD Biosciences) for data acquisition and analysis. Propidium iodide was added to the cells (2 µg/ml) just before flow cytometric analysis (FCA). Gating was done on propidium iodide negative cells to exclude dead cells. Sorting was performed on a FACSVantage (BD Biosciences) equipped with an argon laser.

Cell-mediated cytotoxicity

Tumor targets used were the mastocytoma P815 (H-2Kd,Dd) and TAP-deficient T2Q cells transfected with the Qa1b gene and untransfected T2 cells (51) (kindly provided by Dr. C. Brooks, The Medical School, Newcastle, U.K.). A total of 1 x 106 target cells were labeled with 100 µCi 51Cr (Amersham, Little Chalfont, Buckinghamshire, England) in serum-free medium for 60 min at 37°C. Cells were washed three times. As shown previously, the cytotoxicity of FD17 thymocytes can be triggered by preincubation with mAb 2B4 (52). Cytolytic activity against P815 was tested in the presence of 3S9 mAb or isotype control Ab both at a final concentration of 30 µg/ml. When untransfected T2 cells or Qa1b-transfected T2Q cells were used, these cells were incubated at 26°C with 30 µM Qdm peptide (AMAPRTLLL; kindly provided by Dr. C. Brooks) (53) or with an unrelated peptide (ESIINFEK; kindly provided by Dr. J. Vandekerckhove, Ghent, Belgium) for 18 h before the killing assay. A total of 30 µM peptide was also added during 51Cr labeling and during the cytotoxicity assay. In blocking studies, T2Q target cells loaded with Qdm peptide were preincubated with anti-Qa1b mAb at room temperature for 1 h and assayed for lysis by V{gamma}3 T cells in the presence of 20 µg/ml anti-Qa1b mAb. Graded effector cell numbers were added in duplicate to 103 tumor cells in V-bottom wells of a 96-well plate in a final volume of 100 µl/well. After incubation for 4 h at 37°C, 75 µl supernatant was removed from each well. A total of 225 µl Optiphase Supermix (Wallac, Turku, Finland) was added to the supernatants and radioactivity was measured using a 96-well scintillation counter (Microbeta; Wallac). The spontaneous release of radioactivity was determined in wells without effector cells, and the maximal release in wells in which target cells were lysed by addition of 1% Triton X-100 at the start of incubation. The percentage of specific lysis was calculated using the following equation: 100 x (experimental - spontaneous release)/(maximal - spontaneous release).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ly49E and NKG2 receptors are expressed by TCR V{gamma}3 cells during differentiation in the fetal thymus

Earlier analysis by our group has shown that during fetal life immature TCR V{gamma}3lowHSAhigh cells differentiate in the thymus into mature TCR V{gamma}3 cells with a V{gamma}3highHSAlow phenotype (54). Recently, we have generated two mAbs against NK receptors: mAb 4D12 recognizing Ly49E with cross-reactivity to Ly49C, and mAb 3S9 against NKG2A/C/E (33). To investigate whether Ly49E/C and NKG2 NK receptors are expressed on TCR V{gamma}3 thymocytes, thymocytes from FD15 to FD18 were freshly isolated and analyzed by gating on V{gamma}3+ T cells using flow cytometry. Fig. 1Go shows that FD15 V{gamma}3 thymocytes are immature (HSAhigh) and did not express Ly49E/C or NKG2 receptors. From FD16, part of the TCR V{gamma}3 thymocytes become mature (HSAlow) and Ly49E/C and NKG2 receptors were expressed on a subpopulation of these mature cells. The percentages of mature TCR V{gamma}3 thymocytes expressing Ly49E/C or NKG2 increased from ~35% at FD16 to ~60% for Ly49E/C and ~50% for NKG2 at FD17–18.



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  		FIGURE 1. Ly49E/C and NKG2 expression by TCR V{gamma}3 cells during differentiation in the fetal thymus. FD15–18 B6 thymocytes were freshly isolated and stained with PE-conjugated anti-V{gamma}3 and biotin-conjugated anti-HSA and then revealed with streptavidin-allophycocyanin and FITC-conjugated 4D12 or FITC-conjugated 3S9. Expression was analyzed by flow cytometry by gating on the V{gamma}3+ cells. Expression of Ly49E/C or NKG2A/C/E on HSAlowV{gamma}3+ T cells is shown in parentheses. Results shown are representative of three experiments.

 
The above data show that both Ly49E/C and NKG2 receptors are expressed on a subpopulation of TCR V{gamma}3 thymocytes. A more detailed analysis of the NK receptor expression on thymic TCR V{gamma}3+ lymphocytes is shown in Fig. 2Go. Because the highest percentage of mature TCR V{gamma}3 thymocytes is found at FD17 (data not shown), we focused on this stage of development. As fetal V{gamma}3 thymocytes are the precursors of V{gamma}3+ T cells in the epidermis of adult mice (45), we also analyzed the expression of NK receptors by skin-located V{gamma}3 T cells in parallel. The NK receptor expression was analyzed by gating on V{gamma}3+ cells using flow cytometry. Whereas NKG2 was detected on ~30% of total FD17 TCR V{gamma}3+ thymocytes, NKG2+ cells comprised ~70% of epidermal V{gamma}3+ T cells. mAb 4D12 (anti-Ly49E/C) stained one-third of TCR V{gamma}3+ T cells isolated from FD17 thymus or adult skin. Analysis of both thymic and epidermal V{gamma}3 T cells revealed that mAb 5E6, recognizing Ly49C and I, did not stain V{gamma}3 T cells. Therefore, the present data demonstrate that fetal thymic and skin-located V{gamma}3 T cells expressed Ly49E but not Ly49C. We also failed to detect expression of Ly49A, D, or G2 on V{gamma}3 T lymphocytes. To exclude the possibility that a small subpopulation of TCR V{gamma}3 thymocytes expressed Ly49 receptors, FD17 thymocytes were cultured with rIL-2 for 4 days. This resulted in an increase in the percentage of V{gamma}3 T cells (47). However, no expression of other Ly49 molecules could be observed (data not shown). Further phenotypic analysis revealed that 2B4 and IL-2R{beta} were expressed on the majority of thymic TCR V{gamma}3+ lymphocytes and on all skin-located V{gamma}3 T cells (Fig. 2Go). Neither thymic nor epidermal V{gamma}3 T cells expressed the NK receptor DX5 (data not shown). Approximately 10% of FD17 TCR V{gamma}3+ thymocytes expressed the NK receptor NK1.1, and this percentage increased to ~40% after IL-2 culture (data not shown).



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  		FIGURE 2. Expression of NK receptors on fetal thymic and skin-located V{gamma}3+ T cells. Freshly isolated FD17 B6 thymocytes and epidermal cells prepared from adult B6 mice were stained with PE- or FITC-conjugated anti-V{gamma}3 mAb in combination with FITC-conjugated 3S9, FITC-conjugated 4D12, and biotin-conjugated 12A8, and then revealed with streptavidin-PE, PE-conjugated 5E6, FITC-conjugated 4D11, PE-conjugated anti-2B4, or FITC-conjugated anti-IL-2R{beta} mAbs. Open histograms show the expression of indicated receptors by gating on V{gamma}3+ T cells. Filled histograms represent background staining. Results shown are representative of more than three experiments.

 
Expression of CD94/NKG2 and Ly49E on {gamma}{delta} T cells other than V{gamma}3 T cells

The expression of CD94/NKG2 and Ly49E was also analyzed on neonatal (day 0) thymic and on adult splenic {gamma}{delta} T cells. Because a small percentage of V{gamma}3 cells is still present in neonatal thymus, V{gamma}3+ cells were gated out in these samples. CD94/NKG2 was detected on ~30% of splenic {gamma}{delta} T cells and on only 0.4% of neonatal thymic V{gamma}3-{gamma}{delta}+ T cells (Fig. 3GoA). The low frequency of CD94/NKG2+ neonatal thymic {gamma}{delta} T cells could not be due to their immaturity, because 40% of neonatal V{gamma}3-{gamma}{delta}+ T cells expressed low levels of HSA, representing mature {gamma}{delta} T cells (data not shown). Fig. 3GoA shows that ~3% of adult splenic {gamma}{delta} T cells and <1% of neonatal V{gamma}3-{gamma}{delta}+ T cells expressed Ly49E/C. Because mAb 4D12 recognizes both Ly49E and Ly49C, we performed staining with mAb 4LO3311 alone and double staining with mAbs 4D12 and 4LO3311. Staining with mAb 4LO3311 alone revealed that ~0.2 and 1.6%, respectively, of neonatal thymic and adult splenic {gamma}{delta} T cells expressed Ly49C. By gating on TCR {gamma}{delta}+ splenocytes, costaining demonstrated that 2.1% of these {gamma}{delta} T cells were 4D12 single-positive. As previously reported (33), it is reasonable to assume that only the bright 4D12 single-positive {gamma}{delta} T cells, which represent 0.5% of splenic {gamma}{delta} T cells (Fig. 3GoB, oval area in dot plot), resemble Ly49C-E+ {gamma}{delta} T cells. Because we are not able to distinguish more than three different fluorochromes, we could not combine 4D12 and 4LO3311 mAbs on fetal V{gamma}3-{gamma}{delta}+ thymocytes. In conclusion, these data demonstrate that Ly49E and CD94/NKG2 are less frequently expressed on neonatal thymic and adult splenic {gamma}{delta} T cells compared with V{gamma}3 T cells.



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  		FIGURE 3. Ly49E/C and CD94/NKG2 expression by non-V{gamma}3 {gamma}{delta} T cells. A, Freshly isolated neonatal (day 0) B6 thymocytes were depleted for CD4+ T cells and stained with following mAbs: FITC-conjugated 3S9, FITC-conjugated 4D12, or biotin-conjugated 4LO3311, and PE- or FITC-conjugated anti-V{gamma}3, and PE- or biotin-conjugated anti-{gamma}{delta} TCR, and then revealed with streptavidin-allophycocyanin. Expression of NK receptors on {gamma}{delta} T cells was analyzed by gating on V{gamma}3- thymocytes. Freshly isolated splenocytes from adult mice were depleted for CD4 T cells and B cells. Cells were stained with FITC-conjugated 3S9, FITC-conjugated 4D12, or biotin-conjugated 4LO3311, and PE- or biotin-conjugated anti-{gamma}{delta} TCR, and then revealed with streptavidin-allophycocyanin. The percentages shown in the upper right quadrant of each dot plot represent the expression of the indicated NK receptor on {gamma}{delta}+ T cells. B, Dot plot shows costaining with mAbs 4D12 and 4LO3311 by gating on TCR {gamma}{delta}+ splenocytes. Results are representative of three experiments.

 
V{gamma}3+ thymocytes expressing NK receptors exhibit a memory phenotype

Because it appeared that Ly49E and NKG2A/C/E NK receptors are exclusively expressed on mature TCR V{gamma}3 thymocytes, we examined whether expression of 2B4 and IL-2R{beta} by V{gamma}3 thymocytes also correlated with a mature phenotype. As shown in Fig. 4Go, both 2B4 and IL-2R{beta} receptors were mainly expressed on mature HSAlow TCR V{gamma}3 thymocytes. Furthermore, we questioned whether expression of NK receptors on mature TCR V{gamma}3 thymocytes parallels with a memory phenotype by these cells. Fig. 4Go shows that all Ly49E+, NKG2+, or IL-2R{beta}+ V{gamma}3+ T cells expressed CD44, which is indicative of a memory phenotype. In accordance with this, essentially all Ly49E+, NKG2+, or IL-2R{beta}+ V{gamma}3+ T cells did not express CD25.



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  		FIGURE 4. V{gamma}3+ thymocytes expressing NK receptors exhibit a memory phenotype. FD17 B6 thymocytes were freshly isolated and stained with the following mAbs: FITC-conjugated 4D12 (anti-Ly49E/C), FITC-conjugated 3S9 (anti-NKG2A/C/E), or FITC-conjugated anti-IL-2R{beta} (CD122) in combination with biotin-conjugated anti-HSA, or biotin-conjugated anti-IL-2R{alpha} (CD25), and then revealed with streptavidin-allophycocyanin and PE-conjugated anti-V{gamma}3, or in combination with PE-conjugated anti-CD44 and biotin-conjugated anti-V{gamma}3, and then revealed with streptavidin-allophycocyanin. When PE-conjugated anti-2B4 was used, thymocytes were stained with biotin-conjugated anti-HSA and then revealed with streptavidin-allophycocyanin and FITC-conjugated anti-V{gamma}3. Cells were analyzed by flow cytometry by gating on V{gamma}3+ cells. Results shown are representative of three experiments.

 
Ly49E and NKG2A/C/E expression by TCR V{gamma}3+ lymphocytes is not dependent on MHC class I molecules

Coles et al. (31) demonstrated that expression of Ly49 receptors on memory CD8+ TCR {alpha}{beta} cells is predominantly dependent upon MHC class I expression. To analyze whether expression of Ly49E and CD94/NKG2 receptors on V{gamma}3 T cells is also affected by loss of MHC class I molecules, we compared the expression of these receptors on TCR V{gamma}3 thymocytes isolated from FD17 wild-type (WT) vs {beta}2m-/- B6 mice. As illustrated in Fig. 5Go, we observed similar percentages of Ly49E- and NKG2-positive cells, as well as similar expression levels of these receptors on mature FD17 V{gamma}3 thymocytes from {beta}2m-/- mice compared with WT mice. This shows that Ly49E and NKG2 expression on V{gamma}3 cells is not dependent upon MHC class I expression. Because it has been demonstrated that the expression of Ly49C on NK cells is up-regulated in {beta}2m-/- mice compared with WT mice (55, 56), we wanted to exclude the possibility that the similar percentages of 4D12+ V{gamma}3 T cells in both mice were due to an increased expression of Ly49C in {beta}2m-/- mice. For this purpose, the expression of Ly49C on V{gamma}3 T cells was compared between WT and {beta}2m-/- mice. Fig. 5Go demonstrates that Ly49C is not expressed on FD17 V{gamma}3 thymocytes from both WT and {beta}2m-/- mice. Therefore, these data show that similar frequencies of Ly49E+ V{gamma}3 T cells are present in WT and {beta}2m-/- mice.



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  		FIGURE 5. Comparison of Ly49E and CD94/NKG2 expression on V{gamma}3 T cells from FD17 WT and {beta}2m-/- mice. FD17 thymocytes from WT and {beta}2m-/- mice were freshly isolated and stained with FITC- or PE-conjugated anti-V{gamma}3, biotin-conjugated anti-HSA, and with FITC-conjugated 4D12, FITC-conjugated 3S9 mAbs, or PE-conjugated 5E6. FCA was performed by gating on V{gamma}3+ cells. Results shown are representative of three experiments.

 
CD94/NKG2-mediated inhibition of tumor cell lysis by TCR V{gamma}3+ T cells

To examine whether the CD94/NKG2 receptors expressed on V{gamma}3 T cells are functional, we analyzed their role in the cytotoxic activity of these cells. TCR V{gamma}3+ cells were sorted from FD17 thymocytes after 3 days of IL-2 culture. After an additional culture with IL-2 for 3 days to remove mAbs from the cell surface, V{gamma}3 T lymphocytes were used as effector cells in different cytotoxic assays. As illustrated in Fig. 6GoA, Fc{gamma}R+ P815 target cells were at least four times less susceptible to lysis after pretreatment of V{gamma}3+ T cells with mAb 3S9 compared with isotype control mAb, indicating that cross-linking of CD94/NKG2 results in inhibition of the cytotoxicity of V{gamma}3 cells. Next, we analyzed the cytotoxic activity of CD94/NKG2low and CD94/NKG2high sorted subsets of V{gamma}3+ T cells. Part (~20%) of sorted CD94/NKG2low V{gamma}3 cells up-regulated CD94/NKG2 expression during the additional IL-2 culture, while expression of CD94/NKG2 on sorted CD94/NKG2high V{gamma}3 T cells was stable (data not shown). As demonstrated in Fig. 6GoB, P815 targets were less susceptible to lysis by V{gamma}3+CD94/NKG2high T cells as compared with V{gamma}3+CD94/NKG2low T cells after pretreatment of these effector cells with anti-NKG2 mAb. To obtain more evidence that CD94/NKG2 functions as an inhibitory receptor on V{gamma}3 T cells, we performed a cytotoxic assay using target cells that are able to present the ligand of CD94/NKG2 (53, 57). The ligand for CD94/NKG2 is the Qdm peptide presented in the context of Qa1b molecule. Qdm is derived from the leader sequence of classical MHC class I molecules and forms a complex with the nonclassical MHC Qa1b molecule peptide in a TAP-dependent manner (15, 16, 17, 18, 19). T2Q cells transfected with Qa1b were preincubated in the presence of Qdm peptide or unrelated peptide and were used as target cells. As illustrated in Fig. 6GoC, the lysis of T2Q target cells by V{gamma}3 T cells was clearly inhibited by addition of Qdm peptide but not unrelated peptide. The inhibition of V{gamma}3 killing activity was not due to nonspecific effects of Qdm peptide alone, because untransfected T2 targets incubated with Qdm peptide were efficiently lysed by V{gamma}3 T cells. Addition of anti-Qa1b mAb to T2Q targets loaded with Qdm reversed inhibition of cytolytic activity of V{gamma}3 T cells (Fig. 6GoC). Fig. 6GoD shows that T2Q target cells, preincubated with Qdm, were less susceptible to lysis by the CD94/NKG2high subset of V{gamma}3 T cell compared with the CD94/NKG2low subset of V{gamma}3 T cells. The slight reduction in the killing of T2Q targets incubated with Qdm by CD94/NKG2low V{gamma}3 cells, as compared with the killing of T2Q targets incubated with control peptide, is probably due to low expression of CD94/NKG2 on the majority of these effector cells and to the up-regulation of CD94/NKG2 on part of these cells. T2Q targets incubated with unrelated peptide were equally killed by both effector cells. In conclusion, these data demonstrate the significance of Qa1b/Qdm recognition by CD94/NKG2 and show that CD94/NKG2 expressed on TCR V{gamma}3 T cells functions as an inhibitory receptor following recognition of Qdm presented by Qa1b.



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  		FIGURE 6. CD94/NKG2-mediated inhibition of tumor cell lysis by V{gamma}3 T cells. After culture of FD17 thymocytes with rIL-2 for 3 days, V{gamma}3+ T cells were sorted using FITC-conjugated anti-TCR V{gamma}3 mAb or using PE-conjugated anti-TCR V{gamma}3 mAb combined with FITC-conjugated anti-NKG2 (3S9) mAb. After 3 days of additional rIL-2 culture, V{gamma}3+ T cells were used as effector cells in a 51Cr release assay. Purity of sorted V{gamma}3+, V{gamma}3+CD94/NKG2low, and V{gamma}3+CD94/NKG2high T cells was >98, 77, and 96%, respectively. Cytotoxicity of V{gamma}3+ T cells (A) and V{gamma}3+CD94/NKG2low and V{gamma}3+CD94/NKG2high T cells (B) against Fc{gamma}R+ P815 target cells in the presence of 30 µg/ml anti-NKG2 (3S9) or isotype control (IgG2b) mAbs is shown. C, Cytotoxic activity of V{gamma}3+ T cells against Qa1b-transfected T2Q target cells preincubated with Qdm peptide or unrelated peptide, and against untransfected T2 cells incubated with Qdm peptide. In blocking studies, T2Q target cells loaded with Qdm peptide were incubated with anti-Qa1b mAb. D, Cytotoxicity of V{gamma}3+CD94/NKG2low and V{gamma}3+CD94/NKG2high T cells against Qa1b-transfected T2Q target cells preincubated with Qdm peptide or unrelated peptide.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we identified the cellular distribution of NK receptors on V{gamma}3 T cells, a subset of murine {gamma}{delta} T cells. We demonstrate that the inhibitory NK receptors Ly49E and CD94/NKG2 are expressed on fetal thymic and adult skin-located V{gamma}3 T cells. FCA failed to detect expression of other members of the Ly49 family. This NK receptor repertoire on V{gamma}3 T cells is similar to the expression of NK receptors on fetal NK cells (33). The expression of Ly49E and CD94/NKG2 on V{gamma}3 T cells is correlated with the expression of CD44, 2B4, and IL-2R{beta}, and the absence of CD25. This phenotype of V{gamma}3 T cells resembles that of Tm1 cells. As recently reviewed by Anfossi et al. (42), Tm1 cells are memory CD8+ {alpha}{beta} T cells expressing CD44 and inhibitory NK receptors and are further characterized by a CD25-IL-2R{beta}+2B4+ phenotype. In addition, NK1.1 or DX5 expression does not correlate with the Tm1 cell population (42). Consistent with this phenotype, V{gamma}3 T cells are predominantly NK1.1- and do not express DX5 (data not shown). The expression of Ly49E and CD94/NKG2 on thymic V{gamma}3 T cells contrasts with the failure to detect CD94/NKG2 and Ly49 expression, including Ly49E, on conventional {alpha}{beta} T cells from adult thymus (data not shown) and is also in contrast with the finding that TCR {alpha}{beta} Tm1 cells are only detectable in the periphery of adult mice (31). In addition, only marginal expression of Ly49E could be detected on neonatal thymic V{gamma}3-{gamma}{delta}+ cells and on adult splenic {gamma}{delta} cells. Taken together, this report characterizes a subpopulation of murine {gamma}{delta} T cells, namely V{gamma}3 T cells, displaying hallmarks of a Tm1 phenotype detectable in the fetal thymus.

Ly49E and CD94/NKG2 are expressed on mature HSAlow V{gamma}3 thymocytes but not on immature V{gamma}3 thymocytes, suggesting that Ly49E and CD94/NKG2 expression is initiated at a late stage during V{gamma}3 T cell differentiation. Together with the memory phenotype of Ly49E+ and CD94/NKG2+ V{gamma}3 T cells, these data could indicate that Ly49E and CD94/NKG2 receptor expression is induced following stimulation of mature V{gamma}3 T cells. As reported by others (21, 58, 59, 60), expression of inhibitory NK receptors can be induced following Ag activation. We can only hypothesize about the Ag responsible for induction of NK cell receptors on V{gamma}3 T cells. Havran et al. (61) demonstrated that skin-located V{gamma}3 T cells are stimulated by self-Ags on keratinocytes in a TCR-dependent manner. Because thymic and skin epithelium have the same embryonic origin, it can be hypothesized that thymic TCR V{gamma}3+ thymocytes recognize the same self-Ag expressed on thymic epithelium. The recognition of self-Ags on epithelium could explain why TCR V{gamma}3 T cells express NK cell receptors. TCR V{gamma}3 T cells bear an invariant TCR on their cell surface (43), and therefore it is not clear why only 70% of skin-located V{gamma}3 T cells expressed CD94/NKG2. Because CD94/NKG2 and Ly49E receptors are expressed on overlapping subsets of V{gamma}3 T cells, a small subset of V{gamma}3 T cells was still negative for both Ly49E and CD94/NKG2 (data not shown). Similar findings have been observed with memory CD8+CD44+ TCR {alpha}{beta} cells where only 35% express Ly49A, C, F, G2, and/or I receptors (31). Interestingly, it has been reported by Huard et al. (60) that KIR expression on CD8+ T cells is maintained by continuously triggering of their TCR. In line with this, it could be possible that the expression of CD94/NKG2 or Ly49E receptors on V{gamma}3 T cells is not stable resulting in down-regulation of these receptors, unless V{gamma}3 T cells are continuously exposed to Ag. In contrast to the increased frequency of CD94/NKG2 expression, we observed a 2- to 3-fold reduction of Ly49E expression on skin epithelial V{gamma}3 T cells compared with fetal thymic V{gamma}3 T lymphocytes. It is possible that the decreased expression of Ly49E is related to the increased expression of CD94/NKG2 on skin-located V{gamma}3 T cells. However, double staining of skin-located V{gamma}3 T cells with mAbs 4D12 and 3S9 showed that Ly49E expression and CD94/NKG2 expression on V{gamma}3 T cells are independent of each other (data not shown).

This is the first report demonstrating that the CD94/NKG2 receptor can function as an inhibitory NK receptor in controlling the cytotoxic activity of murine {gamma}{delta} T cells. Engagement of CD94/NKG2 using mAb 3S9 leads to inhibition of cytolysis, suggesting that CD94/NKG2 functions as an inhibitory receptor on V{gamma}3 T cells. This is supported by the finding that Qdm peptide, the ligand for CD94/NKG2, protected lysis of Qa1b-transfected target cells by V{gamma}3 T cells and that addition of blocking Qa1b mAb could reverse this inhibition. In analogy, functional data have demonstrated that the Qdm peptide is necessary to protect Qa1b-transfected target cells from lysis by NK cells (53, 57). Together, these findings demonstrate that the CD94/NKG2 receptor on V{gamma}3 T cells is able to inhibit their cytolytic activity following recognition of Qdm peptide presented by Qa1b. This suggests that V{gamma}3 T cells mainly express the inhibitory CD94/NKG2A receptor rather than the activating CD94/NKG2C and CD94/NKG2E receptors. This is in line with the observation that activating Ly49 receptors, Ly49D and Ly49H, are not expressed on memory {alpha}{beta} T lymphocytes (30, 31, 62). Concerning a possible role for CD94/NKG2 in T cell differentiation, mice transgenic for Ly49A demonstrate an impaired negative selection resulting in the failure to delete potentially self-reactive T cells (63, 64). In contrast, normal T cell development has been observed in mice transgenic for both a human inhibitory KIR and its HLA class I ligand (65). Together with our finding that CD94/NKG2 is expressed at a late stage during V{gamma}3 T cell development and that its expression is correlated with a memory phenotype on V{gamma}3 T cells, we favor the hypothesis that CD94/NKG2 is not involved in thymic selection and development of V{gamma}3 T cells.

Coles et al. (31) have demonstrated that development of Ly49-expressing memory CD8+ TCR {alpha}{beta} cells is MHC class I dependent. Our results show that Ly49E and CD94/NKG2 expression on V{gamma}3 T cells is not dependent upon MHC class I expression. This is in contrast with the finding that V{gamma}3 T lymphocytes develop normally in {beta}2m-/- mice (66). Recently, studies with mice transgenic for KIR2DL3 and its ligand have illustrated that interaction between KIRs and MHC class I molecules promotes the survival of memory CD8 T cells (67). Because binding of MHC class I molecules to inhibitory receptors is thought to be necessary for survival of memory T cells, and because CD94/NKG2 receptors recognize self-MHC class I molecules, although in an indirect manner, this would predict a preferential accumulation of CD94/NKG2+V{gamma}3+ T cells in WT mice compared with {beta}2m-/- mice. As described above, we observed no difference in the frequency of V{gamma}3 T cells expressing CD94/NKG2 in both types of mice. Thus, these data show no evidence for a preferential survival of {gamma}{delta} T cells expressing the CD94/NKG2 inhibitory receptor.

In conclusion, our results demonstrate that expression of NK receptors on V{gamma}3 T cells is correlated with a memory phenotype, such as Tm1 cells, and that CD94/NKG2 ligation inhibits the cytolytic activity of V{gamma}3 T cells.


   Acknowledgments
 
We are grateful to Dr. M. Gately and Dr. S. Gillis for providing us with rIL-2 and rIL-7, respectively; Dr. J. Unkeless, Dr. J. Ortaldo, Dr. M. Nabholz, and Dr. T. Tanaka for providing us with mAbs; Dr. A. Kruisbeek and Dr. A. Geldhof for providing cell lines; Dr. C. Brooks for providing us the T2 and T2Q cell lines and the Qdm peptide; and Dr. J. Vandekerckhove for providing the control peptide. We thank E. Naessens and M. De Smedt for purification of Abs, G. De Smet and C. Collier for animal care, and C. De Boever for artwork.


   Footnotes
 
1 This work was supported by grants from the Research-fund of Ghent University and the Fund for Scientific Research-Flanders (Belgium). F.S. is a research assistant of the Fund for Scientific Research-Flanders. K.V.B. and A.D.C. are Ph.D. students supported by a grant from the Research-fund of Ghent University. Back

2 Address correspondence and reprint requests to Dr. Georges Leclercq, Department of Clinical Chemistry, Microbiology, and Immunology, University of Ghent, University Hospital, Blok A, 4th Floor, De Pintelaan 185, B-9000 Ghent, Belgium. E-mail address: georges.leclercq{at}rug.ac.be Back

3 Abbreviations used in this paper: KIR, killer cell inhibitory receptor; FCA, flow cytometric analysis; FD, fetal day; WT, wild type; Tm1, T memory type 1; {beta}2m; {beta}2-microglobulin. Back

Received for publication August 20, 2001. Accepted for publication January 30, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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