BY55/CD160 acts as a co-receptor in TCR signal transduction of a human circulating cytotoxic effector T lymphocyte subset lacking CD28 expression
Maria Nikolova1,
Anne Marie-Cardine1,
Laurence Boumsell1 and
Armand Bensussan1
1 INSERM 448, Faculté de Médecine de Créteil, 8 rue du général Sarrail, 94010 Créteil, France
The first two authors M. Nikolova and A. Marie-Cardine contributed equally to this work
Correspondence to: A. Marie-Cardine; E-mail: marie-cardine@im3.inserm.fr
Transmitting editor: W. Knapp
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Abstract
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In the present study, we examined the role of the recently identified glycosylphosphatidylinositol (GPI)-anchored cell surface molecule BY55, assigned as CD160, in TCR signaling. CD160 is expressed by most intestinal intraepithelial lymphocytes and by a minor subset of circulating lymphocytes including NK, TCR
and cytotoxic effector CD8bright+CD28 T lymphocytes. We report that CD160, which has a broad specificity for MHC class Ia and Ib molecules, behaves as a co-receptor upon T cell activation. Anti-CD160 mAb enhance the CD3-induced proliferation of freshly isolated CD160-enriched peripheral blood lymphocytes and CD160+ T cell clones. Further, the engagement of CD160 receptors on normal clonal T lymphocyte populations lacking CD4, CD8 and CD28 molecules by MHC class I molecules results in an increased CD3-induced cell proliferation. Further, we found that CD160 co-precipitates with the protein tyrosine kinase p56lck and tyrosine phosphorylated
chains upon TCRCD3 cell activation. Thus, we demonstrate that CD160 provides co-stimulatory signals leading to the expansion of a minor subset of circulating lymphocytes including double-negative CD4/CD8 T lymphocytes and CD8bright+ cytotoxic effector T lymphocytes lacking CD28 expression.
Keywords: cytotoxic T lymphocyte, glycosylphosphatidylinositol-anchored receptors, intestinal intraepithelial lymphocytes, T lymphocyte co-receptor, TCR 
lymphocyte activation
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Introduction
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T lymphocyte activation is controlled by positive signals initiated by the engagement of the TCR and molecules, including the MHC receptors CD4/CD8, CD2, CD28 and integrins. It has been extensively reported that protein tyrosine kinases (PTK) are indispensable signaling molecules for the initiation of T cell activation cascades. Indeed, one of the earliest signaling events following TCR engagement is the tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAM) located within the TCRCD3-associated
chains, which is essential in coupling the TCR to downstream signaling pathways, including hydrolysis of inositol-containing phospholipids, calcium mobilization and activation of the ras/MAP kinases cascade (1,2). It is now well documented that src family PTK (e.g. p56lck) mediate the ITAM phosphorylation of the
chains, allowing their association with the syk family PTK ZAP70 via its tandem src homology 2 (SH2) domains (3). Once recruited, ZAP70 kinase activity is highly increased by both a self and a lck-dependent phosphorylation mechanisms (4). At the outer leaflet of the cell membrane, T cell activation is controlled through contact zones between T cells and antigen-presenting cells where the receptors constitute a mature immunological synapse (IS) or a supramolecular activation cluster (SMAC) (5,6). In the IS/SMAC the receptors are highly organized into two subregions, one internal where the TCR and CD4, together with the kinases lck, fyn and protein kinase C
are located, and one external including the integrin molecules CD18/CD11a and talin which is connected via vinculin to actin filaments (5,6). The IS are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids that are assembled within GPI-enriched microdomains (7). These membrane microdomains (or lipid rafts) also contain cytoplasmic signaling molecules including src family PTK, G proteins and adaptor proteins such as the linker for activation of T cells, LAT (8), but are devoid of most transmembrane proteins (9).
We have previously reported the identification of the GPI-anchored cell membrane receptor BY55, recently assigned as CD160 (10), which is expressed on intestinal intraepithelial T lymphocytes, CD56dim+CD16+ circulating NK lymphocytes, and a small T lymphocytes population including most circulating TCR-
-bearing cells and a minor subset of CD8bright+ lymphocytes mediating cytotoxic activity (1113). CD160 shares a weak homology with KIR2DL4 receptors, and exhibits a broad specificity for MHC class Ia and Ib molecules (14). We previously demonstrated that cross-linking of MHC class I molecules by CD160 enhances the CD3-induced proliferation of a CD4+ T cell clone (14). However, until now CD160 functions on CD160-expressing T lymphocytes were not defined. In the present study we investigated the role of CD160 upon TCR-mediated signal transduction of CD160-bearing T lymphocytes. We show that engagement of CD160 with either specific mAb or its natural ligand, i.e. MHC class I molecules, provides a co-stimulatory signal upon CD3-induced lymphocyte proliferation. Further, we demonstrate that upon CD3 activation CD160 co-precipitates with the PTK p56lck and with tyrosine phosphorylated
chains. Thus, we describe for the first time a unique pathway involving CD160 which potentiates the proliferation of a highly differentiated subset of circulating cytotoxic T lymphocytes.
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Methods
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Cells and cell lines
Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by Ficoll-Isopaque density gradient centrifugation (Pharmacia, Piscataway, NJ). The CD160-enriched peripheral blood T lymphocytes (PB-T lymphocytes) were obtained by negative selection from freshly isolated PBMC using magnetic bead sorting. Briefly, PBMC were stained with anti-CD4, anti-CD19 and anti-CD16 mAb-coupled magnetic beads (Miltenyl Biotec, Bergisch Gladbach, Germany), and the negative fraction was collected using a MiniMACS separation system (Miltenyl Biotec). The CD160-enriched population contained <1% CD4+, CD19+ and CD16+ cells, and 4070% CD160+CD3+ cells. These CD160+ lymphocytes included both CD8bright+TCR
ß+ and TCR
+ subsets, and no CD16+ NK cells. The TCR 
T cell clones DS6 and LSO were obtained as previously described (15,16). The .221-B46 and .221-Cw3 cell lines generated by transfection of the HLA-A, -B and -C-deficient B-EBV cell line 721.221 were kindly provided by Professor A. Moretta (University of Genova, Genova, Italy).
mAb and immunofluorescence analysis
Anti-CD160 mAb BY55 (IgM) and CL1-R2 (IgG1) were described elsewhere (11,14). mAb ANA 3 (CD3, IgG2a), IP26A (TCR
ß, IgG1), BB29 (CD8, IgM), BJ40 (CD48, IgG1) and P296 (CD59, IgG1) were locally produced. The other mAb BW209/2 (CD16, IgG2a) and CD28.1 (CD28, IgG1) were obtained through exchanges during the Fifth International Workshop on Human Leukocyte Differentiation Antigens. Phycoerythrin-conjugated EB6 (CD158a, IgG1), GL183 (CD158b, IgG1), HP-3B1 (CD94, IgG2a), NKH-1 (CD56, IgG1), ECD-conjugated UCHT1 (CD3, IgG1) as well as purified GL183 (CD158b, IgG1) were purchased from Immunotech-Coulter (Marseilles, France). Anti-
rabbit antiserum (17) was kindly provided by Dr O. Acuto (Pasteur Institute, Paris, France). Single-color immunofluorescence staining was performed as previously reported (14). For three-color analysis 3 x 105 cells were stained with anti-CD160 mAb for 30 min at 4°C. Cells were then washed and incubated with FITC-conjugated goat anti-mouse (GAM) IgM (Caltag, San Francisco, CA) followed by a second phycoerythrin-conjugated and third TRI-conjugated specific mAb of IgG isotype. Stained cells were analyzed using a single argon flow cytometry analyzer (Epics XL; Beckman-Coulter, Miami, FL). Instrument standardization was performed using Flow-Set fluorospheres (Beckman-Coulter). Lymphocytes were gated using forward scatter (size) and side scatter (granularity) parameters. Data were analyzed using WinMDI software.
Proliferation assays
For proliferation assays, cells (35 x 105) were cultured in triplicates in 96-well round-bottomed plates (Greiner, Nürtingen, Germany) in a final volume of 0.2 ml culture medium consisting of RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, 100 µg/ml penicillin/streptomycin and 10% pooled human AB serum. When needed, the plates were pre-coated with various concentrations of purified anti-CD3 mAb, as previously described (18). In selected experiments cells were pre-incubated with anti-CD158b mAb (1 µg/ml) and set to proliferate in the presence of anti-CD160 mAb BY55 or an isotype-matched control, and GAM IgG + IgM (Caltag) or in the presence or HLA-Cw3 transfectant cells. Co-cultures of DS6 cells with irradiated (100 Gy) parental or transfected .221 cells were carried out as previously described (14). The anti-CD160 CL1-R2 mAb was used for blocking experiments at 2 µg/ml, whereas it was used at 1 µg/ml for providing co-stimulatory effect. The cells were cultured for 4 days and pulsed with 1 µCi of [3H]thymidine during the last 816 h of culture. [3H]Thymidine incorporation was measured in a liquid scintillation counter (Topcount; Packard, Meriden, CT). Results are presented as the mean ± SD of triplicate cultures.
Immunoprecipitation, in vitro kinase assay and re-immunoprecipitation experiments
Cells (4 x 106/sample) were washed twice with PBS and resuspended in lysis buffer (20 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% Brij 58, 1 mM Na vanadate, 10 mM NaF, 1 mM PMSF, 1 µg/ml aprotinin and 1 µg/ml leupeptin) for 1 h at 4°C. Postnuclear supernatant was then incubated for 2 h at 4°C in a 96-well plate (MaxiSorp; Nunc, Roskilde, Denmark) pre-coated with GAM IgG + IgM (Caltag) plus anti-CD160 (BY55 or CL1-R2), anti-CD48 or anti-CD59 mAb. When needed, cells were pre-activated by incubation with anti-CD3 mAb for 20 min at 4°C cross-linked with GAM IgG + IgM (3 min at 37°C). In vitro kinase assays were performed as previously described (19) and radiolabeled proteins were resolved by SDSPAGE.
For re-immunoprecipitation experiments 32P-labeled proteins from CD160 immunoprecipitates were eluted into heated 1% Triton X-100 lysis buffer supplemented with 0.5% SDS and subsequently diluted 1:10 in 1% Triton X-100 containing lysis buffer. Re-immunoprecipitations of the released material were performed for 90 min at 4°C in a 96-well plate pre-coated with GAM or anti-rabbit Ig and the appropriate antibodies. Following SDSPAGE, the in vitro phosphorylated proteins were detected by autoradiography of dried gels.
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Results
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Anti-CD160 mAb increase the CD3-induced proliferation of CD160-enriched PB-T lymphocytes
We have previously shown that CD160 is expressed by a minor subset of circulating CD8bright+ T cells, but not by CD4+ T cells (12,20). Within the CD8bright+ PB-T lymphocytes population, only the separated CD160+ cells are capable of exhibiting functional cytotoxic activity with any ex vivo stimulation (12,20). Here we show that circulating CD3+CD160+ lymphocytes, that include both TCR
ß CD8bright+ T cells and TCR
CD8dim+ or CD8 cells (11,12), are mainly CD28 and completely include the GL183/CD158b+ T lymphocytes population (Fig. 1).

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Fig. 1. Tri-color analysis of CD160 expression within the CD3+ peripheral blood lymphocytes. PBMC from normal individuals were stained with anti-CD160 mAb (IgM) followed by FITC-conjugated isotype-specific goat anti-mouse, ECD-conjugated anti-CD3 (IgG1) and one of the phycoerythrin-conjugated mAb: anti-CD28 (IgG1) or anti-CD158b (IgG1). Double-gated CD3+ lymphocytes are presented on the dot-plots. This experiment is representative of five studied donors.
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Next we investigated whether CD160 triggering potentiates the proliferation of these circulating cytotoxic effector T lymphocytes. In order to avoid prior cross-linking of CD160 during the positive selection, we performed a negative selection by depleting freshly isolated peripheral blood lymphocytes of the CD4+, CD19+ and CD16+ lymphocytes. The percentage of CD160+CD3+ cells in the different CD160-enriched T lymphocyte populations varied from one individual to the other and corresponded to 4070% of CD160+ T lymphocytes which exhibited, as expected from previous studies (12), anti-CD3 mAb-redirected cytotoxicity (data not shown). The CD160-enriched T cells were then induced to proliferate using several concentrations of immobilized purified anti-CD3 mAb in the absence or presence of each anti-CD160 mAb (BY55 or CL1-R2 mAb). When necessary, an IgM isotype mAb (anti-CD8) was used as control. We observed that the CD3-induced proliferation corresponding to increasing concentrations of anti-CD3 mAb was strongly enhanced in the presence of each anti-CD160 mAb. In contrast, a similar level of cell proliferation was observed following activation of the cells with the anti-CD3 mAb alone or in combination with the anti-CD8 control mAb (Fig. 2). In addition, when cells were incubated with a sub-mitogenic concentration of anti-CD3 mAb (50 ng/ml), the simultaneous cross-linking of CD160 did not induce any cell proliferation. Thus, CD160 triggering alone is not sufficient to promote intracellular events leading to T cell activation and proliferation. Taken together these results suggest that CD160 can exert a co-stimulatory function at the surface of CD160-expressing cells.

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Fig. 2. Anti-CD160 mAb increased the CD3-induced proliferation of normal T lymphocytes. PBMC from a representative donor were enriched in CD160+ T lymphocytes as described in Methods. The CD160-enriched T lymphocytes were stimulated with the indicated concentrations of purified immobilized anti-CD3 mAb in the absence or presence of BY55 (used at 1/400 dilution of ascites) or CL1-R2 (1 µg/ml) anti-CD160 mAb. An IgM anti-CD8 mAb was used as a control (1/400 dilution of ascites). The added mAb were cross-linked with GAM Ig. Both anti-CD160 mAb were tested at various concentrations of purified CL1-R2 or dilutions of BY55 ascites. Their co-stimulatory effect was dose dependent with a maximum at the concentration or dilution used above (data not shown). Results shown are representative of at least three separated experiments performed with different donors and are expressed as mean c.p.m. of triplicate wells.
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Anti-CD160 mAb enhances the CD3-induced proliferation of TCR
+ T cell clones
We previously established that CD160 is not only expressed by a minor subset of CD8+TCR
ß+ T lymphocytes, but also by most TCR
+ circulating T cells (11). To determine at the clonal T cell population level whether CD160 provides intracellular signals upon TCR engagement, we used the previously described TCR
+ T cell clone DS6 (21). DS6 was identified as a double-negative CD4/CD8 T cell clone that expressed functional V
5V
6 TCR. Here, we further demonstrated that DS6 cells expressed the HLA-C-specific receptor CD158b (KIR2DL/S2) and failed to express CD28 (Fig. 3). We previously showed that cell membrane expression of CD160 is rarely detectable on IL-2-dependent NK and T lymphocyte clones (11). In accordance, we observed that CD160 cell membrane expression is down-modulated in freshly isolated peripheral blood lymphocytes activated for a few hours (11). Therefore, DS6 represents a unique T cell clone because CD160 expression increases significantly after 48 h of culture in the absence of IL-2 (Fig. 3). We then evaluated the proliferative responses of CD160high+ DS6 cells stimulated with immobilized anti-CD3 mAb in the presence of anti-CD160 mAb or a control mAb (Fig. 4A and B). DS6 cells express functional CD3 molecules since cross-linking with immobilized anti-CD3 mAb leads to a moderate but significant cell proliferation. The simultaneous cross-linking of CD160 results in a marked enhancement of the CD3-induced proliferation, whereas co-engagement of the GPI-linked cell membrane receptor CD48 has no effect. It should be noted that various concentrations of anti-CD48 mAb were tested and that none of them led to an increased CD3-induced proliferation of DS6 cells, which express high levels of CD48 molecules (data not shown). Under the same experimental conditions we tested a different TCR
-bearing lymphocyte clone termed LSO, which fails to express a detectable level of CD160 even when cultured in the absence of IL-2 (data not shown). The results indicated that the CD3-induced proliferation of LSO is not affected in the presence of anti-CD160 or anti-CD48 mAb (Fig. 4A). We concluded that CD160 engagement provides co-stimulatory signals to the CD3-induced proliferation of TCR
+CD28CD4CD8 DS6 cells. Interestingly, we found that cross-linking of CD158b molecules did not inhibit the positive effect obtained following the subsequent engagement of CD160, whereas it down-regulated the proliferation of DS6 cells in response to anti-CD3 mAb (Fig. 4B).

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Fig. 3. Single-color phenotypic analysis of DS6 T cell clone. DS6 cloned cells were stained with the indicated mAb followed by FITC-conjugated GAM. Isotype controls are presented by the shaded curves. The different curves obtained with the anti-CD160 mAb represent DS6 cells grown in the presence of IL-2 (thin line), in the absence of IL-2 for 24 h (dotted line) or in the absence of IL-2 for 48 h (thick line).
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The CD3-induced proliferation of TCR
+ T cell clones is enhanced in the presence of cells transfected with MHC class I molecules
We further tested whether the triggering of CD160 at the T cell surface by its natural ligand, i.e. the MHC class I molecules, results in a similar effect upon CD3-induced T cell proliferation, as observed using each anti-CD160 mAb. The results presented in Fig. 5 demonstrate that CD3-induced DS6 cell proliferation is enhanced in the presence of MHC class I-transfected cells (.221-B46 cells or .221-Cw3), whereas the HLA-A, -B and -C-deficient parental cell line (.221 cells) has no effect. It should be mentioned that the specificity of DS6 cells is not restricted to the HLA molecules expressed by the transfectants, as they do not proliferate in the absence of immobilized anti-CD3 mAb. Furthermore, the addition of the soluble anti-CD160 mAb CL1-R2, that we previously identified as blocking the binding of MHC class I tetramers on CD160-transfected CHO cells (14), leads to an inhibition of the increased DS6 proliferation observed in the presence of HLA-B46- or HLA-Cw3-transfected cells (Fig. 5). Interestingly, cells transfected with HLA-Cw3 molecules, which bind both CD160 (14) and CD158b (22) molecules, also increased DS6 cell proliferation. However, such enhancement was only observed after pre-incubation of DS6 cells with the anti-CD158b mAb.

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Fig. 5. Cross-linking of CD160 by MHC class I molecules enhances the CD3-induced proliferation of DS6 T cell clone. The DS6 (CD160+) T cell clone was stimulated with 2 x 104 irradiated .221, .221-B46 or .221-Cw3 cells alone or in the presence of immobilized anti-CD3 mAb (1 µg/ml). For blocking experiments, DS6 cells were pre-incubated with 2 µg/ml of purified CL1-R2 mAb or isotype-matched antibody (anti-CD1a mAb) before adding them to the various culture wells. For stimulation in the presence of .221-Cw3 cells, DS6 cells were pre-incubated with soluble anti-CD158b mAb (1 µg/ml) and washed to avoid engagement of CD158b molecules with HLA- Cw3. Results shown are representative of three separate experiments and are expressed as mean c.p.m. ± SD of triplicate wells.
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CD160 co-precipitates with a protein complex comprising the PTK p56lck and tyrosine phosphorylated
chains upon TCRCD3 triggering
To address the molecular basis of CD160 function, in vitro kinase assays were performed on CD160 immunoprecipitates obtained from resting or CD3-activated CD160-enriched PB-T lymphocytes (Fig. 6, left panel). An immunoprecipitate using an isotype-matched mAb (anti-CD34, IgM) was realized on CD3-activated cells and served as negative control. Thus, no radiolabeled material was recovered from anti-CD34 immunoprecipitate obtained from CD3-activated cells following in vitro labeling (Fig. 6A, control lane). In contrast, we observed that CD160 constitutively co-precipitates with radiolabeled phosphoproteins of 56, 80 and 200 kDa in circulating CD3+ lymphocytes (Fig. 6A). These CD160-associated molecules do not appear to be phosphorylated on tyrosine residues as they are not re-immunoprecipitated by an anti-phosphotyrosine mAb (Fig. 6B, PTyr panel). We further observed that activation of the cells with an anti-CD3 mAb resulted in dramatic changes in the pattern of phosphoproteins co-precipitated with CD160. Indeed, CD3 stimulation led to an increased phospholabeling of the proteins in the 56, 80 and 200 kDa range, and, more importantly, to the detection of additional radiolabeled polypeptides of 1623, 27 and 70 kDa within the immunoprecipitates. Importantly, identical phosphoproteins patterns were obtained using both anti-CD160 mAb (e.g BY55 or CL1-R2) for precipitation. In contrast, precipitation of the GPI-linked molecules CD48 and CD59 on CD3-activated cells resulted in the detection of radiolabeled protein complexes distinct from the one observed in association with CD160 (Fig. 6A). Subsequent re-immunoprecipitation of the CD160-associated phosphoproteins from CD3-activated cells using an anti-phosphotyrosine mAb demonstrated that at least the proteins with an apparent molecular mass of 1623, 56 and 200 kDa are tyrosine phosphorylated (Fig. 6B, PTyr panel). In an attempt to identify the tyrosine-phosphorylated molecules that co-precipitate with CD160, re-immunoprecipitation experiments were conducted using antibodies of known specificity (Fig. 6B). We established that the 1623 and 56 kDa phosphoproteins correspond to tyrosine-phosphorylated
chains and to the PTK p56lck respectively. No trace of the PTK p59fyn nor of the adaptor molecule LAT was detected within CD160 immunoprecipitates (not shown). Taken together, these data suggest that a functional signaling complex is formed between CD160, lck and the
chains upon T cell activation.
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Discussion
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We have previously identified the BY55 molecule as a GPI-anchored cell membrane molecule with a single Ig-like domain that is expressed by CD56dim+CD16+ circulating NK lymphocytes (13). Further, the BY55 receptor, that is now assigned as CD160 (10), has been found to exhibit a broad specificity for both classical and non-classical MHC class I molecules (14). In contrast to the genes encoding the killer cell inhibitory receptors that are located together in the leukocyte receptor cluster (23), the gene encoding CD160/BY55 is located on chromosome 1q42.3 (10). Besides NK lymphocytes, CD160 is expressed by most PB-TCR
lymphocytes and by a minor subset of circulating CD8bright+ TCR
ß cells. Within the whole CD8+ PB-T lymphocyte population, only the CD160+ cells that represent 1020% of the cells subset are capable of exhibiting functional cytotoxic activity with any prior ex vivo stimulation (12). In the present study, we report that those CD160+ PB-T lymphocytes are mainly CD28 and include the T lymphocyte population expressing killer cell-like Ig receptor (KIR). More importantly, we found that cross-linked anti-CD160 mAb BY55 (IgM) and CL1-R2 (IgG1) are both capable of enhancing the CD3-induced proliferation of CD160-enriched T lymphocytes. A similar observation was made on a KIR+CD160+CD28 T cell clone cultured either with the anti-CD160 mAb BY55 or with the natural ligand of CD160, i.e. in the presence of MHC class I-transfected cells. Moreover, the results obtained with soluble CL1-R2 mAb in the blocking experiments allowed us to demonstrate that the CD160 co-stimulatory effect observed upon CD3-induced proliferation is due to the MHC class ICD160 interaction. Interestingly, co-engagement of the pan-T cell GPI-linked molecule CD48 did not result in the enhancement of CD3-induced DS6 cell proliferation. However, additional studies using other anti-CD48 mAb will be needed to ascertain whether CD48 triggering mediates no positive signal on the proliferation of CD3-activated DS6 cells. Thus, we report for the first time that CD160 molecules can mediate a co-stimulatory signal upon TCR activation of a minor circulating CD3+CD8+ T cell subset including the memory CD8+KIR+ (24,25) and the CD28 cytotoxic effector T lymphocytes (26). In addition, it appears that the inhibitory signals provided upon KIR ligation (CD158b) do not interfere with the activation pathway delivered through CD160 (see Fig. 4B). However, it should be mentioned that in the presence of the natural ligand HLA-Cw3, which binds to both CD160 and CD158b, a maximal increase of CD158b+CD160+ DS6 clone proliferation in the presence of CD160 mAb was observed when the cells were pre-incubated with the soluble anti-CD158b mAb.
Our attempts to understand the molecular basis underlying CD160 functions resulted in the identification of an oligomeric protein complex, including CD160, in CD3-activated cells. As reported for various GPI-linked receptors (9), we found that the PTK p56lck co-precipitates with CD160 following CD3 stimulation. In addition, tyrosine-phosphorylated
chains were detected within CD160 immunoprecipitates in activated cells. The CD160-associated phosphoproteins of 70 and 200 kDa, according to their apparent molecular mass, might correspond to transducing molecules shown to play key roles in the processes of T cell activation such as ZAP70 or SLP76 (27). Their identification will further clarify the role of CD160 receptors in the generation of T cell signaling cascades.
Importantly, the immunoprecipitation of the pan-T cell GPI-linked molecules CD48 and CD59 led to the detection of protein complexes distinct from the one observed in association with CD160 receptors (Fig. 6A). Almost no trace of phosphorylated
chains was observed in CD48 and CD59 immunoprecipitates. It is therefore tempting to speculate that the association of CD160 to the reported polypeptides is not simply related to the expected localization of CD160 in lipid rafts, but to its particular function on a restricted cell subset. Our results suggest that CD160 molecules exert a co-receptor function through interaction with a fraction of activated p56lck. This latter could then account for the phosphorylation of the
chains, leading to the recruitment of Syk family kinases and to the initiation of downstream activation events. Consequently, the simultaneous triggering of both CD160 and CD3 would result in an increased T cell proliferation when compared to the one observed upon CD3 engagement alone. Importantly, the ligation of CD160 at the cell surface also up-regulates the CD3-induced proliferation of CD4CD8 and CD28 T lymphocytes. Thus, one can hypothesize that the recruitment of CD160 at the cell surface might partially overcome the lack of CD4, CD8 or CD28 co-receptors in these cells. Therefore, CD160 represents a potent candidate for being an alternative co-receptor in the minor subset of CD28 T lymphocytes.
The precise physiological function of CD160 triggered by cross-linked MHC class I molecules is still unknown, although one can consider a particular role in the intestinal intraepithelial T lymphocytes. Indeed, these T lymphocytes have a cytotoxic effector cell phenotype such as CD8+, CD45RO+, CD28 and CD160+ (13). The introduction of a pathogen in the intestinal microenvironment induces pro-inflammatory cytokines that lead to an increase in MHC class I expression in epithelial cells (28) and thus may provide signals through CD160 within the engaged T lymphocytes.
The results on CD28-deficient mice have indicated that CD28 is not required for all T cell responses (29). Therefore, it has been proposed that additional T cell activation pathways exist. Until now, only a few potential candidate molecules on T cells were reported to serve as alternative co-stimulators in the absence of CD28. Amongst these molecules, SLAM, which is expressed on all activated T cells (30), and NKG2D/DAP10 (31,32) were shown to provide signals upon their ligation with specific mAb.
In conclusion, we report for the first time a novel and unique pathway that potentiates T cell expansion of a highly restricted functional lymphocytes subset lacking CD28 and/or CD4 and CD8 expression.
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Acknowledgements
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This work was supported by grants from INSERM and Association de la Recherche contre le Cancer. We would like to thank Dr Oreste Acuto (Pasteur Institute, Paris, France) for helpful discussion.
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Abbreviations
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ISimmunological synapse
GAMgoat anti-mouse
GPIglycosylphosphatidylinositol
ITAMimmunoreceptor tyrosine-based activation motif
KIRkiller cell Ig-like receptor
PB-T lymphocyteperipheral blood T lymphocyte
PBMCperipheral blood mononuclear cell
PTKprotein tyrosine kinase
SMACsupramolecular activation cluster
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