Increased Expression of a Novel Early Activation Surface Membrane Receptor in Cutaneous T Cell Lymphoma Cells

Abstract

Using a newly generated monoclonal antibody we identified the 96 kDa transmembrane receptor SC5 expressed simultaneously on a human Sezary cell line and a minor T cell subset in normal individuals. SC5 antigen was detected mostly on CD45RO+ lymphocytes from both CD4+ and CD8+ subsets as well as on natural killer and B lineage cells. SC5 surface expression increased very early after polyclonal stimulation of CD3+ cells due to the transfer of intracellular SC5 molecules to the cell membrane. Engagement of SC5 receptor by its monoclonal antibody inhibited the anti-CD3-induced proliferation and cytokine secretion of peripheral blood T cells and cell clones, whereas SC5 monoclonal antibody did not affect the cytotoxic activity of CD8+ T cell clones. Extensive phenotypic analysis revealed that the percentage of SC5+ CD4+ circulating lymphocytes in Sezary syndrome patients was significantly increased in comparison with controls (p < 0.01) and correlated with the morphologically detected percentage of Sezary syndrome cells in peripheral blood (p < 0.001). In one patient we clearly demonstrated that the circulating malignant T cells coexpress SC5 molecules. Importantly, ligation of SC5 receptor in a cutaneous T cell lymphoma cell line profoundly inhibited the anti-CD3-induced proliferation. Consequently, the expression of SC5 receptor in the peripheral blood of Sezary syndrome patients may serve not only to detect the presence of circulating malignant CD4+ cells but also as a target for immunotherapy. Using a newly generated monoclonal antibody we identified the 96 kDa transmembrane receptor SC5 expressed simultaneously on a human Sezary cell line and a minor T cell subset in normal individuals. SC5 antigen was detected mostly on CD45RO+ lymphocytes from both CD4+ and CD8+ subsets as well as on natural killer and B lineage cells. SC5 surface expression increased very early after polyclonal stimulation of CD3+ cells due to the transfer of intracellular SC5 molecules to the cell membrane. Engagement of SC5 receptor by its monoclonal antibody inhibited the anti-CD3-induced proliferation and cytokine secretion of peripheral blood T cells and cell clones, whereas SC5 monoclonal antibody did not affect the cytotoxic activity of CD8+ T cell clones. Extensive phenotypic analysis revealed that the percentage of SC5+ CD4+ circulating lymphocytes in Sezary syndrome patients was significantly increased in comparison with controls (p < 0.01) and correlated with the morphologically detected percentage of Sezary syndrome cells in peripheral blood (p < 0.001). In one patient we clearly demonstrated that the circulating malignant T cells coexpress SC5 molecules. Importantly, ligation of SC5 receptor in a cutaneous T cell lymphoma cell line profoundly inhibited the anti-CD3-induced proliferation. Consequently, the expression of SC5 receptor in the peripheral blood of Sezary syndrome patients may serve not only to detect the presence of circulating malignant CD4+ cells but also as a target for immunotherapy. detergent-insoluble glycolipid-enriched fraction killer cell immunoglobulin receptor mycosis fungoides peripheral blood lymphocytes peripheral blood mononuclear cells Sezary syndrome Tlymphocyte immune responses are regulated by functional cell surface molecules providing positive signals that lead to the expansion of antigen-specific clones, and negative signals that prevent excessive stimulation or responsiveness to self-antigens. The signal provided by the engagement of T cell receptors (TCR) must be accompanied by a second positive signal in order to result in optimal T cell response. Recent studies demonstrated that CD3/TCR stimulation leads to a redistribution of the detergent-insoluble glycolipid-enriched membrane fraction (DIG) or raft, which results in the aggregation of TCR/CD3 and DIG-associated signal-transducing molecules (Montixi et al., 1998Montixi C. Langlet C. Bernard A.M. et al.Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains.EMBO J. 1998; 17: 5334-5348https://doi.org/10.1093/emboj/17.18.5334Crossref PubMed Scopus (553) Google Scholar). CD28, which is considered as the major T cell costimulatory receptor (Watts and DeBenedette, 1999Watts T. DeBenedette M. T cell co-stimulatory receptors other than CD28.Curr Opin Immunol. 1999; 11: 286-293https://doi.org/10.1016/s0952-7915(99)80046-6Crossref PubMed Scopus (0) Google Scholar;Yumi et al., 2000Yumi Y.-O. Zhou X.-Y. Toyo-oka K. et al.Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts.J Immunol. 2000; 164: 1251-1259Crossref PubMed Scopus (137) Google Scholar), enhances raft redistribution to the site of TCR engagement (Viola et al., 1999Viola A. Schroeder S. Sakakibara Y. Lanzavecchia A. T lymphocyte co-stimulation mediated by reorganization of membrane microdomains.Science. 1999; 283: 680-682https://doi.org/10.1126/science.283.5402.680Crossref PubMed Scopus (831) Google Scholar). Further, a series of other molecules may provide costimulatory signals to T cells, acting at different time points, affecting different subsets, or promoting distinct effector functions (Shuford et al., 1997Shuford W. Klussman K. Trichler D.D. et al.4-1BB costimulatory signals preferentially induce CD8+ T cell proliferation and lead to amplification in vivo of cytotoxic T cell responses.J Exp Med. 1997; 186: 47-55Crossref PubMed Scopus (608) Google Scholar;Agrawal et al., 1999Agrawal S. Marquet J. Freeman G.J. et al.Cutting edge. MHC class I triggering by a novel cell surface ligand costimulates proliferation of activated human T cells.J Immunol. 1999; 162: 1223-1226PubMed Google Scholar;Hutloff et al., 1999Hutloff A. Dittrich A. Beier K.C. Eljaschewitsch B. Kraft R. Anagnostopoulos I. Kroczek R. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28.Nature. 1999; 397: 263-266https://doi.org/10.1038/16717Crossref PubMed Scopus (1111) Google Scholar;Tamada et al., 2000Tamada K. Shimozaki K. Chapoval A.I. et al.Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway.Nature Med. 2000; 6: 283-289Crossref PubMed Scopus (264) Google Scholar). Negative regulation of T lymphocyte responses can be mediated by CD152-induced signals (Thompson and Allison, 1997Thompson K. Allison J. The emerging role of CTLA-4 as an immune attenuator.Immunity. 1997; 7: 445-450Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar), by apoptosis through members of the tumor necrosis factor (TNF) receptor superfamily (Lenardo et al., 1999Lenardo M. Chan F.K.M. Hornung F. McFarland H. Siegel R. Wang J. Zheng L. Mature T lymphocyte apoptosis-immune regulation in a dynamic and unpredictable antigenic environment.Annu Rev Immunol. 1999; 17: 221-253Crossref PubMed Scopus (822) Google Scholar), or by the inhibitory receptors recently discovered on subsets of natural killer cells and CD8+ T lymphocytes (Mingari et al., 1995Mingari M.C. Vitale C. Cambiaggi A. Sciavetti F. Melioli G. Ferrini S. Poggi A. Cytolytic T lymphocytes displaying natural killer (NK) -like activity: expression of NK-related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TCR-mediated target cell lysis or lymphokine production.Int Immunol. 1995; 7: 697-703Crossref PubMed Scopus (208) Google Scholar,Mingari et al., 1998Mingari M.C. Moretta A. Moretta L. Regulation of KIR expression in human cells: a safety mechanism that may impair protective T-cell responses.Immunol Today. 1998; 19: 155-157Abstract Full Text Full Text PDF Scopus (224) Google Scholar;D'Andrea et al., 1996D'Andrea A. Chang C. Phillips J.H. Lanier L.L. Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I allels.J Exp Med. 1996; 184: 789-794Crossref PubMed Scopus (100) Google Scholar). In this report, we describe a novel 96 kDa transmembrane receptor, SC5, which delineates a minor subset of peripheral blood lymphocytes (PBL) in normal individuals. We found that SC5 was predominantly located in the intracellular compartment of resting T lymphocytes and its surface membrane expression increased rapidly after cell activation. SC5 molecule engagement inhibited the anti-CD3 monoclonal antibody (MoAb) induced proliferation of resting T lymphocytes or T cell clones. Further, we observed that the percentage of SC5+ CD4+ circulating lymphocytes was significantly increased in Sezary syndrome (SS) patients compared to PBL of normal individuals. Importantly, we were able to demonstrate that Sezary cells express SC5 molecules and that their triggering resulted in a strong inhibition of a cutaneous T cell lymphoma (CTCL) cell line proliferation induced by anti-CD3 MoAbs. Thus, SC5 molecule expression may serve to detect the presence of circulating malignant CD4+ cells in SS patients. Moreover, the identification of a cell surface molecule providing negative signals upon ligation constitutes a new tool to investigate the mechanisms of pathologic T cell growth and could be used to prevent it. SC5 MoAb was obtained by immunizing 6-wk-old BALB/C mice with the natural killer cell line Ytindi, using an established protocol (David et al., 1990David V. Bachelez E. Leca G. Degos L. Boumsell L. Bensussan A. Identification of a novel 110 kD structure on a subset of T cell receptor-γδ-bearing cloned lymphocytes.J Immunol. 1990; 144: 1-6PubMed Google Scholar). Hybridoma supernatants were screened for selective reactivity with the immunizing cell line and a CTCL cell line termed Pno (Poszepczynska et al., 2000Poszepczynska E. Bagot M. Echchakir H. et al.Functional characterization of an IL-7-dependent CD4+CD8αα+ Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma.Blood. 2000; 96: 1056-1063PubMed Google Scholar) by indirect immunofluorescence and flow cytometry. The reactive supernatants were further tested on PBL. A hybridoma supernatant reacting with both tumoral cell lines and with a minor PBL subpopulation was selected and cloned twice, and cloned hybridomas were passaged into pristane-primed BALB/C mice to produce ascites. The antibody was termed anti-SC5 MoAb, and its isotype was determined as IgM. The ascites was dialyzed against phosphate-buffered saline (PBS), sterilized by ultrafiltration, and further utilized at a final dilution of 1:200. After informed consent and approval by an ethics committee (CCPPRB, Hôpital Henri Mondor, Creteil), we obtained blood samples from 12 patients with CTCL. Nine patients had an SS with 10%-80% circulating CD3+, CD4+ Sezary cells, whereas three patients had a transformed mycosis fungoides (MF) and presented with disseminated skin tumors with a CD3+, CD4+, CD8- phenotype. The patients had not been previously treated with chemotherapy. Peripheral blood mononuclear cells (PBMC) were isolated by the technique of Ficoll-Isopaque (Pharmacia, Piscataway, NJ) density gradient centrifugation. Human T cell clones GDS.3 (CD3+ TCRαβ+ CD4+ CD8-), DS6 (CD3+ TCRγδ+ CD4- CD8-), LSO (CD3+ TCRγδ+ CD4- CD8-), and JF1 (CD3+ TCRαβ+ CD4- CD8+), described elsewhere (David et al., 1987David V. Bourge J.-F. Gugliemi P. Mathieu-Mahul D. Degos L. Bensussan A. Human T cell clones use a CD3-associated surface antigen recognition structure to exhibit both NK-line and allogeneic cytotoxic reactivity.J Immunol. 1987; 138: 2831-2836PubMed Google Scholar,David et al., 1988David V. Leca E. Vilmer E. et al.Expression of T cell gamma gene by a functionally defined T cell clone.Scand J Immunol. 1988; 27: 473-483Crossref PubMed Scopus (12) Google Scholar;Vilmer et al., 1988Vilmer E. Triebel F. David V. et al.Prominent expansion of circulating lymphocytes bearing γ T-cell receptors, with preferential expression of variable γ genes after allogeneic bone marrow transplantation.Blood. 1988; 72: 841-849PubMed Google Scholar were fed each 8 d with irradiated allogenic PBMC in culture medium consisting of RPMI 1640 (Gibco, Paisley, U.K.), 2 mmol per l L-glutamine, penicillin (100 U per ml), streptomycin (100 mg per ml), 10% heat-inactivated human serum, 50 IU per ml recombinant interleukin-2 (rIL-2) (kindly provided by Sanofi Synthélabo, Labège, France), and 1 µg per ml phytohemagglutinin (PHA) (Wellcome, Beckenham, U.K.). Functional studies were performed at day 7 after the feeding. Standard human leukemic cell lines were mycoplasma-free and maintained in logarithmic growth in complete RPMI medium supplemented with 10% fetal bovine serum and antibiotics. The human CTCL cell clone, Pno, was established from peripheral blood of a CTCL patient and maintained in culture as previously described (Poszepczynska et al., 2000Poszepczynska E. Bagot M. Echchakir H. et al.Functional characterization of an IL-7-dependent CD4+CD8αα+ Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma.Blood. 2000; 96: 1056-1063PubMed Google Scholar). The Pno cell line has a CD3+ Vβ22+ CD4+ CD8αα+ CD25- phenotype and proliferates in response to IL-7 and to anti-CD3 MoAb stimulation. Indirect immunofluorescent staining was performed with hybridoma supernatants or ascites fluid using fluorescein isothiocyanate (FITC) conjugated goat antimouse Ig from Caltag Laboratories (San Francisco, CA). For two-color analysis the immunofluorescent staining was performed by incubating 3 × 105 cells with anti-SC5 MoAb for 30 min at 4°C. Cells were then washed and incubated with FITC-conjugated goat antimouse IgM (Caltag Laboratories) followed by a second PE-, ECD- or TRI-conjugated specific MoAb of IgG isotype. Stained cells were analyzed using a single argon flow cytometer analyzer (Epics XL, Beckman-Coulter, Miami, FL) as described previously (Schiavon et al., 1999Schiavon V. Elhabazi A. Agrawal S. Tawab A. Nikolova M. Boumsell L. Bensussan A. G10.3 monoclonal antibody identifies novel functional cell surface structures expressed by normal B lymphocytes and various malignant cell lines.Tissue Antigens. 1999; 53: 23-32https://doi.org/10.1034/j.1399-0039.1999.530103.xCrossref PubMed Scopus (3) Google Scholar). For intracellular labeling, cells were fixed in PBS 4% p-formaldehyde for 20 min at 4°C, washed, and then permeabilized with staining buffer containing 0.1% saponin. Conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD69, and anti-TCRVβ22 MoAbs were purchased from Immunotech (Marseille, France). Other MoAbs were locally produced or obtained through the exchanges of the Vth International Workshop on white cell differentiation antigens (Boston, MA, November 1994). Two × 107 cells were washed twice in PBS and surface-labeled with Sulfo-NHS-LC biotin (Pierce Europe, Interchim, Montluçon, France), 2 mg per ml in PBS for 20 min at 4°C. After quenching for 20 min with RPMI 1640, cells were washed twice in PBS and resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM Na vanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 µg per ml aprotinin, and 1 µg per 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 immunoplate) precoated with goat antimouse IgG + IgM (Caltag Laboratories) followed by anti-SC5 MoAb or anti-TCRβ (C305 isotype matched MoAb, kindly provided by Dr. A. Weiss, University of California, San Francisco, CA). Immunoprecipitates were washed four times with washing buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM Na vanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride) and precipitated proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis was performed using streptavidin-peroxidase (Immunotech) and the ECL detection system according to the manufacturer's recommendations (Amersham Pharmacia, Orsay, France). For lymphocyte activation, PBMC were cultured in culture medium containing 15% human serum in the presence of 1 µg per ml PHA (Wellcome). For proliferation assays, 5 × 104 cells were cultured in triplicates in 96-well round-bottomed plates (Greiner, Nürtingen, Germany) in a final volume of 0.2 ml culture medium. When needed, the plates were precoated with anti-CD3 MoAb in the indicated dilutions as previously described (Poszepczynska et al., 2000Poszepczynska E. Bagot M. Echchakir H. et al.Functional characterization of an IL-7-dependent CD4+CD8αα+ Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma.Blood. 2000; 96: 1056-1063PubMed Google Scholar). For stimulation of the Pno cell line, precoated anti-CD3 MoAb (2.5 µg per well) was used in combination with 2 ng per ml phorbol 12β-myristate 13α-acetate (PMA, Sigma Biochemicals). Cells were cultured for 4 d and were pulsed with 1 µCi of 3H[TdR] during the last 8–16 h of culture. 3H[TdR] incorporation was measured in a liquid scintillation counter (Topcount; Packard Instrument, Meriden, CT) For the determination of IL-2 production, 105 PBL were stimulated with immobilized anti-CD3 MoAb in the presence of anti-SC5 or control MoAb. After 32 h of culture, 100 μl of supernatant was added to 105 cells from an Il-2-dependent T cell clone. The redirected cytotoxicity assay was carried out as already described (Le Cleach et al., 2000Le Cleach L. Delaire S. Boumsell L. Bagot M. Bourgault-Villada I. Bensussan A. Roujeau J.C. Blister fluid T lymphocytes dring toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors.Clin Exp Immunol. 2000; 119: 225-230https://doi.org/10.1046/j.1365-2249.2000.01119.xCrossref PubMed Scopus (129) Google Scholar). The target murine mastocytoma P815 tumor cells were loaded with 100 µl 51Cr (2.5 mCi per ml) for 90 min at 37°C, washed, and incubated with anti-CD3 for 15 min at room temperature. Effector cells were likewise preincubated with anti-SC5 MoAb (1:200 final dilution of ascites) and added to target cells in the ratio 5:1, in a final volume of 150 µl in 96-well V-bottomed microtiter plates for 4 h. After centrifugation 100 µl aliquots were counted in a gamma-counter to determine 51Cr release. The spontaneous release was always less than 20% of the maximum release (target cells with 1% Nonidet P-40). The percentage of specific 51Cr release was calculated as previously described (David et al., 1987David V. Bourge J.-F. Gugliemi P. Mathieu-Mahul D. Degos L. Bensussan A. Human T cell clones use a CD3-associated surface antigen recognition structure to exhibit both NK-line and allogeneic cytotoxic reactivity.J Immunol. 1987; 138: 2831-2836PubMed Google Scholar). Statistical analysis of the results was performed with Statistica software version 5.0 (StatSoft, Los Angeles, CA). Significant differences between SC5 expression in patients and the control group were evaluated by the Mann–Whitney U test, and data were presented using boxplots. Correlation between the percentage of SC5+ CD4+ cells and the percentage of malignant cells in patients' peripheral blood was evaluated by the Spearman rank order correlations test. Monoclonal antibodies raised against the functional tumor cell line Ytindi were analyzed for their simultaneous reactivity with the immunizing cells and the CTCL cell line Pno (Poszepczynska et al., 2000Poszepczynska E. Bagot M. Echchakir H. et al.Functional characterization of an IL-7-dependent CD4+CD8αα+ Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma.Blood. 2000; 96: 1056-1063PubMed Google Scholar). The SC5-specific MoAb, of IgM isotype, stained both the tumor cells and a minor subset of PBL. The SC5 MoAb reactive molecule was expressed by a variable percentage of CD3+ T cells, never exceeding 20% (mean 10.5%, SD ±5.6). Both CD4+ and CD8+ lymphocytes were stained by anti-SC5 MoAb. A subpopulation of peripheral blood CD56+ natural killer cells was also reactive with anti-SC5 MoAb, though with more important interindividual variations (mean 19.4%, SD ±11.4) (Table I). It should be noted that most SC5+ lymphocytes belonged to the activation/memory pool, over 80% of the SC5-positive cells coexpressing CD45RO (Figure 1). Table I further indicates that anti-SC5 MoAb stained a subpopulation of B cells, practically all monocytes, and 30%-50% of granulocytes.Table IExpression of SC5 in peripheral blood subpopulationsSC5+ cellsCell populationN-testedmean (%)SDTotal Ly1514.16.8CD3+ Ly1010.55.6CD4+ Ly89.45.1CD8+ Ly814.85.1CD56+ Ly1019.411.4CD19+ Ly512.55.7Total Mo5100–Total Gr5455.2 Open table in a new tab As SC5 molecule expression was detected in all IL-2-dependent T lymphocyte clones tested (data not shown), we verified whether its self-surface expression was induced during T lymphocyte activation. Stimulation of PBL with PHA promptly increased the expression of SC5 on CD3+ lymphocytes. Both the percentage of SC5+ T cells and the level of SC5 expression rose very rapidly after stimulation (Figure 2 and data not shown). As early as 4 h after stimulation with PHA, the percentage of SC5+ CD3+ cells increased 2- to 3-fold and reached a maximum after 24–48 h. In the course of 5–7 d of in vitro stimulation SC5 expression returned to the basal level. In parallel we studied the expression of the very early activation antigen CD69 and we observed similar kinetic profiles of CD69 and SC5 on CD3+ lymphocytes. It should be noted that unlike CD69 molecules low levels of SC5 were already present on circulating PBL before stimulation (Figure 1,Figure 2). Activation-induced antigen expression results either from de novo protein synthesis or cell surface export of preexisting intracellular molecules. Therefore, we studied the expression of SC5 in nonactivated permeabilized PBL. As shown in Figure 3, anti-SC5 MoAb stained over 90% of permeabilized lymphocytes versus 16% of the same nonpermeabilized cells. The specificity of anti-SC5 MoAb staining was confirmed by using the isotype-matched MoAb BY55, which detects a GPI-anchored cell surface structure expressed by a subset of circulating lymphocytes (Agrawal et al., 1999Agrawal S. Marquet J. Freeman G.J. et al.Cutting edge. MHC class I triggering by a novel cell surface ligand costimulates proliferation of activated human T cells.J Immunol. 1999; 162: 1223-1226PubMed Google Scholar). A comparable percentage of BY55+ cells was found in permeabilized and nonpermeabilized lymphocytes. As a positive control for the detection of an intracellular epitope, we used an anti-CD3ζ chain MoAb that only labeled permeabilized T lymphocytes. Thus, the surface expression of SC5 is dynamically regulated by the activation state of cells and its induction during T cell activation is due to the enhanced transport of the molecule from an intracellular pool to the surface membrane. In order to characterize the molecular weight of the structure identified by anti-SC5 MoAb, an IL-2-dependent TCRγδ+ clone, termed DS6 (David et al., 1988David V. Leca E. Vilmer E. et al.Expression of T cell gamma gene by a functionally defined T cell clone.Scand J Immunol. 1988; 27: 473-483Crossref PubMed Scopus (12) Google Scholar), was surface labeled with biotin and the cell lysates were immunoprecipitated with either anti-SC5 MoAb or an isotype-matched anti-TCRβ MoAb (as negative control). Anti-SC5 MoAb recognized a single molecule with apparent molecular weight 96 kDa under reducing conditions (Figure 4). As tumor cell lines (the immunizing Ytindi cells) are often characterized by aberrant expression of carbohydrate epitopes (Derappe et al., 1996Derappe C. Haentjens G. Lemaire S. et al.Circulating malignant lymphocytes from Sezary syndrome express high level of glycoproteins carrying b (1–6) N-acetylglucosanine-branched N-linked oligosaccharides.Leukemia. 1996; 10: 138-141PubMed Google Scholar;Schiavon et al., 1999Schiavon V. Elhabazi A. Agrawal S. Tawab A. Nikolova M. Boumsell L. Bensussan A. G10.3 monoclonal antibody identifies novel functional cell surface structures expressed by normal B lymphocytes and various malignant cell lines.Tissue Antigens. 1999; 53: 23-32https://doi.org/10.1034/j.1399-0039.1999.530103.xCrossref PubMed Scopus (3) Google Scholar), we studied the reactivity of anti-SC5 MoAb with the SC5+ natural killer cell line NK3.3 after treatment with neuraminidase or sodium periodate in concentrations destroying known sialylated epitopes (digestion of CD75 epitope on Raji cells was used as a positive control). Expression of SC5 was not affected by neuraminidase or sodium periodate treatment (data not shown). Thus, we excluded a possible carbohydrate nature of the epitope. In order to examine the function of the SC5 molecule in normal T cells, we studied the effect of anti-SC5 MoAb alone or during the proliferative responses induced by immobilized anti-CD3 MoAb. We found that anti-SC5 MoAb alone or in combination with PMA did not induce the proliferation of peripheral blood T lymphocytes (data not shown). Interestingly, when soluble anti-SC5 MoAb was present together with immobilized anti-CD3 MoAb, a significant inhibition of lymphocyte proliferation rate was observed. The inhibition varied between 33% and 66% at day 4, depending on the donor, compared to the effect of an isotype-matched irrelevant control MoAb (data not shown). As SC5 molecules are expressed on monocytes, it was important to determine whether the inhibitory effect was monocyte independent. We studied the proliferation of two T cell clones, GDS.3 and LSO, stimulated by immobilized anti-CD3 MoAb in the presence of anti-SC5 MoAb or an isotype control MoAb. The results obtained with both T cell clones indicated that engagement of SC5 molecules resulted in an inhibition of the proliferation to anti-CD3 MoAb (Figure 5a). It should be noted that the inhibitory effect of anti-SC5 MoAb on T cell proliferation depended on the concentration of the agonistic anti-CD3 MoAb. The inhibition obtained with anti-SC5 MoAb was significant only when T cells were stimulated with optimally diluted anti-CD3 MoAb (Figure 5b). Such inhibitory behavior was also reported for anti-killer cell immunoglobulin receptor (anti-KIR) MoAbs (Cambiaggi et al., 1999Cambiaggi A. Darche S. Guia S. Kourilski P. Abastado J.P. Vivier E. Modulation of T cell functions in KIR3DL3 (CD158b) transgenic mice.Blood. 1999; 94: 2396-2402PubMed Google Scholar). The decrease of T cell proliferation following SC5 engagement could be due to a direct induction of cell death, a perturbation of the IL-2R expression, or the inhibition of cytokine synthesis. We found that anti-SC5 MoAb did not inhibit the IL-2-dependent proliferation of T cell clones (Figure 5a). Furthermore, the addition of rIL-2 to T cells preincubated with anti-SC5 MoAb (ascites diluted to 1:200) in combination with anti-CD3 for 48 h restored their proliferation rate. This would not be the case if the specific MoAb inhibited IL-2R expression or caused cell death (data not shown). Finally, in order to prove that SC5 triggering may inhibit cytokine production, we compared the amount of IL-2 secreted by anti-CD3-activated PBL in the presence or absence of anti-SC5 MoAb, using an IL-2-dependent T cell line. The quantity of IL-2 produced in the presence of anti-SC5 MoAb represented 40%-65% of the control values obtained with an isotype-matched antibody (Figure 5c). Thus, the simultaneous engagement of SC5 and CD3 molecules in T cells results in the decrease in cytokine production. We next studied the effect of anti-SC5 MoAb on T cell effector cytotoxic activity using an anti-CD3 MoAb redirected killing assay against the FcγR+ murine cell line P815. Figure 5(d) shows a representative experiment performed with a cytotoxic T cell clone at an E:T ratio of 5:1. The CD8+ T cell clone JF1, expressing a high amount of SC5 molecules, was induced to kill murine target cells following anti-CD3 MoAb stimulation. The simultaneous targeting of SC5 by its specific MoAb did not influence JF1 cytotoxic activity, regardless of the E:T ratio (data not shown) or the concentration of stimulating anti-CD3 MoAb. As we initially screened anti-SC5 MoAb for its reactivity with the Pno CTCL cell line, we further studied the expression of SC5 molecules in PBL isolated from nine patients with SS. Two-color flow cytometry analysis showed that expression of SC5 was increased in the CD4+ subset (see representative results obtained with one patient in Figure 6a). The percentage of CD4+ SC5+ lymphocytes in SS patients' blood was significantly higher compared to healthy controls (p = 0.0015). As shown in Figure 6(b) practically no overlap existed between the values observed for the SC5/CD4 population in the patients' group (median 32%, range 16%-83%) and in the control group (median 11%, range 3%-12%). In the nine SS patients studied, the expression of SC5 on CD4+ cells correlated with the percentage of circulating malignant cells detected by cytomorphology (r = 0.92, p = 0.0012) (Figure 6c). We also studied SC5 molecule expression in peripheral blood samples from three MF patients without blood involvement. No significant difference in the levels of SC5 expression on peripheral blood CD4+ cells was observed between MF patients and healthy donors. In one SS patient we had previously demonstrated that the circulating malignant cells were clonal lymphocytes with a CD4+ TCRVβ22+ phenotype (Poszepczynska et al., 2000Poszepczynska E. Bagot M. Echchakir H. et al.Functional characterization of an IL-7-dependent CD4+CD8αα+ Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma.Blood. 2000; 96: 1056-1063PubMed Google Scholar). Here we demonstrated that SC5 was coexpressed by the TCRVβ22+ cells, i.e., by the malignant cell population (Figure 7a). It should be noted that the profile of SC5 MoAb binding to TCRVβ22+ cells corresponded to a weak homo