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CD99 Engagement on Human Peripheral Blood T Cells Results in TCR/CD3-Dependent Cellular Activation and Allows for Th1-Restricted Cytokine Production1 Martina Waclavicek,* Otto Majdic,* Thomas Stulnig,† Markus Berger,† Raute Sunder-Plassmann,‡ Gerhard J. Zlabinger,* Thomas Baumruker,¶ Johannes Sto¨ckl,* Christof Ebner,§ Walter Knapp,* and Winfried F. Pickl2* We have assessed the functional effect of CD99 engagement on resting human peripheral blood (PB) T cells. CD99, as detected by the mAb 3B2/TA8, is constitutively expressed on all PB T cells and becomes further up-regulated upon cellular activation. In this study we demonstrate that cross-linking of the CD99 molecule with the agonistic mAb 3B2/TA8 cooperates with suboptimal TCR/CD3 signals, but not with phorbol ester, ionomycin, or CD28 mAb stimulation, to induce proliferation of resting PB T cells. Comparable stimulatory effects were observed with the CD99 mAb 12E7. Characterization of the signaling pathways involved revealed that CD99 engagement leads to the elevation of intracellular Ca21, which is dependent on the cell surface expression of the TCR/CD3 complex. No CD99 mAb-induced calcium mobilization was observed on TCR/CD3-modulated or TCR/CD3-neg- ative T cells. To examine the impact of CD99 stimulation on subsequent cytokine production by T cells, we cross-linked CD99 molecules in the presence of a suboptimal TCR/CD3 trigger followed by determination of intracellular cytokine levels. Signifi- cantly, T cell lines as well as Th1 and Th0 clones synthesized TNF-a and IFN-g after this treatment. In contrast, Th2 clones were unable to produce IL-4 or IFN-g when stimulated in a similar fashion. We conclude that CD99 is a receptor that mediates TCR/CD3-dependent activation of resting PB T cells and specifically induces Th1-type cytokine production in polyclonally acti- vated T cell lines, Th1 and Th0 clones. The Journal of Immunology, 1998, 161: 4671–4678. he type I integral membrane protein CD99 (1–3) is the (12) that is expressed only on T cells, NK cells, and monocytes but product of the MIC2 gene that is located in the pseudo- not on B cells, erythrocytes, or platelets. T autosomal (pairing) region of the human X and Y chro- The function of CD99 is not yet fully understood. The CD99 mosomes (4). Originally CD99 was described as a human thymus protein has, on the one hand, limited regions of similarity to col- leukemia Ag (5), an Ewing‘s sarcoma-specific membrane marker lagen (1, 3, 12); on the other, it is strongly glycosylated, and all molecule (6, 7), and a putative adhesion molecule (termed E2) sugar residues appear to be O-linked (8). The fully sialylated 32- involved in spontaneous rosette formation of T cells with eryth- kDa membrane form of CD99 is thus related to other sialomucin- rocytes (3, 8–10). CD99 is broadly distributed on many cell types, type glycoproteins, such as CD34 or CD43, which represent signal with particularly strong expression on Ewing’s sarcoma cells and transducing cell surface molecules involved in cellular adhesion peripheral primitive neuroectodermal tumors (7, 11). Within the processes (15–22). hemopoietic system, CD99 is expressed on virtually all cell types Signal transduction via CD99 has to date only been demon- except granulocytes (12). The expression density on T-lineage strated in immature thymocytes and Jurkat cells (23, 24). With cells seems to be maturation linked. CD99 has been shown to be thymocytes, CD99 ligation was shown to induce phosphatidylser- highly expressed on cortical thymocytes, whereas further differ- ine exposure at the cell membrane followed by apoptotic cell death entiated medullary thymocytes exhibit relatively weak CD99 ex- of a distinct subset of CD41CD81 thymocytes, a process preceded pression (3, 9). Heterogeneity in CD99 expression and epitope by homotypic adhesion of the very same cell population (23–25). density is also observed on PBLs. With distinct CD99 mAbs, dif- More mature, single-positive thymocytes were not affected by this ferent T cell subsets can be distinguished (10, 13, 14). Interest- treatment, nor were mature peripheral blood (PB)3 T lymphocytes ingly, a restricted epitope of CD99, i.e., CD99R, has been defined (24). The functional consequences of CD99 ligation observed in these experiments were thus restricted to a particular stage of T cell development, and they ultimately resulted in apoptotic cell *Institute of Immunology, University of Vienna, Vienna Austria; †Division of En- death. docrinology and ‡Division of Nephrology and Dialysis, Department of Internal Med- In this paper we analyze the function of CD99 on mature PB T icine III, §Institute of General and Experimental Pathology, University of Vienna, cells and demonstrate growth- and function-promoting stimulatory Vienna, Austria; and ¶Novartis Research Institute, Vienna, Austria effects. Cross-linking of CD99 on resting PB T cells in the pres- Received for publication March 18, 1998. Accepted for publication June 29, 1998. ence of a suboptimal TCR/CD3 trigger leads to their polyclonal The costs of publication of this article were defrayed in part by the payment of page expansion and to Th1-type growth factor production in T cell lines charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and T cell clones. 1 This work was supported by the Fonds zur Fo¨rderung der Wissenschaftlichen For- schung in O¨ sterreich. 2 Address correspondence and reprint requests to Dr. Winfried F. Pickl, Institute of 3 Abbreviations used in this paper: PB, peripheral blood; GAM-IgG, goat anti-mouse 21 21 Immunology, University of Vienna, Borschkegasse 8A, A-1090 Vienna, Austria. E- immunoglobulin G; [Ca ]i, intracellular Ca concentration; TNP, trinitrophenyl; mail address: [email protected] NGFR, nerve growth factor receptor. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 4672 T CELL STIMULATION VIA CD99 Table I. MAbs used in the studya Immunofluorescence analyses For membrane staining, 50 ml of highly purified T cells (1 3 107/ml) were mAb Name Isotype Specificity Source incubated for 30 min at 4°C with the indicated mAbs or an irrelevant m b isotype-matched control mAb (VIAP) used in a concentration of 20 g/ml. VIAP IgG1 Calf intestine — After washing cells twice with ice cold PBS/1% BSA solution, binding of alcaline 9 the primary mAb was visualized using sheep F(ab )2 anti-mouse Ig-FITC phosphatase (SAM; An der Grub, Bio Forschungs, Kaumberg, Austria) as the second- 200-3-G6-4 IgG1 NGFR ATCC (Manassas, VA) step reagent. To analyze surface expression after activation, cells were OKT3 IgG2a CD3 Ortho Diagnostics incubated with PMA (Sigma) in a final concentration of 1027 M and iono- (Raritan, NJ) m b mycin (Sigma; final concentration, 1 M) for 60 h, followed by the stain- VIT3 IgM CD3 — ing procedure as described above. After washing the cells three times with LM2 IgG1 CD11b ATCC b PBS/1% BSA, the membrane fluorescence was analyzed on a FACScan VIM13 IgM CD14 — flow cytometer supported by CellQuest software (Becton Dickinson, San MEM18 IgG1 CD14 Gift of V. Horejsi (Praha, Jose, CA). Czech Republic) 3G8 IgG1 CD16 Caltag (Burlingame, CA) T cell proliferation assays BU12 IgG1 CD19 Gift of D. Hardie (Birmingham, UK) Proliferation assays of highly purified PB T cells derived from healthy 3G10 IgG1 CD25 —b adult volunteers (5 3 104 cells/well) were performed in triplicate in 96- Leu-28 IgG1 CD28 Becton Dickinson well U-bottom tissue culture plates (Costar, Cambridge, MA) in a final (San Jose, CA) volume of 200 ml. Proliferation was induced by the indicated mAbs (5 CD33-4D3 IgG2b CD33 —b mg/ml) cross-linked with GAM-IgG (10 mg/ml; Sigma) and by PMA (Sig- 2 MEM93 IgG1 CD45RA Gift of V. Horejsi ma; final concentration, 10 7 M) or ionomycin (Sigma; final concentration, UCHL1 IgG2a CD45R0 Biotechnische 1 mM). For proliferation experiments with immobilized CD3 mAb, 96-well Forschungs GmbH flat-bottom plates (Costar) were coated overnight at 4°C with 100 mlof (An der Grub, 0.125 to 1.0 mg/ml of purified OKT3 mAb diluted in PBS. The plates were Kaumberg, Austria) washed twice with PBS and subsequently used for the assays. PMA (Sig- L78 IgG1 CD69 Becton Dickinson ma), ionomycin (Sigma), and the mAbs were diluted in RPMI 1640 (Life 3B2/TA8 IgG1 CD99 —b Technologies, Grand Island, MD) supplemented with 10% FCS, 2 mM 12E7 IgG1 CD99 6th Workshop on HLDA L-glutamine, 10 U/ml penicillin, and 100 mg/ml streptomycin. GAM-IgG 132/3C3 IgG2b CD147 —b and the cells were resuspended in RPMI 1640 supplemented with 10% LA45 IgG1 HLA-Class I —b pooled human serum. b VID1 IgG1 HLA-Class II — After 72 h of incubation in a humidified atmosphere with 5% CO2 at MP9-20A4* IgG TNF-a Caltag 37°C, the cells were pulsed with 1 mCi/well of [methyl-3H]TdR (Amer- GZ4 IgG1 IFN-g Boehringer Mannheim sham). Eighteen hours later the cell lysates were harvested on glass-fiber (Mannheim, Germany) filters (Packard, Topcount, Meriden, CT), and radioactivity was determined MP4-25D2* IgG1 IL-4 Caltag on a microplate scintillation counter (Packard). a Table shows clone names of used mouse and rat (*) mAbs, their isotypes, their Determination of cytoplasmic free calcium concentrations corresponding specificities, and their providers. b Our laboratory. Cell culture. The human T cell line Jurkat, subclone E6-1, and J.RT3- T3.5 (both obtained from the American Type Culture Collection, Manas- sas, VA) were grown under standard conditions in RPMI 1640 medium supplemented with 10% heat-inactivated bovine calf serum (HyClone, Lo- gan, UT), penicillin/streptomycin (50 U/ml and 50 mg/ml; Life Technol- Materials and Methods ogies, Gaithersburg, MA), and 2 mM glutamine (Life Technologies) at 37°C in a humidified atmosphere in the presence of 5% CO2.