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 Ca2؉, 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, lines as well as Th1 and Th0 clones synthesized TNF-␣ and IFN-␥ after this treatment. In contrast, Th2 clones were unable to produce IL-4 or IFN-␥ 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 CD99 (1–3) is the (12) that is expressed only on T cells, NK cells, and monocytes but product of the MIC2 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 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 , 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 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 CD4ϩCD8ϩ 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 2ϩ 2ϩ 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 ␮l of highly purified T cells (1 ϫ 107/ml) were mAb Name Isotype Specificity Source incubated for 30 min at 4°C with the indicated mAbs or an irrelevant ␮ 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 Ј 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 10Ϫ7 M and iono- (Raritan, NJ) ␮ 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 ϫ 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 ␮l. Proliferation was induced by the indicated mAbs (5 CD33-4D3 IgG2b CD33 —b ␮g/ml) cross-linked with GAM-IgG (10 ␮g/ml; Sigma) and by PMA (Sig- Ϫ MEM93 IgG1 CD45RA Gift of V. Horejsi ma; final concentration, 10 7 M) or ionomycin (Sigma; final concentration, UCHL1 IgG2a CD45R0 Biotechnische 1 ␮M). For proliferation experiments with immobilized CD3 mAb, 96-well Forschungs GmbH flat-bottom plates (Costar) were coated overnight at 4°C with 100 ␮lof (An der Grub, 0.125 to 1.0 ␮g/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 ␮g/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-␣ Caltag 37°C, the cells were pulsed with 1 ␮Ci/well of [methyl-3H]TdR (Amer- GZ4 IgG1 IFN-␥ 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 ␮g/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. Cell isolation Determination of calcium levels. Jurkat E6–1 cells or J.RT3-T3.5 were 2ϩ ␮ PBMC were isolated from heparinized blood of healthy adult donors by labeled with the fluorescent Ca indicator indo-1/AM (2 M; Molecular standard density gradient centrifugation with Ficoll-Paque (Pharmacia, Probes, Eugene, OR) by incubation at 37°C for 20 min in HBSS supple- ϫ 6 Uppsala, Sweden). Subsequently, T cells were separated by magnetic sort- mented with 10 mM HEPES and 0.5% BSA (HHB; 0.5%). Cells (1 10 ␮ ing using the MACS technique (Miltenyi Biotec, Bergisch Gladbach, Ger- in a volume of 20 l) were incubated with the indicated mAbs at a final ␮ ␮ many), as described previously (26). Purified T cells were obtained by concentration of 0.6 g (diluted with 0.5% HHB to a final volume of 20 l) ␮ depletion of CD11b, CD14, CD16, CD19, CD33, and HLA class II-posi- or with 20 l of 0.5% HHB alone for 18 min at room temperature. Sub- ␮ tive cells with the respective mAbs (Table I). The purity of the T cell sequently, the volume was adjusted to 250 l with 0.5% HHB medium Ͼ followed by a 7-min equilibration period at 37°C in the water bath. Sub- preparations was controlled by flow cytometry and was found to be 98%. 2ϩ sequently, measurement of [Ca ]i by flow cytometry was started at 37°C ␮ Ј constant temperature, and after 1 min, 20 g of cross-linking F(ab )2 of Immunoblots GAM-IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was added, and the measurement was continued for another 4 min. As positive Immunoblots were conducted as described previously (27). Jurkat cells ␮ (1 ϫ 108) were washed with ice-cold PBS followed by solubilization in 1 controls, 2 g of OKT3 mAb was added for CD3 stimulation, or thapsi- ml of lysis buffer (10 mM Tris-HCl (pH 8.3) (Merck, Darmstadt, Ger- gargin (Sigma) was used at a final concentration of 200 nM. Flow cyto- many), 140 mM sodium chloride (Merck), 2 mM EDTA (Sigma, St. Louis, metric analyses were performed on a FACStar Plus flow cytometer (Becton MO), 5 mM iodoacetamide (Sigma), 1% Nonidet P-40 (Pierce, Rockford, Dickinson) with the following settings: excitation by argon laser at 50 mW IL), 1 mM PMSF (Sigma), 15 ␮g/ml aprotinin (Sigma), and 15 ␮g/ml multiline UV, and emission at 530 nm (Fl1; calcium-free indo) and 395 nm leupeptin (Sigma)) for 30 min on ice. Lysates were centrifuged twice for 10 (Fl2, calcium-bound form of indo). The fluorescence ratio Fl2/Fl1, which min at 15,000 rpm in 1.5-ml tubes (Eppendorf, Hamburg, Germany) at 4°C is a direct estimate of the cytoplasmic calcium concentration (30), was to remove insoluble material. computed in real-time by a pulse-processing unit and is expressed as ar- Subsequently, soluble were diluted one-half with sample bitrary units. buffer, heated for 4 min at 96°C, subjected to SDS-PAGE (20 ␮l/slot, IL-2 luciferase reporter gene assay 12.5% gel) (28), and blotted onto nitrocellulose (Bio-Rad, Richmond, CA) (29). Western blots were then incubated with the indicated first Abs (0.2 Jurkat cells (clone 41-19) transfected with an IL-2 promoter (position ␮g/ml) for 3 h, followed by a 1-h incubation step with horseradish perox- Ϫ583 to ϩ40)-luciferase gene construct (31) were cultured in RPMI 1640 idase-conjugated sheep anti-mouse Ig Ab (Amersham, Aylesbury, U.K.) medium (Life Technologies) plus 10% FCS supplemented with 2 mM L- diluted 1/5000. Finally, a chemiluminescence detection system (Amer- glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and geneticin- sham) was used for the visualization of relevant proteins on Kodak X- 418 sulfate (0.8 mg/ml; Gibco). Ninety-six-well flat-bottom plates (Costar) OMAT S films (Eastman Kodak, Rochester, NY). Individual blots were were coated overnight at 4°C with 100 ␮l/well of a 10 ␮g/ml solution of exposed for 30 s to 5 min to gain optimal signal to noise ratio. GAM-IgG (Sigma) diluted in sterile PBS. Subsequently, plates were The Journal of Immunology 4673

washed twice with PBS, and free binding sites were blocked by incubation with RPMI 1640 medium plus 10% FCS for1hat37°C. Afterward, plates were reacted overnight at 4°C with 50 ␮l/well of OKT3 mAb (7.5 ng/ml diluted in PBS) followed by washing the plates once with PBS. Jurkat cells (5 ϫ 104/well/200 ␮l medium) were stimulated in triplicate in these pre- coated plates in the presence of the indicated mAbs (10 ␮g/ml final con-

centration) at 37°C in a 5% CO2 atmosphere. After 4 h, plates were cen- trifuged at 200 ϫ g for 5 min. Supernatants were removed by flicking, and the cells sticking to the plates were lysed by the addition of 25 ␮l/well of lysis buffer (Promega, Madison, WI) and by shaking for 10 min on a Titer- Tek apparatus (Flow Laboratories, Rockville, MD). After transfer of the cell lysates to Microlite plates (Dynatech Laboratories, Chantilly, VA) and the automatic addition of luciferin substrate solution (50 ␮l/well), fluores- cence due to luciferase activity was determined on a Luminoscan RS (Lab Systems, Helsinki, Finland). Arbitrary units obtained upon incubation with different mAbs were related to the values obtained after incubation of cells with medium alone (without further addition of mAbs). The medium value was designated 1. The results are expressed as x-fold costimulation (ϮSD), ϭ according to the formula: x-fold costimulationmAb ODmAb/ODmedium. The level of promoter activity of the medium control corresponds to an OD value of 0.057 Ϯ 0.020 (mean Ϯ SD). FIGURE 1. mAb 3B2/TA8 recognizes CD99. Western blotting of whole cell lysates of Jurkat T cells with 3B2/TA8 mAb. Nonidet P-40 (1%) Generation of T cell lines and T cell clones cell lysates of Jurkat cells were resolved on a 12.5% SDS-PAGE under For generation of PHA/IL-2-dependent blasts, PBMC (1 ϫ 105/well) were nonreducing (A) or reducing (B) conditions, transferred onto nitrocellulose, cultured in RPMI 1640 plus 10% FCS (Life Technologies) supplemented and reacted either with 3B2/TA8 mAb, the HLA class I ␣-chain-specific with antibiotics in the presence of PHA (Sigma; final concentration, 1 mAb LA45, or with the irrelevant isotype-matched control mAb VIAP. ␮g/ml) in 96-well U-bottom culture plates (Costar) for 7 days. Subse- First Abs bound to the membrane were detected with goat anti-mouse Abs quently, every 5 to 7 days 10 U/ml of IL-2 (provided by the Novartis conjugated with horseradish peroxidase. Bound second Abs were visual- Research Institute, Vienna, Austria) plus autologous irradiated (3000 rad, ized by the application of a chemiluminescence detection system. The po- 137 ϭ Cs source) PBMC as feeder cells (ratio of blasts/feeder cells 1:1) were sitions and molecular masses of prestained standard proteins are indicated. added. The cells were cultured for at least 1 mo before the first experiments were performed. EBV-transformed lymphoblastoid B cells (EBV-LCL) were TNP mod- ified by treatment with 2,4,6-trinitrobenzene sulfonic acid (Sigma) as pre- viously described (32). Autologous PBL were stimulated with TNP-mod- Biochemical analyses show that the mAb 3B2/TA8 recognizes two ified EBV-LCL for 7 days in complete medium plus 5% human AB serum. structures with molecular masses of approximately 28 and 32 kDa Upon restimulation and cultivation for 5 days in the presence of IL-2 (10 in whole cell lysates of Jurkat T cells (Fig. 1), which is in agree- U/ml), the outgrowing T cells were cloned by limiting dilution and were ment with previous reports on other CD99 mAbs (3, 8). propagated by weekly restimulation with TNP-modified EBV-LCL and IL-2 (10 U/ml). CD99, as detected by 3B2/TA8 mAb, is expressed on the whole T cell blasts from Bet v 1-specific T cell lines were obtained as previ- PB T cell population (Fig. 2). This is at variance with certain other ously described (33). T cell blasts were seeded in limiting dilution (0.3 CD99- or CD99R-specific mAbs, such as 12E7 or D44, which cells/well) in 96-well U-bottom plates (Nunclone, Nunc, Roskilde, Den- have been described to react only with distinct PB T cell subpopu- mark) in the presence of 105 irradiated (5000 rad) allogeneic PBMC as feeder cells, 1% (v/v) PHA (Life Technologies), and rIL-2 (4 U/well) in lations (10, 12). Following cellular activation, CD99 is up-regu- Ultra culture medium (BioWhittaker, Walkersville, MD). Growing mi- lated, resulting in a uniformly high expression density on the crocultures were then expanded at weekly intervals with fresh feeder whole T cell population (Fig. 2). The kinetics of CD99 up-regu- cells and rIL-2. The specificity of T cell clones was assessed as previ- lation on T cells following polyclonal activation with PMA plus ously described (34). ionomycin, compared with those of CD69 or CD25, are slow and Determination of intracellular cytokines require Ͼ72 h for completion (data not shown).

Ninety-six-well flat-bottom tissue culture plates (Costar) were coated with CD99 cross-linking induces proliferation of PB T cells in the GAM-IgG (Sigma; 10 ␮g/ml) plus a suboptimal concentration of the CD3 mAb OKT3 (20 ng/ml) at 4°C overnight. After two washings with PBS, T presence of a suboptimal TCR/CD3 stimulus ϫ 5 cell lines or clones (1–2 10 /well) were incubated in precoated plates Ligation of CD99 on immature T cells has previously been shown with optimal concentrations (5 ␮g/ml) of CD99 mAb 3B2/TA8, CD28 mAb Leu28, or isotype control mAb recognizing the NGFR. Assays were to induce apoptotic cell death (23–25). With mature PB T cells no set up in a total volume of 200 ␮l/well in RPMI 1640 medium containing such effect was observed (23–25). 5% pooled human serum supplemented with antibiotics and 2 ␮g/ml (final In an attempt to characterize the signaling potential of CD99 on concentration) of brefeldin A (Sigma). After 18 h of incubation at 37°C in mature PB T cells, we incubated resting T cells with the CD99 a5%CO atmosphere, the cells were harvested and analyzed for the pres- 2 mAb 3B2/TA8 in the presence or the absence of a CD3 trigger. ence of intracellular cytokines. For staining, 50 ␮l of the cell suspension (corresponding to 1–2 ϫ 105 cells) were fixed for 30 min at room temper- The mAb 3B2/TA8 alone or cross-linked with GAM-IgG did not ature by the addition of 100 ␮l of FIX solution (An der Grub). Subse- induce T cell proliferation and, in agreement with previous studies, quently, cells were washed once with 4 ml of PBS/1% BSA, resuspended did not induce (data not shown). However, in the pres- ␮ ␮ in 50 l of PBS/1% BSA, permeabilized by the addition of 100 lof ence of a suboptimal TCR/CD3 signal delivered by plate-bound PERM solution (An der Grub), and incubated for 30 min at room tem- perature with the indicated directly conjugated anti-cytokine mAb. Fi- CD3 mAb OKT3, cross-linking of CD99 with 3B2/TA8 mAb and nally, cells were washed twice, resuspended in PBS, and analyzed by GAM-IgG resulted in vigorous proliferation of PB T cells (Fig. 3, flow cytometry. A and B). Compared with a CD3 plus CD28 mAb (Leu28)-based stimulation, the cross-linked 3B2/TA8 mAb requires higher CD3 Results mAb (OKT3) concentrations to obtain equivalent [methyl-3H]TdR PB T cells express CD99 that is up-regulated upon cellular uptake values (Fig. 3A). In contrast, soluble CD3 mAb plus soluble activation CD99 mAb, even when cross-linked with GAM-IgG, did not lead Our mAb 3B2/TA8 was classified as a CD99 mAb at the Sixth to the proliferation of PB T cells (Fig. 3B). Furthermore, stimula- Workshop on Human Leucocyte Differentiation Antigens (35). tion with the IgM-type CD3 mAb VIT3, PMA, ionomycin, or 4674 T CELL STIMULATION VIA CD99

FIGURE 2. Binding pattern of CD99 mAb 3B2/TA8 to resting and ac- tivated T cells. Resting PB T cells were stained with the CD99 mAb 3B2/ TA8, the CD25 mAb 3G10, the CD3 mAb OKT3 (gray profiles), or the irrelevant isotype control mAb VIAP (open profiles, thin lines). Cells were activated with PMA and ionomycin for 60 h, stained, and analyzed by flow cytometry, leading to open histograms (thick lines). Binding of mAbs to individual cells was visualized by an FITC-SAM conjugate, and the fluo- rescence of cells was analyzed on a flow cytometer and is displayed as overlay histograms. The ordinate indicates the number of cells with a given green fluorescence intensity shown on the abscissa. The figure shows one representative experiment of three performed.

FIGURE 3. CD99 mAb 3B2/TA8 induces proliferation of PB T cells in CD28 failed to provide the appropriate signal sufficient to render the presence of suboptimal TCR/CD3 stimulation. A, Highly purified T ϫ 4 PB T cells responsive for CD99 signals (Fig. 3B). cells (5 10 ) were exposed to culture plates coated with the indicated concentrations of CD3 mAb OKT3 and to the soluble CD99 mAb (3B2/ CD99 cross-linking on T cells results in TCR/CD3-dependent TA8; black circles), the isotype-matched control mAb NGFR (open cir- 28 elevation of [Ca2ϩ] cles), or the CD28 mAb (Leu ; black squares). In all experiments mAbs i were cross-linked with GAM-IgG. After 3 days of culture, [methyl-3H]TdR In of the observed mitogenic properties of the CD99 mAb was added for the following 18 h, and thymidine uptake was determined as 3B2/TA8 on T cells, we subsequently analyzed early T cell acti- described in Materials and Methods. The figure shows [methyl-3H]TdR vation events in the human leukemic T cell line Jurkat (36, 37). uptake, in counts per minute, from one representative experiment of six The results in Figure 4A show that intracellular free calcium performed. B, Highly purified T cells (5 ϫ 104) were incubated on either 28 concentrations in Jurkat T cells are significantly increased upon coated (CD3 mAb OKT3, or CD28 mAb Leu ) or uncoated 96-well flat- cross-linking of CD99 with 3B2/TA8 mAb plus GAM-IgG ( p Ͻ bottom tissue culture plates. As indicated in the figure, individual wells ␮ 0.007, by Student’s paired t test). This effect was strictly dependent were supplemented in addition with soluble CD99 mAb 3B2/TA8 (5 g/ ml), the isotype-matched control mAb NGFR (5 ␮g/ml), CD28 mAb Leu28 on GAM-IgG-enhanced cross-linking of the CD99 molecules with (5 ␮g/ml), CD3 mAb OKT3 (1 ␮g/ml), CD3 mAb VIT3 (5 ␮g/ml), iono- mAb 3B2/TA8, since no calcium mobilization was detected upon mycin (1 ␮M), or PMA (10Ϫ7 M). To all wells containing soluble mAbs, incubation with 3B2/TA8 mAb alone (data not shown). Treatment GAM-IgG (20 ␮g/ml) was added as a cross-linker. In proliferation assays of Jurkat T cells with CD3 mAbs plus GAM-IgG consistently led with solid phase CD28 mAb Leu28, all mAbs tested in combination were to a very strong increase in cytoplasmic free calcium levels, provided in a plate-bound form. After 3 days of culture, [methyl-3H]TdR whereas the binding or nonbinding control mAbs did not influence was added for the following 18 h, and thymidine uptake was determined as 2ϩ described in Materials and Methods. The figure shows the [methyl-3H]TdR [Ca ]i (Fig. 4A). Given the fact that CD99-induced proliferation requires TCR/ uptake, in counts per minute (mean Ϯ SEM), of seven experiments. CD3 engagement, we analyzed whether the Ca2ϩ response to CD99 is linked to TCR/CD3-mediated signaling events. We ex- amined TCR/CD3-modulated parental Jurkat cells and the TCR/ CD3-negative Jurkat subclone J.RT3-T3.5. Importantly, in both CD99 cross-linking in the presence of a suboptimal TCR/CD3 cell lines CD99 mAb 3B2/TA8 failed to induce significant changes trigger induces IL-2 promoter activity 2ϩ in [Ca ]i (Fig. 4, B and C), although the cell surface expression Cytokine production by T cells plays an important role in the reg- levels of CD99 are comparable to those on the parental cell line ulation of the immune response (reviewed in Ref. 38). Therefore, 2ϩ (Fig. 4A). The abortive CD3 mAb-induced rise in [Ca ]i observed we wondered whether in addition to its proliferation-inducing ef- in TCR/CD3-modulated Jurkat cells might be due to the residual fects on T cells, CD99 engagement is able to induce cytokine expression of TCR/CD3, as shown by FACS analysis (Fig. 4B). . The Journal of Immunology 4675

FIGURE 4. Cross-linking of CD99 elevates intracellular free Ca2ϩ levels in Jurkat cells, which is dependent on TCR/CD3 expression. De- termination of intracellular free Ca2ϩ levels is shown in TCR/CD3-positive, parental Jurkat cells (A), in TCR/CD3-modulated Jurkat cells (B), and in the TCR/CD3-deficient Jurkat sub- clone J.RT3-T3.5 (C). Jurkat cells were loaded with indo-1 and incubated with CD3 mAb OKT3 (black triangles), CD99 mAb 3B2/TA8 (black squares), the binding isotype-matched control mAb 132/3C3 (CD147, open triangles), or the nonbinding isotype-matched control mAb NGFR (open squares). Thapsigargin (black circles) was used as a positive control in B and C. Cytoplas- mic free Ca2ϩ concentrations were determined by flow cytometry upon cross-linking of mAbs Ј with F(ab )2 of GAM-IgG. The figure shows the time-dependent indo-1 fluorescence ratio of one representative experiment. The time of addition of GAM-IgG or thapsigargin is indicated by the arrow. The right side of the figure shows a pair of overlay histograms for each cell line. Profiles of the irrelevant control mAb VIAP (thick lines, left side of histograms) are overlaid by histograms obtained upon staining with CD99 mAb 3B2/ TA8 or CD3 mAb VIT3 (thin lines). Mean flu- orescence intensity values obtained upon staining with specific mAbs are indicated. In the histo- grams, the ordinate represents the cell number, and the abscissa represents the individual log green fluorescence intensity of a given cell.

For that purpose, Jurkat cells transfected with an IL-2 promoter/ luciferase-gene construct were exposed to both a suboptimal TCR/ CD3 stimulus delivered by the mAb OKT3 and optimal concen- trations of the mAb 3B2/TA8 or the indicated control mAbs. As shown in Figure 5, CD99 mAb 3B2/TA8 cross-linking on Jurkat T cells leads to an approximately 6-fold increase in IL-2 promoter activity ( p Ͻ 0.006, by paired Student’s t test) compared with the isotype-matched binding or nonbinding control mAbs. Anti-CD28 mAb (Leu28) stimulation of Jurkat T cells resulted in a Ͼ10-fold induction of IL-2 promoter activity (Fig. 5). This demonstrates that besides its comitogenic capacity, CD99 mAb 3B2/TA8 also costimulates IL promoter activity leading to gene transcription.

CD99 mAb 3B2/TA8 induces TNF-␣ and IFN-␥ production in polyclonally activated T cell lines, Th1 and Th0 clones Th1 immune responses, even when established, appear to be sen- sitive to modulation by CD28 costimulation (39). Most established Th1 clones continue to require CD28 costimulation for activation (40). To disclose whether CD99 engagement could substitute for FIGURE 5. CD99 ligation elevates IL-2 promoter activity. Human CD28 costimulation, we performed intracellular staining experi- Jurkat T cells transfected with an IL-2 promoter-luciferase gene construct ments for ILs in PB-derived T cell lines and clones. were exposed to suboptimal concentrations of plate-bound CD3 mAb In polyclonally activated T blasts, cross-linkage with the CD99 OKT3 in the presence of CD99 mAb 3B2/TA8. As negative controls, we mAb 3B2/TA8 in the presence of suboptimal concentrations of the used both a nonbinding (VIAP) and a binding (132/3C3; CD147) isotype- 28 CD3 mAb OKT3 led to the production of TNF-␣ in a high pro- matched control mAb. The CD28 mAb (Leu ) served as a positive control. portion of cells (Fig. 6). TNF-␣-producing cells ranged from 12.5 Arbitrary units obtained upon incubation with different mAbs were related to the values obtained after incubation of cells with medium alone (without to 52.5% (mean Ϯ SEM, 28.8 Ϯ 7.3%) and were significantly further addition of mAbs). The medium value was designated 1. The figure increased compared with cells treated with CD3 mAb plus the shows the mean (ϮSD) relative increase in IL-2 promoter activity of nine Ϯ isotype control mAb NGFR (7.8 0.9%). Furthermore, Th1 and independently performed experiments. Values obtained upon incubation Th0 cell clones responded to CD99 costimulation with the pro- with CD99 mAb 3B2/TA8 were compared with those obtained upon in- duction of IFN-␥ (Table II). More importantly, CD99 mAbs failed cubation with VIAP mAb by paired Student’s t test and were significant to induce significant levels of IL-4 in both Th0 and Th2 clones and (p Ͻ 0.006). 4676 T CELL STIMULATION VIA CD99

Table II. Cytokine production by T cell lines and T cell clonesa

CD3 Suboptimal

Mock NGFR 3B2/TA8 Leu28

IFN-␥ Cell lines PHA 96/5966 2.5 12.0 27.7 (2.3) 42.1 (3.5) PHA 96/6820 4.0 4.8 13.0 (2.7) 47.1 (9.8) PHA 96/9163 4.8 11.2 57.5 (5.2) 39.5 (3.5) Th1-Th0 clones VIC7 ND 12.4 46.2 (3.7) 11.3 (Ͻ1) VIC11 ND 2.7 17.1 (6.3) 30.2 (11.2) VIC12 ND 4.4 10.8 (2.5) 15.0 (3.4) VIC14 ND 8.0 23.4 (2.9) 30.0 (3.8) Th2 clones VIC3 ND 4.6 5.4 (1.2) 5.4 (1.2) Ͻ FIGURE 6. CD99 mAb 3B2/TA8 induces TNF-␣ production in PHA/ VIC13 3.5 2.4 4.4 (1.3) 2.4 ( 1) PSD238 0.2 0.3 0.14 (Ͻ1) 0.3 (1) IL-2-dependent T cell lines suboptimally stimulated with CD3 mAb OKT3. PSD218 0.9 0.3 1.40 (1.6) 0.7 (Ͻ1) PHA/IL-2-dependent cell lines were generated as described in Materials PSD266 1.4 0.3 1.76 (1.3) 0.4 (Ͻ1) ϫ and Methods. Five to seven days after the last stimulation cells (1–2 IL-4 105/well) were incubated on uncoated (mock) or mAb-coated (CD3 mAb Cell lines OKT3 plus NGFR, CD99 mAb 3B2/TA8, or CD28 mAb Leu28) 96-well PHA 96/5966 2.5 2.5 3.4 (1.4) 5.0 (2.0) flat-bottom culture plates. They were incubated overnight in RPMI 1640 PHA 96/6820 2.2 3.0 2.3 (Ͻ1) 2.4 (Ͻ1) medium supplemented with 5% HS plus 2 ␮g/ml brefeldin A. The next PHA 96/9163 1.6 2.0 2.8 (1.4) 2.4 (1.2) day, individual cell samples were harvested, fixed, permeabilized, and sub- Th1-Th0 clones Ͻ sequently stained with an FITC-conjugated TNF-␣-specific mAb and a VIC7 ND 3.5 2.9 ( 1) 5.9 (1.7) VIC11 1.9 3.9 3.4 (Ͻ1) 2.9 (Ͻ1) phycoerythrin-conjugated control mAb followed by flow cytometric anal- VIC12 ND 1.5 1.4 (Ͻ1) 7.8 (5.2) ysis. Results are depicted as two parameter dot plots showing red fluores- VIC14 ND 2.4 1.8 (Ͻ1) 13.6 (5.7) cence intensity on the ordinate and green fluorescence intensity on the Th2 clones abscissa. As a negative control for green fluorescence the FITC-conjugated VIC3 ND 3.5 4.1 (1.2) 16.5 (4.7) isotype-matched nonbinding control mAb VIAP was used. Markers were VIC13 6.7 18.4 17.4 (Ͻ1) 27.5 (1.5) set according to the fluorescence characteristics of cells that were incubated PSD238 0.8 5.1 4.7 (Ͻ1) 17.3 (3.4) with the negative control mAbs. Numbers indicate the percentage of single PSD218 0.3 6.3 4.2 (Ͻ1) 19.9 (3.2) positive (green) cells. This figure shows a representative experiment of 14 PSD266 2.2 10.1 11.6 (1.1) 22.5 (2.2) performed. a The table shows the percentage of positive cells upon staining with directly labeled mAbs specific for IFN-␥ or IL-4 as determined by intracellular staining of cytokines followed by FACS analyses. Cells were analyzed either unstimulated (mock) or after overnight stimulation with suboptimal doses of plate bound CD3 mAb OKT3 plus either isotype control mAb NGFR, or CD99 mAb 3B2/TA8 or CD28 mAb were incapable of inducing IFN-␥ production in Th2-restricted Leu-28. Numbers in parentheses display the x-fold increase of positive cells upon engagement of T cells with CD99 or CD28 mAbs compared to NGFR or mock clones (Table II). treatment. ND, not done. This clearly distinguishes CD99 from CD28 costimulation, in- asmuch as CD28-derived signals drive both Th1-restricted cyto- kine responses by Th1 clones and Th2-restricted cytokine produc- In our experiments cross-linking of CD99 with mAb 3B2/TA8 tion by Th2 clones (Table II). In general, intracellular IL and GAM-IgG dramatically reduced the threshold of anti-CD3- production determined at the single cell level correlated well with mediated proliferation of freshly isolated, resting PB T cells (Fig. total cytokine production by these cells as measured by sandwich 3A). In general, CD99-induced [methyl-3H]TdR incorporation ELISA assays (data not shown). Significant induction of cytoplas- reached its maximum between day 3 and day 4 of costimulation, matically stored IL-2 could only be detected by a combination of which was similar to the CD28-based costimulatory kinetics ob- CD3 mAb plus CD28 mAb (data not shown). served in parallel experiments (data not shown). Importantly, the induction of T cell proliferation in combination with a suboptimal Discussion TCR/CD3 stimulus is not a salient feature of our CD99 mAb 3B2/ Ligation of CD99/E2 surface molecules has been recently shown TA8, since similar activity was also observed with the classical to induce apoptotic cell death of immature double-positive thymo- CD99/MIC2 mAb 12E7 (data not shown). Consequently, the strict cytes but not to affect other thymocytes or mature T lymphocytes requirement for pan CD99 mAbs, as reported for ag- (24). Here we demonstrate that cross-linking of CD99 is capable of gregation and apoptosis (23–25), does not seem to apply for the providing a potent costimulus for mature T cells leading to cell induction of T cell mitogenesis. proliferation and Th1-type cytokine production in combination The finding that CD99 does not cosignal with PMA or CD28 with a suboptimal TCR/CD3 signal. Thus, our data support the clearly distinguishes CD99 from other costimulatory molecules view that CD99 molecules on mature PB T cells can transmit a such as CD2 (46–48), CD5 (49–51), and CD47 (52), which are positive activation signal. That the signaling properties of one able to activate T cells in the absence of a TCR/CD3 stimulus. It given surface molecule may depend on the maturational stage of seems that CD99 obviously does not induce a signaling cascade the respective T cell is highly reminiscent of CD2 and CD3 mol- that can substitute a TCR/CD3 stimulus, suggesting a role for ecules. Engagement of these molecules on distinct populations of CD99 only in Ag-dependent stimulation of PB T cells. Further- thymocytes mediates apoptosis (41–43), whereas engagement of more, we show that only a solid phase TCR/CD3 trigger leads to the same receptors on PB T cells provides a strongly activating CD99-based costimulation. Neither CD3 mAb OKT3 provided in signal (44, 45). soluble form, an IgM-type CD3 mAb (VIT3) that is known to elicit The Journal of Immunology 4677 strong Ca2ϩ fluxes in T cells (53), nor calcium ionophore was able duction in either of the cell lines tested was detected only upon to substitute for solid phase TCR/CD3 ligation. CD28 costimulation or in PMA- plus ionomycin-triggered cells. How would solid phase vs solution phase cross-linking of the These results, which are in contrast to those of the IL-2 promoter TCR/CD3 complex influence the costimulatory outcome via an studies, might be explained by cell type-specific slower production unrelated cell surface molecule such as CD99? One possibility or higher consumption rates of IL-2 compared with IFN-␥ or might be that heterologous cross-linking of TCR/CD3 and CD99 TNF-␣. The capability for CD99 costimulation and induction of in solution phase could lead to the disruption of a putative critical IFN-␥ production in Th1/Th0 clones is reminiscent of the signal- association between these two receptors. This hypothesis, how- ing lymphocytic activation molecule described recently (59). At ever, would suggest that the CD99 molecule might functionally variance to signaling lymphocytic activation molecule, CD99 co- and/or sterically be coupled with the TCR/CD3 complex in T cells. stimulation is insufficient, however, to induce a shift in cytokine That CD99 is indeed noncovalently associated in a membrane mi- production of Th2 clones. crodomain with the TCR/CD3 complex of T lymphocytes has been Taken together our data indicate that while CD99 costimulation shown recently by Cerny and co-workers (54). is able to drive Th1 and Th0 clones toward the production of the Further indications for a dependence between the TCR/CD3 Th1-specific cytokine IFN-␥, it is incapable of driving Th2 or Th0 complex and CD99 are provided by our calcium flux experiments. clones to synthesize detectable amounts of IL-4, otherwise induced Multimerization of CD99 molecules leads to a clear-cut elevation by CD28 costimulation. Of importance, allergic diseases have of cytosolic calcium levels in parental Jurkat cells that express the been shown to be associated with down-regulation of IFN-␥-pro- TCR/CD3 complex. However, when Jurkat T cells, after down- ducing cells and the expansion of Th2-type cytokine-producing modulation of TCR/CD3 molecules by overnight incubation with cells, leading to enhanced IgE synthesis (60, 61). Conversely, a CD3-specific mAb, are treated with CD99 mAb 3B2/TA8 plus IFN-␥ has been shown to be one of the dominant factors inhibiting GAM-IgG, no increment in intracellular calcium levels was ob- IL-driven IgE production (62, 63). Thus, our data allow us to sug- served. Note that in these cells also the CD3-mediated calcium flux gest that therapeutically induced activation pathways, such as was strongly compromised. These experiments allow us to suggest CD99-driven costimulation, that effectively augment levels of T that reduced levels of cell surface-expressed TCR/CD3 impair cell-produced IFN-␥ while having no stimulatory effect on Th2- CD99-driven signal transduction in T cells and underline the im- type cells could provide an efficient way to intervene with allergic portance of the TCR/CD3 trigger for CD99 costimulation. diseases (64). To further explore that issue and to rule out modulation-induced negative or regulatory signals as the overall basis for the CD99 Acknowledgments nonresponsiveness, a variant of the Jurkat line (clone J.RT3-T3.5) We thank Ms. Lisbeth Gschwantler and Mr. Klaus Wenhardt for expert lacking TCR/CD3 surface expression was analyzed and was found FACS analyses, and Ms. Saro Ku¨nig for expert technical assistance. We are 2ϩ to be unable to increase [Ca ]i upon CD99 cross-linking. 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