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The Journal of Immunology, 1998, 161: 4671-4678.
Copyright © 1998 by The American Association of Immunologists

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{dagger}, Markus Berger{dagger}, Raute Sunder-Plassmann{ddagger}, Gerhard J. Zlabinger*, Thomas Baumruker, Johannes Stöckl*, Christof Ebner§, Walter Knapp* and Winfried F. Pickl2,*

* Institute of Immunology, University of Vienna, Vienna Austria; {dagger} Division of Endocrinology and {ddagger} Division of Nephrology and Dialysis, Department of Internal Medicine III, § Institute of General and Experimental Pathology, University of Vienna, Vienna, Austria; and Novartis Research Institute, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-negative 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. Significantly, T cell lines as well as Th1 and Th0 clones synthesized TNF-{alpha} and IFN-{gamma} after this treatment. In contrast, Th2 clones were unable to produce IL-4 or IFN-{gamma} 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 activated T cell lines, Th1 and Th0 clones.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The type I integral membrane protein CD99 (1, 2, 3) is the product of the MIC2 gene that is located in the pseudoautosomal (pairing) region of the human X and Y chromosomes (4). Originally CD99 was described as a human thymus leukemia Ag (5), an Ewing‘s sarcoma-specific membrane marker molecule (6, 7), and a putative adhesion molecule (termed E2) involved in spontaneous rosette formation of T cells with erythrocytes (3, 8, 9, 10). CD99 is broadly distributed on many cell types, with particularly strong expression on Ewing’s sarcoma cells and peripheral primitive neuroectodermal tumors (7, 11). Within the hemopoietic system, CD99 is expressed on virtually all cell types except granulocytes (12). The expression density on T-lineage cells seems to be maturation linked. CD99 has been shown to be highly expressed on cortical thymocytes, whereas further differentiated medullary thymocytes exhibit relatively weak CD99 expression (3, 9). Heterogeneity in CD99 expression and epitope density is also observed on PBLs. With distinct CD99 mAbs, different T cell subsets can be distinguished (10, 13, 14). Interestingly, a restricted epitope of CD99, i.e., CD99R, has been defined (12) that is expressed only on T cells, NK cells, and monocytes but not on B cells, erythrocytes, or platelets.

The function of CD99 is not yet fully understood. The CD99 protein has, on the one hand, limited regions of similarity to collagen (1, 3, 12); on the other, it is strongly glycosylated, and all sugar residues appear to be O-linked (8). The fully sialylated 32-kDa membrane form of CD99 is thus related to other sialomucin-type glycoproteins, such as CD34 or CD43, which represent signal transducing cell surface molecules involved in cellular adhesion processes (15, 16, 17, 18, 19, 20, 21, 22).

Signal transduction via CD99 has to date only been demonstrated in immature thymocytes and Jurkat cells (23, 24). With thymocytes, CD99 ligation was shown to induce phosphatidylserine exposure at the cell membrane followed by apoptotic cell death of a distinct subset of CD4+CD8+ thymocytes, a process preceded by homotypic adhesion of the very same cell population (23, 24, 25). More mature, single-positive thymocytes were not affected by this treatment, nor were mature peripheral blood (PB)3 T lymphocytes (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 death.

In this paper we analyze the function of CD99 on mature PB T cells and demonstrate growth- and function-promoting stimulatory effects. Cross-linking of CD99 on resting PB T cells in the presence of a suboptimal TCR/CD3 trigger leads to their polyclonal expansion and to Th1-type growth factor production in T cell lines and T cell clones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation

PBMC were isolated from heparinized blood of healthy adult donors by standard density gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). Subsequently, T cells were separated by magnetic sorting using the MACS technique (Miltenyi Biotec, Bergisch Gladbach, Germany), as described previously (26). Purified T cells were obtained by depletion of CD11b, CD14, CD16, CD19, CD33, and HLA class II-positive cells with the respective mAbs (Table IGo). The purity of the T cell preparations was controlled by flow cytometry and was found to be >98%.


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  		Table I. MAbs used in the study1

 
Immunoblots

Immunoblots were conducted as described previously (27). Jurkat cells (1 x 108) were washed with ice-cold PBS followed by solubilization in 1 ml of lysis buffer (10 mM Tris-HCl (pH 8.3) (Merck, Darmstadt, Germany), 140 mM sodium chloride (Merck), 2 mM EDTA (Sigma, St. Louis, MO), 5 mM iodoacetamide (Sigma), 1% Nonidet P-40 (Pierce, Rockford, IL), 1 mM PMSF (Sigma), 15 µg/ml aprotinin (Sigma), and 15 µg/ml leupeptin (Sigma)) for 30 min on ice. Lysates were centrifuged twice for 10 min at 15,000 rpm in 1.5-ml tubes (Eppendorf, Hamburg, Germany) at 4°C to remove insoluble material.

Subsequently, soluble proteins were diluted one-half with sample buffer, heated for 4 min at 96°C, subjected to SDS-PAGE (20 µl/slot, 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 µg/ml) for 3 h, followed by a 1-h incubation step with horseradish peroxidase-conjugated sheep anti-mouse Ig Ab (Amersham, Aylesbury, U.K.) diluted 1/5000. Finally, a chemiluminescence detection system (Amersham) was used for the visualization of relevant proteins on Kodak X-OMAT S films (Eastman Kodak, Rochester, NY). Individual blots were exposed for 30 s to 5 min to gain optimal signal to noise ratio.

Immunofluorescence analyses

For membrane staining, 50 µl of highly purified T cells (1 x 107/ml) were incubated for 30 min at 4°C with the indicated mAbs or an irrelevant isotype-matched control mAb (VIAP) used in a concentration of 20 µg/ml. After washing cells twice with ice cold PBS/1% BSA solution, binding of the primary mAb was visualized using sheep F(ab')2 anti-mouse Ig-FITC (SAM; An der Grub, Bio Forschungs, Kaumberg, Austria) as the second-step reagent. To analyze surface expression after activation, cells were incubated with PMA (Sigma) in a final concentration of 10-7 M and ionomycin (Sigma; final concentration, 1 µM) for 60 h, followed by the staining procedure as described above. After washing the cells three times with PBS/1% BSA, the membrane fluorescence was analyzed on a FACScan flow cytometer supported by CellQuest software (Becton Dickinson, San Jose, CA).

T cell proliferation assays

Proliferation assays of highly purified PB T cells derived from healthy adult volunteers (5 x 104 cells/well) were performed in triplicate in 96-well U-bottom tissue culture plates (Costar, Cambridge, MA) in a final volume of 200 µl. Proliferation was induced by the indicated mAbs (5 µg/ml) cross-linked with GAM-IgG (10 µg/ml; Sigma) and by PMA (Sigma; final concentration, 10-7 M) or ionomycin (Sigma; final concentration, 1 µM). For proliferation experiments with immobilized CD3 mAb, 96-well flat-bottom plates (Costar) were coated overnight at 4°C with 100 µl of 0.125 to 1.0 µg/ml of purified OKT3 mAb diluted in PBS. The plates were washed twice with PBS and subsequently used for the assays. PMA (Sigma), ionomycin (Sigma), and the mAbs were diluted in RPMI 1640 (Life Technologies, Grand Island, MD) supplemented with 10% FCS, 2 mM L-glutamine, 10 U/ml penicillin, and 100 µg/ml streptomycin. GAM-IgG and the cells were resuspended in RPMI 1640 supplemented with 10% pooled human serum.

After 72 h of incubation in a humidified atmosphere with 5% CO2 at 37°C, the cells were pulsed with 1 µCi/well of [methyl-3H]TdR (Amersham). Eighteen hours later the cell lysates were harvested on glass-fiber filters (Packard, Topcount, Meriden, CT), and radioactivity was determined on a microplate scintillation counter (Packard).

Determination of cytoplasmic free calcium concentrations

Cell culture. The human T cell line Jurkat, subclone E6-1, and J.RT3-T3.5 (both obtained from the American Type Culture Collection, Manassas, VA) were grown under standard conditions in RPMI 1640 medium supplemented with 10% heat-inactivated bovine calf serum (HyClone, Logan, UT), penicillin/streptomycin (50 U/ml and 50 µg/ml; Life Technologies, Gaithersburg, MA), and 2 mM glutamine (Life Technologies) at 37°C in a humidified atmosphere in the presence of 5% CO2.

Determination of calcium levels. Jurkat E6–1 cells or J.RT3-T3.5 were labeled with the fluorescent Ca2+ indicator indo-1/AM (2 µM; Molecular Probes, Eugene, OR) by incubation at 37°C for 20 min in HBSS supplemented with 10 mM HEPES and 0.5% BSA (HHB; 0.5%). Cells (1 x 106 in a volume of 20 µl) were incubated with the indicated mAbs at a final concentration of 0.6 µg (diluted with 0.5% HHB to a final volume of 20 µl) or with 20 µl of 0.5% HHB alone for 18 min at room temperature. Subsequently, the volume was adjusted to 250 µl with 0.5% HHB medium followed by a 7-min equilibration period at 37°C in the water bath. Subsequently, measurement of [Ca2+]i by flow cytometry was started at 37°C constant temperature, and after 1 min, 20 µg of cross-linking F(ab')2 of GAM-IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was added, and the measurement was continued for another 4 min. As positive controls, 2 µg of OKT3 mAb was added for CD3 stimulation, or thapsigargin (Sigma) was used at a final concentration of 200 nM. Flow cytometric analyses were performed on a FACStar Plus flow cytometer (Becton Dickinson) with the following settings: excitation by argon laser at 50 mW multiline UV, and emission at 530 nm (Fl1; calcium-free indo) and 395 nm (Fl2, calcium-bound form of indo). The fluorescence ratio Fl2/Fl1, which is a direct estimate of the cytoplasmic calcium concentration (30), was computed in real-time by a pulse-processing unit and is expressed as arbitrary units.

IL-2 luciferase reporter gene assay

Jurkat cells (clone 41-19) transfected with an IL-2 promoter (position -583 to +40)-luciferase gene construct (31) were cultured in RPMI 1640 medium (Life Technologies) plus 10% FCS supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and geneticin-418 sulfate (0.8 mg/ml; Gibco). Ninety-six-well flat-bottom plates (Costar) were coated overnight at 4°C with 100 µl/well of a 10 µg/ml solution of GAM-IgG (Sigma) diluted in sterile PBS. Subsequently, plates were washed twice with PBS, and free binding sites were blocked by incubation with RPMI 1640 medium plus 10% FCS for 1 h at 37°C. Afterward, plates were reacted overnight at 4°C with 50 µl/well of OKT3 mAb (7.5 ng/ml diluted in PBS) followed by washing the plates once with PBS. Jurkat cells (5 x 104/well/200 µl medium) were stimulated in triplicate in these precoated plates in the presence of the indicated mAbs (10 µg/ml final concentration) at 37°C in a 5% CO2 atmosphere. After 4 h, plates were centrifuged at 200 x g for 5 min. Supernatants were removed by flicking, and the cells sticking to the plates were lysed by the addition of 25 µl/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 µl/well), fluorescence 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 (±SD), 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 ± 0.020 (mean ± SD).

Generation of T cell lines and T cell clones

For generation of PHA/IL-2-dependent blasts, PBMC (1 x 105/well) were cultured in RPMI 1640 plus 10% FCS (Life Technologies) supplemented with antibiotics in the presence of PHA (Sigma; final concentration, 1 µg/ml) in 96-well U-bottom culture plates (Costar) for 7 days. Subsequently, every 5 to 7 days 10 U/ml of IL-2 (provided by the Novartis Research Institute, Vienna, Austria) plus autologous irradiated (3000 rad, 137Cs source) PBMC as feeder cells (ratio of blasts/feeder cells = 1:1) were 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 modified by treatment with 2,4,6-trinitrobenzene sulfonic acid (Sigma) as previously described (32). Autologous PBL were stimulated with TNP-modified EBV-LCL for 7 days in complete medium plus 5% human AB serum. Upon restimulation and cultivation for 5 days in the presence of IL-2 (10 U/ml), the outgrowing T cells were cloned by limiting dilution and were propagated by weekly restimulation with TNP-modified EBV-LCL and IL-2 (10 U/ml).

T cell blasts from Bet v 1-specific T cell lines were obtained as previously described (33). T cell blasts were seeded in limiting dilution (0.3 cells/well) in 96-well U-bottom plates (Nunclone, Nunc, Roskilde, Denmark) 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 Ultra culture medium (BioWhittaker, Walkersville, MD). Growing microcultures were then expanded at weekly intervals with fresh feeder cells and rIL-2. The specificity of T cell clones was assessed as previously described (34).

Determination of intracellular cytokines

Ninety-six-well flat-bottom tissue culture plates (Costar) were coated with GAM-IgG (Sigma; 10 µg/ml) plus a suboptimal concentration of the CD3 mAb OKT3 (20 ng/ml) at 4°C overnight. After two washings with PBS, T cell lines or clones (1–2 x 105/well) were incubated in precoated plates with optimal concentrations (5 µg/ml) of CD99 mAb 3B2/TA8, CD28 mAb Leu28, or isotype control mAb recognizing the NGFR. Assays were set up in a total volume of 200 µl/well in RPMI 1640 medium containing 5% pooled human serum supplemented with antibiotics and 2 µg/ml (final concentration) of brefeldin A (Sigma). After 18 h of incubation at 37°C in a 5% CO2 atmosphere, the cells were harvested and analyzed for the presence of intracellular cytokines. For staining, 50 µl of the cell suspension (corresponding to 1–2 x 105 cells) were fixed for 30 min at room temperature by the addition of 100 µl of FIX solution (An der Grub). Subsequently, cells were washed once with 4 ml of PBS/1% BSA, resuspended in 50 µl of PBS/1% BSA, permeabilized by the addition of 100 µl of PERM solution (An der Grub), and incubated for 30 min at room temperature with the indicated directly conjugated anti-cytokine mAb. Finally, cells were washed twice, resuspended in PBS, and analyzed by flow cytometry.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PB T cells express CD99 that is up-regulated upon cellular activation

Our mAb 3B2/TA8 was classified as a CD99 mAb at the Sixth Workshop on Human Leucocyte Differentiation Antigens (35). Biochemical analyses show that the mAb 3B2/TA8 recognizes two structures with molecular masses of approximately 28 and 32 kDa in whole cell lysates of Jurkat T cells (Fig. 1Go), which is in agreement with previous reports on other CD99 mAbs (3, 8).



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  		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%) cell lysates of Jurkat cells were resolved on a 12.5% SDS-PAGE under nonreducing (A) or reducing (B) conditions, transferred onto nitrocellulose, and reacted either with 3B2/TA8 mAb, the HLA class I {alpha}-chain-specific mAb LA45, or with the irrelevant isotype-matched control mAb VIAP. First Abs bound to the membrane were detected with goat anti-mouse Abs conjugated with horseradish peroxidase. Bound second Abs were visualized by the application of a chemiluminescence detection system. The positions and molecular masses of prestained standard proteins are indicated.

 
CD99, as detected by 3B2/TA8 mAb, is expressed on the whole PB T cell population (Fig. 2Go). This is at variance with certain other CD99- or CD99R-specific mAbs, such as 12E7 or D44, which have been described to react only with distinct PB T cell subpopulations (10, 12). Following cellular activation, CD99 is up-regulated, resulting in a uniformly high expression density on the whole T cell population (Fig. 2Go). The kinetics of CD99 up-regulation on T cells following polyclonal activation with PMA plus ionomycin, compared with those of CD69 or CD25, are slow and require >72 h for completion (data not shown).



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  		FIGURE 2. Binding pattern of CD99 mAb 3B2/TA8 to resting and activated 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 fluorescence 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.

 
CD99 cross-linking induces proliferation of PB T cells in the presence of a suboptimal TCR/CD3 stimulus

Ligation of CD99 on immature T cells has previously been shown to induce apoptotic cell death (23, 24, 25). With mature PB T cells no such effect was observed (23, 24, 25).

In an attempt to characterize the signaling potential of CD99 on mature PB T cells, we incubated resting T cells with the CD99 mAb 3B2/TA8 in the presence or the absence of a CD3 trigger. The mAb 3B2/TA8 alone or cross-linked with GAM-IgG did not induce T cell proliferation and, in agreement with previous studies, did not induce apoptosis (data not shown). However, in the presence of a suboptimal TCR/CD3 signal delivered by plate-bound CD3 mAb OKT3, cross-linking of CD99 with 3B2/TA8 mAb and GAM-IgG resulted in vigorous proliferation of PB T cells (Fig. 3Go, A and B). Compared with a CD3 plus CD28 mAb (Leu28)-based stimulation, the cross-linked 3B2/TA8 mAb requires higher CD3 mAb (OKT3) concentrations to obtain equivalent [methyl-3H]TdR uptake values (Fig. 3GoA). In contrast, soluble CD3 mAb plus soluble CD99 mAb, even when cross-linked with GAM-IgG, did not lead to the proliferation of PB T cells (Fig. 3GoB). Furthermore, stimulation with the IgM-type CD3 mAb VIT3, PMA, ionomycin, or CD28 failed to provide the appropriate signal sufficient to render PB T cells responsive for CD99 signals (Fig. 3GoB).



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  		FIGURE 3. CD99 mAb 3B2/TA8 induces proliferation of PB T cells in the presence of suboptimal TCR/CD3 stimulation. A, Highly purified T cells (5 x 104) were exposed to culture plates coated with the indicated concentrations of CD3 mAb OKT3 and to the soluble CD99 mAb (3B2/TA8; black circles), the isotype-matched control mAb NGFR (open circles), or the CD28 mAb (Leu28; black squares). In all experiments mAbs were cross-linked with GAM-IgG. After 3 days of culture, [methyl-3H]TdR was added for the following 18 h, and thymidine uptake was determined as described in Materials and Methods. The figure shows [methyl-3H]TdR uptake, in counts per minute, from one representative experiment of six performed. B, Highly purified T cells (5 x 104) were incubated on either coated (CD3 mAb OKT3, or CD28 mAb Leu28) or uncoated 96-well flat-bottom tissue culture plates. As indicated in the figure, individual wells were supplemented in addition with soluble CD99 mAb 3B2/TA8 (5 µg/ml), the isotype-matched control mAb NGFR (5 µg/ml), CD28 mAb Leu28 (5 µg/ml), CD3 mAb OKT3 (1 µg/ml), CD3 mAb VIT3 (5 µg/ml), ionomycin (1 µM), or PMA (10-7 M). To all wells containing soluble mAbs, GAM-IgG (20 µg/ml) was added as a cross-linker. In proliferation assays with solid phase CD28 mAb Leu28, all mAbs tested in combination were provided in a plate-bound form. After 3 days of culture, [methyl-3H]TdR was added for the following 18 h, and thymidine uptake was determined as described in Materials and Methods. The figure shows the [methyl-3H]TdR uptake, in counts per minute (mean ± SEM), of seven experiments.

 
CD99 cross-linking on T cells results in TCR/CD3-dependent elevation of [Ca2+]i

In light of the observed mitogenic properties of the CD99 mAb 3B2/TA8 on T cells, we subsequently analyzed early T cell activation events in the human leukemic T cell line Jurkat (36, 37).

The results in Figure 4GoA show that intracellular free calcium concentrations in Jurkat T cells are significantly increased upon cross-linking of CD99 with 3B2/TA8 mAb plus GAM-IgG (p < 0.007, by Student’s paired t test). This effect was strictly dependent on GAM-IgG-enhanced cross-linking of the CD99 molecules with mAb 3B2/TA8, since no calcium mobilization was detected upon incubation with 3B2/TA8 mAb alone (data not shown). Treatment of Jurkat T cells with CD3 mAbs plus GAM-IgG consistently led to a very strong increase in cytoplasmic free calcium levels, whereas the binding or nonbinding control mAbs did not influence [Ca2+]i (Fig. 4GoA).



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  		FIGURE 4. Cross-linking of CD99 elevates intracellular free Ca2+ levels in Jurkat cells, which is dependent on TCR/CD3 expression. Determination of intracellular free Ca2+ 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 subclone 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. Cytoplasmic free Ca2+ concentrations were determined by flow cytometry upon cross-linking of mAbs 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 fluorescence intensity values obtained upon staining with specific mAbs are indicated. In the histograms, the ordinate represents the cell number, and the abscissa represents the individual log green fluorescence intensity of a given cell.

 
Given the fact that CD99-induced proliferation requires TCR/CD3 engagement, we analyzed whether the Ca2+ response to CD99 is linked to TCR/CD3-mediated signaling events. We examined TCR/CD3-modulated parental Jurkat cells and the TCR/CD3-negative Jurkat subclone J.RT3-T3.5. Importantly, in both cell lines CD99 mAb 3B2/TA8 failed to induce significant changes in [Ca2+]i (Fig. 4Go, B and C), although the cell surface expression levels of CD99 are comparable to those on the parental cell line (Fig. 4GoA). The abortive CD3 mAb-induced rise in [Ca2+]i observed in TCR/CD3-modulated Jurkat cells might be due to the residual expression of TCR/CD3, as shown by FACS analysis (Fig. 4GoB).

CD99 cross-linking in the presence of a suboptimal TCR/CD3 trigger induces IL-2 promoter activity

Cytokine production by T cells plays an important role in the regulation of the immune response (reviewed in 38 . Therefore, we wondered whether in addition to its proliferation-inducing effects on T cells, CD99 engagement is able to induce cytokine genes.

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 concentrations of the mAb 3B2/TA8 or the indicated control mAbs. As shown in Figure 5Go, CD99 mAb 3B2/TA8 cross-linking on Jurkat T cells leads to an approximately 6-fold increase in IL-2 promoter activity (p < 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 >10-fold induction of IL-2 promoter activity (Fig. 5Go).



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  		FIGURE 5. CD99 ligation elevates IL-2 promoter activity. Human Jurkat T cells transfected with an IL-2 promoter-luciferase gene construct were exposed to suboptimal concentrations of plate-bound CD3 mAb OKT3 in the presence of CD99 mAb 3B2/TA8. As negative controls, we used both a nonbinding (VIAP) and a binding (132/3C3; CD147) isotype-matched control mAb. The CD28 mAb (Leu28) served as a positive control. 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 figure shows the mean (±SD) relative increase in IL-2 promoter activity of nine independently performed experiments. Values obtained upon incubation with CD99 mAb 3B2/TA8 were compared with those obtained upon incubation with VIAP mAb by paired Student’s t test and were significant (p < 0.006).

 
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-{alpha} and IFN-{gamma} production in polyclonally activated T cell lines, Th1 and Th0 clones

Th1 immune responses, even when established, appear to be sensitive 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 CD28 costimulation, we performed intracellular staining experiments for ILs in PB-derived T cell lines and clones.

In polyclonally activated T blasts, cross-linkage with the CD99 mAb 3B2/TA8 in the presence of suboptimal concentrations of the CD3 mAb OKT3 led to the production of TNF-{alpha} in a high proportion of cells (Fig. 6Go). TNF-{alpha}-producing cells ranged from 12.5 to 52.5% (mean ± SEM, 28.8 ± 7.3%) and were significantly increased compared with cells treated with CD3 mAb plus the isotype control mAb NGFR (7.8 ± 0.9%). Furthermore, Th1 and Th0 cell clones responded to CD99 costimulation with the production of IFN-{gamma} (Table IIGo). More importantly, CD99 mAbs failed to induce significant levels of IL-4 in both Th0 and Th2 clones and were incapable of inducing IFN-{gamma} production in Th2-restricted clones (Table IIGo).



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  		FIGURE 6. CD99 mAb 3B2/TA8 induces TNF-{alpha} production in PHA/IL-2-dependent T cell lines suboptimally stimulated with CD3 mAb OKT3. PHA/IL-2-dependent cell lines were generated as described in Materials and Methods. Five to seven days after the last stimulation cells (1–2 x 105/well) were incubated on uncoated (mock) or mAb-coated (CD3 mAb OKT3 plus NGFR, CD99 mAb 3B2/TA8, or CD28 mAb Leu28) 96-well flat-bottom culture plates. They were incubated overnight in RPMI 1640 medium supplemented with 5% HS plus 2 µg/ml brefeldin A. The next day, individual cell samples were harvested, fixed, permeabilized, and subsequently stained with an FITC-conjugated TNF-{alpha}-specific mAb and a phycoerythrin-conjugated control mAb followed by flow cytometric analysis. Results are depicted as two parameter dot plots showing red fluorescence intensity on the ordinate and green fluorescence intensity on the abscissa. As a negative control for green fluorescence the FITC-conjugated isotype-matched nonbinding control mAb VIAP was used. Markers were set according to the fluorescence characteristics of cells that were incubated with the negative control mAbs. Numbers indicate the percentage of single positive (green) cells. This figure shows a representative experiment of 14 performed.

 

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  		Table II. Cytokine production by T cell lines and T cell clones1

 
This clearly distinguishes CD99 from CD28 costimulation, inasmuch as CD28-derived signals drive both Th1-restricted cytokine responses by Th1 clones and Th2-restricted cytokine production by Th2 clones (Table IIGo). In general, intracellular IL production determined at the single cell level correlated well with total cytokine production by these cells as measured by sandwich ELISA assays (data not shown). Significant induction of cytoplasmatically stored IL-2 could only be detected by a combination of CD3 mAb plus CD28 mAb (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ligation of CD99/E2 surface molecules has been recently shown to induce apoptotic cell death of immature double-positive thymocytes but not to affect other thymocytes or mature T lymphocytes (24). Here we demonstrate that cross-linking of CD99 is capable of providing a potent costimulus for mature T cells leading to cell proliferation and Th1-type cytokine production in combination with a suboptimal TCR/CD3 signal. Thus, our data support the view that CD99 molecules on mature PB T cells can transmit a positive activation signal. That the signaling properties of one given surface molecule may depend on the maturational stage of the respective T cell is highly reminiscent of CD2 and CD3 molecules. Engagement of these molecules on distinct populations of thymocytes mediates apoptosis (41, 42, 43), whereas engagement of the same receptors on PB T cells provides a strongly activating signal (44, 45).

In our experiments cross-linking of CD99 with mAb 3B2/TA8 and GAM-IgG dramatically reduced the threshold of anti-CD3-mediated proliferation of freshly isolated, resting PB T cells (Fig. 3GoA). In general, CD99-induced [methyl-3H]TdR incorporation reached its maximum between day 3 and day 4 of costimulation, which was similar to the CD28-based costimulatory kinetics observed in parallel experiments (data not shown). Importantly, the induction of T cell proliferation in combination with a suboptimal TCR/CD3 stimulus is not a salient feature of our CD99 mAb 3B2/TA8, since similar activity was also observed with the classical CD99/MIC2 mAb 12E7 (data not shown). Consequently, the strict requirement for pan CD99 mAbs, as reported for thymocyte aggregation and apoptosis (23, 24, 25), does not seem to apply for the induction of T cell mitogenesis.

The finding that CD99 does not cosignal with PMA or CD28 clearly distinguishes CD99 from other costimulatory molecules such as CD2 (46, 47, 48), CD5 (49, 50, 51), and CD47 (52), which are able to activate T cells in the absence of a TCR/CD3 stimulus. It seems that CD99 obviously does not induce a signaling cascade that can substitute a TCR/CD3 stimulus, suggesting a role for CD99 only in Ag-dependent stimulation of PB T cells. Furthermore, we show that only a solid phase TCR/CD3 trigger leads to CD99-based costimulation. Neither CD3 mAb OKT3 provided in soluble form, an IgM-type CD3 mAb (VIT3) that is known to elicit strong Ca2+ fluxes in T cells (53), nor calcium ionophore was able to substitute for solid phase TCR/CD3 ligation.

How would solid phase vs solution phase cross-linking of the TCR/CD3 complex influence the costimulatory outcome via an unrelated cell surface molecule such as CD99? One possibility might be that heterologous cross-linking of TCR/CD3 and CD99 in solution phase could lead to the disruption of a putative critical association between these two receptors. This hypothesis, however, would suggest that the CD99 molecule might functionally and/or sterically be coupled with the TCR/CD3 complex in T cells. That CD99 is indeed noncovalently associated in a membrane microdomain with the TCR/CD3 complex of T lymphocytes has been shown recently by Cerny and co-workers (54).

Further indications for a dependence between the TCR/CD3 complex and CD99 are provided by our calcium flux experiments. Multimerization of CD99 molecules leads to a clear-cut elevation of cytosolic calcium levels in parental Jurkat cells that express the TCR/CD3 complex. However, when Jurkat T cells, after down-modulation of TCR/CD3 molecules by overnight incubation with a CD3-specific mAb, are treated with CD99 mAb 3B2/TA8 plus GAM-IgG, no increment in intracellular calcium levels was observed. Note that in these cells also the CD3-mediated calcium flux was strongly compromised. These experiments allow us to suggest that reduced levels of cell surface-expressed TCR/CD3 impair CD99-driven signal transduction in T cells and underline the importance of the TCR/CD3 trigger for CD99 costimulation.

To further explore that issue and to rule out modulation-induced negative or regulatory signals as the overall basis for the CD99 nonresponsiveness, a variant of the Jurkat line (clone J.RT3-T3.5) lacking TCR/CD3 surface expression was analyzed and was found to be unable to increase [Ca2+]i upon CD99 cross-linking. These data suggest that the presence of a sufficient number of TCR/CD3 complexes on the cell surface is required for CD99-mediated signal transduction, a finding strongly reminiscent of the results of earlier studies performed on T cells stimulated via CD2, CD5, or CD47 (52, 55, 56, 57, 58). Whether CD99 in vivo initiates a signaling cascade complementary to TCR/CD3 ligation and potentiates TCR/CD3-induced T cell activation or whether the interaction of CD99 with its putative ligand merely enhances the T cell-APC interaction is still a matter of debate. Our data showing that cross-linking of CD99 alone leads to a clear-cut Ca2+ flux in T cells demonstrate that CD99 per se is a signal-transducing molecule. Thus, according to the experimental settings used in our report, CD99 can be regarded as acting primarily in a costimulatory fashion during T cell activation. Nevertheless, CD99 was originally described as a molecule involved in T cell rosette formation with erythrocytes, suggesting a role for CD99 as an adhesion molecule (9). It will be the subject of future studies to show how the functional interrelationship between CD99 and the TCR/CD3 complex is organized and which function, i.e., costimulation or adhesion, dominates its T cell stimulatory capacity.

CD99 cross-linking not only leads to the polyclonal expansion of PB T cells but also induces cytokine production. Experiments performed with PHA/IL-2 dependent polyclonal T cell lines revealed that CD99 ligation in the presence of a suboptimal TCR/CD3 signal induces the production of TNF-{alpha} and IFN-{gamma}. Similar to the T cell blasts, activation of Th0 and Th1 clones with CD99 plus suboptimal doses of CD3 mAbs induced TNF-{alpha} and IFN-{gamma} production in a considerable number of cells. In contrast, CD99 mAbs either alone or in combination with CD3 mAbs failed to induce IL-4 production in allergen- or TNP-specific Th2 clones. Activation of these clones with CD28 plus CD3 mAbs or with PMA plus ionophore (data not shown), however, led to a clear-cut induction of the Th2-specific cytokine IL-4. Significant IL-2 production in either of the cell lines tested was detected only upon CD28 costimulation or in PMA- plus ionomycin-triggered cells. These results, which are in contrast to those of the IL-2 promoter studies, might be explained by cell type-specific slower production or higher consumption rates of IL-2 compared with IFN-{gamma} or TNF-{alpha}. The capability for CD99 costimulation and induction of IFN-{gamma} production in Th1/Th0 clones is reminiscent of the signaling lymphocytic activation molecule described recently (59). At variance to signaling lymphocytic activation molecule, CD99 costimulation is insufficient, however, to induce a shift in cytokine production of Th2 clones.

Taken together our data indicate that while CD99 costimulation is able to drive Th1 and Th0 clones toward the production of the Th1-specific cytokine IFN-{gamma}, it is incapable of driving Th2 or Th0 clones to synthesize detectable amounts of IL-4, otherwise induced by CD28 costimulation. Of importance, allergic diseases have been shown to be associated with down-regulation of IFN-{gamma}-producing cells and the expansion of Th2-type cytokine-producing cells, leading to enhanced IgE synthesis (60, 61). Conversely, IFN-{gamma} has been shown to be one of the dominant factors inhibiting IL-driven IgE production (62, 63). Thus, our data allow us to suggest that therapeutically induced activation pathways, such as CD99-driven costimulation, that effectively augment levels of T cell-produced IFN-{gamma} while having no stimulatory effect on Th2-type cells could provide an efficient way to intervene with allergic diseases (64).


   Acknowledgments
 
We thank Ms. Lisbeth Gschwantler and Mr. Klaus Wenhardt for expert FACS analyses, and Ms. Saro Künig for expert technical assistance. We are grateful to Paul Breit for photographic artwork.


   Footnotes
 
1 This work was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich. Back

2 Address correspondence and reprint requests to Dr. Winfried F. Pickl, Institute of Immunology, University of Vienna, Borschkegasse 8A, A-1090 Vienna, Austria. E-mail address: Back

3 Abbreviations used in this paper: PB, peripheral blood; GAM-IgG, goat anti-mouse immunoglobulin G; [Ca2+]i, intracellular Ca2+ concentration; TNP, trinitrophenyl; NGFR, nerve growth factor receptor. Back

Received for publication March 18, 1998. Accepted for publication June 29, 1998.


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 Results
 Discussion
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