Costimulatory Element CD63 As an Activation-Linked T Cell

CD63 as an Activation-Linked T Cell Costimulatory Element Katharina Pfistershammer, Otto Majdic, Johannes Stöckl, Gerhard Zlabinger, Stefanie Kirchberger, Peter Steinberger This information is current as and Walter Knapp of October 2, 2021. J Immunol 2004; 173:6000-6008; ; doi: 10.4049/jimmunol.173.10.6000 http://www.jimmunol.org/content/173/10/6000 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD63 as an Activation-Linked T Cell Costimulatory Element1

Katharina Pﬁstershammer, Otto Majdic, Johannes Sto¨ckl, Gerhard Zlabinger, Stefanie Kirchberger, Peter Steinberger, and Walter Knapp2

Dendritic cells (DC) are unique in their capacity to either stimulate or regulate T cells, and receptor/ligand pairs on DC and T cells are critically involved in this process. In this study we present such a molecule, which was discovered by us when analyzing the functional effects of an anti-DC mAb. This mAb, 11C9, reacted strongly with DC, but only minimally with lymphocytes. In MLR it constantly reduced DC-induced T cell activation. Therefore, we assumed that mAb 11C9 primarily exerts its functions by binding to a DC-structure. This does not seem to be the case, however. Preincubation of DC with mAb 11C9 before adding T cells had no inhibitory effect on T cell responses. Retroviral expression cloning identiﬁed the 11C9 Ag as CD63. This lysosomal- associated membrane protein (LAMP-3), is only minimally expressed on resting T cells but can, as we show, quickly shift to the surface upon stimulation. Cross-linkage of that structure together with TCR-triggering induces strong T cell activation. CD63 on

T cells thus represents an alternative target for mAb 11C9 with its binding to activated T cells rather than DC being responsible Downloaded from for the observed functional effects. This efficient CD63-mediated costimulation of T cells is characterized by pronounced induction of proliferation, strong IL-2 production and compared with CD28 enhanced T cell responsiveness to restimulation. Particularly in this latter quality CD63 clearly surpasses several other CD28-independent costimulatory pathways previously described. CD63 thus represents an activation-induced reinforcing element, whose triggering promotes sustained and efficient T cell activation and expansion. The Journal of Immunology, 2004, 173: 6000–6008. http://www.jimmunol.org/ t is generally accepted that efficient activation of resting T family, have been reported to be involved in T cell activation pro- cells requires a secondary signal in addition to signal 1 that cesses (13, 14). I is normally provided via specific engagement of the TCR CD63 is also a member of this family and was up to now mainly complex with a defined peptide-MHC complex (1–3). This signal described as a marker molecule for lysosomal-associated mem- 2 is generally referred to as costimulation and significantly lowers brane protein (LAMP)3 compartments (also termed LAMP-3)(15). the number of TCR complexes needed to be triggered for the in- It was studied for its role in cellular spreading (16) and in the duction of activation and the acceleration of T cell responses (4). pathogenesis of melanoma (17, 18). Furthermore, CD63 is known Characteristic features of an effective costimulation process are as a marker of activation of several cell types including granulo- induction of high levels of IL-2, prevention of anergy, and en- cytes and platelets (19–22). Of particular importance clinically has by guest on October 2, 2021 hanced cell survival (5). become the analysis of CD63 expression on human basophils, The primary costimulatory pathway in T cells is the ligation of which significantly increases upon their activation (23). CD28 on T cells with B7.1/2 on APC (6). Still, mice deficient in In contrast, there are only a few studies concerning the expres- CD28 are able to raise immune responses, indicating that mole- sion of CD63 on T cells and its regulation and function on these cules distinct from CD28 are also able to exert similar effects (7). cells (24–28). We show that CD63 is induced on T cells upon Costimulatory function has been attributed to a number of mole- activation and demonstrate that activation-induced T cell surface cules. Among them, molecules that prolong and favor T cell-APC CD63 has a number of interesting functional effects. Although contact are well established to act in a costimulatory fashion. Two blocking CD63 on T cells with soluble CD63 mAb 11C9 impairs main structures on T cells involved in maintenance of this cellular dendritic cell (DC)-induced T cell proliferation, cross-linking of interaction are CD2 and LFA-1 (8, 9). Activation of LFA-1, a key CD63 via mAb 11C9 delivers a potent costimulatory signal to T structure of the immunological synapse, is one of the first conse- cells. This CD63-based costimulatory signal differs from other quences of a T cell signal (10, 11). Other well-established costimu- CD28 independent costimulatory pathways in its high efficacy latory molecules like OX40, CD27, and 4-1BB belong to the and furthermore increases the responsiveness of T cells to TNFR family (6, 8). A number of non-CD28 costimulators, like restimulation. CD5, CD44, and CD9 have been reported to exert their effects by favoring the association of TCR complexes with lipid rafts (12). In Materials and Methods addition, CD81 and CD82, like CD9 members of the tetraspan Media and reagents Cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies, Paisley, Scotland) supplemented with 2 mM L-glutamine, 100 U/ml peni- Institute of Immunology, Medical University of Vienna, Vienna, Austria cillin, 100 ␮g/ml streptomycin, and 10% FCS (Sigma-Aldrich, Vienna, Austria). Recombinant human GM-CSF and IL-4 were kindly provided by Received for publication March 22, 2004. Accepted for publication August 31, 2004. the Novartis Research Institute (Vienna, Austria). PHA, PMA, ionomycin, The costs of publication of this article were defrayed in part by the payment of page LPS (from Escherichia coli serotype O127-B8), and propidium iodide (PI) charges. This article must therefore be hereby marked advertisement in accordance were obtained from Sigma-Aldrich Chemie (Deisenhofen, Germany). with 18 U.S.C. Section 1734 solely to indicate this fact. CD3/CD28 mAbs coated beads (Dynabeads CD3/CD28 T cell expander) 1 This work was supported by a grant from the Austrian Science Fund (SFB005.2). 2 Address correspondence and reprint requests to Dr. Walter Knapp, Institute of Im- munology, Medical University of Vienna, Borschkegasse 8A, A-1090 Vienna, Aus- 3 Abbreviations used in this paper: LAMP, lysosome-associated membrane protein; tria. E-mail address: [email protected] DC, dendritic cell; mdDC, monocyte-derived DC; PI, propidium iodide.

were obtained from Dynal Biotech (Oslo, Norway). CFSE for proliferation enna, Austria). For the last 18 h [methyl-3H]TdR (ICN Pharmaceuticals, analysis was purchased from Molecular Probes (Eugene, OR). Irvine, CA) was added. Cells were harvested and incorporated [methyl- 3H]TdR was measured on a microplate scintillation counter (Packard; Top- Antibodies count Instrument, Meriden, CT). All T cell proliferation assays were per- Monoclonal Abs 11C9 was obtained by immunizing BALB/c mice with formed in triplicate. ϫ 5 human monocyte-derived DC (mdDC) and selecting for mAbs, which bind For the primary MLR, allogenic, purified T cells (1 10 /well) were to DC and interfere with DC-T cell interactions. cocultured with graded numbers of DC for 5 days in presence of mAbs at ␮ Fab of mAb 11C9 were prepared using the Avidchrom Fab kit (Unisyn a final concentration of 10 g/ml. ϫ 5 Technologies, San Diego, CA) following the manufacturers recommenda- For stimulation with Ab-coated beads T cells (1 10 /well) were cul- tions. Preparations were purified by FPLC superdex gel filtration (Phar- tivated with graded numbers of Dynabeads T cell expander for 4 days in ␮ macia, Uppsala, Sweden) and removal of intact IgG with protein A super- presence of mAbs at a final concentration of 10 g/ml. dex. The quality of the Fab preparation was checked in binding assays with For T cell stimulation with the Bw cell lines, BwCD64/CD63, BwCD64/ fluorochrome-labeled Fc- and Fab-specific Abs. CD80, and the control BwCD64 cells were irradiated (6000 rad) and in- Other murine mAbs that were generated in our laboratory are VIAP (calf cubated with purified anti-CD3 mAb (final concentration 10 ng/ml) or with intestine alkaline phosphatase-specific) used as nonbinding control Ab, mAbs to CD3 and CD28 (final concentration, 10 ng/ml each) and added to ϫ 4 VIT6b (CD1a), VIM12 (CD11b), VIM13 (CD14), CD33-4D3, 1/47 (MHC flat-bottom 96-well plates (2.5 10 cells per well). Purified human T ϫ 4 class II), AAA1 (CD147), VIT4 (CD4), VIT8 (CD8), CD25-3G10, 1-456 cells (5 10 ) were added to each well and cocultivated for 3 days. (CD58), 5-272 (B7-H1), M80 (CD141), CD5-5D7, VIT12 (CD6), CD7- For T cell stimulation with plate-bound Abs, 96-well flat-bottom plates ␮ 6B7, VIT14 (CD27), and 7-480 (CD80). The CD14 mAb MEM18 and the were incubated over night at 4°C with either CD3 (1 g/ml) alone or in ␮ CD3 mAb UCHT-1 were kindly provided by An der Grub (Bio Fors- combination with CD28, CD63, or CD147 Abs (2 g/ml). Plates were chungs, Kaumberg, Austria) and the CD19 mAb (BU12) provided by An- washed and blocked with medium containing FCS for 4 h at room tem- ϫ 4 cell (Bayport, MN). The mAbs CD16-3G8, CD3 (S4.1) Tricolor, CD83 perature. Purified human T cells (5 10 ) were added and T cell proliferation was measured after 96 h of culture. For cytokine measurement

(HB15) and CD86 (BU63) were purchased from Caltag Laboratories (Bur- Downloaded from lingame, CA). mAb speciﬁc for CD64 (32.2) was from American Type culture supernatants were harvested after 72 h and cytokines were mea- Culture Collection (Manassas, VA) and mAbs to CD28 (Leu28), CD69 sured as described (31). ϫ 5 (FN50), and CD63 (H6C5) were from BD Biosciences (Palo Alto, CA). In restimulation experiments, 1 10 puriﬁed T cells per well were For stimulation of T cells the CD3 mAb OKT3 (Ortho Pharmaceutical, cultured for 7 days in presence of the following combinations of plate- Raritan, NJ) was used. bound mAbs: CD3/CD147, CD3/CD28, or CD3/CD63. After 7 days of primary culture, cultured T cells were harvested and restimulated using Fluorescence staining either plate-bound mAbs (CD3/CD28 or CD3/CD63), or different types of

allogenic APC (immature DC, mature DC, or monocytes, ratio 1:5) for http://www.jimmunol.org/ ϫ 5 For flow cytometric analysis, cells (5 10 /ml) were incubated with flu- 2 days. orochrome-conjugated mAbs or unlabeled primary Ab (10 ␮g/ml) for 20 min on ice and washed. For indirect staining, Oregon Green conjugated Measurement of number of cell cycles by CFSE staining anti-mouse Ig (Molecular Probes) was used as a secondary reagent. Stain- ing of Fc receptor-bearing cells was done in the presence of human IgG CFSE labeling was performed as previously described (32). Briefly, T cells ϫ 5 ␮ Abs (20 mg/ml, Beriglobin; Aventis Behring, Vienna, Austria). For double (5 10 /ml) resuspended in PBS were incubated with 5 M CFSE for 4 or triple staining, cells were incubated with Tricolor-conjugated CD3 Ab, min at room temperature. The staining process was stopped by addition of PE-conjugated 11C9 Ab, and FITC-labeled Abs directed against CD25, medium. Cells were washed, resuspended in medium, and used for prolif- MHC class II, CD69, CD28, CD27, CD5, CD6, CD7. Analysis was per- eration assays as described. At the indicated time point cells were harvested and washed in PBS and analyzed by flow cytometry. Dead cells were formed by gating on CD3-positive cells. For staining of intracellular CD63 ϩ ϩ

we used the reagent combination Fix&Perm from An der Grub and fol- excluded by using PI. To assess proliferation of the CD4 and the CD8 by guest on October 2, 2021 lowed the manufacturer’s protocol. Flow cytometric analysis was per- subset, cells were counterstained with Abs to these molecules. formed using a FACSCalibur flow cytometer (BD Immunocytometry Sys- Assessment of apoptosis tem, Palo Alto, CA) supported by CellQuest software (BD Biosciences). For the exclusion of dead cells, PI was used. T cells were incubated with immobilized mAb in 96-well at 37°C for 3 or 7 days. Apoptosis was assessed by staining with FITC-labeled annexin V Cell preparations (Caltag Laboratories) and PI and flow cytometric analysis. Annexin V- Mononuclear cells from peripheral blood were isolated from heparinized positive and PI-negative cells were scored as apoptotic cells. whole blood of healthy donors by standard density gradient centrifugation RT-PCR analysis of Bcl-2 and Bcl-x expression with Ficoll-Paque (Pharmacia). L Monocytes and T cells were separated from PBMC by magnetic sorting T cells were stimulated with immobilized mAb for 72 h and used for RNA as previously described (29). Monocytes were enriched by using biotinyl- preparation. RNA (1 ␮g) was used for cDNAs synthesis using oligo(dT) prim- ated CD14 mAbs VIM13 and MEM18 (purity Ͼ95%). Purified T cells ing. cDNAs were amplified with primers specific for Bcl-2 (5Ј-CTCTT were obtained through negative depletion of CD11b, CD14, CD16, CD19, CAGGGACGGGGTGAA-3Ј and 5Ј-TGGATCCAGGTGTGCAGGTG-3Ј) Ј Ј Ј CD33, and MHC class II-positive cells with the respective mAbs (purity and Bcl-xL (5 -CTGAGGCCCCAGAAGGGACT-3 and 5 -GTCCCTG Ͼ95%). GGGTGATGTGGAG-3Ј), and control primers specific for G3DPH ϩ DCs were generated by culturing CD14 monocytes in the presence of (5Ј-TCAAAGGCATCCTGGGCTACA-3Ј) and 5Ј-GAGGGGAGATCCA GM-CSF (50 ng/ml) and IL-4 (100 U/ml) for 7 days (29). For the gener- GTGTGGTG-3Ј). ation of mature DC, LPS (1 ␮g/ml) was added to DC cultures on day 6 of cultivation. Retroviral expression cloning Activated T cells were generated by culturing PBMC (1 ϫ 106/ml) up cDNA cloning was performed as described by us before (30). Briefly, a to 3 days in the presence of soluble PHA (5 ␮g/ml), plate-bound CD3/ retroviral cDNA library derived from immature and mature mdDC was CD28 mAbs (1 ␮g/ml and 2 ␮g/ml) or allogenic DC (ratio 1:10). expressed in Bw cells. Cells expressing the 11C9 Ag were repeatedly en- Generation of the BwCD64, BwCD64/CD80, and the BwCD64/ riched by MACS. Single cell cultures were obtained by limiting dilution CD63 cell line culture and genomic DNA was prepared from cell clones expressing the 11C9 Ag. The cDNA inserts were PCR amplified from genomic DNA with The generation of the murine thymoma cells Bw5147 (referred to as Bw primers specific for the flanking retroviral sequences using the Expand cells throughout this work) expressing CD64 or CD64/CD80 is described PCR system (Roche Applied Sciences, Mannheim, Germany). The ob- elsewhere (30). To generate the BwCD64/CD63 cell line BwCD64 cells tained PCR products were gel-purified and cloned using Topocloning (In- were retrovirally transduced with the human CD63 cDNA. From the cell vitrogen Life Technologies). Selected plasmids were transfected into 293 T pool, CD63ϩ cells were selected by MACS to obtain the BwCD64/CD63 cells using LipofectAmine to confirm that mAb 11C9 reacts with the pro- cell line. tein encoded by the isolated cDNA. T cell proliferation assays Statistics For T cell proliferation assays, highly purified T cells were cultured with Mann-Whitney U test was used to assess significances. Differences were the respective stimuli in 96-well cell culture plates (Corning; Costar, Vi- considered significant at p Ͻ 0.05. 6002 COSTIMULATION OF HUMAN T CELLS VIA CD63

Results Monoclonal Ab 11C9 inhibits DC-induced T cell proliferation The aim of this study was to identify immunoregulatory cell surface structures involved in DC-T cell interaction. For this purpose mAbs were tested for their capacity to modulate the stimulatory function of DC. mAb 11C9 fulﬁlled these criteria. Its addition to MLR at culture onset consistently reduced the proliferative response of T cells to allogenic DC (Fig. 1). The mean inhibitory effect obtained by addition of mAb 11C9 was 43 Ϯ 15% ( p Ͻ FIGURE 2. Identiﬁcation of the 11C9 Ag by molecular expression clon- 0.05; n ϭ 4). mAb 11C9 exerted its effect independently of the ing. A, PCR recovery of the retroviral cDNA insert from the genomic DNA type of APC used because it was also observed with mature DC, of the 11C9-reactive cell clone. B, 293 T cells transfected with plasmid which are potent T cell activators, and with monocytes, which only DNA containing the 0.9 Kb PCR product (gray histogram) but not control- transfected cells (open histogram) react with mAb 11C9. induce weak T cell stimulation (data not shown).

Monoclonal Ab 11C9 recognizes the CD63 Ag Cellular reaction pattern and time kinetics of expression of The observed functional capacity of mAb 11C9 prompted us to CD63 identify the Ag recognized by this mAb. For this purpose we CD63 has previously been described as a marker of the myeloid screened a retroviral expression library from mdDC (30) with our cell lineage and is routinely used as an activation marker of baso- Downloaded from mAb. Transduced cells expressing the 11C9 Ag were isolated by phil granulocytes (23). Lymphocytes have been reported to be MACS and used for preparation of genomic DNA. A PCR product CD63 negative (24). Using our CD63 mAb 11C9, we found the (0.9 Kb) obtained from a single cell clone (Fig. 2A) was introduced expected expression proﬁle with intracellular and extracellular ex- into an eukaryotic expression vector, and 293 T cells transfected pression of monocytes and DC, intracellular expression in resting with this construct were selectively reacting with mAb 11C9 (Fig. granulocytes, and surface expression of CD63 on granulocytes 2B). The nucleotide sequence of our 11C9 cDNA clone was found only upon activation. Regarding T lymphocytes, we identiﬁed a http://www.jimmunol.org/ ϩ to be identical with the sequence of CD63 (GenBank accession small subset (3–5%) of peripheral blood CD3 T cells that dis- number GI33876593). CD63 is a member of the tetraspan family, plays CD63 on the cell surface. Strong intracellular reactivity with ϩ also known as LAMP-3, LIMP, lysosome-integrated membrane our mAb was found in the majority of the CD3 T cells (Fig. 3A). protein, or melanoma-associated Ag. However, upon activation with PHA, mAbs against CD3, CD3 and CD28, or allogenic APC most T cells were induced to express large amounts of cell surface CD63 (Fig. 3, A and B). Surface CD63 expression was induced to a similar extend on CD4ϩ and CD8ϩ T cells (Fig. 3C). The kinetics of this activation-induced by guest on October 2, 2021 surface expression of CD63 are fast. Expression can be detected after 5 h, similar to the established and widely used early activation marker CD69, although the expression level of CD63 is lower. Activation-induced CD63 expression increases up to 72 h whereas CD69 expression already declines by this time point (Fig. 3D). Interestingly when further analyzing the population of CD63ϩ peripheral T cells in triple staining experiments, we found that these cells were distinct from CD69ϩ and CD25ϩ or MHC class IIϩ T cells (Fig. 3E). These cells thus characterize a population that differs from activated T cells.

CD63 surface expression on APC has no influence on their T cell stimulatory capacity Because our results showed that CD63 is not only found on DC but also strongly expressed on activated T cells, the inhibitory effect of mAb 11C9 could either result from binding to T cells or DC. To clarify this, we first performed a number of experiments to test whether interaction of mAb 11C9 with CD63 on DC is responsible for the reduced proliferative response of T cells in an allo-MLR. First we tested whether interaction of mAb 11C9 with CD63 has an effect on the in vitro differentiation or maturation of mdDC. Presence of our mAb did not interfere with these processes as we could not see any alteration in the expression profile of markers for DC differentiation and maturation (Fig. 4, A and B). In addition a similar inhibitory effect of DC-induced T cell ac- FIGURE 1. Soluble CD63 mAb 11C9 inhibits T cell proliferation in allogenic MLR. Purified T cells were stimulated with graded numbers of tivation was seen when adding Fab of 11C9 instead of whole Ab allogenic DC in the presence or absence of the mAb 11C9 to CD63 and to an allo-MLR further excluding the possibility that a signal de- control Abs to CD58 and B7-H1. Proliferation of T cells was measured on livered by CD63 mAb to DC is responsible for the observed effects day 4 of culture. The experiment shown is representative for four inde- (data not shown). In additional experiments we pretreated DC with pendent experiments. CD63 mAb 11C9 or a control mAb (M80), removed nonbound Ab The Journal of Immunology 6003 Downloaded from http://www.jimmunol.org/

FIGURE 3. Expression of CD63. A, The surface and total (surface and intracellular) expression profile of CD63 was analyzed by flow cytometric by guest on October 2, 2021 analysis of different cell preparations (filled histograms). A control mAb (VIAP) was used in all experiments (open histograms). B, CD63 surface expression on T cells is inducible via TCR triggering. T cells were activated via plate-bound mAb to CD3 and CD28 or by cocultivation with allogenic APC. Surface reactivity with mAb11C9 of activated (filled histograms) and resting T cells (bold line) and control staining with VIAP (dotted line) are shown. C, CD63 surface expression was analyzed on the CD4ϩ and the CD8ϩ subset of T cells activated with CD3/CD28 immobilized mAb. D, Kinetic of the CD69 and CD63 surface expression on T cells. MNC were stimulated with PHA and CD69 or CD63 surface expression of T cells (CD3ϩ) at different time points are shown. E, Fresh PBMNC were analyzed. T cells (CD3ϩ) were measured for coexpression of CD63 with early and late activation markers CD69, CD25, and MHC class II. by washing and added the DC to allogenic T cells. Under these mAb 11C9 are not due to the delivery of a negative signal to DC. conditions, mAb 11C9 had no inhibitory effect on T cell stimula- Therefore it is likely that mAb 11C9 exerts its inhibitory effect by tion (Fig. 4C). interacting with CD63 on T cells. Because Fab of 11C9 has a To study the effects of CD63 surface expression directly we similar effect as whole Ab (data not shown) transduction of a neg- used artificial APC. We used stable transfectants of the murine ative signal to T cells by our Ab is improbable. Interestingly, pres- thymoma cell line Bw expressing the human high affinity Fc re- ence of CD63 mAb 11C9 did not affect T cell activation mediated ceptor CD64 (BwCD64). These cells were further transduced to by CD3/CD28 mAb coated beads (data not shown). Thus it is coexpress either CD63 (BwCD64/CD63) or as a control human likely that mAb 11C9 blocks the interaction of a costimulatory DC CD80 (BwCD64/CD80; Fig. 5A). All transductants can be effi- structure with CD63 on T cells. ciently loaded with mAbs via the high affinity Fc␥R CD64 (30). BwCD64/CD63 cells, like BwCD64 cells loaded with CD3 mAb Cross-linking of CD63 provides a costimulatory signal to T cells alone, cannot induce highly purified T cells to proliferate, indicat- To investigate whether a costimulatory signal can be delivered to ing that CD63 on APC is not able to costimulate the activation of T cells via CD63, the effects of plate bound mAb 11C9 alone or in the TCR complex (Fig. 5B). In contrast, BwCD64/CD80 induced combination with other mAbs were analyzed. Purified T cells were strong T cell proliferation when loaded with CD3 mAb alone. In cultivated with plate-bound CD3 mAb alone or in combination the presence of CD3 and CD28 mAbs, all three cell types were with CD28 mAb, CD63 mAb, or a binding control mAb (AAA1). able to induce T cell proliferation. Expression of CD63 on the CD3 mAb alone only led to a very weak T cell proliferation as did artificial APC, however, had no influence on T cell stimulation the combination of CD3/control mAb. The slightly reduced T cell mediated by cross-linked CD3 and CD28 mAbs (Fig. 5B). proliferation obtained with CD3/control mAb compared with CD3 Taken together our results indicate that CD63 on APC does not mAb alone is probably due to a reduced amount of immobilized act in a costimulatory manner and that the effects observed with CD3 mAb in the presence of a second mAb. Stimulation with CD3 6004 COSTIMULATION OF HUMAN T CELLS VIA CD63 Downloaded from http://www.jimmunol.org/

FIGURE 5. Presence of CD63 on artificial APC has no influence on their capacity to stimulate T cells.A, FACS analysis of BwCD64, BwCD64/ CD80, and BwCD64/CD63 cells using mAb specific for CD64, CD80, and CD63 (gray histograms). Reactivity of the control mAb (open histograms)

is shown. B, Irradiated BwCD64, BwCD64/CD80, and BwCD64/CD63 by guest on October 2, 2021 cells were loaded with Abs as indicated and cocultured with puriﬁed human T cells. Data of one experiment are representative of three independent experiments shown.

even higher induction of the activation markers CD69 and CD25 on T cells (Fig. 6B). Interestingly, in contrast to CD28 mAb, sol- FIGURE 4. The inhibitory effect of mAb 11C9 in an allo MLR is not uble CD63 mAb was not able to enhance CD3-triggered T cell due to its interaction with CD63 on DC. A, CD63 mAb 11C9 does not proliferation (data not shown). As solid phase immobilization to interfere with DC differentiation. Monocytes were cultivated with GM- the plate surface is required, strong cross-linking seems to be nec- CSF and IL-4 for 7 days in presence of mAb 11C9 or a control mAb. DCs were analyzed for expression of surface markers as indicated. B, CD63 essary for costimulation via CD63. mAb 11C9 does not interfere with DC maturation. Immature DC were To further characterize CD63 costimulation, the kinetics of T maturated with LPS in presence of either mAb 11C9 or a control mAb for cell proliferation were studied with the CFSE labeling technique. 2 days. Cells were analyzed for expression of maturation markers. C, Pre- Compared with CD28 costimulation, the number of T cells that did incubation of DC with mAb 11C9 has no effect on their allostimulatory not proliferate was slightly higher. However, the majority of CD63 capacity. Purified T cells were cultured with graded numbers of allogenic costimulated cells did enter cell cycling and underwent compara- DC preincubated over night with mAb11C9 or control Ab M80. ble numbers of cell cycles during 7 days as CD28 costimulated cells (Fig. 6C). At earlier time points we could, however, notice a small delay in the onset of cell cycling in T cells costimulated via mAb together with CD28 mAb led to strong T cell proliferation, CD63 (data not shown). This could be explained by the fact that but, surprisingly, the use of immobilized CD63 mAb in combina- CD63 needs to be induced before stimulation. Similar to costimu- tion with CD3 mAb induced proliferative rates in T cells that were lation via CD28, costimulation via CD63 led to proliferation in ϩ ϩ ϩ as strong. CD63 mAb alone did not induce any T cell proliferation both the CD4 and the CD8 subset (Fig. 6C). Purified CD8 T (Fig. 6A). Plate-bound CD63 mAb was also able to further enhance cells could be also very efficiently activated via immobilized mAb CD28-costimulated T cell proliferation in some but not all exper- to CD3 and CD63 (data not shown) indicating that costimulation ϩ ϩ iments and could induce activation of T cells in combination with of CD8 T cells via CD63 does not require CD4 T cell help. low amounts of CD3/CD28 mAbs that when used alone did not show any effect (data not shown). Costimulation via CD63 induces IL-2 production in T cells T cell stimulation with immobilized CD3 and CD63 mAbs did The induction of sufficient and prolonged IL-2 production is nec- not only result in strong proliferation but also in comparable or essary to generate activated T cells that are fully functional. CD28 The Journal of Immunology 6005

bilized mAb and cell culture supernatant was harvested on day 3. As expected almost no IL-2 was produced by T cells stimulated with CD3 mAb alone. CD3/CD63 mAbs however induced levels of IL-2 comparable to CD3/CD28 mAbs stimulation (Fig. 6D). Furthermore, the presence of both CD28 and CD63 mAb with CD3 mAb resulted in higher IL-2 production compared with T cells costimulated via CD28 only, indicating that CD28 driven costimulation can be further enhanced via CD63. T cells activated via CD3/CD63 also produced IFN-␥ in amounts comparable to CD3/ CD28 stimulated T cells (Fig. 6D). Also puriﬁed CD8ϩ T cells produced comparable amounts of IL-2 and IFN-␥ when costimulated via CD63 and CD28 (data not shown). Taken together, these results demonstrate that regarding proliferation and cytokine-induction, CD63 seems to be able to deliver a costimulatory signal to T cells as potent as CD28. CD63 costimulation leads to induction of survival genes T cells fully activated by signal 1 and signal 2 up-regulate antiapoptotic pathways. Many of the CD28 independent costimulatory pathways, however, have been reported to induce brief T cell ac- Downloaded from tivation followed by apoptosis (5, 35). We therefore compared T cells costimulated via CD28 with CD63 in this aspect. We performed annexin V staining after 3 or 7 days of T cell activation with plate bound mAbs. No signs of enhanced apoptosis of CD3/CD63 mAbs stimulated T cells compared with CD3/CD28 mAbs stimulated T cells could be detected even after prolonged http://www.jimmunol.org/ cultivation (Fig. 7A). Furthermore, induction of the survival genes

Bcl-2 and Bcl-xL was comparable in T cells costimulated via CD28 and CD63 after 72 h of stimulation (Fig. 7B). We could however notice a delayed induction of survival genes when mea-

suring Bcl-2 and Bcl-xL expression at earlier time points (data not shown). This is in line with a delayed onset of proliferation that was observed in T cells upon CD63 costimulation compared with T cells costimulated via CD28. by guest on October 2, 2021 T cells costimulated via CD63 show enhanced responsiveness to restimulation Many of the CD28-independent costimulatory pathways have been reported to induce only brief T cell activation that is followed by unresponsiveness (5, 35). We therefore compared T cells costimulated via CD28 with CD63 regarding their capacity to respond to secondary stimulation. For that purpose, purified T cells were cultivated in presence of different immobilized mAbs. After 7 days the cells were harvested FIGURE 6. Immobilized CD63 mAb 11C9 acts in a costimulatory fash- and restimulated with CD3/CD28 or CD3/CD63 mAbs for another ion. A, Purified T cells were cultivated with immobilized Abs. Proliferation 48 h. To control viability, we also restimulated the T cells with of T cells was measured on day 3. Data of one experiment representative PMA/ionomycin. Although T cells prestimulated with medium or for four independent experiments are shown. The differences between the proliferation induced with CD3 mAb and CD3/CD63 mAb were statisti- CD3 mAb alone showed no or only very weak responsiveness to cally significant (p Ͻ 0.01) and no difference between proliferation induced restimulation, T cells activated during the first round of stimulation via CD3/CD28 and CD3/CD63 mAb was found. B, T cells cultivated with via triggering CD3 and either of the two costimulatory molecules the indicated immobilized Abs for 5 days were analyzed for CD69 and fully responded to secondary stimulation (Fig. 8A). Furthermore, CD25 expression. Reactivity of T cells stimulated with CD3/CD28 (gray cells activated in primary cultures via CD3/CD63 even responded histogram), CD3/CD63 (bold line), and CD3/control (dotted line) mAb in the second stimulation better than CD3/CD28 primed cells ( p Ͻ with indicated Abs is shown. C, Purified T cells were labeled with CFSE 0.01; n ϭ 6). and cultivated with immobilized Abs for 7 days. Proliferation was evalu- We tested next whether this enhanced responsiveness of CD63 ated by FACS analysis. D, Purified T cells were cultivated with immobi- costimulated T cells is also found when restimulating these cells lized Abs. Culture supernatants were harvested on day 3, and IL-2 and with APC. Also in these experiments, we measured higher prolif- IFN-␥ were measured by ELISA. Data of one experiment representative for four are shown. erative response of T cells costimulated via CD63 in the primary stimulation ( p Ͻ 0.05; n ϭ 5) (Fig. 8B). This enhanced responsiveness of T cells preactivated via CD3 and CD63 was obtained and non-CD28 costimulators have been reported to differ in this regardless whether immature DC, mature DC, or monocytes were aspect (33–35). We therefore analyzed the IL-2 production in- used to restimulate T cells. duced by CD3 mAb alone or in combination with CD63 or CD28 Our results indicate that T cells can receive a strong costimu- mAbs. Purified T cells were cultivated in the presence of immo- latory signal via CD63. In many aspects these T cells show no 6006 COSTIMULATION OF HUMAN T CELLS VIA CD63 Downloaded from http://www.jimmunol.org/

FIGURE 8. CD3/CD63 mAb-stimulated T cells show strong secondary responses. For primary stimulation purified T cells were cultivated with immobilized mAbs as indicated for 7 days and used for restimulation experiments. A, T cells were restimulated with the indicated immobilized FIGURE 7. CD63 costimulation induces antiapoptotic pathways. A, Pu- mAb combinations or PMA/ionomycin. Proliferation was measured 48 h rified T cells activated with immobilized mAb were analyzed with Annexin later. B, T cells were restimulated with allogenic APC (ratio 1:5). Prolif- V-FITC. B, Purified T cells were activated with immobilized mAb for 3 eration of T cells was determined 48 h later. by guest on October 2, 2021 days. Induction of survival genes Bcl-2 and Bcl-xL was analyzed by RT-PCR. ture. This effect seems, however, to require interaction with a specific epitope on CD63 as the commercially available CD63 Ab functional differences to T cells activated via the primary costimu- H6C5, while also inhibiting DC induced T cell responses, is not latory molecule CD28. They have however a higher capacity to able to act in a costimulatory fashion with CD3 mAb when pre- respond to secondary stimulation. sented in solid phase bound form (data not shown). There are at least two possibilities how CD63 could be involved Discussion in the transfer of a costimulatory signal from DC to T cells. First, During T cell activation costimulation is an essential process, in- CD63 could be the receptor of a costimulatory signal provided by fluencing the outcome of the immune response: In presence of DC. Although no ligand for CD63 has been described so far, other costimulatory signals T cells become efficiently activated, whereas members of the tetraspan family have recently been reported to be absence of costimulation leads to anergy (1–3). able to act as receptors (36, 37). Therefore it is conceivable that We describe in this study CD63 as a novel potent, activation- CD63 directly interacts with a yet unidentified ligand on DC. In induced costimulatory structure on T cells. We show that although contrast, tetraspans are well known for their multiple cis-interac- only a small percentage of resting T cells displays CD63 on the tions and associations that build up a so called tetraspan network surface, this molecule is quickly induced upon T cell activation in (38–43). Thus binding of mAb 11C9 might alter the interaction of vitro. Activation via TCR triggering alone is sufficient to up-reg- CD63 with a molecule that is a critical receptor for a costimulatory ulate CD63 surface expression. Targeting these CD63 molecules signal delivered by DC. The involvement of a CD63-associated on T cells has functional implications: Binding of intact mAb molecule could also explain the requirement for strong cross-link- 11C9 or its Fab to CD63 on T cells consistently inhibits their ing, achieved by immobilizing mAb 11C9 to plastic surfaces, to responses to allogenic mdDC, whereas cross-linking of CD63 with induce CD63 triggered costimulation in T cells. mAb 11C9 can costimulate the activation of the TCR complex in We show for the first time that CD63 has a costimulatory func- human T cells. These findings lead to the conclusion that CD63 tion on T cells. It shares this capacity with a number of other might be involved in the delivery of a costimulatory signal. molecules aside from CD28, which is generally considered the A yet unidentified structure expressed on APC seems to be re- primary costimulatory molecule on T cells (6, 44). Among these sponsible for this activation via CD63, as blocking of CD63 by molecules are several members of the tetraspan family (6, 13, 14, mAb 11C9 inhibits the T cell response to DC, but not to CD3/ 45–50). Costimulation via molecules distinct from CD28 seems in CD28 mAb-coated beads. Cross-linking of CD63 via immobilized most instances not to be sufficient for inducing full T cell activa- mAb might therefore act in an agonistic manner for this DC struc- tion (6, 33). This was also seen with costimulation via CD9, the The Journal of Immunology 6007 tetraspan molecule most widely examined in this context (35). The Dr. Garry P. Nolan and colleagues for providing the retroviral vector main deficiency in this CD28-independent costimulatory pathway pBMNZ and the packaging cell line Phoenix-E. Prof. Walter Knapp died appears to lie in the induction of IL-2, resulting in insufficient August 30, 2004. We will always remember his enthusiasm for science and antiapoptotic signals, which leads to only brief activation of T cells his mentorship. followed by apoptosis (5, 33–35, 51–53). We did not, however, notice marked differences regarding T cell proliferation, up-regu- References 1. Mueller, D. L., M. K. Jenkins, and R. H. Schwartz. 1989. Clonal expansion lation of activation markers, and, most importantly, IL-2 produc- versus functional clonal inactivation: a costimulatory signalling pathway deter- tion when comparing T cells costimulated via CD28 and via CD63. mines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol. Furthermore, we did not observe enhanced apoptosis induced by 7:445. 2. 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Adaptor protein 3-dependent microtu- We thank Dr. David Segal for critically reading the manuscript and appre- bule-mediated movement of lytic granules to the immunological synapse. Nat. Immunol. 4:1111. ciate the excellent technical assistance of Petra Kohl, Alessandra Mathe, 28. von Lindern, J. J., D. Rojo, K. Grovit-Ferbas, C. Yeramian, C. Deng, G. Herbein, Margarethe Merio, Christoph Klauser, and Claus Wenhardt. We also thank M. R. Ferguson, T. C. Pappas, J. M. Decker, A. Singh, R. G. Collman, and W. A. 6008 COSTIMULATION OF HUMAN T CELLS VIA CD63

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