Critical role of CD81 in cognate TB cell interactions leading to Th2 responses
Jun Deng1,4,
Rosemarie H. Dekruyff2,
Gordon J. Freeman3,
Dale T. Umetsu2 and
Shoshana Levy1
1 Division of Oncology, Department of Medicine, and 2 Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University Medical Center, Stanford, CA 94305, USA 3 Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA 4 Present address: SurroMed, 2375 Garcia Avenue, Mountain View, CA 94043, USA.
Correspondence to: S. Levy; E-mail: levy{at}cmgm.stanford.edu
Transmitting editor: P. W. Kincade
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Abstract
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We previously demonstrated that CD81/ mice fail to develop Th2-biased immune responses and allergen-induced airway hyper-reactivity. Because CD81 is expressed on both activated T and on B cells, we examined the role of CD81 expression by each cell type. We established an in vitro system by backcrossing the CD81 deletion to TCR transgenic (Tg) mice and to BCR Tg mice. Here we demonstrate that CD81 expression by T cells is critical for their induction of IL-4 synthesis by B cells. CD81/ TCR Tg T cells were impaired in IL-4 production compared to CD81+/+ TCR Tg T cells, whereas CD81/ and CD81+/+ BCR Tg B cells induced equivalent amounts of IL-4 in CD81+/+ TCR Tg T cells. CD81/ TCR Tg T cells expressed reduced levels of ICOS, GATA-3, STAT6 and phosphorylated STAT6 when activated by antigen-presenting B cells. Taken together, these results indicate that CD81 expression by T cells greatly enhances cognate TB cell interactions and greatly augments intracellular activation pathways leading to Th2 polarization.
Keywords: cytokines, knockout, Th1/Th2, tetraspanins
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Introduction
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Optimal activation of T cells by antigen-presenting cells (APC) requires at least two signals, the first of which is provided by cognate TCRMHC interaction, and the second by interactions between co-stimulatory molecules on T cells and on APC (1). The best-characterized co-stimulatory complex consists of CD28 on T cells, which interacts with B7-1 (CD80) and B7-2 (CD86) on APC, and ligation of CD28 by B7 molecules dominates over other co-stimulatory interactions in the initiation of T cell responses (25). However, CD28-deficient mice efficiently clear viral infection and reject allografts (6,7), indicating that other molecules, such as CD40 ligand (8), OX40 (9) and ICOS (10,11), effectively provide co-stimulatory signals to T cells. Both OX40 (12,13) and ICOS, which is selectively expressed on Th2-polarized T cells (14,15), predominantly enhance Th2 cytokine production (1618), indicating that co-stimulatory molecules influence the polarization process to Th1 or Th2 phenotypes. In addition, we have previously shown that CD81 is yet another molecule involved in enhancing Th2-biased immune responses (1921).
CD81 (22,23) and other members of the tetraspanin superfamily, CD9 (24), CD37 (25) and CD82 (26), have all been shown to co-stimulate T cell activation. CD81 is widely expressed on a variety of tissues and cell types including B cells, T cells and dendritic cells (27,28). It associates with numerous cell surface molecules in a cell-type dependent manner. On B cells, CD81 associates with the B cell-specific CD19/CD21 molecular complex (29,30). Co-stimulation of the BCR and this complex lowers the threshold for B cell activation. B cells from CD81/ mice express lower levels of CD19 on the cell surface and show deficient cell proliferation in response to engagement of their BCR (21,31), indicating an important role for CD81 in B cell function. In human T cell lines, CD81 associates with CD4, CD8 (32,33) and
4ß1 (VLA-4) integrins (34,35), and engagement of CD81 augments superantigen-induced T cell proliferation and activation (23). In murine T cells, CD81 expression is increased upon activation with anti-CD3 plus anti-CD28 (22) or with phorbol myristate acetate plus ionomycin (28). Moreover, co-engagement of CD81 and CD3 on the surface of murine CD4+ and CD8+ T cells induces CD28-independent proliferation (22). These observations suggest that CD81 may play an important co-stimulatory role in T cell activation.
In vivo, CD81 is required for the optimal induction of Th2-dominated immune responses. CD81/ mice are deficient in T cell-dependent IgG1 production and in Th2 cytokine secretion (21,31). Furthermore, in the context of a murine model of asthma, a Th2-dependent disease, CD81 expression is essential for the development of allergen-induced airway hyper-reactivity (AHR), since AHR fails to develop in CD81/ mice (19). However, naive CD81/ T cells can be polarized towards the Th2 phenotype and can produce normal levels of IL-4 when stimulated in vitro using polyclonal, Th2-inducing conditions (19), indicating that CD81 does not affect the intrinsic ability of T cells to produce Th2 cytokines. These results suggest that CD81 regulates the interaction between T cell and APC in antigen-specific Th2 responses, and the current study was aimed at defining the role of CD81 in cognate T cellAPC interactions that lead to Th2 responses. Using a transgenic (Tg) in vitro system to study T cellAPC interactions we demonstrate that (i) CD81 expressed on T cells is necessary for induction of Th2 immune responses and (ii) the role of CD81 is manifested when B cells, but not splenic adherent cells (SpAC), present the antigen.
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Methods
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Animals
BALB/c CD81/ mice were generated as described previously (19,21). Anti-hen egg lysozyme (HEL) BCR Tg MD4 mice (36) were backcrossed to BALB/cByJ mice for >10 generations (37). In these mice, 6090% B cells express anti-HEL surface IgM and IgD, and secrete anti-HEL IgM, detected by ELISA (37). DO11.10 mice, expressing an MHC class II-restricted ovalbumin (OVA)323339 TCR Tg, were obtained from Dr Susan Webb (Scripps Research Institute, La Jolla, CA) with permission from Dr Denis Loh (Washington University, St Louis, MO). To obtain OVA TCR Tg CD81/ mice, we first crossed CD81+/ mice (CD81/ mice are deficient in reproduction) to DO11.10 homozygous. Offspring CD81+/ OVA TCR+/ F1 mice were crossed with CD81+/ mice to generate OVA TCR Tg CD81/ and wild-type littermates. To identify OVA TCR Tg mice, the animals were bled from the tail vein, and red blood cells were lysed in 0.144 M NH4Cl and 0.017 M Tris, pH 7.2. Intact cells were stained with phycoerythrin (PE)-conjugated anti-CD4 mAb and FITC-conjugated KJ126 anti-OVA TCR mAb and analyzed by FACScan as described below. HEL BCR Tg CD81/ mice and HEL BCR Tg CD81+/+ littermates were generated using similar breeding methods. Mice aged 816 weeks old (age and sex matched) were used in all experiments. All animal protocols were approved by the Stanford University Committee on Animal Welfare.
Antibodies and reagents
Hamster anti-mouse CD81 mAb Eat1 and Eat2 were produced in our laboratory as described previously (28). Anti-CD3, anti-CD28, FITC-conjugated anti-CD3 and anti-CD69, and PE-conjugated anti-CD4, anti-CD25, anti-CD28 and anti-CD81 mAb were obtained from PharMingen (San Diego, CA). OptEIA ELISA kits for mouse IL-4 and IFN-
were also from PharMingen. Hybridoma cells producing the anti-clonotypic antibody KJ1-26 (38) were provided by Dr P. Marrack (National Jewish Medical and Research Center, Denver, CO). Anti-ICOS mAb 7E.17G9 was generated as described (15). Mouse anti-GATA3 mAb, rabbit anti-STAT6 polyclonal IgG, goat anti-actin polyclonal IgG and donkey anti-goat IgG horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). IgG from pooled hamster sera and goat anti-mouse IgG1HRP was from Southern Biotechnology Associates (Birmingham, AL). Goat anti-rabbit IgGHRP was from Tago (Burlingame, CA). HEL and OVA were obtained from Sigma (St Louis, MO), and they were conjugated as described (37).
CD4+ T cell purification
Single-cell suspensions were incubated with anti-mouse CD8 antibody-conjugated magnetic beads at 612°C for 15 min. The cells were passed through an MS column to deplete CD8+ T cells. Unbound cells were further incubated with anti-Thy-1.2 antibody-conjugated magnetic beads and passed through a new MS column. Column bound cells were collected as CD4+ T cells. CD4+ T cell purity, analyzed by FACScan using FITC-conjugated anti-CD4 mAb on a cell sample, was >95%. All antibody-conjugated beads, columns and magnetic separators were purchased from Miltenyi Biotec (Auburn, CA).
Purification of APC
B cells from HEL BCR Tg CD81+/+ and HEL BCR Tg CD81/ mice were prepared by negative selection. Briefly, splenocytes from HEL BCR Tg mice were incubated with anti-mouse Thy-1.2, CD11b and CD11c beads, and passed through an MS column to deplete T cells, phagocytes and dendritic cells; flow-through was collected as the B cell fraction. Samples of purified B cells showed
85% CD19+ cells by FACScan. Purified B cells were treated with mitomycin C (50 µg/ml) for 30 min at 37°C, washed 3 times and used as APC. All antibody-conjugated beads, columns and magnetic separators were purchased from Miltenyi Biotec.
SpAC were prepared as described previously (37).
Polyclonal T cell activation
Purified CD4+ T cells (1 x 106/ml) were stimulated on plate-bound anti-CD3 (2 µg/ml) and anti-CD28 (5 µg/ml) for 7 days, and then re-stimulated at 106 cells/ml on anti-CD3 plus anti-CD28-coated plates. Levels of cytokines in the culture medium, 24 h later, were determined by ELISA. Cell proliferation of polyclonally stimulated T cells was determined by serial dilutions in 96-well plates (1 x 105 to 2.5 x 104 /well) and [3H]thymidine incorporation was measured during the 24 h re-stimulation period.
Stimulation of naive DO11.10 Tg CD4+ T cells
CD4+ T cells (3 x 105/ml) from DO11.10 Tg mice were cultured with APC [HEL BCR Tg B cells (1.5 x 105/ml) or SpAC (1 x 105/ml)] and antigen for 5 days and T cells (3 x 105/ml) were re-stimulated with freshly purified APC on day 5. In some experiments, 10 µg/ml of Eat1, Eat2 or purified hamster serum IgG was added to cultures through the primary and the re-stimulation course. Culture supernatants were harvested 24 h after re-stimulation for analysis of cytokines by ELISA.
T cell proliferation
CD4+ T cells (3 x 105/ml) were cultured in 96-well plates in 200 µl of medium containing a range of concentration of either HELOVA, HEL or OVA as antigens and B cells or SpAC served as APC, as indicated. After 4 days, cells were pulsed with 1 µCi/well [3H]thymidine (Amersham, Arlington Heights, IL) in 50 µl of medium for 20 h. The cells were harvested onto filters using a 96-well harvester (Wallac, Turku, Finland) and read on a 96-well format scintillation counter (Wallac).
Flow cytometry analysis
Cultured cells were washed in PBS/1% BSA and resuspended at 1 x 107 cells/ml in PBS/1% BSA. Then 50 µl (5 x 105 cell) was stained with 1 µg mAb for 30 min on ice, washed twice in PBS/1% BSA, fixed with 2% paraformaldehyde in PBS and analyzed on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The cytometer was calibrated with CaliBRITE beads using the FACSComp program and CellQuest 3.3 was used to generate the plots (beads and software program were obtained from Becton Dickinson)
Cytokine ELISA
IL-4 and IFN-
in culture medium were determined by using OptEIA ELISA kits based on the manufacturers instructions. IL-5 and IL-13 were measure by ELISA as described previously (19).
Western blot analysis
CD4+ T cells from naive OVA TCR Tg CD81/ and CD81+/+ mice were co-cultured with HEL BCR Tg B cells with or without 1 µg/ml HELOVA and re-stimulated with freshly purified B cells on day 5. Cells were harvested on days 0, 3, 5 and 6 of the culture. Cells were lysed on ice in RIPA buffer [1 x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)] for 30 min, and centrifuged at 10,000 g at 4°C for 20 min. Supernatants from equal amount of cells were separated on 10% Trisglycine SDS gels (Invitrogen, San Diego, CA) and transferred onto nitrocellulose membranes. The levels of GATA3, STAT6, phosphorylated STAT6 and actin were detected by incubation with the respective primary antibodies (mouse anti-GATA3 mAb, rabbit anti-STAT6 polyclonal IgG, rabbit anti-phospho-STAT6 polyclonal IgG and goat anti-actin polyclonal IgG), followed by washing in TBS + 0.1% Tween 20 and incubation with HRP-linked secondary antibodies. Blots were visualized by chemiluminescence detection (ECL; Amersham, Little Chalfont, UK) following the instructions of the manufacturer.
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Results
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An anti-CD81 mAb inhibits IL-4 production in antigen-specific TB cell interaction
To understand the cellular mechanisms responsible for the deficient Th2 immune responses in vivo in CD81/ mice, we first examined whether CD81 is involved in antigen-specific TB cell interaction in vitro. Antigen-specific naive B cells from anti-HEL BCR Tg (37) were used as APC to present HELOVA (antigen), in a cognate fashion, to naive OVA TCR Tg CD4+ T cells. We tested the effects of two anti-CD81 mAb on T cell proliferation and IL-4 production in this system (Fig. 1A). In response to antigen stimulation, CD4+ T cells showed antigen-specific dose-dependent proliferation (Fig. 1B) and IL-4 production (Fig. 1C). HEL or OVA alone, which are not presented in a cognate manner by the B cells, did not induce detectable cell proliferation or IL-4 production (data not shown). Addition of the anti-CD81 mAb or control hamster IgG did not affect T cell proliferation (Fig. 1B). However, one of the antibodies, Eat1, markedly and consistently inhibited antigen-dependent IL-4 production, while Eat2 and hamster IgG showed no effect (Fig. 1C). Eat2 had no effect on IFN-
production, while the effect of Eat1 was not consistent, it had either no effect, but in some experiments it slightly reduced the production of this cytokine (data not shown). These results suggest that CD81 is important for antigen-specific TB cell interaction leading to Th2 responses. Since CD81 is expressed on both T cells and B cells (28), it was unclear from this experiment whether the presence of CD81 on T cells, B cells or both cell types is important in antigen-specific TB cell interaction.

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Fig. 1. Anti-CD81 mAb Eat1 inhibits IL-4 production in antigen-specific TB cell interaction. CD4+ cells from naive OVA TCR Tg mice were co-cultured with HEL BCR Tg B cells for 5 days in the presence of the indicated concentrations of HELOVA. T cells were re-stimulated on day 5 for 24 h with freshly purified B cells and the same concentrations of antigen. Eat1, Eat2 or hamster IgG control (10 µg/ml) were present in the cultures during the primary and secondary stimulation (A). Thymidine incorporation during the last 20 h of the primary stimulation (B) and IL-4 production during 24 h re-stimulation (C) were determined. Comparable results were obtained in three independent experiments.
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CD81/ B cells prime naive T cells and induce IL-4 synthesis
CD81 is highly expressed on resting mouse B cells (28) where it associates with the B cell-specific molecular complex that includes CD19 and CD21 (30,39). Previously we immunized chimeric JH/ mice, reconstituted with CD81/ ES cells. These mice showed reduced Th2 immune responses, implicating B cells in this defect (20). To examine the role of CD81 in antigen presentation we tested B cells from OVA-primed CD81/ and CD81+/+ mice for their ability to stimulate OVA TCR Tg T cells to produce IL-4. These experiments revealed that B cells from both groups induced similar, albeit lower, levels of cytokines (data not shown). To increase the number of antigen-specific B cells we generated HEL BCR Tg CD81/ mice and tested their B cells for their ability to stimulate OVA TCR Tg T cells as illustrated in Fig. 2(A). To our surprise, CD81/ and CD81+/+ B cells functioned equally well in stimulating T cell proliferation, and IL-4 and IFN-
production (Fig. 2BD respectively). These results demonstrate that the expression of CD81 on B cells does not influence their ability to present cognate antigen to T cells and to stimulate Th2 cytokine production.

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Fig. 2. CD81/ B cells prime naive T cells and induce IL-4 synthesis. CD4+ T cells (3 x 10 5/ml) were purified from peripheral lymph nodes of naive OVA TCR Tg mice and co-cultured with CD81/ and CD81+/+ HEL BCR Tg B cells (1.5 x 10 5/ml) as illustrated in (A). Incremental concentrations of HELOVA were added to the culture medium as indicated. On day 5, T cells were re-stimulated with fresh CD81/ and CD81+/+ HEL BCR Tg B cells and HELOVA. T cell proliferation during the last 20 h of primary stimulation (B), and IL-4 (C) and IFN- (D) production during 24 h re-stimulation were determined.
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CD81/ T cells have no intrinsic defect in IL-4 production
Previously, we have shown that CD81/ and CD81+/+ T cells are capable of producing similar levels of IL-4 when activated by using Th2-polarizing conditions (19). Here we examined whether CD81 on T cells is required for IL-4 production when the T cells were stimulated with anti-CD3 and anti-CD28 antibodies, in the absence of exogenous IL-4. When naive CD81+/+ and CD81/ T cells were stimulated with plate-bound anti-CD3 plus anti-CD28 mAb (Fig. 3A), CD81/ T cells showed identical levels of cell proliferation (Fig. 3B) and IL-4 secretion as CD81+/+ T cells (Fig. 3C). IFN-
levels were also similar between the groups, 6.21 ± 0.21 ng/ml for the CD81+/+ and 3.41 ± 0.55 ng/ml for the CD81/. This result indicates that CD81/ T cells are intrinsically capable of producing the Th2 cytokine IL-4, even in the absence of Th2-polarizing conditions.

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Fig. 3. CD81/ T cells produce similar levels of IL-4 as do CD81+/+ T cells when stimulated with anti-CD3 plus anti-CD28. Naive CD4+ T cells were purified from CD81/ and CD81+/+ mice and stimulated with plate-bound anti-CD3 (2 µg/ml) plus anti-CD28 (5 µg/ml) for 7 days. On day 7, the cells were re-stimulated using the same condition (A). Cell proliferation (B) and cytokine (IL-4 and IFN- ) (C) production during the 24-h re-stimulation period were determined. Comparable results were obtained in two independent experiments.
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Deficient IL-4 production by naive OVA TCR Tg CD81/ T cells when stimulated in vitro with cognate BCR Tg B cells
To study whether CD81 on naive T cells plays a role during antigen-specific TB cell interaction, we crossed CD81/ mice to DO11.10 OVA TCR Tg mice (40) to obtain OVA TCR Tg CD81/ mice. Naive CD4+ T cells were purified from the peripheral lymph node of CD81+/+ and CD81/ OVA TCR Tg mice and stimulated in vitro with HEL BCR Tg B cells as illustrated in Fig. 4(A). In the presence of HELOVA, CD81/ and CD81+/+ T cells showed similar levels of antigen-dependent cell proliferation (Fig. 4E). Strikingly, while CD81+/+ T cells produced increasing amounts of IL-4, IL-5 and IL-13 with increasing dosage of antigen, CD81/ T cells produced significantly less IL-4, IL-5 and IL-13 (Fig. 4BD respectively). IFN-
secretion was comparable between CD81/ and CD81+/+ T cells at 0.1 mg/ml antigen, but this Th1-type cytokine secreted by the CD81/ T cells was consistently elevated by comparison to that of the CD81+/+ T cells at the higher antigen concentration (Fig. 4F). Similar results were obtained when non-Tg T cells from OVA-primed CD81/ mice were stimulated with HELOVA by HEL BCR Tg B cells. Again, CD81/ T cells also produced considerably less IL-4 than T cells from CD81+/+ mice (data not shown). Taken together these results demonstrate that CD81 on T cells is critical for antigen-specific TB cell interaction in Th2 responses.

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Fig. 4. Deficient IL-4 production by naive OVA TCR Tg CD81/ T cells when stimulated in vitro with cognate BCR Tg B cells. Purified naive CD81/ and CD81+/+ OVA TCR Tg CD4+ T cells were co-cultured with HEL BCR Tg B cells as detailed in Fig. 2(A), as illustrated in (A). T cell proliferation during the last 20 h of primary stimulation (E) and cytokine production [IL-4 (B), IL-5 (C), IL-13 (D) and IFN- , (F)] during 24-h re-stimulation were determined. This experiment is representative of at least four independent experiments. The same results were obtained in additional experiments that utilized negatively purified CD4+ T cells.
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Surface expression of T cell activation markers following activation with cognate BCR Tg B cells
To follow up on the above results, we examined CD81/ and CD81+/+ T cells for differences in cell surface expression of T cell activation markers following co-culture with the cognate B cells (Fig. 4A). CD81/ and CD81+/+ T cells showed identical antigen dose-dependent increases in cell surface expression of the IL-2 receptor (CD25), the co-stimulatory molecule CD28 and the early activation marker CD69 (Fig. 5). However, the percentages of the inducible co-stimulatory molecule (ICOS) positive cells and the levels of its expression were lower in CD81/ than in CD81+/+ T cells following antigen-specific stimulation (Fig. 5). Since recent studies indicate that co-stimulation through ICOS is required for optimal TB cell collaboration and IL-4 production by T cells (18), the reduced ICOS expression on CD81/ T cells may contribute to their deficiency in IL-4 production.

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Fig. 5. Surface expression of T cell activation markers on CD81/ and CD81+/+ T cells following co-culture with B cells. Cells harvested 24 h after re-stimulation (Fig. 4) were analyzed for the expression of CD25, CD28, CD69, ICOS and CD81 on CD4+ cells; shown are the percentage of double-positive cells in the total cell population and the level of ICOS expression of cultures stimulated with 1 µg/ml OVA. Comparable results were obtained in two independent experiments.
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It is of note that resting mouse T cells express very low levels of CD81 and that CD81 is inducible on T cells upon activation (28). After antigen-specific in vitro priming and re-stimulation, the expression of CD81 on CD81+/+ T cells was increased dramatically (Fig. 5). This result suggests that CD81 plays an important, yet undefined, role in T cell activation.
Reduced expression of GATA3 and STAT6, and reduced STAT6 phosphorylation in CD81/ T cells after in vitro stimulation
Since the expression of the Th2-specific transcription factor GATA3 and the phosphorylation of STAT6 are critical steps in Th2 differentiation and cytokine production (41,42), we determined whether CD81/ and CD81+/+ T cells differed in GATA3 expression and STAT6 phosphorylation during TB cell interactions. CD4+ T cells from naive OVA TCR Tg CD81/ and CD81+/+ mice were stimulated in vitro with HEL BCR Tg B cells as described above (Fig. 4A). Cells were harvested on days 0, 3, 5 and 6 of the stimulation, and the expression of GATA3, STAT6 and phosphorylated STAT6 were determined by Western blot and immunostaining. Figure 6 shows that resting T cells express low levels of GATA3 and STAT6, and phosphorylated STAT6 is undetectable. After activation, CD81+/+ T cells show a time-dependent increase in expression of GATA3, STAT6 and phosphorylated STAT6. CD81/ T cells also increase GATA3 expression, but to a considerably lower level. CD81/ T cells show no increase in STAT6 expression or detectable phosphorylated STAT6 (Fig. 6). The percentages of CD81/ and CD81+/+ CD4+ T cells are comparable at each time point, thus the changes seen are not simply due to a reduction in the number of CD4+ T cells. These results indicate that the expression of CD81 on the surface of T cells facilitates co-stimulatory and downstream signaling pathways required for induction of STAT6 expression and phosphorylation and GATA3 expression, and the consequent enhancement of Th2 cytokine production.

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Fig. 6. Reduced expression of GATA3 and STAT6, and reduced STAT6 phosphorylation in CD81/ T cells after in vitro stimulation. CD4+ T cells from naive CD81/ and CD81+/+ OVA TCR Tg mice were co-cultured with HEL BCR Tg B cells with or without 1 µg/ml HELOVA for 5 days and re-stimulated with freshly purified B cells on day 5. Cells were harvested on days 0, 3, 5 and 6 of the cultures, and lysed in RIPA buffer. Cell lysate from equal numbers of cells were separated on 10% Trisglycine SDS gels. The levels of GATA3, STAT6 and phosphorylated STAT6 were determined by immune staining as described in Methods. Actin expression was used as a reference for equal loading. Comparable results were obtained in three independent experiments.
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IL-4 production by CD81/ and CD81+/+ T cells is comparable when primed with antigen-presenting SpAC
So far we have demonstrated that CD81 on T cells is critical for antigen-specific TB cell collaboration and Th2 responses. To determine whether CD81 on T cells is also important for their interactions with other APC, SpAC from wild-type BALB/c mice were used to present antigen to CD81/ and CD81+/+ OVA TCR Tg T cells as illustrated in Fig. 7(A). The proliferation, IL-4 secretion and IFN-
production of CD81/ T cells were similar to those of CD81+/+ T cells (Fig. 7BD respectively). These results suggest that CD81 on T cells is not critical in antigen-specific T cellSpAC interactions leading to Th1 or Th2 responses.

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Fig. 7. IL-4 production by CD81/ and CD81+/+ T cells is comparable when primed with antigen-presenting SpAC. The experiment was performed as described in Fig. 4 except that SpAC (1 x 10 5/ml) were used as APC (A). T cell proliferation (B) and cytokine (C and D) production were determined. Comparable results were obtained in three independent experiments.
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Discussion
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The initial contact between Th precursor cells and APC is critical for Th1/Th2 polarization. Cytokines such as IL-4, IL-12 and their respective receptors have been demonstrated to play a major role in this process. In addition, polarized immune responses involve other cell surface and intracellular molecules that affect immune synapses and downstream signaling events. For example, ICOS, which was discovered only recently, is a molecule essential for Th2 response. Further, our previous (19,21) and current results show that CD81, a tetraspanin molecule, is also critical for the development of Th2 responses. Like ICOS, CD81 preferentially affects Th2 immune responses in vivo and is selectively expressed on activated T cells (Fig. 5). However, unlike ICOS, CD81 is also expressed on APC such as B cells and DC.
The current study demonstrates that CD81 is crucial in the initiation of Th2 immune responses in vitro in unprimed T cells. First, an anti-CD81 mAb, abrogated IL-4 secretion by T cells when the T cells were activated by antigen-specific B cells (Fig. 1C). Second, CD81 expression by T cells (Fig. 4) but not B cells (Fig. 2) played an important role in cognate TB cell interaction leading to Th2 responses. Lack of CD81 expression by T cells led to reduced Th2 cytokine production by OVA-primed (data not shown) and by OVA TCR Tg T cells (Fig. 4) when they were stimulated with antigen-specific B cells. Finally, lack of CD81 on T cells was associated with reduced cell surface expression of ICOS (Fig. 5), reduced levels of intracellular GATA3 and STAT6, and reduced STAT6 phosphorylation (Fig. 6) in the activated T cells.
Phosphorylation of the transcription factor STAT6 and up-regulation of GATA3 expression are critical events in Th2 cell differentiation and cytokine production (42). STAT6-deficient mice show defective Th2 responses, a phenotype similar to IL-4-deficient mice. Moreover, STAT6 activation per se is sufficient for Th2 differentiation because T cells cultured in Th1-polarizing conditions increase IL-4, IL-5 and IL-13 expression, and reduce IFN-
production upon direct induction of STAT6 phosphorylation (43). Activation of STAT6 induces expression of GATA3 and C-Maf, transcription factors expressed only in Th2 differentiated T cells. However, only GATA3 is necessary and sufficient for Th2 differentiation and cytokine production (4446). It is likely that CD81 on T cells acts upstream of STAT6 and GATA3, and provides important co-stimulatory signals that enhance STAT6 phosphorylation and GATA3 expression (Fig. 6). Interestingly, GATA3 expression is considerably reduced but not completely obliterated in the absence of CD81, indicating that phosphorylated STAT6 may be produced in the cells at very low levels or that GATA3 may be induced by additional mechanisms.
It remains to be determined how CD81 on T cells affects their interactions with B cells and how it facilitates intracellular signaling events that lead to Th2 differentiation. CD81 is a tetraspanin molecule; like other members of this family of proteins, it has four transmembrane domains, two extracellular loops and two short intracellular tails. The transmembrane domains and cytoplasmic tails are highly conserved amongst CD81 expressed by different vertebrate species, whereas diversity is found within the large extracellular loop (LEL), located between the third and forth transmembrane regions (47). The anti-CD81 mAb Eat1, which reacts with the LEL [but not Eat2, which reacts with a combinatorial epitope on CD81 (28)] has a striking effect in the in vitro system (Fig. 1A), inhibiting IL-4 production induced in T cells by cognate TB cell interaction (Fig. 1C). Yet, we have previously demonstrated that Eat2 can induce signaling events upon binding to lymphoid cells and promote cell aggregation (28). We believe that the effect of Eat1 in this system is not related to the initiation of a signaling cascade, but to an inhibition of cellcell interaction. Eat1, but not Eat2, was also shown to inhibit cellular interactions in the nervous system, between astrocytes and neurons (48). How CD81 affects cellular interactions is unclear, since the intracellular domains of CD81 are very short and do not contain known phosphorylation motifs. However, the molecule has been shown to associate with a multitude of cellular proteins, and it is likely that CD81 acts by facilitating interactions of its associated molecules and that one or more of these associated molecules actually transduces the signal.
On T cells, CD81 has been shown to associate with the cytoplasmic region of CD4, but not with CD4 that is p56lck bound (33). It has also been shown that activation of p56lck kinase is required for Th2 differentiation (49) and that the engagement of CD4 enhances its phosphorylation (50). It is possible that CD81 on T cells facilitates the interaction of the CD4/TCRpeptide with the MHC class II complex, thereby enhancing activation via p56lck and promoting Th2 differentiation. CD81 also associates with numerous integrins,
3ß1,
4ß1and
6ß1 (34,35,51,52). Recent evidence suggests that CD81 and other tetraspanin molecules may serve as linkers between the extracellular
chain domains of integrins and intracellular signaling molecules such as phosphatidylinositol-4 kinase and protein kinase C (5355). Indeed, we recently demonstrated that CD81 plays a role in LFA-1-mediated adhesion leading to TB cell collaboration in human (23). Thus, CD81 may facilitate intracellular signaling through its associated adhesion molecules.
Increasing attention has been drawn to the function of lipid rafts, detergent-insoluble glycolipid-enriched membrane complexes and their role in immune synapses. Engagement of T cell co-stimulatory molecules appears essential for the recruitment of lipid rafts to the TCR contact site, and for the construction of a platform that facilitates TCR engagement and sustained signal transduction events (56,57). Indeed, several key signaling molecules that act downstream of TCR, including Lck, Fyn, LAT and the GPI-linked CD48 molecule, preferentially partition within lipid rafts (5860). Furthermore, perturbation of the structural integrity of lipid rafts inhibits TCR-induced protein tyrosine phosphorylation and calcium flux (61,62). It has been reported recently that CD81 and several other tetraspanins along with their associated integrins are present in raft-like lipid microdomains (51). Disruption of CD81 on T cells may distort the integrity of lipid rafts, and further inhibit TCR signaling and cytokine production.
Because the type of APC may affect cognate interaction with T cells we tested antigen presentation by non-B cells. Interestingly, lack of CD81 on T cells did not result in biased cytokine production in response to antigen presented by SpAC (Fig. 7C and D). This result is consistent with our previous observation that anti-CD81 mAb enhances IL-4 synthesis in human CD4+ T cells only when CD4+ T cells were cultured with B cells but not when they were cultured with monocytes as APC (63). It has been shown that the type of APC is critical for Th precursor cell polarization. When B cells present cognate antigen they induce Th2 cytokine production, while SpAC preferentially induce Th1 cytokine production (37). It is likely that the different APC provide distinct co-stimulatory signals to the interacting T cells. For example, the ICOS ligand B7RP-1 is expressed mainly on B cells and monocytes, but not on the majority of dendritic cells (11). In turn, activation of ICOS has profound effects on TB cell collaboration and Th2 responses (1618). From our results it is apparent that CD81 is another molecule that is involved in antigen-specific interactions between T and B cells, but not between T cells and other APC. CD81 probably acts upstream of ICOS because ICOS expression on CD81/ T cells is reduced when they are activated with antigen-specific B cells (Fig. 5).
We previously showed that JHD/ mice reconstituted with CD81/ ES cells were deficient in Th2 immune responses (20), suggesting that expression of CD81 on B cell was required for Th2 responses in vivo. The current study demonstrates that expression of CD81 on B cells is in fact not absolutely required for efficient antigen presentation leading to Th2 cytokine production by T cells and that CD81/ B cells, under some circumstances, can effectively present antigen. These results therefore indicate that the deficient Th2 responses seen in the chimeric JHD/ mice was not due to the failure of antigen presentation by CD81/ B cells, but most likely was due to ineffective B cell activation. B cells normally express CD81 constitutively, unlike T cells that express the molecule only upon activation (Fig. 5). In B cells, CD81 is a component of a molecular complex that includes the B cell-specific CD19/CD21 proteins, which has been shown to play a critical role in B cell activation (29,30). Thus, in the in vivo experiments with chimeric mice, we believe that the use of non-cognate antigen in the context of a defective CD19low/CD21/CD81/ complex resulted in impaired activation of CD81/ polyclonal B cells and subsequently in impaired Th2 cytokine production.
In contrast to our previous in vivo study with chimeric mice and non-cognate antigen, the current study utilized cognate antigen and BCR Tg CD81/ B cells that circumvented the important role of CD81 in B cell activation. Under these circumstances, CD81/ B cells presented antigen to T cells and induced Th2 cytokine synthesis. Indeed, studies aimed at analyzing antigen presentation by BCR Tg B cells to high-affinity TCR Tg T cells have shown that enhanced activation and division of both B and T cells occurred in the absence of BCR-mediated antigen internalization (64). By using BCR-mediated antigen uptake and cognate antigen, we showed that B cell activation could develop without the need for the CD19/CD21/CD81 complex, and high-affinity interactions between antigen-presenting BCR Tg B cells and TCR Tg T cells occurred. Thus, in these in vitro experiments, BCR Tg CD81/ B cells primed T cells and induced normal IL-4 production in T cells (Fig. 2).
In summary, our current experiments analyze and focus on the role of CD81 on T cells in the induction of Th2 cytokines. We previously showed that CD81 expression by B cells critically affected B cell activation and subsequent Th2 cytokine synthesis, and we now show that CD81 expression by T cells significantly affects cognate TB cell interaction leading to Th2 immune responses. CD81 may enhance TB cell contact through its associated molecules, and magnify the intracellular signaling leading to STAT6 phosphorylation and GATA3 expression. Thus, CD81 enhances Th2 cytokine production both by directly affecting B cell activation and by directly affecting Th2 cell differentiation. These studies therefore demonstrate the important dual molecular role of CD81 in affecting cognate TB cell interaction and Th2 responses.
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Acknowledgements
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This work was supported by the National Institute of Health Grant AI45900. J. D. was supported by Fellowships from the National Institute of Health Training Grant 5 T32 AI07290-15 Molecular and Cellular Immunobiology and by the American Lung Association of California. We thank Tsipi Shoham and Ron Levy for reviewing the manuscript, and David Walter for his help with the cytokine assays.
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Abbreviations
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AHRairway hyper-reactivity
APCantigen-presenting cell
HELhen egg lysozyme
HRPhorseradish peroxidase
LELlarge extracellular loop of CD81
PEphycoerythrin
SpACsplenic adherent cells
Tgtransgenic
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