Dendritic cell membrane CD83 enhances immune responses by ...

Article

Dendritic cell membrane CD83 enhances immune responses by boosting intracellular calcium release in T lymphocytes Mariana Pereira Pinho, Isabella Katz Migliori, Elizabeth Alexandra Flatow, and José Alexandre M. Barbuto1 Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, Brazil RECEIVED APRIL 30, 2013; REVISED DECEMBER 3, 2013; ACCEPTED DECEMBER 27, 2013. DOI: 10.1189/jlb.0413239

ABSTRACT CD83 is a marker of mDCs directly related to their lymphostimulatory ability. Some data suggest that it has a central role in the immune system regulation, but how this function is performed remains to be determined. This work aimed to analyze the influence of CD83, present in mDCs, in the modulation of calcium signaling in T lymphocytes. Mo were differentiated into iDCs and activated with TNF-␣. iDCs were treated, 4 h before activation, with siRNACD83, to reduce CD83 expression. Purified allogeneic T lymphocytes were labeled with the calcium indicator Fluo-4-AM, and calcium mobilization in the presence of mDCs was analyzed. CD83 knockdown mDCs induced lower calcium signal amplitude in T lymphocytes (29.0⫾10.0) compared with siRNAscrtreated mDCs (45.5⫾5.3). In another set of experiments, surface mDC CD83 was blocked with a specific mAb, and again, decreased calcium signaling in T lymphocytes was detected by flow cytometry and microscopy (fluorescence and confocal). In the presence of antibody, the percentage of responding T cells was reduced from 58.14% to 34.29%. As expected, anti-CD83 antibodies also reduced the proliferation of T lymphocytes (as assessed by CFSE dilution). Finally, in the absence of extracellular calcium, CD83 antibodies abrogated T cell signaling induced by allogeneic mDCs, suggesting that the presence of CD83 in mDC membranes enhances T lymphocyte proliferation by boosting calcium release from intracellular stores in these cells. J. Leukoc. Biol. 95: 755–762; 2014.

Introduction DCs are professional APCs responsible for activating and modulating the adaptive immune response [1]. In the absence of

Abbreviations: CD83Ab⫽specific antibody for CD83, FSC⫽forward-scatter, iDC⫽immature DC, iMAX⫽Lipofectamine RNAiMAX Transfection Reagent, InsP3⫽inositol 1,4,5 trisphosphate, LAT⫽linker for activation of T cells, mDC⫽mature DC, MFI⫽median fluorescence intensity, Mo⫽monocytes, NFAT⫽NF of activated T cell, RFI⫽relative fluorescence intensity, siRNA⫽small interfering RNA, siRNACD83⫽CD83 small interfering RNA, siRNAscr⫽scrambled small interfering RNA, SSC⫽side-scatter

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pathogenic stimuli, iDCs induce anergy of T cells [2, 3]. Inflammatory and pathogenic stimuli induce maturation of DCs, which acquire the ability to stimulate the differentiation and proliferation of naive T cells [4, 5]. During the maturation process of DCs, many molecules, such as CD83, increase their expression [6, 7]. CD83 is a transmembrane protein, mainly known as a marker of mDCs [8], although it is also expressed in activated B [9] and T cells [10], neutrophils [11], thymic epithelial cells [12], and tumor cells [13]. CD83 also has a soluble isoform that can be detected in the serum [14]. Although there is evidence for the existence of a CD83 ligand, at the molecular level, it has never been described [15, 16]. The presence of membrane CD83 in DCs is correlated with an increased stimulation of T cells [17–19], whereas soluble CD83 impairs T cell activation induced by DCs [16, 20, 21]. Although the transmembrane region of CD83 reduces the degradation of CD86 and MHC class II molecules in DCs [22], the exact correlation of these phenomena to the modulation of T cell proliferation by CD83 remains largely undetermined. On the other hand, it is well established that T cell activation depends on calcium signaling [23], the name given to the rapid intracellular increase of free calcium concentration with a determined temporal pattern [24]. Increases in intracellular calcium activate calcineurin [25], which in turn, leads to the activation of NFAT, a transcriptional factor that induces IL-2 expression and drives T lymphocyte proliferation [26, 27]. In T cells, this signaling is caused by the engagement of the TCR, which activates PLC␥, which in turn, generates InsP3, that binds to its receptor in the ER membrane, leading to release of its calcium stores. Release of internal calcium stores generates a sustained calcium influx through calcium release-activated channels [28]. Variations in amplitude, duration, and frequency of calcium signaling affect significantly the pattern of T cell responses [29, 30].

1. Correspondence: Dept. of Immunology, Institute of Biomedical Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 1730 - CEP 05508-000, São Paulo, SP, Brazil. E-mail: [email protected]

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In this paper, we investigated the effects of CD83 expression/modulation by DCs upon the calcium signaling that occurs in T cells stimulated by the former. We measured the intracellular calcium concentration of T cells during immunological synapses with DCs, which had different availabilities of its membrane CD83, blocked either by a specific antibody or knocked down by siRNA treatment.

MATERIALS AND METHODS

PBMC isolation and DC generation After written consent from platelet donors, leukoreduction chambers of apheresis, performed in the Blood Bank from Hospital Oswaldo Cruz (São Paulo, SP, Brazil), were collected. The Institutional Ethics Committee of the Institute of Biomedical Sciences approved the protocol. PBMCs from those chambers were separated by centrifugation over Ficoll-Paque (GE Healthcare, Uppsala, Sweden). PBMCs were resuspended in AIM V medium (Gibco, Grand Island, NY, USA), seeded in 75 cm2 cell-culture flasks, and incubated overnight at 37°C and 5% CO2. After overnight incubation, nonadherent cells were removed, the medium was replaced, and GM-CSF (50 ng/ml; PeproTech, Rocky Hill, NJ, USA) and IL-4 (50 ng/ml; PeproTech) were added. After 5 days, the cells received a maturation stimulus with TNF-␣ (50 ng/ml; PeproTech). mDCs were obtained 48 h after activation. For phenotypic characterization of the cells during the culture, Mo were harvested after nonadherent cell removal at Day 0, and iDCs were harvest before the maturation stimulus, at Day 5.

Determination of the membrane phenotype of DCs Membrane phenotype of the cells during differentiation and maturation of DCs was determined by flow cytometry. For each condition, 2 ⫻ 105 cells were labeled with fluorescence-labeled antibodies specific for the different membrane molecules (HLA-DR, CD14, CD83, CD80, CD86, CD11c; BD Biosciences, San Jose, CA, USA) and analyzed in a FACSCalibur cytometer (BD Biosciences) using the FlowJo software (Version 7.2.4; Tree Star, Ashland, OR, USA). Dead cells were excluded from the analysis by using the LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA). The RFI of the surface markers was calculated by dividing the MFI of the labeled group by the MFI of the unlabeled group.

Transfection of iDCs with siRNA For each group, 5 ␮l iMAX (Invitrogen) was diluted in 95 ␮l Opti-MEM media (Invitrogen) and 1.5 ␮l of a 50-mM siRNA solution was diluted in 98.5 ␮l Opti-MEM. Then, both solutions were mixed, incubated at room temperature for 30 min, and placed in a six-well plate. iDCs were harvest and resuspended in AIM V medium, supplemented with IL-4 and GM-CSF. For each treatment, 1 ⫻ 106 cells (which were resuspended in 1.3 mL medium) were seeded in a six-well plate, pretreated with 200 ␮l siRNA complex. Cells were activated with TNF-␣, 4 h after transfection and analyzed after 48 h.

CD83 blocking To block membrane CD83 in mDCs, 100 ng CD83 mAb (HB15e clone; BD Biosciences) were added to 5 ⫻ 104 cells. After 20 min of incubation at 4°C, DCs were washed twice to remove antibody excess. IgG antibodies were used as control.

mDC staining mDCs were harvest and resuspended at a concentration of 1 ⫻ 106 cells/mL in PBS, supplemented with 0.5% of BSA and 5 mM CellTracker Red CMPTX (Invitrogen). The cells were incubated at 37°C for 15 min, washed twice, and resuspended in the calcium assay buffer (composed of 1 mM CaCl2, 130 mM NaCl, 4.6 mM KCl, 5 mM glucose, and 20 mM

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HEPES) or a KEGTA solution (composed of 10 mM NaCl, 130 mM KCl, 20 mM HEPES, 10 mM EGTA, and 10 mM Na2CO3; Sigma, St. Louis, MO, USA).

Fluo-4-AM loading T cells were isolated from nonadherent PBMCs by negative selection with magnetic beads from the Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. After purification, T cells were resuspended at 1 ⫻ 107 cells/mL in PBS, supplemented with 0.5% of BSA, incubated with 1␮M Fluo-4-AM (Invitrogen) for 30 min at 28°C, washed twice, and resuspended in the calcium assay buffer at 1 ⫻ 106 cells/mL.

Calcium mobilization assay by flow cytometry Fluorescence of T cells stained with Fluo-4-AM was acquired by flow cytometry for 60 s without stimulus. Then, allogeneic DCs stained with CellTracker Red CMPTX were added, and the cells were acquired for an additional 240 s. Ionomycin (Invitrogen) was used as a positive control. Using the FlowJo software (Version 7.2.4; Tree Star), changes in the median of Fluo-4-AM fluorescence in the T cells were calculated.

Calcium mobilization assay by fluorescence and confocal microscopy DCs stained with CellTracker Red CMPTX (Invitrogen) were seeded in a Petri dish, and allogeneic T cells were added while acquiring the video in the fluorescence microscope Nikon Eclipse Ti-S (Nikon, Melville, NY, USA) with NIS-Elements AR software (Nikon) or in the multifoton Zeiss LSM 780 microscope (Zeiss, Oberkochen, Germany) with ZEN confocal software (Zeiss). Images were acquired for at least 10 min.

T cell proliferation assay Purified T cells were labeled with 5 ␮M CFSE (Invitrogen) and cultivated in culture in a 96-well U-bottom plate, with allogeneic mDCs at a 10:1 lymphocyte:DC ratio. After 5 days, the cells were harvest, stained with anti-CD3 antibody (BD Biosciences), and analyzed by flow cytometry. T cell proliferation was assessed by CFSE dilution.

Statistical analysis Statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). The effect of anti-CD83 antibody was analyzed by paired t-test (*P⬍0.05; **P⬍0.01). Comparisons among results obtained from the status of DC differentiation and from CD83 knockdown experiments were performed by one-way ANOVA with the Tukey post-test (*P⬍0.05; **P⬍0.01). All graphs show mean ⫾ sem.

RESULTS

The differentiation of Mo into DCs leads to changes in expression of several surface markers and higher frequency of CD83-positive cells We differentiated Mo from peripheral blood into iDCs by culture for 5 days with GM-CSF and IL-4 and generated mDCs by activating iDCs with TNF-␣ for 48 h. As assessed by flow cytometry, the percentage of positive cells for HLA-DR, one of the human MHC class II molecules, remained above 97% during all of the stages of the culture (Fig. 1A). As expected, the cellular positivity of the Mo marker CD14 decreased after the differentiation and remained low after maturation (Fig. 1A). Mo, obtained from leukoreduction chambers, had very high levels of CD83 expression, both in percentage of positive cells (45.8⫾18.1) and in RFI (20.9⫾5.3; Fig. 1A and B). The per-

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Pinho et al. Dendritic cell CD83 and T cell calcium signaling

Figure 1. Analysis of cellular phenotype during the differentiation of Mo into DCs. Mo, isolated from peripheral blood of a healthy donor, were differentiated into DCs by culture for 7 days in the presence of GM-CSF, IL-4, and activated in the last 2 days with TNF-␣. The frequency of positive cells and the RFI of surface markers in the cells at Days 0 (Mo), 5 (iDC), and 7 (mDC) were measured by flow cytometry. (A) Percentage of positive cells for HLA-DR, CD83, and CD14 during the culture (Mo, n⫽2; iDC, n⫽3; mDC, n⫽3). (B) RFI of CD83 (n⫽3). (C) Percentage of positive cells and RFI of characteristic mDC markers (n⫽6).

centage of CD83-positive cells tended to increase during the differentiation and maturation culture (iDC: 55.4⫾13.3; mDC: 67.7⫾6.6), whereas the RFI decreased from Mo to iDC and increased after the activation stimuli (iDC: 10.2⫾0.4; mDC: 16.3⫾2.4; Fig. 1A and B). The majority of the mDCs was positive for the costimulatory molecules CD80 and CD86 (Fig. 1C).

DC transfection with a siRNACD83 specifically down-regulates CD83 expression DCs were transfected with iMAX and a labeled siRNA, which showed a transfection efficiency of 52.65% (Fig. 2A). To diminish CD83 expression, cells were treated with a specific siRNACD83 or as a control, with a siRNAscr. The viability of

Figure 2. Analysis of efficacy and effect of DC transfection with siRNA. iDCs were transfected with the lipofection agent iMAX complexed with siRNAs with different specificity, activated after 4 h and evaluated after 48 h. (A) Histograms of iMAX-treated mDCs (filled) and (iMAX ⫹ Cy3 labeled GAPDH siRNA)-treated mDCs (unfilled). (B) Viability of the mDCs, recovered after 48 h of treatment, compared with mDCs, treated only with the vehicle (n⫽3; *P⬍0.05; **P⬍0.01). (C) CD83 expression in mDCs treated with iMAX or only with the vehicle (OptiMEM). (D) Histograms of CD83, CD80, CD86, and HLA-DR expression in mDCs treated with just iMAX or with iMAX and specific (siRNACD83) or control siRNA (siRNAscr). FL2/3/4-H, Fluorescence 2/3/4-height.

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Figure 3. CD83 expression significantly decreases using lipofection with specific siRNA compared with the control siRNA. Knockdown of CD83 was made by lipofection of iDCs with iMAX and a CD83-specific siRNA (siRNACD83) or a siRNA without known specificity (siRNAscr). iDCs were activated 4 h after the transfection. Flow cytometry analysis of mDCs, 48 h after transfection, showing (A) frequency of positive cells and (B) RFI of CD83 in mDCs treated with only iMAX or with iMAX and siRNACD83 or siRNAscr (n⫽5; *P⬍0.05).

iMAX-treated mDCs decreased significantly after 48 h of treatment (Fig. 2B). So, to analyze just viable cells, we excluded dead cells from analysis using a viability marker. DCs treated with iMAX had increased CD83 expression (Fig. 2C). CD80 and CD86 were also up-regulated in the presence of iMAX (data not shown). However, the treatment with iMAX ⫹ siRNAscr or iMAX ⫹ siRNACD83 did not change CD80, CD86, and HLA-DR expression compared with the group treated only with iMAX (Fig. 2D). In the iMAX ⫹ siRNACD83-treated group, we observed two subpopulations regarding CD83 expression: one with the same expression of the iMAX ⫹ siRNAscr group but another one with diminished CD83 expression (Fig. 2D). The percentage of CD83-positive cells was reduced significantly from 84.8 ⫾ 5.8 in the iMAX ⫹ siRNAscr group to 68.2 ⫾ 10.6 in the iMAX ⫹ siRNACD83 group (n⫽5; P⬍0.05; Fig. 3A). The RFI of CD83 also seemed to be reduced by the siRNACD83, but the difference did not reach statistical significance (Fig. 3B).

DCs with diminished CD83 expression induced lower calcium signal amplitude in T cells Fluorescence of Fluo-4-AM-labeled T cells was acquired in the flow cytometer for 60 s prior to addition of stimulus. The cells

were stimulated by allogeneic Red-stained mDCs, and the fluorescence was acquired for an additional 240 s. To analyze only the Fluo-4-AM-labeled T cells, we made two consecutive gates: the first, which gated the population with T cell-characteristic FSC and SSC parameters, excluded DCs; the second, which gated the cells that were negative for the Red dye, excluded possible contaminating T cells from the mDC culture (Fig. 4A). Figure 4B shows a representative graph of Fluo-4-AM median fluorescence time variation. The calcium-signaling index of T cells, stimulated by iMAX ⫹ siRNACD83-treated mDCs (29.0⫾10.0), was lower than the calcium-signaling index of T cells stimulated by mDCs treated with iMAX ⫹ siRNAscr (45.5⫾5.3) or mDCs treated only with iMAX (47.1⫾8.4; n⫽5; Fig. 4C).

The blocking of membrane CD83 in DCs by antibodies also reduced the calcium signaling observed in T cells We used another approach to evaluate the role of DC CD83 in calcium signaling in T cells: the blocking of membrane CD83 with specific antibodies. Again, calcium signaling in T cells activated by CD83-modulated DCs achieved lower values (Fig. 5A). CD83Ab-treated mDCs induced in T cells a calcium-signaling

Figure 4. Effect of diminished CD83 expression in mDCs in the calcium signaling of allogeneic T cells. Allogeneic mDCs, labeled with a Red dye, were added to Fluo-4-AM-labeled T cells after they were acquired for 60 s in the flow cytometer. (A) DCs were excluded from the analysis by gating in the population with T cell-characteristic FSC and SSC parameters (left). T cells from the mDC culture were also excluded by gating only the cells that were negative to the Red dye (middle). Then, the changes of Fluo-4-AM fluorescence during the acquired time were analyzed only in the allogeneic T cells (right). (B) A representative graphic illustrating the fluctuation of the median of Fluo4-AM fluorescence shows the increase of MFI after the addiction of mDCs (arrow). (C) Graphic showing calcium-signaling index (which is calculated using the maximum value of the Fluo-4AM MFI before and after the stimuli) of T cells stimulated with iMAX-treated mDCs (n⫽5).


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Figure 5. Diminished calcium-signaling amplitude of T cells stimulated by DCs treated with CD83 antibodies. (A) Representative histograms, obtained by flow cytometry, of Fluo-4-AM MFI of T cells stimulated with mDCs, treated or not with CD83Ab. (B) Graphic showing the calcium-signaling index of T cells that were calculated by subtracting the maximum fluorescence before and after the stimuli with mDCs or ionomycin as a positive control (ionomycin, n⫽2; mDC, n⫽8; mDC⫹IgG, n⫽3; mDC⫹CD83Ab, n⫽8; *P⬍0.05). (C) Flow cytometry histograms showing the calcium signaling in the absence of extracellular calcium.

index of 76.0 ⫾ 17.7 (n⫽8) compared with 144.2 ⫾ 31.5 induced by mDCs treated with IgG (n⫽3) and 112.6 ⫾ 17.8 from mDCs alone (n⫽8; P⬍0.05; Fig. 5B). Calcium signaling in T cells begins with the liberation of calcium from internal stores, which leads to a calcium influx through calcium-released activated channels. To investigate in which step CD83 could be modulating the calcium signaling, T cells were stimulated by mDCs in the absence of extracellular calcium. An increase in fluorescence was still observed when T cells were stimulated by allogeneic mDCs, although with less intensity, but the CD83Ab abolished the measurable response completely (Fig. 5C). To confirm the results obtained by flow cytometry and to evaluate the calcium signaling in single cells, we also evaluated the effect of the CD83Ab by fluorescence microscopy. The maximum fluorescence of T lymphocytes stimulated by CD83Ab-treated mDCs was significantly lower (58.2⫾3.6) than

that of the control group (80.5⫾4.3; P⬍0.0001; Fig. 6A). Next, we used confocal microscopy, a more accurate method, to analyze the effect of CD83Ab further. First, we analyzed the percentage of responding T cells—those that increased at least 15% of its fluorescence after contact with mDCs (Fig. 6B)— and found that it was lower in the presence of antibody (34.3 vs. 58.1% in the control group). Also, the calcium signal amplitude of responding cells was affected by the CD83Ab, which decreased it (mDC: 15.7⫾1.5; mDC⫹CD83Ab: 11.5⫾1.3; P⬍0.05; Fig. 6C).

CD83 antibodies reduced the proliferation of T cells in the presence of mDCs Allogeneic T cells were separated, labeled with CFSE, and cocultured with mDCs for 5 days in the presence or absence of CD83Ab. Proliferation was evaluated by CFSE dilution within CD3-positive cells. The presence of CD83Ab reduced the per-

Figure 6. Fluorescence and confocal analysis of calcium signaling of T cell stimulated by mDCs treated with CD83 antibodies. (A) Analysis by fluorescence microscopy of the maximum fluorescence achieved by T cells that were in contact with mDCs and had a typical profile of fluorescence variation (mDC, n⫽41; mDC⫹CD83Ab, n⫽47). Allogeneic T cells labeled with Fluo-4-AM (green) were incubated with mDCs (red) and were analyzed for calcium signaling by confocal microscopy. (B) A frame of the video (left) showing T cells that were considered in contact with mDCs (arrows) and a sequence of frames of a T cell that had changed its fluorescence after contact with mDCs (right). An increase of at least 15% of fluorescence after contact with DCs was considered as a response. (C) Calciumsignaling index of responding T cells in contact with mDCs, treated or not with CD83 antibodies (mDC, n⫽25; mDC⫹CD83Ab, n⫽24).

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Figure 7. Proliferation analysis of T cells stimulated by mDCs, treated or not with CD83 antibodies. Allogeneic mDCs, with or without CD83 blockage, were cocultured for 5 days with CFSE-labeled T cells. CFSE dilution in T cells was analyzed by flow cytometry. (A) Representative histograms showing T cells with CFSE dilution after coculture with mDC, treated (darker) or not with CD83 antibodies. (B) Bar graph showing the percentage of T cells with CFSE dilution after coculture with mDCs alone (n⫽6), mDCs treated with IgG (n⫽3), or mDCs treated with CD83 antibodies (n⫽6; *P⬍0.05).

centage of T cells with CFSE dilution but did not change the MFI of dividing cells (Fig. 7A). The percentage of T lymphocytes that proliferated significantly decreased from 29.3 ⫾ 4.9 in the mDC group to 21.0 ⫾ 3.3 in the CD83Ab group (Fig. 7B).

DISCUSSION We showed here, for the first time, that CD83, present on mDCs, acts by giving a costimulatory signal that enhances the calcium signaling and consequently, the proliferation of interacting T cells. Furthermore, CD83 seems to increase the percentage of responding cells and enhance the calcium-signaling amplitude of those cells. The first approach to evaluate the CD83 effect in the calcium signaling of T cells was to reduce CD83 expression in mDCs by treating them with a specific siRNA. In our model, however, iDCs already expressed high levels of CD83, and although it was able to knock down the surface levels of CD83 significantly, the decrease of expression was low. This is in agreement with Prechtel et al. [19], who were able to knock down CD83 efficiently from human mDCs but pointed out that CD83 is a stable protein in mDC membrane and that it would be difficult to decrease its expression if they started with a DC that already expressed CD83. Moreover, only 52.65% of iDCs incorporated a labeled siRNA, and treatment with iMAX induced iDC maturation in all experiments. In accordance with the low decrease in surface CD83, the siRNA treatment did not lead to a significant difference in the calcium signaling of T cells. Nevertheless, despite not reaching statistical significance, siRNACD83-treated mDCs induced in T cells lower calcium-signaling indexes in all experiments. As siRNA treatment was a harmful treatment and unable to decrease CD83 expression to low levels, we decided to use another approach: blocking CD83 with specific antibodies. DCs, treated with CD83Ab, did induce in T cells a significant, lower calcium-signaling index. However, it was not clear if this effect 760 Journal of Leukocyte Biology

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was a result of a smaller number of responding cells or of lower calcium signaling of each responding cell. To address this question, T cells were analyzed by fluorescence and confocal microscopy, where we could evaluate the calcium signaling of each individual cell. T cells stimulated with mDCs, treated with CD83Ab, had a significantly reduced calcium-signaling amplitude. In addition, among T cells that were in contact with mDCs, the percentage of responding cells was lower in the presence of CD83Ab. These results show that the calciumsignaling reduction observed by flow cytometry reflects, actually, two different phenomena: a smaller percentage of responding cells and a lower calcium signaling of each responding cell. It was described recently that the transmembrane region of CD83 can increase the stability of CD86 and MHC class II molecules in DCs [22]. If this were the mechanism by which CD83 enhanced calcium signaling in T cells, then we would expect the knockdown of CD83 by siRNA to change HLA-DR and CD86 expression. However, this is not what we observed. Furthermore, the blockage of the extracellular region of CD83 with antibodies, which should not affect the interaction of its transmembrane region with other molecules, also diminished and significantly, T cell calcium signaling. Thus, one could suggest that CD83 may act directly on T cells and not through other known costimulatory molecules. Furthermore, our data indicate that CD83 interferes with the first step of calcium signaling in T cells, that is, the liberation of calcium from internal stores. If CD83 interfered with calcium influx (from an extracellular source), one could expect that no alteration would be observed in the absence of extracellular calcium when CD83 is blocked. However, blockage of surface CD83 in the absence of extracellular calcium abrogated the signaling measured in T cells. This shows that at least the initial effects of CD83 blockage do not depend on external calcium. However, when extracellular calcium was present, a diminished but significant calcium influx was detected, even in the presence of CD83Ab. This could seem contradictory, as the amplification signal (from external calcium sources) depends on the initial release of calcium from internal stores, which we suggested as the target of CD83. However, we cannot assure that in our conditions, blocking was complete. Thus, a very weak signal could still occur, go undetected by our assay, but still be enough to be amplified by the calcium influx, generating the weak signal detected in the presence of extracellular calcium. On the other hand, IL-2, which is one of the most important signals to induce T cell proliferation [24], is strongly reduced in cocultures of T cells and CD83-knockdown mDCs [19]. The expression of IL-2 involves NFAT, which is a calciumsignaling-dependent transcription factor [23]. As CD83 modulates calcium signaling in T cells, its proliferation could be affected by the presence of CD83. In agreement with this, we showed, as described previously [17–19], that CD83 increases T cell proliferation induced by allogeneic mDCs. Interestingly, although the percentage of proliferating T cells decreased when T cells were stimulated by CD83Abtreated mDCs, the cells that did proliferate seemed to do so at

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the same rate as those stimulated by nontreated DCs. These data suggest that CD83 blockage decreases the amount of dividing cells but does not change the number of divisions of stimulated T cells. This is in agreement with the report by Schwarz et al. [31], who found that little calcium entry was sufficient to induce maximum T cell proliferation. So, the lower calcium signaling of each responding cell, observed when CD83 was blocked, seems not to be important to induce difference in the proliferation. This indicates that for T cell proliferation, the most important effect on the calcium signaling induced by CD83 would be the diminished percentage of responding T cells. CD83 is an important modulator of the immune system and may become an important target for immunotherapy. Studies with a recombinant, soluble form of CD83, for example, showed the potential of this molecule to treat autoimmune diseases [32] and to prevent allograft rejection [33]. The understanding of the mechanism of action of CD83 is an important step for the design of better modulatory drugs. As the membrane region of CD83 stabilizes MHC class II molecules and CD86 [22], we can infer that these molecules interact and colocalize in the immunologic synapses. If this is so, CD83 could act by facilitating the interaction between molecules involved in the signal transduction, thus enhancing the signal transduction. This hypothesis could explain, for example, the opposite roles of the membrane and soluble form of CD83. If the membrane CD83 approaches molecules in the immunologic synapses, then the soluble CD83 will bind to the CD83 ligand, preventing its focalization. In this context, a candidate molecule for the interaction with CD83 would be the LAT, which is a transmembrane adaptor protein, present in T cells, required for TCR-mediated activation of PLC␥, the enzyme responsible for triggering the calcium signaling in T cells [34]. LAT appears to be constitutively in lipid rafts [35], whereas TCR is recruited to lipid rafts after activation [36]. Before activation, LAT and TCR are in separate membrane domains, but they colocalize after T cell activation [37], although localization of LAT on lipid rafts is not essential for T cell activation [38]. This raises the question of how LAT and TCR colocalize after T cell activation. Recently, Williamson et al. [39] showed that when T cells were activated, LAT domains were not transported to the activation site: in their model, LAT was recruited from intracellular vesicles, which triggered the signaling. However, they activated T cells with anti-CD3- and anti-CD28coated surfaces—signals that could be insufficient to trigger the approximation of LAT and TCR— but this does not imply that it could not occur in mDC-stimulated T cells. Therefore, the idea that CD83 could interact with LAT seems plausible, is consistent with the data presented here, and therefore, deserves investigation. In conclusion, we show here that different strategies for decreasing membrane CD83 availability on DCs affect calcium signaling in T cells significantly. Furthermore, our data support the hypothesis that this effect is mainly a result of the blocking of the initial calcium release from intracellular stores and might be conveyed by the T cell surface molecule LAT.

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AUTHORSHIP M.P.P. performed the research, collected and interpreted data, and wrote the manuscript. I.K.M. helped perform some experiments and analyzed some results. E.A.F. helped perform some experiments. J.A.M.B. designed the research, discussed the results, and assisted with manuscript preparation.

ACKNOWLEDGMENTS This study was supported by grants (#09/54599-5 and #11/ 01082-5) from the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and from Conselho Nacional de Desenvolvimento Científico e Tecnológico. We thank the Blood Bank of the Hospital Oswaldo Cruz (São Paulo, Brazil) for providing the leukoreduction chambers and the Institute of Biomedical Sciences Research Facilities Center (CEFAP-USP) for helping in the use of the confocal microscope.

DISCLOSURES

The authors declare no conflict of interest.

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KEY WORDS: signaling 䡠 activation 䡠 signal transduction

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