Human T lymphoblasts and activated dendritic cells in the allogeneic mixed leukocyte reaction are susceptible to NK cell-mediated anti-CD83-dependent cytotoxicity
David J. Munster1,
Kelli P. A. MacDonald1,2,
Masato Kato1 and
Derek J. N. Hart1
1 Dendritic Cell Laboratory, Mater Medical Research Institute, Brisbane, Queensland 4101, Australia 2 Present address: Immunoregulation Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Royal Brisbane Hospital, Queensland 4029, Australia
Correspondence to: D. N. J. Hart; E-mail: dhart{at}mmri.mater.org.au
Transmitting editor: D. Tarlinton
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Abstract
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CD83 is a marker of dendritic cell (DC) differentiation/activation and its expression in the mouse thymus contributes to CD4+ T lymphocyte development. Its extrathymic role remains unclear despite the functional effects observed with CD83 fusion proteins or CD83 antibody and recent reports of potential ligands. We investigated the previously observed and presumed functional blockade of the allogeneic mixed leukocyte reaction (MLR) with rabbit polyclonal anti-CD83 (RA83). RA83 inhibition of T lymphocyte proliferation stimulated with allogeneic immature monocyte-derived DC (iMoDC) was confirmed. However, we found it was due to antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells in the responder T cell preparation. The likely targets of the ADCC were MoDC that had up-regulated CD83 during the MLR. Using a 51Cr-release assay, we confirmed that CD83+ MoDC, but not CD83 MoDC, are lysed by NK cells in the presence of RA83. However, prior fixation of the stimulator MoDC in the allogeneic MLR did not abrogate RA83 inhibition, indicating that cells from the responder T lymphocyte preparation, involved in the MLR proliferative response, also expressed CD83. We found, after 34 days of culture with allogeneic MoDC, a subset of CD3+ cells had up-regulated CD83 and CD25. These were blasting T cells and, when isolated from the MLR, were found to be lysed by autologous NK cells in the presence of RA83. Thus, CD83 is expressed by responding T cells as well as by stimulating cells in the MLR and both are susceptible to anti-CD83-mediated ADCC.
Keywords: cellular activation, immunosuppression, mixed leukocyte reaction, T lymphocyte
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Introduction
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CD83 is a member of the Ig superfamily and is expressed on the surface of activated dendritic cells (DC) and B lymphocytes, and, at low levels, on mitogen and phorbol myristate acetate (PMA)-activated T cells (1,2). It is minimally expressed on immature blood DC (BDC) and monocyte-derived DC (MoDC), and up-regulated upon differentiation/activation of DC, along with CD40, CD80/86, HLA-DR, CMRF-44 and CMRF-56 (3). CD83 is also expressed on cytokine-activated neutrophils (4) and is present in the cytoplasmic compartment of immature MoDC (iMoDC); a feature which was used to distinguish them from macrophages (5). A soluble form of CD83 is detectable in normal serum and is released from cell lines and MoDC (6,7).
The function of CD83 is not known, although recent data from CD83 gene-deleted mice suggests that its expression on thymic epithelium contributes to CD4 T lymphocyte development (8). Curiously, DC and CD4+ T lymphocytes from CD83/ mice functioned normally in the allogeneic mixed leukocyte reaction (MLR) and in other in vitro assays, although in vivo B cell function was altered, presumably due to reduced CD4+ T cell help. Human thymic epithelium has been reported to express low levels of CD83 (8), but its selective expression on extrathymic activated leukocytes raises the possibility that it plays a role in migration to, or in adhesive or other interactions with cells in, secondary lymphoid tissue. Although many other members of the Ig superfamily expressed on leukocytes have known (co-) stimulatory or inhibitory signaling functions (e.g. CD80/86, CD28, CTLA-4), the 40-amino-acid cytoplasmic tail of CD83 has no recognizable signaling or other motifs. However, it contains five serine/threonine residues that can potentially be phosphorylated (1). Cramer et al. (9) have suggested that a ligand for CD83 is expressed on murine B cells, whereas Scholler et al. recently claimed that human CD83 is a sialic acid-binding Ig-like lectin adhesion receptor, the counter-receptor for which is a 72-kDa protein expressed on monocytes and a subset of activated or stressed T cells (10). Furthermore, Lechmann et al. have recently reported that a ligand for CD83 is expressed on both immature and activated human DC (11).
The role of CD83 in human DC:lymphocyte interactions has been examined experimentally. It was reported that polyclonal rabbit anti-CD83 (RA83) inhibits the proliferative response of human peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA), to the recall antigen tetanus toxoid (TT) and to allogeneic stimulators (12). Murine anti-CD83 mAb failed to have a significant effect (12,13). It was also reported that RA83 inhibited the B cell proliferative response of T cell-depleted PBMC to CD40 ligand (CD40L) and that it inhibited CD40L + IL10-induced antibody synthesis. In contrast, it has been reported that a murine CD83 fusion protein weakly inhibits the proliferative response to antigen of splenocytes from DO11.10 TCR transgenic mice and that antigen-induced IL-2 expression is reduced by the murine CD83 fusion protein by up to 56% in this model (9). Also, Lechmann et al. reported that ligation of human DC with synthetic CD83 extracellular domain inhibits the MLR and an antigen-specific T cell response (11). These results are difficult to reconcile with the finding that mouse CD83/ DC stimulate a normal allogeneic MLR (8).
Here we define the mechanism involved in the above-mentioned RA83-induced inhibition of T cell proliferation in the MLR.
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Methods
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Anti-CD83 (RA83)
CD83Ig, consisting of the human CD83 extracellular domain fused at the C-terminus to human IgG1-Fc, was purified from medium conditioned by COS-7 cells transfected with pCD83Ig (7). Rabbit polyclonal anti-CD83 serum was prepared by immunization with CD83 fusion proteins, as described (7). The IgG fraction was purified from this serum and from non-immunized rabbit serum (HiTrap Protein A; Amersham Biosciences, Sydney, Australia). Anti-human IgG, anti-mouse serum protein and anti-FCS protein activity was removed from both IgG fractions by passage through columns (HiTrap NHS-activated; Amersham) of immobilized human IgG (Intragam; CSL, Parkville, Australia), mouse serum and FCS proteins. The final preparations, designated RA83 and RAneg respectively, consisted of a single major protein band of 150-kD (non-reducing SDSPAGE) with minor contaminants. On reduction, only two bands were visible, 25 and 50 kDa, corresponding to IgG light and heavy chains. RA83, but not RAneg, bound to the CD83+ Hodgkins lymphoma-derived cell line L428, as shown by flow cytometry after secondary staining with FITCgoat anti-rabbit Ig (Dako, Botany, Australia). RA83, but not RAneg, also bound to CD83Ig or soluble native CD83 antigen captured by the anti-CD83 mAb Hb15a (Beckman Coulter) immobilized on ELISA plates.
Fab fragments of RA83 and RAneg were generated by papain digestion (14). The reactions were stopped with iodoacetamide, dialyzed in PBS, and passed through a HiTrap Protein A column to remove Fc fragments and uncleaved IgG. Unbound protein consisted of a major band at
42 kDa (= Fab) and several lighter bands at 2030 kDa (non-reducing SDSPAGE). Fab derived from whole RA83, but not from RAneg, also stained L428 cells.
Preparation of cells
PBMC were prepared from buffy coats provided by the Australian Red Cross. MoDC were prepared from PBMC after depletion of CD2+ cells by rosetting with neuraminidase-treated sheep erythrocytes. Briefly, the non-rosetted cells (ER), containing 5060% CD14+ monocytes, were cultured at 0.5 x 106 CD14+ cells/ml in medium containing granulocyte macrophage colony stimulating factor (200 U/ml) and IL-4 (50 U/ml; Sigma-Aldrich, Sydney, Australia) (15). Differenti ation of monocytes to iMoDC was confirmed on day 5 by flow cytometric staining to show up-regulation of CD1a and down-regulation of CD14. Maturation/activation was induced by addition of lipopolysaccharide (LPS, 1 µg/ml; Sigma-Aldrich). For some experiments, PBS-washed MoDC were fixed in 2% paraformaldehyde (PFA) for 20 min at ambient temperature, washed in PBS, then in medium twice, incubated in medium overnight at 37°C and washed again.
Rosette-positive cells (ER+) were 8090% CD3+ and were used directly as responder cells in the MLR or were further purified by immunomagnetic depletion after staining with mAb for CD11b, CD14, CD16, CD19 and HLA-DR. These further purified responder cells were >95% CD3+. For some experiments, T cells and NK cells were purified by FACS sorting of FITC phycoerythrin (PE) and FITC PE+ events respectively in the live gate after staining ER+ cells with CD56PE, and FITC-conjugated CD14, CD19, CD34 and HLA-DR.
In some experiments MoDC were compared with BDC. BDC were prepared from PBMC, as described (16).
For the 51Cr-release assay, NK cells were purified by FACS from the normal lymphoid gate by negative selection (FITC, PE) from 3- to 4-day MLR cultures consisting of ER+ cells and allogeneic iMoDC (20:1), after staining with CD3PE, and FITC-conjugated CD14, CD19, CD34 and HLA-DR. When required as targets, T cell blasts were sorted simultaneously by collecting FITC, PE+ events in the high forward scatter blast cell gate (see Fig. 5E). Non-blasting T cells were collected as FITC, PE+ events in the normal lymphoid gate.

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Fig. 5. CD83 expression by T cells in the allogeneic MLR. (A) Time course of percentage CD83+ T cells (diamonds), CD25+ T cells (squares) and CD83+CD25+ T cells (triangles) stimulated by allogeneic iMoDC (one of two similar experiments). Dot-plots of (B and E) forward and side light scatter, and (C, D and FH) fluorescence intensity after (B and C) 0, (D) 3, (EG) 96 and (H) 72 h of culture. Panels (C, D, F and H) are gated on the region shown in (B) and all but (H) also on CD3FITC+ cells. Panel (G) is gated on CD3FITC+ cells in the high forward scatter region shown in (E), which excludes the resting lymphoid cells. Panels (AG) are from the same donor.
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Staining and flow cytometry
Cells were stained with antibodies by incubation for 20 min on ice, washed with 2% FCS in PBS containing 0.05% NaN3 and resuspended in 1% PFA in PBS for flow cytometry (FACSCalibur; BD Biosciences, North Ryde, Australia). The following commercial antibodies were employed for staining cells: CD83FITC, CD83PE and purified CD83 (Hb15a; Beckman Coulter), CD11cPE, CD25PE (BD), CD86FITC and CD86PE (BD PharMingen). Staining with unconjugated mAb was detected with FITC-conjugated sheep anti-mouse antibody, except T cell staining for CD83 was detected with biotinylated anti-mouse Ig (Sigma) followed by streptavidinPECy5 (Dako). Intracellular staining with Ki67FITC (Dako) was performed with Fix & Perm (Caltag, Burlingame, CA), in which case prior surface staining for CD83 was detected with streptavidinallophycocyanin (BD PharMingen).
Lymphocyte stimulation
The one-way MLR was performed in 96-well U-bottomed culture plates with up to 5000 MoDC or BDC per well and 105 allogeneic ER+ or immunomagnetically purified T cells per well. DC were pre-incubated for 10 min at 37°C in the wells with RA83, RAneg or medium alone prior to the addition of T cells. The MLR plate was cultured in a 37°C/5%CO2 incubator for 4 days, pulsed with 1 µCi [3H]thymidine (Amersham) per well, incubated for a further 16 h, harvested (Mach 3M; Tomtec, Hamden, CT) and counted (Trilux 1450; Wallac, Turku, Finland). Mean c.p.m. ± SE for replicate wells are reported without subtraction of counts for stimulators (MoDC) or responders alone.
T cells were also stimulated by culture in U-bottomed 96-well plates pre-coated with purified CD3 (OKT3) and CD28 (Leu28; BD PharMingen) mAb in PBS. After blocking and washing with medium, either RA83 or RAneg (final concentration 5 µg/ml) or medium alone was added, along with 105 T cells, to give 200 µl/well. These were incubated for 96 h, pulsed with 1 µCi [3H]thymidine, harvested and counted as for the MLR.
For the 51Cr-release assay, <106 blasting or non-blasting T cells FACS purified from an MLR culture, iMoDC or LPS-activated MoDC were labeled with 0.1 mCi Na51CrO4 (Amersham), washed, resuspended in MLR conditioned medium and dispensed into 96-well conical culture plates at 2500 cells/well, with the following antibodies: RA83, RAneg, rabbit anti-human thymocyte globulin (ATG; Fresenius Medical Care, Bad Homburg, Germany) or normal rabbit Ig (Intracutan; Fresenius) at 5 µg/ml. Purified NK cells, also resuspended in MLR conditioned medium, were added at up to a 20-fold excess. Wells were made up to 180 µl, centrifuged at 200 g for 3 min, cultured for 4 h, centrifuged again and 25 µl of supernatant was mixed with 150 µl scintillant (Optiphase Supermix; Wallac) for counting (Trilux 1450).
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Results
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Functional effects of anti-CD83 and CD83Ig
Purified rabbit polyclonal IgG anti-CD83 (RA83) was used to investigate the potential contribution of CD83 to DC:T lymphocyte interactions. We confirmed the findings of Armitage et al. (12) that RA83 inhibits the proliferative response of PBMC to TT (data not shown). We established that RA83 inhibited the proliferative response of ER+ responders to both allogeneic BDC (data not shown) and MoDC (see below). MoDC were used as stimulators in all subsequent experiments. The degree of inhibition of [3H]thymidine incorporation varied between donors, plateaued at concentrations of RA83 > 2.5 µg/ml (data not shown) and was dependent on the number of allogeneic stimulators (Fig. 1A). RA83 inhibition was specific for CD83 because it could be overcome by the addition of CD83Ig, but not human IgG, to the MLR (data not shown). CD83Ig added alone did not significantly affect the T cell proliferative response in the MLR. Inhibition was abrogated if the ER+ responders were further purified by immunomagnetic depletion with a cocktail of mAb for CD11b, CD14, CD16, CD19 and HLA-DR, suggesting that another cell in the ER+ responder cell preparation was involved in the inhibition (Fig. 1A and B).

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Fig. 1. Inhibition of MLR with RA83. Proliferative response (c.p.m.) of (A) rosette purified PBMC (ER+, 105/well) or of (B) the same cells further purified by negative immunomagnetic depletion (see Methods) versus number of allogeneic iMoDC/well, in the presence of 5 µg/ml RA83 or RAneg, or nil antibody. The negative control antibody, RAneg, often had a weak inhibitory effect relative to nil-antibody controls, but this was always considerably less than that observed with RA83. Representative example of six experiments. (C) Failure of inhibition of MLR by immunoreactive Fab fragments of RA83 (one experiment). (D) Effect of RA83 and number of added NK cells on the proliferative response of sort-purified T cells (105/well) to allogeneic MoDC. The CD16 function blocking mAb 3G8 reversed the effect of the added NK cells. One of two similar experiments. Error bars all ±2 SE.
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RA83 inhibition is due to NK cell-mediated ADCC of CD83+ targets
Further depletion experiments with each mAb alone, from the above cocktail, suggested that the inhibition was mediated by CD16+ cells, which co-purified with T cells in the rosetting procedure (data not shown). The possibility of an ADCC mechanism for the inhibition was suggested because we could prevent it by either (i) replacement of the RA83 whole IgG antibody with immunoreactive Fab fragments (Fig. 1C) or by (ii) addition of the CD16 FcR-blocking mAb 3G8 (Fig. 1D) (17). This mechanism was confirmed by showing that the addition of purified CD56+ NK cells to wells containing immunomagnetically purified T cells, allogeneic MoDC and RA83, but not RAneg, inhibited the MLR. The reduction in T cell proliferation was dependent on the number of NK cells added, approaching normal blood NK:T ratios (Fig. 1D). We concluded that RA83 did not inhibit the MLR by blocking a functional interaction between CD83 and any potential ligand, but instead enabled NK ADCC of susceptible targets.
CD83+ MoDC are targets of NK cell/RA83-mediated lysis
We considered that the most likely targets of the RA83/NK cell-mediated ADCC in the MLR would be MoDC that had up-regulated surface CD83+ during the culture period. MoDC activated with LPS to express CD83 are potent stimulators of T cell proliferation in the allogeneic MLR and, with ER+ responders, we found this was inhibited by RA83, but not by RAneg (data not shown). We confirmed that RA83-induced lysis of 51Cr-labeled LPS activated MoDC in the presence of allogeneic NK cells isolated from an MLR (Fig. 2A). There was no measurable lysis when RA83 was replaced with RAneg. In contrast, iMoDC were lysed slowly by NK cells with RAneg and this was only minimally enhanced by RA83 (Fig. 2B). CD83 iMoDC used as stimulators in the MLR were expected to become activated, and up-regulate CD83, due to interaction with CD40L expressed on activated responding T lymphocytes (18). However, we found that co-culture of iMoDC with freshly isolated allogeneic T cells did not consistently up-regulate surface CD83 on the MoDC (Fig. 3A) despite consistent proliferative T lymphocyte responses (data not shown). On those occasions when CD83 was up-regulated, the MoDC divided into discrete CD83+ and CD83 subpopulations, which contrasted with the unimodal expression observed after LPS activation (Fig. 3B). At this stage, it was not clear how RA83 inhibited the MLR, when the stimulatory iMoDC failed to up-regulate CD83 during co-culture with responding ER+ cells (Fig. 3A).

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Fig. 2. Effect of RA83 on NK cell-mediated lysis of allogeneic MoDC. NK cells were sort purified from a 3-day MLR culture consisting of ER+ cells stimulated with allogeneic iMoDC (20:1 ratio). (A) LPS-activated MoDC and (B) iMoDC, derived from the same donor, were labeled with 51CrO4 and co-cultured for 4 h with the NK cells at the ratios shown, with either RA83 or RAneg. MoDC lysis was measured as release of 51Cr into the medium (100% = 51Cr released by Triton X-100). Representative example of three experiments.
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Fig. 3. Effect on MoDC CD83 expression of co-culture with allogeneic T cells. (A) Percentage CD83+ MoDC at 0 and 48 h of culture with a 20-fold excess of allogeneic T cells (six independent experiments shown). (B) CD83 expression (x-axis = MFI) for MoDC cultured for 48 h with a 20-fold excess of allogeneic T cells or with LPS at 1 µg/ml. Representative example of three experiments.
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RA83/NK cell ADCC targets are also present in the responder cell preparation
We next tested whether RA83 inhibited the MLR, when the allogeneic iMoDC used as stimulators were prevented from up-regulating surface CD83. This was done by fixing the iMoDC in PFA prior to culture with ER+ responders. As expected, this resulted in reduced T cell proliferation compared to fresh iMoDC from the same donor/MoDC culture (data not shown). However, RA83 once again inhibited 3H incorporation, relative to RAneg (Fig. 4), even though the iMoDC were prevented, by fixation, from up-regulating CD83 and were also not susceptible to NK cell-mediated lysis. We concluded from this data that CD83+ MoDC are not the only targets for ADCC and that CD83+ target cells must also be present in or arise from the responder preparation.

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Fig. 4. Prior fixation of iMoDC did not abrogate RA83-mediated inhibition of MLR. Proliferative responses of ER+ cells to either fresh (solid line) or paraformaldehyde-fixed (broken line) allogeneic MoDC from the same donor/MoDC preparation plotted as a function of the number of MoDC. [3H]Thymidine incorporation in the presence of RA83 is expressed as a percentage of that in the presence of RAneg. Fixation of MoDC was expected to abrogate the blocking effect of RA83 and therefore give values of 100%. One of two similar experiments.
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CD83 expression is not unique to activated DC. B cells can also express CD83 (2) and these were present as minor contaminants in the ER+ responder preparations used above. We established that B cells were not functionally important in the RA83 inhibition of the MLR, by demonstrating that prior immunomagnetic depletion of CD19+ cells, or of HLA-DR+ cells, from ER+ responder preparations had no effect on the inhibition (data not shown).
CD83 has been reported to be expressed by mitogen-activated human lymphocytes (1,6), but there are no reports of CD83 expression on T cells induced by allogeneic stimulation in man. To confirm that the CD83+ targets for RA83-mediated ADCC were in the responder cell preparation we attempted to inhibit T cell proliferation with RA83 in the absence of stimulator antigen-presenting cells (APC). We co-cultured FACS-purified T cells and NK cells in wells coated with CD3 and CD28 mAb. However, we could not inhibit T cell proliferation with RA83 in these experiments (data not shown), suggesting that this stimulus failed to induce CD83 (see below), or in some other way failed to induce sensitive targets or activate the NK cells sufficiently.
CD83 expression by T cells
Using a sensitive three-step staining protocol (see Methods) we investigated the expression of CD83 on T lymphocytes in co-cultures of ER+ and allogeneic MoDC. Low levels of CD83 were rapidly induced on a high proportion of CD3+ T cells, reaching a maximum in 3 h, then declining to near-background levels at 12 h (Fig. 5A and D). Further experiments showed that this 3-h induction of CD83 expression also occurred when the stimulator cells were omitted from the culture. The staining at 3 h was specific for CD83 because it was blocked by pre-incubation of the Hb15a CD83-staining mAb with CD83Ig fusion protein, but not with human IgG. Also, the polyclonal RA83, but not RAneg, positively stained these cells at 3 h (data not shown).
ER+ cultured with allogeneic MoDC for longer periods resulted in the appearance of small, but variable, numbers of CD83+ T cells that also expressed the T cell activation marker CD25. At 96 h a subset of CD83+CD25+CD3+ cells was clearly evident (Fig. 5F). This subset included virtually all of the T cell blasts (judged from the high forward scatter characteristics, Fig. 5G) and very few of the cells in the normal lymphoid gate. Further phenotyping revealed that the majority of CD83+ T cells were CD4+CD8 and stained positively for the intracellular proliferation marker Ki67 (Fig. 5H). We conclude that this CD83+ T cell subset consists mostly of proliferating blasts in the MLR.
In two experiments, we directly compared the CD83 expression induced on T cells stimulated by immobilized CD3 and CD28 mAb with the same T cells stimulated with allogeneic MoDC. CD83 expression was assessed by a less-sensitive single-step staining method using CD83FITC because the three-step method used above suffered high background staining due to uptake of plastic immobilized CD3 and CD28 mAb by T cells. For one donor, after 96 h of culture, the mean fluorescence intensity (MFI) for CD83 for CD3/CD28 mAb-stimulated T blasts was 18.7 (isotype control MFI = 8.8) cf. 24.5 (8.4) for those stimulated with allogeneic MoDC. For the second donor the corresponding MFI values were 20.8 (11.7) cf. 27.8 (10.2) respectively. This lesser degree of CD83 up-regulation on antibody-stimulated T cells compared to allogeneic MoDC-stimulated cells may contribute in part to their lesser susceptibility to RA83 ADCC mentioned earlier.
RA83 also induces NK cell-mediated lysis of CD83+ T cell blasts
We then asked whether RA83-mediated inhibition of the allogeneic MLR was explained, at least in part, by lysis of CD83+ T cells by autologous NK cells. A 24-h delay in addition of RA83 did not abrogate inhibition (Fig. 6). We therefore concluded that the 3-h CD83+ T cell population (Fig. 5A and D) is not a critical target for RA83-mediated inhibition of the MLR. The data in Fig. 6 suggests that the majority of targets of interest appear, as a lesser population, 2448 h after initiating the MLR. We therefore sort purified NK, blasting and non-blasting T cells from 3- to 4-day co-cultures of ER+ and allogeneic iMoDC for ADCC studies. We found, in a 4-h 51Cr-release assay, that T cell blasts were lysed by autologous NK cells in the presence of RA83, but only minimally with RAneg (Fig. 7A). For five donors, percent specific lysis values at 10:1 effector/target ratio were 21% (RA83RAneg, Students t-test P < 0.005), 31% (P < 0.02), 16% (P < 0.1), 16% (P < 0.04) and 5% (P < 0.02). In contrast, the non-blasting T cells purified from MLR cultures were not lysed in the presence of RA83 and NK cells (Fig. 7B). As expected, both T cell blasts and non-blasts were lysed in the presence of ATG (Fig. 7C and D). NK cells activated by culture for 3 days in IL-2 (6000 IU/ml) were equally effective in RA83-mediated ADCC of T blasts, but resting NK cells induced minimal lysis with RA83 (data not shown).

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Fig. 6. Effect on MLR of delayed addition of RA83. Proliferative response (c.p.m.) of 105 ER+ cells/well to 2500 allogeneic iMoDC added at 0 h. RA83, RAneg or medium only were added at the times shown. All wells were pulsed with [3H]thymidine at 96 h and harvested 16 h later. From one of two similar experiments.
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Fig. 7. ADCC of T cell blasts (A and C) and T non-blasts (B and D). Blasting and non-blasting T cells and NK cells were sort purified from a 3-day MLR culture consisting of ER+ cells and allogeneic iMoDC. T cell targets were labeled with 51CrO4 and co-cultured for 4 h with the autologous NK cells at the ratios shown, with either RA83 (solid lines in A and B), ATG (solid lines in C and D), or RAneg or non-immune rabbit globulin-negative control antibodies (broken lines). T cell lysis was measured as release of 51Cr into the medium (100% = 51Cr released by Triton X-100). Panel (A) is representative of five experiments; (BD) from one of two similar experiments.
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We concluded that proliferating T lymphocyte blasts induced by fresh allogeneic stimulators up-regulate CD83 and that RA83-dependent inhibition of the MLR is due to NK cell-mediated ADCC lysis of both these proliferating CD83+ responding T cells and CD83+ stimulators.
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Discussion
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In investigating the role of CD83 in DC stimulation of allogeneic T cells we have shown that RA83 inhibition of the MLR can be explained by NK cell-mediated ADCC lysis of DC and of CD83+ T cell blasts. Although expression of CD83 by mitogen-stimulated T cells was briefly reported in the original description of CD83 (1), attention has focused on its expression by DC. However, our findings in the human system reported here, and of others in the mouse (9), and the recent demonstration of the role of NF-
B in T cell expression of CD83 (19) shift the focus on CD83 to the T cell. Interestingly, the major phenotypic alteration reported recently for the CD83/ mouse is in the T cell subset composition (8). The CD83/ mouse findings indicate that CD83 expression on thymic epithelium is required for the development of CD4+CD8 thymocytes from CD4+CD8+ precursors. This does not explain, however, the role of CD83 present on extrathymic APC and T cell blasts. The proposition that CD83 has an extrathymic role was supported by the recently reported effects of soluble CD83 constructs on T cell stimulation (9,11,20), although we found no such effect with our CD83Ig, a result possibly related to structural differences between the recombinant proteins. Until now, these former reports appeared to conflict with observations of RA83 inhibition. They remain, however, difficult to reconcile with the failure of CD83 gene deletion to affect DC or T lymphocyte function in vitro (8).
CD83 expression has many similarities to the better understood co-stimulatory molecule CD86, including up-regulation on activation of DC (21), the existence of soluble forms (7,22) and expression on activated T cells (23). There are also important differences, which suggest a role for CD83 independent of CD86. Published differences include an unusual mode of regulation at the level of nuclear export of CD83 mRNA (24) and the lack of CD83 on DC-derived CD86+ exosomes (25). We found that iMoDC consistently up-regulated surface CD83 on exposure to LPS, but on co-culture with allogeneic T cells, CD83 was up-regulated in a variable fashion (Fig. 3A). When it was up-regulated only
50% of the MoDC-expressed CD83 (Fig. 3B), even though T cell proliferation consistently occurred along with CD25 and CD83 expression on the T cell blasts. Others have reported that only
50% of MoDC become CD83+ after 2 days of culture with CD40L (26).
It has previously been reported that rabbit polyclonal but not murine monoclonal anti-CD83 inhibits the T cell proliferative response to allogeneic stimulators, as well as to specific antigen (TT), and to PHA, and of B cells to CD40L-induced proliferation (12). This was assumed to be a functional blockade and it was argued that an accessory cell, perhaps an APC, was involved because the T and B cell responses were not inhibited by RA83 if the responding cells were further purified. We confirmed that RA83 inhibited the proliferative response to TT added to cultured PBMC and to allogeneic MoDC cultured with ER+ cells. We also confirmed that further purification of the T cells abrogated the inhibition. However, we found that (i) the accessory cell was the NK cell, (ii) the inhibition was due to ADCC and (iii) the targets included responder CD83+CD25+CD4+ T cells. These findings are consistent with the preservation of DC and T lymphocyte function in vitro despite CD83 gene deletion (8).
Rabbit antibody has been shown to be more effective than murine mAb in ADCC with human NK cells (27). This, isotype differences and the likelihood of more epitopes on CD83 for a polyclonal antibody compared to a mAb could explain previous reports of lack of inhibition with murine CD83 mAb (12,13). We found that NK cells activated with IL-2 were at least as effective as those isolated from MLR cultures, but resting NK cells did not lyse autologous T blasts in the presence of RA83 as effectively. IL-2 secreted by activated T cells in the MLR presumably activates resting NK cells, but DC have also been shown to directly activate NK cells by a contact-dependent mechanism (28).
We found that CD83+ MoDC were resistant to lysis by MLR-activated NK cells in the absence of RA83 (Fig. 2A). iMoDC were lysed slowly by the NK cells and RA83 had little additional effect (Fig. 2B). Such NK cell-mediated natural cytotoxic lysis of iMoDC, but not of activated MoDC, has been observed previously in autologous systems (29). Clearly the resistance of activated MoDC to this natural cytotoxicity can be overcome by RA83 antibody-mediated ligation of the NK cell Fc
R to the MoDC via CD83 antigen.
The CD83+ T cells observed after 3 h of culture (Fig. 5A and D) are probably not NK/ADCC targets as (i) the level of CD83 expression is very low, (ii) the E:T ratio is low (
0.05) because a high proportion of the T cells express CD83 at 3 h (Fig. 5D) and (iii) the NK cells are not sufficiently activated. The transitory appearance of low-levels of CD83 on the surface of resting T cells placed into culture for 3 h was specific for CD83, but may be an in vitro effect. It is not known if it is derived by de novo synthesis or from cytoplasmic stores of CD83. As it occurs in the absence of added MoDC, it is unlikely to be derived from soluble CD83 in the medium. The most likely NK/ADCC targets derived from the responder cell preparation are the subpopulation of CD83+CD25+ proliferating T cells that appears after 48 h (Fig. 5A, F and G). This was supported by the relatively undiminished degree of inhibition when RA83 was added to the MLR after 24 h (Fig. 6). Finally, the mechanism was confirmed by demonstrating RA83 dependent lysis of T cell blasts by autologous NK cells, both purified from 3- to 4-day MLR cultures (Fig. 7A). In contrast, the non-proliferating T cells were resistant to RA83-mediated ADCC, although susceptible to rabbit ATG.
The in vitro immune inhibition experiments described here raise the novel possibility of anti-CD83-mediated immunotherapy directed against both activated APC and activated T cells. To date, antibody-mediated therapeutic immunosuppression in transplantation has been aimed, via CD3 and CD25, at the effector cell rather than at APC. It is likely that anti-CD83 antibodies will target both the activated CD83+ DC and the CD83+ responding T cell (and the B cell). Such an agent, that specifically targets both activated stimulator and activated responder cells, may prove to be a highly effective immunosuppressant.
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Acknowledgements
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This work was supported by the Queensland Cancer Fund, NHMRC (Australia) and the Mater Medical Research Institute. The authors would like to acknowledge the contribution of the Australian Red Cross Blood Service Queensland and their donors, and the assistance of Ken Field (MMRI) in flow cytometry.
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Abbreviations
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ADCCantibody-dependent cellular cytotoxicity
APCantigen-presenting cell
ATGanti-human thymocyte globulin
BDCblood DC
CD40LCD40 ligand
DCdendritic cell
iMoDCimmature MoDC
LPSlipopolysaccharide
MFImean fluorescence intensity
MLRmixed leukocyte reaction
MoDCmonocyte-derived DC
PBMCperipheral blood mononuclear cell
PEphycoerythrin
PFAparaformaldehyde
PHAphytohemagglutinin
RA83rabbit polyclonal anti-CD83 IgG
RAnegnon-immune rabbit polyclonal IgG
TTtetanus toxoid
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