Proliferation Soluble CD83 Proteins That Inhibit T Cell Alternative

Alternative Splicing Generates Putative Soluble CD83 Proteins That Inhibit T Cell Proliferation

This information is current as Diana Dudziak, Falk Nimmerjahn, Georg W. Bornkamm and of September 23, 2021. Gerhard Laux J Immunol 2005; 174:6672-6676; ; doi: 10.4049/jimmunol.174.11.6672 http://www.jimmunol.org/content/174/11/6672 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Alternative Splicing Generates Putative Soluble CD83 Proteins That Inhibit T Cell Proliferation1

Diana Dudziak,2* Falk Nimmerjahn,3† Georg W. Bornkamm,* and Gerhard Laux*

CD83 is expressed on mature dendritic cells and activated lymphocytes and has been implicated to play an important role during T cell development in the thymus. In contrast, not much is known about the function of CD83 in the periphery. Soluble forms of CD83 have been detected in the serum, but neither the function nor the mechanism of how these soluble forms of CD83 are generated are fully understood. In this study, we report the identiﬁcation of four different transcripts of CD83 in unstimulated PBMCs. Sequence analysis demonstrated that the longest form codes for transmembrane CD83 (CD83-TM), whereas the smaller transcripts are splice variants of full-length CD83, coding for putative soluble CD83 proteins. Stimulation of PBMCs with PHA, TNF-␣, or LPS leads to the up-regulation of the full-length CD83 transcript and to a strong down-regulation of two of the three smaller transcripts. The smallest CD83 splice product can be translated efﬁciently into protein, and recombinant soluble CD83 shows a strong inhibitory effect on T cell proliferation in MLRs. Our results suggest that the constitutive production of soluble Downloaded from forms of CD83 under steady-state conditions may have an important function in regulating immune homeostasis. The Journal of Immunology, 2005, 174: 6672–6676.

he surface glycoprotein CD83 has a molecular mass of are rejected after injection into C3H/HeN mice, whereas wild-type 40–45 kDa and belongs to the Ig superfamily (1, 2). It melanoma cells showed tumor growth (16, 17).

consists of an extracellular V-type Ig-like domain at the N The addition of a human CD83-Ig fusion protein or a human http://www.jimmunol.org/ T 4 terminus, one transmembrane (TM) domain, and a short intracel- soluble CD83 protein lacking the TM and cytosolic domain lular cytoplasmic domain of 39 aa (2). CD83 is described as a cell strongly inhibited MLR or DC-mediated allogeneic T cell prolif- surface marker of mature dendritic cells including Langerhans eration, demonstrating that soluble CD83 could have an inhibitory cells in the skin and interdigitating reticulum cells in the T cell function (18). Moreover, soluble CD83 was able to prevent exper- zones of the lymph nodes (2–5). In immature monocyte-derived imental autoimmune encephalomyelitis (EAE) in C57BL/6 mice dendritic cells (MoDCs), CD83 is up-regulated after stimulation (19). Latest studies suggest that soluble CD83 is present in the with LPS, TNF-␣, or CD40L (6–8). Addition of high doses of serum of healthy donors (20–22) and it was proposed that the IL-4, GM-CSF, and TNF-␣ induces the expression of CD83 on mechanism for the generation of soluble CD83 is shedding of cell by guest on September 23, 2021 monocytes, granulocyte-precursor cells, and myelocytes (9, 10). It surface-associated CD83. However, soluble forms of cell surface was also shown that CD83 is expressed on polymorphonuclear proteins can also result from alternative splicing as reported for neutrophils (11) and on murine thymus epithelial cells (12). Fur- several members of the Ig superfamily, e.g., CD80 (B7-1), CD86 thermore, CD83 is expressed on Hodgkin cells (13) and EBV- (B7-2), CTLA-4, or CD28 (23–28). transformed lymphoblastoid cell lines (2, 14). In this study, we report that alternative splicing generates soluble The function of CD83 remains largely unknown. CD83 knock- forms of CD83. Stimulation of freshly isolated PBMCs with PHA, out mice showed a block in the development of CD4ϩ single- LPS, or TNF-␣/IL-1␤ up-regulates transcripts encoding TM CD83 positive T cells (12). CD83 expression on mature dendritic cells and down-regulates transcripts coding for alternatively spliced prod- and activated lymphocytes (1, 2, 10, 15) suggests that CD83 might ucts of CD83. These putative soluble forms of CD83 proteins are be involved in activating immune effector cells. Along these lines, characterized by a partial deletion of the extracellular and the TM melanoma cells that were engineered to express cell surface CD83 domain. Soluble CD83 displayed inhibitory effects in MLRs and, therefore, might have important immunoregulatory functions in vivo.

*Institute of Clinical Molecular Biology and Tumor Genetics, GSF National Research Materials and Methods Center (GSF) for Environment and Health and †Clinical Cooperation Group of Pe- Isolation of human PBMCs and DCs diatric Oncology, GSF and Children’s Hospital of the Technical University, Munich, Germany Human PBMCs were isolated by centrifugation on Ficoll-Paque (Amer- sham Biosciences). Isolated PBMCs were cultured in complete medium Received for publication September 15, 2004. Accepted for publication March (RPMI 1640 medium; Invitrogen Life Technologies) supplemented with 15, 2005. 10% FCS (Dynacyte), 100 U/ml penicillin, 100 ␮g/ml streptomycin, 1 mM The costs of publication of this article were defrayed in part by the payment of page sodium-pyruvate, 2 mM L-glutamine (all from Invitrogen Life Technolo- charges. This article must therefore be hereby marked advertisement in accordance gies) and were left either unstimulated or stimulated with PHA (1 ␮g/ml), with 18 U.S.C. Section 1734 solely to indicate this fact. LPS (1 ␮g/ml), or a mix of TNF-␣ (2.5 ng/ml; Sigma-Aldrich), IL-1␤ (1 1 This work was supported by a grant from Deutsche Forschungsgemeinschaft ng/ml; Sigma-Aldrich), and PGE2 (1 ng/ml; Sigma-Aldrich) for the indi- (SFB455; to F.N.). G.W.B. was supported by Fonds der Chemischen Industrie. cated time. Human DCs were generated as described (4) and matured with 2 ␣ ␤ Address correspondence and reprint requests to Dr. Diana Dudziak at the current LPS (100 ng/ml) or TNF- /IL-1 /PGE2. address: Laboratory of Molecular Immunology, The Rockefeller University, Box 220, 1230 York Avenue, New York, NY 10021. E-mail address: [email protected] Cloning and sequencing of the CD83 mRNA splice products 3 Current address: Laboratory of Molecular Genetics and Immunology, The Rock- Total RNA of PBMCs was extracted using the RNeasy Midi kit (Qiagen). efeller University, 1230 York Avenue, New York, NY 10021 RNA from human tissues was kindly provided by J. Mautner (Clinical 4 Abbreviations used in this paper: TM, transmembrane; MoDC, monocyte-derived Cooperation Group of Pediatric Oncology, GSF, Munich, Germany). Sin- dendritic cell; ORF, open reading frame. gle-stranded cDNA was synthesized from 5 ␮g of total RNA by reverse

transcription using Superscript reverse transcriptase (Invitrogen Life Tech- Results nologies) and an oligo-dT primer (Amersham Biosciences) at 42°C. CD83 Identification of alternatively spliced variants of the CD83 mRNA cDNA was amplified with primers designed to amplify the entire coding sequence of CD83 (5Ј-GCGGGGGAATTCCTCGAGATGTCGCGCGG Recently, it was shown that soluble CD83 variants are present in CCTCCAGCTTC-3Ј and 5Ј-CCCCGGGCGGCCGCTCATACCAGTTCT the serum of healthy individuals and that they are enriched in leu- GTCTTGTGAGGAGTC-3Ј; Metabion). The PCR was performed using PFU DNA Polymerase (Promega) as follows: 94°C for 4 min, than 35 kemia patients (20–22). To examine whether soluble CD83 forms cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 2 min, followed by are generated by alternative splicing, we isolated total RNA from a final extension step at 72°C for 10 min (PerkinElmer). The amplified PBMCs that were either left untreated or treated for 4 or 16 h with fragments were separated on a 2% agarose gel and visualized by PHA (Fig. 1A) or for 16 h with LPS or TNF-␣/IL-1␤, respectively ethidium bromide. After excision from the gel, resulting products were (Fig. 1B). Stimulation of PBMCs with PHA was followed by cloned into the pcDNA3.1 vector. Cloned cDNAs were verified by sequencing (Sequiserve). RNA integrity and cDNA synthesis was proven FACS analysis with an anti-human CD83-PE-labeled Ab showing by amplifying GAPDH cDNA (5Ј-ACCACAGTCCATGCCATCAC-3Ј a strong induction of CD83 on the cell surface of PBMCs 4 h after and 5Ј-TCCACCACCCTGTTGCTGTA-3Ј). PHA addition (Fig. 1C). The amplification of the CD83 coding sequence by RT-PCR using primers flanking the ATG initiation Generation of His-tagged soluble CD83 codon and the translation termination codon revealed the constitutive expression of four transcripts by nonactivated PBMCs, with CD83 splice variants were tagged with a C-terminal histidine hexapep- tide. The cDNAs CD83-TM, CD83-a, CD83-b, and CD83-c were re- sizes of 618, 599, 389, and 282 bp (Fig. 1A, lanes 1, 4, and 7). amplified using PFU DNA polymerase (Promega) with the 5Ј primer Subsequent cloning (Fig. 2, A and B) and sequencing of these (5Ј-GCGGGGGAATTCCTCGAGATGTCGCGCGGCCTCCAGCTTC- fragments identified the largest band as full-length CD83 (CD83- Ј Ј Downloaded from 3 ) and 3 primers inserted the histidine tag upstream of the resulting TM), whereas the smaller fragments were truncated versions of stop codon (5Ј-CCCCGGGCGGCCGCTCAGTGATGGTGATGGTG ATGTACCAGTTCTGTCTTGTGAG-3Ј and 5Ј-CCCCGGGCGGCCG CD83 (Fig. 2, A and B). The 599-bp fragment (CD83-a) lacks bp CTTAATGGTGATGGTGATGAGTAGAAAATAACCAGAGCCAG-3Ј). 364–383; the 389-bp fragment (CD83-b) has a deletion between The PCR products were cloned into the EcoRI/NotI sites of the pcDNA 3.1 bp 154 and 383; and the smallest fragment (CD83-c) misses bp vector and verified by sequencing. 154–489. According to the genomic organization of the human 293-T cells were cultured in DMEM medium supplemented with 10% CD83 gene on chromosome 6 (AL022396 and AL133250) the

FCS (Dynacyte), 100 U/ml penicillin, 100 ␮g/ml streptomycin, 1 mM so- http://www.jimmunol.org/ dium-pyruvate, 2 mM L-glutamine (all from Invitrogen Life Technologies). smaller RNAs are generated by alternative splicing of either the His-tagged variants and membrane CD83-His were transiently transfected last 19 bp of exon 3 (CD83-a), skipping of the complete exon 3 into 293-T cells by calcium phosphate method (29). After transfection, (CD83-b) or exon 3 and 4 (CD83-c). Exon 3 is coding for the 293-T cells were grown for 7 days in serum-free medium (SFMII 293; largest part of the V-type Ig-like domain, and exon 4 is coding for Invitrogen Life Technologies). After harvesting the medium, the soluble CD83 variants were enriched by Ni-NTA beads, purified under native con- the TM domain of CD83. Additionally, we could define the fol- ditions (Qiagen) and analyzed by Western blot. When used in MLR, CD83- lowing intron sizes of intron 1 with 101 bp, intron 2 with 13,454 c-His protein was dialyzed at 4°C overnight against PBS and further pu- bp, intron 3 with 1,900 bp, and intron 4 with 1,352 bp. Because of rified and enriched by gel filtration (Ultrafree-4 filter; Amicon). deletions in the CD83-a and CD83-b splice products the reading

frame changes, generating a novel short amino acid sequence with by guest on September 23, 2021 Western blot analysis an as yet unknown function. Finally, the frame shift results in a Puriﬁed protein (0.5 ␮g) or 15 ␮l of supernatant was electrophoretically stop codon (TAA) at nucleotide position 450 after the translation separated on a 15% SDS polyacrylamide gel under reducing conditions and transferred onto a polyvinylidene diﬂuoride membrane (Hybond P; Amer- sham Biosciences). Blocking was done for1hinTBST buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% Tween 20) containing 5% milk (Merck). Membranes were incubated with either anti-CD83 Ab (1:500 to 1:1000, HB15A; Immunotech) or anti-His Ab (1:500; Roche) in TBST containing 3% dry milk overnight at 4°C. After washing, membranes were incubated with peroxidase-labeled goat anti-mouse IgG Ab (1:2000 to 1:5000; Dianova) for1hatroom temperature. Finally, blots were washed three times. Proteins were visualized using the ECL system (Amersham Biosciences).

FACS analysis To evaluate cell surface expression of PBMCs, cells were washed in FACS buffer (PBS/0.5% BSA/0.02% sodium azide) and incubated with PE-con- jugated mouse anti-human CD83 (HB15A; Coulter Immunotech) or iso- type control IgG2b Abs (BD Biosciences) for 30 min on ice. Flow cytometry was performed on a FACSCalibur cytometer (BD Biosciences) and analyzed with CellQuest acquisition software.

Allogeneic MLR Allogeneic PBMC stimulation was measured in a standard MLR. Stimu- FIGURE 1. Identification of alternatively spliced CD83 mRNA variants. latory cells were inactivated by gamma-irradiation (20 Gy), cells were PBMCs from three healthy volunteers (I–III) were isolated and left either un- washed three times with complete RPMI 1640 medium. Stimulatory cells treated (A, lanes 1, 4, and 7; B, lanes 1 and 4; C, upper part) or were stimulated 5 (1 ϫ 10 /well) were incubated with allogeneic PBMC in 96-well microtiter for4(A, lanes 2, 5, and 8; C, lower part)or16h(A, lanes 3, 6, and 9; B, lanes plates in complete medium at a ratio of 1:1 and 1:10 for 5 days. Purified 2 and 4) with PHA (A) or LPS or TNF-␣/IL-1␤ (B). A total of 1 ϫ 107 cells ␮ recombinant CD83-c-His protein (0.75, 1.5 g/ml) was added immedi- were harvested and total RNA was isolated (A and B); and additionally, 1 ϫ ately. As control, His-tagged neomycin phosphotransferase (NeoR-His, 1.5 105 C A B - ␮g/ml, kindly provided by J. Mautner) or PBS was used. For the determi- cells were used for FACS analysis ( ). and , Total RNA was tran nation of cell proliferation the cells were pulsed with [3H]thymidine (1␮Ci/ scribed into cDNA, and RT-PCR using primers spanning the whole ORF was 5 well; Amersham Biosciences) for the last 16 h. Incorporation of [3H]thy- performed. GAPDH was used as a control. C, A total of 1 ϫ 10 cells were midine into DNA was counted with TopCount NXT counter (Packard incubated with human CD83-PE-labeled Ab (y-axis) and analyzed by flow Instrument). Data are expressed as mean and SD. cytometry. The result represents one of four independent experiments. 6674 ALTERNATIVELY SPLICED CD83 INHIBITS T CELL ACTIVATION

initiation codon of the full-length open reading frame (ORF) of CD83. In the CD83-c splice product, the ORF of the cytoplasmic domain is not changed in comparison to full-length CD83 mRNA (Fig. 2, A and B). These data suggest that alternative CD83 transcripts lacking the TM domain result in putative soluble CD83 variants. The expression of these four CD83 mRNA variants has been observed in 10 of 10 healthy volunteers and in different B cell lines (Raji, BL41, DG75, and ER/EB2–5; data not shown). In contrast to unstimulated PBMCs, the stimulation of cells with PHA for 4–16 h shows an increase of TM CD83 (Fig. 1, A, lanes 2, 3, 5, 6, 8, and 9, and C) and results in nearly complete inhibition of the CD83-b and CD83-c mRNA variants encoding putative soluble CD83. The CD83-a variant seemed to be less sensitive to PHA induced down- regulation. A similar effect was observed when PBMCs were stimulated with LPS or TNF-␣/IL-1␤ (Fig. 1B, lanes 2 and 5). How- ever, LPS treatment did not lead to a pronounced down-regulation of the CD83-c splice product in PBMCs. These data indicate that putative soluble versions of CD83 derived by alternative splicing

are expressed mainly under noninﬂammatory conditions. Downloaded from

Tissue distribution of CD83 splice variants To determine the distribution of the CD83 transcripts in human tissue, we performed RT-PCR using primers spanning the whole ORF. The TM form as well as the splice forms were detected in

tonsils, bone marrow, and testis. TM-associated CD83 was detect- http://www.jimmunol.org/ able in spleen, thymus, brain, kidney, adrenal gland, lung, ovary, and uterus, and only a little expression was found in skin, small intestine, and liver. There was no expression of CD83 variants in heart, pancreas, and skeletal muscle. GAPDH was ampliﬁed as a control (Fig. 3, A and B). Additionally, the presence of CD83 splice variants in human MoDCs was analyzed. In DCs cultured in the presence of GM-CSF and IL-4 (0 h) the CD83-TM and CD83-a and CD83-b variants could be detected. After maturation with TNF-␣/IL-1␤ or LPS for 8 by guest on September 23, 2021 or 24 h, respectively, the CD83-TM transcript was slightly enhanced, whereas the CD83-b form was down-regulated (Fig. 3C). As observed for PBMCs, there was no regulation of the CD83-a variant (Figs. 1A and 3C). The splice product CD83-c was not detectable.

Expression of recombinant soluble CD83 To study the function of the splice products, all cloned splice variants were fused to a hexahistidine-tag at the very C terminus. 293-T cells were transfected with the splice products, and Western blot analysis of the supernatants and Ni-NTA-enriched proteins was performed. Only the in-frame CD83-c splice product missing exon 3 and 4 could be detected as protein in the supernatant of transfected 293-T cells (Fig. 4A). No protein expression was observed for the splice products CD83-a and CD83-b in the supernatant or in total cell extracts of transfected 293-T cells (data not shown). CD83 cell surface expression could only be detected by transfection of 293-T cells with full-length CD83. None of the CD83 splice products was expressed on the cell surface (data not shown). These results indicate that CD83-c is the only splice product expressed as a functional protein and is secreted into the supernatant.

The CD83-c splice product shows an inhibitory effect in MLRs

FIGURE 2. CD83 splice forms and sequences. A, Model of the exon- To further investigate the functionality of the CD83-c splice prod- intron structure of human CD83. The gray boxes represent coding parts of uct, we tested the puriﬁed CD83-c-His protein in allogeneic the CD83 exons and the numbers refer to the base pairs of full-length membrane CD83. Exon 1 is coding for a signaling peptide, exon 2 and exon 3 are coding for the extracellular domain of CD83, exon 4 contains CD83-c, exon 3 and exon 4 are missing. B, Sequence analysis of the CD83 the TM region, and exon 5 contains the intracellular domain of CD83. A splice variants. The splicing in CD83-a and CD83-b leads to a change in and B, In CD83-a, a part of exon 3 is excluded (19 bp); in the splice variant the reading frame, resulting in a new stop codon at nucleotide position 450. CD83-b, exon 3 is completely spliced out; and in the splice product In CD83-c, the ORF is preserved. The Journal of Immunology 6675 Downloaded from

FIGURE 4. Inhibitory effect of recombinant soluble CD83-c in MLRs.

FIGURE 3. http://www.jimmunol.org/ Tissue distribution of CD83 splice variants. CD83 splice prod- A, Purified His-tagged CD83-c protein was analyzed on an SDS-PAGE gel ucts are selectively expressed in unstimulated blood PBMC, bone marrow and visualized by Western Blot either with an anti-human-CD83 (HB15A) (BM), tonsil, and testis. Using primers for the full-length ORF, the expression or an anti-6xHis Ab. B, Allogeneic MLR was performed using freshly of CD83 splice variants was analyzed by RT-PCR in different human tissues isolated human PBMCs from healthy volunteers as described in Materials A B ( and ) or isolated immature MoDCs (0 h) and for 8 and 24 h with LPS or and Methods. Responder cells were cultured with irradiated allogeneic ␣ ␤ C TNF- /IL-1 matured DCs ( ). The amplified fragments were visualized by stimulator cells as indicated. Highly purified recombinant CD83-c-His pro- ethidium bromide staining. As control, PCR with GAPDH primers was done. tein (0.75, 1.5 ␮g/ml) or an irrelevant control protein was added immedi- ately. Results are expressed as mean cpm Ϯ SD. One representative of four MLRs. The CD83-c-His protein was harvested and purified from independent experiments with different human blood donors is shown. serum-free supernatants from transiently transfected 293-T cells. by guest on September 23, 2021 As controls, His-tagged NeoR protein or PBS were used. The pro- mRNAs, the part encoding the TM domain is deleted either by liferation of the cells was measured by [3H]thymidine incorpora- partial (CD83-a) or complete deletion of exon 3 (CD83-b) or exon tion. The addition of increasing amounts of CD83-c-His had an 3 and 4 (CD83-c). Whereas CD83-b and CD83-c mRNAs use inhibitory effect on the proliferation of T cells in these MLRs, common splice donor and splice acceptor sites CD83-a shows an leading to a complete inhibition of proliferation at the highest unconventional splice donor site within exon 3, which was also CD83-c-His concentration (1.5 ␮g/ml). At the same time, un- described for alternatively spliced CD28 mRNA (25). Besides the treated cells or cells treated with an irrelevant protein showed no TM domain, in all three splice products, parts of the extracellular such block in proliferation (Fig. 4B). These data suggest an inhib- V-type Ig-like domain are lost, and furthermore, in CD83-a and itory function of soluble CD83 in the immune system. CD83-b, the coding sequence of the TM domain is changed to an amino acid sequence with unknown function and no similarities to Discussion other known proteins. Although all three forms are highly ex- Soluble forms of cell surface molecules can be generated either by pressed on RNA level, only CD83-c is translated into a detectable splicing as in the case of IL-4R (30), IL-7R (31), or Fas (32); protein after transfection into 293-T cells. At present it cannot be enzymatic cleavage of the extracellular domain as in the case of excluded that these other versions of CD83 are expressed as func- TNF (33) and IL-2R (34); or both as was shown before for IL-6R tional proteins as well and that 293-T cells lack cofactors that (35). For different costimulatory molecules like CD28, CTLA-4, or might be necessary for stabilization of CD83-a and CD83-b. CD86, alternative splicing was described (23–28). These soluble Our data suggest that soluble CD83 expressed under noninflam- counterparts of TM proteins have been described to have important matory conditions has a negative immunomodulatory function pre- functions, e.g., to potentiate proliferation and lymphokine produc- venting unspecific effector cell activation. Soluble CD83 has been tion by human T cells stimulated with an anti-CD3 Ab (24). The identified in the sera of healthy individuals, supporting a role of existence of biological active soluble CD83 has been described this protein variant under steady-state conditions. Interestingly, the recently (20–22) and it was suggested that soluble CD83 is pro- amount of soluble CD83 was elevated in the sera of several leu- duced by enzymatic cleavage of the cognate membrane form (20). kemia patients (21), which might lead to an increased threshold for In this study, we have identified three alternative spliced transcripts T cell activation in these patients. After induction of inflammatory of CD83, named CD83-a, CD83-b, and CD83-c, which are constitu- cytokines by polyclonal activators such as PHA, these inhibitory tively expressed by nonactivated human PBMC in addition to the CD83-splice products are down-regulated, which might relieve full-length transcript encoding the membrane-bound form of CD83. this inhibitory effect. The most potent cells for induction of an The human CD83 gene is a single copy gene organized in five immune response are dendritic cells (36). After induction of den- exons (2) similar to the mouse CD83 gene (12), and the TM do- dritic cell maturation these cells strongly up-regulate CD83. There- main is encoded by exon 4. In all three alternatively spliced CD83 fore, the expression of soluble CD83-c in immature DCs might be 6676 ALTERNATIVELY SPLICED CD83 INHIBITS T CELL ACTIVATION a potential mechanism to suppress immune cell activation under 12. Fujimoto, Y., L. Tu, A. S. Miller, C. Bock, M. Fujimoto, C. Doyle, D. A. Steeber, ϩ steady-state conditions. However, we were not able to detect the and T. F. Tedder. 2002. CD83 expression influences CD4 T cell development in the thymus. Cell 108: 755–767. alternatively spliced mRNAs in human myelocytic dendritic cells 13. Sorg, U. R., T. M. Morse, W. N. Patton, B. D. Hock, H. B. Angus, cultured in the presence of GM-CSF and IL-4. Whereas no CD83 B. A. Robinson, B. M. Colls, and D. N. Hart. 1997. Hodgkin’s cells express protein could be detected on the cell surface (data not shown), at CD83, a dendritic cell lineage associated antigen. Pathology 29: 294–299. 14. Dudziak, D., A. Kieser, U. Dirmeier, F. Nimmerjahn, S. Berchtold, A. Steinkasserer, the RNA level, these cells exclusively expressed the CD83 mRNA G. Marschall, W. Hammerschmidt, G. Laux, and G. W. Bornkamm. 2003. Latent encoding for the TM form. This conforms with the notion that membrane protein 1 of Epstein-Barr virus induces CD83 by the NF-␬B signaling pathway. J. Virol. 77: 8290–8298. IL-4/GM-CSF-treated dendritic cells have been referred to as in- 15. Cramer, S. O., C. Trumpfheller, U. Mehlhoop, S. More, B. Fleischer, and termediate rather than immature DCs (37), suggesting that these A. von Bonin. 2000. Activation-induced expression of murine CD83 on T cells cells are no longer in an immature state. Hock et al. (20) have and identification of a specific CD83 ligand on murine B cells. Int. Immunol. 12: 1347–1351. found enhanced levels of soluble CD83 in the supernatant of 16. Scholler, N., M. Hayden-Ledbetter, A. Dahlin, I. Hellstrom, K. E. Hellstrom, and TNF-␣ or LPS matured dendritic cells using a rabbit polyclonal J. A. Ledbetter. 2002. Cutting edge: CD83 regulates the development of cellular Ab. This finding might be explained by a delayed release of the immunity. J. Immunol. 168: 2599–2602. 17. Yang, S., Y. Yang, J. Raycraft, H. Zhang, S. Kanan, Y. Guo, Z. Ronai, soluble CD83 protein by dendritic cells. Indeed we have shown I. Hellstrom, and K. E. Hellstrom. 2004. Melanoma cells transfected to express that CD83 protein can be found in intracellular vesicles before it is CD83 induce antitumor immunity that can be increased by also engaging CD137. expressed at the cell surface (14). Alternatively, it is possible that Proc. Natl. Acad. Sci. USA 101: 4990–4995. 18. Lechmann, M., D. J. Krooshoop, D. Dudziak, E. Kremmer, C. Kuhnt, under these in vitro culture conditions CD83 is shed from the plasma C. G. Figdor, G. Schuler, and A. Steinkasserer. 2001. The extracellular domain membrane of dendritic cells. However, it is not clear if soluble CD83 of CD83 inhibits dendritic cell-mediated T cell stimulation and binds to a ligand protein generated under these conditions is functional and can sup- on dendritic cells. J. Exp. Med. 194: 1813–1821. Downloaded from 19. Zinser E, M. Lechmann, A. Golka, M. B. Lutz, and A. Steinkasserer. 2004. press T cell proliferation. We are currently addressing the role of Prevention and treatment of experimental autoimmune encephalomyelitis by sol- soluble CD83 in dendritic cells in greater detail. uble CD83. J. Exp. Med. 200: 345–351. Combined with previous studies that identified soluble CD83 in 20. Hock, B. D., M. Kato, J. L. McKenzie, and D. N. Hart. 2001. A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is de- human serum, our studies suggest that the generation of soluble tectable in normal human sera. Int. Immunol. 13: 959–967. CD83 by alternative splicing is tightly regulated and might be an 21. Hock, B. D., L. F. Haring, A. Steinkasserer, K. G. Taylor, W. N. Patton, and J. L. McKenzie. 2004. The soluble form of CD83 is present at elevated levels in important mechanism for immune cell activation. http://www.jimmunol.org/ a number of hematological malignancies. Leuk. Res. 28: 237–241. 22. Senechal, B., A. M. Boruchov, J. L. Reagan, D. N. Hart, and J. W. Young. 2004. Acknowledgments Infection of mature monocyte-derived dendritic cells with human cytomegalovi- We acknowledge the advice and human RNAs provided by J. Mautner. We rus inhibits stimulation of T-cell proliferation via the release of soluble CD83. thank A. Steinkasserer and J. Hauber for helpful discussions. For technical Blood 103: 4207–4215. 23. Inobe, M., N. Aoki, P. S. Linsley, J. A. Ledbetter, R. Abe, M. Murakami, and assistance, we are grateful to G. Marschall. T. Uede. 1996. The role of the B7-1a molecule, an alternatively spliced form of murine B7-1 (CD80), on T cell activation. J. Immunol. 157: 582–588. Disclosures 24. Jeannin, P., G. Magistrelli, J. P. Aubry, G. Caron, J. F. Gauchat, T. Renno, The authors have no financial conflict of interest. N. Herbault, L. Goetsch, A. Blaecke, P. Y. Dietrich, et al. 2000. Soluble CD86 is a costimulatory molecule for human T lymphocytes. Immunity 13: 303–312.

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