The CD83 reporter mouse elucidates the activity of the CD83 in B, T, and populations in vivo

Matthias Lechmann*, Naomi Shuman, Andrew Wakeham, and Tak W. Mak*

Campbell Family Institute for Breast Cancer Research and Ontario Cancer Institute, University of Toronto, Toronto, ON, Canada M5G 2C1

Contributed by Tak W. Mak, July 1, 2008 (sent for review December 11, 2007) CD83 is the major surface marker identifying mature dendritic cells (22). However, truncated splice forms of CD83 have yet to be (DCs). In this study, we report the generation of reporter mice detected in human serum. At least some sCD83 may be generated expressing EGFP under the control of the CD83 promoter. We have by proteolytic cleavage of mCD83 (20). used these mice to characterize CD83 expression by various im- Although the CD83 ligand remains a mystery, analyses of - mune system cell types both in vivo and ex vivo and under targeted CD83-deficient mice have revealed that thymic CD83 steady-state conditions and in response to stimulation with a expression is crucial for the maturation of CD4ϩCD8ϩ Toll-like receptor (TLR) ligand. With those mice we could prove in into CD4ϩ T cells (13, 14). In addition, CD83 may regulate the vivo that the CD83 promoter is highly active in all DCs and B cells intercellular interactions between DCs and peripheral T and B cells in lymphoid organs. Interestingly, this promoter activity in B cells (12, 23–25). In vitro culture of either human or murine lymphocytes mainly depended on the stage of development, is up-regulated in in the presence of sCD83 inhibits their proliferation (26, 27). More the late pre- stage, and was maintained on a high level in all remarkably, the administration of recombinant human CD83 pro- peripheral B cells. We also confirmed that CD83 in those cells is tein can prevent the onset of experimental autoimmune encepha- mainly intracellular but is up-regulated after TLR stimulation. lomyelitis (EAE; a mouse model for multiple sclerosis) and even to Otherwise, CD83 promoter activity in T cells seemed to depend on cure established EAE disease in vivo (28). Another group has ؉ ؉ stimulation and could be found mainly in CD4 CD25 and demonstrated that administration of soluble human Ig-conjugated ؉ ؉ ؉ ؉ CD8 CD25 T cells and in CD4 and CD8 memory cells. In addition, CD83 can delay acute rejection of MHC-mismatched mouse skin we identified the murine homologues of the human CD83 splice allografts (29). To identify the physiological signaling pathways variants. In contrast to those in human, those extremely rare short underlying these effects, it will be necessary to conduct studies using transcripts were never found without the expression of the highly CD83 that has been correctly folded and posttranslationally mod- dominant full-length form. So, the murine CD83 surface expression ified in a living organism. Here, we describe a CD83 knockin mouse is mainly regulated posttranslationally in vivo. Our CD83 reporter generated by positioning a reporter cassette consisting of EGFP mice represent a useful mouse model for monitoring the activation linked to an internal ribosomal entry site (IRES2; ref. 30) right after status and migration of DCs and lymphocytes under various con- the CD83 stop codon. We have examined EGFP expression in ditions, and our results provide much needed clarification of the various tissues of these CD83 reporter mice and have observed true nature of CD83 promoter activity. strong CD83 promoter activity early during the differentiation of B cells and DCs. Moreover, this activity is enhanced by inflammatory ͉ ͉ immune system mouse model intravital microscopy stimuli. In contrast, CD83 promoter activity is weak in naı¨ve CD4ϩ peripheral T cells and very weak in naı¨ve CD8ϩ peripheral T cells. he primary cells mediating adaptive responses are T and B Our CD83 reporter mouse model is suitable for use in any lymphocytes and dendritic cells (DCs). A DC’s ontogeny, state T immunological experiment in which the nature of CD83 signaling IMMUNOLOGY of differentiation, and degree of maturation governs its expression is examined or in which the generation, migration, and/or suppres- of a plethora of membrane-bound and soluble molecules that can sion of DC, T, or B cell activation must be followed. induce immunostimulatory and immunosuppressive responses (1– 3). Maturing DCs up-regulate certain cell surface molecules that Results greatly enhance their ability to present antigen and activate naive Generation of CD83-IRES2-EGFP Mice. ϩ ϩ We created CD83-IRES2- CD4 and CD8 T cells. Among these molecules is CD83, first EGFP mice by using standard methods of homologous DNA described by Zhou et al. (4), CD83 is one of the most useful markers recombination in embryonic stem (ES) cells. The targeting vector for identifying mature DCs capable of activating naı¨ve T cells (5–8). inserted a reporter cassette consisting of EGFP linked to a viral CD83 expression also occurs on certain T cell subsets (9, 10), B cells IRES2 sequence positioned right after the stop codon located in (10–12), and murine thymic epithelial cells (13, 14). Studies of exon 5 of the genomic CD83 gene (Fig. 1A). A neomycin (neo) ␬ CD83 transcription have shown that it is mediated by NF- B during resistance gene introduced after the EGFP stop codon in the the induction of adaptive responses (15, 16). targeting vector enabled selection. The entire coding region of the CD83 is conserved from fish species to mammals (17), with CD83 gene remained intact in the targeting construct. Expression mouse CD83 sharing 63% amino acid identity with human CD83 of the targeted CD83 gene generated a bicistronic transcript (18, 19). To date, two isoforms of CD83 have been reported in humans: a membrane-bound form (mCD83) (5) and a soluble form (sCD83) (20). mCD83 is a highly glycosylated surface protein Author contributions: M.L. and T.W.M. designed research; M.L., N.S., and A.W. performed of the Ig superfamily with a molecular mass of 40–45 kDa (5, 21). research; M.L. and T.W.M. analyzed data; and M.L. and T.W.M. wrote the paper. mCD83 contains an extracellular Ig-like V domain at the N The authors declare no conflict of interest. terminus, a short intracellular cytoplasmic domain of 39 aa, and one Freely available online through the PNAS open access option. transmembrane domain (5). In contrast, sCD83 may contain only *To whom correspondence may be addressed. E-mail: [email protected] or the extracellular Ig-like domain (20). But the origin of sCD83 is not [email protected]. yet clear. In humans, four different splice variants of CD83 have This article contains supporting information online at www.pnas.org/cgi/content/full/ been sequenced. The largest variant encodes mCD83, whereas all 0806335105/DCSupplemental. of the smaller transcripts encode putative soluble forms of CD83 © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806335105 PNAS ͉ August 19, 2008 ͉ vol. 105 ͉ no. 33 ͉ 11887–11892 Downloaded by guest on September 24, 2021 Fig. 1. Generation of CD83-IRES2-EGFP reporter mice. (A) CD83 knockin gene targeting strategy. Untranslated regions, filled boxes; ORFs, open boxes; PCR primers, arrowheads; DT-A, diphtheria toxin subunit A. The Southern blot probes a, b, and neo are indicated by underlining. (B) Southern blot analysis of BamHI- digested DNA from ES cell clones. het, heterozygous; wt, wild type; wt band, filled arrow; mutant band, open arrow. (C) PCR screening of tail DNA of four littermate progeny of CD83-IRES2-EGFPϩ/Ϫ mice backcrossed to C57Bl6 (Bl6) mice. Control, water.

encoding both CD83 and EGFP. It is unknown whether splicing of murine CD83 mRNA occurs; however, our EGFP reporter is still likely to be expressed because, in humans, all splice forms include exon 5. Targeted IRES2-EGFP-neo insertion was confirmed by Southern blot analysis of genomic DNA from ES cell clones (Fig. 1B). The genotypes of offspring generated by crossing heterozygous Fig. 2. EGFP expression in untreated and LPS-stimulated BMDCs. (A) Lack of ϩ/Ϫ CD83-IRES2-EGFP mice to C57BL/6 mice were confirmed by EGFP expression by or granulocytes isolated from freshly extracted BM ϩ Ϫ PCR analysis (Fig. 1C). CD83-IRES2-EGFP / and CD83-IRES2- from either CD83-IRES2-EGFPϩ/Ϫ mice ( line) or WT littermates (Ϫ/Ϫ, dark line). EGFPϩ/ϩ mice were born at the expected Mendelian frequency and This result did not change after stimulation with LPS for 24 h. (B) Increasing ϩ ϩ ϩ Ϫ have thrived and reproduced as well as their WT (Ϫ/Ϫ) littermates. numbers of CD11c EGFP cells in CD83-IRES2-EGFP / BMDC cultures treated loϩint hi No obvious morphological and developmental abnormalities have with GM-CSF for 4, 6, or 10 days. Gray, CD83EGFP ; green, CD83EGFP .(C) ϩ/Ϫ Surface expression of the DC activation markers CD80, CD83, CD86, and MHCII been detected in CD83-IRES2-EGFP or CD83-IRES2- b ϩ ϩ/ϩ (I-A ) on CD11c BMDCs from CD83-IRES2-EGFP mice was compared with the EGFP mice of up to 12 months of age. EGFPlo population on day 8 (gray line) versus EGFPhi cells at 22 h post-LPS stimulation (green line). (D) EGFP expression in bright field plus fluorescence Analysis of Bone Marrow (BM)-Derived DCs (BMDCs) from CD83-IRES2- microscopic image overlays (ϫ20) of CD83-IRES2-EGFPϩ/Ϫ (a and b) and WT (c) EGFP Mice. To confirm the activity of the CD83 promoter and the BMDCs untreated (a) or after 24 h LPS stimulation (a and c). (d) Fluorescence specificity of EGFP expression in our reporter mice, we generated image of WT (Upper) and CD83-IRES2-EGFPϩ/Ϫ (Lower) spleens ex vivo.(E) Kinetics ϩ Ϫ DCs in vitro from BM precursors of CD83-IRES2-EGFPϩ/Ϫ mice. of EGFP versus mCD83 expression as determined by FACS of CD11c and CD11c ϩ/Ϫ Ϫ/Ϫ Only 5% or fewer of cells in freshly isolated BM expressed EGFP. populations of CD83-IRES2-EGFP and CD83-IRES2-EGFP BMDCs that were In contrast, ex vivo fluorescence microscope analysis of spleens either left untreated or stimulated with LPS for the indicated times. (F) Compar- ison of surface CD83 and intracellular (ϩsurface) CD83 after saponin treatment from those mice showed a strong EGFP expression pattern (Fig. Ϫ ϩ Ϫ ϩ with the kinetics of EGFP expression after LPS stimulation. Results shown are one 2D). CD11c Gr1 granulocytes and CD11c CD11b monocytes analysis representative of at least three independent littermate pairs. from freshly extracted BM did not express EGFP, whether or not they were treated with LPS (1 ␮g/ml) for 24 h (Fig. 2A). However, after 8–10 days of in vitro culture, large numbers of proliferating and for 16–24 h and observed (as expected) that EGFP, CD80, CD86, differentiating CD11cϩ BMDCs were observed that showed an and MHCII were all up-regulated on all BMDC populations (Fig. up-regulation of EGFP in a particular subpopulation (Fig. 2B). 2C). However, EGFPhiCD11cϩ BMDCs showed a much greater These EGFPhiCD11cϩ cells also showed high levels of the DC increase in surface levels of CD80, CD86, and MHCII than did activation markers CD80, CD83, CD86, and MHC class II (MH- EGFPlow and EGFPint CD11cϩ cells. No cells showing CD83 CII), whereas EGFPlowϩintCD11cϩ cells showed low to intermedi- expression in the absence of EGFP expression were detected. Thus, ate expression of these markers (Fig. 2C). To activate the cultured EGFP expression increases with DC maturation and the normal BMDCs, we treated them on culture days 8–10 with LPS (1 ␮g/ml) up-regulation of other costimulatory markers in our knockin mice.

11888 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806335105 Lechmann et al. Downloaded by guest on September 24, 2021 We next examined the kinetics of DC activation marker induc- were EGFPϩ (Fig. 3B). Similarly, Ϸ5% of CD4ϩCD25ϩCD127Ϫ tion in CD83-IRES2-EGFPϩ/Ϫ BMDCs. The induction patterns of regulatory T cells (Tregs) present in the thymus expressed EGFP CD80, CD86, and MHCII expression on CD83-IRES2-EGFPϩ/Ϫ (Fig. 3B). Only very low numbers (1% or less) of thymic BMDCs were comparable to those observed for BMDCs obtained CD4ϪCD8ϪTCR␣␤ϩ cells and thymic ␥␦ϩ T cells were EGFPϩ from WT littermates. However, the kinetics of CD80, CD86, and (Fig. 3B). MHCII induction in the knockin BMDCs were slightly different The high level of EGFP expression in the spleen and LNs was from those observed for EGFP expression, in that 21% of largely caused by the B220ϩ B cell population. In the spleen, 65% CD86hiCD11cϩ cells and 35% of MHCIIhiCD11cϩ cells did not of IgMϩIgDϪ and almost 90% of IgMϩIgDϩ B cells showed EGFP express EGFP (Fig. 2C). Nevertheless, after LPS stimulation for expression. Almost all CD23hiCD21loIgMlo follicular B cells and 24 h, fluorescence microscopy revealed bright EGFP fluorescence CD23hiCD21hiIgMhi marginal zone precursor cells (transitional 2, in the cytoplasm of Ͼ90–95% of CD83-IRES2-EGFPϩ/Ϫ BMDCs T2) and Ͼ80% of CD23loCD21loIgMhi transitional 1 (T1) and (Fig. 2D). We then compared the kinetics of EGFP versus CD83 CD23loCD21hiIgMhi marginal zone B cells were EGFPϩ (Fig. 3C). surface expression in LPS-stimulated CD11cϩ and CD11cϪ BM About 20% of CD3ϩCD4ϩ splenic T cells were EGFPϩ but only 3% cells. Significant increases in EGFP and CD83 expression were of CD3ϩCD8ϩ splenic T cells expressed EGFP at all and these were detected in knockin CD11cϩ BMDCs by 2 h post-LPS stimulation EGFPlo. Further examination of T cell subsets revealed that EGFP (Fig. 2E). A small population of EGFPϩCD83Ϫ cells was present was expressed by only 5–10% of CD62LϩCD44Ϫ naı¨ve CD4ϩ and among untreated knockin CD11cϩ BMDCs and in CD11cϩ BM- CD8ϩ T cells, but that almost 60% of central and Ͼ30% of effector DCs that had been LPS-stimulated for only 45 min, but these latter memory CD4ϩ T cells, and 20% of central and Ϸ10% of effector cells very rapidly became CD83ϩ as the LPS stimulation progressed. memory CD8ϩ T cells, were EGFPϩ (Fig. 3C). In general in the After2hofLPStreatment, the up-regulation of both EGFP and spleen, the total EGFP expression in the T cell compartment was CD83 in knockin CD11cϩ BMDCs appeared to be linear (Fig. 2E). always lower than in the B cell compartment. With respect to This up-regulation pattern was confirmed by using real-time PCR splenic DCs, 70% of CD11bϩCD8Ϫ DCs, Ͼ80% of CD11bϪCD8ϩ and Western blotting [supporting information (SI) Fig. S1 A and B]. DCs and 88% of CD11cϩB220ϩ Gr-1ϩ plasmacytoid DCs (pDCs) Surface CD83 or EGFP expression was detected in only a small were EGFPϩ under steady-state conditions (Fig. 3C). In contrast, number of unstimulated CD11cϩ BMDCs, but never in unstimu- only 2–3% of CD11cϪCD11bhi splenocytes were EGFPϩ (Fig. 3C). lated or LPS-stimulated CD11cϪ populations (Fig. 2E). Further In the LNs, the dominant EGFP-expressing population was again FACS analysis investigating surface versus intracellular CD83 ex- the B220ϩ B cells. About 80–90% of IgMϩIgDϪ LN B cells and pression in permeabilized cells confirmed that the EGFP expres- Ͼ90% of IgMϩIgDϩ LN B cells were EGFPϩ (Fig. 3D). Among LN sion correlates the total CD83 expression (Fig. 2F). To demonstrate T cells, Ϸ9–10% of CD3ϩCD4ϩCD25ϪCD69Ϫ naı¨ve T cells but that the immune system was functionally normal in our CD83- Ͻ2% of CD3ϩCD8ϩCD25ϪCD69Ϫ naı¨ve T cells expressed EGFP IRES2-EGFP knockin mice, we performed mixed lymphocyte (Fig. 3D). About 78% of CD4ϩCD25ϩCD69ϩ activated T cells were reactions and measured T cell proliferation. LPS-stimulated BM- EGFPϩ but only 58% of CD8ϩCD25ϩ activated T cells showed DCs from CD83-IRES2-EGFPϩ/Ϫ mice showed the same capacity some level of EGFP expression (Fig. 3D). With respect to LN DCs, as LPS-stimulated BMDCs from control littermates to stimulate almost all (87–100%) were EGFPϩ. Indeed, EGFP expression by BALB/c splenocytes in co-cultures (Fig. S1C). In addition, cultures CD8ϩ DCs and pDCs in LNs was up to one log greater than EGFP of WT and CD83-IRES2-EGFPϩ/Ϫ BMDCs showed identical expression by LN B cells or LN CD8Ϫ DCs (Fig. 3D). Less than 4% yields and equivalent viability, as determined by trypan blue or of CD11cϪCD11bhi LN cells expressed EGFP (Fig. 3D). Annexin V staining (data not shown). We also analyzed cDNAs of Our experiments with CD83-IRES2-EGFPϩ/Ϫ BMDCs in vitro BMDCs, splenocytes, splenic B cells, thymocytes, and BM cells showed a clear expression pattern in which mCD83 was present on from WT and CD83-IRES2-EGFPϩ/Ϫ mice. We identified very low the surface of EGFPϩ cells. However, we were unable to replicate expression levels of spliced variants of the CD83 mRNAs homol- this pattern in vivo when we examined freshly isolated cell popu- ogous to the described human transcripts in those cell types (Fig. lations with a commercially available anti-CD83 antibody (Michel- S2). In our hands, LPS activation of BMDCs did not show a relevant 17) (31). Consistent with data published by other groups (10, 32), shift in the expression profile of those splice forms (Fig. S2B). we found that neither freshly isolated splenic DCs nor CD3ϩCD8ϩ IMMUNOLOGY T cells expressed substantial amounts of mCD83 (Fig. 4A). Fur- CD83 Promoter Activity in Vivo in the Absence of Stimulation. We next thermore, only 10–15% of freshly isolated CD3ϩCD4ϩEGFPϩ T used flow cytometry to analyze CD83 promoter activity in the BM, cells and EGFPϩB220ϩ B cells expressed mCD83. EGFPϪ DCs, T thymus, spleen, and lymph nodes (LNs) of CD83-IRES2-EGFPϩ/Ϫ cells, and B cells showed no anti-CD83 binding (Fig. 4A). mice under steady-state conditions in vivo. About 5% of total BM cells expressed EGFP (Fig. 3A), whereas Ͻ5% of thymic cells were CD83 Promoter Activity After LPS Challenge in Vivo. We next inves- EGFPϩ (Fig. 3B). In the spleen, 35–50% of total splenocytes tigated whether CD83 promoter activity in our reporter mice could expressed EGFP (Fig. 3C). About 50% of LN cells were also be further induced by treatment with a Toll-like receptor (TLR) EGFPϩ (Fig. 3D). These high levels of EGFP expression in the ligand. To this end, we i.p.-injected groups of CD83-IRES2- spleen and LNs were unexpected but consistent in all animals EGFPϩ/Ϫ mice with LPS (100 ␮g/ml PBS) and analyzed their tested. Further investigation of the BM revealed that Ͻ1% of CD8ϩ splenocytes after 22 h. As controls, we i.p-injected CD83-IRES2- T cells but Ϸ20% of CD4ϩ T cells expressed EGFP (Fig. 3A). As EGFPϩ/Ϫ mice with PBS and CD83-IRES2-EGFPϪ/Ϫ littermates well, Ͻ1% of B220intCD43ϩ and up to 3% of B220intCD43Ϫ BM of the knockin mice with LPS or PBS (Fig. 4B). Flow cytometric cells showed CD83 promoter activity (Fig. 3A). All analysis revealed that, despite the high base level of CD83 promoter EGFPϩB220intCD43Ϫ BM cells were also IgMϩ (data not shown). activity in the spleen of unstimulated (PBS) control mice, the More than 90% of B220hi cells in the BM expressed EGFP and number of EGFPϩ cells in this organ almost doubled after LPS Ͼ95% of B220hiIgMϩ IgDϩ B cells were EGFPhi (Fig. 3A). The low injection (Fig. 4B). More detailed screening demonstrated that EGFP expression in the thymus was mainly caused by a population EGFP expression by B220ϩIgMϩ splenic B cells of LPS-stimulated of CD4ϪCD8ϪTCR␥␦ϪTCR␤Ϫ cells; Ϸ40% of these cells were knockin mice increased by Ϸ20% such that Ͼ90% of splenic B cells EGFPϩ (Fig. 3B). Further analysis revealed that this thymic were EGFPϩ. In contrast, only Ϸ70% of B cells in PBS-treated EGFPϩ cell population was composed of 15–35% B220ϩCD11cϪ knockin mice were EGFPϩ. In addition, the total EGFP fluores- B cells, 5% CD11cϩ DCs, and 60% CD11cϪDEC-205ϩ thymic cence in LPS-treated knockin splenic B cells was elevated. Thus, not epithelial cells (data not shown). CD4ϩCD8ϩ thymocytes were only the number of EGFPϩ cells but also the level of EGFP EGFPϪ but up to 5% of CD4ϩCD8Ϫ and CD4ϪCD8ϩ thymocytes expression per individual cell were up-regulated in response to LPS

Lechmann et al. PNAS ͉ August 19, 2008 ͉ vol. 105 ͉ no. 33 ͉ 11889 Downloaded by guest on September 24, 2021 Fig. 3. CD83 promoter activity in murine tissues. Samples of BM, thymus, spleen, and LNs were assessed for the presence of EGFP-expressing cells using flow cytometric analysis of immunostained cells. Results shown are representative of the levels of EGFP expression detected in the indicated gated subpopulations obtained from four or more littermate pairs of CD83-IRES2-EGFPϩ/Ϫ and CD83-IRES2-EGFPϪ/Ϫ mice. , DC, B cell, and T cell subsets and their activation status were determined based on the expression of various cell surface markers. The dot blots show total EGFPϩ cells in BM (A), thymus (B), spleen (C), and LNs (D) as related to forward scatter. The histograms show EGFP expression in the indicated gated cell subpopulations from CD83-IRES2-EGFPϩ/Ϫ mice (dark line) versus their WT littermates (Ϫ/Ϫ, light line) in %max.

(Fig. 4B). CD11cϩ splenic DC populations of LPS-treated knockin Discussion mice demonstrated a similar increase in CD83 promoter activity The murine CD83 promoter has not been precisely defined, which compared with controls. Elevated EGFP was observed in Ͼ90% of ϩ obviated the creation of a transgenic animal. To elucidate CD83 total CD11c splenic DCs in the mutants (Fig. 4B). However, LPS stimulation did not significantly increase EGFP expression in either promoter activity in immune system cells, we generated CD83 CD4ϩ or CD8ϩ T cells of knockin mice (Fig. 4B). No EGFP or knockin reporter mice by using a cloning strategy that minimized CD83 expression was detected in either neutrophils or BM mono- interference with CD83 function (Fig. 1). Expression of the tar- cytes either under steady-state conditions or after LPS injection geted CD83 gene successfully produced a bicistronic transcript (data not shown). encoding both CD83 and EGFP.

11890 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806335105 Lechmann et al. Downloaded by guest on September 24, 2021 Functionally, our knockin BMDCs showed normal viability and T cell activation capacity. When we screened our knockin mice for in vivo CD83-EGFP expression, we noted weak CD83 promoter activity in BM (Fig. 3A) and thymus (Fig. 3B) but surprisingly strong CD83 promoter activity in spleen (Fig. 3C) and LNs (Fig. 3D). The main sources of CD83-EGFP expression in the latter tissues were the resident B cell populations. In contrast to CD83 expression by BMDCs and T cells, CD83 expression by B cells appeared to depend on differentiation stage rather than activation status because all B cell populations in the knockin spleen and LNs were highly positive for EGFP. In BM, pro-B and early pre-B cells showed no EGFP expression (Fig. 3A) but CD83 promoter activity was up-regulated in late pre-B cells. The majority of naı¨ve immature B cells were EGFPϩ, as were nearly 100% of recirculating B cells (Fig. 3A). With respect to the thymus, most thymocytes did not express CD83 during thymic selection (Fig. 3B), but B220ϪCD11cϪDEC-205ϩ thymic stromal cells, which have been previously shown to be CD83ϩ (13), recirculating B cells, and DCs were EGFPϩ (data not shown). In contrast to DCs and B cells, the knockin mice demonstrated an activation-dependent promoter activity in T cells. Only 5–10% of naı¨ve CD4ϩ and CD8ϩ T cells were EGFPϩ, but up to 80% of CD4ϩ and 45% of CD8ϩ-activated CD25ϩ T cells showed increased EGFP expression. In addition, 30% of effector memory cells, 60% of central memory CD4ϩ T cells, and 10–20% of memory CD8ϩ T cells activated the CD83 promoter (Fig. 3 C and D). Lastly, Ϸ5% of thymic Tregs and 30–40% of splenic and LN Tregs were EGFPϩ, implying that CD83 alone cannot be used to distinguish these cells from the large numbers of EGFPϩ-activated conventional T cells in Fig. 4. Correlation between CD83-EGFP and CD83 surface expression and these organs. CD83-EGFP up-regulation after LPS challenge in vivo.(A) Flow cytometric analysis The unexpectedly high level of CD83-EGFP expression we found of CD83 expression (blue) by EGFPϩ versus EGFPϪ splenocytes from untreated in BMDCs stands in contrast to the much lower levels of in vivo CD83-IRES2-EGFPϩ/Ϫ mice versus isotype control (black). PE, phycoerythrin. (B) mCD83 expression reported by other groups (6, 9, 11, 13) and EGFP expression in mouse spleens at 22 h after i.p. injection of LPS (numbers show confirmed by us (Fig. 4A). Only Ϸ10% of freshly isolated EGFPϩ percentage of EGFPϩ cells; gray line, PBS-treated Ϫ/Ϫmice; black line, LPS-treated B cells and EGFPϩCD4ϩ T cells, and no EGFPϩ DCs, showed Ϫ/Ϫ mice; blue line, PBS-treated ϩ/Ϫ mice; red line, LPS-treated ϩ/Ϫ mice). mCD83 expression. Thus, CD83 induction occurs early during an Results shown are one analysis representative of two independent littermate immune response but the protein appears to be confined intracel- pairs. lularly in a much wider spectrum of cells than previously appreci- ated, with major concentrations being found in B cells, DCs, and CD4ϩ T cells. Recent reports have demonstrated that CD83 not We also confirmed next to the by far dominant CD83-TM RNA only colocalizes with MHCII molecules but also modifies their the murine homologues to the human CD83-a, CD83-b, and turnover rate in B cells, T cells, and DCs (10, 35). In addition, CD83 CD83-c forms (Fig. S2). As in human, those shorter splice forms is required for the viability of B and CD4ϩ T cells (10, 35). Thus, were extremely rare transcripts. We could not find a significant the high levels of CD83 promoter activity that we observed in both IMMUNOLOGY difference in ratios of expression profiles before and after stimu- immature and mature B cells may not only prepare these cells for lation with LPS (Fig. S2). So, all EGFP-expressing cells reflected activation but also provide a survival signal. Furthermore, the mainly the expression of CD83-TM, because of the very low RNA number of CD83ϩ B cells and DCs and their relative expression of levels of the short types, whereas no analyzed cell type showed only this marker were increased by LPS treatment of the knockin mice the shorter splice forms. (Fig. 4B). (As expected, there was no further CD83 induction in T After confirming the specificity of our reporter gene under cells, which lack the relevant TLR.) The up-regulation of CD83 ϩ controlled conditions of BMDC culture, we showed that all EGFPhi promoter activity in LPS-treated CD11c DCs was massive com- BMDCs derived from our knockin mice were also positive for pared with that in B cells (Fig. 4B). If CD83 indeed controls the CD80, CD86, and MHCII expression, as expected for mature, fully MHCII turnover rate, this dramatic elevation in CD83 activity ϩ Ϫ could explain why mature DCs are so much more efficient than activated DCs (Fig. 2B). We also identified an EGFP mCD83 Ϫ DC population that represents immature DCs, consistent with immature DCs in presenting antigens. Almost all splenic CD8 DCs were EGFPϩ but expressed much less CD83 per cell than did previous reports in which intracellular pools of CD83 were found CD8ϩ DCs and pDCs. Thus, it may be that CD83 is involved in the in the Golgi complex and endocytic vesicles of immature and efficient cross-presentation of antigens to T cells, a capacity unique mature DCs (33, 34). However, in contrast to the findings of Cao ϩ Ϫ to CD8 DCs (36). et al. (34), we did not detect CD83 promoter activity in CD11c Prazma et al. (10) have reported the up-regulation of CD83 on monocytes/macrophages (Fig. 2A). With respect to TLR engage- CD4ϩ and CD8ϩ T cells. Their observation that CD83 up- ment, LPS stimulation of BMDCs significantly induced EGFP and regulation does not occur until 4–6 h after activation can be easily mCD83 to levels that further increased over time in parallel with explained by results showing that neither CD83 promoter activity CD80, CD86, and MHCII (Fig. 2 B, E, and F). Interestingly, nor preformed intracellular CD83 molecules are present in naı¨ve T although these DC activation markers were all highly induced by cells. Prazma et al. further described a loss of viability of CD83- TLR signaling, they differed in their expression kinetics (Fig. 2C). deficient CD4ϩ T cells. Regulation of MHCII expression is thought BMDCs could be CD86hi and MHCIIhi at the same time as they to limit T cell clonal expansion and thus control T cell responses were EGFPϪ, but CD80ϩ and CD83ϩ cells were invariably EGFPϩ. (37). However, whether CD83 truly regulates MHCII expression

Lechmann et al. PNAS ͉ August 19, 2008 ͉ vol. 105 ͉ no. 33 ͉ 11891 Downloaded by guest on September 24, 2021 and thus T cell responses requires additional investigation. Another 1640/10% FCS medium for 22–24 h. CD83 expression was determined by flow unresolved issue is the definition of the differences between the cytometry and fluorescence microscopy as described below. CD83ϩ and CD83Ϫ memory T cell populations. With our CD83- IRES2-EGFP reporter mice, we should be able to answer these Viability Assays. Cell viability assay was done by using trypan blue with Vi-CELL questions and monitor the activation and migration of DCs and T XR cell viability analyzer with Vi-CELL XR 2.03 software (Beckman Coulter). Cell has been measured by translocation of the membrane phospholipid and B cell subpopulations during immune responses in vivo. phosphatidylserine by using phycoerythrin-conjugated Annexin-V following the protocols of the manufacturer (BD Bioscience). Materials and Methods Generation of CD83-IRES2-EGFP Reporter Mice. To generate the CD83-IRES2- Lymphocyte Activation Assays. For mixed leukocyte reactions, titrated numbers EGFP targeting vector, we used the Expand High Fidelity PCR System (Roche) to (3,800–15,000) of day-11 cultured BMDCs were cocultured for 48–72 h in a amplify two DNA fragments from E14K ES cell (129/Ola) genomic DNA. The long 96-well flat-bottom plate (Falcon) with 2 ϫ 105 splenocytes isolated from BALB/c homology arm was a 6-kb segment starting upstream of CD83 exon 3 and mice (Jackson). Cultures were then pulsed with 1 ␮Ci per well [3H]methyl- containing exon 4 and the ORF region of exon 5, including the CD83 stop codon thymidine (PerkinElmer) overnight for 12–16 h to measure proliferation. Plates (Fig. 1A). The short homology arm was a 2-kb segment containing the 3Ј terminal were harvested with a Filtermate harvester (PerkinElmer), and thymidine incor- part of exon 5 and its downstream sequence. The PCR products were cloned into poration was determined by using Top Count NXT (PerkinElmer). All samples a modified pSPUC࿝LFneoDTA vector containing the diphtheria toxin subunit A were analyzed in triplicate. cassette, an IRES2-EGFP sequence (derived from pIRES2-EGFP; Clontech), fol- lowed by a neo resistance cassette. We electroporated E14K ES cells with the Cloning and Sequencing of CD83 mRNA Splice Products. cDNAs from total RNA targeting construct and selected transfectants in G418. Five ES cell clones that had of isolated murine cells were synthesized as described (22). CD83 cDNA was undergone homologous recombination (as confirmed by Southern blot analysis) amplified with primers binding to exon 1 (5Ј-GCCTCCAGCTCCTGTTTCTA-3Ј) and were used to generate CD83-IRES2-EGFP knockin mice using a standard protocol exon 5 (5Ј-TGGGAAAATGCTTTGTAGTCG-3Ј) as follows: 15 min 94°C, 36–40 cycles ϩ Ϫ (32). CD83-IRES2-EGFP / mice were then backcrossed extensively to C57BL/6 of 30 s 94°C, 30 s 55°C, 90 s 72°C, and a final 10-min 72°C step. The amplified ϩ Ϫ mice (Jackson). CD83-IRES2-EGFP / mice and control littermates from backcross fragments were subcloned into pCR2.1 (Invitrogen) and sequenced. generations F3–F6 were used for all experiments. All animal experiments were performed according to protocols approved by the Animal Care Committee of Flow Cytometric Analysis and Fluorescence Microscopy. Flow cytometry was the University Health Network (Toronto). For Southern blot analyses, we isolated carried out on samples of BM, spleen, thymus, LNs, peritoneal fluid, or BMDCs genomic DNA from E14K ES cells and PCR-amplified DNA segments representing according to standard protocols. All antibodies were purchased from BD PharM- parts of CD83 2 (probe c, 455 bp) and the 3Ј UTR following exon 5 (probe ingen, except anti-CD83 (Michel-17; Ebioscience). Analyses of immunostained a, 674 bp). A neo-specific probe (603 bp) was also used for Southern analysis. cells were performed by using a FACS Canto with CellQuest (BD Bioscience) Germ-line transmission of the mutant allele was determined by PCR screening of software. For fluorescence microscopy, BMDCs derived from CD83-IRES2-EGFPϩ/Ϫ the mice using DNA extracted from tails via ethanol precipitation (WT, 160 bp; mice were stimulated in vitro with LPS as described above and analyzed 22 h later knockin, 280 bp). for EGFP expression with a Zeiss Axiovert 200M inverted fluorescence microscope with deconvolution software (Zeiss). Generation of BMDCs. BMDCs were generated from CD83-IRES2-EGFPϩ/Ϫ and Ϫ Ϫ CD83-IRES2-EGFP / knockin mice and C57BL/6 controls as described (38). BMDCs ACKNOWLEDGMENTS. We thank M. Saunders for scientific editing. M.L. was were either left untreated or incubated with 1 ␮g/ml LPS (Sigma) in RPMI medium supported by German Science Foundation Grant DFG/LE1853/1-1.

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