Inflammatory Lesions (CD80) and B7.2 (CD86) in Vitro and In

Human Muscle Cells Express a Functional Costimulatory Molecule Distinct from B7.1 (CD80) and B7.2 (CD86) In Vitro and in Inflammatory Lesions This information is current as of September 26, 2021. Lüder Behrens, Martin Kerschensteiner, Thomas Misgeld, Norbert Goebels, Hartmut Wekerle and Reinhard Hohlfeld J Immunol 1998; 161:5943-5951; ; http://www.jimmunol.org/content/161/11/5943 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Human Muscle Cells Express a Functional Costimulatory Molecule Distinct from B7.1 (CD80) and B7.2 (CD86) In Vitro and in Inﬂammatory Lesions1

Lu¨der Behrens,* Martin Kerschensteiner,* Thomas Misgeld,* Norbert Goebels,*† Hartmut Wekerle,* and Reinhard Hohlfeld2*†

The B7 family of costimulatory molecules likely includes members distinct from B7.1 (CD80) and B7.2 (CD86). After stimulation with IFN-␥ or TNF-␣, human myoblasts selectively express BB-1, but not B7.1 or B7.2. BB-1 is detected by anti-BB-1, a mAb cross-reacting with B7.1 (but not B7.2) and an as yet undeﬁned costimulatory molecule. The absence of B7.1 and B7.2 in BB-1- positive myoblasts was conﬁrmed by RT-PCR. The molecule detected by anti-BB-1 is functional, because anti-BB-1 mAb and

CTLA4Ig (but not anti-B7.1- or anti-B7.2-specific mAbs) completely inhibit Ag presentation by cytokine-induced myoblasts to Downloaded from HLA-DR-matched Ag-specific CD4؉ T cell lines. Stimulation of myoblasts with IL-4 induces B7.1 and B7.2, as well as BB-1, but with different time kinetics. Stimulation of CD40-positive myoblasts with anti-CD40 mAb selectively induces BB-1, whereas stimulation with CD40L-transfected mouse L cells induces BB-1 and B7.1, with different kinetics. To assess whether BB-1 is expressed in muscle tissue, we investigated 23 muscle biopsy specimens from patients with polymyositis, dermatomyositis, inclusion body myositis, Duchenne muscular dystrophy, and nonmyopathic controls by immunohistochemistry and confocal laser microscopy. We found that, in all inflammatory myopathy cases, but not in normal muscle, many muscle fibers strongly react with http://www.jimmunol.org/ anti-BB-1. In contrast, muscle fibers did not react with B7.1- or B7.2-monospecific mAbs in any of the pathologic specimens or in normal muscle. Our results demonstrate that human muscle cells can be induced to selectively express BB-1, a functional costimulatory molecule distinct from B7.1 and B7.2. This molecule may play an important role in the immunobiology of muscle. The Journal of Immunology, 1998, 161: 5943–5951.

uscle can be a site of desirable and undesirable im- nocytochemical and RT-PCR studies of cultured myoblasts. Fur- mune reactions (1). These are deliberately induced by thermore, we investigated human muscle biopsy specimens for ex- protein- or DNA-based vaccination (2, 3) or develop pression of the B7 family of costimulatory molecules, using mAbs M by guest on September 26, 2021 spontaneously during the course of autoimmune and infectious specific for B7.1 (12) or B7.2 (13), and anti-BB-1 (9), a mAb that muscle diseases (4, 5). Furthermore, local immune reactions can cross-reacts with B7.1 and a hitherto undefined member of the B7 pose a serious problem after intramuscular injection of vectors for family (10, 11). We observed that, whereas normal muscle fibers gene therapy (6, 7). do not express B7 molecules, there is abundant expression of It has been suggested that muscle cells can actively participate BB-1, but not B7.1 or B7.2, in different inflammatory myopathies. in local immune reactions (1). Notably, human myoblasts can Based on these observations, we propose that BB-1 plays an im- present Ags to CD4ϩ T cells (8). However, little is known about portant role in immune reactions in muscle. the costimulatory molecules expressed by muscle cells in vivo and in vitro. Here, we report that human myoblasts can be induced to selectively express BB-1, a functional CTLA4Ig3-binding co- Materials and Methods stimulatory molecule that is distinct from B7.1 (CD80) and B7.2 Myoblast culture (CD86) (9–11). Human myoblasts were isolated from muscle obtained for diagnostic rea- Our evidence for the differential expression and regulation of the sons from patients with suspected myopathy. For in vitro experiments, different B7 molecules in muscle cells relies on functional, immu- myoblasts were obtained from nonmyopathic tissue. Myoblasts were cultured as previously described (8) with some modifications. Briefly, muscle specimens were mechanically dissociated and passed through a steel sieve *Department of Neuroimmunology, Max-Planck Institute of Neurobiology, D-82152 (Tissue Grinder Kit, Sigma, Deisenhofen, Germany). The homogenate was Martinsried, Germany; and †Department of Neurology, Klinikum Grosshadern, Mu- digested in trypsin-EDTA-solution (Life Technologies, Eggenstein, Ger- nich, Germany many) for 30 min at 37°C. The resulting suspension was centrifuged (5 min, 1500 rpm) and washed with PBS. The cells were transferred into Received for publication April 23, 1998. Accepted for publication July 30, 1998. plastic tissue culture flasks (Falcon, Heidelberg, Germany) and incubated The costs of publication of this article were defrayed in part by the payment of page in skeletal muscle cell growth medium (modified MCDB 120 supple- charges. This article must therefore be hereby marked advertisement in accordance mented with 5% FCS, 10 ng/ml epidermal growth factor (EGF), 1 ng/ml with 18 U.S.C. Section 1734 solely to indicate this fact. basic fibroblast growth factor (bFGF), 0.5 mg/ml fetuin, 0.1 mg/ml insulin, 1 This study was supported by the Max-Planck Society, Deutsche Forschungsgemein- 0.4 ␮g/ml dexamethasone, 50 ␮g/ml gentamicin sulfate, 50 ng/ml ampho- schaft (SFB 217, Project C13), and a European Community Grant (Project BMH4- tericin B; PromoCell, Heidelberg, Germany). After 30 min, nonadherent CT96-0893). cells were removed and seeded in new tissue flasks coated with poly-L- 2 Address correspondence and reprint requests to Dr. Reinhard Hohlfeld, Department lysine (Sigma) and laminin (kindly provided by Dr. H. Neumann, MPI of of Neuroimmunology, Max-Planck Institute of Neurobiology, D-821521 Martinsried, Neurobiology, Martinsried, Germany) and cultured at 37°C at 5% CO2 in Germany. E-mail address: [email protected] skeletal muscle cell growth medium (see above). The myoblasts were re- 3 Abbreviations used in this paper: CTLA4Ig, CTL-associated protein 4 Ig; MBP, peatedly purified during the culture period and 1 wk before experiments, myelin basic protein; NCAM, neural cell adhesion molecule; TCL, T cell line. using a magnetic cell separation system (Dynal, Hamburg, Germany) and

anti-CD56 mAb (8) (NCAM/Leu-19; Becton Dickinson, Heidelberg, Ger- myoblasts, the MBP-specific T cell lines SS014 and SS018 were used many). Purity was checked by FACS analysis. Only Ͼ95% pure cultures between days 13 and 15 after the last restimulation with peptide Ag. were used in all experiments. The cultured myoblasts do not contain professional APC (macrophages or dendritic cells), since they are are negative Coculture experiments of myoblasts and T cells for CD11b, CD14, and VCAM by flow cytometry and do not express B7.1 or B7.2 by RT-PCR. However, a small proportion (Ͻ5%) of NCAM-neg- Myoblasts were plated in 96-well flat-bottom plates (Costar, Bodenheim, ϫ 4 ative fibroblasts may be present. To exclude the possibility that fibroblasts Germany) at a density of 2 10 cells/well and induced for 72 h with the ϫ 4 also express BB-1, we isolated and expanded the NCAM-negative cell indicated cytokines. In previous experiments, a cell density of 1–2 10 population and treated them with IFN-␥ or TNF-␣, alone or in combina- myoblasts per flat-bottom well was found optimal for T cell stimulation, tion. The fibroblasts remained negative for B7.1, B7.2, and BB-1 by flow because the myoblasts grow essentially confluent at this seeding density cytometry and did not transcribe B7.1 or B7.2 mRNA by RT-PCR. (8). After induction, myoblasts were irradiated with 50 Gy, washed in The HLA-DR type of the myoblasts was determined by Drs. E. Albert RPMI 1640 (Life Technologies) and cultured in the presence or absence of ϫ 5 and S. Scholz (Labor fu¨r Immungenetik, Ludwig-Maximilians-Universita¨t, Ag and 2 10 HLA-DR-matched or autologous Ag-specific T cells. Mu¨nchen, Germany), using PCR and sequence-specific oligonucleotide Irradiated PBMC, isolated from blood using standard density gradient cen- primers (Dynal). trifugation (Lymphoprep, Nycomed, Oslo, Norway), were used as professional APC in control experiments. T cell proliferation was measured by Culture of CD40L-transfected mouse fibroblasts [3H]TdR incorporation in a gas scintillation counter (Matrix 96 Direct Beta Counter, Packard, Frankfurt, Germany). It should be noted that the absolute CD40L-transfected Ltk cells (kindly provided by Dr. S. Lebecque, Scher- counts measured by this “dry” scintillation system are only ϳ20% of the ing-Plough, Dardilly, France) were cultured in plastic tissue culture flasks counts obtained by liquid scintillation (15); variability and proportions of (Falcon) at 5% CO2 as described previously (14). The culture medium the measured values are identical with both methods. Ag was used at a consisted of 45% DMEM, 45% F10-Nutrient Mix (HAM) with Glu- concentration of 5 ␮g/ml. Abs were used at the indicated concentrations ␮ tamax-I, 10% FCS, 10 mM HEPES, and 50 g/ml penicillin/streptomycin (see mAb section). Downloaded from (all from Life Technologies). For coculture experiments, 3 ϫ 105 myoblasts were incubated with the same number of irradiated (100 Gy) CD40L RNA extraction, cDNA synthesis, and PCR transfectants. Expression of surface molecules was determined by FACS analysis at 24, 48, and 72 h after initiation of cultures. The expression of Total cellular RNA was extracted using an RNA extraction kit (Qiagen, CD40L on transfected L cells and CD40 on myoblasts was checked and Hilden, Germany). The RNA was subsequently treated with DNase I confirmed in all experiments. The CD40L transfectants do not express (Boehringer) for 15 min at 37°C to digest any potentially contaminating B7.1, B7.2, or CD40. Myoblasts are CD40L negative (data not shown). genomic DNA and afterward heated to 95°C for 5 min to inactivate the enzyme. RNA (100 ng) was reverse transcribed using random hexamer http://www.jimmunol.org/ Monoclonal Abs primers (Boehringer) and SuperScript TM Reverse Transcriptase (Life Technologies). Single-stranded cDNA was amplified in a thermal cycler The source and working concentrations of the mAbs used as primary Abs (Biometra Personal Cycler, Biotron, Go¨ttingen, Germany). All reactions for flow cytometry are as follows: anti-CD11b and anti-CD14 (IgG; 10 were conducted in a total volume of 50 ␮l containing 2.5 U Taq Polymer- ␮ ␮ g/ml; Becton Dickinson); anti-VCAM (IgG; 10 g/ml; Novacastra, Dos- ase (Perkin-Elmer, Applied Biosystems, Weiterstadt, Germany), 200 ␮M senheim, Germany); anti-NCAM (Leu-19; IgG; 1:100; Becton Dickinson); of each dNTP (Roth), and 50 pmol of each primer. The individual cycles ␮ anti-MHC class II (L243; hybridoma supernatant; 10 g/ml; American of hot start PCR, after an initial denaturation of 5 min at 93°C, consisted Type Culture Collection (ATCC), Manassas, VA); anti-B7.1 (L307; IgG; of 1 min denaturation at 94°C, 1 min annealing at 60°C or 63°C (B7.1a), ␮ ␮ 10 g/ml; M. Azuma, Tokyo, Japan (12)); anti-B7.2 (IT.2; IgG; 10 g/ml; and 1 min extension at 72°C, followed by a 10-min extension step at 72°C. M. Azuma or PharMingen, Hamburg, Germany (13)); anti-BB-1 (IgM; 10 RNA from normal PBMC, which was not reverse transcribed, was used as ␮ by guest on September 26, 2021 g/ml; NatuTec, Frankfurt, Germany or PharMingen); anti-CD40 (Mab89; a negative control in all PCR experiments. A 25-␮l aliquot of the PCR ␮ ␮ IgG; 10 g/ml; S. Lebecque); and anti-CD40 CEA-5; IgG; 10 g/ml; Natu- reaction was eletrophoretically separated on a 1.7% agarose gel (Roth) Tec). For controls, isotype-matched irrelevant mAbs were substituted at using standard conditions. UV-induced fluorescence of ethidium bromide identical concentration (mouse anti-DNP; IgG or IgM; PharMingen). Di- was used for visualization of amplified products. The correct size of the bands Ј chlorotriazinyl-fluoresceine (DTAF)-conjugated goat F(ab )2 anti-mouse was determined by comparison with a DNA mass standard (␾-X 174, Life IgG and IgM were used as secondary Ab (1:150; Immunotech, Hamburg, Technologies; and pUC8, MBI Fermentas, St. Leon-Rot, Germany). All PCR Germany). Fusion protein human (h) CTLA-4 fused with mouse IgG2a products were sequenced (Medigene, Martinsried, Germany). ␮ (Ancell, Laüfelfingen, Switzerland) was used at 10 g/ml. This protein was The primer sequences were as follows: ␤-actin (accession no. M10277 detected with FITC-conjugated goat anti-mouse Ig heavy and light chain (16)) forward, 5Ј-CCACACCTTCTACAATGAGC-3Ј (exon 3, position (Immunotech). 1485–1504); ␤-actin reverse, 5Ј-ACAGCCTGGATAGCAACGTA-3Ј For inhibition experiments, azide-free anti-MHC II, anti-B7.1, anti- (exon 4, position 2062–2081). ␮ B7.2, anti-BB-1, and CTLA4Ig (Ancell) were used at 5–50 g/ml. Mouse The primers for B7.1, B7.2, and CD40 (accession nos. M27533, IgM anti-DNP mAb was used as an isotype control for anti-BB-1. L25259, and HSCDW40) were designed using published cDNA sequences ␮ For immunohistochemistry, anti-BB-1 was used at 10 g/ml. Anti- (17, 18), as follows: B7.1a forward, 5Ј-GTGGCAACGCTGTCCTGT MHC class I (W6/32; IgG; hybridoma supernatant; ATCC) and class II GGT-3Ј (cDNA-position 450–471); B7.1a reverse, 5Ј-CCAGGAGAGGT ␮ Ј mAbs were used at 2 g/ml. Cy3 F(ab )2 goat anti-mouse IgG and IgM GAGGCAC-3Ј (cDNA-position 842–825); B7.2 forward, 5Ј-CCAAAGC ␮ were used at 2 g/ml as the secondary reagent. CTGAGTGAGCTAGT-3Ј (cDNA position 270–291); B7.2 reverse, 5Ј- Ј Cytokine induction CTTAGGTTCTGGGTAACCGTG-3 (cDNA position 630–610); CD40 forward, 5Ј-TGGGGCTGCTTGCTGACCGC-3Ј (cDNA-position 78–97); For analysis of inducible surface molecules and coculture experiments, CD40 reverse, 5Ј-CCAAAGCCGGGCGAGCATGA-3Ј(cDNA-position myoblasts were cultured in the presence of 100 U/ml rIFN-␥ (Boehringer, 436–417). Mannheim, Germany) and/or 250 U/ml TNF-␣ (provided by Drs. U. Trau- To rule out the presence of an unusual splice variant of B7.1 in myo- gott and M. Mu¨ller-Neumann, BASF/Knoll, Ludwigshafen, Germany) or blasts, two additional primer pairs (B7.1b and -c) were used for amplifi- 500 U/ml rIL-4 (PromoCell) for the time indicated. These concentrations cation of B7.1: B7.1b forward, 5Ј-ACTGGCAAAAGGAGAAGAAA-3Ј were found to induce maximal expression of inducible surface Ag. Ex- (cDNA position 511–530); B7.1b reverse, 5Ј-ATACAGGGCGTA pression was monitored at 24, 48, and 72 h following cytokine induction. CACTTTCC-3Ј (cDNA position 1182–1163) (19); B7.1c forward, 5Ј- For functional studies in cocultures, myoblasts were induced for 72 h with CCTAAGAATTCGAAGCCATGGGCCACACACCGAGG-3Ј (cDNA IFN-␥ and TNF-␣, or for 24 h with IL-4. position 301–335); B7.1c reverse, 5Ј-TGGGCGCAGAGCCAGGATCA-3Ј (cDNA position 642–622) (20). Also, with these additional primers, only Ag-specific T cell lines PBMC but not myoblasts were found to transcribe B7.1. The PCR products obtained from PBMC were confirmed as B7.1 by sequence analysis. PBMC were stimulated with a synthetic peptide of human myelin basic protein (MBP; sequence 86–105; synthesized by Luis Moroder, MPI for Muscle biopsy specimens for immunohistochemistry Biochemistry, Martinsried, Germany), and CD4ϩ Ag-specific T cell lines were isolated using the split-well cloning technique as described previously Diagnostic muscle biopsy specimens were obtained from patients with in- (8). Every 2 wk, T cell cultures were restimulated with Ag plus irradiated flammatory myopathies (six polymyositis, five dermatomyositis, six inclu- (50 Gy) autologous or HLA-DR-matched PBMC. Ag-specific proliferation sion body myositis) and degenerative muscle disease (three Duchenne was measured by [3H]TdR incorporation. For coculture experiments with muscular dystrophy); there were three nonmyopathic controls. The Journal of Immunology 5945 Downloaded from http://www.jimmunol.org/

FIGURE 1. FACS analysis of untreated (nonstimulated) human myoblasts. The myoblasts uniformly express the differentiation marker NCAM (neural cell adhesion molecule). Nonstimulated myoblasts are negative for B7.1, B7.2, and BB-1 and do not bind CTLA4Ig. Open curve represents isotype control. by guest on September 26, 2021 Confocal laser microscopy Twenty-micrometer serial cryostat muscle sections were analyzed. Optical sections were acquired in 0.5-␮m steps along the z-axis with a confocal laser scanning microscope equipped with ϫ40 oil objectives (Leica TCS 4D, Nussloch, Germany). Baseline labeling was revealed with irrelevant mAbs and secondary fluorochrome Cy3-conjugated goat anti-mouse Ig. Fluorescence intensity signals were converted into different colors. Results We investigated the expression of different members of the B7 family of costimulatory molecules in cultured human myoblasts and in muscle tissue. Myoblasts were stimulated a) with IFN-␥ and/or TNF-␣, b) with IL-4, and c) with cross-linking anti-CD40 mAb or CD40L-transfected cells. In all cases, B7 transcription and protein expression were investigated by RT-PCR and FACS analysis. The functional role of B7 molecules induced in myoblasts was investigated in cocultures of myoblasts with MHC-class II- ϩ matched Ag-specific CD4 T cell lines. All functional experi- FIGURE 2. A, Selective induction of BB-1 on human myoblasts treated ments were performed with highly purified NCAM-positive hu- with IFN-␥ and TNF-␣. Purity of the myoblasts is documented by NCAM man myoblasts (8) (see Materials and Methods). expression (upper panel). After 24 h, no B7 molecules are detected. After 72 h, myoblasts express BB-1 and bind CTLA4Ig. Open curves represent ␥ ␣ Myoblasts stimulated with IFN- or TNF- selectively express isotype control. See B for corresponding RT-PCR results. B, Absence of BB-1, a CTLA4Ig-binding costimulatory molecule distinct from B7.1 and B7.2 transcripts in IFN-␥- and TNF-␣-double-induced myoblasts. B7.1 and B7.2 Myoblasts were induced for 72 h. Control PBMC constitutively transcribe For detection of B7 molecule expression, we used mAbs against B7.1, B7.1 and B7.2. The right lane, labeled “Control,” represents RNA that was not reverse transcribed. See A for corresponding FACS data. C, Absence of B7.2, and an mAb named BB-1, which cross-reacts with B7.1 and an alternative transcripts of B7.1 in IFN-␥/TNF-␣-induced myoblasts. To ex- as yet undefined costimulatory molecule (9–11). Without cytokine clude that cytokine-induced myoblasts contain alternative transcripts of treatment, myoblasts did not express B7.1 or B7.2 (Fig. 1). Myoblasts B7.1, we repeated the RT-PCR experiment shown in B with two additional treated with TNF-␣ or IFN-␥, alone or in combination, remained neg- pairs of primers (B7.1b and B7.1c). Only PBMC, but not myoblasts, tran- ative for B7.1 and B7.2, as analyzed by flow cytometry and RT-PCR scribe detectable levels of B7.1 mRNA. The PCR products obtained from (Fig. 2, A and B). In contrast, BB-1 was strongly induced after treat- PBMC were confirmed as B7.1 by sequencing. 5946 COSTIMULATORY MOLECULES IN HUMAN MUSCLE

FIGURE 4. Constitutive expression of CD40 on NCAM-positive myoblasts.

ment of myoblasts with TNF-␣ or IFN-␥, alone or in combination

(Fig. 2A). Induction of BB-1 was maximal after 72 h of cytokine Downloaded from treatment. Thus, after 72 h, the myoblasts remain negative for B7.1 both by immunocytochemistry and RT-PCR, but react with anti-BB-1 and CTLA4Ig. To rule out that anti-BB-1 and CTLA4Ig react with a splice variant of B7.1, we repeated the RT-PCR experiment with

two additional primer pairs (B7.1b and B7.1c; see Materials and http://www.jimmunol.org/ Methods). The results are shown in Fig. 2C. Also, with the additional B7.1 primers, a PCR product was obtained only from PBMC. Sequencing of the PCR products conﬁrmed that they cor- respond to B7.1. Together with results from another group who used yet another set of B7.1 primers (21), our data suggest that B7.1 or B7.1 splice variants are absent in myoblasts.

Myoblasts induced with IL-4 express B7.1, B7.2, and BB-1 with

different time kinetics by guest on September 26, 2021 Next we assessed whether treatment with IL-4 can induce B7.1 or B7.2 in myoblasts. IL-4 has previously been reported to induce B7.1 or B7.2 in B cells, Langerhans cells, and keratinocytes (22– 24). FACS analysis showed that IL-4 induces different members of the B7 family on myoblasts with different kinetics (Fig. 3A). After 24 h, anti-B7.1, anti-B7.2, anti-BB-1, and CTLA4Ig fusion protein all bound to myoblasts. After 72 h, B7.1 and B7.2 expression was reduced to background, whereas BB-1 and CTLA4Ig still bound strongly (Fig. 3A). Consistent with the FACS results, RT-PCR analysis showed that IL-4-induced myoblasts transcribe mRNA for B7.1 and B7.2 (Fig. 3B). Transcription was detected after 24 and 72 h. The FACS data provide further evidence for differential regulation of the different B7 family members in human myoblasts. Although our myoblast preparations are Ͼ95% pure, they may contain a small fraction of NCAM-negative fibroblasts (see Ma- terials and Methods). Therefore, we expanded fibroblasts from muscle tissue and used them as an additional control. As shown in Fig. 3B, these muscle-derived fibroblasts do not transcribe B7.1 or B7.2 and are therefore not the source of the B7 message detected FIGURE 3. A, FACS analysis of IL-4-induced myoblasts. After 24 h, in our myoblasts by RT-PCR. the myoblasts express B7.1, B7.2, and BB-1 and bind CTLA4Ig. After 72 h, B7.1 and B7.2 are no longer detected. However, BB-1 expression and Anti-CD40 stimulation differentially induces B7 molecules in CTLA4Ig binding is still noticeable. See B for corresponding RT-PCR myoblasts results. B, RT-PCR analysis of IL-4-induced myoblasts. IL-4-induced myo- B7 expression is known to be influenced by CD40-CD40L signal- blasts and control PBMC transcribe mRNA for B7.1 and B7.2 after 24 and ing (25–28). We therefore asked whether human myoblasts ex- 72 h. Comparison with A shows that B7.1 and B7.2 were no longer detectable at the protein level after 72 h, although mRNA is still detectable press CD40 and, if so, whether CD40 stimulation would affect B7 at this time point. The discrepancy may be explained by the nonquantitative expression in myoblasts. As shown in Fig. 4, NCAM-positive nature of the RT-PCR results. NCAM-negative control fibroblasts (Fi- myoblasts constitutively and uniformly express CD40. brobl.) do not transcribe B7.1 or B7.2. Lanes labeled “Control” represent To analyze the effects of CD40 stimulation on B7 expression, RNA that was not reverse transcribed. we stimulated myoblasts with the anti-CD40 mAb EA-5 or with The Journal of Immunology 5947

CD40L-transfected mouse fibroblasts. Fig. 5A shows that stimulation with mAb EA-5 induces BB-1 but not B7.1 or B7.2 after 24 and 72 h. In contrast, a nonstimulatory anti-CD40 mAb (Mab89) (29) failed to induce any of the B7 molecules after 24, 48, or 72 h (data not shown). The results of RT-PCR analysis of myoblasts stimulated with mAb EA-5 are consistent with the FACS data (Fig. 5B). Coculture of myoblasts with CD40L transfectants induces weak expression of B7.1 after 24 h, which returns to background after 72 h (Fig. 6A). B7.2 was not expressed. BB-1 expression was maximal after 72 h. A similar staining pattern was seen with CTLA4Ig. The PCR results are consistent with the FACS data (Fig. 6B). Human control fibroblasts did not transcribe B7.1 or B7.2 (Fig. 6B). The CD40L-transfected mouse fibroblasts also did not yield a PCR signal for human B7.1 or B7.2 (not shown). BB-1 on myoblasts costimulates Ag-specific CD4ϩ T cells As previously reported, IFN-␥/TNF-␣-induced human myoblasts can act as facultative APC in vitro and present Ag to CD4ϩ Ag- specific T cell lines (8). To assess the functional role of BB-1 in Downloaded from this system, we cocultured IFN-␥/TNF-␣-double-induced myoblasts with autologous or HLA-DR-matched Ag-specific CD4ϩ T cell lines and measured Ag-induced proliferation in the presence of CTLA4Ig or mAbs against MHC class II, B7.1, B7.2, BB-1, or MHC class I (Fig. 7C). CTLA4Ig (10 ␮g/ml) was completely inhibitory (Fig. 7C). Furthermore, Ag presentation was completely http://www.jimmunol.org/ blocked in the presence of anti-MHC-II mAbs or anti-BB-1, but not in the presence of B7.1- or B7.2-specific mAbs (Fig. 7C). Mouse IgM anti-DNP, which was used as an IgM isotype control for anti-BB-1, was not inhibitory (Fig. 7C). In contrast, anti-B7.1, anti-B7.2, and anti-BB-1 mAbs all effi- ciently blocked the MHC-class II-restricted Ag presentation by control PBMC (Fig. 7A) and by myoblasts treated for 24 h with IL-4 (Fig. 7B). These functional data are consistent with our ob- servation that IL-4-induced myoblasts transiently express B7.1 and by guest on September 26, 2021 B7.2 (Fig. 3A). To corroborate the results of the functional experiments, we repeated the experiment with the same TCL (SS014) and with another MBP-specific CD4ϩ TCL (SS018). Mouse IgM anti-DNP was used as an isotype control for anti-BB-1. As shown in Table I, the results are consistent with those shown in Fig. 7, B and C. Muscle fibers selectively express BB-1 but not B7.1 or B7.2 in inflammatory myopathy lesions To assess whether muscle fibers express BB-1 or any other B7 molecule in muscle tissue, we treated cryostat sections of 23 muscle biopsy specimens from patients with polymyositis, dermatomyositis, inclusion body myositis, and Duchenne muscular dystrophy and from nonmyopathic controls with mAbs against B7.1, B7.2, and the BB-1 Ag for immunohistochemistry. B7.1 and B7.2 were detectable only on mononuclear cells in inflammatory infil- trates, but never on muscle fibers. In contrast, BB-1 was expressed on many muscle fibers in all inflammatory myopathy cases (six polymyositis, five dermatomyositis, six inclusion body myositis). In one case of Duchenne muscular dystrophy, BB-1 was expressed on a few scattered inflammatory cells, but not in muscle fibers. BB-1 was absent in nonmyopathic controls. In the inflammatory cases, the distribution of BB-1 was similar to HLA class I (Fig. 8). (It should be noted that HLA class I and II Ags are not detectable on normal muscle fibers in situ (1, 4)). FIGURE 5. A, FACS analysis of myoblasts stimulated with anti-CD40 mAb EA-5. After 24 h, the myoblasts express BB-1, but not B7.1 or B7.2. Discussion After 72 h, BB-1 expression and CTLA4Ig binding is increased. See B for corresponding RT-PCR results. B, RT-PCR analysis of myoblasts stimulated Our study provides functional, immunocytochemical, RT-PCR, with anti-CD40 mAb EA-5. B7.1 and B7.2 are undetectable after 24 and 72 h and histologic evidence for the differential regulation and expres- (compare A for corresponding FACS data). Control PBMC constitutively tran- sion of different B7 molecules in human muscle cells. Specifically, scribe B7.1 and B7.2, whereas NCAM-negative fibroblasts are negative. 5948 COSTIMULATORY MOLECULES IN HUMAN MUSCLE Downloaded from

FIGURE 7. Differential inhibition of Ag presentation by anti-B7.1, anti- http://www.jimmunol.org/ B7.2, anti-BB-1 mAbs, and CTLA4Ig. Irradiated PBMC (A), IL-4-induced myoblasts (B), and IFN-␥/TNF-␣-double-induced human myoblasts (C) were incubated with or without Ag (MBP peptide 86–106), different anti- B7 mAbs, CTLA4Ig, or anti-MHC class II or class I mAb, and Ag-speciﬁc HLA-DR-matched CD4ϩ T line cells (SS014). With PBMC and IL-4- induced myoblasts, Ag presentation is inhibited by anti-B7.1, anti-B7.2, anti-BB-1, CTLA4Ig, and anti-MHC class II mAbs, whereas Ag presentation by IFN-␥/TNF-␣-induced myoblasts is inhibited only by anti-BB-1,

CTLA4Ig, and anti-MHC class II, but not by anti-B7.1 or anti-B7.2 mAbs. by guest on September 26, 2021 Control mAb against MHC class I and an IgM isotype control mAb (anti- DNP) are not inhibitory. Note different scales for proliferation of T cells in the presence of PBMC (A) and myoblasts (B and C). In separate experiments, similar data were obtained with TCL SS014 and with a different MBP-speciﬁc TCL (SS018; see Table I).

our data demonstrate that, in vitro, myoblasts can be induced to selectively express BB-1, a functional costimulatory molecule distinct from B7.1 and B7.2. Furthermore, we show that, in muscle tissue of patients with various inflammatory myopathies, but not in normal muscle, muscle fibers also selectively express BB-1. The costimulatory molecules B7.1 (CD80) and B7.2 (CD86) are members of the Ig superfamily (30–34). They are expressed mainly on professional APCs and T cells. Although their precise function(s) and relationship with the ligands CD28 and CTLA-4 are still not completely understood, there is universal agreement that the B7 molecules provide critical costimulatory signals to T cells and play an essential role in normal and pathologic immune reactions (30–35). The genes for murine and human B7.1 and B7.2 have been FIGURE 6. A, FACS analysis of myoblasts cocultured with CD40L- cloned (13, 17, 18, 36). It has long been suspected, however, that transfected mouse L cells. After 24 h, there is a weak expression of B7.1 additional functional members of the B7 family exist. Indeed, dis- and weak binding of CTLA4Ig. After 72 h, B7.1 and B7.2 expression is cordant expression of BB-1 and B7 has been noted for activated undetectable, whereas BB-1 expression and CTLA4Ig binding are in- human B lymphocytes (10) and keratinocytes (37). Furthermore, creased. See B for corresponding RT-PCR results. B, RT-PCR analysis of myoblasts cocultured with CD40L-transfected L cells. After 24 h, B7.1, but an alternative CTLA-4-binding molecule was identified on mouse not B7.2 mRNA, is transcribed in myoblasts (this is consistent with the B cells (38). Our present results provide strong additional support FACS data shown in A). After 72 h, no transcripts for B7.1 or B7.2 are for the existence of at least one additional functional member of detectable in myoblasts. Control PBMC constitutively transcribe B7.1 and the B7 family of costimulatory molecules. Presently, our evidence B7.2, whereas NCAM-negative fibroblasts do not. for the differential expression and regulation of the different B7 The Journal of Immunology 5949

Table I. Inhibitory effects of anti-B7 mAbs on Ag presentation by cytokine-induced myoblasts

% Inhibition with mAb Against Induction of SI TCLa Myoblasts (mean cpm)b B7.1 B7.2 BB-1 MHC II IgM-controlc

SS018 IL-4 16.8 (843) 95.9 92.7 97.1 96.4 2.6 SS014 IL-4 10.9 (2461) 96.7 92.9 98.7 98.6 12.2 SS018 IFN/TNF 35.3 (1271) 3.9 5.1 97.9 95.8 0 SS014 IFN/TNF 4.4 (1110) 0 0 96.8 96.0 0

a CD4ϩ T cell lines specific for MBP peptide 86-106. b Absolute counts in the presence of Ag, measured with a direct scintillation counter (see Materials and Methods). SI, stimulation index. c Isotype control for anti-BB-1. molecules in muscle cells relies on the results of immunocyto- and FACS analysis. However, a small proportion of NCAM-neg- chemical and RT-PCR studies, and, perhaps most significantly, on ative fibroblasts may be present. Therefore, we isolated and ex- functional experiments. Our results will provide a basis for future panded the NCAM-negative cell population and used them as a experiments to identify the postulated B7/BB-1 molecule at the control. After stimulation with cytokines, anti-CD40 mAb, or molecular level. One problem that needs to be overcome is that CD40L-transfected L cells, the human fibroblasts remained nega- Downloaded from only relatively small numbers of human myoblasts can be ex- tive for B7.1, B7.2, and BB-1 by flow cytometry and did not tran- panded from diagnostic muscle biopsy tissue, whereas large scribe B7.1 or B7.2 mRNA by RT-PCR. Therefore, the B7 tran- amounts of purified, cytokine-induced myoblasts will be needed scripts and protein detected in our myoblast preparations could not for immunoprecipitation, Western blotting, and protein sequencing have been derived from potentially contaminating NCAM-nega- studies. tive fibroblasts.

We isolated and cultured human myoblasts from adult muscle Our study reveals that, in myoblasts, B7.1, B7.2, and BB-1 http://www.jimmunol.org/ biopsy tissue, using standard protocols (8, 39). Myoblasts express are differentially regulated and expressed. Stimulation with the neural cell adhesion molecule NCAM, which serves as a con- IFN-␥ and/or TNF-␣ selectively induces BB-1, but not B7.1 or venient selection marker for magnetic bead sorting (8, 40). Our B7.2. Recently, the absence of expression of B7.1 and B7.2 in myoblast preparations are Ͼ95% NCAM positive. They do not human myoblasts has also been reported by other groups (21, contain professional APC (macrophages or dendritic cells), since 41). To exclude the possibility that BB-1 and CTLA4Ig recog- they are negative for CD11b, CD14, and VCAM by ﬂow cytom- nize a splice variant of B7.1 on cytokine-induced myoblasts, we etry and do not constitutively express B7.1 or B7.2 by RT-PCR used three different primer pairs for RT-PCR ampliﬁcation of by guest on September 26, 2021

FIGURE 8. Confocal laser microscopy lo- calization of BB-1 (A), MHC class I (C), and MHC class II (D) in human muscle. Serial transverse cryostat sections of a muscle biopsy specimen from a patient with polymyositis were processed as described in Materials and Methods. The control section (B) was treated with nonimmune IgG and IgM control Abs as Ј primary reagents and Cy3 F(ab )2 goat anti- Mouse IgG and IgM as secondary Ab. Fluo- rescence signals were converted into different colors. There was strong, surface-associated expression of BB-1 in all muscle ﬁbers in this area (A), closely resembling the distribution of MHC class I (C). The pattern of MHC class II expression was more patchy (D). In normal muscle, BB-1 and MHC expression is undetectable by immunohistochemistry (not shown). Magniﬁcation ϫ300. 5950 COSTIMULATORY MOLECULES IN HUMAN MUSCLE

B7.1. In all cases, B7.1 was absent from myoblasts (Fig. 2, B myositis, and Duchennne muscular dystrophy and from nonmyo- and C). These results argue against the expression of an alter- pathic controls, using mAbs directed against B7.1, B7.2, and BB-1 native splice form of B7.1 and support the notion that myoblasts for immunohistochemistry and confocal laser microscopy. B7.1 can be induced to express a hitherto undefined B7 molecule and B7.2 were expressed on inflammatory cells in inflammatory recognized by anti-BB-1 and CTLA4Ig. myopathy tissue, but not on muscle fibers. In contrast, BB-1 was In contrast, stimulation with IL-4 induces B7.1 and B7.2, as well strongly expressed on many muscle fibers in the inflammatory my- as BB-1, but with different kinetics. This is consistent with previ- opathies, but not in nonmyopathic control tissue. The distribution ous reports that IL-4 can induce B7.1 and B7.2 in B cells, Lang- of BB-1 resembles that of MHC class I (Fig. 8). erhans cells, and keratinocytes (Refs. 22–24 and 42; reviewed in The induced and selective expression of BB-1 in inflammatory Ref. 30). myopathy tissue is remarkably consistent with the selective induc- The CD40/CD40L pathway is known to play an important role tion and regulation of BB-1 in cultured myoblasts. Since many in controlling B7.1 and B7.2 expression (Refs. 25–28; reviewed in BB-1ϩ fibers are contacted by inflammatory cells, it is likely that Refs. 29, 43, and 44). Because we observed that myoblasts con- both MHC class I and BB-1 are up-regulated under the influence stitutively express CD40, we were interested to see whether stim- of proinflammatory cytokines secreted by inflammatory T cells ulation with anti-CD40 mAb or CD40L-transfected cells can in- and macrophages. It seems that BB-1 plays an important role in the duce BB-1 or other B7 molecules in myoblasts. Again, we found immunobiology of muscle and, hence, may be a target for thera- evidence for selective expression and transcription of the different peutic intervention in autoimmune muscle diseases. B7 molecules. Stimulation with anti-CD40 mAb selectively in- It should be noted, however, that the significance of BB-1 ex- duces BB-1, but not B7.1 or B7.2. In contrast, stimulation with pression in muscle reaches beyond its implications for the patho- Downloaded from CD40L-transfected cells induces B7.1 and BB-1, but with different genesis and treatment of inflammatory myopathies. Muscle is the kinetics. site for many other immune reactions, including muscle infections, As we have shown previously, myoblasts treated with IFN-␥ can graft-vs-host disease, conventional intramuscular vaccination, and, present different protein Ag to Ag-specific CD4ϩ HLA-DR- recently, intramuscular gene transfer by injection of genetically matched T cell lines (8). We used this system to investigate the engineered myoblasts or naked DNA (1, 3, 6, 7, 50). In all these functional role of the B7 molecules inducible on myoblasts. The reactions, the induced expression of BB-1 on muscle fibers should http://www.jimmunol.org/ results show that BB-1, which is selectively induced by treatment play an important role. of myoblasts with IFN-␥/TNF-␣, provides a costimulatory signal Note added in proof. After submission of our manuscript it was to T cells. Proliferation of Ag-specific CD4ϩ T cells is completely reported that mAb BB-1 reacts with MHC class II-associated in- blocked in the presence of anti-BB-1 mAb or CTLA4Ig, but not variant chain CD74 (51). Our results with human muscle cells anti-B7.1 or -B7.2 mAbs. Of importance, the IFN-␥/TNF-␣-in- cannot be explained by the CD74 cross-reactivity of BB-1. First, duced myoblasts do not express B7.1 and B7.2 by FACS analysis B7.1/B7.2 double-negative myoblasts stained with BB-1 and (Fig. 2A), nor do they transcribe B7.1 or B7.2 mRNA by RT-PCR CTLA4Ig (Fig. 2). Second, the costimulation of cultured T cells by (Fig. 2, B and C). The selective expression of BB-1 allows func- B7.1/B7.2 double-negative, BB-1-positive myoblasts was inhib- tional studies of BB-1 in isolation, without coexpression of B7.1 ited not only with BB-1 but also with CTLA4Ig (Fig. 7). Third, the by guest on September 26, 2021 and B7.2 as it is observed in most other systems. By comparison, anti-CD74 mAb BU-45 (kindly provided by Drs. G. Moldenhauer myoblasts treated with IL-4 for 24 h coexpress B7.1, B7.2, and and G. J. Ha¨mmerling, Deutsches Krebsforschungszentrum, Hei- BB-1 (Fig. 3). In this case, Ag presentation to CD4ϩ T cells was delberg, Germany) did not stain cultured myoblasts in FACS anal- inhibited not only by anti-BB-1 mAb, but also by anti-B7.1 and ysis or muscle fibers in tissue sections, whereas BB-1 did (our anti-B7.2 mAbs (Fig. 7B). unpublished results). To investigate whether our in vitro observations with cultured myoblasts are relevant in vivo, we studied the expression of B7 Acknowledgments molecules on mature muscle fibers in muscle biopsy tissue. Muscle fibers are highly differentiated large syncytial cells that contain We are grateful to Dr. Miyuki Azuma (Juntendo University School of hundreds of nuclei. It would therefore not be surprising if the reg- Medicine, Department of Immunology, Tokyo, Japan) for kindly providing the B7.1 and B7.2 mAbs; Dr. Serge Lebecque, Schering-Plough, Dardilly, ulation of surface molecules such as B7 was different in muscle France for his gift of CD40L-transfected L cells; Dr. Harald Neumann, fibers as compared with mononuclear myoblasts. For example, Max-Planck Institute of Neurobiology, Martinsried, Germany for help with whereas cultured human myoblasts constitutively express MHC the design of PCR primers and discussion; Dr. Luis Moroder, Max Planck class I (8), mature muscle fibers in muscle tissue are MHC class I Institute of Biochemistry, Martinsried for peptide synthesis; Dr. Stefano negative, as assessed by immunohistochemical staining (1, 4, 5, Sotgiou for providing T cell clones; and Ms. Martina So¨lch for excellent 45). However, MHC class I molecules are strongly up-regulated on technical assistance. 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