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Costimulatory Ligand B7-H3Synoviocytes: Role of the B7 Family Interactions of T Cells with Fibroblast-Like

Urquhart, Kevin C. Chung and David A. FoxOliver, Peter T. White, Judith L. Endres, Andrew G. Chinh N. Tran, Seth G. Thacker, Deanna M. Louie, Jennifer

http://www.jimmunol.org/content/180/5/2989doi: 10.4049/jimmunol.180.5.2989

2008; 180:2989-2998; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Interactions of T Cells with Fibroblast-Like Synoviocytes: Roleof the B7 Family Costimulatory Ligand B7-H31

Chinh N. Tran,* Seth G. Thacker,* Deanna M. Louie,* Jennifer Oliver,* Peter T. White,*Judith L. Endres,* Andrew G. Urquhart,† Kevin C. Chung,‡ and David A. Fox2*

Fibroblast-like synoviocytes (FLS) and T cells can activate each other in vitro, and in vivo interactions between these cells maybe important in rheumatoid arthritis (RA), yet FLS lack significant expression of CD28 ligands. We sought to identify moleculeshomologous to CD28 ligands that are strongly expressed by FLS, and documented strong B7-H3 expression on FLS and byfibroblasts of other tissues, which was unaffected by a variety of cytokines. Western blot analysis of FLS lysates showed predom-inant expression of the larger, four Ig-like domain isoform of B7-H3. Immunohistological sections of RA synovial tissue showedstrong staining for B7-H3 on FLS. Cells expressing B7-H3 were distinct from but in close proximity to cells that expressed CD45,CD20, and CD3. Confocal microscopy of FLS and T cell cocultures showed localization of B7-H3 in the region of the T cell-FLScontact point, but distinct from the localization of T cell CD11a/CD18 (LFA-1) and FLS CD54 (ICAM-1). Reduction of B7-H3expression on FLS by RNA interference affected interactions of FLS with resting T cells or cytokine-activated T cells. Resting Tcells showed increased production of TNF-�, IFN-�, and IL-2, whereas cytokine-activated T cells showed reduced cytokineproduction relative to control. However, cytokine production by T cells activated through their TCR was not notably altered byknock down of B7-H3. These observations suggest that B7-H3 may be important for the interactions between FLS and T cells inRA, as well as other diseases, and the outcome of such interactions depends on the activation state of the T cell. The Journal ofImmunology, 2008, 180: 2989–2998.

T he rheumatoid arthritis (RA)3 synovial environment com-prises a complex mix of cell types. Invading lymphocytesare in close proximity to resident structural cells. In this

milieu, particular focus has turned to the interaction between themost numerous lymphocyte, the T cell, and the structural cell thatinvades cartilage, the fibroblast-like synoviocyte (FLS) (1–4).

In vitro assays have documented strong association and bidirec-tional signaling between these two cell types. Similar to profes-sional APC-T cell interactions, FLS and T cells in cocultures havebeen shown to interact in Ag-dependent systems. FLS can presentsuperantigen to T cells inducing a proliferative response (2). FLSare also able to take up and present arthritogenic peptide autoan-tigens to HLA-DR4-restricted T cell hybridomas (5). Moreover, Tcells previously stimulated by Ag presented by professional APChave augmented ability to interact with FLS, leading to increasedrelease of IL-15, TNF-�, IL-18, IL-17, and IFN-� (6).

T cells and FLS can also interact and signal in the absence ofexogenous Ag. Naive T cells cultured with FLS cause FLS toincrease production of inflammatory mediators such as IL-6, IL-8,and PGE2 (4). Conversely, T cells have been reported to expressthe activation marker CD69 after coculture with FLS (1).

FLS share features in common with professional APC, and theyexpress many surface glycoproteins related to lymphocyte traffick-ing and signaling, such as VCAM-1 and CD54 (7). The integrinCD11a/CD18 (LFA-1) clusters at the contact point between T celland FLS (8) and T cell adherence to FLS is inhibited by blockingAb to LFA-1 (9). FLS also express significant MHC class II invivo (10), which can be reinduced in vitro (11, 12). However,unlike traditional APC, FLS do not express the classic costimula-tory molecules B7-1 or B7-2 (CD80, CD86) (13, 14), which typ-ically provide the second signal for full T cell activation.

A family of B7-related proteins has been discovered that have vari-able homology to CD80 or CD86. One of these proteins is termedB7-H3, a type I transmembrane protein with �20% sequence homol-ogy to B7-1, B7-2, PD-L1, and ICOS ligand (ICOS-L) (15). HumanB7-H3 was initially described as a costimulatory molecule expressedonly on some activated APC and lymphocytes. Ligation of B7-H3 toa putative receptor on activated T cells enhanced proliferation andIFN-� production. Further genomic analysis of B7-H3, in human andmouse, revealed that the human form can be expressed as two distinctisoforms, whereas the mouse expresses only one isoform (16–18).The human B7-H3 gene contains four Ig-like repeats that can be al-ternatively spliced to express a protein containing either four Ig-like(4IgB7-H3, B7-H3b) or two Ig-like (2IgB7-H3, B7-H3) domains. Themouse gene only contains two Ig-like repeats, and thus only one formis expressed.

Similar to the other B7 family members, subsequent studies haveshown that B7-H3 can mediate inhibitory effects (17, 19, 20) on T cellfunction as well as costimulatory effects (15, 21–23). Unlike B7-1 andB7-2, B7-H3 has been shown to also be expressed on nonhemopoietic

*Rheumatic Disease Core Center and Division of Rheumatology, Department of In-ternal Medicine, †Department of Orthopedic Surgery, and ‡Section of Plastic Surgery,Department of Surgery, University of Michigan Medical School, Ann Arbor, MI48109

Received for publication February 22, 2007. Accepted for publication December11, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grants AR038477 and AR048310 from the NationalInstitutes of Health, and by the University of Michigan Medical Scientist TrainingProgram.2 Address correspondence and reprint requests to Dr. David A. Fox, Room 3918Taubman Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0358.E-mail address: dfox@umich.edu3 Abbreviations used in this paper: RA, rheumatoid arthritis; FLS, fibroblast-like sy-noviocyte; RNAi, RNA interference; OA, osteoarthritis; ICOS-L, ICOS ligand; eGFP,enhanced GFP; Tck, cytokine-activated T cell; Trest, resting T cell.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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cells (17). Although FLS lack expression of the classic costimulatorymolecules B7-1 and B7-2, we hypothesized that FLS might expressB7-H3 and that B7-H3 might participate in T cell-FLS interactions.

Materials and MethodsFLS isolation and culture

All procedures involving specimens obtained from human subjects were per-formed under a protocol approved by the University of Michigan InstitutionalReview Board. FLS were obtained by collagenase (Worthington Biochemical)digestion of human synovial tissue obtained at arthroplasty or synovectomyfrom RA or osteoarthritis (OA) joints. RA diagnosis was based upon the pres-ence of at least four of the seven criteria developed by the American Collegeof Rheumatology for RA (24). The diagnosis of OA was based upon charac-teristic clinical and radiographic features, and confirmed by pathological find-ings at joint surgery. Cells were maintained in CMRL medium (Invitrogen LifeTechnologies) supplemented with 10% FCS (Atlanta Biologicals), 2 mM glu-tamine (Cambrex), 50 U/ml penicillin (Cambrex), and 50 �g/ml streptomycin(Cambrex). FLS were used after passage 4 from primary cultures.

T cell generation

T cells were obtained from peripheral blood by negative selection usingRosetteSep Human T cell Enrichment Cocktail (StemCell Technologies)

and separation on a Ficoll gradient. Briefly, freshly isolated blood fromvolunteers was incubated with kit Ab mix for 20 min at room temperature.Blood was diluted 1/1 with 2% normal calf serum/PBS before overlay ontoroom temperature Ficoll, and spun at 1200 relative centrifugal force (RCF)with the brake off. The buffy coat containing only T cells was recoveredand used for subsequent experiments. T cells were maintained in RPMI1640 (Cambrex) supplemented with 10% FCS (Atlanta Biologicals), 2 mMglutamine (Cambrex), 50 U/ml penicillin (Cambrex), and 50 �g/ml strep-tomycin (Cambrex). Resting T cells (Trest) had been freshly isolated. Cy-tokine-activated T cells (Tck) were stimulated by a mix of 25 ng/mlTNF-�, 100 ng/ml IL-6, and 25 ng/ml IL-2 for 8 days (25). T cells wereactivated with superantigen staphylococcal enterotoxin A (10 ng/ml, Tsea)by coculture with IFN-�-stimulated FLS (2). T cells were also stimulated

FIGURE 1. FLS express the B7 family molecule B7-H3. A, FLS werestimulated with IFN-� for 72 h. FLS were removed from culture wells withEDTA and stained with mouse anti-human B7-H3 Ab (black line histo-gram), mouse anti-human ICOS-L (dashed line histogram), or controlmouse IgG (gray line histogram). B, The panel of Abs was expanded in aseparate experiment to include B7-H3 (black line histogram), PD-L2(dashed line histogram), B7-H1 (gray line histogram), or mouse IgG con-trol (gray-filled histogram). Staining is representative of two experiments.

FIGURE 2. Fibroblasts from various tissue sourcesexpress B7-H3. RA FLS, OA FLS, lung fibroblasts, andskin fibroblasts were stimulated with TNF-�, IFN-�, IL-1�, IL-17, IL-4, or IL-10 for 72 h. Fibroblasts wereremoved from culture wells with EDTA and stainedwith mouse anti-human B7-H3, CD54, or control Ab.Staining is representative of two experiments.

FIGURE 3. Fibroblasts express the 4IgB7-H3. A, Recombinant 4IgB7-H3and 2IgB7-H3 were separated on SDS-PAGE and stained with goat anti-human B7-H3 Ab, after transfer to Immobilon-P. Blots were developed byrabbit anti-goat HRP and ECL. B, RA FLS whole cell lysates were precleared,and then separated on SDS PAGE, blot transferred, and developed with goatanti-human B7-H3 Ab or goat IgG. Lysates from the same RA FLS line wererun in all lanes. These results are representative of two experiments. C, Wholecell lysates from RA FLS, OA FLS, lung fibroblasts, and skin fibroblasts wererun on SDS-PAGE without preclearing, blot transferred, and developed withgoat anti-human B7-H3 Ab or goat IgG. The film exposure duration was 5 s.Staining is representative of two experiments. Numbers shown to the left ofeach panel represent molecular mass marker (kDa).

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by plates coated with a combination of 10 �g/ml mouse anti-humanCD3 and 10 �g/ml mouse anti-human CD28 (TCD3/CD28) for 8 days.

Flow cytometry

Fibroblasts were grown for 48 h in 6- or 12-well plates before stimulationwith cytokines: IFN-� 1000 U/ml, TNF-� 10 ng/ml, IL-1� 1 ng/ml, IL-1720 ng/ml, IL-10 10 ng/ml, or IL-4 10 ng/ml. Cytokine stimulation pro-ceeded for 72 h. Fibroblasts were removed from plates for staining byincubation with 3 mM EDTA in PBS for 20 min at room temperature. Cellswere stained with mouse anti-human Abs specific for CD98, CD54, orB7-H3 (R&D Systems), or with a mouse IgG control. A secondary goatanti-mouse IgG-Alexa Fluor 488 (Molecular Probes) conjugate was used tovisualize fluorescence. Cytometry was performed on a Coulter EPICS XL.

Western blot

Western blot analysis used standard protocols. Fibroblasts were grown in 175-cm2 flasks to confluence and lysed with a buffer containing 1 mM PMSF, 5mM iodoacetamide, 8 mM Tris-HCl, 100 mM NaCl, 0.02% NaN3, and 1%Nonidet P-40 or 1% saponin as the primary detergent. Preclearing was with thefollowing steps: 1) 25 �L of protein G-agarose beads (Invitrogen Life Tech-nologies), 2) 1 �g of mouse IgG (Sigma-Aldrich) followed by 25 �L of pro-tein G-agarose, and 3) 1 �g of goat IgG (Antibodies Inc) followed by tworounds of 25 �L of protein G-agarose. Lysates were run in reducing buffer onTris-glycine 4–20% acrylamide gradient gels (Novex; Invitrogen Life Tech-nologies) and transferred onto an Immobilon-P membrane (Millipore). Pri-

mary Ab, goat anti-human B7-H3 (R&D Systems), was used at a concentra-tion of 1 �g/ml for 1 h at 4°C. Control goat IgG Ab (Sigma-Aldrich) was alsoused at 1 �g/ml. Membranes were washed three times with wash buffer beforedevelopment with rabbit anti-goat-HRP conjugate (Molecular Probes) at 0.2�g/ml for 1 h at 4°C. Membranes were developed by chemiluminescence ECL(Amersham Biosciences).

Immunohistochemistry

RA synovial tissue was fixed in 10% neutral-buffered formalin or frozen inOCT, and sections were mounted onto glass slides. Immunohistochemistrywas performed using standard protocols for dichromatic staining of frozenand paraffin-embedded sections (26). Goat anti-human Abs were anti-B7-H3(R&D Systems), biotinylated anti-B7-H3 (R&D Systems), anti-CD90 (BDBiosciences), and anti-cadherin-11 (R&D Systems). Mouse anti-human Abswere anti-CD3 (R&D Systems), anti-CD68 (DakoCytomation), anti-CD20(Signet Laboratories), anti-CD45 (Signet Laboratories), and anti-cadherin-11 (Zymed Laboratories). Detection Abs and secondary detectionreagents used from Vector Laboratories were biotinylated horse anti-goatIgG, alkaline phosphatase horse anti-mouse IgG, R.T.U. VECTAStainElite ABC Reagent, Vector Blue Substrate, Vector Red Substrate, and AECsubstrate. After staining sections were sealed using Faramount.

Confocal microscopy

FLS were grown on glass coverslips for 48 h. Trest, Tck, or Tsea werecocultured with FLS for various time points. Coverslips were then washed

FIGURE 4. B7-H3 expression within the RA synovium. Sections of three RA and three OA synovial tissue were stained. Sections are representative of eachexperiment and come from RA tissue. A, B7-H3 staining was compared with cadherin-11 and CD90 staining on frozen sections. To control for dichromatic staining,control Ab was developed in blue for B7-H3/cadherin-11 and red for CD90. Images are at a magnification of �20. B, Paraffin-embedded sections of RA synovialtissue were dually stained with goat anti-human B7-H3 (red) and mouse anti-human CD20, CD45, or CD68 (all blue). As a control for B7-H3, goat IgG was used.Images are at a magnification �40. C, Frozen sections were dually stained with goat anti-human B7-H3 (red) and mouse anti-human CD3 or CD80 (both blue).Images are at a magnification �40. D, CD3 (blue) staining was compared with cadherin-11 (red) staining on frozen sections, with goat-IgG control for cadherin-11(red) (top). In addition, CD3 (red) was compared with CD90 (blue), with mouse IgG (blue) control for CD90 (bottom). Images are at a magnification �40.

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with PBS and fixed in 4% paraformaldehyde (Molecular Probes) for 1 h atroom temperature or overnight at 4°C. Coverslips were stained using goatanti-human B7-H3 (R&D Systems), mouse anti-human CD11a, and mouseanti-human CD54. Secondary Abs used were goat anti-mouse IgG AlexaFluor 594, and rabbit anti-goat IgG Alexa Fluor 488. For coculture studiescomparing LFA-1 and ICAM-1 localization, FLS were first transfectedwith an expression vector for a fusion protein of ICAM-1 and enhancedGFP (eGFP), and then plated on glass coverslips. This expression vectorwas termed CD54-eGFP and was created as follows. Primers were con-structed against the sequence described by Accession no. BC015969 toamplify the open reading frame of human ICAM-1 by PCR. A HindIIIrestriction site added to the beginning of the 5� primer and an AgeI siteadded to the tail of the 3� primer facilitated ligation of the amplified frag-ment into the pEGFP-N1 expression vector (Clontech Laboratories). After

ligation, the insert sequence was verified to be free of mutation. This re-sulted in a CD54-eGFP fusion protein expression vector, with ICAM-1 atthe N terminus and eGFP at the C terminus (intracellular) and a shortpeptide linker (GSTPVAT) in between. Transfection was conducted usingthe Adult Human Dermal Fibroblast kit (Amaxa), and the manufacturer’sprotocols were observed. Images were taken using Olympus FluoView 500Laser Scanning Confocal Microscope.

B7-H3 knock down and coculture of T cells and FLS

Three stealth RNA interference (RNAi) and two control RNAi were ob-tained from Invitrogen Life Technologies algorithms based against the se-quence Accession no. AJ583695 from GenBank. Stealth RNAi was pooledin equimolar amounts and used at a working concentration of 10 �M, to

FIGURE 4. (continued)

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knock down B7-H3 expression. Control RNAi was also pooled in equimo-lar amounts and used at a working concentration of 10 �M. FLS weretransfected with RNAi using the Adult Human Dermal Fibroblast kit(Amaxa). For transfection, FLS were removed from plates by trypsiniza-tion and pelleted. FLS were then resuspended in transfection buffer with1–5 �M final concentration of RNAi and placed in cuvettes. FLS wereelectroporated and replated for 48 h before use. As an additional control,FLS were mock transfected without the use of RNAi. Transfected FLSwere allowed to rest for 48 h before being seeded onto plates. FLS were leftto adhere for an additional 48 h after seeding. To evaluate the effect of FLSon T cell cytokine production, Trest, Tck, or TCD3/CD28 were then added toFLS at a ratio of 1:6.66 FLS to T cells. After 8 days, T cells were collectedfrom cocultures by gentle washing with medium. Harvested T cells werewashed twice in medium and then replated at 500,000 cells in 1.5 ml ofmedium. T cells were then stimulated for 12 h with 10 ng/ml PMA and 0.5�M ionomycin. Supernatants were harvested, and TNF-�, IFN-�, and IL-2levels were measured by ELISA (BD Biosciences).

ResultsB7-H3 is expressed by FLS and other fibroblasts

Surface staining of FLS revealed strong expression of B7-H3 bythe resting and IFN-� stimulated cells (Fig. 1A). In contrast, the B7family member ICOS-L (B7-H2) was not detected (Fig. 1A). Ex-pression of B7-1 or B7-2 (CD80 and CD86, respectively) by FLSwas previously reported to be negligible (13, 14). Similar toICOS-L expression, B7-H1 and PD-L2 expression was negligibleon FLS and did not increase with IFN-� stimulation (Fig. 1B). Toassess whether other fibroblasts also selectively expressed B7-H3and whether immunoregulatory cytokines affected B7-H3 expres-sion, we compared B7-H3 expression on RA FLS, OA FLS, skinfibroblasts, and lung fibroblasts. These various fibroblasts were

stimulated, before surface staining, with various immunoregulatorycytokines for 72 h, including IFN-�, TNF-�, IL-1�, and IL-17. As acontrol, CD54 (ICAM-1) expression was also measured. All fourtypes of fibroblasts (RA FLS, OA FLS, skin, and lung) expressed highbasal levels of B7-H3, which was unaffected by cytokine stimulation(Fig. 2). In contrast, CD54 expression increased or decreased depend-ing on the cell type and cytokine (Fig. 2).

B7-H3 expressed by FLS and other fibroblasts is predominantlyfour Ig-like domain

Because human B7-H3 can be alternatively spliced to yield4IgB7-H3 (B7-H3b) or 2IgB7-H3 (B7-H3), Western blot analysiswas used to determine the m.w. of B7-H3 expressed by fibroblasts.We first established that the Ab used for Western blot analysisrecognized both isoforms of B7-H3, detecting a band at slightly�98 kDa for recombinant 4IgB7-H3 and slightly �50 kDa for2IgB7-H3 (Fig. 3A).

Whole cell lysate, from RA FLS lysed in Nonidet P-40 was pre-cleared, separated by SDS-PAGE, blot transferred, and probed withB7-H3 Ab. This revealed only one band at �110 kDa correspondingto glycosylated 4IgB7-H3. Extending the blot development time to3 h did not reveal the presence of a 2IgB7-H3 band near 50 kDa, butnonspecific background staining was visible (Fig. 3B).

To determine whether OA FLS or fibroblasts from skin and lungalso predominantly express 4IgB7-H3, Western blot analysis wasagain used. Whole cell lysates from RA FLS, OA FLS, skin fi-broblasts, or lung fibroblasts were created using saponin as thedetergent (which resulted in better recovery of B7-H3 compared

FIGURE 5. B7-H3 localization at the T cell/FLSsynapse. A, FLS were grown on glass coverslips andcocultured with Tsea, Tck, or Trest for 24 h. Coverslipswere fixed in 4% paraformaldehyde and stained withgoat anti-human B7-H3 (green) and CD3 (red). B, Stain-ing of B7-H3 (green) and CD3 (red) after 2 h of cocul-ture. Images are representative of three experiments.

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with Nonidet P-40, data not shown). To minimize any potentialloss of 2IgB7-H3 signal, this lysate was not precleared before sep-aration on SDS-PAGE. Blot development yielded a strong band at110 kDa for all fibroblast types and a much weaker band at �50kDa (Fig. 3C). This suggests that the four Ig-like domain isoformis the major and almost exclusive isoform of B7-H3 expressed onfibroblasts.

B7-H3 is broadly expressed in RA synovium

To evaluate the expression of B7-H3 in vivo, synovium was ob-tained from arthroplasty or synovectomy of RA joints, and imme-diately fixed in formalin for embedding in paraffin, or frozen inOCT. To locate regions rich in FLS, RA synovial sections werestained with the FLS specific anti-cadherin-11, which was com-pared with B7-H3 staining (Fig. 4A). Cadherin-11 is used by FLSin homotypic adhesive interactions, is expressed by synovial liningFLS, and is important for the overall morphology and developmentof the synovial lining (27). Monochromatic staining of B7-H3 orcadherin-11 showed almost identical patterns of expression. Thestrongest localization of cadherin-11/B7-H3 was to the lining layerof RA synovium, however, cadherin-11/B7-H3 was also found dif-fusely throughout synovial tissue (Fig. 4A). CD90, another fibro-blast marker, was used to show the diffuse expansion of FLS in RAsynovium, but unlike cadherin-11/B7-H3, CD90 localized mostintensely to blood vessel rich regions (Fig. 4A). The immunohis-tochemical studies indicate that, similar to cultured FLS, the FLSin RA synovium express B7-H3.

Dichromatic immunohistochemical staining was performed, tocompare the expression of B7-H3 to immune cell markers. Stain-ing of paraffin sections for B7-H3 and CD20 (B cell), CD45 (he-mopoietic), or CD68 (macrophage) surface structures revealed thatB7-H3 staining was distinct from CD20 and CD45 (Fig. 4B).There was diffuse B7-H3 expression throughout the pannus withslightly stronger localization toward the lining layer and aroundblood vessels. Patches of B cells and hemopoietic cells appeareddistinct from the B7-H3� cells. The macrophage marker CD68showed broad staining that overlapped partially with B7-H3 ex-pression. This most likely represents resident macrophage-like sy-noviocytes and invading macrophages. The control Ab for B7-H3(goat IgG) displayed only a background staining pattern (Fig. 4B).Frozen sections were stained to compare B7-H3 staining to CD3(T cell) or CD80 (APC) staining (Fig. 4C). CD3� T cells wereabundant and in close proximity to B7-H3� cells. CD80 stainingshowed a diffuse pattern, indicating the broad distribution of pro-fessional APC within the synovium. However, at the synovial lin-ing, B7-H3 staining was strong and there was little CD80 signal.

By the staining patterns of B7-H3 and CD3, T cells appear to bejuxtaposed to FLS throughout RA synovial lesions. However, be-cause B7-H3 is not unique to FLS, CD3 staining was comparedwith cadherin-11 and CD90 (Fig. 4D). Similar to the pattern seenwith B7-H3/CD3 (Fig. 4C), cadherin-11/CD3 and CD90/CD3staining showed both well-organized and diffuse T cell regions,with many T cells in close proximity to cadherin-11-positive FLS(Fig. 4D).

B7-H3 localizes to the contact point between FLS and T cells

T cells representing three different T cell activation states werecocultured with FLS. Tsea were T cells that had been stimulatedthrough their TCR by superantigen presented by FLS. Tck wereactivated with a cytokine mix (and not by the TCR), consisting ofIL-6, TNF-�, and IL-2, and represent a population that share char-acteristics similar to T cells isolated from RA synovial fluid (25).Trest were T cells that were purified from peripheral blood and nototherwise stimulated. After 2 or 24 h, the cocultures were fixed

with paraformaldehyde and stained for B7-H3 and CD3. By 24 h,B7-H3 signal could be seen outlining the contact point of both Tckand Tsea on FLS (Fig. 5). At 24 h, Trest adherence to FLS wasmarked, but strong B7-H3 signal at the contact site was not clearlyvisible (Fig. 5A). This localization of B7-H3 to the contact regionwith Tck and Tsea on FLS is clearly distinct as early as 2 h ofcoculture (Fig. 5B).

B7H3 localization at the FLS/Tck point of contact is distinctfrom the zone of CD54/LFA-1 engagement

B7-H3 localization was compared with the localization of CD54(ICAM-1) on FLS and its counter receptor, LFA-1 (CD11a/CD18),on the T cell. FLS were transfected with a CD54-eGFP (green)

FIGURE 6. B7-H3 localization compared with CD54 and LFA-1 local-ization at the T cell/FLS synapse. Images are representative of three ex-periments and digitally zoomed before capture. A, FLS were transfectedwith an expression vector for CD54-eGFP fusion protein (green) beforeplating on glass coverslips and coculture with Tck. Tck were stained withanti-LFA-1 (red). B, Tck were cocultured with FLS and stained for B7-H3(green) and CD54 (red). C, B7-H3 (green) dually stained with anti-LFA-1(red) in cocultures of FLS and Tck.

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expression vector before coculture with Tck. Cocultured cells werefixed and Tck were indirectly stained for CD11a which was visu-alized by means of the fluorochrome Alexa Fluor 594 (red). At thesynapse of Tck and FLS, confocal imaging revealed merged sig-nals (yellow) between CD54 and LFA-1 (Fig. 6A). In contrast,B7-H3 at the FLS/Tck contact point showed a staining pattern thatwas distinct from CD54 (Fig. 6B), as seen by the separation ofgreen and red signals. Similarly no merging of B7-H3 and LFA-1fluorescence was detected (Fig. 6C). The separation of B7-H3from regions dense in LFA-1/CD54 at the contact point betweenFLS and Tck could be temporally dependent. It is possible thatB7-H3 might migrate to and from LFA-1/CD54 dense areas in arapid sequence or at an extremely slow rate not detected at the timepoints selected for confocal imaging. However, we have not ob-served merged signals of B7-H3 with LFA-1 or CD54 at coculturetimes up to 24 h (data not shown).

FIGURE 7. B7-H3 specific RNAi knockdown. FLS were transfectedwith pooled B7-H3 specific RNAi, pooled control RNAi, or mock trans-fected (with no RNAi) before staining with B7-H3 (black line histogram),CD98 (dashed line histogram), or control mouse IgG (gray-filled histo-gram) at 12 days post-transfection. Histograms are representative of fiveexperiments.

FIGURE 8. T cell cytokine production after co-culture with B7-H3 RNAi transfected FLS. FLSwere transfected with pooled B7-H3 RNAi (B7-H3),pooled control RNAi (Control), or mock transfected(No RNA) and cocultured with Trest, Tck, orTCD3/CD28. T cells were harvested after 8 days of co-culture and restimulated with PMA and ionomycin.Supernatants were measured for cytokines byELISA. Error bars represent 95% confidence inter-vals. Data are representative of three experiments.A, TNF-� production. Using two-tailed t test anal-ysis comparing cytokine production from Trest, cul-tured with B7-H3� FLS (B7-H3) to B7-H3� FLS(Control) and with B7-H3� FLS (B7-H3) to mocktransfected (No RNA), yielded p values �0.0021and �2.2 � 10�5, respectively. Similarly, t testanalysis for Tck yielded p values �9.1 � 10�8 and�5.6 � 10�7, respectively. B, IFN-� production.Trest cytokine production yielded p values �1.8 �10�5 and �0.0013, respectively. Tck cytokine pro-duction yielded p values of �2.8 � 10�9 and�1.2 � 10�7, respectively. C, IL-2 production.Trest cytokine production yielded p values �2.5 �10�5 and �0.0005, respectively. Tck cytokine pro-duction yielded p values of �2.4 � 10�5 and�2.7 � 10�6, respectively.

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Distinct functions of B7-H3 in interactions with Trest vs Tck

To assess the role of B7-H3 in cocultures of T cells and FLS,RNAi technology was used to knock down B7-H3 expression inFLS before coculture with T cells. Pools of specific or controlRNAi were transfected into FLS and screened for effective inhi-bition of B7-H3. Effective and specific knock down of B7-H3 per-sisted for at least 12 days post-transfection (Fig. 7). RNAi trans-fection did not adversely affect CD98 expression in mocktransfected or RNAi transfected FLS (Fig. 7).

The following T cells were representative of three activation statescocultured with FLS: Trest were freshly isolated from peripheralblood; Tck were similar to RA synovial T cells (25); TCD3/CD28 wereT cells that had been previously activated through plate-boundanti-CD3/anti-CD28. TCD3/CD28 were used in place of Tsea to rep-resent TCR-stimulated T cells in these experiments, to avoid theneed for exposure of T cells to APC before incubation with B7-H3knockdown FLS, which could potentially confound interpretationof the results. These various types of T cells were cocultured withFLS that had been transfected with pooled B7-H3 RNAi, pooledcontrol RNAi, or mock transfected FLS (no RNAi). After 8 daysof coculture with B7-H3 knockdown FLS, T cells were harvestedand restimulated with PMA and ionomycin for 12 h. Production ofTNF-�, IFN-�, and IL-2 by the T cells was measured by ELISA.Although cytokine production by TCD3/CD28 following coculturewith FLS was not substantially affected by B7-H3 knockdown,striking and opposite effects on cytokine production of Tck andTrest were observed. Trest cocultured with B7-H3� FLS showedincreased levels of cytokine production compared with FLS trans-fected with control RNAi (Fig. 8). In contrast, Tck cocultured withB7-H3� FLS showed reduced cytokine production compared withcoculture with control RNAi FLS (Fig. 8). This suggests thatB7-H3 has an inhibitory effect on Trest and a stimulatory effecton Tck.

DiscussionB7-H3 is inhibitory or costimulatory

To achieve full activation during the response to peptide Ags, Tcells require two stimulatory signals. TCR engagement ofMHC/Ag triggers complex signaling cascades in T cells, but alonethis is insufficient for activation (28–31). A costimulatory signal isrequired in conjunction with TCR signaling to induce clonal pro-liferation and prevent anergy. This additional signal to the TCR istypically provided by a surface structure present on accessorycells, such as B7-1 (CD80), which can engage the CD28 moleculeon the T cell surface (32–36). The discovery of a second receptorfor B7, CTLA-4, which is important in limiting T cell responses,established a molecular basis for the bifunctional role of B7 asboth an activating and inhibitory ligand (37).

Starting from the initial B7-1 costimulatory ligand, a family ofB7 molecules has been discovered. B7-2 (B70, CD86) is a secondligand for both CD28 and CTLA-4. New ligand/receptor pairs in-clude PD-L1 (B7-H1)/PD-1, PD-L2 (B7-DC)/PD-1, ICOS-L (B7-H2, B7h, B7RP-1)/ICOS, and B7-H4/BTLA (38–40). These novelB7 family molecules have documented effects in enhancing T cellfunctions, inhibiting T cell functions, or both.

This duality is further reinforced by the more recent character-ization of the B7-related protein VSIG4. Initially, VSIG4 (CRIg)was described as a complement receptor necessary for bindingenzymatic products of C3 and for effective phagocytosis byKupffer cells (41). In addition, VSIG4 has been also been found toreduce T cell proliferation by CD3/CD28 stimulation in vitro, andto blunt CD8 T cell responses and IFN-� production in vivo (42).This complex pattern of inhibition and/or activation is an impor-

tant characteristic of the B7 family. Thus beyond the their role inimmune activation, the B7 family functions in the complex realmof immune modulation (38–40). This is exemplified by the dis-coveries that CD28 and CTLA-4 not only transduce signals intothe T cell by ligation of B7 family members, but that the signalingis bidirectional with functional signals transmitted into the APC aswell (43, 44). Further complicating immune modulation by B7family members is the more recent discovery that PD-L1 and B7-1can indeed bind to each other, and that this interaction is inhibitoryand signals bidirectionally (45).

The current study has documented expression of a B7 familycostimulatory molecule, B7-H3, on FLS and other fibroblasts,which are not typically viewed as professional APC. Unlike pro-fessional APC, which only express B7-H3 after activation (15),FLS expression is constitutive and uninfluenced by immunoregu-latory cytokines. This constitutive B7-H3 expression is over-whelmingly of the four Ig-like domain isoform and is robust bothin vivo and in vitro.

The literature on the effects of B7-H3 on T cells has not hithertoprovided consensus as to its function. B7-H3 was originally de-scribed in human systems as a two Ig-like domain costimulatorymolecule that increased T cell proliferation, enhanced cytotoxicity,and increased IFN-� production (15). However, a subsequentstudy indicated that the predominant isoform of B7-H3 is the fourIg-like domain form (17). This report did not recapitulate the co-stimulatory effects originally documented. In yet another study,NK lysis of neuroblastoma lines was enhanced by blocking Absagainst 4Ig B7-H3 (46).

Studies of B7-H3 in mice, which only contain one isoform, thetwo Ig-like domain isoform (16, 18), do not resolve this issue.B7-H3 knockout mice showed increased airway hypersensitivityand earlier onset of experimental autoimmune encephalomyelitis(20). Similar results were obtained using B7-H3 blocking Abs,notably that T cells increased cytokine production and experimen-tal autoimmune encephalomyelitis symptoms were exacerbated byB7-H3 blockade (19). In contrast to reports in humans, tumor ex-pression of B7-H3 enhances cytotoxicity (21, 22). And whenB7-H3 knockout mice are used in allograft survival studies, B7-H3-deficient transplants fare better when combined with immuno-suppression (23). Thus far, there appears to be no specific andconsistent association of the two Ig-like or four Ig-like B7-H3domain with activating or inhibitory effects.

We found that B7-H3 expression was associated with FLS richareas and was in close proximity to T cells in the RA pannus.When the T cell-FLS interaction was explored by confocal mi-croscopy, B7-H3 was found at the contact point, but was differ-entially localized from CD54 and LFA-1. This suggested thatB7-H3 could be an important signaling molecule between FLS andT cells. However, knock down of B7-H3 in FLS did not affect theability of FLS to present superantigen to T cells or to respond tocontact with T cells by the secretion of IL-6 and IL-8 (data notshown). Nevertheless, interesting B7-H3 dependent effects on cy-tokine secretion by T cells following coculture with FLS weredocumented, including the differential effects of B7-H3 on cyto-kine production by resting or Tck cells. This suggests that theactivation state of the T cell has significant influence over theoutcome of T cell ligation of B7-H3, and also suggests thatthere may be two receptors for B7-H3 (which is a pattern thatwould be similar to other B7 family ligands). Furthermore, cy-tokine production by TCD3/CD28 did not greatly differ aftercoculture with B7-H3 expressing or knockdown FLS.

A simplistic model to explain our observations would be thatTrest predominately express an inhibitory receptor for B7-H3,

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whereas Tck predominantly express an activating receptor for B7-H3. T cells activated through stimulation by CD3 and CD28 didnot have significant differences in cytokine production followingB7-H3 knockdown, suggesting that this type of activated T cellsdoes not express a functionally significant level of B7-H3 receptor,or that, perhaps, opposing effects of engagement of both positiveand negative receptors are offsetting. So far we have not observedan effect of engagement or interference with B7-H3 signaling onFLS activation, implying that perhaps B7-H3 signaling is not bi-directional in FLS-T cell interactions as compared with the roles ofvarious B7 family molecules in interactions of T cells with pro-fessional APC. However, an alternative possibility is that otherassays need to be used to measure the functional consequences ofB7-H3 engagement, or inhibition, on the B7-H3 expressing cells.

Costimulation in RA

The strong expression of B7-H3 by FLS of the RA pannus raisesinteresting issues regarding costimulation of T cells in RA. It hasbeen suggested that an unusual subset of CD4�CD28null T cells isexpanded in RA (47, 48). These T cells are likely to require co-stimulatory signals distinct from CD80/CD86. There is evidencethat constant exposure to TNF-� (which is abundant in RA) blocksthe transcription of CD28 (49). These CD4�CD28null T cells havebeen shown to be potent producers of cytokines (including TNF-�and IFN-�) (50), and to also express costimulatory molecules nor-mally associated with NK cells (51). It will be interesting to de-termine whether this CD4�CD28null T cell subset interacts withFLS through the B7-H3 molecule.

CD4�CD28null T cells are, of course, not the only inflammatoryT cell subset in RA. Recent trials indicate that the drug abatacept(CTLA-4-IgG fusion protein) has clinical benefit in RA (52). Un-fortunately, as with all current therapy for RA, complete remissionof disease is unlikely. Nonetheless the clinical data proves thatinterference with costimulatory molecules may be an effective ap-proach for treatment of RA, and it is likely that targets other thanCD28 will also be studied. The robust, constitutive and selectiveexpression of B7-H3 by FLS in vivo and in vitro, and its involve-ment in FLS/T cell interactions implies a unique and significantrole for this molecule in the pathogenesis of synovitis. Definitionof its receptors on the T cell, and further insights into their co-stimulatory or inhibitory signaling functions will be required todetermine whether B7-H3 or its receptors are, like CD28, potentialtargets for novel molecular therapeutic agents in RA and otherdiseases.

AcknowledgmentDonna Cash for preparation of the manuscript.

DisclosuresThe authors have no financial conflict of interest.

References1. Miranda-Carus, M. E., A. Balsa, M. Benito-Miguel, C. Perez de Ayala, and

E. Martin-Mola. 2004. IL-15 and the initiation of cell contact-dependent synovialfibroblast-T lymphocyte cross-talk in rheumatoid arthritis: effect of methotrexate.J. Immunol. 173: 1463–1476.

2. Tsai, C., L. A. Diaz, Jr., N. G. Singer, L. L. Li, A. H. Kirsch, R. Mitra,B. J. Nickoloff, L. J. Crofford, and D. A. Fox. 1996. Responsiveness of human Tlymphocytes to bacterial superantigens presented by cultured rheumatoid arthritissynoviocytes. Arthritis Rheum. 39: 125–136.

3. Vallejo, A. N., H. Yang, P. A. Klimiuk, C. M. Weyand, and J. J. Goronzy. 2003.Synoviocyte-mediated expansion of inflammatory T cells in rheumatoid synovitisis dependent on CD47-thrombospondin 1 interaction. J. Immunol. 171:1732–1740.

4. Yamamura, Y., R. Gupta, Y. Morita, X. He, R. Pai, J. Endres, A. Freiberg,K. Chung, and D. A. Fox. 2001. Effector function of resting T cells: activation ofsynovial fibroblasts. J. Immunol. 166: 2270–2275.

5. Tran, C. N., M. J. Davis, L. A. Tesmer, J. L. Endres, C. D. Motyl, C. Smuda,E. C. Somers, K. C. Chung, A. G. Urquhart, S. K. Lundy, et al. 2007. Presentation

of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes.Arthritis Rheum. 56: 1497–1506.

6. Kim, W. U., M. L. Cho, Y. O. Jung, S. Y. Min, S. W. Park, D. J. Min, J. H. Yoon,and H. Y. Kim. 2004. Type II collagen autoimmunity in rheumatoid arthritis.Am. J. Med. Sci. 327: 202–211.

7. Bombara, M. P., D. L. Webb, P. Conrad, C. W. Marlor, T. Sarr, G. E. Ranges,T. M. Aune, J. M. Greve, and M. L. Blue. 1993. Cell contact between T cells andsynovial fibroblasts causes induction of adhesion molecules and cytokines.J. Leukocyte Biol. 54: 399–406.

8. Nakatsuka, K., Y. Tanaka, S. Hubscher, M. Abe, A. Wake, K. Saito, I. Morimoto,and S. Eto. 1997. Rheumatoid synovial fibroblasts are stimulated by the cellularadhesion to T cells through lymphocyte function associated antigen-1/intercellu-lar adhesion molecule-1. J. Rheumatol. 24: 458–464.

9. Krzesicki, R. F., W. E. Fleming, G. E. Winterrowd, C. A. Hatfield, M. E. Sanders,and J. E. Chin. 1991. T lymphocyte adhesion to human synovial fibroblasts: roleof cytokines and the interaction between intercellular adhesion molecule 1 andCD11a/CD18. Arthritis Rheum. 34: 1245–1253.

10. Zimmermann, T., E. Kunisch, R. Pfeiffer, A. Hirth, H. D. Stahl, U. Sack,A. Laube, E. Liesaus, A. Roth, E. Palombo-Kinne, et al. 2001. Isolation andcharacterization of rheumatoid arthritis synovial fibroblasts from primary cul-ture–primary culture cells markedly differ from fourth-passage cells. ArthritisRes. 3: 72–76.

11. Wicks, I. P., T. Leizer, S. O. Wawryk, J. R. Novotny, J. Hamilton, G. Vitti, andA. W. Boyd. 1992. The effect of cytokines on the expression of MHC antigensand ICAM-1 by normal and transformed synoviocytes. Autoimmunity 12: 13–19.

12. Boots, A. M., A. J. Wimmers-Bertens, and A. W. Rijnders. 1994. Antigen-pre-senting capacity of rheumatoid synovial fibroblasts. Immunology 82: 268–274.

13. Matsuoka, N., K. Eguchi, A. Kawakami, M. Tsuboi, Y. Kawabe, T. Aoyagi, andS. Nagataki. 1996. Inhibitory effect of clarithromycin on costimulatory moleculeexpression and cytokine production by synovial fibroblast-like cells. Clin. Exp.Immunol. 104: 501–508.

14. Corrigall, V. M., E. Solau-Gervais, and G. S. Panayi. 2000. Lack of CD80 ex-pression by fibroblast-like synoviocytes leading to anergy in T lymphocytes.Arthritis Rheum. 43: 1606–1615.

15. Chapoval, A. I., J. Ni, J. S. Lau, R. A. Wilcox, D. B. Flies, D. Liu, H. Dong,G. L. Sica, G. Zhu, K. Tamada, and L. Chen. 2001. B7-H3: a costimulatorymolecule for T cell activation and IFN-� production. Nat. Immunol. 2: 269–274.

16. Ling, V., P. W. Wu, V. Spaulding, J. Kieleczawa, D. Luxenberg, B. M. Carreno,and M. Collins. 2003. Duplication of primate and rodent B7-H3 immunoglobulinV- and C-like domains: divergent history of functional redundancy and exon loss.Genomics 82: 365–377.

17. Steinberger, P., O. Majdic, S. V. Derdak, K. Pfistershammer, S. Kirchberger,C. Klauser, G. Zlabinger, W. F. Pickl, J. Stockl, and W. Knapp. 2004. Molecularcharacterization of human 4Ig-B7-H3, a member of the B7 family with fourIg-like domains. J. Immunol. 172: 2352–2359.

18. Sun, M., S. Richards, D. V. Prasad, X. M. Mai, A. Rudensky, and C. Dong. 2002.Characterization of mouse and human B7-H3 genes. J. Immunol. 168:6294–6297.

19. Prasad, D. V., T. Nguyen, Z. Li, Y. Yang, J. Duong, Y. Wang, and C. Dong.2004. Murine B7-H3 is a negative regulator of T cells. J. Immunol. 173:2500–2506.

20. Suh, W. K., B. U. Gajewska, H. Okada, M. A. Gronski, E. M. Bertram,W. Dawicki, G. S. Duncan, J. Bukczynski, S. Plyte, A. Elia, et al. 2003. The B7family member B7-H3 preferentially down-regulates T helper type 1-mediatedimmune responses. Nat. Immunol. 4: 899–906.

21. Luo, L., A. I. Chapoval, D. B. Flies, G. Zhu, F. Hirano, S. Wang, J. S. Lau,H. Dong, K. Tamada, A. S. Flies, et al. 2004. B7-H3 enhances tumor immunityin vivo by costimulating rapid clonal expansion of antigen-specific CD8� cyto-lytic T cells. J. Immunol. 173: 5445–5450.

22. Sun, X., M. Vale, E. Leung, J. R. Kanwar, R. Gupta, and G. W. Krissansen. 2003.Mouse B7-H3 induces antitumor immunity. Gene Ther. 10: 1728–1734.

23. Wang, L., C. C. Fraser, K. Kikly, A. D. Wells, R. Han, A. J. Coyle, L. Chen, andW. W. Hancock. 2005. B7-H3 promotes acute and chronic allograft rejection.Eur. J. Immunol. 35: 428–438.

24. Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries,N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, et al. 1988.The American Rheumatism Association 1987 revised criteria for the classifica-tion of rheumatoid arthritis. Arthritis Rheum. 31: 315–324.

25. Brennan, F. M., A. L. Hayes, C. J. Ciesielski, P. Green, B. M. Foxwell, andM. Feldmann. 2002. Evidence that rheumatoid arthritis synovial T cells are sim-ilar to cytokine-activated T cells: involvement of phosphatidylinositol 3-kinaseand nuclear factor �B pathways in tumor necrosis factor alpha production inrheumatoid arthritis. Arthritis Rheum. 46: 31–41.

26. Coligan, J. E., B. Bierer, D. H. Margulies, E. M. Shevach, W. Strober, andR. Coico. 2005. Current protocols in immunology. In Current Protocols.R. Coico, P. Brown, J. C. Donovan, and A. Kruisbeek, eds. John Wiley & Sons,Hoboken, N.J., p. 100.

27. Valencia, X., J. M. Higgins, H. P. Kiener, D. M. Lee, T. A. Podrebarac,C. C. Dascher, G. F. Watts, E. Mizoguchi, B. Simmons, D. D. Patel, et al. 2004.Cadherin-11 provides specific cellular adhesion between fibroblast-like synovio-cytes. J. Exp. Med. 200: 1673–1679.

28. Mueller, D. L., M. K. Jenkins, and R. H. Schwartz. 1989. Clonal expansionversus functional clonal inactivation: a costimulatory signalling pathway deter-mines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol. 7:445–480.

29. Mueller, D. L., M. K. Jenkins, L. Chiodetti, and R. H. Schwartz. 1990. An in-tracellular calcium increase and protein kinase C activation fail to initiate T cell

2997The Journal of Immunology

by guest on February 2, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

proliferation in the absence of a costimulatory signal. J. Immunol. 144:3701–3709.

30. Roska, A. K., and P. E. Lipsky. 1985. Dissection of the functions of antigen-presenting cells in the induction of T cell activation. J. Immunol. 135: 2953–2961.

31. Mueller, D. L., M. K. Jenkins, and R. H. Schwartz. 1989. An accessory cell-derived costimulatory signal acts independently of protein kinase C activation toallow T cell proliferation and prevent the induction of unresponsiveness. J. Im-munol. 142: 2617–2628.

32. Linsley, P. S., W. Brady, L. Grosmaire, A. Aruffo, N. K. Damle, andJ. A. Ledbetter. 1991. Binding of the B cell activation antigen B7 to CD28costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp.Med. 173: 721–730.

33. Gimmi, C. D., G. J. Freeman, J. G. Gribben, K. Sugita, A. S. Freedman,C. Morimoto, and L. M. Nadler. 1991. B-cell surface antigen B7 provides acostimulatory signal that induces T cells to proliferate and secrete interleukin 2.Proc. Natl. Acad. Sci. USA 88: 6575–6579.

34. Koulova, L., E. A. Clark, G. Shu, and B. Dupont. 1991. The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4� T cells. J. Exp.Med. 173: 759–762.

35. Jenkins, M. K., P. S. Taylor, S. D. Norton, and K. B. Urdahl. 1991. CD28 deliversa costimulatory signal involved in antigen-specific IL-2 production by human Tcells. J. Immunol. 147: 2461–2466.

36. Linsley, P. S., E. A. Clark, and J. A. Ledbetter. 1990. T-cell antigen CD28mediates adhesion with B cells by interacting with activation antigen B7/BB-1.Proc. Natl. Acad. Sci. USA 87: 5031–5035.

37. Linsley, P. S., W. Brady, M. Urnes, L. S. Grosmaire, N. K. Damle, andJ. A. Ledbetter. 1991. CTLA-4 is a second receptor for the B cell activationantigen B7. J. Exp. Med. 174: 561–569.

38. Collins, M., V. Ling, and B. M. Carreno. 2005. The B7 family of immune-regulatory ligands. Genome Biol. 6: 223.

39. Greenwald, R. J., G. J. Freeman, and A. H. Sharpe. 2005. The B7 family revis-ited. Annu. Rev. Immunol. 23: 515–548.

40. Rietz, C., and L. Chen. 2004. New B7 family members with positive and negativecostimulatory function. Am. J. Transplant. 4: 8–14.

41. Helmy, K. Y., K. J. Katschke, Jr., N. N. Gorgani, N. M. Kljavin, J. M. Elliott,L. Diehl, S. J. Scales, N. Ghilardi, and M. van Lookeren Campagne. 2006. CRIg:a macrophage complement receptor required for phagocytosis of circulatingpathogens. Cell 124: 915–927.

42. Vogt, L., N. Schmitz, M. O. Kurrer, M. Bauer, H. I. Hinton, S. Behnke, D. Gatto,P. Sebbel, R. R. Beerli, I. Sonderegger, et al. 2006. VSIG4, a B7 family-relatedprotein, is a negative regulator of T cell activation. J. Clin. Invest. 116:2817–2826.

43. Grohmann, U., C. Orabona, F. Fallarino, C. Vacca, F. Calcinaro, A. Falorni,P. Candeloro, M. L. Belladonna, R. Bianchi, M. C. Fioretti, and P. Puccetti. 2002.CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat. Immunol. 3:1097–1101.

44. Orabona, C., U. Grohmann, M. L. Belladonna, F. Fallarino, C. Vacca, R. Bianchi,S. Bozza, C. Volpi, B. L. Salomon, et al. 2004. CD28 induces immunostimulatorysignals in dendritic cells via CD80 and CD86. Nat. Immunol. 5: 1134–1142.

45. Butte, M. J., M. E. Keir, T. B. Phamduy, A. H. Sharpe, and G. J. Freeman. 2007.Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatorymolecule to inhibit T cell responses. Immunity 27: 111–122.

46. Castriconi, R., A. Dondero, R. Augugliaro, C. Cantoni, B. Carnemolla,A. R. Sementa, F. Negri, R. Conte, M. V. Corrias, L. Moretta, et al. 2004. Iden-tification of 4Ig-B7-H3 as a neuroblastoma-associated molecule that exerts aprotective role from an NK cell-mediated lysis. Proc. Natl. Acad. Sci. USA 101:12640–12645.

47. Schmidt, D., P. B. Martens, C. M. Weyand, and J. J. Goronzy. 1996. The rep-ertoire of CD4�CD28� T cells in rheumatoid arthritis. Mol. Med. 2: 608–618.

48. Schmidt, D., J. J. Goronzy, and C. M. Weyand. 1996. CD4�CD7�CD28� T cellsare expanded in rheumatoid arthritis and are characterized by autoreactivity.J. Clin. Invest. 97: 2027–2037.

49. Bryl, E., A. N. Vallejo, C. M. Weyand, and J. J. Goronzy. 2001. Down-regulationof CD28 expression by TNF-�. J. Immunol. 167: 3231–3238.

50. Duftner, C., C. Dejaco, W. Kullich, A. Klauser, C. Goldberger, A. Falkenbach,and M. Schirmer. 2006. Preferential type 1 chemokine receptors and cytokineproduction of CD28� T cells in ankylosing spondylitis. Ann. Rheum. Dis. 65:647–653.

51. Namekawa, T., M. R. Snyder, J. H. Yen, B. E. Goehring, P. J. Leibson,C. M. Weyand, and J. J. Goronzy. 2000. Killer cell activating receptors functionas costimulatory molecules on CD4�CD28null T cells clonally expanded in rheu-matoid arthritis. J. Immunol. 165: 1138–1145.

52. Kremer, J. M., H. K. Genant, L. W. Moreland, A. S. Russell, P. Emery,C. Abud-Mendoza, J. Szechinski, T. Li, Z. Ge, J. C. Becker, and R. Westhovens.2006. Effects of abatacept in patients with methotrexate-resistant active rheuma-toid arthritis: a randomized trial. Ann. Intern. Med. 144: 865–876.

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