Glomerulonephritis Humoral Responses and Immune Complex

CD100 Enhances Dendritic Cell and CD4+ Cell Activation Leading to Pathogenetic Humoral Responses and Immune Complex Glomerulonephritis This information is current as of September 27, 2021. Ming Li, Kim M. O'Sullivan, Lynelle K. Jones, Timothy Semple, Atsushi Kumanogoh, Hitoshi Kikutani, Stephen R. Holdsworth and A. Richard Kitching J Immunol 2006; 177:3406-3412; ; doi: 10.4049/jimmunol.177.5.3406 Downloaded from http://www.jimmunol.org/content/177/5/3406

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD100 Enhances Dendritic Cell and CD4؉ Cell Activation Leading to Pathogenetic Humoral Responses and Immune Complex Glomerulonephritis1

Ming Li,* Kim M. O’Sullivan,* Lynelle K. Jones,* Timothy Semple,* Atsushi Kumanogoh,† Hitoshi Kikutani,† Stephen R. Holdsworth,* and A. Richard Kitching2*

CD100, a member of the semaphorin family, is a costimulatory molecule in adaptive immune responses by switching off CD72’s negative signals. However, CD100’s potential pathogenetic effects in damaging immune responses remain largely unexplored. We tested the hypothesis that CD100 plays a pathogenetic role in experimental immune complex glomerulonephritis. Daily injection of horse apoferritin for 14 days induced immune complex formation, mesangial proliferative glomerulonephritis and proteinuria ؉/؉ ؊/؊ in CD100-intact (CD100 ) BALB/c mice. CD100-deficient (CD100 ) mice were protected from histological and functional Downloaded from glomerular injury. They exhibited reduced deposition of Igs and C3 in glomeruli, reduced MCP-1 and MIP-2 intrarenal mRNA expression, and diminished glomerular macrophage accumulation. Attenuated glomerular injury was associated with decreased Ag-specific Ig production, reduced CD4؉ cell activation and cytokine production. Following Ag injection, CD4؉ cell CD100 expression was enhanced and dendritic cell CD86 expression was up-regulated. However, in CD100؊/؊ mice, dendritic cell CD86 (but not CD80) up-regulation was significantly attenuated. Following i.p. immunization, CD86, but not CD80, promotes early ؉ Ag-specific TCR-transgenic DO11.10 CD4 cell proliferation and IFN-␥ production, suggesting that CD100 expression enables full http://www.jimmunol.org/ expression of CD86 and consequent CD4؉ cell activation. Transfer of CD100؉/؉ DO11.10 cells into CD100؊/؊ mice resulted in decreased proliferation demonstrating that CD100 from other sources in addition to CD100 from Ag-specific CD4؉ cells plays a role in initial T cell proliferation. Although T cell-B cell interactions also may be relevant, these studies demonstrate that CD100 enhances pathogenetic humoral immune responses and promotes the activation of APCs by up-regulating CD86 expression. The Journal of Immunology, 2006, 177: 3406–3412.

he CD100 (Sema4D) is a 150-kDa transmembrane protein responses are defective in CD100-deficient (CD100Ϫ/Ϫ) mice (7), of the class IV semaphorin subfamily (1–3), identified as whereas adaptive immune responses are significantly enhanced in by guest on September 27, 2021 T an inhibitor of axonal growth in neuronal development (4, CD100 transgenic mice that expressed a truncated form of CD100 5), but also expressed by cells of the immune system and involved (8). In addition, costimulatory signals mediated via CD100 result in immune responses (6–8). CD100 is expressed constitutively on in the activation of Ag-specific T cells by enhancing DC matura- resting T cells, but weakly on resting B cells and APCs (9, 10). tion (9). However, there is limited information on the functional CD100 expression is significantly up-regulated after stimulation role of CD100 in pathological inflammatory responses. One study (1, 9, 10) and mediates intracellular responses via ligation of its in experimental autoimmune encephalomyelitis showed that cell surface receptor CD72, which is expressed on B cells and CD100Ϫ/Ϫ mice were protected from disease (9). It is unclear 3 splenic dendritic cells (DCs) (3, 6, 7, 9, 10). Binding of CD100 to whether this pathogenetic role of CD100 extends to other forms of CD72 induces tyrosine dephosphorylation of CD72, resulting in tissue-specific diseases mediated by immune responses, particu- dissociation of CD72 from Src homology region 2 domain-con- larly humoral responses. taining tyrosine phosphatase-1 (6, 10, 11), the attenuation of neg- Glomerulonephritis (GN) is an important cause of renal disease, ative signaling, and the enhancement of immune responses. Stud- usually caused by injurious adaptive immune responses. The dif- ies in genetically modified mice have shown an important role for ferent types and patterns of injury in GN are due in part to the CD100 in both T and B cell responses. T cell priming and B cell participation of both humoral and cellular effector mechanisms (12). Circulating immune complexes, formed by the interaction of

*Centre for Inflammatory Diseases, Monash University Department of Medicine, soluble Ag with Ab are important in the pathogenesis of number of Clayton, Victoria, Australia; and †Department of Molecular Immunology, Research renal diseases, including lupus nephritis, postinfectious GN, and Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan serum sickness (13), and cause glomerular injury via disruption of Received for publication May 24, 2005. Accepted for publication May 25, 2006. the glomerular architecture, activation of the complement path- The costs of publication of this article were defrayed in part by the payment of page way, and the recruitment of effector leukocytes. Although some charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. studies have shown a role for CD40 and members of the B7 family 1 This work was supported by grants from the National Health and Medical Research in both autoimmune and nonautoimmune GN (14–17), the role of Council of Australia. CD100 in the development of GN is not known. To test the hy- 2 Address correspondence and reprint requests to Dr. A. Richard Kitching, Centre for pothesis that CD100 plays a pathogenetic role in humorally me- Inflammatory Diseases, Monash University Department of Medicine, Monash Med- diated GN, GN was induced by injecting a foreign Ag (apoferritin) ical Centre, Clayton 3168, Victoria, Australia. E-mail address: Richard. ϩ/ϩ [email protected] into CD100 wild-type (CD100 ) mice and mice genetically 3 Abbreviations used in this paper: DC, dendritic cell; GN, glomerulonephritis; PAS, deficient in CD100. This model is characterized by immune periodic acid-Schiff; PI, propidium iodide; MHC II, MHC class II. responses against apoferritin, immune complex deposition in

glomeruli, mesangial cell proliferation, the accumulation of mac- in 96-well plates in triplicate in complete culture medium (10% FCS/RPMI rophages in glomeruli, and proteinuria (18). These studies demon- 1640, supplied with L-glutamine, 2-ME) in the presence or absence of ␮ strate that CD100 plays a pathogenetic role in humorally mediated horse apoferritin (72 h). Cells were pulsed with 0.5 Ci/well thymidine ([3H]TdR) for the last 18 h, and the incorporation of the [3H]TdR was injury affecting the kidney by enhancing DC function and up-reg- ϩ detected with a liquid scintillation beta counter (Wallac 1409; Cambridge ulating CD4 T cell priming and subsequent B cell activation. Scientiﬁc). Results are expressed as follows: stimulation index ϭ stimulated group cpm/unstimulated group cpm. Materials and Methods Measurement of cytokine production by Ag-stimulated Mice and induction of immune complex GN lymphocytes CD100Ϫ/Ϫ BALB/c mice (7) and DO11.10 mice (19) (The Jackson Lab- Splenocytes were prepared as above. After three washes with HF2.5, cells oratory) were bred at Monash Medical Centre (Clayton, Victoria, Austra- were incubated in 24-well plates (4 ϫ 106 cells/ml in 10% FCS/RPMI 1640 lia). Male BALB/c mice (8–12 wk of age) were obtained from Monash with L-glutamine, 2-ME, 72 h with 40 ␮g/ml horse apoferritin). IFN-␥ and University Centre Animal Services. All mice were maintained in speciﬁc IL-4 in culture supernatant were measured by ELISA as previously de- pathogen-free conditions. Studies were approved by the Monash University scribed (23). The Abs used were rat anti-mouse IFN-␥ (R4-6A2; BD (Monash Medical Centre Committee B) Animal Ethics Committee. Four Pharmingen), biotinylated rat anti-mouse IFN-␥ (XMG1.2; BD Pharmin- milligrams of horse spleen apoferritin (Sigma-Aldrich) in 78 ␮l of NaCl ϩ ϩ Ϫ Ϫ gen), rat anti-mouse IL-4 (11B11; American Type Culture Collection), and were injected i.p. daily into CD100 / and CD100 / BALB/c recipients biotinylated rat anti-mouse IL-4 (BVD6, DNAX). IL-10 was measured for 14 days. Experiments ended on day 15. To obtain baseline values, age- ϩ ϩ using a similar protocol, using rat anti-mouse IL-10 capture Ab (BD and sex-matched nonimmunized BALB/c CD100 / and in some experi- Ϫ Ϫ Pharmingen) and biotinylated-rat anti-mouse IL-10 (BD Pharmingen). ments CD100 / mice were used. Histological examination was performed on coded slides, results are expressed as mean Ϯ SEM, and the

Preparation of DCs and flow cytometric analyses of immune Downloaded from significance of differences between groups was determined by unpaired t test or one-way ANOVA according to the data to be analyzed. The GN cells ϩ/ϩ experiment was performed twice with eight mice in each group (CD100 Mouse splenocytes (106 cells) were stained with the appropriate mAbs (see Ϫ/Ϫ and CD100 mice) in the first experiment and seven mice in each group below). Mouse DCs were prepared from the spleens of individual mice in the second. Results are presented from one of two consistent (24). Briefly, each spleen was cut into small fragments, and then suspended experiments. in 1 ml of RPMI 1640-FCS containing 1 mg/ml freshly dissolved colla- Assessment of renal injury, leukocyte infiltration, and chemokine genase type I (Sigma-Aldrich) and 0.2 ml of 0.1% DNase I (Roche). Col- lagenase/DNase digestion was conducted at room temperature for 20 min http://www.jimmunol.org/ mRNA with constant pipetting to facilitate digestion. Dissociation of T cell-DC Tissue sections (3 ␮m) from paraffin-embedded kidney tissue were stained complexes was achieved by adding EDTA (1/10 v of 0.1 M EDTA (pH with periodic acid-Schiff’s reagent (PAS) and deposition of PASϩ material 7.2) for 5 min. Residual stromal fragments were removed by passing sus- assessed (minimum, 50 glomeruli/mouse) using a 0–3ϩ scale: 0, no accu- pensions through a stainless-steel sieve. Samples were kept on ice in a mulation of PASϩ material; 1, mild; 2, moderate; and 3, more severe ac- divalent-metal free medium (EDTA-balanced salt solution-FCS) during cumulation of PASϩ material. Total glomerular cell nuclei were counted FACS analysis. FcRs were blocked by Mouse Fc-Block (BD Pharmingen), (minimum, 20 glomeruli/mouse; expressed as cells per glomerular cross and then 1% BSA in PBS with 5 mM EDTA containing appropriate mAbs was added to 106 cells and incubated for 30 min on ice. Propidium iodide section (c/gcs)). Urinary protein excretion was determined by a modified ␮ Bradford method on urine collected over the final 24 h of experiments. (PI) (1 g/ml; Calbiochem) was added to each sample before analysis. The following mAbs were used for the analyses (all BD Pharmingen Serum creatinine concentrations at the completion of experiments were by guest on September 27, 2021 measured by an enzymatic creatininase assay. Macrophages and neutro- unless noted): R-PE or allophycocyanin/Cy7-conjugated rat anti-mouse phils were demonstrated in glomeruli by three-layer immunoperoxidase CD4 (GK1.5), (FITC)-conjugated rat anti-mouse CD45R/B220 (RA3- staining of periodate-lysine paraformaldehyde-fixed frozen 6-␮m kidney 6B2), FITC-mouse anti-mouse-CD72 (K10.6), PE-rat anti-mouse CD19 sections (20). The primary mAbs were FA11, anti-mouse CD68 (21) for (clone 1D3), PE-hamster anti-mouse CD54 (ICAM-1) (3E2), and allophy- macrophages, and RB6-8C5, anti-Gr-1 (DNAX Research Institute) for neu- cocyanin-rat anti-mouse CD44 (IM7), FITC-rat anti-mouse CD25 (7D4), trophils. Isotype control IgG was used as a negative control. Total kidney PE-hamster anti-mouse CD11c (HL3), and PE-anti-DO11.10 TCR (KJI- RNA prepared as previously described (22) was assessed using the Ribo- 26). The rat anti-mouse CD100 (BMA12) (10), anti-mouse MHC class II Quant System (BD Pharmingen; template set mCK-5c) as previously de- (MHC II) (M5/114; K. Shortman, Walter and Eliza Hall Institute (WEHI), scribed (22), normalized to the housekeeping gene L32, and results are Parkville, Australia), rat anti-mouse CD86, and hamster anti-mouse CD80 expressed as arbitrary units. (GL1 and 16-10A1; both from D. Tarlinton, WEHI) hybridomas were cultured, purified, and labeled with Alexa Fluor-647 (Invitrogen Life Tech- nologies). Annexin V-fluos (Roche) was used to stain apoptotic cells (an- Detection of Ig and complement in glomeruli and serum Ag- ϩ Ϫ specific Abs nexin V PI ) as previously described (25). Flow cytometry was performed on FACScan (DakoCytomation). The following negative con- For deposition of mouse Ig, IgG1 and IgG2a frozen sections (6 ␮m) were trols were used: for CD100, CD80, or CD86, splenocytes from unmanipu- Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ stained using FITC-sheep anti-mouse Ig (Silenus; dilution, 1/100), FITC- lated CD100 / or CD80 / CD86 / mice were used. For other markers, rat anti-mouse IgG1 (BD Pharmingen; dilution, 1/200) and FITC-rat anti- isotype-matched irrelevant mAb were used. Cells fluorescing at levels mouse IgG2a (BD Pharmingen; dilution, 1/100). C3 was detected using above the negative control were considered positive. FITC-conjugated goat anti-mouse C3 (Cappel; dilution, 1/100). Fluores- cence intensity was assessed semiquantitatively (0–3ϩ; minimum, 20 glo- DO11.10 cell preparation, adoptive transfer, and immunization meruli). Titers of mouse anti-horse apoferritin were measured by ELISA on Single-cell suspensions were prepared from lymph nodes of DO11.10 serum collected at the end of experiments (21). Microtiter plates were (BALB/c OVA-specific TCR-transgenic) mice (26). In experiments trans- coated with horse apoferritin (50 ␮g/ml), washed, blocked (1% BSA), and ϩ ϩ ferring purified CD4 T cells, the CD4 T cell population was enriched by then incubated with diluted mouse serum (1 h, 37°C). Mouse IgG and IgG1 positive selection via passage of cell preparations through a magnetic col- were detected with HRP-conjugated sheep anti-mouse IgG or goat anti- umn (MACS Technology; Miltenyi Biotec) according to the manufactur- mouse IgG1 (Amersham Biosciences; 1/2000; and Silenus; 1/4000). For er’s instructions. Cells were then stained with PE-anti-CD11c and PE-anti- IgG2a, plates were blocked with 2% casein, incubated with diluted serum ϩ CD19 and sorted by flow cytometry to further exclude CD11c and (2 h, room temperature), and then 2 ␮g/ml biotinylated rat anti-mouse ϩ CD19 cells. Samples of 105 cells stained with allophycocyanin/Cy7-anti- IgG2a (BD Pharmingen), 1 ␮g/ml ExtrAvidin, biotinylated mouse anti- CD4 and PE-KJI-26 demonstrated that Ն97% of cells expressed the trans- avidin Ab, and 1.1 ␮g/ml ExtrAvidin-peroxidase (all Sigma-Aldrich). genic TCR. For CFSE labeling (27), 2 ϫ 107 cells/ml were resuspended in Measurement of lymphocyte proliferation 0.1% BSA-PBS containing 10 ␮M CFSE (Invitrogen Life Technologies), incubated (37°C, 10 min), and then washed with 2.5% FCS-RPMI 1640 Spleens were removed from mice at the end of the experiments and placed three times. Recipient mice were injected i.v. with 3–5 ϫ 106 (consistent in 2.5% FCS-Hanks medium (HF2.5). Single-cell suspensions were pre- within an experiment) CFSE-labeled CD4ϩ.KJI-26ϩ T cells in FCS-free pared by gently pushing spleens through mesh sieves. Erythrocytes were RPMI 1640, and then immunized i.p the next day with 0.25 mg of OVA in lysed by incubation in Boy’s solution (0.17 M Tris/0.16 M ammonium PBS. Anti-CD86, anti-CD80, or control Ab was administered at a dose of chloride; 1 min at 37°C). A total of 4 ϫ 105 cells per well were incubated 0.1 mg, i.p., 1 day before and 1 day after transfer of CFSE-labeled 3408 CD100 IN GN

Table I. Glomerular inﬂammation is reduced in CD100Ϫ/Ϫ mice with immune complex GN

CD100ϩ/ϩ CD100Ϫ/Ϫ (n ϭ 7) (n ϭ 8) p Value

PASϩ materiala 1.6 Ϯ 0.1 0.9 Ϯ 0.1 Ͻ0.001 Glomerular cells/gcsb 41.8 Ϯ 1.0 34.1 Ϯ 0.5 Ͻ0.0001 Glomerular Iga 2.3 Ϯ 0.1 1.5 Ϯ 0.1 Ͻ0.001 Glomerular IgG1a 2.4 Ϯ 0.2 0.9 Ϯ 0.2 Ͻ0.0001 Glomerular IgG2aa 1.3 Ϯ 0.2 0.3 Ϯ 0.1 Ͻ0.001 Complement (C3)a 2.7 Ϯ 0.2 1.6 Ϯ 0.2 Ͻ0.01 CCL2 (MCP-1)c 6.0 Ϯ 1.4 2.9 Ϯ 0.4 Ͻ0.05 CXCL1 (MIP-2)c 3.0 Ϯ 1.0 1.1 Ϯ 0.1 Ͻ0.05 Macrophages/gcsb 5.7 Ϯ 0.4 4.3 Ϯ 0.3 Ͻ0.01

a Glomeruli were scored under a microscope by using a 0–3 scale, with a score of 0 being equivalent to nonimmunized CD100ϩ/ϩ mice. The value for the accumulation of PASϩ material in nonimmunized CD100ϩ/ϩ mice is 0.27 Ϯ 0.02. b Expressed as cells per glomerular cross section (gcs). The value for nonimmunized CD100ϩ/ϩ mice is 33.2 Ϯ 0.6 cells/gcs and 0.21 Ϯ 0.04 cells/gcs for macrophages. c Expressed in arbitrary units (AU). Values for nonimmunized mice are as follows: CCL2: CD100ϩ/ϩ, 4.5 Ϯ 0.9; CD100Ϫ/Ϫ, 3.7 Ϯ 1.6 AU; CXCL1: CD100ϩ/ϩ, 1.5 Ϯ 0.3; CD100Ϫ/Ϫ, 1.7 Ϯ 0.1 AU.

Ϫ Ϫ Downloaded from DO11.10 cells. Three days after immunization, mice were humanely killed, with GN expressed intrarenal chemokine mRNA. In CD100 / ϩ ϩ ϩ splenocytes were stained, and CD4 , KJI-26 , CFSE cells were kidneys, expression of CCL2 (MCP-1) and CXCL1 (MIP-2) were analyzed. reduced at 14 days, CCL2 being reduced also at 7 days ϩ/ϩ Ϯ Ϫ/Ϫ Ϯ Ͻ Results (CD100 , 8.4 1.9; CD100 , 4.1 0.5 arbitrary units; p 0.05). CCL5 (RANTES) mRNA expression was increased in both Endogenous CD100 enhances immune renal injury, systemic Ig ϩ ϩ Ϫ Ϫ CD100 / and CD100 / mice, but mRNA expression for production, glomerular Ig deposition, and recruitment of http://www.jimmunol.org/ CXCL10 (IFN-␥-inducible protein 10), CCL1 (TCA-3), XCL1 glomerular effectors (lymphotactin), CCL3 (MIP-1␣), and CCL4 (MIP-1␤) were not ϩ ϩ After 14 days, wild-type BALB/c (CD100 / ) mice had devel- increased over unimmunized mice (except CD100Ϫ/Ϫ CCL3 oped renal injury. Proliferation, predominantly mesangial, was (MIP-1␣) at 7 days, not increased compared with CD100ϩ/ϩ mice ϩ prominent in glomeruli with deposition of PAS material and pro- at 7 days; data not shown). teinuria (summarized in Table I). Renal injury in CD100Ϫ/Ϫ mice ϩ was attenuated with reduced accumulation of PASϩ material, glo- Endogenous CD100 enhances CD4 T cell function and merular hypercellularity, and decreased proteinuria. Renal failure survival

is not a feature of this model, and there was no difference in serum Collectively, these results imply an important role for CD100 in by guest on September 27, 2021 creatinine between mice (data not shown). Compared with B cell activation and the development of the subsequent Ab ϩ/ϩ Ϫ/Ϫ CD100 mice with GN, CD100 mice had reduced deposi- response. To understand why humoral responses and therefore tion of total Ig, IgG1, and IgG2a in glomeruli (Fig. 1). Changes in renal injury were reduced in the absence of endogenous CD100, glomerular Ig deposition were reflected in systemic Ag-specific immune responses were further assessed in CD100ϩ/ϩ mice and total Ig, IgG1, and IgG2a titers that were decreased in sera of CD100Ϫ/Ϫ mice. Ag-specific lymphocyte proliferation in mice CD100Ϫ/Ϫ mice with GN (Fig. 2). Complement (C3) was depos- ited in glomeruli in CD100ϩ/ϩ mice and reduced in CD100Ϫ/Ϫ mice. The influx of CD68ϩ macrophages into glomeruli was less severe in CD100Ϫ/Ϫ mice. Neutrophils (Gr-1ϩ cells) were only occasionally observed in glomeruli of mice with GN and diminished in the absence of CD100 (data not shown). CD100ϩ/ϩ mice

FIGURE 1. Attenuated glomerular injury in CD100Ϫ/Ϫ mice. a, Pro- teinuria in CD100Ϫ/Ϫ mice with immune complex GN was lower than that of CD100ϩ/ϩ mice with GN. The dotted line refers to values in nonim- FIGURE 2. Reduced systemic Ag-specific Ab responses in CD100Ϫ/Ϫ munized CD100ϩ/ϩ mice. b, Immunofluorescent staining demonstrated mice in immune complex GN. After injection of apoferritin for 14 days, strong deposition of mouse Ig in CD100ϩ/ϩ mice, but there was reduced titers of serum total Ag-specific IgG, IgG1, and IgG2a (ELISA) were sig- deposition of Ig in glomeruli of CD100Ϫ/Ϫ mice. Magnification, ϫ400. nificantly decreased in CD100Ϫ/Ϫ mice (n ϭ 8) compared with CD100ϩ/ϩ .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .(p Ͻ 0.001. mice (n ϭ 7 ,ءءء The Journal of Immunology 3409 with GN ([3H]TdR incorporation) demonstrated decreased lym- reach significance ( p ϭ 0.051). The proportion of CD100Ϫ/Ϫ.CD4ϩ phocyte proliferation in CD100Ϫ/Ϫ cells when stimulated with T cells undergoing apoptosis (annexin VϩPIϪ cells) was increased in different doses of apoferritin (Fig. 3a). CD100 expressed on T the absence of endogenous CD100 (Fig. 3d). cells has the potential to interact with B cell CD72. However, the reduced humoral response may be at least in part due to Expression of immune cell CD100/CD72 in disease, and alterations in CD4ϩ T cell activation and function in the ab- regulation of DC CD86 by CD100 sence of CD100. CD4ϩ T cell responses in wild-type and CD100Ϫ/Ϫ mice were compared at the end of experiments. Serial studies of immune cells at several time points were per- ϩ IL-10 and IL-4 production by Ag-stimulated CD100Ϫ/Ϫ spleno- formed. CD100 was expressed on both naive CD4 T cells ϩ cytes were decreased compared with CD100ϩ/ϩ mice, but (60.6 Ϯ 4.6%) and on CD19 B cells (76.2 Ϯ 4.1%; n ϭ 8). ϩ IFN-␥ production was not significantly reduced (Fig. 3b). Sim- CD100 expression was up-regulated on CD4 T cells by 72 h after ilar proportions of CD4ϩ cells (CD100ϩ/ϩ, 24.6 Ϯ 1.5%, vs injection of horse apoferritin (Fig. 4, a and b). A higher proportion ϩ CD100Ϫ/Ϫ, 22.4 Ϯ 1.1%) and B220ϩ cells (CD100ϩ/ϩ, 49.2 Ϯ of CD4 T cells were CD100 positive at 72 h. The proportion of ϩ 1.4%, vs CD100Ϫ/Ϫ, 51.0 Ϯ 1.2%) were present at the initiation B cells that were CD100 was unaltered over the course of the ϩ of culture. To examine T cell activation and apoptosis, CD54, model. Only a small proportion of CD4 T cells expressed low CD44, and annexin V expression on splenic CD100ϩ/ϩ and levels of CD72 (data not shown). CD72 was expressed on most B ϩ CD100Ϫ/Ϫ CD4ϩ T cells were measured (Fig. 3c). CD44 ex- cells. A marginally higher proportion of B cells were CD72 24 h pression was decreased on CD100Ϫ/Ϫ.CD4ϩ T cells compared after stimulation (Fig. 5c). ϩ ϩ ϩ with CD100 / mice, but differences in CD54 expression did not To determine whether impaired CD4 T cell function is medi- Downloaded from ated by impaired DC costimulatory function, CD72, CD80, and CD86 expression on CD11cϩ.MHC IIϩ cells was assessed. Pro- portions of CD11cϩ cells were similar in both CD100ϩ/ϩ and CD100Ϫ/Ϫ mice (data not shown). CD72 was expressed on a small proportion of CD11cϩ.MHC IIϩ cells (Fig. 5). Over the first 72 h, ϩ ϩ ϩ

this CD11c .MHC II CD72 population was increased. At 24 h, http://www.jimmunol.org/ this increase was evident only in CD100Ϫ/Ϫ mice. As expected, 24 h following Ag injection, CD86 expression was increased on CD11cϩ.MHC IIϩ cells from CD100ϩ/ϩ mice (Fig. 6, a and b). This increase was attenuated in the absence of endogenous CD100. Expression of CD80 was unchanged in the presence or absence of CD100 (Fig. 6c). by guest on September 27, 2021

FIGURE 3. Decreased T cell responses in CD100Ϫ/Ϫ mice. a, Reduced lymphocyte proliferation. Splenocytes from apoferritin-injected mice were cultured for a further 3 days in the presence of horse apoferritin. Compared with CD100ϩ/ϩ mice (n ϭ 6), lymphocyte proliferation was decreased in CD100Ϫ/Ϫ mice (n ϭ 7). b, Reduced cytokine production. Cytokine pro- FIGURE 4. Expression of CD100 on lymphocytes. a, CD100 is ex- duction from culture supernatants was measured. IL-10 and IL-4 were pressed on both naive CD4ϩ T cells and CD19ϩ B cells (representative diminished in the absence of CD100 (n ϭ 7), but IFN-␥ production was histograms from individual mice). CD100 expression on CD4ϩ cells was not significantly reduced. c, Activation markers were decreased on up-regulated after in vivo immunization (thick line; open fill). The shaded CD100Ϫ/Ϫ.CD4ϩ T cells. CD44 expression on CD100Ϫ/Ϫ.CD4ϩ cells gray peak represents control (CD100Ϫ/Ϫ) cells. b, A greater proportion of (n ϭ 7) was reduced compared with CD100ϩ/ϩ mice (n ϭ 6), but reduc- CD4ϩ T cells became CD100ϩ by 72 h after i.p. apoferritin, and the pro- tions in CD54 expression on CD100Ϫ/Ϫ.CD4ϩ cells did not reach signif- portion of CD4ϩ cells that were also CD100ϩ fell to that of naive mice icance (p ϭ 0.051). d, The proportion of apoptotic CD4ϩ cells (annexin later in the course of disease. c, There were no significant changes in ,ءء .VϩPIϪ) was increased in CD100Ϫ/Ϫ mice, compared with CD100ϩ/ϩ CD100 expression on CD19ϩ B cells during the development of GN .p Ͻ 0.01. p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .mice 3410 CD100 IN GN Downloaded from

FIGURE 6. CD11cϩ cell CD86, but not CD80 expression, was decreased in CD100Ϫ/Ϫ mice. a, The increase in CD86 expression (thick line with open ﬁll) on CD11cϩ.MHC IIϩ cells at 24 h was attenuated in the Ϫ/Ϫ ϩ ϩ ϩ absence of CD100. Control staining (CD86 mice) is represented by the

FIGURE 5. CD72 expression on CD11c .MHC II cells and CD19 ϩ ϩ ϩ http://www.jimmunol.org/ shaded areas. b, The proportion of CD86 CD11c .MHC II cells were cells. a, CD11cϩ.MHC IIϩ cells were examined to detect CD72ϩ cells increased after in vivo stimulation for 24 h, but up-regulation was pre- (thick line; open fill). The shaded gray peak represents staining with the vented in the absence of CD100 (n ϭ 4–8 per group). c, The proportion of isotype Ab control. Histograms were derived from gates set on ϩ ϩ ϩ Ϫ Ϫ CD11c .MHC II cells that were CD80 was not reduced in CD100 / CD11cϩ.MHC IIϩ cells. b, The proportion of CD11cϩ.MHC IIϩ cells that .p Ͻ 0.001 ,ءءء .(mice (n ϭ 4–8 mice per group were CD72ϩ increased after injection of apoferritin (24 h in CD100Ϫ/Ϫ mice), more so at 72 h on both CD100ϩ/ϩ and CD100Ϫ/Ϫ mice. c, CD72 was expressed on most B cells. The proportion of CD19ϩ expressing CD72 was significantly, but marginally increased in both CD100ϩ/ϩ and uli are often affected. The current studies demonstrate a role for CD100Ϫ/Ϫ groups after 24 h, and then fell over time (n ϭ 4–8 mice per CD100 in pathological Ab-mediated injury. In these diseases, Ag- p Ͻ 0.001. specific CD4ϩ cells, generated by APC-CD4ϩ cell interactions by guest on September 27, 2021 ,ءءء ;p Ͻ 0.05 ,ء .(group provide help for B cells that produce pathogenetic Abs. The current studies demonstrate that CD100Ϫ/Ϫ mice developed less se- CD86, but not CD80, is important in early T cell proliferation vere immune renal injury, both histological and functional, in im- and cytokine production following i.p. immunization mune complex GN. Immunized mice developed proliferative GN To determine the functional consequences of the selective increase with mesangial matrix expansion, complement deposition, macro- in CD86 expression in the presence of CD100, transgenic OVA- phage accumulation, and proteinuria. However, CD100Ϫ/Ϫ mice specific CFSE-labeled cells from TCR-transgenic DO11.10 mice developed only mild GN. Immune responses to the nephritogenic were transferred into OVA-immunized BALB/c mice. CD86 ex- Ag were attenuated at several levels, including DC costimulatory pression was important in optimal early proliferation and function molecule expression, CD4ϩ cell activation, and Ig production. (Fig. 7). Proliferation (serial halving of CFSE 72 h after immuni- Attenuation of injury in CD100Ϫ/Ϫ mice resulted from reduced zation) was reduced in recipients treated with anti-CD86 mAbs, T cell-dependent B cell Ab responses to the nephritogenic Ag, but not after anti-CD80 mAb treatment (Fig. 7, a and b). Compared horse apoferritin. Reduced serum Ag-specific Ab levels in the with OVA-immunized control Ab-treated or anti-CD80-treated CD100Ϫ/Ϫ mice paralleled glomerular findings. CD100 is up-ex- mice, in anti-CD86-treated mice more cells remained in an undi- pressed on activated CD4ϩ T cells. Because other studies have vided state and fewer cells had reached the fourth division. Ex vivo demonstrated that CD100 promotes B cell activation by the bind- splenocyte culture demonstrated reduced IFN-␥ production in ing of CD100 to CD72 expressed on B cells (6, 7, 10, 11), it is mice treated with anti-CD86 mAbs (Fig. 7c). probable that at least part of the reduced Ig production in the current studies in CD100Ϫ/Ϫ mice relates to uninhibited negative sig- CD100 from outside the Ag-specific CD4ϩ cell population plays naling in B cells by CD72. Additional experiments defined a role a role in T cell activation for CD100 in the early stages of the immune response. CD100 ϩ ϩ Purified CD4 .CD100 DO11.10 cells were transferred into deficiency results in defective T cell activation (7). The current ϩ ϩ Ϫ Ϫ Ϫ Ϫ CD100 / or CD100 / recipients. In CD100 / recipients, the studies show that, in this disease model, in which humoral immu- rate of proliferation of DO11.10 cells was reduced (Fig. 8, a and nity plays an important role, CD4ϩ cell activation and function b), demonstrating that endogenous CD100 from sources in addi- were suppressed. In the absence of CD100, CD4ϩ cells at day 14 ϩ tion to Ag-specific CD4 cells plays a role in early T cell were more prone to apoptosis, exhibited diminished markers of T proliferation. cell activation, and made less IL-4 and IL-10. In contrast to the findings in CD100Ϫ/Ϫ mice immunized s.c. with keyhole limpet Discussion hemocyanin in Freund’s complete adjuvant (7), IFN-␥ production Pathogenetic Ab responses and immune complexes are features of was not reduced. This may be due to the different route of admin- many nonautoimmune and autoimmune diseases, in which glomer- istration and use of adjuvants in the two studies. Reductions in The Journal of Immunology 3411

FIGURE 8. Reduced proliferation of DO11.10 cells in CD100Ϫ/Ϫ hosts. Puriﬁed CD4ϩ.KJI26ϩ cells from CD100ϩ/ϩ DO11.10 mice were transferred into CD100ϩ/ϩ or CD100Ϫ/Ϫ hosts that were then stimulated with i.p OVA. After 3 days, a lower proportion of CFSE-labeled DO11.10 cells had reached the fourth division (a), and there was a trend toward reduced ءءء ϩ ϩ Ϫ/Ϫ proportions of CD4 .KJI26 DO11.10 cells in CD100 mice (b). , Downloaded from p Ͻ 0.001.

clear. It is possible that transfer of Ag-specific CD100Ϫ/Ϫ cells would have a more substantial effect on limiting proliferation. The current studies focus to a considerable extent on the role of CD100 in affecting the ability of DCs to present Ag to and activate http://www.jimmunol.org/ FIGURE 7. In vivo administration of inhibitory anti-CD86 mAbs, but ϩ CD4 cells. B cells are influenced by CD100, express CD72, not anti-CD80 mAbs inhibited DO11.10 cell activation. a, Three days after ϩ i.p. OVA injection, CFSE-labeled proliferation of donor DO11.10 cells CD80, and CD86, and have the capacity to present Ag to CD4 ϩ/ϩ cells. CD100 may influence the Ag-presenting capacity of B cells, was decreased in anti-CD86 mAb-treated CD100 recipients. Compared ϩ with control IgG or anti-CD80-treated animals, a higher proportion of cells particularly in the generation of memory CD4 cells, because both from anti-CD86-treated animals had not divided and a lower proportion simulations (31) and experimental studies (32) suggest that B cells had reached the fourth division. b, The proportion of CD4ϩ.KJI26ϩ may be important in this process. (DO11.10 OVA-specific) cells from spleens of anti-CD86-treated mice was Renal injury in the current studies is driven primarily by the less than anti-CD80 or control IgG-treated groups. c, IFN-␥ production by deposition of immune complexes in glomeruli. Both CD100 and by guest on September 27, 2021 anti-CD86-treated mice was reduced following 3 days ex vivo splenocyte the tissue receptor for CD100, plexin-B1, are expressed in the ءءء Ͻ ءء Ͻ ء culture (without further Ag stimulation). , p 0.05; , p 0.01; , kidney (6, 33), the former largely in the tubulointerstitial compart- p Ͻ 0.001. ment (K. M. O’Sullivan, M. Li, and A. R. Kitching, unpublished observations). Therefore, although intrarenal CD100-plexin B1 interactions may be relevant to the development of other forms of humoral responses were not mediated via enhanced CD4ϩ.CD25ϩ ϩ immune renal injury, particularly those with significant tubuloin- regulatory T cells, because CD25 expression on CD4 cells was terstitial damage or those involving cell-mediated effector injury Ϫ/Ϫ decreased in CD100 mice (data not shown). (20, 23), they are unlikely to have played any role in this model. Because CD72 is expressed on only a small proportion of CD4ϩ ϩ Although these studies do not address a potential role for the sol- cells, we hypothesized that in vivo CD4 cell CD100 expression uble form of CD100, overexpression of soluble CD100 results in participates in DC activation by switching off CD72-induced neg- enhanced T cell-dependent Ab production (8). In conclusion, the ative signals. CD72 is expressed on DCs (9, 28). Following in vitro current studies implicate CD100 in DC activation by expression of stimulation, costimulatory molecule expression on DCs was re- CD86, leading to a number of effects on the generation of patho- duced in the absence of CD100. The current studies show that, in genetic humoral responses including the activation, cytokine ex- vivo, CD100 is required for optimal expression of CD86 on DCs. pression, and survival of CD4ϩ cells. Although proportions of splenic DCs were similar in the presence and absence of CD100, expression of CD86, but not CD80, was Ϫ/Ϫ Acknowledgments significantly reduced in CD100 mice following injection of the We acknowledge the technical assistance of Alice Wright and Gabrielle disease-initiating Ag. 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