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The Journal of Immunology

CD100 Enhances Dendritic Cell and CD4ⴙ 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 (CD100ⴙ/ⴙ) BALB/c mice. CD100-deficient (CD100ⴚ/ⴚ) mice were protected from histological and functional 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 CD4ⴙ cell activation and cytokine production. Following Ag injection, CD4ⴙ cell CD100 expression was enhanced and dendritic cell CD86 expression was up-regulated. However, in CD100ⴚ/ⴚ 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 CD4ⴙ cell proliferation and IFN-␥ production, suggesting that CD100 expression enables full expression of CD86 and consequent CD4ⴙ cell activation. Transfer of CD100ⴙ/ⴙ DO11.10 cells into CD100ⴚ/ⴚ mice resulted in decreased proliferation demonstrating that CD100 from other sources in addition to CD100 from Ag-specific CD4ⴙ 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.

T

he CD100 (Sema4D) is a 150-kDa transmembrane protein of the class IV semaphorin subfamily (1–3), identified as an inhibitor of axonal growth in neuronal development (4, 5), but also expressed by cells of the immune system and involved in immune responses (6 – 8). CD100 is expressed constitutively on resting T cells, but weakly on resting B cells and APCs (9, 10). CD100 expression is significantly up-regulated after stimulation (1, 9, 10) and mediates intracellular responses via ligation of its cell surface receptor CD72, which is expressed on B cells and splenic dendritic cells (DCs)3 (3, 6, 7, 9, 10). Binding of CD100 to CD72 induces tyrosine dephosphorylation of CD72, resulting in dissociation of CD72 from Src homology region 2 domain-containing tyrosine phosphatase-1 (6, 10, 11), the attenuation of negative signaling, and the enhancement of immune responses. Studies in genetically modified mice have shown an important role for CD100 in both T and B cell responses. T cell priming and B cell

*Centre for Inflammatory Diseases, Monash University Department of Medicine, Clayton, Victoria, Australia; and †Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan Received for publication May 24, 2005. Accepted for publication May 25, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the National Health and Medical Research Council of Australia. 2 Address correspondence and reprint requests to Dr. A. Richard Kitching, Centre for Inflammatory Diseases, Monash University Department of Medicine, Monash Medical Centre, Clayton 3168, Victoria, Australia. E-mail address: Richard. [email protected] 3 Abbreviations used in this paper: DC, dendritic cell; GN, glomerulonephritis; PAS, periodic acid-Schiff; PI, propidium iodide; MHC II, MHC class II.

Copyright © 2006 by The American Association of Immunologists, Inc.

responses are defective in CD100-deficient (CD100⫺/⫺) mice (7), whereas adaptive immune responses are significantly enhanced in CD100 transgenic mice that expressed a truncated form of CD100 (8). In addition, costimulatory signals mediated via CD100 result in the activation of Ag-specific T cells by enhancing DC maturation (9). However, there is limited information on the functional role of CD100 in pathological inflammatory responses. One study in experimental autoimmune encephalomyelitis showed that CD100⫺/⫺ mice were protected from disease (9). It is unclear whether this pathogenetic role of CD100 extends to other forms of tissue-specific diseases mediated by immune responses, particularly humoral responses. Glomerulonephritis (GN) is an important cause of renal disease, usually caused by injurious adaptive immune responses. The different types and patterns of injury in GN are due in part to the participation of both humoral and cellular effector mechanisms (12). Circulating immune complexes, formed by the interaction of soluble Ag with Ab are important in the pathogenesis of number of renal diseases, including lupus nephritis, postinfectious GN, and serum sickness (13), and cause glomerular injury via disruption of the glomerular architecture, activation of the complement pathway, and the recruitment of effector leukocytes. Although some studies have shown a role for CD40 and members of the B7 family in both autoimmune and nonautoimmune GN (14 –17), the role of CD100 in the development of GN is not known. To test the hypothesis that CD100 plays a pathogenetic role in humorally mediated GN, GN was induced by injecting a foreign Ag (apoferritin) into CD100 wild-type (CD100⫹/⫹) mice and mice genetically deficient in CD100. This model is characterized by immune responses against apoferritin, immune complex deposition in 0022-1767/06/$02.00

The Journal of Immunology glomeruli, mesangial cell proliferation, the accumulation of macrophages in glomeruli, and proteinuria (18). These studies demonstrate that CD100 plays a pathogenetic role in humorally mediated injury affecting the kidney by enhancing DC function and up-regulating CD4⫹ T cell priming and subsequent B cell activation.

Materials and Methods Mice and induction of immune complex GN CD100⫺/⫺ BALB/c mice (7) and DO11.10 mice (19) (The Jackson Laboratory) were bred at Monash Medical Centre (Clayton, Victoria, Australia). Male BALB/c mice (8 –12 wk of age) were obtained from Monash University Centre Animal Services. All mice were maintained in specific pathogen-free conditions. Studies were approved by the Monash University (Monash Medical Centre Committee B) Animal Ethics Committee. Four milligrams of horse spleen apoferritin (Sigma-Aldrich) in 78 ␮l of NaCl were injected i.p. daily into CD100⫹/⫹ and CD100⫺/⫺ BALB/c recipients for 14 days. Experiments ended on day 15. To obtain baseline values, ageand sex-matched nonimmunized BALB/c CD100⫹/⫹ and in some experiments CD100⫺/⫺ mice were used. Histological examination was performed on coded slides, results are expressed as mean ⫾ SEM, and the significance of differences between groups was determined by unpaired t test or one-way ANOVA according to the data to be analyzed. The GN experiment was performed twice with eight mice in each group (CD100⫹/⫹ and CD100⫺/⫺ mice) in the first experiment and seven mice in each group in the second. Results are presented from one of two consistent experiments.

Assessment of renal injury, leukocyte infiltration, and chemokine mRNA Tissue sections (3 ␮m) from paraffin-embedded kidney tissue were stained with periodic acid-Schiff’s reagent (PAS) and deposition of PAS⫹ material assessed (minimum, 50 glomeruli/mouse) using a 0 –3⫹ scale: 0, no accumulation of PAS⫹ material; 1, mild; 2, moderate; and 3, more severe accumulation of PAS⫹ material. Total glomerular cell nuclei were counted (minimum, 20 glomeruli/mouse; expressed as cells per glomerular cross section (c/gcs)). Urinary protein excretion was determined by a modified Bradford method on urine collected over the final 24 h of experiments. Serum creatinine concentrations at the completion of experiments were measured by an enzymatic creatininase assay. Macrophages and neutrophils were demonstrated in glomeruli by three-layer immunoperoxidase staining of periodate-lysine paraformaldehyde-fixed frozen 6-␮m kidney sections (20). The primary mAbs were FA11, anti-mouse CD68 (21) for macrophages, and RB6-8C5, anti-Gr-1 (DNAX Research Institute) for neutrophils. Isotype control IgG was used as a negative control. Total kidney RNA prepared as previously described (22) was assessed using the RiboQuant System (BD Pharmingen; template set mCK-5c) as previously described (22), normalized to the housekeeping gene L32, and results are expressed as arbitrary units.

Detection of Ig and complement in glomeruli and serum Agspecific Abs For deposition of mouse Ig, IgG1 and IgG2a frozen sections (6 ␮m) were stained using FITC-sheep anti-mouse Ig (Silenus; dilution, 1/100), FITCrat anti-mouse IgG1 (BD Pharmingen; dilution, 1/200) and FITC-rat antimouse IgG2a (BD Pharmingen; dilution, 1/100). C3 was detected using FITC-conjugated goat anti-mouse C3 (Cappel; dilution, 1/100). Fluorescence intensity was assessed semiquantitatively (0 –3⫹; minimum, 20 glomeruli). Titers of mouse anti-horse apoferritin were measured by ELISA on serum collected at the end of experiments (21). Microtiter plates were coated with horse apoferritin (50 ␮g/ml), washed, blocked (1% BSA), and then incubated with diluted mouse serum (1 h, 37°C). Mouse IgG and IgG1 were detected with HRP-conjugated sheep anti-mouse IgG or goat antimouse IgG1 (Amersham Biosciences; 1/2000; and Silenus; 1/4000). For IgG2a, plates were blocked with 2% casein, incubated with diluted serum (2 h, room temperature), and then 2 ␮g/ml biotinylated rat anti-mouse IgG2a (BD Pharmingen), 1 ␮g/ml ExtrAvidin, biotinylated mouse antiavidin Ab, and 1.1 ␮g/ml ExtrAvidin-peroxidase (all Sigma-Aldrich).

Measurement of lymphocyte proliferation Spleens were removed from mice at the end of the experiments and placed in 2.5% FCS-Hanks medium (HF2.5). Single-cell suspensions were prepared by gently pushing spleens through mesh sieves. Erythrocytes were lysed by incubation in Boy’s solution (0.17 M Tris/0.16 M ammonium chloride; 1 min at 37°C). A total of 4 ⫻ 105 cells per well were incubated

3407 in 96-well plates in triplicate in complete culture medium (10% FCS/RPMI 1640, supplied with L-glutamine, 2-ME) in the presence or absence of 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 detected with a liquid scintillation beta counter (Wallac 1409; Cambridge Scientific). Results are expressed as follows: stimulation index ⫽ stimulated group cpm/unstimulated group cpm.

Measurement of cytokine production by Ag-stimulated lymphocytes Splenocytes were prepared as above. After three washes with HF2.5, cells were incubated in 24-well plates (4 ⫻ 106 cells/ml in 10% FCS/RPMI 1640 with L-glutamine, 2-ME, 72 h with 40 ␮g/ml horse apoferritin). IFN-␥ and IL-4 in culture supernatant were measured by ELISA as previously described (23). The Abs used were rat anti-mouse IFN-␥ (R4-6A2; BD Pharmingen), biotinylated rat anti-mouse IFN-␥ (XMG1.2; BD Pharmingen), rat anti-mouse IL-4 (11B11; American Type Culture Collection), and biotinylated rat anti-mouse IL-4 (BVD6, DNAX). IL-10 was measured using a similar protocol, using rat anti-mouse IL-10 capture Ab (BD Pharmingen) and biotinylated-rat anti-mouse IL-10 (BD Pharmingen).

Preparation of DCs and flow cytometric analyses of immune cells Mouse splenocytes (106 cells) were stained with the appropriate mAbs (see below). Mouse DCs were prepared from the spleens of individual mice (24). Briefly, each spleen was cut into small fragments, and then suspended in 1 ml of RPMI 1640-FCS containing 1 mg/ml freshly dissolved collagenase type I (Sigma-Aldrich) and 0.2 ml of 0.1% DNase I (Roche). Collagenase/DNase digestion was conducted at room temperature for 20 min with constant pipetting to facilitate digestion. Dissociation of T cell-DC complexes was achieved by adding EDTA (1/10 v of 0.1 M EDTA (pH 7.2) for 5 min. Residual stromal fragments were removed by passing suspensions through a stainless-steel sieve. Samples were kept on ice in a divalent-metal free medium (EDTA-balanced salt solution-FCS) during FACS analysis. FcRs were blocked by Mouse Fc-Block (BD Pharmingen), 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 (PI) (1 ␮g/ml; Calbiochem) was added to each sample before analysis. The following mAbs were used for the analyses (all BD Pharmingen unless noted): R-PE or allophycocyanin/Cy7-conjugated rat anti-mouse CD4 (GK1.5), (FITC)-conjugated rat anti-mouse CD45R/B220 (RA36B2), FITC-mouse anti-mouse-CD72 (K10.6), PE-rat anti-mouse CD19 (clone 1D3), PE-hamster anti-mouse CD54 (ICAM-1) (3E2), and allophycocyanin-rat anti-mouse CD44 (IM7), FITC-rat anti-mouse CD25 (7D4), PE-hamster anti-mouse CD11c (HL3), and PE-anti-DO11.10 TCR (KJI26). The rat anti-mouse CD100 (BMA12) (10), anti-mouse MHC class II (MHC II) (M5/114; K. Shortman, Walter and Eliza Hall Institute (WEHI), Parkville, Australia), rat anti-mouse CD86, and hamster anti-mouse CD80 (GL1 and 16-10A1; both from D. Tarlinton, WEHI) hybridomas were cultured, purified, and labeled with Alexa Fluor-647 (Invitrogen Life Technologies). Annexin V-fluos (Roche) was used to stain apoptotic cells (annexin V⫹PI⫺) as previously described (25). Flow cytometry was performed on FACScan (DakoCytomation). The following negative controls were used: for CD100, CD80, or CD86, splenocytes from unmanipulated CD100⫺/⫺ or CD80⫺/⫺CD86⫺/⫺ mice were used. For other markers, isotype-matched irrelevant mAb were used. Cells fluorescing at levels above the negative control were considered positive.

DO11.10 cell preparation, adoptive transfer, and immunization Single-cell suspensions were prepared from lymph nodes of DO11.10 (BALB/c OVA-specific TCR-transgenic) mice (26). In experiments transferring purified CD4⫹ T cells, the CD4⫹ T cell population was enriched by positive selection via passage of cell preparations through a magnetic column (MACS Technology; Miltenyi Biotec) according to the manufacturer’s instructions. Cells were then stained with PE-anti-CD11c and PE-antiCD19 and sorted by flow cytometry to further exclude CD11c⫹ and CD19⫹ cells. Samples of 105 cells stained with allophycocyanin/Cy7-antiCD4 and PE-KJI-26 demonstrated that ⱖ97% of cells expressed the transgenic TCR. For CFSE labeling (27), 2 ⫻ 107 cells/ml were resuspended in 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 three times. Recipient mice were injected i.v. with 3–5 ⫻ 106 (consistent within an experiment) CFSE-labeled CD4⫹.KJI-26⫹ T cells in FCS-free RPMI 1640, and then immunized i.p the next day with 0.25 mg of OVA in PBS. Anti-CD86, anti-CD80, or control Ab was administered at a dose of 0.1 mg, i.p., 1 day before and 1 day after transfer of CFSE-labeled

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CD100 IN GN Table I. Glomerular inflammation is reduced in CD100⫺/⫺ mice with immune complex GN



a

PAS material Glomerular cells/gcsb Glomerular Iga Glomerular IgG1a Glomerular IgG2aa Complement (C3)a CCL2 (MCP-1)c CXCL1 (MIP-2)c Macrophages/gcsb

CD100⫹/⫹ (n ⫽ 7)

CD100⫺/⫺ (n ⫽ 8)

p Value

1.6 ⫾ 0.1 41.8 ⫾ 1.0 2.3 ⫾ 0.1 2.4 ⫾ 0.2 1.3 ⫾ 0.2 2.7 ⫾ 0.2 6.0 ⫾ 1.4 3.0 ⫾ 1.0 5.7 ⫾ 0.4

0.9 ⫾ 0.1 34.1 ⫾ 0.5 1.5 ⫾ 0.1 0.9 ⫾ 0.2 0.3 ⫾ 0.1 1.6 ⫾ 0.2 2.9 ⫾ 0.4 1.1 ⫾ 0.1 4.3 ⫾ 0.3

⬍0.001 ⬍0.0001 ⬍0.001 ⬍0.0001 ⬍0.001 ⬍0.01 ⬍0.05 ⬍0.05 ⬍0.01

a Glomeruli were scored under a microscope by using a 0 –3 scale, with a score of 0 being equivalent to nonimmunized CD100⫹/⫹ mice. The value for the accumulation of PAS⫹ material in nonimmunized CD100⫹/⫹ mice is 0.27 ⫾ 0.02. b Expressed as cells per glomerular cross section (gcs). The value for nonimmunized CD100⫹/⫹ mice is 33.2 ⫾ 0.6 cells/gcs and 0.21 ⫾ 0.04 cells/gcs for macrophages. c Expressed in arbitrary units (AU). Values for nonimmunized mice are as follows: CCL2: CD100⫹/⫹, 4.5 ⫾ 0.9; CD100⫺/⫺, 3.7 ⫾ 1.6 AU; CXCL1: CD100⫹/⫹, 1.5 ⫾ 0.3; CD100⫺/⫺, 1.7 ⫾ 0.1 AU.

DO11.10 cells. Three days after immunization, mice were humanely killed, splenocytes were stained, and CD4⫹, KJI-26⫹, CFSE⫹ cells were analyzed.

Results Endogenous CD100 enhances immune renal injury, systemic Ig production, glomerular Ig deposition, and recruitment of glomerular effectors After 14 days, wild-type BALB/c (CD100⫹/⫹) mice had developed renal injury. Proliferation, predominantly mesangial, was prominent in glomeruli with deposition of PAS⫹ material and proteinuria (summarized in Table I). Renal injury in CD100⫺/⫺ mice was attenuated with reduced accumulation of PAS⫹ material, glomerular hypercellularity, and decreased proteinuria. Renal failure is not a feature of this model, and there was no difference in serum creatinine between mice (data not shown). Compared with CD100⫹/⫹ mice with GN, CD100⫺/⫺ mice had reduced deposition of total Ig, IgG1, and IgG2a in glomeruli (Fig. 1). Changes in glomerular Ig deposition were reflected in systemic Ag-specific total Ig, IgG1, and IgG2a titers that were decreased in sera of CD100⫺/⫺ mice with GN (Fig. 2). Complement (C3) was deposited in glomeruli in CD100⫹/⫹ mice and reduced in CD100⫺/⫺ mice. The influx of CD68⫹ macrophages into glomeruli was less severe in CD100⫺/⫺ mice. Neutrophils (Gr-1⫹ cells) were only occasionally observed in glomeruli of mice with GN and diminished in the absence of CD100 (data not shown). CD100⫹/⫹ mice

FIGURE 1. Attenuated glomerular injury in CD100⫺/⫺ mice. a, Proteinuria in CD100⫺/⫺ mice with immune complex GN was lower than that of CD100⫹/⫹ mice with GN. The dotted line refers to values in nonimmunized CD100⫹/⫹ mice. b, Immunofluorescent staining demonstrated strong deposition of mouse Ig in CD100⫹/⫹ mice, but there was reduced deposition of Ig in glomeruli of CD100⫺/⫺ mice. Magnification, ⫻400. ⴱⴱⴱ, p ⬍ 0.001.

with GN expressed intrarenal chemokine mRNA. In CD100⫺/⫺ kidneys, expression of CCL2 (MCP-1) and CXCL1 (MIP-2) were reduced at 14 days, CCL2 being reduced also at 7 days (CD100⫹/⫹, 8.4 ⫾ 1.9; CD100⫺/⫺, 4.1 ⫾ 0.5 arbitrary units; p ⬍ 0.05). CCL5 (RANTES) mRNA expression was increased in both CD100⫹/⫹ and CD100⫺/⫺ mice, but mRNA expression for CXCL10 (IFN-␥-inducible protein 10), CCL1 (TCA-3), XCL1 (lymphotactin), CCL3 (MIP-1␣), and CCL4 (MIP-1␤) were not increased over unimmunized mice (except CD100⫺/⫺ CCL3 (MIP-1␣) at 7 days, not increased compared with CD100⫹/⫹ mice at 7 days; data not shown). Endogenous CD100 enhances CD4⫹ T cell function and survival Collectively, these results imply an important role for CD100 in B cell activation and the development of the subsequent Ab response. To understand why humoral responses and therefore renal injury were reduced in the absence of endogenous CD100, immune responses were further assessed in CD100⫹/⫹ mice and CD100⫺/⫺ mice. Ag-specific lymphocyte proliferation in mice

FIGURE 2. Reduced systemic Ag-specific Ab responses in CD100⫺/⫺ mice in immune complex GN. After injection of apoferritin for 14 days, titers of serum total Ag-specific IgG, IgG1, and IgG2a (ELISA) were significantly decreased in CD100⫺/⫺ mice (n ⫽ 8) compared with CD100⫹/⫹ mice (n ⫽ 7). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

The Journal of Immunology with GN ([3H]TdR incorporation) demonstrated decreased lymphocyte proliferation in CD100⫺/⫺ cells when stimulated with different doses of apoferritin (Fig. 3a). CD100 expressed on T cells has the potential to interact with B cell CD72. However, the reduced humoral response may be at least in part due to alterations in CD4⫹ T cell activation and function in the absence of CD100. CD4⫹ T cell responses in wild-type and CD100⫺/⫺ mice were compared at the end of experiments. IL-10 and IL-4 production by Ag-stimulated CD100⫺/⫺ splenocytes were decreased compared with CD100⫹/⫹ mice, but IFN-␥ production was not significantly reduced (Fig. 3b). Similar proportions of CD4⫹ cells (CD100⫹/⫹, 24.6 ⫾ 1.5%, vs CD100⫺/⫺, 22.4 ⫾ 1.1%) and B220⫹ cells (CD100⫹/⫹, 49.2 ⫾ 1.4%, vs CD100⫺/⫺, 51.0 ⫾ 1.2%) were present at the initiation of culture. To examine T cell activation and apoptosis, CD54, CD44, and annexin V expression on splenic CD100⫹/⫹ and CD100⫺/⫺ CD4⫹ T cells were measured (Fig. 3c). CD44 expression was decreased on CD100⫺/⫺.CD4⫹ T cells compared with CD100⫹/⫹ mice, but differences in CD54 expression did not

FIGURE 3. Decreased T cell responses in CD100⫺/⫺ 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 CD100⫹/⫹ mice (n ⫽ 6), lymphocyte proliferation was decreased in CD100⫺/⫺ mice (n ⫽ 7). b, Reduced cytokine production. Cytokine production from culture supernatants was measured. IL-10 and IL-4 were diminished in the absence of CD100 (n ⫽ 7), but IFN-␥ production was not significantly reduced. c, Activation markers were decreased on CD100⫺/⫺.CD4⫹ T cells. CD44 expression on CD100⫺/⫺.CD4⫹ cells (n ⫽ 7) was reduced compared with CD100⫹/⫹ mice (n ⫽ 6), but reductions in CD54 expression on CD100⫺/⫺.CD4⫹ cells did not reach significance (p ⫽ 0.051). d, The proportion of apoptotic CD4⫹ cells (annexin V⫹PI⫺) was increased in CD100⫺/⫺ mice, compared with CD100⫹/⫹ mice. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.

3409 reach significance ( p ⫽ 0.051). The proportion of CD100⫺/⫺.CD4⫹ T cells undergoing apoptosis (annexin V⫹PI⫺ cells) was increased in the absence of endogenous CD100 (Fig. 3d). Expression of immune cell CD100/CD72 in disease, and regulation of DC CD86 by CD100 Serial studies of immune cells at several time points were performed. CD100 was expressed on both naive CD4⫹ T cells (60.6 ⫾ 4.6%) and on CD19⫹ B cells (76.2 ⫾ 4.1%; n ⫽ 8). CD100 expression was up-regulated on CD4⫹ T cells by 72 h after injection of horse apoferritin (Fig. 4, a and b). A higher proportion of CD4⫹ T cells were CD100 positive at 72 h. The proportion of B cells that were CD100⫹ was unaltered over the course of the model. Only a small proportion of CD4⫹ T cells expressed low levels of CD72 (data not shown). CD72 was expressed on most B cells. A marginally higher proportion of B cells were CD72⫹ 24 h after stimulation (Fig. 5c). To determine whether impaired CD4⫹ T cell function is mediated by impaired DC costimulatory function, CD72, CD80, and CD86 expression on CD11c⫹.MHC II⫹ cells was assessed. Proportions of CD11c⫹ cells were similar in both CD100⫹/⫹ and CD100⫺/⫺ mice (data not shown). CD72 was expressed on a small proportion of CD11c⫹.MHC II⫹ cells (Fig. 5). Over the first 72 h, this CD11c⫹.MHC II⫹CD72⫹ population was increased. At 24 h, this increase was evident only in CD100⫺/⫺ mice. As expected, 24 h following Ag injection, CD86 expression was increased on CD11c⫹.MHC II⫹ cells from CD100⫹/⫹ 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).

FIGURE 4. Expression of CD100 on lymphocytes. a, CD100 is expressed on both naive CD4⫹ T cells and CD19⫹ B cells (representative histograms from individual mice). CD100 expression on CD4⫹ cells was up-regulated after in vivo immunization (thick line; open fill). The shaded gray peak represents control (CD100⫺/⫺) cells. b, A greater proportion of CD4⫹ T cells became CD100⫹ by 72 h after i.p. apoferritin, and the proportion of CD4⫹ cells that were also CD100⫹ fell to that of naive mice later in the course of disease. c, There were no significant changes in CD100 expression on CD19⫹ B cells during the development of GN. ⴱⴱ, p ⬍ 0.01.

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FIGURE 5. CD72 expression on CD11c⫹.MHC II⫹ cells and CD19⫹ cells. a, CD11c⫹.MHC II⫹ cells were examined to detect CD72⫹ cells (thick line; open fill). The shaded gray peak represents staining with the isotype Ab control. Histograms were derived from gates set on CD11c⫹.MHC II⫹ cells. b, The proportion of CD11c⫹.MHC II⫹ cells that were CD72⫹ increased after injection of apoferritin (24 h in CD100⫺/⫺ mice), more so at 72 h on both CD100⫹/⫹ and CD100⫺/⫺ mice. c, CD72 was expressed on most B cells. The proportion of CD19⫹ expressing CD72 was significantly, but marginally increased in both CD100⫹/⫹ and CD100⫺/⫺ groups after 24 h, and then fell over time (n ⫽ 4 – 8 mice per group). ⴱ, p ⬍ 0.05; ⴱⴱⴱ, p ⬍ 0.001.

CD86, but not CD80, is important in early T cell proliferation and cytokine production following i.p. immunization To determine the functional consequences of the selective increase in CD86 expression in the presence of CD100, transgenic OVAspecific CFSE-labeled cells from TCR-transgenic DO11.10 mice were transferred into OVA-immunized BALB/c mice. CD86 expression was important in optimal early proliferation and function (Fig. 7). Proliferation (serial halving of CFSE 72 h after immunization) was reduced in recipients treated with anti-CD86 mAbs, but not after anti-CD80 mAb treatment (Fig. 7, a and b). Compared with OVA-immunized control Ab-treated or anti-CD80-treated mice, in anti-CD86-treated mice more cells remained in an undivided state and fewer cells had reached the fourth division. Ex vivo splenocyte culture demonstrated reduced IFN-␥ production in mice treated with anti-CD86 mAbs (Fig. 7c). CD100 from outside the Ag-specific CD4⫹ cell population plays a role in T cell activation Purified CD4⫹.CD100⫹ DO11.10 cells were transferred into CD100⫹/⫹ or CD100⫺/⫺ recipients. In CD100⫺/⫺ recipients, the rate of proliferation of DO11.10 cells was reduced (Fig. 8, a and b), demonstrating that endogenous CD100 from sources in addition to Ag-specific CD4⫹ cells plays a role in early T cell proliferation.

Discussion Pathogenetic Ab responses and immune complexes are features of many nonautoimmune and autoimmune diseases, in which glomer-

CD100 IN GN

FIGURE 6. CD11c⫹ cell CD86, but not CD80 expression, was decreased in CD100⫺/⫺ mice. a, The increase in CD86 expression (thick line with open fill) on CD11c⫹.MHC II⫹ cells at 24 h was attenuated in the absence of CD100. Control staining (CD86⫺/⫺ mice) is represented by the shaded areas. b, The proportion of CD86⫹CD11c⫹.MHC II⫹ cells were increased after in vivo stimulation for 24 h, but up-regulation was prevented in the absence of CD100 (n ⫽ 4 – 8 per group). c, The proportion of CD11c⫹.MHC II⫹ cells that were CD80⫹ was not reduced in CD100⫺/⫺ mice (n ⫽ 4 – 8 mice per group). ⴱⴱⴱ, p ⬍ 0.001.

uli are often affected. The current studies demonstrate a role for CD100 in pathological Ab-mediated injury. In these diseases, Agspecific CD4⫹ cells, generated by APC-CD4⫹ cell interactions provide help for B cells that produce pathogenetic Abs. The current studies demonstrate that CD100⫺/⫺ mice developed less severe immune renal injury, both histological and functional, in immune complex GN. Immunized mice developed proliferative GN with mesangial matrix expansion, complement deposition, macrophage accumulation, and proteinuria. However, CD100⫺/⫺ mice developed only mild GN. Immune responses to the nephritogenic Ag were attenuated at several levels, including DC costimulatory molecule expression, CD4⫹ cell activation, and Ig production. Attenuation of injury in CD100⫺/⫺ mice resulted from reduced T cell-dependent B cell Ab responses to the nephritogenic Ag, horse apoferritin. Reduced serum Ag-specific Ab levels in the CD100⫺/⫺ mice paralleled glomerular findings. CD100 is up-expressed on activated CD4⫹ T cells. Because other studies have demonstrated that CD100 promotes B cell activation by the binding of CD100 to CD72 expressed on B cells (6, 7, 10, 11), it is probable that at least part of the reduced Ig production in the current studies in CD100⫺/⫺ mice relates to uninhibited negative signaling in B cells by CD72. Additional experiments defined a role for CD100 in the early stages of the immune response. CD100 deficiency results in defective T cell activation (7). The current studies show that, in this disease model, in which humoral immunity plays an important role, CD4⫹ cell activation and function were suppressed. In the absence of CD100, CD4⫹ cells at day 14 were more prone to apoptosis, exhibited diminished markers of T cell activation, and made less IL-4 and IL-10. In contrast to the findings in CD100⫺/⫺ mice immunized s.c. with keyhole limpet hemocyanin in Freund’s complete adjuvant (7), IFN-␥ production was not reduced. This may be due to the different route of administration and use of adjuvants in the two studies. Reductions in

The Journal of Immunology

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FIGURE 8. Reduced proliferation of DO11.10 cells in CD100⫺/⫺ hosts. Purified CD4⫹.KJI26⫹ cells from CD100⫹/⫹ DO11.10 mice were transferred into CD100⫹/⫹ or CD100⫺/⫺ 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 proportions of CD4⫹.KJI26⫹ DO11.10 cells in CD100⫺/⫺ mice (b). ⴱⴱⴱ, p ⬍ 0.001.

FIGURE 7. In vivo administration of inhibitory anti-CD86 mAbs, but not anti-CD80 mAbs inhibited DO11.10 cell activation. a, Three days after i.p. OVA injection, CFSE-labeled proliferation of donor DO11.10 cells was decreased in anti-CD86 mAb-treated CD100⫹/⫹ recipients. Compared with control IgG or anti-CD80-treated animals, a higher proportion of cells from anti-CD86-treated animals had not divided and a lower proportion had reached the fourth division. b, The proportion of CD4⫹.KJI26⫹ (DO11.10 OVA-specific) cells from spleens of anti-CD86-treated mice was less than anti-CD80 or control IgG-treated groups. c, IFN-␥ production by anti-CD86-treated mice was reduced following 3 days ex vivo splenocyte culture (without further Ag stimulation). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.001.

humoral responses were not mediated via enhanced CD4⫹.CD25⫹ regulatory T cells, because CD25 expression on CD4⫹ cells was decreased in CD100⫺/⫺ mice (data not shown). Because CD72 is expressed on only a small proportion of CD4⫹ cells, we hypothesized that in vivo CD4⫹ cell CD100 expression participates in DC activation by switching off CD72-induced negative signals. CD72 is expressed on DCs (9, 28). Following in vitro stimulation, costimulatory molecule expression on DCs was reduced in the absence of CD100. The current studies show that, in vivo, CD100 is required for optimal expression of CD86 on DCs. Although proportions of splenic DCs were similar in the presence and absence of CD100, expression of CD86, but not CD80, was significantly reduced in CD100⫺/⫺ mice following injection of the disease-initiating Ag. Studies in a DO11.10 TCR transgenic adoptive transfer system demonstrated the functional importance of CD86 expression in early T cell proliferation and cytokine production after i.p. immunization. Although studies showing the functional importance of CD86 in early CD4⫹ cell activation were performed in a TCR transgenic system, it is likely that CD86 plays a similar role in nontransgenic systems (29), including T cell proliferation in C57BL/6, BALB/c, and NOD strains (30). Transfer of CD100⫹/⫹ DO11.10 cells to either CD100⫹/⫹ or CD100⫺/⫺ recipients showed some reduction in proliferation in CD100⫺/⫺ recipients, suggesting that CD100 from sources other than the Agspecific CD4⫹ cells plays a role in early events in T cell activation. The mechanism underpinning this particular observation is not

clear. It is possible that transfer of Ag-specific CD100⫺/⫺ 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 CD4⫹ cells. B cells are influenced by CD100, express CD72, CD80, and CD86, and have the capacity to present Ag to CD4⫹ cells. CD100 may influence the Ag-presenting capacity of B cells, particularly in the generation of memory CD4⫹ cells, because both simulations (31) and experimental studies (32) suggest that B cells may be important in this process. Renal injury in the current studies is driven primarily by the deposition of immune complexes in glomeruli. Both CD100 and the tissue receptor for CD100, plexin-B1, are expressed in the kidney (6, 33), the former largely in the tubulointerstitial compartment (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 immune renal injury, particularly those with significant tubulointerstitial damage or those involving cell-mediated effector injury (20, 23), they are unlikely to have played any role in this model. Although these studies do not address a potential role for the soluble form of CD100, overexpression of soluble CD100 results in enhanced T cell-dependent Ab production (8). In conclusion, the current studies implicate CD100 in DC activation by expression of CD86, leading to a number of effects on the generation of pathogenetic humoral responses including the activation, cytokine expression, and survival of CD4⫹ cells.

Acknowledgments We acknowledge the technical assistance of Alice Wright and Gabrielle Wilson and the assistance of Dr. Paul Hutchinson with flow cytometry.

Disclosures The authors have no financial conflict of interest.

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