Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease

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NATURE MEDICINE • VOLUME 8 • NUMBER 12 • DECEMBER 2002 1405

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The apoptosis of autoreactive lymphocytes in peripheral lym-phoid tissues is an important mechanism for maintaining im-mune tolerance. Mice with the lymphoproliferative (Tnfrsf6lpr,or lpr) mutation in the gene encoding Fas do not properly de-plete autoreactive lymphocytes by activation-induced cell death(AICD)1,2. The hallmark of lpr mutations is an aberrant accumu-lation of CD4–CD8–B220+ double negative T cells (DNTCs)3,4.MRL/lpr mice spontaneously develop a severe autoimmune dis-ease, which resembles human systemic lupus erythematosus(SLE) and is characterized by hyperglobulinemia, production ofvarious auto-antibodies and immune complex-associated end-organ disease of the kidney2,5. Currently, immunotherapeuticapproaches for treating lupus patients are limited. Immuno-therapy strategies in murine autoimmune disease models, haveincluded attempts to prevent T-cell activation by administeringblocking peptides, antibodies or other agents that inhibit T-cellsignaling through the T-cell and costimulatory receptors6–11.Other immunotherapeutic approaches exploit cytokine agonistsand antagonists12–14. Pitfalls of these therapies include the re-quirement for long-term treatment and their inability to selec-tively deplete autoreactive lymphocytes or reverse diseaseprogression.

CD137 (4-1BB), a member of the tumor necrosis factor (TNF)receptor superfamily, is a costimulatory molecule15,16 primarilyexpressed on activated T cells17 and natural killer (NK) cells18. Itsnatural ligand, 4-1BBL, has been detected on macrophages, den-dritic cells and B cells19–21. Agonistic monoclonal antibodies(mAbs) against CD137 have been shown to promote interferon-γ(IFN-γ) production, allograft and established tumor rejectionand CD8+ T-cell responses22–26. Some studies have demonstrated

that these antibodies can also inhibit antigen-induced CD4+ T-cell responses27,28. To address whether an agonistic antibody canalter the course of autoreactive lymphocyte accumulation, adultB6/lpr or MRL/lpr mice were treated with a mAb against CD137(2A). We found that 2A treatment blocks the progression of es-tablished spontaneous lupus-like disease and lymphadenopathyin MRL/lpr mice. 2A treatment may enhance activation-inducedapoptosis of abnormal DNTCs and augment IFN-γ-mediated de-pletion of activated B cells. Finally, this short-term treatmentstrategy markedly prolongs the survival of these mice.

2A reduces the DNTC and B cell populationsAgonistic antibodies to CD137 have been shown to affect bothCD4+ and CD8+ T cells during various immune responses. B6/lprmice suffer from a lymphoproliferative disorder characterized byan accumulation of autoreactive lymphocytes soon after birth.We treated 2–3-month-old B6/lpr mice with 2A to explore therole of CD137 stimulation in activating autoreactive lympho-cytes. We found that 3 weeks after treatment initiation, the per-centage of CD8+ T cells increased about 3–4-fold in spleens of2A-treated mice, whereas CD4+ T-cell percentages remained sim-ilar to those of control mice (Fig. 1a). Furthermore, in 2A-treatedmice total CD4+ T-cell numbers decreased, whereas total CD8+ T-cell numbers increased (Fig. 1b). These changes correlated withthe upregulation of the activation markers CD69 and CD44, andthe downregulation of CD62L in CD8+ but not CD4+ T cells (Fig.1a). These results suggest that CD137 signaling preferentially ac-tivates CD8+ T cells in the absence of Fas signaling.

Two to three weeks after 2A treatment, the percentages andnumbers of splenic DNTCs and B cells were markedly reduced

Costimulatory molecule-targeted antibody therapyof a spontaneous autoimmune disease

YONGLIAN SUN1, HELEN M. CHEN1, SUMIT K. SUBUDHI1, JONATHAN CHEN1, RIMA KOKA1,LIEPING CHEN2 & YANG-XIN FU1

1Department of Pathology and Committee in Immunology, University of Chicago, Chicago, Illinois, USA2Department of Immunology, Mayo Clinic, Rochester, Minnesota, USA

Correspondence should be addressed to Y.-X.F.; email: yfu@midway.uchicago.edu

Published online 11 November 2002; doi:10.1038/nm796

Humans and mice deficient in Fas, a tumor necrosis factor (TNF)-receptor family member, can-not induce apoptosis of autoreactive cells, and consequently develop progressive lymphoprolif-erative disorders and lupus-like autoimmune diseases. Previous studies have shown thatshort-term administrations of agonistic monoclonal antibodies against CD137, another TNF-re-ceptor family member, activate T cells and induce rejection of allografts and established tumors.Here we report that treatment with an agonistic monoclonal antibody to CD137 (2A) blockslymphadenopathy and spontaneous autoimmune diseases in Fas-deficient MRL/lpr mice, ulti-mately leading to their prolonged survival. Notably, 2A treatment rapidly augments IFN-γ pro-duction, and induces the depletion of autoreactive B cells and abnormal double-negative T cells,possibly by increasing their apoptosis through Fas- and TNF receptor–independent mechanisms.This study demonstrates that agonistic monoclonal antibodies specific for costimulatory mole-cules can be used as novel therapeutic agents to delete autoreactive lymphocytes and block autoimmune disease progression.

Fig. 1 Effects of 2A treatment on lymphocyte populations and antibody produc-tion. B6/lpr mice were treated with 2A or control rat IgG 3 times at weekly intervals.a, 2A treatment preferentially activates CD8+ T cells in B6/lpr mice. Splenocytes wereanalyzed 3 wk after the first treatment. b, Total cell numbers of splenocytes, T-cellsubsets, B cells, and DNTCs 3 wk after the first treatment. �, control; �, 2A-treated(n = 3). Error bars represent s.d. c, 2A administration reduces the percentages ofsplenic B cells and Thy1.2+B220+ DNTCs. Numbers representing cell percentages ineach quadrant are expressed as mean ± s.d. (n = 3). d, Administration of 2A de-creases the production of auto-antibodies. Sera were collected 1 wk after the finaltreatment, and total IgG (left) and anti-DNA IgG (right) levels were detected byELISA (n = 5). �, control; �, 2A. *, P < 0.05; **, P < 0.01 by Student’s t-test.

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(Fig. 1b and c). The decreased numbers of B cells paralleled re-duced sera levels of anti-DNA and total immunoglobulin G (IgG)to those of wild-type mice (Fig. 1d).

Depletion of lymphocytes is Fas- and TNFR-independentTo study the mechanisms underlying the diminishment of theDNTC and B-cell populations in secondary lymphoid organs fol-lowing 2A treatment, we explored whether these cells under-went tissue redistribution. The decreases in DNTC and B-cellpopulations were similar in the lymph nodes, bone marrow andperipheral blood, and these cells were not detected in variousnon-lymphoid tissues of 2A-treated B6/lpr mice (data notshown). These results suggest that the decreased number of lym-phocytes in the secondary lymphoid tissues is likely due to de-pletion.

We detected a consistent increase in the percentage of apop-totic DNTCs in 2A-treated mice 5–7 days after treatment (Fig. 2a,left panels). Moreover, when the cells were cultured in vitro for 6 hours, DNTCs from the spleens of 2A-treated mice underwentmore marked apoptosis when compared with control cells (Fig.2a, middle panels). We found that CD69-expressing DNTCs werepreferentially depleted (Fig. 2a, right panels), suggesting thatthese cells may be succumbing to activation-induced cell death.We also detected an approximately 80% increase in the percent-age of apoptotic B cells in 2A-treated mice five days after treat-

ment (15 ± 3.7%) versus control mice (8 ± 1.7%). In addition,ELISPOT assays showed that anti-DNA-antibody secreting B cellswere greatly reduced after 2A treatment (Fig. 2b). These findingsindicate that 2A treatment may selectively delete autoreactive B cells.

To better understand the mechanism responsible for CD137-mediated depletion of DNTC and B-cell populations, we studiedthe role of Fas and TNFR. Although lpr mice express Fas mRNASonly weakly29,30, a strong CD137-costimulatory signal may pro-mote Fas expression on lymphocytes and induce apoptosis. Totest this, we treated Fas-knockout mice with 2A and observedsimilar results as seen in B6/lpr mice, with significant reductionsin both the B-cell and DNTC populations (Fig. 2c, left panels). Byadministering TNFR-Ig (300 µg per mouse weekly) in combina-tion with 2A, we found that TNF blockade did not affect deple-tion of B cells and DNTCs in 2A-treated B6/lpr mice (Fig. 2c, rightpanels). These data suggest that, similarly to superantigen-in-duced T-cell apoptosis31, 2A-induced lymphocyte apoptosis isFas- and TNFR-independent.

2A-mediated reduction of B cells is IFN-γ-dependentIFN-γ may be responsible for the reduction of autoreactive B cells, as 2A treatment significantly increases the number ofIFN-γ-producing T cells (Fig. 2d). IFN-γ can activatemacrophages, which can in turn potentially apoptose activated

CD8+ T gated CD4+ T gated

10± 1.6

42 ± 5.6

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14 ± 3.4

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45 ± 5.2 28 ± 5.2

22 ± 2.3

50 ±1.8 19 ± 5.2

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70 ± 2.2 16 ± 1.4

7 ± 1.1

9 ± 1.7

24± 2.9

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10 ± 3.2

56 ± 6.9 1.7 ± 0.8

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11 ± 3.0 58 ± 8.1

18 ± 2.9

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7 d 14 d 21 d

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lymphocytes32–34. We therefore examined the effects of 2A treat-ment on the CD11b+Gr-1+ macrophage/granulocyte populationsin B6/lpr mice, and found a significant increase in their percent-ages in the spleen (Fig. 2e). Anti-IFN-γ treatment was used incombination with 2A to test whether B-cell depletion was IFN-γ-dependent. We found that the combinatorial treatment reversedthe effects of treating mice with 2A alone, with decreased expan-sion of the CD11b+Gr-1+ population and increased B-cell per-centages (Fig. 2f). Anti-IFN-γ treatment alone showed no effect(data not shown). These results suggest that the depletion of au-toreactive B cells by 2A treatment is IFN-γ-dependent.

To test whether IFN-γ activated macrophages could induce B-cell apoptosis, we cultured B6/lpr mice splenocytes with orwithout peritoneal macrophages in the presence or absence ofvarying doses of IFN-γ. The results showed that IFN-γ enhancesB-cell apoptosis in a dose-dependent manner only whenmacrophages are present (Fig. 2g), suggesting that in the pres-ence of IFN-γ, macrophages greatly enhanced B-cell apoptosis.

2A ameliorates lymphadenopathy in MRL/lpr miceThe ability of 2A treatment to decrease the numbers ofDNTCs, CD4+ T cells and B cells, as well as to reduce auto-an-

Annexin V

IFN-γ

Day 7 Day 21

Ctrl. 2A 2A + α-IFN-γ

Fig. 2 Mechanisms underlying in DNTC and B-cell depletion. a, 2A treatment increases apop-tosis in the DNTC populations. 5–7 d after treatment, splenocytes were directly analyzed (leftpanels) or cultured in vitro for 6 h (middle panels) and then stained with anti-Thy-1 and anti-B220 combined with Annexin V. The fluorescence intensity of Annexin V in Thy-1+B220+ popula-tions is shown. CD69 expressing DNTCs are preferentially deleted by 2A treatment. In rightpanel, splenocytes isolated 2 wk after treatment, were stained with anti-Thy-1 and anti-B220combined with anti-CD69, and CD69 expression in the DNTC population was assessed. b, 2Atreatment (�) decreases anti-DNA-antibody secreting B cells versus control(�). 1 wk after B6/lprmice were treated with 2A, splenocytes were collected and anti-DNA-antibody secreting B-cellnumbers were detected by ELISPOT. Data is cell numbers per 104 B cells. Error bars represent s.d.c, Depletion of DNTCs and B cells is not Fas and TNFR-mediated. Fas–/– mice were treated weeklywith control rat IgG or 2A (left panels), and B6/lpr mice were treated similiarly with the additionof TNFR-Ig (right panels). d, 2A treatment increases IFN-γproduction in T cells. B6/lpr mice weretreated with control IgG or 2A. 1 wk later, splenocytes were stained with Thy-1.2 combined withintracellular staining for IFN-γ, and then analyzed by flow cytometry. e, 2A treatment increasesthe CD11b+GR-1+ population. B6/lpr mice were treated as in Fig. 1c, and splenocytes were ana-lyzed by flow cytometry. f, Blockade of IFN-γ reverses the expansion of the CD11b+GR-1+ popu-lation and the reduction of B cells induced by 2A treatment. B6/lpr mice were treated withcontrol rat IgG, 2A alone, or 2A combined with anti-IFN-γ. Splenocytes were analyzed by flow cy-tometry 2 wk after the first treatment. g, IFN-γ-activated macrophages induce apoptosis of Bcells. B6/lpr splenocytes were cultured with peritoneal macrophages in the presence of indicateddoses of IFN-γ. 18 and 40 h later, splenocytes were collected and stained with Annexin V andcell-surface markers. Annexin V staining of B220 single-positive B cells is shown. All of the aboveresults are representative of 3 experiments.

No IFN-γ

IFN-γ (2.5 ng/ml)

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Fig. 3 Administration of 2A ameliorates lymphadenopathy inMRL/lpr mice. 9–10-wk-old female MRL/lpr mice were treated 3times at weekly intervals. a, Number of palpable peripheral lymphnodes (pLNs) in control (�) and 2A-treated (�) mice (n = 10). b, Weights of the spleen and pooled pLNs, including the inguinal,axillary, cervical LNs, and mesenteric LNs (mLNs), were greatly re-duced in 2A-treated mice (�) compared with control groups (�) 2 mo after initial treatment. c, Sizes of the spleen and peripheralLNs in control (left) and 2A-treated (right) mice. Scale bar, 1 cm. d, Cell numbers of different cellular subsets in the spleen (left) andinguinal LNs (right) of control (�) and 2A-treated mice (�) at 4 moof age (n = 3). *, P < 0.05; **, P < 0.01 by Student’s t-test. Error barsrepresent s.d.

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tibody levels in B6/lpr mice, suggests that 2A might have ther-apeutic potential in treating systemic autoimmune diseasesand lymphadenopathy. MRL/lpr mice have severe lupus-likefeatures and show a more severe lymphoproliferative disorderat a younger age (6–8-wk-old) than B6/lpr mice. By 9–10weeks of age, MRL/lpr mice already have significant numbersof aberrant DNTCs and high serum levels of auto-antibodyanti-DNA IgG.

We treated 9–10-week-old MRL/lpr mice with 200-µg doses of2A or control rat IgG weekly for 3 weeks. In accordance with theresults in B6/lpr mice, the MRL/lpr mice showed preferentialCD8+ T-cell activation and decreased DNTCs and B cells 2–3weeks after 2A treatment. Moreover, one week after treatment

we detected significantly increased numbers of IFN-γ-producingT cells in the spleen and higher serum IFN-γ levels in 2A-treatedmice than control mice (data not shown).

By five months of age, all of the control mice displayed pro-gressively severe lymphadenopathy, but only two out of tenmice in the 2A-treated group developed palpable lymph nodes(LNs) (Fig. 3a). In addition, at four months 2A-treated mice hadconsiderably smaller spleens and peripheral LNs than the con-trol mice (Fig. 3b and c). This correlated with a significant reduc-tion in lymphocyte and total cell numbers in the spleen, andespecially in the peripheral LNs (Fig. 3d). The sharpest declinewas in the number of DNTCs, which are a key component oflymphadenopathy in MRL/lpr mice. These results suggest an ag-

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Total CD4 CD8 DNTC B Total CD4 CD8 DNTC B

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Fig. 4 2A administration completely blocks the develop-ment of skin lesions. Female MRL/lpr mice were treated asdescribed in Fig. 3. a, Skin lesions were blocked in 2A-treated mice (�, barely visible) whereas control mice (�)showed severe skin disease. Skins were scored from 1–3 asdescribed in Methods (n = 10). b and c, At 5 mo of age, con-trol MRL/lpr mice exhibited severe skin lesions (b), in con-trast, 2A-treated mice appeared completely normal (c). dand e, Histological sections of the skin from the posteriorneck area of control (d) and 2A-treated mice (e) that were 5mo of age. Skin tissues were fixed in 10% formalin, and 4–5-µm sections were stained with H&E. Representative picturesare shown. Magnification, ×20.

Fig. 5 2A treatment attenuates renal disease and auto-antibody produc-tion, and prolongs survival. Female MRL/lpr mice were treated as in Fig.3a. a, Urinary proteins in MRL/lpr mice were reduced by 2A treatment.Urinary protein levels were assessed monthly and graded semi-quantita-tively as described in Method Section. �, control; �, 2A. b, At 5 mo ofage, kidneys were collected and fixed in formalin. H&E-stained kidneysections from 4 mice per group were scored for glomerulonephritis. �,no inflammation; �, segmental inflammation; �, global inflammation. c,Control (top panel) and 2A-treated (bottom panel) kidney sectionsstained with H&E. Magnification, ×40. d, Immunofluorescence analysis ofIgG and C3 deposits in the renal glomeruli. Control (top panels) and 2A-treated (bottom panels) kidney sections were stained with FITC-labeled

goat-anti-mouse IgG (left panels) or complement C3 (right panels).Magnification, ×43. Representative pictures are shown. e, 2A treatmentdecreased auto-antibody production in MRL/lpr mice. Sera were collectedbefore the treatment at the age of 2 mo and monthly after treatment ini-tiation. Total IgG and auto-antibody levels were detected by ELISA. f, Ratio of anti-DNA versus total IgG. g, Blockade of IFN-γ reverses 2A-treatment induced decrease of auto-antibody production. MRL/lpr micewere treated with control rat IgG (�), with 2A (�), anti-IFN-γ (�) or both(�). Error bars represent s.d. 1 mo after the first treatment, sera were col-lected, and anti-DNA IgG levels were detected by ELISA (n = 3). h, 2Atreatment significantly prolongs the survival of MRL/lpr mice (n = 10). Ine, f and h, �, control; �, 2A. * P < 0.05; ** P < 0.01.

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Age 3 mo 4 mo 5 mo0%

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onistic mAb against CD137 might be used as a therapeutic agentfor treating lymphoadenopathy.

2A prevents development of skin lesions in MRL/lpr miceMRL/lpr mice always developed a progressive spontaneous cuta-neous disease (Fig. 4a), and by five months of age, virtually all ofthe mice had large plaque-like cutaneous lesions on their poste-rior neck (Fig. 4b). No cutaneous lesions were detected in any ofthe 2A-treated mice (Fig. 4a and c). Histological sections of skin(posterior neck) from control mice revealed substantial epider-mal acanthosis along with dermal chronic inflammatory cell in-filtrates (Fig. 4d). Similar sections in 2A-treated mice, however,had normal architecture and morphology (Fig. 4e). These resultssuggest that 2A treatment was effective in preventing cutaneouslupus-like lesions.

2A treatment attenuates renal disease in MRL/lpr miceKidney diseases are considered to be the primary cause of mor-tality in those afflicted with lupus. We therefore examined theeffects of 2A treatment on kidney function in MRL/lpr mice bymeasuring proteinuria levels monthly. We found that protein-uria was significantly reduced in the 2A-treated mice (Fig. 5a).Kidney pathology in control mice demonstrated severe diffuseglobal proliferative glomerulonephritis involving over 80% oftotal glomeruli, and most of the remaining glomeruli had seg-mental glomerulonephritis (Fig. 5b). Histological sections fromcontrol mice exhibited prominent perivascular inflammatorycell infiltrate consisting predominantly of lymphocytes andplasma cells, as well as intra- and extra-capillary necrotizingand sclerosing lesions in most glomeruli (Fig. 5c). In contrast,kidney sections from 2A-treated mice primarily showed focalproliferative glomerulonephritis including about 40% segmen-tal and less than 40% global involvement, and more than 20%of glomeruli appeared completely normal. Patchy perivascularinfiltrate persisted, but was much reduced compared to controlmice (Fig. 5b and c).

Lupus models are characterized by direct auto-antibody-me-diated tissue injury and the deposition of complement-fixing

immune complexes. The deposition of complement C3 in thekidney is a key pathological finding in lupus nephritis35. Wetherefore examined IgG and complement C3 deposition in thekidney; these were significantly reduced in 2A-treated mice (Fig.5d). These results suggest that an agonistic antibody againstCD137 can be used to treat autoimmune renal diseases.

2A treatment reduces auto-antibodies and prolongs survivalBecause autoantibodies are a hallmark of SLE (refs. 2,36), weexplored the effects of 2A treatment on autoantibody produc-tion in MRL/lpr mice. We found that 2A treatment significantlydecreased the levels of autoantibodies against DNA, and, to alesser extent, total IgG production (Fig. 5e). The ratio of anti-DNA versus total IgG levels in MRL/lpr mice was therefore alsoreduced (Fig. 5f). Increased IgG2a isotype levels are associatedwith disease pathogenesis in lpr models, with IgG2a compris-ing the dominant subclass37. 2A treatment markedly decreasedthe levels of the IgG2a and IgG1 anti-DNA isotypes (Fig. 5e),but not the levels of the IgG2b and IgG3 isotypes (data notshown). In accordance with the IFN-γ-mediated B-cell deple-tion in treated B6/lpr mice, we found that in MRL/lpr mice thecombination of anti-IFN-γ and 2A-treatment reversed the re-duction of auto-antibody IgG anti-DNA levels induced by 2A-treatment alone (Fig. 5g).

2A treatment significantly prolonged the survival ofMRL/lpr mice (Fig. 5h). Most of the control mice died by 24weeks, whereas 2A-treated mice remained healthy for anothertwo months, at which point the experiments were termi-nated. This confirms that an agonistic antibody againstCD137 can be a powerful clinical agent for blocking the pro-gression of spontaneous autoimmune disease and prolongingsurvival.

Therapeutic effects of 2A on advanced disease in MRL/lpr miceIt is important to determine whether such a treatment can beuseful for a clinically detectable autoimmune disease. To testthis, we treated three-month-old MRL/lpr mice that had pal-pable lymphoadenopathy, high levels of auto-antibodies and

3 mo 4.5 mo0

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a b c d

in formalin. H&E-stained kidney sections from 3 mice per group werescored for glomerulonephritis. �, no inflammation; �, segmental inflam-mation; �, global inflammation. *, P < 0.05; **, P < 0.01 by Student’s t-test.

Fig. 6 Therapeutic effects of 2A in MRL/lpr mice with advanced disease. 3-mo-old female MRL/lpr mice were treated 3 times at weekly intervals. a, Number of palpable pLNs in control (�) and 2A-treated (�) mice (n = 5).b, Spleen weights and those of pooled pLNs and mLNs were reduced in 2A-treated mice (�) compared with control groups (�) 6 wk after initial treat-ment (n = 3). c, Auto-antibody production was decreased in 2A-treatedmice. Sera were collected before the treatment and 6 wk after treatmentinitiation. IgG anti-DNA levels were detected by ELISA. �, control; �, 2A (n= 5). d, 2A treatment decreases DNTC and B-cell populations in 3-mo-oldMRL/lpr mice. Splenocytes were analyzed by flow cytometry two wk afterthe first treatment (n = 3). e, 2A treatment reduces urinary proteins. �, con-trol; �, 2A. f, 6 wk after initial treatment, kidneys were collected and fixed

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detectable levels of urine proteins with a weekly dose of 200µg 2A or control rat IgG for three weeks. The 2A treatmentregimen attenuated the progression of lymphadenopathy(Fig. 6a and b), decreased the production of auto-antibodyIgG anti-DNA levels (Fig. 6c) and reduced the DNTC and B-cell populations (Fig. 6d). The treatment also amelioratedrenal function and pathology (Fig. 6e and f). These resultssuggest that this approach could potentially be clinicallyvaluable in treating well-established autoimmune diseases.

DiscussionThere is a lack of effective immunotherapeutic approaches fortreating patients with lupus. In contrast to conventional T-cellcostimulation blockade, we demonstrate here that an agonisticmAb (2A) against a costimulatory molecule, CD137, effectivelyameliorates both lymphoadenopathy and spontaneous autoim-mune diseases in the MRL/lpr strain, a model of human SLE.Most importantly, 2A treatment prolongs long-term survival ofthese mice. The therapeutic effects of 2A treatment were medi-ated by the depletion of DNTCs and the diminishment of au-toreactive B cells.

Several TNF superfamily members have been shown to medi-ate apoptosis. Our study indicates that signaling through CD137reduces the number of abnormal DNTC and autoreactive B cellsin lpr mice, independent of resident Fas and TNF. In MRL/lprmice, a subset of DNTCs (23.7 ± 3.6%) and activated T cells (forCD4+ T cells, 10.4 ± 2.6% and CD8+ T cells, 3.5 ± 1.8%; n = 5) ex-press CD137. Therefore, we believe that an agonistic antibodymay deliver a strong costimulatory signal that promotes AICD ofactive lymphocytes, circumventing the need for Fas-mediatedapoptosis.

Because CD137 is not expressed on the surface of activated Bcells15,17,20, it was surprising to find that the total B-cell and anti-DNA-antibody secreting B-cell numbers were markedly de-creased after 2A treatment. This suggests that B-cell depletionwas likely due to an indirect effect of 2A treatment. 2A treatmentincreased IFN-γ levels as well as the CD11b+/Gr-1+ population,which may be involved in B-cell reduction and auto-antibodyproduction. This is in accordance with previous studies thathave shown that IFN-γ was a potent activator of macrophages,which may subsequently promote the apoptosis of activated Bcells by direct or indirect pathways32–34. Expansion of the Gr-1population by the administration of anti-Gr-1 antibody is ac-companied by a further reduction of B-cell numbers in 2A-treated B6/lpr mice (unpublished data). Taken together, wepropose that CD137 signaling augments T-cell activation, subse-quently inducing T cells to produce high levels of IFN-γ to acti-vate macrophages/granulocytes, which in turn may induce theapoptosis of activated autoreactive B cells, ultimately resultingin decreased auto-antibody production. In addition, the lack ofT-cell help due to the downregulation of the CD4+ T-cell popula-tion could also contribute to reduced auto-antibody production.

Previous studies have demonstrated that IFN-γ- or IFN-γR-de-ficient MRL/lpr mice do not develop autoimmune diseases withthe same extent as normal MRL/lpr mice38–41. Additionally, it hasbeen shown that disrupting IFN-γ-mediated signaling throughtreatment with a cDNA encoding IFN-γR (ref. 14) resulted inmarked disease regression in mice, suggesting that IFN-γ playsan essential role in SLE pathogenesis. Unexpectedly, our studiesshowed that the sudden and robust IFN-γ production resultingfrom CD137 stimulation could contribute to activated autore-active lymphocyte apoptosis, leading to the amelioration of au-

toimmune diseases. This contrasts with the gradual sponta-neous increase of IFN-γ levels observed in MRL/lpr mice over aprolonged period. In support of our hypothesis, Nicoletti et al.found that the prophylactic administration of IFN-γ to 6–8-week-old MRL-lpr mice favorably modulated the histological,serological and clinical signs of disease42. Thus, it remains to bedetermined how the presence or absence of IFN-γ expression atvarious phases of disease progression affects outcome. Taken to-gether, these studies suggest that IFN-γ can have contradictoryeffects in regulating the SLE syndrome.

The goal of immunotherapies for autoimmune diseases is toselectively deplete autoreactive lymphocytes, while avoidingglobal immunosuppression. 2A treatment presents a clear modelof depletion of both the autoreactive B-cell and abnormal DNTCpopulations, without profoundly affecting the normal immuneresponse to exogenous antigens. We found that when B6/lprmice were immunized with SRBC or KLH 4 weeks after 2A treat-ment termination, anti-SRBC and -KLH IgG responses were notreduced compared with control mice (unpublished data). Thissuggests that IgG responses to exogenous antigens were normalafter treatment termination.

In summary, the short-term administration of an agonisticmAb against CD137 (2A) in adult MRL/lpr mice profoundly de-pleted autoreactive B-cell and DNTC populations. Our study hasrevealed a potential dual effect of CD137 in regulating immuneresponses and supports that CD137 signaling initially con-tributes to the early stage of T-cell activation, and later inducesapoptosis of fully activated T cells. Most importantly, 2A treat-ment markedly attenuated lymphoadenopathy and autoim-mune disease, and prolonged survival in these mice. Thetherapeutic use of agonistic antibodies against costimulatorymolecules may therefore constitute a novel clinical approach fortreating autoimmune diseases and lymphoprolifative disorders.

MethodsMice. B6.MRL-Tnfrsf6lpr (B6/lpr), MRL/MpJ-Tnfrsf6lpr (MRL/lpr), MRL.129P2(B6)-Tnfsf6tm1qsa (Fas–/–) mice were purchased from The Jackson Laboratory(Bar Harbor, Maine). C57BL/6 wild-type (B6/wt) mice were purchased fromthe National Cancer Institute (Frederick, Maryland). All animal protocolswere approved by the University Committee on Use and Care of Animals atthe University of Chicago.

In vivo treatment with antibodies. Agonistic monoclonal antibody againstCD137 (2A) was generated as described25. Rat IgG was purchased fromSigma and served as a control antibody. Starting from 2–3 mo of age, micewere injected i.p. with 200 µg/mouse of 2A or rat IgG weekly for 3 wk.

Flow cytometric analysis. A flow-cytometric analysis was performed usingFITC-, PE- or Cy-Chrome-conjugated monoclonal antibodies to mouseCD4, CD8α, Thy1.2, B220, CD69, CD44, CD62L, CD11b, Gr-1 and IFN-γ(BD Pharmingen, San Diego, California). Cells were preincubated with2.4G2 monoclonal antibodies to block FcγR, and were then incubated withthe relevant monoclonal antibodies for 30 min at 4 °C. Finally, cells werewashed twice with 0.2% BSA in PBS and analyzed by FACScan (BDBiosciences, Mountain View, California). Cells were stained with Annexin-V(BD PharMingen) for detection of apoptosis, according to the manufactur-er’s protocol. Intracellular IFN-γ staining was performed as described43. Foranalysis, all the splenocytes are gated in forward versus side scatter for theentire study, and in certain cases, T-cell subsets are further gated as men-tioned in the relevant figures.

Detection of antibodies by ELISA. Serum samples were collected monthlyand examined for presence of autoantibodies to DNA by ELISA as describedbefore43. Experimental values from separate experiments were expressed asmg/ml or normalized to a single MRL-lpr positive-control serum used inevery assay (arbitrarily defined as 100 U).

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Gross pathology. Gross skin pathology was scored monthly. Skin lesions,which consist of alopecia and scab formation, were scored from 0 to 3based on the number of lesions and area: 0, no lesions; 1, one lesion, <0.5cm; 2, two or more lesions, <0.5 cm; 3, multiple lesions, >0.5 cm.Lymphadenopathy was evaluated monthly by counting palpable nodes.Spleen and lymph-node enlargement was assessed 2 mo after treatment.

Proteinuria. Urinary protein levels were assessed using reagent strips forurinalysis (Labstix; Bayer Corporation, Elkhart, Indiana) and graded semi-quantitatively (0, none; 1, 30–100 mg/dl; 2, 100–300 mg/dl; 3, 300-2000mg/dl; 4, >2,000 mg/dl). Each monthly value was determined by samplingand measuring urine on sequential days.

Histopathology. Kidney and skin tissues were collected and immediatelyimmersed in 10% neutral buffered formalin (Fisher, Pittsburgh,Pennsylvania). Formalin-fixed tissue was embedded in paraffin, and 4-µmsections were stained with H&E and evaluated by light microscopy. Kidneysamples were blindly examined for pathology at ×20 and ×40 magnifica-tion. The glomeruli were assessed by counting 200 glomerular cross-sec-tions (GCS) per kidney and scoring each glomerulus as no inflammation,segmental inflammation and global involvement of inflammation.

Immunofluorescent evaluation of IgG and C3 deposits in kidney.Kidneys were embedded in OCT compound and snap-frozen at –70 °C. 4-µm sections were air-dried and fixed with acetone, then pretreated withgoat serum and stained with FITC-labeled anti-mouse IgG (SouthernBiotechnology Associates, Birmingham, Alabama) and anti-mouse C3 Ab(ICN/Cappel, Aurora, Ohio). Fluorescence was examined by UV-fluores-cence microscopy.

Detection of DNA-secreting B cells by ELISPOT. Serial 5-fold dilutions ofsplenocytes were plated in triplicate into 96-well ELISA spot plates (CellularTechnology, Cleveland, Ohio) pre-coated with sperm DNA (250 µg/ml;Sigma) and incubated overnight at 37 °C. Bound anti-DNA IgG was de-tected by incubation with AP-conjugated goat anti-mouse IgG (H+L)(Southern Biotechnology Associates) at room temperature for 3 h. Colordevelopment was performed with nitroblue tetrazolium substrate solution(Sigma).

Blockade of IFN-γ and TNF. For blocking INF-γ, mice were injected i.p.every 4 d with 500 µg/mouse rat IgG or anti-IFN-γ (obtained from asciticfluid collected from RAG-1-deficient mice inoculated with rat hybridomaXMG1.2). For blockade of TNF, mice were injected i.p. with 300 µg TNFRI-hIg (from J. Browning, Biogen, Cambridge, Massachusetts) weekly for 2weeks.

Detection of B-cell apoptosis induced by IFN-γ activated macrophages.B6/lpr splenocytes (5 × 105 per well) were cultured with or without peri-toneal macrophages (1:1) in the presence of different doses of recombinantIFN-γ (BD PharMingen). At various time points, splenocytes were collected,and the percentage of B cells undergoing apoptosis was determined bystaining with FITC-labeled Annexin V combined with PE-Thy1.2 and Cy-chrome-B220.

Statistics. Student’s t-test was used to determine the statistical significanceof differences between groups. Survival of control and 2A-treated femaleMRL/lpr mice was analyzed by the Kaplan–Meier method, and the signifi-cance of differences was determined by the log-rank test.

AcknowledgmentsThis research was in part supported by NIH grants (HD-37104, DK-20595-25,and DK-58891) and JDFI (1-2000-875). J.C. was in part supported by theHoward Hughes Medical Institute undergraduate summer program.

Competing interests statementThe authors declare that they have no competing financial interests.

RECEIVED 15 MARCH; ACCEPTED 4 OCTOBER 2002

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Costimulatory molecule-targeted antibody therapy of a spontaneous autoimmune disease

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