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CD4+CD25+ T-Cells Control Autoimmunity in the Absence of B-Cells

CD4+CD25+ T-Cells Control Autoimmunity in the Absence of B-Cells ORIGINAL ARTICLE CD4 CD25 T-Cells Control Autoimmunity in the Absence of B-Cells Eliana Marin ˜ o, Jeanette Villanueva, Stacey Walters, David Liuwantara, Fabienne Mackay, and Shane T. Grey OBJECTIVE—Tumor necrosis factor ligand family members B-cell–activating factor (BAFF) and a proliferation-inducing li- he members of the tumor necrosis factor (TNF) gand (APRIL) can exert powerful effects on B-cell activation and development, type 1 T-helper cell (Th1) immune responses, and ligand family of molecules B-cell–activating fac- autoimmunity. We examined the effect of blocking BAFF and APRIL tor (BAFF) (also known as BLyS, TNFSF13b) on the development of autoimmune diabetes. T and a proliferation-inducing ligand (APRIL) can exert powerful effects on B-cell development, survival, and RESEARCH DESIGN AND METHODS—Female NOD mice function; T-cell activation; and type 1 T-helper cell (Th1) were administered B-cell maturation antigen (BCMA)-Fc from 9 immune responses and autoimmunity (1). BAFF exists as to 15 weeks of age. Diabetes incidence, islet pathology, and T- both a soluble and a membrane bound molecule and is and B-cell populations were examined. expressed by a wide range of inflammatory-activated cells, RESULTS—BCMA-Fc treatment reduced the severity of insulitis including monocytes, macrophages, dendritic cells, and and prevented diabetes development in NOD mice. BCMA-Fc– T-cells (2). In contrast, APRIL is processed intracellularly treated mice showed reduced follicular, marginal-zone, and and exerts its function as a soluble protein. BAFF and T2MZ B-cells. B-cell reduction was accompanied by decreased APRIL can bind to one of two receptors: B-cell maturation frequencies of pathogenic CD4 CD40 T-cells and reduced Th1 antigen (BCMA) (3) or transmembrane activator and cal- cytokines IL-7, IL-15, and IL-17. Thus, T-cell activation was cium modulator and cyclophylin ligand interactor (TACI) blunted with reduced B-cells. However, BCMA-Fc–treated (3,4), whereas BAFF can also bind to BR3 (otherwise mice still harbored detectable diabetogenic T-cells, suggesting known as BAFF-R) (5). These receptors are found on a that regulatory mechanisms contributed to diabetes preven- tion. Indeed, BCMA-Fc–treated mice accumulated increased wide range of B-cell subsets including immature, transi- CD4 CD25 regulatory T-cells (Tregs) with age. CD4 CD25 tional, mature, memory, and germinal center B-cells, as cells were essential for maintaining euglycemia because their well as on plasma cells (2). Further, activated T-cells can depletion abrogated BCMA-Fc–mediated protection. BCMA-Fc express the receptors BR3 and TACI (4,6). did not directly affect Treg homeostasis given that BAFF has emerged as an important player in the devel- CD4 CD25 Foxp3 T-cells did not express TACI or BR3 recep- opment of autoimmunity. Elevated BAFF and APRIL levels tors and that CD4 CD25 Foxp3 T-cell frequencies were equiv- have been detected in sera from human patients with / / / / alent in wild-type, BAFF , TACI , BCMA , and BR3 rheumatoid arthritis, lupus, and Sjogren’s syndrome (7–9). mice. Rather, B-cell depletion resulted in CD4 CD25 T-cell– Moreover, BAFF-transgenic mice harbor increased titers mediated protection from diabetes because anti-CD25 monoclonal of self-reactive antibodies and develop autoimmune symp- antibody treatment precipitated diabetes in both diabetes-resistant toms very similar to those of lupus and Sjogren’s syn- NOD.MT and BCMA-Fc–treated mice. drome (10,11). Forced expression of BAFF also results in CONCLUSIONS—BAFF/APRIL blockade prevents diabetes. a marked expansion of marginal-zone B-cells (MZBs) BCMA-Fc reduces B-cells, subsequently blunting autoimmune activ- (12)—a B-cell subset associated with autoimmune condi- ity and allowing endogenous regulatory mechanisms to preserve a tions including lupus (13), Sjogren’s syndrome (11), and, prehyperglycemic state. Diabetes 58:1568–1577, 2009 more recently, type 1 diabetes (14,15). Thus, the BAFF/ APRIL system can be considered a proinflammatory path- way associated with the development of autoimmunity (7,8). Indeed, studies designed to explore the therapeutic potential of BAFF pathway blockers for the treatment of autoimmune conditions are underway (16,17). This back- ground makes targeting the BAFF/APRIL system a poten- tial therapeutic candidate for the treatment of type 1 diabetes. This study was undertaken to test the hypothesis From the Immunology and Inflammation Program, Garvan Institute of Medical that targeting the BAFF/APRIL system would have multi- Research, Darlinghurst, NSW, Australia. ple inhibitory effects on the spontaneous development of Corresponding author: Shane T. Grey, [email protected]. Received 31 October 2008 and accepted 16 March 2009. autoimmune diabetes in the NOD model. Published ahead of print at http://diabetes.diabetesjournals.org on 31 March 2009. DOI: 10.2337/db08-1504. © 2009 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, RESEARCH DESIGN AND METHODS and the work is not altered. See http://creativecommons.org/licenses/by C57BL/6, NOD.SCID, and NOD/Lt (NOD) mice were obtained from The Walter -nc-nd/3.0/ for details. and Eliza Hall Institute of Medical Research (WEHI) Kew, Melbourne, The costs of publication of this article were defrayed in part by the payment of page / / Australia. NOD.MT mice were provided by Dr. Serreze (18). BAFF , charges. This article must therefore be hereby marked “advertisement” in accordance / / with 18 U.S.C. Section 1734 solely to indicate this fact. BCMA , and TACI mice were provided by Dr. Susan Kalled (Biogen See accompanying commentary, p. 1479. Idec). BR3 mice were a gift from Dr. Rajewsky (19). All animal experiments 1568 DIABETES, VOL. 58, JULY 2009 ˜ E. MARINO AND ASSOCIATES were approved by the St. Vincent’s Campus Animal Experimentation and ** Ethics Committee. Diabetes incidence studies. NOD mice were administered BCMA-Fc (150 g per treatment) based on previous studies (20). BCMA-Fc is a fusion 0.8 protein—the extracellular portion of BCMA fused to the Fc domain of human IgG. BCMA-Fc was provided by Dr. Susan Kalled (Biogen Idec). Controls were treated with PBS or intravenous globulin (HuIvIg) (150 g). For adoptive 0.6 transfer studies, splenocytes (1  10 ) from pre-diabetic 16-week-old female NOD donors or BCMA-Fc–treated mice were transferred intravenously into NOD.SCID recipients. Glucose levels were monitored twice weekly from 10 0.4 weeks of age onward for BCMA-Fc–treated mice or starting with transfer of splenocytes; a blood glucose level 12.0 mmol/l on two consecutive readings was scored as indicative of diabetes. 0.2 Phenotypic analysis of mononuclear cells. Lymphocytes were isolated and analyzed by flow cytometry exactly as previously described (15). T-cell subpopulations were identified as follows: CD8a (Ly2)(53-6-7) and memory- high low effector cells CD44 CD62L and regulatory T-cells (Tregs) CD4 (L3T4) 20 30 40 50 (GK1.5), CD25 (7D4) and (PC61), and Foxp3 (Foxp3-staining kit; Time (weeks) eBioscience, San Diego, CA). Diabetogenic T-cell clones were identified based on expression of CD4 (H129.19) and CD40 (3/23) as previously described (21). B-cell subpopulations were identified exactly as previously FIG. 1. Administration of BCMA-Fc prevents diabetes in NOD mice. high  int Diabetes incidence was followed for NOD mice administered BCMA-Fc described (15): follicular B-cells (FoB) (CD23 , IgM , and CD21 ), MZBs low high high low (black line) (n  10), HuIvIg (gray line) (n  20), and PBS (broken (CD23 , IgM , and CD21 ), transitional type 1 (T1) cells (CD23 , high low high  line) (n  30) from 9 to 15 weeks of age. **P  0.0041 (Mantel-Cox IgM , and CD21 ), and transitional type 2 (T2MZ) cells (CD23 , IgM , log-rank analysis) for BCMA-Fc treatment vs. HuIvIg; P < 0.0001 for high and CD21 ). Isotype controls included IgG , ; IgG , ; IgG , ; and IgG , 1 1 2b 2a BCMA-Fc treatment vs. PBS. . Flow cytometric analysis was conducted on a FACScalibur flow cytometer (BD Biosciences, San Jose, CA). Cytokine analysis. Cytokine profile of sera samples was performed with a BCMA-Fc treatment was carried out by flow cytometry. LINCOplex mouse 9-plex cytokine kit from Linco Research (St. Charles, MO), Given the well-described requirement of BAFF in the following the manufacturer’s instructions. The assays were carried out at The regulation of steady-state B-cell homeostasis (1), we first University of New South Wales Inflammation Disease Unit in conjunction with examined B-cell populations. As shown in Fig. 2A, all three Taline Hampartzoumian. known BAFF and APRIL receptors were expressed by Histopathology. Formaldehyde-fixed, paraffin-embedded pancreata sections (5 m) were hematoxylin and eosin stained. Insulitis was scored (100  NOD IgM B220 B-cells, suggesting that NOD B-cells magnification) as follows: grade 0, no insulitis; grade 1, peri-insulitis; grade 2, would be sensitive to BAFF/APRIL blockade. Tracking insulitis involving 25% islet; grade 3, insulitis involving 25% islet; and grade IgM B220 cells in the blood during the course of 4, insulitis involving 75% and/or complete islet infiltration. Photos were BCMA-Fc and HuIvIg treatment revealed a steady reduc- taken using a Leica DC300 camera on a Leica DMRB microscope. tion in the peripheral B-cell frequency during the BCMA-Fc Anti-CD25 antibody treatment. Mice were administered the anti-CD25 treatment period, reaching a nadir at 4 weeks (Fig. 2B). monoclonal antibody (mAb) PC61 (200 g) (The Walter and Eliza Hall Institute of Medical Research [WEHI] mAb facility, Melbourne, Australia) fortnightly for a Further analysis conducted at the end of the 9- to 15-week total of four injections. Control mice received 200 g rat IgG  (BD Biosciences). treatment period demonstrated that BCMA-Fc treatment BCMA-Fc–treated NOD mice were first inoculated on the 16th week. reduced the frequencies of mature follicular and MZB NOD.MT mice were administered PC61 beginning at 16 weeks of age. The subsets, as well as the immature T2MZ cells in the spleen frequency of CD25 T-cells was determined by analysis of CD4 CD25 (mAb and pancreatic lymph node (PLN) (Fig. 2D). Similarly, the 7D4) Foxp3 cells. Diabetes incidence was followed as described above. absolute numbers of the follicular, marginal-zone, and Statistical analysis. Statistical significance for mononuclear cell analysis was determined by calculating P values using the Student’s t test (GraphPad Software, T2MZ subsets were reduced by 80 –90% (Fig. 2E). In San Diego, CA). Diabetes incidence studies were graphed as Kaplan-Meier contrast, the frequency and absolute numbers of T1 pre- survival plots and analyzed using the Mantel-Cox log-rank method with 2 degrees cursors in the spleen were less affected by BCMA-Fc of freedom (GraphPad Prism; GraphPad Software). P values represent compari- treatment (Fig. 2D and E), a result consistent with the role son between different treatments as indicated in the figure legends. of BAFF in promoting B-cell development after the T1 checkpoint (22). RESULTS Effect of BCMA-Fc treatment on peripheral T-cell Disrupting the BAFF/APRIL pathway in the preclini- populations and Th1 cytokines. In contrast to its effect cal phase prevents diabetes onset. To test the effect of on B-cell populations, administration of BCMA-Fc from 9 disrupting the BAFF/APRIL pathway before the onset of to 15 weeks of age did not impact the absolute number of hyperglycemia, we injected NOD mice with 150 g peripheral T-cells or CD4 and CD8 T-cell subsets (Fig. BCMA-Fc intraperitoneally (i.p.) twice weekly from 9 to 15 3A). However, the frequency of splenic CD4 and CD8 weeks of age (12 injections over a 6-week period); control T-cells was proportionally increased, presumably as a groups were administered PBS or HuIvIg (150 g i.p.) over result of the decreased number of B-cells (data not de- the same period (Fig. 1). We found that all NOD mice picted). To determine how BCMA-Fc treatment affected treated with PBS or HuIvIg from 9 to 15 weeks of age the activation of effector T-cell clones, we analyzed the developed diabetes with the expected high frequencies. expression of CD44 and CD40 on peripheral CD4 and CD8 There were no significant differences in diabetes incidence T-cell populations. CD44 is expressed by activated T-cells, between PBS- and HuIvIg-treated groups (P 0.1309; n  whereas CD40 has been identified as a marker for highly 10). In contrast, we found that NOD mice treated with diabetogenic T-cell clones (21). BCMA-Fc treatment did high  high BCMA-Fc from 9 to 15 weeks of age were completely not alter the frequency of CD44 CD4 or CD44 CD8 protected from diabetes (diabetes incidence 0 of 10 at 50 T-cells (data not depicted); however, the frequency of weeks of age; P 0.0041, n  10, log-rank vs. HuIvIg). CD4 and CD8 CD40 T-cells was reduced in both the Effect of BCMA-Fc treatment on peripheral B-cell spleen and PLN of BCMA-Fc–treated mice (Fig. 3B). The populations. Examination of peripheral lymphoid popu- reduction in frequency of pathogenic CD40 T-cells in lations before and at the cessation of the 9- to 15-week BCMA-Fc–treated mice was associated with a decrease in DIABETES, VOL. 58, JULY 2009 1569 % of Normoglycemic mice TARGETING THE BAFF/APRIL PATHWAY PREVENTS DIABETES A B C 50 Gating strategy 40 T2MZ BCMA BR3 TACI FoB CD23hi CD23lo MZB FSC T1 12 3 4 56 7 89 Time (weeks of treatment) IgM SPLN PLN PBS HuIvIg BCMA-Fc PBS HuIvIg BCMA-Fc *** T2MZ 4.83% 7% 0.95% T2MZ 1.73% 0.7% 0.48% 20% FoB 28% 23.25% ***3.6% ***3% FoB 20% MZB 2.22% 1.9% MZB 13.7% 12% *** 0.64% ***6.32% T1 T1 0.9% 1.18% 1.2% 4.4% 3% 3.58% IgM IgM SPLN PLN FoB cells MZB cells FoB cells MZB cells 2 0.5 45 45 *** *** *** *** *** 0.4 1.5 *** 30 30 0.3 0.2 15 15 0.5 0.1 0 0 0 0 T2MZ cells T1 cells T2MZ cells T1 cells 0.5 0.5 16 16 *** * *** ** 0.4 0.4 12 12 0.3 0.3 8 8 0.2 0.2 4 4 0.1 0.1 0 0 0 FIG. 2. Effect of BCMA-Fc treatment on peripheral B-cells. A: Expression of BCMA, BR3, and TACI (black line) on IgM B220 NOD splenocytes. Gray line, isotype control. Representative fluorescence-activated cell sorting plots are shown. B: Frequency of IgM B220 cells in peripheral blood of NOD mice treated with BCMA-Fc from 9 to 15 weeks of age (E)(n  5) and HuIvIg control mice (O)(n  5). Time indicates period post–first injection. C: Gating strategy used for identification of B-cell subsets. D: Representative fluorescence-activated cell sorting plots illustrating frequency of B-cell subsets in the spleen (SPLN) and PLN from 16-week-old NOD mice treated with PBS, HuIvIg, or BCMA-Fc from 9 to 15 weeks of age. Numbers represent percentage of total lymphocytes. E: Absolute numbers, calculated from values in D, of B-cell subsets in the spleen and PLN from 16-week-old NOD mice treated with PBS (F), HuIvIg (O), or BCMA-Fc (E). Values from individual mice are shown (n > 8 per group). The bar represents median value. *P < 0.05, **P < 0.01, and ***P < 0.001. FSC, forward light scatter. the circulating levels of interleukin (IL)-7, IL-15, and IL-17 Effect of BCMA-Fc treatment on B-cell repopulation. (Fig. 3C), cytokines critical for the expansion and activa- To examine how BCMA-Fc treatment effected B-cell re- tion of effector T-cells. population, further analysis was carried out in long-term 1570 DIABETES, VOL. 58, JULY 2009 6 6 Cell number (x10 ) Cell number (x10 ) CD21 % IgM+B220+ 6 6 Cell number (x10 ) Cell number (x10 ) CD21 CD23 CD21 ˜ E. MARINO AND ASSOCIATES A B SPLN PLN SPLN CD4+ T cells CD4+ T cells 60 4 NOD HuIvIg BCMA-Fc9-15 100 30% 32% 100 100 1.5% 40 80 80 80 CD4 60 60 (H129.19) 40 40 20 20 0 0 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 100 35% 100 40% 100 3% CD8+ T cells CD8+ T cells 60 4 80 80 80 60 60 60 CD8 3 40 40 40 20 20 20 0 0 0 2 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 CD40 PLN 0 0 NOD HuIvIg BCMA-Fc9-15 100 40% 100 35% 100 1% IL-2 IL-4 IL-7 IL-10 20 10 200 100 80 80 80 *** CD4 60 60 60 (H129.19) 8 80 40 40 40 15 150 20 20 20 6 60 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 10 100 010 10 10 10 010 10 10 10 010 10 10 10 4 40 100 25% 100 38% 100 0.83% 5 50 2 20 80 80 80 60 60 60 CD8 0 0 0 0 40 40 40 20 20 20 IL-12 IL-15 IL-17 TNF-a 300 800 60 0 0 0 * 2 3 4 5 2 3 4 5 2 3 4 5 *** 010 10 10 10 010 10 10 10 010 10 10 10 CD40 200 40 100 20 0 0 0 FIG. 3. Effect of BCMA-Fc on peripheral T-cells and cytokines. A: Absolute number of CD4 (upper panel) and CD8 (lower panel) T-cells from 16-week-old NOD mice treated with PBS (F), HuIvIg (O), or BCMA-Fc (E) from 9 to 15 weeks of age. Values from individual mice are shown (n > 8 per group). B: Representative fluorescence-activated cell sorting plots illustrating frequency of CD4 CD40 (upper panel) and CD8 CD40 (lower panel) T-cells from treated mice at 16 weeks of age. Black line, CD40; gray line, isotype control (n  7 per group). C: Serum cytokine levels in HuIvIg- (O) and BCMA-Fc– (E) treated NOD mice at 16 weeks of age. Values from individual mice are shown (n > 3 per group). Significant differences between sample means are indicated. Bar represents median value. *P < 0.05 and ***P < 0.001. SPLN, spleen. surviving (50 weeks of age) BCMA-Fc–treated mice. Effect of BCMA-Fc treatment on the pancreatic BCMA-Fc–protected mice exhibited normal frequencies infiltrate. We conducted histological analysis of the pan- and absolute numbers of FoB, MZB, T2MZ, and T1 B-cell creata from the BCMA-Fc–protected NOD mice at 16 and subsets in the periphery compared with those in HuIvIg- 50 weeks of age and a comparison with treated control treated control mice (Fig. 4A and B). Although the PLN of mice. Representative histology for each group is shown BCMA-Fc–treated mice harbored frequencies and num- in Fig. 5A, and insulitis scores for these mice are shown in bers of FoB and MZB cells similar to those of control mice, Fig. 5C. Although BCMA-Fc–treated NOD mice did exhibit an increase in both the frequency and the number of T2MZ insulitis at the 16-week time point, the severity was and T1 cells was observed (Fig. 4A and B). These data reduced compared with that in HuIvIg-treated and diabetic demonstrate that BCMA-Fc–treated NOD mice could re- control NOD mice at 16 weeks. Indeed, the frequency of populate their mature B-cell pool. These data also demon- islets exhibiting heavy insulitis (grades 3 and 4) at 16 strate that BCMA-Fc treatment provides long-term weeks was only 20 vs. 45–50% in control groups. Flow protection from diabetes in NOD mice despite the return cytometric analysis of the pancreatic infiltrate revealed of B-cell populations. that the frequency of FoB was markedly reduced (P DIABETES, VOL. 58, JULY 2009 1571 Concentration Concentration 6 6 Cell number (x10 ) Cell number (x10 ) (pg/ml) (pg/ml) TARGETING THE BAFF/APRIL PATHWAY PREVENTS DIABETES SPLN A B HuIvIg BCMA-Fc SPLN SPLN PLN PLN T2MZ 6.6% 5% FoB cells FoB cells T2MZ cells T2MZ cells 80 0.8 0.075 FoB 22% 27% 60 0.6 0.05 16% MZB17% 40 0.4 5 0.025 T1 3% 0.2 3.6% IgM 0 0 0 0 PLN MZB cells MZB cells T1 cells T1 cells 80 0.075 15 0.075 1.9% 0.8% 0.05 10 0.05 25% 23% 0.025 5 0.7% 0.025 1.5% 0.4% 0 0 2% 0 IgM FIG. 4. Effect of BCMA-Fc treatment on B-cell repopulation. A: Representative fluorescence-activated cell sorting plots illustrating frequency of B-cell subsets from NOD mice treated with HuIvIg at 30 weeks of age or BCMA-Fc at >50 weeks of age. Numbers represent percentage of total lymphocytes. B: Absolute numbers of B-cell subsets from HuIvIg- (O) and BCMA-Fc– (E) treated NOD mice. Values calculated from A. Results from individual mice are shown (n > 3 per group). Bar represents median value. *P < 0.05 and **P < 0.01. SPLN, spleen. 0.014; n  5) in the BCMA-Fc–treated mice at 16 weeks Interestingly, 50% (4 of 8) of NOD.SCID mice receiving (Fig. 5D). No changes were observed in the ratios of CD4 splenocytes from BCMA-Fc–treated NOD mice did de- to CD8 T-cells, though their frequencies were increased velop hyperglycemia (P 0.088, NOD vs. BCMA-Fc– (Fig. 5D), again, most probably as a result of the decrease treated splenocytes; n 8). These data demonstrate that in FoB. BCMA-Fc–treated mice still harbored T-cells with self- The proportion of severely infiltrated islets in the BCMA- reactive potential, but these T-cells are unable to precipi- Fc–treated mice did not increase over time, as evidenced by tate diabetes in their BCMA-Fc–treated hosts. analysis of the long-term–protected mice (e.g., 50-week-old Time-dependent accumulation of CD4 CD25 T-cells mice) (Fig. 5A and C). Thus, although BCMA-Fc–treated mice in BCMA-Fc–treated NOD mice. The long-term protec- did exhibit evident insulitis, the severity of insulitis was tion afforded by BCMA-Fc treatment despite the persis- maintained at a level equivalent to that exhibited by prehy- tence of self-reactive T-cells prompted us to investigate perglycemic 8- to 15-week-old NOD mice. possible regulatory mechanisms. Tregs that express the Given that NOD mice exhibit a progressive insulitis from markers CD4 and CD25 can control the progression to the preclinical to hyperglycemic phase, these data suggest overt diabetes in the NOD model (23). We analyzed the that BCMA-Fc treatment has not reversed the autoimmune frequency and number of CD4 CD25 T-cells in the process as determined by the persistence of a mononu- spleen, PLN, and pancreas of BCMA-Fc–treated NOD mice clear cell pancreatic infiltrate but, rather, halted the pro- at 16 weeks of age. As shown in Fig. 6A and B, compared gression to fulminant diabetes. Thus, we questioned with treated control NOD mice, 9- to 15-week BCMA-Fc– whether this related to a change in the nature of the treated mice harbored an increased frequency of splenic insulitic lesion or active regulation. Analysis of the B- and CD4 CD25 T-cells at 16 weeks of age. The majority T-cell subsets infiltrating the pancreas in the protected (90%) of these CD4 CD25 T-cells were also Foxp3 BCMA-Fc–treated mice showed that the frequencies of (Fig. 6C and D), demonstrating that they belonged to the infiltrating B- and T-cell subsets were similar to those of set of Tregs. Analysis of the long-term (50 weeks of age) treated control mice (Fig. 5D). Thus, in long-term–pro- BCMA-Fc–protected mice revealed increased (twofold or tected mice, B-cells are not prohibited from forming a part more) frequencies of CD4 CD25 T-cells in both the of the insulitic lesion. To test whether the BCMA-Fc– spleen and PLN (Fig. 6E). Further, in contrast with the protected NOD mice still harbored T-cells with self-reac- 16-week time point, the increased frequency of Tregs was tive potential, splenocytes from normoglycemic BCMA- also reflected as an increase in the absolute numbers of Fc–treated NOD mice were adoptively transferred into CD4 CD25 T-cells in the spleen and PLN (Fig. 6E). Thus, NOD.SCID recipients (Fig. 5E). As a positive control, other BCMA-Fc–treated mice showed an accumulation of Treg groups received splenocytes from pre-diabetic 16-week- numbers over time. The increased number of Tregs was old mice. We found that 90% (7 of 8) NOD.SCID mice associated with a halting of the autoimmune attack and receiving these control splenocytes developed diabetes. permanent euglycemia. 1572 DIABETES, VOL. 58, JULY 2009 CD21 CD21 6 6 Cell number (x10 ) Cell number (x10 ) 6 6 Cell number (x10 ) Cell number (x10 ) ˜ E. MARINO AND ASSOCIATES A B BCMA-Fc BCMA-Fc NOD. µMT-/- 16-week-old >50-week-old 6-16-week-old ** ** HuIvIg PBS 16-week-old 16-week-old 16 wks-old NOD mice >50 wks-old NOD mice PBS HuIvIg BCMA-Fc HuIvIg BCMA-Fc 0.63% T2MZ 0.88% 0.5% T2MZ 0.63% 0.6% FoB 5.8% 5.8% 9.62% FoB B cells 7.3% 3.51% MZB 1.17% MZB 1.3% 3.7% 2.2% 4.15% T1 T1 1.96% 2.4% 3.4% 1.96% 1.6% IgM IgM 5.46% 9.64% 11% 9% 20% T cells *** 4.87% 2.41% 4% 3% 8% CD8 CD8 FIG. 5. Histology and insulitis scores of BCMA-Fc–treated NOD mice. A: Representative histological section of pancreas from normoglycemic mice treated with BCMA-Fc from 9 to 15 weeks at 16 or >50 weeks of age is shown (upper panels). Sections 0.8 from HuIvIg- and PBS-treated mice at 16 weeks are also shown (lower panels). B: Representative pancreatic sections from an NOD.MT mouse. C: Insulitis scores for treated mice; 0.6 20 –70 islets were scored from four to seven mice per group. Differences in insulitis scores for BCMA-Fc–treated mice at 16 and >50 weeks were significant (**P < 0.01). P values resulted 0.4 comparing insulitis level at grade 4 among HuIvIg control mice. D: Representative fluorescence-activated cell sorting plots illus- 0.2 trating frequency of pancreatic B-cell (upper panel) and T-cell (lower panel) subsets in treated NOD mice at 16 and >50 weeks of age (n > 7 per group). E: Splenocytes (1  10 ) from >50-week-old normoglycemic BCMA-Fc–treated NOD mice 010 20 30 40 50 60 70 (black line) (n  8) and pre-diabetic 16-week-old NOD mice Time (days) (gray line) (n  8) were adoptively transferred (intravenously) to NOD.SCID recipients. Diabetes incidence was then followed over time. P  0.08 (Mantel-Cox log-rank analysis) for BCMA-Fc–treated vs. NOD islets. (A high-quality digital representation of this figure is available in the online issue.) BCMA-Fc–mediated protection from diabetes requires achieve this, mice were administered the anti-CD25 mAb the presence of CD4 CD25 T-cells. We next de- PC61. Control mice received PBS or rat IgG . In prelim- termined whether CD4 CD25 T-cells were required inary experiments, we could show that administration for BCMA-Fc–mediated protection from diabetes. To of a single dose of PC61 (200 g i.p.) induced an DIABETES, VOL. 58, JULY 2009 1573 NOD 8-15 wks-old NOD hyperglycemic BCMA 16 wk-old HuIvIg 16 wks-old BCMA >50 wk-old NOD.µMT-/-6-16wks- old % of Normoglycemic mice CD4 CD21 % of islets CD2 1 CD4 TARGETING THE BAFF/APRIL PATHWAY PREVENTS DIABETES A HuIvIg C PBS BCMA-Fc PBS HuIvIg BCMA-Fc 4 4 4 4 4 4 10 10 10 10 10 8% 5.1% 7.5% 4.5% 15% 6.28% 13.4% 9.03% 10% 3 3 3 3 3 10 10 10 10 10 SPLN 2 2 2 2 2 10 10 10 10 10 1 1 1 1 1 10 10 10 10 10 1.26% 1.82% 1.4% 0 0 0 0 0 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4 4 4 10 10 10 CD25 10% 13% 10.5% 3 3 3 10 10 10 PLN SPLN 2 2 2 10 10 10 1 1 1 *** 10 10 10 *** 0 0 0 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4 4 4 10 10 10 9.8% 9.3% 10.6% 3 3 3 10 10 10 PANC 2 2 2 10 10 10 1 1 1 10 10 10 1 0 0 0 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD 4 SPLN PLN B SPLN PLN E *** *** 60 30 20 50 *** 50 *** 0 0 ** *** 14 0.35 14 ** 0.35 ** 12 12 0.25 10 10 0.25 8 8 0.15 6 6 0.15 4 4 0.05 2 0.05 0 0 0 FIG. 6. CD4 CD25 T-cells in BCMA-FC–treated NOD mice. A: Representative fluorescence-activated cell sorting plot illustrating frequency of CD25-expressing CD4 T-cells from 16-week-old PBS-, HuIvIg-, and BCMA-Fc–treated NOD mice (n > 7 per group) is shown. B: Pooled data showing frequencies and calculated absolute numbers of CD25-expressing CD4 T-cells of PBS- (F), HuIvIg- (O), or BCMA-Fc– (E) treated NOD mice at 16 weeks. Values from individual mice are shown (n > 7 per group). Bar represents median value. C: Representative fluorescence-activated cell sorting plots illustrating frequency of CD4 CD25 Foxp3 cells from 16-week-old PBS-, HuIvIg-, and BCMA-Fc–treated NOD mice. D: Absolute numbers, calculated from C,ofCD4 CD25 Foxp3 cells from PBS- (F), HuIvIg- (O), and BCMA-Fc– (E) treated NOD mice. Values from individual mice are shown. Bar represents median value. E: Cumulative data showing frequency and calculated absolute numbers of CD25-expressing CD4 T-cells from HuIvIg- (O) or BCMA-Fc– (E) treated NOD mice (n > 3 per group). Bar indicates median value. **P < 0.01 and ***P < 0.001. PANC, pancreas; SPLN, spleen. 90% reduction in the frequency of peripheral NOD mice developed hyperglycemia with the expected CD4 CD25 Foxp3 T-cells for 14 days (Fig. 7A). For the frequency (Fig. 7B). In contrast, subsequent administra- experiment, NOD mice were first treated with BCMA-Fc tion of PC61 after BCMA-Fc treatment precipitated diabe- from 9 to 15 weeks of age; at the cessation of BCMA-Fc tes in 100% of mice (P 0.001 BCMA-Fc vs. BCMA-Fc plus treatment, mice were administered the anti-CD25 mAb PC61; n  5). PC61 (200 g i.p.) every 14 days (a total of four injections), Blocking the BAFF/APRIL pathway indirectly modu- and blood glucose levels were monitored. Kaplan-Meier lates CD4 CD25 Foxp3 T-cells. We next focused on survival analysis showed that BCMA-Fc–treated NOD mice determining the mechanism by which BCMA-Fc treatment remained diabetes free, whereas control PC61-treated would effect protection in a CD4 CD25 T-cell– depen- 1574 DIABETES, VOL. 58, JULY 2009 Cell number (x10 ) % CD25-expressing CD25-expressing CD 4 CD25 cells CD4 T cells Foxp3 6 6 Cell number (x10 ) Cell number (x10 ) % CD25-expressing CD25-expressing CD 4 CD25+Foxp3-expressing T cells CD4 T cells CD4 T cells ˜ E. MARINO AND ASSOCIATES 15 15 10 10 5 5 0 0 B CD25-expressing B Cells (B220) CD4+ T cells CD4 T cells BR3 TACI 0.8 FIG. 7. CD25 T-cells are required for BCMA-Fc–mediated protection 0.6 from diabetes. A: Frequency of CD4-expressing CD25 (mAb 7D4) Foxp3 T-cells in peripheral blood of 16-week-old NOD (upper panel) and NOD.MT (lower panel) mice treated with PC61 or isotype control 0.4 (rat IgG , ). Representative fluorescence-activated cell sorting plot is shown (n > 4 per group). B: Diabetes incidence was followed for NOD mice administered BCMA-Fc from 9 to 15 weeks of age (solid black line) 0.2 (n  5), PC61 (broken line) (n  5), or BCMA-Fc from 9 to 15 weeks of age plus PC61 (solid gray line) (n  8). **P  0.001 (Mantel-Cox log-rank analysis) for BCMA-Fc alone vs. BCMA-Fc plus PC61 treatment. 10 40 20 50 30 Time (weeks) dent manner. To examine whether disrupted BAFF or FIG. 8. CD25 T-cells are required for diabetes resistance in B-cell– APRIL signaling would engender an accumulation of deficient NOD mice. A: Left plot: frequencies of splenic CD4 CD25 Foxp3 T-cells in C57BL/6 (F) and NOD (E) mice. Right CD4 CD25 Foxp3 T-cell numbers over time, we exam- plot: frequencies of splenic CD4 CD25 Foxp3 T-cells in BAFF ined the frequency of CD4 CD25 Foxp3 T-cells in mice / / / (F), BR3 (), TACI (E), and BCMA (‚) mice. Values from deficient for BAFF or the BAFF and APRIL receptors individual mice are shown (n > 4 per group). Bar represents median value. Differences are not significant (P > 0.05). B: Expression of BR3 BCMA and TACI, as well as the BAFF receptor BR3. and TACI (black line) on C57BL/6 splenic, B220 B-cells, CD4 T-cells, Though these mutations were on a C57BL/6 background, and CD25-expressing CD4 T-cells. Gray line, isotype control. Repre- we believe that the analysis is valid given that the median sentative fluorescence-activated cell sorting plots are shown. C: Dia- betes incidence was followed for NOD.MT mice (black line) (n frequency of CD4 CD25 Foxp3 T-cells was similar be- 5) and NOD.MT mice administered PC61 (solid gray line) (n  10). tween NOD and C57BL/6 mice (P 0.3513; n  7): 9% *P  0.0181 (Mantel-Cox log-rank analysis) for control vs. PC61 for both strains (Fig. 8A). Also shown in Fig. 8A, the treatment. frequency of peripheral CD4 CD25 Foxp3 T-cells in / / / / BAFF , BR3 , BCMA , and TACI mice was com- parable with that in wild-type mice. Further, to determine relationship between a reduction in B-cells and CD4 CD25 whether BAFF or APRIL could engage Tregs directly, we T-cells, we used B-cell– deficient NOD.MT mice (18). examined TACI and BR3 expression in CD4 CD25 T- NOD.MT mice exhibit a number of features, with regard cells. CD4 CD25 T-cells did not express significant levels to diabetes development, reminiscent of those in BCMA-Fc– of the receptors BR3 or TACI (Fig. 8B) in contrast to treated mice. These include a reduced degree of insulitis (Fig. splenic B-cells. These data indicate that it is unlikely that 5B and C) and resistance to diabetes development (Fig. 8C). targeting the BAFF/APRIL system resulted in increased To assess whether CD4 CD25 T-cells were important in CD4 CD25 Foxp3 T-cells via a direct mechanism. maintaining euglycemia in NOD.MT mice, 16-week-old CD4 CD25 T-cells reign in destructive T-cells in mice were treated with PC61 (200 g i.p. each) every 14 days B-cell– deficient NOD mice. B-cell subpopulations were for a total of four injections and blood glucose levels were severely reduced by BCMA-Fc treatment. To explore the followed. This treatment reduced the frequency of peripheral DIABETES, VOL. 58, JULY 2009 1575 % of CD25+Foxp3-expressing CD4 T cells % of Normoglycemic mice % of CD25+Foxp3-expressing CD4 T cells TARGETING THE BAFF/APRIL PATHWAY PREVENTS DIABETES CD4 CD25 Foxp3 T-cells as observed for B-cell–sufficient reduced incidence of diabetes. Together with our study NOD mice (Fig. 7A). Treatment with PC61 precipitated results, these data indicate that reducing B-cells in auto- diabetes in 80% (P 0.0184; n 10) of NOD.MT mice immune NOD mice is associated with an increased fre- (Fig. 8C)—a dramatic result when compared with data in quency of cells with a Treg phenotype. Significantly, our control NOD.MT mice. Therefore, targeting CD25 reg- present data demonstrate that in the absence of B-cells, ulatory populations triggered diabetes onset in B-cell– defi- endogenous Tregs control the progression of autoimmu- cient NOD.MT mice that are otherwise resistant. nity in NOD mice. This conclusion is further supported by our analysis of NOD.MT mice that lack B-cells and are resistant to diabetes (18). Treatment of NOD.MT mice DISCUSSION with the anti-CD25 mAb PC61 precipitated the onset of diabetes, demonstrating that the hyperglycemia-free state Emerging evidence demonstrates that B-cells can impact multiple stages in the pathogenesis of autoimmune diabe- in NOD.MT mice is also dependent on CD25 regula- tory cells. tes (24). BAFF and APRIL play critical roles in supporting B-cell survival (1), such that BCMA-Fc–treated mice har- These are, to our knowledge, the first results to show that the resistance of B-cell– deficient NOD mice to diabetes can bored a much reduced B-cell pool, providing one potential mechanism by which BCMA-Fc prevented diabetes in be due to the activity of Tregs. However, they are consistent with emerging results from other models of autoimmune NOD mice. BAFF-activated B-cells show an enhanced antigen-presenting cell (APC) capacity (25), and in vivo disease. Indeed, NOD.H-2h4 mice develop spontaneous au- toimmune thyroiditis, whereas B-cell– deficient NOD.H-2h4 BAFF-activated B-cells facilitate heightened Th1 T-cell responses (26). In the NOD model, B-cells undergo a mice are protected from autoimmunity (38). Treatment of B-cell– deficient NOD.H-2h4 with PC61 induced the rapid marked expansion from 9 to 15 weeks of age (15), show an increased capacity to act as APCs during this time onset of autoimmunity (39), similar to the results obtained in NOD.MT mice, suggesting a common mechanism (15,27), and can present captured autoantigen to self- reactive T-cells (15,28,29). The APC function of B-cells is whereby the absence of B-cells allows a regulatory pathway to prevent autoimmunity. A number of possibilities exist to essential for the activation (30) and expansion (31) of the CD4 T-cell repertoire from 9 to 15 weeks of age, i.e., explain this intriguing relationship between B-cells and Tregs. As an example, Olson et al. (40) using a model of before the onset of hyperglycemia. Thus, by reducing the availability of B-cells to act as APCs, BCMA-Fc treatment Crohn’s disease demonstrated that B-cells could block Treg activity in a glucocorticoid-induced tumor necrosis factor curtailed B- and T-cell interactions during the critical 9- to 15-week time period, halting the progression from clini- receptor (GITR)-dependent manner. Thus, activated NOD B-cells may directly suppress the activity of Tregs through cally silent insulitis to overt hyperglycemia. This concept is consistent with our observation that BCMA-Fc–treated mechanisms including expression of GITR ligand (40). Alter- natively, because the number of B-cells increases as disease mice exhibited a reduced proportion of B-cells in associ- ation with a diminished frequency of CD4 CD40 diabe- progresses (15), the opportunity for T-cells to engage activat- ing APCs (e.g., B-cells) (30,31) versus a tolerizing APC (e.g., togenic T-cells and a reduced level of Th1 T-cell– derived cytokines at 16 weeks of age. Additional evidence to macrophages or dendritic cells) (41) may decrease. In this scenario, BCMA-Fc treatment could be protective via two support this hypothesis comes from studies in which NOD mice expressing human CD20 have been treated with an potential mechanisms. First, BCMA-Fc, by reducing B-cells, may alter the balance in the production of effector cells anti-CD20 mAb (32); in this case, reducing B-cells from 9 weeks of age delays diabetes onset. Depletion of B-cells versus Tregs, leading to an accumulation of CD4 CD25 Tregs. Second, without B-cells to expand up self-reactive with anti-CD20 impairs activation of adaptive and autore- active CD4 T-cell responses (32,33), further demonstrat- T-cell clones, endogenous regulatory cells may simply be able to reign in the present autoreactive T-cell pool, prevent- ing the required role for B-cells in the activation of self-reactive CD4 T-cells (30,34). ing the progression to hyperglycemia. These hypotheses should be explored in further studies. Our studies uncover an additional and perhaps unex- pected mechanism by which targeting the BAFF and In this study, we demonstrate that targeting the BAFF and APRIL pathway with BCMA-Fc before the onset of hypergly- APRIL system can prevent diabetes, namely through in- creasing CD4 CD25 T-cells. Tregs expressing CD25 and cemia prevented diabetes in spontaneously diabetic NOD mice. We hypothesize that the major action achieved by Foxp3 can control the development of autoimmune diabe- tes (35), and we found that CD4 CD25 T-cells were blocking BAFF and APRIL was a reduction in peripheral B-cells, thereby limiting their involvement in diabetes patho- required for the continued maintenance of a hyperglyce- mia-free state. Further, these CD4 CD25 T-cells ex- genesis. This prevented diabetes progression through two mechanisms: a dampening of T-cell autoimmune activity and pressed the Treg lineage marker Foxp3, suggesting that they belonged to the set of natural Tregs (36). To address subsequent elaboration of cytokines, most likely achieved by decreasing the availability of B-cells to act as APCs; a second the question of how BCMA-Fc might affect CD4 CD25 T-cell homeostasis, we examined Foxp3 Treg frequencies mechanism also related to decreased B-cells, which in- duced or allowed Tregs to reign in the autoreactive in mice in which BAFF and APRIL signaling were dis- rupted. These data demonstrated that loss of BAFF or potential of diabetogenic T-cells. This second mecha- nism highlights a novel pathway by which targeting APRIL did not increase the frequency of Foxp3 Tregs per se, suggesting that BCMA-Fc altered Treg homeostasis B-cells may provide resistance to autoimmunity. through an indirect mechanism. Of interest, an increased frequency of Tregs was demonstrated in two recent stud- ACKNOWLEDGMENTS ies in which NOD mice were treated with the B-cell depleting agents: anti-CD20 or anti-CD22 mAb, respec- This study was supported by a grant from the Juvenile tively (32,37). Both studies also reported that NOD mice Diabetes Research Foundation International (5-2005-1132) treated between 9 and 15 weeks exhibited a delayed and to S.T.G. E.M. is supported by a National Health and 1576 DIABETES, VOL. 58, JULY 2009 ˜ E. MARINO AND ASSOCIATES 20. Pelletier M, Thompson JS, Qian F, Bixler SA, Gong D, Cachero T, Gilbride Medical Research Council Dora Lush Fellowship. S.T.G. is K, Day E, Zafari M, Benjamin C, Gorelik L, Whitty A, Kalled SL, Ambrose supported by a BioFirst Award from the Office for Science C, Hsu YM. Comparison of soluble decoy IgG fusion proteins of BAFF-R and Medical Research, NSW. and BCMA as antagonists for BAFF. 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CD4+CD25+ T-Cells Control Autoimmunity in the Absence of B-Cells

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Abstract

ORIGINAL ARTICLE CD4 CD25 T-Cells Control Autoimmunity in the Absence of B-Cells Eliana Marin ˜ o, Jeanette Villanueva, Stacey Walters, David Liuwantara, Fabienne Mackay, and Shane T. Grey OBJECTIVE—Tumor necrosis factor ligand family members B-cell–activating factor (BAFF) and a proliferation-inducing li- he members of the tumor necrosis factor (TNF) gand (APRIL) can exert powerful effects on B-cell activation and development, type 1 T-helper cell (Th1) immune responses, and ligand family of molecules B-cell–activating fac- autoimmunity. We examined the effect of blocking BAFF and APRIL tor (BAFF) (also known as BLyS, TNFSF13b) on the development of autoimmune diabetes. T and a proliferation-inducing ligand (APRIL) can exert powerful effects on B-cell development, survival, and RESEARCH DESIGN AND METHODS—Female NOD mice function; T-cell activation; and type 1 T-helper cell (Th1) were administered B-cell maturation antigen (BCMA)-Fc from 9 immune responses and autoimmunity (1). BAFF exists as to 15 weeks of age. Diabetes incidence, islet pathology, and T- both a soluble and a membrane bound molecule and is and B-cell populations were examined. expressed by a wide range of inflammatory-activated cells, RESULTS—BCMA-Fc treatment reduced the severity of insulitis including monocytes, macrophages, dendritic cells, and and prevented diabetes development in NOD mice. BCMA-Fc– T-cells (2). In contrast, APRIL is processed intracellularly treated mice showed reduced follicular, marginal-zone, and and exerts its function as a soluble protein. BAFF and T2MZ B-cells. B-cell reduction was accompanied by decreased APRIL can bind to one of two receptors: B-cell maturation frequencies of pathogenic CD4 CD40 T-cells and reduced Th1 antigen (BCMA) (3) or transmembrane activator and cal- cytokines IL-7, IL-15, and IL-17. Thus, T-cell activation was cium modulator and cyclophylin ligand interactor (TACI) blunted with reduced B-cells. However, BCMA-Fc–treated (3,4), whereas BAFF can also bind to BR3 (otherwise mice still harbored detectable diabetogenic T-cells, suggesting known as BAFF-R) (5). These receptors are found on a that regulatory mechanisms contributed to diabetes preven- tion. Indeed, BCMA-Fc–treated mice accumulated increased wide range of B-cell subsets including immature, transi- CD4 CD25 regulatory T-cells (Tregs) with age. CD4 CD25 tional, mature, memory, and germinal center B-cells, as cells were essential for maintaining euglycemia because their well as on plasma cells (2). Further, activated T-cells can depletion abrogated BCMA-Fc–mediated protection. BCMA-Fc express the receptors BR3 and TACI (4,6). did not directly affect Treg homeostasis given that BAFF has emerged as an important player in the devel- CD4 CD25 Foxp3 T-cells did not express TACI or BR3 recep- opment of autoimmunity. Elevated BAFF and APRIL levels tors and that CD4 CD25 Foxp3 T-cell frequencies were equiv- have been detected in sera from human patients with / / / / alent in wild-type, BAFF , TACI , BCMA , and BR3 rheumatoid arthritis, lupus, and Sjogren’s syndrome (7–9). mice. Rather, B-cell depletion resulted in CD4 CD25 T-cell– Moreover, BAFF-transgenic mice harbor increased titers mediated protection from diabetes because anti-CD25 monoclonal of self-reactive antibodies and develop autoimmune symp- antibody treatment precipitated diabetes in both diabetes-resistant toms very similar to those of lupus and Sjogren’s syn- NOD.MT and BCMA-Fc–treated mice. drome (10,11). Forced expression of BAFF also results in CONCLUSIONS—BAFF/APRIL blockade prevents diabetes. a marked expansion of marginal-zone B-cells (MZBs) BCMA-Fc reduces B-cells, subsequently blunting autoimmune activ- (12)—a B-cell subset associated with autoimmune condi- ity and allowing endogenous regulatory mechanisms to preserve a tions including lupus (13), Sjogren’s syndrome (11), and, prehyperglycemic state. Diabetes 58:1568–1577, 2009 more recently, type 1 diabetes (14,15). Thus, the BAFF/ APRIL system can be considered a proinflammatory path- way associated with the development of autoimmunity (7,8). Indeed, studies designed to explore the therapeutic potential of BAFF pathway blockers for the treatment of autoimmune conditions are underway (16,17). This back- ground makes targeting the BAFF/APRIL system a poten- tial therapeutic candidate for the treatment of type 1 diabetes. This study was undertaken to test the hypothesis From the Immunology and Inflammation Program, Garvan Institute of Medical that targeting the BAFF/APRIL system would have multi- Research, Darlinghurst, NSW, Australia. ple inhibitory effects on the spontaneous development of Corresponding author: Shane T. Grey, [email protected]. Received 31 October 2008 and accepted 16 March 2009. autoimmune diabetes in the NOD model. Published ahead of print at http://diabetes.diabetesjournals.org on 31 March 2009. DOI: 10.2337/db08-1504. © 2009 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, RESEARCH DESIGN AND METHODS and the work is not altered. See http://creativecommons.org/licenses/by C57BL/6, NOD.SCID, and NOD/Lt (NOD) mice were obtained from The Walter -nc-nd/3.0/ for details. and Eliza Hall Institute of Medical Research (WEHI) Kew, Melbourne, The costs of publication of this article were defrayed in part by the payment of page / / Australia. NOD.MT mice were provided by Dr. Serreze (18). BAFF , charges. This article must therefore be hereby marked “advertisement” in accordance / / with 18 U.S.C. Section 1734 solely to indicate this fact. BCMA , and TACI mice were provided by Dr. Susan Kalled (Biogen See accompanying commentary, p. 1479. Idec). BR3 mice were a gift from Dr. Rajewsky (19). All animal experiments 1568 DIABETES, VOL. 58, JULY 2009 ˜ E. MARINO AND ASSOCIATES were approved by the St. Vincent’s Campus Animal Experimentation and ** Ethics Committee. Diabetes incidence studies. NOD mice were administered BCMA-Fc (150 g per treatment) based on previous studies (20). BCMA-Fc is a fusion 0.8 protein—the extracellular portion of BCMA fused to the Fc domain of human IgG. BCMA-Fc was provided by Dr. Susan Kalled (Biogen Idec). Controls were treated with PBS or intravenous globulin (HuIvIg) (150 g). For adoptive 0.6 transfer studies, splenocytes (1  10 ) from pre-diabetic 16-week-old female NOD donors or BCMA-Fc–treated mice were transferred intravenously into NOD.SCID recipients. Glucose levels were monitored twice weekly from 10 0.4 weeks of age onward for BCMA-Fc–treated mice or starting with transfer of splenocytes; a blood glucose level 12.0 mmol/l on two consecutive readings was scored as indicative of diabetes. 0.2 Phenotypic analysis of mononuclear cells. Lymphocytes were isolated and analyzed by flow cytometry exactly as previously described (15). T-cell subpopulations were identified as follows: CD8a (Ly2)(53-6-7) and memory- high low effector cells CD44 CD62L and regulatory T-cells (Tregs) CD4 (L3T4) 20 30 40 50 (GK1.5), CD25 (7D4) and (PC61), and Foxp3 (Foxp3-staining kit; Time (weeks) eBioscience, San Diego, CA). Diabetogenic T-cell clones were identified based on expression of CD4 (H129.19) and CD40 (3/23) as previously described (21). B-cell subpopulations were identified exactly as previously FIG. 1. Administration of BCMA-Fc prevents diabetes in NOD mice. high  int Diabetes incidence was followed for NOD mice administered BCMA-Fc described (15): follicular B-cells (FoB) (CD23 , IgM , and CD21 ), MZBs low high high low (black line) (n  10), HuIvIg (gray line) (n  20), and PBS (broken (CD23 , IgM , and CD21 ), transitional type 1 (T1) cells (CD23 , high low high  line) (n  30) from 9 to 15 weeks of age. **P  0.0041 (Mantel-Cox IgM , and CD21 ), and transitional type 2 (T2MZ) cells (CD23 , IgM , log-rank analysis) for BCMA-Fc treatment vs. HuIvIg; P < 0.0001 for high and CD21 ). Isotype controls included IgG , ; IgG , ; IgG , ; and IgG , 1 1 2b 2a BCMA-Fc treatment vs. PBS. . Flow cytometric analysis was conducted on a FACScalibur flow cytometer (BD Biosciences, San Jose, CA). Cytokine analysis. Cytokine profile of sera samples was performed with a BCMA-Fc treatment was carried out by flow cytometry. LINCOplex mouse 9-plex cytokine kit from Linco Research (St. Charles, MO), Given the well-described requirement of BAFF in the following the manufacturer’s instructions. The assays were carried out at The regulation of steady-state B-cell homeostasis (1), we first University of New South Wales Inflammation Disease Unit in conjunction with examined B-cell populations. As shown in Fig. 2A, all three Taline Hampartzoumian. known BAFF and APRIL receptors were expressed by Histopathology. Formaldehyde-fixed, paraffin-embedded pancreata sections (5 m) were hematoxylin and eosin stained. Insulitis was scored (100  NOD IgM B220 B-cells, suggesting that NOD B-cells magnification) as follows: grade 0, no insulitis; grade 1, peri-insulitis; grade 2, would be sensitive to BAFF/APRIL blockade. Tracking insulitis involving 25% islet; grade 3, insulitis involving 25% islet; and grade IgM B220 cells in the blood during the course of 4, insulitis involving 75% and/or complete islet infiltration. Photos were BCMA-Fc and HuIvIg treatment revealed a steady reduc- taken using a Leica DC300 camera on a Leica DMRB microscope. tion in the peripheral B-cell frequency during the BCMA-Fc Anti-CD25 antibody treatment. Mice were administered the anti-CD25 treatment period, reaching a nadir at 4 weeks (Fig. 2B). monoclonal antibody (mAb) PC61 (200 g) (The Walter and Eliza Hall Institute of Medical Research [WEHI] mAb facility, Melbourne, Australia) fortnightly for a Further analysis conducted at the end of the 9- to 15-week total of four injections. Control mice received 200 g rat IgG  (BD Biosciences). treatment period demonstrated that BCMA-Fc treatment BCMA-Fc–treated NOD mice were first inoculated on the 16th week. reduced the frequencies of mature follicular and MZB NOD.MT mice were administered PC61 beginning at 16 weeks of age. The subsets, as well as the immature T2MZ cells in the spleen frequency of CD25 T-cells was determined by analysis of CD4 CD25 (mAb and pancreatic lymph node (PLN) (Fig. 2D). Similarly, the 7D4) Foxp3 cells. Diabetes incidence was followed as described above. absolute numbers of the follicular, marginal-zone, and Statistical analysis. Statistical significance for mononuclear cell analysis was determined by calculating P values using the Student’s t test (GraphPad Software, T2MZ subsets were reduced by 80 –90% (Fig. 2E). In San Diego, CA). Diabetes incidence studies were graphed as Kaplan-Meier contrast, the frequency and absolute numbers of T1 pre- survival plots and analyzed using the Mantel-Cox log-rank method with 2 degrees cursors in the spleen were less affected by BCMA-Fc of freedom (GraphPad Prism; GraphPad Software). P values represent compari- treatment (Fig. 2D and E), a result consistent with the role son between different treatments as indicated in the figure legends. of BAFF in promoting B-cell development after the T1 checkpoint (22). RESULTS Effect of BCMA-Fc treatment on peripheral T-cell Disrupting the BAFF/APRIL pathway in the preclini- populations and Th1 cytokines. In contrast to its effect cal phase prevents diabetes onset. To test the effect of on B-cell populations, administration of BCMA-Fc from 9 disrupting the BAFF/APRIL pathway before the onset of to 15 weeks of age did not impact the absolute number of hyperglycemia, we injected NOD mice with 150 g peripheral T-cells or CD4 and CD8 T-cell subsets (Fig. BCMA-Fc intraperitoneally (i.p.) twice weekly from 9 to 15 3A). However, the frequency of splenic CD4 and CD8 weeks of age (12 injections over a 6-week period); control T-cells was proportionally increased, presumably as a groups were administered PBS or HuIvIg (150 g i.p.) over result of the decreased number of B-cells (data not de- the same period (Fig. 1). We found that all NOD mice picted). To determine how BCMA-Fc treatment affected treated with PBS or HuIvIg from 9 to 15 weeks of age the activation of effector T-cell clones, we analyzed the developed diabetes with the expected high frequencies. expression of CD44 and CD40 on peripheral CD4 and CD8 There were no significant differences in diabetes incidence T-cell populations. CD44 is expressed by activated T-cells, between PBS- and HuIvIg-treated groups (P 0.1309; n  whereas CD40 has been identified as a marker for highly 10). In contrast, we found that NOD mice treated with diabetogenic T-cell clones (21). BCMA-Fc treatment did high  high BCMA-Fc from 9 to 15 weeks of age were completely not alter the frequency of CD44 CD4 or CD44 CD8 protected from diabetes (diabetes incidence 0 of 10 at 50 T-cells (data not depicted); however, the frequency of weeks of age; P 0.0041, n  10, log-rank vs. HuIvIg). CD4 and CD8 CD40 T-cells was reduced in both the Effect of BCMA-Fc treatment on peripheral B-cell spleen and PLN of BCMA-Fc–treated mice (Fig. 3B). The populations. Examination of peripheral lymphoid popu- reduction in frequency of pathogenic CD40 T-cells in lations before and at the cessation of the 9- to 15-week BCMA-Fc–treated mice was associated with a decrease in DIABETES, VOL. 58, JULY 2009 1569 % of Normoglycemic mice TARGETING THE BAFF/APRIL PATHWAY PREVENTS DIABETES A B C 50 Gating strategy 40 T2MZ BCMA BR3 TACI FoB CD23hi CD23lo MZB FSC T1 12 3 4 56 7 89 Time (weeks of treatment) IgM SPLN PLN PBS HuIvIg BCMA-Fc PBS HuIvIg BCMA-Fc *** T2MZ 4.83% 7% 0.95% T2MZ 1.73% 0.7% 0.48% 20% FoB 28% 23.25% ***3.6% ***3% FoB 20% MZB 2.22% 1.9% MZB 13.7% 12% *** 0.64% ***6.32% T1 T1 0.9% 1.18% 1.2% 4.4% 3% 3.58% IgM IgM SPLN PLN FoB cells MZB cells FoB cells MZB cells 2 0.5 45 45 *** *** *** *** *** 0.4 1.5 *** 30 30 0.3 0.2 15 15 0.5 0.1 0 0 0 0 T2MZ cells T1 cells T2MZ cells T1 cells 0.5 0.5 16 16 *** * *** ** 0.4 0.4 12 12 0.3 0.3 8 8 0.2 0.2 4 4 0.1 0.1 0 0 0 FIG. 2. Effect of BCMA-Fc treatment on peripheral B-cells. A: Expression of BCMA, BR3, and TACI (black line) on IgM B220 NOD splenocytes. Gray line, isotype control. Representative fluorescence-activated cell sorting plots are shown. B: Frequency of IgM B220 cells in peripheral blood of NOD mice treated with BCMA-Fc from 9 to 15 weeks of age (E)(n  5) and HuIvIg control mice (O)(n  5). Time indicates period post–first injection. C: Gating strategy used for identification of B-cell subsets. D: Representative fluorescence-activated cell sorting plots illustrating frequency of B-cell subsets in the spleen (SPLN) and PLN from 16-week-old NOD mice treated with PBS, HuIvIg, or BCMA-Fc from 9 to 15 weeks of age. Numbers represent percentage of total lymphocytes. E: Absolute numbers, calculated from values in D, of B-cell subsets in the spleen and PLN from 16-week-old NOD mice treated with PBS (F), HuIvIg (O), or BCMA-Fc (E). Values from individual mice are shown (n > 8 per group). The bar represents median value. *P < 0.05, **P < 0.01, and ***P < 0.001. FSC, forward light scatter. the circulating levels of interleukin (IL)-7, IL-15, and IL-17 Effect of BCMA-Fc treatment on B-cell repopulation. (Fig. 3C), cytokines critical for the expansion and activa- To examine how BCMA-Fc treatment effected B-cell re- tion of effector T-cells. population, further analysis was carried out in long-term 1570 DIABETES, VOL. 58, JULY 2009 6 6 Cell number (x10 ) Cell number (x10 ) CD21 % IgM+B220+ 6 6 Cell number (x10 ) Cell number (x10 ) CD21 CD23 CD21 ˜ E. MARINO AND ASSOCIATES A B SPLN PLN SPLN CD4+ T cells CD4+ T cells 60 4 NOD HuIvIg BCMA-Fc9-15 100 30% 32% 100 100 1.5% 40 80 80 80 CD4 60 60 (H129.19) 40 40 20 20 0 0 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 100 35% 100 40% 100 3% CD8+ T cells CD8+ T cells 60 4 80 80 80 60 60 60 CD8 3 40 40 40 20 20 20 0 0 0 2 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 CD40 PLN 0 0 NOD HuIvIg BCMA-Fc9-15 100 40% 100 35% 100 1% IL-2 IL-4 IL-7 IL-10 20 10 200 100 80 80 80 *** CD4 60 60 60 (H129.19) 8 80 40 40 40 15 150 20 20 20 6 60 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 10 100 010 10 10 10 010 10 10 10 010 10 10 10 4 40 100 25% 100 38% 100 0.83% 5 50 2 20 80 80 80 60 60 60 CD8 0 0 0 0 40 40 40 20 20 20 IL-12 IL-15 IL-17 TNF-a 300 800 60 0 0 0 * 2 3 4 5 2 3 4 5 2 3 4 5 *** 010 10 10 10 010 10 10 10 010 10 10 10 CD40 200 40 100 20 0 0 0 FIG. 3. Effect of BCMA-Fc on peripheral T-cells and cytokines. A: Absolute number of CD4 (upper panel) and CD8 (lower panel) T-cells from 16-week-old NOD mice treated with PBS (F), HuIvIg (O), or BCMA-Fc (E) from 9 to 15 weeks of age. Values from individual mice are shown (n > 8 per group). B: Representative fluorescence-activated cell sorting plots illustrating frequency of CD4 CD40 (upper panel) and CD8 CD40 (lower panel) T-cells from treated mice at 16 weeks of age. Black line, CD40; gray line, isotype control (n  7 per group). C: Serum cytokine levels in HuIvIg- (O) and BCMA-Fc– (E) treated NOD mice at 16 weeks of age. Values from individual mice are shown (n > 3 per group). Significant differences between sample means are indicated. Bar represents median value. *P < 0.05 and ***P < 0.001. SPLN, spleen. surviving (50 weeks of age) BCMA-Fc–treated mice. Effect of BCMA-Fc treatment on the pancreatic BCMA-Fc–protected mice exhibited normal frequencies infiltrate. We conducted histological analysis of the pan- and absolute numbers of FoB, MZB, T2MZ, and T1 B-cell creata from the BCMA-Fc–protected NOD mice at 16 and subsets in the periphery compared with those in HuIvIg- 50 weeks of age and a comparison with treated control treated control mice (Fig. 4A and B). Although the PLN of mice. Representative histology for each group is shown BCMA-Fc–treated mice harbored frequencies and num- in Fig. 5A, and insulitis scores for these mice are shown in bers of FoB and MZB cells similar to those of control mice, Fig. 5C. Although BCMA-Fc–treated NOD mice did exhibit an increase in both the frequency and the number of T2MZ insulitis at the 16-week time point, the severity was and T1 cells was observed (Fig. 4A and B). These data reduced compared with that in HuIvIg-treated and diabetic demonstrate that BCMA-Fc–treated NOD mice could re- control NOD mice at 16 weeks. Indeed, the frequency of populate their mature B-cell pool. These data also demon- islets exhibiting heavy insulitis (grades 3 and 4) at 16 strate that BCMA-Fc treatment provides long-term weeks was only 20 vs. 45–50% in control groups. Flow protection from diabetes in NOD mice despite the return cytometric analysis of the pancreatic infiltrate revealed of B-cell populations. that the frequency of FoB was markedly reduced (P DIABETES, VOL. 58, JULY 2009 1571 Concentration Concentration 6 6 Cell number (x10 ) Cell number (x10 ) (pg/ml) (pg/ml) TARGETING THE BAFF/APRIL PATHWAY PREVENTS DIABETES SPLN A B HuIvIg BCMA-Fc SPLN SPLN PLN PLN T2MZ 6.6% 5% FoB cells FoB cells T2MZ cells T2MZ cells 80 0.8 0.075 FoB 22% 27% 60 0.6 0.05 16% MZB17% 40 0.4 5 0.025 T1 3% 0.2 3.6% IgM 0 0 0 0 PLN MZB cells MZB cells T1 cells T1 cells 80 0.075 15 0.075 1.9% 0.8% 0.05 10 0.05 25% 23% 0.025 5 0.7% 0.025 1.5% 0.4% 0 0 2% 0 IgM FIG. 4. Effect of BCMA-Fc treatment on B-cell repopulation. A: Representative fluorescence-activated cell sorting plots illustrating frequency of B-cell subsets from NOD mice treated with HuIvIg at 30 weeks of age or BCMA-Fc at >50 weeks of age. Numbers represent percentage of total lymphocytes. B: Absolute numbers of B-cell subsets from HuIvIg- (O) and BCMA-Fc– (E) treated NOD mice. Values calculated from A. Results from individual mice are shown (n > 3 per group). Bar represents median value. *P < 0.05 and **P < 0.01. SPLN, spleen. 0.014; n  5) in the BCMA-Fc–treated mice at 16 weeks Interestingly, 50% (4 of 8) of NOD.SCID mice receiving (Fig. 5D). No changes were observed in the ratios of CD4 splenocytes from BCMA-Fc–treated NOD mice did de- to CD8 T-cells, though their frequencies were increased velop hyperglycemia (P 0.088, NOD vs. BCMA-Fc– (Fig. 5D), again, most probably as a result of the decrease treated splenocytes; n 8). These data demonstrate that in FoB. BCMA-Fc–treated mice still harbored T-cells with self- The proportion of severely infiltrated islets in the BCMA- reactive potential, but these T-cells are unable to precipi- Fc–treated mice did not increase over time, as evidenced by tate diabetes in their BCMA-Fc–treated hosts. analysis of the long-term–protected mice (e.g., 50-week-old Time-dependent accumulation of CD4 CD25 T-cells mice) (Fig. 5A and C). Thus, although BCMA-Fc–treated mice in BCMA-Fc–treated NOD mice. The long-term protec- did exhibit evident insulitis, the severity of insulitis was tion afforded by BCMA-Fc treatment despite the persis- maintained at a level equivalent to that exhibited by prehy- tence of self-reactive T-cells prompted us to investigate perglycemic 8- to 15-week-old NOD mice. possible regulatory mechanisms. Tregs that express the Given that NOD mice exhibit a progressive insulitis from markers CD4 and CD25 can control the progression to the preclinical to hyperglycemic phase, these data suggest overt diabetes in the NOD model (23). We analyzed the that BCMA-Fc treatment has not reversed the autoimmune frequency and number of CD4 CD25 T-cells in the process as determined by the persistence of a mononu- spleen, PLN, and pancreas of BCMA-Fc–treated NOD mice clear cell pancreatic infiltrate but, rather, halted the pro- at 16 weeks of age. As shown in Fig. 6A and B, compared gression to fulminant diabetes. Thus, we questioned with treated control NOD mice, 9- to 15-week BCMA-Fc– whether this related to a change in the nature of the treated mice harbored an increased frequency of splenic insulitic lesion or active regulation. Analysis of the B- and CD4 CD25 T-cells at 16 weeks of age. The majority T-cell subsets infiltrating the pancreas in the protected (90%) of these CD4 CD25 T-cells were also Foxp3 BCMA-Fc–treated mice showed that the frequencies of (Fig. 6C and D), demonstrating that they belonged to the infiltrating B- and T-cell subsets were similar to those of set of Tregs. Analysis of the long-term (50 weeks of age) treated control mice (Fig. 5D). Thus, in long-term–pro- BCMA-Fc–protected mice revealed increased (twofold or tected mice, B-cells are not prohibited from forming a part more) frequencies of CD4 CD25 T-cells in both the of the insulitic lesion. To test whether the BCMA-Fc– spleen and PLN (Fig. 6E). Further, in contrast with the protected NOD mice still harbored T-cells with self-reac- 16-week time point, the increased frequency of Tregs was tive potential, splenocytes from normoglycemic BCMA- also reflected as an increase in the absolute numbers of Fc–treated NOD mice were adoptively transferred into CD4 CD25 T-cells in the spleen and PLN (Fig. 6E). Thus, NOD.SCID recipients (Fig. 5E). As a positive control, other BCMA-Fc–treated mice showed an accumulation of Treg groups received splenocytes from pre-diabetic 16-week- numbers over time. The increased number of Tregs was old mice. We found that 90% (7 of 8) NOD.SCID mice associated with a halting of the autoimmune attack and receiving these control splenocytes developed diabetes. permanent euglycemia. 1572 DIABETES, VOL. 58, JULY 2009 CD21 CD21 6 6 Cell number (x10 ) Cell number (x10 ) 6 6 Cell number (x10 ) Cell number (x10 ) ˜ E. MARINO AND ASSOCIATES A B BCMA-Fc BCMA-Fc NOD. µMT-/- 16-week-old >50-week-old 6-16-week-old ** ** HuIvIg PBS 16-week-old 16-week-old 16 wks-old NOD mice >50 wks-old NOD mice PBS HuIvIg BCMA-Fc HuIvIg BCMA-Fc 0.63% T2MZ 0.88% 0.5% T2MZ 0.63% 0.6% FoB 5.8% 5.8% 9.62% FoB B cells 7.3% 3.51% MZB 1.17% MZB 1.3% 3.7% 2.2% 4.15% T1 T1 1.96% 2.4% 3.4% 1.96% 1.6% IgM IgM 5.46% 9.64% 11% 9% 20% T cells *** 4.87% 2.41% 4% 3% 8% CD8 CD8 FIG. 5. Histology and insulitis scores of BCMA-Fc–treated NOD mice. A: Representative histological section of pancreas from normoglycemic mice treated with BCMA-Fc from 9 to 15 weeks at 16 or >50 weeks of age is shown (upper panels). Sections 0.8 from HuIvIg- and PBS-treated mice at 16 weeks are also shown (lower panels). B: Representative pancreatic sections from an NOD.MT mouse. C: Insulitis scores for treated mice; 0.6 20 –70 islets were scored from four to seven mice per group. Differences in insulitis scores for BCMA-Fc–treated mice at 16 and >50 weeks were significant (**P < 0.01). P values resulted 0.4 comparing insulitis level at grade 4 among HuIvIg control mice. D: Representative fluorescence-activated cell sorting plots illus- 0.2 trating frequency of pancreatic B-cell (upper panel) and T-cell (lower panel) subsets in treated NOD mice at 16 and >50 weeks of age (n > 7 per group). E: Splenocytes (1  10 ) from >50-week-old normoglycemic BCMA-Fc–treated NOD mice 010 20 30 40 50 60 70 (black line) (n  8) and pre-diabetic 16-week-old NOD mice Time (days) (gray line) (n  8) were adoptively transferred (intravenously) to NOD.SCID recipients. Diabetes incidence was then followed over time. P  0.08 (Mantel-Cox log-rank analysis) for BCMA-Fc–treated vs. NOD islets. (A high-quality digital representation of this figure is available in the online issue.) BCMA-Fc–mediated protection from diabetes requires achieve this, mice were administered the anti-CD25 mAb the presence of CD4 CD25 T-cells. We next de- PC61. Control mice received PBS or rat IgG . In prelim- termined whether CD4 CD25 T-cells were required inary experiments, we could show that administration for BCMA-Fc–mediated protection from diabetes. To of a single dose of PC61 (200 g i.p.) induced an DIABETES, VOL. 58, JULY 2009 1573 NOD 8-15 wks-old NOD hyperglycemic BCMA 16 wk-old HuIvIg 16 wks-old BCMA >50 wk-old NOD.µMT-/-6-16wks- old % of Normoglycemic mice CD4 CD21 % of islets CD2 1 CD4 TARGETING THE BAFF/APRIL PATHWAY PREVENTS DIABETES A HuIvIg C PBS BCMA-Fc PBS HuIvIg BCMA-Fc 4 4 4 4 4 4 10 10 10 10 10 8% 5.1% 7.5% 4.5% 15% 6.28% 13.4% 9.03% 10% 3 3 3 3 3 10 10 10 10 10 SPLN 2 2 2 2 2 10 10 10 10 10 1 1 1 1 1 10 10 10 10 10 1.26% 1.82% 1.4% 0 0 0 0 0 10 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4 4 4 10 10 10 CD25 10% 13% 10.5% 3 3 3 10 10 10 PLN SPLN 2 2 2 10 10 10 1 1 1 *** 10 10 10 *** 0 0 0 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4 4 4 10 10 10 9.8% 9.3% 10.6% 3 3 3 10 10 10 PANC 2 2 2 10 10 10 1 1 1 10 10 10 1 0 0 0 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 CD 4 SPLN PLN B SPLN PLN E *** *** 60 30 20 50 *** 50 *** 0 0 ** *** 14 0.35 14 ** 0.35 ** 12 12 0.25 10 10 0.25 8 8 0.15 6 6 0.15 4 4 0.05 2 0.05 0 0 0 FIG. 6. CD4 CD25 T-cells in BCMA-FC–treated NOD mice. A: Representative fluorescence-activated cell sorting plot illustrating frequency of CD25-expressing CD4 T-cells from 16-week-old PBS-, HuIvIg-, and BCMA-Fc–treated NOD mice (n > 7 per group) is shown. B: Pooled data showing frequencies and calculated absolute numbers of CD25-expressing CD4 T-cells of PBS- (F), HuIvIg- (O), or BCMA-Fc– (E) treated NOD mice at 16 weeks. Values from individual mice are shown (n > 7 per group). Bar represents median value. C: Representative fluorescence-activated cell sorting plots illustrating frequency of CD4 CD25 Foxp3 cells from 16-week-old PBS-, HuIvIg-, and BCMA-Fc–treated NOD mice. D: Absolute numbers, calculated from C,ofCD4 CD25 Foxp3 cells from PBS- (F), HuIvIg- (O), and BCMA-Fc– (E) treated NOD mice. Values from individual mice are shown. Bar represents median value. E: Cumulative data showing frequency and calculated absolute numbers of CD25-expressing CD4 T-cells from HuIvIg- (O) or BCMA-Fc– (E) treated NOD mice (n > 3 per group). Bar indicates median value. **P < 0.01 and ***P < 0.001. PANC, pancreas; SPLN, spleen. 90% reduction in the frequency of peripheral NOD mice developed hyperglycemia with the expected CD4 CD25 Foxp3 T-cells for 14 days (Fig. 7A). For the frequency (Fig. 7B). In contrast, subsequent administra- experiment, NOD mice were first treated with BCMA-Fc tion of PC61 after BCMA-Fc treatment precipitated diabe- from 9 to 15 weeks of age; at the cessation of BCMA-Fc tes in 100% of mice (P 0.001 BCMA-Fc vs. BCMA-Fc plus treatment, mice were administered the anti-CD25 mAb PC61; n  5). PC61 (200 g i.p.) every 14 days (a total of four injections), Blocking the BAFF/APRIL pathway indirectly modu- and blood glucose levels were monitored. Kaplan-Meier lates CD4 CD25 Foxp3 T-cells. We next focused on survival analysis showed that BCMA-Fc–treated NOD mice determining the mechanism by which BCMA-Fc treatment remained diabetes free, whereas control PC61-treated would effect protection in a CD4 CD25 T-cell– depen- 1574 DIABETES, VOL. 58, JULY 2009 Cell number (x10 ) % CD25-expressing CD25-expressing CD 4 CD25 cells CD4 T cells Foxp3 6 6 Cell number (x10 ) Cell number (x10 ) % CD25-expressing CD25-expressing CD 4 CD25+Foxp3-expressing T cells CD4 T cells CD4 T cells ˜ E. MARINO AND ASSOCIATES 15 15 10 10 5 5 0 0 B CD25-expressing B Cells (B220) CD4+ T cells CD4 T cells BR3 TACI 0.8 FIG. 7. CD25 T-cells are required for BCMA-Fc–mediated protection 0.6 from diabetes. A: Frequency of CD4-expressing CD25 (mAb 7D4) Foxp3 T-cells in peripheral blood of 16-week-old NOD (upper panel) and NOD.MT (lower panel) mice treated with PC61 or isotype control 0.4 (rat IgG , ). Representative fluorescence-activated cell sorting plot is shown (n > 4 per group). B: Diabetes incidence was followed for NOD mice administered BCMA-Fc from 9 to 15 weeks of age (solid black line) 0.2 (n  5), PC61 (broken line) (n  5), or BCMA-Fc from 9 to 15 weeks of age plus PC61 (solid gray line) (n  8). **P  0.001 (Mantel-Cox log-rank analysis) for BCMA-Fc alone vs. BCMA-Fc plus PC61 treatment. 10 40 20 50 30 Time (weeks) dent manner. To examine whether disrupted BAFF or FIG. 8. CD25 T-cells are required for diabetes resistance in B-cell– APRIL signaling would engender an accumulation of deficient NOD mice. A: Left plot: frequencies of splenic CD4 CD25 Foxp3 T-cells in C57BL/6 (F) and NOD (E) mice. Right CD4 CD25 Foxp3 T-cell numbers over time, we exam- plot: frequencies of splenic CD4 CD25 Foxp3 T-cells in BAFF ined the frequency of CD4 CD25 Foxp3 T-cells in mice / / / (F), BR3 (), TACI (E), and BCMA (‚) mice. Values from deficient for BAFF or the BAFF and APRIL receptors individual mice are shown (n > 4 per group). Bar represents median value. Differences are not significant (P > 0.05). B: Expression of BR3 BCMA and TACI, as well as the BAFF receptor BR3. and TACI (black line) on C57BL/6 splenic, B220 B-cells, CD4 T-cells, Though these mutations were on a C57BL/6 background, and CD25-expressing CD4 T-cells. Gray line, isotype control. Repre- we believe that the analysis is valid given that the median sentative fluorescence-activated cell sorting plots are shown. C: Dia- betes incidence was followed for NOD.MT mice (black line) (n frequency of CD4 CD25 Foxp3 T-cells was similar be- 5) and NOD.MT mice administered PC61 (solid gray line) (n  10). tween NOD and C57BL/6 mice (P 0.3513; n  7): 9% *P  0.0181 (Mantel-Cox log-rank analysis) for control vs. PC61 for both strains (Fig. 8A). Also shown in Fig. 8A, the treatment. frequency of peripheral CD4 CD25 Foxp3 T-cells in / / / / BAFF , BR3 , BCMA , and TACI mice was com- parable with that in wild-type mice. Further, to determine relationship between a reduction in B-cells and CD4 CD25 whether BAFF or APRIL could engage Tregs directly, we T-cells, we used B-cell– deficient NOD.MT mice (18). examined TACI and BR3 expression in CD4 CD25 T- NOD.MT mice exhibit a number of features, with regard cells. CD4 CD25 T-cells did not express significant levels to diabetes development, reminiscent of those in BCMA-Fc– of the receptors BR3 or TACI (Fig. 8B) in contrast to treated mice. These include a reduced degree of insulitis (Fig. splenic B-cells. These data indicate that it is unlikely that 5B and C) and resistance to diabetes development (Fig. 8C). targeting the BAFF/APRIL system resulted in increased To assess whether CD4 CD25 T-cells were important in CD4 CD25 Foxp3 T-cells via a direct mechanism. maintaining euglycemia in NOD.MT mice, 16-week-old CD4 CD25 T-cells reign in destructive T-cells in mice were treated with PC61 (200 g i.p. each) every 14 days B-cell– deficient NOD mice. B-cell subpopulations were for a total of four injections and blood glucose levels were severely reduced by BCMA-Fc treatment. To explore the followed. This treatment reduced the frequency of peripheral DIABETES, VOL. 58, JULY 2009 1575 % of CD25+Foxp3-expressing CD4 T cells % of Normoglycemic mice % of CD25+Foxp3-expressing CD4 T cells TARGETING THE BAFF/APRIL PATHWAY PREVENTS DIABETES CD4 CD25 Foxp3 T-cells as observed for B-cell–sufficient reduced incidence of diabetes. Together with our study NOD mice (Fig. 7A). Treatment with PC61 precipitated results, these data indicate that reducing B-cells in auto- diabetes in 80% (P 0.0184; n 10) of NOD.MT mice immune NOD mice is associated with an increased fre- (Fig. 8C)—a dramatic result when compared with data in quency of cells with a Treg phenotype. Significantly, our control NOD.MT mice. Therefore, targeting CD25 reg- present data demonstrate that in the absence of B-cells, ulatory populations triggered diabetes onset in B-cell– defi- endogenous Tregs control the progression of autoimmu- cient NOD.MT mice that are otherwise resistant. nity in NOD mice. This conclusion is further supported by our analysis of NOD.MT mice that lack B-cells and are resistant to diabetes (18). Treatment of NOD.MT mice DISCUSSION with the anti-CD25 mAb PC61 precipitated the onset of diabetes, demonstrating that the hyperglycemia-free state Emerging evidence demonstrates that B-cells can impact multiple stages in the pathogenesis of autoimmune diabe- in NOD.MT mice is also dependent on CD25 regula- tory cells. tes (24). BAFF and APRIL play critical roles in supporting B-cell survival (1), such that BCMA-Fc–treated mice har- These are, to our knowledge, the first results to show that the resistance of B-cell– deficient NOD mice to diabetes can bored a much reduced B-cell pool, providing one potential mechanism by which BCMA-Fc prevented diabetes in be due to the activity of Tregs. However, they are consistent with emerging results from other models of autoimmune NOD mice. BAFF-activated B-cells show an enhanced antigen-presenting cell (APC) capacity (25), and in vivo disease. Indeed, NOD.H-2h4 mice develop spontaneous au- toimmune thyroiditis, whereas B-cell– deficient NOD.H-2h4 BAFF-activated B-cells facilitate heightened Th1 T-cell responses (26). In the NOD model, B-cells undergo a mice are protected from autoimmunity (38). Treatment of B-cell– deficient NOD.H-2h4 with PC61 induced the rapid marked expansion from 9 to 15 weeks of age (15), show an increased capacity to act as APCs during this time onset of autoimmunity (39), similar to the results obtained in NOD.MT mice, suggesting a common mechanism (15,27), and can present captured autoantigen to self- reactive T-cells (15,28,29). The APC function of B-cells is whereby the absence of B-cells allows a regulatory pathway to prevent autoimmunity. A number of possibilities exist to essential for the activation (30) and expansion (31) of the CD4 T-cell repertoire from 9 to 15 weeks of age, i.e., explain this intriguing relationship between B-cells and Tregs. As an example, Olson et al. (40) using a model of before the onset of hyperglycemia. Thus, by reducing the availability of B-cells to act as APCs, BCMA-Fc treatment Crohn’s disease demonstrated that B-cells could block Treg activity in a glucocorticoid-induced tumor necrosis factor curtailed B- and T-cell interactions during the critical 9- to 15-week time period, halting the progression from clini- receptor (GITR)-dependent manner. Thus, activated NOD B-cells may directly suppress the activity of Tregs through cally silent insulitis to overt hyperglycemia. This concept is consistent with our observation that BCMA-Fc–treated mechanisms including expression of GITR ligand (40). Alter- natively, because the number of B-cells increases as disease mice exhibited a reduced proportion of B-cells in associ- ation with a diminished frequency of CD4 CD40 diabe- progresses (15), the opportunity for T-cells to engage activat- ing APCs (e.g., B-cells) (30,31) versus a tolerizing APC (e.g., togenic T-cells and a reduced level of Th1 T-cell– derived cytokines at 16 weeks of age. Additional evidence to macrophages or dendritic cells) (41) may decrease. In this scenario, BCMA-Fc treatment could be protective via two support this hypothesis comes from studies in which NOD mice expressing human CD20 have been treated with an potential mechanisms. First, BCMA-Fc, by reducing B-cells, may alter the balance in the production of effector cells anti-CD20 mAb (32); in this case, reducing B-cells from 9 weeks of age delays diabetes onset. Depletion of B-cells versus Tregs, leading to an accumulation of CD4 CD25 Tregs. Second, without B-cells to expand up self-reactive with anti-CD20 impairs activation of adaptive and autore- active CD4 T-cell responses (32,33), further demonstrat- T-cell clones, endogenous regulatory cells may simply be able to reign in the present autoreactive T-cell pool, prevent- ing the required role for B-cells in the activation of self-reactive CD4 T-cells (30,34). ing the progression to hyperglycemia. These hypotheses should be explored in further studies. Our studies uncover an additional and perhaps unex- pected mechanism by which targeting the BAFF and In this study, we demonstrate that targeting the BAFF and APRIL pathway with BCMA-Fc before the onset of hypergly- APRIL system can prevent diabetes, namely through in- creasing CD4 CD25 T-cells. Tregs expressing CD25 and cemia prevented diabetes in spontaneously diabetic NOD mice. We hypothesize that the major action achieved by Foxp3 can control the development of autoimmune diabe- tes (35), and we found that CD4 CD25 T-cells were blocking BAFF and APRIL was a reduction in peripheral B-cells, thereby limiting their involvement in diabetes patho- required for the continued maintenance of a hyperglyce- mia-free state. Further, these CD4 CD25 T-cells ex- genesis. This prevented diabetes progression through two mechanisms: a dampening of T-cell autoimmune activity and pressed the Treg lineage marker Foxp3, suggesting that they belonged to the set of natural Tregs (36). To address subsequent elaboration of cytokines, most likely achieved by decreasing the availability of B-cells to act as APCs; a second the question of how BCMA-Fc might affect CD4 CD25 T-cell homeostasis, we examined Foxp3 Treg frequencies mechanism also related to decreased B-cells, which in- duced or allowed Tregs to reign in the autoreactive in mice in which BAFF and APRIL signaling were dis- rupted. These data demonstrated that loss of BAFF or potential of diabetogenic T-cells. This second mecha- nism highlights a novel pathway by which targeting APRIL did not increase the frequency of Foxp3 Tregs per se, suggesting that BCMA-Fc altered Treg homeostasis B-cells may provide resistance to autoimmunity. through an indirect mechanism. Of interest, an increased frequency of Tregs was demonstrated in two recent stud- ACKNOWLEDGMENTS ies in which NOD mice were treated with the B-cell depleting agents: anti-CD20 or anti-CD22 mAb, respec- This study was supported by a grant from the Juvenile tively (32,37). Both studies also reported that NOD mice Diabetes Research Foundation International (5-2005-1132) treated between 9 and 15 weeks exhibited a delayed and to S.T.G. E.M. is supported by a National Health and 1576 DIABETES, VOL. 58, JULY 2009 ˜ E. MARINO AND ASSOCIATES 20. Pelletier M, Thompson JS, Qian F, Bixler SA, Gong D, Cachero T, Gilbride Medical Research Council Dora Lush Fellowship. S.T.G. is K, Day E, Zafari M, Benjamin C, Gorelik L, Whitty A, Kalled SL, Ambrose supported by a BioFirst Award from the Office for Science C, Hsu YM. Comparison of soluble decoy IgG fusion proteins of BAFF-R and Medical Research, NSW. and BCMA as antagonists for BAFF. 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Journal

Diabetes – Pubmed Central

Published: Mar 31, 2009

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