Correction

IMMUNOLOGY Correction for “Anti–IL-7 -α reverses established type 1 diabetes in nonobese diabetic mice by modulating effector T-cell function,” by Li-Fen Lee, Kathryn Logronio, Guang Huan Tu, Wenwu Zhai, Irene Ni, Li Mei, Jeanette Dilley, Jessica Yu, Arvind Rajpal, Colleen Brown, Charles Appah, Sherman Mi- chael Chin, Bora Han, Timothy Affolter, and John C. Lin, which appeared in issue 31, July 31, 2012, of Proc Natl Acad Sci USA (109:12674–12679; first published June 25, 2012; 10.1073/ pnas.1203795109). The authors note that their conflict of interest statement was omitted during publication. The authors declare that all the authors are full-time employees of Pfizer Inc. Pfizer Inc. filed a patent application, with L.F.-L., J.C.L., and W.Z. as co- inventors: US Application Serial No. 13/033,491, entitled “Antagonist Anti-IL-7 Receptor Antibodies and Methods.”

www.pnas.org/cgi/doi/10.1073/pnas.1214896109 CORRECTION

www.pnas.org PNAS | October 2, 2012 | vol. 109 | no. 40 | 16393 Downloaded by guest on September 23, 2021 Anti–IL-7 receptor-α reverses established type 1 diabetes in nonobese diabetic mice by modulating effector T-cell function

Li-Fen Leea,1, Kathryn Logronioa, Guang Huan Tua, Wenwu Zhaia, Irene Nia, Li Meia, Jeanette Dilleya, Jessica Yua, Arvind Rajpala, Colleen Browna, Charles Appaha, Sherman Michael Chin a, Bora Hanb,TimothyAffolterb, and John C. Lina,1 aRinat, Pfizer Inc., South San Francisco, CA 94080; and bDrug Safety R and D, Pfizer Inc., La Jolla, CA 92121

Edited by Lewis L. Lanier, University of California, San Francisco, CA, and approved May 28, 2012 (received for review March 7, 2012) Genetic variation in the IL-7 receptor-α (IL-7R) is associated influence the development of the IFN-γ–expressing CD8+ with susceptibility to human type 1 diabetes (T1D). Here we in- TC1 cells. vestigate the therapeutic efficacy and mechanism of IL-7Rα anti- The activities of CD4+ and CD8+ T effector cells (Teffs) are body in a mouse model of T1D. IL-7Rα antibody induces durable, normally under stringent control by a number of negative regu- complete remission in newly onset diabetic mice after only two lators expressed on their surface. One major negative regulator, to three injections. IL-7 increases, whereas IL-7Rα antibody therapy Programmed Death 1 (PD-1) interacts with its (PD-) + + reduces, the IFN-γ–producing CD4 (TH1) and IFN-γ–producing CD8 (27) to maintain the robust long-term tolerance of Teffs in the T cells. Conversely, IL-7 decreases and IL-7Rα antibody enhances the inflamed tissue (28–30). IL-7 was recently shown to down-regu- inhibitory receptor Programmed Death 1 (PD-1) expression in the late PD-1 in a murine viral infection model (31). It is thus per- effector T cells. Programmed Death 1 blockade reversed the immune tinent to ask whether IL-7 may contribute to the PD-1 down- tolerance mediated by the IL-7Rα antibody therapy. Furthermore, IL- regulation in the NOD mice. α 7Rα antibody therapy increases the frequency of regulatory T cells In this study we investigate how the IL-7/IL-7R pathway contributes to the development of T1D in the NOD mouse without affecting their suppressor activity. The durable efficacy and α IMMUNOLOGY the multipronged tolerogenic mechanisms of IL-7Rα antibody ther- model by using IL-7R blocking antibodies. We also elucidate apy suggest a unique disease-modifying approach to T1D. the cellular and molecular mechanisms that underlie the prom- ising therapeutic efficacy of IL-7Rα antibody therapy.

T cell depletion | programmed death ligand 1 | biologics | adoptive transfer Results IL-7Rα Antibody Treatment Showed Efficacy in the Prevention of ype I diabetes (T1D) in both humans and animal models, Diabetes. To study the role of IL-7/IL-7Rα pathway in T1D, fe- Tsuch as nonobese diabetic (NOD) mice, is a complex, mul- male NOD mice were given either 10 mg/kg of control IgG tifactorial autoimmune disease in which the islet-specific T-cell (mIgG2a or rat IgG1) or an anti–IL-7Rα, 28G9 (of rat IgG1 immune response destroys insulin-producing β-cells in the islets isotype or rIgG1), or 28G9-mIgG2a in which the Fc portion of of Langerhans (1–4). At the clinical onset of diabetes, some the original rIgG1 clone is replaced with that of mouse IgG2a residual β-cells still produce insulin, offering a potential window (Table S1). In the prophylactic treatment paradigm, we admin- for therapeutic intervention to stop the autoimmune destruction istered these different antibodies to NOD mice once weekly and preserve β-cell function (5). from 9 wk of age till the end of the study. We found that 100% of MHC class II are the major genetic loci determining the NOD mice were prevented from diabetes by 28G9-mIgG2a, 67% susceptibility of T1D in human and NOD mice, although MHC (six of nine) by 28G9-rIgG1 compared with 0–20% of mice class II genes alone cannot fully account for genetic pre- treated with rat IgG1 isotype or mouse IgG2a isotype controls, disposition to T1D (6, 7). Recently, a SNP in the IL-7 receptor respectively (Fig. 1A). We also observed a pronounced dose- (IL-7Rα) gene was identified as one of the non-MHC–linked loci dependent effect of 28G9-rIgG1 in the prophylactic paradigm associated with risk of multiple sclerosis (8–10) and T1D (11, (Fig. S1 A–C). 12). IL-7 is a major survival factor implicated in mouse and Histological examination of mice at the end of the pro- human immune homeostasis and disorders (13). IL-7 is essential phylactic experiment revealed that pancreatic islets were heavily for the homeostatic proliferation of naïve T cells and also con- infiltrated by T cells in control IgG-treated mice, but less in those + tributes to that of CD8 memory T cells in mice (14, 15). Pro- treated with 28G9-mIgG2a or with 28G9-rIgG1 (Fig. 1 B and C). vision of exogenous IL-7 or lymphopenia-induced production of Moreover, insulin staining in the islets of the 28G9-mIgG2a– IL-7 can promote the expansion of self-reactive T-cell clones treated mice was significantly higher than that in the control IgG- (16). The IL-7 receptor is composed of two subunits: the com- treated group (Fig. 1D). mon γ-chain and the IL-7Rα chain. In humans, IL-7Rα de- In the of NOD mice, CD4+ and CD8+ T cells ficiency results in the absence of T cells but B-cell counts remain expressed high levels of IL-7Rα, B cells expressed low levels of normal (17), whereas the IL-7Rα KO mice are essentially devoid IL-7Rα, but regulatory T cells (Tregs), NK, and CD11b+ cells of T and B cells (18), suggesting that the effect of IL-7/IL-7Rα signaling in T-cell development is shared between humans and mice. + + Author contributions: L.-F.L. and J.C.L. designed research; L.-F.L., K.L., G.H.T., and T.A. The islet autoreactive CD4 helper T (TH) cells and CD8 performed research; W.Z., I.N., L.M., J.D., J.Y., A.R., C.B., C.A., and S.M.C. contributed cytotoxic T (TC) cells are involved in the immune pathogenesis new reagents/analytic tools; L.-F.L., K.L., G.H.T., B.H., and J.C.L. analyzed data; and L.-F.L. of human T1D and NOD mice (19–25). Recently we showed that and J.C.L. wrote the paper. fl IL-7 can promote the development of IFN-γ–producing TH1 The authors declare no con ict of interest. cells, but not IL-17–producing TH17 cells, from the naïve T cells This article is a PNAS Direct Submission. fi of humans and of the C57BL/6 mice (26). This nding raises an 1To whom correspondence may be addressed. E-mail: Li-Fen.Lee@pfizer.com or John.Lin@ intriguing possibility that IL-7/IL-7Rα pathway may be linked to pfizer.com. T1D risk at least in part through the regulation of TH1 de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. velopment. In addition, it was not known whether IL-7 could 1073/pnas.1203795109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1203795109 PNAS Early Edition | 1of6 A B a bcCD3 CD4 d CD8

100 28G9-mIgG2a 80 28G9-mIgG2a (10 mg/kg) 60 28G9-rIgG1 (10 mg/kg) e fhg

40 Ctrl mIgG2a (10mg/kg) 28G9-rIgG1 Ctrl rIgG1 (10 mg/kg)

Diabetes free (%) 20 0 i jkl

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Fig. 1. IL-7Rα antibody show antidiabetic efficacy in the prophylactic treatment. (A) Diabetes incidence in NOD mice treated with 10 mg/kg of 28G9-mIgG2a (n = 10), 28G9 (rat IgG1), mIgG2a (n = 10), or rat IgG1 (n = 9), starting at 9 wk of age until 29 wk of age, at which time the tissues were analyzed. Data are from one representative experiment of two independent experiments: 28G9-mIgG2a vs. mIgG2a P = 0.001; 28G9-mIgG2a vs. rat IgG1 P < 0.001; 28G9-rIgG1 vs. rat IgG1 P = 0.001; 28G9-rIgG1 vs. mIgG2a P = 0.037; and with P > 0.05 for all of the rest of pair comparison (Log- test). (B) Histological and immunohistochemical analysis of insulitis in mice treated with (a–d) 28G9-mIgG2a, (e–h) 28G9-rIgG1, (i–l) control IgG; (a, e, and i) H&E staining. Immunostaining of distinct cell subsets was performed using mAbs against CD3 (b, f, j), CD4 (c, g, k), and CD8 (d, h, l) at 29 wk of age. (Magnification: 20×.) (C) Intraislet infiltration in 28G9-mIgG2a, 28G9- rIgG1, or isotype control-treated mice was quantified histologically at 20–30 wk of age. The graph shows the fraction of islets with no infiltration (0), peri-insulitis (1), moderate insulitis with <50% islet area infiltrated (2), or severe insulitis with >50% islet area infiltrated (3), respectively. Data are from six to nine mice per group. (D) Insulin-staining score from treated animals, n =4–5 from two experiments. Data in C and D are from one representative experiment of two in- dependent experiments including four animals per group. (E) Absolute cell counts of CD4+, CD8+ from spleen (SPL) and PLN of 28G9-mIgG2a, 28G9-rIgG1, or IgG control-treated mice at 12 wk of age. (F) Absolute cell counts of IFN-γ+CD4+, IL-17+CD4+ from mesenteric (MLN) and PLN of 28G9-mIgG2a, 28G9-rIgG1, or isotype control at 12-wk of age. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with post tests relative to the control IgG group). did not express IL-7Rα (Fig. S2A). We also confirmed that mice (Fig. 2A). These 28G9-mIgG2a–treated mice remained CD4+ and CD8+ naïve, effector memory, and central memory T normoglycemic over a long period compared with the control cells from NOD mice all expressed IL-7Rα compared with the IgG-treated mice (n = 7), none of which could maintain nor- isotype IgG staining (Fig. S2B). In consonance with the cellular moglycemia (Fig. 2A). expression pattern of IL-7Rα, 28G9-mIgG2a, and 28G9-rIgG1 In another cohort of NOD mice, 86% remission rate (n =7) reduced CD4+ and CD8+ T cells in most of the lymphoid tissues was achieved in the newly onset diabetic mice with a short course B (Fig. 1E, and Figs. S1D and S2C). Similarly, the pathogenic TH1 of three injections of 28G9-mIgG2a antibody (Fig. 2 ), com- n and TH17 cells were significantly reduced in the mesenteric pared with 0% remission rate ( = 6) in the isotype control IgG lymph nodes and pancreatic lymph nodes (PLNs) by 28G9- group. We included another clone of IL-7Rα antibody, SB/14 mIgG2a and by 28G9-rIgG1 (Fig. 1F). B and NK cell pop- (BD Biosciences), with minimal binding to mouse Fcγ receptors ulations remained unaffected by any antibody tested in most (Table S1) and found a 63% remission rate (n = 8), which is peripheral tissues (Fig. S2 D and E). These initial data suggested statistically indistinguishable from the efficacy of 28G9-mIgG2a that the IL-7Rα antibody primarily targets CD4+ and CD8+ T (Fig. 2B)(P=0.004 for 28G9-mIgG2a vs. control IgG; P = 0.028 cells in the NOD mice. In later studies described below, however, for SB/14 vs. control IgG; and P = 0.33 for 28G9-mIgG2a vs. SB/ we found that T-cell depletion was not absolutely required for 14, Fisher’s exact test). As expected, the CD4+ and CD8+ T-cell the therapeutic efficacy. counts in the peripheral blood of NOD mice after short-term treatment with 28G9-mIgG2a were significantly reduced relative IL-7Rα Antibody Treatment Induces Durable Disease Remission in to those with isotype control (P < 0.01) (Fig. S3). In contrast, SB/ Newly Established Diabetes. Next, we assessed whether IL-7Rα 14 did not change CD4+ and CD8+ T-cell numbers in the pe- antibody can reverse established diabetes in NOD mice. In one ripheral blood of NOD mice (not significant) (Fig. S3). Both experiment we found that weekly administration of 28G9- 28G9-mIgG2a and SB/14 antibodies showed similar degrees of mIgG2a led to remission in 50% (6 of 12) of newly onset diabetic blockade of IL-7–mediated STAT5 phosphorylation (26). Thus,

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203795109 Lee et al. induced TH1 cell differentiation (Fig. 3A). In contrast, IL-7 alone A + 28G9- Ctrl mIgG2a potently induced IFN-γ–producing cells from CD8 naïve T cells mIgG2a 750 750 in NOD mice similar to that of IL-12 alone (Fig. 3B). Interestingly, γ– 500 500 the presence of IL-12 and IL-7 together further augment IFN- producing cell differentiation from either CD4+ or CD8+ naïve T 250 250 6/12 0/7 cells to a level greater than either cytokine alone. 0

Blood glucose Blood glucose (mg/dL) 0 Blood glucose Blood glucose (mg/dL) 0 20 40 60 80 100 120 140 0 20 40 60 80 100120140 Next, we assessed the effect of IL-7 on the expansion of pre- Days after disease onset Days after disease onset viously polarized TH1orTC1 cells. Under TH1 and TC1-polar- izing conditions, IL-12 expanded TH1 cells as expected (Fig. 3 C B + + 28G9-mIgG2a SB/14 Ctrl mIgG2a and D). When CD4 and CD8 naïve T cells were first stimu-

750 750 750 lated in the presence of IL-7, anti-CD3 plus anti-CD28, and then rested for 48 h followed by restimulation with IL-7, we found 500 500 500 that IL-7 increased the percentage of IFN-γ+ cells originally differentiated from either CD4+ or CD8+ naïve T-cell cultures 250 250 250 6/7 5/8 0/6 (Fig. 3 C and D). These effects of IL-7 on TH1 cell expansion Blood glucose Blood glucose (mg/dL) Blood glucose (mg/dL) 0 0 Blood glucose (mg/dL) 0 were also recapitulated in NOD.BDC2.5 mice (Fig. S4 A and B). + + Days after disease onset Days after disease onset Days after disease onset In contrast, neither IL-17 nor IL-21 T cells (Fig. S4 C–F) was increased by IL-7 treatment in NOD mice but TGF-β plus Fig. 2. Diabetes remission induced by IL-7Rα antibody therapy after disease IL-6 significantly increased IL-17–producing cells in naïve CD4+ onset. (A) Newly onset diabetic NOD mice (based on two consecutive blood- or CD8+ T cells, as expected (Fig. S4 C and D). glucose concentrations over 250 mg/dL) were treated with 10 mg/kg of Furthermore, those cytokines associated with the TH1 (TNF-α, 28G9-mIgG2a (n = 12) or control IgG (n = 7) once a week. Blood glucose was IL-2, and IFN-γ) and T 17 (IL-17A, IL-6, and IL-21) lineage H − monitored. (B). Another cohort of newly onset diabetic NOD mice were were significantly reduced in both CD44+ (Fig. S4H) and CD44 treated with 10 mg/kg of 28G9-mIgG2a (n = 7), or SB/14 (n = 8), or isotype G I – control (n = 6) once a week for 3 wk. Gray-shaded areas indicate the (Fig. S4 and ) cultures from 28G9-mIgG2a treated mice treatment period. 28G9-mIgG2a vs. Ctrl IgG P=0.004; SB/14 vs. Ctrl IgG P = compared with those from isotype control, with the only excep- ’ tion that IL-2 and IL-10 were elevated by 28G9-mIgG2a in the 0.028; 28G9 vs. SB/14 P = 0.33, Fisher s exact test. − CD44 culture (Fig. S4G). the blockade of IL-7 signaling alone appears to be sufficient to PD-1 Can Regulate the Maintenance of in IL-7Rα IMMUNOLOGY – α confer the long-lasting antidiabetic efficacy without affecting the Antibody Treatment. To determine the effect of anti IL-7R on Teff proliferation, equal numbers of carboxyfluorescein succini- circulating T-cell numbers. − midyl ester (CFSE)-labeled Teffs (CD4+CD25 CD44+) isolated Role of IL-7 in Mouse T and Differentiation and Type 1 from BDC2.5.NOD mice treated with IL-7Rα antibody or iso- H C −/− Diabetes. We asked whether IL-7/IL-7R signaling may regulate type controls were adoptively transferred to the NOD.Rag + mice. The Teffs derived from IL-7Rα antibody-treated mice TH1 and TC1 cell differentiation in the NOD mice. Sorted CD4 fi + fi were signi cantly less proliferative than those from control or CD8 naïve T cells from NOD mice were rst cultured under treated mice (Fig. 4A). IL-12 alone, or IL-7 alone, or IL-12 plus IL-7 conditions. IL-12 Then we asked whether IL-7 could affect certain key negative + induced IFN-γ producing cell differentiation in either naïve regulators, such as PD-1, cytotoxic T-lymphocyte antigen 4 CD4+ or CD8+ cultures (Fig. 3 A and B). IL-7 alone slightly (CTLA-4), or related molecules on the Teffs. Indeed, recombinant

A C medium IL-7 IL-12 IL-12 + IL-7 medium IL-7 IL-12 IL-12 + IL-7

5 5 5 5 5 5 5 5 10 10 10 10 10 10 10 10 0.325 1.36 14.3 29.5 14.3 46.4 62.1 61.8 4 4 4 4 4 4 4 4 10 10 10 10 10 10 10 10

3 3 3 3 3 3 3 3 10 10 10 10 10 10 10 10 IFN IFN 2 2 2 2 2 2 2 2 10 10 10 10 10 10 10 10 0 0 0 0 0 0 0 0

2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 0 010 10 10 10 010 10 10 10 010 10 10 10 CD4 CD4 B D medium IL-7 IL-12 IL-12 + IL-7 medium IL-7 IL-12 IL-12 + IL-7 105 105 105 105 105 105 105 105 51.8 70.2 94.4 96.5 18.2 41.3 44.7 54.4 104 104 104 104 104 104 104 104 A>: IFNr A>: A>: IFNr A>:

- 3 3 3 3 103 103 103 103 10 10 10 10 : IFNr

0 0 0 0 0 0 0 0

2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 CD8 CD8

Fig. 3. IL-7 promotes IFN-γ+ cell development from NOD naïve CD4+ and CD8+ T cells. (A) Sorted naïve CD4+ and (B) CD8+ T cells from NOD mice were stimulated for 3 d with anti-CD3/CD28 activation beads in the presence of indicated cytokines. (C and D) After 3 d of culture in the presence of indicated cytokine shown in A, cells were rested for 48 h followed by restimulation with anti-CD3 and anti-CD28 in the presence of different cytokines (e.g., IL-12, IL-7, IL-7+IL-12), or medium as indicated for each individual FACS plot during the expansion period. CD4+ T cells (A and C) or CD8+ T cells (B and D) were analyzed for IFN-γ by flow cytometry.

Lee et al. PNAS Early Edition | 3of6 PLN DLN 500 ** A PLN B -1 PBS 450 51.7 60 mIL7 ** PIL 400 350 Ctrl mIgG2a 0 40

MFI of PD MFI of 300 20 250 cell division (%) division cell 200 28.5 0 150 *** 28G9-mIgG2a PLN 0 100

MFI of PD-1 MFI of 50 CFSE 0 C gated on CD4+ v 4+ 10 5 10 5 D 10 4 10 4 300 3 63 3 60.6 70 -1 10 10 PIL *** PBS 65 2 2 * 10 10 200 0 0 60 0102 103 10 4 10 5 0102 103 10 4 10 5

5 5 55 10 10 PD MFI of 100

10 4 104 50

PD-1+ cells (%) Ctrl

3 43.9 3 28G9 IL-7 10 10 47.6 45 0

10 2 102 40 PLN 0 0

2 3 4 5 2 3 4 5 CD4 010 10 10 10 010 10 10 10 PD-1 PD-1

E F 28G9-mIgG2a SB/14 100 100 28G9 ** 80 80 SB/14 800 PIL 60 Ctrl 60 Ctrl % of % Max % of Max * 40 40 600 20 20

0 2 3 4 5 0 2 3 4 5 400

010 10 10 10 010 10 10 10 PD-1 MFI of : PD-1 : PD-1 Ctrl 100 100 200 28G9 80 80 60 60 0 % of Max PLN of % Max 40 40 20 20 0 0 0102 103 104 105 0102 103 104 105 PD-1 BTLAICOS CTLA-4 GITR : PD-1 : PD-1 PD-1

Fig. 4. IL-7 suppresses, whereas IL-7Rα antibody up-regulates, PD-1 in Teffs. (A) Proliferation of CFSE-labeled T cells isolated from 28G9-mIgG2a or control IgG treated BDC2.5 mice after adoptive transfer into NOD.Rag−/− mice. Flow cytometry was performed 7 d after transfer into NOD.Rag−/− in PLNs and the draining lymph node (DLN). Proliferation percentage was determined by CFSE dilution in CD4+Vβ4+ T cells. (B–E) Expression of PD-1 (x-axis of the FACS plots) by the PIL − T cells or PLNs CD4+FoxP3 (Teff) cells. (B) Mice treated with PBS (gray) or 1 μg of mIL-7 (white) every other day for a total of three injections starting at 9 wk of age. (C) Lymph node T cells were isolated from NOD mice and treated with anti-CD3 and anti-CD28 in the presence of PBS or mIL-7 in vitro for 3 d. (D and E) Mice were treated once a week with control IgG-mIgG2a (gray) or 28G9-mIgG2a (white) starting at 9 wk-of-age (D) or at 12 wk-of-age (E) for 3 consecutive weeks. Graphs are representative of two independent experiments. (F) PD-1 expression in the PLN Teffs. NOD mice were treated with 28G9-mIgG2a, SB/14 or control IgG starting at 9 wk of age, each at 10 mg/kg per week for 4 wk. Mean flouorescence intensity (MFI) results are pooled from five animals in each group. Data are from one representative experiment of two independent experiments. The error bars represent SEM. *P < 0.05 and **P < 0.01 (one-way ANOVA with post tests); ***P < 0.001 (Student t test). mouse IL-7 treatment in vivo led to reduced expression of PD-1 in efficacy of short-term IL-7Rα antibody treatment in the newly the Teff of pancreatic infiltrating lymphocytes (PIL) and PLNs onset diabetes. The NOD mice received a short course of of the NOD mice (Fig. 4B). Similarly, in vitro IL-7 treatment treatment with either 10 mg/kg of SB/14 (treatment duration of suppressed PD-1 expression after T-cell activation (Fig. 4C). which is indicated by the filled bar in Fig. 5A) or 28G9-mIgG2a Conversely, 28G9-mIgG2a treatment of 9-wk-old NOD mice (indicated by open bar in Fig. 5B) once weekly for 3 wk imme- led to an up-regulation of PD-1 in PLN Teffs (Fig. 4D) and the diately after diabetic onset. As expected, such treatment of either increase of PD-1 expression is regulated by 28G9-mIgG2a in IL-7Rα antibody reversed diabetes of these mice for over 3 mo. a dose-dependent manner (Fig. S5). Treatment with 28G9- These IL-7Rα antibody-induced euglycemic mice, upon a single mIgG2a of 12-wk-old NOD mice was associated with a signifi- injection of anti–PD-1 (black arrows in Fig. 5) but not isotype cant increase in the expression of PD-1 in PIL Teffs (Fig. 4E), control IgG (red arrows in Fig. 5), rapidly developed hypergly- without much effect on the PD-1 expression in Tregs of the PIL cemia within 5 d. Thus, the long-term tolerance induced by IL- or PLN (Fig. S6B). This modulatory effect by IL-7Rα antibody is 7Rα antibody therapy likely requires PD-1 function. specific to PD-1 in that other related molecules, such as CTLA-4, inducible T-cell costimulator, glucocorticoid-induced TNF-re- IL-7Rα Antibody Treatment Increases the Frequency, but Not the ceptor, and B- and T-lymphocyte attenuator were not affected Intrinsic Suppressor Activity, of Tregs. To study the effect on by the IL-7Rα antibody treatment (Fig. 4E). Tregs, we initiated antibody treatment in 9-wk-old female NOD Although 28G9-mIgG2a and SB/14 have differential FcγR mice. After 3 wk of once weekly injection, 28G9-mIgG2a signifi- binding (Table S1) and also have distinct effect on T-cell numbers cantly increased the frequency of Tregs in the spleen and in the (Fig. S3), these two antibodies exhibited comparable efficacy in PLN (Fig. S7 A and B), and also the absolute count of Tregs in the reversing newly onset diabetes (Fig. 2B). On the other hand, PD-1 spleen of these mice (Fig. S7C). In our experiment, the majority of expression was consistently increased by 28G9-mIgG2a and by SB/ Foxp3+ cells are found in the CD4+CD25hi T cells although 14 in the Teffs of PLNs at week 9 (Fig. 4F), supporting the notion CD4+CD25lo cells also express some level of Foxp3 (Fig. S7 E that PD-1 up-regulation may contribute to the efficacy of the IL- and F)(32). 7Rα antibodies. These results also suggest that PD-1 up-regulation We asked whether IL-7Rα antibody treatment may modulate by IL-7Rα antibody may not require the engagement of FcγR. the intrinsic suppressor activity of Tregs. Because the CD4+ We tested whether the PD-1 pathway can regulate therapeutic CD25hi T cells have consistently shown greater inhibition indices efficacy in IL-7Rα antibody by taking advantage of the durable than other T-cell populations (33), we purified these cells using

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203795109 Lee et al. A B given; (iii) SB/14, another efficacious clone of IL-7Rα antibody 600 without T-cell–depleting ability, also increased PD-1 expression isotype-3 600 F fi iv α isotype-1 isotype-4 (Fig. 4 ); and nally ( ) long-term tolerance induced by IL-7R isotype-2 isotype-5 A B 400 antibodies can be abrogated by PD-1 blockade (Fig. 5 and ). anti-PD-1-1 anti-PD-1-4 400 Based on our findings that IL-7 can promote TH1 and TC1 cell anti-PD-1-2 anti-PD-1-5 A–D anti-PD-1-3 development in NOD mice (Fig. 3 ) and down-regulates PD- 200 anti-PD-1-6 200 1 expression (Fig. 5 B and C), we propose that IL-7Rα antibody therapy may operate through (i) the reduction of IFN-γ+ Teffs Blood glucose (mg/dL) Blood glucose Blood glucose (mg/dL) ii 0 0 and ( ) increased PD-1 expression in the Teffs at the anatomical 0 25 50 75 100125150175200225250 0 25 50 75 100125150175200225250 site of active inflammation. At the same time, the genetic asso- Days after disease onset Days after disease onset ciation of IL-7R polymorphisms with the risk of human T1D (11, 12) may also be better appreciated from the perspective of these Fig. 5. PD-1 blockade abrogates the antidiabetic efficacy of IL-7Rα anti- two immune mechanisms. body. Anti–PD-1 treatment exacerbates disease in reversal paradigm. Newly Like many discoveries, our findings raise new questions, such diabetic NOD mice were treated with IL-7Rα antibody (A) SB/14 or with (B) as the precise intracellular signaling pathways that mediate PD-1 28G9-mIgG2a for a total of three doses once weekly at 10 mg/kg i.p. The up-regulation, and whether these signals differ from those that treatment period for each IL-7Rα antibody was indicated by the filled and mediate the cell proliferation and IFN-γ expression in the IL-7 the open bars, respectively. Long-term remission of existing diabetes was target cells. Several reports showed that blocking PD-1/PD-L1 observed for over 100 d. The animals reverted to normoglycemia by IL-7Rα signaling by neutralizing antibody or by genetic deletion of PD-1 – antibody were randomly assigned to treatment of either anti PD-1 (n =3,or or PD-L1 exhibited significantly elevated IFN-γ–producing cells rat IgG2a isotype control (isotype, n =2–3) once weekly at 200 μg per mouse. – – in several autoimmune diseases (28, 36, 39 42). Of note, PD-1/ Black arrows indicate the timing of anti PD-1 injections, whereas red arrows PD-L1 signaling was shown to inhibit IFN-γ production during indicate that of isotype IgG injection. naïve T-cell activation. PD-1 or PD-L1–deficient NOD mice displayed significantly higher IFN-γ production, which resulted FACS to study their suppressor activity on Teffs. In the in vitro in the rapid onset of diabetes and the early onset of insulitis (28, coculture system, the CD4+CD25hi Tregs isolated from the pan- 36). A recent report showed that PD-1/PD-L1 signaling converts creatic lymph nodes and the spleen of IL-7Rα antibody-treated human TH1 cells into Tregs in vitro and in vivo, thereby pre- venting human-into-mouse xenogeneic graft-vs.-host disease. animals displayed similar suppressive capacity compared with Recipient of T 1 cells plus T cells expressing PD-L1 had a re- IMMUNOLOGY those from isotype control-treated animals across different ratios H duced number of T-bet+ T cells and an increased number of of Tregs to Teffs (Fig. S7D). Thus, IL-7Rα antibody treatment Foxp3+ T cells (43). In this connection, we also noted an in- increases the frequency and absolute counts of Tregs in some creased frequency and also absolute number of Tregs in certain— lymphoid compartments without altering the intrinsic suppressive but not all—lymphoid compartments in NOD mice treated with activity of Tregs. IL-7Rα antibody. The sparing of Tregs by IL-7Rα antibody Discussion therapy is consistent with the relatively low level of expression of α A α IL-7R in Tregs (Fig. S2 ) (26, 44, 45). The goal of this study was to examine the effects of IL-7R What variables of the newly diabetic mice may have contrib- antibody on NOD mice and to understand the functional con- uted to the durable remission of NOD mice by anti–IL-7Rα?To tribution of IL-7Rα to the T1D pathogenesis. We show here that α begin to address this question, we combined several reversal IL-7R antibody treatment can prevent and delay the pro- experiments and analyzed the age of onset and blood glucose gression of T1D before onset, as well as reversing the newly value at onset of all of the newly onset NOD mice treated with onset diabetes. either isotype control or one of the two anti–IL-7Rs (28G9- Recently we demonstrated that IL-7 can enhance mouse TH1 mIgG2a and SB/14) across several experiments. Our preliminary cell expansion on the C57BL/6 background (26). In this article, post hoc observations raise the possibility that animals with we show that IL-7 promotes NOD mouse TH1 cell development, a less-aggressive course of newly onset diabetes (i.e., with a later fi even in the absence of IL-12, consistent with our previous nd- age of onset and with lower blood-glucose levels at onset) may be ings in human cord-blood cells (26). In addition, we are unique more responsive to the IL-7Rα antibody therapy (Fig. S8 B and E). + γ in demonstrating that IL-7 can promote CD8 IFN- cell de- This possibility will have to be confirmed by future prospective velopment during the differentiation and expansion stage of studies. If substantiated, this theory may have important implica- in vitro culture (Fig. 4 B and D). In type 1 diabetes the de- tions for the clinical development of IL-7Rα antibody therapy in velopment of disease has usually been ascribed to a TH1orTC human T1D. – response (19 25). On the other hand, the exact role of TH17 cells Previously it was reported that, in the preventative paradigm, in the pathogenesis of T1D is not well understood. For example, TNF-α and anti–TNF-α treatments in young vs. adult NOD fi highly puri ed TH17 from BDC2.5.NOD mice were able to animals exhibited opposite outcome (46, 47). The anti-CD3 transfer diabetes to NOD/SCID mice, primarily via conversion to therapy also did not show protection when administered to 4-wk- γ– an IFN- producing TH1-like phenotype, whereas transfer of old, prediabetic NOD mice. Thus, it is reasonable to ask TH1 cells did not result in the generation of TH17 cells (34). IFN- whether, in the preventative paradigm, anti–IL-7Rα might exert γ producing TH1 and TC1 cells are important for T1D because any age-dependent effect on NOD pathogenesis or not. We blockade of IFN-γ or IL-12 (35–37) prevented disease, whereas treated some NOD mice starting at 4 wk of age with 28G9- IL-12 promoted disease in NOD mice (38). In our studies, the mIgG2a or with isotype control IgG for three injections (at 4-, 7-, IL-7Rα–blocking antibody treatment significantly reduced TH1 and 10-wk-old) and found that 50% mice in the 28G9-mIgG2a and TH17 cells (Fig. 1F), as well as TH1 cytokines (IFN-γ and treated group did not develop diabetes, and merely 20% of the TNF-α) and TH17 cytokines (IL-17A, IL-6, and IL-21) (Fig. control IgG treated mice exhibited normaglycemia (Fig. S9). S4G). These changes likely contribute to the therapeutic efficacy. These results further suggest the unique potential of preventing How does IL-7Rα antibody therapy attenuate the activity of the clinical onset of T1D in certain high-risk groups by targeting the islet targeting Teffs? The immune tolerance mediated the IL-7Rα pathway, as oppose to the TNF-α or anti-CD3– through PD-1 may be important in restraining Teff function targeting drugs. based on several lines of evidence: (i) the expression of PD-1 in In conclusion, we show that IL-7 suppressed PD-1 expression the PIL and PLN Teffs was reduced in IL-7–treated mice (Fig. 4 after T-cell activation, and IL-7 can promote IFN-γ+ cell de- B and C); (ii) PD-1 expression was up-regulated in the PLN and velopment in CD4+ and CD8+ T cells, both T-cell subsets of PIL Teffs (Fig. 4 D and E) when 28G9-mIgG2a treatment was which contribute to the pathogenesis of T1D. Anti–IL-7Rα

Lee et al. PNAS Early Edition | 5of6 treatment can prevent diabetes and can also reverse newly Antibody Treatment. For IL-7Rα antibody treatment, mice were injected in- established disease in NOD mice through the combination of traperitoneally with 10 mg/kg or 3 mg/kg body weight of anti–IL-7Rα mAb targeting IFN-γ–producing T cells and keeping pathogenic T once weekly starting at week 9, or after disease onset, as indicated in each fi cells in check via PD-1 up-regulation. Our findings provide the gure. For PD-1 blockade experiments, PD-1 (RMP1-14) (29, 48) were pur- mechanistic insight to the genetic association of IL-7R with hu- chased from eBioscience. In the reversal paradigm, three to four injections of fi α– 28G9-mIgG2a or SB/14 were given in diabetic mice and reverted diabetes-free man T1D, as well as the scienti c rationale for the IL-7R for3mo.Onedoseofanti–PD-1 (200 μg per mouse) was given in the previously blocking antibody as a unique therapy for the patients with T1D. 28G9-mIgG2a or SB/14-treated mice. Animals were monitored for blood-glu- Materials and Methods cose measurement and mice were considered diabetic if blood-glucose levels were >250 mg/dL on two consecutive draws. A detailed description of Ab Mice. NOD.Lt mice were obtained from The Jackson Laboratory and were generation, mouse T-helper cell differentiation, flow cytometry, immunohis- housed in groups of five under specific pathogen-free conditions in Rinat- tochemistry, ELISA, cytometry bead assay, CFSE labeling, adoptive transfer, and Pfizer animal facilities. All animals used were 7- to 8-wk-old females, unless in vitro suppression assay are provided in SI Materials and Methods. specifically noted. All animal experiments were conducted according to the protocols approved by the Institutional Animal Care and Use Committee of ACKNOWLEDGMENTS. We thank M. Gilbert for flow cytometry, M. Karnoub Rinat, Pfizer. for statistical analysis, and D. Malashock and Y. Abdiche for the biosensor assay.

1. Anderson MS, Bluestone JA (2005) The NOD mouse: A model of immune dysregula- 25. Sumida T, et al. (1994) Prevention of insulitis and diabetes in beta 2-microglobulin- tion. Annu Rev Immunol 23:447–485. deficient non-obese diabetic mice. Int Immunol 6:1445–1449. 2. Castaño L, Eisenbarth GS (1990) Type-I diabetes: A chronic autoimmune disease of 26. Lee LF, et al. (2011) IL-7 promotes T(H)1 development and serum IL-7 predicts clinical human, mouse, and rat. Annu Rev Immunol 8:647–679. response to interferon-β in multiple sclerosis. Sci Transl Med 3:93ra68. 3. Tisch R, McDevitt H (1996) Insulin-dependent diabetes mellitus. Cell 85:291–297. 27. Keir ME, Butte MJ, Freeman GJ, Sharpe AH (2008) PD-1 and its ligands in tolerance 4. Lee LF, et al. (2005) The role of TNF-alpha in the pathogenesis of type 1 diabetes in and immunity. Annu Rev Immunol 26:677–704. the nonobese diabetic mouse: Analysis of maturation. Proc Natl Acad Sci 28. Ansari MJ, et al. (2003) The programmed death-1 (PD-1) pathway regulates autoim- USA 102:15995–16000. mune diabetes in nonobese diabetic (NOD) mice. J Exp Med 198:63–69. 5. Chatenoud L, Bluestone JA (2007) CD3-specific antibodies: A portal to the treatment 29. Fife BT, et al. (2006) Insulin-induced remission in new-onset NOD mice is maintained of autoimmunity. Nat Rev Immunol 7:622–632. by the PD-1-PD-L1 pathway. J Exp Med 203:2737–2747. 6. Todd JA, Bell JI, McDevitt HO (1987) HLA-DQ beta gene contributes to susceptibility 30. Okazaki T, Honjo T (2006) The PD-1-PD-L pathway in immunological tolerance. Trends and resistance to insulin-dependent diabetes mellitus. Nature 329:599–604. Immunol 27:195–201. 7. Vyse TJ, Todd JA (1996) Genetic analysis of autoimmune disease. Cell 85:311–318. 31. Pellegrini M, et al. (2011) IL-7 engages multiple mechanisms to overcome chronic viral 8. Gregory SG, et al.; Multiple Sclerosis Genetics Group (2007) Interleukin 7 receptor infection and limit organ pathology. Cell 144:601–613. alpha chain (IL7R) shows allelic and functional association with multiple sclerosis. Nat 32. You S, et al. (2007) Adaptive TGF-beta-dependent regulatory T cells control autoim- Genet 39:1083–1091. mune diabetes and are a privileged target of anti-CD3 antibody treatment. Proc Natl – 9. Hafler DA, et al.; International Multiple Sclerosis Genetics Consortium (2007) Risk Acad Sci USA 104:6335 6340. alleles for multiple sclerosis identified by a genomewide study. N Engl J Med 357: 33. Belghith M, et al. (2003) TGF-beta-dependent mechanisms mediate restoration of 851–862. self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat Med – 10. Lundmark F, et al. (2007) Variation in interleukin 7 receptor alpha chain (IL7R) in- 9:1202 1208. fi fluences risk of multiple sclerosis. Nat Genet 39:1108–1113. 34. Bending D, et al. (2009) Highly puri ed Th17 cells from BDC2.5NOD mice convert into – 11. Concannon P, Rich SS, Nepom GT (2009) Genetics of type 1A diabetes. N Engl J Med Th1-like cells in NOD/SCID recipient mice. J Clin Invest 119:565 572. 35. Nicoletti F, et al. (1996) The effects of a nonimmunogenic form of murine soluble 360:1646–1654. interferon-gamma receptor on the development of autoimmune diabetes in the NOD 12. Todd JA, et al.; Genetics of Type 1 Diabetes in Finland; Wellcome Trust Case Control mouse. Endocrinology 137:5567–5575. Consortium (2007) Robust associations of four new regions from ge- 36. Keir ME, et al. (2006) Tissue expression of PD-L1 mediates peripheral T cell tolerance. J nome-wide analyses of type 1 diabetes. Nat Genet 39:857–864. Exp Med 203:883–895. 13. Surh CD, Sprent J (2008) Homeostasis of naive and memory T cells. Immunity 29: 37. Trembleau S, Penna G, Gregori S, Gately MK, Adorini L (1997) Deviation of pancreas- 848–862. infiltrating cells to Th2 by interleukin-12 antagonist administration inhibits autoim- 14. Tan JT, et al. (2001) IL-7 is critical for homeostatic proliferation and survival of naive T mune diabetes. Eur J Immunol 27:2330–2339. cells. Proc Natl Acad Sci USA 98:8732–8737. 38. Trembleau S, et al. (1995) Interleukin 12 administration induces T helper type 1 cells 15. Tan JT, et al. (2002) Interleukin (IL)-15 and IL-7 jointly regulate homeostatic pro- and accelerates autoimmune diabetes in NOD mice. J Exp Med 181:817–821. liferation of memory phenotype CD8+ cells but are not required for memory phe- 39. Salama AD, et al. (2003) Critical role of the programmed death-1 (PD-1) pathway notype CD4+ cells. J Exp Med 195:1523–1532. in regulation of experimental autoimmune encephalomyelitis. JExpMed198: 16. Calzascia T, et al. (2008) CD4 T cells, lymphopenia, and IL-7 in a multistep pathway to 71–78. autoimmunity. Proc Natl Acad Sci USA 105:2999–3004. 40. Sandner SE, et al. (2005) Role of the programmed death-1 pathway in regulation of 17. Puel A, Ziegler SF, Buckley RH, Leonard WJ (1998) Defective IL7R expression in T(-)B(+) alloimmune responses in vivo. J Immunol 174:3408–3415. fi – NK(+) severe combined immunode ciency. Nat Genet 20:394 397. 41. Sandner SE, et al. (2005) Mechanisms of tolerance induced by donor-specific trans- 18. Peschon JJ, et al. (1994) Early lymphocyte expansion is severely impaired in interleukin fusion and ICOS-B7h blockade in a model of CD4+ T-cell-mediated allograft rejection. fi – 7 receptor-de cient mice. J Exp Med 180:1955 1960. Am J Transplant 5:31–39. 19. Santamaria P (2010) The long and winding road to understanding and conquering 42. Latchman YE, et al. (2004) PD-L1-deficient mice show that PD-L1 on T cells, antigen- – type 1 diabetes. Immunity 32:437 445. presenting cells, and host tissues negatively regulates T cells. Proc Natl Acad Sci USA fi 20. Healey D, et al. (1995) In vivo activity and in vitro speci city of CD4+ Th1 and Th2 cells 101:10691–10696. – derived from the of diabetic NOD mice. J Clin Invest 95:2979 2985. 43. Amarnath S, et al. (2011) The PDL1-PD1 axis converts human TH1 cells into regulatory fi 21. Itoh N, et al. (1993) Mononuclear cell in ltration and its relation to the expression of T cells. Sci Transl Med 3(111):111ra120. major histocompatibility complex antigens and adhesion molecules in pancreas bi- 44. Liu W, et al. (2006) CD127 expression inversely correlates with FoxP3 and suppressive opsy specimens from newly diagnosed insulin-dependent diabetes mellitus patients. J function of human CD4+ T reg cells. J Exp Med 203:1701–1711. Clin Invest 92:2313–2322. 45. Seddiki N, et al. (2006) Expression of interleukin (IL)-2 and IL-7 receptors discriminates 22. Katz JD, Benoist C, Mathis D (1995) subsets in insulin-dependent di- between human regulatory and activated T cells. J Exp Med 203:1693–1700. abetes. Science 268:1185–1188. 46. Cope AP, et al. (1997) Chronic tumor necrosis factor alters T cell responses by atten- 23. Nagata M, Santamaria P, Kawamura T, Utsugi T, Yoon JW (1994) Evidence for the role uating T cell receptor signaling. J Exp Med 185:1573–1584. of CD8+ cytotoxic T cells in the destruction of pancreatic beta-cells in nonobese di- 47. Yang XD, et al. (1994) Effect of tumor necrosis factor alpha on insulin-dependent abetic mice. J Immunol 152:2042–2050. diabetes mellitus in NOD mice. I. The early development of autoimmunity and the 24. Serreze DV, Leiter EH, Christianson GJ, Greiner D, Roopenian DC (1994) Major his- diabetogenic process. J Exp Med 180:995–1004. tocompatibility complex class I-deficient NOD-B2mnull mice are diabetes and insulitis 48. Fife BT, et al. (2009) Interactions between PD-1 and PD-L1 promote tolerance by resistant. Diabetes 43:505–509. blocking the TCR-induced stop signal. Nat Immunol 10:1185–1192.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1203795109 Lee et al.