Therapeutic effect of αCD52 mAb in multiple sclerosis and its animal models is mediated via T-regulatory cells

Abstract

The objective of this study is to determine the mechanism of action of αCD52 mAb treatment in patients with relapsing-remitting multiple sclerosis (RRMS). Experimental autoimmune encephalomyelitis (EAE), an animal model of the disease, was used to address the role of T regulatory cells (Tregs) in the αCD52 maAb-induced suppression of the disease. In-vitro studies on PBMCs from RRMS patients and matched healthy controls (HCs) determined the effect of IL-7 on the expansion of CD4⁺CD25⁺CD127⁺ Tregs and induction of their suppressive phenotype. The present sudy using EAE animal models of MS has shown that mouse (mu) αCD52 mAb suppression of clinical disease was augmented by co-administration of IL-7, and partially reversed by αIL-7 mAb. In-vitro human studies showed that IL-7 induced expansion of CD4⁺CD25⁺CD127⁺ Tregs and increased their Foxp3, GITIR, CD46, CTLA-4, GZMB and perforin expression. αCD52 mAb treatment of mice with RREAE induced expansion of Foxp3⁺CD4⁺ Treg cells and the suppression of IL-17A⁺CD4⁺ and IFN-γ⁺CD4⁺ cells in peripheral immune organs and in CNS infiltrates. The effect was detected immediately after the treatment and maintained over a long-term follow-up. Foxp3⁺CD4⁺ Treg-mediated suppression of IL-17A⁺CD4⁺ and IFN-γ⁺CD4⁺ cells in the spinal cord infiltrates were reversed after inducible Foxp3 depletion in DEREG mice. Our results demonstrated that the therapeutic effect of FDA-approved αCD52 mAb is dependent on the presence of Treg cells.

Introduction

Studies reporting a deficient Treg suppression in RRMS (1) have demonstrated that Tregs from RRMS patients have a decreased ability to suppress T effector (Teff) cell proliferation, which correlates with their decreased Foxp3 and CTLA-4 expression (2). Furthermore, RRMS patients have a lower frequency of CD39⁺ Tregs, which selectively inhibit Th17 cell cytokine secretion (3). Thus, impaired Treg function may contribute to the Th17 autoimmune response in RRMS.

Alemtuzumab (Lemtrada^®) is a humanized monoclonal antibody against the surface CD52 molecule, expressed on all lymphocytes (4). As an effective lymphocyte-depleting therapy, it has been FDA approved as a treatment for aggressive RRMS, for patients who fail first-line therapy (5). The annual relapse rate and sustained accumulation of disability were significantly reduced in Phase III clinical trials (6). αCD52 mAb intravenous treatment has been established as one of the longest-lasting suppressors of RRMS progression (7). It efficiently depletes T and B lymphocytes and, to a lesser extent, monocytes, macrophages, dendritic cells, and NK cells via Ab-dependent cellular cytotoxicity and complement-induced cell lysis (8, 9). Although effectively depleting all circulating CD52-bearing cells, this treatment does not affect hematopoietic stem cells, thus preserving the potential for an immune reconstitution (10). The loang-lasting clinical disease suppression and an improvement in disability scores have led to the hypothesis that in addition to immune cell depletion, the subsequent differential reconstitution of CD4⁺ cell subsets may contribute to a decreasd new brain lesion formation, demonstrated by brain magnetic resonance imaging (MRI) scans in Phase III clinical trials (5). While its efficacy is well established and disease activity suppression lasts for years (11), its mechanisms of action have not been elucidated. αCD52 mAb induces lymphocyte lysis, which confers an immunosuppressive effect. However, in the context of this pharmacologically-induced lymphopenia, our previous studies have identified a differential reconstitution of T cell subsets, characterized by a four-fold increase in the percentage of CD25⁺CD127⁻ Treg cells within the CD4⁺ lymphocytes at month 1, followed by a progressive increase in the numbers of TGF-β⁺, IL-10⁺ and IL-4⁺ regulatory CD4⁺ cells, and the inhibition of IFN-γ⁺ and IL-17A⁺ Teff CD4⁺ cells at months 12 and 24 post-treatment (4). The in-vivo expanded CD4⁺CD25⁺CD127⁻ Treg cells following αCD52 mAb treatment maintained an immunosuppressive Foxp3⁺CD39⁺Granzyme B⁺TGF-β−1⁺ phenotype, which had a regulatory function in human in-vitro studies (12). Several studies have demonstrated that Tregs from alemtuzumab-treated patients suppress IL-17A and IFN-γ production in PBMCs (13) and that alemtuzumab in-vitro treatment of CD4⁺ cells induces increased suppressive function of Tregs by cell-to-cell contact (14).

Our human studies have identified a significant incremental increase in serum IL-7 concentrations from day 7 to 6 months following αCD52 mAb-induced lymphopenia. In-vitro studies have demonstrated the IL-7 induction of STAT5 phosphorylation and Foxp3 expression, and the IL-7-induced proliferation of FOXP3⁺ Treg cells (4).

The current study has confirmed that Tregs mediate the therapeutic effect of αCD52 mAb in animal models of the disease. The therapeutic effect was enhanced by co-administration of IL-7, and partially reversed by co-administration of αIL-7 mAb. An in-vitro human study on PBMCs from RRMS patients revealed that IL-7 induced expansion of CD4⁺C25*CD127⁻ Tregs and increased their Foxp3⁺, glucocorticoid-induced TNFR (GITR), CD46, CTLA-4, perforin and Granzyme B (GZMB) expression, indicating suppressive mechanisms of expanded Treg cells. Studies of EAE animal models confirmed expansion of FoxP3⁺ and IL-10⁺ Tregs and suppression of IL-17⁺ and IFN-γ⁺ and IL-17⁺ CD4⁺ cells in peripheral immune organs and in the CNS of treated mice. We propose that repopulating Tregs in the setting of αCD52 mAb-induced lymphopenia led to the suppression of inflammatory responses in RRMS and in animal models of disease.

Materials and Methods

Mice

Eight to 12-week-old female SJL/J mice and C57BL/6 mice were purchased from Charles River Laboratories. DEREG mice were purchased from Jackson Laboratory. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill and Thomas Jefferson University.

αCD52 mAb

Murine αCD52 IgG2a mAb was provided by Sanofi Genzyme Inc. (Cambridge MA, USA); the antibody production in mice was described by Turner et al. (15).

Actively induced EAE

We used two EAE models: proteolipid protein (PLP)_139–151-induced RR EAE in SJL/J mice and myelin oligodendrocyte glycoprotein (MOG)_35–55-induced chronic EAE in C57BL/6 and DEREG mice.

RR EAE: 8–12-week-old female SJL/J mice were immunized with 50 μg PLP_139–151 peptide per mouse in complete Freund’s adjuvant (CFA) containing Mycobacterium (M.) tuberculosis (4 mg/ml). Pertussis toxin (PTX) (200 ng) was administered i.p. on days 0 and 2 post immunization (p.i.). Mice were monitored daily for the development of clinical signs, and starting from day 12 p.i., 10 mice per group received daily s.c. mu αCD52 mAb (200 μg/day) or IgG2a isotype control (200 μg/day), mu αCD52 mAb + IL-7 (1 μg/day), or mu αCD52 mAb + αIL-7 mAb (150 μg/day) for 5 consecutive days. Clinical scores were assigned daily as follows: 1, limp tail; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and forelimb weakness; 5, moribund mice; and 6, death.

Chronic EAE: 6–18-week-old female C57BL/6 or DEREG Foxp3-DTR (diphtheria toxin receptor) mice were immunized with 50 μg MOG_35–55 (AnaSpec) in CFA consisting of incomplete Freund’s adjuvant (Difco Laboratories) and 5 mg/ml of M. tuberculosis H37RA (Difco Laboratories). 200ng PTX (List Biological) was injected i.p. on days 0 and 2 p.i. 200 μg of mu αCD52 mAb was injected s.c. each day per mouse for 5 days (day 11–15 p.i.). For Foxp3⁺ cell depletion, 200 ng (10 μg/kg) diphtheria toxin (DT) was injected i.p for 4 consecutive days (days 12–15 p.i.). Clinical scores were recorded daily as above.

ELISA

Serum samples were collected from mu αCD52 mAb-treated mice with RREAE on days 0, 17 and 23 p.i. and IL-7 levels were measured using ELISA (R&D Systems, DY407) in duplicate.

Flow cytometry

Blood samples were obtained from 5 untreated RRMS patients and 5 age-, sex- and race-matched HCs who had signed the Thomas Jefferson University-approved IRB consent form. PBMCs were separated using Ficoll gradient. Fresh PBMCs were stained for the following surface markers: CD4, CD25, CD127 (for gating purposes), and Treg markers CTLA-4, CD39 (eBioscience), GITR, CD46 (BD Biosciences). Intracellular staining was performed after stimulation with PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich) for 2 h, and brefeldin A (1:1000 dilution) (eBioscience) was added for an additional 2 h. Cells were fixed, permeabilized, and stained with fluorescein-conjugated Abs against FoxP3, Ahr, granzyme B, perforin (eBioscience), IL-10 (BD Biosciences), as reported previously (4). The percentages of cells expressing each molecule in gated CD4⁺CD25⁺CD127⁻ Tregs were determined using a BD FACSAria Fusion flow cytometer and FlowJo software following 48 hour stimulation with IL-7 (100 ng/ml).

For the flow cytometry EAE experiments, the SJL mice with RR EAE were sacrificed on days 23 and 60 p.i., C57BL/6 and DEREG mice with chronic EAE were sacrificed on day 25 p.i. On the above-mentioned days, blood was collected via cardiac puncture, and mice were perfused with cold 50 ml PBS containing heparin (10 U/ml) (Sigma). PBMCs, lymph nodes (LNs), including superficial cervical, deep cervical, axillary, brachial, and inguinal LNs, spleen, spinal cord, and brain tissue were harvested. Spinal cord and brain tissue was cut into small pieces and digested in PBS-containing Collagenase D (5 mg/ml) (Roche) for 45 min at 37°C with a short vortex every 15 minutes. After digestion, the cells were passed through a strainer and washed with PBS, followed by 38% Percoll (Sigma) gradient separation of the CNS mononuclear cell infiltrates.

The cells were stimulated with PMA, ionomycin for 2 hours and with Brefeldin A (BFA) for an additional 3 hours prior to intracellular staining. The cells were stained with αCD4 (RM4–5, Biolegend), αFoxp3 (FJK16S, eBioscience), αIFN-γ (XMG1.2) and αIL-17A mAb (TC11–18H10.1) from Biolegend. Foxp3 staining was done using Foxp3 Fixation/Permeabilization reagent (eBioscience). Isotype controls were used to determine the background. The percentage of cells expressing each molecule was determined in gated CD4⁺ cells using a BD FACSCalibur™and Canto (BD Biosciences) Flow Cytometer, with FCS Express software (De Novo Software). We determined the percentages of the indicated cell subsets in PBMCs, LN, spleen, brain and spinal cord, and present statistically significant changes.

Statistical analysis

The results of clinical scores and FACS studies were analyzed using two-way ANOVA and Tukey’s multiple comparisons test (GraphPad Software). p values of < 0.05 were considered significant. The comparison of FACS data with two groups was performed using two-tailed Student’s unpaired t-test.

Results

αCD52 mAb treatment suppresses RREAE

Since αCD52 mAb is approved treatment for RR MS, the RR EAE animal model, induced by PLP_139–151 peptide immunization of SJL mice (16, 17), is the most suitable model for determining its mechanisms of action. Administration of murine (mu) αCD52 mAb at 10 mg/kg s.c. for 5 days, starting at the peak of disease (day 12 p.i.), provides an optimal model for studies of the cellular mechanisms underlying its effects demonstrated in humans (15). In addition, we tested to what extent addition of IL-7 to mu αCD52 mAb enhances the therapeutic effect by expanding Tregs, and whether addition of αIL-7 mAb ameliorates the treatment effect by suppressing the expansion of Foxp3⁺ Tregs.

Forty female SJL mice were immunized with PLP_139–151 and divided into 4 groups (10 mice per group): the first group received mu αCD52 mAb (200 μg/day), the second group received isotype control IgG2a (200 μg/day), the third group αCD52 mAb and IL-7 (1 μg/day), and the fourth group αCD52 mAb and αIL-7 mAb (150 μg/day) on day 12 p.i. for 5 consecutive days. Monitoring of clinical scores over 60 days, which corresponds to five human years post treatment (18), demonstrated that mu αCD52 mAb suppressed disease activity and prevented the second flare-up in comparison to isotype control (Fig. 1). IL-7 co-administered with αCD52 mAb further decreased the disease activity, while αIL-7 mAb partially reversed the mu αCD52 mAb treatment effect (Fig. 1). IL-7 measurements in serum of αCD52 mAb-treated mice at day 17 and day 23 p.i. revealed an increased IL-7 concentration, but the results did not reach statistical significance (Supplementary Fig. 1).

FIGURE 1.

mu αCD52 mAb treatment suppresses RR EAE. (A) 40 SJL mice were immunized with PLP_139–151 peptide and divided into 4 groups (10 mice per group) that received mu αCD52 mAb (200 μg/day), isotype control IgG2a (200 μg/day), mu αCD52 mAb + IL-7 (1 μg/day), or mu αCD52 mAb + αIL-7 mAb (150 μg/day) at peak of the disease (day 12 p.i.) for 5 consecutive days. Gray area in the figure indicates treatment. Clinical scores were monitored daily for 60 days. The figure presents mean and SEM per group. mu αCD52 mAb suppressed disease activity in comparison to the isotype control group. The administration of IL-7 in addition to mu αCD52 mAb further suppressed disease, while αIL-7 mAb partially reversed the therapeutic effect. Statistical analysis was performed using two-way ANOVA with multiple comparison post-test. * indicates p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Asterisks indicate p values for comparison between isotype control and αCD52 mAb treated mice; comparison between αCD52 mAb and αCD52 mAb+IL-7 treatment revealed significant decrease in clinical scores at day 47 (adjusted p value 0.04), and comparison between αCD52+αIL-7 mAb treatment revealed a significant increase in clinical scores on days 45–49 (adjusted p values <0.05). The experiment was repeated four times.

IL-7 induces expansion of human CD4⁺CD25⁺CD127⁻ Treg cells and increases their expression of FoxP3 and other suppressive markers

Since our human studies reported a longitudinally progressive increase in serum IL-7 levels following αCD52 mAb treatment from day 7 to 6 months post-therapy, and in-vitro studies documented IL-7 induced Foxp3 expression and Foxp3⁺CD4⁺ cell proliferation (4), we extended the study of the in-vitro IL-7 effect in untreated RRMS patients and matched HCs. Fresh PBMCs from 5 RRMS patients and HCs were stained for CD4, CD25, CD127 (gating) and surface Treg markers CTLA-4, CD39, GITR, CD46, and intracellular Foxp3, IL-10, granzyme B, and perforin expression. Following IL-7 stimulation (100 ng/ml) over 2 days, we found a significantly increased percentage of CD4⁺CD25⁺CD127⁺ Treg cells within CD4⁺ cells in both RRMS patients and HCs (Fig. 2A). Phenotyping of those cells revealed increased expression of Foxp3⁺, GITR⁺, CD46⁺ and co-expressing GITR⁺CTLA-4⁺, and Perforin⁺GZMB⁺ Tregs in RRMS patients (Fig. 2B). The results suggest contact-dependent (GITR, CD46) and secreted molecule (Perforin, GZMB)-mediated suppression, induced by IL-7, in the setting of αCD52 mAb-induced lymphopenia.

FIGURE 2.

IL-7 induces Treg expansion and regulatory phenotype in RRMS patients. PBMCs from 5 untreated RRMS patients and 5 matched HCs were stimulated with recombinant human IL-7 (100 mg/ml) over 2 days and flow cytometry was used to determine (A) frequency of CD4⁺CD25⁺CD127⁺ cells within gated CD4⁺ population; and (B) expression of indicated intracellular and surface markers on gated CD4⁺CD25⁺CD127⁺ cells. Each symbol represent one donor, statistical analysis was performed via unpaired t-test, p<0.05 is considered significant.

mu αCD52 mAb treatment depletes CD4⁺ T cells in the CNS and in peripheral immune organs in RREAE

We first tested to what extent mu αCD52 mAb treatment of RREAE depletes the CD4⁺ lymphocytes, as demonstrated in patients with RRMS. On day 25 p.i., mice were sacrificed, and cells isolated from LNs, spleen, brain and spinal cord. mu αCD52 mAb caused a significant depletion of CD4⁺ T cells in all tested organs (Supplementary Fig. 2A). Percentages of CD4⁺ cells within peripheral immune organs and CNS were significantly decreased, with relative sparing of CD4⁺ cells within LNs organs (Supplementary Fig. 2B), suggestive of lymphocyte survival within LNs, as reported previously (19). We propose that homeostatic proliferation of CD4⁺ cells from LNs may contribute to their reconstitution following αCD52 mAb treatment-induced lymphocytopenia.

mu αCD52 mAb treatment of RR EAE increases the frequency of Foxp3⁺ and IL-10⁺ CD4⁺ cells and decreases IL-17A⁺ and IFN-γ⁺CD4⁺ cells in peripheral immune organs and CNS

Studies of the mechanisms of action of αCD52 mAb in an animal model of the disease examined T cell subsets reconstitution and determined to what extent the results replicate findings on T cell reconstitution in RRMS patients (4).

Twenty SJL mice were immunized with PLP_139–151 and treated with αCD52 mAb or IgG2a isotype control at the peak of the disease for 5 days (days 12–16 p.i.). Mice were sacrificed at day 23 p.i., 7 days following completion of the αCD52 mAb treatment, and cells were isolated from PBMCs, LNs, spleen, brain and spinal cord. The percentages of CD4⁺ T cells expressing Foxp3, IL-10, IL-17A and IFN-γ were determined by flow cytometry. mu αCD52 mAb treatment induced a significant increase in the percentage of regulatory Foxp⁺CD4⁺ cells in LNs and spinal cord (p<0.0001 and p=0.03, respectively, Fig. 3A), as well as an increase in IL-10⁺CD4⁺ cells in PBMCs and LNs (p=0.04 and p=0.02, Fig. 3B), and IL-10⁺Foxp3⁺CD4⁺ cells in LNs (p=0.0006, Fig. 3C). In contrast, mu αCD52 mAb-treated mice had decreased percentages of IL-17A⁺CD4⁺ cells in PBMC, LNs, brain and spinal cord (p<0.0001, p<0001, p=0.007, p=0.01, Fig. 3D), IFN-γ⁺CD4⁺ cells in LNs (p<0.0001, Fig. 3E) and IL-17A⁺IFN-γ⁺CD4⁺ cells in LNs and brain in comparison to the control group at day 23 p.i. (p<0.0001, p=0.004, Fig. 3F). Given the immunodepleting effect of this treatment, absolute numbers of Tregs were decreased in LN, while the absolute number of Foxp3⁺CD4⁺ Tregs was significantly increased in spinal cord infiltrate of αCD52 mAb-treated mice. The treatment significantly decreased absolute cell numbers of IL-17A⁺ and IFN-γ⁺ CD4⁺ cells in most of the tested organs (Supplementary Fig. 3).

FIGURE 3.

mu αCD52 mAb induces increased frequency of Foxp3⁺CD4⁺ cells and decreased IL-17⁺CD4⁺ cells in multiple organs in RREAE. SJL female mice were immunized with PLP_139–151 and treated with αCD52 mAb or IgG2a isotype control as in Fig.1. Mice were sacrificed at day 23 p.i., 5 days following completion of the αCD52 mAb treatment (day 12–16 p.i.). Percentages of cells expressing each molecule in gated CD4⁺ T cells were determined by flow cytometry in PBMC, LN, brain- and spinal cord. (A) αCD52 mAb increased percentages of Foxp3⁺CD4⁺ cells in LN and spinal cord; (B) IL-10⁺CD4⁺ cells in PBMC and LN; (C) IL-10⁺Fop3⁺CD4⁺ T cells in LN. (D) Percentages of IL-17A⁺CD4⁺ cells were suppressed in PBMC, LN, brain and spinal cord; (E) IFN-γ⁺CD4⁺ T cells in LNs; (F) IL-17A⁺IFN-γ⁺CD4⁺ T cells in LNs and brains of treated mice in comparison to the isotype control. Statistical analysis was performed using un-paired t-test. p values are indicated in the figures. Horizontal bars represent mean+SD.

mu αCD52 mAb treatment induces long-term increased percentages of Foxp3⁺ and IL-10⁺ CD4⁺ cells and decreased percentages of IL-17A⁺ and IFN-γ⁺ CD4⁺ cells in multiple organs

In order to address the long-term effect of the mu αCD52 mAb therapy, we studied the response to treatment at day 12–16 p.i. at a late time point (day 60 p.i.). Consistent with the long-lasting suppression of clinical scores (Figure 1), mu αCD52 mAb treatment caused a significant increase in the percentages of Foxp3⁺CD4⁺ Treg cells in LNs, spleen, brain, and spinal cord (p=0.0006, p=0.002, p=0.02, p=0.0008, Fig. 4A), as well as an increased percentages of IL-10⁺CD4⁺ cells in PBMC, LNs and spleen (p=0.002, p=0.004, p=0.003, Fig. 4B), and an increased percentage of Foxp3⁺IL-10⁺ double positive CD4⁺ cells in LNs and spleen in comparison to control mice at day 60 p.i. (p=0.0006, p=0.03, Fig. 4C). In contrast, the percentages of IL-17A⁺CD4⁺ cells were decreased in PBMCs, brain, and spinal cord (p=0.0004, p=0.001, p=0.02, Fig. 4D), IFN-γ⁺CD4⁺ T cells were decreased in the brain (p=0.002, Fig. 4E), and the percentage of IL-17A⁺IFN-γ⁺CD4⁺ cells was decreased in the brains of mu αCD52 mAb-treated mice at day 60 p.i. (p=0.01, Fig. 4F).

FIGURE 4.

mu αCD52 mAb treatment induces long-lasting effect of Foxp3⁺CD4⁺ cells and decreased frequency of IL-17⁺CD4⁺ cells in multiple organs. SJL mice with PLP_139–151-induced RR EAE were treated as in Fig. 1. with mu αCD52 mAb or isotype control (10 mice per group). Mice were sacrificed at day 60 p.i. and cells isolated from PBMCs, LNs, spleen, brain and spinal cord for flow cytometry study. (A) mu αCD52 mAb caused increased percentages of Foxp3⁺CD4⁺ Treg cells in LN, spleen, brain and spinal cord; (B) IL-10⁺CD4⁺ cells in PBMCs, LNs and spleen; and (C) Foxp3⁺IL-10⁺CD4⁺ cells in LN and spleen. (D) The treatment decreased IL-17A⁺CD4⁺ cell percentage in PBMCs, brain, and spinal cord; (E) IFN-γ⁺CD4⁺ cells in brain; and (F) IL-17A⁺ FN-γ⁺CD4⁺ cells in brain in comparison to the isotype control group. Statistical analysis was performed using un-paired t-test. p values are indicated in the figures. Horizontal bars represent mean+SD.

Absolute numbers of Foxp3⁺CD4⁺ cells were increased in LN, spleen, brain and spinal cord; however, the changes were statistically significant only in LN and spinal cord (Supplementary Fig. 4A). The absolute numbers of IL-10⁺CD4⁺ cells were significantly increased in PBMCs, LN, spleen, and IL-10⁺Foxp3⁺ double positive cells in the LN (Supplementary Fig. 4B–C). The numbers of IL-17⁺CD4⁺ cells were significantly decreased in PBMCs and brain, while the IFN-γ⁺ and IL-17⁺IFN-γ⁺ CD4⁺ cells were decreased in the brain infiltrates (Supplementary Fig. 4D–F), indicating a long-term effect on Treg expansion and IL-17⁺ and IFN-γ⁺ CD4⁺ cell depletion in treated mice.

A comparison between the fold changes between percentages of treated and untreated mice at two time points (day 23 and day 60 p.i.) related to acute and long-term clinical effects, indicated that the increase in Foxp3⁺CD4⁺ cells was maintained in PBMC, spleen, brain and spinal cord at day 60 p.i. in comparison to day 23, suggesting that a long-term disease suppression is dependent on the presence of Foxp3⁺CD4⁺ cells. The decrease (fold change between percentages of treated and untreated mice) of IL-17A⁺CD4⁺ cells was maintained in spleen, brain and spinal cord at day 60 p.i., suggesting a treatment-induced long-lasting suppression of Th17 inflammatory responses (Figs 3 and 4).

Mu αCD52 mAb suppresses chronic EAE, which is abrogated by transient inducible depletion of Foxp3⁺T cells

We next tested the therapeutic effect of mu αCD52 mAb in chronic EAE induced in C57Bl/6 mice by MOG_35–55 peptide immunization (6 mice per group). Mu αCD52 mAb administered at the onset of disease (day 11 p.i.) for five days (200 μg/mouse) significantly decreased clinical scores in comparison to untreated mice (PBS) (Fig. 5A).

FIGURE 5.

mu αCD52 mAb suppresses chronic EAE, the effect was abrogated upon inducible depletion of Foxp3⁺CD4⁺ Tregs. (A) 12 C57BL/6 WT mice were immunized with MOG_35–55 peptide and divided into 2 groups (6 mice per group) that received mu αCD52 mAb (200 μg/day), or PBS control vehicle at the onset of disease (day 11 p.i.) for 5 consecutive days. Clinical scores were monitored daily for 20 days and presented as mean ± s.e.m. mu αCD52 mAb suppressed disease activity in comparison to the control group. Statistical analysis was performed using two-way ANOVA. (B) 30 DEREG mice were immunized as above and treated with control PBS, mu αCD52 mAb or mu αCD52 mAb+DT. mu αCD52 mAb was administered at 200 μg/mouse starting at day 11 p.i. for 5 days and DT at 200 ng/mouse daily starting at day 12 p.i. for 4 days. Clinical scores were monitored daily. Statistical analysis was performed using two-way ANOVA with multiple comparison post-test. * indicates p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. The experiments were repeated two times.

In order to directly confirm the critical role of Foxp3⁺ Treg cells in the therapeutic effect of mu αCD52 mAb, we employed inducible transient depletion of Foxp3⁺ Treg cells following DT administration to Foxp3-DTR-GFP knock-in mice (20). These Depletion of REGulatory T cell (DEREG) mice express simian diphtheria toxin receptor (DTR)-eGFP transgene under control of the Foxp3 promoter, allowing for Foxp3⁺ Treg cell depletion following DT administration (21). Rodents are resistant to DT due to their 10⁵ times lower DTR affinity, which was confirmed by DT treatment of mice with EAE, which did not show any toxicity (data not shown). In contrast, transgenic expression of high affinity primate DTR in a specific cell type allows for their specific ablation upon DT injection. DT blocks a protein synthesis and induces rapid apoptotic cell death (22). This system has been widely used to study the effect of transient depletion of specific cell types (CD4, CD19, DCs) in the inflammatory response in mice with DTR transgene under the control of a cell type-specific transcription factor (23). Since this mouse is generated on a C57Bl/6 background, we used the chronic EAE model induced by MOG_35–55 peptide in DEREG mice, and treated them at the onset of disease (days 11–15 p.i.) with mu αCD52 mAb (200 μg/mouse) as in Figure 5A. In order to deplete Foxp3⁺ Tregs, 200 ng DT was administered s.c. daily for 4 days with αCD52 mAb treatment for 5 days on day 11–15 p.i.. Clinical scores were monitored in 10 mice per group daily. We confirmed a significant clinical suppression of chronic EAE following mu αCD52 mAb treatment as demonstrated in C57Bl/6 WT mice. Co-administration of DT, which depleted Foxp3⁺Treg cells, completely ameliorated the therapeutic effect (Fig. 5B).

mu αCD52 mAb treatment depletes CD4⁺ T cells in peripheral immune organs and in spinal cord inflammatory infiltrates in chronic EAE

Studies of the CD4⁺ cell numbers in the WT C57Bl/6 mice LN, spleen and spinal cord (6 mice per group) revealed a significant depletion of absolute CD4⁺ T cell numbers following αCD52 mAb treatment (Supplementary Fig. 5A). Similar results were obtained when determining the percentages of CD4⁺ cells in the LN, spleen and spinal cord inflammatory infiltrates (Supplementary Fig. 5B). In DEREG mice treated with mu αCD52 mAb (4 mice per group), we also detected decreased absolute numbers of CD4⁺ cells in LN, spleen and spinal cord, which were partially reversed in mice that received DT (Supplementary Fig. 5C). Similar results were obtained when determining the percentages of CD4⁺ cells in the spleen, LNs and spinal cord inflammatory infiltrates (Supplementary Fig. 5D).

mu αCD52 mAb treatment of chronic EAE induces Foxp3⁺ Treg expansion and suppression of IL-17A⁺ and IFN-γ⁺CD4⁺ cells, which are abrogated following inducible depletion of Foxp3⁺ Tregs

Flow cytometry studies of the LNs, spleen and spinal cord infiltrates in control (PBS) and mu αCD52 mAb-treated DEREG mice revealed a significant increase in absolute numbers of Foxp3⁺CD4⁺ cells in spleen and LNs of αCD52 mAb-treated mice, which were completely depleted in mice that received mu αCD52 mAb+DT (p=0.01, p=0.02, Fig. 6A). Absolute numbers of pro-inflammatory IL-17A⁺CD4⁺ cells were significantly decreased in spleen (p=0.03) and spinal cord (p=0.01) of mu αCD52 mAb-treated mice, which were partially reversed with DT administration (Fig. 6B). IFN-γ⁺CD4⁺ cell numbers were decreased in spleen, LNs and spinal cord (p=0.02, p=0.07, p=0.001) and were partially reversed in mice that received DT (Fig. 6C). The percentages of Foxp3⁺CD4⁺ cells were increased in spleen and LNs of αCD52 mAb-treated mice (p=0.0006, p=0.001) and were completely depleted following DT administration (Fig 6D). The percentages of IL-17A⁺CD4⁺ (p=0.02, Fig. 6E) and IFN-γ⁺CD4⁺ cells in spinal cord were decreased by mu αCD52 mAb treatment and reversed by DT administration (both p=0003, Fig. 6F).

FIGURE 6.

mu αCD52 mAb induces expansion ofFoxp3⁺CD4⁺ cells and decreases numbers of IL-17⁺CD4⁺ and IFN-γ⁺CD4⁺ cells in multiple organs, which was partially reversed by the Foxp3⁺CD4⁺ cell depletion in DEREG mice. Ten mice per group with chronic EAE were untreated, treated with αCD52 mAb or αCD52 mAb and DT. The mice were sacrificed at day 25 p.i. (A) Absolute numbers of Foxp3⁺ CD4⁺ cells; (B) IL-17A⁺CD4⁺ cells; and (C) IFN-γ⁺CD4⁺ cells in LN, spleen and spinal cord in DEREG mice treated with control PBS, mu αCD52 mAb and mu αCD52 mAb+DT. (D) Percentages of Foxp3⁺CD4⁺ cells in LN and spleen (E) percentages of IL-17A⁺CD4⁺ and IFN-γ⁺CD4⁺ cells in spinal cord of mice treated with control PBS, mu αCD52 mAb and mu αCD52 mAb+DT. Statistical analysis was performed using two-way ANOVA with multiple comparison post-test. p values are indicated in figures. Horizontal bars represent mean+SD.

Discussion

Our published study on alemtuzumab-treated RR MS patients suggested that therapeutic effect is mediated by nearly complete depletion of lymphocytes from the peripheral circulation, and that differential reconstitution of T cell subsets may contribute to the delayed repopulation of CD4⁺ cells. The delayed reconstitution of CD4⁺ cells, resulting from deficient CD4⁺ cell homeostatic proliferation, susceptibility to apoptosis and a relative expansion of immunoregulatory CD4⁺CD25⁺CD127^low Treg, and induced peripheral (i) IL-10⁺CD4⁺ Tregs, is proposed to mediate the alemtuzumab-induced long-lasting clinical disease suppression.

In the present animal study, we show that mu αCD52 mAb causes disease suppression in two animal models, PLP_139–151-induced RR EAE in SJL mice and MOG_35–55-induced chronic EAE in C57BL/6 mice, in comparison to controls, which had a second flare-up in RR EAE and chronic disease progression in the chronic EAE model. These animal models recapitulate some aspects of human disease and provide an opportunity to study CNS infiltrates, which are not accessible in human studies (24).

IL-7, a key cytokine promoting homeostatic proliferation in the setting of lymphopenia (25), is significantly increased in the serum in the longitudinal study of alemtuzumab-treated RR MS patients from day 7 to 6 months post-treatment in comparison to the baseline levels (4). The results suggested its role in the expansion of Tregs following alemtuzumab-induced immunodepletion. Clinical data in the current study from SJL/PLP_139–151 RR EAE mice confirmed that addition of IL-7 to the mu αCD52 mAb enhanced its therapeutic effect, while addition of αIL-7 mAb decreased the αCD52 mAb treatment effect. While the effect of IL-7 is complex and also induces expansion of T effector cells (26), our results provide clinical evidence that administration of IL-7 in the setting of αCD52 mAb-induced lymphopenia promotes Treg expansion and ameliorates clinical disease. The increased IL-7 serum levels of mice treatd with αCD52 mAb did not reach statistical significance, but are in line with above human studies from αCD52 mAb-treated RR MS patients and with the above mice clinical data. The differences in the increase in serum IL-7 in treated mice and patients may be related to species differences in production of this cytokine.

To further test the effect of IL-7 on the expansion and phenotype of Tregs in RRMS patients, we performed an in-vitro study, where CD4⁺CD25⁺CD127^low Treg expansion was demonstrated following IL-7 stimulation in both RRMS patients and matched HCs. IL-7 increased expression of Treg transcription factor Foxp3, and Foxp3-regulated Treg marker GITR, which induces Treg proliferation and expansion (27). IL-7 also increased expression of CD46, whose activation stimulates IL-10 secretion from inducible Tregs, and co-expression of GITR and CTLA-4, and secreted enzymes perforin and GZMB, which mediate Treg suppression via cell lysis. We propose that the phenotype of IL-7-expanded Tregs reflects the mechanism of their suppressive activity upon homeostatic proliferation. Since the phenotype of Tregs detected in alemtuzumab-treated patients after 1 month had also increased GITR, Granzyme B and perforin expression, we propose that the Treg reconstitution is mediated via increased IL-7 serum levels in the setting of treatment-induced lymphocytopenia.

Ex-vivo studies of the CD4⁺ cell subsets in RR EAE have demonstrated that αCD52 mAb induced increased percentages of Foxp3⁺CD4⁺ cells in the peripheral immune organs and in CNS. In addition, we have demonstrated a suppression of IL-17A⁺CD4⁺ cells in PBMCs, LNs, brain and spinal cord in mice with RR EAE 5 days following treatment. Consistent with the long-lasting suppression of the disease, we have also demonstrated an increased percentage of Foxp3⁺CD4⁺ cells in the peripheral immune organs and the CNS, as well as the suppression of IL-17A⁺CD4⁺ cells in the PBMCs and CNS infiltrates at day 60 p.i. These results are consistent with a study by Turner et al. (15), who reported a similar decrease in RREAE and chronic EAE scores following mu αCD52 mAb treatment, and depletion of CD4⁺ cells in spleen and CNS infiltrates. They reported a significant decrease in the number of spleen-derived IL-17A⁺ and IFN-γ⁺ CD4⁺ cells when stimulated in-vitro with MOG_35–55. However, they did not study repopulation of the CD4⁺ cell subsets, particularly Treg cells. Consistent with our data, Pant et al. (28) have reported that mu αCD52 mAb treatemtns of chronic EAE significantly decreased inflammatory infiltrate and demyelination in the spinal cord and increased IL-10, while decreasing IL-17, IFN-γ and RORγτ gene expression in treated mice.

Finally, the reversal of αCD52 mAb treatment effect in DEREG mice with inducible transient depletion of Foxp3⁺ Tregs directly demonstrated a role of Treg cells in the suppression of IL-17A⁺ and IFN-γ⁺CD4⁺ cells in the spinal cord infiltrates and in the therapeutic mechanism of αCD52 mAb. Since Moletedo et al. (29) reported transient generalized lymphopenia restricted to peripheral but not thymic T cells at day 2 post DT administration, which subsequently had increased T cell activation by day 4, we would like to emphasize that our study determined T cell responses at day 9 post DT administration when lymphocyte counts have normalized. While due to limited Treg cell numbers, we did not demonstrate recovery of Treg suppressive function in the in-vitro suppressive assay, clinical suppression of EAE following αCD52 mAb-induced Treg persistence indicates that Tregs functionally suppress IL-17A⁺ and IFN-γ⁺ CD4⁺ responses in-vivo. We did not treat DEREG mice with DT, as that had already been reported by Kurtolos et al. (30), who reported that DT-induced depletion of Treg cells severely exacerbated EAE. The study confirmed Treg control of Teff cells in the CNS of mice with EAE. We chose to use transient selective Treg depletion in DEREG mice with EAE, similar to a study by Buenafe et al. (31), to provide a proof of principle that αCD52 mAb treatment effect is dependent on the presence of Tregs. Since the αCD52mAb effect in DEREG+DT treated mice is similar, but not more severe than in WT mice (30), it is possible that αCD52 mAb therapeutic effect is partially mediated independently of Treg cells,.

In summary, the current results indicate that the mechanism of the long-lasting αCD52 mAb clinical disease suppression is mediated via Treg cell suppression of inflammatory IL-17A⁺ and IFN-γ⁺ CD4⁺ cells, as demonstrated in αCD52 mAb-treated RRMS patients (4).

Alemtuzumab is a very effective long-lasting therapy approved for selected patients with aggressive RRMS, who fail first-line therapies and are at risk of progressive multifocal leukoencephalopathy (PML) associated with αVLA-4 mAb and αCD20 mAb. However, its use is limited due to prevalent side effects, particularly secondary autoimmunity observed in 35% of treated patients. The current study did not address B cell repopulation, which may lead to the expansion of antibody-producing memory B cells and antibody-mediated thyroid disease, thrombocytopenia and glomerulonephritis (32). Our previously reported human study (4) demonstrated that increased frequencies of IL-4-, TGF-β− and IL-10-producing CD4⁺ cells may contribute to B cell maturation (33), regulation of Ab isotypes (34) and the induction of Ab production (35). Future studies of B cell repopulation are needed to understand the induction of secondary autoimmunity and to advise about add-on therapies. In the current therapeutic landscape, αCD52 mAb is a treatment for selected patients.

Key Points:

αCD52 mAb suppresses EAE via Treg expansion

IL-7 induces expansion of CD4⁺CD25⁺CD127⁻ Tregs from RRMS patients

αCD52 mAb induces suppression of IL-17⁺ and IFN-γ⁺ CD4⁺ cell responses

Data Availability Statement

The data generated in this study are available upon request to the corresponding author.