Alemtuzumab long-term immunologic effect

Objective: To analyze changes in T-helper (Th) subsets, T-regulatory (Treg) cell percentages and function, and mRNA levels of immunologically relevant molecules during a 24-month follow-up after alemtuzumab treatment in patients with relapsing-remitting multiple sclerosis (RRMS). Methods: Multicenter follow-up of 29 alemtuzumab-treated patients with RRMS in the Comparison of Alemtuzumab and Rebif Efficacy in Multiple Sclerosis (CARE-MS) I and CARE-MS II trials. Peripheral blood (PB) samples were obtained at months 0, 6, 12, 18, and 24. We evaluated (1) mRNA levels of 26 immunologic molecules (cytokines, chemokines, chemokine receptors, and transcriptional factors); (2) Th1, Th17, and Treg cell percentages; and (3) myelin basic protein (MBP)–specific Treg suppressor activity. Results: We observed 12 relapses in 9 patients. mRNA levels of the anti-inflammatory cytokines interleukin (IL)–10, IL-27, and transforming growth factor–b persistently increased whereas those of proinflammatory molecules related to the Th1 or Th17 subsets persistently decreased after alemtuzumab administration throughout the follow-up period. PB CD41 cell percentage remained significantly lower than baseline while that of Th1 and Th17 cells did not significantly change. A significant increase in Treg cell percentage was observed at month 24 and was accompanied by an increase in Treg cell suppressive activity against MBP-specific Th1 and Th17 cells. Conclusions: The long-lasting therapeutic benefit of alemtuzumab in RRMS may involve a shift in the cytokine balance towards inhibition of inflammation associated with a reconstitution of the PB CD41 T-cell subsets that includes expansion of Treg cells with increased suppressive function. Neurol Neuroimmunol Neuroinflamm 2016;3:e194; doi: 10.1212/NXI.0000000000000194 GLOSSARY BBB5 blood–brain barrier; CARE-MS5 Comparison of Alemtuzumab and Rebif Efficacy in Multiple Sclerosis; CCL5 C-C motif ligand; CCR 5 C-C chemokine receptor; CXCL 5 C-X-C motif ligand; CXCR 5 C-X-C chemokine receptor; EDSS 5 Expanded Disability Status Scale; ELISPOT5 enzyme-linked immunospot; FACS5 fluorescence-activated cell sorting; FoxP35 forkhead box P3; IFN 5 interferon; IL 5 interleukin; mAb 5 monoclonal antibodies; MBP 5 myelin basic protein; MS 5 multiple sclerosis; PB 5 peripheral blood; PBMC 5 peripheral blood mononuclear cells; PPD 5 purified protein derivative of tuberculin; RORC 5 retinoid-related orphan receptor g; RR 5 relapsing-remitting; Tbet 5 T-box expressed in T cells; TGF 5 transforming growth factor; Th 5 T-helper; TNF 5 tumor necrosis factor; Treg 5 T-regulatory cells; VLA 5 very late antigen. Alemtuzumab is a humanized monoclonal antibody that targets the CD52 antigen, expressed mainly on the surface of T and B lymphocytes and less on natural killer cells, monocytes, and dendritic cells. Besides its use in fludarabine-resistant B-cell chronic lymphocytic leukemia, alemtuzumab was tested in 2 phase III trials (Comparison of Alemtuzumab and Rebif *These authors contributed equally to this work. From the Division of Neurology (S.D.M., S.R., A.C., L.D., M.C.) and the Department of Clinical and Biological Sciences (S.D.M., A.C., L.D., M.C.), University of Torino, San Luigi Gonzaga University Hospital, Orbassano; Center for Experimental Research and Medical Studies (CERMS) (S.R., V.B., F.N.), Azienda Ospedaliera Città della Salute e della Scienza di Torino; Department of Molecular Biotechnology and Health Sciences (V.B., F.N.), Università degli Studi di Torino; Multiple Sclerosis Center (E.C.), Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Italy; Department of Neurology (A.V., S.S.-B.), Clinical Hospital Sveti Duh Zagreb; Medical Faculty University (A.V., S.S.-B.), J.J. Strossmayer Osijek; Department of Neurology (M.H., I.A.), Referral Center for Demyelinating Diseases of the Central Nervous System, University Hospital Center Zagreb, Croatia; Department of Neurology and Center of Clinical Neuroscience (D.H.), Charles University in Prague, First Faculty of Medicine and General University Hospital, Czech Republic; and Multiple Sclerosis Study Center (P.A.), AO S. Antonio Abate, Gallarate (VA), Italy. Funding information and disclosures are provided at the end of the article. Go to Neurology.org/nn for full disclosure forms. The Article Processing Charge was paid by Dipartimento di Scienze Cliniche e Biologiche–Torino University. ‡These authors share senior authorship. Coinvestigators are listed on the Neurology® Web site at Neurology.org/nn. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BYNC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially. Neurology.org/nn © 2016 American Academy of Neurology 1 a 2016 American Academy of Neurology. Unauthorized reproduction of this article is prohibited. Efficacy in Multiple Sclerosis [CARE-MS] I and CARE-MS II) in relapsing-remitting (RR) multiple sclerosis (MS). These 2 trials were designed to test the superiority of alemtuzumab vs interferon (IFN)–b-1a, 44 mcg subcutaneously, 3 times a week. Compared with IFN-b, alemtuzumab significantly reduced relapse rate by 49% to 55% and the risk of sustained accumulation of disability over 6 months by 42%. A single therapeutic course of alemtuzumab induces severe Tand B-cell depletion, with lymphocyte repopulation occurring over several months, and an increase of the percentage of T-regulatory cells (Treg). T-helper (Th) subsets are involved in MS pathology, and brain MS lesions are enriched in Th1 and Th17 cells. Th17 and Th1/17 cells (an intermediate subset producing interleukin [IL]–17 and IFN-g) are increased in the peripheral blood (PB) and CSF at the time of a new relapse and cross the blood–brain barrier (BBB) more easily than Th1 cells. Some reports have examined the CD19, CD4, and CD8 subsets and related cytokine changes in a few patients treated with alemtuzumab. No studies have evaluated Treg function in alemtuzumab-treated patients with MS. We organized a multicenter 24-month study to analyze the changes in Th subsets, Treg proportion and function, and mRNA levels of cytokines and other immunologically related molecules during phase III alemtuzumab trials. The long-term follow-up of immune system reconstitution together with clinical and radiologic data could provide practical tools to define the timing of repeated alemtuzumab courses. METHODS Patients and clinical study design. Twentynine patients with RRMS participating in CARE-MS I and II trials in 6 European MS centers entered this study and were evaluated at baseline and for 24 months after alemtuzumab treatment. Inclusion and exclusion criteria were described in the original articles. Patients were treated with 12 mg/d IV alemtuzumab in 2 annual courses (5 administrations at month 0 and 3 administrations at month 12). Patients’ demographic and clinical characteristics are reported in the table. Neurologic assessments, performed by blinded investigators, were done at baseline and repeated every month or in case of relapses. Blood samples were taken at baseline (before first alemtuzumab course) and at months 6, 12 (before second alemtuzumab course), 18, and 24. At each time point, blood cell count, coagulation, thyroid, liver, and kidney function were analyzed in a central facility. Fresh blood was collected in heparin-treated vacutainers and immediately sent to a single coordinating center located at the University of Torino for immunologic testing. All samples were received and processed within 48 hours from the blood withdrawal. Standard protocol approvals, registrations, and patient consents. The institutional review board of the participating centers approved the study and all subjects gave written informed consent (protocol number Bio2009001). Cytokine mRNA analysis. Aliquots (0.5 mL each) of blood were mixed with 1.3 mL RNAlater (Ambion, Life Technologies, Carlsbad, CA) immediately after arrival at the coordinating center and stored at 280°C. To determine mRNA levels, samples were treated as previously described: RNA was extracted with the RiboPure Blood Kit (Ambion) and cDNA obtained with the high-capacity cDNA reverse transcription kit (Applied Biosystems, Life Technologies, Monza, Italy). We assessed the mRNA levels of the following 26 immunologically relevant molecules whose function in MS has been documented by using TaqMan low-density arrays (Applied Biosystems 7900HT real-time PCR system): molecules with proinflammatory function including IL-1b, IL-2, IL-6, IL-12, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-26, IFN-g, T-box expressed in T cells (Tbet), retinoid-related orphan receptor g (RORC), tumor necrosis factor–a (TNF-a), C-C chemokine receptor type 3 (CCR3), CCR4, CCR5, CCR6, C-X-C chemokine receptor type 3 (CXCR3), C-X-C motif ligand 10 (CXCL10), C-C motif ligand 20 (CCL20), and very late antigen 4 (VLA4); and molecules with anti-inflammatory function, including IL-10, IL-27, transforming growth factor–b (TGF-b), and forkhead box P3 (FoxP3). Glyceraldehyde-3 phosphate dehydrogenase served as the housekeeping gene for normalization. The relative expression of each gene was calculated using the comparative threshold cycle method as directed by the manufacturer (User Bulletin No. 2, Applied Biosystems) and expressed in arbitrary units as previously described in detail. Flow cytometry (fluorescence-activated cell sorting [FACS]). PB mononuclear cells (PBMC) were isolated by density gradient centrifugation from heparinized venous blood. PBMC were stained for Treg cells with anti-CD4, anti-CD25, and anti-CD127 monoclonal antibodies (mAb) (Biolegend, San Diego, CA) on the cell surface. For detection of the transcriptional factor FoxP3, cells were fixed with fixation and permeabilization buffers (eBioscience, San Diego, CA) and were then stained with anti-FoxP3 mAb (eBioscience). The Table Demographic data