Methods: Immune cell migration was evaluated using both in vitro and in vivo analysis. Lymphocyte migration in vitro was evaluated in transwell assays using CD4 T cells purified from anti-muCD52-treated mice. In vivo migration was assessed using an acute CNS inflammation model in which mice were injected intracerebroventricularly with lipopolysaccharide (LPS) following anti-muCD52 treatment. Similarly, migration of innate immune cell subsets was evaluated in a peritonitis model of inflammation, in which animals were injected intraperitone- ally with thioglycollate 3 days following anti-muCD52 treatment. The numbers of CNS-infiltrating lymphocytes or peritoneum-infiltrating cells were measured by polychromatic flow cytometry. Results: Immune cells from anti-muCD52-treated mice retained their ability to migrate. In vitro, CD4 T cells were able to migrate in response to SDF-1alpha as efficiently as cells from vehicle-treated animals. In vivo, lymphocytes in anti-muCD52-treated animals were able to migrate into the CNS following local induction of inflamma- tion by LPS. Similarly, innate immune cell migration was not affected by anti-muCD52 treatment, as similar numbers and types of cells were observed in the inflamed peritoneum of anti-muCD52- and vehicle-treated animals. Conclusions: These results indicate that following anti-CD52 treat- ment, immune cells retain their ability to migrate. These findings suggest that anti-CD52 treatment may not compromise immune surveillance, but further studies are required. Study Supported by: Genzyme, a Sanofi company. doi:10.1016/j.jneuroim.2014.08.165 177 Increased neutralization capacity of TNF alpha in sera of MS patients may play a role in — MS pathogenesis and is reversed by therapy with interferon-γ Karin Fainberg a , Keren Regev b , Olga Dorman a , Arnon Karni c a Neuroimmunology Laboratory, Tel Aviv Medical Center, Tel Aviv, Israel; b Neuroimmunology Laboratory, Tel Aviv Medical Center, Tel Aviv, Israel; c Neuroimmunology Laboratory, Tel Aviv Medical Center, Sackler's Medical School, Tel Aviv University, Tel Aviv, Israel Background: High TNFa is found in active MS lesions and in CSF of MS patients it correlates with disease activity. However, TNFa neutralizing agents induced worsening of disease activity in MS studies, and TNFa antagonist therapy, in other inflammatory diseases may cause demyelinative neurological adverse events. These suggest that TNFa neutralization may play a role in inflammatory demyelinating diseases. Objectives: To study TNFα neutralization capacity and levels of anti- TNFa antibodies (Ab) in the sera of RRMS patients. Methods: Twenty six untreated RRMS patients, 27 interferon β1a (IFNb1a) treated RRMS patients and 28 matched healthy controls (HC) participated in the study. The capacity of sera to neutralize TNFaTNFa bioactivity was studied by a neutralization of TNFaTNFa-induced death of L929 cell line. Briefly, 1 ng/ml of rhTNFa was incubated with dilutions of sera from donors for 2 h, 37 °C. Then, 50 μl/well of the serum-rhTNFa mixed solution was added to the cells, with 1 μg/ml Actinomycin D, for overnight, 37 °C, 5% CO 2 . Cell survival was examined by XTT assay, and OD 450 nm measurement. TNFa neutralization was calculated by the percentage of blockade of the TNFa killing effect on the cell line. Serum levels of TNFa and binding anti-TNFa were studied by ELISA and are expressed in OD 450 nm . Results: Sera of untreated RRMS patients had a higher TNF-α neutralization capacity vs. HC. For serum titer of 1:8 the average neutralization of TNFa effect in untreated RRMS was 57.8 ± 20.8% vs. 32.4 ± 20.6% in HC, p = 0.025. For titer of 1:16 the averages of neutralization of TNFa effect were 51.0 ± 19.3% vs. 22.4 ± 22.5%, respectively, p = 0.017, and for titer 1:32: 36.4 ± 12.8% vs. 21.7 ± 16.1%, p = 0.05. In IFNb1a treated patients, TNFα neutralization capacity was similar to HC, e.g. for titer 1:32 the average of TNFa neutralization effect was 20.4 ± 19.0%, p = 0.027 in vs. untreated patients. TNFa was detected more in the HC (38.5%) vs. untreated RRMS (29.4%) and treated patients (17.4%). Anti TNFa binding Ab levels were higher in HC (0.545 ± 0.209) vs. untreated RRMS (0.436 ± 0.118, p = 0.021) and vs. IFNb1a treated patients (0.372 ±0.097, p = 0.037). Conclusions: TNFa neutralization is known to induce MS-like lesions. We found that untreated RRMS patients have an increased TNFa neutralization capacity. These findings suggest that blockade of TNFa signaling may play a role in the pathogenesis of MS. No correlation was found between the TNFa neutralization capacity and the levels of anti TNFa binding Ab. Therapy with IFNb1a normalized the TNFa neutralization capacity. This is an unreported mode of action of IFNb1a. doi:10.1016/j.jneuroim.2014.08.166 385 Lymphocyte subset dynamics following alemtuzumab treatment in patients who relapsed on a prior therapy Lloyd H. Kasper a , Douglas L. Arnold b , Alasdair J. Coles c , Hans-peter Hartung d , Eva Havrdova e , Krzysztof W. Selmaj f , Jeffrey Palmer g , David H. Margolin g , Michael A. Panzara g , D. Alastair S. Compston h a Geisel School of Medicine at Dartmouth, Geisel School of Medicine, Hanover, United States; b Department of Neurology and Neurosurgery, Montréal Neurological Institute, McGill University, Montréal, Québec, Canada; c University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom; d Department of Neurology and Center for Neuropsy- chiatry, Heinrich-Heine University, Düsseldorf, Germany; e Charles University in Prague, First Medical Faculty, Prague, Czech Republic; f Department of Neurology, Medical University of Łódź, Łódź, Poland; g Genzyme, a Sanofi company, Cambridge, MA, United States; h Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom Background: Alemtuzumab, a humanized monoclonal antibody, is approved in over 30 countries for active relapsing–remitting multiple sclerosis (RRMS). Alemtuzumab selectively targets CD52 to deplete circulating T and B lymphocytes, followed by a distinctive pattern of T- and B-cell repopulation. In the phase 3 CARE-MS II study, alemtuzumab had significant positive treatment effects on clinical efficacy in RRMS patients with disease activity despite prior disease-modifying therapy. Objective: To investigate the effect of alemtuzumab on lymphocyte subsets in CARE-MS II (NCT00548405). Methods: Patients (n = 667) were randomized to alemtuzumab (12 mg/day intravenously on 5 consecutive days at study start and on 3 consecutive days 12 months later) or interferon beta-1a (44 mcg subcutaneously 3 times weekly). Blood counts were tested monthly. Circulating lymphocytes were phenotyped by flow cytom- etry quarterly and at Months 1 and 13. Results: Mean alemtuzumab serum concentrations became low or undetectable within ~ 1 month after each course. After alemtuzumab treatment, lymphocyte counts were lowest at the earliest post-treatment time point (1 month); B cells approached baseline levels by Month 6 while T cells repopulated more slowly. Differential depletion/repopulation of lymphocyte subsets led to Abstracts 63