Long-Term Decrease in VLA-4 Expression and Functional Impairment of Dendritic Cells during Natalizumab Therapy in Patients with Multiple Sclerosis

Myeloid and plasmacytoid dendritic cells (mDCs, pDCs) are central to the initiation and the regulation of immune processes in multiple sclerosis (MS). Natalizumab (NTZ) is a humanized monoclonal antibody approved for the treatment of MS that acts by blocking expression of VLA-4 integrins on the surface of leukocytes. We determined the proportions of circulating DC subsets and analyzed expression of VLA-4 expression in 6 relapsing-remitting MS patients treated with NTZ for 1 year. VLA-4 expression levels on pDCs and mDCs decreased significantly during follow-up. In vitro coculture of peripheral blood mononuclear cells and pDCs, with different doses of NTZ in healthy controls (HC) and MS patients showed dose-dependent down-regulation of VLA-4 expression levels in both MS patients and HC, and reduced functional ability to stimulate antigen-specific T-lymphocyte responses. The biological impact of NTZ may in part be attributable to inhibition of transmigration of circulating DCs into the central nervous system, but also to functional impairment of interactions between T cells and DC

Perforin Expression by CD4+ Regulatory T Cells Increases at Multiple Sclerosis Relapse: Sex Differences

Abstract: Multiple sclerosis (MS) represents the leading cause of neurological deficit among young adults, affecting women more frequently than men. In MS, the extent of central nervous system lesions is determined by the net balance between self-reactive and regulatory T-cells at any given time, among other factors, as well as by the effect of inflammatory response. Here, we studied both CD4+ and CD8+ TReg in parallel in blood and CSF during MS relapse. A recruitment of both regulatory CD4+ and CD8+ T cells (TReg) within the cerebrospinal fluid (CSF) takes place during MS relapse. Not previously described, the presence of CD4+ TReg in CSF was higher in women than in men, which could account for the sexual dimorphism in the incidence of MS. A direct correlation between plasma oestradiol (E2) and IL-2 levels was observed, in line with a putative circuit of E2 and perforin expression by CD4+ TReg playing a role in MS. Also, serum IFN-alpha was higher in females, with direct correlation with serum E2 levels. This is the first study to analyze perforin expression by CD4+ TReg in MS, which was greatly enhanced in CSF

<i>In vitro</i> effect of natalizumab (NTZ) on VLA-4 expression on pDCs after 6 days of culture of PBMCs in the presence of antigen-specific stimulus (PPD) in NTZ-treated MS patients at different time-points (baseline, at 48 hours, and at 3 months).

<p>In vitro, VLA-4 expression on the surface of pDCs was greatly enhanced at day 6 with/without PPD, while NTZ was able to down-modulate it in NTZ-treated MS patients. <i>In vitro</i>, we observed that the enhanced expression of VLA-4 on pDCs was diminished in NTZ-treated MS patients more markedly after 3 months of therapy and that their pDCs were more sensitive <i>in vitro</i> to the VLA-4 down-modulating effects of NTZ after therapy. Isotypic IgG4 control is marked in red.</p

Effect of natalizumab (NTZ) on the immunogenic function of pDCs.

<p>CFSE-labelled autologous T lymphocytes from the same patient before NTZ treatment (baseline), at 48 hours, and at 3 months of NTZ therapy were cocultured with PPD-treated pDCs at a pDC∶PBMC ratio of 1∶10. After 6 days, lymphocytes were harvested, and the percentages of proliferating T cells (CFSElow) were calculated. In vitro, NTZ induced reduced CD4+ T-lymphocyte proliferation in PPD-specific responses in 4 individual NTZ-MS patients. The proliferative responses were weaker when we used pDCs from the same patients after 48 hours and 3 months of treatment with respect to baseline, suggesting an ongoing progressive functional impairment.</p

Changes in the in vivo expression of VLA-4 on pDCs before and after 48 hours of natalizumab (NTZ) therapy in MS patients, compared with healthy controls (HC).

<p>Isotypic IgG4 control is marked in red. The example shown is representative for data obtained from the 6 RRMS patients and 10 HC.</p

Demographic and clinical characteristics of patients with relapsing-remitting multiple sclerosis (n = 6).

<p>Definition of relapsing-remitting multiple sclerosis according to Poser et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034103#pone.0034103-Poser1" target="_blank">[25]</a> ARR = annualized relapse rate. EDSS = Expanded Disability Status Scale (Kurtzke).</p

Dose-dependent down-modulation of VLA-4 expression on pDCs in vitro after 6 days of culture of PBMCs from healthy controls with 3 different doses of natalizumab (NTZ)—22 (×1/5), 110 (1×), and 550 µg/mL (5×), respectively—and antigen-specific stimulus (PPD, 50 IU/mL).

<p>The case shown is representative of 3 independent experiments. Isotypic IgG4 control is marked in red. These results were not reproduced in the MS patients tested (n = 3).</p

Proportions of circulating subsets of DCs (mDCs and pDCs) and their expression of adhesion molecules (VLA-4 and LFA-1) at baseline and during natalizumab therapy (NTZ = 6) in MS patients, healthy controls (HC = 10), and untreated RRMS controls (MSC = 4).

<p>Data are presented as mean± standard deviation (SD), median (IQR). *Sig. (2-tailed) with respect to baseline by Wilcoxon Signed Ranks Test. ¥Sig. (2-tailed) with respect to healthy controls by Mann-Whitney U Test. *p<0.05; **p<0.01; ***p<0.001</p