Effect of Natalizumab Treatment on Circulating Plasmacytoid Dendritic Cells: A Cross-Sectional Observational Study in Patients with Multiple Sclerosis

Objectives: Dendritic cells (DCs) serve a critical role both in promoting and inhibiting adaptive immunity. The goal of this study was to investigate the effect of natalizumab (NTZ) treatment on DC numbers, phenotype, and function in patients with multiple sclerosis (MS). Methods: Frequency and phenotype of myeloid and plasmacytoid DCs (MDCs and PDCs, respectively) were analyzed in blood from two separate cohorts of untreated, interferon-treated, or NTZ-treated MS patients. In addition, PDCs were stimulated with CpG-containing oligonucleotides or co-cultured with homologous T cells in the presence or absence of NTZ in vitro to determine functional effects of NTZ treatment. Results: We observed that NTZ treatment was associated with a 25–50% reduction in PDC frequency in peripheral blood as compared to untreated MS patients, while the frequency of MDCs was unchanged. PDCs in NTZ-treated patients displayed a mature, activated phenotype with increased expression of HLA-DR, TLR9, CCR7, IL-6 and IL-12. In contrast, in vitro treatment with NTZ did not increase markers of PDC activation or their ability to induce T cell differentiation. Conclusion: Our study shows that NTZ treatment is associated with a reduced frequency of PDCs in the peripheral circulation, but that PDCs in NTZ-treated individuals display an activated phenotype. Taken together the data suggests that transmigration of activated PDCs is preferentially affected by blockade of integrin α4 leading to an increased frequency of activated PDCs in blood

Effects of natalizumab treatment on plasmacytoid dendritic cells function.

<p>Effects of natalizumab (NTZ) treatment on the functional ability of plasmacytoid dendritic cells (PDCs) to stimulate CD4 cell responses. PDCs were isolated from healthy controls using FACS sorting and stimulated with ODN-2006 for 18 hours in the presence of 15 µg/ml of NTZ or an isotype control and homologous CD4+ T cells were added. CD4+ T cells were isolated after an additional 6 days of incubation and cytokine mRNA expression determined using rt-PCR. Graph shows expression in cultures treated with NTZ or an isotype.</p

Frequency of myeloid and plasmacytoid dendritic cells in peripheral blood.

<p>Frequency of myeloid and plasmacytoid dendritic cells (MDCs and PDCs) was determined in frozen cells from relapsing-remitting MS patients using flow cytometry. A. Dead cells were excluded using a viability dye (LIVE/DEAD), followed by exclusion of cells expressing mature hematopoietic lineages using a Lineage cocktail (CQ). PDCs and MDCs were identified based on their expression of CD123 and CD11c, respectively. B–C. Frequency of MDCs and PDCs in untreated MS patients (UNT) and MS patients treated with natalizumab (NTZ) or interferon-β (IFN) from Cohort 1 (B) and Cohort 2 (C). Graph shows mean, interquartile range, and min-max range. Frequency of PDCs in NTZ-treated patients in relation to duration of treatment in patients from Cohort 1 (filled) or Cohort 2 (open) (D).</p

CD49d expression on plasmacytoid dendritic cells.

<p>Mean fluorescence intensity (MFI) of CD49d on plasmacytoid dendritic cells (PDCs)(A) and frequency of PDCs expressing CD49d (B), in peripheral blood from untreated MS patients (UNT) and MS patients treated with natalizumab (NTZ) or interferon-β (IFN) determined by flow cytometry. Box-and-whisker plots showing mean, interquartile range, and min-max range. Representative histograms showing CD49d staining on PDCs from an UNT (blue open; C, E) and a NTZ-treated (red filled; D, E) patient.</p

Phenotype and cytokine expression of plasmacytoid dendritic cells.

<p>Frequency of plasmacytoid dendritic cells (PDCs) expressing TLR9 (A), HLA-DR (B), and CCR7 (C) in peripheral blood from untreated MS patients (UNT) and MS patients treated with natalizumab (NTZ) or interferon-β (IFN) determined using flow cytometry. Representative histograms showing TLR9 (D) and HLA-DR (E), staining on PDCs from an UNT (blue open) and a NTZ-treated (red filled) patient. Flow plots showing CCR7 staining on CD49d^hi and CD49d^low PDCs from an UNT (F) and a NTZ-treated (G) patient. Expression of IL-6 (H) and IL-12 (p35) (I) mRNA in PDCs isolated from the patients described in A-C using FACS sorting and rt-PCR without ex-vivo stimulation. Graph shows mean, interquartile range, and min-max range (A–C) or mean and SD (H–I).</p

In vitro effects of natalizumab treatment on plasmacytoid dendritic cells.

<p>Effects of natalizumab (NTZ) treatment on plasmacytoid dendritic cells (PDCs) stimulated in vitro with CpG-containing oligonucleotides (open: ODN-2006; gray: ODN-2336). (A) PDCs were isolated from healthy controls using FACS sorting and stimulated in the presence of 15 µg/ml of NTZ or an isotype control for 4 hours. The expression of CD49d, IL-6, IL-12 (p35), TNF, TLR9, CD80, HLA-DR, and CCR7 mRNA was determined using rt-PCR. (B) Total PBMCs were stimulated in the presence of 15 µg/ml of NTZ or an isotype control for 18 hours. Surface expression of CD49d, CD40, CD80, CD83, CD86, CCR7, and HLA-DR was determined on PDCs using flow cytometry. Graphs shows fold change in expression comparing cultures treated with NTZ to cultures treated with an isotype control.</p

Patient demographics.

<p>*Number of donors with a documented relapse during 24 months of follow-up. M = male; F = female. Data is expressed as mean (range) unless otherwise specified. EDSS = Expanded Disability Status Scale. Differences between the groups were determined using ANOVA (age and disease duration), Kruskal-Wallis one-way analysis of variance (EDSS), or Fisher exact test (gender and presence of relapses; p-value shows comparison Untreated vs. Tysabri).</p