Effect of Natalizumab on Circulating CD4⁺ T-Cells in Multiple Sclerosis

<div><p>In multiple sclerosis (MS), treatment with the monoclonal antibody natalizumab effectively reduces the formation of acute lesions in the central nervous system (CNS). Natalizumab binds the integrin very late antigen (VLA)-4, expressed on the surface of immune cells, and inhibits VLA-4 dependent transmigration of circulating immune-cells across the vascular endothelium into the CNS. Recent studies suggested that natalizumab treated MS patients have an increased T-cell pool in the blood compartment which may be selectively enriched in activated T-cells. Proposed causes are sequestration of activated T-cells due to reduced extravasation of activated and pro-inflammatory T-cells or due to induction of VLA-4 mediated co-stimulatory signals by natalizumab. In this study we examined how natalizumab treatment altered the distribution of effector and memory T-cell subsets in the blood compartment and if T-cells in general or myelin-reactive T-cells in particular showed signs of increased immune activation. Furthermore we examined the effects of natalizumab on CD4⁺ T-cell responses to myelin in vitro. Natalizumab-treated MS patients had significantly increased numbers of effector-memory T-cells in the blood. In T-cells from natalizumab-treated MS patients, the expression of TNF-α mRNA was increased whereas the expression of fourteen other effector cytokines or transcription factors was unchanged. Natalizumab-treated MS patients had significantly decreased expression of the co-stimulatory molecule CD134 on CD4⁺CD26^HIGH T-cells, in blood, and natalizumab decreased the expression of CD134 on MBP-reactive CD26^HIGHCD4⁺ T-cells <em>in vitro</em>. Otherwise CD4⁺ T-cells from natalizumab-treated and untreated MS patients showed similar responses to MBP. In conclusion natalizumab treatment selectively increased the effector memory T-cell pool but not the activation state of T-cells in the blood compartment. Myelin-reactive T-cells were not selectively increased in natalizumab treated MS.</p> </div

T-cell counts and integrin expression.

<p>(A) Number of circulating CD4⁺CD8⁻ and CD4⁻CD8⁺ T-cells and surface expression of (B) VLA-4 and (C) CD11a and CD18 on CD4⁺ and CD8⁺ T-cells (median fluorescence intensity, MFI) from 13 untreated and 17 natalizumab treated RRMS patients. (D) mRNA expression of <i>ITGB1</i> and <i>ITGA4</i> measured by quantitative real-time PCR in untreated RRMS (UNT; n = 25) and 50 natalizuamb treated RRMS patients 3 months (T3), 6 months (T6) and 12 months (T12) after initiation of treatment. (E) Histograms show the expression of VLA-4 and the surface-binding of IgG4 on CD4⁺CD8⁻ and CD4⁻CD8⁺ T-cells after incubation of PBMCs from a healthy volunteer with and without natalizumab 25 µg/ml in PBS for 1 hour at 4°C. Boxes represent interquartile range, median value indicated as a line, whiskers represent range, ^o = outliers, * = extremes.</p

mRNA expression of genes associated with T-helper (T_H) cell immune activation and regulatory T-cell activity in CD4+ and CD8+ T-cells from untreated and natalizumab treated MS-patients.

<p>Data are given as median with interquartile range in the parenthesis; statistical analysis was by Mann-Whitney U test and p<0.01 was used as level of significance. When a target was not measurable in more than one sample from a group, data were not applied to the table (NA); when a target was not measured in at least 6 individuals of each group, statistics were not done (ND); NS = not significant.</p

Effect of natalizumab on CD4⁺ T-cell reactivity to myelin basic protein (MBP) <i>in vitro</i>.

<p>PBMCs were stained with CFSE and cultured for 7 days with MBP (30 µg/ml) and with natalizumab (NA; 25 µg/ml), IgG4 (25 µg/ml) or without further supplement for control (CON). Experiments were done with PBMCs from 4 healthy individuals. After staining for cell surface markers, we assessed (A) CD134 expression on CD26^HIGHCD3⁺CD4⁺ T-cells, proliferating in response to MBP and (B) proliferation of CD4⁺ T-cells by flow-cytometry. After stimulation with PMA/ionomycin for five hours, staining for surface-markers, permeabilization and staining for intracellular cytokines, the (C) IL-17 and (D) IFN-γ expression of MPB reactive CD4⁺ T-cells was assessed by flow-cytometry. Boxes represent interquartile range, median value indicated as a line, whiskers represent range, ^o = outliers, * = extremes. Statistics was by paired sample t-test.</p

CD134 and VLA-4 on CD26-defined subsets of CD4⁺ or CD8⁺ T-cells from 13 untreated and 17 natalizumab treated MS patients.

<p>Concentration of CD26 defined CD4⁺ T-cell and CD8⁺ T-cell subsets in the blood. VLA-4 expression on CD26-defined (C) CD4⁺ or (D) CD8⁺ T-cell subsets is given as median fluorescence intensity (MFI) and representative dot-plots used for analysis are shown. (E) CD134 expression was assessed as percentage of positive cells within CD26 defined CD4⁺ T-cells subsets and afterwards the background-staining, assessed in the corresponding combined isotype and fluorescence-minus one control, was subtracted. CD8⁺ T-cell subsets did not stain positive for CD134 and are not shown here. Boxes represent interquartile range, median value indicated as a line, whiskers represent range, ^o = outliers, * = extremes. Statistics was by Mann-Whitney U test, for comparison between the cohorts, and by Wilcoxon's test for comparrison of paired parameters. •• = p<0.005; ••• = p = 0.001. NEG = negative; VLA = very late antigen.</p

Systemic Inflammation in Progressive Multiple Sclerosis Involves Follicular T-Helper, Th17- and Activated B-Cells and Correlates with Progression

<div><p>Pathology studies of progressive multiple sclerosis (MS) indicate a major role of inflammation including Th17-cells and meningeal inflammation with ectopic lymphoid follicles, B-cells and plasma cells, the latter indicating a possible role of the newly identified subset of follicular T-helper (T_FH) cells. Although previous studies reported increased systemic inflammation in progressive MS it remains unclear whether systemic inflammation contributes to disease progression and intrathecal inflammation. This study aimed to investigate systemic inflammation in progressive MS and its relationship with disease progression, using flow cytometry and gene expression analysis of CD4⁺ and CD8⁺T-cells, B-cells, monocytes and dendritic cells. Furthermore, gene expression of cerebrospinal fluid cells was studied. Flow cytometry studies revealed increased frequencies of ICOS⁺T_FH-cells in peripheral blood from relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients. All MS subtypes had decreased frequencies of Th1 T_FH-cells, while primary progressive (PPMS) MS patients had increased frequency of Th17 T_FH-cells. The Th17-subset, interleukin-23-receptor⁺CD4⁺T-cells, was significantly increased in PPMS and SPMS. In the analysis of B-cells, we found a significant increase of plasmablasts and DC-SIGN⁺ and CD83⁺B-cells in SPMS. ICOS⁺T_FH-cells and DC-SIGN⁺B-cells correlated with disease progression in SPMS patients. Gene expression analysis of peripheral blood cell subsets substantiated the flow cytometry findings by demonstrating increased expression of <i>IL21</i>, <i>IL21R</i> and <i>ICOS</i> in CD4⁺T-cells in progressive MS. Cerebrospinal fluid cells from RRMS and progressive MS (pooled SPMS and PPMS patients) had increased expression of T_FH-cell and plasmablast markers. In conclusion, this study is the first to demonstrate the potential involvement of activated T_FH-cells in MS. The increased frequencies of Th17-cells, activated T_FH- and B-cells parallel findings from pathology studies which, along with the correlation between activated T_FH- and B-cells and disease progression, suggest a pathogenic role of systemic inflammation in progressive MS. These observations may have implications for the treatment of progressive MS.</p> </div

Endogenous and Recombinant Type I Interferons and Disease Activity in Multiple Sclerosis

<div><p>Although treatment of multiple sclerosis (MS) with the type I interferon (IFN) IFN-β lowers disease activity, the role of endogenous type I IFN in MS remains controversial. We studied CD4+ T cells and CD4+ T cell subsets, monocytes and dendritic cells by flow cytometry and analysed the relationship with endogenous type I IFN-like activity, the effect of IFN-β therapy, and clinical and magnetic resonance imaging (MRI) disease activity in MS patients. Endogenous type I IFN activity was associated with decreased expression of the integrin subunit CD49d (VLA-4) on CD4+CD26^high T cells (Th1 helper cells), and this effect was associated with less MRI disease activity. IFN-β therapy reduced CD49d expression on CD4+CD26^high T cells, and the percentage of CD4+CD26^high T cells that were CD49d^high correlated with clinical and MRI disease activity in patients treated with IFN-β. Treatment with IFN-β also increased the percentage of CD4+ T cells expressing CD71 and HLA-DR (activated T cells), and this was associated with an increased risk of clinical disease activity. In contrast, induction of CD71 and HLA-DR was not observed in untreated MS patients with evidence of endogenous type IFN I activity. In conclusion, the effects of IFN-β treatment and endogenous type I IFN activity on VLA-4 expression are similar and associated with control of disease activity. However, immune-activating effects of treatment with IFN-β may counteract the beneficial effects of treatment and cause an insufficient response to therapy.</p> </div

Interleukin (IL)-23 receptor (R), IL12 receptor (CD212), and VLA-4 on CD161 defined subsets of CD4⁺ or CD8⁺ T-cells from 13 untreated and 17 natalizumab treated MS patients.

<p>Concentration of (A) CD161^NEG and CD161^POS CD4⁺ T-cells and (E) CD161^NEG, CD161^LOW and CD161^HIGH CD8⁺ T-cells in the blood. VLA-4 expression on CD161 defined (B) CD4⁺ or (F) CD8⁺ T-cell subsets is given as median fluorescence intensity (MFI). IL23R and CD212 expression was assessed as percentage of positive cells within CD161-defined (C and D) CD4⁺ or (G and H) CD8⁺ T-cell subsets, afterwards the unspecific antibody staining, assessed in the corresponding combined isotype and fluorescence-minus one control , was subtracted. Boxes represent interquartile range, median value indicated as a line, whiskers represent range, ^o = outliers, * = extremes. Statistics was by Mann-Whitney U test. • = p<0.01; •• = p<0.005; ••• = p = 0.001.</p

Intracellular cytokine expression in PMA/ionomycin stimulated T-cells from 13 natalizumab (NAT) and 11 untreated (UNT) RRMS patients <i>ex vivo</i>.

<p>Unstimulated peripheral blood mononuclear cells (PBMCs) were incubated for 7 days as negative control in our CFSE assay and stimulated with PMA/ionomycin for five hours, stained for cell surface markers, permeabilized and stained for intracellular cytokines. The cytokine expression was assessed by flow cytometry as percentage of interleukin (IL) -17⁺, interferon (IFN) -γ⁺, IL-17/IFN-γ or tumor necrosis factor (TNF) -α expressing CD8⁻CD3⁺ and CD8⁺CD3⁺ T-cells. Boxes represent interquartile range, median value indicated as a line, whiskers represent range, ^o = outliers, * = extremes. Statistics was by Mann-Whitney U test.</p

T cell activation, <i>CXCL10</i> and <i>MX1</i> expression.

<p>The relationship between the percentage of CD4+CD26^high T cells expressing CD49d or CXCR3 and the expression of <i>MX1</i> and <i>CXCL10</i> mRNA in blood mononuclear cells from untreated patients with relapsing-remitting multiple sclerosis was analysed by Spearman rank correlation coefficients (SRCC).</p