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

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

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

Naïve, memory and effector T-cell subsets in the blood compartment of 13 untreated and 17 natalizumab treated MS patients.

<p>Numbers of circulating naïve (CCR7⁺CD45RA⁺CD27⁺), central memory (CCR7⁺CD45RA⁻CD27⁺), effector (CCR7⁻CD45RA⁺CD27^+/−) and effector memory (CCR7⁻CD45RA⁻CD27^+/−) (A) CD4⁺ T-cell and (B) CD8⁺ T-cell subsets were assessed by flow cytometry. VLA-4 surface expression on circulating naïve (CCR7⁺CD45RA⁺CD27⁺), central memory (CCR7⁺CD45RA⁻CD27⁺), effector (CCR7⁻CD45RA⁺CD27^+/−) and effector memory (CCR7⁻CD45RA⁻CD27^+/−) and (C) CD4⁺ T-cell and (D) CD8⁺ T-cell subsets is given as median fluorescence intensity (MFI). 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. UNT = untreated; NAT = natalizumab treated; NEG = negative; VLA = very late antigen.</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

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

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

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

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