Lymphocyte Subsets Show Different Response Patterns to In Vivo Bound Natalizumab—A Flow Cytometric Study on Patients with Multiple Sclerosis

Natalizumab is an effective monoclonal antibody therapy for the treatment of relapsing- remitting multiple sclerosis (RRMS) and interferes with immune cell migration into the central nervous system by blocking the α4 subunit of very-late activation antigen-4 (VLA-4). Although well tolerated and very effective, some patients still suffer from relapses in spite of natalizumab therapy or from unwanted side effects like progressive multifocal leukoencephalopathy (PML). In search of a routine-qualified biomarker on the effectiveness of natalizumab therapy we applied flow cytometry and analyzed natalizumab binding to α4 and α4 integrin surface levels on T-cells, B-cells, natural killer (NK) cells, and NKT cells from 26 RRMS patients under up to 72 weeks of therapy. Four-weekly infusions of natalizumab resulted in a significant and sustained increase of lymphocyte-bound natalizumab (p<0.001) which was paralleled by a significant decrease in detectability of the α4 integrin subunit on all lymphocyte subsets (p<0.001). We observed pronounced natalizumab accumulations on T and B cells at single measurements in all patients who reported clinical disease activity (n = 4). The natalizumab binding capacity of in vitro saturated lymphocytes collected during therapy was strongly diminished compared to treatment-naive cells indicating a therapy-induced reduction of α4. Summing up, this pilot study shows that flow cytometry is a useful method to monitor natalizumab binding to lymphocytes from RRMS patients under therapy. Investigating natalizumab binding provides an opportunity to evaluate the molecular level of effectiveness of natalizumab therapy in individual patients. In combination with natalizumab saturation experiments, it possibly even provides a means of studying the feasability of patient-tailored infusion intervals. A routine-qualified biomarker on the basis of individual natalizumab saturation on lymphocyte subsets might be an effective tool to improve treatment safety

Cerebrospinal fluid CXLC13 indicates disease course in neuroinfection: an observational study

Abstract Background The chemokine CXCL13 is an intensively investigated biomarker in Lyme neuroborreliosis (LNB). Its role in other neuroinfections is increasingly recognized but less clear. Objective To determine the significance of CXCL13 in established central nervous system (CNS) infections other than LNB by matching cerebrospinal fluid (CSF) CXCL13 elevations with severity of the disease course. Methods We investigated 26 patients with bacterial (n = 10) and viral (n = 16; tick-borne encephalitis, n = 6; varicella zoster infection, n = 10) neuroinfections of whom CSF CXCL13 levels were available twice, from lumbar punctures (LP) performed at admission and follow-up. As outcome classification, we dichotomized disease courses into “uncomplicated” (meningitis, monoradiculitis) and “complicated” (signs of CNS parenchymal involvement such as encephalitis, myelitis, abscesses, or vasculitis). CXCL13 elevations above 250 pg/ml were classified as highly elevated. Results Eight of 26 patients (31%) with both bacterial (n = 4) and viral (n = 4) neuroinfections had a complicated disease course. All of them but only 3/18 patients (17%) with an uncomplicated disease course had CSF CXCL13 elevations > 250 pg/ml at the follow-up LP (p  250 pg/ml. All four patients with a complicated disease course but only one with an uncomplicated disease course had sustained CXCL13 elevations at follow-up. Patient groups did not differ with regard to age, time since symptom onset, LP intervals, type of infections, and anti-pathogen treatments. Conclusion Our study revealed pronounced CXCL13 elevations in CSF of patients with severe disease courses of bacterial and viral neuroinfections. This observation indicates a role of CXCL13 in the CNS immune defense and points at an additional diagnostic value as biomarker for unresolved immune processes leading to or associated with complications

Adaptive Immune Responses in a Multiple Sclerosis Patient with Acute Varicella-Zoster Virus Reactivation during Treatment with Fingolimod

Fingolimod, an oral sphingosine 1-phosphate (S1P) receptor modulator, is approved for the treatment of relapsing forms of multiple sclerosis (MS). The interference with S1P signaling leads to retention particularly of chemokine receptor-7 (CCR7) expressing T cells in lymph nodes. The immunological basis of varicella zoster virus (VZV) infections during fingolimod treatment is unclear. Here, we studied the dynamics of systemic and intrathecal immune responses associated with symptomatic VZV reactivation including cessation of fingolimod and initiation of antiviral therapy. Key features in peripheral blood were an about two-fold increase of VZV-specific IgG at diagnosis of VZV reactivation as compared to the previous months, a relative enrichment of effector CD4+ T cells (36% versus mean 12% in controls), and an accelerated reconstitution of absolute lymphocytes counts including a normalized CD4+/CD8+ ratio and reappearance of CCR7+ T cells. In cerebrospinal fluid (CSF) the lymphocytic pleocytosis and CD4+/CD8+ ratios at diagnosis of reactivation and after nine days of fingolimod discontinuation remained unchanged. During this time CCR7+ T cells were not observed in CSF. Further research into fingolimod-associated VZV reactivation and immune reconstitution is mandatory to prevent morbidity and mortality associated with this potentially life-threatening condition

From natalizumab to fingolimod in eight weeks - Immunological, clinical, and radiological data in quest of the optimal switch

Natalizumab (NZB) discontinuation during a treatment change is associated with recurrence of disease activity in a significant proportion of multiple sclerosis (MS) patients. The immunological basis why disease reactivation occurs in selected patients is unresolved. In search of a prognostic biomarker for a safe and effective transition from NZB to fingolimod, we monitored five parameters related to pharmacokinetic and pharmacodynamic effects of the two drugs in 12 MS patients until six months on fingolimod. Clearance of free and cell-bound NZB, re-expression of alpha-4, and fingolimod-mediated changes on CD8+ and CD4+ T cell subsets showed pronounced interindividual variability. Higher frequencies of memory CD8+ T cells after six months on fingolimod were the sole association with disease reactivation. None of the investigated parameters thus had potential as prognostic biomarker for the outcome of the switch. Our findings rather support the thesis of broad interindividual differences in the immunopathogenesis of MS. (C) 2017 Elsevier Inc. All rights reserve

Correlation results between cell-bound natalizumab and α₄ integrin surface levels.

<p>(A) Anti-natalizumab rfi levels of week 12–48 measurements correlated with anti-α₄ rfi levels from cells collected at baseline (n = 17). Filled symbols highlight patients who reported disease activity. (B) Anti-natalizumab rfi levels from <i>in vitro</i> saturated cells collected at baseline correlated with anti-α₄ rfi levels from natalizumab-naïve cells of the same blood collection (n = 10). FITC, Fluorescein isothiocyanate; NatSat, <i>in vitro</i> treated cells with saturating amounts of natalizumab; NK, natural killer cells; NKT, natural killer T cells; rfi, relative fluorescence intensity; T12–48, time of measurements at weeks 12–48.</p

Patient Characteristics - Clinical Parameters.

<p>Abbreviations: EDSS, Expanded Disability Status Scale; F, female; GA, glatiramer acetate; ID, identification number IFN-ß, Interferon-beta; i.m., intramuscularly; IU, international units; M, male; NAB, natalizumab neutralizing antibodies; s.c., subcutaneously; T0, baseline; w, weeks; y, years.</p>1<p>age at which first MS-specific symptoms were reported.</p>2<p>number of relapses 12 months before natalizumab therapy.</p>3<p>number of relapses during natalizumab therapy.</p>4<p>non-persisting low titre NAB.</p

Flow cytometry in a case with natalizumab neutralizing antibodies (NAB).

<p>Anti-natalizumab-FITC rfi levels (black lines) and anti-α₄-FITC rfi levels (grey lines) on CD3+ T cells (solid line), CD19+ B cells (dashed line), NK cells (dotted line), and NKT cells (dot and dash line) from one patient with non-persistent NAB. The numbers of infusions and EDSS scores are specified in a separate diagram. Each bold arrow plotted onto the x-axis represents 4 weeks. NAB were measured from sera collected at weeks 4, 8, and 24. Frequencies of the CD19+ B cell population appear in brackets [%]. EDSS, expanded disability status scale FITC, Fluorescein isothiocyanate; NAB, natalizumab neutralizing antibodies; NK, natural killer cells; NKT, natural killer T cells; rfi, relative fluorescence intensity.</p

Maximum natalizumab binding capacity and natalizumab saturation before and during natalizumab therapy.

<p>(A) Percent natalizumab saturation of T cells, B cells, NK cells, and NKT cells collected at week 12 (T12, n = 8) during therapy related to maximum natalizumab binding capacity of lymphocytes from the same blood collection after <i>in vitro</i> treatment with saturating amount of natalizumab (100%). (B) Percent maximum natalizumab binding capacity of <i>in vitro</i> saturated T cells, B cells, NK cells, and NKT cells collected at week 12 (T12, n = 8) related to maximum natalizumab binding capacity of <i>in vitro</i> saturated lymphocytes from the same donors collected at baseline (100%). Vertical bars represent 95% confidential intervals. Max, maximum; NAT, natalizumab; NK, natural killer cells; NKT, natural killer T cells; T(x), time of measurement (weeks).</p

Changes in Frequencies of Lymphocyte Subsets during Natalizumab Therapy.

<p>Abbreviations: NK, natural killer; NKT, natural killer T cells; SD, standard deviation;</p><p>*) p<0.05;</p><p>**) p<0.01;</p><p>***) <0.001.</p