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

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

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

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

Patient Characteristics - Means, Medians, and Standard Deviations of Treatment Parameters.

<p>Abbreviations: approx., approximately; SD, standard deviation.</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

Evaluation of natalizumab accumulations on lymphocyte subsets from individual patients at single time-points.

<p>Abbreviations: ID, identification number; NK, natural killer cells; NKT, natural killer T cells; SD, standard deviation; T, time of measurement; Tx/T0, quotient of anti-natalizumab rfi levels of measurements performed during natalizumab therapy (T12,…,48) and background rfi levels from measurements performed at baseline (T0);</p><p>Bold ID numbers highlight patients with reported disease activity; bold results highlight values above cut-off; bold results with plus signs highlight values above cut-off of patients with reported disease activity.</p>1<p>cut-off defined as sum of means plus one standard deviation.</p

Flow cytometric analysis of natalizumab binding to four different lymphocyte subsets from RRMS patients under therapy.

<p>Increase of anti-natalizumab rfi levels (A) and decrease of anti-α₄ rfi levels (B) illustrated for CD3+ T cells (filled diamond), CD19+ B cells (open square), NK cells (open triangle), and NKT cells (filled circle) at weeks 12 (T12, n = 17), 24 (T24, n = 17), 36 (T36, n = 17), 48 (T48, n = 17), and 72 (T72, n = 10) compared to background (anti-natalizumab) respectively baseline (anti-α₄ rfi) levels (T0, n = 17). Significance levels calculated for lymphocyte subset-specific differences of anti-natalizumab rfi levels at each measurement during therapy are summarized in the associated table. Representative FACS plots show anti-natalizumab rfi levels on CD3+ T cells at baseline, i.e. background signaling, (C) and after 12 weeks of natalizumab therapy (D). Anti-natalizumab median fluorescence intensities of the above two FACS plots depicted as overlapping histrograms (E). ECD, electron coupled dye; FITC, Fluorescein isothiocyanate; NK, natural killer cells; NKT, natural killer T cells; n.s., non significant; Rfi, relative fluorescence intensity; T(x), Time of measurement (weeks); triple stars (***) highlight highly significant results (p<0.001); vertical bars represent 95% confidential intervals.</p