Natalizumab therapy decreases surface expression of both VLA-heterodimer subunits on peripheral blood mononuclear cells

Journal of Neuroimmunology 234 (2011) 148–154

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Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Natalizumab therapy decreases surface expression of both VLA-heterodimer subunits on peripheral blood mononuclear cells Andrea Harrer a,⁎,1, Peter Wipfler a,1, Max Einhaeupl b, Georg Pilz a, Katrin Oppermann a, Wolfgang Hitzl b, Shahrzad Afazel a, Elisabeth Haschke-Becher a, Peter Strasser a, Eugen Trinka a, Joerg Kraus a a b

Christian-Doppler-Klinik, Department of Neurology, Paracelsus Medical University, Salzburg, Austria Paracelsus Medical University, Salzburg, Austria

a r t i c l e

i n f o

Article history: Received 20 September 2010 Received in revised form 28 February 2011 Accepted 3 March 2011 Keywords: Natalizumab Multiple sclerosis VLA-4 Beta-1 integrin Adhesion molecule Blood-brain-barrier Flow cytometry

a b s t r a c t Natalizumab interferes with immune cell migration into the central nervous system via blocking the alpha-4 subunit of very-late activation antigen-4 (VLA-4). Occurrence of rare but serious progressive multifocal leukoencephalopathy during prolonged natalizumab therapy of multiple sclerosis (MS) calls for a more detailed understanding of potential coeffects. We longitudinally studied alpha-4 and beta-1 surface levels on blood cells from 18 MS patients by flow cytometry. Expectedly, detectability of natalizumab-blocked alpha-4 was diminished on all investigated cell subsets. In addition, we report a concurrent and significant decrease of beta-1 surface levels on T-cells, B-cells, natural killer cells, and natural killer T cells, but not on monocytes. Uncovering secondary effects of natalizumab is mandatory to increase safety in MS therapy. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) (Lassmann et al., 2007; Noseworthy et al., 2000). Recruitment of activated immune cells across the blood brain barrier (BBB) is considered essential for the initiation of inflammatory brain lesions (Kraus and Oschmann, 2006; von Andrian and Engelhardt, 2003). The monoclonal antibody natalizumab was designed on the basis of progenitor antibodies capable of preventing leukocyte accumulation in experimental autoimmune encephalomyelitis (EAE) and is proposed to block extravasation of autoreactive T cells into the CNS (von Andrian and Engelhardt, 2003; Yednock et al., 1992). It selectively binds to the alpha-4 integrin subunit of very late activation antigen-4 (VLA-4, alpha-4/beta-1 integrin) on immune cells and was shown to impressively reduce relapse frequency and disease progression in patients with relapsing remitting MS (RRMS) (Miller et al., 2003;

⁎ Corresponding author at: Christian-Doppler-Klinik, Department of Neurology, Paracelsus Medical University, Ignaz-Harrer-Strasse 79, A-5020 Salzburg, Austria. Tel.: + 43 662 4483 56033; fax: + 43 662 4483 3734. E-mail address: [email protected] (A. Harrer). 1 Andrea Harrer and Peter Wipfler equally contributed to the manuscript. 0165-5728/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2011.03.001

Polman et al., 2006). However, clinical benefits are counteracted by the risk to develop progressive multifocal leukoencephalopathy (PML) upon a mean treatment duration of 25 months (Clifford et al., 2010). Despite considerable efforts, the overall dimension of the pharmacological activity of natalizumab is unresolved. Moreover, failing efficacy of the small molecule alpha-4 integrin inhibitor CDP323 in a recent phase II study (Polman et al., 2010) is strongly suggestive of yet unknown additional effects of natalizumab treatment in RRMS. Like natalizumab, CDP323 blocks the interaction of alpha-4 with its ligands. In contrast to animal models and in vitro studies, the compound did not show the expected effect of an alpha-4 inhibitor in RRMS patients. In our study we focus on the impact of natalizumab therapy on the heterodimeric VLA-4 integrin's beta-1 subunit of which little is known to date. Beta-1 chains are common to the family of VLA adhesion receptors which are involved in attachment to extracellular matrix proteins and immune cell trafficking. The question is whether deprivation of the major VLA-4 functionality also affects overall surface expression of beta-1 and, as a further consequence, immune surveillance and migration of immune cells to sites of inflammation. We applied flow cytometry to investigate the effect of serial natalizumab infusions on the surface expression of the alpha-4 and beta-1 integrin subunits on peripheral blood mononuclear cells (PBMC) from 18 RRMS patients during the first year of natalizumab treatment and from 18 healthy donors.

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fluorescence-conjugated monoclonal antibodies for 30 min at 4 °C in the dark and fixed with 1% formaldehyde in PBS.

2. Materials and methods 2.1. Participants

2.3. Flow cytometry Eighteen patients (17 females, 1 male; mean age 39.1 years) with clinically definite RRMS according to the revised McDonald criteria (Polman et al., 2005) were included into the study (Table 1). They were recruited at the Department of Neurology at the Paracelsus Medical University, Salzburg. All included patients received natalizumab according to the European Medicines Agency's indications due to active disease despite previous immunomodulatory therapy. To avoid our investigated parameters being affected by prior therapy, the time period between the last immunomodulatory treatment or application of corticosteroid and the first blood sampling was at least eight weeks, and the interval between the last immunosuppressive therapy and the first blood sampling was at least 6 months. Steroid therapy and infections during the study period were exclusion criteria for natalizumab infusions. Patients received infusions of the standard dose of natalizumab (300 mg iv) every four weeks. Routine examinations for neutralizing antibodies (NABs) were performed before the second, third, and seventh natalizumab infusions. Blood was collected before initiation of natalizumab therapy (baseline, n = 18) and after 12 weeks (short-term therapy, n = 13), 24 weeks (intermediate-term therapy, n = 10), and 48 weeks (long-term therapy, n = 8) immediately before subsequent infusions. Blood from healthy controls (n = 18; 12 females, 6 males; mean age 43.1 years) was measured twice in an interval of 3–6 months. All participants gave written informed consent and the study was approved by the local ethics committee (Ethikkommission für das Bundesland Salzburg 415-E774/ 6-2007).

2.2. Sample preparation Venous blood was collected in commercial PBMC enrichment tubes (Becton Dickinson AG, Basel, Switzerland). PBMC were enriched by centrifugation at 880 × g for 20 min and resuspended in staining buffer (PBS pH 7.2, 2.5% fetal calf serum, 0.1% sodium acid) after a final wash in PBS. Blood cells were stained with saturating amounts of

Expression levels of alpha-4 (CD49d [clone HP2/1, FITC]) and beta1 (CD29 [clone 4B4, FITC]) integrin subunits were investigated on four lymphocyte subpopulations (forward/sideward scatter, CD3+ [clone UCHT1, ECD; identification of T cells], CD19+ [clone J3-119, PC7; identification of B cells], CD16/56+ [clones 3 G8 and N901, PE; identification of natural killer (NK, CD3-) and NKT (CD3+) cells]) and monocytes (forward/sideward scatter, CD14+ [clone RM052, PC5]) by 5-color flow cytometry (Cytomics FC500, Beckman Coulter, Vienna, Austria). Isotype-matched antibodies (all IgG1) and CD45+ [clone J33, FITC, PE, ECD, PC5, PC7] were used as negative and positive controls, respectively. With the exception of IgG1-FITC/PE (Exalpha, Watertown, MA, USA) all antibodies were obtained from Beckman Coulter, Vienna, Austria. Lymphocytes and monocytes were analyzed separately because monocytes are markedly different from lymphocytes in size and autofluorescence. We used the parameter median fluorescence intensity of the entire population as main comparative read-out because a clear cut off between beta-1 high and beta-1 low expressing populations was not always possible and because the median is less sensitive to outliers than the mean. For the purpose of a better inter- and intraindividual comparability, relative fluorescence intensity (rfi) levels were calculated from median fluorescence intensities (MFI) of all leukocyte subpopulations as previously described (Kraus et al., 1998). In short, fluorescence intensities of different measurements were corrected for the MFI of negative controls (IgG1) and related to the MFI of positive controls. Fluorescence intensity gains of cellular surface markers generally are a measure of surface expression of the investigated molecule. In the case of natalizumab-blocked alpha-4 integrin the measured decrease in rfi levels does not correspond with surface expression of alpha-4 but with low abundance of unblocked alpha-4. For this reason the term “detectability” was used in the context of natalizumabblocked alpha-4 on PBMC from MS patients under natalizumab therapy, whereas the term “surface expression” was used to refer to

Table 1 Patient characteristics. No

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Sex

w w w w w m w w w w w w w w w w w w

Age (years)

12 months before natalizumab therapy

During natalizumab therapy

Previous therapies

At disease onset

At baseline

Relapses

EDSS at baseline

Relapses

Last EDSS

Time-point

NABs

23 28 32 28 43 29 31 18 40 21 36 38 48 22 23 38 33 25

29 31 59 29 50 53 32 24 55 25 41 40 50 27 33 39 35 47

3 2 3 4 3 2 5 2 5 6 1 4 4 3 4 3 2 2

4.5 4.5 6.0 1.0 6.0 2.5 5.0 0.0 6.0 1.0 1.5 3.0 3.0 0.0 3.0 4.0 3.0 5.0

0 2 0 0 0 0 0 1 0 0 0 0 1 0 0 0 1 0

4.5 4.0 6.0 1.0 6.0 2.5 5.0 0.0 6.0 1.0 1.5 3.0 3.0 0.0 2.5 4.0 3.0 4.5

12 24 0a 24 0a 48 0a 48 0a 48 48 0a 48 0a 48 24 48 48

Negative Positivec Negative Negative Negative Negative Positiveb Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

IFN-ß-1a (i.m.), IFN-ß-1b (s.c.) 22 μg IFN-ß-1a (s.c.), GA IFN-ß-1b (s.c.), GA, IVIG, IFN-ß-1b (s.c.) 22 μg IFN-ß-1a (s.c.), 44 μg IFN-ß-1a (s.c.) 22 μg IFN-ß-1a (s.c.), GA, 44 μg IFN-ß-1a (s.c.) IVIG, IFN-ß-1b (s.c.) IFN-ß-1a (i.m.), GA 22 μg IFN-ß-1a (s.c.), 44 μg IFN-ß-1a (s.c.), IFN-ß-1a (i.m.), GA 22 μg IFN-ß-1a (s.c.) 44 μg IFN-ß-1a (s.c.) GA, 22 μg IFN-ß-1a (s.c.), 44 μg IFN-ß-1a (s.c.), Mitoxantrone IFN-ß-1a (i.m.) 22 μg IFN-ß-1a (s.c.), 44 μg IFN-ß-1a (s.c.) IFN-ß-1a (i.m.), GA, IFN-ß-1b (s.c.) 22 μg IFN-ß-1a (s.c.), 44 μg IFN-ß-1a (s.c.) 22 μg IFN-ß-1a (s.c.), 44 μg IFN-ß-1a (s.c.) 22 μg IFN-ß-1a (s.c.), 44 μg IFN-ß-1a (s.c.) IFN-ß-1a (i.m.), GA

Abbreviations: NABs, neutralizing antibodies; EDSS, Expanded Disability Status Scale; IFN-ß, Interferon-beta; GA, glatiramer acetate; IVIG, intravenous immunoglobulins; i.m., intramuscularly; s.c., subcutaneously. a Baseline data only. b Therapy aborted due to hypersensitivity reaction and high titer NABs; baseline data only. c Non-persisting low titred NABs; 12 weeks data excluded from analysis.

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alpha-4 and beta-1 rfi levels on PBMC from healthy controls and patients prior to natalizumab therapy. 2.4. In vitro binding assay

STATISTICA 6.1 (StatSoft, Inc. (2004)). All analyses were done by one of the authors (W.H.).

3. Results 6

Preincubation of PBMC (1 × 10 cells/ml) from healthy controls with natalizumab (final dilutions: 0, 1, 5, 25, 125, and 625 ng/ml) for 1 h at 4 °C was followed by staining and flow cytometric analysis of alpha-4 (CD49d) and beta-1 (CD29) expression and bound natalizumab (anti-human IgG4 [clone HP6025, FITC], Beckman Coulter) on CD3+ T cells as described above. 2.5. Statistics Data were tested for normality (Kolmogorov–Smirnov-Test) and a repeated measures ANOVA with two-sided, paired Student's t-tests as a post hoc test was applied to test for significance. Two-sided unpaired Student's t-tests were used to compare treatment with control group. A p-value less than 5% was used to indicate a statistically significant difference. All analyses were done using

3.1. Expression patterns of alpha-4 and beta-1 integrins on lymphocyte subsets Flow cytometric analysis of alpha-4 and beta-1 surface expressions on five PBMC subsets from 18 RRMS patients and 18 controls showed high and low beta-1 expressing subpopulations of CD3+ T cells and to a lesser extent of CD19+ B cells in controls and in all but one (#18) RRMS patients (Fig. 1). In contrast, alpha-4 rfi levels appeared homogenous throughout the investigated cell populations and individuals. A single beta-1 population was also observed in NK cells, NKT cells and monocytes. Statistical analysis of the percentages of beta-1 high and low expressing T and B cell subpopulations showed no differences between patient baseline samples and controls. Analysis of fluorescence intensities revealed significantly higher rfi levels on both T cell subpopulations from RRMS patients (CD29 low,

Fig. 1. Varying beta-1 (CD29) expression on CD3+ T cells. Prevailing low and high expressing fractions from RRMS patient #8 (left panel) versus single population from patient #18 (right panel) at baseline (C, D) and after 48 weeks of natalizumab therapy (E, F). Respective isotype controls (A, B). Abbreviations and synonyms: FITC, fluoresceine isothiocyanate; CD29, beta-1 integrin; IgG1-FITC, FITC-labeled isotype control.

A. Harrer et al. / Journal of Neuroimmunology 234 (2011) 148–154

p b 0.01; CD29 high, p b 0.05) with a similar trend on B cells (CD29 low, p b 0.05; CD29 high, p = 0.06) compared to controls (Table 2). 3.2. Baseline expression levels of alpha-4 and beta-1 integrin Comparing alpha-4 and total beta-1 expression levels from untreated patients and controls, we noticed that RRMS patients had significantly higher alpha-4 rfi levels on CD19+ B cells (p b 0.02) and NK cells (p b 0.01) and considerably higher beta-1 rfi levels on NK cells (p b 0.002) than the controls (Fig. 2). Differences in total beta-1 rfi levels of CD3+ T cell (p = 0.056) and CD19+ B cell population were borderline and not significant, respectively (Fig. 2, Table 2). Monocytes from RRMS patients had higher alpha-4 baseline expression levels (p b 0.04) compared to controls, but did not differ in beta-1 expression (Fig. 3A). Evaluating fluorescence intensities between the four lymphocyte subsets we found that alpha-4 expression levels were lowest on CD3+ T cells and beta-1 expression levels were lowest on CD19+ B cells in patients and controls (Fig. 2). A potential gender bias was excluded by analyzing the control group with its female:male ratio of 12:6 showing no gender differences in alpha-4 and beta-1 integrin expression (data not shown). The concurrence of alpha-4 and beta-1 expression levels on each leukocyte subset was determined by relating baseline rfi levels of both chains and calculating beta-1/alpha-4 ratios. We found a ratio of beta1/alpha-4 of 1.4 (RRMS patients) and 1.2 (controls) on CD3+ T cells, 1.4 (both groups) on NK cells, 1.1 (RRMS patients) and 1.2 (controls) on NKT cells, and an opposite ratio of 0.6 (both groups) on CD19+ B cells. The highest ratio between the two integrin subunits was found on myeloid lineage-derived monocytes with 6.6 (RRMS patients) and 7.9 (controls) in favor of beta-1. 3.3. Analysis of alpha-4 and beta-1 integrin levels during natalizumab therapy

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natalizumab did not affect alpha-4 integrin detection until the 24 week infusion. This patient had developed anti-natalizumab neutralizing antibodies (NAB) which resolved over time. We observed a similar though less pronounced pattern evaluating rfi levels of the VLA-4 integrins' beta-1 subunit. All lymphocytic subpopulations showed a significant decrease in beta-1 rfi levels after 12 weeks (CD3+ T cells, p b 0.001; CD19+ B cells, p b 0.01; NK cells, p b 0.0001; NKT cells, p b 0.001) compared to preinfusion PBMC (Fig. 4A–D). Analysis of 12 and 48 weeks' data from patients on long-term natalizumab therapy (n = 8) revealed a further decline after about 1 year (CD3+ T cells, − 17.7%; CD19+ B cells, − 9.1%; NK cells, −26.5%; NKT cells, − 21.0%) (Table 3). The histograms in Fig. 1(E and F) visualize the impact of 48 weeks natalizumab therapy on percentages and fluorescence intensities of beta-1 high and low expressing populations on CD3+ T cells from patients #8 and #18. Comparative analysis of patient baseline samples versus the 48-week follow-up samples (n = 8) revealed a significant decrease in percent beta-1 high and a corresponding increase in percent beta-1 low subpopulations in both, CD3+ (p b 0.001) and CD19+ (p b 0.05) lymphocytes (Table 2). The observed increase in beta-1 low subpopulations was paralleled by significantly decreased rfi levels of CD3+ T cells (p b 0.001) and CD19+ B cells (p b 0.01). Analysis of the total beta-1 rfi levels of CD3+ (p b 0.001) and CD19+ (p b 0.01) populations produced similar results. The beta-1 high subpopulation of CD3+ T cells (p b 0.001) but not CD19+ B cells showed significantly decreased rfi levels (Table 2). Beta-1 integrin on monocytes apparently was not affected by natalizumab during the whole treatment period since we observed neither a significant increase nor decrease in rfi levels (Fig. 3B). Another exception was the 12 week measurement of the patient with non-persistent NAB whose beta-1 rfi levels were largely unchanged after 12 weeks compared to baseline on CD3+ T cells, NK, and NKT cells. 3.4. In vitro binding assay and potential steric effects

Examination of follow-up samples from RRMS patients under short-, intermediate, and long-term natalizumab therapy was done by analyzing total rfi levels and showed a sustained and strong reduction of alpha-4 rfi levels on all investigated leukocyte populations (CD3+ T cells, CD19+ B cells, NK cells, NKT cells, and monocytes) (Fig. 4A–D). The decrease in unblocked alpha-4 rfi levels on lymphocytes approximated to 80% whereas on monocytes the reduction was 55– 60% (Table 3). In one individual (patient #2, Table 1) administration of

To determine whether the observed decrease in beta-1 rfi levels on lymphocytes was a real effect of natalizumab therapy or an artefact due to steric hindrance of antibody binding we performed an in vitro experiment and incubated PBMC from volunteer donors with increasing amounts of natalizumab. The subsequent flow cytometric analysis of CD3+ T cells clearly showed an inverse correlation between increased amounts of cell-bound natalizumab (anti-IgG4)

Table 2 Mean percentages and fluorescence intensities of CD29 high and low expressing T and B cells. Relative fluorescence intensities

Percentage (%) Controls

RRMS patients

Controls

CD3+ T cells

Beta-1 low 45.8 ± 13.4 45.4 ± 18.9 12.6 ± 2.1 Beta-1 high 54.0 ± 13.4 54.4 ± 19.1 56.1 ± 5.3 Total beta-1 100 100 29.3 ± 9.4 CD19+ B cells beta-1 low 55.7 ± 18.0 54.9 ± 19.7 10.0 ± 1.9 beta-1 high 43.5 ± 17.9 42.8 ± 18.9 42.3 ± 15.5 total beta-1 100 100 18.2 ± 6.4 Values represent the means ± standard deviations of 18 RRMS patients or 18 controls. *p b 0.05; **p b 0.01; 1p = 0.056; 2p = 0.06.

CD19+ B cells

beta-1 low beta-1 high total beta-1 beta-1 low beta-1 high total beta-1

**14.7 ± 2.3 *63.6 ± 8.6 1 37.0 ± 13.6 *11.3 ± 1.6 2 51.0 ± 11.2 24.5 ± 13.8

Relative fluorescence intensities

Percentage (%)

CD3+ T cells

RRMS patients

Baseline

48 weeks

Baseline

48 weeks

40.4 ± 15.0 59.6 ± 14.9 100 57.0 ± 19.2 42.2 ± 18.5 100

**46.6 ± 13.7 *52.8 ± 13.3 100 *78.4 ± 18.5 *21.1 ± 18.6 100

15.7 ± 2.5 65.7 ± 7.3 40.5 ± 15.4 11.5 ± 2.2 50.2 ± 9.9 21.9 ± 11.9

***11.5 ± 2.1 ***49.8 ± 4.8 ***25.6 ± 13.2 *8.3 ± 4.15 43.3 ± 18.5 **9.3 ± 3.9

Values represent the means ± standard deviations of 8 RRMS patients at baseline and after 48 weeks of natalizumab therapy. *p b 0.05; **p b 0.01; ***p b 0.001. Abbreviations: RRMS, relapsing-remitting multiple sclerosis. The patient with the single beta-1 population was excluded from analyzing beta-1 low and high expressing populations.

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Fig. 2. Mean baseline relative fluorescence intensities (rfi) of alpha-4 (A) and beta-1 (B) on four lymphocyte subpopulations (CD3+ T cells, CD19+ B cells, NK cells, and NKT cells). Open circles: RRMS patients (n = 18), open squares: controls (n = 18). Bars represent 95% confidential intervals, triple asterisks represent high significance (p b 0.000001); n.s., non significant; NK, natural killer cells; NKT, CD3+ natural killer cells.

and decreased detection of alpha-1. Surface rfi levels of beta-1 remained unaffected irrespective of the amount natalizumab bound to the cells (Fig. 5) demonstrating no steric hindrance of the anti-beta1 detection antibody by natalizumab. 4. Discussion In this study we applied flow cytometry to demonstrate the blocking effect of monthly infused natalizumab on the alpha-4 chains and to investigate any additional effect on surface levels of the beta-1 chains of the heterodimeric VLA-4 molecule. We addressed the question if natalizumab-induced blockade of alpha-4 which renders the adhesion receptor non-functional also affects expression of beta-1. Natalizumab is generally considered a mere blocking antibody which antagonizes the interaction of VLA-4 of immune cells with vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (Man et al., 2009; Pilz et al., 2008). The blocking functionality of natalizumab was achieved by engineering the complementaritydetermining region of the murine parent antibody onto a human IgG4 framework. In contrast to antibodies of the IgG1 subclass, IgG4 do not elicit cytopathic effector functions as they neither bind complement nor have pronounced affinity to Fc receptors on other immune cells (Lutterotti et al., 2008). Obviously natalizumab exerts additional effects as it was also shown to increase numbers of leukocytes, nucleated erythrocytes, and pre-B cells (Krumbholz et al., 2008; Polman et al., 2006) in the

Fig. 3. Relative fluorescence intensities (rfi) of alpha-4 and beta-1 on monocytes. (A) Open circles: RRMS patients (n = 18), open squares: controls (n = 18). (B) Effects of natalizumab therapy on the rfi of alpha-4 (black line) and beta-1 (grey line) on monocytes after 12 weeks (n = 11), 24 weeks (n = 10), and 48 weeks (n = 8) compared to baseline. Bars represent 95% confidential intervals, triple asterisks represent high significance (p b 0.000001); n.s., non significant.

peripheral blood and to reduce endothelial activity by downregulation of VCAM-1 (Millonig et al., 2010). In particular increased peripheral pre-B cells might have specific consequences in respect to PML because B cell lineage-derived mononuclear cells have been identified as potential carriers for JC viruses (Houff et al., 1988). In the CNS, natalizumab was reported to interfere with immune surveillance by inducing a sustained decrease in immune cell numbers in the cerebrospinal fluid (Stuve et al., 2005) and depletion of dendritic cells in cerebral perivascular spaces (del Pilar et al., 2008). The molecular mechanisms underlying the observed shifts in response to natalizumab therapy are largely unresolved. Here, we confirmed that infusions of the standard dose of 300 mg natalizumab effectively blocked the alpha-4 integrin subunit on PBMC from our RRMS patients. This was demonstrated by a lack in fluorescence signals of the alpha-4 detection antibody which has the same epitope specificity as natalizumab. The sustained decrease of alpha-4 detectability of about 80% on lymphocytes and of approximately 55–60% on monocytes during the entire follow-up period was indicative of RRMS patients being on a pharmacological steady-state situation during natalizumab treatment. In addition to remarkably consistent results with a former study (Wipfler et al., 2011), reliability of our method and potential clinical relevance were established with the delayed decrease of alpha-4 detectability in one of our patients until the 24 week measurement which was caused by non-persisting antinatalizumab NAB. The most relevant finding of this study is the observed decrease in beta-1 integrin surface expression on lymphocytes but not on monocytes in response to natalizumab therapy. We found decreased beta-1 surface levels also on NK and NKT cells which are distinct from T and B lymphocytes. Like monocytes, NK cells are components of the

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Fig. 4. The therapeutic blocking and additional effects of natalizumab therapy on the relative fluorescence intensities (rfi) of alpha-4 (black line) and beta-1 (grey line) on CD3+ T cells (A), CD19+ B cells (B), NK cells (C), and NKT cells (D) after 12 weeks (n = 11), 24 weeks (n = 10), and 48 weeks (n = 8) compared to baseline. Bars represent 95% confidential intervals, triple asterisks represent high significance (p b 0.000001); n.s., non significant; NK, natural killer cells ; NKT, CD3+ natural killer cells.

innate immune system whereas NKT cells with their small repertoire of T cell receptors represent a bridge between innate and adaptive immunity (Van Kaer et al., 2011). Beta-1 expression further declined in the course of therapy between 12 and 48 weeks (Table 3) and apparently was unaffected by clinical apparent disease activity as beta-1 expression levels of three patients (#8, 13, and 17) experiencing a relapse during the observation period did not deviate from the general trend. In contrast to alpha-4 and its natalizumab-blocked surface detectability, decreases in beta-1 surface levels indicate a secondary treatment effect at the cellular level and an actual shift in surface expression. A reading artefact due to steric hindrance of the beta-1 detection antibody by proximate natalizumab was ruled out with our in vitro experiment proving that detection of beta-1 was unaffected by natalizumab bound to the alpha-4 subunit. To date we cannot explain the mechanism — if natalizumab affects a genuine downregulation of the molecule or if cells merely shed the redundant beta-1 chain. A certain capacity of natalizumab to alter gene expression in blood cells has been recently demonstrated by transcriptional profiling of whole blood from MS patients (Lindberg et al., 2008). This study provided evidence of natalizumab affecting gene transcription related to immune response, signal transduction, adhesion, and metabolism. However, it did not allow detailed conclusions as the molecular analysis was not performed on individual cell subpopulations.

In our study we report a possible additional effect of natalizumab on cellular adhesion via a decreased beta-1 integrin surface expression on four lymphocyte subpopulations. Moreover, we showed that B cells and NK cells from RRMS patients prior to natalizumab therapy had higher surface levels of alpha-4 and/or beta1 compared to controls indicating increased baseline levels. However, individuals assigned for natalizumab therapy are a special group of RRMS patients because of continuing disease activity despite immunomodulatory therapy. Factors potentially affecting baseline beta-1 expression levels including prior immunomodulatory therapy or disease activity and steroid use were minimized by the study design requiring a treatment- and relapse-free period of at least eight weeks before first blood sampling. Differences in baseline levels between patients and controls were even more pronounced by separately examining fluorescence intensities of beta-1 high and low subpopulations on T and B cells which revealed higher baseline levels in both CD3+ T cells and in the beta-1 low fraction of CD19+ B cells (Table 2). Total beta-1 rfi levels from patients receiving natalizumab since 48 weeks indicated a treatment-induced reduction of beta-1 expression levels below those from controls. In contrast to gender-specific differences recently reported from a study with male mice lacking beta-1 on their myelin basic proteinprimed T cells (Brahmachari and Pahan 2010) we have not observed any gender difference in beta-1 expression analyzing the control group.

Table 3 Mean% decrease in rfi levels of alpha-4 and beta-1 surface expression from the eight RRMS patients receiving natalizumab for one year. Cell type

CD3+ T cells CD19+ B cells NK cells NKT cells Monocytes

Alpha-4

Beta-1

% decrease rfi (SD) compared to baseline

% decrease rfi (SD) compared to baseline

48 weeks compared to 12 weeks

12 weeks

48 weeks

12 weeks

48 weeks

% decrease (rfi)

− 79.11 − 80.62 − 78.88 − 81.10 − 59.43

− 79.12 − 79.15 − 78.20 − 80.50 − 56.20

− 31.67 − 49.48 − 23.93 − 36.34 − 4.50

− 37.29 (14.8)* − 53.99 (15.3)* − 30.28 (7.5)* − 44.00 (14.0)* − 6.0 (24.1)

− 17.7 − 9.1 − 26.5 − 21.0 –

(2.5)* (4.7)* (5.0)* (3.1)* (6.4)*

(7.4)* (4.7)* (6.8)* (6.9)* (20.8)*

(12.9)* (13.2)* (6.4)* (9.6)* (27.4)

Abbreviations: rfi, relative fluorescence intensity; SD, standard deviation; NK, natural killer cells; NKT, CD3+ natural killer cells, *p b 0.01.

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Fig. 5. In vitro natalizumab binding on CD3+ T cells from a control individual. Bound natalizumab (open squares), alpha-4 expression (open rectangle), beta-1 expression (open circles). Values represent mean rfi ± SEM. rfi, relative fluorescence intensity; SEM, standard error of the mean.

We first reported secondary responsiveness of immune cells to natalizumab in a recent study observing decreased levels of leukocyte function antigen-1 (LFA-1) levels on T cells (Wipfler et al., 2011). Here we corroborate this finding by adding another candidate. LFA-1 has been shown to be involved in the development of severe EAE and to play a suppressor role on CNS inflammation and demyelination (Dugger et al., 2009). An important role of beta-1 on T cell trafficking during EAE has recently been shown with beta-1 deficient mice. Beta-1 deficiency resulted in encephalitogenic T cells which were unable to firmly adhere to CNS endothelium (Bauer et al., 2009). Along with alpha-4, beta-1 non-covalently associates with at least nine different alpha-chains representing a panel of VLA integrin adhesion receptor molecules for attachment to various extracellular matrix ligands. Reduced beta-1 levels might affect migration to sites of inflammation, immune surveillance, and presence of immunoregulatory cells at sites of deescalation. The cumulative decrease of alpha-4 availability as well as beta-1 and LFA-1 surface expressions might synergistically play a role in both, the occurrence of opportunistic CNS infections during long-term natalizumab treatment and the further slight decline in relapse rate in the second year of natalizumab treatment (Polman et al., 2006). Taken together, we have clearly demonstrated that besides blocking lymphocyte migration into the CNS, natalizumab exerts additional effects on the VLA-4 molecule resulting in severely reduced levels of beta-1 on all investigated subpopulations of peripheral blood lymphocytes after 12 weeks which further decline with time. Changes in surface availability of beta-1 suggest far reaching consequences with regard to long-term therapeutic effects of natalizumab. Intensive research is required to find answers concerning the molecular mechanisms and to achieve an improved safety profile for natalizumab therapy in the life-long disease MS. Acknowledgment This investigator initiated study was supported by a research grant of Biogen Idec Austria. References Bauer, M., Brakebusch, C., Coisne, C., Sixt, M., Wekerle, H., Engelhardt, B., Fassler, R., 2009. Beta1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity. Proc. Natl Acad. Sci. U.S.A. 106, 1920–1925.

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