M.B. received a travel grant from Biogen Idec, the manufacturer of natalizumab (Tysabri®), which was not related to this manuscript. Furthermore, he received travel grants, speaker’s fees and a research grant from Merck Serono, served on an advisory board for Almirall Hermal GmbH, and received research grants from Sanofi Aventis and Teva Pharmaceutical Industries Ltd. as well as from Novartis. The other authors declare no competing interests. Biogen Idec/Elan had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: FBS MB. Performed the experiments: TFB LD EMH EL FBS. Analyzed the data: TFB LD EMH EL AR ES MB FBS. Contributed reagents/materials/analysis tools: MB EMH EL AR. Wrote the paper: FBS MB.
¶ These authors also contributed equally to this work.
Natalizumab is a recombinant monoclonal antibody raised against integrin alpha-4 (CD49d). It is approved for the treatment of patients with multiple sclerosis (MS), a chronic inflammatory autoimmune disease of the CNS. While having shown high therapeutic efficacy, treatment by natalizumab has been linked to progressive multifocal leukoencephalopathy (PML) as a serious adverse effect. Furthermore, drug cessation sometimes induces rebound disease activity of unknown etiology. Here we investigated whether binding of this adhesion-blocking antibody to T lymphocytes could modulate their phenotype by direct induction of intracellular signaling events. Primary CD4+ T lymphocytes either from healthy donors and treated with natalizumab
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS
Th1 cells, as defined by IFN-γ and TNF-α production, were found to be the predominant subpopulation of T lymphocytes in the CNS and the peripheral blood of MS patients
Effector lymphocytes express elevated levels of adhesion molecules, enabling them to leave the vascular bed across the endothelium. More specifically, their firm arrest to endothelial cells is mediated by integrins. Integrin receptors are composed of non-covalently linked α- and β-chains. A special feature of integrins is their ability of bi-directional signaling
The integrin α-4 (CD49d) is known to dimerize with either integrin β-1 (CD29) or β-7. These integrin subunits are expressed by
Gene set | # genes ingene set (K) | # genes inoverlap (k) | k/K | P value |
LEE_TCELLS3_UP | 106 | 16 | 0.159 | 7.76E-06 |
MOREAUX_TACI_HI_VS_LOW_UP | 422 | 37 | 0.0877 | 1.52E-05 |
GOLDRATH_CELLCYCLE | 34 | 8 | 0.2353 | 6.11E-05 |
HSA04060_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION | 257 | 24 | 0.0934 | 1.61E-04 |
NUCLEAR_RECEPTORS | 40 | 8 | 0.2 | 2.00E-04 |
STEMPATHWAY | 15 | 5 | 0.3333 | 2.68E-04 |
IDX_TSA_UP_CLUSTER3 | 90 | 12 | 0.1333 | 3.10E-04 |
CDC25PATHWAY | 9 | 4 | 0.4444 | 3.29E-04 |
DOX_RESIST_GASTRIC_UP | 44 | 8 | 0.1818 | 3.88E-04 |
GAMMA-UV_FIBRO_UP | 35 | 7 | 0.2 | 4.92E-04 |
Genes were annotated to pathways (C2) using GSEA (Gene Set Enrichment Analysis) database, which showed at least a 0.5 fold [log2] stronger expression in the group of natalizumab-treated CD4+ T cells than in the group of natalizumab-untreated cells according to the respective mean values of three biological replicates.
IL2 | Interleukin 2 |
IL4 | Interleukin 4 |
IL9 | Interleukin 9 |
IL3 | Interleukin 3 (colony-stimulating factor, multiple) |
CSF1 | Colony stimulating factor 1 (macrophage) |
LTB | Lymphotoxin beta (TNF superfamily, member 3) |
LTA | Lymphotoxin alpha (TNF superfamily, member 1) |
CXCL5 | Chemokine (C-X-C motif) ligand 5 |
CCL4 | Chemokine (C-C motif) ligand 4 |
CCL1 | Chemokine (C-C motif) ligand 1 |
CXCR3 | Chemokine (C-X-C motif) receptor 3 |
IL17AR | Interleukin 17A |
ACVR1B | Activin A receptor, type IB |
FLT1 | Fms-related tyrosine kinase 1 |
TNFRSF13C | Tumor necrosis factor receptor superfamily, member 13C |
BMPR2 | Bone morphogenetic protein receptor, type II |
IL22 | Interleukin 22 |
OSM | Oncostatin M |
IL21 | Interleukin 21 |
XCL1 | Chemokine (C motif) ligand 1 |
FLT3LG | Fms-related tyrosine kinase 3 ligand |
CCL28 | Chemokine (C-C motif) ligand 28 |
XCL2 | Chemokine (C motif) ligand 2 |
TNFSF14 | Tumor necrosis factor (ligand) superfamily, member 14 |
Genes of the HSA04060_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION pathway are given which were found to be enriched in samples of natalizumab-treated cells by GSEA pathway annotation. Listed genes exhibited an at least 0.5-fold [log 2] stronger expression in natalizumab-treated than in natalizumab-untreated cells.
Natalizumab is a humanized monoclonal IgG4κ antibody directed against the human integrin α-4 subunit of VLA-4 and was generated to physically interfere with lymphocyte extravasation across the BBB in patients with MS, after the central pathogenic relevance of VLA-4 in EAE had been discovered
To rapidly remove natalizumab in case of PML, patients receive plasma exchange or immunoabsorption to hasten immune reconstitution. However, they regularly develop exuberant CNS inflammatory disease activity a few weeks later, termed immune reconstitution inflammatory syndrome (IRIS) or more specifically PML-IRIS
The reason for the occurrence of peripheral pro-inflammatory T lymphocytes during natalizumab treatment is unknown. Therefore, we decided to explore the capability of this anti-integrin α-4 antibody to modulate the phenotype of T lymphocytes by direct induction of intracellular signaling. And indeed, natalizumab, designed to block CD49d-mediated immune cell extravasation, was found to induce overall mild ‘outside-in’ signals in activated T lymphocytes along with an increased expression of pro-inflammatory cytokines. In line with inter-individual differences with respect to rebound disease and PML-IRIS, not all T cells were equally responsive. Therefore, natalizumab signals directly and – dependent on the immunological situation of individual patients with MS – can serve as a pro-inflammatory costimulus for T cell receptor-activated cells.
All experiments involving MS patients were approved by the Ethics Committee of the Faculty of Medicine at the University of Würzburg. Written informed consent was obtained from all 9 patients prior to collection of blood samples. All of them had RRMS according to the 2005 revision of the McDonald diagnostic criteria
Blood from healthy donors of random age and gender was obtained after written informed consent in accordance with the Declaration of Helsinki, under a protocol that had received Institutional Review Board approval from the Ethics Committee of the Faculty of Medicine at the University of Würzburg. In addition, PBL generated during thrombocytapheresis of blood from healthy donors were kindly provided by the local Department of Transfusion Medicine. Since these blood samples were offered anonymously, the Ethics Committee of the Faculty of Medicine at the University of Würzburg gives a general approval.
PBMC were isolated from EDTA blood samples via centrifugation in Biocoll® separating solution (Biochrom, Berlin, Germany). CD4+ T lymphocytes were enriched by positive selection using a MACS human CD4 microbeads isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Purity of the CD4+ population was typically >85% up to 95%.
For microarray gene expression profiling, 3.0×105 CD4+ T cells were placed in 25 ml CTL medium containing 30 Gy irradiated PBMC (2.5×107), 60 Gy irradiated T2 feeder cells (5.0×106), and OKT-3 (30 ng/ml) for 3 days
Except for microarray experiments, PBMC and CD4+ T lymphocytes (1–5×106 cells/ml) were cultured in complete X-VIVO15 medium, supplemented with 10% heat-inactivated, pooled human AB serum and 100 U/ml penicillin-streptomycin (all from Life Technologies). Jurkat cells (5×106 cells/ml) were kept in complete RPMI1640 medium, containing 5% heat-inactivated FCS. Cells were stimulated for the indicated times (2, 4, 8, 24, 48, 72 h): 1 µg/ml staphylococcus enterotoxin B (SEB, Toxin Technology, Sarasota, FL), anti-CD3/28-Dynabeads® (Life Technologies) or plate-bound anti-CD3/28 mAb (both 5 µg/ml, BD Biosciences, Heidelberg, Germany), T/I (10 ng/ml/2 µM) in the absence or presence of natalizumab (30 µg/ml), controlled by IgG4 (BD Biosciences or Sigma Aldrich) or HP2/1 (Abcam, Cambridge, UK; Abnoby, Heidelberg, Germany; or Beckman Coulter, Krefeld, Germany). For intracellular FACS stainings 1 µg/ml brefeldin A or brefeldin A plus 1.34 µg/ml monensin (for IL-17 and IFN-γ was added for the last 8–12 h.
Gene expression profiling was performed with HG U133 plus 2.0 gene expression arrays (Affymetrix) following the Affymetrix expression analysis technical manual (
Cells were harvested, washed in PBS and FACS buffer (PBS containing 0.1% BSA), blocked with Fc receptor blocking reagent (BD Biosciences) for 10 min at room temperature (RT) and rinsed with FACS buffer. Surface staining was performed using anti-human CD4-FITC and anti-human CD49d-PE (all BD Biosciences), for 30 min at RT, and fixation with 2% paraformaldehyde (PFA) in PBS, for 20 min at RT. For intracellular staining cells were incubated in permeabilization buffer (FACS buffer containing 0.1% saponin), for 10 min at RT, pelleted, resuspended in a small volume of permeabilization buffer, and stained with anti-IFN-γ-FITC, anti-IL-17-APC or anti-IL-2-APC (all Miltenyi Biotech), for 90 min at 4°C. Washed samples were measured on the FACS LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR,
Total RNA from human primary CD4+ T cells was purified using Trizol® (Life Technologies), and cDNA was accomplished by the iScript cDNA synthesis kit (BioRad, Hercules, CA). Quantitative RT-PCR (qRT-PCR) was performed by using an ABI 7000 RT-PCR instrument (Applied Biosystems, Carlsbad, CA). All primers were TaqMan® probes from Applied Biosystems and used according to the manufacturer’s standard protocols. Normalization for each sample was achieved by using the formula: ΔCT = CT(target gene) – CT(B2M). Relative expression was calculated with the formula: 2–ΔCT.
Whole cellular protein lysates were made from 2×107 Jurkat or CD4+ T cells using RIPA buffer and the snap-freezéńthaw method. Nuclear and cytosolic protein extracts were generated using the ProteoJET® Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas Life Sciences, St. Leon-Rot, Germany). An equal amount of total protein was fractionated by 12% SDS-PAGE and electroblotted onto nitrocellulose membrane. For detection, anti-JNK, anti-pJNK, anti-ERK, anti-pERK and anti-NFATc1 antibodies (all from Santa Cruz Biotechnology, Heidelberg, Germany), with corresponding anti-mouse or anti-rabbit peroxidase-coupled secondary antibodies (Sigma Aldrich) were used with an enhanced chemiluminescence system (Pierce, Rockford, IL). To analyze CD49d protein levels in cell culture supernatants, proteins were first concentrated by centrifugation of 300 µl per stimulation condition supernatant through Microcon YM-10 centrifugal filter devices (Millipore, Schwalbach/Ts., Germany). The recovered proteins were fractionated by 8% SDS-PAGE and electroblotted onto nitrocellulose membrane. For detection, two different mAbs against the extracellular domain of CD49d (clone L25, BD Biosciences; clone HP2/1, Beckman Coulter) were used with an anti-mouse peroxidase-coupled secondary antibody (Jackson ImmunoResearch via Dianova, Hamburg, Germany) along with an enhanced chemoluminescence system (BD Biosciences).
PBMC from MS patients were isolated, resuspended at a total of 5×106/ml cells and stimulated with plate-bound anti-CD3/28 (both BD Biosciences) in a 24-well tissue culture plate. Supernatants were collected after 48 h and cytokine measurement of human IFN-γ, IL-2, IL-10, IL-4, IL-21, IL-12/IL-23 p40 and IL-17 was performed using a cytometric bead array (CBA) kit (BD Biosciences). The mixtures were incubated for 3 h; beads were washed and analyzed using flow cytometry (FACS CantoII, BD Biosciences). Cytokine concentration (pg/ml) was calculated using CBA software.
Following stimulation as indicated, 5×104 PBMC per stimulation condition were transferred to microscope slides, using a standard cytospin protocol. Cells were fixed with 3.7% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 5 min at RT, washed two times with phosphate-buffered saline and blocked in PBS containing 5% BSA for 1 h at RT. Subsequently, a primary mouse mAb against CD49d (clone L25) in PBS containing 1% BSA was incubated at 4°C overnight. Then, a Cy3-coupled anti-mouse secondary antibody (Jackson ImmunoResearch) was incubated for 1 h at RT. Nuclei were counterstained with DAPI. Finally, an antifading agent comprised of Mowiol 4–88 (Calbiochem), glycerole and Dabco (Sigma Aldrich) in 0.2 M Tris HCl was added. Negative controls were performed by staining with an isotype-matched IgG2b control antibody (BD Biosciences). The stainings were analyzed by confocal laser scanning microscopy (Leica TCS SP2 equipment, objective lense; HeX PL APO, 40×/1.25–0.75) and LCS software (Leica).
Total protein levels of matrix metalloproteinases MMP-2 and MMP-9 in culture supernatants of 1×107 PBMC in 2 ml complete X-VIVO15 medium as detailed above were measured using commercial ELISA kits (R&D, Wiesbaden-Nordenstadt, Germany) according to the instructions of the manufacturer. Both MMP-2 and MMP-9 were detectable in all supernatants. All samples were analyzed in duplicates. Mean intraassay reproducibility was 7.4% for MMP-2 and 6.2% for MMP-9.
Except for pathway annotation of microarray data, where two-tailed t tests were employed (see above), non-parametric tests were used throughout the study. For comparison of two conditions, a two-tailed Wilcoxon matched-pairs signed rank test or a two-tailed Mann-Whitney test were employed as appropriate. For comparison of more than two time points, Bonferroni adjustment was applied, when indicated. Alternatively, a two-tailed Friedman test or a two-tailed Kruskal-Wallis test were performed, which both were followed by Dunn’s multiple comparisons test, if p values were <0.05. For correlation analysis, Spearman tests were used. A p value <0.05 was considered as statistically significant. GraphPad PRISM 5 software (La Jolla, CA) was used for all analyses.
In order to detect any possible signaling via natalizumab we performed an unbiased screen by gene expression profiling using the HG U133 plus 2.0 gene expression arrays (Affymetrix). RNA was prepared from CD4+ T cells grown and (re-) stimulated in the presence or absence of natalizumab. The Affymetrix expression analysis revealed only mild changes. Nevertheless, several pathways were – positively or negatively – affected, clearly demonstrating a signaling potential of natalizumab (
(A) List of genes that were influenced the most by natalizumab in CD4+ T cells, which were restimulated for 8 h. (B) List of genes that were influenced the most by natalizumab in CD4+ T cells, which were not restimulated.
To analyze the effect of natalizumab on lymphokine expression more closely, important lymphokines, surface molecules, and the key regulators of CD4+ T cell differentiation were compared on a heat map (
(A) RNA expression differences of three independent biological replicates are depicted in heat maps for immunologically relevant genes. Cells had not or had been restimulated with T/I (TI). Both groups had been grown with or without natalizumab (N or Nat). (B) qRT-PCR of RNA used for the microarray (triplicates) was analyzed for RNA levels of IL-2 as well as RORγt, T-BET and FOXP3. These three transcription factors are transcribed from the genes
If natalizumab exerts a direct signaling effect, an influence should become detectable within the time frame of one cell cycle. And indeed, intracellular staining for IL-2 and concomitant FACS analyses demonstrated that CD4+ T cells stimulated with superantigen (SEB) or anti-CD3/28 for only 24 h both expressed significantly more IL-2 in the presence of natalizumab (
(A, B) PBMC were stimulated by SEB or anti-CD3/28 without or in the presence of natalizumab for 24 h. After surface staining for CD4 and intracellular staining for IL-2, cells were analyzed by FACS. (A) Representative dot blots are given. (B) Single values of samples from n = 11 (SEB) and n = 17 (anti-CD3/28) donors are shown. (C, D) Isolated CD4+ T cells were stimulated with T/I (n = 9) or ionomycin (n = 10) for 8 h. Intracellular FACS stainings for IL-2, IL-17, and IFN-γ were performed, and the differences between cultures with and without natalizumab were calculated. Two-tailed Wilcoxon matched-pairs signed rank tests were performed for statistical analysis in (B, C, and D).
In addition to IL-2, expression of IL-17 and IFN-γ under the influence of natalizumab was analyzed on the protein level. In order to achieve sufficient effector lymphokine expression, CD4+ T cells were activated with the chemical compounds TPA and ionomycin, bypassing, but mimicking TCR crosslinking by activating protein kinase C and Ca2+/calcineurin, respectively, for only 24 h. Intracellular FACS staining revealed robust IL-2 expression after T/I stimulation (median 45.6% CD49dhi cells without presence of natalizumab), which was more than ten times higher than that mediated by SEB (median 3.2%) or anti-CD3/28 (median 4.0%) stimulation (
If integrin α-4 can be triggered by natalizumab, footmarks of the induced signaling should be detectable. For molecular analyses, we initially used the human CD4+ T cell line Jurkat. Surface expression of integrin α-4 (
(A) Surface expression of CD49d was verified for Jurkat cells. (B) Intracellular FACS staining for IL-2 revealed increased expression in the presence of natalizumab, when cells were stimulated by T/I (2 concentrations) for 8 h. (C) Western blot analysis of nuclear extracts for NFATc1 after stimulation as indicated for 2 h. (D, E) Immunoblot of proteins extracted from Jurkat cells after stimulation with TPA, ionomycin or both in the presence or absence of natalizumab for 2 h. (D) Detection of pERK and ERK within whole cell extracts. (E) Detection of pJNK and JNK within whole cell extracts. (F) Immunoblot of total proteins extracted from human CD4+ T cells after stimulation with anti-CD3/28– alone or in combination - in the absence or presence of either agonistic anti-CD49d or natalizumab for 2 h. Phosphorylated ERK and total ERK were detected.
Thereafter, we analyzed MAPK pathways described to be touched by integrin signals. Jurkat T cells were stimulated by TPA and/or ionomycin. (i) While the overall expression of ERK was unchanged during natalizumab treatment (
A common feature of receptor engagement and signaling is the concomitant internalization. We sought to follow integrin α-4 expression on the surface of Jurkat T cells when solely natalizumab was added. Clearly, CD49d was reduced by the magnitude of roughly half a log scale within 24 h (
(A) Jurkat cells were treated with natalizumab for 24 h and stained for CD49d expression. (B) A possible interference of natalizumab and the staining antibody for CD49d was evaluated. (C) T/I-activated PBMC incubated with natalizumab for 24 h prior to staining and FACS analyses. Gated on CD4+ T cells, CD49d appeared in three subpopulations. (D) Percentage of CD49dhi cells among all CD4+ cells after stimulation as indicated for 8 h (left column) and normalization of natalizumab-stimulated to natalizumab-unstimulated cells without and with T/I activation for 2, 4 and 8 h (right column). CD49dhi expression was evaluated by FACS. (E) CD4+ T cells were activated with CD3/28 or T/I in the absence or presence of natalizumab for the indicated durations. Ratios of natalizumab-stimulated vs. unstimulated cells were calculated (upper two diagrams). Lower diagram shows percentage of CD49dhi cells among all CD4+ cells after activation with CD3/28 or T/I for 24 h. Same samples as in first columns of upper and middle diagram. (F) CD4+ T cells had been preincubated for 24 h with natalizumab, finally stimulated by T/I for 8 h, stained for CD49d surface and IL-2 intracellular expression and evaluated by FACS. (G) CD4+ T cells were stimulated by T/I for 8 h and stained for intracellular IL-17 and IFN-γ as well as for surface expression of CD49d, analyzed by FACS.
Activation of T cells by T/I for 8 h (without natalizumab) strongly reduced the amount of CD49dhi CD4+ T cells in 4 of 6 samples from independent donors in comparison to unstimulated cells (median 69.6% reduction,
When comparing CD3/28- and T/I-stimulated cells over a longer period of time, the relative reduction of CD49hi T cells due to natalizumab treatment over 24 h was much more pronounced after activation by CD3/28 costimulation vs. T/I treatment in this independent set of samples (Mann-Whitney p<0.0001,
Under all conditions, the CD49dhi population was predominantly reduced upon natalizumab addition. Major IL-2 correlated with CD49dhi expression and was only forced into low and intermediate integrin α-4 expressers by natalizumab (
We next sought to further investigate the fate of integrin α-4 after natalizumab binding. Immunocytochemical staining of CD49d in unstimulated PBMC allowed distinction of different lymphocyte subpopulations according to differential integrin α-4 expression levels, as observed by flow cytometry. CD49d mostly showed a fine granular pattern of membrane staining, with only minor intracellular staining observed under a confocal microscope (
(A–C) Immunocytochemical staining of CD49d and counter-staining of nuclei in PBMC that were either left untreated (A), stimulated with 30 µg/ml natalizumab for 24 h (B) or stimulated with 30 µg/ml IgG4κ isotype control (C). Cells were fixed with 3.7% PFA and permeabilized with 0.1% Triton X-100 before staining. Representative of samples from 4 different donors. (D) PBMC were stimulated with anti-CD3/28 and/or natalizumab as indicated for 24 h. Subsequently, total protein levels of MMP-2 and MMP-9 were determined in cell culture supernatants by commercial ELISA kits. Friedman tests, followed by Dunn’s post tests were performed for statistical analysis.
As only single cells displayed intracellular inclusions after only 24 h of natalizumab stimulation, while the vast majority of cells showed very low CD49d expression levels, we next evaluated potential CD49d secretion into cell culture supernatants. Of note, some members of the integrin family were described to be subject to shedding from the cell surface by metalloproteinases
Although primary CD4+ T cells had been analyzed during the course of the experiments, it was not clear if autoimmune-active cells would react the same way. Therefore, PBMC were isolated from 9 RRMS patients immediately before and 24 h after the very first receipt of natalizumab. To avoid any perturbing influence from separation procedures, no CD4+ T cell isolation was performed. PBMC from both blood withdrawals were immediately analyzed or gently stimulated by plate-bound anti-CD3/28 for 24 h without any further addition of natalizumab. Therefore, any difference observed can be attributed to the infused drug.
In accordance with the findings in CD4+ T cells from healthy donors, which were loaded with natalizumab
Pre- and post-natalizumab blood samples were taken from 9 patients with RRMS. (A-C) PBMC were cultivated with plastic-coated anti-CD3/28 for 24 h. (A) Cells were stained for surface expression of CD4 and CD49d before and after
Natalizumab blocks the firm arrest of leukocytes to the endothelium of the BBB, preventing their extravasation into the CNS with high efficacy. Here we demonstrate that this mAb – named Tysabri® as a drug – additionally has a direct costimulatory effect on T cells, mildly supporting a pro-inflammatory phenotype. This was not only shown
Natalizumab exerts its leukocyte adhesion blocking effect by direct molecular interference with the binding of VLA-4 on leukocytes to VCAM-1 on CNS microvascular endothelium. In addition, integrin α-4 was found to be partially but persistently downregulated on PBMC of MS patients receiving the drug, potentially representing an additional mechanism of adhesion blockade
However, extravasation is only one possible process that is mediated by CD49d and its integrin binding partners β-1 and β-7. Several cellular events are affected in addition to adhesion and migration, including cell differentiation, polarization, activation and survival
Evidence that integrin α-4/β-1 is involved in priming of naive T cells has been provided: upon binding of α-4 activating antibodies, VLA-4 colocalizes with the signaling molecules of the TCR complex and T cells are shifted towards a Th1-type immune response
Prolonged therapeutic CD49d blockade by natalizumab certainly results in unexpected changes of the immune system. Enrichment with leukocytes of different origin in the periphery and mobilization of hematopoietic stem/progenitor cells
Overall, this helps to explain a rebound of disease activity observed in some patients after natalizumab cessation
Furthermore, PML-IRIS in natalizumab-treated patients
Taken together, natalizumab efficiently excludes activated lymphocytes from the CNS by at least two mechanisms, but the activated pro-inflammatory situation in the periphery caused by direct signaling capacities of natalizumab might help to explain some adverse effects of this treatment. Novel integrin α-4 blocking pharmacological agents, which are currently in clinical development, should be carefully evaluated for their signaling potential.
We are indebted to Max Topp and his lab for providing irradiated PBMC and T2 feeder cells for CD4+ T cell culture as well as to the Department of Transfusion Medicine for PBL. We thank our scientific colleague Martin Vaeth for valuable support and helpful discussions as well as Nadine Kehl and Melanie Schott for excellent technical assistance.