Very Late Activation Antigen-4 Research Articles

LFA-1 activation enriches tumor-specific T cells in a cold tumor model and synergizes with CTLA-4 blockade.

The inability of CD8+ effector T cells (Teffs) to reach tumor cells is an important aspect of tumor resistance to cancer immunotherapy. The recruitment of these cells to the tumor microenvironment (TME) is regulated by integrins, a family of adhesion molecules that are expressed on T cells. Here, we show that 7HP349, a small-molecule activator of lymphocyte function–associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrin cell-adhesion receptors, facilitated the preferential localization of tumor-specific T cells to the tumor and improved antitumor response. 7HP349 monotherapy had modest effects on anti–programmed death 1–resistant (anti–PD-1–resistant) tumors, whereas combinatorial treatment with anti–cytotoxic T lymphocyte–associated protein 4 (anti–CTLA-4) increased CD8+ Teff intratumoral sequestration and synergized in cooperation with neutrophils in inducing cancer regression. 7HP349 intratumoral CD8+ Teff enrichment activity depended on CXCL12. We analyzed gene expression profiles using RNA from baseline and on treatment tumor samples of 14 melanoma patients. We identified baseline CXCL12 gene expression as possibly improving the likelihood or response to anti–CTLA-4 therapies. Our results provide a proof-of-principle demonstration that LFA-1 activation could convert a T cell–exclusionary TME to a T cell–enriched TME through mechanisms involving cooperation with innate immune cells.

Homing Genes Expression in Fucosyltransferase VI-Treated Umbilical Cord Blood CD133+ Cells which Expanded on Protein-Coated Nanoscaffolds.

Umbilical cord blood (UCB)-derived hematopoietic stem cells (HSCs) are considered because of their self-renewing, differentiating, proliferating, and readily available properties. Moreover, HSCs' homing to the hematopoietic microenvironment is an important step in their transplantation process. But low content of progenitor cells in one unit of UCB and defect in the bone marrow (BM) homing limit their applications. Hence, we decided to correct this deficiency with ex vivo incubation of CD133+ cells using fucosyltransferase VI and GDP-fucose. Then C-X-C chemokines receptor-4 (CXCR4), very late activation antigen-4 (VLA4), very late activation antigen-5 (VLA5), lymphocyte function-associated antigen-1 (LFA-1), and E-cadherin (E-cad) genes expressions were investigated with the goal of homing evaluation. The purity of MACS isolated CD133+ cells and confirmation of fucosylation were done by flow cytometry, and the viability of cells seeded on protein-coated poly L-lactic acid (PLLA) scaffold was proven via MTT assay. Scanning electron microscopy (SEM), CFU assays, and expression assays of CXCR4, VLA4, VLA5, LFA-1 and E-cad by real-time PCR were performed, too. Flow cytometry data showed that isolated cells were suitable for fucosyltransferase VI (FT-VI) incubation and expansion on nanoscaffolds. MTT, CFU assays, and SEM micrographs demonstrated fibronectin (FN)-collagen-selectin (FCS)-coated scaffold serve as best environment for viability, clonogenicity, and cell attachment. High levels of homing genes expression were also observed in cells seeded on FCS-coated scaffolds. Also, CXCR4 flow cytometry analysis confirmed real-time data. FCS-PLLA scaffolds provided optimal conditions for viability of FT-VI-treated CD133+ cells, and clonogenicity with the goal of improving homing following UCB-HSCs transplantation.

High interindividual variability in the CD4/CD8 T cell ratio and natalizumab concentration levels in the cerebrospinal fluid of patients with multiple sclerosis.

Strongly decreased leucocyte counts and a reduced CD4/CD8 T cell ratio in the cerebrospinal fluid (CSF) of natalizumab (NZB)-treated multiple sclerosis (MS) patients may have implications on central nervous (CNS) immune surveillance. With regard to NZB-associated progressive multi-focal leucoencephalopathy, we aimed at delineating a relationship between free NZB, cell-bound NZB, adhesion molecule (AM) expression and the treatment-associated shift in the CSF T cell ratio. Peripheral blood (PB) and CSF T cells from 15 NZB-treated MS patients, and CSF T cells from 10 patients with non-inflammatory neurological diseases and five newly diagnosed MS patients were studied. Intercellular adhesion molecule-1 (ICAM-1), leucocyte function antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), NZB saturation levels, and T cell ratios were analysed by flow cytometry. NZB concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Lower NZB saturation levels (P<0.02) and a higher surface expression of ICAM-1 and LFA-1 (P<0.001) were observed on CSF CD8 T cells. CSF T cell ratios (0.3-2.1) and NZB concentrations (0.01-0.42 µg/ml) showed a pronounced interindividual variance. A correlation between free NZB, cell-bound NZB or AM expression levels and the CSF T cell ratio was not found. Extremely low NZB concentrations and a normalized CSF T cell ratio were observed in one case. The differential NZB saturation and AM expression of CSF CD8 T cells may contribute to their relative enrichment in the CSF. The reduced CSF T cell ratio appeared sensitive to steady-state NZB levels, as normalization occurred quickly. The latter may be important concerning a fast reconstitution of CNS immune surveillance.

Elevated expression of CX3C chemokine receptor 1 mediates recruitment of T cells into bone marrow of patients with acquired aplastic anaemia

Acquired aplastic anaemia (AA) is a T-cell-mediated, organ-specific autoimmune disease characterized by haematopoietic stem cell destruction in the bone marrow. The exact molecular mechanism of T-cell trafficking into the bone marrow is unclear in AA. Very late activation antigen-4 (VLA-4) and CX3C chemokine receptor 1 (CX3CR1) play active roles in many autoimmune diseases. Therefore, we investigated whether VLA-4 and CX3CR1 also contribute to T-cell migration into the bone marrow in acquired AA. Expression levels of CX3CR1 and VLA-4 and their ligands [fractalkine (CX3CL1) and vascular cell adhesion molecule-1 (VCAM-1)] were examined in 63 patients with AA and 21 healthy control subjects. T-cell chemotaxis and adhesion were analysed in 17 patients with severe AA. We also prospectively evaluated the expression pattern of CX3CR1 during treatment with antithymocyte globulin plus cyclosporine in 11 patients with severe AA. The proportion of peripheral and bone marrow CD4(+) and CD8(+) T cells expressing CX3CR1 and the level of CX3CL1 was increased in patients with AA. However, there was no significant difference in VLA-4 expression or VCAM-1 levels. Functional studies demonstrated that chemotaxis towards autologous bone marrow plasma or soluble CX3CL1 was significantly higher in T cells from AA patients and could be blocked by CX3CR1 inhibitors. CX3CR1-mediated T-cell adhesion was also upregulated in these patients. The expression of CX3CR1 was associated with the efficacy of immunosuppressive therapy. The present findings demonstrate that CX3CR1 plays a pivotal role in recruitment of T cells into the bone marrow in acquired AA and is a potential therapeutic target for treatment of this disorder.

Glatiramer acetate attenuates the pro-migratory profile of adhesion molecules on various immune cell subsets in multiple sclerosis

An altered expression pattern of adhesion molecules (AM) on the surface of immune cells is a premise for their extravasation into the central nervous system (CNS) and the formation of acute brain lesions in multiple sclerosis (MS). We evaluated the impact of glatiramer acetate (GA) on cell-bound and soluble AM in the peripheral blood of patients with relapsing-remitting MS (RRMS). Fifteen patients treated de novo with GA were studied on four occasions over a period of 12 months. Surface levels of intracellular cell adhesion molecule (ICAM)-1, ICAM-3, lymphocyte function-associated antigen (LFA)-1 and very late activation antigen (VLA)-4 were assessed in T cells (CD3(+) CD8(+) , CD3(+) CD4(+) ), B cells, natural killer (NK) cells, natural killer T cells (NK T) and monocytes by five-colour flow cytometry. Soluble E-selectin, ICAM-1, ICAM-3, platelet endothelial cell adhesion molecule (PECAM)-1, P-selectin and vascular cell adhesion molecule (VCAM)-1 were determined with a fluorescent bead-based immunoassay. The pro-migratory pattern in RRMS was verified by comparison with healthy controls and was characterized by up-regulation of LFA-1 (CD3(+) CD4(+) T cells, B cells), VLA-4 (CD3(+) CD8(+) T cells, NK cells), ICAM-1 (B cells) and ICAM-3 (NK cells). Effects of GA treatment were most pronounced after 6 months and included attenuated levels of LFA-1 (CD3(+) CD4(+) ) and VLA-4 (CD3(+) CD4(+) , CD3(+) CD8(+) , NK, NK T, monocytes). Further effects included lowering of ICAM-1 and ICAM-3 levels in almost all immune cell subsets. Soluble AM levels in RRMS did not differ from healthy controls and remained unaltered after GA treatment. The deregulated pro-migratory expression profile of cell-bound AM is altered by GA treatment. While this alteration may contribute to the beneficial action of the drug, the protracted development and unselective changes indicate more secondary immune regulatory phenomena related to these effects.

VLA-4 Blockade by Low Molecular Weight Heparin: A Novel Therapeutic Approach in Sickle Cell Disease.

AbstractAbstract 2113 Background and SignificanceVaso-occlusion in sickle cell disease (SCD) that leads to painful events and complications is caused in part by adhesion of sickled erythrocytes (SSRBCs) to components of the vascular wall and to circulating leukocytes (WBC). Very late activation antigen-4 (VLA-4) or α4β1 integrin expressed on RBCs and WBCs mediates SSRBC-WBC adhesion as well as SSRBCs adhesion to the endothelium via vascular cell adhesion molecule (VCAM-1). VLA-4 blockade has been used to treat pathologic inflammation in multiple sclerosis and Crohn’s Disease by inhibiting the interaction between VLA-4 on the leukocyte and endothelial VCAM-1. Enoxaparin, a low molecular weight heparin (LMWH), has been recently shown to exhibit anti-adhesive properties via VLA-4 inhibition. The potential role of enoxaparin as an anti-adhesive therapy for SCD has not been explored. The objective of this study was to determine if enoxaparin could decrease VLA-mediated SS RBC adhesive interactions. Design and MethodsPeripheral blood samples were obtained from pediatric patients (n=6) with homozygous SCD (5–18 years) at steady state. Normal control samples (n=3) were obtained from healthy volunteers.We evaluated the interaction between VLA-4 on isolated SSRBCs and immobilized VCAM-1 in a flow adhesion assay under physiologic flow conditions. RBCS were isolated from whole blood after ficoll density gradient separation. We observed adhesion of SSRBCs to immobilized VCAM-1 under three conditions: 1) unstimulated, 2) stimulated with Manganese (Mn2+) – an experimentally accepted method of integrin stimulation that puts VLA-4 in its active confirmation and 3) Mn2+ stimulated RBCs pretreated with enoxaparin.SSRBC-WBC aggregates were identified by classical flow cytometry. Whole blood samples were processed under three conditions: 1) unstimulated, 2) stimulated with Mn2+ and 3) stimulated with Mn2+ and pretreated with enoxaparin. Samples were separated on a ficoll density gradient to obtain an enriched WBC layer. Within this WBC layer, components of multicellular aggregates were identified using antibodies staining for WBCs (anti-CD45), or RBCs (anti-glycophorin A), and CD71 for reticulocytes. ResultsMn2+ treatment enhanced VLA-4 adhesion of isolated SSRBCs to immobilized VCAM-1 in 5/6 patient samples. When Mn2+ stimulated samples were treated with enoxaparin, 4/6 patient samples showed a significant decrease in adhesion (p < 0.02).Mn2+ stimulation induced SS RBC-WBC aggregate formation in 3/4 patient samples tested. Both mature red blood cells and reticulocytes were involved in these aggregates. Aggregates were variable in size. All 4 patient samples treated with enoxaparin showed decreased aggregates. 3/4 patient samples showed aggregates decreased to levels at or below that observed in unstimulated samples. ConclusionsOur data show that enoxaparin inhibits adhesion of pediatric SSRBCs to immobilized VCAM-1, presumably via blockade of activated VLA-4 on the SS RBC surface. Further, our data demonstrate that stimulation of VLA-4 can increase adhesive interactions of circulating SSRBCs and WBCs, and that these interactions can be disrupted by enoxaparin. The complications and sequelae in SCD begin in childhood warranting the need to find more treatment strategies for pediatric SCD. The fact that numerous adhesive interactions responsible for vaso-cclusive events in SCD are VLA-4-mediated makes enoxaparin an attractive therapeutic possibility to prevent and treat vaso-occlusive complications in SCD. Disclosures:No relevant conflicts of interest to declare.

CXCR4 and vascular cell adhesion molecule 1 are key chemokine/adhesion receptors in the migration of cytokine‐activated T cells

To examine the migratory properties of cytokine-activated T (Tck) cells. Tck cells were generated by culture of peripheral blood T cells in the presence of interleukin-6 (IL-6), tumor necrosis factor α, and IL-2. Changes in cell surface phenotype were analyzed by flow cytometry. Chemotactic responsiveness was measured using in vitro chemotaxis assays and transendothelial migration through human umbilical vein endothelial cell monolayers. Levels of vascular cell adhesion molecule 1 (VCAM-1) were measured by sandwich enzyme-linked immunosorbent assay. Cytokine stimulation up-regulated the expression of chemokine receptors and integrins on Tck cells, including CXCR4, very late activation antigen 4 (VLA-4), and lymphocyte function-associated antigen 1. Increased expression of CXCR4 and VLA-4 integrin resulted in concentration-dependent chemotaxis to their ligands, stromal cell-derived factor 1 (SDF-1) and VCAM-1, which could be selectively blocked using a specific CXCR4 inhibitor and antibodies against VLA-4. Increased expression of VLA-4 also resulted in increased transendothelial migration of Tck cells, which could be abrogated using blocking antibodies against VLA-4. Tck cells also showed an increased chemotactic response to rheumatoid arthritis (RA) fibroblast-like synoviocytes cultured in vitro, which could be blocked using inhibitors against VLA-4 and CXCR4. The activated phenotype of Tck cells results in increased migratory responsiveness to SDF-1 and soluble VCAM-1, which are among the chemokines and proteins found elevated in the RA synovial joint environment. Cytokine-dependent activation may contribute to RA pathogenicity by promoting T cell recruitment to and retention in the joint, perpetuating the inflammatory cascade in RA.

Natalizumab Treatment Effects Sustained Decrease in Serum Levels of Fibronectin and Soluble Adhesion Molecules sVCAM-1 and sICAM-3 (P02.114)

Objective: To obtain a better understanding of potential additional effector mechanisms of natalizumab we analyzed the influence of short and long-term treatment on soluble adhesion molecules (sAMs) in peripheral blood of MS patients. Background Natalizumab (Tysabri) is a humanized monoclonal antibody used in the treatment of relapsing-remitting multiple sclerosis (MS). It directly binds to the alpha4-integrin subunit of very late activation antigen-4 (VLA-4) and results in decreased detectability of unblocked alpha-4 leading to an inhibition of immune cell extravasation across the blood-brain-barrier. The impressive therapeutic effect of natalizumab is overshadowed by rare but potentially serious side effects such as progressive multifocal leukoencephalopathy. Design/Methods: Serum levels of fibronectin (FN) and 4 different sAMs (soluble intercellular adhesion molecule-1,-2,-3 (sICAM-1,-2,-3) and vascular cell adhesion molecule-1 (sVCAM-1)) were analyzed before (baseline, n=16), 3 months after (short-term, n=16) and 24 months after (long-term, n=14) onset of Natalizumab treatment by fluorescent bead immunoassay and enzyme-linked immunosorbent assay. Results: A significant and sustained decrease in serum concentrations of FN, sICAM-3 and sVCAM-1 was observed during the first 3 months (FN p Conclusions: The observed decreases in serum levels highlight sICAM-3, sVCAM-1, and FN to be affected by natalizumab therapy. Decreases in sVCAM-1 serum levels have already been previously reported. We add sICAM-3 and the soluble form of the extracellular matrix protein FN. Both, sVCAM-1 and FN have been shown to be directly engaged in VLA-4-mediated extravasation of immune cells and sICAM-3 is important in the initiation of immune responses. Decreased serum levels indicate potential therapy-induced interference with fine-tuning and/or feedback mechanisms between endothelium and immune cells with yet unresolved consequences. Supported by: Biogen-Idec Austria. Disclosure: Dr. Kraus has received personal compensation for activities with Biogen Idec, Bayer Pharmaceuticals Corporation, Sanofi-Aventis Pharmaceuticals, Merck-Serono, Novartis, Genzyme Corporation as a consultant. Dr. Kraus has received research support from Biogen Idec, Bayer Pharmaceuticals Corporation, Sanofi-Aventis Pharmaceuticals, Merck Serono, Novartis, Genzyme Corporation. Dr. Oppermann has nothing to disclose. Dr. Pilz has nothing to disclose. Dr. Wipfler has nothing to disclose. Dr. Afazel has nothing to disclose. Dr. Haschke-Becher has nothing to disclose. Dr. Trinka has received personal compensation for activities with UCB Pharma, Eisai Inc., Sunovion, Medtronic, Pfizer Inc, Ever-Neuropharma and GlaxoSmithKline, Inc. as a consultant and/or speaker. Dr. Trinka has received research support from UCB Pharma, Ever-Neuropharma, Medtronic and GlaxoSmithKline, Inc. Dr. Harrer has nothing to disclose.

Antibody to α4 integrin suppresses natural killer cells infiltration in central nervous system in experimental autoimmune encephalomyelitis

Natalizumab inhibits the influx of leukocytes into the central nervous system (CNS) via blockade of alpha-4 subunit of very late activation antigen (VLA)-4. The association of natalizumab therapy with progressive multifocal leukoencephalopathy (PML) suggests a disturbance of CNS immune surveillance in a small percentage of Multiple Sclerosis (MS) patients exposed to the medication. Natural killer (NK) cells are known to play an important role in modulating the evolution of different phases of this lymphocyte mediated disease, and we investigated the effects of natalizumab on the NK cell phenotype and infiltration in the CNS in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Our data show that both resting (from naïve mice) and activated (from EAE mice) NK cells express high levels of VLA-4, and anti-VLA-4 antibody treatment significantly decreases NK cells frequency in the CNS of EAE mice. Moreover, we find that anti-VLA-4 possibly impairs NK cells migratory potential, since unblocked VLA-4 expression levels were downregulated on those NK cells that penetrate the CNS. These data suggest that treatment with antibody to VLA-4 may alter immune surveillance of the CNS by impacting NK cell functions and might contribute to the understanding of the mechanisms leading to the development of PML in some MS patients.

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.

Molecular evidence of transient therapeutic effectiveness of natalizumab despite high-titre neutralizing antibodies

Natalizumab is a humanized monoclonal antibody directed against the alpha-4 integrin subunit of very late activation antigen-4 (VLA-4). Natalizumab neutralizing antibodies (NAB) have been found to significantly reduce beneficial effects of natalizumab treatment in multiple sclerosis. We investigated interactions of NAB with natalizumab by serial measurements of alpha-4 integrin levels on peripheral blood mononuclear cells using flow cytometry. In addition, serum concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1), the endothelial ligand of VLA-4, and serum NAB were serially determined. Natalizumab infusion led to a transient reduction in alpha-4 integrin levels on immune cells and serum sVCAM-1 levels along with serum negativity of NAB lasting for a few days post-infusion. Apparently, the high-dose effect of freshly infused natalizumab resulted in a transient neutralization of NAB possibly involving a transient therapeutic effectiveness.

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

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.

Regulation of acute graft-versus-host disease by human umbilical cord blood derived stromal cells in haploidentical stem cell transplantation in mice through very late activation antigen-4

Human umbilical cord blood derived stromal cells (hUCBDSCs), a novel resource isolated by our laboratory, have been shown to exert an immunologic regulation. Very late activation antigen-4 (VLA-4) has been associated with graft-versus-host disease (GVHD). This study aimed to investigate the possible mechanism by in vitro co-cultured splenocytes of donor mice with hUCBDSCs and in haploidentical stem cell transplantation in mice with acute GVHD. Both hUCBDSCs and human bone marrow stromal cells (hBMSCs) elicited decreased lymphocyte expression of VLA-4, but this decrease was stronger with hUCBDSCs than with hBMSCs (p<0.05). Cotransplantation of bone marrow with hUCBDSCs significantly decreased the expression of VLA-4 compared with control mice (p<0.05). A significant reduction of VLA-4 labeling in the target organs of GVHD was evident in haploidentical mice cotransplanted with hUCBDSCs. Our study shows that hUCBDSCs may protect mouse recipients of haploidentical stem cell transplantation from aGVHD via downregulating the expression of VLA-4.

Adhesion molecules are promising candidates to establish surrogate markers for natalizumab treatment

Background: Natalizumab is the first monoclonal antibody therapy approved for multiple sclerosis (MS). Its therapeutic mechanism is the blockade of the α4-integrin subunit of the adhesion molecule (AM) very late activation antigen-4 (VLA-4), which leads to an inhibition of immune cell extravasation into the central nervous system (CNS). Methods: We investigated changes in the expression levels of unblocked α4-integrin and further AM (intercellular adhesion molecule-1, -2, -3 (cICAM-1, -2, -3), leukocyte function associated antigen-1 (LFA-1)) on peripheral blood mononuclear cells (PBMC) determined by flow cytometry from 25 patients with MS before the first natalizumab infusion and before the fourth infusion. In 15 MS patients AM expression was evaluated every 3 months over 1 year. Results:We found a significant decrease (p < 0.0001) of unblocked α4-integrin cell surface expression on all investigated PBMC subsets (T cells −61.7%, B cells −69.1%, monocytes/macrophages −46.4%) in the blood of MS patients after 3 months of natalizumab treatment. Moreover, a continuous decrease (p < 0.05) of unblocked α4-integrin expression levels was seen after 3, 6, 9, and 12 months. As a secondary effect, expression levels of the other investigated AM were differentially affected. Conclusions:Results show a sustained decrease of unblocked α4-integrin expression not only in all patients but also in all investigated PBMC subsets. This probably results in a continuously decreasing transmigration of PBMC into the CNS and may explain the improved clinical efficacy in the second treatment year and also the increasing risk of progressive multifocal leukoencephalopathy during long-term natalizumab therapy. We conclude that AM expression profiles are promising candidates for the development of a biomarker system to determine both natalizumab treatment response and patients at risk for opportunistic CNS infections.

Effects of Natalizumab on Circulating B Cells, T Regulatory Cells and Natural Killer Cells

Background: Natalizumab is a humanized monoclonal antibody directed against very late activation antigen 4 (VLA-4) and has a potent effect on disease activity in multiple sclerosis. Blockade of VLA-4 with natalizumab may not only interfere with autoimmunological mechanisms but also with central nervous system immune surveillance. Methods: Longitudinal ex vivo and in vitro study to determine the effect of natalizumab on the frequency of distinct immune cells and on the frequency and suppressive function of natural CD4+CD25+ regulatory T cells (Tregs). Results: Natalizumab binding to VLA-4 was more marked for B cells than for T cells (49% reduction in VLA-4-expressing B cells compared to 24.5% reduction of T cells). There was an increase in circulating B cells over T cells (2.6 vs. 1.5 fold, p < 0.001). Natural killer cells increased 1.5-fold (p = 0.01). Natalizumab led to a relative decrease in CD4+CD25+ Tregs from 18.9 to 14.1% (p = 0.04). The impaired suppressive capacity of Tregs was not restored. Conclusion: Natalizumab reduces VLA-4 expression on all investigated immune cells, but changes were most marked for B cells. Further differential effects on immune cells may be relevant to opportunistic central nervous system infections during treatment with natalizumab.

Co-engagement of α 4β 1 integrin (VLA-4) and CD4 or CD8 is necessary to induce maximal Erk1/2 phosphorylation and cytokine production in human T cells

The α 4β 1 integrin VLA-4 (very-late activation antigen–4) and the lineage-specific CD4 and CD8 receptors have been proposed as putative co-stimulatory receptors on T cells. To assess the relative contribution of signaling through the TCR, CD28 and these accessory molecules, we activated human T cells using soluble antibodies recognizing all four of these T-cell receptor classes (CD3, CD28, CD4/CD8, and VLA-4), and we assessed the degree of activation using higher-order flow cytometry detecting intracellular Erk1/2 phosphorylation and production of IL-2 and IFN-γ. We found that: (1) co-stimulation via CD4/CD8, in addition to CD28, is required for optimal T-cell activation; (2) VLA-4 binding consistently potentiates CD4 + and CD8 + T-cell activation; (3) augmentation of T-cell activation through VLA-4 binding is most pronounced following engagement of CD4/CD8. These results confirm that multiple signals, including VLA-4 engagement, are necessary for maximal T-cell activation beyond that induced via the TCR and CD28.

Iatrogenic immunosuppression with biologics in MS

Natalizumab (Tysabri®, Biogen Idec Inc., Cambridge, MA) is a recombinant humanized monoclonal antibody that binds to the α4 chain of the integrin very late activation antigen (VLA)-4 and to α4β7 integrin. It was designed to diminish the adherence of activated leukocytes to biologic membranes and their subsequent migration into peripheral tissues, including the brain and the spinal cord. The clinical effectiveness of natalizumab in patients with relapsing-remitting multiple sclerosis (MS) was evaluated in 2 phase 3 clinical trials (the AFFIRM monotherapy trial1 and the SENTINEL add-on trial with interferon beta-1a [Avonex®, Biogen Idec Inc.]2). In the United States, the Food and Drug Administration first approved natalizumab on November 24, 2004, for the treatment of relapsing forms of MS. Only 3 months later, the manufacturers of natalizumab announced its voluntary withdrawal after 2 patients with MS and 1 patient with Crohn disease who had received natalizumab in clinical trials were diagnosed with progressive multifocal leukoencephalopathy (PML).3–5 PML, a brain demyelinating disorder caused by the human polyomavirus JC, is almost exclusively diagnosed in individuals who experience severe and prolonged immunosuppression. The occurrence of PML in patients with MS treated with natalizumab was unexpected to many because there …

Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions

AbstractLeukocyte adhesion deficiency (LAD)–III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor–stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3–null effector T cells exhibited total loss of inside-out LFA-1 activation by chemokine signals as well as abrogated intrinsic LFA-1 adhesiveness. Surprisingly, VLA-4 in Kindlin-3–null resting or effector lymphocytes retained intrinsic rolling adhesions to vascular cell adhesion molecule-1 and exhibited only partial defects in chemokine-stimulated adhesiveness to vascular cell adhesion molecule-1. Deletion of the putative β1 Kindlin-3 binding site also retained VLA-4 adhesiveness. Thus, our study provides the first evidence that Kindlin-3 is more critical to LFA-1 than to VLA-4–adhesive functions in human lymphocytes.

Junctional adhesion molecule (JAM)‐B supports lymphocyte rolling and adhesion through interaction with α4β1 integrin

Junctional adhesion molecule-A (JAM-A), JAM-B and JAM-C have been implicated in leucocyte transmigration. As JAM-B binds to very late activation antigen (VLA)-4, a leucocyte integrin that contributes to rolling and firm adhesion of lymphocytes to endothelial cells through binding to vascular cell adhesion molecule (VCAM)-1, we hypothesized that JAM-B is also involved in leucocyte rolling and firm adhesion. To test this hypothesis, intravital microscopy of murine skin microvasculature was performed. Rolling interactions of murine leucocytes were significantly affected by blockade of JAM-B [which reduced rolling interactions from 9.1 +/- 2.6% to 3.2 +/- 1.2% (mean +/- standard deviation)]. To identify putative ligands, T lymphocytes were perfused over JAM-B-coated slides in a dynamic flow chamber system. JAM-B-dependent rolling and sticking interactions were observed at low shear stress [0.3 dyn/cm(2): 220 +/- 71 (mean +/- standard deviation) versus 165 +/- 88 rolling (P < 0.001; Mann-Whitney rank sum test) and 2.6 +/- 1.3 versus 1.0 +/- 0.7 sticking cells/mm(2)/min (P = 0.026; Mann-Whitney rank sum test) on JAM-B- compared with baseline], but not at higher shear forces (1.0 dyn/cm(2)). As demonstrated by antibody blocking experiments, JAM-B-mediated rolling and sticking of T lymphocytes was dependent on alpha4 and beta1 integrin, but not JAM-C expression. To investigate whether JAM-B-mediated leucocyte-endothelium interactions are involved in a disease-relevant in vivo model, adoptive transfer experiments in 2,4,-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity reactions were performed in mice in the absence or in the presence of a function-blocking JAM-B antibody. In this model, JAM-B blockade during the sensitization phase impaired the generation of the immune response to DNFB, which was assessed as the increase in ear swelling in untreated, DNFB-challenged mice, by close to 40% [P = 0.037; analysis of variance (anova)]. Overall, JAM-B appears to contribute to leucocyte extravasation by facilitating not only transmigration but also rolling and adhesion.

Effects of Natalizumab Treatment on Foxp3+ T Regulatory Cells

BackgroundNatalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.MethodologyA combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.Principal FindingsBinding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25highCD127lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.ConclusionsWe provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.