Soluble CD40L activates soluble and cell-surface integrin αvß3, α5ß1, and α4ß1 by binding to the allosteric ligand-binding site (site 2).

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvß3 and α5ß1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4ß1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvß3, α5ß1, and α4ß1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.

The tellurium-based immunomodulator, AS101 ameliorates adjuvant-induced arthritis in rats.

Despite undeniable improvement in the management of rheumatoid arthritis (RA), the discovery of more effective, less toxic and, ideally, less immune suppressive drugs are much needed. In the current study, we set to explore the potential anti-rheumatic activity of the non-toxic, tellurium-based immunomodulator, AS101 in an experimental animal model of RA. The effect of AS101 was assessed on adjuvant-induced arthritis (AIA) rats. Clinical signs of arthritis were assessed. Histopathological examination was used to assess inflammation, synovial changes and tissue lesions. Very late antigen-4 (VLA-4)+ cellular infiltration was detected using immunohistochemical staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating anti-cyclic citrullinated-peptide autoantibody (ACPA) and real-time polymerase chain reaction (PCR) was used to measure the in-vitro effect of AS101 on interleukin (IL)-6 and IL-1ß expression in activated primary human fibroblasts. Prophylactic treatment with intraperitoneal AS101 reduced clinical arthritis scores in AIA rats (P < 0·01). AS101 abrogated the migration of active chronic inflammatory immune cells, particularly VLA-4+ cells, into joint cartilage and synovium, reduced the extent of joint damage and preserved joint architecture. Compared to phosphate-buffered saline (PBS)-treated AIA rats, histopathological inflammatory scores were significantly reduced (P < 0·05). Furthermore, AS101 resulted in a marked reduction of circulating ACPA in comparison to PBS-treated rats (P < 0·05). Importantly, AS101 significantly reduced mRNA levels of proinflammatory mediators such as IL-6 (P < 0·05) and IL-1ß (P < 0·01) in activated primary human fibroblasts. Taken together, we report the first demonstration of the anti-rheumatic/inflammatory activity of AS101 in experimental RA model, thereby supporting an alternative early therapeutic intervention and identifying a promising agent for therapeutic intervention.

Blockade of VLA4 sensitizes leukemic and myeloma tumor cells to CD3 redirection in the bone marrow microenvironment.

Redirecting T cells to specifically kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of blinatumomab for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. In hematological malignancies, the bone marrow (BM) niche is protective to leukemic stem cells and has minimized the efficacy of several anti-cancer drugs. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using bispecific antibodies targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia or multiple myeloma cells with BM stromal cells protected tumor cells from bispecific antibody-T cell-mediated lysis in vitro and in vivo. Impaired CD3 redirection cytotoxicity was correlated with reduced T cell effector responses and cell-cell contact with stromal cells was implicated in reducing T cell activation and conferring protection of cancer cells. Finally, blocking the VLA4 adhesion pathway in combination with CD3 redirection reduced the stromal-mediated inhibition of cytotoxicity and T cell activation. Our results lend support to inhibiting VLA4 interactions along with administering CD3 redirection therapeutics as a novel combinatorial regimen for robust anti-cancer responses.

VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1.

When targeted by the tumor-promoting enzyme heparanase, cleaved and shed syndecan-1 (Sdc1) then couples VEGFR2 (also known as KDR) to VLA-4, activating VEGFR2 and the directed migration of myeloma cells. But how VEGFR2 activates VLA-4-mediated motility has remained unknown. We now report that VEGFR2 causes PKA-mediated phosphorylation of VLA-4 on S988, an event known to stimulate tumor metastasis while suppressing cytotoxic immune cells. A key partner in this mechanism is the chemokine receptor CXCR4, a well-known mediator of cell motility in response to gradients of the chemokine SDF-1 (also known as CXCL12). The entire machinery necessary to phosphorylate VLA-4, consisting of CXCR4, AC7 (also known as ADCY7) and PKA, is constitutively associated with VEGFR2 and is localized to the integrin by Sdc1. VEGFR2 carries out the novel phosphorylation of Y135 within the DRY microswitch of CXCR4, sequentially activating Gαißγ, AC7 and PKA, which phosphorylates S988 on the integrin. This mechanism is blocked by a syndecan-mimetic peptide (SSTNVEGFR2), which, by preventing VEGFR2 linkage to VLA-4, arrests tumor cell migration that depends on VLA-4 phosphorylation and stimulates the LFA-1-mediated migration of cytotoxic leukocytes.

Age-related tumor growth in mice is related to integrin α 4 in CD8+ T cells.

Cancer incidence increases with age, but paradoxically, cancers have been found to grow more quickly in young mice compared with aged ones. The cause of differential tumor growth has been debated and, over time, attributed to faster tumor cell proliferation, decreased tumor cell apoptosis, and/or increased angiogenesis in young animals. Despite major advances in our understanding of tumor immunity over the past 2 decades, little attention has been paid to comparing immune cell populations in young and aged mice. Using mouse colon adenocarcinoma model MC38 implanted in young and mature mice, we show that age substantially influences the number of tumor-infiltrating cytotoxic CD8+ T cells, which control cancer progression. The different tumor growth pace in young and mature mice was abrogated in RAG1null mice, which lack mature T and B lymphocytes, and upon selective depletion of endogenous CD8+ cells. Transcriptome analysis further indicated that young mice have decreased levels of the Itga4 gene (CD49d, VLA-4) in tumor-infiltrating lymphocytes when compared with mature mice. Hypothesizing that VLA-4 can have a tumor-protective effect, we depleted the protein, which resulted in accelerated tumor growth in mature mice. These observations may explain the paradoxical growth rates observed in murine cancers, point to the central role of VLA-4 in controlling tumor growth, and open new venues to therapeutic manipulation.

PI3Kγ Activates Integrin α4 and Promotes Immune Suppressive Myeloid Cell Polarization during Tumor Progression.

Immunosuppressive myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) accumulate in tumors where they inhibit T cell-mediated antitumor immune responses and promote tumor progression. Myeloid cell PI3Kγ plays a role in regulating tumor immune suppression by promoting integrin α4-dependent MDSC recruitment to tumors and by stimulating the immunosuppressive polarization of MDSCs and TAMs. Here, we show that integrin α4 promotes immunosuppressive polarization of MDSCs and TAMs downstream of PI3Kγ, thereby inhibiting antitumor immunity. Genetic or pharmacological suppression of either PI3Kγ or integrin α4 blocked MDSC recruitment to tumors and also inhibited immune suppressive myeloid cell polarization, thereby reducing expression of IL10 and increasing expression of IL12 and IFNγ within tumors. Inhibition of PI3Kγ or integrin α4 within tumors stimulated dendritic cell and CD8+ T-cell recruitment and maturation, as well as tumor cell cytotoxicity in vivo, thereby inhibiting tumor growth. As blockade of PI3Kγ or integrin α4 prevents accumulation of MDSC and reduces myeloid cell expression of immunosuppressive factors that stimulate tumor immune escape, these results highlight PI3Kγ and integrin α4 as targets for the design of cancer therapeutics. Cancer Immunol Res; 5(11); 957-68. ©2017 AACR.

Increased Mobilization of CD45+CD34+VLA-4+ Cells in Acute Viral Myocarditis Induced by Coxsackievirus B3.

OBJECTIVES: Bone marrow-derived cells (BMCs) have recently been identified to play a vital role in repairing damaged myocardium; however, it is not known whether or not mobilization of BMCs is involved in the pathogenesis of acute viral myocarditis (VMC). Thus, we analyzed the expression of CD45+CD34+VLA-4+ cells and vascular cell adhesion protein-1 (VCAM-1) in a murine model of acute VMC. METHODS: Male BALB/c mice were intraperitoneally infected with coxsackievirus B3 to establish acute VMC. The frequency of CD45+CD34+VLA-4+ cells in the heart, peripheral blood, and bone marrow was examined by flow cytometry 3, 7, 14, and 28 days after injection. Cardiac VCAM-1 and pathology scores were determined by immunohistochemistry, and myocardial VCAM-1, IL-1ß, and TNF-α were analyzed by RT-PCR and Western blot. RESULTS: In mice with acute VMC, the CD45+CD34+VLA-4+ cell population in the heart was significantly increased by day 7 and then decreased; in contrast, the CD45+CD34+VLA-4+ cell population decreased in the bone marrow and peripheral blood by day 3 and then increased. High expression of VCAM-1 was detected in the heart in parallel with CD45+CD34+VLA-4+ cell expression. CONCLUSIONS: In mice with acute VMC, VCAM-1-induced CD45+CD34+VLA-4+ cell mobilization into the injured heart is involved in the repair of injured myocardium.

Impaired Transmigration of Myeloid-Derived Suppressor Cells across Human Sinusoidal Endothelium Is Associated with Decreased Expression of CD13.

Human monocytic myeloid-derived suppressor cells (MO-MDSCs) within the hepatic compartment suppress inflammation and impair immune surveillance in liver cancer. It is currently not known whether recruitment of MO-MDSCs from blood via hepatic sinusoidal endothelium (HSEC) contributes to their enrichment within the hepatic compartment. We compared the transmigratory potential of MO-MDSCs and monocytes after adhesion to hepatic endothelial monolayers in flow-based assays that mimic in vivo shear stress in the sinusoids. Despite comparable binding to HSEC monolayers, proportionally fewer MO-MDSCs underwent transendothelial migration, indicating that the final steps of extravasation, where actin polymerization plays an important role, are impaired in MO-MDSCs. In this article, we found reduced levels of CD13 on MO-MDSCs, which has recently been reported to control cell motility in monocytes, alongside reduced VLA-4 expression, an integrin predominantly involved in adherence to the apical side of the endothelium. CD13 and VLA-4 blocking and activating Abs were used in flow-based adhesion assays, live-cell imaging of motility, and actin polymerization studies to confirm a role for CD13 in impaired MO-MDSC transmigration. These findings indicate that CD13 significantly contributes to tissue infiltration by MO-MDSCs and monocytes, thereby contributing to the pathogenesis of hepatic inflammation.

Positron Emission Tomography Imaging of Macaques with Tuberculosis Identifies Temporal Changes in Granuloma Glucose Metabolism and Integrin α4ß1-Expressing Immune Cells.

Positron emission tomography and computed tomography imaging (PET/CT) is an increasingly valuable tool for diagnosing tuberculosis (TB). The glucose analog [18F]fluoro-2-deoxy-2-d-glucose ([18F]-FDG) is commonly used in PET/CT that is retained by metabolically active inflammatory cells in granulomas, but lacks specificity for particular cell types. A PET probe that could identify recruitment and differentiation of different cell populations in granulomas would be a useful research tool and could improve TB diagnosis and treatment. We used the Mycobacterium-antigen murine inflammation model and macaques with TB to identify [64Cu]-labeled CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A), a high affinity peptidomimetic ligand for very late Ag-4 (VLA-4; also called integrin α4ß1) binding cells in granulomas, and compared [64Cu]-LLP2A with [18F]-FDG over the course of infection. We found that [64Cu]-LLP2A retention was driven by macrophages and T cells, with less contribution from neutrophils and B cells. In macaques, granulomas had higher [64Cu]-LLP2A uptake than uninfected tissues, and immunohistochemical analysis of granulomas with known [64Cu]-LLP2A uptake identified significant correlations between LLP2A signal and macrophage and T cell numbers. The same cells coexpressed integrin α4 and ß1, further supporting that macrophages and T cells drive [64Cu]-LLP2A avidity in granulomas. Over the course of infection, granulomas and thoracic lymph nodes experienced dynamic changes in affinity for both probes, suggesting metabolic changes and cell differentiation or recruitment occurs throughout granuloma development. These results indicate [64Cu]-LLP2A is a PET probe for VLA-4, which when used in conjunction with [18F]-FDG, may be a useful tool for understanding granuloma biology in TB.

Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate.

Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL-/-) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL-/- B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration.

VHL promotes immune response against renal cell carcinoma via NF-κB-dependent regulation of VCAM-1.

Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule assigned to the activated endothelium mediating immune cells adhesion and extravasation. However, its expression in renal carcinomas inversely correlates with tumor malignancy. Our experiments in clear cell renal cell carcinoma (ccRCC) cell lines demonstrated that von Hippel Lindau (VHL) loss, hypoxia, or PHD (for prolyl hydroxylase domain-containing proteins) inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-inducible factor and dependent on the nuclear factor κB signaling pathway. Conversely, VHL expression leads to high VCAM-1 levels in ccRCC, which in turn leads to better outcomes, possibly by favoring antitumor immunity through VCAM-1 interaction with the α4ß1 integrin expressed in immune cells. Remarkably, in ccRCC human samples with VHL nonmissense mutations, we observed a negative correlation between VCAM-1 levels and ccRCC stage, microvascular invasion, and symptom presentation, pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression.

The α4ß1 Homing Pathway Is Essential for Ileal Homing of Crohn's Disease Effector T Cells In Vivo.

BACKGROUND: The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohn's disease (CD) are still unclear, and clinical outcome data from patients with inflammatory bowel disease treated with the anti-α4ß7 integrin antibody vedolizumab suggest differences between ulcerative colitis and CD. METHODS: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo. RESULTS: Despite in vitro blockade of CD Teff adhesion to mucosal vascular addressin cell adhesion molecule-1 (MadCAM-1) and in contrast to previous observations in ulcerative colitis, anti-α4ß7 treatment did not result in reduced Teff cell homing to the colon in vivo. However, the integrin α4ß1 was expressed in higher levels on Teffs from patients with CD compared with controls, while its expression in the peripheral blood declined, and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to vascular cell adhesion molecule-1 (VCAM-1) was blocked by inhibition of α4 and α4ß1 in vitro. Moreover, in vivo homing of CD Teffs to the ileum was reduced by inhibition of α4 and α4ß1 integrins, but not α4ß7 integrins. CONCLUSIONS: Our findings suggest that Teff cell homing to the ileum through the axis α4ß1-VCAM-1 is an essential and nonredundant pathway in CD in vivo, possibly affecting efficacy of clinical treatment with antiadhesion compounds.

MFG-E8-derived peptide attenuates adhesion and migration of immune cells to endothelial cells.

Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) plays an immunomodulatory role in inflammatory diseases. MFG-E8-derived short peptide (MSP68) greatly reduces neutrophil infiltration and injury in the lung during sepsis. In this study, we examined the effect of MSP68 on chemotaxis of various immune cells and its regulatory mechanism. Bone marrow-derived neutrophils (BMDNs) from C57BL/6 mice, human monocyte THP-1 cell line, and human T lymphocyte Jurkat cell line were used for adhesion and migration assays using a Transwell method in the presence of MSP68. Treatment with MSP68 significantly inhibited the BMDN and THP-1 cell but not Jurkat cell adhesion on the TNF-α-stimulated pulmonary artery endothelial cell (PAEC) monolayer dose-dependently. MSP68 also significantly reduced BMDN adhesion on VCAM-1-coated wells dose dependently. Surface plasmon resonance (SPR) analysis revealed that MSP68 efficiently recognized integrin α4ß1 (receptor for VCAM-1) at the dissociation constant (KD) of 1.53 × 10-7 M. These findings implicate that MSP68 prevents neutrophil adhesion to the activated endothelial cells by interfering with the binding between integrin α4ß1 on neutrophils and VCAM-1 on endothelial cells. Moreover, MSP68 significantly attenuated the migration of BMDN and THP-1 cells but not Jurkat cells to their chemoattractants. Pretreatment with MSP68 inhibited the transmigration of BMDNs across the PAECs toward chemoattractants, fMLP, MIP-2, and complement fragment 5a (C5a) dose-dependently. Finally, we identified that the activation of p38 MAPK in BMDNs by fMLP was inhibited by MSP68. Thus, MSP68 attenuates extravasation of immune cells through the endothelial cell lining into inflamed tissue, implicating MSP68 to be a novel, therapeutic agent for inflammatory diseases caused by excessive immune cell infiltration.

IL-10+ Innate-like B Cells Are Part of the Skin Immune System and Require α4ß1 Integrin To Migrate between the Peritoneum and Inflamed Skin.

The skin is an important barrier organ and frequent target of autoimmunity and allergy. In this study, we found innate-like B cells that expressed the anti-inflammatory cytokine IL-10 in the skin of humans and mice. Unexpectedly, innate-like B1 and conventional B2 cells showed differential homing capacities with peritoneal B1 cells preferentially migrating into the inflamed skin of mice. Importantly, the skin-homing B1 cells included IL-10-secreting cells. B1 cell homing into the skin was independent of typical skin-homing trafficking receptors and instead required α4ß1-integrin. Moreover, B1 cells constitutively expressed activated ß1 integrin and relocated from the peritoneum to the inflamed skin and intestine upon innate stimulation, indicating an inherent propensity to extravasate into inflamed and barrier sites. We conclude that innate-like B cells migrate from central reservoirs into skin, adding an important cell type with regulatory and protective functions to the skin immune system.

Early Development in the Peritoneal Cavity of CD49dhigh Th1 Memory Phenotype CD4+ T Cells with Enhanced B Cell Helper Activity.

The Th cells that regulate peritoneal B-1 cell functions have not yet been well characterized. To address this question, we investigated peritoneal CD4(+) T cells, observed a high frequency of the conjugates of B-CD4(+) T cells in the peritoneal cavity, and identified a population of CD49d(high)CD4(+) T cells that constituted about half of all CD4(+) T cells in the peritoneal cavity, but were rarely found in other compartments. Peritoneal CD49d(high)CD4(+) T cells were CD44(high)CD62L(low); expressed integrin α4ß1 and CXCR3; and rapidly secreted IFN-γ, TNF-α, and IL-2, showing features of proinflammatory Th1 cells. Peritoneal CD49d(high)CD4(+) T cells developed spontaneously, were detected at the age of 12 d, and showed stem cell-like properties. Their development was observed in mice deficient for signaling lymphocytic activation molecule-associated protein, but not in athymic nude mice and mice lacking in expression of MHC class II on thymic epithelial cells. Peritoneal CD49d(high)CD4(+) T cells were more resistant to irradiation and more sensitive to NAD-induced cell death than CD49d(low)CD4(+) T cells. Notably, peritoneal CD49d(high)CD4(+) T cells also showed some characteristics of follicular Th cells, such as the expression of programmed cell death 1, ICOS, IL-21, and CXCR5. Moreover, peritoneal CD49d(high)CD4(+) T cells enhanced the secretion of IgM Abs by B-1a cells and IgG Abs by splenic B cells. These data suggest that peritoneal CD49d(high)CD4(+) T cells may be innate-like CD4(+) T cells, which develop early and have a dual capacity to support both humoral and cellular immunity.

Defective inflammatory monocyte development in IRF8-deficient mice abrogates migration to the West Nile virus-infected brain.

IRF8 (interferon-regulatory factor-8) plays a critical role in regulating myeloid cell differentiation. However, the role of this transcription factor in the development of Ly6C+ inflammatory monocytes and their migration to the infected brain has not been examined. We have previously shown that West Nile virus (WNV) infection of wild-type (WT) mice triggers a significant increase in numbers of Ly6C+ monocytes in the bone marrow. These cells traffic via the blood to the infected brain, where they give rise to proinflammatory macrophages. Here, we show that WNV-infected IRF8-deficient (IRF8-/-) mice had significantly reduced numbers of Ly6C+ monocytes in the periphery, with few of these cells found in the blood. Furthermore, low numbers of inflammatory monocyte-derived macrophages were observed in the brains of IRF8-/- mice throughout infection. Adoptive transfer of IRF8-/- Ly6C+ monocytes demonstrated that these cells were intrinsically unable to traffic to the inflamed brain. Low expression of the chemokine receptor CCR2 and integrin VLA-4 by IRF8-/- monocytes likely contributed to this defect, as the interactions between these proteins and their ligands are critical for monocyte egress and migration to inflammatory foci. These data highlight a critical role for IRF8 in inflammatory monocyte differentiation and migration during WNV infection.

Therapeutic uses of anti-α4-integrin (anti-VLA-4) antibodies in multiple sclerosis.

Multiple sclerosis (MS) is a disorder of putative autoimmune origin, where immune cells invade the central nervous system and cause damage by attacking the myelin sheath of nerve cells. The blockade of the integrin very late antigen-4 (VLA-4) with the monoclonal antibody natalizumab has become the most effective therapy against MS since its approval in 2004. It is assumed that the inhibition of VLA-4-mediated immune cell adhesion to the endothelium of the blood-brain barrier (BBB) alleviates pathogenic processes of MS and, therefore, reduces disease severity and burden. Not all approaches to treat additional immune-mediated disorders (e.g. Rasmussen encephalitis and neuromyelitis optica) with natalizumab have been successful, but allowed researchers to gain additional insight into mechanisms of specific immune cell subsets' migration through the BBB in the human system. While the long-term efficacy and general tolerability of natalizumab in MS are clear, the over 400 cases of natalizumab-associated progressive multifocal leukoencephalopathy (PML) have been of great concern and methods of risk stratification in patients have become a major area of research. Modern risk stratification includes established factors such as treatment duration, previous immune-suppressive therapy, and anti-John Cunningham virus (JCV) antibody seropositivity, but also experimental factors such as anti-JCV antibody titers and levels of L-selectin. Today, anti-VLA-4 therapy is reserved for patients with highly active relapsing-remitting MS and patients are monitored closely for early signs of potential PML.

VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAM-adhesion of TH17 cells.

The focus of this study is the characterization of human T cell blood-brain barrier migration and corresponding molecular trafficking signatures. We examined peripheral blood and cerebrospinal fluid immune cells from patients under long-term anti-very late antigen-4 (VLA-4)/natalizumab therapy (LTNT) and from CNS specimens. LTNT patients' cerebrospinal fluid T cells exhibited healthy central-/effector-memory ratios, but lacked CD49d and showed enhanced myeloma cell adhesion molecule (MCAM) expression. LTNT led to an increase of PSGL-1 expression on peripheral T cells. Although vascular cell adhesion molecule-1 (VLA-4 receptor) was expressed at all CNS barriers, P-selectin (PSGL-1-receptor) was mainly detected at the choroid plexus. Accordingly, in vitro experiments under physiological flow conditions using primary human endothelial cells and LTNT patients' T cells showed increased PSGL-1-mediated rolling and residual adhesion, even under VLA-4 blockade. Adhesion of MCAM(+)/TH17 cells was not affected by VLA-4 blocking alone, but was abrogated when both VLA-4 and MCAM were inhibited. Consistent with these data, MCAM(+) cells were detected in white matter lesions, and in gray matter of multiple sclerosis patients. Our data indicate that lymphocyte trafficking into the CNS under VLA-4 blockade can occur by using the alternative adhesion molecules, PSGL-1 and MCAM, the latter representing an exclusive pathway for TH17 cells to migrate over the blood-brain barrier.