Contributions
Abstract: P1075
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Immunology
Background: Active migration of proinflammatory T cells from the peripheral blood (PB) across the central nervous system (CNS) barriers is critical to multiple sclerosis (MS) pathogenesis. Integrins, which are heterodimeric alpha/beta transmembrane proteins, facilitate leucocyte transmigration by mediating cell-cell or cell-extracellular matrix protein interactions. Beta1 integrin heterodimers have been associated with the migration of PB lymphocytes across the CNS barriers, as supported by the clinical efficacy of Natalizumab, a monoclonal antibody targeting integrin alpha4beta1. However, due to widespread expression of alpha4beta1 across leucocyte subsets, Natalizumab treatment is associated with impaired immune surveillance and susceptibility to severe viral infections. Therefore, our goal is to identify novel beta1 integrin partners involved in the specific migration of pathogenic T cells across the CNS vasculature in order to solve this unmet clinical need.
Methods and Results: Whole cell lysate proteomic analysis reveals that proinflammatory TH17 cells express integrin alpha8, which heterodimerizes exclusively with integrin beta1. Here we show through qPCR, western blot (WB), flow cytometry (FC) and immunofluorescence (IF) that alpha8 is specifically expressed by activated CD4+ and CD8+ T cells and is upregulated in pro-inflammatory conditions in healthy controls. By FC we demonstrate that alpha8 is increased on MS patient T cells ex-vivo, as compared to healthy controls. IF reveals that alpha8+ T cell infiltrates are found in the brains of MS patients and mice with experimental autoimmune encephalomyelitis (EAE). Furthermore, we demonstrate via RT-PCR, WB and IF that both BBB and BMB endothelial cells (ECs) express the main ligand of alpha8, nephronectin (NPNT). Blockade of the alpha8 binding site decreases TH1 and TH17, but not TH2 cell migration across a monolayer of BBB-ECs in vitro. Moreover, therapeutic i.p. injections of alpha8 blocking peptide, as compared to control peptide, ameliorates clinical severity and limits proinflammatory T lymphocyte infiltration into the CNS in MOG35-55 actively induced EAE mice.
Conclusions: These data highlight an important role for alpha8 in mediating pro-inflammatory T lymphocyte migration across the CNS microvasculature, suggesting that this integrin may be an effective therapeutic target to prevent disease activity and progression in MS.
Disclosure: This investigation was supported by a CIHR Project Bridge Grant to A.P.; and an endMS Doctoral Studentship from the Multiple Sclerosis Society of Canada (MSSC), a Doctoral Scholarship from the Fonds de recherche du Québec - Santé (FRQS), and a CIHR Neuroinflammation Training Program studentship to E.M.G. B.B was supported by an FWO Post-Doctoral Fellowship. M.C. an MSSC Doctoral Scholarship. E.P. is supported by a FRQS and MSSC Post-Doctoral Fellowship. E.M.G., B.B., E.P., S.G., M.A.L., L.B., S.L., M.C., S.T., P.D., and A.P. have no additional disclosures related to this project.
Abstract: P1075
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Immunology
Background: Active migration of proinflammatory T cells from the peripheral blood (PB) across the central nervous system (CNS) barriers is critical to multiple sclerosis (MS) pathogenesis. Integrins, which are heterodimeric alpha/beta transmembrane proteins, facilitate leucocyte transmigration by mediating cell-cell or cell-extracellular matrix protein interactions. Beta1 integrin heterodimers have been associated with the migration of PB lymphocytes across the CNS barriers, as supported by the clinical efficacy of Natalizumab, a monoclonal antibody targeting integrin alpha4beta1. However, due to widespread expression of alpha4beta1 across leucocyte subsets, Natalizumab treatment is associated with impaired immune surveillance and susceptibility to severe viral infections. Therefore, our goal is to identify novel beta1 integrin partners involved in the specific migration of pathogenic T cells across the CNS vasculature in order to solve this unmet clinical need.
Methods and Results: Whole cell lysate proteomic analysis reveals that proinflammatory TH17 cells express integrin alpha8, which heterodimerizes exclusively with integrin beta1. Here we show through qPCR, western blot (WB), flow cytometry (FC) and immunofluorescence (IF) that alpha8 is specifically expressed by activated CD4+ and CD8+ T cells and is upregulated in pro-inflammatory conditions in healthy controls. By FC we demonstrate that alpha8 is increased on MS patient T cells ex-vivo, as compared to healthy controls. IF reveals that alpha8+ T cell infiltrates are found in the brains of MS patients and mice with experimental autoimmune encephalomyelitis (EAE). Furthermore, we demonstrate via RT-PCR, WB and IF that both BBB and BMB endothelial cells (ECs) express the main ligand of alpha8, nephronectin (NPNT). Blockade of the alpha8 binding site decreases TH1 and TH17, but not TH2 cell migration across a monolayer of BBB-ECs in vitro. Moreover, therapeutic i.p. injections of alpha8 blocking peptide, as compared to control peptide, ameliorates clinical severity and limits proinflammatory T lymphocyte infiltration into the CNS in MOG35-55 actively induced EAE mice.
Conclusions: These data highlight an important role for alpha8 in mediating pro-inflammatory T lymphocyte migration across the CNS microvasculature, suggesting that this integrin may be an effective therapeutic target to prevent disease activity and progression in MS.
Disclosure: This investigation was supported by a CIHR Project Bridge Grant to A.P.; and an endMS Doctoral Studentship from the Multiple Sclerosis Society of Canada (MSSC), a Doctoral Scholarship from the Fonds de recherche du Québec - Santé (FRQS), and a CIHR Neuroinflammation Training Program studentship to E.M.G. B.B was supported by an FWO Post-Doctoral Fellowship. M.C. an MSSC Doctoral Scholarship. E.P. is supported by a FRQS and MSSC Post-Doctoral Fellowship. E.M.G., B.B., E.P., S.G., M.A.L., L.B., S.L., M.C., S.T., P.D., and A.P. have no additional disclosures related to this project.