Contributions
Abstract: 102
Type: Young Scientific Investigators' Session
Abstract Category: Pathology and pathogenesis of MS - Immunology
Immune surveillance in the central nervous system (CNS) is essential to control infections. However, infiltration is also part of pathology in disorders such MS. It is not known yet how immune surveillance is conducted in the absence of inflammation. This is important for designing more specific therapeutics for MS allowing immune surveillance (i.e. to control infections), while inhibiting pathology-associated inflammatory immune cell invasion. We and others have shown that during inflammation (e.g. MS relapse), mainly CD4 central-memory T-cells (CM) expressing the lymphoid-homing chemokine receptor (CCR) 7 are found in the cerebrospinal fluid (CSF) of patients, whereas under homeostatic conditions (e.g. MS remission, natalizumab treatment, or non-inflammatory CNS diseases like hydrocephalus) mainly CD4+ CCR7- effector-memory T-cells (EM) are present in the CSF. We therefore investigated the process (adhesion, diapedesis) of CD4 T-cell migration through non-inflamed primary human brain microvascular endothelial cells (HBMEC) in vitro under physiological blood flow conditions by video microscopy with sorted CD4 T-cell subsets. We also employed microarray-based candidate searches, qPCR, immune-histology, and -cytology. In line with our previous findings, only CD4 T-cells of the EM phenotype were able to adhere to non-inflamed HBMEC, via VLA-4/VCAM-1 interactions. These cells also showed enhanced crawling (directed locomotion and -velocity) over HBMEC. Most importantly, only EM T cells were able to perform diapedesis through a non-inflamed HBMEC layer. Further analysis revealed that CCR5 as well as granzyme A&K expression defines EM T-cells that are exclusively able to cross the BBB under homeostatic conditions as compared to CM and CCR5- EM. To investigate the need for these specific granyzmes, granzyme K+ NK-CD56bright cells were compared to their granzyme K- A+B+ CD56dim counterparts and only CD56bright cells were able to migrate through the non-inflamed BBB. T-cell adhesion could be impeded by CCR5 antagonists, diapedesis could be blocked by MMP-inhibitors. Ex vivo flow-cytometry analyses of CSF, as well as brain tissue of patients with non-inflammatory conditions support our hypothesis, as these infiltrates were dominated by CD4 granzyme A+K+ CCR5+ EM T cells. The next steps will be to elucidate by which mechanism CCR5+ granzyme A+K+ EM cells migrate over the non-inflamed BBB and how this process can be enhanced or spared by future therapeutic concepts.
Disclosure: Funding: This study was funded by the DFG (SFB128, B1 to NS and Z2 to HW).
Disclosures: SH, JB, JR, PO, ASM, OG, MK, and WS have no conflicts of interest. TSH has received travel support from Biogen and Novartis. NS has received travel funding by Genzyme and Novartis. CCG received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis, and Bayer. HW has received honoraria for acting as a member of Scientific Advisory Boards for Biogen, Sanofi-Genzyme, Merck Serono, Novartis, Evgen, MedDay Pharmaceuticals, Roche Pharma AG); speaker honoraria and travel support (Biogen, Sanofi-Genzyme, Merck Serono, Novartis, Roche Pharma AG, Alexion, Cognomed, F.Hoffmann-LA Roche Ltd, TEVA, WebMD Global, Gemeinnützige Hertie-Stiftung, Peervoice, Swiss Multiple Sclerosis Society); compensation as consultant (Biogen, Novartis, Sanofi-Genzyme, Abbvie, Actelion, IGES, GlaxoSmithKline GmbH, Roche Pharma AG, Swiss Multiple Sclerosis Society) an received research support by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgesellschaft (DFG), Else Kröner Fresenius Foundation, Fresenius Foundation, Hertie Foundation, NRW Ministry of Education and Research, Interdisciplinary Center for Clinical Studies (IZKF) Muenster and RE Children's Foundation, Biogen, GlaxoSmithKline GmbH, Roche Pharma AG, Sanofi-Genzyme and Novartis.
Abstract: 102
Type: Young Scientific Investigators' Session
Abstract Category: Pathology and pathogenesis of MS - Immunology
Immune surveillance in the central nervous system (CNS) is essential to control infections. However, infiltration is also part of pathology in disorders such MS. It is not known yet how immune surveillance is conducted in the absence of inflammation. This is important for designing more specific therapeutics for MS allowing immune surveillance (i.e. to control infections), while inhibiting pathology-associated inflammatory immune cell invasion. We and others have shown that during inflammation (e.g. MS relapse), mainly CD4 central-memory T-cells (CM) expressing the lymphoid-homing chemokine receptor (CCR) 7 are found in the cerebrospinal fluid (CSF) of patients, whereas under homeostatic conditions (e.g. MS remission, natalizumab treatment, or non-inflammatory CNS diseases like hydrocephalus) mainly CD4+ CCR7- effector-memory T-cells (EM) are present in the CSF. We therefore investigated the process (adhesion, diapedesis) of CD4 T-cell migration through non-inflamed primary human brain microvascular endothelial cells (HBMEC) in vitro under physiological blood flow conditions by video microscopy with sorted CD4 T-cell subsets. We also employed microarray-based candidate searches, qPCR, immune-histology, and -cytology. In line with our previous findings, only CD4 T-cells of the EM phenotype were able to adhere to non-inflamed HBMEC, via VLA-4/VCAM-1 interactions. These cells also showed enhanced crawling (directed locomotion and -velocity) over HBMEC. Most importantly, only EM T cells were able to perform diapedesis through a non-inflamed HBMEC layer. Further analysis revealed that CCR5 as well as granzyme A&K expression defines EM T-cells that are exclusively able to cross the BBB under homeostatic conditions as compared to CM and CCR5- EM. To investigate the need for these specific granyzmes, granzyme K+ NK-CD56bright cells were compared to their granzyme K- A+B+ CD56dim counterparts and only CD56bright cells were able to migrate through the non-inflamed BBB. T-cell adhesion could be impeded by CCR5 antagonists, diapedesis could be blocked by MMP-inhibitors. Ex vivo flow-cytometry analyses of CSF, as well as brain tissue of patients with non-inflammatory conditions support our hypothesis, as these infiltrates were dominated by CD4 granzyme A+K+ CCR5+ EM T cells. The next steps will be to elucidate by which mechanism CCR5+ granzyme A+K+ EM cells migrate over the non-inflamed BBB and how this process can be enhanced or spared by future therapeutic concepts.
Disclosure: Funding: This study was funded by the DFG (SFB128, B1 to NS and Z2 to HW).
Disclosures: SH, JB, JR, PO, ASM, OG, MK, and WS have no conflicts of interest. TSH has received travel support from Biogen and Novartis. NS has received travel funding by Genzyme and Novartis. CCG received speaker honoraria and travel expenses for attending meetings from Genzyme, Novartis, and Bayer. HW has received honoraria for acting as a member of Scientific Advisory Boards for Biogen, Sanofi-Genzyme, Merck Serono, Novartis, Evgen, MedDay Pharmaceuticals, Roche Pharma AG); speaker honoraria and travel support (Biogen, Sanofi-Genzyme, Merck Serono, Novartis, Roche Pharma AG, Alexion, Cognomed, F.Hoffmann-LA Roche Ltd, TEVA, WebMD Global, Gemeinnützige Hertie-Stiftung, Peervoice, Swiss Multiple Sclerosis Society); compensation as consultant (Biogen, Novartis, Sanofi-Genzyme, Abbvie, Actelion, IGES, GlaxoSmithKline GmbH, Roche Pharma AG, Swiss Multiple Sclerosis Society) an received research support by the German Ministry for Education and Research (BMBF), Deutsche Forschungsgesellschaft (DFG), Else Kröner Fresenius Foundation, Fresenius Foundation, Hertie Foundation, NRW Ministry of Education and Research, Interdisciplinary Center for Clinical Studies (IZKF) Muenster and RE Children's Foundation, Biogen, GlaxoSmithKline GmbH, Roche Pharma AG, Sanofi-Genzyme and Novartis.