Flow Cytometric Immunophenotyping of the Blood and CSF of Patients with Multiple Sclerosis (P02.081)

Abstract

Objective: To monitor intrathecal immune responses and to investigate their role in pathogenesis of multiple sclerosis (MS). Background Immune cells in the cerebrospinal fluid (CSF) better reflect intrathecal inflammation than peripheral blood. However, low cellularity and susceptibility to apoptosis makes immunophenotyping of CSF challenging. Design/Methods: CSF and concomitant peripheral blood was collected in prospective manner in untreated patients (N=36) who presented for the diagnostic work-up of neuroimmunological disorder. Cells were isolated from 10-15cc of CSF within 15 min of collection. An optimized 12 color flow cytometric immunophenotyping (FCI) protocol was used to identify immune subpopulations (CD4, CD8 T cells, B cells, CD56bright- and CD56dim NK cells, monocytes, myeloid and plasmacytoid dendritic cells (DCs)) and their activation markers. FCI results were analyzed in an unbiased manner with the absolute cell count in blood and CSF and with the diagnosis established by the clinicians. Results: Upon unblinding, our cohort consisted of clinically isolated syndrome (CIS, N=2), relapsing-remitting MS (RR-MS, N=18), primary progressive MS (PP-MS, N=4), secondary progressive MS (SP-MS, N=4), non-inflammatory neurological diseases (NIND, N=4) and other inflammatory neurological diseases (OIND, N=5) controls. CSF was enriched for T cells, HLA-DR+ (i.e. activated) T cells and CD56bright NK cells in comparison to blood. We observed positive correlations between blood and CSF B cells, and blood and CSF HLA-DR+ T cells and terminally-differentiated double negative T cells, suggesting a link between systemic and intrathecal immune activation. Intrathecal differences between MS patients and controls consisted of elevated B cell to monocyte ratio (p=0.0004) and elevated CD4/CD8 T cell ratio (p=0.0007). Conclusions: The CSF immunophenotyping has been adapted to all NIB clinical protocols, including interventional trials, so updated results will be presented. We expect that with expanded cohort and longitudinal data, CSF immunophenotyping will provide essential information about intrathecal immune responses in neuroimmunological diseases. Supported by: NIH Intramural Research Program. Disclosure: Dr. Han has nothing to disclose. Dr. Salgado has nothing to disclose. Dr. Alfahad has nothing to disclose. Dr. Bielekova has received royalty payments from NIH.