טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBlumenfeld Kan Shiri
SubjectAssessment of the Immuno-Modulatory Effects of Fingolimod
on B Cell Functions in Multiple Sclerosis
DepartmentDepartment of Medicine
Supervisor Professor Ariel Miller
Full Thesis textFull thesis text - English Version


Abstract

Background- Multiple Sclerosis (MS) is a chronic disease of the central nervous system, characterized by inflammation and neurodegeneration. The importance of B cells in MS pathology is becoming evident. Fingolimod, the first oral therapy for relapsing remitting patients with MS (PwMS), inhibits lymphocyte egress from secondary lymphoid organs, resulting in lymphopenia. Considering the major role of B cells in MS, it is important to understand the immunomodulatory effects of current MS therapies on B cells; however, only little is known on how Fingolimod affects B cells.

Aim- Assessment of the immunomodulatory effects of Fingolimod on B cells subset profile and functions, as part of its mode of action in PwMS.

Methods- 41 RRMS patients were recruited before Fingolimod treatment initiation; blood was collected at baseline and following 3 months and PBMCs, B and T cells isolated. Clinical data and lipid profile was collected for 1 year follow up. Flow cytometry was used to assess the effect of Fingolimod on immune and B cell subsets frequencies, markers associated with regulatory function and antigen presentation, and the cytokine profile of B cells alone or in a co-culture with T cells. Proliferation of T cells in a co-culture was done using CFSE, and IL2 secretion assessed by ELISA. Levels of plasma IgM and IgG were also assessed by ELISA. B cell migration was assessed by a transwell system. Some assessments were repeated in vitro on primary B cells derived from 49 untreated PwMS. For the drug effect on RNA, protein and viability, 16 lymphoblastoid cell lines from untreated PwMS were used and assessed by RTPCR, western blot and XTT, respectively.

Results- Three months therapy reduced the proportion of CD4 T cells and B cells and increased proportion of monocytes. The proportion of memory B cells was reduced, while transitional B cells were strongly increased, accompanied by an increase in several regulatory subset including IL10 cells. Fingolimod therapy reduced percentages of ICAM B cells, associated with antigen presentation and increased percentages of activated CD69 cells. Several B cell cytokines were elevated following therapy including IL10, TGFβ, LTα, TNFα, IFNγ and IL4 with the overall profile becoming more anti-inflammatory. A reduction in the percentage of CXCR4 B cells and migration capacity of B cells towards CXCL12 was observed following therapy. B cells co-cultured with T cells showed increased ratio of TGFβ to TNFα or LTα . The percentage of TGFβ T cells co-cultured with B cells was increased, as well as their ratio to LTα , TNFα or IFNγ T cells. After Fingolimod therapy, the stimulation of 2 T cell proliferation by B cells was abolished in line with reduced IL2 secretion. Fingolimod therapy increased plasma HDL levels in treated patients. In vitro, Fingolimod reduced S1PR1 in B cells and showed anti-apoptotic effects mediated through Bcl2.

Conclusion- Fingolimod mediates immunomodulation of proportions and functions of B cell subsets, towards a more regulatory, anti-inflammatory profile. These effects are part of the beneficial mode of action of Fingolimod in PwMS and may be significant for other B cellmediated autoimmune diseases.