Peripheral Regulatory B Cell Phenotype in Multiple Sclerosis Patients Open Access

Abstract

Multiple Sclerosis (MS) is a demyelinating autoimmune disease. Though the exact pathogenesis of the disease is not clear, both T cells and B cells play key roles. B regulatory cells (Bregs) are a small subset of B cells that produce the anti-inflammatory cytokine interleukin 10 (IL-10). We found that there is an increase in B cell number and Naïve B cell number in MS patients (1347 ± 159 cells/μL, average ± SEM) compared to healthy controls (935±129 cells/μL). Our lab has created a fusion protein of granulocyte macrophage colony stimulating factor and interleukin 15, named GIFT15, which induces a Breg phenotype in naïve B cells. Treatment of peripheral blood mononuclear cells with GIFT15 results in proliferation of B cells and increased production of IL-10. These findings establish a baseline of B cell phenotype in MS patients and essential for future therapeutic treatments of MS with GIFT15.

Table of Contents

Table of Contents

Introduction 1

Overview of Multiple Sclerosis 1

B Cell Subsets 3

GIFT15 4

Aims 6

Methods 6

Sample Collection 6

Reagents 7

GIFT15 Production 7

PBMC Isolation 7

PBMC Culture 8

ELISA 9

CFSE 9

Intracellular Cytokine Staining 9

Statistical Analysis 10

Results 11

Whole Blood Analysis 11

Patient Subset Analysis 11

PBMC Culture 13

CFSE Proliferation 15

Intracellular Cytokine Staining 16

Discussion 18

Accomplishment of Aims 18

Future Direction 20

Conclusion 21

References 22

List of Figures

Figure 1. Signaling pathway of GIFT15 5

Figure 2. Whole blood gating strategy 10

Figure 3. Analysis of whole blood samples for MS and HC 12

Figure 4. MS patient drug distributions 12

Figure 5. IL-10 concentration in GIFT15 PBMC supernatant peaks at 3 days 14

Figure 6. The concentration of IL-10 significantly increases in the supernatant of GIFT15 cultured PBMCs 14

Figure 7. CFSE proliferation assay of HC PBMCs shows expansion of GIFT15 cultured CD19⁺ lymphocytes 15

Figure 8. CFSE proliferation assay of HC PBMCs shows a slight expansion of GMCSF + IL-15 cultured CD19^- lymphocytes 16

Figure 9. ICS of HC PBMCs cultured in CM, GIFT15, or GMCSF + IL-15 17

List of Tables

Table 1. Subset analysis of MS patients on no medication, Glatiramer Acetate, and Interferon 12

About this Honors Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Health Sciences, Immunology Biology, Cell
Keyword	Regulatory B cell B cell Multiple Sclerosis Breg Interleukin 10 GIFT15
Committee Chair / Thesis Advisor	Galipeau, Jacques, Emory University
Committee Members	Beck, Christopher, Emory University Soria, Jose, Emory University Yedvobnick, Barry, Emory University

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