Neuroantigen-specific Cd8+ T Cells in Experimental Autoimmune Encephalitis (eae)

Roy J. and Lucille A. Carver College of Medicine Medical Student Research Day September 4, 2015 Sponsored by: Medical Student Research Club and Medical Student Research Council Funds provided by University of Iowa Student Government Free to University of Iowa Students Neuroantigen-specific CD8+ T cells in experimental autoimmune encephalitis (EAE) Tina Arkee, B.S., Alexander Boyden, PhD, and Nitin Karandikar, MD, PhD 1 University of Iowa Medical Scientist Training Program 2 Department of Pathology, University of Iowa BACKGROUND: Multiple sclerosis (MS) in an immune-mediated demyelinating disease of the central nervous system that affects more than two million people worldwide. Though its etiology is still unclear, the mechanisms underlying MS can be studied using experimental autoimmune encephalitis (EAE) animal models, in which MS-like ascending paralysis is induced by immunizing mice with myelin proteins or their derived peptides (such as MOG, PLP, and MBP). EAE studies suggest that neuroantigen-specific CD4+ T cells mediate EAE/MS pathology. However, CD8+ T cells may also play a pathogenic role, as they outnumber CD4+ T cells in MS lesions. Interestingly, evidence from our laboratory demonstrates that neuroantigen-specific CD8+ T cells play a novel regulatory role in EAE/MS. The purpose of this research experience was to become familiar with the EAE model and the potential regulatory role CD8+ T cells play therein. HYPOTHESIS: EAE induction results in neuroantigen-specific, IFNγ-producing CD8+ T cells that are capable of playing a regulatory role in clinical disease. METHODS: Active EAE induction: Female C57BL/6 mice were immunized (s.c.) with 50-100μg of OVA323-339, MOG35-55, or PLP178-191 in CFA on day 0. Pertussis toxin (250ng) was additionally injected i.p. on days 0 and 2. Mice were monitored for clinical disease according to a 5 point scale of ascending paralysis. CFSE proliferation assays: Splenocytes were harvested from EAE mice, labeled with 0.25μM CFSE, and cultured in the presence of cognate or non-cognate antigen (MOG35-55 or PLP178-191), ConA or media alone for five days. CFSE dilution as a measure of T cell proliferation was subsequently assessed via flow cytometry. Intracellular cytokine staining: Splenocytes were cultured in the presence of antigen for 5 days as described previously. On day 5, cells were stimulated with leukocyte activation cocktail (BD) for 4 hours, stained intracellularly using Foxp3 detection kit buffers (eBioscience) and assessed for IFNγ production via flow cytometry. CD8+ T cell isolation: Active EAE was induced with 100μg of OVA323-339 or PLP178-191 as described. Spleens and inguinal lymph nodes were harvested 16 days post-immunization, and cells were cultured in the presence of cognate antigen and hIL-2 for 3 days. αCD8 (Ly2) magnetic beads (Miltenyi) were used to isolate CD8+ T cells. Population purity was assessed by flow cytometry. RESULTS: IFNγ-producing CD8+ T cells can be detected in the spleens of EAE mice. When compared to noncognate controls or media alone, stimulating splenocytes from EAE mice with cognate antigen induced a higher fraction of proliferation as well as an increase in IFNγ-producers within the CD8+ T cell population. Attempts to isolate the CD8+ T cells from culture proved difficult, although the qualities of these cells (which are neuroantigenspecific in nature) shows the potential for discerning effector regulatory function capability in future protection