Identifying Potentially Autoaggressive CD8 T Cell Receptors in Multiple Sclerosis Brain Lesions
Gintarė Kemežytė, Zita Aušrelė Kučinskienė
Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease, where T cells attack the brain and the spinal cord. Tissue infiltrating CD8+ T cells are significant to the pathogenesis of MS, because they were detected in different brain regions and may persist for many years. However, their target antigens are still unknown. Over the past years several new technologies were developed and that now allows us to distinguish potentially autoaggressive T cells from irrelevant cells, to isolate single T cells from frozen biopsy samples, to clone their T cell receptor (TCR) a- and P-chains, and to functionally express the receptors in vitro. Then “revived” T cells can be used to investigate the antigens.
The aim of this prot ect was to investigate the TCR repertoire of autoaggressive CD8+ T cells in brain lesions of an MS patient MS4.
For immunohistochemical staining and laser microdissection, 10 ^m brain cryostat sections were double stained by cyanine 3 (Cy3) labelled anti CD8 antibodies and fluorescein isothiocyanate (FITC) labelled anti Vpi antibodies. The slides were examined for CD8 and Vpi double positive cells using P.A.L.M. Microbeam-Z microscope. The cells were marked electrontcally, mi t crodissected and laser pressure catapulted into single reaction tubes. Single cells were analyzed for expression of expanded TCR P-chains by single cell PCR protocols. Positive cells were further examined for matching a-chains by a multiplex PCR protocol that allows amplification of the entire human TCR alpha chain repertoire.
In total 612 CD8 and Vpi double positive cells and 732 CD8 positive, but Vpi negative cells were isol ated. From these we characterized seven functionally rearranged TCR p-chains. Analysis of the corresponding a-chains is currently in progress.
Identification of functional TCR’s from MS brain lesions may be used to resurrect them in vitro and to detect the antigens in subsequent experiments. Discovering such target antigens of autoaggressive T cells may help us to better understand the pathogenesis of MS, to identify new diagnostic markers and to im t prove our therapeutic opportunities.
Keywords: multiple sclerosis, T cell, receptor, polymerase chain reaction.