Identifying Potentially Autoaggressive CD8 T Cell Receptors in Multiple Sclerosis Brain Lesions

Identifying Potentially Autoaggressive CD8 T Cell Receptors in Multiple Sclerosis Brain Lesions

Gintarė Kemežytė, Zita Aušrelė Kučinskienė

Summary

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.