Methodological Approach to Identify and Isolate Disease Relevant Brain-infiltrating T Cell Clones in Multiple Sclerosis Patients

Abstract

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system considered to be mediated by T cells. Two main approaches have been employed to study pathogenic T cells in MS in the past. The first approach focused on the functional phenotype of peripheral blood T cells, which were generated with myelin proteins, but for which a pathogenic role was otherwise unclear. Based on the ‘skewed’ TCR repertoire of brain infiltrating T cells, the second approach focused on clonally expanded T cells identified by expressing particular TCRs. This second approach is not biased by assumptions about autoantigens and considers that clonally expanded T cells are probably disease relevant. However, the main limitation in the few reported studies was the inability to further characterize the functional phenotype and specificity of infiltrating T cells since only frozen autopsy tissue was available. At least one group currently attempts to “revive” brain-infiltrating T cells by expressing TCR alpha and beta chains in recombinant systems that may allow to dissect the specificity but most likely not the functional phenotype of the original T cell clones (TCC). Here we present a new strategy consisting in a) identifying clonally expanded, brain-infiltrating T cells from autopsy tissue of a MS patient, who died after very aggressive disease course and b) subsequent isolation of the relevant TCC from the patient’s cerebrospinal fluid (CSF). An actively demyelinating lesion based on reduced density of myelinated fibers and myelin degradation products within infiltrating macrophages was identified. Using deep sequencing approaches, we sequenced the TCR variable beta (V-beta) chains expressed by T cells infiltrating this lesion. We identified 312 different lesion-infiltrating TCC. Both CD4 (32%) and CD8 (68%) TCC were present and clonally expanded with frequencies up to 30%. In a following step, we were able to isolate 14 of the most frequent CD4+ and CD8+ TCC using as a cell source in vitro PHA-expanded CSF T cells. T cells expressing V-beta families of interest were sorted and cloned by limiting dilution. These TCC are currently being characterized for functional phenotype and antigen specificity by using a combination of two complementary unbiased methods, synthetic combinatorial peptide libraries and an autologous brain-derived cDNA library, which is expressed in suitable antigen presenting cells.