Clonal expansions of CD8(+) T cells dominate the T cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction

Abstract

Clonal composition and T cell receptor (TCR) repertoire of CD4(+) and CD8(+) T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells. Individual CD4(+) or CD8(+) T cells were isolated from frozen sections of lesional tissue by micromanipulation and subjected to single target amplification of TCR-beta gene rearrangements. This strategy allows the assignment of a TCR variable region (V region) sequence to the particular T cell from which it was amplified. Sequence analysis revealed that in both cases investigated, the majority of CD8(+) T cells belonged to few clones. One of these clones accounted for 35% of CD8(+) T cells in case 1. V region sequence comparison revealed signs of selection for common peptide specificities for some of the CD8(+) T cells in case 1. In both cases, the CD4(+) T cell population was more heterogeneous. Most CD4(+) and CD8(+) clones were represented in perivascular infiltrates as well as among parenchymal T cells. In case 2, two of the CD8(+) clones identified in brain tissue were also detected in peripheral blood. Investigation of the antigenic specificities of expanded clones may help to elucidate their functional properties.

Figure 1

Schematic overview of sections of the two blocks of brain tissue (A and B) analyzed for case 1. Nos. 1–4 designate MS lesions from which T cells were micromanipulated. Hatched lesional areas show signs of ongoing myelin destruction. Block A also contained an old lesion (SCAR) showing astroglial scarring, and only a little infiltration by T cells or other signs of inflammation.

Figure 2

Detection of clone 10 (identified in brain tissue of case 2) in peripheral blood T cells by CDR3 spectratyping analysis. mRNA extracted from FACS^®-sorted CD8⁺ T cells was reverse transcribed. TCR-β cDNA was amplified using 1 Cβ- and 26 Vβ-specific primers. PCR products were used as templates in runoff reactions with fluorescently labeled Jβ-specific primers, and runoff products were analyzed on an automated DNA sequencer. The products shown were amplified with the Vβ14-Cβ primer pair and labeled with an Jβ1S1-specific primer in the runoff reaction. The spectratypes correspond to the blood samples obtained 12 mo (top) and 31 mo (bottom) after brain surgery. The prominent peaks marked by the arrows potentially represented the rearrangement of clone 10 (same V-J combination and CDR3 length). Direct sequencing of the Vβ14-Jβ1S1 PCR products resulted in mixed sequences that potentially contained the CDR3 sequence of clone 10. The products were cloned into a plasmid vector. After transformation of Escherichia coli and sequence analysis of plasmid DNA from bacterial colonies, the V region sequence of clone 10 was identified in 7 of 26 colonies (“12 months”) and 7 of 12 colonies (“31 months”).