Table 1.
Summary of Single Cell PCR Analysis of T Cells Micromanipulated from MS Lesions of Cases 1 and 2
| Samples analyzed | Positive for at least onespecific PCR product | Total no. of potentially functional rearrangements | |
|---|---|---|---|
| Case 1 | 547 single parenchymal T cells | 141/547 (26%) | 133 |
| 107 samples of perivascular cells | 60/107 (56%) | 86 | |
| Case 2 | 251 single parenchymal T cells | 88/251 (35%) | 76 |
| 33 samples of perivascular cells | 26/33 (79%) | 46 | |
| Controls | 90 micromanipulated GFAP+ astrocytes | 0/90 | — |
| 90 buffer samples | 0/90 | — | |
| 90 water controls | 0/90 | — | |
| 90 single FACS®-sorted T cells | 66/90 (73%) | 58 |
Frozen sections of MS lesions were used to micromanipulate samples of 5–10 cells from perivascular infiltrates and single parenchymal T cells located outside of perivascular infiltrates. TCR-β gene rearrangements were amplified from these samples. Single astrocytes micromanipulated from adjacent sections, aliquots of the buffer covering the sections during the micromanipulation procedure, and control tubes containing PCR buffer but no cells served as negative controls. Single T cells sorted from blood of healthy donors were used to control for the efficiency of single target amplification. Among the 448 rearrangements that were amplified in total from micromanipulated and control T cells and for which the reading frame could be unequivocally determined, 49 were nonfunctional (11%; i.e., either stop codon in CDR3, rearrangement of a pseudogene, or out-of-frame rearrangement). As this study focused on the detection of potentially functional rearrangements, additional bands amplified from single cells that may potentially represent nonfunctional rearrangements were not necessarily sequenced. Therefore, our results probably underestimate the prevalence of nonfunctional rearrangements in human T cells. To exclude the possibility that a substantial number of rearrangements was derived from γ/δ instead of α/β T cells (potentially functional TCR-β gene rearrangements were described in γ/δ T cells [reference 65]), adjacent tissue sections were stained for TCR-γ/δ or -α/β. In both cases, γ/δ T cells accounted for <3% of all T cells.