Clonal analysis of graft-infiltrating lymphocytes from renal and cardiac biopsies dominant rearrangements of TcRβ genes and persistence of dominant rearrangements in serial biopsies

Graft-infiltrating lymphocytes from both human renal and cardiac allografts were propagated in interleukin 2 in order to evaluate rearrangements in the T-cell receptor (TcR) β-chain genes. Individual biopsies from renal allografts during episodes of cellular rejection were examined as well as multiple biopsies of heart transplant patients from whom endomyocardial samples were taken prior to, during, and after episodes of rejection. TcRβ-chain rearrangements were evaluated in Southern blots using DNA extracted from interleukin 2-propagated cells and digested with restriction endonucleases permitting assessment of rearrangements to both Cβa and Cβ2. Rearrangements shared among greater than 5% of the “bulk” culture appear as nongermline bands when hybridized with a Cβ probe. Single-cell progeny were generated from limiting dilution, and the rearrangements among the cloned progeny compared to the “bulk” of the cultured progeny of graft-infiltrating lymphocytes. The results indicate that “dominant” rearrangements are a common feature of renal allograft-infiltrating lymphocytes (14 of 15 cases examined). Since the number of cells which can be recovered from a given cardiac biopsy may be limiting, evaluation of clonal dominance from these cultures is more difficult to evaluate. However, sharing of “dominant” rearrangements among multiple biopsies from the same cardiac allograft patient indicates an in vivo selection for T cells with the same receptor rearrangement. Analysis of individual clones showed 3133 clones from a renal allograft sharing the “dominant” rearrangement noted in the bulk culture, but none of these “dominant” clones showed antidonor specificity. Of particular note is that these clones were not the progeny of a single cell, as there was not sharing of the second rearrangement noted in each clone. Thus, the “dominance” noted in the bulk culture was not the result of outgrowth of a single rapidly dividing cell, but rather a reflection of some advantage to cells with a similar rearrangement of TcRβ. One in fifteen clones from a cardiac allograft shared the rearrangement noted in the bulk culture from which it was derived. This clone showed a donor-specific proliferative activity which was also noted in another biopsy that shared the same dominant restriction pattern and donor-specific proliferative activity. Taken together, these results indicate