Retrovirus-mediated gene transfer in primary T lymphocytes: Influence of the transduction/selection process and of ex vivo expansion on the T cell receptor beta chain hypervariable region repertoire

We have initiated a phase I/II clinical trial, involving the use of herpes simplex thymidine kinase gene (HS-tk)-expressing donor primary T cells, in order to modulate the graft-versus-host disease (GvHD) occurring after allo geneic hematopoietic stem cell transplantation. The preparation of gene-mod ified T cells (TkTCs) required a 12-day ex vivo culture comprising an initi al OKT3 and IL-2 stimulation, a retrovirus-mediated transduction, and a 7-d ay selection step in the presence of G418 and IL-2. The low transduction ef ficiency as well as the culture conditions may significantly alter the dive rsity of the T cell repertoire. We therefore examined the T cell repertoire of HS-tk-expressing T cell samples from 11 different donors by the Immunos cope method. This method analyzes the hypervariable region of the T cell re ceptor beta chain (TCRBV) by amplifying the complementarity-determining reg ion 3 (CDR3) and determining size diversity. In all examined samples (four of which were infused into patients), all TCRBV subfamilies were represente d with, however, a significant skewing within a minority of subfamilies. Ki netic studies demonstrated that this skewing appeared between day 7 and day 12, with dates of appearance variable from one subfamily to another. In ad dition, the repertoire analysis of two different culture products, harveste d and produced at different times from the same donors, suggested that some repertoire abnormalities could be donor specific. Quantitative analysis re vealed no major modifications in gene usage, even in skewed TCRBV subfamili es, with a few clonal expansions concerning a limited number of TCRBV subfa milies. Importantly, identical abnormalities were found in control cells gr own in parallel under similar conditions but not transduced or selected, th us demonstrating that these abnormalities were not related to the transduct ion or the selection process, but rather to the ex vivo culture. The initia l stimulus used for T cell activation is a major source of TCRBV perturbati on, since replacing the OKT3 + IL-2 stimulus by CD3 + CD28 monoclonal antib ody-coated beads prevented the occurrence of alterations. Overall, the HS-f k-expressing T cells used in our clinical trial exhibit limited TCR reperto ire skewing that is not due to the transduction/selection procedure. Howeve r, future T cell gene transfer protocols for clinical trials should be desi gned to take into account or possibly prevent such T cell repertoire altera tions.