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 allogeneic hematopoietic stem cell transplantation. The preparation of gene-modified T cells (TkTCs) required a 12-day ex vivo culture comprising an initial OKT3 and IL-2 stimulation, a retrovirus-mediated transduction, and a 7-day selection step in the presence of G418 and IL-2. The low transduction efficiency as well as the culture conditions may significantly alter the diversity 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 Immunoscope method. This method analyzes the hypervariable region of the T cell receptor beta chain (TCRBV) by amplifying the complementarity-determining region 3 (CDR3) and determining size diversity. In all examined samples (four of which were infused into patients), all TCRBV subfamilies were represented with, however, a significant skewing within a minority of subfamilies. Kinetic studies demonstrated that this skewing appeared between day 7 and day 12, with dates of appearance variable from one subfamily to another. In addition, the repertoire analysis of two different culture products, harvested and produced at different times from the same donors, suggested that some repertoire abnormalities could be donor specific. Quantitative analysis revealed no major modifications in gene usage, even in skewed TCRBV subfamilies, with a few clonal expansions concerning a limited number of TCRBV subfamilies. Importantly, identical abnormalities were found in control cells grown in parallel under similar conditions but not transduced or selected, thus demonstrating that these abnormalities were not related to the transduction or the selection process, but rather to the ex vivo culture. The initial stimulus used for T cell activation is a major source of TCRBV perturbation, since replacing the OKT3 + IL-2 stimulus by CD3 + CD28 monoclonal antibody-coated beads prevented the occurrence of alterations. Overall, the HS-tk-expressing T cells used in our clinical trial exhibit limited TCR repertoire skewing that is not due to the transduction/selection procedure. However, future T cell gene transfer protocols for clinical trials should be designed to take into account or possibly prevent such T cell repertoire alterations.