Retrovirus mediated gene transduction of human T-cell subsets.
OBJECTIVE
Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukaemia. Graft versus host disease (GVHD) is a potentially life threatening complication of AlloBMT mediated by the T cells contained within the graft. In order to be able to control GVHD, the allogeneic T cells may be transduced with a suicide gene such as herpes simplex virus thymidine kinase (HSV-tk). For this strategy to be successful, all subsets of T cells should be transduced to a similar extent. Also, the transduction protocol should not induce expression of unwanted homing receptors, nor should it lead to unwanted skewing of the T-cell receptor repertoire. We have studied the transduction efficiency of naïve and memory subsets of CD4+ and CD8+ T cells, and examined the transduced T-cell subsets for possible changes in T-cell receptor (TCR) repertoire and homing receptor expression.


METHODS
The cells were transduced using a Moloney murine retroviral vector carrying a conjugate of the genes encoding the truncated form of the cell surface marker, low affinity nerve growth factor receptor (DeltaLNGFR) and HSV-tk. Transduction efficiency and homing receptor expression were quantified by flow cytometry. TCR repertoire was determined by spectratyping.


RESULTS
We obtained a transduction efficiency of 30-50% of the cells, with no difference between the T-cell subsets. Cell surface receptors responsible for homing to skin, gastrointestinal tract or lymph nodes were practically absent at the end of 2 weeks in culture. The activation procedure seemed to favour the expansion of certain T-cell clones over polyclonal populations. However, there was no difference in the TCR repertoire between transduced and non-transduced cells.


CONCLUSIONS
Changes in the composition of the T-cell subsets at the end of the cell culture were the results of the activation, and not the suicide gene transduction. The transduced T cells did not express unwanted homing receptors.
Hedfors
Ida Aagård
IA
Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway.
Beckstrøm
Karen Johanne
KJ
Benati
Claudia
C
Bonini
Chiara
C
Brinchmann
Jan E
JE
D016026:Bone Marrow Transplantation; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors; D006086:Graft vs Host Disease / Q000276:immunology* / Q000517:prevention & control*; D006561:Herpes Simplex / Q000235:genetics; D006801:Humans; D016201:Receptors, Lymphocyte Homing / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics; D016176:T-Lymphocyte Subsets; D013937:Thymidine Kinase / Q000235:genetics; D014161:Transduction, Genetic; D014184:Transplantation, Homologous
D016201:Receptors, Lymphocyte Homing; D013937:Thymidine Kinase