Targeted in vitro and in vivo gene transfer into T lymphocytes: potential of direct inhibition of allo-immune activation.
BACKGROUND
Successful inhibition of alloimmune activation in organ transplantation remains one of the key events in achieving a long-term graft survival. Since T lymphocytes are largely responsible for alloimmune activation, targeted gene transfer of gene of cyclin kinase inhibitor p21 into T cells might inhibit their aberrant proliferation. A number of strategies using either adenoviral or lentiviral vectors linked to mono or bispecific antibodies directed against T cell surface markers/cytokines did not yield the desired results. Therefore, this study was designed to test if a CD3promoter-p21 chimeric construct would in vitro and in vivo transfer p21 gene to T lymphocytes and result in inhibition of proliferation. CD3 promoter-p21 chimeric constructs were prepared with p21 in the sense and antisense orientation. For in vitro studies EL4-IL-2 thyoma cells were used and for in vivo studies CD3p21 sense and antisense plasmid DNA was injected intramuscularly in mice. Lymphocyte proliferation was quantified by 3H-thymidine uptake assay; IL-2 mRNA expression was studied by RT-PCR and using Real Time PCR assay, we monitored the CD3, p21, TNF-alpha and IFN-gamma mRNA expression.


RESULTS
Transfection of CD3p21 sense and antisense in mouse thyoma cell line (EL4-IL-2) resulted in modulation of mitogen-induced proliferation. The intramuscular injection of CD3p21 sense and antisense plasmid DNA into mice also modulated lymphocyte proliferation and mRNA expression of pro-inflammatory cytokines.


CONCLUSIONS
These results demonstrate a novel strategy of in vitro and in vivo transfer of p21 gene to T cells using CD3-promoter to achieve targeted inhibition of lymphocyte proliferation and immune activation.
Khanna
Ashwani K
AK
Department of Medicine, Division of Cardiology, University of Maryland, Baltimore, MD 21201, USA. akhanna@medicine.umaryland.edu
Mehra
Mandeep R
MR
D000818:Animals; D017252:CD3 Complex / Q000276:immunology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016373:DNA, Antisense / Q000378:metabolism; D015870:Gene Expression; D007376:Interleukin-2 / Q000235:genetics; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009928:Organ Specificity; D010957:Plasmids / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D017252:CD3 Complex; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016373:DNA, Antisense; D007376:Interleukin-2; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins