Cellular mediated by genetically modified antigen-specific

Antibody-Dependent Cytotoxicity (ADCC) human T Abstract In the context of transplantation, donor and virus-specific T-lymphocytes infusions have demonstrated the dramatic potential of T cells as immune effectors. Unfortunately, most attempts to exploit the T cell immune system against non-viral malignancies in the syngeneic setting have been disappointing. In contrast treatments based on monoclonal antibodies (Abs) have been clinically successful and have demonstrated the clinical relevance of several antigens as therapeutic targets and the importance of the antibody dependant cellular cytotoxicity (ADCC) pathway. In the present study we considered the possibility of arming specific T cells with a receptor that would enable them to mediate ADCC. After transduction with a CD16/ γ receptor gene, CD4+ and CD8+ cytotoxic T lymphocytes displayed stable expression of the CD16 receptor at their surface. In the absence of Ab, CD16/ γ expression did not affect the capacity of specific T-lymphocytes to kill their target following "natural" T cell receptor recognition. When tested against the autologous B lymphoblastoid cell line (BLCL) coated with anti-CD20 mAb, the newly expressed Fc receptor enabled the T cells to kill the BLCL through ADCC. Adoptive transfer of such newly designed immune effector may be considered to increase antibody efficiency by harnessing the immune potential of T cells. gene The resulting CD16/ -transduced T cells retained their natural function (proliferation and cytotoxicity) through TCR recognition of cognate allo- or viral-antigens and were able to kill Ab-coated autologous tumor target cells by ADCC mediated by the transduced chimeric CD16/ γ receptor. added 3 days after the second One day thereafter, retroviral transduction was performed by mixing 2 × 10 6 T cells (in 2 ml RPMI, 8% HS supplemented with 80 UI IL-2/ml) with 2 ml of retroviral supernatant and 8µg/ml polybrene and then centrifuging at 2400 x g for 90 minutes at 34°C. As a control, the T cells were incubated with nontransfected Phoenix-ampho cell supernatant (nontransduced control CTL). The day after transduction half of the medium was changed. Transduction efficiencies were assessed 3 days later and a third stimulation was performed 7 days after the second, in the presence of IL-2 and with an identical T:B ratio (4:1). and resuspended in 50 µl of 1X BD TM Phosflow Perm/Wash buffer for 20 min at RT. The following monoclonal antibodies (mAbs) were used : PE-mouse anti- human TNF α (Mab1, BD Biosciences Pharmingen), PE-mouse anti- human INF γ (B27, BD Biosciences Pharmingen) or with mouse IgG1 (BD Biosciences Pharmingen) as a negative control. Cells were stained at RT for 20 min with 50 µl of the aforementioned PE-mAbs diluted 1:50 in 1X BD TM Phosflow Perm/Wash buffer. The cells were then pelleted, washed in 1X BD TM Phosflow Perm/Wash buffer followed by two further absence of target cells, the soluble mAb was unable to induce significant TNF production by the clone, at concentrations of up to 1000 µg/ml. Three independent experiments were performed with 3 different CD16/ γ -transduced T cell clones and for three cytokines (TNF α , IFN γ and IL-2), leading to the same conclusion. The same results were also observed when testing human serum at a concentration of up to 50%.