Eradication of B-Lymphoma by Allogeneic Anti-Third Party Host Non-Reactive CTLs: Growing Evidence for TCR Independent Reactivity Against B-Cell Malignancies.

Establishment of cell lines capable of killing malignant cells, in the absence of alloreactivity against normal hematopoietic host cells, represents a most desirable goal in bone marrow transplantation (BMT) and in cancer immunotherapy. We have recently shown, that allogeneic anti third party CTLs depleted of alloreactivity against host cells, are endowed with a potent anti B-CLL reactivity in vitro and in-vivo (Arditi F et al. Blood. 2004 Jul 6, Epub ahead of print). While the ‘conventional’ killing of target cells of the stimulator cell origin could be blocked by anti-CD3 antibody (OKT3), the lysis of the B-CLL cells was not inhibited by this antibody, suggesting a TCR independent cytotoxicity. Annexin staining revealed that this killing is mediated by apoptosis. In the present study we extended our finding and investigated the potential of anti-third party CTLs to eradicate other B cell malignancies, testing in-vitro their capacity to kill 3 different B-cell lymphoma lines. Apoptosis of tumor cells was followed by annexin staining, and reduction in cell number was determined by cytofluorimetry of CD20+ positive cells. Human anti-third party CTLs were prepared as previously described by Arditi et al. and cultured for different periods with two human B cell Burkitt’s lymphoma lines (BL-44, and Namalwa) and one Mantle cell lymphoma line (Grantas). In order to avoid cross reactivity between the lymphoma lines and the stimulators used for preparing CTLs, their HLA Class 1 was deliberately mismatched. As previously found for B-CLL, in 4 different experiments the CTLs lysed a mean of 93%±8.4% of targets of stimulator origin within 4 hours. Upon addition of anti OKT3 antibody cytotoxicity was reduced to 25±26. In contrast, substantial killing of lymphoma lines was initially detected only after 24 hours and peaked at 72 hours (In 3 experiments a mean of 99%±0.5%, 80±20 and 62±31 was found for Grantas, Namlva and BL-44, respectively). This killing could be blocked by anti-LFA1 antibody when tested against the Grantas line targets (26±22% killing) but it was not inhibited by the OKT3 antibody (98%±2 killing), suggesting a TCR independent mechanism. The unique activation status of in-vitro expanded CTLs which express high levels of FasL and other death molecules might be responsible for the killing of B cell malignancies, provided initial binding is sufficiently prolonged, to allow for apoptosis to occur. Thus, adhesion mediated by LFA1 might contribute the additional avidity required to compensate for the lack of TCR recognition, so as to initiate close contact upon which the apoptotic machinery can be triggered. Taken together, these results suggest that the use of allogeneic host non-reactive anti-third party CTLs might be extended from B-CLL to include lymphoma patients. The potential of this surprisingly strong GVT reactivity as a new therapeutic approach should be further explored in preclinical models and for a wide range of hematopoietic malignancies.