We hypothesize that immune tolerance after allogeneic hematopoietic cell transplantation is maintained by an active cellular mechanism that includes Treg CD4+CD25+ cells. In the dog model of mixed hematopoietic chimerism, adoptive immunotherapy with donor lymphocyte infusion (DLI) that successfully converted recipients to all-donor chimerism required the breaking of immune tolerance to target antigens. We reasoned that depletion of Treg cells might break tolerance and could improve the therapeutic efficacy of subsequent DLI. Denileukin diftitox (DAB389IL-2, Ontak) is a fusion protein consisting of the translocation and ADP-ribosylase domains of diphtheria toxin and interleukin 2. DAB389IL-2 binds to the high affinity IL-2 receptor (CD25, CD122 and CD132) expressed on the cell surface of activated T cells. Some Treg cells express the high affinity IL-2 receptor. Although DAB389IL-2 has been shown to deplete alloreactive T cells, we asked if DAB389IL-2 could deplete Treg cells without depleting alloreactive T cells in dog mixed lymphocyte cultures (MLC). First we asked if DAB389IL-2 could induce specific cell death of activated T cells. Five days after initiation of a one-way, dog leukocyte antigen (DLA)-mismatched MLC, DAB389IL-2 was added to the MLC for 24 hours and cells were pulsed with 3H-thymidine. At 10−11 and 10−10 M [DAB389IL-2] there was 34% and 92% inhibition of cell proliferation, respectively. To determine if DAB389IL-2 could deplete Treg cells, we established DLA-mismatched MLC (n=5) with cyclosporine (CSP) 400ng/mL (Martin, PJ et al. BBMT, 2004) and added DAB389IL-2 in half-log increments from 10−11 to 10−9.5 M on day 4 of MLC. On day 5, culture medium was changed to remove DAB389IL-2 and cells were analyzed for expression of CD4 and CD25. Flow cytometric analysis showed that CSP treated MLC had reduced CD4+CD25bright and CD4+CD25dim by 90 ± 3.3% and 64 ± 10%, respectively, compared to no CSP. DAB389IL-2 [10−11 M] treatment of MLC with CSP decreased CD4+CD25bright and dim populations 31 ± 12% and 30 ± 4%, respectively, compared to no DAB389IL-2. On day 10 of the culture, cells were tested in secondary (2°) MLC to determine the effect of depleting CD4+CD25+ cells with DAB389IL-2. 2° MLC was performed without the addition CSP or DAB389IL-2. After 3 days of 2° MLC, cells were assayed for proliferation. Compared to controls with no CSP or DAB389IL-2 treatment during 1° MLC, depletion of CD4+CD25+ cells with DAB389IL-2 increased 2° allo-specific proliferation 36.2 ± 26%. When CSP was present in the 1° MLC, DAB389IL-2 treatment on day 4 increased subsequent 2° MLC allo-specific T cell proliferation 86 ± 22%. These results are consistent with the hypothesis that DAB389IL-2 can efficiently eliminate Treg (CD4+CD25+) cells, particularly when Treg cells are generated in the presence of CSP.

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