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Donor-derived hematopoiesis was assessed in 17 patients who received allogeneic marrow grafts from HLA-matched siblings between 1971 and 1980. Complete blood counts were normal or near normal in all patients except one. Chimerism analyses, using either dual-color XY-chromosome fluorescence in situ hybridization (FISH) or analysis of variable number tandem repeat loci, indicated that 15 out of 16 patients had greater than 97% donor-derived hematopoiesis, whereas 1 patient had indeterminate chimerism. All 12 recipients of grafts from female donors exhibited polyclonal hematopoiesis by X-linked clonal analysis with the use of molecular probes. Of the 17 recipients, 9 exhibited a less than 1.0-kilobase shortening of granulocyte telomere length compared with their respective donors, according to terminal restriction fragment analysis or flow-FISH with a fluorescein-labeled peptide nucleic acid probe. These data suggest that under standard transplantation conditions, the stem cell proliferative potential is not compromised during hematopoietic reconstitution.

We hypothesize that immune tolerance after allogeneic hematopoietic cell transplantation is maintained by an active cellular mechanism that includes T reg 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 T reg cells might break tolerance and could improve the therapeutic efficacy of subsequent DLI. Denileukin diftitox (DAB 389 IL-2, Ontak) is a fusion protein consisting of the translocation and ADP-ribosylase domains of diphtheria toxin and interleukin 2. DAB 389 IL-2 binds to the high affinity IL-2 receptor (CD25, CD122 and CD132) expressed on the cell surface of activated T cells. Some T reg cells express the high affinity IL-2 receptor. Although DAB 389 IL-2 has been shown to deplete alloreactive T cells, we asked if DAB 389 IL-2 could deplete T reg cells without depleting alloreactive T cells in dog mixed lymphocyte cultures (MLC). First we asked if DAB 389 IL-2 could induce specific cell death of activated T cells. Five days after initiation of a one-way, dog leukocyte antigen (DLA)-mismatched MLC, DAB 389 IL-2 was added to the MLC for 24 hours and cells were pulsed with 3 H-thymidine. At 10 −11 and 10 −10 M [DAB 389 IL-2] there was 34% and 92% inhibition of cell proliferation, respectively. To determine if DAB 389 IL-2 could deplete T reg cells, we established DLA-mismatched MLC (n=5) with cyclosporine (CSP) 400ng/mL (Martin, PJ et al. BBMT, 2004) and added DAB 389 IL-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 DAB 389 IL-2 and cells were analyzed for expression of CD4 and CD25. Flow cytometric analysis showed that CSP treated MLC had reduced CD4 + CD25 bright and CD4 + CD25 dim by 90 ± 3.3% and 64 ± 10%, respectively, compared to no CSP. DAB 389 IL-2 [10 −11 M] treatment of MLC with CSP decreased CD4 + CD25 bright and dim populations 31 ± 12% and 30 ± 4%, respectively, compared to no DAB 389 IL-2. On day 10 of the culture, cells were tested in secondary (2°) MLC to determine the effect of depleting CD4 + CD25 + cells with DAB 389 IL-2. 2° MLC was performed without the addition CSP or DAB 389 IL-2. After 3 days of 2° MLC, cells were assayed for proliferation. Compared to controls with no CSP or DAB 389 IL-2 treatment during 1° MLC, depletion of CD4 + CD25 + cells with DAB 389 IL-2 increased 2° allo-specific proliferation 36.2 ± 26%. When CSP was present in the 1° MLC, DAB 389 IL-2 treatment on day 4 increased subsequent 2° MLC allo-specific T cell proliferation 86 ± 22%. These results are consistent with the hypothesis that DAB 389 IL-2 can efficiently eliminate T reg (CD4 + CD25 + ) cells, particularly when T reg cells are generated in the presence of CSP.