Hematopoietic stem cell transplantation across major genetic barriers: tolerance induction by megadose CD34 cells and other veto cells.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used overcomes major genetic barriers. In vitro studies suggest that veto cells, within the population of hematopoietic progenitors, facilitate this favorable outcome. Tolerance induction can be further enhanced by other veto cells. Perhaps the most potent veto cell is the CD8(+) CTL. However, this cell is also associated with marked GVHD, which can be separated from the veto activity by generating anti-third party CTLs under IL-2 deprivation.

Transplants across human leukocyte antigen barriers.

Clinical experience with full haplotype-mismatched stem cell transplants has a 20-year history. Early results in leukemia patients were disappointing because of a high incidence of severe graft-versus-host disease (GvHD) in T-replete transplants or high rejection rates in T-cell-depleted transplants. The breakthrough came with introduction of a megadose T-cell-depleted progenitor cell transplant following a high-intensity conditioning regimen and the realization that donor natural killer (NK) cell alloreactivity also plays a role in facilitating engraftment and in preventing relapse. Treating end-stage patients inevitably confounded clinical outcome in early pilot studies. Today, high-risk acute leukemia patients are treated at less advanced stages of disease, receive a reasonably well-tolerated conditioning regimen, and benefit from advances in post-transplant immunological reconstitution. These factors have markedly reduced transplant-related mortality. Overall, event-free survival (EFS) and transplant-related mortality (TRM) compare favorably with reports from unrelated matched transplants. T-cell-depleted megadose stem cell transplant from a mismatched family member, who is immediately available, can now be offered as a viable option to candidates with high-risk acute leukemias.

Hematopoietic stem cell transplantation across major genetic barriers: tolerance induction by megadose CD34 cells and other veto cells.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used ("megadose" transplants) overcomes major genetic barriers. In vitro studies suggest that veto cells, within the population of hematopoietic progenitors, facilitate this favorable outcome. Thus, when purified CD34(+) cells were added to bulk mixed-lymphocyte reactions (MLRs) they suppressed CTLs against the donor's stimulators, but not against stimulators from a third party. This tolerizing activity depends on cell contact and can be blocked by the caspase inhibitor BD-FMK, suggesting that the effector host T cells are deleted by apoptosis upon interaction with the CD34(+) cells. Early myeloid CD33(+) cells generated by short-term ex vivo expansion of CD34(+) cells also exhibit veto activity, and these cells can be grown in large numbers. Tolerance induction can be further enhanced by other veto cells. Perhaps the most potent veto cell is the CD8+ CTL. However, this cell is also associated with marked GVHD (graft-versus-host disease. GVHD can be separated from the veto activity by generating anti-third party CTLs under IL2 deprivation. Under such selective pressure only the stimulated clones which make IL2 can survive, while anti-host clones die. In vivo studies show that such anti-third party veto CTLs can be used safely for tolerance induction without GVHD.

Immune regulatory activity of CD34+ progenitor cells: evidence for a deletion-based mechanism mediated by TNF-alpha.

Previous studies suggest that cells within the CD34(+) hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti-IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti-tumor necrosis factor-alpha (anti-TNF-alpha) and not by anti-transforming growth factor-beta (anti-TGF-beta), suggesting a potential role for TNF-alpha in the regulatory activity of CD34+ cells.

Crossing the HLA barriers.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used ("megadose" transplants) overcomes major genetic barriers. In vitro studies suggest that veto cells, within the population of hematopoietic progenitors, facilitate this favorable outcome. Thus, when purified CD34+ cells were added to bulk mixed-lymphocyte reactions (MLRs), they suppressed CTLs against donor's stimulators but not against stimulators from a third party. This tolerizing activity depends on cell contact and can be blocked by the caspase inhibitor BD-FMK, suggesting that the effector host T cells are deleted by apoptosis upon interaction with the CD34+ cells. Early myeloid CD33+ cells generated by short-term ex vivo expansion of CD34+ cells also exhibit veto activity, and these cells can be grown in large numbers. Tolerance induction can be further enhanced by other veto cells. Perhaps the most potent veto cell is the CD8+ CTL. However, this cell is also associated with marked graft-versus-host disease (GVHD). GVHD can be separated from the veto activity by generating anti-third party CTLs under IL2 deprivation. Under such selective pressure, only the stimulated clones which make IL2 can survive, while anti-host clones die. In vivo studies show that such anti-third party veto CTLs can be used safely for tolerance induction without GVHD.

Tolerance induction by megadose hematopoietic progenitor cells: expansion of veto cells by short-term culture of purified human CD34(+) cells.

Stem cell-dose escalation is one way to overcome immune rejection of incompatible stem cells. However, the number of hematopoietic precursors required for overcoming the immune barrier in recipients pretreated with sublethal regimens cannot be attained with the state-of-the-art technology for stem cell mobilization. This issue was addressed by the observation that cells within the human CD34(+) population are endowed with veto activity. In the current study, we demonstrated that it is possible to harvest about 28- to 80-fold more veto cells on culturing of purified CD34(+) cells for 7 to 12 days with an early-acting cytokine mixture including Flt3-ligand, stem cell factor, and thrombopoietin. Analysis of the expanded cells with fluorescence-activated cell-sorter scanning revealed that the predominant phenotype of CD34(+)CD33(-) cells used at the initiation of the culture was replaced at the end of the culture by cells expressing early myeloid phenotypes such as CD34(+)CD33(+) and CD34(-)CD33(+). These maturation events were associated with a significant gain in veto activity as exemplified by the minimal ratio of veto to effector cells at which significant veto activity was detected. Thus, whereas purified unexpanded CD34(+) cells exhibited veto activity at a veto-to-effector cell ratio of 0.5, the expanded cells attained an equivalent activity at a ratio of 0.125. The availability of novel sources of veto cells such as those in this study might contribute to the realization of immunologic tolerance in "minitransplants," without any risk of graft-versus-host disease.