Bone marrow transplantation offers great promise for treating a number of disease states. However, the widespread application of this approach is dependent upon the development of less toxic methods to establish chimerism and avoid graft-versus-host disease (GVHD). CD8+/TCR− facilitating cells (FCs) have been shown to enhance engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. In the present studies, we have identified the main subpopulation of FCs as plasmacytoid precursor dendritic cells (p-preDCs). FCs and p-preDCs share many phenotypic, morphological, and functional features: both produce IFN-α and TNF-α, both are activated by toll-like receptor (TLR)-9 ligand (CpG ODN) stimulation, and both expand and mature after Flt3 ligand (FL) treatment. FL-mobilized FCs, most of which express a preDC phenotype, significantly enhance engraftment of HSCs and induce donor-specific tolerance to skin allografts. However, p-preDCs alone or p-preDCs from the FC population facilitate HSC engraftment less efficiently than total FCs. Moreover, FCs depleted of preDCs completely fail to facilitate HSC engraftment. These results are the first to define a direct functional role for p-preDCs in HSC engraftment, and also suggest that p-preDCs need to be in a certain state of maturation/activation to be fully functional.

Nonmyeloablative conditioning has significantly reduced the morbidity associated with bone marrow transplantation. The donor hemopoietic cell lineage(s) responsible for the induction and maintenance of tolerance in nonmyeloablatively conditioned recipients is not defined. In the present studies we evaluated which hemopoietic stem cell-derived components are critical to the induction of tolerance in a total body irradiation-based model. Recipient B10 mice were pretreated with mAbs and transplanted with allogeneic B10.BR bone marrow after conditioning with 100–300 cGy total body irradiation. The proportion of recipients engrafting increased in a dose-dependent fashion. All chimeric recipients exhibited multilineage donor cell production. However, induction of tolerance correlated strictly with early production of donor T cells. The chimeras without donor T cells rejected donor skin grafts and demonstrated strong antidonor reactivity in vitro, while possessing high levels of donor chimerism. These animals lost chimerism within 8 mo. Differentiation into T cells was aborted at a prethymic stage in recipients that did not produce donor T cells. Moreover, donor Ag-driven clonal deletion of recipient T cells occurred only in chimeras with donor T cells. These results demonstrate that donor T cell production is critical in the induction of transplantation tolerance and the maintenance of durable chimerism. In addition, donor T cell production directly correlates with the deletion of potentially alloreactive cells.

Chronic malaria infection is characterized by polyclonal B cell activation, hyperglobulinemia, and elevated titers of autoantibodies. We have recently identified the cysteine-rich interdomain region 1α (CIDR1α) of the Plasmodium falciparum erythrocyte membrane protein 1 as a T cell-independent polyclonal B cell activator and Ig binding protein. Here, we show that, although the binding affinity of CIDR1α to human IgM and IgG is relatively low, B cell activation still proceeds. CIDR1α rescues tonsillar B cells from apoptosis, and increases the proportion of cycling cells. Comparison of the impact on naive and memory B cell compartment indicated that CIDR1α preferentially activates memory B lymphocytes. Analysis of the gene expression profiles induced by CIDR1α and anti-Ig activation using a cDNA microarray demonstrated a low degree of homology in the signatures imposed by both stimuli. The microarray data correlate with the functional analysis demonstrating that CIDR1α activates various immunological pathways and protects B cells from apoptosis. Together, the results provide evidence for a role of malaria in preferentially activating the memory B cell compartment. The polyclonal B cell activation and augmented survival induced by CIDR1α is of relevance for understanding the mechanisms behind the increased risk of Burkitt’s lymphoma in malaria endemic areas.

We evaluated the relative contribution of the humoral and cellular arms of the immune response to bone marrow cells transplanted into sensitized recipients. We report here for the first time that humoral immunity contributes predominantly to allosensitization. Although the major role for nonmyeloablative conditioning is to control alloreactive host T cells in nonsensitized recipients, strikingly, none of the strategies directed primarily at T-cell alloreactivity enhanced engraftment in sensitized mice. In evaluating the mechanism behind this barrier, we found that humoral immunity plays a critical role in the rejection of allogeneic marrow in sensitized recipients. Adoptive transfer of as little as 25 L serum from sensitized mice abrogated engraftment in secondary naive recipients. With the use of MT mice as recipients, we found that T-cell-mediated immunity plays a secondary but still significant role in allorejection. Targeting

CD8-positive/T-cell receptor-negative (CD8 ؉ /TCR ؊ ) graft facilitating cells (FCs) are a novel cell population in bone marrow that potently enhance engraftment of hemopoietic stem cells (HSCs). Previously, we showed that the CD11c ؉ /B220 ؉ / CD11b ؊ plasmacytoid-precursor dendritic cell (p-preDC) FC subpopulation plays a critical but nonredundant role in facilitation. In the present study, we investigated the mechanism of FC function. We report that FCs induce antigen-specific CD4 ؉ / CD25 ؉ /FoxP3 ؉ regulatory T cells ( IntroductionRecently, a great deal of interest has focused on the therapeutic potential of cell-based therapies to induce tolerance. Of greatest interest is the subpopulation of bone marrow-derived plasmacytoidprecursor dendritic cells (p-preDCs) and the regulatory T cells (Tregs) that they induce. A major limitation to the use of p-preDCs and Tregs in vivo has been the failure to identify an approach to prevent them from losing their tolerogenic properties and becoming immunogenic after transplantation. We recently demonstrated that CD8␣-positive/T-cell receptor-negative (CD8␣ ϩ /TCR Ϫ ) graft facilitating cells (FCs) enhance the engraftment of hematopoietic stem cells (HSCs) and tolerance induction in allogeneic recipients. [1][2][3] FCs suppress graft-versus-host disease (GVHD) in vivo by producing CD4 ϩ /CD25 ϩ /FoxP3 ϩ Tregs 4 and induce Tregs in vitro in the presence of CpG. 5 The majority of CD8␣ ϩ /TCR Ϫ FCs share the B220 ϩ /CD11c ϩ /CD11b Ϫ p-preDC phenotype, and we have demonstrated the first in vivo engraftment-enhancing and tolerancepromoting effect of the p-preDC FC subpopulation. 2 Although removal of p-preDC FCs from total FCs completely abrogates facilitation, p-preDC FCs alone do not replace FCs to provide the full in vivo biologic effect of facilitation. The mechanism of FC function has yet to be precisely characterized.CD4 ϩ /CD25 ϩ /FoxP3 ϩ Tregs play a critical role in the maintenance of self-tolerance. 6 Defects in Treg development or homeostasis result in systemic autoimmunity, 7 whereas adoptive transfer of Tregs as a therapeutic method can control ongoing autoimmune diseases. [8][9][10] Recently, several studies have demonstrated an important role for Tregs in mediating transplantation tolerance in animal models, 11-14 but little is known about the mechanism of Treg development and homeostasis. [15][16][17] p-preDCs may be important in the generation of Tregs, as evidenced by their potential to facilitate engraftment of HSCs 2,18 and to prolong heart allograft survival. 19,20 In addition, in vitro activation of p-preDCs with CpGoligodeoxynucleotides (CpG-ODNs) induces the production of Tregs in vitro. 5,21 We therefore evaluated whether the mechanism of FC function in vivo is to induce Tregs.In the present study, we first evaluated whether FCs enhance allogeneic HSC engraftment in diabetes-prone nonobese diabetic (NOD) mice. Second, we investigated whether FCs induce the production of Tregs and examined their function using in vivo transplantation models and in vitr...