Striking augmentation of hematopoietic cell chimerism in noncytoablated allogeneic bone marrow recipients by flt3 ligand and tacrolimus

The influence of granulocyte-macrophage colony-stimulating factor (GM- CSF) and the recently identified hematopoietic stem-progenitor cell mobilizing factor flt3 ligand (FL) on donor leukocyte microchimerism in noncytodepleted recipients of allogeneic bone marrow (BM) was compared. B10 mice (H2b) given 50 x 106 allogeneic (B10.BR [H2(k)]) BM cells also received either GM-CSF (4 μg/day s.c.), FL (10 μg/day i.p.), or no cytokine, with or without concomitant tacrolimus (formerly FK506; 2 mg/kg) from day 0. Chimerism was quantitated in the spleen 7 days after transplantation by both polymerase chain reaction (donor DNA [major histocompatibility complex class II; I-E(k)]) and immunohistochemical (donor [I-E(k+)] cell) analyses. Whereas GM-CSF alone significantly augmented (fivefold) the level of donor DNA in recipients' spleens, FL alone caused a significant (60%) reduction. Donor DNA was increased 10-fold by tacrolimus alone, whereas coadministration of GM-CSF and tacrolimus resulted in a greater than additive effect (28-fold increase). A much more striking effect was observed with FL + tacrolimus (>125-fold increase in donor DNA compared with BM alone). These findings were reflected in the relative numbers of donor major histocompatibility complex class II+ cells (many resembling dendritic cells) detected in spleens, although quantitative differences among the groups were less pronounced. Evaluation of cytotoxic T lymphocyte generation by BM recipients' spleen cells revealed that FL alone augmented antidonor immunity and that this was reversed by tacrolimus. Thus, although FL may potentiate antidonor reactivity in nonimmunosuppressed, allogeneic BM recipients, it exhibits potent chimerism-enhancing activity when coadministered with recipient immunosuppressive therapy. This property of FL may offer considerable potential for the augmentation of microchimerism, with therapeutic implications for organ allograft survival and tolerance induction

Qualitative and quantitative abnormalities in splenic dendritic cell populations in NOD mice

The phenotype and function of splenic DC populations from diabetes-prone NOD mice were chara-cterized and compared to DC from diabetes-resistant strains in the presence or absence of Flt3 ligand (FL) treatment. NOD mice were found to have significantly fewer CD8α(+) DC than both B10.BR and C57BL/6 mice, and this defect was reversed by FL treatment. Freshly isolated CD8α(+) and CD8α(−) DC from all three strains were found to express similar levels of costimulatory molecules and this was similar in both FL-treated and untreated animals. IL-12 p40 production was significantly lower in purified CD11c(+) DC from NOD mice compared to DC from C57BL/6 or B10.BR mice. CD8α(+) DC isolated from NOD mice produced lower levels of IL-12p40 than CD8α(+) DC from C57CBL/6 and this was dependent on the nature of the stimulus given. In contrast both CD8α(+) and CD8α(−) DC from FL-treated mice produced high levels of IL-12p40 following activation, but only the CD8α(−) DC produced IL-12p70. Functionally, freshly isolated CD8α(−) DC were more stimulatory than CD8α(+) DC in a primary allogeneic mixed lymphocyte reaction. However, DC maturation resulted in increased T cell stimulatory capacity for both DC subsets, and this pattern was seen in all strains. These results demonstrate significant differences in phenotype and function of splenic NOD CD8α(+) DC, and further suggest that FL treatment may reverse some of these abnormalities

Site-specific regulation of tissue dendritic cell function by granulocyte–macrophage colony-stimulating-factor

Tissue dendritic cells (DC) are usually associated with phagocytic function but poor T-cell immunostimulatory capacity. Following activation, dendritic cells are stimulated to leave tissue sites and migrate to lymphoid tissue, acquiring immunostimulatory capacity during the process. We provide evidence that the immunostimulatory capacity of tissue DC, but not spleen cells, can be affected in situ by granulocyte–macrophage colony-stimulating-factor (GM-CSF). Initially it was found that islet cells from non-obese diabetic and BALB/c mice, which produce GM-CSF, showed significantly higher immunostimulatory capacity than islets from C3H and C57BL/6 mice, which do not produce GM-CSF. Second, pretreatment of nonobese diabetic mice with anti-GM-CSF antibody significantly reduced the immunostimulatory capacity of islet cells, but not spleen cells, although it had no effect on the numbers of cells expressing DC-associated antigens. Therefore the immunostimulatory function of islet DC is partially dependent on GM-CSF. By contrast, spleen DC immunostimulatory function does not show the same dependence on GM-CSF. This may affect the ability of dendritic cells to stimulate autoimmune responses or tolerance

Mucosal CD8α(+) DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties

Repetitive stimulation of naïve T cells by immature splenic dendritic cells (DC) can result in the differentiation of T-cell lines with regulatory properties. In the present study we identified a population of DC in the mucosae that exhibit the plasmacytoid phenotype, secrete interferon-α (IFN-α) following stimulation with oligodeoxynucleotides containing certain cytosine-phosphate-guanosine (CpG) motifs and can differentiate naïve T cells into cells that exhibit regulatory properties. Although these DC appear to be present in both spleen and mesenteric lymph nodes (MLN), only CpG-matured DC from the MLN (but not the spleen) were able to differentiate naïve T cells into T regulatory 1-like cells with regulatory properties. The activity of these DC failed to sustain robust T-cell proliferation and thereby enhanced the suppressive efficacy of CD4(+) CD25(+) T regulatory cells. These DC are the major CD8α(+) DC population in the Peyer's patches (PP). Given their significant presence in mucosal tissue, we propose that these DC may provide a mechanistic basis for the homeostatic regulation in the gut by eliciting regulatory cell suppressor function and poorly supporting T helper cell proliferation at a site of high antigenic stimulation like the intestine