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

Flt-3 Ligand Increases Microchimerism But Can Prevent the Therapeutic Effect of Donor Bone Marrow in Transiently Immunosuppressed Cardiac Allograft Recipients

C3H (H2(k)) mice received 50 x 10(6) B10 (H2(b)) bone marrow (BM) cells either alone or with fit-3 ligand (FL) (10 mu g/day), tacrolimus (2 mg/kg/day), or both agents for 7 days, Donor MHC class II+ (IA(b+)) cells were quantitated in spleens by immunohistochemical analysis, and donor class II DNA detected in BM by PCR, Donor cells were rare in the BM alone and BM + FL groups, whereas there was a substantial increase in chimerism in the BM + tacrolimus group, Addition of FL to BM + tacrolimus led to a further eightfold increase in donor cells and enhanced donor DNA compared with the BM + tacrolimus group, This increase in donor cells was almost 500-fold compared with BM alone, C3H recipients of B10 heart allografts given perioperative B10 BM and tacrolimus (days 0-13) exhibited a markedly extended median graft survival time (MST, 42 days) compared with those given tacrolimus alone (MST, 22 days), Addition of FL (10 mu g/day; 7 days) to BM + tacrolimus prevented the beneficial effect of donor BM (MST, 18 days), BM alone or BM + FL resulted in uniform early heart graft failure (MST < 8 days), Functional studies revealed maximal antidonor MLR and CTL activities in the BM- and BM + FL-treated groups, with minimal activity in the tacrolimus-treated groups, Thus, dramatic growth factor-induced increases in chimerism achieved under cover of immunosuppression may result in augmented antidonor T cell reactivity and reduced graft survival after immunosuppressive drug withdrawal, With FL, this map reflect striking augmentation of immunostimulatory dendritic cells

Impact of Flt3 ligand on donor-derived antigen-presenting cells and alloimmune reactivity in heart graft recipients given adjuvant donor bone marrow

The influence of the haematopoietic growth factor Flt-3 ligand (FL) on the incidence and function of donor major histocompatibility complex (MHC) class II+ cells in the lymphoid tissues of non-cytoablated recipients of heart allografts and donor bone marrow (BM) cells was investigated. C3H (H2k) mice received a nonvascularized B10 (H2b) heart allograft in the dorsal ear pinna, followed by an i.v. influsion of 50 × 106 donor BM cells. They were given FL (10 μg/day i.p., ×7 days), tacrolimus (2mg/kg/day i.p., ×13 days) or both agents immediately following heart transplantation (HTx) and were killed 10 or 21 days later. Their BM cells were propagated in vitro in granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 for 5 days to promote the growth of dendritic cells (DC). Donor DC were identified by immunocytochemical staining. Spleens were harvested, and donor (IAb+) cells enumerated by immunohistochemical analysis. Donor MHC class II DNA was detected in spleens and cultured BM-derived cells by reverse transcriptase-polymerase chain reaction (RT-PCR). A striking increase in donor MHC class II+ cells was noted in both the spleen and BM of the BM + tacrolimus-treated group compared to either the BM alone, or BM + FL-treated groups. Addition of FL treatment to BM + tacrolimus led to a further increase in donor cells in spleen (three-fold at 10 days, and two-fold at 21 days) The increase in donor cells at 10 days was almost 140-fold compared to that with donor BM alone. PCR analysis at this time revealed enhanced donor DNA in the BM + FL + tacrolimus group compared to that in the BM + tacrolimus group. FL treatment augmented mixed leucocyte reactions (MLR) and cytotoxic T lymphocyte (CTL) activity of host spleen cell against donor alloantigens. These effects were reversed by tacrolimus administration. Histopathology of heart grafts from tacrolimus-treated animals at 10 and 21 days showed absence or substantial reduction in cellular infiltration, and the preservation of viable myocardium. By contrast, in untreated mice, or animals given BM or BM + FL alone, there was marked cellular infiltration, and features of accelerated rejection. Donor-derived DC could be propagated in vitro from the BM of heart transplant recipients given donor BM, especially from mice that also received tacrolimus ± FL. At day 21, donor-derived cells could only be propagated from the BM + FL + tacrolimus-treated group. These findings show that numbers of donor antigen presenting cells (APC) or their progenitors can be markedl