Cytoreductive protocols are integral both as conditioning regimens for bone marrow (BM) transplantation and as part of therapies for malignancies, but their associated comorbidities represent a long-standing clinical problem. In particular, they cause myeloablation that debilitates the physiological role of mesenchymal stem and precursor cells (MSPCs) in sustaining hematopoiesis. This review addresses the damaging impact of cytoreductive regimens on MSPCs. In addition, it discusses prospects for alleviating the resulting iatrogenic comorbidities. New insights into the structural and functional dynamics of hematopoietic stem cell (HSC) niches reveal the existence of "empty" niches and the ability of the donor-derived healthy HSCs to outcompete the defective HSCs in occupying these niches. These findings support the notion that conditioning regimens, conventionally used to ablate the recipient hematopoiesis to create space for engraftment of the donor-derived HSCs, may not be a necessity for allogeneic BM transplantation. In addition, the capacity of the MSPCs to cross-talk with HSCs, despite major histocompatibility complex disparity, and suppress graft versus host disease indicates the possibility for development of a conditioning-free, MSPCs-enhanced protocol for BM transplantation. The clinical advantage of supplementing cytoreductive protocols with MSPCs to improve autologous hematopoiesis reconstitution and alleviate cytopenia associated with chemo and radiation therapies for cancer is also discussed.

The major therapeutic modality for type 1 diabetes mellitus (T1DM) remains sustaining euglycemia by exogenous administration of insulin. Based on a new understanding of bone marrow structural and functional dynamics, a conditioning-free bone marrow transplantation (BMT), with reduced adverse effects, opens the possibility for evaluating beta cell regeneration and restoration of euglycemia by induction of allogeneic chimerism in patients T1DM, as shown in a mouse model. With this therapeutic modality, donor bone marrow (BM) selection based on T1DM-predisposing and preventive phenotypes will improve treatment outcomes by limiting the risk of exacerbating the autoimmune processes in the BM recipient.

Physiologically sufficient β cell regeneration can be achieved by the induction of hematopoietic chimerism in a type 1 diabetes mellitus (T1DM) mouse model. However, pancytopenia and graft-versus-host disease (GVHD) limits the clinical adaptation of this modality. In this review, we discuss new perceptions on the induction of chimerism, without bone marrow (BM) recipient conditioning, via supplementation of mesenchymal stem cells (MSCs) to support engraftment of allogeneic HSCs. The use of haploidentical, gender-matched, predisposing T1DM genotype-free HSCs in combination with MHC-disparate MSCs could lead to the development of a safe protocol for the induction of hematopoietic chimerism for the treatment of T1DM.

Previously, we established a model in which physiologically adequate function of the autologous β cells was recovered in non-obese diabetic (NOD) mice after the onset of hyperglycemia by rendering them hemopoietic chimera. These mice were termed antea-diabetic. In the current study, we addressed the role of T regulatory (Treg) cells in the mechanisms mediating the restoration of euglycemia in the antea-diabetic NOD model. The data generated in this study demonstrated that the numbers of Treg cells were decreased in unmanipulated NOD mice, with the most profound deficiency detected in the pancreatic lymph nodes (PLNs). The impaired retention of the Treg cells in the PLNs correlated with the locally compromised profile of the chemokines involved in their trafficking, with the most prominent decrease observed in SDF-1. The amelioration of autoimmunity and restoration of euglycemia observed in the antea-diabetic mice was associated with restoration of the Treg cell population in the PLNs. These data indicate that the function of the SDF-1/CXCR4 axis and the retention of Treg cells in the PLNs have a potential role in diabetogenesis and in the amelioration of autoimmunity and β cell regeneration in the antea-diabetic model. We have demonstrated in the antea-diabetic mouse model that lifelong recovery of the β cells has a strong correlation with normalization of the Treg cell population in the PLNs. This finding offers new opportunities for testing the immunomodulatory regimens that promote accumulation of Treg cells in the PLNs as a therapeutic approach for type 1 diabetes (T1D).

This report describes age-related alterations of dendritic cells (DC) distribution in nude athymic mice in vivo and reversal of certain age-dependent defects by an in vivo administration of hematopoietic growth factor FLT3 ligand (FLT3L). There are decreased percentages of CD11c+ DC in the bone marrow and spleen and a reduced expression of MHC class II and CD86 molecules on DC in old nude mice. The decreased levels of CD11c+ DC were due to the CD8α− DC subset. The distribution of CD11c+ CD8α+ DC in the lymphoid tissues was not different in young and old mice. The effect of in vivo administration of FLT3L on the generation and distribution of DC in the lymphoid tissues in young and old nude mice was also evaluated. Although, FLT3L had a higher inductive potential on the expansion of DC from the bone marrow in the elderly mice, the total level of CD11c+ DC in the young animals was still significantly higher as compared to the old animals. Interestingly, FLT3L induced a pronounced redistribution and accumulation of MHC class II+ DC in the lymphoid tissues in old mice, markedly increased the accumulation of CD8α− DC in the bone marrow in both young and old nude mice, and elevated both CD8α− and CD8α+ DC in the spleen in young mice. However, only the level of CD8α+ DC was up regulated in the spleen in old athymic mice after FLT3L-based therapy. In summary, abnormalities in DC generation and distribution in old athymic mice could be, in part, circumvented by the in vivo administration of FLT3L.

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of beta cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft-bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous beta cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity-free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoictic chimerism can abrogate destruction of beta cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft-bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous 0 cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity-free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

The adaptation of allogeneic chimerism in treatment of autoimmune diabetes has been shown as a promising approach in numerous studies in both experimental and clinical settings. Establishment of hemopoietic chimerism in NOD mice is the most adequate animal model to study mechanisms involved in the multiple aspects of the curative effects of chimerism in autoimmunity-prone individuals. However, there are some discrepancies in the current literature for parameters and criteria used to characterize chimerism in the NOD model. This study was aimed to standardize the criteria for the different pathological stages of diabetogenesis in chimeric versus unmanipulated NOD mice. We report two well-defined scoring systems and a new Index N for the assessment of the pathological characteristics of diabetogenesis and GVHD in chimeric NOD mice. Also, we have demonstrated that, in the NOD model, recipient conditioning resulting in as low as 1% of chimerism is sufficient to promote engraftment of the BM donor-specific islets of Langerhans.

The process of hematopoietic stem and progenitor cell (HSPC) seeding in recipient bone marrow (BM) early after transplantation is not fully characterized. In vivo tracking of HSPCs, labeled with PKH dyes, through an optical window surgically implanted on the mouse femur revealed that transplanted cells cluster in the recipient BM. Within the first day after intravenous injection, 86 +/- 6% of the cells seeded in clusters (p < 0.001 versus scattered cells) in the endosteal surfaces of the epiphyses. The primary clusters were formed by concomitant seeding of 6-10 cells over an area of approximately 70 microm, and secondarily injected cells did not join the already existing clusters but formed new clusters. Major antigen-disparate HSPCs participated in formation of the primary clusters, and T lymphocytes were also incorporated. After 4 to 5 days, some cellular clusters were observed in the more central regions of the BM, where the brightness of PKH fluorescence decreased, indicating cellular division. These later clusters were classified as secondary, assuming that the mechanisms of migration in the BM might be different from those of primary seeding. Some clusters remained in the periphery of the BM and retained bright fluorescence, indicating cellular quiescence. The number of brightly fluorescent cells in the clusters decreased exponentially to two to three cells after 24 days (p < 0.001). The data suggest that the hematopoietic niche is a functional unit of the BM stromal microenvironment that hosts seeding of a number of transplanted cells, which form a cluster. This may be the site where auxiliary non-HSPC cells, such as T lymphocytes, act in support of HSPC engraftment.

636 Bone marrow (BM) chimerism is one way to achieve donor specific tolerance and is a treatment of choice for a large spectrum of malignant and genetic disorders. Graft versus host disease (GVHD) (the GVHD occurrence reach up to 100% in more than 2 Ag disparity combination) and graft failure (engraftment of physiologic numbers of highly purified SC in MHC-disparate recipients has not been reported to date) are two life-threatening complications which are associated with conventional bone marrow transplantation (BMT). The protocol to propagate the cells with ability to promote allogeneic chimerism without causing GVHD is elaborated and might solve the current obstacles to the widespread application of BMT as a curative approach. BM cells from 5-Fluoruoracil-treated (5 FU) (150mg/kg, a single IV injection) mice and splenocytes from animals treated with 5 FU, plus FLT3-Ligand (FL) by 10 daily SC injections of 10μg, and 7 daily SC injections of 7.5μg of G-CSF were cultured in the presence of GM-CSF and TNFα for 8 days, and splenocytes were cultured with SCF, FL, GM-CSF, IL-7, IL-12, and TNFα for 10 days. An in vivo murine model of mixed allogeneic reconstitution with a ratio of 1:1, syngeneic to allogeneic, T-cell depleted bone marrow cells(5×106 B10RAMB + 5×106 BRRAMB → B10) was used to evaluate the function of cultured cells to enhance allogeneic chimerism. T-cell depletion of donor and host BM was accomplished using rabbit anti-mouse brain (RAMB) polyclonal serum. The above chimeras show very low levels of donor chimerism (≤16%). Facilitation was considered to have occurred if ≥75% donor chimerism resulted. The co-administration of 0.25×106 cultured cells for recipient reconstitution enhanced donor chimerism to levels of ≥75% in all animals. The donor chimerism is durable and there is no GVHD during 6 month of observation. Five percent of the cells generated in culture have the FC phenotype(CD8+/αβTCR-) and are positive for lymphoid dendritic cell (LDC) markers as well. We suggest that cells with the described phenotype(CD8+/ αβTCR-/ B7.2+/ CD11c+) may represent the facilitating cell population and that FC may be a subpopulation of LDC. The yield of the FC, generated ex-vivo, in BM and splenocyte culture, is sufficient for 1 and 200 recipients for allogeneic reconstitution respectively. We have elaborated a method to generate ex-vivo cells with ability to enhance the allogeneic chimerism. The further studies are needed to purify the FC from the culture cell populations, and to explore the hypothesis that FC belongs to the LDC hematopoietic cell compartment.

318 Bone marrow stem cell chimerism induces donor-specific tolerance to solid organ and cellular grafts without immunosuppressive therapy. Transplantation of purified hematopoietic stem cells (HSC) would offer the optimal approach to achieve chimerism yet avoid GVHD. Although HSC engraft readily in MHC-matched recipients, they require an accessory facilitating cell (FC) to engraft across MHC-barriers. We have previously characterized the FC population in murine bone marrow as CD8+/TCR-/NK-. We recently determined that both FC and HSC are most efficiently mobilized into peripheral blood (PB) by combined treatment with Flt3 ligand (FL) and G-CSF, while mobilization with G-CSF alone was less efficient. The aim of this study was to compare the engraftment-potential of FC and HSC mobilized by single and combined growth factor administration. Donor mice (B10.BR) received daily injections with 10μg FL, 7.5μg G-CSF or FL plus G-CSF. FL was given from day 1 to 10 and G-CSF from day 4 to 10. PB was obtained from growth factor-treated animals on day 10 and from untreated control mice and the percentage of FC(CD8+/TCR-) and primitive HSC(lineage-/Sca-1+/c-kit+) was analyzed by flow cytometry. C57BL/10SnJ recipients (n=3 to 7 per group) were conditioned with 950cGy TBI and reconstituted with PB containing 1 ×106, 2.5×106 and 5 ×106 PBMNC from mobilized or untreated donors, respectively. Engraftment was confirmed by flow cytometry after 1 and 6 months following transplantation. TableAs few as 1 × 106 PBMNC mobilized with FL alone or FL plus G-CSF rescued 83% and 100% of animals from TBI-induced aplasia, respectively. In striking contrast, only 17% of recipients reconstituted with PB from G-CSF-treated donors were alive 30 days after TBI. All control animals died within 12 days. Even at a PBMNC dose of 5 × 106, only 33% of recipients transplanted with PB mobilized with G-CSF alone survived. Flow cytometric analysis after 1 month showed > 90% donor chimerism in engrafted animals. After > 6 months, 67% of recipients treated with FL and FL plus G-CSF were alive and donor chimerism was > 99%. The presence of multiple hematopoietic lineages in these animals demonstrates HSC engraftment. We present in this study for the first time that allogeneic recipients can be rescued from TBI-induced aplasia with as few as 1 × 106 PBMNC mobilized by FL and FL plus G-CSF.

We have previously identified a cellular population in murine bone marrow that facilitates engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex (MHC) barriers without causing graft-versus-host disease. Here we investigated the effect of flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) on the mobilization of facilitating cells (FC) and HSC into peripheral blood (PB). Mice were injected with FL alone (day 1 to 10), G-CSF alone (day 4 to 10), or both in combination. The number of FC (CD8(+)/alpha betaTCR-/gamma deltaTCR-) and HSC (lineage-/Sca-1(+)/c-kit+) was assessed daily by flow cytometry. Lethally irradiated allogeneic mice were reconstituted with PB mononuclear cells (PBMC). FL and G-CSF showed a highly significant synergy on the mobilization of FC and HSC. The peak efficiency for mobilization of FC (21-fold increase) and HSC (200-fold increase) was reached on day 10. Our data further suggest that the proliferation of FC and HSC induced by FL in addition to the mobilizing effect mediated by G-CSF might be responsible for the observed synergy of both growth factors. Finally, the engraftment potential of PBMC mobilized with FL and G-CSF or FL alone was superior to PBMC obtained from animals treated with G-CSF alone. Experiments comparing the engraftment potential of day 7 and day 10 mobilized PBMC indicate that day 10, during which both FC and HSC reached their maximum, might be the ideal time point for the collection of both populations.

FLT3 ligand (FL) is a recently described hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic progenitors. We have investigated the effect of FL on murine hematopoiesis and dendritic cell (DC) generation and accumulation in lymphoid tissues and liverin vivoandin vitroevaluating the morphologic, phenotypic, and functional characteristics of these DC. We have observed extramedullary hematopoiesis in the mouse spleen with all lineages of hematopoietic cells represented after the administration of FL. Injection of FL results in a time-dependent and reversible accumulation of DC in the spleen, bone marrow, lymph nodes, and liver. Both flow cytometry and immunohistochemistry revealed a significant accumulation of DC in these tissues. Results of mixed leukocyte reaction suggested that these cells, isolated from murine bone marrow or spleen, were active as antigen presenting cells. Furthermore, cultivation of splenic and marrow cells with GM-CSF and IL-4 gave rise to large numbers of functionally active mature DC. Thus, the results of this study suggest that FL is a promising growth factor that stimulates the generation of large number of DC and may be a useful cytokine for the immunotherapy of cancer.

Cytotoxic T lymphocytes (CTL) are an important component of the host’s immune response to cancer1,2. A number of genes encoding tumor-associated antigens (TAA) and their peptide products which are recognized by CTL in the context of major histocompatibility complex (MHC) class I molecules have recently been identified3,4. Our group has focused on the translation of these new insights into the development and application of novel immunotherapies.

Dendritic cells (DCs) are considered the most effective antigen-presenting cells (APCs) for primary immune responses. Since presentation of antigens to the immune system by appropriate professional APCs is critical to elicit a strong immune reaction and DCs seem to be quantitatively and functionally defective in the tumor host, DCs hold great promise to improve cancer vaccines. Even though they are found in lymphoid organs, skin and mucosa, the difficulty of generating large numbers of DCs has been a major limitation for their use in vaccine studies. A simple method for obtaining DCs from mouse bone marrow cells cultured in the presence of GM-CSF + interleukin 4 is now available. In four different tumor models, mice injected with DCs grown in GM-CSF plus interleukin 4 and prepulsed with a cytotoxic T lymphocyte-recognized tumor peptide epitope developed a specific cytotoxic T lymphocyte response and were protected against a subsequent tumor challenge with tumor cells expressing the relevant tumor antigen. Moreover, treatment of day 5-14 tumors with peptide-pulsed DCs resulted in sustained tumor regression in five different tumor models. These results suggest that presentation of tumor antigens to the immune system by professional APCs is a promising method to circumvent tumor-mediated immunosuppression and is the basis for ongoing clinical trials of cancer immunotherapy with tumor peptide-pulsed DCs.