Plasmacytoid precursor dendritic cells facilitate allogeneic hematopoietic stem cell engraftment.

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-alpha and TNF-alpha, 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.

Analysis of popular press articles concerning postpartum depression: 1998-2006.

The purpose of the study was to analyze the content of popular press magazine articles that focused on postpartum depression, published from 1998-2006. Replicating earlier research by Martinez, Johnson-Robledo, Ulsh, and Chrisler, 2000, 47 articles were identified and their content analyzed in the areas of etiology, symptoms, treatment, resources, and demographic assumptions about readers. Popular press magazines contained contradictory information about the definition, prevalence, onset, duration, symptoms, and treatment of postpartum mood disorders. Health care providers should be proactive in directing childbearing women to factual sources of information on postpartum depression.

Addition of cyclophosphamide to T-cell depletion-based nonmyeloablative conditioning allows donor T-cell engraftment and clonal deletion of alloreactive host T-cells after bone marrow transplantation.

BACKGROUND: Bone marrow (BM) chimerism has been shown to have a beneficial effect on allograft survival. We recently found that production of donor T-cells was highly correlated with induction of tolerance in minimally conditioned chimeras. In the present studies, we demonstrate that nonmyeloablative conditioning and BM cell infusion modulate innate and adaptive host immune responses. METHODS: Chimeras were generated by bone marrow transplantation (B10.BR to B10). Recipients were preconditioned with T-cell depleting antibodies and total body irradiation with or without cyclophosphamide. Donor-specific tolerance was tested by skin grafting. RESULTS: Transfer of tolerant splenocytes to immunocompetent secondary recipients did not transfer tolerance, nor did infusion of tolerant CD4+/CD25+ T-cells into chimeras without donor T-cell production, demonstrating that linked suppression is an unlikely mechanism in tolerance induction in the context of BM cell infusion. The addition of a single dose of cyclophosphamide to the conditioning enhanced engraftment and tolerance. This was associated with production of donor T-cells and effective clonal deletion, and a significant reduction in activated recipient plasmacytoid dendritic cells (pDC) and natural killer (NK) cells. Chimeras without donor T-cell production that eventually lost their chimerism did not generate an antidonor humoral response, whereas unconditioned controls infused with similar numbers of BM cells did, indicating that infusion of donor BM cells into conditioned recipients induced immune deviation for adaptive B-cell immunity, preventing sensitization to major histocompatibility complex (MHC) alloantigens. CONCLUSIONS: These results demonstrate that recipient T-cells, pDC, and NK cells contribute to the host barrier for establishing chimerism, implicate deletional tolerance as the mechanism for total body irradiation-based nonmyeloablative conditioning for BM transplantation, and show a beneficial effect of BM cells in preventing sensitization to MHC alloantigens.

Endogenous bone marrow derived cells express retinal pigment epithelium cell markers and migrate to focal areas of RPE damage.

PURPOSE: The aim of the present study was to investigate whether bone marrow-derived cells (BMCs) can be induced to express retinal pigment epithelial (RPE) cell markers in vitro and can home to the site of RPE damage after mobilization and express markers of RPE lineage in vivo. METHODS: Adult RPE cells were cocultured with green fluorescence protein (GFP)-labeled stem cell antigen-1 positive (Sca-1(+)) BMCs for 1, 2, and 3 weeks. Cell morphology and expression of RPE-specific markers and markers for other retinal cell types were studied. Using an animal model of sodium iodate (NaIO(3))-induced RPE degeneration, BMCs were mobilized into the peripheral circulation by granulocyte-colony stimulating factor, flt3 ligand, or both. Immunocytochemistry was used to identify and characterize BMCs in the subretinal space in C57BL/6 wild-type (wt) mice and GFP chimeric mice. RESULTS: In vitro, BMCs changed from round to flattened, polygonal cells and expressed cytokeratin, RPE65, and microphthalmia transcription factor (MITF) when cocultured in direct cell-cell contact with RPE. In vivo, BMCs were identified in the subretinal space as Sca-1(+) or c-kit(+) cells. They were also double labeled for GFP and RPE65 or MITF. These cells formed a monolayer on the Bruch membrane in focal areas of RPE damage. CONCLUSIONS: Thus, it appears that BMCs, when mobilized into the peripheral circulation, can home to focal areas of RPE damage and express cell markers of RPE lineage. The use of endogenous BMCs to replace damaged retinal tissue opens new possibilities for cell replacement therapy in ophthalmology.

Graft facilitating cells are derived from hematopoietic stem cells and functionally require CD3, but are distinct from T lymphocytes.

OBJECTIVE: We previously demonstrated that CD8(+)/TCR(-) bone marrow cells facilitate engraftment of HSC in allogeneic recipients without causing graft-vs-host disease. Whether facilitating cells (FC) develop from T cells or represent a distinct lineage has not been determined. METHODS: In the present studies, we characterized the lineage derivation of FC, defined the role for the CD3 complex in allogeneic facilitation, and demonstrated syngeneic facilitation by FC but not T cells. RESULTS: We demonstrate for the first time that FC development and function is independent of T cells and cannot be replaced by them. Purified GFP(+) HSC transplanted in syngeneic recipients produce GFP(+) FC, which facilitate in secondary transplants, confirming that FC are derived from HSC. In addition, FC, but not T cells, potently facilitate the engraftment of suboptimal numbers of HSC in syngeneic recipients. Notably, FC contain the transcripts for CD3 epsilon and CD3 delta, but not TCR alpha or TCR beta, excluding the possibility of T-cell contamination. Genetic mutations that generate a functional deficiency in CD3 signaling significantly impair FC function in allogeneic facilitation (p=0.006). CONCLUSION: Taken together, these data clearly distinguish FC from T cells. Moreover, they indicate that FC require the CD3 epsilon gene to facilitate allogeneic HSC engraftment. The unique function(s) of FC make them an attractive focus for new cell-based therapeutic approaches to enhance HSC engraftment while reducing toxicity, especially when limiting numbers of HSC are available.

Costimulatory blockade of CD154-CD40 in combination with T-cell lymphodepletion results in prevention of allogeneic sensitization.

Sensitization is a critical unresolved challenge in transplantation. We show for the first time that blockade of CD154 alone or combined with T-cell depletion prevents sensitization. Allogeneic skin grafts were rejected by recipients treated with anti-alphabeta T-cell receptor (TCR), anti-CD154, anti-OX40L, or anti-inducible costimulatory pathway (ICOS) mAb alone with a kinetic similar to untreated recipients. However, the production of anti-donor MHC antibody was prevented in mice treated with anti-CD154 mAb only, suggesting a specific role for the CD154-CD40 pathway in B-cell activation. The impairment of T cell-dependent B-cell responses by blocking CD154 occurs through inhibiting activation of T and B cells and secretion of IFN-gamma and IL-10. Combined treatment with both anti-CD154 and anti-alphabeta TCR abrogated antidonor antibody production and resulted in prolonged skin graft survival, suggesting the induction of both T- and B-cell tolerance with prevention of allogeneic sensitization. In addition, we show that the tolerance induced by combined treatment was nondeletional. Moreover, these sensitization-preventive strategies promote bone marrow engraftment in recipients previously exposed to donor alloantigen. These findings may be clinically relevant to prevent allosensitization with minimal toxicity and point to humoral immunity as playing a dominant role in alloreactivity in sensitized recipients.

TNF-alpha is critical to facilitate hemopoietic stem cell engraftment and function.

The use of tolerogenic cells as an approach to induce tolerance to solid organ allografts is being aggressively pursued. A major limitation to the clinical application of cell-based therapies has been the ability to obtain sufficient numbers and also preserve their tolerogenic state. We previously reported that small numbers of bone marrow-derived CD8+/TCR- graft facilitating cells (FC) significantly enhance hemopoietic stem cell (HSC) engraftment in allogeneic and syngeneic recipients. Although the majority of FC resemble precursor plasmacytoid dendritic cells (p-preDC), p-preDC do not replace FC in facilitating function. In the present studies, we investigated the mechanism of FC function. We show for the first time that FC significantly enhance HSC clonogenicity, increase the proportion of multipotent progenitors, and prevent apoptosis of HSC. These effects require direct cell:cell contact between FC and HSC. Separation of FC from HSC by transwell membranes completely abrogates the FC effect on HSC. p-preDC FC do not replace FC total in these effects on HSC function. FC produce TNF-alpha, and FC from TNF-alpha-deficient mice exhibit impaired facilitation in vivo and loss of the in vitro effects on HSC. Neutralizing TNF-alpha in FC similarly blocks the FC effect. The antiapoptotic effect of FC is associated with up-regulation of Bcl-3 transcripts in HSC and blocking of TNF-alpha is associated with abrogation of up-regulation of Bcl-3 transcripts. These data demonstrate a critical role for TNF-alpha in mediating FC function. FC may have a significant impact upon the safe use of chimerism to establish tolerance to transplanted organs and tissue.

Systemically transferred hematopoietic stem cells home to the subretinal space and express RPE-65 in a mouse model of retinal pigment epithelium damage.

Stem cell regeneration of damaged tissue has recently been reported in many different organs. Since the loss of retinal pigment epithelium (RPE) in the eye is associated with a major cause of visual loss - specifically, age-related macular degeneration - we investigated whether hematopoietic stem cells (HSC) given systemically can home to the damaged subretinal space and express markers of RPE lineage. Green fluorescent protein (GFP) cells of bone marrow origin were used in a sodium iodate (NaIO(3)) model of RPE damage in the mouse. The optimal time for adoptive transfer of bone marrow-derived stem cells relative to the time of injury and the optimal cell type [whole bone marrow, mobilized peripheral blood, HSC, facilitating cells (FC)] were determined by counting the number of GFP(+) cells in whole eye flat mounts. Immunocytochemistry was performed to identify the bone marrow origin of the cells in the RPE using antibodies for CD45, Sca-1, and c-kit, as well as the expression of the RPE-specific marker, RPE-65. The time at which bone marrow-derived cells were adoptively transferred relative to the time of NaIO(3) injection did not significantly influence the number of cells that homed to the subretinal space. At both one and two weeks after intravenous (i.v.) injection, GFP(+) cells of bone marrow origin were observed in the damaged subretinal space, at sites of RPE loss, but not in the normal subretinal space. The combined transplantation of HSC+FC cells appeared to favor the survival of the homed stem cells at two weeks, and RPE-65 was expressed by adoptively transferred HSC by four weeks. We have shown that systemically injected HSC homed to the subretinal space in the presence of RPE damage and that FC promoted survival of these cells. Furthermore, the RPE-specific marker RPE-65 was expressed on adoptively transferred HSC in the denuded areas.

Humoral immunity is the dominant barrier for allogeneic bone marrow engraftment in sensitized recipients.

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 microL serum from sensitized mice abrogated engraftment in secondary naive recipients. With the use of microMT mice as recipients, we found that T-cell-mediated immunity plays a secondary but still significant role in allorejection. Targeting of T cells in sensitized B-cell-deficient microMT mice enhanced alloengraftment. Moreover, both T- and B-cell tolerance were achieved in sensitized recipients when allochimerism was established, as evidenced by the acceptance of second donor skin grafts and loss of circulating donor-specific Abs. These findings have important implications for the management of sensitized transplant recipients and for xenotransplantation in which B-cell reactivity is a predominant barrier.

Flt3-ligand-mobilized peripheral blood, but not Flt3-ligand-expanded bone marrow, facilitating cells promote establishment of chimerism and tolerance.

Facilitating cells (CD8+/TCR-) (FCs) enhance engraftment of limiting numbers of hematopoietic stem cells (HSCs). The primary component of FCs is precursor-plasmacytoid dendritic cells (p-preDCs), a tolerogenic cell expanded by Flt3-ligand (FL). In this study, we evaluated the function and composition of FL-expanded FCs. FL treatment resulted in a significant increase of FCs in bone marrow (BM) and peripheral blood (PB). When FL-expanded FCs were transplanted with c-Kit+/Sca-1+/Lin- (KSL) cells into allogeneic recipients, BM-FCs exhibited significantly impaired function whereas PB-FCs were potently functional. A significant upregulation of P-selectin expression and downregulation of VCAM-1 (vascular cell adhesion molecule 1) were present on FL-expanded PB-FCs compared with FL BM-FCs. Stromal cell-derived factor-1 (SDF-1), and CXCR4 transcripts were significantly increased in FL PB-FCs and decreased in FL BM-FCs. Supernatant from FL PB-FCs primed HSC migration to SDF-1, confirming production of the protein product. The FL PB-FCs contained a predominance of p-preDCs and natural killer (NK)-FCs, and NK-FCs were lacking in FL BM-FCs. The impaired function for BM-FCs was restored within 5 days after cessation of treatment. Taken together, these data suggest that FCs may enhance HSC homing and migration via the SDF-1/CXCR4 axis and adhesion molecule modulation. These findings may have implications in development of strategies for retaining function of ex vivo manipulated FCs and HSCs.

NK cells play a critical role in the regulation of class I-deficient hemopoietic stem cell engraftment: evidence for NK tolerance correlates with receptor editing.

The role that NK cells play in the rejection of hemopoietic stem cell (HSC) and tolerance induction has remained controversial. In this study, we examined whether NK cells play a direct role in the rejection of HSC. Purified HSC from MHC class II-deficient mice engrafted readily in congenic mice, while HSC from class I-deficient donors (beta(2)-microglobulin(-/-) (beta(2)m(-/-))) failed to engraft. Recipient mice lacking CD8(+), CD4(+), or T cells also rejected HSC from class I-deficient donors, pointing directly to NK cells as the effector in rejection of HSC. Recipients, deficient in or depleted of NK cells, engrafted readily with beta(2)m(-/-) HSC. Expression of the activating Ly-49D and inhibitory Ly-49G2 receptors on recipient NK cells was significantly decreased in these beta(2)m(-/-)-->B6 chimeras, and the proportion of donor NK cells expressing Ly-49D was also significantly decreased. Notably, beta(2)m(-/-) chimeras accepted beta(2)m(-/-) HSC in second transplants, demonstrating that NK cells in the chimeras had been tolerized to beta(2)m(-/-). Taken together, our data demonstrate that NK cells play a direct role in the regulation of HSC engraftment, and down-regulation and/or deletion of specific NK subsets in mixed chimeras can contribute to the induction of NK cell tolerance in vivo. Moreover, our data show that bone marrow-derived elements significantly contribute to NK cell development and tolerance.

Production of donor T cells is critical for induction of donor-specific tolerance and maintenance of chimerism.

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.

Facilitating cells as a venue to establish mixed chimerism and tolerance.

Graft rejection and the toxicity associated with the use of non-specific immunosuppression remain the major limitations in pediatric solid organ transplantation. The induction of tolerance in transplant recipients is an elusive but achievable goal that will decrease the dependence on immunosuppressive agents. BMT is associated with a robust form of donor-specific transplantation tolerance. It achieves a state of chimerism, defined as the presence of donor marrow cells in the recipient. The two major toxicities in conventional bone marrow transplantation that have prevented its clinical application to induce tolerance are the toxicity of ablative conditioning and GVHD. Two forms of chimerism exist: full chimerism and mixed chimerism. In full chimerism, the hematopoietic system of the recipient is replaced by that of the donor following ablative conditioning. Full chimerism is associated with a relatively impaired immunocompetence for primary immune responses and an increased risk of GVHD. In addition, the 7-10% regimen-related mortality associated with ablation could not be accepted in solid organ allograft recipients. In mixed chimerism the donor hematopoietic system co-exists with that of the recipient. Mixed chimerism induces donor-specific tolerance and is associated with superior immunocompetence and a relative resistance to GVHD compared with full chimerism. Moreover, it can be achieved with partial conditioning, thereby reducing the regimen-related morbidity associated with myeloablation. Approaches to establish mixed chimerism using non-myeloablative-conditioning regimens have been aggressively pursued over the past decade. Mixed chimerism can be safely established with minimal conditioning, resulting in a significant reduction in risk compared with ablative conditioning. GVHD is the final hurdle that has prevented the widespread application of chimerism to induce tolerance. Donor T cells are the primary effector cells for GVHD. Although T cell depletion of the donor marrow avoids GVHD, it results in an increase in the rate of graft failure in MHC-disparate recipients. The dichotomy between GVHD and T cell depletion graft failure has recently been dissociated by the discovery of CD8+/TCR- graft FC. Purified HSC engraft readily in syngeneic recipients but not in MHC-disparate allogeneic recipients. The addition of small numbers of facilitating cells permits durable HSC engraftment in allogeneic recipients and avoids GVHD. Using FC to promote HSC engraftment following non-myeloablative conditioning could be a promising approach to establish tolerance in solid organ transplantation. This invited review focuses on recent developments in stem cell chimerism and tolerance that could bring the use of this approach to induce tolerance to solid organ transplantation one step closer to reality.