A comparison of murine T-cell–depleted adult bone marrow and full-term fetal blood cells in hematopoietic engraftment and immune reconstitution

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 364-371 ◽  
Author(s):  
Benny J. Chen ◽  
Xiuyu Cui ◽  
Gregory D. Sempowski ◽  
Maria E. Gooding ◽  
Congxiao Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Blood Cells ◽  
Immune Reconstitution ◽  
Full Term ◽  
Fetal Blood ◽  
Adult Bone

Umbilical cord blood has been increasingly used as a source of hematopoietic stem cells. A major area of concern for the use of cord blood transplantation is the delay in myeloid and lymphoid recovery. To directly compare myeloid and lymphoid recovery using an animal model of bone marrow and cord blood as sources of stem cells, hematopoietic engraftment and immune recovery were studied following infusion of T-cell–depleted adult bone marrow or full-term fetal blood cells, as a model of cord blood in a murine allogeneic transplantation model (C57BL/6 [H-2b] → BALB/c [H-2d]). Allogeneic full-term fetal blood has poorer radioprotective capacity but greater long-term engraftment potential on a cell-to-cell basis compared with T-cell–depleted bone marrow. Allogeneic full-term fetal blood recipients had decreased absolute numbers of T, B, and dendritic cells compared with bone marrow recipients. Splenic T cells in allogeneic full-term fetal blood recipients proliferated poorly, were unable to generate cytotoxic effectors against third-party alloantigens in vitro, and failed to generate alloantigen-specific cytotoxic antibodies in vivo. In addition, reconstituting T cells in fetal blood recipients had decreased mouse T-cell receptorδ single-joint excision circles compared with bone marrow recipients. At a per-cell level, B cells from fetal blood recipients did not proliferate as well as those found in bone marrow recipients. These results suggest that full-term fetal blood can engraft allogeneic hosts across the major histocompatibility barrier with slower hematopoietic engraftment and impaired immune reconstitution.

Enhancement of T Cell Development in Murine Bone Marrow Transplant Recipients by Transfer of the IL-7 or SCF Gene into Marrow-Derived Mesenchymal Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3572-3572
Author(s):  
Brile Chung ◽  
Dullei Min ◽  
Mark Krampf ◽  
Won Jong Ju ◽  
Kenneth I. Weinberg
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
T Cell Development ◽  
Cell Development ◽  
Allogeneic Bmt

Abstract The ability of the thymus to generate T cells diminishes with increasing age, the use of chemotherapy, bone marrow transplantation (BMT), anti-retroviral therapy for HIV, and graft-versus-host disease (GVHD) which can lead to a major clinical problem. Therefore, developing a clinically relevant strategy for the rapid development of T lymphocytes is crucial for treating immune deficiency. Stem cell factor (SCF: also known as kit ligand) and interleukin-7 (IL-7) are stroma–derived cytokines that induce proliferation, differentiation, and survival of developing immature T cells in the thymus. Studies have shown that administration of recombinant human IL-7 following murine BMT resulted in improved thymopoiesis and immune function. However, our previous studies have shown that that IL-7 treatment post-HSCT to enhance immune reconstitution in the allogeneic setting may have adverse effects because of the dual role of IL-7 in supporting both thymopoiesis and mature T lymphocyte expansion. Therefore it raises the question of whether IL-7 treatment after allogeneic BMT will increase the frequency or severity of GVHD. The purpose of this study was to examine whether: administration of IL-7 and SCF with infusion of mature T cell depleted (TCD) BM cells can induce enhancement of donor-derived immune reconstitution more rapidly than treatment with either cytokine alone and whether IL-7 and SCF are synergistic and partially complementary signals for the proliferation, survival, and differentiation of immature T cells. To evaluate the combinatory effect of IL-7 and SCF in T cell development following BMT, we developed a gene therapy approach using retrovirally-mediated transduction of BM-derived mesenchymal stem cells (MSC) with the human IL-7 or murine SCF gene (soluble isoform). C57BL/6J (CD45.2) recipient mice were irradiated (1300 cGy) and co-transplanted with 1 × 10 6 T cell depleted (TCD) bone marrow cells from congenic donor B6.SJL mice (CD45.1) and different doses (0.1 × 10 6 or 0.3 × 10 6) of eGFP (control), IL-7, SCF, or combination of IL-7 and SCF MSC. At day 30 following BMT, we observed that transplantation of both IL-7 and SCF MSC resulted in significantly higher numbers of donor-derived thymocytes and peripheral lymphocytes than either IL-7 or SCF MSC transplantation alone. Most noticeably, the number of donor-derived immature and mature T cells recovered from the animals receiving transplantation of 0.1 × 10 6 IL-7 MSC and 0.3 × 10 6 SCF MSC was similar to that of animals receiving 0.3 × 10 6 IL-7 MSC alone, demonstrating that the reduced proliferative signals produced by 0.1 × 10 6 IL-7 MSC can be compensated by co-transplantation of 0.3 × 10 6 SCF MSC. Moreover, transplantation of IL-7 and SCF MSC significantly increased the number of donor-derived common lymphoid progenitors (CLP [Lin-, Sca-1 low, Thy1-, c-Kit low, IL-7R+]) in the BM, suggesting that transplanted CLPs are induced to differentiate or expand more rapidly in response to IL-7 and SCF and may have contributed to increased immune reconstitution. Collectively, our findings demonstrate that IL-7 and SCF gene therapy may be a therapeutically useful method to promote enhancement of T cell development in de novo. Furthermore, the experiments resulted in important knowledge about complementary signals provided between IL-7 and SCF, and suggest various doses of IL-7 and SCF therapy may enhance development of T cells with limited expansion of mature T cells responsible for causing GVHD in allogeneic BMT setting.

Hematopoietic Reconstitution of Mice Cord Blood Transplantation Combined with Bone Marrow Transient Hematopoietic Progenitor Cell

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4688-4688
Author(s):  
Jun Shi ◽  
Kazuma Ikeda ◽  
Maeda Yosinobu ◽  
Yinghua Yuan ◽  
Yehua Yu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Lymphocytes ◽  
Cord Blood ◽  
Progenitor Cells ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Hematopoietic Progenitor Cell ◽  
Fetal Blood

Abstract Abstract 4688 Umbilical cord blood has been increasingly used as a source of hematopoietic stem cells. However, fetal blood recipients had slower hematopoietic engraftment and impaired immune reconstitution. To accelerate myeloid and lymphoid recovery, we used an animal model of newborn blood modeled for cord blood (CB), along with transiently reconstituting progenitor cells from congenic bone marrow with recipients, as sources of stem cells. According to the previous reports that murine transiently reconstituting progenitor cells express the c-kit molecule, but not Sca-1 and lymphohematopoietic lineage markers, Lin-sca-1-c-kit+(c-kit+) were isolated by MACS method. c-kit+ cells population consisted of exclusively of medium- or large-sized blast-like cells, which displayed relatively low proliferative potential in vitro than Lin-sca-1+ (sca-1+) population. After transplantation of CB from DBA/2 mice (H-2d/d, CD45.2), with or without graded numbers of either c-kit+ or sca-1+ cells isolated from BDF1 mice (H-2d/b, CD45.1) bone marrow into lethally irradiated CD45.2 congenic BDF1 mice, hematopoietic engraftment were dynamic investigated. The intermingled transplantation of CB and c-kit+ cells or sca-1+ cells at the dosages of 1×104 or 2.5×104 or 5×104 to recipient mice leads to the quantity of white blood cells and platelets increased to 1×109/L and 1×1012/L at day12, whereas the injection of CB alone resulted in day17. By 2 weeks post-transplantation, congenic BM-derived cells were dominantly found in granulocytes and B lymphocytes, while host cells were dominantly found in T lymphocytes in CB transplantation combined either with c-kit+cells or sca-1+ cells. In cotransplantation with CB and c-kit+cells – engrafted surviving mice, the degree of donor CB cells in the peripheral blood increased progressively over time, while congenic donor BM-derived cells decreased gradually. After 60 weeks cotransplantation with CB and c-kit+ cells, a complete chimerism frequency of CB–derived cells continued to maintain in granulocytes and B lymphocytes, while T lymphocytes were dominantly derived from CB. On the other hand, congenic bone marrow or host-derived cells were the dominant population and CB-derived cells in the peripheral blood were less than 10% after 60 weeks cotransplantation with CB and sca-1+cells. In conclusion, the cotransplantation of CB and congenic c-kit+ cells was able to accelerate early hematopoietic recovery due to congenic marrow cells. But complete or main chimerism of cord blood was formed without or with fewer residual cells of host origin and congenic BM origin in long-term multilineage reconstitution. Thus, this cotransplant model in vivo may be to bring useful information for improving hematopoietic and immune reconstitution in fetal blood recipients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Engineering Regenerative Thymic Tissues to Restore Long-Term T Cell Lymphopoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5092-5092
Author(s):  
Hui Gai ◽  
Rafa Gras~Pena ◽  
Yogendra Verma ◽  
Vincent Fateh ◽  
Kazuya Ikeda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Immune Reconstitution ◽  
Thymic Epithelial Cells ◽  
Tcr Repertoire ◽  
Thymic Stromal Cells

Abstract The thymus is a primary lymphoid organ that plays a critical role in the development of adaptive T cell immunity and central tolerance. Bone marrow-derived lymphoid progenitor cells migrate into the thymus and interact with thymic epithelial cells (TECs) through sequential positive and negative selection to mature. Thymus-educated mature T cells express a diverse, MHC-restricted and self-tolerant T cell receptor (TCR) repertoire that protects against infection and prevents autoimmunity. Patients born with congenital thymic aplasia, due to 22q11 Deletion Syndrome, or mutations in TBX1, FOXN1 or CHD7, present with complete absence of T cells and a severe combined immunodeficiency (SCID)-like phenotype. Bone marrow transplantation does not cure the thymic defect in these patients and severe infections occur within the first year of life if left untreated. Allogenic thymus transplantation has provided proof of principle that HLA-unmatched pediatric donor thymic tissues can lead to successful immune reconstitution with the emergence of a diverse TCR V-beta repertoire. However, post-transplant organ-specific autoimmunity remains a major concern. Currently allogeneic thymus transplantation is no longer available in the US leaving a deadly therapeutic void for patients born without thymus. Patient-specific or histocompatible thymic tissues derived from pluripotent stem cells could address the critically unmet need, and also a broader range of clinical applications including immune reconstitution post hematopoietic stem cell transplantation (HSCT) and tolerance induction for solid donor organs. The thymus contains two major non-hematological components: the thymic stromal cells and the extracellular matrix (ECM). The thymic stromal layer is composed of thymic epithelial cells and mesenchymal cells. The thymic ECM forms a three-dimensional (3D) network to provide physical support and nutrition to thymic stromal cells. Methods: To address the need for histocompatible regenerative thymic tissues, we aim to differentiate fully functional thymic epithelial progenitor cells (TEPCs) from human pluripotent stem cells (hPSCs) and further generate 3D transplantable organoids using engineered matrix proteins that mimic the native thymic microenvironment. Results: We have developed a novel platform to generate hPSC-derived TEPCs by dissecting the key signaling pathways that govern human thymic ontogeny. These hPSC-derived TEPCs express the defining markers of TEPC-fate, such as FoxN1, Cytokeratin 8, Cytokeratin 5, Delta-like Canonical Notch Ligand 4 (DLL4) and MHC class II. Previous studies have shown FoxN1 to be the master regulator controlling thymic development, however, little is known about its regulatory network. Elucidating and validating the factors that initiate and maintain FoxN1 expression is the key to successfully engineer sustainable thymic tissues. We have identified a combination of morphogens that can maintain the expression of FoxN1, DLL4 and AIRE of primary TECs in culture. To gain insight into the composition of primary thymic ECM proteins and adapt their characteristics beyond the features of commercially available 3D hydrogels, we analyzed a series of human fetal thymic tissues using whole transcriptome analysis. Our current work focuses on adapting our 2D culture protocol to sustain hPSC-TEPCs in 3D matrix-based organoids. Ongoing studies test the capacity of hPSC-TECPs to promote T cell maturation and the development of a diverse TCR repertoire in an athymic xenograft mouse model (NSG-FoxN1null). Conclusions: hPSC can be differentiated in vitro into TEPC-fate and developed into thymic organoids using custom-designed protein matrices. Studies to test sustainability and functionality of the engineered thymic organoids in vivo are currently under way. Disclosures No relevant conflicts of interest to declare.

scholarly journals Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3360-3362 ◽  
Author(s):  
Karen M. Pastos ◽  
William B. Slayton ◽  
Lisa M. Rimsza ◽  
Linda Young ◽  
Martha C. Sola-Visner
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Conditioned Media ◽  
Valuable Source ◽  
Cd34 Cells ◽  
Adult Bone

Abstract Umbilical cord blood (CB) is a valuable source of stem cells for transplantation, but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)–derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this, we cultured CB- and PB-CD34+ cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo, and the addition of CM inhibited their maturation. In contrast, CB-MKs reached highest ploidy in CM without rTpo, and high rTpo concentrations (> 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines.

Donor DC Subsets Polarize Donor T-Cell Immune Responses in Allogeneic BMT.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 573-573
Author(s):  
Jian-Ming Li ◽  
Cynthia Giver ◽  
Doug McMillan ◽  
Wayne Harris ◽  
David L. Jaye ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Hematopoietic Cell ◽  
Post Transplant ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Introduction: Impaired or inappropriate immune reconstitution after allogeneic bone marrow transplantation (BMT) can lead to infection, graft-versus-host disease (GvHD) and leukemia relapse. We have previously reported that BM contains two populations of dendritic cell (DC) subsets, CD11b+ DC and CD11b− DC, and that CD11b depleted donor BM promoted increased donor T-cell chimerism and increased graft-versus-leukemia (GvL) activity in C57BL/6 → B10BR transplants [BBMT, 2004, 10: 540]. To explore the mechanism by which CD11b-depletion improved allo-reactivity, we performed allogeneic hematopoietic cell transplants using defined populations of donor stem cells, DCs, and T-cells in a MHC mis-matched BMT model. Methods: We transplanted FACS purified populations of 50,000 GFP+ CD11b- DC or CD11b+ DC in combination with 5,000 FACS purified Lin- Sca-1+ c-kit+ hematopoietic stem cells (HSC) and 300,000 or 1,000,000 congenic spleen T-cells from C57BL/6 donors into C57BL/6[H-2Kb], B10BR[H-2Kk] and PL/J[H-2Ku] recipients. Proliferation of CFSE stained donor T-cells was measured at 72 hours post-transplant. FACS cytometric bead array and intracellular cytokine staining measured serum and intracellular cytokines in donor T-cells. Results: The initial proliferation and Ki-67 expression of CFSE labeled donor T-cells in allogeneic recipients were much higher than in syngeneic recipients (homeostatic proliferation). Confocal microscopy showed co-localization of donor DC subsets with donor T-cells in the recipient spleens at 3 and 10 days post-transplant. In the allogeneic transplant settings, donor T-cells co-transplanted with CD11b- DC showed increased IFN-γ synthesis at 3 and 10 days post-transplant compared to donor T-cells co-transplanted with HSC plus CD11b+ DC or HSC alone. Increased proliferation of donor T-cells led to increased donor T-cell chimerism at day 10, 30, 60, and day105 post-transplant among recipients of CD11b- DC compared to recipients of HSC alone or HSC plus CD11b+ DC (Figure 1). Transplantation of spleen T-cells and CD11b- DC did not increase GvHD, but was associated with full donor chimerism. In contrast, transplantation of allogeneic CD11b+ DC led to persistence and expansion of residual host T-cells (Figure 2), increased numbers of donor CD4+CD25++Foxp3+ T-cells, and higher serum level of IL-10 supporting early post-transplant expansion of donor T regulatory cells (Treg). Conclusions: Donor CD11b- DC promoted immune reconstitution by polarizing donor T-cells to Th1 immune responses associated with increased IFN-γ synthesis and donor T-cell proliferation, while donor CD11b+ DC suppressed immune reconstitution by inhibiting donor T-cell allogeneic immune responses. These data support a novel paradigm for the regulation of post-transplant immunity and suggest clinical methods to test the hypothesis that manipulation of the DC content of a hematopoietic cell allograft regulates post transplant immunity in the clinical setting. Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells]

Immunosupressive Property of Mesenchymal Stem Cells (MSCs) in Mixed Lymphocyte Reactions In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4313-4313 ◽  
Author(s):  
Jianyong Li ◽  
Lijuan Meng ◽  
Yu Zhu ◽  
Hua Lu ◽  
Changgeng Ruan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
T Cell Subsets ◽  
Bone Marrow Mscs ◽  
Human Bone Marrow Mscs ◽  
Mixed Lymphocyte Reactions ◽  
Activation Phase

Abstract Meesnchymal stem cells (MSCs) were successfully used in the prevention and treatment of graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. To further explore the immunosuppressive property of human bone marrow (MSCs) in alloantigen-induced mixed lymphocyte reactions (MLRs) in vitro, human bone marrow MSCs and lymphocytes were prepared from healthy volunteers. MSCs were expanded in vitro in Mesencult serum free media. MSCs were cocultured with one-way MLRs and bidirectional MLRs, responder cells were labeled with carboxyfluorescein diacetate- succinimidyl ester (CFSE) in bidirectional MLRs. Cell Counting Kit-8(CCK-8)kit was used in cell proliferation detection, T-cell subsets were analyzed by flow cytometry (FCM). The results showed that MSCs were positive for CD105, CD73, CD13, CD90 and were negative for hematopoietic cell markers. In one-way MLRs, MSCs down-regulated alloantigen-induced lymphocyte expansion in a dose-dependent and MHC-independend manner. In two-way MLRs, MSCs suppressed proliferation of CFSE positive cells. T cell subsets were changed: Th2 and Tc2 were down-regulated. Th2 was reduced from 1.70% to 0.65%, and Tc2 reduced from 1.10% to 0.47%, while Th1 and Tc1 were unaffected. T cells that became CD69+, which was an early activation marker, were significantly up-regulated from 7.14% to 26.12% and CD4+CD25+T regulatory cells (CD4+CD25+Tr) were up-regulated from 4.04% to 6.19%, which indicating that suppression did not interfere with activation phase of T cells and might be mediated by CD4+CD25+Tr partly. We conclede that MSCs down-regulated alloantigen-induced lymphocyte expansion. The immunosupressive effect might involve in post-activation phase of T cells. CD4+CD25+Tr might contribute to the suppressory activity of MSCs.

Mesenchymal Stem Cells from the Wharton’s Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3650-3650
Author(s):  
Kent W. Christopherson ◽  
Tiki Bakhshi ◽  
Shamanique Bodie ◽  
Shannon Kidd ◽  
Ryan Zabriskie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Cord Blood Cells

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

Pure Hematopoietic Stem Cells Reconstitute Immunity in Allogeneic Hosts Superior to Bone Marrow: Immunosuppression by Subclinical Graft-Versus-Host Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4869-4869
Author(s):  
Gabriel J. Tsao ◽  
Jessica A. Allen ◽  
Kathryn Logronio ◽  
Laura C. Lazzeroni ◽  
Judith A. Shizuru
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Graft Versus Host Disease ◽  
Immune Reconstitution ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Allele Specific

Abstract Antigen specific immune responses are known to be impaired following allogeneic hematopoietic cell transplantation (HCT). Some clinical studies suggest that graft T cell depletion for the prevention of graft-versus-host disease (GVHD) leads to poorer immune recovery, while other correlate GVHD with delayed immune reconstitution. Our studies sought to examine the degree to which the co-transplantation of GVHD-inducing mature cells mediated protective immunity post-HCT. We compared the transplant of FACS purified hematopoietic stem cells (HSC: cKit+Thy1.1loSca+Lin-) with bone marrow (BM) between congenic (BA to B6Ly5.2), minor-antigen mismatched (BA to BALB.B), haploidentical (BAxSWR F1 to BALB/cxSWR F1) and MHC-mismatched (BA to BALB/c) donor and host pairs. We show that grafts composed solely of purified HSC give uniformly superior lymphoid reconstitution across all mismatched pairs, both qualitatively and quantitatively. Although absolute blood lymphocytes counts were increased in recipients of BM compared to HSC, lymphoid reconstitution as measured by lymph node size, counts and architecture was significantly improved in the HSC groups regardless of minor or major mismatches between donor and host. Proliferative responses to the allele specific peptides of the antigen hen egg lysozyme were also significantly increased in the HSC as compared BM recipients (p=0.028), with fully MHC mismatched BM recipient cells showing almost no proliferative response. The use of MHC allele specific antigens also revealed that T cell responses post-HCT are dominated by donor-restricted elements. These data suggest that subclinical GVHD mediated by mature cells in the donor BM result in impaired immune reconstitution. While there may be an increase in absolute lymphocyte numbers, this increase does not correlate with an increase in immune cell function. These findings provide important insight into the benefits of purified HSC for preventing post-HCT infectious complication in addition to its known GVHD prophylaxis benefits. Our data highlights the benefit of transplanting a pure HSC population, as the relatively small number of T cells that are found in mouse bone marrow or that remain in the graft after T cell depletion in humans can still result in immune impairment due to subclinical GVHD.

FTY720 Promotes Engraftment of Allogeneic Donor Bone Marrow Stem Cells but Impairs Thymopoiesis Resulting in Delayed Graft Rejection.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3530-3530
Author(s):  
Patricia A Taylor ◽  
Ryan M Kelly ◽  
Michael J Ehrhardt ◽  
Bruce R. Blazar
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Graft Rejection ◽  
Absolute Number ◽  
Cell Lineages ◽  
Single Positive ◽  
High Level

Abstract Abstract 3530 Poster Board III-467 FTY720 (FTY), a sphingosine-1-phosphate receptor agonist, inhibits lymphocyte egress from lymphoid tissues although the complete mechanism of its immunomodulatory effects is not fully understood. We previously published that FTY inhibited but did not prevent graft-versus-host disease by multiple mechanisms. Using the same dose and schedule (3 mg/kg orally d0-28) we evaluated FTY for its effect on allogeneic bone marrow (BM) engraftment in sublethally-irradiated mice. C57BL/6 mice were irradiated with 5.0 Gy total body irradiation (TBI) on day -1, and received 107 T-cell depleted BALB/c BM cells on day 0. At 5 wks, FTY-treated mice had a mean 84% ± 4% (mean ± SEM, n=47) donor chimerism in peripheral blood leukocytes (PBL) versus 5% ± 2% in water-treated controls (n=38, p<0.001). However, engraftment promotion was transient in most mice. PBL phenotyping at 3 months revealed that mean donor chimerism decreased to 22% ± 6%. Of the 32 mice that were >90% donor at 5 wks, only 6 were >50% donor at 3 months indicating that even high level donor chimeras were subject to delayed graft rejection. We found that although FTY promoted robust donor engraftment in the NK, myeloid and B cell lineages in BM, spleen, and lymph nodes by the first week after transplantation, thymopoiesis was severely impaired at 1 month resulting in near absent donor (and also host) thymic T cell production. FTY-treated mice had very low thymocyte cellularity (<7×106, n=10). Most thymocytes (65-85%) were host CD4 or CD8 single positive T cells. We hypothesized that upon cessation of FTY, which prevents thymocyte egress, the mature host single positive T cells were released into the periphery and mediated delayed graft rejection. Consistent with this hypothesis, the in vivo depletion of host T cells but not host NK cells, at the time of cessation of FTY treatment, abrogated the loss of the donor graft indicating that host T cells were responsible for delayed graft rejection. Also consistent with our hypothesis, and demonstrating the immune competence of the host T cells retained in the thymus, the adoptive transfer of thymocytes from FTY-treated engrafted mice into lethally-irradiated C57BL/6 recipients mediated donor BALB/c BM rejection. To further examine the mechanism of early and robust albeit transient engraftment promotion in some cell lineages, but near absent thymopoiesis, we evaluated the absolute number of donor lin−Sca-1+cKit+ stem cells in the BM at 1 month. For these experiments, an engrafted control was deemed to be a more useful comparator than water-treated mice that rejected their graft. To ensure an engrafted control using the same TBI and allogeneic cell dose parameters, control mice were given peri-transplant injections of anti-CD4 and anti-CD8, a strategy that depletes host T cells and results in durable high level donor chimeras. Consistent with reports that FTY supports migration and bone marrow homing of stem cells, FTY-treated mice had a 4.9-fold increase in the absolute number of donor lin−Sca-1+cKit+ stem cells in the BM compartment compared to anti-CD4/8-treated mice. We hypothesized that the lack of donor thymopoiesis was the result of common lymphoid progenitors being trapped in the BM compartment and unable to migrate to and/or enter the thymus. Consistent with this hypothesis, FTY-treated mice had 125-fold fewer donor-type linlocKithiCD25− early thymic progenitors (ETPs) compared to anti-CD4/8-treated control mice. In contrast to FTY-treated mice, anti-CD4/8-treated mice had evidence of vigorous donor thymopoiesis. Collectively these data indicate that although FTY supports donor stem cell migration/homing to the BM and early donor NK, myeloid and B cell engraftment, the block in donor thymopoiesis and retention of thymic host T cells result in only transient engraftment in most sublethally-irradiated mice. These data have important implications in the use of FTY in BMT and further warrant examination of thymopoiesis in patients receiving FTY for immune suppression. Disclosures: No relevant conflicts of interest to declare.

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