Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

Preclinical Study for the Use of Abatacept to Prevent Rejection of Allogeneic CD34+ Cells in a Xenograft Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4271-4271
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Vitalyi Senyuk ◽  
Elisa Bonetti ◽  
Nadim Mahmud ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
B Cells ◽  
Xenograft Model ◽  
Costimulatory Blockade ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract The aims of this study were to analyze the role of T cells on the engraftment of allogeneic CD34+ cells after transplantation in immunodeficient nonobese diabetic/ltsz-scid/scid (NOD/SCID) IL2 receptor gamma chain knockout (NSG) mice and to test the in-vivo ability of abatacept (CTLA4-Ig) in preventing graft failure. Human CD34+ cells (0.2x106 cells/animal) were co- transplanted with allogeneic CD3+ T cells into sublethally irradiated NSG mice at ratios ranging from 1:50 to 1:0.5, or without T cells as a control. The engraftment of huCD45+ cell subsets in the bone marrow and spleen was measured by flow cytometry after 4-8 weeks. An expansion of T cells without engraftment of CD34+ cells was detected in each group of mice transplanted with CD34:T cells at ratios ≥ 1:0.5. To test whether T cells prevented any engraftment of CD34+ cells, or caused rejection after initial CD34+ cell homing in the bone marrow, kinetics experiments were performed by analyzing the marrow and spleen of mice at 1,2 or 4 weeks after transplant of CD34+ and T cells at 1:1 ratio. These experiments showed that at two weeks after transplant, CD34+ cells had repopulated the bone marrow but not the spleen, while T cells were found primarily in the spleen. Instead, in mice sacrificed at 4 weeks after transplant the marrow and the spleen contained only T cells and the CD34+ cells had been rejected. Based on our previous in-vitro studies showing that CD34+ cell immunogenicity is mainly dependent on B7:CD28 costimulatory signaling, we then tested whether costimulatory blockade with abatacept (CTLA4-Ig, Bristol Myers Squibb) would block stem cell rejection. Three groups of mice were transplanted with CD34+ and allo-T cells at 1:1 ratio and injected with Abatacept at 250 ug i.p. every other day from: a) day -1 to +28, b) day -1 to day +14 or c) day +14 to +28, then the animals were sacrificed at day +56 (8 weeks) after transplant to assess the engraftment. In Group a) the overall engraftment of huCD45+ cells was only <10%, but Abatacept completely prevented T cell-mediated stem cell rejection with >98% huCD45+ cells of myeloid or B cell lineage and <1% T cells in the marrow and spleen. In Group b) 70% of huCD45+ cells both in the marrow and spleen were T cells, and the remaining fraction of myeloid or B cells were derived from CD34+ cells. In Group c), instead, 100% of huCD45+ cells were T cells, with complete rejection of CD34+ cells. T cells recovered from the spleen of mice in groups b) and c) were also tested as responders in MLC stimulated with the original CD34+ cells and showed a brisk proliferation, consistent with lack of tolerance. Finally, another group of mice that received Abatacept from day -1 to + 28 was rechallenged with a boost of CD34+ cells on day +28 to test whether the low CD34+ cell engraftment was secondary to a latent rejection or partial stem cell exhaustion. The CD34+ cell boost resulted in a full hematopoietic recovery with 37% huCD45+CD3- cells, including myeloid and B cells, as well as CD34+ cells in the bone marrow and spleen. In this preclinical xenograft model we demonstrated that costimulatory blockade with Abatacept at the time of allogeneic transplant of human CD34+ cells can prevent T cell mediated rejection provide the basis for the future non-myeloablative protocols for incompatible stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

C75 Fatty Acid Synthesis (FAS) Inhibitor Has Potent Immunosuppressive Activity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2156-2156 ◽  
Author(s):  
Vitalyi Senyuk ◽  
Dolores Mahmud ◽  
Annie L. Oh ◽  
Pritesh R. Patel ◽  
Damiano Rondelli
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Fatty Acid ◽  
T Cell ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Fatty acid synthesis (FAS) or oxidation (FAO) are important regulatory pathways in immune response. In fact, FAS plays a pivotal role in antigen presentation and T cells activation and FAO leads to fatty acid degradation which has been previously shown to regulate hematopoietic stem cell maintenance. Here we hypothesized that FAS can be a new target to suppress T cell alloimmune responses in solid organ or stem cell transplantations. Therefore, we tested if the FAS inhibitor C75 could suppress T cell alloreactivity without impairing normal hematopoiesis. The immuno-suppressive (IS) effect of moderate FAS inhibition was demonstrated in mixed leukocyte cultures (MLC) where C75 at 10 mkg/ml significantly reduced T cell proliferation and prevented the expansion of CD3+CD25+ and CD3+CD69+ T cells. In T cells stimulated by alloantigen, C75 also induced the downregulation of NF-kB gene expression and the upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene involved in ubiquitination and degradation of NF-kB protein. When compared to other standard IS agents, such as anti-thymocyte globulin (ATG), Cyclosporine A, Rapamycin or inhibitor of FAO Etomoxir, C75 showed similar anti-T cell activity. The same dose of C75 (10 mkg/ml) did not cause apoptotic death of human CD34+ cells in vitro, nor affected CD34+ cell clonogenicity in vitro. In fact, C75 increased the number of BFU-E and CFU-GM colonies (P < 0.05). We observed that the expression of de novo DNA methyltrasferases DNMT3A and DNMT3B, which are important regulators of stem cell renewal, was strongly reduced in CD34+ cells co-cultured for 3 days with allogeneic T cells. On the contrary, in the presence of C75 the expression of DNMT3A and DNMT3B was not different from baseline control. To test the in-vivo effect of C75 we utilized a xenograft model of stem cell rejection where 2 x 105 human CD34+ cells and HLA incompatible T lymphocytes were injected in immunodeficient nonobese diabetic/ltsz-scid/scid - IL2 receptor gamma chain knockout (NSG) mice at 1:1 ratio. Four weeks after transplantation, control NSG mice showed complete rejection of huCD45+CD34+ cells and the expansion of T cells in the marrow and spleen. NSG mice treated with intra-peritoneum injections of C75 every 3 days for 2 weeks, instead, showed 10-15% human CD45+ myeloid cells in the marrow and spleen at week 4 after transplant, suggesting at least a partial effect on preventing rejection of incompatible stem cells. We showed here that moderate FAS inhibition may represent a novel immunosuppressive strategy and our findings will prompt preclinical investigations exploiting the effect of FAS inhibitors alone or in combination with standard IS agents in models of allogeneic transplantation or bone marrow failure. Disclosures No relevant conflicts of interest to declare.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor–negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1735-1738 ◽  
Author(s):  
Stephanie Bridenbaugh ◽  
Linda Kenins ◽  
Emilie Bouliong-Pillai ◽  
Christian P. Kalberer ◽  
Elena Shklovskaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
Cd8 Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8+ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8+ cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8α+CD3ϵ+ T-cell receptor– negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34+ cells. In vitro, cFCs cocultured with CD34+ cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8+CD3+TCR− cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

scholarly journals Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for the Amplification and Self-Renewal of Repopulating Stem Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3736-3749 ◽  
Author(s):  
Wanda Piacibello ◽  
Fiorella Sanavio ◽  
Antonella Severino ◽  
Alessandra Danè ◽  
Loretta Gammaitoni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Hematopoietic Cells ◽  
Scid Mice ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

Adoptive Transfer of In Vitro Generated T Cell Precursors Enhances Donor T Cell Reconstitution and Graft-Versus-Tumor Activity in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 63-63 ◽  
Author(s):  
Johannes L. Zakrzewski ◽  
Adam A. Kochman ◽  
Sidney X. Lu ◽  
Theis H. Terwey ◽  
Theo D. Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Hematopoietic Stem ◽  
T Cell Reconstitution

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of &gt;95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.

Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4295-4295
Author(s):  
Jae-Hung Shieh ◽  
Tsann-Long Su ◽  
Jason Shieh ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Culture System ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

Rapid and efficient homing of human CD34+CD38−/lowCXCR4+stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3283-3291 ◽  
Author(s):  
Orit Kollet ◽  
Asaf Spiegel ◽  
Amnon Peled ◽  
Isabelle Petit ◽  
Tamara Byk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Immunodeficient Mice ◽  
Human Cd34 ◽  
Kinase C ◽  
Cd34 Cells

Abstract Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34+CD38−/lowCXCR4+ cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34−CD38−/lowLin− cells were not detected. Moreover, whereas freshly isolated CD34+CD38+/high cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34+CD38+ cells. Homing of enriched human CD34+ cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34+CD38−/lowCXCR4+cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34+ cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Gαiproteins, inhibited SDF-1–mediated in vitro transwell migration but not adhesion or in vivo homing of CD34+ cells. Homing of human CD34+ cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34+CD38−/lowCXCR4+cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.

Regulatory T Cells Do Not Affect Human Hematopoietic Stem Cell Engraftment and Prevent T Cell Alloreactivity Against CD34+ Cells: A Preclinical Study.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4870-4870
Author(s):  
Dolores Mahmud ◽  
Sandeep Chunduri ◽  
Nadim Mahmud ◽  
Lennert Van Den Dries ◽  
John J. Maciejewski ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Regulatory T Cells ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Intracellular Expression

Abstract We have previously demonstrated that allogeneic blood T cells stimulate cord blood (CB) CD34+ cell differentiation into professional antigen presenting cells (APC) in-vitro and in-vivo (Abbasian J, Blood2006:108:203–208). In this study we immunomagnetically selected human CD4+CD25+ regulatory T cells (Tregs) and showed that >80% of these cells were positive for FoxP3 intracellular expression. Then we tested whether Tregs may affect CB CD34+ cell clonogenic activity in-vitro an in-vivo, and if co-incubation of Tregs and CD34+ cells may modify the phenotype and function of Tregs. A colony-forming cells (CFU-C) assay performed with CD34+ cells mixed with allogeneic Tregs at 1:2 ratio resulted in comparable numbers of Granulocyte- Macrophage CFU (CFU-GM), burst-forming unit-erythroid (BFU-E) and CFU-Mix as compared to cultures with CD34+ cells alone (p=0.2, p=0.5 and p=0.5, respectively)(n=3 exps). Human CD34+ cells were co-transplanted with human CD4+CD25+ allogeneic Tregs into NOD/SCID mice at 1:1 and 1:2 ratio. After 6 weeks mice marrow was harvested and showed a 1.3±1.1% (n=3 mice) and 1.6±0.8% (n=4 mice) engraftment of huCD45+ cells, respectively, which was comparable to the engraftment observed in control animals transplanted with CD34+ cells alone (1.4±0.4). In addition, among the engrafted huCD45+ cells similar proportion of CD33+ myeloid cells, CD14+ monocytes and CD1c+ dendritic cells were observed in the three groups of animals. Mixed lymphocyte culture (MLC) experiments showed that irradiated CD34+ cells stimulated brisk proliferative responses of CD4+CD25- cells (S:R=1:2), but did not induce any proliferation of Tregs (n=3 exps). After incubation with CD34+ cells in the presence of IL2, on average >80% CD4+CD25+ cells maintained the intracellular expression of FoxP3 and surface expression of CD62L and CD152 (n=3 exps). Then, Tregs autologous to CD34+ cells were isolated from the CB CD34- cell fraction while allogeneic Tregs were isolated from healthy individuals’ peripheral blood. When 2.5 x 104 autologous or allogeneic Tregs were added to an MLC with 2.5 x 104 irradiated CD34+ stimulator cells and allogeneic responders at 1:2 ratio, they suppressed T cell alloreactivity to CD34+ cells on average by 68±14% and 41±16%, respectively (n=3 exps). Our findings suggest that co-transplantation of CD34+ cells and autologous or allogeneic Tregs may allow normal stem cell engraftment while limiting T cell alloreactivity. These results will prompt the design of new strategies in allogeneic hematopoietic stem cell transplantation, particularly in an HLA incompatible setting.

An in-Vivo Model of T Cell-Mediated Rejection of Human Hematopoietic CD34+ Stem Cells Using NOD/SCID γnull (NOG) Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4474-4474
Author(s):  
Benedetta Nicolini ◽  
Dolores Mahmud ◽  
Nadim Mahmud ◽  
Giuseppina Nucifora ◽  
Damiano Rondelli
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Mouse Model ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Abstract 4474 We have previously demonstrated that human CD34+ cells include subsets of antigen presenting cells capable of stimulating anti-stem cell T cell alloreactivity in-vitro. In this study we transplanted human CD34+ cells and allogeneic T cells in a NOD/SCID γnull (NOG) mouse model and evaluated the occurrence of stem cell rejection as well as xenogeneic graft-versus-host disease (GVHD) following the infusion of different doses of T cells. After sublethal irradiation NOG mice were cotransplanted with 2×105 CD34+ cells and HLA mismatched CD4+CD25- T cells at 1:0 (control), 1:2 or 1:10 CD34+ cell: T cell ratio (n=5-10 mice per group). Hematopoietic stem cell and T cell engraftment was assessed in the bone marrow and in the spleen 6 weeks following transplantation or earlier in case the animals died. Control mice transplanted with CD34+ cells alone showed a high level of stem cell engraftment (huCD45+ cells: 60±10%) in the bone marrow, encompassing CD19+ B cells (64±4%), CD34+ cells (18±1%), CD33+ myeloid cells (7±1%), CD14+ monocytes (3±1%), and no T cells within huCD45+ cells. In contrast, mice that were transplanted with CD34+ cells and 4×105 (1:2 ratio) or 2×106 (1:10 ratio) T cells had only 9±2% and 3±1% huCD45+ cells, respectively, in the bone marrow (p=0.01). Moreover, marrow samples of mice cotransplanted with CD34+ cells and T cells at 1:2 or 1:10 ratio included >98% huCD3+ T cells and no CD34+ cells. Spleen engraftment of huCD45+ cells was lower (25±8%) in control mice (1:0 ratio) as compared to 66±10% and 36±11% in 1:2 and 1:10 groups, respectively (p=0.05). As observed in the marrow, also the spleen of animals receiving CD34+ and T cells included >98% CD3+ T cells. Among the T cells, both in the marrow and in the spleen of mice in the 1:2 and 1:10 ratio groups, 60-70% were CD4+CD8- cells, 22-25% CD8+CD4- cells, 1-3% CD56+ cells, and 2-5% CD4+CD25+ cells. In mice receiving 4 ×105 T cells (1:2 ratio), on average 12±6% of the T cells in the bone marrow and spleen were CD4+CD8+. Only mice receiving 2×106 T cells (1:10 ratio) showed GVHD. This was demonstrated by fur changes, reduced survival (p=0.02) and weight loss (p=0.0001) compared to control mice or mice receiving a lower dose of T cells (1:2 ratio). The marrow engraftment of CD3+ cells with disappearance of CD34+ cells in mice receiving low doses of allogeneic T cells, in the absence of evident xenogeneic GVHD, suggests that NOG mouse model represents a useful tool to study human stem cell rejection. This model will be also utilized to investigate new strategies of immunosuppressive cell therapy applied to stem cell transplantation in an HLA mismatched setting. Disclosures: No relevant conflicts of interest to declare.

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