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.

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.

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 In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4040-4048 ◽  
Author(s):  
M Rosenzweig ◽  
DF Marks ◽  
H Zhu ◽  
D Hempel ◽  
KG Mansfield ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
T Cell Differentiation ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

Engraftment of CLL-Derived T Cells in NSG Mice Is Feasible, Can Support CLL Cell Proliferation, and Eliminates the Need for Third Party Antigen Presenting Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 975-975 ◽  
Author(s):  
Piers EM Patten ◽  
Shih-Shih Chen ◽  
Davide Bagnara ◽  
Rita Simone ◽  
Sonia Marsilio ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Third Party ◽  
Cd4 Cells ◽  
Cd8 Cells ◽  
Nsg Mice ◽  
Cell Engraftment ◽  
Cfse Dilution ◽  
Cd34 Cells

Abstract Abstract 975 We recently described a xenograft model of chronic lymphocytic leukemia (CLL) using NOD/SCID/γcnull (NSG) mice. Adoptive transfer of primary patient PBMCs into these mice results in engraftment and proliferation of CLL cells if autologous activated T cells are present. To date, such CLL-derived T cell proliferation has been achieved by co-transfer of third party antigen presenting cells (APCs). Unfortunately, in this setting, mice succumb to graft-versus-host disease, due to complex interactions between CLL cells, alloAPCs, T cells, and the xenogeneic host. We hypothesized that alternative strategies of autologous T cell activation might refine the model, ultimately providing longer CLL cell engraftment and animal survival. We describe 3 approaches to achieving engraftment and activation of CLL-derived T cells that support B cell growth in vivo. First, we activated CLL CD3+ cells isolated from PBMCs of 4 patients with anti-CD3/28 beads for 72 hours in vitro. Cells were then mixed with CFSE-labeled PBMCs from the same patient at varying ratios (1:50 to 1:1000 CD3+ cells:CLL PBMCs) and injected intraorbitally (io) into a total of 17 mice. CD4, CD8 and CD19 cell engraftment, identified by a human CD45 lymphocyte gate (hCD45), and proliferation, assessed by CFSE dilution of labeled cells, were monitored weekly. All mice demonstrated detectable CD3+ and CD5+CD19+ cells from week (wk) 1. The percent (%) CD3+ cells, as a proportion of hCD45, increased weekly in all mice receiving anti-CD3/28-activated cells. While overall % CD5+CD19+ cells decreased weekly, the % proliferating increased and strongly correlated with increasing % of T cells (r2=0.7799, p<0.0001, 45 evaluable data pairs). At the time of reporting (up to 5 wks follow up), 4 animals have died. Death correlated with high % circulating CD8+ (mean 57.7% vs. 14.2% prior to death, p<0.0001), but not CD4+ cells (mean 31.0% vs 36.3%, p=n/s). Spleen immunohistochemistry analyses revealed follicles containing CD20+ CLL cells, based on L chain restriction and RT-PCR for the leukemic IGHV-D-J. Follicles were infiltrated with both CD4+ and CD8+ cells. These findings were the same as found with our published model. Our second approach involved engraftment of solely CLL-derived peripheral blood T cells, without associated autologous PBMCs or alloAPCs. Positively selected CD3+ populations from 2 patients were activated with anti-CD3/28 beads, expanded in vitro, and 5–10×106 such cells injected into mice io. Cells from both patients yielded engraftment of CD4+ and CD8+ cells by wk 2. We then utilized two mice engrafted with cells from one patient, 1 with 70% and 1 with 30% CD4+ cells to compare the effect of CD4+ T cells in CLL cell growth. Injection of CFSE-labeled PBMCs into the mouse with high % of CD4 cells showed a clear circulating CFSE+CD5+CD19+ population at wk 1. By wk 2, 95% of the leukemic B cells had divided with 60% showing ≥6 divisions. In the mouse with low % CD4, CD5+CD19+ cell engraftment was suboptimal, never being more than 100 detectable events in analysis from any single bleed. No clear CFSE dilution pattern was apparent. Finally, we have assessed the development of CLL-derived T cells from autologous bone marrow CD34+ cells of 4 CLL cases. 48hr old neonatal NSG mice received CD34+ cells (≤1×106) intrahepatically. In 1 of the 4 animals, CD19+ cells emerged at 2 months but by month 4 the predominant hCD45+ population was CD3+ (60% of total hCD45). Subsequent io injection of CFSE-labeled PBMCs from the same subject demonstrated engraftment and immediate proliferation (detected by wk1 following injection) of both the io transferred CD3+ and CD5+CD19+ populations. In conclusion, we demonstrate that engraftment and expansion of CLL-derived T cells is feasible in NSG mice both as in vitro activated cells or as descendants of CD34+ cells and that the presence of CLL cells and third party APCs is not required. Such T cells are able to support engraftment and proliferation of CLL B cells from the same CLL subject. Strategies to reduce excessive numbers of T-cell subsets which may prevent early death of animals are underway. Elimination of third party APCs will permit easier dissection of the pathophysiology within this model, allowing for a better understanding of CLL cell kinetics and mechanism of action of novel therapeutics to treat this disease. Disclosures: No relevant conflicts of interest to declare.

Enhanced Myeloid and T Cell Development and Improved Functionality of Cord Blood Xenografts In the NSGS Mouse.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3729-3729
Author(s):  
Mark Wunderlich ◽  
Pietro Presicce ◽  
Fu-Sheng Chou ◽  
Claire Chougnet ◽  
Julio Aliberti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
B Cell ◽  
Human Cells ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Human T Cells ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Abstract 3729 The advent of NOD/SCID mouse strains lacking interleukin-2 receptor gamma (IL2RG) function (NSG and NOG mice) has greatly improved efforts to develop xenograft models of human hematopoiesis. Notably, these mice exhibit markedly reduced adaptive and innate immunity and are well suited for stable engraftment of human CD34+ cells. Importantly, these mice also allow for the development of human T cells, setting them apart from other immunodeficient strains. As a result, NSG and NOG mice are increasingly used for the building of model systems to study HIV infection, graft versus host disease, and immunity. While significant improvements have been realized, IL2RG knockouts lack robust myeloid components, and lymphoid function is likely to be suboptimal due to problems with B cell differentiation defects and delayed appearance of T cells. Recently, we have generated an NSG mouse strain with transgenic expression of several human myelo-supportive cytokines (SCF, GM-CSF, and IL-3), the NSGS mouse. In our initial characterization and study of this novel strain, we found a significant improvement in engraftment of AML cell lines and patient samples relative to NSG mice. In the current study we have extended these findings to include xenografts obtained using umbilical cord blood CD34+ cells (UCB). We have found the NSGS mouse to be equal to the NSG as a host for long-term stable engraftment of these cells when saturating cells doses are used, and superior when limiting numbers of CD34+ cells are injected. While CD34+ levels are much lower in established grafts in primary NSGS recipients compared to NSG mice, possibly as a result of continual mobilization of these cells by the cytokines, human cells were readily detected in secondary NSGS recipients, indicating maintenance of a primitive stem/progenitor cell compartment in vivo. Bone marrow of NSGS mice was predominantly composed of human myeloid cells, while the NSG mice show primarily CD19+ B cells. In contrast, the lineage of the human cells in the peripheral blood were very similar in these two strains, with both showing a gradual switch from myeloid to B cell dominance between weeks 3 and 7 post engraftment. Surprisingly, human T cells were found in the PB of transplanted adult NSGS mice as early as 8 weeks post engraftment, a full 8 weeks before T cells were detectable in NSG mice engrafted with the same UCB sample in parallel. These CD3+ cells presumably develop from the CD34+ stem cells and not from contaminating CD3+ T cells, since FACS-sorted CD3−CD34+ UCB samples produced the same result. Furthermore, normal donor human T cells did not exhibit any advantage in engraftment, cycling, or expansion in NSGS mice when compared to NSG mice in models of GVHD. Characterization of the T cells generated from human CD34+ xenografts revealed CD4+ and CD8+ subpopulations with phenotypes resembling activated, naïve, and memory T cell subsets. CD3+ spleen cells cultured ex vivo were responsive to activation by PHA/IL-2 stimulation and were susceptible to HIV-1 infection. Finally, humanized NSGS mice immunized with a toxoplasmosis extract were able to mount a response to virulent toxoplasmosis infection sufficient to significantly prolong survival while humanized NSG mice or non-humanized NSGS did not. This difference could not be attributed simply to T cell levels, because several of the NSG mice had comparable CD3+ populations at the time of exposure to antigen and subsequent challenge. While T cells are likely to be required for a response to toxoplasmosis challenge, the increased myeloid and dendritic cell populations generated in the NSGS mouse may prove to be equally critical for the functionality of UCB CD34+ xenografts. Disclosures: No relevant conflicts of interest to declare.

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.

RAGE-mediated T cell metabolic reprogramming shapes T cell inflammatory response after stroke

2021 ◽  
pp. 0271678X2110671
Author(s):  
Yueman Zhang ◽  
Fengshi Li ◽  
Chen Chen ◽  
Yan Li ◽  
Wanqing Xie ◽  
...  
Keyword(s):  
T Cells ◽  
Fatty Acid ◽  
T Cell ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Conditional Knockout ◽  
Metabolic Reprogramming ◽  
Stroke Recovery ◽  
Post Stroke ◽  
Soluble Rage

The metabolic reprogramming of peripheral CD4+ T cells that occurs after stroke can lead to imbalanced differentiation of CD4+ T cells, including regulation of T cells, and presents a promising target for poststroke immunotherapy. However, the regulatory mechanism underlying the metabolic reprogramming of peripheral CD4+ T cell remains unknown. In this study, using combined transcription and metabolomics analyses, flow cytometry, and conditional knockout mice, we demonstrate that the receptor for advanced glycation end products (RAGE) can relay the ischemic signal to CD4+ T cells, which underwent acetyl coenzyme A carboxylase 1(ACC1)-dependent metabolic reprogramming after stroke. Furthermore, by administering soluble RAGE (sRAGE) after stroke, we demonstrate that neutralization of RAGE reversed the enhanced fatty acid synthesis of CD4+ T cells and the post-stroke imbalance of Treg/Th17. Finally, we found that post-stroke sRAGE treatment protected against infarct volume and ameliorated functional recovery. In conclusion, sRAGE can serve as a novel immunometabolic modulator that ameliorates ischemic stroke recovery by inhibiting fatty acid synthesis and thus favoring CD4+ T cells polarization toward Treg after cerebral ischemia injury. The above findings provide new insights for the treatment of neuroinflammatory responses after ischemia stroke.

scholarly journals Crispr Engineering in CD34+ Progenitors Reveals Cis-Acting Regulatory Regions Mediating 3D Interactions and Stem Cell Fate Decisions

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1466-1466
Author(s):  
Mira Jeong ◽  
Yong Lei ◽  
Ivan Bochkov ◽  
Muhammad S Shamim ◽  
Anna G Guzman ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
T Cells ◽  
Hoxa Cluster ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract DNA methylation is a critical regulator of cis-regulatory elements that can impact the distribution of epigenetic regulators and transcription factors. We have mapped DNA methylation changes in human CD34+ cells and downstream progeny to detect changes in DNA methylation with differentiation. Some large regions of low DNA methylation emerge with differentiation, which we call dynamic (d-) canyons. In order to determine whether these correlate with changes in the 3D genome, we generated 6 high-resolution Hi-C contact maps from CD34+CD38- cells, Adult bone marrow CD34+ cells, 7 days cultured CD34+ cells, CD71+CD36+CD235+ Erythroid cells, CD4+ T cells and AML patient blast cells. We generated ~ 1 billion mapped reads in order to create each 3D map of the genome at kilobase resolution. We identified several sites of d-canyons which correlate with changes in Hi-C loops. To study the functional role of d-canyons, we performed CRISPR/CAS9 mediated genome engineering targeting three sites of interest in CD34+ progenitors. We designed sgRNAs to delete several d-canyon regions that exhibited cell-type specific methylation and dynamic H3K27ac marking. First, we targeted a novel putative regulatory region of the RUNX1 gene, a critical master regulator of hematopoiesis that is frequently mutated in human leukemia. From genome-wide DNA methylation profiling, we identified a d-canyon located in the first intron of RUNX1, which overlaped with a H3K27ac peak in human CD34+CD38- hematopoietic progenitor cells. DNA methylation in this region is further depleted in T cells and increased in AML cells, suggesting a role for regulating RUNX1 in specific cell types. To study its function, we designed 2 sgRNAs to delete a 1.8 kb d-canyon. CRISPR/Cas9-mediated deletion of this region resulted in ablation of RUNX1 expression in cord blood hematopoietic stem cells and a significant decrease of engraftment activity in NSG mice along with an increase of erythroid colony forming ability in in vitro assays. In addition, we identified d-canyons upstream of the master regulators GATA2 and in the HOXA cluster. We deleted 1.7kb d-canyon upstream of GATA2 gene, finding that it resulted in increased self-renewal of CD34+CD38- cells in NSG xenografts. In contrast, when we deleted one a d-canyon located ~2Mb upstream of the HOXA cluster, within a HiC loop signal present only in CD34+CD38- cells, we observed in decreased CD34+CD38- cell self-renewal but a significant increase of CD34+CD38+ differentiated progenitors in an in vitro culture system, as well as in NSG mice. Colony forming assay showed decreased colony size and numbers. Finally, we identified a d-canyon associated with TCF3, also known as E2A, a transcription factor involved in B and T cell lineage differentiation. We designed 4 sgRNAs to delete 1.5kb d-canyon edge regions within the second intron of TCF3. Removal of this region resulted in a significant decrease of CD19+ B cells, but an increase of CD3+ T cells in NSG mice. Taken together, these results suggest the functional importance of d-canyons for orchestrating genome architecture and fate decisions of hematopoietic stem cells. These findings advance our understanding of the relationship between DNA methylation changes and loop interactions, providing new insights into the potential impact of potential aberrant DNA methylation and chromatin structure. Disclosures No relevant conflicts of interest to declare.

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