Regulatory T Cells of G-CSF Mobilized Stem Cell Donors Qualify for Clinical Applications,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4055-4055
Author(s):  
Sya N. Ukena ◽  
Sarvari Velaga ◽  
Goudeva Lilia ◽  
Philipp Ivanyi ◽  
Arnold Ganser ◽  
...  
Keyword(s):  
Clinical Trials ◽  
T Cells ◽  
Stem Cell ◽  
Regulatory T Cells ◽  
Conflicts Of Interest ◽  
Interleukin 2 ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cell Numbers

Abstract Abstract 4055 Recent clinical studies demonstrate the high potency of human regulatory T cells (Tregs) to control graft-versus-host disease following hematopoietic stem cell transplantation (SCT). Isolation of Tregs after recombinant G-CSF induced mobilization of stem cells would simplify the design of clinical trials in allogeneic SCT. However, there is growing evidence that G-CSF also exerts profound immune modulatory effects in the adaptive immune system. Therefore, we analyzed Tregs isolated by FACS based cell sorting from stem cell donors before (n=8) and after (n=13) G-CSF administration regarding their phenotype, stability, functional and expansion properties. Absolute CD4+ T cell (3.2 fold increase of mean after G-CSF; p<0.05) and CD4+CD25hi Treg cell numbers (4.1 fold increase of mean after G-CSF; p<0.01) were significantly increased after G-CSF administration. Analysis of the Foxp3 TSDR demethylation level displays a stable Foxp3 phenotype of G-CSF encountered Tregs (mean value 97.1% vs. 95.0 % after G-CSF administration). Moreover, the CD4+CD25hi Tregs of G-CSF treated SC donors suppress the proliferation of effector T cells with no significant differences to Tregs isolated from healthy donors before G-CSF treatment (mean values 42.1% vs. 49.9% after G-CSF administration at a Treg/ T effector cell ratio of 1:1). In vitro expansion of Tregs isolated after G-CSF application with anti-CD3 and anti-CD28 dynabeads in the presence of interleukin-2 led to comparable cell numbers as for the stimulation of control Treg cells. However, differences could be detected for the thymic derived marker molecule CD31 and those associated with activation (LAP, CD69, CD62L) and migration (CxCR3) as detected by FACS analysis. Our results show no significant differences regarding suppressive function and stability of Tregs isolated from stem cell donors before and after G-CSF administration. These data support the application of G-CSF mobilized Tregs for clinical trials which in turn opens up new possibilities for the adoptive transfer of Tregs in HSCT. Disclosures: No relevant conflicts of interest to declare.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. 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.

scholarly journals IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo

Blood ◽  
2006 ◽  
Vol 108 (5) ◽  
pp. 1571-1579 ◽  
Author(s):  
Emmanuel Zorn ◽  
Erik A. Nelson ◽  
Mehrdad Mohseni ◽  
Fabrice Porcheray ◽  
Haesook Kim ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Nk Cells ◽  
Low Dose ◽  
Fold Increase ◽  
Foxp3 Expression ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem

IL-2 plays a critical role in the maintenance of CD4+CD25+ FOXP3+ regulatory T cells (Tregs) in vivo. We examined the effects of IL-2 signaling in human Tregs. In vitro, IL-2 selectively up-regulated the expression of FOXP3 in purified CD4+CD25+ T cells but not in CD4+CD25- cells. This regulation involved the binding of STAT3 and STAT5 proteins to a highly conserved STAT-binding site located in the first intron of the FOXP3 gene. We also examined the effects of low-dose IL-2 treatment in 12 patients with metastatic cancer and 9 patients with chronic myelogenous leukemia after allogeneic hematopoietic stem cell transplantation. Overall, IL-2 treatment resulted in a 1.9 median fold increase in the frequency of CD4+CD25+ cells in peripheral blood as well as a 9.7 median fold increase in FOXP3 expression in CD3+ T cells. CD56+CD3- natural killer (NK) cells also expanded during IL-2 therapy but did not express FOXP3. In vitro treatment of NK cells with 5-aza-2′-deoxycytidine restored the IL-2 signaling pathway leading to FOXP3 expression, suggesting that this gene was constitutively repressed by DNA methylation in these cells. Our findings support the clinical evaluation of low-dose IL-2 to selectively modulate CD4+CD25+ Tregs and increase expression of FOXP3 in vivo.

scholarly journals IL-2 administration increases CD4+CD25hi Foxp3+ regulatory T cells in cancer patients

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2409-2414 ◽  
Author(s):  
Mojgan Ahmadzadeh ◽  
Steven A. Rosenberg
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Interleukin 2 ◽  
Selective Inhibition ◽  
High Dose ◽  
Protein Levels ◽  
And Function ◽  
The Impact

Abstract Interleukin-2 (IL-2) is historically known as a T-cell growth factor. Accumulating evidence from knockout mice suggests that IL-2 is crucial for the homeostasis and function of CD4+CD25+ regulatory T cells in vivo. However, the impact of administered IL-2 in an immune intact host has not been studied in rodents or humans. Here, we studied the impact of IL-2 administration on the frequency and function of human CD4+CD25hi T cells in immune intact patients with melanoma or renal cancer. We found that the frequency of CD4+CD25hi T cells was significantly increased after IL-2 treatment, and these cells expressed phenotypic markers associated with regulatory T cells. In addition, both transcript and protein levels of Foxp3, a transcription factor exclusively expressed on regulatory T cells, were consistently increased in CD4 T cells following IL-2 treatment. Functional analysis of the increased number of CD4+CD25hi T cells revealed that this population exhibited potent suppressive activity in vitro. Collectively, our results demonstrate that administration of high-dose IL-2 increased the frequency of circulating CD4+CD25hi Foxp3+ regulatory T cells. Our findings suggest that selective inhibition of IL-2-mediated enhancement of regulatory T cells may improve the therapeutic effectiveness of IL-2 administration. (Blood. 2006;107:2409-2414)

IL-9 Production by Regulatory T Cells Recruits Mast Cells That Are Essential for Regulatory T Cell-Induced Immune-Suppression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2782-2782
Author(s):  
Anna Maria Wolf ◽  
Dominik Wolf ◽  
Andrew McKenzie ◽  
Marcus Maurer ◽  
Alexander R Rosenkranz ◽  
...  
Keyword(s):  
T Cells ◽  
Mast Cells ◽  
Regulatory T Cells ◽  
Adoptive Transfer ◽  
Conflicts Of Interest ◽  
Cell Populations ◽  
Perfect Agreement ◽  
Immunosuppressive Cell

Abstract Abstract 2782 Tipping the balance between effector and regulatory cell populations is of critical importance in the pathogenesis of various autoimmune disorders. Both, mast cells (MC) and regulatory T cells (Treg) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MC and Treg have been shown to play a protective role. We recently provided evidence that adoptive transfer of wild-type (wt) Treg into wt recipients almost completely prevents development of NTS. We here show that Treg transfer induces a profound increase of MC in the kidney draining lymph nodes (LN). In contrast, transfer of wt Treg into animals deficient in MC, which are characterized by an exaggerated susceptibility to NTS, do not prevent acute renal inflammation. Blocking the pleiotropic cytokine IL-9, which is known to be critically involved in MC recruitment and proliferation, by means of an antagonizing monoclonal antibody in animals receiving wt Treg abrogated protection from NTS. Moreover, we provide clear evidence that Treg-derived IL-9 is critical for MC recruitment as mediators of their full immune-suppressive potential, as adoptive transfer of IL-9 deficient Treg failed to protect from NTS. In line with our hypothesis, absence of Treg-derived IL-9 does not induce MC accumulation into kidney-draining LN, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Treg, as shown by in vitro testing of their functional capacities. Finally, we observed a significantly decreased expression of the MC chemoattractant Cxcl-1 in the LN of mice receiving IL-9 deficient Treg as compared to mice receiving wt Treg or control CD4+CD25− T cells, which might at least in part explain the deficient MC recruitment under these conditions. In summary, our data provide the first evidence that the immunosuppressive effects of adoptively transferred Treg depend on IL-9-mediated recruitment of MC to the kidney draining LN in NTS. This data is in perfect agreement with our previous report showing that CCR7-mediated LN occupancy of Treg is a prerequisite for their immune-suppressive potential and furthers adds a piece of information to the functional understanding of the in vivo anti-inflammatory effects of Treg. Disclosures: No relevant conflicts of interest to declare.

Enhancement of Megakaryocyte Interactions with the Osteoblastic Hematopoietic Stem Cell Niche Improves Engraftment Efficiency Following Hematopoietic Stem Cell Transplantation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 211-211
Author(s):  
Timothy S. Olson ◽  
Satoru Otsuru ◽  
Ted Hofmann ◽  
Edwin M. Horwitz
Keyword(s):  
Stem Cell ◽  
Growth Factor ◽  
Hematopoietic Stem Cell ◽  
Stem Cell Niche ◽  
Structural Changes ◽  
Conflicts Of Interest ◽  
Imatinib Treatment ◽  
Hematopoietic Stem ◽  
Cell Niche

Abstract Abstract 211 Bone marrow (BM) radioablation produces structural changes in the endosteal osteoblastic stem cell niche, a critical site of hematopoietic stem cell (HSC) engraftment following HSC transplantation (HSCT). We have previously shown that total body irradiation (TBI) in wildtype (WT) mice induces migration of recipient megakaryocytes to the niche and an expansion of niche osteoblasts that supports HSC engraftment following transplantation. We have also demonstrated that c-MPL-deficient (mpl−/−) recipients have decreases in total megakaryocytes (35% of WT), the percentage of megakaryocytes migrating to the endosteum (<20% of WT), and niche osteoblast expansion (<50% of WT) following TBI, leading to profound deficits in long-term (LT)-HSC engraftment following HSCT. We now present data examining mechanisms by which megakaryocytes facilitate both niche osteoblast expansion post-TBI and donor HSC engraftment following HSCT, and a therapeutic strategy utilizing these mechanisms to enhance donor HSC engraftment. The decrease in total megakaryocytes and absent thrombopoietin (TPO) signaling in mpl−/− mice resulted in a 90% reduction in post-TBI mpl−/− versus WT BM levels of platelet-derived growth factor beta (PDGFβ), a known osteoblast growth factor. In vitro, megakaryocytes cultured together or across a transwell membrane markedly enhanced osteoblast growth (> 2.5 fold, p < 0.001), but PDGFβ signaling inhibition completely abrogated megakaryocyte-driven osteoblast growth. In vivo, inhibition of PDGF receptor signaling in WT mice via imatinib treatment resulted in near complete blockade of TBI-induced osteoblast expansion, and imatinib treatment of primary recipients resulted in diminished LT-HSC engraftment in secondary transplant assays. Blockade of CD41 integrin-mediated adhesion of megakaryocytes in WT recipient BM blocked TBI-induced megakaryocyte migration to the endosteal niche and severely abrogated LT-HSC engraftment efficiency. However, in contrast to c-MPL deficiency, CD41 blockade did not decrease PDGFβ expression or niche osteoblast expansion, suggesting that in addition to PDGFβ-dependent effects on niche expansion, the megakaryocyte migration to the niche itself is also required to efficiently engraft HSC. Mice with decreased GATA-1 expression (Gata-1tm2sho/J), have a large increase in total BM megakaryocytes a >2-fold (p < 0.001) increase in PDGFβ levels, and greatly increased expansion of osteoblast and other mesenchymal elements 48 hours post-TBI compared to WT mice. However, Gata-1tm2sho/J megakaryocytes have known defective terminal differentiation and function including decreased platelet production, and Gata-1tm2sho/J primary recipients did not engraft LT-HSC more efficiently than WT primary recipients, demonstrating the need for fully functional megakaryocytes, and not only increased PDGFβ-induced mesenchymal proliferation, to foster HSC engraftment. Finally, we have examined whether TPO administration prior to radioablation and HSCT can enhance host megakaryocyte effects on the niche and HSC engraftment. TPO administration for 5 days prior to radioablation, resulted in a significant increase in BM megakaryocytes and a 50% increase in niche osteoblast expansion. Furthermore, competitive secondary transplantation assays demonstrated that TPO- versus sham-treatment of primary recipients prior to TBI and BM transplant, resulted in increased initial engraftment at 24 hours post-primary transplant (40% increase, p < 0.05) increased short-term HSC and progenitor engraftment 3–6 weeks following secondary transplant (4–20 fold increase, p < 0.02), and sustained LT-HSC engraftment at 28 weeks post-transplant in 47% versus 7% (p < 0.05) of secondary recipients of TPO- versus sham-treated primary recipient BM, respectively. Taken together, our results demonstrate that host megakaryocytes facilitate efficient HSC engraftment following TBI and HSCT through PDGFβ-dependent enhancement of niche osteoblast expansion and through direct interactions of megakaryocytes with the niche. TPO-treatment of transplant recipients prior to radioablation and stem cell infusion enhances these megakaryocyte-dependent pathways and subsequent donor HSC engraftment efficiency, providing a clinically applicable strategy to enhance niche function and stem cell engraftment following clinical transplantation. Disclosures: No relevant conflicts of interest to declare.

Anti-Thymocyte Globulin (ATG) Differentially Depletes naïve and Memory T Cells and Permits Memory-Type Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4670-4670
Author(s):  
Chang-Qing Xia ◽  
Anna Chernatynskaya ◽  
Clive Wasserfall ◽  
Benjamin Looney ◽  
Suigui Wan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Memory T Cells ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem

Abstract Abstract 4670 Anti-thymocyte globulin (ATG) has been used in clinic for the treatment of allograft rejection and autoimmune diseases. However, its mechanism of action is not fully understood. To our knowledge, how ATG therapy affects naïve and memory T cells has not been well investigated. In this study, we have employed nonobese diabetic mouse model to investigate how administration of anti-thymocyte globulin (ATG) affects memory and naïve T cells as well as CD4+CD25+Foxp3+ regulatory T cells in peripheral blood and lymphoid organs; We also investigate how ATG therapy affects antigen-experienced T cells. Kinetic studies of peripheral blood CD4+ and CD8+ T cells post-ATG therapy shows that both populations decline to their lowest levels at day 3, while CD4+ T cells return to normal levels more rapidly than CD8+ T cells. We find that ATG therapy fails to eliminate antigen-primed T cells, which is consistent with the results that ATG therapy preferentially depletes naïve T cells relative to memory T cells. CD4+ T cell responses post-ATG therapy skew to T helper type 2 (Th2) and IL-10-producing T regulatory type 1 (Tr1) cells. Intriguingly, Foxp3+ regulatory T cells (Tregs) are less sensitive to ATG depletion and remain at higher levels following in vivo recovery compared to controls. Of note, the frequency of Foxp3+ Tregs with memory-like immunophenotype is significantly increased in ATG-treated animals, which might play an important role in controlling effector T cells post ATG therapy. In summary, ATG therapy may modulate antigen-specific immune responses through modulation of naïve and memory T cell pools and more importantly through driving T cell subsets with regulatory activities. This study provides important data for guiding ATG therapy in allogenieic hematopoietic stem cell transplantation and other immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

Unique Role of mTOR Inhibitor, Everolimus in Inducible Regulatory T Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4349-4349
Author(s):  
Tokiko Nagamura-Inoue ◽  
Yuki Yamamoto ◽  
Seiichiro Kobayashi ◽  
Kazuo Ogami ◽  
Kiyoko Izawa ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Mtor Inhibitor ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Foxp3 Expression ◽  
Induction Rate ◽  
The Impact

Abstract Abstract 4349 Background: Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Unbalance between Tregs and effector T cells is involved in graft-versus-host disease (GvHD) and other autoimmune disorders. Adoptive use of inducible Tregs (iTregs) is a candidate immunosuppressive therapy, and major concern has been focused on sustained expression of Foxp3 in iTregs. We previously reported that iTregs can be efficiently expanded from cord blood (CB)-derived CD4+ T cells in the presence of IL2, TGFb and a mTOR inhibitor, Everolimus (Eve). However, the effect of Eve on in vitro induction of iTreg remains to be elucidated. Here we studied the impact of Eve on CB-CD4+ T cells. Methods: CD4+ T cells were prepared from CB with a purity of >95% and put into the flask coated with anti-CD3/CD28 MAb. For Treg induction, these cultures were supplemented with IL2, IL-2/TGFb, IL2/TGFb/Eve, or IL2/Eve and kept for two weeks. The resulting CD4+ T cells including variable proportion of iTregs were subjected to mixed lymphocyte reaction (MLR) along with CFSE-labeled autologous responder T cells and allogeneic dendritic cells (DCs) as stimulator. Results: The basal proportion of CD25+Foxp3+ cells in CB-CD4+ T cells was 0.60 ± 0.59%. After two weeks, the induction rate of CD25+Foxp3+CD4+ T cells was higher in the culture with IL2/TGFb/Eve than that with IL2/TGFb, but Eve itself could not significantly induce iTregs in the absence of TGFb (Figure1.). The iTreg ratio (CD25+Foxp3+ cells/total CD4+ T cells) was 79.3 ± 17.4% in the culture with IL2/TGFb/Eve, 53.1 ± 23.8% with IL2/TGFb, 35.5±18.6% with IL2/Eve and 22.7 ± 18.6% with IL2, respectively. There was no significant relationship between the dose of Eve and the iTreg ratio, but the highest ratio and induction rate of iTregs were observed at 10nM Eve. Thus, an average of 2.95 ± 2.8 ×107 iTregs was obtained from 5 ×104 CB-CD4+ T cells after two weeks of culture with IL2/TGFb/Eve. The iTreg-rich population cultured with IL2/TGFb/Eve and IL2/TGFb, but not IL2 alone, efficiently inhibited MLR triggered by allogeneic DCs (Figure 2.). These iTregs were also active in MLR using allogeneic responder T cells. Interestingly, IL2/Eve-treated CB-CD4+ T cells also inhibited MLR, irrespective of the low or moderate iTreg ratio. The inhibitory effect on MLR was much less observed by another mTOR inhibitor, rapamycin, rather than Eve (Figure2). Expression of CD26 on CD4+ T cells was inversely correlated to Foxp3 expression and significantly down-regulated by TGFb with or without Eve. Discussion: Treatment of CB-CD4+ T cells with IL2/TGFb/Eve results in the efficient ex vivo expansion of functional iTregs. Eve enhanced TGFb induction of Foxp3 expression, but did not induce Foxp3 expression by itself. mTOR is a complex of TORC1 and 2. Rapamycin is reported to inhibit TORC1, while Eve inhibits both of them, at general dose. In recent report, mTOR-deficient T cells (TORC1/2, not TORC1 alone) displayed normal activation and IL-2 production upon initial stimulation, but failed to differentiate into effecter T cells, instead, differentiated into Tregs. Although the direct mechanism to inhibit MLR by CB-CD4+ T cells treated with Eve remained to be elucidated, these results suggested the aberrant pathways of immunological inhibition. Disclosures: No relevant conflicts of interest to declare.

MiR-29a Maintains Hematopoietic Stem Cell Self-Renewal and Is Required For Myeloid Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1190-1190
Author(s):  
Wenhuo Hu ◽  
James Dooley ◽  
Stephen S. Chung ◽  
Safak Yalcin ◽  
Yu Sup Shin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Colony Formation ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Null Mouse ◽  
Cell Cycle Regulators ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract microRNAs (miRNAs) are important regulators of both embryonic and adult tissue stem cell self-renewal. We previously showed that ectopic expression of miR-29a, a miRNA highly expressed in HSCs as well as in human acute myeloid leukemia (AML) stem cells, in immature mouse hematopoietic cells is sufficient to induce a myeloproliferative disorder that progresses to AML. During the early phase of this disease, miR-29a induces aberrant self-renewal of committed myeloid progenitors, strongly suggesting a role for miR-29a in regulating HSC self-renewal. In order to determine the role of miR-29a in HSC function, we have evaluated our recently described miR-29a/b1 null mouse. Homozygous deletion of miR-29a/b1 resulted in reduced bone marrow cellularity and reduced colony forming capacity of hematopoietic stem and progenitor cells (HSPCs). The phenotype was mediated specifically by miR-29a since miR-29b expression was not significantly altered in HSCs and reconstitution of miR-29a/b1 null HSPCs with miR-29a, but not miR-29b, rescued in vitro colony formation defects. Self-renewal defects were observed in miR-29a deficient HSCs in both competitive and non-competitive transplantation assays, and these deficits were associated with increased HSC cell cycling and apoptosis. Gene expression studies of miR-29a deficient HSCs demonstrated widespread gene dysregulation including a number of up-regulated miR-29a target genes including DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6, Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient HSCs resulted in partial restoration of colony formation, providing functional validation that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a loss also abrogated leukemogenesis in the MLL-AF9 retroviral AML model. Together, our results demonstrate that miR-29a positively regulates HSC self-renewal and is required for myeloid leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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