Gordon Watkins Douglas researched cervical cancer, breach delivery, and treatment of high blood pressure during pregnancy [5] in the US during the twentieth century. He worked primarily at Bellevue Hospital Center in New York, New York. While at Bellevue, he worked with William E. Studdiford to develop treatments for women who contracted infections as a result of illegal abortions performed throughout the US in unsterile environments. Douglas also established the first contraception [6] and pregnancy [5] termination clinic at Bellevue Hospital shortly after the legalization of abortion [7] as a result of the 1973 US Supreme Court ruling in Roe v. Wade [8]. Furthermore, Douglas showed that fetal and maternal cells exchange between the pregnant woman and fetus [9] during pregnancy [5], which led to the later development of non-invasive prenatal testing in the early twenty-first century.

Hematopoietic stem cells (HSC) reside in hypoxic niches (∼1-4% O2), yet, HSC studies are typically performed using cells isolated in ambient air (∼20% O2). Inhibiting ambient air exposure/harm of cells, which we termed Extra Physiologic Oxygen Shock/Stress (EPHOSS), by collecting/processing human cord blood (hCB) or mouse bone marrow (mBM) stem cells in hypoxia (3% O2), enhances recovery of phenotypic/functional long-term repopulating HSC (LT-HSC) and is mechanistically linked, in part, to the mitochondrial permeability transition pore (MPTP), Reactive Oxygen Species (ROS) and cyclophilin D. We hypothesized that Dipeptidyl Peptidase 4 (DPP4), an enzyme that N terminally cleaves and modifies the function of select proteins leading to alterations in homing/engraftment of HSC, may be altered by EPHOSS and involved in EPHOSS effects on HSC. Proteomic and bioinformatic analysis identified 1) many unexpected intracellular/secreted proteins with DPP4 truncation (T) sites and 2) specific, as well as overlapping, modifications in phosphorylated and differential protein signaling of T-cytokines (GM-CSF and IL-3) compared to their full length (FL) forms leading to diversified regulation of signaling/function in normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding compared to their FL forms with significant, and reciprocal, blunting of functional activity of both factors in vitro and in vivo. To investigate effects of DPP4 on EPHOSS, and vice versa, mBM was harvested (air/ hypoxia) with a DPP4 inhibitor (DPA), or from DPP4 K/O mice. This resulted in significant increases in the number of phenotypic LT-HSC (p=.017) in air, suggesting that DPP4 inhibition blunts EPHOSS mediated loss of phenotypic LT-HSC. Also, the percentage of DPP4+ cells was increased in primitive fractions of mBM or hCB, (LSK ∼15%, LSKCD150 40%, CD34+CD38- ∼10%, CD34+CD38-CD45RA-CD90+CD49F+ ∼40% p=.007) and further enhanced 15- 20% when cells are isolated in hypoxia (p=.005). Unexpectedly, LT-HSC ROS levels (mitochondrial/total) were not diminished in DPA or DPP4 K/O groups harvested in air despite the increase in phenotypic LT-HSC over air harvest alone, suggesting a non ROS/MPTP mechanism. In conclusion, DPP4 expression/activity serves heretofore unknown roles in the regulation/signaling of multiple protein types as well as cellular responses to EPHOSS and hematopoiesis.

Dipeptidyl peptidase 4 (DPP4)/CD26 truncates certain proteins, and this posttranslational modification can influence their activity. Truncated (T) colony-stimulating factors (CSFs) are decreased in potency for stimulating proliferation of hematopoietic progenitor cells (HPCs). T-CXCL12, a modified chemokine, is inactive as an HPC chemotactic, survival, and enhancing factor for replating or ex-vivo expansion of HPCs. Moreover, T-CSFs and T-CXCL12 specifically downmodulates the positively acting effects of their own full-length molecule. Other chemokines have DPP4 truncation sites. In the present study, we evaluated effects of DPP4 inhibition (by Diprotin A) or gene deletion of HPC on chemokine inhibition of multicytokine-stimulated HPC, and on chemokine-enhancing effects on single CSF-stimulated HPC proliferation, as well as effects of DPP4 treatment of a number of chemokines. Myelosuppressive effects of chemokines with, but not without, a DPP4 truncation site were greatly enhanced in inhibitory potency by pretreating target bone marrow (BM) cells with Diprotin A, or by assaying their activity on dpp4/cd26(-/-) BM cells. DPP4 treatment of myelosuppressive chemokines containing a DPP4 truncation site produced a nonmyelosuppressive molecule, but one which had the capacity to block suppression by that unmodified chemokine both in vitro and in vivo. Additionally, DPP4 treatment ablated the single cytokine-stimulated HPC-enhancing activity of CCL3/MIP-1α and CCL4/MIP-1β, and blocked the enhancing activity of each unmodified molecule, in vitro and in vivo. These results highlight the functional posttranslational modulating effects of DPP4 on chemokine activities, and information offering additional biological insight into chemokine regulation of hematopoiesis.

Although hematopoietic stem cells (HSC) are the best characterized and the most clinically used adult stem cells, efforts are still needed to understand how to best ex vivo expand these cells. Here we present our unexpected finding that OCT4 is involved in the enhancement of cytokine-induced expansion capabilities of human cord blood (CB) HSC. Activation of OCT4 by Oct4-activating compound 1 (OAC1) in CB CD34(+) cells enhanced ex vivo expansion of HSC, as determined by a rigorously defined set of markers for human HSC, and in vivo short-term and long-term repopulating ability in NSG mice. Limiting dilution analysis revealed that OAC1 treatment resulted in 3.5-fold increase in the number of SCID repopulating cells (SRCs) compared with that in day 0 uncultured CD34(+) cells and 6.3-fold increase compared with that in cells treated with control vehicle. Hematopoietic progenitor cells, as assessed by in vitro colony formation, were also enhanced. Furthermore, we showed that OAC1 treatment led to OCT4-mediated upregulation of HOXB4. Consistently, siRNA-mediated knockdown of HOXB4 expression suppressed effects of OAC1 on ex vivo expansion of HSC. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and has important roles in multiple aspects of cancer aggressiveness. Thus targeting STAT3 promises to be an attractive strategy for treatment of advanced metastatic tumors. Although many STAT3 inhibitors targeting the SH2 domain have been reported, few have moved into clinical trials. Targeting the DNA-binding domain (DBD) of STAT3, however, has been avoided due to its ‘undruggable‘ nature and potentially limited selectivity. In a previous study, we reported an improved in silico approach targeting the DBD of STAT3 that resulted in a small-molecule STAT3 inhibitor (inS3-54). Further studies, however, showed that inS3-54 has off-target effect although it is selective to STAT3 over STAT1. In this study, we describe an extensive structure and activity-guided hit optimization and mechanistic characterization effort, which led to identification of an improved lead compound (inS3-54A18) with increased specificity and pharmacological properties. InS3-54A18 not only binds directly to the DBD and inhibits the DNA-binding activity of STAT3 both in vitro and in situ but also effectively inhibits the constitutive and interleukin-6-stimulated expression of STAT3 downstream target genes. InS3-54A18 is completely soluble in an oral formulation and effectively inhibits lung xenograft tumor growth and metastasis with little adverse effect on animals. Thus inS3-54A18 may serve as a potential candidate for further development as anticancer therapeutics targeting the DBD of human STAT3 and DBD of transcription factors may not be ‘undruggable‘ as previously thought.

Simple efforts are needed to enhance cord blood (CB) transplantation. We hypothesized that short-term exposure of CD34(+) CB cells to 39.5°C would enhance their response to stromal-derived factor-1 (SDF-1), by increasing lipid raft aggregation and CXCR4 expression, thus leading to enhanced engraftment. Mild hyperthermia (39.5°C) significantly increased the percent of CD34(+) CB that migrated toward SDF-1. This was associated with increased expression of CXCR4 on the cells. Mechanistically, mild heating increased the percent of CD34(+) cells with aggregated lipid rafts and enhanced colocalization of CXCR4 within lipid raft domains. Using methyl-β-cyclodextrin (MβCD), an agent that blocks lipid raft aggregation, it was determined that this enhancement in chemotaxis was dependent upon lipid raft aggregation. Colocalization of Rac1, a GTPase crucial for cell migration and adhesion, with CXCR4 to the lipid raft was essential for the effects of heat on chemotaxis, as determined with an inhibitor of Rac1 activation, NSC23766. Application-wise, mild heat treatment significantly increased the percent chimerism as well as homing and engraftment of CD34(+) CB cells in sublethally irradiated non-obese diabetic severe combined immunodeficiency IL-2 receptor gamma chain d (NSG) mice. Mild heating may be a simple and inexpensive means to enhance engraftment following CB transplantation in patients.

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

A hyaluronic-acid-rich node and duct system (HAR-NDS) was found on the surface of internal organs of mice, and inside their blood and lymph vessels. The nodes (HAR-Ns) were filled with immune cells of the innate system and were especially enriched with mast cells and histiocytes. They also contained hematopoietic progenitor cells (HPCs), such as granulocyte-macrophage, erythroid, multipotential progenitors, and mast cell progenitors (MCPs). MCPs were the most abundant among the HPCs in HAR-Ns. Their frequency was fivefold higher than that of the MCPs in bone marrow. In addition, the system contained pluripotent stem cells (PSCs) capable of producing CD45(-)Flk1(+) hemangioblast-like cells, which subsequently generated various types of HPCs and differentiated blood cells. Although HAR-Ns did not appear to harbor enough number of cells capable of long-term reconstitution or short-term radioprotection of lethally irradiated recipients, bone marrow cells were able to engraft in the HAR-NDS and reconstitute hematopoietic potentials of the system. PSCs and HPCs were consistently found in intravenous, intralymphatic, and intestinal HAR-ND. We infer that PSCs and HPCs reside in the HAR-ND and that this novel system may serve as an alternative means to traffic immature and mature blood cells throughout the body.

Hematopoietic stem cells (HSCs) reside in hypoxic niches within the bone marrow (BM). Yet, all HSC studies have been performed to date with cells immediately isolated in non-physiologic ambient air, whether or not they are subsequently processed in low oxygen tension. By collecting/manipulating BM in physiologically native conditions of hypoxia where all procedures are performed inside a hypoxic chamber, we demonstrate that brief exposure of mouse BM or human cord blood (CB) to ambient oxygen decreases recovery of phenotypically-defined and functional self-renewing long-term repopulating HSC and concomitantly increases numbers of progenitor cells, a phenomenon we term Extra Physiologic Oxygen Shock/Stress (EPHOSS). This new phenomenon is exquisitely sensitive to oxygen and great care must be taken to ensure all reagents, solutions, plastics, and anything that will come into contact with the cell suspension, is extensively pre-equilibrated in hypoxia. Up to 5-fold greater numbers of long-term (LT)-HSCs (CD34-CD150+Lin-Sca1+c-kit+CD41-CD48- or CD34-CD135-Lin-Sca1+c-kit+) could be recovered from mouse BM harvested in 3% O2 compared to BM harvested in air, or even BM harvested in 3% O2 and then exposed to air for as little as 30 min before analysis, even if subsequently returned to hypoxia. There was a concomitant decrease in short term-HSCs and multipotent progenitors when BM was harvested in hypoxia, an effect associated with decreased functional cytokine-stimulated colony formation of hematopoietic progenitor cells (HPC: CFU-GM, BFU-E, and CFU-GEMM). Similarly, if human CB was harvested under similar low oxygen conditions, a 3-fold increase in recovered HSCs (Lin-CD34+CD38-CD45RA+CD90+CD49f+) could be achieved compared to CB harvested in air. Using a custom mouse respirator to conduct competitive repopulating transplant experiments completely in a 3% O2 environment revealed an increase in competitive repopulating units (CRUs) up to more than 42 fold was recovered when BM is harvested in hypoxic conditions compared to air harvested BM in primary recipients, thus demonstrating the beneficial effects of hypoxic harvest on functional/transplantable HSCs with secondary transplant capability. These data strongly support the surprising conclusion that, until now, the true numbers of HSCs and the transplantation potency of BM and CB has been routinely and consistently underestimated because of rapid initiation of differentiation of LT-HSCs in ambient air. We present evidence linking mitochondrial function and cyclophilin D to EPHOSS. Genetically or pharmacologically suppressing cyclophilin D function, or p53 gene deletion link production of reactive oxygen species (ROS) to induction of the mitochondrial permeability transition pore (MPTP) as a molecular mechanism of EPHOSS, where rapid ROS generation in HSCs after exposure to “hyperoxic” room air initiates an irreversible cascade of differentiation signals (see illustration). We present additional evidence from gene knock-out model studies implicating roles for miR210 and Hif-1a in EPHOSS. The MPTP inhibitor, cyclosporine A, protects phenotypically-defined as well as functional and transplantable HSCs from EPHOSS during collection in air resulting in at least a 3-fold increase in HSC recovery as well as increased transplantation potency. Thus, pharmacological mitigation of EPHOSS during HSC collections for use in patient transplantation procedures may be clinically advantageous. Because cyclosporine A is already in use clinically, this EPHOSS-reducing strategy may be readily and easily tested for efficacy in a hospital setting. Because many different adult stem cells exist naturally in hypoxic niches, EPHOSS is likely relevant to other stem cells routinely harvested in air. Evidence suggests that aged HSCs may be more sensitive to the deleterious effects of EPHOSS than young HSCs. We propose that metabolic profiling of stem cells, including cancer stem cells, may not accurately represent the metabolism, behavior, and responses of these cells as they exist in their native hypoxic environments because they are harvested and studied in air. Thus, experimental designs that include a consideration of EPHOSS effects may be required to obtain a more complete understanding of stem cell metabolism and biology especially as it relates to stem cell aging or responses of cancer stem cells to chemotherapy.

We reported that Dipeptidylpeptidase 4 (DPP4) truncates GM-CSF, G-CSF, IL-3, EPO and CXCL12 (Broxmeyer et al, 2012, Nature Med 18:1786). Truncated (TR)-CSFs are decreased in hematopoietic progenitor cell (HPC) stimulating activity, and TR-CXCL12/SDF-1 is inactive as an HPC chemotactic, survival or expansion factor. Moreover, TR-molecules block positively acting effects of their own full length (FL)-molecule. Many chemokines have DPP4 TR sites. To determine a role for DPP4 truncation on negatively acting cytokines, we assessed in vitro effects of FL, TR, and FL plus TR myelosuppressive CCL3/MIP-1α, CCL2/MCP-1, CXCL4/PF4, CXCL5/ENA-74, CXCL6/GCP-2, GXCL8/IL-8, CXCL9/MIG, and CXL10/IP-10, and non-myelosuppressive CCL4/MIP-1β and CCL5/RANTES on multi-cytokine stimulated HPC. Myelosuppressive XCL1/Lymphotoxin, without a DPP4 TR site, was also assessed. Myelosupressive chemokines with, but not without, a DPP4-TR site were 100-1000 fold enhanced in suppressor potency by pretreating target BM cells with Diprotin A, a DPP4 inhibitor, or by assaying activity on dpp4 -/- marrow. Non myelosuppressive chemokines remained non suppressive. DPP4-treatment of chemokines with myelosuppressive activity produced non-myelosuppressive molecules that blocked suppression by their FL-chemokine, likely through receptor interference. In vitro effects were reproduced in vivo after IV injection of FL, TR, and FL plus TR CCL3, CXCL8, and CXCL9. CCL3 is myelosuppressive for immature subsets of HPC responsive to stimulation by multiple cytokines, but CCL3 and non-myelosuppressive CCL4 enhance single cytokine (GM-CSF or M-CSF) stimulated colony formation in vitro by more mature HPC (Broxmeyer et al, 1989, JEM 170:1583; 1990, Blood 76:1110). We now show that DPP4-TR CCL3 and CCL4 lose their enhancing activity for GM-CSF and M-CSF stimulated HPC in vitro and in vivo, and TR-CCL3 and -CCL4 each block enhancement by FL CCL3 and CCL4. This highlights intricate in vitro and in vivo influences of DPP4 that should be considered when evaluating regulatory cytokine/chemokine effects and means to modulate hematopoiesis for biological and clinical insight.

Dipeptidylpeptidase 4 (DPP4) is a serine peptidase with enzymatic activity leading to the N terminal cleavage of select penultimate amino acids of proteins. We previously published that the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, as well as the ability of DPP4 T proteins to induce signaling that FL factors can not, have not been previously investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating the here to fore unknown ability of DPP4 cleavage of proteins to alter not only cellular function, but intracellular signaling of growth factors leading to miRNA expression, phosphorylation, and global induction of proteins that the FL form of the protein does not induce. Additionally, and unexpectedly, a 1:1 mixture of the DPP4 T protein and their FL counterpart leads to both overlapping signaling between the FL and T, as well as unique signaling, that is not induced by the FL or T protein alone. This suggests multifaceted, important, and currently unappreciated roles that the DPP4 truncation of proteins may play in the regulation of normal and malignant hematopoiesis as well as other physiologic and pathophysiologic states.

Umbilical cord blood (UCB) is a viable source of hematopoietic stem cells (HSC) for transplantation to treat patients with malignant and non-malignant disease. Although the limitation of low stem cell dose in single UCB transplantation has been improved upon by the use of double UCB units, this has not resulted in a shortened time to engraftment. Therefore efforts are needed to enhance the speed of engraftment of these cells. Mild heating (39.5°C) of mature hematopoietic cells increases membrane fluidity, resulting in increased lipid raft aggregation, and priming cells for increased sensitization of responsiveness to cytokine signaling. We hypothesized that exposing CD34+ UCB cells to 39.5°C would enhance their capacity to respond to SDF-1/CXCL12, a chemokine secreted in the bone marrow microenvironment that strongly attracts HSCs and progenitor cells, by increasing lipid raft aggregation and CXCR4 expression, thus leading to better engraftment following transplantation. To determine if this mild heating had any effect on engraftment in vivo, NSG mice received 4Gy TBI followed one day later with 20,000 or 80,000 CD34+ UCB cells incubated for 4 hours at 37°C or 39.5°C prior to i.v. injection. Percent chimerism in the blood was determined through the detection of human CD45 by flow cytometry at 1, 2, 4 and 6 months following transplantation. Mild heat treatment significantly increased the percent chimerism following transplantation by 1.7 fold at 1 month (when 80,000 cells were used) and by 1.8 fold at 2 months (when 20,000 cells were used), without significant increases in percent chimerism at 4 or 6 months. This suggests that the effects of heat treatment are most likely occurring early following transplantation, potentially due to an increase in homing of the heated CD34+ UCB cells to the bone marrow microenvironment. To begin to test this hypothesis, CD34+ UCB and Mo7e cells (a human megakaryoblastic leukemia cell line) were exposed to 39.5°C for 4 hours followed by a chemotaxis assay. Exposure of the CD34+ UCB cells to 39.5°C manifested in a significant increase in the percent of cells that migrated towards 12.5 (1.8 fold increase), 25 (1.9 fold increase), 50 (2.2 fold increase), 100 (2.3 fold increase) and 200 ng/mL SDF-1 (2.5 fold increase). Mild heating also significantly increased the percent of Mo7e cells that migrated towards 12.5 (1.8 fold increase), 25 (1.4 fold increase), 50 (1.5 fold increase), 100 (1.3 fold increase), and 200 ng/mL SDF-1 (1.5 fold increase). Increases in chemotaxis towards SDF-1 following mild heating was associated with a significant, but modest, increase (1.3 fold as determined by MFI) in expression of the SDF-1 receptor, CXCR4, on the surface of CD34+ UCB cells as detected by flow cytometry. In order to optimize SDF-1 signaling, CXCR4 must be incorporated into the cholesterol-enriched lipid rafts of the cellular membrane followed by lipid raft aggregation. Mild heating increased the percent of Mo7e cells with aggregated lipid rafts from 13 percent for cells incubated for 4 hours at 37°C to 52% for cells incubated for 4 hours at 39.5C. By using a dose of MbCD (1.25mM), an agent that can deplete cholesterol in the cellular membrane thus blocking lipid raft aggregation, it was determined that the enhancement in chemotaxis seen following mild heating was dependent upon lipid raft aggregation in both CD34+ UCB and Mo7e cells. Overall, mild heating may be a simple and inexpensive novel adjuvant therapy to enhance engraftment following UCB transplantation in patients. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play a vital role in replenishment of blood cells. In addition to growth factors, energy metabolism plays an important role in cellular proliferation. Oxidative phosphorylation that occurs in the mitochondria is the major source of ATP. In this study, we have investigated the role of peroxisome proliferator-activated-γ coactivator-1α (PGC-1α), a major regulator of mitochondrial biogenesis, in hematopoiesis. PGC-1α is expressed in HSC/HPCs. Loss of PGC-1α minimally affects basal hematopoiesis; however, it significantly impairs stress hematopoiesis. Recovery of hematopoiesis poststress involves rapid proliferation of HSC/HPCs. Growth factors stimulate HSC/HPC proliferation in a dose-dependent manner and this response is modulated by oxygen tension. Although severe hypoxic conditions inhibit HSC/HPC proliferation, mild hypoxia enhances the clonogenic potential; however, the mechanism underlying this phenomenon remains largely unknown. Our studies demonstrate that PGC-1α-mediated mitochondrial biogenesis is critical for the increased clonogenic potential of progenitors under mild hypoxia. Metabolic programming and increased glucose uptake can drive rapid progenitor cell proliferation under relatively low oxygen tension only if the HPC has the capacity to increase PGC-1α expression and mitochondrial biogenesis. Loss of PGC-1α also impairs the long-term repopulating potential of HSCs. Our findings may have therapeutic applications for rapid recovery of blood cells following myeloablation.

DPP4 (CD26) is a dipeptidyl peptidase that functions by enzymatically cleaving the penultimate proline, alanine or select other amino acids such as serine of proteins, resulting in functional alterations of the protein. We recently published that many cytokines, chemokines and growth factors have putative DPP4 truncations sites and that DPP4 specifically was able to truncate some colony stimulating factors such as GM-CSF and IL-3 with resultant blunting of their activity. However, the mechanism of action of the truncated factors is still unknown and requires further investigation. The expression, and activity, of DPP4 is relevant in normal and malignant hematopoiesis as we have data showing that CD34+ umbilical cord blood cells (UCB) as well as Acute Myelogenous Leukemia (AML) patient samples express active DPP4. Further, specific inhibition of DPP4 increases homing and engraftment of both human UCB and mouse bone marrow cells after transplantation in mice indicating the therapeutic potential of DPP4 activity altering compounds. Due to its potential importance in disease states, and their subsequent treatment, it is relevant to study how the activity of DPP4 alters the functions of the molecules it cleaves, and subsequently their interactions with each other.

The transcriptional repressor Bcl6 is a critical arbiter of Th cell fate, promoting the follicular Th lineage while repressing other Th cell lineages. Bcl6-deficient (Bcl6−/−) mice develop a spontaneous and severe Th2-type inflammatory disease, thus warranting assessment of Bcl6 in regulatory T cell (Treg) function. Bcl6−/− Tregs were competent at suppressing T cell proliferation in vitro and Th1-type colitogenic T cell responses in vivo. In contrast, Bcl6−/− Tregs strongly exacerbated lung inflammation in a model of allergic airway disease and promoted higher Th2 responses, including systemic upregulation of microRNA-21. Further, Bcl6−/− Tregs were selectively impaired at controlling Th2 responses, but not Th1 and Th17 responses, in mixed chimeras of Bcl6−/− bone marrow with Foxp3−/− bone marrow. Bcl6−/− Tregs displayed increased levels of the Th2 transcription factor Gata3 and other Th2 and Treg genes. Bcl6 potently repressed Gata3 transcriptional transactivation, providing a mechanism for the increased expression of Th2 genes by Bcl6−/− Tregs. Gata3 has a critical role in regulating Foxp3 expression and functional fitness of Tregs; however, the signal that regulates Gata3 and restricts its transactivation of Th2 cytokines in Tregs has remained unexplored. Our results identify Bcl6 as an essential transcription factor regulating Gata3 activity in Tregs. Thus, Bcl6 represents a crucial regulatory layer in the Treg functional program that is required for specific suppression of Gata3 and Th2 effector responses by Tregs.

Enhancement of hematopoietic recovery after radiation, chemotherapy, or hematopoietic stem cell (HSC) transplantation is clinically relevant. Dipeptidylpeptidase (DPP4) cleaves a wide variety of substrates, including the chemokine stromal cell-derived factor-1 (SDF-1). In the course of experiments showing that inhibition of DPP4 enhances SDF-1-mediated progenitor cell survival, ex vivo cytokine expansion and replating frequency, we unexpectedly found that DPP4 has a more general role in regulating colony-stimulating factor (CSF) activity. DPP4 cleaved within the N-termini of the CSFs granulocyte-macrophage (GM)-CSF, G-CSF, interleukin-3 (IL-3) and erythropoietin and decreased their activity. Dpp4 knockout or DPP4 inhibition enhanced CSF activities both in vitro and in vivo. The reduced activity of DPP4-truncated versus full-length human GM-CSF was mechanistically linked to effects on receptor-binding affinity, induction of GM-CSF receptor oligomerization and signaling capacity. Hematopoiesis in mice after radiation or chemotherapy was enhanced in Dpp4(-/-) mice or mice receiving an orally active DPP4 inhibitor. DPP4 inhibition enhanced engraftment in mice without compromising HSC function, suggesting the potential clinical utility of this approach.

Nuclear transcription factor Stat3 is important for proper regulation of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) proliferation, survival, and cytokine signaling responses. A new, noncanonical role for Stat3 in mitochondrial function has been discovered recently. However, there is little information on the role(s) of mitochondrial Stat3 in HSC/HPC function, especially potential effects of Stat3/mitochondrial dysregulation in human diseases. We investigated hematopoietic cell-targeted deletion of the STAT3 gene in HSCs/HPCs with a focus on mitochondrial function. We found that STAT3(-/-) mice, which have a very shortened lifespan, dysfunctional/dysregulated mitochondrial function and excessive reactive oxygen species production in HSCs/HPCs that coincides with pronounced defects in function. These animals have a blood phenotype with similarities to premature aging and to human diseases of myelodysplastic syndrome and myeloproliferative neoplasms such as erythroid dysplasia, anemia, excessive myeloproliferation, and lymphomyeloid ratio shifts. We show herein that the lifespan of STAT3(-/-) animals is lengthened by treatment with a reactive oxygen species scavenger, which lessened the severity of the blood phenotype. These data suggest a need for more detailed studies of role(s) of Stat3 in HSC/HPC mitochondrial function in human diseases and raise the idea that mitochondrial Stat3 could be used as a potential therapeutic target.