Heather A. O'Leary

Dipeptidylpeptidase 4 negatively regulates colony-stimulating factor activity and stress hematopoiesis

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.

Implications of DPP4 modification of proteins that regulate stem/progenitor and more mature cell types

Dipeptidylpeptidase (DPP) 4 has the potential to truncate proteins with a penultimate alanine, proline, or other selective amino acids at the N-terminus. DPP4 truncation of certain chemokines, colony-stimulating factors, and interleukins have recently been linked to regulation of hematopoietic stem/progenitor cells, more mature blood cells, and other cell types. We believe that the potential role of DPP4 in modification of many regulatory proteins, and their subsequent effects on numerous stem/progenitor and other cell-type functions has not been adequately appreciated. This review addresses the potential implications of the modifying effects of DPP4 on a large number of cytokines and other growth-regulating factors with either proven or putative DPP4 truncation sites on hematopoietic cells, and subsequent effects of DPP4-truncated proteins on multiple aspects of steady-state and stressed hematopoiesis, including stem/progenitor cell, and more mature cell, function.

The role of dipeptidyl peptidase 4 in hematopoiesis and transplantation

Purpose of reviewDipeptidyl peptidase 4 (DPP4, CD26) is a protease that cleaves selected amino acids at the N-terminal penultimate position and has the potential to alter the protein function. The regulation and roles of DPP4 activity are not well understood; therefore, the purpose of this review is to discuss the recent literature regarding DPP4 regulation, as well as the variety of molecules it may affect, and their potential clinical applications. Recent findingsRecent insight into the number of proteins that have DPP4 sites, and how DPP4 truncation may alter hematopoiesis based on the protein full length vs. truncated state, has shown that DPP4 truncation of colony-stimulating factors (CSFs) alters their function and that the activity of these CSFs can be enhanced when DPP4 activity is inhibited. DPP4 inhibition has recently been used in a clinical trial to attempt to enhance the engraftment of cord blood cells, and an endogenous DPP4 inhibitor tissue factor pathway inhibitor has been discovered, increasing our understanding of the potential importance of DPP4. SummaryDPP4 plays a role in regulating the activity of CSFs and other cytokines involved in hematopoiesis. This information may be useful for enhancing hematopoietic cell transplantation, blood cell recovery after stress, and for understanding the physiology and pathophysiology of blood and other cell systems.

The importance of hypoxia and extra physiologic oxygen shock/stress for collection and processing of stem and progenitor cells to understand true physiology/pathology of these cells ex vivo.

Purpose of reviewHematopoietic stem (HSCs) and progenitor (HPCs) cells reside in a hypoxic (lowered oxygen tension) environment, in vivo. We review literature on growth of HSCs and HPCs under hypoxic and normoxic (ambient air) conditions with a focus on our recent work demonstrating the detrimental effects of collecting and processing cells in ambient air through a phenomenon termed extra physiologic oxygen shock/stress (EPHOSS), and we describe means to counteract EPHOSS for enhanced collection of HSCs. Recent findingsCollection and processing of bone marrow and cord blood cells in ambient air cause rapid differentiation and loss of HSCs, with increases in HPCs. This apparently irreversible EPHOSS phenomenon results from increased mitochondrial reactive oxygen species, mediated by a p53-cyclophilin D–mitochondrial permeability transition pore axis, and involves hypoxia inducing factor-1&agr; and micro-RNA 210. EPHOSS can be mitigated by collecting and processing cells in lowered (3%) oxygen, or in ambient air in the presence of, cyclosporine A which effects the mitochondrial permeability transition pore, resulting in increased HSC collections. SummaryOur recent findings may be advantageous for HSC collection for hematopoietic cell transplantation, and likely for enhanced collection of other stem cell types. EPHOSS should be considered when ex-vivo cell analysis is utilized for personalized medicine, as metabolism of cells and their response to targeted drug treatment ex vivo may not mimic what occurs in vivo.

Concise Review: Role of DEK in Stem/Progenitor Cell Biology

Understanding the factors that regulate hematopoiesis opens up the possibility of modifying these factors and their actions for clinical benefit. DEK, a non‐histone nuclear phosphoprotein initially identified as a putative proto‐oncogene, has recently been linked to regulate hematopoiesis. DEK has myelosuppressive activity in vitro on proliferation of human and mouse hematopoietic progenitor cells and enhancing activity on engraftment of long‐term marrow repopulating mouse stem cells, has been linked in coordinate regulation with the transcription factor C/EBPα, for differentiation of myeloid cells, and apparently targets a long‐term repopulating hematopoietic stem cell for leukemic transformation. This review covers the uniqueness of DEK, what is known about how it now functions as a nuclear protein and also as a secreted molecule that can act in paracrine fashion, and how it may be regulated in part by dipeptidylpeptidase 4, an enzyme known to truncate and modify a number of proteins involved in activities on hematopoietic cells. Examples are provided of possible future areas of investigation needed to better understand how DEK may be regulated and function as a regulator of hematopoiesis, information possibly translatable to other normal and diseased immature cell systems. STEM Cells 2013;31:1447–1453

508. IL-33/ST2 Triggering of IL-9-Secreting T Cells: From Proteomics to Therapeutics

As one of the most validated immunotherapies to date, allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative option for high-risk hematological malignancies, particularly acute myeloid leukemia (AML). The immunotherapeutic activity of allo-HCT is known as the graft-vs-leukemia (GVL) activity. However, GVL activity is often accompanied by T-cell reactivity to allo-antigens in normal host tissues, which leads to graft-versus-host disease (GVHD), another major cause of death after HCT. Therefore, there is a great unmet need to improve the current process of allo-HCT through increasing the GVL activity and decreasing GVHD. We have shown that an elevated plasma level of soluble (s)ST2 in HCT patients is a risk factor for severe GVHD. ST2 blockade reduces sST2-producing T cells while maintaining protective membrane (m)ST2-expressing T cells such as type 2 T cells and regulatory T cells during aGVHD. A novel IL-9 producing T helper subset, Th9, expresses mST2. Furthermore, Th9 cells and IL-9 producing CD8 cytotoxic (Tc9) cells have higher antitumor activity than Th1 and Tc1 cells in melanoma models. Interestingly, we found that the addition of IL-33 during T9 differentiation (T9IL-33) increased expression of mST2 and PU.1, a transcription factor that promotes IL-9 production in both CD4 and CD8 T cells. Adoptive transfer of T9IL-33 cells with bone marrow cells in a murine model of HCT resulted in less severe GVHD compared to transfer of T9IL-33 cells generated from ST2−/− or IL-9−/− T cells. Furthermore, cytolytic molecules implicated in anti-leukemic activity (granzyme B and perforin) were upregulated in WT T9IL-33 cells while ST2−/− T9IL-33 cells did not. WT T9IL-33 cells also exhibited higher anti-leukemic activity when cultured with a retrovirally transduced MLL-AF9 leukemic cells in comparison to ST2−/− T9IL-33 in in vitro cytolytic assays. In vivo GVL experiments with MLL-AF9 AML and adoptive transfer of T9IL-33 cells resulted in increased survival compared to syngeneic mice, allo-HCT mice transferred with T1 cells, or T9 cells or T9IL-33 cells generated from ST2−/− or IL-9−/− T cells (Figure 1Figure 1). Human T9 cells are poorly studied. Here we demonstrate that IL-33 has the same impact on human T cells through enhancing IL-9 and Granzyme B production compared to T9 cells as well as demonstrated higher in vitro anti-leukemic cytolytic activity when incubated with MOLM14, an aggressive AML tumor cell line expressing FLT3/ITD mutations. Importantly, CD8α expression was upregulated in WT T9IL-33 (both CD4 and CD8) cells in comparison to ST2−/− T9IL-33 cells, and CD8α blockade with neutralizing antibody during allogeneic specific T9IL-33 differentiation reduced cytotoxicity of both murine T9IL-33, and human T9IL-33 cells as compared to the cell blocked with isotype control, suggesting that CD8α was associated with MHC-restricted cytolytic activity in T9IL-33 cells. Altogether, our observations demonstrated that adoptive transfer of T9IL-33 cells represents a promising cellular therapy following HCT.View Large Image | Download PowerPoint Slide

ID: 95: IL-33/ST2 TRIGGERING OF IL-9–SECRETING T CELLS: FROM PROTEOMICS TO THERAPEUTICS

As one of the most validated immunotherapies to date, allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative option for high-risk hematological malignancies, particularly acute myeloid leukemia (AML). The immunotherapeutic activity of allo-HCT is known as the graft-vs-leukemia (GVL) activity. However, GVL activity is often accompanied by T-cell reactivity to allo-antigens in normal host tissues, which leads to graft-versus-host disease (GVHD), another major cause of death after HCT. Therefore, there is a great unmet need to improve the current process of allo-HCT through increasing the GVL activity and decreasing GVHD. We have shown that an elevated plasma level of soluble (s)ST2 in HCT patients is a risk factor for severe GVHD. ST2 blockade reduces sST2-producing T cells while maintaining protective membrane (m)ST2-expressing T cells such as type 2 T cells and regulatory T cells during aGVHD. A novel IL-9 producing T helper subset, Th9, expresses mST2. Furthermore, Th9 cells and IL-9 producing CD8 cytotoxic (Tc9) cells have higher antitumor activity than Th1 and Tc1 cells in melanoma models. Interestingly, we found that the addition of IL-33 during T9 differentiation (T9IL-33) increased expression of mST2 and PU.1, a transcription factor that promotes IL-9 production in both CD4 and CD8 T cells. Adoptive transfer of T9IL-33 cells with bone marrow cells in a murine model of HCT resulted in less severe GVHD compared to transfer of T9IL-33 cells generated from ST2−/− or IL-9−/− T cells. Furthermore, cytolytic molecules implicated in anti-leukemic activity (granzyme B and perforin) were upregulated in WT T9IL-33 cells while ST2−/− T9IL-33 cells did not. WT T9IL-33 cells also exhibited higher anti-leukemic activity when cultured with a retrovirally transduced MLL-AF9 leukemic cells in comparison to ST2−/− T9IL-33 in in vitro cytolytic assays. In vivo GVL experiments with MLL-AF9 AML and adoptive transfer of T9IL-33 cells resulted in increased survival compared to syngeneic mice, allo-HCT mice transferred with T1 cells, or T9 cells or T9IL-33 cells generated from ST2−/− or IL-9−/− T cells. Human T9 cells are poorly studied. Here we demonstrate that IL-33 has the same impact on human T cells through enhancing IL-9 and Granzyme B production compared to T9 cells as well as demonstrated higher in vitro anti-leukemic cytolytic activity when incubated with MOLM14, an aggressive AML tumor cell line expressing FLT3/ITD mutations. Importantly, CD8α expression was upregulated in WT T9IL-33 (both CD4 and CD8) cells in comparison to ST2−/− T9IL-33 cells, and CD8α blockade with neutralizing antibody during allogeneic specific T9IL-33 differentiation reduced cytotoxicity of both murine T9IL-33, and human T9IL-33 cells as compared to the cell blocked with isotype control, suggesting that CD8α was associated with MHC-restricted cytolytic activity in T9IL-33 cells. Altogether, our observations demonstrated that adoptive transfer of T9IL-33 cells represents a promising cellular therapy following HCT.