scholarly journals Epigenetic Plasticity of Type II Innate Lymphoid Cells in the Lower Gastrointestinal Tract

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1921-1921
Author(s):  
Sonia J. Laurie ◽  
Danny W. Bruce ◽  
Hemamalini Bommiasamy ◽  
Melodie P Noel ◽  
Joseph P. Foster ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Research Funding ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Type Ii ◽  
Hematopoietic Stem ◽  
Chip Sequencing ◽  
Equity Ownership ◽  
The Impact

Though hematopoietic stem cell transplantation (HSCT) is the preferred treatment for a variety of blood malignancies, its use is limited by the development of acute graft-versus-host disease (aGvHD). Type II innate lymphoid cells (ILC2s) are immune cells that play an important role in maintaining mucosal homeostasis, and our lab has previously shown that ILC2s in the gastrointestinal tract (GI) are sensitive to conditioning therapy prior to HSCT. Strikingly, we have demonstrated that the infusion of activated donor ILC2s markedly reduces aGvHD-associated mortality. We therefore wanted to investigate the mechanism of the loss of protective ILC2s from the GI tract. We hypothesized that ILC2s fail to repopulate the gut after HSCT due to inflammatory environmental cues that convert ILC2 precursors to an alternate, ILC1- or ILC3-like fate. Thus, we evaluated the impact of cytokines associated with commitment on murine ILC2s by exposing them to cytokines that may promote differentiation to an ILC1 or ILC3 fate (IL-1b/IL-12/IFN-γ and TGF-b/IL-6/IL-23, respectively). We found ILC2 cells acquired the ability to secrete TNF and IL-17 after in vitro skewing (with these lineage-defining cytokines. To test the ability of these "ex-ILC2" cells to home to other tissues in vivo, GFP-ILC2s were infused into recipients at the time of transplantation. We tracked GFP-ILC2s to the liver and spleen, where they made IFN-g and IL-17 and expressed transcription factors associated with the ILC1 and ILC3 lineages (Figure 1). Next we assessed the ability of cytokines alter ILC2 fate via epigenetic reprogramming by using ChIP-sequencing to evaluate the presence of histone marks that may underlie cellular plasticity. We show that these changes are associated with alterations in epigenetic marks around pioneer, lineage-determining factors. We therefore chose to test a screen of compounds known to modulate a variety of epigenetic targets to ask if they can maintain or convert ILC2s to alternate fates and identified a number of compounds that target bromodomains, methyltransferases, and histonedeacetylases, respectively, that alter the viability and differentiation of ILC2s into an "ex-ILC2"-like phenotype. Preliminary work suggests that maintenance ofG9a expression is able to rescue the loss of ILC2s, which is being tested in vivo. Taken together, these data provide new insights into mechanisms by which innate lymphoid cell precursors are epigenetically regulated, providing novel approaches to treating aGvHD following HSCT. Figure 1 Disclosures Davis: Triangle Biotechnology: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Pattenden:Triangle Biotechnology, Inc.: Equity Ownership, Other: Inventor on intellectual property. Serody:Merck: Research Funding; GlaxoSmithKline: Research Funding.

Death Receptor 3 (DR3) Is Expressed By Innate Lymphoid Cells (ILC) and Ligation By Tumor Like Antigen-1 (TL1A) Leads To Costimulation and Significant ILC Expansion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 782-782
Author(s):  
Michael R. Verneris ◽  
Yong-Oon Ahn ◽  
Matthew A Weeres ◽  
Bruce R Blazar ◽  
Jeffrey S. Miller
Keyword(s):  
T Cells ◽  
Death Receptor ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Lymphoid Tissues ◽  
Fetal Life ◽  
Hematopoietic Stem ◽  
Tnf Superfamily ◽  
Death Receptor 3 ◽  
The Impact

Abstract ROR-gt expressing innate lymphoid cells (ILCs) are unique lymphocytes that play important roles in the immune system and have applications to hematopoietic cell transplantation. In fetal life, ILCs orchestrate lymph node organogenesis, while in adults they facilitate the repair of damaged lymphoid structures and mediate mucosal immunity. ILCs function through the production of IL-22 and the expression of TNF-superfamily molecules (lymphotoxin, BAFF, and OX40L) that act on stroma or other lymphocytes, respectively. ILCs are almost exclusively found in the secondary lymphoid tissues (i.e., lymph nodes) and are essentially absent from the peripheral blood, making the study of these cells difficult and clinical application nearly impossible. To overcome these limitations, we devised a method to generate ILCs from hematopoietic stem cells (HSCs) cultured on irradiated stroma in the presence of cytokines (IL-7, IL-15, SCF and FLT3L) [Blood 11:4052-5, 2011]. After ∼21 days of culture, functional ILCs and conventional NK cells differentiate. While this system robustly leads to the ILCs differentiation, GMP-compatible methods of expansion will be required for clinical translation. We sought to identify factors involved in ILC growth and proliferation. After screening gene expression arrays on purified ILCs, the TNF superfamily receptor, known as Death Receptor 3 (DR3, TNFRSF25) was selected for further study. DR3 expression was confirmed using quantitative PCR. Compared to cNK cells, ILCs expressed significant quantities of mRNA for DR3 (p<0.001). While DR3 contains death receptor signaling domains, under some conditions it can also mediate T cell growth. We assessed DR3 functionality in purified ILCs by stimulating them with recombinant TL1A (TNSF15), the only reported ligand for DR3. Consistent with known effect of TL1A on NF-kB activation in T cells, NK-kB phosphorylation was also observed in ILCs. To determine the impact of TL1A on ILC function, IL-22 production was tested. When TL1A was added to ILCs, there was surprisingly no IL-22 production by intracellular cytokine staining or ELISA. We reasoned that TL1a may costimulate ILC activation and combined TL1A with IL-1b and IL-23, known to activate IL-22 production in ILCs. Compared to IL-1b and IL-23, the addition of TL1A led to a significantly higher percentage of IL-22 producing ILCs (9.3% vs. 23.3%, n=8, p<0.001) and more IL-22 production as determined by ELISA (3399 pg/ml vs. 8757 pg/ml, n=3, p<0.001). Nearly identical results were obtained for IL-8 production (p<0.001). Prior studies in T cells show that DR3 signaling increases the expression of the high affinity IL-2 receptor (CD25). While TL1A alone did not increase CD25 on resting ILCs and IL1b + IL-23 only marginally increased CD25 expression (∼1.7x increase from baseline), the combination of TL1A and IL1b + IL-23 led to significantly higher amounts of CD25 on the surface of ILCs (∼3.8x induction). We then tested whether IL-2 could be used to expand ILCs in vitro. Purified ILCs were treated in the following conditions: media (control), TL1A alone, IL1b + IL-23, or the combination of TL1A + IL-1b + IL-23 for 16 hours. Cells were then washed and cultured in media containing IL-2 (1,000U/ml). In short term (5 day) cultures, there was significantly more proliferation with TL1A + IL-1b + IL-23 (14.4% vs. 21.2% vs. 17.4% vs. 42%, n=7, p<0.001) as measured by CSFE dilution. When ILCs were cultured for 14 days, TL1A + IL-1b + IL-23 resulted in significantly greater expansion than IL1-b + IL23 cultured cells (39.3x vs. 14x, n=7, p=0.007). Since TL1A has been associated with skewing of T cells to IL-17 production and the onset of inflammatory conditions (Crohn’s disease), we assessed the expanded ILCs for the loss of IL-22 and/or the acquisition of IL-17. ILCs expanded in the presence of TLA1 + IL-1b + IL-23 had no change in their surface phenotype or capacity to produce IL-22. Importantly, neither IL-17 nor changes in RORgt or AHR mRNA expression were detected after expansion. Collectively, these studies identify a novel axis where DR3/TL1A signaling costimulates IL1b and IL-23 induced production of IL-22 and results in the expression of IL-2R (CD25) along with the associated proliferative response to IL-2. These studies significantly advance our ability to devise GMP-compliant methods to generate ILCs and pave the way for adoptive transfer experiments using ILCs in humans. Disclosures: Miller: Coronado Biosciences: Scientific Advisory Board Other.

IL-22 Is an Intestinal Stem Cell Growth Factor, and IL-22 Administration in Vivo Reduces Morbidity and Mortality in Murine GvHD

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 651-651
Author(s):  
Caroline A Lindemans ◽  
Anna Mertelsmann ◽  
Margaret Helen O'Connor ◽  
Jarrod A Dudakov ◽  
Robert Jenq ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Paneth Cell ◽  
Innate Lymphoid Cells ◽  
Lymphoid Cells ◽  
Morbidity And Mortality ◽  
Intestinal Stem Cell ◽  
Epithelial Stem Cells ◽  
Hematopoietic Stem

Abstract Graft versus host disease (GVHD) remains a major limitation of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and gut GVHD specifically is a major cause of GVHD-related morbidity and mortality. Little is known about regulation of the intestinal stem cell (ISC) compartment in gut GVHD. We have found that Interleukin-22 (IL-22) produced by innate lymphoid cells is important for ISC recovery after transplant. However, the mechanism of action and specific cellular targets of IL-22 leading to ISC recovery are poorly understood. Using clinically modeled LP into C57BL/6 (B6) minor antigen-mismatched HSCT (H-2 into H-2b), we found that daily treatment with recombinant murine (rm)IL-22 (4ug, intraperitoneal injection) starting day seven after transplant led to reduced intestinal pathology from GVHD without altering alloreactive immunity. Both overall GVHD pathology and epithelial apoptosis scores were significantly lower three weeks post-BMT in rmIL-22-treated mice with GVHD compared to PBS-treated controls (p<0.001). We observed that mice treated with rmIL-22 (and no pharmacologic immunosuppression) had increased numbers of Lgr5+ ISCs and significantly greater ISC proliferation (p<0.01). This was not due to IL-22-dependent changes in the ISC niche, as Paneth cell numbers, Paneth cell-derived growth factors (EGF, Wnt3), and stroma-derived growth factors (Rspo3) were all unchanged after IL-22 administration. However, the antimicrobial proteins Reg3β and Reg3γ were both upregulated by qPCR in small intestine (SI) of rmIL-22-treated mice (p<0.01 and p<0.001 respectively), although this did not result in consistent changes in the gut microbial flora. To evaluate direct effects on epithelial regeneration, we performed intestinal organoid culture assays in the presence of IL-22. Organoids generated from SI and large intestine (LI) crypts of wild-type B6 mice demonstrated substantially increased size after seven days of culture with IL-22 (p<0.001, SI, Fig. 1A; p<0.05, LI). Co-culturing crypts with innate lymphoid cells (ILC), potent producers of IL-22 in vivo, led to increased organoid size as well. Furthermore, culture with IL-22 significantly increased organoid budding (new crypt formation), resulting in increased organoid expansion with serial passaging in the presence of IL-22 (1ng/ml) suggesting that IL-22 could directly increase ISC expansion. Indeed, IL-22 culture led to increased organoid EDU incorporation and expansion of Lgr5+ ISCs after culture of SI crypts from Lgr5-GFP reporter mice (p< 0.001, Fig. 1D). Demonstrating a direct effect on ISCs, IL-22 led to STAT3 phosphorylation specifically in Lgr5+ cells and resulted in increased budding of organoids cultured from isolated single SI ISCs after only four days in culture (p<0.01). To investigate the translational potential for use in humans, we tested a human IL-22 dimer/Fc fusion molecule (F-652, Generon Corp., Shanghai) on mouse intestinal crypts and found that F-652 significantly increased the size of SI and LI organoids. Using the LP into B6 allo-HSCT model described above, we found that every other day subcutaneous (SQ) treatment with 100 ug/kg F-652 starting day seven post-BMT led to significant improvement in both clinical GVHD score (P<0.0001) and survival (p<0.05, Fig. 1C). In summary, we found that IL-22 and innate lymphoid cells can bridge immune function and tissue regeneration by acting directly on epithelial stem cells. IL-22 and F-652 therapy may represent a novel approach to promote intestinal recovery in patients with GVHD without increasing post-transplant immunodeficiency. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Ibrutinib for Bridging to Allogeneic Hematopoietic Stem Cell Transplantation (alloHCT) in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL) Is Safe and Effective: First Results of a Survey By the Chronic Malignancy and the Lymphoma Working Parties of the EBMT

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4657-4657 ◽  
Author(s):  
Peter Dreger ◽  
Mauricette Michallet ◽  
Jennifer Hoek ◽  
Ariane Boumendil ◽  
Mohamad Sobh ◽  
...  
Keyword(s):  
Stem Cell ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Chronic Gvhd ◽  
Disease Status ◽  
Observation Time ◽  
Lymphocytic Leukemia ◽  
Hematopoietic Stem ◽  
The Impact

Abstract BACKGROUND: The advent of the Bruton's tyrosine kinase inhibitor ibrutinib has improved the outlook of patients with CLL and MCL failing chemoimmunotherapy (CIT). However, the impact of ibrutinib on the feasibility and safety of a subsequent alloHCT is unknown. Here we present results of the ibrutinib cohort of an ongoing EBMT survey on the outcome of alloHCT following prior exposure to pathway inhibitors (PI) in patients with CLL or lymphoma (EBMT study code LWP 2013-N-03/CMWP 44204425). DESIGN: Eligible were patients aged >18 years registered with the EBMT data office for a planned alloHCT for CLL or lymphoma after pre-exposure to ibrutinib at any time before transplant. Baseline patient, disease, and transplant data were collected from MED-A forms. Centers were requested to provide additional treatment and follow-up information. Statistical analysis used Gray's test to assess the impact of baseline characteristics on the cumulative incidence of relapse (REL) in a competing risk framework. RESULTS: As of July 4, 2016, 38 patients (84% male) were evaluable in the ibrutinib cohort. Diagnosis was CLL in 28 patients, MCL in 9 patients, and follicular lymphoma (FL) in 1 patient. The median age was 51 (33-68) years and the median number of treatment lines prior to ibrutinib 2 (1-9). Eight of the 9 patients with MCL but none of the other patients had a prior autoHCT. Patients had been on ibrutinib for a median of 190 (39-432) days. In 2 patients, ibrutinib had been stopped because of disease progression >100d before transplant, whereas the interval between ibrutinib withdrawal and alloHCT was 15-100d in 30%, 4-14d in 51%, and 0-1d in 14% of the patients. Of the CLL patients, 43% had a TP53 lesion, and 87% and 79% met at least one of the 2007 and 2014 EBMT criteria for high-risk CLL, respectively, including PI failure in 29%. Disease status at alloHCT was sensitive in 78% of the CLL patients, and in 60% of the patients with lymphoma. Conditioning was reduced-intensity in 60% of the transplants and included in-vivo T cell depletion with ATG (71%) or alemtuzumab (11%) in the majority of cases. Donors were identical siblings in 26%, matched unrelated in 66%, and partially matched unrelated in 8%, with PBSC (89%) being the predominant stem cell source (bone marrow 8%, cord blood 3%). The median time to reach neutrophils of >0.5/nl and platelets of >20/nl was 17 (10-26) and 15 (10-46) d post transplant, respectively. Acute GVHD grade 2-4 (3-4) was observed in 37% (10%) of 30 evaluable patients, and limited and extensive chronic GVHD occurred in 24% and 16% of 25 patients at risk. With a median observation time of survivors of 8 (1-24) months, there were only 2 non-relapse deaths, translating into a 1-year non-relapse mortality (NRM) of 6% (95%CI 0-15%). 1-year REL, progression-free survival, and overall survival was 36%, 61%, and 73% for CLL, and 14%, 75%, and 75% for lymphoma. In the 25 evaluable patients with CLL, PI-sensitive compared to refractory disease status at alloHCT tended to be associated with a lower 1-y REL (29% vs 60%; p 0.071), whereas prior PI failure, TP53 status, duration of ibrutinib exposure, interval between ibrutinib withdrawal and alloHCT, and conditioning intensity had no significant impact on REL. CONCLUSIONS: Ibrutinib for bridging to alloHCT for CLL and MCL does not appear to adversely affect engraftment, GVHD risk, and NRM. Patients with CLL still responding to ibrutinib at the time of alloHCT might benefit from ibrutinib bridging as our preliminary results indicate that also after PI exposure sensitive disease translates into a lower risk of relapse. Therefore, ibrutinib may improve the perspective of CIT-refractory patients scheduled for alloHCT. The optimum timing of ibrutinib administration in the interrelation to alloHCT in CLL and MCL needs to be defined by additional studies. Disclosures Dreger: Gilead: Consultancy; Janssen: Consultancy; Novartis: Speakers Bureau; Gilead: Speakers Bureau; Novartis: Consultancy; Roche: Consultancy. Michallet:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria. Berg:Celgene: Other: Travel Funding; Astellas: Other: Travel Funding; Alexion: Other: Travel Funding. Niederwieser:Novartis Oncology Europe: Research Funding, Speakers Bureau; Amgen: Speakers Bureau. Montoto:Gilead: Research Funding; Roche: Honoraria. Schetelig:Sanofi: Honoraria.

scholarly journals Modeling the Cellular Origin of EVI1 + MLL-AF9-Driven Acute Myeloid Leukemia (AML)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2210-2210
Author(s):  
Hugues-Etienne Chatel-Soulet ◽  
Sabine Juge ◽  
Frederik Otzen Bagger ◽  
Alexandar Tzankov ◽  
Mineo Kurokawa ◽  
...  
Keyword(s):  
Steady State ◽  
Pharmaceutical Company ◽  
Research Funding ◽  
Median Latency ◽  
Hematopoietic Stem ◽  
Cellular Origin ◽  
Type Iv ◽  
Multipotent Progenitors ◽  
The Impact

Abstract We previously reported that transplantation of hematopoietic stem cells (HSC) expressing a doxycycline (DOX)-regulated AML-associated MLL-AF9 fusion transgene can induce an invasive and chemoresistant disease in mice formed by tumour cells that express the transcription factor EVI1, a known marker of poor prognosis in AML and some solid cancers. To better understand the association of EVI1 and the cellular origin of the disease we analyzed Evi1-IRES-GFP reporter mice (female, n=8, 8-10 weeks old) and found that not only the, quiescent long-term hematopoietic stem cell (LT-HSC : lineage marker-negative (lin -) cKit +Sca1 +(= LSK), CD34 -CD135 -CD150 +CD48 -, 24±3.7%, n=8) compartment, but also more proliferating multipotent progenitors such as MPP1 (LSK, CD34 +CD135 -CD150 +CD48 -, 23±4.6%, n=8), MPP2 (LSK, CD34 +CD135 -CD150 +CD48 +, 6±2.3%, n=8) and MPP3 (LSK, CD34 +CD135 -CD150 -CD48 +, 2±1%, n=8) contain a significant number of cells that express abundant Evi1 ("Evi1 high") at steady state. Notably, we did not observe any significant changes in numbers of Evi1 + cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments 5 days after DOX-mediated induction of the iMLL-AF9 fusion. To address the impact of Evi1 on clonogenic growth of iMLL-AF9-expressing LT-HSC, we plated Evi1 high and Evi1 low naïve cells in methylcellulose (MC) and found that upon addition of DOX, Evi1 high cells formed more colonies with an invasive morphology ("type IV") compared to Evi1 low cells (n=11, p&lt;0.05). Immunophenotypically, cells from Evi1 high cell-derived colonies retained a more immature phenotype, reflected by higher cKit +Sca1 + expression (n=11, p&lt;0.05). Plated Evi1 high cells formed Evi1 + colonies whereas Evi1 low lost Evi1 expression (n=11, p&lt;0.05). To address the differential transformation susceptibility in vivo, we transplanted identical numbers of naïve steady-state Evi1 + iMLL-AF9 LT-HSC and MPP1 into irradiated syngeneic recipients. While recipients of Evi1 + MPP1 cells developed an invasive AML earlier than Evi1 + LT-HSC-transplanted mice (n=27, median latency: 96.5 vs. 146.5d, n.s.), very similar disease phenotypes were observed. In contrast, transplants of Evi1 - MPP1 or LT-HSC resulted in a significantly delayed disease induction (n=31, median latency: &gt;200d; LT-HSC: n.s.; MPP1: p&lt;0.05). Although Evi1 + cell-induced disease did present with more extensive organ infiltration by leukemic blasts than Evi1 - AML the phenotypes were similar. We also wondered whether modulation of the HSC compartment by exogenous factors may change Evi1 expression and affect AML induction. We found that 2 days after a single injection (200mg/kg) of recombinant mouse thrombopoietin (mTPO) the number of LT-HSC (n=23; 647 vs. 1165/10 6 lin - cells, p&lt;0.05), but not of MPP1, significantly increased. Similarly, a single application of the synthetic mTPO receptor agonist Romiplostim (RP, 200mg/kg) resulted after 48h in an increase of LT-HSC (n=14; 647 vs. 1459/10 6 lin - cells, p&lt;0.0005). Likewise, a single dose (10mg/kg) of polyinosinic:polycytidylic acid (pI:pC) also significantly increased the number of LT-HSC (n=9; 460 vs. 2300/10 6 lin - cells, p&lt;0.005) but not of MPP1 after 24h. In contrast, 5-Fluorouracil (5-FU; 150mg/kg) did not significantly change the number of LT-HSC and MPP1, 3- and 6-days post-injection. However, only mTPO and RP but not pI:pC or 5-FU significantly increased the fraction of Evi1 high expressing LT-HSC (23 vs. 50%, 23 vs. 49%; n=29, p&lt;0.0001) and MPP1 (22 vs. 47%, 22 vs- 48%; n=29, p&lt;0.0001). Transplantation of identical numbers of iMLL-AF9 LT-HSC and MPP1 isolated 2 days after mTPO application to the donors into irradiated syngeneic recipients resulted in a significantly faster induction of Evi1 + AML than controls (n=19, MPP1: 35 vs. 96.5d, p&lt;0.0001; LT-HSC: 41 vs 146.5d, p&lt;0.0001). Currently ongoing single-cell RNA sequencing experiments of LT-HSC and MPP1 with and without in vivo mTPO stimulation conditionally expressing the iMLL-AF9 fusion should provide some mechanistic insights into increased susceptibility for EVI1 + AML. Our results so far demonstrate that the dynamics of the HSC compartment critically affects the cellular origin and biology of MLL-AF9 driven AML. Disclosures Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau.

scholarly journals Extracellular Tyrosyl-tRNA Synthetase Is a Potent Stimulator of Thrombocytopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1476-1476
Author(s):  
Sachiko Kanaji ◽  
Taisuke Kanaji ◽  
My-Nuong Vo ◽  
Alessandro Zarpellon ◽  
Ryan Shapiro ◽  
...  
Keyword(s):  
Research Funding ◽  
Ex Vivo ◽  
Trna Synthetase ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Equity Ownership

Abstract Aminoacyl-tRNA synthetases (aaRSs) are enzymes with a key role in the first step of protein synthesis by catalyzing the esterification of a specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. During evolution, eukaryotic aaRSs have acquired additional domains and motifs conferring non-canonical functions beyond translation, such as expressing multiple cytokine activities. Repurposing aaRSs often requires an activation step and the first reported example was for human tyrosyl-tRNA synthetase (YRS), which is abundant in platelets and released from their α-granules upon thrombin or arachidonic acid stimulation. As shown by previous work, activated YRS (YRSACT) - created by natural proteolysis, alternative splicing, or rational mutagenesis - can express the activity of different cytokines. In the current study, we demonstrate that recombinant YRSACT rendered active by the gain-of-function mutation Tyr341Ala exhibits a previously unrecognized role in megakaryocytopoiesis and thrombocytopoiesis. When administered in vivo in C57BL/6 wild type (WT) mice, recombinant YRSACT caused platelet increase both under baseline conditions as well as in a model of immune-mediated thrombocytopenia in which mice are made thrombocytopenic by injection of rat anti-mouse glycoprotein (GP) Ib monoclonal IgG. When WT mouse bone marrow (BM) cells were cultured ex vivo for 3 days, YRSACT treatment increased the number of megakaryocytes by 3.0-fold, particularly of megakaryocytes with 16N ploidy. This effect was independent of thrombopoietin (TPO) signaling because YRSACT could support the expansion of c-mpl-/- (TPO receptor knock-out) mouse megakaryocytes. YRSACT had no effect on purified mouse CD41+ or Sca1+ hematopoietic progenitor cells, indicating that YRS-dependent stimulation likely required the contribution of other cells present in BM cultures. When mouse BM cells were stimulated with different doses of YRSACT, the number of F4/80+ monocyte/macrophages as well as of megakaryocytes increased in a dose-dependent manner. Mechanistic analysis revealed YRSACT targets the Toll-like receptor (TLR) pathway signaling through MyD88 in monocyte/macrophages, thereby enhancing release of cytokines that influence megakaryocyte development. In vitro binding assay showed that YRSACT is capable of binding to TLR2 and TLR4. The effect of YRSACT was attenuated in the BM cells derived from TLR2-/- mice and was abolished in MyD88-/- mice. Among the cytokines with synthesis induced by YRSACT, IL-6 plays a pivotal role in megakaryocyte development. Thus, we tested the effect of YRSACT on megakaryocytes obtained by culturing BM cell derived from IL-6-/- mice and found that no effect was apparent. The stimulatory effect of YRSACT on megakaryocytopoiesis was confirmed with human CD41+ megakaryocyte progenitors differentiated from CD34+ hematopoietic stem cells derived from peripheral blood. In conclusion, we have documented a previously unrecognized activity of YRSACT that results in enhanced megakaryocytopoiesis and platelet production. These studies document a mechanistically distinct aaRS-directed hematological activity that highlights new potential approaches to stimulating platelet production for treating thrombocytopenia and for improving ex vivo preparation of platelet concentrates for transfusion. Disclosures Belani: aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties. Do:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Yang:aTyr Pharma: Consultancy, Patents & Royalties, Research Funding. Schimmel:aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals Advances in the Gene Therapy of Patients with Fanconi Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1022-1022 ◽  
Author(s):  
Juan A. Bueren ◽  
Susana Navarro ◽  
Wei Wang ◽  
Rebeca Sanchez-Dominguez ◽  
Eva Merino ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fanconi Anemia ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Insertion Site ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Equity Ownership

Abstract Fanconi anemia (FA) is a DNA repair syndrome characterized by bone marrow failure, congenital abnormalities and cancer predisposition. Based on previous experimental results showing the in vivo proliferative advantage of gene corrected FA patients' hematopoietic stem cells (HSCs; Rio, Navarro et al. Blood 2017) a gene therapy trial in non-conditioned FA-A patients was initiated in 2016. Six patients have been treated to-date using fresh and cryopreserved CD34+ cells mobilized to peripheral blood with G-CSF and plerixafor, and transduced with the PGK-FANCA.Wpre* lentiviral vector. Cell doses infused in four patients with a follow-up of at least 12 months varied from 0.6 to 1.4 million CD34+ cells/kg. Transduction efficacies of these samples, determined as vector copies per cell, ranged from 0.17 to 0.53 copies/cell. Despite the absence of patients' conditioning, a marked in vivo expansion of gene-corrected cells was observed in all hematopoietic cell lineages analyzed in BM and PB. Significantly, up to 44% of corrected cells were determined in total PB cells at the most recent follow-up visit (24 month) in the first treated patient. Insertion site analyses in PB cells showed an oligoclonal pattern of hematopoietic reconstitution, and revealed engraftment of multipotent corrected HSCs and no evidence of insertion-site mediated clonal expansion. Functional studies showed significant increases in the resistance of BM progenitors to mitomycin C in all treated patients. Additionally, patients with higher levels of corrected cells also showed significant increases in the chromosomal stability of T cells exposed to diepoxybutane. Finally, analyses discriminating the presence of corrected and uncorrected PB cells in these patients showed marked increases in the total number of corrected leukocytes, contrasting to progressive decreases of uncorrected cells. Our studies demonstrate for the first time that lentiviral-mediated gene therapy results in progressive engraftment and phenotypic correction of HSCs in non-conditioned FA patients, suggesting that this gene therapy approach may constitute a low-toxicity option for the treatment and prevention of BMF in patients with FA. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Casado:Rocket Pharmaceuticals Inc: Patents & Royalties. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH: Employment; German Cancer Research Center: Employment; bluebird bio: Consultancy. Rio:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals Inc: Honoraria, Patents & Royalties.

scholarly journals Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 368-368
Author(s):  
Jonathan Hoggatt ◽  
Pratibha Singh ◽  
Tiffany Tate ◽  
Peter V. Kharchenko ◽  
Amir Schajnovitz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Knockout Mice ◽  
Peripheral Blood ◽  
Research Funding ◽  
Gene Set Enrichment Analysis ◽  
Hematopoietic Stem ◽  
Equity Ownership

Abstract Hematopoietic stem cells (HSCs) are at the apex of lifelong blood cell production. Recent clonal analysis studies suggest that HSCs are heterogeneous in function and those that contribute to homeostatic production may be distinct from those that engraft during transplant. We developed a rapid mobilization regimen utilizing a unique CXCR2 agonist (an N-terminal truncated MIP-2a) and the CXCR4 antagonist AMD3100. A single subcutaneous injection of both agents together resulted in rapid mobilization in mice with a peak progenitor cell content in blood reached within 15 minutes. This mobilization was equivalent to a 5-day regimen of G-CSF. This rapid mobilization is the result of synergistic signaling, and was blocked in CXCR4 or CXCR2 knockout mice, confirming receptor and mechanism specificity. Mobilization is caused by synergistic release of MMP-9 from neutrophils and mobilization was blocked in MMP-9 knockout mice, mice treated with an anti-MMP-9 antibody, TIMP-1 transgenic mice, or mice where neutrophils were depleted in vivo using anti-GR-1 antibody. In vivo confocal imaging of mice demonstrated that the mobilization regimen causes a rapid and transient increase in bone marrow vascular permeability, "opening the doorway" for hematopoietic egress to the peripheral blood. Transplantation of 2x106 peripheral blood mononuclear cells (PBMC) from the rapid regimen resulted in a 4 or 6 day quicker recovery of neutrophils and platelets, respectively, compared to a G-CSF mobilized graft (n=12 mice per group, P<0.01). In limiting dilution competitive transplants, the rapid regimen demonstrated a greater than 2-fold enhancement in competitiveness (n=30 mice/treatment group, 2 individual experiments, P<0.001). Additionally, in secondarily transplanted mice, competitiveness of the rapidly mobilized graft increased as measured by contribution to chimerism, while G-CSF mobilized grafts remained static (n=16 mice/group, P<0.01). Surprisingly, despite robust enhancement in both short and long-term engraftment by the rapidly mobilized graft, phenotypic analysis of the blood of mobilized mice for CD150+ CD48- Sca-1+ c-kit+ Lineage neg (SLAM SKL) cells, a highly purified HSC population, showed lower numbers of phenotypically defined HSCs than in the G-CSF group. These data suggested that a unique subset of "highly engraftable" HSCs (heHSCs) are mobilized by the rapid regimen compared to G-CSF. However, as our earlier studies were performed using grafts that contained the total PBMC fraction (similar to the clinical apheresis product) we could not rule out the potential contribution of accessory cells to the enhanced engrafting ability of the heHSCs. Therefore, in 3 independent experiments, we mobilized large cohorts of mice with the rapid regimen or G-CSF and sorted SLAM SKL cells from the PBMC fraction and competitively transplanted equal numbers of SLAM SKL cells from either the rapid regimen or G-CSF and tracked contribution to chimerism over 36 weeks. Remarkably, the heHSCs from the rapid regimen demonstrated a 2-fold enhancement in competitiveness compared to SLAM SKL cells from the G-CSF group (n=17 mice/group, P<0.0004). While appreciation for HSC heterogeneity has grown, methods are lacking for prospectively isolating differing HSC populations with known biologic function, to study molecular heterogeneity. Like panning for gold, we sought to use the differential mobilization properties of our rapid regimen and G-CSF as a "biologic sieve" to isolate the heterogeneous HSC populations from the blood. We again flow sorted SLAM SKL cells from mice mobilized with the rapid regimen or G-CSF and performed RNA-Seq analysis of the purified populations. The heHSCs mobilized by the rapid regimen had a unique transcriptomic signature compared to G-CSF mobilized or random HSCs acquired from bone marrow (P<0.000001). Strikingly, gene set enrichment analysis (GSEA) demonstrated that the heHSCs had a gene signature highly significantly clustered to that of fetal liver HSCs, further demonstrating the selective harvesting of a subset of highly engraftable stem cells. Our results mechanistically define a new mobilization strategy, that in a single day can mobilize a graft with superior engraftment properties compared to G-CSF, and selectively mobilize a novel population of heHSCs with an immature molecular phenotype capable of robust long-term engraftment. Disclosures Hoggatt: Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Scadden:Magenta Therapeutics: Consultancy, Equity Ownership; GlaxoSmithKline: Research Funding; Harvard University: Patents & Royalties. Pelus:GlaxoSmithKline: Consultancy.

scholarly journals Rapid Expansion of Gene-Marked Lymphocytes in X-SCID Dogs after AMD3100+G-CSF-Based Hematopoietic Stem/Progenitor Cell Mobilization and Intravenous Injection of a Common γ-Chain Expressing Foamy Viral Vector

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1348-1348
Author(s):  
Frieda Chan ◽  
Olivier Humbert ◽  
Troy Torgerson ◽  
Nicholas Hubbard ◽  
Patricia O'Donnell ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Progenitor Cells ◽  
Research Funding ◽  
Immune Reconstitution ◽  
Viral Vector ◽  
Gamma Chain ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Equity Ownership

Abstract In both humans and canines, X-linked severe combined immunodeficiency disease (XSCID) is caused by mutations in the interleukin-2 receptor gamma chain gene (IL2RG) which results in a lack of response to common gamma-chain (gammaC) dependent cytokines and abnormal development of T and B lymphocytes, and natural killer (NK) cells. Death from infections usually occurs before 1 year of age unless allogeneic hematopoietic cell transplantation (HCT) is performed. While HCT is successful if an HLA-matched sibling donor is available, transplants from mismatched and unrelated donors are associated with greater morbidity and overall survival can be as low as 50%. To circumvent these complications, several clinical trials are testing the possibility of utilizing blood and marrow stem cells from the patient for ex vivo gene therapy to treat X-SCID. Although these trials show promising results, they require expensive GMP cell manufacturing that are not accessible to many patients, and may also necessitate low level of conditioning to improve engraftment of gene-corrected cells. With these limitations in mind, we have explored in vivo gene therapy as a treatment for X-SCID. We previously showed that foamy virus vectors (FVs), exhibit a potentially more favorable integration profile compared to lenti- and gamma-retroviral vectors. In vivo delivery of a gammaC-FV in dogs resulted in immune reconstitution with gene-corrected T cells in dogs but the treated animals still developed infections and had low levels of immunoglobulin levels. We hypothesized that an increased transduction of hematopoietic stem/progenitor cells in vivo might result in more rapid and sustained immune reconstitution. Thus, in the current study, we used cG-CSF and AMD3100 to mobilize hematopoietic stem/progenitor cells into the peripheral blood prior to in vivo injection with a FV expressing the gammaC gene driven by a PGK promoter (PGK-gammaC-FV). We mobilized two X-SCID dogs at ~3 weeks of age with 5ug/kg of cG-CSF bi-daily from day -4 to -1 prior to FV injection, and with 4mg/kg of AMD3100 on the morning of the injection with 4x10e8 IU of PGK-gammaC-FV. Our mobilization protocol resulted in a 10-fold increase in CD34+ cells in the peripheral blood of mobilized X-SCID dogs as compared to a unmobilized normal littermate control (Figure 1 A). Lymphocyte recovery and gene marking in the mobilized animals was significantly improved as compared to animals that were previously injected with similar doses of either PGK-gammaC-FV or EF1a-gammaC-FV but without mobilization. As illustrated in Figure 1B-C, lymphocyte counts expanded to ~3000 cells/uL with ~75% gene marking in the mobilized animals treated with PGK-gammC-FV within 30 days, as compared to <1500 cells/uL with <5% gene marking in unmobilized dogs treated with EF1a-gammaC-FV and to <1000 cells/uL with <50% gene marking in unmobilized dogs treated with PGK-gammaC-FV at all time points post-therapy. The expansion of CD3+ T-cells at 6 weeks post injection for the mobilized dogs was about 2700 cells/uL, as compared to <380 cells/uL in the PGK-gammaC-FV and <210 cells/uL in the EF1a-gammaC-FV unmobilized dogs. Notably, in human clinical trials, CD3 T cell counts were <250 cells/uL following transplantation with autologous CD34+ cells modified with EF1a-gammaC-SIN gamma-retrovirus (Hacein-Bey-Abina, NEJM, 2014). In conclusion, mobilization with cG-CSF and AMD3100 prior to in vivo injection of PGK-gammaC-FV substantially improved the lymphocyte expansion and immune reconstitution in X-SCID dogs and resulted in a higher level of gene marking in myeloid cells (about 1%) at one-month post injection than seen in our previous studies in unmobilized dogs. These results suggest remarkable potential for an accessible and portable approach for treatment of human X-SCID clinical trials using combination of hematopoietic stem/progenitor cells mobilization and in vivo foamy viral vector delivery. Disclosures Adair: Rocket Pharmaceuticals: Consultancy, Equity Ownership. Scharenberg:bluebird bio: Consultancy, Equity Ownership, Research Funding; Alpine Immune Sciences: Consultancy. Kiem:Rocket Pharmaceuticals: Consultancy, Equity Ownership, Research Funding.

scholarly journals CD5 Surface Expression Marks Intravascular Human Innate Lymphoid Cells That Have a Distinct Ontogeny and Migrate to the Lung

2021 ◽  
Vol 12 ◽  
Author(s):  
Arlisa Alisjahbana ◽  
Yu Gao ◽  
Natalie Sleiers ◽  
Elza Evren ◽  
Demi Brownlie ◽  
...  
Keyword(s):  
Surface Expression ◽  
Innate Lymphoid Cells ◽  
Immune Defense ◽  
Lymphoid Cells ◽  
Humanized Mouse Model ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Effector Cytokines

Innate lymphoid cells (ILCs) contribute to immune defense, yet it is poorly understood how ILCs develop and are strategically positioned in the lung. This applies especially to human ILCs due to the difficulty of studying them in vivo. Here we investigated the ontogeny and migration of human ILCs in vivo with a humanized mouse model (“MISTRG”) expressing human cytokines. In addition to known tissue-resident ILC subsets, we discovered CD5-expressing ILCs that predominantly resided within the lung vasculature and in the circulation. CD5+ ILCs contained IFNγ-producing mature ILC1s as well as immature ILCs that produced ILC effector cytokines under polarizing conditions in vitro. CD5+ ILCs had a distinct ontogeny compared to conventional CD5- ILCs because they first appeared in the thymus, spleen and liver rather than in the bone marrow after transplantation of MISTRG mice with human CD34+ hematopoietic stem and progenitor cells. Due to their strategic location, human CD5+ ILCs could serve as blood-borne sentinels, ready to be recruited into the lung to respond to environmental challenges. This work emphasizes the uniqueness of human CD5+ ILCs in terms of their anatomical localization and developmental origin compared to well-studied CD5- ILCs.

Ellagitannins, Gallotannins and their Metabolites- The Contribution to the Anti-Inflammatory Effect of Food Products and Medicinal Plants

2019 ◽  
Vol 25 (37) ◽  
pp. 4946-4967 ◽  
Author(s):  
Anna K. Kiss ◽  
Jakub P. Piwowarski
Keyword(s):  
Immune Response ◽  
Gastrointestinal Tract ◽  
Gut Microbiota ◽  
Health Effects ◽  
Biological Effects ◽  
Food Products ◽  
Anti Inflammatory ◽  
The Impact

The popularity of food products and medicinal plant materials containing hydrolysable tannins (HT) is nowadays rapidly increasing. Among various health effects attributable to the products of plant origin rich in gallotannins and/or ellagitannins the most often underlined is the beneficial influence on diseases possessing inflammatory background. Results of clinical, interventional and animal in vivo studies clearly indicate the antiinflammatory potential of HT-containing products, as well as pure ellagitannins and gallotannins. In recent years a great emphasis has been put on the consideration of metabolism and bioavailability of natural products during examination of their biological effects. Conducted in vivo and in vitro studies of polyphenols metabolism put a new light on this issue and indicate the gut microbiota to play a crucial role in the health effects following their oral administration. The aim of the review is to summarize the knowledge about HT-containing products’ phytochemistry and their anti-inflammatory effects together with discussion of the data about observed biological activities with regards to the current concepts on the HTs’ bioavailability and metabolism. Orally administered HT-containing products due to the limited bioavailability of ellagitannins and gallotannins can influence immune response at the level of gastrointestinal tract as well as express modulating effects on the gut microbiota composition. However, due to the chemical changes being a result of their transit through gastrointestinal tract, comprising of hydrolysis and gut microbiota metabolism, the activity of produced metabolites has to be taken into consideration. Studies regarding biological effects of the HTs’ metabolites, in particular urolithins, indicate their strong and structure-dependent anti-inflammatory activities, being observed at the concentrations, which fit the range of their established bioavailability. The impact of HTs on inflammatory processes has been well established on various in vivo and in vitro models, while influence of microbiota metabolites on silencing the immune response gives a new perspective on understanding anti-inflammatory effects attributed to HT containing products, especially their postulated effectiveness in inflammatory bowel diseases (IBD) and cardiovascular diseases.

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