scholarly journals CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1054-1054 ◽  
Author(s):  
Hongxing Liu
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Lymph Nodes ◽  
Conflicts Of Interest ◽  
Sh2 Domain ◽  
Janus Kinase ◽  
Gingival Hyperplasia ◽  
Hematopoietic Stem ◽  
T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

Comparative Analysis on Cellular Components of Bone Marrow Microenvironment in Normal and Aberrant Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4808-4808
Author(s):  
Young-Ho Lee ◽  
Young-hee Kwon ◽  
Kyoujung Hwang ◽  
Hyunju Jun ◽  
Byungbae Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Conflicts Of Interest ◽  
T Cell Subsets ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Stromal Cell Derived Factor ◽  
And Control ◽  
Cellular Components

Abstract Abstract 4808 Background: It is now evident that hematopoietic stem cells (HSCs) reside preferentially at the endosteal region within the bone marrow (BM) where bone-lining osteoblasts are a key cellular component of the HSC niche that directly regulates HSC fate. We investigated the microenvironmental differences including osteoblastic activities and HSC components in myeloproliferative (chronic myeloid leukemia, CML) and hypogenerative disease (aplastic anemia, AA) as well as normal control (NC). Methods: The immunohistochemistry for osteonectin, osteocalcin, stromal cell derived factor (SDF, CXCL12), T cell, T helper/inducer cell, T suppressor/cytotoxic cell, hematopoietic stem/progenitor (CD34, CD117) and megakaryocytes was performed on BM biopsy specimens from 10 AA patients, 10 CML patients and 10 NC (lymphoma without BM involvement). The positive cells for immunohistochemical stainings except osteocalcin on each slide were calculated on 10 high power fields (HPF, ×400), and then corrected by the cellularity. The positive cells for osteocalcin were counted on the peritrabecular line on each slide, and then corrected by the mean length measured. Results: The CD34+ cells (p=0.012) and megakaryocytes (p<0.0001) were significantly lower in AA than in NC, but CD117+ cells was comparable in AA, CML, and control samples. The osteonectin+ cells (p=0.0003) were lower in CML than in AA and NC, however the osteocalcin+ cells showed wide variation (0-903/2035um) and no significant difference. The SDF+ cells (p<0.0001) was significantly higher in AA and very lower in CML, compared with NC. The counts for T cell and T cell subsets were significantly lower in CML than in NC, and higher in AA than in NC (p<0.0001). Conclusions: Cellular components of BM microenvironment in 2 hematologic diseases representative of myeloproliferation (CML) and hyporegeneration (AA) respectively are quite different. Further studies would be required to explore the role of these components for hematopoiesis and the rationale for therapeutic application. Disclosures: No relevant conflicts of interest to declare.

Hhex Is a Critical Gene In The Development Of Normal and Malignant Lymphoid Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3788-3788
Author(s):  
Charnise Goodings ◽  
Stephen B. Smith ◽  
Elizabeth Mathias ◽  
Elizabeth Smith ◽  
Rati Tripathi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Transgenic Mice ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Conditional Knockout ◽  
Hematopoietic Stem ◽  
Direct Transcriptional Target ◽  
Cell Counts ◽  
Transcriptional Target

Abstract Hematopoietically expressed homeobox (Hhex) is a T-cell oncogene. It is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. We discovered that HHEX is a direct transcriptional target of an LIM domain Only-2 (LMO2)-associated protein complex. HHEX clusters with LMO2-overexpressing T-ALLs and is especially overexpressed in Early T-cell Precursor (ETP) – ALL where it is a direct transcriptional target of LMO2. To further understand Hhex's function, we induced a conditional knockout in floxed Hhex mice with the Vav-iCre transgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhex in all hematopoietic tissues. Thymocytes from conditional knockouts showed a normal pattern of development. Most impressively, Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice (figure 1). Hhex conditional knockouts (Hhex cKOs) also had a significant decrease in mature B cells in the spleen and bone marrow. Interestingly, hematopoietic stem and progenitor cells plated on OP9-GFP or OP9-DL1 stromal cells showed proliferative defects and incomplete differentiation towards both B and T lineage. Also under stress conditions such as sublethal irradiation and competitive bone marrow transplants, Hhex conditional knockouts show a marked defect in both B and T lineages but an increase in early progenitor populations. Our experiments show that Hhex is a critical transcription factor in lymphoid development and in LMO2-induced T-ALL.Figure 1Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic miceFigure 1. Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regulation of Notch Signaling By O-Glycans during Lymphopoiesis and Myelopoiesis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2170-2170
Author(s):  
Ankit Tanwar ◽  
Pamela Stanley
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
B Cell ◽  
Notch Signaling ◽  
Target Genes ◽  
Myeloid Cell ◽  
T Cell Subsets ◽  
Hematopoietic Stem ◽  
Glcnac Transferase

Abstract Introduction Notch signaling is essential for the optimal generation of T, B and myeloid cells. Epidermal growth factor-like (EGF) repeats in the extracellular domain of Notch receptors are modified by O-fucose and O-GlcNAc glycans transferred by protein O-fucosyltransferase 1 (POFUT1) and EGF O-GlcNAc-transferase (EOGT), respectively. EOGT promotes Notch ligand binding and Notch signaling in the developing retina in mice, and its loss is the basis of Adam's Oliver Syndrome 4 (AOS4) in humans. The loss of POFUT1 in humans leads to the Dowling Degos Disease 2 (DDD2). Thus, O-fucose and O-GlcNAc glycans may have different functions in Notch signaling, reflected in different requirements for T, B or myeloid cell development. Methods: Eogt control, Eogt null, Pofut1 floxed and Vav1-iCre transgenic mice were used to generate single and compound mutant mice with inactive Eogt, Pofut1 or both Eogt and Pofut1. Antibody markers of hematopoietic stem progenitor cell (HSPC), lymphoid and myeloid subsets were used to identify different T, B and myeloid cell subsets by flow cytometry using the Cytek TM Aurora Flow Cytometer. FCS files were analyzed using FlowJo software (BD). CD45.1+ (B6.SJL-Ptprc a Pepc b/BoyJ #002014) congenic mice were used for bone marrow transfer. Expression of NOTCH1 and binding of soluble Notch ligands to DN T cells was analyzed by flow cytometry. Expression of different Notch target genes was determined using qRT-PCR anlaysis. Results: Eogt null mice exhibited altered production of certain T cell subsets in thymus and B cell subsets in spleen, most similar to alterations observed in mice lacking LFNG, MFNG and RFNG. This phenotype was cell-autonomous as Eogt null bone marrow cells transferred defective T and B cell development to irradiated recipient mice. More severe defects in hematopoiesis were observed in mice conditionally lacking Pofut1 in hematopoietic stem cells (HSC) via Vav1-iCre. However , deletion of both O-fucose and O-GlcNAc glycans together in Eogt:Pofut1 double knockout HSC, led to the most severe decrease in common lymphoid and myeloid progenitors in bone marrow. In thymus, the dramatic reduction in T cell subsets observed in Pofut1 cKO mice was reduced further (in DN2 subsets) in Eogt:Pofut1 dKO mice. In spleen, there was a significantly greater decrease in follicular B and other B cell populations, and a greater increase in CD11b/c+ and Gr1+ myeloid cells in Eogt:Pofut1 dKO mice. Splenomegaly occurred in both Pofut1 cKO and Eogt:Pofut1 dKO mice, with a greater increase in extramedullary hematopoiesis in the dKO spleen. Binding of soluble DLL4-Fc was &gt;90% reduced in Pofut1 cKO DN T cells, and not further reduced in dKO cells, while NOTCH1 expression at the cell surface was only slightly reduced. The relative expression of Notch target genes including Hes1, CD25, cMyc, and Deltex1 was similarly reduced in Pofut1 cKO and Eogt:Pofut1 dKO DN T cell progenitors. Conclsuion: The combined data suggest that O-fucose and O-GlcNAc glycans act in an additive fashion to optimize Notch signaling in lymphoid and myeloid differentiation. Keywords: Notch Signaling, O-Glycans, Protein O-fucosyltransferase 1 (POFUT1), O-GlcNAc transferase (EOGT), Lymphopoiesis, Myelopoiesis. Disclosures Stanley: Aviceda Therapeutics, Inc.: Consultancy, Current holder of stock options in a privately-held company.

RUNX1C-Mediated Reprogramming Of Human Bone Marrow Stromal Cells into Early Blood Progenitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2463-2463
Author(s):  
Weihong Yin ◽  
Christopher D Porada ◽  
Stephen Walker ◽  
Colin Bishop ◽  
Graca Almeida-Porada
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Dermal Fibroblasts ◽  
Hematopoietic Stem ◽  
Surface Marker Expression ◽  
Somatic Cell Reprogramming ◽  
Hematopoietic Lineage

Abstract Somatic cell reprogramming to the hematopoietic lineage, either through a pluripotent state or directly, opens the possibility of production of a ready source of autologous hematopoietic stem cells (HSC) that can be used to treat/cure a wide variety of blood disorders. While it has previously been shown that dermal fibroblasts (HFF) can be directly reprogrammed to the hematopoietic lineage, the efficiency was relatively low and the resultant hematopoietic cells lacked multilineage differentiative potential. Stro1(+) isolated stromal progenitors (SIPs) can easily be isolated from the bone marrow (BM) and expanded ex-vivo to obtain clinically significant numbers of cells. In similarity to HSC, SIPs are derived from the mesoderm, and are intimately linked with HSC specification during ontogeny. As such, they are likely to be epigenetically closer to HSC than HFF, and therefore good candidates for reprogramming into hematopoietic cells. To verify the uniqueness of SIPs for reprogramming, we transduced SIPs and HFF with OCT4 and/or RUNX1C, a master transcription factor (TF) that triggers the developmental onset of definitive hematopoiesis, in the following combinations: 1) OCT4 alone; 2) RUNX1C alone; or 3) OCT4+RUNX1C. We then performed a timeline of gene/cell surface marker expression (using microarray, qRT-PCR, and flow cytometry) from day 3-16 post-transduction. Visual inspection of the cultures showed that, while reprogrammed colonies began to appear in SIPs cultures at day 9, no colonies were seen during this time period in HFF cultures. Flow cytometry and molecular analyses of colonies obtained from OCT4+RUNX1C combination demonstrated that expression of CD41, the earliest marker of commitment to the hematopoietic lineage, commenced within only 3-4 days and peaked at day 5-6, by which time ∼20% of SIPs expressed this marker. This peak in CD41 expression coincided with commencement of expression of CD34 and CD45, and maximal induction of several hematopoiesis-specific TFs and phenotypic markers such as PU.1, HOXB4, GATA2, MIXL, WNT3, KDR, CDX4, which occurred at 1-3 logs higher levels in SIPs than HFF. Further studies demonstrated that the chromatin remodeling function of OCT4 could be replaced with the histone methyltransferase inhibitor Bix-01294, with the combination of RUNX1C and Bix-01294 inducing levels of CD34 and CD41 expression by day 5 that were similar to those achieved with RUNX1C plus OCT4. The present studies thus take several important steps towards making the promise of producing autologous hematopoietic cells for transplantation via direct reprogramming a reality. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Probing Co-Operating Somatic Mutations in MLL-ENL Driven Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2855-2855
Author(s):  
Amol Sanjay Ugale ◽  
Sofia Adolfsson ◽  
Petter Säwén ◽  
Monika Dudenhöffer-Pfeifer ◽  
David Bryder
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Acute Leukemia ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Cell Generation ◽  
Driver Mutations ◽  
Human Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Missense mutations and other chromosomal perturbations underlie the heterogeneity of acute leukemia. While Mixed lineage leukemia-1 (MLL1/KMT2A) translocations are recurrent in human acute leukemia, most if not all MLL fusions are permissive for either acute myeloid (AML), acute lymphoid (ALL) or mixed/biphenotypic leukemia (MLL). At the same time, previous work have suggested that MLL rearranged leukemias require few additional mutations to induce transformation(1, 2). The MLL-ENL translocation is apart from being associated with AML also found in human acute T cell leukemia (T-ALL). Despite this, but consistent with most previous observations in mice, we failed to observe development of ALL even when inducing MLL-ENL in multiple early hematopoietic progenitors(3). We hypothesized that this could be caused by a compromised T cell generation induced by the MLL-ENL translocation product. Culturing bone marrow Granulocyte-Monocyte-Lymphoid Progenitor (GMLPs) on OP9-DL1 stromal cells revealed that MLL-ENL blocked differentiation at a stage that phenotypically corresponds to a primitive DN1 stage. In vivo, short-term induction of MLL-ENL led to deregulation of T cell differentiation, with an evident block at the DN2/3 stage. As such a compromise in T cell generation could omit T-ALL development from immature T cell progenitors in the bone marrow, we next assessed the leukemic capacity of defined lymphoid progenitor cells at different stages of development. The latent myeloid potential of DN1 cells and early B lymphoid progenitors (BLPs), a property lost upon further differentiation of these cells, was sufficient to confer potent leukemia initiating activity for AML. By contrast, mice transplanted with of later stages of T cell and B cell progenitors failed to associate with development of disease. The discrepancy of lineage assignment between MLL translocations in established human leukemia and attempts to mimic these in the mouse might be due to a requirement of necessary co-mutations. Although MLL fusions typically associate with few secondary mutations, previous studies have established that activating somatic mutations in the RAS pathway are frequently co-occurring with MLL rearranged leukemias. Therefore, we next investigated whether mutation order might influence on the developing murine leukemia. Co-expression of KRASG12D in GMLPs led to a significant reduction in disease latency compared to MLL-ENL alone. However, disease was still restricted to the myeloid lineage. In strikingly contrast, but in agreement with previous reports, mice receiving KRASG12D expressing GMLPs in the absence of MLL-ENL expression developed T-ALL with a median latency of 143 days and with roughly a 30% penetrance. The onset of MLL-ENL expression 5 weeks post transplantation in KRASG12D expressing GMLPs also gave rise to T-ALL, but with a shorter mean latency (111 days) and higher (50%) penetrance. The sequence of acquisition of somatic mutations therefore can influence not only on disease latency, but also on the lineage assignment of the developing leukemia. Finally, to identify novel somatic mutations occurring during MLL-ENL induced leukemogenesis, we generated paired leukemic samples by transplanting leukemia initiating cells from individual donor mice into separate cohorts of recipients, followed by whole genome and exome sequencing upon development of AML. While MLL-ENL induced AML associated with very few secondary mutations, targeted re-sequencing of a panel of variants validated a few single nucleotide variants from leukemic samples, including amongst others driver mutations in PTPN11 and RAS known from studies of human leukemia. Further studies are now undertaken to obtain functional insights into the mode of action of the identified somatic mutations. References 1. Andersson AK, Ma J, Wang J, Chen X, Gedman AL, Dang J, et al. The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias. Nature genetics. 2015;47(4):330-7. 2. Cancer Genome Atlas Research N. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. The New England journal of medicine. 2013;368(22):2059-74. 3. Ugale A, Norddahl GL, Wahlestedt M, Sawen P, Jaako P, Pronk CJ, et al. Hematopoietic stem cells are intrinsically protected against MLL-ENL-mediated transformation. Cell reports. 2014;9(4):1246-55. Disclosures No relevant conflicts of interest to declare.

In Vivo Assessment of T Effector, T Regulatory Cell, Dendritic Cell Interaction in Lymphoid Tissue Using Intravital Imaging During Acute GvHD

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 818-818
Author(s):  
Kaifeng Lisa Lin ◽  
Michelle L West ◽  
James Coghill ◽  
Stephen Jones ◽  
James E Bear ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Random Walk ◽  
T Cell ◽  
T Cell Activation ◽  
Lymphoid Tissue ◽  
Conflicts Of Interest ◽  
Activation Process ◽  
Hematopoietic Stem ◽  
T Effector Cells

Abstract Abstract 818 Graft-versus-host disease (GvHD) is a complication that results from minor and/or major MHC incompatibilities between the donor and the recipient after hematopoietic stem cell transplantation (HSCT). Although the importance of donor T effector cells (Teffs) in inducing GvHD is well-established, the early events by which host antigen presenting cells (APCs) activate allogeneic T cells are not well described. In pathogen specific immunity, after T cells migrate to the lymph node, they initially form brief contacts with dendritic cells (DCs). This phase lasts for 6–8 hours, which is termed random-walk. Following this phase, T cells establish long-lasting arrest on DCs. At least 6 hours of stable T cell-DC interaction are required for na•ve T cells to undergo clonal expansion. After the phase of long-lasting contact, T cells start to proliferate and expand. At the same time (20∼24 hrs), their interactions with DCs become very short again. It is not clear if allogeneic T cells follow a similar tri-phasic activation process. In addition, studies on autoimmunity have shown that Tregs can prevent T cell activation by prolonged interaction with APCs. However, the mechanisms by which donor regulatory T cells (Tregs) regulate T effector responses in GvHD in lymphoid tissue has not described previously. In this study, we have used intravital microscopy to monitor the movement of donor na•ve T cells and Tregs within lymph nodes after bone marrow transplantation (BMT) in a C57BL/6 to BALB/c murine model (Figure 1A, DCs are green, T cells are red). We have found that donor na•ve T cells do not follow the triphasic activation process demonstrated by von Andrian's group for pathogen-specific transgenic T cells. Instead, a substantial minority of MHC mismatched allogeneic T cells demonstrate a diminished velocity within 4 hours of adoptive transfer. These data indicate that prolonged interactions with host DCs develop rapidly post transplant. The velocity of allogeneic T cells increased at 18 hours post BMT. Peak velocity of Teff cells occurred 24 hours post BMT, consistent with the movement pattern of activated syngeneic T cells.Figure 1.Figure 1. We have also observed that although the speed of donor Treg movement is very similar to na•ve T cell 4∼6 hours after BMT, the velocity of donor Tregs is much slower than the velocity of Teffs 18 hours post BMT. This slow speed persists even at 24 hours post BMT, indicating that Tregs form very stable contacts with host DCs. Interestingly, donor na•ve T cells move significantly faster when transplanted with Tregs at a 2:1 (T:Treg) ratio (Figure 1B), suggesting that Tregs interfere with interactions between na•ve T cells and DCs. We conclude that allogeneic na•ve T cells do not go through a random-walk phase in order to interact with host DCs early post HSCT. The transplantation of Tregs with donor T cells and bone marrow cells significantly increased the velocity of donor na•ve T cells, probably diminishing the interaction of Teff cells with DCs after BMT. We believe that the large number of APCs able to present antigen in the allogeneic setting allows for rapid activation of donor Teff cells and precludes the necessity of the random-walk phase characteristic of pathogen-specific T cells. Disclosures: No relevant conflicts of interest to declare.

Lmo2 Overexpression and Arf Loss Induce Myeloid Differentiation in Primitive Thymocytes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 47-47
Author(s):  
Louise M. Treanor ◽  
Sheng Zhou ◽  
Taihe Lu ◽  
Charles G Mullighan ◽  
Brian P. Sorrentino
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Myeloid Differentiation ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Blast Cells

Abstract Abstract 47 Lmo2 overexpression and Arf loss induce myeloid differentiation in primitive thymocytes LMO2 is a hematopoietic transcription factor that is deregulated as a consequence of chromosomal translocations in T-cell leukemia. Recently we reported that Lmo2 overexpression collaborates with loss of the p19Arf tumor suppressor to induce central T cell leukemias in mice, in part by conferring an increase in self-renewal and engraftment potential in thymic repopulating cells (Treanor LM et al, Blood 2011). Primary recipients were transplanted with Lmo2-transduced, Arf−/− DN2 (CD4−CD8−CD44+CD25+) thymocytes that were cultured on OP9-DL1 cells for 20 days before transplant. Primary recipients engrafted and secondary transplants were later performed. Several secondary recipients developed acute myelogenous leukemia originating from the thymocyte graft. These mice had elevated white blood counts between 200–500×103/μl and their spleen, thymus, bone marrow and peripheral blood contained 90% mCherry+, Gr1+, Mac1+ cells. Both the spleen and liver were infiltrated with myeloperoxidase positive blast cells and pathological review confirmed acute myeloid leukemia. Tertiary irradiated hosts transplanted with these cells developed a Gr1+ tumor with the same phenotype as the secondary animal. All of the tumor cells observed had a high expression level of the mCherry vector indicating that Lmo2 was expressed in the blast cells. Vector integration site clonality analyses confirmed that the vector was present in the blast cells and was from the same clone in the primary, secondary and tertiary recipient. This data led to the hypothesis that enforced Lmo2 expression and Arf loss may reprogram DN2 thymocytes to obtain myeloid differentiation potential. Transduced DN2 thymocytes were then assayed for myeloid colony formation in semisolid cultures containing cytokines that are specific for myeloid differentiation. Initially CD4−CD8− thymocytes were selected from Arf+/+ and Arf−/− thymi, transduced with either control vector or Lmo2 and cultured on OP9-DL1 stromal cells for 20 days. At day 20 the thymocytes were sorted for the vector positive DN2 population and 5×104 of these DN2 thymocytes were plated into these semisolid cultures. Only thymocytes that contained the Lmo2 vector were able to form myeloid colonies and this colony forming ability was greatly enhanced by the absence of Arf (n=3) as shown in figure 1. Moving to an in vivo assay, sublethally irradiated Rag2−/−γc−/−were transplanted with 2×105 transduced DN2 thymocytes. Three weeks after transplant the spleen, bone marrow and peripheral blood contained greater than 50% mCherry+ cells and of these cells between 3%-10% were Mac1+Gr1+ double positive. Vector+ (mCherry+) cells were sorted from the bone marrow and plated in semisolid culture with myeloid cytokines. After seven days the cultures were positive for myeloid colonies that were mCherry+, Gr1+, Mac1+. These in vivo and in vitro assays demonstrate that Lmo2 induces myeloid potential in DN2 thymocytes. These data indicate that Lmo2 expression combined with loss of the Arf locus may recapitulate a hematopoietic stem cell (HSC) “state” in the DN2 thymocytes as HSCs express relatively high levels of Lmo2 and do not express p19Arf due to Bmi1-mediated epigenetic suppression. The novel reprogramming events that we now report could have relevance to early thymic precursor leukemia, in which various degrees of myeloid conversion are noted. We recently documented high amounts of Lmo2 mRNA expression in pediatric early thymic precursor leukemia by expression array analysis in 11/12 cases. Disclosures: No relevant conflicts of interest to declare.

Abnormalities of the Bone Marrow Immune Microenvironment in Patients with Prolonged Isolated Thrombocytopenia after Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4602-4602
Author(s):  
Yang Song ◽  
Yuan Kong ◽  
Min-Min Shi ◽  
Yu-Qian Sun ◽  
Yu Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Microenvironment ◽  
Hematopoietic Stem ◽  
Cell Subpopulations ◽  
T Cell Subpopulations

Abstract Background:Prolonged Isolated Thrombocytopenia (PT), is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and defined as the engraftment of all peripheral blood cell lines other than a PLT count ≤20×10E+9/L or dependence on PLT transfusions for more than 90 days after allo-HSCT. Nevertheless, the mechanisms underlying PT remain unclear. Recent studies have presumed that the mechanism of PT might be similar, at least in part, to that of Immune Thrombocytopenia (ITP). BM immune microenvironment is considered to be involved in the regulation of hematopoiesis, and also influence the production of platelets. There is growing evidence that activated CD8+ T cells in the bone marrow (BM) of patients with ITP might suppress megakaryocyte apoptosis, leading to impaired platelet production. In our previous study, we also found the deregulated T cells responses in BM were associated with ITP patients. Therefore, we hypothesized aberrant immune microenvironment may also influence the production of platelet after allo-HSCT, contributing to the occurrence of PT, so we conducted a study to analyze the alteration of T cell subpopulations and cytokines in BM micro-environment of allotransplant patients. Aims:To compare the cellular compositions and function of T cells in BM microenvironment between patients with PT and good graft function (GGF) after allo-HSCT. Methods:Using a prospective nested case-control study, the T cell subpopulations in BM were analyzed by flow cytometry in 15 patients with PT, 30 matched patients with GGF after allo-HSCT, and 15 healthy donors (HDs). The fractions of T cells, including Th1, Tc1,Th2, Tc2 ,Th17 and Treg were identified as CD3+CD8-IFN-gama+, CD3+CD8-IFN-gama+, CD3+CD8+IL4+, CD3+CD8+IL-4+, CD3+CD8-IL17A+ and CD3+CD4+CD25+Foxp3+, respectively. The levels of IFN-gama, IL-4 and IL-17A in BM plasma were detected by cytometric beads assay. Results: The demographic and clinical characteristics were similar between allo-HSCT patients with PT and those with GGF. The T cell subset analysis revealed that the proportion of CD8+ T cells in BM was higher in PT patients. The in vitro cytokine stimulated tests demonstrated a significant higher proportion of Th1 in PT patients (29.8% ±13.0% vs. 21.7%±12.2%, P=0.01), and we also found an elevated percentage of Tc1 in PT patients when compared with GGF (39.3% ±19.3% vs. 23.0% ± 14.0%, P=0.01). Meanwhile, the similar percentage of Th2 and Tc2 were found in PT patients. The type-1/ type-2 response ratio was calculated by the percentages of Th1/Th2 and Tc1/Tc2. A significant elevation in the ratio of Tc1/Tc2 (37.3 vs. 22.1 vs. 15.6, P<0.05) was observed in PT when compared with those in GGF and HDs, whereas the ratio of Th1/Th2 did not differ from GGF. Moreover, we also found the significant elevated percentage of Th17 (3.1% ±2.1% vs. 1.1%± 0.7%, P<0.01) and the similar percentage of Treg in PT patients compared with GGF, leading to a higher ratio of Th17/Treg (0.9 vs. 0.6 vs. 0.3, P<0.05). The changes of IFN-gama, IL-4 and IL-17A levels in BM plasma detected by cytometric beads assay were in accordance with the intracellular cytokine results analyzed by flow cytometry. Summary/Conclusion: Our study demonstrated that the abnormal BM immune microenvironment including the higher percentage of Th1, Tc1, and Th17 cells in patients with PT, suggesting that the dysfunction of T cells response in BM immune microenvironment may contribute to the occurrence of PT after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bone Marrow Memory CD8+ T Cells Play a Supporting Role in Hematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4370-4370
Author(s):  
Sulima Geerman ◽  
Fernanda M Pascutti ◽  
Sudeep Bhushal ◽  
Martijn A. Nolte
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Lymphoid Organ ◽  
Strong Impact ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Memory Cd8 T Cells

Abstract The bone marrow (BM) has an important function as primary lymphoid organ through the process of hematopoiesis. This process is sustained by hematopoietic stem cells (HSCs) and their life-long production of new blood cells, which is made possible by their unique ability to self-renew. Next to this, BM also functions as a secondary lymphoid organ, as it can mediate primary T cell responses against invading pathogens. We and others have shown that activated T cells can influence the hematopoietic process through the production of pro-inflammatory cytokines. This indicates that adaptive immune responses and hematopoiesis are intertwined in the BM, though most of the cellular and molecular interactions that occur during this crosstalk are yet unknown. Based on previous observations that T cell deficient mice have altered hematopoiesis and that depletion of T cells from allogeneic BM grafts compromises HSC engraftment, we questioned to what extent BM T cells can directly affect the function of HSCs. To test this, we sorted and co-cultured murine BM T cells with HSCs (Lin-Sca-1+c-Kit+CD48-CD150+). We found that particularly BM CD8+ central memory (CD44+CD62L+) T cells (Tcm) enhance the capacity of HSCs to self-renew. Furthermore, we found that TCR-transgenic mice, which do not have memory T cells, have lower numbers of HSCs, which could subsequently be increased by transferring BM CD8+ Tcm. Remarkably, an increase in HSC numbers was also observed when HSCs were cultured with only supernatant derived from BM CD8+ Tcm. Moreover, HSCs cultured with supernatant from BM CD8+ Tcm and later transplanted in myeloablated hosts displayed a strongly enhanced ability to restore hematopoiesis. Importantly, the strong impact of BM T cells on HSCs was not only apparent in the steady state situation, but also following a viral infection with either acute or chronic lymphocytic choriomeningitis virus (LCMV). We could establish that both acute and chronic LCMV-specific CD8+ T cells or supernatant from these cells could increase the HSC self-renewal capacity. In conclusion, our findings demonstrate that BM memory CD8+ T cells can positively influence the function of HSC through soluble mediators. We postulate that this process is particularly relevant after immune activation, in order to protect and/or restore the HSC pool and the subsequent hematopoietic recovery. We are currently using a proteomics approach to identify these soluble mediator produced by CD8+ T cells. Disclosures No relevant conflicts of interest to declare.

Double Haploidentical Hematopoietic Stem Cell Transplantation (HSCT) Results In Successful Engraftment of Bone Marrow From Both Donors without Graft Versus Host or Graft Versus Graft Effects.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1453-1453
Author(s):  
Chandra Biswas ◽  
Daniel Rittenberg ◽  
Xiaoling Luo ◽  
Christopher Sauter ◽  
Dolores Grosso ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Low Dose ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Mixed Chimerism ◽  
Mouse Strains ◽  
Hematopoietic Stem ◽  
Graft Versus Host

Abstract Abstract 1453 The use of haploidentical donors extends the potential clinical application of HSCT. However, relapse of resistant malignancy contributes to low success rates in high risk patients. Relapse may be due to the ability of leukemic cells to immunologically escape a single donor's GVL effects. We hypothesized that the use of two haploidentical donors, each targeting a different recipient haplotype, will increase anti- leukemia activity after double haploidentical SCT. We performed murine studies to establish new single haploidentical (SH) and double-haploidentical (DH) murine models that mimic the possible scenarios which might be encountered clinically rather that using more traditional Parent → F1 models. We first established a haploidentical transplant model using two different hybrid mouse strains as donor and recipient in the experiments. Lethally irradiated B6CBAF1 (H2Kb/k) recipients were transplanted with T cell depleted (TCD) bone marrow (BM) from B6D2F1 (H2Kb/d) donors. Recipient mice harvested at days 28, 42 and 56, showed more than 90% donor cell engraftment, including donor derived lymphopoiesis and myelopoiesis, without evidence of graft versus host disease. Subsequently, lethally irradiated B6CBAF1 (H2Kb/k) recipients were transplanted with TCD-BM from two haploidentical donors (DH model) including B6SJF1 (H2Kb/s) (donor 1 - D1) and B6D2F1 (H2Kb/d) (donor 2 - D2). We observed recipients for 90 days and all mice survived without evidence of GVHD or weight loss. Analyses of blood collected retro-orbitally at day 90 revealed that recipients of DH transplants had significantly higher WBC and neutrophil counts than recipients of SH HSCT from either D1 or D2 respectively. DH recipients consistently showed successful engraftment with mixed chimerism in both bone marrow and spleen. There was no difference in thymopoiesis, B cell and myeloid cell reconstitution compared to SH transplants. In contrast, the number of splenic T cells was higher in SH recipients of D1 marrow (B6SJF1). We then explored the effects of low dose T cell infusions (1×105) on chimerism of donor cells. Low dose T cell infusion from either D1 or D2 did not affect the BM cellularity, but did increase the degree of dominance of that donor's cells in the BM and spleen. A similar outcome was observed when this study was extended to other models such as B6C3 + C3D2F1 → B6D2F1 and B6CBAF1 + B6SJF1 → CB6F1 models where all recipients of DH transplants survived without evidence of GVHD. Recipients of TCD DH transplants were challenged with P815 tumor cells. We used B6SJF1 (D1) + B6CBAF1 (D2) → B6D2F1 model in tumor experiments. Interestingly, recipients of TCD-DH transplants exhibited a significantly better survival than recipients of D1 SH or D2 SH transplants. However, after a low dose T cell infusion (1 ×105), recipients of D2 BM survived significantly better than recipients of D1 BM. In contrast to the TCD model, recipients of DH BM + DH T cells show similar probability of survival with recipients of D2 SH BM + D2 SH T cells. We conclude that TCD DH HSCT results in successful engraftment of both types of BM cells. Additionally, infusion of low dose haploidentical T cells improves the anti-tumor effect without stimulating GVHD. Double haploidentical HSCT may be an ideal platform to enhance GVL effects after transplantation. Disclosures: No relevant conflicts of interest to declare.

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