The Extent in Telomere Attrition and the Number of Affected Leukocyte Subsets Distinguishes Patients with Different Myeloproliferative Neoplasms (MPN).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1913-1913
Author(s):  
Dirk de Beer ◽  
Elke Beutler ◽  
Adrian Emanuel Schmidt ◽  
Lucie Kopfstein ◽  
Elisabeth Oppliger Leibundgut ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Telomere Length ◽  
Progenitor Cell ◽  
Myeloproliferative Neoplasms ◽  
Healthy Controls ◽  
Cell Of Origin ◽  
Hematopoietic Stem ◽  
Telomere Attrition ◽  
Mutational Status

Abstract Abstract 1913 Poster Board I-936 Background: Myeloproliferative neoplasms (MPN) are clonal disorders with an origin of the disease in a hematopoietic stem or progenitor cell. Except for chronic myeloid leukemia (CML), the diagnosis for Philadelphia-chromosome negative (Ph-neg.) MPN is less straightforward. Although many patients with a Ph-neg. MPN can be identified by mutations in JAK2 and/or TET2, the categorization into primary or secondary cythosis or specifically into polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) can be difficult. Since telomeres can be used to estimate the mitotic history of cells, our aims were to evaluate 1) whether the telomere length can be used to distinguish clonal from polyclonal hematopoiesis, 2) whether telomere attrition correlates to the mutational status of JAK2 and 3) whether the extent of telomere shortening in different subsets of leukocytes can point to the originating level of the hematopoietic stem or progenitor cell. Patients and Methods: So far, 32 patients with Ph-neg. MPN diagnosed according to WHO criteria (range: 32 – 85 years; PV: n=19, ET n=5, PMF n=8; JAK2V617F positive n=22, JAK2V617F negative n=16) and 11 patients with secondary erythrocythosis (range 39 – 61 years) were included in this study. 400 healthy individuals (range 0-102 years) served as controls. After informed consent peripheral blood was taken from the patients to measure the telomere length in subsets of leukocytes by automated multicolor flow-FISH. In order to correct for the age-dependent decline in telomere length, telomere length differences to the 50th percentile of the healthy cohort (DeltaTel) were calculated. Telomere length values below the 10th percentile of those from healthy donors were considered as substantially affected by telomere attrition. The mutational status of the JAK2V617F was assessed by allele-specific real time quantitative PCR. Results: The mean telomere length in granulocytes from patients with MPN was considerably shorter compared to healthy controls (mean ± STD DeltaTel: 2.73kb ± 1.20kb), whereas there was no remarkable difference in lymphocytes (0.70kb ± 0.81kb). Furthermore, we found significant DeltaTel between granulocytes from patients with MPN and with secondary erythrocythosis (mean ± STD: 2.73kb ± 1.20kb vs. 1.66kb ± 0.894kb, p<0.0001). Regarding the JAK2V617F mutational status we found no difference for the average DeltaTel (mean ± STD: 2.35kb ± 1.25kb (JAKV617F+), 2.28kb ± 1.12kb (JAKV617F-), p=0.84). Most interestingly, there was a clear difference in the average DeltaTel in granulocytes from patients with different types of MPN (ANOVA p=0.017). The most striking DeltaTel was seen between patients with PMF and patients with PV and ET (PMF: 3.72kb ± 0.30kb, PV and ET: 2.40kb ± 1.18kb, p=0.005). In addition, in patients with PMF most subsets of leukocytes demonstrated substantial telomere length differences compared to healthy controls (Granulocytes: 2.06kb ± 0.55kb, p=0.0001, T-cells: 0.84kb ± 1.07kb, p=0.016, B-cells: 1.62kb ± 1.29kb, p=0.0001, NK-cells: 0.95kb ± 1.49kb, p=0.039), whereas in patients with ET, except for the granulocyte subset, no significant DeltaTel values were detected (Granulocytes: 1.01kb ± 0.856kb, p=0.014, T-cells: -0.435kb ± 0.723, p=0.34, B-cells: 0.26kb ± 1.00kb, p=0.65, NK-cells: 0.30kb ± 0.86kb, p=0.58). In patients with PV one to three leukocyte subsets showed substantial DeltaTel, but in varying combinations. Conclusion: In our ongoing study we were able to confirm shorter telomeres in granulocytes of MPN patients compared to telomeres in granulocytes of healthy controls and of patients with secondary erythrocythosis. Based on the telomere length attrition patients with a clonal cythosis can be distinguished from such patients with a secondary cythosis, which could help diagnostically in uncertain cases. No correlation was found between the extent of telomere attrition and the JAK2V617F mutational status. The extremely short telomeres found in most subsets of leukocytes from patients with PMF could point to a very early hematopoietic stem cell as the cell of origin, whereas for ET and PV with only one or a few subsets of leukocytes affected by telomere attrition the cell of origin could be a hematopoietic stem cell at a somewhat later stage. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunosenescent Characteristics of T Cells in Young Patients Following Haploidentical Stem Cell Transplantation from Parental Donors

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3283-3283
Author(s):  
Ga Hye Lee ◽  
Kyung Taek Hong ◽  
Jung Yoon Choi ◽  
Hee Young Shin ◽  
Won-Woo Lee ◽  
...  
Keyword(s):  
Dna Damage ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Telomere Length ◽  
Cell Transplantation ◽  
Cd8 T Cells ◽  
Hematopoietic Stem

Introduction: Pediatric and adolescent patients in need of allogeneic hematopoietic stem cell transplantation generally receive stem cells from older, unrelated or parental donors when a sibling donor is not available. Despite encouraging clinical outcomes, it has been suggested that immune reconstitution accompanied by increased replicative stress and a large difference between donor and recipient age may worsen immunosenescence in pediatric recipients. Therefore, in this study paired samples were collected at the same time from donors and recipients of haploidentical hematopoietic stem cell transplantation (HaploSCT). Methods: We conducted flow cytometry-based phenotypic and functional analyses and telomere length measurements of 21 paired T-cell sets from parental donors and children who received T cell-replete HaploSCT with post-transplant cyclophosphamide (PTCy) at Seoul National University Children's Hospital between February 2014 and January 2017. The conditioning regimen was comprised of targeted busulfan (total target area under the curve, 75,000 mg•h/L) with intensive pharmacokinetic monitoring, fludarabine and cyclophosphamide. Results: Fourteen pediatric, adolescent, and young adult patients with malignant disease and seven with nonmalignant disease were included with a median post-transplantation period of 16.9 months (range, 12.4-38.8). Senescent T cells, CD28- or CD57+ subsets of both CD4+ and CD8+ T cells, were significantly expanded in patients compared with parental donors. Further, not only CD4+CD28- T cells, but also CD4+CD28+ T cells showed reduced cytokine production capacity and impaired polyfunctionality compared with parental donors, whereas their TCR mediated proliferation capacity was comparable. Of note, the telomere length in patient T cells was preserved, or even slightly longer, in senescent T cells compared with donor cells. We also found that the patients had a higher level of γ-H2AX-expressing CD28- senescent T cells compared with the donors, which is used as a DNA damage marker. Regression analysis showed that senescent features of CD4+ and CD8+ T cells in patients were influenced by donor age and the frequency of CD28- cells, respectively. Conclusions: Our data suggest that T cells undergo premature immunosenescent changes and exhibit functional defects in pediatric HaploSCT recipients. Further, there is an increased level of DNA damage in patient CD4+ T cells compared to those of parental donors. Therefore, long-term, comprehensive immune monitoring of these patients is necessary. Disclosures No relevant conflicts of interest to declare.

Runx1-Cbfb Interacts with CHD7, a Chromatin Modifying Enzyme with a Potential Role in Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1300-1300
Author(s):  
Jingmei Hsu ◽  
Chung-Tsai Lee ◽  
Scott Gerber ◽  
Shuqian Yu ◽  
Nancy A. Speck
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Heart Defects ◽  
Genetic Alterations ◽  
Transactivation Domain ◽  
Hematopoietic Stem

Abstract Abstract 1300 Mutations in RUNX1 and CBFB are among the most common genetic alterations in hematologic malignancies, including acute myeloid and lymphoid leukemia (AML, ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome and myeloproliferative neoplasms. Loss of Runx1-CBFb causes a failure of hematopoietic stem cell emergence during embryogenesis. Critical roles for Runx1-CBFb in adult hematopoiesis include hematopoietic stem and progenitor homeostasis, and lymphoid and megakaryocytic differentiation. We took an unbiased co-immunoprecipitation and mass spectrometry approach to identify Runx1-CBFb co-regulators in T cells, and identified chromodomain helicase binding protein 7 (CHD7) as a potential interacting partner. CHD7 is an ATP-dependent chromatin remodeling protein that primarily occupies enhancer and promoter regions. Autosomal dominant mutations in CHD7 cause CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). It was shown that CHD7 interacts with Sox2, and its occupancy correlates with H3K4me1/2 modifications and P300 binding at enhancer regions, and H3K4me3 marks at promoters. We confirmed the interaction of endogenous Runx1 and CHD7 in T cells. We demonstrate that the Runx1 transactivation domain, which is critical at all stages of hematopoiesis, is required for the CHD7 interaction. To elucidate an in vivo function for CHD7 in hematopoiesis, we generated a conditional pan-hematopoietic Chd7 deletion in mice using a floxed Chd7 allele and Vav1-Cre. Deletion of Chd7 in hematopoietic cells appears to cause no lineage specific defects. However, CHD7 deficient bone marrow cells had a competitive advantage in T cell reconstitution as compared to wild type cells, suggesting a role for CHD7 in restraining T cell numbers in the adult. Determining how CHD7 exerts its functions should shed light on underlying mechanisms in hematopoietic stem cell formation, T cell development, and hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sars-Cov-2 T-Cell Response in Allogeneic Hematopoietic Stem Cell Recipients Following Two Doses of BNT162b2 Vaccine

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2895-2895
Author(s):  
Beatrice Clemenceau ◽  
Thierry Guillaume ◽  
Marianne Coste-Burel ◽  
Pierre Peterlin ◽  
Alice Garnier ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cellular Immunity ◽  
Cell Response ◽  
Humoral Response ◽  
T Cell Response ◽  
Healthy Controls ◽  
Hematopoietic Stem ◽  
Hla Dr

Abstract Introduction: Virus-specific humoral and cellular immune responses act synergistically to protect from viral infection. In our recent observational monocentric study of 117 hematopoietic stem cell adult recipients, we found that 54% and 83 % patients achieved a humoral response after two doses of BNT162b2 anti-SARS-CoV-2 messenger RNA vaccine (Pfizer BioNTech), respectively. Here, we evaluated the T-cell response against the SARS-Cov-2 spike protein after two doses of BNT162b2 vaccine in some allografted patients from the same cohort and compared these results to those from healthy controls. Methods: To quantify SARS-CoV-2 specific T-cells, we used an INFg ELISpot assay that detects these cells after activation of peripheral blood mononuclear cells (PBMC) with 3 peptide pools covering the whole protein sequence of the spike glycoprotein (Prot _S1; _S+ and _S PepTivator peptide pools, Miltenyi Biotec, Bergisch Gladbach, Germany). EBV and CMV specific T-cells were also quantified as controls. The immunophenotype of PBMC was determined by flow cytometry, after dead cell exclusion, with monoclonal antibodies identifying the following surface antigens: CD45, CD3, CD14, CD19 and HLA-DR. The frequencies of spot-forming units (SFU) were reported as per 10 6 CD3+ T-cells. Results: Samples from 46 allografted patients (acute myeloblastic leukemia, N=27, myelodysplastic syndrome, N=19) and 16 healthy controls were available. Characteristics of the population are given in Table 1. All fully vaccinated healthy donors became seropositive and developed a positive T-cell response to spike peptide pools even though variable frequencies were observed. The median response was 195 SFU/10 6 T-cells. By comparison, the frequency of EBV-specific T-cells was 774 SFU/10 6 T-cells (Figure 1). In the group of patients, 78% (n=36/46) had achieved a humoral response after the second dose of vaccine. Among these humoral responders (HR), 89% (n=32/36) also had a positive anti-spike T-cell response with variable frequencies (median =119 SFU/10 6 T-cells. For 8 patients, this T cell response was higher than that of controls (&gt;800 SFU/10 6 T-cells) (Figure 1), which is equivalent to more than 1 specific T-cell per microliter of blood (Figure 2). The humoral responders (HR) who did not develop a T-cell response (11%, n=4/36) had a median time from transplant to vaccination of 523 days compared to 1032 days for cellular responder patients. Among the 10 patients who were non humoral responders (NHR) (22%, n=10/46), 4 (40%) developed a cellular immunity, including one with a very high T cell response (1333 SFU/10 6 T-cells). As expected, the absence of humoral response was observed in patients who were within one year of the transplant. Of note, somehow unexpectedly, patients often presented a high frequency of EBV- and CMV-specific T cells (Figures 1 & 2). As expected, PBMC immunophenotypic analysis revealed that CD3+ frequencies were lower in patients compared to those of controls but were similar between HR and NHR. NHR had very low frequencies of B cells and interestingly, they had an elevated frequency of CD14+ monocytes with low/neg HLA-DR expression potentially corresponding to myeloid-derived suppressor cells (MDSCs) (Figure 3). Conclusion: In this series, 89% of allografted patients who developed an anti-spike humoral response also presented an anti-SARS-Cov-2 cellular immunity. Interestingly, anti-SARS-Cov-2 specific T-cells could be detected in 40% of NHR patients. Although a larger group of patients is required to confirm these results, it remains to be determined whether this T-cell response is protective against SARS-Cov-2 infection as previously demonstrated for CMV (Litjens et al, 2017). Finally, the role of potential immunosuppressive MDSCs must be explored in patients who develop no sign of T-cell response after vaccination. Figure 1 Figure 1. Disclosures Moreau: Oncopeptides: Honoraria; Celgene BMS: Honoraria; Sanofi: Honoraria; Abbvie: Honoraria; Janssen: Honoraria; Amgen: Honoraria.

scholarly journals The Determinative Role of Hematopoietic Stem and Progenitor Cell Graft Composition on the Engraftment Kinetics after HSCT

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1794-1794
Author(s):  
Giulia Barbarito ◽  
Beruh Dejene ◽  
Gopin Saini ◽  
Rachana Patil ◽  
David C. Shyr ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Blood Cell ◽  
Board Of Directors ◽  
Progenitor Cell ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cell Dose ◽  
Graft Composition

Abstract The importance of hematopoietic stem and progenitor cell (HSPC) dose in the outcome of hematopoietic stem cell transplant (HSCT) has been demonstrated by analyses of the threshold dose of CD34+ cells required to achieve donor engraftment, usually defined as endpoints of donor-derived neutrophil (PMN) or platelet (PLT) absolute numbers, and RBC transfusion independence. We recently described the heterogeneous HSPC composition of prospectively obtained umbilical cord blood samples and lack of correlation between CD34+ cell dose and the frequency of hematopoietic stem cells (HSC)(Mantri S et al, Blood Adv 2020). We describe here a pilot analysis of the relationship between HSPC graft composition and engraftment after HSCT. The study population is comprised of 17 children (3.4-22 years, median 13 years) treated with αβT/CD19B - cell depleted mobilized peripheral blood stem cell (PBSC) HSCT (αβhaplo-HSCT). Eight patients had acute lymphoblastic leukemia, seven had acute myeloid leukemia, and two myelodysplastic syndrome. All patients received serotherapy with Thymoglobulin and Rituximab, and a myeloablative conditioning consisting of either TBI 1200 cGy in combination with Fludarabine/Thiotepa (15 patients), Melphalan/Thiotepa (1 patient), or TBI (400 cGy) with Fludarabine/Thiotepa (1 patient receiving a second HSCT). Only 2/17 patients received 1-2 doses of G-CSF before Day+30. The CD34+ Lin- cells in the PBSC products were analyzed for HSPC composition, including HSC (CD38- CD90+ CD45RA-), Multipotent Progenitors (MPP, CD38- CD90- CD45RA-), Common Myeloid Progenitors (CMP, CD38+ CD123+ CD45RA-), Granulocyte-Monocyte Progenitors (GMP, CD38+ CD123+ CD45RA+), Megakaryocyte-Erythroid Progenitors (MEP, CD38+ CD123- CD45RA-) and Common Lymphoid Progenitors (CLP, CD38+ CD127+). Similar HSPC analyses were performed on marrow obtained at Days +30, +60, and +90 post-HSCT. The CD34+ cell dose in the grafts ranged from 8.5-40 x 10e6/kg recipient body weight (mean 15.6). The median time to Absolute Neutrophil Count (ANC) &gt; 500 or 1000/mm 3 were Days +12 and +14, respectively, and for PLT &gt; 50K or 100K/mm 3 Days +14 and +15, respectively. The time to either PMN or PLT engraftment did not correlate with the HSC dose. In contrast, the GMP dose was predictive of PMN engraftment: 7/8 patients receiving the highest GMP dose achieved ANC &gt; 500 at 0-3 days before the median day of engraftment, while PMN engraftment was delayed by 1-5 days beyond the median in 6 of the 9 receiving the lowest GMP dose (χ 2 = 8.14, p=0.004)(Fig A). Of the patients with the highest MEP dose, 7/8 achieved PLT &gt;50K/mm 3 0-4 days before the median, while 5/9 receiving the lowest dose engrafted 1-6 days beyond the median (χ 2 4.9, p=0.026) (Fig B). In the first 100 days post-HSCT, naïve CD4+ T-cells were all CD31+ CD45RA+ Recent Thymic Emigrants (RTE), indicating that they were newly produced T cells and not adoptively transferred from the αβhaplo-HSCT. The HSC dose did not correlate with the number of naïve CD4+ T cells until Day+180 (Fig C). Like PMN and PLT recovery, early T lymphoid recovery after HSCT was mainly derived from infused CLP, and likely switched to HSC-derived lymphopoiesis by Day +180. Consistent with this concept, HSC dose in the PBSC products was unrelated to the numbers of committed progenitors, e.g., CMP, MEP, CLP, in early (Day +30) post-transplant marrow samples. These data are consistent with clonal analyses of patients receiving lentiviral gene therapy and murine experiments demonstrating prolonged steady-state contribution of committed progenitors to peripheral blood cell maintenance (Biasco L et al, Cell Stem Cell 2016; Sun J et al, Nature 2014). While post-HSCT PMN or PLT numbers are frequently equated with "HSC engraftment" and naïve T-cell numbers with HSC-derived immune reconstitution, these early events reflect blood cell production by committed progenitors, which are variably present in the grafts. Although CD34+ cell dose is currently used to predict post-transplant engraftment and to qualify stem cell products for release, more accurate clinical predictions may be determined by the HSPC grafts' composition. Further, engineering of the progenitor composition of clinical HSCT products, e.g., co-transplantation of additional committed progenitors like GMP or CLP, could be strategically used to control post-transplant lymphohematopoietic recovery. Figure 1 Figure 1. Disclosures Parkman: Jasper Biotech: Consultancy. Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Low telomerase activity in CD4+ regulatory T cells in patients with severe chronic GVHD after hematopoietic stem cell transplantation

Blood ◽  
2011 ◽  
Vol 118 (18) ◽  
pp. 5021-5030 ◽  
Author(s):  
Yutaka Kawano ◽  
Haesook T. Kim ◽  
Ken-ichi Matsuoka ◽  
Gregory Bascug ◽  
Sean McDonough ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Regulatory T Cells ◽  
Stem Cell Transplantation ◽  
Telomere Length ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Telomerase Activity ◽  
Hematopoietic Stem

Abstract CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in the control of chronic graft-versus-host disease (cGVHD). In this study, we examined telomere length and telomerase activity of Treg and conventional CD4+ T cells (Tcon) in 61 patients who survived more than 2 years after allogeneic hematopoietic stem cell transplantation. Cell proliferation and expression of Bcl-2 were also measured in each subset. Treg telomere length was shorter and Treg telomerase activity was increased compared with Tcon (P < .0001). After transplantation, Treg were also more highly proliferative than Tcon (P < .0001). Treg number, telomerase activity, and expression of Bcl-2 were each inversely associated with severity of cGVHD. These data indicate that activation of telomerase is not sufficient to prevent telomere shortening in highly proliferative Treg. However, telomerase activation is associated with increased Bcl-2 expression and higher Treg numbers in patients with no or mild cGVHD. In contrast, patients with moderate or severe cGVHD have fewer Treg with lower levels of telomerase activity and Bcl-2 expression. These results suggest that failure to activate Treg telomerase may restrict proliferative capacity and increase apoptotic susceptibility, resulting in the loss of peripheral tolerance and the development of cGVHD.

Very Short Telomeres As a Novel Mechanism Of Donor-Cell Derived Leukemia After Cord Blood Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1245-1245
Author(s):  
Bogdan Dumitriu ◽  
Yasutaka Ueda ◽  
Sachiko Kajigaya ◽  
Danielle Townsley ◽  
Jennifer Cuellar Rodriguez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Telomere Length ◽  
Genomic Instability ◽  
Donor Cell ◽  
Comparative Genomic ◽  
Hematopoietic Stem ◽  
Telomere Attrition ◽  
Post Transplant

Abstract Donor-cell derived leukemia (DCL) is a rare but serious complication of hematopoietic stem cell transplantation (HSCT), reported in 5% of all relapses following allogeneic HSCT. Only 12 cases of DCL after umbilical cord blood (UCB) HSCT are reported in the literature. Multiple mechanisms have been proposed for DCL, including occult donor leukemia, impaired immunity, drug toxicity, or a bone marrow “leukemogenic milieu”. We propose a novel mechanism of leukemogenesis, mediated by very short telomeres of donor cells with subsequent severe telomere attrition in vivo, genomic instability, and progression to complex cytogenetics acute myeloid leukemia (AML), based on study of a patient who underwent UCB HSCT for myelodysplasia (MDS). The patient was a 44-year-old woman, subsequently shown to have a T354M mutation in GATA2, who presented at age 19 years with multiple infections and MDS. At age 41, due to progression of MDS to AML, she received induction chemotherapy and underwent a single 4/6 HLA-matched UCB HSCT. She had delayed engraftment. achieving an absolute neutrophil count of 500/ul more than 100 days post-transplant. Chimerism studies demonstrated 100% donor cell at all time-points post-transplant. Two years and eight months after HSCT she presented with pancytopenia, circulating myeloblasts, and 50% myeloblasts in bone marrow, indicating recurrence of with AML. However, cytogenetics revealed complex abnormalities, t(2p;3q) and an interstitial deletion of 5q, male cells, indicating donor origin of AML. Telomere length of the transplanted cord blood cells measured by Q-PCR showed a severe decrease in length to 7.7 kb compared with an average length of 13 kb in control UCB (n=12). Moreover the telomere length decreased precipitously to 6.6 kb 2 years after transplantation and 5.6 kb at the time of diagnosis of AML. Single telomere length assay (STELA) was used to assess chromosome-specific telomere length. Very short telomeres (<3.0 kb) were present in the UCB before transplant that increased in proportion post-transplant before progression to AML. In contrast, very short telomeres were not present in control UCB STELA. Sequencing of cord blood-derived myeloid cells showed no mutation in TERT or TERC. Comparative genomic hybridization confirmed the complex cytogenetics. Telomere attrition occurs in the first year after HSCT, presumably due to proliferative stress on the stem cell compartment. In comparison to bone marrow, UCB have longer telomeres, but HSC number may be lower than in a BM inoculum, especially for adult recipients. In our case, initial telomere length of UCB was extremely short, for unclear reasons but possibly related to low cord blood cell viability. The long time that was needed for engraftment generated additional telomere attrition with accumulation of very short telomere and genomic instability leading to development of leukemia. Screening UCB for telomere length prior to HSCT might prove useful in identifying situations at risk for graft failure or malignant transformation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hematopoietic progenitor cell regulation by CD4+CD25+ T cells

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4934-4943 ◽  
Author(s):  
Maite Urbieta ◽  
Isabel Barao ◽  
Monica Jones ◽  
Roland Jurecic ◽  
Angela Panoskaltsis-Mortari ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Cell Activity ◽  
Innate Immune ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract CD4+CD25+FoxP3+ regulatory T cells (Tregs) possess the capacity to modulate both adaptive and innate immune responses. We hypothesized that Tregs could regulate hematopoiesis based on cytokine effector molecules they can produce. The studies here demonstrate that Tregs can affect the differentiation of myeloid progenitor cells. In vitro findings demonstrated the ability of Tregs to inhibit the differentiation of interleukin-3 (IL-3)/stem cell factor (colony-forming unit [CFU]-IL3)–driven progenitor cells. Inhibitory effects were mediated by a pathway requiring cell-cell contact, major histocompatibility complex class II expression on marrow cells, and transforming growth factor-β. Importantly, depletion of Tregs in situ resulted in enhanced CFU-IL3 levels after bone marrow transplantation. Cotransplantation of CD4+FoxP3+gfp Tregs together with bone marrow was found to diminish CFU-IL3 responses after transplantation. To address the consequence of transplanted Tregs on differentiated progeny from these CFU 2 weeks after hematopoietic stem cell transplantation, peripheral blood complete blood counts were performed and examined for polymorphonuclear leukocyte content. Recipients of cotransplanted Tregs exhibited diminished neutrophil counts. Together, these findings illustrate that both recipient and donor Tregs can influence hematopoietic progenitor cell activity after transplantation and that these cells can alter responses outside the adaptive and innate immune systems.

scholarly journals Data Driven Computational Modeling of Hematopoiesis in Myelodysplastic Syndromes Unveils Differences in Hematopoietic Stem Cell Kinetics Compared to Age-Matched Healthy Controls

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4354-4354
Author(s):  
Michèle Kyncl ◽  
Lisa Bast ◽  
Lynette Henkel ◽  
Fabian Theis ◽  
Robert A.J. Oostendorp ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Progenitor Cell ◽  
Fixed Number ◽  
Healthy Controls ◽  
Hematopoietic Stem ◽  
Cell Compartments ◽  
Acute Myeloid

Abstract Background: Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis, peripheral cytopenias and risk of transformation to acute myeloid leukemia (AML). Recently, HSC in MDS have been shown to carry acquired distinct genetic and epigenetic mutations, most frequently in epigenetic regulators or splicing machinery factors, which are considered disease-initiating events and are likely responsible for the dysplastic features observed in this disease. Hierarchies of hematopoiesis have been established for healthy HSC and their progeny but it is unclear whether MDS HSC follow the same patterns. Clonal hierarchies in MDS have been inferred on the basis of mutational data, these only constitute a snapshot of hematopoiesis in the patient at a given time and may not accurately reflect the kinetics of the disease. To understand how clonal dominance of MDS over healthy HSC is achieved, we sought to identify differences in proliferation kinetics and lineage fate decisions of MDS HSC in comparison to healthy hematopoiesis and to fit this real data into a mathematical model of MDS hematopoiesis. Methods: Lower-risk MDS (according to IPSS-R) bone marrow (BM) samples from patients harboring an SF3B1 mutation (n = 5) or higher-risk MDS BM samples from patients with an ASXL1 mutation (n = 5) were chosen for analysis to limit heterogeneity and allow for better comparability. Healthy age-matched control BM samples (n = 5) were obtained from patients undergoing routine hip replacement surgery. Distribution of stem and progenitor cell compartments (HSC, MPP, MLP, CMP, GMP, MEP) in BM on day 0 were determined by multiparameter flow cytometry. Cells were labeled with violet cell trace and HSC (Lin-CD34+CD38-CD90+CD45RA-) were sorted by flow cytometry. A fixed number of HSC as well single HSC cell cultures were set up in vitro in serum-free medium with defined growth factors (FL, KL, TPO, IL-6, G-CSF, GM-CSF and EPO). Cell divisions, proliferation rates, cell death and lineage maturation were determined by flow cytometry on days 1, 3, 5, 7 and 9. Clonogenic capacity was probed by standard CFU assays in methylcellulose on day 0 and day 7. Additional integration of genomic sequencing analyses of bulk BM and HSC was used to assess the contribution of the malignant clone to the population of proliferating cells and to each hematopoietic lineage. Results: While CFU numbers at day 0 were significantly reduced for all MDS samples compared to healthy controls, ASXL1 mutated MDS BM samples contained significantly higher numbers of HSC than SF3B1 MDS or healthy control BM samples. SF3B1 MDS samples had significantly larger CMP and GMP compartments compared to healthy controls or ASXL1 mutated samples. In cultures started with a fixed number of HSC, no apparent differences in proliferation kinetics were observed between MDS and healthy samples over time. However, cell trace labeling and sorting of single HSC into 96 well plates revealed clear differences between MDS and healthy HSC. Overall, MDS HSC were less robust, with fewer wells of the 96 plate containing cells after sorting than for healthy HSC samples. In contrast, the remaining HSC from both both ASXL1 and SF3B1 mutated MDS showed a significantly higher cell division rate than healthy HSC in vitro, suggesting MDS HSC are more active than their normal counterparts. Genotyping analyses to assess single MDS HSC contributions to proliferation are ongoing and data are currently being fitted to mathematical models. Conclusion: Detailed analysis of hematopoietic stem and progenitor cell compartments as well as time-resolved tracking of HSC revealed significant differences between MDS and healthy HSC kinetics in vitro. Integration of this data into a comprehensive mathematical model of MDS hematopoiesis may aid in dissecting the mechanisms of progression of MDS towards acute myeloid leukaemia (AML). Disclosures Götze: Takeda: Honoraria, Other: Travel aid ASH 2017; Celgene: Honoraria, Research Funding; Novartis: Honoraria; JAZZ Pharmaceuticals: Honoraria.

scholarly journals Reduced dose of PTCy followed by adjuvant α-galactosylceramide enhances GVL effect without sacrificing GVHD suppression

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Makoto Nakamura ◽  
Yusuke Meguri ◽  
Shuntaro Ikegawa ◽  
Takumi Kondo ◽  
Yuichi Sumii ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell Subsets ◽  
Inkt Cells ◽  
Early Recovery ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Reduced Dose ◽  
Graft Versus Leukemia ◽  
Novel Strategy

AbstractPosttransplantation cyclophosphamide (PTCy) has become a popular option for haploidentical hematopoietic stem cell transplantation (HSCT). However, personalized methods to adjust immune intensity after PTCy for each patient’s condition have not been well studied. Here, we investigated the effects of reducing the dose of PTCy followed by α-galactosylceramide (α-GC), a ligand of iNKT cells, on the reciprocal balance between graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect. In a murine haploidentical HSCT model, insufficient GVHD prevention after reduced-dose PTCy was efficiently compensated for by multiple administrations of α-GC. The ligand treatment maintained the enhanced GVL effect after reduced-dose PTCy. Phenotypic analyses revealed that donor-derived B cells presented the ligand and induced preferential skewing to the NKT2 phenotype rather than the NKT1 phenotype, which was followed by the early recovery of all T cell subsets, especially CD4+Foxp3+ regulatory T cells. These studies indicate that α-GC administration soon after reduced-dose PTCy restores GVHD-preventing activity and maintains the GVL effect, which is enhanced by reducing the dose of PTCy. Our results provide important information for the development of a novel strategy to optimize PTCy-based transplantation, particularly in patients with a potential relapse risk.

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