scholarly journals Generation of Synthetic T-ALL By De Novo Transformation of Human Cord Blood Progenitors with a 4-Oncogene Cocktail

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3675-3675
Author(s):  
Manabu Kusakabe ◽  
Claire Shanna ◽  
Xuehai Wang ◽  
Catherine Jenkins ◽  
Vincenzo Giambra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Human Cells ◽  
Post Transplant ◽  
Cell Counts ◽  
Flow Cytometric

Abstract Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of blood cancer that can arise in both children and adults. Numerous studies have explored the effects of putative T-ALL oncogenes in mouse models and have contributed significantly to our understanding of disease pathogenesis. Nonetheless, it is clear there are important differences between mouse and human cells, particularly with respect to cellular transformation, and additional work is therefore needed to generate more accurate models of human disease. We sought here to create human T-ALL in the lab from normal CB progenitors by lentiviral transduction with a combination of known T-ALL oncogenes. Methods: Human CD34+ hematopoietic progenitor cells were isolated from pooled cord blood by magnetic bead/flow cytometric sorting (MACS/FACS). Sorted cells were then transduced by lentiviral vectors encoding a combination of four known T-ALL oncogenes including activated NOTCH1. NOTCH1 virus was marked with a GFP reporter (N1/GFP) while the other three accessory oncogenes were marked with a Cherry reporter (3xOnc/Cherry). Transduced cells were cultured on OP9-DL1 stromal feeders briefly prior to transplantation into NOD/SCID-IL2Rg-null (NSG) mice to assess leukemogenesis, or for longer periods to study their behavior in vitro. Results: Initial transduction efficiencies were typically 3-5% for each virus with 1-2% doubly-transduced N1/GFP+, 3xOnc/Cherry+ cells (hereafter referred as 4xOnc cells). After 28 days culture in vitro, the 4xOnc population reproducibly expanded and outcompeted singly- and non-transduced populations, accounting for more than 70% of cells in mixed cultures. By absolute cell counts, non-transduced cells stopped expanding within the first few weeks; however, 4xOnc cells kept expanding even after 6 weeks of culture. To test leukemogenesis in vivo, CD45+ cells were FACS sorted after 10 days of culture on OP9-DL1 feeders (including doubly-, singly-, and non-transduced populations) and injected intrahepatically into NSG neonates. Engraftment of human cells was followed monthly by flow cytometry of peripheral blood. Engraftment of GFP+ Cherry+ 4xOnc cells was first detected 2 months after transplantation whereas no engraftment of singly- or non-transduced cells was detected. The level of engraftment was below 5% and did not increase substantially even after 6 months following transplantation. At day 203 post-transplant, the primary recipient was sacrificed and 4xOnc cells were recovered from bone marrow, spleen and thymus where the levels of engraftment were approximately 10%. 4xOnc cells from the primary recipient were then serially transplanted into secondary recipients. Engraftment of 4xOnc cells in secondary recipients was observed 5 weeks after transplant. Unlike the primary recipient, however, the percentage of 4xOnc cells in the peripheral blood of secondary recipients gradually increased and these animals developed clinically morbid disease by 20 weeks post-transplant. At the time of necropsy, splenomegaly, lymphadenopathy, and enlarged thymus were observed and the bone marrow contained 80-90% 4xOnc cells. By flow cytometric analyses, 4xOnc cells expressed CD2, CD3, CD7, CD38, and TdT supporting acute T-cell leukemia. Also, TCR gamma clonality assay was performed with genomic DNA from 4xOnc cells from secondary recipients and revealed of 5-7 distinct clonal populations. These in vitro and in vivo findings were observed with multiple experimental replicates and with different pools of cord blood. Conclusion: Our in vitro and in vivo results suggest that NOTCH1, in combination with 3 accessory oncogenes are sufficient to transform normal human blood cells into clonal T-ALL-like malignant cells. Although we cannot exclude the possibility of the spontaneous acquisition of additional co-operating genetic or epigenetic abnormalities, this model provides a significant step forward to reveal the mechanisms involved in human T-ALL pathogenesis. Disclosures No relevant conflicts of interest to declare.

Differences In the Short Term Granulocyte and Platelet Producing Activity of Cord Blood and Mobilized Peripheral Blood Transplants Revealed by Quantitative Repopulation Assays In NOD/SCID-IL-2Rγc−/− Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3733-3733
Author(s):  
Alice M.S. Cheung ◽  
Connie J. Eaves
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Human Cells ◽  
Aldh Activity ◽  
Differential Distribution ◽  
Post Transplant ◽  
Mobilized Peripheral Blood

Abstract Abstract 3733 Human G-CSF-mobilized peripheral blood (mPB) progenitor harvests collected by leukapheresis have become the usual transplant of choice when a suitable normal histocompatible or unrelated adult donor is available for patients requiring a transplant. Unfortunately, for most such patients such a donor is not available and, for these individuals, an autotransplant or unrelated cord blood (CB) unit provide potential alternatives. Autologous harvests are frequently compromised by prior exposure to chemotherapy, and the relatively small size of a single CB unit, can also pose significant problems, particularly in adult recipients. These typically involve pronounced delays in neutrophil recovery and almost always a protracted and/or poor recovery in platelet output. Interestingly, although the simultaneous transplantation of multiple CB units has successfully reduced graft failure, timely hematopoietic recovery is still not commonly achieved, especially for the platelets. There is thus a compelling need to understand the types of repopulating cells that are present in different mPB and CB harvests and how these may vary numerically and biologically. Such information is key to devising better surrogate predictors of adequate engrafting potential and as a first step towards developing new approaches to improve the utility of CB and poor mPB harvests for clinical transplant applications. To pursue these issues, we have developed new assays for quantifying different types of human cells with different durabilities of platelet and granulocyte generating activity in sublethally irradiated NOD/SCID-IL-2Rγc−/− (NSG) mice and have now used these assays to compare the numbers and output properties of different phenotypically characterized subpopulations of cells present in CB and normal mPB samples. Consistent with previous reports, we found that >90% of the week-3 bone marrow repopulating activity in transplanted mice was contained within the Lin−ALDH+ fraction regardless of the source of the human cells tested. However, a substantial proportion of early (week 2) platelet repopulating activity came from the Lin−ALDH− fraction of both CB and mPB (30% for CB and 61% for mPB, decreasing to 12% and 16% respectively by week 3). In vitro CFU-Mk (and CFU-GM/BFU-E/CFU-GEMM) assays reflected a similar differential distribution between the Lin−ALDH+ and Lin−ALDH− cells from the two sources, suggesting possible overlap in the corresponding in vitro and in vivo endpoints. Interestingly, at 3 weeks post-transplant, we found the Lin−ALDH+ cells from mPB produced more of the GM cells than the platelets as compared to the Lin−ALDH+ cells from CB. Limiting dilution assays revealed that the actual frequency of week-3 GM-repopulating cells (GMRC-3wks) and platelet-repopulating cells (PRC-3wks) in Lin−ALDH+ cells in mPB (1/2500, 95% CI=1/1500–1/4200 and 1/3300, 95% CI=1/1900–1/5600) is actually about 3-fold lower (p<0.05) than that in CB (1/780, 95% CI=1/470–1/1300 and 1/1300, 95% CI=1/800–1/2000). Thus, the average mature cell output per RC of either type is correspondingly higher in mPB than in CB (≂f8-fold for GMRC-3wks and 4-fold for PRC-3wks). Based on these findings and knowledge of the median cell dose used for clinical transplants of mPB and a single CB unit, we estimated that adults transplanted with the latter would receive ≂f35-fold less GMRC-3wks and PRC-3wks than what would be given in a typical mPB transplant. In summary, our results show that discontinued ALDH activity identifies many of the cells in both mPB and CB that are responsible for very rapid (within 2–3 weeks) but transient production of mature myeloid cells and platelets post-transplant versus those with persistent ALDH activity that are required for the more prolonged output of these cells (>3 weeks). In addition, our results demonstrate that the 3-week RCs in mPB and CB differ in their average clonal outputs of mature GM cells and platelets in vivo, suggesting ontogeny-related changes in the mechanisms that determine this functional parameter. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

Evidence for An Anti-Cancer Barrier in a Mixed Lineage Leukemia Mouse Model in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3347-3347
Author(s):  
Sylvia Takacova ◽  
Jiri Bartek ◽  
Lucie Piterkova ◽  
Robert K. Slany ◽  
Vladimir Divoky
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Mouse Model ◽  
Peripheral Blood ◽  
Myeloid Cells ◽  
Mixed Lineage Leukemia ◽  
Cell Counts ◽  
Potential Tumor Suppressor

Abstract Mixed Lineage Leukemia (MLL) mutations identify a unique group of acute leukemias with distinct biological and clinical features. Although the role of MLL in leukemogenesis has been extensively studied, a precise mechanism regarding the leukemogenic potential of MLL mutations is not known. We generated a switchable MLL-ENL-ERtm mouse model, in which the MLL-ENL oncogene has been introduced by homologous recombination and is controlled by the endogenous MLL promoter, thus, expressed at physiological levels. Due to fusion with the estrogen receptor ligand binding domain (ERtm), the MLL-ENL-ERtm protein activity is dependent on continuous provision of tamoxifen or 4-hydroxytamoxifen. The MLL-ENL-ERtm mice have developed a myeloproliferative disorder (MPD) characterized by persistent mature neutrophilia after 484,5 +/− 75,68 days of latency on a tamoxifen diet, in association with high white cell counts in peripheral blood, splenomegaly and occasionally with anemia. Blood smears showed large numbers of mature myeloid elements consisting of 40–80% neutrophils (non-segmented forms in abundance), admixed with immature myeloid elements, 3–11% monocytes and 2–6% myeloblasts. The phenotype of MPD also involved myelomonocytic proliferation with 35% immature monocytic cells in one animal and severe anemia with increased numbers of immature erythroid cells in peripheral blood in another animal. Hematoxylin- and eosin-stained sections of the bone marrow from MLL-ENL-ERtm mice revealed expansion of myeloid cell population with no signs of progressive dysplasia. We observed massive infiltration of myeloid cells (positive for myeloperoxidase) into spleen with various degree of loss of normal splenic architecture depending on disease progression. FACS profiles of both bone marrow and spleen cells showed a typical pattern of granulocyte/macrophage/monocyte surface marker expression (CD34-CD43+Mac- 1+Gr-1+CD16/32+). In vitro evaluation of hematopoetic progenitors derived from bone marrow of leukemic mice at the terminal stage of the disease revealed decreased numbers of BFU-Es and increased numbers of CFU-GMs and CFU-Gs compared to matched controls. These results correlated with the expansion of the myelomonocytic and reduction of the erythroid compartment observed in the bone marrow of these animals. The average size (cellularity) of the mutant myeloid colonies was much smaller than the colonies derived from the wild-type controls, which could be caused by a partial block of terminal differentiation of myeloid progenitors in vitro. In vivo, MLL-ENL leads to expansion of differentiated myeloid cells in our model. High penetrance and long latency of leukemia in our model permits the study of early leukemia development. Our model revealed that MLL-ENL - induced myeloproliferation occurs as early as twelve weeks after MLL-ENL-ERtm activation in the bone marrow and infiltrates the spleen with a consequent decrease in lymphoid B220+CD19+IgM+ cells. Using the TUNEL assay on bone marrow sections, we observed induction of apoptosis in the highly proliferative bone marrow compartment compared to matched controls. These results suggest activation of a potential tumor suppressor mechanism by MLL-ENL in early stages of leukemia. We are currently investigating potential tumor suppressor pathways that might be involved in MLL-ENL - induced apoptosis in preleukemia.

Clinical and Cost Outcomes of Double Umbilical Cord Blood Versus Bone Marrow and Peripheral Blood Unrelated Hematopoietic Stem Cell Transplants.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 967-967
Author(s):  
Guillermo López-González ◽  
Charles F. LeMaistre ◽  
Carlos Bachier ◽  
Brad Smith ◽  
Ka Wah Chan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Clinical Outcomes ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cell Counts ◽  
Transplant Center

Abstract Umbilical cord blood (UCB) is a viable source of hematopoietic stem cells (HSC) however, because of the increased risk of nonengraftment associated with low total nucleated cell counts (TNC) per kg, double UCB (DCB) transplants are used in larger patients (pts). Minimal data has been collected on cost analysis and clinical outcomes comparing DCB to peripheral blood (PB) and bone marrow (BM) unrelated donor (UD) transplants. Thus, we conducted a retrospective review of DCB transplants (n=30) from January 2004 through April 2008 compared to BM (n= 23, 30%) and PB (n = 54, 70%) UD transplants during the same time period. Median age was 21 yrs (range 13–66) for DCB recipients and 45 yrs (range 0.4–67) for UD pts. UD pts were HLA typed at A, B, C, DR, and DQ and matched with donors at 9(10) (21%) and 10(10) (79%) loci [all mismatches at class I alleles]. Pts were considered for DCB transplant if a suitable 10/10 or 9/10 donor could not be identified. DCB recipients had HLA matches of 6/6, 5/6/, 4/6 and 3/6 as follows: (cord 1/ cord 2): (0.0%/6.7%), (20.0%/30.0%), (76.7%/63.3%), (3.3%/0.0%) [HLA disparity between each UCB unit and recipient was not necessarily at the same loci]. The median TNC/kg in DCB pts was 2.03x107 (range 7.9x106 – 9.3x108). Full intensity preparative regimens (BuCY2 or TBI³ 1200 cGy based) were used in 43% of UD and 50% of DCB pts. Median day to ANC>500/ul for DCB and UD pts was 23 days (range 6–66) and 15 days (range 7–52), and platelet >20,000/ul was 52 days (range 7–130) and 19 days (range 9–63), respectively. Incidences of acute GVHD ≥2 and chronic GVHD in the UD and DCB pts were 61.0% and 51% vs. 53.3% and 45%, respectively. Median follow-up was 143 days (range 12–847) for the DCB group and 242 days (range 34–1506) for the UD pts. Estimated overall survival (OS) at 1 year was 61% (95% CI: 48% to 71%) for UD pts vs. 55% (95% CI: 34% to 72%) in DCB pts (p = NS). Estimated transplant related mortality (TRM) at 100 days for UD pt was 6.5% (95% CI: 2.8% to 14.9%) and 16.7% (95% CI: 7.3% to 35.5%) (p=0.09) in DCB pts. There were 48 total deaths; 14 (46.7%) in the DCB pts and 34 (44.2%) in the UD pts. In both the DCB and UD groups, disease relapse (21.4% and 23.5%) and infection (28.6% and 14.7%) caused the majority of deaths. All facility transplant related costs were reviewed from the transplant center thru the first year post transplant, excluding pretransplant workup and care not received at the transplant center. The total median costs for DCB transplants were significantly more than the UD costs at D30, D100 and 1yr post transplant by differences of $42,067, $50,806, and $52,297, respectively (p<0.05 for each time interval). The median length of stay for initial hospitalization at start of preparative regimen was 22 days (2–144) in the UD pts and 29 days (6–103) in the DCB pts, and 60% of total median costs occurred by D30 in the DCB group compared to 52% in the UD group. In conclusion, DCB provides a viable option for HSC with comparable clinical outcomes to UD transplants, however DCB transplants are more expensive primarily because of inpatient costs in the first 30 days.

In Vitro Expansion of Human Hematopoietic Cells with Delayed but Sustained Multi-Lineage Repopulating Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1270-1270
Author(s):  
Alice M.S. Cheung ◽  
Stefan Wohrer ◽  
Paul H. Miller ◽  
Suzan Imren ◽  
Shabnam Rostamirad ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Cord Blood ◽  
Stromal Cell ◽  
Human Cells ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Post Transplant ◽  
Nsg Mice

Abstract Abstract 1270 In vivo expansion of hematopoietic stem cells (HSCs) involves local interactions with stimuli generated from non-hematopoietic niche environments, but the full spectrum of molecular mechanisms responsible have remained elusive. Initial experiments in mice showed that highly purified HSCs from adult mouse bone marrow are consistently expanded 3–5-fold with full maintenance of their long term (≥6 months), serially transplantable, multi-lineage repopulating ability when cultured for 7 days in serum-free UG26 stromal-cell conditioned medium (CM) supplemented with 100 ng/ml mouse Steel Factor (SF) and 20 ng/ml mouse IL-11. To explore the potential effects of this CM on HSCs in human cord blood, we conducted an initial experiment in which CD34+CD38− cells were cultured for 7 days in UG26 CM supplemented with 100 ng/ml human Flt3-ligand, 100 ng/ml human SF, and 20 ng/ml each of human IL-3, IL-6 and G-CSF. The results of limiting dilution transplants of the cultured cells in intravenously injected NSG mice showed retention of input numbers of cells with equivalent robust 6-month lympho-myeloid repopulating activity. To characterize the initial target cells and determine whether their proliferative responses might be predictive of their self-renewal behavior, we set up single cell cultures with the CD49f+ subset of CD34+CD38−CD45RA−CD90+Rho−cells with the 5 growth factors in the presence or absence of CM. Under both conditions, 7/13 and 4/13 input cells, respectively, died within the first 72 hours in culture. The subsequent rate of proliferation of the survivors was similar with all completing a first division after 96 hours and a second division 24–48 hours later. By day 8, clones of variable sizes were noted (6–1100 and 4–200 cells/clone, respectively). Clones generated under the same conditions were pooled and injected intravenously into 2 NSG mice each. We then looked for the presence of human cells in the mice by analysis of serial bone marrow aspirates starting 3 weeks post-transplant. Human cells were detected in only one of each of the 2 pairs of mice and, interestingly, in both cases, no evidence of human cells was detectable until 3 months post-transplant. In the positive mouse injected with cells generated in the absence of CM, this repopulation was transient, peaking at ∼0.1% of the mouse bone marrow compartment at 4 months post-transplant and undetectable a month later. In contrast, in the positive recipient of cells from the cultures that contained CM, both lymphoid and myeloid human cells reached much higher levels (together making up ∼20% of the mouse bone marrow compartment) which were maintained for another 3 months when the mouse was sacrificed. Transplants of cells obtained at this time from the marrow gave positive repopulation of secondary mice. In a subsequent experiment, in which similar cultures were initiated with CD34+ cord blood cells, evidence of a late continuing effect of the CM was obtained with a net absolute expansion of CD34+CD45RA−CD90+ cell numbers during the interval between 12 and 21 days in vitro. These findings highlight the important potential of as yet unidentified secreted stromal cell factors to stabilize the stem cell state in HSCs stimulated to proliferate in vitro by growth factors that favor their self-renewal. Disclosures: No relevant conflicts of interest to declare.

A Practical Erythroid Assay in Humanized Xenograft Mouse Model

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2446-2446
Author(s):  
Jun Hayakawa ◽  
Matthew Hsieh ◽  
Naoya Uchida ◽  
Kareem Washington ◽  
Oswald Phang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Cells ◽  
Human Origin ◽  
Post Transplant ◽  
Hemoglobin Switching ◽  
Cd34 Cells ◽  
High Level

Abstract Xenografting immunodeficient mice has been employed as a surrogate human hematopoietic stem cell (HSC) assay, however, erythroid output has not been reliably reported, limiting the usefulness of this model for erythroid disorders. We have previously demonstrated that busulfan preconditioning is sufficient to produce stable, high level engraftment of human cells in NOD/SCID/IL2Rγ null mice. Importantly, this high level engraftment can be achieved with low mortality, substantially reducing the number of animals required for experiments requiring long-term follow-up. Supplementation with human holo-transferrin (Tf) allows the detection human erythrocytes in this chimeric mouse assay at low levels, providing a potential model for the study of disorders affecting human red blood cells (2007 ASH meeting #3594). In the current work, we extend our observations and establish practical in vivo erythroid assay system of human HSCs in the humanized mouse model by the addition of an in vitro culture. Bone marrow (BM) from humanized mice containing 24.8±8.7% human cells (n=6) was first exposed to recombinant human (rHu) SCF+IL3 for 3 days in order to specifically enrich for human cells in the mixed chimera. After 3 days, human cells comprised 68.1±8.5% of the culture. The enriched cells were then cultured with rHu EPO+SCF+TGF-β for 7 additional days and then in rHu EPO+SCF for 7 days. After culture, and 98.9±1.47% of cells were of human origin. After centrifugation, the pellets were visibly red. Cells were assayed with both human CD71+ and GPA+ by flow cytometry: 64.0±7.44% were CD71+ and 69.2±7.02% were GPA+, and human α, β, and γ globin were confirmed by hemoglobin electrophoresis and mass spectrometry. In order to determine the utility of this approach, we tested 3 possible applications of this methodology: gene marking erythroid progeny, modeling of human hemoglobinopathies, and modeling of hemoglobin switching. We first transplanted human cord blood (CB) CD34+ cells after lentiviral transduction with a vector encoding GFP following busulfan conditioning. Three months post-transplant, bone marrow was harvested and placed in the in vitro culture. After in vivo culture, 98.9% of cells were of human origin and 60.7% were CD71+ and 72.3% were GPA+. The majority of CD71+ or GPA+ cells were GFP+ (82.3% and 87.7%, respectively). We subsequently transplanted human PB CD34+ cells derived from individuals with sickle cell trait (SCT) as we have previously demonstrated that these cells, unlike those from individuals with sickle cell disease, can be safely mobilized and processed. Further, the percentage of HbS expressed can be reliably measured. Three months post transplant, HbS was easily detectable by hemoglobin electrophoresis. Finally, we sought to address whether this model accurately reflects human erythropoiesis by examining hemoglobin switching after transplanting either CB expressing HbF or PB HSCs expressing HbA and monitoring the output of HbF and HbA over time. Early post-transplant, bone marrow derived from CB recipients expressed predominantly HbF after culture whereas that derived from PB HSC recipients expressed predominantly HbA. HbF declined during follow up and was replaced by HbA over 6 months of follow up from CB recipients, whereas HbA expression remained stable from PB HSC recipients. The time course of hemoglobin switching is similar to human ontogeny. In summary, our practical approach to model human erythropoiesis in the xenograft mouse should prove useful in the both the study of human erythroid disorders as well as therapeutic interventions.

scholarly journals Human Cd25+Cd4+ T Regulatory Cells Suppress Naive and Memory T Cell Proliferation and Can Be Expanded in Vitro without Loss of Function

2001 ◽  
Vol 193 (11) ◽  
pp. 1295-1302 ◽  
Author(s):  
Megan K. Levings ◽  
Romina Sangregorio ◽  
Maria-Grazia Roncarolo
Keyword(s):  
T Cell ◽  
Peripheral Blood ◽  
T Regulatory Cells ◽  
Transforming Growth Factor ◽  
Cytotoxic T Lymphocyte ◽  
Cell Receptor ◽  
Major Advance ◽  
Loss Of Function

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4+ Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25+CD4+ Tr cells in humans has not been reported. Here we show that human CD25+CD4+ Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25+CD4+ Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25+CD4+ Tr cells strongly inhibited the proliferative responses of both naive and memory CD4+ T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25+CD4+ Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25+CD4+ Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

scholarly journals Absolute Lymphocyte Count (ALC) Less Than 100 Predicts Adverse Transplant Outcomes with Rabbit Thymoglobulin in Patients Undergoing Matched Unrelated Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4527-4527
Author(s):  
Dipenkumar Modi ◽  
Malini Surapaneni ◽  
Seongho Kim ◽  
Lois Ayash ◽  
Asif Alavi ◽  
...  
Keyword(s):  
T Cell ◽  
Relapse Rate ◽  
Peripheral Blood ◽  
Conditioning Regimen ◽  
Advisory Board ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Transplant Outcomes ◽  
Gvhd Prophylaxis

Introduction: Rabbit thymoglobulin, an in-vivo T-cell depleting agent, is widely used as a part of GVHD prophylaxis regimen. Current dosing of thymoglobulin is often weight based and does not consider patient related factors. This results in highly variable exposure of thymoglobulin. Although higher doses (>7mg/kg) of thymoglobulin have shown to reduce the risk of GVHD, it is associated with increased rate of opportunistic infections and disease recurrence. Conversely, lower dose (2.5mg/kg) of thymoglobulin is associated with increased risk of GVHD. Thus, optimum dosing of thymoglobulin remains undefined. We hypothesized that recipient peripheral blood ALC on the first day of thymoglobulin infusion would interact with the dose of thymoglobulin administered and predict post-transplant outcomes. We plan to identify association of thymoglobulin dose with the ALC on the first day of thymoglobulin. Methods: We retrospectively evaluated clinical outcomes of adult patients (pts) who underwent matched unrelated donor AHSCT and received tacrolimus, mycophenolate (cellcept) and thymoglobulin as GVHD prophylaxis. Thymoglobulin was given at a total dose of 4.5mg/kg in divided fashion (0.5mg/kg on day -3, 1.5mg/kg on day -2 and 2.5mg/kg on day -1). The objectives were to determine rate of GVHD, overall survival (OS), relapse-free survival (RFS), relapse rate and non-relapse mortality (NRM) following AHSCT using Cox proportional hazard regression and competing risk models. Results: Between January 2005 and December 2017, 217 pts underwent AHSCT. The most common indications for AHSCT were AML (n=95, 44%), MDS (n=57, 26%), non-Hodgkin's lymphoma (n=23, 11%), and ALL (n=22, 10%). Median age of pts was 60 years (range, 18-79). All pts received peripheral blood stem cells. Ninety-eight pts (45%) received full intensity conditioning regimen and 119 pts (55%) received reduced intensity regimen. The median ALC on the first day of thymoglobulin administration was 200 K/cubic millimeter. The 6-month cumulative incidence rate (CIR) of grade III-IV acute GVHD was 14.8% and the 2-year CIR of chronic extensive GVHD was 35.4%. With a median follow up of 3.82 years for surviving patients, the 2-year RFS, OS, relapse and NRM were 50%, 57.1, 20.1%, and 30.2%, respectively. CMV and EBV reactivation rates were 37% and 11%, respectively. Four pts developed CMV disease. By our lowest ALC cutoff of 100 K/cubic millimeter, pts were divided into two groups (ALC ≤ 100 vs. ALC > 100). Multivariable analysis revealed that ALC > 100 was associated with significantly superior OS (HR 0.51, 95% CI 0.33-0.79, p=0.002), RFS (HR 0.49, 95% CI 0.33-0.74, p=0.001) and lower NRM (SHR 0.57, 95% CI 0.34-0.97, p=0.038) and marginally lower relapse rate (SHR 0.57, 95% CI 0.31-1.05, p=0.070). In addition, higher infused total nucleated cells was associated with higher NRM (SHR 1.70, 95% CI 1.02-2.83, p=0.041). No impact of disease risk index, KPS, conditioning regimen, infused CD34 cells on NRM, relapse, RFS or OS was observed. Conclusion: Our study indicates that ALC ≤ 100 is associated with adverse post-transplant outcomes when thymoglobulin dose of 4.5mg/kg is used for in-vivo T cell depletion. This finding may indicate that in pts with lower ALC, thymoglobulin dose may need to be adjusted to optimize its efficacy and avoid toxicities. In the future prospective studies, which evaluate dose reduction of thymoglobulin in pts with low ALC need to be planned to confirm these results. Disclosures Deol: Agios: Other: Advisory board; Novartis: Other: Advisory board; Kite: Other: Advisory board.

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