Decentralized Manufacture of TCR-Alpha/Beta and CD19 Depleted Haploidentical Stem Cell Grafts for Children within a Multicenter Phase I/II Clinical Trial

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2172-2172
Author(s):  
Michael Schumm ◽  
Matthias K. Eyrich ◽  
Markus Wiesneth ◽  
Halvard Bönig ◽  
Peter J. Lang ◽  
...  
Keyword(s):  
Stem Cell ◽  
B Cells ◽  
Research Funding ◽  
Pediatric Patients ◽  
Acute Gvhd ◽  
Round Robin ◽  
Cell Numbers ◽  
Cd34 Cells ◽  
Alpha Beta

Abstract Several single center experiences have shown favorable outcomes using in-vitro depletion of T cell receptor (TCR)-alpha/beta cells and B cells. For the first time we show the manufacturing results of stem cell grafts from haploidentical family donors for 30 pediatric patients with various hematological and non-hematological malignancies and non-malignant diseases within a prospective, multi-center phase I/II clinical trial utilizing the CliniMACS plus System (Miltenyi Biotec, Germany) in combination with a reduced conditioning (www.clinicaltrialsregister.org; 2011-005562-38). The in-vitro T cell depletions of the grafts were performed by four different laboratories under fixed conditions. The grafts were sent to six treatment centers. Methods: Donors received G-CSF for mobilization of stem cells according to local hospital routine followed by leukaphereses that were depleted from TCRab+ and CD19+ cells according to manufacturer's instructions (CliniMACS plus System, Miltenyi Biotec) and as approved by local authorities. For quality purposes the sponsor performed regularly round robin tests to ensure provision of comparable results regarding the residual number of TCRab+ cells in the grafts. The transplants should be composed of a maximum of 7.5 x 108 nucleated cells/mL with targeted ≥ 4 x 106 CD34+ cells/kg, ≤ 25 x 103 TCRab+ cells/kg, ≤ 1 x 105 CD20+ cells/kg and a CD34+ cell viability of ≥ 95%. In case the targeted value for CD34+ cells/kg was not reached, the number of TCRab+ cells/kg was allowed to exceed by up to the four-fold until 4 x 106 CD34+ cells/kg per transplant have been isolated. One transplant could consist of up to three single aphereses depleted of TCRab+ and CD19+ cells. These products were to be infused directly after separation or following cryopreservation. Pooling of aphereses from a single donor was not allowed. Results: 30 pediatric patients, median age 7 years (range, 1 - 17 years) received a total of 43 TCRab/CD19 depleted haploidentical stem cell products. 17 patients got one infusion, 11 two and one patient three infusions. The stem cell products contained in median 9.46 x 106/kg (range, 1.35 - 54.9) CD34+ cells, 8.4 x 103/kg (range, 0.62 - 40.6) TCRab+ cells and 0.32 x 105/kg (range, 0.037 - 1.7) CD20+ cells. Log depletion for TCRab+ cells and B cells was 4.75 (range, 1.2 - 5.33) and 3.43 (range, 0 - 3.93), respectively. In addition significant numbers of NK and TCRgd+ cells/kg were preserved: 3.9 x 107 (median; range, 0.11 - 18.2), 0.67 x 107 (median; range, 0.05 - 4.0), respectively, summing up to a median number of 6.99 x 106(range, 0.42 - 39.7) total CD3+ cells/kg. Viability of CD34+ cells was 97.9% (median, range 91.5 - 100). All patients received transplants (consisting of up to three single consecutive products) with the targeted CD34+ cell dose - median 14.6 x 106 cells/kg (range, 4 - 54.9) and with less than the maximal allowed number of 1x105 TCRab+ cells/kg. Three single products exceeded targeted TCRab cell numbers but remained within the defined limit of the transplant of 1 x 105 cells/kg in order to meet the specification of ≥ 4 x 106 CD34+ cells/kg. In six single products the B cell numbers were above the specified targeted limit (max. 1.7 x 105/kg). Four products had a viability of CD34+ cells between 91.5 and 95%. Of 30 treated patients no patient experienced acute GVHD°III-IV. Only one patient had acute GVHD°II. The transplant of this patient fulfilled the targeted specifications for total CD34+, TCRab+ and CD20+ cells/kg. Round robin tests were performed prior to study start and during the enrollment period. The identified issues were addressed prior to study start and subsequent tests revealed a uniform performance of the manufacturing centers. Conclusion: 43 stem cell products were manufactured and released by 4 manufacturing laboratories for 30 pediatric patients in 6 hospitals within a clinical study investigating TCR alpha/beta and CD19 depleted haploidentical stem cell transplantation after reduced intensity conditioning. A highly effective depletion of TCRab + cells and B cells with comparable results was shown in all laboratories as controlled by frequent evaluation in round robin tests. Limited exceedances of the targeted release criteria of TCRa/b+ cells were acceptable to the physicians and had no clinical impact. Disclosures Bönig: Miltenyi Biotec: Consultancy, Honoraria, Research Funding. Bader:Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Medac: Consultancy, Research Funding. Aktas:Miltenyi Biotec: Employment. Dresing:Miltenyi Biotec: Employment. Karitzky:Miltenyi Biotec: Employment. Holtkamp:Miltenyi Biotec: Employment. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-Patentholder of TcRalpha/beta depletion technology.

TCR-Alpha/Beta and CD19 Depleted Haploidentical Stem Cell Transplantation Following Reduced Intensity Conditioning in Children: First Results of a Prospective Multicenter Phase I/II Clinical Trial

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 389-389 ◽  
Author(s):  
Peter J. Lang ◽  
Paul G. Schlegel ◽  
Roland Meisel ◽  
Ansgar S. Schulz ◽  
Johann Greil ◽  
...  
Keyword(s):  
Stem Cell ◽  
Research Funding ◽  
Graft Failure ◽  
Immune Reconstitution ◽  
Acute Gvhd ◽  
Reduced Intensity Conditioning ◽  
Gvhd Prophylaxis ◽  
Cd34 Cells ◽  
Alpha Beta ◽  
Reduced Intensity

Abstract We report the first prospective, multi-center, open-label, single-arm phase I/II clinical trial that assesses the safety and feasibility of stem cell transplantation with TCRalpha/beta and CD19-depleted haploidentical grafts generated with the CliniMACS plus System (Miltenyi Biotec, Germany) in combination with a reduced-intensity conditioning in pediatric patients suffering from various malignant and non-malignant diseases (www.clinicaltrialsregister.org; 2011-005562-38). All patients received single agent MMF as short-term GVHD prophylaxis (40mg/kg/day for 30 days). The speed of immune reconstitution was measured in two core labs using standardized methods and the MACSQuant flow cytometry device (Miltenyi Biotec, Germany). Results: Thirty patients from six hospitals were treated (13 female, 17 male; median age 7 years, range 1 - 17 years). Of the 30 recipients, 10 had ALL, 8 had AML, 6 had solid tumors (soft tissue sarcomas and neuroblastomas), 3 had MDS/MPS, and 1 each with lysosomal storage disorder, SCID, and Wiskott Aldrich syndrome. Disease status in acute leukemias/MDS was: CR1 (n=4), relapsed/refractory (n=17). 5/6 patients with solid tumors had relapsed metastatic disease. The conditioning regimen consisted of 15 or 30 mg ATG (Fresenius/Grafalon) or 7 Gy total nodal irradiation, 160 mg/m2 fludarabine, 10 mg/kg thiotepa, and 140 mg/m2 melphalan. The median number of CD34+ cells, TCRalpha/beta+ cells and CD20+ cells infused was 14.6 x 106 (range, 4 - 54.9), 14 x 103 (range, 0.62 - 40.6) and 0.55 x 105 (range, 0.04 - 1.85), respectively. In addition, significant numbers of NK and TCRgd+ cells/kg were infused - 6.67 x 107 (median; range, 0.68 - 18.2) and 1.58 x 107 (median; range, 0.13 - 4.7), respectively. All 30 patients tolerated the infusion of haploidentical stem cell grafts well. Twenty-five patients had primary engraftment of ANC > 500 cells/µL at a median of 12 days (range, 10 - 18) and PLT > 20,000 cells/µL at a median of 15 days (range, 11 - 27). Peripheral T-cell chimerism at the time of engraftment was completely donor in 19/25 patients (76%), mixed in 3 (12%), and not measured in three. Five patients experienced primary graft failure and 2 had secondary graft failure. All except of one were successfully re-transplanted. None of the recipients developed severe acute GVHD grades III - IV. Only 1 patient had acute GVHD grade II that started on day 22. The vast majority of patients (96.7%) experienced no or only grade I acute GVHD despite minimal GVHD prophylaxis after transplantation. Samples from 24/25 patients with primary engraftment were evaluable for immune reconstitution (Figure 1). On day 28, the majority of WBC were NK cells (median 309 cells/µL; range, 64 - 1026). The second main type of cells were CD3+ cells (median 151 cells/µL; range, 9 - 953), mostly TCRgd+ (median 87 cells/µL; range, 7 - 891). At day 100, TCRab+ cells equalled TCgd+ cells (median 108 vs. 116 cells/µL). B cells recovered more slowly, with a median of 255.5 cells/µL (range, 1 - 1218) on day 63. ADV reactivation contributed most to infectious complications following transplantation. In total, 16/30 patients had ADV DNAemia or were positive in stool. Additionally, seven patients were tested positive for CMV (blood or urine). BK virus was present in 5 patients with 3 patients experiencing cystitis. No EBV reactivation was observed. Two patients had bacterial sepsis, 1 moderate, 1 fatal (due to non-engraftment).No fatal viral infection occurred within 100 days. One molecular relapse was observed within 100 days post transplantation that was treated with blinatumomab. Two of the 30 transplanted patients died within 100 days after transplantation: 1 patient due to sepsis following graft failure (non-relapse mortality) and 1 due to relapse. On day 100, chimerism was completely donor in 20 patients and mixed in two. Conclusions: The CliniMACS depletion system of TCRab+ and CD19+ cells yielded a large number of CD34+ cells, NK cells and TCRgd+ cells, that could be infused safely into pediatric patients with minimal risk of severe acute GVHD. The immune reconstitution was rapid and there was no TRM associated with viral or fungal infections. Coupled with a reduced-intensity regimen, the overall TRM was low. Longer follow up will provide essential information on chronic GVHD and survival outcomes. Figure 1 Immune Reconstitution after transplantation of TCR-alpha/beta and CD19 depleted haploidentical stem cell grafts Figure 1. Immune Reconstitution after transplantation of TCR-alpha/beta and CD19 depleted haploidentical stem cell grafts Disclosures Bader: Riemser: Research Funding; Neovii Biotech: Research Funding; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Medac: Consultancy, Research Funding. Karitzky:Miltenyi Biotec: Employment. Holtkamp:Miltenyi Biotec: Employment. Siewert:Miltenyi Biotec: Employment. Bönig:Miltenyi Biotec: Consultancy, Honoraria, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-Patentholder of TcRalpha/beta depletion technology.

scholarly journals Effect of the Neddylation Inhibitor Pevonedistat on Normal Hematopoietic Stem Cell Subsets and Immune Cell Composition

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4787-4787
Author(s):  
Fatemeh Majidi ◽  
Oumaima Stambouli ◽  
Ron-Patrick Cadeddu ◽  
Simon Kai Brille ◽  
Jasmin Ewert ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Low Dose ◽  
Tumor Stem Cell ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Biphasic Effect

Abstract Introduction: Antitumor activity of the neddylation inhibitor pevonedistat has been documented in several hematologic and non-hematologic malignancies. Unexpectedly, Zhou et al (PNAS, 2016) discovered a dose-dependent biphasic effect of pevonedistat in solid tumor cell lines. While micromolar concentrations inhibited tumor cell growth, low nanomolar concentrations significantly increased cell proliferation and tumor stem cell self-renewal both in vitro and in vivo. The effect of low-dose pevonedistat has not yet been explored in the field of hematopoietic stem cell transplantation. Therefore, we evaluated how pevonedistat affects the viabiilty, growth and proportions of CD34 + cell subpopulations. In view of the emerging role of neddylation in the regulation of both innate and adaptive immunity, we also investigated the influence of pevonedistat on T-cell activation to explore a potentially beneficial effect on posttransplant immune complications. Methods and Results: Using the WST-1 assay we confirmed the biphasic effect of pevonedistat on normal mobilized CD34 + cells. Incubation for 72 h with 0.1 µM pevonedistat significantly increased metabolic activity as a surrogate parameter for proliferation, while 1.0 µM pevonedistat showed a cytotoxic effect. We explored the underlying mechanism for the low-dose effect. Since Zhou et al. previously showed that pevonedistat can promote tumor stem cell proliferation by inducing EGFR homodimerization, we used a proximity ligation assay and found that 0.1 µM pevonedistat induced EGFR homodimerization in normal mobilized CD34 + cells, too. In addition to homodimerization, we also looked at phosphorylation at Tyr1068, a marker of EGFR activation. By flow cytometry, we showed that phosphorylation was increased by 0.01 µM and 0.1 µM pevonedistat. Using an ELISA-based transcription assay, we also observed a biphasic effect of pevonedistat on c-Myc expression, which is regarded as a marker of 'stemness'. Incubation with pevonedistat for 72 hrs at 0.01 and 0.1 µM stimulated expression of c-Myc, whereas incubation at 1.0 µM downregulated c-Myc. Fractions of hematopoietic stem and progenitor cell (HSPC) subpopulations were measured in CD34 + cells from cord blood after incubation with 0.01, 0.1 and 1.0 µM pevonedistat. Flow cytometry was performed using antibodies against CD34, CD45RA and CD133, as well as 7-AAD for testing cell viability. Exposure to pevonedistat for 72 hrs at 0.1 µM caused an increase in the number of CD34 + cells compared to vehicle-treated CD34+ cells at 72 h as well as compared to initial number of CD34+ cells, whereas 1.0 µM caused a significant decrease. The absolute number of multipotent progenitors (MPP) (CD34 +CD133 +CD45RA -) remained relatively stable at all concentrations, while lympho-myeloid progenitors (LMPP) (CD34+CD133+CD45RA+) and late progenitors (LP) (CD34+CD133-CD45RA+) increased slightly with 0.1 µM pevonedistat compared with controls. However, a significant decrease in LMPP and LP cell numbers was observed at 1.0 µM. Different concentrations of pevonedistat were tested for their capability to modulate allogeneically stimulated T cell activation in a multi-donor mixed lymphocyte reaction (mdMLR) assay in vitro. Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (MSC-EV) were used as internal immuno-modulatory and non-immuno-modulatory controls in the assay. After 5 days, alterations in the immune cell composition were analyzed by flow cytometry. Pevonedistat was not toxic for MNCs in the mdMLR. However, it decreased the number of activated (CD25high CD54+) CD4+ cells and CD8+ cells. Conclusions: One of the problems in the post-transplant period is a rapid decline in MPP numbers, associated with increased risk of engraftment failure. We showed that low-dose pevonedistat (0.1 µM) is capable of increasing the number of CD34 + cells in vitro while keeping the absolute number of MPPs stable. This finding, together with the observed increase in c-Myc expression, suggests that pevonedistat may help to preserve 'stemness' of CD34+ donor cells, thus supporting engraftment of hematopoietic stem and progenitor cells. Furthermore, the immunosuppressive effects revealed by mdMLR suggest that low-dose pevonedistat may also play a useful immunomodulatory role in the post-transplant setting to potentially reduce the risk of graft-versus-host disease. Figure 1 Figure 1. Disclosures Majidi: Takeda: Research Funding. Germing: Jazz Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria. Zeiser: Incyte, Mallinckrodt, Novartis: Honoraria, Speakers Bureau. Gattermann: Celgene: Honoraria; Takeda: Research Funding; Novartis: Honoraria.

Prevention of Graft-Versus-Host Disease by Inhibition of Lymphocyte Trafficking Using a CCR5 Antagonist

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 673-673 ◽  
Author(s):  
Ran Reshef ◽  
Selina M. Luger ◽  
Alison W. Loren ◽  
Noelle V. Frey ◽  
Steven C. Goldstein ◽  
...  
Keyword(s):  
Research Funding ◽  
Acute Gvhd ◽  
Off Label Use ◽  
Lymphocyte Trafficking ◽  
Off Label ◽  
Gvhd Prophylaxis ◽  
Cd34 Cells ◽  
Novel Strategy ◽  
Dose Levels

Abstract Abstract 673 Inhibition of lymphocyte trafficking early after allogeneic stem cell transplantation (SCT) could limit T cell interactions with antigen-presenting cells and migration to target tissues. This represents a novel strategy to prevent GvHD without interfering with GvL activity. CCR5 is a chemokine receptor expressed on effector T-cells and immature dendritic cells and binds 3 ligands - CCL3, CCL4 and RANTES (CCL5). Accumulating evidence from animal models and clinical observations implicates CCR5 as pivotal in the pathogenesis of GvHD. Genomic analyses suggest that the same CCR5 polymorphisms that confer resistance to HIV infection also correlate with a lower susceptibility to acute GvHD. Maraviroc (MVC; Selzentry®, Pfizer) is the first oral CCR5 antagonist in clinical use. We hypothesized that modulating T-cell trafficking early after allogeneic SCT via CCR5 blockade would limit GvHD. We therefore performed preclinical and clinical testing of MVC as GvHD prophylaxis. Our goals were to 1) determine in vitro activity of MVC on chemotaxis, 2) determine the feasibility, safety and appropriate dose of MVC as part of GvHD prophylaxis, and 3) demonstrate biological activity of MVC through immune pharmacodynamic assays. In vitro, MVC fully inhibited CCR5 internalization by CCL3 and RANTES even at concentrations as low as 1 μM. Using RANTES as a chemotactic trigger, MVC caused dose-dependent inhibition of lymphocyte chemotaxis by up to 53% at MVC 1mM. To address concerns that MVC might impair hematopoiesis, we demonstrated that CCR5 was not expressed on the surface of bone marrow- and peripheral blood-derived CD34+ cells. Moreover, when CD34+ cells were plated in methylcellulose, formation of CFU-GEMM and CFU-GM was not affected by the presence of MVC 1μM; CFU-E and BFU-E were slightly decreased compared to controls. Based on these and other data, we enrolled 19 pts in a phase I/II study of reduced intensity conditioned allogeneic SCT with MVC GvHD prophylaxis. Pts received fludarabine 120mg/m2 and IV busulfan 6.4 mg/kg followed by peripheral blood stem cells from matched related (n=6), matched unrelated (n=10) and 1-antigen mismatched unrelated (n=3) donors. In addition to standard GvHD prophylaxis with tacrolimus and methotrexate, MVC at escalating dose levels was given from day -2 to +30. Median age was 63 (range 21–74). Indications for SCT were AML/MDS (9), NHL (4), myelofibrosis (2), CLL, aplastic anemia, Hodgkin lymphoma and myeloma (1 each). Pharmacokinetic analysis on 6 pts at each dose revealed that the 300 mg and 150 mg bid dose levels resulted in mean Cavg of 536 and 118 ng/ml, respectively. 3/6 patients at 150mg did not reach the targeted minimum Cavg (100 ng/ml), while the 300mg dose level resulted in adequate Cavg in 6/6 patients and was used as the phase II dose. MVC was well tolerated; 3 pts did not complete the entire course because of transient LFT abnormalities (1) or mucositis (2). The median time to ANC>500/μL was 15 d (range 10–21) and to platelets>20k/μL was 13 d (range 11–24) with no graft rejections. The median donor chimerism at day 100 was 97% (range 83–100%). A day 100-landmark analysis in evaluable pts demonstrated that the cumulative incidence of acute GvHD grade 2–4 was 27% (grade 3–4; 9%) in this high-risk population. Importantly, by day 100 all cases of acute GvHD involved only the skin without liver or intestinal involvement. At a median follow up of 186 days, 3/19 patients relapsed (2 AML, 1 NHL) and 6/19 patients died (3 disease-related, 1 neutropenic sepsis, 1 SOS, 1 unrelated). There were no GvHD-related deaths. To explore potential mechanisms, we tested the capacity of patient serum to inhibit CCR5 internalization and chemotaxis. Patient serum from multiple time points (trough, 1, 2, 3, 4, 6 hr post dose) effectively prevented internalization of CCR5 by RANTES. In addition, in vitro chemotaxis of normal donor T-cells in response to RANTES was significantly impaired in the presence of patient serum from day 0 (on MVC) as compared to day 60 (off MVC). In summary, inhibition of lymphocyte trafficking to peripheral tissues represents a novel strategy to modulate and possibly reduce acute GvHD in allogeneic SCT. MVC at 300mg bid was well tolerated and biologically active in pharmacodynamic assays. Patients receiving MVC exhibited limited GvHD by day 100 without excessive relapses. The phase II portion of the trial is ongoing. Disclosures: Off Label Use: Off label use of maraviroc (Selzentry) will be discussed. Frey:Pfizer, Inc.: Speakers Bureau. Vonderheide:Pfizer, Inc.: Research Funding. Porter:Pfizer, Inc.: Research Funding.

Low Dose Lenalidomide and Dexamethasone Induction Followed by Autologous Transplantation In Untreated Patients with Myeloma Is Associated with High Response Rates and Preservation of CD8, but Not CD4 or NK Cellular Immunity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1862-1862 ◽  
Author(s):  
Simon J. Harrison ◽  
Amit S Khot ◽  
Tsin Tsin ◽  
Andy Hsu ◽  
Karen Chen ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Induction Therapy ◽  
Research Funding ◽  
Low Dose ◽  
Cell Function ◽  
Nk Cell ◽  
Newly Diagnosed ◽  
Cell Numbers ◽  
Cd34 Cells

Abstract Abstract 1862 Multiple Myeloma (MM) and its treatment are associated with impaired humoral and cellular immunity (CI). Lenalidomide (Len) is thought to mediate its anti-MM effect in part via stimulation of in vivo T cell, NK cell and NKT cell activity. However, as we have previously demonstrated (Hsu et al, Blood, 2011;117:1605), the immunostimulatory effects of Len are substantially abrogated by co-administration of high doses of Dexamethasone (Dex). It is unknown whether the efficacy of the Len/Dex induction regimen is maintained in newly diagnosed MM if the dose of Dex is lowered to preserve CI. In a prospective study (PMCC HREC #05/56), we examined both anti-MM responses and CI using a regimen of low dose Len and Dex induction followed by consolidation with autologous stem cell transplant (ASCT) in previously untreated patients with MM. Methods: Twenty patients were enrolled. Induction therapy was with four 28 day (d) cycles of Len 15 mg d1 − 21 and Dex 20 mg weekly followed by hematopoietic stem cell (HSC) mobilization with cyclophosphamide 2 – 4 gm/m2 and G-CSF 10mcg/kg/day, and a melphalan 140 – 200mg/m2 conditioned ASCT. Maintenance with Rev 25mg d1-21 of a 28d cycle was commenced on d21-35 post ASCT until progression. Interim assessments of response were made post-induction, post-ASCT and on 31/05/2011 using IMWG uniform response criteria. Assessments of CI were undertaken at enrolment and again at the end of 4 cycles of induction by flow cytometric analysis of peripheral blood lymphocyte subsets (CD19+, CD3+, CD4+, CD8+, CD4+/CD25+/CD127+ Treg, CD3−/ CD16+/CD56+ NK cells), T cell proliferation to allogeneic stimulators (ratio 1:1) in a 7d mixed lymphocyte reaction (MLR) and analysis of NK function by cytotoxicity against K562 targets. All data were compared to age-matched controls, and analysed for statistical significance using a student T-test. Results: Clinical responses: All patients have completed induction and ASCT. Median age=57.5 yrs, (range 44 – 70); male= 15; IgG = 10, IgA = 6 and light chain = 4. Stage at enrolment ISS1 = 9, ISS2 = 9 and ISS3 = 2. Median harvested CD34+ cells=10.8×106 /kg (range 4.9 – 40.6). The median number of CD34+ cells infused = 4.8×106 / kg (range 2.9 – 11.5) and median time to recovery of neutrophils > 0.5 ×109/L and platelets > 20 ×109/L was 12d (range 9 – 18) and 11d (range 8 – 25) respectively. The post-induction, overall response rate was 85%, very good partial response (VGPR)=20%, partial response (PR)= 65%, stable disease =5%, and 10% were refractory. Post-autograft responses improved to complete response (CR)=10%, VGPR= 40% and PR=35%. At a median follow-up of 17 months (range 5 – 29 months), the best response achieved to date is 35% CR, 25% VGPR and 30% PR (Fig 1). Immunology: CI data is available on 19 patients. Compared-to age matched controls, patients at enrolment had reduced CD3+ (p≤0.05), CD4+ (p≤0.01) (Fig 2)and Treg (P≤0.01) numbers and reduced NK cell function (p<0.05); however CD8+ T cell, NK cell and B cell numbers were normal. Following four cycles of Len/Dex therapy, CD3+, CD4+ and Treg numbers all increased, but remained below those of normal controls. Conversely, circulating B lymphocyte numbers fell substantially (P≤0.01) and NK cell function remained significantly impaired (p<0.05) following induction therapy. However, functional analysis of CD3+ (Fig 3), CD4+ and CD8+ proliferation in an MLR at enrolment and following induction therapy was identical to that of controls. Conclusion: This novel treatment regimen of low dose Len and low dose Dex followed by ASCT in untreated patients with MM is associated with high response rates and successful HSC collection. The depth of response progressively improved following ASCT. Cellular immunology in MM patients at diagnosis shows specific reductions in CD4+ and Treg cell numbers, and NK cell function that is not rescued by Len therapy when co-administered with Dex, even at low doses, which induced a further reduction in CD19+ cells. However despite these deficiencies, preserved proliferative capacity of both CD4+ and CD8+ T-cells is seen at diagnosis and following this induction therapy, suggesting that with the low dose Len-Dex regimen, the immune environment is conducive to novel strategies that are aimed at inducing adaptive anti-MM immune responses. Disclosures: Harrison: Celgene: Honoraria, Research Funding. Off Label Use: Low dose lenalidomide in newly diagnosed myeloma. Neeson:Celgene: Research Funding. Prince:Celgene: Honoraria, Research Funding.

scholarly journals The APC/C Coactivator Cdh1 Controls Self-Renewal and Differentiation of Human and Murine HSPCs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2650-2650
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Andrea Schmidts ◽  
Marie Follo ◽  
Dagmar Wider ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract Introduction: The balance between differentiation and self-renewal in hematopoietic stem and progenitor cells (HSPCs) is crucial for homeostasis and lifelong blood cell production. Differentiation is predominantly initiated in the G1 phase of the cell cycle when the E3 ligase anaphase-promoting complex or cyclosome (APC/C) is highly active. Its coactivator Cdh1 determines substrate specificity and mediates proteasomal degradation. Relevant target proteins are associated with cell fate decisions in G1/G0, and there is growing evidence that Cdh1 is an important regulator of differentiation. While this has already been demonstrated in neurons, muscle cells or osteoblasts, little is known about the role of APC/CCdh1 in hematopoiesis. Here we report on the function of Cdh1 in human and murine HSPCs in vitro and in vivo. Methods: Human CD34+ cells from the peripheral blood of G-CSF mobilized donors were exposed to different cytokine combinations and gains or losses of surface marker expression during cell division were determined. By using the established culture conditions Cdh1 expression was detected in distinct hematopoietic lineages and developmental states. CD34+ cells were transduced with a lentivirus to deplete Cdh1 by stably expressing shRNA and was then used for in vitro differentiation in liquid culture or CFU assay. In a second miR-based RNAi approach murine BM cells were depleted of Cdh1 and used for competitive transplantation assays. Complementary xenotransplantation of human Cdh1-depleted CD34+cells was carried out with NSG mice. Results: The stimulation of freshly thawed CD34+ cells with cytokines led to cell cycle entry and proliferation. Self-renewing cells preserved CD34 expression for up to 7 cell divisions with a low proliferation rate. In contrast, during granulopoiesis and erythropoiesis cells divided more frequently with rapid down-regulation of CD34. Cdh1 expression was tightly connected to differentiation status and proliferation properties. In vitro cultured CD34+ cellsand those from BM of healthy human donors showed the highest Cdh1 level compared to moderate or low expression in lymphoid and myeloid cells. Cdh1 is highly expressed at the transcriptional and translational level during both self-renewal and also when cells were directed toward erythroid differentiation. Therefore, high Cdh1 expression is characteristic of immature hematopoietic cells and differentiating precursors. The knockdown of Cdh1 (Cdh1-kd) did not affect proliferation or viability as detected by CFSE staining and measuring the cell cycle length via live-cell imaging. However, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with a lower frequency of glycophorin A+ cells. The functional relevance of Cdh1 depletion was verified in CFU assays. Cells with Cdh1-kd formed fewer primary colonies but significantly more secondary colonies, indicating a preference for self-renewal over differentiation. After competitive transplantation Cdh1-depleted murine BM cells showed a significant enhancement in the repopulation of PB, BM and spleen at week 3, while there was no change in cell cycle properties. However, after 8 weeks chimerism in each of the compartments was reduced to that of the control cells. Accordingly, higher LK and LSK frequencies supported the engraftment of Cdh1-depleted cells at week 3, but there was a significant decrease at week 8 compared to control cells, suggestive of stem cell exhaustion. The Cdh1 level also affected cell differentiation in vivo. After 8 weeks the population of B cells (B220+) was increased in transplanted Cdh1-kd cells and the frequency of mature granulocytes (CD11b+ Gr1high) was reduced. Consistently, human Cdh1-depleted CD34+ cells engrafted to a much higher degree in the murine BM 8 and 12 weeks after xenotransplantation, as shown by a higher frequency of human CD45+ cells. Moreover, the increase of human CD19+ B cells with Cdh1-kd confirmed the results of the competitive transplantation. Conclusions: Loss of the APC/C coactivator Cdh1 supports repopulation of murine HSPCs after transplantation with a lymphoid-biased differentiation, and was confirmed in xenotranplantation experiments. In the long-term, Cdh1 loss led to exhaustion of primitive LK and LSK population, highlighting the role of Cdh1 as a critical regulator of HSPC self-renewal and differentiation. Disclosures Engelhardt: Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.

scholarly journals Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2730-2737 ◽  
Author(s):  
Colin H. Martin ◽  
Petter S. Woll ◽  
Zhenya Ni ◽  
Juan Carlos Zúñiga-Pflücker ◽  
Dan S. Kaufman
Keyword(s):  
Stem Cell ◽  
B Cells ◽  
Progenitor Cells ◽  
Embryonic Stem ◽  
Cell Development ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Developmental Potential ◽  
Cd34 Cells

Abstract Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.

scholarly journals Impact of Induction Treatment on Stem Cell Collection: A COVID Related Study

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4848-4848
Author(s):  
Brad Rybinski ◽  
Ashraf Z. Badros ◽  
Aaron P. Rapoport ◽  
Mehmet Hakan Kocoglu
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Research Funding ◽  
Median Number ◽  
Cell Transplant ◽  
Cd34 Cells ◽  
Significant Difference ◽  
Number Of Cycles ◽  
Time Required ◽  
Stem Cell Collection

Abstract Introduction: Standard induction therapy for multiple myeloma consists of 3-6 cycles of bortezomib, lenalidomide, and dexamethasone (VRd) or carfilzomib, lenalidomide and dexamethasone (KRd). Receiving greater than 6 cycles of a lenalidomide containing regimen is thought to negatively impact the ability to collect sufficient CD34+ stem cells for autologous stem cell transplant (Kumar, Dispenzieri et al. 2007, Bhutani, Zonder et al. 2013). Due to the COVID-19 pandemic, at least 20 patients at University of Maryland Greenebaum Comprehensive Cancer Center (UMGCC) had transplant postponed, potentially resulting in prolonged exposure to lenalidomide containing induction regimens. Here, in the context of modern stem cell mobilization methods, we describe a retrospective study that suggests prolonged induction does not inhibit adequate stem cell collection for transplant. Methods: By chart review, we identified 56 patients with multiple myeloma who received induction with VRd or KRd and underwent apheresis or stem cell transplant at UMGCC between 10/1/19 and 10/1/20. Patients were excluded if they received more than 2 cycles of a different induction regimen, had a past medical history of an inborn hematological disorder, or participated in a clinical trial of novel stem cell mobilization therapy. We defined 1 cycle of VRd or KRd as 1 cycle of "lenalidomide containing regimen". In accordance with routine clinical practice, we defined standard induction as having received 3-6 cycles of lenalidomide containing regimen and prolonged induction as having received 7 or more cycles. Results: 29 patients received standard induction (Standard induction cohort) and 27 received prolonged induction (Prolonged induction cohort) with lenalidomide containing regimens. The median number of cycles received by the Standard cohort was 6 (range 4-6), and the median number of cycles received by the Prolonged cohort was 8 (range 7-13). The frequency of KRd use was similar between patients who received standard induction and prolonged induction (27.58% vs. 25.93%, respectively). Standard induction and Prolonged induction cohorts were similar with respect to clinical characteristics (Fig 1), as well as the mobilization regimen used for stem cell collection (p = 0.6829). 55/56 patients collected sufficient stem cells for 1 transplant (≥ 4 x 10 6 CD34 cells/kg), and 40/56 patients collected sufficient cells for 2 transplants (≥ 8 x 10 6 CD34 cells/kg). There was no significant difference in the total CD34+ stem cells collected at completion of apheresis between standard and prolonged induction (10.41 and 10.45 x 10 6 CD34 cells/kg, respectively, p = 0.968, Fig 2). Furthermore, there was no significant correlation between the number of cycles of lenalidomide containing regimen a patient received and total CD34+ cells collected (R 2 = 0.0073, p = 0.5324). Although prolonged induction did not affect final stem yield, prolonged induction could increase the apheresis time required for adequate collection or result in more frequent need for plerixafor rescue. There was no significant difference in the total number of stem cells collected after day 1 of apheresis between patients who received standard or prolonged induction (8.72 vs. 7.96 x 10 6 cells/kg, respectively, p = 0.557). However, patients who received prolonged induction were more likely to require 2 days of apheresis (44% vs. 25%, p = 0.1625) and there was a trend toward significance in which patients who received prolonged induction underwent apheresis longer than patients who received standard induction (468 vs 382 minutes, respectively, p = 0.0928, Fig 3). In addition, longer apheresis time was associated with more cycles of lenalidomide containing regimen, which neared statistical significance (R 2 = 0.0624, p = 0.0658, Fig 4). There was no significant difference between standard and prolonged induction with respect to the frequency of plerixafor rescue. Conclusions: Prolonged induction with lenalidomide containing regimens does not impair adequate stem cell collection for autologous transplant. Prolonged induction may increase the apheresis time required to collect sufficient stem cells for transplant, but ultimately clinicians should be re-assured that extending induction when necessary is not likely to increase the risk of collection failure. Figure 1 Figure 1. Disclosures Badros: Janssen: Research Funding; J&J: Research Funding; BMS: Research Funding; GlaxoSmithKline: Research Funding.

scholarly journals Lower Hematopoietic Progenitor Cell Counts and Yields at Subsequent Donations Is Influenced By a Shorter Inter-Donation Interval between the First and Subsequent Mobilizations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1962-1962
Author(s):  
Sandhya R. Panch ◽  
Brent R. Logan ◽  
Jennifer A. Sees ◽  
Bipin N. Savani ◽  
Nirali N. Shah ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Time Interval ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Peripheral Blood Cd34 ◽  
Cell Counts ◽  
Cd34 Cells

Introduction: Approximately 7% of unrelated hematopoietic stem cell (HSC) donors are asked to donate a subsequent time to the same or different recipient. In a recent large CIBMTR study of second time donors, Stroncek et al. incidentally found that second peripheral blood stem cell (PBSC) collections had lower total CD34+ cells, CD34+ cells per liter of whole blood processed, and CD34+ cells per kg donor weight. Based on smaller studies, the time between the two independent PBSC donations (inter-donation interval) as well as donor sex, race and baseline lymphocyte counts appear to influence CD34+ cell yields at subsequent donations. Our objective was to retrospectively evaluate factors contributory to CD34+ cell yields at subsequent PBSC donation amongst NMDP donors. Methods. The study population consisted of filgrastim (G-CSF) mobilized PBSC donors through the NMDP/CIBMTR between 2006 and 2017, with a subsequent donation of the same product. evaluated the impact of inter-donation interval, donor demographics (age, BMI, race, sex, G-CSF dose, year of procedure, need for central line) and changes in complete blood counts (CBC), on the CD34+ cell yields/liter (x106/L) of blood processed at second donation and pre-apheresis (Day 5) peripheral blood CD34+ cell counts/liter (x106/L) at second donation. Linear regression was used to model log cell yields as a function of donor and collection related variables, time between donations, and changes in baseline values from first to second donation. Stepwise model building, along with interactions among significant variables were assessed. The Pearson chi-square test or the Kruskal-Wallis test compared discrete variables or continuous variables, respectively. For multivariate analysis, a significance level of 0.01 was used due to the large number of variables considered. Results: Among 513 PBSC donors who subsequently donated a second PBSC product, clinically relevant decreases in values at the second donation were observed in pre-apheresis CD34+ cells (73.9 vs. 68.6; p=0.03), CD34+cells/L blood processed (32.2 vs. 30.1; p=0.06), and total final CD34+ cell count (x106) (608 vs. 556; p=0.02). Median time interval between first and second PBSC donations was 11.7 months (range: 0.3-128.1). Using the median pre-apheresis peripheral blood CD34+ cell counts from donation 1 as the cut-off for high versus low mobilizers, we found that individuals who were likely to be high or low mobilizers at first donation were also likely to be high or low mobilizers at second donation, respectively (Table 1). This was independent of the inter-donation interval. In multivariate analyses, those with an inter-donation interval of >12 months, demonstrated higher CD34+cells/L blood processed compared to donors donating within a year (mean ratio 1.15, p<0.0001). Change in donor BMI was also a predictor for PBSC yields. If donor BMI decreased at second donation, so did the CD34+cells/L blood processed (0.74, p <0.0001). An average G-CSF dose above 960mcg was also associated with an increase in CD34+cells/L blood processed compared to donors who received less than 960mcg (1.04, p=0.005). (Table 2A). Pre-apheresis peripheral blood CD34+ cells on Day 5 of second donation were also affected by the inter-donation interval, with higher cell counts associated with a longer time interval (>12 months) between donations (1.23, p<0.0001). Further, independent of the inter-donation interval, GCSF doses greater than 960mcg per day associated with higher pre-apheresis CD34+ cells at second donation (1.26, p<0.0001); as was a higher baseline WBC count (>6.9) (1.3, p<0.0001) (Table 2B). Conclusions: In this large retrospective study of second time unrelated PBSC donors, a longer inter-donation interval was confirmed to be associated with better PBSC mobilization and collection. Given hematopoietic stem cell cycling times of 9-12 months in humans, where possible, repeat donors may be chosen based on these intervals to optimize PBSC yields. Changes in BMI are also to be considered while recruiting repeat donors. Some of these parameters may be improved marginally by increasing G-CSF dose within permissible limits. In most instances, however, sub-optimal mobilizers at first donation appear to donate suboptimal numbers of HSC at their subsequent donation. Disclosures Pulsipher: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Honoraria. Shaw:Therakos: Other: Speaker Engagement.

scholarly journals Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4363-4363
Author(s):  
Alexandre Janel ◽  
Nathalie Boiret-Dupré ◽  
Juliette Berger ◽  
Céline Bourgne ◽  
Richard Lemal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Confocal Microscopy ◽  
Hematopoietic Stem Cell ◽  
Biological Samples ◽  
Mesenchymal Cells ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Cd34 Cells

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

Growth and Differentiation of Human Stem Cell Factor/Erythropoietin-Dependent Erythroid Progenitor Cells In Vitro

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3658-3668 ◽  
Author(s):  
Birgit Panzenböck ◽  
Petr Bartunek ◽  
Markus Y. Mapara ◽  
Martin Zenke
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Large Cell ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Peripheral Blood Stem Cells ◽  
Erythroid Progenitor ◽  
Cell Numbers

Abstract Stem cell factor (SCF) and erythropoietin (Epo) effectively support erythroid cell development in vivo and in vitro. We have studied here an SCF/Epo-dependent erythroid progenitor cell from cord blood that can be efficiently amplified in liquid culture to large cell numbers in the presence of SCF, Epo, insulin-like growth factor-1 (IGF-1), dexamethasone, and estrogen. Additionally, by changing the culture conditions and by administration of Epo plus insulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34+ peripheral blood stem cells and human bone marrow and can be isolated, amplified, and differentiated in vitro under the same conditions. Thus, highly homogenous populations of SCF/Epo-dependent erythroid progenitors can be obtained in large cell numbers that are most suitable for further biochemical and molecular studies. We demonstrate that such cells express the recently identified adapter protein p62dok that is involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21cip1 and p27kip1 that are highly induced when cells differentiate. Thus, the in vitro system described allows the study of molecules and signaling pathways involved in proliferation or differentiation of human erythroid cells.

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