scholarly journals Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 400-409 ◽  
Author(s):  
S Siena ◽  
M Bregni ◽  
B Brando ◽  
N Belli ◽  
F Ravagnani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cancer Therapy ◽  
Large Scale ◽  
Autologous Transplantation ◽  
Hematopoietic Progenitor Cell ◽  
Direct Immunofluorescence ◽  
High Dose ◽  
Hematopoietic Progenitors ◽  
Flow Cytometry Assay ◽  
Cd34 Cells

Optimum methods of harvesting circulating hematopoietic progenitors for autologous transplantation to support myeloablative cancer therapy are still uncertain, mostly because of the lack of an assay for marrow- repopulating stem cells. The CFU-GM assay, the commonly used indirect indicator of the quality of the graft, is poorly standardized and provides results evaluable only retrospectively. Based on the knowledge that hematopoietic progenitors express CD34 and CD33 differentiation antigens, we developed a dual-color direct immunofluorescence flow cytometry assay with the aim of replacing the CFU-GM assay advantageously. For this purpose, we applied both assays to 157 blood samples obtained daily throughout 20 different recoveries from pancytopenia induced by high-dose cyclophosphamide or etoposide cancer therapy with or without recombinant human GM colony-stimulating factor (rhGM-CSF). The appearance of CD34+ cells in the circulation indicated that hematopoietic progenitors had increased to more than 500 CFU- GM/mL, a level clinically adequate for large-scale harvest by leukapheresis. Total CD34+ cells correlated well with CFU-GM (r = .89), and data could be fitted by a linear regression line described by the equation y = 388.3 + 64.0x, where y = CFU-GM/mL and x = CD34+ cells per microliter. Moreover, in a series of six patients treated with myeloablative chemoradiotherapy, early hematopoietic recovery of marrow functions was predicted more accurately by the number of transplanted CD34+/CD33+ cells than by either total nucleated cells, CFU-GM, CD34+/CD33- cells, or CD34-/CD33+ cells. Data presented in this article favor clinical use of the CD34/CD33 flow cytometry assay to guide harvesting of circulating hematopoietic progenitors for autologous transplantation and contribute to better understanding of the role played by circulating hematopoietic progenitor cell subsets in marrow recovery after myeloablative cancer therapy.

scholarly journals Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients

Blood ◽  
1991 ◽  
Vol 77 (2) ◽  
pp. 400-409 ◽  
Author(s):  
S Siena ◽  
M Bregni ◽  
B Brando ◽  
N Belli ◽  
F Ravagnani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cancer Therapy ◽  
Large Scale ◽  
Autologous Transplantation ◽  
Hematopoietic Progenitor Cell ◽  
Direct Immunofluorescence ◽  
High Dose ◽  
Hematopoietic Progenitors ◽  
Flow Cytometry Assay ◽  
Cd34 Cells

Abstract Optimum methods of harvesting circulating hematopoietic progenitors for autologous transplantation to support myeloablative cancer therapy are still uncertain, mostly because of the lack of an assay for marrow- repopulating stem cells. The CFU-GM assay, the commonly used indirect indicator of the quality of the graft, is poorly standardized and provides results evaluable only retrospectively. Based on the knowledge that hematopoietic progenitors express CD34 and CD33 differentiation antigens, we developed a dual-color direct immunofluorescence flow cytometry assay with the aim of replacing the CFU-GM assay advantageously. For this purpose, we applied both assays to 157 blood samples obtained daily throughout 20 different recoveries from pancytopenia induced by high-dose cyclophosphamide or etoposide cancer therapy with or without recombinant human GM colony-stimulating factor (rhGM-CSF). The appearance of CD34+ cells in the circulation indicated that hematopoietic progenitors had increased to more than 500 CFU- GM/mL, a level clinically adequate for large-scale harvest by leukapheresis. Total CD34+ cells correlated well with CFU-GM (r = .89), and data could be fitted by a linear regression line described by the equation y = 388.3 + 64.0x, where y = CFU-GM/mL and x = CD34+ cells per microliter. Moreover, in a series of six patients treated with myeloablative chemoradiotherapy, early hematopoietic recovery of marrow functions was predicted more accurately by the number of transplanted CD34+/CD33+ cells than by either total nucleated cells, CFU-GM, CD34+/CD33- cells, or CD34-/CD33+ cells. Data presented in this article favor clinical use of the CD34/CD33 flow cytometry assay to guide harvesting of circulating hematopoietic progenitors for autologous transplantation and contribute to better understanding of the role played by circulating hematopoietic progenitor cell subsets in marrow recovery after myeloablative cancer therapy.

Quantization of CD34+ Peripheral Blood Hematopoietic Progenitors for Autografting in Cancer Patients

1993 ◽  
Vol 16 (5_suppl) ◽  
pp. 80-82 ◽  
Author(s):  
M. Di Nicola ◽  
S. Siena ◽  
M. Bregni ◽  
F. Peccatori ◽  
M. Magni ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Autologous Transplantation ◽  
High Dose Chemotherapy ◽  
Direct Immunofluorescence ◽  
High Dose ◽  
Hematopoietic Progenitors ◽  
Flow Cytometry Assay ◽  
Real Time Information ◽  
Time Information

After myeloablative regimens, combined reinfusion of peripheral blood hematopoietic circulating progenitor cells (CPC) and bone marrow, yields a very rapid hematopoietic recovery. Therefore, based on the knowledge that CPC express the CD34 and CD33 differentiation antigen, we have developed a direct immunofluorescence flow cytometry assay to detect the peak of CPC in the peripheral blood of patients treated with high dose chemotherapy and growth factors. This assay, compared to CFU-GM assay, has the following advantages: 1) easy to do 2) standardized method 3) real time information on CPC number. This work illustrates the practical aspects of this assay and substantiate the widespread use of the CD34/33 flow cytometry assay to guide harvesting of circulating hematopoietic progenitors for autologous transplantation.

scholarly journals Human peripheral blood hematopoietic progenitors are optimal targets of retroviral-mediated gene transfer

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1418-1422 ◽  
Author(s):  
M Bregni ◽  
M Magni ◽  
S Siena ◽  
M Di Nicola ◽  
G Bonadonna ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cancer Patients ◽  
Resistance Gene ◽  
Peripheral Blood ◽  
Large Scale ◽  
Human Peripheral Blood ◽  
High Dose ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem

Abstract Hematopoietic progenitor cells circulate in the peripheral blood (PB) of cancer patients during the recovery phase that follows treatment with high-dose cyclophosphamide followed by hematopoietic growth factor infusion. We report that when PB progenitors were exposed in vitro to filtered supernatant from cell line PA317-N2, producing amphotropic helper-free N2 vector at conventional titers, successful retroviral- mediated transfer of neomycin resistance gene was documented by polymerase chain reaction in 93% of day 14 myelomonocytic colonies. Under the same conditions, gene transfer was achieved in 22% of steady- state bone marrow-derived myelomonocytic colonies. Neo-resistance gene transfer was documented also in a CD34+/cyclophosphamide-resistant precursor to granulocyte-macrophage colonies, an undifferentiated progenitor close to the hematopoietic stem cell. Neither cocultivation with vector-producing cells nor high vector titer were stringent requisites for efficient gene transfer. The large-scale availability of PB hematopoietic progenitors in cancer patients, together with the high gene transfer rate achieved under safe and clinically feasible conditions, support an optimal approach for gene transfer procedures into the human hematopoietic system.

AMD3100 + G-CSF Improves Hematopoietic Progenitor Cell (HPC) Collection in Patients with Hodgkin’s Disease (HD).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1979-1979 ◽  
Author(s):  
Amanda Cashen ◽  
Steven Devine ◽  
Ravi Vij ◽  
John DiPersio
Keyword(s):  
Autologous Transplantation ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cell ◽  
Platelet Recovery ◽  
Cd34 Cells ◽  
First Remission ◽  
Grade Ii ◽  
Historical Controls ◽  
Better Than ◽  
Count Recovery

Autologous stem cell transplantation is indicated for patients with HD who have primary refractory disease or who relapse after a first remission. For these patients, as for other patients undergoing autologous transplantation, the number of CD34+ cells infused is a reliable predictor of neutrophil and platelet engraftment, and doses ≥ 5 x 106 CD34+ cells/kg are associated with faster count recovery. However, among the 98 patients with HD who have undergone G-CSF-alone mobilization at our institution in the past five years, 22% did not achieve a minimum HPC collection of 2 x 106 CD34+ cells/kg in ≤ 5 apheresis procedures, and only 15% achieved a collection ≥ 5 x 106 CD34+ cells/kg. AMD3100 mobilizes HPCs by reversibly inhibiting the interaction of CXCR4 and SDF-1α. It has been shown to improve HPC mobilization in patients with multiple myeloma and non-Hodgkin’s lymphoma. Here we present results for the first ten HD patients treated with a mobilization regimen of AMD3100 + G-CSF. To date, ten patients with relapsed (8) or refractory (2) HD have been mobilized with G-CSF (10 ug/kg/d) + AMD3100 (240 ug/kg/d) beginning on day 4. Apheresis was performed 11 hours after each AMD3100 dose. The first dose of AMD3100 produced a median (range) 3.0 (1.9–5.1) fold increase in the number of circulating CD34+ cells. Six patients (60%) achieved a collection of ≥ 5 x 106 CD34+ cells/kg, and all patients collected > 2 x 106 CD34+ cells/kg (range, 3.6–9.4 x 106 CD34+ cells/kg). The median (range) number of apheresis procedures performed per patient was 2 (1–4). No grade II-IV adverse events were ascribed to AMD3100. Eight patients have been transplanted with G-CSF + AMD3100 mobilized cells. All have had prompt and stable engraftment, with median neutrophil recovery at day +9 (9–11) and median platelet recovery at day +20 (15–23). Two patients had very early engraftment, with absolute neutrophil count greater than 100 on day +7. We conclude that AMD3100 + G-CSF is a well-tolerated and effective mobilization regimen in patients with HD. All patients (100%, 95% CI 69%-100%) mobilized with AMD3100 + G-CSF achieved the minimum collection of 2 x 106 CD34+ cells/kg, and a significantly higher proportion of patients (60%, 95% CI 26%–88%) achieved the goal collection of ≥ 5 x 106 CD34+ cells/kg than did the historical controls (15%). Importantly, the median collection in the first two days of pheresis was 5.4 x 106 CD34+ cells/kg, which is significantly better than historical controls, who collected a median 3.0 x 106 CD34+ cells/kg in the first two days of pheresis (p=0.014). Our results demonstrate that the mobilization regimen of AMD3100 + G-CSF can improve the number of HPCs collected and decrease the number of days of pheresis in HD patients. This regimen will be pursued further in this patient population.

Carfilzomib and Bortezomib Containing Regimens Yield High Rates Of MRD Negative Autologous Hematopoietic Progenitor Cell (HPC) Grafts In Multiple Myeloma (MM) and High Risk Smoldering Myeloma (SMM)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2030-2030
Author(s):  
Nishant Tageja ◽  
Neha Korde ◽  
Constance Yuan ◽  
Kristen Cole ◽  
Jennifer Hsu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
High Risk ◽  
Tumor Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cell ◽  
Myeloma Cells ◽  
Cd34 Cells ◽  
The Mean

Abstract Background Regimens incorporating modern anti-myeloma drugs, such as carfilzomib (CFZ) and bortezomib (BOR), produce rapid, deep and durable responses in newly diagnosed myeloma patients but their effect on collection of autologous HPC is not well known, including minimal residual disease (MRD) testing of stem cell grafts. Employing older induction regimens (such as VAD), less sensitive flow cytometry techniques detected circulating myeloma cells in 38-46% of autologous HPC grafts (Stewart, et al. JCO. 2001 and Bourhis, et al. Haematologica. 2007). We hypothesized that the use of modern CRd combination therapy including Carfilzomib (CFZ)-Lenalidomide (LEN)-Dexamethasone (DEX) would significantly lower the rates of HPC product contamination. Methods Thirty-six patients, including 29 with MM and 7 with high-risk SMM, underwent HPC mobilization and collection following induction with CRd (n=30), LEN-BOR-DEX (RVd, n=4), Cyclophosphamide-BOR-DEX (CyBorD, n=1) and Cyclophosphamide-BOR-Prednisone (CyBorP, n=1). For HPC mobilization, all patients received 5 days of filgrastim at 10-16 mcg/kg/dose. A combination of the patient’s weight and a peripheral blood CD34 count after 4 doses was used to determine the likelihood of collecting > 4 x106 CD34+ cells/ kg in a single apheresis procedure after a fifth filgrastim dose, according to a previously published algorithm from our institution. Only subjects predicted to require > 1 apheresis by the algorithm received Plerixafor (PLX) at 240 mcg/kg/dose on the fifth day along with the fifth filgrastim dose. HPC collection occurred on day 6, 8 hours after the last mobilizing agent(s) administration. Product contamination with myeloma cells (i.e. MRD status) was evaluated using multi-parameter flow cytometry with a minimum of 3 x 106 events obtained (sensitivity detection rate 1 x 10-5) to examine expression of 9 antigens by the plasma cells. Results The median age at mobilization was 56.2 years (range 40-73) and 19 (53%) were male. At the time of HPC collection, 20 (55%) patients were in sCR/CR/nCR, 11 (30%) had VGPR with 4 PR (11%) and 1 SD (3%). The mean CD34+ cells in the peripheral blood were 33/uL on day 5 and 55/uL on day 6 for the whole cohort. Thirteen (36%) patients did not need PLX. Interestingly, the mean CD34+ count dropped by a mean of 2% from D5 to D6 in patients not receiving PLX while, as expected, it increased by 304% in those who did. The median number of CD34+ cells collected was 6.86 million/kg (range 2.6-12.5) for the whole cohort, (6.6 million/kg without PLX and 7.52 million with PLX p=0.46). Thirty-three of 36 patients (92%) achieved a collection of > 4 million cells /kg in a single apheresis procedure. The 30 patients treated with CRd had a median of 5 (range = 3-7) prior cycles containing LEN with a median of 12 days (range 1-34) between mobilization and last LEN dose. Only 2 of 36 (5%) products were found to have evidence of tumor cell contamination (i.e. MRD positive) using sensitive multiparameter flow cytometry, one patient in PR after 6 cycles of CRd and a second patient in CR after 5 cycles of RVd. Conclusions Modern anti-myeloma therapies, such as CRd and RVd, allow adequate HPC collection in a single apheresis procedure in most cases and improve the quality of the HPC product with greatly reduced tumor cell contamination compared to historical controls. Indeed, 34/36 (94%) patients treated with modern anti-myeloma therapy collected an MRD negative HPC product. Future prospective studies are needed to assess whether autologous stem cell transplants (ASCT) using tumor-free HPC products collected in the era of modern induction therapies have better outcomes. Disclosures: No relevant conflicts of interest to declare.

Rituximab plus gemcitabine, ifosfamide, oxaliplatin (R-GIFOX) as salvage therapy for recurrent Hodgkin lymphoma

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 8579-8579
Author(s):  
G. Corazzelli ◽  
F. Frigeri ◽  
G. Marcacci ◽  
G. Capobianco ◽  
M. Arcamone ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Therapeutic Potential ◽  
Prognostic Score ◽  
Autologous Transplantation ◽  
Disease Free Survival ◽  
Dose Intensity ◽  
High Dose ◽  
Free Survival ◽  
Safe Delivery ◽  
Cd34 Cells

8579 Background: Gemcitabine (G), ifosfamide (Ifo), oxaliplatin (Ox) and rituximab (R) have been accounted of cross-synergy in preclinical and early clinical studies in Hodgkin lymphoma (HL). We assessed activity, toxicity and stem cells (SCs) mobilizing capacity of a bi-weekly salvage combination with these agents in HL recurring after conventional or high dose therapy (HDT). Methods: Patients were scheduled to receive 3 R-GIFOX courses followed by SCs mobilization and HDT if elegible for autologous transplantation (ASCT) or 3 more courses if not. R-GIFOX consisted of R 375 mg/m2 D1, G 1000 mg/m2 D2, Ox 130 mg/m2 D3 and Ifo 5 g/m2 D3, as a 24-h single infusion, G-CSF 5 mcg/kg/d DD 7–11 (10 mcg/kg/d, 3rd course until SCs mobilization). Results: Twenty-one patients (median age 33 yrs, r 22–64) with relapsed (n = 16) [post-ASCT (n=6), <12 mo.s (n=7), > 12 mo.s (n=3)] or primary progressive (n = 5) HL, were prospectively accrued. Ten patients (48%) had received ≥ 2 previous CHT lines and 15 (78%) had GHLSG recurring HL prognostic score ≥ 2. Eighty-three total courses were delivered (median 3, r 3–6). CTCAE v3.0 G4 thrombocytopenia occurred in 18% of courses, G4 infection in 11%. Ifosfamide was withdrawn at the 4th course in 2 patients, both aged 64 yrs, due to tachyarrhythmia and encephalopathy. Actual dose intensity of the first 3 courses was 82%, 86%, 92 % for G, Ifo and Ox, respectively. The overall response according to FDG-PET/IWC criteria after 3 courses was 86%, with 2 partial and 16 complete responses (CRs) (76%; CR=10, CRu=6). Four CRs were achieved among the 6 patients with post-ASCT relapses. Eight of 14 eligible patients had effective CD34+ cells harvest [median 4,35 × 106/kg (r 2,91–11.45)] and proceeded to subsequent ASCT. Five ’bad mobilizers’ had previously undergone radiation therapy (n=3) and radioimmunotherapy (n=2). At 42 mo.s. Failure Free Survival was 57%. At a median f.u. of 12 mo.s for CRs, Disease Free Survival was 79% in patients eligible for ASCT and 41% in those unfit treated with additional R-GIFOX. Conclusions: R-GIFOX retains an attractive therapeutic potential in recurring HL, enabling pre-ABMT cytoreduction and mobilization, and also a safe delivery of a full salvage program to patients unfit for HDT. No significant financial relationships to disclose.

scholarly journals Aberrant Plasma Cell Contamination of Peripheral Blood Stem Cell Autografts, Assessed by Next-Generation Flow Cytometry, Is a Negative Predictor for Deep Response Post Autologous Transplantation in Multiple Myeloma; A Prospective Study in 199 Patients

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4047
Author(s):  
Ioannis V. Kostopoulos ◽  
Evangelos Eleftherakis-Papaiakovou ◽  
Pantelis Rousakis ◽  
Ioannis Ntanasis-Stathopoulos ◽  
Chrysanthi Panteli ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Plasma Cells ◽  
Autologous Transplantation ◽  
Predictive Marker ◽  
Induction Treatment ◽  
High Dose ◽  
Next Generation ◽  
Potential Biomarker

High-dose chemotherapy with autologous stem cell support (ASCT) is the standard of care for eligible newly diagnosed Multiple Myeloma (MM) patients. Stem cell graft contamination by aberrant plasma cells (APCs) has been considered a possible predictive marker of subsequent clinical outcome, but the limited reports to date present unclear conclusions. We prospectively estimated the frequency of graft contamination using highly sensitive next-generation flow cytometry and evaluated its clinical impact in 199 myeloma patients who underwent an ASCT. Contamination (con+) was detected in 79/199 patients at a median level 2 × 10−5. Its presence and levels were correlated with response to induction treatment, with 94%, 71% and 43% achieving CR, VGPR and PR, respectively. Importantly, con+ grafts conferred 2-fold and 2.8-fold higher patient-risk of not achieving or delaying reaching CR (4 vs. 11 months) and MRD negativity (5 vs. 18 months) post ASCT, respectively. Our data also provide evidence of a potentially skewed bone marrow (BM) reconstitution due to unpurged grafts, since con+ derived BM had significantly higher prevalence of memory B cells. These data, together with the absence of significant associations with baseline clinical features, highlight graft contamination as a potential biomarker with independent prognostic value for deeper responses, including MRD negativity. Longer follow-up will reveal if this corresponds to PFS or OS advantage.

Myeloablative Conditioning of Patients with Myelofibrosis with Myeloid Metaplasia Using i.v. Treosulfan and Autologous Peripheral Blood Progenitor Cell Transplantation (PBPCT) with High Doses of CD34+ Cells Results in Hematologic Responses - 32 Months Follow-Up of Three Patients and Short-Term Results of a New Study.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5515-5515
Author(s):  
Stefan Fruehauf ◽  
Eike C. Buss ◽  
Julian Toplay ◽  
Hans H. Kreipe ◽  
Anthony D. Ho
Keyword(s):  
Multicenter Study ◽  
Autologous Transplantation ◽  
Conditioning Regimen ◽  
Allogeneic Transplantation ◽  
High Dose ◽  
Post Transplantation ◽  
Myeloid Metaplasia ◽  
Cd34 Cells ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder. Myeloablation with high-dose treosulfan and autologous PBPCT provides a palliative approach. A minimum of 5 x 106 CD34+ cells/kg was collected in all patients after G-CSF-priming (16 μg/kg/d for 4 days). Myeloablation consisted of treosulfan (total dose 42 g/m2). To date we have transplanted 3 patients, all female on an individual basis. We observed a prolonged time to reconstitution of leucocytes &gt; 1/nl post transplantation of 28 days (#1, #2) and 38 days (#3). The observation period post transplantation is 32 months now. Patient #1, who required erythrocyte transfusions twice weekly pretransplant received her last erythrocyte transfusion on day 56; her last Hb-value is 9.5 g/dl. The second patient (#2) recovered to platelet counts higher than pre transplantation (58/nl) at 3 months (143/nl) and had 125/nl at last follow-up. Pat. #3 showed a marked reduction of max. spleen size and a rise in Hb from 9 g/dl to 12 g/dl after 12 months, the last Hb value at 26 months was 7.6 g/dl and she received about 3 erythrocyte transfusions per month again. She then underwent allogeneic transplantation from her sister and is currently alive and well. Three male patients were treated in a new one-armed, multicenter study applying the aforementioned protocol. In two patients we observed again a prolonged reconstitution period of 28 (S1) and 44 (S2) days. In one patient (S1) leukocytosis and thrombocytosis resolved while transfusion dependent anemia persisted. In the second patient (S2) transfusion dependent anemia persisted. He received a backup transplantation but still remains transfusion-dependent. Patient S3 had a critical thrombocytopenia of 16/nl before transplantation and proved to be refractory to thrombocyte transfusions post-transplantation. He developed cerebral hemorrhage and died. The conditioning regimen was well tolerated and despite the prolonged aplasia period neutropenic fever was rare (median 1 day, range 0–6 days). The overall response rate was 60%, the mortality rate was 17% which is both compatible with previous data of autologous transplantation in MMM patients undergoing busulfan conditioning. This multicenter study is continuing to recruit patients (http://leukaemie.krebsinfo.de/kn_home/Studien/studie_101.html).

CXCR7 Functions Together with CXCR4 in SDF-1/CXCL12 Induced CD34+ Hematopoietic Progenitor Cell Cycling Through β-Arrestin 2-Dependent Akt Activation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3448-3448
Author(s):  
Frederic Torossian ◽  
Aurélie Chabanon ◽  
Denis Clay ◽  
Bernadette Guerton ◽  
Adrienne Anginot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Stem ◽  
Akt Activation ◽  
Respective Role ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cell Cycling

Abstract Abstract 3448 Introduction SDF-1/CXCL12 chemokine exhibits a well-known effect on retention, migration and homing of hematopoietic stem/progenitor cells (HSC/HP). We have previously demonstrated that it is also a key regulator of hematopoiesis homeostasis, acting, at low concentrations, as a survival and cell cycle promoting factor for human CD34+ HP. It has long been considered that CXCR4 was responsible for SDF-1 induced biological effects until the recent discovery of its second receptor, CXCR7. In the present study, we explored the respective role of CXCR4 and CXCR7 in the cell cycling and survival promoting effect of SDF-1/CXCL12 on human CD34+HP. Material and Methods We used CD34+ HP purified from the peripheral blood (PB) of healthy un-mobilized donors since they are mainly in G0. This allows to study the role of CXCR4 and CXCR7 receptors in 0.5ng/ml SDF-1/CXCL12 induced G0-G1 transition in synchronized quiescent cells. Gene expression was detected by RT-QPCR. Protein expression was detected and quantified using confocal microscopy, flow cytometry, immunoblotting and immunoprecipitation. Cell cycling experiments were performed using a Ki67 antibody and CXCR7 binding assay was performed using SDF-1/CXCL12AF647. Neutralization experiments were performed using a specific CXCR4 antibody or CXCR7 chemical inhibitors, a kind gift from ChemoCentryx, Inc (CCX771 and CCX733) and their respective controls (IgG and CCX704). Results Flow cytometry and confocal analysis showed that CXCR7 and CXCR4 are differentially distributed in PB CD34+ cells. In contrast to CXCR4 that is present at both the plasma membrane and intracellular level, CXCR7 expression is mainly restricted to the intracellular compartment. Confocal analysis suggested the presence of CXCR4/CXCR7 heterodimers on these cells the presence of which were confirmed by immunoprecipitation in a HP cell line. Despite its very low expression at the surface of CD34+ cells, we found that CXCR7 is capable of binding to exogenous SDF-1/CXCL12. Indeed, pretreatment with CXCR7 antibody or a chemical inhibitor reduces the mean fluorescence of bound fluorescent SDF-1/CXCL12AF647, a fully functional and specific chemokine with similar effects compared to unlabeled SDF-1/CXCL12. Neutralizing either CXCR4 or CXCR7 in PB CD34+ cells strongly reduced Akt activation induced by SDF-1/CXCL12 (0.5 ng/ml) as well as the percentage of cells in cycle (G1 and S + G2/M), colony formation and cell survival. This demonstrates that both receptors cooperate in SDF-1/CXCL12 induced functional effects. We further analyzed the respective role of CXCR4 and CXCR7 in SDF-1/CXCL12 signalization. In contrast to CXCR4, CXCR7 is reported not to activate G protein signaling pathways in response to SDF-1/CXCL12. However, it can transduce cell signaling through the β-arrestin pathway. In the present study, we showed that CXCR7 and β-arrestin 2 colocalize near the plasma membrane in freshly purified PB CD34+ cells, suggesting that CXCR7 is constitutively activated. After SDF-1/CXCL12 treatment, the majority of β-arrestin 2 was translocated to the nucleus and only a partial colocalization persisted in the cytoplasm. Using neutralizing antibodies and specific inhibitors, we showed that β-arrestin 2 nuclear translocation was dependent on both CXCR7 and CXCR4 receptors. Reducing β-arrestin 2 expression using siRNA decreased SDF-1/CXCL12 induced Akt activation in PB CD34+cells indicating the involvement of β-arrestin 2 in this process. Conclusion Altogether, our results demonstrate for the first time the role of CXCR7 together with CXCR4 in SDF-1/CXCL12-induced CD34+ cell cycling/proliferation. They also suggest the involvement of β-arrestin 2 as signalling hubs, downstream of both receptors. Disclosures: No relevant conflicts of interest to declare.

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