Early hemopoietic progenitors in the peripheral blood of patients with severe aplastic anemia (SAA) after treatment with antilymphocyte globulin (ALG), cyclosporin-A and G-CSF

Abstract

ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n A. Bacigalupo et al. 134 administration of G-CSF in SAA patients leads to mobilization of hemopoietic progenitors, which are then available for analysis and can actually be harvested at weekly leukophereses and cryopreserved. 14,15 Human LTC-ICs share many of the characteristics of murine LTC-ICs, which have proven in vivo long-term repopulating ability. Materials and Methods Patients Clinical data on the patients are presented in Response Patients were classified as complete responders if they were transfusion independent with a hemoglobin level of ≥ 11 g/dL, a neutrophil count greater than 1.5ϫ10 9 /L, and a platelet count greater than 100ϫ10 9 /L; partial responders were transfusion independent with a hemoglobin level of ≥ 8 g/dL, a neutrophil count of >0.5ϫ10 9 /L, and a platelet count of >20ϫ10 9 /L. Persistence of transfusion dependence was taken as evidence of no response. Normal donors Sixteen leukaphereses from 7 normal donors treated with G-CSF (10 ug/kg/day for 5 days) were used as controls. These volunteers underwent the procedure as matched donors for their HLA-identical sibling. Informed consent was obtained from SAA patients and normal donors, and investigations were approved by our Ethical Committee. Flow cytometry PB cells recovered from each leukaphereses were processed with a work station Coulter Q-Prep (Coulter Corporated Hialeah, FL) in order to lyse erythrocytes and to fix white blood cells. Cell surface antigens were detected by direct immunofluorescence using a CD34 HPCA-2 for progenitor cells (Becton Dickinson, Mountainview, CA). Fluorescence was analyzed with an XL Coulter. Isotypically matched mouse Ig directly conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) were used as negative controls in all experiments. Overall, 10 4 events were acquired on an FS (Forward Scatter) x SS (Side Scatter) diagram on all populations except platelets and debris. Immediately after staining, cells were sorted on a Coulter Epics 753 dual laser flow cytometer (Coulter Electronics, Hialeah, FL) equipped with a CICERO high-speed computer (Cytomation, Fort Collins, CO). FITC and PE were excited using the 488nm wavelength from a dedicated 5 W argon laser. In this staining protocol, it was still possible to obtain CD34 + HLA-DR + cells, even though both HLA-DR (IgG1 isotype, Becton-DickinsonImmunocytometry System) and CD15 were FITC conjugated (FITC-conjugated mouse antihuman-LeuM1, Becton-Dickinson). 18 Functional assays Light density cells from each leukapheresis were assayed for clonogenic precursors. Briefly 10 5 MNC were plated in: A. 1.1 mL consisting of Iscove's modified Dulbecco's medium (IMDM, GIBCO BRL, Life Technologies LTD, UK) + 0.9% methylcellulose (Sigma Chemical, St.Louis, MO, USA) + 30% fetal calf serum (FCS, Gibco) + 100 ng of rhGM-CSF (Sandoz, Basel, CH); B. 1.5 ml consisting of IMDM + 0.9%methylcellulose + 30% FCS + 1% bovine serum albumin (BSA, Sigma) + 10 -4 M mercaptoethanol (ME, Sigma) + rhGM-CSF (10 ng) + rhIL3 (10 ng Sandoz) + rhG-CSF (10 ng) + rhEPO (4U Cilag AG, Schaffhauser, CH) + SCF (50 ng Genzyme Corporation, Cambridge, UK). After 14 days of incubation in a humidified atmosphere at 37°C in 5% CO 2 , colonies were classified and counted using an inverted microscope (Zeiss, Germany). Sorted cells were cultured in the presence of PIXY and SCF as described. 18 MNC and freshly sorted cells were cultured in suspensions containing different cytokines and scored according to previously established criteria. 19 LTC-IC assays were performed by seeding an aliquot (usually 5ϫ10 6 MNC/flask) of light density cells over a feeder layer of irradiated (1.5 cGy) normal marrow cells. These were subcultured from adherent layers of previously established 4-week-old LTCs. LTC-ICs were maintained for 3 days at 37°C, then switched to 33°C and fed weekly by replacing half the growth medium (IMDM + 12.5% horse serum (HS, Gibco) + 12.5% FCS + 10 -4 M ME + 10 -6 M hydrocortisone, Sigma) containing half of the nonadherent cells with fresh growth medium. After 5 weeks, adherent cells were trypsinized and combined with the nonadherent fraction. These harvested cells were washed and assayed for clonogenic precursors in standard methylcellulose cultures (B). As previously reported, 14 the number of LTC-ICs present in the starting cell suspension was shown to be equal to the total CFC content of the 5-week-old LTC assay divided by 4. Statistical analysis Chi-square, Student's t-test and Mann-Whitney rank sum test were used to analyze the data. Results Patients All patients completed the designed course of IS treatment and underwent leukaphereses, which © F e r r a t a S t o r t i F o u n d a t i o n Early hemopietic progenitors in PB of treated SAA patients 135 were scheduled weekly starting on day +30 of treatment. One patient who did not become transfusion independent after a first course was treated again with a second course of ALG, CyA and G-CSF, and then a third time with CyA and G-CSF. He therefore underwent 3 courses of leukaphereses ( Flow cytometry Flow cytometry demonstrated a higher frequency of CD3 + cells Committed progenitors Samples from each apheresis were assayed for in vitro granulocytic, erythroid and multipotent progenitors (CFU-GM, BFU-E and CFU-GEMM). In our experiments we were able to grow mainly CFU-GM, with very low numbers of other colonies (in percentages: 25-100% CFU-GM, 0-30% BFU-E and 0-19% CFU-GEMM). Five patients showed significant increments in white blood cell (WBC) counts (>10ϫ10 9 /L, Early progenitors HPP-CFC were studied in 4 patients (#1, 4, 5, 6); they grew at a frequency of 0-3.4/10 5 MNC, in one case in the absence of committed progenitors. In addition, we studied LTC-ICs in 18 leukaphereses from 4 patients: in 7/18 samples LTC-ICs were present at low frequency (range: 0.4-2/10 6 MNC) when compared with collections from G-CSF-treated normal donors (range: 5.4-130/10 6 MNC); p<0.00001) Cell sorting Cells derived from 3 aphereses were sorted into CD34 + DR + and CD34+DR -CD15 -; these were then cultured in vitro with different cytokine combinations. CD34 + DR + cells showed evident increments in GM, GEMM, BFU-E and HPP colony formation Discussion It has been always difficult to design in vitro colony assays in patients with SAA, mainly because of the lack of bone marrow cells to initiate such studies. We have described the presence of hemopoietic progenitors in the peripheral blood of many SAA patients after treatment with ALG-CyA and prolonged administration of rhG-CSF. 13 By performing weekly leukaphereses in such patients, one can collect large numbers of cells which can be cryopreserved and are, at the same time, available for in vitro assays. The aim of the present study was to assess phenotypic and functional properties of such PBHP. As regards the phenotype, SAA PB cells contained large numbers of CD3 + cells, significantly more than 136 controls (p=0.006), with an inverted CD4/CD8 ratio. This is in keeping with reports of an excess of suppressor cells in SAA, When SAA cells were plated in vitro we grew prevalently CFU-GM in numbers which were significantly lower than in normal controls, with a median 28-fold reduction. BFU-E and CFU-GEMM were seen less frequently in both SAA patients and normal controls, possibly as a result of in vivo triggering with G-CSF. LTC-ICs were studied in 18 leukaphereses and scored in 7 of these, though in small numbers: they were significantly fewer than in controls (0-2 vs 5-130/10 6 MNC). In one of these patients LTC-ICs were present in the blood in the absence of peripheral blood and marrow CFU-GM, and this was also seen for HPP colonies. Therefore primitive progenitors may be present in the absence of committed progenitors, as previously suggested by hematologic reconstitution in most SAA patients in spite of no colony formation from bone marrow cells. © F e r r a t a S t o r t i F o u n d a t i o n or whether their survival is limited. When we assessed the total yield of leukaphereses from SAA patients we recovered reduced numbers of CFU-GM (p=0.05), although SAA patients underwent a median of 5 vs 2 leukaphereses performed with normal controls. It is difficult to say whether these cryopreserved cells are suitable for an autotransplant, a possibility one may wish to consider in the case of poor or absent hematologic reconstitution or in the case of evolution of the disease into overt myelodysplasia or leukemia. Highdose chemotherapy has been shown do induce complete remissions when given to SAA patients, though at a cost of prolonged periods of neutropenia; 24 the infusion of autologous cryopreserved progenitors may shorten the duration of chemotherapy-induced aplasia. The total CFU-GM content recovered from leukaphereses was in the range reported to allow hemopoietic reconstitution if infused after ablative chemo-radiotherapy, and so was the LTC-IC content in 2 out of 4 patients studied. Another important issue may be to consider blood progenitor cell mobilization as a prediction or as a measure of clinical improvement. Indeed two complete responders (#5 and #6) mobilized high numbers of CFU-GM; one patient (#2) mobilized very few CFU-GM and showed no response. In patient #3 clonogenic progenitors appeared in the blood after the second course of IS treatment and he achieved a durable response after the third course. This suggests that the presence of committed and early progenitors in the blood during IS treatment may be predictive of clinical response, in keeping with data reported by Finally, early hemopoietic progenitors mobilized with G-CSF may reseed the marrow 26 and contribute to hematologic recovery in SAA patients undergoing treatment with ALG and Cy A, as indicated by the high response rate seen with this regimen and a survival rate greater than 90%. 9 It may be possible to prove this hypothesis by using the neomycin resistance gene to mark circulating progenitors. 27 In conclusion, this study suggests that very early progenitors are found in the peripheral blood of patients with SAA after prolonged G-CSF administration and after treatment with IS therapy. The number of such early precursors is small, and the majority of cells exhibit the features of committed progenitors. It remains to be determined whether these cells contribute to hematologic recovery and whether they can be used for autografting. Early hemopietic progenitors in PB of treated SAA patients References 137 © F e r r a t a S t o r t i F o u n d a t i o