Alessandra Fortuna

Generation and functional characterization of human dendritic cells derived from CD34 ˛ cells mobilized into peripheral blood: comparison with bone marrow CD34 ˛ cells

Dendritic cells (DCs) are the most powerful professional antigen‐presenting cells (APC), specializing in capturing antigens and stimulating T‐cell‐dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady‐state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony‐stimulating factor (G‐CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony‐forming unit‐dendritic cells (CFU‐DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM‐CSF and TNF‐α with or without SCF and FLT‐3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early‐acting growth factors SCF and FLT‐3L with IL‐4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+CD14− cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL‐4‐stimulated DCs was significantly higher than those generated in the absence of IL‐4. Furthermore, autologous serum collected during G‐CSF treatment was more efficient than fetal calf serum (FCS) or two different serum‐free media for large‐scale production of DCs. Thus, our comparative studies indicate that G‐CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early‐acting and intermediate‐late‐acting colony‐stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.

Expression and functional role of c-kit ligand (SCF) in human multiple myeloma cells.

Summary. In this study we investigated the proliferation of three well‐documented MM lines and 10 bone marrow samples from myeloma patients in response to rh‐SCF alone and combined with Interleukin‐6 (IL‐6), IL‐3 and IL‐3/GMCSF fusion protein PIXY 321. Neoplastic plasma cells were highly purified (>90%) by immunomagnetic depletion of T, myeloid. monocytoid and NK cells. The number of S‐phase cells was evaluated after 3 and 7d of liquid culture by the bromodeoxyuridine (BRDU) incorporation assay. The proliferation of RPMI 8226 and U266 cell lines was also assessed by a clonogenic assay. All the experiments were performed in serum‐free conditions. RPMI 8226 cell line was not stimulated by SCF which also did not augment the proliferative activity of IL‐6, IL‐3 and PIXY‐321. Conversely, SCF addition resulted in 2.4‐fold increase of the number of U266 colonies and in a higher number of U266 and MT3 cells in S‐phase (24.5 ± 2% SEM v 14.5 ± 1% SEM and 32 ± 3% SEM v 21 ± 4% SEM, respectively; P < 0.05). The c‐kit ligand also enhanced the proliferation of MT3 and U266 cells mediated by the other cytokines. Anti‐SCF polyclonal antibodies completely abrogated the proliferative response of MT3 cells to exogenous SCF and markedly reduced the spontaneous growth of the same cell line. Reverse transcriptase‐polymerase chain reaction amplification (RT‐PCR) did detect SCF mRNA in MT3 and RPMI 8226 cells. Moreover, secreted SCF was found, in a biologically active form, in the supernatant of the two cell lines by the M07e proliferation assay.

SELECTION AND TRANSPLANTATION OF AUTOLOGOUS HEMATOPOIETIC CD34+ CELLS FOR PATIENTS WITH MULTIPLE MYELOMA

Here we review our recent experience addressing the issue of positive selection and transplantation of hematopoietic CD34+ cells to reduce neoplastic contamination in peripheral blood (PB) autografts from patients with multiple myeloma (MM). We evaluated PB samples from 30 pretreated MM patients following the administration of high dose cyclophosphamide (Cy; 7g/m2 or 4g/m2) and granulocyte-colony stimulating factor (G-CSF), for collection of circulating stem cells (PBSC) to support hematopoietic reconstitution following myeloablative radio-chemotherapy. Twenty six patients showed adequate mobilization of CD34+ progenitor cells and were submitted to PBSC collection. Circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, cryopreserved, and used to reconstitute BM function after myeloablative therapy in 13 patients. The median purity of the enriched CD34+ cell population was 89.5% (range 51-94%) with a 75-fold increase compared to the pretreatment samples. The median overall recovery of CD34+ cells and CFU-GM was 58% (range 33-95%) and 45% (range 7-100%), respectively. Positive selection of CD34+ cells resulted in 2.5-3 log of plasma cells and CD19+ B-lineage cells depletion as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene demonstrated the persistence of minimal residual disease (MRD) in 5 out of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (TBI, 1000 cGy) and high dose Melphalan (140 mg/m2) or Melphalan (200 mg/m2) alone. They received a median of 5 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis: the median time to 0.5 x 10(9) neutrophils, 20 and 50 x 10(9) platelets/L of PB was 10, 11 and 12 days, respectively. When we analyzed the immunological reconstitution of this group of patients, we observed a rapid and full recovery of total lymphocyte and NK cell count, although the absolute CD4+ cell count was lower than pretreatment level. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (=13) receiving unmanipulated PBSC following the same pretransplant conditioning regimen. The results of this trial demonstrate that positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3 log and provides a cell suspension capable of restoring a normal hematopoiesis after a TBI-containing conditioning regimen. Based on this pilot trial, we have recently started a clinical study involving a double autotransplant, conditioned with melphalan (200 mg/m2) followed by melphalan (140 mg/m2) and busulphan (14 mg/kg), supported by the reinfusion of highly purified CD34+ cells.

BIOLOGICAL CHARACTERIZATION OF CD34+ CELLS MOBILIZED INTO PERIPHERAL BLOOD

We evaluated 18 acute myeloblastic leukaemia (AML) and myelodysplastic syndrome (MDS) patients with abnormal karyotype at diagnosis who underwent peripheral blood stem cell (PBSC) transplantation. To evaluate the presence of residual tumour cells, bone marrow (BM) samples and PBSC collections were analysed by cytogenetics and in selected cases also by fluorescence in situ hybridisation (FISH) and molecular studies. All patients were considered to be in morphologic and cytogenetic complete remission (CR) at the time of mobilisation. Seven patients showed neoplastic cells in PBSC harvest and/or BM specimen before reinfusion. Cytogenetic studies revealed contamination in apheretic collections in one patient only, while three patients had BM but not PBSC contamination. Three more patients had leukaemic cells both in the BM and PBSC. All but one (with only BM contamination) of these patients relapsed within 9 months. However, five more patients relapsed after transplantion: in four cases there was no cytogenetic sign of contamination either in PBSC or BM cells and in one case no molecular evidence was revealed either. This study suggests that, whereas the presence of leukaemic cells in autologous grafts correlates with a poor prognosis, the lack of detection of tumour cells is not always predictive of long-term disease-free survival. More importantly, PBSC collections from AML patients are not contaminated by leukaemic cells if the BM is disease-free.

T cell alloreactivity induced by normal G-CSF-mobilized CD34+ blood cells

In this study, the hypothesis that a subset of granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ blood cells may actively induce an allogeneic T cell response in vitro was tested. Circulating CD34+ cells were purified to ⩾98% by high gradient magnetic separation and then analyzed for the coexpression of HLA-DR, the common β-chain of the leukointegrin family CD18 and costimulatory molecules CD80 (B7–1) and CD86 (B7–2). These antigens were expressed on average on: 94.9 ± 2.5%, 64.4 ± 15.4%, 0% and 1.9 ± 1.2% CD34+ blood cells, respectively. Irradiated CD34+ cells induced a high proliferative response of allogeneic, but not autologous, purified CD4+ and CD8+ T cells in primary mixed leukocyte culture (MLC). An average three-fold lower CD4+ and CD8+ T cell response was induced by mononuclear cells from G-CSF-treated donors. A lower frequency of allostimulating cells among mononuclear cells rather than among CD34+ cells in the apheresis was documented by limiting dilution assay (LDA). As previously observed with marrow, sorted CD34+/CD18+ cells induced the proliferation of allogeneic T cells in MLC, while CD34+/CD18− cells, which were >94% HLA-DR+ and contained both committed (CFU-C) and early (LTC-IC) hematopoietic progenitors, stimulated allogeneic T cells poorly. Three-color staining cytofluorimetry indicated that expression of CD80 and CD86 were upregulated in 6.9 ± 4.9 and 10.7 ± 2.6% CD34+ blood cells respectively, after 24–30 h of culture with autologous or allogeneic mononuclear cells, or with CD4+, or CD8+ T cells, but not with medium alone. Moreover, the upregulation of CD86 was observed on CD34+/CD18+ rather than on CD34+/CD18− cells after 30 h in MLC. Blocking experiments demonstrated that preincubation of stimulator and responder cells with anti-CD80 plus anti-CD86 monoclonal antibodies induced a 84 ± 8% inhibition of CD34+ cell allostimulating activity after 6 days in primary MLC. These results suggest that G-CSF-mobilized CD34+ hematopoietic progenitors with alloantigen presenting function express CD18 and may upregulate CD80 and CD86 upon interaction with T cells. Since activation of B7 costimulatory molecules represents an active costimulatory pathway on G-CSF-mobilized CD34+ cells, the blockade of these molecules or, alternatively, the use of selected non-immunogenic CD34+/CD18− blood stem cells may represent a new strategy for reducing graft rejection and overcoming HLA barriers in allogeneic stem cell transplantation.

C-Kit Ligand (SCF) in Human Multiple Myeloma Cells

Here we review our recent experience addressing the role of SCF in multiple myeloma (MM). We first investigated the proliferation of MM cell lines and bone marrow samples from myeloma patients in response to rh-SCF alone and combined with Interleukin-6 (IL-6), IL-3, and IL-3/GM-CSF fusion protein PIXY 321. Neoplastic plasma cells were highly purified (>90%) by immunomagnetic depletion of T, myeloid, monocytoid and NK cells. The number of S-phase cells was evaluated after 3 days of liquid culture by the bromodeoxyuridine (BRDU) incorporation assay. The proliferation of RPMI 8226 and U266 cell lines was also assessed by a clonogenic assay. All the experiments were performed in serum-free conditions. RPMI 8226 cell line was not stimulated by SCF which also did not augment the proliferative activity of IL-6, IL-3 and PIXY-321. Conversely, SCF addition resulted in 2.4-fold increase of the number of U266 colonies and in a higher number of U266 and MT3 cells in S-phase. The c-kit ligand also enhanced the proliferation of MT3 and U266 cells mediated by the other cytokines. Anti-SCF polyclonal antibodies completely abrogated the proliferative response of MT3 cells to exogenous SCF and markedly reduced the spontaneous growth of the same cell line. Reverse transcriptase-polymerase chain reaction amplification (RT-PCR) did detect SCF mRNA in MT3 and RPMI 8226 cells. Moreover, secreted SCF was found, in a biologically active form, in the supernatant of the two cell lines by the MO7e proliferation assay. These results suggest that an autocrine proliferative loop may be operative in MT3 cell line. When tested on fresh myeloma samples, SCF increased the number of S-phase plasma cells (4.7 +/- 1.6% vs 3.4 +/- 1.3% in control cultures; p = 0.02). Significant proliferation was also induced by IL6, IL-3 and PIXY-321. The addition of SCF significantly enhanced the proliferation of myeloma cells responsive to IL-6. Preliminary experiments performed on circulating plasma cells and myeloma precursors further supported the role of SCF on the proliferation of the neoplastic clone in MM.

Concomitant and sequential administration of recombinant human granulocyte colony-stimulating factor and recombinant human interleukin-3 to accelerate hematopoietic recovery after autologous bone marrow transplantation for malignant lymphoma.

PURPOSE To assess the safety, tolerability, and hematopoietic efficacy of sequential and concomitant administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human interleukin-3 (rhIL-3), to accelerate reconstitution of hematopoiesis following myeloablative chemotherapy and autologous bone marrow transplantation (ABMT) for heavily pretreated lymphoma patients. PATIENTS AND METHODS Fifty-four consecutive patients with refractory or relapsed non-Hodgkins lymphoma (NHL; n = 30) and Hodgkins disease (HD; n = 24) were studied. Two different conditioning regimens were used for ABMT: carmustine, cyclophosphamide, etoposide, and cytarabine (BAVC) and carmustine, melphalan, etoposide, and cytarabine (BEAM) for NHL and HD, respectively. Patients were enrolled sequentially onto one of three treatment groups: group 1, G-CSF (5 micrograms/kg/d subcutaneously [SC]) from day +1 after reinfusion of autologous marrow (n = 23); group 2, G-CSF from day +1 combined with IL-3 (10 micrograms/kg/d SC) from day +6 (n = 22, overlapping schedule); and group 3, G-CSF treatment discontinued at day +6 before initiation of IL-3 administration (n = 9, sequential schedule). In the three groups, growth factor(s) was administered until the granulocyte count was greater than 0.5 x 10(9)/L for 3 consecutive days. RESULTS The study cytokines were generally well tolerated. No side effects were observed when G-CSF was given alone. Four of 31 patients (12.9%) who received SC IL-3 had one severe adverse event defined as World Health Organization (WHO) grade 3 to 4 toxicity (fever, n = 2; pulmonary toxicity, n = 2) and were withdrawn from the study. Groups 2 and 3 did not differ as for treatment tolerability, whereas we observed a trend toward a faster hematopoietic recovery when IL-3 was administered concomitant with G-CSF from day 6 (ie, group 2). Pooled together, patients who received IL-3 showed a median time to achieve a granulocyte count greater than 0.1 and greater than 0.5 x 10(9)/L of 8 and 11 days, respectively. The median time to an unsupported platelet count greater than 20 and 50 x 10(9)/L was 15 and 20 days, respectively, and only one patient did not reach a normal platelet count. The median number of days to hospital discharge was 16 after ABMT (range, 12 to 29). When the hematologic reconstitution of patients in groups 2 and 3 was compared with that of patients in group 1, the addition of IL-3 resulted in a significant improvement of multilineage hematopoietic recovery, lower transfusion requirements, a lower number of documented infections, and shorter hospitalizations. CONCLUSION We conclude that the combination of G-CSF and IL-3 is safe and well tolerated in intensively pretreated lymphoma patients, undergoing ABMT and results in rapid hematopoietic recovery following myeloablative chemotherapy.

Interleukin‐11 (IL‐11) acts as a synergistic factor for the proliferation of human myeloid leukaemic cells

Summary. Interleukin‐11 is a stromal cells derived cytokine which stimulates the proliferation of primitive haemopoietic progenitor cells. For this paper we have studied the constitutive expression of IL‐11 mRNA in a panel of well‐known leukaemic cell lines and samples from AML patients at diagnosis. Moreover, the same cellular populations were evaluated for their proliferative response to recombinant‐human‐(r‐hu) IL‐11 alone and combined with r‐hu‐IL‐3, granulocyte‐macrophage colony stimulating factor (GM‐CSF) and stem cell factor (SCF, c‐kit ligand). The colony‐forming ability of HL60, K562, KG1 cells and eight fresh AML cell populations was assessed by a clonogenic assay in methylcellulose. In eight additional AML cases the number of S‐phase leukaemic cells induced by IL‐11 was determined by the bromodeoxyuridine (BRDU) incorporation assay after 3 d of liquid culture.

Immunotoxins containing saporin linked to different CD2 monoclonal antibodies: In vitro evaluation

In this study we describe immunotoxins prepared with different CD2 monoclonal antibodies (mAbs) and a ribosome‐inactivating protein, saporin. The CD2 immunotoxins were tested on different models. Anti‐CD2–saporin conjugates inhibited protein synthesis by a neoplastic CD2+ cell line (SKW‐3) and by an interleukin 2 dependent polyclonal CD2+ lymphoid cell culture (T lymphoblasts), with IC50s ranging from 10‐13m to 10‐11m (as saporin). Similar results were obtained with proliferation inhibition tests (3H‐thymidine incorporation) on phytohaemagglutinin (PHA) driven lymphoid cultures and on mixed lymphocyte culture activated lymphocytes. Moreover a CD2–ricin A chain conjugate was less effective than an analogous immunotoxin containing the same CD2 mAb and saporin in inhibiting lymphocyte proliferation induced by PHA (IC50 approximately 10‐9m as ricin A chain versus 10‐12m as saporin). The conjugates were not toxic on bone marrow stem cells. These results suggest that CD2–saporin immunotoxins could represent an effective tool for CD2+ lymphomas or leukaemias, and for T‐dependent immune disorders, such as transplanted organ rejection and graft‐versus‐host disease.

Selective expansion of normal haemopoietic progenitors from chronic myelogenous leukaemia marrow.

CD34+ and CD34+ DR− cells from the bone marrow (BM) of chronic‐phase chronic myelogenous leukaemia (CML) patients at diagnosis were tested for their colony‐forming ability in response to early and intermediate‐late colony stimulating factors (CSFs). Molecular analysis revealed that 55.6 ± 9% SD of CD34+DR− colonies, in which actin and ABL mRNA were detectable, expressed the product of the BCR‐ABL gene. The percentage and the clonogenic efficiency of CML DR− cells were significantly lower than those of comparable DR− cells from normal donors. However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, IL‐11 and IL‐3, used as single factors in the presence of erythropoietin (EPO) and was almost entirely due to erythroid progenitors. Conversely, optimal stimulation of CD34+DR− cells from normal donors required co‐incubation with three or more CSFs. Stroma‐noncontact long‐term cultures were then established in the presence of exogenous CSFs and human irradiated allogeneic stromal layers or the murine stromal cell line M2‐10B4, engineered to produce G‐CSF and IL‐3. In these cultures the combination of SCF and IL‐3 induced a 25.4 ± 5 SD, 40 ± 6 SD and 20.5 ± 6 SD fold increase of colony‐forming unit cells (CFU‐C), at weeks 2, 4 and 5, respectively. At the same time‐points the number of primitive long‐term culture initiating cells (LTC‐IC) showed a 4 ± 2 SD, 3.3 ± 1.5 SD and 2.3 ± 1 SD fold increase compared to baseline values. BCR‐ABL mRNA analysis of single colonies demonstrated that 27 ± 9% SD and 7 ± 3% SD CFU‐C at weeks 4 and 5, respectively, expressed the fusion gene, whereas leukaemic LTC‐IC disappeared from the culture by week 2. These results suggest that leukaemic CD34+DR− cells have a different pattern of response to CSFs than normal cells. In addition, we established culture conditions which allow selective expansion of benign haemopoietic cells coexisting with leukaemic progenitors.