RESUMO
Bortezomib, a proteasome inhibitor, has shown immunosuppressive activity in animal models of GVHD. In this study, we evaluated the effects of Bortezomib on the survival of monocytes, a major circulating source of DCs. PBMCs or purified CD14+ monocytes were cultured for 24 h with Bortezomib (0.1-100 ng/ml). Apoptosis was demonstrated on the basis of detection of phosphatydilserine. Bortezomib induced a significant dose-dependent depletion (P=0.008) of monocytes in PBMC preparations, with <1% CD14+ cells remaining at doses >or=5 ng/ml. Moreover, Bortezomib decreased the survival of purified monocytes within 24 h (P=0.004) (n=6). Monocyte loss was due to apoptosis (effective dose 50%, ED(50), 1-10 ng/ml). In addition, both immature and mature monocyte-derived DC underwent apoptosis following exposure to Bortezomib. Kinetic experiments showed that apoptosis increased at 16 h through 24 h of culture. However, short term (4 h) incubation with Bortezomib irreversibly committed monocytes to undergo apoptosis at 24, 72 and 144 h. Instead, Bortezomib induced no apoptosis of purified CD19+ B, CD3+ T lymphocytes and CD34+ progenitor cells (ED(50) >50 ng/ml). The inhibitory effect of Bortezomib on professional APCs, such as monocytes and DCs, suggests its possible use in GVHD prophylaxis.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Células Dendríticas/efeitos dos fármacos , Imunossupressores/farmacologia , Monócitos/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Bortezomib , Linhagem Celular Tumoral , Células Cultivadas , Células Dendríticas/citologia , Humanos , Monócitos/citologiaRESUMO
TH2-inducing dendritic cells (DC2) are commonly identified as negative for lineage markers and positive for HLA-DR and CD123 expression. More recently, normal blood DC2 were shown also to be positive for BDCA-2 and BDCA-4 antigens. The aim of this study was to evaluate whether BDCA-2 expression on DC2 is impaired in patients undergoing an allogeneic hematopoietic stem cell transplantation (HSCT) and in healthy donors treated with G-CSF for HSC mobilization. Flow cytometry assays for DC2 detection using either a triple staining with anti-HLA-DR PerCP, anti-Lin(+) anti-CD34 FITC and anti-CD123 PE monoclonal antibodies (mAbs), or a double staining with anti-HLA-DR PE and anti-BDCA-2 FITC mAbs were compared in blood samples from patients who underwent an allogeneic HSCT (n = 30) or from healthy donors before (n = 11) and after (n = 8) G-CSF mobilization, as well as in healthy donors' leukapheresis products (n = 12) or bone marrow (n = 4). Staining of BDCA-2(+) cells with other markers such as anti-CD38, anti-CD54 and anti-CD58 were also performed. Median values of CD123(+) DC2 and BDCA-2(+) DC2 were not statistically different in the blood of patients previously treated with chemotherapy, nor in the blood or bone marrow of heathy donors. Also, a 5 day G-CSF treatment did not affect BDCA-2 or adhesion molecule expression on healthy donors' blood DC2 significantly. A correlation between all the results (n = 65) obtained with the two assays was demonstrated in a linear regression curve (r = 0.914) (P = 0.00001). BDCA-2 is a marker highly specific for DC2 that is not downregulated by chemotherapy or G-CSF treatment. Therefore, the anti-BDCA-2 mAb can be efficiently combined with other mAbs and used in studies addressing the role of DC2 in the allogeneic HSCT setting.
Assuntos
Anticorpos Monoclonais , Células Dendríticas/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Lectinas Tipo C/metabolismo , Biomarcadores/análise , Células Sanguíneas/citologia , Células Sanguíneas/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Estudos de Casos e Controles , Contagem de Células/métodos , Células Dendríticas/citologia , Células Dendríticas/patologia , Citometria de Fluxo/métodos , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/terapia , Humanos , Lectinas Tipo C/imunologia , Leucaférese , Glicoproteínas de Membrana , Receptores Imunológicos , Transplante Homólogo/métodosRESUMO
In this study we compared the lymphocyte reconstitution in 13 multiple myeloma (MM), nine acute myeloid leukemia (AML) and 10 chronic myeloid leukemia (CML) patients after allogeneic G-CSF-mobilized PBSC transplantation from HLA-identical siblings. Conditioning regimens included standard total body irradiation + cyclophosphamide (CY), or busulphan + CY, whereas VP-16 was added in patients with advanced disease. Overall comparable numbers of mononuclear cells, CD34+ cells and CD3+ T cells were infused in each group. A significantly higher CD3+ T cell number was observed in MM and AML than in CML patients 1 month after transplant. However, MM patients showed a faster and better recovery of CD4+ T cells than both AML and CML patients at 3 months (P = 0.01 and P = 0.01, respectively) and 12 months (P = 0.01 vs AML, while P = NS vs CML) after transplant, and had a CD4:CD8 ratio > 1 with a median CD4+ T cell value > 400/microl 1 year after transplant. Development of acute graft-versus-host disease (GVHD) did not affect CD4:CD8 ratios but patients who experienced acute GVHD > grade I had lower CD4+ and CD8+ T cell numbers at all time points. However, after excluding patients with GVHD > grade I, MM patients still showed a significantly higher CD4+ T cell value than patients with myeloproliferative diseases 1 year after transplant. These findings suggest that although allogeneic PBSC transplantation induces rapid immune reconstitution, different kinetics may occur among patients with hematological malignancies. In particular, the rapid reconstitution of CD4+ T cells in MM patients may contribute to the low transplant-related mortality achieved in this disease.