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Effect of cryopreservation on adhesion molecule expression in peripheral blood derived CD34+ cells
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Abstracts/Experimental Hematology 28 (2000) 31–131
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
IDENTIFICATION OF A 85.0Ⲑ124.5 KD MOLECULE WITH ANTI-HOMOAGGREGATION ACTIVITY ON KG1a CELL SURFACE BY A SINGLE CHAIN ANTIBODY FROM A PHAGE DISPLAY LIBRARY Jianhua Sui*, Yixin He, Xueying Jiang*, Zengxuan Song Institute of hematology, Peking Union Medical College & Chinese Academy of Medical Sciences (PUMC&CAMS) Hematopoietic cell surface molecule plays a crucial role in the regulation of hematopoiesis. To identify and characterize novel hematopoietic stemⲐprogenitor cell-surface molecules and their antibodies, we chose human hematopoietic progenitor cell line KG1a as a model system, and a single chain antibody (scFv) phage display library was constructed from the spleen cells of mice immunized with intact KG1a cells. The library was selected by using KG1a cells as panning antigen, and after 4 rounds biopanning, 27 of 126 cones showed strong and selective positive reaction to the KG1a cells in phage ELISA. Diversity analysis of these clones by BstNI fingerprinting revealed four distinct scFv fragments. Two of them, named 5C1 and 1B3 showed significant binding to KG1a cells by flow cytometry, but no or weak cross-reactivity to nonhematopoietic and other leukemia cell lines. These two scFvs were sequenced and further expressed in E.coli., and purified as soluble scFv. With the purified scFvs, the effects of them on KG1a cell line were investigated. The scFv 5C1 was able to block homoaggregation of KG1a cells in a dose-dependent manner. To identify the KG1a cell surface molecule, which bound to the scFv 5C1, proteins from KG1a cell lysates were analyzed by Western blotting. Two bands with apparent molecular weights of 85.0 and 124.5 KDa were recognized by the 5C1 scFv. The results suggest that the 85.0Ⲑ124.5KDa molecule may be an adhesive apparatus necessary for homotypic adhesion. It encourages us to further study the nature and function of the 5C1 antigen. The results also indicate the advantages of phage display antibody technology in searching for hematopoietic stemⲐprogenitor cell-surface molecules. 112
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
EFFECT OF ACTIVATED ADHERENT CELL CONDITIONED MEDIUM ON HUMAN CD34⫹ CELL RECRUITMENT IN SHORT- AND LONG TERM CULTURES E. Wunder*, D. Robay*, L. Mazini*, C. Zanetti*, Ph. Hénon Institut de Recherche en Hématologie et Transfusion, Mulhouse, France Highly purified human CD34⫹ cells produced up to 500⫻ less CFU-GM in short term culture (STC), and 10⫻ less week-5 CAFC in murine FBMD-1 feeder long term culture (LTC) than MNC as calculated per input CD34⫹ cells, according to previous results (1). Cocultured CD34⫺ cells, separated from CD34⫹ cells by a dialysis membrane, re-established efficient colony growth in STC. We now tested efficiency of conditioned media from normal blood adherent cells (ACS) after stimulation with 7ngⲐml GM-CSF for 48h. Addition of ACS from 9 healthy donors enhanced growth of CFU-GM in STC of purified mobilized blood CD34⫹ cells at individually variable degree (2,5–168.8% additional colony yield). 82% additional colonies resulted from pooled supernatants. When
this pooled ACS was added weekly at 1% vⲐv to CD34⫹ cells growing in LTC, the yield of weeks-5 CAFC raised from 67,1 to 1234,6 CAFC per 105 CD34⫹ cells (determined by limiting dilution method and extrapolated); neutralizing antibodies against G-CSF and SCF did not change this enhancement. Fractions bound to anion-exchange columns enhanced early CAFC (week-2), while unbound fractions late (week-5) CAFC. We conclude that the supportive effect of mature adherent cells must be due to secreted factors, which can be separated by their different net electric charge. (1) Mature accessory cells influence long term growth of human hematopoietic progenitors on a murine stromal cell feeder layer. L. Mazini, E. Wunder, H. Sovalat et al.; Stem Cells 16, 404-412 (1998) 113
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Lymphoproliferative Disorders
EFFECT OF CRYOPRESERVATION ON ADHESION MOLECULE EXPRESSION IN PERIPHERAL BLOOD DERIVED CD34⫹ CELLS H. Sovalat*, M. Ojeda1*, Y. Arkam1*, H. Lewandowsky1*, V. Chabouté1*, Ph. Hénon Institut de Recherche en Hématologie et Transfusion, 1Department of Hematology, Mulhouse, France Homing of stem cells to the bone marrow after grafting is essential for hematopoietic recovery and supposedly depends on cellular adhesion molecules (CAMs). Since cryopreservation may affect expression of CAMs, members from integrin-(CD11a, CD49d), immunoglobulin-(CD54, CD58), selectin-(CD62L), and CD44 families were analyzed on CD34⫹ cells in fresh leukapheresis products (LKP) and after their cryopreservation. Two parameters were compared: the percentage of CD34⫹ cells with CAM expression, and the degree of CAM antigen expression; the latter can be estimated by quantitating the number of molecules of monoclonal antibody (MAb) that bind to the cell surface, and is called antibody-binding capacity (ABC). In fresh LKP 100% of CD34⫹ cells were positive for CD44 and CD58; the majority of CD34⫹ cells coexpressed CD11a, and CD49d; 73% coexpressed CD62L (L-selectin). CD54 was expressed with remarkable inter-individual variability. In thawed LKP the percentage of CD34⫹ cells coexpressing adhesion molecules CD54, CD11a was slightly (but not significantly) lower than in fresh samples. When the ABC is compared, generally a slightly lower expression of CAMs was observed. A marked difference was found for expression of CD62L; in the thawed LKP the surface antigen density and fraction of CD62⫹ cells was significantly reduced. Our results show that freezing-thawing process influences on qualitative change of the graft reflected by the decrease of CAMs on CD34⫹ cells, especially L-selectin. This might hamper homing, and possibly lead to suboptimal duration of aplasia. 114
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Lymphoproliferative Disorders
ENDOSTATIN INDUCES TUMOR STABILIZATION AFTER CHEMO- OR ANTI-CD20 THERAPY OF HIGHGRADE NON-HODGKIN’S LYMPHOMA (NHL) F. Bertolini, L. Fusetti*, P. Mancuso*, A. Gobbi*, C. Corsini*, P. F. Ferrucci*, G. Martinelli*, G. Pruneri* Hematology-Oncology, Experimental Oncology and Pathology, European Institute of Oncology, Milan, Italy. Both chemotherapy and chimeric anti-CD20 monoclonal antibodies are effective agents against B-cell NHL. However, patients
111
Abstracts/Experimental Hematology 28 (2000) 31–131
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
IDENTIFICATION OF A 85.0Ⲑ124.5 KD MOLECULE WITH ANTI-HOMOAGGREGATION ACTIVITY ON KG1a CELL SURFACE BY A SINGLE CHAIN ANTIBODY FROM A PHAGE DISPLAY LIBRARY Jianhua Sui*, Yixin He, Xueying Jiang*, Zengxuan Song Institute of hematology, Peking Union Medical College & Chinese Academy of Medical Sciences (PUMC&CAMS) Hematopoietic cell surface molecule plays a crucial role in the regulation of hematopoiesis. To identify and characterize novel hematopoietic stemⲐprogenitor cell-surface molecules and their antibodies, we chose human hematopoietic progenitor cell line KG1a as a model system, and a single chain antibody (scFv) phage display library was constructed from the spleen cells of mice immunized with intact KG1a cells. The library was selected by using KG1a cells as panning antigen, and after 4 rounds biopanning, 27 of 126 cones showed strong and selective positive reaction to the KG1a cells in phage ELISA. Diversity analysis of these clones by BstNI fingerprinting revealed four distinct scFv fragments. Two of them, named 5C1 and 1B3 showed significant binding to KG1a cells by flow cytometry, but no or weak cross-reactivity to nonhematopoietic and other leukemia cell lines. These two scFvs were sequenced and further expressed in E.coli., and purified as soluble scFv. With the purified scFvs, the effects of them on KG1a cell line were investigated. The scFv 5C1 was able to block homoaggregation of KG1a cells in a dose-dependent manner. To identify the KG1a cell surface molecule, which bound to the scFv 5C1, proteins from KG1a cell lysates were analyzed by Western blotting. Two bands with apparent molecular weights of 85.0 and 124.5 KDa were recognized by the 5C1 scFv. The results suggest that the 85.0Ⲑ124.5KDa molecule may be an adhesive apparatus necessary for homotypic adhesion. It encourages us to further study the nature and function of the 5C1 antigen. The results also indicate the advantages of phage display antibody technology in searching for hematopoietic stemⲐprogenitor cell-surface molecules. 112
Monday, July 10, 2000 (16:00–17:00) Poster Session II: Stem Cell Biology
EFFECT OF ACTIVATED ADHERENT CELL CONDITIONED MEDIUM ON HUMAN CD34⫹ CELL RECRUITMENT IN SHORT- AND LONG TERM CULTURES E. Wunder*, D. Robay*, L. Mazini*, C. Zanetti*, Ph. Hénon Institut de Recherche en Hématologie et Transfusion, Mulhouse, France Highly purified human CD34⫹ cells produced up to 500⫻ less CFU-GM in short term culture (STC), and 10⫻ less week-5 CAFC in murine FBMD-1 feeder long term culture (LTC) than MNC as calculated per input CD34⫹ cells, according to previous results (1). Cocultured CD34⫺ cells, separated from CD34⫹ cells by a dialysis membrane, re-established efficient colony growth in STC. We now tested efficiency of conditioned media from normal blood adherent cells (ACS) after stimulation with 7ngⲐml GM-CSF for 48h. Addition of ACS from 9 healthy donors enhanced growth of CFU-GM in STC of purified mobilized blood CD34⫹ cells at individually variable degree (2,5–168.8% additional colony yield). 82% additional colonies resulted from pooled supernatants. When
this pooled ACS was added weekly at 1% vⲐv to CD34⫹ cells growing in LTC, the yield of weeks-5 CAFC raised from 67,1 to 1234,6 CAFC per 105 CD34⫹ cells (determined by limiting dilution method and extrapolated); neutralizing antibodies against G-CSF and SCF did not change this enhancement. Fractions bound to anion-exchange columns enhanced early CAFC (week-2), while unbound fractions late (week-5) CAFC. We conclude that the supportive effect of mature adherent cells must be due to secreted factors, which can be separated by their different net electric charge. (1) Mature accessory cells influence long term growth of human hematopoietic progenitors on a murine stromal cell feeder layer. L. Mazini, E. Wunder, H. Sovalat et al.; Stem Cells 16, 404-412 (1998) 113
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Lymphoproliferative Disorders
EFFECT OF CRYOPRESERVATION ON ADHESION MOLECULE EXPRESSION IN PERIPHERAL BLOOD DERIVED CD34⫹ CELLS H. Sovalat*, M. Ojeda1*, Y. Arkam1*, H. Lewandowsky1*, V. Chabouté1*, Ph. Hénon Institut de Recherche en Hématologie et Transfusion, 1Department of Hematology, Mulhouse, France Homing of stem cells to the bone marrow after grafting is essential for hematopoietic recovery and supposedly depends on cellular adhesion molecules (CAMs). Since cryopreservation may affect expression of CAMs, members from integrin-(CD11a, CD49d), immunoglobulin-(CD54, CD58), selectin-(CD62L), and CD44 families were analyzed on CD34⫹ cells in fresh leukapheresis products (LKP) and after their cryopreservation. Two parameters were compared: the percentage of CD34⫹ cells with CAM expression, and the degree of CAM antigen expression; the latter can be estimated by quantitating the number of molecules of monoclonal antibody (MAb) that bind to the cell surface, and is called antibody-binding capacity (ABC). In fresh LKP 100% of CD34⫹ cells were positive for CD44 and CD58; the majority of CD34⫹ cells coexpressed CD11a, and CD49d; 73% coexpressed CD62L (L-selectin). CD54 was expressed with remarkable inter-individual variability. In thawed LKP the percentage of CD34⫹ cells coexpressing adhesion molecules CD54, CD11a was slightly (but not significantly) lower than in fresh samples. When the ABC is compared, generally a slightly lower expression of CAMs was observed. A marked difference was found for expression of CD62L; in the thawed LKP the surface antigen density and fraction of CD62⫹ cells was significantly reduced. Our results show that freezing-thawing process influences on qualitative change of the graft reflected by the decrease of CAMs on CD34⫹ cells, especially L-selectin. This might hamper homing, and possibly lead to suboptimal duration of aplasia. 114
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Lymphoproliferative Disorders
ENDOSTATIN INDUCES TUMOR STABILIZATION AFTER CHEMO- OR ANTI-CD20 THERAPY OF HIGHGRADE NON-HODGKIN’S LYMPHOMA (NHL) F. Bertolini, L. Fusetti*, P. Mancuso*, A. Gobbi*, C. Corsini*, P. F. Ferrucci*, G. Martinelli*, G. Pruneri* Hematology-Oncology, Experimental Oncology and Pathology, European Institute of Oncology, Milan, Italy. Both chemotherapy and chimeric anti-CD20 monoclonal antibodies are effective agents against B-cell NHL. However, patients