[Establishment of long-term culture system for human B cells derived from peripheral blood mononuclear cells activated via soluble CD40 ligand].
OBJECTIVE
To develop a method for long-term culture of human B cells from peripheral blood mononuclear cells (PBMCs) by means of human soluble CD40 ligand (sCD40L), and to investigate the proliferation and antigen-presenting function of the CD40-activated B cells.


METHODS
Peripheral blood sample of 30 ml was collected from a healthy person. PBMCs were isolated and cultured in the presence of sCD40L. Flow cytometry was used to examine the proliferation, DNA cycle, and cell surface markers: CD86, CD80, major histocompatibility complex (MHC) class II, and MHC Class I of the B cells. The cells cultured for 45 days were divided into 2 groups: Group 1 incubated with the fluorescein isothiocyanate (FITC)-conjugated hepatitis-B core antigen (HBcAg-FITC) and Group 2 used as control group. Eighteen hours later cytometry and fluorescence microscopy were used to detect the peptide pulsing.


RESULTS
The B cells could be cultured up to 50 days in the sCD40L culture system. The ratio of B cells in the PBMCs was 8.21% at the beginning, and increased to 70.67% by day 47, and the B cell absolute count was 6.56 x 10(5) at the beginning and increased to 8.61 x 10(6) at day 50, about 10 times higher. sCD40L promoted a strong up-regulation of cell surface markers. The expression rates of CD80, CD86, MHC-II, and MHC-I of the sCD40L-activated B cells were 83.97%, 84.73%, 99.59%, and 98.70% respectively. Flow cytometry showed that 98.10% of the B cells co-incubated with HBcAg-FITC were loaded with HBcAg. Fluorescence microscopy showed that the HBcAg was in the cytoplasm of the B cells.


CONCLUSIONS
A human sCD40L culture system has been developed with the ability to culture human peripheral blood B cells, thus realizing the long-term proliferation and activation of human peripheral blood B cells that function as antigen-presenting cells.
Wu
Chao
C
Department of Infectious Diseases, Drum Tower Clinical College of Nanjing Medical University, Nanjing 210008, China.
Liu
Yong
Y
Zhao
Qi
Q
Chen
Guang-Mei
GM
Chen
Jun-Hao
JH
Yan
Xiao-Min
XM
Zhou
Yi-Hua
YH
Huang
Zu-Hu
ZH
D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D023201:CD40 Ligand / Q000378:metabolism*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans
D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D023201:CD40 Ligand