Autogeneic rna-electroporated CD40-ligand activated b-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo.
Since dendritic cells (DCs) constitute only 0.1-0.5% of human peripheral blood mononuclear cells (PBMCs), and generation of DCs from monocytes or stem cells is difficult and expensive, we choose B-lymphocytes as an alternative, cost-effective source of antigen presenting cells (APC).


OBJECTIVE
To induce specific CTLs response ex vivo by CD40L activated B-cells (CD40-B) transfected with hepatocellular carcinoma (HCC) total RNA.


METHODS
To induce CD40-B PBMCs of patients with HCC were isolated by Ficoll technique and cultured in RMPI 1640 supplemented with 10% fetal calf serum (FCS), sCD40L (2 microg/ml), recombinant human interleukin-4 (IL-4) (4 ng/ml). The expression of CD80 and CD86 was evaluated by flow cytometry. The level of interleukin-12 (IL-12) produced by cultured B-lymphocytes was measured using enzyme-linked immunosorbent assay (ELISA). HCC patient's T-lymphocytes were obtained from PBMCs cultured in RMPI 1640 supplemented with 10% FCS, 2 ng/mL IL-4 and 10 ng/ml IL-7. CD40-B transfected with tumor total RNA isolated from HCC cells were used to induce specific CTL proliferation. The level of IFN-gamma was measured using ELISA and the expression of CD8 was determined by FCAS. Specific cytotoxicity was measured using MTT method.


RESULTS
The results show that the activated B-lymphocytes were easily expandable and formed large clones, and a high expression of CD80/CD86 and a high IL-12 secretion by CD40-B was registered. CD40-B transfected with tumor total RNA can induce CTLs to express CD8 and generate IFN-gamma at high levels. Compared to the control group, the specific cytotoxicity of CTLs was up-regulated.


CONCLUSIONS
These findings demonstrate that CD40-B-cells electroporated with total RNA derived from carcinoma cells can be used as alternative APC for the induction of antigen-specific CD8(+) T-cell responses, which might be used in HCC immunotherapy.
Shen
S
S
Department of Oncology, the Affiliated Drum Tower Hospital, Medical School of Naniing University and Clinical Cancer Institute of Nanjing University, Naniing, PR China.
Xu
Z
Z
Qian
X
X
Ding
Y
Y
Yu
L
L
Liu
B
B
D000938:Antigen-Presenting Cells / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D018122:B7-1 Antigen / Q000378:metabolism; D051940:B7-2 Antigen / Q000378:metabolism; D019013:CD40 Antigens / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D006528:Carcinoma, Hepatocellular / Q000276:immunology*; D002478:Cells, Cultured; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis; D018664:Interleukin-12 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008113:Liver Neoplasms / Q000276:immunology*; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis
D018122:B7-1 Antigen; D051940:B7-2 Antigen; D019013:CD40 Antigens; D012333:RNA, Messenger; D018664:Interleukin-12; D007371:Interferon-gamma