Prolonged expression of MHC class I - peptide expression in bone marrow derived retrovirus transfected matured dendritic cells by continuous centrifugation in the presence of IL-4

Abstract

Background & objectives: Dendritic cells (DCs) are potent antigen presenting cells which proceed from immature to a mature stage during their differentiation. There are several methods of obtaining long lasting mature antigen expressing DCs and different methods show different levels of antigen expressions. We investigated bone marrow derived DCs for the degree of maturation and genetically engineered antigen presentation in the presence of interleukin-4 (IL-4) as a maturity enhancer.

Methods: DCs and transfected retrovirus were cultured together in the presence of granulocyte-macrophage colony stimulating factor (GMCSF)-IL4, GMCSF +IL4, lipopolysaccharide (LPS). B 7.1, B7.2 and CD11c were measured by the degree of immune fluorescence using enhanced green fluorescent protein (EGFP) shuttled retrovirus transfected antigen. Degree of MHC class I molecule with antigen presentation of antigen was also evaluated by fluorescence activated cell sorting. The antigen presenting capacity of transfected DCs was investigated. Bone marrow DCs were generated in the presence of GMCSF and IL-4 in vitro. Dividing bone marrow cells were infected with EGFP shuttled retrovirus expressing SSP2 by prolonged centrifugation for three consecutive days from day 5, 6 and 7 and continued to culture in the presence of GMSCF and IL-4 until day 8.

Results: IL-4 as a cytokine increased the maturation of retrovirus transfected DCs by high expression of B 7-1 and B 7-2. Also, IL-4 induced DC enhanced by the prolonged centrifugation and it was shown by increased antigen presentation of these dendric cells as antigen presenting cell (APC). Cytolytic effects were significantly higher in cytotoxic T cell response (CTLs) mixed with transfected DCs than CTLs mixed with pulsed DCs.

Interpretation & conclusions: There was an enhanced antigen presentation by prolonged expression of antigen loaded MHC class I receptors in DCs in the presence of IL-4 by prolonged centrifugation.

Fig. 1

For phenotype analysis, 10⁶ DCs were incubated with anti- CD80 (B7.1), anti CD86 (B7.2) and double stained with CD11c directly labelled antibody. For B7-1 and B7-2 double staining, cells were cultured with GMCSF alone, (A2, B2) SSP2 transfected cells in the presence of GMCSF alone, SSP2 transfected cells in the presence of GMCSF with IL-4 and (A4 and B4) LPS (positive control) respectively. Values are mean ± SD.

Fig. 2

Shows percentage of DCs expressing B7.1 and B7.2 with CD11c positive cells in each different culture situations (GMCSF alone, SSP2 transfected, SSP2 transfected in the presence of IL-4, and LPS).

Fig. 3

Degree of MHC class I expression in CD 11c dendritic cells were seen in each different culture situations GMCSF alone, GMCSF with SSP2 transfected DCs with IL-4 and without IL-4 And positive control LPS. Mean fluorescence intensity of double stained cells is given. An istotype antibody was used as control. The experiment was repeated several times, one representative result is shown.

Fig. 4A.

Shows the expression of recombinant SSP2 protein (34 kD protein) after PCR synthesis by agarose gel electrophoresis. It was detected as 34 kD protein. Protein expression is shown in soluble and insoluble lysates of transfected DCs, transfected RL tumour cells as a control, non-transfected DCs and non-transfected RL tumour cells. Tumour cell line is also used in this experiment for the comparison among different types of antigen presenting cells. Synthesized SSP2 protein was used as the positive control. M, Marker; L, SSP2 injected line.

Fig. 4B.

CTLs from BALB/c mice were analyzed for its cytolytic activity of the matched antigen-pulsed target cells by ⁵¹Cr-release assay. For antigen specific CTL induction, CTLs were cultured with A and C peptides for 7 days. For cytolytic assay, CTLs were recultured with peptide-pulsed and transfected DCs and transfected P815 tumour target cells and mock transfected for the analysis for Cr-release. These antigens specific CTLs identified and killed significantly higher number of peptide transfected targets than the pulsed targets. Cytolytic activity of transfected target cells is significantly more than the RL♂ mock cells. Data represent one of the three experiments.

Fig. 5

Enhanced transfection efficacy of retrovirus transfected dendritic cells by prolonged centrifugation. DCs were infected with EGFP shuttled retrovirus vector and green fluorescence was measured by FACS. Cells were compensated for each staining (CD11c and EGFP) and number of CD11c positive EGFP expressing cells is given. Kinetic study of CD11c expressing DCs was done from day 8 to day 12. Data show the mean fluorescence intensity with time.