T cells recognizing EBV-epitopes arose in co-culture of peripheral blood mononuclear cells from EB-infected patients and dendritic cells loaded with LMP2-mixed peptides / 中国实验血液学杂志

ABSTRACT

The latent membrane protein 2 (LMP2) is a kind of protein expressed by EBV-infected cells. This study was aimed to investigate whether the stimulation of peripheral blood mononuclear cells with peptides induces EBV-specific cytotoxic T lymphocytes (CTL). The peptides were mixture of LMP2 protein and available for people with different HLA types. Peripheral blood sample was collected from a patient with EBV-associated hemophagocytic syndrome. The mononuclear cells were isolated and cultured to obtain dendritic cells (DCs). Immature DCs were pulsed with MIX-LMP2 and added with different maturation-promoting factors. The auto-T lymphocytes were stimulated weekly with the harvested mature DCs loaded with MIX-LMP2, and totally for two times. Part of isolated lymphocytes was cultured without any stimulation as control. T-cell receptor (TCR) beta spectratyping was used to analyze the distribution of different T cell subgroups before and after culture. The phenotype of T lymphocytes was determined by flow cytometry. The IFN-gamma assay was used to estimate specific cytoxic activity of the cultured T cells. The results showed that the distribution of TCRbeta was changed according to analysis of TCR spectratypes. From the distribution of gene families of TCRbeta, the T lymphocytes were oligoclonal before culture, but shifted to a polyclonal after culture in vitro like the normalization of TCR diversity, suggesting the subgroups of lymphocyte could return to normal. The percentage of CD3+, CD3+CD8+ CD3+ CD45RA- CD45 RO+ on T lymphocytes from freshly isolated mononuclear cells were 70.73%, 42.99%, 27.56% respectively. After being stimulated twice with DC loaded with MIX-LMP2, they further increased to 95.17%, 52.54% and 81.41%. The percentages of CD3-CD56+ NK cells and CD4+CD35+ FOXP3+ regulation T cells seldom changed, from 2.12%, 0.03% to 2.35%, 0.02% respectively. The increase of CD3+CD45RA-CD45RO+ cells obviously indicated that most naive T cells could be activated. ELISA for IFN-gamma showed that when DCs loaded with LMP2 peptide were used as target cells, IFN-gamma level secreted by the T cells stimulated with LMP2 peptide-pulsed DCs was 805+/-16 pg/ml and 1729+/-49 pg/ml, the IFN-gamma level secreted by T cells stimulated twice with LMP2 peptide-pulsed DCs was 956+/-23 pg/ml and 2325+/-58 pg/ml respectively at effector-target ratios of 101 and 102. They were both significantly higher than that secreted by T cells without any stimulation (441+/-27 pg/m and 557+/-19 pg/ml) (p<0.05). But DCs unpulsed with LMP2 peptide were used as target cells, there were no significant differences between the T cells stimulated with LMP2 peptide-pulsed DCs and the T cells without stimulation (p>0.05). It is concluded that the antigen specific T cells recognizing EBV epitopes can be obtained by using DCs pulsed with MIX-LMP2 peptide in vitro, meanwhile the distribution of T cell subgroups can be changed and normalized.