Many viral epitope specific T cell receptors (TCRs) in MHC-matched individuals have been demonstrated to involve conserved amino acid motifs in β chain complementarity-determining region 3 (CDR3). However, it is not sure whether the conserved motifs can also be found in TCR α chain. In previous studies, we developed a modified method to enlarge the percentage of cytomegalovirus (CMV) pp65 peptide-specific CD8+ T cells in PBMC by continuous peptide stimulation in vitro, which provides sufficient number of specific T cells for detection. In this study, we further analyzed the restrictive usage of TCR Vα and Vβ gene families and investigated the CDR3 gene sequence of pp65 peptide-specific CD8+ T cells. Analysis of CDR3 spectratypes suggested a restricted usage of TCR α chain AV8, AV12, AV21, AV31 families and TCR β chain BV3, BV14, BV21, BV23, BV11 families in donor CD8+ T cells stimulated by pp65 peptide. The sequences of these T cells involved similar sequence (TX) G (X) A in CDR3 region of TCR α chain and L (XT) G (X) A in TCR β chain.
CMV-specific immunity is essential for control of human cytomegalovirus (HCMV) infection. Stem cell trans-plantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the ex vivo production of cells for adoptive transfer is labor intensive and expensive. We report here a modified peptide stimulation method using CMV-specific epitope peptides to stimulate PBMCs for generation of CMV-specific CTLs. This method permits efficient amplification of CMV-specific CTLs and provides a large number of cells for FACS analysis from a single blood sample. Significantly, it achieves high frequencies of tetramer staining of CD8+ T cells allowing the data of different individuals to be easily compared and sequentially evaluated. Thus, this approach expands and selects HLA-restricted CMV-pp65-reactive T-cell lines of high specificity for potential adoptive immunotherapy.
Guangping RuanLi MaQian WenWei LuoMingqian ZhouXiaoning Wang
Objective To study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hIL-2/mGM-CSF). Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coli) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein /L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×106 IU/mg and 7.1×106 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hIL-2/mGM-CSF used in immune therapy.
