Efficient cloning and expression of HLAclass I cDNA in human B‐lymphoblastoidcell lines

Abstract

Analysis of HLA restriction specificity is one of the important steps in characterizing T cell clones. This usually requires either a panel of HLA-typed cells or HLA cDNA transfectants. Although preparation of HLA cDNA transfectants is laborious, utilization of transfectants is advantageous when a suitable panel is not available due to linkage disequilibrium or rarity of the HLA allele of interest. In this report, we describe an efficient and rapid HLA cloning and expression system. Three sets of PCR primers specific for HLA-A, B and C loci were designed by extensively sequencing 5'- and 3'- untranslated regions of HLA class I genes. The PCR-amplified products were introduced into modified Phoenix retrovirus vectors containing a puromycin resistant gene under the control of a LTR promotor. Gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was produced and infected into B-lymphoid cell lines. Following expansion in selection media, more than 80% of cells expressed transduced HLA at a comparable level to that normally expressed. These results indicate that locus-specific PCR cloning and utilization of GALV-pseudotyped retroviral vector can be an effective and relatively efficient tool for constructing a panel of different HLA transfectants.

Epstein‐Barr virus (EBV) latent membrane protein‐1‐specific cytotoxic T lymphocytes targeting EBV‐carrying natural killer cell malignancies

“…All three were cultured as previously described [6]. HEK-293T cells (American Type Culture Collection, Manassas, VA) and Phoenix-GALV cells [49] (kind gifts from Dr. Kiem, Fred Hutchinson Cancer Research Center; and Dr. Nolan, Stanford University, Stanford, CA) were cultured as previously described. Retroviral transduction of HLA genes was performed as detailed earlier [50].…”

Efficient Generation of Antigen-Specific Cytotoxic T Cells Using Retrovirally Transduced CD40-Activated B Cells

“…The resultant fragment containing puromycin-resistance gene and CMV immediate early promotor was ligated into the LZRSpBMN vector, which was designated as pLBPC. The pp65 gene was inserted into the pLBPC (pLBPC-pp65 (14), as shown in Fig. 2) to prepare retroviral vector containing pp65 that is selectable by puromycin.…”

The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206

“…LCLs were transduced with retroviral vectors carrying individual HLA cDNAs by a method described previously. 18 HLA cDNA-transfected cell lines are referred to by their 'cell line name/HLA allele' designation (e.g. 721.221/A*0201).…”