Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes

AIM: To establish a simplified method for generating peptide-major histocompatibility complex (MHC) class I tetramers.METHODS: cDNAs encoding the extracellular domain of human lymphocyte antigen (HLA)-A*0201 heavy chain (A2) and β2-microglobulin (β2m) from total RNA extracted from leukocytes of HLA-A2+ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and β2m proteins were expressed in Escherichia coli strain BL21(DE3) and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain β2m and HLA-A2-restricted peptide antigens. The refolded A2monomers were biotinylated with a commercial biotinylation enzyme (BirA) and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column.The tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4:1. Flow cytometry was used to confirm the expected tetramer staining of CD8+ T cells.RESULTS: Recombinant genes for HLA-A*0201 heavy chain (A2) fused to a BirA substrate peptide (A2-BSP) and mature β2m from HLA-A2+ donor leukocytes were successfully doned and highly expressed in E. coli. Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of β2m and peptides loaded with either human cytomegalovirus pp65495-503 peptide (NLVPMVATV,NLV; designated as A2-NLV) or influenza virus matrix protein Mp58-66 peptide (GILGFVFTL, GIL; designated as A2-GIL). Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplication as revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8+ T cells from a HLA-A2+ donor,but failed to bind to those from a HLA-A2- donor.CONCLUSION: The procedure is simple and efficient for generating peptide-MHC tetramers.

Computer navigation-aided joint-preserving resection and custom-made endoprosthesis reconstruction for bone sarcomas:long-term outcomes

Background:Computed tomography(CT)and magnetic resonance imaging(MRI)data can be fused to identify the tumor boundaries.This enables surgeons to set close but tumor-free surgical margins and excise the tumor more precisely.This study aimed to report our experience in performing computer navigation-aided joint-preserving resection and custom-made endoprosthesis reconstruction to treat bone sarcoma in the diaphysis and metaphysis of the femur and tibia.Methods:Between September 2008 and December 2015,24 patients with bone sarcomas underwent surgical resection and joint-sparing reconstruction under image-guided computer navigation.The cohort comprised 16 males and eight females with a median age of 19.5 years(range:12-48 years).The tumor location was the femoral diaphysis in three patients,distal femur in 19,and proximal tibia in two.The tumors were osteosarcoma(n=15),chondrosarcoma(n=3),Ewing sarcoma(n=3),and other sarcomas(n=3).We created a pre-operative plan for each patient using navigation system software and performed navigation-aided resection before reconstructing the defect with a custom-made prosthesis with extracortical plate fixation.Results:Pathological examination verified that all resected specimens had appropriate surgical margins.The median distance from the tumor resection margin to the joint was 30 mm(range:13-80 mm).The median follow-up duration was 62.5 months(range:24-134 months).Of the 24 patients,21 remain disease free,one is alive with disease,and two died of the disease.One patient developed local recurrence.Complications requiring additional surgical procedures occurred in six patients,including one with wound hematoma,one with delayed wound healing,one with superficial infection,one with deep infection,and two with mechanical failure of the prosthesis.The mean Musculoskeletal Tumor Society score at the final follow-up was 91%(range:80%-100%).The 5-and 10-year implant survival rates were 91.3%and 79.9%,respectively.Conclusions:Computer navigation-aided joint-preserving resection and custom-made endoprosthesis reconstruction with extracortical plate fixation is a reliable surgical treatment option for bone sarcoma in the diaphysis and metaphysis of the femur and tibia.