A Novel Co-culture System To Reveal The Heterogeneity Of Antigen Specificity Among Major HLA-A2 Subtypes In Chinese Population

A novel co-culture system to reveal the heterogeneity ofantigen specificity among major HLA-A2 subtypes inChinese populationPh.D.candidate:Shengjun LuSupervisor:Prof.Xiongwen WuMajor histocompatibility complexes (MHC) is employed in presentingantigenic peptides to T lymphocytes and elicites immune responses by inducingproliferation and activation of antigen specific T lymphocytes.In this process,antigenic peptide/MHC compounds (pMHC) instead of MHC itself alone act as theligands of T cell receptors.Difference in the antigen specificity among serologicalallotypes of MHC (eg.HLA-A2 vs HLA-A24) is well known,which can be shownby lymphocyte proliferation in mixed lymphocyte culture (MLC),an in vitro modelof allograft transplantation.Nevertheless,it is not addressed yet that whether there isalso difference in antigen specificity among subtypes of a serologically typed MHC(eg.HLA-A*0201 vs HLA-A*0206).Understanding of this issue will contributemuch to vaccine design strategy and benefit donor selection in clinical unrelatedtransportation.HLA-A2 is one of the most prevalent serological HLA-A types in variousethnic populations.Individuals with HLA-A2 antigen count for some 50% inChinese people.The most frequent allele of HLA-A2 is HLA-A*0201.Otherprominent HLA-A2 subtypes in Chinese population include HLA-A*0203,HLA-A*0206,HLA-A*0207.These HLA-A2 subtypes are nearly identical in theirpeptide sequences,with only 1-3 amino acids substitutions among them. Nevertheless,some data implies that they functionally differ in the properties of theirantigen presentation process and in their specificity of CTL recognition.With theobstacle of the features of MHC (with multiple gene,high polymorphism andhaplotype inherence),no proper experimental system has been developed yet tostudy the heterogeneity of major HLA-A2 subtypes in their antigen specificity.Experimental evidence is absent about whether they are identical or not in theirantigen specificity.The aim of this study is to explore an experimental system toreveal this issue.A divalent HLA-A*0201/IgG1-Fc molecule (HLA-A*0201 dimer) consisting ofthe extracellular domains of HLA-A*0201 and the Fc-CH1 segment of human IgG1gamma chain,is employed in this study.The peptide/HLA-A*0201 dimer was ableto be introduced as an allogeneic epitope onto HLA-A2-ve monocytes via interactionof the Fc and FcR.Autologous PBLs proliferation can be observed after co-culturedwith the dimer-loaded HLA-A2-ve monocyte.This data indicates that the co-culturesystem can be used to show the antigen specificity of a single pMHC epitope.A modified MLC,lymphocytes-autologous monocytes co-culture (LAMC) isdeveloped to show the heterogeneity of major HLA-A2 subtypes in their antigenspecificity.A few improvements are made in LAMC.Firstly,an additionalHLA-A*0201 molecule is attached to the stimulating cell of other HLA-A2 subtypethan HLA-A*0201.Secondly,the effector cells are autologous lymphocytes insteadof allogeneic ones used in conventional MLC.Appearantly,the lymphocyteproliferation in the LAMC indicates the heterogeneity of HLA-A*0201 and thegiven HLA-A2 subtype in their antigen specificity.Counts of proliferating cells in the LAMC is applied but not directely used inshowing the antigen specificity heterogeneity,for a single pMHC epitope ownslimited ability to promote lymphocyte proliferation,which may be submerged by thevariation of lymphocyte proliferation among individual samples.To avoid this problem,we introduce the proliferation efficiency (Pe) to redress the absolute countsof proliferating cells,the Pe is figured out with a formula listed below.In which,'Tf'means the proliferating PBLs counts in the LAMC with the peptide/HLA-A*0201dimer loaded autologous monocytes (HLA-A2+ve);'Bf'is the backgroundproliferating frequency in the LAMC of the native HLA-A2+ve monocyte andautologous PBLs;'Pf'is the positive proliferating frequency of the HLA-A2+vePBLs in MLC HLA-A2-ve monocytes instead of autologous monocytes.Pe=(Tf-Bf)/(Pf-Bf)With a self-peptide Tyr368-376 (NH₂-YMDGTMSQV-COOH) fromintracellular tyrosinase,the LAMC shows the heterogeneity of major HLA-A2subtypes in their antigen specificity.The Pe is observed significantly differentbetween HLA-A*0201 and HLA-A*0203 (with Pe values of 0.012±0.109 vs 0.124±0.067,P≤0.05),and between HLA-A*0201 and HLA-A*0206 (with Pe values of0.012±0.109 vs 0.225±0.184,P≤0.01);no statistical Pe difference betweenHLA-A*0201 and HLA-A*0207 is observed (with Pe values of 0.012±0.109 vs0.050±0.109,P＞0.05).Our observations suggest antigen specificity ofHLA-A*0201 and HLA-A*0203,and that of HLA-A*0201 and HLA-A*0206 aredifferent,antigen specificity of HLA-A*0201 and HLA-A*0207 is not observeddifferent.