The Role Of T Cells In The Pathogenesis Of Ankylosing Spondylitis

BackgroundAnkylosing spondylitis(AS)is a chronic inflammatory disease with worldwide high prevalence.Forty-five years ago,the prevalence of AS was associated with the expression of a human leukocyte antigen(HLA)class I surface molecule,HLA-B27,and more than 90%of AS patients carry at least one HLA-B27 allele.Besides HLA-B27,genome-wide association studies(GWASs)identified polymorphism in endoplasmic reticulum aminopeptidase 1(ERAP1)as a risk factor for AS,and ERAP1 variants only influence the susceptibility of patients carrying the HLA-B27 allele.ERAP1 functions to process peptides for class Ⅰ MHC antigen presentation.Although this linkage proves that AS is an autoimmune disease strongly associated with MHC-I HLA-B27 antigen presentation of arthritogenic peptide,the role played by αβ T cells in AS remains elusive.There are two major enigmas1)disease manifestation remains nearly intact when CD8 T cells are depleted in animal models whereas CD4 T cells can transfer the disease;2)if CD4 T cells drive pathogenesis,it is unclear how MHC-I HLA-B27 activates these MHC-II antigen recognizing CD4 T cellsAlso,in consistent with previous clinical studies,we also found the overall differences in T cell phenotypes between AS patients and healthy subjects are rather moderateMethodsAided by the analytical power of next-generation deep sequencing of TCRβ repertoire,we analyzed the TCR repertoires of T cell sub-populations from AS patients with varying disease activities.Although TCRβ repertoire sequencing has emerged as a popular technology in recent years,its clinical application is limited by the complexity of the TCR repertoire.Due to the life-long battle with environmental pathogens,meaningful information is usually muffled by noise from irrelevant clonal changes of T cells.Hence,the quantification of T cell responses against specific antigens is the central urgency facing the application of Rep-seq to clinical monitoring,especially for antigen-unknown conditions such as AS.To address this challenge,we developed several bioinformatics tools to evaluate T cell responses from global repertoire changes:Asymptotic diversity profileDiversity indices such as richness,Shannon index and Simpson index are frequently used to evaluate the T cell clonal frequency distributions in the repertoire and represent the state of T cell clonal expansion and selection.However,these types of analyses based on different diversity indices can yield qualitatively different results.Actually,the difference of those various diversity indices is the usage of different weights on abundant clonotypes In order to guarantee the reliability of diversity analysis,we used the Hill-based diversity to developed asymptotic diversity profiles using continuous weights Top[N]similarity indexSimilarity indices such as jaccard index and morisita index are frequently used to evaluate the clonotype overlap between two repertoires and represent common T cell response.Previous studies analyzed the clonotype expansion and the clonotype overlap seperately.In our study,we developed a new similarity analysis method,the Top[N]index,to evaluate the overlap of the expanded clonotypes and represent the clonal expansion under common T cell response the "Motif Analysis" algorithm to discriminate antigen-specific T cell responsesRecently,the comprehensive structural biology studies clearly suggest that not every amino acid of CDR3 is required to execute TCR-pMHC interaction.Instead,antigen recognition relies on hotspots formed by only a few(3-4)amino acids in the CDR3 loop Hence,it is conceivable that traditional similarity analysis mistakenly excludes TCRs that recognize a specific autoantigen with the same hotspot but varied amino acids in other positions.To improve the accuracy of antigen specificity-based similarity measurement,we developed a new computational framework to search for TCRs sharing the same antigen specificity in repertoires-the Motif Analysis.Based on antigen-recognizing CDR3 motifs,the "Motif Analysis" algorithm was critical for us to identify TCRs sharing the same antigen specificity without sequence homology.This method was generated and validated with over 400 T cell repertoires.This tool enables the reliable and unbiased dissection of antigen-specific T cell responses from the global repertoire,which can be generally applied to and empower various repertoire-based clinical immune monitoring, such as those for cancer immunotherapiesResultsUsing those bioinformatic approaches developed in house,we investigated TCR repertoires in sorted CD8+,CD4+CD45RA+,and CD4+CD45RO+T cells from a clinical cohort of AS patients with various pathological activities.Several interesting results were obtained1)AS disease progression is associated with CD4+CD45RA+T cell clonal expansion2)CD4+CD45RO+and CD8+T cells are stimulated by shared antigens among AS patients with active disease3)AS patients have an antigen specificity profile that is distinct from that of other T cell-mediated autoimmune diseases;also,CD4 and CD8 T cells from AS patients have no difference in antigen specificity4)The above results enabled us to discover a novel T cell population consisting of both CD4 and CD8 T cells expressing identical TCRs,herein termed CD4/8 T cells CD4/8 T cells are highly enriched and expanded in active AS patients.These results provide evidence on the T cell clone side to reveal the potential role of CD4/8 T cells in the etiology of AS developmentConclusionsAbove results provides direct evidence to support three major conclusions1)for individual patients,expansion of CD4+CD45RA+T cells is associated with the activity of AS,which suggests that αβ3 T cell activation may mediate AS disease progression.2)for highly active disease,CD4+CD45RO+ and CD8+T cells are likely to be stimulated by certain shared autoantigens;3)AS pathological CD4 and CD8 T cells might share the same antigen specificity by virtue of expressing the same TCRs.Together,these findings reconcile the major controversy existing in the AS field:both CD4 and CD8 T cells are critical for disease manifestation;and,pathological CD4 T cells might recognize HLA-B27 because they carry the exactly same TCR as CD8 T cellsAbove all,although our analyses do not have the power to suggest any characteristics of arthritogenic antigen,it provides direct evidence that AS progression is associated with both CD4 and CD8 T cell expansion in patients.Furthermore,we discovered that these pathology-associated CD4 and CD8 T cells share identical CDR3 and TCR sequences,which are defined as CD4/8 T cells.