<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Ligation of CD28 with ligands such as CD80 or CD86 provides a critical second signal alongside antigen presentation by class II major histocompatibility complex expressed on antigen-presenting cells through the T cell antigen receptor for naïve T cell activation. A number of studies suggested that CD28 plays an important role in the pathogenesis of various human diseases. Recent genome-wide association studies (GWASs) identified<jats:italic>CD28</jats:italic>as a susceptibility locus for lymphocyte and eosinophil counts, multiple sclerosis, ulcerative colitis, celiac disease, rheumatoid arthritis, asthma, and primary biliary cholangitis. However, the primary functional variant and molecular mechanisms of disease susceptibility in this locus remain to be elucidated. This study aimed to identify the primary functional variant from thousands of genetic variants in the<jats:italic>CD28</jats:italic>locus and elucidate its functional effect on the CD28 molecule.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Among the genetic variants exhibiting stronger linkage disequilibrium (LD) with all GWAS-lead variants in the<jats:italic>CD28</jats:italic>locus, rs2013278, located in the Rbfox binding motif related to splicing regulation, was identified as a primary functional variant related to multiple immunological traits. Relative endogenous expression levels of<jats:italic>CD28</jats:italic>splicing isoforms (CD28i and CD28Δex2) compared with full-length CD28 in allele knock-in cell lines generated using CRISPR/Cas9 were directly regulated by rs2013278 (P < 0.05). Although full-length CD28 protein expressed on Jurkat T cells showed higher binding affinity for CD80/CD86, both CD28i and CD28Δex2 encoded loss-of-function isoforms.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>The present study demonstrated for the first time that<jats:italic>CD28</jats:italic>has a shared disease-related primary functional variant (i.e., rs2013278) that regulates the CD28 alternative splicing that generates loss-of-function isoforms. They reduce disease risk by inducing anergy of effector T cells that over-react to autoantigens and allergens.</jats:p></jats:sec>