The Journal of Immunology The CD6 Multiple Sclerosis Susceptibility Allele Is Associated with Alterations in CD4 + T Cell Proliferation David M. Kofler,* ,†,‡,1 Christopher A. Severson, ‡,1 Narine Mousissian,* ,†,‡ Philip L. De Jager, ‡,x,1 and David A. Hafler* ,†,‡,x,1 Genome-wide association studies have revealed a large number of genetic associations with autoimmune diseases. Despite this prog- ress, the mechanisms underlying the contribution of allelic variants to the onset of immune-related diseases remain mostly unknown. Our recent meta-analysis of genome-wide association studies of multiple sclerosis (MS) identified a new susceptibility locus tagged by a single nucleotide polymorphism, rs17824933 ( p = 3.8 3 10 29 ), that is found in a block of linkage disequilibrium containing the CD6 gene. Because CD6 plays an important role in maintenance of T cell activation and proliferation, we examined the biologic phenotypes of the risk-associated allele. In this article, we report that the MS susceptibility allele in CD6 is associated with decreased expression of full-length CD6 in CD4 + and CD8 + T cells. As a consequence, proliferation is diminished during long-term activation of CD4 + T cells from subjects with the risk allele. Selective knockdown of full-length CD6 using exon 5-specific small interfering RNA induces a similar proliferation defect of CD4 + T cells from subjects homozygous for the protective allele. Exon 5 encodes for the extracellular binding site of the CD6 ligand ALCAM, which is required for CD6 stimulation. In CD4 + T cells from subjects with the risk allele, exon 5 is consistently underexpressed, thereby providing a mechanism by which the allele affects proliferation of CD4 + T cells. These findings indicate that the MS risk allele in the CD6 locus is associated with altered pro- liferation of CD4 + T cells and demonstrate the influence of a disease-related allelic variant on important immunological character- istics. The Journal of Immunology, 2011, 187: 3286–3291. M ultiple sclerosis (MS) is an inflammatory disease of the CNS that shares elements of its genetic architecture with other autoimmune diseases (1). Pathologically, there are perivenular infiltrates of CD4 + and CD8 + T cells in the CNS white matter and meninges with demyelinating lesions and loss of axons in both white and gray matter (2, 3). There are also marked changes in systemic immune function with loss of regu- latory T cell (Treg) function and increases in myelin-reactive CD4 + T cells (4–6). Interestingly, unbiased genome-wide associ- ation scans have identified susceptibility loci almost exclusively in regions containing genes with immune function, including CD6, CD25, CD40, CD58, HLA A, HLA B, HLA-DRB1, IL2RA, IL7R, IL12A, IRF8, STAT3, and TNFRSF1A (7–11). The identification of susceptibility loci is a major step forward in MS, but we do not yet understand how allelic variation influences immune function. This transition remains a major challenge in the understanding of MS and of other human autoimmune diseases. CD6 is a 130-kDa type 1 transmembrane glycoprotein expressed on the surface of CD4 + and CD8 + T cells and, to a lesser extent, on B cells. CD6 stimulation plays an important role in the mainte- nance of T cell activation (12–19) because blocking interactions between CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM) result in diminished proliferation of T cells (12). The ALCAM-binding site is located at the membrane- proximal scavenger receptor cysteine-rich (SRCR) domain 3 of CD6 and is present in most of the five intracellular and three extracellular isoforms of CD6 that have been described (20–22). The CD6Dd3 splice variant, created by excluding exon 5, is lacking the membrane-proximal SRCR domain 3 with the ALCAM-binding site (23). The MS risk allele rs17824933 G is located in intron 1 of the CD6 gene (7) and is found at a frequency of 44% of subjects of European ancestry from the PhenoGenetic Project, a cohort of .1200 healthy subjects recruited from the Greater Boston Metropolitan area that supports the functional characterization of genetic variation; subjects homozygous for the risk allele (with an rs17824933 GG genotype) are found at 7.6% frequency in this population. Materials and Methods Subjects The Brigham and Women’s Hospital PhenoGenetic Cohort with a collec- tion of 1200 healthy control subjects served as basis for subject recruitment and sample collection. These subjects were genotyped and had the fol- lowing characteristics: female/male sex ratio was 60:40%; race distribution was 14% African American, 12% Asian American, 68% white, and 6% Hispanic; smoking history was 82% never smokers, 9% former smokers, and 9% smokers; mean age was 24.3 y (range, 18–50 y); mean body mass index was 22.5 (range, 13–50); and MHC was 51% HLA A2, 20% HLA A3, 23% HLA-DR2, and 20% HLA-DR1 and -DR4. All subjects have been comprehensively genotyped for allelic variants related to suscepti- bility to autoimmune diseases. The study was conducted in compliance with the Declaration of Helsinki. Approval was obtained from the local ethics committee before study initiation, and written informed consent was obtained from all patients before performing any study procedures. *Department of Neurology, Yale School of Medicine, New Haven, CT 06520; † Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520; ‡ Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and x Broad Institute of Harvard University and Massachusetts Institute of Technology, Cambridge, MA 02142 1 D.M.K., C.A.S., P.L.D.J., and D.A.H. contributed equally to this article. Received for publication March 3, 2011. Accepted for publication July 18, 2011. This work was supported by the Deutsche Forschungsgemeinschaft (Grant KO 4034/ 1-1 to D.M.K.). Address correspondence and reprint requests to Dr. David A. Hafler, Department of Neurology, Yale School of Medicine, New Haven, CT 06520. E-mail address: david. hafler@yale.edu The online version of this article contains supplemental material. Abbreviations used in this article: ALCAM, activated leukocyte cell adhesion mol- ecule; MS, multiple sclerosis; rhALCAM-Fc, recombinant human ALCAM-Fc fusion protein; siRNA, small interfering RNA; SNP, single nucleotide polymorphism; SRCR, scavenger receptor cysteine-rich; Treg, regulatory T cell. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100626