It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune conditions suggest that the CD6-CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.C D6 is a marker of T cells and an important T-cell regulator (1). Recent genome-wide association studies also identified CD6 as a risk gene for multiple sclerosis (MS) (2-5), an autoimmune disease in which T cells play a vital role in the pathogenesis. CD6 is composed of three extracellular domains (domains 1, 2, and 3), and it functions by interacting with its ligand(s) (6). The domain 3 of CD6 has been shown to be the site that the identified CD6 ligand, CD166, also known as ALCAM (activated leukocyte cell adhesion molecule), binds to (7). However, anti-CD166 antibodies only partially blocked the binding of thymic epithelial cells to CD6-overexpressing COS cells, and mAbs blocking CD6-CD166 interactions do not abolish CD6 function (8, 9). Itolizumab, an anti-CD6 mAb developed in Cuba and approved in India for treating psoriasis, reduces pathogenic T-cell responses in patients with psoriasis, but this mAb binds to domain 1 of CD6 instead of domain 3, and it does not interfere with the CD6-CD166 interaction. Interestingly, UMCD6, a mouse antihuman CD6 mAb that we found highly effective in treating encephalomyelitis (EAE) in CD6 humanized mice, also fails to block the CD6-CD166 interaction. All these studies suggest the existence of an additional CD6 ligand, other than CD166, that binds to domain 1 of CD6, and could be critical for CD6 function in autoimmune conditions. Further studies using a CD6 fusion protein as a bait to pull down CD6-binding proteins from synovial fibroblast surface proteins showed the binding of three polypeptides (10). One of these polypeptides was identified as CD166, and the identities of the other two were unknown (11). A mAb termed 3A11 was developed, and the antigen recognized by this mAb was identified as the new ligand of ...

None of the work contained in this thesis would have been possible without the help and support of the members of the Cooper and Rosenthal Labs, the TOLAB team members, or the study participants and their families. Special thanks to Frida Ceja for helping me collect the phenotypic drug sensitivity data in Uganda; Patrick Tumwebaze, Oswald Byaruhanga, and Thomas Katairo for their support collecting and analyzing data in Uganda; Melissa Conrad for being a great genotyping and research mentor; and Jenny Legac for her constant guidance and support. I am extremely grateful to Dominican University and the UC Berkeley Minority Health/Global Health Disparities Research Fellowship for helping support my travel to Tororo, Uganda in 2015, 2016, and 2017 to gather data critical to this thesis. Lastly, I would like to thank my mentors, Roland Cooper and Philip Rosenthal, for letting me work in their labs, travel to Uganda, and helping me grow as a research scientist.

Limitations of checkpoint inhibitor cancer immunotherapy include induction of autoimmune syndromes and resistance of many cancers. Since CD318, a novel CD6 ligand, is associated with aggressiveness and metastatic potential of human cancers, we tested the effect of an anti-CD6 monoclonal antibody, UMCD6, on killing of cancer cells by human lymphocytes. UMCD6 augmented killing of breast, lung or prostate cancer cells through direct effects on both CD8+ T cells and natural killer (NK) cells, increasing cancer cell death and lowering cancer cell survival in vitro more robustly than monoclonal antibody checkpoint inhibitors that interrupt the PD-1/PD-L1 axis. UMCD6 also augmented in vivo killing by human peripheral blood lymphocytes of a human breast cancer line xeno-transplanted into immunodeficient mice. Mechanistically, UMCD6 upregulated the expression of the activating receptor NKG2D and down-regulated expression of the inhibitory receptor NKG2A on both NK cells and CD8+ T cells, with concurrent increases in perforin and granzyme-B production. The combined capabilities of an anti-CD6 monoclonal antibody to control autoimmunity through effects on CD4+ lymphocyte differentiation, while enhancing killing of cancer cells through distinct effects on CD8+ and NK cells, opens a potential new approach to cancer immunotherapy that would suppress rather than instigate autoimmunity.

Background Abnormalities in lymphocyte surface markers and functions have been described in systemic sclerosis (SSc), but conflicting results abound, and these studies often examined patients with heterogeneous disease duration, severity, clinical phenotype, and concurrent immunosuppressive agents. We studied a clinically homogeneous group of early diffuse cutaneous SSc patients not exposed to immunosuppressive drugs who were enrolled in a clinical trial and compared their immune parameters to healthy control subjects. Methods Lymphocyte subsets were enumerated by multi-parameter flow cytometry of peripheral blood mononuclear cells at baseline visit. Production of the cytokines IL-4 and IL-17 was measured by intracellular flow cytometry following T cell activation. Results SSc patients had increased percentages of CD4+ T cells but lower percentages of CD8+ T cells versus controls. The CD28-negative population was expanded in SSc, in the CD4 subset. Striking expansion of CD319+ T cells was noted among the CD4+ cells, in which they were barely detectable in healthy subjects. Frequencies of IL-4 producing cells did not differ between SSc and controls, but expansion of IL-17 producing cells was observed in SSc. A higher proportion of CD319+ cells produced cytokines, compared to other CD4+ cells. Numbers of activated T cells, regulatory T cells, and B cells were similar in SSc and control groups. Circulating follicular helper but not peripheral helper T cells were slightly expanded in SSc. Conclusion In this carefully selected group of early diffuse cutaneous SSc patients, analysis of immune cell parameters has identified abnormalities that likely reflect disease pathogenesis and that are candidate biomarkers for sub-classification and targeted treatment. The CD4+CD319+ (SLAM-F7+) cells are cytotoxic and oligoclonal, were recently shown to be a dominant T cell population in perivascular lymphocytic infiltrates in SSc skin, actively secrete cytokines, and are emerging as a target for novel treatments of SSc.