Background/Purpose: CD6, an important regulator of T cells, has one known ligand, CD166, but studies performed during the treatment of autoimmune conditions and in vitro experiments 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 this ligand is hitherto unknown.

Methods: To determine the identity of the antigen recognized by mAb 3A11, we investigated HBL-100 cell surface proteins pulled down by this mAb by mass spectrum (MS) analysis. We then probed whole HBL-100 cell lysates with an anti-CD318 Ab in western blot and assessed CD318 expression levels on HBL-100 cells by flow cytometry before and after IFNγ stimulation. We next studied transfected MDA-468 cells that overexpress CD318 after doxycycline induction and transfected MDA-468 cells knocked down for CD318 expression using shRNA, by flow cytometry using a commercial anti-CD318 mAb and mAb 3A11. We also studied binding of CD6 to HT-1080 sarcoma cells in which expression of CD166 but not CD318 was selectively ablated by CRISP-R technology, and measured binding of rCD318 to CHO-CD6 transfectants. We then assessed the role of CD318 in autoimmunity in vivo using CD318-knockout mice and the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis.

Results: CD318-related peptides were abundant in mAb 3A11 precipitates from HBL-100 cells. Mab 3A11 and anti-CD318 immunoprecipitated identical bands at 130kDa, and binding of each mAb to the cell surface was identically upregulated by pre-exposure of cells to IFNγ. Moreover, each mAb bound to CD318 in western blots. In a CD318-inducible system, staining with mAb 3A11 resulted in exactly the same pattern as seen with anti-CD318 mAbs, while in CD318 knockdown cells neither mAb showed detectable staining. An HT-1080 CD166 KO cell line was developed that expresses CD318 but not CD166 -binding of CD6 to the surface of these cells was attenuated but still evident, and was further reduced by rCD318 in a dose-dependent manner. Recombinant CD6 precipitated a protein from lysates of these cells that was recognized by anti-CD318. Recombinant CD318 bound to human CD6-expressing CHO cells but not control CHO cells. Moreover, CD318 KO mice had markedly attenuated disease severity of EAE, with reduced antigen-specific Th1 and Th17 responses and significantly decreased inflammation and CD4+ T cell infiltration in the central nervous system.

Conclusion: These data establish CD318 as a novel second ligand of CD6, and indicate a previously unknown role for CD318 in regulation of T cell driven autoimmunity. The engagement of CD6 by CD318 is an unusual example of a ligand-receptor interaction between a lymphocyte-specific cell surface glycoprotein that can participate in T cell activation (CD6) and a molecule (CD318) that is found only on cells that are traditionally considered not to be components of the immune system. This interaction points to an ability of T cells to specifically recognize distinct signals from “non-immune system” tissue cells that may be important in organ-targeted autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd318-is-a-new-ligand-for-cd6/