Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model.

O-GlcNAcylation is a reversible post-translational modification that regulates numerous cellular processes, including embryonic development as well as immune responses. However, its role in inflammation remains ambiguous. This study was designed to examine the role of O-GlcNAcylation in rheumatoid arthritis (RA) and its regulation using human RA patient-derived synovial fibroblasts (RASFs). The efficacy of penta-O-galloyl-beta-D-glucose (PGG), a potent anti-inflammatory molecule, in regulating inflammatory processes in human RASFs was also evaluated. Human synovial tissues and RASFs exhibited higher expression of O-GlcNAcylation compared to their non-diseased counterparts. Pretreatment of RASFs with Thiamet G, an inhibitor of O-GlcNAcase, markedly increased the O-GlcNAc-modified proteins and concomitantly inhibited the IL-1ß-induced IL-6 and IL-8 production in human RASFs in vitro. Pretreatment of human RASFs with PGG (0.5-10 µM) abrogated IL-1ß-induced IL-6 and IL-8 production in a dose-dependent manner. Immunoprecipitation analysis showed that PGG inhibited O-GlcNAcylation of TAB1 to reduce its association with TGF ß-activated kinase 1 (TAK1) and its autophosphorylation, an essential signaling step in IL-1ß-induced signaling pathways. Molecular docking in silico studies shows that PGG occupies the C174 position, an ATP-binding site in the kinase domain to inhibit TAK1 kinase activity. Oral administration of PGG (25 mg/kg/day) for 10 days from disease onset significantly ameliorated rat adjuvant-induced (AIA) in rats. PGG treatment reduced the phosphorylation of TAK1 in the treated joints compared to AIA joints, which correlated with the reduced disease severity and suppressed levels of serum IL-1ß, GM-CSF, TNF-α, and RANKL. These findings suggest O-GlcNAcylation as a potential therapeutic target and provide the rationale for testing PGG or structurally similar molecule for their therapeutic efficacy.

CD6 is a target for cancer immunotherapy.

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 the 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, and prostate cancer cells through direct effects on both CD8+ T cells and NK cells, increasing cancer cell death and lowering cancer cell survival in vitro more robustly than monoclonal antibody checkpoint inhibitors that interrupt the programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) axis. UMCD6 also augmented in vivo killing by human peripheral blood lymphocytes of a human breast cancer line xenotransplanted into immunodeficient mice. Mechanistically, UMCD6 upregulated the expression of the activating receptor NKG2D and downregulated 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 capability 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.

Real time visualization of cancer cell death, survival and proliferation using fluorochrome-transfected cells in an IncuCyte® imaging system.

Cancer immunotherapy is a rapidly advancing and viable approach to treating cancer along with more traditional forms of therapy. Real-time cell analysis technologies that examine the dynamic interactions between cancer cells and the cells of the immune system are becoming more important for assessment of novel therapeutics. In this report, we use the IncuCyte® imaging system to study the killing potential of various immune cells on cancer cell lines. The IncuCyte® system tracks living cells, labeled by a red fluorescent protein, and cell death, as indicated by the caspase-3/7 reagent, which generates a green fluorescent signal upon activation of apoptotic pathways. Despite the power of this approach, obtaining commercially fluorescent cancer cell lines is expensive and limited in the range of cell lines that are available. To overcome this barrier, we developed an inexpensive method using a lentiviral construct expressing nuclear localized mKate2 red fluorescent protein to stably label cancer cells. We demonstrate that this method is effective in labeling a wide variety of cell lines, allowing for analyses of different cancers as well as different cell lines of the same type of cancer.

Attenuation of Murine Collagen-Induced Arthritis by Targeting CD6.

OBJECTIVE: CD6 is an important regulator of T cell function that interacts with the ligands CD166 and CD318. To further clarify the significance of CD6 in rheumatoid arthritis (RA), we examined the effects of targeting CD6 in the mouse model of collagen-induced arthritis (CIA), using CD6-knockout (CD6-KO) mice and CD6-humanized mice that express human CD6 in lieu of mouse CD6 on their T cells. METHODS: We immunized wild-type (WT) and CD6 gene-KO mice with a collagen emulsion to induce CIA. For treatment studies using CD6-humanized mice, mice were immunized similarly and a mouse anti-human CD6 IgG (UMCD6) or control IgG was injected on days 7, 14, and 21. Joint tissues were evaluated for tissue damage, leukocyte infiltration, and local inflammatory cytokine production. Collagen-specific Th1, Th9, and Th17 responses and serum levels of collagen-specific IgG subclasses were also evaluated in WT and CD6-KO mice with CIA. RESULTS: The absence of CD6 reduced 1) collagen-specific Th9 and Th17, but not Th1 responses, 2) the levels of many proinflammatory joint cytokines, and 3) serum levels of collagen-reactive total IgG and IgG1, but not IgG2a and IgG3. Joint homogenate hemoglobin content was significantly reduced in CD6-KO mice with CIA compared to WT mice with CIA (P < 0.05) (reduced angiogenesis). Moreover, treating CD6-humanized mice with mouse anti-human CD6 monoclonal antibody was similarly effective in reducing joint inflammation in CIA. CONCLUSION: Taken together, these data suggest that interaction of CD6 with its ligands is important for the perpetuation of CIA and other inflammatory arthritides that are T cell driven.

Citrullinated Inhibitor of DNA Binding 1 Is a Novel Autoantigen in Rheumatoid Arthritis.

OBJECTIVE: To explore the intrinsic role of inhibitor of DNA binding 1 (ID-1) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and to investigate whether ID-1 is citrullinated and autoantigenic in RA. METHODS: RA patient serum ID-1 levels were measured before and after infliximab treatment. RA FLS were transfected with a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 construct targeting ID-1 to examine the effects of ID-1 deletion. RA synovial fluid (SF) and homogenized synovial tissue (ST) were immunoprecipitated for ID-1 and measured for citrullinated residues using an enzyme-linked immunosorbent assay and Western blotting. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on in vitro-citrullinated recombinant human ID-1 (cit-ID-1) to localize the sites of citrullination. Normal and RA sera and SF were analyzed by immunodot blotting for anti-citrullinated protein antibodies (ACPAs) to cit-ID-1. RESULTS: RA patient serum ID-1 levels positively correlated with several disease parameters and were reduced after infliximab treatment. RA FLS displayed reduced growth and a robust increase in interleukin-6 (IL-6) and IL-8 production upon deletion of ID-1. ID-1 immunodepletion significantly reduced the levels of citrullinated residues in RA SF, and citrullinated ID-1 was detected in homogenized RA ST (n = 5 samples; P < 0.05). Immunodot blot analyses revealed ACPAs to cit-ID-1 but not to native ID-1, in RA peripheral blood (PB) sera (n = 30 samples; P < 0.001) and SF (n = 18 samples; P < 0.05) but not in normal PB sera. Following analyses of LC-MS/MS results for citrullination sites and corresponding reactivity in immunodot assays, we determined the critical arginines in ID-1 for autoantigenicity: R33, R52, and R121. CONCLUSION: Novel roles of ID-1 in RA include regulation of FLS proliferation and cytokine secretion as well as autoantigenicity following citrullination.

CD318 is a ligand for CD6.

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.