Anti-CTLA-4 treatment suppresses hepatocellular carcinoma growth through Th1-mediated cell cycle arrest and apoptosis.

Inhibiting the cytotoxic T-lymphocyte-associated protein-4 (CTLA-4)-mediated immune checkpoint system using an anti-CTLA-4 antibody (Ab) can suppress the growth of various cancers, but the detailed mechanisms are unclear. In this study, we established a monoclonal hepatocellular carcinoma cell line (Hepa1-6 #12) and analyzed the mechanisms associated with anti-CTLA-4 Ab treatment. Depletion of CD4+ T cells, but not CD8+ T cells, prevented anti-CTLA-4 Ab-mediated anti-tumor effects, suggesting dependence on CD4+ T cells. Anti-CTLA-4 Ab treatment resulted in recruitment of interferon-gamma (IFN-g)-producing CD4+ T cells, called T-helper 1 (Th1), into tumors, and neutralization of IFN-g abrogated the anti-tumor effects. Moreover, tumor growth suppression did not require major histocompatibility complex (MHC)-I or MHC-II expression on cancer cells. In vitro studies showed that IFN-g can induce cell cycle arrest and apoptosis in tumor cells. Taken together, these data demonstrate that anti-CTLA-4 Ab can exert its anti-tumor effects through Th1-mediated cell cycle arrest and apoptosis.

S-531011, a Novel Anti-Human CCR8 Antibody, Induces Potent Antitumor Responses through Depletion of Tumor-Infiltrating CCR8-Expressing Regulatory T Cells.

Although regulatory T cells (Treg) are inhibitory immune cells that are essential for maintaining immune homeostasis, Tregs that infiltrate tumor tissue promote tumor growth by suppressing antitumor immunity. Selective reduction of tumor-infiltrating Tregs is, therefore, expected to activate antitumor immunity without affecting immune homeostasis. We previously reported that selective Treg depletion targeted by a C-C motif chemokine receptor 8 (CCR8) resulted in induction of strong antitumor immunity without any obvious autoimmunity in mouse models. Thus, herein, we developed a novel humanized anti-CCR8 monoclonal antibody, S-531011, aimed as a cancer immunotherapy strategy for patients with cancer. S-531011 exclusively recognized human CCR8 among all chemokine receptors and showed potent antibody-dependent cell-mediated cytotoxicity activity toward CCR8+ cells and neutralization activity against CCR8-mediated signaling. We observed that S-531011 reduced tumor-infiltrating CCR8+ Tregs and induced potent antitumor activity in a tumor-bearing human-CCR8 knock-in mouse model. Moreover, combination therapy with S-531011 and anti-mouse programmed cell death 1 (PD-1) antibody strongly suppressed tumor growth compared with anti-PD-1 antibody alone with no observable adverse effects. S-531011 also depleted human tumor-infiltrating Tregs, but not Tregs derived from human peripheral blood mononuclear cells. These results suggest that S-531011 is a promising drug for inducing antitumor immunity without severe side effects in the clinical setting.

Sulfated glycosaminoglycans and non-classically secreted proteins, basic FGF and epimorphin, coordinately regulate TGF-ß-induced cell behaviors of human scar dermal fibroblasts.

BACKGROUND: Upon skin injuries, dermal fibroblasts actively produce transforming growth factor-ß (TGF-ß), which leads to the formation of α-smooth muscle actin (αSMA)-positive granulation tissues. The hyperplasia or incomplete regression of these tissues subsequently causes scar formation in the skin, where sulfated glycosaminoglycans (GAGs), side chains of unique proteoglycans, are supposed to play important roles. OBJECTIVE: The aim of this study is to clarify the effects of sulfated GAGs on dermal cell behaviors triggered by the TGF-ß signaling, along with its possible regulators basic fibroblast growth factor (bFGF) and cell surface epimorphin. bFGF and epimorphin might regulate the TGF-ß-induced αSMA expression, they could exert such effects only in specific cellular contexts, given that they lack conventional signal sequences for extracellular localization. METHODS: Human scar-derived dermal fibroblasts (HSFs) were treated with TGF-ß alone, TGF-ß plus bFGF, and TGF-ß plus cell surface expression of epimorphin. The effects of GAGs on the expression of αSMA and the cellular morphology were then investigated. RESULTS: A highly sulfated chondroitin sulfate (CS-E) or its substitute (heparinoid) had marked inhibitory effects on TGF-ß-mediated changes in HSF behaviors. We found that heparinoid can directly associate with TGF-ß, bFGF and epimorphin. We also found that bFGF downregulated αSMA, which was attenuated by heparinoid, whereas epimorphin augmented αSMA expression, which was further amplified by heparinoid. CONCLUSIONS: TGF-ß, bFGF and epimorphin in the extracellular microenvironment cooperatively affect HSF behaviors under the control of a highly sulfated chondroitin sulfate. These results provide important evidence towards understanding the regulation of TGF-ß-induced HSF behaviors.

Membrane translocation of t-SNARE protein syntaxin-4 abrogates ground-state pluripotency in mouse embryonic stem cells.

Embryonic stem (ES) and induced pluripotent stem (iPS) cells are attractive tools for regenerative medicine therapies. However, aberrant cell populations that display flattened morphology and lose ground-state pluripotency often appear spontaneously, unless glycogen synthase kinase 3ß (GSK3ß) and mitogen-activated protein kinase kinase (MEK1/2) are inactivated. Here, we show that membrane translocation of the t-SNARE protein syntaxin-4 possibly is involved in this phenomenon. We found that mouse ES cells cultured without GSK3ß/MEK1/2 inhibitors (2i) spontaneously extrude syntaxin-4 at the cell surface and that artificial expression of cell surface syntaxin-4 induces appreciable morphological changes and mesodermal differentiation through dephosphorylation of Akt. Transcriptome analyses revealed several candidate elements responsible for this, specifically, an E-to P-cadherin switch and a marked downregulation of Zscan4 proteins, which are DNA-binding proteins essential for ES cell pluripotency. Embryonic carcinoma cell lines F9 and P19CL6, which maintain undifferentiated states independently of Zscan4 proteins, exhibited similar cellular behaviors upon stimulation with cell surface syntaxin-4. The functional ablation of E-cadherin and overexpression of P-cadherin reproduced syntaxin-4-induced cell morphology, demonstrating that the E- to P-cadherin switch executes morphological signals from cell surface syntaxin-4. Thus, spontaneous membrane translocation of syntaxin-4 emerged as a critical element for maintenance of the stem-cell niche.