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Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our in vitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer. [ABSTRACT FROM AUTHOR]

Earlier, we have shown that GM-CSF-exposed CD8a2 DCs that express low levels of pro-inflammatory cytokines IL-12 and IL-1&bgr; can induce Foxp3+ Tregs leading to suppression of autoimmunity. Here, we examined the differential effects of IL-12 and IL-1&bgr; on Foxp3 expression in T cells when activated in the presence and absence of DCs. Exogenous IL-12 abolished, but IL-1&bgr; enhanced, the ability of GM-CSF-exposed tolerogenic DCs to promote Foxp3 expression. Pre-exposure of DCs to IL-1&bgr; and IL-12 had only a modest effect on Foxp32 expressing T cells; however, T cells activated in the absence of DCs but in the presence of IL-1&bgr; or IL-12 showed highly significant increase and decrease in Foxp3+ T cell frequencies respectively suggesting direct effects of these cytokines on T cells and a role for IL-1&bgr; in promoting Foxp3 expression. Importantly, purified CD4+CD25+ cells showed a significantly higher ability to maintain Foxp3 expression when activated in the presence of IL-1&bgr;. Further analyses showed that the ability of IL-1&bgr; to maintain Foxp3 expression in CD25+ T cells was dependent on TGF-b1 and IL-2 expression in Foxp3+Tregs and CD252 effectors T cells respectively. Exposure of CD4+CD25+ T cells to IL- 1&bgr; enhanced their ability to suppress effector T cell response in vitro and ongoing experimental autoimmune thyroidits in vivo. These results show that IL-1&bgr; can help enhance/maintain Tregs, which may play an important role in maintaining peripheral tolerance during inflammation to prevent and/or suppress autoimmunity. [ABSTRACT FROM AUTHOR]

TNF receptor superfamily comprises many T-cell costimulatory receptors, including TNFRSF1, TNFRSF2, TNFRSF4 (OX40), TNFRSF9 (4-1BB), TNFRSF18 (GITR), and TNFRSF7 (CD27). Signaling through these costimulatory stimulatory receptors can promote conventional T-cell (Tconv) proliferation, and effector functions in an antigen-dependent manner. Thus, agonistic antibodies and ligands for OX40, 4-1BB, GITR, and CD27 have been tested for inducing T-cell-mediated antitumor responses in several cancers. However, recently emerging reports show critical role for TNFR signaling in regulatory T-cell (Treg) differentiation and expansion, which might suppress effector T-cell proliferation and functions. Here, we show preferential over expression of TNFR2, OX40, 4-1BB, and GITR in Treg cells over Tconv cells, and the ability of OX40L and GITRL to induce selective proliferation of Treg cells, but not Tconv cells, in an antigen-independent manner. We describe the standard protocols used for Affymetrix gene expression profiling, T-cell isolation, and Cell Trace Violet-based cell proliferation assay.