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Edited by Luke O'Neill SEC2, a major histocompatibility complex class II (MHC II)dependent T-cell mitogen, binds MHC II and T-cell receptor (TCR) V␤s to induce effective co-stimulating signals for clonal T-cell expansion. We previously characterized a SEC2 mutant with increased recognition of TCR V␤s, ST-4, which could intensify NF-B signaling transduction, leading to IL-2 production and T-cell activation. In this study, we found that in contrast to SEC2, ST-4 could induce murine CD4 ؉ T-cell proliferation in a V␤8.2and V␤8.3-specific manner in the absence of MHC II ؉ antigenpresenting cells (APCs). Furthermore, although IL-2 secretion in response to either SEC2 or ST-4 stimulation was accompanied by up-regulation of protein kinase C (PKC), inhibitor of B (IB), ␣ and ␤ IB kinase (IKK␣/␤), IB␣, and NF-B in mouse splenocytes, only ST-4 could activate CD4 ؉ T cells in the absence of MHC II ؉ APCs through the PKC/NF-B signaling pathway. The PKC inhibitor AEB071 significantly suppressed SEC2/ST-4-induced T-cell proliferation, CD69 and CD25 expression, and IL-2 secretion with or without MHC II ؉ APCs. Further, SEC2/ST-4-induced changes in PKC/NF-B signaling were significantly relieved by AEB071 in a dose-dependent manner. Using Lck siRNA, we found that Lck controlled SEC2/ST-4-induced phosphorylation of PKC. We also demonstrated that the IL-2R/STAT5 pathway is essential for SEC2/ST-4-induced T-cell activation. Collectively, our data demonstrate that an enhanced ST-4-TCR interaction can compensate for lack of MHC II and stimulate MHC II-free CD4 ؉ T-cell proliferation via PKC/NF-B and IL-2R/STAT5 signaling pathways. Compared with SEC2, intensified PKC/NF-B and IL-2R/STAT5 signals induced by ST-4 lead to enhanced T-cell activation. The results of this study will facilitate better understanding of TCR-based immunotherapies for cancer.

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CD8 + T cells can switch between fatty acid catabolism and mitochondrial energy metabolism to sustain expansion and their cytotoxic functions. ST-4 is a TCR-enhanced mutant derived from superantigen staphylococcal enterotoxin C2 (SEC2), which can hyperactivate CD4 + T cells without MHC class II molecules. However, whether ST-4/SEC2 can enhance metabolic reprogramming in CD8 + T cells remains poorly understood. In this study, we found that ST-4, but not SEC2, could induce proliferation of purified CD8 + T cell from BALB/c mice in Vb8.2-and -8.3-specific manners. Results of gas chromatography-mass spectroscopy analysis showed that fatty acid contents in CD8 + T cells were increased after ST-4 stimulation. Flow cytometry and Seahorse analyses showed that ST-4 significantly promoted mitochondrial energy metabolism in CD8 + T cells. We also observed significantly upregulated levels of gene transcripts for fatty acid uptake and synthesis, and significantly increased protein expression levels of fatty acid and mitochondrial metabolic markers of mTOR/PPARg/SREBP1 and p38-MAPK signaling pathways in ST-4activated CD8 + T cells. However, blocking mTOR, PPARg, SREBP1, or p38-MAPK signals with specific inhibitors could significantly relieve the enhanced fatty acid catabolism and mitochondrial capacity induced by ST-4. In addition, blocking these signals inhibited ST-4-stimulated CD8 + T cell proliferation and effector functions. Taken together, our findings demonstrate that ST-4 enhanced fatty acid and mitochondria metabolic reprogramming through mTOR/PPARg/SREBP and p38-MAPK signaling pathways, which may be important regulatory mechanisms of CD8 + T cell activation. Understanding the effects of ST-4-induced regulatory metabolic networks on CD8 + T cells provide important mechanistic insights to superantigen-based tumor therapy.

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As a biological macromolecule, the superantigen staphylococcal enterotoxin C2 (SEC2) is one of the most potent known T-cell activators, and it induces massive cytotoxic granule production. With this property, SEC2 and its mutants are widely regarded as immunomodulating agents for cancer therapy. In a previous study, we constructed an MHC-II-independent mutant of SEC2, named ST-4, which exhibits enhanced immunocyte stimulation and antitumor activity. However, tumor cells have different degrees of sensitivity to SEC2/ST-4. The mechanisms of immune resistance to SEs in cancer cells have not been investigated. Herein, we show that ST-4 could activate more powerful human lymphocyte granule-based cytotoxicity than SEC2. The results of RNA-seq and atomic force microscopy (AFM) analysis showed that, compared with SKOV3 cells, the softer ES-2 cells could escape from SEC2/ST-4-induced cytotoxic T-cell-mediated apoptosis by regulating cell softness through the CDC42/MLC2 pathway. Conversely, after enhancing the stiffness of cancer cells by a nonmuscle myosin-Ⅱ-specific inhibitor, SEC2/ST-4 exhibited a significant antitumor effect against ES-2 cells by promoting perforin-dependent apoptosis and the S-phase arrest. Taken together, these data suggest that cell stiffness could be a key factor of resistance to SEs in ovarian cancer, and our findings may provide new insight for SE-based tumor immunotherapy.