The FSS treatment of T cells in combination with soluble and bead-bound CD3/CD28 antibodies increased the activation of signaling proteins essential for T cell activation, such as zeta-chain-associated protein kinase-70 (ZAP70), nuclear factor of activated T cells (NFAT), nuclear factor kappa B (NF-κB), and AP-1 (activator protein 1). The FSS treatment also enhanced the expression of the cytokines tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), and interferon gamma (IFN-γ), which are necessary for sustained T cell activation and function. The enhanced activation of T cells by FSS was calcium dependent. The calcium signaling was controlled by the mechanosensitive ion channel Piezo1, as GsMTx-4 and Piezo1 knockout reduced ZAP70 phosphorylation by FSS.
For proper T cell activation, the transcription factors NFAT, NF-κB, and AP-1 need to be activated for the transcription of important proteins and cytokines, such as tumor necrotic factor alpha (TNF-α), interleukin 2 (IL-2), and interferon gamma (IFN-γ) [24]. The activation of each transcription factor was measured after 1 h of stimulation with FSS and CD3/CD28 antibodies. NFAT activation of Jurkat cells was measured by quantifying the colocalization of NFAT with the nucleus using confocal microscopy, since only the active conformation of NFAT reveals a nuclear localization signal [25]. The colocalization was measured via antibody staining for NFAT and DAPI staining of the nucleus. When the Jurkat cells were treated with CD3/CD28 antibodies and FSS, a significant increase in NFAT-nucleus colocalization was observed relative to untreated, or antibody-only treated Jurkat cells (Fig. 2A). NF-κB activation was quantified by measuring the phosphorylation of NF-κB using flow cytometry, as NF-κB phosphorylation is indicative of its activation [26]. AP-1 activation was quantified by measuring the phosphorylation of cFOS, since cFOS must be phosphorylated to form the AP-1 complex [27]. FSS in combination with CD3/CD28 antibodies significantly increased the activation of NF-κB and AP-1 compared to untreated and antibody-only treated Jurkat cells (Fig. 2B, C).
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