T cells are key players in the adaptive immune system that eliminates pathogens from the body [1]. The T cell receptor (TCR) first recognizes antigenic peptides presented by major histocompatibility complex (MHC) components, followed by the transduction of various signaling pathways to initiate immune responses such as cell proliferation, cytokine production, differentiation, and selection [1]. As a result of various immune responses, invading pathogens are removed. Adaptor proteins are critical regulators of immune responses [2–4]. They interact with signaling molecules and positively or negatively modulate signal transduction [2–4]. Signal-transducing adaptor protein-2 (STAP-2) is a novel adaptor protein that has been isolated as a colony-stimulating factor-1 receptor (c-fms)-binding protein [5]. Previous studies showed that STAP-2 modulates various immune responses [6] such as the signal transducer and activator of transcription 3 (STAT3)/STAT5-related signaling [7–10], FS-7-associated surface antigen (Fas)-mediated apoptosis in T cells [11], high-affinity immunoglobulin E (IgE) receptor [Fc epsilon receptor I (FcεRI)]-mediated signaling in mast cell [12, 13], toll-like receptor (TLR)-mediated signaling in macrophages [14], integrin-mediated adhesion of T cells [15], stromal cell-derived factor-1α (SDF-1α)-mediated T cell migration [16], and TCR-mediated T cell activation (Figure 1) [17]. STAP-2 is a positive regulator of T cells via its interaction with CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) and lymphocyte-specific protein tyrosine kinase (LCK) (Figure 2) [17]. Experimental autoimmune encephalomyelitis (EAE), which is widely known as a mouse model of multiple sclerosis, revealed that STAP-2 knockout (KO) mice had significantly lower clinical scores than wild-type mice and a higher clinical score in STAP-2 transgenic (Tg) mice [17]. Therefore, disrupting the interaction between STAP-2 and CD3ζ ITAMs is likely to help regulate TCR-mediated signaling and may be a new therapeutic target of T cell-mediated immune diseases. This perspective summarizes the function of an inhibitory peptide [STAP-2 inhibitor (iSP2)] which targets STAP-2/CD3ζ ITAM binding in vivo and in vitro.

Because the target is STAP-2/CD3ζ ITAM binding, which is a typical intracellular protein-protein interaction (PPI), it is very difficult to inhibit this PPI using small molecules or antibodies [18–20]. Recently, the discovery of peptide drugs has become remarkable. Peptides are now promising tools for targeting PPI as recent research has shown that they can be structured to have high stability, better binding affinity, and membrane permeability [21]. Therefore, peptides are suitable tools to inhibit PPI. Prior to constructing an iSP2, the important sequence for STAP-2/CD3ζ ITAM binding has been identified. A series of deletion mutants of STAP-2 were generated, and they were used to pull down the assay with Sepharose beads conjugated with the ITAM peptide. After the critical part and sequence for the interactions between STAP-2 and CD3ζ ITAMs were determined, the possible interfering peptide by adding cell-penetrating octa-arginine sequence to the STAP-2-CD3ζ ITAM binding site was constructed. This was further optimized by shortening the sequence without losing the capacity to inhibit T cell function [22]. The optimized and control peptides are shown as “iSP2” and “iCont”, respectively, in this perspective. iSP2 contains five AA [STAP-2-based 5 AA sequence (WPVIL)] corresponding to the C-terminal proline-lich region of STAP-2 [22].

Since STAP-2 is important for TCR-mediated T cell activation, including TCR-mediated signaling, cell proliferation, and IL-2 production [17], the inhibition of STAP-2 functions by iSP2 was investigated. In the human leukemia-derived cell line Jurkat and human peripheral blood mononuclear cells, iSP2 shows an inhibitory effect on TCR-mediated signaling, cell proliferation, and IL-2 production [22]. iSP2 effects on murine T lymphoma EL-4 cells and murine CD4⁺ T cells were tested. Consistent with its suppressive effect on human-derived cells, iSP2 has the capacity to attenuate TCR-mediated signaling, cell proliferation, and IL-2 production [22]. Collectively, these results indicate that iSP2 functions as an iSP2 for T cells by blocking the interaction between STAP-2 and CD3ζ ITAMs. In drug discovery research, the off-target responses or cell-specific functions of a drug are important in terms of cytotoxicity or adverse events [23]. Regarding its possible effects on other signaling pathways in T cells, iSP2 did not affect cell proliferation upon stimulation with phorbol myristate acetate (PMA)/ionomycin, indicating that it has a TCR signaling-specific function [22]. When the function of iSP2 was analyzed using non-T cells, iSP2 failed to affect spontaneous proliferation in the human prostate cancer cell line DU145 and murine melanoma cell line B16F10, as well as FcεRI-mediated IL-6 production in bone marrow-derived basophils [22]. Thus, iSP2 is likely to exert T cell-specific suppressive functions.

As iSP2 negatively regulates not only human but also murine T cells [22], the therapeutic potential of iSP2 in T cell-mediated immune diseases was analyzed. In the previous study using an EAE model, STAP-2 KO mice exhibited significantly lower clinical scores while STAP-2 Tg mice exhibited higher scores than wild-type mice [17]. During EAE development, STAP-2 messenger RNA (mRNA) expression is enhanced and STAP-2 in CD4⁺ T cells contributes to the activation and expansion of autoreactive myelin oligodendrocyte glycoprotein (MOG)-specific T cells [17]. Thus, STAP-2 is likely involved in worsening the severity of EAE. EAE is widely recognized as a mouse model of multiple sclerosis and is induced by immunization with MOG peptide emulsified with complete Freund’s adjuvant, followed by an intravenous injection of pertussis toxin. Approximately 12–14 days after immunization, autoreactive immune cells infiltrate the central nervous system (CNS) and induce demyelination; thus, multiple sclerosis-like symptoms are detected [24–27]. Using this model, the role of iSP2 in EAE development was examined. After treatment with iSP2 following MOG immunization, the peak disease severity was significantly lower than that in iCont-treated mice [22]. Histologically, hematoxylin and eosin staining and Luxol fast blue staining revealed that immune cell infiltration and spinal cord demyelination during EAE development were significantly lower in iSP2-treated mice than in iCont-treated mice [22]. To test whether iSP2 modulates the T cell response, CNS-infiltrating T cells was analyzed. T helper (Th) 1 and Th17 cells are critical for the pathogenesis of EAE. They are first activated in peripheral tissues, after which they migrate across the blood-brain barrier to enter the CNS. Infiltrated Th1 and Th17 produce their specific cytokines, namely interferon (IFN)-γ and IL-17, respectively [28–30]. These cytokines not only directly damage myelin and cause neuroinflammation, but also induce the migration and activation of glial cells, followed by additional neuroinflammation and axonal damage. Thus, Th1 and Th17 cells play direct and indirect critical roles in pathogenesis. Therefore, the CNS-infiltrating Th1 and Th17 in iSP2- and iCont-treated mice were analyzed. In iSP2-treated mice, the absolute number and proportion of both Th1 and Th17 cells were significantly attenuated compared to iCont-treated mice [22], indicating that iSP2 suppressed the activation of autoreactive T cells, contributing to lower disease severity. To investigate the mechanism by which iSP2 regulates EAE pathogenesis, single-cell RNA-sequencing of CNS-infiltrating cells from iSP2- and iCont-treated mice was performed. Based on the expression of cell markers, pathogenic T cell-related gene expression decreased in iSP2-treated mice [22]. Flow cytometric analysis also confirmed that iSP2 treatment suppressed the migration of immune cells to the CNS [22]. Collectively, these results indicate that iSP2 suppresses the pathogenesis of EAE by reducing Th1 and Th17 cell activation and immune cell infiltration (Figure 3).