STAP-1-derived peptide suppresses TCR-mediated T cell activation and ameliorates immune diseases by inhibiting STAP-1–LCK binding

Abstract

Signal-transducing adaptor protein-1 (STAP-1) is an adaptor protein specifically expressed in immune cells, such as T cells. We previously demonstrated that STAP-1 positively upregulates T cell receptor (TCR)-mediated T cell activation by interacting with LCK and phospholipase C-γ1 and affecting autoimmune demyelination and airway inflammation. In this study, we aimed to generate a new STAP-1-derived peptide, iSP1, to inhibit the STAP-1–LCK interaction. We also analyzed its function in vitro and in vivo. iSP1 successfully interfered with STAP-1–LCK binding and suppressed TCR-mediated signal transduction, interleukin-2 production, and human and murine T cell proliferation. Additionally, iSP1 prevented the progression of experimental autoimmune encephalomyelitis by inhibiting Th1 and Th17 cell infiltration. Our findings suggest iSP1 as a new therapeutic immunomodulatory agent for T cell-mediated autoimmune diseases.

Introduction

T cells are key drivers of the adaptive immune system. By recognizing the antigen-derived peptides presented by major histocompatibility complexes (MHCs) on antigen-presenting cells, T cells initiate immune responses, such as cell proliferation, cell differentiation, and soluble factor production, to maintain homeostasis¹ Upon T cell receptor (TCR) engagement, immunoreceptor tyrosine-based activation motifs (ITAMs) in CD3ζ are phosphorylated by LCK, followed by the recruitment of zeta-associated protein 70 (ZAP-70) to the TCR/CD3 complex and phosphorylation by LCK. Activated ZAP-70 promotes LAT recruitment and phosphorylates SLP-76, subsequently recruiting Vav, NCK, GAD5, and ITK. ITK further phosphorylates phospholipase C (PLC)-γ1, finally leading to the induction of interleukin (IL)-2 expression via the transcription factor, nuclear factor of activated T cells (NFAT).^2–6 In this manner, T cells are activated to initiate immune responses.

Signal-transducing adaptor protein-1 (STAP-1) is an adaptor protein that is a key regulator of immune cell signaling. STAP-1 was identified as a B cell receptor (BCR) downstream signaling molecule that regulates BCR-mediated signaling by interacting with TEC; in addition, it is only expressed in secondary lymphoid tissues, such as the spleen and lymph nodes.⁷ STAP-1 mutation is observed in autosomal dominant hypercholesterolemia.^8,9 STAP-1 contains a pleckstrin homology (PH) domain in its N-terminal region and an Src homology 2 (SH2) domain in its C-terminal region. STAP-1 SH2 domain binds to the non-T cell activation linker (NTAL), c-KIT, and c-FMS.^7,10 We recently reported that STAP-1 binds to LCK, ITK, and phospholipase C (PLC)-γ1 as scaffolds to promote TCR-mediated signaling. STAP-1 deficiency impairs T cell activation, including cell proliferation, cytokine production, and TCR-mediated signaling. Moreover, STAP-1 deficiency ameliorates experimental autoimmune encephalomyelitis (EAE) and antigen-induced airway inflammation.¹¹ We previously demonstrated that STAP-1 is critical for TCR-mediated immune responses in vivo and in vitro.¹¹ Therefore, STAP-1 inhibition is a potential therapeutic strategy for autoimmune diseases. However, STAP-1 is an adaptor protein and forming protein-protein interactions (PPIs), which are difficult to inhibit by small molecule. In this study, we employed peptide as a tool for inhibiting PPIs due to its capability to cell penetration and target PPIs. Based on the strategy, we generated an inhibitory peptide to interfere with STAP-1–LCK binding and investigated its effects on T cell function. The optimized peptide successfully inhibited TCR-mediated activation of human and murine T cells and ameliorated EAE in STAP-1 knockout (KO) mice. Our data suggest the potential of targeting the STAP-1–LCK interaction as a new strategy for autoimmune disease treatment.

Materials and methods

Mice and cells

C57BL/6 mice were purchased from Sankyo Labo service (Hokkaido, Japan). All animal studies were approved by Hokkaido university Animal Ethics Committee. All mice were housed and bred in the Pharmaceutical Sciences Animal Center of Hokkaido University under specific pathogen-free conditions. A human T lymphoma cell line, Jurkat, and a murine T lymphoma cell line, EL-4, were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 50 μM 2-Mercaptoethanol. Human PBMCs were purchased from Lonza (Tokyo, Japan). Murine CD4⁺ T cells were purified from spleen using EasySep mouse CD4⁺ T Cell Isolation Kit (STEMCELL Technologies Inc., Vancouver, Canada). Human PBMCs and murine CD4⁺ T cells were cultured in the same medium as in Jurkat and EL-4 cells.

Peptides

All peptides were synthesized by GL Biochem (Shanghai, China). The peptide sequences used in this study are shown in Table 1.

Table 1.

List of the STAP-1 peptides

Peptide	Sequence
SP1-12/31	RRRRRRRRGGRRIFQERLKITALPLYFEGF
SP1-26/43	RRRRRRRRGGPLYFEGFLLIKRSGYREYE
SP1-41/59	RRRRRRRRGGEYEHYWTELRGTTLFFYTD
SP1-58/77	RRRRRRRRGGTDKKSIIYVDKLDIVDLTCL
SP1-73/92	RRRRRRRRGGDLTCLTEQNSTEKNCAKFTL
SP1-88/106	RRRRRRRRGGAKFTLVLPKEEVQLKTENT
SP1-44/48	RRRRRRRRGGHYWTE
SP1-47/54	RRRRRRRRGGTELRGTTL
SP1-52/59	RRRRRRRRGGTTLFFYTD
SP1-52/56	RRRRRRRRGGTTLFF
SP1-55/59	RRRRRRRRGGFFYTD
SP1-52/56-1A	RRRRRRRRGGATLFF
SP1-52/56-2A	RRRRRRRRGGTALFF
SP1-52/56-3A	RRRRRRRRGGTTAFF
SP1-52/56-4A	RRRRRRRRGGTTLAF
SP1-52/56-5A	RRRRRRRRGGTTLFA

Antibodies

Anti-human CD3 (clone OKT3) and anti-human CD28 (clone CD28.8) mAbs were purchased from BioLegend (San Diego, California, USA). Anti-mouse CD3 (clone 145-2C11) and anti-mouse CD28 (clone PV-1) mAbs were purchased from American Type Culture Collection (Manassas, Virginia, USA) and Bio X Cell (West Lebanon, New Hampshire, USA), respectively. Anti-Myc was purchased from Sigma-Aldrich (StLouis, Missouri, USA). Anti-phospho-PLC-γ1 (Tyr783), anti-phospho-ZAP70 (Tyr319), Anti-phospho-ERK (Thr202/Tyr204) and anti-ZAP70 Abs were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Anti-phospho-LCK (Tyr394) Ab was purchased from Invitrogen (Waltham, Massachusetts, USA). Anti-ERK, anti-β-actin and anti-PLC-γ1 were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Anti-LCK was purchased from R&D Systems (Minneapolis, Minnesota, USA). FITC anti-mouse CD3ε (clone 17A2), PE/Cyanine7 anti-mouse-CD4 (clone GK1.5), PerCP/Cyanine5.5 anti-mouse CD45 (clone 30-F11), APC anti-mouse IFN-γ (clone XMG1.2) mAbs and Zombie Red were purchased from BioLegend (San Diego, California, USA). PE anti-mouse IL-17 (clone ebio17B7) was purchased from eBioscience (San Diego, CA).

ELISA

Jurkat cells, EL-4 cells and murine CD4⁺ T cells were stimulated with immobilized anti-CD3 (3 μg/ml) and anti-CD28 (1 μg/ml) for indicated periods. Jurkat cells were also stimulated with PMA (50 ng/ml) and Ionomycin (1 μg/ml). Human and mouse IL-2 levels in culture supernatants were measured using ELISA kits (BioLegend, San Diego, California, USA).

Proliferation assay

Jurkat cells and EL-4 cells were cultured with or without inhibitory peptides in RPMI 1640 medium supplemented with 10% FBS and 50 μM 2-mercaptoethanol, and proliferation was analyzed using WST-8 reagent (Dojindo Laboratories, Kumamoto, Japan) as described previously¹². CD4⁺ T cells were purified using EasySep mouse CD4⁺ T cell isolation kit (STEMCELL Technologies Inc., Vancouver, Canada) and stimulated with immobilized anti-CD3 (10 μg/ml) and anti-CD28 (3 μg/ml) mAbs in the presence or absence of inhibitory peptides in RPMI 1640 medium supplemented with 10% FBS and 50 μM 2-mercaptoetanol. After 96 h stimulation, T cell proliferation was analyzed using the WST-8 reagent. Human PBMCs were stimulated with immobilized anti-CD3 (10 μg/ml) and anti-CD28 (3 μg/ml) mAbs for 48 h, and cell proliferation was monitored by a CellTiter-Glo 2.0 cell viability assay (Promega, Madison, Wisconsin, USA).

Immunoblotting and immunoprecipitation

After stimulation with anti-CD3 and anti-CD28, the cell lysates were prepared using lysis buffer (50 mM Tris-HCl (pH7.4), 0.15 M NaCl, 1% NP-40, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM NaF). The samples were boiled with SDS sample buffer, and the lysates were analyzed by SDS-PAGE and transferred to PVDF membranes. The membranes were first blocked with 5% skim milk TBS-T or 2% BSA TBS-T and incubated with primary Abs. Proteins reactive with the primary Abs were visualized with HRP-conjugated secondary Abs (GE Healthcare Bio-Sciences, Uppsala, Sweden) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, Bedford, Massachusetts, USA). Densitometrical analysis was performed using ImageJ program (NIH, Bethesda, Maryland, USA). For Immunoprecipitation, Myc-tagged hSTAP-1 expressing Jurkat cells were treated with inhibitors for 30 min. Subsequently, cell lysates were incubated with anti-Myc antibody, followed by incubation with nProtein A Sepharose 4 Fast Flow (GE Healthcare Bio-Sciences). The beads were boiled in 1×SDS sample buffer, and coimmunoprecipitated proteins were detected by Western blot analysis. Densitometric analysis was performed using ImageJ program (National Institutes of Health, Bethesda, Maryland, USA).

Experimental autoimmune encephalomyelitis

Male and female mice were s.c. immunized with 100 μg MOG (Sigma-Aldrich) emulsified in CFA (BD, Sparks, Maryland, USA) plus 500 μg Mycobacterium tuberculosis H37Ra (BD). At the times of immunization and 2 d later, mice were i.v. injected with 400 ng Pertussis toxin (LIST Biological Laboratories, Inc). For therapeutic treatment, from 8 d after the first MOG treatment, mice were i.v. injected with inhibitors (40 µg/mouse) every other day for 13 d. Disease severity of EAE was monitored using the criteria as previously described.¹³ Formalin-fixed paraffin-embedded sections of spinal cords were stained by H&E or Luxol fast blue staining procedure for analysis of inflammation or demyelination, respectively.

Purification of mononuclear cells from CNS

For analysis of Th1 and Th17 cells in the CNS, the brain and spinal cord were first homogenized, the cells were resuspended in 30% Percoll, and the cell suspension was overlayed onto 70% Percoll. After centrifugation, interface between 30% and 70% Percoll were collected and used as mononuclear cells for detection of Th1 and Th17 in CNS of EAE mice.

Intracellular staining

The mononuclear cells in CNS were firstly stimulated with PMA (50 ng/mL) plus Ionomycin (1 μg/ml) in the presence of brefeldin A (5 μg/ml) for 5 h, and the cells were stained with Zombie Red, followed by anti-CD3, CD4 and CD45. After that, the cells were treated with IC Fixation buffer (eBiosciences, San Diego, California, USA) for 30 min, and stained with anti-IFN-γ and anti-IL-17 in Permeabilization buffer (eBiosciences, San Diego, California, USA). Th1 and Th17 cells were detected by flowcytometry. For analysis, samples were gated on live cells (Zombie-Red negative cells), CD45⁺, CD3⁺ and CD4⁺ in this order. Then, frequency and absolute number of IFN-γ or IL-4 positive cells were measured.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6.02. A Mann-Whitney U test, Sidak multiple comparisons test and Dunnett’s multiple comparisons test were employed. Outliers were identified by the ROUT method in the GraphPad Prism. Data were considered significant at P < 0.05. Data were shown as mean ± SEM.

Results

Phenotypical screening of STAP-1 peptides suppressing T cell function

We previously demonstrated that the PH domain, but not the SH2 domain, of STAP-1 interacts with LCK ¹¹. Here, we generated a series of STAP-1 PH domain-derived peptides to narrow down the sequence needed to suppress T cell function (Fig. 1A). After treatment of the human leukemia T cell line, Jurkat, with peptides, spontaneous proliferation was analyzed using the WST-8 system. As shown in Fig. 1B, N41-59 peptide suppressed cell proliferation in a concentration-dependent manner. However, N12-31, N26-43, N58-77 have high cytotoxicity from the concentration of 3 μM. Also, N73-92 has high cytotoxicity at the concentration of 100 μM. On the other hand, N41-59 peptide did not show significant cytotoxicity up to the concentration of 100 μM. (Fig. S1). Moreover, N88-106 did not affect proliferation at concentrations up to 100 μM. Therefore, only the N41-59 peptide phenotypically suppressed cell proliferation without cytotoxicity. We further narrowed down the N41-59 sequence by splitting it into three peptides (N44-48, N47-54, and N52-59) and analyzed their effects on cell proliferation. As shown in Fig. 1C, only N52-59 peptide significantly suppressed cell proliferation. To determine whether N52-59 can be further optimized, we split N52-59 peptide into 2 peptides (N52-56 and N55-59). As shown in Fig. 1D, N52-56 successfully suppressed cell proliferation. Hereafter, we focused on the N52-56 peptide, termed as iSP1, as a candidate STAP-1-derived inhibitory peptide. Next, to determine the sequence contributing to the suppression of T cell function, we generated point mutants of the N52-56 peptide. SP1-52/56-3A, SP1-52/56-4A, and SP1-52/56-5A did not suppress growth (Fig. 1E), suggesting that sequence “LFF” is critical for regulating T cell function. As SP1-52/56-5A exhibited no effect on cell proliferation, it was used as “iCont” in this study.

Figure 1.

Identification of human STAP-1 sequences which effectively inhibits STAP-1-LCK binding. (A) Scheme of truncated forms of STAP-1 PH domain-derived peptide used for cell proliferation assay. Octa-arginine (R8) sequence is conjugated to each PH domain-derived sequence. (B) Jurkat cells were preincubated with 6 peptides for 30 min and spontaneous cell proliferation was analyzed. Data are shown as mean ± SEM of 3 to 6 independent experiments (n =3 to 6) by Dunnett’s multiple comparisons test. (C) Left panel, Sequence of R8-conjugated N44-48, N47-54, and N52-59 peptides. Right panel, Jurkat cells were preincubated with the 3 peptides for 30 min and spontaneous proliferation was analyzed. Data are shown as mean ± SEM of 3 or 4 independent experiments (n = 3 to 4). *P < 0.05, **P < 0.01, by Dunnett’s multiple comparisons test. (D) Left panel, Sequence of R8-conjugated N52-59 and N55-59 peptides. Right panel, Jurkat cells were preincubated with the 3 peptides for 30 min and spontaneous proliferation was analyzed. Data are shown as mean ± SEM of 3 independent experiments (n =3). **P < 0.01, by Dunnett’s multiple comparisons test. (E) Left panel, Sequence of R8 STAP-1 N52-56 (R8-SP1-52/56) and its point mutants (R8-SP1-52/56-1A to 5A). Right panel, Jurkat cells were preincubated with the 3 peptides for 30 min and spontaneous proliferation was analyzed. Data are shown as mean ± SEM of 3 or 4 independent experiments (n = 3 to 4). **P < 0.01, ***P < 0.001, ****P < 0.0001, by Dunnett’s multiple comparisons test.

iSP1 inhibits TCR-mediated activation of human-derived T cells

As iSP1 consists of a part of the STAP-1 PH domain (N52/56) with a cell-penetrating octa-arginine sequence (RRRRRRRRGG [R8]), we tested whether iSP1 penetrates the cell membrane using the fluorescein isothiocyanate (FITC)-conjugated iSP1 peptide (FITC-iSP1) and FITC-conjugated iSP1 peptide without R8 sequence (FITC-R8(-)-iSP1). Flow cytometry analysis showed that FITC-R8(-)-iSP1 treatment exhibited almost no positivity, whereas FITC-iSP1 treatment clearly exhibited FITC positivity compared to the treatment without peptide, indicating that iSP1 penetrated the T cell membrane and was delivered to the intracellular molecules in the presence of cell-penetrating R8 sequence (Fig. 2A). To investigate the effects of iSP1 on human T cell functions, we analyzed the proliferation of iCont- and iSP1-treated Jurkat cells. As shown in Fig. 2B, cell proliferation was significantly suppressed by iSP1 treatment compared to that with iCont treatment. Furthermore, IL-2 production following TCR stimulation was suppressed by iSP1 treatment in a concentration-dependent manner (Fig. 2C). However, PMA+ionomycin-induced IL-2 production was not suppressed by iSP1 treatment (Fig. 2D), suggesting that iSP1 functions in a TCR-dependent manner. We also investigated the inhibitory effect of iSP1 on the phosphorylation of downstream molecules induced by TCR engagement. Notably, iSP1, but not iCont, inhibited TCR-mediated signal transduction (Fig. 2E), including the phosphorylation of LCK and ZAP-70. To confirm the functions of iSP1 in human samples, we used human peripheral blood mononuclear cells (PBMCs). iSP1 suppressed TCR-mediated cell proliferation (Fig. 2F) and STAP-1–LCK/STAP-1–ITK interaction (Fig. 2G). Collectively, these findings suggest that iSP1 impairs T cell activation in a TCR-dependent manner.

Figure 2.

iSP1 inhibits TCR-mediated human T cell activation. (A) Jurkat cells were treated with FITC-conjugated R8-iSP1 (FITC-iSP1) or FITC-conjugated R8-free-iSP1 (FITC-R8(-)-iSP1) peptide for 30 min, and the uptake of peptides was detected by flowcytometry analysis. (B) Jurkat cells were preincubated with iCont or iSP1 for 30 min, and spontaneous proliferation was analyzed. Data are shown as mean ± SEM of 3 independent experiments (n = 3). *P < 0.05, by Sidak’s multiple comparisons test. (C, D)Jurkat cells were preincubated with iCont or iSP1 for 30 min and stimulated with anti-CD3/anti-CD28 mAb (C) or PMA/Ionomycin (D) for 48 h (C) or 24 h (D), and IL-2 production in the supernatant was analyzed by ELISA. Data are shown as mean ± SEM of 4 independent experiments (n = 4). ***P < 0.001, ***P < 0.0001, by Sidak’s multiple comparisons test. (E) Jurkat cell were preincubated with iCont or iSP1 for 30 min and stimulated with anti-CD3/anti-CD28 for indicated periods, and TCR signal transduction was analyzed by Western blotting (left panel), and band intensity was quantified by Image J software (right panel). Data are shown as mean ± SEM of 5 independent experiments (n = 5). *P < 0.05, by Dunnett’s multiple comparisons test. (F) Human PBMCs were preincubated with iCont or iSP1 for 30 min and stimulated with immobilized anti-CD3/anti-CD28 for 48 h, Cell proliferation was evaluated by CellTiter-Glo Luminescent Cell Viability Assay. Data are shown as mean ± SEM of 3 independent experiments (n = 3). ****P < 0.0001, by Sidak’s multiple comparisons test. (G)Myc-tagged hSTAP-1-expressing Jurkat cells were preincubated with iCont or iSP1 for 30 min and stimulated with anti-CD3/anti-CD28 for indicated periods. Subsequently, association of Myc-tagged hSTAP-1 with endogenous LCK was analyzed by coimmunoprecipitation assay, followed by Western blotting. For immunoprecipitation experiment, rabbit IgG was used as isotype control.

iSP1 inhibits TCR-mediated activation of murine T cells

Next, we tested whether iSP1 inhibits TCR-mediated T cell responses in the murine T lymphoma cell line, EL-4. EL-4 cells treated with iSP1, but not iCont, exhibited suppressed cell proliferation (Fig. 3A). Furthermore, TCR-induced IL-2 proliferation was inhibited in iSP1-pretreated EL-4 cells compared to that in iCont-pretreated cells in a concentration-dependent manner (Fig. 3B). We further investigated the functions of iSP1 in murine CD4⁺ T cells purified from splenocytes. TCR-mediated cell proliferation was significantly suppressed by iSP1 treatment compared to that by iCont treatment (Fig. 3C). Moreover, IL-2 production upon TCR stimulation was inhibited in CD4⁺ T cells (Fig. 3D). As these T cell functions are initiated by the phosphorylation of TCR signaling molecules, such as LCK and ZAP-70, we analyzed whether iSP1 is involved in the downregulation of TCR signaling. Indeed, CD4⁺ T cells treated with iSP1, but not iCont, exhibited significantly suppressed phosphorylation of TCR signaling molecules upon TCR stimulation (Fig. 3E). Therefore, as shown in Figs 2 and 3, iSP1 inhibits T cell activation in both human and murine T cells by inhibiting STAP-1–LCK interaction.

Figure 3.

iSP1 also inhibits TCR-mediated murine T cell activation. (A) EL-4 cells were preincubated with iCont or iSP1 for 30 min, and spontaneous proliferation was evaluated by WST-8. Data are shown as mean ± SEM of 3 independent experiments (n = 3). *P < 0.001, by Sidak’s multiple comparisons test. (B)EL-4 cells were preincubated with iCont or iSP1 for 30 min and stimulated with immobilized anti-CD3/anti-CD28 mAb for 48 h, and IL-2 production in the supernatant was analyzed by ELISA. Data are shown as mean ± SEM of 4 independent experiments (n = 4). **P < 0.01, by Sidak’s multiple comparisons test. (C)CD4⁺T cells were preincubated with iCont or iSP1 for 30 min and stimulated with immobilized anti-CD3/anti-CD28 mAb for 96 h. Cell proliferation was evaluated by WST-8. Data are shown as mean ± SEM of 4 independent experiments (n = 4). ****P < 0.0001, by Sidak’s multiple comparisons test. (D)CD4⁺T cells were preincubated with iCont or iSP1 for 30 min and stimulated with immobilized anti-CD3/anti-CD28 mAb for 48 h. IL-2 production in the supernatant was analyzed by ELISA. Data are shown as mean ± SEM of 4 independent experiments (n = 4). ***P < 0.001, ****P < 0.0001, by Sidak’s multiple comparisons test. (E)CD4⁺T cells were preincubated with iCont or iSP1 for 30 min and stimulated with anti-CD3/anti-CD28 for indicated periods, and TCR signal transduction was analyzed by western blotting (left panel), and band intensity was quantified by Image J software (right panel). Data are shown as mean ± SEM of 4 independent experiments (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001 by Dunnett’s multiple comparisons test.

iSP1 does not affect non-T cell functions

Non-specific functions of drugs can lead to severe adverse effects in clinical settings. Therefore, we investigated whether iSP1 exhibits any non-T cell functions. As shown in Fig. S2, iSP1 did not inhibit the spontaneous proliferation of Ramos human B lymphoma cells and HeLa human epithelial-like cells derived from cervical cancer (Fig. S2A and B), as well as no inhibition of IgM-mediated IL-6 and IL-10 production in iSP1-treated murine B cells compared with iCont-treated B cells (Fig. S2C and D).

iSP1 ameliorates the pathogenesis of EAE

We examined the role of iSP1 in modulating T cell responses in vivo using an EAE model, which is also widely used as a mouse model of multiple sclerosis (MS). In EAE and MS, myelin-specific autoreactive T cells are primed by peripheral lymphocytes and migrate to the central nervous system (CNS) across the blood–brain barrier, where they produce cytokines inducing inflammation and axonal/neuronal damage.¹⁴ Immunization of mice with myelin oligodendrocyte glycoprotein (MOG_35-55) and complete Freund’s adjuvant led to the induction of EAE at the peak point, 12–16 d after immunization. We previously showed the low disease severity in STAP-1 KO mice.¹¹ In this study, we investigated whether iSP1 ameliorates the pathogenesis of EAE. iSP1 was administered every alternate day on days 8–20 after MOG immunization (Fig. 4A). During this period, clinical scores were evaluated as previously described¹⁵. Notably, clinical scores were significantly lower in the iSP1-treated mice than in the iCont-treated mice (Fig. 4B). Also, area under the curve was significantly lower in iSP1-adminstered mice than in iCont-administered mice (Fig. 4C). Furthermore, hematoxylin and eosin (H&E) and Luxol Fast Blue staining showed that immune cell infiltration and demyelination were suppressed in iSP1-treated mice (Fig. 4D), indicating that iSP1 attenuates the progression of EAE. Previously, we reported that STAP-1 expression exacerbates EAE progression by promoting the differentiation of Th1 and Th17 cells, which produce the key cytokines, interferon (IFN)-γ and IL-17, respectively.¹¹ Therefore, we analyzed CD4⁺ T cells in CNS at the peak of the disease. Both the frequencies and absolute numbers of Th1 and Th17 cells among the CD4⁺ cells were significantly lower in iSP1-treated mice than in iCont-treated mice (Figs 4E and F). These findings suggest that iSP1 ameliorates the pathogenesis of EAE by inhibiting pathogenic Th1 and Th17 cell infiltration.

Figure 4.

iSP1 suppresses the pathogenesis of EAE via downregulating the infiltration of Th1 and Th17 cells into CNS. (A) Procedure of therapeutic treatment of iSP1 in EAE mice. (B) Clinical score of EAE mice therapeutically treated with iCont or iSP1. Data are shown as mean ± SEM of 4 independent experiments (iCont: n = 20, iSP1: n = 20). *P < 0.05, by Sidak’s multiple comparisons test. (C) Area under curve of EAE clinical score. Data are shown as mean ± SEM of 4 independent experiments (iCont: n = 20, iSP1: n = 20). *P < 0.05, by Mann-Whitney U test. (D) Left panels, H&E staining of spinal cords and cell infiltration areas in iCont- or iSP1-treated mice. Yellow dash line shows cell infiltration areas. Data are shown as mean ± SEM of 2 independent experiments (n = 5). *P < 0.05, by Mann-Whitney U test. Right panels, Luxol fast blue staining of spinal cord and demyelinating areas in iCont- or iSP1-treated mice. Yellow dash line shows demyelinating areas. Data are shown as mean ± SEM of 2 independent experiments (n = 5). *P < 0.05, by Mann-Whitney U test. (E) Left panel, absolute number of IFN-γ expressing CD4⁺ T cells from the CNS in iCont- or iSP1-treated EAE mice. Right panel, absolute number of IL-17 expressing CD4⁺ T cells from the CNS in iCont- or iSP1-treated EAE mice. Data shown are at the point of day14 after MOG immunization. Data are shown as mean ± SEM of 2 independent experiments (n =10 to 14), by Mann-Whitney U test. (F) Left panel, frequency of IFN-γ expressing CD4⁺T cells from the CNS in iCont- or iSP1-treated EAE mice. Right panel, frequency of IL-17 expressing CD4⁺T cells from the CNS in iCont- or iSP1-treated EAE mice. Data shown are at the point of day14 after MOG immunization. Data are shown as mean ± SEM of 2 independent experiments (n = 10 to 12). *P < 0.05, **P < 0.01, by Mann-Whitney U test.

Discussion

In this study, we identified a five-amino acid sequence (52/56; TTLFF) in the human STAP-1 PH domain as a promising inhibitor of the STAP-1–LCK interaction. STAP-1-derived peptide iSP1 suppressed TCR-mediated T cell activation, cell proliferation, IL-2 production, and signal transduction in both human and murine T cells. Notably, iSP1 inhibited disease progression in the CD4⁺ T cell-mediated EAE mouse model. Our findings suggest iSP1 as a candidate peptide to treat T cell-mediated autoimmune diseases. STAP-1 affects immune responses in invariant natural killer T (iNKT) cells,¹⁶ mast cells (data not shown), chronic myeloid leukemia stem cells,¹⁷ and B cells.¹⁸ Moreover, we recently showed STAP-1 as a novel regulator of TCR-mediated signaling that initiated adaptive immune responses. STAP-1 constitutively interacts with ITK, and LCK is recruited to the STAP-1–ITK complex upon antigen–MHC complex recognition by TCR. PLC-γ1 is also recruited to the STAP-1–ITK complex in a TCR-dependent manner, suggesting that STAP-1 functions as a scaffold to form ITK–LCK and ITK–PLC–γ1 complexes to mediate T cell activation. Previously, we revealed that STAP-1 PH domain binds to LCK and PLC-γ1 after TCR ligation¹¹. Therefore, pharmacological inhibition of STAP-1–LCK binding is a potential strategy to suppress T cell activation and mitigate T cell-mediated autoimmune diseases. We previously reported that the STAP-1 PH domain binds to STAT5,^17,19 and that STAP-2, another STAP family member, binds to BRK, c-FMS, PYK2, EGFR, TRAF3, and STAT5 in its PH domain.^20–25 Therefore, STAP-1 and STAP-2 PH domains have many binding partners, and blockade of PH domain-mediated binding leads to off-target effects. Consequently, we controlled the STAP-1 PH domain-derived peptide without affecting its ability to inhibit STAP-1–LCK binding and suppress T cell functions. We also identified a 5-amino acid sequence critical for the STAP-1–LCK interaction and TCR-mediated signaling. Disease in the EAE model, which exhibited some characteristics of MS such as inflammation and axonal damage, was mediated by myelin-specific T cells. EAE model is a T cell-dependent model as T cell-deficient mice do not develop EAE.^26,27 Here, EAE model mice intravenously treated with iSP1 exhibited significantly lower clinical scores than those treated with iCont, indicating that iSP1 shows both in vitro and in vivo efficacy. Moreover, mice treated with iSP1 did not show any abnormal phenotypes within the effective concentration range as seen in STAP-1 KO mice (data not shown). These findings suggest iSP1 as an effective and tolerable therapeutic for T cell-mediated autoimmune diseases, such as MS.

Recently, development of peptide drugs targeting protein–protein interactions (PPIs) has attracted the attention of various pharmaceutical companies as peptides penetrate cell membranes and target PPIs, which are generally not targeted by conventional small molecules.^28–30 Some peptide drugs are currently in the clinical stages of development. For example, synthetic cyclic peptide LUNA18 is an oral peptide that interferes with RAS–GEF binding. LUNA18 is currently being tested for clinical stages of solid carcinoma.³¹ ST101 is a CCAAT/enhancer binding protein β (C/EBPβ) antagonist that inhibits C/EBPβ dimerization. Effects of ST101 on gastrointestinal tract cancer are also being investigated.³² In this study, we identified a novel five-amino acid sequence (TTLFF) in STAP-1 PH domain and demonstrated its potential therapeutic application for T cell-mediated autoimmune diseases. TTLFF is preserved in humans and mice; therefore, iSP1 suppressed TCR-dependent activation in both murine and human T cells in the same manner. To ensure iSP1 application from basic research to clinical treatment, further optimization of its absorption, distribution, metabolism, excretion, and toxicity (ADMET) is necessary as cell-penetrating R8 sequence in iSP1 is unstable and easy to dissociate in vivo.^30,33 Cell-penetrating druggable peptides, such as cyclic oral and stapled peptides, can be used to enhance the draggability of iSP1.^34,35 Recently, we demonstrated that STAP-2-derived peptide, which interferes with STAP-2–CD3ζ ITAM binding, suppresses TCR-mediated T cell activation and EAE pathogenesis.¹² Therefore, combination of STAP-1- and STAP-2-derived peptides may exhibit great potential for MS treatment.

Previously, we demonstrated that STAP-1 is involved in airway inflammation.¹¹ Antigen-induced airway inflammation was attenuated in STAP-1 KO mice compared to that in WT mice; therefore, iSP1 may also aid in the treatment of airway inflammation. In antigen-induced airway inflammation, total cell and eosinophil numbers in BALF were significantly lower in STAP-1 KO mice than in WT mice.¹¹ Additionally, IL-5, IL-13, and IgE levels in BALF were significantly lower in STAP-1 KO mice than in WT mice.¹¹ However, the mechanisms by which STAP-1 affects Th2 responses to airway inflammation remain unclear. Future studies should investigate these mechanisms to determine the applicability of iSP1 for bronchial asthma treatment.

Author contributions

Y. S., K. K., S. K., and J. K. performed experiments and analyzed the data. All authors discussed the entire results. Y. K., R. M., K. O., J. K., and T. M helped supervise the project. Y. S, K. O., J. K., and T. M. designed the project and wrote the manuscript with input from all co-authors. All authors read and approved the final manuscript.

Supplementary material

Supplementary material is available at ImmunoHorizons online.

Funding

This study was supported by AMED under grant number JP23ym016801j0002, in part JSPS KAKENHI under grant number 19H03364 (T.M.) and 24K09781 (J.K.).

Conflicts of interest

The authors have no financial conflicts of interest.

Data availability

This is our future plan. We do not have any available data at the present time.