Previously we generated the recombinant protein and a synthetic peptide of the cytoplasmic domain of the cytotoxic T lymphocyte antigen-4 (CTLA-4) in conjugation with a CPP dNP2 (dNP2-ctCTLA-4) (36–38). This protein and peptide showed successful immune modulatory effects on T cell activation during autoimmune or allergic disease with an increase of Foxp3 expressing Treg cells (36, 38–40). Building on this knowledge, we aimed to investigate whether the dNP2-ctCTLA-4 could be effective in treating psoriasis with increased Treg cells. As both Th17 (CD4) and Tc17 (CD8) cells are important pathogenic cells in psoriasis, we examined the functional effects of the dNP2-ctCTLA-4 peptide on mouse CD4 and CD8 T cells in vitro. In our experiments, we cultured TCRαβ⁺CD4⁺CD25^-CD62L^highCD44^low naïve CD4 T cells under Treg condition (TGF-β, IL-2) with or without the peptide. The results demonstrated a significant increase in CD25⁺Foxp3⁺ Treg cells when the peptide was present ( Figures 1A, B ). Furthermore, when we cultured these naïve CD4 T cells under Th17+IL-2 conditions (TGF-β, IL-6, IL-1β, IL-23, IL-2), dNP2-ctCTLA-4 exhibited inhibitory effects on IL-17 production while simultaneously increasing Foxp3 expression ( Figures 1C, D ). Similar observations were made with TCRαβ⁺CD8⁺CD25^-CD62L^highCD44^low naïve CD8 T cells, which were differentiated under Treg condition (TGF-β, IL-2) in the presence of dNP2-ctCTLA-4. The peptide treatment led to a significant increase in CD25⁺Foxp3⁺ CD8 T cells ( Figures 1E, F ). Moreover, when naïve CD8 T cells were cultured under Tc17+IL-2 conditions (TGF-β, IL-6, IL-1β, IL-23, IL-2), the peptide demonstrated a reduction in IL-17 production while promoting Foxp3 expression ( Figures 1G, H ). To investigate whether dNP2-ctCTLA-4 can intrinsically increase TGF-β mediated Foxp3 expression, naïve CD8 T cells were stimulated with anti-CD3 and TGF-β in the presence or absence of dNP2-ctCTLA-4 peptide. The dNP2-ctCTLA-4 peptide reduced SMAD2 linker region phosphorylation and the amount of p-ERK. This result is consistent with our previous research confirmed in the splenocytes (38), suggesting that dNP2-ctCTLA-4 peptide intrinsically enhances TGF-β signaling in CD8 T cells, leading to an increase in Foxp3⁺ T cells ( Figure 1I ). In summary, our findings demonstrate that the dNP2-ctCTLA-4 peptide has the ability to induce Foxp3 expression in both CD4 and CD8 T cells under various stimulatory conditions. These results suggest the therapeutic potential of the peptide in modulating Th17/Tc17 responses associated with psoriasis.

To assess the potential of the dNP2-ctCTLA-4 peptide in inducing Treg cells and alleviating psoriatic skin inflammation, we conducted in vivo experiments using the well-established imiquimod (IMQ)-induced psoriasis mouse model (41), which closely resembles human psoriasis (42). The IMQ-induced psoriasis mouse model was established by daily application of IMQ to the back skin of mice until day-5. Concurrently, the mice received intraperitoneal administration of 100 μg of the dNP2-ctCTLA-4 peptide three times daily from day-1 to 5 ( Figure 2A ). Our findings demonstrated that the dNP2-ctCTLA-4 peptide significantly inhibited the progression of psoriasis ( Figure 2B ) and effectively improved the psoriasis area and severity index (PASI) score ( Figure 2C ). Histological analysis of dorsal tissue samples revealed a reduction in epidermal hyperplasia and immune cell infiltration ( Figure 2D ). Flow cytometry analysis of skin-draining lymph nodes (SDLN) showed a notable increased in the proportion of Foxp3⁺ CD4 T cells, accompanied by a reduction in IL-17A-producing γδ T cells ( Figures 2E, F ). Taken together, these results highlight the potential of the dNP2-ctCTLA-4 peptide to induce Foxp3⁺ Treg cells in vivo, consequently ameliorating psoriasis-like skin inflammation. This induction of Treg cells leads to a significantly increased Treg/Teff ratio ( Figure 2G ), underscoring the therapeutic efficacy of the dNP2-ctCTLA-4 peptide in the treatment of psoriatic skin inflammation.

CTLA-4-Ig (Abatacept) is a US Food and Drug Administration (FDA)-approved drug primarily used for rheumatoid arthritis treatment. It consists of the extracellular domain of CTLA-4 fused with the Fc region of an immunoglobulin (43, 44). By binding to B7 molecules, CTLA-4-Ig effectively inhibits the co-stimulatory signal of T cells, thereby preventing their activation (45). In contrast, the dNP2-ctCTLA-4 peptide utilized in our study employs the cytoplasmic domain of CTLA-4 to modulate intracellular signaling and result in Foxp3-expressing Treg cells. Considering the distinct domains of CTLA-4 targeted by these two agents ( Figure 3A ), we hypothesized that they would exhibit different mechanisms of action in treating psoriasis. To evaluate this hypothesis, equal doses of CTLA-4-Ig and dNP2-ctCTLA-4 peptide were administered to mice with IMQ-induced psoriatic skin inflammation, following the same protocol as in the previous experiment ( Figure 3B ). As shown in the figure, CTLA-4-Ig did not effectively inhibit psoriatic skin inflammation, while the dNP2-ctCTLA-4 peptide significantly reduced psoriasis symptoms and PASI score ( Figures 3C, D ). Additionally, only the group treated with the dNP2-ctCTLA-4 peptide demonstrated a decrease in epidermal layer thickness and infiltration of inflammatory cells ( Figure 3E ). These compelling findings suggest that the dNP2-ctCTLA-4 peptide, which specifically targets the intracellular signaling events related to the cytoplasmic domain of CTLA-4, would be more effective in regulating psoriatic skin inflammation compared to CTLA-4-Ig. The differential mechanisms of action of these two agents provide further insights into their distinct therapeutic potential for managing psoriasis.

To investigate the impact of dNP2-ctCTLA-4 peptide and CTLA-4-Ig on lymphoid cells in psoriasis-like skin inflammation, we conducted a flow cytometry analysis. Intriguingly, the dNP2-ctCTLA-4 peptide significantly induced CD25⁺ Foxp3⁺ Treg cells, whereas CTLA-4-Ig could not in SDLN ( Figures 4A, B ). Neither treatment showed a statistically significant tendency to inhibit the production of IL-17A and IFN-γ in CD4 T cells ( Figure 4B ). Although the dNP2-ctCTLA-4 peptide resulted in a slightly reduced tendency in the proportion of IL-17A-producing γδ T cells ( Figures 4C, D ), it exhibited a significantly increased Treg/Teff ratio compared to CTLA-4-Ig, confirming its potential as an effective regulator of psoriatic skin inflammation ( Figure 4E ). Consistent with these findings, the dNP2-ctCTLA-4 peptide also demonstrated a significant reduction in the absolute cell number of infiltrated CD45⁺ lymphocytes into the skin tissue, as compared to control group ( Figures 4F, G ). However, it is worth noting that neither CTLA-4-Ig nor the dNP2-ctCTLA-4 peptide appeared to reduce the proportion of IL-17⁺ γδ T cells in the skin. Overall, this result suggests that CTLA-4-Ig might not be an effective molecule for controlling psoriatic skin inflammation, whereas the dNP2-ctCTLA-4 peptide could serve as a promising novel therapeutic option, as it exhibits a mechanism of increasing the Treg/Teff ratio.

To gain further insights into the distinct mechanisms underlying T cell regulation by dNP2-ctCTLA-4 peptide and CTLA-4-Ig, we performed experiments utilizing FACS-sorted naïve CD4 T cells derived from 2D2 mice. These cells were stimulated with MOG_35-55 peptide in the presence of APCs to induce Th17 and Treg cells, while being exposed to either the dNP2-ctCTLA-4 peptide or CTLA-4-Ig. Our findings revealed noteworthy disparities between the two agents. Specifically, the dNP2-ctCTLA-4 peptide demonstrated induction of Foxp3⁺ Treg cells under Th17 differentiation conditions (IL-6, TGF-β), which was not observed with CTLA-4-Ig ( Figures 5A, B ). Intriguingly, CTLA-4-Ig exhibited a tendency to inhibit IL-17⁺ CD4 T cells, a response not observed with the dNP2-ctCTLA-4 peptide. We further examined key functional molecules in Treg functions including CD25 and CD39. Notably, the dNP2-ctCTLA-4 peptide significantly upregulated both CD25 and CD39 expression in Foxp3⁺ Treg cells compared to CTLA-4-Ig ( Figures 5C, D ). Moreover, the dNP2-ctCTLA-4 peptide induced CD25⁺ Foxp3⁺ Treg cells under Treg conditions, while CTLA-4-Ig failed to elicit a similar response ( Figures 5E, F ). We observed an increase in the CD25^high CTLA-4^high population within Foxp3⁺ Treg cells under Treg conditions when treated with the dNP2-ctCTLA-4 peptide, but not with CTLA-4-Ig ( Figures 5G, H ). Notably, the expression level of CTLA-4 tended to be increased in Foxp3⁺ CD4 T cells by the dNP2-ctCTLA-4 peptide, whereas CTLA-4-Ig did not elicit a similar effect ( Figures 5I, J ). Collectively, these findings highlight that the dNP2-ctCTLA-4 peptide induces Foxp3⁺ Treg cells with enhanced Treg characteristics, while CTLA-4-Ig suppresses Th17 responses. Thus, our results suggest that the mechanisms underlying the actions of CTLA-4-Ig and the dNP2-ctCTLA-4 peptide are fundamentally different which provides a plausible explanation for the divergent outcomes observed in the psoriasis experiments.

Female C57BL/6 mice aged 8 to 10 weeks were purchased from Daehan Biolink (DBL, Korea), while 2D2 TCR-transgenic mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were housed and bred in a specific pathogen-free animal facility at Hanyang University under controlled conditions of constant temperature (21 ± 1°C), humidity (50 ± 5%), and a 12-hour light/dark cycle, and provided with regular chow and autoclaved water. All mouse experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committees of Hanyang University (Animal Experiment Approval Number: 2022-0002A) and all mice studies were randomized in a blinded manner in psoriasis mouse models.

To induce psoriasis, 8- to 10-week-old female C57BL/6 mice purchased from Daehan Biolink (DBL, Korea) were used. On Day 0, hair was removed from the dorsal skin using a clipper and hair removal cream. Starting from Day 1, 40 mg of 5% Aldara Cream (3M Pharmaceuticals, Leicestershire, United Kingdom) was topically applied to the dorsal skin for 4 to 5 consecutive days. Psoriasis was induced under deep anesthesia with zoletil (30 mg/kg; Virvac, Carros, France) and rompun (10 mg/kg; Bayer Korea, Ansan, Korea). During disease induction, 100 μg of either dNP2-ctCTLA-4 or CTLA-4-Ig were administered intraperitoneally three times a day. On day 5 or 6, mice were sacrificed and the severity of inflammation in each tissue was assessed using flow cytometry (FACS) or histological analysis with hematoxylin and eosin (H&E) staining. Skin lesion severity was evaluated using the PASI scoring criteria, which considers three signs of erythema, scaling, and thickening, with a scale ranging from 0 (no symptoms) to 4 (very marked). To investigate the effect on ears, we applied 20 mg of 5% Aldara cream daily for six days, and administered 100 μg of protein via intraperitoneal injection. We measured the thickness of the ears using a micrometer (Mitutoyo, Kawasaki, Japan) every day and analyzed the data on day 7.

To acquire single cells, the fat was initially scraped off, and the dorsal skin tissue of the mouse was then separated into the epidermal and dermal layers at 37°C for 90 minutes using 2.4 U/ml of dispase II (04 942 078 001; Roche). After separation, the tissue was cut into small pieces and digested in DMEM media containing 1 mg/ml of collagenase D (11 088 866 001; Sigma-Aldrich), 0.2 mg/ml of DNase I (10 104 159 001; Sigma-Aldrich), and 2% FBS (S 001 01; Welgene) at 225 rpm for 1 hour at 37°C. The enzymatic reaction was stopped by adding 0.5 M ethylenediaminetetraacetic acid (EDTA), followed by filtering through a 40 µm cell strainer and washing with PBS. Single cells were finally obtained, and flow cytometry analysis was performed after antibody staining.

Cells isolated from each tissue were analyzed using flow cytometry. Cells were restimulated with Cell Stimulation Cocktail (00-4975-03; eBioscience) to determine intracellular cytokine levels for 4 h at 37°C. To exclude dead cells, cells were stained with the Zombie Aqua Fixable Viability Kit (423101; BioLegend) at room temperature for 10 min. After washing with PBS, the surface proteins were stained with specific monoclonal antibodies for 20 min at 4°C. After washing with PBS, cells were fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set (00-5523-00; eBioscience) for 30 min at room temperature. Finally, intracellular cytokine proteins were stained with monoclonal antibodies for 30 min at room temperature. FACS Canto II or FACS Symphony A3 was used to acquire data and FlowJo software version 10.8.0 was used to analyze the data.

For histological analysis of mouse back skin samples, the tissues were fixed in 4% paraformaldehyde overnight at 4°C, followed by embedding in paraffin. Sections of the skin tissue blocks were prepared and stained with hematoxylin and eosin to reveal the cellular architecture. The resulting H&E-stained sections were then mounted with a cover glass using a mounting solution. Subsequently, the sections were observed under an inverted microscope (Leica DMi8, Leica Microsystems), and images were captured and analyzed using Image J software version 2.0.0

Naïve CD4 T cells (TCRαβ⁺CD4⁺CD25^-CD62L^highCD44^low) or naïve CD8 T cells (TCRαβ⁺CD8⁺CD25^-CD62L^highCD44^low) were isolated from splenocytes of 8- to 10-week-old C57BL/6 mice using a FACSAria Fusion (BD Biosciences). Purified naïve CD4 T cells were activated with plate-bound anti-CD3 (2 μg/ml; 553057; BD Pharmingen) and anti-CD28 (2 μg/ml; 553294; BD Pharmingen) antibodies and differentiated with the following cytokine cocktails in a 96-well plate for 3 or 4 days: 20 U/ml of IL-2 (212-12; Peprotech), 2 ng/ml of TGF-β (240-B-002; R&D Systems) for Treg differentiation; or 5 μg/ml of anti-IL-4 (11B11; 16-7041-85; eBioscience), 5 μg/ml of anti-IFN-γ (XMG1.2; 16-7311-85; eBioscience), 30 ng/ml of IL-6 (406-ML; R&D Systems), 2 ng/ml of TGF-β, 20 ng/ml of IL-1β (401-ML-010; R&D Systems), 20 ng/ml of IL-23 (1887-ML-101; R&D Systems), 20 U/ml of IL-2 for Th17 differentiation. Additionally, cells were co-treated with 2 μM of dNP2-ctCTLA-4. Purified naïve CD8 T cells were activated with plate-bound anti-CD3 (3 μg/ml) and anti-CD28 (3 μg/ml) antibodies and differentiated with the following cytokine cocktails in a 96-well plate for 3 days: 25 U/ml of IL-2, 0.5 ng/ml of TGF-β for Treg differentiation; or 5 μg/ml of anti-IL-4, 5 μg/ml of anti-IFN-γ, 30 ng/ml of IL-6, 2 ng/ml of TGF-β, 20 ng/ml of IL-1β, 20 ng/ml of IL-23, 20 U/ml of IL-2 for Tc17 differentiation. Additionally, cells were co-treated with 2 μM of dNP2-ctCTLA-4.

Naïve CD4 T cells (CD4⁺TCR-Vβ11⁺CD25^-CD62L^highCD44^low) were isolated from splenocytes of 8- to 10-week-old 2D2 mice using the CD4 T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions, followed by FACS sorting. The antigen-presenting cells were irradiated with a 3500 rad gamma ray and seeded with the sorted naïve CD4 T cells at a 5:1 ratio. T cells were activated using 40 μg/ml of MOG_35-55 (AnyGen, Korea), and differentiated using the following cytokine cocktails in a 96-well flat bottom plate for 3 days: 5 μg/ml of anti-IL-4 (11B11; 16-7041-85; eBioscience), 5 μg/ml of anti-IFN-γ (XMG1.2; 16-7311-85; eBioscience), 30 ng/ml of IL-6 (406-ML; R&D Systems), 2 ng/ml of TGF-β (240-B-002; R&D Systems) for Th17 differentiation; or 5 ng/ml of TGF-β for Treg differentiation. Additionally, cells were co-treated with dNP2-ctCTLA-4 or CTLA-4-Ig.

Naïve CD8 T cells were isolated and stimulated with 5μg/ml of anti-CD3/CD28 mAb and 5 ng/ml of TGF-β at 37°C, 30 minutes. Afterward, the cells were washed with PBS and lysed with RIPA buffer (Cell Signaling, Beverly, MA) containing 1 μM of NaF and 1 μM of PMSF for 30 minutes on ice. Immunoblotting was performed on PVDF membranes (Bio-Rad) using the following primary antibodies: phospho-Smad2, phospho-ERK, and β-actin mouse mAb.

dNP2-conjugated recombinant proteins were purified as previously described (36). Briefly, dNP2-ctCTLA-4 was purified by Ni-NTA affinity chromatography (Qiagen, Chatsworth, CA, USA) and desalted using a PD-10 Sephadex G-25 column (GE Healthcare, Uppsala, Sweden). To obtain highly purified proteins, an additional ion-exchange protein purification step was performed using SP Sepharose High Performance (GE Healthcare, Uppsala, Sweden). For the peptide form of dNP2-ctCTLA-4 (from AnyGen, Korea), the peptide was synthesized by solid-phase peptide synthesis as previously described (38). The purity of the final peptide products was more than 95%.

To extract RNA from ear tissue, we used TRI Reagent RT (Molecular Research Center, Inc.) following the manufacturer’s protocol. The extracted RNA was then quantified using a UV spectrophotometer (CellTAGen, Inc., Seoul, Korea). Subsequently, cDNA synthesis was performed using a ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). For quantitative RT-PCR analysis, we used iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) with a Bio-Rad CFX Connect real-time PCR detection system. To ensure accuracy, we normalized expression levels to CD45.

The following monoclonal antibodies were used for cell surface staining: anti-mouse CD45 (30-F11; BioLegend), anti-mouse TCRβ (H57-597; eBioscience), anti-mouse TCRγδ (eBioGL3 (GL-3, GL3); eBioscience), anti-mouse Vγ1 (2.11; BioLegend), anti-mouse CD4 (RM4-5; BioLegend), anti-mouse CD25 (PC61; BioLegend), anti-mouse CD27 (LG.3A10; BD Biosciences), anti-mouse CD39 (Duha59; BioLegend), anti-mouse CD44 (IM7; BioLegend), anti-mouse CD62L (MEL-14; eBioscience). The following antibodies were used for intracellular staining: anti-mouse Foxp3 (FJK-16s; eBioscience), anti-RORγt (Q31-415 378; BD Biosciences), anti-mouse IL-17A (eBio17B7; eBioscience), anti-mouse IFN-γ (XMG1.2; eBioscience), anti-mouse CTLA-4 (UC10-4B9; eBioscience).

Statistical analysis was conducted using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA). Mann–Whitney test, Kruskal–Wallis test and Brown-Forsythe and Welch ANOVA tests were employed for data analysis. The data are presented as mean ± S.D. or ± S.E.M., and p ≤ 0.05 was considered statistically significant. Additional information regarding sample size and specific statistical analyses can be found in the corresponding figure legend.