This was a prospective, Single-arm observational cohort study conducted at Shanghai Skin Disease Hospital. The study was approved by the ethics committees of Shanghai Skin Disease Hospital and conducted in accordance with the Declaration of Helsinki.

The inclusion criteria were adults with a PASI score ≥3 considered to be in need of treatment with biologics. Exclusion criteria were missing baseline data; patients who did not provide follow-up information after screening or who were noncompliant with medication; patients with uncontrolled internal medical conditions, such as diabetes; and pregnant individuals. A total of 22 patients with plaque psoriasis were enrolled for the main analysis (Figure 1). All patients received anti–IL-17A monoclonal antibody ixekizumab (Taltz^®, Eli Lilly and Co.), with an initial dose of 160 mg at week 0, followed by 80 mg at weeks 2, 4, 6, 8, 10, 12, 16, 20, and 24. Venous blood samples were collected from the participants at baseline and at weeks 4, 12, and 24. Written informed consent was obtained from all participants prior to inclusion.

PBMCs were isolated from patient blood samples using Ficoll-Paque Plus (GE Healthcare, Anaheim, CA), following the manufacturer’s instructions.

To assess cytokine secretion, PBMCs were treated in vitro with Cell Stimulation Cocktail (#00-4975-03, eBioscience) for 4 hours before staining. Single-cell suspensions were pre-incubated with Fc Receptor Blocking Solution (#422302, BioLegend) for 10 minutes at room temperature. To discriminate dead cells, the cells were stained with Fixable Viability Stain 780 (FVS780, #565388, BD Biosciences) for 15 minutes at room temperature. Subsequently, the cells were stained for 30 minutes with surface marker antibodies in FACS buffer (PBS containing 2% fetal bovine serum) at 4 ˚C. For intracytoplasmic cytokines (ICs) detection, the cells were fixed with IC Fixation Buffer (#00-8222-49, eBioscience) for 30 minutes at 4 ˚C. For intranuclear transcription factor analysis, cells were fixed and permeabilized with Fixation/Permeabilization Diluent and Concentrate (#00–5123-43, eBioscience) for 40 minutes at 4 ˚C. Intracellular antibodies were applied in Permeabilization Buffer (#00-8333-56, eBioscience) for 30 minutes at 4 °C. Data acquisition was performed on a BD FACSCelesta™ Cell Analyzer and analyses were conducted using FLOWJO software (Tree Star, Ashland, or USA). The following human antibodies were used: BV650-conjugated anti-CD3 (clone SK7, #564003, BD Biosciences), BV786-conjugated anti-CD4 (clone SK3, #563881, BD Biosciences), FITC-conjugated anti-CD4 (clone RPA T4, #300506, Biolegend), APC-conjugated anti-CD4 (clone RPA-T4, #300514, eBioscience), AF700-conjugated anti-CD8 (clone SK1, # 564116, Biolegend), BV605-conjugated anti-CD8 (clone SK1, #344723, BD Biosciences), PE-conjugated anti-CD25 (clone BC96, #12-0259-80, eBioscience), APC-conjugated anti-Foxp3 (clone BC96, #17-0529-42, eBioscience), PE-conjugated anti-PROCR (clone RCR- 401, #351904, Biolegend), FITC-conjugated anti-PDPN (clone NC-08, #337026, Biolegend), BV605-conjugated anti-CTLA-4 (clone BNI3, #369610, Biolegend), PerCP-Cy 5.5-conjugated anti-BTLA (clone MIH26, #344514, Biolegend), BV421-conjugated anti-TIGIT (clone A15153G, #372710, Biolegend), AF700-conjugated anti-PD-1 (clone EH12.2H7, #329952, Biolegend), BV510-conjugated anti-LAG-3 (clone 11C3C65, #369318, Biolegend), BV421-conjugated-IL-17A (clone N49-653, #562933, BD Biosciences), PerCP-eFluor 710-conjugated- B7-H6 (clone JAM1EW, #46-6526-42, eBioscience). Gate strategies for T cell subsets are illustrated in Supplementary Figures S1–S3.

Data normality was assessed using the Shapiro–Wilk test. Continuous variables are presented as mean ± standard deviation (SD).

For normally distributed data, comparisons between two independent groups were performed using the two-tailed unpaired Student’s t-test. For non-normally distributed data, the Mann–Whitney U test was applied. Comparisons across more than two groups were conducted using one-way ANOVA (with Bonferroni correction) or the Kruskal–Wallis test followed by Dunn’s multiple comparisons test, as appropriate.

Correlation analyses were performed using Pearson or Spearman correlation coefficients depending on data distribution. Receiver operating characteristic (ROC) curve analysis was used to evaluate the discriminative performance of baseline immunological parameters for early response, and area under the curve (AUC) values with 95% confidence intervals (CI) were calculated. Categorical variables were compared using the chi-square test or Fisher’s exact test when appropriate.

Statistical analyses were performed using GraphPad Prism or SPSS software. Differences were considered statistically significant at p < 0.05.

The assessment of treatment effect in psoriasis primarily relies on measures such as the PASI and affected body surface area (BSA). In this study, the main efficacy outcome was defined as the PASI50 response rate after 4 weeks of treatment. Additional efficacy outcomes included changed in BSA and DLQI, Physician Global Assessment (PGA)0/1 (‘clear’/’almost clear’) response rates, PASI75, PASI90, PASI100 at weeks 12 and 24. As there is no universally accepted definition of early clinical response in psoriasis, based on existing literature evaluating short-term treatment outcome (44–46), Early Responders (ER) were defined as patients who achieved PASI50 at week 4. Patients who did not achieve PASI50 at week 4 were classified as Non-Early Responders (NER).

Baseline characteristics did not differ significantly between groups, including patients with and without available week 4 PASI data (Table 1; Supplementary Table S1). The overall cohort had a mean PASI score of 13.2 ± 7.6 and mean BSA of 17.4 ± 12.6%. The mean disease duration was 13.1± 7.7 years.

Following ixekizumab treatment, sustained clinical improvement was observed. The mean PASI score decreased to 5.6 ± 3.1 at week 4 and further declined to 2.3 ± 2.8 and 1.5 ± 2.4 at weeks 12 and 24, respectively. Similarly, mean BSA decreased to 10.0 ± 6.2% at week 4, and 4.1 ± 5.2% at week 12, and 2.6 ± 3.7% at week 24. PGA 0/1 response rates increased overtime, reaching 62.5% at week 12, and 72.7% at week 24. The mean percentage improvement in PASI from baseline was 50.5%, 82.4%, and 91.7% at weeks 4, 12, and 24, respectively (Table 2). Importantly, the median PASI improvement at week 4 was 49.1%, supporting PASI50 as a clinically reasonable and distribution-based cutoff for defining early response.

Following ixekizumab treatment, the percentage of Th17 cell proportion among circulating CD4⁺ T cells decreased from 0.67% to 0.23% by week 24 (p=0.0158) (Figures 2A, B). However, no significant correlation was observed between Th17 cell proportion and either disease duration or PASI score (Figures 2C, D). Notably, baseline Th17 cell proportion was significantly negatively correlated with PASI score improvement at week 4 (r=-0.8301, 95% CI [-0.9418– -0.5529], p<0.0001) (Figure 2E), although no such correlation was found at weeks 12 or 24 (Figures 2F, G). Similarly, Tc17 cell proportion in circulating CD8⁺ T cells decreased after ixekizumab treatment, with significant difference (p=0.0417) at week 12 (Figures 3A, B). Baseline Tc17 cell proportion was significantly negatively correlated with PASI score improvement at week 4 (r=-0.6631, 95% CI [-0.8811– -0.2125], p=0.0085), while no significant correlation was found between Tc17 cell proportion and PASI score, disease duration, or therapeutic efficacy at weeks 12 and 24 (Figures 3C–G).

Patients receiving ixekizumab did not exhibit any significant alteration in the percentage of Treg cells among peripheral CD4⁺ T cells, nor was this percentage correlated with PASI score, disease duration, or treatment efficacy (Supplementary Figures S4A–G). However, the Th17/Treg ratio was significantly reduced by week 24 (p=0.0103) (Supplementary Figure S4H), but the baseline Th17/Treg ratio was not associated with disease duration or PASI score (Supplementary Figures S4I, J). Notably, the baseline Th17/Treg ratio was negatively correlated with PASI score improvement at week 4 (r=-0.6416, 95% CI [-0.8684– -0.1929], p=0.0099) (Supplementary Figure S4K), but it did not influence therapeutic efficacy at weeks 12 and 24 (Supplementary Figures S4L, M).

LAG-3 expression on circulating CD4⁺ T cells decreased from 3.08% to 0.51% at week 24 (p=0.0121) (Figures 4A, B). While LAG-3 expression on CD4⁺ T cells tended to increase over the disease course (Figure 4C), it showed no correlation with PASI scores (Figure 4D). Furthermore, baseline LAG-3 expression was negatively correlated with PASI score improvement at week 4 (r=-0.5605, 95% CI [-0.8335– -0.0677], p=0.0297) (Figure 4E), although it did not influence therapeutic efficacy at weeks 12 and 24 (Figures 4F, G).

Similarly, LAG-3 expression on circulating CD8⁺ T cells decreased from 2.48% to 0.47% by week 24 after treatment (p=0.0307) (Figures 5A, B). However, no significant association was found between LAG-3 expression on circulating CD8⁺ T cells and disease duration or PASI score (Figures 5C, D). Baseline LAG-3 expression on circulating CD8⁺ T cells was negatively correlated with PASI score improvement at week 4 (r=-0.5929, 95% CI [-0.8523– -0.0992], p=0.0222) (Figure 5E), although it did not influence therapeutic efficacy at weeks 12 and 24 (Figures 5F, G).

TIGIT expression on CD4⁺ T cells tended to decrease after ixekizumab treatment, although the changes were not statistically significant (Figures 6A, B). Additionally, there appeared to be a positive correlation between TIGIT expression on CD4⁺ T cells and disease duration (Figure 6C). However, TIGIT expression on CD4⁺ T cells was not associated with PASI score at baseline (Figure 6D). Baseline TIGIT expression on CD4⁺ T cells was negatively associated with early therapeutic efficacy (r=-0.5238, 95% CI [-0.8169– -0.0158], p=0.0450), but it did not significantly affect efficacy at weeks 12 and 24 (Figures 6E–G). Furthermore, there was no significant change in TIGIT expression on circulating CD8⁺ T cells (Supplementary Figure S5).

B7-H6, a recently identified ligand of the B7 family, has been explored in various solid cancers. By binding to the NK cell receptor NKp30, B7-H6 regulates innate immune responses but may also limit antitumor T-cell responses through NK cell recognition (34, 47). Psoriasis patients exhibited a decrease in B7-H6 expression on circulating CD4⁺ T cells following ixekizumab treatment at week 24 (p=0.0424) (Figures 7A, B). However, no associations were found between B7-H6 expression and disease duration or PASI score (Figures 7C, D). Additionally, B7-H6 expression did not impact therapeutic efficacy (Figures 7E–G). A similar change trend was observed in B7H6 expression on CD8⁺ T cells (Supplementary Figure S6).

CTLA-4, expressed on activated T cells, B cells, and monocyte-derived DCs, inhibits T cell activation by competing with CD28 for binding to B7 family molecules (48). CTLA-4⁺ T cells are more abundant in psoriatic lesional skin compared to healthy skin, with expression levels negatively correlated with disease severity (49). BTLA, a member of the CD28 immunoglobulin superfamily, functions as an inhibitory receptor that suppresses T and B cell activation and cytokine secretion (50, 51). PROCR is expressed in many cell types, including hematopoietic stem cells and tumor cells. Recent studies demonstrated that PROCR also binds other ligands, including TCR present on a subset of Vδ2−γδ T cells (52). PDPN, expressed on keratinocytes and inflammatory cells such as monocytes, promotes IL-17 secretion in inflammatory skin diseases (53, 54). PD-1, expressed on activated and exhausted T cells, inhibits T cell proliferation, polarization, and cytokine production via interaction with PD-Ligand (L)1 and PD-L2 (55). In psoriasis, CD4⁺ and CD8⁺ T cells from PBMCs show decreased PD-1 expression (56, 57). However, there was neither significant alteration of CTLA-4, BTLA, PROCR, PDPN, and PD-1 expression on circulating T cells nor any correlation with the disease severity (Supplementary Figures S7-S11). Nonetheless, CTLA-4 expression on CD8⁺ T cells was positively correlated with disease duration (Supplementary Figure 7J), and BTLA expression on CD4⁺ T cells was negatively correlated with duration of disease duration (Supplementary Figure S8C).

Given the significant correlations between baseline Th17 and Tc17 proportions, LAG-3 expression, and early PASI score improvement (Figures 2E, 4E, 5E, 6E), we investigated whether these markers could distinguish early and non-early responders. The expression of LAG-3 on CD4⁺ (p=0.0096) and CD8⁺ T (p=0.0093) cells, and the Th17 (p=0.0041) and Tc17 cell (p=0.0090) proportion were significantly higher in non-early responders than in early responders. (Figures 8A–H). No significant differences were observed in TIGIT expression between the groups (Figures 8I–L). Furthermore, ROC curves were used to assess the predictive value of immune checkpoint molecule expression for early efficacy. The AUC values for LAG-3 expression on CD4⁺ and CD8⁺ T cells, and the Th17 and Tc17 cell proportion were 0.893 (p= 0.011, 95% CI [0.7315–1.000]), 0.893 (p= 0.011, 95% CI [0.6899–1.000]), 0.946 (p= 0.004, 95% CI [0.8299–1.000]), and 0.893 (p= 0.011, 95% CI [0.7249–1.000]), respectively, indicating that these biomarkers may serve as potential predictors of early efficacy for ixekizumab. Additionally, optimal predictive thresholds for LAG-3 expression on CD4⁺ and CD8⁺ T cells, and the Th17 and Tc17 cell proportion were 3.295 (J = 0.625), 0.805 (J = 0.857), 0.570 (J = 0.875), and 0.495 (J = 0.714), respectively (Figures 8B, D, F, H).