Fig 2: ICOS expression in primary Sjögren’s syndrome. (A) Comparison of ICOS expression in studied groups. Relative ICOS gene expression was determined by the 2−ΔΔCt method using GAPDH as a reference gene. (B) Relative expression of ICOS according to the polymorphism (IVS1 + 173 T/C) and (C) (c.1624 C/T). (D) Analysis of T cells in peripheral blood in controls subjects and patients. (E) Representation of mean fluorescence intensity (MFI) for ICOS. (F) Percentage of CD3+ CD4+ ICOS+ T cells in IVS1 + 173 T/C carriers. (G) MFI for ICOS in IVS1 + 173 T/C carriers. (H) Percentage of CD3+ CD4+ ICOS+ T cells in c.1624 C/T carriers. (I) MFI for ICOS in c.1624 C/T carriers. The level of significance is represented by * p < 0.05, ** p < 0.01, Mann–Whitney U test, ns = not significative.

Fig 3: Distribution of ICOS expression in pSS with focus score and antibodies. (A) Comparison of ICOS membrane expression (MFI) patients with =1, =2, and 4 ≥ focus score. (B) ICOS MFI according to the IgG serum levels (low < 700 mg/dL, normal IgG = 700–1600 mg/dL, and high IgG ≥ 1600 mg/dL). (C) (ICOS MFI according to the Ro/La antibodies positive or negative. (D) mRNA levels of ICOS according to focus score. (E) ICOS mRNA expression according to the IgG serum levels. (F) mRNA levels of ICOS according to Ro/La antibodies positive. Quantification of mRNA was performed using real-time PCR and the results are expressed as relative units (RU). The level of significance is represented by * p < 0.05, Kruskal–Wallis test and Mann–Whitney U-test, ns = not significative.

Fig 4: Co-inhibitory receptors exert distinct quantitative and qualitative control over T cell re-activation(A) Experimental design for blockade of co-inhibitory receptors.(B) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later mice were randomized to receive either 0.5 mg isotype pool (1:1 ratio of rat IgG1 and rat IgG2a), anti-Lag3, or anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells were analyzed for Nr4a3-Blue versus Nr4a3-Red analysis 4 h after peptide re-challenge.(C and D) Summary data from (B) detailing the percentage of responders (percent of Nr4a3-Blue+Red+, C) or median Nr4a3-Blue within Nr4a3-Blue+Red+ CD4+ T cells (D) in isotype (n = 5), anti-Lag3 (n = 6), or anti-PD1 (n = 6) treated mice. Bars represent mean ± SEM, dots represent individual mice. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test.(E) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later, mice were randomized to receive 0.8 mg isotype pool, 0.8 mg anti-Lag3, or 0.8 mg anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells expression of Nr4a3-Blue versus Nr4a3-Red 4 h after peptide re-challenge in pre-sorted (top) and sorted (bottom) populations.(F) RNA was extracted from the sorted populations and 3′ mRNA-seq performed. PCA of the normalized expression data identified 3 clusters, n = 2.(G) KEGG pathway analysis of DEG between isotype and anti-PD1 treated groups.(H) Z score heatmap analysis of log2 transformed and normalized counts displaying the 69 DEG between isotype and anti-PD1 groups, in isotype, anti-PD1, or anti-Lag3 groups.(I) Tg4 Nr4a3-Tocky Il10-GFP mice were immunized s.c. with 80 μg of [4Y] MBP. 24 h later mice received 0.5 mg rat IgG2a or or anti-PD1 30 min prior to re-challenge with 8 μg [4Y] MBP peptide. Splenic CD4+ T cells were analyzed for Nr4a3-Blue versus Nr4a3-Red analysis 4 h after peptide re-challenge. Gates were set on responding T cells.(J) Summary data from (I) for cells responding to dose 2 (n = 4). Bars represent mean ± SEM. ∗p < 0.05 by unpaired t test.(K) Histograms showing expression of Nr4a3, OX40, GITR and ICOS in responding T cells (indicated by gates in I) between isotype (gray)- or anti-PD1 (blue)-treated cells.(L) Summary data of the median of expression of the stated markers in responding T cells, n = 4. Bars represent mean ± SEM. Statistical analysis by unpaired t test. ∗∗p < 0.01, ∗∗∗p < 0.001.(M) Analysis of intracellular IRF8 in CD4+ Tg4 T cells from isotype (gray)- or anti-PD1 (blue)-treated mice, n = 4. Bars represent mean ± SEM. Statistical analysis by unpaired t test. ∗p < 0.05..(N) Analysis of intracellular STAT4 in isotype or anti-PD1 treated mice. Bars represent mean ± SEM, n = 4.Please also see Figures S4 and S5 and Table S3.

Fig 5: Lower ratio of PD‐1+CD8/ICOS+Tregs in advanced hepatitis B virus‐associated hepatocellular carcinoma (HBV‐HCC) patients indicates functional exhaustion. (A, B) Intracellular staining for perforin, granzyme B, and Ki67 in different PD‐1+CD8/ICOS+Tregs ratios from advanced HBV‐HCC patients (n = 60). (C, D) Percentage of expression of tissue‐resident memory T cells (TRM), granzyme B, and Ki67 of TRM in different PD‐1+CD8/ICOS+Tregs ratios from advanced HBV‐HCC patients (n = 60). p‐values were obtained by the Kruskal–Wallis ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001.