Fig 2: Immune cells L‐PHA MFI over different time points. Boxplots of L‐PHA MFI on immune cells expressed as percentage fold change in T, B, and NK cells. The change in MFI of L‐PHA was compared between baseline and 6 months in the high‐dose arm (blue) and the low‐dose arm (gray) in (A) CD3+ (P = 0.007), (B) CD4+ (P = 0.005), (C) CD8+ (P = 0.026) T cells, (D) CD19+ B cells (P = 0.178), (E) CD56bright (P = 0.020), and (F) CD56dim, (p = 0.028) NK cells. Nonparametric analysis of covariate (ANCOVA) with baseline as covariate (n = 38). Abbreviations: BL, baseline, V6, 6 months, V12, 12 months, V18, 18 months.

Fig 3: Screening of immune checkpoints on CD8+ TILs in cholangiocarcinoma microenvironment. (A) Representative flow plots of immune checkpoints on CD8+ TILs. The proportion of Lag3+ cells in CD8+ TILs of tumor tissue, paratumor tissue, and normal tissue from one patient. (B-I) Quantitative analysis of immune checkpoints on CD8+ TILs. PD-1 (B), Tim-3 (C), Lag3 (D), Tigit (E), BTLA (F), CTLA4 (G), CD244 (H), and CD160 (I) were detected by flow cytometric analysis respectively. The connecting lines indicated that the tissue came from one patient (n = 15). (J) Statistical analysis of immune checkpoints on CD8+ TILs in cholangiocarcinoma tissue. Experiments were performed independently at least 3 times. Paired two-tailed student's t-test was used to compare two non-independent groups. Experimental data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.Abbreviations: TIL, tumor-infiltrating lymphocyte; PD-1, programmed death-1; Tim-3, T-cell immunoglobulin and mucin domain-3; Tigit, T cell immunoglobulin and ITIM domain; Lag3, lymphocyte activation gene 3; BTLA, B and T lymphocyte attenuator; CTLA4, cytotoxic T lymphocyte-associated protein-4; SEM, standard error of the mean.

Fig 4: The expression of CD8, IFN-?, and TNF-a in LSG tissues. The molecules of CD8 (green), IFN-? (orange), and TNF-a (red) were detected by immunofluorescence in the paraffin-embedded sections of LSG tissue of a pSS patient with focus score 2.9, and DAPI was used to counterstain the nuclei. The red arrows show CD8+ T cells with positive IFN-? and positive TNF-a in the Merge picture. Left scale bars = 100 µm, right scale bars = 50 µm

Fig 5: CD8+ T cell counts in LSG tissues and its correlations with disease parameters. A–C Representative images of three groups of patients: control patients (n = 6, A), pSS patients who were salivary gland 0≤ FS <1 (n = 12, B), and pSS patients who were salivary gland FS ≥1 (n = 11, C). D Comparations between the three groups. E Correlations of CD8+ T cell counts with disease parameters in pSS patients. Spearman correlation coefficients (r) and p values are indicated. CD8+ T cell counts had significant positive correlations with IgG, RF, ANA, and IFN-γ and had a negative correlation with complement C3. Left scale bars = 200 μm, right scale bars = 50 μm. Horizontal lines represent median and interquartile range, *p < 0.05, ***p < 0.001. IFN-γ: pg/mL; IgG: mg/dL; ESR: mm/h; RF: IU/mL; C3: mg/dL; C4: mg/dL. ANA titres graded at 4 levels (1:320, 1:1000, 1:3200, and 1:10000)