Fig. 2.

Intrahepatic naive-like CD4⁺ T cells are present in inflammatory liver diseases and expanded in patients with PSC.

(A) Graphical abstract of the workflow on cryopreserved cells derived from patients with liver diseases of different aetiologies. (B) Frequencies of naive and naive-like CD4⁺ T cells from blood and liver, respectively, of the same patients determined by classical hierarchical gating for CCR7 and CD45RA. PSC showed the highest frequencies in both liver (PSC vs. ALD, p = 0.0039; PSC vs. LRM, p = 0.0205) and blood (PSC vs. ALD, p = 0.0039). Data in (B) is presented as median (IQR). Statistical significance within blood or liver was assessed by Kruskal-Wallis test (Liver: p = 0.0018; Blood: p = 0.0049). (C) PCA of multi-parameter flow cytometry data of intrahepatic CD4⁺ T cells from late-stage PSC (grey) and ALD (red). Permutational analysis of variance on Euclidean distance was performed to determine statistical differences between the groups (p = 0.001; R2 = 8.4%). (D) Random forest classifier for intrahepatic CD4⁺ T cells of PSC and ALD. Discrimination was assessed by AUC, specificity and sensitivity. Statistical significance of the classifier performance was tested by Mason’s and Graham’s non-parametric test. p <0.05 was considered statistically significant. (E) Populations of intrahepatic CD4⁺ T cells identified to best discriminate between PSC and ALD: identified by iteratively removing the features statistically proven to be less relevant than random samples generated by permutation of the original variable values. (F) UMAP projection of 4,072 intrahepatic T cells of late-stage PSC and ALD. Projection of naive-like DEG core signature, extracted from the atlas, is shown. Cells with matching transcriptomes are highlighted in red. Contribution of underlying disease to the cluster of naive-like CD4⁺ T cells is shown in the respective colour. (G) Volcano plot of DEG between intrahepatic naive-like CD4⁺ T cells from late-stage PSC and ALD, indicating similarity of gene expression. Lines indicate cut-off of statistical significance (p <0.05) and logarithmic fold-change of expression (|logFC| >0.7). See also Fig. S2 and Tables S2-3. ALD, alcohol-related liver disease; DEG, differentially expressed gene; LRM, liver resection margin; NASH, non-alcoholic steatohepatitis; PCA, principle component analysis; PSC, primary sclerosing cholangitis; UMAP, uniform manifold approximation and projection. Graphical abstract was created with BioRender.com.