Demographic and clinical characteristics of the subjects
The demographic and clinical characteristics of PBC patients and HCs are shown in Table 1. A total of 74 PBC patients and 74 age- and sex-matched HCs were enrolled in the study. Significant differences were observed in serum levels of alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), transaminase, albumin (ALB), total bilirubin (TBIL) and platelet count (PLT) between PBC patients and HCs. Among PBC patients, 12 were treatment-naïve, and 33 (44.6%) responded to UDCA therapy according to Paris I (for cirrhotic PBC patients) and Paris II criteria (for noncirrhotic PBC patients).
Table 1
Demographic and clinical characteristics of the subjects.
Characteristics | PBC (n = 74) | HCs (n = 74) | P value |
Age, years | 57 (48–66) | 54 (48–62) | 0.052 |
Female gender, n (%) | 67 (90.5) | 68 (91.2) | 1.000 |
ALP, U/L | 144 (99–236) | 70 (55–83) | < 0.001 |
GGT, U/L | 67 (36–140) | 14 (13–18) | < 0.001 |
ALT, U/L | 29 (18–44) | 15 (13–20) | < 0.001 |
AST, U/L | 33 (27–50) | 20 (17–22) | < 0.001 |
ALB, g/L | 40.3 (34.2–43.7) | 42.7 (40.9–44.8) | 0.001 |
TBIL, µmol/L | 17.6 (11.7–26.3) | 12.6 (11.0-15.4) | < 0.001 |
PLT, 10^9/L | 168 (90–216) | 235 (191–262) | < 0.001 |
IgG, mg/dl | 1490 (1270–1730) | / | / |
IgM, mg/dl | 227 (122–355) | / | / |
AMA-M2 (+), n (%) | 64 (86.5) | / | / |
Cirrhosis, n (%) | 28 (37.8) | / | / |
Treatment-naive, n (%) | 12 (16.2) | / | / |
UDCA responders#, n (%) | 33 (44.6) | / | / |
P values refer to comparisons between PBC patients and healthy controls. |
#Paris I and Paris II criteria were performed in cirrhotic and non-cirrhotic PBC patients to identify UDCA responders, respectively. |
PBC, primary biliary cholangitis; HCs, healthy controls; ALP, alkaline phosphatase; GGT, gamma-glutamyl transpeptidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin; TBIL, total bilirubin; PLT, platelet count; IgG, immunoglobulin G; IgM, immunoglobulin M; AMA-M2, anti-mitochondrial M2 antibody; UDCA, ursodeoxycholic acid. |
Circulating TCRγδ low cells were significantly decreased in PBC patients compared with HCs
γδ T cells were defined as CD3 + TCRγδ + cells in the study. The representative flow cytometric analysis demonstrated two distinct subsets based on the CD3/γδTCR complex: the TCRγδhigh and TCRγδlow subsets (Fig. 1a). The frequency of circulating TCRγδlow cells among CD3 + cells was significantly higher than that of TCRγδhigh cells among CD3 + cells both in PBC patients (median 2.75% vs. 0.94%) and HCs (median 5.84% vs. 0.88%) (both p < 0.001).
The frequency and absolute number of circulating TCRγδlow cells were significantly decreased in PBC patients (n = 74) compared with HCs (n = 74) (p < 0.001), but the frequency and number of circulating TCRγδhigh cells were similar between the two groups (p > 0.05) (Fig. 1b).
The frequency of circulating TCRγδ low cells was correlated with disease severity and UDCA response
We found a significant positive correlation between the frequency of circulating TCRγδlow cells and the serum level of PLT (p = 0.001, r = 0.396), but no correlations were found between the frequency of circulating TCRγδlow cells and the levels of TBIL, ALP or aspartate aminotransferase (AST) (Fig. 1c). Furthermore, the frequency and absolute number of circulating TCRγδlow cells were significantly decreased in cirrhotic PBC patients (n = 28) compared with noncirrhotic PBC patients (n = 46) (Fig. 1d).
Additionally, subgroup analysis showed that the frequency and absolute number of circulating TCRγδlow cells were the lowest in UDCA treatment-naïve PBC patients, followed by nonresponders and responders. A significantly decreased frequency and number of circulating TCRγδlow cells were detected in treatment-naïve patients (n = 12) and UDCA nonresponders (n = 29) compared with UDCA responders (n = 33) (Fig. 1e, p < 0.05).
The proportion of Vδ2 T cells in the circulating TCRγδ low subset was decreased in PBC patients
We further detected the expression of Vδ1 and Vδ2 in the TCRγδhigh and TCRγδlow subsets (Fig. 2a). Approximately 75.1% of TCRγδhigh cells were Vδ1 T cells and 19.3% were Vδ1−Vδ2− T cells in HCs. The proportion of Vδ1 T cells in the TCRγδhigh subset was not different between HCs and PBC patients (p > 0.05, Fig. 2b).
In contrast, the majority of TCRγδlow cells were Vδ2 T cells in HCs. The proportion of Vδ2 T cells in the TCRγδlow subset was decreased (89.4% vs. 97.4%), and the proportion of Vδ1−Vδ2− T cells was increased (7.3% vs. 2.1%) in PBC patients compared with HCs (p < 0.001, Fig. 2c).
Circulating TCRγδ low cells exhibited similar apoptotic and proliferative phenotypes and enhanced CXCR6 expression in PBC patients
To explain the reduction in TCRγδlow cells in PBC patients, we assessed the apoptosis, proliferation, and expression of liver-homing chemokine receptors of γδ T cells (Fig. 3a). No difference was observed in the expression of annexin-V (AV) and Ki67 in either circulating TCRγδhigh cells or TCRγδlow cells between HCs and PBC patients (p > 0.05, Fig. 3b and 3c).
Compared with TCRγδhigh cells, the expression of liver-homing chemokine receptors, including CXCR6 and CXCR3, was significantly higher in TCRγδlow cells in both HCs and PBC patients (all p < 0.001). The expression of CXCR6 in TCRγδhigh cells and TCRγδlow cells was significantly increased in PBC patients compared with HCs (p < 0.05, Fig. 3d), but the expression of CXCR3 in TCRγδhigh cells and TCRγδlow cells was comparable between the two groups (p > 0.05, Fig. 3e).
Circulating TCRγδ low cells demonstrated an activated phenotype and enhanced GZMB production in PBC patients
The expression of activation markers, including CD69, HLA-DR, and CD25, in circulating γδ T cells was analyzed in PBC patients and HCs. Circulating TCRγδhigh cells were more activated than TCRγδlow cells in both HCs and PBC patients. Both the circulating TCRγδhigh cells and TCRγδlow cells in PBC were more activated than those in HCs, characterized by higher expression of CD69 and HLA-DR (p < 0.05, Fig. 4a).
In addition, circulating TCRγδhigh cells produced higher GZMB but lower IFN-γ and TNF-α than TCRγδlow cells in both HCs and PBC patients. We found an increased GZMB production of TCRγδlow cells in PBC patients (n = 28) compared with HCs (n = 30) (p < 0.05, Fig. 4c). The production of IFN-γ and TNF-α by TCRγδlow cells in PBC patients was lower than that in HCs, but the results remained statistically insignificant (p > 0.05, Fig. 4c).