The Relationship between CXCR6+CD8+T cells and Clinicopathological Parameters in Patients with primary biliary cholangitis

The Relationship between CXCR6+CD8+T cells and Clinicopathological Parameters in Patients with primary biliary cholangitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Relationship between CXCR6+CD8+T cells and Clinicopathological Parameters in Patients with primary biliary cholangitis Huilian Shi, Xiangtao Xu, Shuangshuang Wang, Qinlei Chen, Fan Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4426292/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Aug, 2024 Read the published version in Hepatology International → Version 1 posted 5 You are reading this latest preprint version Abstract Background CXCR6 + CD8 + T cells have been implicated in the pathogenesis of various liver and autoimmune diseases. However, their involvement in Primary Biliary Cholangitis (PBC) has not been eluciated. Methods We used immunohistochemistry and flow cytometry to quantify CXCR6 + CD8 + T cells in hepatic tissue and peripheral blood samples obtained from CXCR6 + CD8 + T cells obtained from PBC patients. Then, we performed comprehensive statistical analyses to access the correlation between the abundance of these cells and clinical as well as pathological data across different stages of PBC. Results Our research revealed that CXCR6 + cell frequencies in CD3 + CD8 + T cells from PBC patients significantly exceeded that of healthy controls (HCs) (2.24% vs. 0.61%, p < 0.01). A similar pattern emerged for hepatic CXCR6 + CD8 + T cell counts, which were notably higher in the PBC cohort compared to HCs. Our cohort consisted of 118 PBC patients, categorized into 62 early-stage (E-PBC) and 56 late-stage (L-PBC) cases. Notably, significant disparities existed between these groups in terms of liver enzyme and lipid profile levels (p 0.05). Intriguingly, the quantity of hepatic CXCR6 + CD8 + T cells per high power field (HPF) was significantly elevated in both E-PBC and L-PBC patients as opposed to normal liver samples, indicating a substantial increase in these cells across all stages of PBC (p = 0.000). Spearman’s rank correlation analysis using showed a positive correlation between CXCR6 + CD8 + T cell counts and serum levels of Alkaline Phosphatase (AKP) and Gamma-Glutamyl Transferase (GGT), while revealing a negligible correlation with Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST). Subsequent findings indicated significant variances in CXCR6 + cell numbers not only among different PBC stages but also across various degrees of inflammation and fibrosis (p ≤ 0.007). In a follow-up study post-Ursodeoxycholic Acid (UDCA) treatment, stark differences were identified in biochemical and immunohistochemical profiles between responder (31 patients) and non-responder (33 patients) groups (p < 0.05). A Wilcoxon rank-sum test further demonstrated a significant difference in the level of hepatic CXCR6 + CD8 + T cells between these two response groups (p = 0.002). Conclusion CXCR6 + CD8 + T cells play a vital role in the pathogenesis of PBC, exhibiting correlations with the extent of inflammation, staging of liver fibrosis, and response to pharmacological interventions in PBC patients. Primary biliary cirrhosis CXCR6 + CD8 + T cell༛Autoimmune liver disease༛Clinical and pathological data Figures Figure 1 Figure 2 Figure 3 1.Introduction Primary Biliary Cholangitis (PBC) is a chronic and progressive non-suppurative inflammatory condition characterized by damage to the epithelial cells of the bile ducts. It represents an organ-specific autoimmune liver disease that can ultimately lead to bile stasis, liver fibrosis, and potentially cirrhosis[ 1 ]. With advancements in diagnostic techniques, there has been a noted rise in the global incidence of PBC, with reported annual rates ranging from 0.9 to 5.8 per 100,000 individuals. Notably, the incidence in women over the age of 40 is as high as 155.8 per 100,000[ 2 ]. PBC typically begins insidiously, primarily manifesting as fatigue and pruritus, with a majority of patients already in the cirrhotic stage at the time of diagnosis[ 3 ]. Currently, the recommended treatment for PBC is Ursodeoxycholic acid (UDCA) at a dose of 13 to 15 mg per kilogram of body weight per day. However, approximately one-third of patients respond poorly to UDCA treatment, particularly those in advanced stages of the disease who have an even higher proportion of nonresponse[ 4 ]. Consequently, the prevention and treatment of PBC are of paramount importance. To date, the pathogenesis of PBC remains incompletely understood. Emerging research emphasizes the pivotal role of immune responses in liver damage, with particular attention to the role of alterations in immune system function in the disease mechanism of PBC. Current evidence underscores the significant involvement of CD8 + T cell responses in the pathogenic process of PBC. Previous studies have suggested elevated levels ofboth CD8 + T cell abundance and activation state in PBC, with these factors correlating with disease severity[ 5 ]. However, the regulation of CD8 + T cells in PBC patients remains a puzzle yet to be fully solved. Among these, a subset of CD8 + T cells expressing CXCR6 has sparked interest. Past studies have indicated that these cells, expressing the chemokine receptor CXCR6, can become activated upon lipid stimulation, leading to an aggressive attack on normal liver cells and exacerbating liver inflammation[ 6 ]. The proactive cytotoxicity of this particular group of CD8 + T cells has drawn widespread attention due to its implications. Literature suggests that CXCR6 + CD8 + T cells serve as predictive biomarkers for immune infiltration and therapeutic outcomes in hepatocellular carcinoma, dominating the immune microenvironment and regarded as potential therapeutic biomarkers for immunotherapy in primary liver cancer[ 7 ]. However, research on CXCR6 + CD8 + T cells in the realm of PBC is limited. Therefore, our research aims to explore the quantity of circulating and hepatic CXCR6 + CD8 + T cells in PBC, and their relationship with clinical data, disease severity, and drug treatment response in PBC patients. 2.Methods Patients Patients diagnosed with PBC underwent confirmation and follw-up at the Affiliated Hospital of Nanjing University of Chinese Medicine from 2021 to 2023. This study received ethical approval from the Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine, with the approval number 2021NL-101-03. All procedures carried out were in strict compliance with the ethical guidelines of the responsible committee on human experimentation, at both institutional and national levels, and adhered to the principles of the Helsinki Declaration of 1975, as amended in 2008. Informed consent was secured from all participants prior to their inclusion in the study. Inclusion Criteria: Based on the practice guidelines of the American Association for the Study of Liver Diseases (AASLD) [ 8 ], patients who meet two or more of the following three criteria were diagnosed with PBC: ① Serological biochemical changes related to cholestasis, with elevated levels of alkaline phosphatase (ALP) or gamma-glutamyltransferase (GGT), without explicable causes.②Presence of anti-mitochondrial antibodies (AMA) with a positive titer.③Liver biopsy showing non-suppurative cholangitis and the destruction of the medium and small bile ducts. Exclusion Criteria:①Intrahepatic local bile duct obstruction or extrahepatic biliary obstruction. ②Other liver diseases such as alpha-1 antitrypsin deficiency, viral hepatitis, alcoholic liver disease, drug-induced liver injury, and intrahepatic cholestasis of pregnancy.③ Autoimmune hepatitis, primary sclerosing cholangitis, and other autoimmune diseases such as Sjögren's syndrome, systemic lupus erythematosus, and other systemic diseases associated with liver damage. Based on hematoxylin and eosin (H&E) staining of liver biopsy samples, patients with PBC are classified into two stages: advanced PBC, indicating patients with cirrhosis, and early-stage PBC, indicating patients without cirrhosis. The biochemical response to UDCA treatment in both advanced and early-stage PBC patients is assessed in accordance with the Paris I and Paris II criteria [ 9 ], respectively. Follow-up assessments are conducted over a six-month period, with comprehensive biochemical testing and peripheral blood flow cytometry both before and after treatment. Clinical and laboratory assessments Blood samples were collected from all study participants. Prior to initiating treatment, serological, biochemical, and immunological testing was conducted. Each patient underwent a clinical examination along with routine liver function tests. The clinical laboratory conducted a complete blood count, assessing white blood cells (WBC), platelets (PLT), hemoglobin (Hb), and measured serum levels of various enzymes and markers including aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total bilirubin (TBil), direct bilirubin (Dbil), total bile acids (TbA), total cholesterol (CHOL), triglycerides (TG), low-density lipoprotein (LDL), and lipoprotein (a) (LP(a)). The serological presence of anti-mitochondrial antibodies types 2, 4, and 9 (AMA2, AMA4, AMA9) was determined using immunofluorescence (IF) analysis. Flow Cytometry Blood samples collected from the study participants were analyzed for the quantity and phenotypes of circulating CXCR6 + CD8 + T cells using flow cytometry. Antibodies including anti-CD8 and anti-CXCR6-PE, procured from Biolegend (San Diego, CA, USA), were utilized for the staining process., both of which were. Flow cytometric analysis was conducted using a FACSAria II flow cytometer (BD Biosciences, CA, USA), with subsequent data analysis conducted by FlowJo software. Immunohistochemistry and Immunofluorescence Immunofluorescence staining was meticulously performed on paraffin-embedded liver tissue sections. The primary antibodies used included a human-specific CXCR6 antibody (Catalog No. NLS1102, BSA-Free, from NovusBio) and a CD8 alpha Monoclonal Antibody (Clone C8/144B, Catalog No. MA5-13473, sourced from Thermo Fisher Scientific). These were subsequently detected with respective secondary antibodies: a CY3-conjugated goat anti-rabbit (provided by Servicebio, Wuhan, China) for CXCR6, and an AF488-conjugated goat anti-mouse (also from Servicebio, Wuhan, China) for CD8 alpha. Following counterstaining with DAPI, the stained sections were visualized under a NIKON Eclipse Ti fluorescent microscope. Cell density analysis for the markers of interest, CXCR6 and CD8 alpha, was quantified using the ImageJ software. The quantification was deliberately kept non-specific to encompass a range of cell types identifiable by these markers. For a more detailed analysis of specific cell populations highlighted by these markers, further elaboration can be provided as needed. Statistical Analysis All statistical analyses were conducted using SPSS software, version 20.0. The normality of continuous variables was assessed using the Shapiro-Wilk test. Normally distributed continuous variables are presented as means ± standard deviations (SD), whereas non-normally distributed continuous variables are expressed as medians (P25, P75). Categorical data are shown as counts (%). For group comparisons, the Student's t-test was applied to normally distributed continuous variables, the Mann-Whitney U test was utilized for non-normally distributed continuous variables, and the chi-square (χ2) test was used for categorical data. The significance level was set at α = 0.05. 3.Results 3.1 Higher Peripheral/heptatic CXCR6 + CD8 + T Cells From Patients With PBC Our initial investigation focused on assessing the prevalence of CXCR6 + CD3 + CD8 + T cells within the peripheral blood mononuclear cells (PBMCs) from PBC patients, in comparison to HCs. As depicted in Figs. 1 A and 1 B, the results of flow cytometry analyses for CXCR6 + CD8 + T cells in HCs and PBC patients, respectively. We observed a significantly higher frequency of CXCR6 + cells within the CD3 + CD8 + T cell population in PBC patients compared to HCs (2.24 ± 1.2% versus 0.61 ± 0.28%, p < 0.01, n = 10). However, the presence and extent of these T cells in the liver remained unclear. To address this, we subsequently employed immunofluorescence techniques to investigate the infiltration of CXCR6 + CD8 + T cells in hepatic tissue. Our findings revealed a notably increased quantity of CXCR6 + CD8 + T cells in the livers of PBC patients compared to the HC group. These outcomes suggest that CXCR6 + CD8 + T cells may contribute to the pathogenesis of PBC. 3.2 CXCR6 + CD8 + T Cells show difference in Patients with PBC (early-stage PBC and late-stage PBC) We enrolled a total of 118 PBC patientsbased on elevated cholestatic liver enzymes and typical histological features in liver biopsy specimens. According to the Ludwig classification [ 10 ], patients were categorized in two groups:early-stage PBC (E-PBC, 62 cases) and late-stage PBC (L-PBC, 56 cases). Significant statistical differences were observed between the two groups in markers such as ALT, AST, G, A, AKP, GGT, TBI, DBI, and TBA (P < 0.05). Similarly, there were significant statistical differences in blood lipids, including LDL and Lipa (P < 0.05). However, there were no statistically significant differences between the two groups in terms of gender, age, WBC, Hb, PLT, CHOL, TG, high-density lipoprotein (HDL), ferritin, antinuclear antibodies (ANA), and anti-mitochondrial antibodies (AMA2, AMA4, AMA9) (P > 0.05) (Table 1 ). It is noteworthy that immunohistochemical analysis of hepatic CXCR6 + CD8 + T cells in the portal vein area revealed a significant increase in their numbers in both early-stage and late-stage PBC patients compared to normal livers (Fig. 2 A, B, C). Furthermore, compared to E-PBC patients (n = 21), L-PBC patients (n = 25) exhibited a marked increase in the number of CXCR6 + CD8 + T cells, with a significant statistical difference in the expression of this specific T cell subset between the two groups (z=-5.667, P = 0.000), as detailed in Table 1 . This suggests a strong correlation between CXCR6 + CD8 + T cells and the severity of PBC, potentially implicating them in the progression of the disease.. The clinical manifestations of PBC typically include elevated GGT and AKP levels, and severe cases may also present with elevated ALT and AST levels. To explore the relationship between CXCR6 + CD8 + T cells and liver inflammation, Spearman correlation analysis was conducted, revealing a positive correlation between CXCR6 and AKP (r = 0.3150, P = 0.003) and GGT (r = 0.6839, P < 0.000) levels. However, the correlation with ALT (r = 0.1097, P = 0.4679) and AST (r = 0.1771, P = 0.2391) was minimal. These results indicate a stronger correlation between the abundance of this specific T cell subset and elevated AKP and GGT levels, suggesting their involvement in bile duct injury processes, leading to significant increases in AKP and GGT (Fig. 2 D, E, F, and G ). In conclusion, CXCR6 + CD8 + T cells isolated from patients with late-stage PBC patients exhibited inflammatory activity, suggesting their potential role in the pathological process of PBC. Table 1 Summarizes the main clinical and biochemical characteristics of the 118 patients with E-PBC and L-PBC E-PBC(n = 62) L-PBC(n = 56) t / Χ 2 /z P Age(year) 53.76 ± 8.84 53.05 ± 11.83 t = 0.369 0.713 Sex Male 9(14.5) 8(14.3) Χ 2 = 0.001 0.972 female 53(85.5) 48(85.7) WBC(×10^9/L) 4.80 ± 1.76 5.01 ± 1.75 t=-0.613 0.541 Hb(g/L) 123.98 ± 16.20 121.69 ± 14.57 t = 0.707 0.481 PLT(×10^9/L ) 173.31 ± 77.22 169.62 ± 71.22 t = 0.245 0.807 ALT(U/L) 43(26, 104.5) 97(45, 141) z=-2.760 0.006 AST(U/L) 40(26, 78) 66(43, 120) z=-3.956 0.000 A(g/L) 40.04 ± 4.48 38.20 ± 4.44 t = 2.243 0.027 G(g/L) 27.40 ± 5.53 30.50 ± 6.70 t =-2.751 0.007 AKP(U/L) 134(104, 179) 280(173, 353) z=-4.692 0.000 GGT(U/L) 116(62, 218) 330(134, 675) z=-4.223 0.000 TBI(µmol/L) 11.86(7.88, 19.83) 21.61(13.73, 49.63) z=-3.614 0.000 DBil(µmol/L) 2.6(1.72,6.35) 7.9(4.15, 32.64) z=-4.403 0.000 TBA(umol/L) 16.2(7.55, 32.65) 28.2(12.3, 96.9) z=-3.422 0.001 CHOL(mmol/L) 4.57(3.61, 5.29) 5.06(3.99, 6.27) z=-1.719 0.086 TG(mmol/L) 1.4(0.95, 1.64) 1.33(0.85, 2.09) z=-0.927 0.345 HDL(mmol/L) 1.48(1.14, 1.67) 1.50(1.28, 1.99) z=-1.268 0.205 LDL(mmol/L) 2.38(1.79, 2.84) 2.85(2.03, 3.52) z=-2.038 0.042 Lipa(mg/dL) 60(24, 94) 41(24, 94) z=-1.774 0.076 Ferritin(µg/L) 100.5(47.4, 164.85) 131.9(62.7, 243.4) z=-0.546 0.585 The number of CXCR6 + CD8 + T cell in liver(n/HPF ) 56(14, 93.5)*༈n = 21༉ 139(82, 181.5)*༈n = 25༉ z=-3.849 0.000 ANA quantification 320(100, 320) 320(320, 400) Z=-0.481 0.631 AMA2 Neg 28(45.2) 18(32.1) Χ 2 = 2.096 0.148 Pos 34(54.8) 38(67.9) AMA4 Neg 44(83) 38(88.4) Χ 2 = 0.546 0.460 Pos 9(17) 5(11.6) AMA9 Neg 47(88.7) 41(95.3) Χ 2 = 1.671 0.434 Pos 5(9.4) 2(4.7) P values refer to comparisons between E-PBC and L-PBC patients PBC primary biliary cholangitis,, ALP alkaline phosphatase, GGT gamma-glutamyl transpeptidase, ALT alanine aminotransferase, AST aspartate aminotransferase, ALB albumin, TBIL total bilirubin, PLT platelet count,AMA-M2 ,4,9,anti-mitochondrial M2,4 and 9 antibody.,TG triglyceride,TBA total bile acids,CHOL cholesterol,HDL high-density lipoprotein, LDL Low-density lipoprotein, Lipa lipoprotein a 3.3 CXCR6 + CD8 + T Cells Isolated From Patients With L-PBC Show a Higher Activity of Inflammation and Fibrosis Degree Due to the differential infiltration of CXCR6 + CD8 + T cells across various stages of PBC, our aim was to elucidate the relationship between these specialized T cells and the degree of liver inflammation and fibrosis. Employing HE and Masson's staining techniques, we observed a positive correlation between CXCR6 + CD8 + T cells and liver inflammation and fibrosis (Fig. 3 A). Statistical analysis revealed significant differences in the abundance of CXCR6-positive cells between different states of PBC (E-PBC and L-PBC) (Fig. 3 B, P = 0.000), as well as significant statistical differences between different grades of inflammation (G grades) and fibrosis (S grades) (with H = 27.119, P = 0.000 and H = 12.147, P = 0.007, respectively). These findings strongly suggest that the involvement of CXCR6 + CD8 + T cells in the processes of inflammation and fibrosis in PBC. 3.4 The elevation of CXCR6 + CD8 + T cells is considered one of the factors contributing to poor response in PBC. The aforementioned results demonstrate a significant correlation between the quantity of CXCR6 + CD8 + T cells and the levels of inflammation, liver fibrosis, and the disease progression of PBC. Here, the relationship between these T cells and the response to UDCA treatment was explored. According to the AASLD guidance [ 11 ], patients undergoing standard UDCA treatment were monitored for six months. A total of 64 patients were included in the follow-up, and categorized into responder and non-responder groups, with 31 patients identified as responders and 33 as non-responders. Through single-factor analysis, notable statistical variances were observed between the two groups in various biochemical indicators, including ALT, AST, G, AKP, GGT, TBI, Dbi, and TBA, as well as Hb, HDL, and LDL (p < 0.05) (Table 2 ). Employing rank sum tests, it was discerned that the levels of specialized CXCR6 + CD8 + T cells in immunohistochemistry exhibited significant disparities between responders and non-responders (Z= -3.057, p = 0.002) (Fig. 4). The count of hepatic CXCR6 + CD8 + T cells per HPF was notably higher in UDCA responders (n = 5) compared to UDCA non-responders (n = 5), indicating an association between CXCR6 + CD8 + T cells and responsiveness to UDCA treatment. Table 2 Summarization of the main clinical and biochemical characteristics of the 64 patients in UDCA non-responders compared with UDCA responders UDCA responders(n = 31 ) UDCA non-responders (n = 33) t / Χ 2 /z P Age(year) 51.55 ± 11.30 53.39 ± 10.00 t=-0.393 0.491 Sex Male 4(12.9) 3(9.1) 0.238 0.625 Female 27(87.1) 30(90.9) WBC(10^9/L) 5.10 ± 1.65 4.99 ± 1.86 t = 0.223 0.825 Hb(g/L) 129.4 ± 16.70 116.64 ± 14.92 t = 2.942 0.005 PLT(10^9/L ) 165.39 ± 81.90 177.75 ± 78.06 t=-0.568 0.572 ALT(U/L) 26.5(18,49.25) 77(43,110) z=-3.279 0.001 AST(U/L) 28(24,42.5) 61(43,88) z=-4.824 0.000 A(g/L) 40.56 ± 4.54 38.31 ± 3.94 t = 0.575 0.038 G(g/L) 27.38 ± 5.00 31.11 ± 5.56 t=-2.817 0.006 AKP(U/L) 115.5(82,135) 330(210,405) z=-6.872 0.000 GGT(U/L) 68(38.75,113.75) 360(155,675) z=-5.179 0.000 TBI(µmol/L) 9.50(5.90,12.4) 22.64(12.75,49.63) z=-2.935 0.003 DBil(µmol/L) 1.72(1.18,2.64) 8.39(3.82,32.64) z=-3.849 0.000 TBA(umol/L) 13.65(8,27.45) 48.3(20.2,97.9) z=-3.649 0.000 CHOL(mmol/L) 4.53(3.52,5.20) 5.98(5.06,8.22) z=-3.337 0.001 TG(mmol/L) 1.32(1.02,2.21) 1.56(0.85,2.18) z=-0.613 0.540 HDL(mmol/L) 1.33(1.15,1.58) 1.94(1.57,2.29) z=-3.705 0.000 LDL(mmol/L) 2.49(1.73,3.14) 2.94(2.19,3.61) z=-2.647 0.008 Lipa(mg/dL) 91(47.5,188.25) 41(14,94) z=-0.975 0.330 Ferritin(µg/L) 60.5(27.65,132.15) 88.7(42.8,177.9) z=-1.365 1.172 The number of CXCR6 + CD8 + T cell in liver (n/HPF ) 76(18,132)(n = 5) 149(80.25,191.25)(n = 5) z=-2.245 0.0025 Paris I and Paris II criteria were utilized in late-stage (stage III–IV) and early-stage (stage I–II) PBC patients, respectively, to identify responders to UDCA. The data are presented as medians with interquartile ranges. Abbreviations: PBC: Primary Biliary Cholangitis；ALP: Alkaline Phosphatase；GGT: Gamma-Glutamyl Transpeptidase；ALT: Alanine Aminotransferase；AST: Aspartate Aminotransferase；ALB: Albumin；TBIL: Total Bilirubin；PLT: Platelet；TG: Triglyceride；TBA: Total Bile Acids；CHOL: Cholesterol；HDL: High-Density Lipoprotein；LDL: Low-Density Lipoprotein；Lipa: Lipoprotein A Discussion PBC is a chronic, progressive liver disease with an unidentified etiology, primarily characterized by cholestasis and liver damage. The pathogenesis of PBC is multifaceted, with the immune response playing a key role in its pathological process[12]. Our study indicates that x patients with advanced PBC are more susceptible to hepatocyte and bile duct damage compared to those in the early stages. This increased susceptibility results in elevated levels of serum indicators, such as ALT and GGT. HE staining has revealed significant increase in the infiltration of CXCR6+CD8+T cells around the hepatic bile duct region in patients with advanced PBC. This indicates a higher likelihood of severe hepatitis, extensive bile duct damage, and increased fibrosis in the later stages of the disease. These results are consistent with the findings of Zhao, et al[13], who reported an increase in CD8+ T cells in L-PBC patients, accompanied by lymphoid follicle formation, bile duct damage, and fibroblast proliferation. Collectively, these pathological characteristics show the potential involvement of CXCR6+CD8+T cells in the pathogenesis of PBC. It is well-known that CD8+ T cells are composed of various subtypes, yet the specific mechanisms by which these subtypes act in PBC remain unclear. Recent research highlights the pivotal role of CXCR6+ CD8+ T cells in the progression of non-alcoholic steatohepatitis (NASH), particularly their ability to directly destroy liver cells, which has greatly attracted our attention[14]. In this study, we observed a significant increase in the number of CD8+ T cells expressing C-X-C motif chemokine receptor 6 (CXCR6) both in the circulating blood and liver tissues of PBC patients, a novel finding not previously documented. CXCR6, a member of the CXC chemokine subfamily, is primarily distributed on the surface of different types of various T lymphocytes[15]. Previous studies have shown that CXCR6 is preferentially expressed on memory T cells as well as activated Th1 and Tc1 effector T cell subgroups[16]. In oncology, CXCR6 expression is crucial for the anti-tumor activitie=s of CD8+ T cells[17,18]. In addition, CXCR6+ CD8+ T cells accumulate significantly in the peripheral blood of elderly individuals, suggesting a potential role in the progression of neurodegenerative diseases[19]. In hepatology, these cells also play an important role in liver cancer and represent a potential therapeutic target for primary liver cancer[20]. In our in-depth study, we have found that the escalation inCXCR6+CD8+ T cells throughout the advancement of PBC presents a potential risk factor. Our analysis has observed a notable surge in circulating CXCR6+CD8+ T cells among PBC patients, which is closely associated with both disease severity and the efficacy of UDCA treatment. Particularly, within the periportal regions of PBC patients’ livers, a marked increase in intrahepatic CXCR6+CD8+ T cells was observed. Our statistical analysis further confirmed a correlation between the abundance of these cells, the biomarkers of cholestasis, and the response to UDCA treatment. Although previous studies have highlighted the importance of NKT cells and gamma-delta (γδ) T cells in PBC[21,22], the function of CXCR6+CD8+ T cells has not been fully explored. Early studies suggest that CXCR6 is a key chemotactic factor in the liver, and its dependent aggregation of NK T cells leads to increased inflammation and fibrosis[23,24]. However, the precise function of CXCR6+CD8+ T cells in PBC remains unclear. Studies indicate that these cells can produce a variety of cytokines, especially IFN-γ, TNF-α, and GZMB, demonstrating their antiviral, pro-inflammatory, and cytotoxic capabilities[14]. It is noteworthy that many PBC patients display abnormal lipid metabolism, consistent with the findings of Zhang et al[25]., which pointed out minimal hepatic steatosis and higher levels of high-density lipoprotein cholesterol (HDL-C) in PBC patients. Our study also found significant differences in CHOL, TG, and HDL-C among PBC patients at different stages. In the study of NASH pathogenesis, it is known that CXCR6+CD8+ T cells not only exhibit cytotoxicity driven by lipids but also engage in "auto-aggressive killing" of normal cells. We hypothesize that within the pathogenesis of PBC, CXCR6+CD8+ T cells might activate in an environment of lipid metabolism disorder, thereby releasing cytokines to enhance inflammatory responses. At the same time, these cells may aggravate liver damage by "auto-aggressive" behaviors, fostering the spread of inflammation and fibrosis. In summary, CXCR6+CD8+ T cells may wield significant influence in PBC development, contributing to the immunopathological cascade through cytotoxic effects, inflammatory mediator secretion, and interactions with other immune cells. Nevertheless, the detailed content of these mechanisms requires further scientific inquiry. This study has several limitations that warrant discussion. Firstly, the lack of sufficient fresh PBC liver samples precluded the utilization of flow cytometry for a detailed analysis of both the quantity and functionality of CD8+ T cell subsets within the liver. We anticipate that future studies can use advanced technologies, such as single-cell RNA sequencing, to delve more deeply into the diverse functions of CXCR6-related T cell subsets in PBC. Secondly, this study was designed as a cross-sectional study. If it were possible to implement a larger sample size and conduct long-term tracking of the changes in CXCR6+CD8+ T cell levels before and after UDCA treatment, more rich and meaningful data might be obtained. Overall, the progression of chronic liver disease, from persistent inflammation to fibrosis and potentially carcinogenesis, presents a formidable challenge in the development of new therapeutic strategies. Our research results provide empirical evidence regarding the activity of the CXCR6+CD8+ T cell subset in PBC patients, particularly their localization within the liver. The different expressions of this subset at various stages of PBC and its correlation with the response to UDCA treatment need further exploration. A thorough understanding of the role played by CXCR6+CD8+ T cells in PBC is crucial for revealing the pathogenic mechanisms underlying this condition and for the development of related therapeutic interventions. We look forward to future studies that will shed light on the functions and regulatory mechanisms of CXCR6+CD8+ T cells in PBC through more experiments and clinical data. Declarations Acknowledgements The authors especially thank Dr. Weiting Lu for her assistance in observing and choosing the microphotographs, and Dr. Shuangshuang Wang for her help in the immunohistochemistry experiment. Author contributions Huilian Shi and Fei Qiao designed, revised and finalized the manuscript. Weiting Lu for her help in observing and choosing the microphotographs. Shuangshuang Wang performed the experiment, Xiangtao Xu 、Haiyan Guo analyzed the data and drafted the manuscript.Doctors Qinlei Chen and Fanzhang collected the blood sample and clinical data. All authors read and approved the fnal manuscript. Funding This research was funded by the Natural Science Foundation of Jiangsu Province BK20221421 (Huilian Shi). Availability of data and material Not applicable. Code availability Not applicable. Conflict of interest The authors declare that there are no competing interests associated with the manuscript. Ethics approval All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. The ethics committee of the Affiliated Hospital of Nanjing University of Chinese Medicine reviewed and approved the study. Consent to participate Written informed consent was obtained from all subjects enrolled in the study. Consent for publication Written informed consent for publication was obtained from all participants. References Houri Inbal,Hirschfield Gideon M,Primary Biliary Cholangitis. Pathophysiology [J] Clin Liver Dis. 2024;28:79–92. Faisal Muhammad Salman,Gonzalez, Humberto C, Gordon Stuart C. Primary Biliary Cholangitis: Epidemiology, Diagnosis, and Presentation.[J].Clin Liver Dis, 2024, 28: 63–77. European Association for the Study of the Liver. EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis. J Hepatol. 2017;67:145–72. Corpechot C, Carrat F, Bahr A, et al. The efect of ursodeoxycholic acid therapy on the natural course of primary biliary cirrhosis. Gastroenterology. 2005;128:297–303. Zhang STXW, Li, et al. Downregulation of Programmed Death-1 Pathway Promoting CD8 + T Cell Cytotoxicity in Primary Biliary Cholangitis. [J] Dig Dis Sci. 2022;67:2981–93. Dudek Michael,Pfister Dominik,Donakonda Sainitin. Auto-aggressive CXCR6 CD8 T cells cause liver immune pathology in NASH.[J]. Nature. 2021;592:444–9. Gou Haoxian,Liu Shenglu,Liu Linxin. Obeticholic acid and 5β-cholanic acid 3 exhibit anti-tumor effects on liver cancer through CXCL16/CXCR6 pathway[. J] Front Immunol. 2022;13:1095915. Lindor KD, Bowlus CL, Boyer J, et al. Primary biliary cholangitis: 2018 practice guidance from the American association for the study of liver diseases. Hepatology. 2019;69:394–419. Chazouilleres 9CC, Poupon O. Early primary biliary cirrhosis: biochemical response to treatment and prediction of long-term outcome. J Hepatol. 2011;55:1361–7. Ludwig J, Dickson ER, McDonald GS. Staging of chronic nonsuppurative destructive cholangitis (syndrome of primary biliary cirrhosis). Virchows Arch Pathol Anat Histol. 1978;379:103–12. Lindor KD, Bowlus CL, Boyer J, Levy C, Mayo M. Primary biliary cholangitis: 2018 practice guidance from the American association for the study of liver diseases. The American association for the study of liver diseases practice guidelines. Hepatology. 2019;69(1):394–419. Hebbandi Nanjundappa Roopa,Shao Kun,Krishnamurthy Prasanna. Invariant natural killer T cells in autoimmune cholangiopathies: Mechanistic insights and therapeutic implications[. J] Autoimmun Rev. 2023;23:103485. Zhao S-X. Emperipolesis mediated by CD8 T cells correlates with biliary epithelia cell injury in primary biliary cholangitis.[J]. J Cell Mol Med. 2020;24:1268–75. Li Wen-Cong,Fu Na. Dudek Michael,Pfister Dominik,Donakonda Sainitin. Auto-aggressive CXCR6 CD8 T cells cause liver immune pathology in NASH.[J]. Nature. 2021;592:444–9. Heesch Kira,Raczkowski Friederike,Schumacher Valéa. The function of the chemokine receptor CXCR6 in the T cell response of mice against Listeria monocytogenes[. J] PLoS One. 2014;9:e97701. Andrea TS-W, Espinosa Diego J et al. A. The chemokine receptor CXCR6 is required for the maintenance of liver memory CD8⁺ T cells specific for infectious pathogens.[J].J Infect Dis, 2014, 210: 1508-16. Mossanen Jana C, Alexander, et al. CXCR6 Inhibits Hepatocarcinogenesis by Promoting Natural Killer T- and CD4 T-Cell-Dependent. Control Senescence [J] Gastroenterol. 2019;156:1877–e18894. Kee Ji-Ye,Ito Aya,Hojo Shozo et al. Chemokine CXCL16 suppresses liver metastasis of colorectal cancer via augmentation of tumor-infiltrating natural killer T cells in a murine model[. J] Oncol Rep, 2013, 29: 975–82. Li T, Hongqi, et al. CXCR6-based immunotherapy in autoimmune, cancer and inflammatory infliction[. J] Acta Pharm Sin B. 2022;12:3255–62. Xiao-Ying GQZY-J et al. CXCR6 upregulation contributes to a proinflammatory tumor microenvironment that drives metastasis and poor patient outcomes in hepatocellular carcinoma.[J].Cancer Res, 2012, 72: 3546-56. Chen S, Lv T, Sun G, et al. Reciprocal alterations in circulating and hepatic gamma-delta T cells in patients with primary biliary cholangitis. Hepatol Int. 2022;16(1):195–206. Hydes TJ, Blunt MD, Naftel J, et al. Constitutive Activation of Natural Killer Cells in Primary Biliary Cholangitis. Front Immunol. 2019;10:2633. Liepelt Anke,Wehr Alexander,Kohlhepp, Marlene, et al. CXCR6 protects from inflammation and fibrosis in NEMO mice.[J]. Biochim Biophys Acta Mol Basis Dis. 2019;1865:391–402. Wehr Alexander,Baeck Christer,Heymann Felix. Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fibrosis[. J] J Immunol. 2013;190:5226–36. Zhang Y, Hu X, Chang J, et al. The liver steatosis severity and lipid characteristics in primary biliary cholangitis. BMC Gastroenterol. 2021;21(1):395. Cite Share Download PDF Status: Published Journal Publication published 12 Aug, 2024 Read the published version in Hepatology International → Version 1 posted Editorial decision: Major Revisions Needed 14 Jun, 2024 Reviewers agreed at journal 25 May, 2024 Reviewers invited by journal 24 May, 2024 Editor assigned by journal 16 May, 2024 First submitted to journal 15 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4426292","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":306556088,"identity":"69a7a6f3-e467-434d-a9b6-abe69ab95fe4","order_by":0,"name":"Huilian Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACCcY2BgYDCx4GBuaDDxIqJOTkidQiAdTClmzw4YyFsWEDQS0MbGCSgYHHTHJmW0UiwwECOuRnN7c95imQkOGf3ZYgzTtPIoGxgfnhoxt4tBjcOdhuzAN0mMSdwweMebdJ5LEzsBkb5+DTIpHYJg3SwnAjLSEZqKWYsYGHTRqfFvkZUC3yN3IMDvPOkUhsOEBAC8MNqBaDGzmGjTMbiNBiANQiOQeoxfBGWjLDh2MSxobNBPwiPyP9mcSbPzb2cjeSj/9IqKmTk2dvfvgYr8MwATNpykfBKBgFo2AUYAEAvGlGGEkWz58AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2437-2573","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Huilian","middleName":"","lastName":"Shi","suffix":""},{"id":306556089,"identity":"692723f6-2a9d-4fd1-82c4-3dcde892d395","order_by":1,"name":"Xiangtao Xu","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiangtao","middleName":"","lastName":"Xu","suffix":""},{"id":306556090,"identity":"64a1ca6a-0a9b-4d17-b652-7d615eb7e2e1","order_by":2,"name":"Shuangshuang Wang","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuangshuang","middleName":"","lastName":"Wang","suffix":""},{"id":306556091,"identity":"aa13a159-3894-40b4-b7c4-df164f954ad9","order_by":3,"name":"Qinlei Chen","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qinlei","middleName":"","lastName":"Chen","suffix":""},{"id":306556092,"identity":"d5848365-e51d-477e-b805-8cc1ddcb6d18","order_by":4,"name":"Fan Zhang","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Zhang","suffix":""},{"id":306556093,"identity":"ba79374c-98ea-4804-9a62-0cb4dd4b6d27","order_by":5,"name":"Haiyan Guo","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Guo","suffix":""},{"id":306556094,"identity":"79d611fc-2cda-4973-a240-462922d0b7b1","order_by":6,"name":"Weiting Lu","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weiting","middleName":"","lastName":"Lu","suffix":""},{"id":306556095,"identity":"2579e288-f087-4181-aad6-1f0eef636840","order_by":7,"name":"Fei Qiao","email":"","orcid":"","institution":"Affiliated Hospital of Nanjing University of Chinese Medicine: Jiangsu Province Academy of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Qiao","suffix":""}],"badges":[],"createdAt":"2024-05-15 15:49:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4426292/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4426292/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12072-024-10715-0","type":"published","date":"2024-08-12T15:58:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58143998,"identity":"b53b8c37-1ec8-4a5b-adfe-b32c95a25ada","added_by":"auto","created_at":"2024-06-11 18:22:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":235111,"visible":true,"origin":"","legend":"\u003cp\u003eHigher Peripheral/heptatic CXCR6+CD8+T Cells From Patients With PBC\u003c/p\u003e\n\u003cp\u003e(A, B, and D) depict representative flow cytometry plots, along with the absolute counts (displayed at the bottom) of peripheral CXCR6+CD8+T cells isolated from both patients with Primary Biliary Cholangitis (PBC, n=5) and healthy controls (HCs, n=5). (C) showcases the results of immunofluorescence staining used to identify CXCR6+CD8+T cells in hepatic tissue samples from both PBC patients (n=8) and healthy control subjects (HC, n=8). In these images, CD8+ is marked in red, CXCR6+ in green, and co-stained cells in both colorsindicate the presence of CXCR6+CD8+T cells. The images were captured at a magnification of 400×, with a scale bar representing 50µm.\u003c/p\u003e","description":"","filename":"FIgure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4426292/v1/4bbe4bc39d5deb30aaba5f27.jpg"},{"id":58143995,"identity":"5a0cd417-98a5-4307-a202-1161789fd1ff","added_by":"auto","created_at":"2024-06-11 18:22:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":470314,"visible":true,"origin":"","legend":"\u003cp\u003eCXCR6+CD8+T Cells play an important role in L-PBC and in E-PBC patients.\u003c/p\u003e\n\u003cp\u003eA-C:HE and Immunohistochemistry staining of CXCR6+CD8+T cell in the liver of patients with L-PBC and in E-PBC(n=8);magnification: 40x,scale bar:250µm. D-G:The number of hepatic CXCR6+CD8+T cells/HPF was positively correlated with ALP and GGT, but not correlated with the level of ALT and AST(E-PBC:n=21, L-PBC:n=25 ).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4426292/v1/f28cf300fcaaa4475f47115c.jpg"},{"id":58145348,"identity":"b73352da-3ac6-49bd-af37-14e54a63e6d3","added_by":"auto","created_at":"2024-06-11 18:30:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":346421,"visible":true,"origin":"","legend":"\u003cp\u003eCXCR6+CD8+T Cells Isolated from PBC Patients Show Higher Correlation with Inflammation and Fibrosis Degree\u003c/p\u003e\n\u003cp\u003eA: Masson, HE, and Immunohistochemistry staining of CXCR6+CD8+T cells in the livers of PBC patients with stage S0 and stage S4, with a magnification of 20× and a scale bar of 500 µm. B: The frequencies and numbers of hepatic CXCR6+CD8+T cells detected in E-PBC patients (n=21) compared with L-PBC (n=25). C and D: The correlation of hepatic CXCR6+CD8+T cell population with pathological parameters in patients with PBC. p \u0026lt; 0.001 by the Student’s t-test indicates statistical significance. Correlations were calculated using Spearman's correlation analysis.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4426292/v1/54736e14138f69afa89bb5ff.jpg"},{"id":63071574,"identity":"570c7c31-8b2a-4104-a370-b928ddc8d655","added_by":"auto","created_at":"2024-08-22 20:08:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1689812,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4426292/v1/a9fe792e-ecf5-4338-a423-250ed64a3ae1.pdf"}],"financialInterests":"","formattedTitle":"The Relationship between CXCR6+CD8+T cells and Clinicopathological Parameters in Patients with primary biliary cholangitis","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003ePrimary Biliary Cholangitis (PBC) is a chronic and progressive non-suppurative inflammatory condition characterized by damage to the epithelial cells of the bile ducts. It represents an organ-specific autoimmune liver disease that can ultimately lead to bile stasis, liver fibrosis, and potentially cirrhosis[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. With advancements in diagnostic techniques, there has been a noted rise in the global incidence of PBC, with reported annual rates ranging from 0.9 to 5.8 per 100,000 individuals. Notably, the incidence in women over the age of 40 is as high as 155.8 per 100,000[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PBC typically begins insidiously, primarily manifesting as fatigue and pruritus, with a majority of patients already in the cirrhotic stage at the time of diagnosis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, the recommended treatment for PBC is Ursodeoxycholic acid (UDCA) at a dose of 13 to 15 mg per kilogram of body weight per day. However, approximately one-third of patients respond poorly to UDCA treatment, particularly those in advanced stages of the disease who have an even higher proportion of nonresponse[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consequently, the prevention and treatment of PBC are of paramount importance.\u003c/p\u003e \u003cp\u003eTo date, the pathogenesis of PBC remains incompletely understood. Emerging research emphasizes the pivotal role of immune responses in liver damage, with particular attention to the role of alterations in immune system function in the disease mechanism of PBC. Current evidence underscores the significant involvement of CD8\u0026thinsp;+\u0026thinsp;T cell responses in the pathogenic process of PBC. Previous studies have suggested elevated levels ofboth CD8\u0026thinsp;+\u0026thinsp;T cell abundance and activation state in PBC, with these factors correlating with disease severity[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the regulation of CD8\u0026thinsp;+\u0026thinsp;T cells in PBC patients remains a puzzle yet to be fully solved. Among these, a subset of CD8\u0026thinsp;+\u0026thinsp;T cells expressing CXCR6 has sparked interest. Past studies have indicated that these cells, expressing the chemokine receptor CXCR6, can become activated upon lipid stimulation, leading to an aggressive attack on normal liver cells and exacerbating liver inflammation[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The proactive cytotoxicity of this particular group of CD8\u0026thinsp;+\u0026thinsp;T cells has drawn widespread attention due to its implications. Literature suggests that CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells serve as predictive biomarkers for immune infiltration and therapeutic outcomes in hepatocellular carcinoma, dominating the immune microenvironment and regarded as potential therapeutic biomarkers for immunotherapy in primary liver cancer[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, research on CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in the realm of PBC is limited. Therefore, our research aims to explore the quantity of circulating and hepatic CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in PBC, and their relationship with clinical data, disease severity, and drug treatment response in PBC patients.\u003c/p\u003e"},{"header":"2.Methods","content":"\u003cp\u003e \u003cb\u003ePatients\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePatients diagnosed with PBC underwent confirmation and follw-up at the Affiliated Hospital of Nanjing University of Chinese Medicine from 2021 to 2023. This study received ethical approval from the Ethics Committee of the Affiliated Hospital of Nanjing University of Chinese Medicine, with the approval number 2021NL-101-03. All procedures carried out were in strict compliance with the ethical guidelines of the responsible committee on human experimentation, at both institutional and national levels, and adhered to the principles of the Helsinki Declaration of 1975, as amended in 2008. Informed consent was secured from all participants prior to their inclusion in the study.\u003c/p\u003e \u003cp\u003eInclusion Criteria: Based on the practice guidelines of the American Association for the Study of Liver Diseases (AASLD) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], patients who meet two or more of the following three criteria were diagnosed with PBC: ① Serological biochemical changes related to cholestasis, with elevated levels of alkaline phosphatase (ALP) or gamma-glutamyltransferase (GGT), without explicable causes.②Presence of anti-mitochondrial antibodies (AMA) with a positive titer.③Liver biopsy showing non-suppurative cholangitis and the destruction of the medium and small bile ducts. Exclusion Criteria:①Intrahepatic local bile duct obstruction or extrahepatic biliary obstruction. ②Other liver diseases such as alpha-1 antitrypsin deficiency, viral hepatitis, alcoholic liver disease, drug-induced liver injury, and intrahepatic cholestasis of pregnancy.③ Autoimmune hepatitis, primary sclerosing cholangitis, and other autoimmune diseases such as Sj\u0026ouml;gren's syndrome, systemic lupus erythematosus, and other systemic diseases associated with liver damage.\u003c/p\u003e \u003cp\u003eBased on hematoxylin and eosin (H\u0026amp;E) staining of liver biopsy samples, patients with PBC are classified into two stages: advanced PBC, indicating patients with cirrhosis, and early-stage PBC, indicating patients without cirrhosis. The biochemical response to UDCA treatment in both advanced and early-stage PBC patients is assessed in accordance with the Paris I and Paris II criteria [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], respectively. Follow-up assessments are conducted over a six-month period, with comprehensive biochemical testing and peripheral blood flow cytometry both before and after treatment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eClinical and laboratory assessments\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBlood samples were collected from all study participants. Prior to initiating treatment, serological, biochemical, and immunological testing was conducted. Each patient underwent a clinical examination along with routine liver function tests. The clinical laboratory conducted a complete blood count, assessing white blood cells (WBC), platelets (PLT), hemoglobin (Hb), and measured serum levels of various enzymes and markers including aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total bilirubin (TBil), direct bilirubin (Dbil), total bile acids (TbA), total cholesterol (CHOL), triglycerides (TG), low-density lipoprotein (LDL), and lipoprotein (a) (LP(a)). The serological presence of anti-mitochondrial antibodies types 2, 4, and 9 (AMA2, AMA4, AMA9) was determined using immunofluorescence (IF) analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow Cytometry\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBlood samples collected from the study participants were analyzed for the quantity and phenotypes of circulating CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells using flow cytometry. Antibodies including anti-CD8 and anti-CXCR6-PE, procured from Biolegend (San Diego, CA, USA), were utilized for the staining process., both of which were. Flow cytometric analysis was conducted using a FACSAria II flow cytometer (BD Biosciences, CA, USA), with subsequent data analysis conducted by FlowJo software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemistry and Immunofluorescence\u003c/b\u003e \u003c/p\u003e \u003cp\u003eImmunofluorescence staining was meticulously performed on paraffin-embedded liver tissue sections. The primary antibodies used included a human-specific CXCR6 antibody (Catalog No. NLS1102, BSA-Free, from NovusBio) and a CD8 alpha Monoclonal Antibody (Clone C8/144B, Catalog No. MA5-13473, sourced from Thermo Fisher Scientific). These were subsequently detected with respective secondary antibodies: a CY3-conjugated goat anti-rabbit (provided by Servicebio, Wuhan, China) for CXCR6, and an AF488-conjugated goat anti-mouse (also from Servicebio, Wuhan, China) for CD8 alpha. Following counterstaining with DAPI, the stained sections were visualized under a NIKON Eclipse Ti fluorescent microscope. Cell density analysis for the markers of interest, CXCR6 and CD8 alpha, was quantified using the ImageJ software. The quantification was deliberately kept non-specific to encompass a range of cell types identifiable by these markers. For a more detailed analysis of specific cell populations highlighted by these markers, further elaboration can be provided as needed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll statistical analyses were conducted using SPSS software, version 20.0. The normality of continuous variables was assessed using the Shapiro-Wilk test. Normally distributed continuous variables are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD), whereas non-normally distributed continuous variables are expressed as medians (P25, P75). Categorical data are shown as counts (%). For group comparisons, the Student's t-test was applied to normally distributed continuous variables, the Mann-Whitney U test was utilized for non-normally distributed continuous variables, and the chi-square (χ2) test was used for categorical data. The significance level was set at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"3.Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Higher Peripheral/heptatic CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T Cells From Patients With PBC\u003c/h2\u003e\n \u003cp\u003eOur initial investigation focused on assessing the prevalence of CXCR6\u0026thinsp;+\u0026thinsp;CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells within the peripheral blood mononuclear cells (PBMCs) from PBC patients, in comparison to HCs. As depicted in Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, the results of flow cytometry analyses for CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in HCs and PBC patients, respectively. We observed a significantly higher frequency of CXCR6\u0026thinsp;+\u0026thinsp;cells within the CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell population in PBC patients compared to HCs (2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% versus 0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, n\u0026thinsp;=\u0026thinsp;10). However, the presence and extent of these T cells in the liver remained unclear. To address this, we subsequently employed immunofluorescence techniques to investigate the infiltration of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in hepatic tissue. Our findings revealed a notably increased quantity of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in the livers of PBC patients compared to the HC group. These outcomes suggest that CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells may contribute to the pathogenesis of PBC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T Cells show difference in Patients with PBC (early-stage PBC and late-stage PBC)\u003c/h2\u003e\n \u003cp\u003eWe enrolled a total of 118 PBC patientsbased on elevated cholestatic liver enzymes and typical histological features in liver biopsy specimens. According to the Ludwig classification [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e], patients were categorized in two groups:early-stage PBC (E-PBC, 62 cases) and late-stage PBC (L-PBC, 56 cases). Significant statistical differences were observed between the two groups in markers such as ALT, AST, G, A, AKP, GGT, TBI, DBI, and TBA (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, there were significant statistical differences in blood lipids, including LDL and Lipa (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, there were no statistically significant differences between the two groups in terms of gender, age, WBC, Hb, PLT, CHOL, TG, high-density lipoprotein (HDL), ferritin, antinuclear antibodies (ANA), and anti-mitochondrial antibodies (AMA2, AMA4, AMA9) (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). It is noteworthy that immunohistochemical analysis of hepatic CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in the portal vein area revealed a significant increase in their numbers in both early-stage and late-stage PBC patients compared to normal livers (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, B, C). Furthermore, compared to E-PBC patients (n\u0026thinsp;=\u0026thinsp;21), L-PBC patients (n\u0026thinsp;=\u0026thinsp;25) exhibited a marked increase in the number of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells, with a significant statistical difference in the expression of this specific T cell subset between the two groups (z=-5.667, P\u0026thinsp;=\u0026thinsp;0.000), as detailed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. This suggests a strong correlation between CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells and the severity of PBC, potentially implicating them in the progression of the disease..\u003c/p\u003e\n \u003cp\u003eThe clinical manifestations of PBC typically include elevated GGT and AKP levels, and severe cases may also present with elevated ALT and AST levels. To explore the relationship between CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells and liver inflammation, Spearman correlation analysis was conducted, revealing a positive correlation between CXCR6 and AKP (r\u0026thinsp;=\u0026thinsp;0.3150, P\u0026thinsp;=\u0026thinsp;0.003) and GGT (r\u0026thinsp;=\u0026thinsp;0.6839, P\u0026thinsp;\u0026lt;\u0026thinsp;0.000) levels. However, the correlation with ALT (r\u0026thinsp;=\u0026thinsp;0.1097, P\u0026thinsp;=\u0026thinsp;0.4679) and AST (r\u0026thinsp;=\u0026thinsp;0.1771, P\u0026thinsp;=\u0026thinsp;0.2391) was minimal. These results indicate a stronger correlation between the abundance of this specific T cell subset and elevated AKP and GGT levels, suggesting their involvement in bile duct injury processes, leading to significant increases in AKP and GGT (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, E, F, and \u003cstrong\u003eG\u003c/strong\u003e). In conclusion, CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells isolated from patients with late-stage PBC patients exhibited inflammatory activity, suggesting their potential role in the pathological process of PBC.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummarizes the main clinical and biochemical characteristics of the 118 patients with E-PBC and L-PBC\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eE-PBC(n\u0026thinsp;=\u0026thinsp;62)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eL-PBC(n\u0026thinsp;=\u0026thinsp;56)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003et\u003c/em\u003e/\u003cem\u003e\u0026Chi;\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e/z\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAge(year)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.76\u0026thinsp;\u0026plusmn;\u0026thinsp;8.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.05\u0026thinsp;\u0026plusmn;\u0026thinsp;11.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et\u0026thinsp;=\u0026thinsp;0.369\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.713\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9(14.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8(14.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026Chi;\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.972\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003efemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53(85.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48(85.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eWBC(\u0026times;10^9/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et=-0.613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.541\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHb(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e123.98\u0026thinsp;\u0026plusmn;\u0026thinsp;16.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e121.69\u0026thinsp;\u0026plusmn;\u0026thinsp;14.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et\u0026thinsp;=\u0026thinsp;0.707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.481\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePLT(\u0026times;10^9/L )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e173.31\u0026thinsp;\u0026plusmn;\u0026thinsp;77.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e169.62\u0026thinsp;\u0026plusmn;\u0026thinsp;71.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et\u0026thinsp;=\u0026thinsp;0.245\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.807\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eALT(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43(26, 104.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97(45, 141)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-2.760\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAST(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40(26, 78)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e66(43, 120)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.956\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eA(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.04\u0026thinsp;\u0026plusmn;\u0026thinsp;4.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.20\u0026thinsp;\u0026plusmn;\u0026thinsp;4.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.243\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.027\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eG(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.40\u0026thinsp;\u0026plusmn;\u0026thinsp;5.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.50\u0026thinsp;\u0026plusmn;\u0026thinsp;6.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003et\u003c/em\u003e=-2.751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAKP(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134(104, 179)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e280(173, 353)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-4.692\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eGGT(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116(62, 218)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e330(134, 675)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-4.223\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTBI(\u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.86(7.88, 19.83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.61(13.73, 49.63)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.614\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDBil(\u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.6(1.72,6.35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.9(4.15, 32.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-4.403\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTBA(umol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.2(7.55, 32.65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.2(12.3, 96.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.422\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCHOL(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.57(3.61, 5.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.06(3.99, 6.27)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-1.719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.086\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTG(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.4(0.95, 1.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.33(0.85, 2.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-0.927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.345\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHDL(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.48(1.14, 1.67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.50(1.28, 1.99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-1.268\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.205\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLDL(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.38(1.79, 2.84)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.85(2.03, 3.52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-2.038\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.042\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLipa(mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60(24, 94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41(24, 94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-1.774\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.076\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eFerritin(\u0026micro;g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.5(47.4, 164.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e131.9(62.7, 243.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-0.546\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.585\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eThe number of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell in liver(n/HPF )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e56(14, 93.5)*༈n\u0026thinsp;=\u0026thinsp;21༉\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139(82, 181.5)*༈n\u0026thinsp;=\u0026thinsp;25༉\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.849\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eANA quantification\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e320(100, 320)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e320(320, 400)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eZ=-0.481\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.631\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAMA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28(45.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18(32.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e\u0026Chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;2.096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.148\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34(54.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38(67.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAMA4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44(83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38(88.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e\u0026Chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.546\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.460\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9(17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5(11.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAMA9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47(88.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41(95.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e\u0026Chi;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;1.671\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.434\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5(9.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2(4.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e values refer to comparisons between E-PBC and L-PBC patients\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ePBC\u003c/em\u003e primary biliary cholangitis,, ALP alkaline phosphatase, GGT gamma-glutamyl transpeptidase, ALT alanine aminotransferase, AST aspartate aminotransferase, ALB albumin, TBIL total bilirubin, PLT platelet count,AMA-M2 ,4,9,anti-mitochondrial M2,4 and 9 antibody.,TG triglyceride,TBA total bile acids,CHOL cholesterol,HDL high-density lipoprotein, LDL Low-density lipoprotein, Lipa lipoprotein a\u003c/p\u003e\n \u003cp\u003e3.3 CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T Cells Isolated From Patients With L-PBC Show a Higher Activity of Inflammation and Fibrosis Degree\u003c/p\u003e\n \u003cp\u003eDue to the differential infiltration of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells across various stages of PBC, our aim was to elucidate the relationship between these specialized T cells and the degree of liver inflammation and fibrosis. Employing HE and Masson\u0026apos;s staining techniques, we observed a positive correlation between CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells and liver inflammation and fibrosis (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Statistical analysis revealed significant differences in the abundance of CXCR6-positive cells between different states of PBC (E-PBC and L-PBC) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, P\u0026thinsp;=\u0026thinsp;0.000), as well as significant statistical differences between different grades of inflammation (G grades) and fibrosis (S grades) (with H\u0026thinsp;=\u0026thinsp;27.119, P\u0026thinsp;=\u0026thinsp;0.000 and H\u0026thinsp;=\u0026thinsp;12.147, P\u0026thinsp;=\u0026thinsp;0.007, respectively). These findings strongly suggest that the involvement of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in the processes of inflammation and fibrosis in PBC.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.4 The elevation of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells is considered one of the factors contributing to poor response in PBC.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe aforementioned results demonstrate a significant correlation between the quantity of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells and the levels of inflammation, liver fibrosis, and the disease progression of PBC. Here, the relationship between these T cells and the response to UDCA treatment was explored. According to the AASLD guidance [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], patients undergoing standard UDCA treatment were monitored for six months. A total of 64 patients were included in the follow-up, and categorized into responder and non-responder groups, with 31 patients identified as responders and 33 as non-responders.\u003c/p\u003e\n \u003cp\u003eThrough single-factor analysis, notable statistical variances were observed between the two groups in various biochemical indicators, including ALT, AST, G, AKP, GGT, TBI, Dbi, and TBA, as well as Hb, HDL, and LDL (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Employing rank sum tests, it was discerned that the levels of specialized CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in immunohistochemistry exhibited significant disparities between responders and non-responders (Z= -3.057, p\u0026thinsp;=\u0026thinsp;0.002) (Fig. 4). The count of hepatic CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells per HPF was notably higher in UDCA responders (n\u0026thinsp;=\u0026thinsp;5) compared to UDCA non-responders (n\u0026thinsp;=\u0026thinsp;5), indicating an association between CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells and responsiveness to UDCA treatment.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u003cstrong\u003eSummarization of the main clinical and biochemical characteristics of the 64 patients in UDCA non-responders compared with UDCA responders\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUDCA responders(n\u0026thinsp;=\u0026thinsp;31 )\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUDCA non-responders (n\u0026thinsp;=\u0026thinsp;33)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003et\u003c/em\u003e/\u003cem\u003e\u0026Chi;\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e/z\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAge(year)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51.55\u0026thinsp;\u0026plusmn;\u0026thinsp;11.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e53.39\u0026thinsp;\u0026plusmn;\u0026thinsp;10.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et=-0.393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.491\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4(12.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3(9.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e0.625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27(87.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30(90.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eWBC(10^9/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et\u0026thinsp;=\u0026thinsp;0.223\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.825\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHb(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e129.4\u0026thinsp;\u0026plusmn;\u0026thinsp;16.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e116.64\u0026thinsp;\u0026plusmn;\u0026thinsp;14.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et\u0026thinsp;=\u0026thinsp;2.942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003ePLT(10^9/L )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e165.39\u0026thinsp;\u0026plusmn;\u0026thinsp;81.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e177.75\u0026thinsp;\u0026plusmn;\u0026thinsp;78.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et=-0.568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.572\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eALT(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.5(18,49.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77(43,110)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAST(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28(24,42.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61(43,88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-4.824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eA(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.56\u0026thinsp;\u0026plusmn;\u0026thinsp;4.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.31\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et\u0026thinsp;=\u0026thinsp;0.575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.038\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eG(g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.38\u0026thinsp;\u0026plusmn;\u0026thinsp;5.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.11\u0026thinsp;\u0026plusmn;\u0026thinsp;5.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003et=-2.817\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eAKP(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e115.5(82,135)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e330(210,405)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-6.872\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eGGT(U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68(38.75,113.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e360(155,675)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-5.179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTBI(\u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.50(5.90,12.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.64(12.75,49.63)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-2.935\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDBil(\u0026micro;mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.72(1.18,2.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.39(3.82,32.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.849\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTBA(umol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.65(8,27.45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.3(20.2,97.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.649\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCHOL(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.53(3.52,5.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.98(5.06,8.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.337\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTG(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.32(1.02,2.21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.56(0.85,2.18)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-0.613\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.540\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eHDL(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.33(1.15,1.58)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.94(1.57,2.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-3.705\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLDL(mmol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.49(1.73,3.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.94(2.19,3.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-2.647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLipa(mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e91(47.5,188.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41(14,94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-0.975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.330\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eFerritin(\u0026micro;g/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.5(27.65,132.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e88.7(42.8,177.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-1.365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.172\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eThe number of CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell in liver (n/HPF )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76(18,132)(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e149(80.25,191.25)(n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ez=-2.245\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0025\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eParis I and Paris II criteria were utilized in late-stage (stage III\u0026ndash;IV) and early-stage (stage I\u0026ndash;II) PBC patients, respectively, to identify responders to UDCA. The data are presented as medians with interquartile ranges.\u003c/p\u003e\n \u003cp\u003eAbbreviations: PBC: Primary Biliary Cholangitis；ALP: Alkaline Phosphatase；GGT: Gamma-Glutamyl Transpeptidase；ALT: Alanine Aminotransferase；AST: Aspartate Aminotransferase；ALB: Albumin；TBIL: Total Bilirubin；PLT: Platelet；TG: Triglyceride；TBA: Total Bile Acids；CHOL: Cholesterol；HDL: High-Density Lipoprotein；LDL: Low-Density Lipoprotein；Lipa: Lipoprotein A\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePBC is a chronic, progressive liver disease with an unidentified etiology, primarily characterized by cholestasis and liver damage. The pathogenesis of PBC is multifaceted, with the immune response playing a key role in its pathological process[12]. Our study indicates that x patients with advanced PBC are more susceptible to hepatocyte and bile duct damage compared to those in the early stages. This increased susceptibility results in elevated levels of serum indicators, such as ALT and GGT. HE staining has revealed significant increase in the infiltration of CXCR6+CD8+T cells around the hepatic bile duct region in patients with advanced PBC. This indicates a higher likelihood of severe hepatitis, extensive bile duct damage, and increased fibrosis in the later stages of the disease. These results are consistent with the findings of Zhao, et al[13], who reported an increase in CD8+ T cells in L-PBC patients, accompanied by lymphoid follicle formation, bile duct damage, and fibroblast proliferation. Collectively, these pathological characteristics show the potential involvement of CXCR6+CD8+T cells in the pathogenesis of PBC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is well-known that CD8+ T cells are composed of various subtypes, yet the specific mechanisms by which these subtypes act in PBC remain unclear. Recent research highlights the pivotal role of CXCR6+ CD8+ T cells in the progression of non-alcoholic steatohepatitis (NASH), particularly their ability to directly destroy liver cells, which has greatly attracted our attention[14]. In this study, we observed a significant increase in the number of CD8+ T cells expressing C-X-C motif chemokine receptor 6 (CXCR6) both in the circulating blood and liver tissues of PBC patients, a novel finding not previously documented. CXCR6, a member of the CXC chemokine subfamily, is primarily distributed on the surface of different types of various T lymphocytes[15]. Previous studies have shown that CXCR6 is preferentially expressed on memory T cells as well as \u0026nbsp;activated Th1 and Tc1 effector T cell subgroups[16]. In oncology, CXCR6 expression is crucial for the anti-tumor activitie=s of CD8+ T cells[17,18]. In addition, CXCR6+ CD8+ T cells accumulate significantly in the peripheral blood of elderly individuals, suggesting a potential role in the progression of neurodegenerative diseases[19]. In hepatology, these cells also play an important role in liver cancer and represent a potential therapeutic target for primary liver cancer[20].\u003c/p\u003e\n\u003cp\u003eIn our in-depth study, we have found that the escalation inCXCR6+CD8+ T cells throughout the advancement of PBC presents a potential risk factor. Our analysis has observed a notable surge in circulating CXCR6+CD8+ T cells among PBC patients, which is closely associated with both disease severity and the efficacy of UDCA treatment. Particularly, within the periportal regions of PBC patients\u0026rsquo; livers, a marked increase in intrahepatic CXCR6+CD8+ T cells was observed. Our statistical analysis further confirmed a correlation between the abundance of these cells, the biomarkers of cholestasis, and the response to UDCA treatment. Although previous studies have highlighted the importance of NKT cells and gamma-delta (\u0026gamma;\u0026delta;) T cells in PBC[21,22], the function of CXCR6+CD8+ T cells has not been fully explored. Early studies suggest that CXCR6 is a key chemotactic factor in the liver, and its dependent aggregation of NK T cells leads to increased inflammation and fibrosis[23,24]. However, the precise function of CXCR6+CD8+ T cells in PBC remains unclear. Studies indicate that these cells can produce a variety of cytokines, especially IFN-\u0026gamma;, TNF-\u0026alpha;, and GZMB, demonstrating their antiviral, pro-inflammatory, and cytotoxic capabilities[14]. It is noteworthy that many PBC patients display abnormal lipid metabolism, consistent with the findings of Zhang et al[25]., which pointed out minimal hepatic steatosis and higher levels of high-density lipoprotein cholesterol (HDL-C) in PBC patients. Our study also found significant differences in CHOL, TG, and HDL-C among PBC patients at different stages. In the study of NASH pathogenesis, it is known that CXCR6+CD8+ T cells not only exhibit cytotoxicity driven by lipids but also engage in \u0026quot;auto-aggressive killing\u0026quot; of normal cells. We hypothesize that within the pathogenesis of PBC, CXCR6+CD8+ T cells might activate in an environment of lipid metabolism disorder, thereby releasing cytokines to enhance inflammatory responses. At the same time, these cells may aggravate liver damage by \u0026quot;auto-aggressive\u0026quot; behaviors, fostering the spread of inflammation and fibrosis. In summary, CXCR6+CD8+ T cells may wield significant influence in PBC development, contributing to the immunopathological cascade through cytotoxic effects, inflammatory mediator secretion, and interactions with other immune cells. Nevertheless, the detailed content of these mechanisms requires further scientific inquiry.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations that warrant discussion. Firstly, the lack of sufficient fresh PBC liver samples precluded the utilization of flow cytometry for a detailed analysis of both the quantity and functionality of CD8+ T cell subsets within the liver. We anticipate that future studies can use advanced technologies, such as single-cell RNA sequencing, to delve more deeply into the diverse functions of CXCR6-related T cell subsets in PBC. Secondly, this study was designed as a cross-sectional study. If it were possible to implement a larger sample size and conduct long-term tracking of the changes in CXCR6+CD8+ T cell levels before and after UDCA treatment, more rich and meaningful data might be obtained. Overall, the progression of chronic liver disease, from persistent inflammation to fibrosis and potentially carcinogenesis, presents a formidable challenge in the development of new therapeutic strategies. Our research results provide empirical evidence regarding the activity of the CXCR6+CD8+ T cell subset in PBC patients, particularly their localization within the liver. The different expressions of this subset at various stages of PBC and its correlation with the response to UDCA treatment need further exploration. A thorough understanding of the role played by CXCR6+CD8+ T cells in PBC is crucial for revealing the pathogenic mechanisms underlying this condition and for the development of related therapeutic interventions. We look forward to future studies that will shed light on the functions and regulatory mechanisms of CXCR6+CD8+ T cells in PBC through more experiments and clinical data.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors especially thank Dr. Weiting Lu for her assistance in observing and choosing the microphotographs, and Dr. Shuangshuang Wang for her help in the immunohistochemistry experiment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHuilian Shi and Fei Qiao designed, revised and finalized the manuscript. Weiting Lu for her help in observing and choosing the microphotographs. Shuangshuang Wang performed the experiment, Xiangtao Xu\u0026nbsp;、Haiyan Guo analyzed the data and drafted the manuscript.Doctors Qinlei Chen and Fanzhang collected the blood sample and clinical data. All authors read and approved the fnal manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Natural Science Foundation of Jiangsu Province BK20221421 (Huilian Shi).\u003c/p\u003e\n\u003cp\u003eAvailability of data and material Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e \u0026nbsp;Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflict of interest\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003eThe authors declare that there are no competing interests associated with the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. The ethics committee of the Affiliated Hospital of Nanjing University of Chinese Medicine reviewed and approved the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to participate\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003eWritten informed consent was obtained from all subjects enrolled in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWritten informed consent for publication was obtained from all participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHouri Inbal,Hirschfield Gideon M,Primary Biliary Cholangitis. Pathophysiology [J] Clin Liver Dis. 2024;28:79\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaisal Muhammad Salman,Gonzalez, Humberto C, Gordon Stuart C. Primary Biliary Cholangitis: Epidemiology, Diagnosis, and Presentation.[J].Clin Liver Dis, 2024, 28: 63\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Association for the Study of the Liver. EASL Clinical Practice Guidelines: the diagnosis and management of patients with primary biliary cholangitis. J Hepatol. 2017;67:145\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorpechot C, Carrat F, Bahr A, et al. The efect of ursodeoxycholic acid therapy on the natural course of primary biliary cirrhosis. Gastroenterology. 2005;128:297\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang STXW, Li, et al. Downregulation of Programmed Death-1 Pathway Promoting CD8\u0026thinsp;+\u0026thinsp;T Cell Cytotoxicity in Primary Biliary Cholangitis. [J] Dig Dis Sci. 2022;67:2981\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDudek Michael,Pfister Dominik,Donakonda Sainitin. Auto-aggressive CXCR6 CD8 T cells cause liver immune pathology in NASH.[J]. Nature. 2021;592:444\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGou Haoxian,Liu Shenglu,Liu Linxin. Obeticholic acid and 5β-cholanic acid 3 exhibit anti-tumor effects on liver cancer through CXCL16/CXCR6 pathway[. J] Front Immunol. 2022;13:1095915.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindor KD, Bowlus CL, Boyer J, et al. Primary biliary cholangitis: 2018 practice guidance from the American association for the study of liver diseases. Hepatology. 2019;69:394\u0026ndash;419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChazouilleres 9CC, Poupon O. Early primary biliary cirrhosis: biochemical response to treatment and prediction of long-term outcome. J Hepatol. 2011;55:1361\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLudwig J, Dickson ER, McDonald GS. Staging of chronic nonsuppurative destructive cholangitis (syndrome of primary biliary cirrhosis). Virchows Arch Pathol Anat Histol. 1978;379:103\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindor KD, Bowlus CL, Boyer J, Levy C, Mayo M. Primary biliary cholangitis: 2018 practice guidance from the American association for the study of liver diseases. The American association for the study of liver diseases practice guidelines. Hepatology. 2019;69(1):394\u0026ndash;419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHebbandi Nanjundappa Roopa,Shao Kun,Krishnamurthy Prasanna. Invariant natural killer T cells in autoimmune cholangiopathies: Mechanistic insights and therapeutic implications[. J] Autoimmun Rev. 2023;23:103485.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao S-X. Emperipolesis mediated by CD8 T cells correlates with biliary epithelia cell injury in primary biliary cholangitis.[J]. J Cell Mol Med. 2020;24:1268\u0026ndash;75. Li Wen-Cong,Fu Na.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDudek Michael,Pfister Dominik,Donakonda Sainitin. Auto-aggressive CXCR6 CD8 T cells cause liver immune pathology in NASH.[J]. Nature. 2021;592:444\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeesch Kira,Raczkowski Friederike,Schumacher Val\u0026eacute;a. The function of the chemokine receptor CXCR6 in the T cell response of mice against Listeria monocytogenes[. J] PLoS One. 2014;9:e97701.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrea TS-W, Espinosa Diego J et al. A. The chemokine receptor CXCR6 is required for the maintenance of liver memory CD8⁺ T cells specific for infectious pathogens.[J].J Infect Dis, 2014, 210: 1508-16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMossanen Jana C, Alexander, et al. CXCR6 Inhibits Hepatocarcinogenesis by Promoting Natural Killer T- and CD4 T-Cell-Dependent. Control Senescence [J] Gastroenterol. 2019;156:1877\u0026ndash;e18894.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKee Ji-Ye,Ito Aya,Hojo Shozo et al. Chemokine CXCL16 suppresses liver metastasis of colorectal cancer via augmentation of tumor-infiltrating natural killer T cells in a murine model[. J] Oncol Rep, 2013, 29: 975\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi T, Hongqi, et al. CXCR6-based immunotherapy in autoimmune, cancer and inflammatory infliction[. J] Acta Pharm Sin B. 2022;12:3255\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao-Ying GQZY-J et al. CXCR6 upregulation contributes to a proinflammatory tumor microenvironment that drives metastasis and poor patient outcomes in hepatocellular carcinoma.[J].Cancer Res, 2012, 72: 3546-56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Lv T, Sun G, et al. Reciprocal alterations in circulating and hepatic gamma-delta T cells in patients with primary biliary cholangitis. Hepatol Int. 2022;16(1):195\u0026ndash;206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHydes TJ, Blunt MD, Naftel J, et al. Constitutive Activation of Natural Killer Cells in Primary Biliary Cholangitis. Front Immunol. 2019;10:2633.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiepelt Anke,Wehr Alexander,Kohlhepp, Marlene, et al. CXCR6 protects from inflammation and fibrosis in NEMO mice.[J]. Biochim Biophys Acta Mol Basis Dis. 2019;1865:391\u0026ndash;402.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWehr Alexander,Baeck Christer,Heymann Felix. Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fibrosis[. J] J Immunol. 2013;190:5226\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Hu X, Chang J, et al. The liver steatosis severity and lipid characteristics in primary biliary cholangitis. BMC Gastroenterol. 2021;21(1):395.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"hepatology-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hepi","sideBox":"Learn more about [Hepatology International](https://www.springer.com/journal/12072)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/hepi/default.aspx","title":"Hepatology International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Primary biliary cirrhosis, CXCR6 + CD8 + T cell༛Autoimmune liver disease༛Clinical and pathological data","lastPublishedDoi":"10.21203/rs.3.rs-4426292/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4426292/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells have been implicated in the pathogenesis of various liver and autoimmune diseases. However, their involvement in Primary Biliary Cholangitis (PBC) has not been eluciated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe used immunohistochemistry and flow cytometry to quantify CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells in hepatic tissue and peripheral blood samples obtained from CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells obtained from PBC patients. Then, we performed comprehensive statistical analyses to access the correlation between the abundance of these cells and clinical as well as pathological data across different stages of PBC.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur research revealed that CXCR6\u0026thinsp;+\u0026thinsp;cell frequencies in CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells from PBC patients significantly exceeded that of healthy controls (HCs) (2.24% vs. 0.61%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A similar pattern emerged for hepatic CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell counts, which were notably higher in the PBC cohort compared to HCs. Our cohort consisted of 118 PBC patients, categorized into 62 early-stage (E-PBC) and 56 late-stage (L-PBC) cases. Notably, significant disparities existed between these groups in terms of liver enzyme and lipid profile levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with no notable differences observed in gender, age, blood counts, cholesterol levels, or autoantibodies (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Intriguingly, the quantity of hepatic CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells per high power field (HPF) was significantly elevated in both E-PBC and L-PBC patients as opposed to normal liver samples, indicating a substantial increase in these cells across all stages of PBC (p\u0026thinsp;=\u0026thinsp;0.000). Spearman\u0026rsquo;s rank correlation analysis using showed a positive correlation between CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cell counts and serum levels of Alkaline Phosphatase (AKP) and Gamma-Glutamyl Transferase (GGT), while revealing a negligible correlation with Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST). Subsequent findings indicated significant variances in CXCR6\u0026thinsp;+\u0026thinsp;cell numbers not only among different PBC stages but also across various degrees of inflammation and fibrosis (p\u0026thinsp;\u0026le;\u0026thinsp;0.007). In a follow-up study post-Ursodeoxycholic Acid (UDCA) treatment, stark differences were identified in biochemical and immunohistochemical profiles between responder (31 patients) and non-responder (33 patients) groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A Wilcoxon rank-sum test further demonstrated a significant difference in the level of hepatic CXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells between these two response groups (p\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCXCR6\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells play a vital role in the pathogenesis of PBC, exhibiting correlations with the extent of inflammation, staging of liver fibrosis, and response to pharmacological interventions in PBC patients.\u003c/p\u003e","manuscriptTitle":"The Relationship between CXCR6+CD8+T cells and Clinicopathological Parameters in Patients with primary biliary cholangitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 18:22:34","doi":"10.21203/rs.3.rs-4426292/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2024-06-14T20:51:25+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-05-25T05:20:53+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-25T02:20:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-16T09:36:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Hepatology International","date":"2024-05-15T11:49:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"hepatology-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hepi","sideBox":"Learn more about [Hepatology International](https://www.springer.com/journal/12072)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/hepi/default.aspx","title":"Hepatology International","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dd0a0078-1d35-4c8f-9f41-e24f5e5ec9b4","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T19:42:40+00:00","versionOfRecord":{"articleIdentity":"rs-4426292","link":"https://doi.org/10.1007/s12072-024-10715-0","journal":{"identity":"hepatology-international","isVorOnly":false,"title":"Hepatology International"},"publishedOn":"2024-08-12 15:58:14","publishedOnDateReadable":"August 12th, 2024"},"versionCreatedAt":"2024-06-11 18:22:34","video":"","vorDoi":"10.1007/s12072-024-10715-0","vorDoiUrl":"https://doi.org/10.1007/s12072-024-10715-0","workflowStages":[]},"version":"v1","identity":"rs-4426292","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4426292","identity":"rs-4426292","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}