1. Introduction
Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis [1], is widely recognized as an autoimmune cholestatic liver disease [2, 3]. PBC is a chronic and slowly progressive liver disease that leads to autoimmune-mediated nonsuppurative cholangitis mainly affecting small to medium-sized intrahepatic bile ducts, with subsequent cholestasis, ductopenia, cirrhosis, occasionally hepatocellular carcinoma, and eventually liver failure [4, 5].
The etiology of PBC is complex. The combination of genetic risk, environmental triggers are probably responsible for PBC development and progression. In terms of immunopathology, PBC is characterized by the presence of disease-specific anti-mitochondrial antibodies (AMAs) and a high frequency of autoreactive CD4+ [6] and CD8+ T cells [7].
In fact, despite some remarkable improvements in defining the immunological characteristics, data on the mechanisms of biliary tract injury remain insufficient [6]. Therefore, understanding the immunological mechanisms of PBC is critical for seeking more targeted therapeutic options and providing effective care for patients.
LAMP family is a set of conserved transmembrane glycoproteins mainly located in prelysosomes and lysosomes. Human LAMP-2A is an isoform of LAMP-2 due to alternative splicing [8]. Former studies have suggested that LAMP-2A plays an important role in immune system regulation. LAMP-2A functions in delivering proteins from the cytoplasm to lysosomes [9, 10] and is an essential component for regulating and maintaining CD4+ T cell activation-induced responses in mice [11].
CD4+ T cells from patients with PBC show differential T cell receptor (TCR) repertoires and T cell activation [12], but the mechanism through which specific T cell subsets participate in the natural progression of the disease remains obscure [13, 14]. To address this issue, we comprehensively evaluated the frequency, activation status, proliferative ability, and function of naïve CD4+ T cells in patients with PBC and controls. We report that CD4+ T cells from patients with PBC demonstrate hyperactivity, which is probably caused by high LAMP-2A expression of naïve CD4+ T cells. Additionally, LAMP-2A expression levels of naïve CD4+ T cells could indirectly reveal the activity of the disease, avoiding the inconvenience of liver biopsy. By modification of abnormal LAMP-2A expression, the CD4+ T cell hyperactivity significantly reversed. As a result, this kind of hyperactivity-caused intrahepatic bile ducts injury could also be ameliorated. Taken together, these data have broad implications for our understanding of PBC immunopathogenesis, and LAMP-2A may serve as a therapeutic target for the treatment of the disease.
2. Materials and Methods
2.1. Patient Enrollment and Sampling
In this study, 42 newly diagnosed patients with PBC and 20 HCs were enrolled from January 2019 to October 2019 in Xijing Hospital of Digestive Diseases. The diagnostic criteria of PBC were based on the guidelines of AASLD in 2018 [3] and EASL in 2017 [15] and were as follows: (1) biochemical evidence of cholestasis based on ALP elevation, after the exclusion of other causes leading to biliary obstruction; (2) the presence of AMA, or other PBC-specific autoantibodies (sp100, gp210) if AMA is negative; and (3) histologic evidence of chronic nonsuppurative cholangitis and destruction of interlobular bile ducts. Patients who met two of the three criteria could be diagnosed with PBC. No patients were diagnosed with PBC-AIH overlap [16], other autoimmune diseases, or other liver-related diseases, including hepatitis B, hepatitis C, alcoholic disease, and non-alcoholic fatty liver disease. No patients had taken glucocorticoids or immunosuppressive drugs for 6 months before diagnosis. The Institutional Research Ethics Committee of Xijing Hospital of Digestive Diseases approved this study (KY20173316-1), and written informed consent was obtained from each participant. Peripheral blood and paraffin-embedded liver tissues were collected for analysis.
2.2. Antibodies
The following antibodies were used in flow cytometry: fluorescein isothiocyanate (FITC)-conjugated anti-CD4, allophycocyanin (APC)-conjugated anti-CD8, APC-Cy7-conjugated anti-HLA-DR, phycoerythrin (PE)-conjugated anti-ICOS, peridinin chlorophyll protein (PerCP)-conjugated anti-CD45RA, PerCP-Cy5.5-conjugated anti-IFN-γ, Brilliant Violet 421-conjugated donkey anti-rabbit IgG, and PE-Cy7-conjugated anti-CD3 were purchased from BioLegend (San Diego, CA, USA); PE-conjugated anti-CD25, Brilliant Violet 510-conjugated anti-IL-17, and Alexa Fluor 647-anti-Foxp3 were purchased from BD Biosciences and BD Pharmingen™ (San Diego, CA, USA); PE-Cy7-conjugated anti-IL-4 was purchased from eBioscience (San Diego, CA, USA); and rabbit anti-human LAMP-2A was purchased from Abcam (Cambridge, UK).
2.3. Flow Cytometry and Ex Vivo Stimulation
Fc receptors on human cells were pre-blocked with Human TruStain FcX™ (BioLegend, San Diego, CA, USA). For cell surface staining (CD3, CD4, CD8, CD25, HLA-DR, ICOS, and CD45RA), whole blood was incubated with fluorochrome-conjugated monoclonal antibodies followed by BD FACS™ Lysing Solution for 10 minutes. Then, the cells were fixed with Fixation/Permeabilization Solution (BD Biosciences, San Diego, CA, USA) and permeabilized with Perm/Wash™ buffer (BD Biosciences, San Diego, CA, USA). After that, the cells were blocked by 5% BSA (Sigma, St. Louis, MO, USA) with 2% normal goat serum (BOSTER, Wuhan, China) and incubated with excess amount of primary polyclonal antibody against LAMP-2A overnight at 4 °C according to the established protocol [17]. After washing, the cells were further incubated with secondary fluorochrome-conjugated antibody. After the final wash, the cells were acquired with the BD FACSCanto™ II flow cytometer and analyzed by FCS Express7 (De Novo™ Software, Los Angeles, CA, USA).
For intracellular cytokine staining (IFN-γ, IL-4, and IL-17), whole blood was diluted 1:1 with RPMI-1640 (Gibco, Carlsbad, CA, USA) and stimulated by Cell Stimulation Cocktail (eBioscience, San Diego, CA, USA) for 4 hours at 37 °C and 5% CO2 before staining. Unstimulated samples served as controls.
All incubation periods except for LAMP-2A staining were performed at 25 °C in the dark. Appropriate FMO-isotype controls were used.
2.4. Cell Isolation and Purification
PBMCs were isolated by gradient centrifugation with Lymphoprep™ (Stemcell Technologies, Vancouver, British Columbia, Canada). Heparinized whole blood was diluted 1:1 with RPMI-1640 (Gibco, Carlsbad, CA, USA) containing 2% fetal bovine serum (Kibbutz Beit Haemek, Israel) and placed on top of the Lymphoprep™ carefully. After spinning, the PBMCs were collected at the interface between plasma and Lymphoprep™. Then, the cells were incubated with BD Pharm Lyse™ Lysing Buffer and washed twice with medium for further use.
Naïve CD4+ T cells were negatively selected from PBMCs with human Naïve CD4+T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The purity of the isolated cell population was more than 90%.
2.5. Cell Culture
Isolated naïve CD4+ T cells were cultured in X-VIVO15 (Lonza, Basel, Switzerland) with Penicillin/Streptomycin Solution (Hyclone, South Logan, UT, USA) in a 5% CO2 incubator at 37 °C. Where appropriate, the cells were activated with ImmunoCult™ Human CD3/CD28 T Cell Activator (Stemcell Technologies, Vancouver, British Columbia, Canada) for 4 days.
2.6. Transfection of T Cells
HEK293T cells were seeded in a cell culture dish in DMEM complete medium one day before transfection. Cells were co-transfected with the pHelper 1.0, pHelper 2.0 plasmid and either the GV493 vector or an empty vector using Lipofectamine (Genechem, Shanghai, China). The supernatant of HEK293T cells was collected 48 hours after transfection. Human CD4+ T cells were transfected with the supernatant using the Easy-T T Cell-Specific Lentivirus Transfection Kit (Genechem, Shanghai, China) following the manufacturer’s instructions.
2.7. Proliferation and Apoptosis Assay
Proliferation assay was assessed by staining using the CFSE Cell Division Tracker Kit (BioLegend, San Diego, CA, USA). Apoptosis was assessed with an Annexin V-PE Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA). The cells were acquired with a flow cytometer and analyzed.
2.8. Enzyme-linked Immunosorbent Assays
Cell supernatants were collected, and IFN-γ and IL-2 concentrations were measured with Human IFN-γ and IL-2 ELISA Kits (4A Biotech, Beijing, China) according to the manufacturer’s instructions.
2.9. Statistical Analysis
All values are expressed as the mean ± standard deviation. The two-tailed unpaired Student’s t-test and Mann-Whitney U test were used to evaluate differences in continuous variables between groups depending on the distribution of the data. Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA). A p value less than 0.05 was considered statistically significant.
3. Results
3.1. Patient Demographics
In total, 42 newly diagnosed PBC patients were included in this study. There were no significant differences in gender or age between these two cohorts, although the PBC cohort predominantly comprised middle-aged and elderly women. The demographic and clinical features of the PBC patients at baseline are listed in Table 1.
Of the 42 PBC patients, 35 paraffin-embedded liver tissues were available from ultrasound-guided needle liver biopsies. These 35 patients were divided into stage I-II (n = 19), stage III- IV (n = 16) respectively, based on Ludwig’s classification [18].
3.2. CD4+ T Cells from Patients with PBC Show Increased Phenotypic Activation
Whole blood was obtained from the 42 subjects with PBC and 20 healthy volunteers included as controls, and the frequencies of peripheral CD4+ T cells were analyzed using multiparameter flow cytometry. As shown in Figure 1a, there was no significant difference in the percentage of CD4+ T cells in the total T cells of peripheral blood from PBC patients compared to that of HCs (58.65 ± 11.69% vs. 53.27 ± 8.91%, p = 0.0888). While the proportions of naïve CD4+ T cell subsets showed a dramatic decrease (31.43 ± 20.00% vs. 40.89 ± 13.38%, p = 0.0296), as shown in Figure 1b. To explore the activation status of PBC patients, the expression of mid- and late-activation markers (i.e., ICOS and HLA-DR, respectively) was used to determine the activated CD4+ T cell phenotype. The results indicated that the proportions of ICOS+and HLA-DR+ CD4+ T cell subsets were significantly higher than those in HCs (21.08 ± 11.52% vs. 14.36 ± 6.41%, p = 0.0244; and 15.23 ± 10.84% vs. 9.22 ± 7.62%, p = 0.0077, respectively), as shown in Figure1c.