3.3. CD4+ T Cells from Patients with PBC Show Multiple-Cytokine Activation
PMA with ionomycin was used to stimulate the whole blood in vitro, as shown in Figure 3a. The frequencies of CD4+ T cells producing interferon-γ (IFN-γ), interleukin-4 (IL-4), and IL-17 (defined as Th1/Th2/Th17 subsets, respectively) were measured, as shown in Figure 2. Consistent with the former results of increased phenotypic activation, Th1/Th2/Th17 cell frequencies were greater in PBC (Th1, 12.51 ± 9.06% vs. 7.34 ± 7.79%, p = 0.0075; Th2, 3.39 ± 4.18% vs. 2.21 ± 3.57%, p = 0.0253; and Th17, 6.00 ± 5.04% vs. 3.15 ± 2.25%, p = 0.0240), as shown in Figure 3c. In summary, these two results suggested that CD4+ T cells from PBC patients showed a higher activation status compared to those from HCs.
3.4. LAMP-2A Expression is Significantly Increased in Activated CD4+ T Cells
The whole blood was stained by an excess amount of anti-LAMP-2A antibody and subsequently conjugated with fluorescent Brilliant Violet 421. The mean fluorescence intensity (MFI) in certain T cell subsets is proportional to the amount of total cellular LAMP-2A [17]. The results showed that the MFI in HLA-DR+CD4+ T cells was significantly greater than in HLA-DR CD4+ T cells, either from PBC patients or HCs (PBC, 2231.03 ± 1239.19 vs. 1652.06 ± 1001.79, p = 0.0294; HC, 1644.66 ± 916.89 vs. 1079.30 ± 465.46, p = 0.0337), as shown in Figure 3a. For ICOS-marked cells, the results were not dramatic but also showed a consistent trend (PBC, 1711.64 ± 941.06 vs. 1409.06 ± 787.78, p = 0.0531; HC, 1368.30 ± 623.62 vs. 1085.61 ± 475.18, p = 0.1158), as shown in Figure 3b. This indicated that LAMP-2A may participate in the initiation and maintenance of CD4+T cells activation responses.
3.5. LAMP-2A expression in Naïve CD4+ T Cells from PBC is Significantly Higher than in HCs
To investigate the role of LAMP-2A in CD4+ T cell hyperactivity in PBC patients, LAMP-2A expression was detected in non-activated CD4+ T cells. The results suggested that LAMP-2A expression in HLA-DR CD4+ T cells from PBC patients was significantly higher than that from HCs (1652.06 ± 1001.79 vs. 1079.30 ± 465.46, p = 0.0249), and ICOS CD4+ T cells showed the same trend (1411.98 ± 771.37 vs. 1084.18 ± 488.16, p = 0.0673), as shown in Figure 3c. Furthermore, we found that the level of LAMP-2A in naïve CD4+ T cells from PBC patients was dramatically higher than that from HCs (2033.21 ± 925.56 vs. 1493.61 ± 714.10, p = 0.0296), as shown in Figure 3d.
3.6. LAMP-2A Expression in Naïve CD4+ T Cells from PBC Are Related to Disease Severity
The relationship between LAMP-2A expression in naïve CD4+ T cells and PBC clinical features were investigated in our research. Given that PBC predominantly occurred in the middle-aged and elder women, we tried to determine the influence of gender and age profile on LAMP-2A expression in naïve CD4+ T cells, as shown in Figure 3e, f. While these differences were statistically insignificant (Female vs. male, 2049.45 ± 907.34 vs. 1952.03 ± 1086.04, p = 0.6855; age < 58-year patients vs. age ≥ 58-year patients, 1941.18 ± 1002.53 vs. 2125.24 ± 856.34, p =0.3143). Additionally, as Figure 3g shows, there were no significant differences between AMA positive versus AMA negative patients (AMA, 2022.19 ± 926.21 vs. 2080.04 ± 985.01, p = 0.9617; AMA-M2, 1941.78 ± 915.30 vs. 2216.07 ± 952.78, p = 0.4006). A variety of biochemical indicators could reflect the disease status and were related to the prognosis of the disease. But we found LAMP-2A expression in naïve CD4+ T cells is independent of these biochemical indicators, including ALT, AST, ALP, GGT, TBil and Alb, as shown in Figure 3h.
Interestingly, LAMP-2A expression in naïve CD4+ T cells were higher in patients in stage III-IV than in stage I-II (2387.480 ± 1023.36 vs. 1505.54 ± 685.33, p = 0.0108), as shown in Figure 3i. We also further found that LAMP-2A expression in naïve CD4+ T cells in cirrhotic group was dramatically higher than the non-cirrhotic group (2185.61 ± 988.85 vs. 1430.38 ± 673.96, p = 0.0384), as shown in Figure 3j. Therefore, we speculated that hyperactivity of CD4+ T cells from PBC patients caused by high expression level of LAMP-2A in naïve CD4+ T cells may related to PBC clinical severity.
3.7. Naïve CD4+ T Cells from Patients with PBC Show Enhanced Function after Stimulation in Vitro
To further verify the ability of naïve CD4+ T cells from PBC patients to activate, we isolated naïve CD4+T cells with immunomagnetic beads and then activated them with CD3/CD28 T Cell Activator in vitro. We found that naïve CD4+ T cells from PBC patients showed a greater ability to proliferate (64.99 ± 16.03% vs. 54.57 ± 9.91%, p = 0.0432), as shown in Figure 4a. Additionally, annexin V+ 7-AADsubsets were defined as apoptotic cells. There were no significant differences between the cells from PBC patients and HCs (10.58 ± 0.82% vs. 8.68 ± 1.43%, p = 0.2936), as shown in Figure 4b. Activation induced cytokine production in the cell supernatant; for example, IL-2 levels were dramatically higher in cells from PBC patients (5.80 ± 1.31 ng/ml vs. 4.52 ± 1.57 ng/ml, p = 0.0257), IFN-γ levels were not dramatic but also showed a consistent trend (10.27 ± 1.33 ng/ml vs. 9.05 ± 1.45 ng/ml, p = 0.0923), as shown in Figure 4c.
3.8. Silencing of LAMP-2A Expression Prevents Excessive Activation of Naïve CD4+ T Cells from Patients with PBC
To determine the important role of LAMP-2A in the hyperactivity of naïve CD4+ T cells from PBC patients, we investigated the effects of silencing LAMP-2A mRNA expression in naïve CD4+ T cells. We transduced naïve CD4+ T cells from PBC patients with retrovirus expressing a control or LAMP-2A-specific short hairpin RNA (shRNA) and puromycin resistance, which was used to select cells. After activation in vitro, silencing of LAMP-2A expression significantly reduced activation-induced IFN-γ and IL-2 production (IFN-γ, 10.27 ± 1.33 ng/ml vs. 1.56 ± 0.45, p < 0.0001; IL-2, 5.80 ± 1.31 ng/ml vs. 0.83 ± 0.52, p < 0.0001) as well as proliferation (64.99 ± 16.03% vs. 14.34 ± 10.62%, p < 0.0001) of naïve CD4+ T cells from PBC patients, as shown in Figure 4a, c, d. The impaired T cell responses caused by silencing the LAMP-2A mRNA were not due to increased apoptosis, as no significant differences were found in annexin V binding (10.58 ± 0.82% vs. 11.80 ± 0.98%, p = 0.3747), as shown in Figure 4b. These data indicated that decreasing LAMP-2A expression could prevent naïve CD4+ T cells from PBC patients from being too susceptible to activation.