关键词: 刚地弓形虫, 感染, LAG-3, 蜕膜免疫细胞, 细胞因子

Abstract:

Objective To investigate the effect of Toxoplasma gondii infection on the expression of lymphocyte-activation gene 3 (LAG-3) and related functional molecules on the surface of decidual T cells at the maternal-fetal interface, and to examine its potential association with adverse pregnancy outcomes. Methods Mononuclear cells were isolated from healthy individuals’ decidual tissues in the first trimester and divided into three groups, including the control group, T. gondii infection group, and infection + LAG-3 inhibitorr (10 μmol/L Relatlimab) group, of 1.0 × 10⁷ cells each group. Cells in the control group were given no treatments, while cells in the infection and infection + LAG-3 inhibitor groups were infected with T. gondii tachyzoites at a tachyzoite-to-cell ratio of 1 ∶ 3 and co-cultured in vitro for 19 h prior to cell harvesting. The expression of LAG-3 and intracellular functional molecules interferon gamma (IFN-γ) and granzyme B was detected on the surface of decidual CD4⁺ T (dCD4⁺ T) and CD8⁺ T (dCD8⁺ T) cells using flow cytometry. Human dCD4⁺ and dCD8⁺ T cells were isolated from aforementioned mononuclear cells with CD4/CD8 sorting magnetic beads and purified, and assigned into the control and T. gondii infection groups to quantify LAG-3, IFN-γ and granzyme B expression in presence of T. gondii infection alone. Human dCD4⁺ and dCD8⁺ T cells were divided into the infection and infection + LAG-3 inhibitor groups to examine the effect of T. gondii infection on IFN-γ and granzyme B expression in decidual T cells following blockade of LAG-3. Cells in the control group were given no treatments, while cells in the infection and infection + LAG-3 inhibitor groups (1.0 × 10⁷ cells each group) were infected with T. gondii tachyzoites at a tachyzoite-to-cell ratio of 1 ∶ 3 and co-cultured in vitro for 19 h prior to cell harvesting. Total protein was extracted from cells, and the expression of LAG-3, IFN-γ, and granzyme B proteins was determined in dCD4⁺ and dCD8⁺ T cells using Western blotting assays. All statistical analyses were performed using the software GraphPad Prism 10.3.0. Differences of means between groups were tested for statistical significance with independent-sample Student’s t-test. Results Flow cytometry detected lower proportions of LAG-3-positive human dCD4⁺ [(17.30 ± 1.50)% vs. (27.13 ± 1.45)%; t = 19.750, P < 0.01] and dCD8⁺ T cells [(22.20 ± 2.98)% vs. (35.50 ± 3.60)%; t = 6.389, P < 0.05] in the infection group than those in the control group, higher proportions of IFN-γ-positive human dCD4⁺ T [(7.15 ± 6.90)% vs. (5.46 ± 0.47)%; t = 6.230, P < 0.05] and dCD8⁺ T cells [(3.94 ± 0.27)% vs. (3.02 ± 0.48)%; t = 7.101, P < 0.05] in the infection group than in the control group, higher proportions of IFN-γ-positive human dCD4⁺ T [(8.51 ± 0.36)% vs. (7.15 ± 6.90)%; t = 13.350, P < 0.01] and dCD8⁺ T cells [(5.83 ± 1.01)% vs. (3.94 ± 0.27)%; t = 4.73, P < 0.05] in the infection + LAG-3 inhibitor group than in the infection group. Similarly, higher percentages of granzyme B-positive human dCD4⁺ [(78.30 ± 1.01)% vs. (72.30 ± 1.5)%; t = 8.260, P < 0.05] and dCD8⁺ T cells [(49.00 ± 6.44)% vs. (39.12 ± 10.25)%; t = 5.164, P < 0.05] were detected in the infection group than in the control group, and higher proportions of granzyme B-positive human dCD4⁺ (87.00% ± 0.65%) and dCD8⁺ T cells (57.30% ± 3.93%) were seen in the infection + LAG-3 inhibitor group than in the infection group (t = 18.710, P < 0.01; t = 5.636, P < 0.05). Western blotting assay determined lower relative LAG-3 protein expression in human primary dCD4⁺ [(0.89 ± 0.06) vs. (1.09 ± 0.04); t = 10.790, P < 0.01] and dCD8⁺ T cells [(0.78 ± 0.06) vs. (0.99 ± 0.10); t = 4.503, P < 0.05] in the infection group than in the control group, and higher relative IFN-γ and granzyme B protein expression in dCD4⁺ [IFN-γ: (0.85 ± 0.05) vs. (0.64 ± 0.04), t = 16.77, P < 0.01; granzyme B: (0.75 ± 0.05) vs. (0.61 ± 0.09), t = 5.141, P < 0.05] and dCD8⁺ T cells [IFN-γ: (0.94 ± 0.08) vs. (0.66 ± 0.07), t = 6.193, P < 0.05; granzyme B: (0.80 ± 0.08) vs. (0.44 ± 0.09), t = 4.446, P < 0.05] in the infection group than in the control group. Similarly, higher IFN-γ and granzyme B protein expression was determined in dCD4⁺ [IFN-γ: (0.97 ± 0.06) vs. (0.77 ± 0.08), t = 13.940, P < 0.01; granzyme B: (0.96 ± 0.04) vs. (0.70 ± 0.08), t = 10.250, P < 0.01] and dCD8⁺ T cells [IFN-γ: (1.15 ± 0.11) vs. (0.86 ± 0.07), t = 5.346, P < 0.05; granzyme B: (1.03 ± 0.30) vs. (0.78 ± 0.14), t = 10.520, P < 0.01] in the infection + LAG-3 inhibitor group than in the infection group. Conclusion T. gondii infection in the first trimester may down-regulate the expression of LAG-3 on the surface of dCD4⁺ T and dCD8⁺ T cells, thereby up-regulating the expression of functional molecules IFN-γ and granzyme B, which may be an important mechanism responsible for adverse pregnancy outcomes.

Key words: Toxoplasma gondii, Infection, LAG-3, Decidual immune cell, Cytokine