目的 探索刚地弓形虫感染对早孕期母胎界面蜕膜T细胞表面淋巴细胞活化基因-3（LAG-3）和相关功能分子表达水平的影响，探讨其与不良妊娠结局发生的潜在关系。方法 取健康人早孕期蜕膜组织分离单个核细胞，设对照组、弓形虫感染组和感染 + LAG-3抑制剂组（每组1.0 × 10⁷个细胞），对照组不做任何处理，感染组和感染 + LAG-3抑制剂（10 μmol/L Relatlimab）组按照弓形虫速殖子与细胞1∶3的比例进行感染，体外共培养19 h后收集细胞。采用流式细胞术检测蜕膜CD4⁺ T（dCD4⁺ T）、dCD8⁺ T细胞表面LAG-3及胞内功能分子γ干扰素（IFN-γ）、颗粒酶B的表达水平。使用CD4、CD8分选磁珠从单个核细胞中分离纯化获得人dCD4⁺ T、dCD8⁺ T细胞，分别设对照组、弓形虫感染组，检测单纯感染情况下LAG-3、IFN-γ、颗粒酶B的表达情况；将dCD4⁺ T、dCD8⁺ T细胞均分为感染组和感染 + LAG-3抑制剂组，检测阻断LAG-3后弓形虫感染对蜕膜T细胞内IFN-γ和颗粒酶B表达水平的影响。对照组不做任何处理，感染组和感染 + 抑制剂组按照弓形虫速殖子与细胞为1∶3的比例进行感染（每组1.5 × 10⁷个细胞），体外共培养19 h后收集细胞并提取蛋白，利用蛋白质免疫印迹（Western blotting）检测蜕膜CD4⁺ T、CD8⁺ T细胞LAG-3、IFN-γ和颗粒酶B的表达水平。利用GraphPad Prism 10.3.0软件进行数据分析，组间数据分析采用独立样本Student’s t检验。结果 流式细胞术检测人蜕膜单个核细胞结果显示，人dCD4⁺ T、dCD8⁺ T细胞感染组LAG-3阳性细胞数分别为（17.30 ± 1.50）%、（22.20 ± 2.98）%，均低于对照组的（27.13 ± 1.45）%、（35.50 ± 3.60）%（t = 19.750，P < 0.01；t = 6.389，P < 0.05）。人dCD4⁺ T、dCD8⁺ T细胞感染组IFN-γ阳性率分别为（7.15 ± 6.90）%、（3.94 ± 0.27）%，均高于对照组的（5.46 ± 0.47）%、（3.02 ± 0.48）%（t = 6.230、7.101，均P < 0.05）；感染 + LAG-3抑制剂组分别为（8.51 ± 0.36）%、（5.83 ± 1.01）%，均高于感染组（t = 13.350，P < 0.01；t = 4.372，P < 0.05）。人dCD4⁺ T、dCD8⁺ T细胞中颗粒酶B感染组的阳性率为（78.30 ± 1.01）%、（49.00 ± 6.44）%，均高于对照组的（72.30 ± 1.50）%、（39.12 ± 10.25）%（t = 8.260、5.164，均P < 0.05）；感染 + LAG-3抑制剂组分别为（87.00 ± 0.65）%、（57.30 ± 3.93）%，均高于感染组（t = 18.710，P < 0.01；t = 5.636，P < 0.05）。Western blotting检测结果显示，人dCD4⁺ T、dCD8⁺ T细胞感染组LAG-3蛋白相对表达水平分别为0.89 ± 0.06、0.78 ± 0.06，与对照组的1.09 ± 0.04、0.99 ± 0.10相比均明显降低（t = 10.790，P < 0.01；t = 4.503，P < 0.05）。人dCD4⁺ T、dCD8⁺ T细胞感染组胞内IFN-γ蛋白相对表达水平分别为0.85 ± 0.05、0.94 ± 0.08，高于对照组的0.64 ± 0.04、0.66 ± 0.07（t = 16.770，P < 0.01；t = 6.193，P < 0.05）；人dCD4⁺ T、dCD8⁺ T细胞感染组颗粒酶B表达水平为0.75 ± 0.05、0.80 ± 0.08，与对照组的0.61 ± 0.09、0.44 ± 0.09相比明显上调（t = 5.141、t = 4.446，均P < 0.05）。dCD4⁺ T、dCD8⁺ T细胞感染 + LAG-3抑制剂组胞内IFN-γ蛋白表达水平为0.97 ± 0.06、1.15 ± 0.11，与感染组的0.77 ± 0.08、0.86 ± 0.07相比明显上调（t = 13.940，P < 0.01；t = 5.346，P < 0.05）；dCD4⁺ T、dCD8⁺ T细胞感染 + LAG-3抑制剂组颗粒酶B表达水平分别为0.96 ± 0.04、1.03 ± 0.30，均高于感染组的0.70 ± 0.08、0.78 ± 0.14（t = 10.250、10.520，均P < 0.01）。结论 早孕期弓形虫感染可下调dCD4⁺ T、dCD8⁺ T细胞表面LAG-3的表达水平，进而上调功能分子IFN-γ和颗粒酶B的表达水平，这是导致不良妊娠结局发生的重要机制之一。

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.