Fig 1.
LAG3 expression is up-regulated in infiltrating CD4+ T cells and Treg cells from liver tissue of AE patients.
(A) Heat map presentation of differentially expressed genes related to T cell function, and selected up-regulated genes including negative and positive regulation of T cell activation. (B) Representative immunohistochemical staining of LAG3 (left panel 100×, enlarged 400× on the right panel) in the liver tissue sections from patients with AE. (C) The percentage of positive staining area was calculated to evaluate the expression of LAG3 (n = 19). (D) The percentage of liver CD4+ or CD8+ T cells expressing LAG3 in paired CLT versus DLT from AE patients by flow cytometry (n = 8). (E) Representative images from immunofluorescence co-staining of DAPI (blue), CD4 (green), LAG3 (red) and merged image on the liver tissue sections from patients with AE (n = 8). Boxed areas show × 200 magnification of histological images. Arrows indicate CD4+LAG3+ T cells. Lesion is delimited with a white line. (F) Heatmap representing relative expression of selected marker genes in each cluster in CLT, DLT and PB from AE patients (n = 4, accession no. HRA000553). CLT, “close” liver tissue; DLT, “distant” liver tissue; PB, peripheral blood. Data were analyzed using paired Student’s t tests or one-way ANOVA test. All data are presented as mean ± SD. *P < 0.05, **P <0.01, n.s., P > 0.05.
Fig 2.
LAG3 is mainly expressed on Th2 and Treg cells in liver of E. multilocularis-infected mice after weeks 24 of infection.
(A) Representative flow cytometry plot and percentage of IL-4, IL-10 and TGF-β1 production by CD4+ T cells in liver from mice after 24 weeks of infection (5–6 mice per group). (B) MFI of GATA3 expression by LAG3+ or LAG3- CD4+ T cells in liver from mice after 24 weeks of infection (5 mice per group). (C) Representative flow cytometry plot and percentage of Treg cells (CD4+CD25+Foxp3+) by LAG3+ or LAG3- CD4+ T cells in the liver from mice after 24 weeks of infection (6 mice per group). (D) MFI of Ki67 expression by LAG3+ or LAG3- CD4+ T cells in liver from mice after 24 weeks of infection (5 mice per group). All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., P > 0.05.
Fig 3.
LAG3 deficiency delays disease progression by promoting Th1 cell differention in liver of E. multilocularis-infected mice after weeks 24 of infection.
(A) Establishment of a protocol in the late stage of E. multilocularis-infected mice. (B) Representative images of metacestode tissue in the liver from WT and LAG3-KO mice after 24 weeks of infection. Metacestode tissues are circled by the yellow line. (C) Lesion weight in the liver from WT and LAG3-KO mice after 24 weeks of infection (10 mice per group). (D) Percentage and absolute numbers of CD4+T cells in the liver from WT and LAG3-KO mice after 24 weeks of infection (5 mice per group). (E) Percentage of Tn and Tem in CD4+ T cells in the liver from WT and LAG3-KO mice after 24 weeks of infection (5 mice per group). (F, G) Representative flow cytometry plot and percentage of IFN-γ, TNF-α, IL-4 and IL-10 production by CD4+ T cells in the liver from WT and LAG3-KO mice after 24 weeks of infection (5–6 mice per group). (H, I) Representative flow cytometry plot and percentage of Treg (CD4+CD25+Foxp3+) cells in the liver from WT and LAG3-KO mice after 24 weeks of infection (5–6 mice per group). KO, knockout; WT, wild type; Tn, naive T cells (CD44-CD62L+); Tem, effector T cells (CD44+CD62L-). All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., P > 0.05.
Fig 4.
Increased Th1 cytokine production by LAG3-deficient CD4+ T cell following E. multilocularis protoscoleces antigen stimulation in vitro.
(A, B) Representative flow cytometry plot and percentage of IFN-γ, IL-4 and IL-10 production by liver CD4+ T cells from WT and LAG3-KO mice with E. multilocularis protoscoleces antigen stimulation in vitro. (C, D) Representative flow cytometry plot, percentage and absolute numbers of liver Treg (CD4+CD25+Foxp3+) cells from WT and LAG3-KO mice with E. multilocularis protoscoleces antigen stimulation in vitro. KO, knockout. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, n.s., P > 0.05.
Fig 5.
LAG3-deficient CD4+ T cell develop into Th1 cells upon adoptive transfer into E. multilocularis infected CD4 KO mice.
(A) Establishment of adoptive transfer mouse model. Splenocytes were isolated from WT (CD45.1) and LAG3-KO (CD45.2) mice, mixed, and then transferred to E. multilocularis-infected CD4-KO recipient mice. (B, C) Percentage and absolute number of transferred CD4+ T cells, CD4+ Tn and CD4+ Tem in liver and spleen of E. multilocularis-infected CD4-KO mice that received adoptive transfer. (D, E) Representative flow cytometry plot and percentage of IFN-γ, TNF-α, IL-4, IL-10 production by transferred CD4+ T cells in liver and spleen from E. multilocularis-infected CD4-KO mice. (F, G) Representative flow cytometry plot and MFI of T-bet and GATA-3 expression by transfered CD4+ T cells in the liver and spleen from E. multilocularis-infected CD4-KO mice. (H) Percentage of Treg cells (CD4+CD25+Foxp3+) in the liver and spleen from E. multilocularis-infected CD4-KO mice that received adoptive transfer. KO, knockout; WT, wild type. All data are presented as mean ± SD. (n = 3) *P < 0.05, **P < 0.01, ***P < 0.001, n.s., P > 0.05.
Fig 6.
LAG3 deficiency enhance Th1 cells and decrease Treg upon adoptive transfer into wild-type mice followed by E. multilocularis infection.
(A) Establishment of adoptive transfer mouse model. CD4+ Tn cells were isolated from spleen of LAG3-KO (CD45.2) mice and transferred to WT (CD45.1) recipient mice one day before E. multilocularis infection. (B) Percentage of CD4+ Tn and CD4+ Tem in the liver from E. multilocularis-infected WT recipient mice (CD45.1) with transferred by LAG3-KO (CD45.2) cells. (C, D) Representative flow cytometry plot and percentage of IFN-γ, IL-4 and IL-10 production by CD4+ T cells in the liver from E. multilocularis-infected WT recipient mice (CD45.1) with transferred by LAG3-KO (CD45.2) cells. (E, F) Representative flow cytometry plot and percentage of Treg cells (CD4+CD25+Foxp3+) in the liver from E. multilocularis-infected WT recipient mice (CD45.1) with transferred by LAG3-KO (CD45.2) cells. KO, knockout; WT, wild type; Tn, naive T cells. All data are presented as mean ± SD. *P < 0.05, ***P < 0.001, n.s., P > 0.05.
Table 1.
Liver samples from AE patients used for immunological studies.