Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 1. Recipient CC1 signaling mitigates hepatic IRI and suppresses NF-kB p65 in mouse OLT. Mouse WT livers subjected to 18 hours of cold storage were transplanted into WT or CC1KO syngeneic recipients (n ¼ 6–8/group). OLT/serum samples were analyzed 6 hours after reperfusion. The sham group (n ¼ 5) underwent the same procedures except for OLT. (A) Representative H&E and TUNEL staining. DAPI, 40,6-diamidino-2-phenylindole. Scale bars ¼ 100 mm. (B) Suzuki’s histologic grading of liver IRI and quantification of TUNELþ cells/HPF. (C) sAST/sALT and (D) lactate dehydrogenase (LDH) levels (U/L). Quantitative reverse transcription polymerase chain reaction–assisted OLT detection of (E) IFN-gamma, IL6, IL17, and gran- zyme B/perforin 1, and (F) TLR4, IL1b, and TNF-a (n ¼ 6–7/group). Data normalized to hypoxanthine guanine phosphor- ibosyltransferase gene expression. (G) Western blot–assisted detection of phosphorylated (p)-IkBa, IkBa, p-NF-kB p65, NF-kB p65, and vinculin (VCL). The relative intensity ratio of p-IkBa/IkBa and p-NF-kB p65/NF-kB p65 (n ¼ 4/group) is shown. Data are shown as mean ± standard error of the mean *P < .05, **P < .01, Student t test.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: TUNEL Assay, Staining, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 2. CC1 signaling enhances TIM-3 expression and suppresses CD4þ T-cell inflammatory signature. (A) Representative (n ¼ 4/group) flow cytometry of CC1 and TIM-3 expression in peripheral blood lymphocytes from naive WT and CC1KO mice. TIM-3þ frequency in (B) WT CC1þ and CC1 CD4þ T cells and in (C) WT and CC1KO CD4þ and CD8þ T cells. (D) Repre- sentative (n ¼ 4/group) flow cytometry of CC1 and TIM-3 expression in activated peripheral blood lymphocytes from WT and CC1KO mice subjected to hepatic IRI. TIM-3þ frequency in (E) WT CC1þ and CC1 CD4þ T cells and in (F) WT and CC1KO CD4þ and CD8þ T cells. (G) CC1þ frequency in naive vs post-IRI in WT CD4þ T cells. (H) TIM-3þ frequency in naive vs post-IRI in WT and CC1KO CD4þ T cells. (I) Quantitative reverse-transcription polymerase chain reaction–assisted detection of IFN-g, T-box protein (T-bet) expressed in T cells, IL6, IL17, IL22, and TNF-a in CD4þ T cells from WT and CC1KO mice before and after stimulation (n ¼ 6/group). White square: WT; black square: CC1KO mouse. Data shown as mean ± standard error of the mean. *P < .05, ****P < .0001, Student t test.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: Expressing, Cytometry, Reverse Transcription, Polymerase Chain Reaction

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 4. Enhanced T cell–specific TIM-3 alleviates IRI-OLT and suppresses Kupffer cell NF-kB p65 in CC1-deficient re- cipients. WT livers after 18 hours of cold storage were transplanted into WT, CC1KO, T cell–specific TIM-3Tg/CC1KO, and TIM-3Tg/CC1KO þ anti–TIM-3 antibody (n ¼ 6–8/group). OLT/serum samples were analyzed at 6 hours. (A) Representative H&E and TUNEL staining. DAPI, 40,6-diamidino-2-phenylindole. Scale bars ¼ 100 mm. (B) Suzuki’s histologic grading of liver IRI and quantification of TUNELþ cells/HPF. (C) sAST/sALT (U/L). Quantitative reverse-transcription polymerase chain reaction–assisted detection of (D) IFN-gamma, IL6, and IL17, and (E) TLR4, IL-1b, and TNF-a in OLT (n ¼ 6/group). Data in D and E were normalized to hypoxanthine guanine phosphoribosyltransferase gene expression. (F) Western blot-assisted detection of CC1, phosphorylated (p)-NF-kB p65, NF-kB p65, and vinculin (VCL). The relative intensity ratio of p-NF-kB p65/NF-kB p65 (n ¼ 3–4/group) is shown. (G) Representative NF-kB staining in OLT. Arrows indicate nuclear NF-kB locali- zation in nonparenchymal cells. Scale bars ¼ 100 mm. (H) Representative C-type lectin domain family 4 member F (CLEC4F; Kupffer cell) and p-NF-kB p65 staining. Arrowheads indicate Kupffer cells augmenting p-NF-kB p65. Scale bars ¼ 100 mm (left panels) and 20 mm (enlarged images). Data are shown as mean ±standard error of the mean. *P < .05, **P < .01, ***P < .01, Student t test.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: TUNEL Assay, Staining, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 5. Donor liver CC1 deficiency compromises T cell–specific TIM-3 regulation in CC1-deficient recipients. (A) CC1KO livers after 18 hours of cold storage were transplanted into CC1KO or TIM-3Tg/CC1KO mice. OLT/serum samples were analyzed at 6 hours (n ¼ 6/group). The sham group (n ¼ 5) underwent the same procedures, except for OLT. (B) Representative H&E staining. Scale bars ¼ 100 mm. (C) Suzuki’s histologic grading of liver IRI and sAST/sALT (U/L). (D) Representative C-type lectin domain family 4 member F (CLEC4F; Kupffer cells) and phosphorylated (p)-NF-kB p65 staining in OLT. DAPI, 40,6- diamidino-2-phenylindole. Arrowheads indicate p-NF-kB p65þ cells. Scale bars ¼ 100 mm. Quantative reverse-transcription polymerase chain reaction–assisted detection of (E) IFN-gamma, IL6, and IL17 (n ¼ 6/group), and (F) TLR4, IL-1b, and TNF-a (n ¼ 6/group) in OLT. Data were normalized to hypoxanthine guanine phosphoribosyltransferase (HPRT) gene expression. (G) Western blot–assisted detection of CC1, p-NF-kB p65, NF-kB p65, and b-actin. The relative intensity ratio of p- NF-kB p65/NF-kB p65 (n ¼ 3/group) is shown. Data are shown as mean ± standard error of the mean. ***P < .001, Student t test.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: Staining, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 6. Perioperative increase of CC1 promotes anti-inflammatory phenotype in human OLT. (A) Pretransplant (after cold storage) and posttransplant (2 hours after reperfusion) hepatic biopsy specimens were collected from OLT patients. Post-/pre- CC1 ratios were analyzed at the gene (n ¼ 27) and protein (n ¼ 50) levels. Relationship between post-/pre-CC1 gene ratio and (B) CD154, CD28, IFN-gamma, and IL-17, (C) TLR2, TLR4, TLR9, and CD68, and (D) cathepsin G and HO-1 gene expression with b-actin normalization (n ¼ 27), *P < .05, **P < .01; nonparametric Spearman’s method.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: Gene Expression

Journal: Gastroenterology

Article Title: T Cell CEACAM1-TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans.

doi: 10.1053/j.gastro.2023.07.004

Figure Lengend Snippet: Figure 7. Perioperative increase of CC1 attenuates hepatocellular injury and improves rejection-free human OLT survival. Post-/pre- CC1 ratios were assessed by Western blots with b-actin normalization. (A) OLT patients were divided into low (n ¼ 25) and high (n ¼ 25) post-/pre-CC1 ratio groups, based on the median value of the CC1 ratio (cutoff ¼ 1.05). (B) Representative Western blots and case patient-related clinical parameters (case patients 1 and 2: low post-/pre-CC1 ratio, case patients 3 and 4: high post-/pre-CC1 ratio). (C) sAST/sALT at POD 17. (D) Representative CD4/CC1 staining in OLT. Arrows: CC1-negative CD4þ T cells; arrowheads: CC1-positive CD4þ T cells. Scale bars ¼ 100 mm. DAPI, 40,6-diamidino-2-phenylindole. (E) Incidence of EAD. (F) The cumulative rejection rate (Kaplan-Meier method). The solid line indicates high and dotted line low post-/pre-CC1 ratio in human OLT. Data shown as mean ± standard error of the mean. *P < .05, Mann-Whitney U test in C, Fisher’s exact test in E, and log-rank test in F.

Article Snippet: Human liver samples were stained with rabbit anti-CD4 (ab133616/ EPR6855, Abcam) and rat anti-CC1 antibody (MAB6480/ 723629, R&D Systems).

Techniques: Western Blot, Staining, MANN-WHITNEY