CD107a expression in liver isolated CD8+ T-cells.

<p>(A) In vitro CD107a expression on liver (DBD and DCD) CD8+ T-cells was analysed after a co-culture with K562 cell line. Mann-Whitney U or Spearman correlation test was used (B) Gating of liver mononuclear cells to analyse CD107a expression on CD8+ T-cells. ns = not significant, * p<0.05.</p

The dominant T-cell subset of the liver is memory CD8 T-cells.

<p>Hepatic mononuclear cells (HMC) isolated from liver perfusate of living donors (LD, n = 11) were compared to matched PBMC. (A) Dot plots of CD4 and CD8 T-cells isolated from blood, through collagenase digestion and perfusion of the same liver. (B) Analysis of the combined expression of CD3, CD4, CD8, CD45RO and CD62L; on CD4+ (left) and CD8+ (right) T-cells (Mann-Whitney U test); on (C, D) CD4 (left) and CD8 (right) CD45RA+CD62L+ naïve (C) and CD45RO+CD62L- memory (D) CD69+ T-cells (Mann-Whitney U test); (E) CD127 expression on memory CD45RO+CD62L- CD4 (left) and CD8 (right) T-cells (Student t-test). ns = non-significant, *** p<0.001, ** p<0.01.</p

The donation status of the liver allograft influences the cytokine production in culture.

<p>Cytokines in the supernatants of day-seven HMC culture were measured. Left column: levels of IL-2 (A), TNF-α (B), IFN-γ(B), IFN-in the supernatants of day-seven HMC culture were measured. Left column: levels of IL-2 (A), TNF-ells. Numbers indicate epatocellular injury iα (B), IFN-γ(B), IFN-in the supernatants of day-seven HMC un-stimulated and stimulated HMC. Mann Whitney U or Kruskal Wallis test was performed. ns = non-significant, * p<0.05, **p<0.01, ***p<0.001.</p

Intrahepatic T-cells respond to HMGB1 <i>in vitro</i>.

<p>(A, B) Biopsies obtained from liver allografts before (left) and one-hour after (right) reperfusion. (A) Haematoxylin and eosin staining of a representative biopsy from DBD or DCD livers showing hepatocellular necrosis (plain arrow) and neutrophil aggregation (arrowhead) in post-reperfusion samples; (B) Immuno-histochemical staining for HMGB1 of the biopsies shown in (A) reveals an increased proportion of hepatocytes showing nuclear expression of HMGB1 in post-reperfusion samples compared to the pre-reperfusion counterparts but no cytoplasmic expression; (C) Production of IFN-γ from T-cells of a representative donor co-cultured with syngeneic DC at the ratio of 20:1 for 7 days. Percentage of IFN-γ-producing cells gated on CD8+CD3+ T-cells in unstimulated cultures (negative control), in presence of anti-CD3/CD28 (positive control) or in presence of HMGB1 at 100ng/ml; (D) Proliferative response of T-cells in unstimulated cultures (negative control), in presence of anti-CD3/CD28 (positive control) or in presence of HMGB1 at 100ng/ml. 1 = negative, 3 = positive control and 2 = HMGB1; (E) Quantitative analysis of IFN-γ concentration in supernatants of cultures. NT = negative control, S = aCD3/CD28 stimulation, HMGB1 = cultures stimulated in presence of 100 or 10ng/ml of HMGB1. Depiction of IFN-γ concentration in pg/ml (left) and ratio of increase reported to the negative control (right). Kruskal Wallis test was used.</p

Clinical parameters of recipients.

<p>Clinical parameters of recipients.</p

Cytokine production of T-cells in peripheral blood, LD, DBD and DCD livers.

<p>Hepatic mononuclear cells isolated from liver perfusate of living donors (LD) and matched PBMC were stimulated <i>ex vivo</i> and their cytokine production (n = 9 to 17) was assessed by intracellular staining. Frequency of IL-2 (A), IFN-γ (B) and IL-17 producing CD4 (left) and CD8 (right) T-cells (C). ** p<0.01, *** p<0.001. Furthermore, HMC isolated from DBD, DCD or LD livers have been stimulated <i>ex vivo</i> and cytokine production assessed by intracellular staining. Gating strategy determined using unstimulated cells. Numbers indicate percentage of cells detected; (D-F) Cytokine production of HMC isolated from indicated liver donors. Frequency of CD4 or CD8 T-cells expressing IL-2 (D), IFN-γ, E) and IL-17 (F). Statistical analysis: Mann Whitney U or Kruskal Wallis test was applied, ns = non-significant, **p<0.01, ***p<0.001.</p