1. Volume 39, Issue 6, Pages 910-917 (December 2003)
Specific, functional effector/memory CD8+ T cells are found in the liver post-vaccination 
Nektarios Dikopoulos, Ieva Jomantaite, Reinhold Schirmbeck, Jörg Reimann 
Journal of Hepatology 
Volume 39, Issue 6, Pages (December 2003)
DOI: /S (03)

2. Fig. 1
HBsAg-specific CD8+ T cells primed by different vaccination protocols migrate to the liver. (A) B6 mice were immunized intramuscularly with either HBsAg particles (mixed with immune-stimulating ODN), or pCI/S plasmid DNA encoding HBsAg. Liver MNC were isolated 12 days post-vaccination and stained for CD8 and intracellular IFNγ after a 5 h ex vivo restimulation with the Kb-binding S190–197 epitope. The dot blots show the CD8+ IFNγ+ cell population. (B) The numbers of specific liver and splenic CD8+ IFNγ+ T cells/105 CD8+ T cells and the numbers of specific CD8+ IFNγ+ T cells per organ (×103) (±SEM) after in vitro restimulation with the Kb-binding S190–197 HBsAg epitope were measured in three individual mice per group. (C) Liver MNC and spleen cells from F1 BALB/c x B6 (H-2bxd) mice immunized with pCI/S plasmid DNA intramuscularly were restimulated in vitro with five immunogenic, H-2 class I-binding HBsAg peptides with the epitope and restriction specificities listed. The mean numbers of specific CD8+ IFNγ+ T cells/105 CD8+ T cells (±SEM) of three individual mice are shown.
Journal of Hepatology  , DOI: ( /S (03) )

3. Fig. 2
CD8+ T cells primed for different antigens migrate to the liver. B6 mice were immunized intramuscularly with plasmid DNA encoding HBcAg of HBV, ovalbumin (OVA), or the large T-Ag of SV40. Liver MNC and spleen cells were isolated 12 days post-vaccination and restimulated in vitro for 4 h with peptides of the respective epitope specificity listed. The numbers of specific CD8+ IFNγ+ T cells/105 CD8+ T cells in three individual mice per group was determined. Mean values (±SEM) are shown.
Journal of Hepatology  , DOI: ( /S (03) )

4. Fig. 3
Kinetics of appearance of primed CD8+ T cells in liver and spleen after plasmid DNA vaccination. F1 BALB/c x B6 (H-2bxd) mice were immunized intramuscularly with pCI/S plasmid DNA encoding HBsAg. Liver MNC and spleen cells were isolated and restimulated in vitro with the different antigenic peptides of HBsAg (Kb-binding S190–197 VWLSVIWM, Ld-binding S28–39 IPQSLDSWWTSL, Kd-binding S199–208 WYWGPSLYSI and the Dd-binding S201–209 WGPSLYSIL). The number of specific CD8+ IFNγ+ T cells/105 CD8+ T cells in the liver or the spleen from three individual mice at the indicated time points post-vaccination was determined. Mean values (±SEM) are shown.
Journal of Hepatology  , DOI: ( /S (03) )

5. Fig. 4
Surface phenotype of primed, specific liver CD8+ T cells. F1 BALB/c x B6 (H-2bxd) mice were immunized intramuscularly with pCI/S plasmid DNA. Liver MNC were isolated 12 days or 25 days post-vaccination. Cells were co-stained with FITC-conjugated anti-CD8 mAb, PE-conjugated Ld/S28–39 tetramers and biotinylated mAbs for the surface markers listed above. The small HBsAg-specific (tet+) CD8+ T cell population indicated in the dot-blot diagram was compared to the non-specific (tet−) CD8+ T cell population of the liver. Data from one representative experiment are shown in the dot blot. The mean percentages of cells that express the respective surface marker within both T cell subsets analyzed in three individual mice (±SEM) are shown in the table.
Journal of Hepatology  , DOI: ( /S (03) )

6. Fig. 5
Primed CD8+ T cells are not eliminated in the liver. Normal B6 mice and congenic B6 lpr/lpr mice (deficient for CD95 signalling) were immunized intramuscularly with pCI/S plasmid DNA. Liver MNC and spleen cells from both strains were isolated at the indicated time-points post-vaccination and restimulated in vitro with the Kb-binding S190–197 HBsAg epitope. The number of specific CD8+ IFNγ+ T cells/105 CD8+ T cells within the liver (A); and spleen (B) was determined in three individual mice per group and per time point. Mean values (±SEM) are shown.
Journal of Hepatology  , DOI: ( /S (03) )

7. Fig. 6
Intrahepatic primed CD8+ T cells show specific cytotoxicity. BALB/c mice were immunized intramuscularly with pCI/S plasmid DNA. Liver MNC and spleen cells were isolated 12 days post-vaccination, and restimulated in vitro with HBsAg-presenting, syngeneic stimulator cells. The specific cytolytic reactivity was determined in a 4 h 51Cr-release assay using P815/S transfectants as targets.
Journal of Hepatology  , DOI: ( /S (03) )

8. Fig. 7
HBsAg-specific, primed CD8+ T cells do not have to traffic through the spleen to reach the liver. B6 mice were either splenectomized (Sx) or sham operated. Two days later mice were immunized intramuscularly with pCI/S plasmid DNA. Their liver MNC were isolated 12 days post-vaccination. After a 4 h in vitro restimulation with the Kb-binding S190–197 peptide, the numbers of specific CD8+ IFNγ+ T cells/105 CD8+ T cells were determined for three individual mice per group. Mean values (±SEM) are shown.
Journal of Hepatology  , DOI: ( /S (03) )

9. Fig. 8
HBsAg-specific CD8+ T cells in liver or spleen can reconstitute specific CD8+ T immunity in syngeneic, naı̈ve hosts. B6 mice were immunized intramuscularly with pCI/S plasmid DNA. T cells from liver and spleen of vaccinated mice were isolated 12 days post-vaccination and transferred into syngeneic, naı̈ve adoptive hosts. Each host mouse received the hepatic or splenic T cell population from one donor mouse. The transplanted mice were rested for 40 days (but not immunized). Thereafter, liver MNC and spleen cells were isolated. The numbers of S190–197-specific CD8+ IFNγ+ T cells/105 CD8+ T cells were determined for three individual mice per group. Mean values (±SEM) are shown.
Journal of Hepatology  , DOI: ( /S (03) )