1. Volume 135, Issue 3, Pages 980-988 (September 2008)
B7-H1 on Hepatocytes Facilitates Priming of Specific CD8 T Cells But Limits the Specific Recall of Primed Responses 
Christian Wahl, Petra Bochtler, Lieping Chen, Reinhold Schirmbeck, Jörg Reimann 
Gastroenterology 
Volume 135, Issue 3, Pages (September 2008)
DOI: /j.gastro
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2. Figure 1
Surface phenotype of hepatocytes (HC). The surface phenotype of freshly isolated, purified, scatter gated, 7-AAD− HC was determined using the indicated, conjugated mAbs (and appropriate isotype controls). Data from an individual, representative B6 mouse (from 7 individual mice independently analyzed) are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

3. Figure 2
Surface phenotype of OT-I CD8 T cells primed by peptide-pulsed dendritic cells (DC) or hepatocytes (HC). (A) Purified HC or DC (104 cells/well) presenting the Kb-binding OVA257–264 peptide SIINFEKL (1 μg/mL/106 cells) were cocultured for 3 days with purified, naive, splenic CD8 OT-I T cells (105 cells/well). Harvested CD8 T cells were washed and surface stained with the conjugated mAbs listed (or appropriate isotype controls). Data from a representative (out of 4 independent) experiment(s) are shown. (B) Kinetics of appearance of PD-1, B7-H1, and CD80 on the surface of CD8 T cells during priming. Purified HC or DC presenting the antigenic OVA257–264 peptide were cocultured for 24 hours or 48 hours with naive CD8 OT-I T cells. Harvested CD8 T cells were surface stained with the conjugated mAbs listed (or appropriate isotype controls). Data from 1 (out of 2 independent) experiment(s) are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

4. Figure 3
Peptide-presenting hepatocytes (HC) or DC prime naïve OT-I RAG−/− CD8 T cells. (A) Carboxy-fluorescein-diacetate-succinimidyl ester (CFSE)-labeled CD8 T cells (105 cells/well) cocultured for 48 hours with peptide (SIINFEKL)-pulsed DC or HC (104 cells/well). Data from a representative (out of 3 independent) experiment(s) are shown. (B) Kinetics of IFN-γ release by CD8 T cells primed by HC or DC presenting either the cognate (SIINFEKL) or an unrelated control (HBV) peptide. Pulsed HC or DC were cocultured with naive CD8 T cells. IFN-γ in the supernatants harvested at the indicated time points was determined by ELISA. Mean values of triplicates (+SEM) of a representative (out of 3 independent) experiment(s) are shown. (C) Dose-dependent IFN-γ and TNF-α release by CD8 T cells cocultured with HC or DC pulsed with increasing doses of the SIINFEKL peptide. Cytokines were determined in the 72-hour supernatant by ELISA. Mean values of triplicates (+SEM) of a representative (out of 6 independent) experiment(s) are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

5. Figure 4
B7-H1 and PD-1 blockade down modulate CD8 T-cell priming by peptide-presenting hepatocytes (HC) or DC. (A) Naive CD8 T cells were cocultured with HC or DC pulsed with 10 μg/mL SIINFEKL peptide. In some groups, 5 μg/mL of a blocking anti-B7-H1 and/or anti-PD-1 antibody were added. IFN-γ and IL-2 were determined in 72-hour supernatants by ELISA. Mean values of triplicates (+SEM) of 1 representative (out of 3 independent) experiment(s) are shown. *P < .05; **P < .01. (B) Naive OT-I CD8 T cells were cocultured with SIINFEKL peptide-pulsed HC in the presence of the indicated concentration of anti-B7-H1 or anti-PD-1 antibody. IFN-γ and IL-2 were determined in 72-hour supernatants by ELISA. Mean values of triplicates (+SEM) of 1 representative (out of 2 independent) experiment(s) are shown. (C) B7-H1 and PD-1 blockade limit CD8 T-cell proliferation. Left panel: Carboxy-fluorescein-diacetate-succinimidyl ester (CFSE) profiles of CD8 T cells cocultured for 48 hours with peptide-pulsed HC (with or without a 20 U/mL IL-2 supplement) in the presence of 5 μg/mL anti-B7-H1 or anti-PD-1 blocking antibody or an unrelated, isotype-matched control antibody. Surface expression of the IL-2 receptor α-chain (CD25) and β-chain (CD122) by OT-I CD8 T cells harvested from 48-hour cultures. Shaded histograms represent cells stained with conjugated anti-CD25 or anti-CD122 antibodies; open histograms represent cells stained with conjugated isotype control antibodies. Intracellular staining for IL-2 produced by CD8 T cells cocultured for 48 hours with peptide-pulsed HC in the presence or absence of anti-B7-H1 antibody. (D) IFN-γ release by CD8 T cells cocultured for 24 hours with peptide-pulsed HC in the presence or absence of anti-B7-H1 antibody.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

6. Figure 5
B7-H1-deficient HC inefficiently prime CD8 T cells. (A) OT-I CD8 T cells (105 cells/well) were cocultured with SIINFEKL peptide-pulsed HC (104 cells/well) from wild-type (wt) or B7-H1-deficient (B7-H1−/−) B6 mice. Proliferation in 2-day cultures was assayed by measuring 6-hour 3H-thymidine uptake. (B) IFN-γ and IL-2 were determined by ELISA in supernatants harvested at the indicated time points of culture. Mean values of triplicates (+SEM) of a representative (of 3 independent) experiment(s) are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions

7. Figure 6
The B7-H1/PD-1 interaction down modulates cytokine production and proliferation of CD8 T blasts specifically restimulated by peptide-pulsed HC (or DC). (A) OT-I CD8 T blasts activated by peptide-pulsed, splenic DC were harvested, washed, purified by MACS, and cocultured for 72 hours with peptide-pulsed HC or DC in the presence of 5 μg/mL anti-B7-H1 or anti-PD-1 blocking antibody. IFN-γ in supernatants was determined by ELISA. Mean values of triplicates (+SEM) of 1 representative (of 3 independent) experiment(s) are shown. *P < .05. (B) CD8 T blasts (105 cells/well) were cocultured with peptide (SIINFEKL)-pulsed HC (104 cells/well) from wild-type (wt) or B7-H1-deficient (B7-H1−/−) B6 mice. Proliferation was measured after 2 days of culture by 6-hour 3H-thymidine uptake. IFN-γ in supernatants harvested at the indicated time points were determined by ELISA. Mean values of triplicates (+SEM) of a representative (out of 3 independent) experiment(s) are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2008 AGA Institute Terms and Conditions