Intrahepatic accumulation and activation of COR93-specific CD8⁺ T cells in HBV transgenic mice is independent of T cell homing to the lymph nodes.

<p>Groups of 3–4 lineage 1.3.32 HBV-transgenic mice were treated with anti-CD62L antibodies (αCD62L) at 16 and 4 hours before adoptive transfer of naïve COR93-specific CD8⁺ BC10 T cells. 1 hour later, the mice were sacrificed and organs were harvested to analyze the frequency (A) and CD69 expression (B) of the transferred T cells in the liver, lymph nodes, and spleen. The data represent mean ± SD of three mice.</p

Kinetics of COR93-specific T cell activation.

<p>The kinetics of activation marker expression by COR93-specific CD8⁺ T cells in the liver (white), lymph nodes (gray), and spleen (black) were examined at indicated time points after adoptive transfer of 2×10⁷ of spleen cells from BC10.3 TCR transgenic mice into HBV-transgenic mice lineage 1.3.32 (A and B) and nontransgenic mice infected with 2×10⁷ pfu vaccinia virus expressing the HBV core protein (cVac) (C and D). COR93-specific CD8⁺ T cells were identified as CD8⁺CD45⁺ cells and the fraction of CD69 and CD25 expressing COR93-specific CD8⁺ T cells were depicted. The data represent mean ± SD of at least three mice.</p

Depletion of myeloid dendritic cells (mDCs) diminishes CD40 activation induced functional differentiation of HBV-specific CD8⁺ T cells.

<p>(A)–(D): Four groups of 3 CD11c-DOG mice were treated with saline (NaCl)+control liposome (NaCl-L), Diphtherial Toxin (DTX)+NaCl-L, NaCl+clodronate liposome (CLL) and DTX+CLL, and then sacrificed for the analysis of intrahepatic professional antigen presenting cell (pAPC) population. DTX or saline (NaCl) was administered 1 and 3 days before sacrifice, while CLL or NaCl-L was administered 2 days before sacrifice. The number of myeloid dendritic cells (F480⁺CD11c⁺), lymphoid dendritic cells (F480⁻CD11c⁺), Kupffer cells (F480⁺CD11c⁻), and B cells (B220⁺) were measured by FACS analysis. The percent of control group (NaCl+NaCl-L) in each group (n = 3) was expressed as a bar, and the absolute number of each cell population in the control group (n = 3) was also depicted. (E)–(H); Groups of 3–4 CD11c-DOG HBV transgenic mice were treated with DTX, CLL or DTX+CLL, and then received agonistic anti-CD40 antibody (αCD40). CD11c-DOG HBV-transgenic mice that were treated with NaCl, NaCl-L or NaCl+NaCl-L served as controls for DTX, CLL, or DTX+CLL treated mice, respectively. DTX or NaCl was administered every other day beginning from 3 days before αCD40 administration. CLL or NaCl-L was administered once 2 days before αCD40 administration. On day 1 after αCD40 administration, 2–3×10⁶ of purified COR93-specific naïve CD8⁺ T cells were adoptively transferred into each group. The mice were sacrificed on day 7 after adoptive transfer of COR93-specific naïve CD8⁺ T cells, and the absolute numbers of intrahepatic COR93-specific CD8⁺ T cells, IFNγ-producing CD8⁺ T cells, and Granzyme B positive CD8⁺ T cells, and ALT activity in DTX, CLL, and DTX+CLL treated mice (n = 3–4 for each group) were divided by the corresponding numbers in their respective control group (n = 3–4). Numbers above each bar represent the control values for each condition.</p

Induction of functional COR93-specific CD8⁺ T cell responses in HBV transgenic mice by CD40 activation.

<p>(A)–(D) HBV transgenic mice were treated with either saline (white) or agonistic anti-CD40 antibodies (αCD40) (black), and 16 hours later, 2×10⁷ of spleen cells harvested from BC10.3 TCR transgenic mice (containing approximately 3–5×10⁶ of COR93-specific naive T cells) were adoptively transferred into the HBV transgenic recipients. Mice were sacrificed on day 7 after adoptive transfer to analyze intrahepatic COR93-specific CD8⁺ T cell for the total number (A), the fraction of in vitro IFNγ producing (B), ex vivo GrB expressing (C), and PD-1 expressing (D) CD8⁺ T cells. The data represent mean ± SD of three mice. *P<0.05, **P<0.01, ***P<0.001. (E) Serum alanine aminotransferase (sALT) activity in the HBV transgenic mice described in (A)–(D) is expressed as units/liter. sALT activity in HBV transgenic mice that were treated with αCD40, but did not receive BC10.3 TCR transgenic spleen cells was also shown in gray bars. The data represent mean ± SD of three mice. *P<0.05, **P<0.01, ***P<0.001. (F) Effect of COR93-specific CD8⁺ T cell responses on HBV gene expression in the liver. Northern blot analysis of 20 µg of total liver RNA isolated from the same mice. GAPDH was used to normalize the amount of RNA bound to membrane.</p

Functional characterization of COR93-specific CD8⁺ T cells.

<p>COR93-specific CD8⁺ T cells were analyzed for their ability to expand, produce IFNγ, and express Granzyme B (GrB) in the liver (white), lymph nodes (gray), and spleen (black) at various time points after adoptive transfer to HBV transgenic mice lineage 1.3.32 (left panel) and cVac infected nontransgenic recipients (right panel). Figures (A) and (F) show the frequency COR93-specific CD8⁺ T cells (i.e. CD8⁺CD45.1⁺ cells), while Figure (B) and (G) show the absolute number of COR93-specific CD8⁺ T cells. Figures (C) and (H) show the fraction of in vitro IFNγ -producing COR93-specific CD8⁺ T cells, and Figures (D) and (I) show the fraction of ex vivo Granzyme B expressing CD8⁺ T cells. Figures (E) and (J) show the fraction of PD-1 positive CD8⁺ T cells. The data represent mean ± SD of three mice.</p

Expression and phenotype of HBV-specific CD8⁺ T cells in T cell receptor (TCR) transgenic mice.

<p>Spleen cells were isolated from TCR transgenic mouse lineages BC10.3 and 6C2.36 and the frequencies of K^b-restricted COR93-specific and L^d-restricted ENV28-specific CD8⁺ T cells were analyzed using peptide-pulsed K^b-, and L^d-dimers respectively (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003490#ppat-1003490-g001" target="_blank">Figure 1A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003490#ppat-1003490-g001" target="_blank">Figure 1F</a>). Splenic COR93- and ENV28-specific CD8⁺ T cells from respective TCR transgenic mice were gated by CD8⁺ and COR93-dimer and ENV28-dimer staining as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003490#ppat-1003490-g001" target="_blank">Figure 1A and 1F</a>, and analyzed for the expression of CD45.1, CD62L, CD44, CD69, and CD25 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003490#ppat-1003490-g001" target="_blank">Figures 1B–1D and 1G–1I</a>). Spleen cells from BC10.3 and 6C2.36 TCR transgenic mice were stimulated with COR93 and ENV28 peptide for 5 hours in vitro and CD8⁺ T cells were analyzed for the expression of IFNγ and Granzyme B (GrB) by intracellular cytokine staining (1E and 1J).</p

Infection with recombinant vaccinia viruses induces functional differentiation of COR93-specific CD8⁺ T cells in HBV transgenic mice.

<p>Groups of 3–4 HBV transgenic mice were treated with either saline (NaCl) or 2×10⁷ pfu of recombinant vaccinia viruses expressing the HBV core antigen (cVac), and one hour later, received 3–5×10⁶ of COR93-specific naïve CD8⁺ T cells. Groups of 3–4 nontransgenic mice were also infected with 2×10⁷ pfu of cVac and then received 3–5×10⁶ of COR93-specific naïve CD8 T cells. In addition, groups of 3–4 HBV transgenic mice were infected with the same titer of cVac without receiving COR93-specific naïve CD8⁺ T cells. On days 3 and 7, transferred COR93-specific CD8⁺ T cells were analyzed for expansion (A), IFNγ production (B) and Granzyme B expression (C) in the liver of HBV transgenic mice treated with saline (white bars), HBV transgenic mice infected with cVac (black bars) and nontransgenic mice infected with cVac (blue bars). The T cell responses in HBV transgenic mice were correlated with the degree of liver disease monitored by serum ALT activity (D) and HBV gene expression in the liver monitored by Northern Blot analysis (E). sALT activity and HBV gene expression in HBV transgenic mice that were infected with cVac without receiving COR93-specific naïve T cells were also shown in (D; red bar) and (E). The data represent mean ± SD of three to four mice (n = 3–4). *P<0.05, **P<0.01, ***P<0.001.</p

Naïve COR93-specific CD8⁺ T cells are activated by HBV expressing hepatocytes.

<p>CD8⁺ T cells were isolated from BC10.3 TCR transgenic mice. Hepatocytes, liver sinusoidal endothelial cells (LSECs), liver dendritic cells (DCs), and Kupffer cells (KCs) were purified from HBV transgenic mice lineage 1.3.32 and nontransgenic littermates as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003490#s4" target="_blank">Materials and Methods</a>, and then incubated in culture medium (Ex Vivo; left column) or pulsed with COR93-peptide (+COR93 peptide; right column) for 1 hour. After washing, 2×10⁵ of hepatocytes (A and F) or 1×10⁶ of purified LSECs (B and G), liver DCs (C and H) or Kupffer cells (D and I) from HBV transgenic mice were cocultured with 3×10⁵ of CD8⁺ T cells isolated form BC10.3 TCR transgenic mice. 16 hours later, cells were harvested and CD69 expression on COR93-specific CD8⁺ T cells was monitored by flow cytometric analysis (FACS). A representative histogram illustrating CD69 expression on COR93-specific CD8⁺ T cells after coculture with each cell population from HBV-transgenic mice lineage was shown in panels (A)–(D) and (F)–(I). Experiments were performed three times for each cell population, and bar graphs representing mean ± SD of the three experiments were shown in Figures (E) and (J). Bars represent the percentage of CD69 on COR93-specific CD8⁺ T cells after coculturing with Hepatocytes, LSECs, DCs, and KCs isolated from HBV transgenic mice lineage 1.3.32 (black bars) or with Hepatocytes and LSECs isolated from nontransgenic littermates (white bars). *P<0.05, **P<0.01, ***P<0.001.</p

Intrahepatic accumulation and activation of COR93-specific CD8⁺ T cells in HBV transgenic mice.

<p>2×10⁷ of spleen cells were isolated from BC10 TCR transgenic mice, and adoptively transferred into groups of 3–4 lineage 1.3.32 HBV transgenic mice and MUP-core 50 (MC50) transgenic mice. Lineage 1.3.32 replicates HBV and expresses HBcAg in their hepatocytes and secretes virus particles and HBeAg, a secreted viral protein that is highly cross-reactive with HBcAg. Lineage MC50 expresses only HBcAg under the mouse major urinary protein (MUP) promoter, and its expression is restricted to the hepatocytes. 1 hour after adoptive transfer of naïve COR93-specific CD8⁺ BC10 T cells, the mice were sacrificed and organs were harvested to analyze the frequency (A) and CD69 expression (B) of the transferred T cells in the liver, lymph nodes, and spleen. The data represent mean ± SD of three mice.</p

PD-1 deficient COR93-specific CD8⁺ T cells develop cytolytic ability in the liver of HBV transgenic mice.

<p>HBV transgenic mice were adoptively transferred with spleen cells from wild type (white) or PD-1 deficient (black) BC10.3 TCR transgenic mice. Mice were sacrificed on day 7 after adoptive transfer to analyze intrahepatic COR93-specific CD8⁺ T cell for the total number (A), the fraction of in vitro IFNγ producing (B), and ex vivo GrB expressing (C) CD8⁺ T cells. Serum alanine aminotransferase (sALT) activity in the same HBV transgenic mice is expressed as units/liter (D). The data represent mean ± SD of three mice. (E) Northern blot analysis of total liver RNA isolated from the same mice. GAPDH was used to normalize the amount of RNA bound to membrane. *P<0.05, **P<0.01, ***P<0.001.</p