Adoptive transfer of splenocytes to study cell-mediated immune responses in hepatitis C infection using HCV transgenic mice

Abstract

Background: Hepatitis C virus (HCV) is a major cause of chronic hepatitis and a health problem affecting over 170 million people around the world. We previously studied transgenic mice that express HCV Core, Envelope 1 and Envelope 2 proteins predominantly in the liver, resulting in steatosis, liver and lymphoid tumors, and hepatocellular carcinoma. Herein, the immune-mediated cell response to hepatitis C antigens was evaluated by adoptive transfers of carboxyfluorescein succinimidyl ester (CFSE) labelled splenocytes from HCV immunized mice into HCV transgenic mice.

Results: In comparison to non-transgenic mice, there was a significant decrease in the percentage of CFSE-labeled CD4+ and CD8+ T cells in transgenic mouse peripheral blood receiving adoptive transfers from immunized donors. Moreover, the percentage of CFSE-labeled CD4+ and CD8+ T cells were significantly higher in the spleen of transgenic and non-transgenic mice when they received splenocytes from non-immunized than from immunized mice. On the other hand, the percentages of CD4+ and CD8+ T cells in the non-transgenic recipient mouse lymph nodes were significantly higher than the transgenic mice when they received the adoptive transfer from immunized donors. Interestingly, livers of transgenic mice that received transfers from immunized mice had a significantly higher percentage of CFSE labeled T cells than livers of non-transgenic mice receiving non-immunized transfers.

Conclusions: These results suggest that the T cells from HCV immunized mice recognize the HCV proteins in the liver of the transgenic mouse model and homed to the HCV antigen expression sites. We propose using this model system to study active T cell responses in HCV infection.

Figure 1

Humoral immune responses of the donor mice immunized with HCV immunogens as determined by ELISA. Seven mice were immunized with HCV immunogens containing HCV plasmid DNA, HCV recombinant polyprotein and montanide. Mice were immunized three times intramuscularly and boosted twice with the same vaccine. After the third immunization, serum samples were collected, serially diluted and tested for reactivity with HCV core, E1 and E2 protein. Sera were collected from the mice pre-immunization were used as a baseline. Immunized mice had significant increase in total IgG, IgG1, and IgG2a after the third immunization at 1:900 antibody titer (* P < 0.05).

Figure 2

CD4⁺ T cell proliferation response of HCV-immunized mice. The splenocytes were stained with CFSE dye and incubated with different stimulants for 4 days. Cells were stained for surface markers using anti-CD3⁺ and CD4⁺-antibodies and tested using flow cytometry. (A) Unstimulated cells showing no proliferation, (B) CE1E2 protein-stimulated cells showing proliferation of the cells which is indicated by the shift of fluoresecence in the cell population (circle), (C) Core peptide stimulated cells showing proliferation. Daughter cells contain half the fluorescent intensity of the parent cell.

Figure 3

CD8⁺ T cells cytolytic activity in the immunized mice as demonstrated by IFN-γ intracellular staining. Two weeks after the last HCV vaccine immunization, cultured splenocytes were unstimulated (A), stimulated with CE1E2 protein (B), core peptide (C), or vaccinia HCV poly (D). Cells were cultured for 18 hrs in the presence of brefeldin A then stained intracellularly with anti-IFN-γ antibody and surface stained with anti-CD3⁺ and anti-CD8⁺ antibodies to be analyzed by flow cytometry. Percentages in the upper right quadrant represent the frequency of CD3⁺8⁺ T lymphocytes expressing IFN-γ. The P value for significant differences was < 0.05.

Figure 4

Detection of CD4⁺ and CD8⁺ T lymphocyte responses to HCV vaccine in immunized mice using IFN-γ ELISPOT assay. ELISPOT counts (spot-forming units [SFUs]/1 × 10⁶) in response to core, E1 and E2 protein, Core peptides, or vaccinia HCV poly. Spot forming cell (SFC) frequencies are shown after subtraction of background with unstimulated cells or empty vaccinia stimulated cells. Cells were incubated with core, E1 and E2 protein, Core peptides, or vaccinia HCV poly for 48 hrs before measuring IFN-γ ELISPOT responses. Spot forming cell (SFC) frequency per million cells is indicated for each immunized and non-immunized donor mice. The P value was < 0.05.

Figure 5

Immunofluoresent analysis of CFSE labeled splenocytes before injection. A) CFSE unlabeled splenocytes showing no CFSE staining. B) CFSE labeled splenocytes showing green fluorescent cells. Scale bar = 50 μm.

Figure 6

Flow cytometric analysis of recipient mouse blood 24 hrs and 7 days post-adoptive transfer. A) The percentage of CFSE CD4⁺ and CD8⁺ T cells in the blood of the recipient mice 24 hrs post-injection. The × axis indicates the donor and recipient mouse groups (n = 7) and the Y axis indicate the percentage of the CFSE⁺ CD4⁺ or CD8⁺ T cells B) The percentage of donor CD4⁺ and CD8⁺ T cells in the blood seven days after the injection. The cells were surface stained with anti-CD3⁺ and anti-CD4⁺ antibodies or anti-CD3⁺ and anti-CD8⁺ and analyzed by flow cytometry (P <0.001).

Figure 7

Flow cytometric analysis of recipient mouse spleens and lymph nodes. A) The percentage of CD4⁺ and CD8⁺ T cells in the spleens of mice receiving immunized and non- immunized donor cells. B) The percentage of CD4⁺ and CD8⁺ T cells in the lymph nodes of the recipient mice. The cells were surface stained with anti-CD3⁺ and anti-CD4⁺ antibodies or anti-CD3⁺ and anti-CD8⁺ and analyzed by flow cytometry (P <0.001).

Figure 8

Flow cytometric analysis of recipient mouse livers. The percentage of CD4⁺ and CD8⁺ T cells in the liver of mice receiving immunized and non-immunized donor cells was detected by FACS. The cells were surface stained with anti-CD3⁺ and anti-CD4⁺ antibodies or anti-CD3⁺ and anti-CD8⁺ and analyzed by flow cytometry (P <0.001).

Figure 9

Histological alterations in livers from transgenic and non-transgenic mice injected with CFSE-labeled splenocytes from immunized mice. A) Immunofluorescent analysis of frozen liver sections (5 μm thick) of a transgenic mouse showing CFSE labeled cells scattered over all the liver section. The fluorescent cells are indicated by arrows. B) H&E stained liver section of transgenic mouse showing steatosis. There is infiltration of lymphocytes in the liver which is concentrated close to hepatic steatosis (indicated by arrows). C) Immunofluorescence analysis of frozen liver sections (5 μm thick) of non-transgenic mouse showing no CFSE labeled cells over the liver section. D) H&E staining of liver section of non-transgenic mouse showing normal histology of the liver and no lymphocyte infiltration. Scale bar = 50 μm.

Figure 10

Histological alterations in livers from transgenic and non-transgenic mice injected with CFSE-labeled splenocytes from non-immunized mice. A) Immunofluorescent analysis of frozen liver sections (5 μm thick) of a transgenic mouse showing few CFSE labeled cells scattered over all the liver section. B) H&E stained liver section of transgenic mouse showing steatosis. There is no infiltration of lymphocytes in the liver. C) Immunofluorescence analysis of frozen liver sections (5 μm thick) of non-transgenic mouse showing no CFSE labeled cells over the liver section. D) H&E staining of liver sections of non-transgenic mouse showing normal histology of the liver and no lymphocyte infiltration. Scale bar = 50 μm.