IL-21 is pivotal in determining age-dependent effectiveness of immune responses in a mouse model of human hepatitis B (original) (raw)
Different disease outcomes occur in young and adult HBVtgRag mice following adoptive transfer of adult splenocytes. Adoptive transfer of splenocytes from adult (8- to 10-week-old) wild-type mice into adult (8- to 10-week-old) Rag1–/– C57BL/6 HBVEnv mice (HBVEnvRag mice) or Rag1–/– C57BL/6 HBVRpl mice (HBVRplRag mice) revealed a robust, HBV-dependent inflammatory response and liver injury after adoptive transfer. The mice developed an acute hepatitis, beginning 4–5 days after adoptive transfer, as evidenced by a rise in plasma alanine transaminase (ALT), a measure of hepatic necrosis (Figure 1, A and B). Histological analysis of H&E-stained liver sections from these mice revealed significant HBV-dependent portal and intraparenchymal inflammation as well as hepatic necrosis (Figure 1, C, D, and F).
Transfer of adult splenocytes into young and adult HBVtgRag mice results in a difference in disease outcome. Depiction of plasma ALT in young and adult (A) HBVEnvRag or (B) HBVRplRag mice after adoptive transfer of adult wild-type C57BL/6 mouse splenocytes. Error bars show mean ± SEM. Data are representative of at least 3 separate experiments where N ≥ 5 mice. Sections from formalin-fixed, paraffin-embedded liver tissue were taken 8 days after adoptive transfer of adult, syngeneic splenocytes and stained for H&E. (C) Composite score of necrosis, portal inflammation, and intraparenchymal inflammation as read by an unbiased pathologist. Error bars depict mean ± SEM; N ≥ 5 mice. Statistical significance was determined using the ANOVA with Tukey’s multiple comparison test. **P < 0.01. Representative liver H&E stains from (D) adult HBVEnvRag, (E) young HBVEnvRag, and (F) adult HBVtg-negative Rag1–/– mice. Arrows point to portal tract inflammation adjacent to the portal vein (PV) and bile duct (BD). Asterisks indicate necrotic hepatocytes. Scale bar: 30 mm. Statistical significance was determined using the unpaired 2-tailed t test. (G) IL-10, IL-17, IFN-γ, IL-4, and IL-12 cytokine levels were determined by ELISpot assay on liver-derived lymphocytes from adult or young HBVEnvRag mice 8 days after transfer of adult syngeneic splenocytes. Samples were pooled from n = 4 mice; error bars represent mean ± SEM from at least 3 separate wells.
To assess the contribution of the age of the resident immune-priming environment in the HBVEnvRag or HBVRplRag mice to the immune response to HBV, we adoptively transferred splenocytes from adult (8- to 10-week-old) wild-type mice into young (3- to 4-week-old) HBVEnvRag or HBVRplRag mice. This experiment revealed a strikingly different HBV-dependent inflammatory response. The young transgenic mice that received adult splenocytes never developed ALT elevation and exhibited only minimal HBV-dependent hepatic inflammation, without evidence of hepatic necrosis (Figure 1, A–C, E, and F). No ALT elevation or hepatic inflammation was observed in Rag1–/– mice after adoptive transfer (Figure 1F; data not shown). A composite hepatic inflammation score that grades portal inflammation, intraparenchymal inflammation, and necrosis revealed that the adult HBVEnvRag mice had significantly increased hepatic inflammation as compared with that of the young HBVEnvRag mice 8 days after adoptive transfer of splenocytes (Figure 1C). Both the young and adult HBVEnvRag mice had increased hepatic inflammation scores as compared with those of Rag1–/– mice (Figure 1C).
ELISpot analysis of liver lymphoid cells at the peak of hepatitis in the adult mice (8 days after adoptive splenocyte transfer) revealed HBV-dependent production of IL-12, IFN-γ, and IL-4 but little detectable IL-17 or IL-10 (Figure 1G). Surprisingly, ELISpot analysis of liver lymphoid cells extracted from young mice 8 days after adoptive splenocyte transfer revealed HBV-dependent cytokine production similar to that in adult mice, suggesting these young mice are initiating an immune response to the viral proteins. The liver lymphoid cells from young mice also revealed HBV-dependent production of IL-12, IFN-γ, and IL-4 and little detectable IL-17 or IL-10, although adult mice produced more IL-4 and IFN-γ (Figure 1G). We determined that cytokine production was HBV antigen-dependent by including control, Rag1–/– HBV transgene-negative C57BL/6 mice in adoptive transfer experiments (ref. 9 and Supplemental Figure 4B; supplemental material available online with this article; doi:10.1172/JCI44198DS1).
Adult HBVtgRag mice generate antibody to hepatitis B core antigen and hepatitis B surface antigen and clear hepatitis B surface antigen from circulation; young HBVtgRag mice only generate antibody to hepatitis B core antigen and do not clear hepatitis B surface antigen. When an effective immune response resulting in viral clearance is mounted in humans infected with HBV, hepatitis B surface antigen (HBsAg) disappears from the blood, as antibodies against hepatitis B core antigen (HBcAb) and HBsAg (HBsAb) are generated. In patients who become chronically infected with HBV, HBcAb is generated, but HBsAg remains detectable in the blood and HBsAb is not generated (3). Because these serum profiles of HBsAg, HBsAb, and HBcAb are signatures of viral clearance and persistence in human HBV infection, we determined the presence of these markers in the plasma of HBVtgRag mice after splenocyte transfer.
Adoptive transfer of splenocytes from adult wild-type mice into adult HBVEnvRag or HBVRplRag mice resulted in the generation of an immune response that led to life-long clearance of HBsAg from plasma, beginning 14 days after adoptive splenocyte transfer (Figure 2, A and B). In addition, these mice produced antibodies to HBsAg beginning 28 days after adoptive transfer (Figure 2, C and D), and the HBVRplRag mice produced antibodies to hepatitis B core antigen (HBcAg) beginning 12 days after adoptive transfer (Figure 2E).
Transfer of adult splenocytes into young and adult HBVtgRag mice results in a difference in HBsAg clearance and HBsAb responses. Depiction of (A and B) the percentage of mice with detectable circulating HBsAg and (C and D) plasma HBsAb titer in young and adult (A and C) HBVEnvRag or (B and D) HBVRplRag mice after adoptive transfer of adult wild-type C57BL/6 mouse splenocytes. Error bars show mean ± SEM where N ≥ 5 mice. (E) Presence of HBcAb in plasma of adult or young HBVRplRag mice after adoptive transfer of adult, wild-type C57BL/6 mouse splenocytes (n = 4 mice). Statistical significance was determined using the unpaired 2-tailed t test. Data are representative of at least 3 separate experiments.
However, adoptive transfer of splenocytes from adult wild-type mice into young HBVEnvRag or HBVRplRag mice resulted in failure to clear HBsAg from circulation and failure to produce HBsAb, despite their immune response to HBV (Figure 2, A–D). Notably, however, young HBVRplRag mice did generate an antibody response to HBcAg (Figure 2E).
Taken together, the effective immune responses and their kinetics observed in the adult HBVtgRag mice are comparable to those seen in adult humans experiencing acute, self-limited infection (refs. 3 and 4; Figure 3A). On the other hand, the immune responses and their kinetics observed in the young HBVtgRag mice are strikingly similar to those seen in young humans who develop persistent HBV infection (refs. 3 and 4; Figure 3B). Thus, this mouse model of primary HBV infection recapitulates many of the key differences in viral clearance during early life and adulthood in humans.
Patterns of serologic and molecular markers in this age-dependent model of primary HBV infection. Typical levels of ALT, HBsAg, HBsAb, and HBcAb are shown in (A) adult HBVtgRag mice that received adult splenocytes by adoptive transfer and (B) young HBVtgRag mice that received adult splenocytes by adoptive transfer. The intensity of the responses, as a function of time after infection, is indicated schematically.
Analysis of young and adult HBVtgRag mice (both HBVEnvRag and HBVRplRag mice) revealed minimal differences in antigen expression in the plasma or liver. Both age groups demonstrate overlapping plasma levels and hepatocyte expression of HBsAg, demonstrating that the observed age-related differences in immune response likely do not result from differences in antigen expression (Supplemental Figure 1). Additionally, the differences in disease and antigen clearance in young and adult HBVtgRag mice is not mouse strain-specific, since these differences were also observed in HBVEnvRag mice on a B10.D2 (H-2d) background after syngeneic splenocyte transfer (Supplemental Figure 2).
Adult HBVEnvRag recipient mice have more hepatic CD8+ T cells and T follicular helper cells. To investigate differences in the HBV-specific cellular immune responses in young and adult HBVEnvRag mice that might account for the dichotomous, age-dependent disease outcomes, we analyzed hepatic lymphocyte repertoires after adoptive transfer of adult splenocytes. We observed a significant, early increase in the percentages and absolute numbers of T cells, CD8+ T cells, and T follicular helper (TFH) cells but not of CD4+ T cells, B cells, NK, or NKT cells in the livers of adult versus young mice 8 days after adoptive transfer of splenocytes (Figure 4A and Supplemental Figure 3). There was a compensatory increase in the percentage of NK cells in the young HBVEnvRag mice, but absolute NK cell numbers were equivalent in young and adult livers (Supplemental Figure 3). Notably, there was no difference in the percentages of hepatic T regulatory cells during the critical early period after adoptive splenocyte transfer (8 days and 22 days; Figure 4B).
Adult HBVEnvRag recipient mice after adoptive transfer have more CD8+ T cells and TFH cells and elicit a more diverse and long-lived HBV-specific T cell response in the liver. (A) The frequency of lymphocyte populations from the livers of adult and young HBVEnvRag mice 8 days after splenocyte transfer. TFH cells are defined as CD4+, CXCR5+, ICOS+ cells. B cells are defined as CD19+, B220+ cells. Error bars depict mean ± SEM; n = 4 mice. Statistical significance was determined using the unpaired 2-tailed t test. (B) Frequency of T regulatory cells in adult and young HBVEnvRag mouse liver-derived lymphocytes on days 8 and 21 after transfer of wild-type splenocytes. Samples were pooled from N ≥ 4 mice. Bars show percentages of CD4+-gated cells that are CD25+and FoxP3+. IFN-γ ELISpot results from young and adult HBVEnvRag mouse liver-derived lymphocytes (C) 8 days, (D) 3 months, or (E) 1 year after adoptive transfer. Cells were stimulated with HBV envelope peptide pools as described in Supplemental Figure 3 (pool 0 = no peptide). Dotted and solid lines indicate baseline IFN-γ levels for adult and young mice, respectively. A positive response was considered to be more than twice that of baseline. Lymphocytes were combined 1:1 with Rag1–/– splenocytes (to function as APCs); background IFN-γ levels from splenocytes are indicated by striped bars. Data are representative of at least 2 experiments; samples were pooled from N ≥ 4 mice.
Adult HBVEnvRag mice elicit a more robust, diverse, and long-lived HBV-specific T cell response in the liver. It is generally accepted that the adaptive immune response to HBV is highly indicative of the course of disease. Correlative clinical studies in patients show that acute, self-limited hepatitis B is associated with a strong polyclonal and multispecific CD8+ T cell response (which can be shown in peripheral blood) early in infection. These responses involve both MHC class II–restricted CD4+ helper T cells and MHC class I–restricted CD8+ CTLs. The antiviral CTL response is directed against multiple epitopes within the HBV core, polymerase, and envelope proteins. By contrast, in chronic carriers of HBV, such initial virus-specific T cell responses are weak and exhibit narrow epitopic complexity, at least as assayed in cells from the peripheral blood (14, 15).
To examine the strength and epitopic diversity of the HBV-specific T cell response in the age-dependent mouse model of primary HBV infection, we tested liver-, spleen-, and lymph node–derived lymphoid cells in an ELISpot assay using pools of peptides, spanning the entire HBV envelope proteins (Supplemental Figure 4A). Eight days after adoptive transfer of adult splenocytes into adult HBVEnvRag mice, liver-derived T cells produced IFN-γ in response to multiple HBV envelope peptides (Figure 4C). The epitopic diversity of liver T cell responses was still seen at 3 months after adoptive transfer (Figure 4D). After 1 year, the adult HBVEnvRag mice retained a T cell response to 2 peptide pools (Figure 4E). In young HBVEnvRag mice reconstituted with adult splenocytes, ELISpot assays also revealed the presence of HBV-specific T cell responses, which were generally weaker and less diverse than in their adult counterparts (Figure 4, C–E). Specifically, 8 days after adoptive transfer of adult splenocytes into the young HBVEnvRag transgenic mice, the liver-derived T cells contained fewer IFN-γ–producing cells and responded to fewer peptide pools, largely concentrated at the C terminus of the envelope protein (Figure 4C). In the young mice, these relatively narrow T cell responses were still present 3 months after adoptive transfer (Figure 4D), but after 1 year, no appreciable responses to the peptide pools were observed (Figure 4E). In contrast, after adoptive transfer of wild-type splenocytes, no significant IFN-γ production was observed from HBVEnvRag splenocytes or regional lymph node–derived cells or from Rag1–/– control mice in response to the peptide pools (Supplemental Figure 4, B–D).
Our antigenic peptide library consists of overlapping 15–amino acid peptides, which can be presented by both MHC class I and class II molecules. The relative contributions of CD8+ and CD4+ T cell responses were therefore investigated during the HBV-specific immune responses observed in our system. Intracellular IFN-γ staining of liver-derived lymphoid cells from the HBVEnvRag mice reconstituted with splenocytes revealed that liver-derived CD8+, CD4+, and CD4+8+ T cells all contributed to the HBV-specific T cell response (Supplemental Figure 5A).
Liver lymphocytes from adult HBVEnvRag mice after adoptive transfer produce IL-21 from TFH cells in an HBV-dependent manner and have increased numbers of IgG-expressing B cells. The blunted HBV-specific T and B cell responses in our young versus adult transgenic mice, together with the discovery that the young mice have significantly fewer TFH cells in the liver, stimulated comparative analysis of known TFH cell factors involved in the generation of antibody responses and T cell expansion. IL-21 is a type 1 cytokine, produced by TFH cells, that is critical for plasma cell generation, antibody isotype switching, and regulating immunoglobulin production and that can also promote expansion of CD8+ T cells (16–18). In models of acute and chronic infection of lymphocytic choriomeningitis virus (LCMV), IL-21 was found to be important in rescuing “exhausted” T cells after chronicity had been established, although it was not required for the specific CD8+ T cell response or viral control (19–21). Because these known properties of IL-21 might explain some of the immune defects in the young mice, we investigated the production and role of IL-21 in our model of HBV infection.
Eight days after adoptive splenocyte transfer, the adult HBVEnvRag mice had a 4.3-fold increase in Il21 mRNA in liver-derived CD4+ T cells (Figure 5A), as well as a substantial increase in the number of IL-21–producing, liver-derived lymphoid cells (Figure 5B), compared with that in the young HBVEnvRag mice. The HBV-dependent increase in Il21 mRNA occurred mostly in the CD4+ T cell subset that expressed the chemokine receptor, CXCR5 (78% of these CXCR5+CD4+ T cells were ICOS+; Figure 5C). Thus, the majority of IL-21–producing cells were TFH cells. In contrast, we did not detect an increase in HBV-dependent Il21 transcripts or production of IL-21 protein in the spleen (Figure 5, A and B).
Liver lymphocytes from adult HBVEnvRag mice after adoptive transfer produce more IL-21 from TFH cells and have increased numbers of IgG-expressing B cells. (A) Il21 mRNA levels relative to those of GAPDH in CD4+ (CD4 enrich) and CD4– (CD4 deplete) fractions from liver-derived lymphocytes or splenocytes of adult or young HBVEnvRag or adult Rag1–/– mice 8 days after adoptive transfer of splenocytes. Error bars depict duplicate wells, samples were pooled from N ≥ 6 mice. (B) IL-21 protein expression determined by ELISpot on liver-derived lymphocytes and splenocytes 8 days after transfer. Samples were pooled from N ≥ 6 mice. (C) Il21 transcripts from unsorted liver lymphocytes, CXCR5–CD4+ sorted cells, CXCR5+CD4+ sorted cells (not determined for Rag1–/– due to low cell number), and CXCR5–CD4– cells from HBVEnvRag and Rag1–/– mice 8 days after transfer. Sorted cells were also CD19–DAPI–. Error bars depict triplicate wells; samples were pooled from N ≥ 4 mice. ND, not determined. (D) Frequency of B cells (plasmablasts and plasma cells) in adults and young HBVEnvRag mice 3 weeks after splenocyte transfer. The left plots show B220 versus CD44 expression on TCRβ– populations, and the right plots show IgM versus IgG1 on TCRβ–, CD44hi, B220lo populations. The percentage of IgG1-, IgG2b-, IgG3-, and IgM-expressing B cells from (E) liver-derived lymphocytes and (F) splenocytes. Error bars depict mean ± SEM; N ≥ 4 mice. Statistical significance was determined using the unpaired 2-tailed t test.
Analysis of immunoglobulin expression on plasma cells and plasmablasts in the liver and spleen of adult and young HBVEnvRag mice 3 weeks after adoptive splenocyte transfer revealed that the adult mice exhibited a significant increase in the number of plasma cells and plasmablasts that were differentiated into IgG1-, IgG2b-, and IgG3-expressing cells (Figure 5, D–F). This B cell differentiation and class-switch difference occurred to a lesser extent in the spleen than in the liver (Figure 5, E and F).
IL-21 receptor on transferred splenocytes is necessary to generate an immune response that correlates with viral clearance. To test whether an IL-21–dependent immune response is necessary to generate the HBV antigen clearance seen in adult HBVEnvRag mice, we used splenocytes from IL-21 receptor–deficient (IL-21R–deficient) mice as donor cells in adoptive transfer experiments (17). These experiments showed that lack of IL-21R on adoptively transferred splenocytes resulted in immune responses in adult HBVEnvRag mice that resembled the responses in young HBVEnvRag mice and resulted in HBV antigen persistence. More specifically, lack of IL-21R on donor splenocytes prevented hepatitis (ALT elevation) during the peak of the adaptive immune response (day 7) but did not appear to influence hepatitis during the “NKT cell phase” of disease (refs. 9 and 10; Figure 6A). Furthermore, lack of IL-21R on donor splenocytes resulted in the absence of clearance of circulating HBsAg (Figure 6B) and the absence of HBsAb production (Figure 6C). Finally, ELISpot analysis using the pools of HBV envelope peptides demonstrated that the absence of IL-21R on donor splenocytes caused a weaker and less diverse T cell response to HBV envelope peptides 8 weeks after adoptive transfer (Figure 6D). Intracellular IFN-γ staining of liver-derived lymphoid cells from HBVEnvRag mice 2 months after adoptive transfer of wild-type or IL-21R–deficient splenocytes revealed that lack of IL-21R on donor cells resulted in a greater reduction in the strength and diversity of the CD8+ T cell response than of the CD4+ T cell response (Supplemental Figure 5).
Adult HBVEnvRag mice fail to clear HBsAg, fail to produce HBsAb, and have a weaker and less diverse HBV-specific T cell response in the absence of IL-21R on transferred splenocytes. Adult HBVEnvRag mice were adoptively transferred with either wild-type or IL-21R–deficient C57BL/6 splenocytes. (A) Mice were monitored for plasma ALT (no ALT differences were observed at later time points; data not shown). Horizontal bars indicate SEM. (B) The percentage of mice with detectable circulating HBsAg is shown. (C) Mice were monitored for HBsAb titer in the plasma (N ≥ 7 mice). Horizontal bars indicate SEM. (D) HBV-specific T cell responses at 2 months after transfer as measured by the IFN-γ ELISpot response of liver-derived lymphocytes stimulated with peptide pools; samples were pooled from N ≥ 4 mice. Statistical significance was determined using the unpaired 2-tailed t test.
Increased IL-21 expression is part of an effective immune response to HBV in adults acutely infected with HBV who clear the virus. The mouse model and the data presented thus far led us to hypothesize that increased IL-21 expression is a key part of a primary immune response to HBV that results in viral clearance and immunity in adults and that increased IL-21 expression would not occur in a primary immune response to HBV in newborn or young individuals who do not clear the virus. Although it is not ethical to obtain blood or liver tissue from newborns or young children acutely infected with HBV, we can obtain blood from adults acutely infected with HBV with evidence of hepatitis as well as from patients chronically infected with HBV (who were likely infected young), during a hepatitic flare of chronic infection. Both groups typically have similar and overlapping plasma viral titers and ALT elevation.
We therefore tested this hypothesis by isolating RNA from PBMCs from adult patients acutely infected with HBV and hepatitis, adult patients chronically infected with HBV during a hepatitic flare, adult patients chronically infected with HBV without active hepatitis, and uninfected adult individuals. Strikingly, there was a significant 7-fold increase in relative expression of IL21 mRNA in PBMCs from patients acutely infected with HBV who cleared the virus as compared with PBMCs from patients chronically infected with HBV who have hepatitic flares but fail to clear the virus (Figure 7). Notably, we also did not observe increased IL21 mRNA in healthy individuals or in inactive chronic carriers (low ALT and low or undetectable viral load; Figure 7). Since all the studied patients with acute hepatitis B (5 patients) cleared the virus (>90% of adults clear), we have not yet been able to study adult patients who fail to clear. This will be a primary objective of future human studies.
Patients acutely infected with HBV express more IL21 mRNA in their PBMCs compared with patients with chronic HBV infection during actively flaring disease, inactive chronic HBV carriers, and healthy individuals. IL21 mRNA relative to that of GAPDH in PBMCs taken from patients with acute HBV infection (high viral load, high ALT, IgM core Ab+, HBsAg+, and clinical history of exposure); patients with chronic HBV infection with a flare of disease (high ALT, high viral load, HBsAg+, and known history of chronic infection); patients with chronic HBV infection (low ALT, low viral load, and HBsAg+); and healthy, uninfected patients (low ALT and HBsAg–) is shown. Error bars depict mean ± SEM; N ≥ 5. Statistical significance was determined using the ANOVA with Tukey’s post-hoc test. *P < 0.05.