Liver type I regulatory T cells suppress germinal center formation in HBV-tolerant mice

Abstract

The liver plays a critical role in inducing systemic immune tolerance, for example, during limiting hypersensitivity to food allergy and in rendering acceptance of allotransplant or even hepatotropic pathogens. We investigated the unknown mechanisms of liver tolerance by using an established hepatitis B virus (HBV)-carrier mouse model, and found that these mice exhibited an antigen-specific tolerance toward peripheral HBsAg vaccination, showing unenlarged draining lymph node (DLN), lower number of germinal centers (GC), and inactivation of GC B cells and follicular T helper (Tfh) cells. Both in vivo and in vitro immune responses toward HBsAg were suppressed by mononuclear cells from HBV-carrier mice, which were CD4(+) Foxp3(-) type 1 regulatory T (Tr1)-like cells producing IL-10. Using recipient Rag1(-/-) mice, hepatic Tr1-like cells from day 7 of HBV-persistent mice acquired the ability to inhibit anti-HBV immunity 3 d earlier than splenic Tr1-like cells, implying that hepatic Tr1-like cells were generated before those in spleen. Kupffer cell depletion or IL-10 deficiency led to impairment of Tr1-like cell generation, along with breaking HBV persistence. The purified EGFP(+)CD4(+) T cells (containing Tr1-like cells) from HBV-carrier mice trafficked in higher numbers to DLN in recipient mice after HBsAg vaccination, and subsequently inactivated both Tfh cells and GC B cells via secreting IL-10, resulting in impaired GC formation and anti-HB antibody production. Thus, our results indicate Tr1-like cells migrate from the liver to the DLN and inhibit peripheral anti-HBV immunity by negatively regulating GC B cells and Tfh cells.

Keywords: peripheral tolerance; unresponsive.

Fig. 1.

HBV-specific regulatory cells mediate HBV-induced immune tolerance. (A) Splenocytes from HBV-carrier or control mice were transferred (intravenously) (2 × 10⁷) into naive mice. Serum anti-HBs levels were measured 1 wk after HBsAg vaccination. (B) Mice were treated as in A, serum antiovalbumin IgG levels were determined by ELISA. (C) HBV-carrier WT or HBV-carrier Rag1^−/− mice received a transfer (intravenously) of splenocytes from HBsAg-immunized mice (2 × 10⁷) (called “anti-HBV” here). (D) HBV-carrier Rag1^−/− mice received splenocytes from either HBV-carrier mice (2 × 10⁷) (called “HBV” here), anti-HBV (2 × 10⁷), or a mixture of HBV with anti-HBV at a 1:1 ratio (total 4 × 10⁷ cells). (E) Naive mice received a transfer (intravenously) of splenocytes from control mice or anti-HBV, followed by 6 μg of HBV plasmid injection. Serum HBsAg levels were measured (C–E) at indicated time points. (F) ³H-TdR incorporation was used to detect specific inhibition by splenocytes from HBV-carrier mice. Results represent two to four independent experiments (n = at least 3 per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2.

HBV-specific regulatory cells are IL-10–producing Tr1-like cells. (A) CD4⁺NK1.1⁻ cells were sorted from splenocytes of control or HBV-carrier mice, and the remaining cells without CD4⁺NK1.1⁻ cells were called non-CD4⁺ cells. Recipient Rag1^−/− mice received CD4⁺ cells and non-CD4⁺ cells in various combinations. (B) Naive recipient mice were divided into four groups: two groups received an intravenous transfer of total splenocytes (2 × 10⁷) from control or HBV-carrier mice, and the others received an intravenous transfer of CD4⁺ cells (0.4 × 10⁷) or non-CD4 cells (1.6 × 10⁷) from HBV-carrier mice, respectively. Three days after cells transfer, recipient Rag1^−/− mice were immunized with HBsAg vaccine twice within a 2-wk interval and then anti-HBs levels were detected for the treatment shown in A and B at day 7 after the last HBsAg vaccination. (C) Liver MNCs or splenocytes from control or HBV-carrier mice were cultured with anti-CD3 plus anti-CD28 antibodies. After 96 h, supernatants were assessed for IL-10 and IFN-γ levels. (D) Treatment was performed as in C. IL-10 expression levels in CD4⁺ or CD4⁻ cells from liver MNCs of HBV-carrier or control mice was determined by flow cytometry. (E and F) Statistical analysis of IL-10 expression in CD4⁺Foxp3⁻ or CD4⁺Foxp3⁺ cells (E) and in CD4⁻ cells (F). (G) Treatment was performed as in C. Hepatic CD4⁺ T cells were purified from control or HBV-carrier mice, and then supernatant levels of IL-10 and IFN-γ were detected. ND, not detected; ns, no significance. Results represent two to three independent experiments (n = at least 3 per group). *P < 0.05 and **P < 0.01.

Fig. 3.

KC-derived IL-10 is critical for generation of Tr1-like cells. (A) IL-10 and IFN-γ levels in culture supernatant of hepatic MNCs from different mice. (B) Naive CD4⁺ T cells were cocultured with KC cells from control or HBV-carrier mice for 3 d. CD4⁺ T cells were isolated from culture and activated with anti-CD3/CD28 in vitro. Four days later, secreted IL-10 in supernatant and Tr1 phenotype (CD4⁺Foxp3⁻IL-10⁺) cells were assessed. (C) Naive CD4⁺ T cells were cocultured with KCs from HBV-carrier mice in the presence or absence of anti–IL-10R mAb (10 μg/mL) or IL-10^−/− HBV mice and then treated as in B. (D) Naive mice were transferred with total splenocytes (2 × 10⁷) or purified CD4⁺ T cells (4 × 10⁶) from control, HBV-carrier, KC-depleted HBV, or IL-10^−/− HBV mice. One day later, mice were immunized with HBsAg. (E) The treatment was performed as in Fig. 1D. Serum HBsAg levels were measured 1 wk after donor cell transfer. KCs depletion was obtained by intravenous injection of clodronate liposome (125 μL) once on day −2 before hydrodynamic injection of HBV plasmid. ND, not detected. Results represent two independent experiments (n = 3–6 per group). *P < 0.05 and **P < 0.01.

Fig. 4.

GC B cells and Tfh cells are low in the DLN of HBV-carrier mice after HBsAg vaccination. At 12 d after HBsAg vaccination at the tibialis anterior muscle, lymphocytes from the DLN (popliteal LN) were stained with different antibodies. (A) The DLN was photographed (Left) and total cells were counted (Right). (B) DLN sections were stained with Biotin-PNA; GCs are shown (Scale bar, 200 μm). (C) GC B cell (B220⁺Gl-7⁺Fas⁺) frequency and (D) Bcl-6 expression levels were examined. The MNC amount of the popliteal LN was too low from control mice without immunization with HBsAg (red symbol), so we put MNCs from two mice into one tube for intracellular staining by flow cytometry. (E) Tfh cell (CD4⁺CXCR5⁺PD-1⁺) frequency and (F) CD44⁺ ICOS⁺ cell percentage among CD4⁺ T cells were measured. Results represent three independent experiments (n = at least 3 per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.

Hepatic Tr1-like cells inactivate Tfh and GC B cells in DLN of HBV-carrier mice after HBsAg vaccination. (A and B) Recipient Rag1^−/− mice received liver MNCs (1 × 10⁷) or splenocytes (1 × 10⁷) isolated at different time points (days 1, 7, and 10) from HBV-plasmid–injected mice. Serum anti-HBs antibody levels (A) and GC B cells (B) were determined after multiple HBsAg vaccination. (C) Seven days after HBV plasmids injection, hepatic or splenic CD4⁺ T cells were purified from 56 HBV-injected mice by MACS. Recipient Rag1^−/− mice received CD4⁺ cells (0.5 × 10⁷) and non-CD4⁺ cells (2.0 × 10⁷) in various combinations. Then, the recipient mice were treated as shown in Fig. S9A. (L, liver MNCs; ND, not detected; S, splenocytes). (D) EGFP⁺CD4⁺ T cells from liver MNCs of HBV-carrier or control mice were adoptively transferred into naive recipient mice, which were then immunized with HBsAg vaccine 1 d later. After 4 wk, flow cytometry was used to analyze EGFP⁺CD4⁺ T-cell number in the DLN. (E and F) Hepatic CD4⁺ T cells from HBV-carrier (2 wk) mice were cocultured with purified B cells (E) or sorted EGFP⁺CD4⁺ T cells (F) from DLN of HBsAg-immunized mice in presence of anti–IL-10R or not. GC B cell or EGFP⁺ Tfh cell frequency was measured after 3 d. Results represent two independent experiments (n = at least 3 per group). *P < 0.05 and **P < 0.01.

Fig. 6.

IL-10 plays a crucial role in Tr1-like cells-mediated systemic tolerance. (A and B) Treatment was performed as in Fig. 3E; IL-10 and IFN-γ levels were determined in culture supernatants of sorted CD4⁺ T cells from liver MNCs of control, HBV-carrier, or IL-10^−/−HBV mice. (C) GC B-cell frequency was measured in DLN from HBV-carrier or IL-10^−/− HBV-injected mice after HBsAg vaccination. (D) Splenic CD4⁺ T cells and splenocytes without CD4⁺ T cells (called non-CD4⁺ cells), from WT, HBV-carrier, or IL-10^−/− HBV mice, were transferred to recipient Rag1^−/− mice in various combinations. ND, not detected. Results represent two independent experiments (n = at least 3 per group). *P < 0.05.