IL-6 contributes to an immune tolerance checkpoint in post germinal center B cells

Abstract

The generation of a B cell repertoire involves producing and subsequently purging autoreactive B cells. Receptor editing, clonal deletion and anergy are key mechanisms of central B cell tolerance. Somatic mutation of antigen-activated B cells within the germinal center produces a second wave of autoreactivity; but the regulatory mechanisms that operate at this phase of B cell activation are poorly understood. We recently identified a post germinal center tolerance checkpoint, where receptor editing is re-induced to extinguish autoreactivity that is generated by somatic hypermutation. Re-induction of the recombinase genes RAG1 and RAG2 in antigen-activated B cells requires antigen to engage the B cell receptor and IL-7 to signal through the IL-7 receptor. We demonstrate that this process requires IL-6 to upregulate IL-7 receptor expression on post germinal center B cells. Diminishing IL-6 by blocking antibody or haplo-insufficiency leads to reduced expression of the IL-7 receptor and RAG and increased titers of anti-DNA antibodies following immunization with a peptide mimetope of DNA. The dependence on IL-6 to initiate receptor editing is B cell intrinsic. Interestingly, estradiol decreases IL-6 expression thereby increasing the anti-DNA response. Our data reveal a novel regulatory cascade to control post germinal center B cell autoreactivity.

Figure 1. Reduced RAG/IL-7R expression after anti-IL-6 treatment in DWEYS-MAP immunized BALB/c mice

BALB/c mice (3-month-old) were immunized with DWEYS-MAP and boosted a week later (see Material and Methods). Anti-IL-6 antibody or isotype control (3–5 mice each group) was administrated to mice intravenously at day 8 and day 14 following the initial immunization. Spleens were collected at day 17. Antigen specific B cells were sorted as shown in (A) and RNA was extracted for qPCR to assess RAG (B) and IL-7R (C) mRNA. Spleen sections were frozen on the same day and immunohistochemistry was performed to detect RAG (left column, staining in green) and DWEYS peptide reactivity (mid column, tetramer staining in blue) levels (D and E).

Figure 1. Reduced RAG/IL-7R expression after anti-IL-6 treatment in DWEYS-MAP immunized BALB/c mice

BALB/c mice (3-month-old) were immunized with DWEYS-MAP and boosted a week later (see Material and Methods). Anti-IL-6 antibody or isotype control (3–5 mice each group) was administrated to mice intravenously at day 8 and day 14 following the initial immunization. Spleens were collected at day 17. Antigen specific B cells were sorted as shown in (A) and RNA was extracted for qPCR to assess RAG (B) and IL-7R (C) mRNA. Spleen sections were frozen on the same day and immunohistochemistry was performed to detect RAG (left column, staining in green) and DWEYS peptide reactivity (mid column, tetramer staining in blue) levels (D and E).

Figure 2. Elevated serum anti-dsDNA IgG titer after anti-IL-6 treatment in DWEYS-MAP immunized BALB/c mice

BALB/c mice (3-month-old) were immunized with DWEYS-MAP and boosted a week later (see Material and Methods). Anti-IL-6 antibody or isotype control (10 mice each group) was injected to mice intravenously at day 8 and day 14 following the initial immunization. Serum was collected at week 6 post initial immunization. ELISA was performed to assess IgG titer of anti-dsDNA autoantibody (A) and anti-DWEYS-peptide antibody levels (B). The experiment was performed twice. Data are from a representative experiment.

Figure 3. Reduced RAG/IL-7R expression in DWEYS-MAP immunized IL-6^+/− mice

IL-6 heterozygous (IL-6^+/−) mice (3-month old) and age-matched wild type BALB/c mice were immunized with DWEYS-MAP and boosted a week later (see Material and Methods). Antigen specific B cells were sorted on day 17 following the initial immunization and RNA was extracted for qPCR to check RAG and IL-7R mRNA (A). Spleen sections were frozen on the same day of sorting and immunohistochemistry was performed to detect RAG protein levels (B).

Figure 4. B cell production of IL-6 is required for re-induction of receptor editing. BALB/c mice (3-month-old) were immunized with DWEYS-MAP and boosted a week later. RNA was extracted from splenocytes at day 17 for qPCR to assess IL-6 mRNA level

(A). Splenic B cells from 3-month-old IL-6^+/− mice or age-matched wild type BALB/c mice were purified and adoptively transferred to μMT mice by intravenous injections. 3 weeks later, the recipient mice were immunized with DWEYS-MAP and boosted a week later (see Material and Methods). Spleens were collected at day 17. Antigen specific B cells were isolated and RNA was extracted for qPCR to assess RAG and IL-7R mRNA (B). Spleen sections were frozen on the same day and immunohistochemistry was performed to detect RAG protein levels (C).

Figure 4. B cell production of IL-6 is required for re-induction of receptor editing. BALB/c mice (3-month-old) were immunized with DWEYS-MAP and boosted a week later. RNA was extracted from splenocytes at day 17 for qPCR to assess IL-6 mRNA level

(A). Splenic B cells from 3-month-old IL-6^+/− mice or age-matched wild type BALB/c mice were purified and adoptively transferred to μMT mice by intravenous injections. 3 weeks later, the recipient mice were immunized with DWEYS-MAP and boosted a week later (see Material and Methods). Spleens were collected at day 17. Antigen specific B cells were isolated and RNA was extracted for qPCR to assess RAG and IL-7R mRNA (B). Spleen sections were frozen on the same day and immunohistochemistry was performed to detect RAG protein levels (C).

Figure 5. Estrogen downregulates RAG expression through IL-6/IL-7R

BALB/c mice (3-month-old) were ovariectomized and implanted with either estradiol (E2) or placebo pellets subcutaneously. Mice were rested 7–10 days and then immunized with DWEYS-MAP as described in Material and Methods. On day 17, antigen-specific B cells were isolated and RNA was extracted for qPCR to assess mRNA levels of RAG, IL-7R and IL-6.

Figure 6. Estrogen increases serum anti-dsDNA IgG titer in DWEYS-immunized BALB/c mice

BALB/c mice (3-month-old) were ovariectomized and implanted with either estradiol (E2) or placebo pellets subcutaneously. Mice were rested 7–10 days and then immunized with DWEYS-MAP as described in Material and Methods. On week 6 following the initial immunization, serum was collected to detect anti-dsDNA autoantibody (A) and anti-DWEYS-peptide antibody (B) levels by ELISA.