IFN-I promotes T-cell-independent immunity and RBC autoantibodies via modulation of B-1 cell subsets in murine SCD

Abstract

The pathophysiology of sickle cell disease (SCD) is characterized by hemolytic anemia and vaso-occlusion, although its impact on the adaptive immune responses remains incompletely understood. To comprehensibly profile the humoral immune responses, we immunized SCD mice with T-cell-independent (TI) and T-cell-dependent (TD) antigens (Ags). Our study showed that SCD mice have significantly enhanced type 2 TI (TI-2) immune responses in a manner dependent on the level of type I interferons (IFN-I), while maintaining similar or decreased TD immune responses depending on the route of Ag administration. Consistent with the enhanced TI-2 immune responses in SCD mice, the frequencies of B-1b cells (B-1 cells in humans), a major cell type responding to TI-2 Ags, were significantly increased in both the peritoneal cavity and spleens of SCD mice and in the blood of patients with SCD. In support of expanded B-1 cells, elevated levels of anti-red blood cell (anti-RBC) autoantibodies were detected in both SCD mice and patients. Both the levels of TI-2 immune responses and anti-RBC autoantibodies were significantly reduced after IFN-I receptor (IFNAR) antibody blockades and in IFNAR1-deficient SCD mice. Moreover, the alterations of B-1 cell subsets were reversed in IFNAR1-deficient SCD mice, uncovering a critical role for IFN-I in the enhanced TI-2 immune responses and the increased production of anti-RBC autoantibodies by modulating the innate B-1 cell subsets in SCD. Overall, our study provides experimental evidence that the modulation of B-1 cells and IFN-I can regulate TI immune responses and the levels of anti-RBC autoantibodies in SCD.

Graphical abstract

Figure 1.

A normal antibody response to TD Ag in SCD mice: intraperitoneally (IP). (A) Kinetics of NP-specific IgM and IgG antibody levels after immunization of SS mice (▲) and control AS mice (●) with 50 μg of NP-CGG in alum IP. The levels of antibodies were quantified by enzyme-linked immunosorbent assay (ELISA) with plasma collected on the dates indicated after immunization. (B-C) The levels of NP-specific (B)/total (C) IgM, IgG1, IgG2b, IgG2c, and IgG3 antibodies in SS mice (▲) and AS mice (●) were measured with plasma collected before immunization (D0) and on day 14 (D14) after immunization with 50 μg of NP-CGG in alum by ELISA and Luminex assay, respectively. Combined data from 2 independent experiments: AS (n = 10) and SS (n = 8). (D) Representative flow cytometry plots for GL7 and FAS expression gated on B220⁺ cells in the spleens from SS mice and AS mice immunized with 50 μg of NP-CGG in alum. Splenocytes were analyzed before immunization (D0), on day 7 after primary immunization (D7), and on day 5 after boost (mD5). (E) Quantification of GCB cells (GL7⁺FAS⁺B220⁺) in splenocytes from SS mice and AS mice (n = 4 for D0; n = 9 and 6 for D7; n = 6 for mD5); combined data from 3 independent experiments. (F) Representative immunofluorescence images of splenic sections from AS and SS mice. Spleens were collected before immunization (D0) or on day 7 after immunization with 50 μg of NP-CGG in alum. Data are representative images from at least 3 mice per group. IgD, red; PNA, green; CD3, magenta; scale bar, 200 μM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

Figure 2.

TD immune response was route dependent in SCD mice. (A) Kinetics of NP-specific IgM and IgG antibody levels after immunization of SS mice (▲) and control AS mice (●) with 50 μg of NP-CGG in alum IM. The levels of antibodies were quantified by ELISA with plasma collected on the dates indicated after immunization. (B) The levels of NP-specific IgM, IgG1, IgG2b, IgG2c, and IgG3 antibodies in SS mice (▲) and AS mice (●) were measured with sera collected before immunization (D0) and on D14 after immunization with 50 μg of NP-CGG in alum by ELISA. Combined data from 2 independent experiments: AS (n = 8) and SS (n = 9). (C) Representative flow cytometry plots for GL7 and FAS expression gated on B220⁺ cells in the draining LNs from SS mice and AS mice immunized with 50 μg of NP-CGG. Cells from LNs were analyzed before immunization (D0) and on day 5 after boost (mD5). (D) Quantification of GCB cells (GL7⁺FAS⁺B220⁺) in the draining LNs from SS mice and AS mice (n = 4 and 5 for D0; n = 6 for D7; n = 11 and 7 for mD5); combined data from 2 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

Figure 3.

Enhanced TI immune response in SCD mice despite low MZB cells. (A) Kinetics of NP-specific IgM and IgG antibody levels after immunization of SS mice (▲) and control AS mice (●) with 100 μg of NP-Ficoll IP. The levels of antibodies were quantified by ELISA with plasma collected on the dates indicated after immunization. (B) The levels of NP-specific IgM, IgG1, IgG2b, IgG2c, and IgG3 antibodies in SS mice (▲) and AS mice (●) were measured with plasma collected before immunization (D0) and on D14 after immunization with 100 μg of NP-Ficoll by ELISA. Combined data from 2 independent experiments: AS (n = 8) and SS (n = 9). (C) Representative flow cytometry plots for CD23 and CD21 expression gated on B220⁺AA4.1^– cells in the spleens from mice of the indicated genotype in naïve (D0) and immunized with 100 μg of NP-Ficoll 5 days earlier (D5). (D) The quantification of the frequencies of MZB cells in the spleens from mice of the indicated genotype in naïve (D0) or immunized with 100 μg NP-Ficoll 5 days earlier (D5) (n = 4). Data are representative from 2 independent experiment. (E) Representative immunofluorescent images of the spleen sections from naïve mice, mice immunized with 50 μg of NP-CGG in alum 7 days earlier, and with 100 μg of NP-Ficoll 5 days earlier of the indicated genotype. MZB cell layer was marked by yellow borders, showing a significantly reduced MZB cells in SS mice. Data are representative images from at least 3 mice per group. IgM, red; MOMA1, green; scale bar, 100 μM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 4.

Altered levels of B-1 cells and increased levels of autoantibodies in SCD mice. (A) Flow cytometry plots showing a gating strategy used for the identification of B-1 cell subsets in the peritoneal cavity; peritoneal B-2 cells (IgM⁺CD23⁺CD19^loCD11b^–CD5^–, blue), B-1b cells (IgM⁺CD23^–CD19⁺CD11b⁺CD5^lo-neg, red rectangle), and B-1a cells (IgM⁺CD23^–CD19⁺CD11b⁺CD5⁺, green). (B) Quantification of B-1 cell subsets in the peritoneal cavity from SS mice (▲) and AS mice (●). Data were combined from 2 independent experiments (n = 9). (C) Flow cytometry plots showing a gating strategy used for the identification of B-1 cell subsets in the spleens from naïve mice of the indicated genotype. To exclude non–B-1 cells in the CD23^–CD19⁺ cell gate, CD43 was added to the splenic staining; B-1b cells (B220⁺CD23^–CD19⁺CD43⁺CD11b^lo-negCD5^lo-neg, red rectangle) and B-1a cells (B220⁺CD23^–CD19⁺CD43⁺CD11b^lo-negCD5⁺, green). (D) Quantification of B-1 cell subsets in the spleens from the indicated genotype. Data were combined from 3 independent experiments (n = 10). (E) Flow cytometry plots for CD19 and CD23 expression gated on IgM⁺ cells in the peritoneal cavity from SS mice and AS mice immunized 5 days earlier with 100 μg of NP-Ficoll. (F) Quantification of B-1 cell subsets in the peritoneal cavity from SS mice (▲) and AS mice (●) immunized with 100 μg of NP-Ficoll 5 days earlier. Data were combined from 2 independent experiments (n = 5). (G) Quantification of B-1 cell subsets in the spleens from SS mice (▲) and AS mice (●) immunized with 100 μg of NP-Ficoll 5 days earlier. Data were combined from 2 independent experiments (n = 10). (H) Flow cytometry plots showing gating strategy for autoantibody positive RBC. (I) Quantification of autoantibody positive RBC and MFI in naïve SS mice (▲) and AS mice (●). Data were combined from 2 independent experiments (n = 18). (J) Quantification of B-1 cell subsets in the peritoneal cavity from AS and SS mice treated with PBS or Milli-Q water (H₂O) every 2 days for 2 weeks (n = 3). (K) The levels of NP-specific IgG in response to NP-Ficoll were measured with plasma collected from AS and SS mice treated with PBS or Milli-Q water (n = 3). (L) Quantification of autoantibody positive RBC in AS and SS mice treated with PBS or Milli-Q water (n = 6). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. MFI, mean fluorescence intensity.

Figure 4.

Altered levels of B-1 cells and increased levels of autoantibodies in SCD mice. (A) Flow cytometry plots showing a gating strategy used for the identification of B-1 cell subsets in the peritoneal cavity; peritoneal B-2 cells (IgM⁺CD23⁺CD19^loCD11b^–CD5^–, blue), B-1b cells (IgM⁺CD23^–CD19⁺CD11b⁺CD5^lo-neg, red rectangle), and B-1a cells (IgM⁺CD23^–CD19⁺CD11b⁺CD5⁺, green). (B) Quantification of B-1 cell subsets in the peritoneal cavity from SS mice (▲) and AS mice (●). Data were combined from 2 independent experiments (n = 9). (C) Flow cytometry plots showing a gating strategy used for the identification of B-1 cell subsets in the spleens from naïve mice of the indicated genotype. To exclude non–B-1 cells in the CD23^–CD19⁺ cell gate, CD43 was added to the splenic staining; B-1b cells (B220⁺CD23^–CD19⁺CD43⁺CD11b^lo-negCD5^lo-neg, red rectangle) and B-1a cells (B220⁺CD23^–CD19⁺CD43⁺CD11b^lo-negCD5⁺, green). (D) Quantification of B-1 cell subsets in the spleens from the indicated genotype. Data were combined from 3 independent experiments (n = 10). (E) Flow cytometry plots for CD19 and CD23 expression gated on IgM⁺ cells in the peritoneal cavity from SS mice and AS mice immunized 5 days earlier with 100 μg of NP-Ficoll. (F) Quantification of B-1 cell subsets in the peritoneal cavity from SS mice (▲) and AS mice (●) immunized with 100 μg of NP-Ficoll 5 days earlier. Data were combined from 2 independent experiments (n = 5). (G) Quantification of B-1 cell subsets in the spleens from SS mice (▲) and AS mice (●) immunized with 100 μg of NP-Ficoll 5 days earlier. Data were combined from 2 independent experiments (n = 10). (H) Flow cytometry plots showing gating strategy for autoantibody positive RBC. (I) Quantification of autoantibody positive RBC and MFI in naïve SS mice (▲) and AS mice (●). Data were combined from 2 independent experiments (n = 18). (J) Quantification of B-1 cell subsets in the peritoneal cavity from AS and SS mice treated with PBS or Milli-Q water (H₂O) every 2 days for 2 weeks (n = 3). (K) The levels of NP-specific IgG in response to NP-Ficoll were measured with plasma collected from AS and SS mice treated with PBS or Milli-Q water (n = 3). (L) Quantification of autoantibody positive RBC in AS and SS mice treated with PBS or Milli-Q water (n = 6). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. MFI, mean fluorescence intensity.

Figure 5.

IFN-I enhances TI immune response by modulating B-1 cell subsets in SCD mice. (A) Schematic diagram exhibiting the administration of SS mice with IFNAR1–blocking antibody or mouse IgG1 isotype control as indicated with red arrows. Blue arrows indicated the dates of IP injection of mice with NP-Ficoll and of bleeding for ELISA assays. (B) The levels of NP-specific IgM and IgG in response to NP-Ficoll were measured with plasma collected from SS mice after the treatment with IFNAR1–blocking antibody as indicated in panel A. (C) The levels of NP-specific IgM and IgG in response to NP-Ficoll were measured with plasma collected from SS mice and Ifnar1^–/– SS mice. (D) Flow cytometry plots showing gating strategy for autoantibody positive RBC. (E) Quantification of autoantibody positive RBC and MFI in naïve SS mice (n = 7) and Ifnar1^–/– SS mice (n = 8). (F) Flow cytometry plots showing a gating strategy used for the identification of B-1 cell subsets in the peritoneal cavity from SS mice and Ifnar1^–/– SS mice as shown in Figure 4A. (G) Quantification of B-1 cell subsets in the peritoneal cavity from naïve SS mice and Ifnar1^–/– SS mice (n = 5). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.

Figure 6.

Altered frequencies of MZB cells and B-1 cells in patients with SCD. (A) Flow cytometry plots showing a gating strategy used for the identification of MZB cells in human peripheral blood; MZB cells (dump^–CD19⁺CD27⁺IgM⁺IgD⁺). MZB cells can be further divided into IgM^hi and IgM^lo subsets. (B) Quantification of MZB cells in peripheral blood from race- and age-matched healthy donors (HDs) (n = 11) and patients with SCD (n = 15; see supplemental Table 1 for patient characteristics). (C) Flow cytometry plots showing a gating strategy used for the identification of B-1 cells in human peripheral blood; B-1 cells (CD3^-CD19⁺CD20⁺CD27⁺CD43⁺). (D) Quantification of B-1 cells in peripheral blood from race- and age-matched HDs and patients with SCD (same cohort as MZB cell study). (E) Flow cytometry plots showing gating strategy for autoantibody positive RBC. (F) Quantification of autoantibody positive RBC and MFI in HDs (●) and patients with SCD (▲). (G) Correlation between autoantibody positive RBC and peripheral B-1 cells. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.