Tolerogenic function of Blimp-1 in dendritic cells

Abstract

Blimp-1 has been identified as a key regulator of plasma cell differentiation in B cells and effector/memory function in T cells. We demonstrate that Blimp-1 in dendritic cells (DCs) is required to maintain immune tolerance in female but not male mice. Female mice lacking Blimp-1 expression in DCs (DCBlimp-1(ko)) or haploid for Blimp-1 expression exhibit normal DC development but an altered DC function and develop lupus-like autoantibodies. Although DCs have been implicated in the pathogenesis of lupus, a defect in DC function has not previously been shown to initiate the disease process. Blimp-1(ko) DCs display increased production of IL-6 and preferentially induce differentiation of follicular T helper cells (T(FH) cells) in vitro. In vivo, the expansion of T(FH) cells is associated with an enhanced germinal center (GC) response and the development of autoreactivity. These studies demonstrate a critical role for Blimp-1 in the tolerogenic function of DCs and show that a diminished expression of Blimp-1 in DCs can result in aberrant activation of the adaptive immune system with the development of a lupus-like serology in a gender-specific manner. This study is of particular interest because a polymorphism of Blimp-1 associates with SLE.

Figure 1.

Deletion of Blimp-1 in DCs and phenotype of DCBlimp-1^ko mice. (A) Expression of Blimp-1 in DCs and plasma cells by Western blotting. Cell lysates from 5 × 10⁵ BM-DCs, splenic DCs, and plasma cells of control mice were loaded (top). DCs from spleens of control and DCBlimp-1^ko mice were used. The level of Blimp-1 was compared in splenic DCs from control (CRE⁻) and Blimp-1^ko (CRE⁺) female and male mice. Representative data from three independent experiments are shown. (B) Anti-dsDNA IgG in serum of control and DCBlimp-1^ko mice by ELISA. Mean ± SD of three independent experiments is shown. n = 8. The bottom shows representative pictures of ANA. (C) Isotype-specific anti-dsDNA ELISA. Mean ± SD of three independent experiments is shown. n = 7. (D) 24-h urine samples were collected from 8–10-mo-old control and DCBlimp-1^ko mice and protein was measured. IgG deposition and histology of kidneys from 8-mo-old control and DCBlimp-1^ko mice. Pictures are representative of each strain. Bars: (IgG) 20 µm; (histology) 40 µm. Horizontal bars indicate mean. (E) Anti-dsDNA and anti-ENA5 IgG in serum from 6–8-mo-old control and DCBlimp-1^ko female and male mice by ELISA. Mean ± SD of three independent experiments is shown. n > 10.

Figure 2.

Increased expression of IL-6 in Blimp-1^ko DCs from female mice and IL-6–dependent autoantibody generation. Splenic DCs (A) and BM-DCs (B) were prepared as described in Materials and methods and cultured with or without LPS stimulation. IL-6 in the supernatant was measured by ELISA. Mean ± SD of three independent experiments is shown. n = 6. (C) IL-6 secretion by purified splenic DCs of control, DCBlimp-1^ko, and IL-6^+/− DCBlimp-1^ko. Mean ± SD of three independent experiments is shown. n = 5. (D) Serum from 4-mo-old mice was obtained, and the level of dsDNA and ENA5 IgG was measured as described in Materials and methods. Each dot represents an individual mouse and horizontal bars indicate mean.

Figure 3.

Characterization of ANA IgG and GC response. (A) Sequence analysis of ANA IgG from hybridomas. Hybridomas were generated by splenocytes of 4-mo-old DCBlimp-1^ko mice. Total heavy and light chain of ANA-positive IgG was amplified and sequenced. Mutations were determined by comparison with the mouse genomic sequence database. Numbers in each pie graph represent the number of clones categorized by the number of mutation (n = 4). (B) Spontaneous GC formation in the spleen of 6–10-wk-old DCBlimp-1^ko mice. GC (PNA⁺ B220⁺, asterisks) was analyzed by IHC. Pictures are representative images (bars, 100 µm). On the right, GL-7⁺B220⁺ GC B cells were quantified by flow cytometry as depicted in representative pictures. Each dot represents an individual mouse and horizontal bars indicate means of three independent experiments.

Figure 4.

Increased T_FH cells in DCBlimp-1^ko mice in vivo and in vitro. (A) 6–10-wk-old control and DCBlimp-1^ko mice were sacrificed and T_FH (Lin (B220/CD11b/Gr-1)⁻, TCR-β/CD4/CXCR5/PD-1⁺) were analyzed by flow cytometry. Total T_FH cell number was calculated and graphed on the right. Each dot represents an individual mouse and horizontal bars indicate means of three independent experiments. (B) In vitro differentiation of T_FH cells. Naive CD4⁺ T cells and splenic DCs were co-cultured and activated as described in Materials and methods. After 4 d, cells were harvested and the frequency of T_FH cells was analyzed by flow cytometry. The top panel displays representative flow cytometry and the bottom is a summarized table (five independent experiments, n = 9). Total RNA was prepared from purified CD4⁺ T cells from each culture conditions, and the Bcl-6 level was measured by qPCR. The level of Bcl-6 of in vivo T_FH cells (T_FH cells sorted 12 d after NP immunization) was used as a positive control. Mean ± SD of three independent experiments is shown (n = 6).

Figure 5.

Decreased GC and T_FH cells in IL-6^+/− DCBlimp-1^ko mice. Spleens were harvested from 6–8-wk-old mice, and GC B cells and T_FH cells were enumerated by flow cytometry. Number of cells was calculated as percentage of positive cells × total splenocytes. Mean ± SD of three independent experiments is shown (n = 5).