CTLA-4 expression by B-1a B cells is essential for immune tolerance

CTLA-4 is an important regulator of T-cell function. Here, we report that expression of this immune-regulator in mouse B-1a cells has a critical function in maintaining self-tolerance by regulating these early-developing B cells that express a repertoire enriched for auto-reactivity. Selective deletion of CTLA-4 from B cells results in mice that spontaneously develop autoantibodies, T follicular helper (Tfh) cells and germinal centers (GCs) in the spleen, and autoimmune pathology later in life. This impaired immune homeostasis results from B-1a cell dysfunction upon loss of CTLA-4. Therefore, CTLA-4-deficient B-1a cells up-regulate epigenetic and transcriptional activation programs and show increased self-replenishment. These activated cells further internalize surface IgM, differentiate into antigen-presenting cells and, when reconstituted in normal IgH-allotype congenic recipient mice, induce GCs and Tfh cells expressing a highly selected repertoire. These findings show that CTLA-4 regulation of B-1a cells is a crucial immune-regulatory mechanism.

(F) Graph shows the overview of the gene sets annotation enrichment analysis results for differentially expressed genes between splenic B-1a cells from control and CKO mice. x-axis represents the median fold change on logarithmic scale, and y-axis represents the negative logarithm of the adjusted p-value (the higher, the more significant). The area of the displayed circles is proportional to the number of genes assigned to the term. All the terms with the negative logarithm of the adjusted p-value larger than two were shown.

Legend:
Hematoxylin and eosin (H&E) stained sections from a CKO mouse exhibiting cutaneous pathology. The dermis is occasionally expanded by dense and sometimes hyalinized eosinophilic matrix (black asterisks; a, b). In the overlying epidermal epithelium there is occasional intra-epidermal clefting (red asterisks, a'), or clefting at the dermal-epidermal junction (red asterisks, b'). a' is a higher magnification of a. b' is a higher magnification of b. (B) Elisa assay shows the rheumatoid factor antibody levels in serum of control or CKO mice at the indicated age. Each dot represents data for an individual mouse, n=7-15 mice per group, nonparametric Wilcoxon one-way test.
(C) Pictures of three representative mice (7-month-old) are shown. They were derived from the same litter but expressed different genotype.
(D) Representative images of H&E stained skin sections from a CKO mouse show the cutaneous pathology. The dermis is occasionally expanded by dense and sometimes hyalinized eosinophilic matrix (black asterisks; a, b). In the overlying epidermal epithelium there is occasional intra-epidermal clefting (red asterisks, a'), or clefting at the dermalepidermal junction (red asterisks, b'). a' is a higher magnification of a. b' is a higher magnification of b. Magnification: a= 1.25x; a'= 20x; b= 4x; b'= 10x Scale bar: a= 500μm; a'= 50μm; b= 200μm; b'= 100μm.
(E) Representative images of H&E stained sections from CKO mice show the inflammation in various organs. Inflammation in the intestine varied from mild (a) to moderate (b) and was composed of neutrophils and/or lymphocytes. Additional organs affected by inflammatory influx included the liver (c) and the skin of the ear (pinna; d) Magnification: a-d= 40x Scale bar: a-d= 20μm.              Column 1, the list of CDR3 peptides; Column 2, for each CDR3 peptide, their encoded V(D)J sequence; Column 3, out of 7 CKO mice, number of mice in which this particular V(D)J sequence are found in both IgM hi B-1a and IgM neg B-1a cells; Column 4 and 5, nucleotides added or deleted in CDR3 junctions; Column 6, the specificity for each V(D)J IgH sequence.

Supplementary Table 3: TCRβ sequencing summary of non-Tfh T cells and Tfh cells.
Phenotypically defined T cell subsets were sorted from indicated mice. The TCRβ cDNA library was generated from each subset and sequenced. Total of 14 separately prepared TCRβ libraries were sequenced with each derived from about 2x10 4 sorted T subset from an individual mouse. Tfh cells (CD19 neg myeloid neg CD3 + CD4 + PD-1 hi CXCR5 + ), non-Tfh CD4 T cells (CD19 neg myeloid neg CD3 + CD4 + PD-1 neg CXCR5 neg ), non CD4 T cells (CD19 neg myeloid neg CD3 + CD4 neg PD-1 neg CXCR5 neg ). CB.17 recipients refer the chimeric mice reconstituted with CTLA-4-deficient B-1a cells from CKO mice. D50 value refers the D50 metric analysis quantifying the TCRβ CDR3 nucleotide sequence diversity for each T cell sample.