Variant-to-function mapping in lupus links IL12A to the expansion of disease-associated B cells with a cytotoxic program

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by over 200 risk variants identified through genome-wide association studies. While the majority of these are non-coding variants with unresolved functions, elucidating their mechanisms is critical for prioritizing therapeutic targets with increased clinical success. Several SLE risk loci span genes involved in the IL-12 signaling pathway. However, for most of them the causal variants, their definitive target genes, and their cellular consequences remain unestablished. Concurrently the expansion of double-negative 2 (DN2) B cells is a hallmark of SLE, but whether IL-12 and/or genetic risk functionally drive DN2 cells is unclear. In this study, we integrated candidate risk variants at the 3q25.33 risk locus with regulatory maps of a B cell line, identifying risk variant rs485499 located within a putative enhancer 39kb downstream of IL12A, and overlapping an open chromatin region in primary B cells stimulated with a DN2-skewing cocktail. Using CRISPR-based tools in a B cell line, we validated this region as an enhancer, rs485499 as a likely causal variant and established IL12A as its definitive target gene. Individuals homozygous for the rs485499 risk allele exhibited elevated IL12A production in naive B cells and presented with an expanded DN2 population in peripheral blood, compared to non-risk allele carriers. Mechanistically, we found the transcription factor IRF4 preferentially binds the rs485499 risk allele, driving IL12A upregulation. In vitro recombinant IL-12A promoted DN2 differentiation an effect that is abrogated by IL-12 inhibition with ustekinumab, establishing a causal IL-12-driven DN2 B cell expansion axis. Finally, we reveal that DN2 B cells inherently possess a previously unrecognized cytotoxic function that is potentiated by the IL-12 signaling axis. This cytotoxic profile is further supported by SLE patient data, identifying it as a bona fide effector state of DN2 B cells. Collectively, these findings identify rs485499 as a likely causal variant within this locus and establish a functional link between genetic risk and DN2 B cell expansion, validating this subset as a key pathogenic driver armed with a newly identified cytotoxic program. Furthermore, we identify the IL-12-IFNy-DN2 axis as a promising therapeutic target, providing a mechanistic rationale for future subset-specific interventions in SLE.