Background/Purpose: Systemic lupus erythematosus (SLE) is characterized by an increase in T-bet⁺ IgD⁻CD27⁻ double negative 2 (DN2) B cells, attributed to heightened TLR7 signaling. Identifying an inhibitory signal capable of suppressing DN2 B cell development could provide therapeutic benefits. Here, we investigated whether activation of the aryl hydrocarbon receptor (AhR) via its natural agonist can inhibit T-bet⁺ B cell development both in vitro and in vivo.

Methods: SLE patients meeting ACR/EULAR classification criteria were recruited with IRB approval. Kynurenine (KYN), an AhR agonistic ligand, was added in vitro to B cells from SLE patients under  DN2 B cell polarizing condition. In lupus-prone BXD2 mice, KYN was administered via intraperitoneal injection. B cell-specific AhR knockout mice (AhR B-KO) were generated by crossing B6-Ahr^f/f mice with Cd19.Cre mice, with AhR^WT Cd19.Cre (AhR B-WT) mice serving as controls. T-bet⁺ B cell development was induced by biweekly injections of the TLR7 agonist R848 over 5 weeks. Phenotype analysis was conducted using flow cytometry, and gene expression was assessed via real-time qPCR.  Animal procedures were approved by the IACUC committee.

Results: Naïve (p=0.01) and DN (p=0.01) B cells from SLE patients (n=6) exhibited significantly decreased AhR compared to healthy controls (HC)(n=3). Additionally, the AhR target gene CYP1A1 was significantly decreased in SLE (p=0.004)(n=10) versus HC (n=8). Culture of B cells with KYN in the presence of the DN2 cocktail resulted in a significant increase in CYP1A1 expression (p=0.0312), accompanied by decreased T-bet expression in both IgD⁺ naïve B cells (p=0.0047) and IgD⁻ B cells (p=0.0024)(n=14). In vivo treatment of R848-injected BXD2 mice with KYN significantly suppressed the percentage of CD11c⁺Ki67⁺CD138⁺ plasma B cells (p=0.0027) and CD11c⁺T-bet⁺ B cells in the germinal center (GL7⁺Fas⁺) subset (p=0.025)(n=4 per group). Conversely, in R848-administered AhR B-KO mice (n=6), there was a significant increase in the percentage of Fcrl5⁺T-bet⁺ B cells in both the follicular CD23⁺⁺CD21⁺ (p=0.018) and the marginal zone CD23⁻CD21⁺ B cell subsets (p=0.014), compared to control AhR B-WT mice (n=6).  AhR deficiency in B cells was associated with a significant increase in CD80⁺CXCR3⁺ populations at both IgD⁺ (p=0.005) and IgD⁻ (p=0.023) stages.

Conclusion: These findings suggest that impaired AhR regulatory program may contribute to abnormal activation phenotypes that drive DN2 B cell development in SLE. B cell-specific AhR deficiency leads to heightened T-bet⁺ B cell generation starting from the IgD⁺ naïve stage of B-cell development. Additionally, increased CD80 and CXCR3 expression in AhR-deficient B cells indicates the crucial role of AhR program in limiting TLR7-stimulated B cell interactions with Th1 CD4 T cells, ultimately promoting the development of CD138⁺ antibody-secreting B cells.  As KYN was effective in suppressing activated B cells including Fcrl5⁺T-bet⁺ B cells, CD80⁺CXCR3⁺ B cells, and plasma B cells in vivo, these results underscore the potential of AhR activation by natural agonistic ligands such as KYN to inhibit autoreactive B cell development in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/aryl-hydrocarbon-receptor-as-an-intrinsic-novel-checkpoint-that-inhibits-tlr7-induced-b-cell-activation-in-sle/