1741: Mutated Nod2 Controls T Cell Function and Promotes Uveitis in a Blau Syndrome Mouse Model

Leah Huey¹, Kylie Koney¹, Emily Vance¹, Chia-Feng Tsai², Rosalie K. Chu², Marina A. Gritsenko², Ellen Lee¹, Holly Rosenzweig¹ and Ruth Napier¹, ¹Oregon Health & Science University and VA Portland Health Care System, Portland, OR, ²Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA

Background/Purpose: Mutations in the microbial signaling molecule NOD2 cause granulomatous uveitis, arthritis, and dermatitis in Blau Syndrome. We previously showed a novel role for Nod2 within CD4+ T cells in protection against IL-17-mediated experimental autoimmune uveitis (EAU). Here, we wanted to determine if mice expressing the Nod2 mutation in Blau Syndrome (R334Q) have altered T cell function or disease in uveitis.

Methods: Uveitis was induced in wild type (WT), Nod2^-/- mice and mice with heterozygous expression of the Nod2 mutation R314Q (corresponding to R334Q in humans) referred to as Blau mice; or in lymphopenic Nod2^+/+Rag1^-/- mice reconstituted with WT or Blau CD4+ T cells by immunization with the retinal autoantigen interphotoreceptor retinoid-binding protein (IRBP). Uveitis was assessed by clinical scoring and histology. IL-17 production by ocular-infiltrating T cells was assessed by flow cytometry. RT-qPCR was used to determine NOD2 expression in Jurkat T cells after TCR stimulation with monoclonal antibodies to CD3 and CD28. Mass spectrometry-based global phosphoproteomics was performed on purified CD4+ T cells 20 minutes post-TCR-activation treatment. Data were analyzed by Mann-Whitney or ANOVA, and p< 0.05 were considered significant.

Results: Similar to Nod2-deficient mice (Nod2^-/-), Blau mice developed exacerbated uveitis with increased IRPB-specific Th17 cells in the eye compared to WT mice. Blau T cells had enhanced pathogenicity as lymphopenic mice reconstituted with Blau CD4+ T cells developed worse uveitis than those reconstituted with WT CD4+ T cells. Nod2 is expressed by T cells upon autoantigen recognition, yet the role of Nod2 in T cell activation is unknown. Following overnight TCR stimulation, Nod2^-/- and Blau CD4+ T cells produced less IL-2 compared to WT, indicating Nod2 controls early T cell activation events. In support, NOD2 mRNA expression was increased within 20 minutes of TCR stimulation in Jurkat T cells. Analysis of global protein phosphorylation events at 20 minutes post stimulation of purified Nod2^-/- and Blau CD4⁺ T cells revealed altered phosphorylation kinetics of the T cell-activating transcription factors Nfkb1 and Nfatc compared to activated WT T cells. We also detected enhanced phosphorylation of the Th17 differentiation mediators Runx1 and IL-21r in Nod2^-/- and Blau mice compared to WT, supporting a role for Nod2 in controlling IL-17 signaling in response to TCR stimulation.

Conclusion: Our data show that a Blau Syndrome-associated Nod2 mutation (R334Q) results in loss-of-function in Nod2, which similar to Nod2 deficiency (Nod2^-/-), leads to exacerbated T cell-mediated uveitis and defective T cell activation and signaling. Together, our work suggests that a T cell-targeted therapy may be an effective treatment for Blau Syndrome.

Disclosures: L. Huey, None; K. Koney, None; E. Vance, None; C. Tsai, None; R. Chu, None; M. Gritsenko, None; E. Lee, None; H. Rosenzweig, None; R. Napier, None.