Disruption of Nod2 within T cells causes uveitis: Implications for Blau Syndrome

Abstract

Purpose : Nod2 (nucleotide-binding oligomerization domain-containing 2) is an important anti-microbial receptor. However, mutations in NOD2 result in Blau Syndrome, typified by uveitis. We recently uncovered a T cell-intrinsic mechanism for Nod2 in suppression of Th17-immunity and experimental autoimmune uveitis (EAU). Nod2 is induced upon antigen-recognition in CD4⁺ T cells; hence we examined how Nod2 disruption controls T cell receptor (TCR)-triggered responses under homeostatic conditions in Nod2^-/- mice as well as in patients with Blau Syndrome.

Methods : Naive (CD62L^hiCD44^-) CD4⁺ T cells purified from WT vs. Nod2^-/- mice were differentiated under Th17 polarizing conditions (CellXVivo) and Th17 cells quantified by flow cytometry. For TCR-ligation, purified memory (CD62L^-CD44^hi) CD4⁺ T cells from naive mice were stimulated with anti-CD3/CD28 antibodies, and activation state (CD69) and cytokine production were measured by flow cytometry (n=4 mice/genotype x 3 experiments). Data were analyzed by two-tailed Mann-Whitney U test, and p<0.05 was considered significant. Peripheral blood mononuclear cells from Blau Syndrome patients (heterozygote NOD2 1147G->A mutation, n=2) or healthy controls (n=3) were stimulated with PMA/ionomycin.

Results : Nod2 was dispensable for conventional Th17 differentiation of naïve, mouse T cells. Rather, Nod2 functioned within memory (antigen-experienced) CD4⁺ T cells, with Nod2^-/- memory cells producing significantly greater amounts of IL-17, RORγt and CD69 after continued TCR-stimulation. Transcriptional analysis of early TCR-signaling events revealed a unique profile involving IL23r, Ccl25, Ccr7, and Cxcl2 regulation by Nod2 in memory cells. Importantly, continued TCR-stimulation of CD4⁺ T cells from Blau Syndrome patients recapitulated the exacerbated IL-17 phenotype. Further immunoprofiling indicated enhanced cell surface expression of CCR7 in Blau patients vs. controls, which was further increased upon TCR-ligation; whereas production of cytokines IL-2, IFNg and TNF was impaired based on kinetics of TCR-activation.

Conclusions : Our data support Nod2 as a homeostatic factor in T cells, which suppresses responses of antigen-experienced Th17 cells and uveitis. Thus, loss of proper Nod2 function within T cells could be involved in the pathology of Blau Syndrome; thereby presenting new therapeutic options for patients.

This is a 2021 ARVO Annual Meeting abstract.