Background/Purpose: Microbial sensing molecule nucleotide-binding oligomerization-domain containing protein 2 (NOD2) is expressed by CD4+ T cells and plays a novel T cell-intrinsic role within CD4+ T cells in protecting against Th17-mediated experimental uveitis and arthritis. It is known that CD4+ T cells from Nod2-deficient (Nod2^-/-) mice have increased IL-17A responses and decreased IL-2 compared to WT mice. However, it is unknown how NOD2 mutations that cause the rheumatic disease Blau Syndrome affect T cell function. Here, we investigated how mutated NOD2 controls T cell activation and cytokine production in Blau patients and Blau knock-in mice (Nod2^R314Q).

Methods: Peripheral blood mononuclear cells from Blau patients or healthy controls were treated with T cell receptor (TCR) activator, anti-CD3, and co-stimulatory agent, anti-CD28, either overnight or for 5 days followed by a 4-hour incubation with anti-CD28 or phorbol myristate acetate (PMA) and ionomycin. After incubation, cytokine expression was quantified by ELISA and flow cytometry in supernatants and cells, respectively. Patient plasma IL-2 was quantified by ELISA. Splenocytes from wildtype (WT) naïve mice or Blau knock-in mice expressing Nod2^R314Q, a mutation in Nod2 known to cause Blau in humans, were stimulated with T cell activators and analyzed for cytokine production by ELISA and flow cytometry. Data were analyzed by unpaired two-sided student T test, and p< 0.05 were considered significant.

Results: Blau patients had decreased plasma IL-2 compared to control subjects (29 pg/mL vs. 320 pg/mL). TCR-activation of Blau patients’ CD4+ T cells resulted in lower production of IL-2 after 5 days, supporting of a role for mutated NOD2 in controlling T cell function. Conversely, Blau patient T cell incubation with PMA and ionymin, a T cell stimulant which bypasses the TCR, resulted in similar levels of IL-2, indicating NOD2 plays a role directly downstream of the TCR. In corroboration, TCR-activation of T cells from naïve Blau Nod2^R314Q mice resulted in decreased IL-2 production compared to WT mice. Mechanistically, freshly isolated and overnight stimulated CD4+ T cells from naïve Blau Nod2^R314Q mice had increased expression of Nur77 (T cell activation molecule directly proportional to the strength of TCR engagement) and Ki67 (indicator of proliferation) compared to WT T cells, yet similar levels of CD69 (early TCR-activation molecule) and CD25 (IL-2 receptor alpha). Cumulatively, these data suggest that NOD2^R314Q expression results in dysregulated T cell responses downstream of TCR-specific activation including enhanced TCR-signaling strength, increased proliferation, and reduced IL-2 production.

Conclusion: Our data suggest that defective IL-2 signaling is driving aspects of systemic inflammation in Blau patients as similar patients with loss-of-function mutations in IL-2 signaling have unchecked CD4+ T cell proliferation and develop systemic autoimmunity. Additionally, our data indicate that Nod2^R314Q expression may be altering TCR signaling strength, resulting in a dysregulation of T cell activation. Thus, further studies investigating how NOD2 is controlling TCR signaling will contribute to novel T cell-targeted therapeutics for Blau patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mutated-nod2-controls-il-2-production-in-blau-syndrome-patients-and-mice/