Irina Proekt

The non-canonical NF-κB pathway, in which NFKB2 plays a central role, is critical for lymphoid organ development and immune homeostasis. Pathogenic NFKB2 variants have been strongly associated with a broad spectrum of human pathologies, notably immunodysregulation syndromes, characterized by recurrent infections, autoantibody production and endocrine abnormalities. The mechanisms underlying the resulting immune defects remain incompletely understood. In the present study, we characterized the immune phenotype of a novel rat model harboring a Nfkb2 non-sense mutation located in the c- terminal region of the gene (c.2602T>A; p.Tyr867*, Nfkb2 ^p.Tyr867* ). This variant has been identified in patients with a familial form of common variable immunodeficiency (CVID) presenting with autoimmunity manifestations. Using this model, we demonstrate that mutated NFKB2 resulted in profound alterations of the thymic microenvironment, including reduced K5 ⁺ medullary thymic epithelial cells and decreased AIRE expression, accompanied by altered thymocyte development and impaired thymic regulatory T populations. Peripheral lymphoid organization was also profoundly affected, with absence of lymph nodes, defects in the splenic marginal zone, and alterations of CD4 ⁺ and CD8 ⁺ T-cell homeostasis, including impaired Treg populations and reduced ICOS expression on CD4 ⁺ T cells. The B-cell compartment was markedly altered, with impaired B-cell maturation, characterized by accumulation of IgM ⁺ IgD ⁻ transitional cells and a marked reduction of IgD ⁺ mature B cells. These abnormalities were associated with impaired humoral immunity and complete absence of circulating immunoglobulins. Collectively, our findings define a systemic combined immunodeficiency phenotype in the Nfkb2-mutated rat, highlighting its utility for studying the role of non-canonical NF-kB signaling in immune system organization and adaptative immunity.

The Nr4a nuclear hormone receptors are transcriptionally upregulated in response to antigen recognition by the T cell receptor (TCR) in the thymus and are implicated in clonal deletion, but the mechanisms by which they operate are not clear. Moreover, their role in central tolerance is obscured by redundancy among the Nr4a family members and by their reported functions in Treg generation and maintenance. Here we take advantage of competitive bone marrow chimeras and the OT-II/RIPmOVA model to show that Nr4a1 and Nr4a3 are essential for the upregulation of Bcl2l11/BIM and thymic clonal deletion by self-antigen. Importantly, thymocytes lacking Nr4a1/3 acquire an anergy-like signature after escaping clonal deletion and Treg lineage diversion. We further show that the Nr4a family helps mediate a broad transcriptional program in self-reactive thymocytes that resembles anergy and may operate at the margins of canonical thymic tolerance mechanisms to restrain self-reactive T cells after thymic egress.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production ( Rag2 ^-/- and μMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn ^-/- IgD ^+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire ^-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.

The developmental programs that generate a broad repertoire of regulatory T cells (T_reg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature T_reg cells were generated through two distinct developmental programs involving CD25⁺ T_reg cell progenitors (CD25⁺ T_regP cells) and Foxp3^lo T_reg cell progenitors (Foxp3^lo T_regP cells). CD25⁺ T_regP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3^lo T_regP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3^lo T_regP cells. The development of both CD25⁺ T_regP cells and Foxp3^lo T_regP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25⁺ T_regP cells and Foxp3^lo T_regP cells arose by coopting negative-selection programs and positive-selection programs, respectively. T_reg cells derived from CD25⁺ T_regP cells, but not those derived from Foxp3^lo T_regP cells, prevented experimental autoimmune encephalitis. Our findings indicate that T_reg cells arise through two distinct developmental programs that are both required for a comprehensive T_reg cell repertoire capable of establishing immunotolerance.