Gene Regulatory Events in Establishing Mature T Cell Tolerance

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Inborn errors of immunity (IEI) can affect global cellular regulatory systems. We looked at two projects where IEI can cause devastating disease. For the first project, we explored how cellular functional mutations in HEM1/2 can affect immunity. The mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 are global regulators of metabolism and cell signaling. mTORC2, comprising of mTOR, RICTOR, and other proteins, activates downstream kinases downstream to promote T cell survival, proliferation, and differentiation. Similarly, actin is a global regulator of cellular behavior and immune synapse formation. The WAVE regulatory complex (WRC) is protein complex made of HEM1/2 and other proteins, and is involved in the formation of the actin cytoskeleton. It is also known that WRC components can have noncanonical roles in cellular processes beyond actin filament nucleation and that the HEM1/2 protein exists, and likely functions, outside of the WRC complex. We showed that in human patients with immunodeficiency and immune hyperactivation, the loss-of-function mutations in NCKAP1L, the gene encoding HEM1, disrupt WRC-mediated actin polymerization and abrogate activation of cellular survival and growth. The resulting autosomal recessive mutation affects multiple hematopoietic lineages and leads to bacterial and viral infections, atopic disease, autoimmunity, cytokine overproduction, and lymphoproliferative disease. We demonstrated that HEM1 and the WRC maintain the cortical actin network, which restricts cytokine secretion and lytic granule release. We also showed that HEM1 plays a key binding role in WRC activation. Our findings suggest a broader effect of genetic HEM1 deficiency on the cytokine repertoire and cellular effector function that should be addressed in future work. Finally, we identified an interaction between HEM1 and RICTOR that is essential for mTORC2 regulation. We posit that HEM1 independently coordinates WRC-mediated actin nucleation and mTORC2 catalytic activity in response to signals that activate both protein complexes during T cell activation and possibly during B and NK cell activation. These data could explain how mTORC2 is activated downstream of actin-generated membrane tension and can negatively regulate the WRC. Because mTORC2 exerts similar roles in all lymphocytes, and because their activation involves actin-dependent regulation, it is likely that B and NK cell abnormalities contribute to immunopathology in the HEM1-deficient patients. Our study elucidated a human congenital disorder caused by loss of HEM1 and highlighted potential routes for immunological therapy. The second project looked at combined immunodeficiencies due to germline biallelic mutations in CD27 or CD70, which is characterized by increased susceptibility to bacterial and viral infections, impaired humoral immunity and hypogammaglobulinemia. The major pathogenic threat to these patients is Epstein Barr Virus (EBV), causing chronic viremia and severe diseases like lymphoma. A growing number of experts emphasize the need to implement immunological and EBV screening. However, there are no current definitions or reference values of immunological parameters or biomarkers in patients with malignancies prior to treatment. In our study, a majority of CD27- and of CD70-deficient patients presented with EBV-associated lymphoproliferation or lymphoma at a young age, suggesting the inability to control EBV infection is a strong indication to raise attention among physicians taking care of these patients. Besides lymphoma, a high number of CD27- and CD70-deficient patients experience autoinflammatory symptoms. So far, no specific disease biomarkers exist, so clinical awareness is of paramount importance in patients with signs of autoinflammation, especially with atypical presentation or unusual/lack of response where CD27/CD70 defects should be considered. The spectrum of EBV-associated diseases in patients with IEIs results from defective CD8+ T-cell activation, expansion and/or cytotoxicity that compromise immune-mediated control of EBV infection. Patients with CD27 or CD70 mutations have increased CD8+ T-cells and naive CD4+ T-cells, but reduced proportions of memory T and B cell and EBV-specific CD8+ T-cells. We found CD27-deficient memory CD8+ T cells have reduced production of cytokines and cytotoxic molecules and increased apoptosis in vitro. CD27-CD70 interactions are important for expansion of EBV-specific T-cells, evidenced by lack of expansion of CD27-deficient T-cells in response to CD70-expressing EBV+ B-cells, and impaired T-cell expansion, including EBV-specific T-cells, to CD70-deficient B-cells. Collectively, these cellular defects likely manifest as impaired cytotoxic T-cell-mediated control of EBV-infected B cells, resulting in EBV-associated disease. These cellular and functional defects, together with a lack of detectable expression of CD27 or CD70 on patient immune cells, could be used as biomarkers for the early identification and putative diagnosis of individuals with inactivating mutations in CD27 or CD70. Interestingly, the spectrum of clinical manifestations varied between patients in the same family, including identification of asymptomatic individuals with biallelic CD27 mutations, indicating that additional mechanisms including environmental factors may contribute to the variable penetrance of this genetic disease. It remains unclear whether CD27/CD70 have cell-intrinsic roles in tumorigenesis which may explain the difference in lymphoma occurrence. While hematopoietic stem cell transplantation (HSCT) is the only cure for individuals with refractory or relapsed malignancies, treatment strategies in less severely affected patients differ broadly. The excellent outcome after HSCT in CD27- or CD70-deficient patients with severe disease manifestations emphasizes that HSCT needs timely consideration as a definitive treatment, especially in patients with malignant transformation to lymphoma.