Gene Regulatory Events in Establishing Mature T Cell Tolerance

先天性免疫缺陷（IEI）可影响全球细胞调节系统。我们研究了IEI可能导致毁灭性疾病的两个项目。在第一个项目中，我们探索了HEM1/2细胞功能突变如何影响免疫。雷帕霉素复合体1 （mTORC1）和mTORC2的机制靶点是代谢和细胞信号传导的全局调节因子。mTORC2由mTOR、RICTOR等蛋白组成，激活下游激酶，促进T细胞存活、增殖和分化。同样，肌动蛋白是细胞行为和免疫突触形成的全局调节剂。WAVE调节复合体（WRC）是由HEM1/2和其他蛋白质组成的蛋白质复合体，参与肌动蛋白细胞骨架的形成。我们还知道，WRC成分在肌动蛋白丝成核之外的细胞过程中可能具有非规范的作用，HEM1/2蛋白存在于WRC复合体之外，并可能发挥作用。我们发现，在人类免疫缺陷和免疫过度激活患者中，编码HEM1的基因NCKAP1L的功能缺失突变破坏了wrc介导的肌动蛋白聚合，并取消了细胞存活和生长的激活。由此产生的常染色体隐性突变影响多种造血谱系，并导致细菌和病毒感染、特应性疾病、自身免疫、细胞因子过度产生和淋巴细胞增生性疾病。我们证明HEM1和WRC维持皮层肌动蛋白网络，这限制了细胞因子的分泌和溶解颗粒的释放。我们还发现HEM1在WRC激活中起关键的结合作用。我们的研究结果表明，遗传HEM1缺陷对细胞因子库和细胞效应功能的影响更广泛，应该在未来的工作中加以解决。最后，我们发现HEM1和RICTOR之间的相互作用对mTORC2调控至关重要。我们假设HEM1独立地协调wrc介导的肌动蛋白成核和mTORC2的催化活性，以响应T细胞激活和B细胞和NK细胞激活过程中激活蛋白复合物的信号。这些数据可以解释mTORC2如何在肌动蛋白产生的膜张力下游被激活，并可以负向调节WRC。由于mTORC2在所有淋巴细胞中发挥相似的作用，并且由于它们的激活涉及肌动蛋白依赖性调节，因此B细胞和NK细胞异常可能导致hem1缺陷患者的免疫病理。我们的研究阐明了一种由HEM1缺失引起的人类先天性疾病，并强调了免疫治疗的潜在途径。

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.