The molecular role of the invariant chain/CD74 and its proteolytic degradation in dendritic cell function and anti-mycobacterial immune recognition

在抗原呈递细胞中，不变性链（CD74）在MHCII类（MHCII）复合物的组装和靶向中起着核心作用。在抗原处理室中，CD74的管腔结构域通过顺序蛋白水解被降解，从而释放MHCII。最后的CD74膜结合n端片段（NTF）经历了信号肽肽酶样2a （SPPL2a）的膜内蛋白水解。SPPL2a-/-小鼠表现出脾脏B细胞成熟阻滞和树突状细胞（dc）缺失。最近，根据特定DC亚群的缺失，确定了3例sppl2a缺陷的人类分枝杆菌病孟德尔易感性（MSMD）患者。在这两种情况下，积累的未切割的CD74 NTF诱导了所描述的表型。然而，由该片段引发的病理生理序列仍不完全清楚。在小鼠SPPL2a-/- B细胞中，我们发现中枢存活途径，特别是B细胞抗原受体的下游，受到干扰。虽然可能有其他影响，但我们的研究结果表明，CD74 ntf诱导的相关受体的错误运输代表了一个核心机制。在本项目中，我们计划更详细地分析sppl2a缺乏对树突状细胞功能的影响。初步结果表明，SPPL2a-/- dc在细胞因子反应方面表现出明显的变化，特别是在抗分枝杆菌免疫识别方面。通过表征这种功能缺陷，我们想要阐明除了DC减少外，这是如何导致sppl2a缺陷患者和小鼠的MSMD表型的。与我们在B细胞中的发现类似，我们注意到某些质膜蛋白的细胞分布，例如c型凝集素受体Dectin-1，在SPPL2a-/- dc中发生改变，导致细胞表面水平降低。我们假设参与病原体识别或DC分化的其他受体可能会受到CD74 NTF诱导的错误运输。我们希望使用假设驱动和无偏倚方法的组合系统地分析这一点，并将确定这在多大程度上解释了SPPL2a-/- dc的功能缺陷。使用Dectin-1作为模型，我们将调查哪些贩运路线主要由CD74 NTF调制。为了了解NTF在分子水平上的作用，我们进行了IP-MS筛选，并确定了该片段的几个潜在相互作用伙伴，这些伙伴在膜运输中起着记录作用。在多步骤验证策略中，我们的目标是确认这些假定的相互作用，并分析它们与SPPL2a-/- dc细胞表型的功能相关性。由于SPPL2a代表了一个有希望的治疗靶点，以消耗自身免疫中的B细胞和/或树突状细胞，因此从翻译的角度来看，本研究提供的对分子机制的更好理解也将相关。

The invariant chain (CD74) plays a central role in assembly and targeting of MHC class II (MHCII) complexes in antigen-presenting cells. In antigen-processing compartments, the luminal domain of CD74 is degraded by sequential proteolysis thereby releasing MHCII. The final CD74 membrane-bound N-terminal fragment (NTF) undergoes intramembrane proteolysis by Signal peptide peptidase-like 2a (SPPL2a) as we demonstrated before. SPPL2a-/- mice exhibit an arrest of splenic B cell maturation and a loss of dendritic cells (DCs). Recently, three SPPL2a-deficient human patients with Mendelian Susceptibility to Mycobacterial Disease (MSMD) based on the depletion of certain DC subsets were identified. In both contexts, the accumulating uncleaved CD74 NTF induces the described phenotypes. However, the pathophysiological sequence triggered by this fragment remains incompletely understood. In murine SPPL2a-/- B cells, we found that central survival pathways, in particular downstream of the B cell antigen receptor, were disturbed. Though additional effects may contribute, our findings indicate that a CD74 NTF-induced mis-trafficking of the involved receptors represents a central mechanism. In the proposed project, we plan to analyse the impact of SPPL2a-deficiency on dendritic cell function in more detail. Prelimininary results indicate that SPPL2a-/- DCs exhibit distinct changes in cytokine responses, in particular with regard to anti-mycobacterial immune recognition. By characterising this functional deficit, we want to elucidate how this contributes to the MSMD phenotype of SPPL2a-deficient patients and mice in addition to the DC reduction. In analogy with our findings in B cells, we noticed that the cellular distribution of certain plasma membrane proteins, exemplified by the C-type lectin receptor Dectin-1, is altered in SPPL2a-/- DCs leading to reduced levels at the cell surface. We hypothesise that further receptors involved in pathogen recognition or DC differentiation may undergo mis-trafficking induced by the CD74 NTF. We want to analyse this systematically using a combination of hypothesis-driven and unbiased approaches and will determine to what extent this accounts for the functional deficits of SPPL2a-/- DCs. Using Dectin-1 as a model, we will investigate which trafficking routes are primarily modulated by the CD74 NTF. To understand effects by the NTF at the molecular level, we have performed an IP-MS screen and identified several potential interaction partners of this fragment with a documented role in membrane trafficking. In a multistep validation strategy, we aim to confirm these putative interactions and to analyse their functional relevance with regard to the cellular phenotypes of SPPL2a-/- DCs. Since SPPL2a represents a promising therapeutic target to deplete B cells and/or dendritic cells in autoimmunity, a better understanding of the molecular mechanism as provided by this study will be relevant also from a translational point of view.