Objective
Increased expression of HLA class I and antigen presentation to the immune system are key steps for immune-induced β-cell killing. The transcription activator NLRC5 has been shown in other cell types of mouse models to regulate the expression of genes involved in the HLA class I antigen-presentation machinery and in triggering HLA class I-dependent CD8+ T-cell activation. The multiple effects of NLRC5 are cell- and context-dependent and little is known of its role in human pancreatic β-cells in the context of T1D. NLRC5 expression is up-regulated in β-cells from donors with T1D and we observed that IFNα up-regulates NLRC5 expression in human β-cells, while NLRC5 silencing down-regulates IFNα-induced expression of HLA class I and other genes involved in antigen presentation, besides preventing the alternative splicing changes triggered by IFNα (own preliminary data). This double role of NLRC5 on HLA class I expression and regulation of alternative splicing suggests that this transcription factor may play a key role both in the generation of neoantigens and in their presentation to the immune system. We presently propose the following comprehensive aims to identify the role and regulation of NLRC5 in human β-cells:
Aim 1 – To clarify the role of IFNα-induced NLRC5 on the regulation of HLA class I and related genes in human β-cells. This aim focuses on the regulation of NLRC5 expression and on NLRC5’s role on the regulation of HLA class I and other genes involved in antigen presentation and immune responses.
Aim 2 – To clarify the role of IFNα-induced NLRC5 in the regulation of alternative splicing and neoantigen generation in human β-cells. We will approach this goal by determining expression of all genes and splice variants of purified human β-cells following inactivation of NLRC5 and exposure to IFNα. We will then compare the NLRC5-regulated β-cell splice variants against gene expression from 16 other tissues to identify potential novel β-cell neoantigens that are recognized by the immune system in T1D.
Aim 3 – To define the impact of NLRC5 on the crosstalk between human β-cells and CD8+ T cells. This will be accomplished by using co-culture between human β-cells derived from inducible-pluripotent stem cells (exposed or not to IFNα and with previous inhibition of NLRC5 by siRNA) and human CD8+ T cells.
Background Rationale
The early steps of the process that will eventually lead to type 1 diabetes (T1D) involve local release of pro-inflammatory mediators (cytokines) at the pancreatic islet level. One of these mediators is interferon-α (IFNα). In line with this, pancreatic islets obtained from living donors with recent onset T1D have a significant increase in the expression of IFN-stimulated genes, while inhibition of IFNα signalling prevents the development of T1D in animal models. Of particular relevance, T1D is also prevented in patients who spontaneously develop blocking antibodies against IFNα. Our recent findings indicate that exposure of human pancreatic islets to IFNα induces three hallmarks of T1D, namely endoplasmic reticulum stress (a form of stress that contributes to β-cell death), release of chemokines (proteins that attract cells of the immune system that will harm the β-cells) and up-regulation of HLA class I (which facilitates presentation of β-cell autoantigens to the immune system, particularly the cytotoxic CD8+ T-cells and consequently accelerates pancreatic β-cell killing). This induction of HLA class I is long-lasting and, together with induction of endoplasmic reticulum stress and changes in alternative splicing (a process by which a gene can generate different messenger RNAs and proteins) may contribute to presentation of novel β-cell antigens (“neoantigens”) to the immune system. However, the molecular mechanisms regulating HLA class I expression in β-cells and neoantigen presentation – decisive steps for CD8+ T-cell-mediated β-cell death and the triggering of T1D – remain to be clarified. The transcription activator NLRC5 has been shown in other cell types and in mouse models to regulate the expression of genes involved in the HLA class I antigen-presentation machinery and in triggering HLA class I-dependent CD8+ T-cell activation. The multiple effects of NLRC5 are cell- and context-dependent and little is known of its role in pancreatic β-cells in the context of T1D. NLRC5 expression is up-regulated in β-cells from donors with T1D and we observed that IFNα up-regulates NLRC5 expression in human β-cells, while NLRC5 silencing down-regulates IFNα-induced expression of HLA class I and other genes involved in antigen presentation, besides preventing the alternative splicing changes triggered by IFNα (own preliminary data). This double role of NLRC5 on HLA class I expression and regulation of alternative splicing suggests that this transcription factor may play a key role both in the generation of neoantigens and in their presentation to the immune system. We presently intend to identify the role and regulation of NLRC5 in human β-cells and to clarify how this leads to increased expression of HLA class I and antigen presentation to the immune system, a key step for immune-induced β-cell killing.
Description of Project
Type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing pancreatic β-cells. The very early steps of this process involve local release of pro-inflammatory mediators (cytokines) from immune cells that infiltrate the pancreatic islets where β-cells are found. One of these mediators – a key one – is interferon-α (IFNα). Indeed, pancreatic islets obtained from living donors with recent onset T1D have a significant increase of IFN-stimulated genes and inhibition of IFNα signalling prevents T1D development in animal models. Our recent findings indicate that exposure of human pancreatic islets to IFNα induce three hallmarks of T1D, namely endoplasmic reticulum stress (a form of stress that contributes to β-cell death), release of chemokines (proteins that attract cells of the immune system that will harm the β-cells) and up-regulation of HLA class I (which facilitates presentation of β-cell autoantigens to the immune system, particularly to the cytotoxic CD8+ T-cells that do most of the pancreatic β-cell killing). This induction of HLA class I is long-lasting and, together with induction of cellular stress and changes in alternative splicing (a process by which a gene can, after transcription, generate different messenger RNAs and proteins) may contribute to presentation of novel β-cell antigens (“neoantigens”) to the immune system. However, the molecular mechanisms regulating HLA class I expression in β-cells and neoantigen presentation – decisive steps for CD8+ T-cell-mediated β-cell death and the triggering of T1D – remain to be clarified.
The transcription activator NLRC5 has been shown in other cell types and in mouse models to regulate the expression of genes involved in the HLA class I antigen-presentation machinery and in triggering HLA class I-dependent CD8+ T-cell activation. The multiple effects of NLRC5 are cell- and context-dependent and little is known on its role in pancreatic β-cells in the context of T1D. NLRC5 expression is up-regulated in β-cells from donors with T1D and we observed that IFNα up-regulates NLRC5 expression in human β-cells, while NLRC5 inhibition down-regulates IFNα-induced expression of HLA class I and other genes involved in antigen presentation, besides preventing the alternative splicing changes triggered by IFNα (own preliminary data). This double role of NLRC5 on HLA class I expression and regulation of alternative splicing suggests that this transcription factor plays a key role both in the generation of neoantigens and in their presentation to the immune system. We presently propose the following comprehensive aims to identify the role and regulation of NLRC5 in human β-cells:
Aim 1 – To clarify the role of IFNα-induced NLRC5 on the regulation of HLA class I and related genes in human β-cells. This aim focuses on the regulation of NLRC5 expression and on NLRC5’s role on the regulation of HLA class I and other genes involved in antigen presentation and immune responses.
Aim 2 – To clarify the role of IFNα-induced NLRC5 in the regulation of alternative splicing and neoantigen generation in human β-cells. We will approach this goal by determining expression of all genes and splice variants of human β-cells following inactivation of NLRC5 and exposure to IFNα. We will then compare the NLRC5-regulated β-cell splice variants against gene expression from 16 other tissues to identify potential novel β-cell neoantigens that are recognized by the immune system in T1D.
Aim 3 – To define the impact of NLRC5 on the crosstalk between human β-cells and CD8+ T cells. This will be accomplished by using co-culture between human β-cells derived from inducible-pluripotent stem cells (exposed or not to IFNα and with previous inhibition of NLRC5 by siRNA) and human CD8+ T cells.
Anticipated Outcome
We expect that the presently proposed project will generate the following anticipated outcomes:
1. Defining how the cytokine IFNα regulates expression of NLRC5, a key transcription factor for the induction of HLA class I and the presentation of β-cell antigens to the immune system;
2. Clarifying the most relevant genes and gene pathways regulated by NLRC5 in IFNα-treated human β-cells, with focus on the regulation of HLA class I;
3. A comparison between the NLRC5-regulated splice variants in human β-cells against splice variants present in 16 other human tissues, allowing us to identify β-cell specific variants that may function as neoantigens in T1D;
4. Evaluating on whether these β-cell potential neoantigens are recognized by immune CD8+ T-cells from patients affected by T1D, which may open the way for new diagnostic tests and the identification of targets for induction of tolerance (in other words, allowing us to identify which are β-cell antigens that must be “forgotten” by the immune system to convince it to stop attacking β-cells);
5. Developing a relevant platform for testing interactions between human β-cells and CD8+ T-cells and, using this platform, defining whether NLRC5 inhibition suffices to prevent CD8+ T-cell activation and β-cell killing.
Relevance to T1D
Pioneering research by the applicants introduced a paradigm shift in the field, indicating that the “battle” leading to β-cell loss in diabetes is fought, to a large extent, inside the β-cells, and leads to a “dialogue” with the invading immune cells that amplifies the immune assault. A key component of this dialogue is the presentation of β-cell neoantigens in the context of HLA class I to the immune system. Most of the research efforts for the discovery of novel treatments for T1D has focused on the immune system, and innovative therapies that protect β-cells and block this deleterious β-cell-immune system dialogue remain to be discovered. The presently proposed approach, focusing on the mechanisms that lead human pancreatic β-cells to up-regulate HLA class I and present autoantigens to the immune system, will clarify a key step in the “misguided dialog” between β-cells and the immune system that eventually culminates in T1D. It will also point to potentially relevant targets for β-cell protection in the early stages of the disease and for the induction of immune tolerance to key β-cell antigens.