A Cre-driven allele-conditioning line to interrogate CD4+ conventional T cells.

CD4+ T cells share common developmental pathways with CD8+ T cells, and upon maturation, CD4+ T conventional T (Tconv) cells lack phenotypic markers that distinguish these cells from FoxP3+ T regulatory cells. We developed a tamoxifen-inducible ThPOKCreERT2.hCD2 line with Frt sites inserted on either side of the CreERT2-hCD2 cassette, and a Foxp3Ametrine-FlpO strain, expressing Ametrine and FlpO in Foxp3+ cells. Breeding these mice resulted in a CD4conviCreERT2-hCD2 line that allows for the specific manipulation of a gene in CD4+ Tconv cells. As FlpO removes the CreERT2-hCD2 cassette, CD4+ Treg cells are spared from Cre activity, which we refer to as allele conditioning. Comparison with an E8IiCreERT2.GFP mouse that enables inducible targeting of CD8+ T cells, and deletion of two inhibitory receptors, PD-1 and LAG-3, in a melanoma model, support the fidelity of these lines. These engineered mouse strains present a resource for the temporal manipulation of genes in CD4+ T cells and CD4+ Tconv cells.

DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1-5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1ß and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease6-12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.

NLRC3 is an inhibitory sensor of PI3K-mTOR pathways in cancer.

NLRs (nucleotide-binding domain and leucine-rich repeats) belong to a large family of cytoplasmic sensors that regulate an extraordinarily diverse range of biological functions. One of these functions is to contribute to immunity against infectious diseases, but dysregulation of their functional activity leads to the development of inflammatory and autoimmune diseases. Cytoplasmic innate immune sensors, including NLRs, are central regulators of intestinal homeostasis. NLRC3 (also known as CLR16.2 or NOD3) is a poorly characterized member of the NLR family and was identified in a genomic screen for genes encoding proteins bearing leucine-rich repeats (LRRs) and nucleotide-binding domains. Expression of NLRC3 is drastically reduced in the tumour tissue of patients with colorectal cancer compared to healthy tissues, highlighting an undefined potential function for this sensor in the development of cancer. Here we show that mice lacking NLRC3 are hyper-susceptible to colitis and colorectal tumorigenesis. The effect of NLRC3 is most dominant in enterocytes, in which it suppresses activation of the mTOR signalling pathways and inhibits cellular proliferation and stem-cell-derived organoid formation. NLRC3 associates with PI3Ks and blocks activation of the PI3K-dependent kinase AKT following binding of growth factor receptors or Toll-like receptor 4. These findings reveal a key role for NLRC3 as an inhibitor of the mTOR pathways, mediating protection against colorectal cancer.

The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development.

Z-DNA-binding protein 1 (ZBP1) is an innate immune sensor of nucleic acids that regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating receptor-interacting Ser/Thr kinase 3 (RIPK3), caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate immune sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we generated Zbp1ΔZα2/ΔZα2 mice that express ZBP1 lacking the Zα2 domain and demonstrate that this domain is critical for influenza A virus-induced PANoptosis and underlies perinatal lethality in mice in which the RIP homotypic interaction motif domain of RIPK1 has been mutated (Ripk1mRHIM/mRHIM). Deletion of the Zα2 domain in ZBP1 abolished influenza A virus-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1mRHIM/mRHIM mice from perinatal lethality caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in several physiological functions and establish a link between Z-RNA sensing via the Zα2 domain and promotion of influenza-induced PANoptosis and perinatal lethality.

T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event.

Type 1 diabetes is a T cell-mediated autoimmune disease, characterized by lymphocytic infiltration of the pancreatic islets. It is currently thought that islet antigen specificity is not a requirement for islet entry and that diabetogenic T cells can recruit a heterogeneous bystander T cell population. We tested this assumption directly by generating T cell receptor (TCR) retrogenic mice expressing two different T cell populations. By combining diabetogenic and nondiabetogenic or nonautoantigen-specific T cells, we demonstrate that bystander T cells cannot accumulate in the pancreatic islets. Autoantigen-specific T cells that accumulate in islets, but do not cause diabetes, were also unaffected by the presence of diabetogenic T cells. Additionally, 67% of TCRs cloned from nonobese diabetic (NOD) islet-infiltrating CD4(+) T cells were able to mediate cell-autonomous islet infiltration and/or diabetes when expressed in retrogenic mice. Therefore, islet entry and accumulation appears to be a cell-autonomous and tightly regulated event and is governed by islet antigen specificity.

Guanylate Binding Proteins Regulate Inflammasome Activation in Response to Hyperinjected Yersinia Translocon Components.

Gram-negative bacterial pathogens utilize virulence-associated secretion systems to inject, or translocate, effector proteins into host cells to manipulate cellular processes and promote bacterial replication. However, translocated bacterial products are sensed by nucleotide binding domain and leucine-rich repeat-containing proteins (NLRs), which trigger the formation of a multiprotein complex called the inflammasome, leading to secretion of interleukin-1 (IL-1) family cytokines, pyroptosis, and control of pathogen replication. Pathogenic Yersinia bacteria inject effector proteins termed Yops, as well as pore-forming proteins that comprise the translocon itself, into target cells. The Yersinia translocation regulatory protein YopK promotes bacterial virulence by limiting hyperinjection of the translocon proteins YopD and YopB into cells, thereby limiting cellular detection of Yersinia virulence activity. How hyperinjection of translocon proteins leads to inflammasome activation is currently unknown. We found that translocated YopB and YopD colocalized with the late endosomal/lysosomal protein LAMP1 and that the frequency of YopD and LAMP1 association correlated with the level of caspase-1 activation in individual cells. We also observed colocalization between YopD and Galectin-3, an indicator of endosomal membrane damage. Intriguingly, YopK limited the colocalization of Galectin-3 with YopD, suggesting that YopK limits the induction or sensing of endosomal membrane damage by components of the type III secretion system (T3SS) translocon. Furthermore, guanylate binding proteins (GBPs) encoded on chromosome 3 (GbpChr3 ), which respond to pathogen-induced damage or alteration of host membranes, were necessary for inflammasome activation in response to hyperinjected YopB/-D. Our findings indicate that lysosomal damage by Yersinia translocon proteins promotes inflammasome activation and implicate GBPs as key regulators of this process.

Central nervous system destruction mediated by glutamic acid decarboxylase-specific CD4+ T cells.

High titers of autoantibodies against glutamic acid decarboxylase (GAD) 65 are commonly observed in patients suffering from type 1 diabetes as well as stiff-person syndrome (SPS), a disorder that affects the CNS, and a variant of SPS, progressive encephalomyelitis with rigidity and myoclonus. Although there is a considerable amount of data focusing on the role of GAD65-specific CD4(+) T cells in type 1 diabetes, little is known about their role in SPS. In this study, we show that mice possessing a monoclonal GAD65-specific CD4(+) T cell population (4B5, PA19.9G11, or PA17.9G7) develop a lethal encephalomyelitis-like disease in the absence of any other T cells or B cells. GAD65-reactive CD4(+) T cells were found throughout the CNS in direct concordance with GAD65 expression and activated microglia: proximal to the circumventricular organs at the interface between the brain parenchyma and the blood-brain barrier. In the presence of B cells, high titer anti-GAD65 autoantibodies were generated, but these had no effect on the incidence or severity of disease. In addition, GAD65-specific CD4(+) T cells isolated from the brain were activated and produced IFN-gamma. These findings suggest that GAD65-reactive CD4(+) T cells alone mediate a lethal encephalomyelitis-like disease that may serve as a useful model to study GAD65-mediated diseases of the CNS.

Determining distinct roles of IL-1α through generation of an IL-1α knockout mouse with no defect in IL-1ß expression.

Interleukin 1α (IL-1α) and IL-1ß are the founding members of the IL-1 cytokine family, and these innate immune inflammatory mediators are critically important in health and disease. Early studies on these molecules suggested that their expression was interdependent, with an initial genetic model of IL-1α depletion, the IL-1α KO mouse (Il1a-KOline1), showing reduced IL-1ß expression. However, studies using this line in models of infection and inflammation resulted in contrasting observations. To overcome the limitations of this genetic model, we have generated and characterized a new line of IL-1α KO mice (Il1a-KOline2) using CRISPR-Cas9 technology. In contrast to cells from Il1a-KOline1, where IL-1ß expression was drastically reduced, bone marrow-derived macrophages (BMDMs) from Il1a-KOline2 mice showed normal induction and activation of IL-1ß. Additionally, Il1a-KOline2 BMDMs showed normal inflammasome activation and IL-1ß expression in response to multiple innate immune triggers, including both pathogen-associated molecular patterns and pathogens. Moreover, using Il1a-KOline2 cells, we confirmed that IL-1α, independent of IL-1ß, is critical for the expression of the neutrophil chemoattractant KC/CXCL1. Overall, we report the generation of a new line of IL-1α KO mice and confirm functions for IL-1α independent of IL-1ß. Future studies on the unique functions of IL-1α and IL-1ß using these mice will be critical to identify new roles for these molecules in health and disease and develop therapeutic strategies.

RIPK1 Distinctly Regulates Yersinia-Induced Inflammatory Cell Death, PANoptosis.

Bacterial pathogens from the genus Yersinia cause fatal sepsis and gastritis in humans. Innate immune signaling and inflammatory cell death (pyroptosis, apoptosis, and necroptosis [PANoptosis]) serve as a first line of antimicrobial host defense. The receptor-interacting protein kinase 1 (RIPK1) is essential for Yersinia-induced pyroptosis and apoptosis and an effective host response. However, it is not clear whether RIPK1 assembles a multifaceted cell death complex capable of regulating caspase-dependent pyroptosis and apoptosis or whether there is cross-talk with necroptosis under these conditions. In this study, we report that Yersinia activates PANoptosis, as evidenced by the concerted activation of proteins involved in PANoptosis. Genetic deletion of RIPK1 abrogated the Yersinia-induced activation of the inflammasome/pyroptosis and apoptosis but enhanced necroptosis. We also found that Yersinia induced assembly of a RIPK1 PANoptosome complex capable of regulating all three branches of PANoptosis. Overall, our results demonstrate a role for the RIPK1 PANoptosome in Yersinia-induced inflammatory cell death and host defense.

Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer.

Interferon regulatory factor 1 (IRF1) regulates diverse biological functions, including modulation of cellular responses involved in tumorigenesis. Genetic mutations and altered IRF1 function are associated with several cancers. Although the function of IRF1 in the immunobiology of cancer is emerging, IRF1-specific mechanisms regulating tumorigenesis and tissue homeostasis in vivo are not clear. Here, we found that mice lacking IRF1 were hypersusceptible to colorectal tumorigenesis. IRF1 functions in both the myeloid and epithelial compartments to confer protection against AOM/DSS-induced colorectal tumorigenesis. We further found that IRF1 also prevents tumorigenesis in a spontaneous mouse model of colorectal cancer. The attenuated cell death in the colons of Irf1-/- mice was due to defective pyroptosis, apoptosis, and necroptosis (PANoptosis). IRF1 does not regulate inflammation and the inflammasome in the colon. Overall, our study identified IRF1 as an upstream regulator of PANoptosis to induce cell death during colitis-associated tumorigenesis.

Rapid identification of MHC class I-restricted antigens relevant to autoimmune diabetes using retrogenic T cells.

The method described herein provides a novel strategy for the rapid identification of CD8(+) T cell epitopes relevant to type 1 diabetes in the context of the nonobese diabetic (NOD) mouse model of disease. Obtaining the large number of antigen-sensitive monospecific T cells required for conventional antigen discovery methods has historically been problematic due to (1) difficulties in culturing autoreactive CD8(+) T cells from NOD mice and (2) the large time and resource investments required for the generation of transgenic NOD mice. We circumvented these problems by exploiting the rapid generation time of retrogenic (Rg) mice, relative to transgenic mice, as a novel source of sensitive monospecific CD8(+) T cells, using the diabetogenic AI4 T cell receptor on NOD.SCID and NOD.Rag1(-/-) backgrounds as a model. Rg AI4 T cells are diabetogenic in vivo, demonstrating for the first time that Rg mice are a means for assessing the pathogenic potential of CD8(+) T cell receptor specificities. In order to obtain a sufficient number of Rg CD8(+) T cells for antigen screens, we optimized a method for their in vitro culture that resulted in a approximately 500 fold expansion. We demonstrate the high sensitivity and specificity of expanded Rg AI4 T cells in the contexts of (1) specific peptide challenge, (2) islet cytotoxicity, and (3) their ability to resolve previously defined mimotope candidates from a positional scanning peptide library. Our method is the first to combine the speed of Rg technology with an optimized in vitro Rg T cell expansion protocol to enable the rapid discovery of T cell antigens.

Prevention of autoimmune diabetes by ectopic pancreatic ß-cell expression of interleukin-35.

Interleukin (IL)-35 is a newly identified inhibitory cytokine used by T regulatory cells to control T cell-driven immune responses. However, the therapeutic potential of native, biologically active IL-35 has not been fully examined. Expression of the heterodimeric IL-35 cytokine was targeted to ß-cells via the rat insulin promoter (RIP) II. Autoimmune diabetes, insulitis, and the infiltrating cellular populations were analyzed. Ectopic expression of IL-35 by pancreatic ß-cells led to substantial, long-term protection against autoimmune diabetes, despite limited intraislet IL-35 secretion. Nonobese diabetic RIP-IL35 transgenic mice exhibited decreased islet infiltration with substantial reductions in the number of CD4(+) and CD8(+) T cells, and frequency of glucose-6-phosphatase catalytic subunit-related protein-specific CD8(+) T cells. Although there were limited alterations in cytokine expression, the reduced T-cell numbers observed coincided with diminished T-cell proliferation and G1 arrest, hallmarks of IL-35 biological activity. These data present a proof of principle that IL-35 could be used as a potent inhibitor of autoimmune diabetes and implicate its potential therapeutic utility in the treatment of type 1 diabetes.

On the pathogenicity of autoantigen-specific T-cell receptors.

OBJECTIVE: Type 1 diabetes is mediated by T-cell entry into pancreatic islets and destruction of insulin-producing beta-cells. The relative contribution of T-cells specific for different autoantigens is largely unknown because relatively few have been assessed in vivo. RESEARCH DESIGN AND METHODS: We generated mice possessing a monoclonal population of T-cells expressing 1 of 17 T-cell receptors (TCR) specific for either known autoantigens (GAD65, insulinoma-associated protein 2 (IA2), IA2beta/phogrin, and insulin), unknown islet antigens, or control antigens on a NOD.scid background using retroviral-mediated stem cell gene transfer and 2A-linked multicistronic retroviral vectors (referred to herein as retrogenic [Rg] mice). The TCR Rg approach provides a mechanism by which T-cells with broad phenotypic differences can be directly compared. RESULTS: Neither GAD- nor IA2-specific TCRs mediated T-cell islet infiltration or diabetes even though T-cells developed in these Rg mice and responded to their cognate epitope. IA2beta/phogrin and insulin-specific Rg T-cells produced variable levels of insulitis, with one TCR producing delayed diabetes. Three TCRs specific for unknown islet antigens produced a hierarchy of insulitogenic and diabetogenic potential (BDC-2.5 > NY4.1 > BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before full T-cell reconstitution (days 42-56). Remarkably, as few as 1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice. CONCLUSIONS; Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and beta-cell destruction on their own and that autoreactivity does not necessarily imply pathogenicity.

Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.

Although T-cell receptor (TCR) transgenic as well as knockout and knockin mice have had a large impact on our understanding of T-cell development, signal transduction and function, the need to cross these mice delays experiments considerably. Here we provide a methodology for the rapid expression of TCRs in mice using 2A peptide-linked multicistronic retroviral vectors to transduce stem cells of any background before adoptive transfer into RAG-1(-/-) mice. For simplicity, we refer to these as retrogenic mice. We demonstrate that these retrogenic mice are comparable to transgenic mice expressing three commonly used TCRs (OT-I, OT-II [corrected] and AND). We also show that retrogenic mice expressing male antigen-specific TCRs (HY, MataHari and Marilyn) facilitated the analysis of positive and negative selection in female and male mice, respectively. We examined various tolerance mechanisms in epitope-coupled TCR retrogenic mice. This powerful resource could expedite the identification of proteins involved in T-cell development and function.

Generation of T-cell receptor retrogenic mice.

T-cell receptor (TCR) transgenic (Tg) mice have revolutionized our understanding of many aspects of T-cell biology. Whereas they provide an almost unlimited source of T cells with a single specificity, breeding them onto different backgrounds and/or new knockout/knock-in mouse models is often time-consuming (6 months to several years), which can make the process costly and can significantly delay research. This protocol describes a new method for expressing defined TCR-alpha and TCR-beta proteins from a single 2A peptide-linked multicistronic retroviral vector in mice, using retrovirus-mediated stem cell gene transfer. We refer to these as 'retrogenic' (Rg) mice ('retro' from retrovirus and 'genic' from Tg) to avoid confusion with traditional transgenic mice. We have successfully used this approach to express over 50 different TCRs on several different mouse backgrounds in as little as 6 weeks.

Diabetes incidence is unaltered in glutamate decarboxylase 65-specific TCR retrogenic nonobese diabetic mice: generation by retroviral-mediated stem cell gene transfer.

TCR transgenic mice are valuable tools for dissecting the role of autoantigen-specific T cells in the pathogenesis of type 1 diabetes but are time-consuming to generate and backcross onto congenic strains. To circumvent these limitations, we developed a new approach to rapidly generate mice expressing TCR using retroviral-mediated stem cell gene transfer and a novel picornavirus-like 2A peptide to link the TCR alpha- and beta-chains in a single retroviral vector. We refer to these as retrogenic (Rg) mice to avoid confusion with conventional transgenic mice. Our approach was validated by demonstrating that Rg nonobese diabetic (NOD)-scid mice expressing the diabetogenic TCRs, BDC2.5 and 4.1, generate clonotype-positive T cells and develop diabetes. We then expressed three TCR specific for either glutamate decarboxylase (GAD) 206-220 or GAD 524-538 or for hen egg lysozyme 11-25 as a control in NOD, NOD-scid, and B6.H2(g7) mice. Although T cells from these TCR Rg mice responded to their respective Ag in vitro, the GAD-specific T cells exhibited a naive, resting phenotype in vivo. However, T cells from Rg mice challenged with Ag in vivo became activated and developed into memory cells. Neither of the GAD-reactive TCR accelerated or protected mice from diabetes, nor did activated T cells transfer or protect against diabetes in NOD-scid recipients, suggesting that GAD may not be a primary target for diabetogenic T cells. Generation of autoantigen-specific TCR Rg mice represents a powerful approach for the analysis of a wide variety of autoantigens.