The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells.

Intestinal microfold cells (M cells) are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. The mechanisms of M-cell differentiation are poorly understood, as the rarity of these cells has hampered analysis. Exogenous administration of the cytokine RANKL can synchronously activate M-cell differentiation in mice. Here we show the Ets transcription factor Spi-B was induced early during M-cell differentiation. Absence of Spi-B silenced the expression of various M-cell markers and prevented the differentiation of M cells in mice. The activation of T cells via an oral route was substantially impaired in the intestine of Spi-B-deficient (Spib(-/-)) mice. Our study demonstrates that commitment to the intestinal M-cell lineage requires Spi-B as a candidate master regulator.

Autoinflammatory Diseases Due to Defects in Degradation or Transport of Intracellular Proteins.

The number of human inborn errors of immunity has now gone beyond 430. The responsible gene variants themselves are apparently the cause for the disorders, but the underlying molecular or cellular mechanisms for the pathogenesis are often unclear. In order to clarify the pathogenesis, the mutant mice carrying the gene variants are apparently useful and important. Extensive analysis of those mice should contribute to the clarification of novel immunoregulatory mechanisms or development of novel therapeutic maneuvers critical not only for the rare monogenic diseases themselves but also for related common polygenic diseases. We have recently generated novel model mice in which complicated manifestations of human inborn errors of immunity affecting degradation or transport of intracellular proteins were recapitulated. Here, we review outline of these disorders, mainly based on the phenotype of the mutant mice we have generated.

Heme ameliorates dextran sodium sulfate-induced colitis through providing intestinal macrophages with noninflammatory profiles.

The local environment is crucial for shaping the identities of tissue-resident macrophages (Mϕs). When hemorrhage occurs in damaged tissues, hemoglobin induces differentiation of anti-inflammatory Mϕs with reparative function. Mucosal bleeding is one of the pathological features of inflammatory bowel diseases. However, the heme-mediated mechanism modulating activation of intestinal innate immune cells remains poorly understood. Here, we show that heme regulates gut homeostasis through induction of Spi-C in intestinal CX3CR1high Mϕs. Intestinal CX3CR1high Mϕs highly expressed Spi-C in a heme-dependent manner, and myeloid lineage-specific Spic-deficient (Lyz2-cre; Spicflox/flox ) mice showed severe intestinal inflammation with an increased number of Th17 cells during dextran sodium sulfate-induced colitis. Spi-C down-regulated the expression of a subset of Toll-like receptor (TLR)-inducible genes in intestinal CX3CR1high Mϕs to prevent colitis. LPS-induced production of IL-6 and IL-1α, but not IL-10 and TNF-α, by large intestinal Mϕs from Lyz2-cre; Spicflox/flox mice was markedly enhanced. The interaction of Spi-C with IRF5 was linked to disruption of the IRF5-NF-κB p65 complex formation, thereby abrogating recruitment of IRF5 and NF-κB p65 to the Il6 and Il1a promoters. Collectively, these results demonstrate that heme-mediated Spi-C is a key molecule for the noninflammatory signature of intestinal Mϕs by suppressing the induction of a subset of TLR-inducible genes through binding to IRF5.

Anticancer effects of chemokine-directed antigen delivery to a cross-presenting dendritic cell subset with immune checkpoint blockade.

BACKGROUND: Cancer peptide vaccines show only marginal effects against cancers. Immune checkpoint inhibitors (ICIs) show significant curative effects in certain types of cancers, but the response rate is still limited. In this study, we aim to improve cancer peptide vaccination by targeting Ag peptides selectively to a dendritic cell (DC) subset, XCR1-expressing DCs (XCR1+ DCs), with high ability to support CD8+ T-cell responses. METHODS: We have generated a fusion protein, consisting of an Ag peptide presented with MHC class I, and an XCR1 ligand, XCL1, and examined its effects on antitumour immunity in mice. RESULTS: The fusion protein was delivered to XCR1+ DCs in an XCR1-dependent manner. Immunisation with the fusion protein plus an immune adjuvant, polyinosinic:polycytidylic acids (poly(I:C)), more potently induced Ag-specific CD8+ T-cell responses through XCR1 than the Ag peptide plus poly(I:C) or the Ag protein plus poly(I:C). The fusion protein plus poly(I:C) inhibited the tumour growth efficiently in the prophylactic and therapeutic tumour models. Furthermore, the fusion protein plus poly(I:C) showed suppressive effects on tumour growth in synergy with anti-PD-1 Ab. CONCLUSIONS: Cancer Ag targeting to XCR1+ DCs should be a promising procedure as a combination anticancer therapy with immune checkpoint blockade.

The mechanism of action of Spi-B in the transcriptional activation of the interferon-α4 gene.

Plasmacytoid dendritic cells (pDCs) are characterized by an exclusive expression of nucleic acid sensing Toll-like receptor 7 (TLR7) and TLR9, and production of high amounts of type I interferon (IFN) in response to TLR7/9 signaling. This function is crucial for both antiviral immunity and the pathogenesis of autoimmune diseases. An Ets family transcription factor, i.e., Spi-B (which is highly expressed in pDCs) is required for TLR7/9 signal-induced type I IFN production and can transactivate IFN-α promoter in synergy with IFN regulatory factor-7 (IRF-7). Herein, we analyzed how Spi-B contributes to the transactivation of the Ifna4 promoter. We performed deletion and/or mutational analyses of the Ifna4 promoter and an electrophoretic mobility shift assay (EMSA) and observed an Spi-B binding site in close proximity to the IRF-7 binding site. The EMSA results also showed that the binding of Spi-B to the double-stranded DNA probe potentiated the recruitment of IRF-7 to its binding site. We also observed that the association of Spi-B with transcriptional coactivator p300 was required for the Spi-B-induced synergistic enhancement of the Ifna4 promoter activity by Spi-B. These results clarify the molecular mechanism of action of Spi-B in the transcriptional activation of the Ifna4 promoter.

Cholera toxin B induces interleukin-1ß production from resident peritoneal macrophages through the pyrin inflammasome as well as the NLRP3 inflammasome.

Cholera toxin B (CTB) is a subunit of cholera toxin, a bacterial enterotoxin secreted by Vibrio cholerae and also functions as an immune adjuvant. However, it remains unclear how CTB activates immune cells. We here evaluated whether or how CTB induces production of a pro-inflammatory cytokine, interleukin-1ß (IL-1ß). CTB induced IL-1ß production not only from bone marrow-derived macrophages (BMMs) but also from resident peritoneal macrophages in synergy with O111:B4-derived lipopolysaccharide (LPS O111:B4) that can bind to CTB. Meanwhile, when prestimulated with O55:B5-derived LPS (LPS O55:B5) that fails to bind to CTB, resident peritoneal macrophages, but not BMMs, produced IL-1ß in response to CTB. The CTB-induced IL-1ß production in synergy with LPS in both peritoneal macrophages and BMMs was dependent on ganglioside GM1, which is required for internalization of CTB. Notably, not only the NLRP3 inflammasome but also the pyrin inflammasome were involved in CTB-induced IL-1ß production from resident peritoneal macrophages, while only the NLRP3 inflammasome was involved in that from BMMs. In response to CTB, a Rho family small GTPase, RhoA, which activates pyrin inflammasome upon various kinds of biochemical modification, increased its phosphorylation at serine-188 in a GM1-dependent manner. This phosphorylation as well as CTB-induced IL-1ß productions were dependent on protein kinase A (PKA), indicating critical involvement of PKA-dependent RhoA phosphorylation in CTB-induced IL-1ß production. Taken together, these results suggest that CTB, incorporated through GM1, can activate resident peritoneal macrophages to produce IL-1ß in synergy with LPS through novel mechanisms in which pyrin as well as NLRP3 inflammasomes are involved.

Involvement of exchange protein directly activated by cAMP and tumor progression locus 2 in IL-1ß production in microglial cells following activation of ß-adrenergic receptors.

Endogenous noradrenaline (NA) has multiple bioactive functions and, in the central nervous system (CNS), has been implicated in modulating neuroinflammation via ß-adrenergic receptors (ß-ARs). Microglia, resident macrophages in the CNS, have a central role in the brain immune system and have been reported to be activated by NA. However, intracellular signaling mechanisms of the AR-mediated proinflammatory responses of microglia are not fully understood. Using a rapid and stable in vitro reporter assay system to evaluate IL-1ß production in microglial BV2 cells, we found that NA and the ß-AR agonist isoproterenol upregulated the IL-1ß reporter activity. This effect was suppressed by ß-AR antagonists. We further examined the involvement of EPAC (exchange protein directly activated by cAMP) and TPL2 (tumor progression locus 2, MAP3K8) and found that inhibitors for EPAC and TPL2 reduced AR agonist-induced IL-1ß reporter activity. These inhibitors also suppressed NA-induced endogenous Il1b mRNA expression and IL-1ß protein production. Our results suggest that EPAC and TPL2 are involved in ß-AR-mediated IL-1ß production in microglial cells, and extend our understanding of its intracellular signaling mechanism.

Programmed cell death ligand 1 disruption by clustered regularly interspaced short palindromic repeats/Cas9-genome editing promotes antitumor immunity and suppresses ovarian cancer progression.

Programmed cell death ligand 1 (PD-L1) on tumor cells suppresses anti-tumor immunity and has an unfavorable prognostic impact in ovarian cancer patients. We herein report the pathophysiological and therapeutic impacts of PD-L1 disruption in ovarian cancer. PD-L1 was genetically disrupted in the murine ovarian cancer cell line ID8 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. PD-L1 knockout (KO) and control ovarian cancer cells were intraperitoneally inoculated into syngeneic mice, and survival and tumor dissemination were evaluated. Survival times were significantly longer in the PD-L1-KO ID8-inoculated groups than in their control groups, and its therapeutic benefit was enhanced in combination with the cisplatin treatment. Tumor weights and ascites volumes were significantly lower in the PD-L1-KO ID8 groups than in their control groups. Immunohistochemical and immunofluorescence analyses showed that intratumoral CD4+ T cells, CD8+ T cells, NK cells and CD11c+ M1 macrophages were significantly increased, whereas regulatory T cells were significantly decreased in the PD-L1-KO ID8 groups compared with those in their control groups. The intratumoral mRNA expression of interferon-γ, tumor-necrosis factor-α, interleukin (IL)-2, IL-12a, CXCL9 and CXCL10 was significantly stronger, while that of IL-10, vascular endothelial growth factor, CXCL1 and CXCL2 was significantly weaker in the PD-L1-KO ID8 groups. These results indicate that CRISPR/Cas9-mediated PD-L1 disruption on tumor cells promotes anti-tumor immunity by increasing tumor-infiltrating lymphocytes and modulating cytokine/chemokine profiles within the tumor microenvironment, thereby suppressing ovarian cancer progression. These results suggest that PD-L1-targeted therapy by genome editing may be a novel therapeutic strategy for ovarian cancer.

Discovery of DSP-1053, a novel benzylpiperidine derivative with potent serotonin transporter inhibitory activity and partial 5-HT1A receptor agonistic activity.

We have previously shown that SMP-304, a serotonin uptake inhibitor with weak 5-HT1A partial agonistic activity, may act under high serotonin levels as a 5-HT1A antagonist that improves the onset of paroxetine in the rat swimming test. However, SMP-304 is mostly metabolized by CYP2D6, indicating limited efficacy among individuals and increased side effects. To reduce CYP2D6 metabolic contribution and enhance SERT/5-HT1A binding affinity, we carried out a series of substitutions at the bromine atom in the left part of the benzene ring of SMP-304 and replaced the right part of SMP-304 with a chroman-4-one. This optimization work led to the identification of the antidepressant candidate DSP-1053 as a potent SERT inhibitor with partial 5-HT1A receptor agonistic activity. DSP-1053 showed low CYP2D6 metabolic contribution and a robust increase in serotonin levels in the rat frontal cortex.

Transcription factor IRF8 plays a critical role in the development of murine basophils and mast cells.

Basophils and mast cells play critical roles in host defense against pathogens and allergic disorders. However, the molecular mechanism by which these cells are generated is not completely understood. Here we demonstrate that interferon regulatory factor-8 (IRF8), a transcription factor essential for the development of several myeloid lineages, also regulates basophil and mast cell development. Irf8(-/-) mice displayed a severe reduction in basophil counts, which was accounted for by the absence of pre-basophil and mast cell progenitors (pre-BMPs). Although Irf8(-/-) mice retained peripheral tissue mast cells, remaining progenitors from Irf8(-/-) mice including granulocyte progenitors (GPs) were unable to efficiently generate either basophils or mast cells, indicating that IRF8 also contributes to the development of mast cells. IRF8 appeared to function at the GP stage, because IRF8 was expressed in GPs, but not in basophils, mast cells, and basophil/mast cell-restricted progenitor cells. Furthermore, we demonstrate that GATA2, a transcription factor known to promote basophil and mast cell differentiation, acts downstream of IRF8. These results shed light on the pathways and mechanism underlying the development of basophils and mast cells.

Transcriptional control of dendritic cell differentiation.

Dendritic cells (DCs) are professional antigen presenting cells involved critically not only in provoking innate immune responses but also in establishing adaptive immune responses. Dendritic cells are heterogenous and divided into several subsets, including plasmactyoid DCs (pDCs) and several types of conventional DCs (cDCs), which show subset-specific functions. Plasmactyoid DCs are featured by their ability to produce large amounts of type I interferons (IFNs) in response to nucleic acid sensors, TLR7 and TLR9 and involved in anti-viral immunity and pathogenesis of certain autoimmune disorders such as psoriasis. Conventional DCs include the DC subsets with high crosspresentation activity, which contributes to anti-viral and anti-tumor immunity. These subsets are generated from hematopoietic stem cells (HSCs) via several intermediate progenitors and the development is regulated by the transcriptional mechanisms in which subset-specific transcription factors play major roles. We have recently found that an Ets family transcription factor, SPI-B, which is abundantly expressed in pDCs among DC subsets, plays critical roles in functions and late stage development of pDCs. SPI-B functions in cooperation with other transcription factors, especially, interferon regulatory factor (IRF) family members. Here we review the transcription factor-based molecular mechanisms for generation and functions of DCs, mainly by focusing on the roles of SPI-B and its relatives.

Critical roles of a dendritic cell subset expressing a chemokine receptor, XCR1.

Dendritic cells (DCs) consist of various subsets that play crucial roles in linking innate and adaptive immunity. In the murine spleen, CD8α(+) DCs exhibit a propensity to ingest dying/dead cells, produce proinflammatory cytokines, and cross-present Ags to generate CD8(+) T cell responses. To track and ablate CD8α(+) DCs in vivo, we generated XCR1-venus and XCR1-DTRvenus mice, in which genes for a fluorescent protein, venus, and a fusion protein consisting of diphtheria toxin receptor and venus were knocked into the gene locus of a chemokine receptor, XCR1, which is highly expressed in CD8α(+) DCs. In both mice, venus(+) cells were detected in the majority of CD8α(+) DCs, but they were not detected in any other cells, including splenic macrophages. Venus(+)CD8α(+) DCs were superior to venus(-)CD8α(+) DCs with regard to their cytokine-producing ability in response to TLR stimuli. In other tissues, venus(+) cells were found primarily in lymph node (LN)-resident CD8α(+), LN migratory and peripheral CD103(+) DCs, which are closely related to splenic CD8α(+) DCs, although some thymic CD8α(-)CD11b(-) and LN CD103(-)CD11b(-) DCs were also venus(+). In response to dsRNAs, diphtheria toxin-treated XCR1-DTR mice showed impaired CD8(+) T cell responses, with retained cytokine and augmented CD4(+) T cell responses. Furthermore, Listeria monocytogenes infection and anti-L. monocytogenes CD8(+) T cell responses were defective in diphtheria toxin-treated XCR1-DTRvenus mice. Thus, XCR1-expressing DCs were required for dsRNA- or bacteria-induced CD8(+) T cell responses. XCR1-venus and XCR1-DTRvenus mice should be useful for elucidating the functions and behavior of XCR1-expressing DCs, including CD8α(+) and CD103(+) DCs, in lymphoid and peripheral tissues.

Spi-B is critical for plasmacytoid dendritic cell function and development.

Plasmacytoid dendritic cells (pDCs), originating from hematopoietic progenitor cells in the BM, are a unique dendritic cell subset that can produce large amounts of type I IFNs by signaling through the nucleic acid-sensing TLR7 and TLR9 (TLR7/9). The molecular mechanisms for pDC function and development remain largely unknown. In the present study, we focused on an Ets family transcription factor, Spi-B, that is highly expressed in pDCs. Spi-B could transactivate the type I IFN promoters in synergy with IFN regulatory factor 7 (IRF-7), which is an essential transcription factor for TLR7/9-induced type I IFN production in pDCs. Spi-B-deficient pDCs and mice showed defects in TLR7/9-induced type I IFN production. Furthermore, in Spi-B-deficient mice, BM pDCs were decreased and showed attenuated expression of a set of pDC-specific genes whereas peripheral pDCs were increased; this uneven distribution was likely because of defective retainment of mature nondividing pDCs in the BM. The expression pattern of cell-surface molecules in Spi-B-deficient mice indicated the involvement of Spi-B in pDC development. The developmental defects of pDCs in Spi-B-deficient mice were more prominent in the BM than in the peripheral lymphoid organs and were intrinsic to pDCs. We conclude that Spi-B plays critical roles in pDC function and development.

A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1ß production in tissue-resident macrophages.

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1ß (IL-1ß), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1ß production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1ß production, indicating that IRE1α is required for CT- or CTB-induced IL-1ß production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

SpiB regulates the expression of B-cell-related genes and increases the longevity of memory B cells.

Generation of memory B cells is one of the key features of adaptive immunity as they respond rapidly to re-exposure to the antigen and generate functional antibodies. Although the functions of memory B cells are becoming clearer, the regulation of memory B cell generation and maintenance is still not well understood. Here we found that transcription factor SpiB is expressed in some germinal center (GC) B cells and memory B cells and participates in the maintenance of memory B cells. Overexpression and knockdown analyses revealed that SpiB suppresses plasma cell differentiation by suppressing the expression of Blimp1 while inducing Bach2 in the in-vitro-induced germinal center B (iGB) cell culture system, and that SpiB facilitates in-vivo appearance of memory-like B cells derived from the iGB cells. Further analysis in IgG1+ cell-specific SpiB conditional knockout (cKO) mice showed that function of SpiB is critical for the generation of late memory B cells but not early memory B cells or GC B cells. Gene expression analysis suggested that SpiB-dependent suppression of plasma cell differentiation is independent of the expression of Bach2. We further revealed that SpiB upregulates anti-apoptosis and autophagy genes to control the survival of memory B cells. These findings indicate the function of SpiB in the generation of long-lasting memory B cells to maintain humoral memory.

Critical role of IkappaB Kinase alpha in TLR7/9-induced type I IFN production by conventional dendritic cells.

A plasmacytoid dendritic cell (DC) can produce large amounts of type I IFNs after sensing nucleic acids through TLR7 and TLR9. IkappaB kinase alpha (IKKalpha) is critically involved in this type I IFN production through its interaction with IFN regulatory factor-7. In response to TLR7/9 signaling, conventional DCs can also produce IFN-beta but not IFN-alpha in a type I IFN-independent manner. In this study, we showed that IKKalpha was required for production of IFN-beta, but not of proinflammatory cytokines, by TLR7/9-stimulated conventional DCs. Importantly, IKKalpha was dispensable for IFN-beta gene upregulation by TLR4 signaling. Biochemical analyses indicated that IKKalpha exerted its effects through its interaction with IFN regulatory factor-1. Furthermore, IKKalpha was involved in TLR9-induced type I IFN-independent IFN-beta production in vivo. Our results show that IKKalpha is a unique molecule involved in TLR7/9-MyD88-dependent type I IFN production through DC subset-specific mechanisms.

Preparation and Inflammasome Activation of Murine Bone Marrow-Derived and Resident Peritoneal Macrophages.

Cholera toxin (CT), secreted by Vibrio cholerae, not only causes cholera but also functions as an immune adjuvant. Macrophages, which respond to a variety of immune adjuvants, are quite heterogenous and their development and function depend on the tissues where they are localized. We have characterized the effects of the B subunit of CT (CTB) on two types of murine macrophages, that is, bone marrow-derived macrophages (BMMs) and resident peritoneal macrophages (rPMs). CTB could induce production of interleukin-1ß (IL-1ß) from both macrophages in synergy with lipopolysaccharides. However, underlying molecular mechanisms for IL-1ß induction were different. Here, we describe the protocols for preparation and stimulation of BMMs and rPMs.

Conventional Type 1 Dendritic Cells in Intestinal Immune Homeostasis.

Dendritic cells (DC) play critical roles in linking innate and adaptive immunity. DC are heterogenous and there are subsets with various distinct functions. One DC subset, conventional type 1 DC (cDC1), can be defined by expression of CD8α/CD103 in mice and CD141 in humans, or by expression of a chemokine receptor, XCR1, which is a conserved marker in both mice and human. cDC1 are characterized by high ability to ingest dying cells and to cross-present antigens for generating cytotoxic CD8 T cell responses. Through these activities, cDC1 play crucial roles in immune responses against infectious pathogens or tumors. Meanwhile, cDC1 involvement in homeostatic situations is not fully understood. Analyses by using mutant mice, in which cDC1 are ablated in vivo, revealed that cDC1 are critical for maintaining intestinal immune homeostasis. Here, we review the homeostatic roles of cDC1, focusing upon intestinal immunity.

Augmentation of Stimulator of Interferon Genes-Induced Type I Interferon Production in COPA Syndrome.

OBJECTIVE: Coatomer subunit alpha (COPA) syndrome, also known as autoinflammatory interstitial lung, joint, and kidney disease, is caused by heterozygous mutations in COPA. We identified a novel COPA variant in 4 patients in one family. We undertook this study to elucidate whether and how the variant causes manifestations of COPA syndrome by studying these 4 patients and by analyzing results from a gene-targeted mouse model. METHODS: We performed whole-exome sequencing in 7 family members and measured the type I interferon (IFN) signature of the peripheral blood cells. We analyzed the effects of COPA variants in in vitro experiments and in Copa mutant mice that were generated. RESULTS: We identified a heterozygous variant of COPA (c.725T>G, p.Val242Gly) in the 4 affected members of the family. The IFN score was high in the members carrying the variant. In vitro analysis revealed that COPA V242G, as well as the previously reported disease-causing variants, augmented stimulator of interferon genes (STING)-induced type I IFN promoter activities. CopaV242G/+ mice manifested interstitial lung disease and STING-dependent elevation of IFN-stimulated gene expression. In CopaV242G/+ dendritic cells, the STING pathway was not constitutively activated but was hyperactivated upon stimulation, leading to increased type I IFN production. CONCLUSION: V242G, a novel COPA variant, was found in 4 patients from one family. In gene-targeted mice with the V242G variant, interstitial lung disease was recapitulated and augmented responses of the STING pathway, leading to an increase in type I IFN production, were demonstrated.

Heterozygous missense variant of the proteasome subunit ß-type 9 causes neonatal-onset autoinflammation and immunodeficiency.

Impaired proteasome activity due to genetic variants of certain subunits might lead to proteasome-associated autoinflammatory syndromes (PRAAS). Here we report a de novo heterozygous missense variant of the PSMB9 proteasome subunit gene in two unrelated Japanese infants resulting in amino acid substitution of the glycine (G) by aspartic acid (D) at position 156 of the encoded protein ß1i. In addition to PRAAS-like manifestations, these individuals suffer from pulmonary hypertension and immunodeficiency, which are distinct from typical PRAAS symptoms. The missense variant results in impaired immunoproteasome maturation and activity, yet ubiquitin accumulation is hardly detectable in the patients. A mouse model of the heterozygous human genetic variant (Psmb9G156D/+) recapitulates the proteasome defects and the immunodeficiency phenotype of patients. Structurally, PSMB9 G156D interferes with the ß-ring-ßring interaction of the wild type protein that is necessary for 20S proteasome formation. We propose the term, proteasome-associated autoinflammatory syndrome with immunodeficiency (PRAAS-ID), to indicate a separate category of autoinflammatory diseases, similar to, but distinct from PRAAS, that describes the patients in this study.