Quiescent Tissue Stem Cells Evade Immune Surveillance.

Stem cells are critical for the maintenance of many tissues, but whether their integrity is maintained in the face of immunity is unclear. Here we found that cycling epithelial stem cells, including Lgr5+ intestinal stem cells, as well as ovary and mammary stem cells, were eliminated by activated T cells, but quiescent stem cells in the hair follicle and muscle were resistant to T cell killing. Immune evasion was an intrinsic property of the quiescent stem cells resulting from systemic downregulation of the antigen presentation machinery, including MHC class I and TAP proteins, and is mediated by the transactivator NLRC5. This process was reversed upon stem cell entry into the cell cycle. These studies identify a link between stem cell quiescence, antigen presentation, and immune evasion. As cancer-initiating cells can derive from stem cells, these findings may help explain how the earliest cancer cells evade immune surveillance.

Targeted demethylation and activation of NLRC5 augment cancer immunogenicity through MHC class I.

Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer.

NLRC5/MHC class I transactivator: A key target for immune escape by SARS-CoV-2.

Antigen presentation to CD8+ T cells by MHC class I molecules is essential for host defense against viral infections. Various mechanisms have evolved in multiple viruses to escape immune surveillance and defense to support viral proliferation in host cells. Through in vitro SARS-CoV-2 infection studies and analysis of COVID-19 patient samples, we found that SARS-CoV-2 suppresses the induction of the MHC class I pathway by inhibiting the expression and function of NLRC5, a major transcriptional regulator of MHC class I genes. In this review, we discuss the molecular mechanisms for suppression of the MHC class I pathway and clinical implications for COVID-19.

FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA.

Major histocompatibility complex (MHC) class I and II molecules play critical roles in the activation and regulation of adaptive immunity through antigen presentation to CD8+ and CD4+ T cells, respectively. Strict regulation of MHC expression is critical for proper immune responses. CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, is a master regulator of MHC class II (MHC-II) gene transcription. Although it has been known that CIITA activity is regulated at the transcriptional and protein levels, the mechanism to determine CIITA protein level has not been elucidated. Here, we show that FBXO11 is a bona fide E3 ligase of CIITA and regulates CIITA protein level through ubiquitination-mediated degradation. A nonbiased proteomic approach for CIITA-binding protein identified FBXO11, a member of the Skp1-Cullin-1-F-box E3 ligase complex, as a binding partner of CIITA but not MHC class I transactivator, NLRC5. The cycloheximide chase assay showed that the half-life of CIITA is mainly regulated by FBXO11 via the ubiquitin-proteasome system. The expression of FBXO11 led to the reduced MHC-II at the promoter activity level, transcriptional level, and surface expression level through downregulation of CIITA. Moreover, human and mouse FBXO11-deficient cells display increased levels of MHC-II and related genes. In normal and cancer tissues, FBXO11 expression level is negatively correlated with MHC-II. Interestingly, the expression of FBXO11, along with CIITA, is associated with prognosis of cancer patients. Therefore, FBXO11 is a critical regulator to determine the level of MHC-II, and its expression may serve as a biomarker for cancer.

The balance between nuclear import and export of NLRC5 regulates MHC class I transactivation.

Major histocompatibility complex (MHC) class I molecules play an essential role in regulating the adaptive immune system by presenting antigens to CD8 T cells. CITA (MHC class I transactivator), also known as NLRC5 (NLR family, CARD domain-containing 5), regulates the expression of MHC class I and essential components involved in the MHC class I antigen presentation pathway. While the critical role of the nuclear distribution of NLRC5 in its transactivation activity has been known, the regulatory mechanism to determine the nuclear localization of NLRC5 remains poorly understood. In this study, a comprehensive analysis of all domains in NLRC5 revealed that the regulatory mechanisms for nuclear import and export of NLRC5 coexist and counterbalance each other. Moreover, GCN5 (general control non-repressed 5 protein), a member of HATs (histone acetyltransferases), was found to be a key player to retain NLRC5 in the nucleus, thereby contributing to the expression of MHC class I. Therefore, the balance between import and export of NLRC5 has emerged as an additional regulatory mechanism for MHC class I transactivation, which would be a potential therapeutic target for the treatment of cancer and virus-infected diseases.

Regulation of MHC Class I Expression in Lung Epithelial Cells during Inflammation.

Lung infections are a perennial leading cause of death worldwide. The lung epithelium comprises three main cell types: alveolar type I (AT1), alveolar type II (AT2), and bronchiolar cells. Constitutively, these three cell types express extremely low amounts of surface MHC class I (MHC I) molecules, that is, <1% of levels found on medullary thymic epithelial cells (ECs). We report that inhalation of the TLR4 ligand LPS upregulates cell surface MHC I by ∼25-fold on the three subtypes of mouse lung ECs. This upregulation is dependent on Nlrc5, Stat1, and Stat2 and caused by a concerted production of the three IFN families. It is nevertheless hampered, particularly in AT1 cells, by the limited expression of genes instrumental in the peptide loading of MHC I molecules. Genes involved in production and response to cytokines and chemokines were selectively induced in AT1 cells. However, discrete gene subsets were selectively downregulated in AT2 or bronchiolar cells following LPS inhalation. Genes downregulated in AT2 cells were linked to cell differentiation and cell proliferation, and those repressed in bronchiolar cells were primarily involved in cilium function. Our study shows a delicate balance between the expression of transcripts maintaining lung epithelium integrity and transcripts involved in Ag presentation in primary lung ECs.

IFN-λ Enhances Constitutive Expression of MHC Class I Molecules on Thymic Epithelial Cells.

Regulation of MHC class I (MHC I) expression has been studied almost exclusively in hematolymphoid cells. We report that thymic epithelial cells (TECs), particularly the medullary TECs, constitutively express up to 100-fold more cell surface MHC I proteins than epithelial cells (ECs) from the skin, colon, and lung. Differential abundance of cell surface MHC I in primary ECs is regulated via transcription of MHC I and of genes implicated in the generation of MHC I-binding peptides. Superior MHC I expression in TECs is unaffected by deletion of Ifnar1 or Ifngr1, but is lessened by deletion of Aire, Ifnlr1, Stat1, or Nlrc5, and is driven mainly by type III IFN produced by medullary TECs. Ifnlr1 -/- mice show impaired negative selection of CD8 thymocytes and, at 9 mo of age, present autoimmune manifestations. Our study shows unanticipated variation in MHC I expression by ECs from various sites and provides compelling evidence that superior expression of MHC I in TECs is crucial for proper thymocyte education.

MHC class I transactivator NLRC5 in host immunity, cancer and beyond.

The presentation of antigenic peptides by major histocompatibility complex (MHC) class I and class II molecules is crucial for activation of the adaptive immune system. The nucleotide-binding domain and leucine-rich repeat receptor family members CIITA and NLRC5 function as the major transcriptional activators of MHC class II and class I gene expression, respectively. Since the identification of NLRC5 as the master regulator of MHC class I and class-I-related genes, there have been major advances in understanding the function of NLRC5 in infectious diseases and cancer. Here, we discuss the biological significance and mechanism of NLRC5-dependent MHC class I expression.

Class I transactivator, NLRC5: a central player in the MHC class I pathway and cancer immune surveillance.

Major histocompatibility complex (MHC) class I and class II molecules play critical roles in the activation of the adaptive immune system by presenting antigens to CD8+ and CD4+ T cells, respectively. Although it has been well known that CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, as a master regulator of MHC class II gene expression, the mechanism of MHC class I gene transactivation was unclear. Recently, another NLR protein, NLRC5 (NLR family, CARD domain-containing 5), was identified as an MHC class I transactivator (CITA). NLRC5 is a critical regulator for the transcriptional activation of MHC class I genes and other genes involved in the MHC class I antigen presentation pathway. CITA/NLRC5 plays a crucial role in human cancer immunity through the recruitment and activation of tumor killing CD8+ T cells. Here, we discuss the molecular function and mechanism of CITA/NLRC5 in the MHC class I pathway and its role in cancer.

NLRC5/MHC class I transactivator is a target for immune evasion in cancer.

Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and ß2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.

Microbiota regulates type 1 diabetes through Toll-like receptors.

Deletion of the innate immune adaptor myeloid differentiation primary response gene 88 (MyD88) in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D) results in microbiota-dependent protection from the disease: MyD88-negative mice in germ-free (GF), but not in specific pathogen-free conditions develop the disease. These results could be explained by expansion of particular protective bacteria ("specific lineage hypothesis") or by dominance of negative (tolerizing) signaling over proinflammatory signaling ("balanced signal hypothesis") in mutant mice. Here we found that colonization of GF mice with a variety of intestinal bacteria was capable of reducing T1D in MyD88-negative (but not wild-type NOD mice), favoring the balanced signal hypothesis. However, the receptors and signaling pathways involved in prevention or facilitation of the disease remained unknown. The protective signals triggered by the microbiota were revealed by testing NOD mice lacking MyD88 in combination with knockouts of several critical components of innate immune sensing for development of T1D. Only MyD88- and TIR-domain containing adapter inducing IFN ß (TRIF) double deficient NOD mice developed the disease. Thus, TRIF signaling (likely downstream of Toll-like receptor 4, TLR4) serves as one of the microbiota-induced tolerizing pathways. At the same time another TLR (TLR2) provided prodiabetic signaling by controlling the microbiota, as reduction in T1D incidence caused by TLR2 deletion was reversed in GF TLR2-negative mice. Our results support the balanced signal hypothesis, in which microbes provide signals that both promote and inhibit autoimmunity by signaling through different receptors, including receptors of the TLR family.

TANK-binding kinase 1 (TBK1) controls cell survival through PAI-2/serpinB2 and transglutaminase 2.

The decision between survival and death in cells exposed to TNF relies on a highly regulated equilibrium between proapoptotic and antiapoptotic factors. The TNF-activated antiapoptotic response depends on several transcription factors, including NF-κB and its RelA/p65 subunit, that are activated through phosphorylation-mediated degradation of IκB inhibitors, a process controlled by the IκB kinase complex. Genetic studies in mice have identified the IκB kinase-related kinase TANK-binding kinase 1 (TBK1; also called NAK or T2K) as an additional regulatory molecule that promotes survival downstream of TNF, but the mechanism through which TBK1 exerts its survival function has remained elusive. Here we show that TBK1 triggers an antiapoptotic response by controlling a specific RelA/p65 phosphorylation event. TBK1-induced RelA phosphorylation results in inducible expression of plasminogen activator inhibitor-2 (PAI-2), a member of the serpin family with known antiapoptotic activity. PAI-2 limits caspase-3 activation through stabilization of transglutaminase 2 (TG2), which cross-links and inactivates procaspase-3. Importantly, Tg2(-/-) mice were found to be more susceptible to apoptotic cell death in two models of TNF-dependent acute liver injury. Our results establish PAI-2 and TG2 as downstream mediators in the antiapoptotic response triggered upon TBK1 activation.

Macrophage phenotype controls long-term AKI outcomes--kidney regeneration versus atrophy.

The mechanisms that determine full recovery versus subsequent progressive CKD after AKI are largely unknown. Because macrophages regulate inflammation as well as epithelial recovery, we investigated whether macrophage activation influences AKI outcomes. IL-1 receptor-associated kinase-M (IRAK-M) is a macrophage-specific inhibitor of Toll-like receptor (TLR) and IL-1 receptor signaling that prevents polarization toward a proinflammatory phenotype. In postischemic kidneys of wild-type mice, IRAK-M expression increased for 3 weeks after AKI and declined thereafter. However, genetic depletion of IRAK-M did not affect immunopathology and renal dysfunction during early postischemic AKI. Regarding long-term outcomes, wild-type kidneys regenerated completely within 5 weeks after AKI. In contrast, IRAK-M(-/-) kidneys progressively lost up to two-thirds of their original mass due to tubule loss, leaving atubular glomeruli and interstitial scarring. Moreover, M1 macrophages accumulated in the renal interstitial compartment, coincident with increased expression of proinflammatory cytokines and chemokines. Injection of bacterial CpG DNA induced the same effects in wild-type mice, and TNF-α blockade with etanercept partially prevented renal atrophy in IRAK-M(-/-) mice. These results suggest that IRAK-M induction during the healing phase of AKI supports the resolution of M1 macrophage- and TNF-α-dependent renal inflammation, allowing structural regeneration and functional recovery of the injured kidney. Conversely, IRAK-M loss-of-function mutations or transient exposure to bacterial DNA may drive persistent inflammatory mononuclear phagocyte infiltrates, which impair kidney regeneration and promote CKD. Overall, these results support a novel role for IRAK-M in the regulation of wound healing and tissue regeneration.

Regulation of intestinal microbiota by the NLR protein family.

The human intestine harbors a diverse microbial community consisting of a large number of bacteria and other micro-organisms that have co-evolved with the host intestinal immune system. During this process, microbiota and the host immune system shape one another by various mechanisms to achieve a successful symbiotic relationship. An increasing amount of evidence suggests that dysbiosis--the breakdown of such harmonized colonization--may result in infectious and inflammatory disorders, and recent advances in our studies indicate that receptors such as Toll-like receptors and NLR (nucleotide-binding oligomerization domain-like receptor; or nucleotide-binding domain- and leucine-rich repeat-containing receptor) proteins that detect micro-organisms and their products play a critical role in maintaining intestinal homeostasis. In this review, we summarize the role of NLR proteins in the regulation of intestinal microbiota. NLR proteins belong to a diverse family of cytoplasmic microbial sensors, mutations of which are involved in various disorders, including inflammatory bowel diseases. Understanding of the different roles of NLR family proteins in the intestine is, therefore, an important step towards the development of therapeutics against digestive diseases.

Cutting edge: impaired MHC class I expression in mice deficient for Nlrc5/class I transactivator.

MHC class I and class II are crucial for the adaptive immune system. Although regulation of MHC class II expression by CIITA has long been recognized, the mechanism of MHC class I transactivation has been largely unknown until the recent discovery of NLRC5/class I transactivator. In this study, we show using Nlrc5-deficient mice that NLRC5 is required for both constitutive and inducible MHC class I expression. Loss of Nlrc5 resulted in severe reduction in the expression of MHC class I and related genes such as ß(2)-microglobulin, Tap1, or Lmp2, but did not affect MHC class II levels. IFN-γ stimulation could not overcome the impaired MHC class I expression in Nlrc5-deficient cells. Upon infection with Listeria monocyogenes, Nlrc5-deficient mice displayed impaired CD8(+) T cell activation, accompanied with increased bacterial loads. These findings illustrate critical roles of NLRC5/class I transactivator in MHC class I gene regulation and host defense by CD8(+) T cell responses.

NLRC5 cooperates with the RFX transcription factor complex to induce MHC class I gene expression.

Tight regulation of MHC class I gene expression is critical for CD8 T cell activation and host adaptive-immune responses. The promoters of MHC class I genes contain a well-conserved core module, the W/S-X-Y motif, which assembles a nucleoprotein complex termed MHC enhanceosome. A member of the nucleotide-binding domain, leucine-rich repeat (NLR) protein family, NLRC5, is a newly identified transcriptional regulator of MHC class I genes. NLRC5 associates with and transactivates the proximal promoters of MHC class I genes, although the molecular mechanism of transactivation has not been understood. In this article, we show that NLRC5-mediated MHC class I gene induction requires the W/S and X1, X2 cis-regulatory elements. The transcription factors RFX5, RFXAP, and RFXANK/B, which compose the RFX protein complex and associate with the X1 box, cooperate with NLRC5 for MHC class I expression. Coimmunoprecipitation experiments revealed that NLRC5 specifically interacts with the RFX subunit RFXANK/B via its ankyrin repeats. In addition, we show that NLRC5 can cooperate with ATF1 and the transcriptional coactivators CBP/p300 and general control nonderepressible 5, which display histone acetyltransferase activity. Taken together, our data suggest that NLRC5 participates in an MHC class I-specific enhanceosome, which assembles on the conserved W/S-X-Y core module of the MHC class I proximal promoters, including the RFX factor components and CREB/ATF1 family transcription factors, to promote MHC class I gene expression.

Epigenetic regulation of major histocompatibility complexes in gastrointestinal malignancies and the potential for clinical interception.

BACKGROUND: Gastrointestinal malignancies encompass a diverse group of cancers that pose significant challenges to global health. The major histocompatibility complex (MHC) plays a pivotal role in immune surveillance, orchestrating the recognition and elimination of tumor cells by the immune system. However, the intricate regulation of MHC gene expression is susceptible to dynamic epigenetic modification, which can influence functionality and pathological outcomes. MAIN BODY: By understanding the epigenetic alterations that drive MHC downregulation, insights are gained into the molecular mechanisms underlying immune escape, tumor progression, and immunotherapy resistance. This systematic review examines the current literature on epigenetic mechanisms that contribute to MHC deregulation in esophageal, gastric, pancreatic, hepatic and colorectal malignancies. Potential clinical implications are discussed of targeting aberrant epigenetic modifications to restore MHC expression and 0 the effectiveness of immunotherapeutic interventions. CONCLUSION: The integration of epigenetic-targeted therapies with immunotherapies holds great potential for improving clinical outcomes in patients with gastrointestinal malignancies and represents a compelling avenue for future research and therapeutic development.

Brucella-driven host N-glycome remodeling controls infection.

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

Proteasomal degradation of Nod2 protein mediates tolerance to bacterial cell wall components.

The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock.