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Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

Twenty-six patients with rheumatoid disease affecting the cervical spine underwent surgical treatment for neck pain, neurological deficit, or both. Atlantoaxial subluxation (n = 13), subaxial subluxation (n = 7) and vertical migration of the odontoid (n = 6) were treated. Arthrodesis with autologous bone was augmented with wire, Ransford loop, Hartshill rectangle or Magerl technique. Pain relief occurred in 92% of patients. Neurological deficit improved in 89% and was unchanged in the remainder. Radiographic stability was achieved in all but one patient. Posterior surgery effectively relieved pain and neurological deficit, and the complications encountered did not jeopardize the outcome. [ABSTRACT FROM PUBLISHER]

Programmed death receptor 1 (PD-1) is an inhibitory receptor on T cells shown to restrain T-cell proliferation. PD-1 immune checkpoint blockade has emerged as a highly promising approach in cancer treatment. Much of our understanding of the function of PD-1 is derived from in vitro T-cell activation assays. Here we set out to further investigate how T cells integrate inhibitory signals such as PD-1 in vitro using the PD-1 agonist, PD-1 ligand 1 (PD-L1) fusion protein (PD-L1.Fc), coimmobilized alongside anti-CD3 agonist monoclonal antibody (mAb) on plates to deliver PD-1 signals to wild-type and PD-1 -/- CD8 + T cells. Surprisingly, we found that the PD-L1.Fc fusion protein inhibited T-cell proliferation independently of PD-1. This PD-L1.Fc inhibition was observed in the presence and absence of CD28 and interleukin-2 signaling. Binding of PD-L1.Fc was restricted to PD-1-expressing T cells and thus inhibition was not mediated by the interaction of PD-L1.Fc with CD80 or other yet unknown binding partners. Furthermore, a similar PD-1-independent reduction of T-cell proliferation was observed with plate-bound PD-L2.Fc. Hence, our results suggest that the coimmobilization of PD-1 ligand fusion proteins with anti-CD3 mAb leads to a reduction of T-cell engagement with plate-bound anti-CD3 mAb. This study demonstrates a nonspecific mechanism of T-cell inhibition when PD-L1.Fc or PD-L2.Fc fusion proteins are delivered in a plate-bound coimmobilization assay and highlights the importance of careful optimization of assay systems and reagents when interpreting their influence on T-cell proliferation., (© 2023 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

Mucosal-associated invariant T (MAIT) cells, natural killer T (NKT) cells, and γδT cells are collectively referred to as 'unconventional T cells' due to their recognition of non-peptide antigens and restriction to MHC-I-like molecules. However, the factors controlling their widely variable frequencies between individuals and organs are poorly understood. We demonstrated that MAIT cells are increased in NKT or γδT cell-deficient mice and highly expand in mice lacking both cell types. TCRα repertoire analysis of γδT cell-deficient thymocytes revealed altered Trav segment usage relative to wild-type thymocytes, highlighting retention of the Tcra-Tcrd locus from the 129 mouse strain used to generate Tcrd -/- mice. This resulted in a moderate increase in distal Trav segment usage, including Trav1, potentially contributing to increased generation of Trav1-Traj33 + MAIT cells in the Tcrd -/- thymus. Importantly, adoptively transferred MAIT cells underwent increased homeostatic proliferation within NKT/gdT cell-deficient tissues, with MAIT cell subsets exhibiting tissue-specific homing patterns. Our data reveal a shared niche for unconventional T cells, where competition for common factors may be exploited to collectively modulate these cells in the immune response. Lastly, our findings emphasise careful assessment of studies using NKT or γδT cell-deficient mice when investigating the role of unconventional T cells in disease., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between in vitro and in vivo gene integration rates. Homology-directed repair occurs primarily in the S/G 2 phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton's tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G 1 resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

X-linked agammaglobulinemia (XLA) is a monogenic primary immune deficiency characterized by very low levels of immunoglobulins and greatly increased risks for recurrent and severe infections. Patients with XLA have a loss-of-function mutation in the Bruton's tyrosine kinase ( BTK ) gene and fail to produce mature B lymphocytes. Gene editing in the hematopoietic stem cells of XLA patients to correct or replace the defective gene should restore B cell development and the humoral immune response. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform to precisely target integration of a corrective, codon-optimized BTK complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic BTK under transcriptional control of its endogenous regulatory elements. Each integrated copy of this cDNA in BTK-deficient K562 cells produced only 11% as much BTK protein as the wild-type gene. The donor cDNA was modified to include the terminal intron of the BTK gene. Successful integration of the intron-containing BTK donor led to a nearly twofold increase in BTK expression per cell over the base donor. However, this donor variant was too large to package into an adeno-associated viral vector for delivery into primary cells. Donors containing truncated variants of the terminal intron also produced elevated expression, although to a lesser degree than the full intron. Addition of the Woodchuck hepatitis virus posttranscriptional regulatory element led to a large boost in BTK transgene expression. Combining these modifications led to a BTK donor template that generated nearly physiological levels of BTK expression in cell lines. These reagents were then optimized to maximize integration rates into human hematopoietic stem and progenitor cells, which have reached potentially therapeutic levels in vitro . The novel donor modifications support effective gene therapy for XLA and will likely assist in the development of other gene editing-based therapies for genetic disorders.

Mass cytometry (CyTOF) is a technology that has revolutionised single-cell biology. By detecting over 40 proteins on millions of single cells, CyTOF allows the characterisation of cell subpopulations in unprecedented detail. However, most CyTOF studies require the integration of data from multiple CyTOF batches usually acquired on different days and possibly at different sites. To date, the integration of CyTOF datasets remains a challenge due to technical differences arising in multiple batches. To overcome this limitation, we developed an approach called CytofRUV for analysing multiple CyTOF batches, which includes an R-Shiny application with diagnostic plots. CytofRUV can correct for batch effects and integrate data from large numbers of patients and conditions across batches, to confidently compare cellular changes and correlate these with clinically relevant outcomes., Competing Interests: MT, CT, TT, LL, DG, TS No competing interests declared, (© 2020, Trussart et al.)

Apoptosis-regulating BCL-2 family members, which can promote malignant transformation and resistance to therapy, have become prime therapeutic targets, as illustrated by the striking efficacy in certain lymphoid malignancies of the BCL-2-specific inhibitor venetoclax. In other lymphoid malignancies, however, such as the aggressive mantle cell lymphoma (MCL), cell survival might rely instead or also on BCL-2 relative MCL-1. We have explored MCL-1 as a target for killing MCL cells by both genetic and pharmacologic approaches. In several MCL cell lines, MCL-1 knockout with an inducible CRISPR/Cas9 system triggered spontaneous apoptosis. Accordingly, most MCL cell lines proved sensitive to the specific MCL-1 inhibitor S63845, and MCL-1 inhibition also proved efficacious in an MCL xenograft model. Furthermore, its killing efficacy rose on combination with venetoclax, the BCL-X L -specific inhibitor A-1331852, or Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which reduced pro-survival signals. We also tested the MCL-1 inhibitor in primary samples from 13 MCL patients, using CD40L-expressing feeder cells to model their microenvironmental support. Notably, all unstimulated primary MCL samples were very sensitive to S63845, but the CD40L stimulation attenuated their sensitivity. Mass cytometric analysis revealed that the stimulation likely conveyed protection by elevating BCL-X L and MCL-1. Accordingly, sensitivity of the CD40L-stimulated cells to S63845 was substantially restored by co-treatment with venetoclax, the BCL-X L -specific inhibitor or ibrutinib. Overall, our findings indicate that MCL-1 is very important for survival of MCL cells and that the MCL-1 inhibitor, both alone and together with ibrutinib, venetoclax or a BCL-X L inhibitor, offers promise for novel improved MCL therapies.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize antigens derived from riboflavin biosynthesis. In addition to anti-microbial functions, human MAIT cells are associated with cancers, autoimmunity, allergies and inflammatory disorders, although their role is poorly understood. Activated MAIT cells are well known for their rapid release of Th1 and Th17 cytokines, but we have discovered that chronic stimulation can also lead to potent interleukin (IL)-13 expression. We used RNA-seq and qRT-PCR to demonstrate high expression of the IL-13 gene in chronically stimulated MAIT cells, and directly identify IL-13 using intracellular flow cytometry and multiplex bead analysis of MAIT cell cultures. This unexpected finding has important implications for IL-13-dependent diseases, such as colorectal cancer (CRC), that occur in mucosal areas where MAIT cells are abundant. We identify MAIT cells near CRC tumors and show that these areas and precancerous polyps express high levels of the IL-13 receptor, which promotes tumor progression and metastasis. Our data suggest that MAIT cells have a more complicated role in CRC than currently realized and that they represent a promising new target for immunotherapies where IL-13 can be a critical factor., (© 2019 The Authors Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

FOXP3 + regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1 + (NRP1 + ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1 - Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4 + T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein., (© 2019 Australian and New Zealand Society for Immunology Inc.)

Regulatory T (T reg ) cells maintain immunological tolerance in steady-state and after immune challenge. Activated T reg cells can undergo further differentiation into an effector state that highly express genes critical for T reg cell function, including ICOS, TIGIT and IL-10, although how this process is controlled is poorly understood. Effector T reg cells also specifically express the transcriptional regulator Blimp-1 whose expression overlaps with many of the canonical markers associated with effector T reg cells, although not all ICOS + TIGIT + T reg cells express Blimp-1 or IL-10. In this study, we addressed the role of Blimp-1 in effector T reg cell function. Mice lacking Blimp-1 specifically in T reg cells mature normally, but succumb to a multi-organ inflammatory disease later in life. Blimp-1 is not required for T reg cell differentiation, with mutant mice having increased numbers of effector T reg cells, but regulated a suite of genes involved in cell signaling, communication and survival, as well as being essential for the expression of the immune modulatory cytokine IL-10. Thus, Blimp-1 is a marker of effector T reg cells in all contexts examined and is required for the full functionality of these cells during aging., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The lung presents a highly oxidative environment, which is tolerated through engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor nuclear factor erythroid-2-related factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major cancer driver. We demonstrate that inactivation of Keap1 and Pten in the mouse lung promotes adenocarcinoma formation. Notably, metabolites identified in the plasma of Keap1 f/f /Pten f/f tumor-bearing mice indicate that tumorigenesis is associated with reprogramming of the pentose phosphate pathway. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumor regression was achieved utilizing immune checkpoint inhibition. Thus, our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung tumors harboring KEAP1/NRF2 pathway alterations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Targeting survival mechanisms of immune cells may provide an avenue for immune intervention to dampen unwanted responses (e.g. autoimmunity, immunopathology and transplant rejection) or enhance beneficial ones (e.g. immune deficiency, microbial defence and cancer immunotherapy). The selective survival mechanisms of the various immune cell types also avails the possibility of specific tailoring of such interventions. Here, we review the role of the BCL-2 anti-apoptotic family members (BCL-2, BCL-XL, BCL-W, MCL-1 and A1) on cell death/survival of the major immune cell types, for example, T, NK, B, dendritic cell (DC) lineages. There is both selectivity and redundancy among this family. Selectivity comes partly from the expression levels in each of the cell types. For example, plasmacytoid DC express abundant BCL-2 and are susceptible to BCL-2 antagonism or deficiency, whereas conventional DC express abundant A1 and are susceptible to A1 deficiency. There is, however, also functional redundancy; for example, overexpression of MCL-1 can override BCL-2 antagonism in plasmacytoid DC. Moreover, susceptibility to another anti-apoptotic family member can be unmasked, when one or other member is removed. These dual principles of selectivity and redundancy should guide the use of antagonists for manipulating immune cells.

Natural killer cells constitute potent innate lymphoid cells that play a major role in both tumor immunosurveillance and viral clearance via their effector functions. A four-stage model of NK cell functional maturation has been established according to the expression of CD11b and CD27, separating mature NK (mNK) cells into distinct populations that exhibit specific phenotypic and functional properties. To identify genetic factors involved in the regulation of NK cell functional maturation, we performed a linkage analysis on F 2 (B6.Rag1 -/- × NOD.Rag1 -/- intercross) mice. We identified six loci on chromosomes 2, 4, 7, 10, 11, and 18 that were linked to one or more mNK cell subsets. Subsequently, we performed an in silico analysis exploiting mNK cell subset microarray data, highlighting various genes and microRNAs as potential regulators of the functional maturation of NK cells. Together, the combination of our unbiased genetic linkage study and the in silico analysis positions genes known to affect NK cell biology along the specific stages of NK cell functional maturation. Moreover, this approach allowed us to uncover a novel candidate gene in the regulation of NK cell maturation, namely Trp53 Using mice deficient for Trp53 , we confirm that this tumor suppressor regulates NK cell functional maturation. Additional candidate genes revealed in this study may eventually serve as targets for the modulation of NK cell functional maturation to potentiate both tumor immunosurveillance and viral clearance., (Copyright © 2017 by The American Association of Immunologists, Inc.)

The pro-survival proteins of the BCL-2 family regulate the survival of all cells, and genetic deletion models for these proteins have revealed which specific BCL-2 family member(s) is/are critical for the survival of particular cell types. A1 is a pro-survival BCL-2-like protein that is expressed predominantly in haematopoietic cells, and here we describe the characterisation of a novel mouse strain that lacks all three functional isoforms of A1 (A1-a, A1-b and A1-d). Surprisingly, complete loss of A1 caused only minor defects, with significant, although relatively small, decreases in γδTCR T cells, antigen-experienced conventional as well as regulatory CD4 T cells and conventional dendritic cells (cDCs). When examining these cell types in tissue culture, only cDC survival was significantly impaired by the loss of A1. Therefore, A1 appears to be a surprisingly redundant pro-survival protein in the haematopoietic system and other tissues, suggesting that its targeting in cancer may be readily tolerated.

The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3 + regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.

Major histocompatibility complex class II (MHC II) expression is tightly regulated, being subjected to cell type-specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHC II expression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHC II in thymic epithelial cells (TECs). March8(-/-) mice have elevated MHC II at the surface of cortical TECs and autoimmune regulator (AIRE)(-) medullary TECs (mTECs), but not AIRE(+) mTECs. Despite this, thymic and splenic CD4(+) T cell numbers and repertoires remained unaltered in March8(-/-) mice. Notably, the ubiquitination of MHC II by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83(anu/anu) mice) have impaired CD4(+) T cell selection, but deleting March8 in Cd83(anu/anu) mice restored CD4(+) T cell selection to normal levels. Therefore, orchestrated regulation of MHC II surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4(+) T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation., (© 2016 Liu et al.)

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak., (© 2016 Lee et al.; Published by Cold Spring Harbor Laboratory Press.)

γδ T cells respond to molecules upregulated following infection or cellular stress using both TCR and non-TCR molecules. The importance of innate signals versus TCR ligation varies greatly. Both innate-like IL-17-producing γδ T (γδT-17) and IFN-γ-producing γδ T (γδT-IFNγ) subsets tune the sensitivity of their TCR following thymic development, allowing robust responses to inflammatory cytokines in the periphery. The remaining conventional γδ T cells retain high TCR responsiveness. We determined homeostatic mechanisms that govern these various subsets in the peripheral lymphoid tissues. We found that, although innate-like γδT-17 and γδT-IFNγ cells share elements of thymic development, they diverge when it comes to homeostasis. Both exhibit acute sensitivity to cytokines compared with conventional γδ T cells, but they do not monopolize the same cytokine. γδT-17 cells rely exclusively on IL-7 for turnover and survival, aligning them with NKT17 cells; IL-7 ligation triggers proliferation, as well as promotes survival, upregulating Bcl-2 and Bcl-xL. γδT-IFNγ cells instead depend heavily on IL-15. They display traits analogous to memory CD8(+) T cells and upregulate Bcl-xL and Mcl-1 upon cytokine stimulation. The conventional γδ T cells display low sensitivity to cytokine-alone stimulation and favor IL-7 for their turnover, characteristics reminiscent of naive αβ T cells, suggesting that they may also require tonic TCR signaling for population maintenance. These survival constraints suggest that γδ T cell subsets do not directly compete with each other for cytokines, but instead fall into resource niches with other functionally similar lymphocytes., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Defects in apoptosis can cause autoimmune disease. Loss-of-function mutations in the 'death receptor' FAS impair the deletion of autoreactive lymphocytes in the periphery, leading to progressive lymphadenopathy and systemic lupus erythematosus-like autoimmune disease in mice (Fas(lpr/lpr) (mice homozygous for the lymphoproliferation inducing spontaneous mutation)) and humans. The REL/nuclear factor-κB (NF-κB) transcription factors regulate a broad range of immune effector functions and are also implicated in various autoimmune diseases. We generated compound mutant mice to investigate the individual functions of the NF-κB family members NF-κB1, NF-κB2 and c-REL in the various autoimmune pathologies of Fas(lpr/lpr) mutant mice. We show that loss of each of these transcription factors resulted in amelioration of many classical features of autoimmune disease, including hypergammaglobulinaemia, anti-nuclear autoantibodies and autoantibodies against tissue-specific antigens. Remarkably, only c-REL deficiency substantially reduced immune complex-mediated glomerulonephritis and extended the lifespan of Fas(lpr/lpr) mice. Interestingly, compared with the Fas(lpr/lpr) animals, Fas(lpr/lpr)nfkb2(-/-) mice presented with a dramatic acceleration and augmentation of lymphadenopathy that was accompanied by severe lung pathology due to extensive lymphocytic infiltration. The Fas(lpr/lpr)nfkb1(-/-) mice exhibited the combined pathologies caused by defects in FAS-mediated apoptosis and premature ageing due to loss of NF-κB1. These findings demonstrate that different NF-κB family members exert distinct roles in the development of the diverse autoimmune and lymphoproliferative pathologies that arise in Fas(lpr/lpr) mice, and suggest that pharmacological targeting of c-REL should be considered as a strategy for therapeutic intervention in autoimmune diseases.

FASL/FAS signaling imposes a critical barrier against autoimmune disease and lymphadenopathy. Mutant mice unable to produce membrane-bound FASL (FasL(Δm/Δm)), a prerequisite for FAS-induced apoptosis, develop lymphadenopathy and systemic autoimmune disease with immune complex-mediated glomerulonephritis. Prior to disease onset, FasL(Δm/Δm) mice contain abnormally high numbers of leukocytes displaying activated and elevated NF-κB-regulated cytokine levels, indicating that NF-κB-dependent inflammation may be a key pathological driver in this multifaceted autoimmune disease. We tested this hypothesis by genetically impairing canonical or non-canonical NF-κB signaling in FasL(Δm/Δm) mice by deleting the c-Rel or NF-κB2 genes, respectively. Although the loss of NF-κB2 reduced the levels of inflammatory cytokines and autoantibodies, the impact on animal survival was minor due to substantially accelerated and exacerbated lymphoproliferative disease. In contrast, a marked increase in lifespan resulting from the loss of c-REL coincided with a striking reduction in classical parameters of autoimmune pathology, including the levels of cytokines and antinuclear autoantibodies. Notably, the decrease in regulatory T-cell numbers associated with loss of c-REL did not exacerbate autoimmunity in FasL(Δm/Δm)c-rel(-/-) mice. These findings indicate that selective inhibition of c-REL may be an attractive strategy for the treatment of autoimmune pathologies driven by defects in FASL/FAS signaling that would be expected to circumvent many of the complications caused by pan-NF-κB inhibition.

FoxP3(+) regulatory T (Treg) cells comprise a highly dynamic population that restrains autoreactivity. Although complete or long-term depletion of Foxp3(+) CD4(+) Treg cells in adult mice has been shown to result in chronic inflammation and autoimmune disease, the impact of transient Treg-cell depletion on self-reactive responses is poorly defined. A new study published in this issue of the European Journal of Immunology [Eur. J. Immunol. 2014. 44: 3621-3631] shows that, although transient depletion of Treg cells in mice is swiftly followed by recovery of Treg-cell numbers, the "rebounded" population fails to maintain tolerance, culminating in severe autoimmune gastritis. This commentary explores new questions about the quantitative and qualitative aspects of Treg-cell function in immunological tolerance raised by this study and others., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Thymic epithelial cells (TECs) are critical for T cell development and self-tolerance but are gradually lost with age. The existence of thymic epithelial progenitors (TEPCs) in the postnatal thymus has been inferred, but their identity has remained enigmatic. Here, we assessed the entire adult TEC compartment in order to reveal progenitor capacity is retained exclusively within a subset of immature thymic epithelium displaying several hallmark features of stem/progenitor function. These adult TEPCs generate mature cortical and medullary lineages in a stepwise fashion, including Aire+ TEC, within fetal thymus reaggregate grafts. Although relatively quiescent in vivo, adult TEPCs demonstrate significant in vitro colony formation and self-renewal. Importantly, 3D-cultured TEPCs retain their capacity to differentiate into cortical and medullary TEC lineages when returned to an in vivo thymic microenvironment. No other postnatal TEC subset exhibits this combination of properties. The characterization of adult TEPC will enable progress in understanding TEC biology in aging and regeneration., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Regulatory T (TReg) cells constitute an essential counterbalance to adaptive immune responses. Failure to maintain appropriate TReg cell numbers or function leads to autoimmune, malignant and immunodeficient conditions. Dynamic homeostatic processes preserve the number of forkhead box P3-expressing (FOXP3(+)) TReg cells within a healthy range, with high rates of cell division being offset by apoptosis under steady-state conditions. Recent studies have shown that TReg cells become specialized for different environmental contexts, tailoring their functions and homeostatic properties to a wide range of tissues and immune conditions. In this Review, we describe new insights into the molecular controls that maintain the steady-state homeostasis of TReg cells and the cues that drive TReg cell adaptation to inflammation and/or different locations. We highlight how differing local milieu might drive context-specific TReg cell function and restoration of immune homeostasis, and how dysregulation of these processes can precipitate disease.

Foxp3⁺ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⁺ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

Activation of apoptosis through transcriptional induction of Puma and Noxa has long been considered to constitute the critical (if not sole) process by which p53 suppresses tumor development, although G1/S boundary cell-cycle arrest via induction of the CDK inhibitor p21 has also been thought to contribute. Recent analyses of mice bearing mutations that impair p53-mediated induction of select target genes have indicated that activation of apoptosis and G1/S cell-cycle arrest may, in fact, be dispensable for p53-mediated tumor suppression. However, the expression of Puma, Noxa, and p21 was not abrogated in these mutants, only reduced; therefore, the possibility that the reduced levels of these critical effectors of p53-mediated apoptosis and G1/S-cell-cycle arrest sufficed to prevent tumorigenesis could not be excluded. To resolve this important issue, we have generated mice deficient for p21, Puma, and Noxa (p21-/-puma-/-noxa-/- mice). Cells from these mice were deficient in their ability to undergo p53-mediated apoptosis, G1/S cell-cycle arrest, and senescence. Nonetheless, these animals remained tumor free until at least 500 days, in contrast to p53-deficient mice, which had all succumbed to lymphoma or sarcoma by 250 days. Interestingly, DNA lesions induced by γ-irradiation persisted longer in p53-deficient cells compared to wild-type or p21-/-puma-/-noxa-/- cells, and the former failed to transcriptionally activate several p53 target genes implicated in DNA repair. These results demonstrate beyond a doubt that the induction of apoptosis, cell-cycle arrest, and possibly senescence is dispensable for p53-mediated suppression of spontaneous tumor development and indicate that coordination of genomic stability and possibly other processes, such as metabolic adaptation, may instead be critical., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Dysregulation of the "intrinsic" apoptotic pathway is associated with the development of cancer and autoimmune disease. Bak and Bax are two proapoptotic members of the Bcl-2 protein family with overlapping, essential roles in the intrinsic apoptotic pathway. Their activity is critical for the control of cell survival during lymphocyte development and homeostasis, best demonstrated by defects in thymic T-cell differentiation and peripheral lymphoid homeostasis caused by their combined loss. Because most bak(-/-)bax(-/-) mice die perinatally, the roles of Bax and Bak in immunological tolerance and prevention of autoimmune disease remain unclear. We show that mice reconstituted with a Bak/Bax doubly deficient hematopoietic compartment develop a fatal systemic lupus erythematosus-like autoimmune disease characterized by hypergammaglobulinemia, autoantibodies, lymphadenopathy, glomerulonephritis, and vasculitis. Importantly, these mice also develop a multiorgan autoimmune disease with autoantibodies against most solid glandular structures and evidence of glandular atrophy and necrotizing vasculitis. Interestingly, similar albeit less severe pathology was observed in mice containing a hematopoietic compartment deficient for only Bak, a phenotype reminiscent of the disease seen in patients with point mutations in BAK. These studies demonstrate a critical role for Bak and an ancillary role for Bax in safeguarding immunological tolerance and prevention of autoimmune disease. This suggests that direct activators of the intrinsic apoptotic pathway, such as BH3 mimetics, may be useful for treatment of diverse autoimmune diseases.