Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.

Single Cell Transcriptomics Implicate Novel Monocyte and T Cell Immune Dysregulation in Sarcoidosis.

Sarcoidosis is a systemic inflammatory disease characterized by infiltration of immune cells into granulomas. Previous gene expression studies using heterogeneous cell mixtures lack insight into cell-type-specific immune dysregulation. We performed the first single-cell RNA-sequencing study of sarcoidosis in peripheral immune cells in 48 patients and controls. Following unbiased clustering, differentially expressed genes were identified for 18 cell types and bioinformatically assessed for function and pathway enrichment. Our results reveal persistent activation of circulating classical monocytes with subsequent upregulation of trafficking molecules. Specifically, classical monocytes upregulated distinct markers of activation including adhesion molecules, pattern recognition receptors, and chemokine receptors, as well as enrichment of immunoregulatory pathways HMGB1, mTOR, and ephrin receptor signaling. Predictive modeling implicated TGFß and mTOR signaling as drivers of persistent monocyte activation. Additionally, sarcoidosis T cell subsets displayed patterns of dysregulation. CD4 naïve T cells were enriched for markers of apoptosis and Th17/Treg differentiation, while effector T cells showed enrichment of anergy-related pathways. Differentially expressed genes in regulatory T cells suggested dysfunctional p53, cell death, and TNFR2 signaling. Using more sensitive technology and more precise units of measure, we identify cell-type specific, novel inflammatory and regulatory pathways. Based on our findings, we suggest a novel model involving four convergent arms of dysregulation: persistent hyperactivation of innate and adaptive immunity via classical monocytes and CD4 naïve T cells, regulatory T cell dysfunction, and effector T cell anergy. We further our understanding of the immunopathology of sarcoidosis and point to novel therapeutic targets.

Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.

The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here, we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.

T-Cell Molecular Modulation Responses in Atherosclerosis Anergy.

Atherosclerosis continues to be a major cause of death in patients with cardiovascular diseases. The cooperative role of immunity has been recently considered in atherosclerotic plaque inflammation, especially adaptive immune response by T cells. In this review, we examine the possible role of T cells in atherosclerosis-mediated inflammation and conceivable therapeutic strategies that can ameliorate complications of atherosclerosis. The cytokines secreted by T-lymphocyte subsets, different pathophysiological profiles of microRNAs (miRs), and the growth factor/receptor axis have diverse effects on the inflammatory cycle of atherosclerosis. Manipulation of miRNA expression and prominent growth factor receptors involved in inflammatory cytokine secretion in atherosclerosis can be considered diagnostic biomarkers in the induction of anergy and blockade of atherosclerotic development. This manuscript reviews immunomodulation of T cells responses in atherosclerosis anergy.

Pan-Cancer Analysis Reveals Disrupted Circadian Clock Associates With T Cell Exhaustion.

Although dysfunctional circadian clock has emerged as a hallmark of cancer, fundamental gaps remain in our understanding of the underlying mechanisms involved. Here, we systematically analyze the core genes of the circadian clock (CLOCK, ARNTL, ARNTL2, NPAS2, NR1D1, NR1D2, CRY1, CRY2, RORA, RORB, RORC, PER1, PER2, and PER3) across a broad range of cancers. To our surprise, core negative regulators (PER1, PER2, PER3, CRY1, and CRY2) are consistently downregulated, while core positive regulators show minimal alterations, indicating disrupted circadian clock in cancers. Such downregulation originates from copy number variations where heterozygous deletion predominates. The disrupted circadian clock is significantly associated with patient outcome. Further pathway enrichment analysis suggests that the circadian clock widely impacts 45 pathways such as the Ras signaling pathway and T cell receptor signaling pathway. By using state-of-the-art immune cell deconvolution and pathway quantification, we demonstrate that abnormal circadian clock contributes to T cell exhaustion and global upregulation of immune inhibitory molecules such as PD-L1 and CTLA-4. In summary, the rhythm of the circadian clock is disrupted in cancers. Abnormal circadian clock linked with immune evasion may serve as a potential hallmark of cancer.

Exhausted-like Group 2 Innate Lymphoid Cells in Chronic Allergic Inflammation.

Mammalian group 2 innate lymphoid cells (ILC2s) are responsible for the early production of type 2 cytokines at mucosal barriers upon exposure to allergen. Inflammatory tissue environmental cues can influence ILC2 activity, and this cellular population can be further categorized into subtypes with additional or alternative functions. Subtypes can include trained (or 'memory-like') ILC2s, which recall previous allergic inflammation, inflammatory ILC2s, which acquire the ability to produce IL-17, and ex-ILC2s, which produce ILC1 cytokines. However, the functional states of ILC2s at sites of chronic or severe inflammation are not well characterized. Here, we discuss the emergence of ILC2s with 'exhausted'-like signatures, and argue that their hyporesponsiveness to stimulation and expression of inhibitory receptors is relevant in mammalian chronic allergic inflammation.

Ikaros prevents autoimmunity by controlling anergy and Toll-like receptor signaling in B cells.

The establishment of a diverse B cell antigen receptor (BCR) repertoire by V(D)J recombination also generates autoreactive B cells. Anergy is one tolerance mechanism; it renders autoreactive B cells insensitive to stimulation by self-antigen, whereas Toll-like receptor (TLR) signaling can reactivate anergic B cells. Here, we describe a critical role of the transcription factor Ikaros in controlling BCR anergy and TLR signaling. Mice with specific deletion of Ikaros in mature B cells developed systemic autoimmunity. Ikaros regulated many anergy-associated genes, including Zfp318, which is implicated in the attenuation of BCR responsiveness by promoting immunoglobulin D expression in anergic B cells. TLR signaling was hyperactive in Ikaros-deficient B cells, which failed to upregulate feedback inhibitors of the MyD88-nuclear factor κB signaling pathway. Systemic inflammation was lost on expression of a non-self-reactive BCR or loss of MyD88 in Ikaros-deficient B cells. Thus, Ikaros acts as a guardian preventing autoimmunity by promoting BCR anergy and restraining TLR signaling.

Biology and therapy of primary mediastinal B-cell lymphoma: current status and future directions.

Primary mediastinal B-cell lymphoma (PMBCL) is a distinct disease closely related to classical nodular sclerosing Hodgkin lymphoma. Conventional diagnostic paradigms utilising clinical, morphological and immunophenotypical features can be challenging due to overlapping features with other B-cell lymphomas. Reliable diagnostic and prognostic biomarkers that are applicable to the conventional diagnostic laboratory are largely lacking. Nuclear factor kappa B (NF-κB) and Janus kinase/signal transducers and activators of transcription (JAK-STAT) signalling pathways are characteristically dysregulated in PMBCL and implicated in several aspects of disease pathogenesis, and the latter pathway in host immune evasion. The tumour microenvironment is manipulated by PMBCL tumours to avoid T-cell mediated destruction via strategies that include loss of tumour cell antigenicity, T-cell exhaustion and activation of suppressive T-regulatory cells. R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) and DA-EPOCH-R (dose-adjusted etoposide, prednisolone, vincristine, cyclophosphamide, doxorubicin, rituximab) are the most common first-line immunochemotherapy regimens. End of treatment positron emission tomography scans are the recommended imaging modality and are being evaluated to stratify patients for radiotherapy. Relapsed/refractory disease has a relatively poor outcome despite salvage immunochemotherapy and subsequent autologous stem cell transplantation. Novel therapies are therefore being developed for treatment-resistant disease, targeting aberrant cellular signalling and immune evasion.

Natural killer cells target and differentiate cancer stem-like cells/undifferentiated tumors: strategies to optimize their growth and expansion for effective cancer immunotherapy.

Natural killer (NK) cells are known to select and differentiate cancer stem-like cells/undifferentiated tumors via lysis, and secreted/membrane bound IFN-γ and TNF-α respectively, resulting in the control of tumor growth. Several in vivo mouse models including humanized-BLT mice have been used to study the biology and significance of NK cells in selection/differentiation of stem-like tumors within the context of a reconstituted human immune system. In addition, we discuss the evidence and significance of NK cell loss at the pre-neoplastic stage. Therefore, because of their indispensable role in targeting CSCs/undifferentiated tumors, NK-cells should be placed high in the armamentarium of tumor therapy.

PD-1-PD-L1 immune-checkpoint blockade in malignant lymphomas.

Tumor cells can evade immune surveillance through overexpressing the ligands of checkpoint receptors on tumor cells or adjacent cells, leading T cells to anergy or exhaustion. Growing evidence of the interaction between tumor cells and microenvironment promoted the emergence of immune-checkpoint blockade. By targeting programmed cell death-1 (PD-1) pathway, cytotoxic activity of T cell is enhanced significantly and tumor cell lysis is induced subsequently. Currently, various antibodies against PD-1 and programmed death-ligand 1 (PD-L1) are under clinical studies in lymphomas. In this review, we outline the rationale for investigation of PD-1-PD-L1 immune-checkpoint blockade in lymphomas and discuss their prospect of applications in clinical treatment.

Altered toll-like receptor responsiveness underlies a dominant heritable defect in B cell tolerance in autoimmune New Zealand Black mice.

Systemic lupus erythematosus is a debilitating autoimmune disease in which autoantibodies and autoreactive T cells destroy kidneys and other organs. Disease is clinically and genetically heterogeneous, suggesting that underlying mechanisms vary between patients. We previously used an autoantibody transgenic mouse reporter system to examine the effect of different autoimmune backgrounds on B-cell tolerance, failure of which is a fundamental defect in lupus. We identified a defect consistent with reversible anergy induced by endotoxin stimulation of B cells from Ig transgenic New Zealand Black (NZB) mice. Herein we report that the tolerance defect is revealed by TLR7 and TLR9 as well as TLR4 ligands, with additive effect, and is partially reversed by Mek inhibition. Gene expression analysis reveals significant differences in transcription of multiple TLR pathway genes and ptpn22 in stimulated NZB compared to B6 B cells. Additionally, the defect is detected in Ig transgenic NZB F1 hybrid strains (NZBxNZW)F1 and (B6xNZB)F1. These results implicate an inherited defect wherein NZB anergic B cells maintain coordinated TLR/BCR signaling that permits autoantibody production. Agents targeting these pathways may have therapeutic benefit in the subset of lupus patients that manifest similar defects in B-cell regulation.

NFAT2 is a critical regulator of the anergic phenotype in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a clonal disorder of mature B cells. Most patients are characterised by an indolent disease course and an anergic phenotype of their leukaemia cells, which refers to a state of unresponsiveness to B cell receptor stimulation. Up to 10% of CLL patients transform from an indolent subtype to an aggressive form of B cell lymphoma over time (Richter´s syndrome) and show a significantly worse treatment outcome. Here we show that B cell-specific ablation of Nfat2 leads to the loss of the anergic phenotype culminating in a significantly compromised life expectancy and transformation to aggressive disease. We further define a gene expression signature of anergic CLL cells consisting of several NFAT2-dependent genes including Cbl-b, Grail, Egr2 and Lck. In summary, this study identifies NFAT2 as a crucial regulator of the anergic phenotype in CLL.NFAT2 is a transcription factor that has been linked with chronic lymphocytic leukaemia (CLL), but its functions in CLL manifestation are still unclear. Here the authors show, by analysing mouse CLL models and characterising biopsies from CLL patients, that NFAT2 is an important regulator for the anergic phenotype of CLL.

T Cell Exhaustion: An Epigenetically Imprinted Phenotypic and Functional Makeover.

A recent article in Cell demonstrates that the absence of a single DNA methyltransferase, Dnmt3a, prevents cytotoxic T cells from acquiring the hypofunctional or exhausted phenotype typically seen in chronic viral infections and tumors. Upon establishing a causal relationship between exhaustion-associated epigenetic changes and reduced CD8+ T cell function, the authors provided mechanistic evidence that exhaustion constitutes a specific differentiation program.

Frequency and functional characterization of exhausted CD8+ T cells in chronic lymphocytic leukemia.

OBJECTIVES: The phenotypic and functional properties of Tim-3+ /PD-1+ /CD8+ cells as exhausted T cells were investigated in chronic lymphocytic leukemia (CLL). METHODS: Frequency of CD8+ /Tim-3+ /PD-1+ exhausted cells was determined by flow cytometry. For functional analysis, magnetic beads-isolated CD8+ T cells were stimulated with PHA and PMA/ionocymin to assess their proliferative responses and cytokine production by MTT and ELISA, respectively. Cytotoxic activity of isolated CD8+ T cells was determined using CD107a degranulation assay. RESULTS: The proportion of exhausted CD8+ T cells was significantly higher in CLL compared to controls. Isolated CD8+ T cells from CLL showed functional defects in proliferation, degranulation, and cytokines production. While IL-2, TNF-α, and IFN-γ were significantly lower in CLL patients, IL-10 was higher in the patients group. Patients with progressive clinical stages showed higher frequency and dysfunction of exhausted CD8+ T cells. CONCLUSION: Targeting immune inhibitory receptors to restore the function of tumor surrounding T cells could be helpful for immunotherapy of CLL.

Systematic characterization of basophil anergy.

BACKGROUND: Cohort studies indicated that in certain individuals the basophils do not respond toward allergens due to a desensitization of their Fc epsilon receptor pathway. Cause and functional role as well as the implications on allergic reactions, however, are not clear yet. METHODS: A cross-sectional study was carried out in the tropical urban environment of Singapore, where the allergic response is dominated by a single allergen (house dust mite; HDM). Blood samples were collected from 476 individuals and analyzed comprehensively to correlate the functional state of their basophils with the clinical state as well as the composition of the cellular and soluble plasma components. RESULTS: Inactivation of basophils ('basophil anergy') was observed in about 10% of the cohort. It was associated with a downregulation of basophil Syk and an apparent reduction in the incidence of allergic rhinitis. Correlations on the cohort level suggest that it represents a transitional state to be passed through during the interconversion of responder and nonresponder state. CONCLUSIONS: Basophil anergy thus seems to function as activation barrier to prevent unwanted reactions against minor allergens. It may therefore be relevant for diagnostic purposes or therapeutic interventions of allergic diseases.

Increased endogenous antigen presentation in the periphery enhances susceptibility to inflammation-induced gastric autoimmunity in mice.

How the immune system maintains peripheral tolerance under inflammatory conditions is poorly understood. Here we assessed the fate of gastritogenic T cells following inflammatory activation in vivo. Self-reactive T cells (A23 T cells) specific for the gastric H+ /K+ ATPase α subunit (HKα) were transferred into immunosufficient recipient mice and immunised at a site distant to the stomach with adjuvant containing the cognate HKα peptide antigen. Activation of A23 T cells by immunisation did not impact on either immune tolerance or protection from gastric autoimmunity in wild-type BALB/c mice. However, increased presentation of endogenously derived HKα epitopes by dendritic cells (DCs) in the gastric lymph node of IE-H+ /K+ ß transgenic mice (IEß) reduces A23 T-cell tolerance to gastric antigens after inflammatory activation, with subsequent development of gastritis. While HKα-specific A23 T cells from immunised wild-type mice were poorly responsive to in vitro antigen specific activation, A23 T cells from immunised IEß transgenic mice were readily re-activated, indicating loss of T-cell anergy. These findings show that DCs of gastric lymph nodes can maintain tolerance of pathogenic T cells following inflammatory stimulation and that the density of endogenous antigen presented to self-reactive T cells is critical in the balance between tolerance and autoimmunity.

IgD attenuates the IgM-induced anergy response in transitional and mature B cells.

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire.

Nuclear calcium is required for human T cell activation.

Calcium signals in stimulated T cells are generally considered single entities that merely trigger immune responses, whereas costimulatory events specify the type of reaction. Here we show that the "T cell calcium signal" is a composite signal harboring two distinct components that antagonistically control genomic programs underlying the immune response. Using human T cells from healthy individuals, we establish nuclear calcium as a key signal in human T cell adaptogenomics that drives T cell activation and is required for signaling to cyclic adenosine monophosphate response element-binding protein and the induction of CD25, CD69, interleukin-2, and γ-interferon. In the absence of nuclear calcium signaling, cytosolic calcium activating nuclear factor of activated T cells translocation directed the genomic response toward enhanced expression of genes that negatively modulate T cell activation and are associated with a hyporesponsive state. Thus, nuclear calcium controls the T cell fate decision between a proliferative immune response and tolerance. Modulators of nuclear calcium-driven transcription may be used to develop a new type of pro-tolerance immunosuppressive therapy.

Peroxisome proliferator-activated receptor alpha deficiency impairs regulatory T cell functions: Possible application in the inhibition of melanoma tumor growth in mice.

Regulatory T (Treg) cells are important to induce and maintain immunological self-tolerance. Although the progress accomplished in understanding the functional mechanism of Treg cells, intracellular molecules that control the mechanisms of their suppressive capacity are still on investigation. The present study showed that peroxisome proliferator-activated receptor-alpha deficiency impaired the suppressive activity of Treg cells on CD4+CD25- and CD8+ T cell proliferation. In Treg cells, PPARα gene deletion also induced a decrease of migratory abilities, and downregulated the expression of chemokine receptors (CCR-4, CCR-8 and CXCR-4) and p27KIP1 mRNA. Treg cells from PPARα-/- mice also lost their anergic property. Since low Treg activity, as observed in PPARα-/- mice, is known to be associated with the inhibition of tumor growth, we inoculated these mice with B16 melanoma cells and assessed tumor proliferation. In PPARα-/- mice, cancer growth was significantly curtailed, and it was correlated with high expression of granzyme B and perforin mRNA in tumor bed. Degranulation of cytolytic molecules by CD8+ T cells, assessed by a perforin-release marker CD107a expression, was higher in PPARα-/- mice than that in wild-type mice. Tumor-infiltrating lymphocytes (TIL) in melanoma tumors in PPARα-/- mice exhibited high pro-inflammatory Th1 phenotype. Consistently, adoptive transfer into lymphopenic RAG2-/- mice of total PPARα-/-splenic T cells inhibited more the growth rate of B16 tumor than the wild type splenic T cells. Our findings suggest that PPARα deficiency, by diminishing Treg cell functions and upregulating pro-inflammatory T cell phenotype, exerts an in vivo anti-cancer properties.

Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development.

Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education.