AIM2 controls microglial inflammation to prevent experimental autoimmune encephalomyelitis.

The role of the PYHIN family member absent in melanoma 2 (AIM2), another important inflammasome sensor, in EAE remains unclear. In this study, we found that AIM2 negatively regulates the pathogenesis of EAE independent of inflammasome activation. AIM2 deficiency enhanced microglia activation and infiltration of peripheral immune cells into the CNS, thereby promoting neuroinflammation and demyelination during EAE. Mechanistically, AIM2 negatively regulates the DNA-PK-AKT3 in microglia to control neuroinflammation synergistically induced by cGAS and DNA-PK. Administration of a DNA-PK inhibitor reduced the severity of the EAE. Collectively, these findings identify a new role for AIM2 in controlling the onset of EAE. Furthermore, delineation of the underlying inflammasome-independent mechanism highlights cGAS and DNA-PK signaling as potential targets for the treatment of heterogeneous MS.

Cytoplasmic DNA sensing by KU complex in aged CD4+ T cell potentiates T cell activation and aging-related autoimmune inflammation.

Aging is associated with DNA accumulation and increased homeostatic proliferation of circulating T cells. Although these attributes are associated with aging-related autoimmunity, their direct contributions remain unclear. Conventionally, KU complex, the regulatory subunit of DNA-dependent protein kinase (DNA-PK), together with the catalytic subunit of DNA-PK (DNA-PKcs), mediates DNA damage repair in the nucleus. Here, we found KU complex abundantly expressed in the cytoplasm, where it recognized accumulated cytoplasmic DNA in aged human and mouse CD4+ T cells. This process enhanced T cell activation and pathology of experimental autoimmune encephalomyelitis (EAE) in aged mice. Mechanistically, KU-mediated DNA sensing facilitated DNA-PKcs recruitment and phosphorylation of the kinase ZAK. This activated AKT and mTOR pathways, promoting CD4+ T cell proliferation and activation. We developed a specific ZAK inhibitor, which dampened EAE pathology in aged mice. Overall, these findings demonstrate a KU-mediated cytoplasmic DNA-sensing pathway in CD4+ T cells that potentiates aging-related autoimmunity.

Pathology of macrophage activation syndrome in humanized NSGS mice.

"Humanized" immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.

Human DNA-PK activates a STING-independent DNA sensing pathway.

Detection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection. Whether there are additional DNA sensing pathways, and how such pathways might function, remains controversial. We show here that humans-but not laboratory mice-have a second, potent, STING-independent DNA sensing pathway (SIDSP). We identify human DNA-dependent protein kinase (DNA-PK) as the sensor of this pathway and demonstrate that DNA-PK activity drives a robust and broad antiviral response. We show that the E1A oncoprotein of human adenovirus 5 and the ICP0 protein of herpes simplex virus 1 block this response. We found heat shock protein HSPA8/HSC70 as a target for inducible phosphorylation in the DNA-PK antiviral pathway. Last, we demonstrate that DNA damage and detection of foreign DNA trigger distinct modalities of DNA-PK activity. These findings reveal the existence, sensor, a specific downstream target, and viral antagonists of a SIDSP in human cells.

Sensitivity of CD3/CD28-stimulated versus non-stimulated lymphocytes to ionizing radiation and genotoxic anticancer drugs: key role of ATM in the differential radiation response.

Activation of T cells, a major fraction of peripheral blood lymphocytes (PBLCS), is essential for the immune response. Genotoxic stress resulting from ionizing radiation (IR) and chemical agents, including anticancer drugs, has serious impact on T cells and, therefore, on the immune status. Here we compared the sensitivity of non-stimulated (non-proliferating) vs. CD3/CD28-stimulated (proliferating) PBLC to IR. PBLCs were highly sensitive to IR and, surprisingly, stimulation to proliferation resulted in resistance to IR. Radioprotection following CD3/CD28 activation was observed in different T-cell subsets, whereas stimulated CD34+ progenitor cells did not become resistant to IR. Following stimulation, PBLCs showed no significant differences in the repair of IR-induced DNA damage compared with unstimulated cells. Interestingly, ATM is expressed at high level in resting PBLCs and CD3/CD28 stimulation leads to transcriptional downregulation and reduced ATM phosphorylation following IR, indicating ATM to be key regulator of the high radiosensitivity of resting PBLCs. In line with this, pharmacological inhibition of ATM caused radioresistance of unstimulated, but not stimulated, PBLCs. Radioprotection was also achieved by inhibition of MRE11 and CHK1/CHK2, supporting the notion that downregulation of the MRN-ATM-CHK pathway following CD3/CD28 activation results in radioprotection of proliferating PBLCs. Interestingly, the crosslinking anticancer drug mafosfamide induced, like IR, more death in unstimulated than in stimulated PBLCs. In contrast, the bacterial toxin CDT, damaging DNA through inherent DNase activity, and the DNA methylating anticancer drug temozolomide induced more death in CD3/CD28-stimulated than in unstimulated PBLCs. Thus, the sensitivity of stimulated vs. non-stimulated lymphocytes to genotoxins strongly depends on the kind of DNA damage induced. This is the first study in which the killing response of non-proliferating vs. proliferating T cells was comparatively determined. The data provide insights on how immunotherapeutic strategies resting on T-cell activation can be impacted by differential cytotoxic effects resulting from radiation and chemotherapy.

Genetic Variants in Immune-Related Pathways and Breast Cancer Risk in African American Women in the AMBER Consortium.

Background: Constitutional immunity shaped by exposure to endemic infectious diseases and parasitic worms in Sub-Saharan Africa may play a role in the etiology of breast cancer among African American (AA) women.Methods: A total of 149,514 gene variants in 433 genes across 45 immune pathways were analyzed in the AMBER consortium among 3,663 breast cancer cases and 4,687 controls. Gene-based pathway analyses were conducted using the adaptive rank truncated product statistic for overall breast cancer risk, and risk by estrogen receptor (ER) status. Unconditional logistic regression analysis was used to estimate ORs and 95% confidence intervals (CIs) for single variants.Results: The top pathways were Interleukin binding (P = 0.01), Biocarta TNFR2 (P = 0.005), and positive regulation of cytokine production (P = 0.024) for overall, ER+, and ER- cancers, respectively. The most significant gene was IL2RB (P = 0.001) for overall cancer, with rs228952 being the top variant identified (OR = 0.85; 95% CI, 0.79-0.92). Only BCL3 contained a significant variant for ER+ breast cancer. Variants in IL2RB, TLR6, IL8, PRKDC, and MAP3K1 were associated with ER- disease. The only genes showing heterogeneity between ER- and ER+ cancers were TRAF1, MAP3K1, and MAPK3 (P ≤ 0.02). We also noted genes associated with autoimmune and atopic disorders.Conclusions: Findings from this study suggest that genetic variants in immune pathways are relevant to breast cancer susceptibility among AA women, both for ER+ and ER- breast cancers.Impact: Results from this study extend our understanding of how inherited genetic variation in immune pathways is relevant to breast cancer susceptibility. Cancer Epidemiol Biomarkers Prev; 27(3); 321-30. ©2018 AACR.

PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity.

BACKGROUND: PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance. OBJECTIVE: We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients. METHODS: Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency. RESULTS: We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells. CONCLUSION: Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.

Antibodies to DNA repair proteins in headache with neurological deficits and cerebrospinal fluid lymphocytosis (HaNDL) patients.

Autoimmune mechanisms have been implicated in the pathogenesis of headache and neurological deficits with cerebrospinal fluid lymphocytosis (HaNDL). Pooled sera of five HaNDL patients and 30 controls (10 multiple sclerosis patients, 10 migraine patients, 10 healthy controls) were screened by protein macroarray. All sera were also individually subjected to immunoprecipitation with neuroblastoma cells and the bound antigens were identified by mass spectrometry. Antibodies to three DNA repair proteins (mitogen-activated protein kinase-4, DNA-dependent protein kinase catalytic subunit, DNA excision repair protein ERCC-6) were identified by both macroarray and immunoprecipitation methods in 3/5 HaNDL sera, but in none of the controls. The presence of DNA repair protein antibodies indicates DNA damage and provides further support for the inflammatory etiology of HaNDL.

Identification of HLA ligands and T-cell epitopes for immunotherapy of lung cancer.

INTRODUCTION: Lung cancer is the most common cancer worldwide. Every year, as many people die of lung cancer as of breast, colon and rectum cancers combined. Because most patients are being diagnosed in advanced, not resectable stages and therefore have a poor prognosis, there is an urgent need for alternative therapies. Since it has been demonstrated that a high number of tumor- and stromal-infiltrating cytotoxic T cells (CTLs) is associated with an increased disease-specific survival in lung cancer patients, it can be assumed that immunotherapy, e.g. peptide vaccines that are able to induce a CTL response against the tumor, might be a promising approach. METHODS: We analyzed surgically resected lung cancer tissues with respect to HLA class I- and II-presented peptides and gene expression profiles, aiming at the identification of (novel) tumor antigens. In addition, we tested the ability of HLA ligands derived from such antigens to generate a CTL response in healthy donors. RESULTS: Among 170 HLA ligands characterized, we were able to identify several potential targets for specific CTL recognition and to generate CD8+ T cells which were specific for peptides derived from cyclin D1 or protein-kinase, DNA-activated, catalytic polypeptide and lysed tumor cells loaded with peptide. CONCLUSIONS: This is the first molecular analysis of HLA class I and II ligands ex vivo from human lung cancer tissues which reveals known and novel tumor antigens able to elicit a CTL response.

DNA double strand break repair defects, primary immunodeficiency disorders, and 'radiosensitivity'.

PURPOSE OF REVIEW: It is important to assess 'radiosensitivity' in patients suspected of immunodeficiency because underlying DNA double strand break (DSB) repair defects have considerable impact on V(D)J recombination, class switching and lymphocyte maturation, leading to increased infections and cancer risk. In addition, the phenotype of 'radiosensitivity' may identify patients with increased toxicity to radiation and chemotherapeutic agents and could impact upon their preparation for stem cell transplantation. To date, the gold standard for evaluating 'radiosensitivity' has been the colony-survival assay (CSA), which reflects the efficiency of DNA repair of DSBs as it impacts upon replication and cell survival. Other methods measure other aspects of DNA repair; however, their limited specificity often leads to false negatives for predicting 'radiosensitivity', especially clinical radiosensitivity. Lastly, clinical awareness of an overarching syndrome of DSB repair disorders, XCIND, could help to raise diagnostic levels of suspicion and, thereby, identify additional patients with new forms of immunodeficiency, cancer susceptibility and radiosensitivity. RECENT FINDINGS: Within the past year, three new radiosensitivity disorders of DSB repair have been described, involving deficiencies of RNF168, RAD50, and DNA-PKcs. These are truly translational advances because they validate laboratory models and allow new patients to be identified. SUMMARY: Recognizing compromised genome stability is important but difficult. We review the evidence for correlations between DSB repair, abnormal colony formation, clinical radiosensitivity and other laboratory methods.

p53-Independent apoptosis disrupts early organogenesis in embryos lacking both ataxia-telangiectasia mutated and Prkdc.

The ataxia-telangiectasia mutated (ATM) protein and the nonhomologous end-joining (NHEJ) pathway play crucial roles in sensing and repairing DNA double-strand breaks in postnatal cells. However, each pathway is dispensable for early embryogenesis. Loss of both ATM and Prkdc/Ku is synthetically lethal, but neither the developmental processes perturbed nor the mechanisms of lethality have been determined by previous reports. Here, we show that ATM and Prkdc collaborate to maintain genomic stability during gastrulation and early organogenesis, a period of rapid proliferation and hypersensitivity to DNA damage. At E7.5 to E8.5, ATM(-/-)Prkdcscid/scid embryos displayed normal proliferation indices but exhibited excessive apoptosis and elevated expression of Ser15-phosphorylated p53. Thus, this crucial regulatory residue of p53 can be phosphorylated in the absence of ATM or Prkdc. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that apoptosis is p53 independent in these in ATM(-/-)Prkdcscid/scid embryos. Because mice with combined disruptions of ATM and other NHEJ components (ligase IV, Artemis) are viable, our data suggest a novel NHEJ-independent function for Prkdc/Ku that is required to complete early embryogenesis in the absence of ATM.