�����ƽ��

BACKGROUND The goal of antiretroviral therapy (ART) is to suppress HIV-1 replication and reconstitute CD4+ T cells. Here, we report on HIV-infected individuals who had a paradoxical decline in CD4+ T cells despite ART-mediated suppression of plasma HIV-1 load (pVL). We defined such an immunological outcome as extreme immune decline (EXID). METHODS EXID's clinical and immunological characteristics were compared to immunological responders (IRs), immunological nonresponders (INRs), healthy controls (HCs), and idiopathic CD4+ lymphopenia (ICL) patients. T cell immunophenotyping and assembly/activation of inflammasomes were evaluated by flow cytometry. PBMC transcriptome analysis and genetic screening for pathogenic variants were performed. Levels of cytokines/chemokines were measured by electrochemiluminescence. Luciferase immunoprecipitation system and NK-mediated antibody-dependent cellular cytotoxicity (ADCC) assays were used to identify anti-lymphocyte autoantibodies. RESULTS EXIDs were infected with non-B HIV-1 subtypes and after 192 weeks of consistent ART-mediated pVL suppression had a median CD4+ decrease of 157 cells/mul, compared with CD4+ increases of 193 cells/mul and 427 cells/mul in INR and IR, respectively. EXID had reduced naive CD4+ T cells, but similar proportions of cycling CD4+ T cells and HLA-DR+CD38+CD8+ T cells compared with IR and INR. Levels of inflammatory cytokines were also similar in EXID and INR, but the IL-7 axis was profoundly perturbed compared with HC, IR, INR, and ICL. Genes involved in T cell and monocyte/macrophage function, autophagy, and cell migration were differentially expressed in EXID. Two of the 5 EXIDs had autoantibodies causing ADCC, while 2 different EXIDs had an increased inflammasome/caspase-1 activation despite consistently ART-suppressed pVL. CONCLUSIONS EXID is a distinct immunological outcome compared with previously described INR. Anti-CD4+ T cell autoantibodies and aberrant inflammasome/caspase-1 activation despite suppressed HIV-1 viremia are among the mechanisms responsible for EXID.

TET enzymes are dioxygenases that promote DNA demethylation by oxidizing the methyl group of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Here, we report a close correspondence between 5hmC-marked regions, chromatin accessibility and enhancer activity in B cells, and a strong enrichment for consensus binding motifs for basic region-leucine zipper (bZIP) transcription factors at TET-responsive genomic regions. Functionally, Tet2 and Tet3 regulate class switch recombination (CSR) in murine B cells by enhancing expression of Aicda, which encodes the activation-induced cytidine deaminase (AID) enzyme essential for CSR. TET enzymes deposit 5hmC, facilitate DNA demethylation, and maintain chromatin accessibility at two TET-responsive enhancer elements, TetE1 and TetE2, located within a superenhancer in the Aicda locus. Our data identify the bZIP transcription factor, ATF-like (BATF) as a key transcription factor involved in TET-dependent Aicda expression. 5hmC is not deposited at TetE1 in activated Batf-deficient B cells, indicating that BATF facilitates TET recruitment to this Aicda enhancer. Our study emphasizes the importance of TET enzymes for bolstering AID expression and highlights 5hmC as an epigenetic mark that captures enhancer dynamics during cell activation.

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca-1) cells to reconstitute aged BM and rejuvenate the aged heart, and examined the underlying molecular mechanisms. BM Sca-1+ or Sca-1- cells from young (2-3 months) or aged (18-19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca-1+ , young Sca-1- , old Sca-1+ , and old Sca-1- . Four months later, expression of rejuvenation-related genes (Bmi1, Cbx8, PNUTS, Sirt1, Sirt2, Sirt6) and proteins (CDK2, CDK4) was increased along with telomerase activity and telomerase-related protein (DNA-PKcs, TRF-2) expression, whereas expression of senescence-related genes (p16INK4a , P19ARF , p27Kip1 ) and proteins (p16INK4a , p27Kip1 ) was decreased in Sca-1+ chimeric hearts, especially in the young group. Host cardiac endothelial cells (GFP- CD31+ ) but not cardiomyocytes were the primary cell type rejuvenated by young Sca-1+ cells as shown by improved proliferation, migration, and tubular formation abilities. C-X-C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP+ ) cells isolated from young Sca-1+ chimeric hearts. Protein expression of Cxcr4, phospho-Akt, and phospho-FoxO3a in endothelial cells derived from the aged chimeric heart was increased, especially in the young Sca-1+ group. Reconstitution of aged BM with young Sca-1+ cells resulted in effective homing of functional stem cells in the aged heart. These young, regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells.

Both targeted therapy and immunotherapy have been used successfully to treat melanoma, but the development of resistance and poor response rates to the individual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles, but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein, we describe the development and characterization of a novel, immunogenic variant of the BrafV600ECdkn2a-/-Pten-/- YUMM1.1 tumor model that expresses the immunogen, ovalbumin (YOVAL1.1). We demonstrate that, unlike parental tumors, YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy. Importantly, YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK, responding in a manner consistent with human BRAFV600E melanoma. The YOVAL1.1 melanoma model is transplantable, immunogenic and sensitive to clinical therapies, making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma.

Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress, with activation of the IRE1alpha-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however, the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress, mainly affecting neutrophils, monocytes, and macrophages. An overactive IRE1alpha-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state, with increased glycolytic rates and mitochondrial function, associated with exaggerated production of TNF and IL-6. This hyper-metabolic state, seen in CF macrophages, is reversed by inhibiting the RNase domain of IRE1alpha, thereby decreasing the increased glycolic rates, mitochondrial function and inflammation. Altogether, our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover, the IRE1alpha-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages, which is associated with the exaggerated inflammatory response. Modulating ER stress, metabolism and inflammation, by targeting IRE1alpha, may improve the metabolic fitness of macrophages, and other immune cells in CF and other immune-related disorders.

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.

Intestinal fibrosis is a serious complication in inflammatory bowel disease (IBD). Despite the remarkable success of recent anti-inflammatory therapies for IBD, incidence of intestinal fibrosis and need for bowel resection have not significantly changed. To clarify the contribution of haematopoietic-derived cells in intestinal fibrosis, we prepared bone marrow (BM) chimeric mice (chimeras), which were reconstituted with BM cells derived from enhanced green fluorescent protein (EGFP)-transgenic mice or CC chemokine receptor 2 (CCR2)-deficient mice. After 2 months of transplantation, BM chimeras were treated with azoxymethane/dextran sodium sulphate. During chronic inflammation, CCR2+ BM-derived monocyte and fibrocyte infiltration into the colon and CC chemokine ligand 2 production increased, leading to colon fibrosis in EGFP BM chimeras. In CCR2-deficient BM chimeras, monocyte and fibrocyte numbers in the colonic lamina propria significantly decreased, and colon fibrosis was attenuated. In colon tissue, mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 but not of collagen I, transforming growth factor-beta1 or matrix metalloproteinases was significantly different between the two chimeras. CCR2+ monocytes and fibrocytes showed high Timp1 mRNA expression. Our results suggest that infiltrating CCR2+ monocytes and their progenies, fibrocytes, promote colon fibrosis by inhibiting collagen degradation through TIMP-1 production.

Type 1 diabetes (T1D) is an autoimmune disease in which a strong inflammatory response causes the death of insulin-producing pancreatic beta-cells, while inefficient regulatory mechanisms allow that response to become chronic. Ethyl pyruvate (EP), a stable pyruvate derivate and certified inhibitor of an alarmin-high mobility group box 1 (HMGB1), exerts anti-oxidant and anti-inflammatory properties in animal models of rheumatoid arthritis and encephalomyelitis. To test its therapeutic potential in T1D, EP was administered intraperitoneally to C57BL/6 mice with multiple low-dose streptozotocin (MLDS)-induced T1D. EP treatment decreased T1D incidence, reduced the infiltration of cells into the pancreatic islets and preserved beta-cell function. Apart from reducing HMGB1 expression, EP treatment successfully interfered with the inflammatory response within the local pancreatic lymph nodes and in the pancreas. Its effect was restricted to boosting the regulatory arm of the immune response through up-regulation of tolerogenic dendritic cells (CD11c+CD11b-CD103+) within the pancreatic infiltrates and through the enhancement of regulatory T cell (Treg) levels (CD4+CD25highFoxP3+). These EP-stimulated Treg displayed enhanced suppressive capacity reflected in increased levels of CTLA-4, secreted TGF-beta, and IL-10 and in the more efficient inhibition of effector T cell proliferation compared to Treg from diabetic animals. Higher levels of Treg were a result of increased differentiation and proliferation (Ki67+ cells), but also of the heightened potency for migration due to increased expression of adhesion molecules (CD11a and CD62L) and CXCR3 chemokine receptor. Treg isolated from EP-treated mice had the activated phenotype and T-bet expression more frequently, suggesting that they readily suppressed IFN-gamma-producing cells. The effect of EP on Treg was also reproduced in vitro. Overall, our results show that EP treatment reduced T1D incidence in C57BL/6 mice predominantly by enhancing Treg differentiation, proliferation, their suppressive capacity, and recruitment into the pancreas.

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40{\%} of antigen-specific SED B cells bind antigen within 2 h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines.

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer, early events of tumorigenesis have already taken place. Similarly, development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article, we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model, human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1), referred to as NPM1c. Following engraftment into immunodeficient mice, transduced HSPCs give rise to human myeloid leukemia, whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML, with CD123+ leukemic stem or initiating cells (LSC), resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics.

In the inner ear, endolymph fluid surrounds the organ of Corti, which is important for auditory function; notably, even slight environmental changes mediated by trauma or infection can have significant consequences. However, it is unclear how the immune response is modulated in these tissues. Here, we report the local immune surveillance role of cleaved cochlin LCCL (Limulus factor C, Cochlin, and Lgl1) during Pseudomonas aeruginosa infection in the cochlea. Upon infection, the LCCL domain is cleaved from cochlin and secreted into the perilymph. This cleaved fragment sequesters infiltrating bacteria in the scala tympani and subsequently recruits resident immune cells to eliminate the bacteria. Importantly, hearing loss in a cochlin knockout mouse model is remedied by treatment with a cochlin LCCL peptide. These findings suggest cleaved cochlin LCCL constitutes a critical factor in innate immunity and auditory function and may be a potential therapeutic target to treat chronic otitis media-induced hearing loss.

CD4 T cells play a critical role in promoting the development of autoimmunity in type 1 diabetes. The diabetogenic CD4 T cell clone BDC-2.5, originally isolated from a NOD mouse, has been widely used to study the contribution of autoreactive CD4 T cells and relevant Ags to autoimmune diabetes. Recent work from our laboratory has shown that the Ag for BDC-2.5 T cells is a hybrid insulin peptide (2.5HIP) consisting of an insulin C-peptide fragment fused to a peptide from chromogranin A (ChgA) and that endogenous 2.5HIP-reactive T cells are major contributors to autoimmune pathology in NOD mice. The objective of this study was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with the 2.5HIP Ag (2.5HIP-coupled PLG NPs) can tolerize BDC-2.5 T cells. Infusion of 2.5HIP-coupled PLG NPs was found to prevent diabetes in an adoptive transfer model by impairing the ability of BDC-2.5 T cells to produce proinflammatory cytokines through induction of anergy, leading to an increase in the ratio of Foxp3+ regulatory T cells to IFN-gamma+ effector T cells. To our knowledge, this work is the first to use a hybrid insulin peptide, or any neoepitope, to re-educate diabetogenic T cells and may have significant implications for the development of an Ag-specific therapy for type 1 diabetes patients.