Renal tubular epithelial cells are constitutive non-cognate stimulators of resident T cells.

Understanding the roles of different cell types in regulating T cell homeostasis in various tissues is critical for understanding adaptive immunity. Here, we show that RTECs (renal tubular epithelial cells) are intrinsically programmed to polyclonally stimulate proliferation of kidney αß T cells by a cell-cell contact mechanism that is major histocompatibility complex (MHC) independent and regulated by CD155, αVß3-integrin, and vitronectin. Peripheral CD4 and CD8 are resistant to RTEC-mediated stimulation, while the minor subset of double-negative (DN) T cells are responsive. This functional property of RTEC is discovered by using a coculture system that recapitulates spontaneous in vivo polyclonal proliferation of kidney T cells, which are mainly comprised of central memory T (TCM) and effector memory T (TEM) cells. This robust cell-intrinsic stimulatory role of RTECs could be underlying the steady-state spontaneous proliferation of kidney T cells. The results have conceptual implications for understanding roles of different cell types in regulating systemic and organ-specific T cell homeostasis.

WDR82-Mediated H3K4me3 Is Associated with Tumor Proliferation and Therapeutic Efficacy in Pediatric High-Grade Gliomas.

Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.

Reducing stigma and increasing competence working with mental illness: Adaptation of a contact-based program for osteopathic medical students to a virtual, active learning format.

PURPOSE: Contact-based education, offering meaningful contact with individuals living in recovery with mental illness, reduces stigma. This study evaluated the effectiveness of the National Alliance on Mental Illness Provider Education Program (NAMI PEP) when implemented as a curricular requirement across two cohorts of third-year osteopathic medical students, comparing traditional, passive learning and active, online delivery formats. MATERIALS AND METHODS: Participants were two cohorts of third-year medical students (Cohort 1 n = 186; Cohort 2 n = 139; overall N = 325) who completed questionnaires measuring affect, beliefs, and behaviors toward patients with mental illness at pre-program, 1-week follow-up, and 6-month follow-up. For Cohort 1, the existing community-based NAMI PEP was implemented. For Cohort 2, the program was adapted to an online, active learning format tailored to medical students, and an additional 3-month follow-up assessment was added to better identify intermediate-term effects. RESULTS: The NAMI PEP was associated with longitudinal improvements in target outcomes, with enhanced effects with the adapted curriculum in Cohort 2. At 6-month follow-up, students reported less stigma, fewer stereotyping negative attitudes, and lower anxiety treating patients with mental illness. They also reported increased confidence integrating psychiatry into routine care and increased competence in principles of collaborative mental health treatment. CONCLUSIONS: This study demonstrates the longitudinal effectiveness of the NAMI PEP across two cohorts of medical students, with strengthened effects observed when the program is tailored to contemporary medical education.

Expression of unique gene signature distinguishes TCRαß+ /BCR+ dual expressers from CD3+ CD14+ doublets.

Increasing evidence shows pathophysiological significance of rare immune cells, necessitating the need for reliable and proper methods for their detection and analysis. We have recently identified a new lymphocyte that coexpresses lineage markers of T- and B-cells including T cell receptor and B cell receptor (called dual expressers, DEs). Because of the peculiar phenotype of DEs, we used multiple techniques to authenticate their identity (fluorescence-activated cell sorting [FACS], scRNAseq, EBV cell lines, and imaging flow cytometry). In an recent article published in this journal, Burel and colleagues successfully detected DEs using FACS and imaging microscopy. Yet they claimed, based on the profile of what they called naturally occurring CD3+ CD14+ T cell/monocyte complexes that the scRNAseq signature of DEs resembles that of cell-cell complexes. Serious flaws in their analysis, however, invalidate their conclusions. Unlike the CD3+ CD14+ complexes, DEs have a distinct identity due to expression of a unique set of signature genes. Without a clear explanation, Burel and colleagues excluded these genes from their analysis, thereby effectively stripped DEs from their identity. Inclusion of these genes as described in this communication restores the identity of DEs. Moreover, contrary to the claim of Burel and colleagues, B- and T-cell specific genes are similarly expressed in DE cells.

Itraconazole Exerts Its Antitumor Effect in Esophageal Cancer By Suppressing the HER2/AKT Signaling Pathway.

Itraconazole, an FDA-approved antifungal, has antitumor activity against a variety of cancers. We sought to determine the effects of itraconazole on esophageal cancer and elucidate its mechanism of action. Itraconazole inhibited cell proliferation and induced G1-phase cell-cycle arrest in esophageal squamous cell carcinoma and adenocarcinoma cell lines. Using an unbiased kinase array, we found that itraconazole downregulated protein kinase AKT phosphorylation in OE33 esophageal adenocarcinoma cells. Itraconazole also decreased phosphorylation of downstream ribosomal protein S6, transcriptional expression of the upstream receptor tyrosine kinase HER2, and phosphorylation of upstream PI3K in esophageal cancer cells. Lapatinib, a tyrosine kinase inhibitor that targets HER2, and siRNA-mediated knockdown of HER2 similarly suppressed cancer cell growth in vitro Itraconazole significantly inhibited growth of OE33-derived flank xenografts in mice with detectable levels of itraconazole and its primary metabolite, hydroxyitraconazole, in esophagi and tumors. HER2 total protein and phosphorylation of AKT and S6 proteins were decreased in xenografts from itraconazole-treated mice compared to xenografts from placebo-treated mice. In an early phase I clinical trial (NCT02749513) in patients with esophageal cancer, itraconazole decreased HER2 total protein expression and phosphorylation of AKT and S6 proteins in tumors. These data demonstrate that itraconazole has potent antitumor properties in esophageal cancer, partially through blockade of HER2/AKT signaling.

Ablation of Sam68 in adult mice increases thermogenesis and energy expenditure.

Genetic deletion of Src associated in mitosis of 68kDa (Sam68), a pleiotropic adaptor protein prevents high-fat diet-induced weight gain and insulin resistance. To clarify the role of Sam68 in energy metabolism in the adult stage, we generated an inducible Sam68 knockout mice. Knockout of Sam68 was induced at the age of 7-10 weeks, and then we examined the metabolic profiles of the mice. Sam68 knockout mice gained less body weight over time and at 34 or 36 weeks old, had smaller fat mass without changes in food intake and absorption efficiency. Deletion of Sam68 in mice elevated thermogenesis, increased energy expenditure, and attenuated core-temperature drop during acute cold exposure. Furthermore, we examined younger Sam68 knockout mice at 11 weeks old before their body weights deviate, and confirmed increased energy expenditure and thermogenic gene program. Thus, Sam68 is essential for the control of adipose thermogenesis and energy homeostasis in the adult.

A reply to "TCR+/BCR+ dual-expressing cells and their associated public BCR clonotype are not enriched in type 1 diabetes".

We have recently identified a novel lymphocyte that is a dual expresser (DE) of TCRαß and BCR. DEs in T1D patients are predominated by a public BCR clonotype (clone-x) that encodes a potent autoantigen that cross-activates insulin-reactive T cells. Betts and colleagues were able to detect DEs but alleged to not detect high DE frequency, clone-x, or similar clones in T1D patients. Unfortunately, the authors did not follow our methods and when they did, their flow cytometric data at two sites were conflicting. Moreover, contrary to their claim, we identified clones similar to clone-x in their data along with clones bearing the core motif (DTAMVYYFDYW). Additionally, their report of no increased usage of clone-x VH/DH genes by bulk B cells confirms rather than challenges our results. Finally, the authors failed to provide data verifying purity of their sorted DEs, making it difficult to draw reliable conclusion of their repertoire analysis. This Matters Arising Response paper addresses the Japp et al. (2021) Matters Arising paper, published concurrently in Cell.

Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Multiple genetic programs contribute to CD4 T cell memory differentiation and longevity by maintaining T cell quiescence.

While memory T-cells represent a hallmark of adaptive immunity, little is known about the genetic mechanisms regulating the longevity of memory CD4 T cells. Here, we studied the dynamics of gene expression in antigen specific CD4 T cells during infection, memory differentiation, and long-term survival up to nearly a year in mice. We observed that differentiation into long lived memory cells is associated with increased expression of genes inhibiting cell proliferation and apoptosis as well as genes promoting DNA repair response, lipid metabolism, and insulin resistance. We identified several transmembrane proteins in long-lived murine memory CD4 T cells, which co-localized exclusively within the responding antigen-specific memory CD4 T cells in human. The unique gene signatures of long-lived memory CD4 T cells, along with the new markers that we have defined, will enable a deeper understanding of memory CD4 T cell biology and allow for designing novel vaccines and therapeutics.

GEAMP, a novel gastroesophageal junction carcinoma cell line derived from a malignant pleural effusion.

Gastroesophageal junction (GEJ) cancer remains a clinically significant disease in Western countries due to its increasing incidence, which mirrors that of esophageal cancer, and poor prognosis. To develop novel and effective approaches for prevention, early detection, and treatment of patients with GEJ cancer, a better understanding of the mechanisms driving pathogenesis and malignant progression of this disease is required. These efforts have been limited by the small number of available cell lines and appropriate preclinical animal models for in vitro and in vivo studies. We have established and characterized a novel GEJ cancer cell line, GEAMP, derived from the malignant pleural effusion of a previously treated GEJ cancer patient. Comprehensive genetic analyses confirmed a clonal relationship between GEAMP cells and the primary tumor. Targeted next-generation sequencing identified 56 nonsynonymous alterations in 51 genes including TP53 and APC, which are commonly altered in GEJ cancer. In addition, multiple copy-number alterations were found including EGFR and K-RAS gene amplifications and loss of CDKN2A and CDKN2B. Histological examination of subcutaneous flank xenografts in nude and NOD-SCID mice showed a carcinoma with mixed squamous and glandular differentiation, suggesting GEAMP cells contain a subpopulation with multipotent potential. Finally, pharmacologic inhibition of the EGFR signaling pathway led to downregulation of key downstream kinases and inhibition of cell proliferation in vitro. Thus, GEAMP represents a valuable addition to the limited number of bona fide GEJ cancer cell lines.

Zika virus knowledge, contraception use, and lessons learned from a Dominican Republic pilot study.

OBJECTIVE: To assess knowledge of the Zika virus (ZIKV), use of contraceptives, and sources of health information in rural communities in the Dominican Republic. METHODS: Over 4 days in March 2017, a research team traveled to four rural communities in the Dominican Republic to provide healthcare services. Overall, 90 men and women consented to a voluntary verbal 12-question survey. RESULTS: Of the participants, 55% were not certain whether ZIKV is transmitted sexually; 75% of participants were either not sure or thought ZIKV was not present in their community. Charlas (informal discussions led by community health workers) were cited as the most common source for public health information. Prevalence of contraceptive use was 26.6% hormonal and 1.1% long-acting reversible contraception (LARC); 30.0% cited no use of contraception. CONCLUSION: Significant deficits in ZIKV knowledge, underutilization of LARCs, and socioeconomic factors exist that constrain the application of WHO recommendations for preventing ZIKV infection. Additional and more robust surveys are needed to assess public health education and interventions, critical for disease prevention in communities facing current and future epidemics.

A Public BCR Present in a Unique Dual-Receptor-Expressing Lymphocyte from Type 1 Diabetes Patients Encodes a Potent T Cell Autoantigen.

T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.

Potentially inappropriate medication prescribing in the elderly: Is the Beers Criteria relevant in the Emergency Department today?

STUDY OBJECTIVE: To investigate the frequency of Beers Criteria (BC) medication and opioid use in patients age 65 years and older arriving in the Emergency Department. METHODS: We performed a retrospective observational study of a convenience sample of 400 patients, age 65 years and older, arriving to and discharged solely from the Emergency Department. We examined 400 sequential patient charts with visit dates April-July 2017, for the presence of a Beers Criteria medication or opioid prescription. We also examined each chart for nine specific chief complaints, including return visits and subsequent admissions. RESULTS: Of the 400 patients included in this study, 304 patients (76%; 95% confidence interval [CI] 72% to 80%) had at least 1 prescription at the index ED visit for an "avoid" or "use with caution" Beers Criteria medication. Of these patients, 194 (64%; 95% CI 58% to 69%) had ≥2 Beers medication prescriptions and 122 patients (40%; 95% CI 35% to 46%) had ≥3 Beers medication prescriptions. We found no difference in the number of patients with a chief complaint of interest between the BC medication list (28%) and lacking a BC medication (29%) (p-value = 1). No patients returned in the next 7 days for a medication-related complaint. CONCLUSION: The results of this study call into question the routine application of lists without high-quality evidence to critique the prescribing of certain medications. Further patient-oriented study of the relevance of the Beers Criteria list, especially in light of the changed face of medication profiles and populations, is called for.

Global Reduction of H3K4me3 Improves Chemotherapeutic Efficacy for Pediatric Ependymomas.

BACKGROUND: Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. METHODS: Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. RESULTS: H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells increased cell response to chemotherapy. CONCLUSIONS: Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Autophagy, Cell Viability, and Chemoresistance Are Regulated By miR-489 in Breast Cancer.

It is postulated that the complexity and heterogeneity in cancer may hinder most efforts that target a single pathway. Thus, discovery of novel therapeutic agents targeting multiple pathways, such as miRNAs, holds promise for future cancer therapy. One such miRNA, miR-489, is downregulated in a majority of breast cancer cells and several drug-resistant breast cancer cell lines, but its role and underlying mechanism for tumor suppression and drug resistance needs further investigation. The current study identifies autophagy as a novel pathway targeted by miR-489 and reports Unc-51 like autophagy activating kinase 1 (ULK1) and lysosomal protein transmembrane 4 beta (LAPTM4B) to be direct targets of miR-489. Furthermore, the data demonstrate autophagy inhibition and LAPTM4B downregulation as a major mechanism responsible for miR-489-mediated doxorubicin sensitization. Finally, miR-489 and LAPTM4B levels were inversely correlated in human tumor clinical specimens, and more importantly, miR-489 expression levels predict overall survival in patients with 8q22 amplification (the region in which LAPTM4B resides).Implications: These findings expand the understanding of miR-489-mediated tumor suppression and chemosensitization in and suggest a strategy for using miR-489 as a therapeutic sensitizer in a defined subgroup of resistant breast cancer patients. Mol Cancer Res; 16(9); 1348-60. ©2018 AACR.

HER2 Overexpression Triggers an IL1α Proinflammatory Circuit to Drive Tumorigenesis and Promote Chemotherapy Resistance.

Systemic inflammation in breast cancer correlates with poor prognosis, but the molecular underpinnings of this connection are not well understood. In this study, we explored the relationship between HER2 overexpression, inflammation, and expansion of the mammary stem/progenitor and cancer stem-like cell (CSC) population in breast cancer. HER2-positive epithelial cells initiated and sustained an inflammatory milieu needed to promote tumorigenesis. HER2 induced a feedforward activation loop of IL1α and IL6 that stimulated NFκB and STAT3 pathways for generation and maintenance of breast CSC. In mice, Il1a genetic deficiency delayed MMTV-Her2-induced tumorigenesis and reduced inflammatory cytokine expression as well as CSC in primary tumors. In clinical specimens of human breast tumor tissues, tissue microarray analysis revealed a strong positive correlation between IL1α/IL6 expression and CSC-positive phenotype. Pharmacologic blockade of IL1α signaling reduced the CSC population and improved chemotherapeutic efficacy. Our findings suggest new therapeutic or prevention strategies for HER2-positive breast cancers.Significance: IL1α signaling driven by HER2 promotes chronic inflammation needed to support cancer stem-like cell maintenance in HER2-positive breast cancers. Cancer Res; 78(8); 2040-51. ©2018 AACR.

Complement Component C1q Programs a Pro-Efferocytic Phenotype while Limiting TNFα Production in Primary Mouse and Human Macrophages.

Deficiency in complement component C1q is associated with an inability to clear apoptotic cells (efferocytosis) and aberrant inflammation in lupus, and identification of the pathways involved in these processes should reveal important regulatory mechanisms in lupus and other autoimmune or inflammatory diseases. In this study, C1q-dependent regulation of TNFα/IL-6 expression and efferocytosis was investigated using primary mouse bone marrow-derived macrophages and human monocyte-derived macrophages. C1q downregulated LPS-dependent TNFα production in mouse and human macrophages. While prolonged stimulation with C1q (18 h) was required to elicit a dampening of TNFα production from mouse macrophages, the human macrophages responded to C1q with immediate downregulation of TNFα. IL-6 production was unchanged in mouse and upregulated by human macrophages following prolonged stimulation with C1q. Our previous studies indicated that C1q programmed enhanced efferocytosis in mouse macrophages by enhancing expression of Mer tyrosine kinase and its ligand Gas6, a receptor-ligand pair that also inhibits proinflammatory signaling. Here, we demonstrated that C1q-dependent programming of human macrophage efferocytosis required protein synthesis; however, neither Mer nor the related receptor Axl was upregulated in human cells. In addition, while the C1q-collagen-like tails are sufficient for promoting C1q-dependent phagocytosis of antibody-coated targets, the C1q-tails failed to program enhanced efferocytosis or dampen TNFα production. These data further elucidate the mechanisms by which C1q regulates proinflammatory signaling and efferocytosis in macrophages, functions that are likely to influence the progression of autoimmunity and chronic inflammation.

A novel double-negative feedback loop between miR-489 and the HER2-SHP2-MAPK signaling axis regulates breast cancer cell proliferation and tumor growth.

Human epidermal growth factor receptor 2 (HER2 or ErBb2) is a receptor tyrosine kinase overexpressed in 20-30% of breast cancers and associated with poor prognosis and outcome. Dysregulation of several microRNAs (miRNAs) plays a key role in breast cancer progression and metastasis. In this study, we screened and identified miRNAs dysregualted in HER2-positive breast cancer cells. Our molecular study demonstrated that miR-489 was specifically downregulated by the HER2-downstream signaling, especially through the MAPK pathway. Restoration or overexpression of miR-489 in HER2-positive breast cancer cells significantly inhibited cell growth in vitro and decreased the tumorigenecity and tumor growth in xenograft mice. Mechanistically, we found that overexpression of miR-489 led to the decreased levels of HER2 and SHP2 and thus attenuated HER2-downstream signaling. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2-SHP2-MAPK and miR-489 signaling pathways form a mutually inhibitory loop. Using quantitative real-time PCR analysis and Fluorescent in situ hybridization technique (FISH), we found that miR-489 was expressed at significantly lower level in tumor tissues compared to the adjacent normal tissues. Downregulation of miR-489 in breast cancers was associated with aggressive tumor phenotypes. Overall, our results define a double-negative feedback loop involving miR-489 and the HER2-SHP2-MAPK signaling axis that can regulate breast cancer cell proliferation and tumor progression and might have therapeutic relevance for HER2-positive breast cancer.

Transplant-induced reactivation of murine cytomegalovirus immediate early gene expression is associated with recruitment of NF-κB and AP-1 to the major immediate early promoter.

Reactivation of latent human cytomegalovirus is a significant infectious complication of organ transplantation and current therapies target viral replication once reactivation of latent virus has already occurred. The specific molecular pathways that activate viral gene expression in response to transplantation are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. Murine cytomegalovirus (MCMV) is a valuable model for studying latency and reactivation of CMV in vivo. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces reactivation of immediate early (IE) gene expression and epigenetic reprogramming of the major immediate early promoter (MIEP) within 48 h. We hypothesize that these events are mediated by activation of signalling pathways that lead to binding of transcription factors to the MIEP, including AP-1 and NF-κB. Here we show that transplantation induces rapid activation of several members of the AP-1 and NF-κB transcription factor family and we demonstrate that canonical NF-κB (p65/p50), the junD component of AP-1, and nucleosome remodelling complexes are recruited to the MIEP following transplantation. Proteomic analysis of recipient plasma and transcriptome analysis of kidney RNA identified five extracellular ligands, including TNF, IL-1ß, IL-18, CD40L and IL-6, and three intracellular signalling pathways associated with reactivation of IE gene expression. Identification of the factors that mediate activation of these signalling pathways may eventually lead to new therapies to prevent reactivation of CMV and its sequelae.

Double-Negative αß T Cells Are Early Responders to AKI and Are Found in Human Kidney.

Ischemia-reperfusion injury (IRI) is a major cause of AKI, and previous studies established important roles for conventional CD4(+) T cells, natural killer T cells, and CD4(+)CD25(+)FoxP3(+) Tregs in AKI pathogenesis. We recently identified CD4(-)CD8(-) (double-negative; DN) T cells as an important subset of αß T cell receptor-positive cells residing in mouse kidney. However, little is known about the pathophysiologic functions of kidney DN T cells. In this study, we phenotypically and functionally characterized murine kidney DN T cells in the steady state and in response to IRI. Unlike CD4(+) and CD8(+) T cells, DN T cells in the steady state expressed high levels of CD69, CD28, and CD40L; differentially expressed IL-27 and IL-10 anti-inflammatory cytokines; spontaneously proliferated at a very high rate; and suppressed in vitro proliferation of activated CD4(+) T cells. Within the first 3-24 hours after IRI, kidney DN T cells expanded significantly and upregulated expression of IL-10. In adoptive transfer experiments, DN T cells significantly protected recipients from AKI by an IL-10-dependent mechanism. DN T cells also made up a large fraction of the T cell compartment in human kidneys. Our results indicate that DN T cells are an important subset of the resident αß(+) T cell population in the mammalian kidney and are early responders to AKI that have anti-inflammatory properties.