MicroRNAs regulate T-cell production of interleukin-9 and identify hypoxia-inducible factor-2a as an important regulator of T helper 9 and regularoty T-cell differentiation

MicroRNAs (miRNAs) regulate many aspects of helper T cell (Th) development and function. Here we found that they are required for the suppression of interleukin‐9 (IL‐9) expression in Th9 cells and other Th subsets. Two highly related miRNAs (miR‐15b and miR‐16) that we previously found to play an important role in regulatory T (Treg) cell differentiation were capable of suppressing IL‐9 expression when they were over‐expressed in Th9 cells. We used these miRNAs as tools to identify novel regulators of IL‐9 expression and found that they could regulate the expression of Epas1, which encodes hypoxia‐inducible factor (HIF)‐2α. HIF proteins regulate metabolic pathway usage that is important in determining appropriate Th differentiation. The related protein, HIF‐1α enhances Th17 differentiation and inhibits Treg cell differentiation. Here we found that HIF‐2α was required for IL‐9 expression in Th9 cells, but its expression was not sufficient in other Th subsets. Furthermore, HIF‐2α suppressed Treg cell differentiation like HIF‐1α, demonstrating both similar and distinct roles of the HIF proteins in Th differentiation and adding a further dimension to their function. Ironically, even though miR‐15b and miR‐16 suppressed HIF‐2α expression in Treg cells, inhibiting their function in Treg cells did not lead to an increase in IL‐9 expression. Therefore, the physiologically relevant miRNAs that regulate IL‐9 expression in Treg cells and other subsets remain unknown. Nevertheless, the analysis of miR‐15b and miR‐16 function led to the discovery of the importance of HIF‐2α so this work demonstrated the utility of studying miRNA function to identify novel regulatory pathways in helper T‐cell development

The Blood of Healthy Individuals Exhibits CD8 T Cells with a Highly Altered TCR Vb Repertoire but with an Unmodified Phenotype

CD8 T cell clonal expansions (TCE) have been observed in elderly, healthy individuals as well in old mice, and have been associated with the ageing process. Both chronic latent and non-persistent viral infections have been proposed to drive the development of distinct non-functional and functional TCE respectively. Biases in TCR Vβ repertoire diversity are also recurrently observed in patients that have undergone strong immune challenge, and are preferentially observed in the CD8 compartment. Healthy adults can also exhibit CD8 T cells with strong alterations of their CDR3 length distribution. Surprisingly, no specific investigations have been conducted to analyze the CD8 T cell repertoire in normal adults, to determine if such alterations in TCR Vβ repertoire share the features of TCE. In this study, we characterized the phenotype and function of the CD8 population in healthy individuals of 25–52 years of age. All but one of the EBV-positive HLA-B8 healthy volunteers that were studied were CMV-negative. Using a specific unsupervised statistical method, we identified Vβ families with altered CDR3 length distribution and increased TCR Vβ/HPRT transcript ratios in all individuals tested. The increase in TCR Vβ/HPRT transcript ratio was more frequently associated with an increase in the percentage of the corresponding Vβ+ T cells than with an absence of modification of their percentage. However, in contrast with the previously described TCE, these CD8+ T cells were not preferentially found in the memory CD8 subset, they exhibited normal effector functions (cytokine secretion and cytotoxic molecule expression) and they were not reactive to a pool of EBV/CMV/Flu virus peptides. Taken together, the combined analysis of transcripts and proteins of the TCR Vβ repertoire led to the identification of different types of CD8+ T cell clone expansion or contraction in healthy individuals, a situation that appears more complex than previously described in aged individuals

A sequence-independent in vitro transposon-based strategy for efficient cloning of genomes of large DNA viruses as bacterial artificial chromosomes

Bacterial artificial chromosomes (BACs) derived from genomes of large DNA viruses are powerful tools for functional delineation of viral genes. Current methods for cloning the genomes of large DNA viruses as BACs require prior knowledge of the viral sequences or the cloning of viral DNA fragments, and are tedious because of the laborious process of multiple plaque purifications, which is not feasible for some fastidious viruses. Here, we describe a novel method for cloning the genomes of large DNA viruses as BACs, which entails direct in vitro transposition of viral genomes with a BAC cassette, and subsequent recovery in Escherichia coli. Determination of insertion sites and adjacent viral sequences identify the BAC clones for genetic manipulation and functional characterization. Compared to existing methods, this new approach is highly efficient, and does not require any information on viral sequences or cloning of viral DNA fragments, and plaque purifications. This method could potentially be used for discovering previously unidentified viruses

Workshop on Immunizations in Older Adults: Identifying Future Research Agendas

Goals for immunization in older adults may differ from those in young adults and children, in whom complete prevention of disease is the objective. Often, reduced hospitalization and death but also averting exacerbation of underlying chronic illness, functional decline, and frailty are important goals in the older age group. Because of the effect of age on dendritic cell function, T cell-mediated immune suppression, reduced proliferative capacity of T cells, and other immune responses, the efficacy of vaccines often wanes with advanced age. This article summarizes the discussion and proceedings of a workshop organized by the Association of Specialty Professors, the Infectious Diseases Society of America, the American Geriatrics Society, the National Institute on Aging, and the National Institute of Allergy and Infectious Diseases. Leading researchers and clinicians in the fields of immunology, epidemiology, infectious diseases, geriatrics, and gerontology reviewed the current status of vaccines in older adults, identified knowledge gaps, and suggest priority areas for future research. The goal of the workshop was to identify what is known about immunizations (efficacy, effect, and current schedule) in older adults and to recommend priorities for future research. Investigation in the areas identified has the potential to enhance understanding of the immune process in aging individuals, inform vaccine development, and lead to more-effective strategies to reduce the risk of vaccine-preventable illness in older adults.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79340/1/j.1532-5415.2010.02772.x.pd

Nutrition, diet and immunosenescence

Ageing is characterized by immunosenescence and the progressive decline in immunity in association with an increased frequency of infections and chronic disease. This complex process affects both the innate and adaptive immune systems with a progressive decline in most immune cell populations and defects in activation resulting in loss of function. Although host genetics and environmental factors, such as stress, exercise and diet can impact on the onset or course of immunosenescence, the mechanisms involved are largely unknown. This review focusses on identifying the most significant aspects of immunosenescence and on the evidence that nutritional intervention might delay this process, and consequently improve the quality of life of the elderly

NPEPPS Is a Druggable Driver of Platinum Resistance

There is an unmet need to improve the efficacy of platinum-based cancer chemotherapy, which is used in primary and metastatic settings in many cancer types. In bladder cancer, platinum-based chemotherapy leads to better outcomes in a subset of patients when used in the neoadjuvant setting or in combination with immunotherapy for advanced disease. Despite such promising results, extending the benefits of platinum drugs to a greater number of patients is highly desirable. Using the multiomic assessment of cisplatin-responsive and -resistant human bladder cancer cell lines and whole-genome CRISPR screens, we identified puromycin-sensitive aminopeptidase (NPEPPS) as a driver of cisplatin resistance. NPEPPS depletion sensitized resistant bladder cancer cells to cisplatin in vitro and in vivo. Conversely, overexpression of NPEPPS in sensitive cells increased cisplatin resistance. NPEPPS affected treatment response by regulating intracellular cisplatin concentrations. Patient-derived organoids (PDO) generated from bladder cancer samples before and after cisplatin-based treatment, and from patients who did not receive cisplatin, were evaluated for sensitivity to cisplatin, which was concordant with clinical response. In the PDOs, depletion or pharmacologic inhibition of NPEPPS increased cisplatin sensitivity, while NPEPPS overexpression conferred resistance. Our data present NPEPPS as a druggable driver of cisplatin resistance by regulating intracellular cisplatin concentrations.</p

Tolerance induction in memory CD4 T cells is partial and reversible

Memory T cells respond rapidly in part because they are less reliant on heightened levels of costimulatory molecules. This enables rapid control of secondary infecting pathogens but presents challenges to efforts to control or silence memory CD4 T cells, for example in antigen specific tolerance strategies for autoimmunity. We have examined the transcriptional and functional consequences of re‐activating memory CD4 T cells in the absence of an adjuvant. We find that memory CD4 T cells generated by infection or immunisation survive secondary activation with antigen delivered without adjuvant, regardless of their location in secondary lymphoid organs or peripheral tissues. These cells were, however, functionally altered following a tertiary immunisation with antigen and adjuvant, proliferating poorly but maintaining their ability to produce inflammatory cytokines. Transcriptional and cell cycle analysis of these memory CD4 T cells suggest they are unable to commit fully to cell division potentially because of low expression of DNA repair enzymes. In contrast, these memory CD4 T cells could proliferate following tertiary reactivation by viral re‐infection. These data indicate that antigen specific tolerogenic strategies must examine multiple parameters of T cell function, and provide insight into the molecular mechanisms that may lead to deletional tolerance of memory CD4 T cells

A Novel Cre Recombinase Imaging System for Tracking Lymphotropic Virus Infection In Vivo

BACKGROUND:Detection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells. METHODOLOGY/PRINCIPAL FINDINGS:Murine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow. CONCLUSIONS/SIGNIFICANCE:The use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections

Gammaherpesvirus-Driven Plasma Cell Differentiation Regulates Virus Reactivation from Latently Infected B Lymphocytes

Gammaherpesviruses chronically infect their host and are tightly associated with the development of lymphoproliferative diseases and lymphomas, as well as several other types of cancer. Mechanisms involved in maintaining chronic gammaherpesvirus infections are poorly understood and, in particular, little is known about the mechanisms involved in controlling gammaherpesvirus reactivation from latently infected B cells in vivo. Recent evidence has linked plasma cell differentiation with reactivation of the human gammaherpesviruses EBV and KSHV through induction of the immediate-early viral transcriptional activators by the plasma cell-specific transcription factor XBP-1s. We now extend those findings to document a role for a gammaherpesvirus gene product in regulating plasma cell differentiation and thus virus reactivation. We have previously shown that the murine gammaherpesvirus 68 (MHV68) gene product M2 is dispensable for virus replication in permissive cells, but plays a critical role in virus reactivation from latently infected B cells. Here we show that in mice infected with wild type MHV68, virus infected plasma cells (ca. 8% of virus infected splenocytes at the peak of viral latency) account for the majority of reactivation observed upon explant of splenocytes. In contrast, there is an absence of virus infected plasma cells at the peak of latency in mice infected with a M2 null MHV68. Furthermore, we show that the M2 protein can drive plasma cell differentiation in a B lymphoma cell line in the absence of any other MHV68 gene products. Thus, the role of M2 in MHV68 reactivation can be attributed to its ability to manipulate plasma cell differentiation, providing a novel viral strategy to regulate gammaherpesvirus reactivation from latently infected B cells. We postulate that M2 represents a new class of herpesvirus gene products (reactivation conditioners) that do not directly participate in virus replication, but rather facilitate virus reactivation by manipulating the cellular milieu to provide a reactivation competent environment