Howell F. Moffett

Comprehensive Profiling of Epstein-Barr Virus MicroRNAs in Nasopharyngeal Carcinoma

ABSTRACT Epstein-Barr Virus (EBV) establishes a long-term latent infection and is associated with a number of human malignancies that are thought to arise from deregulation of different stages of the viral life cycle. Recently, a large number of microRNAs (miRNAs) have been described for EBV, and it has been suggested that their expression may vary between the different latency states found in normal and malignant tissue. To date, however, no technique has been utilized to comprehensively and quantitatively test this idea by profiling expression of the EBV miRNAs in primary infected tissues. We describe here a multiplex reverse transcription-PCR assay that allows the profiling of 39 of the 40 known mature EBV miRNAs from as little as 250 ng of RNA. With this approach, we present a comprehensive profile of EBV miRNAs in primary nasopharyngeal carcinoma (NPC) tumors including estimates of miRNA copy number per tumor cell. This is the first comprehensive profiling of EBV miRNAs in any EBV-associated tumor. In contrast to previous suggestions, we show that the BART-derived miRNAs are present in a wide range of copy numbers from ≤103 per cell in both primary tumors and the widely used NPC-derived C666-1 cell line. However, we confirm the hypothesis that the BHRF1 miRNAs are not expressed in NPC. Lastly, we demonstrate that EBV miRNA expression in the widely used NPC line C666-1 is, with some caveats, broadly representative of primary NPC tumors.

Heat Shock Protein-Mediated Cross-Presentation of Exogenous HIV Antigen on HLA Class I and Class II

Strong CD4+ and CD8+ T cell responses are considered important immune components for controlling HIV infection, and their priming may be central to an effective HIV vaccine. We describe in this study an approach by which multiple CD4+ and CD8+ T cell epitopes are processed and presented from an exogenously added HIV-1 Gag-p24 peptide of 32 aa complexed to heat shock protein (HSP) gp96. CD8+ T cell recognition of the HSP/peptide complex, but not the peptide alone, was inhibited by brefeldin A, suggesting an endoplasmic reticulum-dependent pathway. This is the first report to describe efficient processing and simultaneous presentation of overlapping class I- and class II-restricted epitopes from the same extracellularly added precursor peptide complexed to HSP. Given previous reports of the strong immunogenicity of HSP/peptide complexes, the present data suggest that HSP-complexed peptides containing multiple MHC class I- and class II-restricted epitopes represent potential vaccine candidates for HIV and other viral infections suitable to induce effective CTL memory by simultaneously providing CD4 T cell help.

Stable inhibitory activity of regulatory T cells requires the transcription factor Helios

How T cells maintain their identity Although best known for their pathogen-fighting prowess, T lymphocytes also ensure that the immune response does not run amok. A subset of T cells called regulatory T cells (Tregs) performs this function by, for example, making sure T cells only attack pathogens and not self. T cells can exhibit plasticity in their functions in the face of an inflammatory stimulus. Kim et al. sought to identify the molecules that ensure the stable maintenance of Tregs. Using genetically modified mice, they found that both CD4+ and CD8+ Tregs require the transcription factor Helios to stably maintain their identity. Science, this issue p. 334 Regulatory T cells require the transcription factor Helios to stably maintain their identity. The maintenance of immune homeostasis requires regulatory T cells (Tregs). Given their intrinsic self-reactivity, Tregs must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3+ CD4 and Qa-1–restricted CD8 Tregs results in defective regulatory activity and autoimmunity in mice. Helios-deficient Tregs develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 Tregs also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes Tregs in the face of inflammatory responses provides a genetic explanation for a core property of Tregs.

Beyond Help: Direct Effector Functions of Human Immunodeficiency Virus Type 1-Specific CD4+ T Cells

ABSTRACT The immune correlates of protection in human immunodeficiency virus type 1 (HIV-1) infection remain poorly defined, particularly the contribution of CD4+ T cells. Here we explore the effector functions of HIV-1-specific CD4+ T cells. We demonstrate HIV-1 p24-specific CD4+-T-cell cytolytic activity in peripheral blood mononuclear cells directly ex vivo and after enrichment by antigen-specific stimulation. We further show that in a rare long-term nonprogressor, both an HIV-1-specific CD4+-T-cell clone and CD4+ T cells directly ex vivo exert potent suppression of HIV-1 replication. Suppression of viral replication was dependent on cell-cell contact between the effector CD4+ T cells and the target cells. While the antiviral effector activity of CD8+ T cells has been well documented, these results strongly suggest that HIV-1-specific CD4+ T cells are capable of directly contributing to antiviral immunity.

Genomic basis for the convergent evolution of electric organs

Only one way to make an electric organ? Electric fish have independently evolved electric organs that help them to communicate, navigate, hunt, and defend themselves. Gallant et al. analyzed the genome of the electric eel and the genes expressed in two other distantly related electric fish. The same genes were recruited within the different species to make evolutionarily new structures that function similarly. Science, this issue p. 1522 Multiple divergent fish lineages have used the same evolutionary toolkit to produce electric organs. Little is known about the genetic basis of convergent traits that originate repeatedly over broad taxonomic scales. The myogenic electric organ has evolved six times in fishes to produce electric fields used in communication, navigation, predation, or defense. We have examined the genomic basis of the convergent anatomical and physiological origins of these organs by assembling the genome of the electric eel (Electrophorus electricus) and sequencing electric organ and skeletal muscle transcriptomes from three lineages that have independently evolved electric organs. Our results indicate that, despite millions of years of evolution and large differences in the morphology of electric organ cells, independent lineages have leveraged similar transcription factors and developmental and cellular pathways in the evolution of electric organs.

Multiple effector functions mediated by human immunodeficiency virus-specific CD4(+) T-cell clones.

ABSTRACT Mounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cells contribute to effective antiviral control, but their functional characteristics and the precise epitopes targeted by this response remain to be defined. In this study, we generated CD4+ T-cell clones specific for Gag from HIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells. Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG (amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examined induced cell proliferation, gamma interferon (IFN-γ) secretion, and cytolytic activity. Cytolysis was abrogated by concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediated mechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker for cytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate that T helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be a component of the antiviral effect of these cells.

Not miR-ly small RNAs: big potential for microRNAs in therapy.

RNA interference (RNAi) describes a set of natural processes in which genes are silenced by small RNAs. RNAi has been widely used as an experimental tool that has recently become the focus of drug development efforts to treat a variety of diseases and disorders. Like all molecular therapies, in vivo delivery is the major hurdle to realizing therapeutic RNAi. Several strategies have been developed that increase small RNA half-life in the blood, facilitate transduction across biological membranes, and mediate cell-specific delivery. Importantly, these strategies permit targeting of mRNAs as well as microRNAs (miRNAs), a class of small RNAs encoded in the genome. miRNAs are required for multiple developmental and cellular processes. Dysfunction of miRNAs can result in a host of pathologies, suggesting that miRNAs are potential targets of therapy. Recent studies of miRNA function in immune-specific pathways indicate that specific miRNAs might be exploited as therapeutic targets to treat immune disorders, including autoimmunity, allergy, and hematopoietic cancers.

MicroRNA Expression Profiling Identifies Activated B Cell Status in Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70+ and IgVH unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL.

Fine specificity and cross-clade reactivity of HIV type 1 Gag-specific CD4+ T cells.

Despite growing evidence that HIV-1-specific CD4(+) T helper (Th) cells may play a role in the control of viremia, discrete Th cell epitopes remain poorly defined. Furthermore, it is not known whether Th cell responses generated using vaccines based on clade B virus sequences will elicit immune responses that are effective in regions of the world where non-clade B viruses predominate. To address these issues we isolated CD4(+) T cell clones from individuals with vigorous HIV-1-specific Th cell responses and identified the minimum epitopes recognized. The minimum peptide length required for induction of CD4(+) T cell proliferation, IFN-gamma secretion, and cytolytic activity ranged from 9 to 16 amino acids in the five epitopes studied. Cross-clade recognition of the defined epitopes was examined for variant peptides from clades A, B, C, D, and AE. Over half the variant epitopes (17 of 32) exhibited impaired recognition, defined as less than 50% of the IFN-gamma secretion elicited by B clade consensus sequence. There was no evidence for antagonistic activity mediated by the variant peptides, and despite strong responses there was no escape of autologous virus from Th responses in the epitopes we studied. Abrogated recognition of variant CD4(+) T cell epitopes presents a potential obstacle to vaccine development.

The microRNA miR-31 inhibits CD8+ T cell function in chronic viral infection

During infection, antigen-specific T cells undergo tightly regulated developmental transitions controlled by transcriptional and post-transcriptional regulation of gene expression. We found that the microRNA miR-31 was strongly induced by activation of the T cell antigen receptor (TCR) in a pathway involving calcium and activation of the transcription factor NFAT. During chronic infection with lymphocytic choriomeningitis virus (LCMV) clone 13, miR-31-deficent mice recovered from clinical disease, while wild-type mice continued to show signs of disease. This disease phenotype was explained by the presence of larger numbers of cytokine-secreting LCMV-specific CD8+ T cells in miR-31-deficent mice than in wild-type mice. Mechanistically, miR-31 increased the sensitivity of T cells to type I interferons, which interfered with effector T cell function and increased the expression of several proteins related to T cell dysfunction during chronic infection. These studies identify miR-31 as an important regulator of T cell exhaustion in chronic infection.