Thomas N. Denny

Tissue-Specific Genetic Control of Splicing: Implications for the Study of Complex Traits

Numerous genome-wide screens for polymorphisms that influence gene expression have provided key insights into the genetic control of transcription. Despite this work, the relevance of specific polymorphisms to in vivo expression and splicing remains unclear. We carried out the first genome-wide screen, to our knowledge, for SNPs that associate with alternative splicing and gene expression in human primary cells, evaluating 93 autopsy-collected cortical brain tissue samples with no defined neuropsychiatric condition and 80 peripheral blood mononucleated cell samples collected from living healthy donors. We identified 23 high confidence associations with total expression and 80 with alternative splicing as reflected by expression levels of specific exons. Fewer than 50% of the implicated SNPs however show effects in both tissue types, reflecting strong evidence for distinct genetic control of splicing and expression in the two tissue types. The data generated here also suggest the possibility that splicing effects may be responsible for up to 13 out of 84 reported genome-wide significant associations with human traits. These results emphasize the importance of establishing a database of polymorphisms affecting splicing and expression in primary tissue types and suggest that splicing effects may be of more phenotypic significance than overall gene expression changes.

Phenotypic properties of transmitted founder HIV-1

Defining the virus–host interactions responsible for HIV-1 transmission, including the phenotypic requirements of viruses capable of establishing de novo infections, could be important for AIDS vaccine development. Previous analyses have failed to identify phenotypic properties other than chemokine receptor 5 (CCR5) and CD4+ T-cell tropism that are preferentially associated with viral transmission. However, most of these studies were limited to examining envelope (Env) function in the context of pseudoviruses. Here, we generated infectious molecular clones of transmitted founder (TF; n = 27) and chronic control (CC; n = 14) viruses of subtypes B (n = 18) and C (n = 23) and compared their phenotypic properties in assays specifically designed to probe the earliest stages of HIV-1 infection. We found that TF virions were 1.7-fold more infectious (P = 0.049) and contained 1.9-fold more Env per particle (P = 0.048) compared with CC viruses. TF viruses were also captured by monocyte-derived dendritic cells 1.7-fold more efficiently (P = 0.035) and more readily transferred to CD4+ T cells (P = 0.025). In primary CD4+ T cells, TF and CC viruses replicated with comparable kinetics; however, when propagated in the presence of IFN-α, TF viruses replicated to higher titers than CC viruses. This difference was significant for subtype B (P = 0.000013) but not subtype C (P = 0.53) viruses, possibly reflecting demographic differences of the respective patient cohorts. Together, these data indicate that TF viruses are enriched for higher Env content, enhanced cell-free infectivity, improved dendritic cell interaction, and relative IFN-α resistance. These viral properties, which likely act in concert, should be considered in the development and testing of AIDS vaccines.

Influence of HLA-C expression level on HIV control

Thwarting HIV Multiple genome-wide association studies have revealed that human leukocyte antigen (HLA) genes of the major histocompatibility complex locus have the strongest impact on HIV. In particular, a single-nucleotide polymorphism 35 base pairs upstream of HLA-C shows significant association with viral load and protection against HIV. How HLA-C mediates these effects is unknown. Apps et al. (p. 87) now demonstrate that increasing surface expression of HLA-C is associated with reduced viral load and reduced rate of progression to low CD4+ T cell counts in African and European Americans. High HLA-C expression likely promoted improved HIV control through a more effective cytotoxic CD8+ T cell response. In contrast to HIV infection, high HLA-C expression was associated with a higher risk of the inflammatory bowel disease, Crohns disease. Increased levels of human leukocyte antigen C are associated with control of HIV infection but increased susceptibility to Crohn’s disease. A variant upstream of human leukocyte antigen C (HLA-C) shows the most significant genome-wide effect on HIV control in European Americans and is also associated with the level of HLA-C expression. We characterized the differential cell surface expression levels of all common HLA-C allotypes and tested directly for effects of HLA-C expression on outcomes of HIV infection in 5243 individuals. Increasing HLA-C expression was associated with protection against multiple outcomes independently of individual HLA allelic effects in both African and European Americans, regardless of their distinct HLA-C frequencies and linkage relationships with HLA-B and HLA-A. Higher HLA-C expression was correlated with increased likelihood of cytotoxic T lymphocyte responses and frequency of viral escape mutation. In contrast, high HLA-C expression had a deleterious effect in Crohn’s disease, suggesting a broader influence of HLA expression levels in human disease.

High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.

Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (V(H) and V(L)) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig V(H) and V(L) genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable V(H) or V(L) genes. The utility of these Ig gene expression cassettes was established using synthetic V(H) or V(L) genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using V(H) and V(L) genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning.

Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated

Many HIV-1 envelope-reactive antibodies shortly after HIV-1 transmission may arise from crow-reactive memory B cells previously stimulated by non-HIV-1 host or microbial antigens

Inhibition of Human Immunodeficiency Virus Type 1 Transcription by Chemical Cyclin-Dependent Kinase Inhibitors

ABSTRACT Cyclin-dependent kinases (cdks) have recently been suggested to regulate human immunodeficiency virus type 1 (HIV-1) transcription. Previously, we have shown that expression of one cdk inhibitor, p21/Waf1, is abrogated in HIV-1 latently infected cells. Based on this result, we investigated the transcription of HIV-1 in the presence of chemical drugs that specifically inhibited cdk activity and functionally mimicked p21/Waf1 activity. HIV-1 production in virally integrated lymphocytic and monocytic cell lines, such as ACH2, 8E5, and U1, as well as activated peripheral blood mononuclear cells infected with syncytium-inducing (SI) or non-syncytium-inducing (NSI) HIV-1 strains, were all inhibited by Roscovitine, a purine derivative that reversibly competes for the ATP binding site present in cdks. The decrease in viral progeny in the HIV-1-infected cells was correlated with a decrease in the transcription of HIV-1 RNAs in cells treated with Roscovitine and not with the non-cdk general cell cycle inhibitors, such as hydroxyurea (G1/S blocker) or nocodazole (M-phase blocker). Cyclin A- and E-associated histone H1 kinases, as well as cdk 7 and 9 activities, were all inhibited in the presence of Roscovitine. The 50% inhibitory concentration of Roscovitine on cdks 9 and 7 was determined to be ∼0.6 μM. Roscovitine could selectively sensitize HIV-1-infected cells to apoptosis at concentrations that did not impede the growth and proliferation of uninfected cells. Apoptosis induced by Roscovitine was found in both latent and activated infected cells, as evident by Annexin V staining and the cleavage of the PARP protein by caspase-3. More importantly, contrary to many apoptosis-inducing agents, where the apoptosis of HIV-1-infected cells accompanies production and release of infectious HIV-1 viral particles, Roscovitine treatment selectively killed HIV-1-infected cells without virion release. Collectively, our data suggest that cdks are required for efficient HIV-1 transcription and, therefore, we propose specific cdk inhibitors as potential antiviral agents in the treatment of AIDS.

Effects of one year of supplementation with zinc and other micronutrients on cellular immunity in the elderly.

The objective of this study was to determine the effects of a year of Zn supplementation on Zn concentrations in circulating cells and on cellular immune functions in the elderly. Subjects, aged 60-89, were given a placebo, 15 mg Zn, or 100 mg Zn daily for 12 months. All subjects also received a multivitamin/mineral supplement that contained no additional Zn. Blood samples were drawn and immune functions assessed prior to and at 3, 6, 12, and 16 months after beginning Zn supplementation. Subject diets were also assessed at each visit. Dietary folate, pyridoxine, alpha-tocopherol, copper, zinc, and magnesium were consistently below recommended intakes. Although plasma Zn increased significantly in the 100 mg Zn treatment group, concentrations of Zn in erythrocytes, mononuclear cells, polymorphonuclear leukocytes, and platelets were not significantly increased by zinc supplementation. Natural killer cell activity was transiently enhanced by the 100 mg/day dose of Zn. There was a progressive improvement in delayed dermal hypersensitivity (DDH) and in lymphocyte proliferative responses to two mitogens; this may have been due to one or more components of the multivitamin/mineral supplement administered to all study subjects. The enhancement of DDH was significantly greater in the placebo group than in either zinc treatment group. Thus, zinc had a beneficial effect on one measure of cellular immune function while simultaneously having an adverse effect on another measure of cellular immunity.

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination.

Background During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection. Methods and Findings To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject. Conclusion The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

Depressed interleukin-12-producing activity by monocytes correlates with adverse clinical course and a shift toward Th2-type lymphocyte pattern in severely injured male trauma patients

OBJECTIVE To determine the effect of major trauma on the cytokine-producing activity of monocytes and CD4+ T cells in a homogeneous cohort of patients as well as to determine the relationship between monocyte and T-lymphocyte responses and clinical outcome. SETTINGS Surgical intensive care units of a trauma center and flow cytometry and experimental laboratories at a teaching hospital. DESIGN Prospective cohort clinical study with measurements of white cell cytokine-producing activity on days 2, 5, and 10 postinjury. The number of cytokine-producing CD14+ monocytes, CD4+, and CD8+ T cells were determined in whole blood using flow cytometry combined with the intracellular cytokine staining method. Basal and lipopolysaccharide-stimulated interleukin (IL)-12, tumor necrosis factor-alpha, IL-6, and IL-1alpha production by monocytes as well as basal and phorbol 12-myristate 13-acetate plus ionomycin-stimulated interferon-gamma, IL-4, and tumor necrosis factor-alpha production by T cells were determined on days 2, 5, and 10 postinjury and compared with similar measurements made in healthy control subjects. PATIENTS Twelve randomly selected black, male patients were enrolled in the study: mean injury severity score, 26; mean age, 35 yrs; mean Glasgow Coma Scale score, 13; systemic inflammatory response syndrome, 92%; sepsis, 42%; bronchial infection, 42%; and adult respiratory distress syndrome 25%. MAIN RESULTS After lipopolysaccharide stimulation, the number of IL-12-, tumor necrosis factor-alpha-, IL-1alpha-, and IL-6-producing CD14+ monocytes was 40% to 70% lower in trauma patients on postinjury days 2, 5, and 10 than in healthy control subjects. After phorbol 12-myristate 13-acetate stimulation, the number of IL-4-producing CD4+ cells increased three-fold in the trauma patients compared with healthy control subjects. In contrast, the number of interferon-gamma- or tumor necrosis factor-alpha-producing CD4+ and CD8+ T cells was not different between the patients and control subjects. The Th1/Th2 ratio was significantly lower in patients on all postinjury days than in the control subjects. A statistically significant inverse correlation was found between the number of IL-12-producing monocytes and IL-4-producing CD4+ T cells in trauma patients (p =.007, r2 =.47). This correlation was absent in control subjects. The degree of depressed capacity of monocyte IL-12 production on day 2 postinjury showed a statistically significant correlation with the development of adult respiratory distress syndrome, sepsis, or infections and also with the duration of systemic inflammatory response syndrome and sepsis. CONCLUSIONS Major trauma results in an early and marked decrease in monocyte cytokine-producing activity. The trauma-induced depression in IL-12 production by the mononuclear phagocyte system may promote T-cell commitment toward a Th2 pattern early after trauma. The appearance of the Th2 pattern is the result of elevated numbers of IL-4-producing cells without major alterations in T-cell interferon-gamma-producing capacity. The degree of alterations in monocyte and T-cell responses on day 2 postinjury correlates with the development of adverse clinical outcomes and the subsequent duration of the inflammatory response.

Phenotypic and Functional Profile of HIV-Inhibitory CD8 T Cells Elicited by Natural Infection and Heterologous Prime/Boost Vaccination

ABSTRACT Control of HIV-1 replication following nonsterilizing HIV-1 vaccination could be achieved by vaccine-elicited CD8+ T-cell-mediated antiviral activity. To date, neither the functional nor the phenotypic profiles of CD8+ T cells capable of this activity are clearly understood; consequently, little is known regarding the ability of vaccine strategies to elicit them. We used multiparameter flow cytometry and viable cell sorts from phenotypically defined CD8+ T-cell subsets in combination with a highly standardized virus inhibition assay to evaluate CD8+ T-cell-mediated inhibition of viral replication. Here we show that vaccination against HIV-1 Env and Gag-Pol by DNA priming followed by recombinant adenovirus type 5 (rAd5) boosting elicited CD8+ T-cell-mediated antiviral activity against several viruses with either lab-adapted or transmitted virus envelopes. As it did for chronically infected virus controllers, this activity correlated with HIV-1-specific CD107a or macrophage inflammatory protein 1β (MIP-1β) expression from HIV-1-specific T cells. Moreover, for vaccinees or virus controllers, purified memory CD8+ T cells from a wide range of differentiation stages were capable of significantly inhibiting virus replication. Our data define attributes of an antiviral CD8+ T-cell response that may be optimized in the search for an efficacious HIV-1 vaccine.