Longitudinal transcriptomic characterization of the immune response to acute hepatitis C virus infection in patients with spontaneous viral clearance.

Most individuals exposed to hepatitis C virus (HCV) become persistently infected while a minority spontaneously eliminate the virus. Although early immune events influence infection outcome, the cellular composition, molecular effectors, and timeframe of the host response active shortly after viral exposure remain incompletely understood. Employing specimens collected from people who inject drugs (PWID) with high risk of HCV exposure, we utilized RNA-Seq and blood transcriptome module (BTM) analysis to characterize immune function in peripheral blood mononuclear cells (PBMC) before, during, and after acute HCV infection resulting in spontaneous resolution. Our results provide a detailed description of innate immune programs active in peripheral blood during acute HCV infection, which include prominent type I interferon and inflammatory signatures. Innate immune gene expression rapidly returns to pre-infection levels upon viral clearance. Comparative analyses using peripheral blood gene expression profiles from other viral and vaccine studies demonstrate similarities in the immune responses to acute HCV and flaviviruses. Of note, both acute dengue virus (DENV) infection and acute HCV infection elicit similar innate antiviral signatures. However, while transient in DENV infection, this signature was sustained for many weeks in the response to HCV. These results represent the first longitudinal transcriptomic characterization of human immune function in PBMC during acute HCV infection and identify several dynamically regulated features of the complex response to natural HCV exposure.

Immune activation alters cellular and humoral responses to yellow fever 17D vaccine.

BACKGROUND: Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. METHODS: We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. RESULTS: We showed that YF-17D-induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D-neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. CONCLUSION: Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. TRIAL REGISTRATION: Registration is not required for observational studies. FUNDING: This study was funded by Canada's Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development.

CD80 and CD86 IgC domains are important for quaternary structure, receptor binding and co-signaling function.

CD86 and CD80, the ligands for the co-stimulatory molecules CD28 and CTLA-4, are members of the Ig superfamily. Their structure includes Ig variable-like (IgV) domains, Ig constant-like (IgC) domains and intracellular domains. Although crystallographic studies have clearly identified the IgV domain to be responsible for receptor interactions, earlier studies suggested that both Ig domains are required for full co-signaling function. Herein, we have used deletion and chimeric human CD80 and CD86 molecules in co-stimulation assays to study the impact of the multimeric state of IgV and IgC domains on receptor binding properties and on co-stimulatory function in a peptide-specific T cell activation model. We report for the first time the presence of CD80 dimers and CD86 monomers in living cells. Moreover, we show that the IgC domain of both molecules inhibits multimer formation and greatly affects binding to the co-receptors CD28 and CTLA-4. Finally, both IgC and intracellular domains are required for full co-signaling function. These findings reveal the distinct but complementary roles of CD80 and CD86 IgV and IgC domains in T cell activation.

Diminished Th17 (not Th1) responses underlie multiple sclerosis disease abrogation after hematopoietic stem cell transplantation.

OBJECTIVE: To define changes in phenotype and functional responses of reconstituting T cells in patients with aggressive multiple sclerosis (MS) treated with ablative chemotherapy and autologous hematopoietic stem cell transplantation (HSCT). METHODS: Clinical and brain magnetic resonance imaging measures of disease activity were monitored serially in patients participating in the Canadian MS HSCT Study. Reconstitution kinetics of immune-cell subsets were determined by flow cytometry, whereas thymic function was assessed using T-cell receptor excision circle analyses as well as flow cytometry measurements of CD31+ recent thymic emigrants (RTEs). Functional assays were performed to track central nervous system-autoreactive antigen-specific T-cell responses, and the relative capacity to generate Th1, Th17, or Th1/17 T-cell responses. RESULTS: Complete abrogation of new clinical relapses and new focal inflammatory brain lesions throughout the 2 years of immune monitoring following treatment was associated with sustained decrease in naive T cells, in spite of restoration of both thymic function and release of RTEs during reconstitution. Re-emergence as well as in vivo expansion of autoreactive T cells to multiple myelin targets was evident in all patients studied. The reconstituted myelin-specific T cells exhibited the same Th1 and Th2 responses as preablation myelin-reactive T cells. In contrast, the post-therapy T-cell repertoire exhibited a significantly diminished capacity for Th17 responses. INTERPRETATION: Our results indicate that diminished Th17 and Th1/17 responses, rather than Th1 responses, are particularly relevant to the abrogation of new relapsing disease activity observed in this cohort of patients with aggressive MS following chemoablation and HSCT.

A conserved polylysine motif in CD86 cytoplasmic tail is necessary for cytoskeletal association and effective co-stimulation.

T cell activation requires both antigen specific and co-stimulatory signals that include the interaction of CD28 with its ligands CD80 and CD86. These signals are delivered by antigen presenting cells (APC) in the context of the immunological synapse (IS). Reorganization of the cytoskeleton is required for the formation and maintenance of the IS. Our results show that a highly conserved polylysine motif in CD86 cytoplasmic tail, herein referred to as the K4 motif, is responsible for the constitutive association of CD86 to the cytoskeleton in primary human APC as well as in a murine APC model. This motif is not involved in initial APC:T cell conjugate formation but mutation of the K4 motif affects CD86 reorientation at the IS. Importantly, APCs expressing CD86 with mutated K4 motif are severely compromised in their capacity to trigger complete T cell activation upon peptide presentation as measured by IL-2 secretion. Altogether, our results reveal the critical importance of the cytoskeleton-dependent CD86 polarization to the IS and more specifically the K4 motif for effective co-signaling.

Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses.

Correlates of immune-mediated protection to most viral and cancer vaccines are still unknown. This impedes the development of novel vaccines to incurable diseases such as HIV and cancer. In this study, we have used functional genomics and polychromatic flow cytometry to define the signature of the immune response to the yellow fever (YF) vaccine 17D (YF17D) in a cohort of 40 volunteers followed for up to 1 yr after vaccination. We show that immunization with YF17D leads to an integrated immune response that includes several effector arms of innate immunity, including complement, the inflammasome, and interferons, as well as adaptive immunity as shown by an early T cell response followed by a brisk and variable B cell response. Development of these responses is preceded, as demonstrated in three independent vaccination trials and in a novel in vitro system of primary immune responses (modular immune in vitro construct [MIMIC] system), by the coordinated up-regulation of transcripts for specific transcription factors, including STAT1, IRF7, and ETS2, which are upstream of the different effector arms of the immune response. These results clearly show that the immune response to a strong vaccine is preceded by coordinated induction of master transcription factors that lead to the development of a broad, polyfunctional, and persistent immune response that integrates all effector cells of the immune system.

p56Lck tyrosine kinase enhances the assembly of death-inducing signaling complex during Fas-mediated apoptosis.

Although the death-inducing signaling complex (DISC) is rapidly assembled, several lines of evidence suggest that formation of this complex is not the first consequence of cell surface CD95 (Fas) stimulation but rather a later step in this process. Activation of Fas triggers a cascade of signaling events that culminate in cellular apoptosis. Tyrosine kinases are critical effectors in T cell activation. However, their functional involvement in death receptor-mediated apoptosis is unknown. Here, we used p56(Lck)-deficient cells to show that CD95-induced cell death is highly dependent on p56(Lck) activity and its localization within plasma membrane. We found that p56(Lck) acts upstream of the mitochondria; in the absence of p56(Lck), Bid cleavage and the release of cytochrome c were severely impaired. Moreover, p56(Lck)-deficient cells or cells expressing an inactive form of p56(Lck) displayed defective formation of the DISC post CD95 stimulation. In vivo reconstitution of thymocytes from p56(lck)-deficient mice, which are resistant to apoptosis, with p56(Lck) restored Fas-mediated cell death. Our results support a novel model whereby sensitivity to apoptosis is regulated through quantitative changes in the stoichiometry of DISC components triggered by p56(Lck) activation and localization.

Prospect for an Entamoeba histolytica Gal-lectin-based vaccine.

Entamoeba histolytica is the aetiological agent of invasive amoebiasis, the third leading parasitic cause of mortality in the world. The disease can be easily cured by chemotherapy; however, prevention, mainly in the form of vaccination, could greatly decrease the incidence of the disease, and possibly help in its eradication. The parasite's surface galactose and N-acetyl-d-galactosamine-inhibitable adherence lectin (Gal-lectin) is highly antigenic and is the most promising subunit vaccine candidate. We have generated a Gal-lectin-based DNA vaccine and tested its immunogenicity in mice. Although further optimization will probably be required, this vaccine could help in the generation of an amoebiasis DNA vaccine for use in humans.

Gerbil interleukin-18 and caspase-1: cloning, expression and characterization.

We are reporting the molecular cloning of gerbil interleukin-18 (IL-18) and caspase-1. The cDNAs encoding the molecules were cloned by cross-species reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'/3' rapid amplification of cDNA ends (RACE). COS-7 cells transfected with plasmids encoding pro-IL-18 and caspase-1 precursor expressed the two proteins intracellularly. When the cells were lysed in the presence of dithiothreitol, caspase-1 precursor became active and converted pro-IL-18 into mature IL-18. A partially purified preparation of gerbil mature IL-18 was bioactive, as it stimulated the proliferation of gerbil spleen cells in a dose-dependent manner.

Construction and immunogenicity of a codon-optimized Entamoeba histolytica Gal-lectin-based DNA vaccine.

Invasive amebiasis caused by Entamoeba histolytica is the third leading parasitic cause of mortality, and there are no vaccines available to help control the disease. The galactose-adherence lectin (Gal-lectin) is the parasite's major molecule allowing it to adhere to colonic mucin for colonization and to target cells for tissue destruction. It is immunodominant and is regarded as the most promising candidate molecule to be included in a subunit vaccine against amebiasis. In this study, we are reporting the construction of a codon-optimized DNA vaccine encoding a portion of the Gal-lectin heavy subunit that includes the carbohydrate recognition domain (CRD), and its in vivo testing in mice. The vaccine stimulated a Th1-type Gal-lectin-specific cellular immune response as well as the development of serum antibodies that recognized a recombinant portion of the heavy subunit, and that inhibited the adherence of trophozoites to target cells in vitro.

Molecular cloning and expression of gerbil granulocyte/macrophage colony-stimulating factor.

Using a combination of cross species reverse transcriptase-polymerase chain reaction and 3' rapid amplification of cDNA ends techniques, we cloned the cDNA encoding gerbil granulocyte/macrophage colony-stimulating factor (GM-CSF). The open reading frame had 81% nucleotide identity with its mouse counterpart, while the mature protein had 80% homology with mature mouse GM-CSF. COS-7 cells transfected with gerbil GM-CSF cDNA secreted high levels of bioactive GM-CSF, as their supernatant stimulated gerbil bone-marrow cell proliferation and colony formation in semi-solid medium.