Generation of Potent and Stable Human CD4+ T Regulatory Cells by Activation-independent Expression of FOXP3

Therapies based on enhancing the numbers and/or function of T regulatory cells (Tregs) represent one of the most promising approaches to restoring tolerance in many immune-mediated diseases. Several groups have investigated whether human Tregs suitable for cellular therapy can be obtained by in vitro expansion, in vitro conversion of conventional T cells into Tregs, or gene transfer of the FOXP3 transcription factor. To date, however, none of these approaches has resulted in a homogeneous and stable population of cells that is as potently suppressive as ex vivo Tregs. We developed a lentivirus-based strategy to ectopically express high levels of FOXP3 that do not fluctuate with the state of T-cell activation. This method consistently results in the development of suppressive cells that are as potent as Tregs and can be propagated as a homogeneous population. Moreover, using this system, both naive and memory CD4+ T cells can be efficiently converted into Tregs. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4+ Tregs, which can be used for further biological study and developed for antigen-specific cellular therapy applications

Novel HIV-1 Recombinant Forms in Antenatal Cohort, Montreal, Quebec, Canada

Near full-length genomes of 4 unclassified HIV-1 variants infecting patients enrolled in an antenatal cohort in Canada were obtained by sequencing. All 4 variants showed original recombination profiles, including A1/A2/J, A1/D, and A1/G/J/CRF11_cpx structures. Identification of these variants highlights the growing prevalence of unique recombinant forms of HIV-1 in North America

HIV-1 Genetic Diversity in Antenatal Cohort, Canada

Non-B HIV-1 was consistent with patients’ area of origin

Structural Basis for Broad Neutralization of Hepatitis C Virus Quasispecies

Monoclonal antibodies directed against hepatitis C virus (HCV) E2 protein can neutralize cell-cultured HCV and pseudoparticles expressing envelopes derived from multiple HCV subtypes. For example, based on antibody blocking experiments and alanine scanning mutagenesis, it was proposed that the AR3B monoclonal antibody recognized a discontinuous conformational epitope comprised of amino acid residues 396–424, 436–447, and 523–540 of HCV E2 envelope protein. Intriguingly, one of these segments (436–447) overlapped with hypervariable region 3 (HVR3), a domain that exhibited significant intrahost and interhost genetic diversity. To reconcile these observations, amino-acid sequence variability was examined and homology-based structural modelling of E2 based on tick-borne encephalitis virus (TBEV) E protein was performed based on 413 HCV sequences derived from 18 subjects with chronic hepatitis C. Here we report that despite a high degree of amino-acid sequence variability, the three-dimensional structure of E2 is remarkably conserved, suggesting broad recognition of structural determinants rather than specific residues. Regions 396–424 and 523–540 were largely exposed and in close spatial proximity at the surface of E2. In contrast, region 436–447, which overlaps with HVR3, was >35 Å away, and estimates of buried surface were inconsistent with HVR3 being part of the AR3B binding interface. High-throughput structural analysis of HCV quasispecies could facilitate the development of novel vaccines that target conserved structural features of HCV envelope and elicit neutralizing antibody responses that are less vulnerable to viral escape

Molecular analysis of the T-Cell Receptor b chain repertoire during HIV infection

Human Immunodeficiency Virus Type 1 (HIV-1) infection is characterized by relentless depletion of cells belonging to the CD4+ helper-inducer subset, leading to the appearance in the patient of a lethal state of functional immunodeficiency. Many mechanisms have been proposed to account for the virus-induced destruction of T cells, including direct cytopathic effects of the virus, induction of apoptosis, and killing of infected or bystander CD4+ T cells by the cytotoxic immune response. Also, since HIV-1 is a retrovirus, it has been proposed that, like mouse mammary tumor viruses (MMTV), it could encode a protein with superantigenic properties, which could, by itself, sequentially anergize and delete CD4+ T cells expressing specific T cell receptor (TCR) V$beta$ determinants.In order to reveal the existence of such molecules in HIV infection, different cohorts of HIV-infected subjects were studied by PCR and flow cytometry, including HIV-discordant monozygotic twins, HIV-infected and uninfected children from HIV-infected mothers, and patients undergoing primary, acute HIV infection. The presence of TCR $beta$ chain repertoire perturbations not normally observed in uninfected subjects was revealed in each of these systems. These results supported the existence of an HIV-associated superantigen (SAg).However, high-level V$beta$-specific expansions of CD8+ T cells were also observed in acutely-infected patients, and in HIV-infected children from HIV-infected mothers. These expansions were oligoclonal, and comprised cells that possessed cytotoxic activity directed against HIV-infected cells, suggesting that these cell populations were part of the primary immune response to HIV-1 infection. These expansions were also transient, encompassing defined expansion and decline phases. In some cases, disappearance of these highly expanded effectors occurred while circulating virus titers were still extremely high, and in absence of viral mutation within the cognate CTL epitopes. This suggested that disappearance of these cells resulted from a process of clonal exhaustion, a mechanism by which differentiated peripheral cytotoxic effectors are deleted in presence of overwhelming levels of antigen.Transient, high-level V$beta$-specific expansions of cytolytic T cells might represent a fundamental characteristic of primary antiviral responses not only in the context of HIV infection, but also in other acute viral diseases. As well, clonal deletion constitutes a new mechanism of viral escape that could result in the base-line CTL repertoire being progressively restricted during the course of HIV infection and HIV-associated disease. (Abstract shortened by UMI.

The effects of glucocorticoids on the replication of the human immunodeficiency virus type 1 in vitro /

Several retroviruses, like the mouse mammary tumor virus (MMTV), possess glucocorticoid responsive elements (GREs) that enable them to directly interface with the glucocorticoid receptor (GR) signal transduction pathway, resulting in an enhanced replication rate in presence of hormone. We have first demonstrated that glucocorticoid agonists dexamethasone (10$sp{-9}$ M) and cortisol (10$sp{-7}$ M) can stimulate human immunodeficiency virus type 1 (HIV-1) replication in de novo HIV-1-infected lymphoid cell lines, and that the magnitude of this induction correlated with relative levels of GR and GR message present in responder and non-responder cell lines. DNA sequence analysis has revealed the presence of a dozen conserved GRE-like motifs distributed throughout the HIV-1 genome. By using an electrophoretic mobility shift assay, we have demonstrated that purified GR can specifically interact with at least two of these sequences: the first one is positioned in the HIV-1 long terminal repeat (LTR), in a region referred to as the negative regulatory element (NRE); the second one is located intragenically, within the vif open reading frame. To assess the functional nature of these potential GREs, we subcloned then into a MMTV-LTR-luciferase construct from which endogenous GREs had been previously deleted. The activity of these recombinants was measured in a GR-negative cell line in presence and absence of co-transfected GR expression vector, and with or without glucocorticoid treatment. Our results showed that the two HIV-1 sequences we tested were capable of significantly enhancing the baseline transcription of the reporter gene, and conferred mild glucocorticoid inducibility to the recombinant MMTV promoter. These data suggest that HIV-1 contains discrete functional GREs that might serve to modulate the rate of viral transcription and replication, or control some aspects of latency. This has important clinical significance, since adjuvant corticosteroid therapy ha

Pathogenesis of Hepatitis C During Pregnancy and Childhood

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