Scott E. VanCompernolle

Antimicrobial Peptides from Amphibian Skin Potently Inhibit Human Immunodeficiency Virus Infection and Transfer of Virus from Dendritic Cells to T Cells

ABSTRACT Topical antimicrobicides hold great promise in reducing human immunodeficiency virus (HIV) transmission. Amphibian skin provides a rich source of broad-spectrum antimicrobial peptides including some that have antiviral activity. We tested 14 peptides derived from diverse amphibian species for the capacity to inhibit HIV infection. Three peptides (caerin 1.1, caerin 1.9, and maculatin 1.1) completely inhibited HIV infection of T cells within minutes of exposure to virus at concentrations that were not toxic to target cells. These peptides also suppressed infection by murine leukemia virus but not by reovirus, a structurally unrelated nonenveloped virus. Preincubation with peptides prevented viral fusion to target cells and disrupted the HIV envelope. Remarkably, these amphibian peptides also were highly effective in inhibiting the transfer of HIV by dendritic cells (DCs) to T cells, even when DCs were transiently exposed to peptides 8 h after virus capture. These data suggest that amphibian-derived peptides can access DC-sequestered HIV and destroy the virus before it can be transferred to T cells. Thus, amphibian-derived antimicrobial peptides show promise as topical inhibitors of mucosal HIV transmission and provide novel tools to understand the complex biology of HIV capture by DCs.

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel.

Deciphering the signaling pathways that govern stimulation of naïve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of naïve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.

Helicobacter pylori vacuolating cytotoxin inhibits activation-induced proliferation of human T and B lymphocyte subsets

Helicobacter pylori are Gram-negative bacteria that persistently colonize the human gastric mucosa despite the recruitment of immune cells. The H. pylori vacuolating cytotoxin (VacA) recently has been shown to inhibit stimulation-induced proliferation of primary human CD4+ T cells. In this study, we investigated effects of VacA on the proliferation of various other types of primary human immune cells. Intoxication of PBMC with VacA inhibited the stimulation-induced proliferation of CD4+ T cells, CD8+ T cells, and B cells. VacA also inhibited the proliferation of purified primary human CD4+ T cells that were stimulated by dendritic cells. VacA inhibited both T cell-induced and PMA/anti-IgM-induced proliferation of purified B cells. Intoxication with VacA did not alter the magnitude of calcium flux that occurred upon stimulation of CD4+ T cells or B cells, indicating that VacA does not alter early signaling events required for activation and proliferation. VacA reduced the mitochondrial membrane potential of CD4+ T cells, but did not reduce the mitochondrial membrane potential of B cells. We propose that the immunomodulatory actions of VacA on T and B lymphocytes, the major effectors of the adaptive immune response, may contribute to the ability of H. pylori to establish a persistent infection in the human gastric mucosa.

Expression and Function of Formyl Peptide Receptors on Human Fibroblast Cells

The migration of polymorphonuclear leukocytes from the blood to sites of infection in tissues is a hallmark of the innate immune response. Formylated peptides produced as a byproduct of bacterial protein synthesis are powerful chemoattractants for leukocytes. Formyl peptides bind to two different G protein-coupled receptors (formyl peptide receptor (FPR) and the low affinity formyl peptide receptor-like-1 (FPRL1)) to initiate a signal transduction cascade leading to cell activation and migration. Our analysis of expressed sequences from many cDNA libraries draws attention to the fact that FPRs are widely expressed in nonlymphoid tissues. Here we demonstrate that FPRs are expressed by normal human lung and skin fibroblasts and the human fibrosarcoma cell line HT-1080. The expression on fibroblasts of receptors for bacteria-derived peptides raises questions about the possible function of these receptors in nonleukocyte cells. We studied the function of FPRs on fibroblasts and find that stimulation with fMLP triggers dose-dependent migration of these cells. Furthermore, fMLP induces signal transduction including intracellular calcium flux and a transient increase in F-actin. The fMLP-induced adhesion and motility of fibroblasts on fibronectin require functional protein kinase C and phosphatidylinositol 3-kinase. This first report of a functional formyl peptide receptor in cells of fibroblast origin opens new possibilities for the role of fibroblasts in innate immune responses.

CCR5 Expression Levels Influence NFAT Translocation, IL-2 Production, and Subsequent Signaling Events during T Lymphocyte Activation

Ligands of CCR5, the major coreceptor of HIV-1, costimulate T lymphocyte activation. However, the full impact of CCR5 expression on T cell responses remains unknown. Here, we show that compared with CCR5+/+, T cells from CCR5−/− mice secrete lower amounts of IL-2, and a similar phenotype is observed in humans who lack CCR5 expression (CCR5-Δ32/Δ32 homozygotes) as well as after Ab-mediated blockade of CCR5 in human T cells genetically intact for CCR5 expression. Conversely, overexpression of CCR5 in human T cells results in enhanced IL-2 production. CCR5 surface levels correlate positively with IL-2 protein and mRNA abundance, suggesting that CCR5 affects IL-2 gene regulation. Signaling via CCR5 resulted in NFAT transactivation in T cells that was blocked by Abs against CCR5 agonists, suggesting a link between CCR5 and downstream pathways that influence IL-2 expression. Furthermore, murine T cells lacking CCR5 had reduced levels of intranuclear NFAT following activation. Accordingly, CCR5 expression also promoted IL-2-dependent events, including CD25 expression, STAT5 phosphorylation, and T cell proliferation. We therefore suggest that by influencing a NFAT-mediated pathway that regulates IL-2 production and IL-2-dependent events, CCR5 may play a critical role in T cell responses. In accord with our prior inferences from genetic-epidemiologic studies, such CCR5-dependent responses might constitute a viral entry-independent mechanism by which CCR5 may influence HIV-AIDS pathogenesis.

Small molecule inhibition of hepatitis C virus E2 binding to CD81

The hepatitis C virus (HCV) is a causal agent of chronic liver infection, cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. CD81 is a receptor for HCV envelope glycoprotein E2. Although the binding of HCV-E2 with CD81 is well documented the role of this interaction in the viral life cycle remains unclear. Host specificity and mutagenesis studies suggest that the helix D region of CD81 mediates binding to HCV-E2. Structural analysis of CD81 has enabled the synthesis of small molecules designed to mimic the space and hydrophobic features of the solvent-exposed face on helix D. Utilizing a novel bis-imidazole scaffold a series of over 100 compounds has been synthesized. Seven related, imidazole-based compounds were identified that inhibit binding of HCV-E2 to CD81. The inhibitory compounds have no short-term effect on cellular expression of CD81 or other tetraspanins, do not disrupt CD81 associations with other cell surface proteins, and bind reversibly to HCV-E2. These results provide an important proof of concept that CD81-based mimics can disrupt binding of HCV-E2 to CD81.

Anti-CD81 activates LFA-1 on T cells and promotes T cell-B cell collaboration.

CD81 is expressed on human T cells at all stages of development. CD81 is physically associated with CD4 and CD8 and antibodies against CD81 generate signals which influence thymocyte adhesion and proliferation. Here we evaluate the function of CD81 on mature T cells. We employ a system in which B cells present superantigen to autologous T cells and find that anti‐CD81 promotes T cell–B cell collaboration. Anti‐CD81 induces T cell–B cell adhesion of peripheral blood lymphocytes which is partially mediated by LFA‐1. CD81 engagement promotes LFA‐1‐dependent T cell activation, IL‐2 production and proliferation. The antibody 5A6 was uniquely potent in exerting these effects compared to another antibody to CD81 or to antibodies that react with other tetraspanins expressed on T cells, anti‐CD53 or anti‐CD82. CD81‐derived signals rapidly induce high‐avidity LFA‐1 as measured by cell binding to recombinant ICAM‐3‐coated fluorescent microspheres or by cell adhesion to ICAM‐3‐coated plastic. 5A6 activation of LFA‐1 does not expose the high‐affinity conformation epitope recognized by monoclonal antibody 24.

Despite Biased TRBV Gene Usage against a Dominant HLA B57-Restricted Epitope, TCR Diversity Can Provide Recognition of Circulating Epitope Variants

The role of epitope-specific TCR repertoire diversity in the control of HIV-1 viremia is unknown. Further analysis at the clonotype level is important for understanding the structural aspects of the HIV-1 specific repertoire that directly relate to CTL function and ability to suppress viral replication. In this study, we performed in-depth analysis of T cell clonotypes directed against a dominantly recognized HLA B57-restricted epitope (KAFSPEVIPMF; KF11) and identified common usage of the TCR β-chain TRBV7 in eight of nine HLA B57 subjects examined, regardless of HLA B57 subtype. Despite this convergent TCR gene usage, structural and functional assays demonstrated no substantial difference in functional or structural avidity between TRBV7 and non-TRBV7 clonotypes and this epitopic peptide. In a subject where TRBV7-usage did not confer cross-reactivity against the dominant autologous sequence variant, another circulating TCR clonotype was able to preferentially recognize the variant peptide. These data demonstrate that despite selective recruitment of TCR for a conserved epitope over the course of chronic HIV-1 infection, TCR repertoire diversity may benefit the host through the ability to recognize circulating epitope variants.

Production of Specific mRNA Transcripts, Usage of an Alternate Promoter, and Octamer-Binding Transcription Factors Influence the Surface Expression Levels of the HIV Coreceptor CCR5 on Primary T Cells

Surface levels of CCR5 on memory CD4+ T cells influence HIV-1/AIDS susceptibility. Alternative promoter usage results in the generation of CCR5 mRNA isoforms that differ based on whether they contain or lack the untranslated exon 1. The impact of exon 1-containing transcripts on CCR5 surface expression is unknown. In this study, we show that the increased cell surface expression of CCR5 on primary T cells is associated with selective enrichment of exon 1-containing transcripts. The promoter that drives exon 1-containing transcripts is highly active in primary human T cells but not in transformed T cell lines. The transcription factors Oct-1 and -2 inhibit and enhance, respectively, the expression of exon 1-containing transcripts and CCR5 surface levels. However, polymorphisms at homologous octamer-binding sites in the CCR5 promoter of nonhuman primates abrogate the binding of these transcription factors. These results identify exon 1-containing transcripts, and the cis-trans factors that regulate the expression levels of these mRNA isoforms as key parameters that affect CCR5 surface expression levels, and by extension, susceptibility to HIV/AIDS among humans, and possibly, the observed interspecies differences in susceptibility to lentiviral infection.

Helicobacter pylori VacA Toxin Inhibits Human Immunodeficiency Virus Infection of Primary Human T Cells

ABSTRACT Human CD4+ T cells are major targets for human immunodeficiency virus (HIV) infection. Resting T cells are resistant to HIV infection unless activated through the T-cell receptor (TCR) or by cytokine signals. How T-cell signaling promotes susceptibility of T cells to HIV infection remains poorly understood. Here we demonstrate that the VacA toxin produced by Helicobacter pylori can inhibit HIV infection of primary T cells, stimulated through the TCR or by cytokines alone. This activity of VacA was dependent on its ability to form membrane channels. VacA suppressed HIV infection of T cells at a stage after viral entry, post-reverse transcription and pre-two-long-terminal-repeat circle formation, similar to the cytokine signaling inhibitor rapamycin. Mechanistically, neither VacA nor rapamycin inhibited the activation of cytokine signal transduction components (STAT5, p42/44 mitogen-activated protein kinase, or p38), but both blocked activation of key regulatory proteins required for G1 cell cycle transition. In contrast to rapamycin, VacA did not suppress phosphorylation of p70 S6 kinase but caused mitochondrial depolarization and ATP depletion within primary T cells. These results suggest that VacA inhibits T-cell activation and HIV infection via a novel mechanism. Identifying the host cell targets of VacA could be useful for elucidating the HIV life cycle within primary T cells.