David Stephany

HIV Envelope-CXCR4 Signaling Activates Cofilin to Overcome Cortical Actin Restriction in Resting CD4 T Cells

Binding of the HIV envelope to the chemokine coreceptors triggers membrane fusion and signal transduction. The fusion process has been well characterized, yet the role of coreceptor signaling remains elusive. Here, we describe a critical function of the chemokine coreceptor signaling in facilitating HIV infection of resting CD4 T cells. We find that static cortical actin in resting T cells represents a restriction and that HIV utilizes the Galphai-dependent signaling from the chemokine coreceptor CXCR4 to activate a cellular actin-depolymerizing factor, cofilin, to overcome this restriction. HIV envelope-mediated cofilin activation and actin dynamics are important for a postentry process that leads to viral nuclear localization. Inhibition of HIV-mediated actin rearrangement markedly diminishes viral latent infection of resting T cells. Conversely, induction of active cofilin greatly facilitates it. These findings shed light on viral exploitation of cellular machinery in resting T cells, where chemokine receptor signaling becomes obligatory.

Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.

Cardiac allograft survival across major histocompatibility complex barriers in the rhesus monkey following T lymphocyte-depleted autologous marrow transplantation. III. Late allograft rejection.

We have studied organ allograft survival in rhesus monkeys conditioned with myeloablative total-body irradiation and T cell-depleted autologous bone marrow transplantation then given a heterotopic MHC-mismatched cardiac allograft in the immediate postmyeloablative period. This model has enabled us to investigate the role of T cells in vascularized organ allograft rejection. We previously reported (1) that recipients of marrow depleted of T cells below a critical threshold (0.16% residual marrow T cells, or 0.14×l05 infused T cells/kg) experienced a period of freedom from acute rejection associated with a profound nonspecific immune deficiency (determined by skin grafting). Resolution of the nonspecific immune deficiency was associated with late graft rejection. In the present report, we correlate the results of peripheral immune reconstitution studies and direct immunohistochemical analysis with allograft status in order to study T cell subsets involved in late rejection. We report that, in contrast with CD8+/CD28−T cells, CD16+ NK cells, and CD20+ B cells, late allograft rejection was associated with the return of peripheral CD4+ T cells and CD8+/CD28+ T cells, suggesting a critical role for one or both of these subsets in late allograft rejection in this model.

Fluorescence flow cytometry in the study of lymphoid cell receptors.

Publisher Summary This chapter describes the way measurements can be made, using cell surface receptors for the Fc portion of IgG (FcR) as an example. In homogeneous cell populations, radioactive assays for receptor number and affinity give meaningful results. However, in heterogeneous cell populations, for example human peripheral blood leukocytcs and murine splenocytcs, average numbers of receptors per cell or average affinities of receptors have little meaning. Direct measures of receptor number and affinity on only those subsets of cells that express the receptor are required. These can be provided by fluorescence assays, especially when the fluorescence is measured quantitatively on a large number of cells by flow cytometry. By using multicolor flow cytometric analyses, receptors can be quantitated on individual subsets of cells, and those subsets can be further identified by virtue of their staining with a second marker, in a single measurement.

Interleukin‐5 does not influence differential transcription of transmembrane and soluble isoforms of IL‐5Rα in vivo

Abstract:  Interleukin‐5 (IL‐5) promotes signal transduction and expansion of eosinophil colonies in bone marrow via interactions with its heterodimeric receptor (IL‐5R). Two variants encoding soluble forms of the alpha subunit (sIL‐5Rα) have been described, although the signals promoting and/or limiting differential transcription remain to be clarified. Objectives: Our intent was to explore the role of IL‐5 in regulating differential transcription of these splice variants in vivo. Methods: We have designed a quantitative reverse transcriptase‐polymerase chain reaction assay to detect transcripts encoding the transmembrane, soluble 1 and 2 forms of IL‐5Rα in two strains of wild‐type (BALB/c and C57BL/6) and corresponding IL‐5 gene‐deleted mice. Wild‐type mice respond to S. mansoni infection with a gradual increase in serum IL‐5 and eosinophilia, which is not observed in IL‐5 gene‐deleted mice. Results and conclusions: We find that IL‐5 is not necessary for differential splicing to occur in vivo, as all three forms of the IL‐5Rα are detected in both strains of IL‐5 gene‐deleted mice, with ratios of transcript expression (transmembrane : soluble 1 : soluble 2) that were indistinguishable from their wild‐type counterparts. Differential splicing does vary markedly between strains, potentially because of local effects of strain‐specific polymorphisms.

Activated platelet-T-cell conjugates in peripheral blood of patients with HIV infection: coupling coagulation/inflammation and T cells.

Background:Despite successfully suppressed viremia by treatment, patients with high levels of biomarkers of coagulation/inflammation are at an increased risk of developing non-AIDS defining serious illnesses such as cardiovascular diseases. Thus, there is a relationship between persistent immune activation and coagulation/inflammation, although the mechanisms are poorly understood. Platelets play an important role in this process. Although interactions between platelets and elements of the innate immune system, such as monocytes, are well described, little is known about the interaction between platelets and the adaptive immune system. Design:We investigated the interaction of a component of the coagulation system, platelets, and the adaptive immune system T cells. Methods:Healthy controls and combination antiretroviral therapy (cART)-treated HIV-infected patients with viral loads of less than 40 copies/ml for more than 15 months were analysed for platelet–T-cell conjugate formation. Results:Platelets can form conjugates with T cells and were preferentially seen in CD4+ and CD8+ T-cell subsets with more differentiated phenotypes [memory, memory/effector and terminal effector memory (TEM)]. Compared with healthy controls, these conjugates in patients with HIV infection were more frequent, more often composed of activated platelets (CD42b+CD62P+), and were significantly associated with the D-dimer serum levels. Conclusion:These data support a model in which platelet–T-cell conjugates may play a critical role in the fast recruitment of antigen-experienced T cells to the place of injury. This mechanism can contribute in maintaining a state of coagulation/inflammation observed in these patients contributing to the pathology of the disease.

Coordinate-Enhanced Expression of Three Adhesion Molecules (LFA-3, CD2, and LFA-1) and Three Other Molecules (4B4, UCHL1, and Pgp-1) Defines a Human T Cell Subset Containing Memory Cells and Characterized by Enhanced γ Interferon Production

Abstract: The present report summarizes our experimental findings regarding phenotypic and functional differences between two reciprocal subsets of human peripheral blood T cells: memory cells versus naive cells. Human memory T cells are phenotypically distinguishable from naive T cells by increased expression of six cell-surface molecules (LFA-3, CD2, LFA-1, 4B4, UCHL1, and Pgp-1). Neonatal cord blood consists almost exclusively of naive cells expressing lower levels of these markers, which increase in expression following phytohemagglutin (PHA) activation. As expected, only the high-expressing T cells in adults proliferate in response to recall antigens. Memory cells produce greater than five times more γ interferon with PHA activation than naive cells, although they produce equivalent amount of IL2. Our results integrate findings reported from multiple laboratories regarding different markers (e.g., 4B4, 2H4, HB-11, UCHL1, and Pgp-1), and demonstrate that these subsets represent different maturational stages of T cells rather than different T-cell lineages.