Luann Borowski

DC-SIGN Is a Receptor for Human Herpesvirus 8 on Dendritic Cells and Macrophages

Human herpesvirus 8 (HHV-8) causes Kaposi’s sarcoma and pleural effusion lymphoma. In this study, we show that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN; CD209) is a receptor for HHV-8 infection of myeloid DCs and macrophages. DC-SIGN was required for virus attachment to these cells and DC-SIGN-expressing cell lines. HHV-8 binding and infection were blocked by anti-DC-SIGN mAb and soluble DC-SIGN, and mannan, a natural ligand for DC-SIGN. Infection of DCs and macrophages with HHV-8 led to production of viral proteins, with little production of viral DNA, similar to HHV-8 infection of vascular endothelial cells. Infection of DCs resulted in down-regulation of DC-SIGN, a decrease in endocytic activity, and an inhibition of Ag stimulation of CD8+ T cells. We propose that DC-SIGN serves as a portal for immune dysfunction and oncogenesis caused by HHV-8 infection.

Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+ T-Lymphocyte Reactivity during Combination Antiretroviral Therapy in HIV-1-Infected Patients with Advanced Immunodeficiency

ABSTRACT The long-term efficacy of combination antiretroviral therapy may relate to augmentation of anti-human immunodeficiency virus type 1 (HIV-1) CD8+ T-cell responses. We found that prolonged treatment of late-stage HIV-1-infected patients with a protease inhibitor and two nucleoside reverse transcriptase inhibitors failed to restore sustained, high levels of HIV-1-specific, HLA class I-restricted, cytotoxic-T-lymphocyte precursors and gamma interferon (IFN-γ) production by CD8+ T cells. In some patients, particularly those initiating three-drug combination therapy simultaneously rather than sequentially, there were early, transient increases in the frequency of anti-HIV-1 CD8+ T cells that correlated with decreases in HIV-1 RNA and increases in T-cell counts. In the other patients, HIV-1-specific T-cell functions either failed to increase or declined from baseline during triple-drug therapy, even though some of these patients showed suppression of plasma HIV-1 RNA. These effects of combination therapy were not unique to HIV-1 specific T-cell responses, since similar effects were noted for CD8+T cells specific for the cytomegalovirus pp65 matrix protein. The level and breadth of CD8+ cell reactivity to HLA A*02 HIV-1 epitopes, as determined by IFN-γ production and HLA tetramer staining after combination therapy, were related to the corresponding responses prior to treatment. There was, however, a stable, residual population of potentially immunocompetent HIV-1-specific T cells remaining after therapy, as shown by tetramer staining of CD8+CD45RO+ cells. These results indicate that new strategies will be needed to target residual, immunocompetent HIV-1-specific CD8+ T cells to enhance the effectiveness of antiretroviral therapy in patients with advanced immunodeficiency.

Induction of Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+ and CD4+ T-Cell Reactivity by Dendritic Cells Loaded with HIV-1 X4-Infected Apoptotic Cells

ABSTRACT T-cell responses to X4 strains of human immunodeficiency virus type 1 (HIV-1) are considered important in controlling progression of HIV-1 infection. We investigated the ability of dendritic cells (DC) and various forms of HIV-1 X4 antigen to induce anti-HIV-1 T-cell responses in autologous peripheral blood mononuclear cells from HIV-1-infected persons. Immature DC loaded with HIV-1 IIIB-infected, autologous, apoptotic CD8− cells and matured with CD40 ligand induced gamma interferon production in autologous CD8+ and CD4+ T cells. In contrast, mature DC loaded with HIV-1 IIIB-infected, necrotic cells or directly infected with cell-free HIV-1 IIIB were poorly immunogenic. Thus, HIV-1-infected cells undergoing apoptosis serve as a rich source of X4 antigen for CD8+ and CD4+ T cells by DC. This may be an important mechanism of HIV-1 immunogenicity and provides a strategy for immunotherapy of HIV-1-infected patients on combination antiretroviral therapy.

Delivery of Liposome-Encapsulated HIV Type 1 Proteins to Human Dendritic Cells for Stimulation of HIV Type 1-Specific Memory Cytotoxic T Lymphocyte Responses

An important aspect of vaccine development involves delivery of antigens to antigen-presenting cells for the induction of potent antigen-specific T lymphocyte responses. We investigated the effect of a cationic liposome, lipofectin, on delivery of whole proteins to human dendritic cells (DCs) derived from blood mononuclear cells by culture in interleukin 4 and granulocyte-monocyte colony-stimulating factor for stimulation of human immunodeficiency virus type 1 (HIV-1)-specific memory cytotoxic T lymphocyte (CTL) responses. Delivery of HIV-1 Gag, Pol, and Env proteins to DCs by lipofectin stimulated greater anti-HIV-1 memory CTL responses in cells from HIV-1-infected subjects than those induced by DCs loaded with protein alone. The CTLs were CD8+ and HLA class I restricted. Antigen presentation was enhanced by chloroquine, but blocked by brefeldin A and peptide aldehyde inhibitors of proteasomes, indicating that the classic MHC class I cytosolic pathway was used for processing and presentation of HIV-1 protein by the DCs. Stimulation of anti-HIV-1 CTLs by this safe, inexpensive, and broadly applicable approach may be used in DC-based therapies for HIV-1 infection.

Priming of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cell responses by dendritic cells loaded with HIV-1 proteins

Proteins may serve as ideal CD8(+) T cell immunogens for human immunodeficiency virus type 1 (HIV-1) if they can be delivered to and processed through the human leukocyte antigen class I pathway. This study shows that human blood monocyte-derived dendritic cells loaded with liposome-complexed HIV-1 proteins and matured with CD40 ligand can prime CD8(+) T cells to HIV-1 in vitro. Whole HIV-1 protein in liposome may be an effective immunogen for HIV-1 vaccine protocols.

Primary Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses Induced by Myeloid Dendritic Cells

ABSTRACT Induction of an antigenically broad and vigorous primary T-cell immune response by myeloid dendritic cells (DC) in blood and tissues could be important for an effective prophylactic or therapeutic vaccine to human immunodeficiency virus type 1 (HIV-1). Here we show that a primary CD8+ T-cell response can be induced by HIV-1 peptide-loaded DC derived from blood monocytes of HIV-1-negative adults and neonates (moDC) and by Langerhans cells (LC) and interstitial, dermal-intestinal DC (idDC) derived from CD34+ stem cells of neonatal cord blood. Optimal priming of single-cell gamma interferon (IFN-γ) production by CD8+ T cells required CD4+ T cells and was broadly directed to multiple regions of Gag, Env, and Nef that corresponded to known and predicted major histocompatibility complex class I epitopes. Polyfunctional CD8+ T-cell responses, defined as single-cell production of more than one cytokine (IFN-γ, interleukin 2, or tumor necrosis factor alpha), chemokine (macrophage inhibitory factor 1β), or cytotoxic degranulation marker CD107a, were primed by moDC, LC, and idDC to HIV-1 Gag and reverse transcriptase epitopes, as well as to Epstein-Barr virus and influenza A virus epitopes. Thus, three major types of blood and tissue myeloid DC targeted by HIV-1, i.e., moDC, LC, and idDC, can prime multispecific, polyfunctional CD8+ T-cell responses to HIV-1 and other viral antigens.

Assessing Immunophenotyping Performance: Proficiency-Validation for Adopting Improved Flow Cytometry Methods

The continuous improvement and evolution of immune cell phenotyping requires periodic upgrading of laboratory methods and technology. Flow cytometry laboratories that are participating in research protocols sponsored by the NIAID are required to perform “switch” studies to validate performance before methods for T‐cell subset analysis can be changed.

Maturation of dendritic cells for enhanced activation of anti‐HIV‐1 CD8+ T cell immunity

Maturation of dendritic cells (DC) to enhance their capacity to activate T cell immunity to HIV‐1 is a key step in immunotherapy of HIV‐1 infection with DC. We compared maturation of DC derived from HIV‐1‐uninfected subjects and infected subjects on antiretroviral therapy (ART) or ART naïve by CD40 ligand (CD40L) and combinations of TLR3 ligand polyinosinic:polycytidylic acid [poly(I:C)] and inflammatory cytokines IFN‐γ, IFN‐α, IL‐1β, and TNF‐α. The greatest levels of virus‐specific IFN‐γ production by CD8+ T cells were stimulated by DC treated with CD40L, followed by DC treated with the poly(I:C)‐cytokine combination. The highest levels of IL‐12p70 were produced by DC treated with CD40L + IFN‐γ, followed by CD40L and the poly(I:C)‐cytokine combination. Neutralization of IL‐12p70 indicated that it was only partially involved in direct enhancement of antiviral CD8+ T cell activity. DC stimulation of antiviral CD8+ T cell reactivity was enhanced by activated CD4+ T cells at low concentrations but was suppressed at higher CD4+ T cell concentrations. Maturation of DC with CD40L obviated the need for CD4+ T cell help and overcame this suppressive activity. Finally, we showed that DC from HIV‐1‐infected subjects on ART, which were treated with the poly(I:C)‐cytokine combination, retained the capacity to produce IL‐12p70 and activate anti‐HIV‐1 CD8+ T cell responses after restimulation with CD40L, with or without IFN‐γ. Thus, DC from HIV‐1‐infected subjects can be engineered with CD40L or a poly(I:C)‐cytokine combination for enhancing CD8+ T cell responses to HIV‐1, which has potential applications in HIV‐1 immunotherapy.

Comparison of Interlaboratory Variation in Absolute T-Cell Counts by Single-Platform and Optimized Dual-Platform Methods

Previous studies have reported that the adoption of a single‐platform flow cytometry cell counting method resulted in lower interlaboratory variation in absolute T cell counts as compared to predicate dual‐platform flow cytometry methods which incorporate independent automated lymphocyte counts (Schnizlein‐Bick et al., Clin Diagn Lab Immunol 2000;7:336–343; Reimann et al., Clin Diagn Lab Immunol 2000;7:344–351). In the present study, we asked whether use of a single‐platform method could reduce variation in absolute cell counts across the laboratories in the Multicenter AIDS Cohort Study (MACS) (n = 4), as suggested by the studies cited.

Dendritic cells reveal a broad range of MHC class I epitopes for HIV-1 in persons with suppressed viral load on antiretroviral therapy.

BACKGROUND HIV-1 remains sequestered during antiretroviral therapy (ART) and can resume high-level replication upon cessation of ART or development of drug resistance. Reactivity of memory CD8(+) T lymphocytes to HIV-1 could potentially inhibit this residual viral replication, but is largely muted by ART in relation to suppression of viral antigen burden. Dendritic cells (DC) are important for MHC class I processing and presentation of peptide epitopes to memory CD8(+) T cells, and could potentially be targeted to activate memory CD8(+) T cells to a broad array of HIV-1 epitopes during ART. PRINCIPAL FINDINGS We show for the first time that HIV-1 peptide-loaded, CD40L-matured DC from HIV-1 infected persons on ART induce IFN gamma production by CD8(+) T cells specific for a much broader range and magnitude of Gag and Nef epitopes than do peptides without DC. The DC also reveal novel, MHC class I restricted, Gag and Nef epitopes that are able to induce polyfunctional T cells producing various combinations of IFN gamma, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1 beta and the cytotoxic de-granulation molecule CD107a. SIGNIFICANCE There is an underlying, broad antigenic spectrum of anti-HIV-1, memory CD8(+) T cell reactivity in persons on ART that is revealed by DC. This supports the use of DC-based immunotherapy for HIV-1 infection.