Detection of IgG3 antibodies specific to the human immunodeficiency virus type 1 (HIV-1) p24 protein as marker for recently acquired infection.

Reducing the risk of human immunodeficiency virus type 1 (HIV-1) transmission is still a public health priority. The development of effective control strategies relies on the quantification of the effects of prophylactic and therapeutic measures in disease incidence. Although several assays can be used to estimate HIV incidence, these estimates are limited by the poor performance of these assays in distinguishing recent from long-standing infections. To address such limitation, we have developed an assay to titrate p24-specific IgG3 antibodies as a marker of recent infection. The assay is based on a recombinant p24 protein capable to detect total IgG antibodies in sera using a liquid micro array and enzyme-linked immunosorbent assay. Subsequently, the assay was optimised to detect and titrate anti-p24 IgG3 responses in a panel of sequential specimens from seroconverters over 24 months. The kinetics of p24-specific IgG3 titres revealed a transient peak in the 4 to 5-month period after seroconversion. It was followed by a sharp decline, allowing infections with less than 6 months to be distinguished from older ones. The developed assay exhibited a mean duration of recent infection of 144 days and a false-recent rate of ca. 14%. Our findings show that HIV-1 p24-specific IgG3 titres can be used as a tool to evaluate HIV incidence in serosurveys and to monitor the efficacy of vaccines and other transmission control strategies.

Development of potent class II transactivator gene delivery systems capable of inducing de novo MHC II expression in human cells, in vitro and ex vivo.

Class II transactivator (CIITA) induces transcription of major histocompatibility complex (MHC) II genes and can potentially be used to improve genetic immunotherapies by converting non-immune cells into cells capable of presenting antigens to CD4+ T cells. However, CIITA expression is tightly controlled and it remains unclear whether distinct non-immune cells differ in this transactivator regulation. Here we describe the development of gene delivery systems capable of promoting the efficient CIITA expression in non-immune cell lines and in primary human cells of an ex vivo skin explant model. Different human cell types undergoing CIITA overexpression presented high-level de novo expression of MHC II, validating the delivery systems as suitable tools for the CIITA evaluation as a molecular adjuvant for gene therapies.

Linked in: immunologic membrane nanotube networks.

Membrane nanotubes, also termed tunneling nanotubes, are F-actin-based structures that can form direct cytoplasmic connections and support rapid communication between distant cells. These nanoscale conduits have been observed in diverse cell types, including immune, neuronal, stromal, cancer, and stem cells. Until recently, little was known about the mechanisms involved in membrane nanotube development in myeloid origin APCs or how membrane nanotube networks support their ability to bridge innate and adaptive immunity. New research has provided insight into the modes of induction and regulation of the immune process of "reticulation" or the development of multicellular membrane nanotube networks in dendritic cells. Preprogramming by acute type 1 inflammatory mediators at their immature stage licenses mature type 1-polarized dendritic cells to reticulate upon subsequent interaction with CD40 ligand-expressing CD4(+) Th cells. Dendritic cell reticulation can support direct antigen transfer for amplification of specific T cell responses and can be positively or negatively regulated by signals from distinct Th cell subsets. Membrane nanotubes not only enhance the ability of immature dendritic cells to sense pathogens and rapidly mobilize nearby antigen-presenting cells in the peripheral tissues but also likely support communication of pathogen-related information from mature migratory dendritic cells to resident dendritic cells in lymph nodes. Therefore, the reticulation process facilitates a coordinated multicellular response for the efficient initiation of cell-mediated adaptive immune responses. Herein, we discuss studies focused on the molecular mechanisms of membrane nanotube formation, structure, and function in the context of immunity and how pathogens, such as HIV-1, may use dendritic cell reticulation to circumvent host defenses.

Dengue virus-infected human dendritic cells reveal hierarchies of naturally expressed novel NS3 CD8 T cell epitopes.

Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8(+) T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75-100%), compared to the others (0-50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

Dendritic cells pulsed with apoptotic squamous cell carcinoma have anti-tumor effects when combined with interleukin-2.

OBJECTIVES: Dendritic cells, the most potent of the antigen-presenting cells, have been widely studied as a promising tool for antitumor immunotherapies. However, little has been determined about the efficacy of dendritic cell-based therapy for the treatment of squamous cell carcinoma (SCC) because there are no known SCC-specific antigens. Recent reports indicate that dendritic cells can acquire antigens in the form of apoptotic cells and induce cytotoxic T-lymphocyte responses. The aim of this study was to test the feasibility of adoptive dendritic cell immunotherapy against SCC by using apoptotic tumor cells as a source of tumor antigens. STUDY DESIGN: A poorly immunogenic SCC line KLN 205 was used to make subcutaneous tumors on the flank of DBA2/J syngeneic mice. Bone marrow-derived dendritic cells were pulsed with ultraviolet B-irradiated (apoptotic) KLN 205 cells in vitro and transferred to the opposite flank subcutaneously. Some of the animals received simultaneous intraperitoneal injections of low-dose interleukin-2. RESULTS: When combined with interleukin-2, adoptive transfers of dendritic cells that were pulsed with apoptotic SCC significantly suppressed the tumor growth (P <.001) without notable side effects. Splenic T cells of treated mice produced greater amounts of interferon-gamma when restimulated with the relevant tumor (P <.001) as compared with control groups, indicative of an effective T-cell-mediated systemic immune response. CONCLUSION: Adoptive transfer of dendritic cells pulsed with apoptotic tumor cells as a source of tumor antigens, can elicit effective antitumor responses in the poorly immunogenic SCC model when combined with interleukin-2.

Dendritic cells prolong tumor-specific T-cell survival and effector function after interaction with tumor targets.

Tumor specific CTLs are susceptible to tumor-mediated activated induced cell death (AICD) after target engagement. The presence of dendritic cells (DCs) at the site of tumors correlates with an improved prognosis in patients with a variety of histological tumor types. We examined whether DCs can modify the survival of tumor specific CTLs during encounter with tumor targets. HLA-A2+ gp100-specific CD8+ CTLs were used as effectors against gp100+, A2+ melanoma FEM-X, and Mel526 (A2-) targets as well as the melanoma target Mel397. Cytolytic assays and [3H]DNA fragmentation (JAM) assays were used to evaluate CTL specificity and tumor-mediated AICD, respectively. A functional assay, ARK (activity of rescued killer cells), was developed to measure cytolytic activity of surviving CTLs after a 12-h coincubation with tumor. In JAM assays, the CTLs proved more susceptible to apoptosis when exposed to the relevant tumors FEM-X and Mel526 than the irrelevant Mel397 (37 and 23% versus 3%; P < .001). The addition of human A2+ monocyte-derived immature DCs significantly (P < 0.001) limited this tumor-induced death of CTLs. In ARK assays, the presence of DCs decreased tumor-mediated suppression of CTLs, with increases in cytolytic function of CTLs reaching up to 2-fold. These findings suggest that DCs may play an important role during the effector phase of the immune response by enhancing the survival and function of CTLs in the tumor microenvironment.

Interleukin-18 (IL-18) synergizes with IL-2 to enhance cytotoxicity, interferon-gamma production, and expansion of natural killer cells.

We investigated the in vitro effects of combining interleukin-18 (IL-18) and IL-2 on human lymphocytes. The combined use of these two cytokines synergistically enhanced the proliferation, cytolytic activity, and interferon-gamma production of peripheral blood mononuclear cells. Phenotypic analysis revealed a preferential expansion of CD56+CD3- cells and an up-regulation of IL-2 receptor-alpha expression on natural killer cells. Isolated natural killer cells showed a substantial increase in proliferation and cytotoxicity compared with CD4+ and CD8+ T cells. The combined use of IL-18 and IL-2 should be considered a viable strategy to induce an antitumor response in vivo.

Stable transduction of the interleukin-2 gene into human natural killer cell lines and their phenotypic and functional characterization in vitro and in vivo.

A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2-dependent NK-92 and IL-2-independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 and neor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/10(6) cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.