An Alternative and Effective HIV Vaccination Approach Based on Inhibition of Antigen Presentation Attenuators in Dendritic Cells

BACKGROUND: Current efforts to develop HIV vaccines that seek to stimulate immune responses have been disappointing, underscoring the inability of natural immune responses to control HIV-1 infection. Here we tested an alternative strategy to induce anti-HIV immune responses by inhibiting a host's natural immune inhibitor. METHODS AND FINDINGS: We used small interfering RNA (siRNA) to inhibit suppressor of cytokine signaling (SOCS) 1, a key negative regulator of the JAK/STAT pathway, and investigated the effect of this silencing on the ability of dendritic cells (DCs) to induce anti-HIV-1 immunity. We found that SOCS1-silenced DCs broadly induced enhanced HIV-1 envelope (Env)-specific CD8(+) cytotoxic T lymphocytes and CD4(+) T helper cells, as well as antibody responses, in mice. Importantly, SOCS1-silenced DCs were more resistant to HIV Env-mediated suppression and were capable of inducing memory HIV Env-specific antibody and T cell responses. SOCS1-restricted signaling, as well as production of proinflammatory cytokines such as interleukin-12 by DCs, play a critical role in regulating the anti-HIV immune response. Furthermore, the potency of HIV DNA vaccination is significantly enhanced by coimmunization with SOCS1 siRNA expressor DNA. CONCLUSIONS: This study demonstrates that SOCS1 functions as an antigen presentation attenuator to control both HIV-1-specific humoral and cellular responses. This study represents the first, to our knowledge, attempt to elicit HIV-specific T cell and antibody responses by inhibiting a host's antigen presentation attenuator, which may open a new and alternative avenue to develop effective therapeutic and prophylactic HIV vaccines

Twist-2 Controls Myeloid Lineage Development and Function

Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2–deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPα. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-γ (IFNγ) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells

Dendritic cell vaccination and immune monitoring

We exploited dendritic cells (DC) to vaccinate melanoma patients. We recently demonstrated a statistical significant correlation between favorable clinical outcome and the presence of vaccine-related tumor antigen-specific T cells in delayed type hypersensitivity (DTH) skin biopsies. However, favorable clinical outcome is only observed in a minority of the treated patients. Therefore, it is obvious that current DC-based protocols need to be improved. For this reason, we study in small proof of principle trials the fate, interactions and effectiveness of the injected DC

SOCS1 restricts dendritic cells’ ability to break self tolerance and induce antitumor immunity by regulating IL-12 production and signaling

DC-based tumor vaccine research has largely focused on enhancing DC maturation/costimulation and antigen presentation in order to break tolerance against self tumor-associated antigens. DC immunization can activate autoreactive T cells but rarely causes autoimmune pathologies, indicating that self tolerance at the host level is still maintained in the vaccinated hosts. This study in mice reveals a novel regulatory mechanism for the control of self tolerance at the host level by DCs through the restriction of positive cytokine feedback loops by cytokine signaling inhibitor SOCS1. The study further finds the requirement of persistent antigen presentation by DCs for inducing pathological autoimmune responses against normal tissues and tumor, which can be achieved by silencing SOCS1 to unleash the unbridled signaling of IL-12 and the downstream cytokine cascade. However, the use of higher-affinity self peptides, enhancement of DC maturation, and persistent stimulation with cytokines or TLR agonists fail to break tolerance and induce pathological antitumor immunity. Thus, this study indicates the necessity of inhibiting SOCS1, an antigen presentation attenuator, to break self tolerance and induce effective antitumor responses

Enhanced gp120-Specific Antibody and T Cell Responses Induced by SOCS1-Silenced DCs

<p>Groups of C57BL/6 mice were immunized with gp120 (SF162) protein-pulsed, transduced BM-derived DCs (1 × 10⁶ cells/mouse) twice at a weekly interval, followed by PolyI:C or R837 stimulation (30 μg/mouse) in vivo three times on days 1, 3, and 5 after each DC immunization, and sera and splenocytes were collected from each group of mice 14 d later. HIV gp120-specific IgG subclass titers (A) from the pooled sera of each group (4–6 mice/group) were quantified by capture ELISA. CD8⁺ T cells (B) and CD4⁺ T cells (C) isolated from pooled splenocytes were used for IFN-γ ELISPOT assays stimulated with gp120 proteins. Intracellular IFN-γ staining of CD8⁺ T cells from the pooled splenocytes were also performed (D). Representative data from one of three experiments are presented. NS, no stimulation. *<i>P</i> < 0.01, LV-SOCS1-siRNA-DCs versus LV-GFP-siRNA-DCs.</p

Comparison of gp120-Specific Antibody and T Cell Responses Induced by Protein Immunization and SOCS1-Silenced DCs

<p>Groups of C57BL/6 mice were immunized with gp120 protein (20 μg/ml)-pulsed, transduced BM-derived DCs (1 × 10⁶ cells/mouse) or the same amount of gp120 protein formulated in IFA (20 μg/mouse) twice at a weekly interval. All of the mice were injected with PolyI:C or R837 (30 μg/mouse) in vivo three times on days 1, 3, and 5 after each immunization, and sera and splenocytes were collected from each group of mice 14 d later. HIV gp120-specific IgG subclass titers (A) from the pooled sera of each group (4–6 mice/group) were quantified by capture ELISA. CD8⁺ T cells (B) and CD4⁺ T cells (C) isolated from pooled splenocytes were used for IFN-γ ELISPOT assays stimulated with gp120 proteins. Representative data from one of three experiments are presented. *<i>P</i> < 0.01, gp120 protein + IFA versus gp120-pulsed LV-SOCS1-siRNA-DCs.</p

Resistance of SOCS1-Silenced DCs to HIV gp120-Mediated Suppression

<div><p>(A) Effects of gp120 on cytokine production by DCs. BM-DCs were transfected with SOCS1-siRNA or a SOCS1-siRNA mutant oligonucleotide as described previously [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0030011#pmed-0030011-b024" target="_blank">24</a>], and then cultured with or without SF162 gp120 (20 μg/ml) or LPS (100 ng/ml), and cytokine levels were determined at the different times of cultures, as indicated.</p> <p>(B–E) Effects of gp120 on DC antigen presentation in vivo. Transfected BM-DCs were pulsed with OVA, incubated with or without gp120 for 2 d, and then stimulated with LPS (100 ng/ml) ex vivo overnight. Mice were then immunized with the transduced DCs twice, following three in vivo LPS stimulations. OVA-specific antibody IgG (B) and IgG1 (C) titers and frequencies of IFN-γ-producing OVA-specific CD8⁺ T cells (D) and CD4⁺ T cells (E) were examined 2 wk after the second DC immunization. Data are representative of two repeats.</p></div