Aoshuang Chen

Cutting Edge: Immunosuppressant as Adjuvant for Tolerogenic Immunization

Vaccination for autoimmune and alloimmune diseases has long been an attractive idea. Yet, there is no suitable adjuvant to forcefully steer the immune response toward tolerance. In this study we show that dexamethasone, a potent glucocorticoid immunosuppressant, can function as a tolerogenic adjuvant when applied together with peptide immunogen. BALB/c mice with pre-established delayed-type hypersensitivity to hen OVA were immunized with an OVA-derived, MHC II-restricted peptide (OVA323–339) in the presence of dexamethasone. The treatment caused long-term desensitization in treated animals to hen OVA via a dexamethasone-dependent tolerogenic mechanism that blocks maturation of dendritic cells and expands OVA323–339-specific CD4+CD25+Foxp3+ regulatory T cells in vivo. Similar treatment of NOD mice using dexamethasone and an insulin-derived, MHC II-restricted peptide (B:9–23) prevented predisposed spontaneous diabetes. Remarkably, in both models, dexamethasone-augmented immunization induced long-term persistent, Ag-specific regulatory T cells responsive to recall Ags. These results reveal for the first time the potential usefulness of immunosuppressants as tolerogenic adjuvants.

The adjuvant effects of co-stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination

Because DNA vaccines on their own tend to induce weak immune responses in humans, adjuvant methods are needed in order to improve their efficacy. The co‐stimulatory molecules 4‐1BBL, OX40L, and CD70 have been shown to induce strong T cell activities; therefore, in this study, we investigated whether they may be used as molecular adjuvants for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcDS2) in eliciting strong cellular and memory responses. Compared to mice immunized with pcDS2 alone, addition of the co‐stimulatory molecules increased T cell proliferation and an HBsAg‐specific antibody response that was marked with a higher ratio of IgG2a/IgG1. Importantly, pcDS2 plus these co‐stimulatory molecules elicited a higher level of IFN‐γ and IL‐4 in CD4+ T cells and a higher level of IFN‐γ in CD8+ T cells. In addition, a significantly robust antigen‐specific cytotoxic T lymphocyte (CTL) response and the production of long‐term memory CD8+ T cells were also observed in the groups immunized with pcDS2 plus 4‐1BBL, OX40L, or CD70. Consistently, as late as 100 days after immunization, upregulated expressions of BCL‐2, Spi2A, IL‐7Ra, and IL‐15Ra were still observed in mice immunized with pcDS2 plus these co‐stimulatory molecules, suggesting the generation of memory T cells in these groups. Together, these results suggest that the co‐stimulatory molecules 4‐1BBL, OX40L, or CD70 can enhance the immunogenicity of HBsAg DNA vaccines, resulting in strong humoral, cellular, and memory responses. This approach may lead to an effective therapeutic vaccine for chronic hepatitis B virus (HBV) infection. Copyright

Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.

Dexamethasone promotes tolerance in vivo by enriching CD11cloCD40lo tolerogenic macrophages

We previously showed that antigen immunization in the presence of the immunosuppressant dexamethasone (a strategy we termed “suppressed immunization”) could tolerize established recall responses of T cells. However, the mechanism by which dexamethasone acts as a tolerogenic adjuvant has remained unclear. In the present study, we show that dexamethasone enriches CD11cloCD40lo macrophages in a dose‐dependent manner in the spleen and peripheral lymph nodes of mice by depleting all other CD11c+CD40+ cells including dendritic cells. The enriched macrophages display a distinct MHC class II (MHC II)loCD86hi phenotype. Upon activation by antigen in vivo, CD11cloCD40lo macrophages upregulate IL‐10, a classic marker for tolerogenic antigen‐presenting cells, and elicit a serum IL‐10 response. When presenting antigen in vivo, these cells do not elicit recall responses from memory T cells, but rather stimulate the expansion of antigen‐specific regulatory T cells. Moreover, the depletion of CD11cloCD40lo macrophages during suppressed immunization diminishes the tolerogenic efficacy of the treatment. These results indicate that dexamethasone acts as a tolerogenic adjuvant partly by enriching the CD11cloCD40lo tolerogenic macrophages.

FK506 as an adjuvant of tolerogenic DNA vaccination for the prevention of experimental autoimmune encephalomyelitis

DNA vaccination is a strategy that has been developed primarily to elicit protective immunity against infection and cancer.

Enhanced Antitumor Responses Elicited by Combinatorial Protein Transfer of Chemotactic and Costimulatory Molecules

Thus far, immunotherapies based on one or a few immunostimulatory molecules have shown limited antitumor efficacy. This highlights the need to use multiple immunostimulatory molecules, to target different immune cells, including immunosuppressive cells, simultaneously. Consequently, in this study, we delivered intratumorally via protein transfer four molecules, including the chemotactic molecules secondary lymphoid tissue chemokine and Fas ligand and the costimulatory molecules 4-1BBL and TNF-related activation-induced cytokine. Secondary lymphoid tissue chemokine and Fas ligand together can attract an array of immune cells and induce apoptosis in CD4+CD25+ regulatory T cells (Treg), whereas 4-1BBL and TRANCE together can stimulate T cells and dendritic cells (DCs). We show that the transfer of all four molecules increases tumor-infiltrating neutrophils, DCs, and CD4+ and CD8+ T cells and decreases intratumoral Treg. We show that the treatment favors the generation of a Th1 cytokine milieu at the tumor site, which is attributed not only to an increase in IL-12-producting DCs and IFN-γ-producing CD8+ T cells, but also to a decrease in IL-10-producing Treg. Importantly, in the L5178Y lymphoma model, we show that compared with transfer of the chemotactic molecules alone or the costimulatory molecules alone, transfer of all four molecules demonstrates stronger antitumor responses against established tumors. Furthermore, we show that the antitumor responses elicited by transfer of all four molecules are mediated by long-term, systemic antitumor immunity. Hence, this study demonstrates for the first time that combinatorial use of chemotactic and costimulatory molecules provides a useful strategy for enhancing antitumor responses.

HMGB1 in Hormone-Related Cancer: a Potential Therapeutic Target

High-mobility group box 1 (HMGB1) is a dynamic nuclear protein participating in transcription, chromatin remodelling, and DNA recombination and repair processes. Accumulating evidence indicates that its function now extends beyond the nucleus, notably its extracellular role in inflammation. HMGB1 is implicated as a late mediator of sepsis and is also believed to promote atherosclerosis and other inflammatory diseases such as rheumatoid arthritis and systemic lupus erythematosus. Interestingly, deregulation of HMGB1 is shown to be associated with the hallmarks of cancer development. Moreover, several clinical studies have shown that HMGB1 is a promising biomarker for a variety of cancer types. In this review, we provide novel insights into the role and mechanisms of HMGB1, in particular, to hormone-related cancers and its potential to serve as a therapeutic target.

Development and evaluation of a fluorescent antibody-drug conjugate for molecular imaging and targeted therapy of pancreatic cancer

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC’s utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC’s efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer.

Cutting Edge: Dexamethasone Potentiates the Responses of Both Regulatory T Cells and B-1 Cells to Antigen Immunization in the ApoE−/− Mouse Model of Atherosclerosis

The immunosuppressant dexamethasone was shown to preferentially deplete CD4+ effector T cells while sparing regulatory T cells (Tregs) in vivo. In the current study, we show that it also preferentially depletes B-2 cells while sparing B-1 cells. In the ApoE−/− mouse model of atherosclerosis, in which both Tregs and B-1 cells are thought to play an atheroprotective role, we show that HSP60-targeted immunization in the presence of dexamethasone raises Ag-reactive Tregs and B-1 cells concomitantly and reduces the severity of atherosclerosis. These results indicate that dexamethasone is an adjuvant that potentiates both the Treg and B-1 responses to immunogens. This study shows that B-1 cells with a specificity for a disease-relevant Ag can be raised in vivo by immunization.

Tolerogenic DNA Vaccine for Prevention of Autoimmune Ovarian Disease

DNA vaccines have been widely used to induce immune responses against molecular targets. In this study, we explored the possibility of using DNA vaccine combined with the immunosuppressant FK506 (tacrolimus) to antigen-specifically suppress unwanted immune responses and prevent autoimmune ovarian disease. To that end, we immunized C57BL/6 mice with a DNA vaccine encoding mouse zona pellucida 3 (ZP3) together with FK506. The immunization induced ZP3-specific CD4+CD25+Foxp3+ regulatory T cells (Treg), which suppressed the induction of ZP3-specific delayed-type hypersensitivity in the animals. Significantly, the immunization also protected the animals from experimentally induced autoimmune ovarian disease. These results suggest that DNA vaccination in the presence of FK506 may be used to induce Treg cells and prevent AOD.