Youmin Kang

Induction of regulatory T cells by physiological level estrogen.

Naturally occurring CD4+CD25+ regulatory T cells (Treg) exert an important role in mediating maternal tolerance to the fetus during pregnancy, and this effect might be regulated via maternal estrogen secretion. Although estrogen concentration in the pharmaceutical range has been shown to drive expansion of CD4+CD25+ Treg cells, little is known about how and through what mechanisms E2 within the physiological concentration range of pregnancy affects this expansion. Using in vivo and in vitro mouse models in these experiments, we observed that E2 at physiological doses not only expanded Treg cell in different tissues but also increased expression of the Foxp3 gene, a hallmark for CD4+CD25+ Treg cell function, and the IL‐10 gene as well. Importantly, our results demonstrate that E2, at physiological doses, stimulated the conversion of CD4+CD25− T cells into CD4+CD25+ T cells which exhibited enhanced Foxp3 and IL‐10 expression in vitro. Such converted CD4+CD25+ T cells had similar regulatory function as naturally occurring Treg cells, as demonstrated by their ability to suppress naïve T cell proliferation in a mixed lymphocyte reaction. We also found that the estrogen receptor (ER) exist in the CD4+CD25− T cells and the conversion of CD4+CD25− T cells into CD4+CD25+ T cells stimulated by E2 could be inhibited by ICI182,780, a specific inhibitor of ER(s). This supports that E2 may directly act on CD4+CD25− T cells via ER(s). We conclude that E2 is a potential physiological regulatory factor for the peripheral development of CD4+CD25+ Treg cells during the implantation period in mice. J. Cell. Physiol. 214: 456–464, 2008.

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

Depleting Intratumoral CD4+CD25+ Regulatory T Cells via FasL Protein Transfer Enhances the Therapeutic Efficacy of Adoptive T Cell Transfer

One strategy for improving adoptive therapy is preconditioning the host immune environment by depleting CD4(+)CD25(+) regulatory T cells (Treg) suppressive to antitumor responses. Given that Treg increase, or selectively accumulate, within tumors and are sensitive to FasL-mediated apoptosis, we test here the hypothesis that inducing apoptosis of intratumoral Treg using FasL may improve adoptive T cell therapy. We show that FasL applied intratumorally via protein transfer decreases intratumoral Treg via inducing apoptosis in these cells. Significantly, we show that the use of FasL prior to the infusion of tumor-reactive CD8(+) T cells enhances the therapeutic efficacy of adoptive T cell transfer against established tumors, which is mediated by persistent, systemic antitumor immunity. Intratumoral FasL protein transfer also results in neutrophil infiltration of tumor. However, we show that intratumoral immunodepletion of neutrophils does not abolish the effect of FasL on adoptive transfer. Rather, the effect of FasL is completely abolished by cotransfer of Treg, isolated from the tumor-draining lymph nodes. Hence, our study shows for the first time that using FasL to predeplete intratumoral Treg provides a useful means for optimizing adoptive therapy.

The effects of IL-6 and TNF-α as molecular adjuvants on immune responses to FMDV and maturation of dendritic cells by DNA vaccination

Various approaches have been developed to improve efficacy of DNA vaccination, such as the use of plasmid expressing cytokine as a molecular adjuvant. In this study, we investigated whether co-inoculation of a construct expressing either IL-6 or TNF-alpha as the molecular adjuvant with FMDV DNA vaccine, pcD-VP1, can increase immune responses. Compared to the group immunized with pcD-VP1 alone, the co-inoculation with either molecular adjuvant induced a higher ratio of IgG2a/IgG1, higher levels of expression of IFN-gamma in CD4+ and CD8+ T cells, IL-4 in CD4+ T cells, and in vivo antigen-specific cytotoxic response. Both adjuvants induced maturation of dendritic cells, suggesting a correlation between the initiating innate response and subsequent activating adaptive immune responses. Together, the results demonstrate that IL-6 and TNF-alpha used as molecular adjuvants can enhance the antigen-specific cell-mediated responses elicited by VP1 DNA vaccine.

Levamisole is a potential facilitator for the activation of Th1 responses of the subunit HBV vaccination.

Chemical compounds activating innate responses may present potential adjuvants for the vaccine development. Levamisole (LMS), demonstrated as a potent adjuvant for DNA and viral killed vaccines in our previous studies, may activate such responses. To confirm this notion, LMS combined with the recombinant HBsAg (rHBsAg) was investigated. Compared to the vaccination with rHBsAg alone, LMS could up-regulate the expressions of TLR7&8, MyD88, IRF7 and their downstream pro-inflammatory cytokines including IFN-alpha and TNF-alpha, which promote DCs activation. Strikingly, we find that the combination of LMS and alum adjuvant synergistically enhances immunogenicity of rHBsAg and leads to a robust cell-mediated response demonstrated by the higher level of IgG2a/IgG1, T cell proliferation, and importantly, a high level of antigen-specific CTL and IFN-gamma production within these activated CD8(+) T cells. The achieved robust responses are at a comparative level with CpG+alum used as a positive control adjuvant in mice. The combination of LMS+alum with rHBsAg may provide a cost-effective, safe, and effective therapy to treat those individuals chronically infected by HBV, since antigen-specific cellular immunity is implicated for the clearance of HBV chronic infection.

Induction of Adaptive T Regulatory Cells That Suppress the Allergic Response by Coimmunization of DNA and Protein Vaccines

Allergen-induced immediate hypersensitivity (AIH) is a health issue of significant concern. This robust inflammatory reaction is initiated by the allergen-specific T cell responsiveness. Severe lesion reactions on skin are consequential problem requiring medical treatment. Effective Ag-specific treatments or preventions are lacking. Using a rodent model of AIH induced by flea allergens, we first report that coimmunization of DNA and protein vaccines encoding the flea salivary specific Ag-1 ameliorated experimental AIH, including Ag-induced wheal formation, elevated T cell proliferation, and infiltration of lymphocytes and mast cells to the site of allergen challenge. The amelioration of AIH was directly related to the induction of a specific population of flea antigenic specific T cells exhibiting a CD4+CD25−FoxP3+ phenotype, a characteristic of regulatory T (TREG) cells. These TREG cells expressing IL-10, IFN-γ, and the transcriptional factor T-bet after Ag stimulation were driven by a tolerogenic MHC class II+/CD40low dendritic cell population that was induced by the coimmunization of DNA and protein vaccines. The tolerogenic dendritic cell could educate the naive T cells into CD4+CD25−FoxP3+ TREG cells both in vitro and in vivo. The study identified phenomenon to induce an Ag-specific tolerance via a defined Ag vaccinations and lead to the control of AIH. Exploitation of these cellular regulators and understanding their induction provides a basis for the possible development of novel therapies against allergic and related disorders in humans and animals.

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.

Mushroom lectin enhanced immunogenicity of HBV DNA vaccine in C57BL/6 and HBsAg-transgenic mice

DNA vaccination is a promising strategy for activating immune responses against hepatitis B virus (HBV) infection. However, the accumulated data have shown that DNA vaccination alone generates weak immune responses. To enhance the immunogenicity of HBV DNA vaccine, lectin purified from pleurotus ostreatus (POL) was used as adjuvant of HBV DNA vaccine for C57BL/6 and HBV surface antigen transgenic (HBVsAg-Tg) mice. Our data demonstrate that low dose of POL (1 μg/mouse) in conjunction with HBV DNA vaccine stimulated stronger HBV-specific delayed-type hypersensitivity (DTH) responses and higher HBV-specific IgG level than that in high dose of POL groups (5 μg/mouse and 10 μg/mouse). POL activated strong Th2 and Tc1 cell responses in immunized C57BL/6 and HBVsAg-Tg mice. POL as adjuvant of HBV DNA vaccine effectively enhanced HBV surface protein antibody (HBVsAb) and decreased HBVsAg level for HBV Tg mice treatment. Furthermore, POL infiltrated more lymphocytes excluding Th1, Th2 and Tc1 cell subtypes to liver of HBVsAg-Tg mice. Together, these results suggest that POL as adjuvant enhanced immunogenicity of HBV DNA vaccination and effectively stimulated immune reaponse for HBsAg-Tg mice treatment. Our findings implicate the potential of mushroom lectin as adjuvant of HBV DNA vaccine.

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.

CD40-expressing plasmid induces anti-CD40 antibody and enhances immune responses to DNA vaccination.

Various approaches have been used to improve the efficacy of DNA vaccination, including the incorporation of molecular adjuvants. Because the CD40 ligand–CD40 interaction plays a major role in initiating immune responses, we sought to develop a molecular adjuvant targeting this interaction.