[show abstract][hide abstract] ABSTRACT: Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier.
We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8(+) and CD4(+) memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8(+) memory T cells and production of IFNγ plus CD4(+) memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes.
These experiments show that vault nanocapsules induced strong anti-OVA CD8(+) and CD4(+) T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8(+) and CD4(+) T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.
[show abstract][hide abstract] ABSTRACT: Aim
Antibody-mediated rejection (AMR) is a clinical problem of solid organ transplantation. Recipients producing anti-HLA antibodies (Ab) are at a higher risk for acute and chronic AMR. Anti-HLA Ab contribute to the process of AMR by binding to the HLA molecules on endothelial cells (EC) and triggering signal transduction pathways leading to cell survival and proliferation. We and others have reported that ligation of HLA-I with Ab on EC contributes to the process of transplant vasculopathy. However, the role of HLA-II Ab in this process is unknown.
Primary human aortic EC were infected with recombinant adenovirus pAd/PL-DEST encoding CIITA, the regulator of HLA-II expression, or treated with TNFa and IFNg. HLA-II expression was determined by FACS using Ab. CIITA infected or cytokine-treated EC were stimulated with anti-HLA-II Ab, phosphorylation was detected by Western Blot, and proliferation was measured by CSFE labeling and flow cytometry.
Infection of EC with adenovirus-CIITA induced a 3 to 5-fold increase in HLA-II expression, including DP, DQ, and DR. Similarly, cytokine treatment of EC mediated a 2-fold increase in HLA-II expression on EC. Ligation of HLA-II antigens on CIITA infected and cytokine treated EC stimulated a marked increase in phosphorylation of Src and FAK, activation of mTORC1 pathway including S6K, S6RP, and 4E-BP1, and activation of mTORC2 signaling, Akt and ERK. The highest phosphorylation level was seen using 1.0 μg/ml of Ab. Ligation of HLA with class II mAb on EC also stimulated cell proliferation.
In this study we found that CIITA adenovirus infection and cytokine treatment can induce HLA-II expression on EC. Ligation of class II with Ab on EC triggered intracellular signal cascades promoting cell survival and proliferation. Our results provide novel methods to study the mechanisms of HLA-II Ab-mediated alterations in endothelial cells. These studies also suggest that HLA-II Ab may contribute to the process of chronic rejection.