Construction of Artificial Antigen Presenting Cell for Hepatitis B Virus Research

Up to 20% of adults in Taiwan are chronic carriers of hepatitis B. These chronic carriers have a high risk of developing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Worldwide deaths from HBV-associated liver cancer exceed one million per year. Thus, development of an effective treatment for chronic hepatitis B (CHB) patients is of medical importance. Many studies suggest that viral replication in chronic hepatitis B patients could be inhibited by activated HBV-specific cytotoxic T cells. Therefore, adoptive transfer activated HBV specific CTLs into CHB patients represents a promising method to treat CHB. For adoptive immunotherapy, one commonly used approach for induction and expansion of antigen-specific CTLs has been based on the use of antigen-loaded dendritic cells as antigen presenting cells. However, generation and maintenance of DCs is costly and cumbersome. An alternative source of APC, artificial antigen-presenting cell (aAPC), is therefore designed to stimulate the expansion and acquisition of optimal therapeutic features of T cells.
Efficient T-cell activation requires stimulation of a combination of signals. Antigen encountered T cells receive the first signal through engagement of the T-cell receptor with peptide：MHC (major histocompatibility complex) complexes on APCs. At the same time, a second signal, the co-stimulatory signal, is delivered by the same APC.
In this study, we aimed to develop aAPC systems to expand HBV-specific CTLs. The fibroblast-based aAPCs were engineered to express a single chain HBs28-39-β2 microglobulin-H2Ld complex (signal 1), a membrane anchored anti-4-1BB single- chain Fv fragments (scFv) (signal 2) as well as T-cell growth and stimulating factor, IL-2 or IL-15. Expression of HBs-H2Ld and anti-4-1BB scFv protein was detected by FACS analysis of aAPCs. We also demonstrated that these aAPCs release a significant amount of biologically active IL-2 and IL-15. In one experiment, we found that these aAPCs could induce a small percentage (~ 3%) HBs-specific CTLs in vitro as detected by MHC pentamer staining. However, in other experiments HBs-β2m- H2Ld-expressing target cells were not sensitized for lysis by HBs-specific CTLs in a 51Cr-release assay. Further experiments are required to resolve this discrepancy.

In the second part of the study, we isolated human peripheral mononuclear cells, and proved that their proliferative activity was preserved after cryopreservation. We also set up techniques to culture plastic-adherent blood monocytes and stimulated their differentiation into CD83+ cells with immunophenotypic and morphologic characteristic of mature dendritic cells.