Improving T-Lymphocyte Recognition of Vaccinia Virus Antigens
The purpose of this study is to analyze the role of vaccinia virus (VACV) protein X in classical and in TAP-independent recognition of VACV infected cells by CD8+ T lymphocytes. In this study, bone marrow dendritic cells (BMDC) (antigen presenting cells) were isolated from wild-type C57BL/6 and TAP knockout (TAPKO) mice. The BMDCs were each infected with three VACV: delta X, revertant X and IPTG-inducible X. Splenocytes were isolated from the spleen of a C57BL/6 mouse infected with VACV and used as effector T-lymphocytes. BMDCs and splenocytes were co-cultured in medium with Brefeldin A, an inhibitor of protein transport to the plasma membrane. CD8+ T-lymphocytes in co-culture were labeled with the anti-CD8+ antibody conjugated to an APC fluorophore. As a measure of the activation of CD8+ T-lymphocytes, the cytokine interferon gamma (IFNγ+) was labeled with an anti-IFNγ+ antibody conjugated to a PE fluorophore. The labeled co-cultured samples were passed through a flow cytometer and data were analyzed using FlowJo software.
Data show that BMDCs infected with delta X VACV lead to higher CD8+ T-cell activation than BMDCs infected with revertant X in both C57BL/6 and TAPKO BMDCs. There was no significant difference between the T-cell activation with the IPTG-inducible X and revertant X infected BMDCs. The same results were obtained in vivo.
The high percentage of antigen presentation in BMDCs from C57BL/6 and TAPKO mice infected with delta X VACV was not expected since the X gene was deleted. This may indicate that protein X may inhibit the function of membrane proteins required for antigen presentation or for T-cell activation. Further studies will be done to determine if protein X plays a role in either function. Several viruses and tumors interfere with the classical antigen presentation pathway by inactivating TAP. It is important to find novel routes of antigen presentation that can potentially lead to development of vaccines which provide long-lasting, potent, and effective immune responses to viral and bacterial infections.