Theileria annulata-transformed cell lines are efficient antigen-presenting cells for in vitro analysis of CD8 T cell responses to bovine herpesvirus-1

Abstract

Continuously growing cell lines infected with the protozoan parasite Theileria annulata can readily be established by in vitro infection of leukocytes with the sporozoite stage of the parasite. The aim of the current study was to determine whether such transformed cell lines could be used as antigen presenting cells to analyse the antigenic specificity of bovine CD8 T cell responses to viral infections. Bovine herpes virus 1 (BHV-1), which is known to induce CD8 T cell responses, was used as a model. T. annulata- transformed cells were shown to express high levels of CD40 and CD80 and were susceptible to infection with BHV-1, vaccinia and canarypox viruses. The capacity of the cells to generate antigen-specific CD8 T cell lines was initially validated using a recombinant canarypox virus expressing a defined immunodominant T. parva antigen (Tp1). Autologous T. annulata-transformed cells infected with BHV-1 were then used successfully to generate specific CD8 T cell lines and clones from memory T cell populations of BHV-1-immune animals. These lines were BHV-1-specific and class I MHC-restricted. In contrast to previous studies, which reported recognition of the glycoproteins gB and gD, the CD8 T cell lines generated in this study did not recognise these glycoproteins. Given the ease with which T. annulata-transformed cell lines can be established and maintained in vitro and their susceptibility to infection with poxvirus vectors, these cell lines offer a convenient and efficient in vitro system to analyse the fine specificity of virus-specific CD8 T cell responses in cattle.

Identification of immediate early gene products of bovine herpes virus 1 (BHV-1) as dominant antigens recognized by CD8 T cells in immune cattle

“…Animals vaccinated with a DNA plasmid encoding gB were also shown to generate a gB-specific CD8 T-cell response [35]. However, the CD8 T cell lines used in the current study did not have detectable reactivity with cells transfected with the gB, gC or gD genes, and in a previous study we showed that the same cell lines did not recognize cells infected with recombinant vaccinia viruses expressing these glycoproteins [25]. Previous antigen screening by Denis et al, [24] was based on cytotoxicity assays using virus-stimulated whole PBMC as effectors and was confined to four glycoproteins expressed Table 3.…”

“…The use of T. annulata-infected cells as antigen-presented cells to generate the CD8 T-cell lines is unlikely to have resulted in a preferential bias in the specificity of the detected response. They express high levels of both class I and class II MHC proteins on their cell surface and support fully productive infection with BHV-1, resulting in cell lysis and release of infectious virus; they also show surface expression of BHV-1 glycoproteins following infection with the virus [25]. Hence, they are expected to express the full repertoire of BHV-1 proteins and present them efficiently to CD8 T cells.…”

“…The Cooper and Iowa strains of BHV-1 used in the study (kindly provided by Moredun Scientific, Roslin, UK) and the methods used to culture and quantify the virus are described elsewhere [25]. All of the in vitro analyses of Tcell responses used the Cooper strain.…”

“…The method used for establishing CD8 T-cell lines specific for BHV-1 is described in detail elsewhere [25]. Briefly, aliquots of 4Â10 6 PBMC from immunized animals were cultured in 24-well plates with 2Â10 5 gamma-irradiated autologous T. annulata-infected cells infected with BHV-1.…”

“…This appears to have been partly due to the poor sensitivity of assays using ex vivo-derived T cells and the lack of reliable in vitro systems for the maintenance and enrichment of virus-specific CD8 T cells. Recently, we developed a system to derive virus-specific CD8 T-cell lines from BHV-1-immune cattle using Theileria annulata-transformed cell lines infected with the virus as stimulator cells [25]. Herein we describe the use of CD8 T-cell lines generated using this system to identify CD8 T cell target antigens in BHV1.…”