1a.Objectives (from AD-416)
The innate response of inflammatory cells in animals infected with foot-and-mouth disease virus (FMDV) has begun to reveal the immune evasion capabilities of this acute virus. Analysis of the natural killer cell (NK cell) response in FMDV infection has led to indications that the virus has evolved the capacity to induce short term, non-responsive state in these cells in swine. This project will now focus on the innate response in cattle. Should funds become available, we will continue analysis of swine responses as well.
The objectives of this project are 1: To determine methods of activation of NK cells in response to FMDV infection. 2: To design soluble gamma-delta T cell receptor (sTCR) of bovine and to investigate the viral antigen binding capability of gamma-delta T cells . Antigens identified through the sTCR will be studied regarding their capacity to stimulate gamma-delta T cells.
1b.Approach (from AD-416)
1. Methods of activation of NK cells in response to FMDV infection will be determined through the analysis of the activation/suppression of NK cells. ARS, PIADC will provide Warsaw Univ. samples from infected bovine for analysis at PIADC. 2. Based on these data, Warsaw Univ. will design and assemble in vitro, soluble gamma-delta T cell receptors, screen the sTCR for antigen binding to delineate the nature of antigen type and assess the activation status of gamma-delta T cells with identified antigens. ARS, PIADC will screen the sTCR for FMDV antigen binding and examine the activation status of gamma-delta T cells following stimulation with FMDV.
1. Assessment of in vitro NK cell cytotoxic activity. The K562 cell line was identified as a sensitive target for porcine or bovine NK cells. These cells were transfected with the green fluorescent protein encoding gene to generate K562-pmaxFP. These cells will be used to identify target cells via flow cytometry, 2. Studies were initiated on the construction of soluble gammadelta T cell receptor (TCR). Bovine WC1+ cells have been expanded from the interleukin (IL) IL-12 and IL-15 proliferation and are being subcloned for use in spectratyping.
This project was monitored through email and telephone exchange, as well as site visits by University of Warsaw staff to ARS, PIADC.