Location: Foreign Animal Disease Research2012 Annual Report
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.
3. Progress Report:
This goal of this research project is to understand the innate response of Foot-and-Mouth Disease Virus (FMDV) infection in cattle through the determination of methods to activate gammadelta T cells and (natural killer) NK cells against FMDV infected targets and to design a soluble gammadelta T cell receptor (sTCR) of bovine and to investigate the viral antigen binding capability of the gammadelta T cells. Antigens identified by the soluble TCR will be studied regarding their capacity to stimulate gamma/delta T cells independently. During FY 2012, we tested various biophospho-antigens for their capability to stimulate bovine gammadelta T cells as has been reported in human and mouse studies. The purpose of experiments is to find suitable biophospho-antigens that are able to stimulate bovine gammadelta T cells and can be optimized for use in enhancing innate immunity in the bovine. These antigens were shown to activate gammadelta T cells to increase the expression of cytokines with only a marginal degree of proliferation. Over the life of the project we expect to derive soluble T cell receptors (sTCR) of gamma/delta T cells. These soluble receptors will allow us to show whether the gamma/delta T cells are involved in FMDV antigen recognition and if so what antigens these are and if they could be used in targeting FMDV. Since we have observed a moderate increase in activation following culture of gammadelta T cells in the presence of biophospho-antigens, in FY 2013 we plan to follow up this result by performing cytotoxicity assays. Once we have solved the problems with establishment of highly purified gamma/delta T cells for cloning, we will diversity of the TCR gamma/delta TCR in the clones raised previously. This will enable us to single out the dominating gamma/delta T cells which will form the basis of soluble TCR construction. Impact: This work will help determine if the early, innate gamma/delta T cell responses to FMDV infection in cattle are mediated by an antigen nonspecific reactivity where the TCR is acting as a pathogen recognition receptor (PRR) or if there is antigen specificity involved in this T cell activation. Technology transfers include; Indicator cells (K562-pmaxFP) have been developed and can be transferred to other users for analysis of T cell activation. Publications for the reporting period include; 1. Toka FN and WT Golde. Cell mediated innate responses of cattle and swine during virus infection: A unique landscape of innate immunity. Manuscript submitted to Veterinary Immunology & Immunopathology. 2. R. Waters, P. Dar, J. Patch, M. Kenney, R. Glabman, F. Toka, and W. Golde. Analysis of bovine natural killer (NK) cell and ¿d T cell mediated cytotoxicity following infection with foot-and-mouth disease virus (FMDV). Abstract - Submitted to the European Veterinary Immunology Workshop, Edinburgh, 2-4 September 2012.