This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To target the Wisconsin National Primate Research Center's (WNPRC) mission to develop treatments for human disease, generate knowledge of primate biology, and to facilitate the progress of research by providing expertise and resources to external scientists, we are developing a methodology that we will use to determine the abundance and in situ localization of CD4 T cells specific to simian immunodeficiency virus (SIV) in tissues during SIV infection. RESULTS: We initiated these studies by working to develop methods to stain antigen specific CD4 T cells in mice. In collaboration with Dr. Marc Jenkins and Dr. T. Dileepan at the University of Minnesota we are using mice infected with bacteria that are engineered to express a peptide that triggers a peptide-specific CD4 T cell response for which MHC class II tetramers are available. We are staining nasal associated lymphoid tissues (NALT) and spleen tissues because peptide-specific CD4 T cells accumulate at these sites. We stained tissues with class II tetramers at several different concentrations, for different lengths of incubation time, at different temperatures, and using several amplification techniques. We were successful at identifying conditions that allowed us to detect antigen-specific CD4 T cells cells stained with tetramers in the NALT. These finding will be presented at the Immunology 2011 meeting and are presently being written up for publication. In addition, Dr. Nancy Wilson at the WNPRC made substantial progress developing class II reagents for use in SIV-infected macaques. She has now successfully produced, purified, and tetramerized rhesus macaque class II molecules. Lara Vojnov at the WNPRC has begun to test these reagents in tissues from SIV infected rhesus macaques. In addition, Dr. Wilson continues to expand the repertoire of class II tetramer reagents to include additional class II alleles. Details of Dr. Wilson's work is presented below. Gag102-113 (QIVQRHLVVE), along with a linker region was engineered N terminal of DRB*w606, between the leader sequence and start of translation. DRA*010401 was engineered into the other expression site for the bi-cistronic vector pBAC10. These were done using the method of Kozono, et al. This plasmid was co-transformed with linear DNA encoding the rest of the baculovirus genome and baculovirus was generated on SF9 cells, grown to high volume and titered. Hi5 cells were expanded and infected with baculovirus encoding this construct. Supernatant was collected after 6 days of infection and monomer was purified using an affinity column to which 5 mg/ml of L243 antibody had been covalently linked. The monomer was biotinylated and conjugated to Streptavidin APC. The cells used for testing the tetramer were cryopreserved CD4+ T cell clones generated in our lab (Giraldo-Vela, et al). Unfortunately the amount of tetramer produced was small, so we have only a limited amount available for testing.