This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Although a number of techniques have recently been developed for the identification of antigen-specific cells, the ability to isolate viable antigen-specific cells remains limited, especially for CD4+ T cells. Here we describe a novel approach to the isolation of live antigen-specific CD4+ T cells using CD40L and CD69 surface staining and demonstrate its utility for isolating rhesus macaque CD4 T cells. Critical to the success of the technique was staining for CD40L concurrent with antigen stimulation. In absence of anti-CD40L antibody during antigen stimulation, rhesus CD40L was rapidly endocytosed and inaccessible to antibody added following antigen stimulation. Isolation of CD4 T cells based on surface marker upregulation has the following advantages: 1) whole antigen or peptide pools can be used to stimulate cells, therefore there is no need to do epitope mapping and MHC class II restriction; and 2) surface activation marker upregulation occurs on both CD4 T cell effector and central memory populations. In contrast, most central memory CD4 T cells do not secrete cytokines such TNFalpha and IFNgamma and will not be detected by techniques based on their secretion. The methodology described here therefore opens new avenues of investigation for nonhuman primate models of human diseases and may prove beneficial in the evaluation of vaccine protocols when the induced response is largely central memory T cell restricted.
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