Functional Analysis of CD4+ T Cells

The functional analysis of CD4⁺ T cell responses has been published previously [66 (link)]. Briefly, CD4⁺ T cells were MACS-purified from the draining lymph nodes with anti-CD4 (Miltenyi Biotec, Auburn, CA). Subsequently, isolated CD4⁺ T cells were used to directly measure cytokine gene expression by qPCR. Alternatively, 2 x 10⁵ CD4⁺ T cells/well were cultured in 96-well U-bottom plates in the presence of 5μg E651 peptide and 3 x 10⁵ naïve splenocytes as antigen-presenting cells. IL-21 production in the cultures was measured three days later by either qPCR. IFN-γ protein was measured by ELISA with an anti-mouse IFN-γ capture antibody, followed by an biotinylated anti-mouse IFN-γ detection antibody, and subsequently streptavidin-conjugated horseradish peroxidase (SA-HRP) and TMB substrate (both BD Bioscience, San Diego, CA).