Anti cd4 or anti cd8 conjugated microbeads

Mouse spleens were collected and processed into single-cell suspensions. For cell depletion, splenocytes were incubated with anti-CD4- or anti–CD8-conjugated microbeads (Miltenyi Biotec), and the CD4 or CD8 T cells were negatively selected by VarioMACS separation columns (Miltenyi Biotech). FACS analysis of the remaining CD4 or CD8 T cells revealed <5% purity (not shown).

HSV-specific T cell responses were analyzed as previously described42 (link). At day 6 post-infection, CD4⁺ and CD8⁺ T cells were isolated from the draining lymph nodes of mice infected intravaginally with 10⁶ pfu of TK- HSV-2 using anti-CD4- or anti-CD8-conjugated microbeads (Miltenyi Biotech) according to the manufacturer’s instructions. 1 × 10⁵ CD4⁺ and CD8⁺ T cells were co-cultured with 2 × 10⁵ splenocytes as antigen presenting cells and restimulated with various amounts of heat-inactivated HSV-2 or HSV glycoprotein B (gB) peptide (SSIEFARL) at 37 °C for 72 h. IFN-γ production in supernatants was measured by ELISA.