HLA-Restricted CD4+ T Cell Responses

Briefly, an ELISPOT plates (Human IFN-γ ELISPOT set, BD, USA) were coated with IFN-γ capture antibody and incubated overnight at 4°C. The plates were washed 3 times with phosphate-buffered saline (PBS) and then blocked with RPMI 1640. After 2h the blocking, the plates were washed with PBS. The 5×10⁵ CD4⁺ T cells were co-incubated with peptide-pulsed or not 5×10⁴ aAPCs for 20h. The cells were removed and the plates were washed 3 times with 0.05% Tween 20/PBS using microplate washer (405LSR; BioTek). Biotinylated detection antibody for IFN-γ was added and incubated for 2h at 37°C. After 4 times washes with 0.05% Tween 20/PBS, avidin-horseradish peroxidase was added and plates were incubated for 1h at 37°C. The plates were washed 4 times with 0.05% Tween 20/PBS, followed by 2 washes with PBS and the addition of AEC substrate (BD) per well. The reaction was stopped by washing with deionized water, and the plates were dried overnight. The spot forming units were counted using an AID ELISPOT Reader System (AID Diagnostika GmbH). The frequencies of an HLA allotype-restricted CD4⁺ T cell response to antigens were calculated as [(response to aAPCs expressing HLA pulsed with peptide pools) − (response to aAPCs expressing HLA)] − [(response to aAPCs pulsed with peptide pools) − (response to aAPCs)], as previously described (32 (link), 33 (link)).