Skip to main content
. 2014 Jun 3;97(11):1119–1127. doi: 10.1097/TP.0000000000000113

FIGURE 4.

FIGURE 4

PBMCs can present HLA-A2 monomer derived peptides via HLA-DR1 with no measurable semi-direct alloreactivity. A–B, 1×105 Typed HLA-DR1+/HLA-A2- PBMCs were incubated with different concentrations of HLA-A2 monomers or pulsed with an HLA-A2 peptide and cocultured with or without 5×103 4.44 T-cells; 48 hr after incubation, supernatants were collected and IFN-γ measured. C–D, PBMCs were incubated with different concentrations of HLA-A2 monomers and cocultured with either the indirect recognizing clone 4.44 (gray circles) or with the direct recognizing clone 1E2 (white circles) for 48 hr. IFN-γ and proliferation were analyzed as previously described. E, Different PBMC numbers were incubated with 25 μg/mL HLA-A2 in the presence of 5×103 4.44 for 24 or 48 hr. Supernatants were then harvested and IFN-γ measured. F, To test the minimal amount of T-cells needed to detect a response, 4.44 were titrated, and 25 μg/mL HLA-A2 monomer was added in combination with 1×105 HLA-typed PBMCs for 48 hr. Supernatants were then collected and IFN-γ measured. G–H, PBMCs were cocultured with 4.44 T-cells in the presence of 25 μg/mL HLA-A2 for 24 hr with or without anti-HLA-DR monoclonal antibodies (mAb). The reciprocal dilution of the HLA-DR mAb is set on the x-axis. All experiments were conducted at least three times. Shown is the mean+SD of one experiment in triplicate. Dashed line represents the detection limit of the ELISA used. ND, not detectable.