Antigen-presenting cells efficiently process tumor antigens linked to chemokines and facilitate peptide presentation through MHC class I molecules. (A) Naive C57BL/6 splenocytes were incubated overnight with 1 μg/mL MIP3α-gp100, MC148-gp100, MC148D-gp100, or gp100 protein alone. The cells were subsequently washed, irradiated, and then cocultured with immune effector splenocytes from C57BL/6 mice (immunized with hgp100_25-33/IFA). IFN-γ release was measured after overnight incubation. Effector-cell specificity was validated using splenocytes pulsed with 1 μg/mL hgp100_25-33 or an irrelevant A20 peptide, or incubating with B16 melanoma or control A20 lymphoma cells. (B) APCs (splenocytes) from BALB/c mice also cross-present exogenous OFA-iLRP antigen. iDCs were incubated with 0.1 μg/mL MIP3α-mOFA. Control DCs treated with MIP3α fused with an irrelevant tumor antigen, gp100 (MIP3α), alone or mixed with free unlinked MC148-D-mOFA (MIP3α+ OFA) failed to stimulate T cells. The specificity of effector T cells shown by their response to splenocytes directly pulsed with OFA-iLRP–specific MHC class I peptide (iLR_58-66), but not control MOPC315 MHC class II peptide (NP peptide). The P value is the comparison between MIP3α-mOFA and MIP3α+ mOFA. Control for effector cells mixed with untreated splenocytes is shown (E + T). (C) Chemokine fusions also presented to MHC class I in vivo. Splenocytes from mice immunized with pMIP3α-gp100 or pmDF2β-gp100; control vaccine pMIP3α-D-gp100; or PBS were irradiated without additional peptide pulsing and mixed with activated effector cells (splenocytes) from pmel-1 mice. Specificity and activity of effector pmel-1 cells have been tested on APCs pulsed with human gp100_25-33 peptide (hgp100_25-33). A representative experiment is shown from 4 (A-B) and 2 (C) independent experiments, all yielding similar results. Data are averages of triplicate wells, and error bars represent standard deviation of the mean.