Figure 2.
Figure 2. Electroporation of monocyte-derived DCs from HIV-1–seropositive individuals with various gag mRNA results in efficient expression, secretion, and presentation of Gag protein and peptides. (A) iDCs from an HIV-1–seropositive individual (patient P1) were mock electroporated or electroporated with hHxB-2 gag mRNA and further matured for 24 hours. Afterward they were analyzed for intracellular Gag protein. Histogram overlay showing anti-Gag fluorescence of mock-electroporated control mDCs (open histogram) and hHxB-2 gag mRNA-electroporated mDCs (filled histogram). The example shown was representative for patients P1, P3, P4, and P10. (B) Gag protein secretion was measured in the supernatant 24 hours after electroporation and maturation. This experiment was performed and is shown for patients P1, P3, P5, P7, and P8. mDC-mock indicates mock-electroporated mDCs; mDC-hHxB-2 gag, mDCs electroporated with hHxB-2 gag mRNA. (C) iDCs from patient P15 were electroporated with hHxB-2 gag mRNA or mock electroporated, further matured for 24 hours, and the antigen-processing and -presenting capacity was investigated in IFN-γ ELISPOT using a CD8+ Gag-specific T-cell line. The HIV-1 Gag77-85 peptide was used as a positive control and the HTLV-I Tax11-19 peptide as a negative control. The numbers are GMs ± SE (quadruplicate wells) of IFN-γ SFCs per 60 000 responder cells. (D) iDCs from an HIV-1–seropositive individual (patient P14) were mock electroporated or electroporated with autologous proviral gag mRNA and further matured for 24 hours. Afterward they were analyzed for intracellular Gag protein. Histogram overlay showing anti-Gag fluorescence of mock-electroporated control mDCs (open histogram) and pv-gag mRNA-electroporated mDCs (filled histogram). (E) Gag p24 protein secretion was measured by ELISA in the supernatant of pv-gag mRNA-electroporated mDCs (DC-pv-gag) and v-gag mRNA-electroporated mDCs (DC-v-gag). This experiment was performed and is shown for patients P9, P12, P13, P21, and P22. Gag p24 protein secretion of mock-electroporated mDCs was below background (data not shown). (F) iDCs from an HIV-1–seropositive patient (P12) were mock electroporated, electroporated with autologous pv-gag or v-gag mRNA, and further matured. After 24 hours, these mDCs were cocultured in an ELISPOT assay with a Gag peptide-specific CD8+ T-cell line. Peptide controls and SFCs are the same as in panel C.

Electroporation of monocyte-derived DCs from HIV-1–seropositive individuals with various gag mRNA results in efficient expression, secretion, and presentation of Gag protein and peptides. (A) iDCs from an HIV-1–seropositive individual (patient P1) were mock electroporated or electroporated with hHxB-2 gag mRNA and further matured for 24 hours. Afterward they were analyzed for intracellular Gag protein. Histogram overlay showing anti-Gag fluorescence of mock-electroporated control mDCs (open histogram) and hHxB-2 gag mRNA-electroporated mDCs (filled histogram). The example shown was representative for patients P1, P3, P4, and P10. (B) Gag protein secretion was measured in the supernatant 24 hours after electroporation and maturation. This experiment was performed and is shown for patients P1, P3, P5, P7, and P8. mDC-mock indicates mock-electroporated mDCs; mDC-hHxB-2 gag, mDCs electroporated with hHxB-2 gag mRNA. (C) iDCs from patient P15 were electroporated with hHxB-2 gag mRNA or mock electroporated, further matured for 24 hours, and the antigen-processing and -presenting capacity was investigated in IFN-γ ELISPOT using a CD8+ Gag-specific T-cell line. The HIV-1 Gag77-85 peptide was used as a positive control and the HTLV-I Tax11-19 peptide as a negative control. The numbers are GMs ± SE (quadruplicate wells) of IFN-γ SFCs per 60 000 responder cells. (D) iDCs from an HIV-1–seropositive individual (patient P14) were mock electroporated or electroporated with autologous proviral gag mRNA and further matured for 24 hours. Afterward they were analyzed for intracellular Gag protein. Histogram overlay showing anti-Gag fluorescence of mock-electroporated control mDCs (open histogram) and pv-gag mRNA-electroporated mDCs (filled histogram). (E) Gag p24 protein secretion was measured by ELISA in the supernatant of pv-gag mRNA-electroporated mDCs (DC-pv-gag) and v-gag mRNA-electroporated mDCs (DC-v-gag). This experiment was performed and is shown for patients P9, P12, P13, P21, and P22. Gag p24 protein secretion of mock-electroporated mDCs was below background (data not shown). (F) iDCs from an HIV-1–seropositive patient (P12) were mock electroporated, electroporated with autologous pv-gag or v-gag mRNA, and further matured. After 24 hours, these mDCs were cocultured in an ELISPOT assay with a Gag peptide-specific CD8+ T-cell line. Peptide controls and SFCs are the same as in panel C.

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