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. 2017 Oct 2;14(10):3312-3321.
doi: 10.1021/acs.molpharmaceut.7b00367. Epub 2017 Aug 28.

Direct Loading of iTEP-Delivered CTL Epitope Onto MHC Class I Complexes on the Dendritic Cell Surface

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Free PMC article

Direct Loading of iTEP-Delivered CTL Epitope Onto MHC Class I Complexes on the Dendritic Cell Surface

Shuyun Dong et al. Mol Pharm. .
Free PMC article

Abstract

Cytotoxic T lymphocyte (CTL)-mediated immune responses are the primary defense mechanism against cancer and infection. CTL epitope peptides have been used as vaccines to boost CTL responses; however, the efficacy of these peptides is suboptimal. Under current vaccine formulation and delivery strategies, these vaccines are delivered into and processed inside antigen-presenting cells such as dendritic cells (DCs). However, the intracellular process is not efficient, which at least partially contributes to the suboptimal efficacy of the vaccines. Thus, we hypothesized that directly loading epitopes onto MHC class I complexes (MHC-Is) on the DC surface would significantly improve the efficacy of the epitopes because the direct loading bypasses inefficient intra-DC vaccine processing. To test the hypothesis, we designed an immune-tolerant elastin-like polypeptide (iTEP)-delivered CTL vaccine containing a metalloproteinase-9 (MMP-9)-sensitive peptide and an CTL epitope peptide. We found that the epitope was released from this MMP-sensitive vaccine through cleavage by DC-secreted MMP-9 outside of the DCs. The released epitopes were directly loaded onto MHC-Is on the DC surface. Ultimately, the MMP-sensitive vaccine strikingly increased epitope presentation by DCs by 7-fold and enhanced the epitope-specific CD8+ T-cell response by as high as 9.6-fold compared to the vaccine that was uncleavable by MMP. In summary, this novel direct-loading strategy drastically boosted vaccine efficacy. This study offered a new avenue to enhance CTL vaccines.

Keywords: CTL vaccine; DC presentation; MHC-I; MMP; direct loading; epitope; iTEP.

Figures

Figure 1
Figure 1
The iTEP-delivered, MMP-9-sensitive vaccine that enables direct epitope-loading. The vaccine, which contains a MMP-9 cleavage site, is able to take advantage of MMP-9 ((shown as red scissors) secreted from and surrounding DCs to release CTL epitopes. The MMP-9 cleave the epitopes from the vaccines and facilitate an accumulation of the epitopes around DCs. The direct epitope loading occurs when these epitopes, at high concentration, replace the predecessor epitopes bound with MHC-I on DC surfaces.
Figure 2
Figure 2
Characterization of iTEP-derived iTEP vaccines and their response to MMPs. (A) SDS-PAGE gel showing the identity and purity of iTEP and iTEP-derived vaccines. (B–D) Turbidity profiles (OD350) of iTEP (B), iTEP-pOVA (C), and iTEP-sMMP-pOVA (D) when 25 μM samples were heated and then cooled between 20 °C and 80 °C in PBS and 1M NaCl. (E) Enzymatic digestion of iTEP-pOVA and iTEP-sMMP-pOVA by recombinant active MMP-2 or MMP-9. iTEP vaccines were labeled with NHS-Fluorescein and treated with enzymes at a 1:1000 ratio of enzyme to substrate at 37 °C for 16 h. The enzyme-digested products were resolved by SDS-PAGE, and the image was recorded under UV. The position of the fragment of pOVA generated by MMP-2/9 is shown by an arrow.
Figure 3
Figure 3
DCs secrete MMP-9 to cleave iTEP-sMMP-pOVA and release pOVA epitope. (A) Gelatin zymography analysis showing that like Raw264.7 cells, DC2.4 cells and BMDCs from C57BL/6 mouse greatly secreted MMP-9 but not MMP-2. 50 μg protein from DC2.4 cell medium, 2.5 μg protein from BMDC medium, 12.5 μg protein from Raw264.7 cell medium, 20 ng MMP-9, or 10 ng MMP-2 were resolved by SDS-PAGE containing 0.1% gelatin. (B) Gelatin zymography analysis of MMP-9 secreted by DC2.4 cells with different inhibitors. Lane2-4, 50 μg protein from DC2.4 cell medium was treated with PMSF, 1,10 phenothroline, or EDTA for 30 min before loading to the gelatin gel. Lane 5, 50 μg protein from DC2.4 cell medium without treatment was resolved by gelatin gel and incubated with developing buffer containing 10 μM GM6001 before Coomassie brilliant blue staining. (C) Effect of inhibitors on enzymatic digestion of iTEP-sMMP-pOVA by DC-cultured medium. DC-cultured medium was pretreated with inhibitors as in (B) at 37 °C for 2 h. Then fluorescein-labeled iTEP-sMMP-pOVA was added and incubated at 37 °C for 16 h. The enzyme-digested products were resolved by SDS-PAGE, and the image was recorded under UV. The position of the fragment of pOVA generated by MMP-2/9 is shown by an arrow.
Figure 4
Figure 4
iTEP-sMMP-pOVA more effectively presents pOVA to DCs than iTEP-pOVA. (A) Histograms of flow cytometric analysis. DC2.4 cells were stained with anti-mouse pOVA bound H-2Kb after the cells were incubated with 5 μM iTEPs or pOVA. (B) Mean fluorescence intensity (MFI) of H-2Kb-pOVA on DC2.4 cells after different treatments. (C) Stimulation of B3Z cells via presentation of pOVA by DC2.4 cells which were pre-incubated with different reagents at 0.2 μM or 1 μM. The data in all panels are representative of 3 independent experiments. Bars, mean+SD, n=3. Analysis of variance (Student's t-test). ★★P<0.01.
Figure 5
Figure 5
pOVAs released from iTEP-sMMP-pOVA directly load onto DC for antigen presentation which is dependent on DC-secreted MMP-9. (A) Comparison of uptake of iTEP-pOVA vs iTEP-sMMP-pOVA by DC cells. DC2.4 cells were incubated with 10 μM of fluorescein-labeled iTEP vaccines in FBS free medium at 37 °C from 10 min to 4 h. The normalized cellular fluorescence intensity was recorded and compared. (B) Stimulation of B3Z cells via direct loading of pOVA on H-2Kb of DC 2.4 cells. The iTEP vaccines were incubated with RPMI medium without FBS, supernatant of DC-cultured medium without column concentration, or activated MMP-9 at 37 °C for 16 h before loading to DC2.4 cells on ice. After 30 min of ice incubation, the DC2.4 cells were washed, fixed, and then mixed with B3Z cells for B3Z activation. (C). Effect of MMP inhibitors on direct loading of pOVA released from iTEP-sMMP-pOVA. The experiment procedure was similar to (B) except that DC-cultured medium was treated with inhibitors at 37 °C for 2 h before incubation with iTEP-sMMP-pOVA. The data in all panels are representative of 3 independent experiments. Bars, mean+SD, n=3. Analysis of variance (Student's t-test). ★ P<0.05. ★★ P<0.01.

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