Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide In Vitro

Abstract

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

Figure 1.. Cross-presentation of OVA-SLP and MS-OVA mainly follows a cytosolic pathway.

a) Graphical illustration of cellular targets of the inhibitors used to interfere with cross-presentation pathways. b) Schematic representation of the inhibitor experiment. Antigen presenting cells (APCs) were pre-treated for 30 min with the following inhibitors: 100 μM leupeptin (cathepsin inhibitor), 10 μM MG-132 (proteasome inhibitor), 10 μg/ml Brefeldin A (BFA, inhibits ER to Golgi transport), before incubation with either 25 μg/96-well of PLGA microsphere-encapsulated ovalbumin (OVA) (MS-OVA) or 10 μg/ml of an OVA-derived synthetic long peptide (OVA-SLP). After 2 hours, APCs were fixed with PFA and cross-presentation was quantified using a B3Z T-cell hybridoma assay. c+d) Antigen cross-presentation of MS-OVA (left panels) and OVA-SLP (right panels) by BMC2 (c) and MutuDC cells (d). B3Z T-cell activation was evaluated in a colorimetric LacZ assay by measuring the optical density (OD) on an ELISA plate reader (OD_570nm-OD_620nm). Data was normalized to the respective control condition in the presence of antigen without inhibitor treatment. Pooled results from four independent experiments are shown as mean with standard deviation and each independent experiment was performed with three technical replicates. Statistical evaluation of mean differences was performed using an one-way ANOVA with post-hoc Dunnet’s test to compare inhibitor treated samples to the respective control condition, with * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure 2.. Sec22b knockout does not alter normal surface expression of MHC class I.

a) Graphical illustration of CRISPR/Cas9 gene editing strategy used to generate Sec22b knockout (KO) cells. Antigen presenting cells (APCs) were transduced with lentiviral vectors containing Sec22b-specific single guide RNAs (sgRNA1 and 2) targeting exon 1 and exon 2 of Sec22b, respectively, selected for successful viral integration, and further subcloned to obtained single cell-derived KO clones. b+c) KO of Sec22b was confirmed on the protein level after cell lysis by western blot analysis using an anti-Sec22b antibody (expected molecular weight ~24kDa) for wildtype (WT), non-targeting sgRNA control 1 (NT1) and 2 (NT2) and Sec22b-targeted cells (sgRNA1 and sgRNA2; Sec22b KO clone 1 and 2, respectively) with BMC2 (b) and MutuDC cells (c). An anti-actin antibody was used as control for equal protein loading. Western blot shown is representative of three independent experiments. d+e) APCs were harvested and stained for H-2K^b to analyse MHC class I surface expression by flow cytometry on BMC2 (c) and MutuDC cells (d) (gating strategy Supp. Fig 2a). Graphs show median fluorescence intensity (MFI) of the pooled data from three (c) or four (d) independent experiments, illustrated as mean with standard deviation. Each independent experiment was performed with one technical replicate. f+g) BMC2 (f) and MutuDC cells (g) were externally pulsed with indicated concentrations of the ovalbumin-derived, H-2K^b-restricted T-cell epitope SIINFEKL for 1 hour, washed 3 times, and then co-cultured with B3Z T-cell hybridomas for 18 hours. B3Z T-cell activation was evaluated in a colorimetric LacZ assay by measuring the optical density (OD) on an ELISA plate reader (OD_570nm-OD_620nm). Graphs show pooled data with mean with standard deviation from six independent experiments with three technical replicates. Statistical evaluation of mean differences was performed using a one-way ANOVA followed by Tukey test (c-d) or a two-way ANOVA followed by Tukey test (e-f). Results were only indicated as significant if Sec22b KO APCs targeted with either sgRNA1 or sgRNA2 were significantly different from both NT controls, with * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure 3.. APCs lacking Sec22b efficiently cross-present MS-OVA and OVA-SLP.

a+b) Antigen uptake by BMC2 (a) and MutuDC cells (b) was evaluated using PLGA microspheres containing fluorescent quantum dots (MS-QD; 583nm; right panel). Antigen presenting cells (APCs) were incubated with MS-QD at indicated temperatures for 2 hours before antigen uptake was analyzed by flow cytometry (gating strategy Supp. Fig. 2d). Graphs show median fluorescence intensity (MFI) and display the pooled results of three independent experiments as mean with standard deviation, performed with one technical replicate. c+d) Cross-presentation efficiency in Sec22b knockout (KO) BMC2 cells (c) and MutuDC cells (d) with indicated concentrations of PLGA microsphere-encapsulated OVA (MS-OVA; left panels) or an ovalbumin (OVA)-derived synthetic long peptide (OVA-SLP; right panels) for either pre-incubation of 2 hours with the antigen followed by addition of primary OT-1 T-cells for 4 hours in presence of brefeldin A (top panels) or co-incubation of 18 hours in the presence of B3Z hybridoma cells (bottom panels). APCs transduced with non-targeting sgRNAs (NT control 1 and 2) were used as control. Primary OT-1 T-cell activation was evaluated by intracellular cytokine staining for IFN-γ followed by flow cytometry. Graphs show the mean percentage of IFN-γ positive events from the total population of CD8⁺ T-cells. One representative of three independent experiments with standard deviation is shown (gating strategy Suppl. Fig. 2c). Each independent experiment was performed with two technical replicates. B3Z T-cell activation was evaluated in a colorimetric LacZ assay by measuring the optical density (OD) on an ELISA plate reader (OD_570nm-OD_620nm). Graphs show the pooled data as mean with standard deviation from five (c, bottom left) or three (c, bottom right + d, bottom right) or seven (d, bottom left) independent experiments. All independent experiments were performed with three technical replicates. Statistical evaluation of mean differences was performed on pooled data from all replicates using a one-way ANOVA followed by Tukey test on the 37°C phagocytosis condition (a+b) or by a two-way ANOVA followed by Tukey test (c+d). Results were indicated as significant if KO APCs targeted with either sgRNA1 or sgRNA2 were significantly different from both NT controls, with * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure 4.. Stx4 knockout does not affect MHC class I surface expression on both BMC2 and MutuDC cells.

a) Graphical illustration of CRISPR/Cas9 gene editing strategy used to generate Stx4 knockout (KO) cells. Antigen presenting cells (APCs) were transduced with lentiviral vectors containing Stx4-specific single guide RNAs (sgRNA1 and 2) targeting exon 2 and exon 3 of Stx4, respectively, selected for successful viral integration, and further subcloned to obtained single cell-derived KO clones. b-c) KO of Stx4 was confirmed on the protein level after cell lysis by western blot analysis using an anti-Stx4 antibody (expected molecular weight ~34kDa) for wildtype (WT), non-targeting sgRNA control 1 (NT1) and 2 (NT2) and Stx4-targeted cells (sgRNA1 and sgRNA2; Stx4 KO clone 1 and 2, respectively) with BMC2 (b) and MutuDC cells (c). An anti-actin antibody was used as control for equal protein loading. Western blot shown is representative of two independent experiments. d+e) APCs were harvested and stained for H-2K^b to analyze MHC class I surface expression by flow cytometry on BMC2 (d) and MutuDC cells (e). Graphs show median fluorescence intensity (MFI) of the pooled data from four (d) or three (e) independent experiments, illustrated as mean with standard deviation (gating strategy Suppl. Fig. 2a). Each independent experiment was performed with one technical replicate. f+g) BMC2 (f) and MutuDC cells (g) were externally pulsed with indicated concentrations of the ovalbumin-derived, H-2K^b-restricted T-cell epitope SIINFEKL for 1 hour, washed 3 times, and then co-cultured with B3Z T-cell hybridomas for 18 hours. B3Z T-cell activation was evaluated in a colorimetric LacZ assay by measuring the optical density (OD) on an ELISA plate reader (OD_570nm-OD_620nm). Graphs show the pooled data as mean with standard deviation from four (f+g) independent experiments. Each independent experiment was performed with three technical replicates. Statistical evaluation of mean differences was performed using a one-way ANOVA followed by Tukey multiple comparisons test (c-d) or a two-way ANOVA followed by Tukey test (e-f). Results were indicated as significant if Stx4 KO APCs targeted with either sgRNA1 or sgRNA2 were significantly different from both NT controls, with * = p < 0.05; *** = p < 0.001.

Figure 5.. Stx4 KO has no major effect on cross-presentation of OVA-SLP and MS-OVA.

a+b) Antigen uptake by BMC2 (a) and MutuDC cells (b) was evaluated using PLGA microspheres containing fluorescent quantum dots (MS-QD; 583nm). Antigen presenting cells (APCs) were incubated with MS-QD at indicated temperatures for 2 hours before antigen uptake was analyzed by flow cytometry (gating strategy Suppl. Fig. 2d). Graphs show median fluorescence intensity (MFI) and summarize the results of three independent experiments as mean with standard deviation. Each independent experiment was performed with one technical replicate. c+d) Cross-presentation efficiency in Stx4 knockout (KO) BMC2 cells (c) and MutuDC cells (d) with indicated concentrations of PLGA microsphere-encapsulated OVA (MS-OVA; left panels) or an ovalbumin (OVA)-derived synthetic long peptide (OVA-SLP; right panels) for either 2 hours of pre-incubation with the antigen followed by addition of primary OT-1 T-cells for 4 hours in presence of brefeldin A (top panels) or co-incubation of 18 hours in the presence of B3Z hybridoma cells (bottom panels). APCs transduced with non-targeting sgRNAs (NT control 1 and 2) were used as control. Primary OT-1 T-cell activation was evaluated by intracellular cytokine staining for IFN-γ followed by flow cytometry. Graphs show the mean percentage of IFN-γ positive events from the total population of CD8⁺ T-cells from one representative of three independent experiments with standard deviation (gating strategy Suppl. Fig. 2c). Each independent experiment was performed with two technical replicates. B3Z T-cell activation was evaluated in a colorimetric LacZ assay by measuring the optical density (OD) on an ELISA plate reader (OD_570nm-OD_620nm). Graphs show the pooled data as mean with standard deviation from three (c, bottom right; d, bottom panels) or five (c, bottom left) independent experiments. Each independent experiment was performed with three technical replicates. Statistical evaluation of mean differences was performed on pooled data using a one-way ANOVA followed by Tukey test on the 37°C condition (a+b) or by a two-way ANOVA followed by Tukey post-hoc test (c+d). Results were indicated as significant if KO APCs targeted with either sgRNA1 or sgRNA2 were significantly different from both NT controls, with * = p < 0.05; ** = p < 0.01; *** = p < 0.001.