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

Therapeutic vaccines that induce potent CTL responses are of crucial importance for immunotherapy of cancer (1). CTLs can kill their target cells after recognition of endogenous peptides that are presented on MHC class I molecules via the direct presentation pathway (2). To acquire their full CTL effector functions, naive CD8⁺ T cells need initial priming by professional APCs, such as dendritic cells (DCs) and macrophages (3, 4). APCs can present MHC class I–associated peptides in combination with costimulatory signals after their activation by pathogen- or danger-associated molecular patterns, a mechanism exploited by adjuvants in vaccine formulations (5). Besides the direct presentation of endogenous Ags, APCs have the unique property to present peptides derived from exogenous sources in the context of MHC class I (3, 4). This process is referred to as “cross-presentation” and represents the exclusive pathway by which CTLs can be primed against tissue-specific Ags, such as those present in cancer or Ags associated with intracellular pathogens that either do not infect APCs or interfere with the direct presentation pathway (3, 6).

In general, cross-presentation takes place after endocytosis of exogenous Ags, after which internalized cargo reaches the endosomal compartment. From here, Ags can follow two distinct cross-presentation pathways (3, 4). In the vacuolar pathway, Ags are processed by lysosomal proteases in an endosomal/lysosomal compartment after acidification, in which generated peptides are also loaded onto MHC class I molecules for cell surface presentation (3, 4). In the cytosolic pathway, Ags leave the endosome via an ER-associated protein degradation–associated protein translocon to the cytoplasm and are degraded into peptides by the proteasome (7, 8). Next, generated peptides can again follow two distinct routes. In the phagosome-to-cytosol pathway, Ags enter the direct presentation pathway via the ER and Golgi (3, 4, 6). Alternatively, in the phagosome-to-cytosol-to-phagosome pathway, the peptides are transported back into the endosome by the TAP and are then loaded on MHC class I inside the endosomal compartment (6). In both cases, the MHC class I/peptide complexes are then transported to the surface for cross-priming of CTLs.

In the cytosolic pathway, the delivery of ER-resident proteins of the ER-associated protein degradation machinery (such as the translocon Sec61/p97), the TAP transporter, and components of the peptide-loading complex to the endosomes are essential for efficient cross-presentation. There is general consensus that this delivery occurs via an ER–Golgi intermediate compartment (ERGIC), which is normally known for trafficking proteins from the ER to the Golgi (4, 9, 10). The fusion between ERGIC vesicles and the endosome is mediated by SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) proteins, present on both the ERGIC and the endosomal membrane (11). It has previously been suggested that the vesicle SNARE (v-SNARE) protein Sec22b is crucial for the endosomal delivery of ER-resident proteins for efficient cross-presentation (9, 10, 12, 13). However, this role of Sec22b remains controversial, as it has also been challenged (14). Interestingly, in current literature the target SNARE protein syntaxin 4 (Stx4) has been suggested as the endosomal interaction partner of Sec22b for mediating the fusion of ERGIC vesicles with the endosomal membrane, but experimental evidence for a role of Stx4 in cross-presentation is still pending.

In this study, we further explore the roles of Sec22b and Stx4 in the context of two candidate vaccine formulations that have the potential to cross-prime CTL responses, for example, in cancer immunotherapy (1). First, poly(lactic-co-glycolic acid) (PLGA) microspheres (MSs) are among the most frequently explored polymer-based vaccine formulations due to their efficient uptake by APCs, their biodegradability, and their overall safety (Food and Drug Administration approved since 1989) (15, 16). PLGA-MSs show ideal properties to encapsulate protein, peptide, or cancer cell lysate-derived Ags (17, 18) and their potential to cross-prime Ag-specific CTL responses after controlled and sustained release of their content has been demonstrated in various preclinical and clinical studies (15, 16). Second, synthetic long peptides (SLPs) are promising agents in vaccination, as they are safe, easy to administer and, compared with recombinant proteins, easier to produce (19, 20). By definition, SLPs are 15–50 aa in length (21), and they were shown to cross-prime CTL responses more efficiently than shorter peptide sequences that rather induced tolerance (22–24). Because vaccine formulations based on protein or peptide Ags require cross-presentation by APCs to prime CTL responses, characterizing the intracellular pathways and molecular mechanisms involved in the related processes remains important to understand and to further optimize current therapeutic strategies (25).

Previous studies have already elucidated important details about the cross-presentation pathways used by vaccine formulations based on PLGA-MS–encapsulated proteins (16, 26, 27) and SLPs (20, 22). Because in both cases cross-presentation was shown to depend on a cytosolic pathway (21, 27–31), we were interested to evaluate the role of the SNARE proteins Sec22b and Stx4 in related processes. We therefore used CRISPR/Cas9-mediated genome editing to generate homozygous knockouts (KOs) of Sec22b and Stx4 in two established C57BL/6-derived APC cell lines and evaluated their effect on cross-presentation of the PLGA-MS–encapsulated model Ag OVA (MS-OVA) and an OVA-derived SLP (OVA-SLP). In contrast to previous findings, we did not observe an essential role of Sec22b in the cross-presentation of both Ag types in this system. In addition, we were, to our knowledge, the first to directly address the role of Stx4 in the context of cross-presentation. Similar to our findings with Sec22b KOs, we found no evidence for a major role of Stx4 KO in the cross-presentation of both MS-OVA and OVA-SLP under the conditions tested in the current study. Overall, our study points toward SNARE protein redundancy in the cytosolic pathways of cross-presentation.

C57BL/6J (H-2^b) mice were obtained from Charles River Laboratories and kept under specific pathogen-free conditions during the experiments. C57BL/6-Tg(TcraTcrb)1100Mjb/Crl (OT-1) mice were initially obtained from Charles River Laboratories and further maintained at the Erasmus MC animal facility under specific pathogen-free conditions. All experiments using ex vivo cell material were approved by the local authorities in the Netherlands (Centrale Commissie Dierproeven, license no. AVD101002016793). Mice were sacrificed at 6–15 wk of age.

The murine macrophage cell line BMC2 (H-2^b) (32) was originally obtained from K. Rock and cultured in RPMI 1640 (Life Technologies) supplemented with 25 mM HEPES. B3Z hybridoma T cells were a gift from N. Shastri (33) and were cultured in RPMI 1640 (Life Technologies). MutuDC2114 (MutuDC) cells were contributed by H. Acha-Orbea (34) and cultured in IMDM (Life Technologies) supplemented with 35 mM HEPES (Life Technologies) and 50 µM 2-ME (Life Technologies). HEK293T cells (American Type Culture Collection, CRL-3216) were cultured in DMEM (Life Technologies). OT-1 T cells were cultured in RPMI 1640 (Life Technologies) supplemented with 35 mM HEPES (Life Technologies), 2 mM l-glutamine (Lonza), 1 mM sodium pyruvate (Sigma-Aldrich), 1× nonessential amino acids (Life Technologies, 100x), and 50 µM 2-ME (Life Technologies). Bone marrow–derived DCs (BMDCs) were cultured in RPMI 1640 (Life Technologies) supplemented with 5 µM 2-ME (Life Technologies) and 20 ng/ml recombinant mouse GM-CSF (PeproTech). All cell culture media were supplemented with 10% heat-inactivated FCS (Life Technologies) and 100 U/ml penicillin/streptomycin (Lonza). All cells were cultured at 37°C and in 5% CO₂. BMC2 and HEK293T cells were detached using 0.05% trypsin/EDTA (Life Technologies). MutuDC cells and BMDCs were detached using 5 and 1 mM EDTA/PBS (Merck), respectively.

For preparation of BMDCs, femurs and tibias of C57BL/6J mice were taken and their bone marrow was isolated by crushing the bones in a mortar with PBS. Single-cell suspensions were generated using a 70-µm cell strainer and subsequently cultured in BMDC medium on non–cell culture-treated petri dishes. Medium was refreshed after 4 d and cells were harvested for use in experiments after 7 d.

Splenocytes from OT-1 mice were obtained and mononuclear cells were isolated using Ficoll (Cytiva). Next, cells were stimulated with 10⁻⁸ M SIINFEKL (S8L) peptide (OVA_257–264; AntiBodyChain) as well as 5 ng/ml of both mouse IL-7 and mouse IL-15 (PeproTech). On day 5, live and dead cells were separated by using Ficoll. The remaining CD8⁺ T cells were allowed to rest in the presence of mouse IL-7 and mouse IL-15 for 48 h before being used in the cross-presentation assays.

Single-guide RNAs (sgRNAs) were designed using Benchling (an online platform for molecular biology design and analysis; https://www.benchling.com) and cloned into the lentiCRISPR v2 plasmid (Addgene, no. 52961) for all cell lines, except MutuDC/Stx4 KO cells, for which sgRNAs were cloned into lentiGuide-Puro (Addgene, no. 52963). The following sequences were used: Sec22b sgRNAs targeting exon 1: sgRNA1 (5′-TGTGGCGGACGGCCTTCCGC-3′) and targeting exon 2: sgRNA2 (5′-GCTTCCAAGGTACATCGGGT-3′); STX4 sgRNAs targeting exon 2: sgRNA1 (5′-GGCGACAGGACCCACGAGTTG-3′) (BMC2 and MutuDC) and targeting exon 3: sgRNA2 for BMC2 (5′-GAGATGAGGTTCGAGTCGCGC-3′) and sgRNA2 for MutuDC (5′-GTGAGGTTCGAGTCGCGCTGG-3′). Nontargeting (NT) control sgRNAs were NT control 1 (5′-GCGAGGTATTCGGCTCCGCG-3′) and NT control 2 (5′-ATGTTGCAGTTCGGCTCGAT-3′), as extracted from Sanjana et al. (35). To clone the different sgRNAs, we annealed the forward oligonucleotide with the reverse, which were then ligated into the cut lentiviral backbone plasmid (lentiCRISPR v2 or lentiGuide-Puro). First, both plasmids were cut using Esp3l (New England Biolabs) for 2 h at 37°C, put on agarose gel, and purified from the gel (QIAquick gel extraction kit, Qiagen) according to the manufacturers’ instructions to obtain the linearized vector. Annealed oligonucleotides and cut vectors were then ligated using T4 DNA ligase according to the manufacturer’s instructions (Life Technologies). Ligation reactions were transformed into homebrew-competent Escherichia coli (Stbl3, originally from Thermo Fisher Scientific, genotype: F⁻mcrB mrrhsdS20(r_B⁻, m_B⁻) recA13 supE44 ara-14 galK2 lacY1 proA2 rpsL20(Str^R) xyl-5 λ⁻leumtl-1). Transformation reactions were plated on Luria–Bertani agar plates (Sigma-Aldrich) with 100 µg/ml ampicillin (Sigma-Aldrich) and incubated overnight at 37°C. Single colonies were selected and grown in Luria–Bertani (Sigma-Aldrich) overnight with 100 µg/ml ampicillin. Plasmids were extracted by using a QIAprep spin miniprep kit (Qiagen) according to the manufacturer’s instructions. Plasmids were checked for the correct insertion of the sgRNA by Sanger sequencing at GATC-Eurofins using the hU6 primer (5′-GACTATCATATGCTTACCGT-3′). To generate lentivirus containing the plasmids with either Sec22b or Stx4 KO sgRNAs, 6 × 10⁵ HEK293T cells per well were seeded in a six-well plate and grown to 70–80% confluency. The cells were then transfected with 500 ng of packing plasmid (psPAX2, Addgene, no. 12260), 50 ng of envelope protein plasmid (pCMV-VSV-G, Addgene, no. 8454), and 500 ng of expression vector using Lipofectamine 2000 (Invitrogen), following the manufacturers’ instructions. Lentivirus-containing supernatant was collected 24 and 48 h posttransfection and filtered using a 0.2-µm sterile filter to remove cell debris. To transduce BMC2 cells with Sec22b KO and Stx4 KO sgRNAs, respectively, 6 × 10⁵ cells per well were plated in a six-well plate 18 h before transduction. The next day, fresh medium containing 8 µg/ml Polybrene (Sigma-Aldrich) and 500 µl of filtered lentivirus supernatant was added. The plates were then centrifuged at 2000 rpm for 90 min at 37°C. To transduce MutuDC with Sec22b KO sgRNAs, 5 × 10⁶ cells were resuspended in 1 ml of lentivirus containing supernatant in the presence of 8 µg/ml Polybrene, which was stirred every 30 min for 2 h at 37°C. To target MutuDC with Stx4 KO sgRNAs, the cells were first targeted with a Cas9-expressing vector, lentiCas9-Blast (Addgene, no. 52962). This plasmid was delivered as a lentivirus, which was made in HEK293T cells as follows: HEK293T cells were plated as described above. Cells were transfected with 1000 ng of packaging plasmid (psPAX2), 100 ng of envelope protein plasmid (pCMV-VSV-G), and 1000 ng of expression vector using TransIT-VirusGEN (Mirus Bio) following the manufacturer’s instructions. Supernatant containing lentivirus was collected after 48 h and filtered as described above. MutuDC cells were transduced as described above with Sec22b KO sgRNAs and selected after 3 d using 5 µg/ml blasticidin (InvivoGen). Monoclonal cultures were obtained by limited dilution, and Cas9 expression of monoclonal cell lines was confirmed on Western blot (data not shown). MutuDC cells expressing Cas9 were then transduced with plasmids containing either Stx4 KO or NT control sgRNAs in lentiGuide-Puro in lentivirus made as described above with TransIT-VirusGEN. For all Sec22b and Stx4 KO cell lines, 3 d after transduction, APCs were selected using medium containing 6 µg/ml (BMC2) and 4 µg/ml (MutuDC) puromycin (Sigma-Aldrich). Monoclonal cultures were obtaining by limited dilution, and clones were further characterized on the genetic level using Sanger sequencing and protein level by Western blotting.

DNA was isolated from BMC2 and MutuDC cells using a QIAamp DNA mini kit (Qiagen, no. 51306). Samples were amplified via PCR using Phusion high-fidelity DNA polymerase (Life Technologies) according to the manufacturer’s instructions. The following primers were used: Sec22b exon 1 forward, 5′-GGAGAAGAAGGGACAGTGA-3′, reverse, 5′-ACGAGCAAACGGTAAAAGA-3′; Sec22b exon 2 forward, 5′-GCTTTGTGTGATGTGTGTT-3′, reverse, 5′-GCCCCTACTGTGATATTCTT-3′; STX4 exons 2 and 3 forward, 5′-CACGACTGTGGATGGTGAAAGG-3′, reverse, 5′-TAAGTGTCACTCTAGTCCGCCC-3′. PCR-amplified products were loaded on agarose gels to check for a band at the expected size: 236 bp for Sec22b exon 1, 254 bp for Sec22b exon 2, and 536 bp for Stx4. PCR products were sent for Sanger sequencing at GATC-Eurofins using the forward and reverse primers, respectively. Results were aligned against the reference sequence of Sec22b (C57BL/6, ENSMUSG00000027879) or STX4 (C57BL/6, ENSMUSG00000030805) to evaluate cutting of the CRISPR/Cas9 machinery using the program CLC Workbench v7.

To confirm the absence of Sec22b or Stx4 protein in our KO cell lines, we lysed ∼1 × 10⁶ cells on ice using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in H₂O), supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein levels were determined using a Bradford protein assay (Sigma-Aldrich) and loaded on 4–15% polyacrylamide Tris-glycine gels (Bio-Rad) that were run in Tris/glycine/SDS (25 mM Tris, 192 mM glycine, 0.1% SDS [pH 8.3]; Bio-Rad) buffer. After transfer of separated proteins to polyvinylidene difluoride membranes using the Trans-Blot Turbo system (Bio-Rad), the membranes were blocked using 5% blocking grade nonfat milk (Bio-Rad) in PBS containing 0.05% Tween 20 (Bio-Rad) (PBST) and incubated overnight at 4°C in PBST containing 2.5% blocking grade nonfat milk with primary Abs (anti-Sec22b, 1:400, mouse anti-mouse, SC101267, Santa Cruz; anti-STX4, 1:3,000, rabbit anti-mouse, ab184545, Abcam; anti-actin, 1:3,000, rabbit anti-mouse, A2066, Sigma-Aldrich; anti–β-actin, 1:10,000, mouse anti-human, ab6276, Abcam). After washing using PBST, blots were incubated for 2 h at room temperature with secondary Abs (IRDye 800CW, goat anti-mouse, 926-32210; IRDye 680RD, goat anti-rabbit, 926-68071; Westburg Life Sciences; 1:7500 dilution) in the presence of PBST containing 2.5% blocking grade nonfat milk. After washing again with PBST, the fluorescent signal was measured with the Odyssey CLx imaging system (LI-COR Biosciences) and analyzed with Image Studio Lite v5.2.5.

PLGA-MSs were generated as previously described (26) with slight modifications. For Ag encapsulation, 50 mg of OVA (grade V, Merck) was dissolved in 1 ml of 0.1 M NaHCO₃ (aqueous phase) and emulsified with 1 g of PLGA in 20 ml of dichloromethane (organic phase) using a digital microtip sonicator. PLGA-MSs labeled with fluorescent quantum dots (MS-QDs; emission wavelength 583 nm) were generated by encapsulation of 50 mg of OVA as described above and by addition of QD583 into the dichloromethane phase of the spray-drying process (27). The obtained dispersion was immediately spray-dried with the Mini Spray Dryer 290 (BÜCHI Labortechnik) at a flow rate of 1 ml/min and inlet/outlet temperatures of 25/23°C. Spray-dried MSs were washed out of the spray-dryer’s cyclone with 0.05% poloxamer 188 (Merck) and collected on a cellulose acetate membrane filter. PLGA-MSs were dried under vacuum at room temperature and subsequently stored under desiccation at 4°C. A fresh stock of MS-OVA or MS-QDs was prepared for every experiment by weighing 4 mg of MS-OVA and subsequent resuspension in APC medium to a concentration of 4 mg/ml. This suspension was sonicated for 1–2 min to ensure proper homogenization of MS-OVA.

To evaluate the uptake of Ags, APCs were plated at 2.5 × 10⁵ cells per well in a 24-well plate. APCs were incubated with 150 µg of MS-QDs per well for 2 h at either 37°C in medium or at 4°C in PBS supplemented with 0.5% BSA. APCs were detached and phagocytosis was evaluated using flow cytometry. To measure cross-presentation, APCs were plated in round-bottom 96-well plates (2.5 × 10⁴ APCs for 18-h incubation [B3Z assay] and 1.0 × 10⁵ APCs for 2-h incubation [OT-1 T cell assay]). Ags (OVA_257–264 or S8L, AnaSpec; OVA-SLP, OVA_252–271, LEQLESIINFEKLTEWTSSN, GenScript; PLGA-MS containing OVA [MS-OVA]) were added at the indicated concentrations. For the inhibition assay, APCs were incubated for 30 min with either 100 µM leupeptin (Sigma-Aldrich), an inhibitor of cysteine, serine, and threonine proteases; 10 µM MG-132 (Merck), a proteasome inhibitor; or 10 µg/ml brefeldin A (BFA; Sigma-Aldrich) before adding indicated amounts of the Ags and incubating for an additional 2 h. APCs were washed twice with PBS before fixing with 1% paraformaldehyde (PFA; diluted in PBS) for 10 min at 4°C. Cells were then washed once with PBS. Excess PFA was quenched by adding 0.2 M glycine for 5 min at room temperature. Thereafter, APCs were washed three times with PBS before adding 1.0 × 10⁵ B3Z hybridoma cells. As a positive control and for peptide titration experiments to determine overall MHC class I surface expression, APCs were incubated for 1 h with S8L and washed with PBS three times before adding 1.0 × 10⁵ B3Z hybridoma cells. To determine cross-presentation after 18 h of incubation with Ags, B3Z cells were added simultaneously to the wells. All conditions tested in the B3Z assays were performed in technical triplicates. For the 2-h time points, APCs were incubated with Ag for 2 h before adding 1.0 × 10⁵ OT-1 T cells per well in the medium of the APC cell line. OT-1 T cells also received protein transport inhibitor (1:1000, BD Biosciences) and the assay was incubated for 4 h at 37°C. All conditions tested in the OT-1 assays were performed in technical duplicates. Afterward, OT-1 T cell activation was quantified by intracellular cytokine staining for IFN-γ using flow cytometry. After an 18-h incubation with APCs and B3Z hybridomas, cells were washed once with PBS and chlorophenol red-β-d-galactopyranoside (CPRG) (Sigma-Aldrich, 59767) substrate in a PBS buffer containing 0.13% IGEPAL (Sigma-Aldrich), and 9 mM MgCl₂ (Merck) was added to detect β-galactosidase activity as a measure of T cell activation. Substrate conversion was measured at an OD of 570 nm and a control at OD 620 nm on a VersaMax ELISA microplate reader. The final OD was calculated by subtracting the values at 620 nm from those at 570 nm. Plates were measured every 30–60 min until the positive control condition (10⁻⁶ M S8L) reached a calculated OD of 1.0. For the inhibition assay, values were normalized to the control condition, which was APCs plus Ag without inhibitors, showing the highest amount of cross-presentation possible for BMC2 cells, MutuDC cells, or BMDCs with the respective Ag (MS-OVA or OVA-SLP). Control conditions were then normalized to their respective means.

To investigate the expression levels of H-2K^b and H-2D^b on APCs, the cells were detached, washed to remove all serum, and resuspended in FACS buffer containing 0.5% BSA and 0.1% sodium azide. Next, the cells were stained in the presence of FACS buffer for 30 min on ice (anti–H-2D^b-PE, 1:200, mouse anti-mouse, 12-5999-81; anti–H-2K^b-PE, 1:200, 12-5958-80; Life Technologies). Hereafter, cells were washed with a surplus of FACS buffer to remove excess Ab. Samples were gated on live cells using forward scatter (FSC)/side scatter (SSC), followed by singlet gating using FSC area and FSC height. To measure the activation of OT-1 T cells by intracellular cytokine staining of IFN-γ, the T cells were stained extracellularly for 20 min at 4°C in FACS buffer (anti–CD8a-eF450, 1:80, Thermo Fisher Scientific, 48-0081-82), washed twice with FACS buffer, and fixed using intracellular fixation buffer (Thermo Fisher Scientific, 00-8833-56). T cells were then washed once with permeabilization buffer (Thermo Fisher Scientific, 00-8222-49) and stained intracellularly in presence of permeabilization buffer for 45 min at 4°C (anti–IFN-γ-allophycocyanin, 1:160, Thermo Fisher Scientific, 17-7311-82). Samples were washed once with permeabilization buffer, once with FACS buffer, and then resuspended in FACS buffer for subsequent measurements. One technical replicate was used per experiment. Live T cells were gated on FSC/SSC, then on singlets using FSC area and FSC height, followed by CD8 gating using CD8⁺ and SSC, and, finally, IFN-γ⁺ cells were gated on CD8⁺ and IFN-γ⁺ (for all gating strategies, see Supplemental Fig. 2). All samples were run on the FACSCanto II (BD Biosciences) and analyzed using FlowJo (v10; BD Life Sciences).

Unless otherwise stated, data are shown as the mean ± SD. Unless indicated otherwise, the median fluorescence intensity (MFI) of flow cytometry data was used for quantification. Statistical analyses of results were done using GraphPad Prism v9. Methods for statistical testing are listed in the respective figure legends.

To investigate the role of Sec22b and Stx4 in the cross-presentation of PLGA-MS–encapsulated Ags and SLPs, we decided to select two murine APC lines: the macrophage cell line BMC2 (32) and the CD8⁺ DC line MutuDC (34). Both cell lines have previously been used to explore the mechanisms of cross-presentation (32, 34, 36–39), and they efficiently cross-present the H-2K^b–restricted, chicken OVA–derived epitope S8L (OVA_257–264) after incubation with PLGA-MS–containing full-length OVA (MS-OVA) or a S8L-containing 20-mer SLP (OVA_252–271; OVA-SLP). As Sec22b has previously been assigned a role in the cytosolic pathway of cross-presentation, we initially evaluated which intracellular pathways were used for the cross-presentation of MS-OVA and OVA-SLP by the APC lines selected for this study. We therefore studied cross-presentation of MS-OVA and OVA-SLP in the presence of specific inhibitors that interfere with key steps of Ag processing and intracellular transport (Fig. 1A). To allow time for the inhibitors to act before adding the Ags, we preincubated APC lines with the compounds for 30 min, before adding the two different OVA-based Ags, respectively (Fig. 1B). After 2 h of incubation, APCs were fixed and cocultured with the S8L-specific T cell hybridoma cell line B3Z (33), using T cell activation as a measure for cross-presentation efficiency. These experiments revealed that cross-presentation of MS-OVA and OVA-SLP by BMC2 macrophages was sensitive to the reversible proteasome inhibitor MG-132 (40, 41) and the secretory pathway inhibitor BFA (36, 42) (Fig. 1C), with the interesting observation that cross-presentation of OVA-SLP was preferentially inhibited by interfering with proteasomal activity. In contrast, treatment with leupeptin, an inhibitor of cysteine, serine, and threonine proteases important for endosomal processing (36, 43–45), did not affect the cross-presentation efficiency detected for both Ags tested. We therefore concluded that MS-OVA and OVA-SLP follow the cytosolic pathway of cross-presentation in BMC2 cells. Also in the case of MutuDC cells, cross-presentation of the same Ags was sensitive to MG132 and BFA, but not to leupeptin, again indicating a dependency on the cytosolic pathway (Fig. 1D). Similar results were obtained when performing the same experiment with primary wild-type BMDCs (Supplemental Fig. 1). Overall, it was therefore evident that presentation of MS-OVA and OVA-SLP by both APC lines largely depended on the cytosolic pathways of cross-presentation. We therefore considered our experimental setup as a valid model to study the role of Sec22b and Stx4 in the cross-presentation of these Ags.

To investigate the role of Sec22b in the cross-presentation of MS-OVA and OVA-SLP, we used CRISPR/Cas9-mediated genome editing to generate homozygous KO clones for both BMC2 and MutuDC cells. To reduce the risk of potential off-target effects in our experiments, we targeted both exons 1 and 2 of Sec22b, respectively, with two different, nonhomologous sgRNAs (Fig. 2A). We thereby generated two independent Sec22b KO clones for both APC lines that we aimed to use for our cross-presentation experiments. As controls, we transduced wild-type APCs with NT sgRNAs to ensure that the experimental procedure of generating Sec22b KO clones had no additional Sec22b-independent effects on the phenotype of APCs. After lentiviral targeting of APCs, single-cell clones (Sec22b KOs) or NT pools of cells (controls) were tested by Western blot analysis to evaluate Sec22b expression. This experiment revealed that although both BMC2 (Fig. 2B) and MutuDC cells (Fig. 2C) showed prominent expression of Sec22b for wild-type and NT controls at the expected size of ∼24 kDa, this expression was completely absent in single-cell clones targeted with Sec22b-specific sgRNAs. These results demonstrated that we successfully generated two independent Sec22b KO clones for both BMC2 and MutuDC cells, respectively.

As functional MHC class I surface expression is an important parameter for evaluation of cross-presentation efficiency, we next wanted to ensure that Sec22b KO did not affect the overall amount of MHC class I surface expression of targeted APCs. For this purpose, we stained the cells with fluorescently labeled Abs against the two C57BL/6-specific MHC class I alleles H-2K^b and H-2D^b and analyzed their surface expression by flow cytometry (Fig. 2D, 2E, Supplemental Fig. 2A–C). Of note, we did not observe any alteration of MHC class I expression when comparing wild-type APCs with cells that were modified with NT control sgRNAs, demonstrating that introducing the CRISPR/Cas9 machinery alone had no effect on MHC class I surface levels. Although both Sec22b KO clones of BMC2 cells (Fig. 2D) and one clone of MutuDC cells (Fig. 2E) showed a small reduction in the overall H-2K^b surface expression, this phenotype was not significant. In support of this notion, we also did not observe a significant reduction of H-2D^b expression that was monitored in parallel (Supplemental Fig. 2B, 2C).

To independently validate these results, we used B3Z T cell hybridoma activation as a functional readout to quantify the overall MHC class I–restricted Ag presentation capacity by Sec22b KO cells and NT controls (Fig. 2F, 2G). APCs were externally pulsed with titrated amounts of the minimal peptide epitope S8L that binds to surface H-2K^b without requiring internalization and further processing. After removing unbound peptides, pulsed cells were cocultured with B3Z T cell hybridoma cells overnight. When comparing Sec22b KO clones with the NT controls, we observed no differences in B3Z activation for BMC2 cells line. Similar, for MutuDC cells, and despite a slight but significant reduction observed for KO clone 1, Sec22b KO overall did not affect the capacity of cells to activate B3Z hybridomas. We therefore decided to continue with both BMC2 and MutuDC clones to evaluate possible effects of Sec22b KO on the cross-presentation of MS-OVA and OVA-SLP.

Before investigating the role of Sec22b in cross-presentation, we evaluated Ag uptake as a critical first step of the cross-presentation pathway. MS-QDs were used to evaluate the phagocytic abilities of Sec22b KO APCs. Cells were incubated with MS-QDs for 2 h before samples were analyzed by flow cytometry. This experiment showed that equal amounts of MS-QDs were phagocytosed by BMC2 cells, when comparing Sec22b KO clones with either NT controls or wild-type cells (Fig. 3A). The same assay performed at 4°C showed strongly reduced fluorescence signals, indicating that MS-QD uptake by APCs depended on an active cellular process, as expected. Although BMC2 cells lacking Sec22b showed unaltered levels of phagocytosis, it appeared that Sec22b KO in MutuDC cells led to a slight reduction in the overall phagocytosis capacity (Fig. 3B).

As a next step, we evaluated the effect of Sec22b KO on the cross-presentation of MS-OVA and OVA-SLP using two different T cell assays as a readout for the quantification of H-2K^b/S8L surface presentation. First, we tested the cross-presentation efficiency of BMC2 cells using primary H-2K^b/S8L–restricted T cells from TCR transgenic OT-1 mice as a readout. In the light of recent studies that showed an effect of Sec22b deficiency on the cross-presentation of different OVA-derived Ags (9, 13, 14), we used a similar experimental setup with restricted time for Ag processing and presentation. BMC2 cells were incubated with Ag for 2 h before coincubation with OT-1 T cells for an additional 4 h in the presence of BFA. Next, the percentage of IFN-γ⁺ CD8⁺ T cells was quantified by flow cytometry as a measure of T cell activation. Interestingly, in BMC2 cells Sec22b KO had no consistent effect on the cross-presentation efficiency observed for both MS-OVA and OVA-SLP, compared with the NT controls (Fig. 3C, top panels). To independently validate these finding, we repeated our experiments using the B3Z hybridoma assay. In this setting, wild-type and Sec22b KO APCs were incubated with Ag and simultaneously cocultured with B3Z hybridoma cells for 18 h. Again, these experiments revealed that overall Sec22b KO did not interfere with cross-presentation of both Ags (Fig. 3C, bottom panels).

To further investigate the role of Sec22b in the cross-presentation of MS-OVA and OVA-SLP, we performed the same set of experiments with MutuDC cells (Fig. 3D). Similar to our experiments with BMC2 cells, we did not observe a consistent reduction in the cross-presentation capacity of Sec22b KO MutuDC cells when incubating them with MS-OVA or OVA-SLP for 2 h followed by addition of primary OT-1 T cells. Although Sec22b KO clone 1 consistently showed reduced cross-presentation of MS-OVA, this effect appeared to be unrelated to Sec22b expression, as the independently generated KO clone 2 was able to cross-present normally (Fig. 3D, top panels). The small reduction seen for overall H-2K^b expression of KO clone 1 may explain this reduction (Fig. 2G). Next, we again wanted to independently validate these results by coincubating Sec22b KO MutuDC cells with Ag and B3Z T cell hybridoma cells for 18 h. This assay showed robust cross-presentation of both MS-OVA and OVA-SLP that was not consistently different compared with the NT controls (Fig. 3D, bottom panels). Interestingly, cross-presentation of MS-OVA rather seemed slightly increased in Sec22b KO MutuDC cells. Taken together, we did not observe a consistent cross-presentation–related phenotype in Sec22b KO APCs under the conditions tested in this study.

In the recent literature, Sec22b has been suggested to mediate membrane fusion events in collaboration with the target SNARE Stx4 (4, 9, 46). For the cytosolic pathways of cross-presentation, however, experimental data regarding a functional role of Stx4 are still pending. In this study, we therefore aimed to experimentally evaluate the role of Stx4 in the context of Ag cross-presentation. Using a similar CRISPR/Cas9-mediated genome editing strategy as used to make the Sec22b KO APC clones, we generated two independent homozygous Sxt4 KO clones for the two APC cell lines BMC2 and MutuDC. Again, two nonhomologous sgRNAs were designed that targeted exon 2 and exon 3 of Stx4, respectively (Fig. 4A). After introducing the CRISPR/Cas9 machinery and the sgRNAs using a lentiviral approach, successfully targeted cells were selected by puromycin or blasticidin resistance, and APCs were subcloned to obtain single-cell clones. Next, the targeted cells were evaluated by Western blot analysis for their Stx4 protein expression (Fig. 4B, 4C). Although both wild-type BMC2 and MutuDC cells, as well as respective controls targeted with two different nontargeting sgRNAs, showed a prominent Stx4-specific band at the expected molecular mass of 34 kDa, no residual expression of Stx4 was observed in APC clones targeted with Stx4-specific sgRNAs. We therefore concluded that we successfully generated viable Stx4 KO clones of both BMC2 and MutuDC cells, which allowed us to study the role of Stx4 in the cross-presentation of MS-OVA and OVA-SLP.

We next evaluated possible effects of Stx4 KO on the overall surface expression of MHC class I, as a prerequisite for Ag cross-presentation. APCs were stained for H-2K^b and H-2D^b, respectively, and analyzed by flow cytometry (Fig. 4D, 4E, Supplemental Fig. 3). This experiment demonstrated that Stx4 KO did not alter MHC class I surface expressing on both APC lines tested. Although there was a slight trend toward reduced H-2K^b and H-2D^b surface expression levels in BMC2 cells targeted with Stx4 KO sgRNAs 1 and 2, these differences were not significant. To independently validate these results, we again used T cell activation as a second readout to evaluate surface H-2K^b expression. Therefore, APCs were externally pulsed with titrated amounts of the minimal T cell epitope S8L, followed by washing steps to remove unbound peptide and incubation with B3Z hybridoma cells. Similar to the initial characterization of Sec22b KO clones (Fig. 2F, 2G), we did not detect major differences between Stx4 KO BMC2 clones and NT controls (Fig. 4F). The Stx4 KO clones in MutuDC cells were similar to the NT controls, although a small reduction was observed for the limiting peptide concentrations that was consistent for both KO clones (Fig. 4G). Next, we continued to evaluate the role of Stx4 in the cross-presentation of MS-OVA and OVA-SLP in the following experiments.

Similar to the characterization of Sec22b KO APCs, we initially investigated Ag uptake by Stx4 KO clones as a first, essential requirement for cross-presentation. Wild-type, NT control, and Stx4 KO APCs were incubated with fluorescent MS-QDs for 2 h and uptake efficiency was quantified by flow cytometry (Fig. 5A, 5B). The results of these experiments revealed that Stx4 KO did not alter the capacity to efficiently take up MS-QDs in both BMC2 (Fig. 5A) and MutuDC cells (Fig. 5B). The fluorescence intensity after incubating APCs at 4°C was strongly reduced, indicating that the MS-QD–specific signal was based on active endocytosis.

Next, we evaluated the effect of Stx4 KO on the cross-presentation of MS-OVA and OVA-SLP in both APC cell lines, using primary OT-1 T cell activation and B3Z hybridoma as a measure of cross-presentation efficiency. Initially, BMC2 cells were incubated for 2 h with Ag, before OT-1 T cells were added for an additional 4 h (Fig. 5C, top panels). These experiments revealed that cross-presentation of both MS-OVA and OVA-SLP was not compromised by Stx4 KO BMC2 cells. We next aimed to validate these findings using the B3Z hybridoma assay as an independent readout for cross-presentation (Fig. 5C, bottom panels). For these experiments, BMC2 cells and B3Z hybridoma cells were cocultured for 18 h in the presence or absence of MS-OVA or OVA-SLP. Interestingly, although cross-presentation of OVA-SLP was not consistently affected by Stx4 KO (Fig. 5C, bottom right panel), the activation of B3Z hybridomas after incubation with MS-OVA was significantly reduced in both Stx4 KO clones (Fig. 5C, bottom left panel). Therefore, although we did not observe this in the OT-1 T cell assay, it appears that under specific experimental conditions (Ag type, time of incubation, readouts system) Stx4 might play a nonredundant role in cross-presentation.

Next, we evaluated the effects of Stx4 KO on the cross-presentation of MS-OVA and OVA-SLP in MutuDC cells, using the same set of experiments. Of note, we did not observe any reduction in T cell activation (OT-1 T cell and B3Z hybridoma assay), indicating that cross-presentation was not affected by Stx4 KO in any of the conditions tested (Fig. 5D). This led us to the conclusion that Stx4 depletion in the two APC lines tested had overall very limited effect on the cross-presentation of MS-OVA and OVA-SLP.

The contribution of specific SNARE proteins to cross-presentation is an ongoing subject of discussion (9, 13, 14, 47), and we therefore set out to specifically investigate the role of Sec22b and Stx4 in related pathways. Initially, we confirmed the dependence of MS-OVA and OVA-SLP cross-presentation by BMC2 macrophages and MutuDC cells on the cytosolic pathway, which was an important validation of previous findings (21, 27–31) and essential to qualify our experimental setup. Of note, both APC lines have been used as model systems reflecting aspects of in vivo–relevant mechanisms of cross-presentation (32, 34, 36–39). Still, our experiments point toward cell line and Ag-specific pathway preferences, as the inhibitory effect of MG-132 on OVA-SLP presentation seemed more pronounced in BMC2 cells compared with MutuDC cells. This was accompanied by a remarkably reduced effect of BFA treatment under the same condition, indicating that cross-presentation of OVA-SLP in BMC2 cells but not in MutuDC cells might have primarily relied on the indirect cytosolic pathway (i.e., phagosome-to-cytosol-to-phagosome) (3, 4, 6). When comparing the cross-presentation efficiency of MS-OVA in the presence of MG132 and BFA between BMC2 cells and MutuDC cells, it was evident that both inhibitors led to a comparable reduction, arguing for a contribution of the direct cytosolic pathway (i.e., phagosome-to-cytosol) in this system (6). Although DCs are generally considered the most relevant cross-presenting cell type in vivo (3), especially for cell-associated Ags, macrophages can process and cross-present exogenous Ag delivered by vaccine formulations (48, 49). For example, cross-priming of CD8⁺ T cells of MS-OVA was only compromised after depleting both DCs and macrophages in vivo (26). In addition, a similar dependence on the cytosolic pathways was also observed when performing the same experiments with primary BMDCs, indicating that this was not specific for the cell lines selected for this study (Supplemental Fig. 1). Of note, some PLGA-based polymers have previously been described to grant encapsulated Ags artificial access to the direct MHC class I presentation pathway by inducing endosomal membrane rupture (31, 50). However, for the MS-OVA formulation used in our study, this mechanism has previously been excluded (27).

The v-SNARE protein Sec22b has been found on the ERGIC as well as on phagosomal/endosomal membranes, positioning it at the right place for mediating membrane fusion events between the ERGIC and Ag-containing endosomes (9, 51–53). For these reasons, the role of Sec22b in cross-presentation has been investigated before by Cebrian et al. (9). Using short hairpin RNAs (shRNAs) targeting Sec22b mRNA, this study reported that knockdown of Sec22b protein in vitro decreased the cross-presentation efficiency of different types of OVA-based Ags, a finding that has been confirmed by another study (9, 12). Additionally, two groups have separately generated a conditional KO mouse for Sec22b in DCs via the use of Cre expression under control of the CD11c promoter (13, 14). Alloatti et al. (13) showed ex vivo and in vivo that Sec22b expression was essential for efficient cross-presentation of various OVA-based model Ags. Conversely, a similar conditional Sec22b KO model generated by Wu et al. (14) showed no defect in cross-presentation as tested ex vivo. A critical factor in both models might have been variable residual Sec22b expression, on either a cellular (shRNA approach) or a population level (conditional KO) (13, 14, 47). In contrast to previous studies, we therefore made use of CRISPR/Cas9-mediated genome editing technology to generate homozygous KOs for Sec22b and Stx4, respectively, which allowed us (to our knowledge) for the first time to study cross-presentation in the complete absence of residual protein expression. To circumvent possible off-target effects that were discussed as confounding factors in some of the above studies (14), we used two distinct sgRNAs targeting both Sec22b and Stx4 in different exons, respectively. Using this model, we showed that Sec22b depletion had no consistent effect on the cross-presentation of MS-OVA and OVA-SLP in this system, which was in line with similar experiments performed by Wu et al. (14), but in contrast to earlier findings by Cebrian et al. (9) and Alloatti et al. (13). Importantly, our results do not exclude a redundant role of Sec22b in the delivery of ER proteins to the endosome that might depend on the type and concentration of Ag as well as cell type–specific factors and the time allowed for Ag processing that might determine pathway vulnerabilities. In both cases, other v-SNARE proteins might have rescued the cross-presentation–related phenotype of Sec22b KO APCs, as a certain degree of redundancy has been reported among SNARE proteins of the same family (54–56). This has particularly been demonstrated for Sec22b and its yeast homolog Sec22p (56, 57). The SNARE protein Ykt6p has been shown to substitute for Sec22p in the early secretory pathway in yeast (57). It is therefore possible that the murine homolog Ykt6 might replace Sec22b to promote the delivery of ER-resident proteins to Ag-containing endosomes. However, Ykt6 could not be detected in phagosomes from both wild-type and Sec22b knockdown BMDCs containing latex beads (9), but this might be different for phagosomes containing MS-OVA or OVA-SLP. Other SNARE proteins that could possibly take over a Sec22b-related function include Vamp2, which has been demonstrated to be interchangeable with Sec22b in biochemical interaction studies (58). For future studies, it would therefore be interesting to study Sec22b KO APCs lacking additional SNARE proteins to evaluate possible redundancies in Ag cross-presentation.

The SNARE protein Stx4 is commonly found on the plasma membrane of cells where it mediates vesicle fusion events (9, 11, 56, 59–61). In DCs, however, Stx4 is also present on early phagosomes where it interacts with Sec22b, most likely traveling along during the uptake of Ags (9, 46). Although frequently associated with vesicle fusion (11), to our knowledge the role of Stx4 in the cross-presentation of any type of Ag has never been addressed experimentally, making our study the first to explore possible effects of Stx4 KO on cross-presentation. In contrast to previous findings using an shRNA approach to deplete Stx4, the KO clones generated in this study were viable and did not show any clear phenotype compared with wild-type APCs (9). When analyzing the consequences of Stx4 KO on cross-presentation we observed no major phenotype in both BMC2 and MutuDC cells, as both MS-OVA and OVA-SLP were cross-presented with at least the efficiency of the NT controls. Reduced cross-presentation that we observed for MS-OVA in Stx4 KO BMC2 cells using the B3Z hybridoma assay was not evident when using primary OT-1 T cells as a readout. Because MS-OVA uptake and related cross-presentation pathway dependencies observed in our inhibitor experiments were similar between BMC2 and MutuDC cells (Figs. 1C, 1D, 3A, 5A), the different T cell readouts might have accounted for the varying effects of the Stx4 KO in this condition. Although both were restricted to H-2K^b/S8L, B3Z hybridoma activation requires long coincubation (18 h) with APCs to accumulate β-galactosidase, possibly integrating differences in cross-presentation that might occur late after Ag uptake. Activation of OT-1 T cells in contrast is measured shortly (4 h) after coincubation with APCs, therefore representing a snapshot of the initial hours of cross-presentation. Given their primary origin, OT-1 T cells might in addition require fewer MHC/peptide complexes per APC for full activation, which could also explain the overall stronger activation of OT-1 T cells already at lower Ag concentrations. At the same time, this effect could have favored B3Z hybridomas in our setup to trace subtle effects of Stx4 KO in MS-OVA–treated BMC2 cells.

Overall, our results argue against a major role of Stx4 in the cross-presentation settings tested in this study, indicating the presence of alternative mechanisms and/or compensatory mechanisms (e.g., SNARE protein redundancy, alternative pathways) that might have rescued cross-presentation in Stx4 KO APCs. Possible redundancies of Stx4 with other SNARE proteins might be supported by indirect evidence suggesting that Stx4, together with SNAP-23, can interact with several other SNARE proteins, including VAMP2, VAMP4, VAMP7, and VAMP8 (58, 59, 61). Additionally, Stx5 was reported to interact with Sec22b in the context of ER-to-Golgi trafficking, but it could not directly be linked to cross-presentation, as Stx5 knockdown reduced steady-state surface MHC class I expression and interfered with T cell activation (9). Similarly, Sec22b/Stx18 interactions were shown to regulate phagocytosis by macrophages, but a connection to ERGIC trafficking during cross-presentation has not been established (51, 52). Further investigation of the network of specific SNARE proteins involved in cross-presentation therefore remains important to fully elucidate possible redundancies present in this pathway.

In this study, we did not find evidence for a role of Sec22b and Stx4 in most of the experimental conditions tested, highlighting that the previously suggested model of Sec22b/Stx4 function might not fully reflect the complexity of cross-presentation mechanisms present in the various types of APCs and with different Ags. The results of this study led us to three general conclusions: 1) SNARE proteins involved in the cytosolic pathways of cross-presentation might be redundant; 2) the molecular machinery of cross-presentation, including the selection of specific SNARE proteins involved, may vary by APC and Ag type or concentration/time after uptake; and 3) other compensatory mechanisms in the absence of specific SNARE proteins (e.g., use of alternative pathways) might further complicate experimental interpretations. Furthermore, we cannot exclude the presence of compensatory effects as a direct consequence of SNARE protein depletion in our system that would not be present in the wild-type situation. Overall, our study adds to the current discussion about the role of SNARE proteins in the cytosolic pathways of cross-presentation and highlights the need of a differentiated cell and Ag type–specific evaluation of the mechanisms involved. In addition, our results contribute to a better understanding of the cell biological mechanisms involved in the cross-presentation of PLGA-MS and SLP-based Ag delivery systems, which might eventually help to further improve their potential to elicit potent and lasting CTL response in cancer vaccination.

The authors have no financial conflicts of interest.

We thank Marcus Groettrup for providing PLGA microspheres containing the model Ag OVA, Manzhi Zhao and Ling Li for advice and technical support, and Peter D. Katsikis for critical input and helpful suggestions for this manuscript. Finally, we express our gratitude to the Department of Immunology at the Erasmus MC for generous financial support and research space.