Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/bc
Cross-Linked Peptide Nanoclusters for Delivery of Oncofetal Antigen as a Cancer Vaccine Alexandra N. Tsoras and Julie A. Champion* School of Chemical & Biomolecular Engineering and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, United States S Supporting Information *
ABSTRACT: Peptide subunit vaccines are desirable because they increase control over the immune response and safety of the vaccine by reducing the risk of off-target responses to molecules other than the target antigen. The immunogenicity of most peptides, however, is low. Peptide nanoclusters (PNC) are proposed as a subunit peptide vaccine delivery system made completely of cross-linked peptide antigen that could improve the immunogenicity of a peptide vaccine. Proof of concept is demonstrated with oncofetal antigen (OFA), an immature laminin receptor protein expressed by many hematologic cancer cells but not by healthy cells. Peptide epitopes from this protein, called OFA 1, 2, and 3, were synthesized into PNC as a potential cancer peptide vaccine delivery system. PNC were formed by desolvation and stabilized with disulfide bonds using a trithiol cross-linker. Cysteines were added to the C-terminus of each peptide to assist in this cross-linking step, denoted OFA 1C, 2C, and 3C PNC. OFA 2C was found to form the smallest PNC, 148 ± 15 nm in diameter and stable in solution. This size is in the range where particles are readily internalized by dendritic cells (DCs) and may also passively diffuse to regional lymph nodes. OFA 2C PNC and soluble OFA 2C were internalized similarly by DCs in vitro, but only PNC resulted in significant peptide presentation by DCs. This indicates the potential for PNC to improve immune activation against this antigen. Additionally, PNC displayed higher retention at the intradermal injection site in vivo than soluble peptide, allowing more time to interact with DCs in an area of increased DC activity. While offering traditional nanoparticle benefits such as increased DC recognition, slower diffusion, and potential for multivalent cellular interactions, PNC also maximize antigen delivered per particle while minimizing off-target material delivery because the antigens are the main building blocks of the particle. With these properties, PNC are a delivery system with potential to increase peptide subunit vaccine immunogenicity for OFA and other peptide antigens.
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INTRODUCTION
immunogenicity and rapid clearance from the body, reducing the likelihood to generate a strong immune response. As the use of inert subunits of a pathogen becomes a more desired form of vaccination, new technologies have been developed to increase immune responses to these materials in the attempt to match the level of immune response from full inactivated or attenuated vaccine platforms. There are multiple approaches for increasing immunogenicity of subunit vaccines. While administration of adjuvants, such as alum or Montanide, has been shown to provide the necessary stimulation of innate immunity,4 their lack of specificity has also led to many adverse
Subunit vaccines are attractive because of their potential to increase safety of vaccines and allow more control over the immune response of the host.1 This is crucial for vaccines needed to combat highly variable viral and bacterial strains that threaten pandemics.2 Similarly, cancer vaccines require significantly more control over the immune response to enable a strong and narrowly targeted response toward cancer cells but not healthy cells.3 The subunit components of a vaccine are generally surface protein antigens or peptide epitopes from surface proteins of a pathogen or cancer cell. Peptide vaccines are ideal subunit vaccines because of their facile and affordable synthesis and their potential for multivalent targeting or multiple epitope administration.2 The challenge with peptide vaccines is that their extremely small size results in low © XXXX American Chemical Society
Special Issue: Bioconjugate Materials in Vaccines and Immunotherapies Received: January 30, 2018
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DOI: 10.1021/acs.bioconjchem.8b00079 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry local and systemic effects including inflammation, toxicity, immunosuppression, and allergy.1,5 Different adjuvants activate specific innate pathways that do not always induce an effective adaptive immune response for a particular antigen. Particle-based carrier systems can act as adjuvants while also serving as a protective delivery system that enhances recognition and internalization by phagocytic antigen presenting cells (APCs).4 Carrier materials like lipids,6 polymers,7,8 gas vesicle nanoparticles (GVNPs),9 virus-like particles (VLPs),10 and metal organic frameworks11 have been used to stabilize, modulate delivery, and increase immunogenicity of peptide antigens. Conjugation of antigen to nanoparticulates,12 membrane proteins,13 universal T cell epitopes,14,15 or other materials with DC targeting capabilities such as CpG oligodeoxynucleotides,16 heat shock proteins,17 or DC-targeting peptide (DCpep)18 have also been shown to improve protective immune responses and decrease side effects by inducing trafficking to lymph nodes or targeting specific immune cell receptors. Self-assembling β-sheet peptide frameworks have been extensively studied to modulate humoral immune responses to antigens incorporated into the peptide framework.19−23 Similarly, α-helical peptide structures have also been shown to induce cell-mediated immune responses.24 These materials insert an antigen sequence into the expressed self-assembling protein sequence and have been shown to retain this self-assembled structure with antigen incorporation. However, the kinetics of assembly are difficult to control and there can be a lack of structural precision with the incorporation of different peptides.24 Additionally, successful expression or synthesis and display of the epitope may be highly dependent on the properties of each antigen. While protein-based carrier-adjuvants, such as GVNPs, VLPs, selfassembling protein constructs, and conjugate proteins and peptides have shown promise, pre-existing immunity to these protein carriers can induce epitope suppression.25 The use of protein constructs to encapsulate or adsorb peptides also reintroduces the potential for off target reactions against these carrier materials. In general, encapsulation, incorporation, or adsorption of antigen to carrier materials reduces the overall percentage of the material that is truly the desired target antigen.1,5 Furthermore, many systems involve only encapsulation or surface adsorption of the antigen to the carrier material, but it has been shown that immune responses increase when delivery systems contain antigen on both the inside and outside of the particle.26 This design is difficult to form with a general process as many encapsulating or adsorption carrier materials depend on electrostatic interactions with antigen, which varies with each antigen.5 Recombinant Oncofetal Antigen (OFA) and OFA peptide epitopes have been shown to induce activation of CD4+ Th1 and CD8+ T-cells,27,28 making OFA a promising subunit cancer vaccine or immunotherapy.4,27−30 OFA is a highly conserved protein antigen expressed in many types of cancer cells including breast, head/neck, and hematologic malignancies, but it is not detectably expressed in healthy cells.29,30 This protein is a laminin receptor that is expressed as an immature monomer in fetuses during early gestation and induces maternal immune responses. Immunosuppressive cells work to inhibit fetal rejection during the early gestation period. It is altered by mid- to late gestation into a mature dimerized form that is nonimmunogenic. The immature monomeric OFA protein is re-expressed in cancer cells and has been determined to play an important role in metastasis.30
Three peptide sequences from OFA immature laminin receptor protein (OFA-iLRP) were previously identified as Tcell epitopes,4 and are arbitrarily named here OFA 1 (OFAiLRP58−66: LLLAARAIV, m.w. 938.6 g/mol), OFA 2 (OFAiLRP146−154: ALCNTDSPL, m.w. 932.4 g/mol), and OFA 3 (OFA-iLRP177−185: MLAREVLRM, m.w. 1117.6 g/mol). OFA 1, 2, and 3, were selected to represent the epitopes with distinct regions of recognition that displayed the highest binding affinity or detectable spontaneous T-cell response.27 These peptide antigens are presented on major histocompatibility complex I (MHC I) haplotype human leukocyte antigen A2.1 (HLAA2.1).27 Like many peptide vaccines, one of the largest obstacles facing OFA peptides as subunit vaccines is their low immunogenicity.4 To create a peptide nanoparticulate system that could improve peptide subunit vaccines, we sought to fabricate crosslinked peptide nanoclusters (PNC) with antigen on both the inside and outside while minimizing unnecessary materials that have the potential for off target responses. As nanoparticles, PNC should provide the same general benefits including slower diffusion from the injection site because of their larger size, and increased likelihood of internalization because APCs, and in particular dendritic cells (DCs), can phagocytose objects in the 20−2000 nm range, with a preference for 200 nm) by JAWS II murine DCs over 24 h, in contrast to OFA 2C internalization.32 However, different surface charge and hydrophobicity and 30× difference in size between OFA 2C and OVA could significantly affect internalization of these soluble antigens. Additionally, other materials including gold, silica, and polymer nanoparticles have been reported to be internalized more and reach maximum uptake faster when in the 20−50 nm range than when >50 nm.51−53 Although rapid uptake can be important, many studies only measure internalization after 24 h or later and some studies do not report a correlation between size and APC uptake.50,54 Given the different rates of uptake and very different sizes of soluble and PNC OFA 2C, it is possible that they have different mechanisms of internalization. Burgdorf and Kurtz suggest that nanoparticulate and soluble antigens may have different fates upon internalization.55 Internalized nanoparticle antigens are more likely to be crosspresented through delayed acidification and therefore delayed lysosomal protease activity and production of peptides available for MHC II presentation, allowing time for phagosomal escape and cross-presentation. Furthermore, Chang et al., provide an example of cross-linked protein nanoparticles of similar sizes to OFA 2C PNC inducing this delayed acidification indicative of cross-presentation.32 Although the exact mechanism of internalization was not evaluated, differences in uptake rates between soluble peptide and PNCs suggest their uptake route, and eventual internalization fate, may be different. These different
promote iDC progression into mature DCs (mDCs) for the next 1−2 days. Maturation of iDCs into mDCs generally occurs in the body when DCs internalize foreign antigens and receive chemical signals to present these antigens to other immune cells and promote adaptive immunity.49 To compare how much peptide was internalized by DCs in PNC and soluble form, 25 μg/mL OFA 2C-FITC PNC or soluble OFA 2C + 10% OFA 2-FITC were incubated with iDCs during the first 20 h after maturation cytokines were added. Figure 3a shows the amount of internalized fluorescent peptide after 4 and 20 h at 37 °C. Extracellular FITC fluorescence was quenched with trypan blue to ensure fluorescence detected was only from internalized peptide (Figure S6). After 4 h, a significant amount of peptide was already internalized by DCs in both the soluble and PNC wells; however, more soluble peptide than PNC was internalized at this time point. The amount of soluble peptide uptake appeared to have maximized after 4 h in the soluble wells, whereas PNC continued to be internalized at 20 h. After 20 h, the amount of peptide internalized was the same for both soluble and PNC wells. These results indicate a marginally slower uptake of PNC, but an overall significant amount of uptake equivalent to soluble peptide uptake after 20 h. Significant internalization of soluble peptide and PNC and was further confirmed through confocal microscopy of M3 DCs after 24 h of incubation with OFA 2C-FITC (Figure 3b). Lysosomal-associated membrane protein 1 (LAMP1) was also labeled in confocal images to assess intracellular location of the peptide. Images reveal that there is significant green peptide signal not colocalized with lysosomes, suggesting that peptide can avoid lysosomal degradation and is available for cross presentation on MHC I through intracellular trafficking from the cytosol to the endoplasmic reticulum. It was also confirmed E
DOI: 10.1021/acs.bioconjchem.8b00079 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 4. (a) Live images of representative intradermal injection sites of mice injected with either OFA 2C PNC-NIR or soluble OFA 2C + 5% NIRtagged OFA 2. Images were taken over several time points (0, 12, 24, and 54 h) using In Vivo Imaging System (IVIS) Spectrum CT. Fluorescence was scaled to highest intensity image (OFA 2C PNC, 0 h). Fluorescent peptide can still be seen retained in injection site up to 54 h post injection, whereas soluble peptide is not visible by 12 h. (b) Quantified region of interest total fluorescence retention, area under the curve (AUC), normalized to the 0 h time point of each mouse. **p < 0.01.
the cell surface unless they are actively presenting intracellular antigen.24 Therefore, while the HLA-A2.1 surface molecules could have been presenting other intracellular peptides, these background HLA-A2.1 levels should be similar across groups since all groups were exposed to the same media and maturation cytokines. Peptide positive cells could be caused by nonspecific surface adsorbed peptide, but since levels of both OFA 2C and HLA-A2.1 increase on the surfaces of only DCs pulsed with PNC, we infer that the peptide being presented was OFA 2C. Figure S8 confirms that only PNC-incubated DCs display a significantly higher percentage of double positive cells compared to the soluble-incubated and control DCs. This result suggests a higher likelihood that OFA 2C peptide was processed and presented as a foreign intracellular antigen on its presenting MHC I haplotype, HLA-A2.1, compared to soluble peptide. Internalization and presentation of OFA 2C soluble peptide versus PNC generally aligns with the guidelines of Benne et al., proposing that PNC create effective DC interactions from a peptide that would otherwise be too small for optimum presentation. This indicates that OFA 2C PNC may more effectively induce processing steps necessary by DCs to present antigen for the activation of T-cells and ultimately promote an adaptive immune response to OFA. DC activation and transition to mature antigen presenting cells is an important step in producing an effective immune response. DC CD80 surface expression was measured after incubation with 25 μg/mL of OFA 2C soluble peptide or PNC. Figure 3e shows that CD80 levels were upregulated significantly more on cells incubated with PNCs than both soluble peptide and untreated cells. These results support the claim that DCs may better recognize PNCs as foreign and receive necessary stimulation from PNCs to more efficiently activate to transition into mature, antigen-presenting cells. This self-adjuvanting effect has been observed with the use of other nanoparticle systems in delivering peptide antigens.6,9,23 PNCs display similar abilities to increase DC activation, reducing the need for adjuvants in vaccine formulations. In Vivo PNC Retention. It is vital for any vaccine or immunotherapy to promote interactions between the administered antigens and APCs. Intradermal injection is a potent vaccine delivery method to target an area where DCs localize and are most active, as the skin is a common route of infection.56 Longer retention of antigen in an intradermal injection site increases the time available to interact with resident DCs and provide persisting antigen exposure. OFA 2C
expected fates could promote cross-presentation, which induces cellular immune responses desirable for cancer vaccines. Though delivery systems can control antigen uptake, the amount of uptake by DCs does not directly correlate to strength of the subsequent immune response. While some APCs, like macrophages, maximize uptake to maximize clearance of foreign antigen, DCs only internalize enough antigen necessary to initiate an adaptive immune response.56 Moreover, Benne et al. developed general guidelines to predict expected DC interactions based on antigen size, which suggest that smaller antigens will be internalized the most, with slightly decreasing uptake for larger antigens. However, internalized antigens will be presented by DCs more when they fall within an optimal medium size range as opposed to very small or very large antigens.57 Therefore, DC internalization of antigen, while necessary for presentation, is not directly correlated with successful presentation. The goal of the PNCs is to facilitate sufficient antigen uptake to ultimately be released from the particle during DC processing mechanisms and presented on MHC I molecules to activate antigen-specific T cell responses. To evaluate DC presentation of OFA 2C, 25 μg/mL OFA 2C-biotin PNC or soluble OFA 2C + 10% OFA 2-biotin was incubated with iDCs during the first 24 h of maturation. To make OFA 2-biotin, OFA 2 (not OFA 2C) was C-terminally modified with the addition of a lysine to which biotin was conjugated. This modification was chosen to most closely mimic OFA 2C while enabling evaluation of antigen presentation by labeling of surface OFA 2-biotin. mDCs were then labeled with fluorescently tagged streptavidin to identify any biotinylated peptide on the surface of the cell. Levels of HLA-A2.1, which presents OFA peptides on the cell surface, was also measured by fluorescent antibody labeling. OFA 2C presentation was determined by measuring the percentage of DCs that displayed significant levels of surface OFA 2C peptide fluorescence and HLA-A2.1 surface expression (peptide+ and HLA-A2.1+). Figure 3c reports that only DCs incubated with OFA 2C PNC show a significant number of DCs with peptide on their surfaces. This is noteworthy because although soluble peptide and PNC internalization levels were similar, surface peptide was detected only on DCs incubated with PNC. Furthermore, Figure 3d reveals that PNC-incubated DCs also express higher levels of HLA-A2.1 than the control or soluble, while soluble peptide did not induce significant upregulation of HLA-A2.1. MHC I molecules do not reach F
DOI: 10.1021/acs.bioconjchem.8b00079 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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optimized for other antigenic peptides for application as a vaccine nanoparticle delivery system for a wide variety of cancer or infectious diseases.
PNC fall within the optimal size range for lymph node drainage from an injection site (20−200 nm) where lymph noderesident DCs are also highly active antigen transporters.31 Furthermore, particles within that range should also diffuse more slowly than soluble peptide, increasing exposed time to skin-resident DCs. To understand how formulation of OFA 2C peptide into PNC affects retention at the injection site, an in vivo peptide tracking study was performed. Groups of three BALB/c mice were injected intradermally at the base of the tail with OFA 2C-NIR PNC or soluble OFA 2C + 5% OFA 2Cy5.5. Mice were imaged over several time points to monitor injection sites for retention of fluorescent OFA 2C peptide. Major organs and the draining inguinal lymph nodes were also harvested after the mice were sacrificed to measure distribution of peptide; however, fluorescence levels were too low to detect significant distribution away from the injection site. Figure 4 shows both qualitatively and quantitatively that there was significantly more retention of OFA 2C PNC at the injection site than soluble OFA 2C. These findings confirm that the increase in size of the OFA 2C vaccine delivery system results in slower diffusion and clearance from the injection site, which could allow more time for DC internalization, processing, and activation.
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EXPERIMENTAL PROCEDURES PNC Synthesis. Peptide nanoclusters were synthesized using desolvation. Peptides OFA 1C, 2C, and 3C (Genscript) were solubilized in hexafluoroisopropanol (HFIP) (Sigma) at 5 mg/mL. 100 μL of the peptide solution was mixed on a stirplate at 1200 rpm in a 17 × 60 mm glass vial. Under mixing, 12.83 mg trimethylolpropane tris(3-mercaptopropionate) (trithiol) (Sigma) was added to the solution. 2 mL diethyl ether (DEE) (Sigma) was added dropwise (1 mL/min) to the vial with a syringe pump. After 50 min of continual mixing, the solution was centrifuged for 7 min at 18,400 rcf. The supernatant was aspirated and the remaining pellet was resuspended in Milli-Q water (OFA 2C), 150 mM L-arginine (OFA 2C variations) or 1% Tween 80 (OFA 1C, 3C). 10% FITC-tagged or 10% biotin-tagged OFA 2 (Genscript) were incorporated into the initial peptide concentration when synthesizing PNC for uptake and presentation studies, respectively. 5% Cy-5.5-tagged OFA 2 was incorporated into peptide concentration for synthesis of PNC for in vivo particle tracking analysis. Fabrication procedure for PNC variations was identical to that described above. PNC Characterization. Peptide nanocluster size, polydispersity index (PDI), and zeta potential were analyzed using dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS). Additional information on specifics of DLS data acquisition is found in Table S2. Scanning electron microscope (SEM) images were obtained using a Hitachi SU8230 SEM. Nanoclusters were suspended in water and drop cast on an aluminum stub and sputter coated with Au/Pd (Hummer Sputter Coater). Atomic force microscopy (AFM) images were obtained using a Veeco AFM. PNC were lyophilized and cast onto atomically flat mica to image. Yield % of peptide captured within PNC during desolvation was determined by extracting supernatant from the nanocluster desolvation solution. This supernatant was evaporated and leftover residue was resuspended in ethanol. LC-MS (Micromass Quattro LC) was used to create a standard curve of known concentrations of OFA 2C in ethanol. The final volume of the leftover solution was recorded and the concentration of unincorporated peptide was determined by LC-MS using the standard curve. The total amount of leftover peptide was subtracted from the starting amount to calculate the percentage of peptide formed into nanoclusters incorporated into the PNC. In Vitro Dendritic Cell Uptake, Surface Presentation, and Activation Evaluation. MUTZ-3 dendritic cells (M3 DC) (obtained from Dr. James Varani, University of Michigan School of Medicine as part of an ongoing collaboration) were cultured in M3 media (MEMα with ribonucleosides, Lglutamine, phenol red (Gibco), supplemented with 20% heat inactivated fetal bovine serum (h.i. FBS), 10% 5637 conditioned medium, and 1% Pennicillin/Streptomycin). 5637 cells (a kind gift from Dr. James Varani, University of Michigan School of Medicine) are a feeder cell line that produce cytokines necessary for M3 DC growth.23 5637 medium consisted of 90% RPMI 1640 (Gibco) and 10% h.i. FBS. To condition media, 106 5637 cells were cultured in T-75 flasks with 10 mL media/flask for 3 days or until about 70%
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CONCLUSION OFA peptide epitopes were successfully synthesized into PNC made entirely of cross-linked peptide antigen to serve as a delivery system of a cancer peptide vaccine. This is the first demonstration of PNC formation from peptides. OFA 1C, 2C, and 3C PNC were formed by desolvation. OFA 2C PNC were determined to be the best PNC for improvement of an OFA cancer vaccine because they remained stable the longest in solution and their size of 148 nm was smallest and fell well within the target range of 100−300 nm. OFA 2C PNC were evaluated in vitro and in vivo in comparison to soluble OFA 2C. DCs internalized OFA 2C PNC and soluble similarly over 20 h. This could be due to a different mechanism of internalization, which may also imply different downstream processing of the peptide. However, mechanistic studies are needed to evaluate this directly. DC presentation of peptide was different for PNC and soluble OFA 2C. A significant number of DCs displayed both OFA 2C peptide and its presenting MHC I haplotype, HLA-A2.1, only when incubated with PNC and not soluble peptide. This suggests the potential for PNC to induce better presentation, activation of T-cells, and T-cell killing of cancer cells, which should be evaluated in future work. It should also be acknowledged that the C-terminal modification of the OFA 2 epitope into OFA 2C may have epitope-specific effects on presentation. Any modifications necessary for PNC synthesis from a particular peptide antigen should be carefully assessed to identify those that would have the least harmful or most beneficial impact on antigen presentation. Finally, increased in vivo retention of PNC at an intradermal injection site demonstrated that PNC diffuse more slowly and have a higher residence time in this area of high DC activity, which could result in increased DC interactions and subsequent immune cell activation. Based on the data presented, OFA 2C PNC demonstrate the potential to promote more effective immune cell activation and ultimately serve as a therapeutic cancer vaccine. Evaluation of OFA 2C PNC in transgenic mice expressing human HLA-A2.1 is necessary to compare the immune response with traditionally adjuvanted soluble OFA 2C.58 More broadly, the PNC fabrication strategy can be G
DOI: 10.1021/acs.bioconjchem.8b00079 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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resuspended in SFB, and examined for fluorescence via a Beckman Coulter CytoFLEX. In Vivo PNC Tracking. All animal work was compliant with the NIH Guide for the Care and Use of Laboratory Animals and all protocols and procedures employed were reviewed and approved by the Georgia Institute of Technology Institutional Animal Care and Use Committee (A16021). Mice were kept on an alfalfa-free diet for 2 weeks prior to and throughout the experiment to minimize autofluorescence that may interfere with imaging. Mice (n = 3) were put under anesthesia with 5% isoflurane, shaved, and depilated around the injection site. They were intradermally injected with 30 μL of 670 μg/mL OFA 2C soluble peptide or PNC containing 5% Cy-5.5-tagged OFA 2 in pharmaceutical saline with 3.5 mg/mL L-arginine to the left of the base of the tail. Live near-infrared fluorescent imaging was conducted while mice were under anesthesia at 0, 12, 24, and 54 h with IVIS Spectrum In Vivo Imaging System to monitor fluorescence at the injection site. Fluorescence was quantified using Living Image Software. Statistical Analysis. Comparisons between groups for uptake study in vitro were compared using two-way ANOVA with a confidence interval of 95% varying in treatment and time. Comparisons between groups in presentation study in vitro were performed using one-way ANOVA with a confidence interval of 95%. Comparisons in vivo were performed using a Student’s unpaired two-tailed t test with a confidence interval of 95%. All above analysis was performed using Graphpad Prism software; *p < 0.05, **p < 0.01, ***p < 0.001. Power analysis of in vivo PNC retention study was performed using G*Power software (power = 88.2%). A power of >80% was considered to be a robust study.59
confluency was reached. All cells were incubated under humidified conditions at 37 °C, 5% CO2. On Day 0, M3 were seeded at 2 × 105 cells/mL in 24 (1 mL) or 6 (2 mL) well plates (for uptake and presentation studies, respectively) in M3 media without the addition of 5637 conditioned media but supplemented with 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 10 ng/mL interleukin (IL)-4, and a low dose of tumor necrosis factor (TNF)-α (2.5 ng/mL) (Peprotech). On day 3, 500 μL (24 well plate) or 1 mL (6 well plate) of cytokine supplemented media (as described above) was added to each well. On day 6−7, 750 μL (24 well plate) or 1.5 mL (6 well plate) of cytokine supplemented media was added to each well with a high dose of TNF-α (75 ng/mL) to induce maturation. By day 6−7, differentiated DCs were larger, extremely clumped together and had many dendritic extensions and nonspherical morphology. Twenty-five μg/mL of either soluble peptide or PNC were also added to the wells at the same maturation induction time. After 4 and 20 or 24 h of incubation (for uptake or presentation studies, respectively), cells were collected to conduct flow cytometric analysis. For uptake studies, after 4 or 20 h, cells were centrifuged at 300 g for 10 min. They were resuspended in trypan blue to quench external fluorescence and examined for internal fluorescence via a Beckman Coulter CytoFLEX. For confocal microscopy experiments, 167 μg/mL of soluble or PNC OFA 2C-FITC were incubated with differentiated DCs for 24 h in an 8 well chamber slide coated with poly(L-lysine). Cells were fixed with ice cold methanol for 20 min at 4 °C, washed with PBS, and permeablized with 0.1% Triton-X 100 in PBS for 20 min at room temperature. Cells were washed again with PBS and blocked with 1% BSA in PBS for 1 h at room temperature. To label lysosomal-associated membrane protein 1 (LAMP1) (a.k.a. CD107a), anti-CD107A antibodies (ThermoFisher) were incubated with cells under gentle mixing at room temperature for 2 h, and then incubated with AlexaFluor-680 secondary antibodies (ThermoFisher) for 1 h. Cell nuclei were stained with Hoechst 33342 (Invitrogen) for 20 min at room temperature. Cells were mounted in 0.1% phenylenediamine in 50% glycerol/50% phosphate buffer (pH 8.5) and imaged with a Zeiss LSM 700 Confocal Microscope. For surface presentation, after 24 h cells were collected and centrifuged at 300 g for 10 min. They were resuspended in standard flow buffer (SFB; PBS with 1% h.i. FBS). Cells were blocked with nonspecific mouse antibody isotypes IgG2a and IgG2b (Invitrogen) for 30 min at room temperature. They were then labeled with 4 μL 1:10 diluted streptavidin-allophycocyanin (eBioscience) and 8 μL phycoerythrin-anti-HLA-A2 antibody (BD Pharmingen) for 30 min at room temperature protected from light. Cells were washed once in SFB and fixed with 3.7% formaldehyde for 10 min. Cells were resuspended in SFB and examined for fluorescence via a Beckman Coulter CytoFLEX. For the DC activation experiment, cells were collected and centrifuged at 300 g for 10 min after 48 h of PNC or soluble peptide incubation. They were resuspended in blocking buffer (10 μg/mL Rabbit IgG (Sigma) in PBS) and incubated at room temperature for 1 h. Cells were centrifuged again and incubated with anti-CD80 (ThermoFisher) antibodies in SFB for 2 h at room temperature under gentle mixing. Then cells were centrifuged and labeled with secondary AlexaFluor-680 secondary antibodies (ThermoFisher) in SFB for 1 h under the same conditions. Cells were centrifuged a final time,
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00079. Additional size and morphology characterization of PNC, confirmation of efficient fluorescence quenching, additional cell fluorescence information from in vitro studies (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Julie A. Champion: 0000-0002-0260-9392 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Benovus Bio, Inc. is gratefully acknowledged for providing OFA peptides. Dr. James Varani, Dr. Roscoe Warner, and Shannon McClintock from the University of Michigan School of Medicine are gratefully acknowledged for their contributions to in vitro experimental design and shared MUTZ-3 and 5637 cell lines as part of an ongoing collaboration. David Bostwick of H
DOI: 10.1021/acs.bioconjchem.8b00079 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
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the Georgia Institute of Technology Bioanalytical Mass Spectrometry Facility is thanked for providing LC-MS data. This work was performed in part at the Georgia Tech Institute for Electronics and Nanotechnology, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (Grant No. ECCS1542174). We wish to acknowledge the core facilities at the Parker H. Petit Institute for Bioengineering and Bioscience at the Georgia Institute of Technology for the use of their shared equipment, services and expertise.
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DOI: 10.1021/acs.bioconjchem.8b00079 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.bioconjchem.8b00079 Bioconjugate Chem. XXXX, XXX, XXX−XXX
