HLA-typed peripheral blood mononuclear cells (PBMCs)
Twenty-one HLA-typed donor PBMCs were selected using an in-house algorithm called “SampPick”17. The preselected donors were purchased from Cellular Technology Limited (CTL) ePBMC library. The PBMCs were stored in vapor–liquid nitrogen until further use.
Peptides and proteins
The individual SaCas9 peptides (>90% purity), with amino acid sequences specified in Fig. S1, were purchased from GenScript. The individually lyophilized 15-mer peptides were initially dissolved in 100% Dimethyl sulfoxide (DMSO) (Fisher Scientific) at a concentration of 10 mg/mL and further diluted with 10% DMSO in CTL test media at a final concentration of 1 mg/mL. Overlapping 15-mer peptides were staggered by five amino acids. Thus, the first peptide covered amino acids 1–15 of the SaCas9 sequence, the second amino acids 6–20, the third amino acids 11–25, and so on. The last peptide is a 13-mer peptide due to the length of the SaCas9 protein. Ten individual peptides were pooled, each containing 10 consecutive 15-mer peptides (except for pool 21, which contains nine peptides). Aliquots of pooled peptides were stored at −80 °C. SaCas9 and SpCas9 (Cas9 from Staphylococcus pyrogens) proteins were endotoxin-free products manufactured by Editas Medicine as previously described6. We also obtained a laboratory-grade SpCas9 from a commercial supplier as a control. Peptides and proteins were used at 10 μg/mL concentration for all experiments.
Cell culture
Cryopreserved peripheral blood mononuclear cells (PBMCs), which were purchased from CTL, were thawed by following the supplier’s instructions. Briefly, Cryopreserved PBMCs were thawed at 37 °C by placing in a water bath for 8 min and flipping the cryovial twice to resuspend the cells. The resuspended cells were added in 10 mL of prewarmed RMNI-1640 with 1X Antiaggregate wash medium (CTL). The cells were centrifuged at room temperature at 330 g for 10 min and the supernatant was discarded. To completely remove the freezing media, we washed the cells again with 10 mL of antiaggregate wash medium. The PBMCs were then cultured in CTL Test Medium (CTL) supplemented with L-glutamine (1%, Thermo Fisher) and costimulatory reagent (BD Bioscience) CD28/CD49d costimulatory antibody at 10 μL/mL.
In vitro peptide stimulation
1.5 million PBMCs were stimulated with individual peptide pools, the full-length SaCas9 protein or Staphylococcal Enterotoxin B (SEB) (positive control, 1 μg/mL) (Sigma). The negative control (unstimulated) samples were treated with 10% DMSO in CTL Test media (DMSO working concentration 0.01%). All samples were incubated for 18 h at 37 °C, 5% CO2 in a 24 well plate. For analysis of antigen-induced intracellular markers (IFN-γ, TNF-α, and IL-2) 10 μg/mL of brefeldin A (Sigma) was added 6 h postantigen stimulation.
Intracellular cytokine staining/flow cytometry
After stimulation, cells were collected, washed twice with 1X PBS (Thermo Fisher), and stained with fluorescently conjugated monoclonal antibodies from BD Bioscience for extracellular markers CD3 (APC-Cy7, clone SK7), CD4 (BV711, clone SK3), CD8 (BV786, clone RPA-T8) for 30 minutes at 4 °C. Antibodies were used at a dilution of 1:60. To exclude dead cells from analysis, LIVE/DEAD Fixable Aqua Dead Cell Stain (405 nm excitation) (Thermo Fisher Scientific) was added. Afterwards, cells were fixed and permeabilized using the eBiosciences FoxP3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Samples were stained for 30 min at 4 °C using fluorescently conjugated monoclonal antibodies from BD Bioscience for intracellular markers: IFN-γ (BV421, clone B27), TNF-α (PE-CF594, Mab11), and IL-2 (BV650, clone 5344.111). All antibodies were used at a dilution of 1:60. Cells were finally resuspended in 300 μL Flow Cytometry staining buffer (PBS + 1% FCS ice cold) and measured on a BD LSR Fortessa X20 flow cytometer. Compensation was performed using tubes of Ultra Comp eBeads (Thermo Fisher), individually stained with each fluorophore and compensation matrices were calculated with FACSdiva.
Flow cytometric analysis
Flow cytometry data collection was performed with BD FACSDiva (v6) and analysis was performed using the FlowJo software version 10.5.3. Lymphocytes were gated based on FSC verse SSC profile, and doublets were excluded using FSC-A plotted against FSC-Height (FSC-H). Once single cells were selected, the samples were further gated for alive cells, then CD3+ , CD4+ T-cells for analysis of various intracellular markers (IFN-γ, TNF-α, and IL-2). Unstimulated cells were used as controls.
In vitro profiling of peptides displayed by MHC-II after uptake by monocyte-derived dendritic cells (DCs)
The study was performed by ProImmune (Oxford, UK) using the ProPresent Antigen Presentation Assay (see20,21,22,23,24). Monocytes were isolated from the mononuclear cell fraction of peripheral blood samples by positive selection and differentiated in vitro into immature DCs. The immature DCs were: i) Cultured in the presence of SaCas9. ii) Matured in the presence of the same protein. The mature DCs were harvested and lysed. Immune-affinity columns were used to isolate MHC-II-DR molecules from the cell lysate. The MHC-II-bound peptides were eluted and processed for further analysis by high-resolution sequencing-MS (LC-MS/MS). The resulting data were analyzed using sequence analysis software referencing the Swiss-Prot Human Proteome Database with the incorporated test item sequence(s). LC-MS/MS-based identification of peptide sequences was based on scoring algorithms and statistical significance determination. The likelihood of peptides to be real identities is described by their Expect Value (EV) and by the False Discovery Rate (FDR) obtained by searching an unrelated database. The EV considers specific features of the experimental MS/MS spectrum, e.g., the number of peaks that match predicted fragments from a peptide sequence in the database. The EV provides a statistical significance for the peptide identification and it is defined as being reflective of the number of assignments with equal (or better) scores that are expected to occur by chance alone. The lower this value, the higher is the probability the assignment is correct. EV ≤ 0.05 was used to report significant hits as recommended by current guidelines for documentation of peptide and protein identification by mass spectrometry24. A false-discovery rate (FDR) is determined by repeating the search using identical searching parameters against a “decoy” database, where the sequences have been reversed or randomized. The number of matches that are found with the decoy database is an estimate of the number of false positives that are present in the results from the real or “target” database. The FDR can be expressed as the total false positives divided by the sum of true positives and false positives. Identifications reported with EV ≤ 0.05 typically have FDRs < 1%.
Statistical test to determine positive responders
Positive responses were determined using a one-sided Fisher’s Exact Test. For each donor, each gating strategy identified a cell count for each biomarker for each pool for CD4cells. In addition, for each donor, there was a cell count for the unstimulated sample for CD4+ for each gating strategy. In addition, each pool including the unstimulated well had a specific count of total CD4+ cells for that donor/pool (including unstimulated) combination. Using Fisher’s exact test, a p value was calculated for the one-sided hypothesis that the ratio of cells counted to total cells for a particular biomarker were higher than the ratio of cells counted to total cell for the unstimulated values for that Donor/CD cell type. These p values were adjusted for multiple comparisons using the “Holm-Bonferroni” method. Responses with a false-discovery rate < 0.05 were considered positive responders for that cell-type/pool/marker.
Data analysis
Data Analysis was done in R v3.6.1. Base R functions were used for Fisher testing. Graphics were created using ggplot2 (v3.3.0), cowplot (v1.0.0), reshape2 (v1.4.4). Code is available from the corresponding author with a request. Flow Cytometry Data Analysis was performed with FlowJo (v10.5.3). SampPick Software to select a representative donor cohort can be found at https://www.github.com/fda/SampPick.
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
