Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4+ T-cells

Figures

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  Fig. 1. SaCas9 specific peptide pools and their T-cells responses in North American population. a, A comparison of allele frequencies in the North American population (blue), the cohort of donors selected by the SampPick algorithm for the flow cytometry assay (red), and the donors included in the MAPPs assay (green). b, Responses of CD4+ T-cells obtained from 21 donors to peptide pools from SaCas9 and three full-length Cas9 proteins are shown. The three Cas9 proteins are SaCas9 from Editas (Ed-Sa), SpCas9 from Editas (Ed-Sp) and control SpCas9 from a commercial vendor (CO-Sp). Significant responses were identified using a one-sided Fisher’s exact test comparing the cell counts for IFN-ƴ (red), TNF-α (blue), and IL-2 (green) in CD4+ T-cells as compared to unstimulated samples. P values were adjusted according to the Bonferonni-Holm method. c, Percent of donors responsive to each of the peptide pools and Sa- and SpCas9 proteins (see b). Donors were considered responders if at least one of the three cytokines (IFN-ƴ, TNF-α, or IL-2) were significantly higher than cell counts for the unstimulated samples.

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  Fig. 2. Differences in T -cell activation across the donors. a The number of peptide-pools that induced CD4+ T-cell activation in PBMCs from each donor is shown. The peptide pools that elicit responses are color coded by peptide-pool number. b Heatmap showing the Log fold change for each biomarker compared to unstimulated controls (i.e., cells were not exposed to any Cas9 protein or peptide) in CD4+ cells.

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  Fig. 3. Identification MHC I and MHC-II-mediated T -cell immune responses after peptide stimulation. a Upper panels, MHC-II alleles that are associated with CD4+ activation. An MHC was deemed to be associated with a T-cell response if cells from at least one donor with that MHC allele was activated by a peptide pool. b Upper panels, MHC-II alleles that are associated with CD4+ activation selected based on the rule that an MHC allele is associated with T-cell activation only if all donors with that allele have a positive response for a peptide pool. Binary plots show which MHC alleles are considered to be responders to each pool. a, b Lower panels. Promiscuity plots for data presented in the upper panels. The promiscuity plot shows the number of MHC variants each peptide pool is associated with, weighted for the frequency with which each allele occurs in the NA population.

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  Fig. 4. Peptides Identified in a MAPPs Assay. a Peptides identified in a MAPPs assay are shown with their positions on the SaCas9 protein (depicted on the X-axis). The peptides identified are shown individually for each of 18 donors. The HLA-DRB1 alleles associated with the donors are depicted on the Y-axis. The peptides are stacked to show multiple peptides detected at each position on the Cas9 sequence for each donor. b The number of unique, continuous SaCas9 peptides detected on DCs from each donor. c Results of the MAPPs assay (colored lines) are overlayed on the results of the flow cytometry-based T-cell proliferation assay (pink areas). We assumed that donors were a match if they shared at least one HLA allele.