Fig. 2: Modular antigen presentation on pAPCs through kinetically driven peptide replacement. | Nature Communications

Fig. 2: Modular antigen presentation on pAPCs through kinetically driven peptide replacement.

From: Polymerised superparamagnetic antigen presenting cell lymphocyte capture for enriching tumour reactive T-cells and neoantigen identification

Fig. 2

A A Schematic illustrating the exogenous peptide exchange process. B The percentage of H2-kb-SIINFEKL+ pAPCs following incubation in various concentrations of SIINFEKL for 5 min. P values from left to right, ****p < 0.0001, ***p = 0.0002, ***p = 0.0003, **p = 0.0039 (Independent biological replicates n = 3). C Flow cytometric analysis of H2-Kb SIINFEKL+ pAPCs following incubation with 2 mg/mL SIINFEKL at incubation time from 1 to 15 min. D A schematic illustrating antigen presentation by live APCs or pAPCs following antigen pulsing. E Live APCs were pulsed with ovalbumin, OVA248–274 (27 amino acids), or SIINFEKL peptide for 4 h. For transfection, live APCs were transfected with SIINFEKL-containing plasmid for 48 h. Each group underwent flow cytometric analysis to characterize H2-Kb SIINFEKL+ cells. P values from left to right, ****p < 0.0001, ****p < 0.0001, ****p < 0.0001, NS p = 0.8382 (Independent biological replicates n = 3). F SIINFEKL antigen presentation over a 48 h period for SIINFEKL-pulsed live APC and SIINFEKL-loaded pAPCs. Live APCs were pulsed with 1 µg/mL of SIINFEKL for 4 h and pAPCs were incubated with 2 mg/mL of SIINFEKL for 5 min prior to incubation. P values, ****p < 0.0001 (Independent biological replicates n = 3). G A schematic illustrating the experimental design for assessing peptide exchange efficiency on pAPCs with MHC binders of low binding affinity. pAPCs were pre-loaded with SIINFEKL as a model strong binder. The extent of SIINFEKL peptide replacement upon incubation with various concentration of H PB1703-711 or I E648-57 peptides. The replacement efficiency was determined by the proportion of H2-kb-SIINFEKL- cells. P values in (H) from left to right, ****p < 0.0001, ****p < 0.0001, ***p = 0.0002. P values in (I) from left to right, ****p < 0.0001, ****p < 0.0001, **p = 0.004 (Independent biological replicates n = 3). J–L Validation of pAPC antigen presentation through a T cell activation assay via flow cytometric analysis of CD69 levels. OT-I specific T cells derived from OT-I transgenic mice and splenocytes derived from PB1703–711-vaccinated mice were incubated with SIINFEKL-loaded pAPCs or pAPCs following PB1703-711 peptide replacement for 24 h prior to analysis of T cell activation. P values in (K) from left to right, ****p < 0.0001, ****p < 0.0001. P values in (L) from top to bottom, *p = 0.0106, ***p = 0.0003, NS p = 0.5813 (Independent mice replicates n = 3). The error bars represent mean ± standard deviation. Statistical analyses were performed by two-sided Student’s t tests.

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