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. 2024 Dec 6;9:241. doi: 10.1038/s41541-024-01040-6

Fig. 4. Identification and tracking of T cell responses against the P36 and P52 candidate peptides.

Fig. 4

a Schematics of peptide pooling and screening strategy. For identifying the candidate immunogenic peptides from the P36 or 52 peptide library, BALB/cJ mice were gene gun primed with DNA encoding P36 or P52 on days 0 and 2. Mice were later boosted again with P36 or P52 on day 14 or 28. Finally, mice were sacrificed 14 days post-boosting and splenocytes were subjected to IFN-γ ELISPOT. b Individual mice ELISPOT data for an identified peptide from P36 peptide library (Peptide 71). c Individual mice ELISPOT data was compiled for three identified P52 peptide pools (Pool A, Pool B, Pool C). d BALB/cJ mice were gene-gun primed with protective cocktails of DNA encoding both CSP plus either P36 or P52 at day 0. Mice were later received a reactivation dose of 2 K Py-RAS followed by spleen harvest for IFN-γ ELISPOT of Peptide 71 and Pool A*, Pool C. e Number of IFN-γ positive spots per million splenocytes for P36 Peptide 71, P52 Pool A* and P52 Pool C. f, g Splenocytes from the mice of panel d were in vitro stimulated either with P36 Peptide 71 for CSP + P36 immunized group (f) or with P52 Pool A* and P52 Pool C for CSP + P52 immunized group (g) and Tem/e population among both CD4+ (i) and CD8+ T (ii) cells were tracked for IFN-γ production. For ELISPOT results are normalized against wells treated with equivalent volume of DMSO. For flow cytometry fluorescence minus one (FMO) control was used to gate on the IFN-γ expressing CD8+ and CD4+ T cells (S Fig. 2, panel d). To quantify T cell activation following antigen stimulation, peptide antigen(s) were not added to unstimulated wells. Frequency of T cells activation following either P36 or P52 antigen stimulation was compared with the column graph on right side of each panel using the marker IFN-γ. N = 3-4 mice per group for figures (b, c). N = 5 mice per group for panels d–g. Data are representative from two or three independent experiments. Data are the mean ± SEM. Data were analyzed by the Mann-Whitney test. P < 0.05 is considered significant. * P < 0.05, ** P < 0.01. Panel 4a made with Biorender.