Fig 1: TAC-OVA suppresses endogenous immune responses to OVA challenge irrespective of the spleen. (A-G) C57BL/6 mice were untreated or treated with TAC squeezed with PBS (Empty TAC) or OVA (TAC-OVA) on days -7 and -4. Mice were immunized on day 0 with CFA and OVA. 7 days later, immune responses were assessed in the draining LNs. (B) Number of IL-2 and (C) IFNγ producing cells were assayed by ELISpot after restimulation with (B) OVA protein or (C) SIINFEKL peptide. B-C are n = 3 for naïve and n=5 for other groups. Secretion of (D, E) Th1 and (F, G) Th17 associated cytokines after OVA protein restimulation. (D–G) show n=4 pools of 2 mice each per group. (H-J) C57BL/6 mice underwent sham surgery or splenectomy. Mice were either untreated or treated with TAC-OVA and challenged with OVA in CFA. OVA-specific immune responses were assessed in the draining LNs after 7 days by ELISpot. (I) IFNγ and (J) IL-2 producing cells after restimulation with (I) SIINFEKL peptide or (J) OVA protein. (I-J) n = 4-5 per group. (A-J) TAC dose was 1x109 and OVA squeeze concentration was 100μM. Mean +/- SD. Mann-Whitney U Test. Representative of at least 2 independent experiments.
Fig 2: Acidity inhibits T-cell function. Assessment of T-cell proliferation. (A) Representative histograms and summary data showing CTV dilution of stimulated OT-I T cells at 72 h in pH 7.4 vs. 6.6 media. (B) Thymidine incorporation in pH 7.4 and 6.6 at 72 h post-stimulation. (C) Assessment of T-cell migration. Migration of OT-I T cells in pH 7.4 vs. 6.6 media according to CXCL10 serial dilution and flow cytometry staining of CXCR3 on CD8+ T cells 2 h post-incubation at pH 7.4 vs. 6.6. Two experiments were performed and each experiment contained two biological replicates for each CXCL10 concentration. (D) Representative histograms and summary graph of flow stained pmel T cells at 24, 48, 72 and 96 h post-stimulation for the activation markers CD25 and CD69 in pH 7.4 vs. 6.6 media. (E) Supernatant from stimulated pmel T cells analyzed via LEGENDplex for secreted IL-2, TNF-α, and IFN-γ levels after 24, 48, 72, and 96 h in pH 7.4 vs. 6.6 media. (F) Representative dot plot and summary graph of stimulated OT-I T cells in pH 7.4 vs. 6.6 media stained for intracellular IFN-γ (p < 0.0431). Assessment of T-cell reactivity via IFN-γ production. (G) Summary data showing the coculture of OT-I T cells at a 10:1 ratio with either MB49OVA, IFN-γ-pretreated MB49OVA, MB49, or IFN-γ-pretreated MB49 in pH 7.4 vs. 6.6 media. Results are one of two to four independent experiments, n = 2–5. Analysis by independent t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
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