s+16a nanobody does not directly alter IFN-γ production ex vivo.

Wild type B6 mice were either naïve or infected with 200CFU TAS2010 i.v. At week 5 p.i., splenocytes were harvested and re-stimulated ex vivo in the presence of indicated concentration of s+16a nanobody at 2×10⁶ cells per well in 200μl (n = 4). The nanobody was present during the entirety of the re-stimulation protocol, either with (A) PMA and ionomycin for 4h with brefeldin A, or (B) a pool of Salmonella peptides (5pept, as per Fig 5) for 18 h with brefeldin A added in the final 4 h. At the end of the incubation period, re-stimulated cells were intracellularly stained for IFN-γ. No statistically significant difference (one-way ANOVA with Bonferroni post-tests) was observed between re-stimulated cells treated with different concentration of s+16a nanobody. (C) IFN-γ-eYFP^in/in mice were infected with 200CFU TAS2010 i.v. for 12 weeks. Shown is the geometric mean fluorescence intensity (gMFI) of IFN-γ-eYFP in both CD69⁺ and CD69^- CD4⁺ T cells in mice that were pre-injected with either 50μg of s+16a nanobody or equal volume of PBS 15-20min immediately prior to euthanasia and tissue collection (n = 6–8). The liver was then perfused with PBS to remove circulating cells. No statistically significant difference (Student t-test) was observed between s+16a or PBS pre-injected mice. Symbols represent data from individual mice, mean±SEM shown.

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