Fig. 4: The CD103-targeted adjuvant effectively primes Th17 cells following i.n immunizations. | Mucosal Immunology

Fig. 4: The CD103-targeted adjuvant effectively primes Th17 cells following i.n immunizations.

From: ADP-ribosylating adjuvant reveals plasticity in cDC1 cells that drive mucosal Th17 cell development and protection against influenza virus infection

Fig. 4

Adoptive transfer i.v of CFSE-labeled OT II TCR Tg CD4 T cells (CD45.1+) to WT C57Bl/6 mice (CD45.2+) was followed by a single i.n immunization with 5 μM OVA, CTA1-II-DD or CTA1-II-CD103 the next day (equimolar doses of p323). On day 5 mice (n = 5) were analyzed for OT II cell proliferation in the mLN (mediastinal lymph node) by assessing the dilution of CFSE-label by flow cytometry. The enzymatically inactive CTA1R9K-II-CD103 fusion protein was evaluated for its adjuvant activity. The frequency of expanding OTII cells of all CD4 T cells is given as mean % ±SD of 3–5 mice in each group and pooled data from three independent experiments is shown. a The ability to prime OT II CD4 T cells in the draining mLN at different days post-immunization was assessed as in a. OTII cells were injected i.v. on days 1, 4, or 8 after i.n immunizations with the fusion proteins and values are given as means ±SD of 5 mice in each group (n = 2) (b). The differentiation into Th17 or Th1 OT II cells in the mLN following i.n immunization is given as the frequency of rorγt+, CCR6+ or Tbet+ of all OT II CD4 T cells. Values in each category are given as mean % ±SD of all OT II cells, as indicated, and these are representative of three independent experiments with 2–6 mice in each group (c). IL-17A and IFNγ-specific ELISPOT analysis of OT II CD4 T cell recall responses to p323 peptide was performed in triplicates by freshly isolated mLN cells following a single i.n immunization of the respective fusion protein.Values are given as mean±SD SFC/105 cells of three independent experiments with 3-5 mice per group (d). Statistical significance was calculated using ANOVA with Dunnett’s posttest (a, b) or Student’s t test (c). p values are given as; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Finally, OT II cells from i.n immunized mice were subjected to a total RNAseq analysis using pooled samples from mLN from three mice. The heat map shows relative expression of Th17-relevant genes (rorc, rora, IL-17a, IL-22, Ccr6) in the different groups; OVA, CTA1-I/II-CD103 and CTA1-I/II-DD. This is a representative analysis of two independent experiments giving similar results (e).

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