Fig. 2: The CD103-targeted CTA1-adjuvant acts through cross-presenting cDC1 cells effectively priming CD8 T cells and CTLs. | Mucosal Immunology

Fig. 2: The CD103-targeted CTA1-adjuvant acts through cross-presenting cDC1 cells effectively priming CD8 T cells and CTLs.

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

Fig. 2

C57Bl/6 mice were i.n. administered with 5μM of OVA, CTA1-I/II-DD or CTA1-I/II-CD103, and 20 h later the frequency of migrating CD103+ CD11b− cDC1 cells expressing SIINFEKL-peptide + MHC I complexes in the mLN was assessed using flow cytometry and a labeled complex-specific Mab and an isotype control Mab. MFI values are given as means±SD of three independent experiments (a). An adoptive transfer model with CFSE-labeled OT-I TCR Tg donor CD8 T cells (CD45.1+) injected into WT C57Bl/6 (CD45.2+) mice followed by a single i.n priming immunization with an equimolar dose (SIINFEKL peptide) of ovalbumin (OVA), CTA1-I/II-DD or CTA1-I/II-CD103. At 4 days post-immunization CD45.1+ OT I CD8 T cell proliferation was assessed in freshly isolated mLN cells by flow cytometry. The CFSE-dilution profiles were determined and values are given as the mean frequency ± SD of proliferating OT I cells of all CD8 T cells, proliferation index, and frequency of responding OT I CD8 T cells, as indicated (see M & M section) Representative dot-blots of three independent experiments giving similar results and values are given as means ± SD of 3–5 mice in each group (b). Determination of SIINFEKL-specific IFNy-ELISPOTs in OT I CD8 T cells stimulated with recall ovalbumin at 5 days following i.n immunizations with the fusion proteins. Values are given as means ± SD of 5 mice in each group and one representative experiment of three is shown (c). Induction of CTL responses in C57BL6 mice following i.n. immunization with a 5 μM dose of OVA, CTA1-I/II-DD, CTA1-I/II-DD, or CTA1- II-CD103 (control w/o SIINFEKL peptide) fusion protein. After 1 week mice were injected i.v. with CFSE-labeled splenocytes pulsed with 1 μM SIINFEKL peptide (Target cells) or un treated control splenocytes (Bystander cells). After 20 h freshly isolated mLN (mediastinal lymph node) cells were analyzed by flow cytometry for specific lysis of peptide expressing target cells . Values are means ± SD of two independent experiments giving similar results with 2–3 mice per group (d). Statistical significance was calculated using ANOVA with Dunnett’s post-test (a, b, c) or Student’s t test (d). ns; not significant; p values *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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