Fig. 4

Inhibition of anti-AAV8 capsid cellular and humoral responses with SVP[Rapa] co-administration. a CD8 T cell infiltrates in the liver. Livers from animals treated in Fig. 1a were collected after killing on day 53 and evaluated for CD8 mRNA expression by quantitative PCR using the ΔΔC_t method relative to housekeeping gene and to average of untreated mice. b–d Male C57BL/6 mice were treated with 4 × 10¹² vg kg⁻¹ of AAV8-VP1 vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, spleens were collected for B and T cell assays. b Analysis of T cell recall responses after overnight stimulation with an AAV8 peptide pool in splenocytes measured by IFN-γ ELISpot and c anti-AAV8 IgG and IgM secreting B cell responses in splenocytes measured by B ELISpot. d Frequency of B220⁺ CD19⁺ B cells in spleens measured by flow cytometry. e–i Male C57BL/6 mice were treated with 4 × 10¹² vg kg⁻¹ of AAV8-luc vector together with SVP[Rapa] or with SVP[empty] control. 14 days later, animals were sacrificed. e Gating of germinal center (GC) B cells (CD95⁺ GL7⁺) are shown in representative flow cytometry plot. Cells were gated on B220⁺IgD⁻ cells. Shown is a mouse from the SVP[empty] control group. f Frequency of GC B cells (CD95⁺ GL7⁺) in lymph nodes of treated mice determined by flow cytometry as shown in e; g frequency of CD25⁺ FoxP3⁺ regulatory T cells in lymph nodes; h frequency of CXCR5⁺ PD1⁺ Foxp3⁺ follicular regulatory T (Tfr) cells and i frequency of CXCR5⁺PD1⁺FoxP3⁻ follicular helper T (Tfh) cells in lymph nodes. SVP[Rapa] treatment consisted of 200 µg of rapamycin. Data are shown as mean ± s.d. Statistical analyses were performed by one-way ANOVA with Tukey post hoc test in a and by unpaired, two-tail t-test in b–d, f–i (n = 5 in a–d, n = 20 in f–i. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)