Figure 3. AAV-mediated expression of rat IL-34 inhibits antidonor cellular responses.

(A) Cells were transduced with AAV–IL-34 or control AAV-GFP at the indicated MOI and analyzed for IL-34 expression 24 hours later (solid thick black line: AAV–IL-34 MOI 10² labeled with anti-Myc; solid thin gray line: AAV–IL-34 MOI 10; dotted black line: AAV-GFP MOI 10² labeled with anti-Myc Ab; and filled gray area: AAV–IL-34 MOI 10² with isotypic control Ab; 1 representative experiment of 4). (B) IL-34 protein was quantified by ELISA in AAV–IL-34 versus AAV-GFP–treated rats 15 days after infection. Serum of nontreated naive animals was used as a control for IL-34 endogenous expression (n = 3). (C) Supernatants of AAV–IL-34 or AAV-GFP–transduced cells were tested for suppression of CD4⁺CD25^– T cell proliferation in response to allogeneic pDCs and analyzed for CFSE dilution after 5 days. CD8⁺ Tregs were used as a positive control for suppression (n = 3 experiments performed in duplicate). Results are expressed as the mean ± SEM of the normalized percentage of proliferation versus proliferation in the absence of CD8⁺ Tregs (100%). Representative histogram is shown. Two-way repeated-measures ANOVA with Bonferroni’s post test versus AAV-GFP–transduced cell supernatant dilution, *P < 0.05. (D) Serial dilution of sera from AAV–IL-34 or AAV-GFP–treated (not shown) rats or naive animals (dotted line) were tested for suppression of CD4⁺CD25^– T cell proliferation in response to allogeneic pDCs and analyzed for CFSE dilution after 5 days. The proportion of dividing CD4⁺CD25^– T cells with pDCs only represented 80% of the cells on day 5 and was given a value of 100 in each experiment. Results are expressed as the mean ± SEM of the relative proportion of dividing CD4⁺CD25^– T cells. CD8⁺ Tregs were used as positive control of suppression (n = 3 experiments performed in duplicate). Two-way repeated-measures ANOVA with Bonferroni’s post test versus serum from naive rats, *P < 0.05, ***P < 0.001.