Journal: The Journal of Experimental Medicine

Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice

doi: 10.1084/jem.20212443

Figure Lengend Snippet: The reduced IL-10 production in IL-24–deficient Th17 cells is a cell-intrinsic property. (a) Gene dose effect through titrating down the genetic availability of Il24 product directly impacts IL-10. Naive T cells from Il24 +/+ , Il24 +/− , and Il24 −/− mice were cultured for 3 d under Th17 conditions and then subjected to intracellular cytokine staining for IL-17 and IL-10. Representative flow cytometry plots (left) and quantification of relative fractions of cells that were either single- or double-positive for the indicated cytokines, normalized to Il24 +/+ levels for each population (right), summarizing 10 independent experiments (mean ± SEM). Relative fractions were derived by dividing the frequency of the indicated cytokine-expressing subset (among CD4 + cells) by the corresponding frequency of the Il24 +/+ (wild-type) genotype for each experiment. Therefore, for each subset, the relative fraction for Il24 +/+ itself was defined as 1, while a relative fraction lower than 1 indicates a decrease of that particular subset compared with the wild-type population in a given genotype and vice versa. Asterisks indicate significance level of Dunnett’s multiple comparison test following two-way ANOVA (****, P < 0.0001). (b and c) Acute ablation of IL-24 reduces IL-10 expression in Th17 cells. (b) Naive Cas9 transgenic T cells were cultured in Th17 differentiation conditions. After retroviral transduction with gRNAs targeting Il24 or control (directed at the GFP transgene) on day 1, Th17 cells were analyzed for intracellular IL-10 on day 3. Representative FACS plots (left, pregated for viable and Thy1.1 + transduced CD4 + cells) and summary of four independent experiments (mean ± SEM) normalized to control (right). (c) Naive T cells from wild-type mice were cultured in Th17 differentiation conditions. Th17 cells were treated with siRNA targeting IL-24 or control siRNA (Ctrl) on day 1 and analyzed for intracellular IL-10 on day 3. Representative FACS plots (left, pregated for viable CD4 + cells) and summary (mean ± SEM) of four independent experiments (right). (b and c) Relative fraction indicates IL-10 + frequency (among CD4 + cells) for the respective treatment group divided by the corresponding IL-10 + frequency of the control group ( gfp guide and control siRNA, respectively). Asterisks indicate significance level of two-tailed t tests (****, P < 0.0001; **, P < 0.01). (d and e) Absence of IL-24 receptors does not impact IL-10. Naive T cells from wild-type, Il20rb −/− , and Il24 −/− mice were cultured for 3 d under Th17 conditions and then subjected to (d) intracellular cytokine staining for IL-17 and IL-10 with representative cytometry plots (left, pregated for viable CD4 + T cells) and summary (mean ± SEM) of five independent experiments (right, indicated as relative fractions for each subset normalized to wild-type, as described above), as well as (e) RT-qPCR analysis for Il10 expression, summarized from seven independent experiments with data normalized to wild-type expression levels (mean ± SEM). Asterisks indicate significance level (****, P < 0.0001; *, P < 0.05) of Sidak’s multiple comparison test (d) and Tukey’s multiple comparison test (e). (f) Il10 expression is impervious to external IL-24. Naive T cells from Il24 +/+ and Il24 −/− mice were cultured for 3 d under Th17 conditions in the presence or absence of exogenous murine or human recombinant IL-24, or anti–IL-24 antibodies from rat or goat or their respective isotype controls, followed by RT-qPCR for Il10 . Summary of two independent experiments (mean ± SEM). (g) Availability of natively secreted IL-24 does not impact Il10 expression in coculture. Naive T cells from congenically marked Il24 +/+ and Il24 −/− as well as from mixed bone marrow chimeric mice with 1:1 ratio of the two compartments were cocultured for 3 d under Th17 conditions and then sorted apart for individual RT-qPCR analysis of Il10 expression. Summary of seven independent experiments (mean ± SEM). In panels f and g, asterisks indicate significance levels of Sidak’s multiple comparison test (****, P < 0.0001; ***, P < 0.001).

Article Snippet: The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

Techniques: Cell Culture, Staining, Flow Cytometry, Derivative Assay, Expressing, Transgenic Assay, Transduction, Two Tailed Test, Cytometry, Quantitative RT-PCR, Recombinant