Production of IL-17A and IL-17F protein in BT co-cultures requires B cell and CD4 T cell interaction.

<p>Measurement of IL-17A (A) and IL-17F (B) by ELISA in supernatants from 25,000 B cells/well co-cultured with 25,000 PBMC/well and stimulated with α-IgM and SAg for three days. Measurement of IL-17A (C) and IL-17F (D) by ELISA in supernatants from 25,000 B cells/well and 25,000 CD4 T cells/well cultured alone or 25,000 B cells cultured with increasing numbers of CD4 T cells in the presence of α-IgM and SAg or carrier (Control) for three days. Data are the means ± standard deviation of three B cell and CD4 T cell donor pools and two independent experiments.</p

SR2211, an RORγ inverse agonist, and Calcitriol, a Vitamin D3 receptor agonist, block production of IL-17A and IL-17F in BT co-cultures.

<p>Profiles of SR2211 (0.370–10 μM; A) or Calcitriol (0.15–4.1 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured (B cell Proliferation, PBMC Cytotoxicity, Secreted IgG, sIL-17A, sIL-17F, sIL-2, sIL-6, and sTNF-alpha) are indicated along the x-axis. Data are presented as the Log₁₀ ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.</p

Summary of well-characterized compound targets that preferentially regulated production of both IL-17A and IL-17F, or had selectivity for IL-17A or IL-17F^a.

a<p>Data from a screen of 144 pharmacologic modulators are summarized to show compound classes and targets that regulated production of both IL-17A and IL-17F or had selectivity for IL-17A or IL-17F in BT co-cultures. The number of agents with selectivity for a specific target is shown in parentheses next to the total number of agents in the screen for that target. Class/targets were only included if greater than 50% of agents specific for a class/target had effects on IL-17A and/or IL-17F. Agents that inhibited both IL-17A and IL-17F, or preferentially inhibited IL-17A or IL-17F had readout values less than log₁₀ ratio −0.2 and were observed at 2 or more non-cytotoxic doses. *Agents against these targets selectively inhibited IL-17A or IL-17F at 2 or more relatively low doses and inhibited both IL-17A and IL-17F at 2 or more higher doses.</p

PGE1 and PGE2 stimulate production of IL-17A and impair production of IL-17F.

<p>Profiles of PGE1 (0.37–3.33 μM; A) and PGE2 (0.37–3.33 μM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. PGE1 and PGE2 increased production of IL-17A and IL-6, but inhibited IL-17F, IL-2, and TNFα production. Data are presented as the Log₁₀ ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.</p

IL-17A and IL-17F are predominantly expressed by CD4 T cells in a BT co-culture model of human B cell-dependent T cell responses.

<p>FACS analysis gating strategy for cell types present in BT co-cultures after stimulation for three days with α-IgM and SAg (A). Gated cell populations are listed above the FACS plots and the percentage of cells present in each gate relative to the parent population is shown. To detect intracellular cytokine expression, BT co-cultures were treated for 5 hours with PMA, ionomycin, and monensin, and then stained with antibodies specific to IL-17A (A), IL-17F (B), or with an isotype control antibody common to the isotype of α-IL-17A and α-IL-17F (C).</p

Erythromycin and Wortmannin at select doses inhibit IL-17A and IL-17F without modulating other parameters.

<p>Profiles of erythromycin (3.3–90 μM; A) or wortmannin (1.5–13.7 nM; B) added to BT co-cultures stimulated with α-IgM and SAg for three days. Erythromycin inhibited IL-17A and IL-17F production but did not affect B cell proliferation, secreted IgG, IL-2, IL-6, or TNFα, whereas wortmannin selectively inhibited IL-17A and IL-17F only at lower doses. Data are presented as the Log₁₀ ratio of treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.</p

Torin-1, CP-690,550, and Axitinib are examples of compounds that regulated production of IL-17A, IL-17F or both IL-17A and IL-17F.

<p>Profiles of Torin-1 (0.460–12.3 nM; A), CP-690,550 (0.041–1.11 μM; B), or Axitinib (0.333–9 μM; C) added to BT co-cultures stimulated with α-IgM and SAg for three days. Parameters measured are indicated along the x-axis. Torin-1 inhibits IL-17A more potently than IL-17F (A), whereas CP-690,550 inhibits IL-17F more potently than IL-17A (B). Similarly, Axitinib blocks IL-17F production but does not affect IL-17A (C). Data are presented as the Log₁₀ ratio of drug-treated stimulated cells compared to control stimulated cells. The gray area above and below the y-axis origin indicates the 95% significance envelope for control samples based on historical data.</p

IL-17F is the most strongly induced gene in BT co-cultures after three days of stimulation in a model of T cell-dependent B cell activation^a.

a<p>The top ten induced genes as determined by microarray analysis in BT co-cultures stimulated with α-IgM and SAg for three days compared to the same cells co-cultured for three days without stimulation. Data are from 3 independent replicates with 3 different donor pools.</p

CD4 T cells increase expression of several Th17-associated genes after three days of stimulation in a model of T cell-dependent B cell activation^a.

a<p>The top ten induced genes as determined by quantitative RT-PCR in CD4 T and B cell populations purified by FACS from BT co-cultures stimulated for three days with α-IgM and SAg stimulation. Gene expression values (2^−Ct) were normalized to b-actin gene expression and presented as the fold change average ± standard deviation of cells sorted from 3 independent donor pools of BT co-cultures compared to cells isolated from non-stimulated BT co-cultures.</p

Summary of compound targets that stimulated production of IL-17A and/or IL-17F^a.

a<p>Data from the screen summarized to show compound targets that increased production of both IL-17A or IL-17F, or had selectivity for IL-17A or IL-17F in BT co-cultures. Effects of agents on IL-17A and/or IL-17F had readout values greater than log₁₀ ratio 0.15 and were observed at 2 or more non-cytotoxic doses. The number of agents with selectivity for a specific target is shown in parentheses next to the total number of agents in the screen for that target.</p