DC10-induced CD4⁺ regulatory T cell suppression of OVA-specific IgG1-secreting B cell responses.

<p>CD4⁺ lung cells magnetically sorted from lung single cell suspensions generated 3 wk after treatment of asthmatic mice with saline (Sal.) or DC10 were <b>(A)</b> analyzed for expression of the regulatory T cell (Treg) markers CD25 and Foxp3 by FACS. (<b>B)</b> These CD4⁺ T cells, known to be rich in activated allergen-specific Treg, were co-cultured with asthmatic lung cells for 24 h at the indicated B cell:CD4⁺ T cell ratios, and then they were transferred into ELISPOT plates for 5 h to assess their impact on OVA-specific IgG1 B cells. Positive and negative control wells contained lung cells alone as a source of IgG1-secreting B cells, and lung cells that had been co-cultured with CD4⁺ T cells from saline-treated asthmatic animals (sal. CD4), respectively. <b>(C)</b> We also magnetically-sorted CD25⁺Foxp3⁺ Treg from the lungs of DC10-treated asthmatic (DC10-iTreg; inset, 58% CD25⁺Foxp3⁺ cells) mice and assessed their abilities to reduce the IgG1 response as in panel B, setting up the cells at a 1:1 iTreg:lung cell ratio, but also assessing the impact of neutralizing anti-IL-10 and/or -TGFβ antibodies as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190414#pone.0190414.g004" target="_blank">Fig 4</a>. The purified DC10-induced Treg suppressed IgG1 secretion, but neutralization of IL-10 and/or TGFβ had no significant impact on this suppression (p>0.05). (<b>D)</b> We generated DC10-induced Treg <i>in vitro</i>, as noted in the Materials and Methods, and sorted CD25⁺ natural Treg (nTreg) from the lungs and spleens of normal mice, and then injected 1x10⁶ of these cell transtracheally into asthmatic mice. Two days later we generated single cell suspensions of lung and MLN cells from the treated mice and assessed these in 5 h ELISPOT assays for OVA-specific IgG1-secreting B cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190414#pone.0190414.g003" target="_blank">Fig 3</a>. Each bar in the graphs represents the mean (±SEM) of 4 wells. These results are representative of three independent experiments, with n = 3 or 4/group. Statistical analyses were performed using one-way ANOVA assays with Tukey’s <i>post-hoc</i> testing (panels B & C) or <i>t</i>-testing (panel D). *, **, *** and NS signify p<0.05, 0.01, 0.001 and p>0.05, respectively.