Cell counting on the MLNs 48 hr after the last OVA challegenge (a) and 72 hr after the second sensitization (b)

Cell counting on the MLNs 48 hr after the last OVA challegenge (a) and 72 hr after the second sensitization (b). Physique S4. Representative circulation cytometry gating strategy for Tfh phenotyping in the spleen. Populace selection, SSC x FSC (a), CD3+CD4+CXCR5+PD1+ Foxp3+ gating strategy (b). Physique S5. Representative circulation cytometry gating strategy for Th phenotyping in the MLNs and spleen. Populace selection, SSC x FSC (a). Representative CD3+CD4+IL4+/GATA3+ gating strategy in the MLNs (b). Representative CD3+CD4+IL17A+/RORT+ gating strategy in the MLNs. IMM-157-268-s001.pdf (851K) GUID:?7928B188-F693-4CD0-BAAE-CC999A05D7BC Summary Asthma and obesity present increasing incidence, and their concomitance is usually a reason for concern, as obese individuals are usually resistant to standard asthma treatments and have more exacerbation episodes. Obesity affects several features in the lungs during asthma onset, shifting the T helper type 2 (Th2)/eosinophilic response towards a Th17/neutrophilic profile. Moreover, those individuals can present reduced atopy and delayed cytokine production. However, the impact of obesity on follicular helper T (Tfh) cells and B cells that could potentially result in antibody production disturbances are still unclear. Therefore, we aimed to assess the peripheral response to ovalbumin (OVA) in a concomitant model of obesity and asthma. Pulmonary allergy was induced, in both slim and obese female BALB/c mice, through OVA sensitizations and difficulties. Mediastinal lymph nodes (MLNs) and spleen were processed for immunophenotyping. Lung was utilized Cinnamic acid for standard allergy analysis. Obese\allergic mice produced less anti\OVA IgE and more IgG2a than slim\allergic mice. Dendritic cells (CD11c+?MHCII high) expressed less CD86 and more PDL1 in obese\allergic mice compared with lean\allergic mice, in the MLNs. In the mean time, B cells (CD19+?CD40+) were more frequent and the amount of PDL1/PD1+ cells was diminished by obesity, with the opposite effects in the spleen. Tfh cells (CD3+?CD4+?CXCR5+?PD1+) expressing FoxP3 were more frequent in obese mice, associated with the predominance of Th (CD3+?CD4+) cells expressing interleukin\4/GATA3 PRP9 in the MLNs and interleukin\17A/ROR (phycoerythrin\Cy7 clone: XMG1.2), anti\IL\17 (Alexa 488 clone: TC11\18H10); anti\FOXP3 (Alexa Fluor 488 clone: MF23), anti\CD11c (BV 510 clone: HL3), anti\MHC\II (allophycocyanin clone: AMS 321), anti\CD80 (fluorescein isothiocyanate clone: 16\10A1). Samples were fixed in paraformaldehyde after labeling. Data acquisition was performed in FACS\Canto II (BD) and analyzed on flowjo (TreeStar, Ashland, OR). Statistical analysisData were analyzed using graph pad prism 6.0 (San Diego, CA). Numerical data were analyzed using KolmogorovCSmirnov normality test. Weight gain analysis along time was performed with two\way analysis of variance, coupled with Tukey’s multiple comparison test. For four\column graphs, parametric data were analyzed with one\way analysis of variance, Cinnamic acid followed by Bonferroni post\test; for non\parametric data a KruskalCWallis test was performed, followed by Dunn’s test. For two\column graphs, parametric data were analyzed using unpaired Student’s contamination.47 There is crosstalk between B cells and DCs during antigen presentation and activation of T naive cells, promoting Cinnamic acid their differentiation into the different T\cell profiles. Among them, Tfh cells are polarized by IL\21 and IL\6 stimuli, resulting in their increased CXCR5 expression, a chemokine receptor for CCL13, expressed in the germinal Cinnamic acid centers.8, 48 Tfh cells mutually interact with B cells contributing for high\affinity antibody production in plasma cells.49 Additionally, recent studies have exhibited that specific regulatory B cells dampen Tfh differentiation through their elevated PDL1 expression in the B\cell marginal zone,27, 50 our data demonstrate this function, as the higher frequency of PDL1high B cells was accompanied by a reduced differentiation of Tfh cells in the spleen. However, in the MLNs the low frequency of regulatory B cells (CD19+?CD40+?PDL1high) did not result in a higher Tfh cell differentiation, possibly due to the regulatory profile of the interacting\DCs (CD11c+?MHCII+?PDL1+). In fact, it has already been exhibited that Tfh cell differentiation is dependent on CD86 co\activation by DCs.51 Despite the higher quantity of splenic Tfh cells, they presented reduced PD1 expression. Although we have not evaluated the function of these cells, it has already been exhibited that Tfh cells require PD1 expression to maintain cytokine production and their normal function.52 Hence, the impaired PD1 expression in Tfh cells could be.