Bars represent the geometric mean fluorescence intensity of chemokine receptors CXCR5, CXCR3, and CCR6; error bars represent SD. with the progression of asymptomatic illness to Chagas heart disease. Asymptomatic individuals are known to develop balanced Th1/Th2/Th17/Treg adaptive immune reactions, with effector cells generating proinflammatory cytokines such as IFN-, TNF- and IL-17, and regulatory cells generating IL-10, while individuals with the cardiac form of chronic Chagas disease show a mainly proinflammatory cytokine profile with limited production of IL-10 (Gomes et al., 2003; Vitelli-Avelar et al., 2008; Dutra et al., 2014). Numerous B cell problems have also been reported in Tfh cells. As for Th1, Th2 and Th17 cells (Acosta-Rodriguez E et al., 2007), the chemokine receptors CXCR3 and CCR6 have been used to define functionally distinct cTfh cell subsets (Morita et al., 2011; Schmitt et al., 2014). In humans, CD4+CD45RO+CXCR5+CXCR3+CCR6- (cTfh1) cells are known to Rabbit Polyclonal to NDUFA4 secrete mostly IFN-, while CD4+CD45RO+CXCR5+CXCR3-CCR6- (cTfh2) cells secrete IL-4, IL-5, IL-13 and IL-21, and CD4+CD45RO+CXCR5+CXCR3-CCR6+ (cTfh17) cells secrete mainly IL-17, IL-21, and IL-22. Dysregulation of the cTfh cell compartment has been reported in autoimmune diseases, cancer and infections associated with abnormal B cell responses (Boswell et al., 2014; Ueno et al., 2015). However, whether Tfh cells contribute to the generation of the abnormal B cell responses observed during contamination remains to be elucidated (Minoprio et al., 1988; Fernndez et al., 2014). The aim of this work was to provide an insight in the adaptive immune response elicited by in humans. To this end, various peripheral blood CD4+CD45RO+CXCR5+ cell subsets were assessed in adult patients with different clinical forms of chronic Chagas disease. The results showed growth of CCR6+ cTfh cells and decreased cTfh2 cells in infected patients, regardless of the clinical status of the disease. Other phenotypic changes observed include growth of cTfh17 cells and decreased cTfh1 cells in asymptomatic patients -but not in patients with chagasic dilated cardiomyopathy-, which suggest that dysregulation of Tfh cells might contribute to Chagas disease progression. Materials and Methods Study Population A total of 31 adult participants were enrolled at the Clinical Facilities of Instituto HG-9-91-01 Nacional de Parasitologa Dr. Mario Fatala Chabn, Buenos Aires. The clinical status of enrolled individuals was HG-9-91-01 determined by physical examination and clinical tests, including serologic testing for contamination, electrocardiography, chest radiography, and echocardiography. Individuals who had positive serology for were grouped according to the classification of Kuschnir et al. (1985) into: (a) patients with no clinical manifestations of heart disease, a normal electrocardiogram and conserved cardiac silhouette in chest X-rays and echocardiogram (ASYMP; Kuschnir group 0; = 12; mean age SD = 48.83 12.37 years) and (b) patients with chronic Chagas dilated cardiomyopathy (CCC; Kuschnir group 2; = 5; mean age SD = 48.4 7.50); clinically healthy individuals with a negative test for contamination (CTRL, = 14; mean age SD = 41.71 13.36) served as noninfected controls. Age of controls was matched as much as made possible to that of the infected individuals for each assay. A signed informed consent was obtained from all participants. All the research was conducted in accordance with the Declaration of Helsinki and the Council for International Businesses of Medical Sciences (CIOMS). The study protocol was approved by the HG-9-91-01 Review Board of Instituto Nacional de Parasitologa Dr. Mario Fatala Chabn, Administracin Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbrn (IRB00006651). Isolation of PBMC and Flow Cytometry HG-9-91-01 Assays Freshly isolated peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation on Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) and stained with mAbs against CD4-FITC (Biolegend), CD45RO-PerCp-Cy5.5 (Biolegend), CXCR5-APC (eBioscience), CCR7-PE-Cy7 (BD Pharmingen), PD1-Pac Blue (Biolegend), CXCR3-PE-Cy7 (Biolegend), CCR6-PE (Biolegend), CD19-PE-Cy7 (BD Pharmingen), IgD-PE (BD Pharmingen), and IgG-APC (BD Pharmingen), following the instructions of manufacturers. Cell viability was monitored in all samples by incorporating Fixable Viability Dye e-Fluor 780 (Invitrogen, Carlsbad, CA, USA) to HG-9-91-01 the stains. At least 200,000 events per sample were collected at a FACSAria II flow cytometer (BD Immunocytometry Systems).