The cells were isolated using gradient centrifugation, cryopreservation, and resuscitation. Once activated via in vitro stimulation, the PBMCs were harvested and immunostained with the respective fluorochromeconjugated antibodies for flow cytometry analysis. In separate procedure, whole blood was collected from test participants and immunostained with fluorochromeconjugated monoclonal antibodies for t-follicular helper cell immunophenotyping via flow cytometry. For statistical analysis, the association between cytokine production phenotypes was calculated and assessed by linear regression. The frequency of circulating t-follicular helper cells between type 1 diabetic patients and healthy controls was compared using an unpaired two-tailed Student’s t-test. The effects of age, sex and time of collection on the association with type 1 diabetes were dealt by the experimental design used in this study and, therefore, not included as independent …show more content…
Consistent with this increase in production, an increase in the frequency of t-follicular helper cells was also observed. Moreover, the differential expression of IL-21 in type 1 diabetes patients was investigated to determine if the observed increase in frequency could be caused by an altered T central memory (Tcm) cell compartment. There was no evidence of an altered production; with the frequency of IL-21+ cells among the Tcm subset still significantly increased in type 1 diabetes patients. As t-follicular helper cells are known to produce IL-21, this association suggests that IL-21 production by t-follicular helper cells may be an aetiological factor in type 1 diabetes. In contrast to IL-21, there was no convincing support for a differential frequency of IL-17 and IFN-γ-producing cells within them CD4+ T cell subset in type 1 diabetes patients as compared with