Increased frequency of PD-1hiCXCR5- T cells and B cells in patients with newly diagnosed IgA nephropathy

Recent research has identified a population of PD-1hiCXCR5− ‘peripheral helper’ T (Tph) cells that simulate plasma cell differentiation by interactions between IL-21 and SLAMF5. However, the alteration of circulating Tph and CD138+ B in IgA nephropathy (IgAN) remains poorly understood. Flow cytometry analysis was used to measure the frequency of circulating PD-1hiCXCR5− T cells and CD138+ B cells in 37 patients with IgAN and 23 healthy controls (HCs). Estimated glomerular filtration rate (eGFR), 24 h urinary protein and serum cytokine concentrations were measured. The percentage of different subsets of circulating PD-1hiCXCR5− T cells and CD138+ B cells were significantly higher in patients with IgAN compared to HCs. Pretreatment, the percentage of different subsets of circulating PD-1hiCXCR5− T cells and CD138+ B cells were negatively correlated with eGFR, the percentage of circulating CD138+ B cells was positively correlated with 24-h urinary protein concentration, and the percentage of circulating PD-1hiCXCR5−, CD28+ and ICOS+ T cells. Posttreatment, the percentage of different subsets of circulating PD-1hiCXCR5− T cells and CD138+ B cells and serum IL-21 concentration were significantly reduced. Different subsets of circulating PD-1hiCXCR5− T cells contribute to the progression and pathogenesis of IgAN by regulating the differentiation of CD138+ B cells through a combination of surface molecules.

Treatment and follow up. Treatment for patients with IgAN and 24-h urinary protein >1 g included pednisone (PDN, Tianyao Pharmaceuticals, Tianjin, China) 1 mg/kg/d for the first two months, gradually decreasing to a maintenance dose of 10 mg/d over the next 6 months. Benazepril (10 mg/d) or valsartan (80 mg/d, Novartis Pharma, Beijing, China) was administered to other patients.
Aspirin (100 mg/d, Bayer, Germany) or dipyridamole (100 mg/d, Yunpeng Pharmaceutical, Shanxi, China) was provided to patients with a high risk of clotting.
Patients visited the clinic monthly and were followed up for at least 8-12 weeks after the initiation of treatment.
Blood sampling. Fasting  www.nature.com/scientificreports www.nature.com/scientificreports/ Statistical analysis. Statistical analyses were performed using GraphPad Prism version 5.01 software. Data are reported as median and range and were analyzed using the Kruskal-Wallis H non-parametric test. The relationship between variables was analyzed using Spearman's rank correlation test. All tests were two-sided and a P-value of <0.05 was considered significant.

Results
Characteristics of the study subjects. This study included 37 patients with IgAN and 23 age-and gender-matched HCs. Demographic and clinical characteristics of the study subjects are shown in Table 1. There were no significant differences in age, gender, leukocyte and lymphocyte counts, serum uric acid, triglyceride, cholesterol, or albumin levels, or microscopic hematuria between patients with IgAN and HCs. However, 24 h urinary www.nature.com/scientificreports www.nature.com/scientificreports/ protein concentration was higher and eGFR was lower in patients with IgAN, suggesting that patients with IgAN had impaired kidney function.

Associations between different subsets of PD1
Some research shows that T cells with a high expression of programmed cell death protein-1 (PD-1) have impaired cytotoxicity [22][23][24][25] , while other reports suggest that PD-1 + T cells induce high levels of autoantibody production by activating antigen-specific autoreactive B cells and promoting the survival of long-lived plasma cells 26 . In the present study, the percentage of different subsets of circulating PD-1 hi CXCR5 − T cells and CD138 + B cells was significantly higher in patients with IgAN compared to HCs, and the percentage of circulating CD138 + B cells was positively correlated with the percentage of circulating PD1 hi CXCR5 − , CD28 + PD-1 hi CXCR5 − , and ICOS + PD-1 hi CXCR5 − T cells. These data suggest that PD-1 hi CXCR5 − T cells support B-cell responses and antibody production by mechanisms that are dependent on costimulation through CD28 or includible costimulator (ICOS). Accordingly, previous studies demonstrated that CD28 is a costimulatory receptor that binds CD80 and CD86 and plays an important role in T cell-B cell interactions 27 , and ICOS costimulation in necessary for T cell proliferation and is involved in humoral immune responses (B cell germinal center formation) 28 .
IL-21 production is mostly restricted to Tfh cells, and is important for Tfh cell differentiation 29 . However, some evidence shows that PD-1 hi CXCR5 − T (Tph) cells also express and release IL-21 9 to recruit B cells and Tfh cells and promote the production of autoantibodies 30,31 . One report showed that plasma cell differentiation can be induced in vitro by Tph cells through IL-21 secretion and surface molecule interaction 9 . In this study, we found a positive correlation between the percentage of circulating PD-1 hi CXCR5 − and IL21 + PD-1 hi CXCR5 − T cells and serum IL-21 concentrations. Most importantly, serum IL21 concentrations were significantly higher in patients with IgAN than HCs. www.nature.com/scientificreports www.nature.com/scientificreports/ Corticosteroids are widely used as immunotherapy as they inhibit the T/B response and the production of cytokines. In this study, corticosteroid treatment of patients with IgAN significantly reduced the percentage of circulating PD-1 hi CXCR5 − T cells and CD138 + B cells, as well as serum IL-21 concentration; these findings align with those of a previous study 11 . Th1 cells selectively produce IFN-γ and IL-2 and increase cell-mediated immunity. Th2 cells selectively produce IL-4 and IL-10 and are responsible for antibody production. IL-17A is a characteristic cytokine of Th17 cells with a key role in the pathogenesis of chronic inflammatory diseases and autoimmune responses 32 . In this study, serum IL-4, IL-10, IL-17A, and IFN-γ concentrations were significantly higher in patients with IgAN compared to HCs. We speculate that Th1 responses may be involved in the mechanism of IgAN and induce anti-inflammatory Th2 cells, which feedback to down regulate proinflammatory responses during the pathogenesis of IgA. This hypothesis is consistent with previous reports 33 . After treatment, serum IL-10 and IL-4 concentrations were significantly increased in patients with IgAN, while the concentrations of other cytokines were unchanged. Taken together, these data suggest that pro-inflammatory Th1 and Th17 responses may be involved in the pathogenesis of IgAN and the anti-inflammatory Th2 response may predominate after corticosteroid treatment. We recognize that our study has limitations, including a small sample size and the lack of functional investigations exploring different types of PD-1 hi CXCR5 − T cells and B cells. Thus, further studies in a larger population are warranted.
In conclusion, our study revealed that patients with IgAN have higher levels of circulating PD-1 hi CXCR5 − T cells and B cells than HCs, and the percentage of these cells is correlated with disease severity. These findings offer new insights into understanding the pathogenesis of IgAN. Furthermore, the high expression of PD-1 on Tph cells in patients with IgAN represents a potential strategy for therapeutic targeting.