IL-21 production by CD4+ effector T cells and frequency of circulating follicular helper T cells are increased in type 1 diabetes patients

Abstract

Aims/hypothesis: Type 1 diabetes results from the autoimmune destruction of insulin-secreting pancreatic beta cells by T cells. Despite the established role of T cells in the pathogenesis of the disease, to date, with the exception of the identification of islet-specific T effector (Teff) cells, studies have mostly failed to identify reproducible alterations in the frequency or function of T cell subsets in peripheral blood from patients with type 1 diabetes.

Methods: We assessed the production of the proinflammatory cytokines IL-21, IFN-γ and IL-17 in peripheral blood mononuclear cells from 69 patients with type 1 diabetes and 61 healthy donors. In an additional cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. IL-21 and IL-17 production was also measured in peripheral blood mononuclear cells (PBMCs) from a subset of 46 of the 62 donors immunophenotyped for Tfh.

Results: We found a 21.9% (95% CI 5.8, 40.2; p = 3.9 × 10(-3)) higher frequency of IL-21(+) CD45RA(-) memory CD4(+) Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). Consistent with this finding, we found a 14.9% increase in circulating Tfh cells in the patients (95% CI 2.9, 26.9; p = 0.016).

Conclusions/interpretation: These results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target.

Fig. 1

IL-21 production is increased in type 1 diabetes patients. (a) Gating strategy for the delineation of IL-21⁺ CD45RA⁻, IFN-γ⁺ CD45RA⁻ and IL-17⁺ CD45RA⁻ CCR6⁺ CD4⁺ memory effector T cell subsets. (b) Scatter plot depicts the distribution (geometric mean ± 95% CI) of IL-21⁺ cells among memory CD45RA⁻ CD4⁺ T cells. The frequency of IL-21⁺ cells was compared between 61 type 1 diabetes patients and 53 healthy donors (p = 3.9 × 10⁻³). (c, d) Scatter plots depict the frequency of IFN-γ⁺ (c) and IL-17⁺ (d) cells in a sample of 62 type 1 diabetes patients and 54 healthy donors following in vitro stimulation with 100 ng/ml PMA and ionomycin. (e, f) Frequency of IFN-γ⁺ (e) and IL-17⁺ (f) cells was also assessed in a subset of 24 type 1 diabetes patients and 15 healthy controls following in vitro stimulation with a higher concentration (500 ng/ml) of ionomycin. The p values were calculated by linear regression of the log-transformed data, including batch as a covariate. Horizontal bars represent the geometric mean (±95% CI) obtained from the transformation of the log-transformed data: (Y = exp[Y]) of each group. Additional data from the statistical analysis are provided in Table 3. HC, healthy control; T1D, type 1 diabetic patient. *p < 0.05, **p < 0.01

Fig. 2

Frequency of IFN-γ⁺ HELIOS⁻ CD45RA⁻ FOXP3⁺ CD4⁺ Tregs is not altered in type 1 diabetes patients. (a) Gating strategy for the HELIOS⁻ CD45RA⁻ FOXP3⁺ CD4⁺ Treg subset. FACS gating plots depict data from one illustrative donor. (b) Scatter plot depicts the distribution (geometric mean ± 95% CI) of IFN-γ⁺ cells in the HELIOS⁻CD45RA⁻ FOXP3⁺ CD4⁺ Treg subset. The frequency of IFN-γ⁺ cells was compared between type 1 diabetes patients (n = 62) and healthy donors (n = 54; p = 0.79). The p values were calculated by linear regression of the log-transformed data, including batch as a covariate. HC, healthy control; T1D, type 1 diabetic patient

Fig. 3

Increased frequency of Tfh cells within the CXCR5⁺ CD4⁺ memory cell subset of type 1 diabetes patients as compared with healthy controls. (a) Gating strategy for the delineation the Tfh memory effector T cell subset defined as the frequency of PD-1⁺ CCR6⁻ cells out of CD45RA⁻ CXCR5⁺ CD4⁺ T cells. (b) Scatter plot depicts the distribution (geometric mean ± 95% CI) of Tfh cells (defined as % PD-1⁺ of CCR6⁻ CXCR5⁺) in a cohort of type 1 diabetes patients (n = 30) and healthy controls (n = 32; p = 0.016). Frequency of circulating Tfh cells was assessed using fresh whole blood from type 1 diabetes patients and healthy donors enrolled from the CBR, matched for sex and 5 year age bands. The p value was calculated using an unpaired two-tailed t test. HC, healthy control; T1D, type 1 diabetic patient. *p < 0.05