Impaired survival of regulatory T cells in pulmonary sarcoidosis

Abstract

Background: Impaired regulatory T cell (Treg) function is thought to contribute to ongoing inflammatory responses in sarcoidosis, but underlying mechanisms remain unclear. Moreover, it is not known if increased apoptotic susceptibility of Tregs may contribute to an impaired immunosuppressive function in sarcoidosis. Therefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of Tregs in sarcoidosis.

Methods: Patients with pulmonary sarcoidosis (n = 58) were included at time of diagnosis. Tregs were analyzed in broncho-alveolar lavage fluid and peripheral blood of patients and healthy controls (HC).

Results: In sarcoidosis patients no evidence was found for a relative deficit of Tregs, neither locally nor systemically. Rather, increased proportions of circulating Tregs were observed, most prominently in patients developing chronic disease. Sarcoidosis circulating Tregs displayed adequate expression of FoxP3, CD25 and CTLA4. Remarkably, in sarcoidosis enhanced CD95 expression on circulating activated CD45RO(+) Tregs was observed compared with HC, and proportions of these cells were significantly increased. Specifically sarcoidosis Tregs--but not Th cells--showed impaired survival compared with HC. Finally, CD95L-mediated apoptosis was enhanced in sarcoidosis Tregs.

Conclusion: In untreated patients with active pulmonary sarcoidosis, Tregs show impaired survival and enhanced apoptotic susceptibility towards CD95L. Increased apoptosis likely contributes to the insufficient immunosuppressive function of sarcoidosis Tregs. Further research into this field will help determine whether improvement of Treg survival holds a promising new therapeutic approach for chronic sarcoidosis patients.

Fig. 1

Increased proportions of circulating Tregs in patients developing chronic sarcoidosis. Treg proportions were determined in PB of HCs and SRC patients. a Representative flow cytometry analysis of an HC and SRC patient to determine Tregs in PB. b PB Treg proportions. c Subgroup analyses of PB Treg proportions at time of diagnosis in patients undergoing disease resolution, or developing (non-) active chronic disease. Statistics: Horizontal lines indicate the median and significance was determined using a Mann–Whitney U test, ** p < 0.01. PB peripheral blood, HC healthy control, SRC sarcoidosis

Fig. 2

Adequate expression of FoxP3, CD25 and CTLA4 on sarcoidosis circulating Tregs. FoxP3, CD25 and CTLA4 expression was determined on circulating CD25^int-highFoxP3^high Tregs of HC and SRC patients by flow cytometry. a–c. Mean fluorescence intensity of FoxP3 (a), CD25 (b) and CTLA4 (c). Mean fluorescence intensity was standardized to average expression in healthy control peripheral blood cells. Statistics: Horizontal lines indicate the median and significance was determined using a Mann–Whitney U test, * p < 0.05 ** p < 0.01. FoxP3 forkhead box P3, CTLA4 cytotoxic T lymphocyte antigen 4, HC healthy control, SRC sarcoidosis

Fig. 3

Activated CD45RO⁺ Tregs from sarcoidosis patients highly express CD95. The proportions of CD45RO and CD95 expressing Th cells and Tregs were determined in PB of HCs and SRC patients. a Representative flow cytometry analysis of an HC and SRC patient to determine the proportions CD45RO⁺, CD95^high and double positive Tregs (gated on CD3⁺CD4⁺CD25^int-highCD127^low.). b Proportions of CD45RO⁺ and CD95^high Th cells and Tregs. c Correlation between proportions CD45RO⁺ Tregs and CD95^high Tregs in PB. Open dots represent HC Tregs and closed dots represent SRC Tregs. d Mean fluorescence intensity of CD95 on CD45RA⁺ and CD45RO⁺ Tregs. Statistics: Horizontal lines indicate the median and significance was determined using a Mann–Whitney U test, * p < 0.05. Correlation was analysed using Spearman’s rank-order correlation test. Regression line with R and p-value are shown in the plot. Th T helper, CD95 Fas; death receptor, PB peripheral blood, HC healthy control, SRC sarcoidosis

Fig. 4

Impaired survival of sarcoidosis Tregs. Isolated Tregs (purity >97 %) were cultured with recombinant human IL-2. a Percentage alive Tregs at 72 hours of culture. Horizontal line indicates the median. Significance was determined using a Mann–Whitney U test, **p < 0.01. b Representative flow cytometry analysis of an HC and SRC patient after 12 hours co-culture with autologous Th cells to determine Treg survival and apoptosis. c Survival (above) and apoptosis (below) graph of Tregs cultured for 48 hours with autologous Th cells. Dots indicate mean +/− SEM of 3 HCs and 3 SRC patients. One representative experiment is shown of 3 independent experiments. HC healthy control, SRC sarcoidosis

Fig. 5

Increased sensitivity of sarcoidosis Tregs towards CD95L-mediated apoptosis. Freshly isolated Tregs were analysed for apoptotic susceptibility towards soluble CD95L. a Representative flow cytometry analysis of an HC and SRC patient after 20 hours of culture with IL-2 only or with IL-2 and soluble CD95L. Numbers indicate percentage of CD25⁺FoxP3⁺ Tregs in culture. Induced apoptosis by CD95L was calculated, using the following formula: ((% CD25⁺FoxP3⁺ Tregs cultured with IL-2 - % CD25⁺FoxP3⁺ Tregs cultured with IL-2 and CD95L)/(% CD25⁺FoxP3⁺ Tregs cultured with IL-2))*100 %. b Percentage Tregs in culture of 9 HCs and 7 SRC patients after 20 hours. Paired data is shown of the Treg cultures without or with soluble CD95L. c Percentage CD95L-induced apoptosis in HC and SRC Tregs. Statistics: Horizontal lines indicate the median. Significance was determined using a Mann–Whitney U test (c) or Wilcoxon matched pairs test (b), *p < 0.05 **p < 0.01. CD95L CD95 ligand, HC healthy control, SRC sarcoidosis