Limits...
Expansion of regulatory GITR+CD25 low/-CD4+ T cells in systemic lupus erythematosus patients.

Nocentini G, Alunno A, Petrillo MG, Bistoni O, Bartoloni E, Caterbi S, Ronchetti S, Migliorati G, Riccardi C, Gerli R - Arthritis Res. Ther. (2014)

Bottom Line: CD4+CD25 low/-GITR+ cells were isolated with magnetic separation, and their phenotype was compared with that of CD4+CD25 high GITR- cells.Phenotypic analysis demonstrated that CD4+CD25 low/-GITR+ cells display regulatory T-cell (Treg) markers, including FoxP3, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor-beta (TGF-β), and interleukin (IL)-10.Functional experiments demonstrated that CD4+CD25 low/-GITR+ cells exert a higher inhibitory activity against both autologous and heterologous cells as compared with CD4+CD25 high GITR- cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: CD4+CD25 low/-GITR+ T lymphocytes expressing forkhead box protein P3 (FoxP3) and showing regulatory activity have been recently described in healthy donors. The objective of the study was to evaluate the proportion of CD4+CD25 low/-GITR+ T lymphocytes within CD4+ T cells and compare their phenotypic and functional profile with that of CD4+CD25 high GITR- T lymphocytes in systemic lupus erythematosus (SLE) patients.

Methods: The percentage of CD4+CD25 low/-GITR+ cells circulating in the peripheral blood (PB) of 32 patients with SLE and 25 healthy controls was evaluated with flow cytometry. CD4+CD25 low/-GITR+ cells were isolated with magnetic separation, and their phenotype was compared with that of CD4+CD25 high GITR- cells. Regulatory activity of both cell subsets was tested in autologous and heterologous co-cultures after purification through a negative sorting strategy.

Results: Results indicated that CD4+CD25 low/-GITR+ cells are expanded in the PB of 50% of SLE patients. Expansion was observed only in patients with inactive disease. Phenotypic analysis demonstrated that CD4+CD25 low/-GITR+ cells display regulatory T-cell (Treg) markers, including FoxP3, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor-beta (TGF-β), and interleukin (IL)-10. In contrast, CD4+CD25 high GITR- cells appear to be activated and express low levels of Treg markers. Functional experiments demonstrated that CD4+CD25 low/-GITR+ cells exert a higher inhibitory activity against both autologous and heterologous cells as compared with CD4+CD25 high GITR- cells. Suppression is independent of cell contact and is mediated by IL-10 and TGF-β.

Conclusions: Phenotypic and functional data demonstrate that in SLE patients, CD4+CD25 low/-GITR+ cells are fully active Treg cells, possibly representing peripheral Treg (pTreg) that are expanded in patients with inactive disease. These data may suggest a key role of this T-cell subset in the modulation of the abnormal immune response in SLE. Strategies aimed at expanding this Treg subset for therapeutic purpose deserve to be investigated.

Show MeSH

Related in: MedlinePlus

Percentages of circulating CD4+CD25low/-GITR+and CD4+CD25highGITR−cells in HC are different from those in SLE patients. Percentage of CD4+CD25low/-GITR+(A), CD4+CD25highGITR−(B) and CD4+CD25highGITR+(C) in CD4+ T lymphocytes evaluated with flow-cytometry analysis of anti-GITR- and anti-CD25-stained CD4+ T lymphocytes, isolated from PB of 25 HC and 32 SLE patients, is shown. Horizontal lines indicate mean percentage. P values are according to Mann-Whitney U test comparing differences in HC and SLE. The percentage of patients with more than 1.4% CD4+CD25low/-GITR+ cells (90th percentile of HC) is also indicated (A). Percentage of CD4+CD25low/-GITR+(D) and CD4+CD25highGITR−(E) in CD4+ T cells purified from freshly isolated PB of HC, patients with inactive disease identified by an SLEDAI =0 (n =13), and patients with active disease identified by an SLEDAI >0 (n =19) was evaluated. Bars indicate mean ± SEM. n.s., P >0.05, *P <0.05 and ***P <0.001, according to Kruskal-Wallis test comparing active SLE, inactive SLE, and HC. (F) Correlation between levels of CD4+CD25low/-GITR+ and CD4+CD25highGITR− in CD4+ cells purified from freshly isolated SLE patients (Spearman ρ = −0.5; P <0.01). (G) Distribution of CD4+CD25highGITR− cells according to the expansion of CD4+CD25low/-GITR+ cells. SLE patients with a percentage of these cells higher than 1.4% (90th percentile of the distribution in HC) were defined as having expansion of the CD4+CD25low/-GITR+ cells (16 of 32 patients). P <0.001 according to χ2 test on raw data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4209023&req=5

Fig2: Percentages of circulating CD4+CD25low/-GITR+and CD4+CD25highGITR−cells in HC are different from those in SLE patients. Percentage of CD4+CD25low/-GITR+(A), CD4+CD25highGITR−(B) and CD4+CD25highGITR+(C) in CD4+ T lymphocytes evaluated with flow-cytometry analysis of anti-GITR- and anti-CD25-stained CD4+ T lymphocytes, isolated from PB of 25 HC and 32 SLE patients, is shown. Horizontal lines indicate mean percentage. P values are according to Mann-Whitney U test comparing differences in HC and SLE. The percentage of patients with more than 1.4% CD4+CD25low/-GITR+ cells (90th percentile of HC) is also indicated (A). Percentage of CD4+CD25low/-GITR+(D) and CD4+CD25highGITR−(E) in CD4+ T cells purified from freshly isolated PB of HC, patients with inactive disease identified by an SLEDAI =0 (n =13), and patients with active disease identified by an SLEDAI >0 (n =19) was evaluated. Bars indicate mean ± SEM. n.s., P >0.05, *P <0.05 and ***P <0.001, according to Kruskal-Wallis test comparing active SLE, inactive SLE, and HC. (F) Correlation between levels of CD4+CD25low/-GITR+ and CD4+CD25highGITR− in CD4+ cells purified from freshly isolated SLE patients (Spearman ρ = −0.5; P <0.01). (G) Distribution of CD4+CD25highGITR− cells according to the expansion of CD4+CD25low/-GITR+ cells. SLE patients with a percentage of these cells higher than 1.4% (90th percentile of the distribution in HC) were defined as having expansion of the CD4+CD25low/-GITR+ cells (16 of 32 patients). P <0.001 according to χ2 test on raw data.

Mentions: The percentage of CD4+ Tregs characterized by GITR expression and low/negative levels of CD25 (CD4+CD25low/-GITR+), recently described in HC [36], was evaluated in SLE patients with flow cytometry (Figure 1A). As in HC [36], the levels of expression of CD25 in this subset (formally CD25−) were higher than those in CD25−GITR− cells (effectors), as shown by both real-time PCR and flow cytometry (Figure 1B,C). SLE patients with a percentage of CD4+CD25low/-GITR+ cells higher than 1.4% (90th percentile of the distribution in HCs) were defined as having an expansion of CD4+CD25low/-GITR+ cells (number 16; 50%). Consequently, the mean value of circulating CD4+CD25low/-GITR+ Tregs in SLE was significantly higher than detected in HCs (Figure 2A). This result was in striking contrast with that observed in CD4+CD25highGITR− and CD4+CD25highGITR+ Tregs, which were in lower proportion and equal in SLE patients, respectively (Figure 2B,C).Figure 1


Expansion of regulatory GITR+CD25 low/-CD4+ T cells in systemic lupus erythematosus patients.

Nocentini G, Alunno A, Petrillo MG, Bistoni O, Bartoloni E, Caterbi S, Ronchetti S, Migliorati G, Riccardi C, Gerli R - Arthritis Res. Ther. (2014)

Percentages of circulating CD4+CD25low/-GITR+and CD4+CD25highGITR−cells in HC are different from those in SLE patients. Percentage of CD4+CD25low/-GITR+(A), CD4+CD25highGITR−(B) and CD4+CD25highGITR+(C) in CD4+ T lymphocytes evaluated with flow-cytometry analysis of anti-GITR- and anti-CD25-stained CD4+ T lymphocytes, isolated from PB of 25 HC and 32 SLE patients, is shown. Horizontal lines indicate mean percentage. P values are according to Mann-Whitney U test comparing differences in HC and SLE. The percentage of patients with more than 1.4% CD4+CD25low/-GITR+ cells (90th percentile of HC) is also indicated (A). Percentage of CD4+CD25low/-GITR+(D) and CD4+CD25highGITR−(E) in CD4+ T cells purified from freshly isolated PB of HC, patients with inactive disease identified by an SLEDAI =0 (n =13), and patients with active disease identified by an SLEDAI >0 (n =19) was evaluated. Bars indicate mean ± SEM. n.s., P >0.05, *P <0.05 and ***P <0.001, according to Kruskal-Wallis test comparing active SLE, inactive SLE, and HC. (F) Correlation between levels of CD4+CD25low/-GITR+ and CD4+CD25highGITR− in CD4+ cells purified from freshly isolated SLE patients (Spearman ρ = −0.5; P <0.01). (G) Distribution of CD4+CD25highGITR− cells according to the expansion of CD4+CD25low/-GITR+ cells. SLE patients with a percentage of these cells higher than 1.4% (90th percentile of the distribution in HC) were defined as having expansion of the CD4+CD25low/-GITR+ cells (16 of 32 patients). P <0.001 according to χ2 test on raw data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4209023&req=5

Fig2: Percentages of circulating CD4+CD25low/-GITR+and CD4+CD25highGITR−cells in HC are different from those in SLE patients. Percentage of CD4+CD25low/-GITR+(A), CD4+CD25highGITR−(B) and CD4+CD25highGITR+(C) in CD4+ T lymphocytes evaluated with flow-cytometry analysis of anti-GITR- and anti-CD25-stained CD4+ T lymphocytes, isolated from PB of 25 HC and 32 SLE patients, is shown. Horizontal lines indicate mean percentage. P values are according to Mann-Whitney U test comparing differences in HC and SLE. The percentage of patients with more than 1.4% CD4+CD25low/-GITR+ cells (90th percentile of HC) is also indicated (A). Percentage of CD4+CD25low/-GITR+(D) and CD4+CD25highGITR−(E) in CD4+ T cells purified from freshly isolated PB of HC, patients with inactive disease identified by an SLEDAI =0 (n =13), and patients with active disease identified by an SLEDAI >0 (n =19) was evaluated. Bars indicate mean ± SEM. n.s., P >0.05, *P <0.05 and ***P <0.001, according to Kruskal-Wallis test comparing active SLE, inactive SLE, and HC. (F) Correlation between levels of CD4+CD25low/-GITR+ and CD4+CD25highGITR− in CD4+ cells purified from freshly isolated SLE patients (Spearman ρ = −0.5; P <0.01). (G) Distribution of CD4+CD25highGITR− cells according to the expansion of CD4+CD25low/-GITR+ cells. SLE patients with a percentage of these cells higher than 1.4% (90th percentile of the distribution in HC) were defined as having expansion of the CD4+CD25low/-GITR+ cells (16 of 32 patients). P <0.001 according to χ2 test on raw data.
Mentions: The percentage of CD4+ Tregs characterized by GITR expression and low/negative levels of CD25 (CD4+CD25low/-GITR+), recently described in HC [36], was evaluated in SLE patients with flow cytometry (Figure 1A). As in HC [36], the levels of expression of CD25 in this subset (formally CD25−) were higher than those in CD25−GITR− cells (effectors), as shown by both real-time PCR and flow cytometry (Figure 1B,C). SLE patients with a percentage of CD4+CD25low/-GITR+ cells higher than 1.4% (90th percentile of the distribution in HCs) were defined as having an expansion of CD4+CD25low/-GITR+ cells (number 16; 50%). Consequently, the mean value of circulating CD4+CD25low/-GITR+ Tregs in SLE was significantly higher than detected in HCs (Figure 2A). This result was in striking contrast with that observed in CD4+CD25highGITR− and CD4+CD25highGITR+ Tregs, which were in lower proportion and equal in SLE patients, respectively (Figure 2B,C).Figure 1

Bottom Line: CD4+CD25 low/-GITR+ cells were isolated with magnetic separation, and their phenotype was compared with that of CD4+CD25 high GITR- cells.Phenotypic analysis demonstrated that CD4+CD25 low/-GITR+ cells display regulatory T-cell (Treg) markers, including FoxP3, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor-beta (TGF-β), and interleukin (IL)-10.Functional experiments demonstrated that CD4+CD25 low/-GITR+ cells exert a higher inhibitory activity against both autologous and heterologous cells as compared with CD4+CD25 high GITR- cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: CD4+CD25 low/-GITR+ T lymphocytes expressing forkhead box protein P3 (FoxP3) and showing regulatory activity have been recently described in healthy donors. The objective of the study was to evaluate the proportion of CD4+CD25 low/-GITR+ T lymphocytes within CD4+ T cells and compare their phenotypic and functional profile with that of CD4+CD25 high GITR- T lymphocytes in systemic lupus erythematosus (SLE) patients.

Methods: The percentage of CD4+CD25 low/-GITR+ cells circulating in the peripheral blood (PB) of 32 patients with SLE and 25 healthy controls was evaluated with flow cytometry. CD4+CD25 low/-GITR+ cells were isolated with magnetic separation, and their phenotype was compared with that of CD4+CD25 high GITR- cells. Regulatory activity of both cell subsets was tested in autologous and heterologous co-cultures after purification through a negative sorting strategy.

Results: Results indicated that CD4+CD25 low/-GITR+ cells are expanded in the PB of 50% of SLE patients. Expansion was observed only in patients with inactive disease. Phenotypic analysis demonstrated that CD4+CD25 low/-GITR+ cells display regulatory T-cell (Treg) markers, including FoxP3, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), transforming growth factor-beta (TGF-β), and interleukin (IL)-10. In contrast, CD4+CD25 high GITR- cells appear to be activated and express low levels of Treg markers. Functional experiments demonstrated that CD4+CD25 low/-GITR+ cells exert a higher inhibitory activity against both autologous and heterologous cells as compared with CD4+CD25 high GITR- cells. Suppression is independent of cell contact and is mediated by IL-10 and TGF-β.

Conclusions: Phenotypic and functional data demonstrate that in SLE patients, CD4+CD25 low/-GITR+ cells are fully active Treg cells, possibly representing peripheral Treg (pTreg) that are expanded in patients with inactive disease. These data may suggest a key role of this T-cell subset in the modulation of the abnormal immune response in SLE. Strategies aimed at expanding this Treg subset for therapeutic purpose deserve to be investigated.

Show MeSH
Related in: MedlinePlus