Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia

Cellular Immunology xxx (2015) xxx–xxx

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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm

Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia Li Yan, Rong Fu, Hui Liu, Huaquan Wang, Chunyan Liu, Ting Wang, Weiwei Qi, Jing Guan, Lijuan Li, Zonghong Shao ⇑ The Department of Hematology, General Hospital of Tianjin Medical University, Tianjin, China

a r t i c l e

i n f o

Article history: Received 6 February 2015 Revised 2 April 2015 Accepted 5 April 2015 Available online xxxx Keywords: Aplastic anemia Regulatory T cells CTLA4

a b s t r a c t Severe aplastic anemia is a rare autoimmune disease characterized by severe pancytopenia and bone marrow failure, which is caused by activated T lymphocytes. Tregs are believed to control development and progression of autoimmunity by suppressing autoreactive T cells. This study aims at understanding the quantity and function of peripheral Tregs in SAA. The expression of related biomarkers on Treg cell surface were determined by flow cytometry. The frequency of Tregs and the expression of CTLA-4 in SAA was significantly decreased than that in normal controls. The expression of perforin in SAA was significantly increased than that in controls, while expression of CD39, CD73 and GITR in Tregs did not show any significant difference between the two groups. These data revealed that CTLA-4 could be responsible for Treg abnormalities in SAA, but suppression mediated by perforin, CD39, CD73 and GITR, and survival capability of single Treg cell may not be injured. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Acquired severe aplastic anemia (SAA) is a rare disease characterized by severe pancytopenia and bone marrow failure. Although the exact etiology is still unknown, previous studies have showed that it is an immune-mediated disease with destruction of hematopoietic cells by activated T cells [1,2]. In recent years, many studies have demonstrated dysregulation T cell function, especially of cytotoxic T lymphocytes (CTLs) leads to bone marrow failure in SAA [3,4]. However, it remains unclear that how T cell become activated. Regulatory T cells (Tregs) is a special subset of T cells reported first by Sakaguchi in 1995 [5]. One of the major characteristics of Tregs is immunosuppression, which performed as inhibiting the activation and proliferation of CD4+ and CD8+ T cells, preventing the antigen presenting process of antigen presenting cells (APC), and mediating target cell death directly. Studies have shown that Tregs played a pivotal role in controlling adaptive immune responses and maintenance of self-tolerance whether in mice or humans. Tregs express a variety of cell surface molecules, while some of them have a relatively close relationship with its ⇑ Corresponding author at: Department of Hematology, General Hospital of Tianjin Medical University, 154 Anshandao, Heping District, Tianjin 300052, China. Tel.: +86 02260362086; fax: +86 02260362085. E-mail address: [email protected] (Z. Shao).

function, such as CD25 (IL-2Ra chain), FoxP3 (fork head box protein 3), CTLA-4 (Cytotoxic T lymphocyte antigen, CD152), GITR (glucocorticoid-induced tumor necrosis factor receptor). Previous studies have shown that FoxP3 is required for Tregs to exert their suppressive ability and the ectopic expression of this transcription factor by lymphoid and non-lymphoid cells is able to confer suppressive function, making FoxP3 a critical regulator of Tregs function [6–8]. These FoxP3+ cells express high levels of CD25 and low levels of CD127 (FoxP3+ CD25high CD127low CD4+ T cells) [9]. CD127 expression is associated with acquisition of Tregs function [10]. It inversely correlates with FoxP3 expression and Tregs suppressive activity [11]. With the gradual understanding of Tregs, now more and more researchers use CD4+ CD25+ CD127dim as the molecular markers of Tregs. There is accumulating evidence that impaired function of Tregs has been implicated in the development of several common autoimmune diseases [12,13], including SAA [14,15]. Until now, most reports of Treg abnormality in aplastic anemia have shown that the numbers of circulating Tregs decreased in most patients. In this study, we intended to examine the number and functional changes of peripheral blood CD4+ CD25+ CD127dim Tregs in untreated and recovery SAA patients and normal controls, analyze its possible mechanism in pathogenesis of SAA, and then provide a theoretical basis for cell targeted therapy of SAA.

http://dx.doi.org/10.1016/j.cellimm.2015.04.001 0008-8749/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

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L. Yan et al. / Cellular Immunology xxx (2015) xxx–xxx

2. Materials and methods 2.1. Study subjects A total of forty-five (thirty-one males, fourteen females) patients with a median age of 27 years (range 5–73 years) were included in the study. This cohort of patients included twentyone (twelve males, nine females) SAA cases who were previously untreated and twenty-four cases (nineteen males, five females) with recovering-SAA (R-SAA). The diagnosis of SAA was established by bone marrow biopsy, bone marrow smear, and peripheral blood cell count according to International AA Study Group Criteria [2]. SAA was defined by following criteria: (1) bone marrow cellularity <25% or 25–50% with <30% residual (hematopoietic) cells; (2) two of the following three: neutrophils <0.5  109/L, platelets <20  109/L, and reticulocytes <20  109/L [16,17]; (3) no evidence of malignant disease, no extensive iron deposition, storage disease, myelofibrosis, and no

other autoimmune diseases. Bone marrow cytogenetic studies were performed in all patients, and the clinical characteristics of patients are listed in Table 1. Recovering SAA patients were defined as substantial improvement after therapy with rabbit antithymocyte globulin (rATG) (Fresenius, Germany), decreasing dose of cyclosporine (CsA) [18], glucocorticoid and hematopoietic stimulating factors (recombinant human erythropoietin, granulocyte colony-stimulating factor, recombinant human thrombopoietin, and/or IL-11 in combination), and all the patients became completely transfusion independent. The duration of erythrocytes/platelets transfusion independency in 25%, 50% and 75% patients was 86/87, 140/126 and 196/183 days after ATG therapy, respectively. The median duration that neutrophils, erythrocytes and platelets returned to normal level was 35 days, 11 months and 12.5 months after ATG therapy, respectively. Among all the R-SAA patients, fourteen have achieved complete response (CR), while the other ten were partial response (PR) according to Camitta criteria (1976).

Table 1 Clinical and demographic parameters of patients participating in the study. Diagnosis

Number

Age (years)

ANC (109/L)

Hb (g/L)

Untreated SAA

21

32(5–73)

0.62 ± 0.82

64.38 ± 13.68

Recovering SAA

24

26(7–55)

2.95 ± 1.87

126.21 ± 33.58

PLT (109/L)

Ret%

Bone marrow Karyotype

Therapy

Duration (months)

15.05 ± 13.33

0.39 ± 0.30 2.45 ± 1.85

Not previously treated except for transfusions Treated with combination indicated

2(1–3)

128.96 ± 62.93

Male 46, XY [20] Female 46, XX [20] Male 46, XY [20] Female 46, XX[20]

32(6–116)

Values are expressed as mean ± SD. Age and duration values are mid (min, max). ANC: absolute neutrophil count; Hb: hemoglobin; PLT: platelet; Ret: reticulocyte. Duration of ‘‘untreated SAA’’: from the onset of disease to time of Tregs measurement; duration of ‘‘recovering SAA’’: from the ATG therapy to time of Tregs measurement.

A

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Fig. 1. Decreased frequencies of CD4+ CD25+ CD127dim Tregs in PB from patients with SAA. (A) The frequencies of Tregs in SAA patient. (B) The frequencies of Tregs in normal control. (C) Percentage of CD4+ T cells in lymphocyte population of SAA (n = 18), R-SAA (n = 23) and normal control (n = 31). (D) Percentage of Tregs in CD4+ T cells of untreated SAA (n = 20), R-SAA (n = 25) and normal control (n = 31). (E) Percentage of Tregs in lymphocytes of SAA (n = 18), R-SAA (n = 23) and normal control (n = 31). (F) Absolute Treg numbers of SAA (n = 19), PR-SAA (n = 8), CR-SAA (n = 14) and normal control (n = 31). Data were expressed as mean ± SD. ⁄P < 0.05; ⁄⁄P < 0.01.

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

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Normal values of regulatory T cells counts were determined by thirty-one age-matched healthy volunteers aged 18–71 years old. The study was approved by the Ethics Committee of the Tianjin Medical University. Informed written consent was obtained from all patients or their parents in accordance with the Declaration of Helsinki. 2.2. Reagents and instruments The fluorophore-conjugated monoclonal antibodies: CD4PerCP, CD25-FITC, CD127-APC, mouse IgG1-PE, CTLA-4-PE, Perforin-PE, CD39-PE, CD73-PE and erythrocyte lytic solution were from BD Biosciences (Franklin Lakes, NJ, USA), GITR-PE was from R&D systems (Minneapolis, MN, USA). Lysing Solution was purchased from BD Biosciences (Franklin Lakes, NJ, USA). 2.3. Measurement of Tregs and expression of biomarkers Circulating CD4+ T cells and regulatory T cells were identified by flow cytometric analysis. Briefly, fresh EDTA anticoagulant blood samples (100 lL) were immunostained with above-mentioned antibodies in TruCount tubes (BD). After incubation in the dark for 30 min at 4 °C, cells were incubated with 2 mL erythrocyte lytic solution (BD PharMingen) for 10 min at room temperature and washed three times with phosphate buffer solution (PBS). Finally, a minimum of 50,000 cells were acquired on a FACS-Calibur flow

cytometer (BD Biosciences) and analyzed by CellQuest softwara version 3.1 software. Among mononuclear cells, regulatory T cells are identified as CD4+ CD25+ CD127dim. The expression of CTLA-4, Perforin, CD39, CD73 and GITR were analyzed based on isotype control with mouse IgG1. Cytogenetic analysis was performed on unstimulated shortterm bone marrow cultures. Analysis of 20 R-banded metaphases was carried out and reported according to the International System for Human Cytogenetic Nomenclature. 2.4. Statistical analysis All analyses were performed using SPSS 21.0 software (SPSS Science). Data were presented as mean ± standard deviation (SD). The significance of the differences was assessed by One-way ANOVA. Spearman’s rank correlation test was used for correlated data. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Decreased frequency and absolute number of Tregs in PB of patients with SAA To detect the frequencies of Tregs in SAA, we analyzed the frequencies of CD4+ CD25+ CD127dim Tregs (gating on CD4+ T cell population and lymphocyte population) in PB samples obtained from

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Fig. 1 (continued)

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

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Fig. 2. (A) Decreased expression of CTLA-4 in PB Tregs from patients with SAA. (B) The expression of CTLA-4 in PB Tregs from patients with SAA (n = 16), R-SAA (n = 19) and normal control (n = 23). (C) The mean fluorescence intensity of CTLA-4+ Tregs of SAA (n = 16), R-SAA (n = 19) and normal control (n = 22). (D) The expression of CTLA-4 in Tregs multiplied by the percentage of Tregs in CD4+ T cells, i.e. the percentage of CTLA-4+ Tregs in CD4+ T cells from patients with SAA (n = 16), R-SAA (n = 19) and normal control (n = 23). (E) The mean fluorescence intensity of CTLA-4+ Tregs multiplied by the percentage of Tregs in CD4+ T cells of SAA (n = 16), R-SAA (n = 19) and normal control (n = 22). Data were expressed as mean ± SD. ⁄P < 0.05; ⁄⁄P < 0.01.

SAA patient (Fig. 1A) and normal control (Fig. 1B). The percentage of CD4+ T cells in lymphocyte population of SAA, R-SAA and normal

control group was (27.80 ± 12.51)%, (25.60 ± 10.03)%, and (36.23 ± 5.80)%, respectively (Fig. 1C). All of the SAA patients had

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

L. Yan et al. / Cellular Immunology xxx (2015) xxx–xxx

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significantly decreased frequencies of Tregs compared with normal controls (Fig. 1D). The percentage of Tregs in CD4+ T cells of untreated SAA patients was (2.94 ± 0.65)%, which was obviously lower than that of recovery patients (4.07 ± 1.17)% and normal controls (4.98 ± 1.38)% (P < 0.01); and the difference between the latter two groups also had statistical significance (P < 0.05). It indicates that the changes in quantities observed in Tregs are related with IST. Moreover, the percentage of Tregs in lymphocytes of SAA, R-SAA and normal control was (0.95 ± 0.57)%, (1.05 ± 0.49)%, and (1.78 ± 0.47)%, respectively (Fig. 1E). We also calculated the absolute Tregs numbers in SAA, R-SAA (PR), R-SAA (CR) and normal control group, which were (0.90 ± 0.83)  107/L, (0.97 ± 0.80)  107/L, (3.93 ± 2.50)  107/L and (4.43 ± 1.18)  107/L, respectively (Fig. 1F). The number of Tregs in SAA patients were substantially decreased compared with R-SAA and normal controls (P < 0.01), and there was a prominent difference between PR-SAA and CR-SAA group (P < 0.01), which further confirmed that the quantities of Tregs in SAA patients improved significantly after intensive immune suppressive treatment (IST).

substantially and significantly decreased when compared with normal control group (15.00 ± 8.30)% (P < 0.05), but had no significant difference with R-SAA group (8.61 ± 4.47)% (Fig. 2B). While the mean fluorescence intensity of CTLA-4+ Tregs of SAA, R-SAA and normal control were (27.40 ± 15.19), (23.37 ± 10.84) and (41.10 ± 20.75), with the significant difference between R-SAA and normal control (Fig. 2C). The SAA group had lower mean fluorescence intensity of CTLA-4+ Tregs compared with normal control but without significant difference. More interestingly, when multiplied by the percentage of Tregs in CD4+ T cells, the expression of CTLA-4 in Tregs within three groups had a little change, which meant the percentage of CTLA4+ Tregs in CD4+ T cells of SAA (0.26 ± 0.16)% and R-SAA group (0.34 ± 0.19)% was much lower than that of normal control group (0.64 ± 0.34)% (P < 0.01) (Fig. 2D). When multiplied by the percentage of Tregs in CD4+ T cells, the mean fluorescence intensity of CTLA-4+ Tregs of SAA, R-SAA and normal control became (0.80 ± 0.50), (0.94 ± 0.60) and (1.78 ± 0.86). SAA and R-SAA group were both lower than normal control, with significant difference (P < 0.01) (Fig. 2E).

3.2. CTLA-4 expression on Tregs in SAA and healthy controls

3.3. Perforin expression on Tregs in SAA and healthy controls

Based on the gate of CD4+ CD25+ CD127dim Tregs and isotype control used by mouse anti-human IgG antibody, we analyzed the expression percentage and mean fluorescence intensity of a series of different functional molecules (Fig. 2A). The results showed that the number of CTLA-4+ Tregs expressed as a fraction of total Tregs from SAA group was (9.11 ± 5.57)%, which was

The number of Perforin+ Tregs expressed as a fraction of total Tregs from SAA group was (3.80 ± 2.54)%, which was substantially and significantly increased when compared with normal control group (1.88 ± 1.55)% (P < 0.05), and R-SAA group (2.78 ± 2.52)% but had no significant difference (Fig. 3A). While the mean fluorescence intensity of Perforin+ Tregs of SAA, R-SAA and normal control

Fig. 3. The expression of perforin in PB Tregs from patients with SAA. (A) The expression of perforin in PB Tregs from patients with SAA (n = 19), R-SAA (n = 25) and normal control (n = 26). (B) The mean fluorescence intensity of perforin+ Tregs of SAA (n = 20), R-SAA (n = 25) and normal control (n = 25). (C) The expression of perforin in Tregs multiplied by the percentage of Tregs in CD4+ T cells, i.e. the percentage of perforin+ Tregs in CD4+ T cells from patients with SAA (n = 19), R-SAA (n = 25) and normal control (n = 26). (D) The mean fluorescence intensity of perforin+ Tregs multiplied by the percentage of Tregs in CD4+ T cells of SAA (n = 20), R-SAA (n = 25) and normal control (n = 26). Data were expressed as mean ± SD. ⁄P < 0.05; ⁄⁄P < 0.01.

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

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L. Yan et al. / Cellular Immunology xxx (2015) xxx–xxx

were (24.45 ± 21.28), (13.66 ± 7.36) and (22.14 ± 19.30), without any significant difference within three groups (Fig. 3B). Nevertheless, when multiplied by the percentage of Tregs in CD4+ T cells, the expression of Perforin in Tregs of SAA, R-SAA and normal control became (0.10 ± 0.06)%, (0.11 ± 0.09)% and (0.09 ± 0.09)%. The value of SAA group was no longer higher than that of normal control (Fig. 3C). When multiplied by the percentage of Tregs in CD4+ T cells, the mean fluorescence intensity of Perforin+ Tregs of SAA, R-SAA and normal control became (0.76 ± 0.71), (0.56 ± 0.33) and (1.00 ± 0.96). SAA and R-SAA group both had a little decreased tendency compared with normal control group, but without significant difference (Fig. 3D). 3.4. CD39 expression on Tregs in SAA and healthy controls The percentage of CD39+ Treg in total Tregs of SAA, R-SAA and normal control were (19.60 ± 15.44)%, (17.94 ± 13.80)% and (14.11 ± 13.05)%. SAA group was a bit higher than normal control but without significant difference (Fig. 4A). The mean fluorescence intensity of CD39+ Tregs of SAA was (49.51 ± 33.38), which was almost the same as R-SAA group (52.36 ± 27.85) and normal control group (50.52 ± 23.08) (Fig. 4B). On the contrary, when multiplied by the percentage of Tregs in CD4+ T cells, the expression of CD39 in Tregs of SAA (0.56 ± 0.56)% became a little lower than R-SAA (0.73 ± 0.58)% and normal control (0.70 ± 0.75)% instead, but still without significant difference (Fig. 4C). When multiplied by the percentage of Tregs in CD4+ T cells, the mean fluorescence intensity of CD39+ Tregs of SAA group

A

was (1.38 ± 1.04), which was significantly decreased when compared with normal control group (2.51 ± 1.51) (P < 0.05), and RSAA group (2.10 ± 1.21) but had no significant difference (Fig. 4D). 3.5. CD73 expression on Tregs in SAA and healthy controls The percentage of CD73+ Treg in total Tregs of SAA, R-SAA and normal control were (6.56 ± 3.67)%, (8.42 ± 5.65)% and (7.93 ± 3.63)%. SAA group was a bit lower than R-SAA and normal control group, but without significant difference (Fig. 5A). Similar to the percentage, the mean fluorescence intensity of CD73+ Tregs of SAA (36.52 ± 35.23) was a bit lower than R-SAA group (43.89 ± 27.72) and normal control group (61.28 ± 48.46), but without significant difference (Fig. 5B). Moreover, when multiplied by the percentage of Tregs in CD4+ T cells, the expression of CD73 in Tregs of SAA (0.19 ± 0.10)% became much lower than R-SAA (0.34 ± 0.24)% (P < 0.05) and normal control (0.39 ± 0.22)% (P < 0.01) (Fig. 5C). When multiplied by the percentage of Tregs in CD4+ T cells, the mean fluorescence intensity of CD73+ Tregs of SAA group was (1.04 ± 0.97), which was significantly decreased when compared with normal control group (3.06 ± 2.97) (P < 0.05), and R-SAA group (1.67 ± 0.88) but had no significant difference (Fig. 5D). 3.6. GITR expression on Tregs in SAA and healthy controls The percentage of GITR+ Treg in total Tregs of SAA, R-SAA and normal control were (20.17 ± 15.59)%, (17.38 ± 12.23)% and

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Fig. 4. The expression of CD39 in PB Tregs from patients with SAA. (A) The expression of CD39 in PB Tregs from patients with SAA (n = 17), R-SAA (n = 25) and normal control (n = 24). (B) The mean fluorescence intensity of CD39+ Tregs of SAA (n = 17), R-SAA (n = 25) and normal control (n = 24). (C) The expression of CD39 in Tregs multiplied by the percentage of Tregs in CD4+ T cells, i.e. the percentage of CD39+ Tregs in CD4+ T cells from patients with SAA (n = 18), R-SAA (n = 25) and normal control (n = 24). (D) The mean fluorescence intensity of CD39+ Tregs multiplied by the percentage of Tregs in CD4+ T cells of SAA (n = 18), R-SAA (n = 25) and normal control (n = 24). Data were expressed as mean ± SD. ⁄P < 0.05; ⁄⁄P < 0.01.

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

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L. Yan et al. / Cellular Immunology xxx (2015) xxx–xxx

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Fig. 5. The expression of CD73 in PB Tregs from patients with SAA. (A) The expression of CD73 in PB Tregs from patients with SAA (n = 18), R-SAA (n = 25) and normal control (n = 24). (B) The mean fluorescence intensity of CD73+ Tregs of SAA (n = 17), R-SAA (n = 25) and normal control (n = 24). (C) The expression of CD73 in Tregs multiplied by the percentage of Tregs in CD4+ T cells, i.e. the percentage of CD73+ Tregs in CD4+ T cells from patients with SAA (n = 18), R-SAA (n = 25) and normal control (n = 24). (D) The mean fluorescence intensity of CD73+ Tregs multiplied by the percentage of Tregs in CD4+ T cells of SAA (n = 18), R-SAA (n = 25) and normal control (n = 24). Data were expressed as mean ± SD. ⁄P < 0.05; ⁄⁄P < 0.01.

(15.78 ± 9.48)%. SAA group was a bit higher than normal control but without significant difference (Fig. 6A). The mean fluorescence intensity of GITR+ Tregs of SAA was (33.93 ± 22.29), which was almost the same as R-SAA group (27.88 ± 12.20) and normal control group (39.85 ± 20.80) (Fig. 6B). On the contrary, when multiplied by the percentage of Tregs in CD4+ T cells, the expression of GITR in Tregs of SAA (0.56 ± 0.40)% became a little lower than R-SAA (0.66 ± 0.52)% and normal control (0.73 ± 0.44)% instead, but still without significant difference (Fig. 6C). When multiplied by the percentage of Tregs in CD4+ T cells, the mean fluorescence intensity of GITR+ Tregs of SAA group was (1.00 ± 0.61), which was significantly decreased when compared with normal control group (1.83 ± 0.95) (P < 0.01). R-SAA group (1.06 ± 0.51) was lower than normal control as well (P < 0.01) (Fig. 6D).

3.7. Correlation of Tregs quantity and functional biomarkers expression with clinical parameters In SAA and R-SAA patients, the percentage of Tregs in CD4+ T cells was positively correlated with the level of absolute neutrophil count (r = 0.533; P = 0.000) (Fig. 7A), hemoglobin (r = 0.337; P = 0.024) (Fig. 7B), platelet count (r = 0.355; P = 0.017) (Fig. 7C), and proportion of reticulocytes in peripheral blood (r = 0.393; P = 0.008) (Fig. 7D). This indicates that quantity of Tregs was positively correlated with therapeutic effect.

The mean fluorescence intensity of perforin+ Tregs was negatively correlated with the level of hemoglobin (r = 0.303; P = 0.043) (Fig. 7E) and proportion of reticulocytes (r = 0.312; P = 0.039) (Fig. 7F). There was no significant relationship between the other biomarkers expression with clinical parameters.

4. Discussion As one of the key lymphocyte subpopulation in induction of immune tolerance, Tregs are believed to control development and progression of autoimmunity by suppressing autoreactive T cells. Accumulating data provided evidence that impaired function of Tregs contributed to the development of autoimmunity diseases, such as autoimmune hepatitis [19,20], type 1 diabetes [21], multiple sclerosis [22], rheumatoid arthritis [23], and systemic lupus erythematosus [24]. With the discovery that FoxP3 plays a central role in the differentiation and maintenance of Treg cells [6,7], the use of flow cytometry-based analysis of FoxP3 expression in T cells became the gold standard for defining Treg cells. However, it then became evident that FoxP3 can also be expressed by effector T cells following activation [25], raising the possibility that any assessment of Treg cell number or function may include recently activated effector T cells in the Treg cell population. Furthermore, as FoxP3 is a nuclear protein, assessment of its expression in T cells requires fixation and permeabilization of the cells, resulting in an inability to obtain viable cells for further functional analysis. In

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

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control

Fig. 6. The expression of GITR in PB Tregs from patients with SAA. (A) The expression of GITR in PB Tregs from patients with SAA (n = 18), R-SAA (n = 24) and normal control (n = 24). (B) The mean fluorescence intensity of GITR+ Tregs of SAA (n = 18), R-SAA (n = 24) and normal control (n = 24). (C) The expression of GITR in Tregs multiplied by the percentage of Tregs in CD4+ T cells, i.e. the percentage of GITR+ Tregs in CD4+ T cells from patients with SAA (n = 18), R-SAA (n = 25) and normal control (n = 24). (D) The mean fluorescence intensity of GITR+ Tregs multiplied by the percentage of Tregs in CD4+ T cells of SAA (n = 18), R-SAA (n = 25) and normal control (n = 24). Data were expressed as mean ± SD. ⁄P < 0.05; ⁄⁄P < 0.01.

the past few years, additional markers, such as CD127 (also known as IL-7Ra) [11], have been identified that assist in the distinction of effector T cells from Treg cells and facilitate the experimental purification of Treg cells [26]. An increasing number of studies have shown that SAA is an autoimmune disease caused by dysregulation of immune cell subsets, especially T lymphocytes [1,27,28]. There are several immune abnormalities associated with pathogenesis of SAA, including imbalance of DC subsets (elevated DC1), enhancement of DC function, insufficiency of regulatory T cell, imbalance of Th1/Th2 subsets (enhanced Th1), increase of type I lymphoid factors (IL-2, IFN-c), and decrease of NK cell proportion [29]. Previous data have shown inadequate numbers of PB Tregs in patients with AA [14], and our results confirmed this fact over again. Furthermore, the quantities of Tregs in AA patients could improve substantially after IST based on ATG + CsA. Considering the role of Tregs in AA, the decreasing dose of CsA seems to allow a better clinical effect [18]. Nevertheless, little is known regarding the function of Tregs in AA because of the attack of autologous T cells on BM hematopoietic progenitors. Recent studies have revealed several impairments of Tregs in AA. Kordasti and his colleagues have shown the reduction in absolute number and frequency of Tregs which also correlates with disease severity, and sorted Tregs from AA patients were unable to suppress cytokine secretion (both IL-2 and IFN-c) by autologous effector T cells [15]. Another study also reported the decreased frequencies of Tregs in PB and BM, reversed ratio of Treg frequencies of BM versus PB, the abnormality of migratory

potential, and defective immunosuppression on Teff cells in vitro [30]. Tregs probably use multiple suppressive mechanisms, and the importance of each one may vary depending on the environment and the context of immune responses [31]. The objective of our study was to determine the quantity and function of Tregs in SAA patients by measuring different biomarkers on cell surface after Tregs was defined as CD4+ CD25+ CD127dim. The role of CTLA-4 in immuno-regulation has been extensively investigated. Despite its constitutive expression on Treg, its precise contribution to their suppressive function has not been fully defined. A recent study in mice has shown that CTLA-4 is crucial for the suppressive function of Foxp3+ in vitro and in vivo [32]. CTLA-4 expressed by Tregs can modulate CD80 and CD86 expression by dendritic cells and thereby inhibit the activation of effector T cells. But in humans, it is intriguing that only CD45RO+ cells express CTLA-4 at high levels [33]. It remains to be determined whether the action of CTLA-4 on APCs is indispensable for the in vitro suppressive function of Tregs [26]. Our results showed a significant reduction of CTLA-4 expression in Treg from patients with SAA compared with Treg from healthy controls, indicating that CTLA-4 could be responsible for Treg abnormalities in SAA patients. It has been suggested that human Tregs can be suppressive in vitro even in the absence of APCs, indicating that target cell suppression can occur through direct contact between Tregs and effector T cells [26]. Some Tregs may differentiate to kill or inactivate

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

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B

Fig. 7. Correlation of Tregs quantity and functional biomarkers expression with clinical parameters. (A) The quantity of Tregs with absolute neutrophil count (n = 45, r = 0.533, P = 0.000). (B) The quantity of Tregs with hemoglobin (n = 45, r = 0.337, P = 0.024). (C) The quantity of Tregs with platelet count (n = 45, r = 0.355, P = 0.017). (D) The quantity of Tregs with reticulocytes proportion (n = 45, r = 0.393, P = 0.008). (E) The mean fluorescence intensity of perforin+ Tregs with hemoglobin (n = 45, r = 0.303, P = 0.043). (F) The mean fluorescence intensity of perforin+ Tregs with reticulocytes proportion (n = 45, r = 0.312, P = 0.039).

responder T cells by secreting granzyme/perforin or immunosuppressive cytokines (such as IL-10) [31]. Alternatively, absorption of cytokines by Tregs may induce apoptosis in responder T cells [34]. Tregs might kill responder T cells or APC through cell-to-cell contact by a granzyme- or perforin-dependent mechanism. Our results revealed a significant increment in perforin expression in Tregs from SAA patients compared with Tregs from healthy controls, which may be a kind of compensatory mechanism for the weakness of CTLA-4 mediate suppression. Even so, the value of SAA group became approximately equal to normal control when

multiplied by the percentage of Tregs, suggesting that perforin-dependent suppression of Tregs in SAA patients may not be powerful enough because of the inadequate number of whole Treg cells. Tregs might also kill responder T cells or APC through delivery of a negative signal to responder T cells. Possible negative signals include up-regulation of intracellular cyclic AMP, which leads to inhibition of T cell proliferation and IL-2 production, or the generation of pericellular adenosine catalyzed by CD39 and CD73 expressed by Tregs [35]. Inflammation promoted by extracellular nucleotides is subject to regulation by some members of the

Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001

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ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, such as CD39 and CD73 [36]. CD39 is also named ENTPD1 and degrades ATP to ADP and then to adenosine monophosphate (AMP) [36,37]. An ecto-50 -nucleotidase named CD73 and adenosine deaminase, among many others, contribute to the further catabolism of extracellular nucleotides [37,38]. CD73 degrades 50 -AMP to adenosine, which perhaps has anti-inflammatory properties, thus, contributing to the prevention of excessive inflammation through this pathway [39]. Tumor necrosis factor receptor superfamily member 18 (TNFRSF18) also known as activation-inducible TNFR family receptor (AITR) or glucocorticoid-induced TNFR-related protein (GITR), is an agonistic antibody in humans which is encoded by the TNFRSF18 gene. This gene encodes a member of the TNF-receptor superfamily. The encoded receptor has been shown to have increased expression upon T cell activation, and it is thought to play a key role in dominant immunological self-tolerance maintained by Tregs. Knockout studies in mice also suggest the role of this receptor is in the regulation of CD3-driven T cell activation and programmed cell death. A recent study demonstrated that low frequencies of GITR+ Tregs showed a higher susceptibility to apoptosis in ex vivo and in vitro, suggesting that GITR is a marker of Treg that would be primarily involved in cell survival rather than in their suppressor function [40]. In our results, expression of CD39, CD73 and GITR in Tregs from SAA patients is not decreased compared with Tregs from healthy controls, suggesting that the suppression mediated by CD39, CD73 and GITR, and survival capability of single Treg cell may not be injured. However, the expression of these three biomarkers in Tregs from SAA group became decreased when multiplied by the percentage of Tregs, reflecting that suppressive function of whole Tregs in SAA patients may still cannot be made up to normal condition because of the inadequate number of whole Treg cells. These results provide us an inspiration that adoptive transfer of Tregs into SAA patient may become one kind of treatment, as long as an effective in vitro culture system of Tregs could be established in future.

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Please cite this article in press as: L. Yan et al., Abnormal quantity and function of regulatory T cells in peripheral blood of patients with severe aplastic anemia, Cell. Immunol. (2015), http://dx.doi.org/10.1016/j.cellimm.2015.04.001