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Frequencies of PD-1- positive T CD3+CD4+, T CD3+CD8+ and B CD19+ lymphocytes in female patients with Graves' disease and healthy controls– preliminary study
Molecular and Cellular Endocrinology xxx (2017) 1e6
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Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study c, Aleksandra Pyzik a, *, Ewelina Grywalska a, Beata Matyjaszek-Matuszek b, Agata Smolen d a ski Dawid Pyzik , Jacek Rolin a
Department of Immunology, Medical University of Lublin, Lublin, Poland Department of Endocrinology, Medical University of Lublin, Lublin, Poland Department of Epidemiology, Medical University of Lublin, Lublin, Poland d Department of Orthopedics and Traumatology of the Locomotor System, Medical University of Lublin, Lublin, Poland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 September 2016 Received in revised form 5 March 2017 Accepted 7 March 2017 Available online xxx
PD-1 maintains tolerance and inhibits autoimmune responses. Graves' disease (GD) is one of the most frequent autoimmune diseases of unclear etiology. The aim of this study was to evaluate the percentage and absolute counts of PD-1 positive T and B cells in newly diagnosed, untreated patients with hyperthyroidism due to GD. The study group included 30 patients and the control group comprised of 20 ageand sex-matched healthy individuals. Results showed significantly higher frequencies and absolute counts of PD-1 positive CD3þCD4þ T cells, CD3þCD8þ T cells and CD19þB cells in patients with GD in comparison to the healthy volunteers. Moreover, higher mean fluorescence intensity of PD-1 was found on CD3þCD4þ T cells, CD3þCD8þ T cells and CD19þB cells in the study group than in the control group. These results suggest that PD-1 protein might involved in the pathogenesis of GD. © 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Graves' disease PD-1 CD279 Hyperthyroidism Lymphocytes
1. Introduction Graves' disease (GD) is a chronic, organ specific autoimmune disorder of the thyroid of unclear etiology. It is one of the most frequent autoimmune diseases with the average incidence of 14/ 100,000 per year and it is still growing (McLeod and Cooper, 2012). In iodine-sufficient areas, GD comprises about 70e80% of all cases of hyperthyroidism (Karasek and Lewinski, 2003). GD is directly caused by the loss of tolerance to self antigens, mainly to the thyroid stimulating hormone receptor (TSHR). It is caused, among others, by impaired function of regulatory T cells (Tregs, CD4þCD25þhighFoxp3þ) and/or their depletion. Such process results in the imbalance between effector and regulatory cells. The surviving T helper cells (mainly Th2), recognize “self“ antigens as
* Corresponding author. Department of Immunology, Medical University of Lublin, Chod zki 4a, 20-093 Lublin. Poland. E-mail addresses: [email protected] (A. Pyzik), ewelina.grywalska@ gmail.com (E. Grywalska), [email protected] (B. Matyjaszek-Matuszek), agata. ), [email protected] (D. Pyzik), jacek.rolinski@ [email protected] (A. Smolen ski). gmail.com (J. Rolin
“foreign”, which in turn induces the production of cytokines (IL-2, IL-4, IL-5) that stimulate B cells (CD19þ) to produce TSHR autoantibodies (TSHR-Ab). After binding with TSHR these activate adenylate cyclase and stimulate the thyroid gland to produce hormones, which leads to the clinical manifestation of hyperthyroidism (Ploski et al., 2011; Hen et al., 2012; Klatka et al., 2012). Due to difficulties in achieving euthyreosis or remission in conservative treatment, predisposition to relapsing hyperthyroidism, high and volatile risk of thyroid-associated orbitopathy, finding ethiopathogenesis of this disease will translate into a successful therapy. PD-1 (CD279) is an immunoreceptor, a 50e55 kDa type protein of 288 amino acids which is a member of the CD28 receptor family and the immunoglobulin-like superfamily. To date, more than 30 PD-1 single-nucleotide polymorphisms (SNPs) have been detected and found to be linked to the development of such human autoimmune diseases as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS) and DM1 (Prokunina et al., 2002; Helms et al., 2003; Nielsen et al., 2003; Kroner et al., 2005; Lee et al., 2006; Okazaki and Honjo, 2007). PD-1 is expressed on the surface of activated T cells, B cells, NK cells, monocytes and some dendritic cell (DC) subsets. If, unbound it can
http://dx.doi.org/10.1016/j.mce.2017.03.006 0303-7207/© 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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A. Pyzik et al. / Molecular and Cellular Endocrinology xxx (2017) 1e6
Abbreviations GD AITD DM1 TSHR Tregs ITregs TSHR-Ab SNPs RA SLE MS
Graves' disease autoimmune thyroid disease diabetes mellitus type 1 thyroid stimulating hormone receptor regulatory T cells (CD4þCD25þhighFoxp3þ) induced regulatory T cells thyroid stimulating hormone receptor autoantibodies single-nucleotide polymorphisms rheumatoid arthritis systemic lupus erythematosus multiple sclerosis
be detected in the cytoplasm of regulatory T cells (Foxp3þ) and naïve CD4þ. PD-1 does not play an enzymatic role e it serves as an adapter transmitting signal to the cell (Grzywnowicz and Giannopoulos, 2012; Kong and Flynn, 2014). Regulation of the immune response by PD-1 starts with its binding with its ligand PD-L1 (B7-H1; CD 274) or PD-L2 (B7-DC; CD273). The main role of this protein is to maintain tolerance especially the peripheral one and to inhibit autoimmune responses by down-regulating the proliferation and production of cytokines by T cells and to control damage of healthy tissues during infection (Riley, 2009). This pathway may also prevent acquiring effector characteristics via autoreactive naïve T cells. Interaction of PD-1 and PD-L1 on naïve T cells may stimulate induced regulatory T cells (iTregs) as well as maintain and regulate their function. However, PD-L1 expressed on regulatory T cells may also inhibit PD-1 expression on T cells and DCs. Blocking the PD-1/PD-L1 pathway increases the response against an antigen presented by DCs. 2. Aim The aim of the study was to evaluate the percentage and number of PD-1 positive T CD3þ cells (CD4þ and CD8þ) and B cells (CD19þ) in the patients newly diagnosed with hyperthyroidism in the course of GD and to find correlations between the increased expression of this molecule and the thyroid stimulating hormone (TSH) levels, free thyroid hormones (FT3, FT4) or TRAb, anti-TPO, and clinical symptoms.
DC TSH FT3 FT4 anti-TPO PBS PBMCs MFI PD-L1 TRAIL EAE APC
dendritic cell thyroid stimulating hormone free triiodothyronine free thyroxine thyroperoxidase antibodies phosphate-buffered saline peripheral blood mononuclear cells mean fluorescence intensity programmed death-ligand 1 TNF-related apoptosis inducing ligand experimental autoimmune encephalomyelitis antigen presenting cell
and laboratory tests (elevated FT3 and FT4 with suppressed TSH levels). Whereas, GD was diagnosed basing on the positive tests for TRAb. The tests were performed in the Radiology and Nuclear Medicine Department of the Medical University of Lublin with radioimmunoassay. TRAb >1.5 U/l was regarded as positive. 3.2. Material The study material was peripheral blood collected from the basilic vein into tubes containing 10 ml of heparin (aspiration and vacuum systems, Sarstedt, Germany). The collected material was immediately tested in the Department of Clinical Immunology of the Medical University of Lublin by the flow cytometric method. 3.3. Isolation of peripheral blood mononuclear cells (PBMCs) The peripheral blood was diluted 1:1 with a 0.9% phosphatebuffered saline (PBS) without calcium (Caþ) or magnesium (Mgþ) ions (Biochrome AG, Germany). The diluted material was then overlaid on 3 ml of Gradisol L preparation (Aqua Medica, Poland) with specific gravity of 1.077 g/ml and subjected to density gradient centrifugation at 700 g for 20 min. The obtained fraction of PBMCs was collected with a Pasteur pipette and washed twice in PBS without Caþ and Mgþions for 5 min. Next, the cells were suspended in 1 ml PBS without Caþ and Mgþ and counted in the Neubauer chamber and their viability was determined by a trypan blue dye exclusion assay (0.4% Trypan Blue Solution, Sigma Aldrich, Germany).
3. Material and methods 3.4. Flow cytometric analysis 3.1. Patients and control group The study group included 30 females with newly diagnosed GD treated in the Department of Endocrinology of the Medical University of Lublin and Outpatient Clinic of Teaching Hospital No. 4 in Lublin. The mean age of the patients was 41.6 ± 16.28 years (min. 20, max. 56). The control group included 20 females between 19 and 53 years of age (average 39.15 ± 13.73) with normal thyroid hormone levels. The patients who had a positive history of autoimmune co-morbidities, suffered from allergies, experienced symptoms of infection in the last 4 weeks before the study, took medication affecting the immune system. The patients who had undergone blood transfusion were excluded from the study from both groups. All participants gave written consent of their participation in the study. The study protocol was accepted by the Bioethics Committee of the Medical University in Lublin. Hyperthyroidism was diagnosed basing on the clinical picture
PBMCs obtained from density gradient centrifugation were then subjected to 2- or 3-color staining with monoclonal antibodies according to the recommendations of the manufacturer. The suspension of cells was distributed 1 106 per tube and incubated at room temperature with monoclonal antibodies for 20 min (20 ml antibodies/tube): anti-CD19 FITC/anti-PD-1 PE, anti-CD3 Pe-Cy5/ anti-CD4 FITC/anti-PD-1 PE, anti-CD3 Pe-Cy5/anti-CD8 FITC/antiPD-1 PE. In Table 1 we present the list of antibodies used to determine PD-1 status of lymphocytes T CD3þ CD4þ, T CD3þCD8þ and B CD19þ, along with their isotype controls and catalogue numbers. After the incubation, the cells were washed twice with buffered PBS (700 g, 5 min) and immediately analyzed with the FACS Calibur flow cytometer (Becton Dickinson, US). Data acquisition was performed with FACS Diva Software 6.1.3 (BD) with 30,000 cells analyzed in each run. The analysis of the data was
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
A. Pyzik et al. / Molecular and Cellular Endocrinology xxx (2017) 1e6
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Table 1 The list of antibodies used to determine PD-1 status of lymphocytes T CD3þ CD4þ, T CD3þCD8þ and B CD19þ, along with their isotype controls and catalogue numbers. Antibody
Fluorochrome
Name
Catalogue number
Anti- CD19
FITC
FITC Mouse Anti-Human CD19 Clone HIB19 (RUO)
Anti-CD3
Pe-Cy5
PE-Cy™5 Mouse Anti-Human CD3 Clone HIT3a (RUO)
Anti- CD4
FITC
Mouse Anti-Human CD4 Clone RPA-T4 (RUO)
Anti- CD8
FITC
Mouse Anti-Human CD8 Clone HIT8a (RUO)
Anti- PD1
PE
Mouse Anti-Human CD279 Clone MIH4 (RUO)
Anti- CD45/14
FITC/PE
CD45 FITC/CD14 PE (2D1, M4P9)
Isotype Control IgG1 К BD
FITC
FITC Mouse IgG1, k Isotype Control Clone MOPC-21
Isotype Control IgG1 К BD
PE
PE Mouse IgG1, k Isotype Control Clone MOPC-21 (RUO)
555412 BD Biosciences, 555341 BD Biosciences, 555346 BD Biosciences, 555634 BD Biosciences, 557946 BD Biosciences, 342408 BD Biosciences, 555748 BD Biosciences, 555749 BD Biosciences,
carried out with CellQuest Pro Software (Becton Dickinson, US). The results of the cytometric analysis were shown as a number of cells stained with monoclonal antibodies conjugated to fluorescent dyes and as the mean fluorescence intensity (MFI) which represents the intensity of cell surface antigen expression. 4. Statistical analysis The results were statistically analyzed. Since the ratio scale was applied, the values of the parameters were expressed as the arithmetic mean, standard deviation, median, minimum and maximum values and/or lower and upper quartiles and the range of variability. Due to the skewed distribution of the measured parameters evaluated with the Shapiro-Wilk test or the heterogeneity of variances evaluated with the F-test, the Mann-Whitney U test was employed to compare the values between the groups. Whereas, the Spearman's rank correlation coefficient was applied to evaluate the correlations between the studied parameters. The error was set at 5% and the significance at p < 0.05. Statistical analyses were performed using Statistica v. 10.0 software (StatSoft, Poland). 5. Results The flow cytometry test showed a significantly higher percentage and the absolute number of PD-1 positive CD3þCD4þ, CD3þCD8þ T cells and CD 19þB cells in the study group compared
USA USA USA USA USA USA USA USA
with the control one. Similarly, the MFI of PD-1 on CD3þCD4þ, CD3þCD8þ T cells and CD19þ B cells in GD patients was significantly higher than in the control group. Comparison of the flow cytometric analysis results are presented in Table 2. The representative histograms of PD-1 expression on the surface of T CD3þ/CD4þ, T CD3þ/CD8þ and B CD19þ cells in a newly diagnosed, treatment-naive patient with GD and in a healthy person are presented in Figs. 1 and 2. No significant difference was found between the study and the control groups in terms of mean lymphocytosis and it amounted to 1.89 ± 0.45 and 1.89 ± 0.42, respectively. Moreover, a higher percentage of CD3þCD4þ T cells and a lower percentage of CD3þCD8þ T cells was found in the study than in the control group and the percentage of CD19 þ B cells was similar in both groups (Table 2). The mean levels of hormones in the study group were: TSH 0.039 ± 0.017 mIU/l, FT4- 47.60 ± 20.75 pmol/l, FT335.35 ± 35.59 pmol/l, TRAb - 12.33 ± 9.98 IU/l and anti-TPO 1916.57 ± 2767.98 IU/l. In the control group mean TSH was 1.49 ± 0.364 mIU/l, FT4-1.56 ± 0.46 pmol/l, FT3-3.07 ± 1.25 pmol/l, TRAb e 1.02 ± 0.21 IU/l and anti-TPO - 5.28 ± 2.37 IU/l. Other correlations were also analyzed such as between the PD-1 expression and the clinical picture (the number of symptoms and signs), anti-TPO and anti-TSH and the levels of TSH, FT3 and FT4 as well as the mean lymphocytosis. However, no significant correlation was found between the PD-1 expression and any of the studied parameters.
Table 2 Comparison of the flow cytometric analysis results of the basic subclass of peripheral blood lymphocytes and expression of PD-1 on CD3þCD4þ, CD3þCD8þ T cells and CD19þcells in the study and control groups. Parameter
Study group
Control group
p
CD3þ T cells [%] CD19þB cells [%] CD3þ/CD4þ T cells [%] CD3þ/CD8þ T cells [%] CD4þ/CD8þ T cells ratio CD3þ/CD4þ/PD-1 T cells [%] CD3þ/CD4þ/PD-1 T cells number [103/mm3] MFI of PD-1 on CD3þ/CD4þ T cells CD3þ/CD8þ/PD-1 T cells [%] CD3þ/CD8þ/PD-1 T cells number [103/mm3] MFI of PD-1 on CD3þ/CD8þ T cells CD19þ/PD-1 B cells [%] CD19þ/PD-1 B cells number [103/mm3] MFI of PD-1 on CD19 þ B cells
69.16 ± 4.06 12.05 ± 3.51 53.03 ± 7.55* 26.90 ± 3.69* 2.02 ± 0.48* 35.15 ± 11.10* 0.35 ± 0.13* 163.32 ± 60.63* 21.20 ± 9.27* 0.11 ± 0.06* 141.48 ± 34.63* 13.10 ± 2.45* 0.03 ± 0.01* 139.06 ± 33.08*
68.26 ± 3.84 11.25 ± 2.50 44.46 ± 2.50* 34.36 ± 3.29* 1.30 ± 0.16* 5.35 ± 1.54* 0.04 ± 0.02* 34.33 ± 18.01* 3.6 ± 1.45* 0.02 ± 0.01* 22.85 ± 13.11* 1.67 ± 0.84* 0.00 ± 0.00 41.62 ± 17.07*
0.372 0.422 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
*p < 0.05 compared with the control group; MFI- median fluorescence intensity.
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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Fig. 1. Sample analysis of PD-1þ/CD4þ T lymphocytes, PD-1þ/CD8þ T lymphocytes and PD-1þ/CD19 þ B lymphocytes in a newly diagnosed, treatment-naive patient with GD.
6. Discussion Our study has shown increased percentage and number of CD279-positive CD3þCD4þ, CD3þCD8þ T cells and CD19þ B cells in patients at GD diagnosis. Literature to date has stressed the role of PD-1 and its ligands in the pathogenesis of autoimmune diseases, allergies, infections, neoplasms and in transplantology (Helms et al., 2003; Siwiec and Majdan, 2015). Many studies have suggested that PD-1 and its ligands play an important immunoregulatory function. Such authors like Salama et al. (2003), Latchman et al. (2004) showed that PD-1 inhibits the proliferation of naïve and effector T cells as well as of autoreactive T cells and the production of cytokines, which plays a key role in the maintenance of central and peripheral tolerance (Salama et al., 2003; Latchman et al., 2004). Recent years have witnessed many studies on the function of PD-1 in human autoimmune diseases. Despite this fact, no data on its role in the pathogenesis of GD e as one of the most prevalent autoimmune diseases e have been found. First such study on mice was conducted by Nishimura et al. (1999). The authors showed that PD-1 deficiency translates into the spontaneous development of lupus-like diseases with a late onset and deposition of IgG3 in renal
glomeruli and dilated cardiomyopathy related to the production of antibodies against cardiac troponin I (Nishimura et al., 1999). While, Ansari et al. (2003) observed that non-obese prediabetic mice administered with anti-PD-1/anti-PD-L1 quickly developed diabetes due to pancreatitis and the production of proinflammatory cytokines by activated T cells (Ansari et al., 2003). The authors also proved that PD-1/PD-L1 interaction plays an important role both in the onset and the progression of diabetes in this group of animal models. This is how the involvement of this protein in downregulation of immune response was corroborated. It was proven that PD-1 deficiency fosters the development of autoimmune diseases due to no inhibition of activated lymphocyte proliferation (Goła˛ bek and Wo zniakowska-Ge˛ sicka, 2010). Therefore, it may be assumed that the cells producing antibodies in autoimmune diseases may escape the immune system. Increased PD-1 expression on T cells was also observed in other autoimmune disorders such as €gren's syndrome (Hatachi et al., 2003; Kobayashi et al., RA or Sjo 2005). Wan et al. found in the serum and synovial fluid of RA patients an additional soluble PD-1 splicing variant which blocks PD-1 and deteriorates the course of the disease (Wan et al., 2006). This soluble form of the protein was also observed in aplastic anemia along with increased T cell proliferation (Wu et al., 2009). What is
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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Fig. 2. Sample analysis of PD-1þ/CD4þ T lymphocytes, PD-1þ/CD8þ T lymphocytes and PD-1þ/CD19 þ B lymphocytes in a healthy person.
more, in the serum of RA patients some authors found antibodies against PD-L1, which correlates with the disease progression (Dong et al., 2003). Dong et al. (1999) in their study on ligands for the PD-1 receptor observed their ability to promote the activation and proliferation of T cells at an early activation stage by unidentified receptors (Dong et al., 1999). This stimulatory effect mainly refers to T helper cells, which was corroborated in RA patients where the antagonistic antibody binding to PD-L1 stimulated the activation and proliferation of resting T cells, increased cytokine production and aggravated activated T cell apoptosis through the activation of caspase-3 and expression of TRAIL (Dong et al., 2003). Salama et al. (2003) confirmed that PD-1 blockade leads to clinical deterioration of the patients with experimental autoimmune encephalomyelitis (EAE) (Salama et al., 2003). 7. Conclusions This study showed a significantly higher percentage and number of PD-1 positive CD3þ CD4þ, CD3þ CD8þ T cells and CD19þ B cells in the study group than in the control group. Described impaired PD-1 expression in GD also observed in other autoimmune diseases shed a light on its role in the pathogenesis of such
disorders. Although our findings imply that PD-1 may play a role in pathogenesis of GD, this study did not provide mechanistic data to support this. Nevertheless, the results of this preliminary research provided a novel insight into the role ofPD-1 in GD development. We recommend that further studies are needed to elucidate whether the normalization of FT4 and FT3 levels correlates with the decrease in the number of PD-1 positive cells and/or the MFI of PD1, which may lead to the conclusion that potential PD-1/PD-L1 blockade may constitute a novel therapeutic option in GD. Funding This work was supported by research grants no. N N402 682440, no. UMO-2011/01/N/NZ6/01762 and no. UMO-2012/05/B/NZ6/ 00792 of the Polish National Science Centre and MNsd 524, MNmb 523 and DS460 of the Medical University of Lublin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of interest The authors declare that there is no conflict of interest that
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study c, Aleksandra Pyzik a, *, Ewelina Grywalska a, Beata Matyjaszek-Matuszek b, Agata Smolen d a ski Dawid Pyzik , Jacek Rolin a
Department of Immunology, Medical University of Lublin, Lublin, Poland Department of Endocrinology, Medical University of Lublin, Lublin, Poland Department of Epidemiology, Medical University of Lublin, Lublin, Poland d Department of Orthopedics and Traumatology of the Locomotor System, Medical University of Lublin, Lublin, Poland b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 September 2016 Received in revised form 5 March 2017 Accepted 7 March 2017 Available online xxx
PD-1 maintains tolerance and inhibits autoimmune responses. Graves' disease (GD) is one of the most frequent autoimmune diseases of unclear etiology. The aim of this study was to evaluate the percentage and absolute counts of PD-1 positive T and B cells in newly diagnosed, untreated patients with hyperthyroidism due to GD. The study group included 30 patients and the control group comprised of 20 ageand sex-matched healthy individuals. Results showed significantly higher frequencies and absolute counts of PD-1 positive CD3þCD4þ T cells, CD3þCD8þ T cells and CD19þB cells in patients with GD in comparison to the healthy volunteers. Moreover, higher mean fluorescence intensity of PD-1 was found on CD3þCD4þ T cells, CD3þCD8þ T cells and CD19þB cells in the study group than in the control group. These results suggest that PD-1 protein might involved in the pathogenesis of GD. © 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BYNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Graves' disease PD-1 CD279 Hyperthyroidism Lymphocytes
1. Introduction Graves' disease (GD) is a chronic, organ specific autoimmune disorder of the thyroid of unclear etiology. It is one of the most frequent autoimmune diseases with the average incidence of 14/ 100,000 per year and it is still growing (McLeod and Cooper, 2012). In iodine-sufficient areas, GD comprises about 70e80% of all cases of hyperthyroidism (Karasek and Lewinski, 2003). GD is directly caused by the loss of tolerance to self antigens, mainly to the thyroid stimulating hormone receptor (TSHR). It is caused, among others, by impaired function of regulatory T cells (Tregs, CD4þCD25þhighFoxp3þ) and/or their depletion. Such process results in the imbalance between effector and regulatory cells. The surviving T helper cells (mainly Th2), recognize “self“ antigens as
* Corresponding author. Department of Immunology, Medical University of Lublin, Chod zki 4a, 20-093 Lublin. Poland. E-mail addresses: [email protected] (A. Pyzik), ewelina.grywalska@ gmail.com (E. Grywalska), [email protected] (B. Matyjaszek-Matuszek), agata. ), [email protected] (D. Pyzik), jacek.rolinski@ [email protected] (A. Smolen ski). gmail.com (J. Rolin
“foreign”, which in turn induces the production of cytokines (IL-2, IL-4, IL-5) that stimulate B cells (CD19þ) to produce TSHR autoantibodies (TSHR-Ab). After binding with TSHR these activate adenylate cyclase and stimulate the thyroid gland to produce hormones, which leads to the clinical manifestation of hyperthyroidism (Ploski et al., 2011; Hen et al., 2012; Klatka et al., 2012). Due to difficulties in achieving euthyreosis or remission in conservative treatment, predisposition to relapsing hyperthyroidism, high and volatile risk of thyroid-associated orbitopathy, finding ethiopathogenesis of this disease will translate into a successful therapy. PD-1 (CD279) is an immunoreceptor, a 50e55 kDa type protein of 288 amino acids which is a member of the CD28 receptor family and the immunoglobulin-like superfamily. To date, more than 30 PD-1 single-nucleotide polymorphisms (SNPs) have been detected and found to be linked to the development of such human autoimmune diseases as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS) and DM1 (Prokunina et al., 2002; Helms et al., 2003; Nielsen et al., 2003; Kroner et al., 2005; Lee et al., 2006; Okazaki and Honjo, 2007). PD-1 is expressed on the surface of activated T cells, B cells, NK cells, monocytes and some dendritic cell (DC) subsets. If, unbound it can
http://dx.doi.org/10.1016/j.mce.2017.03.006 0303-7207/© 2017 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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Abbreviations GD AITD DM1 TSHR Tregs ITregs TSHR-Ab SNPs RA SLE MS
Graves' disease autoimmune thyroid disease diabetes mellitus type 1 thyroid stimulating hormone receptor regulatory T cells (CD4þCD25þhighFoxp3þ) induced regulatory T cells thyroid stimulating hormone receptor autoantibodies single-nucleotide polymorphisms rheumatoid arthritis systemic lupus erythematosus multiple sclerosis
be detected in the cytoplasm of regulatory T cells (Foxp3þ) and naïve CD4þ. PD-1 does not play an enzymatic role e it serves as an adapter transmitting signal to the cell (Grzywnowicz and Giannopoulos, 2012; Kong and Flynn, 2014). Regulation of the immune response by PD-1 starts with its binding with its ligand PD-L1 (B7-H1; CD 274) or PD-L2 (B7-DC; CD273). The main role of this protein is to maintain tolerance especially the peripheral one and to inhibit autoimmune responses by down-regulating the proliferation and production of cytokines by T cells and to control damage of healthy tissues during infection (Riley, 2009). This pathway may also prevent acquiring effector characteristics via autoreactive naïve T cells. Interaction of PD-1 and PD-L1 on naïve T cells may stimulate induced regulatory T cells (iTregs) as well as maintain and regulate their function. However, PD-L1 expressed on regulatory T cells may also inhibit PD-1 expression on T cells and DCs. Blocking the PD-1/PD-L1 pathway increases the response against an antigen presented by DCs. 2. Aim The aim of the study was to evaluate the percentage and number of PD-1 positive T CD3þ cells (CD4þ and CD8þ) and B cells (CD19þ) in the patients newly diagnosed with hyperthyroidism in the course of GD and to find correlations between the increased expression of this molecule and the thyroid stimulating hormone (TSH) levels, free thyroid hormones (FT3, FT4) or TRAb, anti-TPO, and clinical symptoms.
DC TSH FT3 FT4 anti-TPO PBS PBMCs MFI PD-L1 TRAIL EAE APC
dendritic cell thyroid stimulating hormone free triiodothyronine free thyroxine thyroperoxidase antibodies phosphate-buffered saline peripheral blood mononuclear cells mean fluorescence intensity programmed death-ligand 1 TNF-related apoptosis inducing ligand experimental autoimmune encephalomyelitis antigen presenting cell
and laboratory tests (elevated FT3 and FT4 with suppressed TSH levels). Whereas, GD was diagnosed basing on the positive tests for TRAb. The tests were performed in the Radiology and Nuclear Medicine Department of the Medical University of Lublin with radioimmunoassay. TRAb >1.5 U/l was regarded as positive. 3.2. Material The study material was peripheral blood collected from the basilic vein into tubes containing 10 ml of heparin (aspiration and vacuum systems, Sarstedt, Germany). The collected material was immediately tested in the Department of Clinical Immunology of the Medical University of Lublin by the flow cytometric method. 3.3. Isolation of peripheral blood mononuclear cells (PBMCs) The peripheral blood was diluted 1:1 with a 0.9% phosphatebuffered saline (PBS) without calcium (Caþ) or magnesium (Mgþ) ions (Biochrome AG, Germany). The diluted material was then overlaid on 3 ml of Gradisol L preparation (Aqua Medica, Poland) with specific gravity of 1.077 g/ml and subjected to density gradient centrifugation at 700 g for 20 min. The obtained fraction of PBMCs was collected with a Pasteur pipette and washed twice in PBS without Caþ and Mgþions for 5 min. Next, the cells were suspended in 1 ml PBS without Caþ and Mgþ and counted in the Neubauer chamber and their viability was determined by a trypan blue dye exclusion assay (0.4% Trypan Blue Solution, Sigma Aldrich, Germany).
3. Material and methods 3.4. Flow cytometric analysis 3.1. Patients and control group The study group included 30 females with newly diagnosed GD treated in the Department of Endocrinology of the Medical University of Lublin and Outpatient Clinic of Teaching Hospital No. 4 in Lublin. The mean age of the patients was 41.6 ± 16.28 years (min. 20, max. 56). The control group included 20 females between 19 and 53 years of age (average 39.15 ± 13.73) with normal thyroid hormone levels. The patients who had a positive history of autoimmune co-morbidities, suffered from allergies, experienced symptoms of infection in the last 4 weeks before the study, took medication affecting the immune system. The patients who had undergone blood transfusion were excluded from the study from both groups. All participants gave written consent of their participation in the study. The study protocol was accepted by the Bioethics Committee of the Medical University in Lublin. Hyperthyroidism was diagnosed basing on the clinical picture
PBMCs obtained from density gradient centrifugation were then subjected to 2- or 3-color staining with monoclonal antibodies according to the recommendations of the manufacturer. The suspension of cells was distributed 1 106 per tube and incubated at room temperature with monoclonal antibodies for 20 min (20 ml antibodies/tube): anti-CD19 FITC/anti-PD-1 PE, anti-CD3 Pe-Cy5/ anti-CD4 FITC/anti-PD-1 PE, anti-CD3 Pe-Cy5/anti-CD8 FITC/antiPD-1 PE. In Table 1 we present the list of antibodies used to determine PD-1 status of lymphocytes T CD3þ CD4þ, T CD3þCD8þ and B CD19þ, along with their isotype controls and catalogue numbers. After the incubation, the cells were washed twice with buffered PBS (700 g, 5 min) and immediately analyzed with the FACS Calibur flow cytometer (Becton Dickinson, US). Data acquisition was performed with FACS Diva Software 6.1.3 (BD) with 30,000 cells analyzed in each run. The analysis of the data was
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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Table 1 The list of antibodies used to determine PD-1 status of lymphocytes T CD3þ CD4þ, T CD3þCD8þ and B CD19þ, along with their isotype controls and catalogue numbers. Antibody
Fluorochrome
Name
Catalogue number
Anti- CD19
FITC
FITC Mouse Anti-Human CD19 Clone HIB19 (RUO)
Anti-CD3
Pe-Cy5
PE-Cy™5 Mouse Anti-Human CD3 Clone HIT3a (RUO)
Anti- CD4
FITC
Mouse Anti-Human CD4 Clone RPA-T4 (RUO)
Anti- CD8
FITC
Mouse Anti-Human CD8 Clone HIT8a (RUO)
Anti- PD1
PE
Mouse Anti-Human CD279 Clone MIH4 (RUO)
Anti- CD45/14
FITC/PE
CD45 FITC/CD14 PE (2D1, M4P9)
Isotype Control IgG1 К BD
FITC
FITC Mouse IgG1, k Isotype Control Clone MOPC-21
Isotype Control IgG1 К BD
PE
PE Mouse IgG1, k Isotype Control Clone MOPC-21 (RUO)
555412 BD Biosciences, 555341 BD Biosciences, 555346 BD Biosciences, 555634 BD Biosciences, 557946 BD Biosciences, 342408 BD Biosciences, 555748 BD Biosciences, 555749 BD Biosciences,
carried out with CellQuest Pro Software (Becton Dickinson, US). The results of the cytometric analysis were shown as a number of cells stained with monoclonal antibodies conjugated to fluorescent dyes and as the mean fluorescence intensity (MFI) which represents the intensity of cell surface antigen expression. 4. Statistical analysis The results were statistically analyzed. Since the ratio scale was applied, the values of the parameters were expressed as the arithmetic mean, standard deviation, median, minimum and maximum values and/or lower and upper quartiles and the range of variability. Due to the skewed distribution of the measured parameters evaluated with the Shapiro-Wilk test or the heterogeneity of variances evaluated with the F-test, the Mann-Whitney U test was employed to compare the values between the groups. Whereas, the Spearman's rank correlation coefficient was applied to evaluate the correlations between the studied parameters. The error was set at 5% and the significance at p < 0.05. Statistical analyses were performed using Statistica v. 10.0 software (StatSoft, Poland). 5. Results The flow cytometry test showed a significantly higher percentage and the absolute number of PD-1 positive CD3þCD4þ, CD3þCD8þ T cells and CD 19þB cells in the study group compared
USA USA USA USA USA USA USA USA
with the control one. Similarly, the MFI of PD-1 on CD3þCD4þ, CD3þCD8þ T cells and CD19þ B cells in GD patients was significantly higher than in the control group. Comparison of the flow cytometric analysis results are presented in Table 2. The representative histograms of PD-1 expression on the surface of T CD3þ/CD4þ, T CD3þ/CD8þ and B CD19þ cells in a newly diagnosed, treatment-naive patient with GD and in a healthy person are presented in Figs. 1 and 2. No significant difference was found between the study and the control groups in terms of mean lymphocytosis and it amounted to 1.89 ± 0.45 and 1.89 ± 0.42, respectively. Moreover, a higher percentage of CD3þCD4þ T cells and a lower percentage of CD3þCD8þ T cells was found in the study than in the control group and the percentage of CD19 þ B cells was similar in both groups (Table 2). The mean levels of hormones in the study group were: TSH 0.039 ± 0.017 mIU/l, FT4- 47.60 ± 20.75 pmol/l, FT335.35 ± 35.59 pmol/l, TRAb - 12.33 ± 9.98 IU/l and anti-TPO 1916.57 ± 2767.98 IU/l. In the control group mean TSH was 1.49 ± 0.364 mIU/l, FT4-1.56 ± 0.46 pmol/l, FT3-3.07 ± 1.25 pmol/l, TRAb e 1.02 ± 0.21 IU/l and anti-TPO - 5.28 ± 2.37 IU/l. Other correlations were also analyzed such as between the PD-1 expression and the clinical picture (the number of symptoms and signs), anti-TPO and anti-TSH and the levels of TSH, FT3 and FT4 as well as the mean lymphocytosis. However, no significant correlation was found between the PD-1 expression and any of the studied parameters.
Table 2 Comparison of the flow cytometric analysis results of the basic subclass of peripheral blood lymphocytes and expression of PD-1 on CD3þCD4þ, CD3þCD8þ T cells and CD19þcells in the study and control groups. Parameter
Study group
Control group
p
CD3þ T cells [%] CD19þB cells [%] CD3þ/CD4þ T cells [%] CD3þ/CD8þ T cells [%] CD4þ/CD8þ T cells ratio CD3þ/CD4þ/PD-1 T cells [%] CD3þ/CD4þ/PD-1 T cells number [103/mm3] MFI of PD-1 on CD3þ/CD4þ T cells CD3þ/CD8þ/PD-1 T cells [%] CD3þ/CD8þ/PD-1 T cells number [103/mm3] MFI of PD-1 on CD3þ/CD8þ T cells CD19þ/PD-1 B cells [%] CD19þ/PD-1 B cells number [103/mm3] MFI of PD-1 on CD19 þ B cells
69.16 ± 4.06 12.05 ± 3.51 53.03 ± 7.55* 26.90 ± 3.69* 2.02 ± 0.48* 35.15 ± 11.10* 0.35 ± 0.13* 163.32 ± 60.63* 21.20 ± 9.27* 0.11 ± 0.06* 141.48 ± 34.63* 13.10 ± 2.45* 0.03 ± 0.01* 139.06 ± 33.08*
68.26 ± 3.84 11.25 ± 2.50 44.46 ± 2.50* 34.36 ± 3.29* 1.30 ± 0.16* 5.35 ± 1.54* 0.04 ± 0.02* 34.33 ± 18.01* 3.6 ± 1.45* 0.02 ± 0.01* 22.85 ± 13.11* 1.67 ± 0.84* 0.00 ± 0.00 41.62 ± 17.07*
0.372 0.422 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
*p < 0.05 compared with the control group; MFI- median fluorescence intensity.
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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Fig. 1. Sample analysis of PD-1þ/CD4þ T lymphocytes, PD-1þ/CD8þ T lymphocytes and PD-1þ/CD19 þ B lymphocytes in a newly diagnosed, treatment-naive patient with GD.
6. Discussion Our study has shown increased percentage and number of CD279-positive CD3þCD4þ, CD3þCD8þ T cells and CD19þ B cells in patients at GD diagnosis. Literature to date has stressed the role of PD-1 and its ligands in the pathogenesis of autoimmune diseases, allergies, infections, neoplasms and in transplantology (Helms et al., 2003; Siwiec and Majdan, 2015). Many studies have suggested that PD-1 and its ligands play an important immunoregulatory function. Such authors like Salama et al. (2003), Latchman et al. (2004) showed that PD-1 inhibits the proliferation of naïve and effector T cells as well as of autoreactive T cells and the production of cytokines, which plays a key role in the maintenance of central and peripheral tolerance (Salama et al., 2003; Latchman et al., 2004). Recent years have witnessed many studies on the function of PD-1 in human autoimmune diseases. Despite this fact, no data on its role in the pathogenesis of GD e as one of the most prevalent autoimmune diseases e have been found. First such study on mice was conducted by Nishimura et al. (1999). The authors showed that PD-1 deficiency translates into the spontaneous development of lupus-like diseases with a late onset and deposition of IgG3 in renal
glomeruli and dilated cardiomyopathy related to the production of antibodies against cardiac troponin I (Nishimura et al., 1999). While, Ansari et al. (2003) observed that non-obese prediabetic mice administered with anti-PD-1/anti-PD-L1 quickly developed diabetes due to pancreatitis and the production of proinflammatory cytokines by activated T cells (Ansari et al., 2003). The authors also proved that PD-1/PD-L1 interaction plays an important role both in the onset and the progression of diabetes in this group of animal models. This is how the involvement of this protein in downregulation of immune response was corroborated. It was proven that PD-1 deficiency fosters the development of autoimmune diseases due to no inhibition of activated lymphocyte proliferation (Goła˛ bek and Wo zniakowska-Ge˛ sicka, 2010). Therefore, it may be assumed that the cells producing antibodies in autoimmune diseases may escape the immune system. Increased PD-1 expression on T cells was also observed in other autoimmune disorders such as €gren's syndrome (Hatachi et al., 2003; Kobayashi et al., RA or Sjo 2005). Wan et al. found in the serum and synovial fluid of RA patients an additional soluble PD-1 splicing variant which blocks PD-1 and deteriorates the course of the disease (Wan et al., 2006). This soluble form of the protein was also observed in aplastic anemia along with increased T cell proliferation (Wu et al., 2009). What is
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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Fig. 2. Sample analysis of PD-1þ/CD4þ T lymphocytes, PD-1þ/CD8þ T lymphocytes and PD-1þ/CD19 þ B lymphocytes in a healthy person.
more, in the serum of RA patients some authors found antibodies against PD-L1, which correlates with the disease progression (Dong et al., 2003). Dong et al. (1999) in their study on ligands for the PD-1 receptor observed their ability to promote the activation and proliferation of T cells at an early activation stage by unidentified receptors (Dong et al., 1999). This stimulatory effect mainly refers to T helper cells, which was corroborated in RA patients where the antagonistic antibody binding to PD-L1 stimulated the activation and proliferation of resting T cells, increased cytokine production and aggravated activated T cell apoptosis through the activation of caspase-3 and expression of TRAIL (Dong et al., 2003). Salama et al. (2003) confirmed that PD-1 blockade leads to clinical deterioration of the patients with experimental autoimmune encephalomyelitis (EAE) (Salama et al., 2003). 7. Conclusions This study showed a significantly higher percentage and number of PD-1 positive CD3þ CD4þ, CD3þ CD8þ T cells and CD19þ B cells in the study group than in the control group. Described impaired PD-1 expression in GD also observed in other autoimmune diseases shed a light on its role in the pathogenesis of such
disorders. Although our findings imply that PD-1 may play a role in pathogenesis of GD, this study did not provide mechanistic data to support this. Nevertheless, the results of this preliminary research provided a novel insight into the role ofPD-1 in GD development. We recommend that further studies are needed to elucidate whether the normalization of FT4 and FT3 levels correlates with the decrease in the number of PD-1 positive cells and/or the MFI of PD1, which may lead to the conclusion that potential PD-1/PD-L1 blockade may constitute a novel therapeutic option in GD. Funding This work was supported by research grants no. N N402 682440, no. UMO-2011/01/N/NZ6/01762 and no. UMO-2012/05/B/NZ6/ 00792 of the Polish National Science Centre and MNsd 524, MNmb 523 and DS460 of the Medical University of Lublin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of interest The authors declare that there is no conflict of interest that
Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006
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Please cite this article in press as: Pyzik, A., et al., Frequencies of PD-1- positive T CD3þCD4þ, T CD3þCD8þ and B CD19þ lymphocytes in female patients with Graves' disease and healthy controlse preliminary study, Molecular and Cellular Endocrinology (2017), http://dx.doi.org/10.1016/ j.mce.2017.03.006