Assay of T- and NK-cell subsets and the expression of NKG2A and NKG2D in patients with new-onset systemic lupus erythematosus
This study aims to explore the percentage of T-cell and NK-cell subsets, the expression of NKG2A and NKG2D on CD3+ T cells and CD3−CD56+ NK cells on the total lymphocytes in new-onset systemic lupus erythematosus (SLE) patients, and explore clinical significance of these cell subsets. Thirty-two SLE patients and 32 normal controls were enrolled. Flow cytometry was used to count T- and NK-cell subsets and to detect the expression of NKG2A and NKG2D on CD3+ T cells and CD3−CD56+ NK cells in patients with new-onset SLE. Results show that CD4+ T (t = 2.04, P < 0.05), CD4+/CD8+ T cell (t = 2.66, P < 0.05), CD4+ CD25+ T (t = 2.48, P < 0.05), CD3+CD56+ natural killer T (NKT) (t = 40.05, P < 0.01), CD3−CD56+CD16+ NK-cell subsets (t = 3.50, P < 0.01) were significantly decreased, CD8+ T-cell subsets was significantly increased in patients with new-onset SLE (t = 3.80, P < 0.01), as compared with healthy controls. CD8+ T-cell subset was significantly increased in patients with vasculitis (t = 2.47, P < 0.05), and CD3−CD56+CD16+ NK was increased in patients with arthritis (t = 3.21, P < 0.01). However, no statistically significant correlation was found among different PBMC subsets and SLEDAI activity scores. Patients with SLE had a lower expression of NKG2A (U = 2.42, P < 0.05) as well as NKG2A/NKG2D ratio (t = 2.61, P < 0.05) and a higher expression of NKG2D (t = 2.21, P < 0.05) on CD3+ T cells, compared with normal controls. However, they had a higher expression of NKG2A (t = 2.59, P < 0.05) as well as NKG2A/NKG2D ratio (t = 49.45, P < 0.01) and a lower expression of NKG2D (t = 3.05, P < 0.01) on CD3−CD56+ NK cells. Taken together, the findings indicate the decreased CD4+ T-cell, CD4+/CD8+ T-cell, CD4+CD25+ T-cell, CD3+CD56+ NKT-, and CD3−CD56+CD16+ NK-cell subsets, increased CD8+ T-cell subsets as well as the abnormal expression of NKG2A and NKG2D on CD3+ T and CD3−CD56 + NK cells may play a role in the etiology of SLE.
KeywordsNK-cell subset NKG2A NKG2D Systemic lupus erythematosus T-cell subset
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, characterized by a multitude of autoantibody production, complement activation and immune-complex deposition, causing tissue and organ damage . At present, the pathogenesis of SLE remains unclear. However, there is a large body of evidence that immunological disturbances play a key role in the pathogenesis of SLE [2, 3, 4]. It would be interesting to highlight the influence of autoantibodies on the immunological status of patients with SLE including leuko-/lymphocytopenia. Immunological characteristics of SLE are hyperactive B cells and abnormal T cells, which lead to the production of autoantibodies against components of the cell nucleus. It is generally accepted that T and B lymphocytes play key roles at multiple stages in the onset and progression of SLE. Recently, a possible role of natural killer (NK) cells in the pathogenesis of SLE has been suggested [5, 6, 7], which are lymphocytes belonging to the innate immune system, playing an important role in innate immunity and adaptive immunity . However, the current studies about the amount of T-cell subsets and NK-cell subsets in SLE are in disagreement.
More than ten NK cell receptors have been discovered so far , among which, NKG2A is inhibitory while NKG2D is activating. Adverse regulative signals are transmitted by NKG2A and NKG2D. Normally, the expression of NKG2A and NKG2D incline to balance. Patients with SLE have low numbers of NK cells in the peripheral blood [10, 11]. However, the role of NK cell receptors in SLE has been rarely studied. In the present study, we apply flow cytometry (FCM) to count T- and NK-cell subsets, and detect the expression of NKG2A/NKG2D on CD3+ T cells and CD3−CD56+ NK cells in patients with new-onset SLE.
Materials and methods
Thirty-two SLE patients (M/F = 2/30), fulfilling the ACR criteria , were included in the study, as well as 32 race-, gender- and age-matched healthy volunteers from the same geographical area.
Characteristics of controls and patients with systemic lupus erythematosus
Controls (n = 32)
SLE patients (n = 32)
29.74 ± 12.20
32.97 ± 13.20
SLEDAI (median and range)
The baseline demographic and clinical data were collected from hospital records and reviewed by experienced physicians. The data included age, gender, and medication. Disease activity was determined by calculating the SLE disease activity index (SLEDAI) . Routine laboratory investigation included full blood count; erythrocyte sedimentation rate; serum levels of C-reactive protein; serum concentrations of complement factors C3 and C4; anti-dsDNA using an immunoblotting technique; serum IgG, IgM, and IgA; and 24-h urinary protein levels.
Blood samples from all subjects were stained and analyzed within 6 h of the collection. A whole blood lysing method, using monoclonal antibodies that had been directly conjugated to fluorescein (FITC), phycoerythrin (PE), and Cy-chrome (Cy), was carried out to stain white blood cells. Lymphocyte subset analyses of patients and of normal controls were performed by direct three-color immunofluorescence and flow cytometry (FACScalibur, BD, USA).
Mouse IgG1 monoclonal antibodies with appropriate isotype controls were obtained as follows: anti-human CD3(Cy5), anti-human FITC-CD56, anti-human FITC-CD4, anti-human PE-CD4 , anti-human PE-CD8, anti-human FITC-CD25 (BD Pharmingen Company, USA), anti-human PE-NKG2A, and anti-human PE-NKG2D (R&D System Company, USA).
Winmdi 2.9 software was used to obtain percentages of lymphocyte subsets. For each sample, a lymphocyte gate was set on the basis of linear forward and side scatter. NK cells were defined as CD3−CD56+ lymphocytes while T cells were the CD3+ populations.
All statistical analyses were performed by SPSS 10.01 (SPSS Inc., Chicago, IL, USA). Test of the normal distribution of the data was made using the Kolmogorow–Smirnow test. If the data were normally distributed, it was described as mean ± SD. Otherwise, it was described as median and P25 ∼ P75. The significance of the results was analyzed by Student's t test or the Mann–Whitney U test. Probability level less than 0.05 were used as a criterion of significance.
The percentages of T cells and NK Cells in total lymphocytes
The percentages of T cells and NK cells in total lymphocytes in patients and controls
(n = 32)
(n = 32)
CD3+ T cells
72.79% ± 10.57%
69.94% ± 7.79%
CD4+ T cells
35.87% ± 9.91%
40.28% ± 7.14%
CD8+ T cells
33.59% ± 11.62%
24.30% ± 7.53%
1.31% ± 0.85%
1.85% ± 0.78%
CD4+CD25+ T cells
3.99% ± 1.57%
5.38% ± 2.76%
CD3+CD56+ NKT cells
2.40% ± 1.51%
5.12% ± 3.92%
0.78% ± 0.54%
0.93% ± 0.64%
CD3−CD56+D16+ NK cells
5.86% ± 4.77%
10.99% ± 6.79%
CD3−CD56−CD16+ NK cells
1.36% ± 0.90%
1.58% ± 0.97%
No statistically significant correlation was found among different PBMC subsets and SLEDAI activity scores.
When major clinical characteristics were considered, we found that there was a significant difference in CD8+ T cells between patients with and without vasculitis (44.53% ± 14.58%, n = 5; 31.58% ± 10.05%, n = 27; t = 2.47, P < 0.05). Similarly, we observed a significant difference in CD3−CD56+CD16+ NK cells between patients with and without arthritis (6.87% ± 5.02%, n = 24; 2.68% ± 2.03%, n = 8; t = 3.21, P < 0.01). However, different PBMC subsets did not differ between patients with and without fever, nor was any difference found when different T- and NK-cell subsets were compared between patients with and without rash, alopecia, mucosal ulcers, pleurisy, pericarditis, myositis, and nephritis.
The expression of NKG2A and NKG2D on T and NK cells
The prevalence of NKG2A and NKG2D on CD3+ T cells in patients and controls
SLE patients(n = 32)
3.12 ± 2.61
49.47 ± 12.91
6.65 ± 6.08
Controls(n = 32)
43.65 ± 7.43
11.69 ± 9.06
The prevelance of NKG2A and NKG2D on CD3−CD56+ NK cells in patients and controls
SLE patients(n = 32)
23.36 ± 16.37
86.36 ± 11.83
27.12 ± 18.21
Controls(n = 32)
14.60 ± 9.88
93.13 ± 4.20
15.63 ± 10.46
In SLE, the most common changes in T- and NK-cell subsets are a reduction of CD4+ T cells and an elevation of CD8+ T cells with imbalance of CD4+/CD8+ ratio . Spinozzi et al.  reported that CD8+ T cells in SLE patients was mostly made up of γδT cell subpopulation, a kind of contrasuppressor T lymphocytes, which cannot suppress but help CD4+ T cells activation. γδT cell subpopulation disappeared and clinical and pathological remission SLE was often obtained after in vivo immunosuppressive therapy. Jiang et al.  pointed out that patients with active SLE had a markedly low percentage of T4+2H4+ cells, which cannot induce CD8+ T to inhibit B cells. Therefore, a reduction of CD4+ T cells and an elevation of CD8+ T cells in SLE still lead to B hyperactivity.
Recent studies have suggested that CD4+CD25+ T cells, also called regulatory T cells, exhibit immune suppressive activity and also play a critical role in the maintenance of self-tolerance [17, 18, 19, 20]. Some studies concerning the role of CD4+CD25+ T cells in lupus revealed decreased frequencies of these cells in SLE patients [21, 22, 23] and lupus mice . Similar results had been observed in our research.
NKT cells are a subset of T cells that share properties of natural killer cells and conventional T cells. NKT cells are involved in a number of pathological conditions and have been implicated in the pathogenesis of autoimmune diseases . In our study, NKT cells were decreased in SLE patients than in controls, which was similar to previous studies [26, 27].
NK cells were once regarded as effector cells of natural immunity. However, they are now revealing themselves as multifunctional regulatory cells that are present throughout the body. The role of NK cells in autoimmunity is attracting increased attention, although the picture is clouded by a broad range of data that presents disease-promoting  as well as disease-protective roles . Our results further verify that patients with SLE have low numbers of NK cells circulating in the blood, which were in accordance with previous reports[10, 11]. Genetic factors  and apoptosis  are related to NK cell deficiency.
This study found out that the association of different T- and NK-cell subsets and SLEDAI activity scores was not statistically significant. There were few studies to data discussing the correlation. We suppose that the pathogenesis of SLE is complicated, and different clinical subtypes and individuals have the different immune disorder. Moreover, various cell subsets mutually stimulate and/or inhibit under the different immune state. Therefore, the percentage of only one kind of T- and NK-cell subsets in total lymphocytes does not represent SLEDAI activity scores.
Our study found that patients with vasculitis have an increased CD8+ T-cell percentage, and patients with arthritis have an increased CD3−CD56+CD16+ NK-cell percentage. Vasculitis is inflammation of blood vessels and may affect both arteries and veins, which is diagnosed primarily by history and clinical examination. There were few studies to data discussing the relationship between clinical characteristics and PBMC subsets. The result indicates CD8+ T and CD3−CD56+CD16+ NK cells may serve as a predictor of inflammation in SLE.
The term “NK cell receptors” is used to describe the growing number of activating and inhibitory cell-surface receptors known to be expressed by NK cells, while in some cases also expressed by other immune cell. NKG2A, NKG2C, and NKG2E are highly homologous lectin-like NK cell receptors that form heterodimers with the CD94 molecule . NKG2A, containing immunoreceptor tyrosine-based inhibition motif, is inhibitory, whereas the others are stimulatory. These receptors recognize peptides derived from the leader sequence of many classical MHC class I molecules, embedded in the groove of a specialized non-classical class I molecule called HLA-E (in humans) or Qa-1 (in mice) . NKG2D, belonging to C-type lectin containing immunoreceptor tyrosine-based activation motif, which is activating, shares little sequence homology with the NKG2 receptor family members, and does not appear to pair with CD94 . Unlike the activating members of the Ly49 and NKG2 families, NKG2D uses DAP10, rather than DAP12, as an adapter signaling molecule . Interestingly, the diverse ligands recognized by NKG2D include multiple members of at least two gene families, all of which are only distantly related to MHC class I molecules. Patients with SLE have low numbers of NK cells circulating in the blood [10, 11]. However, the role of NK cell receptors in SLE has been rarely studied. We have attempted to answer this question by determining NKG2A/NKG2D expression on CD3+ T lymphocytes and CD3−CD56+ NK cells in patients with SLE, which is the novel aspect of the study. What is more, we recruited patients with new-onset SLE in order to eliminate the treatment and medicine influence on results.
In this study, we found that the expression of NKG2A on CD3+ T cells in SLE patients was significantly decreased, while NKG2D was significantly increased. It was reported that  NKG2A resists NK as well as CTL-mediated lysis in vitro, while NKG2D functioned as a costimulatory receptor in the adaptive immune system (CD8+ T cells) or as both a primary recognition structure and a costimulatory receptor in the innate immune system (NK cells and macrophages) . Our results, together with those from previous studies indicated that the abnormal expression of NKG2A and NKG2D on CD3+ T cells may participate in the pathogenesis of SLE. The cytolytic activity and cytokine production of NK cells is tightly regulated by an array of activating and inhibitory receptors on the cell surface . Our detecting results of NKG2A and NKG2D on NK cells confirmed several groups' reports, which pointed out that the functional activity of NK cells was diminished in SLE [7, 37].
To summarize, our findings indicate that the decreased CD4+/CD8+ T cell, CD4+ T, CD4+CD25+ T-, NKT-, and NK-cell subsets, increased CD8+ T-cell subsets, as well as altered expression of NKG2A and NKG2D on CD3+ T and CD3−CD56+ NK cells may play an important role in the regulation of NK cell activities and pathogenesis of SLE.
This work was supported by grants from the National Natural Science Foundation of China (30771848) and the key program of National Natural Science Foundation of China (30830089).
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