Evaluation of regulatory T-cells and autoimmunity in IgA deficiency
Habib Soheili1, Asghar Aghamohammadi1,2 ,Shervin shahin pour1, Hassan Abolhassani1 , Armin
Hirbod 1, Narges Arandi1, Mahomud Tavassoli1, Nima Parvaneh1, Nima Rezaei 1,2
1- Research Center for Immunodeficiencies, Pediatrics
Center of Excellence, Children’s
Medical Center, Tehran University of Medical Sciences, Tehran, Iran.
2- Molecular Immunology Research, Center, and Department of Immunology, School of
Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Corresponding Author: Asghar Aghamohammadi
Address: Children’s Medical Center Hospital, 62 Qarib St., Keshavarz Blvd., Tehran 14194,
Iran
Tel: + 98 21 6642 8998
Fax: + 98 21 6692 3054
Email: aghamohammadi@tums.ac.ir
Abstract
Objective: Selective IgA deficiency (SIGAD) is the most common primary antibody deficiency,
characterized by significant decreased in serum levels of IgA in the presence of normal IgG and
IgM. Abnormalities of CD4+CD25highforkhead box P3 (FoxP3)+ regulatory T cells (T-reg)
have been associated with autoimmune and inflammatory disorders. We hypothesized that IgA
deficiency with autoimmunity might be associated by T-reg abnormalities.
Methods: In order to evaluate relation between autoimmunity and regulatory T cells in IgA
deficiency, we study 26 IgA deficient patients (aged 4–17 years) with serum IgA levels less
than 7 mg/d. Also in this study 26 controls (aged 4–17 years) were included. Regulatory T cells
were measured by flowcytometry using T-reg markers including CD4+ CD25+ FoxP3+.
Result: The mean percent of CD4+ CD25+ FoxP3+ regulatory T cell from all CD4+ cells was
4.08±0.86 in healthy controls which was higher than SIGAD patient significantly (2.93±1.3; pvalue= 0.003). We set a cut of point (2.36%) for regulatory T cell level which was two standard
deviations lower than the mean of normal controls. According to this cut point and in order to
verification of effects of regulatory T cell in clinical manifestation of SIGAD patients, we
classified patients into two groups; group1 (G1) with T-reg<2.36% and group 2 (G2) with Treg>2.36%.Sixteen patients (9 males and 7 females) were included in G1 and remaining 10
patients (7males and 3 females) were classified in G2. The mean age of G1 was significantly
higher rather than G2 (11.90±3.9 vs. 8.05±3.35;p-value=0.018). Autoimmunity were recorded in
9 patients (53.3%) of G1 in contrast only 1 patient in G2 presented autoimmunity (10%; pvalue=0.034). Class switching defect was recorded in 40% o f patients in G1 which meaningfully
different from G2 in which had no any report of such defect (p-value=0.028).
Conclusion: We have demonstrated decreased proportions of T-reg in IgA deficiency patients,
particularly in those with signs of chronic inflammation. Decreased proportions of Treg are
suggested to be path genetically important in autoimmunity, and our results suggest that Treg
may have a similar role in IgA deficiency.
Keywords: autoimmunity, chronic inflammation, IgA deficiency, regulatory T cells
Introduction
Selective IgA deficiency (IgAD) is the most common primary immunodeficiency disorder and is
characterized by decreased serum IgA concentration of <0.07 g/l and normal serum IgM and IgG
levels[1].
The defect is presumed to result from impaired switching to IgA or a maturational failure of IgAproducing lymphocytes. Many of these
individuals have no apparent disease, whereas selected patients suffer from recurrent mucosal
infections, allergies and autoimmune diseases[2] . In one retrospective series, 28 percent of 127
patients with IgA deficiency had evidence of autoimmunity [3]. We propose that maybe low
levels of regulatory T-cells or insufficiency of them leads to these autoimmune diseases in IgA
deficient patients.
Treg-cells are T-cells with an immunosuppressive phenotype, several different types of role
Treg-cells (regulatory T-cells) play in maintenance of self-tolerance[4] which includes anergy,
apopotosis, immune deviation and ignorance. among several different types of Treg-cells,
CD4_CD25_regulatory Treg-cells play important roles in the maintenance of self-tolerance and
the control of autoimmunity[5-7]. It has been shown that the majority of CD4_CD25_ TReg are
of thymic origin [8-10] nd neonatal thymectomy leads to the spontaneous development of
autoimmune diseases including gastritis and thyroiditis [11, 12].
Thus, T reg cells are essential to the maintenance of self-tolerance and the prevention of
autoimmune diseases. There are no studies considering t-reg cells in IgA deficient patients who
develop autoimmune diseases therefore we performed this study determine amount of
CD4_CD25_regulatory Tcells in IgA deficient patients who has developed autoimmune diseases
and also determine them in IgA deficient patients who has not developed autoimmune diseases,
another group is consist of patients who has developed only autoimmunity and the last is our
control group including people with no primary immunodeficiency diseases and autoimmunity.
then we compare the amount of regulatory Tcells in these groups in order to find out if t-reg cells
are responsible for autoimmune manifestations in IgA deficiency or not.
Materials and methods
Patients and Study population
In this case-control study our population is divided into three groups, two groups are case and
one control:
1. Patients who have IgA deficiency with or without autoimmunity.
2. Patients with autoimmunity.
3. People with neither primary immunodeficiency nor autoimmunity.
For making the first group In this study, we reviewed the hospital records of 40 diagnosed
patients with IgAD whom are being treated at Children’s Medical Center. The diagnosis of IgAD
was made according to the diagnostic
criteria of PAGID (the Pan-American Group for Immunodeficiency) and ESID (the European
Society for Immunodeficiencies)[13, 14] including IgA levels under 7 mg/dl with normal
serum IgG and IgM in a male or female patient more than 4 years of age, in whom other
causes of immunodeficiency have been excluded. Besides, the patient frequently presents normal
IgG antibody response to vaccination.
IgA deficient patients were visited when they referred to Children’s Medical Center and
diagnosis of autoimmunity was made according to signs and symptoms and also laboratory tests.
A questionnaire was developed and they were filled during visiting and interviewing patients,
containing all the patient’s demographic information, including date of birth, first clinical
presentation, age at onset of symptoms, age at diagnosis, history of recurrent and chronic
infections, laboratory tests, autoimmunity, malignancy and other complications. For making the
second group we reviewed the hospital records of diagnosed patients with autoimmunity that is
being treated at Children’s Medical Center. They were referred to Children’s Medical Center and
the questionnaire was also filled for them therefore patients with autoimmunity based on signs
and symptoms and laboratory tests included.
Sample preparation
Five milliliter (5 ml) of heparinzed blood sample was collected from all subjects. Peripheral
blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on ficoll
(Histoprep, 35423 Lich, Germany). Cells were washed once with RPMI 1640 and prepared for
surface staining.
Flowcytometric staining
For surface staining, 1×106 cells were resuspended in 100 µl flow cytometry staining buffer
(eBioscience, CA, USA). Cells were incubated with FITC-labeled anti-CD4 (clone RPA-T4,
eBioscience, CA, USA) and PE-labeled anti-CD25 (clone BC96, eBioscience, CA, USA)
antibodies for 30 min at 4 ˚C in dark. For intracellular staining, after permeabilization with
Fixation/Permeabilization buffer (eBioscience, CA, USA), PE-Cy5-labeled anti-FOXP3 antibody
(clone PCH101) (eBioscience, CA, USA) was added and incubated for 30 min at 4 ˚C in dark.
FITC and PE-conjugated mouse IgG1 and PE-Cy5 conjugated rat IgG2a antibodies were used as
the isotype control antibodies.
Flow cytometry was carried out using a Partec flow cytometer (Partec GmbH, Germany) and
lymphocytes were gated based on their forward and side scatter.
Data were analyzed with FlowMax software. The percentage of Treg was measured by
calculating the percentage of CD25+ FOXP3+ double positive cells within CD4+ gate.
Statistical analysis
Statistical analysis on collected data was processed by SPSS software (Version 16.0). Chi square
analysis was used for binomial parameters and student t-test was used for comparison means
parameters between groups. For evaluating independent association of each predictor factors
with following response of patients to MTX multiple logistic regressions were used. Differences
were considered statistically significant when the p value was<0.05
Results:
Finally, 26 patients (16 males, 10 females) with mean age of 10.17 ±4.9 years were enrolled in
this study. Demographic and immunological data participants as showed in Table 1. The mean
percent of CD4+ CD25+ FoxP3+ regulatory T cell from all CD4+ cells was 4.08±0.86 in healthy
controls which was higher than SIGAD patient significantly (2.40±1.7; p-value= 0.003). Based
on the strategy of this study we set a cut of point (2.36%) for regulatory T cell level which was
two standard deviations lower than the mean of normal controls. According to this cut point and
in order to verification of effects of regulatory T cell in clinical manifestation of SIGAD patients,
we classified patients into two groups; group1 (G1) with T-reg<2.36% and group 2 (G2) with Treg>2.36%.
Sixteen patients (61.5%; 9 males and 7 females) were included in G1 and remaining 10 patients
(38.4%; 7males and 3 females) were classified in G2. Patients characteristic and percents of Treg from all CD4+ cells and percents of T-reg pbcms were compared between two groups in
table2. The mean age of G1 was significantly higher rather than G2 (11.82±4.7 vs. 8.05±3.35; pvalue=0.016). Moreover patients in G1 had more delay in diagnosis (4.4±1.4 vs. 2.66±2.5; pvalue=0.05). Autoimmunity were recorded in 9 patients (56.2%) of G1 (autoimmune hemolytic
anemia in 3 cases, vitiligo in 3 case, autoimmune tyroiditis in 1 case, combination of vitiligo and
ulcerative colitis in 1 case) in contrast only 1 patient in G2 present type 2 diabetes mellitus (10%;
p-value=0.029). CD19 levels in G1 and G2 were 11.63±9.0 and 15.6±3.4 respectively (p-value=
0.026). Class switching defect was recorded in 46.6% of patients in G1 which meaningfully
different from G2 in which had no any report of such defect (p-value=0.019).
Discussion
The purpose of this study was to evaluate t-reg cells in IgA deficient patients who develop
autoimmune diseases and comparing the amount of t-reg cells in in these patients with healthy
people and we found that the mean percent of CD4+ CD25+ FoxP3+ regulatory T cell from all
CD4+ cells in healthy controls was higher than IGAD patients. Treg cells play a critical role in
the maintenance of selftolerance by suppressing, in a dominant manner, immune activation of
self-aggressive Teffector cells [15]. Upregulation of Treg cell function or increases in the
numbers of cells might be beneficial for treating autoimmune diseases and allergies and for
preventing allograft rejection. Conversely, inhibiting Treg cell function or decreasing Treg cell
numbers might boost immunity against tumors and microorganisms.
Different types of Treg-cells have been described including naturally arising CD4+CD25+ Tregcells, IL (interleukin)-10-secreting Tr1 cells, TGF-β (transforming growth factor-β)- secreting
Th3 cells, CD8+CD28− T-cells, CD8+CD122+ T-cells, γ δ T-cells and NK (natural killer) Tcells [16].Treg cells can both differentiate in the thymus and emigrate into the periphery as fully
functional natural suppressor cells, or they can be induced in the periphery from naive T-cell
precursors. The differentiation of Treg cells from naive CD4 T cells occurs when exposed to
TGF-b and IL-2 [17].
Treg cells are characterized by the expression of the transcription factor FOXP3, [18,19,20]
which is induced by TGF-b in the presence of IL-2 [21].
Absence of FOXP3 in patients with immune dysregulation, polyendocrinopathy, enteropathy, Xlinked (IPEX) syndrome [22] results in lack of functional Treg cells. Although humans with
IPEX (immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome) lack
Foxp3+ cells and subsequently develop severe autoimmune disease [23,24].
CD4+Foxp3+ Treg-cells can suppress the proliferation and cytokine production of effector Tcells through production of inhibitory cytokines, most notably IL-10 and TGF-β.[25].
Suppression by cytolysis of effector T-cell killing by Treg-cells through the release of granzyme
B and perforin has also been reported [26,27]. A third mode of action is suppression by
metabolic disruption such as cytokinedeprivation- mediated apoptosis of effector CD4+ Tcells[28], and intracellular or extracellular release of adenosine nucleosides which leads to
suppression of effector T-cells [29,30].
Overexpression of FOXP3 in conventional T cells directs them to a Treg cell phenotype with
suppressive activity, leading to a state of immune deficiency [31, 32].The majority of Treg cells
express high levels of CD25 (IL-2 receptor a) [15], suggesting a major influence of IL-2 for the
long-term maintenance and competitiveness of these cells.
According to our results there is a significant decrease in amount of regulatory T cells in patients
with IgAD comparing with healthy people. Regulatory T cells constitute 1-10% of thymic and
peripheral CD4+ T cells in humans and mice, and arise during normal thymic lymphocyte
development [33]. The development of mature and effective antibody responses occurs in two
phases (reviewed in report by Durandy) [34]. First, rearrangement of immunoglobulin genes
occurs in B-lymphocyte precursors in primary lymphoid tissue resulting in the IgM repertoire.
This phase occurs independently of T lymphocytes and antigen. Patients with defects in these
processes, such as Bruton’s X-linked agammaglobulinaemia, present in infancy and childhood
with severe bacterial infections. The second phase occurs in the germinal centres of secondary
lymphoid tissue such as the spleen, lymph nodes and tonsils, and is dependent on antigen and
collaboration between B cells and CD4+ T cells. Here class switch recombination (CSR) enables
the production of IgA, IgG subclasses and IgE, and somatic hypermutation (SH) results in the
generation and selection
of antibodies with the highest binding affinity for antigen. Failure of CSR and SH by known and
hypothesized mechanisms is thought to underlie the development of most primary antibody
deficiency syndromes such as IgAD, and there is a theory about the association of this IgAD
with abnormalities in T-cell regulation, which could be responsible for both IgAD and
autoimmunity [35].
Schneider et al recently demonstrated that in CCR7 knockout mice, CD41CD251Foxp31 Treg
cells were unable to home to lymph nodes and were unable to suppress antigen-induced T-cell
responses [36].
Kriegel et al. found that the suppressive capacity of CD4+CD25+ T-cells in co-cultures with
CD4+CD25− T-cells and OKT3 (also called muromonab) or PHA (phytohaemagglutinin) was
impaired in patients with APS-II (autoimmune polyglandular syndrome type II) [37].
Furthermore, Treg-cells from normal donors could suppress responders from patients with APSII, whereas APS-II Treg-cells could not suppress CD4+CD25− T-cells from normal donors,
indicating that the failure of APS-II Treg-cells to suppresswas due to a defect in the Treg-cells
rather than increased resistance of APS-II responders to Treg-cell-mediated suppression. Similar
findings were made in patients with myasthenia gravis; in contrast with normal patients,
CD4+CD25+ thymocytes from patients with myasthenia gravis were unable to suppress the
proliferative response of CD4+CD25− thymocytes to allogenic stimulation. The amount of
Foxp3 mRNA from patients was reduced 2-fold compared with young adult and newborn
CD4+CD25+ thymocytes[38]. CD4+CD25hi T-cells from patients with multiple sclerosis were
found to suppress CD4+CD25− T-cell proliferation and IFN-γ production less effectively than
CD4+CD25hi cells from normal donors co-cultured with CD4+CD25− T-cells and anti-CD3
antibody[39]. Finally, qualitative and/or quantitative differences in CD4+CD25+ T-cells were
also obtained in patients with psoriasis, autoimmune liver disease and systemic lupus
erythematosus [40,41,42].
Additional reports from patients with ulcerative colitis, primary Sjogren’s syndrome and
autoimmunethyroid disease further highlight the lack of consensus on the role of Treg-cells in
autoimmune pathology in humans; peripheral CD4+CD25+ T-cells from patients with primary
Sjogren’s syndrome were not lower than age-matched controls and were effective at suppressing
the in vitro proliferation of effectors [43]. Colonic CD4+CD25+ T-cells from patients with
ulcerative colitis were able to suppress the in vitro proliferation of colonic effector T-cells and
the proportion of Treg-cells increased with disease activity leading to the suggestion “that their
suppressive capacity was being influenced by the in vivo environment” [44]. It has subsequently
been shown that patients with ulcerative colitis have significantly increased frequencies of
CD25+ and Foxp3+ lymphocytes in the lamina propria [45]. There was a significantly higher
number of CD4+Foxp3+ T-cells in the peripheral blood of patients with autoimmune thyroid
disease than normal controls and there was no difference in the proportion of CD4+Foxp3+ Tcells in the peripheral blood compared with thyroid disease in patientswith autoimmune thyroid
disease [46].
According to our results in table 2 we concluded that t-reg and IgA concentration of serum do
not relate together directly because in table 2 we have divided patients into two groups based on
cut of point (2.36%) for regulatory T cell level and as shown in the table there is a significant
difference in percent of t-reg between these two groups (P-value = 0.001) but there is no
significant difference in concentration of IgA of the serum.(P-value= 0.68)
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Table1- Comparison of immunological data of 26 SIGAD patients and 26 healthy controls
Parameters
SIGAD patients Healthy controls P-value
(26 patients)
(26 patients)
IgG
1163.2±421.8
1803.1±562.0
0.47
IgM
113.9±72.5
95.1±74.2
0.36
IgA
4.6±3.3
45.9±18.6
<0.001
CD3
56.0±22.5
59.1±24.0
0.20
CD4
33.2±17.8
34.0±12.9
0.57
CD8
21.8±7.4
23.3±11.5
0.71
CD19
12.8±9.2
15.8±7.7
0.59
Percent of T-reg
2.40±1.7
4.08±0.86
0.003
Percent of pbcms T-reg
1.05±0.31
1.54±0.33
0.005
Table2- Comparison of clinical and immunological characteristics between two groups of
SIGAD patients
Parameters
G1 (16 patients)
G2(10 patients)
P-value
Sex (m/f)
9/7
7/3
0.65
Age at onset ±SD (years)
3.7±2.5
3.4±2.0
0.76
Age at diagnosis ±SD (years)
8.24±4.3
6.07±2.35
0.21
Current Age ±SD (years)
11.82±4.7
8.05±3.35
0.016
Follow-up period ±SD (years)
3.6±2.0
1.75±1.2
0.07
Delay in diagnosis ±SD (years)
4.7±1.9
2.66±2.5
0.05
Percent of T-reg ±SD
1.81±1.46
4.60±1.90
0.001
Percent of pbcms T-reg ±SD
0.69±0.48
1.65±0.54
0.54
Autoimmunity (%)
9(56.2)
1(10)
0.029
Specific antibody deficiency (%)
5(31.2)
1(10)
0.26
IgG2 deficiency (%)
1(6.2)
0
0.67
IgG3 deficiency (%)
1(6.2)
0
0.67
Pneumonia (%)
6(37.5)
0
0.042
Bronchiectasis (%)
3(18.7)
0
0.26
Allergy (%)
7(43.7)
4(40)
0.63
IgG ±SD
1130.1±427.5
1241.6±468.2
0.76
IgM ±SD
134.8±92.6
83.8±34.9
0.07
IgA ±SD
4.3±3.8
5.11±4.07
0.65
CD3 ±SD
57.9±12.2
57.6±14.4
0.61
CD4 ±SD
31.24±8.14
34.3±10.17
0.28
CD8 ±SD
21.6±7.1
22.0±4.5
0.13
CD19 ±SD
11.63±9.0
15.6±3.4
0.026
Dn 27+ 19+ ±SD
0.93±0.42
1.75±0.73
0.14
Class switching defect (%)
7(46.6)
0
0.019