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Central European Journal of Immunology
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vol. 38

Clinical immunology
Examination of correlation between vitamin D3 (25-OHD3) concentration and percentage of regulatory T lymphocytes (FoxP3) in children with allergy symptoms

Bolesław Kalicki
Sławomir Lewicki
Wanda Stankiewicz
Anna Jung
Agnieszka Rustecka
Monika Turkowska
Piotr Rutkowski
Paweł Bodera
Robert Zdanowski

(Centr Eur J Immunol 2013; 38 (1): 70-75)
Online publish date: 2013/04/17
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The immune tolerance of organism is more than passiveness, ignoring antigens and leaving them alone. It is also active recognition of self antigens by T lymphocytes and issuing orders banning the executive cells for aggressive behavior against their own tissues. Maintaining of this condition is possible due to the interaction of the lymphocyte with antigen in the early stages of its development. Cells that have not acquired tolerance in central lymphoid organs, are deleted or become anergic in peripheral lymphoid organs [1]. A crucial role in the formation and maintenance of immune tolerance is played by the defined

populations of lymphocytes T. This phenotypically heterogeneous group of cells regulates the immune system, participates in the control and inhibition of excessive immune responses (especially against their own antigens), play an important role in tumor immunology, transplantation immunology and the pathogenesis of multiple disorders, including autoimmune diseases [2-5].

Natural Tregs (nTregs) constitute 5-10% of peripheral CD4+ cell pool with co-expression of CD25 receptor; their survival and functioning depend on IL-2. The transcription factor FoxP3 is considered a specific nTregs marker, which is crucial for the development of a regulatory cell activity. Constant and high co-expression of CD25 surface antigen differs them from the activated T cells CD4+ CD8+, which have a lower and transient expression of this marker [6-9].

In this heterogeneous population of regulatory lymphocytes (CD4+), besides nTregs there are also sub-populations of induced regulatory T cells, such as Th3, Tr1 cells and anergic cells included. Stimulation of naive CD4+ cells in vitro in the presence of exogenous IL-10 leads to the differentiation of Tr1 population which, following antigenic stimulation produces large amounts of IL-10 and is capable of inhibiting the activity of Th1 and Th2 cells in vivo. Another stimulator of Tr1 cell differentiation in vitro is interferon  [10-12]. Oral administration of antigen stimulates the activity of another Tregs cell subpopulation – Th3. These cells produce TGF- (transforming growth factor ), immunosuppressive cytokine and a “switch” for the synthesis of IgA. The final population of Tregs cells are anergic T cells which have the ability to inhibit T cell responses in vitro and in vivo through a mechanism that modify the function of antigen presenting cells (APC)

[6, 12, 13].

In recent years, studies were published showing a relationship between the concentration of metabolites of vitamin D and the incidence of autoimmune diseases such as diabetes mellitus type 1 and multiple sclerosis [14-17]. Gathered evidence confirmed that vitamin D might prevent the development of these diseases, by increasing the number or the effect of the naturally occurring Tregs cells [15, 16].

In the world literature, there are few reports about the relationship between vitamin D levels and the amount of regulatory T-cell subpopulations. This study aimed to examine the correlation of vitamin D3 (25-OHD3) with the percentage of Tregs lymphocytes (FoxP3+) in children with allergic symptoms. The studies are preliminary and will be the basis to conduct further experiments that expand our knowledge in this field.

Material and methods

The study was conducted on a group of 19 children who were treated from January to April 2012 in the Paediatric, Nephrology and Allergology Clinic, Military Institute of Medicine in Warsaw. In all examined children there was a tendency to airway obstruction confirmed by clinical trial and in some cases by the spirometry test. In the same time, the concentration of 25-OHD3 in the serum was determined in all patients and the information concerning diet, supply of vitamins and living conditions including sun exposure were collected during the interview. In all children the profile of immune cells (CD19, CD4, CD8, CD25/FoxP3), and NK cells (CD16/56) were determined.

Based on interview information, clinical symptoms, level of immunoglobulin E (IgE) in the blood serum and the results of skin prick tests (SPT) (made in some patients), children were divided into two groups: a control group (without clinical signs of allergy, with low IgE level) and a group with clinical symptoms of atopic allergy (with elevated IgE level, diagnosed asthma and/or atopic dermatitis). Characteristics of these groups of patients are shown in Table 1; all values are expressed as mean (± SE). There were no statistical differences between the groups referred to characteristics.

25-hydroxyvitamin D3 (25-OHD3) and IgE were determined in serum samples by commercially available enzyme immunoassay kits according to the manufacturer’s instructions.

Regulatory T cell percentages were determined by surface and intracytoplasmic staining peripheral blood mo­nonuclear cells with fluorochrome-coniugated mouse antibodies against: CD4-PerCP, CD25-APC and FoxP3-PE (BD Biosciences), according to the manufacturer’s protocol. For examination of lymphocyte subsets, samples were labeled with antibodies against: CD3, CD4, CD8, CD19 and CD16/56 (IMK Test, BD Biosciences). The samples were analyzed by flow cytometry using appropriate isotype controls. Cytometry analyses were performed using FACS Calibur Flow Cytometer equipped with CellQuest Software (BD Biosciences).

The obtained results are presented as mean values and standard errors. Nature of the data distribution was checked using the Shapiro-Wilk test. For comparison between separate groups, t test was used if the distribution of the collected data was a normal distribution and the homogeneity of variance was maintained. In other cases – the Mann-Whitney U test were used. In order to investigate the de­pendence of the parameters (concentration of 25-OHD3 and the percentage of regulatory cells) analysis were performed using the Pearson correlation statistical method.

The significance level was p < 0.05.

The study is part of a research project which was approved by the Bioethics Committee WIL 108/12.


In the obtained results there was significantly lower mean concentration of total IgE in serum of children with no symptoms of allergy (control group) in comparison to allergic group (Table 2). Although in the control group mean concentration of this immunoglobulin was tenfold lower, the difference was not statistically significant. This is probably due to the large differences between individuals (especially in the group with symptoms of allergy), and a small, highly diverse in terms of coexisting diseases in control group. Similarly obserwation was found with the average concentration of vitamin D3 (25-OHD3). However the differences were not so significant and the lower concentrations was found in the control group (Table 2).

In the study subpopulation of regulatory T cells was defined as: CD4+, CD25++ and nuclear factor FoxP3+ expression. To define this, the cells in the population of mononuclear cells were firstly visualized for CD4 surface marker (Gate 1, R1). Next within this population (G1) were determined cells with high expression of CD25 and expression of intracellular nuclear factor FoxP3 (Gate 2, R2). Typical cytogram from the investigation is shown in Fig. 1, and the average number and percentages of regulatory cells in the blood of the children presented in Table 3.

In this study we also define the size of main subpopulations of lymphocytes. Average percentages of B cells,

T and NK cells (CD19+, CD4+, CD8+ and CD16/56+) are shown in Fig. 2. There was a statistically significant decrease in CD4 lymphocytes in children with the recognized allergy compared to the control group.

In a further step of the research a relation between the concentration of 25-OHD3 and percentage of regulatory cells (CD4+, CD25+ FoxP3+) was analyzed. The results are shown in Fig. 3 – for children with clinically recognized allergy and in Fig. 4 for the control group. The obtained data show that there is a positive correlation between Tregs cells and 25-OHD3 only in children with allergic symptoms. The correlation factor was 0.772 in this case, a significance level of p = 0.0008. A similar dependence was not revealed in the group of children without allergy symptoms.


Hypersensitivity and allergy are diseases associated with malfunction of the immune system. Chronic allergic inflammation, in the past called allergy, is excessive and changed response of the immune system, that definition was introduced by K. von Pirquet in 1906 [18]. Currently it is used as a synonym for temporary or permanent disturbances of immunoregulation causing incorrectly addressed immune response against a variety of allergens, leading to tissue damage. These diseases are called “civilization diseases” and affect increasing numbers of occupants, including children [19, 20]. Disturbing is the fact that the age of children catching severe forms of allergic diseases is decreasing. Recent studies have shown that there is subpopulation of

T cells called regulatory cells (Tregs) responsible for the strength and scope regulation of the immune system response. Numerous studies have demonstrated that an increased Tregs subpopulation percentage reduces the sensitivity of the immune system to external or changed in the disease process antigens, which allows the growth of tumors [21, 22]. On the other hand decreased Tregs percentage increases the strength of immune response, which may lead to chronic inflammatory diseases and autoimmune diseases [23-25]. Therefore it seems to be important to find factors which can affect the level and function of the cells Tregs.

In the project, preliminary study was attempted to examine the relationship between serum 25-OH vitamin D3 level and the value of Tregs in the blood cells of children with clinical symptoms of allergy. The control group was children hospitalized at the Clinic due to diseases with obturation, but without symptoms of allergies. The clinical diagnosis was a criterion for group division. Those children whose IgE levels were more than 2.5 times higher than the upper limit of normal for the particular age group were qualified to allergic group. This level is usually correlated with atopic diseases where IgE antibodies play a crucial role in response to the sensitizing antigen [26-29].

In performed experiments no statistically significant differences in both average number of leukocytes in the blood, and their respective populations (lymphocytes/monocytes/ neutrophil/eosinophil/basophils) were observed. Also we didn’t noticed significant differences in the mean concentration of 25-OHD3 and the average number and percentage of regulatory cells in the blood between groups. Lymphocyte phenotyping revealed only statistically significant decrease of CD4 percentage in allergy group. Interesting information was provided by examination of correlation between serum 25-OHD3 and the percentage of Tregs in the blood. There was no correlation in the control group (children without allergy), while in the group of children with allergies a strong, proportional correlation between serum 25-OHD3 and the percentage of Tregs in the blood have been shown. Similar results in the studies of the correlation of these factors in multiple sclerosis were obtained [30]. There are also studies that do not support these observation. The research of Royal group’s showed an inverse correlation with 25-OHD and directly proportional relation of Tregs and 25-(OH)2D/25-OHD ratio [31]. Therefore, there is no conclusive data demonstrating the relationships between vitamin D metabolites and size of the sub-population of regulatory cells in the case of multiple sclerosis. It seems that these discrepancies may be the result of the lack of a clear methodology for determining of regulatory cell subpopulations.

Many other studies suggest an inverse relationship between low vitamin D levels in patients and increased incidence of various diseases, such as diseases of the skeletal system, cardiovascular, infectious diseases and autoimmune diseases [32-36]. Despite the high interest in the role of Tregs and vitamin D there is a lack of research on their significance in allergic diseases.

In the literature there is a lot of information about vitamin D deficiency and its negative effects (airway hyperresponsiveness, impaired respiratory function and resistance to steroid therapy), that may result from [37]. It is known that T cells play a key regulatory role in T-cell homeostasis and promote a correct immune response [38, 39]. It is widely believed that immunological basis of chronic allergic inflammatory according to inappropriate responses of T-cell helper 2 (Th2) to common allergens. Cytokines such as IL-4 and IL-13 associated with Th2 are responsible for IgE production in response to allergens, and IL-5 (also associated with Th2) contributes to eosinophilic inflammation, which is one of the characteristic features of allergic diseases [40, 41].

Given the above information and the results of the study (our own and others authors) it can be presumed that there is a potential for preventing, or at least reducing the symptoms of allergic diseases through proper supplementation of vitamin D.

There is still some open issues. Is the severity of allergy symptoms in the early spring season is only associated with an increased risk for allergens exposure, or is it also results from the low levels of vitamin D3 (small sun exposure during the winter)? Does it matter that our study was performed between January and April? Why the correlation occurs in the group of allergic children only and was not observed in the control group (children without symptoms of allergy)? Are there any other immunological factors (changes in intracellular and surface markers, signal transduction disturbances, etc.), which may be useful in the diagnosis and treatment of atopic diseases? All these questions require further, much more detailed researches.

The results cannot be basis for the final conclusions (mainly due to small number of participants in both groups) but allow to have a hope for promising results in the future. The above data might suggest that Tregs cells are involved in T cell responses to allergic inflammation, and can play a possible role as a limiting element of this process. Also very important (especially in children) is the possibility to find an easy, cheap and effective method for reducing the allergic symptoms throught the adequate vitamin D3 supplementation.


 1. Gołąb J, Jakóbisiak M, Lasek W (2004): Immunologia. PWN press. Warsaw.

 2. Kręcisz B, Chomiczewska D, Kieć-Świerczyńska M (2009): The role of regulatory t cells in allergic contact dermatitis. Med Pr 60: 315-319 [Article in Polish].

 3. Lewkowicz P, Lewkowicz N, Tchorzewski H (2005): Limfocyty T regulatorowe CD4+CD25+: fizjologia i rola tych komórek w modulowaniu odpowiedzi immunologicznej. Post Hig Med Dosw 59: 362-370.

 4. Domagała-Kulawik J, Hoser G, Dąbrowska M, et al. (2011): Fas+ lymphocytes and CD4+/CD25+ cells in peripheral blood of never smoking patients with chronic obstructive pulmonary disease. Centr Eur J Immunol 36: 226-232.

 5. Stankiewicz W, Rongies W, Bodera P, et al. (2011): Im­mu­noregulatory disorders in irritable bowel syndrome. Centr Eur J Immunol 36: 267-274.

 6. Maggi E, Cosmi L, Liotta F, et al. (2005): Thymic regulatory T cells. Autoimmun Rev 4: 579-586.

 7. Fehervari Z, Sakaguchi S (2004): CD4(+) Tregs and immu­necontrol. J Clin Invest 114: 1209-1217.

 8. Baecher-Allan C, Brown JA, Freeman GJ, et al. (2001): CD4+CD25+ regulatory cells in human peripheral blood.

J Immunol 167: 1245-1253.

 9. Jonuleit H, Schmitt E, Stassen M, et al. (2001): Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood.

J Exp Med 193: 1285-1294.

10. Roncarolo MG, Gregori S, Battaglia M, et al. (2006): Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev 212: 28-50.

11. Ryba M, Myśliwska J (2010): Limfocyty T CD4+CD25+ Foxp3+: naturalnie występujące limfocyty T regulatorowe. Pediatr Endocrinol Diabetes Metab 16: 289-294.

12. Roncarolo MG, Bacchetta R, Bordignon C, et al. (2001): Type 1 T regulatory cells. Immunol Rev 182: 68-79.

13. Weiner HL (2001): Induction and mechanism of action of transforming growth factor-beta-secreting T3 regulatory cells. Immunol Rev 182: 207-214.

14. Holick MF (2007): Vitamin D deficiency. N Engl J Med 357: 266-281.

15. Shoenfeld N, Amital H, Shoenfeld Y (2009): The effect of melanism and vitamin D synthesis on the incidence of autoimmune disease. Nat Clin Pract Rheumatol 5: 99-105.

16. Arnson Y, Amital H, Shoenfeld Y (2007): Vitamin D and autoimmunity: new aetiological and therapeutic considerations. Ann Rheum Dis 66: 1137-1142.

17. Prietl B, Pilz S, Wolf M, et al. (2010): Vitamin D supplementation and regulatory T cells in apparently healthy subjects: vitamin D treatment for autoimmune diseases? Isr Med Assoc J 12: 136-139.

18. Żukiewicz-Sobczak W, Krasowska E, Zwoliński J, et al. (2012): Allergic diseases – current state of knowledge. Postep Derm Alergol 29: 451-455.

19. Nahhas M, Bhopal R, Anandan Ch, et al. (2012): Prevalence of allergic disorders among primary school-aged children in Madinah, Saudi Arabia: two-stage cross-sectional survey. PLoS One 7: 1932-6203.

20. Punekar YS, Sheikh A (2009): Establishing the incidence and prevalence of clinician-diagnosed allergic conditions in children and adolescents using routinely collected data from general practices. Clin Exp Allergy 39: 1209-1216.

21. Kim J, Lahl K, Hori S, et al. (2009): Cutting edge: depletion of Foxp3+ cells leads to induction of autoimmunity by specific ablation of regulatory T cells in genetically targeted mice. J Immunol 183: 7631-7634.

22. Heinze E, Chan G, Mory R, et al. (2011): Tumor suppressor and T-regulatory functions of Foxp3 are mediated through separate signaling pathways. Oncol Lett 2: 665-668.

23. Nanke Y, Ishiguro N, Yago T, et al. (2012): The role of regulatory T cells in mice with experimental autoimmune uveitis – a preliminary study. Centr Eur J Immunol 37: 102-105.

24. Sakaguchi S, Sakaguchi N, Shimizu J, et al. (2001): Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity and transplantation tolerance. Immunol Rev 182: 18-32.

25. Maczyńska I, Baskiewicz-Masiuk M, Ratajczak-Stefańska V, et al. (2009): CD4+ CD25high regulatory T cells and CD4+ CD69+ T cells in peripheral blood of patients with cutaneous lupus erythematosus. Centr Eur J Immunol 34: 213-217.

26. Karki R, Jung MA, Kim KJ, et al. (2012): Inhibitory effect of nelumbo nucifera (Gaertn.) on the development of atopic dermatitis-like skin lesions in NC/Nga mice. Evid Based Complement Alternat Med 2012: 1-7.

27. Ikegawa Y, Sato S, Lim G, et al. (2012): Amelioration of the progression of an atopic dermatitis-like skin lesion by silk peptide and identification of functional peptides. Biosci Biotechnol Biochem 76: 473-477.

28. Akbari O, Stock P, De Kruyff RH, et al. (2003): Role of regulatory T cells in allergy and asthma. Curr Opin Immunol 15: 627-633.

29. Szczawińska-Popłonyk A, Bręborowicz A (2012): Vitamin D impact on immune functions: implications for preventive strategy of allergic disease? Postep Derm Alergol 29: 176-181.

30. Smolders J, Menheere P, Thewissen M, et al. (2010): Regulatory T cell function correlates with serum 25-hydroxyvitamin D, but not with 1,25-dihydroxyvitamin D, parathyroid hormone and calcium levels in patients with relapsing re-

mitting multiple sclerosis. J Steroid Biochem Mol Biol 12: 243-236.

31. Royal W III, Mia Y, Li H, et al. (2009): Peripheral blood regulatory T cell measurements correlate with serum vitamin D levels in patients with multiple sclerosis. J Neuroimmunol 213: 135-141.

32. Plum LA, De Luca HF (2010): Vitamin D, disease and therapeutic opportunities. Nat Rev Drug Discov 9: 941-955.

33. Boucher BJ, Witham MD (2011): Should we be giving enhanced vitamin D intakes at all? J R Coll Physicians Edinb 41: 324-329.

34. Hollis BW (2011): Short-term and long-term consequences and concerns regarding valid assessment of vitamin D deficiency: comparison of recent food supplementation and clinical guidance reports. Curr Opin Clin Nutr Metab Care 14: 598-604.

35. Hart PH, Gorman S, Finlay-Jones JJ (2011): Modulation of the immune system by UV radiation: more than just the effects of vitamin D? Nat Rev Immunol 11: 584-596.

36. Hart PH (2012): Vitamin D supplementation, moderate sun exposure, and control of immune diseases. Discov Med 13: 397-404.

37. Sandhu MS, Casale TB (2010): The role of vitamin D in asthma. Ann Allergy Asthma Immunol 105: 191-199.

38. Nouri-Aria KT (2009): Foxp3 expressing regulatory T-cells in allergic disease. Adv Exp Med Biol 665: 180-194.

39. Chambers ES, Hawrylowicz CM (2011): The impact of vitamin D on regulatory T cells. Curr Allergy Asthma Rep 11:


40. Kay AB (2001): Allergy and allergic diseases. N Engl J Med 344: 30-37.

41. Robinson DB (2009): Regulatory T cells and asthma. Clin Exp Allergy 39: 1314-1323.
Copyright: © 2013 Polish Society of Experimental and Clinical Immunology This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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