Asthma is a chronic inflammatory disorder of the airways characterized by variable airflow obstruction and presence of airway hyperresponsiveness that leads to typical symptoms like recurrent episodes of wheezing, breathlessness, chest tightness and coughing . CD4+ T cells are one of the most important cells in the pathogenesis of airway inflammation in asthma. CD4+ T cells are traditionally divided into Th1 and Th2 subsets based on profile of their cytokines production [2, 3]. Th2 cells, which are characterized by IL-4, IL-5, IL-9 and IL-13 cytokine production, predominate in asthma, whereas Th1 cells producing IFN-γ, IL-2 and TNF have rarely been associated with asthma [2, 3]. More recently new subsets of CD4+ T cells have been described: anti-inflammatory (suppressive) T regulatory (Treg) cells and proinflammatory Th17 cells [2, 3].
CD4+ Treg cells can be divided into natural regulatory T cells (nTreg cells) and inducible or adaptive regulatory T cells (iTreg cells) . nTreg got their name because they are always present in the body and constantly perform their function during normal surveillance of self-antigens . They arise in thymus and leave it fully active, ready to inhibit immunological responses. They constitute the population of long-living cells, resident in peripheral organs and one of their main activities is protection against autoimmunological disorders . The first described marker of these cells was CD25 with a more recent marker of nTreg cells being the constitutively expressed transcription factor Foxp3 . Therefore CD4+Foxp3+ or CD4+CD25+Foxp3+ cells are often regarded as nTreg cells, in spite of the fact that newer markers have also been proposed . However it should be noted that we still lack precise and specific markers of nTreg cells .
iTreg, in contrast to nTreg, obtain their regulatory ability in peripheral tissues. Naïve or mature CD4+ cells are activated by dendritic cells in the secondary lymphoid organs . The main function of iTreg is therefore to counteract excessive or the pathological inflammatory response [7, 8]. There are no good surface markers of iTreg and according to some authors they lack CD25 expression (CD4+CD25- cells)  whereas others have shown that CD4+CD25+ iTreg can be generated from naïve CD4+ cells after antigen stimulation in the presence of IL-2 and TGF-β . Furthermore it was shown that TGF-β-induced iTreg cells may express Foxp3  and that Foxp3+ Treg cells are a rich source of TGF-β [11–13]. These latter cells are also known as Th3 cells. Phenotypic characterization of Th3 cells is very difficult as not all TGF-β-secreting T cells are Th3 cells and not all Th3 cells express Foxp3 [11–13].
Tr1 cells are another subset of iTreg cells, which is difficult to study in great details as we still lack precise markers, which could help us to reliably distinguish IL-10-secreting Treg cells from other T cells . Tr1 cells produce IL-10, TGF-β and little or no IL-2, IL-4 and can be sometimes defined as: IL-10+IL-5+IL-4-IL-2+/-IFN-γ+. Many studies have demonstrated that IL-10-secreting T cells did not express Foxp3 [15–17] but there are also reports of subsets of Foxp3-expressing Treg cells with the ability to secrete IL-10 [18, 19]. Phenotypic characterization of Tr1 cells could be therefore defined as CD4+IL-10+IL-4-.
The percentage of CD4+CD25high cells was decreased in bronchoalveolar lavage (BAL) fluid of pediatric asthma patients . Also Foxp3 protein expression was decreased within blood CD4+CD25high cells . However, Smith et al. have recently shown an increased percentage of CD4+Foxp3+ and CD4+CD25+CD127- cells in BAL fluid of moderate to severe asthma . In another study the percentage of CD4+Foxp3+ cells in BAL increased after allergen provocative test in asthmatic patients .
Th17 cells are a new population of proinflammatory CD4+ T cells which are important during activation of the inflammatory response against bacterial infection for example . Th17 cells have an opposite function to Treg cells (especially iTreg cells) in relation to Th1 and Th2 cells. Interestingly, TGF-β in the presence of IL-6 induced the development of Th17 cells from naïve T cells whereas TGF-β alone induced the development of CD4+CD25+Foxp3+ Treg cells [25, 26]. Mouse Th17 cells produce large amounts of IL-17, but no IL-4, IFN-γ or Foxp3 and contribute to airway hyper-responsiveness [25, 27]. Several studies showed a possible involvement of Th17 cells in the pathogenesis of asthma. The number of IL-17 positive cells in sputum and BAL as well as the expression of mRNA for IL-17 in sputum cells is increased in asthma [28, 29]. The levels of IL-17 in sputum correlated with hyperresponsiveness to methacholine in asthmatic subjects . Increased levels of IL-17 in sputum were also seen in patients with allergic rhinitis after a nasal allergen challenge . Interestingly, two recent reports showed the opposite results [32, 33]. In the first study an increased expression of IL-17 in the bronchial epithelium was observed in severe asthma but not in mild or moderate asthma  whereas increased expression of IL-17 was reported in mild-moderate, but not severe asthma, in the second study .
In this study we isolated CD4+
cells from BAL fluid of asthmatics and age-matched control subjects and after short in vitro
stimulation we measured simultaneously the expression of up to six parameters in these cells:
set 1: TGF-β, FoxP3, CD25, IFN-γ, IL-4, TNF-α;
set 2: IL-10, FoxP3, CD25, IFN-γ, IL-4, MIP-1β;
set 3: IL-17A, IL-8, IFN-γ, IL-4, MIP-1β.
The aim of our study was to analyze the incidence of different subsets of CD4+ T cells in BAL fluid of asthmatics and healthy subjects, in particular CD4+CD25+Foxp3+ cells, CD4+Foxp3+ cells, CD4+IL-10+IL-4- cells, CD4+TGF-β+ cells, CD4+TGF-β+Foxp3+ cells and CD4+IL-17+ cells together with the co-expression of various cytokines: IFN-γ - a marker of Th1 cells, IL-4 – a marker of Th2 cells, IL-8 (CXCL8) – a chemokine for neutrophils, MIP-1β (CCL4) – a chemokine for monocytes and macrophages and TNF-α - a proinflammatory cytokine.