Background
Chronic inflammation in the lung with airway hyper-responsiveness is one of the major characteristics of asthma [
1]. Asthma is a highly heterogeneous disease comprised of distinct clinical, immunological, and genetic phenotypes [
2‐
4]; however, the pathogenesis of asthma has been classically characterized as elevated Th2-type inflammatory responses to antigen. These elevated Th2-type cells have also been found in the blood of asthma patients, indicating that immune cells responsible for chronic inflammation in the lung circulate in the blood [
5‐
8].
The normal response to a harmless allergen is tolerance, but asthmatic patients can respond with elevated Th2-type immune responses. Th2-type CD4
+ T cells secrete IL-4, IL-5, and IL-13, which play important downstream roles in asthma pathogenesis [
9]. IL-4 induces IgE class-switching and expression of vascular cell adhesion molecule-1 on endothelial cells [
10,
11]. IL-5 is crucial for the activation of eosinophils and their migration into the lung [
12]. IL-13 is associated with various important events during the effector phase of asthma including airway hyper-responsiveness, mucus hyper-production, and airway remodeling [
13,
14]. However, the high level of clinical heterogeneity of asthma suggests that the pathogenesis of asthma must not be solely driven by Th2-type immune responses [
15]. In almost all patients with asthma, one can find a counter-regulatory population, regulatory T cells (Tregs), that are capable of suppressing inflammatory responses [
16‐
18]. In addition, CD8
+ T cells can also contribute to the etiopathology of asthmatic inflammation [
19,
20]. Overall, T cells can play a central role in the initiation, progression, and exacerbation of asthma. However, the underlying mechanisms of the chronic inflammation in the lung and the levels of contribution by different T cell subsets remain to be fully elucidated.
Antigen-experienced T cells are phenotypically classified into effector and memory T cell populations, the latter being subdivided into CCR7
− effector memory T cells (Tem) and CCR7
+ central memory T cells (Tcm) [
21]. It has been previously reported that memory T cells are associated with chronic inflammatory diseases [
22,
23]. However, the specific subpopulations of human memory T cells that are responsible for chronic allergic disorders, including asthma, have not been well characterized. This is partly due to variations in the phenotypes of pathogenic T cells in asthma patients. It is further exacerbated by patient-intrinsic factors, such as differences in offending allergens, as well as environmental changes, which can affect timing of allergen exposure (e.g., perennial vs. seasonal allergy). Furthermore, the number of memory T cells recoverable from lungs of asthma patients is extremely limited. Despite these complicating factors, it is imperative to find which T cell subsets, especially which subset of memory T cells, are associated with chronic inflammation in the lungs of asthma patients.
To this end, we hypothesized that T cells in atopic asthma patients display unique phenotypes and functions that can support chronic inflammation in the lung. We utilized fresh whole blood from atopic asthma patients and non-asthmatic control subjects as a source of T cells for investigation. Although T cells in the peripheral blood may not be the same as those in the lungs of asthma patients, their altered phenotypes and functions could also be associated with the pathogenesis of asthma [
23‐
27]. We found that T cells in the blood of adult atopic asthma patients display several unique phenotypic and functional features. More importantly, some of the new features found in this study correlate with asthma severity, supporting the clinical relevance of these altered phenotypes and functions in atopic asthma patients. Further clinical data analysis concluded that corticosteroids do not affect these altered phenotypes or functions of T cells in atopic asthma. Data from this study could thus help us extend our knowledge of the pathophysiology of human asthma and potentially contribute to the rational design of new therapeutic approaches for asthma in the future.
Discussion
A persistent allergic inflammation in the lower airway may require an abundant presence of memory T cells [
22,
23,
49] that can readily respond to allergens that are intermittently available throughout the year. In both murine models of allergic asthma and asthma patients, CD4
+ memory T cells are involved in recurrent episodes of inflammation [
23‐
25,
50]. Accordingly, we found a significant increase of circulating CD45RA
−CD45RO
+ memory CD4
+ T cells in atopic asthma patients, compared to non-asthmatic control subjects. In this study, however, we further found that atopic asthma patients have a significant increase in memory CD4
+ T cells that express CCR7, but not CCR7
− memory CD4
+ T cells.
Both Tem and Tcm circulate in the blood. In contrast to Tem, CCR7
+ Tcm cells can migrate to the lymph nodes and can quickly proliferate in response to infiltrating antigen-presenting cells (APCs). Thus, Tcm cells are also considered reactive memory cells [
21,
51,
52]. They also can acquire an effector-like phenotype with the secretion of cytokines and chemokines [
21,
52]. It is therefore possible that such long-lived CD4
+ Tcm cells found in the blood of asthma patients could play an important role in the chronic inflammation in the lower airway in response to a variety of allergens that are intermittently available year-round. It was also important to note that the absolute numbers of CD4
+ Tcm cells were also greater in atopic asthma patients than non-asthmatic control subjects. Therefore, the increase of CD4
+ Tcm cells in atopic asthma patients was not due to a decrease of CD4
+ Tem cells in their blood.
The roles of CCR4
+ T cells in the pathogenesis of asthma are still controversial in both a murine model of asthma and asthma patients [
29,
33,
43,
53‐
57]. The increase of CCR4
+ CD4
+ T cells in asthma patients has been previously reported [
29,
33]. However, data from other studies indicate that the proportion of CCR4
+ CD4
+ T cells in peripheral blood or in the lungs does not always correlate with the severity of asthma [
43,
57]. In our study, we found that atopic asthma patients have more circulating CCR4
+ CD4
+ T cells and this was mainly due to the increase of CD4
+ Tcm cells. In line with this, the difference in the frequency of CCR4
+ CD4
+ T cells between atopic asthma patients and control subjects was even greater when we compared them in Tcm cells. The inverse correlation between the frequency of CCR4
+ CD4
+ Tcm cells and asthma severity further support that CCR4
+ CD4
+ Tcm cells could play an important role in the pathogenesis of atopic asthma. This increase of CCR4
+ CD4
+ Tcm cells can be seen across atopic asthma subtypes and severities. It was also important to note that current therapy (i.e. corticosteroids, β-agonists, leukotriene inhibitors, and combinations thereof) was not capable of reducing the frequency of either total CD4
+ Tcm or CCR4
+ CD4
+ Tcm cells in atopic asthma patients. A previous study reported that corticosteroid treatment slightly decreased the percentage of CCR4
+ total T cells, but it was performed with patients that had mild and stable asthma [
58]. Our findings raise a fundamental question concerning the mechanisms responsible for the increased numbers of CCR4
+ CD4
+ Tem cells in atopic asthma patients. Nonetheless, our data might also be highlighting the possible reasons behind the ineffectiveness of current therapies (i.e., corticosteroids).
In line with the increase of CCR4+ T cells in patients, T cells from atopic asthma patients secreted more of IL-5 and IL-13 than T cells from non-asthmatic control subjects. Our data also show that there was no significant difference in the frequencies of CXCR3+ (for Th1), CXCR5+ (for Th21), or CCR6+ (for Th17) CD4+ T cells in the blood of patients and control subjects. Consistent with the similar frequencies of T cells expressing such chemokine receptors, T cells from patients and control subjects also secreted similar amounts of IFNγ, IL-21, IL-17, TNFα and IL-22.
Atopic asthma patients have a higher percentage of CRTH2
+ cells, but this is only in the CD4
+ Tcm cell compartment. Such increase in patients was not observed when we analyze the frequency of CRTH2
+ cells in total CD4
+ T cells or in total memory CD4
+ T cells. This might explain inconsistent results from previous studies of the frequency of CRTH2
+ cells in asthma patients [
5,
35]. However, the increase of CRTH2
+ CD4
+ Tcm in patients was less significant than the increase of CCR4
+ CD4
+ Tcm cells. In addition, the frequency of CRTH2
+ CD4
+ T cells or CRTH2
+ CD4
+ Tcm cells did not show a significant correlation with any clinical variables, including ACT and % predicted FEV1 scores.
Consistent with the previously published data, we found that patients have an increased frequency of CD4
+ Tregs as assessed by measuring the frequency of CD25
+/high, Foxp3
+, CTLA4
+, Foxp3
+CD25
high, and Foxp3
+CTLA-4
+ CD4
+ T cells [
39]. Such increases in patients could be a natural process to counteract ongoing inflammatory responses, although corticosteroid treatment might also increase Treg frequency [
59,
60]. However, the percentages of Foxp3
+IL-10
+ CD4
+ T cells in the two groups of subjects were similar, and this is in line with the data from a previous study [
61]. Only a few patients showed increased frequency of Foxp3
+IL-10
+ CD4
+ T cells compared to other patients. The amounts of IL-10 secreted from T cells also showed a similar pattern to what was observed for the frequency of Foxp3
+IL-10
+ CD4
+ T cells. This suggests that Tregs in asthma patients might not be fully functional, as previously reported [
37‐
39]. We were not able to test the suppressive function of Tregs due to the limited amounts of blood samples collected from patients. The frequency of Foxp3
+IL-10
+ CD4
+ T cells did not correlate with asthma severity (data not shown). Interestingly, we found that the frequency of CTLA4
+ T cells correlated with the ACT scores. This suggested that fractions of Tregs in patients might still display certain levels of suppressive functions via the action of CTLA4, an inhibitory molecule, even though they may not be fully functional [
39].
Integrins play key roles in adhesion of leukocytes to walls of blood vessels associated with inflammation and in migration of leukocytes to inflamed tissues [
62,
63]. Integrins present on leukocyte surface belong to a large family of heterodimeric glycoproteins, which in the active conformation are composed of 2 noncovalently associated α and β subunits. Currently, 18 α and 8 β subunits are identified, which are associated in a restricted manner to create 24 heterodimers for specific ligand binding [
64]. Among those, both α4 and CD11a, an α chain of LFA-1, are known to play important roles in leukocyte migration to lung [
46‐
48,
65,
66]. A previous study also reported that IL-5 could increase VCAM-1 expression that can facilitate α4
+ leukocyte migration to inflamed lung and airways [
42]. One could thus expect an increase of α4
+ CD4
+ T cells in asthma patients. Interestingly, however, patients and control subjects have similar frequencies of circulating α4
+ CD4
+ T cells. However, we found that the frequency of α4
+ CD4
+ T cells significantly correlated with asthma severity, as assessed with the ACT scores. In contrast to α4
+ and CD11a
+ CD4
+ T cells, patients have significant reductions in the percentages of β7
+ CD4
+ and CD8
+ T cells. The clinical relevance of the decrease of β7
+ T cells in asthma is not clear at this moment. β7 is generally known to play an important role in lymphocyte migration into guts [
67,
68], and T cells in the lungs of asthmatic and non-asthmatic control subjects express only low level of α4β7 [
43]. However, others have also reported that β7 along with α4 can contribute to T cell and eosinophil accumulation in BAL and to airway inflammation in the absence of CCL19 and CCL21 [
44,
45].
Authors’ contributions
MW and KU carried out experiments and analyzed data. WY and JE helped with experiments and data analysis. BL and MM provided clinical samples. KU, HJ, MM, and SO designed this study and analyzed the data. MW, KU, HJ, and SO wrote this manuscript. All authors read and approved the final manuscript.