Decreased XCL1 and XCR1 expression by allergic asthmatic Treg
Subjects included 11 HC, 11 mild-to-severe AA, and 7 mild-to-moderate NA (). CD4+CD25highCD127low/− Treg and CD4+CD25− cells (responder T cells) were isolated by flow cytometry sorting. Purity of Treg and responder T cells was confirmed with intracellular staining for Foxp3 to be more than 95 and 99%, respectively (). To confirm our preliminary QT-PCR data (), we performed flow cytometry analysis of XCL1/XCR1 protein expression in Treg among three subject groups. Our results showed a reduced expression of XCL1/XCR1 in Treg from AA subjects compared with HC and NA in both percentage of XCL1-positive cells and the mean fluorescent intensity (MFI) (). We also examined responder T cells that showed significantly lower expression of XCL1/XCR1 compared with Treg, but no significant differences among three subject groups (), suggesting that the reduction in XCL1 expression in AA was specific to Treg.
Furthermore, we collected BAL from allergen-challenged AA, NA, and HC subjects to investigate the possible involvement of XCL1 in airway inflammation in AA. We first examined XCL1 levels in BAL via ELISA. We found that XCL1 levels from AA BAL were significantly lower than those from HC and NA subjects (). We subsequently purified Treg from these BAL samples via flow cytometry sorting to analyze their XCL1 expression. Due to the small numbers of Treg obtained from BAL, we performed QT-PCR analysis of XCL1 mRNA expression in these cells instead of examining XCL1/XCR1 protein expression. Similar numbers of Treg were used for each subject in PCR analysis. Our results showed that Treg from AA subjects expressed significantly lower levels of XCL1 mRNA transcripts compared with HC and NA subjects (). Altogether, these data suggested that the reduced XCL1 expression in Treg in AA was associated with airway inflammation in the disease.
Allergic asthmatic Treg exhibited suboptimal suppressive and cytotoxic activities
Standard 3H incorporation assays were used to evaluate suppressive activities of Treg against autologous responder T cells. Responder T cells were exposed to plate-bound anti-CD3 Abs as TCR stimuli in the presence of irradiated CD3-depleted APCs from allogeneic HC and autologous Treg. To avoid potential individual variations in proliferative responses of responder T cells in the interpretation of suppressive function of Treg, the degree of suppression of responder T cell proliferation by Treg was quantitated by percentage reduction in 3H uptake in cocultures of Treg and responder T cells compared with cultures of responder T cells alone for each subject. Treg from both AA subjects and HC exhibited suppressive activities against responder T cell proliferation (). However, AA Treg showed markedly reduced suppressive activities against autologous responder T cell proliferation in comparison to HC Treg. This phenomenon was observed at both 1:1 and 1:4 ratios of Treg to responder T cells (). Interestingly, Treg from NA subjects showed no significant changes in suppressive activities against autologous responder T cell proliferation compared with HC Treg (), suggesting that reduced Treg-mediated suppression is specific to AA.
FIGURE 2 Quantitation of in vitro suppressive and cytotoxic activities of Treg among three subject groups. A, Bar graphs showed representatives of suppression assays in HC, AA, and NA subjects. Dot plot graphs showed summary of degree of suppression at 1:4 and (more ...)
In addition to their ability to suppress cell proliferation, Treg have recently been shown to possess potent cytotoxicity against a wide range of target cells upon TCR stimulation by either anti-CD3/CD28 Abs or anti-CD3/CD46 Abs (23
). Therefore, this functional property of Treg was also examined in AA subjects by a flow-based killing assay previously described by Grossman et al. (24
). Treg (effectors) were cultured for 4 days with IL-2 or with additional stimuli and IL-2 while responder T cells (targets) were activated with IL-2 and plate-bound anti-CD3/CD28 Abs for 4 days (please see Materials and Methods
for detailed stimulation protocols). At the beginning of the assays, responder T cells were labeled with CFSE and incubated with Treg. Responder T cell death was detected by 7-AAD staining of CFSE-labeled cells before flow cytometry analysis. As previously demonstrated, anti-CD3/CD46 stimulation of Treg and anti-CD3/CD28 stimulation of responder T cells showed optimal killing of responder T cells by Treg (24
). Thus, this method of activation was chosen to examine Treg function. We have performed killing assays with anti-CD3/CD28 stimulation of Treg and killings assays with unactivated responder T cells and found suboptimal cytotoxicity, similar to studies described by Grossman et al. (Ref. 23
; data not shown). The 4-hour killing assays at 1:1 ratio of Treg to autologous responder T cells showed evidence of responder T cell death, which increased with IL-2 and anti-CD3/CD46 stimulation of Treg compared with Treg from cultures with IL-2 alone, in both HC and NA subjects (). Surprisingly, killing was undetectable in AA subjects when Treg were not activated with anti-CD3/CD46 Abs (). By either adding anti-CD3/CD46 Abs or increasing cell ratio to 10:1 of Treg to autologous responder T cells, responder T cell death became apparent in AA subjects but failed to reach the level from both HC and NA subjects under the same experimental conditions (). Along with findings in suppression assays, these results suggested a reduced regulatory function of Treg from AA subjects.
To rule out the possibility that the suboptimal suppression and cytotoxicity observed above resulted from decreased susceptibility of responder T cells to Treg activities, we have performed suppression assays and killing assays with mixed allogeneic T cells from HC and AA subjects. Treg or responder T cells from each AA subject were paired with their HC counterparts in suppression and killing assays. AA Treg showed reduced killing and suppression of allogeneic responder T cells from HC subjects, whereas HC Treg exerted optimal cytotoxicity and suppression against allogeneic AA responder T cells (). These data suggested that the reduced suppression and killing observed in this study resulted from defective Treg function in AA.
Reduced Treg function in allergic asthmatics correlated with decreased XCL1 expression
Expression of molecules implicated in Treg-mediated suppression and cytotoxicity was examined next by intracellular staining on purified Treg. Our analysis focused on the expression of Foxp3, IL-10, TGF-β, granzyme A, and granzyme B. Treg from all subject groups express similar numbers of Foxp3+ cells (>95%); however, Treg from AA subjects expressed significantly lower levels of Foxp3 than HC and NA subjects via MFI analysis (). IL-10 and TGF-β expression by Treg were similar among HC, AA, and NA subjects (). However, upon receiving anti-CD3/CD46 stimuli, AA Treg expressed markedly lower levels of granzyme A, in terms of granzyme A+ cells and MFI, and lower levels of granzyme B, in terms of MFI, than did HC and NA subjects (). Correlation analysis showed that Foxp3 expression positively correlated with Treg-mediated suppression of responder T cell proliferation, whereas granzyme A expression positively correlated with Treg-mediated cytotoxicity against responder T cells in AA subjects (). Furthermore, XCL1 expression positively correlated with expression of Foxp3 and granzyme A and granzyme B (). We have performed correlation analysis with XCR1 expression and found no significant correlations with Treg function (data not shown). Altogether, these data suggested that lower level of XCL1 was associated with dysfunctional Treg activity in AA.
FIGURE 3 Expression of effector molecules of Treg function among three subject groups. A, Foxp3 expression by Treg in AA, HC, and NA subjects. B, IL-10 expression by Treg in AA, HC, and NA subjects. C, TGF-β expression by Treg in AA, HC, and NA subjects. (more ...)
Correlation analysis of selected parameters of Treg function, clinical assessment of allergic asthma, and XCL1 expression by Treg
XCL1 enhanced Treg-mediated suppression and cytotoxicity in allergic asthmatics
XCL1 has been shown to improve immune suppression (18
). Along with this finding, the association between reduced Treg function and decreased XCL1 expression in AA Treg prompted us to examine the hypothesis that exogenous XCL1 could possibly improve Treg activity in AA. We introduced XCL1 in suppression assays and killing assays of Treg against responder T cells from AA subjects. The optimal dose of XCL1 was determined to be 200 ng/ml (), which was similar to those concentrations of XCL1 used by previous studies (17
). For suppression assays, XCL1 was added in cell cultures at the beginning of the 7-day assay. For killing assays, XCL1 was added at the beginning of the 4-day activation period of Treg with IL-2 and plate-bound anti-CD3/CD46 Abs. Suppression assay results showed a significant enhancement of AA Treg suppression of autologous responder T cell proliferation at 1:1 cell ratio in the presence of XCL1 (). Further cultures of AA Treg/responder T cell alone in the presence of XCL1 indicated no significant effects of XCL1 on either Treg or responder T cell proliferation, suggesting that XCL1 modulated suppressive activity of Treg rather than proliferative potential of responder T cells or Treg (). Killing assays of AA Treg against autologous responder T cells also showed a significantly elevated level of responder T cell death in cultures with XCL1 (). This phenomenon was not due to XCL1 effects on cell viability, because analysis of both Treg and responder T cells before and after 4-day cultures by trypan blue exclusion assay or propidium iodide/annexin staining showed no significant signs of XCL1-induced cell death (data not shown).
FIGURE 4 Modulation of Treg-mediated suppression and cytotoxicity by XCL1. A, Effects of exogenous XCL1 and several other chemokines on Treg-mediated suppression of responder T cell proliferation at different concentrations. Representative data of cocultures of (more ...)
Because chemokines possess shared signaling pathways, we also tested the specificity of the XCL1 effect by introducing other chemokines such as CCL1, CCL5, CCL17, and CCL22 into suppression and killing assays. These chemokines were chosen due to their suggested involvement in Treg function and AA. Interestingly, CCL1, CCL17, and CCL22 did not enhance regulatory activities of AA Treg (). CCL5, a chemokine associated with T cell activation and proliferation (25
), as well as asthma exacerbation (27
) in similar experimental settings, resulted in further reduction in AA Treg-mediated suppression and cytotoxicity (). Cultures of CCL5 with Treg or responder T cell alone also suggested that CCL5 possibly reduced Treg function by acting via expansion/activation of responder T cells, because CCL5 incubation induced a significant increase in responder T cell proliferation but not Treg proliferation ().
To confirm the role of XCL1 in modulating Treg function, we performed suppression assays and killing assays with XCL1 and XCR1 neutralizing Abs. We performed these assays on HC subjects that exhibited normal Treg function. These blocking reagents were added at day 0 in suppression assays and at the beginning of the 4-day activation period of Treg in killing assays. In suppression assays, both neutralizing Abs reduced Treg suppressive function against responder T cell proliferation (). Killing assays with XCL1 or XCR1 neutralizing Abs also resulted in a reduction in cytotoxicity of Treg against target cells (). Similar experiments were performed with “negative control” IgG1 Abs (BD Biosciences) that did not show any influences on regulatory activities of Treg ().
XCL1 up-regulated expression of XCR1/XCL1 and molecules associated with Treg-mediated suppression and cytotoxicity in allergic asthmatics
In association with these functional findings, we examined the effects of recombinant XCL1 on the expression of Foxp3, IL-10, TGF-β, granzyme A, and granzyme B, as well as XCL1/XCR1 by Treg. For Foxp3, IL-10, TGF-β, and XCL1/XCR1 detection, Treg were incubated with XCL1 in C medium with IL-2 supplement for 4 days before analysis. For granzyme A and granzyme B detection, Treg were incubated with XCL1 and anti-CD3/CD46 Abs in C medium with IL-2 supplement for 4 days. Exogenous XCL1 enhanced the expression of Foxp3, IL-10, TGF-β, granzyme A, and granzyme B by Treg (). On the contrary, similar experiments performed with other chemokines such as CCL1, CCL5, CCL17, and CCL22 did not show any significant modulatory effects of these chemokines on the expression of these proteins. Interestingly, incubation of Treg with XCL1 up-regulated their XCL1 and XCR1 expression (), suggesting that XCL1 may act on Treg via a positive feedback, autocrine mechanism.
FIGURE 5 Modulation of expression of effector molecules of Treg function by XCL1. A, Effects of XCL1 and several other chemokines on Foxp3 expression of Treg in AA subjects. B, Effects of XCL1 and several other chemokines on IL-10 expression of Treg in AA subjects. (more ...)
Regulatory activities and XCL1 expression in allergic asthmatic Treg were enhanced by steroid usage and inhibited by exogenous IL-4
We next examined possible correlations between the reduction in Treg function and disease parameters of asthma such as IgE levels, disease severity, FEV1, disease duration, and allergic status. We found no significant correlation between Treg function and IgE level, disease duration, or allergic status (data not shown). Treg from AA subjects with moderate to severe asthma had lower suppressive and cytolytic function compared with those from mild AA subjects. However, the differences were not statistically significant (data not shown). Degrees of suppression and killing were positively correlated with FEV1 (). We did not analyze the correlation between inhaled corticosteroid usage and Treg function because blood samples from these subjects were obtained after they had been on a 48-h steroid withdrawal. Thus, their medication status might not represent the effects of inhaled corticosteroid on immune cells in peripheral blood.
To test whether steroids could enhance Treg function, and to assess the possible biological and clinical relevance of the ability of XCL1 to enhance Treg function in AA subjects, we examined AA Treg function in AA subjects with asthma exacerbation who were treated with oral corticosteroid (OCS). AA subjects (AA1, AA2, AA3, AA4, and AA5) were effectively treated with OCS, and their blood samples were analyzed at day 2 of 5-day treatment with OCS at 40 mg per day. Studies of Treg function from these samples showed a significant increase in Treg activity (). We also tested ex vivo incubation of high dose prednisone (1000 ng/ml) with Treg from AA subjects that were on 48-h steroid withdrawal and found that ex vivo treatment of AA Treg with steroids also enhanced their suppressive and cytotoxic activities (). On the contrary, addition of high-dose IL-4 (1000 U/ml) to suppression and killing assays of Treg obtained from OCS-treated AA subjects reversed the steroid-mediated functional enhancement of Treg ().
FIGURE 6 Modulation of Treg function and XCL1 expression by steroids and exogenous IL-4. A, Suppression assays and killing assays of Treg against responder T cells in HC subjects (n = 11), sarcoidosis subjects (n = 5), sinusitis on high-dose OCS subjects (n = (more ...)
To test whether steroids and IL-4 might enhance Treg function via modulation of their XCL1/XCR1 expression, we examined XCL1/XCR1 levels in Treg from AA subjects before and after OCS treatment, as well as Treg from OCS treated subjects that were subsequently incubated with high dose IL-4. XCL1 and XCR1 expression in Treg from AA subjects were significantly elevated after OCS treatment (). In contrast, exogenous IL-4 suppressed XCL1/XCR1 expression by Treg (). Altogether, these findings suggested opposite roles of steroids and IL-4 on the maintenance of Treg function, possibly via modulation of XCL1/XCR1 expression of Treg.