Allergic asthma is characterized by airway hyperresponsiveness and mucosal inflammation mediated by CD4+
Th2 cells. Thus, the suppression of Th2 cytokines (IL-4, IL-5, IL-9, and IL-13) has been shown to be plausible therapy to suppress airway inflammation and hyperresponsiveness (28
). In this regard, Tregs have been shown to be potent immunomodulators of a disrupted immune response as seen in asthma. In this study we found that adoptive transfer of GFP-labeled NTregs and iTregs isolated from the lung or spleen of naive GFP-transgenic mice migrated to the lymph nodes and to the lungs, and reversed AHR for at least 4 weeks in CRA-sensitized and -challenged mice. Moreover, we found that GFP-labeled CD4+
Tregs differentiated into CD4+
Tregs in the lungs of recipients that received adoptive transfer of L25−
cells. The functional difference and suppressive capacity of these TR
1 cells compared with NTregs appears to be mediated by the differential expression of PD-1, TGF-β, IL-10, and secretion of IFN-γ.
Several investigators reported that naturally occurring CD4+
Tregs are anergic or have nominal to absent proliferatory capacity in vitro
). However, we found that NTregs are able to proliferate in the periphery in response to repeated antigen exposure in vivo
. This could be supported by the findings of Walker and colleagues, that Tregs are able to divide in the peripheral and suppress effector T cells (34
). Moreover, literature has suggested that TR
1 cells proliferate poorly with T cell receptor (TCR) stimulation (35
), and recent findings suggest that cytokines, such as IL-10 and IL-15, are critical for stimulating their proliferation in vitro
). However, TR
1 cells suppress immune responses in vitro
and in vivo
and this mechanism is partially dependent on the production of IL-10 and TGF-β (37
). Moreover, little is known about the proliferatory capacity of TR
1 cells in vivo
. In this study, we for the first time report that iTregs differentiate into TR
1 cells in vivo
under repeated exposure to antigen. These TR
1 cells expressed Foxp3 and high levels of mRNA transcripts of IL-10 and TGF-β, and had robust proliferative capacity. Our data also suggest that the S25+
cells have less proliferative capacity in vivo
compared with L25+
cells, but spleen Tregs have significant capacity to suppress effector T cells under in vivo
conditions. Both Treg types from lung and spleen tissue were substantially suppressive even at the one-eighth ratio of the splenocytes and Tregs.
We also found that the GFP-labeled NTregs and iTregs isolated from the lungs of recipients that received GFP-labeled S25+
cells expressed significantly higher levels of mRNA transcripts of Nrp-1 than GFP-labeled NTregs and iTregs isolated from the lungs of recipients that received GFP-labeled L25+
cells. These data suggest that the expression of Nrp-1 on these splenic Tregs may serve as a marker and/or a functional mechanism to distinguish this subset of Tregs from lung-derived Tregs. It has been shown that the expression of Nrp-1 on activated Tregs may serve as a unique cell surface marker to define Tregs from other activated T cells (38
). Also, the expression of Nrp-1 promotes prolonged cell-to-cell interactions with Tregs and immature dendritic cells than CD4+
T-lymphocytes, resulting in high sensitivity to limited amounts of antigen (39
Tregs were also found to be highly suppressive in the lymph nodes of patients with cervical cancer (40
). The function and characteristics of Tregs may be dependent on anatomical location and the extracellular microenvironment (40
). These reports may explain why Tregs from lung tissue proliferate more than spleen-derived Tregs. In addition, our data suggest that Tregs may have inherent tissue-specific capacity to migrate back to the tissue of origin after adoptive transfer. We found that a high percentage of lung adoptively transferred NTregs and iTregs migrated back to the lung and specifically less than 5,000 of the lung iTregs migrated to the spleen (Figure E5A–E5H). Conversely, a high percentage of the adoptively transferred spleen NTregs and iTregs migrated to the lung, but more than 20,000 of these cells were isolated from the spleen of recipient mice. Unraveling these different subsets of Tregs and their function, characteristics, and movement of these cells warrants further investigation, but it elucidates that these cells have high proliferatory capacity in vivo
and migrate to sites of inflammation. In addition, we found 34,000 to 42,000 GFP-labeled Tregs migrated to the lymph nodes of adoptively transferred recipients; however, in the lymph nodes of CRA-sensitized and -challenged mice we found 47,000 to 60,000 CD4+
T cells (Figure E5I). Moreover, these CD4+
T cells were not Foxp3+
cells, which suggests these cells were probably Th2 and/or Th1 cells (data not shown).
The histological changes in the lung and airway inflammation paralleled the changes in the AHR to methacholine. The CRA-sensitized and -challenged mice exhibited the hallmarks of asthma, including profuse peribronchial and perivascular inflammation, epithelial cell damage resulting from massive inflammatory cell infiltration, goblet cell hyperplasia with mucous hypersecretion, collagen deposition, and smooth muscle cell hyperplasia. The CRAAT+ animals exhibited a reversal of the changes in the histological features with minimal inflammation. Studies have shown that the epithelial layer of the airway can repair and rebuild after asthmatic episodes; however, the time period and how this mechanism occurs remains to be elucidated. The airway remodeling in the CRAAT− resulted from the elevated levels of eosinophils and minimal levels of neutrophils as seen in the BALF. These associated changes in the asthmatic airways appear to be the result of a reduction in the number and/or impaired function of Tregs in the lung of these CRAAT− mice. In normal mice we isolated 40,000 to 43,000 Tregs; however, CRA sensitization resulted in a significant reduction of fewer than 20,000 Tregs in the lung at Day 33. Additionally, at Day 68 there were fewer than 10,000 Tregs in the lungs of CRAAT− mice (Figure E6). Conversely, the adoptive transfer of 100,000 Tregs ameliorated the clinical changes seen in the airways of these allergic asthma-induced mice (Figure E6). The BALF from CRAAT+ mice showed minimal to absence of eosinophils, which was consistent with the histology of normal bronchial parenchyma. The therapeutic effect of the adoptive transfer of Tregs helped to alleviate the characteristics of airway inflammation and AHR, and the morphologic changes were nearly restored to the PBS control level. It is, however, unclear whether the onset of allergic airway inflammation from CRA sensitization impairs the function of Tregs or if this impairment results in a reduction in the number of Tregs in the lung; in all of our sensitized mice, airway inflammation and AHR developed when the number of Tregs was reduced in the airways. Moreover, an adoptive transfer of 100,000 Tregs was very effective in reversing AHR and airway inflammation (Figures E7A and E7B).
Tregs are characterized by a canon of the cell surface expression markers to include: CD4, CD25, CD69, CD62L, ICOS, and GITR. The GFP-labeled iTregs, which upregulated CD25 and Foxp3 isolated from lungs of recipient mice that received S25− and L25− cells, expressed high levels of PD-1. Interestingly, the lung CD4+CD25+ cells obtained from the recipients of S25− group showed nearly 100% PD-1 immunopositive cells compared with about 75% in the S25+ recipient group (). We do not know the number of PD-1–positive cells required for long-lasting reversal of AHR. However, it is tempting to speculate that the differential expression of PD-1 could be one of the reasons underlying the functional differences between these Treg subsets. Therefore, PD-1 could be a strong candidate for characterization of inducible naive T-regulatory cells and critically involved in their suppressive function.
The PD-1 (CD279) receptor is 55-kD type I transmembrane protein of the Ig superfamily, with an extracellular region having one V-like domain. Recently, two new members of the B7 family, B7-H1 (PD-L1) (CD274) and B7-DC (PD-L2) (CD273), were identified as ligands for PD-1. PD-1–PD-L1 belongs to the CD28-B7 signaling family and this interaction leads to downregulation of T cell activity (41
). In the studies by Latchman and colleagues the interaction of PD-L1 and PD-1 leads to cell cycle arrest in G0/G1 but does not increase cell death (42
). Studies in C57BL/6 mice have shown that mice lacking PD-1 developed a lupus-like arthritis and glomerulonephritis (41
). In this study, we found that PD-1 is moderately upregulated after antigen exposure in Tregs isolated from lung tissue from CRA-sensitized and -challenged mice without adoptive transfer. In contrast, PD-1 was expressed at significantly higher levels in CD4+
Tregs isolated from recipient mice of L25−
cells than recipient mice of L25+
cells. These data suggest that PD-1 could be a critical mechanism involved in reversal of AHR and airway inflammation by inducible naive T-regulatory cells.
Therefore, in a separate study we examined the role of PD-1 in allergic asthma and Treg function with an adoptive transfer of Tregs and administered an anti PD-1 antibody (aPD-1ab) in CRA-sensitized and -challenged mice. We found that the therapeutic effect of the adoptive transfer of Tregs was blocked after the administration of the aPD-1ab. AHR was exacerbated in these CRA-sensitized and CRA-challenged mice with aPD-1ab and the deleterious effect of airway inflammation paralleled the CRA-sensitized mice without adoptive transfer therapy (unpublished data, H.S.M., D.K.A.). These data suggest that PD-1 has a role with Tregs and their ability to suppress AHR and airway inflammation. The mechanism of how PD-1 works clearly needs more investigation but nevertheless PD-1 may work in concert with other costimulatory molecules, such as CTLA-4 and/or ICOS.
ICOS is a marker that is upregulated on activated T cells in the presence of antigen. ICOS-ICOSL pathways are essential for the secretion of IL-10 (44
). In this study, CD4+
cells isolated from the lungs of the recipients of NTregs and iTregs were ICOShigh
and this expression was significantly greater than lung CD4+
T cells from mice without adoptive transfer. In a recent report, ICOShigh
T cells were tightly linked to IL-10 production; ICOSmed
T cells secreted IL-4, IL-5, and IL-13; and ICOSlow
T cells were associated with secretions of IL-2, IL-3, IL-6, and IFN-γ (45
). In our study, donor cells expressed low to undetectable levels of IL-10 and TGF-β, but high expression of ICOS on the lung CD4+
T cells isolated from the adoptive transfer recipients showed a tight correlation with the high secretion of IL-10 that was seen in the BALF and IL-10 mRNA transcripts. Hence, it is highly likely that high expression of ICOS seen on the TR
1 cells isolated from the lungs of adoptive transfer recipients and expression of IL-10 mRNA transcripts and BALF IL-10 may act as a switch mechanism for the secretion of IL-10.
Since the renaissance of T-regulatory cells, the mechanisms that govern the function and characterization of Tregs remain elusive. The most widely accepted marker for Tregs is Foxp3. The precise function of Foxp3 is not known, but an absence of Foxp3 in humans results in IPEX syndrome (46
). Patients with IPEX syndrome share many phenotypic features with scurfy mice, exhibiting mutation in Foxp3 and lacking Tregs. However, it remains elusive if these patients and scurfy mice also lack the inducible ability of a naive T cell to differentiate into a TR
In this study we sought to evaluate if this perplexing T-cell subset expressed Foxp3 for regulating AHR. Naive Tregs were found to constitutively express Foxp3, and undifferentiated CD4+
T cells did not express Foxp3, However, Foxp3 mRNA transcripts and intracellular protein were upregulated, parallel with CD25+
, in the lung CD4+
cells post adoptive transfer. Lung CD4+
cells from CRA-sensitized and -challenged mice without adoptive transfer expressed Foxp3 at low to undetectable levels and these mice continued to have the elevated AHR and severe airway inflammation. Foxp3 is crucial for the suppressive capacity of Treg activity in allergic asthma and autoimmune diseases. Subjects with myasthenia gravis have normal density of Tregs similar to healthy subjects, but there was a severe functional defect in their regulatory capacity together with decreased expression of Foxp3 (48
). In patients with multiple sclerosis CD4+
Tregs had reduced levels of Foxp3 expression, which was increased after treatment with a copolymer-1 (49
). Thus, Foxp3 appears to be crucial for Treg suppression.
In summary, we delineated a direct role that an adoptive transfer of Tregs has the therapeutic property of reversing the deleterious effects of allergic airway inflammation and AHR in a CRA murine model of allergic asthma. Specifically, the inducible Tregs from either spleen or lungs that have high PD-1 completely reversed AHR to methacholine and lung inflammation. However, the role of Tregs secreting IL-10 and TGF-β and expressing PD-1 and Nrp-1 to modulate allergic asthma remains to be elucidated; nevertheless this specialized subset of T cells clearly provides clinical implications to prevent the pathogenesis of asthma.