T Cells in the Pathogenesis and Prevention of Asthma


asthmaBy Dale T. Umetsu, MD, PhD

Allergic asthma is an immunologic disease that is caused by adaptive immune responses to environmental allergens. Asthma is thus characterized by the presence of allergen-specific immunoglobulin (Ig)E, which is produced during adaptive immune response and by the presence of allergen-specific CD4+ Th2 cells producing interleukin (IL)-4, IL-5, and IL-13.[1] Th2 cells are also generated during adaptive immune responses, and are thought to play a central role in orchestrating the inflammation in asthma, because IL-4 causes isotype switch to IgE; IL-5 enhances the growth and differentiation of eosinophils; and IL-13 causes airway hyperresponsiveness (AHR) -- a cardinal feature of asthma.[2] The development of these adaptive immune responses with IgE and Th2 cells reflects allergen sensitization, which is a significant risk factor for the development of asthma.

Regulatory T Cells

Although Th2 cells have been the most frequently studied T cell in asthma, there are other CD4+ T cells that can influence inflammation in the airways. For example, CD4+ TReg cells can inhibit inflammation in the airways. These regulatory T cells are generally specific for allergens and produce inhibitory cytokines, such as IL-10 and transforming growth factor (TGF)-beta. TReg cells producing IL-10 have been shown to be present at high frequency in the peripheral blood of nonallergic healthy individuals, but are quite rare in allergic individuals.[3] On the other hand, allergic individuals have more IL-4 producing Th2 cells compared with nonallergic individuals. The goal of allergen immunotherapy is to reduce the number of Th2 cells and increase the number of allergen-specific TReg cells.[4]

There is another CD4+ T-cell type that has been shown to play a critical role in asthma, and that is the natural killer T (NKT) cell. NKT cells constitute a subset of lymphocytes that expresses characteristics of both natural killer cells (part of the innate immune system, the phylogenetically ancient system that has hardwired recognition receptors, such as toll-like receptors and complement receptors) and conventional T cells (part of the adaptive immune system).[5]

NKT Cells

NKT cells are either CD4+ or are double-negative cells, meaning that they lack both CD4 and CD8. In humans a small subset of NKT cells expresses the CD8 marker. Unlike natural killer cells, which do not have T-cell receptors (TCRs), NKT cells express TCRs, and most NKT cells express the same TCR, a conserved or invariant TCR, called Valpha14 in mice and Valpha24 in humans. Unlike the TCRs of conventional adaptive CD4 T cells, which recognize peptides of proteins in the context of major histocompatibility complex (MHC) class II molecules on antigen-presenting cells, the TCR of NKT cells recognize glycolipid antigens in the context of the nonpolymorphic, class I-like protein called CD1d on antigen-presenting cells. Moreover, when NKT cells are activated through this invariant TCR, NKT cells rapidly produce large amounts of IL-4 and interferon gamma (IFN-gamma), and they produce these cytokines much more rapidly than do conventional T cells.

The rapid production of cytokines by NKT cells is a manifestation of innatelike immunity that endows NKT cells with the capacity to critically amplify adaptive immune responses, by activating of T cells, natural killer cells, macrophages, and B cells. Furthermore, the rapid production of cytokines by NKT cells has been shown to regulate adaptive immunity involved in the development of inflammatory diseases, such as infectious diseases, cancer, autoimmune disease, transplant tolerance, and colitis.

NKT Cells in Allergen-Induced Airway Hyperreactivity

Because NKT cells produce large amounts of IL-4 and IL-13, the role of NKT cells in the development of asthma was examined, first in a mouse model. The development of allergen-induced AHR was examined in CD1d-/- mice, which lack the restriction element of NKT cells. Although they were wild-type mice, when they were sensitized and then challenged with ovalbumin (OVA) as allergen and developed severe AHR, surprisingly, the CD1d-/- mice failed to develop AHR when sensitized and challenged in the same way.[6,7] Similar results were obtained with another NKT-cell-deficient mouse strain, Jalpha18-/- mice, which lacks the invariant TCR of NKT cells -- and therefore lacks NKT cells. These mice also failed to develop AHR, indicating that NKT cells are required for the development of AHR measured in several different ways, in several different mouse strains.

In these initial studies, OVA and other protein antigens were used to induce AHR. However, NKT cells do not recognize proteins; rather, as mentioned, they respond to glycolipid antigens. In these initial studies, it is likely that the NKT cells were activated by an endogenous glycolipid that was expressed in the inflammatory environment induced by Th2 cells and protein antigens. On the other hand, if NKT cells were activated directly with a glycolipid, could the NKT cells alone induce AHR? To answer that question, NKT cells were activated with the glycolipid alpha-galactosylceramide which has been shown to specifically activate NKT cells. Within 2 hours following administration of alpha-galactosylceramide to normal mice, there was a measurable increase in AHR, which peaked at 24 hours, at levels of AHR comparable to that seen with OVA as the allergen.[8] Moreover, the AHR induced with alpha-galactosylceramide was associated with a significant inflammatory response in the airways. This inflammation did not occur in CD1d-/- mice treated with alpha-galactosylceramide, nor in the control-treated mice. Therefore, the activation of NKT cells was sufficient for the development of AHR because the direct activation of NKT cells by alpha-galactosylceramide induced all of the features that typify the hyperreactivity and inflammation induced by protein allergen.

Glycolipid Antigens

It is clear that NKT cells did not evolve in response to alpha-galactosylceramide, which is derived from a marine sponge, and that alpha-galactosylceramide is not an important environmental allergen in humans. However, it is possible that environmental glycolipid antigens might activate human NKT cells. For example, bacterial glycolipid antigens isolated from Sphingomonas bacteria can activate NKT cells and induce AHR. Thus, when mice were challenged with Sphingomonas glycolipid, they developed severe AHR and airway inflammation.[8] Although Sphingomonas does not infect humans, these results suggested that glycolipids from other pathogens might be able to activate pulmonary NKT cells and cause wheezing. Moreover, it has been shown that human NKT cells can recognize lipids from cypress tree pollen, suggesting that NKT cells that recognize environmental allergens may play an important role in human asthma.

NKT Cells in Human Asthma

To examine the role of NKT cells in human asthma, 14 patients with moderate-to-severe persistent asthma were studied. Ten of the asthma patients were on corticosteroids to control their symptoms, whereas 4 (noncompliant) asthmatic patients had not received corticosteroids for more than 3 months prior to enrollment. The mean age of the asthmatic patients was 44 years. The mean forced expiratory volume in the first second (FEV1) of the 10 patients on steroids was 84% of predicted, and the mean FEV1 of the 4 patients not on corticosteroid therapy was 71% of predicted. The mean total serum immunoglobulin (Ig)E for all 14 patients was 325 U/mL. Seventy-one percent of the patients were allergic as defined by positive skin tests to common allergens, suggesting that most but not all of the patients had the most common form of allergic asthma. Six normal healthy subjects were also enrolled, and bronchoalveolar lavage (BAL) fluid as well as endobronchial biopsies were obtained from these subjects.

About 15% of the BAL fluid cells from the patients with asthma were classified as lymphocytes with the majority of these cells identified as CD4+. The cells in BAL fluid were stained with CD1d tetramers loaded with alpha-galactosylceramide, which are known to specifically bind to the invariant TCR of NKT cells, or with an antibody, 6B11, which recognizes the CDR3 region of the alpha chain of the invariant TCR of human NKT cells. The staining of the BAL fluid cells with these reagents indicated that a large number of NKT cells were present in the BAL fluid, as defined by expression of the invariant TCR of NKT cells. In the 14 patients with asthma, 45% to 85% of the CD4 T cells were NKT cells, as defined by expression of the invariant TCR of NKT cells.[9]

To confirm the results observed by flow cytometry, biopsy specimens from patients with asthma were also examined. Confocal laser scanning microscopy found similar results as those seen with flow cytometry, principally that many of the cells in the lamina propria expressed CD4 and the invariant TCR of NKT cells. Because the healthy adults had virtually no NKT cells in BAL fluid, a control population with airway inflammation (patients with sarcoidosis) was also examined. These patients are known to have large numbers of CD4 T cells in their lung tissue, which are thought to be Th1 cells because they secrete IFN-gamma. In the biopsy sections from the sarcoidosis patients, many CD4 T cells were identified, but none of these cells expressed the invariant TCR of NKT cells.

To further confirm the presence of NKT cells in the BAL fluid samples from the nonsarcoidosis patients, semiquantitative reverse-transcriptase polymerase chain reaction testing (RT-PCR) for the invariant TCR of NKT cells, Valpha24 and Vbeta11, was performed. These studies demonstrated that mRNA for Valpha24 and Vbeta11 was expressed in BAL fluid cells of patients with asthma. However, mRNA for Valpha23 (an irrelevant TCR) was not expressed by these cells. In contrast, BAL fluid cells from patients with sarcoidosis or from normal individuals did not express mRNA for Valpha24, Vbeta11, or Valpha23. These studies confirmed that NKT cells, expressing the invariant TCR, are present in the lungs of patients with asthma, and suggest that the presence of NKT cells in the lungs is very specific for asthma, but play no role in sarcoidosis. Studies from other groups have confirmed these findings.[10]

NKT Cells in the Peripheral Blood

Although the number of NKT cells was increased in the lungs of patients with asthma, there was no measurable change in the number and phenotype of the NKT cells in the peripheral blood of patients with asthma. About half of the NKT cells in the peripheral blood of patients with asthma were CD4+; half were double-negative; and a small population was CD8+. This relationship was also true for the normal nonasthmatic individuals and for the patients with sarcoidosis. However, in BAL fluid of patients with asthma, > 90% of the NKT were CD4+. This suggested that there was a preferential recruitment or expansion of the CD4+ subset of NKT cells in the lungs in patients with asthma.

The cytokines produced by the NKT cells from the BAL fluid of asthma patients was also examined. The NKT cells were stimulated with phorbol myristate acetate and ionomycin, or with alpha-galactosylceramide, IL-4, and IL-13, but very little IFN-gamma was rapidly produced. In contrast, NKT cells from the peripheral blood of asthma patients produced an unrestricted cytokine profile. This suggests that there is a preferential recruitment or expansion of a subset of NKT cells in the lungs of patients with asthma, and this subset is CD4+ and produces Th2 cytokines, a subset that has the phenotype of Th2 cells.

Conclusions and Clinical Implications

Several different types of CD4+ T cells, including Th2 cells, regulatory T cells, and NKT cells producing IL-4 and IL-13, regulate asthma. In mice, the presence of NKT cells is required, and in some situations, is sufficient, for the development of AHR. In human patients, many of the CD4+ T cells in the lungs of asthmatic patients but not sarcoidosis patients are NKT cells. NKT cells in the lungs of asthmatic individuals produce IL-4 and IL-13, but not IFN-gamma, which is very similar to the profile of Th2 cells. We believe that the role of NKT cells in asthma has been underappreciated, presumably because the technology for identifying these cells was not available until recently.

The idea that NKT cells play an important role in asthma has a number of clinical implications. First, about 10% to 30% of patients with asthma have a steroid-resistant form of asthma, which may be due to the fact that, unlike Th2 cells and eosinophils, NKT cells are steroid-resistant. Therefore, therapies that target NKT cells may provide a very effective approach to controlling asthma. However, improved therapies for asthma will depend on the completion of many more studies of NKT cells, including studies to understand the relationship between NKT cells and conventional CD4+ T cells, and how NKT cells are activated and turned off, the endogenous glycolipids that activate NKT cells, and the exogenous glycolipids from plants or pathogens that activate NKT cells.


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