CD4+ Invariant T-Cell–Receptor+ Natural Killer T Cells in Bronchial Asthma
Omid Akbari, Ph.D., John L. Faul, M.D., Elisabeth G. Hoyte, M.S.N., Gerald J. Berry, M.D., Jan Wahlström, M.D., Ph.D., Mitchell Kronenberg, Ph.D., Rosemarie H. DeKruyff, Ph.D., and Dale T. Umetsu, M.D., Ph.D.
Background Bronchial asthma is associated with an inflammatoryprocess that is characterized by the presence in the airwaysof large numbers of CD4+ T cells producing interleukin-4 andinterleukin-13. However, the CD4 antigen is expressed not onlyby class II major histocompatibility complex (MHC)–restrictedCD4+ T cells, but also by a newly identified subgroup of T cells,CD1d-restricted natural killer T cells. These cells expressa conserved (invariant) T-cell receptor and have a potent immunoregulatoryfunction. Because mouse models of allergic asthma indicate thatnatural killer T cells are required for the development of allergen-inducedairway hyperreactivity, we hypothesized that natural killerT cells play an important role in human asthma.
Methods We used CD1d-tetramers, antibodies specific for naturalkiller T cells, as well as reverse-transcriptase–polymerase-chain-reactionanalysis of the invariant T-cell receptor of natural killerT cells to assess the frequency and distribution of naturalkiller T cells in the lungs and in the circulating blood of14 patients with asthma.
Results About 60 percent of the pulmonary CD4+CD3+ cells inpatients with moderate-to-severe persistent asthma were notclass II MHC–restricted CD4+ T cells but, rather, naturalkiller T cells. The natural killer T cells expressed an invariantT-cell receptor and produced type 2 helper cytokines. In contrast,the CD4+ T cells found in the lungs of patients with sarcoidosiswere conventional CD4+CD3+ T cells, not natural killer T cells.
Conclusions Together with studies in mice indicating a requirementfor natural killer T cells in the development of allergen-inducedairway hyperreactivity, our results strongly suggest that CD4+natural killer T cells play a prominent pathogenic role in humanasthma.
Asthma is characterized by airway inflammation dominated bythe presence of eosinophils and CD4+ T lymphocytes.1,2 The pulmonaryCD4+ cells in patients with asthma produce predominantly thetype 2 helper (Th2) cytokines interleukin-4, interleukin-5,and interleukin-13, which play essential roles in asthma byenhancing the growth, differentiation, and recruitment of eosinophils,basophils, mast cells, and IgE-producing B cells and by directlyinducing airway hyperreactivity,3,4,5 a cardinal feature ofasthma. Thus, class II major histocompatibility complex (MHC)–restrictedCD4+ Th2 T cells, which have been detected in the airways ofvirtually all patients with asthma, are thought to play an essentialrole in the pathogenesis of bronchial asthma.6,7
The CD4 cell surface marker is expressed not only by conventionalclass II–restricted CD4+ T cells but also by natural killerT cells, a newly described, unique subgroup of lymphocytes thatexpress features of both classic T cells and natural killercells. In humans, natural killer T cells express CD4, CD8 (asmall subgroup), or neither (i.e., negative for both CD4 andCD8 surface markers, also called double-negative cells). Manynatural killer T cells express a highly restricted repertoireof T-cell receptors consisting of V14-J18 (in mice) and V24-J18(in humans) and are called invariant T-cell receptor–positivenatural killer T cells (invariant natural killer T cells).8This T-cell receptor endows invariant natural killer T cellswith the unique property of responding to glycolipid antigens,rather than peptide antigens presented by the nonpolymorphicclass I MHC–like protein CD1d, expressed on antigen-presentingcells. Furthermore, on activation, invariant natural killerT cells rapidly produce large quantities of both type 1 helper(Th1)–biased (interferon-) and Th2-biased cytokines (interleukin-4),which enhance the function of dendritic cells, natural killercells, and B cells, as well as the function of conventionalCD4+ and CD8+ T cells.9 This rapid production of cytokines byinvariant natural killer T cells is a manifestation of innate-likeimmunity and provides invariant natural killer T cells withthe capacity to link innate and adaptive immune responses andcritically regulate adaptive immunity and a host of inflammatorydiseases.10,11,12,13,14,15,16 However, the role of invariantnatural killer T cells in humans is not completely understood.To investigate whether these invariant natural killer T cellshave an important role in human asthma, we studied the frequencyand distribution of CD1d-restricted invariant natural killerT cells in the lungs and peripheral blood of patients with persistentasthma.
Methods
Study Population
The panel on medical human subjects of Stanford University,the committee on clinical investigation of Children's HospitalBoston, and the internal review board of the Karolinska Institutein Stockholm approved the study, and written informed consentwas obtained from the 25 patients enrolled. Of these, the 14patients who had asthma were lifelong nonsmokers who had receiveda diagnosis of moderate-to-severe persistent asthma.
Study Procedures
All study patients and healthy controls underwent blood drawingand fiberoptic bronchoscopy. Before transoral fiberoptic bronchoscopy(BF-XT 20 or BF-IT 30 bronchoscope, Olympus) was performed,spirometry was performed (before and after the administrationof albuterol) with the use of equipment and procedures thatmet the guidelines of the American Thoracic Society.17 Patientsand controls were required to have a baseline forced expiratoryvolume in one second (FEV1) of more than 40 percent of the predictedvalue. For entry into the study, patients with asthma were requiredto have variable airflow obstruction as documented by a variabilityof more than 30 percent during serial recordings of the peakexpiratory flow rate2 and had to demonstrate both an increaseof 250 ml and an increase of 12.5 percent in FEV1 after treatmentwith inhaled albuterol. Transoral fiberoptic bronchoscopy wasperformed as previously described.17 Peripheral-blood mononuclearcells were obtained from whole blood from donors and was processedas described in the Supplementary Appendix (available with thefull text of this article at www.nejm.org).
Statistical Analysis
The statistical analysis was performed with InStat software,version 3.05 (GraphPad). The data are reported as means ±SD.Comparisons among the four groups included in the study —patients with asthma treated with corticosteroids, those withasthma not treated with corticosteroids, those with sarcoidosis,and control subjects — were performed with the Kruskal–Wallistest, with the use of Dunn's method for multiple comparisons.P values of less than 0.05 were considered to indicate statisticalsignificance.
Results
We studied 14 patients with moderate-to-severe persistent asthma,6 controls, and 5 patients with sarcoidosis, a respiratory inflammatorydisease in which large numbers of CD4+ Th1 cells are presentin the lungs18,19 (Table 1). No patient who had asthma had hadan exacerbation of the disease or had received oral corticosteroidtherapy or theophylline within the three months before entryinto the study. The four patients with asthma who had not receivedinhaled corticosteroids within three months or longer beforeentry into the study had a mean predicted FEV1 of 71 percent,indicating clinically significant asthma (Table 1). Patientswith atopic asthma had higher serum total IgE levels (mean,361 IU per milliliter) than both patients who did not have atopicasthma (mean, 53 IU per milliliter) and controls (mean, 21 IUper milliliter). Although patients with asthma who had receivedcorticosteroids had a higher mean serum IgE level (mean, 331IU per milliliter) than those with asthma not treated with corticosteroids(mean, 118 IU per milliliter), this difference was not significant.
Table 1. Characteristics of Patients with Asthma and Results of Radioallergosorbent Testing and Studies of Lung Function.
The six control subjects were all asymptomatic volunteers withnormal lung function without evidence of variable airflow obstruction,according to serial peak-flow measures. The five patients withsarcoidosis had stage II disease (lymphadenopathy and parenchymallung findings) with bilateral hilar lymphadenopathy with evidenceof reticulonodular shadowing or pulmonary infiltrates on high-resolutioncomputed tomography (thin sections, 1 mm thick) of the lung.No patient with sarcoidosis had a history of erythema nodosum.All five were white (race was determined by physicians in thisstudy), and all had noncaseating granulomas on transbronchialbiopsy with negative fungal and acid-fast smears and cultures.The average duration of disease in these patients was six months.None had received treatment with oral or inhaled corticosteroidsor other immunosuppressive agents.
Bronchoalveolar Lavage Findings
When specimens of bronchoalveolar-lavage fluid obtained fromall study patients and controls were examined for the presenceof CD4+ cells, CD8+ cells, and invariant natural killer T cells,we expected and found a higher total cell count in specimensfrom patients with asthma or sarcoidosis than in those fromcontrols (Table 2). We also noted an increase in the proportionof lymphocytes in patients with asthma (13 percent) and in patientswith sarcoidosis (21 percent), as compared with controls (7percent), but these differences did not reach significance.In both the asthma and sarcoidosis groups, the majority of lymphocyteswere CD4+. We then examined the bronchoalveolar-lavage fluidfor the presence of invariant natural killer T cells using CD1dtetramers loaded with -galactosylceramide, which specificallybind to the invariant T-cell receptor of invariant natural killerT cells,20 and with the monoclonal antibody 6B11, which specificallyrecognizes the CDR3 region of the V24-J18 T-cell receptor ofhuman invariant natural killer T cells.21 Both reagents staineda large number of cells in the bronchoalveolar-lavage fluidobtained from patients with asthma, indicating that invariantnatural killer T cells were present in the lungs of these patients(Figure 1 in the Supplementary Appendix). By contrast, virtuallyno invariant natural killer T cells were detectable in the bronchoalveolar-lavagefluid from either controls or patients with sarcoidosis.
Table 2. Analysis of Cells in the Bronchoalveolar-Lavage Fluid.
Because invariant natural killer T cells can express the CD4cell surface marker, and because large numbers of CD4+ cellsare known to be present in the lungs of patients with asthma,we measured the fraction of the CD4+ T cells in bronchoalveolar-lavagefluid of patients with asthma that were invariant natural killerT cells. Surprisingly, we found that a large fraction of theseCD4+ T cells were invariant natural killer T cells. In patientswith asthma, 45 to 86 percent (mean, 63 percent) of the CD4+cells expressed the invariant T-cell receptor V24, as determinedwith the use of tetramer staining (Figure 1A, and Table 2 inthe Supplementary Appendix), whereas in patients with sarcoidosis(Figure 1B) and controls (Figure 1C), less than 1 percent ofthe CD4+ cells expressed the invariant T-cell receptor V24.
Figure 1. Analysis of CD4+ Cells in Bronchoalveolar-Lavage Fluid from Patients with Asthma (Panel A), Patients with Sarcoidosis (Panel B), and Controls (Panel C) for Expression of the Invariant T-Cell Receptor of Invariant Natural Killer T Cells.
Specimens were stained with anti-CD3 monoclonal antibody, -galactosylceramide–loaded CD1d tetramers, and anti-CD4 monoclonal antibody. Dot plots were generated after gating on CD3+ cells and represent the expression of CD4 relative to -galactosylceramide–loaded CD1d tetramers. Arrows on the y axis indicate increasing fluorescein on staining with tetramers. Arrows on the x axis indicate increasing staining with anti-CD4.
Similar results were obtained with the use of direct immunofluorescenceand confocal laser scanning microscopy of biopsy specimens frompatients with asthma (Figure 2A through 2D). A photomicrographof one biopsy specimen (Figure 2A) shows the typical featuresof bronchial asthma — thickening of the basement membrane(lamina reticularis), epithelial disruption, and the presenceof a mononuclear cell infiltrate, including invariant naturalkiller T cells, in the submucosa (lamina propria). In findingson confocal laser microscopy (Figure 2B), nearly all the lymphocytesin the lamina propria express both CD4 and the invariant T cellreceptor V24; in contrast, in patients with sarcoidosis, thelymphocytes express CD4 but not V24 and therefore are not invariantnatural killer T cells (Figure 2C).
Figure 2. CD4+ Invariant Natural Killer T (NKT) Cells in the Airways of Patients with Asthma, but Not Patients with Sarcoidosis.
The left-hand portion of Panel A shows an endobronchial-biopsy specimen obtained from a patient with asthma with typical features of chronic asthma, including thickening of the basement membrane (lamina reticularis) (arrowhead), epithelial disruption, and cell infiltrates in the submucosa and lamina propria. On the right-hand side, a section from the same specimen shows staining of cells immediately beneath the lamina reticularis with fluorescein isothiocyanate–conjugated (FITC) antibody (monoclonal antibody 6B11) against the invariant natural killer T-cell receptor (arrow). Laser confocal images of bronchial-biopsy specimens from a patient with asthma are shown in Panel B and from a patient with sarcoidosis in Panel C. The CD4+ cells from the patient with bronchial asthma are invariant NKT cells, but those from the patient with sarcoidosis are not. A lung-biopsy specimen was stained with phycoerythrin-conjugated CD4 (red), and FITC-conjugated 6B11 monoclonal antibody (blue). The overlay results (pink) indicate that almost none of the CD4+ lymphocytes from the patient with sarcoidosis but nearly all of the CD4+ infiltrating lymphocytes from the patient with asthma coexpressed the invariant T-cell receptor. Panel D shows the percentage of CD3+ cells that are invariant NKT cells in bronchoalveolar-lavage fluid from 11 patients with asthma and 5 patients with sarcoidosis. Patients 11, 12, 13, and 14 did not receive corticosteroids. Cells were stained with anti-CD3 and -galactosylceramide–loaded CD1d tetramers. The bars represent the percentages of CD3+ cells that are positive or negative for CD1d tetramers in each of the four patients.
Analysis of the bronchoalveolar-lavage fluid obtained from patientswith asthma indicated that 58 to 86 percent (mean, 74 percent)of the CD3+ cells were invariant natural killer T cells (Figure 2D),whereas in patients with sarcoidosis, less than 2 percent ofthe CD3+ cells were invariant natural killer T cells (Figure 2D,and Table 2 in the Supplementary Appendix). The number of invariantnatural killer T cells in the lungs of the 14 patients withasthma did not appear to be significantly reduced with inhaledcorticosteroid therapy: 10 of these patients had been treatedwith potent inhaled corticosteroids for six months or longerbefore they underwent bronchoscopy, yet the majority of thepulmonary CD3+ cells from the patients (Patients 1, 2, 3, 4,8, 9, and 10) (Figure 2D) expressed the invariant T-cell receptorof invariant natural killer T cells, a finding similar to thatobserved in patients who had not been treated with corticosteroids(Patients 11, 12, 13, and 14).
To confirm the results of our study performed with the use ofCD1d tetramers and the natural killer T-cell–specificantibody, we also performed semiquantitative reverse-transcriptase–polymerase-chain-reactionanalysis. This molecular analysis demonstrated a high expressionof the messenger RNA (mRNA) for the invariant T-cell receptorof invariant natural killer T cells in the lungs of patientswith asthma. The mRNA for V24 and V11 (the invariant T-cellreceptor of natural killer T cells), but not V23 (an irrelevantT-cell receptor), was strongly expressed in cells from the bronchoalveolar-lavagefluid from patients with asthma (Figure 3), but not in thosefrom patients with sarcoidosis or controls. Together, thesestudies conducted with several different approaches indicatethat CD4+ invariant natural killer T cells are virtually absentfrom the lungs of controls and patients with sarcoidosis butare present in high numbers in the lungs of patients with asthma.
Figure 3. Messenger RNA for the Invariant T-Cell Receptor of Invariant Natural Killer T (NKT) Cells Expressed in Cells Obtained by Bronchoalveolar-Lavage from Patients with Asthma.
Cells from Patients 1 through 6 with asthma, Patients 21 and 25 with sarcoidosis, and two controls (Subjects 15 and 17) were analyzed for the expression of V24, V11 (invariant T-cell receptor), and V23 (irrelevant T-cell receptor) by reverse-transcriptase polymerase chain reaction (as described in the Supplementary Appendix). For each patient or control, expression was quantitated by amplification with 35, 30, and 25 cycles (loaded on gel left to right for each patient or control, as indicated by the slope of the triangle). To assess RNA loading, -actin was measured in the same subjects. Messenger RNA from purified invariant NKT cells was used as a standard for expression of V24, V23, and V11.
Although the invariant natural killer T cells in the lungs ofpatients with asthma were distinct from conventional class II–restrictedCD4+ T cells in expressing an invariant T-cell receptor, theinvariant natural killer T cells were similar to CD4+ Th2 cellsin producing interleukin-4 and interleukin-13. We found thatthe invariant natural killer T cells in the lungs of patientswith asthma produced both interleukin-4 and interleukin-13 butlittle interferon- on intracellular cytokine staining afteractivation with phorbol myristyl acetate and ionomycin (Figure 4A)or by measurement of cytokines in supernatants after activationwith -galactosylceramide, which specifically activates invariantnatural killer T cells (Figure 4B). In contrast, invariant naturalkiller T cells in the peripheral blood of all the patients withasthma or sarcoidosis and the controls produced all three cytokines(Figure 4C). Furthermore, in the bronchoalveolar-lavage fluidof patients with asthma, the vast majority (>95 percent)of the invariant natural killer T cells coexpressed CD4+ (Figure 4D),whereas in the peripheral blood of the patients with sarcoidosisand controls, only about 40 percent of the invariant naturalkiller T cells were CD4+ cells (approximately 50 percent ofthe invariant natural killer T cells were negative for bothCD4 and CD8, and approximately 3 percent were CD8+) (Figure 4E).These results suggest that one subgroup of invariant naturalkiller T cells (those producing Th2 cytokines and expressingCD4) is selectively recruited or expanded in the lungs of patientswith bronchial asthma but not in the lungs of patients withsarcoidosis.
Figure 4. Expression of Interleukin-4, Interleukin-13, and Interferon- by Invariant Natural Killer (NKT) T Cells in Bronchoalveolar-Lavage Fluid and Peripheral Blood.
Cells from bronchoalveolar-lavage fluid (Panel A) and purified invariant NKT cells isolated from peripheral blood (Panel C) were stimulated with phorbol myristyl acetate and ionomycin and stained for intracellular cytokines, as described in the Methods section. The open histograms represent the expression of cytokines by invariant NKT cells; the solid histograms depict staining with isotype control antibody. Panel B shows the production of interleukin-4, interleukin-13, and interferon- by bronchoalveolar lavage from patients with asthma after culture with -galactosylceramide or vehicle control. Supernatants were removed after 48 hours and analyzed by enzyme-linked immunosorbent assay. Panel D shows the percentage of all invariant NKT cells in the bronchoalveolar-lavage fluid from six patients with asthma (Panel D) and in peripheral blood from patients with asthma, patients with sarcoidosis, and controls (Panel E) that expressed CD4, CD8, or neither. The bars represent the percentage of invariant NKT cells that are CD4+CD8+ or CD4–CD8– (double negative) in each patient or control.
Discussion
Our studies show that CD4+ and CD3+ invariant natural killerT cells are abundant in the lungs of patients with chronic asthmabut are virtually absent from the lungs of controls and patientswith sarcoidosis. We confirmed previous work6,7 showing thatT cells in the lungs of patients with asthma expressed the CD4cell surface marker and produced Th2 cytokines, interleukin-4and interleukin-13, but not interferon- — that is, thatthese T cells have the typical cytokine profile of conventionalCD4+ Th2 lymphocytes. However, we showed that a great proportion(63 percent) of the pulmonary CD4+ T cells in patients withmoderate-to-severe persistent asthma (and 73 percent of theCD3+ cells) expressed an invariant T-cell receptor and thusare invariant natural killer T cells, rather than conventionalTh2 lymphocytes. The profusion of pulmonary invariant naturalkiller T cells in patients with asthma that we detected is surprising,but this finding mirrors those in mouse models of allergic asthmashowing an essential role for invariant natural killer T cellsin the development of allergen-induced airway hyperreactivity.15,16Moreover, it is surprising that invariant natural killer T cellsare present in the lungs of patients with asthma but not inthe lungs of patients with sarcoidosis, a multisystem disorderpredominantly involving the lungs. Both patients with sarcoidosisand those with asthma have large numbers of CD4+ T cells intheir lungs, but in patients with asthma the T cells secreteinterleukin-4 and interleukin-13, whereas in patients with sarcoidosisthe T cells secrete interferon- rather than interleukin-4 andinterleukin-13.22,23
The large number of invariant natural killer T cells in thelungs of patients with asthma is striking, especially giventhe fact that these cells constitute less than 0.1 percent ofthe mononuclear cells and less than 1 percent of the CD4+ Tcells in the peripheral blood.24 In addition, our finding thatmore than 90 percent of the invariant natural killer T cellsin the lungs of patients with asthma are CD4+ cells, whereasonly about 50 percent of the invariant natural killer T cellsin the peripheral blood are CD4+ cells, suggests that a subgroupof invariant natural killer T cells is recruited and enrichedin the lung, leading to levels in the lung that are 100 timesthe levels in the peripheral blood. The preferential recruitmentof invariant natural killer T cells may be related to a differentialexpression of chemokine receptors on the subgroup of CD4+ cellsthat are invariant natural killer T cells — a subgroupthought preferentially to produce interleukin-4 and interleukin-13.25,26,27,28Accordingly, our study indicates that the immunology of asthmamust be studied not by the examination of peripheral blood but,rather, by the evaluation of cells from within the lung. Thisprinciple may also hold true for other diseases in which invariantnatural killer T cells have been reported to play an importantrole.
To identify invariant natural killer T cells, we used CD1d tetramersloaded with -galactosylceramide, monoclonal antibody 6B11, orboth, currently considered the most sensitive and specific reagentsfor detecting invariant natural killer T cells. We found thatother reagents, such as antibody to the T-cell receptors V24and V11, although effective in identifying resting invariantnatural killer T cells in peripheral blood, were less sensitivethan CD1d tetramers and monoclonal antibody 6B11 in detectinginvariant natural killer T cells in bronchoalveolar-lavage fluid.This finding might be due to the fact that the invariant naturalkiller T cells in the lungs of patients with asthma are partiallyactivated, even in stable asthma, and that the T-cell–receptorexpression on invariant natural killer T cells is greatly down-regulatedafter the activation of invariant natural killer T cells.29However, levels of V24 and V11 mRNA were highly expressed incells in bronchoalveolar-lavage fluid (Figure 3), a findingconsistent with the idea that T-cell receptor down-regulationreduces the sensitivity of detection of invariant natural killerT cells with anti-V24 and anti-V11 antibody. We cannot excludethe possibility that even with the use of CD1d tetramers, monoclonalantibody 6B11, or both to identify invariant natural killerT cells in bronchoalveolar-lavage fluid, the frequency of invariantnatural killer T cells in the lungs of patients with asthmamight be underestimated because of T-cell receptor down-regulation.
CD4+ invariant natural killer T cells in the lungs of patientswith asthma express an invariant T-cell receptor that recognizesglycolipid antigens that are now being defined.30 These antigensappear to be highly conserved in mice and humans and includethe synthetic glycolipid -galactosylceramide, the self-glycolipidisoglobotrihexosylceramide (iGb3),31,32 bacterial glycosphingolipids,33,34and glycolipids from plant pollens.35 However, we propose thatself-glycolipids such as iGb3, which may be exposed in the lungsas a result of pulmonary inflammation or lung injury, can activateinvariant natural killer T cells, leading to airway inflammationand asthma. Alternatively, exogenous glycolipids, such as thosefrom inhaled plant pollens, may activate invariant natural killerT cells in the lungs and cause asthma. Identifying the glycolipidsrecognized by the invariant T-cell receptor of natural killerT cells, and understanding the processes by which glycolipidsare generated and activate invariant natural killer T cells,will probably provide important insights into asthma pathogenesisand perhaps reveal a host of new pathways amenable to new treatmentsspecifically for asthma.
In summary, we found that a large fraction of the CD4+ T cellsin the lungs of patients with asthma, but not in the lungs ofpatients with sarcoidosis, express the invariant T-cell receptorof invariant natural killer T cells, a newly described subgroupof T cells with immunoregulatory function. Together with studiesin mice indicating the requirement of invariant natural killerT cells for the development of allergen-induced airway hyperreactivity,our results strongly suggest that invariant natural killer Tcells in asthma represent a new paradigm in which CD4+ invariantnatural killer T cells, in concert with conventional CD4+ Tcells, produce interleukin-4 and interleukin-13, driving thedevelopment of inflammation in bronchial asthma. If invariantnatural killer T cells do indeed play a prominent role in thepathogenesis of asthma, therapies for asthma that target pulmonaryinvariant natural killer T cells may be highly effective.
Supported by grants from the National Institutes of Health (PO1AI054456,RO1 AI26322, and R01 HL69507, to Dr. Umetsu; RO1 CA52511, toDr. Kronenberg; and MO1-RR00070, to the Stanford UniversityMedical Center General Clinical Research Center), the AmericanLung Association of California (to Dr. Akbari), and the SwedishHeart–Lung Foundation (to Dr. Wahlström).
Dr. Umetsu reports having received consulting fees from TelosPharmaceuticals and owning equity in Innate Immunity. Dr. Faulreports having received consulting fees and lecture fees fromMerck, Pfizer, GlaxoSmithKline, and Boehringer Ingelheim andresearch support from Merck; and Dr. DeKruyff, consulting feesfrom Telos Pharmaceuticals. No other potential conflict of interestrelevant to this article was reported.
We are indebted to Maria Wikén for performing some ofthe staining; to the tetramer facility at the National Instituteof Allergy and Infectious Diseases, National Institutes of Health,for providing CD1d tetramers; and to Mark Exley for providinginvaluable reagents.
Source Information
From the Division of Immunology, Children's Hospital Boston, and the Department of Pediatrics, Harvard Medical School — both in Boston (O.A., R.H.D., D.T.U.); the Division of Pulmonary and Critical Care, Department of Medicine (J.L.F.), the Department of Pediatrics (E.G.H., D.T.U.), and the Department of Pathology (G.J.B.), Stanford University, Stanford, Calif.; the Division of Respiratory Medicine and Department of Medicine, Karolinska Institute, Stockholm (J.W.); and the Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, Calif. (M.K.). Drs. Akbari and Faul contributed equally to this article.
Address reprint requests to Dr. Umetsu at the Division of Immunology, Children's Hospital Boston, Harvard Medical School, Karp Research Laboratories, 1 Blackfan Cir., Rm. 10127, Boston, MA 02115, or at dale.umetsu{at}childrens.harvard.edu.
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14-J
) and Th2-biased cytokines (interleukin-4), which enhance the function of dendritic cells, natural killer cells, and B cells, as well as the function of conventional CD4+ and CD8+ T cells.9 This rapid production of cytokines by invariant natural killer T cells is a manifestation of innate-like immunity and provides invariant natural killer T cells with the capacity to link innate and adaptive immune responses and critically regulate adaptive immunity and a host of inflammatory diseases.10,11,12,13,14,15,16 However, the role of invariant natural killer T cells in humans is not completely understood. To investigate whether these invariant natural killer T cells have an important role in human asthma, we studied the frequency and distribution of CD1d-restricted invariant natural killer T cells in the lungs and peripheral blood of patients with persistent asthma. 
11 (the invariant T-cell receptor of natural killer T cells), but not V
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