Invariant natural killer T cells and asthma: Immunologic reality or methodologic artifact?
2010; Elsevier BV; Volume: 126; Issue: 4 Linguagem: Inglês
10.1016/j.jaci.2010.06.041
ISSN1097-6825
AutoresCollin Brooks, Robert Weinkove, Ian F. Hermans, Christine J. van Dalen, Jeroen Douwes,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoTo the Editor:A role for invariant natural killer T (iNKT) cells in the cause of asthma has been shown in mice,1Akbari O. Stock P. Meyer E. Kronenberg M. Sidobre S. Nakayama T. et al.Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity.Nat Med. 2003; 9: 582-588Crossref PubMed Scopus (600) Google Scholar but the evidence in human subjects is equivocal.2Akbari O. Faul J.L. Hoyte E.G. Berry G.J. Wahlstrom J. Kronenberg M. et al.CD4+ invariant T-cell-receptor+ natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 1117-1129Crossref PubMed Scopus (360) Google Scholar, 3Hamzaoui A. Rouhou S.C. Grairi H. Abid H. Ammar J. Chelbi H. et al.NKT cells in the induced sputum of severe asthmatics.Mediators Inflamm. 2006; 2006: 71214Crossref PubMed Scopus (44) Google Scholar, 4Thomas S.Y. Lilly C.M. Luster A.D. Invariant natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 2613-2616Crossref PubMed Scopus (68) Google Scholar, 5Vijayanand P. Seumois G. Pickard C. Powell R.M. Angco G. Sammut D. et al.Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease.N Engl J Med. 2007; 356: 1410-1422Crossref PubMed Scopus (175) Google Scholar Matangkasombut et al,6Matangkasombut P. Marigowda G. Ervine A. Idris L. Pichavant M. Kim H.Y. et al.Natural killer T cells in the lungs of patients with asthma.J Allergy Clin Immunol. 2009; 123: 1181-1185Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar in a recent issue of the Journal, provided further evidence of higher iNKT cell numbers (up to 64% of all CD3+ T cells) in bronchoalveolar lavage fluid of subjects with severe asthma compared with those seen in subjects with well-controlled asthma, who in turn had higher numbers than seen in nonasthmatic subjects. If true, this might have major implications for our understanding of asthma's pathology.Some of the discrepancies between studies might be due to the use of different cytofluorimetric gating strategies when assessing iNKT cells in bronchoalveolar lavage fluid. It has already been suggested4Thomas S.Y. Lilly C.M. Luster A.D. Invariant natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 2613-2616Crossref PubMed Scopus (68) Google Scholar, 5Vijayanand P. Seumois G. Pickard C. Powell R.M. Angco G. Sammut D. et al.Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease.N Engl J Med. 2007; 356: 1410-1422Crossref PubMed Scopus (175) Google Scholar that the iNKT cells identified in the initial study2Akbari O. Faul J.L. Hoyte E.G. Berry G.J. Wahlstrom J. Kronenberg M. et al.CD4+ invariant T-cell-receptor+ natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 1117-1129Crossref PubMed Scopus (360) Google Scholar are likely to be alveolar macrophages on the basis of forward and side scatter characteristics. The use of a simple, 2-step CD3+SSClow strategy (as subsequently used by Matangksasombut et al6Matangkasombut P. Marigowda G. Ervine A. Idris L. Pichavant M. Kim H.Y. et al.Natural killer T cells in the lungs of patients with asthma.J Allergy Clin Immunol. 2009; 123: 1181-1185Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) allows exclusion of large cells, such as macrophages, but is not capable of excluding nonspecific binding to nonviable cells and debris. To investigate the effectiveness of this 2-step strategy, we compared the detection of iNKT cells in induced sputum (IS) of 25 subjects with physician-confirmed asthma and 19 nonasthmatic subjects by using flow cytometry against a more stringent approach, which involved gating out doublets and nonviable cells.Fifty-four subjects provided informed consent and underwent spirometry, skin prick testing, and sputum induction with hypertonic saline.7Gibson P.G. Wlodarczyk J.W. Hensley M.J. Gleeson M. Henry R.L. Cripps A.W. et al.Epidemiological association of airway inflammation with asthma symptoms and airway hyperresponsiveness in childhood.Am J Respir Crit Care Med. 1998; 158: 36-41Crossref PubMed Scopus (199) Google Scholar Four asthmatic subjects and 5 nonasthmatic subjects did not provide an adequate IS sample and were excluded, and we did not collect a sample from a subject with severe asthma whose FEV1 value was too low to safely undergo hypertonic saline challenge. The clinical characteristics of subjects successfully completing sputum induction (81.5%) are presented in Table I. The study was approved by the Upper South A Regional Ethics Committee, New Zealand. IS was incubated with dithiothreitol (Sputasol; Oxoid Ltd, Hants, England), filtered through a 60-μm filter, counted, and assessed for viability by using trypan blue. Cells obtained were washed and resuspended in RPMI. Cells for flow cytometry were blocked with human polyclonal IgG and stained for 30 minutes on ice. iNKT cells were stained with either phycoerythrin-conjugated CD1d tetramers loaded with α-galactosylceramide (a gift from Vincenzo Cerundolo, University of Oxford, Oxford, United Kingdom) or phycoerythrin-conjugated clone 6B11 antibody, which recognizes the CDR3 region of the invariant Vα24JαQ T-cell receptor chain. Other reagents used were anti-CD3–fluoresein isothiocyanate, anti-CD19–peridinin-chlorophyll-protein, and anti-CD45–allophycocyanin (BD Biosciences, Franklin Lakes, NJ). 4′-6-Diamidino-2-phenylindole was added to allow exclusion of dead cells. Flow cytometric analyses were conducted with a BD LSR II and FlowJo software (TreeStar, Inc, Ashland, Ore) with a median of 115,892 events (interquartile range [IQR], 83,973-198,337 events) analyzed per sample. Preliminary experiments comparing 6B11+ and tetramer-positive staining of peripheral blood–derived CD3+ lymphocytes showed strong correlation between these iNKT-specific reagents (r2 = 0.97). They also showed that blood-derived iNKT cells could be successfully detected with either reagent when spiked into a sputum sample at less than 10 cells per 200,000 sputum cells (data not shown).Table IClinical characteristics of participants who successfully completed sputum inductionAsthmatic subjectsNonasthmatic subjectsP valueNo. (female)25 (14)19 (11)Asthma severity (no.) Intermittent32% (8) Mild persistent24% (6) Moderate persistent44% (11)Age (y)28 (25.5-41)33 (24-43).8495Atopy (no.)80% (20)52.6% (10).1007FEV1 (% predicted)93 (82-98)102 (93-111).0062FEV1/FVC ratio0.8 (0.71-0.81)0.85 (0.83-0.87).0040Sputum eosinophils (%)0.99 (0.24-3.67)0 (0-0.46).0008Values are expressed as numbers, percentages, or medians (IQRs). Asthma classification is based on Global Initiative for Asthma guidelines. P values were calculated by using the Mann-Whitney U test or Fisher exact test.FVC, Forced vital capacity. Open table in a new tab Using the more stringent cytofluorimetric gating strategy, we found little evidence of an increased presence of iNKT cells in asthmatic subjects. In particular, in no sample did more than 0.57% of all T cells express the invariant Vα24-Jα18 T-cell receptor by using either tetramer or 6B11, and no significant difference in iNKT cell frequency between asthmatic and nonasthmatic subjects was found (median of 0.07% [IQR, 0% to 0.17%] vs 0.06% (IQR, 0% to 0.20%); P = .75, Mann-Whitney U test; Fig 1, A). However, a reanalysis of our data using the gating strategy published by Matangkasombut et al6Matangkasombut P. Marigowda G. Ervine A. Idris L. Pichavant M. Kim H.Y. et al.Natural killer T cells in the lungs of patients with asthma.J Allergy Clin Immunol. 2009; 123: 1181-1185Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar showed very different results, with up to 16.9% of the CD3+SSClow population now labeled tetramer positive or 6B11+ in asthmatic subjects. A similar increase in iNKT numbers was detected in nonasthmatic subjects, such that (as with the previous gating strategy) no significant difference was observed between groups (median of 3.38% [IQR, 1.07% to 5.71%] vs 2.29% [IQR, 1.09% to 3.35%]; P = .18; Fig 1, B). Subsequent analysis after subdividing asthmatic subjects on the basis of severity (Table I) or control also showed no statistical differences when using either gating strategy. Further examination of CD3+ tetramer/6B11+ events detected by using the 2-step strategy in both IS and peripheral blood confirmed that most were 4′-6-diamidino-2-phenylindole positive and therefore nonviable and were smaller in size (forward scatter) than expected for viable lymphocytes (Fig 1, C). Exclusion of these nonviable events led to detection of similar numbers of iNKT cells (<0.5% of CD3+SSClow events) compared with the more stringent method. Using a similar approach for iNKT cell analysis, we found equally discordant results for 18 peripheral blood samples of nonasthmatic volunteers (Fig 1, C).These results suggest that iNKT reagents bind in a nonspecific fashion to nonviable cells. Suboptimal gating strategies might therefore lead to overestimation of iNKT cell numbers because of the misidentification of nonviable cells as iNKT cells. Our data also highlight the requirement for adequate precautions to be taken when using flow cytometry to detect any rare cell populations and not only iNKT cells because nonspecific binding to dead cells and debris might have significant effects on observed cell frequencies.Our findings have several limitations, and results should therefore be interpreted with caution. First, the asthmatic population consists of subjects with mild-to-moderate asthma only, and results might be different for subjects with severe asthma, although 5 asthmatic subjects in the present study (20%) had poorly controlled asthma.Second, cells were obtained from IS and therefore have generally lower viability (68% in our study, with no significant difference between population groups) than bronchoalveolar lavage fluid. However, our experiments with peripheral blood (Fig 1, C) show that reagent binding to dead cells and debris is still apparent in high-viability tissues.Third, IS samples represent cell populations present in the central rather than distal airways.Finally, we conducted some of our experiments with the 6B11 antibody rather than the CD1d tetramer. However, preliminary experiments showed that this antibody provided comparable results to the tetramer8Im J.S. Kang T.J. Lee S.B. Kim C.H. Lee S.H. Venkataswamy M.M. et al.Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections.Clin Immunol. 2008; 127: 214-224Crossref PubMed Scopus (36) Google Scholar and in many cases improved the quality of staining by reducing nonspecific staining. The use of unloaded tetramers as controls, as applied by others,2Akbari O. Faul J.L. Hoyte E.G. Berry G.J. Wahlstrom J. Kronenberg M. et al.CD4+ invariant T-cell-receptor+ natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 1117-1129Crossref PubMed Scopus (360) Google Scholar, 6Matangkasombut P. Marigowda G. Ervine A. Idris L. Pichavant M. Kim H.Y. et al.Natural killer T cells in the lungs of patients with asthma.J Allergy Clin Immunol. 2009; 123: 1181-1185Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar could be perceived as a strength. However, loaded and unloaded tetramers might vary in their nonspecific binding propensity, particularly if they do not come from the same lot or have been handled differently.9Krueger L.A. Nugent C.T. Hampl J. Identification of human antigen-specific T cells using MHC class I and class II tetramers.Curr Protoc Cytom. 2004; (Chapter 6:Unit 6 18)PubMed Google Scholar Therefore unless it can be demonstrated that this is not the case, there is a real possibility that the methodologic issues described above are not adequately addressed by the use of unloaded tetramers.In summary, our study emphasizes the importance of stringent gating strategies in analyzing iNKT cells and suggests that simple gating strategies might lead to false-positive results, possibly explaining the mixed evidence regarding the role of iNKT cells in asthma. Our study also further supports previous evidence4Thomas S.Y. Lilly C.M. Luster A.D. Invariant natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 2613-2616Crossref PubMed Scopus (68) Google Scholar, 5Vijayanand P. Seumois G. Pickard C. Powell R.M. Angco G. Sammut D. et al.Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease.N Engl J Med. 2007; 356: 1410-1422Crossref PubMed Scopus (175) Google Scholar suggesting that iNKT cells are not increased in the central airways in subjects with mild-to-moderate asthma. Although we found no difference in iNKT cell numbers between asthmatic and nonasthmatic subjects, we do not rule out the possibility, as suggested by animal models, that iNKT cells might play a role in at least some asthma phenotypes. However, until the methodologic difficulties surrounding the detection of iNKT cells in lung-derived tissue are adequately addressed, the true importance of iNKT cells in the pathology of human asthma will remain unknown. To the Editor: A role for invariant natural killer T (iNKT) cells in the cause of asthma has been shown in mice,1Akbari O. Stock P. Meyer E. Kronenberg M. Sidobre S. Nakayama T. et al.Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity.Nat Med. 2003; 9: 582-588Crossref PubMed Scopus (600) Google Scholar but the evidence in human subjects is equivocal.2Akbari O. Faul J.L. Hoyte E.G. Berry G.J. Wahlstrom J. Kronenberg M. et al.CD4+ invariant T-cell-receptor+ natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 1117-1129Crossref PubMed Scopus (360) Google Scholar, 3Hamzaoui A. Rouhou S.C. Grairi H. Abid H. Ammar J. Chelbi H. et al.NKT cells in the induced sputum of severe asthmatics.Mediators Inflamm. 2006; 2006: 71214Crossref PubMed Scopus (44) Google Scholar, 4Thomas S.Y. Lilly C.M. Luster A.D. Invariant natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 2613-2616Crossref PubMed Scopus (68) Google Scholar, 5Vijayanand P. Seumois G. Pickard C. Powell R.M. Angco G. Sammut D. et al.Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease.N Engl J Med. 2007; 356: 1410-1422Crossref PubMed Scopus (175) Google Scholar Matangkasombut et al,6Matangkasombut P. Marigowda G. Ervine A. Idris L. Pichavant M. Kim H.Y. et al.Natural killer T cells in the lungs of patients with asthma.J Allergy Clin Immunol. 2009; 123: 1181-1185Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar in a recent issue of the Journal, provided further evidence of higher iNKT cell numbers (up to 64% of all CD3+ T cells) in bronchoalveolar lavage fluid of subjects with severe asthma compared with those seen in subjects with well-controlled asthma, who in turn had higher numbers than seen in nonasthmatic subjects. If true, this might have major implications for our understanding of asthma's pathology. Some of the discrepancies between studies might be due to the use of different cytofluorimetric gating strategies when assessing iNKT cells in bronchoalveolar lavage fluid. It has already been suggested4Thomas S.Y. Lilly C.M. Luster A.D. Invariant natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 2613-2616Crossref PubMed Scopus (68) Google Scholar, 5Vijayanand P. Seumois G. Pickard C. Powell R.M. Angco G. Sammut D. et al.Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease.N Engl J Med. 2007; 356: 1410-1422Crossref PubMed Scopus (175) Google Scholar that the iNKT cells identified in the initial study2Akbari O. Faul J.L. Hoyte E.G. Berry G.J. Wahlstrom J. Kronenberg M. et al.CD4+ invariant T-cell-receptor+ natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 1117-1129Crossref PubMed Scopus (360) Google Scholar are likely to be alveolar macrophages on the basis of forward and side scatter characteristics. The use of a simple, 2-step CD3+SSClow strategy (as subsequently used by Matangksasombut et al6Matangkasombut P. Marigowda G. Ervine A. Idris L. Pichavant M. Kim H.Y. et al.Natural killer T cells in the lungs of patients with asthma.J Allergy Clin Immunol. 2009; 123: 1181-1185Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) allows exclusion of large cells, such as macrophages, but is not capable of excluding nonspecific binding to nonviable cells and debris. To investigate the effectiveness of this 2-step strategy, we compared the detection of iNKT cells in induced sputum (IS) of 25 subjects with physician-confirmed asthma and 19 nonasthmatic subjects by using flow cytometry against a more stringent approach, which involved gating out doublets and nonviable cells. Fifty-four subjects provided informed consent and underwent spirometry, skin prick testing, and sputum induction with hypertonic saline.7Gibson P.G. Wlodarczyk J.W. Hensley M.J. Gleeson M. Henry R.L. Cripps A.W. et al.Epidemiological association of airway inflammation with asthma symptoms and airway hyperresponsiveness in childhood.Am J Respir Crit Care Med. 1998; 158: 36-41Crossref PubMed Scopus (199) Google Scholar Four asthmatic subjects and 5 nonasthmatic subjects did not provide an adequate IS sample and were excluded, and we did not collect a sample from a subject with severe asthma whose FEV1 value was too low to safely undergo hypertonic saline challenge. The clinical characteristics of subjects successfully completing sputum induction (81.5%) are presented in Table I. The study was approved by the Upper South A Regional Ethics Committee, New Zealand. IS was incubated with dithiothreitol (Sputasol; Oxoid Ltd, Hants, England), filtered through a 60-μm filter, counted, and assessed for viability by using trypan blue. Cells obtained were washed and resuspended in RPMI. Cells for flow cytometry were blocked with human polyclonal IgG and stained for 30 minutes on ice. iNKT cells were stained with either phycoerythrin-conjugated CD1d tetramers loaded with α-galactosylceramide (a gift from Vincenzo Cerundolo, University of Oxford, Oxford, United Kingdom) or phycoerythrin-conjugated clone 6B11 antibody, which recognizes the CDR3 region of the invariant Vα24JαQ T-cell receptor chain. Other reagents used were anti-CD3–fluoresein isothiocyanate, anti-CD19–peridinin-chlorophyll-protein, and anti-CD45–allophycocyanin (BD Biosciences, Franklin Lakes, NJ). 4′-6-Diamidino-2-phenylindole was added to allow exclusion of dead cells. Flow cytometric analyses were conducted with a BD LSR II and FlowJo software (TreeStar, Inc, Ashland, Ore) with a median of 115,892 events (interquartile range [IQR], 83,973-198,337 events) analyzed per sample. Preliminary experiments comparing 6B11+ and tetramer-positive staining of peripheral blood–derived CD3+ lymphocytes showed strong correlation between these iNKT-specific reagents (r2 = 0.97). They also showed that blood-derived iNKT cells could be successfully detected with either reagent when spiked into a sputum sample at less than 10 cells per 200,000 sputum cells (data not shown). Values are expressed as numbers, percentages, or medians (IQRs). Asthma classification is based on Global Initiative for Asthma guidelines. P values were calculated by using the Mann-Whitney U test or Fisher exact test. FVC, Forced vital capacity. Using the more stringent cytofluorimetric gating strategy, we found little evidence of an increased presence of iNKT cells in asthmatic subjects. In particular, in no sample did more than 0.57% of all T cells express the invariant Vα24-Jα18 T-cell receptor by using either tetramer or 6B11, and no significant difference in iNKT cell frequency between asthmatic and nonasthmatic subjects was found (median of 0.07% [IQR, 0% to 0.17%] vs 0.06% (IQR, 0% to 0.20%); P = .75, Mann-Whitney U test; Fig 1, A). However, a reanalysis of our data using the gating strategy published by Matangkasombut et al6Matangkasombut P. Marigowda G. Ervine A. Idris L. Pichavant M. Kim H.Y. et al.Natural killer T cells in the lungs of patients with asthma.J Allergy Clin Immunol. 2009; 123: 1181-1185Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar showed very different results, with up to 16.9% of the CD3+SSClow population now labeled tetramer positive or 6B11+ in asthmatic subjects. A similar increase in iNKT numbers was detected in nonasthmatic subjects, such that (as with the previous gating strategy) no significant difference was observed between groups (median of 3.38% [IQR, 1.07% to 5.71%] vs 2.29% [IQR, 1.09% to 3.35%]; P = .18; Fig 1, B). Subsequent analysis after subdividing asthmatic subjects on the basis of severity (Table I) or control also showed no statistical differences when using either gating strategy. Further examination of CD3+ tetramer/6B11+ events detected by using the 2-step strategy in both IS and peripheral blood confirmed that most were 4′-6-diamidino-2-phenylindole positive and therefore nonviable and were smaller in size (forward scatter) than expected for viable lymphocytes (Fig 1, C). Exclusion of these nonviable events led to detection of similar numbers of iNKT cells (<0.5% of CD3+SSClow events) compared with the more stringent method. Using a similar approach for iNKT cell analysis, we found equally discordant results for 18 peripheral blood samples of nonasthmatic volunteers (Fig 1, C). These results suggest that iNKT reagents bind in a nonspecific fashion to nonviable cells. Suboptimal gating strategies might therefore lead to overestimation of iNKT cell numbers because of the misidentification of nonviable cells as iNKT cells. Our data also highlight the requirement for adequate precautions to be taken when using flow cytometry to detect any rare cell populations and not only iNKT cells because nonspecific binding to dead cells and debris might have significant effects on observed cell frequencies. Our findings have several limitations, and results should therefore be interpreted with caution. First, the asthmatic population consists of subjects with mild-to-moderate asthma only, and results might be different for subjects with severe asthma, although 5 asthmatic subjects in the present study (20%) had poorly controlled asthma. Second, cells were obtained from IS and therefore have generally lower viability (68% in our study, with no significant difference between population groups) than bronchoalveolar lavage fluid. However, our experiments with peripheral blood (Fig 1, C) show that reagent binding to dead cells and debris is still apparent in high-viability tissues. Third, IS samples represent cell populations present in the central rather than distal airways. Finally, we conducted some of our experiments with the 6B11 antibody rather than the CD1d tetramer. However, preliminary experiments showed that this antibody provided comparable results to the tetramer8Im J.S. Kang T.J. Lee S.B. Kim C.H. Lee S.H. Venkataswamy M.M. et al.Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections.Clin Immunol. 2008; 127: 214-224Crossref PubMed Scopus (36) Google Scholar and in many cases improved the quality of staining by reducing nonspecific staining. The use of unloaded tetramers as controls, as applied by others,2Akbari O. Faul J.L. Hoyte E.G. Berry G.J. Wahlstrom J. Kronenberg M. et al.CD4+ invariant T-cell-receptor+ natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 1117-1129Crossref PubMed Scopus (360) Google Scholar, 6Matangkasombut P. Marigowda G. Ervine A. Idris L. Pichavant M. Kim H.Y. et al.Natural killer T cells in the lungs of patients with asthma.J Allergy Clin Immunol. 2009; 123: 1181-1185Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar could be perceived as a strength. However, loaded and unloaded tetramers might vary in their nonspecific binding propensity, particularly if they do not come from the same lot or have been handled differently.9Krueger L.A. Nugent C.T. Hampl J. Identification of human antigen-specific T cells using MHC class I and class II tetramers.Curr Protoc Cytom. 2004; (Chapter 6:Unit 6 18)PubMed Google Scholar Therefore unless it can be demonstrated that this is not the case, there is a real possibility that the methodologic issues described above are not adequately addressed by the use of unloaded tetramers. In summary, our study emphasizes the importance of stringent gating strategies in analyzing iNKT cells and suggests that simple gating strategies might lead to false-positive results, possibly explaining the mixed evidence regarding the role of iNKT cells in asthma. Our study also further supports previous evidence4Thomas S.Y. Lilly C.M. Luster A.D. Invariant natural killer T cells in bronchial asthma.N Engl J Med. 2006; 354: 2613-2616Crossref PubMed Scopus (68) Google Scholar, 5Vijayanand P. Seumois G. Pickard C. Powell R.M. Angco G. Sammut D. et al.Invariant natural killer T cells in asthma and chronic obstructive pulmonary disease.N Engl J Med. 2007; 356: 1410-1422Crossref PubMed Scopus (175) Google Scholar suggesting that iNKT cells are not increased in the central airways in subjects with mild-to-moderate asthma. Although we found no difference in iNKT cell numbers between asthmatic and nonasthmatic subjects, we do not rule out the possibility, as suggested by animal models, that iNKT cells might play a role in at least some asthma phenotypes. However, until the methodologic difficulties surrounding the detection of iNKT cells in lung-derived tissue are adequately addressed, the true importance of iNKT cells in the pathology of human asthma will remain unknown.
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