Invariant Natural Killer T Cells in Bronchial Asthma
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《新英格兰医药杂志》
To the Editor: We have not found a remarkably high proportion of CD1d-restricted natural killer T cells (>60 percent of CD3+ cells) in bronchoalveolar-lavage specimens from patients with allergic asthma, as reported by Akbari et al. (March 16 issue).1 In five patients with allergic asthma, we found that only 0.4 to 2.1 percent of lymphocytes in bronchoalveolar-lavage fluid were natural killer T cells, on the basis of costaining with 6B11 and V24 antibodies (see Figure 1 of the Supplementary Appendix, available with the full text of this letter at www.nejm.org). We believe this discrepancy may result from fact that the authors did not gate using characteristic lymphocyte forward-scatter (size) and side-scatter (granularity) properties before flow analysis, consequently including large granular cells that stained nonspecifically (Figure 1). A majority of CD4-staining cells in ungated bronchoalveolar-lavage specimens stain with 6B11, but only a minority of CD4+ cells within the lymphocyte gate stain with 6B11. Furthermore, since 6B11 was generated against a peptide epitope in the CDR3 region of V24, staining with 6B11 in the absence of V24 staining may indicate non–CD1d-restricted T-cell or B-cell epitopes.2,3,4 It is also unclear how 6B11 can stain natural killer T cells accurately if the V24 chain is down-regulated, as suggested by the authors. Although we observed that CD1d-restricted natural killer T cells were enriched in the bronchoalveolar-lavage specimens, as compared with peripheral blood, we did not observe a dramatic number of natural killer T cells in the bronchoalveolar-lavage fluid of our patients with asthma.
Figure 1. Flow Analysis of CD1d-Restricted Natural Killer Cells from Bronchoaveolar-Lavage Fluid and Blood with and without Lymphocyte Gating.
In Panel A, 6B11 stains a sizable proportion of ungated CD4 cells obtained by bronchoalveolar lavage but only a small proportion of CD4 lymphocytes. The scatter plot and percentage of total cells in the lymphocyte gate are shown for a representative patient with asthma. The ungated flow plots (indicated with right arrow) and lymphocyte-gated flow plots (indicated with down arrow) are shown, and the percentage of ungated cells that stain for 6B11 and CD4 is indicated by the boxed population. Most of the cells that costain for 6B11 and CD4 in the ungated population were found in the side-scatter–high population, indicated by backgating on 6B11+CD4+ cells and displaying their forward-scatter characteristics (FSC) and side-scatter characteristics (SSC). For the lymphocyte-gated population, there are very few 6B11+CD4+ cells. Numbers in the corners of the panels indicate the percentage of cells found in each quadrant. In Panel B, the scatter plot for cells gated on CD3 alone is shown for a representative patient with asthma. When gated on CD3 and large cells together, almost all cells stain with the IgG2a isotype control, demonstrating high levels of background staining in this cell population. In Panel C, the level of 6B11 staining is low in both ungated and lymphocyte-gated CD4 populations in blood. The scatter plot for peripheral blood is shown for a representative patient with asthma. The ungated and lymphocyte-gated flow plots show 6B11 and CD4 staining. As opposed to the results with bronchoalveolar-lavage fluid, the level of 6B11 staining is low in both ungated and lymphocyte-gated populations in the peripheral blood, which may be related to the absence of alveolar macrophages in the peripheral blood. In Panel D, clones of natural killer T (NKT) cells costain for 6B11 and V24. These clones were grown in interleukin-2 and irradiated feeders of peripheral-blood mononuclear cells.2 The scatter plot is shown for a representative clone of a natural killer T cell. The gated population includes lymphocytes and excludes irradiated feeders. The lymphocyte-gated population shows a lack of staining for isotype controls (IgG2a vs. IgG1) and staining for the canonical natural killer T-cell receptor alpha-chain rearrangement (V24 vs. 6B11). CD1d-restricted clones of natural killer T cells are found in the lymphocyte gate and costain with 6B11 and V24 but not the respective isotype controls.
Seddon Y. Thomas, Ph.D.
Massachusetts General Hospital
Boston, MA 02114
Craig M. Lilly, M.D.
University of Massachusetts Medical Center
Worcester, MA 01655
Andrew D. Luster, M.D., Ph.D.
Massachusetts General Hospital
Boston, MA 02114
aluster@partners.org
References
Akbari O, Faul JL, Hoyte EG, et al. CD4+ invariant T-cell-receptor+ natural killer T cells in bronchial asthma. N Engl J Med 2006;354:1117-1129.
Thomas SY, Hou R, Boyson JE, et al. CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells. J Immunol 2003;171:2571-2580.
Kukreja A, Cost G, Marker J, et al. Multiple immuno-regulatory defects in type-1 diabetes. J Clin Invest 2002;109:131-140.
Tahir SM, Cheng O, Shaulov A, et al. Loss of IFN-gamma production by invariant NK T cells in advanced cancer. J Immunol 2001;167:4046-4050.
To the Editor: Akbari and colleagues analyzed the nature of pulmonary CD4+ T cells in patients with moderate-to-severe asthma. The authors provide data indicating that the majority of these cells (about 60 percent) are represented by the population of invariant natural killer T cells. These cells, in contrast to conventional T cells, express a conserved T-cell receptor (V24–J18), which is capable of recognizing glycolipids.
We have reported that the frequency of invariant natural killer T cells is significantly higher in bronchoalveolar-lavage fluid from children with severe asthma than in fluid from controls.1 The interesting point is that in childhood the frequency of these cells is still relatively low, especially among patients under four years of age (0.2 percent), and that the proportion of invariant natural killer T cells increases progressively in older children (1.2 percent) (unpublished data). It is possible that a high frequency of invariant natural killer T cells in the lungs of children could predict more severe disease in adult life, suggesting that the detection of invariant natural killer T cells in the airways might be a marker for disease prognosis. Cohort and long-term studies are required to confirm this hypothesis.
Nhan Pham-Thi, M.D.
Jacques de Blic, M.D.
H?pital Necker
75015 Paris, France
Maria C. Leite-de-Moraes, Ph.D.
Centre National de la Recherche Scientifique UMR 4187
75015 Paris, France
leite.de.moraes@necker.fr
References
Pham-Thi N, de Blic J, Le Bourgeois M, Dy M, Scheinmann P, Leite-de-Moraes MC. Enhanced frequency of immunoregulatory invariant natural killer T cells in the airways of children with asthma. J Allergy Clin Immunol 2006;117:217-218.
The authors reply: Thomas et al. and Pham-Thi et al. confirm our findings that natural killer T cells are present in increased numbers in the lungs, but not in the peripheral blood, of patients with asthma. Both sets of correspondents note a lower number of pulmonary invariant natural killer T cells in patients with asthma than we did, very probably because they studied either patients with mild, intermittent asthma or children with wheezing, rather than patients with moderate-to-severe persistent asthma, such as those in our study. Nevertheless, these studies can be interpreted to mean that the number of pulmonary invariant natural killer T cells in asthma correlates with the severity or chronicity of disease, but this possibility remains to be studied directly.
Thomas et al. suggest that some of our results may be due to nonspecific staining of cells in bronchoalveolar-lavage fluid, since they found that their control antibody nonspecifically stained cells from such fluid as strongly as did the antibody that is specific for natural killer T cells. We avoided such nonspecific staining by performing multiple steps, well beyond what Thomas et al. did, to ensure that our staining was specific. For example, we processed our preparations of bronchoalveolar-lavage fluid to remove cell clumps and mucus, thoroughly blocked Fc receptors, and gated out the highly granular cells, which as noted by Thomas et al., appear to stain nonspecifically. Because of space limitations, some of the details of these methods were not included in the article.
Moreover, as we noted, we stained with CD1d tetramers loaded with -galactosylceramide (the gold standard for identifying and specifically staining natural killer T cells) and gated on CD3+ T cells to eliminate from the analysis alveolar macrophages, red cells, and noncellular debris, which appear to be present in the samples analyzed by Thomas et al. and which could substantially reduce the apparent number of natural killer T cells. Furthermore, we examined samples of bronchoalveolar-lavage fluid from patients with sarcoidosis, which did not show natural killer T cells, confirming that our staining was specific.
Finally, we examined bronchial-biopsy specimens with confocal laser scanning microscopy, used quantitative reverse-transcriptase–polymerase-chain-reaction analysis, and used functional assays to demonstrate the specific presence of pulmonary natural killer T cells in asthma. These measures together make it very likely that our results are accurate and reliable.
Thomas et al. also ask how 6B11 antibody can stain natural killer T cells accurately if the V24 chain is down-regulated. Other investigators have observed this situation in the past,1,2 suggesting that the V24 antibody is less sensitive than the 6B11 antibody or CD1d tetramers.
Omid Akbari, Ph.D.
Harvard Medical School
Boston, MA 02115
John L. Faul, M.D.
Connolly Hospital
Dublin 15, Ireland
Dale T. Umetsu, M.D., Ph.D.
Harvard Medical School
Boston, MA 02115
dale.umetsu@childrens.harvard.edu
References
Brigl M, van den Elzen P, Chen X, et al. Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors. J Immunol 2006;176:3625-3634.
Gurney KB, Yang OO, Wilson SB, Uittenbogaart CH. TCR gamma delta+ and CD161+ thymocytes express HIV-1 in the SCID-hu mouse, potentially contributing to immune dysfunction in HIV infection. J Immunol 2002;169:5338-5346.
Figure 1. Flow Analysis of CD1d-Restricted Natural Killer Cells from Bronchoaveolar-Lavage Fluid and Blood with and without Lymphocyte Gating.
In Panel A, 6B11 stains a sizable proportion of ungated CD4 cells obtained by bronchoalveolar lavage but only a small proportion of CD4 lymphocytes. The scatter plot and percentage of total cells in the lymphocyte gate are shown for a representative patient with asthma. The ungated flow plots (indicated with right arrow) and lymphocyte-gated flow plots (indicated with down arrow) are shown, and the percentage of ungated cells that stain for 6B11 and CD4 is indicated by the boxed population. Most of the cells that costain for 6B11 and CD4 in the ungated population were found in the side-scatter–high population, indicated by backgating on 6B11+CD4+ cells and displaying their forward-scatter characteristics (FSC) and side-scatter characteristics (SSC). For the lymphocyte-gated population, there are very few 6B11+CD4+ cells. Numbers in the corners of the panels indicate the percentage of cells found in each quadrant. In Panel B, the scatter plot for cells gated on CD3 alone is shown for a representative patient with asthma. When gated on CD3 and large cells together, almost all cells stain with the IgG2a isotype control, demonstrating high levels of background staining in this cell population. In Panel C, the level of 6B11 staining is low in both ungated and lymphocyte-gated CD4 populations in blood. The scatter plot for peripheral blood is shown for a representative patient with asthma. The ungated and lymphocyte-gated flow plots show 6B11 and CD4 staining. As opposed to the results with bronchoalveolar-lavage fluid, the level of 6B11 staining is low in both ungated and lymphocyte-gated populations in the peripheral blood, which may be related to the absence of alveolar macrophages in the peripheral blood. In Panel D, clones of natural killer T (NKT) cells costain for 6B11 and V24. These clones were grown in interleukin-2 and irradiated feeders of peripheral-blood mononuclear cells.2 The scatter plot is shown for a representative clone of a natural killer T cell. The gated population includes lymphocytes and excludes irradiated feeders. The lymphocyte-gated population shows a lack of staining for isotype controls (IgG2a vs. IgG1) and staining for the canonical natural killer T-cell receptor alpha-chain rearrangement (V24 vs. 6B11). CD1d-restricted clones of natural killer T cells are found in the lymphocyte gate and costain with 6B11 and V24 but not the respective isotype controls.
Seddon Y. Thomas, Ph.D.
Massachusetts General Hospital
Boston, MA 02114
Craig M. Lilly, M.D.
University of Massachusetts Medical Center
Worcester, MA 01655
Andrew D. Luster, M.D., Ph.D.
Massachusetts General Hospital
Boston, MA 02114
aluster@partners.org
References
Akbari O, Faul JL, Hoyte EG, et al. CD4+ invariant T-cell-receptor+ natural killer T cells in bronchial asthma. N Engl J Med 2006;354:1117-1129.
Thomas SY, Hou R, Boyson JE, et al. CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells. J Immunol 2003;171:2571-2580.
Kukreja A, Cost G, Marker J, et al. Multiple immuno-regulatory defects in type-1 diabetes. J Clin Invest 2002;109:131-140.
Tahir SM, Cheng O, Shaulov A, et al. Loss of IFN-gamma production by invariant NK T cells in advanced cancer. J Immunol 2001;167:4046-4050.
To the Editor: Akbari and colleagues analyzed the nature of pulmonary CD4+ T cells in patients with moderate-to-severe asthma. The authors provide data indicating that the majority of these cells (about 60 percent) are represented by the population of invariant natural killer T cells. These cells, in contrast to conventional T cells, express a conserved T-cell receptor (V24–J18), which is capable of recognizing glycolipids.
We have reported that the frequency of invariant natural killer T cells is significantly higher in bronchoalveolar-lavage fluid from children with severe asthma than in fluid from controls.1 The interesting point is that in childhood the frequency of these cells is still relatively low, especially among patients under four years of age (0.2 percent), and that the proportion of invariant natural killer T cells increases progressively in older children (1.2 percent) (unpublished data). It is possible that a high frequency of invariant natural killer T cells in the lungs of children could predict more severe disease in adult life, suggesting that the detection of invariant natural killer T cells in the airways might be a marker for disease prognosis. Cohort and long-term studies are required to confirm this hypothesis.
Nhan Pham-Thi, M.D.
Jacques de Blic, M.D.
H?pital Necker
75015 Paris, France
Maria C. Leite-de-Moraes, Ph.D.
Centre National de la Recherche Scientifique UMR 4187
75015 Paris, France
leite.de.moraes@necker.fr
References
Pham-Thi N, de Blic J, Le Bourgeois M, Dy M, Scheinmann P, Leite-de-Moraes MC. Enhanced frequency of immunoregulatory invariant natural killer T cells in the airways of children with asthma. J Allergy Clin Immunol 2006;117:217-218.
The authors reply: Thomas et al. and Pham-Thi et al. confirm our findings that natural killer T cells are present in increased numbers in the lungs, but not in the peripheral blood, of patients with asthma. Both sets of correspondents note a lower number of pulmonary invariant natural killer T cells in patients with asthma than we did, very probably because they studied either patients with mild, intermittent asthma or children with wheezing, rather than patients with moderate-to-severe persistent asthma, such as those in our study. Nevertheless, these studies can be interpreted to mean that the number of pulmonary invariant natural killer T cells in asthma correlates with the severity or chronicity of disease, but this possibility remains to be studied directly.
Thomas et al. suggest that some of our results may be due to nonspecific staining of cells in bronchoalveolar-lavage fluid, since they found that their control antibody nonspecifically stained cells from such fluid as strongly as did the antibody that is specific for natural killer T cells. We avoided such nonspecific staining by performing multiple steps, well beyond what Thomas et al. did, to ensure that our staining was specific. For example, we processed our preparations of bronchoalveolar-lavage fluid to remove cell clumps and mucus, thoroughly blocked Fc receptors, and gated out the highly granular cells, which as noted by Thomas et al., appear to stain nonspecifically. Because of space limitations, some of the details of these methods were not included in the article.
Moreover, as we noted, we stained with CD1d tetramers loaded with -galactosylceramide (the gold standard for identifying and specifically staining natural killer T cells) and gated on CD3+ T cells to eliminate from the analysis alveolar macrophages, red cells, and noncellular debris, which appear to be present in the samples analyzed by Thomas et al. and which could substantially reduce the apparent number of natural killer T cells. Furthermore, we examined samples of bronchoalveolar-lavage fluid from patients with sarcoidosis, which did not show natural killer T cells, confirming that our staining was specific.
Finally, we examined bronchial-biopsy specimens with confocal laser scanning microscopy, used quantitative reverse-transcriptase–polymerase-chain-reaction analysis, and used functional assays to demonstrate the specific presence of pulmonary natural killer T cells in asthma. These measures together make it very likely that our results are accurate and reliable.
Thomas et al. also ask how 6B11 antibody can stain natural killer T cells accurately if the V24 chain is down-regulated. Other investigators have observed this situation in the past,1,2 suggesting that the V24 antibody is less sensitive than the 6B11 antibody or CD1d tetramers.
Omid Akbari, Ph.D.
Harvard Medical School
Boston, MA 02115
John L. Faul, M.D.
Connolly Hospital
Dublin 15, Ireland
Dale T. Umetsu, M.D., Ph.D.
Harvard Medical School
Boston, MA 02115
dale.umetsu@childrens.harvard.edu
References
Brigl M, van den Elzen P, Chen X, et al. Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors. J Immunol 2006;176:3625-3634.
Gurney KB, Yang OO, Wilson SB, Uittenbogaart CH. TCR gamma delta+ and CD161+ thymocytes express HIV-1 in the SCID-hu mouse, potentially contributing to immune dysfunction in HIV infection. J Immunol 2002;169:5338-5346.