Research ArticleAutoimmunity

Lymphocyte-driven regional immunopathology in pneumonitis caused by impaired central immune tolerance

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Science Translational Medicine  05 Jun 2019:
Vol. 11, Issue 495, eaav5597
DOI: 10.1126/scitranslmed.aav5597
  • Fig. 1 Clinical, radiographic, and pulmonary function abnormalities of APECED pneumonitis.

    (A) Clinical symptoms associated with APECED pneumonitis assessed by a standardized questionnaire. Chronic cough is classified as dry (gray shaded area) and with sputum production (black shaded area) (n = 21). (B) Radiographic features of APECED pneumonitis assessed by noncontrast chest computed tomography (CT) (n = 21). (C) Abnormalities in pulmonary function testing (n = 12) and 6-min walk test (n = 7) in patients with active APECED pneumonitis. DLCO, diffusing capacity of the lungs for carbon monoxide. (D to I) Representative radiographic abnormalities of APECED pneumonitis on chest CT imaging. GGO predominate early on (D to F). As disease progresses, bronchiectasis is prominent (G and H) and can lead to recurrent infections, including cavitary pulmonary nontuberculous mycobacteria (NTM) infection (I).

  • Fig. 2 BPIFB1 and KCNRG autoantibodies and the homozygous c.967_979del13 AIRE mutation associate with the development of APECED pneumonitis.

    (A and B) Kaplan-Meier curves illustrating the relationship between the presence of autoantibodies against BPIFB1 (A) or KCNRG (B) and the time to development of pneumonitis. P values are based on the log-rank test. The tables show the sensitivity and specificity of the corresponding autoantibodies (n = 50). (C) Kaplan-Meier curve illustrating the relationship between carrying homozygous c.967_979del13 AIRE mutations (group A), carrying a heterozygous c.967_979del13 AIRE mutation (group B), and carrying no c.967_979del13 AIRE mutations (group C) with the time to development of pneumonitis (n = 50). P1 represents the P value between groups A and B; P2 represents the P value between groups A and C, and P3 represents the P value between groups B and C. P values are based on the log-rank test. (D) Proposed diagnostic algorithm aimed at promoting earlier diagnosis of APECED pneumonitis. Aab, autoantibody.

  • Fig. 3 Accumulation of activated neutrophils in the airways of patients with APECED pneumonitis.

    (A) Neutrophils in the BAL of patients with APECED with pneumonitis (n = 5) and healthy controls (n = 4). Percentage of neutrophils within total CD45+ leukocytes (left) and total number of neutrophils per lavage (right). (B) Concentration of CXC neutrophil-targeted chemokines in the BAL of patients with APECED with pneumonitis (n = 5) and healthy controls (n = 4). (C) Neutrophil activation phenotype in BAL of patients with APECED with pneumonitis (n = 4 to 5) and healthy controls (n = 4). Shown are summary data (left) on mean fluorescence intensity and representative fluorescence-activated cell sorting histograms (right) for CD45, CD66b, CD63, CD18, b558, and CD16. (D) Concentration of the neutrophil products MPO and MMP-9 in the BAL of patients with APECED with pneumonitis (n = 5) and healthy controls (n = 4). (E) LDH concentration in the BAL of patients with APECED with pneumonitis (n = 5) and healthy controls (n = 4). (F) Neutrophils in the BAL of Aire+/+ and Aire−/− mice (n = 8 to 10 per group; three independent experiments). Percentage of neutrophils within total CD45+ leukocytes (left) and total number of neutrophils per lavage (right). (G) Concentration of the CXC neutrophil-targeted chemokines CXCL1 and CXCL2 in the BAL of Aire+/+ and Aire−/− mice (n = 7 to 10 per group; three independent experiments). Differences between groups in all panels were determined using Mann-Whitney test with the exception of the differences between groups in the left panel of (F) (% of neutrophils within total CD45+ leukocytes) and on the right side of the graph in (G) (CXCL2 concentration), which were determined using unpaired t test with Welch’s correction. *P < 0.05, **P < 0.01, and ***P < 0.001. All quantitative data represent means ± SEM.

  • Fig. 4 Basement membrane thickening and lymphocyte infiltration in intraepithelial and submucosal tissue is seen in endobronchial biopsies of patients with APECED pneumonitis.

    (A) Representative image of hematoxylin and eosin (H&E) staining of an endobronchial biopsy from a patient with APECED pneumonitis (patient 3; table S1). Black arrows indicate a thickened basement membrane. (B) Mean basement membrane thickness measured in micrometers (n = 4). The dotted lines outline the range of reported basement membrane thickness seen in patients with asthma (31). (C to F) Representative images of immunohistochemical staining with the lymphocyte markers CD3 (C), CD4 (D), CD8 (E), and CD20 (F) from the same endobronchial biopsy. Scale bars, 150 μm.

  • Fig. 5 Lymphocyte infiltration in bronchiolar, peribronchiolar, and interstitial lung tissue underlies APECED pneumonitis.

    Representative images of H&E staining (A and F) and immunohistochemical staining with the lymphocyte markers CD3 (B), CD4 (C), CD8 (D), and CD20 (E) obtained from resected lung tissue of a patient with APECED with pneumonitis (patient 1; table S1). Panel A is generated from the same data shown in (6). Scale bars, 50 μm.

  • Fig. 6 Lung disease in patients with secondary AIRE deficiency exhibits shared features with APECED pneumonitis.

    (A) Shown is the autoantibody immunoreactivity against BPIFB1 and KCNRG as light units (LU) using the luciferase immunoprecipitation systems (LIPS) immunoassay in 62 patients with thymoma with (n = 13) or without (n = 49) lung involvement, and in 27 recombination-activating gene (RAG)–deficient patients with (n = 19) or without (n = 8) lung involvement. Dotted lines represent the cutoff values for determining autoantibody seropositivity. (B) Representative chest CT image from a patient with thymoma with lung disease. (C and D) Representative images from an endobronchial (C) and transbronchial (D) biopsy of the same patient with thymoma with lung disease. Shown are H&E and immunohistochemical staining with the lymphocyte markers CD3, CD4, CD8, and CD20. Scale bars, 100 μm. (E) Representative chest CT image from a RAG-deficient patient with lung disease.

  • Fig. 7 Lymphocyte deficiency ameliorates airway neutrophil accumulation and lung tissue injury in Aire−/− mice.

    (A) Representative images of H&E staining of Aire+/+, Aire−/−, Aire−/−Tcra−/−, and Aire−/−Ighm−/− mouse lung and of immunohistochemical staining with the lymphocyte markers CD3 and B220 in Aire−/− mouse lung. Original magnification, 40×. Scale bars, 200 μm. n = 6 to 10 per group; one to three independent experiments. (B) Neutrophils in the BAL. Percentage of neutrophils within total CD45+ leukocytes (top; *P < 0.05) and total number of neutrophils per lavage (bottom; **P < 0.01 and ***P < 0.001). n = 7 to 10 per group; three to four independent experiments. (C) Lymphocyte populations in lung tissue. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n = 7 to 10 per group; three independent experiments. (D) Lung histology scores. ****P < 0.0001. n = 6 to 10 per group; three independent experiments. Differences between groups were determined using one-way analysis of variance (ANOVA) with Tukey’s correction for multiple comparisons. Quantitative data represent means ± SEM.

  • Fig. 8 Combination lymphocyte-directed immunomodulation remits pneumonitis in patients with APECED.

    (A) Number of patients reporting cough at baseline and at 1 and 6 months after treatment initiation (n = 5). (B) Percent residual radiographic abnormalities calculated at 1 and 6 months after treatment initiation relative to the pretreatment radiographic abnormalities (n = 5). Statistical analysis of comparison data was performed by paired t test. (C) Representative coronal chest CT images at baseline and at 1 and 6 months after treatment initiation (patient 2; table S2). (D) 3D reconstructed CT images at baseline and at 1 and 6 months after treatment initiation (patient 5; table S2). (E to H) Pulmonary function assessed by measurements at baseline and after treatment initiation of % oxygen desaturation during the 6-min walk (n = 4) (E), the 6-min walk distance in meters (n = 4) (F), the ratio of forced expiratory volume at 1 s to forced vital capacity (FEV1/FVC) (n = 3) (G), and DLCO (n = 3) (H). (I) Shown is the autoantibody immunoreactivity against BPIFB1 and KCNRG as light units (LU) using the LIPS immunoassay at baseline and at 1 and 6 months after treatment initiation (n = 4).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/495/eaav5597/DC1

    Supplementary Materials and Methods

    Fig. S1. Enlarged mediastinal lymph nodes in patients with APECED pneumonitis.

    Fig. S2. Association of autoantibody reactivity against cytokines and tissue-specific autoantigens and the time to development of pneumonitis in patients with APECED.

    Fig. S3. Serum inflammatory markers in patients with APECED pneumonitis.

    Fig. S4. Immunoglobulin concentrations in patients with APECED pneumonitis.

    Fig. S5. Leukocyte subsets other than neutrophils do not exhibit increased accumulation in the airways of patients with APECED pneumonitis.

    Fig. S6. Primary lymphoid follicle and germinal center formation in lung tissue of patients with APECED pneumonitis.

    Fig. S7. Intraepithelial lymphocyte infiltration in Aire−/− mouse lung tissue.

    Fig. S8. Schematic illustration of the lymphocyte-directed immunomodulation used in this study.

    Fig. S9. Treatment response assessed by radiography in patient 1.

    Fig. S10. Treatment response assessed by radiography in patient 2.

    Fig. S11. Treatment response assessed by radiography in patient 3.

    Fig. S12. Treatment response assessed by radiography in patient 4.

    Fig. S13. Treatment response assessed by radiography in patient 5.

    Fig. S14. Lymphocyte-directed immunomodulation causes rapid depletion of B, but not T, lymphocytes in peripheral blood of patients with APECED with pneumonitis.

    Fig. S15. Lymphocyte-directed immunomodulation improves salivary production in patients with APECED with Sjogren’s-like syndrome.

    Fig. S16. Lymphocyte-directed immunomodulation caused resolution of nail dystrophy in one of the three patients with APECED with this condition.

    Table S1. Demographic and geographic origin characteristics of our patients with APECED.

    Table S2. Demographic, clinical, and radiographic response characteristics of the five patients with APECED pneumonitis who received lymphocyte-directed immunomodulation.

    Table S3. Patients with APECED pneumonitis do not carry serum autoantibodies against MDA5.

    Table S4. Percent of lymphocyte subsets within corresponding lymphocytes in the peripheral blood of patients with APECED with or without pneumonitis.

    Table S5. Absolute numbers of lymphocyte subsets in the peripheral blood of patients with APECED with or without pneumonitis.

    Table S6. Standardized pulmonary clinical history questionnaire used for the evaluation of clinical features of APECED pneumonitis in our study.

    Table S7. Clinical, radiographic, lung biopsy, and autoantibody features of pneumonitis in the 21 affected patients with APECED included in this study.

    Data file S1. Primary data.

    References (4858)

  • The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Enlarged mediastinal lymph nodes in patients with APECED pneumonitis.
    • Fig. S2. Association of autoantibody reactivity against cytokines and tissue-specific autoantigens and the time to development of pneumonitis in patients with APECED.
    • Fig. S3. Serum inflammatory markers in patients with APECED pneumonitis.
    • Fig. S4. Immunoglobulin concentrations in patients with APECED pneumonitis.
    • Fig. S5. Leukocyte subsets other than neutrophils do not exhibit increased accumulation in the airways of patients with APECED pneumonitis.
    • Fig. S6. Primary lymphoid follicle and germinal center formation in lung tissue of patients with APECED pneumonitis.
    • Fig. S7. Intraepithelial lymphocyte infiltration in Aire−/− mouse lung tissue.
    • Fig. S8. Schematic illustration of the lymphocyte-directed immunomodulation used in this study.
    • Fig. S9. Treatment response assessed by radiography in patient 1.
    • Fig. S10. Treatment response assessed by radiography in patient 2.
    • Fig. S11. Treatment response assessed by radiography in patient 3.
    • Fig. S12. Treatment response assessed by radiography in patient 4.
    • Fig. S13. Treatment response assessed by radiography in patient 5.
    • Fig. S14. Lymphocyte-directed immunomodulation causes rapid depletion of B, but not T, lymphocytes in peripheral blood of patients with APECED with pneumonitis.
    • Fig. S15. Lymphocyte-directed immunomodulation improves salivary production in patients with APECED with Sjogren’s-like syndrome.
    • Fig. S16. Lymphocyte-directed immunomodulation caused resolution of nail dystrophy in one of the three patients with APECED with this condition.
    • Table S1. Demographic and geographic origin characteristics of our patients with APECED.
    • Table S2. Demographic, clinical, and radiographic response characteristics of the five patients with APECED pneumonitis who received lymphocyte-directed immunomodulation.
    • Table S3. Patients with APECED pneumonitis do not carry serum autoantibodies against MDA5.
    • Table S4. Percent of lymphocyte subsets within corresponding lymphocytes in the peripheral blood of patients with APECED with or without pneumonitis.
    • Table S5. Absolute numbers of lymphocyte subsets in the peripheral blood of patients with APECED with or without pneumonitis.
    • Table S6. Standardized pulmonary clinical history questionnaire used for the evaluation of clinical features of APECED pneumonitis in our study.
    • Table S7. Clinical, radiographic, lung biopsy, and autoantibody features of pneumonitis in the 21 affected patients with APECED included in this study.
    • References (4858)

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    Other Supplementary Material for this manuscript includes the following:

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