Mass cytometry identifies characteristic immune cell subsets in bronchoalveolar lavage fluid from interstitial lung diseases

Abstract

Immune cells have been implicated in interstitial lung diseases (ILDs), although their phenotypes and effector mechanisms remain poorly understood. To better understand these cells, we conducted an exploratory mass cytometry analysis of immune cell subsets in bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF), connective-tissue disease (CTD)-related ILD, and sarcoidosis, using two panels including 64 markers. Among myeloid cells, we observed the expansion of CD14⁺ CD36^hi CD84^hiCCR2^- monocyte populations in IPF. These CD14⁺ CD36^hi CD84^hi CCR2^- subsets were also increased in ILDs with a progressive phenotype, particularly in a case of acute exacerbation (AEx) of IPF. Analysis of B cells revealed the presence of cells at various stages of differentiation in BALF, with a higher percentage of IgG memory B cells in CTD-ILDs and a trend toward more FCRL5⁺ B cells. These FCRL5⁺ B cells were also present in the patient with AEx-IPF and sarcoidosis with advanced lung lesions. Among T cells, we found increased levels of IL-2R⁺ TIGIT⁺ LAG3⁺ CD4⁺ T cells in IPF, increased levels of CXCR3⁺ CD226⁺ CD4⁺ T cells in sarcoidosis, and increased levels of PD1⁺ TIGIT⁺ CD57⁺ CD8⁺ T cells in CTD-ILDs. Together, these findings underscore the diverse immunopathogenesis of ILDs.

Keywords: FCRL5; connective-tissue disease-related interstitial lung disease; idiopathic pulmonary fibrosis; mass cytometry (CyTOF); monocyte; sarcoidosis.

Figure 1

Graphical abstract of the study. Bronchoalveolar lavage fluid (BALF) samples were collected from patients with idiopathic pulmonary fibrosis (IPF), connective-tissue disease (CTD)-related ILD, and sarcoidosis. Following CD45 barcoding for individual sample identification, BALF cells were analyzed with a T cell panel (35 markers) and B cell/myeloid cell panel (32 markers) using mass cytometry. The expansion of CD14⁺ CD36^hi CD84^hi monocyte populations was found in IPF and ILDs with a progressive phenotype. FCRL5⁺ B cells were increased in CTD-ILDs, acute exacerbation (AEx) of IPF, and sarcoidosis with advanced lung lesions.

Figure 2

Characterization of myeloid cell subsets in BALF from patients with IPF, CTD-ILD, and sarcoidosis. (A) UMAP plots of concatenated samples visualizing the distribution of CD11b⁺CD11c⁺ myeloid cell subpopulations in BALF from patients with IPF, CTD-ILD, and sarcoidosis. Monocytes are defined by CD64⁺CD14⁺, CCR2⁺ macrophages (Mp) by CCR2⁺ CD64⁺ CD14^–, Alveolar Mp by CD64⁺CD206⁺, dendritic cells (DC) by CD64^– CD206^– CD11c⁺ HLA-DR⁺, unidentified cells by CD64^– CD206^– CD11c^lo HLA-DR^–. (B) The proportions of myeloid cell subpopulations in IPF, CTD-ILD, and sarcoidosis. Graphical plots represent individual samples. Statistical differences were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. n.s. not significant. (C) Citrus network tree visualizing the hierarchical relationship and intensity of each marker between identified myeloid cell populations gated by CD45⁺CD11b⁺ CD11c⁺ from IPF (n = 8), CTD-ILD (n = 11), and sarcoidosis (n = 10). Clusters with significant differences were represented in red, and those without significant differences in blue. Circle size reflects the number of cells within a given cluster. (D) Heatmap illustrating the expression markers across different clusters of myeloid cells as determined by the Citrus analysis. (E) Citrus-generated violin plots for eight representative and differentially regulated populations. Each cluster number (C#) corresponds to the number shown in panel (C). All differences in abundance were significant at a false discovery rate < 0.01.

Figure 3

The proportion of CD14⁺CD36^hiCD84^hi monocytes was correlated with disease progression. (A) UMAP plots showing CD14, CCR2, CD36, and CD84 expression in myeloid cells. Red triangles indicate CD14+ CCR2– cell subpopulations. (B) The proportion of CD14⁺ CD36^hi CD84^hi monocytes (cluster #6064 defined by the Citrus analysis in Figure 2C ) abundance in myeloid cell populations in individual samples and (C) the correlation with disease progression. *** p < 0.001. Information for disease with clinical progression is also shown. IPF, idiopathic pulmonary fibrosis; CTD-ILD, connective-tissue disease-related interstitial lung disease; AEx, acute exacerbation; SSc, systemic sclerosis; SjS, Sjogren syndrome; SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; DM, dermatomyositis; MCTD, mixed connective tissue disease; na, not accessed.

Figure 4

Characterization of B cell subsets in BALF from patients with IPF, CTD-ILD, and sarcoidosis. (A) Percentage of B cells and plasma cells in CD45⁺ BALF cells. (B) t-stochastic neighborhood embedding (t-SNE) plots of concatenated samples visualizing the distribution of B cell sub-populations in CD64^–CD3^– and CD19⁺ or CD138⁺ gated B cells in BALF from patients with IPF, CTD-ILD, sarcoidosis. Naive B cells are defined by CD19⁺IgD⁺, IgM memory B cells: CD19⁺ IgM⁺ CD27⁺, IgG memory B cells: CD19⁺ IgG⁺ CD27⁺, IgA memory B cells: CD19⁺ IgA⁺ CD27⁺, CD11c^– double negative (DN) B cells: CD19⁺ CD11c^– IgD^– CD27^–, CD11c⁺ DN B cells: CD19⁺ CD11c⁺ IgD^–CD27^–, plasmablasts: CD19⁺ CD27⁺ CD38⁺ CD138^–, plasma cells: CD19^– CD138⁺ and IgG⁺ or IgA⁺. (C) Percentage of B cell sub-populations in IPF, CTD-ILD, and sarcoidosis. Graphical plots represent individual samples. (D) Percentage of FCRL5 expressing B cells in total B cells. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. (E) The proportion of each B cell subsets in FCRL5 expressing B cells. (F) Representative chest CT images of patients with sarcoidosis exhibiting a high percentage of FCRL5 B cells and a low percentage of FCRL5 B cells in BALF.

Figure 5

Characterization of T cell subsets in BALF from patients with IPF, CTD-ILD, and sarcoidosis. (A) The proportion of T cells (defined by CD2⁺CD3⁺) among CD45⁺ BALF cells and the CD4⁺ T cell/CD8⁺ T cell ratio from IPF (n = 8), CTD-ILD (n = 13), and sarcoidosis (n = 10). ns: not significant. (B) UMAP plots of concatenated samples visualizing the distribution of CD2⁺CD3⁺ T cell differentiation in BALF from patients with IPF, CTD-ILD, and sarcoidosis. Central memory (CM) T cells are defined by CCR7⁺ CD45RO⁺ CD28⁺ Fas⁺, Transitional memory (TM) by CCR7^– CD45RO⁺ CD28⁺ Fas⁺, Effector memory (EM) by CCR7^– CD45RO⁺ CD28^– Fas⁺, Terminal effector (TE) by CCR7^– CD45RO^+/– Fas^–, Effector memory RA (EMRA) by CCR7^– CD45RO^– CD45RA⁺ Fas^+/–. Arrows indicate the trajectory of T-cell differentiation. DN: CD4^– CD8^– double negative, DP: CD4⁺ CD8⁺ double positive. (C) Percentage of T cell subpopulations in IPF, CTD-ILD, and sarcoidosis. Graphical plots represent individual samples. Statistical differences were analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. ns. not significant, ** p < 0.01, **** p < 0.0001. (D) The Citrus network tree displays the hierarchical relationship and intensity of each marker among the T-cell populations in BALF from IPF (n = 8), CTD-ILD (n = 13), and sarcoidosis (n = 10). (E) Heatmap illustrating the expression markers across different clusters of T cells as determined by the Citrus analysis. (F) Citrus-generated violin plots for three representative and differentially regulated populations. Each cluster number (C#) corresponds to the number shown in panel (D). All differences in abundance are significant at a false discovery rate < 0.01.

Figure 6

Immunological phenotypes in a patient with acute exacerbation of IPF. (A) Chest computed tomography images of the patient upon admission reveal the emergence of bilateral diffuse ground glass opacities superimposed on a honeycomb pattern, accompanied by peripheral traction bronchiectasis primarily in the basal lungs. (B) Comparison of BALF cell differentiation and CD4/CD8 ratio between patients with a patient experiencing an acute exacerbation (AEx) of the condition and the other cases of IPF. (C) Citrus-generated plots for myeloid sub-populations in IPF patients with stable condition and AEx. Each cluster number (C#) corresponds to the number shown in Figure 2C . (D) A uniform manifold approximation and projection (UMAP) of myeloid cells (CD45⁺CD11b⁺CD11c⁺ gated) showing cell distributions and each marker expression in BALF cells from concatenated samples with AEx and other cases of IPF. (E) t-SNE plots visualizing the distribution of T cell subpopulations in BALF T cells (gated as CD45⁺CD2⁺CD3⁺) from patients with AEx and other cases of IPF. Double negative (DN) T cells were defined as CD4^–CD8^– T cells. A red arrow indicates expansion of the CD57-CD7+CD44+PD-1-CD4 T cell subpopulation in AEx-IPF.

Figure 7

Immunological phenotypes in BALF cells from patients with CTD-ILD. (A) t-SNE plots visualizing the distribution of T cell subpopulations in BALF T cells (gated as CD45⁺CD2⁺CD3⁺) from patients with CTD-ILD. DN: CD4–CD8– double negative T cells. (B)The ratio of CD28^– CD4 T cells/CD28⁺ CD4 T cells defined in t-SNE plots in each disease. (C) The Citrus network tree displays the hierarchical relationship and intensity of each marker among the T-cell populations in BALF from Sjogren syndrome-related ILD (n = 3) and dermatomyositis associated-ILD (n = 3). (D) Heatmap illustrating the expression markers across different clusters of T cells as determined by the Citrus analysis. (E) Citrus-generated violin plots for five representative and differentially regulated populations. Each cluster number (C#) corresponds to the number shown in panel (C). All differences in abundance are significant at a false discovery rate < 0.01. (F) UMAP plots visualizing the distribution of CD11b⁺CD11c⁺ myeloid cell subpopulations in BALF from patients with CTD-ILD. Monocytes are defined by CD64⁺CD14⁺, CCR2⁺ macrophages (Mp) by CCR2⁺ CD64⁺ CD14^–, Alveolar Mp by CD64⁺CD206⁺, dendritic cells (DC) by CD64^– CD206^– CD11c⁺ HLA-DR⁺, unidentified cells by CD64^– CD206^– CD11c^lo HLA-DR^–. (G) The proportions of myeloid cell subpopulations in CTD-ILD.