Graphical abstract of the study.

Bronchoalveolar lavage fluid (BALF) samples were collected from patients with Pneumocystis jirovecii pneumonia (PCP), cytotoxic drug-related interstitial lung disease (DI-ILD), and immune-checkpoint inhibitor-related ILD (ICI-ILD). Subsequently, BALF cells were analyzed using mass cytometry with a T cell panel (35 markers) and B cell/myeloid cell panel (32 markers) following CD45 barcoding for sample identification. The study found that there was a significant increase in the expansion of CD16+ T cells in patients with PCP, with the highest CD16 expression observed in a fatal case of PCP.

Characteristics of the study population.

Serum levels of beta-D-glucan and KL-6 from patients with PCP, DI-ILD, and ICI-ILD.

(A) Serum levels of beta-D-glucan from patients with Pneumocystis jirovecii pneumonia (PCP), cytotoxic drug-related interstitial lung disease (DI-ILD), and immune-checkpoint inhibitor-related ILD (ICI-ILD). (B) A receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic utility of beta-D-glucan for PCP, with values from the DI-ILD group used as a reference. The area under the curve was determined to be 0.8929. (C) Serum levels of KL-6.

Characterization of T cell subsets in BALF from patients with PCP, DI-ILD, and ICI-ILD.

(A) Percentage of T cells (defined as CD2+CD3+) in CD45+ BALF cells and CD4/CD8 ratio in T cells from patients with Pneumocystis jirovecii pneumonia (PCP), cytotoxic drug-related interstitial lung disease (DI-ILD), and immune-checkpoint inhibitor-related ILD (ICI-ILD). (B) Uniform manifold approximation and projection (UMAP) of concatenated samples visualizing the distribution of T cell subpopulations. Central memory (CM) T cells were 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. (D) Citrus network tree visualizing the hierarchical relationship and intensity of each marker between identified T cell populations gated by CD45+CD2+ CD3+ from PCP (n = 7), DI-ILD (n = 9), and ICI-ILD (n = 5). 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. (E) Heatmap demonstrates the expression of various markers in different clusters of T cells, as identified through the Citrus analysis. (F) Citrus-generated violin plots for six representative and differentially regulated populations. Each cluster number (#) corresponds to the number shown in panel (D). All differences in abundance were significant at a false discovery rate < 0.01.

Characterization of myeloid cell subsets in BALF from patients with PCP, DI-ILD, and ICI-ILD.

(A) Uniform manifold approximation and projection (UMAP) of concatenated samples visualizing the distribution of myeloid cell sub-populations in CD3− CD11b+ CD11c+gated myeloid cells in BALF from patients with IPF, CTD-ILD, sarcoidosis. Monocytes were defined by CD64+ CD14+, CCR2+ macrophages (Mp) by CD64+ CD14− CCR2+, Alveolar Mp by CD64+ CDI4 CCR2 CD206+, dendritic cells (DC) by CD64−CD14−CD206−CD11c+ HLA-DR+, Unidentified subsets by CD64− CD11b+/− CD11c+/− CD14−CD206−. (B) Percentage of myeloid cell sub-populations in PCP (n = 7), DI-ILD (n = 9), and ICI-ILD (n = 9). Graphical plots represent individual samples. (C) Citrus network tree visualizing the hierarchical relationship and intensity of each marker between identified myeloid cell populations gated by CD45+ CD3− CD11b+ CD11c+from PCP (n = 6), DI-ILD (n = 9), and ICI-ILD (n = 9). 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 demonstrates the expression of various markers in different clusters of myeloid cells, as identified through the Citrus analysis. (E) Citrus-generated violin plots for six representative and differentially regulated populations. Each cluster number (#) corresponds to the number shown in panel (C). All differences in abundance were significant at a false discovery rate < 0.01.

Characterization of B cell subsets in BALF from patients with PCP, DI-ILD, and ICI-ILD.

(A) Percentages 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 subpopulations in CD64−CD3− and CD19+ or CD138+ gated B cells in BALF from patients with PCP, DI-ILD, and ICI-ILD. Naive B cells are defined by CD19+IgD+, IgM B cells: CD19+ IgM+, IgG B cells: CD19+ IgG+, IgA B cells: CD19+ IgA+, plasmablasts: CD19+ CD27+ CD38+ CD138−, plasma cells: CD19− CD138+and IgG+ or IgA+. (C) Percentages of B cell subpopulations in PCP (n = 7), DI-ILD (n = 9), and ICI-ILD (n = 9). Graphical plots represent individual samples. (D) Twodimensional dot plots depicting FCRL5-expressing B cells within a gated population of B cells defined as CD64−CD3− and CD19+ or CD138+. Percentages of FCRL5-expressing B cells within the total B cell population are also shown.

Immunological phenotypes in a fatal case of PCP.

(A) Chest computed tomography images of the patient upon admission reveal the emergence of bilateral diffuse ground glass opacities and infiltration in both lungs. (B) Comparison of T cell percentage and CD4/CD8 ratio between a fatal case and surviving cases of PCP. (C) t-stochastic neighborhood embedding (t-SNE) plots illustrating the distribution of T cell subpopulations in BALF T cells (gated as CD45+CD2+CD3+) from a fatal case and surviving cases of PCP. Double negative (DN) T cells were defined as CD4 CD8 T cells. (D) Percentages of each T-cell subpopulation. Red dots represent the value of the fatal case. (E) Mean CD16 intensity in the T cell population. (F) Mean CD16 intensity in the myeloid cell population. (G) The correlation matrix in PCP cases. Pearson r values are shown in each square.

Mass cytometry antibody panels.

A T cell gate (CD2+CD3+) and Citrus analysis of T cell populations in BALF cells from PCP, DI-ILD, and ICI-ILD.

The Citrus analysis of myeloid cell populations in BALF cells from PCP, DI-ILD, and ICI-ILD.