Figure 1. Distinct T cell and B cell landscapes in LLNs from ILD patients compared with matched controls.

a. Representative flow cytometry gating for total T cells in LLNs. Cells were pre-gated on live cells. b. Frequencies of T cells in live cells in control vs ILD LLNs. Statistical significance was tested by the Wilcoxon rank sum test. Differential diagnosis and experimental batch are noted by the color and symbol as shown in the legend. c-e. PCA of total T cells based on fluorescence intensities of 19 parameters: CD154, FOXP3, HLA-DR, CCR7, CD25, ICOS, CD127, GATA3, Vα7.2, PBS-57 loaded CD1d tetramer, CD4, CD8, Vδ, PD1, RORγt, BCL6, CD38, CD45RA and CD137. An analysis was run with 10,000 cells, 8,000 cells, and 10,000 cells for batch1, batch2, and batch 3, respectively. f. Box plots comparing the median fluorescence intensity of individual T cell markers between ILD and controls, based on all samples in batches 1–3. Fold change was calculated relative to the mean values of control samples, separately for each batch. Parameters that are significantly associated with ILD based on a likelihood-ratio test on each logistic regression coefficient are shown. Non-significant parameters are shown in Extended Figure 1. P-values were adjusted by Benjamini-Hochberg correction. g. Representative flow cytometry gating for B cells in LLNs. Cells were pre-gated on live cells. h. Frequencies of B cells in live cells in control vs ILD LLNs. Statistical significance was tested by the Wilcoxon rank sum test. i-k. PCA of B cells based on fluorescence intensities of 12 parameters: CXCR4, HLA-DR, CD10, IgD, CD1c, CD44, CD138, CD11C, CD24, CD27, CD38, IgM. An analysis was run with 10,000 cells for each batch, separately. l. Box plots comparing the median fluorescence intensity of individual B cell markers between ILD and controls, based on all samples in batches 1–3. Calculations of fold changes and adjusted p-values were done as described above (f). P<0.05 (*), P<0.01 (**), P<0.001 (***), P<0.0001 (****).