Identification of microenvironmental cell subtypes by single-cell expression profiling and imaging analysis. (A) Left, detection of follicular T (Tf) cells in DLBCL tissue based on indicated markers using multiplexed immunofluorescence imaging analysis and the Mantra system. Representative images taken from 10 patient samples analyzed are shown. We assigned each fluorescent signal to the preset pseudocolor (CD20: red; CD3: green; ICOS: cyan). Most CD3⁺ T cells (green) express ICOS protein (cyan) in disease-free cases, while not in relapsed cases. Note that the frequencies of CD20⁺ lymphoma B cells (red) and overall CD3⁺ T cells (green) were comparable between them. Right, Box and Whisker plot (dots indicate outliers) shows frequency of ICOS+ Tf cells, which were digitally counted in 5 fields. P values were calculated using the Wilcoxon rank-sum test. (B) Left, comparable imaging identified CD68⁺ DCs/macrophages (yellow) expressed CD11c (cyan) in favorable prognostic case. Note that CD68⁺ DC/macrophages (yellow) did not express CD11c in relapsed cases. Right, the proportion of CD11c⁺ CD68⁺ cells among total cells in 5 fields, shown as a Box and Whisker plot (dots indicate outliers), in relapsed vs disease-free cases, as calculated by the Wilcoxon rank-sum test. (C) Characterization of FGFR1⁺ cells (purple) based on staining with the lineage-specific markers CD3 (T cells), CD68 (DC/macrophages), and CD20 (B cells). (D) Multispectral imaging shows steady-state LN (upper) and reactive LN (lower) specimens stained with indicated markers. ICOS, CD11c, and FGFR1 are enriched during GC formation.