Fig. 1. Evaluation of peritumoral tertiary lymphoid structure (TLS) according to density and maturity by hematoxylin/eosin (H&E) staining and immunohistochemistry (IHC) of CD21 and CD23.

a Representative sections for counting peritumoral TLSs (arrowheads) within peritumoral regions. b Representative images of TLSs in each maturation category as evaluated by H&E and IHC. Early TLSs (E-TLSs) are vague dense lymphocytic aggregations without CD21 or CD23 signals. Primary follicle-like TLSs (PFL-TLSs) are definite round- or oval-shaped clusters of small lymphocytes with CD21 signals, but no CD23 signals. Secondary follicle-like TLSs (SFL-TLSs) are follicles with a definite germinal centre formation, comprising large lymphocytes with clear cytoplasm, and having both CD21 and CD23 signals. c Distribution of TLS density and proportion of each TLS maturation category among 316 oesophageal cancer patients. d Proportion of TLS maturity levels according to TLS density. Red lines and the numbers next to them show median values. e, f TLS density according to pT (e) and pStage (f). Scale bars: a = 500 µm; b = 100 µm. Comparisons were made by Mann–Whitney U test.