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. 2021 Jul 12;9(7):e002101. doi: 10.1136/jitc-2020-002101

Figure 2.

Figure 2

PD-1+CXCR5−CD4+ Th-CXCL13 might be involved in the formation of TLSs associated with NPC tumors. (A) Expression of the indicated Th cytokines and chemokines in sorted PD-1+CXCR5−CD4+ Th-CXCL13 in tumor of NPC (n=4) after 12 hours of resting, measured using a bead-based immunoarray. (B) Statistical analysis showing the correlation between the percentages of PD-1+CXCR5−CD4+ TILs and Th-CXCL13 cells analyzed by FACS analysis in tumor of NPC (n=50). (C) MIHC staining of a representative tumor section showing the coexpression of CD4+ (orange), CXCL13+ (magenta) and PD-1+ (green), with nuclei counterstained with DAPI (blue). (D) The expression of CXCR5 on CD3+ T, CD4+ T, CD8+ T and CD19+ B cells among PBMCs of patients with NPC. The same experiment was repeated five times. (E) Migration of CXCR5+CD19+ B, CXCR5+CD4+ T, CXCR5+CD8+ T, CXCR5−CD19+ B, CXCR5−CD8+ T and CXCR5−CD4+ T cells from PBMCs (n=5) in response to recombinant human CXCL13. Chemotaxis abilities of CXCR5+ subset, compared with CXCR5− subset, were demonstrated, in which the chemotaxis index of CXCR5− subsets was set as ‘1’. (F) MIHC staining of a representative tumor section showing CD4+ (green), CXCL13+ (magenta), CD20+ (cyan), CXCR5+ (red), PD-1+ (yellow) and DAPI (blue). (G) MIHC staining of a representative tumor section showing PD-1+ (Red), Bcl-6+ (Green) and CD21+ (Orange), with nuclei counterstained with DAPI (blue). (H) Survival analysis of the cohort stratified by CD4+ cells, PD-1+ cells, CXCR5+ cells and CXCL13+ cells identified by IHC staining, which the threshold used to define high and low is 8, 3, 6, and 4, respectively. (I) Survival analysis of the cohort stratified by CD4+PD-1+CXCR5+, CD4+PD-1+CXCR5−, CD4+PD-1-CXCR5+ and CD4+PD-1−CXCR5− cells subsets among CD4+ TILs identified by MIHC staining and Halo analysis software, which the threshold used to define high and low is 0.997, 4.50, 32.1, and 42.5, respectively. FACS, fluorescence-activated cell sorting; IHC, immunohistochemistry; MIHC, multiplex immunohistochemical; NPC, nasopharyngeal carcinoma; PBMC, peripheral blood mononuclear cell; TIL, tumor-infiltrating lymphocyte; TLS, tertiary lymphoid structure.