The unique expression of CXCL13 in human CD4⁺ GC-Th cells. (A) Expression of CXCL13 in GC-Th cells and other T-cell subsets measured by a cDNA array technique. Data shown are in relative units (naive T cells equal one). *Average up-regulation of CXCL13 mRNA in GC-Th cells versus naive T cells plus or minus standard deviation from 3 independent measurements. (B) Quantitative RT-PCR examination of CXCL13 in freshly isolated GC-Th cells, monocyte-derived mature dendritic cells, and freshly isolated CD19⁺ tonsil B cells. The copy numbers of B-lymphocyte chemoattractant (BLC) were normalized for copy numbers for GAPDH. Relative expression levels are shown (B cells equal 1). (C) CXCL13 is the major chemokine expressed by GC-Th cells. Real-time RT-PCR was used to examine 20 chemokines (in 4 groups: “Th1/inflammation,”“Th2,”“tissue-specific,” and “lymphoid” based on their expression sites or functions); GAPDH and β-actin were used as internal controls. Expression levels are shown in relative SYBR green fluorescence of the gene products to GAPDH products (GAPDH intensity = 100%). (D) CXCL13 expression in GC-Th cells was visualized by an in situ immunohistology technique. Frozen tonsil sections were stained with anti-CXCL13 antibody or control mouse IgG1 and then with secondary antibody conjugated with PE (red). Sections were further stained with anti–CD57-FITC for GC-Th cells (green). A confocal microscope system (Bio-Rad MRC 1024UV and Nikon Diaphot 300 microscope) was used to analyze the sections. (E-F) Many GC-Th cells are in physical contact with dendritic cells and GC-B cells. Frozen tonsil sections were stained with fluorescent monoclonal antibodies to CD11C (red for dendritic cells and some macrophages), CD57 (green for GC-Th cells), CD38 (red), and CD19 (blue). Please note that green (CD57⁺) cells are GC-Th cells and red cells are dendritic cells in panel E. Purple (CD38⁺CD19⁺) cells are GC-B cells and red (CD38⁺CD19^–) cells are plasma cells in panel F. Arrows indicate several examples of GC-Th cells that are in direct contact with dendritic cells or GC-B cells. The images were taken at × 200 magnification.