Figure 1. The expression of CXCR5, CCR7, and ELC/CCL19. Pe- ripheral blood cells (A) or tonsillar cells (B) were stained with anti-CD4, anti-CXCR5, and anti-CCR7, and analyzed by flow cytometry as indi- cated. (C) PBLs were incubated at 378C with 300 ng/ml ELC/CCL19. At the time points indicated, cells were removed, placed on ice, and stained with anti-CD4 and anti-CCR7 mAbs. The expression of CCR7 on CD41 cells was analyzed and is shown as the percentage of control staining of cells not exposed to ELC. (D) Tonsillar cryostat sections were stained using anti-ELC mAb and the tyramide signal amplification. Ar- rowheads indicate that ELC polarized to the luminal site of HEVs. onstrated that the vast majority of CD41CXCR51 cells coexpress CD45RO (13) and CCR7 (Fig. 1 A), thus iden- tifying them as a subpopulation of the recently described TCM (12). In contrast, .50% of CD41CXCR51 cells iso- lated from tonsils lack CCR7, suggesting that most T cells downregulate CCR7 once they are within lymphoid or- gans (Fig. 1 B). As ligand-induced receptor internalization might contribute to this process, we exposed PBLs to ELC. As shown in Fig. 1 C, CCR7 gets downregulated on pe- ripheral blood T cells to z50–60% of the initial levels. This observation suggests that in addition to ligand-induced re- ceptor uptake, other mechanisms such as TCR activation contribute to CCR7 downregulation. When analyzing the expression of ELC within the tonsil, we found this chemokine expressed not only on DCs (not shown) as re- ported earlier (14), but also on the HEVs. High power magnification revealed that ELC polarizes to the luminal site of the HEVs (Fig. 1 D). This observation indicates that, in addition to SLC/CCL21, ELC/CCL19 may contribute to transendothelial migration. CXCR51 CD4 Cells Localize to B Cell Follicles and GCs to Express ICOS and CD40 Ligand. We next studied ex- pression of CXCR5 on T cells using tonsillar cryosections applying anti-CXCR5 and anti-CD4 mAbs. CXCR5, known to be required for the formation of B cell follicles (4), is highly expressed on all B cells of the follicular mantle and on T cells that are closely associated with B cells in the light zone of GCs, the compartment of B cell affinity ma- turation. In addition, some T cells that are closely related to B cell follicles, but outside these B cell–rich areas, also ex- press CXCR5 (Fig. 2 A), thus confirming our earlier ob- servations (4). When using Abs specific for BLC/CXCL13, a ligand for CXCR5 that induces migration of T cells in vitro (15), we observed that expression of this chemokine is restricted to the follicular mantle and the light zone of the Figure 2. CXCR51CD4 cells localize to follicles and express costimulatory molecules. Tonsillar cryostat sections were stained with anti-CD4 (green) and anti-CXCR5 (red; A) or with anti-BLC/CXCL13 (B). M, marginal zone; T, T zone; DZ, dark zone; LZ, light zone. (C) CD4 cells were sorted from tonsils, activated with PMA (50 ng/ml) for 15 min, transferred to ice, and stained with the Abs indicated. (D) CD41CD45RO1CXCR51 cells were sorted from tonsils and subjected to reverse transcription PCR using ICOS-specific primers (lane labeled cDNA). Using genomic DNA (gDNA) as a template, the PCR yielded a fragment of z2 kb in size, indicating an intronic sequence between the primers used and demonstrating the purity of the cDNA. M, marker. 1547 Breitfeld et al.