tion of chemokines, has been renamed to CXCL13 (6). In addition to the chemokine system, various members of the lymphotoxin (LT)/TNF family and their receptors were found to substantially participate in lymphoid organ organ- ogenesis (7). Their effects on regular lymphoid organ ar- chitecture can be attributed, at least in part, to their ability to induce the expression of various chemokines in lym- phoid organs including BLC, secondary lymphoid tissue chemokine (SLC), and EBV-induced molecule 1 ligand chemokine (ELC [8]). In addition, it was recently shown that chemokines can induce a positive feedback loop on LT expression. Data derived from BLC gene-targeted mice demonstrate that BLC induces B cells to upregulate mem- brane-bound LTa1b2, a cytokine known to induce follic- ular dendritic cell (FDC) development and the expression of BLC (5). Taken together, these data demonstrate a piv- otal role for CXCR5 and its ligand in directing FDCs and B cells to form primary and secondary B cell follicles. In contrast to B cells, recently entered T cells do not mi- grate to follicles but stay in close contact with antigen-pre- senting dendritic cells (DCs) to screen these cells for pre- sented peptides recognized by the T cell antigen receptor. In most cases, no specific peptide is encountered and the majority of T cells leave the LN within a few hours. How- ever, if T cells are activated by antigen-presenting cells, a proliferation and differentiation program is initiated giving rise to effector T cells either migrating to places of inflam- mation or to the B cell follicles to participate in the forma- tion of germinal centers (GCs [9, 10]). A fraction of acti- vated T cells develop into memory cells, allowing a fast and effective immune response once rechallenged with the same antigen (11). Based on the expression of the chemokine homing receptor CCR7, it has been suggested that T memory cells fall into one of two pools: CCR72 effector memory T cells (TEM) endowed to migrate to inflamed tis- sue, and CCR71 central memory T cells (TCM) with the potential to home to lymphoid organs (12). We now show that CXCR51 Th cells represent a novel subpopulation of Th cells that localize to B cell follicles and GCs to provide the support that B cells need during their differentiation program in order to efficiently produce Igs. Materials and Methods Abs. In addition to Abs described elsewhere (13), mAbs di- rected against CD95, IL-2, and IFN-g, and mAbs specific for various human Igs were purchased from BD PharMingen. mAbs specific for human BLC/CXCL13 and ELC/CCL19 were ob- tained from R&D Systems; the anti-CCR7 mAb (clone 3D12) was produced in our laboratory and has been described previ- ously (12). Immunohistology and Flow Cytometry. Flow cytometry and im- munohistology were done as described previously (4, 13). In addi- tion, immunohistology using anti-BLC/CXCL13 mAb and anti- ELC/CCL19 was done by enhancing signals with the tyramide signal amplification system (NEN Life Science Products/Dupont). Cytokine Production. PBLs were isolated by Ficoll den- sity centrifugation and stained with anti-CD4–FITC, anti- CD45RO–PE, and anti-CXCR5–biotin/streptavidin–Cychrome 1546 Follicular B Helper T Cells (clone RF8B2). Using flow cytometry (FACS Vantage™; Becton Dickinson), CD41CD45RO1 cells were sorted into two frac- tions: CXCR52 and CXCR51 cells. Sorted cells were cultured in RPMI/10% FCS (105 cells/500 ml) and stimulated with plastic- bound anti-CD3 mAb (20 mg/ml). After 24 h, cell culture super- natants were collected and stored at 2808C. The presence of var- ious cytokines within the supernatants was analyzed using various cytokine-specific ELISAs (Endogen). Detection of intracellular cytokines was done as described elsewhere (12). Ab Production. As described for peripheral blood T cells above, CD41CD45RO1CXCR51 and CD41CD45RO1CXCR52 cells were also isolated from tonsils. In addition, CD191 B cells were isolated from the same tonsils by means of flow cytometry sorting. To assess the capability of both T cell subsets to support Ig secre- tion and isotype switching, B cells were cultured either alone or in the presence of the same number of sorted T cells for 11 d. Super- natants were collected and analyzed for the presence of various Ig isotypes using pairs of isotype-specific Abs and ELISA (BD Phar- Mingen). Memory Response. Using Ficoll, PBLs were isolated from in- dividuals who had not been immunized with tetanus toxin (TT) during the last 2 yr. Cells were plated for 2 h in RPMI/5% auto- logous serum. Nonadherent lymphocytes were withdrawn, and adherent macrophages were collected, irradiated (50 Gy), and stored on ice until further use. Lymphocytes were stained with CD4, CD45RO, and CXCR5 Abs and sorted into the two frac- tions described above. Sorted T cells were titrated and cocul- tured with irradiated autologous macrophages (100,000/well) in RPMI/5% autologous serum in the presence or absence of TT (1:800; Wyeth). In some experiments, sorted T cells were also cultured with irradiated heterologous macrophages. After 6 d of incubation, [3H]thymidine was added to the medium (0.025 mCi/well) for 18 h. Cells were harvested and the amount of in- corporated [3H]thymidine was determined. CCR7 Downregulation. White blood cells (5 3 106/ml) were incubated at 378C (RPMI/10% fetal bovine serum/5 mM Hepes, pH 7.4) in the presence or absence of 300 ng/ml of ELC/CCL19 (R&D Systems). After various incubation periods, aliquots were withdrawn, stored on ice, and stained with anti-CD4 and anti- CCR7 mAbs. Expression levels of CCR7 on CD41 T cells were determined by flow cytometry. Determination of Inducible Costimulator Expression by Reverse Transcription PCR. Total RNA from CD41CD45RO1 CXCR51 cells was isolated with Trizol (Life Technologies). cDNA was generated with Superscript II reverse transcriptase (Boehringer) using random hexamer primers. Primers were de- signed based on the inducible costimulator (ICOS) cDNA se- quence (sequence data available from EMBL/GenBank/DDBJ under accession no. AF218312). The PCR was performed using 5 U Taq polymerase, 1.5 mM MgCl, and 0.6 mM of each primer (primer A, 59-TGCCATTCTCAGTTATCC; primer B, 59- ACATGTATTCACCGTTAGG) in an amplifier (model 225; MJ Research) applying the following parameters: denaturation at 948C for 4 min, and addition of Taq polymerase at 888C followed by 35 amplification cycles with 15 s denaturation at 948C, anneal- ing at 558C for 30 s, and a 1-min extension at 728C. Using the cDNA as template, these primers generated a 298-bp product. Results Peripheral Blood CXCR51 CD4 Cells Are a Subpopulation of TCM. CXCR5 expression was studied on peripheral blood and tonsillar T cells. Flow cytometry on PBLs dem-