GC. Based on the reticular appearance of the staining, BLC1 cells seem to be mainly FDCs (Fig. 2 B). As CXCR51 Th cells reside in GCs, tonsillar CD4 cells were isolated by MACS and tested for the presence of in- tracellular stores of CD154 (CD40 ligand [CD40L]), a molecule known to be crucial for B cell activation and Ab class switching (16). After a 15-min activation with PMA (50 ng/ml)/ionomycin (800 ng/ml), a process known to translocate these molecules to the cell surface, we found a large fraction of CD41CXCR51 cells to express CD154 (Fig. 2 C). It has been recently demonstrated that ICOS, a novel member of the CD28 family of costimulatory molecules, is also highly expressed on Th cells located in follicles and GCs (17). Therefore, we were interested in whether CXCR51 CD4 cells coexpress ICOS. Indeed, using reverse transcription PCR on sorted CD41CD45RO1 CXCR51 tonsillar cells, we found high levels of ICOS- specific transcripts in this cell population (Fig. 2 D). In ad- dition, preliminary data obtained by flow cytometry ap- plying ICOS-specific Abs (provided by Dr. Kroczek, Robert Koch Institute, Berlin, Germany) demonstrate a marked correlation between the expression of CXCR5 and ICOS. CXCR51 CD4 Cells Produce Few Cytokines but Support Ig Production. As CXCR51 CD4 cells express costimulatory molecules, we were interested to see whether CXCR52 and CXCR51 helper cells produce different sets of cyto- kines. According to the expression of CXCR5, peripheral blood CD41CD45RO1 cells were sorted into two frac- tions: CXCR51 and CXCR52 cells. After a 24-h stimula- tion period the cytokine profile was analyzed. Within the scope of our experiments we noticed no differences with regard to IL-2 production. However, we observed de- creased levels of IL-10, and found dramatically reduced levels of IFN-g, IL-5 (Fig. 3, A and B), and IL-4 (not shown) in the CXCR51 population compared with the CXCR52 fraction. These findings suggest that CD41 CXCR51 cells represent a non-Th1/Th2 subset of effector T cells that is involved in B cell help. As CXCR51 CD4 cells migrate to B cell follicles in lym- phoid organs where they express ICOS, which is known to superinduce IL-10, a B cell differentiation factor, and CD40L, we looked into whether these cells support B cells to secret Ig. To that end, tonsillar CD41CD45RO1 cells were sorted into two fractions as described above for pe- ripheral blood cells and cultured in the presence of sorted CD191 B cells isolated from the same tonsil. As shown in Fig. 3 C, B cells produced comparable low amounts of both IgA and IgG when cultured for 11 d without T cells or with the CXCR52 fraction. In contrast, coculture of B cells with CXCR51CD45RO1 CD4 cells resulted in an z10-fold increase in the production of IgA and IgG Abs. With regard to the production of IgM, differences between the two Th cell subsets were less obvious. Compared with B cell cultures without T cells, addition of CXCR52 CD4 cells induced a 5-fold increase in IgM, whereas addition of CXCR51 cells yielded a 15-fold increase in the production 1548 Follicular B Helper T Cells Figure 3. CXCR51CD4 cells support Ig production. PBLs were sorted into two fractions: CD41CD45RO1CXCR52 (black bars) and CD41CD45RO1CXCR51 (white bars). Sorted cells were stimulated with plastic-bound anti-CD3 for 24 h and the amount of cytokines pro- duced was determined by ELISA. Similar results were obtained in two additional experiments. (B) Peripheral blood CD41CD45RO1 cells were sorted, activated with PMA/ionomycin, and examined for intracellular production of cytokines. (C) Tonsillar CD191 B cells were sorted and cocultured for 11 d with sorted CD4 cells from the same tonsil before Ab production was analyzed by ELISA. CD41CD45RO1CXCR52, black bars; CD41CD45RO1CXCR51, white bars; no T cells, shaded bars. Similar results were obtained in a second experiment of this isotype. These data suggest that CD41CXCR51 cells represent the classical Th cells supplying B cells with the costimulatory signals required for Ig production. Memory CD4 Cells Are within the CXCR52 Fraction. As nearly all CD41CXCR51 T cells coexpress CD45RO (13), a molecule found on memory T cells, we were inter- ested in studying whether the expression of CXCR5 is as- sociated with T cell memory. Memory function analysis was performed on sorted peripheral blood T cells derived from donors who had not been immunized with TT during the last 2 yr. Coculture of sorted T cells with irradiated au- tologous antigen-presenting cells loaded with TT revealed that memory cells were principally found within the CXCR52 fraction (Fig. 4 A). In contrast, both populations responded equally well to allogenic antigen-presenting cells