Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production

Abstract

Chemokines and their receptors have been identified as major regulators controlling the functional organization of secondary lymphoid organs. Here we show that expression of CXC chemokine receptor 5 (CXCR5), a chemokine receptor required for B cell homing to B cell follicles, defines a novel subpopulation of B helper T cells localizing to follicles. In peripheral blood these cells coexpress CD45RO and the T cell homing CC chemokine receptor 7 (CCR7). In secondary lymphoid organs, CD4(+)CXCR5(+) cells lose expression of CCR7, which allows them to localize to B cell follicles and germinal centers where they express high levels of CD40 ligand (CD40L), a costimulatory molecule required for B cell activation and inducible costimulator (ICOS), a recently identified costimulatory molecule of the CD28 family. Thus, when compared with CD4(+)CD45RO(+)CXCR5(-) cells, CD4(+)CD45RO(+)CXCR5(+) tonsillar T cells efficiently support the production of immunoglobulin (Ig)A and IgG. In contrast, analysis of the memory response revealed that long-lasting memory cells are found within the CD4(+)CD45RO(+)CXCR5(-) population, suggesting that CXCR5(+)CD4 cells represent recently activated effector cells. Based on the characteristic localization within secondary lymphoid organs, we suggest to term these cells "follicular B helper T cells" (T(FH)).

Figure 1

The expression of CXCR5, CCR7, and ELC/CCL19. Peripheral blood cells (A) or tonsillar cells (B) were stained with anti-CD4, anti-CXCR5, and anti-CCR7, and analyzed by flow cytometry as indicated. (C) PBLs were incubated at 37°C 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 CD4⁺ 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. Arrowheads indicate that ELC polarized to the luminal site of HEVs.

Figure 2

CXCR5⁺CD4 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) CD4⁺CD45RO⁺CXCR5⁺ 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 ∼2 kb in size, indicating an intronic sequence between the primers used and demonstrating the purity of the cDNA. M, marker.

Figure 4

Memory response in CD4⁺CD45RO⁺CXCR5⁻ cells. (A) PBLs were sorted as indicated and cultured with autologous serum and irradiated autologous macrophages in the presence or absence of TT for 7 d. For the last 24 h of culture, [³H]thymidine was added to the medium and the amount of incorporated ³H was determined (every point represents the mean of 12 replicas). Similar results were obtained in a second experiment. In a third experiment, we also observed a weak proliferative response in the TT-treated CXCR5⁺ fraction at the highest T cell count tested. (B) The same as described for A, with the exception that TT was not added and allogenic (allo) macrophages were substituted for autologous macrophages. Data shown are representative of three experiments. (C) CXCR5⁻ and CXCR5⁺ tonsillar CD4 cells were analyzed for the expression of CD95 by flow cytometry.

Figure 3

CXCR5⁺CD4 cells support Ig production. PBLs were sorted into two fractions: CD4⁺CD45RO⁺CXCR5⁻ (black bars) and CD4⁺CD45RO⁺CXCR5⁺ (white bars). Sorted cells were stimulated with plastic-bound anti-CD3 for 24 h and the amount of cytokines produced was determined by ELISA. Similar results were obtained in two additional experiments. (B) Peripheral blood CD4⁺CD45RO⁺ cells were sorted, activated with PMA/ionomycin, and examined for intracellular production of cytokines. (C) Tonsillar CD19⁺ B cells were sorted and cocultured for 11 d with sorted CD4 cells from the same tonsil before Ab production was analyzed by ELISA. CD4⁺CD45RO⁺CXCR5⁻, black bars; CD4⁺CD45RO⁺CXCR5⁺, white bars; no T cells, shaded bars. Similar results were obtained in a second experiment