Human CD57+ germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

Abstract

Background: The function of CD57+ CD4+ T cells, constituting a major subset of germinal center T (GC-Th) cells in human lymphoid tissues, has been unclear. There have been contradictory reports regarding the B cell helping function of CD57+ GC-Th cells in production of immunoglobulin (Ig). Furthermore, the cytokine and co-stimulation requirement for their helper activity remains largely unknown. To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57+ GC-Th cells in inducing B cell Ig synthesis.

Results: We demonstrated that CD57+ GC-Th cells are highly efficient in helping B cell production of all four subsets of Ig (IgM, IgG, IgA and IgE) compared to other T-helper cells located in germinal centers or interfollicular areas. CD57+ GC-Th cells were particularly more efficient than other T cells in helping GC-B cells but not naive B cells. CD57+ GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and class switch recombination in developing B cells. IgG1-3 and IgA1 were the major Ig isotypes induced by CD57+ GC-Th cells. CD40L, but not IL-4, IL-10 and IFN-gamma, was critical in CD57+ GC-Th cell-driven B cell production of Ig. However, IL-10, when added exogenously, significantly enhanced the helper activity of CD57+ GC-Th cells, while TGF-beta1 completely and IFN-gamma partially suppressed the CD57+ GC-Th cell-driven Ig production.

Conclusions: CD57+CD4+ T cells in the germinal centers of human lymphoid tissues are the major T helper cell subset for GC-B cells in Ig synthesis. Their helper activity is consistent with their capacity to induce AID and class switch recombination, and can be regulated by CD40L, IL-4, IL-10 and TGF-beta.

Figure 1

Distribution of CD4⁺ T helper cells in tonsils. Frozen tonsil sections were stained with anti-IgD (PE, red) or anti-CD57 (FITC, green) and isotype control antibodies in panel group A to show the background staining of the system. In panel group B, sections were stained with anti-CD57 (FITC), anti-IgD (PE) and anti-CD4 (APC). In panel group C, sections were stained with anti-CD57 (FITC), anti-CD69 (PE) and anti-CD4 (APC). Two different sections were shown in each group of panels. Stained sections were analyzed with a confocal microscope. GC-Th cells can be divided into CD57⁺ and CD57^- T cells, both of which are CD69⁺. A few CD69⁺ or CD57⁺ T cells are found outside of GC. Most CD4⁺ T cells in the interfollicular areas (IFA or T cell-rich zone) are CD57^- and CD69^-. GCs are surrounded by the ring of mantle zones (MZ) filled with IgD⁺ cells. A representative set of images from three different specimens are shown.

Figure 2

Isolation of human tonsil T cell and B cell subsets examined in this study. T cell subsets and B cell subsets were isolated from tonsils as described in the materials and methods section and were used in this study. The frequencies of the populations in total tonsil CD4⁺ T or CD19⁺ B population are 15–25% for CD4⁺CD57⁺ GC-Th cells, 50–60% for CD57^-CD69⁺ T cells, 20–30% for CD57^-CD69^- T cells, 40–60% for CD19⁺CD38^-IgD⁺ naïve B cells and 30–40% for CD19⁺CD38⁺IgD^- GC-B cells.

Figure 3

CD57⁺ GC-Th cells are more efficient than other tonsil CD4⁺ T cell subsets in helping B cells. (A) CD4⁺ T cell subsets were cultured with total tonsil CD19⁺ B cells for 7 days in the presence of SEB. Naïve B cells (C) or GC-B cells (D) were cultured with equal numbers of CD57⁺GC-Th cells or other T cell subsets (CD57^-, CD57^-CD69⁺ and CD57^-CD69^- T cells) for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 5 independent experiments were combined and the averages are shown with standard errors. Relative production levels to CD57⁺ GC-Th cells are shown. *Significant differences from CD57⁺ GC-Th cells. The absolute Ig production levels (ng/ml) in panel A (GC-Th + Total B cells) were 5737 ± 1764 (IgM), 2111 ± 1185 (IgG), 577 ± 186 (IgA), and 4.8 ± 2.1 (IgE). The absolute Ig production levels (ng/ml) in panel B (GC-Th cells + naïve B cells) were 2045 ± 697 (IgM), 63 ± 21 (IgG), 40 ± 23 (IgA), and 2.9 ± 1.2 (IgE). The average levels (ng/ml) of Ig produced in the cultures of GC-Th cells and GC B cells were 750 ± 279 (IgM), 175 ± 52 (IgG), 51 ± 13 (IgA), and 1.0 ± 0.5 (IgE). (D) Isotype composition of the Ig induced by CD57⁺ GC-Th cells. Naïve B cells or GC-B cells were cultured with equal numbers of CD57⁺GC-Th cells or CD57^-CD69⁺ T cells for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 4 independent experiments were combined and the averages are shown with standard errors. *Significant differences between naïve and GC-B cells, but not between the two T cell subsets, were observed.

Figure 4

CD57⁺ GC-Th cells have the capacities to induce AID expression and to support CSR in B cells. IgD⁺CD38^- naïve B cells were cultured with CD57⁺ GC-Th cells for indicated time periods followed by RT-PCR analysis for (A) AID expression and (B) CSR. The sizes of specific PCR products are 152 bp (IgM); 416 bp (IgG1, G2, G3), 904 bp (IgG4); 904 bp (IgA1); 891 bp (IgA2); and 179 bp (IgE). Shown are productive recombination products. (C) The expression kinetics of AID and productive IgG3 transcripts over an 8 day period are shown together in a graph. In this panel, normalized expression levels calculated after dividing the levels of AID amplification by β-actin levels are shown. The time gap to reach the peak levels of the expression between AID and productive IgG3 transcripts is shown by an arrow. Representative data from at least three independent experiments are shown (panels A and B). (D) Identification of extrachromosomal reciprocal DNA recombination products. Naïve B cells were cultured with CD57⁺ GC-Th cells for indicated time periods and were processed to isolate genomic DNA. Fresh GC-B cells were examined for positive controls. The switch circles were detected by a nested PCR method. Representative data out of three independent experiments are shown. (E) Detection of switch circles by a DC-PCR technique. Naive B cells, CD38⁺ GC-B cells and naïve B cells cultured with GC-Th cells for 5 days were examined for the presence of γ3 and α1/2 switch circles.

Figure 5

CD40L and cytokines in regulation of the helper activity of CD57⁺ GC-Th cells. (A and B) Effects of endogenous CD40L and cytokines on the helper activity of CD57⁺ GC-Th cells were determined. In cultures of CD57⁺ GC-Th cells with naïve or GC-B cells, neutralizing antibodies to IL-4, IL-10, IFN-γ or CD40L or control antibodies (mouse IgG1) were added. *Significant differences from the control group (control antibody). (C and D) Effects of exogenously added cytokines on the helper activity of CD57⁺ GC-Th cells were determined. To cultures of CD57⁺ GC-Th cells with naïve or GC-B cells, IL-4, IL-10, IFN-γ and TGF-β1 were added separately. Cells were cultured for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Relative Ig secretion levels (the medium control = 1) obtained from 9 independent experiments were combined, and averages and standard errors are shown. *Significant differences from the control group (medium).