A Subset of CXCR5+CD8+ T Cells in the Germinal Centers From Human Tonsils and Lymph Nodes Help B Cells Produce Immunoglobulins

Abstract

Recent studies indicated that CXCR5⁺CD8⁺ T cells in lymph nodes could eradicate virus-infected target cells. However, in the current study we found that a subset of CXCR5⁺CD8⁺ T cells in the germinal centers from human tonsils or lymph nodes are predominately memory cells that express CD45RO and CD27. The involvement of CXCR5⁺CD8⁺ T cells in humoral immune responses is suggested by their localization in B cell follicles and by the concomitant expression of costimulatory molecules, including CD40L and ICOS after activation. In addition, CXCR5⁺CD8⁺ memory T cells produced significantly higher levels of IL-21, IFN-γ, and IL-4 at mRNA and protein levels compared to CXCR5^-CD8⁺ memory T cells, but IL-21-expressing CXCR5⁺CD8⁺ T cells did not express Granzyme B and perforin. When cocultured with sorted B cells, sorted CXCR5⁺CD8⁺ T cells promoted the production of antibodies compared to sorted CXCR5^-CD8⁺ T cells. However, fixed CD8⁺ T cells failed to help B cells and the neutralyzing antibodies against IL-21 or CD40L inhibited the promoting effects of sorted CXCR5⁺CD8⁺ T cells on B cells for the production of antibodies. Finally, we found that in the germinal centers of lymph nodes from HIV-infected patients contained more CXCR5⁺CD8⁺ T cells compared to normal lymph nodes. Due to their versatile functional capacities, CXCR5⁺CD8⁺ T cells are promising candidate cells for immune therapies, particularly when CD4⁺ T cell help are limited.

Keywords: B cell; C-X-C chemokine receptor type 5; CD8 T cell; Follicular; Tfh-like cell.

Figure 1

CD8⁺ T cells localized in B cell follicles in tonsils and lymph nodes express CXCR5. The expression of CXCR5 on CD8 T cells in tonsils, lymph nodes and PBMCs was shown in the representative histogram graphs (A) and summary data (B, n = 8). Immunofluorescence staining of CD3⁺ T cells (green) and CD8⁺ T cells (red) (C, n = 5), CD8⁺ T cells (green) and CXCR5 (red) (D, n = 5) in paraffin-embedded tonsil tissues. Data are expressed as the mean ± SD, and compared with Mann-Whitney test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2

Expression of co-stimulated molecules on CXCR5⁺ CD8⁺ memory T cells from tonsils, lymph nodes and PBMCs. Upon stimulation with CD3/CD28 dynabeads for 8 h, the expression of CD40L and ICOS on CXCR5⁺ and CXCR5⁻ CD8⁺ memory T cells from tonsils, lymph nodes and PBMCs was detected by flow cytometry (A,B). The data are representative of thirteen or fifteen independent experiments, and were analyzed by two-tailed unpaired t-test (C). Error bar denote s.e.m. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance.

Figure 3

The expression of cytokines by CXCR5⁺ CD8⁺ memory T Cells from tonsils, lymph nodes and PBMCs. The mononuclear cells from tonsils, lymph nodes and blood were cultured with or without PMA plus ionomycin in the presence of BFA for 6 h. FACS analysis of IL-21, IL-4, and IFN-γ expression in CXCR5⁺ and CXCR5⁻ CD8⁺ memory T Cells (A–C). Data represent mean ± SD, and compared with two-tailed unpaired t-test (D, n = 11). *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance.

Figure 4

Sorted CXCR5⁺ CD8⁺ T cells expressed higher levels of cytokines at protein and mRNA levels than CXCR5⁻ CD8⁺ T cells. Tonsil CD8⁺ T cells were isolated using the appropriate microbeads, and the purity of the cells was ≥98%. CXCR5⁺ CD8⁺ and CXCR5⁻ CD8⁺ T cells from tonsil CD8⁺ T cells were further sorted by flow cytometry (A). Purified CD8⁺, sorted CXCR5⁺ CD8⁺ and CXCR5⁻ CD8⁺ T cells were stimulated with PMA and ionomycin. The supernatants from the cultures were analyzed by ELISA for the production of IL-21, IFN-γ, and IL-4 (B). The frequency of IL-21 and IFN-γ-producing cells was assessed by ELISPOT. The left panel shows representative counting of spot-forming cells (SFC) and the right panel shows the frequency of IL-21 and IFN-γ-producing cells as the mean with individual data points (C). FACS analysis of IL-21, and IFN-γ expression in CD8⁺, CXCR5⁺CD8⁺, CXCR5⁻CD8⁺ T cells (D). The levels of IL-21, IFN-γ and GAPDH mRNA in CD8⁺, CXCR5⁺CD8⁺, CXCR5⁻CD8⁺ T cells were determined by PCR (E), and the ratio of IL-21 or IFN-γ to GAPDH were quantified by densitometry (F). Statistical significance was compared with Mann–Whitney test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

CXCR5⁺ CD8⁺ T Cells provide help to B cells for the production of immunoglobulins. Physical contact between CD8⁺ T Cells (red) and B cells (green) in tonsil sections, and scale bars, 50μm. (A, n = 5). Sorted tonsil B cells and sorted CD8⁺ T Cells at the ratio of 1:1, 1:5, and 1:10 were co-cultured with or without α-CD3/CD28 dynabeads for 10 days (B,C). Sorted B cells were co-cultured with fresh CD8⁺ or fixed CD8⁺ T Cells, fresh CD4⁺ or fixed CD4⁺ T Cells at the ratio of 5:1 in the presence of α-CD3/CD28 dynabeads for 10 days (D). Sorted B cells and CD8⁺, CXCR5⁺CD8⁺, CXCR5⁻CD8⁺T Cells at the ratio of 5:1 were co-cultured with or without anti-IL-21 and anti-CD40L in the presence of α-CD3/CD28 dynabeads for 10 days (E). The supernatants from the different co-cultures were analyzed by ELISA for the production of IgG, IgM, and IgA. Data are expressed as the mean ± SD, and compared with Mann–Whitney test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance.

Figure 6

The lymph nodes from HIV patients had more CD8⁺CXCR5⁺ T cells than those from normal lymph nodes. Immunofluorescence of CD3⁺ T cells (green), CD8⁺ T cells (red) and CXCR5 (green) in the germinal centers of lymph node sections (scale bars, 50 μm) from normal individuals (A,C) and HIV patients (B,D). The summary data represented the distribution of CD3⁺CD8⁺ T cells and CD8⁺CXCR5⁺ T cells from normal and HIV patient's lymph nodes (E,F; n = 15), and were analyzed by Mann-Whitney test. ***P < 0.001.