CD1d-restricted help to B cells by human invariant natural killer T lymphocytes

Abstract

Invariant natural killer T (NKT) cells are a highly conserved subset of T lymphocytes expressing a semi-invariant T cell receptor (TCR), which is restricted to CD1d and specific for the glycosphingolipid antigen alpha-galactosylceramide. Their ability to secrete a variety of cytokines, which in turn modulate the activation of cells of both innate and acquired immune responses, suggests that invariant NKT cells exert a regulatory role mainly via indirect mechanisms. A relevant question is whether invariant NKT cells can directly help B cells. We document here that human invariant NKT cells are as efficient as conventional CD4+ Th0 lymphocytes in promoting proliferation of autologous memory and naive B lymphocytes in vitro, and in inducing immunoglobulin production. Help to B cells by invariant NKT cells is CD1d-dependent and delivered also in the absence of alpha-galactosylceramide, suggesting that NKT cells recognize an endogenous ligand presented by CD1d on B cells. The two major subsets of invariant NKT cells, CD4+ and double negative (CD4-CD8-), express comparable levels of CD40 ligand and cytokines, but differ in helper functions. Indeed, both subsets induce similar levels of B cell proliferation, whereas CD4+ NKT cells induce higher levels of immunoglobulin production. These results suggest a direct role for invariant NKT cells in regulating B lymphocyte proliferation and effector functions.

Figure 1.

Human CD4⁺ inv. NKT cell clones induce CD1d-dependent proliferation of naive and memory B lymphocytes in the presence and in the absence of α-GalCer. (A) Expression of CD1d and CD27 on freshly isolated B lymphocytes from one representative healthy donor out of three analyzed. (B) CFDA-SE-dilution on CD27⁺ and CD27⁻ B lymphocytes (CD20⁺CD3⁻) after 5 d in culture with the indicated reagents, alone (panel I) or together with CD4⁺ inv. NKT cells in the absence (panel II) or in the presence (panel III) of α-GalCer. Samples run in the presence of isotype-matched control mAbs gave no differences above medium. Results are from one experiment out of three, in which three CD4⁺ NKT cell clones were tested in parallel giving comparable results. For each sample, 3 × 10⁵ events in the lymphocyte region were acquired.

Figure 2.

Human CD4⁺ inv. NKT cell clones provide CD1d-dependent help to B lymphocytes for immunoglobulin production. IgM (A) and IgG1 (B) released by B lymphocytes cultured with irradiated CD4⁺ inv. NKT cell clones, the indicated stimuli and a neutralizing anti-CD1d or an isotype control mAb. Values are the mean ± SE from three independent experiments, each performed with three CD4⁺ inv. NKT cell clones. P values between anti-CD1d- and isotype control-treated samples are indicated above the lines.

Figure 3.

Human CD4⁺ and DN inv. NKT cell clones help similarly B cell proliferation, but only CD4+ NKT cell clones sustain immunoglobulin production. The expression of CD40L (A) and intracellular cytokines (B) was compared on conventional CD4⁺ Th0 cells, CD4⁺, and DN inv. NKT cells. For all samples, 10⁴ events in the lymphocyte region were acquired. Results are from one clone representative of six for each group. B lymphocytes proliferation (C), IgM (D), and IgG1 (E) production after stimulation by irradiated CD4⁺ T cells (gray bars), CD4⁺ (open bars), or DN (black bars) inv. NKT cells in the presence of the indicated stimuli (basal levels have been subtracted). Values are the mean ± SE from three independent experiments in which six clones for each group were tested. P values between samples activated with the three groups of clones are indicated.

Figure 4.

Human CD4⁺CCR7⁺CD62L⁺ inv. NKT cells expand in response to α-GalCer. CCR7 and CD62L (A) expression on CD4⁺Vα24⁺Vβ11⁺ and CD4⁻Vα24⁺Vβ11⁺ PBMCs, before and after 2 wk in culture with α-GalCer. Expression of CCR7 and CD62L on total CD4 lymphocytes is also shown. Expansion of inv. NKT cells (B) from the CD4⁺CCR7⁺CD62L⁺ subset sorted from PBMCs or tonsils and stimulated with α-GalCer and irradiated autologous B lymphocytes. Sorting was performed according to the indicated gates. For each sample 2 × 10⁵ to 2 × 10⁶ events in the lymphocyte region were acquired. Results are from one out of three representative donors.