Freshly isolated Valpha24+ CD4+ invariant natural killer T cells activated by alpha-galactosylceramide-pulsed B cells promote both IgG and IgE production

Abstract

CD1d-restricted invariant natural killer T (iNK T) cells activated by their experimental ligand alpha-galactosylceramide (alpha-GC) can produce both T helper 1 (Th1) and Th2 cytokines and display regulatory functions. Recent studies identified CD4(+) and CD4(-) CD8(-) double-negative (DN) iNK T cells as the two major components of the human population and suggest that they display a Th2 and a Th1 profile, respectively. We compared the Th2-promoting activity of freshly isolated human CD4(+) and DN iNK T cells in terms of their capacity to induce Ig production by autologous B cells. Secretion of IgG and IgE but not IgM was enhanced by the CD4(+) T cell subset (including iNK T cells) but not by its DN counterpart. iNK T cells were directly responsible for this pro-Th2 effect, as demonstrated by the requirement for both alpha-GC stimulation and CD1d presentation, as well as by its disappearance upon iNK T cell depletion. Interaction with iNK T cells led to progressive accumulation of isotype-switched and activated B cells. Myeloid dendritic cells (DC) completely block the induction of Ig production in co-culture. This dominant inhibitory effect of myeloid DC was concomitant with a specific loss of interleukin (IL)-4 production by CD4(+) iNK T but not by conventional T cells. These data support the conclusion that, conversely to the interferon (IFN)-gamma-producing DN human iNK T cell population, interleukin (IL)-4-producing CD4(+) iNK T cells can activate and help B cells to produce both IgG and IgE through a CD1d-dependent mechanism, in keeping with a functional Th1/Th2 dichotomy between these subsets.

Fig. 1

IgE and IgG production by α-galactosylceramide (α-GC)-loaded B cells is enhanced by CD4⁺ T lymphocytes but not by their double-negative (DN) counterpart. Purified CD4⁺ or DN lymphocyte fractions (15 × 10⁴ cells) were co-cultured in medium containing interleukin (IL)-2 with 5 × 10⁴ B cells either non-loaded or loaded with α-GC. (a) Similar fold expansion of DN invariant natural killer T (iNK T) cells and CD4⁺ iNK T cells in response to α-GC-loaded B cells. Expansion was calculated after a 7-day culture by dividing the number of Vα24-positive T cells in co-cultures with α-GC-loaded B cells by their number in co-cultures with non-loaded B cells. The total number of iNK T cells recovered ranged from 0·75 × 10³−25·7 × 10³ cells/well for CD4⁺ fraction (n = 4) and from 0·8 × 10³ to 17·5 × 10³ cells/well for their DN counterpart (n = 4), depending on their frequency in fresh peripheral blood lymphocytes (which varied considerably between individuals). Data are mean values ± s.e.m. of experiments performed with PBL obtained from four healthy donors. (b) CD4⁺ lymphocytes promote Ig production by α-GC-loaded B cells. Concentrations of IgM, IgG and IgE in supernatants were determined after a 12-day culture. Data are from experiments performed with cells from four to six healthy donors, each symbol corresponding to one donor; significant differences (P < 0·02 and P < 0·03, respectively, according to Wilcoxon's test) were found for IgE and IgG only. (c) Comparison of IgE levels in supernatants of B cells co-cultured with CD4⁺ or DN cells in the presence or absence of α-GC. Data are means ± s.e.m. from one typical experiment of three.

Fig. 2

Promotion of IgE and IgG production by CD4⁺ T lymphocytes in response to α-galactosylceramide (α-GC)-loaded B cells is mediated by invariant natural killer T (iNK T) cells. Purified CD4⁺ lymphocyte fractions (15 × 10⁴ cells) were co-cultured in interleukin (IL)-2-containing medium with 5 × 10⁴ α-GC-loaded B cells (a,b) or with 5 × 10⁴ B cells and the TSST1 superantigen (c). Supernatant concentrations of IgG and IgE were determined as above. Data are means ± s.e.m. from four to six wells (one typical experiment of three). (a) CD1d-dependence of the CD4⁺/α-GC-loaded B cell cooperation in Ig production. Blocking anti-CD1d monoclonal antibody (20 µg/ml) was added or not to α-GC-loaded B cells before co-culture. (b) Vα24-depleted CD4⁺ lymphocytes fail to help B cells to produce Ig. Depletion of Vα24⁺ cells from CD4⁺ cells was achieved by magnetic cell sorting. (c) CD4⁺ lymphocytes activated by the TSST1 superantigen do not help B cells to produce Ig.

Fig. 3

CD4⁺ invariant natural killer T (iNK T) lymphocytes enhance the proliferation of the memory B cell compartment. Purified 5(6)-carboxyfluoresceine diacetate succinimidyl ester (CFSE)-labelled CD19⁺ B cells were preincubated with α-galactosylceramide (α-GC) and co-cultured with CD4⁺ or iNK T-depleted CD4⁺ cells for 8–10 days. Data are means ± s.e.m. from four experiments. (a) Frequency of CD27⁺ cells among CD19⁺ lymphocytes before and after culture. The increase of CD27⁺ cell frequency along culture depends on CD4⁺ iNK T cells. (b, c) Proliferation of memory B cells depends on CD4⁺ iNK T cells. Proliferation of gated CD19⁺ B cells was analysed on day 10 according to CD27 expression and to dilution of CFSE staining. Dot plots show percentages of cells in each quadrant, and absolute number of dividing memory (CD27⁺) cells (CFSE dilution ≥ 1 division) are shown between brackets.

Fig. 4

Comparison of interleukin (IL)-4 and interferon (IFN)-γ expression by CD4⁺ and double-negative (DN) freshly drawn invariant natural killer T (iNK T) cells. Cells were prepared and stimulated as described in the Materials and methods section and analysed by flow cytometry. (a) A representative experiment; (b) data from five experiments. Differences for IL-4 expression between CD4⁺ and DN iNK T cells were significant by the paired t-test. (P < 0·006). There was no significant difference in terms of IFN-γ production. No cytokines were detected in unstimulated cells.

Fig. 5

Myeloid dendritic cells (DC) block Ig production promoted by CD4⁺ invariant natural killer T (iNK T) cells. CD4⁺ T lymphocyte fractions (15 × 10⁴ cells) were co-cultured in medium containing interleukin (IL)-2 with 5 × 10⁴ α-galactosylceramide (α-GC)-loaded B cells in the presence or not of 5 × 10⁴ α-GC-loaded myeloid DC. (a) Myeloid DC block the CD4⁺ cell-induced Ig production of α-GC-loaded B cells. Supernatant concentrations of IgG and IgE were determined on day 12 of co-culture. Data are arithmetic means from three separate experiments. (b,c,d) Myeloid DC modify the pattern of interferon (IFN)-γ and IL-4 production by CD4⁺ iNK T and conventional CD4⁺ T cells (Vα24^–) co-cultured with α-GC-loaded B cells. After 10–13 days of culture, iNK T cells and conventional T cells were analysed for intracytoplasmic IL-4 and IFN-γ expression as described in the Materials and methods section. Dot plots show percentages of cells of either population that produced the relevant cytokine. Data are from experiments performed with cells from six healthy donors; significant differences were found for IL-4 and IFN-γ expression by CD4⁺ iNK T cells (P < 0·02 and P < 0·03, respectively) and IFN-γ expression by conventional T cells (P < 0·002), according to paired t-tests.