Invariant natural killer T cells in lupus patients promote IgG and IgG autoantibody production

Abstract

IgG autoantibodies, including antibodies to double-stranded DNA (dsDNA), are pathogenic in systemic lupus erythematosus (SLE), but the mechanisms controlling their production are not understood. To assess the role of invariant natural killer T (iNKT) cells in this process, we studied 44 lupus patients. We took advantage of the propensity of PBMCs from patients with active disease to spontaneously secrete IgG in vitro. Despite the rarity of iNKT cells in lupus blood (0.002-0.05% of CD3-positive T cells), antibody blockade of the conserved iNKT TCR or its ligand, CD1d, or selective depletion of iNKT cells, inhibited spontaneous secretion of total IgG and anti-dsDNA IgG by lupus PBMCs. Addition of anti-iNKT or anti-CD1d antibody to PBMC cultures also reduced the frequency of plasma cells, suggesting that lupus iNKT cells induce B-cell maturation. Like fresh iNKT cells, expanded iNKT-cell lines from lupus patients, but not healthy subjects, induced autologous B cells to secrete antibodies, including IgG anti-dsDNA. This activity was inhibited by anti-CD40L antibody, as well as anti-CD1d antibody, confirming a role for CD40L-CD40 and TCR-CD1d interactions in lupus iNKT-cell-mediated help. These results reveal a critical role for iNKT cells in B-cell maturation and autoantibody production in patients with lupus.

Keywords: Autoantibodies; B cells; IFN-γ; Lupus; iNKT cells.

Fig. 1. Spontaneous immunoglobulin secretion by SLE patient PBMCs correlates with disease activity

PBMCs from SLE patients (n=23) were cultured for 10 days in medium devoid of human serum, after which IgG was measured by ELISA in the culture supernatants. (A) Correlation between level of spontaneous IgG production and disease activity (SLEDAI score) was analyzed with the Spearman's rank correlation test (r_s=0.6022, P=0.0024). (B) Comparison of spontaneous IgG production between patients with inactive disease (SLEDAI<6, n=16) and active disease (SLEDAI ≥6, n=7) using the Mann-Whitney test (**, P<0.01). Horizontal lines represent mean ± SD. (C) Comparison of spontaneous IgG levels between patients treated with no or low dose prednisone treatment (<10 mg/day, n=16) and higher dose treatment (≥10 mg/day, n=7) using the Mann-Whitney test. Each data point represents one individual sample. * indicates P<0.05.

Fig. 2. Spontaneous immunoglobulin production by SLE PBMCs is dependent on iNKT cells

PBMCs from SLE patients were cultured for 10 days in the presence of various neutralizing antibodies or their isotypes, IgG (A and C, n=5), IgM and IgA (B, n=3) in culture supernatants was measured by ELISA (D) iNKT cells as a percentage of CD3⁺ T cells before and after anti-TCRVα24 depletion in one experiment representative of 5 performed. (E) iNKT cells were depleted from lupus PBMCs with anti-TCRVα24 antibody coated beads (TCRVα24DEP) and spontaneous IgG production by iNKT-depleted PBMCs was measured by ELISA (n=5). (F) B cell ELISPOT assay was used to detect anti-dsDNA IgG antibody forming cells (AFC) among SLE PBMCs in the presence or absence of the indicated antibody (n=4). The number of anti-dsDNA IgG AFC per 10⁶ PBMCs is shown. (G) Frequency of plasma cells in lupus PBMCs cultured for one week in the presence or absence of anti-iNKT or anti-CD1d antibody (n=3). (A, C and E) Data are shown as the mean ± SD of five samples pooled from five independent experiments. (B) Data are shown as the mean ±SD of three samples pooled from three independent experiments. (F) Data are shown as the mean ± SD of four samples pooled from four independent experiments. * indicates P<0.05, ** indicates P<0.01. (Paired t-test used).

Fig. 3. iNKT cells are rare in lupus patients but expand after αGalCer stimulation

(A) Frequency of iNKT cells and CD4/CD8 distribution on fresh iNKT cells in SLE patients and healthy individuals (n=13 for SLE patients, n=18 for healthy subjects). (B) Cytokine production by iNKT cells was analyzed by intracellular staining of PBMCs stimulated with αGalCer for 6 days (n=7 for healthy subjects, n=10 for SLE patients except for IL-10 where n=4 for SLE patients). (C) Total IgG in the supernatant of SLE PBMCs cultured in the presence of blocking antibody against IFNγ. Data are representative of 4 independent experiments. (D) Characterization of expanded iNKT cells (n=3) by staining with anti-TCRVα24/TCRVβ11, anti-iNKT (clone 6B11) and human CD1d-αGalCer tetramer. Data are representative of 3 independent experiments. (E) Expression of CD4 and CD8 on expanded iNKT cells from healthy subjects (n=5) and SLE patients (n=5). Data are shown as the mean ± SD of five samples pooled from five independent experiments. (F) IFNγ and IL-4 expression in activated iNKT cells. Left: Expanded iNKT cells stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4 hours were analyzed by FACS for intracellular IFNγ. Plots are representative of 5 independent experiments. Right: Expanded iNKT cells (5×10⁴ cells/well) were stimulated with plate-bound anti-CD3 mAb for 24 hours. IFNγ and IL-4 in the supernatants were measured by ELISA. The results represent mean values of triplicate cultures from 9 lupus patients and 5 healthy subjects. Each symbol represents one individual sample. (G) Expression of CXCR5 and PD-1 in expanded iNKT cells by flow cytometric analysis. Data shown are representative of 3 independent experiments with SLE patients and healthy subjects. * indicates P<0.05, ** indicates P<0.01. (Paired t-test used).

Fig. 4. Expanded iNKT cells from SLE patients, but not healthy subjects, can induce IgG production

(A) B cells were cultured for 10 days with expanded autologous iNKT cells from healthy subjects (n=5) or lupus patients (n=5) in the presence or absence of anti-CD1d or isotype control antibody. Immunoglobulin in the culture supernatants was measured by ELISA. (B) B cells from healthy subjects were co-cultured with autologous iNKT cells or iNKT cells from SLE patients for 10 days in the presence of anti-CD1d or isotype control antibody. Alternatively, B cells from SLE patients were co-cultured with autologous iNKT cells or iNKT cells from healthy donors. IgG in the supernatant was measured by ELISA. (C) B cells from SLE patients were stimulated with PWM and SAC in the presence or absence of anti-CD1d, anti-iNKT (6B11) or anti-iNKT TCR (TCRVα24/TCRVβ11) antibodies for 10 days. (D) To assess the ability of iNKT cells from lupus patients to induce Ig class switching, sorted IgM⁺CD27^-CD19⁺ naïve B cells were cultured with autologous iNKT cells for 10 days in the presence or absence of anti-CD1d antibody. Antibodies in the supernatant were measured by ELISA. Left panel shows total IgG and IgM, and right panel shows anti-dsDNA IgG. Antibodies in the supernatant were measured by ELISA. (A) Data are shown as mean ± SD of five samples pooled from five independent experiments. (B-D) Data are shown as mean ± SD of three samples pooled from three independent experiments. * indicates P<0.05, ** indicates P<0.01. (Paired t-test used).

Fig. 5

TCRVα24-TCRVβ11- conventional T cells from SLE patients fail to induce antibody production. Conventional CD4⁺ T cells were purified from two sources as described in Materials and Methods. These cells, or purified iNKT cells, were cultured with autologous B cells for 10 days in the presence or absence of anti-CD1d or isotype control antibody. The level of IgG, IgM, IgA and anti-dsDNA IgG immunoglobulins in the culture supernatants was determined by ELISA. Data are shown as mean ± SD of three samples pooled from three independent experiments. * indicates P<0.05. (Paired t-test used).

Fig. 6. Ability of expanded lupus iNKT cells to help B cells is dependent on CD40L-CD40 interaction

Purified expanded iNKT cells from lupus patients were cultured with autologous B cells for 10 days in the presence or absence of anti-CD40L, anti-CD1d or isotype control antibody. The level of IgG, IgM, IgA and anti-dsDNA IgG immunoglobulins in the culture supernatants was determined by ELISA. Data are shown as mean ± SD of three samples pooled from three independent experiments. * indicates P<0.05. (Paired t-test used).