Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys

Abstract

Double negative (DN) T cells are expanded in patients with systemic lupus erythematosus (SLE) and stimulate autoantibody production as efficiently as CD4(+) T cells. In this study, we demonstrate that DN T cells from patients with SLE produce significant amounts of IL-17 and IFN-gamma, and expand when stimulated in vitro with an anti-CD3 Ab in the presence of accessory cells. Furthermore, IL-17(+) and DN T cells are found in kidney biopsies of patients with lupus nephritis. Our findings establish that DN T cells produce the inflammatory cytokines IL-17 and IFN-gamma, and suggest that they contribute to the pathogenesis of kidney damage in patients with SLE.

FIGURE 1

An abnormally high fraction of T cells from patients with SLE produce IL-17. IL-17 expression was quantified in T cells, either immediately after isolation (A), or after 5-day culture in the absence (B) or presence (C) of plate-bound anti-CD3. At day 6, intracellular staining was performed. Cumulative data from 14 patients with SLE (black bars) and 12 healthy controls (white bars) is presented. *p<0.05. D. Freshly isolated T cells were treated with brefeldin A and stimulated for 3 hours with PMA and ionomycin before staining for flow cytometry. Representative dot plots (IL-17-Alexa fluor 647 vs. FSC; Total T cells, CD4+ T cells, DN T cells) from a healthy control and a patient with SLE are depicted. The gate was set according to an isotype control antibody. E. T cells stimulated during 5 days with plate-bound CD3 in the presence of accessory cells were harvested and stained for flow cytometry analysis. Representative histograms of a control and a SLE patient are shown. Dotted line represents isotype control staining.

FIGURE 2

Double negative cells expand significantly following anti-CD3 stimulation. T cells were incubated for 5 days with autologous accessory cells. TCRαβ⁺ cells were gated and single positive (CD4⁺CD8⁻; CD4⁻CD8⁺) and DN (CD4⁻CD8⁻) cells were quantified in 8 patients with SLE and 7 healthy controls. The percentage of DN (A) and CD4 (B) T cells from normal subjects and SLE patients in the absence (white bars) and presence (black bars) of plate-bound anti-CD3. Representative samples from a control and a patient are presented (C). * p < 0.05.

FIGURE 3

Double negative cells produce IFN-γ and TNF-α. T cells were stimulated with plate-bound anti-CD3 in the presence of accessory cells. At day 6, cytokine expression was determined by intracellular cytokine staining. Representative histograms comparing IL-2 (A), IFN-γ (B), and TNF-α (C) expression in DN (red lines) and CD4⁺ (blue lines) cells from control individuals (upper panels) and patients with SLE (lower panels). Shaded area represents isotype control. The black bar indicates the area considered positive. On the right panels, cumulative data from 6 patients and 6 controls is presented as mean + SEM. White bars represent CD4⁺ T cells; black bars DN T cells. * p < 0.05.

FIGURE 4

TCR γδ+ T cells and NKT cells are not involved in the increased IL-17 production of SLE patients. IL-17 expression was analyzed in recently isolated T cells. Live lymphocytes were gated according to FSC and SSC characteristics. Next, T cells were identified by the CD3 marker. TCR γδ and intracellular IL-17 expression was analyzed in the CD4⁻ CD8⁻ cell subset (DN T cells). Representative plots of a control and a patient (A), as well as cumulative data from 6 SLE patients and 4 controls (B) are shown (*p=0.05). C. Kidney sections from four SLE lupus nephritis patients were stained with mouse anti-human TCR γδ-FITC and rabbit anti-human IL-17 followed by goat anti-rabbit Texas Red. Slides were scanned in a confocal microscope and analyzed for the presence of TCR γδ+ cells. A representative field is shown. D. Expression of the NKT cell-associated TCR Vα24 was investigated in CD4⁺ and DN T cells from patients and controls (n=8). Although the frequency of the positive population varied among individuals, it remained confined to the CD4+ cell subset. Representative histograms of a control and a patient are shown. Shaded area indicates isotype control; blue line CD4+ T cells; red line DN T cells. The area considered positive is indicated by the black bar. E. Kidney sections from patients with lupus nephritis (n=4) were stained with mouse anti-human TCR Vα24 and rabbit anti-human CD3 followed by goat anti-mouse-Texas Red and goat anti-rabbit-FITC. Few NKT cells were observed. Shown is a representative field in which two NKT cells are marked with white circles.

FIGURE 5

IL-17 and IL-23 are detected in affected kidneys from patients with SLE. A. Frozen sections were incubated with either rabbit Ig (control; upper left panel), rabbit anti-human IL-17 (upper right panel), or rabbit anti-human IL-23 (lower panels). Goat anti-rabbit labeled with Alexa fluor 488 was used as secondary antibody. B. Sections were stained with rabbit anti-human IL-17 and mouse anti-human CD3. Goat-anti rabbit-Alexa fluor 488 and goat anti-mouse-Texas Red were used as secondary antibodies and DAPI was used for nuclear staining. White bar represents 5 μm.

FIGURE 6

DN cells are interspersed with CD4⁺ and CD8⁺ T cells in cellular infiltrates of SLE kidney biopsies. Frozen sections were stained with rabbit anti-human CD3 and goat anti-human CD4 mixed with goat anti-human CD8. Goat-anti rabbit-Alexa fluor 488 and goat anti-mouse-Texas Red were used as secondary antibodies and DAPI was used for nuclear staining (A). In B, arrows point to large (~15 μm) CD4/8⁺ CD3⁻ cells, most probably macrophages. Arrowheads indicate conventional single positive cells in which CD3 and either CD4 or CD8 colocalized. DN T cells are circled. White bar measures 50 μm.