Stimulation of T cell autoreactivity by anomalous expression of NKG2D and its MIC ligands in rheumatoid arthritis

Abstract

Effector T cell responses can be modulated by competing positive or negative signals transduced by natural killer (NK) cell receptors. This raises the possibility that dominant T cell stimulation might promote autoimmune reactions. In rheumatoid arthritis (RA), the severity of autoimmune and inflammatory joint disease correlates with large numbers of CD4+CD28- T cells, which are scarce in healthy individuals. For poorly defined reasons, these T cells are autoreactive, implying that they may contribute to disease manifestations. CD4+CD28- T cells in peripheral blood and synovial tissue of RA patients were found to express NKG2D, a costimulatory receptor that is absent on normal CD4 T cells. NKG2D was induced by tumor necrosis factor alpha and IL-15, which are abundant in inflamed synovia and RA patient sera. RA synoviocytes aberrantly expressed the stress-inducible MIC ligands of NKG2D, which stimulated autologous CD4+CD28- T cell cytokine and proliferative responses. Peripheral blood serum samples of RA patients contained substantial amounts of synoviocyte-derived soluble MICA, which failed to induce down-modulation of NKG2D because of the opposing activity of tumor necrosis factor alpha and IL-15. These results suggest that a profound dysregulation of NKG2D and its MIC ligands may cause autoreactive T cell stimulation, thus promoting the self-perpetuating pathology in RA and possibly other autoimmune diseases.

Fig. 1.

Preferential expression of NKG2D on the CD4⁺CD28^– T cell subset in RA patients. (A and B) Three-color flow cytometry shows that CD4⁺ T cells among normal PBL are negative for NKG2D, whereas significant proportions (25% in this representative sample) from RA patients are positive. (C and D) Among gated CD4⁺ T cells from RA PBL, NKG2D expression is preferentially associated with the CD28^– subset. The sample numbers tested are indicated in the text. (E and F) This association is confirmed with freshly isolated CD4⁺CD28^– synovial T lymphocytes (STL). The results shown are representative of five samples. Numbers in dot plots indicate percentages of cells in quadrants.

Fig. 2.

Induction of NKG2D on CD4 T cells by IL-15 and TNF-α. (A and B) Exposure of normal PBL to IL-15 or TNF-α results in induced expression of NKG2D on gated CD3⁺CD4⁺ T cells. (C) This induction is markedly accelerated with CD4⁺NKG2D^– T cells sorted from RA PBL. Data for TNF-α not shown. Numbers in dot plots indicate percentages of NKG2D positive and negative cells.

Fig. 3.

Cytokine-induced expression of NKG2D mRNA. Exposure of CD4⁺ T cells sorted from normal PBL for 6 days to IL-15 results in de novo induction of NKG2D mRNA. The NKL NK cell line serves as a positive control. This result confirms that the absence of NKG2D on uninduced CD4 T cells is not caused by a lack of its DAP10 partner protein, which is required for surface expression.

Fig. 4.

Expression of MIC and NKG2D on proliferating RA synoviocytes and lymphocytes, respectively. Micrographs of frozen sections from RA synovium stained for MIC (A and C) and NKG2D (B and D) by brown diaminobenzidine peroxidase substrate. Nuclei are counterstained with hematoxylin. A and B display parts of the synovial lining layer and underlying sublining areas. C and D show lymphocytic aggregates within sublining areas. (E) Two-color immunofluorescence staining of cell suspensions from RA synovium with anti-MIC and, after permeabilization, with anti Ki-67. (F) Identically treated cell suspensions from osteoarthritis synovium showed no MIC expression.

Fig. 5.

NKG2D-mediated costimulation and autoreactivity of CD4⁺CD28^–NKG2D⁺ T cells. (A) Immunofluorescence staining of the RA-2 synovial T lymphocyte (STL) clone 4. (B) Secretion of IFN-γ and TNF-α by RA-2-STL after stimulation with solid-phase anti-CD3 and anti-NKG2D mAbs. (C) Stimulation of T cell proliferation by anti-CD3 together with anti-NKG2D mAbs. (D) Secretion of IFN-γ upon stimulation with irradiated autologous but not allogeneic synoviocytes. T cell to stimulator cell ratios were 1:1.

Fig. 6.

Failure of soluble MIC in RA patient serum to down-modulate NKG2D due to the presence of TNF-α and IL-15. (Upper) Two-color staining of gated CD3⁺CD4⁺ T cells among RA PBL incubated with control serum or serum from a breast tumor patient containing soluble MIC (serum BT 450–85; ref. 17) in the absence or presence of anti-MIC mAb. (Lower) Incubation of RA PBL with autologous RA patient serum containing soluble MIC in the absence or presence of anti-TNF-α and anti-IL-15 mAbs. See the text for further explanations.