Murine DX5+NKT Cells Display Their Cytotoxic and Proapoptotic Potentials against Colitis-Inducing CD4+CD62Lhigh T Cells through Fas Ligand

Abstract

Introduction: It has been previously shown that immunoregulatory DX5⁺NKT cells are able to prevent colitis induced by CD4⁺CD62L^high T lymphocytes in a SCID mouse model. The aim of this study was to further investigate the underlying mechanism in vitro.

Methods: CD4⁺CD62L^high and DX5⁺NKT cells from the spleen of Balb/c mice were isolated first by MACS, followed by FACS sorting and cocultured for up to 96 h. After polyclonal stimulation with anti-CD3, anti-CD28, and IL-2, proliferation of CD4⁺CD62L^high cells was assessed using a CFSE assay and activity of proapoptotic caspase-3 was determined by intracellular staining and flow cytometry. Extrinsic apoptotic pathway was blocked using an unconjugated antibody against FasL, and activation of caspase-3 was measured.

Results: As previously shown in vivo, DX5⁺NKT cells inhibit proliferation of CD4⁺CD62L^high cells in vitro after 96 h coculture compared to a CD4⁺CD62L^high monoculture (proliferation index: 1.39 ± 0.07 vs. 1.76 ± 0.12; P = 0.0079). The antiproliferative effect of DX5⁺NKT cells was likely due to an induction of apoptosis in CD4⁺CD62L^high cells as evidenced by increased activation of the proapoptotic caspase-3 after 48 h (38 ± 3% vs. 28 ± 3%; P = 0.0451). Furthermore, DX5⁺NKT cells after polyclonal stimulation showed an upregulation of FasL on their cell surface (15 ± 2% vs. 2 ± 1%; P = 0.0286). Finally, FasL was blocked on DX5⁺NKT cells, and therefore, the extrinsic apoptotic pathway abrogated the activation of caspase-3 in CD4⁺CD62L^high cells.

Conclusion: Collectively, these data confirmed that DX5⁺NKT cells inhibit proliferation of colitis-inducing CD4⁺CD62L^high cells by induction of apoptosis. Furthermore, DX5⁺NKT cells likely mediate their cytotoxic and proapoptotic potentials via FasL, confirming recent reports about iNKT cells. Further studies will be necessary to evaluate the therapeutical potential of these immunoregulatory cells in patients with colitis.

Figure 1

Flow cytometry analysis of the spleen CD3⁺DX5⁺NKT, CD8⁺ T, CD4⁺CD62L^high, and CD4⁺CD62L_low cells of Balb/c mice after separation by MACS and FACS sorting (a). Proliferation (b) and proliferation index (c) of CFSE-labeled CD4⁺CD62L^high cells after 48 h and 96 h of monoculture or coculture with CD8⁺ T cells or CD3⁺ DX5⁺NKT. Results are given as mean + SEM. Experiments were repeated at least three times (^∗P < 0.05).

Figure 2

Intracellular flow cytometry detection of IFN-γ in CD4⁺CD62L^high (a) and CD4⁺CD62L_low cells (b) after 4 h and 10 h of monoculture or coculture with CD8⁺ T cells or CD3⁺DX5⁺NKT. Results are given as mean + SEM. Experiments were repeated at least three times (^∗P < 0.05).

Figure 3

Intracellular flow cytometry analysis of caspase-3 in CD4⁺CD62L^high cells after 10 h and 48 h of monoculture or coculture with CD8⁺ T or CD3⁺DX5⁺NKT cells. Results are given as mean + SEM. Experiments were repeated at least three times (^∗P < 0.05).

Figure 4

Flow cytometry analysis FasL expression of CD8 T and CD3⁺DX5⁺NKT cells after 4 h and 10 h monoculture or coculture with CD4⁺CD62L^high cells. Results are given as mean + SEM. Experiments were repeated at least three times (^∗P < 0.05).

Figure 5

Intracellular flow cytometry analysis of caspase-3 in CD4⁺CD62L^high cells after 48 h of coculture with CD3⁺DX5⁺NKT cells and pretreatment with either FasL block or isotype control. Results are given as mean + SEM. Experiments were repeated at least three times (^∗P < 0.05).