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. 2015 Feb 3;4(5):e1003011. doi: 10.1080/2162402X.2014.1003011

Figure 2.

Figure 2.

For figure legend, see next page. Figure 2 (see previous page). IL-18 alone enhances IFNγ production by purified Vδ2neg γδ T cells in a TCR-dependent manner. Human Vδ2neg γδ T-cell clones including (A) Vγ4Vδ5 or (B) Vγ8Vδ3 purified from human PBMCs were cultured through polyclonal activation and incubated with various concentrations of anti-CD3 in the presence or absence of IL-18 or IL-1β for 24 h at 37°C; then, IFNγ secretion was measured by ELISA from cell culture supernatants (mean ± SD; n = 3). The same procedure was applied to purified Vδ2neg γδ T polyclonal cell lines isolated from kidney transplant patients, including (C) Vδ1+Vδ3, or (D) Vδ1, or (E) Vδ5 γδ T cells. Polyclonal cell lines were incubated with various concentrations of γδ TCR agonists (anti-Vδ1, or anti-Vδ3, or anti-CD3 antibodies) in the presence or absence of either IL-18 or IL-1β for 24 h at 37°C. Anti-CD57 was used as negative control for TCR stimulation. IFNγ secretion was then measured by ELISA from cell culture supernatants (mean ± SD; n = 3). (F) Vδ2 γδ T polyclonal cell lines were isolated from PBMCs of 2 healthy donors and cultured through polyclonal activation using 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and IL-2, then incubated with various concentrations of γδ TCR agonist anti-Vδ2 in the presence or absence of either IL-18 or IL-1β for 24 h at 37°C. IFNγ secretion was then measured by ELISA from cell culture supernatants (mean ± SD; n = 3). (G) A Vδ1 γδ T polyclonal cell line was treated with anti-CD3 in the presence or absence of various cytokines alone or in combination for 24 h at 37°C. IFNγ secretion was then measured by ELISA from cell culture supernatants (mean ± SD; n = 3). (H) A Vδ1 γδ T polyclonal cell line treated with anti-CD3 and cell culture supernatant was used to monitor IFNγ, IL-18, or IL-12 secretion by ELISA. LPS/ATP-treated THP-1 cells were used as positive control for cytokine secretion (mean ± SD; n = 3). (I) The various Vδ2neg γδ T-cell clone and polyclonal cell line responses to IL-18 are represented as fold induction of IFNγ secretion by IL-18 (IL-18 + TCR agonist/TCR agonist).