Tumor-derived macrophage migration inhibitory factor (MIF) inhibits T lymphocyte activation

Abstract

Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine that is considered a pro-inflammatory cytokine. However, our studies show that MIF, when produced in super-physiological levels by a murine neuroblastoma cell line (Neuro-2a) exceeding those normally seen during an immune response, inhibits cytokine-, CD3-, and allo-induced T-cell activation. MIF is also able to inhibit T cells that have already received an activation signal. The T-cell inhibitory effects of culture supernatants from neuroblastoma cells were reversed when the cells were transfected with dicer-generated si-RNA to MIF. When T cells were activated in vitro by co-culture with interleukin (IL)-2 and IL-15 and analyzed for cytokine production in the presence or absence of MIF-containing culture supernatant, inhibition of T-cell proliferation and induced cell death were observed even as the treated T cells produced high levels of interferon-gamma (IFN-gamma). The inhibitory effects of MIF were partially reversed when lymphocytes from IFN-gamma knockout mice were tested. We propose that the high levels of MIF produced by neuroblastoma cause activation induced T-cell death through an IFN-gamma pathway and may eliminate activated T cells from the tumor microenvironment and thus contribute to escape from immune surveillance.

Fig. 1

Loss of MIF expression by d-siRNA specific for MIF. (A) Cell lysates from AGN2a and AGN2a/CD80−137L cells that were untreated (lane 1), treated with transfection reagent alone (lane 2), transfected with d-siRNA specific for MIF (lane 3), or transfected with control d-siRNA specific for GFP (lane 4) using the GeneSilencer transfection system (described in Section 2) were prepared from cells collected 60−72 h after transfection and whole cell lysates fractionated by polyacrylamide gel electrophoresis. The electrophoresed proteins were transferred to membranes and detected with anti-MIF polyclonal antibody. (B) 2 × 10⁴ AGN2a cells treated with transfection reagent alone (mock), treated with MIF-siRNA (MIF-siRNA), treated with GFP-siRNA, or untreated, AN2a, were cultured in 1 ml of c-DMEM for 48 h, supernatant collected and MIF concentration determined by ELISA. Average concentration and standard deviation from triplicate wells is shown. Only MIF-siRNA treated tumor showed significantly lowered MIF production when compared to mock transfection (P < 0.05, Student's paired t-test, used in all figures upon F-test for variance unless otherwise noted). (C) To demonstrate functional MIF activity, peritoneal macrophages loaded in capillary tubes were cultured in media alone (cntrl) or in day 3 culture supernatants from AGN2a cells (AGN2a), AGN2a cells transfected with d-siRNA for MIF (+MIF siRNA), AGN2a cells transfected with d-siRNA for GFP (+GFP siRNA), or from AGN2a cells treated with the transfection reagents in the absence of added nucleic acid (T-cntrl). Following 24 h of incubation in these culture supernatants, macrophage-loaded capillary tubes were removed from triplicate tissue culture wells and cells that had migrated out of the capillary tubes counted. Data plotted in all figures is the average and standard deviation from triplicate assay wells unless otherwise noted. Only control and MIF siRNA-treated tumor differed significantly from mock-transfected tumor (P < 0.05). The results are representative of three experiments.

Fig. 2

Tumor-derived MIF directly inhibits immune activation of T cells. (A) Purified T cells from B6 mice, 1 × 10⁵, were incubated with 50 U/ml IL-2 and 12.5 ng/ml IL-15. [³H]Thymidine was added after 72−96 h of culture and the cells harvested after overnight thymidine incorporation. Incorporated average counts per minute (cpm) from triplicate wells (error bars indicate standard deviations) treated with cytokines only (cntrl) or wells containing dilutions of day 3 culture supernatants from non-transfected AGN2a (wild type), AGN2a transfected with d-siRNA for MIF (MIF siRNA), or AGN2a transfected with d-siRNA for GFP (GFP siRNA) are shown. Dilutions of the culture supernatants are indicated on the right of the figure. GFP and wild-type differed significantly from MIF si-RNA (P < 0.02) while the 1:100 dilution of MIF siRNA treated cell culture supernatant did not significantly differ from the control. (B) The amounts of MIF in culture supernatants from AGN2a (solid bars) and from U2OS/MIF cells were normalized by ELISA and then used in the same thymidine incorporation assay. Average thymidine incorporation and standard deviation of triplicate wells are shown, representative of three experiments. MIF from either source resulted in significant loss of proliferative activity (P < 0.01) at 10 pg/ml, while differences at 1 pg/ml were not statistically significant, demonstrating dose dependency.

Fig. 3

Anti-CD3 and allo-immune activation is blocked by MIF. (A) T cells were stimulated with immobilized anti-CD3 antibody in the presence of serial dilutions of culture supernatants (s/n) from U2OS/MIF cells (closed bars) or wild-type U2OS cells (open bars). The concentrations of MIF listed on the axis are taken from ELISA analysis of U2OS/MIF supernatants. The wild-type U2OS supernatants (open) represent equivalent dilutions of supernatant, not MIF concentration. Control (gray) did not contain tumor cell culture supernatant. Averages and standard deviations of triplicate wells are shown. Cells were cultured for 48 h, pulsed with [³H]thymidine and harvested on day 3. In this experiment only 400 and 40 pg/ml MIF differed significantly from control (P < 0.01). (B) Allogeneic T-cell proliferation was induced by co-culturing 1 × 10⁵ T cells from B6 mice with 1 × 10⁴ irradiated strain A-derived AGN2a/CD80−137L cells, AGN2a/CD80−137L transfected with d-siRNA for MIF (+MIF siRNA), AGN2a/CD80−137L transfected with d-siRNA for GFP (+GFP siRNA), or AG-N2a/CD80−137L treated with transfection reagents alone (T-cntrl). Thymidine incorporation by T cells cultured without irradiated tumor cells is also shown (T cells only). [³H]TdR was added 96 h after initiation of co-culture and triplicate wells harvested after overnight incubation. The data shown in each plot are representative of three independent experiments. In comparison to T cells cultured with AGN2a/CD80−137L cells, MIF siRNA treatment differed significantly (P < 0.02), while GFP siRNA or transfection control did not. T cells cultured alone did incorporate thymidine (P < 0.04 compared to AGN2a/CD80−137L) illustrating the inhibitory effects of tumor cells in the assay.

Fig. 4

MIF inhibits activated T cells. Splenic T lymphocytes were cultured for 72 h in IL-2 and IL-15, as before. U2OS/MIF (open) or AGN2a (filled) culture supernatant with MIF concentrations normalized by ELISA was then added on day 3, and the cultures re-fed with fresh IL-2 and IL-15 at this time. The cultures were also re-fed with fresh IL-2 and IL-15 at this time. [³H]thymidine was added on day 6 and cells harvested to determine thymidine uptake on day 7. Thymidine incorporation of cells cultured in cytokines and diluted U2OS wild-type supernatant or media (U2OS wt, hatched, cDMEM, gray) is also shown. Average counts from three wells and standard deviations are shown. In comparison to cDMEM control, significant differences were only seen for AGN2a-derived supernatant containing 10 pg/ml MIF (AGN2a P < 0.02, U2OS/MIF P = 0.058), while both supernatants differed significantly at 1000 pg/ml MIF (P < 0.01). Data are representative of two experiments.

Fig. 5

MIF inhibits the numeric expansion of cytokine activated T cells. (A) B6 T cells were cultured with IL-2 and IL-15 in the absence of culture supernatants (cDMEM, open circle), in the presence of U2OS/MIF supernatant diluted to 10 ng/ml or 10 pg/ml MIF as determined by ELISA (closed diamond and closed triangle, respectively), AGN2a supernatant diluted to 10 ng/ml or 10 pg/ml (open diamond and open triangle, respectively) or with wild-type U2OS supernatant at the same dilution as the U2OS/MIF supernatant (open square). Data shows total viable cells in culture as determined by vital dye exclusion. Values plotted are the average of triplicate wells, and standard deviations greater than 10% are shown. 10 ng/ml MIF from either source differed significantly from U2OS control on day 2 (P < 0.01), and at 10 ng/ml on day 4 (P < 0.01). (B) Cultured T cells were analyzed by flow cytometery upon exposure to culture supernatants. Open symbols show the percentage of cells at the indicated time points that stained positive with annexin V and the closed symbols show the percentage of cells that gated positive for both annexin V and propidium iodide (PI) staining. Cells were treated with 10 ng/ml MIF from AGN2a culture supernatant (circles), U2OS/MIF culture supernatant (triangles), or media alone (diamonds).

Fig. 6

Analysis of cytokines produced by MIF-treated T cells. B6 T cells were cultured in the presence of IL-2 and IL-15 alone (striped) or with control U2OS (open), U2OS/MIF (gray), or AGN2a (black) culture supernatants normalized to the indicated MIF concentrations by ELISA. After 72 h of T-cell culture, IFN-γ (panel A) and TNF-α (panel B) levels in the culture supernatants were determined using a flow cytometric bead array. A representative experiment of more than three independent experiments is shown.

Fig. 7

IFN-γ contributes to MIF effects on T cells. T cells from (A) wild-type (wt) or (B) IFN-γ knockout B6 mice were cultured with IL-2 and IL-15 as described previously. MIF, derived from culture supernatants of U2OS/MIF cells, was added at the indicated concentrations. Positive controls consisted of wells containing no tumor cell culture supernatant (cDMEM) or supernatants from the parental U2OS cell line at a dilution that matched the dilution of U2OS/MIF. [³H]Thymidine incorporation was assessed after 3 days in culture. Average counts from triplicate wells and standard deviations are shown. Results are representative of three experiments. In panel B, recombinant mouse IFN-γ was added at 1 μg/ml (filled) or not added (open) at the beginning of the assay as indicated.