Expression of macrophage migration inhibitory factor by neuroblastoma leads to the inhibition of antitumor T cell reactivity in vivo

Abstract

Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8(+) and CD4(+) T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8(+) T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8(+) T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.

FIGURE 1

Generation of MIF knockdown (MIFKD) AGN2a cells using shRNA. MIF knockdown AGN2a cells (MIFKD-AGN2a) and AGN2a cells transduced with a non-functional shRNA construct (Control) were analyzed for MIF mRNA expression by real-time PCR (C), and the results are expressed as fold decrease in expression versus expression in wild-type AGN2a cells (see Materials and Methods for details). Culture supernatants from wild-type AGN2a cells (AGN2a), MIF knockdown AGN2a cells (MIFKD-AGN2a), and the control-transduced AGN2a cells were analyzed for MIF protein content in western blot assays (B) and ELISA assays (C). The data are from one of three replicate experiments. ** p<0.01.

FIGURE 2

Immunofluorescent analysis of MIFKD AGN2a cells. AGN2a (A) and MIFKD AGN2a (B) were incubated with anti-MIF antibody followed by secondary alexa fluor 568-conjugated antibody. Panel C contains AGN2a cells stained with IgG control antibody. The results are from one of two replicate experiments.

FIGURE 3

In vitro growth rate of MIF knockdown AGN2a cells. 10⁴ AGN2a cells (Wild-type, Control, or MIFKD) were seeded in culture. At the indicated times, the cultures were assessed for absolute numbers of viable cells. The data are from one of three replicate experiments. ** p<0.01.

FIGURE 4

Survival of mice inoculated with wild-type AGN2a, control-transduced AGN2a, or MIF knockdown AGN2a cells. A/J mice were inoculated subcutaneously with 10⁴ (A), 5×10⁴ (B), or 10⁶ (C) wild-type AGN2a (AGN2a), control siRNA (Control AGN2a), or MIFKD AGN2a cells and followed for survival. Mice were considered moribund and euthanized when tumor size exceeded 250 mm2. The data are the combined results of 2–3 experiments (n=10–20 total mice per group).

FIGURE 5

Survival of T cell-depleted mice inoculated with wild-type AGN2a or MIF knockdownAGN2a cells. The T cell-depleted mice were inoculated subcutaneously with 10⁴ (A), 5×10⁴ (B), or 10⁶(C) AGN2a or MIFKD AGN2a cells and followed for survival. Mice were considered moribund and euthanized when tumor size exceeded 250 mm2. The data are the combined results of two experiments in panel A (n=10 mice/group), and from one experiment in panels B and C (n=5 mice/group).

FIGURE 6

Immunization of mice with MIF knockdown AGN2a cells increased the survival of tumor challenged mice and increased CD8 tumor immunity. As depicted in the experimental design (A), A/J mice were given 2 weekly subcutaneous immunizations (vaccine) with irradiated wild-type or MIFKD AGN2a tumor cells. One week after the second immunization, the mice were challenged subcutaneously with 5×10⁴ (B) or 10⁵ (C) viable AGN2a cells. Mice were considered moribund and euthanized when tumor size exceeded 250 mm2. Five days after the second immunization, some mice (n=4 mice/group) were euthanized and draining lymph nodes (dLNs) and spleens harvested, cells from each tissue pooled, and CD8⁺ cells isolated by immunomagnetic sorting. The CD8⁺ cells were tested for reactivity againstAGN2a cells in IFN-γ ELISPOT assays (D). The survival curves represent are the combined results of two experiments (n=10 mice/group), and the ELISPOT results are from one of three replicate experiments. ** p<0.01.

FIGURE 7

Immune cells captured at the site of tumor cell inoculation through the use of a collagen matrix (Matrigel). A/J mice were inoculated subcutaneously with irradiated wild-type AGN2a, MIFKD AGN2a, CD80⁺CD13L⁺ AGN2a (AGN2a-80/137L), or MIFKD AGN2a-80/137L cells suspended in Matrigel. Five days after inoculation, the Matrigel plugs were harvested, processed, and the cells counted. In panels A (AGN2a) and C (AGN2a-80/137L) immune cell numbers were assessed by flow cytometry using CD45 to distinguish infiltrating immune cells from residual tumor cells. In panels B (AGN2a) and D (AGN2a-80/137L) CD8⁺ cells were isolated by immunomagnetic sorting and tested for reactivity against AGN2a cells in IFN-γ ELISPOT assays. The results in panels A and C are the combined data of two independent experiments, and the data in panels B and D are from one of two replicate experiments.**p<0.01.

FIGURE 8

Influence of tumor-derived MIF on annexin V binding to tumor-infiltrating T cells. A/J micewere subcutaneously vaccinated with irradiated CD80⁺CD13L⁺ AGN2a (AGN2a-80/137L) cells. Seven days later, the mice were subcutaneously inoculated with 300 μl of Matrigel containing viable AGN2a or MIFKD AGN2a cells (top histograms in A, B and C), or viable AGN2a-80/137L or MIFKD AGN2a-80/137L cells (bottom histograms in A, B and C). Five days after tumor cell/Matrigel inoculation, the Matrigel plugs (i.e., tumors) and spleens were harvested and processed to obtain single cell suspensions. The tumor-infiltrating cells and splenocytes were stained with combinations of cells surface markers, annexin V and propidium iodide (PI), and the cells were analyzed by flow cytometry. Histograms depicting annexin V staining of PI-negative cells are shown for gated CD3⁺ (A), CD8⁺ (B) and CD4⁺ (C) cells. Each histogram is from the pooled cells of three mice, and the results are representative of three independent experiments.

FIGURE 9

Neutralizing MIF-specific antibody failed to reverse suppressed T cell proliferation induced by AGN2a culture supernatants. Purified A/J T cells were plated at a density of 25,000 cells per well and activated by addition of anti-CD3/28-conjugated beads (75,000/well). AGN2a cell culture supernatants were diluted with media and added to each well at the indicated final dilutions. For supernatants treatedwith MIF-specific antibody or mouse IgG₁ control antibody, supernatants were pre-incubated with antibody at 100μg/ml for 2 hours at room temperature and then added to the T cells. After 72 hours, the cultures were pulsed for 4 hr with ³H-thymidine. The data is shown as mean cpm ± standard deviation of triplicate wells, and the results are representative of 3 separate experiments.