Myeloid-derived suppressor cell development is regulated by a STAT/IRF-8 axis - PubMed (original) (raw)

Myeloid-derived suppressor cell development is regulated by a STAT/IRF-8 axis

Jeremy D Waight et al. J Clin Invest. 2013 Oct.

Abstract

Myeloid-derived suppressor cells (MDSCs) comprise immature myeloid populations produced in diverse pathologies, including neoplasia. Because MDSCs can impair antitumor immunity, these cells have emerged as a significant barrier to cancer therapy. Although much research has focused on how MDSCs promote tumor progression, it remains unclear how MDSCs develop and why the MDSC response is heavily granulocytic. Given that MDSCs are a manifestation of aberrant myelopoiesis, we hypothesized that MDSCs arise from perturbations in the regulation of interferon regulatory factor-8 (IRF-8), an integral transcriptional component of myeloid differentiation and lineage commitment. Overall, we demonstrated that (a) Irf8-deficient mice generated myeloid populations highly homologous to tumor-induced MDSCs with respect to phenotype, function, and gene expression profiles; (b) IRF-8 overexpression in mice attenuated MDSC accumulation and enhanced immunotherapeutic efficacy; (c) the MDSC-inducing factors G-CSF and GM-CSF facilitated IRF-8 downregulation via STAT3- and STAT5-dependent pathways; and (d) IRF-8 levels in MDSCs of breast cancer patients declined with increasing MDSC frequency, implicating IRF-8 as a negative regulator in human MDSC biology. Together, our results reveal a previously unrecognized role for IRF-8 expression in MDSC subset development, which may provide new avenues to target MDSCs in neoplasia.

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Figures

Figure 1

Figure 1. Irf8 expression is downregulated in tumor-induced MDSC subsets.

Irf8 mRNA levels in purified myeloid subsets from spleen of non-tumor-bearing or 4T1 (A) or AT-3 (B) tumor–bearing mice (≥1,000 mm3). The gating strategy for MDSC subset purification is shown in Supplemental Figure 1. Irf8 mRNA levels were measured through qPCR (n = 3 separate mice/group, *P < 0.01) and are representative of two separate experiments. For both panels, the data are expressed as the mean ± SEM of the fold changes relative to the first bar set at 1. (C) Upper panel: quantification of total splenocytes from Irf8–/– or WT littermate controls (8–10 weeks of age) (n = 5 each, *P < 0.01). Lower panel: CD11b and Gr-1 coexpression on splenocytes from Irf8–/– mice versus age-matched WT controls (n = 3 mice each, *P < 0.002). (D) Representative contour plot of Ly6C and Ly6G expression in the gated CD11b+ cells from WT and Irf8–/– mice in C. (E) Quantification of the percentages of granulocytic and monocytic subsets (*P < 0.003). (F) Total numbers of monocytic and granulocytic cells (*P < 0.001).

Figure 2

Figure 2. _Irf8_-deficient mice harbor MDSC-like cells.

(A) Ability of CD11b+Gr-1+ cells isolated from WT or Irf8–/– mice to inhibit anti-CD3–stimulated T cells at the indicated cell densities (n = 3 determinations, *P < 0.02; T cells plus anti-CD3 mAb control, average cpm, 127,151). (B) Ability of purified CD11b+Gr-1+ cells from Irf8–/– mice to suppress alloreactive (H-2b anti–H-2d) T cell proliferation relative to the control, incubated with CD11b+Gr-1+ cells from WT mice. The CD11b+Gr-1+ cells matched the haplotype of the responder T cells and were tested at the indicated lymphocyte to myeloid cell ratios. Proliferation in A and B was measured through 3H-thymidine uptake and is representative of 2 separate experiments. (C) AT-3 tumor cells were co-mixed with or without CD11b+Gr-1+ cells derived from Irf8–/– mice, and tumor growth was recorded. The results represent the mean ± SEM (n = 8 recipient mice/group pooled from 2 separate experiments; *P = 0.002). (D) AT-3 tumor growth rate in WT versus Irf8–/– mice (n = 5 recipient mice/group; P = 0.02).

Figure 3

Figure 3. The gene expression profile of CD11b+Gr-1hi cells from Irf8–/– mice resembles that of tumor-bearing mice.

(A) Mouse whole-genome microarray analysis using purified splenic CD11b+Gr-1hi cells from WT, Irf8–/–, and AT-3 tumor–bearing mice. Three sets of comparisons were made to obtain differentially expressed genes (>2-fold change; P < 0.01): _Irf8–/–_ (KO) versus WT; AT-3 versus WT; and _Irf8–/–_ versus AT-3. (**B**) Hierarchical clustering of genes down- (green) or upregulated (red) from each sample (>2-fold change; P < 0.01). Each group was set up in biological duplicates. (C) As in Figure 2C, except that AT-3 tumor cells were co-mixed with the purified granulocytic fraction from Irf8–/– mice. Purified CD11b+Gr-1+ cells from WT mice were included as a control. The results represent the mean ± SEM (n = 8 WT; n = 7 Irf8–/–; *P = 0.005).

Figure 4

Figure 4. MDSC accumulation is diminished in _Irf8_-Tg mice.

(A) Splenocyte counts (on the right y axis) from AT-3 tumor–bearing WT mice or _Irf8_-Tg mice of the indicated founder line. The data are shown for mice with larger tumor volumes (>1,200 mm3) to illustrate the impact of Irf8 under the most advanced tumor conditions. *P < 0.003. (**B**) Relationship between tumor size and splenocyte number for all mice, including those with smaller tumor volumes (<1,200 mm3) to generate a broader curve (Spearman _r_ value = 0.59; *_P_ = 0.008 for WT; not significant for either Tg line). Each symbol represents a single mouse. (**C**) Quantification of CD11b+Gr-1+ cells from bone marrow of WT or _Irf8_-Tg mice with (TB) or without (NTB) tumor growth (>1,200 mm3). (D) Representative contour plots of Ly6C and Ly6G expression on the CD11b-gated splenic fraction. (E) Upper panel: quantification of splenic granulocytic and monocytic subsets using a gating strategy similar to that described in Supplemental Figure 1 (n = 4 mice each; in the case the Tg group, 3 from line 370 and 1 from line 371). Lower panel: tumor sizes in individual mice. (F) Granulocytic (left y axis) and monocytic (right y axis) MDSC subsets plotted in relation to their respective tumor sizes from E. *P < 0.04 for WT subsets; not significant for Tg subsets. The data are reported as the mean ± SEM for the number of mice or experiments shown.

Figure 5

Figure 5. Intratumoral MDSC accumulation is also diminished in _Irf8_-Tg mice.

Flow analysis of the indicated myeloid populations from primary tumor tissue of WT or _Irf8_-Tg mice at endpoint, as shown in Figure 4, A or C. Total live cells were first gated on the CD45+ leukocyte fraction. The gated CD45+ fraction was then plotted in relation to the myeloid markers shown in a manner similar to that in Supplemental Figure 1. The data are illustrated for total MDSCs (A), MDSC subsets (B and C), macrophages (D), and total DCs (E). (F) Ability of CD11b+Gr-1+ cells from AT-3 tumor–bearing WT or AT-3 tumor–bearing _Irf8_-Tg mice to suppress T cell proliferation in response to immobilized anti-CD3 mAb. T cells (1 × 105/well) and CD11b+Gr-1+ cells (5 × 104/well) (n = 3 determinations; *P < 0.01; T cells + anti-CD3 mAb without any CD11b+Gr-1+ cells, 23,212 ± 1,865).

Figure 6

Figure 6. Influence of Irf8 enhancement on tumor growth during immunosurveillance or immunotherapy.

(A) Tumor growth rate of AT-3 cells in WT versus _Irf8_-Tg mice (n = 9 for WT and n = 10 for Tg line 370). (B) WT or Tg mice (line 370) were treated with either anti-Ly6G mAb or an isotype control after the tumors became palpable. The data are representative of 2 experiments (n = 10 mice/group). *P = 0.009 for differences in tumor growth rate between anti-Ly6G–treated _Irf8_-Tg mice versus all other cohorts. (C) As in B, except that the mice were treated with either anti–CTLA-4 mAb or an isotype control. The data are representative of 2 separate experiments (n = 5 mice/group). *P < 0.04 for differences in tumor sizes between anti–CTLA-4–treated and vehicle-treated _Irf8_-Tg mice at days 18, 21, 27, and 30; P < 0.05 at day 15. (D) Tumor growth rate of 4T1 cells in WT versus _Irf8_-Tg mice (Tg line 370). (E) Spontaneous lung metastasis was quantified at endpoint tumor volumes for mice in D (n = 12 WT; n = 14 Tg). (E) H&E-stained lung tissues were used to quantify metastasis. (F) Representative H&E-stained lung images (left; original magnification, ×20) and representative images of anti–Gr-1 staining (1 of 3 mice tested), analyzed through IHC (right; original magnification, ×400). The arrows indicate examples of discrete foci; Tu, tumor; Pa, parenchyma. H&E analysis comfirmed myeloid morphology. (G) Quantification of IHC data in F, based on the average number of stained cells per high-power field (×400) from 5 random sections of each slide. The data represent the mean ± SEM of 3 mice per group.

Figure 7

Figure 7. Irf8 enhancement slows autochthonous tumor growth.

(A) Tumor growth rate in double-Tg versus single-Tg mice, as determined after tracking the single largest tumor. Each symbol denotes a tumor measurement in a single mouse over time (n = 13 mice each; all _Irf8_-Tg mice from line 370). (B) Kaplan-Meier plot of the data in A, based on time to progression to approximately 50% of maximal tumor growth as a surrogate endpoint. (C) Each data point represents flow analysis of the indicated myeloid subset from single-Tg or double-Tg mice at endpoint. (D) Myeloid/tumor admixture experiments, as in Figure 2. AT-3 cells were mixed with splenic CD11b+Gr-1+ cells recovered from single-Tg, double-Tg, or non-tumor-bearing mice (WT), and tumor growth was recorded. The cells from single-Tg mice, but not those of double-Tg mice, showed a significant (*P < 0.001) increase in the AT-3 tumor growth rate relative to cells from WT mice (n = 5 mice/group; one of 2 separate experiments). The data are expressed as the mean ± SEM for the indicated number of mice. (E) Quantification of lung metastasis using histology (termed cohort 1) or CD11b+Gr-1+ cell frequency using flow cytometry analysis of total lung digests (termed cohort 2) from single-Tg or double-Tg mice. NTB, non-tumor-bearing WT mice. Both cohorts were matched based on age and primary tumor burden, as indicated in Results. (F) Representative H&E-stained images of metastatic foci in MTAG mice (upper left, original magnification, ×40; upper right, original magnification, ×100) and flow plots of CD11b+Gr-1+ cell populations (bottom panel).

Figure 8

Figure 8. IRF-8 selectively modulates CD11b+Gr-1+ MDSC frequency under autochthonous tumor growth conditions.

(A–E) Spearman correlation r values and P values for each genotype, reflecting the indicated comparisons. Each symbol represents a single mouse based on a new series of experiments. The percentages signify the fraction of each subset relative to the total splenocyte population, whereas the tumor volume denotes the summation of all tumors from an individual single- or double-Tg mouse at endpoint. (F) The endpoint data points in A, D, and E were converted to absolute cell counts (*P < 0.02; NS, not significant).

Figure 9

Figure 9. MDSC-associated myelopoietic growth factors inhibit Irf8 expression through STAT-dependent mechanisms.

(A) Irf8 mRNA levels of purified bone marrow–derived CD11b+Gr-1+ cells after treatment with G-CSF (50 ng/ml) (*P < 0.03; the data are representative of 3 separate experiments). As in B, except that the cells were incubated in the absence or presence of FLLL32 (2 μM for 24 hours) (C) Similar to A, except that Irf8 mRNA levels of purified subsets recovered after treatment of BALB/c mice with G-CSF (10 μg/day) or vehicle for 5 consecutive days (n = 3 mice each, *P < 0.001). (D) As in A, except that Irf8 mRNA levels of cells after treatment with GM-CSF (50 ng/ml) (*P < 0.05). (E) As in D, except that the cells were incubated in the absence or presence of pimozide (1 μM for 24 hours). (F) ChIP analysis of the Irf8 promoter in J774.2 cells in the absence (UT) or presence of G-CSF (upper and lower gels) or GM-CSF (lower gel) for 60 minutes to identify a “G-CSF/STAT3/IRF-8 axis” or a “GM-CSF/STAT5/IRF-8 axis” (1 of 3 separate experiments for each).

Figure 10

Figure 10. Relationship between MDSC frequency and IRF-8 expression.

(A) The percentage of CD33+HLA-DR– cells in the peripheral blood of stage III/IV breast cancer patients and matched healthy controls was determined using multicolor flow cytometry. (B) Quantification of the IRF-8 levels in A, based on the methods described in Methods and Supplemental Figure 6. Correlation between the frequencies of the cells in A versus the IRF-8 levels in B for the controls (C) or patients (D). In all panels, each data point represents an individual specimen. Kaplan-Meier plots for progression-free survival (E) or overall survival (F) of all patients stratified into 2 MDSC cohorts based on the MDSC load. The median was used to define high (n = 14) versus low (n = 16) patient subgroups.

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References

    1. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol. 2012;12(4):253–268. doi: 10.1038/nri3175. - DOI - PMC - PubMed
    1. Ostrand-Rosenberg S, Sinha P, Beury DW, Clements VK. Cross-talk between myeloid-derived suppressor cells (MDSC), macrophages, and dendritic cells enhances tumor-induced immune suppression. Semin Cancer Biol. 2012;22(4):275–281. doi: 10.1016/j.semcancer.2012.01.011. - DOI - PMC - PubMed
    1. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol. 2008;181(8):5791–5802. - PMC - PubMed
    1. Sawanobori Y, et al. Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice. Blood. 2008;111(12):5457–5466. doi: 10.1182/blood-2008-01-136895. - DOI - PubMed
    1. Sinha P, Chornoguz O, Clements VK, Artemenko KA, Zubarev RA, Ostrand-Rosenberg S. Myeloid-derived suppressor cells express the death receptor Fas and apoptose in response to T cell-expressed FasL. Blood. 2011;117(20):5381–5390. doi: 10.1182/blood-2010-11-321752. - DOI - PMC - PubMed

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