Myeloid-Derived Suppressor Cell Survival and Function Are Regulated by the Transcription Factor Nrf2

Abstract

Tumor-induced myeloid-derived suppressor cells (MDSC) contribute to immune suppression in tumor-bearing individuals and are a major obstacle to effective immunotherapy. Reactive oxygen species (ROS) are one of the mechanisms used by MDSC to suppress T cell activation. Although ROS are toxic to most cells, MDSC survive despite their elevated content and release of ROS. NF erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates a battery of genes that attenuate oxidative stress. Therefore, we hypothesized that MDSC resistance to ROS may be regulated by Nrf2. To test this hypothesis, we used Nrf2(+/+)and Nrf2(-/-)BALB/c and C57BL/6 mice bearing 4T1 mammary carcinoma and MC38 colon carcinoma, respectively. Nrf2 enhanced MDSC suppressive activity by increasing MDSC production of H2O2, and it increased the quantity of tumor-infiltrating MDSC by reducing their oxidative stress and rate of apoptosis. Nrf2 did not affect circulating levels of MDSC in tumor-bearing mice because the decreased apoptotic rate of tumor-infiltrating MDSC was balanced by a decreased rate of differentiation from bone marrow progenitor cells. These results demonstrate that Nrf2 regulates the generation, survival, and suppressive potency of MDSC, and that a feedback homeostatic mechanism maintains a steady-state level of circulating MDSC in tumor-bearing individuals.

Figure 1. Nrf2 decreases survival time of tumor-bearing mice

Nrf2^+/+ and Nrf2^−/− mice on the BALB/c or C57BL/6 backgrounds were injected with 4T1 mammary carcinoma or MC38 colon carcinoma, respectively. Mice were followed weekly for primary tumor growth (A) and survival time (B). Tumor diameter was calculated as the average measurement of tumor length and width. Data were pooled from two independent experiments. Tumor growth and survival time were tested for statistical significance by Mann-Whitney and log-rank test, respectively.

Figure 2. Nrf2 enhances MDSC suppressive activity and the quantity of tumor-infiltrating MDSC

(A) Nrf2 enhances MDSC-mediated CD4⁺ T cell suppression. MDSC from the peripheral blood of 4T1-bearing BALB/c Nrf2^+/+ and Nrf2^−/− mice were assayed for their ability to suppress the antigen-activation of transgenic CD4⁺ (DO11.10) T cells. (B) Nrf2 enhances MDSC production of H₂O₂. MDSC from BALB/c Nrf2^+/+ and Nrf2^−/− mice with 4T1 tumors were incubated with Amplex Red reagent, stimulated with PMA, and assayed for H₂O₂ production over time. (C) Nrf2^+/+ and Nrf2^−/− MDSC suppress CD4⁺ T cell activation by producing arginase and H₂O₂. MDSC from the peripheral blood of 4T1-bearing BALB/c Nrf2^+/+ and Nrf2^−/− mice were assayed for their ability to suppress the antigen-activation of transgenic CD4⁺ (DO11.10) T cells in the presence of nor-NOHA, L-NMMA, catalase, and sodium pyruvate. (D) Nrf2 enhances the quantity of tumor-infiltrating MDSC. Each circle represents an individual mouse. Figures A, B, and D were analyzed by Students t test, Mann-Whitney test, and Wilcoxon-rank sign test, respectively. Figures A, B, and C represent one of two experiments, each with one Nrf2^+/+ and one Nrf2^−/− mouse per experiment. Data from figure D were pooled from 5 independent experiments; **p<.01, *p<.05.

Figure 3. Nrf2 decreases intracellular MDSC oxidative stress

(A) Circulating MDSC from tumor-bearing Nrf2-sufficient mice contain less intracellular ROS than MDSC from tumor-bearing Nrf2-deficient mice. Circulating MDSC were harvested from 4T1-bearing BALB/c Nrf2^+/+ and Nrf2^−/− mice, stained with DCFDA, and for Gr1 and CD11b. Gr1⁺CD11b⁺ cells were gated and analyzed by flow cytometry for DCFDA fluorescence. Left histogram: MDSC from representative individual Nrf2^+/+ and Nrf2^−/− mice; right graph: average MCF of DCFDA staining for MDSC from six Nrf2^+/+ and five Nrf2^−/− mice. (B) Nrf2 decreases intracellular ROS in MDSC differentiated in vitro from bone marrow progenitor cells. Bone marrow cells from tumor-free BALB/c Nrf2^+/+ or Nrf2^−/− mice were cultured under conditions favoring MDSC differentiation, and the resulting cells were stained with 7AAD and DCFDA, and for Gr1 and CD11b. 7AAD⁻ Gr1⁺CD11b⁺ cells were gated and analyzed by flow cytometry for DCFDA fluorescence. Left histogram: MDSC from representative individual Nrf2^+/+ and Nrf2^−/− mice; right graph: average MCF of DCFDA staining of MDSC from three Nrf2^+/+ and three Nrf2^−/− mice. Data were tested for statistical significance by Student’s t test.

Figure 4. Nrf2 protects MDSC from apoptosis

(A) Nrf2 decreases apoptosis in circulating MDSC of tumor-bearing mice. Circulating MDSC were harvested from 4T1-bearing BALB/c Nrf2^+/+ and Nrf2^−/− mice, and stained for Gr1, CD11b, and with Annexin V and propidium iodide (PI) or 7AAD, and analyzed by flow cytometry. Live Gr1⁺CD11b⁺ MDSC (PI⁻ or 7AAD⁻) were gated and assessed for Annexin V. Left panel: MDSC from representative individual mouse; right graph: average % annexin V⁺Gr1⁺CD11b⁺ MDSC from six Nrf2^+/+ and five Nrf2^−/− mice. (B) Nrf2 decreases apoptosis in MDSC differentiated in vitro from bone marrow progenitor cells. MDSC derived from bone marrow cell cultures were harvested, stained, and analyzed as in panel A. Averaged data are from one of three independent experiments with one Nrf2^+/+ and one Nrf2^−/− mouse per experiment. Data were tested for statistical significance using Student’s t test.

Figure 5. Nrf2 deficiency enhances MDSC proliferation

MDSC were differentiated in vitro from the bone marrow of tumor-free Nrf2^+/+ and Nrf2^−/− BALB/c and C57BL/6 mice. The resulting cells were harvested, counted, and stained for Ly6G, Ly6C, and CD11b, and analyzed by flow cytometry. PMN-MDSC and M-MDSC were identified as LyG⁺Ly6C^−/lowCD11b⁺ and Ly6G^−/lowLy6C⁺CD11b⁺ cells, respectively. (A) Top: Quantity of total cells, percent of cells that are MDSC, and absolute number of MDSC pre-culture and after in vitro differentiation (post-culture). Data are representative of one of three independent experiments with one Nrf2^+/+ and one Nrf2^−/− mouse per experiment. Bottom: Ratio of Nrf2^−/− to Nrf2^+/+ MDSC from the three independent experiments. A value >1 indicates that there is more proliferation in the absence of Nrf2. (B) Nrf2 deficiency preferentially enhances differentiation of PMN-MDSC from bone marrow progenitor cells. MDSC of panel A were gated and analyzed for PMN-MDSC and M-MDSC. Top: Representative staining of M-MDSC and PMN-MDSC from individual Nrf2^+/+ and Nrf2^−/− mice. Bottom: Average ratio of PMN-MDSC to M-MDSC from the three independent experiments.