HMGB1 enhances immune suppression by facilitating the differentiation and suppressive activity of myeloid-derived suppressor cells

Abstract

Chronic inflammation often precedes malignant transformation and later drives tumor progression. Likewise, subversion of the immune system plays a role in tumor progression, with tumoral immune escape now well recognized as a crucial hallmark of cancer. Myeloid-derived suppressor cells (MDSC) are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. Thus, MDSCs may define an element of the pathogenic inflammatory processes that drives immune escape. The secreted alarmin HMGB1 is a proinflammatory partner, inducer, and chaperone for many proinflammatory molecules that MDSCs develop. Therefore, in this study, we examined HMGB1 as a potential regulator of MDSCs. In murine tumor systems, HMGB1 was ubiquitous in the tumor microenvironment, activating the NF-κB signal transduction pathway in MDSCs and regulating their quantity and quality. We found that HMGB1 promotes the development of MDSCs from bone marrow progenitor cells, contributing to their ability to suppress antigen-driven activation of CD4(+) and CD8(+) T cells. Furthermore, HMGB1 increased MDSC-mediated production of IL-10, enhanced crosstalk between MDSCs and macrophages, and facilitated the ability of MDSCs to downregulate expression of the T-cell homing receptor L-selectin. Overall, our results revealed a pivotal role for HMGB1 in the development and cancerous contributions of MDSCs.

Figure 1. HMGB1 is ubiquitously present in the tumor microenvironment, is secreted by MDSC, and activates the NF-κB signaling pathway in MDSC

A, 4T1, CT26, B78H1, AT3 and MC38 tumor cells were cultured in serum-free medium and their supernatants assessed by western blot for secreted HMGB1. Lysates of wild type and HMGB1-knocked-out MEF cells served as positive and negative controls. B, BALB/c 4T1-induced Gr1⁺CD11b⁺ MDSC were obtained from the blood of tumor-bearing mice, stained for Gr1 and CD11b to assess purity, and co-cultured with transgenic CD4⁺ (DO11.10) or CD8⁺ (clone4) splenocytes. Splenocytes were activated with OVA or HA peptide for DO11.10 and clone4 cells, respectively. T cell proliferation was measured by [³H] thymidine incorporation. Data are expressed as cpm of triplicate cultures. C, LPS-treated and untreated macrophages and MDSC, and excised, dissociated tumors of BALB/c (4T1, CT26) and C57BL/6 (B78HI, AT3, MC38) mice were cultured overnight in serum-free medium. Resulting supernatants were assessed by western blot for HMGB1. D, Leukocytes from tumor-free BALB/c mice were treated with or without HMGB1 and stained for Gr1, CD11b, and pNF-κB. Gr1⁺CD11b⁺ cells were gated and analyzed for pNF-κB. Data are from one of three, two, three, and three independent experiments for panels A, B, C, and D, respectively.

Figure 2. HMGB1 drives the differentiation of MDSC from bone marrow progenitor cells

Bone marrow cells were harvested from the femurs of healthy BALB/c mice and cultured with IL-6 and GM-CSF with or without ethyl pyruvate, glycyrrhizin, or vehicle. After four days of culture, the absolute number of MDSC was determined. A, HMGB1 western blot of supernatants of bone marrow cultures. B, Gating logic of Gr1^midCD11b⁺ MDSC from pre-cultured and post-cultured bone marrow cells. C, Absolute number of Gr1^midCD11b⁺ MDSC in the bone marrow cultures after incubation with ethyl pyruvate or glycyrrhizin. D, MDSC generated in the bone marrow cultures were assessed for suppressive activity against antigen-specific MHC-restricted transgenic CD4⁺ and CD8⁺ T cells. E, Bone marrow cells were cultured under MDSC differentiation conditions (GM-CSF+IL-6) ± ethyl pyruvate (EP) and analyzed for the percent of c-kit⁺ (CD117⁺) progenitor cells. p values were obtained by Student's t test. Data are from one of three and two independent experiments for panels A-C and D-E, respectively.

Figure 3. HMGB1 contributes to the ability of MDSC to suppress antigen-driven T cell activation

Splenocytes from CD4⁺ DO11.10 TcR or CD8⁺ clone4 TcR transgenic mice were co-cultured with irradiated 4T1-induced MDSC from BALB/c mice and cognate peptide (OVA or HA peptide for DO11.10 and clone 4 T cells, respectively). A, T cell proliferation was measured by [³H]-thymidine incorporation in the presence of titered amounts of ethyl pyruvate or vehicle control. B, Ethyl pyruvate does not directly affect T cell activation. Transgenic DO11.10 and clone4 T cells were activated with cognate peptide in the presence of titered amounts of ethyl pyruvate. Data are from one of two independent experiments. p values were obtained by Student's t test comparing ethyl pyruvate treated samples versus the respective diluent control samples.

Figure 4. HMGB1 increases MDSC production of IL-10 and MDSC-macrophage crosstalk

A, Co-cultures of 4T1-induced BALB/c MDSC and macrophages from tumor-free mice were incubated with or without ethyl pyruvate or glycyrrhizin, and the supernatants were assayed by ELISA for IL-10. B, MDSC from 4T1-tumor-bearing BALB/c and BALB/c IL-10^−/− mice and peritoneal macrophages from tumor-free BALB/c mice were co-cultured and the supernatants were assayed by ELISA for IL-10. ND indicates non-detectable levels of protein. Data are from one of six and three independent experiments for panels A and B, respectively. p values were obtained by single-factor ANOVA.

Figure 5. HMGB1 facilitates down-regulation of IL-6 and MDSC production of IL-1β, but does not alter MDSC-mediated down-regulation of macrophage production of IL-12

Co-cultures of 4T1-induced BALB/c MDSC and macrophages from tumor-free mice were incubated with or without ethyl pyruvate or glycyrrhizin and the supernatants were assayed by ELISA for IL-12, IL-6, and IL-1β. ND indicates non-detectable levels of protein. Data are from one of four, five, and two independent experiments for IL-12, IL-6, and IL-1β, respectively. p values were obtained by Student's t test.

Figure 6. Tumor-bearing mice treated with mAbs to HMGB1 or with A Box have reduced levels of MDSC

A, C57BL/6 and BALB/c mice were inoculated s.c. with 5 x 10⁵ MC38 colon carcinoma cells or in the mammary fat pad with 7 x 10³ 4T1 mammary carcinoma cells, respectively. Mice were given recombinant A box (300μg/mouse) or vehicle (PBS) three times per week starting when tumors were first palpable (day 7-9 post inoculation). p values were obtained by log rank test. B, C57BL/6 mice were inoculated as in panel A. Treatment with 2G7 (5μg/200μl/mouse, 3x/week), irrelevant IgG, or A Box was started on day 10-13 when tumors were first palpable. Treatment was terminated on day 45 and blood leukocytes were analyzed by flow cytometry for total (Gr1⁺CD11b⁺), monocytic (MO; CD11b⁺Ly6G^-Ly6C⁺), and granulocytic (PMN; CD11b⁺Ly6G⁺Ly6C⁻) MDSC. Mice were sacrificed on day 50 when their tumors were approximately the same size, and spleen and tumor-infiltrating leukocytes (CD45⁺ cells) were analyzed by flow cytometry. n = 7 (blood, control-treated for 2G7), 4 (A Box, PBS-treated), 6 (tumor-infiltrating and spleen, control-treated; blood, 2G7-treated), 4 (tumor-infiltrating and spleen, 2G7-treated), and 4 (A Box-treated) mice/group. Data for 2G7 and their control-treated mice are pooled from two independent experiments; data for A Box and their control-treated mice are from a single experiment.

Figure 7. HMGB1 down-regulates T cell expression of L-selectin

A, Twenty-nine days after tumor inoculation the MC38 tumor-bearing mice from figure 6A were sacrificed and blood leukocytes were analyzed by flow cytometry for L-selectin expression and compared to blood leukocytes from tumor-free C57BL/6 mice. Representative histograms showing L-selectin expression from gated CD45⁺CD3⁺CD4⁺ and CD45⁺CD3⁺CD8⁺ T cells from tumor-free, A box-treated, or control-treated (PBS) C56BL/6 tumor-bearing mice. B, Average percent ± SD of CD45⁺CD3⁺CD4⁺ or CD45⁺CD3⁺CD8⁺ T cells expressing L-selectin. n= 5 mice/group (PBS-treated and tumor-free groups); n= 3 mice/group (A Box-treated group). p values were obtained by Student's t test. Data are from one of two independent experiments. C, Gr1⁺CD11b⁺ cells from tumor-free (left-hand panels) or tumor-bearing (right-hand panels) mice were incubated in vitro for zero, two, or four 4 hours with exogenous HMGB1 (left-hand panels) or ethyl pyruvate (right-hand panels), and stained for Gr1, CD11b, and ADAM17. Gated Gr1⁺CD11b⁺ cells were analyzed for ADAM17 expression. Graphs represent MCF of ADAM17 on Gr1⁺CD11b⁺ cells. Data are representative of three independent experiments.