Tumor-expressed inducible nitric oxide synthase controls induction of functional myeloid-derived suppressor cells through modulation of vascular endothelial growth factor release - PubMed (original) (raw)

. 2012 Jun 1;188(11):5365-76.

doi: 10.4049/jimmunol.1103553. Epub 2012 Apr 23.

Falguni Parikh, Esther Lopez-Rivera, Yared Hailemichael, Amelia Clark, Ge Ma, David Cannan, Marcel Ramacher, Masashi Kato, Willem W Overwijk, Shu-Hsia Chen, Viktor Y Umansky, Andrew G Sikora

Affiliations

Tumor-expressed inducible nitric oxide synthase controls induction of functional myeloid-derived suppressor cells through modulation of vascular endothelial growth factor release

Padmini Jayaraman et al. J Immunol. 2012.

Abstract

Inducible NO synthase (iNOS) is a hallmark of chronic inflammation that is also overexpressed in melanoma and other cancers. Whereas iNOS is a known effector of myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, its pivotal position at the interface of inflammation and cancer also makes it an attractive candidate regulator of MDSC recruitment. We hypothesized that tumor-expressed iNOS controls MDSC accumulation and acquisition of suppressive activity in melanoma. CD11b(+)GR1(+) MDSC derived from mouse bone marrow cells cultured in the presence of MT-RET-1 mouse melanoma cells or conditioned supernatants expressed STAT3 and reactive oxygen species (ROS) and efficiently suppressed T cell proliferation. Inhibition of tumor-expressed iNOS with the small molecule inhibitor L-NIL blocked accumulation of STAT3/ROS-expressing MDSC, and abolished their suppressive function. Experiments with vascular endothelial growth factor (VEGF)-depleting Ab and recombinant VEGF identified a key role for VEGF in the iNOS-dependent induction of MDSC. These findings were further validated in mice bearing transplantable MT-RET-1 melanoma, in which L-NIL normalized elevated serum VEGF levels; downregulated activated STAT3 and ROS production in MDSC; and reversed tumor-mediated immunosuppression. These beneficial effects were not observed in iNOS knockout mice, suggesting L-NIL acts primarily on tumor- rather than host-expressed iNOS to regulate MDSC function. A significant decrease in tumor growth and a trend toward increased tumor-infiltrating CD8(+) T cells were also observed in MT-RET transgenic mice bearing spontaneous tumors. These data suggest a critical role for tumor-expressed iNOS in the recruitment and induction of functional MDSC by modulation of tumor VEGF secretion and upregulation of STAT3 and ROS in MDSC.

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Figures

Figure 1

Figure 1. Tumor -expressed iNOS is required for accumulation and functional activity of _ex-vivo_-derived CD11b+GR1+ MDSC

A. Representative FACs plots of T cell (CD4 and CD8) proliferation as measured by CFSE dilution. B. Time course of accumulation of bone-marrow derived CD11b+GR1+ cells cultured in medium alone, in the presence of MT-RET conditioned supernatant, and in transwell co-culture with MT-RET cells. C. Dose response curve of ability of sorted ex-vivo MT-RET supernatant-derived CD11b+GR1+ MDSC(harvested on day 4 in culture)to inhibit CD4 and CD8 T cell proliferation. D. Relative accumulation (day 5-6) of CD11b+GR1+ MDSC in MT-RET transwell co-culture +/− 1mM L-NIL. E. Relative accumulation of MDSC(day 5)in the presence of MT-RET-conditioned supernatants alone, or MT-RET sups + 1mM L-NIL added to the tumor cells (L-NIL-tumor) and/or to the bone marrow cell culture (L-NIL-BM). Each graph summarizes data from at least 3 experiments.

Figure 2

Figure 2. iNOS inhibition suppresses upregulation of ROS production and STAT3 activation by tumor-derived soluble mediators

Representative histograms (A.) and quantitative summary from at least 3 experiments (B.) of effect of L-NIL on MT-RET-1-mediated ROS production by ex-vivo-derived MDSC (gated on CD11b+GR1+ cells)on day 6 after initial culture.C. Anti phospho-STAT3 antibody staining of permeablized cells is upregulated in CD11b+GR1+ MDSC in MT-RET / bone marrow cell transwell culture, and reversed by 1mM L-NIL (p≤0.05) on day 3 after initial cultures were set up. D. Ability of MT-RET supernatant to induce CD4 and CD8 T cell suppressive activity of ex-vivo-derived MDSC(harvested on day 4 in culture)is abolished by incubation of MT-RET cells with 1mM L-NIL. Each graph summarizes data from at least 3 experiments.

Figure 3

Figure 3. Tumor-secreted VEGF mediates iNOS-dependent cross-talk required for MDSC accumulation

A. Relative VEGF levels in 48 hour supernatants derived from MT-RET-1 cells cultured in the presence or absence of 1mM L-NIL. B. Expression of VEGF-R-1 and 3 in CD11b+GR1+ MDSC derived ex-vivo from wild type bone marrow cultured with MT-RET-1 supernatants for 5 days. C. In vivo concentration of VEGF, IL-6, G-CSF and IL-1β in serum of untreated or L-NIL-treated wild-type mice on day 21 after MT-RET-1 tumor injection L-NIL treatment decreases VEGF in both serum from RET tumor bearing mice and RET tumor supernatants compared to their untreated counterparts (p≤0.05). VEGF concentration in serum was compiled from at least 3 experiments with n=5 mice per group. **D.**Ability of the indicated concentrations of anti-VEGF neutralizing antibody to block MT-RET supernatant-induced MDSC accumulation _ex vivo_at 5 days in culture. Neutralization of VEGF leads to significantly decreased accumulation of MDSC in BM cultures (p≤0.05). E. Ability of the indicated concentrations of recombinant murine VEGF to reverse L-NIL mediated suppression of MT-RET-1 supernatant-induced MDSC accumulation _ex-vivo_at 5 days in culture. Each graph summarizes data from at least 3 experiments.

Figure 4

Figure 4. Pharmacologic iNOS inhibition or genetic ablation of host iNOS significantly reduce accumulation of MDSC in transplantable MT-RET-1 tumors

A. Gating strategy and representative FACS plots of MDSC from tumor, spleen, and bone marrow of untreated and L-NIL-treated mice bearing transplantable MT-RET melanoma with percentages of GR1 Hi or Int populations. B. Relative percentage of CD11b+GR1+ MDSC populations in the tumor, spleen and bone marrow of untreated, L-NIL-treated, and iNOS-KO mice on day 21-23 after MT-RET-1 injection. Numbers are presented as percentage of total (high + intermediate) CD11b+ GR1+ double-positive cells. Tumors from L-NIL-treated mice have significantly decreased numbers ofCD11b+GR1+ cells *p≤0.01).C. Nitrotyrosine expressed by CD11b+GR1+ MDSC from spleens of untreated, L-NIL treated or iNOSknockoutMT-RET-1-bearing mice on day 22 normalized to isotypecontrol. L-NIL treated mice show decreased nitrotyrosine expression as compared to WT mice (*p≤0.01).Each graphrepresents pooled data from at least 3-5 experiments with n=5 mice per group.

Figure 5

Figure 5. CD11b+Gr1+ MDSC sorted from spleens of untreated MT-RET-1-bearing mice, but not L-NIL-treated mice, inhibit CD4 and CD8 T cell proliferation

A. Relative proliferation of CD3/CD28-stimulated CD4 and CD8 T cells in the presence of different ratios of sorted CD11b+GR1+MDSC from spleens (left) or TIL (right) ofuntreated and L-NIL-treated miceon day 21 after MT-RET-1 injection orB. Relative proliferation of CD8 (top) and CD4 (bottom) T cells in the presence of 1:1 CD11b+Gr1+ MDSC or CD11b-GR1-cells sorted from splenocytes of iNOSKO mice, L-NIL-treated mice, or untreated mice in the presence or absence of 1mM L-NIL added to the sorted MDSC prior to the final wash step. Each graph shown above represents pooled data from at least 3 experiments with n=5 mice per group.L-NIL treated MDSC lose their ability to suppress T cell proliferation compared to WT (*p≤0.05)**C.**Effect of L-NIL or genetic ablation of host iNOS on ROS production in vivo by CD11b+GR1+ MDSC from spleenon day 21 after MT-RET injection. L-NIL treatment leads to decreased production of ROS from splenic GR1+Cd11b+ MDSC. D. L-NIL treatment _in vivo_downregulates activated (phosphorylated) STAT3 expression and pSTAT3/ total STAT3 ratio in MT-RET-1 tumors by Western blot. Each graph represents pooled data from at least 3 experiments.

Figure 6

Figure 6. Pharmacologic iNOS inhibition reverses systemic T cell decline in tumor-bearing mice and enhances intratumoral accumulation of CD4 and CD8 T cells

A. Growth of MT-RET untreated and “early” (day 4) L-NIL-treated melanoma in wild-type and RAG-KO mice demonstrates modest T/B cell-dependent anti-tumor effect. Treatment of established (day 14) tumors does not lead to decreases in tumor growth.B. Absolute and relative number of tumor-infiltrating CD4 and CD8 T cells in untreated and L-NIL-treated (WT or iNOS-KO) MT-RET-bearing mice on day 21. Each graph shown represents pooled data from at least 3 experiments with n=5 mice per group. **C.**Cytotoxicity assay against EL-4 target cells with effector cells from splenocytes or TIL of control or L-NIL treated mice (harvested on day 21 after MT-RET injection, enriched for gp100/trp2 specific T cells). CTL efficacy is expressed as percentage of target cell populations lysed. Graph is representative of 2 experiments with n=5 mice per group.

Figure 7

Figure 7. iNOS inhibition suppresses melanoma growth and reduces number of MDSC in ret transgenic tumor bearing mice

A. H& E staining from axial and coronal sections of heads of tumor bearing ret transgenic mice.B.Representative plots showing percentage of MDSC populations gated on CD45+ cells in tumor and metastatic LN.C.Treatment with L-NIL (10 mice per group) induces a decrease in the weight of primary tumorD.CD8 T cells were quantified in both tumor and metastatic LN from both untreated and L-NIL treated mice and expressed as percentage of CD45+ leukocytes. NO was detected intracellularly in cells from treated (L-NIL) and non-treated (control) tumor bearing mice by flow cytometry, and results presented as percentage of NO+ cells within total Gr1+CD11b+ MDSC.

Figure 8

Figure 8

Model of iNOS-mediated VEGF production in control of tumor-directed recruitment and functional maturation of MDSC.

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