MDSC Function Research Articles

Impact of the adenosine receptor A2BR expressed on myeloid cells on immune regulation during pregnancy.

During pregnancy, the maternal immune system must carefully balance protection against pathogens with tolerance toward the semiallogeneic fetus. Dysfunctions of the immune system can lead to severe complications such as preeclampsia, fetal growth restriction, or pregnancy loss. Adenosine plays a role in physiological processes and plasma-level increase during pregnancy. The adenosine receptor A2B (A2BR), which is expressed on both, immune and nonimmune cells, is activated by high adenosine concentrations, achieved during pregnancy. We investigated the impact of A2BR expressed on myeloid cells on immune regulation during pregnancy using a mouse model with myeloid deficiency for A2BR. We demonstrate systemic changes in myeloid and lymphoid cell populations during pregnancy in A2BR-KO (Adora2B923f/f-LysMCre) mice with increased monocytes, neutrophils, and T cells but decreased B cells as well as altered T-cell subpopulations with decreased Th1 cells and Tregs and increased Th17 cells. Lack of A2BR on myeloid cells caused an increased systemic expression of IL-6 but decreased systemic accumulation and function of MDSC and reduced numbers of uterine natural killer cells. The pregnancy outcome was only marginally affected. Our results demonstrate that A2BR on myeloid cells plays a role in immune regulation during pregnancy, but the clinical impact on pregnancy remains unclear.

Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2.

An important, but poorly understood, aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities.

Schlafen4+-MDSC in Helicobacter-induced gastric metaplasia reveals role for GTPases.

MDSCs express SCHLAFEN 4 (SLFN4) in Helicobacter-infected stomachs coincident with spasmolytic polypeptide-expressing metaplasia (SPEM), a precursor of gastric cancer. We aimed to characterize SLFN4+ cell identity and the role of Slfn4 in these cells. Single-cell RNA sequencing was performed on immune cells sorted from PBMCs and stomachs prepared from uninfected and 6-month H. felis-infected mice. Knockdown of Slfn4 by siRNA or PDE5/6 inhibition by sildenafil were performed in vitro. Intracellular ATP/GTP levels and GTPase activity of immunoprecipitated Slfn4 complexes were measured using the GTPase-Glo assay kit. The intracellular level of ROS was quantified by the DCF-DA fluorescent staining, and apoptosis was determined by cleaved Caspase-3 and Annexin V expression. Gli1CreERT2 x Slfn4 fl/fl mice were generated and infected with H. felis. Sildenafil was administered twice over 2 weeks by gavaging H. felis infected mice ~4 months after inoculation once SPEM had developed. Slfn4 was highly induced in both monocytic and granulocytic MDSCs from infected stomachs. Both Slfn4 +-MDSC populations exhibited strong transcriptional signatures for type-I interferon responsive GTPases and exhibited T cell suppressor function. SLFN4-containing protein complexes immunoprecipitated from myeloid cell cultures treated with IFNa exhibited GTPase activity. Knocking down Slfn4 or PDE5/6 inhibition with sildenafil blocked IFNa induction of GTP, SLFN4 and NOS2. Moreover, IFNa induction of Slfn +-MDSC function was inhibited by inducing their reactive oxygen species (ROS) production and apoptosis through protein kinase G activation. Accordingly, in vivo disruption of Slfn4 in Gli1CreERT2 x Slfn4 fl/fl mice or pharmacologic inhibition by sildenafil after Helicobacter infection also suppressed SLFN4 and NOS2, reversed T cell suppression and mitigated SPEM development. Taken together, SLFN4 regulates the activity of the GTPase pathway in MDSCs and precludes these cells from succumbing to the massive ROS generation when they acquire MDSC function.

Phase 1b/2 trial of pepinemab plus avelumab as second line combination immunotherapy for patients with metastatic pancreatic adenocarcinoma.

TPS4195 Background: Monotherapy with immune checkpoint inhibitors (ICIs) is not effective in metastatic pancreatic adenocarcinoma (PDAC), likely due to the unique immunosuppressive tumor microenvironment (TME) of PDAC, thus safe and novel strategies to overcome resistance and facilitate adaptive immune responses are a major unmet need. Preclinical and clinical studies demonstrated that antibody blockade of semaphorin 4D (SEMA4D) promotes tumoral infiltration and activation of DCs and CD8+ T cells and reverses immunosuppression, including attenuation of MDSC recruitment and function, leading to enhanced efficacy of ICIs without increasing toxicity. Pepinemab, a SEMA4D blocking antibody, in combination (combo) with avelumab provided clinical benefit in some patients with ICI-resistant and PD-L1-low NSCLC (NCT03268057). Pepinemab is also being evaluated in combo with pembrolizumab in patients with metastatic squamous cell carcinoma of the head and neck (NCT04815720). The principal goals of this proof-of-concept study are to investigate the safety, efficacy, and TME biomarker changes of treatment with the combo of pepinemab plus avelumab in patients with metastatic PDAC. Methods: This single-arm, open-label study (NCT05102721) is evaluating the safety, tolerability, and efficacy of pepinemab in combo with avelumab in patients diagnosed with metastatic PDAC who have either progressed on or are otherwise intolerant to first line chemotherapy. Patients must have received 5FU or gemcitabine-based first line therapy with evidence of intolerance or treatment failure, including progression both during or after completing first-line treatment. Accrual for the Phase 1b portion follows a Bayesian Optimal Interval Design (BOIN), beginning at the highest dose combo; 20 mg/kg pepinemab and 800 mg avelumab Q2W. Phase 2 begins after 16 subjects are enrolled at MTD, permitting a Simon’s two stage assessment of futility prior to closing the Phase 1b period. The primary objective for Phase 1b is tolerability, and the primary objective for Phase 2 is therapeutic efficacy compared to historical second-line systemic chemotherapy. The trial anticipates a total evaluable cohort of 40 subjects and is powered to detect a response rate of 23% or greater, with alpha set at 0.1 and 80% power. Exploratory objectives include robust correlative analysis of immune, stromal, and genomic profiling of baseline and on-treatment tumor biopsies to ascertain mechanisms of treatment response and failure. Patient reported outcomes are incorporated throughout the trial to assess disease specific symptoms associated with pancreatic cancer and cancer cachexia. One patient has been enrolled as of Feb 14, 2023. Clinical trial information: NCT05102721 .

Tryptophan diet promotes myeloid-derived suppressor cell response via aryl hydrocarbon receptor in Sjogren’s syndrome

Abstract Myeloid-derived suppressor cells (MDSCs) are heterogeneous myeloid cell populations with immunosuppressive capacity, which contribute to immune regulation and tolerance induction. We previously reported impaired MDSC function in patients with primary Sjögren’s syndrome (pSS) and mice with experimental SS (ESS). In this study, we first found that aryl hydrocarbon receptor (AhR) was highly expressed by human and murine polymorphonuclear MDSCs (PMN-MDSCs). Indole-3-propionic acid (IPA), a natural AhR ligand produced from dietary tryptophan, significantly promoted PMN-MDSC differentiation and their suppressive function on CD4 +T cells. In contrast, feeding tryptophan-free diet resulted in decreased PMN-MDSC response, a phenotype that could be reversed by IPA supplementation. Notably, AhR expression was reduced in PMN-MDSCs during ESS development, while AhR antagonism resulted in exacerbated ESS pathology and dysregulated T effector cells, which could be phenocopied by tryptophan-free diet. Interferon regulatory factor 4 (IRF4), a repressive transcription factor, was upregulated in PMN-MDSCs during ESS progression. ChIP analysis revealed the binding capacity of IRF4 to the promoter region of AhR, while IRF4 deficiency markedly enhanced AhR-mediated PMN-MDSC responses. Furthermore, tryptophan-enriched diet or dietary supplementation of IPA markedly ameliorated salivary glandular pathology in ESS mice with restored MDSC immunosuppressive function. Together, our results identify a novel function of AhR in modulating PMN-MDSC response and demonstrate the therapeutic potential of targeting AhR for the treatment of pSS.

Abstract CT111: Phase 1/2 study of pepinemab, an inhibitor of semaphorin 4D, in combination with pembrolizumab as first-line treatment of recurrent or metastatic head and neck cancer (KEYNOTE B84)

Abstract Purpose: Immunosuppressive myeloid cells in the tumor microenvironment (TME) limit the efficacy of immune checkpoint inhibitors (ICIs) in head and neck squamous cell carcinoma (HNSCC). Preclinical and clinical studies demonstrated that antibody blockade of semaphorin 4D (SEMA4D) promotes tumor infiltration and activation of DCs and CD8+ T cells and reverses immunosuppression, including attenuation of MDSC recruitment and function, leading to enhanced efficacy of ICIs. Pepinemab, a humanized SEMA4D blocking antibody, in combination (combo) with avelumab provided clinical benefit in some patients with ICI-resistant and PD-L1-low NSCLC. Pembrolizumab (pembro) is approved as monotherapy or in combo with chemo for the first-line treatment of recurrent or metastatic (R/M) HNSCC. More effective treatments are, however, needed to increase the frequency and duration of responses. The primary hypothesis of this proof-of-concept study is that pepinemab in combo with pembro will yield increased clinical benefit compared to the reported activity for pembro monotherapy in R/M HNSCC. Methods: KEYNOTE B84 (NCT04815720) is a multicenter, single-arm open-label study evaluating the safety, efficacy, and PK/PD of pepinemab in combo with pembro as first-line treatment of R/M HNSCC. Subjects with measurable disease per RECIST1.1, ECOG PS of 0 or 1, and PD-L1 CPS ≥ 0 are eligible; subjects who have received prior ICIs or other systemic treatment for R/M HNSCC are excluded. The study includes a safety run-in (n=3) and dose-expansion phase (maximum n=62). Pepinemab, previously found to be well-tolerated in combo with other ICIs, will be evaluated initially at the highest intended dose of 20 mg/kg, in combo with 200 mg pembro, both administered i.v. Q3W. The dose expansion phase will include an equal distribution of subjects who have tumor PD-L1 combined positive scores (CPS) of <20 and ≥20. The primary efficacy endpoint is ORR, and the secondary endpoints are PFS, DoR, and OS, as well as exploratory biomarker analyses. Pre- and on-treatment biopsies will be collected for evaluation of immune contexture in TME. Results: The safety run-in phase (n=3) was successfully completed. The combo was well tolerated with no DLTs observed. One of the three patients had a confirmed complete response, a second patient progressed with several SAEs attributed to comorbidities (diabetes) deemed unrelated to treatment, and the third patient has not yet completed tumor evaluation. The safety monitoring committee has recommended that the dose-expansion phase be initiated at 20 mg/kg pepinemab and 200 mg pembro Q3W. Conclusions: The ongoing KEYNOTE B84 study completed the initial safety run-in phase. Phase 2 will evaluate pepinemab, a SEMA4D inhibitor, as a novel strategy to potentially overcome resistance and enhance activity of pembro in R/M HNSCC. Citation Format: Terrence L. Fisher, Elizabeth E. Evans, Crystal Mallow, Amber Foster, Megan Boise, Ernest Smith, John E. Leonard, Marya F. Chaney, J. Thaddeus Beck, Steven Hager, Nabil F. Saba, Conor Steuer, Douglas Adkins, Barbara Burtness, Maurice Zauderer. Phase 1/2 study of pepinemab, an inhibitor of semaphorin 4D, in combination with pembrolizumab as first-line treatment of recurrent or metastatic head and neck cancer (KEYNOTE B84) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT111.

Phase 1/2 study of pepinemab, an inhibitor of semaphorin 4D, in combination with pembrolizumab as first-line treatment of recurrent or metastatic head and neck cancer (KEYNOTE-B84).

e18033 Background: Immunosuppressive myeloid cells in the tumor microenvironment (TME) limit the efficacy of immune checkpoint inhibitors (ICIs) in head and neck squamous cell carcinoma (HNSCC). Preclinical and clinical studies demonstrated that antibody blockade of semaphorin 4D (SEMA4D) promotes tumor infiltration and activation of DCs and CD8+ T cells and reverses immunosuppression, including attenuation of MDSC recruitment and function, leading to enhanced efficacy of ICIs. Pepinemab, a SEMA4D blocking antibody, in combination (combo) with avelumab provided clinical benefit in some patients with ICI-resistant and PD-L1-low NSCLC. Pembrolizumab (pembro) is approved as monotherapy or in combo with chemo for the first-line treatment of recurrent or metastatic (R/M) HNSCC, representative of a class of tumors that express higher levels of SEMA4D and MDSC. The primary hypothesis of this proof-of-concept study is that pepinemab in combo with pembro will yield increased clinical benefit compared to the reported activity for pembro monotherapy in R/M HNSCC. Methods: KEYNOTE-B84 (NCT04815720) is a single-arm open-label study evaluating the safety, efficacy, and PK/PD of pepinemab in combo with pembro as first-line treatment of R/M HNSCC. Subjects with measurable disease per RECIST1.1, ECOG 0 or 1, are eligible; subjects who have received prior ICIs or other systemic treatment for R/M HNSCC are excluded. The study includes a safety run-in (n = 3) and dose-expansion phase (maximum n = 62). Pepinemab, previously found to be well-tolerated in combo with other ICIs, will be evaluated initially at the highest intended dose of 20 mg/kg, in combo with 200 mg pembro, both administered i.v. Q3W. The dose expansion phase plans to include nearly equal distribution of subjects who have tumor PD-L1 combined positive scores (CPS) of < 20 and ≥20. The primary efficacy endpoint is ORR, and the secondary endpoints are PFS, DoR, and OS, as well as exploratory biomarker analyses. Pre- and on-treatment biopsies will be collected for evaluation of immune contexture in TME. Results: The safety run-in phase (n = 3) is complete, and the combo appeared to be well-tolerated with no DLTs observed; SMC approved continuation to dose expansion. Strikingly, two of the three patients have been observed to experience a complete response (CR). Biomarker analysis revealed that tumors in both responders expressed low levels of PD-L1 (CPS < 20). The third patient progressed prior to first scan, suffering several unrelated SAEs attributed to a pre-existing co-morbidity. Conclusions: The ongoing KEYNOTE-B84 study completed the initial safety run-in phase, with two of three subjects achieving CR. Phase 2 will evaluate pepinemab, a SEMA4D inhibitor, as a novel strategy to potentially overcome resistance and enhance activity of pembro in R/M HNSCC. Clinical trial information: NCT04815720.

Abnormal immune function of MDSC and NK cells from chronic phase CML patients restores with tyrosine kinase inhibitors

BackgroundMyeloid-derived suppressor cell (MDSC) -mediated immune suppression, and natural killer (NK) and/or T cell-mediated immune responses play important roles in Chronic myeloid leukemia (CML). However, detailed regulation mechanisms of these immune cells in CML have not been fully elucidated. MethodsThe proportion of MDSCs and effector NK cells in newly diagnosed CML patients, patients during TKI treatment, and healthy donors (HD) were detected using flow cytometry. Serum levels and mRNA expression of Arg1 and iNOS in newly diagnosed CML patients, patients during TKI treatment, and HD were measured by ELISA and qPCR, respectively. Effect of CML serum or peripheral blood mononuclear cells (PBMCs) on HD derived CD3+ T cell proliferation was evaluated by CFSE-labeled HD CD3+ T cells co-cultured with PBMCs and serum from CML patients. Effect of CML serum on NK cells killing activity was evaluated via detecting apoptosis of K562 cells. ResultsProportion of Gr-MDSCs and the serum levels of Arg1 and iNOS were significantly increased in patients at diagnosis, and reduced following TKI treatment. However, the proportion of effector NK cells were decreased in patients at diagnosis, and increased following TKI treatment. Serum and PBMC from CML patients suppressed HD derived T cell proliferation in vitro. Additionally, serum from CML patients enhanced HD derived NK cell killing activity in vitro, while the addition of Arg1 inhibitor to CML serum suppressed this phenomenon. ConclusionsGr-MDSCs and effector NK cells play an important role in the pathogenesis of CML, inhibiting the function of MDSC and restoring the function of NK cells is expected to be a therapeutic strategy for CML.

Phase 1/2 Study of Pepinemab in Combination with Pembrolizumab as First-Line Treatment of Advanced, Recurrent or Metastatic Head and Neck Cancer (KEYNOTE B84)

<h3>Purpose/Objective(s)</h3> Immunosuppressive myeloid cells in the tumor microenvironment (TME) limit the efficacy of immune checkpoint inhibitors (ICIs) in head and neck squamous cell carcinoma (HNSCC). Preclinical and clinical studies demonstrated that antibody blockade of semaphorin 4D (SEMA4D) promotes tumor infiltration and activation of DCs and CD8+ T cells and reverses immunosuppression, including attenuation of MDSC recruitment and function, leading to enhanced efficacy of ICIs. Pepinemab, a humanized SEMA4D blocking antibody, in combination with avelumab provided clinical benefit in some patients with difficult to treat ICI-resistant and PD-L1-low NSCLC. Pembrolizumab is approved as monotherapy or in combination with chemotherapy for the first-line treatment of recurrent or metastatic (R/M) HNSCC. More effective treatments are, however, needed to increase the frequency and duration of responses. The primary hypothesis of this proof-of-concept study is that pepinemab in combination with pembrolizumab will yield increased clinical benefit compared to the reported activity for pembrolizumab monotherapy in R/M HNSCC. <h3>Materials/Methods</h3> KEYNOTE B84 (NCT04815720) is a multicenter, single-arm open-label study to evaluate the safety, efficacy, and PK/PD of pepinemab in combination with pembrolizumab as first-line treatment of R/M HNSCC. Subjects with measurable disease per RECIST1.1, and an ECOG PS of 0 or 1, are eligible; subjects who have received prior ICIs or other systemic treatment for R/M HNSCC are excluded. The study will include a safety run-in phase (n=3-6) and dose-expansion phase (maximum n=62). Pepinemab, previously found to be well-tolerated in combination with other ICIs, will be evaluated initially at the highest intended dose of 20 mg/kg, in combination with 200 mg pembrolizumab, both administered i.v. Q3W. The dose expansion phase will include an equal distribution of subjects who have tumor PD-L1 combined positive scores (CPS) of <20 and ≥20. The primary efficacy endpoint is ORR, and the secondary endpoints include PFS, DoR, and OS, as well as exploratory biomarker analyses. Pre- and on-treatment biopsies will be collected for evaluation of immune contexture in TME. <h3>Results</h3> Enrollment has been initiated at several of 18 participating sites. Preliminary data from the safety run-in phase will be evaluated at a safety monitoring review meeting planned for December 2021. In addition, multiplex immunohistochemistry panels have been established to immunophenotype cells in the TME, including CD8+ T cells, DCs, MDSCs, Tregs, monocytes, and macrophages. <h3>Conclusion</h3> There remains a clear unmet need for more effective treatment options to overcome immunosuppressive factors in the TME. The KEYNOTE B84 study will evaluate pepinemab, a SEMA4D inhibitor, as a novel strategy to potentially overcome resistance and enhance activity of pembrolizumab in R/M HNSCC.

Abstract P047: Inactivation of IFN signaling drive immunosuppressive MDSC function and can be therapeutically targeted

Abstract Myeloid-derived suppressor cells (MDSC) are pathologically activated neutrophils and monocytes with potent immune suppressive activity that promote tumor progression and impair the efficacy of anti-tumor therapeutics. Herein we show that type I interferons (IFN1) receptor dampen immune suppressive activity of MDSC. Downregulation of the IFNAR1 is found in MDSC from cancer patients and mouse tumor models. Downregulation of IFNAR1 depends on the activation of the p38 protein kinase and is required for activation of the immune suppressive phenotype moreover genetic stabilization of IFNAR1 in tumor bearing mice reduces suppressive activity of MDSC and promote antitumor effect. Stabilizing IFNAR1 using inhibitor of p38 combined with the interferon induction therapy elicits a robust anti-tumor effect that is completely dependent on IFNAR1 expression on MDSC. Thus, inhibition of MDSC immune suppressive function can be exploited therapeutically. Citation Format: Emilio Sanseviero, Kevin Alicea-Torres, Yulia Nefedova, Serge Y. Fuchs, Dmitry I. Gabrilovich. Inactivation of IFN signaling drive immunosuppressive MDSC function and can be therapeutically targeted [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P047.

Hedgehog transcriptional effector GLI mediates mTOR-Induced PD-L1 expression in gastric cancer organoids.

Tumors evade immune surveillance by expressing Programmed Death-Ligand 1 (PD-L1), subsequently inhibiting CD8+ cytotoxic T lymphocyte function. Response of gastric cancer to immunotherapy is relatively low. Our laboratory has reported that Helicobacter pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Hedgehog (Hh) signaling pathway. The PI3K/AKT/mTOR pathway is activated in gastric cancer and may have immunomodulatory potential. We hypothesize that Hh signaling mediates mTOR-induced PD-L1 expression. Patient-derived organoids (PDOs) were generated from gastric biopsies and resected tumor tissues. Autologous organoid/immune cell co-cultures were used to study the immunosuppressive function of MDSCs. NanoString Digital Spatial Profiling (DSP) of immune-related protein markers using FFPE slide-mounted tissues from gastric cancer patients was performed. DSP analysis showed infiltration of immunosuppressive MDSCs expressing Arg1, CD66b, VISTA and IDO1 within cancer tissues. Orthotopic transplantation of patient derived organoids (PDOs) resulted in the engraftment of organoids and the development of histology similar to that observed in the patient's tumor tissue. PDO/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of PMN-MDSCs within these co-cultures sensitized the organoids to anti-PD-1/PD-L1-induced cancer cell death. Rapamycin decreased phosphorylated S6K, Gli2 and PD-L1 expression in PDO/immune cell co-cultures. Transcriptional regulation of PD-L1 by GLI1 and GLI2 was blocked by rapamycin. In conclusion, the PDO/immune cell co-cultures may be used to study immunosuppressive MDSC function within the gastric tumor microenvironment. The mTOR signaling pathway mediates GLI-induced PD-L1 expression in gastric cancer.

Integrated transcriptional-phenotypic analysis captures systemic immunomodulation following antiangiogenic therapy in renal cell carcinoma patients.

BackgroundThe combination of immune checkpoint blockade (ICB) with standard therapies is becoming a common approach for overcoming resistance to cancer immunotherapy in most human malignancies including metastatic renal cell carcinoma (mRCC). In this regard, insights into the immunomodulatory properties of antiangiogenic agents may help designing multidrug schedules based on specific immune synergisms.MethodsWe used orthogonal transcriptomic and phenotyping platforms combined with functional analytic pipelines to elucidate the immunomodulatory effect of the antiangiogenic agent pazopanib in mRCC patients. Nine patients were studied longitudinally over a period of 6 months. We also analyzed transcriptional data from The Cancer Genome Atlas (TCGA) RCC cohort (N = 571) to assess the prognostic implications of our findings. The effect of pazopanib was assessed in vitro on NK cells and T cells. Additionally, myeloid‐derived suppressor (MDSC)‐like cells were generated from CD14+ monocytes transfected with mimics of miRNAs associated with MDSC function in the presence or absence of pazopanib.ResultsPazopanib administration caused a rapid and dramatic reshaping in terms of frequency and transcriptional activity of multiple blood immune cell subsets, with a downsizing of MDSC and regulatory T cells in favor of a strong enhancement in PD‐1 expressing cytotoxic T and Natural Killer effectors. These changes were paired with an increase of the expression of transcripts reflecting activation of immune‐effector functions. This immunomodulation was marked but transient, peaking at the third month of treatment. Moreover, the intratumoral expression level of a MDSC signature (MDSC INT) was strongly associated with poor prognosis in RCC patients. In vitro experiments indicate that the observed immunomodulation might be due to an inhibitory effect on MDSC‐mediated suppression, rather than a direct effect on NK and T cells.ConclusionsThe marked but transient nature of this immunomodulation, peaking at the third month of treatment, provides the rationale for the use of antiangiogenics as a preconditioning strategy to improve the efficacy of ICB.

The receptor for advanced glycation endproducts (RAGE) decreases survival of tumor-bearing mice by enhancing the generation of lung metastasis-associated myeloid-derived suppressor cells

Metastatic cancer has a poor prognosis. Novel pharmacologic targets need to be identified. The receptor for advanced glycation endproducts (RAGE) is a pattern recognition receptor constitutively expressed in the lungs. Absence of overt disease in RAGE null mice suggests that RAGE is unnecessary or redundant in health. We report that RAGE null tumor-bearing mice have reduced lung metastasis and improved survival. Bone marrow chimera studies suggest that hematopoietic cell RAGE is an important contributor to these effects. Deletion of RAGE reduces both the quantity and suppressive activity of tumor-induced MDSC. Protein and mRNA studies suggest that RAGE contributes to the generation and function of MDSC including expression of the alarmins S100A8/A9 and activity of inducible nitric oxide synthase, arginase-1, and NF-κB. These findings demonstrate the important role of RAGE in determining the quantity and function of tumor-associated MDSC and suggest RAGE as a pharmacologic target for patients with metastatic disease.

Gemcitabine treatment modulates myelopoiesis progenitors and monocytes to promote lung metastasis

Abstract Metastasis-associated macrophages (MAMs) play important roles in tumor metastasis through the formation of a pre-metastatic niche. In mouse models of lung metastasis, interstitial macrophages (IMs) markedly accumulate in the lungs and differentiate into MAMs. Regarding the origin of IMs/MAMs, tissue-resident macrophages and bone marrow (BM)-derived classical monocytes (BM-Mo) contribute to the pool of MAMs. Chemotherapy offers long-term clinical benefits to many cancer patients. However, several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis by multiple mechanisms. Our recent studies suggest that Gemcitabine (GEM) promotes the immunosuppressive function of monocytic MDSC (M-MDSC) in the tumor microenvironment via the tumor-derived GM-CSF and efferocytosis signaling. In this study, we hypothesize that host responses to GEM treatment enhance the accumulation of BM-derived monocytes in lung, which promotes lung metastasis. Multi-dose of GEM treatment induces the increase of BM LSK cells and multipotent progenitors (MPPs) in tumor-free mice, which is associated with the increase of treatment induce mitochondrial ROS (mtROS). Thus, the percentage of BM Ly6C+ monocytes is significantly increased because of increased progenitor monocyte differentiation potential and monocyte proliferation. Further, GEM treatment increases the percentages of lung CCR2+Ly6C+ monocytes and IMs. Consequently, GEM pre-treated mice have a significantly higher number of pulmonary metastases compared to untreated mice in the experimental metastasis model. These results suggest a novel mechanism for chemotherapy induced metastasis via the modulation of myelopoiesis progenitors.

Targeting PARP-1 with metronomic therapy modulates MDSC suppressive function and enhances anti-PD-1 immunotherapy in colon cancer

BackgroundPoly(ADP-ribose) polymerase (PARP) inhibitors (eg, olaparib) are effective against BRCA-mutated cancers at/near maximum tolerated doses by trapping PARP-1 on damaged chromatin, benefitting only small patient proportions. The benefits of targeting...

Isolation and Functional Characterization of Myeloid-Derived Suppressor Cells in Infections Under High Containment.

The current absence of markers unique to MDSC, particularly those expanded during human infection, necessitate concurrent demonstration of their suppressive capacity to ensure unequivocal identification. This is further complicated by the array of heterogeneous markers used to characterize MDSC in various conditions and models. Standardization of phenotypic and functional characterization, as well as isolation, from infectious biological samples of patients, are critical for accurately reporting MDSC dynamics, function, organ abundance, and establishment of their therapeutic value in infectious diseases. To illustrate, we report on our established method for MDSC isolation from bronchoalveolar lavage fluid and peripheral blood of pulmonary TB patients, as well as functional impact on T cells by measuring T cell activation, proliferation, and cytokine production.

MiR-21 regulates immunosuppression mediated by myeloid-derived suppressor cells by impairing RUNX1-YAP interaction in lung cancer

BackgroundMyeloid-derived suppressor cells (MDSCs) are known suppressors of antitumor immunity and contribute to immunosuppressive microenvironment during tumor development including lung cancer. Accumulating evidence shows microRNAs (miRNAs) affect tumor-expanded MDSC accumulation and function in tumor microenvironment and favor solid tumor growth. Herein, we aim to characterize the role of miR-21 in regulating the accumulation and activity of MDSCs in lung cancer.MethodsThe proportions of MDSCs, T helper cells (Th), and cytotoxic T lymphocytes (CTL) were evaluated by flow cytometric analyses of peripheral blood and tumor tissues collected from Lewis lung-cancer-bearing mice. T cell proliferation assay was performed in CD4+ or CD8+ T cells cocultured with MDSCs. MDSC apoptosis was examined by flow cytometric analysis. The levels of IL-10, TGF-β, and GM-CSF in mouse serum were determined by ELISA. miR-21 targeting RUNX1 and RUNX1 interaction with YAP were evaluated by RIP, dual-luciferase reporter gene, and ChIP assays.ResultsMiR-21 inhibition by its antagomir reduced the proportion of MDSCs, increased the proportion of Th and CTL in peripheral blood and tumor tissues of Lewis lung-cancer-bearing mice, protected Th and CTL from the suppression of MDSCs, increased apoptosis of MDSCs, but reduced IL-10, TGF-β and GM-CSF levels in mouse serum. RUNX1 could transcriptionally inhibit the YAP expression, whereas miR-21 targeting RUNX1 led to elevated YAP expression levels. Mechanistic investigation showed that miR-21 maintained MDSC accumulation in tumor microenvironment and promoted immunosuppressive ability of MDSCs in Lewis lung-cancer-bearing mice by down-regulating RUNX1and up-regulating YAP.ConclusionsTaken together, the study provides evidence that targeting miR-21 in MDSCs may be developed as an immunotherapeutic approach to combat lung cancer development.

Abstract 3414: Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis

Abstract Myeloid derived suppressor cells (MDSCs) antagonize antitumor immune responses, and limit the efficacy of immune based therapies for cancer. As a result, MDSCs have garnered attention as therapeutic targets. Unfortunately, there has been limited success in translating agents targeting MDSCs to the clinic. Brd4 is an epigenetic reader and is itself a therapeutic target in oncology due to its ability to regulate the expression of oncogenes such as Myc. In addition, Brd4 is known to regulate inflammatory cytokine production and innate immune responses including myeloid cell function. As a result, it was hypothesized that inhibition of Brd4 would impact MDSC function or expansion. In multiple tumor models (EMT6, 4T1, LLC, and C26) Brd4 inhibitors, both experimental (JQ1) and those in clinical development (PLX51107 and PLX2853) significantly reduced the abundance of total, PMN, and M-MDSC subsets within the tumor and spleen as measured by flow cytometry and IHC (p&amp;lt; 0.05). Furthermore, Nanostring gene expression analysis of EMT6 tumors identified a significant reduction in the abundance signature of neutrophils and macrophages with PLX51107 further confirming a reduction of myeloid cells in the presence of a Brd4 inhibitor (p&amp;lt; 0.01). The Nanostring analysis also showed a significant reduction of IL6 mRNA in PLX51107 treated tumors, and this was confirmed at the protein level by ELISA in the serum of EMT6 and 4T1 tumor bearing mice (p&amp;lt; 0.05). However, exogenous IL6 could not rescue MDSC levels in vivo or in vitro suggesting Brd4 inhibition might be directly affecting MDSCs. This was confirmed by showing that PLX51107 significantly inhibited the expansion of MDSCs from mouse bone marrow cells or healthy donor PBMCs cultured in IL6 and GMCSF as measured by flow cytometry (p&amp;lt;0.05). Furthermore, mouse and FACS sorted MDSCs from patients with melanoma or bladder cancer were found to undergo apoptosis when treated with PLX51107 as measured by flow cytometry (annexin and caspase3, p&amp;lt; 0.05), IF, and western blot. In addition, a pancaspase inhibitor rescued both mouse and patient derived MDSCs from apoptosis in the presence of PLX51107. Further investigation with specific caspase inhibitors showed this effect relied mostly on activation of the intrinsic apoptosis pathway and caspase9. The importance of Brd4 to MDSC survival was further confirmed using a LysMCre Brd4 floxed mouse model. Finally, depletion of MDSCs in vivo with PLX51107 significantly enhanced the efficacy of anti-PDL1 therapy as compared to either agent alone (p&amp;lt; 0.05 in EMT6, 4T1, and LLC tumor models). These results identify Brd4 and the TXNIP/ASK1 apoptosis pathway as novel regulators of MDSC survival, and provide evidence to further investigate Brd4 inhibitors in combination with immune based therapies for solid tumors. Citation Format: Andrew Stiff, Himanshu Savardekar, Robert Wesolowski, Megan Duggan, Luke Scarberry, Brooke Benner, Darren Wethington, Gabby Lapurga, Steven Sun, Jack Hedberg, Logan Good, William E. Carson. Brd4 inhibition enhances checkpoint therapy by inducing MDSC apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3414.

GCN2 drives macrophage and MDSC function and immunosuppression in the tumor microenvironment.

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

Danger-associated extracellular ATP counters MDSC therapeutic efficacy in acute GVHD

AbstractMyeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD), donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer, resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen–induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival, indicating in vivo biologic effects, no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1β (IL-1β), we expected that IL-1β KO donor MDSCs would be superior in subverting GVHD, but such MDSCs proved inferior relative to WT. IL-1β release and IL-1 receptor expression was required for optimal MDSC function, and exogenous IL-1β added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1β could diminish survival in GVHD. However, loss of inflammasome activation and IL-1β release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1β signaling, improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum, the ATP release site, synergized with WT MDSCs, as did regulatory T-cell infusion, which we showed reduced but did not eliminate MDSC inflammasome activation, as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments.