Levels Of Myeloid-derived Suppressor Cells Research Articles

Metformin generates profound alterations in systemic and tumor immunity with associated antitumor effects

BackgroundMetformin is a commonly used antidiabetic medication which has demonstrated promise as an anticancer agent alone and in combination with conventional treatment regimens. There is increasing evidence that metformin can...

Abstract 507: Association of circulating myeloid derived suppressor cells with systemic lymphopenia following chemoradiotherapy in glioblastoma

Abstract Systemic lymphopenia is common among glioblastoma (GBM) patients after chemoradiotherapy and is observed up to six months to 1 year after irradiation. Systemic lymphopenia is associated with a worse survival outcome in GBM. To delineate the underlying mechanisms of lymphopenia in GBM, we prospectively evaluated peripheral blood of GBM patients treated with standard radiation therapy (RT) and concurrent temozolomide as well as conducted in vivo experiments using a syngeneic murine GBM model. Among 20 patients prospectively evaluated, 10 patients (50%) developed lymphopenia and the other 10 did not. The RNA sequencing analysis of the peripheral blood mononuclear cell (PBMC) showed an elevated level of myeloid-derived suppressor cells (MDSCs) regulatory genes including Arg-1 in lymphopenic patients compared to non-lymphopenic patients after chemoradiotherapy. The single-cell RNA sequencing (scRNA-Seq) analysis of two selected phenotypical patients also demonstrated that a severe lymphopenic patient had elevated levels of two types of monocytic MDSCs (M-MDSCs) after RT compared to a non-lymphopenic patient. Immunophenotyping analysis of the PBMCs using flow cytometry confirmed reductions of CD4 and CD8 T cells as well as increased polymorphonuclear MDSCs (PMN-MDSCs) and M-MDSCs in lymphopenic patients compared to non-lymphopenic patients. The in vitro T cell proliferation and functional assays showed that MDSCs from lymphopenic patients exhibited were more suppressive compared to MDSCs from non-lymphopenic patients. Using a syngeneic orthotopic mouse (C57BL/6) GBM (GL261) model, we demonstrated that fractionated RT (2 Gy/day x 5) reduced CD4/CD8 cells and increased MDSCs compared to sham irradiated mice. The MDSCs from the irradiated mice also had increased Arg-1 expression. The addition of Arg-1 inhibitor (Arg1i) or phosphodiesterase type 5 inhibitor (PDE5i) to RT blocked the radiation-induced increase of MDSCs and reduction of CD4/CD8 cells. The combination of either Arg1i or PDE5i also enhanced the survival of mice with orthotopic tumors when compared to mice treated with RT alone or drug alone. In conclusion, we found that RT can induce an increase in circulating MDSCs in both patients and mice and the resulting circulating MDSCs can suppress T-cell proliferation and function. Inhibition of MDSCs using either Arg1i or PDE5i during RT may prevent systemic lymphopenia and affect disease control, which should warrant further investigation in clinical trials. Citation Format: Subhajit Ghosh, Matthew Inkman, Jin Zhang, Sukrutha Thotala, Ekaterina Tikhonova, Natalia Miheecheva, Felix Frenkel, Ravshan Ataullakhanov, Xiaowei Wang, Dennis Hallahan, Jiayi Huang, Dinesh Thotala. Association of circulating myeloid derived suppressor cells with systemic lymphopenia following chemoradiotherapy in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 507.

Abstract 1023: Understanding the anti-tumor activities of daratumumab in natural killer/T cell lymphoma (NKTCL)

Abstract Background: NKTCL is an aggressive and heterogenous type of non-Hodgkin's lymphoma that is characterized by malignant proliferation of cytotoxic natural killer (NK) or T cells and is associated with Epstein-Barr virus (EBV). Previous studies have shown that NKTCL tumors variably express CD38. Thus daratumumab (DARA), a human IgGκ monoclonal antibody targeting CD38 with direct on-tumor and immunomodulatory mechanism of action, was hypothesized to be a novel therapeutic option for patients with relapsed or refractory (R/R) NKTCL. The open-label, single-arm, multicenter, phase 2 study NKT2001 (NCT02927925) assessed the safety and efficacy of DARA in patients with R/R NKTCL, with an overall response rate of 25% and a 55 day median duration of response (DOR). Baseline serum EBV and CD38 expression levels did not show a clear correlation with clinical response. One NKTCL patient whose tumors did not express CD38 responded to treatment, suggesting that the immunomodulatory activities of DARA may be sufficient to confer clinical benefit. To understand the suboptimal response rate and short DOR, we interrogated the environmental, viral, and immune profile of NKTCL patients from NKT2001 in the context of DARA anti-tumor activity. Methods: CD38 expression was assessed by immunohistochemistry. Patient immune landscapes were assessed by cytometry by time-of-flight (CyTOF), multi-parameter flow cytometry (MPFC), clonal sequencing, and serum EBV level measurements. Results: Based on CyTOF and MPFC, high levels of baseline naive CD38- B cells with low percentage of CD38+ B cell populations (including transitional B, plasmablast, and plasma cells) were found to be significantly correlated with clinical response. However, subsequent B and T cell receptor sequencing did not detect clear relationships between pre-existing or treatment-emergent EBV-related B or T cell clones with clinical response. CyTOF analyses demonstrated a reduction of CD38+ NK cells, NK T cells, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) with DARA treatment. Nevertheless, persisting NK and NK T cells maintained cell surface markers indicative of functionality. Total Tregs and MDSCs levels did not change despite diminished CD38 expression, suggesting that DARA did not reverse the immune suppression mediated by Tregs or MDSCs. In contrast, there was a trend towards a decrease in CD4/CD8 ratio from baseline in responders but not in nonresponders, suggesting modulation of the immune environment is crucial for DARA anti-tumor activities in NKTCL. Conclusion: These results demonstrate that DARA can lead to clinical response in patients with NKTCL by modulating the immune environment. Citation Format: Tianyuan Zhou, Min Qing, Yann Abraham, Tatiana Perova, Cheryl Sweeney, Maria Krevvata. Understanding the anti-tumor activities of daratumumab in natural killer/T cell lymphoma (NKTCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1023.

Soluble interleukin-2 receptor as a predictive and prognostic marker for patients with familial breast cancer.

The incidence of breast cancer increases annually, and it has become common within families of breast cancer patients. Interleukin-2 activates cytotoxic T lymphocytes, which are important for cancer immunity. To identify markers of increased familial breast cancer risk, soluble interleukin-2 receptor levels and immunologic factors were investigated in familial breast cancer and non-familial breast cancer patients. Of 106 untreated breast cancer patients in this study, 24 had familial breast cancer and 82 had non-familial breast cancer. The patients' soluble interleukin-2 receptor, interleukin-10, vascular endothelial growth factor, interleukin-17, regulatory T cell, myeloid-derived suppressor cell, white blood cell, and C-reactive protein levels, and their neutrophil-to-lymphocyte ratios were measured, and their prognoses were compared according to the soluble interleukin-2 receptor levels. Additionally, postoperative tissues from the patients with high soluble interleukin-2 receptor levels were stained with programmed cell death ligand 1 and cluster of differentiation 8. The soluble interleukin-2 receptor level in the familial breast cancer patients was significantly higher, and it showed significantly stronger correlations with the neutrophil-to-lymphocyte ratio and the interleukin-10, vascular endothelial growth factor, interleukin-17, regulatory T cell, myeloid-derived suppressor cell, white blood cell, and C-reactive protein levels, than in the non-familial breast cancer patients. The regulatory T cell and myeloid-derived suppressor cell levels were significantly higher in the patients with high soluble interleukin-2 receptor levels, and the overall survival and disease-free-survival rates were significantly worse for the familial breast cancer patients than for the non-familial breast cancer patients. Triple-negative breast cancer tissues from the familial breast cancer patients with high soluble interleukin-2 receptor levels stained well for programmed cell death ligand 1 and cluster of differentiation 8. Soluble interleukin-2 receptor levels can be used to predict the prognosis of familial breast cancer patients. Prospectively identifying patients who are less likely to have non-familial breast cancer is vital for improving their overall survival.

Abstract 359: Molecular signatures associated with long-term response to apalutamide (APA) in patients (pts) with metastatic castration-sensitive prostate cancer (mCSPC) in TITAN

Abstract Background: TITAN, a phase 3 placebo (PBO)-controlled study in a broad population of pts with mCSPC (NCT02489318), demonstrated that the addition of APA to androgen deprivation therapy (ADT) significantly improved radiographic progression-free survival (rPFS) (median rPFS in the APA and PBO groups was not reached and was 22.1 months, respectively) and overall survival (OS) compared with PBO + ADT (Chi N Engl J Med 2019). This exploratory analysis investigated molecular signatures of pts with mCSPC who had long-term responses to APA. Methods: Pts (n = 222) in the biomarker cohort of TITAN were characterized as long-term responders (LTR) or early progressors (EP) by separating rPFS into quartiles. The last quartile with longest rPFS (APA, n = 28; PBO, n = 28) represented LTR. The first quartile, with shortest rPFS (APA, n = 17; PBO, n = 21) represented EP. Gene expression profiles of TITAN pts were generated from archival primary prostate tumors; data were summarized based on 110 predefined gene signatures. Mean signature scores between LTR and EP groups were compared within treatment groups using 2 sample t tests to evaluate whether effects were prognostic or specific to APA treatment. Signatures were also evaluated for association with outcome using Cox proportional hazard models in each treatment group. Results: At primary analysis, with median 22.7 months follow-up, median rPFS was, respectively, not reached and 32.9 months in APA and PBO pts in the LTR group, and 10.9 and 3.7 months in APA and PBO pts in the EP group. A higher level of expression (above median) of either polycomb repressor complex 2 activation or the Decipher signature was more frequently associated with EP in both groups. LTR in the APA treatment group, but not those in the PBO group, had lower expression of transcriptional signatures describing myeloid-derived suppressor cells (MDSCs) and cell cycle progression. These results suggest that pts with mCSPC who have relatively lower levels of MDSCs in the tumor, indicative of a less immune-suppressed tumor microenvironment, may have long-term benefit from treatment with APA. Conclusions: Consistent clinical benefit was observed with the addition of APA to ADT in both EP and LTR. Although the data require confirmation in larger studies, these molecular signatures may have utility in selecting pts with mCSPC who may derive the most benefit from APA and other androgen signaling inhibitors. Citation Format: Justin Lucas, Neeraj Agarwal, Felix Feng, Clemente Aguilar-Bonavides, Shibu Thomas, Michael Gormley, Suneel Mundle, Sabine Brookman-May, Sharon A. McCarthy, Anders Bjartell, Hirotsugu Uemura, Simon Chowdhury, Kim N. Chi. Molecular signatures associated with long-term response to apalutamide (APA) in patients (pts) with metastatic castration-sensitive prostate cancer (mCSPC) in TITAN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 359.

CD33/CD3-Bispecific T-Cell Engaging (BiTE®) Antibody Constructs Efficiently Target Monocytic CD14+ hla-DRlow IDO+ aml-MDSCs

Acute Myeloid Leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 25%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. In this study we sought to investigate whether the CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with documented pre-clinical activity against AML blasts can also target CD33+ AML-MDSCs by redirecting and activating T-cells and thereby enhance AMG 330 mediated anti-leukemic activity.First, we assessed the presence of circulating CD14+ cells that co-express CD33 but lack HLA-DR expression (HLA-DRlo) in patients with newly diagnosed AML. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14+HLA-DRlo cells was significantly increased in untreated AML patients as compared to healthy controls (28,98±4,19%, n=13 versus 3,28±0,75%, n=37) but did not correlate with the number of AML blasts. Purified CD14+HLA-DRlo cells suppressed in vitro T-cell proliferation in a concentration-dependent manner allowing us their denomination as MDSCs.Interestingly, we also observed that primary AML blasts and human AML cell lines (THP, OCI-AML, and HL-60) induced HLA-DRlo cells from healthy donor-derived monocytes that were T-cell suppressive and expressed the immune regulatory indoleamine-2,3-dioxygenase (IDO). Functional and phenotypic reprogramming of monocytes was cell-contact independent.Using ex vivo co-culturing models of primary AML blasts/AML cell lines (=target cells) and T-cells (=effector cells) we observed AMG 330-triggered antibody construct-dependent cell-mediated cytotoxicity/ADCC. Short-term (three to six days) treatment of AML patient-derived peripheral blood mononuclear cells (PBMCs) with 10 or 100 pM AMG 330 led to T-cell activation based on proliferation and on increased expression/production of CD25, CD69, CD137, CD154, IL-2, and IFN-g. The initial T-cell frequency ranging from 0,16 to 14,30% had no impact on their responsiveness. Moreover, we observed a bystander activation of NK-cells. The addition of AML-MDSCs into these co-cultures did not negatively impact ADCC activity. On the contrary, T-cells formed immunological synapses with AML-MDSCs and triggered their elimination (also in presence of AML blasts) overcoming their T-cell suppressive effect. MDSC levels in AML-PBMCs had no effect on AMG 330-mediated T-cell (and bystander cell) activation, which was further corroborated in experiments where the total CD14+ cell compartment (comprising AML-MDSCs) was removed prior AMG 330 application.However, we did observe an impact of MDSCs on BiTE® mediated T-cell proliferation, which has been shown as highly relevant for clinical activity of BiTE® antibody constructs (Zugmaier et al. Blood 2015). Here we found a 1,68-fold enhanced (p=0.001) T-cell expansion in AML-PBMCs treated with 100 pM AMG 330 following CD14+ cell depletion. Given the importance of T-cell proliferation for clinical activity this finding warrants further exploration.Taken together, our results suggest that anti-CD33/CD3 bispecific BiTE® antibody constructs may achieve anti-leukemic efficacy not only through direct T cell mediated cytotoxicity against AML blasts but also through circumventing immune evasion via MDSCs targeting. Although therapeutic targeting of MDSCs in patients has not yet been successfully accomplished, bystander killing of CD33+ MDSCs via anti-CD33/CD3-bispecific BiTE® antibody constructs could represent a very promising approach to increase the anti-leukemic T-cell response in AML patients and to reverse immune evasion. DisclosuresMougiakakos:AMGEN: Research Funding. Kischel:AMGEN: Employment. Michael:AMGEN: Employment. Dos Santos:Amgen Inc.: Employment. Jitschin:AMGEN: Research Funding. Mackensen:AMGEN: Research Funding.

Immunological discrepancy in aged mice facilitates skin allograft survival.

More and more aged people are undergoing organ transplantation. Understanding aging effects on immunity will be helpful for post-transplantation care and adjustment of immunosuppressants for aged recipients. A mouse model, using C3H mice as donors and aged/young C57BL/10J mice as recipients, was employed to study aging effects on immunity. The results showed that frequency of myeloid-derived suppressor cells (MDSC) and level of TGF-β was higher in aged mice than in young mice (4.4 ± 1.4% versus 1.6 ± 1.1%, p = 0.026 for MDSC; 21.04 ± 3.91 ng/ml versus 15.26 ± 5.01 ng/ml, p = 0.026 for TGF-β). In vivo, skin allograft survived longer on the aged than on young mice (19.7 ± 5.2 days versus 11.9 ± 4.1 days, p = 0.005). When entinostat was applied to block MDSC, the survival of skin allografts on aged mice was shorten to 13.5 ± 4.7 days which was not different from the survival on young mice (p = 0.359). In conclusion, allogeneic immunity was different in aged from young mice in high frequency of MDSC and high serum level of TGF-β. Blocking the function of MDSC reversed the low immunity in aged mice and caused skin allograft rejection similar to young recipients.

Dickkopf-1: A Promising Target for Cancer Immunotherapy.

Clinical studies in a range of cancers have detected elevated levels of the Wnt antagonist Dickkopf-1 (DKK1) in the serum or tumors of patients, and this was frequently associated with a poor prognosis. Our analysis of DKK1 gene profile using data from TCGA also proves the high expression of DKK1 in 14 types of cancers. Numerous preclinical studies have demonstrated the cancer-promoting effects of DKK1 in both in vitro cell models and in vivo animal models. Furthermore, DKK1 showed the ability to modulate immune cell activities as well as the immunosuppressive cancer microenvironment. Expression level of DKK1 is positively correlated with infiltrating levels of myeloid-derived suppressor cells (MDSCs) in 20 types of cancers, while negatively associated with CD8+ T cells in 4 of these 20 cancer types. Emerging experimental evidence indicates that DKK1 has been involved in T cell differentiation and induction of cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms of DKK1 affecting cancers and immune cells have received great attention. This review introduces the rapidly growing body of literature revealing the cancer-promoting and immune regulatory activities of DKK1. In addition, this review also predicts that by understanding the interaction between different domains of DKK1 through computational modeling and functional studies, the underlying functional mechanism of DKK1 could be further elucidated, thus facilitating the development of anti-DKK1 drugs with more promising efficacy in cancer immunotherapy.

Determinants of response to checkpoint inhibition in ovarian cancer.

e17538 Background: Despite evidence that ovarian cancer (OC) is an immunogenic disease, single-agent checkpoint inhibitors have shown limited response. A clinical trial was conducted evaluating dose-dense paclitaxel and pembrolizumab in platinum-resistant OC. Our aim was to evaluate for biomarkers of response to this combination. Methods: Immunohistochemistry was performed to identify PD–L1 and tumor infiltrating lymphocytes (TIL) in pretreatment formalin fixed paraffin embedded tissue. We performed flow cytometry on matched blood to identify presence of immunosuppressive myeloid-derived suppressor cells (MDSCs) in circulation. We performed T-cell receptor (TCR) sequencing on tumor and matched blood to evaluate whether an oligoclonal or polyclonal immune response would be associated with outcomes. On selected patients we assessed peripheral T-cell responses to treatment using an interferon-ɣ enzyme-linked immunospot (IFN-ɣ ELISpot) assay between excellent and poor responders. Cox proportional hazards model was used to estimate hazards ratios (HR) for response in relation to each biomarker. Kaplan-Meier analysis and log-rank test were used to evaluate associations between progression free survival (PFS), overall survival (OS) and each biomarker. Results: Analysis was limited to 34 patients with tumor available and one RECIST response evaluation. Odds of response trended higher (OR = 4.1) in patients with PD-L1 (+) than patients with PD-L1 (-) tumors (p = 0.061). When restricting analysis to best response of CR/PR versus PD, the odds was 13.0 (p = 0.041). Patients with PD-L1(+) tumors had a HR for progression 0.56 (0.26-1.22; p = 0.144). Presence of TILs was classified as 0-3 (0-1 low, 2-3 high). High TIL presence was not associated with response, but was associated with improved OS and trend toward improved PFS; HR 0.29 (0.09-0.90, p = 0.022) and HR 0.47 (0.21-1.05, p = 0.06). Pre- and post-treatment levels of MDSCs were not associated with response or survival, though a trend was noted; a one unit increase in pretreatment MDSCs leads to 5% decrease in the odds of being responder. Immunoseq analysis of TCR repertoire pre- and post-treatment suggested that in increase in productive clonality was associated with increased odds of being a responder; this was not statistically significant. Notably, IFN-ɣ ELISpot assay demonstrated a significant T-cell response against neoantigens post-treatment in patients who had a partial response and long overall survival. Post treatment T-cell response showed a significantly mixed response to neoantigens in the patient analyzed who had progressive disease. Conclusions: Finding biomarkers predicting benefit from checkpoint inhibitors has been challenging. Though inconclusive, these data suggest biomarkers that may be associated with this combination. T-cell response to neoantigens in patients with response to treatment suggests neoantigens as a potential target for therapeutic direction.

Identification of tumor-infiltrating lymphocyte subpopulations correlated with patient prognosis in esophageal squamous cell carcinoma.

ObjectiveTo identify biomarkers related to esophageal squamous cell carcinoma (ESCC) prognosis by analyzing genetic variations and the infiltration levels of tumor-infiltrating lymphocytes (TILs) in patients.MethodsThe clinical features of 61 patients with ESCC were collected. DNA panel sequencing was performed to screen differentially expressed genes (DEGs). Transcriptome sequencing was performed to identify gene expression profiles, and subsequent enrichment analysis of DEGs was conducted using Metascape.ResultsWe identified 488 DEGs between patients with ESCC with distinct prognoses that were mainly enriched in the human immune response, fibrinogen complex, and protein activation cascade pathways. Among patients with ESCC treated with postoperative chemotherapy, those with a high infiltration level of myeloid-derived suppressor cells (MDSCs) had longer overall survival (OS), and OS was positively correlated with the infiltration level of T helper type 2 (Th2) cells among patients treated without chemotherapy after surgery. Additionally, in the case of MDSCs >0.7059 or Th2 cells <0.6290, patients receiving postoperative chemotherapy had a longer OS than those treated without chemotherapy following surgery.ConclusionThe level of MDSCs or Th2 cells can be used as a biomarker for assessing the prognosis of patients with ESCC treated with or without postoperative chemotherapy, respectively.

PMN-MDSC Frequency Discriminates Active Versus Latent Tuberculosis and Could Play a Role in Counteracting the Immune-Mediated Lung Damage in Active Disease.

Tuberculosis (TB), due to Mycobacterium tuberculosis infection, is still the principal cause of death caused by a single infectious agent. The balance between the bacillus and host defense mechanisms reflects the different manifestations of the pathology. Factors defining this variety are unclear and likely involve both mycobacterial and immunological components. Myeloid derived suppressor cells (MDSC) have been shown to be expanded during TB, but their role in human TB pathogenesis is not clear. We evaluated the frequency of circulating MDSC by flow-cytometry in 19 patients with active TB, 18 with latent TB infection (LTBI), and 12 healthy donors (HD) as control. Moreover, we investigated the capacity of MDSC to modulate the mycobactericidal activity of monocytes. The association between MDSC level and TB chest X-ray severity score was analyzed. We observed that, unlike active TB, polymorphonuclear (PMN)-MDSC are not expanded in LTBI patients, and, by performing a receiver operating characteristic (ROC) curve analysis, we found that PMN-MDSC frequency supported the discrimination between active disease and LTBI. Interestingly, we observed an association between PMN-MDSC levels and the severity of TB disease evaluated by chest X-ray. Specifically, PMN-MDSC frequency was higher in those classified with a low/mild severity score compared to those classified with a high severity score. Moreover, PMN-MDSC can impact mycobacterial growth by inducing ROS production in Bacillus Calmette et Guerin (BCG)-infected monocytes. This effect was lost when tested with M. tuberculosis (MTB), In conclusion, our data indicate that the elevated frequency of PMN-MDSC in IGRA-positive individuals is associated with active TB. Our findings also pointed out a beneficial role of PMN-MDSC during human active TB, most likely associated with the limitation of inflammation-induced tissue damage.

Yu-Ping-Feng Formula Exerts Antilung Cancer Effects by Remodeling the Tumor Microenvironment through Regulating Myeloid-Derived Suppressor Cells.

Yu-Ping-Feng (YPF) formula is a classical prescription used for enhancing the body's immunity function in traditional Chinese medicine (TCM). In clinical practice, the YPF formula has been reported to exhibit antilung cancer and immunomodulatory effect. However, the relationship between them remains unclear. The present study aimed to investigate the antilung cancer effect of the YPF formula and its immune-related mechanisms. The C57BL/6 tumor-bearing mice model was established and randomly divided into the YPF group and the control group. Tumor volume, spleen weight, and survival in both groups were measured and evaluated during 28 days of consecutive intervention. Flow cytometry was used to detect the proportion of immune cell subsets. Myeloid-derived suppressor cells (MDSCs) were induced in vitro from bone marrow cells. After intervention by the YPF formula, CCK-8 and flow cytometry analyses were performed to detect proliferation and apoptosis of MDSCs. A coculture system containing T cells and MDSCs was established to further study the role of MDSCs in the regulation of T-cell subsets proportion by the YPF formula. The expressions of MDSCs-related genes and proteins were detected by RT-PCR and Western blotting. The results showed the YPF formula inhibited tumor growth, reduced spleen weight, and prolonged the survival of mice. Besides, the proportions of MDSCs subsets and Regulatory T (Treg) in the YPF group decreased, whereas those of CD4+T and CD8+T increased both in vitro and in vivo. CCK-8 and flow cytometry demonstrated that the YPF formula could inhibit proliferation and promote apoptosis of MDSCs. The coculture experiments further confirmed that MDSCs served a critical role in regulating the tumor microenvironment by the YPF formula. RT-PCR and Western blotting indicated that the levels of MDSCs' activation and proliferation-related proteins and genes were downregulated in the YPF group. Therefore, our results demonstrated that the YPF formula could promote apoptosis and inhibit the proliferation of MDSCs. As a result, the negative regulatory effect on the positive immune cells induced by MDSCs was weakened, thus achieving the antilung cancer effect by remodeling the tumor microenvironment.

Novel TGFβ Inhibitors Ameliorate Oral Squamous Cell Carcinoma Progression and Improve the Antitumor Immune Response of Anti-PD-L1 Immunotherapy.

TGFβ is a key regulator of oral squamous cell carcinoma (OSCC) progression, and its potential role as a therapeutic target has been investigated with a limited success. This study evaluates two novel TGFβ inhibitors as mono or combinatorial therapy with anti-PD-L1 antibodies (α-PD-L1 Ab) in a murine OSCC model. Immunocompetent C57BL/6 mice bearing malignant oral lesions induced by 4-nitroquinoline 1-oxide (4-NQO) were treated for 4 weeks with TGFβ inhibitors mRER (i.p., 50 μg/d) or mmTGFβ2-7m (10 μg/d delivered by osmotic pumps) alone or in combination with α-PD-L1 Abs (7× i.p. of 100 μg/72 h). Tumor progression and body weight were monitored. Levels of bioactive TGFβ in serum were quantified using a TGFβ bioassay. Tissues were analyzed by immunohistology and flow cytometry. Therapy with mRER or mmTGFβ2-7m reduced tumor burden (P < 0.05) and decreased body weight loss compared with controls. In inhibitor-treated mice, levels of TGFβ in tumor tissue and serum were reduced (P < 0.05), whereas they increased with tumor progression in controls. Both inhibitors enhanced CD8+ T-cell infiltration into tumors and mRER reduced levels of myeloid-derived suppressor cells (P < 0.001). In combination with α-PD-L1 Abs, tumor burden was not further reduced; however, mmTGFβ2-7m further reduced weight loss (P < 0.05). The collagen-rich stroma was reduced by using combinatorial TGFβ/PD-L1 therapies (P < 0.05), enabling an accelerated lymphocyte infiltration into tumor tissues. The blockade of TGFβ signaling by mRER or mmTGFβ2-7m ameliorated in vivo progression of established murine OSCC. The inhibitors promoted antitumor immune responses, alone and in combination with α-PD-L1 Abs.

Comments on the ambiguity of selected surface markers, signaling pathways and omics profiles hampering the identification of myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSC) are important immune-regulatory cells but their identification remains difficult. Here, we provide a critical view on selected surface markers, transcriptional and translational pathways commonly used to identify MDSC by specific, their developmental origin and new possibilities by transcriptional or proteomic profiling. Discrimination of MDSC from their non-suppressive counterparts is a prerequisite for the development of successful therapies. Understanding the switch mechanisms that direct granulocytic and monocytic development into a pro-inflammatory or anti-inflammatory direction will be crucial for therapeutic strategies. Manipulation of these myeloid checkpoints are exploited by tumors and pathogens, such as M. tuberculosis (Mtb), HIV or SARS-CoV-2, that induce MDSC for immune evasion. Thus, specific markers for MDSC identification may reveal also novel molecular candidates for therapeutic intervention at the level of MDSC.

Myeloid-Derived Suppressor Cells in Immune Microenvironment Promote Progression of Esophagogastric Junction Adenocarcinoma.

Adenocarcinoma of the esophagogastric junction (AEG) is a fatal disease. Accumulating evidence indicates that, for a comprehensive understanding of AEG, studies should be conducted not only to investigate tumor cells, but also the tumor microenvironment (TME). In this study, we collected AEG patient data from The Cancer Genome Atlas, and used the CIBERSORT algorithm to analyze tumor-infiltrating immune cell profiles. The levels of CD8+ T cells and M0 and M2 macrophages were relatively high in AEG tissues. M2 macrophages were abundant in G3 tumors, and neutrophils were associated with poor prognosis. Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immunosuppressive cells which share a similar origin to neutrophils and macrophages. We further analyzed the levels of MDSCs in AEG patients and healthy donors (HD) using flow cytometry. MDSC levels were elevated at tumor sites, with polymorphonuclear MDSCs (PMN-MDSCs) being the predominant subtype. Circulating MDSCs partly represented cells at the tumor site. We observed that PMN-MDSC levels at tumor sites were positively correlated with advanced staging, low grade, lymph node metastasis, and HER2− status. Immunohistochemistry and immunofluorescence analyses indicated that activation of the STAT3 and NF-κB pathways in MDSCs may be a potential mechanism for cancer progression. Our studies provided a comprehensive perspective involving tumor-infiltrating immune cells, and detailed insights into the proportion of MDSCs in AEG and their clinical significance. Together, these findings may improve our current understanding of cancer progression involving tumor-infiltrating immune cells in the TME.

165P Baseline circulating myeloid-derived suppressor cells correlate with neutrophil-to-lymphocyte ratio and overall survival in advanced non-small cell lung cancer patients treated with immune checkpoint inhibitors

Background: Immune Checkpoint Inhibitors (ICI) have become a care standard in advanced non-small cell lung cancer (aNSCLC). Myeloid-Derived Suppressor Cells (MDSC) are potent immunity suppressors and may represent both a potential prognostic and a predictive biomarker. Neutrophil-to-Lymphocyte Ratio (NLR) is an inflammatory biomarker with a prognostic role in NSCLC. We hypothesized that MDSC levels correlate with NLR and overall survival (OS) in aNSCLC.

Immune Cell Profiling of Peripheral Blood as Signature for Response During Checkpoint Inhibition Across Cancer Types.

Despite encouraging results with immune checkpoint inhibition (ICI), a large fraction of cancer patients still does not achieve clinical benefit. Finding predictive markers in the complexity of the tumor microenvironment is a challenging task and often requires invasive procedures. In our study, we looked for putative variables related to treatment benefit among immune cells in peripheral blood across different tumor types treated with ICIs. For that, we included 33 patients with different solid tumors referred to our clinical unit for ICI. Peripheral blood mononuclear cells were isolated at baseline, 6 and 20 weeks after treatment start. Characterization of immune cells was carried out by multi-color flow cytometry. Response to treatment was assessed radiologically by RECIST 1.1. Clinical outcome correlated with a shift towards an effector-like T cell phenotype, PD-1 expression by CD8+T cells, low levels of myeloid-derived suppressor cells and classical monocytes. Dendritic cells seemed also to play a role in terms of survival. From these findings, we hypothesized that patients responding to ICI had already at baseline an immune profile, here called ‘favorable immune periphery’, providing a higher chance of benefitting from ICI. We elaborated an index comprising cell types mentioned above. This signature correlated positively with the likelihood of benefiting from the treatment and ultimately with longer survival. Our study illustrates that patients responding to ICI seem to have a pre-existing immune profile in peripheral blood that favors good outcome. Exploring this signature can help to identify patients likely to achieve benefit from ICI.

Immunosuppressive Microenvironment Revealed by Immune Cell Landscape in Pre-metastatic Liver of Colorectal Cancer.

Background: Colorectal cancer, the fourth leading cause of cancer mortality, is prone to metastasis, especially to the liver. The pre-metastatic microenvironment comprising various resident stromal cells and immune cells is essential for metastasis. However, how the dynamic evolution of immune components facilitates pre-metastatic niche formation remains unclear.Methods: Utilizing RNA-seq data from our orthotopic colorectal cancer mouse model, we applied single sample gene set enrichment analysis and Cell type Identification By Estimating Relative Subsets Of RNA Transcripts to investigate the tumor microenvironment landscape of pre-metastatic liver, and define the exact role of myeloid-derived suppressor cells (MDSCs) acting in the regulation of infiltrating immune cells and gene pathways activation. Flow cytometry analysis was conducted to quantify the MDSCs levels in human and mice samples.Results: In the current work, based on the high-throughput transcriptome data, we depicted the immune cell infiltration pattern of pre-metastatic liver and highlighted MDSCs as the dominant altered cell type. Notably, flow cytometry analysis showed that high frequencies of MDSCs, was detected in the pre-metastatic liver of orthotopic colorectal cancer tumor-bearing mice, and in the peripheral blood of patients with stage I–III colorectal cancer. MDSCs accumulation in the liver drove immunosuppressive factors secretion and immune checkpoint score upregulation, consequently shaping the pre-metastatic niche with sustained immune suppression. Metabolic reprogramming such as upregulated glycolysis/gluconeogenesis and HIF-1 signaling pathways in the primary tumor was also demonstrated to correlate with MDSCs infiltration in the pre-metastatic liver. Some chemokines were identified as a potential mechanism for MDSCs recruitment.Conclusion: Collectively, our study elucidates the alterations of MDSCs during pre-metastatic niche transformation, and illuminates the latent biological mechanism by which primary tumors impact MDSC aggregation in the targeted liver.

β‐glucan therapy converts the inhibition of myeloid‐derived suppressor cells in oral cancer patients

The myeloid-derived suppressor cells (MDSCs) frequently have a high expansion in cancer patients. This research explored whether administration of β-glucan could increase anti-tumor immunity in oral squamous cell carcinoma (OSCC) patients. This study evaluated the MDSC level of circulating blood as CD33+ /CD11b+ /HLA-DR-/low by flow cytometry in 30 healthy donors (HDs, group I), in 48 oral squamous cell carcinoma (OSCC) patients before and after 14-day preoperative administration of β-glucan (group II), and in 52 OSCC patients without taking β-glucan (group III). A significantly higher mean MDSC level was observed in 100 OSCC patients than in 30 HDs (p<.001). There was a significant reduction of the mean MDSC level in group II patients after taking β-glucan (p<.001). Moreover, we discovered a significantly higher recurrence-free survival (RFS) in group II than in group III patients (p=.026). Finally, the multivariate Cox regression further identified the MDSC level ≤1% and administration of β-glucan as more favorable prognostic factors for OSCC patients. Preoperative administration of β-glucan can augment anti-tumor immunity and increase RFS rate via subversion of suppressive function of MDSC in OSCC patients.

Increased incidence of venous thromboembolism with cancer immunotherapy

Cancer immunotherapy is associated with several immune-related adverse events, but the relationship between immunotherapy and venous thromboembolism has not been thoroughly studied. We conducted a retrospective cohort study of 1,686 patients who received immunotherapy for a variety of malignancies to determine the incidence of venous thromboembolism and the impact of venous thromboembolism on survival. To examine the potential role of inflammation in venous thromboembolism, we also profiled immune cells and plasma cytokines in blood samples obtained prior to initiation of immunotherapy in a sub-cohort of patients treated on clinical trials who subsequently did (N = 15), or did not (N = 10) develop venous thromboembolism. Venous thromboembolism occurred while on immunotherapy in 404/1686 patients (24%) and was associated with decreased overall survival [HR=1.22 (95% CI 1.06-1.41), p<0.008]. Patients that developed venous thromboembolism had significantly higher pretreatment levels of myeloid-derived suppressor cells (5.382 ± 0.873 vs. 3.341 ± 0.3402, mean ± SEM; p=0.0045), interleukin 8 (221.2 ± 37.53 vs. 111.6 ± 25.36, mean ± SEM; p=0.016), and soluble vascular cell adhesion protein 1 (1210 ± 120.6 vs. 895.5 ± 53.34, mean ± SEM; p=0.0385). These findings demonstrate that venous thromboembolism is an underappreciated and important immune-related adverse event associated with cancer immunotherapy, and may implicate an interleukin 8 and myeloid-derived suppressor cell-driven pathway in pathogenesis.