Tcf-1 protects anti-tumor TCR-engineered CD8+ T-cells from GzmB mediated self-destruction.

BACKGROUND: T-cell longevity is undermined by antigen-driven differentiation programs that render cells prone to attrition through several mechanisms. CD8 + T cells that express the Tcf-1 transcription factor have undergone limited differentiation and exhibit stem-cell-like replenishment functions that facilitate persistence. We engineered human CD8 + T cells to constitutively express Tcf-1 and a TCR specific for the NY-ESO-1 cancer-associated antigen. Co-engineered cells were assessed for their potential for adoptive cellular immunotherapy. METHODS: Tcf-1 mRNA encoding TCF-1B and TCF-1E isoforms, along with GzmB expression were assessed in CD62L + CD57 -, CD62L - CD57 -, and CD62L - CD57 + CD8 + T cells derived from normal donor lymphocytes. The impact of stable Tcf-1B expression on CD8 + T-cell phenotype, anti-tumor activity, and cell-cycle activity was assessed in vitro and in an in vivo tumor xenograft model. RESULTS: TCF-1B and TCF-1E were dynamically regulated during self-renewal, with progeny of recently activated naïve T cells more enriched for TCF-1B mRNA. Constitutive TCF-1B expression improved the survival of TCR-engineered CD8 + T cells upon engagement with tumor cells. Tcf-1B prohibited the acquisition of a GzmB High state, and protected T cells from apoptosis associated with elicitation of effector function, and promoted stem cell-like characteristics. CONCLUSIONS: Tcf-1 protects TCR-engineered CD8 + T cells from activation induced cell death by restricting GzmB expression. Our study presents constitutive Tcf-1B expression as a potential means to impart therapeutic T cells with attributes of persistence for durable anti-tumor activity.

Comparing thermal stress reduction strategies that influence MDSC accumulation in tumor bearing mice.

Myeloid derived suppressor cells (MDSCs) are a diverse collection of immune cells that suppress anti-tumor immune responses. Decreasing MDSCs accumulation in the tumor microenvironment could improve the anti-tumor immune response and improve immunotherapy. Here, we examine the impact of physiologically relevant thermal treatments on the accumulation of MDSCs in tumors in mice. We found that different temperature-based protocols, including 1) weekly whole-body hyperthermia, 2) housing mice at their thermoneutral temperature (TT, ~30 °C), and 3) housing mice at a subthermoneutral temperature (ST,~22 °C) while providing a localized heat source, each resulted in a reduction in MDSC accumulation and improved tumor growth control compared to control mice housed at ST, which is the standard, mandated housing temperature for laboratory mice. Additionally, we found that low dose ß-adrenergic receptor blocker (propranolol) therapy reduced MDSC accumulation and improved tumor growth control to a similar degree as the models that relieved cold stress. These results show that thermal treatments can decrease MDSC accumulation and tumor growth comparable to propranolol therapy.

Blockade of Host ß2-Adrenergic Receptor Enhances Graft-versus-Tumor Effect through Modulating APCs.

Allogeneic hematopoietic cell transplantation is a potential curative therapy for hematologic malignancies. Host APCs are pivotal to the desired graft-versus-tumor (GVT) effect. Recent studies have shown that ß2-adrenergic receptor (ß2AR) signaling can have an important impact on immune cell function, including dendritic cells (DCs). In this article, we demonstrate that pretreatment of host mice with a ß2AR blocker significantly increases the GVT effect of donor CD8+ T cells by decreasing tumor burden without increasing graft-versus-host disease. ß2AR-deficient host mice have significantly increased effector memory and central memory CD8+ T cells and improved reconstitution of T cells, including CD4+Foxp3+ regulatory T cells. Notably, ß2AR deficiency induces increased CD11c+ DC development. Also, ß2AR-deficient bone marrow-derived DCs induce higher CD8+ T cell proliferation and improved tumor killing in vitro. Metabolic profiling shows that ß2AR deficiency renders DCs more immunogenic through upregulation of mTOR activity and reduction of STAT3 phosphorylation. Altogether, these findings demonstrate an important role for host ß2AR signaling in suppressing T cell reconstitution and GVT activity.

Contribution of Immune Cells to Glucocorticoid Receptor Expression in Breast Cancer.

Breast cancer (BC) patients experience increased stress with elevated cortisol levels, increasing risk of cancer recurrence. Cortisol binds to a cytoplasmic receptor, glucocorticoid receptor (GR) encoded by GR gene (NR3C1). We hypothesized that not only cancer cells, but even immune cells in the tumor microenvironment (TME) may contribute to GR expression in bulk tumor and influence prognosis. To test this, mRNA expression data was accessed from METABRIC and TCGA. "High" and "low" expression was based on highest and lowest quartiles of NR3C1 gene expression, respectively. Single-cell sequencing data were obtained from GSE75688 and GSE114725 cohorts. Computer algorithms CIBERSORT, Gene Set Enrichment Analysis and TIMER were used. GR-high BC has better median disease-free and disease-specific survival. Single cell sequencing data showed higher GR expression on immune cells compared to cancer and stromal cells. Positive correlation between GR-high BC and CD8+ T-cells was noted. In GR-high tumors, higher cytolytic activity (CYT) with decreased T-regulatory and T-follicular helper cells was observed. High GR expression was associated with lower proliferation index Ki67, enriched in IL-2_STAT5, apoptosis, KRAS, TGF-ß signaling, and epithelial-to-mesenchymal transition. Immune cells significantly contribute to GR expression of bulk BC. GR-high BC has a favorable TME with higher CYT with favorable outcomes.

T Cell-Derived CD70 Delivers an Immune Checkpoint Function in Inflammatory T Cell Responses.

The CD27-CD70 pathway is known to provide a costimulatory signal, with CD70 expressed on APCs and CD27 functions on T cells. Although CD70 is also expressed on activated T cells, it remains unclear how T cell-derived CD70 affects T cell function. Therefore, we have assessed the role of T cell-derived CD70 using adoptive-transfer models, including autoimmune inflammatory bowel disease and allogeneic graft-versus-host disease. Surprisingly, compared with wild-type T cells, CD70-/- T cells caused more severe inflammatory bowel disease and graft-versus-host disease and produced higher levels of inflammatory cytokines. Mechanistic analyses reveal that IFN-γ induces CD70 expression in T cells, and CD70 limits T cell expansion via a regulatory T cell-independent mechanism that involves caspase-dependent T cell apoptosis and upregulation of inhibitory immune checkpoint molecules. Notably, T cell-intrinsic CD70 signaling contributes, as least in part, to the inhibitory checkpoint function. Overall, our findings demonstrate for the first time, to our knowledge, that T cell-derived CD70 plays a novel immune checkpoint role in inhibiting inflammatory T cell responses. This study suggests that T cell-derived CD70 performs a critical negative feedback function to downregulate inflammatory T cell responses.

Host-Derived Serine Protease Inhibitor 6 Provides Granzyme B-Independent Protection of Intestinal Epithelial Cells in Murine Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic cell transplantation (allo-HCT) that limits the therapeutic potential of this treatment. Host antigen-presenting cells (APCs) play a vital role in activating donor T cells that subsequently use granzyme B (GzmB) and other cytotoxic molecules to damage host normal tissues. Serine protease inhibitor 6 (Spi6), known as the sole endogenous inhibitor of GzmB, has been implicated in protecting T cells and APCs against GzmB-inflicted damage. In this study we used murine models to examine the previously unknown role of host-derived Spi6 in GVHD pathogenesis. Our results indicated that host Spi6 deficiency exacerbated GVHD as evidenced by significantly increased lethality and clinical and histopathologic scores. Using bone marrow chimera system, we found that Spi6 in nonhematopoietic tissue played a dominant role in protecting against GVHD and was significantly upregulated in intestinal epithelial cells after allo-HCT, whereas Spi6 in hematopoietic APCs surprisingly suppressed alloreactive T cell response. Interestingly, the protective effect of Spi6 and its expression in intestinal epithelial cells appeared to be independent of donor-derived GzmB. We used in silico modeling to explore potential targets of Spi6. Interaction tested in silico demonstrated that Spi6 could inhibit caspase-3 and caspase-8 with the same functional loop that inhibits GzmB but was not capable of forming stable interaction with caspase-1 or granzyme A. Using an in vitro co-culture system, we further identified that donor T cell-derived IFN-γ was important for inducing Spi6 expression in an intestinal epithelial cell line. Altogether, our data indicate that host Spi6 plays a novel, GzmB-independent role in regulating alloreactive T cell response and protecting intestinal epithelial cells. Therefore, enhancing host-derived Spi6 function has the potential to reduce GVHD.

Key role of Dkk3 protein in inhibition of cancer cell proliferation: An in silico identification.

Dkk3 is a member of Dkk family proteins, regulating Wnt signaling. Dkk3 plays different roles in human and mouse tumors. Dkk3 predominantly act as a tumor suppressor, however several reports revealed that Dkk3 could accelerate cancer cell proliferation. Herein, we aimed at launching an in silico study to determine Dkk3 structure and its interactions with Kremen and LRP as Wnt signaling receptors as well as EGF receptor. Using various softwares a model was built for Dkk3 molecule. Different protein modeling approaches along with model refinement processes were employed to arrive at the final model. To achieve the final complex of Dkk3 with Kremen, LRP and EGFR molecules protein-protein docking servers were employed. Model assessment softwares indicated the high quality of the finally refined Dkk3 3D structure, indicating the accuracy of modeling and refinement process. Our results revealed that Dkk3 is capable of interacting with Kremen, LRP and EGFR with comparable binding energies. Dkk3 efficiently interacts with LRP, Kremen and EGF receptor and may be a promising protein in cancer therapy by blocking Wnt and EGFR downstream signaling.

Increasing proliferation of murine adipose tissue-derived mesenchymal stem cells by TNF-α plus IFN-γ.

The objective of the current study was to assess the effects of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) along with their simultaneous application on proliferation and pluripotency genes of murine adipose tissue-derived mesenchymal stem cells (AT-MSCs). The proliferation, doubling time (DT), colony-forming unit-fibroblast (CFU-F), pluripotency genes expression, and proliferation-related immunomodulatory markers of MSCs were analyzed upon activation with TNF-α (10 ng/ml), IFN-γ (10 ng/ml) and both TNF-α and IFN-γ (5 ng/ml + 5 ng/ml). Pluripotency genes including Oct-4, Sox-2, and Nanog as well as proliferation-associated immunomodulatory cytokines such as insulin-like growth factor 1 (IGF-1) and transforming growth factor-ß (TGF-ß) expression were evaluated using real-time PCR. Surface expression of Qa2 (HLA-G) was analyzed by flow cytometry. Pretreatment of MSCs with TNF-α plus IFN-γ led to significantly increased proliferation, DT and CFU-F as well as expression of pluripotency genes in AT-MSCs (p < 0.01). MSCs expressed more IGF-1, TGF-ß, and Qa2 upon activation with TNF-α plus IFN-γ and IFN-γ. MSCs expressed significantly decreased amounts of TGF-ß and Qa2 in presence of TNF-α. TNF-α combined with IFN-γ may be improved the proliferation of AT-MSCs. Conversely, expanded MSCs pointed out low levels of the immunomodulatory marker, s especially Qa2 in the presence of TNF-α. In conclusion, we showed that TNF-α together with IFN-γ increased the proliferation of MSCs and slightly enhanced the expression of pluripotency genes.

Effects of DKK-3, a Wnt signaling inhibitor, on dendritic cell phenotype and T cell polarization.

Wnt signaling plays crucial roles in regulation of a wide range of processes in different cell types including immune cells and, in particular, dendritic cells and T cells. Growing indications point out that Wnt pathway components modulate the both innate and adaptive immune responses through regulating DC functions. We investigated the effects of recombinant DKK-3 protein on the phenotype and biological functions of bone marrow-derived DCs (BM-DCs) as well as T cell polarization. The phenotype and the cytokine production of BM-derived DCs in the presence DKK-3 were analyzed using flow cytometry and ELISA, respectively. Also, capability of DCs to activate T cells was evaluated by CFSE-labeled splenocytes. Regulatory T cell induction, T cell polarization, and cytokine secretion were assessed by flow cytometry and ELISA in splenocytes cultured in the presence of DKK-3. Our results showed that the expression of CD86 and CD40 increased in the DKK-3-treated DC, while the expression of PDL-1 and PDL-2 diminished. Furthermore, the presence of DKK-3 decreased IL-10 and IL-4 production and increased IFN-gamma production by treated DCs.DKK-3. Moreover, DKK-3 shifted naive CD4 T cells towards TH1 cells through up-regulation of T-bet and down-regulation of GATA-3. Our results, therefore, suggest that DKK-3 protein has the ability to promote the generation of Th1-immunostimulatory DCs from its precursors.

The association of arylendosulfatase 1 (SULF1) gene polymorphism with recurrent miscarriage.

PURPOSE: One of the most common problems in reproductive medicine is recurrent miscarriage (RM). There is increasing evidence showing genetic susceptibility of women is an important risk factor in the occurrence of RM. In recent years, there is a growing interest in sulfate and its role in fetal development. A novel mechanism of SULF1 has been demonstrated for modifying the activities of some growth factors and signalling molecules that have major roles during embryogenesis. The aim of present study was to evaluate the association of SULF1 gene polymorphism (rs6990375 G > A) in Iranian patients with RM. METHODS: We established a case-control study of 200 Iranian women: 100 patients with the history of two or more RM as cases and 100 healthy women with at least two cases of successful pregnancy and no history of miscarriage as controls. The polymorphism was examined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. RESULTS: The genotypic analysis between case and controls showed significant differences (p-value = 0.000). Allelic analysis showed no significant correlation (Χ2 = 3.36, p-value = 0.066). The heterozygous genetic variant was significantly higher among healthy women (OR = 12.67, 95% CI = 6.47-24.79). CONCLUSIONS: Our data showed that rs6990375 polymorphism of SULF1 gene could be among one of the factors related to RM in Iranian women. Further evaluation of this polymorphism may be important and need further studies.

The potential role of iNKT cells in experimental allergic encephalitis and multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disorder associated with neurological signs and chronic inflammatory demyelination of the central nervous system (CNS). MS has been thought as Th1 (T helper) and Th17 cells mediated disease, but cells of the innate immune system play an important role both in the initiation and progression of MS. The invariant Natural Killer T (iNKT) cells are the unique innate lymphocytes subtype involved in inflammation and autoimmune disorders and secretes cytokines such as interferon gamma (IFN-γ), Interleukin (IL)-10, IL-4 and IL-13. A reduction in number or defect in function of iNKT cells has been associated with an increased prevalence of autoimmune disorders indicating that iNKT cells have an immune-regulatory role in autoimmune disorders. Also, the protective role of iNKT cells has been extensively studied in EAE and the results of these studies show that iNKT cells might be a target for therapeutic purposes, but needs more extensive studies of their biology. In this review, we will attempt to show the protective role of iNKT cells in the pathogenesis of EAE and human disease.

T Cell Exhaustion Markers in Multiple Myeloma Patients are Lower After Physical Activity Intervention.

PURPOSE: There is compelling evidence that CD4+ and CD8+T cells are dysfunctional in multiple myeloma, compromising their ability to control disease progression. Pre-clinical models suggest that exercise represents a non-pharmacologic means to reduce immune exhaustion, but no studies to date have examined the relationship between an exercise intervention and biomarkers of immune exhaustion in multiple myeloma patients. PATIENTS AND METHODS: The current study includes 24 multiple myeloma patients who participated in a six-month physical activity intervention, consisting of supervised strength training (n = 12) and unsupervised home-based walking arms (n = 12). Comprehensive flow cytometry was utilized to assess the frequency of CD4+ and CD8+T cells and subpopulations expressing the markers of exhaustion PD-1, TIGIT, TIM3 and/or LAG3. Ratios of exhausted to non-exhausted cell populations, and percentages of exhausted to total populations of the same lineage, were calculated for the baseline and final timepoints. RESULTS: Eighteen of 20 exhaustion measures were lower at the end of the intervention than at baseline, and several were significantly or borderline significantly reduced in the entire sample or in one of the arms. The entire sample saw improvements in the ratios of CD4+ TIGIT+ to non-exhausted CD4+ (0.7 [0.6] to 0.6 [0.4], P = .04) and CD8+ PD1+ to non-exhausted CD8+ (1.8 [2.6] to 1.5 [2.0], P = .06), and in total exhausted CD8+ as a percent of total CD8+ (72.9 [21.9] to 68.3 [19.6], P < .01). CONCLUSIONS: This pilot study suggests that physical activity induces changes in MM patients' immune systems, potentially rendering a less exhausted T cell state.

Exportin 1 governs the immunosuppressive functions of myeloid-derived suppressor cells in tumors through ERK1/2 nuclear export.

Myeloid-derived suppressor cells (MDSCs) are a main driver of immunosuppression in tumors. Understanding the mechanisms that determine the development and immunosuppressive function of these cells could provide new therapeutic targets to improve antitumor immunity. Here, using preclinical murine models, we discovered that exportin 1 (XPO1) expression is upregulated in tumor MDSCs and that this upregulation is induced by IL-6-induced STAT3 activation during MDSC differentiation. XPO1 blockade transforms MDSCs into T-cell-activating neutrophil-like cells, enhancing the antitumor immune response and restraining tumor growth. Mechanistically, XPO1 inhibition leads to the nuclear entrapment of ERK1/2, resulting in the prevention of ERK1/2 phosphorylation following the IL-6-mediated activation of the MAPK signaling pathway. Similarly, XPO1 blockade in human MDSCs induces the formation of neutrophil-like cells with immunostimulatory functions. Therefore, our findings revealed a critical role for XPO1 in MDSC differentiation and suppressive functions; exploiting these new discoveries revealed new targets for reprogramming immunosuppressive MDSCs to improve cancer therapeutic responses.

Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via ß2-adrenergic receptor (ß2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using ß2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

Downregulation of IRF8 in alveolar macrophages by G-CSF promotes metastatic tumor progression.

Tissue-resident macrophages (TRMs) are abundant immune cells within pre-metastatic sites, yet their functional contributions to metastasis remain incompletely understood. Here, we show that alveolar macrophages (AMs), the main TRMs of the lung, are susceptible to downregulation of the immune stimulatory transcription factor IRF8, impairing anti-metastatic activity in models of metastatic breast cancer. G-CSF is a key tumor-associated factor (TAF) that acts upon AMs to reduce IRF8 levels and facilitate metastasis. Translational relevance of IRF8 downregulation was observed among macrophage precursors in breast cancer and a CD68hiIRF8loG-CSFhi gene signature suggests poorer prognosis in triple-negative breast cancer (TNBC), a G-CSF-expressing subtype. Our data highlight the underappreciated, pro-metastatic roles of AMs in response to G-CSF and identify the contribution of IRF8-deficient AMs to metastatic burden. AMs are an attractive target of local neoadjuvant G-CSF blockade to recover anti-metastatic activity.

Galectin-3 predicts acute GvHD and overall mortality post reduced intensity allo-HCT: a BMT-CTN biorepository study.

Identifying plasma biomarkers early after allo-HCT may become crucial to prevent and treat severe aGvHD. We utilized samples from 203 allo-HCT patients selected from the Blood & Marrow Transplant Clinical Trials Network (BMT CTN) to identify new biomarker models to predict aGvHD and overall mortality. Two new biomarkers (Gal-3 and LAG-3), and previously identified biomarkers (ST2/IL33R, IL6, Reg3A, PD-1, TIM-3, TNFR1) were screened. Increased Gal-3 levels measured at Day +7 post-transplant predicted the development of aGvHD (grade 2-4) in the total population [AUC: 0.602; P = 0.045] while higher Day +14 levels predicted overall mortality due to toxicity among patients receiving reduced intensity conditioning [P = 0.028] but not myeloablative conditioning. Elevated LAG-3 levels (Day +21) were associated with less severe aGvHD [159.1 ng/mL vs 222.0 ng/mL; P = 0.046]. We developed a model utilizing Gal-3, LAG-3, and PD-1 levels at Days +14 and +21 with an improved performance to predict aGvHD and overall non-relapse mortality. We confirmed four informative biomarkers (Reg3A, ST2, TIM-3, and TNFR1) predict severe aGvHD at day +14 and day +21 (grade 3-4). In conclusion, the combination of Gal-3 alone or in combination with LAG-3, and PD-1 is a new informative model to predict aGvHD development and overall non-relapse mortality after allo-HCT.

Galectin-3 expression in donor T cells reduces GvHD severity and lethality after allogeneic hematopoietic cell transplantation.

Abundant donor cytotoxic T cells that attack normal host organs remain a major problem for patients receiving allogeneic hematopoietic cell transplantation (allo-HCT). Despite an increase in our knowledge of the pathobiology of acute graft versus host disease (aGvHD), the mechanisms regulating the proliferation and function of donor T cells remain unclear. Here, we show that activated donor T cells express galectin-3 (Gal-3) after allo-HCT. In both major and minor histocompatibility-mismatched models of murine aGvHD, expression of Gal-3 is associated with decreased T cell activation and suppression of the secretion of effector cytokines, including IFN-γ and GM-CSF. Mechanistically, Gal-3 results in activation of NFAT signaling, which can induce T cell exhaustion. Gal-3 overexpression in human T cells prevents severe disease by suppressing cytotoxic T cells in xenogeneic aGvHD models. Together, these data identify the Gal-3-dependent regulatory pathway in donor T cells as a critical component of inflammation in aGvHD.

Spatiotemporal assessment of immunogenomic heterogeneity in multiple myeloma.

Spatial heterogeneity is a common phenomenon in metastatic solid tumors and an evolving concept in multiple myeloma (MM). The interplay between malignant plasma cells (PCs) and the microenvironment has not yet been analyzed in MM. For this purpose, we performed bone marrow aspirates and imaging-guided biopsies of corresponding lesions in newly diagnosed MM (NDMM) and relapsed/refractory MM (RRMM) patients. PCs were isolated and subjected to whole-exome sequencing (WES). Non-PCs were studied with next-generation flow (NGF) and T-cell receptor sequencing (TCRseq) to analyze the connection between malignant and nonmalignant cells in the bone marrow and in lesions. Although we observed a strong overlap from WES, NGF, and TCRseq in patients with intramedullary disease, WES revealed significant spatial heterogeneity in patients with extramedullary disease. NGF showed significant immunosuppression in RRMM compared with NDMM as indicated by fewer myeloid dendritic cells, unswitched memory B cells, Th9 cells, and CD8 effector memory T cells but more natural killer and regulatory T cells. Additionally, fewer T-cell receptor (TCR) sequences were detected in RRMM compared with NDMM and healthy individuals. After induction therapy, TCR repertoire richness increased to levels of healthy individuals, and NGF showed more regulatory T cells and myeloid-derived suppressor cells, regardless of depth of response. Clinical significance of imaging-guided biopsies of lesions was demonstrated by detection of monoclonal PCs in patients without measurable residual disease (MRD) in aspirates from the iliac crest as well as identification of secondary primary malignancies in MRD- patients. Furthermore, site-specific clones with different drug susceptibilities and genetically defined high-risk features were detected by our workflow.

Isolation of human and mouse myeloid-derived suppressor cells for metabolic analysis.

Metabolic reprogramming is associated with myeloid-derived suppressor cell (MDSC) immunosuppressive function. Here, we outline the process for acquiring MDSCs from human and murine sources for subsequent analysis of fatty acid oxidation, oxidative phosphorylation, and glycolysis using the Seahorse XFe 96 Analyzer. Murine MDSCs can be isolated directly from tumor-bearing mice or derived through IL-6 and GM-CSF culture of bone marrow cells from non-tumor-bearing mice. To generate human MDSCs, peripheral blood mononuclear cells (PBMCs) can be cultured with IL-6 and GM-CSF. For complete details on the use and execution of this protocol, please refer to Mohammadpour et al. (2021).