Granulocytic Myeloid-derived Suppressor Cells Research Articles

Single-cell profiling reveals a conserved role for hypoxia-inducible factor signaling during human craniotomy infection.

Neurosurgeries complicated by infection are associated with prolonged treatment and significant morbidity. Craniotomy is a common neurosurgical procedure; however, the cellular and molecular signatures associated with craniotomy infection in human subjects are unknown. A retrospective study of over 2,500 craniotomies reveals diverse patient demographics, pathogen identity, and surgical landscapes associated with infection. Leukocyte profiling in patient tissues from craniotomy infection characterizes a predominance of granulocytic myeloid-derived suppressor cells that may arise from transmigrated blood neutrophils, based on single-cell RNA sequencing (scRNA-seq) trajectory analysis. Single-cell transcriptomic analysis identifies metabolic shifts in tissue leukocytes, including a conserved hypoxia-inducible factor (HIF) signature. The importance of HIF signaling was validated using a mouse model of Staphylococcus aureus craniotomy infection, where HIF inhibition increases chemokine production and leukocyte recruitment, exacerbating tissue pathology. These findings establish conserved metabolic and transcriptional signatures that may represent promising future therapeutic targets for human craniotomy infection in the face of increasing antimicrobial resistance.

LTBR acts as a novel immune checkpoint of tumor-associated macrophages for cancer immunotherapy.

Tumor-associated macrophages (TAMs) greatly contribute to immune checkpoint inhibitor (ICI) resistance of cancer. However, its underlying mechanisms and whether TAMs can be promising targets to overcome ICI resistance remain to be unveiled. Through integrative analysis of immune multiomics data and single-cell RNA-seq data (iMOS) in lung adenocarcinoma (LUAD), lymphotoxin β receptor (LTBR) is identified as a potential immune checkpoint of TAMs, whose high expression, duplication, and low methylation are correlated with unfavorable prognosis. Immunofluorescence staining shows that the infiltration of LTBR+ TAMs is associated with LUAD stages, immunotherapy failure, and poor prognosis. Mechanistically, LTΒR maintains immunosuppressive activity and M2 phenotype of TAMs by noncanonical nuclear factor kappa B and Wnt/β-catenin signaling pathways. Macrophage-specific knockout of LTBR hinders tumor growth and prolongs survival time via blocking TAMimmunosuppressive activity and M2 phenotype. Moreover, TAM-targeted delivery of LTΒR small interfering RNA improves the therapeutic effect of ICI via reversing TAM-mediated immunosuppression, such as boosting cytotoxic CD8+ T cells and inhibiting granulocytic myeloid-derived suppressor cells infiltration. Taken together, we bring forth an immune checkpoint discovery pipeline iMOS, identify LTBR as a novel immune checkpoint of TAMs, and propose a new immunotherapy strategy by targeting LTBR+ TAMs.

Targeting myeloid-derived suppressor cells promotes antiparasitic T-cell immunity and enhances the efficacy of PD-1 blockade

Immune exhaustion corresponds to a loss of effector function of T cells that associates with cancer or chronic infection. Here, our objective was to decipher the mechanisms involved in the immune suppression of myeloid-derived suppressor cells (MDSCs) and to explore the potential to target these cells for immunotherapy to enhance checkpoint blockade efficacy in a chronic parasite infection. We demonstrated that programmed cell-death-1 (PD-1) expression was significantly upregulated and associated with T-cell dysfunction in advanced alveolar echinococcosis (AE) patients and in Echinococcus multilocularis-infected mice. PD-1 blockade ex vivo failed to reverse AE patients’ peripheral blood T-cell dysfunction. PD-1/PD-L1 blockade or PD-1 deficiency had no significant effects on metacestode in mouse model. This was due to the inhibitory capacities of immunosuppressive granulocytic MDSCs (G-MDSCs), especially in the liver surrounding the parasite pseudotumor. MDSCs suppressed T-cell function in vitro in an indoleamine 2, 3 dioxygenase 1 (IDO1)-dependent manner. Although depleting MDSCs alone restored T-cell effector functions and led to some limitation of disease progression in E. multilocularis-infected mice, combination with PD-1 blockade was better to induce antiparasitic efficacy. Our findings provide preclinical evidence in support of targeting MDSC or combining such an approach with checkpoint blockade in patients with advanced AE.

Tumor necrosis factor regulates leukocyte recruitment but not bacterial persistence during Staphylococcus aureus craniotomy infection

BackgroundCraniotomy is a common neurosurgery used to treat intracranial pathologies. Nearly 5% of the 14 million craniotomies performed worldwide each year become infected, most often with Staphylococcus aureus (S. aureus), which forms a biofilm on the surface of the resected bone segment to establish a chronic infection that is recalcitrant to antibiotics and immune-mediated clearance. Tumor necrosis factor (TNF), a prototypical proinflammatory cytokine, has been implicated in generating protective immunity to various infections. Although TNF is elevated during S. aureus craniotomy infection, its functional importance in regulating disease pathogenesis has not been explored.MethodsA mouse model of S. aureus craniotomy infection was used to investigate the functional importance of TNF signaling using TNF, TNFR1, and TNFR2 knockout (KO) mice by quantifying bacterial burden, immune infiltrates, inflammatory mediators, and transcriptional changes by RNA-seq. Complementary experiments examined neutrophil extracellular trap formation, leukocyte apoptosis, phagocytosis, and bactericidal activity.ResultsTNF transiently regulated neutrophil and granulocytic myeloid-derived suppressor cell recruitment to the brain, subcutaneous galea, and bone flap as evident by significant reductions in both cell types between days 7 to 14 post-infection coinciding with significant decreases in several chemokines, which recovered to wild type levels by day 28. Despite these defects, bacterial burdens were similar in TNF KO and WT mice. RNA-seq revealed enhanced lymphotoxin-α (Lta) expression in TNF KO granulocytes. Since both TNF and LTα signal through TNFR1 and TNFR2, KO mice for each receptor were examined to assess potential redundancy; however, neither strain had any impact on S. aureus burden. In vitro studies revealed that TNF loss selectively altered macrophage responses to S. aureus since TNF KO macrophages displayed significant reductions in phagocytosis, apoptosis, IL-6 production, and bactericidal activity in response to live S. aureus, whereas granulocytes were not affected.ConclusionThese findings implicate TNF in modulating granulocyte recruitment during acute craniotomy infection via secondary effects on chemokine production and identify macrophages as a key cellular target of TNF action. However, the lack of changes in bacterial burden in TNF KO animals suggests the involvement of additional signals that dictate S. aureus pathogenesis during craniotomy infection.

γ-aminobutyric acid receptor B signaling drives glioblastoma in females in an immune-dependent manner.

Sex differences in immune responses impact cancer outcomes and treatment response, including in glioblastoma (GBM). However, host factors underlying sex specific immune-cancer interactions are poorly understood. Here, we identify the neurotransmitter γ-aminobutyric acid (GABA) as a driver of GBM-promoting immune response in females. We demonstrated that GABA receptor B (GABBR) signaling enhances L-Arginine metabolism and nitric oxide synthase 2 (NOS2) expression in female granulocytic myeloid-derived suppressor cells (gMDSCs). GABBR agonist and GABA analog promoted GBM growth in females in an immune-dependent manner, while GABBR inhibition reduces gMDSC NOS2 production and extends survival only in females. Furthermore, female GBM patients have enriched GABA transcriptional signatures compared to males, and the use of GABA analogs in GBM patients is associated with worse short-term outcomes only in females. Collectively, these results highlight that GABA modulates anti-tumor immune response in a sex-specific manner, supporting future assessment of GABA pathway inhibitors as part of immunotherapy approaches.

Host derived macrophage migration inhibitory factor expression attenuates anti-tumoral immune cell accumulation and promotes immunosuppression in the tumor microenvironment of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a significant public health concern worldwide. Immunomodulatory targets in the HNSCC tumor microenvironment are crucial to enhance the efficacy of HNSCC immunotherapy. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that has been linked to poor prognosis in many cancers, but the mechanistic role of MIF in HNSCC remains unclear. Using a murine orthotopic oral cancer model in Mif+/+ or Mif−/− mice, we determined the function of host derived MIF in HNSCC tumor development, metastasis as well as localized and systemic tumor immune responses. We observed that Mif−/− mice have decreased tumor growth and tumor burden compared to their wild-type counterparts. Flow cytometric analysis of immune populations within the primary tumor site revealed increased Th1 and cytotoxic T cell recruitment to the HNSCC tumor microenvironment. Within the tumors of Mif−/− mice, MIF deletion also enhanced the effector function of anti-tumoral effector CD8+ T cells as well as Th1 cells and decreased the accumulation of granulocytic myeloid derived suppressor cells (g-MDSCs) in the tumor microenvironment. Furthermore, MDSCs isolated from tumor bearing mice chemotactically respond to MIF in a dose dependent manner. Taken together, our results demonstrate a chemotactic and immunomodulatory role for host derived MIF in promoting HNSCC and suggest that MIF targeted immunomodulation is a promising approach for HNSCC treatment.

Dual roles of CD11b+CD33+HLA-DR-/lowCD14- myeloid-derived suppressor cells with a granulocytic morphology following allogeneic hematopoietic stem cell transplantation: from inflammation promoters to immune suppressors within 90 days.

Granulocytic myeloid-derived suppressor cells (G-MDSCs) show fast recovery following allogeneic hematopoietic stem cell transplantation (allo-HSCT) constituting the major part of peripheral blood in the early phase. Although G-MDSCs mediate immune suppression through multiple mechanisms, they may also promote inflammation under specific conditions. G-MDSCs were isolated from 82 patients following allo-HSCT within 90 days after allo-HSCT, and their interactions with autologous CD3+ T-cells were examined. T-cell proliferation was assessed by flow cytometry following CFSE staining, while differentiation and interferon-γ secretion were characterized using chemokine receptor profiling and ELISpot assays, respectively. NK cell cytotoxicity was evaluated through co-culture with K562 cells. An aGVHD xenogeneic model in humanized mice was employed to study the in vivo effects of human leukocytes. Furthermore, transcriptional alterations in G-MDSCs were analyzed via RNA sequencing to investigate functional transitions. G-MDSCs promoted inflammation in the early-stage, by facilitating cytokine secretion and proliferation of T cells, as well as their differentiation into pro-inflammatory T helper subsets. At day 28, patients with a higher number of G-MDSCs exhibited an increased risk of developing grades II-IV aGvHD. Besides, adoptive transfer of G-MDSCs from patients at day 28 into humanized mice exacerbated aGvHD. However, at day 90, G-MDSCs led to immunosuppression, characterized by upregulated expression of indoleamine 2,3-dioxygenase gene and interleukin-10 secretion, coupled with the inhibition of T cell proliferation. Furthermore, transcriptional analysis of G-MDSCs at day 28 and day 90 revealed that 1445 genes were differentially expressed. These genes were associated with various pathways, revealing the molecular signatures of early post-transplant differentiation in G-MDSCs. In addition, genes linked to the endoplasmic reticulum stress were upregulated in patients without aGvHD. The acquisition of immunosuppressive function by G-MDSCs may depend on the activation of CXCL2 and DERL1 genes. Our findings revealed the alteration in the immune characteristics of G-MDSCs within the first 90 days post-allo-HSCT. Moreover, the quantity of G-MDSCs at day 28 may serve as a predictive indicator for the development of aGvHD.

Tregs, gMDSCs, and levels of soluble MICA as prognostic biomarkers for combined NEO-201 and pembrolizumab.

2530 Background: The humanized IgG1 monoclonal antibody NEO-201 binds to Core 1 and/or extended Core 1 O-glycans expressed by several human solid and blood tumors, as well as neutrophils, and mediates killing of cancer cells, neutrophils, regulatory T cells (Tregs) and granulocytic myeloid-derived suppressor cells (gMDSCs) via ADCC and CDC. Resistance to PD-1/PD-L1 blockade may be due to the accumulation of Tregs and gMDSCs in the tumor microenvironment (TME). NEO-201 was proven to bind and reduce the amount of circulating Tregs in cancer patients. This supports the rationale of the ongoing phase II clinical trial (NCT03476681) evaluating the activity of NEO-201 with pembrolizumab in adults with solid tumors resistant to prior checkpoint inhibitors. Furthermore, elevated serum levels of soluble MICA (sMICA) have been correlated with decreased NK cell activity. Methods: Cancer patients were treated each cycle with 3 doses of NEO-201 1.5mg/kg every 2 weeks and pembrolizumab 400mg IV every 6 weeks and imaged for response every 2 cycles. PBMCs and serum from cancer patients pre- and at multiple time points post-treatment were used to evaluate the percentage of circulating gMDSCs and Tregs (flow cytometry) and sMICA levels (ELISA). Results: NEO-201 recognizes naïve Tregs (nTregs: CD3+/CD4+/CD45RA+/Foxp3low cells) but not effector Tregs (eTregs: CD3+/CD4+/CD45RA-/Foxp3high cells). NEO-201 also binds to gMDSCs, defined as HLA-DRneg/CD33+/CD15+/ CD14neg/CD66b+ cells. In patients with durable SD, a patient with HNSCC (SD >11 months) showed >50% reduction of nTregs and >90% of gMDSCs at C3D1 (beginning of cycle 3) compared to baseline. One patient with cervical cancer (SD >8 months) showed 40% reduction of nTregs at C3D1 compared to baseline, while gMDSCs trended down to baseline levels at C3D1 after initial increase. Conversely, we observed a general uptrend of nTregs and gMDSCs in patients with PD after treatment. Median serum levels of sMICA pre-treatment were 33-fold higher in patients with PD compared to patients with SD. Levels of sMICA remained elevated in patients with PD and low in patients with SD at all time points post-treatment. Conclusions: Depletion of circulating Tregs and gMDSCs may prevent their accumulation in the TME and enhance the efficacy of pembrolizumab in subjects with tumors resistant to checkpoint inhibitors. The decrease in circulating nTregs and gMDSCs after treatment with NEO-201 and pembrolizumab was associated with durable SD. High levels of sMICA can impair ADCC mediated by NEO-201, resulting in poor clinical response. Low levels of sMICA in patients with SD, together with the reduction of both circulating nTregs and gMDSCs, could be favorable prognostic markers for clinical benefit following treatment with NEO-201 and pembrolizumab. Ongoing enrollment in this clinical trial will validate these findings in larger cohorts. Clinical trial information: NCT03476681 .

Biomarker analysis of zanzalintinib in clear cell renal cell carcinoma from STELLAR-001.

4545 Background: Zanzalintinib (zanza) is a novel oral multi-targeted TKI that inhibits several receptors, including VEGFR, MET, and TAM kinases. In an expansion cohort of previously treated patients (pts) with clear cell renal cell carcinoma (ccRCC) from the phase 1b STELLAR-001 study (NCT03845166), single-agent zanza showed encouraging activity (ORR, 38%; disease control rate, 88%), with responses seen in VEGFR TKI–pretreated pts, and a manageable safety profile (Pal, IKCS NA 2023:Abs 1). Here, we investigate biomarkers associated with zanza from the STELLAR-001 ccRCC expansion cohort. Methods: Adult pts (N=32) with advanced ccRCC, ECOG ≤1, and 1–3 prior systemic anticancer therapies received zanza 100 mg once daily until unacceptable toxicity or no longer deriving clinical benefit. Circulating plasma biomarkers and immune cell populations in blood were assessed at baseline and on-study. IHC for AXL, c-Met, phosphorylated Met, and VEGFR2 was performed on archival tissue. Associations between response to zanza and biomarker levels (at baseline and treatment-related changes) were investigated by comparing zanza responders (CR/PR; n=10) and non-responders (SD/PD; n=19). The relation between baseline biomarker levels and prior exposure to VEGFR TKI was also evaluated. P values were determined using a Wilcoxon test ( P<0.05 considered significant). Results: Zanza elicited significant changes in soluble biomarkers related to angiogenesis, tumor growth and metastasis, and immune modulation, including an increase in the circulating ligands VEGF ( P<0.001) and GAS6 ( P<0.001); decreases in ANG-1 ( P<0.001) and ANG-2 ( P<0.001), and the soluble angiogenic receptors, VEGFR2 ( P<0.001) and TIE-2 ( P<0.001). A decrease in levels of the chemokine, RANTES ( P<0.001), and an increase in IFNγ ( P=0.039) and the pro-apoptotic protein, granzyme B ( P<0.001) were also observed. An analysis of immune cell subsets showed that zanza raised activated cytotoxic T cell numbers ( P=0.002). Reductions in monocytes ( P=0.001), along with immunosuppressive subsets (total MDSCs [ P=0.013] and granulocytic MDSCs [ P=0.014]) were also observed. Response to zanza was associated with lower baseline levels of plasma biomarkers including CRP ( P=0.025) and HGF ( P=0.017), but not with any tissue biomarkers or zanza-mediated changes in immune cells or plasma biomarkers tested. Lower baseline levels of VEGFR2 were observed in pts with prior VEGFR TKI exposure ( P=0.042). Conclusions: Zanza in pts with advanced ccRCC leads to modulation of plasma biomarkers associated with angiogenesis and peripheral immune cell subsets consistent with its expected mechanism of action. The reduction in immunosuppressive immune cells and activation of effector immune cells by zanza support the rationale for combining zanza with immune checkpoint inhibitors. Given the small sample sizes, further investigation in larger studies is warranted. Clinical trial information: NCT03845166 .

Robust pancreatic tumor suppression by a novel combination treatment with anti-PD-1 immune checkpoint antibody and stroma modifying RNA oligonucleotide STNM01 in mice.

e14630 Background: Tumor stromal remodeling is an obstacle for immune checkpoint blockade therapies. STNM01 is a synthetic RNA oligonucleotide that represses carbohydrate sulfotransferase 15 (CHST15), which is responsible for tumor stromal remodeling. We have previously reported that STNM01 monotherapy by intratumoral injection repressed tumor stromal remodeling while increase tumor-infiltrating T lymphocytes (TILs) in pancreatic tumor-bearing mice. STNM01 was shown to increase TILs in patients with unresectable pancreatic cancer in a Phase I/IIa trial. In the present study, we investigated whether STNM01-mediated TIL enhancement improves anti-tumor effect of anti-PD-1 immune checkpoint antibody by combination treatment in murine models of pancreatic tumor. Methods: In mouse pancreatic cancer KPC and Pan02 subcutaneous syngeneic tumor models, mice were divided into 4 groups for treatment; 1) control, 2) STNM01 monotherapy, 3) anti-PD-1 antibody monotherapy, and 4) combination therapy with STNM01 and anti-PD-1 antibody. Intratumoral injections with 0.9-1.0 mg/mL of STNM01 or control and intraperitoneal administrations of anti-PD-1 antibody or control (5mg/kg) were then performed twice a week for 2 weeks. Mice were sacrificed after 2 week-treatment from baseline, and anti-tumor effects were evaluated by immunohistochemistry for KPC and flowcytometry for Pan02 model, respectively. Results: In the KPC-implanted syngeneic mouse model, combination treatment with intratumoral STNM01 and systemic anti-PD-1 antibody synergistically and robustly suppressed tumor growth as mean % tumor growth inhibition was over 80%. Tumor-infiltrating CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to anti-PD-1 monotherapy group. In the Pan02-implanted syngeneic mouse model, the combination treatment with STNM01 and anti-PD-1 significantly augmented necrosis area of tumor. Flowcytometry analyses showed that tumor-infiltrating CD3+, CD4+ and CD8+ T cells significantly increased in the anti-PD-1 plus STNM01 combination therapy group compared to control group. Although FoxP3+ Treg did not change, the ratio of CD8 to FoxP3 was significantly high only in the anti-PD-1 plus STNM01 combination therapy group. Notably, Ly6C+Ly6G+ granulocytic MDSCs dramatically diminished in the anti-PD-1 plus STNM01 combination therapy group, while anti-PD-1 monotherapy had no impact on tumoral MDSCs. Conclusions: The present work represents the first report of robust synergy between systemic anti-PD-1 antibody and single stroma modifying agent through intratumoral route of administration. Combination usage of STNM01 would provide a novel and clinically feasible therapeutic option to trigger remarkable effect of immune checkpoint inhibitors to this most hard-to-treat solid tumor.

CCL9/CCR1 axis-driven chemotactic nanovesicles for attenuating metastasis of SMAD4-deficient colorectal cancer by trapping TGF-β

SMAD4 deficiency in colorectal cancer (CRC) is highly correlated with liver metastasis and high mortality, yet there are few effective precision therapies available. Here, we show that CCR1+-granulocytic myeloid-derived suppressor cells (G-MDSCs) are highly infiltrated in SMAD4-deficient CRC via CCL15/CCR1 and CCL9/CCR1 axis in clinical specimens and mouse models, respectively. The excessive TGF-β, secreted by tumor-infiltrated CCR1+-G-MDSCs, suppresses the immune response of cytotoxic T lymphocytes (CTLs), thus facilitating metastasis. Hereby, we develop engineered nanovesicles displaying CCR1 and TGFBR2 molecules (C/T-NVs) to chemotactically target the tumor driven by CCL9/CCR1 axis and trap TGF-β through TGF-β-TGFBR2 specific binding. Chemotactic C/T-NVs counteract CCR1+-G-MDSC infiltration through competitive responding CCL9/CCR1 axis. C/T-NVs-induced intratumoral TGF-β exhaustion alleviates the TGF-β-suppressed immune response of CTLs. Collectively, C/T-NVs attenuate liver metastasis of SMAD4-deficient CRC. In further exploration, high expression of programmed cell death ligand-1 (PD-L1) is observed in clinical specimens of SMAD4-deficient CRC. Combining C/T-NVs with anti-PD-L1 antibody (aPD-L1) induces tertiary lymphoid structure formation with sustained activation of CTLs, CXCL13+-CD4+ T, CXCR5+-CD20+ B cells, and enhanced secretion of cytotoxic cytokine interleukin-21 and IFN-γ around tumors, thus eradicating metastatic foci. Our strategy elicits pleiotropic antimetastatic immunity, paving the way for nanovesicle-mediated precision immunotherapy in SMAD4-deficient CRC.

Abstract PO1-18-04: Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968)

Abstract Background: Higher levels of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancers are associated with increased likelihood of pathologic complete response (pCR) to chemotherapy and improved outcomes. DNA methyltransferase inhibitors (DNMTi) can augment immune responses to cancers by upregulating tumor antigen and MHC expression, decreasing numbers and activity of myeloid derived suppressor cells (MDSC), and increasing responsiveness of T lymphocytes. We have shown that the DNMTi decitabine augments the effectiveness of immunotherapy against murine triple negative breast cancer (TNBC) using murine 4T1 and E0771 mammary carcinoma models. Methods: In a single-arm phase 2 study, patients with HER2-negative breast cancer who were candidates for neoadjuvant chemotherapy (NCT) received decitabine (15 mg/m2 × 4 doses over 5 days) followed by 2 doses of pembrolizumab (pembro) (200 mg, 2 weeks apart) – collectively, window immunotherapy – prior to starting NCT. Two research biopsies were obtained: 1 prior to the window immunotherapy and 1 afterwards, prior to starting NCT. Biopsies were analyzed using established procedures to quantify sTILs and PD-L1 expression. Patients proceeded to NCT and tumor resection per standard of care and were followed up for immune-related adverse events (irAEs) and response. The primary endpoint was change in sTILs. Key secondary endpoints were occurrence of irAEs and pCR following neoadjuvant treatment. After the study was opened, reported results of a phase 3 trial (KEYNOTE 522) led to allowing patients with TNBC to receive additional pembro (200 mg q3w) concurrently with standard NCT and adjuvantly following resection (Cohort A2). Results: 46 patients (median age 54.5 yrs, range 28-72; 71.7% White, 28.3% Black; 100% female) were enrolled and treated on study. 21 patients had TNBC and did not receive neoadjuvant pembro concurrently with NCT nor adjuvant pembro (Cohort A), 7 patients had TNBC and did receive neoadjuvant and/or adjuvant pembro (Cohort A2), and 18 patients were ER+ or PR+ and received neither concurrent nor adjuvant pembro (Cohort B). Blood samples collected after decitabine administration before the first pembro dose showed a 59% decrease (P< 0.01) in monocytic MDSCs compared to baseline; decrease in granulocytic MDSCs was not statistically significant. 37 patients had paired biopsies adequate for sTIL evaluation with a mean change from 23.4% to 30.3% (absolute change 6.9%, P< 0.001). Cohorts A/A2 experienced an absolute sTIL increase of 7.4% (P< 0.01); Cohort B experienced absolute sTIL increase of 6.1% (P=0.01). PD-L1 expression in tumors (H-score using MoAb 22C3 clone) increased by 43% (P< 0.01) across all cohorts. 16 of the 39 patients (41.0%) who proceeded to resection achieved pCR (n=12 of 27 [44.4%] in Cohorts A/A2 and n=4 of 13 [30.8%] in Cohort B). The most frequently reported irAEs were adrenal insufficiency (n=5, 10.9%), maculopapular rash (n=3, 6.5%), and hypothyroidism (n=3, 6.5%). Conclusions: Treatment in the pre-neoadjuvant window with decitabine and pembro could potentially sensitize breast cancers to standard NCT by recruitment of TILs to the tumor tissue. The treatment was well -tolerated at the tested doses. Funding: financial support and drug (pembrolizumab) were provided by Merck Citation Format: Harry Bear, Xiaoyan Deng, Dipankar Bandyopadhyay, Michael Idowu, Maciej Kmieciak, Monique Williams, Giovanni Archer, Lindsey Gwaltney, Patrick Dillon, Daniel Flora, Daniel Stover, Andrew Poklepovic, Mary Hackney, Masey Ross, Hetal Vachhani, Raphael Louie, Kandace McGuire, Amelia Grover, Tasnim Rahman, Amber Hendrix. Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-04.

The clinical association of programmed death-1/PD-L1 axis, myeloid derived suppressor cells subsets and regulatory T cells in peripheral blood of stable COPD patients.

Myeloid-derived suppressor cells (MDSCs) have crucial immunosuppressive role in T cell dysfunction in various disease processes. However, the role of MDSCs and their impact on Tregs in COPD have not been fully understood. The aim of the present study is to investigate the immunomodulatory role of MDSCs and their potential impact on the expansion and function of Tregs in COPD patients. Peripheral blood samples were collected to analyze circulating MDSCs, Tregs, PD-1/PD-L1 expression to assess the immunomodulatory role of MDSC and their potential impact on the expansion and function of Treg in COPD. A total of 54 COPD patients and 24 healthy individuals were enrolled in our study. Flow cytometric analyses were performed to identify granulocytic MDSCs (G-MDSCs), monocytic MDSCs (M-MDSCs), Tregs, and the expression of PD-1/PD-L1(L2) on MDSCs and Tregs in peripheral blood. Our results revealed a significantly higher percentage of G-MDSCs and M-MDSCs (p < 0.001) in COPD patients compared to the healthy controls. Additionally, a significantly higher proportion of peripheral blood Tregs was observed in COPD patients. Furthermore, an increased expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on Tregs (p < 0.01) was detected in COPD patients. The expression of PD-1 on CD4+ Tcells and Tregs, but not CD8+Tcells, was found to be increased in patients with COPD compared to controls. Furthermore, an elevated expression of PD-L1 on M-MDSCs (p < 0.01) was also observed in COPD patients. A positive correlation was observed between the accumulation of M-MDSCs and Tregs in COPD patients. Additionally, the percentage of circulating M-MDSCs is positively associated with the level of PD-1 (r = 0.51, p < 0.0001) and CTLA-4 (r = 0.42, p = 0.0014) on Tregs in COPD. The recruitment of MDSCs, accumulation of Tregs, and up-regulation of CTLA-4 on Treg in COPD, accompanied by an increased level of PD-1/PD-L1, suggest PD-1/PD-L1 axis may be potentially involved in MDSCs-induced the expansion and activation of Treg at least partially in COPD.

Abstract 7297: Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA

Abstract The V-domain Ig Suppressor of T cell Activation (VISTA) is expressed on cells of the myeloid and lymphoid lineages and is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. Furthermore, a tripeptide small molecule antagonist of VISTA, CA170, exhibited potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. We have previously demonstrated that KRAS or EGFR vaccines are effective in preventing lung tumorigenesis driven by either KRAS or EGFR in a pure prevention setting but are less effective in mice carrying existing lesions. Here, we determined if combining an MHC class II-restricted multi-antigen vaccine targeting KRAS or EGFR with CA170 will provide enhanced efficacy in preventing lung cancer progression. Using tetracycline-inducible KRAS and EGFR transgenic mice, we found that lung tumor development was significantly suppressed when CA170, delivered by aerosol inhalation to minimize systemic toxicity, was combined with either KRAS or EGFR MHCII-directed peptide vaccines using a post-initiation model. Flow cytometry and single-cell RNA sequencing (scRNA-seq) revealed that CA170 significantly increases tumor infiltrating CD8+ T cells and enhances effector-memory T cell frequencies and functions of both CD4+ and CD8+ T cells. These changes coincide with significant reductions in G-MDSCs (granulocytic myeloid-derived suppressor cells) and Foxp3+ Treg populations within lung tumors from mice treated with CA170. We found that the KRAS and EGFR preventive vaccines primarily induced the expansion of CD4+ effector T cells. VISTA antagonism by CA170 revealed strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory, and regulatory T cells, and drive an adaptive T cell tumor-specific immune response that enhances the efficacy of the KRAS and EGFR preventive vaccines in transgenic mouse models of lung cancer. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Ming You. Inhibition of lung tumorigenesis and potentiation of cancer preventive vaccines by a small molecule CA170 targeting the immune checkpoint protein VISTA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7297.

Abstract 7521: Tumor NLRP3 inflammasome activity mediates resistance to Anti-PD-1 immunotherapy in advanced gastroesophageal cancer

Abstract The addition of anti-PD-1 immunotherapy to the therapeutic armamentarium of advanced gastroesophageal (GE) cancer patients has resulted in modest improvements in clinical outcomes for a subset of patients. More robust predictive biomarkers to guide patient management and to select optimal combination immunotherapy strategies for the individual patient are needed. Our previous studies indicate that the tumor-intrinsic NLRP3 inflammasome-HSP70 signaling axis promotes the recruitment of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) and drives resistance to anti-PD-1 immunotherapy. The KEYLargo phase II clinical trial evaluated the efficacy of pembrolizumab (pembro) in combination with capecitabine/oxaliplatin (CAPOX) in HER2-negative advanced GE cancer patients. An 8-day lead-in period of pembro monotherapy in 36 advanced GE cancer patients allowed for the acquisition of blood and tumor tissue specimens to determine which immune-mediated alterations within the tumor microenvironment correlated with resistance to anti-PD-1 immunotherapy. Baseline and on-treatment specimens were collected for HSP70 ELISA, immunofluorescence microscopy, spatial transcriptomics, NLRP3 FISH, IHC, and NLRP3-ASC proximity ligation assays (PLA) to measure NLRP3 pathway activity levels. All data was correlated with best objective response, progression-free survival (PFS), and overall survival (OS). Tumor-infiltrating CD8+ T cells and PMN-MDSCs were increased in responders and non-responders, respectively. While PD-L1 CPS failed to associate with response, elevated baseline NLRP3 PLA activity correlated with increased tumor-infiltrating PMN-MDSCs (P = 0.001) and resistance to pembro/CAPOX therapy (P = 0.01). Only non-responding tumors exhibited an increase in NLRP3 PLA activity and NLRP3 amplification. Transcriptional studies revealed tumors with enhanced NLRP3 activity to exhibit decreased expression of the NLRC5 transactivator, MHC class I antigen presentation genes, and an increase in the NK cell negative regulator, HLA-G. Baseline plasma HSP70 levels correlated with tumor NLRP3 activity (P = 0.043) and with resistance to pembro/CAPOX therapy (P = 0.001). Elevated baseline NLRP3 PLA activity and amplification were associated with diminished PFS (P = 0.008) and OS (P = 0.009). In additional studies, pharmacologic inhibition of the NLRP3 inflammasome suppressed PMN-MDSC recruitment, upregulated MHC class I, and reversed anti-PD-1 resistance in an orthotopic model of GE cancer. Together, these findings implicate tumor-intrinsic NLRP3-HSP70 signaling as an important mediator of anti-PD-1 resistance in advanced GE cancer. The NLRP3-HSP70 pathway is a promising source of therapeutic targets and biomarkers capable of improving treatment outcomes for this patient population. Additional clinical studies are warranted. Citation Format: Nagendra Yarla, Kaylee Howell, Y-Van Nguyen, Donna Niedwiecki, Hope Uronis, John Strickler, Nicholas C. DeVito, Bala Theivanthiran, Brent A. Hanks. Tumor NLRP3 inflammasome activity mediates resistance to Anti-PD-1 immunotherapy in advanced gastroesophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7521.

Abstract 5086: Functional profiling in lung tumor explants show contributions of the tumor microenvironment to response of Dato-DXdex vivo

Abstract Background: Antibody drug conjugates (ADCs) have demonstrated significant antitumor activity against multiple treatment refractory cancers. While Trastuzumab deruxtecan, a HER2 directed ADC, has shown impressive clinical activity in HER2-expressing cancers, the ability to predict responses of ADCs by IHC has been limited. Dato-DXd is a TROP2 directed ADC that is currently being studied in a number of registrational phase 3 trial incld. TROPION-Lung01. In the phase 1b TROPION-Pan tumor 01 trial (TP01) response to Dato-DXd did not correlate to TROP2 IHC expression. Here we investigate molecular determinants of sensitivity to Dato-DXd using short-term microfluidic culture of patient derived organotypic tumor spheroids (PDOTS). Methods: Surgical NSCLC cases collected from Brigham and Women’s Hospital and St. Elizabeth’s Medical Center under an IRB approved protocol were studied. PDOTS were generated as previously described. Ex vivo response was assessed by live/dead imaging, TROP2 antigen density on EPCAM+ cells by flow cytometry (FCM) and by immunofluorescence (IF). T cell and myeloid cell populations were analyzed by FCM of CD45+ cells isolated during tumor preparation. For a subset of samples single cell RNA sequencing (scRNAseq) was performed. Results:Eleven NSCLC explants were studied. Two specimens were excluded due to excess of fibrotic material. Response to Dato-DXd was measured by change in raw live cell area compared to control samples. Median response to Dato-DXd was -48.5% (ranging from +33.9% to -77.8%). Using a cut-off of -40%, four cases were characterized as non-responders (NR) and 5 as responders (R). Tumor TROP2 expression by IF ranged from 0 to 3+ with no relationship between TROP2 expression and response being observed. TROP2 staining positivity by IF didn’t correlate to Dato-DXd sensitivity ex vivo. TROP2 FCM, on the other hand discriminated between responsive and non-responsive models in 100% of the cases, using a cutoff of MFI &amp;gt;13000. Furthermore, we observed an increase of tumor CD8 abundance in Dato-DXd R compared to NR (median %CD8 in R 10.3% (range 5.1%-62.5%); median %CD8 of live cells in NR 2.3% (range 1.2%-3.7%) p-value 0.029). Conversely, we also observed a trend of higher granulocytic myeloid-derived suppressor cells abundance in NR compared to R (NR median %gMDSC of CD15+ cells 84.1% (range 73.4%-90.2%) ; R median %gMDSC 61.7% (range 54.3%-90.6%); p-value 0.069). In 2 cases with high and low TROP2 MFI available scRNAseq data showed TROP2 expression exclusively in tumor cells, with further analysis ongoing. Conclusion: By functionally profiling NSCLC PDOTS that retain immune cells, we correlated multiple parameters with ex vivo response to Dato-DXd. Our results have important implications for precision deployment of ADCs and suggest that target tumor antigen density in conjunction with addition immune cell features may refine patient selection strategy. Citation Format: Satoru Yasuda, Elena Ivanova, Ha V. Vo, Sophie Kivlehan, Iliana Gjeci, Minh Ha, Patrick Lizotte, Raphael Bueno, David A. Barbie, Cloud P. Paweletz. Functional profiling in lung tumor explants show contributions of the tumor microenvironment to response of Dato-DXdex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5086.

Abstract B087: Targeting myeloid derived suppressor cell migration decreases tumor progression in a murine model of ovarian cancer

Abstract Introduction: High grade serous ovarian, fallopian tube and primary peritoneal carcinoma (HGSC) has an immunosuppressive and tumor promoting immune microenvironment. In this study, we have targeted the migration of myeloid derived suppressor cells (MDSC) through chemical inhibition of the CXCR2 chemotactic receptor to reverse the tumor-induced immunosuppression.Methods: ID8 cells with a p53 mutation (ID8-P53null) were used as a murine model of HGSC. ID8-p53null cells were injected intraperitoneally into immune intact C57/Bl6 mice, with 10 mice per group. After 7 days, mice were treated with cisplatin (cis, 1mg/kg, i.p., weekly), a CXCR2 inhibitor (CXCR2i, 4mg/kg, i.p., daily) or a combination of the 2 treatments. Necropsy was performed on day 29. The tumors were weighed, and the number of metastatic sites was quantified. The spleen and the tumor were resected, digested and flow cytometry was performed to evaluate the effects of treatment on the tumor immune microenvironment (TIME) as well as any systemic changes (evaluated by immune cells in the spleen). Granulocytic MDSC (G-MDSC) and monocytic MDSCs (M-MDSC) were defined as CD11b+Ly6G+,Ly6Clo and CD11b+Ly6G−Ly6Chi, respectively. One-way ANOVA with Tukey multiple comparisons was used for statistics with p&amp;lt;0.05 as the cut off for significance. Results: Tumor weights were significantly less in cis treated vs control mice (0.064g vs 0.220g, p &amp;lt;0.001) and CXCR2i treated vs control mice (0.171g vs 0.220g, p=.003). There was no additive effect of the CXCR2i to the cis treatment (cis 0.064g vs cis + CXCR2i 0.049g, p=0.62). The number of metastatic sites as a measurement of dissemination also decreased with cis or CXCR2i (number of sites: control: 47 vs cis: 12.7 vs CXCR2i: 32.5 vs cis + CXCR2i: 10.2). When the TIME was evaluated by flow cytometry, there were no significant differences in the immune populations including T-cells, NK cells, myeloid cells, and macrophages. However, there was a trend for cis to increase both the G-MDSC and M-MDSC populations and this effect was inhibited with the combination treatment of cis and CXCR2i. For the G-MDSC population control: 0.6% vs cis: 0.9% vs cis+CXCR2i: 0.5% and M-MDSC population control 3.4% vs cis: 6.8% vs cis+CXCR2i: 2.6%. Conclusion: CXCR2 is a chemotactic receptor present on MDSCs. We targeted this receptor to decrease the migration and infiltration of MDSCs into the ovarian cancer TIME. The CXCR2i decreased tumor progression in the ID8-p53null model of ovarian cancer. The TIME was evaluated by flow cytometry, and there was a trend for cis to increase the suppressive MDSC populations and this increase was inhibited by the CXCR2i. These exciting data suggest that traditional chemotherapy may lead to a more immunosuppressive TIME, which could have implications for future recurrence and treatment. Blocking the migration of immunosuppressive cells into the TIME may have a long-term impact on survival in HGSC. Survival studies in this murine model of HGSC are currently underway. Citation Format: Nicole Marjon, Railey Mikeska, Elizabeth R. Woodruff, Ritsuko Iwanaga, Tomomi Yamamoto, Benjamin G. Bitler. Targeting myeloid derived suppressor cell migration decreases tumor progression in a murine model of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B087.

Granulocytic myeloid-derived suppressor cell activity during biofilm infection is regulated by a glycolysis/HIF1a axis.

Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI). A primary contributor to infection chronicity is an expansion of granulocytic myeloid-derived suppressor cells (G-MDSCs), which are critical for orchestrating the antiinflammatory biofilm milieu. Single-cell sequencing and bioinformatic metabolic algorithms were used to explore the link between G-MDSC metabolism and S. aureus PJI outcome. Glycolysis and the hypoxia response through HIF1a were significantly enriched in G-MDSCs. Interfering with both pathways in vivo, using a 2-deoxyglucose nanopreparation and granulocyte-targeted Hif1a conditional KO mice, respectively, attenuated G-MDSC-mediated immunosuppression and reduced bacterial burden in a mouse model of S. aureus PJI. In addition, single-cell RNA-Seq (scRNA-Seq) analysis of granulocytes from PJI patients also showed an enrichment in glycolysis and hypoxia-response genes. These findings support the importance of a glycolysis/HIF1a axis in promoting G-MDSC antiinflammatory activity and biofilm persistence during PJI.

Decreased circulating myeloid-derived suppressor cell count at the engraftment is one of the risk factors for multiple myeloma relapse after autologous hematopoietic stem cell transplantation

Recent studies demonstrated that myeloid-derived suppressor cells (MDSCs) are involved in the pathogenesis and progression of multiple myeloma (MM). Nevertheless, data on the quantitative and functional changes in MDSCs during standard MM treatment remain poorly understood. Here, we determined that monocytic MDSCs (M-MDSC; CD14+HLA-DRlow/–) and granulocytic MDSCs (PMN-MDSC; Lin–HLA-DR–CD33+CD66b+) in MM patients in remission following induction therapy (IT) were significantly increased, while early MDSCs (E-MDSCs; Lin–HLA-DR–CD33+CD66b–) were decreased compared to the donor group. In progression, MM patients had the most pronounced decrease in E-MDSCs and enhanced levels of PMN-MDSCs. IT was accompanied with a decrease in the expression of arginase-1 (Arg-1). In MM patients with relapse or resistance to IT, Arg-1+ cell frequency in M-MDSCs and E-MDSCs, as well as PD-L1+ M-MDSCs, was increased, which may facilitate tumor immunosuppression. G-CSF administration led to a significant increment in the MDSC subsets. At the engraftment, circulating M-MDSC and PMN-MDSCs were temporarily increased, with a gradual decline to the pre-transplant levels in 12 months. The percentage of E-MDSCs was decreased at the leukocyte recovery. Patients with a higher (>Me) M-MDSC count at the engraftment had a shorter post-transplant leukopenia duration (Me 11 vs. 13 days; pU = 0.0086). The advanced MM stage, depth of response, and lower relative count of circulating E-MDSCs at the engraftment were independent risk factors associated with a lower progression-free survival.The obtained data allow us to hypothesize that MDSCs may play a positive role at the stage of leukocyte recovery by ameliorating the long-term anti-tumor response in MM.

CXCR2 inhibition in G-MDSCs enhances CD47 blockade for melanoma tumor cell clearance

The use of colony-stimulating factor-1 receptor (CSF1R) inhibitors has been widely explored as a strategy for cancer immunotherapy due to their robust depletion of tumor-associated macrophages (TAMs). While CSF1R blockade effectively eliminates TAMs from the solid tumor microenvironment, its clinical efficacy is limited. Here, we use an inducible CSF1R knockout model to investigate the persistence of tumor progression in the absence of TAMs. We find increased frequencies of granulocytic myeloid-derived suppressor cells (G-MDSCs) in the bone marrow, throughout circulation, and in the tumor following CSF1R deletion and loss of TAMs. We find that G-MDSCs are capable of suppressing macrophage phagocytosis, and the elimination of G-MDSCs through CXCR2 inhibition increases macrophage capacity for tumor cell clearance. Further, we find that combination therapy of CXCR2 inhibition and CD47 blockade synergize to elicit a significant anti-tumor response. These findings reveal G-MDSCs as key drivers of tumor immunosuppression and demonstrate their inhibition as a potent strategy to increase macrophage phagocytosis and enhance the anti-tumor efficacy of CD47 blockade in B16-F10 melanoma.