Changes In Immune Cells Research Articles

Persistent transcriptional changes in cardiac adaptive immune cells following myocardial infarction: New evidence from the re-analysis of publicly available single cell and nuclei RNA-sequencing data sets

IntroductionChronic immunopathology contributes to the development of heart failure after a myocardial infarction. Both T and B cells of the adaptive immune system are present in the myocardium and have been suggested to be involved in post-MI immunopathology. MethodsWe analyzed the B and T cell populations isolated from previously published single cell RNA-sequencing data sets (PMID: 32130914, PMID: 35948637, PMID: 32971526 and PMID: 35926050), of the mouse and human heart, using differential expression analysis, functional enrichment analysis, gene regulatory inferences, and integration with autoimmune and cardiovascular GWAS. ResultsAlready at baseline, mature effector B and T cells are present in the human and mouse heart, having increased activity in transcription factors maintaining tolerance (e.g. DEAF1, JDP2, SPI-B). Following MI, T cells upregulate pro-inflammatory transcript levels (e.g. Cd11, Gzmk, Prf1), while B cells upregulate activation markers (e.g. Il6, Il1rn, Ccl6) and collagen (e.g. Col5a2, Col4a1, Col1a2). Importantly, pro-inflammatory and fibrotic transcription factors (e.g. NFKB1, CREM, REL) remain active in T cells, while B cells maintain elevated activity in transcription factors related to immunoglobulin production (e.g. ERG, REL) in both mouse and human post-MI hearts. Notably, genes differentially expressed in post-MI T and B cells are associated with cardiovascular and autoimmune disease. ConclusionThese findings highlight the varied and time-dependent dynamic roles of post-MI T and B cells. They appear ready-to-go and are activated immediately after MI, thus participate in the acute wound healing response. However, they subsequently remain in a state of pro-inflammatory activation contributing to persistent immunopathology.

BET inhibition sensitizes innate checkpoint inhibitor resistant melanoma to anti-CTLA-4 treatment.

Approximately 50% of melanoma patients fail to respond to immune checkpoint blockade (ICB), and acquired resistance hampers long-term survival in about half of initially responding patients. Whether targeting BET reader proteins, implicated in epigenetic dysregulation, can enhance ICB response rates and durability, remains to be determined. Here we show elevated BET proteins correlate with poor survival and ICB responses in melanoma patients. The BET inhibitor IBET151, combined with anti-CTLA-4, overcame innate ICB resistance however, sequential BET inhibition failed against acquired resistance in mouse models. Combination treatment response in the innate resistance model induced changes in tumor-infiltrating immune cells, reducing myeloid-derived suppressor cells (MDSCs). CD4+ and CD8+ T cells showed decreased expression of inhibitory receptors, with reduced TIM3, LAG3, and BTLA checkpoint expression. In human PBMCs invitro, BET inhibition reduced expression of immune checkpoints in CD4+ and CD8+ T cells, restoring effector cytokines and downregulating the transcriptional driver TOX. BET proteins in melanoma may play an oncogenic role by inducing immune suppression and driving T cell dysfunction. The study demonstrates an effective combination for innately unresponsive melanoma patients to checkpoint inhibitor immunotherapy, yet highlights BET inhibitors' limitations in an acquired resistance context.

Pharmacokinetics/pharmacodynamics of benralizumab in chronic rhinosinusitis with nasal polyps: Phase III, randomized, placebo-controlled OSTRO trial.

Benralizumab, a humanized, afucosylated monoclonal antibody against the interleukin 5 receptor, α subunit, causes rapid depletion of eosinophils by antibody-dependent cellular cytotoxicity. We investigated the pharmacokinetic and pharmacodynamic effects of benralizumab in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) from the phase III OSTRO trial. Patients received a placebo or 30 mg of benralizumab by subcutaneous injection every 8weeks (first three doses every 4weeks) to week 48; a subset of patients continued in an extended follow-up period to assess treatment durability to week 80. Serum benralizumab concentrations and blood eosinophil and basophil counts were assessed to week 80. Biomarker assessments were performed on nasal polyp tissue biopsies at week 56 and nasal lining fluid at weeks 24 and 56 to examine changes in immune cells and inflammatory mediators. Among 185 patients in this analysis, 93 received benralizumab. Serum benralizumab concentrations reached a steady state by week 24 (median concentration 385.52 ng mL-1); blood eosinophils were almost fully depleted and blood basophils were reduced between weeks 16 and 56. Nasal polyp tissue eosinophils decreased with benralizumab from 57.6 cells mm-2 at baseline to 0 cells mm-2 at week 56 (P< .001 vs placebo), and tissue mast cells were numerically reduced. In nasal lining fluid, eosinophil-derived neurotoxin was significantly reduced at weeks 24 and 56 (P< .001) and interleukin-17 at week 56 (P< .05) with benralizumab. Benralizumab treatment led to rapid, sustained, nearly complete depletion of eosinophils from blood and nasal polyp tissue in patients with CRSwNP.

Persistent immune shifts: Unveiling the legacy of vasopressin-triggered preeclampsia in offspring

Abstract Preeclampsia (PreE), a hypertensive disorder in pregnancy, is the global leading cause of maternal-fetal morbidity and mortality. Previously, we noted immune cell changes at gestational day 18 (GD18) in offspring of PreE. While offspring cardiovascular and metabolic risks are increasingly studied, there is comparatively less investigation into the persistence of immune cell changes post-PreE in offspring. Our study aims to assess the persistence of immune cell changes observed in PreE-affected offspring on GD18 (term in mice) postnatally into adulthood. PreE was induced in virgin C57BL/6J female mice by infusion of vasopressin (AVP) or saline via subcutaneous infusion throughout gestation. Offspring were euthanized at postnatal week 1, 7, or 12 for experimental outcomes. Offspring body and heart weight were recorded, and total urine protein was quantified. Flow cytometry was used to evaluate immune cell populations. Total T cells, as well as CD4+ and CD8+ T cells, were increased in the offspring of PreE postnatally. Surprisingly, there was also an increase in total B cells and isotype-switched B cells, while macrophage populations were decreased in offspring from AVP-induced dams. Our results confirm that immune cell alterations triggered by PreE persist in offspring postnatally. Ongoing research focuses on unraveling the implications of these immune cell changes in potentially shaping responses to pathogens and the development of autoimmune diseases later in life.

Abstract 2007: Ischemic And Non-ischemic Cardiovascular Events Differentially Regulate Cross Disease Communication With Cancer

Cancer and cardiovascular disease (CVD) are the top two leading causes of death worldwide, exhibiting cross-disease communication that increases morbidity. Cancer patients are at significantly increased risk for CVD and recent studies by our lab and others indicate that CVDs like myocardial infarction (MI) or heart failure can in turn accelerate tumor growth. We previously showed this occurs through changes in the innate immune system or via tumor-promoting factors secreted from the damaged myocardium, which together have established the nascent field of Reverse CardioOncology. We hypothesized that ischemic and non-ischemic CVD injuries have distinctly different mechanisms underlying mammary tumor growth. To mimic these events, we modeled non-cardiac hind limb ischemic injury by femoral artery ligation (FAL) and non-ischemic cardiac injury by cardiac cryoinjury (CCI). To test our hypothesis, we introduced primary E0771 mouse mammary cancer cells into the mammary fat pad three days prior to FAL or CCI. Measurements of tumor over time showed significant increases in tumor growth after both FAL and CCI when compared to sham surgeries. However, only the ischemic injury, FAL, induced similar changes in immune cells to what we observed after MI (induced by left anterior descending coronary artery [LAD] ligation). This included significant increases in myeloid populations and decreased lymphocytes within the circulation. Additionally, flow cytometry of immune cells within tumors of mice exposed to FAL showed significantly increased myeloid cells and macrophages, as well as increased immunosuppressive T-regulatory cells. In contrast, while CCI showed significantly increased tumor burden and circulating leukocytes, there were no differences in the above-mentioned subsets of immune cells within the circulation or within the tumor. These data suggest that ischemic injury, via FAL or LAD, induces an immunosuppressive environment within the tumor that accelerates cancer growth that is distinct from the non-ischemic cardiac injury induced by CCI. Taken together, our data suggest that the immune reprogramming after ischemic CVDs is a risk factor for tumor growth and these data help to elucidate the mechanisms that underlie cancer risk in CVD patients.

Cerebral and Peripheral Immune Cell Changes following Rodent Juvenile Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the leading causes of death and disability. TBI is associated with neuroinflammation, but temporal changes in immune and inflammatory signaling following TBI have not been fully elucidated. Furthermore, there have been no previous studies on changes in immune cell populations following TBI via the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA). The current study aimed to determine the time course changes to inflammatory marker mRNA expression in the acute period following TBI in juvenile rats and to determine acute changes to brain and circulating immune cell populations. For this study, post-natal day (PND)-30 male Long Evans rats sustained a TBI or Sham TBI and were euthanized at 0, 3, 6, 12, 24, or 96 h post-injury. Prefrontal cortex and hippocampus samples were used to determine mRNA expression changes of inflammatory factors. The mRNA expression of the pro-inflammatory cytokine TNF-α was significantly elevated at 6 h post-injury in both regions evaluated. To evaluate immune cell populations, male Long Evans rats were euthanized at 48 h post-injury, and brain and blood samples were used for cell sorting by marker-specific antibodies. In the peripheral blood, there was an elevation in CD3+ total T cells, CD45R+ total B cells, and CD3+CD4+ helper T cells in the TBI subjects. However, there were no changes to natural killer cells or CD3+CD8+ cytotoxic T cell populations. In the brain, there was a reduction in CD11b/c+ monocytes/macrophages, but no changes in other immune cell populations. At 48 h post-injury, the TBI subjects also demonstrated expansion of the thymic medulla. These changes in the cerebral and blood immune cell populations and thymic medulla expansion may implicate the subacute recovery timeframe as a vulnerable window for the immune system in the pediatric population.

Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models

BackgroundChitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1).MethodsFemale BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling.ResultsChitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs.ConclusionsChitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.

Identification of candidate hub genes for COVID-19-induced idiopathic pulmonary fibrosis by bioinformatic analysis

Background: Idiopathic pulmonary fibrosis (IPF) is an incurable lung disease characterized by progressive scarring leading to alveolar stiffness, and reduced lung capacity, which might be induced by Coronavirus Disease 2019 (COVID-19). This study aims to explore the molecular mechanism of COVID-19-induced IPF and find the immune changes in the process of IPF. Methods: The data from the GEO database were processed by using the R algorithm package to explore the correlation between COVID-19 and IPF. Differential analysis, Venn analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, immune infiltration analysis, transcriptional analysis, and protein-protein analysis were performed to find the candidate hub genes, related pathways, and immune cell changes in IPF. Results: 69 differentially expressed genes (DGEs) were identified by differential analysis, including 27 up-regulated genes and 42 down-regulated genes. 15 hub genes were identified, which are CEA Cell Adhesion Molecule 8 (CEACAM8), Cathelicidin Antimicrobial Peptide (CAMP), Ribosomal Protein L9 (RPL9), Lactotransferrin (LTF), CD177 Molecule (CD177), Annexin A3 (ANXA3), CD3 Delta Subunit Of T-Cell Receptor Complex (CD3D), Granulin Precursor (GRN), Granzyme A (GZMA), Granzyme K (GZMK), Interleukin 1 Receptor Type 2 (IL1R2), Lipocalin 2 (LCN2), Matrix Metallopeptidase 9 (MMP9), Neural EGFL Like 2 (NELL2) and Ribosomal Protein L4 (RPL4), among which ANXA3, IL1R2, MMP9, LCN2, NELL2 might be the most significant candidate genes for COVID-19-induced IPF. The transcriptional factors of hub genes were found to be related to Sp1 Transcription Factor (SP1), Signal Transducer And Activator Of Transcription 1 (STAT1), RELA Proto-Oncogene, NF-KB Subunit (RELA), and Nuclear Factor Kappa B Subunit 1 (NFKB1). Immune cell analysis revealed that Plasma cells, Macrophages MO, Dendritic cells activated, Mast cells resting, and Eosinophils were significantly elevated in the IPF compared with healthy subjects. Conclusions: The findings shed light on the risk factors of COVID-19 in the development of IPF, and immune changes in COVID-19 and IPF. The shared DEGs might be prognostic in the treatment of IPF.

Unveiling the hub genes in the SIGLECs family in colon adenocarcinoma with machine learning.

Despite the recognized roles of Sialic acid-binding Ig-like lectins (SIGLECs) in endocytosis and immune regulation across cancers, their molecular intricacies in colon adenocarcinoma (COAD) are underexplored. Meanwhile, the complicated interactions between different SIGLECs are also crucial but open questions. We investigate the correlation between SIGLECs and various properties, including cancer status, prognosis, clinical features, functional enrichment, immune cell abundances, immune checkpoints, pathways, etc. To fully understand the behavior of multiple SIGLECs' co-evolution and subtract its leading effect, we additionally apply three unsupervised machine learning algorithms, namely, Principal Component Analysis (PCA), Self-Organizing Maps (SOM), K-means, and two supervised learning algorithms, Least Absolute Shrinkage and Selection Operator (LASSO) and neural network (NN). We find significantly lower expression levels in COAD samples, together with a systematic enhancement in the correlations between distinct SIGLECs. We demonstrate SIGLEC14 significantly affects the Overall Survival (OS) according to the Hazzard ratio, while using PCA further enhances the sensitivity to both OS and Disease Free Interval (DFI). We find any single SIGLEC is uncorrelated to the cancer stages, which can be significantly improved by using PCA. We further identify SIGLEC-1,15 and CD22 as hub genes in COAD through Differentially Expressed Genes (DEGs), which is consistent with our PCA-identified key components PC-1,2,5 considering both the correlation with cancer status and immune cell abundance. As an extension, we use SOM for the visualization of the SIGLECs and show the similarities and differences between COAD patients. SOM can also help us define subsamples according to the SIGLECs status, with corresponding changes in both immune cells and cancer T-stage, for instance. We conclude SIGLEC-1,15 and CD22 as the most promising hub genes in the SIGLECs family in treating COAD. PCA offers significant enhancement in the prognosis and clinical analyses, while using SOM further unveils the transition phases or potential subtypes of COAD.

Abstract CT065: Modakafusp alfa shows intratumoral immune pharmacodynamics (PD) in a subset of patients (pts) in a phase 1b study in advanced/metastatic solid tumors

Abstract Background: Modakafusp alfa (moda), a novel immunocytokine, is an innate immune enhancer comprising an anti-CD38 IgG4 antibody fused to 2 attenuated interferon (IFN) α2b molecules. Moda is being evaluated in phase 1/2 trials for multiple myeloma and in a phase 1/2 trial in solid tumors. Here, we present PD data in tumor biopsies and peripheral blood (PB) from the phase 1/2 study of moda as monotherapy in pts with advanced/metastatic solid tumors (NCT04157517). Methods: A total of 21 pts were treated with moda 0.1-1.5 mg/kg every 3 weeks. Tumor biopsy samples were collected at screening and on cycle 2, day 2 (C2D2). Biopsies were evaluated by multiplex immunofluorescence (using a Multiomyx platform) and RNASeq. PB samples were collected pre-dose on C1D1 and at timepoints after treatment. PB samples were evaluated by CyTOF to assess PD changes in immune cells. Results: No clinical tumor responses (partial or better) were observed in the 21 pts. Analysis of tumor biopsies revealed two subgroups defined by differences in upregulation of CD38 expression after moda treatment, with differential intratumoral PD changes. The CD38 gene has IFN response elements within its promoter region. Thus, correlation of tumor PD with changes in CD38 expression is of interest because, along with being a target of moda, an increase in CD38 expression may indicate drug activity in the tumor microenvironment. Interestingly, in the subset of pts in whom an increase in the percentage of CD38+ lymphocytes in response to moda was reported, there were also increases in CD8 and CD4 T cell activation, (%CD69) as well as CD8 T cell cytotoxic function (%GzB). Additionally, moda led to increase in CD14+CD16+ intermediate monocytes, indicating an enhanced myeloid response in several pts, and minimal changes in FoxP3+ Tregs regardless of changes in CD38 expression. Moda treatment led to an increase in activated and cytolytic natural killer (NK) and CD8 T cells in all pts. This was accompanied by increased NK and CD8 T cell proliferation (%Ki67) in PB, without sustained increase in exhaustion markers. CD4 T cells were also activated in response to moda and there was no increase in circulating FoxP3+ Tregs. Enhanced myeloid cell response, measured by increased proliferation of monocytes and dendritic cells, along with upregulation of costimulatory molecules, were also seen. Importantly, immune PD changes in the PB were seen in most pts and there were no immune PD differences identified between the two patient subsets defined in the biopsy analysis. Conclusion: PD biomarker data from this phase 1/2 trial in pts with advanced solid tumors demonstrated that moda enhances innate and adaptive immune activation in PB and within tumors. The presence of a differential intratumoral PD response in a subset of pts with elevated CD38+ cells highlights that tumor intrinsic factors may play a role in driving moda’s activity in tumors. Citation Format: Gurpanna Saggu, Janet L. Markman, Min Young Lee, Faith Dunbar, Adarsh Joshi, Shining Wang, Sabrina Collins. Modakafusp alfa shows intratumoral immune pharmacodynamics (PD) in a subset of patients (pts) in a phase 1b study in advanced/metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT065.

Abstract LB441: An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and nearly 50% of CRC patients develop liver metastasis during the course of their disease. Treatment of colorectal cancer liver metastasis (CRLM) has been particularly challenging due to tolerogenic nature of liver immune microenvironment as well as an eventual development of therapy resistance to standard chemotherapy regimen. One promising area of cancer immunotherapy is oncolytic virotherapy that can selectively destroy tumor cells and stimulate anti-tumor immune response. In this study, we systemically administered mSJ-650, an intravenous compatible oncolytic Wyeth strain vaccinia virus that expresses human complement regulatory protein CD55 and murine GM-CSF in place of viral thymidine kinase gene and has a deletion of the viral K2L gene, and evaluated its anti-tumor efficacy in CRLM. Mouse CRLM was induced by intrasplenic injection of 5×105 MC38-OVA cancer cells. Following tumor nodules formation in the liver, low dose (1×106 pfu) or high dose (3×106 pfu) of mSJ-650 was administered multiple times via tail vein and liver was harvested for evaluation of anti-tumor efficacy. Flow cytometry analysis was performed to investigate changes in hepatic and tumor immune microenvironment upon mSJ-650 treatment. Liver and tumor tissue were stained for immunofluorescence imaging of viral replication within the tumor cells and subsequent oncolysis by the virus. Systemic administration of mSJ-650 showed potent anti-tumor efficacy and led to significant reduction in metastatic nodule formation in liver even at low dose. The anti-tumor efficacy was mediated by tumor cell-specific viral distribution and replication, followed by lytic egress of mSJ-650 from infected tumor cells. Anti-tumor efficacy of mSJ-650 also accompanied a dramatic shift in immunologically tolerant hepatic microenvironment to immune-stimulatory one by decreasing proportion of myeloid-derived suppressor cells, Kupffer cells, and monocyte-derived macrophages (MDMs) in the liver while increasing CD8+ T cell infiltration by 2~3 folds. Moreover, mSJ-650 treatment was associated with phenotypic change in hepatic immune cells, as evidenced by Kupffer cell and MDM polarization to M1 phenotype and elevated expression of activation markers and cytotoxic molecules in CD8+ T cells. Changes in hepatic immune microenvironment were also observed in tumor nodules, especially in those completely surrounded by adjacent liver tissue, suggesting that anti-tumor immune response in tumor bed is potentiated by reprogramming of tumor-adjacent hepatic immune microenvironment. Systemic administration of mSJ-650 demonstrated superior anti-tumor efficacy in CRLM which was mediated by direct tumor cell killing and tumor-adjacent hepatic immune microenvironment reprogramming by the virus. Citation Format: Eunhye Kim, Yeonsoo Yang, Solchan Won, Namhee Lee, Songyi Lee, Mi-Ju Park, Byung-Jin Jung, Yun-Kyoung Hong, Hang-Rae Kim, Dong-Sup Lee, Keunhee Oh. An intravenous compatible oncolytic vaccinia virus treated hepatic metastasis of colon cancer through extensive cancer cell killing and activation of cytotoxic effector differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB441.

Abstract CT100: Safety and efficacy of AZD8853, an anti-growth and differentiation factor 15 (GDF15) antibody, in patients (pts) with advanced/metastatic solid tumors: First-in-human study

Abstract Background: GDF15 is overexpressed in solid tumors, with immunosuppressive effects on dendritic cells (DCs), T cells and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. AZD8853 is an IgG1 monoclonal antibody that neutralizes GDF15. This Phase 1/2a, open-label study assessed AZD8853 monotherapy in previously treated advanced/metastatic non-small-cell lung cancer, microsatellite-stable colorectal cancer (MSS-CRC) and urothelial carcinoma (UC) (NCT05397171). Methods: Eligible pts were ≥18 years old with ≥1 measurable lesion per RECIST v1.1 and ECOG PS 0/1. AZD8853 was given IV Q3W until disease progression, unacceptable toxicity or consent withdrawal. The primary objective was safety including dose-limiting toxicities (DLTs). Secondary objectives included efficacy, pharmacokinetics (PK) and pharmacodynamics (PD [free serum GDF15]). Exploratory objectives included peripheral immune PD. Results: During dose escalation, 16 pts (15 MSS-CRC, 1 UC) received AZD8853 (300 mg, n=3; 1000 mg, n=6; 3000 mg, n=7). By June 6, 2023, all pts discontinued treatment and the study. Thirteen pts (81.3%) had treatment-emergent adverse events (TEAEs; Table); the most common were diarrhea (31.3%), abdominal pain (31.3%) and decreased appetite (25%). Eight pts (50%) had Grade ≥3 TEAEs and 6 (37.5%) had serious TEAEs; none treatment-related. There were no DLTs. No pts had responses by RECIST; 5 (31.3%) had stable disease and 11 (68.8%) had disease progression as best response. AZD8853 PK was linear with a half-life of 5-10 days, supporting Q3W dosing. GDF15 suppression was transient. There was no evidence of ctDNA clearance or a dose-dependent change in peripheral immune cells including CD8+ and CD4+ T cells, DCs and MDSCs. Conclusions: AZD8853 was well tolerated. No objective responses or PD effects were seen and GDF15 suppression was not sustained, therefore no further development is planned. TABLE 1. NAND Table. AZD8853 safety AZD8853 300 mg 1000 mg 3000 mg Total Safety, n (%) N=3 N=6 N=7 N=16 TEAEs 3 (100) 4 (66.7) 6 (85.7) 13 (81.3) Grade ≥3 TEAEs 1 (33.3) 3 (50.0) 4 (57.1) 8 (50.0) TRAEs 1 (33.3) 0 2 (28.6) 3 (18.8) Grade ≥3 TRAEs 0 0 0 0 Serious TEAEs 1 (33.3) 3 (50.0) 2 (28.6) 6 (37.5) TEAEs leading to discontinuation of AZD8853 0 1 (16.7)* 0 1 (6.3) TEAEs leading to death 0 1 (16.7)* 0 1 (6.3) DLTs, n (%) N=3 N=6 N=6 † N=15 DLTs 0 0 0 0 *Death due to serious AE of cardiac arrest, unrelated to AZD8853; pt had a history of coronary artery disease, coronary stenting, and pulmonary embolism.†One pt discontinued due to clinical progression prior to completing the DLT evaluation period and was not included in the DLT-evaluable set. DLTs, dose-limiting toxicities; TEAEs, treatment-emergent adverse events, TRAEs, treatment-related adverse events Citation Format: Benedito A. Carneiro, Olumide Gbolahan, Albiruni Abdul Razak, John F. Hilton, Arthur Lambert, John Hood, Michael Pluta, Veronique Bragulat, Elhan Sanai, Rakesh Kumar, Duncan Jodrell, Patricia LoRusso. Safety and efficacy of AZD8853, an anti-growth and differentiation factor 15 (GDF15) antibody, in patients (pts) with advanced/metastatic solid tumors: First-in-human study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT100.

Abstract 6800: The tumor progression mechanism of increasing CXCL16-CXCR6 pathway within the tumor microenvironment in TNBC

Abstract Background: Triple negative breast cancer (TNBC) is characterized rapid progression, higher relapse, and poor prognosis, so the establishment of an effective therapeutic target is required. CXCL16, a chemokine abundantly present in the TNBC tumor microenvironment (TME), is secreted by tumor-associated macrophages (TAM) and is known to promote tumor growth and metastasis. However, the exact mechanism through which CXCL16 facilitates tumor growth and metastasis remains unclear. This study aims to study the role of CXCL16 in the tumor progression and confirm the anti-tumor effects of CXCL16 neutralization in TNBC model. Method: MDA-MB-231 human TNBC cell lines were utilized for in vitro validation, and a syngeneic model involving 4T1 mouse TNBC cell lines and Balb/c mice was used for in vivo studies. Results: Stimulation of TNBC cells with recombinant human CXCL16 protein (rhCXCL16) activates the tumor growth signaling pathways, including pAKT and STAT3, as well as EMT markers. These results shows that CXCL16 can promote tumor growth and metastasis. Treatment with CXCL16 neutralizing antibodies inhibited the promotion of cancer growth and metastasis by rhCXCL16. Additionally, combination treatment of CXCL16 antibodies with PD-1 antibodies in the 4T1 syngeneic model increased the proportion of CD8+ T cells and CD80+ M1 like macrophages. These results shows that neutralization of CXCL16 can regulate polarization of macrophage and proportion of T cells. These regulation and changes of immune cells allow neutralization of CXCL16 control the TME to resist tumor cells, suggesting that optimal condition can be constructed to maximize the effectiveness of anti-cancer drugs. Conclusion: This study confirms that the CXCL16-CXCR6 pathway within the TME triggers tumor growth and metastasis, while neutralization of CXCL16 can inhibit tumor progression. This effect is attributed to the increase in CD8+ T cell and M1 macrophage proportions within the TME. In conclusion, these research findings propose the potential of CXCL16 as a novel biomarker and therapeutic target for TNBC. Citation Format: Yun sun Lee, Cham Han Lee, Seong Yun Ha, Sang Hyeob Lee, Jin Yi Yang, Young Sang Kim, Sung Moo Kim, Seong Keun Kim. The tumor progression mechanism of increasing CXCL16-CXCR6 pathway within the tumor microenvironment in TNBC [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 6800.

Abstract 4184: Change of oncological features on tumor and immune cells after anti-PD-1 antibody to pancreatic cancer orthotopic model in CD34+ HSC based humanized mice

Abstract (a) To investigate novel therapeutic agents in pancreatic cancer, animal models that recapitulate the features of the human immune system are necessary. We aim to analyze an orthotopic model in CD34+HSC-based humanized mice and to prove that is an indispensable model for immune-oncology therapy research. (b) We established an orthotopic tumor model using luciferase-expressing BxPC3 cells in humanized NSG mice. After inoculation of tumor cells, anti-PD-1 Ab was administered to mice 5 times a week by I.P. injection. Tumor formation and progression were monitored using IVIS. To compare the subsets and immune-check point receptors of human immune cells in blood and tumor tissue, Flow cytometry and immunohistopathology were used. Additionally, we compared the distribution of various cells included in the tumor and the mRNA expression level of each cell through a single cell RNA sequence. We also establish primary cell lines and organoids with the humanized mouse tumor for further evaluation. (c) After inoculation of BxPC3_Luc cell line into mice pancreas, we monitored tumor growth using a noninvasive imaging method of IVIS. When we compared the tumor growth between the anti-PD-1 treatment and control, there was no significant difference. Using FACS, we analyzed the various markers of human immune cells in mouse blood and tumor tissue. In blood, PD-1 expression of CD8+ T cells decreased, and Tim-3 expression increased in anti-PD-1 group. And, PD-1 expression of CD4+ T cells decreased in anti-PD-1 group. In tumor tissue, we also verified the decrease of PD-1 expression on tissue infiltrating CD8+ T cells and CD4+ T cells in the anti-PD-1 group. Especially in the more responsive mouse among anti-PD-1 group, activation markers in cytotoxic T cells (CD8+), helper T cells (CD4+), and regulatory T cells (CD4+FOXP3+CD25+) increased, and also, M1 macrophage (CD86+) expression was increased. Furthermore, we compared the mRNA expression levels of various cells in the tumor microenvironment according to anti-PD-1 Ab treatment through single cell RNA seq analysis. It was confirmed that the expression of genes related to proliferation and growing decreased, while the expression of genes related to apoptosis increased in cancer cells. Finally, we constructed primary cancer cells and organoids from tumor tissue and confirmed that the immune-oncological characters of these models are maintained after passaging. (d) We successfully established a pancreatic orthotopic model in a humanized mouse and analyzed the changes in immune cells and tumor cells after anti-PD-1 treatment. In addition, by obtaining tumor tissue-based preclinical models, we laid the foundation for future immune-oncologic experiments. We hope that these attempts and results will help in the development and drug verification of new immunotherapy agents for refractory solid tumors such as pancreatic cancer. Citation Format: Sujin Park, Eun-young Koh, Inha Yoo, Ji Hye Jeong, Eunsung Jun. Change of oncological features on tumor and immune cells after anti-PD-1 antibody to pancreatic cancer orthotopic model in CD34+ HSC based humanized mice [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 4184.

Abstract 1598: Individualized longitudinal assessment reveals therapy-induced tumor and microenvironment dynamics in preclinical pancreatic cancer models

Abstract Background &amp; Aim: Progression and therapeutic resistance in pancreatic cancer depends on the ability of cancer cells to adopt specific cell states. These phenotypes largely depend on environmental tissue niches. Here, we investigated therapy-induced tumor dynamics in vivo at single-cell spatial and temporal resolution in different subtypes of PDAC using several genetically engineered mouse models, which reflect distinct human PDAC ecosystems. Methods &amp; Results: We established orthotopic tumor models of pancreatic cancer with distinct tumor microenvironment (TME) formation (reactive vs deserted stroma), which were monitored for tumor growth to define tumor response patterns under MAPK inhibition. Serial ultrasound-guided biopsy sampling was performed to collect tissues from the same tumors at pre-treatment, responsive and recurrence periods.Transcriptomic analysis of biopsies using the Mfuzz algorithm delineated highly dynamic gene and pathway activities during treatment. Reactive, but not deserted, TME showed an upregulation of immune-related pathways (antigen presentation, T cell receptor, NK cytotoxicity) immediately after MAPK inhibition in regressed tumors that dropped dramatically during recurrence of tumors, in which upregulation of TGF-b signaling was observed. In the deserted stroma model, on the other hand, an enrichment in drug metabolic pathways during recurrence was observed.CAF cluster deconvolution analysis revealed an increase in a LRRC15+ CAF cluster, which was TGF-b-driven and immunosuppressive, during recurrence period only in the reactive model. The dynamic changes of immune cells and CAFs were validated at protein level by multiplex immunofluorescent imaging. Cmputational spatial analysis revealed a tumor-encapsulating and CD8+ cytotoxic T cell-repelling pattern exhibited by LRRC15+FAP+ CAFs, implying a potential role of this CAF subset in T cell suppression during recurrence in reactive subtype. Using spectral cell sorting with CellView image technology, specific CAF subsets, tumor cells as well as immune cell subsets based on 30+ markers and cell morphology were isolated, characterized and assessed by ex vivo functional assays to confirm the immunosuppressive and tumor protective mechanism mediated by CAFs. Conclusion: Longitudinal in vivo assessment of distinct PDAC subtypes provides deep mechanistic insights to accurately unlock specific response patterns of tumors with heterogeneous TME dynamics upon therapeutic perturbation, leveraging identification of key nodes for intervention. Citation Format: Jens T. Siveke, Rui Fang, Phyllis Fung-Yi Cheung, Jiajin Yang, Juanfei Peng, Kristina Althoff, Konstantinos Savvatakis. Individualized longitudinal assessment reveals therapy-induced tumor and microenvironment dynamics in preclinical pancreatic cancer models [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 1598.

Abstract 2893: Inhibition of ADAMTS4 and 5 to regulate the tumor microenvironment

Abstract Background: Triple-negative breast cancer accounts for approximately 10~15% of all breast cancers and is characterized by the absence of ER, PR, and HER2 receptors, making targeted therapies challenging due to the lack of biomarkers. ADAMTSs (ADAM metallopeptidase with thrombospondin) are complex extracellular proteases known for their roles in facilitating cancer cell proliferation and safeguarding tumor functions. This enzyme is secreted by cancer cells and various immune cells, influencing the tumor microenvironment. ADAMTSs modulate cellular adhesion, migration, proliferation, and vascular development by cleaving components of the extracellular matrix and regulatory factors such as chemokines and cytokines. Our aim is to positively manipulate the tumor microenvironment of triple-negative breast cancer by inhibiting ADAMTSs, presenting a new strategy for treating this challenging type of breast cancer that lacks targeted therapeutic options. Results: To confirm the anticancer effects of a protease inhibitor concurrently targeting ADAMTS4 and 5, a co-culture of triple-negative breast cancer cells (MDA-MB-157) and THP-1 was established to artificially create a tumor microenvironment. Subsequently, a wound healing assay was conducted, revealing that treatment with the ADAMTS4 and 5 dual inhibitor effectively inhibited cancer cell migration. This observed anticancer effect is attributed to the inhibition of CXCL16 secretion by the ADAMTS4 and 5 dual inhibitor. To verify the anti-tumor mechanisms of ADAMTS4 and 5, MDA-MB-157 cells, a triple-negative breast cancer cell line, were treated with IFNγ and TNFα to promote CXCL16 secretion artificially. Upon treatment with varying concentrations of the ADAMTS4,5 dual inhibitor, it was confirmed that the secretion of CXCL16 was inhibited in a dose-dependent manner. In a mouse model with transplanted 4T1 breast cancer cells, treatment with the ADAMTS4,5 dual inhibitor resulted in the suppression of CXCL16 secretion, leading to an increase in M1 macrophages. Additionally, it was observed that the inhibitor induced CD8+ T cell activation. These regulation and changes of immune cells through inhibition of ADAMTS4 and 5 control the TME to resist tumor cells. Conclusion: ADAMTSs are known as protein-degrading enzymes responsible for the degradation, modification, and regulation of proteins. Recent research has revealed that ADAMTSs play a crucial role in the proliferation and survival of cancer. Activation of ADAMTS4 and 5 has been shown to promote the secretion of CXCL16, leading to the differentiation of macrophages into tumor-associated macrophages. Specifically, ADAMTS4 and 5 convert membrane-bound CXCL16 into a soluble form, exacerbating the tumor microenvironment of triple-negative breast cancer. Therefore, inhibiting ADAM4,5 is intended to change or maintain TME in a suitable state for anti-cancer therapy, which could lead to a new strategy for anti-cancer therapy. Citation Format: Cham han Lee, Seong Keun Kim, Sung Moo Kim, Young Sang Kim, Sang Hyeob Lee, Seong Yun Ha, Yun Sun Lee, Jin Yi Yang. Inhibition of ADAMTS4 and 5 to regulate the tumor microenvironment [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 2893.

Abstract 6860: Fluorescent tracking reveals that radiation therapy influences the migration and plasticity of Tregs within the tumor microenvironment

Abstract Radiation therapy (RT) has long been a cornerstone in cancer treatment, yet the mechanisms underlying its impact on the immune system remain incompletely understood. RT is recognized for its ability to induce DNA damage within cancer cells, leading to cancer cell death. For those cancer cells that do not die as a result of radiation, tumor infiltration by immune cells is closely associated with treatment outcomes. T cell infiltration is a dynamic process, with subsets taking up residence in the tumor while the majority recirculate through lymph nodes into the peripheral blood. T regulatory cells (Tregs) have been recognized as potent suppressors of anti-tumor immune responses, dampening immune-mediated clearance of cancer cells. While RT can initially contribute to an improved anti-tumor immune response, this beneficial effect is often counterbalanced by the presence of suppressive Tregs. Tregs can infiltrate the tumor microenvironment, ultimately impeding and overwhelming the immune response generated by RT. Our objective was to gain insights into the migratory patterns of Tregs to and from tumor sites and to elucidate how RT influences the dynamics of Treg recirculation. Utilizing the established photoconvertible Kaede mouse model we can track photoconverted cells following RT. Following photoconversion, photoconverted T cells were observable within the tumor and subsequently moved to the TdLN and on to other NdLN at later time points. In the TdLN of mice bearing MC38, Moc1, and Moc2 tumors, Tregs represented a significantly higher proportion of photoconverted cells compared to non-Treg CD4 or CD8 T cells. To validate their active departure from the tumor site, mice underwent FTY720 treatment, resulting in a reduction in both photoconverted Treg and non-Treg migration to the TdLN. When RT was administered to the tumor, there was a decrease in photoconverted Treg and non-Treg CD4 T cells within the TdLN, consistent with the expected cytotoxic effects on the irradiated tumor upstream. To focus on recruitment back from the TdLN, mice were analyzed after photoconversion of the TdLN. This revealed that a majority of TdLN Tregs exhibited a preference for relocating to the tumor, with only a minority migrating to the NdLN. The newly entered Tregs were analyzed in the tumor, at D1 and D3 post RT, and we found that these Tregs exhibited increased expression of CCR8, CD69, KLRG1 and Ly6C as compared to those in non-irradiated tumors, suggesting that these are recent entrants with limited suppressive capacity. These findings illuminate the intricate dynamics between RT, Tregs, and the use of site-specific photoconversion to track immune cell movement and changes over time to understand treatment responses. Our ongoing studies focus on the interplay between the Treg and the tumor environment that dictates the success of CD8 T cell mediated control of residual cancer cells following RT. Citation Format: David J. Friedman, Tiffany Blair, Gwen Kramer, Aanchal Preet Kaur, Jason Baird, Alejandro Alice, Megan Potuznik, Marka R. Crittenden, Michael J. Gough. Fluorescent tracking reveals that radiation therapy influences the migration and plasticity of Tregs within the tumor microenvironment [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 6860.

The impact of ovarian stimulation on the human endometrial microenvironment.

How does ovarian stimulation (OS), which is used to mature multiple oocytes for ART procedures, impact the principal cellular compartments and transcriptome of the human endometrium in the periovulatory and mid-secretory phases? During the mid-secretory window of implantation, OS alters the abundance of endometrial immune cells, whereas during the periovulatory period, OS substantially changes the endometrial transcriptome and impacts both endometrial glandular and immune cells. Pregnancies conceived in an OS cycle are at risk of complications reflective of abnormal placentation and placental function. OS can alter endometrial gene expression and immune cell populations. How OS impacts the glandular, stromal, immune, and vascular compartments of the endometrium, in the periovulatory period as compared to the window of implantation, is unknown. This prospective cohort study carried out between 2020 and 2022 included 25 subjects undergoing OS and 25 subjects in natural menstrual cycles. Endometrial biopsies were performed in the proliferative, periovulatory, and mid-secretory phases. Blood samples were processed to determine serum estradiol and progesterone levels. Both the endometrial transcriptome and the principal cellular compartments of the endometrium, including glands, stroma, immune, and vasculature, were evaluated by examining endometrial dating, differential gene expression, protein expression, cell populations, and the three-dimensional structure in endometrial tissue. Mann-Whitney U tests, unpaired t-tests or one-way ANOVA and pairwise multiple comparison tests were used to statistically evaluate differences. In the periovulatory period, OS induced high levels of differential gene expression, glandular-stromal dyssynchrony, and an increase in both glandular epithelial volume and the frequency of endometrial monocytes/macrophages. In the window of implantation during the mid-secretory phase, OS induced changes in endometrial immune cells, with a greater frequency of B cells and a lower frequency of CD4 effector T cells. The data underlying this article have been uploaded to the Genome Expression Omnibus/National Center for Biotechnology Information with accession number GSE220044. A limited number of subjects were included in this study, although the subjects within each group, natural cycle or OS, were homogenous in their clinical characteristics. The number of subjects utilized was sufficient to identify significant differences; however, with a larger number of subjects and additional power, we may detect additional differences. Another limitation of the study is that proliferative phase biopsies were collected in natural cycles, but not in OS cycles. Given that the OS cycle subjects did not have known endometrial factor infertility, and the comparisons involved subjects who had a similar and robust response to stimulation, the findings are generalizable to women with a normal response to OS. OS substantially altered the periovulatory phase endometrium, with fewer transcriptomic and cell type-specific changes in the mid-secretory phase. Our findings show that after OS, the endometrial microenvironment in the window of implantation possesses many more similarities to that of a natural cycle than does the periovulatory endometrium. Further investigation of the immune compartment and the functional significance of this cellular compartment under OS conditions is warranted. Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (R01AI148695 to A.M.B. and N.C.D.), Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD109152 to R.A.), and the March of Dimes (5-FY20-209 to R.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or March of Dimes. All authors declare no conflict of interest.

Immunological Analysis of Postoperative Delirium after Thoracic Aortic Surgery.

Delirium is a recognized neurological complication following cardiac surgery and is associated with adverse clinical outcomes, including elevated mortality and prolonged hospitalization. While several clinical risk factors for post-cardiac surgery delirium have been identified, the pathophysiology related to the immune response remains unexamined. This study was conducted to investigate the immunological factors contributing to delirium in patients after thoracic aortic surgery. We retrospectively evaluated 43 consecutive patients who underwent thoracic aortic surgery between July 2017 and June 2018. These patients were categorized into 2 groups: those with delirium and those without it. All clinical characteristics were compared between groups. Blood samples were collected and tested on the day of admission, as well as on postoperative days 1, 3, 7, and 30. Levels of helper T cells (CD4), cytotoxic T cells (CD8), B cells (CD19), natural killer cells (CD56+CD16++), and monocytes (CD14+CD16-) were measured using flow cytometry. The median patient age was 71 years (interquartile range, 56.7 to 79.0 years), and 21 of the patients (48.8%) were male. Preoperatively, most immune cell counts did not differ significantly between groups. However, the patients with delirium exhibited significantly higher levels of interleukin-6 and lower levels of tumor necrosis factor-alpha (TNF-α) than those without delirium (p<0.05). Multivariate analysis revealed that lower TNF-α levels were associated with an increased risk of postoperative delirium (p<0.05). Postoperative delirium may be linked to perioperative changes in immune cells and preoperative cytokine levels. Additional research is required to elucidate the pathophysiological mechanisms underlying delirium.

Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

BackgroundWhile T cell-activating immunotherapies against recurrent head and neck squamous cell carcinoma (HNSCC) have shown impressive results in clinical trials, they are often ineffective in the majority of patients. NK cells are potential targets for immunotherapeutic intervention; however, the setback in monalizumab-based therapy in HNSCC highlights the need for an alternative treatment to enhance their antitumor activity.MethodsSingle-cell RNA sequencing (scRNA-seq) and TCGA HNSCC datasets were used to identify key molecular alterations in NK cells. Representative HPV-positive ( +) and HPV-negative ( −) HNSCC cell lines and orthotopic mouse models were used to validate the bioinformatic findings. Changes in immune cells were examined by flow cytometry and immunofluorescence.ResultsThrough integration of scRNA-seq data with TCGA data, we found that the impact of IL6/IL6R and CCL2/CCR2 signaling pathways on evasion of immune attack by NK cells is more pronounced in the HPV − HNSCC cohort compared to the HPV + HNSCC cohort. In orthotopic mouse models, blocking IL6 with a neutralizing antibody suppressed HPV − but not HPV + tumors, which was accompanied by increased tumor infiltration and proliferation of CD161+ NK cells. Notably, combining the CCR2 chemokine receptor antagonist RS504393 with IL6 blockade resulted in a more pronounced antitumor effect that was associated with more activated intratumoral NK cells in HPV − HNSCC compared to either agent alone.ConclusionsThese findings demonstrate that dual blockade of IL6 and CCR2 pathways effectively enhances the antitumor activity of NK cells in HPV-negative HNSCC, providing a novel strategy for treating this type of cancer.