Changes In Immune Microenvironment Research Articles

Regulation of vascular remodeling by immune microenvironment after the establishment of autologous arteriovenous fistula in ESRD patients.

Autogenous arteriovenous fistula (AVF) is the preferred dialysis access for receiving hemodialysis treatment in end-stage renal disease patients. After AVF is established, vascular remodeling occurs in order to adapt to hemodynamic changes. Uremia toxins, surgical injury, blood flow changes and other factors can induce inflammatory response, immune microenvironment changes, and play an important role in the maintenance of AVF vascular remodeling. This process involves the infiltration of pro-inflammatory and anti-inflammatory immune cells and the secretion of cytokines. Pro-inflammatory and anti-inflammatory immune cells include neutrophil (NEUT), dendritic cell (DC), T lymphocyte, macrophage (Mφ), etc. This article reviews the latest research progress and focuses on the role of immune microenvironment changes in vascular remodeling of AVF, in order to provide a new theoretical basis for the prevention and treatment of AVF failure.

Abstract 187: Macrophage instead of leukemia cell expressed PD-L1 drives T cell exhaustion and immune evasion of BCRABL1 chronic myeloid leukemia

Abstract BCR-ABL1 fusion gene is the hall marker of chronic myeloid leukemia (CML). Currently, the immune microenvironment of BCRABL1 CML remains largely unexplored. Our recent investigation in a transgenic mouse model of BCRABL1 CML revealed a continuous increase in CD11b+Ly6Cint PMN-MDSC with the progression of the disease. Concurrently, there was an increase in CD11b+Ly6Chigh M-MDSC and F4/80+ macrophages. However, The levels of CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD49b+ NK cells, which are essential for tumor-killing significantly decreased. This immune phenotype was consistently observed in both primary leukemia induction and following serial transplantation of cells from primary leukemic mice. Intriguingly, only recipient mice subjected to sublethal irradiation developed the disease, while mice with intact immune system remained disease-free, which underscores the vital role of the immune system in the development of CML. The predominant presence of CD11b+Ly6Cint PMN-MDSC in the peripheral blood, bone marrow and spleen implies these cells may be at least partially consisted of CML leukemia cells. To examine the hypothesis, qRT-PCR and immunofluorescence (IF) staining were conducted. In both assays, Ly6G+ cells from BCRABL1 mice coexpressed the BCRABL1 oncogene. Furthermore, when co-cultured with CD4 T cells, the division of T cells was significantly inhibited, implying immune suppression by the Ly6G+ PMN-MDSCs and/or CML leukemia cells. In addition, the immuosuppressive pathway genes including Arg1, Hif1a, Enptd1, TGFβ were significantly upregulated. To investigate the possible role of the PD1/PDL1 axis in the BCRABL1 model, flow cytometry analysis was conducted. Unexpectedly, PDL1 was barely expressed in CML leukemia cells, whereas the expression of PDL1 in M-MDSCs and macrophages significantly increased, accompanied by remarkable upregulation of PD1 expression on CD4 T cells and CD8 T cells during leukemia progression. In other words, PDL1 expression on macrophages, rather than leukemia cells, was found to induce T cell exhaustion. For translational purpose, mice transplanted with the primary CML leukemia cells were treated with chemotherapy drugs ponatinib alone or in combination with the anti-PD1 antibody. Compared with the chemotherapy alone group, the mice in the combinational therapy group exhibited rapid remission and better disease control. Most importantly, the disease relapse occurred at a slower rate after treatment discontinuation, suggesting the value of combinational therapy in achieving treatment free remission. In summary, our study not only revealed the dynamic changes of immune microenvironment in the BCRABL1 CML mouse model, but also revealed the different mechanisms underlying immune evasion of BCR-ABL1 CML leukemia cells, which sheds light on a combinational treatment for achieving the treatment free remission of CML. Citation Format: Xiaocui Lu, Hui Fang, Xuexiu Fang, Atsuko Matsunaga, Kebin Liu, Jorge Cortes, John K. Cowell, Tianxiang Hu. Macrophage instead of leukemia cell expressed PD-L1 drives T cell exhaustion and immune evasion of BCRABL1 chronic myeloid leukemia [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 187.

Neoadjuvant toripalimab plus CapeOX in patients with localized dMMR/MSI-H gastric or esophagogastric junction adenocarcinoma (GC/EGJC): Results from the cohort C of phase II NICE trial.

349 Background: Surgery is the cornerstone of curative therapy for localized GC or EGJC and perioperative chemotherapy seems ineffective for those with dMMR/MSI-H tumors. Chemotherapy may booster antitumor immunity. There is a compelling need to investigate the efficacy of perioperative immunochemotherapy for those with dMMR/MSI-H tumors. Methods: NICE trial is a multicenter, multi-cohort phase II trial (NCT04744649) investigating the safety and efficacy of toripalimab combined with CapeOX regimen as perioperative treatment for localized GC or EGJC of PD-L1 CPS≥5 (IHC 22C3) (cohort A), EBV-positive (cohort B), and dMMR/MSI-H (cohort C). Among those cohorts, the cohort A was compared with the control group of perioperative CapeOX regimen in a randomized controlled trial manner, while the cohort B and C were single arm. In present study, we report the findings of Cohort C, and the patients (pts) received toripalimab 240 mg combined with standard dose CapeOX regimen q3 weeks for 4 cycles before and after surgery. The eligible tumor stages were cT3-4aNxM0 or cT2N+M0 diagnosed by chest and abdominal CT combined with staging laparoscopy as well as negative peritoneal cytology. The primary endpoint was major pathologic response (MPR, <10% viable cells) rate. Multiplex fluorescence immunohistochemistry (mIHC) was utilized to explore changes of immune microenvironment. Results: Between March 2021 and April 2023, 15 pts with dMMR/MSI-H localized GC/GEJC were recruited in Cohort C. Tumor stages were cT3N0(n=2), cT3N1-3 (n=2), cT4aN0 (n=1) and cT4aN1-3 (n=10). 14 pts completed all 4 cycles therapy preoperatively and one patient completed 2 cycles therapy because of AEs. None had disease progression, while one achieved a complete clinical response at radiology and endoscopy and refused surgery and the other 14 pts underwent resection. The R0 resection rate was 100% (14/14). The MPR rate was 92.9% (13/14) and the residual tumor percentage was 33.8% in the other one patient (cT4aN2). pCR rate was 78.6% (11/14). Therapy-related grade 3/4 AEs occurred in 5 pts of safety population (n=15). One patient occurred grade 3 immune-related liver toxicity after 2 cycles treatment and recovered after steroids therapy. No pts died during the perioperative treatment, and one patient died of COVID-19 238 days after surgery without relapse. No disease relapse was observed in all pts. The mIHC revealed a significant reduction in CD20+ B cells, CD3+ T cells, NK dim cells, and M1 macrophages within tumor area after treatment for patients who reached pCR. Conclusions: Neoadjuvant toripalimab and CapeOX regimen for the localized dMMR/MSI-H GC/GEJC is safety with high MPR rate. These data indicate that neoadjuvant immunochemotherapy may potentially become one of the treatment options for these patients. Clinical trial information: NCT04744649 .

An oncolytic system produces oxygen selectively in pancreatic tumor cells to alleviate hypoxia and improve immune activation

IntroductionHypoxia is one of the important reasons for the poor therapeutic efficacy of current pancreatic cancer treatment, and the dense stroma of pancreatic cancer restricts the diffusion of oxygen within the tumor. MethodsA responsive oxygen-self-supplying adv-miRT-CAT-KR (adv-MCK) cascade reaction system to improve hypoxia in pancreatic cancer is constructed. We utilized various experiments at multiple levels (cells, organoids, in vivo) to investigate its effect on pancreatic cancer and analyzed the role of immune microenvironment changes in it through high-throughput sequencing. ResultsThe adv-MCK system is an oncolytic adenovirus system expressing three special components of genes. The microRNA (miRNA) targets (miRTs) enable adv-MCK to selectively replicate in pancreatic cancer cells. Catalase catalyzes the overexpressed hydrogen peroxide in pancreatic cancer cells to generate endogenous oxygen, which is catalyzed by killerRed to generate singlet oxygen (1O2) and further to enhance the oncolytic effect. Meanwhile, the adv-MCK system can specifically improve hypoxia in pancreatic cancer, exert antitumor effects in combination with photodynamic therapy, and activate antitumor immunity, especially by increasing the level of γδ T cells in the tumor microenvironment. ConclusionThe responsive oxygen-self-supplying adv-MCK cascade reaction system combined with photodynamic therapy can improve the hypoxic microenvironment of pancreatic cancer and enhance antitumor immunity, which provides a promising alternative treatment strategy for pancreatic cancer.

Sustained Liver HBsAg Loss and Clonal T- and B-Cell Expansion upon Therapeutic DNA Vaccination Require Low HBsAg Levels.

Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination.

Abstract B037: A pharmacodynamic pilot study of DS8201a in patients with HER2 expressing advanced solid tumors

Abstract Background: DS-8201a (Trastuzumab (T) deruxtecan(D)) is an antibody-drug conjugate consisting of the HER2-targeting antibody T covalently bound via a lysosomal protease-cleavable linker to the novel topoisomerase 1 (Top1) inhibitor D with activity in tumors with low HER2 IHC scores of 1+ or 2+. The primary purpose of this study is to investigate the pharmacodynamic (PD) mechanisms underlying the activity of DS-8201a in advanced solid tumors, including those with low HER2 expression. Methods: This multicenter pilot study enrolled patients (pts) with advanced solid tumors expressing HER2 as defined by CLIA-certified labs i.e., a HER2 IHC score of 1+ or greater or ERBB2 amplifications (amp) or mutations (mut). DS-8201a was administered at 5.4 mg/kg intravenously once every 3 weeks, in 21-day cycles (C). PD biomarkers of Top1 target engagement by D payload, consequent DNA damage and repair (DDR), and immune microenvironment changes were analyzed in mandatory tumor biopsies collected at baseline, post-dose C1 and pre-dose C3. Blood samples for biomarker analyses were taken at several time points. Safety and overall response were also evaluated. Results: As of June 16, 2023, a total of 48 pts have been enrolled with diverse HER2 status distribution: 11 IHC 1+, 20 IHC 2+, 6 IHC 3+, 7 ERBB2 amp, 3 ERBB2 mut, and 1 with both IHC 1+ and ERBB2 amp. Median number of prior lines of systemic therapy was 3.5 (range 1-13) and 11 pts received prior Top1 inhibitor therapy. Grade (Gr) 3 or higher treatment-related adverse events (trAEs) were reported in 12 pts (25%). The most common Gr3/4 trAEs were myelosuppression. There were 7 pts (15%) with Gr2 and 1 pt (2%) with Gr3 pneumonitis. One pt with Gr2 pneumonitis was retreated after trAE resolution with steroid therapy and continued for another 9 Cs prior to progression. Of the evaluable patients, 1 pt (2%) had complete response (cervical, HER2 2+), 6 pts (13%) had confirmed partial response (PR) (uterine, 2+; ovarian, 2+; bladder, ERBB2 mut; uterine carcinosarcoma, 2+; 2 head and neck cancers, 1+ and 2+), 4 pts (8%) had unconfirmed PR (breast, 1+; extramammary Paget’s disease, 2+; pancreas, ERBB2 amp; uterine, 2+), and 17 pts (35%) had stable disease as their best response. Median progression free survival was 3.4 months (range 0.5-17.2). Top1 target engagement and induction of nuclear biomarkers of DDR (RAD51, pNBS1) were observed in more than half of the paired biopsies tested to date. DDR biomarker response was associated with both HER2 1+ and 2+ status. PD analyses are currently ongoing. Conclusion: DS-8201a demonstrated responses in various tumor types outside of breast cancer with HER2 low/ERBB2 alteration status. Preliminary PD data demonstrated expected Top1 target engagement and DDR induction, the extent of which is possibly independent of the degree of HER 2 staining. Analyses are currently underway to further define the effect of DS-8201a in advanced solid tumors. Citation Format: Sarah J Shin, Geraldine O'Sullivan-Coyne, Elizabeth K Lee, Arjun Mittra, Cora N Sternberg, Mary Jane Ong, Deborah Wilsker, Katherine Ferry-Galow, Jared Foster, Lamin Juwara, Naoko Takebe, Cheryl A Pickett, Ralph Parchment, James Doroshow, Alice Chen. A pharmacodynamic pilot study of DS8201a in patients with HER2 expressing advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B037.

Single-Cell RNA Sequencing Reveals Contradictory Immune Microenvironment in Hemophagocytic Syndrome

Hemophagocytic lymphohistiocytosis (HLH), a formidable and rare clinical syndrome impacting multiple organs, manifests as a perilous condition. However, the intricate pathophysiological mechanisms underlying HLH remain yet to be fully elucidated. In this study, we obtained peripheral blood samples from 9 HLH patients spanning the years 2018 to 2022. Employing single-cell RNA sequencing (scRNA-seq), we conducted a comprehensive analysis of the alterations in the immune microenvironment, delving into the role of EBV infection in HLH and its influence on pathogenesis. By comparing with health donor peripheral blood , we identified a total of 11 significant immune cell populations among 72,779 single-cell transcriptomes, comprising 20,337 cells from healthy controls and 52,442 cells from HLH patients. Notably, we found that the proportion of T cells increased significantly, and other immune cells decreased in HLH patients. In terms of function, we found that the overall T cell killing ability was weakened, and the factor secretion ability was enhanced. Concerning myeloid cells, the impact of CD16+ monocytes on T cells exhibited a pronounced increase, along with an enhanced ability to secrete IL-18 factors, potentially serving as the main source of cells promoting T cell function. Different subpopulations of T cells have significant changes in the pathogenesis of HLH. Remarkably, both CD4+ T cells and CD8+ T cells exhibited HLA-DR+GZMK+ cell subgroups with upregulated IFN-γ production and response, suggesting their association with the hyperinflammatory state in HLH. A correlation analysis between the proportion of HLA-DRA+ GZMK+ T cells and the severity score revealed a significant correlation (p<0.05), with the ROC curve further affirming their predictability in assessing high inflammatory activity. Among CD4+ T cells, the proportion of Treg cells and the secretion of inhibitory factors such as TGFb1 decreased. Additionally, we noted the presence of a functionally impaired subset of CBLB+CD8+ T cells, indicating the considerable variability in T cell subset functionality in HLH. Next, a comprehensive analysis of the alterations in myeloid cells, B cells, and NK cells in HLH patients was conducted. We observed a substantial reduction in the number of DC cells and immune-related functions within various myeloid cell subsets. Regarding B cells, an increase in anergy B cells was observed, whereas the population of plasma cells, an effective B cell type, exhibited a decrease. In the context of NK cells, there was a significant increase in the proportion of NK cell subsets associated with IFN-γ secretion. Moreover, EBV-infected T cells in EBV-associated HLH were identified as the principal source of IFN-γ. Through single-cell EBV-CALL analysis, we found that IFN-γ secretion was primarily attributed to EBV-infected T cells, subsequently influencing CD16+ monocytes. Additionally, IL-16 production was predominantly observed in EBV-infected CD16+ monocytes, thereby impacting lymphocytes. Moreover, Monocytes were stimulated by EBV-infected CD4+ T cells and Atypical T cells, leading to the secretion of a pro-inflammatory factors such as MIF. Furthermore, our investigation unveiled that EBNA2 could promote MYC into the nucleus and enhance the proliferation and IFN-γ secretion of T cells. In conclusion, our comprehensive analysis of the immune microenvironment changes following the onset of HLH has yielded valuable insights and novel perspectives for understanding the underlying mechanisms and potential clinical treatments of this condition.

Single-cell transcriptome analysis of liver immune microenvironment changes induced by microplastics in mice with non-alcoholic fatty liver

Recent studies have discovered that tiny particles of microplastics (MPs) at the nano-scale level can enter the body of organisms from the environment, potentially causing metabolic ailments. However, further investigation is required to understand the alterations in the immune microenvironment associated with non-alcoholic fatty liver disease (NAFLD) occurrence following exposure to MPs.Experiments were performed using mice, which were given a normal chow or high-fat diet (NCD or HFD, respectively) plus free drinking of sterile water with or without MPs, respectively. Employing an impartial technique known as unbiased single-cell RNA-sequencing (scRNA-seq), the cellular (single-cell) pathology landscape of NAFLD and related changes in the identified immune cell populations induced following MPs plus HFD treatment were assessed. The results showed that mice in the HFD groups had remarkably greater NAFLD activity scores than those from the NCD groups. Moreover, administration of MPs plus HFD further worsened the histopathological changes in the mice's liver, leading to hepatic steatosis, inflammatory cell infiltrations and ballooning degeneration. Following the construction of a sing-cell resolution transcriptomic atlas of 43,480 cells in the mice's livers of the indicated groups, clear cellular heterogeneity and potential cell-to-cell cross-talk could be observed. Specifically, we observed that MPs exacerbated the pro-inflammatory response and influenced the stemness of hepatocytes during HFD feeding. Importantly, treatment with MPs significantly increase the infiltration of the infiltrating liver-protecting Vsig4+ macrophages in the liver of the NAFLD mouse model while remarkably decreasing the angiogenic S100A6+ macrophage subpopulation. Furthermore, mice treated with MPs plus HFD exhibited significantly increased recruitment of CD4+ cells and heightened exhaustion of CD8+ T cells than those from the control group, characteristics typically associated with the dysregulation of immune homeostasis and severe inflammatory damage. Overall, this study offers valuable perspectives into comprehending the potential underlying cellular mechanisms and regulatory aspects of the microenvironment regarding MPs in the development of NAFLD.

A Macrophage-Related Gene Signature for Identifying COPD Based on Bioinformatics and ex vivo Experiments.

This study aims to investigate the association between immune cells and the development of COPD, while providing a new method for the diagnosis of COPD according to the changes in immune microenvironment. In this study, the "CIBERSORT" algorithm was used to estimate the tissue infiltration of 22 types of immune cells in GSE20257 and GSE10006. The "limma" package was used for differentially expressed analysis. The key modules associated with vital immune cells were identified using WGCNA. GO and KEGG enrichment analysis revealed the biological functions of the candidate genes. Ultimately, a novel diagnostic prediction model was constructed via machine learning methods and multivariate logistic regression analysis based on GSE20257. Furthermore, we examined the stability of the model on one internal test set (GSE10006), three external test sets (GSE8545, GSE57148 and GSE76925), one single-cell transcriptome dataset (GSE167295), macrophages (THP-M cells) and lung tissue from COPD patients. M0 macrophages (AUC > 0.7 in GSE20257 and GSE10006) were considered as the most important immune cells through exploring the immune microenvironment landscapes in COPD patients and healthy controls. The differentially expressed genes from GSE20257 and GSE10006 were divided into six and five modules via WGCNA, respectively. The green module in GSE20257 (cor = 0.41, P < 0.001) and the brown module in GSE10006 (cor = 0.67, P < 0.001) were highly correlated with M0 macrophages and were selected as key modules. Forty-one intersected genes obtained from two modules were primarily involved in regulation of cytokine production, regulation of innate immune response, specific granule, phagosome, lysosome, ferroptosis, and other biological processes. On the basis of the candidate genetic markers further characterized via the "Boruta" and "LASSO" algorithm for COPD, a diagnostic model comprising CLEC5A, FTL and SLC2A3 was constructed, which could accurately distinguish COPD patients from healthy controls in multiple datasets. GSE20257 as the training set has an AUC of 0.916. The AUCs of the internal test set and three external test sets were 0.873, 0.932, 0.675 and 0.688, respectively. Single-cell sequencing analysis suggested that CLEC5A, FTL and SLC2A3 were expressed in macrophages from COPD patients. The expressions of CLEC5A, FTL and SLC2A3 were up-regulated in THP-M cells and lung tissue from COPD patients. According to the variations of immune microenvironment in COPD patients, we constructed and validated a novel macrophage M0-associated diagnostic model with satisfactory predictive value. CLEC5A, FTL and SLC2A3 are expected to be promising targets of immunotherapy in COPD.

Changes of immune microenvironment in head and neck squamous cell carcinoma in 3D-4-culture compared to 2D-4-culture

BackgroundThe immune system plays a crucial role in initiating, progressing, and disseminating HNSCC. This study aims to investigate the differences in immune microenvironments between 2D-4-culture and 3D-4-culture models of head and neck squamous cell carcinoma (HNSCC) cells (FaDu), human fibroblasts (HF), human monocytes (THP-1), and human endothelial cells (HUVEC).MethodsFor the 3D-4-culture model, FaDu:HF:THP-1 (2:1:1) were inoculated in an ultra-low attachment culture plate, while HUVECs were placed in a transwell chamber. The ordinary culture plate was used for the 2D-4-culture model. Tumor-associated macrophage markers (CD163), tumor-associated fibroblast markers (FAP), and epithelial-mesenchymal transition (EMT) were detected by western blot. Inflammatory cytokines (IL-4, IL-2, CXCL 10, IL-1 β, TNF-α, CCL 2, IL-17 A, IL-6, IL-10, IFN-γ, IL-12 p 70, CXCL 8, TGFβ1) in the supernatant were measured by flow cytometry. HUVEC migration was observed under a microscope. The 3D spheres were stained and observed with a confocal microscope. CCK8 assay was used to detect the resistance of mixed cells to cisplatin in both 2D-4-culture and 3D-4-culture.ResultsAfter three days of co-culture, the 3D-4-culture model showed increased expression levels of CD163 and FAP proteins (both P < 0.001), increased expression of E-cadherin protein and N-cadherin protein expression (P < 0.001), decreased expression of vimentin (P < 0.01) and Twist protein (P < 0.001). HUVEC migration significantly increased (P < 0.001), as did the concentrations of IP-10, MCP-1, IL-6, and IL-8 (all P < 0.001). Confocal microscopy showed that 3D-4-culture formed loose cell clusters on day 1, which gradually became a dense sphere surrounded by FaDu cells invading the inside. After co-culturing for 24 h, 48 h, and 72 h, the resistance of mix cells to cisplatin in 3D-4-culture was significantly higher than in 2D-4-culture (P < 0.01 for all).ConclusionCompared to 2D-4-culture, 3D-4-culture better simulates the in vivo immune microenvironment of HNSCC by promoting fibroblast transformation into tumor-associated fibroblasts, monocyte transformation into tumor-associated macrophages, enhancing endothelial cell migration ability, partial EMT formation in HNSCC cells, and is more suitable for studying the immunosuppressive microenvironment of HNSCC.Graphical abstract

Exploring Psoriasis Inflammatory Microenvironment by NanoString Technologies.

Psoriasis is a chronic inflammatory skin disease whose molecular mechanisms and microenvironment are poorly understood. We performed gene expression analysis through the nCounter® PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA) on 22 FFPE punch biopsies from 19 psoriasis-affected patients. A subset of five cases was analyzed before (T0) and after 6 months (T6) of treatment with dimethyl fumarate (DMF) to address immune microenvironment changes. Molecular comparisons according to biopsy site and age of onset showed a different distribution of innate immune cells (mast cells, macrophages, NK cells, and DC cells) and pathways (complement regulation and transporter functions). The analysis according to PASI (Psoriasis Area and Severity Index) led to non-significant results, suggesting no link between molecular expression profile and clinical amount of skin disease. In DMF-treated patients, we observed a strong immunomodulatory effect after treatment: A subversion of exhausted CD8 T cells, NK CD56dim cells, Tregs, neutrophils, CD45+ cells, T cells, B cells, and macrophages was reported between the two analyzed time-points, as well as the reduction in pro-inflammatory pathways and molecules, including cytotoxicity, pathogen defense, antigen processing, adhesion, cell cycle, chemokines, cytokines, and interleukins. The inflammatory psoriatic microenvironment can be modulated using DMF with encouraging results, achieving an immune-tolerant and non-inflammatory condition through the regulation of both innate and adaptive immunity.

Impact of PRaG Therapy on Immune Microenvironment of Bilateral Subcutaneous Tumor Model of Colon Cancer

Immune microenvironment is closely related to the efficacy of PD-1 inhibitors. The immune microenvironment contains a variety of immune cells, including effector T cells (cytotoxic CD8+T cells and effector CD4+T cells), dendritic cells (DC), and Myeloid-derived suppressor cells (MDSCs). The antitumor effects of PRaG therapy have been confirmed in bilateral subcutaneous transplantation tumor model of colon cancer. But the impact of PRaG therapy on immune microenvironment of such model is unclear. Therefore, the study continued to reveal the changes of immune microenvironment in mice. 80 male Balb/c mice aged 6-8 weeks were divided into five groups: control group, PD-1 inhibitor group, radiation group, radiation + PD-1 inhibitor group, and radiation + PD-1 inhibitor +GM-CSF (PRaG therapy) group. Bilateral subcutaneous tumor model of colon cancer in mice was constructed. 3×105 CT26.WT cells were inoculated subcutaneously in the right thigh root, and then the left thigh root 3 days later. Right subcutaneous tumor was selected for radiotherapy of 8 Gy×3. GM-CSF (100ng, i.p.) was given on the 1st day and PD-1 inhibitor (0.25mg/kg, i.p.) was given on the 2nd day after radiotherapy with one cycle every 3 days. On day 15, the spleen, left inguinal lymph node and left subcutaneous tumor of mice were collected. The proportion of immune cells was detected by flow cytometry. Compared with other groups, PRaG therapy decreased the proportion of cDC1 in left inguinal lymph node, increased the proportion of cDC2 in left subcutaneous tumor and left inguinal lymph node. Moreover, PRaG therapy increased the proportion of CD8+ effector memory T cells and CD226+CD8+T cells in left inguinal lymph nodes. Finally, PRaG therapy increased the proportion of CD4+, CD8+ central memory T cells and CD69+CD8+T cells and reduced the proportion of M-MDSCs in spleen. PRaG therapy can improve the immune microenvironment of spleen, unirradiated tumors and inguinal draining lymph nodes of bilateral subcutaneous tumor model of colon cancer in mice.

Identification of immune microenvironment changes, immune-related pathways and genes in male androgenetic alopecia.

Although androgenetic alopecia (AGA) is classified as a non-inflammatory alopecia, histological evidence of microinflammation has long been recognized. However, changes in the immune microenvironment, immune-related pathways and the expression of immune-related genes (IRGs) involved in AGA remain unclear. The microarray gene expression data (GSE36169) from patients with male AGA were analyzed. gene set enrichment analysis (GSEA) among statistically changed genes was done. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses among differentially expressed genes were performed. differentially expressed genes were screened to identify IRGs based on the ImmPort database. The cytohubba-MCC plugin of Cytoscape was applied to screen hub immune genes. The infiltration levels of 28 immune cells were quantified adopting single-sample GSEA (ssGSEA) algorithm. The microarray gene expression data (GSE90594) of male AGA was analyzed to validate hub IRGs genes and differential infiltrated immune cells. The ssGSEA revealed γδT cell, central memory CD8+ T cell, mast cell, immature B cell, activated CD8+ T cell, effector memory CD4+ T cell, eosinophil and neutrophil were significantly increased infiltration in the bald scalp. GSEA showed statistically changed genes were most enriched in immune related pathways, including innate immune system, adaptive immune system, cytokine signaling, interferon-γ signaling, interferon signaling and interleukins signaling. The 4 hub IRGs, including matrix metallopeptidase 9, protein tyrosine phosphatase receptor type C, bone morphogenetic protein 2, and thrombospondin 1, were enriched in the pathways of allograft rejection, coagulation and interferon-γ response. In summary, we proposed that the increase in γδ T cells, central memory CD8+ T cells, activated CD8+ T cell as well as the infiltration of mast cells contributed to immune microenvironment changes in male AGA. The 4 hub IRGs may be involved in the development and progression of hair loss in male AGA through interferon-γ signal pathways.

The clinical, genetic, and immune landscape of meningioma in patients with NF2-schwannomatosis.

NF2-schwannomatosis is the most common genetic predisposition syndrome associated with meningioma. Meningioma in NF2-schwannomatosis is a major source of morbidity and mortality. This is due to accumulative tumor burden in patients with synchronous schwannomas and ependymomas, sometimes including complex collision tumors. Balancing the impact of multiple interventions against the natural history of various index tumors, and the ongoing risk of de novo tumors over an individual's lifetime makes decision-making complex. The management of any given individual meningioma is often different from a comparable sporadic tumor. There is typically a greater emphasis on conservative management and tolerating growth until a risk boundary is reached, whereby symptomatic deterioration or higher risk from anticipated future treatment is threatened. Management by high-volume multidisciplinary teams improves quality of life and life expectancy. Surgery remains the mainstay treatment for symptomatic and rapidly enlarging meningioma. Radiotherapy has an important role but carries a higher risk compared to its use in sporadic disease. Whilst bevacizumab is effective in NF2-associated schwannoma and cystic ependymoma, it has no value in the management of meningioma. In this review, we describe the natural history of the disease, underlying genetic, molecular, and immune microenvironment changes, current management paradigms, and potential therapeutic targets.

Construction of a prognostic model of acute myeloid leukemia associated with immunogenic cell death

ABSTRACT Background Immunogenic cell death (ICD)is a kind of regulatory cell death, which causes a series of antigen-specific adaptive immune responses by generating and emitting some danger signals or damage-associated molecular patterns (DAMPs). At present, little is known about the prognostic value of ICD and its related processes in acute myeloid leukemia (AML). The aim of the study was to explore the relationship between ICD and tumor immune microenvironment changes in AML. Research Design & Methods In the study, AML samples were divided into two groups by consensus clustering analysis, and then gene enrichment analysis and GSEA analysis were performed on the ICD high expression group. Furthermore, CIBERSORT was used to analyze the tumor microenvironment and immune characteristics of AML. Finally, a prognostic model related to ICD was constructed by using univariate and multivariate regression analysis. Results ICD was divided into two groups according to the level of ICD gene expression. The ICD high expression group was associated with good clinical results and high levels of immune cell infiltration. Conclusions The study constructed and verified the prognostic characteristics of AML related to ICD, which has important value in predicting the overall survival time of AML patients.

Abstract 2415: NBTXR3 radio-enhancing nanoparticle achieves a more robust antitumor immune response when combined with proton radiotherapy than photon radiotherapy

Abstract Introduction: Immunoradiotherapy combining radiotherapy and immune checkpoint blockade, has achieved impressive treatment outcomes. We have previously demonstrated that photon (XRT) and proton (PRT) localized radiotherapy in combination with immune checkpoint blockade and NBTXR3, a radioenhancing nanoparticle, could induce a systemic antitumor immune response. However, no studies have compared the treatment efficacy of PRT and XRT immunoradiotherapy. Here, we compared the capabilities of PRT and XRT when combined with NBTXR3 and PD1 blockade for inducing an antitumor immune response. Methods: 344SQR cells were inoculated in the right and left hindlimbs of 129Sv/Ev mice on day 0 and day 4 to establish primary and secondary tumors, respectively. Mice were divided into five treatment groups: 1) untreated control; 2) XRT+αPD1; 3) NBTXR3+XRT+αPD1; 4) PRT+αPD1; 5) NBTXR3+PRT+αPD1. The primary tumors were intratumorally injected with NBTXR3 nanoparticles on day 7, followed by two fractions of 12 Gy XRT or PRT on days 8 and 9 (total dose of 24 Gy). Two hundred μg of αPD1 was given to the mice on days 7, 10, 14, 21, 28, 35, and 42 through intraperitoneal injection. We assayed whole tumor RNAs with a Nanostring pan-cancer immune panel to profile the tumor immune microenvironment changes. In addition, immune cells extracted from the tumors were analyzed with single-cell RNA sequencing (scRNAseq). The survivor mice treated with the NBTXR3+PRT+αPD1 were rechallenged with 344SQR and 344SQP cells. Results: PRT+ αPD1 resulted in significantly better control of the primary tumors than XRT+ αPD1. No significant difference was observed in the volume of the secondary tumors treated with XRT+ αPD1 and the control. In contrast, PRT+ αPD1 resulted in a pronounced abscopal effect. Adding NBTXR3 to XRT+ αPD1 and PRT+ αPD1 improved the control of both the primary and secondary tumors. In addition, NBTXR3+PRT+αPD1 achieved significantly slower growth of the two tumors and more prolonged survival than NBTXR3+XRT+αPD1. Remarkably, NBTXR3+PRT+αPD1 eradicated both primary and secondary tumors in 40% of the treated mice, while no mice were cured by NBTXR3+XRT+αPD1. Nanostring analysis and scRNAseq revealed better activation and infiltration of antitumoral immune cells to the two tumors when treated with PRT than XRT. The survivor mice treated with NBTXR3+PRT+αPD1 effectively rejected tumor growth after rechallenge with 344SQR and 344SQP cells. Conclusions: When combined with NBTXR3, PRT with αPD1 exhibited significantly better treatment efficacy than XRT with αPD1. The superior efficacy of NBTXR3+PRT+αPD1 is accompanied by a more robust immune activation and immune cell infiltration in irradiated and unirradiated tumors. Citation Format: Yun Hu, Sébastien Paris, Narayan Sahoo, Qi Wang, Qianxia Wang, Hampartsoum B. Barsoumian, Jordan Da Silva, Célia Bienassis, Ailing Huang, Nahum Puebla-Osorio, Saumil Gandhi, Quynh-Nhu Nguyen, Jing Wang, Maria A. Cortez, James W. Welsh. NBTXR3 radio-enhancing nanoparticle achieves a more robust antitumor immune response when combined with proton radiotherapy than photon radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2415.

Tumor immune microenvironment changes are associated with response to neoadjuvant chemotherapy and long-term survival benefits in advanced epithelial ovarian cancer: A pilot study.

Little is known about the association between efficacy of neoadjuvant chemotherapy (NACT)/survival and the dynamic change of tumor immune environment (TIME) during treatment in epithelial ovarian cancer (EOC). This study investigated the TIME landscape of treatment-naive EOC tumors using multiplex immunofluorescence and associated the TIME before and after platinum-based NACT with treatment efficacy and prognosis in 33 patients with advanced EOC. NACT significantly increased the density of CD8+ T cells (P = 0.033), CD20+ B cells (P = 0.023), CD56 NK cells (P = 0.041), PD-1+ cells (P = 0.042), and PD-L1+CD68+ macrophages (P = 0.005) in the tissue specimens. Response to NACT was evaluated using CA125 response and chemotherapy response score (CRS). Compared with the non-responders, the responders displayed a larger proportion of tumors showing increase in the infiltration of CD20+ cells (P = 0.046) and in the M1/M2 ratio (P = 0.038) as well as fewer tumors showing increase in the infiltration of CD56bright cells (P = 0.041). No association was found between pre-NACT TIME and response to NACT. Density of pre-NACT CD8+ cells was positively associated with longer progression-free survival (PFS) (P = 0.011) and overall survival (OS) (P = 0.048). Post-NACT CD20+ and CD163+ macrophages (M2) infiltrates were associated with prolonged (P = 0.005) and shortened PFS (P = 0.021), respectively. Increase in the density of CD4+ T cells was predictive for longer PFS (P = 0.022) and OS (P = 0.023). In the multivariate analysis, high density of CD8+ cells pre-NACT (P = 0.042) were independently associated with improved OS.

Randomized phase II trial in prostate cancer with hormone-sensitive OligometaSTatic relapse: Combining stereotactic ablative radiotherapy and durvalumab (POSTCARD GETUG P13): Study protocol

BackgroundAs in other solid tumors, increasing evidence indicates that patients diagnosed with a limited number of prostate cancer metastases, so-called oligometastases, have a better prognosis than patients with extensive metastatic disease. Stereotactic body radiotherapy (SBRT) is now considered an option in this population.Programmed death-1 (PD-1) and its ligands (PD-L1) are targeted by immune checkpoint inhibitors. Preclinical studies have shown that the tumor immune microenvironment changes when combining radiotherapy with immunotherapy, especially with hypofractionated radiotherapy.The oligometastatic setting appears to be the most relevant clinical situation for evaluating the immune response generated by radiotherapy and immune checkpoint inhibitors in patients with an intact immune system.We hypothesize that durvalumab will enhance the immune response following SBRT targeting oligometastatic lesions. Our purpose is to demonstrate, via a randomized 2:1 phase II trial, that SBRT (3 fractions) with durvalumab in oligometastatic hormone-sensitive prostate cancer patients would improve progression-free survival in patients with prostate cancer with up to 5 metastases compared to patients who exclusively received SBRT. MethodsThis is a multicentric randomized phase II study in French academic hospitals. Patients with prostate cancer and up to 5 metastases (lymph node and/or bone) were randomized into a 2:1 ratio between Arm A (experimental group), corresponding to durvalumab and SBRT to the metastases, and Arm B (control group), corresponding to SBRT alone to the metastases. The study aims to accrue a total of 96 patients within 3 years. The primary endpoint is two-year progression-free survival and secondary endpoints include androgen deprivation therapy-free survival, quality of life, toxicity, prostate cancer specific survival, overall survival, and immune response. DiscussionThe expected benefit for the patients in the experimental arm is longer life expectancy with acceptable toxicity. We also expect our study to provide data for better understanding the synergy between immunotherapy and radiotherapy in oligometastatic prostate cancer.

Understanding the mechanisms underlying obesity in remodeling the breast tumor immune microenvironment: from the perspective of inflammation.

Obesity is a well-known modifiable risk factor for breast cancer and is considered a poor prognostic factor in pre- and post-menopausal women. While the systemic effects of obesity have been extensively studied, less is known about the mechanisms underlying obesity-associated cancer risk and the local consequences of obesity. Thus, obesity-induced inflammation has become the focus of research interest. Biologically, the development of cancer involves a complex interaction with numerous components. As the tumor immune microenvironment changes due to obesity-triggered inflammation, an increase in infiltration occurs for proinflammatory cytokines and adipokines, as well as adipocytes, immune cells, and tumor cells in the expanded adipose tissue. Complicated cellular-molecular crosstalk networks change critical pathways, mediate metabolic and immune function reprogramming, and have a significant role in tumor metastasis, proliferation, resistance, angiogenesis, and tumorigenesis. This review summarizes recent research findings on how inflammatory mediators in the in situ tumor microenvironment regulate the occurrence and development of breast cancer in the context of obesity. We analyzed the heterogeneity and potential mechanisms of the breast cancer immune microenvironment from the perspective of inflammation to provide a reference for the clinical transformation of precision targeted cancer therapy.

Abstract OT2-08-03: Immunogenicity of pembrolizumab and doxorubicin in a phase I trial for patients with metastatic triple negative breast cancer

Abstract Background: Combination of immune checkpoint inhibitor pembrolizumab and chemotherapy are standard of care therapy for patients with programmed death-ligand 1 positive (PD-L1+) metastatic triple negative breast cancer (mTNBC). However, a greater understanding of how immune checkpoint inhibitors and chemotherapies synergize to yield anti-tumor T cell responses is needed. The current phase I study evaluated the immunogenicity of doxorubicin plus pembrolizumab in patients with mTNBC. Patients and Methods: Patients with mTNBC, no prior anthracycline use, and 0-2 lines of prior systemic chemotherapies received pembrolizumab 200 mg IV and doxorubicin 50-60 mg/m2 IV every 3 weeks for 6 cycles followed by pembrolizumab maintenance until disease progression or intolerance. Patients were not selected based on PD-L1 expression. The primary objectives were safety and objective response rate per RECIST 1.1. Peripheral blood samples were collected at baseline, Cycle 2 Day 1 (C2D1), and post Cycle 3 for analysis by high parameter flow cytometry. Results: Ten patients were enrolled between March 2016 and November 2019. Best responses included one patient with complete response (CR), five with partial responses (PR), two with stable disease (SD), and one with progression of disease (PD). Flow cytometry showed increased CD3+ T cells (p=0.03) from pre-treatment to C2D1 with substantial inter-patient variation in CD3+ T cells. The expansion of proliferative exhausted-like PD-1+ CD8+ T cell population was identified in 8/9 patients, and exhausted CD8+ T cells were significantly expanded from pre-treatment to C2D1 in the patient with CR (p=0.01). Notably, frequencies of PD-1 high proliferative CD8+ T cells contracted significantly from C2D1 to post Cycle 3 (p=0.005), with a return to near baseline frequencies in the majority of the patients. In the context of all identified T cell subsets, PD-1hi proliferative CD8+ T cells demonstrated the greatest increase in fold change from pre-treatment to C2D1. In contrast, PD-1lo proliferative CD8+ T cells demonstrated the greatest decrease in fold change from pre-treatment to C3D1. Conclusion: Anthracycline-naïve patients with mTNBC treated with the combination of pembrolizumab and doxorubicin yielded robust peripheral blood T cell responses. Further studies dissecting the dynamics and durability of anti-tumor T cell responses and concurrent tumor immune microenvironment changes are needed to optimize combined immune checkpoint inhibitor and chemotherapy treatment for patients with mTNBC. Citation Format: Colt A. Egelston, Weihua Guo, Susan E. Yost, Xuan Ge, Jin Sun Lee, Paul H. Frankel, Yujie Cui, Christopher Ruel, Daniel Schmolze, Mireya Murga, Aileen Tang, Norma Martinez, Misagh Karimi, George Somlo, Peter Lee, James Waisman, Yuan Yuan. Immunogenicity of pembrolizumab and doxorubicin in a phase I trial for patients with metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-08-03.