Immune Cell Proliferation Research Articles

Abstract PO2-27-07: The Role of HCMV Infection in Facilitating Brain Metastasis through the Creation of a Suppressive Tumor Microenvironment in Breast Cancer Patients

Abstract Human Cytomegalovirus (HCMV) infection has emerged as a noteworthy factor in breast cancer patients, particularly those experiencing brain metastasis. Despite being prevalent and often asymptomatic, HCMV's contribution to tumor progression remains enigmatic. Notably, a high proportion of breast cancer patients with brain metastasis exhibit HCMV positivity in both primary tumors and metastatic lesions. This study aims to elucidate the influence of HCMV infection on brain metastasis by investigating its impact on the tumor microenvironment. Our findings reveal a distinctive immune suppressive tumor microenvironment in HCMV-positive breast cancer patients. This environment is characterized by impaired NK cell function and an elevated accumulation of TH2 cells. To comprehend the role of HCMV in promoting brain metastasis, we have developed a murine breast tumor model using MCMV, enabling us to mirror the clinical trajectory of tumor progression and metastasis. The integration of a GFP reporter gene into the MCMV genome facilitates the tracking of infected cancer cells. Within this model, we observe a significant increase in brain metastasis coupled with reduced survival rates. Single-cell sequencing analysis uncovers diminished NK cell presence and activation, along with heightened TH2 cell accumulation within the tumor microenvironment—results that parallel the clinical observations in breast cancer patients. GSEA analysis underscores the secretion of inhibitory cytokines by MCMV-infected cancer cells, contrasting with their non-infected counterparts. Collectively, our study reveals that CMV infection contributes to brain metastasis in breast cancer by generating inhibitory cytokines. This hampers the recruitment and proliferation of immune cells within the tumor microenvironment and concurrently impairs the cytotoxic capabilities of NK cells. These insights shed light on the intricate interplay between viral infection and tumor progression, offering potential avenues for therapeutic exploration. Citation Format: Wenjuan Dong, Xin Wang, Kun Han, Akshjot Puri, Amna Irfan, Wei Qian, Liliana Guzman, Roberto Rosato, Jianting Sheng, Hong Zhao, Jenny Chang, Stephen Wong. The Role of HCMV Infection in Facilitating Brain Metastasis through the Creation of a Suppressive Tumor Microenvironment in Breast Cancer Patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-27-07.

Abstract PO3-25-07: Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics

Abstract Background Tertiary lymphoid structures (TLS) are ectopic lymphoid organs playing a role in adaptive antitumor immune response. They were shown to be associated with favorable outcome and appears to be a promising biomarker for response to immune checkpoint inhibitors (ICI). Yet, there is no consensus for their detection and quantification. Here we aimed to derive a specific TLS signature using high resolution spatial transcriptomics data from a large series of triple negative breast cancer (TNBC) samples and to assess its clinical relevance including response to ICI. Methods Spatial transcriptomics (ST) was performed on a series of 94 early-stage TNBC surgical samples with detailed clinicopathological and outcome data. TLS and tumor infiltrating lymphocytes (TILs) were scored using hematoxylin and eosin and double immunohistochemistry (CD3/CD20) stained slides by a dedicated breast pathologist. Regression based on morphological annotations and deconvolution methods estimated the fraction of gene expression related to TLS in each ST spot allowing to derive specific TLS signature. Its prognostic and predictive value of response to ICI was assessed using publicly available breast cancer (METABRIC, SCAN-B, and I-SPY2) and other tumor type datasets treated with ICI (Bareche et al. Ann Oncol, 2022) from bulk gene expression data. Results Deconvolution of the TLS compartments using the ST gene expression data showed that TLS are enriched in all B cell subsets, CD4+ (central) memory and naïve T cells, as well as mast cells. A functional analysis showed an enrichment of ‘vascular endothelial cell proliferation’, ‘mitotic G2/M transition checkpoint’, ‘V/D/J recombination’ and ‘regulation of cell chemotaxis to fibroblast growth factor’ suggesting that TLS aggregates are associated with angiogenesis, immune cell proliferation, adaptative humoral response and tissue remodeling. A comparison of gene expression data between TLS and non-aggregated TILs led to the development of a specific TLS gene signature comprising B and T cell-specific genes, immunoglobulin genes as well as genes associated with TLS initiation. Its projection on tumor slides overlapped with the regions annotated as TLS by the pathologist, demonstrating its specificity for TLS detection. As expected, the highest levels of the TLS signature were observed in the immunomodulatory TNBC molecular subtype and fully inflamed tumors, whereas the lowest levels were observed in the mesenchymal molecular subtype and immune desert tumors (p< 0.05). Of note, high levels of the TLS signature were associated with better prognosis in TNBC patients from METABRIC (p=6.5 10−5) and SCAN-B datasets (p=1.6 10−3) as well as with higher pathological complete response rates in all breast cancer patients treated with neoadjuvant pembrolizumab in the I-SPY2 study (p=0.026). Similar results were found in other tumor types treated with immunotherapy in the metastatic setting including metastatic melanoma, pancreatic and bladder cancers (PFS, p=6 10−6). Of interest, the TLS signature outperformed the predictive value of other immune-related signatures including TILs suggesting a key role of TLS in obtaining a sustainable, adaptive antitumor immune response in the vicinity of the tumor area. Conclusion By leveraging the potential of spatial transcriptomics, we have successfully developed a unique TLS gene signature that demonstrates a strong correlation with the response to ICI in breast cancer and various other tumor types. This TLS gene signature outperforms other immune biomarkers, underscoring the pivotal role of TLS in eliciting a robust antitumor immune response. The universality of this TLS signature makes it a potent biomarker capable of identifying patients who would benefit from immunotherapy, pending further validation. Citation Format: Xiaoxiao Wang, David Venet, Frédéric Lifrange, Denis Larsimont, Mattia Rediti, Linnea Stenbeck, Floriane Dupont, Ghizlane Rouas, Andrea Joaquin Garcia, David Gacquer, Ligia Craciun, Laurence Buisseret, Nayanika Bhalla, Yuvarani Masarapu, Kim Thrane, Eva Gracia Villacampa, Lovisa Franzén, Sami Saarenpää, Linda Kvastad, Joakim Lundeberg, Françoise Rothé, Christos Sotiriou. Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-07.

Spatio-temporal Phenotyping of Murine Embryos with Ultrahigh-plex Antibody Panels for Characterization of Early Development

Abstract The murine embryo model is one of the most common animal models for studying developmental biology. Embryonic development is a highly regulated process with spatial and temporal expression of proteins for regional specialization and tissue-specific functions. Application of spatial proteomics to developing embryos enables us to understand cellular organization and interactions within tissues along with regulatory mechanisms at the single-cell level. In this study, we applied a 36-plex panel, specifically designed for analysis of mouse embryos at different stages of development using the PhenoCycler™-Fusion (PCF) 2.0 Platform. This panel was developed by selecting biomarkers for immune cell lineages, immune activation, proliferation, structural and neuronal components. Purified antibodies were tagged to PhenoCycler barcodes and verified for specificity and reproducibility. After successful validation, the panel was deployed on the FFPE mouse embryos and ultrahigh-plex whole-slide imaging was performed using the PCF 2.0 platform. Unsupervised clustering analysis and deep phenotyping were performed to generate a high-resolution spatial atlas of mouse embryos at different stages of development. The tools developed as part of this study can be used to conduct in-depth research on various aspects of organogenesis and to study the spatio-temporal dynamics of cellular and molecular expression in developing embryos.

Identification of key mitochondria-related genes and their relevance to the immune system linking Parkinson's disease and primary Sjögren's syndrome through integrated bioinformatics analyses

BackgroundMitochondria are the metabolic hubs of cells, regulating energy production and antigen presentation, which are essential for activation, proliferation, and function of immune cells. Recent evidence indicates that mitochondrial antigen presentation may have an impact on diseases such as Parkinson's disease (PD) and autoimmune diseases. However, there is limited knowledge about the mechanisms that regulate the presentation of mitochondrial antigens in these diseases. MethodsIn the current study, RNA sequencing was performed on labial minor salivary gland (LSG) from 25 patients with primary Sjögren's syndrome (pSS) and 14 non-pSS aged controls. Additionally, we obtained gene expression omnibus datasets associated with PD patients from NCBI Gene Expression Omnibus (GEO) databases. Single-sample Gene Set Enrichment Analysis (ssGSEA), ESTIMATE and Spearman correlations were conducted to explore the association between mitochondrial related genes and the immune system. Furthermore, we applied weighted Gene Co-expression Network Analysis (WGCNA) to identify hub mitochondria-related genes and investigate the correlated networks in both diseases. Single cell transcriptome analysis, immunohistochemical (IHC) staining and quantitative real-time PCR (qRT–PCR) were used to verify the activation of the hub mitochondria-related pathway. Pearson correlations and the CIBERSORT algorithms were employed to further reveal the correlation between hub mitochondria-related pathways and immune infiltration. ResultsThe transcriptome analysis revealed the presence of overlapping mitochondria-related genes and mitochondrial DNA damage in patients with pSS and PD. Reactive oxygen species (ROS), the senescence marker p53, and the inflammatory markers CD45 and Bcl-2 were found to be regionally distributed in LSGs of pSS patients. WGCNA analysis identified the STING pathway as the central mitochondria-related pathway closely associated with the immune system. Single cell analysis, IHC staining, and qRT-PCR confirmed the activation of the STING pathway. Subsequent, bioinformatic analysis revealed the proportion of infiltrating immune cells in the STING-high and STING-low groups of pSS and PD. Furthermore, the study demonstrated the association of the STING pathway with innate and adaptive immune cells, as well as functional cells, in the immune microenvironment of PD and pSS. ConclusionOur study uncovered a central pathway that connects mitochondrial dysfunction and the immune microenvironment in PD and pSS, potentially offering valuable insights into therapeutic targets for these conditions.

On Connective Tissue Mast Cells as Protectors of Life, Reproduction, and Progeny.

The connective tissue mast cell (MC), a sentinel tissue-residing secretory immune cell, has been preserved in all vertebrate classes since approximately 500 million years. No physiological role of the MC has yet been established. Considering the power of natural selection of cells during evolution, it is likely that the MCs exert essential yet unidentified life-promoting actions. All vertebrates feature a circulatory system, and the MCs interact readily with the vasculature. It is notable that embryonic MC progenitors are generated from endothelial cells. The MC hosts many surface receptors, enabling its activation via a vast variety of potentially harmful exogenous and endogenous molecules and via reproductive hormones in the female sex organs. Activated MCs release a unique composition of preformed and newly synthesized bioactive molecules, like heparin, histamine, serotonin, proteolytic enzymes, cytokines, chemokines, and growth factors. MCs play important roles in immune responses, tissue remodeling, cell proliferation, angiogenesis, inflammation, wound healing, tissue homeostasis, health, and reproduction. As recently suggested, MCs enable perpetuation of the vertebrates because of key effects-spanning generations-in ovulation and pregnancy, as in life-preserving activities in inflammation and wound healing from birth till reproductive age, thus creating a permanent life-sustaining loop. Here, we present recent advances that further indicate that the MC is a specific life-supporting and progeny-safeguarding cell.

Antioxidant Properties of Brassica Rapa L. Seeds and Immunostimulant Effect on Immunity Broilers Cells (Gallus Gallus Domesticus)

Background: In the past few decades, researchers have focused on finding the benefits of natural substances derived from plants. Objective: This study aimed to evaluate the use of Brassica rapa seeds in poultry feed as an antioxidant and immunostimulant of host defenses. Methods: We prepared three extracts using ethanol, Ethyl Acetate, and water. Spectrophotometric methods determined the total phenolic and flavonoid content in the three extracts. Antioxidant activity was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and β-Carotene bleaching methods. An assessment of their immunostimulant activities in vitro was performed on chicken immunity cell proliferation (splenocytes, thymocytes, and Bursa cells) and IgY production. method: We prepared three different extracts using ethanol, Ethyl Acetate, and water. The total phenolic and flavonoid content in the three extracts was determined by spectrophotometric methods.Antioxidant activity has been assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and β-Carotene bleaching methods. Assessment of their immunostimulant activities in vitro was tested on chicken immunity cell proliferation (splenocytes, thymocytes, and Bursa cells) and IgY production Results: The total phenolic contents ranged from 462.5 to 794.8 mg /g of extract, the order of TPC was as follows: EtOAc > EtOH >Water. Flavonoid contents were, 63.7 mg in EtOH extract, 81.2 mg in EtOAc extract, and 108.7 mg/g Extract in aqueous extract. By DPPH, the IC50s of EtOH, EtOAc, and water, were 1.8μg/ml, 2.4μg/ml, and 1.5μg/ml, respectively. Using the Ferric- Reducing Antioxidant Power (FRAP), we remarked that the EtOH and EtOAc extracts have important antioxidant powers. The ABTS assay indicated that EtOH and water had the highest activities with IC50s of 0.19 and 0.07, respectively. Finally, by the β-carotene bleaching test, we observed that the IC50 of the EtOH, EtOAc, and water were 62.1 μg/mL; 72.7 μg/mL, and 45.8 μg/mL, respectively. The results obtained indicate that Brassica aqueous extract stimulates humoral immunity by stimulating splenocyte (B and T-lymphocytes) and Bursa cell (B-lymphocyte) proliferation by more than 200% of response vs control. In addition, the aqueous extract highly stimulated the function of bursa cells by 208% of the reaction. In the same conditions, we recorded a stimulation of cellular immunity mediated by thymocytes by an increase in cell proliferation (352.7% of response) implicated in virus protection. These extracts also possessed an antimicrobial effect against diverse microorganisms such as coliforms and Staphylococcus. The FT-IR spectrum indicated that the hydroxyl group (phenol), hydroxybenzoic acid family, carbohydrate molecules such as glucopyranose, a carbonyl ester group, hydrocarbon chains (alkyls groups)) were present. result: The total phenolic contents ranged from 462.5 to 794.8 mg /g of Extract, the order of TPC was as follows: EtOAc > EtOH >Water. Flavonoid contents were, 63.7 mg in EtOH extract, 81.2 mg in EtOAc extract, and 108.7 mg/g Extract in aqueous extract.By DPPH, the IC50s of EtOH, EtOAc, and water, were respectively 1.8µg/ml, 2.4µg/ml, and 1.5µg/ml. Using the ferric-reducing antioxidant power (FRAP) we remarked that the EtOH and EtOAc extracts have important antioxidant powers. The ABTS assay indicated that EtOH and water had the highest activities with IC50s of 0.19 and 0.07 respectively. Finally, by the β-carotene bleaching test, we observed that the IC50 of the EtOH, EtOAc, and water were 62.1 µg/mL; 72.7 µg/mL, and 45.8 µg/mL respectively. The results obtained indicate that Brassica aqueous extract stimulates humoral immunity by stimulating splenocyte (B and T-lymphocytes) and Bursa cell (B-lymphocyte) proliferation by more than 200% of response vs control. In addition, the aqueous extract stimulated highly the function of bursa cells by 208% of the response. In the same conditions, we recorded a stimulation of cellular immunity mediated by thymocytes by an increase in cell proliferation (352.7% of response) implicated in virus protection. These extracts possessed also an antimicrobial effect against diverse microorganisms such as coliforms and Staphylococcus. The FT-IR spectrum indicated that the hydroxyl group (phenol), hydroxybenzoic acid family, carbohydrate molecules such as glucopyranose, a carbonyl ester group, hydrocarbon chains (alkyls groups)) were present. Conclusion: This result could have interesting applications in the poultry feed industry to enhance the performance of animal development.

Abstract LB311: Multimodal spatial profiling reveals the emergence of an immune suppressive microenvironment at the initial stages of high-grade serous ovarian cancer development

Abstract Introduction: Despite the clear genetic evidence linking serous tubal intraepithelial carcinoma (STIC) and high-grade serous ovarian carcinoma (HGSOC), the specific conditions and events that promote the progression of STIC lesions into invasive disease remain poorly understood. Method: As a critical initial step, we have assembled a cohort of incidental p53 signatures, STIC lesions, and STIC with concurrent HGSOC. We have performed extensive multi-modal analysis using multiplexed tissue imaging and spatial transcriptomics that identify features of the immune system that play a vital role in the early steps of HGSOC development. We have processed 43 specimens using highly multiplexed tissue imaging at single-cell resolution (cyclic immunofluorescence, CyCIF), and 35 specimens for micro-regional spatial transcriptomics using the GeoMx (Whole Transcriptome, Nanostring) on over 450 pathologist-annotated regions of interest. Results: Our data suggests an immune-cold environment and T-cell dysfunction in STIC lesions, including incidental STIC. One of the significant immune populations identified was CD103+ tissue-resident memory T cells (TRM). In incidental p53 signatures, activation of TRM was rare, similar to FT, which may indicate the absence of sensing “tumor antigen” by these TRM. CyCIF analysis also revealed that most incidental STIC lesions (7/9 cases) overexpress major histocompatibility complex (MHC) class I compared to the normal epithelium, especially both HLA complex, HLA-A and HLA-E. Most STICs showed extensive intra-lesion heterogeneity, with some STICs remaining HLA-A and HLA-E negative. Incidental p53 signatures, on the other hand, were mostly HLA-A and HLA-E negative and, when positive, had only a few cells expressing HLA. We hypothesized that there might be a natural selection of HLA-E-positive clones as STICs progress to HGSOC. Consistent with this, HGSOC showed further overexpression of HLA-E. However, HLA-E heterogeneity was still observed in the invasive tumor, with both positive and negative clones co-existing. Geomx data suggested that the interferon signaling pathway is upregulated in the epithelial of HLA-E positive STIC and cancer clones compared to HLA-E negative clones. In turn, both HLA-A and HLA-E might be overexpressed in the epithelial of HLA-E-positive STICs. Overall, we showed that the response in interferon (IFN) α and γ, NF-KB, and IL-6-induced STAT-3 pathways were upregulated in both STIC lesions and carcinoma. The role of these pathways, especially STAT-3 pathway, has been shown in other aneuploid cancers in promoting immune escape, cell proliferation and migration, chemoresistance and inhibiting apoptosis. We have shown the co-localization of cGAS and BAF, a marker for micronuclei rupture, by super-resolution 3D imaging to confirm one of the mechanisms of IFN activation. Conclusion: Taken together, these data may indicate chromosomal instability is one of the mechanisms that is driving the IFN-signaling pathway and, hence, a potential selective advantage for HLA-E-positive clones in tumorigenesis, leading to inhibiting NK cell surveillance followed by reducing T cell infiltration. Citation Format: Tanjina Kader, Jia-Ren Lin, Shannon Coy, Clemens Hug, Yu-An Chen, Roxanne J. Pelletier, Mariana Leon, John Lee, Yi-Lin Xu, Clarence Yapp, Natalie Shih, Gabriel Mingo, Euihye Jung, Srishti Rathore, Judith Agudo, Charles Drescher, Peter K. Sorger, Ronny Drapkin, Sandro Santagata. Multimodal spatial profiling reveals the emergence of an immune suppressive microenvironment at the initial stages of high-grade serous ovarian cancer development [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 LB311.

Therapeutic potential of interleukin-21 in cancer.

Interleukin-21 (IL-21) is an immunostimulatory cytokine which belongs to the common gamma-chain family of cytokines. It plays an import role in the development, differentiation, proliferation, and activation of immune cells, in particular T and natural killer (NK) cells. Since its discovery in 2000, IL-21 has been shown to regulate both adaptive and immune responses associates with key role in antiviral and antitumor responses. Recent advances indicate IL-21 as a promising target for cancer treatment and encouraging results were obtained in preclinical studies which investigated the potency of IL-21 alone or in combination with other therapies, including monoclonal antibodies, checkpoint inhibitory molecules, oncolytic virotherapy, and adoptive cell transfer. Furthermore, IL-21 showed antitumor effects in the treatment of patients with advanced cancer, with minimal side effects in several clinical trials. In the present review, we will outline the recent progress in IL-21 research, highlighting the potential of IL-21 based therapy as single agent or in combination with other drugs to enhance cancer treatment efficiency.

CD19-targeted chimeric antigen receptor T cell therapy in two patients with multiple sclerosis

Progressive multiple sclerosis (MS) is characterized by compartmentalized smoldering neuroinflammation caused by the proliferation of immune cells residing in the central nervous system (CNS),including B cells. Although inflammatory activity can be prevented by immunomodulatory therapies during early disease, such therapies typically fail to halt disease progression. CD19 chimeric antigen receptor (CAR)-T cell therapies have revolutionized the field of hematologic malignancies. Although generally considered efficacious, serious adverse events associated with CAR-T cell therapies such as immune effector cell-associated neurotoxicity syndrome (ICANS) have been observed. Successful use of CD19 CAR-T cells in rheumatic diseases like systemic lupus erythematosus and neuroimmunological diseases like myasthenia gravis have recently been observed, suggesting possible application in other autoimmune diseases. Here, we report the first individual treatment with a fully human CD19 CAR-T cell therapy (KYV-101) in two patients with progressive MS. CD19 CAR-T cell administration resulted in acceptable safety profiles for both patients. No ICANS was observed despite detection ofCD19 CAR-T cells in the cerebrospinal fluid. In case 1, intrathecal antibody production in the cerebrospinal fluid decreased notably after CAR-T cell infusion and was sustained through day 64. CD19 CAR-T cell administration in progressive MS resulted in an acceptable safety profile. CAR-T cell presence and expansion were observed in the cerebrospinal fluid without clinical signs of neurotoxicity, which, along with intrathecal antibody reduction, indicates expansion-dependent effects of CAR-T cells on CD19+ target cells in the CNS. Larger clinical studies assessing CD19 CAR-T cells in MS are warranted. Both individual treatments as well the generated data were not based on external funding.

The involvement of interferon regulatory factor 8 in regulating the proliferation of haemocytes in oyster Crassostrea gigas

Interferon regulatory factor 8 (IRF8) is an important transcriptional regulatory factor involving in multiple biological process, such as the antiviral immune response, immune cell proliferation and differentiation. In the present study, the involvement of a previously identified IRF8 homologue (CgIRF8) in regulating haemocyte proliferation of oyster were further investigated. CgIRF8 mRNA transcripts were detectable in all the stages of C. gigas larvae with the highest level in D-veliger (1.76-fold of that in zygote, p < 0.05). Its mRNA transcripts were also detected in all the three haemocyte subpopulations of adult oysters with the highest expression in granulocytes (2.79-fold of that in agranulocytes, p < 0.01). After LPS stimulation, the mRNA transcripts of CgIRF8 in haemocytes significantly increased at 12 h and 48 h, which were 2.04-fold and 1.65-fold (p < 0.05) of that in control group, respectively. Meanwhile, the abundance of CgIRF8 protein in the haemocytes increased significantly at 12 h after LPS stimulation (1.71-fold of that in seawater, p < 0.05). The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgIRF8 protein in haemocytes. After the expression of CgIRF8 was inhibited by the injection of CgIRF8 siRNA, the percentage of EdU positive haemocytes, the proportion of granulocytes, and the mRNA expression levels of CgGATA and CgSCL all declined significantly at 12 h after LPS stimulation, which was 0.64-fold (p < 0.05), 0.7-fold (p < 0.05), 0.31-fold and 0.54-fold (p < 0.001) of that in the NC group, respectively. While the expression level of cell proliferation-related protein CgCDK2, CgCDC6, CgCDC45 and CgPCNA were significantly increased (1.99-fold, and 2.41-fold, 3.76-fold and 4.79-fold compared to that in the NC group respectively, p < 0.001). Dual luciferase reporter assay demonstrated that CgIRF8 was able to activate the CgGATA promoter in HEK293T cells after transfection of CgGATA and CgIRF8. These results collectively indicated that CgIRF8 promoted haemocyte proliferation by regulating the expression of CgGATA and other related genes in the immune response of oyster.

Identification of miR-29a as a novel biomarker for lumpy skin disease virus exposure in cattle

ABSTRACT Micro RNAs (miRNAs) have been implicated in the regulation of maturation, proliferation, differentiation, and activation of immune cells. In this study, we demonstrated that miR-29a antagonizes IFN-γ production at early times post-LSDV infection in cattle. miR-29a was predicted to target upstream IFN-γ regulators, and its inhibition resulted in enhanced IFN-γ production in sensitized peripheral blood mononuclear cells (PBMCs). Further, stimulation of PBMCs with LSDV antigen exhibited lower levels of miR-29a, concomitant with a potent cell-mediated immune response (CMI), characterized by an increase in LSDV-specific CD8+ T cell counts and enhanced levels of IFN-γ, which eventually facilitated virus clearance. In addition, a few immunocompromised cattle (developed secondary LSDV infection at ~ 6 months) that failed to mount a potent cell-mediated immune response, were shown to maintain higher miR-29a levels. Furthermore, as compared to the sensitized crossbred cattle, PBMCs from sensitized Rathi (a native Indian breed) animals exhibited lower levels of miR-29a along with an increase in CD8+ T cell counts and enhanced levels of IFN-γ. Finally, we analysed that a ≥ 60% decrease in miR-29a expression levels in the PBMCs of sensitized cattle correlated with a potent CMI response. In conclusion, miR-29a expression is involved in antagonizing the IFN-γ response in LSDV-infected cattle and may serve as a novel biomarker for the acute phase of LSDV infection, as well as predicting the functionality of T cells in sensitized cattle. In addition, Rathi cattle mount a more potent CMI response against LSDV than crossbred cattle.

Recent Advances on Small-Molecule Inhibitors of Lipocalin-like Proteins.

Lipid transfer proteins (LTPs) are crucial players in nonvesicular lipid trafficking. LTPs sharing a lipocalin lipid transfer domain (lipocalin-like proteins) have a wide range of biological functions, such as regulating immune responses and cell proliferation, differentiation, and death as well as participating in the pathogenesis of inflammatory, metabolic, and neurological disorders and cancer. Therefore, the development of small-molecule inhibitors targeting these LTPs is important and has potential clinical applications. Herein, we summarize the structure and function of lipocalin-like proteins, mainly including retinol-binding proteins, lipocalins, and fatty acid-binding proteins and discuss the recent advances on small-molecule inhibitors for these protein families and their applications in disease treatment. The findings of our Perspective can provide guidance for the development of inhibitors of these LTPs and highlight the challenges that might be faced during the procedures.

Abstract 6396: Ex vivo 3D micro tumor testing to assess chemotherapy and immunotherapy responses for NSCLC patients

Abstract Background: Systemic treatment options for NSCLC patients consists of chemo-, targeted or immunotherapy. Although predictive biomarkers can guide treatment decisions, response prediction for chemo- and immunotherapy is still a challenge. Recent studies showed the potential of functional testing to assess patient sensitivity to drugs in various cancers, but NSCLC is lacking. This study reports the feasibility of ex vivo 3D micro lung tumor testing with native tumor microenvironment (TME) to enable treatment sensitivity profiling for all drug classes in parallel. Methods: Fresh NSCLC tumor tissue was collected from pleural fluid stage IVa/b (N=24) or resected primary tumor for stage I-III disease (N=11). Tumor clusters were isolated and exposed to drugs, while preserving the TME. Thirteen therapies were included, including cisplatin, pembrolizumab, osimertinib, and positive control for immune sensitivity (SEA). Morphological features were extracted from high-throughput 3D imaging data. Chemo- and targeted therapy sensitivity was based on AUC comparison of fitted dose-response curves. Immunotherapy sensitivity was classified on percentage and statistical significance of tumor killing, immune cell proliferation and cytokine profiling. Results: The overall assay success rate for solid samples was 64% (7/11), with 100% (4/4) success after protocol optimization. For pleural fluids, the overall success rate was 33% (8/24), caused by many samples arriving without tumor content (13/24, 54%). Excluding these samples, a technical success rate of 73% (8/11) was obtained. The assay was robust and reproducible (mean Z’ = 0.38, CV = 16.1%). Results were delivered in two weeks and on average, eight treatments were tested in parallel per sample. Patient-specific response profiles were observed for chemo-, targeted and immunotherapy. General immunotherapy sensitivity (SEA) was measured in 50% (6/12) of the tissues. Distinct responses were observed for the different immunotherapies: 33% (4/12), 17% (2/12) and 8% (1/12) of the patients responded to pembrolizumab, atezolizumab and/or ipilimumab, respectively. Strong response to osimertinib was observed in a patient with EGFR exon 19 mutation (AUC = 0.26) versus resistance in a WT patient (AUC=0.92). Conclusion: Ex vivo 3D micro tumor testing using liquid and solid tissues is feasible for NSCLC patients with resected primary tumor or pleuritis carcinomatosa. Distinct patient sensitivity profiles for chemo-, targeted and immunotherapies were measured. A preliminary correlation of targeted therapy response with mutation status was found. Discussion: The ex vivo 3D micro tumor testing platform is proven predictive for platinum-based therapy. The current study established the methods to accurately measure ex vivo NSCLC responses to all drug classes to correlate with clinical measures and further validate the platform for guiding treatment decisions. Citation Format: Esmee Koedoot, Timothy J. Sijsenaar, Lieke J. Ceton, Dieudonné J. van der Meer, Thomas J. van Brakel, Peter van Zwam, Erica Geraedts, Willemijn Vader, Egbert F. Smit, Laurie C. Steinbusch, Christi M. Steendam. Ex vivo 3D micro tumor testing to assess chemotherapy and immunotherapy responses for NSCLC patients [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 6396.

Abstract 77: Real-time multiparametric assessment of immune cell-mediated cytotoxicity

Abstract A hallmark of effective immunotherapy is the reliance on the cytotoxic effects of immune cells, such as T-cells and natural killer (NK) cells, on tumor lesions. Cell cluster formation is an important sign of activation, and proliferation, as well as an indication of the killing capability of immune cells. Understanding and modulating the mechanisms underlying the activation, proliferation, and clustering of immune cells is essential for the development of innovative approaches to promote immune cell-induced cytotoxicity and address tumor progression. Here, we report a novel kinetic immuno-oncology assay using the Agilent xCELLigence RTCA eSight, which can simultaneously and quantitatively assess effector/immune cell clustering and proliferation through brightfield live cell time-lapse imaging and immune cell-mediated cytotoxicity of target/cancer cells through real-time monitoring cancer cell viability over a chronic treatment. In this study, we validated the assay using 2 cell models, including CD19 CAR T-cells against Raji cells and NK-92 cells against A549 cells, in a 96-well plate format. The activation, clustering, and proliferation of CD19 CAR T-cells and NK-92 at different Effector: Target (E: T) ratios, as well as the sequential killing of the target cancer cells, were plotted using imaging and cellular-impedance-based readouts throughout the entire assay, respectively. Our results showed 1) the clustering and proliferation of the effector cells could be accurately assessed by brightfield without the facilitation of fluorescent label using the image analysis module of the system; 2) the killing efficacy of the effector cells quantified by the impedance readout highly correlated to their clustering and proliferation assessed by the live-cell imaging; 3) cellular impedance provided a sensitive readout CAR-T killing of immobilized Raji cell count, independent of fluorescent labeling; 4) the kinetics of cluster formation/proliferation and killing efficacy of CD19 CAR T-cells were different from NK cells. The capability of multiparametric assessment of immune cell-medicated cytotoxicity from both effector and target cell perspectives of the assay provides a simple and real-time workflow to understand the correlation and interaction between effector and target cells, which could be an addition of immense value to the development of innovative immunotherapies. Citation Format: Grace Yang, Yan Lu, Tian Wang, Peifang Ye, Xiaoyu Zhang. Real-time multiparametric assessment of immune cell-mediated cytotoxicity [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 77.

Abstract 6970: identification of spatially enriched cancer cell- and immune cell-regulatory multi-marker-defined prognostic cancer associated fibroblast subsets of human colon cancer

Abstract Introduction: Subsets of cancer-associated fibroblasts (CAFs) in colorectal cancer remain poorly characterized preventing their use as biomarkers and drug targets. Results: Integrated scRNA seq profiling and multiplex staining identified four human CAF subsets These were A1 (PDGFRA+/FAP-/TF), PDGFRAhighA2 (PDGFRA+/FAP+/TF-), PDGFRAlowA2 (PDGFRA-/FAP+/TF-) and A3 (PDGFRA+/FAP-/TF+) (TF: Tissue Factor).Spatial analyses identified spatial associations indicative of specific functional interactions of fibroblast subsets with cancer cells or immune cells. Explorative analyses in six CRC cases suggested, in general, favorable effects of FAP-negative subsets (A1 and A3) on immune features and cancer cell proliferation, whereas FAP-positive subsets PDGFRAhighA2 and PDGFRAlowA2 overall showed pro-tumoral associations. To extend findings, niches were quantitated in U-CAN cohort of approximately 200 stage I-IV cases with around 80% stage II/III. Spatial screening generated more than 1600 different multi-marker-defined tissue metrics. Analyses identified a higher average of A1 density in areas surrounding proliferating vs non-proliferating T-cells, and a higher average PDGFRAlowA2 peri-T-cell density in the low proliferating T-cell group. Regarding cancer cell proliferation, high proliferating cancer islands showed lower peri-epithelial density of A3 and PDGFRAlowA2 cells as compared to low proliferating cancer islands. As for T-cell cancer island infiltration, high peri-cancer island density of A1 and PDGFRAhighA2 was associated with high T-cell cancer island infiltration. Additional analyses related CAF metrics to stage and survival. Density of PDGFRAlow A2 cells increased with stage (I-III), whereas density of A1 showed an opposite pattern with an inverse association with stage (I-III). Regarding survival associations, these were in general stronger for metrics collected in peripheral regions of tumors. Among prognosis signals related to niches referred to above, it was noted that A1 density in peri-cancer regions was significantly associated with good prognosis. Interestingly, this association was not detected when A1 density was scored in total stroma. Among all metrics, the strongest metrics were A1 density in 20μm A1 expansion areas (good prognosis) and T-reg density in the 10μm A3 expansion areas (bad prognosis) (P&amp;lt;0.001; univariate Cox-regression); both in peripheral regions. Conclusion: Four novel CRC CAF subsets, defined by PDGFRalpha, FAP and TF, was identified by integrated single cell RNA seq analyses and multiplex staining with associated digital image analyses. Non-random spatial distribution of these subsets and compartment-specific prognosis associations are compatible with distinct and different functions of these subsets. Citation Format: Linglong Huang, Mercedes Herrera, Jonas Sjölund, Vladimir Chocoloff, Simon Joost, Rasul M. Tabiev, Lina Wik Leiss, Carina Strell, Luis Nunes, Artur Mezheyeuski, David Edler, Anna Martling, Fredrik Pontén, Bengt Glimelius, Tobias Sjöblom, Maria Kasper, Kristian Pietras, Arne Östman. identification of spatially enriched cancer cell- and immune cell-regulatory multi-marker-defined prognostic cancer associated fibroblast subsets of human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6970.

Abstract 5302: CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors

Abstract INTRODUCTION: CD137 is a TNF receptor superfamily (TNFRSF9) costimulatory receptor expressed by T cells and NK cells. Clustering-mediated CD137 signaling enhances immune cell survival, proliferation, cytokine production and memory formation. CD40 (previously TNFRSF5) is a related superfamily member expressed primarily on B cells and antigen-presenting cells (APCs) which upon clustering is also a key modulator of T cell responses. Mesothelin (MSLN) is highly upregulated on tumor cells in ovarian and pancreatic cancer and select other solid tumour histologies. To avoid systemic toxicity, CB699 is designed to be immunologically active in the presence of any two of its three binding targets, thus enhancing both immune-immune and tumour-immune crosstalk. We present here the preclinical pharmacology of CB699. EXPERIMENTAL PROCEDURES: Binding of CB699 to CD40, CD137 or MSLN was measured by surface plasmon resonance and by flow cytometry. CD137 and/or CD40 agonism, immune cell activation and cytokine secretion were evaluated in vitro using reporter gene assays and primary cell coculture assays in the presence/absence of MSLN-expressing tumor cells. The ability of CB699 to enhance tumor cell killing was characterized in 3D spheroid cocultures. CB699 toxicology and toxicokinetics was assessed in a dose range-finding study in cynomolgus macaques. CB699 was also assessed in vivo with a human CD34+ cell engrafted mouse model. RESULTS: CB699 binds to primary and immortalized cells that express CD40, CD137 or MSLN, with low nanomolar affinity. CB699 also binds to cells from primary disaggregated human solid tumors. CB699 induces CD137 reporter cell activity in cocultures containing cells expressing either CD40 or MSLN. Additionally, CB699 induces CD40 reporter cell activity in cocultures containing cells expressing either CD137 or MSLN. CB699 induces IL-2 secretion and upregulation of CD86 on B cells in primary cocultures containing healthy human PBMCs and MSLN expressing cancer cells. Enhanced tumor cell killing was observed in spheroid cultures containing CB699 and was coupled with immune cell expansion. CB699 showed significant pharmacology in vivo with immune cell engrafted mice. Finally, CB699 was well tolerated in a dose range finding non-human primate toxicology study. CONCLUSIONS: CB699 is a novel MSLN-binding CD137 and CD40 dual agonist that selectively enhances immune cell activity by enhancing both T cell and APC signaling axes. CB699 is positioned to enter clinical development. Citation Format: Andrew J. Pierce, Phillip M. Brailey, Sophie Archer, Holly Spencer, Minjung Song, Timothy Wong, Phillip D. Bartlett, Kieran Williams, Lukas Jasaitis, Philip Bland-Ward. CB699: A novel mesothelin-binding Humabody® CD40 and CD137 dual-agonist for enhancing immune cell responses against MSLN+ tumors [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 5302.

Multiply restimulated human cord blood-derived Tregs maintain stabilized phenotype and suppressive function and predict their therapeutic effects on autoimmune diabetes

BackgroundRegulatory T cells (Tregs) are involved in the maintenance of immune homeostasis and immune regulation. Clinical trials on the adoptive transfer of Tregs have been ongoing for > 10 years. However, many unresolved issues remain in the production of readymade Treg products and selection of patients. Hence, this study aimed to develop a method to expand off-the-shelf Tregs derived from umbilical cord blood (UCB-Tregs) in vitro without changing their phenotype and inhibitory function. In addition, the study intended to design an approach to precisely select patients who are more likely to benefit from the adoptive Treg transfer therapy.MethodsUCB-Tregs were isolated and cultured in a medium containing human recombinant IL-2 and rapamycin and then multiply restimulated with human T-activator CD3/CD28 dynabeads. The phenotype and suppressive capacity of Tregs were assessed on days 18 and 42. The relationship between the suppressive function of UCB-Tregs in vitro and clinical indicators was analyzed, and the ability of the in vitro suppressive capacity to predict the in vivo therapeutic effects was evaluated.ResultsUCB-Tregs expanded 123-fold and 5,981-fold at 18 and 42 days, respectively. The suppressive function of UCB-Tregs on the proliferation of immune cells at 42 days was not significantly different compared with that of UCB-Tregs obtained at 18 days. The suppression rate of UCB-Tregs to PBMCs was negatively correlated with the course of diabetes. Moreover, the high-suppression group exhibited a better treatment response than the low-suppression group during the 12-month follow-up period.ConclusionsMultiply restimulated UCB-Tregs expanded at a large scale without any alterations in their classical phenotypic features and inhibitory functions. The suppressive function of Tregs in vitro was negatively correlated with the disease duration. The present study revealed the possibility of predicting the in vivo therapeutic effects via the in vitro inhibition assay. Thus, these findings provided a method to obtain off-the-shelf Treg products and facilitated the selection of patients who are likely to respond to the treatment, thereby moving toward the goal of precision treatment.

TIGIT regulates CD4+ T cell immunity against polymicrobial sepsis.

Sepsis is one of the major causes of death and increased health care burden in modern intensive care units. Immune checkpoints have been prompted to be key modulators of T cell activation, T cell tolerance and T cell exhaustion. This study was designed to investigate the role of the negative immune checkpoint, T cell immunoglobulin and ITIM domain (TIGIT), in the early stage of sepsis. An experimental murine model of sepsis was developed by cecal ligation and puncture (CLP). TIGIT and CD155 expression in splenocytes at different time points were assessed using flow cytometry. And the phenotypes of TIGIT-deficient (TIGIT-/-) and wild-type (WT) mice were evaluated to explore the engagement of TIGIT in the acute phase of sepsis. In addition, the characteristics were also evaluated in the WT septic mice pretreated with anti-TIGIT antibody. TIGIT and CD155 expression in tissues was measured using real-time quantitative PCR and immunofluorescence staining. Proliferation and effector function of splenic immune cells were evaluated by flow cytometry. Clinical severity and tissue injury were scored to evaluate the function of TIGIT on sepsis. Additionally, tissue injury biomarkers in peripheral blood, as well as bacterial load in peritoneal lavage fluid and liver were also measured. The expression of TIGIT in splenic T cells and NK cells was significantly elevated at 24 hours post CLP.TIGIT and CD155 mRNA levels were upregulated in sepsis-involved organs when mice were challenged with CLP. In CLP-induced sepsis, CD4+ T cells from TIGIT-/- mice shown increased proliferation potency and cytokine production when compared with that from WT mice. Meanwhile, innate immune system was mobilized in TIGIT-/- mice as indicated by increased proportion of neutrophils and macrophages with potent effector function. In addition, tissue injury and bacteria burden in the peritoneal cavity and liver was reduced in TIGIT-/- mice with CLP induced sepsis. Similar results were observed in mice treated with anti-TIGIT antibody. TIGIT modulates CD4+ T cell response against polymicrobial sepsis, suggesting that TIGIT could serve as a potential therapeutic target for sepsis.

The mechanism of Langchuangding in treatment of systemic lupus erythematosus via modulating TLR7-IRF7-IFNα pathway

ObjectSystemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by aberrant activity of the immune system. Plasmacytoid dendritic cells (pDCs) which the main producer of activated type I interferon, are related to SLE disease activity. To investigate the mechanism of Langchuangding (LCD) improving SLE based on TLR7-IRF7-IFNα pathway. MethodsSLE patients were randomly divided into Chinese medicine combined with western medicine (CWM) group and western medicine (WM) group, to observe the effect of LCD. The percent of pDCs in peripheral blood of SLE patients were detected by flow cytometry, and the influence of LCD on gene expression in SLE patients were detected by gene microarray. Mouse bone marrow cells were differentiated into dendritic like cells (DLC), then divided into Blank, immune complex (IC), LCD and dexamethasone (DXM) group. Employed RT-qPCR to detect MyD88, and IRF7 mRNA, and western blotting to determinate TLR7, MyD88, and p-IRF7 proteins. The IFNα in SLE patients were detected by enzyme-linked immunosorbent assay (ELISA). Employ dual luciferase to observe the interferon stimulated response element (ISRE) gene. ResultspDCs in WM group was higher than that of CWM group. The plasma IFNα in CWM group was significantly lower than that in WM group. The gene microarray showed that the gene expression of IFNα related signaling pathway in peripheral blood mononuclear cell (PBMC) and genes related to activation and proliferation of immune cells were down-regulated after LCD treatment. The DLCs MyD88, and IRF7 mRNA were down-regulated, TLR7, MyD88, and p-IRF7 proteins were significantly reduced, and the supernatant IFNα was significantly decreased in LCD group. LCD were mildly inhibited activation of ISRE in 293T cells. ConclusionsIn certain degree, LCD is beneficial to SLE patients. LCD therapy SLE may be through TLR7 signaling pathway, and IRF7 may be a promising therapeutic target for the treatment of SLE.

Cold Atmospheric Plasma: An Emerging Immunomodulatory Therapy

AbstractCold atmospheric plasma (CAP) is a novel technology that generates a unique combination of reactive oxygen and nitrogen species (ROS/RNS), electric fields, and UV radiation. CAP has shown promise in regulating the immune system and has potential clinical applications in wound healing, cancer treatment, and infection control. This review provides an overview of the immunological regulation activity of CAP, highlighting its substantial impact on cytokines production, immune cell phagocytosis, and immune cell proliferation. CAP has also been demonstrated to have potent therapeutic effect in anti‐inflammation, wound repair, viral and bacterial infections. Furthermore, CAP has been investigated as an adjuvant therapy for tumor treatment, eliciting a robust antitumor immune response and remarkable synergistic effects in diverse combination therapies. Further research is needed to fully understand the mechanisms underlying the effects of CAP on the immune system and to optimize its clinical application.