Immune Cell Subtypes Research Articles

Evaluation of the Local and Peripheral Immune Responses in Patients with Cystic Echinococcosis.

Cystic echinococcosis (CE) cysts may persist for decades because of immune modulation mechanisms. Here, we characterize the cysts and the blood immune responses in patients with CE. We enrolled 61 patients with CE and 19 control subjects. We received tissue samples from seven patients with CE and a control subject requiring liver cystectomy. The immunohistochemistry evaluation of the immune cell subtypes and cytokines in the pericysts and surrounding liver and the antigen B (AgB)-specific response analysis of whole blood were performed. In CE, the pericyst and the surrounding liver parenchyma showed aggregates of CD3+ T lymphocytes, mainly CD4+. B lymphocyte aggregates were present in the liver tissue. Monocytes/granulocytes were rarely observed. Th2 cytokine expression was scarce, whereas IFN-γ expression was present in the CE tissues. The control subject did not show an inflammatory infiltrate. The IL-4-specific response to AgB was increased in the patients with CE compared to the control, and this result was confirmed in a larger cohort (p = 0.003), whereas the IFN-γ-response was similar between the two groups (p = 0.5570). In patients with CE, CD4+ lymphocytes infiltrate the pericyst and the surrounding liver tissue with a low IL-4/IL-13 expression level and a moderate IFN-γ expression level; moreover, an IL-4 parasite-specific response is detected in the periphery. These results support adventitia involvement in CE immunopathogenesis.

A phase 1 study of REGN6569, a GITR mAb, in combination with cemiplimab in patients (pts) with advanced solid tumor malignancies: Initial dose-escalation results.

2650 Background: REGN6569 is a fully human immunoglobulin G1 monoclonal antibody (mAb) that is highly specific for glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR). GITR is expressed on several immune cell subtypes, notably regulatory T cells (Tregs), and activated natural killer (NK) cells. REGN6569 demonstrated greater in vitro antibody-dependent cell-mediated cytotoxicity against GITR-expressing Tregs, as compared to GITR-expressing CD8+ T cells. Mouse studies showed that REGN6569 + cemiplimab (anti-PD-1) combination treatment achieved longer-term tumor responses compared with either drug alone. Methods: This is a first-in-human study (NCT04465487) evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of REGN6569 administered intravenously (IV) every 3 weeks (Q3W) + cemiplimab 350 mg IV Q3W, in pts with advanced solid tumors for which immune checkpoint inhibitor therapies have not been approved or are not available. The study includes a dose-escalation part with 5 dose levels (DL1-DL5) for REGN6569 (“4+3” design), with an initial dose of REGN6569 monotherapy given as a safety lead-in followed by REGN6569 + cemiplimab in subsequent doses. Results: As of the data cutoff date Oct 20, 2023, the dose-escalation part has been completed. 29 pts (median age 58.0 years, 62.1% male) were treated with REGN6569 + cemiplimab, up to the 1200 mg dose level (DL5), across many solid tumor types. The most common tumor type was colorectal cancer (34.5%). One pt (40 mg DL2) experienced a dose-limiting toxicity (Grade 3 hepatic failure); maximum tolerated dose was not reached. Twelve pts (41.4%) had a Grade ≥3 treatment-emergent adverse event (TEAE) and 16 pts (55.2%) had a treatment-related TEAE (any grade). The most frequent TEAEs (any grade) were arthralgia (24.1%), infusion-related reactions, and abdominal pain (20.7% each). There were no treatment-related deaths; 19 (65.5%) pts had disease progression leading to death. Two pts achieved ongoing partial responses by investigator assessment with REGN6569 + cemiplimab treatment: 1 pt with mucoepidermoid tumor of the parotid gland treated with 120 mg (DL3) REGN6569 and 1 pt with B3 thymoma treated with 400 mg (DL4) REGN6569, with duration of responses, 5.6 and 10.4 months, respectively. Full receptor occupancy on circulating Tregs was observed in all dose cohorts following REGN6569 treatment. Increased frequency (~10–50%) of proliferating NK cells in peripheral blood was observed post REGN6569 treatment across all dose cohorts. Conclusions: In this dose-escalation study, REGN6569 was administered up to 1200 mg (DL5) in combination with cemiplimab with one dose-limiting toxicity. The study has progressed to dose-expansion cohorts in anti–PD-1-resistant head and neck cancer, with pts treated with REGN6569 (DL5) + cemiplimab. Clinical trial information: NCT04465487 .

Tumour mutation burden and infiltrating immune cell subtypes influenced the breast cancer prognosis.

Most of its issues are still undecided on the relationship between tumour mutation burden (TMB) and immune-related genes in the breast cancer. This study explores their relationship based on gene mutation and transcription data in The Cancer Genome Atlas (TCGA) database, and the effects of immune cells in TMB and tumour microenvironments on prognosis of breast cancer patients. Cases were divided into low-TMB and high-TMB subgroups. Differentially expressed immune-related genes were identified in different TMB subgroups, and patient prognosis was predicted by gene function enrichment analysis, invasive immune cells and different clinical pathological features were compared among different TMB subgroups. A total of 986 mutation data from breast cancer patients were obtained. Compared with low-TMB group, the survival period of high-TMB group was relatively longer. A total of 337 differential expression genes were identified in this study. Of these genes, seven differentially expressed immune-related genes were associated with prognosis. In the high-TMB group, activated CD4+ memory T cells and other cells had high expression, the expression ratio of memory B cells and other cells in low-TMB group was high. TMB-related immunological infiltration characteristics showed meaningful value for prognosis prediction for breast cancer patients. Differentially expressed immune-related genes in TMB subgroups provide important information on the survival prediction.

Exploring shared molecular signatures and regulatory mechanisms in nonalcoholic steatohepatitis and inflammatory bowel disease using integrative bioinformatics analysis.

The co-existence of inflammatory bowel disease (IBD) and non-alcoholic steatohepatitis (NASH) has raised interest in identifying shared molecular mechanisms and potential therapeutic targets. However, the relationship between these two diseases remains unclear and effective medical treatments are still lacking. Through the bioinformatics analysis in this study, 116 shared differentially expressed genes (SDEGs) were identified between IBD and NASH datasets. GO and KEGG pathway analyses revealed significant involvement of SDEGs in apoptotic processes, cell death, defense response, cytokine and chemokine activity, and signaling pathways. Furthermore, weighted gene co-expression network analysis (WGCNA) identified five shared signature genes associated specifically with IBD and NASH, they were CXCL9, GIMAP2, ADAMTS5, GRAP, and PRF1. These five genes represented potential diagnostic biomarkers for distinguishing patients with diseases from healthy individuals by using two classifier algorithms and were positively related to autophagy, ferroptosis, angiogenesis, and immune checkpoint factors in the two diseases. Additionally, single-cell analysis of IBD and NASH samples highlighted the expression of regulatory genes in various immune cell subtypes, emphasizing their significance in disease pathogenesis. Our work elucidated the shared signature genes and regulatory mechanisms of IBD and NASH, which could provide new potential therapies for patients with IBD and NASH.

Enhanced Proteomic Coverage in Tissue Microenvironment by Immune Cell Subtype Library-Assisted DIA-MS

Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, six immune cell subtype-specific spectral libraries were established from sorted cluster of differentiation markers, CD8+, CD4+ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 μg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7419 proteins compared to directDIA analysis (6978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundance immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was used to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25–3%) in the tumor, our results illuminate downregulation in the adaptive immune signaling pathways (such as C-type lectin receptor signaling, and chemokine signaling), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC model. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.

HMGB1: A New Target for Ischemic Stroke and Hemorrhagic Transformation.

Stroke in China is distinguished by its high rates of morbidity, recurrence, disability, and mortality. The ultra-early administration of rtPA is essential for restoring perfusion in acute ischemic stroke, though it concurrently elevates the risk of hemorrhagic transformation. High-mobility group box 1 (HMGB1) emerges as a pivotal player in neuroinflammation after brain ischemia and ischemia-reperfusion. Released passively by necrotic cells and actively secreted, including direct secretion of HMGB1 into the extracellular space and packaging of HMGB1 into intracellular vesicles by immune cells, glial cells, platelets, and endothelial cells, HMGB1 represents a prototypical damage-associated molecular pattern (DAMP). It is intricately involved in the pathogenesis of atherosclerosis, thromboembolism, and detrimental inflammation during the early phases of ischemic stroke. Moreover, HMGB1 significantly contributes to neurovascular remodeling and functional recovery in later stages. Significantly, HMGB1 mediates hemorrhagic transformation by facilitating neuroinflammation, directly compromising the integrity of the blood-brain barrier, and enhancing MMP9 secretion through its interaction with rtPA. As a systemic inflammatory factor, HMGB1 is also implicated in post-stroke depression and an elevated risk of stroke-associated pneumonia. The role of HMGB1 extends to influencing the pathogenesis of ischemia by polarizing various subtypes of immune and glial cells. This includes mediating excitotoxicity due to excitatory amino acids, autophagy, MMP9 release, NET formation, and autocrine trophic pathways. Given its multifaceted role, HMGB1 is recognized as a crucial therapeutic target and prognostic marker for ischemic stroke and hemorrhagic transformation. In this review, we summarize the structure and redox properties, secretion and pathways, regulation of immune cell activity, the role of pathophysiological mechanisms in stroke, and hemorrhage transformation for HMGB1, which will pave the way for developing new neuroprotective drugs, reduction of post-stroke neuroinflammation, and expansion of thrombolysis time window.

Biofilms and core pathogens shape the tumor microenvironment and immune phenotype in colorectal cancer

ABSTRACT Extensive research has explored the role of gut microbiota in colorectal cancer (CRC). Nonetheless, metatranscriptomic studies investigating the in situ functional implications of host-microbe interactions in CRC are scarce. Therefore, we characterized the influence of CRC core pathogens and biofilms on the tumor microenvironment (TME) in 40 CRC, paired normal, and healthy tissue biopsies using fluorescence in situ hybridization (FISH) and dual-RNA sequencing. FISH revealed that Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. Dual-RNA sequencing demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors in CRC samples with high bacterial activity. In addition, bacterial activity correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells in CRC samples. Specifically, Bacteroides fragilis and Fusobacterium nucleatum correlated with the infiltration of neutrophils and CD4+ T-cells, respectively. The collective bacterial activity/biomass appeared to exert a more significant influence on the TME than core pathogens, underscoring the intricate interplay between gut microbiota and CRC. These results emphasize how biofilms and core pathogens shape the immune phenotype and TME in CRC while highlighting the need to extend the bacterial scope beyond CRC pathogens to advance our understanding and identify treatment targets.

Potential role of CTNNA3 and FRMPD4 in vascular tumorous thrombosis of colon adenocarcinoma.

Vascular tumorous thrombosis is a crucial pathological feature of malignant tumors that is closely associated with lymph node metastasis and is considered a form of tumor micrometastasis. Two downregulated genes, catenin alpha 3 (CTNNA3) and FERM and PDZ domain-containing 4 (FRMPD4), were selected by analyzing the differential expression of vascular tumorous thrombus in colon adenocarcinoma and paracancerous tissues. Further investigation revealed their potential role in the development of vascular tumorous thrombosis in colon adenocarcinomas. Candidate genes for vascular tumorous thrombosis in colon adenocarcinoma were screened using GSE127069, and pan-cancer verification and immune infiltration analysis were performed. The relationship between gene expression and vascular tumorous thrombosis was analyzed based on the level of gene mutations using cBioPortal. Finally, the collected clinical samples were used to verify expression. CTNNA3 and FRMPD4 were expressed at low levels in the vascular tumorous thrombosis of colon adenocarcinoma and positively correlated with microsatellite instability. They are also closely related to the immune microenvironment and the infiltration of immune cell subtypes. Based on gene mutation analysis, gene deletion is suggested to be related to vascular invasion indicators. Finally, protein and messenger ribonucleic acid (mRNA) expression of CTNNA3 and FRMPD4 were downregulated in the vascular tumorous thrombosis samples of colon adenocarcinoma compared to normal glands from paracancerous tissues. Our study suggests that CTNNA3 and FRMPD4 could be promising biomarkers for vascular tumorous thrombosis in colon adenocarcinoma, potentially enabling the identification of micrometastases in this type of cancer. These findings suggest a novel strategy for the detection and management of colon adenocarcinomas.

Immune cell signatures and inflammatory mediators: unraveling their genetic impact on chronic kidney disease through Mendelian randomization

Prior research has established associations between immune cells, inflammatory proteins, and chronic kidney disease (CKD). Our Mendelian randomization study aims to elucidate the genetic causal relationships among these factors and CKD. We applied Mendelian randomization using genetic variants associated with CKD from a large genome-wide association study (GWAS) and inflammatory markers from a comprehensive GWAS summary. The causal links between exposures (immune cell subtypes and inflammatory proteins) and CKD were primarily analyzed using the inverse variance-weighted, supplemented by sensitivity analyses, including MR-Egger, weighted median, weighted mode, and MR-PRESSO. Our analysis identified both absolute and relative counts of CD28 + CD45RA + CD8 + T cell (OR = 1.01; 95% CI = 1.01–1.02; p < 0.001, FDR = 0.018) (OR = 1.01; 95% CI = 1.00–1.01; p < 0.001, FDR = 0.002), CD28 on CD39 + CD8 + T cell(OR = 0.97; 95% CI = 0.96–0.99; p < 0.001, FDR = 0.006), CD16 on CD14–CD16 + monocyte (OR = 1.02; 95% CI = 1.01–1.03; p < 0.001, FDR = 0.004) and cytokines, such as IL-17A(OR = 1.11, 95% CI = 1.06–1.16, p < 0.001, FDR = 0.001), and LIF-R(OR = 1.06, 95% CI = 1.02–1.10, p = 0.005, FDR = 0.043) that are genetically predisposed to influence the risk of CKD. Moreover, the study discovered that CKD itself may causatively lead to alterations in certain proteins, including CST5(OR = 1.16, 95% CI = 1.09–1.24, p < 0.001, FDR = 0.001). No evidence of reverse causality was found for any single biomarker and CKD. This comprehensive MR investigation supports a genetic causal nexus between certain immune cell subtypes, inflammatory proteins, and CKD. These findings enhance the understanding of CKD's immunological underpinnings and open avenues for targeted treatments.

CXCR6 expression correlates with radiotherapy response and immune context in triple-negative breast cancer-experimental studies.

The chemokine receptor CXCR6 is critical for sustained tumor control mediated by CD8+ cytotoxic T cells (CTLs) in tumors. Previous studies have shown that ionizing radiation induces an inflamed immune contexture by upregulating CXCR6. However, the clinical significance of CXCR6 expression in triple-negative breast cancer (TNBC) and its correlation with radiotherapy remains unknown. This study aimed to clarify the prognostic value of CXCR6 and its role in the breast tumor microenvironment (TME). The messenger RNA and protein expression of CXCR6 in human TNBC and their association with survival were analyzed. The role of CXCR6 in the immune context was investigated using a combination of single-cell RNA sequencing, bulk transcriptome sequencing data, and fluorescence-based multiplex immunohistochemistry (mIHC) techniques. Elevated CXCR6 expression correlated with better clinical outcomes and superior response to adjuvant radiotherapy and immunotherapy in TNBC. CXCR6 fostered an immunostimulatory microenvironment characterized by upregulated cytotoxic markers. We also found that CXCR6 plays a crucial role in regulating the differentiation of CD8+ T cells and the intercellular communication of immune cell subtypes, thus shaping the TME. This study highlights the emerging role of CXCR6 in shaping the TME and targeting CXCR6 may be a promising strategy for improving the effectiveness of radiotherapy and immunotherapy in TNBC.

Abstract PO5-25-03: Immune cell infiltrate profiles in primary Triple Negative Breast Cancer and co-relation with treatment response and survival outcomes

Abstract Introduction Tumor microenvironment (TME) in breast cancer is a heterogeneous landscape with multiple cell types, each modulating the tumor progression and the disease prognosis. These cell types include lymphocytes, RBCs, stromal cells, macrophages, cancer-associated fibroblasts (CAFs), and cancer stem cells. Each cell type in the TME influences tumor progression and clinical outcomes for patients in a good or bad way, depending on the interaction with tumor cells. Tumor-infiltrating lymphocytes (TILs) are shown to be prognostic for therapy response in many cancers in the western cohorts. Our recent study showed that a higher proportion of Indian TNBC patients had intra-tumoral TILs compared to Western cohorts, and these are predictive of better response to NACT and longer disease-free survival in patients, making it a distinct finding from Western reports. In the follow-up study, we are characterizing infiltrating immune cell subtypes and their comparative association with treatment response in TNBC patients. Results from preliminary analysis and comparative analysis between responders and non-responders will be presented here. We speculate that the pro- and anti-tumorigenic immune cell population and its spatial information would help us understand TME and its precise role in response to therapy. Methods A retrospective cohort of breast cancer patients from a single one-surgeon breast cancer clinic with a uniform treatment strategy was evaluated for this study. FFPE primary tissue samples with variable TILs scores (moderate to high), taken from patients who showed complete response to the NACT treatment (n=6) and non-responding group (n=8) determined by H&amp;E stained slides from our cohort, were further stained for immune cells (Pan-B cell, Pan-T cells, Pan-macrophages) and T cells (CD4; Helper T cell marker, CD8; Cytotoxic T cell marker), FOXP; Regularly CD4+ cell marker) and PanCK (Pan-tumor marker) and counterstained with DAPI for multiplex imaging. Multispectral images for these slides were obtained using the Leica Aperio Versa system. Parameters such as individual cell types of identification and scoring, spatial proximity to tumor cells, and individual cell types will be determined by HALO software using the High plex module. Results A total of 14 TNBC samples were stained for the T-cell subtypes panel and Immune cell subtype s panel to determine the TIME. DAPI stain was used for nuclear counting based the parameters such as threshold intensity, nuclear size, roundness, and aggressiveness. The cytoplasmic size was selected as 2um beyond the outside nuclear boundary. Using Halo-Indica software, around 50000-300000 cells/tissue were evaluated across all samples from mIF whole slide scans of biopsy samples. Cell count was validated by cross-checking individual ROIs and manual cell count using Fiji. A Highplex module was used to identify individual signal positivity for each marker based on threshold intensity and cell completeness percent. Preliminary data showed a higher proportion of CD8+ cells and macrophages population are spatially closer to the tumor cells in Responders compared to non-responders. Conclusion Immune profiles are distinct among responding and non-responding patients in the Indian TNBC cohort. The association of these profiles with long-term survival in patients and various clinical and pathological parameters will help us determine the clinical relevance of these profiles and identify distinct immune subgroups as prognostic in Indian TNBCs. Citation Format: Pooja Vaid, Devaki Kelkar, LS Shashidhara, C B Koppiker, Madhura Kulkarni. Immune cell infiltrate profiles in primary Triple Negative Breast Cancer and co-relation with treatment response and survival outcomes [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 PO5-25-03.

Abstract PO4-24-12: Premenopausal breast cancers show higher proportion of immunologically cold tumors and are associated with distinct immune cell infiltrate

Abstract Background Breast cancers (BC) in the young women tend to have poor outcomes and higher proportion of aggressive subtypes. Immunotherapy has shown promising results lately, especially for aggressive categories like triple negative breast cancers (TNBC). Success of the immunotherapy is shown to be dependent on the tumor immune microenvironment (TIME) which plays important role in disease progression and response to therapy. Though numerous studies have demonstrated immune hot tumors are associated with better prognosis, mechanism of immunologically hot TIME is not yet deciphered. Material and methods Gene expression data derived from RNA sequencing (n=40) of primary BC was used to divide them into immune hot and cold tumors by using 15 gene signature (Wang et al., Sci. Adv. 2021). Average expression of the hot and cold (hot=12 and cold=3) genes was derived for each tumor and median value of their ratio was used to categorize them into immunologically hot and cold tumors. Association of the hot and cold tumors with clinical characteristics was examined. CIBERSORT algorithm was used to identify immune cell subtypes and Kaplan Meier survival analysis was performed to assess the prognostic significance. Similar analysis was replicated in TCGA (n=1083) dataset. Results Equal proportion of immune hot and cold tumor was observed in the cohort. Higher proportion of cold tumors were observed in younger and premenopausal (p=0.002) patients. Subtype analysis showed higher proportion of cold tumors in TNBC compared to hormone receptor positive tumors (p&amp;lt; 0.0001). No significant difference was observed in other clinicopathological characteristics between the groups. Further analysis of cold tumors for immune cell subtypes, categorized by the menopausal status showed higher proportion of plasma cells and CD4 memory resting cells and lower proportion of M2 macrophages and B memory cell in premenopausal (n=129, TCGA) compared postmenopausal (n=322, TCGA) tumors. In addition, premenopausal cold tumors were associated with better overall survival compared to postmenopausal group (p=0.001). Conclusion Younger premenopausal women with BC are likely to have more immunologically cold tumors. Though circulating levels of sex steroid hormones is the predominant difference between the pre and postmenopausal women, its influence on TIME and therapeutic significance must be explored. Citation Format: Vidya P Nimbalkar, Snijesh VP, Savitha Rajarajan, Annie Alexander, Rakesh Ramesh, B S Srinath, Jyothi S Prabhu. Premenopausal breast cancers show higher proportion of immunologically cold tumors and are associated with distinct immune cell infiltrate [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 PO4-24-12.

Abstract PO2-25-05: Spatial multiplexing of protein biomarkers for immune cell profiling of the tumor microenvironment with ChipCytometry

Abstract Understanding the spatial distribution of key immune cell populations is critical in advancing our understanding of cancer and the development of novel therapeutics. Historically, the spatial analysis of the tumor microenvironment (TME) has been limited to relatively low-plex immunohistochemical (IHC) or immunofluorescent (IF) assays, which were inadequate for deep immune cell profiling of the TME. Here, we present the analysis of fresh-frozen human breast tissue using spatial multiplexing technology called ChipCytometry, which combines iterative immuno-fluorescent staining using open-source antibodies for quantitative measure of virtually unlimited protein biomarkers on the same sample and high dynamic range imaging to facilitate quantitative phenotyping with single-cell resolution. In addition to traditional pathology review of the high-plex dataset, subsequent image analysis can be done using open-source or commercial software for advanced spatial analyses, including cluster analysis, neighborhood analysis and advanced data visualization. The results show precise expression levels for each of the protein biomarkers in the assay for each individual cell within the sample while maintaining spatial information about each cell. Dozens of immune cell subtypes were identified and quantified based on protein expression profiles. Spatial analysis of the samples revealed quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for in-depth immune profiling in clinical samples. The ChipCytometry platform enables simultaneous detection of multiple protein biomarkers on a single tissue section for deep immune cell profiling in the TME. Combined with the single-cell spatial information, such datasets provide an opportunity for the discovery of new complex multiplexed biomarker signatures to inform therapeutic development and personalized medicine. Citation Format: Benton Berigan. Spatial multiplexing of protein biomarkers for immune cell profiling of the tumor microenvironment with ChipCytometry [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 PO2-25-05.

New Classification of Rheumatoid Arthritis Based on Immune Cells and Clinical Characteristics.

Rheumatoid arthritis (RA) is a chronic systemic immune disease characterized by joint synovitis, but there are differences in clinical manifestations and serum test results among different patients. This is a bioinformatics study. We first obtained the gene expression profile of RA and normal synovium from the database, and screened the differentially expressed immune related genes for enrichment analysis. Subsequently, we classified RA into three subtypes by unsupervised clustering of serum gene expression profiles based on immune enrichment scores. Then, the enrichment and clinical characteristics of different subtypes were analyzed. Finally, according to the infiltration of different subtypes of immune cells, diagnostic markers were screened and verified by qRT-PCR. C1 subtype is related to the increase of neutrophils, C-reactive protein and erythrocyte sedimentation rate, and joint pain is more significant in patients. C2 subtype is related to the expression of CD8+T cells and Tregs, and patients have mild joint pain symptoms. The RF value of C3 subtype is higher, and the expression of various immune cells is increased. CD4 T cells, NK cells activated, macrophages M1 and neutrophils are immune cells significantly infiltrated in synovium and serum of RA patients. IFNGR1, TRAC, IFITM1 can be used as diagnostic markers of different subtypes. In this study, RA patients were divided into different immune molecular subtypes based on gene expression profile, and immune diagnostic markers were screened, which provided a new idea for the diagnosis and treatment of RA.

Abstract 3014: Bulk And Single-cell Characterization Of The Immune Microenvironment Of Abdominal Aortic Aneurysm

Background: Abdominal aortic aneurysm (AAA), characterized by localized dilation of the abdominal aorta, involves an immune microenvironment that plays a crucial role in disease development. This study aimed to investigate immune cell heterogeneity and functional dynamics in AAA using single-cell RNA sequencing (scRNA-seq) analysis of AAA content and healthy aorta samples. Methods: By analyzing scRNA-seq datasets from AAA content and healthy aorta samples, along with bulk datasets comprising AAA and healthy aortic tissues, we comprehensively profiled the immune microenvironment. Results: The analysis revealed five major cell types in both AAA contents and healthy aortic tissues. Further integrative analysis identified 18 distinct subpopulations, unraveling specific immune cell subtypes within AAA. These subtypes were distinguished based on tissue infiltration, functional variations, immune dynamics, metabolic changes, and communication patterns. Notably, B cells and plasma cells were found to contribute to AAA and thrombus formation. Additionally, cytotoxic CD8+ T cells showed enrichment in cytokine and chemokine receptor interactions, indicating their involvement in AAA development. Two pro-inflammatory macrophage subtypes (LY6E+ and CCL5+) and two pro-inflammatory neutrophil subtypes specific to AAA emerged as significant findings. Particularly, MME+ neutrophils exhibited heightened immune receptor activity and infiltrated AAA tissues, as supported by bulk dataset analysis. Metabolically active, these neutrophils played a vital role in regulating CXCL signaling, enabling effective cell-to-cell communication among immune cells. Conclusions: This study provides a comprehensive understanding of the diverse immune cell populations within AAA and healthy aorta tissues. The findings shed light on their functional characteristics, developmental trajectories, metabolic differences, and intercellular communication patterns, significantly advancing our knowledge of AAA formation and progression.

Causal effects of immune cells in glioblastoma: a Bayesian Mendelian Randomization study.

Glioblastoma (GBM) is a highly malignant brain tumor, and immune cells play a crucial role in its initiation and progression. The immune system's cellular components, including various types of lymphocytes, macrophages, and dendritic cells, among others, engage in intricate interactions with GBM. However, the precise nature of these interactions remains to be conclusively determined. In this study, a comprehensive two-sample Mendelian Randomization (MR) analysis was conducted to elucidate the causal relationship between immune cell features and the incidence of GBM. Utilizing publicly available genetic data, we investigated the causal associations between 731 immune cell signatures and the risk of GBM. Subsequently, we conducted a reverse Mendelian randomization analysis to rule out reverse causation. Finally, it was concluded that there is a unidirectional causal relationship between three subtypes of immune cells and GBM. Comprehensive sensitivity analyses were employed to validate the results robustness, heterogeneity, and presence of horizontal pleiotropy. To enhance the accuracy of our results, we concurrently subjected them to Bayesian analysis. After conducting MR analyses, we identified 10 immune phenotypes that counteract glioblastoma, with the most protective being FSC-A on Natural Killer T cells (OR = 0.688, CI = 0.515-0.918, P = 0.011). Additionally, we found 11 immune cell subtypes that promote GBM incidence, including CD62L- HLA DR++ monocyte % monocyte (OR = 1.522, CI = 1.004-2.307, P = 0.048), CD4+CD8+ T cell % leukocyte (OR = 1.387, CI = 1.031-1.866, P = 0.031). Following the implementation of reverse MR analysis, where glioblastoma served as the exposure variable and the outcomes included 21 target immune cell subtypes, we discerned that only three cell subtypes (CD45 on CD33+ HLA DR+ CD14dim, CD33+ HLA DR+ Absolute Count, and IgD+ CD24+ B cell Absolute Count) exhibited a unidirectional causal association with glioblastoma. Our study has genetically demonstrated the close relationship between immune cells and GBM, guiding future clinical research.

Expression and Localization Profiles of Tight Junction Proteins in Immune Cells Depend on Their Activation Status.

The ability of the immune system to combat pathogens relies on processes like antigen sampling by dendritic cells and macrophages migrating through endo- and epithelia or penetrating them with their dendrites. In addition, other immune cell subtypes also migrate through the epithelium after activation. For paracellular migration, interactions with tight junctions (TJs) are necessary, and previous studies reported TJ protein expression in several immune cells. Our investigation aimed to characterize, in more detail, the expression profiles of TJ proteins in different immune cells in both naïve and activated states. The mRNA expression analysis revealed distinct expression patterns for TJ proteins, with notable changes, mainly increases, upon activation. At the protein level, LSR appeared predominant, being constitutively present in naïve cell membranes, suggesting roles as a crucial interaction partner. Binding experiments suggested the presence of claudins in the membrane only after stimulation, and claudin-8 translocation to the membrane occurred after stimulation. Our findings suggest a dynamic TJ protein expression in immune cells, implicating diverse functions in response to stimulation, like interaction with TJ proteins or regulatory roles. While further analysis is needed to elucidate the precise roles of TJ proteins, our findings indicate important non-canonical functions of TJ proteins in immune response.

Spatial Distribution of Macrophage and Lymphocyte Subtypes within Tumor Microenvironment to Predict Recurrence of Non-Muscle-Invasive Papillary Urothelial Carcinoma after BCG Immunotherapy.

Non-muscle-invasive papillary urothelial carcinoma (NMIPUC) of the urinary bladder is the most common type of bladder cancer. Intravesical Bacille Calmette-Guerin (BCG) immunotherapy is applied in patients with a high risk of recurrence and progression of NMIPUC to muscle-invasive disease. However, the tumor relapses in about 30% of patients despite the treatment, raising the need for better risk stratification. We explored the potential of spatial distributions of immune cell subtypes (CD20, CD11c, CD163, ICOS, and CD8) within the tumor microenvironment to predict NMIPUC recurrence following BCG immunotherapy. Based on analyses of digital whole-slide images, we assessed the densities of the immune cells in the epithelial-stromal interface zone compartments and their distribution, represented by an epithelial-stromal interface density ratio (IDR). While the densities of any cell type did not predict recurrence, a higher IDR of CD11c (HR: 0.0012, p-value = 0.0002), CD8 (HR: 0.0379, p-value = 0.005), and ICOS (HR: 0.0768, p-value = 0.0388) was associated with longer recurrence-free survival (RFS) based on the univariate Cox regression. The history of positive repeated TUR (re-TUR) (HR: 4.93, p-value = 0.0001) and T1 tumor stage (HR: 2.04, p-value = 0.0159) were associated with shorter RFS, while G3 tumor grade according to the 1973 WHO classification showed borderline significance (HR: 1.83, p-value = 0.0522). In a multivariate analysis, the two models with a concordance index exceeding 0.7 included the CD11c IDR in combination with either a history of positive re-TUR or tumor stage. We conclude that the CD11c IDR is the most informative predictor of NMIPUC recurrence after BCG immunotherapy. Our findings highlight the importance of assessment of the spatial distribution of immune cells in the tumor microenvironment.

Upregulated Matrisomal Proteins and Extracellular Matrix Mechanosignaling Underlie Obesity-Associated Promotion of Pancreatic Ductal Adenocarcinoma.

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFβ, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing.

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.