Immune Cell Populations Research Articles

TMIC-78. CHEMOTHERAPY INDUCED IMMUNOGENIC RESPONSE IN GLIOMA AND IMPLICATIONS FOR THERAPEUTIC STRATEGIES

Abstract INTRODUCTION Many glioblastoma (GBM) treatments fail because they treat tumor cells in isolation and ignore the surrounding microenvironment. In our phase 1b clinical trial of delivery of Topotecan (TPT) via Convection Enhanced Delivery (CED) in glioma, we demonstrated effective tumor cell elimination accompanied by a robust inflammatory response. We hypothesized that TPT induces an immunogenic response resulting in an unfavorable inflammatory landscape. METHODS To characterize chemotherapy’s effect on myeloid cells, we analyzed pre- and post-treatment biopsies from our trial using bulk RNA sequencing to measure canonical myeloid markers. We validated survival benefit of CED in vivo in our tumor model and characterized post-treatment tissue using single cell RNA sequencing (scRNA seq) after 7 days of treatment. We treated slice cultures generated from surgical samples of GBM with TPT and performed scRNA seq and histologic analysis to assess microenvironment changes after 24 hours of treatment. We used several in vitro assays to characterize the direct and indirect mechanisms of myeloid activation. RESULTS Bulk RNA sequencing of post treatment biopsies demonstrates upregulation of markers associated with activated and exhausted immune cell populations such as IBA1, CD68, MSR1, MARCO and loss of markers associated with homeostatic microglia such as P2RY12 and TMEM119. Similar results are found within 7 days of treatment in vivo, however, are not found in human derived samples treated acutely. Treatment of in vitro mono and co-culture systems demonstrate a tumor cell death dependent activation of myeloid cell populations via immunogenic molecules. CONCLUSIONS Chemotherapies eliminate proliferating glioma cells but also induce inflammation. While many investigators have tried to leverage this activation, our results suggest that chronic inflammation contributes to poor prognosis and recurrence. The identification of molecules responsible for this activation point to new therapeutic targets aimed at controlling treatment-induced inflammation, as part of a combinatorial approach to treat GBM.

TMIC-71. ELUCIDATING IMMUNE MICROENVIRONMENTAL DISTINCTIONS BETWEEN IDH-MUTANT ASTROCYTOMA AND OLIGODENDROGLIOMA USING SINGLE-CELL TRANSCRIPTIONAL PROFILING

Abstract Despite exhibiting longer overall survival that IDH-wt counterparts, IDH-mutant gliomas remain uniformly deadly and impact patients in the prime of adult life. The immune microenvironment of IDH-mutant gliomas remains incompletely characterized, particularly with regard to distinctions between IDH-mutant astrocytoma and IDH-mutant, 1p/19q codeleted oligodendroglioma. To address this knowledge gap, we subjected dissociated tumor suspensions derived from 7 astrocytoma and 7 oligodendroglioma clinical samples to flow cytometry-based sorting for the pan-immune marker, CD45, and the microglial marker, P2RY12. This process yielded both microglial and non-microglial immune cell populations, which were then analyzed by scRNA sequencing. This workflow revealed a single large cluster of microglia, characterized by expression of Galectin-1 and lysosomal Lipase A, which was present at a significantly higher level in oligodendroglioma. While the biological significance of this cluster is unclear, gene set enrichment analysis exploring phenotypic differences between microglia in astrocytoma and oligodendroglioma revealed significant increases in expression of gene sets related to antigen presentation in astrocytoma. Parallel analysis of the non-microglial immune fraction showed an increase in the expression of pro-inflammatory gene sets, such as genes related to cytokine secretion, within the CD8 T-cell compartment of astrocytomas. We also observed upregulation of the expression of inflammatory gene sets within the dendritic cells of astrocytomas. Taken together, these changes suggest that the immune compartment of astrocytoma is more active and inflamed, and targeting the immune system in these tumors may represent a potential therapeutic avenue.

SURG-48. LASER INTERSTITIAL THERMAL THERAPY REMODELS THE TUMOR IMMUNE LANDSCAPE IN A MOUSE GLIOMA MODEL

Abstract BACKGROUND Laser interstitial thermal therapy (LITT), a minimally invasive surgical technique for tumor ablation, is increasingly being used to treat primary and recurrent glioma. While the primary goal of using LITT has been to destroy tumor tissue, little is known about the impact of LITT on the overall immune response. To investigate changes in the tumor immune landscape, we utilized a mouse model of LITT and applied it to a poorly immunogenic mouse model of glioma. METHODS SB28 glioma cells were implanted in the right frontal lobe of C57BL/6 mice, and on day 7, an optical fiber is inserted into the tumor, paired with a thermocouple for real-time temperature monitoring. A 30W laser was activated in pulses to maintain a peri-lesional temperature of 44C for 150-180 seconds. The tumor and LITT-associated immune response was analyzed by multiparameter flow cytometry, single cell RNA-seq, and multiplex immunofluorescence. RESULTS LITT (versus sham control) triggered an increase in neutrophils in the tumor during the acute phase at day 3 following treatment. Total immune cell numbers increased over time in both sham- and LITT-treated mice, but there was a significant increase at post-LITT day 7 in select adaptive immune cell populations including type 1 conventional dendritic cells, NK cells, and gamma delta T cells. Monocytes/macrophages comprised the majority of the immune infiltrate, and multiplex immunofluorescence revealed these populations localized to the perimeter of the LITT lesion. scRNA-seq also revealed enrichment of a Fabp5- and Gpnmb-expressing macrophage subset following LITT. CONCLUSIONS LITT modifies the tumor immune infiltrate with increases in neutrophils as well as select adaptive cell and macrophage subsets. Future studies will explore adjunct therapies to further augment the immune response in the setting of LITT.

RBIO-03. A SMALL ANIMAL RADIATION RESEARCH PLATFORM (FLASH-SARRP) TO MEASURE IMMUNOLOGICAL EFFECT OF ULTRA HIGH DOSE RATE (FLASH) RADIOTHERAPY

Abstract Patients who undergo radiation therapy (RT), particularly fractionated courses, often develop lymphopenia. Clinical outcome for various cancer types, including brain tumors, identifies lymphopenia as a negative prognostic factor. The mechanism for RT induced lymphopenia is unclear. Simulated models suggest the volume of blood flowing through a radiation field could play a crucial role in death of radiosensitive lymphocytes. FLASH RT is ultrahigh dose rate administration of RT, >100 times faster dose rate than standard RT that has been observed to improve the therapeutic ratio in preclinical models by preferential normal tissue sparing. Hypothesized mechanisms include chemical reactions depleting oxygen in well oxygenated normal cells creating radioresistance, differing pro-inflammatory signaling impacting tumor and normal tissue, and less impact on circulating immune cells consequent to decreased proportion exposed during a treatment session which may enhance antineoplastic response. We designed the novel FLASH kV x-ray cabinet system for preclinical laboratory research (FLASH-SARRP) to deliver ultra-high dose rates up to 100 Gy/s as well as conventional dose rates of 1.0 Gy/s to facilitate comparative studies. A custom designed docking system was developed to reproducibly position and immobilize mouse for stereotactic radiation of the brain. Dose and dose rate measurements were performed with calibrated Gafchromic EBT3 film in solid water with 0.025 mm additional copper filter. FLASH-SARRP system delivered kV x-rays at an average dose-rate of 79.2±2.1Gy/s corresponding to 10 mm depth in the immobilized mouse brain. For the delivery of 15 Gy, treatment duration was 0.2 seconds for FLASH RT administration whereas it was 244 seconds for conventional dose rate. We successfully designed a high-precision platform to study partial brain x-ray FLASH effects in mice and studies are ongoing to assess differential impact on normal brain tissue, circulating cytokines, and immune cell populations for single dose and fractionated administration of FLASH and standard radiotherapy.

Synovial Fluid Immune Cell Composition Following Intraarticular Fracture May Contribute to Posttraumatic Osteoarthritis

Intra-articular ankle fracture (IAF) often leads to post-traumatic osteoarthritis (PTOA), resulting in significant long-term morbidity. While previous research has focused on the inflammatory cytokines and matrix metalloproteinases within the synovial fluid fracture hematoma (SFFH), the immune cell populations within SFFH that contribute to PTOA development remain underexplored. This study aimed to characterize the immune cell populations in SFFH to better understand their role in the inflammatory response and potential for inducing lasting cartilage damage. Twenty-four patients with IAF underwent surgical ankle aspiration to collect SFFH, which was analyzed using polychromatic flow cytometry. The analysis revealed that 72.8% of the CD45+ cells were lymphocytes, predominantly CD3+ T cells (76.5%), with 42.1% being CD4+ and 39.2% CD8+ T cells. Additionally, monocytes accounted for 21.2% of CD45+ cells, with small populations of natural killer cells and myeloid-derived suppressor cells also present. These findings emphasize the predominance of T cells, particularly CD4+ subsets, in the immune response following IAF. Understanding these dynamics is essential for developing targeted interventions to prevent PTOA. Future research should focus on elucidating the specific roles of these immune cell populations in PTOA progression and exploring potential therapeutic strategies.

Nuclear EGFR in breast cancer suppresses NK cell recruitment and cytotoxicity.

Natural Killer (NK) cells can target and destroy cancer cells, yet tumor microenvironments typically suppress NK cell recruitment and cytotoxicity. The epidermal growth factor receptor (EGFR) is a potent oncogene that can activate survival, migration, and proliferation pathways, and clinical data suggests it may also play an immunomodulating role in cancers. Recent work has demonstrated a novel role for nuclear EGFR (nEGFR) in regulating transcriptional events unique from the kinase domain. Using a novel peptide therapeutic (cSNX1.3) that inhibits retrograde trafficking of EGFR and an EGFR nuclear localization mutant, we discovered that nEGFR suppresses NK cell recruitment and cytotoxicity. RNA-Seq analysis of breast cancer cells treated with cSNX1.3 or modified to lack a nuclear localization sequence (EGFRΔNLS) revealed the EGF-dependent induction of NK activating receptor ligands, while kinase inhibition by erlotinib did not impact these genes. NanoString analysis of tumor-bearing WAP-TGFα transgenic mice treated with cSNX1.3 demonstrated an increase in immune cell populations and activating genes. Additionally, immunohistochemistry confirmed an increase in NK cells upon cSNX1.3 treatment. Finally, cSNX1.3 treatment was found to enhance NK cell recruitment and cytotoxicity in vitro. Together, the data demonstrate a unique immunomodulatory role for nEGFR.

Identification of immune-associated genes for the diagnosis of ulcerative colitis-associated carcinogenesis via integrated bioinformatics analysis

BackgroundUC patients suffer more from colorectal cancer (CRC) than the general population, which increases with disease duration. Early colonoscopy is difficult because ulcerative colitis-associated colorectal cancer (UCAC) lesions are flat and multifocal. Our study aimed to identify promising UCAC biomarkers that are complementary endoscopy strategies in the early stages.MethodsThe datasets may be accessed from the Gene Expression Omnibus and The Cancer Genome Atlas databases. The co-expressed modules of UC and CRC were determined via weighted co-expression network analysis (WGCNA). The biological mechanisms of the shared genes were exported for analysis using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. To identify protein interactions and hub genes, a protein-protein interaction network and CytoHubba analysis were conducted. To evaluate gene expression, external datasets and experimental validation of human colon tissues were utilized. The diagnostic value of core genes was examined through receiver operating characteristic (ROC) curves. Immune infiltration analysis was employed to investigate the associations between immune cell populations and hub genes.ResultsThree crucial modules were identified from the WGCNA of UC and CRC tissues, and 33 coexpressed genes that were predominantly enriched in the NF-κB pathway were identified. Two biomarkers (CXCL1 and BCL6) were identified via Cytoscape and validated in external datasets and human colon tissues. CRC patients expressed CXCL1 at the highest level, whereas UC and CRC patients showed higher levels than the controls. The UC cohort expressed BCL6 at the highest level, whereas the UC and CRC cohorts expressed it more highly than the controls. The hub genes exhibited significant diagnostic potential (ROC curve > 0.7). The immune infiltration results revealed a correlation among the hub genes and macrophages, neutrophils and B cells.ConclusionsThe findings of our research suggest that BCL6 and CXCL1 could serve as effective biomarkers for UCAC surveillance. Additionally, they demonstrated a robust correlation with immune cell populations within the CRC tumour microenvironment (TME). Our findings provide a valuable insight about diagnosis and therapy of UCAC.

Unique T cell signatures associated with reduced Chlamydia trachomatis reinfection in a highly exposed cohort.

Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection (STI) in the United States, despite effective antibiotics. Information regarding natural immunity to CT will inform vaccine design. The objectives of this study were to determine immune cell populations and functional features associated with reduced risk of CT reinfection or endometrial CT infection. PBMCs were collected from a cohort of CT-exposed women who were tested for CT and other STIs at the cervix and endometrium (to determine ascension) and were repeatedly tested over the course of a year (to determine reinfection). Mass cytometry identified major immune populations and T cell subsets. Women with CT had increased CD4+ effector memory T cells (TEM) compared to uninfected women. Specifically, Th2, Th17, and Th17 DN CD4+ TEM were increased. Th17 and Th17 DN CD4+ central memory T cells (TCM) were increased in women who did not experience follow-up CT infection, suggesting that these cells may be important for protection. These data indicate that peripheral T cells display distinct features that correlate with natural immunity to CT and suggest that the highly plastic Th17 lineage plays a role in protection against reinfection.

Host genetics and gut microbiota influence lipid metabolism and inflammation: potential implications for ALS pathophysiology in SOD1G93A mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the progressive loss of motor neurons, with genetic and environmental factors contributing to its complex pathogenesis. Dysregulated immune responses and altered energetic metabolism are key features, with emerging evidence implicating the gut microbiota (GM) in disease progression. We investigated the interplay among genetic background, GM composition, metabolism, and immune response in two distinct ALS mouse models: 129Sv_G93A and C57Ola_G93A, representing rapid and slow disease progression, respectively.Using 16 S rRNA sequencing and fecal metabolite analysis, we characterized the GM composition and metabolite profiles in non-transgenic (Ntg) and SOD1G93A mutant mice of both strains. Our results revealed strain-specific differences in GM composition and functions, particularly in the abundance of taxa belonging to Erysipelotrichaceae and the levels of short and medium-chain fatty acids in fecal samples. The SOD1 mutation induces significant shifts in GM colonization in both strains, with C57Ola_G93A mice showing changes resembling those in 129 Sv mice, potentially affecting disease pathogenesis. ALS symptom progression does not significantly alter microbiota composition, suggesting stability.Additionally, we assessed systemic immunity and inflammatory responses revealing strain-specific differences in immune cell populations and cytokine levels.Our findings underscore the substantial influence of genetic background on GM composition, metabolism, and immune response in ALS mouse models. These strain-specific variations may contribute to differences in disease susceptibility and progression rates. Further elucidating the mechanisms underlying these interactions could offer novel insights into ALS pathogenesis and potential therapeutic targets.

Assessing sepsis-induced immunosuppression to predict positive blood cultures

IntroductionBacteremia is a life-threatening condition that can progress to sepsis and septic shock, leading to significant mortality in the emergency department (ED). The standard diagnostic method, blood culture, is time-consuming and prone to false positives and false negatives. Although not widely accepted, several clinical and artificial intelligence-based algorithms have been recently developed to predict bacteremia. However, these strategies require further identification of new variables to improve their diagnostic accuracy. This study proposes a novel strategy to predict positive blood cultures by assessing sepsis-induced immunosuppression status through endotoxin tolerance assessment.MethodsOptimal assay conditions have been explored and tested in sepsis-suspected patients meeting the Sepsis-3 criteria. Blood samples were collected at ED admission, and endotoxin (lipopolysaccharide, LPS) challenge was performed to evaluate the innate immune response through cytokine profiling.ResultsClinical variables, immune cell population biomarkers, and cytokine levels (tumor necrosis factor [TNFα], IL-1β, IL-6, IL-8, and IL-10) were measured. Patients with positive blood cultures exhibited significantly lower TNFα production after LPS challenge than did those with negative blood cultures. The study also included a validation cohort to confirm that the response was consistent.DiscussionThe results of this study highlight the innate immune system immunosuppression state as a critical parameter for sepsis diagnosis. Notably, the present study identified a reduction in monocyte populations and specific cytokine profiles as potential predictive markers. This study showed that the LPS challenge can be used to effectively distinguish between patients with bloodstream infection leading to sepsis and those whose blood cultures are negative, providinga rapid and reliable diagnostic tool to predict positive blood cultures. The potential applicability of these findings could enhance clinical practice in terms of the accuracy and promptness of sepsis diagnosis in the ED, improving patient outcomes through timely and appropriate treatment.

Myeloid derived suppressor cells mediate hepatocyte proliferation and immune suppression during liver regeneration following resection.

Liver regeneration following resection is a complex process relying on coordinated pathways and cell types in the remnant organ. Myeloid-Derived Suppressor Cells (MDSCs) have a role in liver regeneration-related angiogenesis but other roles they may play in this process remain to be elucidated. In this study, we sought to examine the effect of G-MDSCs on hepatocytes proliferation and immune modulation during liver regeneration. Global gene expression profiling of regenerating hepatocytes in mice with CD11b+Ly6G+ MDSCs (G-MDSCs) depletion revealed disrupted transcriptional progression from day one to day two after major liver resection. Key genes and pathways related to hepatocyte proliferation and immune response were differentially expressed upon MDSC depletion. Hepatocytes cellularity increased when co-cultured with G-MDSCs, or treated with amphiregulin, which G-MDSCs upregulate during regeneration. Cytometry by time-of-flight (CyTOF) analysis of the intra-liver immune milieu upon MDSC depletion during regeneration demonstrated increased natural killer cell proportions, alongside changes in other immune cell populations. Taken together, these results provide evidence that MDSCs contribute to early liver regeneration by promoting hepatocyte proliferation and modulating the intra-liver immune response, and illuminate the multifaceted role of MDSCs in liver regeneration.

Blood integrin and cytokine producing T cells are associated with stage and genetic risk score in age-related macular degeneration.

Age-related macular degeneration (AMD) remains a leading cause of vision loss in the geriatric population. There are age-related changes in peripheral blood leukocyte composition, but their significance for AMD remains unclear. We aimed to determine changes in immune cell populations in blood of AMD patients. A standardized 31 parameter flow cytometry analysis was conducted on peripheral blood mononuclear cells from 59 patients with early and advanced AMD and 39 controls without AMD older than 65 years. Fundus photography and optical coherence tomography were used to classify disease stages and a custom genotype array was used to compute an AMD genetic risk score based on 52 AMD disease risk variants (GRS-52). A generalized linear regression model corrected for age, sex, and smoking status revealed that AMD patients showed decreased frequencies of CD4-positive T helper cell population expressing Integrin Alpha E (CD103) (Padj = 0.019). We further noted that early AMD was characterized by increased interleukin-4 (IL-4)-producing CD4+ T helper cells (Padj = 0.013; <0.001), as well as IL-4-producing cytotoxic CD8+ T cells (Padj = 0.016; <0.001). Reclassification of samples based on the GRS-52 revealed that IL-17-producing T cells decreased incrementally across GRS-52 categories. In AMD, alterations in peripheral blood leukocyte populations are associated with genetic risk score and disease stage and include specifically IL-4 and IL-17A cytokine-producing and CD103 integrin expressing T cell populations.

Microglial activation without peripheral immune cell infiltration characterises mouse and human cerebral small vessel disease.

Cerebral small vessel diseases (SVDs) involve diverse pathologies of the brain's small blood vessels, leading to cognitive deficits. Cerebral magnetic resonance imaging (MRI) reveals white matter hyperintensities (WMHs), lacunes, microbleeds and enlarged perivascular spaces in SVD patients. Although correlations of MRI and histopathology help to understand the pathogenesis of SVD, they do not explain disease progression. Mouse models, both genetic and sporadic, are valuable for studying SVD, but their resemblance to clinical SVD is unclear. The study examined similarities and differences between mouse models of SVDs and human nonamyloid SVD specimens. We analysed four mouse models of SVD (hypertensive BPH mice, Col4a1 mutants, Notch3 mutants and Htra1-/- mice) at different stages for changes in myelin, blood-brain barrier (BBB) markers, immune cell populations and immune activation. The observations from mouse models were compared with human SVD specimens from different regions, including the periventricular, frontal, central and occipital white matter. Postmortem MRI followed by MBP immunostaining was used to identify white matter lesions (WMLs). Only Notch3 mutant and hypertensive BPH mice showed significant changes in myelin basic protein (MBP) immunostaining, correlating with MRI patterns. These changes were linked to altered microglial morphology and focal plasma protein staining around blood vessels, without peripheral immune cell infiltration. In human specimens, both normal-appearing white matter (NAWM) and WMLs lacked peripheral cell infiltration. However, WMLs displayed altered microglial morphology, reduced myelin staining and occasional fibrinogen staining around arterioles and venules. Our data show that Notch3 mutants and hypertensive BPH/2J mice recapitulate several features of human SVD, including microglial activation, focal sites of demyelination and perivascular plasma protein leakage without peripheral immune cell infiltration.

High mitochondrial DNA levels accelerate lung adenocarcinoma progression.

Lung adenocarcinoma is a common aggressive cancer and a leading cause of mortality worldwide. Here, we report an important in vivo role for mitochondrial DNA (mtDNA) copy number during lung adenocarcinoma progression in the mouse. We found that lung tumors induced by KRASG12D expression have increased mtDNA levels and enhanced mitochondrial respiration. To experimentally assess a possible causative role in tumor progression, we induced lung cancer in transgenic mice with a general increase in mtDNA copy number and found that they developed a larger tumor burden, whereas mtDNA depletion in tumor cells reduced tumor growth. Immune cell populations in the lung and cytokine levels in plasma were not affected by increased mtDNA levels. Analyses of large cancer databases indicate that mtDNA copy number is also important in human lung cancer. Our study thus reports experimental evidence for a tumor-intrinsic causative role for mtDNA in lung cancer progression, which could be exploited for development of future cancer therapies.

Immunologic Profiling of CSF in Subarachnoid Neurocysticercosis Reveals Specific Interleukin-10-Producing Cell Populations During Treatment.

Subarachnoid neurocysticercosis (SANCC) is the most severe form of Taenia solium CNS infection and accounts for the majority of neurocysticercosis-associated mortality. Inflammation is important in the treatment of SANCC because overactivity can lead to serious complications, but excessive suppression may be counterproductive toward parasite eradication. A relative abundance of CSF IL-10 to IL-12 has been associated with increased treatment duration for patients with SANCC, suggesting that IL-10 plays an important role in this disease process. To better understand SANCC immunology and the major sources of IL-10 during anthelmintic treatment, we took an unbiased and comprehensive approach to phenotype the immune cell populations in the CSF and peripheral blood of patients with SANCC. Eight samples of CSF cells collected from 5 patients with SANCC during treatment were evaluated using single-cell RNA sequencing. Matched CSF and peripheral blood mononuclear cells from 4 patients were assessed using flow cytometry. Staining for extracellular and intracellular markers allowed for the characterization of IL-10-producing T cells. The CSF during SANCC contains a diversity of immune cell populations including multiple myeloid and lymphoid populations. Although there were changes in the composition of CSF cells during treatment, the largest population at both early and late time points was CD4+ T cells. Within this population, we identified 3 sources of IL-10 unique to SANCC CSF compared with controls: natural regulatory T cells (nTregs), induced regulatory T cells (iTregs), and Th17 cells. The abundance and phenotype of these IL-10-producing populations differed between CSF and blood in patients with SANCC, but iTregs were the single most productive population in the CSF. During treatment, these IL-10 producers persisted in consistent proportions despite decreases in parasite antigen over time. This profile of immune cell populations in the CSF provides a comprehensive blueprint of the local and systemic immunology associated with SANCC. The identification of IL-10-producing cells in the CSF and peripheral blood deepens our understanding of the immunosuppressive phenotype that deters SANCC treatment success. Finally, the discovery that these IL-10 producers persist throughout treatment highlights the endurance of these populations in the CNS.

Myeloid subsets impede the efficacy of anti-PD1 therapy in patients with advanced gastric cancer (WJOG10417GTR study).

Gastric cancer (GC) is one of the most common and deadly malignant diseases worldwide. Despite revolutionary advances, the therapeutic efficacy of anti-PD1/PDL1 monoclonal antibodies in advanced GC is still low due to the emergence of innate and acquired resistance to treatment. Myeloid cells represent the majority of human immune cells. Therefore, their increase, decrease, and abnormality could have a significant impact on the patient's immune system and the progression of cancer, and reprogramming, inhibiting, and eliminating the tumor-supportive types may improve the immunological situation and efficacy of immunotherapy. However, the significance of myeloid cells in anti-PD1/PDL1 therapy remains unclear in GC. In the WJOG10417GTR study on GC, we sought to identify myeloid determinants that could predict anti-PD1 therapeutic efficacy and also serve as potential therapeutic targets. We collected tumor tissues and peripheral blood from 96 patients with advanced GC before and 1 month after anti-PD1 nivolumab monotherapy, and the isolated whole leucocytes were analyzed by flow cytometry for various immune cell populations, including many myeloid subsets. Then, the relationship between the cellular levels and progression-free survival (PFS) or overall survival (OS) was statistically analyzed. We found that high levels of several myeloid subsets expressing molecules that have been targeted in drug discovery but not yet approved for clinical use were significantly associated with shorter PFS/OS as compared with low levels: PDL1+ and CTLA4+ myeloid subsets within tumors at baseline, PDL1+, B7H3+ and CD115+ myeloid subsets in peripheral blood at baseline, and LAG3+, CD155+ and CD115+ myeloid subsets in peripheral blood at post-treatment. This study revealed that these myeloid subsets are significant risk factors in nivolumab therapy for advanced GC. Targeting them may be useful as diagnostic biomarkers to predict potential anti-PD1 therapeutic efficacy, and also as therapeutic targets for accelerating the development of new drugs to improve clinical outcomes in immunotherapy for GC.

Mechanism and Therapeutic Targets of Circulating Immune Cells in Diabetic Retinopathy.

Diabetic retinopathy (DR) continues to be the leading cause of preventable vision loss among working-aged adults, marked by immune dysregulation within the retinal microenvironment. Typically, the retina is considered as an immune-privileged organ, where circulating immune cells are restricted from entry under normal conditions. However, during the progression of DR, this immune privilege is compromised as circulating immune cells breach the barrier and infiltrate the retina. Increasing evidence suggests that vascular and neuronal degeneration in DR is largely driven by the infiltration of immune cells, particularly neutrophils, monocyte-derived macrophages, and lymphocytes. This review delves into the mechanisms and therapeutic targets associated with these immune cell populations in DR, offering a promising and innovative approach to managing the disease.

Incretin-responsive human pancreatic adipose tissue organoids: a functional model for fatty pancreas research.

Infiltration of adipocytes into the pancreatic parenchyma has been linked to impaired insulin secretion in individuals with increased genetic risk of T2D and prediabetic conditions. However, the study of this ectopic fat depot has been limited by the lack of suitable in vitro models. Here, we developed a novel 3D model of functionally mature human pancreatic adipose tissue organoids by aggregating human pancreatic adipose tissue-derived stromal vascular fraction (SVF) cells into organoids and differentiating them over 19 days. These organoids carry biological properties of the in situ pancreatic fat, presenting levels of adipogenic markers comparable to native pancreatic adipocytes and improved lipolytic and anti-lipolytic response compared to conventional 2D cultures. The organoids harbour a small population of immune cells, mimicking in vivo adipose environment. Furthermore, they express GIPR, allowing investigation of incretin effects in pancreatic fat. In accordance, GIP and the dual GLP1R/GIPR agonist tirzepatide stimulate lipolysis but had distinct effects on the expression of proinflammatory cytokines. This novel adipose organoid model is a valuable tool to study the metabolic impact of incretin signalling in pancreatic adipose tissue, revealing potential therapeutic targets of incretins beyond islets. The donor-specific metabolic memory of these organoids enables examination of the pancreatic fat-islet crosstalk in a donor-related metabolic context.

Natural killer cells are required for the recruitment of CD8+ T cells and the efficacy of immune checkpoint blockade in melanoma brain metastases

BackgroundBrain metastases (BrM) affect up to 60% of patients with metastatic melanoma and are associated with poor prognosis. While combined immune checkpoint blockade of programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) demonstrates intracranial efficacy in a proportion of patients with melanoma, the responses are rarely durable, particularly in patients with symptomatic BrM. The brain is an immune-specialized organ and immune responses are regulated differently to the periphery.MethodsUsing our previously established two-site model of melanoma BrM with concomitant intracranial and extracranial tumors, in which clinically observed efficacy of the combined PD-1/CTLA-4 (PC) blockade can be reproduced, we here explored the role of natural killer (NK) cells in BrM, using functional studies, immunophenotyping and molecular profiling.ResultsWe demonstrate that NK cells are required for the intracranial efficacy of PC blockade. While both perforin and interferon gamma were necessary for the PC blockade-dependent control of intracranial tumor growth, NK cells isolated from intracranial tumors demonstrated only a limited cancer cell killing ability, and PC blockade did not alter the abundance of NK cells within tumors. However, the depletion of NK cells in PC blockade-treated mice led to tumor molecular profiles reminiscent of those observed in intracranial tumors that failed to respond to therapy. Furthermore, the depletion of NK cells resulted in a strikingly reduced abundance of CD8+ T cells within intracranial tumors, while the abundance of other immune cell populations including CD4+ T cells, macrophages and microglia remained unaltered. Adoptive T cell transfer experiments demonstrated that PC blockade-induced trafficking of CD8+ T cells to intracranial tumors was chemokine-dependent. In line with this, PC blockade enhanced intratumoral expression of several T cell-attracting chemokines and we observed high expression levels of cognate chemokine receptors on BrM-infiltrating CD8+ T cells in mice, as well as in human BrM. Importantly, the depletion of NK cells strikingly reduced the intratumoral expression levels of T cell attracting chemokines and vascular T cell entry receptors that were upregulated following PC blockade.ConclusionOur data demonstrate that NK cells underpin the efficacy of PC blockade in BrM by orchestrating the "responder" molecular profile in tumors, and by controlling the intratumoral abundance of CD8+ T cells through regulation of multiple key molecular mediators of T cell trafficking.

Effect of anterior quadratus lumborum block with ropivacaine on the immune response after laparoscopic surgery in colon cancer: a substudy of a randomized clinical trial

BackgroundSurgery induces a temporal change in the immune system, which might be modified by regional anesthesia. Applying a bilateral preoperative anterior quadratus lumborum block has proven to be a safe...