Changes In Immune Cell Populations Research Articles

Early-Life Respiratory Syncytial Virus (RSV) Infection Triggers Immunological Changes in Gut-Associated Lymphoid Tissues in a Sex-Dependent Manner in Adulthood.

Severe respiratory syncytial virus (RSV) infection during early life has been linked to gut dysbiosis, which correlates with increased disease severity and a higher risk of developing asthma later in life. However, the impact of such early-life RSV infections on intestinal immunity in adulthood remains unclear. Herein, we show that RSV infection in 3-week-old mice induced persistent differential natural killer (NK) and T cell profiles within the lungs and gastrointestinal (GI) lymphoid tissues (GALT) in adulthood. Notably, male mice exhibited more pronounced RSV-induced changes in immune cell populations in both the lungs and GALT, while female mice displayed greater resilience. Importantly, early-life RSV infection was associated with the chronic downregulation of CD69-expressing T lymphocytes, particularly T regulatory cells in Peyer's patches, which could have a significant impact on T cell functionality and immune tolerance. We propose that RSV infection in early life is a trigger for the breakdown in immune tolerance at mucosal surfaces, with potential implications for airways allergic disease, food allergies, and other GI inflammatory diseases.

Bioinformatics analysis of therapeutic targets for idiopathic pulmonary fibrosis and exploration of immune cell infiltration patterns

Idiopathic pulmonary fibrosis (IPF) is a severe progressive lung disease characterized by fibrotic changes in lung tissue, with limited treatment options. This study analyzed gene expression data from three gene expression omnibus datasets (GSE2052, GSE53845, and GSE110147) using R and LIMMA to identify differentially expressed genes (DEGs) in IPF samples. We identified 215 DEGs, comprising 106 upregulated and 109 downregulated genes. Weighted gene coexpression network analysis revealed five gene modules, with the module eigengene yellow showing the strongest correlation with IPF. Functional enrichment analysis of 40 consensus genes in this module indicated their significant involvement in extracellular matrix (ECM) organization. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed pathways related to protein digestion, cell adhesion molecules, and the advanced glycation end product–receptor for advanced glycation end product signaling pathway. Based on protein–protein interaction network analysis, collagen type XV alpha 1 chain (COL15A1) and collagen type VI alpha 3 chain (COL6A3) were identified as upregulated hub genes in IPF, which were regulated by microRNAs and transcription factors. Immune cell infiltration analysis showed significant changes in immune cell populations in IPF samples, with increases in memory B cells, plasma cells, and M0 macrophages and decreases in CD8 T cells, and resting natural killer cells. Potential drugs targeting COL15A1 and COL6A3 were predicted using multiple databases, revealing compounds such as (+)-JQ1, aristolochic acid I, and dexamethasone with promising binding potential. These findings suggest that COL15A1 and COL6A3 are central hub genes in IPF, are associated with ECM organization and immune response, and serve as therapeutic targets for IPF.

Extracorporeal photopheresis vs. systemic immunosuppression for immune-related adverse events: Interim analysis of a prospective two-arm study

BackgroundCheckpoint inhibitor-induced steroid-refractory (sr) and steroid-dependent (sd) immune-related adverse events (irAE) account for about 11 % of irAE. Although these patients face worse outcomes due to irAE mortality and/or sustained immunosuppression, which impairs anti-tumor response, there is no established second-line treatment based on prospective trial data. MethodsThis prospective comparative study investigates outcomes of extracorporeal photopheresis (ECP), an immunomodulating therapy, versus second-line immunosuppressants (SLI) in sr/sd-irAE. The primary endpoint was longitudinal change in immunophenotype; secondary endpoints were outcome of irAE and tumor response. Patient demographics, quality of life (EORTC QLQ-C30; global health status (GHS/QoL)) and longitudinal blood samples were analyzed at baseline; in weeks 1, 4, 8, and 12. ResultsAt interim analysis, 21 patients (11 ECP, 10 SLI) with 7 different sr/sd-irAE were included. Compared with the SLI group, the ECP group demonstrated a higher clinical response rate of irAE (93 % vs. 80 %; 95 % CI 0.83–1.92; P = 0.54) and a better GHS/QoL score throughout all follow-up visits. ECP patients showed a numerically higher overall survival (23 vs. 12 months; 95 % CI 0.02–3.02; P = 0.27) and lower cancer progression rates (33 % vs. 67 %; 95 % CI 0.09–1.60; P = 0.52). Immunophenotyping revealed changes in immune cell populations and the regulation of immune checkpoints. There were no significant safety issues in either treatment group. ConclusionThis prospective comparative study supports the clinical efficacy of ECP in the treatment of sr/sd-irAE in comparison to the SLI cohort. Thus, ECP represents a potential treatment option for this indication, given its good safety profile while maintaining anti-tumor response. Trial RegistrationClinicalTrials.gov, NCT05700565, https://classic.clinicaltrials.gov/ct2/show/NCT05700565.

Cigarette smoking prolongs inflammation associated with influenza infection and delays its clearance in mice.

Epidemiological studies have shown that smoking is associated with increased incidence of severe viral infections leading to hospitalization. Moreover, studies in experimental models have identified impaired antiviral responses and altered inflammatory responses, yet it is unclear how the effects of smoke exposure and influenza A infection interact and how this varies over the course of infection. We hypothesized that smoking would exacerbate innate immune responses against influenza. To test this, female BALB/c mice were exposed to cigarette smoke or air twice a day for 24-28 days and (mock) infected with H3N2 influenza A on day 21 while smoking continued. Three and seven days after infection, changes in immune cell populations, the transcriptome, and viral clearance in lung tissue were analyzed. After influenza A infection, smoke-exposed mice lost significantly more weight than air-exposed controls, indicating that smoking resulted in more severe disease. Immune cell and lung tissue transcriptome analysis revealed that neutrophil infiltration was prolonged and macrophage activation dysregulated after infection of smoke-exposed mice compared to air-exposed controls. Expression of genes in IL-6 and interferon pathways was similarly longer active. In parallel, we observed lower clearance of influenza virus in smoke-exposed mice after infection compared to air-exposed controls, indicating ineffective antiviral responses. Altogether, the data from our mouse model indicate that cigarette smoke exposure prolongs innate immune responses against influenza A. The results from this study help to explain the susceptibility of current smokers to severe influenza A disease.

Bone Marrow and Peripheral Blood Mononuclear Cell Phenotype Changes after Cultivation and Autologous Infusion in Patients with Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a condition affecting the liver and immune system. In this study, the impact of autologous bone marrow-derived mononuclear cell (BM-MNC) transplantation on PBC patients was investigated. Sixteen eligible PBC patients participated at the National Scientific Medical Center in Astana, Kazakhstan, between 2017 and 2022, and BM-MNCs were harvested from their anterior iliac crest. After isolating and cultivating the BM-MNCs, they were infused back into the patient's peripheral veins. Changes in BM-MNC and peripheral blood mononuclear cell (PB-MNC) phenotypes were assessed before and after a 24-hour cultivation period and 72 hours post-transplantation. We monitored liver function parameters over 6-month intervals and conducted flow cytometry analysis to assess CD markers on BM-MNCs before and after cultivation and PB-MNCs before and after transplantation. Statistical analysis included the Friedman test for liver parameters and the Wilcoxon signed-rank test for BM-MNC and PB-MNC comparisons. Our findings revealed significant reductions in liver function tests after multiple transplantations. Flow cytometry analysis before and after a 24-hour culture and autologous BM-MNC infusion revealed the expansion of specific cell populations, with significant increases in CD3+, CD4+, CD16+, CD20+, CD25+, CD34+, CD105+, CD73+, СD117+, and CD34+populations, while CD4+25+, CD34+105+, and CD4+FOXP3+ populations decreased. Interestingly, a contradictory finding was observed with a decrease in bone marrow CD34+105+ cell lines (P=0.03) alongside an increase in peripheral CD34+105+ population (P=0.03). In summary, our study shows that BM-MNC transplantation in PBC patients leads to changes in immune cell populations and liver function. These findings suggest potential therapeutic applications of BM-MNC transplantation in managing PBC and offer insights into the dynamics of immune cells associated with this treatment approach.

Time to resolution of airway inflammation caused by bronchoalveolar lavage in healthy horses.

Bronchoalveolar lavage (BAL) is a common procedure for evaluation of the equine lower airways. Time to resolution of post-BAL inflammation has not been clearly defined. Residual inflammation, evident by changes in immune cell populations and inflammatory cytokines, will resolve by 72 hours after BAL. Six adult, healthy, institution-owned horses. Randomized, complete cross-over design. Each horse underwent 3 paired BALs, including a baseline and then 48, 72, and 96 hours later, with a 7-day washout between paired BALs. Each sample underwent cytological evaluation and cytokine concentrations were determined by a commercially available multiplex bead immunoassay. Statistical analysis was performed by multilevel mixed-effects Poisson regression analysis. Data are reported as marginal means and 95% confidence interval (CI). Neutrophil, eosinophil and mast cell percentages were not significantly different at any time points. Macrophage percentages were higher at 72 hours (45.0 [95% CI, 41.6-48.4]%) and 96 hours (45.3 [95% CI, 42.9-47.7]%) vs baseline (37.4 [95% CI, 33.5-41.4]%; P < .001 and P = .01, respectively), and at 72 hours and 96 hours vs 48 hours (31.9 [95% CI, 28.1-35.6]%; P < .001). Neutrophil percentage was not significantly increased at 48 hours (P = .11). Interleukin (IL)-6 concentration was increased at 72 hours (5.22 [95% CI, 3.44-6.99] pg/mL) vs 48 hours (4.38 [95% CI, 2.99-5.78] pg/mL; P < .001). Significant lung inflammation was not detected at 72 and 96 hours, suggesting that repeating BAL at 72 hours or more can be done without concern of residual inflammation.

Isolating Immune Cells from Mouse Brain and Skull.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Endoplasmic reticulum stress-related gene expression patterns in glioblastoma models identified by single-cell RNA sequencing: Implications for prognosis and treatment

Objective: Endoplasmic reticulum (ER) stress has therapeutic potential for a variety of malignancies, including glioma. In this study, bioinformatics was used to analyze ER stress-related genes (ERGs) in glioblastoma (GBM), explore their functions and pathways, construct prognostic models, and explore new treatment strategies. Methods: Various bioinformatics algorithms were utilized to screen for ERGs and construct a risk model. According to the expression of ERGs, different subtypes were classified using the consensus clustering method. Gene Set Variation Analysis (GSVA) and Gene Set Enrichment Analysis (GSEA) were performed on the subtypes. Based on screened risk genes, GBM patients were divided into Train and Test groups in a 1:1 ratio. The prognostic model was validated through Receiver Operating Characteristic (ROC) curve analysis and independent prognostic analysis. The model was further validated by comparing the risk scores between high-risk and low-risk groups, and comparisons were made in terms of survival time, immune microenvironment, and pathway regulation. Drug sensitivity was used to screen drugs for low- and high- risk group, and single-cell RNA sequencing (scRNA-seq) analysis were utilized to explore the expression distribution of risk genes in GBM. Results: According to the ERGs, GBM samples can be divided into two groups with significant differences. Cluster A showed better survival rates compared to Cluster B. GSVA and GSEA analysis revealed that Cluster A was mainly enriched in glutamate receptor signaling pathway, synaptic transmission between neurons, postsynaptic density membrane, postsynaptic membrane, and synaptic vesicle membrane functions. It is worth noting that 8 ERGs were screened as model genes, which can effectively and independently predicate the survival risk of GBM patients with high accuracy and discrimination ability. Subsequently, changes in immune cell populations were observed in high-risk and low-risk groups, with differences in memory B cells and resting CD4 memory T cells between the high-risk and low-risk groups. The high-risk group had higher levels of memory B cells, while the low-risk group had higher levels of resting CD4 memory T cells. Furthermore, potential therapeutic strategies were identified, with BI-2536, Daporinad, SB505124, UMI-77, and Vorinostat identified for the low-risk group, while AZD8055, Camptothecin, Gemcitabine, PD0325901, and Topotecan identified for the high-risk group. scRNA-seq identified Ribosomal Protein L10 (RPL10) as one of the eight ER stress-related genes, primarily expressed in malignant cells of various tumors. Conclusion: This study identified eight ERGs and constructed a risk model based on bioinformatics analysis, which can be used for prognosis prediction and drug screening.

Dynamic changes in immune cell populations by AXL kinase targeting diminish liver inflammation and fibrosis in experimental MASH.

Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB- NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

Immune Activation Promotes Cardiac Remodeling in a Mouse Model of Takotsubo Cardiomyopathy

Takotsubo Cardiomyopathy (TCM) is an acute, life-threatening condition characterized by a drop in ejection fraction (EF) and apical ballooning of the left ventricle (LV). TCM is most frequently observed in women and is associated with an emotional or physical stressor. Importantly, with medical intervention, cardiac dysfunction is transient, and EF is often restored over weeks to months. Despite this, patients who experience TCM are at an increased risk of additional cardiovascular events and premature mortality. The precise etiology of TCM is unknown but a wave of excess circulating catecholamines is thought to be a trigger. Furthermore, the cause for increased cardiovascular risk post-TCM is unknown. The goal of this study is to understand how the immune system may contribute to TCM and the associated cardiovascular event risk. To induce TCM, mice were given a single dose of 200mg/kg isoproterenol (ISO), i.p. and cardiac function was assessed by conscious echocardiography (ECG). An initial study compared the effect of ISO between sexes (n=4-6). Though ISO induced a drop in EF in both sexes after 24 hours (p&lt;0.001 compared to vehicle (Veh)), females exhibited lower EF (46.64±3.11%) than males (68.36±2.62%, p&lt;0.0001). This mirrors the increased prevalence of clinically defined TCM in females in human populations. For this reason, female mice were used for the following experiments. A time course study demonstrated that EF begins to normalize by Day 3 (78.78±2.70 v 90.80±1.00%, p&lt;0.001) and is restored to baseline levels by Day 7 (90.65±1.17%). Flow cytometry was used to identify changes in immune cell populations in the heart on Days 0, 1, 3 and 7 (n=4-5). Neutrophils and inflammatory monocytes increased on Day 1, returning to baseline by Day 3. Dendritic cells and macrophages were elevated on Day 3 post-ISO, with a specific increase in CCR2+ TIM4- recruited inflammatory macrophages (p&lt;0.0001). Across all subsets, the number of immune cells returned to baseline levels by Day 7. Due to the robust immune response observed, we tested the specific hypothesis that transfer of immune cells from mice who have experienced TCM would induce cardiac dysfunction in naïve recipient mice. Donor mice were treated with either Veh or ISO and 1 day later splenocyte isolation and transfer was performed. Importantly, transfer of splenocytes from mice who had TCM induced a significant drop in EF the following day in the recipient mice (86.30±1.08%, p=0.001, n=10) concurrent with an increase in LV inner diameter and volume during systole. A final study tested the hypothesis that a history of TCM would increase risk for future cardiovascular disease. Veh or ISO-treated mice were allowed to recover for 10 days. Tail-cuff plethysmography showed no significant difference in baseline blood pressure and minipumps were implanted to deliver a constant infusion of Angiotensin II (Ang II, 140ng/kg/min). Mice with a history of TCM exhibited higher blood pressures compared to controls (135.11±3.32 v 116.9±6.41mmhg, n=6/9, p&lt;0.05) by week 2 of Ang II infusion. These results together demonstrated that TCM is associated with a robust immune response and points to a possible causal contribution of immune activation to the cardiac dysfunction observed in TCM. Furthermore, a history of TCM promotes a hypertensive phenotype, but additional studies are needed to test whether this is through immunological mechanisms. AHAPOST837441 DJF, 19EIA34480023-MSM, and 1R01 HL161212-MSM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Predictive biomarkers and initial analysis of maternal immune alterations in postpartum preeclampsia reveal an immune-driven pathology.

Postpartum preeclampsia (PPPE) is an under-diagnosed condition, developing within 48 hours to 6 weeks following an uncomplicated pregnancy. The etiology of PPPE is still unknown, leaving patients vulnerable and making the identification and treatment of patients requiring postpartum care an unmet need. We aimed to understand the immune contribution to PPPE at the time of diagnosis, as well as uncover the predictive potential of perinatal biomarkers for the early postnatal identification of high-risk patients. Placentas were collected at delivery from uncomplicated pregnancies (CTL) and PPPE patients for immunohistochemistry analysis. In this initial study, blood samples in PPPE patients were collected at the time of PPPE diagnosis (48h-25 days postpartum; mean 7.4 days) and compared to CTL blood samples taken 24h after delivery. Single-cell transcriptomics, flow cytometry, intracellular cytokine staining, and the circulating levels of inflammatory mediators were evaluated in the blood. Placental CD163+ cells and 1st trimester blood pressures can be valuable non-invasive and predictive biomarkers of PPPE with strong clinical application prospects. Furthermore, changes in immune cell populations, as well as cytokine production by CD14+, CD4+, and CD8+ cells, suggested a dampened response with an exhausted phenotype including decreased IL1β, IL12, and IFNγ as well as elevated IL10. Understanding maternal immune changes at the time of diagnosis and prenatally within the placenta in our sizable cohort will serve as groundwork for pre-clinical and clinical research, as well as guiding clinical practice for example in the development of immune-targeted therapies, and early postnatal identification of patients who would benefit from more thorough follow-ups and risk education in the weeks following an uncomplicated pregnancy.

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

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

(9) - High-Dimensional Lung Tissue Imaging Reveals Temporal Changes in Immune Cell Populations and Cell Interactions During Progression of Chronic Lung Allograft Dysfunction (CLAD)

(9) - High-Dimensional Lung Tissue Imaging Reveals Temporal Changes in Immune Cell Populations and Cell Interactions During Progression of Chronic Lung Allograft Dysfunction (CLAD)

Abstract 3388: Targeting liver specific βII-Spectrin to prevent the development of metabolic dysfunction associated steatohepatitis (MASH) through CEACAM1 axis

Abstract Background: Obesity and pro-inflammatory alteration of the gut microbiome are risk factors for gastrointestinal (GI) cancers, including liver cancer (HCC). Dysregulated TGF-β signaling and loss of CEACAM1 activity are implicated in fatty liver disease. Clinically, 40% of human liver and GI cancers have genetic alterations in the TGF-β pathway members. CEACAM1 expression is significantly decreased in the liver of patients with Metabolic dysfunction-associated steatohepatitis (MASH). In contrast, in hepatocellular carcinoma (HCC), high CEACAM1 is associated with invasion, metastasis and poor prognosis. We hypothesized that TGF-β signaling and βII-Spectrin modulate MASH and HCC through CEACAM1. Methods: Wild type and the liver-specific βII-Spectrin (Sptbn1) knockout (LSKO) mice fed a high-fat diet (HFD) or Western diet (WD) and analyzed for Ceacam1 expression, changes in immune cell population, liver inflammation, and cancer. We used Alphafold and molecular modeling to identify the CEACAM1 and SPTBN1 interaction sites. Results: Single cell sequencing analysis of MASH mice liver revealed that Ceacam1 expression is significantly decreased in hepatocytes. Interestingly, we found that CEACAM1 levels are restored to normal in tissues from liver-specific βII-Spectrin knockout (LSKO) mice. Transcriptomic and multiplex imaging analysis suggest that proinflammatory TGF-β signaling is activated and infiltration of CD11b+ cells is increased in MASH liver. These phenotypes are blocked in LSKO mice. Furthermore, siRNA-knockdown of βII-Spectrin blocks diet-induced obesity, fibrosis, lipid accumulation, tissue damage, and cancer susceptibility. We have also found that CEACAM1 interacts with the Smad3 adaptor, βII-Spectrin. Furthermore, structural analysis of CECAM1 and βII-Spectrin revealed critical residues involved in the interaction. Conclusion: These data suggest that βII-Spectrin regulates CEACAM1 expression in the liver and is a potential target for therapeutic intervention to treat MASH and prevent progression to HCC. Citation Format: Krishanu Bhowmick, Kazufumi Ohshiro, Xiyan Xiang, Sahara John, Xiaochun Yang, Nicole Beauchemin, Mohammad I. Hassan, Taj Mohammad, Lopa Mishra. Targeting liver specific βII-Spectrin to prevent the development of metabolic dysfunction associated steatohepatitis (MASH) through CEACAM1 axis [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 3388.

Abstract 4305: Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice

Abstract Cancer is a complex disease that involves both malignant cells and the surrounding microenvironment. The tumor microenvironment (TME) is comprised of various components, including cancer-associated fibroblasts, immune cells, blood vessels, and extracellular matrix elements, all of which interact with cancer cells. It is increasingly recognized that the components of the TME are a critical determinant of therapeutic response and resistance, and as such targeting the TME has emerged as a promising strategy in cancer therapy. Syngeneic models are necessary to study the TME, and more specifically the tumor infiltrating immune cell populations, as these models allow us to monitor various aspects of the immune response. Another important aspect to consider is tumor heterogeneity, which can cause issues during analysis due to immune cell populations not being evenly distributed throughout the tumor. The decision of whether to analyze whole tumors or halved tumors when monitoring changes in immune cell populations is an important consideration. When considering multiple endpoints from a single tumor sample, halved tumors are oftentimes considered as we remain mindful of the 3Rs (replacement, reduction and refinement) in animal research. As such, it is important to understand the effects of splitting samples and how it can affect the data that is generated. To determine the effects of analyzing whole and halved tumor samples on immune cell subsets, studies were designed to subcutaneously implant either CT26.WT or 4T1-Luc2-1A4 cells into female BALB/cAnNHsd mice and harvest tumor samples after the completion of treatment. Mice were treated with isotype control (Clone MPC-11) or anti-mCTLA-4 (Clone 9D9). At the time of sample collection, tumors were kept whole intact or halved and analyzed via flow cytometry to quantify lymphoid and myeloid immune cell subsets within tumor samples. The data revealed differences in immune cell subset populations for both isotype vs. anti-CTLA-4 treated tumors and whole vs. halved tumors. Changes in the lymphoid cell compartment were observed for whole vs. halved tumors of ± ~25-100% and ± ~0-40% for isotype treated and of ± ~0-30% and ± ~25-2600% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively. Changes in the myeloid cell compartment of ± ~5-45% and ± ~10-65% for isotype treated and of ± ~15-100% and ± ~0-70% for anti-CTLA-4 treated CT26.WT or 4T1-Luc2-1A4 tumors, respectively, were also observed. Additionally, differences were observed between the two tumor halves, not just between whole and halved tumors. Taken together, the data support that splitting tumor samples can negatively impact the analysis of the immune cell populations. Tumors can be split if required to perform multiple analyses, however, this should be done with caution, as it can result in trend differences and increases variability within the dataset. Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Sheri Barnes, David Draper, Scott Wise. Analysis of tumor heterogeneity in syngeneic models; CT26.WT colon carcinoma and 4T1-Luc2-1A4 breast carcinoma in female BALB/cAnNHsd mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4305.

Abstract 5358: The influence of extracellular vesicles on immune cells in PDAC

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is bad. The tumor microenvironment consists of an abundance of immunosuppressive immune cells and anti-inflammatory cytokines that contribute to local and systemic immunosuppression in PDAC. Tumor cells can influence the tumor microenvironment and, with that, the present immune cells through the secretion of cytokines or chemokines or the secretion of extracellular vesicles (EVs). EVs are a media of communication among the tumor cells themselves, but also with other cells of the body, with the goal to promote tumor expansion and metastasis and decrease any anti-tumor-directed forces of the immune system. Our lab has shown that those EVs play a significant role in tumor growth and metastasis in PDAC. We have found MUC1 to be an important marker for tumor-derived EVs (tdEVs), associated with their activity and influence on cell proliferation and tumor growth in vitro and in vivo. Determining which immune cell populations are most targeted and impacted by tdEVs is essential to improving our understanding of the role of EVs in PDAC. Methods: TdEVs were isolated from murine pancreatic cancer cell lines through differential ultracentrifugation. EVs were then labeled with a membrane dye, which allows tracking of the EVs in the mouse. We designed an in vivo study to investigate the effects that tdEVs have on different immune cell populations, as well as tumor growth and metastasis. This study has two distinct phases: a 3-week “Pre-Education Phase”, with injection of either MUC1+ or MUC1KO EVs, is followed by orthotopic implantation of tumor cells in the pancreas. Data: We hypothesized that tdEVs impact effector immune cells in PDAC, leading to phenotypic, transcriptional, and functional changes that result in a decreased anti-tumor immune response. The highest uptake of EVs was found in macrophages and dendritic cells (DCs). Furthermore, most immune cells preferably take up MUC1+ as compared to MUC1KO EVs. After three weeks of EV education, RNA sequencing of the liver showed differential gene expression in a variety of genes related to processes associated with B cells. Treatment with MUC1+ EVs downregulated B cell-related processes in the liver, B cell differentiation, proliferation and activity are reduced. Several genes coding for immunoglobulin parts were affected as well. Immune cell profiling of the blood and spleen showed that MUC1+ EVs reduce NK and macrophage numbers in the spleen as compared to MUC1KO EV treatment after three weeks. Additionally, EV-treatment increased CD8+ T cell numbers in the blood but decreased CD8+ T cells in the spleen. Conclusion: We have shown that tdEVs induce phenotypic and transcriptional changes in immune cell populations and alter immune cell frequencies in vivo. Ongoing research in our lab involves analysis of phenotypic and transcriptional changes after tumor implantation as well as functional differences in EV-treated immune cells in vitro and in vivo. Citation Format: Clara S. Mundry, Ying Huang, Paul M. Grandgenett, Michael A. Hollingsworth. The influence of extracellular vesicles on immune cells in PDAC [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 5358.

Abstract 1351: Preclinical characterization of BMS-986288, a novel non-fucosylated (NF) anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) Probody® therapeutic

Abstract Introduction: Blockade of the CTLA-4 pathway with ipilimumab (IPI), alone and in combination with nivolumab (anti-programmed death-1 antibody), has shown clinical benefit in multiple tumor types. However, not all tumors respond to IPI ± nivolumab, and peripheral effects can lead to immune-related adverse events. To enhance the therapeutic index of CTLA-4-directed therapy, a proprietary Probody® therapeutics technology platform was used to develop a modified version of IPI. BMS-986288 is an anti-CTLA-4 antibody that combines a Probody® therapeutic masking peptide and protease-cleavable linker, designed to localize anti-CTLA-4 activity to the tumor microenvironment, with an NF Fc region hypothesized to enhance antigen-presenting cell (APC)-mediated T-cell priming and regulatory T cell (Treg) modulation via increased binding to the FcγRIIIA (CD16) receptor. Here, we present the characterization of the mechanism of action and pharmacodynamic (PD) response of BMS-986288 in preclinical models. Methods: APC-mediated T-cell priming and antigen-specific responses were evaluated in a superantigen model using peripheral blood mononuclear cells (PBMCs) and human CTLA-4 knock-in (KI) mice. Antitumor activity and immune cell population changes were assessed in an MC38 tumor model implanted in human CTLA-4 KI mice. Intratumoral Treg depletion was further investigated using patient-derived dissociated tumor samples as a physiologically relevant model. Peripheral PD effects and tolerability of BMS-986288 were evaluated in a non-human primate (NHP) Ad5 vaccine model. Results: Protease-cleaved BMS-986288 demonstrated enhanced APC-mediated T-cell priming compared with IPI in superantigen-stimulated PBMCs. Similarly, administration of superantigen staphylococcal enterotoxin B (SEB) peptide elicited an increased antigen-specific T-cell receptor (Vβ8+) T-cell response after treatment with cleaved BMS-986288 vs IPI in human CTLA-4 KI mice. In the MC38 tumor-bearing mouse model, BMS-986288 showed enhanced antitumor activity as compared to IPI, with tumor clearance in all mice. Although BMS-986288 enhanced intratumoral CTLA-4+ Treg depletion in the MC38 model, limited Treg depletion was observed in the patient-derived dissociated tumor model with an NF anti-CTLA-4 antibody without the masking peptide, potentially reflecting differences in CTLA-4 expression on human vs mouse Tregs. BMS-986288 reduced PD effects in the peripheral blood compartment and increased tolerability in NHP relative to IPI. Conclusion: BMS-986288 leverages unique characteristics to differentiate it from IPI, enhancing APC-mediated T-cell priming and anti-tumor activity while also reducing peripheral activity in preclinical models. These data support an ongoing phase 1/2 clinical trial of BMS-986288 (NCT03994601) in patients with advanced solid tumors. Citation Format: Amy Jhatakia, Mohammed Nasser, Anandaroop Mukhopadhyay, Kyeongah Kang, Courtni Newsome, Neha Gupta, Remie Gail Z. Mandawe, Felix Findeisen, Jack A. Lohre, Leslie Leung, Yun Wei, Joshua Dobroff, Karen Price, John Engelhardt, Mark Selby, Alan J. Korman, Nicholas Wilson. Preclinical characterization of BMS-986288, a novel non-fucosylated (NF) anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) Probody® therapeutic [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 1351.

Fecal microbiota transplantation stimulates type 2 and tolerogenic immune responses in a mouse model

ObjectivesClostridioides difficile infection (CDI) is the leading hospital-acquired infection in North America. While previous work on fecal microbiota transplantation (FMT), a highly effective treatment for CDI, has focused on colonization resistance mounted against C. difficile by FMT-delivered commensals, the effects of FMT on host gene expression are relatively unexplored. This study aims to identify transcriptional changes associated with FMT, particularly changes associated with protective immune responses. MethodsGene expression was assessed on day 2 and day 7 after FMT in mice after antibiotic-induced dysbiosis. Flow cytometry was also performed on colon and mesenteric lymph nodes at day 7 to investigate changes in immune cell populations. ResultsFMT administration after antibiotic-induced dysbiosis successfully restored microbial alpha diversity to levels of donor mice by day 7 post-FMT. Bulk RNA sequencing of cecal tissue at day 2 identified immune genes, including both pro-inflammatory and Type 2 immune pathways as upregulated after FMT. RNA sequencing was repeated on day 7 post-FMT, and expression of these immune genes was decreased along with upregulation of genes associated with restoration of intestinal homeostasis. Immunoprofiling on day 7 identified increased colonic CD45+ immune cells that exhibited dampened Type 1 and heightened regulatory and Type 2 responses. These include an increased abundance of eosinophils, alternatively activated macrophages, Th2, and T regulatory cell populations. ConclusionThese results highlight the impact of FMT on host gene expression, providing evidence that FMT restores intestinal homeostasis after antibiotic treatment and facilitates tolerogenic and Type 2 immune responses.

Single-cell RNA sequencing reveals the heterogeneity of hepatic non-parenchymal cell responses to chronic PFO5DoDA exposure in male mice

Perfluoroalkyl ether carboxylic acids (PFECA) have emerged as novel alternatives to legacy per- and polyfluoroalkyl substances (PFAS). Existing research has revealed hepatoxicity induced by various PFAS, including PFECA. However, these studies have primarily focused on overall changes in whole liver tissue, particularly in hepatocytes, with the impact of PFAS on diverse liver non-parenchymal cells (NPCs) still inadequately understood. In the present study, we examined the heterogeneous responses of hepatic NPCs following exposure to perfluoro-3,5,7,9,11-pentaoxadodecanoic acid (PFO5DoDA), a type of PFECA, by administering PFO5DoDA (5 μg/L)-contaminated water to male mice for one year. Single-cell RNA sequencing (scRNA-seq) of 15 008 cells from the liver identified 10 distinct NPC populations. Notably, although relative liver weight remained largely unchanged following exposure to 5 μg/L PFO5DoDA, there was an observed increase in proliferating cells, indicating that proliferating NPCs may contribute to the hepatomegaly frequently noted in PFAS-exposed livers. There was also a considerable alteration in the composition of hepatic NPCs. Specifically, the total number of B cells decreased substantially, while many other cells, such as monocytes and macrophages, increased after PFO5DoDA exposure. In addition, interactions among the hepatic NPC populations changed variously after PFO5DoDA exposure. The findings emphasize the heterogeneity in the responses of hepatic NPCs to PFO5DoDA exposure. Taken together, the changes in immune cell populations and their intercellular interactions suggest that PFO5DoDA disrupts immune homeostasis in the liver. These findings offer new insights into the cellular mechanisms of PFAS-induced liver damage.

The impact of age and sex on the inflammatory response during bone fracture healing.

Inflammation is thought to be dysregulated with age leading to impaired bone fracture healing. However, broad analyses of inflammatory processes during homeostatic bone aging and during repair are lacking. Here, we assessed changes in inflammatory cell and cytokine profiles in circulation and in bone tissue to identify age- and sex-dependent differences during homeostasis and repair. During homeostatic aging, male mice demonstrated accumulation of CD4+ helper T cells and CD8+ cytotoxic T cells within bone while both pro-inflammatory "M1" and anti-inflammatory "M2" macrophage numbers decreased. Female mice saw no age-associated changes in immune-cell population in homeostatic bone. Concentrations of IL-1β, IL-9, IFNγ, and CCL3/MIP-1α increased with age in both male and female mice, whereas concentrations of IL-2, TNFα, TNFR1, IL-4, and IL-10 increased only in female mice - thus we termed these "age-accumulated" cytokines. There were no notable changes in immune cell populations nor cytokines within circulation during aging. Sex-dependent analysis demonstrated slight changes in immune cell and cytokine levels within bone and circulation, which were lost upon fracture injury. Fracture in young male mice caused a sharp decrease in number of M1 macrophages; however, this was not seen in aged male mice nor in female mice of any age. Injury itself induced a decrease in the number of CD8+ T cells within the local tissue of aged male and of female mice but not of young mice. Cytokine analysis of fractured mice revealed that age-accumulated cytokines quickly dissipated after fracture injury, and did not re-accumulate in newly regenerated tissue. Conversely, CXCL1/KC-GRO, CXCL2/MIP-2, IL-6, and CCL2/MCP-1 acted as "fracture response" cytokines: increasing sharply after fracture, eventually returning to baseline. Collectively, we classify measured cytokines into three groups: (1) age-accumulated cytokines, (2) female-specific age-accumulated cytokines, and (3) fracture response cytokines. These inflammatory molecules represent potential points of intervention to improve fracture healing outcome.