Changes In Immune Cell Populations Research Articles

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<0.001 compared to vehicle (Veh)), females exhibited lower EF (46.64±3.11%) than males (68.36±2.62%, p<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<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<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<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.

Abstract A085: Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment

Abstract Breast cancer is the most common cancer diagnosis worldwide, and an unfortunately high number of women die every year due to a lack of effective therapeutic strategies for metastatic breast cancer. Patients with brain metastases in particular have a significantly poorer prognosis than women with metastasis to other sites (e.g., lung, liver, bone). Previous studies by our group identified a ligand-receptor complex in the tumor microenvironment (TME) that promotes metastasis of breast cancer to the brain. This interaction between platelet-derived growth factor-BB (PDGFB), produced by cancerous breast epithelial cells, and its receptor, platelet-derived growth factor receptor beta (PDGFRβ), expressed by mesenchymal cells, is also prognostic of breast cancer metastasis to the brain in patients. The mechanism of how the PDGFB-PDGFRβ pathway mediates brain metastasis is still unclear and is the focus of our current work. To further analyze the biological effects of PDGFB, our group evaluated gene expression changes between primary human breast tumors expressing high PDGFB versus those expressing low PDGFB and found significantly more immune signaling in the low-expressing tumors. These results led us to hypothesize that the PDGFB-PDGFRβ pathway creates an immunosuppressive microenvironment, allowing for increased metastasis of cancerous cells. To test this, flow cytometry was completed on the brains, spleens, lungs, and livers of control mice and mutant mice expressing stromal-specific hyperactive PDGFRβ following intracardiac injection of PDGFB-expressing mammary tumor cells. No significant changes in immune cell populations were observed in non-tumor-bearing mice. However, in the brains of tumor-bearing mutant mice, there are significant changes to the myeloid lineage. Specifically, analysis at day 10 post-injection (prior to detectable metastatic lesions) revealed diminished CD45+CD11b+ populations. Segregating by CD45hi/CD45lo confirmed a profound decrease in CD45loCD11b+F4/80+ microglia, likely a tumor-suppressive population. Looking at day 16 post-injection (detectable metastatic lesions), there is a shift towards an increase in CD45+ CD11b+GR1+ infiltrating myeloid-derived suppressor cells (MDSCs) in experimental mice, likely a tumor-promoting population. Minimal changes were observed in the lungs and livers at any time point. Confirmatory immunofluorescence staining showed a decrease in tumor-infiltrating microglia (Iba1+) and a significant increase in tumor-infiltrating peripheral macrophages (F4/80+), mirroring the flow data. This experiment was then repeated to test the contribution of the ligand (PDGFB), where a significant increase of infiltrating microglia (Iba1+) was observed upon tumor cell knockdown of PDGFB. Together, these results suggest that the PDGFB-PDGFRβ pathway creates an immunosuppressive environment in the brain TME at least in part through reorchestration of the myeloid populations. Citation Format: Alexis A Mossing, Johnathon G Schiebel, Rebecca L Packard, Amrendra Kumar, Sarah A Steck, Nathaniel S Grabinski, Katie A Thies, Bri Wasik, Tasneem Ariswala, Gary Tozbikian, Johnathan Godbout, Steven T Sizemore, Paul R Lockman, Anna E Vilgelm, Gina M Sizemore. Stromal PDGFRβ hyperactivation modulates the myeloid lineage in the brain metastatic microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A085.

Modakafusp Alfa Demonstrates Type I Interferon-Mediated Innate and Adaptive Immune Enhancement in a Phase 1/2 Study in Patients with Relapsed and/or Refractory Multiple Myeloma (RRMM)

Modakafusp alfa (moda), a novel immunocytokine, is an innate immunity enhancer comprising two attenuated interferon (IFN) α2b molecules fused to an anti-CD38 IgG4 monoclonal antibody backbone, driving preferential IFNα signalling in CD38-expressing innate and adaptive immune cells as well as myeloma cells. Here, we present pharmacodynamic (PD) data from the first-in-human Phase 1/2 study of moda as a monotherapy in patients with RRMM (iinnovate-1; NCT03215030). A total of 37 patients treated with moda at 1.5 or 3 mg/kg every 4 weeks were included in the analysis. Peripheral blood (PB) and bone marrow (BM) samples were collected during screening or pre-dose on cycle 1 day 1 (C1D1) and at multiple timepoints after treatment. BM and PB samples were evaluated by cytometry time of flight (CyTOF) and bulk RNA sequencing (RNAseq) to assess pharmacodynamic changes in immune cell populations, cell activation, and gene expression. Administration of moda led to enhanced innate immune cell activation and cytotoxic function as demonstrated by increased proportions of CD69+ and granzyme B+ in peripheral NK cells analyzed by CyTOF. This was accompanied by a decrease in the proportion of TIGIT+ NK cells, indicating reduced inhibitory signaling. Furthermore, enhanced proliferation (%Ki67+) of NK cells was observed in both BM and PB. Moda also impacted myeloid cell populations in BM and PB. Dendritic cells (DCs) and monocytes in PB showed increased surface expression of the co-stimulatory molecule CD86, suggesting a potential for enhanced antigen presentation and co-stimulation ability. Increased proliferation (%Ki67) of DCs and monocytes was detected in both PB and BM, accompanied by a decrease in numbers of peripheral DCs which may indicate recruitment or homing to secondary lymphoid organs. Additionally, RNAseq analysis revealed that treatment with moda led to upregulation of CD68 expression in both PB and BM, indicating a proinflammatory M1 macrophage type response. Finally, CyTOF analysis also indicated that moda significantly impacted adaptive immunity. We observed enhanced activation (%CD69, %PD1 and %CD40L) and cytotoxic function (%granzyme B) of peripheral CD8 T cells, and increased CD8 T cell proliferation (%Ki67 CD8) in both PB and BM samples. Enhanced activation of peripheral CD4 T cells (%CD69, %CD40L) was also detected. Consistent with activation of the adaptive immune system, there was a reduction in naïve CD8 T cells accompanied by an increase in CD8 T effector memory cells in PB. Analysis of exhaustion markers (TIGIT, PD-1) in cycle 1 (C1D8, C1D15, and C2D1 predose) showed that moda did not induce CD8 T cell exhaustion in either PB or BM, and there was no change in FoxP3+ Treg cells. Moda-induced innate and adaptive immune cell phenotypic changes were evaluated for correlation with clinical response at timepoints with sufficient number of evaluable samples. While none of the associations were statistically significant after correcting for multiplicity testing (using the false discovery rate method), we observed trends warranting further investigation. There was a greater increase of CD40L+ NK cells and PD1+ CD8 cells in responders vs. non-responders at C1D2. Additionally, at C1D15 responders showed a lower increase in proliferation (%Ki67+) of classical monocytes, NK cells, and myeloid DCs in PBas compared to non-responders. A similar trend was also observed in the BM classical monocytes. Even though the peak of Ki67 expression was observed at C1D8, diminished increases of proliferating cells at C1D15 in responders was a surprising observation that needs further consideration. The PD biomarker data from this first-in-human clinical trial demonstrated that treatment with moda enhances both innate and adaptive immune cell activation in PB and BM. Importantly, moda-mediated immune activation did not result in T cell exhaustion during cycle 1, as evaluated in both PB and BM. We have observed interesting trends of immune phenotypic changes that may correlate with response. Follow-up analyses of the correlation of biomarkers with clinical response are in progress and will be presented. Further clinical trials are underway (iinnovate-2, NCT05556616; iinnovate-3, NCT05590377) to evaluate moda's novel immune activating mechanism in combination with standard of care anti-myeloma therapies.

Sex differences in microglia function in aged rats underlie vulnerability to cognitive decline

Aging is associated with a significant shift in immune system reactivity (“inflammaging”), as basal inflammation increases but protective responses to infection are compromised. The immune system exhibits considerable sex differences, which may influence the process of inflammaging, including immune cell activation and behavioral consequences of immune signaling (i.e., impaired memory). Here, we test the hypothesis that sex differences in immune aging may mediate sex differences in cognitive decline. Aged male and female rats received peripheral immune stimulation using lipopolysaccharide (LPS), then molecular, cellular, and behavioral outcomes were assessed. We observed that LPS-treated aged male rats showed cognitive impairment and increased neuroinflammatory responses relative to adult males. In contrast, aged female rats did not display these aging-related deficits. Using transcriptomic and flow cytometry analyses, we further observed significant age- and sex- dependent changes in immune cell populations in the brain parenchyma and meninges, indicating a broad shift in the neuroinflammatory environment that may potentiate these behavioral effects. Ovariectomized aged female rats were also resistant to inflammation-induced memory deficits, indicating that ovarian hormones are not required for the attenuated neuroinflammation in aged females. Overall, our results indicate that males have amplified inflammatory priming with age, which contributes to age-associated cognitive decline. Our findings highlight sexual dimorphism in mechanisms of aging, and suggest that sex is a crucial consideration for identifying therapies for aging and neuroinflammation.

A single targeted gamma-ray irradiation induced an acute modulation of immune cells and related cytokines in EMT6 mouse-bearing tumour model.

A complicated interplay between radiation doses, tumour microenvironment (TME), and host immune system is linked to the active participation of immune response. The effects of single targeted 2 Gy and 8 Gy gamma-ray irradiations on the immune cell population (lymphocytes, B-cells, T-cells, neutrophils, eosinophils, and macrophages) in EMT6 mouse-bearing tumour models was investigated. The effects of both irradiation doses in early (96 hours) and acute phase (5 to 11 days) post-irradiation on immune parameters were monitored in blood circulation and TME using flow cytometry. Simultaneously, selected cytokines related to immune cells within the TME were measured using multiplex ELISA. A temporary reduction in systemic total white blood count (TWBC) resulted from an early phase (96 hours) of gamma-ray irradiation at 2 Gy and 8 Gy compared to sham control group. No difference was obtained in the acute phase. Neutrophils dominated among other immune cells in TME in sham control group. Eosinophils in TME was significantly increased after 8 Gy treatment in acute phase compared to sham control (p< 0.005). Furthermore, the increment of tumour necrosis (TNF)-α, eotaxin and interleukin (IL)-7 (p< 0.05) in both treatment groups and phases were associated with anti-tumour activities within TME by gamma-ray irradiation. The temporary changes in immune cell populations within systemic circulation and TME induced by different doses of gamma-ray irradiation correlated with suppression of several pro-tumorigenic cytokines in mouse-bearing EMT6 tumour models.

Clinical Efficacy of Bushen Yiqi Fuzheng Decoction Combined with Sunitinib in the Treatment of Postoperative Patients with Renal Cell Carcinoma and Its Influence on Their Immune Function.

This study aimed to investigate the effect of Bushen Yiqi Fuzheng decoction combined with sunitinib on the prognosis, clinical efficacy and immune function of patients with renal cell carcinoma (RCC) after surgery. A total of 120 patients who experienced RCC after surgery were randomly divided into the observation and control groups in this prospective study, with 60 cases in each group. The therapeutic effect, improvement of clinical symptoms, changes of immune function-related indicators and adverse reactions during medication were recorded. The changes in immune cell population, midkine (MK), interleukin 35 (IL-35), hypoxia-inducible factor 2alpha (HIF-2α), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), carcinoembryonic antigen (CEA), osteopontin (OPN), ferritin (FERR) and beta2-microglobulin (β2-MG) levels were measured. The Karnofsky performance status (KPS) score of patients was recorded. The total effective rate of the observation group (95%) was better than that of the control group (85%, p < 0.05). After treatment, the changes of immune function indexes in the control group were not obvious. The indexes related to immune function in the observation group significantly decreased. Significant differences were observed in the cluster of differentiation 3+ (CD3+), cluster of differentiation 4+ (CD4+), cluster of differentiation 8+ (CD8+) and CD4+/CD8+ between the two groups after treatment. The incidence of adverse reactions in the observation group was lower than that of the control group. The KPS of the observation group was higher than that of the control group. Before treatment, no differences were observed in the MK, IL-35, HIF-2α, CEA, OPN, FERR, β2-MG, MMP-9 and TIMP-1 levels between the two groups. After treatment, the levels of the above parameters were lower than those before treatment, especially in the observation group. Bushen Yiqi Fuzheng decoction combined with sunitinib can significantly improve the clinical efficacy and postoperative immune function of RCC patients after surgery and down-regulate MMP-9 and TIMP-1 levels in the serum, which is beneficial to the prognosis of patients.

Intraoperative cell salvage: The impact on immune cell numbers.

Patient outcomes are influenced by many confounding factors peri-operatively, including the type of surgery, anaesthesia, transfusion, and immune competence. We have previously demonstrated (in-vitro) that compared to allogeneic blood transfusion (ABT), intraoperative cell salvage (ICS) improves immune competence. The peri-operative immune response is complex. Altered or impaired immune responses may predispose patients to develop adverse outcomes (i.e., post-operative wound infection, pneumonia, urinary tract infection etc.) Surgical patients may develop infection, even without the confirmed presence of a definite microbiological pathogen. With all these factors in mind it is important to consider changes in immune cell numbers (and sub-populations) and functional capacity during peri-operative transfusion. In this TRIMICS-Cell (Transfusion Related Immune Modulation and Intraoperative Cell Salvage-Cell numbers) study (n = 17, October 2018-November 2019) we prioritized and analysed peri-operative changes in the number and proportions of immune cell populations and sub-populations (B cells (CD20+), NK (natural killer) cells (CD56+), monocytes (CD14+), T cells (total CD3+ and sub-populations: T helper cells (CD4+), cytotoxic T cells (CD8+), effector T cells (CD4+ CD127+), activated effector T cells (CD4+ CD25+ CD127+) and regulatory T cells (CD4+ CD25+ CD127-)), plasmacytoid dendritic cells (pDC; Lineage-, HLA-DR+, CD11c-, CD123+), classical dendritic cell (cDC) (Lineage-, HLA-DR+, CD11c+), and cDC activation (Lineage-, HLA-DR+, CD11c+), co-stimulatory/adhesion molecules and pDC (CD9+, CD38+, CD80+, CD83+, CD86+, CD123+). Firstly we analysed the whole cohort of study patients and secondly according to the relevant transfusion modality (i.e., three study groups: those who received no transfusion, received ICS only (ICS), or both ICS and allogeneic packed red blood cells (pRBC) (ICS&RBC)), during major orthopaedic surgery. For the whole study cohort (all patients), changes in immune cell populations were significant: leucocytes and specifically neutrophils increased post-operatively, returning towards pre-operative numbers by 48h post-operatively (48h), and lymphocytes reduced post-operatively returning to pre-operative numbers by 48h. When considering transfusion modalities, there were no significant peri-operative changes in the no transfusion group for all immune cell populations studied (cell numbers and proportions (%)). Significant changes in cell population numbers (i.e., leucocytes, neutrophils and lymphocytes) were identified in both transfused groups (ICS and ICS&RBC). Considering all patients, changes in immune cell sub-populations (NK cells, monocytes, B cells, T cells and DCs) and functional characteristics (e.g., co-stimulation markers, adhesion, activation, and regulation) were significant peri-operatively and when considering transfusion modalities. Interestingly DC numbers and functional capacity were specifically altered following ICS compared to ICS&RBC and pDCs were relatively preserved post-operatively following ICS. A transient peri-operative alteration with recovery towards pre-operative numbers by 48h post-surgery was demonstrated for many immune cell populations and sub-populations throughout. Immune cell sub-populations and functional characteristics were similar peri-operatively in those who received no transfusion but changed significantly following ICS and ICS&RBC. Interesting changes that require future study are a post-operative monocyte increase in the ICS&RBC group, changes in cDC considering transfusion modalities, and possibly preserved pDC numbers post-operatively following ICS. Future studies to assess changes in immune cell sub-populations, especially during peri-operative transfusion, while considering post-operative adverse outcomes, is recommended.

Fibrocytes enhance mammary gland fibrosis in obesity.

Obesity rates continue to rise, and obese individuals are at higher risk for multiple types of cancer, including breast cancer. Obese mammary fat is a site of chronic, macrophage-driven inflammation, which enhances fibrosis within adipose tissue. Elevated fibrosis within the mammary gland may contribute to risk for obesity-associated breast cancer. To understand how inflammation due to obesity enhanced fibrosis within mammary tissue, we utilized a high-fat diet model of obesity and elimination of CCR2 signaling in mice to identify changes in immune cell populations and their impact on fibrosis. We observed that obesity increased a population of CD11b+ cells with the ability to form myofibroblast-like colonies in vitro. This population of CD11b+ cells is consistent with fibrocytes, which have been identified in wound healing and chronic inflammatory diseases but have not been examined in obesity. In CCR2-null mice, which have limited ability to recruit myeloid lineage cells into obese adipose tissue, we observed reduced mammary fibrosis and diminished fibrocyte colony formation in vitro. Transplantation of myeloid progenitor cells, which are the cells of origin for fibrocytes, into the mammary glands of obese CCR2-null mice resulted in significantly increased myofibroblast formation. Gene expression analyses of the myeloid progenitor cell population from obese mice demonstrated enrichment for genes associated with collagen biosynthesis and extracellular matrix remodeling. Together these results show that obesity enhances recruitment of fibrocytes to promote obesity-induced fibrosis in the mammary gland.

Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors

BackgroundImmunologically cold tumors with an ‘immune desert’ phenotype lack tumor-infiltrating lymphocytes (TILs) and are typically impervious to systemic immune checkpoint blockade (ICB). Intratumoral treatment of tumors with immunomodulatory agents can...