High-dimensional Flow Cytometry Research Articles

EXTH-12. POTENTIATING THE EFFICACY OF IMMUNE CHECK-POINT INHIBITORS IN GLIOBLASTOMA BY INHIBITION OF CXCL12

Abstract BACKGROUND The tumor immune microenvironment (TIME) in glioblastoma (GBM) is rich in CXCL12, a chemokine known for stimulating angiogenesis. CXCL12 also controls immune cell trafficking and promotes polarization to an immunosuppressive phenotype. We postulated that inhibiting CXCL12 will modulate the immunosuppressive TIME in GBM, thereby augmenting the efficacy of immunotherapy agents like immune checkpoint inhibitors (ICIs). We examined the immunomodulatory and therapeutic efficacy of CXCL12 inhibition, using a small molecule NOX-A12, with and without ICIs in pre-clinical murine GBM models. METHODS B6 immunocompetent mice bearing intracranial (i.c.) or subcutaneous (s.c.) SB28 tumors received either vehicle, NOX-A12, anti-PD1 and anti-CTLA4 (dual ICI) or NOX-A12 + dual ICI (combination). Tumor growth was measured by IVIS imaging and immune cells in brain were analyzed using high dimensional flow cytometry. Survival was analyzed using Kaplan-Meier curves and log rank test. RESULTS Combination treatment resulted in tumor regression and long-term survival in 40% of s.c. tumor bearing mice compared to 10% treated with dual ICI only. Three of 4 mice rechallenged with contralateral s.c. tumor remained tumor free while all naive mice reached endpoint. TIME from treated s.c. tumors revealed increase in CD4 and effector memory CD8 T cells by combination treatment compared to dual ICI. In i.c. GBM tumors, while no survival benefit or growth inhibition was seen, combination treatment induced an early increase in effector memory CD8 T cells and PD-L1+ B cells, and a later increase in MHC-II+ microglia compared to dual ICI. CONCLUSION Inhibition of CXCL12 enabled the increase of effector cytotoxic T cells by ICI, improving survival in the s.c. GBM model, but not in i.c. GBM model. This might be due to differences in CXCL12 effect between extra and intra-CNS tumor or due to robust immune response causing excessive brain edema and death. These will be further explored in ongoing and future studies.

IMMU-39. CAR T CELLS TARGETING ICAM-1 SHOW A SURVIVAL BENEFIT IN BOTH SYNGENEIC AND XENOGRAFT GLIOMA MOUSE MODELS

Abstract BACKGROUND Chimeric antigen receptor (CAR) T-cell therapy, while effective against hematological malignancies, has faced significant hurdles in its application to solid tumors. Challenges in CAR T-cell therapy for glioblastoma include the immunosuppressive tumor microenvironment, the limited infiltration of CAR T-cells into the tumor, heterogeneous target expression and antigen escape. In this study, we focused on targeting Intracellular adhesion molecule-1 (ICAM-1), a protein expressed on both glioma cells and tumor-associated cells like macrophages and endothelial cells. METHODS Glioblastoma and normal brain tissue microarray sections (TMA) were immunohistochemically analyzed for ICAM-1 expression. Second-generation human and murine ICAM-1-targeting CAR T cells were generated by lentiviral or retroviral transduction of T cells from healthy human donors or murine splenocytes. Their efficacy was evaluated in co- culture assays with human and murine glioma and endothelial cell lines. Syngeneic and xenograft orthotopic glioma mouse models were used to assess the in vivo activity of CAR T cells. Ex vivo analysis of these tumors included examination of changes in the tumor microenvironment using high-dimensional flow cytometry. RESULTS Immunohistochemical staining of TMA revealed a significant upregulation of ICAM-1 in human glioblastoma tissue compared to normal brain tissue. Single-cell RNA sequencing data from glioblastoma specimens showed a high level of ICAM-1 expression in tumor- associated macrophages. Human and murine ICAM-1-targeting CAR T cells displayed strong lysis of ICAM-1-expressing glioma and endothelial cells in vitro. Intratumoral application of CAR T cells resulted in a survival benefit in both syngeneic and xenograft glioma mouse models. Ex vivo analysis of the tumor microenvironment revealed treatment-induced changes from our CAR T cell therapy and indicates potential for further enhancements. CONCLUSION Our study demonstrates that ICAM-1-targeting CAR T cells improve survival in human and murine glioma mouse models. Flow cytometry of the tumor microenvironment reveals therapy-induced changes, for future improvements of the CAR T cell therapy.

Systemic inflammation accelerates neurodegeneration in a rat model of Parkinson's disease overexpressing human alpha synuclein.

Increasing efforts have been made to elucidate how genetic and environmental factors interact in Parkinson's disease (PD). In the present study, we assessed the development of symptoms on a genetic PD rat model that overexpresses human α-synuclein (Snca+/+) at a presymptomatic age, exposed to a pro-inflammatory insult by intraperitoneal injection of lipopolysaccharide (LPS), using immunohistology, high-dimensional flow cytometry, constant potential amperometry, and behavioral analyses. A single injection of LPS into WT and Snca+/+ rats triggered long-lasting increase in the activation of pro-inflammatory microglial markers, monocytes, and T lymphocytes. However, only LPS Snca+/+ rats showed dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc), associated with a reduction in the release of evoked dopamine in the striatum. No significant changes were observed in the behavioral domain. We propose our double-hit animal as a reliable model to investigate the mechanisms whereby α-synuclein and inflammation interact to promote neurodegeneration in PD.

Potential and challenges of clinical high-dimensional flow cytometry: A call to action.

Clinical biomarker strategies increasingly integrate translational research to gain new insights into disease mechanisms or to define better biomarkers in clinical trials. High-dimensional flow cytometry (HDFCM) holds the promise to enhance the exploratory potential beyond traditional, targeted biomarker strategies. However, the increased complexity of HDFCM poses several challenges, which need to be addressed in order to fully leverage its potential and to align with current regulatory requirements in clinical flow cytometry. These challenges include among others extended timelines for assay development and validation, the necessity for extensive knowledge in HDFCM, and sophisticated data analysis strategies. However, no guidelines exist on how to manage such challenges in adopting clinical HDFCM. Our CYTO 2024 workshop "Potential and challenges of clinical high-dimensional flow cytometry" aimed to find consensus across the pharmaceutical industry and broader scientific community on the overall benefits and most urgent challenges of HDFCM in clinical trials. Here, we summarize the insights we gained from our workshop. While this report does not provide a blueprint, it is a first step in defining and summarizing the most pressing challenges in implementing HDFCM in clinical trials. Furthermore, we compile current efforts with the goal to overcome some of these challenges. As such we bring the scientific community and health authorities together to build solutions, which will accelerate and simplify the full adoption of HDFCM in clinical trials.

High-dimensional Immune Profiles and Machine Learning May Predict Acute Myeloid Leukemia Relapse Early following Transplant.

Identification of early immune signatures associated with acute myeloid leukemia (AML) relapse following hematopoietic stem cell transplant (HSCT) is critical for patient outcomes. We analyzed PBMCs from 58 patients with AML undergoing HSCT, focusing on T cell subsets and functional profiles. High-dimensional flow cytometry coupled with Uniform Manifold Approximation and Projection dimensionality reduction and PhenoGraph clustering revealed distinct changes in CD4+ and CD8+ T cell populations in 16 patients who relapsed within 1 y of HSCT. We observed increased IL-2, IL-10, and IL-17-producing CD4+ T cells, alongside decreased CD8+ T cell function early in relapsing patients. Notably, relapsing patients exhibited increased TCF-1intermediate cells, which lacked granzyme B or IFN-γ production in the CD4+ T cell compartment. We then developed a supervised machine learning algorithm that predicted AML relapse with 90% accuracy within 30 d after HSCT using high-throughput assays. The algorithm leverages condensed immune phenotypic data, alongside the ADASYN algorithm, for data balancing and 100 rounds of XGBoost supervised learning. This approach holds potential for detecting relapse-associated immune signatures months before clinical manifestation. Our findings demonstrate a distinct immunological signature potentially capable of predicting AML relapse as early as 30 d after HSCT.

Methotrexate treatment hampers induction of vaccine-specific CD4 T cell responses in patients with IMID

ObjectivesMethotrexate (MTX) is one of the most commonly used medications to treat rheumatoid arthritis (RA). However, the effect of MTX treatment on cellular immune responses remains incompletely understood. This raises...

Increased peritoneal TGF-β1 is associated with ascites-induced NK-cell dysfunction and reduced survival in high-grade epithelial ovarian cancer.

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients.

Potential immunomodulatory effects of CAS+IMD monoclonal antibody cocktail in hospitalized patients with COVID-19

Passive administration of SARS-CoV-2 neutralizing monoclonal antibodies (mAbs), such as CAS+IMD (Casirivimab+Imdevimab) antibody cocktail demonstrated beneficial effects on clinical outcomes in hospitalized patients with COVID-19 who were seronegative at baseline and outpatients. However, little is known about their impact on the host immunophenotypes. We conducted an immunoprofiling study in 46 patients from a single site of a multi-site trial of CAS+IMD in hospitalized patients. We collected longitudinal samples during October 2020∼April 2021, prior to the emergence of the Delta and Omicron variants and the use of COVID-19 vaccines. All collected samples were analyzed without exclusion and post-hoc statistical analysis was performed. We examined the dynamic interplay of CAS+IMD with host immunity applying dimensional reduction approach on plasma proteomics and high dimensional flow cytometry data. Using an unbiased clustering method, we identified unique immunophenotypes associated with acute inflammation and disease resolution. Compared to placebo group, administration of CAS+IMD accelerated the transition from an acute inflammatory immunophenotype, to a less inflammatory or "resolving" immunophenotype, as characterized by reduced tissue injury, proinflammatory markers and restored lymphocyte/monocyte imbalance independent of baseline serostatus. Moreover, CAS+IMD did not impair the magnitude or the quality of host T cell immunity against SARS-CoV-2 spike protein. Our results identified immunophenotypic changes indicative of a possible SARS-CoV-2 neutralizing antibodies-induced anti-inflammatory effect, without an evident impairment of cellular antiviral immunity, suggesting that further studies of Mabs effects on SAS-CoV-2 or other viral mediated inflammation are warranted. Regeneron Pharmaceuticals Inc and federal funds from the Department of Health and Human Services; Administration for Strategic Preparedness and Response; Biomedical Advanced Research and Development Authority, under OT number: HHSO100201700020C.

Regional Analysis of the Immune Microenvironment in Human Endometrium.

Endometrial immune cells are essential for maintaining homeostasis and the endometrial receptivity to embryo implantation. Understanding regional variations in endometrial immune cell populations is crucial for comprehending normal endometrial function and the pathophysiology of endometrial disorders. Despite previous studies focusing on the overall immune cell composition and function in the endometrium, regional variations in premenopausal women remain unclear. Endometrial biopsies were obtained from four regions (anterior, posterior, left lateral, and right lateral) of premenopausal women undergoing hysteroscopy with no abnormalities. A 15-color human endometrial immune cell-focused flow cytometry panel was used for analysis. High-dimensional flow cytometry combined with a clustering algorithm was employed to unravel the complexity of endometrial immune cells. Additionally, multiplex immunofluorescent was performed for further validation. Our findings revealed no significant variation in the distribution and abundance of immune cells across different regions under normal conditions during the proliferative phase. Each region harbored similar immune cell subtypes, indicating a consistent immune microenvironment. However, when comparing normal regions to areas with focal hemorrhage, significant differences were observed. An increase in CD8+ T cells highlights the impact of localized abnormalities on the immune microenvironment. Our study demonstrates that the endometrial immune cell landscape is consistent across different anatomical regions during the proliferative phase in premenopausal women. This finding has important implications for understanding normal endometrial function and the pathophysiology of endometrial disorders.

Immunophenotyping of Peripheral Blood Cells in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib.

Chronic lymphocytic leukemia (CLL) is a B-cell-derived hematologic malignancy whose progression depends on active B-cell receptor (BCR) signaling. Despite the spectacular efficacy of Ibrutinib, an irreversible inhibitor of Bruton tyrosine kinase (BTK), resistance can develop in CLL patients, and alternative therapeutic strategies are therefore required. Cancer immunotherapy has revolutionized cancer care and may be an attractive approach in this context. We speculated that characterizing the immune responses of CLL patients may highlight putative immunotherapeutic targets. Here, we used high-dimensional spectral flow cytometry to compare the distribution and phenotype of non-B-cell immune populations in the circulating blood of CLL patients treated with Ibrutinib displaying a complete response or secondary progression. Although no drastic changes were observed in the composition of their immune subsets, the Ibrutinib-resistant group showed increased cycling of CD8+ T cells, leading to their overabundance at the expense of dendritic cells. In addition, the expression of 11 different surface checkpoints was similar regardless of response status. Together, this suggests that CLL relapse upon Ibrutinib treatment may not lead to major alterations in the peripheral immune response.

B cells critical for outcome in high grade serous ovarian carcinoma.

Recent work has shown evidence for the prognostic significance of tumor infiltrating B cells (B-TIL) in high grade serous ovarian carcinoma (HGSOC), the predominant histological subtype of ovarian cancer. However, it remains unknown how the favorable prognosis associated with B-TIL relates to the current standard treatments of primary debulking surgery (PDS) followed by chemotherapy or (neo-)adjuvant chemotherapy (NACT) combined with interval debulking surgery. To address this, we analyzed the prognostic impact of B-TIL in relationship to primary treatment and tumor infiltrating T cell status in a highly homogenous cohort of HGSOC patients. This analysis involved a combined approach utilizing histological data and high-dimensional flow cytometry analysis. Our findings indicate that while HGSOC tumors pre-treated with NACT are infiltrated with tumor-reactive CD8+ and CD4+ TIL subsets, only B-TIL and IgA plasma blasts confer prognostic benefit in terms of overall survival. Importantly, the prognostic value of B-TIL and IgA plasma blasts was not restricted to patients treated with NACT, but was also evident in patients treated with PDS. Together, our data point to a critical prognostic role for B-TIL in HGSOC patients independent of T cell status, suggesting that alternative treatment approaches focused on the activation of B cells should be explored for HGSOC.

CytoKernel: Robust kernel embeddings for assessing differential expression of single cell data.

High-throughput sequencing of single-cell data can be used to rigorously evlauate cell specification and enable intricate variations between groups or conditions. Many popular existing methods for differential expression target differences in aggregate measurements (mean, median, sum) and limit their approaches to detect only global differential changes. We present a robust method for differential expression of single-cell data using a kernel-based score test, cytoKernel. cytoKernel is specifically designed to assess the differential expression of single cell RNA sequencing and high-dimensional flow or mass cytometry data using the full probability distribution pattern. cytoKernel is based on kernel embeddings which employs the probability distributions of the single cell data, by calculating the pairwise divergence/distance between distributions of subjects. It can detect both patterns involving aggregate changes, as well as more elusive variations that are often overlooked due to the multimodal characteristics of single cell data. We performed extensive benchmarks across both simulated and real data sets from mass cytometry data and single-cell RNA sequencing. The cytoKernel procedure effectively controls the False Discovery Rate (FDR) and shows favourable performance compared to existing methods. The method is able to identify more differential patterns than existing approaches. We apply cytoKernel to assess gene expression and protein marker expression differences from cell subpopulations in various publicly available single-cell RNAseq and mass cytometry data sets. The methods described in this paper are implemented in the open-source R package cytoKernel, which is freely available from Bioconductor at http://bioconductor.org/packages/cytoKernel.

Predictive and pharmacodynamic biomarkers for combination therapy in stage III-IV melanoma: A Simon phase II trial (NCT03999749).

222 Background: The evaluation of biomarkers that predict response and immune-related adverse events (irAEs) is crucial for optimizing melanoma treatment with Immune checkpoint inhibitors (ICIs). We report predictive and pharmacodynamic biomarkers for response and irAEs using high-dimensional flow cytometry in a phase II trial combining nivolumab (3 mg/kg), ipilimumab (1 mg/ml), and the IL-6 receptor inhibitor tocilizumab (4 mg/kg) in unresectable/advanced melanoma patients. Methods: We analyzed serum and blood from melanoma patients at baseline, weeks 7 and 37, categorized them by irAE severity and clinical response. Patients were categorized based on the severity of irAEs (grades 3-5) (TOX), the absence of toxicity or grades 1-2 irAE (NT), clinical benefit (CB), and non-clinical benefit (NCB). Immune monitoring regimen included ELISA for IL-6, gp130, IL-23, osteopontin, and Luminex oncological panel (LXSAHM-22). We employed multi-parametric flow cytometry panels to examine myeloid cells, cytotoxic T cells, and checkpoint protein expression on immune cells. We processed ~1 million PBMCs per sample using various antibodies, with data acquisition performed via Sony’s ID7000 cytometer. The data underwent quality control, normalization PBMCs per patient, concatenation, dimensionality reduction, and clustering in FlowJo. Results: Among the 70 patients administered treatment, we observed a best overall response rate of 57%. Grade 3-5 irAEs occurred in 22% of patients by week 24. Patients manifesting irAEs exhibited notably higher baseline Th17 cell counts (NT=181±18 vs. Tox=311±49, p<0.01) and CD26+ Naive CD4 T-cell populations (NT=5838±801 vs. Tox=9632±1343, p<0.04), suggesting their potential role as predictive markers for toxicity. Additionally, in vitro investigations led us to identify CD26 co-stimulation as a preliminary step in TH17 cell differentiation. Key pharmacodynamic markers included a significant elevation in Treg cells (NCB=1286±71 vs. CB=1492±95, p=0.02) and reduction in serum gp130 levels (NCB=104±5 vs. CB=83±5 ng/ml, p<0.01) at week 7 in addition to IL-6 (NCB=7.5±1.5 vs. CB=4.6±1.1 pg/ml, p<0.01) and osteopontin (NCB=95±15 vs. CB=25±3 ng/ml, p<0.01) by week 37 when compared to baseline. Conclusions: Our findings suggest that Th17 cells can serve as a toxicity biomarker for combination therapy with ipilimumab, nivolumab and tocilizumab. Circulating Treg cells and serum osteopontin have emerged as promising pharmacodynamic biomarkers. Clinical trial information: NCT03999749 .

Timing and location dictate monocyte fate and their transition to tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells whose phenotypes and functions are shaped by factors that are incompletely understood. Herein, we asked when and where TAMs arise from blood monocytes and how they evolve during tumor development. We initiated pancreatic ductal adenocarcinoma (PDAC) in inducible monocyte fate-mapping mice and combined single-cell transcriptomics and high-dimensional flow cytometry to profile the monocyte-to-TAM transition. We revealed that monocytes differentiate first into a transient intermediate population of TAMs that generates two longer-lived lineages of terminally differentiated TAMs with distinct gene expression profiles, phenotypes, and intratumoral localization. Transcriptome datasets and tumor samples from patients with PDAC evidenced parallel TAM populations in humans and their prognostic associations. These insights will support the design of new therapeutic strategies targeting TAMs in PDAC.

Rapid assay development for low input targeted proteomics using a versatile linear ion trap.

Advances in proteomics and mass spectrometry enable the study of limited cell populations, where high-mass accuracy instruments are typically required. While triple quadrupoles offer fast and sensitive low-mass accuracy measurements, these instruments are effectively restricted to targeted proteomics. Linear ion traps (LITs) offer a versatile, cost-effective alternative capable of both targeted and global proteomics. Here, we describe a workflow using a new hybrid quadrupole-LIT instrument that rapidly develops targeted proteomics assays from global data-independent acquisition (DIA) measurements without needing high-mass accuracy. Using an automated software approach for scheduling parallel reaction monitoring assays (PRM), we show consistent quantification across three orders of magnitude in a matched-matrix background. We demonstrate measuring low-level proteins such as transcription factors and cytokines with quantitative linearity below two orders of magnitude in a 1 ng background proteome without requiring stable isotope-labeled standards. From a 1 ng sample, we found clear consistency between proteins in subsets of CD4 + and CD8 + T cells measured using high dimensional flow cytometry and LIT-based proteomics. Based on these results, we believe hybrid quadrupole-LIT instruments represent an economical solution to democratizing mass spectrometry in a wide variety of laboratory settings.

Proteomic and phenotypic characteristics of memory-like natural killer cells for cancer immunotherapy

BackgroundHuman and mouse natural killer (NK) cells have been shown to develop memory-like function after short-term exposure to the cocktail of IL-12/15/18 or to overnight co-culture with some tumor cell...

The effect of early life cytomegalovirus infection on the immune profile of children

Cytomegalovirus (CMV) infection has a life-long impact on the immune system, particularly on memory T cells. However, the effect of early life CMV infection on the phenotype and functionality of T cells in infants and especially longitudinal changes occurring during childhood have not been explored in detail.The phenotype and functionality of peripheral blood CD8+ and CD4+ T cells from children infected with CMV in early life (< 6 months of age) was analyzed using high-dimensional flow cytometry. Samples from CMV IgG-seropositive (CMV+) children were collected at 6 months and 6 years of age and compared to samples from CMV-seronegative (CMV-) children.Early life CMV infection caused multiple alterations within T cells. These include downregulation of CD28 expression and upregulation of CD57 expression within both CD27+ early and CD27- late effector memory CD8+ and CD4+ T-cells at 6 months of age. Of these changes, only alterations within the highly differentiated late effector memory compartment persisted at the age of 6 years.Early life CMV-infection has a distinct impact on developing CD8+ and CD4+ memory T cell compartments. It appears to induce both temporary as well as longer-lasting alterations, which may affect the functionality of the immune system throughout life.

IMMU-06. CAR DESIGN SHAPES THE BRAIN TUMOR IMMUNE MICROENVIRONMENT: IMPACT ON ACTIVITY AND PERSISTENCE OF ANTI-GLIOMA ADOPTIVE T CELL IMMUNOTHERAPY

Abstract BACKGROUND B7-H3 CAR T-cells effectively eradicate brain tumors in xenograft models and have proven safe in clinical trials, yet no objective responses were observed in patients. The limited efficacy is partly attributed to the immunosuppressive tumor microenvironment (TME), a characteristic not fully recapitulated in investigations using xenograft models. In this study, we set to optimize B7-H3 CAR structure and examine the effects of CAR design on the brain TME using an immunocompetent glioma model. METHODS We generated a panel of fully murine B7-H3 CARs with variations in transmembrane, co-stimulatory, and activation domains. This enabled us to comprehensively investigate their effector functions within the intact immune system. High-dimensional flow cytometry and single-cell RNA sequencing were then used to investigate changes in the brain TME following CAR T-cell therapy. RESULTS While five out of six B7-H3 CARs with single co-stimulatory domains exhibited potent functionality in vitro, optimizing co-stimulation and signaling failed to enhance their anti-glioma efficacy in vivo. To further enhance therapeutic effectiveness and persistence, we incorporated 4-1BB and CD28 co-stimulation through transgenic expression of 4-1BBL on CD28-based CAR T-cells. Although this design significantly improved anti-glioma efficacy in vitro, it conferred no additional benefits in vivo. Analysis of the TME revealed that CAR T-cell therapy influenced the immune composition of the brain TME. Successful anti-tumor responses were dictated by the recruitment, activation, and spatial localization of unique inflammatory ‘immune hubs’ containing specific subsets of macrophages and endogenous T-cells. Interestingly, depletion of brain macrophages using CSF1R inhibitor markedly inhibited CAR T-cell anti-tumor activity. CONCLUSION Our study highlights the critical role of CAR structural design and its modulation of the TME in mediating the efficacy of CAR T-cell therapy in brain tumors. Yet, more specific targeting and/or reprogramming of the TME is warranted for the successful design of effective adoptive immunotherapies for high-grade glioma.

A high-dimensional platform for observing neutrophil-parasite interactions.

Diarrheal diseases with infectious etiology remain a major cause of death globally, particularly in low-income countries. Entamoeba histolytica is a pathogenic protozoan parasite that is the causative agent of amebiasis. Amebiasis has a wide presentation in clinical severity with many factors, including the bacterial microbiota, contributing to this variation. The innate immune response also plays a critical role in regulating the severity of E. histolytica infection, with neutrophils reported to have a protective role. Despite this, the precise mechanism of how neutrophils mediate amebic killing is poorly understood. Thus, modern platforms that allow for inquiry of granulocyte-ameba interactions will increase our understanding of this disease. Herein, we describe an assay for neutrophil killing of E. histolytica by utilizing high-dimensional spectral flow cytometry. Neutrophils were isolated from wild-type 5-week-old C57BL/6 mice and co-cultured with E. histolytica at various multiplicity of infections (MOIs). After co-culture, neutrophils and E. histolytica were stained for spectral flow cytometry. Cell populations were identified using surface markers and fluorescence minus one (FMO) controls. We have previously shown that animals colonized with a component of the human microbiota, Clostridium scindens, were protected from E. histolytica. This protection was associated with elevated neutrophil count. Here, we explored amebic killing capacity and observed that neutrophils from animals with C. scindens possessed heightened amebic killing compared with controls. Thus, this study establishes a novel platform that can provide an in-depth analysis of granulocyte-parasite interactions in various contexts, including during alteration of the intestinal microbiota.IMPORTANCEThe tools for studying host immune cell-E. histolytica interactions are limited. Factors, such as parasite heterogeneity, infectivity, and difficulties with culture systems and animal models, make interrogation of these interactions challenging. Thus, Entamoeba researchers can benefit from next-generation models that allow for the analysis of both host and parasite cells. Here, we demonstrate the use of a novel platform that allows for the determination of parasite-host cell interactions and customizable high-dimensional phenotyping of both populations. Indeed, spectral flow cytometry can approach >40 markers on a single panel and can be paired with custom-developed parasite antibodies that can be conjugated to fluorochromes via commercially available kits. This platform affords researchers the capability to test highly precise hypotheses regarding host-parasite interactions.

HETEROGENEOUS EXPANSION OF POLYMORPHONUCLEAR MYELOID-DERIVED SUPPRESSOR CELLS DISTINGUISHES HIGH-RISK SEPSIS IMMUNOPHENOTYPES IN UGANDA.

Background: Understanding of immune cell phenotypes associated with inflammatory and immunosuppressive host responses in sepsis is imprecise, particularly in low- and middle-income countries, where the global sepsis burden is concentrated. In these settings, elucidation of clinically relevant immunophenotypes is necessary to determine the relevance of emerging therapeutics and refine mechanistic investigations of sepsis immunopathology. Methods: In a prospective cohort of adults hospitalized with suspected sepsis in Uganda (N = 43; median age 46 years [IQR 36-59], 24 [55.8%] living with HIV, 16 [37.2%] deceased at 60 days), we combined high-dimensional flow cytometry with unsupervised machine learning and manual gating to define peripheral immunophenotypes associated with increased risk of 60-day mortality. Results: Patients who died showed heterogeneous expansion of polymorphonuclear myeloid-derived suppressor cells, with increased and decreased abundance of CD16 - PD-L1 dim and CD16 bright PD-L1 bright subsets, respectively, significantly associated with mortality. While differences between CD16 - PD-L1 dim cell abundance and mortality risk appeared consistent throughout the course of illness, those for the CD16 bright PD-L1 bright subset were more pronounced early after illness onset. Independent of HIV co-infection, depletion of CD4 + T cells, dendritic cells, and CD56 - CD16 bright NK cells were significantly associated with mortality risk, as was expansion of immature, CD56 + CD16 - CD11c + NK cells. Abundance of T cells expressing inhibitory checkpoint proteins (PD-1, CTLA-4, LAG-3) was similar between patients who died versus those who survived. Conclusions: This is the first study to define high-risk immunophenotypes among adults with sepsis in sub-Saharan Africa, an immunologically distinct region where biologically informed treatment strategies are needed. More broadly, our findings highlight the clinical importance and complexity of myeloid derived suppressor cell expansion during sepsis and support emerging data that suggest a host-protective role for PD-L1 myeloid checkpoints in acute critical illness.