Fresh Peripheral Blood Mononuclear Cells Research Articles

Evaluating the combined and individual cytotoxic effect of beauvericin, enniatin B and ochratoxin a on breast cancer cells, leukemia cells, and fresh peripheral blood mononuclear cells

Beauvericin (BEA), Enniatin B (ENN B), and Ochratoxin A (OTA) are mycotoxins produced by fungi species. Their main effect on several organs and systems is associated with chronic exposure going from immunotoxicity, estrogenic disorders, and renal failure to cancer (in animals and humans). OTA belongs to Group 1 according to the International Agency for Research in Cancer (IARC) and it has legislated limited values; not happening for BEA nor ENN B. Exposure to mixtures of mycotoxins occurs through food intake in daily consumption. The aim of this study was to evaluate the implication of BEA, ENN B, and OTA individually and combined in producing cytotoxicity in cells for immunological studies and cancer cell lines (human leukemia cells (HL-60), fresh human peripheral blood mononuclear cells (PBMCs), and human breast cancer (MDA-MB-231) cells). Cells were treated for 4 h and 24 h at different concentrations of BEA, ENN B, and OTA, respectively. Viability assays were carried out by flow cytometry using DAPI (4′,6-diamindino-2-phenylindole, dihydrochloride) as a viability dye and the potential effects of synergism, addition, and antagonism were assessed through the Chou and Talalay method. Individual OTA treatment exerted the greatest cytotoxicity for PBMC cells (IC50 0.5 μM) while ENN B for HL-60 (IC50 0.25 μM) and MDA-MB-231 (IC50 0.15 μM). In binary combination [ENN B + OTA] resulted in exerting the greatest cytotoxicity for HL-60 and MDA-MB-231 cells; while [BEA + OTA] in PBMC cells. The triple combination resulted in being highly cytotoxic for PBMC cells compared to HL-60 and MDA-MB-231 cells. In summary, PBMC cells were the most sensible cells for all three mycotoxins and the presence of OTA in any of the combinations had the greatest toxicity causing synergism as the most common cytotoxic effect.

BIOM-01. CARBONIC ANHYDRASE IX INTERPLAY WITH LOCAL AND SYSTEMIC IMMUNE RESPONSE IN PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Brain tumors (BT) remain the leading cause of death among childhood cancers. There is an urgent need to identify novel targets in these patients. BT progression depends on the adaptive responses to hypoxia. Carbonic anhydrase IX (CAIX) helps cancer cells survive in a hypoxic environment and can be potentially as a therapeutic target. The role of CAIX in the interplay between the tumor and immune cells requires in-depth investigation to design effective therapies. METHODS Fresh BT, blood, and intraoperative cerebrospinal fluid (iCSF) samples were collected prospectively from pediatric patients, who underwent brain tumor surgery at Vilnius University Hospital Santaros Klinikos from March 2022 to December 2023. All tumor types were included. Immunohistochemistry (IHC) and flow cytometry (FC) were performed to analyze the expression of CAIX and selected immunological markers in the fresh tumor, iCSF and peripheral blood mononuclear cells (PBMCs). Soluble CAIX content in serum and iCSF was assessed by ELISA. In addition, a retrospective analysis of previously diagnosed pediatric glioma specimens was performed by IHC. RESULTS Twelve patients with heterogeneous BT types have been enrolled prospectively and 17 patients with glioma were reviewed retrospectively. Glioma accounted for 72% (21/29) of all samples. IHC showed that 57% of gliomas were positive for CAIX as well as a moderate correlation between the CAIX score and the expression of CD44, CD206, and CCR2 markers. PD-1/PD-L1 expression was negligible. In the prospective cohort, 83% (10/12) of BT tissue was positive for CAIX by IHC and FC. Soluble CAIX was detectable in 40% of iCSF samples, but not in serum. We found a correlation trend of CAIX expression in the fresh tumor cells and iCSF with increased CCR2 expression on CD45+ PBMCs. CONCLUSIONS This interim analysis showed a correlation between CAIX tumor expression and immune biomarkers, prompting further investigation by enrolling more patients.

Abstract 658: Small molecule allosteric regulators of eIF4E activity target proteomic dysregulation and demonstrate potent antiproliferative activity in multiple myeloma models whilst sparing normal immune cells

Abstract Multiple myeloma is characterized by significant dysregulation of protein translation. The eukaryotic translation initiation factor 4E (eIF4E), a rate determining element of oncogene translation, is highly expressed in multiple myeloma cells compared to normal plasma cells and is associated with enhanced levels of key oncogenic drivers such as MYC. We have developed potent small molecule allosteric regulators of eIF4E activity by aligning medicinal chemistry tools with patient directed pharmacology to optimize and identify orally bioavailable, potent compounds with in vivo activity. To identify sensitive cancer models and markers associated with response to PIC eIF4E regulators, we screened the 302-cell line Eurofins OncoPanel™, followed by gene expression profiling comparing the most and least sensitive cell lines and QIAGEN Ingenuity Pathway Analysis. Consistent with the role of eIF4E, we identified proteomic dysregulation as a marker of sensitivity to PIC eIF4E regulators. In addition, the multiple myeloma cell line MM.1R was amongst the most sensitive cell lines. Proteomic analysis of PIC eIF4E regulator treated MM.1R cells identified significant changes, both increases and decreases, in approximately 8% of the 7800 proteins detected by label free mass spectrometry. QIAGEN Ingenuity Pathway Analysis of significantly altered proteins identified enrichment of proteins involved in the G2/M checkpoint, cell cycle control of chromosomal replication, and DNA repair as well as protein translation, EIF4E regulation and mTOR signaling. PIC eIF4E regulators significantly decreased cell viability of both the steroid sensitive MM.1S and steroid resistant MM.1R multiple myeloma cell lines, which was associated with significantly reduced MYC protein levels. Consistent with earlier reported findings and the mechanistic impacts of PIC eIF4E regulators, reduced MYC expression was associated with a significant decrease in hyper-phosphorylated 4EBP1. In contrast to cancer cells, PIC eIF4E regulator treatment did not affect viability of B, T, NK and plasma cell populations in freshly isolated peripheral blood mononuclear cells (PBMCs) at exposures that cause significant cell death in multiple myeloma cells. Furthermore, CD4+ T-cells isolated from fresh PBMCs demonstrated proliferative responses following stimulation (ImmunoCult™) in the presence of PIC eIF4E regulators. The impact on key oncogenic drivers and potent antiproliferative activity in multiple myeloma models with minimal effects on normal immune cell populations suggests that targeting eIF4E activity with PIC allosteric eIF4E regulators could offer an attractive therapeutic strategy in multiple myeloma. Citation Format: Lisa-Marie Sturla, Xuemei Zhang, Yagmur Kan, Richard Gedrich, Katherine Bowdish, Christopher VanDeusen. Small molecule allosteric regulators of eIF4E activity target proteomic dysregulation and demonstrate potent antiproliferative activity in multiple myeloma models whilst sparing normal immune cells [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 658.

Ultrasound and microbubble mediated T cell modulation in peripheral blood mononuclear cells

Ultrasound (US)-stimulated microbubbles are emerging as a revolutionary therapeutic technique, notably within the field of cancer immunotherapy. While this approach has been shown to modify cell permeability and to trigger local anti-tumor effects, the interactions between vibrating microbubbles and T-cells is not well understood. Here, we explore the biophysical impact of microbubbles on T-cell permeability and viability, setting the stage for potential advancements in cellular immunotherapy. Following the activation of fresh human peripheral blood mononuclear cells (PBMCs), cells were incubated with FITC-dextran (10kDa)—used here as a surrogate drug– and exposed in the presence of DefinityTM(1:217–cell:bubble ratio) US (1MHz, N = 1000, PRF = 5 ms for 2 minutes) over a range of peak-negative pressures (208-563kPa). Viability was assessed immediately and post-treatment concurrently with FITC macromolecule uptake using flow cytometry. T-cell population was identified using CD3 and CD4 antibodies (60% CD3 + and 20% CD4+). All conditions examined resulted > 95% viability. Results indicated a significant increase in viably permeated PBMCs with higher acoustic pressures, ranging from 0% to 30% (sham-corrected). When analyzed separately both CD4 + and CD4- T-cells exhibited similar permeability rates (0%–37.5%). The 563 kPa condition yielded the highest permeability with minimal viability loss. These findings open avenues for enhancing the efficacy of cellular immunotherapy for solid tumors.

OMIP-101: 27-color flow cytometry panel for immunophenotyping of major leukocyte populations in fixed whole blood.

This 27-color flow cytometry antibody panel allows broad immune-profiling of major leukocyte subsets in human whole blood (WB). It includes lineage markers to identify myeloid and lymphoid cell populations including granulocytes, monocytes, myeloid dendritic cells (mDCs), natural killer (NK) cells, NKT-like cells, B cells, conventional CD4 and CD8 T cells, γδ T cells, mucosa-associated invariant T (MAIT) cells and innate lymphoid cells (ILC). To further characterize each of these populations, markers defining stages of cell differentiation (CCR7, CD27, CD45RA, CD127, CD57), cytotoxic potential (perforin, granzyme B) and cell activation/proliferation (HLA-DR, CD38, Ki-67) were included. This panel was developed for quantifying absolute counts and phenotyping major leukocyte populations in cryopreserved, fixed WB collected from participants enrolled in large multi-site tuberculosis (TB) vaccine clinical trials. This antibody panel can be applied to profile major leukocyte subsets in other sample types such as fresh WB or peripheral blood mononuclear cells (PBMCs) with only minor additional optimization.

CD177 drives the transendothelial migration of Treg cells enriched in human colorectal cancer.

Regulatory T (Treg) cells regulate immunity in autoimmune diseases and cancers. However, immunotherapies that target tumor-infiltrating Treg cells often induce unwanted immune responses and tissue inflammation. Our research focussed on exploring the expression pattern of CD177 in tumor-infiltrating Treg cells with the aim of identifying a potential target that can enhance immunotherapy effectiveness. Single-cell RNA sequencing (scRNA-seq) data and survival data were obtained from public databases. Twenty-one colorectal cancer patient samples, including fresh tumor tissues, peritumoral tissues and peripheral blood mononuclear cells (PBMCs), were analysed using flow cytometry. The transendothelial activity of CD177+ Treg cells was substantiated using in vitro experiments. ScRNA-seq and flow cytometry results indicated that CD177 was exclusively expressed in intratumoral Treg cells. CD177+ Treg cells exhibited greater activation status and expressed elevated Treg cell canonical markers and immune checkpoint molecules than CD177- Treg cells. We further discovered that both intratumoral CD177+ Treg cells and CD177-overexpressing induced Treg (iTreg) cells had lower levels of PD-1 than their CD177- counterparts. Moreover, CD177 overexpression significantly enhanced the transendothelial migration of Treg cells in vitro. These results demonstrated that Treg cells with higher CD177 levels exhibited an enhanced activation status and transendothelial migration capacity. Our findings suggest that CD177 may serve as an immunotherapeutic target and that overexpression of CD177 may improve the efficacy of chimeric antigen receptor T (CAR-T) cell therapy.

Skin-Grafting and Dendritic Cell "Boosted" Humanized Mouse Models Allow the Pre-Clinical Evaluation of Therapeutic Cancer Vaccines.

Vaccines have been hailed as one of the most remarkable medical advancements in human history, and their potential for treating cancer by generating or expanding anti-tumor T cells has garnered significant interest in recent years. However, the limited efficacy of therapeutic cancer vaccines in clinical trials can be partially attributed to the inadequacy of current preclinical mouse models in recapitulating the complexities of the human immune system. In this study, we developed two innovative humanized mouse models to assess the immunogenicity and therapeutic effectiveness of vaccines targeting human papillomavirus (HPV16) antigens and delivering tumor antigens to human CD141+ dendritic cells (DCs). Both models were based on the transference of human peripheral blood mononuclear cells (PBMCs) into immunocompromised HLA-A*02-NSG mice (NSG-A2), where the use of fresh PBMCs boosted the engraftment of human cells up to 80%. The dynamics of immune cells in the PBMC-hu-NSG-A2 mice demonstrated that T cells constituted the vast majority of engrafted cells, which progressively expanded over time and retained their responsiveness to ex vivo stimulation. Using the PBMC-hu-NSG-A2 system, we generated a hyperplastic skin graft model expressing the HPV16-E7 oncogene. Remarkably, human cells populated the skin grafts, and upon vaccination with a DNA vaccine encoding an HPV16-E6/E7 protein, rapid rejection targeted to the E7-expressing skin was detected, underscoring the capacity of the model to mount a vaccine-specific response. To overcome the decline in DC numbers observed over time in PBMC-hu-NSG-A2 animals, we augmented the abundance of CD141+ DCs, the specific targets of our tailored nanoemulsions (TNEs), by transferring additional autologous PBMCs pre-treated in vitro with the growth factor Flt3-L. The Flt3-L treatment bolstered CD141+ DC numbers, leading to potent antigen-specific CD4+ and CD8+ T cell responses in vivo, which caused the regression of pre-established triple-negative breast cancer and melanoma tumors following CD141+ DC-targeting TNE vaccination. Notably, using HLA-A*02-matching PBMCs for humanizing NSG-A2 mice resulted in a delayed onset of graft-versus-host disease and enhanced the efficacy of the TNE vaccination compared with the parental NSG strain. In conclusion, we successfully established two humanized mouse models that exhibited strong antigen-specific responses and demonstrated tumor regression following vaccination. These models serve as valuable platforms for assessing the efficacy of therapeutic cancer vaccines targeting HPV16-dysplastic skin and diverse tumor antigens specifically delivered to CD141+ DCs.

Higher cytotoxic activities of CD8+ T cells and natural killer cells from peripheral blood of early diagnosed lung cancer patients

IntroductionCytotoxic (CD8+) and natural killer (NK) cells play critical roles in anti-tumor immunity. Dysfunction in these cells is considered as one of the extrinsic mechanisms for tumor relapse.AimWe aimed in this study to assess cytotoxic activities of CD8 + T and NK cells in the peripheral blood from lung cancer patients before and after induction of chemotherapy.Subjects and methodsHealthy (n = 5) volunteers and lung cancer patients (n = 15:5 before, 5 during, and 5 after induction of chemotherapy) were recruited. Flow cytometry was used to analyze the numbers of CD8 + T cells, NK and CD56+T cells and their intracellular expression of granzyme B (GzB) in fresh peripheral blood mononuclear cells (PBMCs) and after 72 h of their culture in vitro and stimulation with 5 µg/ml Concanavalin A (Con A) and 50ng/ml IL-2). In addition, the plasma levels of inflammatory cytokines were measured using luminex.ResultsAfter culture, significant increases in the number of GzB expressing cells gated on CD3+, CD4+, CD8 + and NKCD8 + T cells in the PBMCs from lung cancer patients before induction of chemotherapy as compared to control individuals as well as patients during and after induction of chemotherapy. Serum levels of IL-1 and CXCL8 in patients before induction of chemotherapy showed 37- and 40-fold increases, respectively, as compared to control individuals. Both GzB expression and cytokines levels in patients during and after chemotherapy were similar.ConclusionPolyclonal stimulation of PBMCs can restore the cytolytic activities of cytotoxic CD8 and NK cells from lung cancer patients even after chemotherapy.

Age-related changes in energy metabolism in peripheral mononuclear blood cells (PBMCs) and the brains of cognitively healthy seniors

Mitochondrial dysfunction is a hallmark of cellular senescence and many age-related neurodegenerative diseases. We therefore investigated the relationship between mitochondrial function in peripheral blood cells and cerebral energy metabolites in young and older sex-matched, physically and mentally healthy volunteers. Cross-sectional observational study involving 65 young (26.0 ± 0.49 years) and 65 older (71.7 ± 0.71 years) women and men recruited. Cognitive health was evaluated using established psychometric methods (MMSE, CERAD). Blood samples were collected and analyzed, and fresh peripheral blood mononuclear cells (PBMCs) were isolated. Mitochondrial respiratory complex activity was measured using a Clarke electrode. Adenosine triphosphate (ATP) and citrate synthase activity (CS) were determined by bioluminescence and photometrically. N-aspartyl-aspartate (tNAA), ATP, creatine (Cr), and phosphocreatine (PCr) were quantified in brains using 1H- and 31P-magnetic resonance spectroscopic imaging (MRSI). Levels of insulin-like growth factor 1 (IGF-1) were determined using a radio-immune assay (RIA). Complex IV activity (CIV) (− 15%) and ATP levels (− 11%) were reduced in PBMCs isolated from older participants. Serum levels of IGF-1 were significantly reduced (− 34%) in older participants. Genes involved in mitochondrial activity, antioxidant mechanisms, and autophagy were unaffected by age. tNAA levels were reduced (− 5%), Cr (+ 11%), and PCr (+ 14%) levels were increased, and ATP levels were unchanged in the brains of older participants. Markers of energy metabolism in blood cells did not significantly correlate with energy metabolites in the brain. Age-related bioenergetic changes were detected in peripheral blood cells and the brains of healthy older people. However, mitochondrial function in peripheral blood cells does not reflect energy related metabolites in the brain. While ATP levels in PBMCs may be be a valid marker for age-related mitochondrial dysfunction in humans, cerebral ATP remained constant.

Peripheral blood mononuclear cell mitochondrial dysfunction in acute alcohol-associated hepatitis.

Patients with acute alcohol-associated hepatitis (AH) have immune dysfunction. Mitochondrial function is critical for immune cell responses and regulates senescence. Clinical translational studies using complementary bioinformatics-experimental validation of mitochondrial responses were performed in peripheral blood mononuclear cells (PBMC) from patients with AH, healthy controls (HC), and heavy drinkers without evidence of liver disease (HD). Feature extraction for differentially expressed genes (DEG) in mitochondrial components and telomere regulatory pathways from single-cell RNAseq (scRNAseq) and integrated 'pseudobulk' transcriptomics from PBMC from AH and HC (n=4 each) were performed. After optimising isolation and processing protocols for functional studies in PBMC, mitochondrial oxidative responses to substrates, uncoupler, and inhibitors were quantified in independent discovery (AH n=12; HD n=6; HC n=12) and validation cohorts (AH n=10; HC n=7). Intermediary metabolites (gas-chromatography/mass-spectrometry) and telomere length (real-time PCR) were quantified in subsets of subjects (PBMC/plasma AH n=69/59; HD n=8/8; HC n=14/27 for metabolites; HC n=13; HD n=8; AH n=72 for telomere length). Mitochondrial, intermediary metabolite, and senescence-regulatory genes were differentially expressed in PBMC from AH and HC in a cell type-specific manner at baseline and with lipopolysaccharide (LPS). Fresh PBMC isolated using the cell preparation tube generated optimum mitochondrial responses. Intact cell and maximal respiration were lower (p≤.05) in AH than HC/HD in the discovery and validation cohorts. In permeabilised PBMC, maximum respiration, complex I and II function were lower in AH than HC. Most tricarboxylic acid (TCA) cycle intermediates in plasma were higher while those in PBMC were lower in patients with AH than those from HC. Lower telomere length, a measure of cellular senescence, was associated with higher mortality in AH. Patients with AH have lower mitochondrial oxidative function, higher plasma TCA cycle intermediates, with telomere shortening in nonsurvivors.

The activity of methylene blue against asexual and sexual stages of Plasmodium vivax.

Methylene blue (MB) is an alternative for combating drug-resistant malaria parasites. Its transmission-blocking potential has been demonstrated in vivo in murine models, in vitro, and in clinical trials. MB shows high efficacy against Plasmodium vivax asexual stages; however, its efficacy in sexual stages is unknown. In this study, we evaluated the potential of MB against asexual and sexual forms of P. vivax isolated from the blood of patients residing in the Brazilian Amazon. An ex vivo schizont maturation assay, zygote to ookinete transformation assay, direct membrane feed assay (DMFA), and standard membrane feed assay (SMFA) using P. vivax gametocytes with MB exposure were performed. A cytotoxicity assay was also performed on freshly collected peripheral blood mononuclear cells (PBMCs) and the hepatocyte carcinoma cell line HepG2. MB inhibited the P. vivax schizont maturation and demonstrated an IC50 lower than that of chloroquine (control drug). In the sexual forms, the MB demonstrated a high level of inhibition in the transformation of the zygotes into ookinetes. In the DMFA, MB did not considerably affect the infection rate and showed low inhibition, but it demonstrated a slight decrease in the infection intensity in all tested concentrations. In contrast, in the SMFA, MB was able to completely block the transmission at the highest concentration (20 µM). MB demonstrated low cytotoxicity to fresh PBMCs but demonstrated higher cytotoxicity to the hepatocyte carcinoma cell line HepG2. These results show that MB may be a potential drug for vivax malaria treatment.

Assessment of immunostimulatory responses to the antimiR-22 oligonucleotide compound RES-010 in human peripheral blood mononuclear cells.

microRNA-22 (miR-22) is a key regulator of lipid and energy homeostasis and represents a promising therapeutic target for NAFLD and obesity. We have previously identified a locked nucleic acid (LNA)-modified antisense oligonucleotide compound complementary to miR-22, designated as RES-010 that mediated robust inhibition of miR-22 function in cultured cells and in vivo. In this study we investigated the immune potential of RES-010 in human peripheral blood mononuclear cells (PBMCs). We treated fresh human peripheral blood mononuclear cells isolated from six healthy volunteers with different concentrations of the RES-010 compound and assessed its proinflammatory effects by quantifying IL-1β, IL-6, IFN-γ, TNF-α, IFN-α2a, IFN-β, IL-10, and IL-17A in the supernatants collected 24h of treatment with RES-010. The T-cell activation markers, CD69, HLA-DR, and CD25 were evaluated by flow cytometry after 24 and 144h of treatment, respectively, whereas cell viability was assessed after 24h of treatment with RES-010. Our results show that RES-010 compound does not induce any significant immunostimulatory responses in human PBMCs in vitro compared to controls, implying that the proinflammatory potential of RES-010 is low.

Circulating Progenitor Cells Are Associated With Bioprosthetic Aortic Valve Deterioration: A Preliminary Study.

Background Mechanisms underlying bioprosthetic valve deterioration are multifactorial and incompletely elucidated. Reparative circulating progenitor cells, and conversely calcification-associated osteocalcin expressing circulating progenitor cells, have been linked to native aortic valve deterioration. However, their role in bioprosthetic valve deterioration remains elusive. This study sought to evaluate the contribution of different subpopulations of circulating progenitor cells in bioprosthetic valve deterioration. Methods and Results This single-center prospective study enrolled 121 patients who had peripheral blood mononuclear cells isolated before bioprosthetic aortic valve replacement and had an echocardiographic follow-up ≥2 years after the procedure. Using flow cytometry, fresh peripheral blood mononuclear cells were analyzed for the surface markers CD34, CD133, and osteocalcin. Bioprosthetic valve deterioration was evaluated by hemodynamic valve deterioration (HVD) using echocardiography, which was defined as an elevated mean transprosthetic gradient ≥30 mm Hg or at least moderate intraprosthetic regurgitation. Sixteen patients (13.2%) developed HVD during follow-up for a median of 5.9 years. Patients with HVD showed significantly lower levels of reparative CD34+CD133+ cells and higher levels of osteocalcin-positive cells than those without HVD (CD34+CD133+ cells: 125 [80, 210] versus 270 [130, 420], P=0.002; osteocalcin-positive cells: 3060 [523, 5528] versus 670 [180, 1930], P=0.005 respectively). Decreased level of CD34+CD133+ cells was a significant predictor of HVD (hazard ratio, 0.995 [95% CI, 0.990%-0.999%]). Conclusions Circulating levels of CD34+CD133+ cells and osteocalcin-positive cells were significantly associated with the subsequent occurrence of HVD in patients undergoing bioprosthetic aortic valve replacement. Circulating progenitor cells might play a vital role in the mechanism, risk stratification, and a potential therapeutic target for patients with bioprosthetic valve deterioration.

Evaluation of Epcoritamab and Rituximab Combination in Preclinical Models of B-Cell Non-Hodgkin's Lymphoma (NHL)

Background: Epcoritamab (Epco) is a CD3 bispecific antibody that induces selective, potent T-cell-mediated killing of CD20-positive malignant B-cells. In the phase 1/2 monotherapy study, subcutaneous administered Epco showed a favorable safety profile and promising efficacy, including complete responses in heavily pretreated patients with relapsed / refractory non-Hodgkin's lymphoma (Hutchings et al, Lancet 2021; Thieblemont et al, EHA 2022, LB2364; EPCORE NHL-1). Ongoing clinical studies combining Epco with standard-of-care therapies including combinations with Rituximab show promising preliminary anti-tumor activity in diffuse large B-cell lymphoma and follicular lymphoma (Falchi et al, ASCO 2022 abstracts 7523 and 7524; EPCORE NHL-2 arm 1 and 2; NCT04663347). Here we evaluated the competition for CD20 binding and functional interactions between these agents and modeled its impact on Epco, T-cell, and tumor cell trimer formation that drives Epco clinical activity. Aims: Investigate the preclinical binding and functional interaction between Epco and Rituximab to establish the potential for the combination of these agents in the treatment of B-cell NHL. Methods: Competition between Epco and Rituximab for binding to CD20 cells was assessed by Flow Cytometry after incubating the single labeled agent in the presence of increasing concentrations of the competitor and vice-versa (across 0.1 - 300 ug/ml evaluated for each agent), on a range of CD20 expressing lymphoma cell lines. Epco-induced-T cell activation and redirected T cell cytotoxicity was determined by co-culturing T cells with lymphoma cells with varying expression of CD20. Rituximab antibody-dependent cellular cytotoxicity (ADCC) activity was assessed by incubating fresh peripheral blood mononuclear cells with lymphoma cells in the presence of a CD3 control Epco antibody (DuoBody-ctrlxCD20). Data from binding and cytotoxicity was used to quantify the degree of binding curve shifts at different Rituximab:Epco concentrations as well as the impact on predicted trimer formation and cytotoxicity, based on modeling. Results: In binding assays, the addition of Rituximab resulted in a dose-dependent reduction of Epco binding to CD20 expressing lymphoma cell lines, with a significant reduction of Epco binding observed at clinically relevant Epco concentrations in cells with high CD20 expression levels (Figure 1). Similar binding interference interactions were observed for binding of Epco to cell lines with low and intermediate CD20 expression levels. Binding of Rituximab to cell lines expressing high CD20 levels was not significantly reduced by clinically relevant concentrations of Epco. In Epco functional studies, the addition of increasing concentrations of Rituximab did not affect Epco cytotoxic activity at clinically relevant range of Rituximab doses tested (Figure 2). Similarly, no effect of Rituximab on Epco-mediated T cell activation was observed at clinically relevant concentrations of Rituximab and Epco. ADCC activity at clinically relevant concentrations of Rituximab (10-250 ug/ml) was not impacted by incubation with the CD3 control Epco antibody (DuoBody-ctrlxCD20), even at concentrations exceeding the Epco clinical concentrations. Modeling and simulation of in vitro data showed that at clinically relevant concentration of Epco, the presence of clinically relevant concentration of Rituximab does not meaningfully impact Epco trimer formation, and the predicted trimer formation remains above EC90 relative to that needed for 50% cell kill. Conclusions: Our studies showed that while Rituximab exerts significant interference on Epco binding to CD20-expressing cells, tumor-cell cytotoxicity by Epco is minimally affected by clinically relevant concentrations of Rituximab. This conclusion is supported by preclinical modeling and simulations showing that Epco activity driven by trimer formation is not meaningfully impacted by clinical concentrations of Rituximab. Furthermore, we found a limited effect of Epco on Rituximab binding to CD20 and no impact on ADCC activity by Rituximab, thereby supporting potential synergy in the combination between these agents. Results from this study confirm the ability to combine Epco with CD20 antibodies, specifically with Rituximab, and the potential for synergistic activity of this combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 13214: Circulating Progenitor Cells Are Associated With Bioprosthetic Aortic Valve Deterioration

Introduction: Mechanisms underlying bioprosthetic valve deterioration (BVD) are multifactorial and incompletely elucidated. Reparative circulating progenitor cells (CPCs), and conversely calcification-associated osteocalcin expressing CPCs (CPC-OCN), have been linked to native aortic valve deterioration. However, their role in BVD remains elusive. This study sought to evaluate the contribution of different subpopulations of CPCs in BVD. Methods: This prospective study enrolled 121 patients who had peripheral blood mononuclear cells (PBMC) isolated prior to bioprosthetic aortic valve replacement (surgical 104, transcatheter 17) and had an echocardiographic follow-up ≥2 years after the procedure. Using flow cytometry, fresh PBMC were analyzed for the surface markers CD34, CD133, and osteocalcin (OCN). BVD was evaluated by hemodynamic valve deterioration (HVD) using echocardiography, which was defined as an elevated mean transprosthetic gradient ≥30mmHg and/or at least moderate intraprosthetic regurgitation. Multivariable models were constructed to evaluate the association of each subpopulation of CPCs and other clinical characteristics with HVD. Results: Sixteen patients (13.2%) developed HVD during follow-up for a median of 5.9 years. Patients with HVD showed significantly lower levels of reparative CD34 + CD133 + cells and higher levels of OCN + cells than those without HVD (CD34 + CD133 + cells: 125 [80, 210] vs 270 [130, 420], P=0.002; OCN + cells: 3060 [523, 5528] vs 670 [180, 1930], P=0.005 respectively). Decreased level of CD34 + CD133 + cells was a significant predictor of HVD. Conclusions: Circulating levels of CD34 + CD133 + cells and OCN + cells were significantly associated with the subsequent occurrence of HVD in patients undergoing bioprosthetic aortic valve replacement. CPCs might play a vital role in the mechanism, risk stratification and a potential therapeutic target for patients with bioprosthetic valve deterioration.

GSK3β and mTORC1 Represent 2 Distinct Signaling Markers in Peripheral Blood Mononuclear Cells of Drug-Naive, First Episode of Psychosis Patients.

Schizophrenia is characterized by a complex interplay between genetic and environmental risk factors converging on prominent signaling pathways that orchestrate brain development. The Akt/GSK3β/mTORC1 pathway has long been recognized as a point of convergence and etiological mechanism, but despite evidence suggesting its hypofunction, it is still not clear if this is already established during the first episode of psychosis (FEP). Here, we performed a systematic phosphorylation analysis of Akt, GSK3β, and S6, a mTORC1 downstream target, in fresh peripheral blood mononuclear cells from drug-naive FEP patients and control subjects. Our results suggest 2 distinct signaling endophenotypes in FEP patients. GSK3β hypofunction exhibits a promiscuous association with psychopathology, and it is normalized after treatment, whereas mTORC1 hypofunction represents a stable state. Our study provides novel insight on the peripheral hypofunction of the Akt/GSK3β/mTORC1 pathway and highlights mTORC1 activity as a prominent integrator of altered peripheral immune and metabolic states in FEP patients.

P1108: HIGH COMPLETE RESPONSE RATE FOLLOWING POINT-OF-CARE ANTI CD19 CAR T-CELL THERAPY IN PATIENTS WITH RELAPSED/REFRACTORY FOLLICULAR LYMPHOMA

Background: Patients with relapsed/refractory follicular lymphoma (R/R FL) often experience multiple relapses and require various lines of therapy. The ELARA and ZUMA-5 trials demonstrated high response rates along with acceptable safety profiles. We perform a phase 1b/2 single-center clinical trial of autologous point-of-care (POC) academic anti-CD19 chimeric antigen receptor (CAR) T-cells for patients with R/R FL treated with at least 2 lines of systemic therapy (NCT02772198). Aims: To report outcomes of POC CAR T-cell therapy in patients with R/R FL. Methods: Adults with R/R FL underwent a single leukapheresis procedure. Fresh peripheral blood mononuclear cells were isolated, activated, and transduced with a gammaretrovirus encoding for a CD19 CAR (based on an FMC63-derived ScFv, a CD28 costimulatory domain, and a CD3-ζ signaling domain). Lymphodepletion included fludarabine 25 mg/m2 over 3 days (days −4 to −2) and cyclophosphamide 900 mg/m2 once (day −2), followed by infusion of 1×106/kg CAR T-cells in the inpatient setting. Primary endpoints were response (by PET-CT, per Lugano criteria) at day 28, best response, and safety. Secondary endpoints included overall survival, progression-free survival (PFS), and production feasibility. Last follow-up was as of 02/2022. Results: All 19 patients enrolled received CAR T-cell infusion in a median of 11 days (IQR 10-11) after leukapheresis. The median age was 61 years (IQR 52-66). Five (26%) patients had Karnofsky performance status < 90%. Disease stage at enrollment was III-IV in 16 (84%) patients. Two (11%) patients had bulky disease; 8 (42%) had LDH > upper limit of normal; and 16 (84%) had Follicular Lymphoma International Prognostic Index ≥ 3. Disease status at enrollment was progressive disease (n=14, 74%), stable disease (n=3, 16%), or partial response (PR; n=2, 11%). Twelve patients (64%) were refractory to last treatment. Disease grade at most recent lymph node biopsy was 1 (n=3, 16%), 2 (n=11, 58%), or 3a (n=5, 26%). The median time from FL diagnosis was 3.9 years (IQR 2.5-4.6). Sixteen (84%) patients had progression of disease within 24 months of initial therapy. The number of prior therapies was ≥ 4 in 6 (32%) patients; and 5 (26%) patients underwent prior autologous transplantation. Grade III-IV cytokine release and immune effector cell-associated neurotoxicity syndromes occurred in 1 (5%) and 4 (21%) patients, respectively. One patient was infected with COVID-19 on the 5th day following cell infusion and was admitted to the intensive care unit. One patient had grade 3 atrial fibrillation. Severe neutropenia (absolute neutrophil count <500/µL), thrombocytopenia (platelets <50K/µL) and anemia (hemoglobin <10g/dl) occurred in 15 (79%), 5 (26%), and 7 (37%) patients, respectively. No bleeding events or death were recorded following cell infusion. Response was evaluated in all patients. Overall response rate on day 28 was 84% (79% complete response [CR]). One patient with PR on day 28 achieved a CR after a year of follow-up. Three patients (16%) continued to progress following CAR infusion. All patients were alive at the last follow-up (median follow-up, 11.5 months [IQR 4-21]). One-year PFS was 74% (95% CI, 53-100). The median duration of response (DOR) was not reached (95% CI, 12.5-not reached). Estimated DOR at 1-year was 89% (95% CI, 71-100). Image:Summary/Conclusion: Point-of-Care anti-CD19 CAR T-cell therapy, performed following a very short production time, induced high CR rate with an acceptable safety profile in a cohort of patients with high-risk R/R FL.

236 Systemic mitochondrial dysfunction in monogenic Parkinson’s disease as a potential biomarker for stratification

To investigate the use of PBMCs as a biomarker of mitochondrial dysfunction in Parkinson disease (PD) fresh peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll gradient separation from PD patients with pathogenic biallelic parkin mutations as well as healthy controls (HC). PBMC mitochondrial function was analysed using Seahorse XF respirometry and flow cytometric assessment of mitochondrial membrane potential (Δψm) using TMRE staining.Nine parkin PD patients (average age 45.2yrs, 66% male) and 41 healthy controls (average age 57.8yrs, 58% female) were sampled for the study. Parkin patients had increased oxygen consumption related to proton leak, reduced maximal respiration and reduced spare respiratory after correction for age (p&lt;0.01). Additionally Parkin patients were found to have reduced resting Δψm and reduced oxygen consumption related to ATP production (p&lt;0.01). Basal oxygen consumption was similar between Parkin patients and healthy controls.These findings are in keeping with parkin’s role in removing dysfunctional mitochondria through mitophagy. The finding of normal basal oxygen consumption suggests that dysfunction is most pronounced when cells are under stress, during high ATP demand. This ‘signature’ of mitochondrial dysfunction could be applied to idiopathic PD patients to define a mitochondrial subgroup, in which personalised, stratified therapeutics could be targeted.p.campbell@ucl.ac.uk

Involvement of small ncRNAome in fresh peripheral blood mononuclear cells of human T-cell leukemia virus type I- associated myelopathy/tropical spastic paraparesis patients and viral carriers

Involvement of small ncRNAome in fresh peripheral blood mononuclear cells of human T-cell leukemia virus type I- associated myelopathy/tropical spastic paraparesis patients and viral carriers

Mitochondrial and Glycolytic Capacity of Peripheral Blood Mononuclear Cells Isolated From Diverse Poultry Genetic Lines: Optimization and Assessment.

Cellular metabolic preference is a culmination of environment, nutrition, genetics, and individual variation in poultry. The Seahorse XFe24 analyzer was used to generate foundational immune cellular metabolic data in layer, broiler, and legacy genetic strains using fresh chicken peripheral blood mononuclear cells (PBMCs). Baseline mitochondrial respiration [oxygen consumption rate (OCR)] and glycolytic activity [extracellular acidification rate (ECAR)] were determined in modern commercial laying hen (Bovans White) and broiler (Ross 308) lines, as well as the highly inbred lines of Iowa State University (L8, Fayoumi M-15.2, Spanish, Ghs-6), partially inbred broiler line, and advanced intercrosses of broiler by Fayoumi M-15.2 and broiler by Leghorn lines. Commercial broiler vs. Bovans layer and unvaccinated vs. vaccinated Bovans layer immune cell metabolic potential were compared following an in-assay pathway inhibitor challenge. Titrations consistently showed that optimal PBMC density in laying hens and broilers was 3 million cells per well monolayer. Assay media substrate titrations identified 25 mM glucose, 1 mM glutamine, and 1 mM sodium pyruvate as the optimal concentration for layer PBMCs. Pathway inhibitor injection titrations in Bovans layers and broilers showed that 0.5 μM carbonyl cyanide-4 phenylhydrazone (FCCP) and 1 μM oligomycin were optimal. Baseline OCR and ECAR were significantly affected by genetic line of bird (p < 0.05), with the dual-purpose, L8 inbred line showing the highest OCR (mean 680 pmol/min) and the partially inbred broiler line showing the greatest ECAR (mean 74 mpH/min). ECAR metabolic potential tended to be greater in modern layers than broilers (p < 0.10), indicating increased ability to utilize the glycolytic pathway to produce energy. OCR was significantly higher in vaccinated than unvaccinated hens (p < 0.05), while baseline ECAR values were significantly lower in vaccinated Bovans laying hens, showing increased oxidative capacity in activated immune cells. These baseline data indicate that different genetic strains of birds utilized the mitochondrial respiration pathway differently and that modern commercial lines may have reduced immune cell metabolic capacity compared with legacy lines due to intense selection for production traits. Furthermore, the Seahorse assay demonstrated the ability to detect differences in cellular metabolism between genetic lines and immune status of chickens.