Cell Populations In Peripheral Blood Research Articles

P-306 GnRH agonists exert a positive influence on regulatory immune cells in the endometrium of women with adenomyosis

Abstract Study question How does treatment with GnRH-agonist (GnRH-a) prior to frozen embryo transfer (FET) in women with adenomyosis affect immune cell populations in endometrium and peripheral blood? Summary answer GnRH-a induces a decrease in monocytes and an increase in CD4+T-cells in peripheral blood and a decrease classical monocytes and T-regulatory cells in the endometrium What is known already Adenomyosis is associated with lower implantation and higher miscarriage rates. Observational studies have reported improved pregnancy outcomes in women with adenomyosis after assisted reproductive technologies (ART) and pre-treatment with GnRH-a prior to FET. However, the specific mechanisms of GnRHa action beyond central hormonal suppression remain unclear, but may be mediated through local and/or systemic immunomodulation. Study design, size, duration Prospective cohort study including infertile women with adenomyosis and GnRH-a treatment ≥3 months prior to FET. Pre- and post-treatment analysis of blood and endometrium samples were compared. Recruitment is ongoing with n = 30 women included thus far, 16 of whom have completed the study, while the remaining are still undergoing GnRH-a therapy. Participants/materials, setting, methods Adenomyosis was diagnosed sonographically if ≥ 2 criteria published by the MUSA group were met. Peripheral blood and endometrial samples were obtained before and after treatment with GnRH-a. Plasma cells and uterine Natural Killer (uNK) cells in endometrial biopsies were quantified with immunhistochemistry (IHC), and leukocyte populations in all samples were analysed using flow cytometry (FC). Pre- and post-treatment levels of immune cells and percentage of leukocyte populations were compared using Wilcoxon rank-sum tests (p < 0.05 significant) Main results and the role of chance Interim analysis of immune cell populations in peripheral blood and endometrium of the 16 women who already completed the study was performed. At baseline, IHC showed elevated plasma cell concentrations indicating chronic endometritis in 3/30 patients (10%), while uNK cells were elevated (>300/mm2) in 13/30 patients (43.3%). FC analysis of peripheral blood showed a significant decrease in percentage of monocytes (p = 0.01) and increase in percentage of T-cells (p = 0.03), specifically concentration of CD4+T-cells (p = 0.03), after treatment with GnRH-a as compared to pre-treatment levels. In the endometrium, following GnRH-a therapy, we observed a decrease in both the percentage of classical monocytes (p = 0.03) and T-regulatory cells (p = 0.03). All 16 participants who completed the study already underwent FET, resulting in ten pregnancies (62.5%), although four resulted in miscarriage, while the outcome of the remaining pregnancies is still pending. Limitations, reasons for caution These are preliminary results whose generalizability needs to be confirmed in a larger study population. No correction for multiple comparisons was performed and significant findings have to be interpreted with caution. Wider implications of the findings Elevated uNK on IHC cells seen at baseline may be associated with implantation failure, miscarriage. CD4+ and DN T-cells are important mediators of reproductive immune tolerance. In women with adenomyosis, GnRH-a may have a positive impact on these regulatory immune cells in peripheral blood as well as in the endometrium. Trial registration number Not applicable

Flow Cytometry as the Tool to Define Peripheral Blood Leukocyte Signatures in Acute EBV Infection.

Primary Epstein-Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. The infection has a wide spectrum of clinical findings and can lead to serious complications in patients with certain underlying immunological dysfunctions. We retrospectively investigated peripheral white blood cell populations' surface marker characteristics in IM using a comprehensive flow cytometry marker panel. Twenty-one cases of IM and seventeen EBV-seropositive cases without IM serving as controls were included. We observed novel alterations in lymphocyte, neutrophil, and monocyte populations. In addition to increased activated cytotoxic T cells and low B cells, we demonstrated high T-large granular lymphocyte (T-LGL) populations in IM cases. Furthermore, despite T cells' increased HLA-DR expression, another activation marker, CD11b, was lower in T-LGL populations. Monocytes showed increased CD16 expression; CD64 was higher in neutrophils. Our findings point to monocyte and neutrophil activation which may account for acute clinical features and may contribute to the understanding of IM immunobiology. Furthermore, they may serve as a useful tool in investigating inherited and post-transplant conditions characterized by deficiencies in controlling EBV infection.

INFECTION CONTROL IN COVID-19 PATIENTS BASED ON POLYMORPHISMS OF TMPRSS2 (rs12329760), FGB (rs1800790), AND NOS3 (rs2070744) GENES

Objective of this study is to investigate the potential of anti-infective protection in patients with COVID-19 by analyzing the absolute and relative number of the main populations of immunocompetent peripheral blood cells depending on the polymorphism of the genes of transmembrane serine protease 2 (TMPRSS2, rs12329760), fibrinogen beta (FGB, rs1800790) and endothelial nitric oxide synthase (NOS3, T-786C, rs2070744). Materials and methods. A total of 204 patients with mild, moderate, and severe COVID-19-associated pneumonia were included in the single-center study. Among the patients were 51.97% (106) women and 48.03% (98) men. Among the patients, there were 51.97% (106) women and 48.03% (98) men, with an average age of 55.93±8.75 years. Anti-infective protection was assessed based on an extended complete blood count (CBC) with the calculation of the main populations of immunocompetent cells. The polymorphism of the TMPRSS2 (rs12329760), FGB (rs1800790) and NOS3 (rs2070744) genes was investigated by real-time polymerase chain reaction (Real Time PCR). Results. There were no differences in the absolute and relative number of most populations of immunocompetent peripheral blood cells between the genotypes of the TMPRSS2 (rs12329760) gene. The absolute and relative number of immunocompetent peripheral blood cell populations between the genotypes of the FGB (rs1800790) gene also did not differ significantly. However, in AA genotype carriers, there was a tendency to decrease the number of neutrophilic granulocytes due to mature segmented nucleated forms against the background of an increase in eosinophilic granulocytes by 27.27% (p=0, 038) and 55.55% (p=0.007) and agranulocytes due to lymphocyte sprouting and monocytes by 8.87-20.09% that implies a more severe course of the disease and a stronger stress of non-specific immunity than in G-allele holders. Conclusions. Inflammatory changes in the complete blood count of the main populations of immunocompetent cells in patients with COVID-19 do not show a consistent dependence on the genotypes of the TMPRSS2 (rs12329760) and NOS3 (rs2070744) genes. However, the presence of the AA genotype of the FGB gene (rs1800790) in patients with COVID-19 is associated with a more severe course of the disease and increased stress on the monocyte-macrophage system.

Cyclophosphamide-free mobilisation increases safety while preserving the efficacy of autologous haematopoietic stem cell transplantation in refractory Crohn's disease patients.

Autologous haematopoietic stem cell transplantation [AHSCT] is a therapeutic option for refractory Crohn's disease [CD]. However, high adverse event rates related to chemotherapy toxicity and immunosuppression limit its applicability. This study aims to evaluate AHSCT's safety and efficacy using a cyclophosphamide (Cy)-free mobilisation regimen. A prospective observational study included 14 refractory CD patients undergoing AHSCT between June 2017 and October 2022. The protocol involved outpatient mobilisation with G-CSF 12-16 μg/kg/daily for 5 days, and optional Plerixafor 240 μg/d (1-2 doses) if the CD34+ cell count target was unmet. Standard conditioning with Cy and anti-thymocyte globulin was administered. Clinical, endoscopic, and radiological assessments were conducted at baseline and during follow-up. All patients achieved successful outpatient mobilisation (7 patients needed Plerixafor) and underwent transplantation. Median follow-up was 106 weeks (IQR 52-348). No mobilisation-related serious adverse events (SAEs) or CD worsening occurred. Clinical and endoscopic remission rates were 71% and 41.7% at 26 weeks, 64% and 25% at 52 weeks, and 71% and 16.7% at the last follow-up. The percentage of patients who restarted CD therapy for clinical relapse and/or endoscopic/radiological activity was 14% at 26 weeks, 57% at 52 weeks, and 86% at the last follow-up. Peripheral blood cell populations and antibody levels post-AHSCT were comparable to Cy-based mobilisation. Cy-free mobilisation is safe and feasible in refractory CD patients undergoing AHSCT. Although relapse occurs in a significant proportion of patients, clinical and endoscopic responses are achieved upon CD-specific therapy reintroduction.

Single-Cell Profiling Reveals Immune Aberrations in Progressive Idiopathic Pulmonary Fibrosis.

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5' single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

Abstract 4010: Assessing impacts of tumor burden and CAR-T cell dosage on the toxicity and efficacy profile of CD19 CAR-T therapy in a PBMC-humanized mouse model

Abstract Chimeric antigen receptor T-cell (CAR-T) therapy is a significant advancement in treating hematological malignancies, yet it faces challenges due to its variable therapeutic responses and the risk of severe toxicities. This study delves into the effects of tumor burden and CAR-T cell doses on the toxicity profile of the therapy, employing a PBMC-humanized mouse model engrafted with luciferase-labeled Raji B cell lymphoma (Raji-luc). By exploring high vs. low tumor burden and high vs. low CAR-T dose scenarios, we aim to gain insights into the dynamic relationship between these factors and CAR-T-induced toxicity and efficacy. In the first experiment, we established PBMC humanized mouse models with a high tumor burden and a low tumor burden. 10 days (high burden) or 7 days (low burden) after the Raji-luc inoculation, mice were engrafted with 10M human PBMCs and dosed with CD19 autologous CAR-Ts. In mice with low tumor burden, CAR-T treatment demonstrated significant efficacy, as evidenced by IVIS imaging, while the high tumor burden model exhibited reduced efficacy. CAR-T treatment in Raji-bearing mice did not induce weight loss in either high or low burden models. CD19 CAR-T cells effectively reduced the target cell population in peripheral blood, with greater expansion observed in the higher tumor burden model. We also evaluated human cytokine levels post-CAR-T treatment, revealing higher cytokine induction in the high tumor burden model, peaking at 2 days post-treatment. Secondly, in a CAR-T dose-response study, we treated PBMC-humanized Raji-bearing mice with 10M, 15M, and 20M CAR-T cells. Higher CAR-T cell doses (15M and 20M) resulted in observable toxicity measured by body weight loss, while the 10M dose did not. All CAR-T doses effectively slowed tumor growth and induced significant CAR-T cell expansion. Selected human cytokines, such as IL-5 and RANTES, demonstrated a dose-response correlation with CAR-T treatment. Additionally, IL-6 and IL-10 were significantly correlated with tumor burden rather than CAR-T doses. The differential cytokine responses observed in our study provide valuable insights into the utility of the PBMC-humanized mouse model for investigating the biological responses associated with CD19 CAR-T therapy. Our findings underscore the utility of the PBMC-humanized mouse model in assessing variability in toxicity and cytokine responses to CAR-T therapy. This model offers valuable insights into the factors influencing CAR-T treatment outcomes and provides a platform for planning more precise treatment and enhancing the safety and efficacy of CAR-T therapy. Citation Format: Won Lee, Destanie Rose, James G. Keck, Jiwon Yang. Assessing impacts of tumor burden and CAR-T cell dosage on the toxicity and efficacy profile of CD19 CAR-T therapy in a PBMC-humanized mouse model [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 4010.

SYSTEMIC INFLAMMATORY INDICES IN PATIENTS WITH MALIGNANT GLIOMAS AND EFFECTS OF PLATELET SECRETOME IN VITRO.

To date, no significant clinical progress has been achieved in the treatment of brain malignant gliomas (MG), and the active search for non-invasive circulating biomarkers continues. The prognostic significance of the ratio of the main peripheral blood cell populations of patients with MG is evaluated. Considerable attention is paid to the secretome of platelets (Pt) of peripheral blood. To evaluate the indicators of the peripheral blood cell population ratios in patients with brain MG and to study the influence of the secretome of Pt (SPt) of the peripheral blood of patients with brain MG in cell cultures in vitro. We studied samples of peripheral blood from patients with glioma CNS WHO grade G2 (n = 5), G3 (n = 12), and G4 (n = 20). The peripheral blood cell counts were analyzed in the preoperative period on an automatic hematology analyzer. The in vitro study of SPt was performed on the U251 human glioblastoma cell line cultured with SPt from MG patients or SPt pre-incubated with anti-TGF-β1 antibody. Cell cultures were observed for 72 h, and mitotic index (MI) was calculated. In MG patients, the count of peripheral blood leukocytes and neutrophils increased (p < 0.05). The neutrophil-to-lymphocyte ratio (NLR) and systemic immune-inflammation index (SII) increased by 2-3 times compared to control. Nevertheless, correlation analysis did not reveal significant relationships between quantitative indicators of peripheral blood cells and the tumor malignancy degree in MG patients. The MI in U251 cells increased under the influence of SPt from patients with MG (p < 0.021), correlated with the tumor degree of malignancy (r = 0.246, p = 0.014). Pre-incubation of SPt with anti-TGF-β1 antibody tends to neutralize this promitotic effect. In MG patients, the integral indicators of NLR and SII increased but no significant relationship with the degree of tumor malignancy was found. In U251 cells, promitotic effects of SPt of MG patients partially decreased by anti-TGF-β1 antibody.

P127 Sphingosine-1-Phosphate Receptor Blockade with Etrasimod alters Lymphocyte Trafficking in Crohn's Disease: Insights from High-Dimensional Phenotypic Mapping

Abstract Background The blockade of lymphocyte trafficking from the lymph nodes (LNs) towards the intestinal mucosa is an effective approach for the treatment of ulcerative colitis (UC). Etrasimod is an oral, once-daily (QD), selective sphingosine 1-phosphate (S1P)1,4,5 receptor (S1PR) modulator, which is currently also under investigation for Crohn’s disease (CD). The therapeutic mechanism of action of S1PR modulators in the context of inflammatory bowel disease (IBD) is not completely understood. It is hypothesized that both B and T- cell populations are affected by this treatment and are retained in the peripheral LNs, reducing circulating lymphocytes and resulting in fewer immune cells available to traffic in the gastrointestinal tract. Etrasimod has been reported to induce a partial and selective reduction of lymphocyte subsets in the blood of healthy subjects, but the direct effect of S1PR modulators on LN leukocytes has not been evaluated. The aim of this study was to investigate changes in leukocyte subpopulations in peripheral LNs and peripheral blood (PB) in response to S1PR inhibition with etrasimod in patients with CD. Methods CD patients with moderate to severe disease activity, participating in the Phase 2, double-blinded, non-placebo-controlled portion of the CULTIVATE trial, were eligible for this sub-study. At baseline and after 14 weeks of etrasimod induction treatment (2 or 3 mg/day), 10cc of PB and 4 ultrasound guided biopsy samples of an inguinal LN were obtained. The isolated peripheral blood mononuclear cells (PBMCs) and LN leukocyte populations, were analyzed at single cell level via high-dimensional immunophenotyping through mass cytometry (CyTOF) at both timepoints. Results We observed that in the LNs, the numbers/percentages of naïve, central and effector memory T-helper cells, as well as, of CD8+ naïve T-cells were increased after treatment with etrasimod. There was a decrease in the proportion of these cell populations in peripheral blood. Circulating naïve and memory B-cells were reduced, while these subsets where also slightly reduced in the LN compartment. Innate immune cell populations were not significantly altered. Conclusion Etrasimod induction treatment resulted in an increase of both naïve and central memory T-lymphocyte subsets in inguinal LNs, with a corresponding decrease in the periphery. The effect on the frequency of B-cells was not consistent in the LNs and PB, while innate immunotypes and T-regs, were not significantly affected. Despite the limited sample size, these selective effects were consistent with previously published PB immunophenotyping studies with etrasimod. This study was conducted as a collaboration between Amsterdam UMC and Pfizer.

Transplantation of Donor-Recipient Chimeric Cells Restores Peripheral Blood Cell Populations and Increases Survival after Total Body Irradiation-Induced Injury in a Rat Experimental Model.

Current therapies for acute radiation syndrome (ARS) involve bone marrow transplantation (BMT), leading to graft-versus-host disease (GvHD). To address this challenge, we have developed a novel donor-recipient chimeric cell (DRCC) therapy to increase survival and prevent GvHD following total body irradiation (TBI)-induced hematopoietic injury without the need for immunosuppression. In this study, 20 Lewis rats were exposed to 7 Gy TBI to induce ARS, and we assessed the efficacy of various cellular therapies following systemic intraosseous administration. Twenty Lewis rats were randomly divided into four experimental groups (n = 5/group): saline control, allogeneic bone marrow transplantation (alloBMT), DRCC, and alloBMT + DRCC. DRCC were created by polyethylene glycol-mediated fusion of bone marrow cells from 24 ACI (RT1a) and 24 Lewis (RT11) rat donors. Fusion feasibility was confirmed by flow cytometry and confocal microscopy. The impact of different therapies on post-irradiation peripheral blood cell recovery was evaluated through complete blood count, while GvHD signs were monitored clinically and histopathologically. The chimeric state of DRCC was confirmed. Post-alloBMT mortality was 60%, whereas DRCC and alloBMT + DRCC therapies achieved 100% survival. DRCC therapy also led to the highest white blood cell counts and minimal GvHD changes in kidney and skin samples, in contrast to alloBMT treatment. In this study, transplantation of DRCC promoted the recovery of peripheral blood cell populations after TBI without the development of GVHD. This study introduces a novel and promising DRCC-based bridging therapy for treating ARS and extending survival without GvHD.

Breastfeeding and Neonatal Age Influence Neutrophil-Driven Ontogeny of Blood Cell Populations in the First Week of Human Life.

The first few days of life are characterized by rapid external and internal changes that require substantial immune system adaptations. Despite growing evidence of the impact of this period on lifelong immune health, this period remains largely uncharted. To identify factors that may impact the trajectory of immune development, we conducted stringently standardized, high-throughput phenotyping of peripheral white blood cell (WBC) populations from 796 newborns across two distinct cohorts (The Gambia, West Africa; Papua New Guinea, Melanesia) in the framework of a Human Immunology Project Consortium (HIPC) study. Samples were collected twice from each newborn during the first week of life, first at Day of Life 0 (at birth) and then subsequently at Day of Life 1, 3, or 7 depending on the randomization group the newborn belongs to. The subsequent analysis was conducted at an unprecedented level of detail using flow cytometry and an unbiased automated gating algorithm. The results showed that WBC composition in peripheral blood changes along patterns highly conserved across populations and environments. Changes across days of life were most pronounced in the innate myeloid compartment. Breastfeeding, and at a smaller scale neonatal vaccination, were associated with changes in peripheral blood neutrophil and monocyte cell counts. Our results suggest a common trajectory of immune development in newborns and possible association with timing of breastfeeding initiation, which may contribute to immune-mediated protection from infection in early life. These data begin to outline a specific window of opportunity for interventions that could deliberately direct WBC composition, and with that, immune trajectory and thus ontogeny in early life. This trial is registered with NCT03246230.

Modified Biejia Jianwan decoction restrains PD-L1-mediated immune evasion through the HIF-1α/STAT3/NF-κB signaling pathway

Ethnopharmacological relevanceModified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its underlying functional mechanism still remains unknown. Aim of the studyThe study aimed to explore the anti-hepatocarcinogenic effects of M-BJJW, specifically its influence on PD-L1-mediated immune evasion in hypoxic conditions, and elucidate the related molecular mechanisms in HCC. Materials and methodsTo investigate the therapeutic efficacy and mechanisms underlying M-BJJW's effects on HCC, we employed a diethylnitrosamine (DEN)-induced rat model maintained for 120 days. Following model establishment, flow cytometry was utilized to assess the distribution of immune cell populations in peripheral blood, spleens, and tumor tissues after M-BJJW administration. Simultaneously, enzyme-linked immunosorbent assays (ELISA) were conducted to analyze cytokine profiles in serum samples. Immunohistochemistry was employed to determine the expression levels of crucial proteins within tumor tissues. Furthermore, HCC cells exposed to CoCl2 underwent Western blot analysis to validate the expression levels of HIF-1α, PD-L1, STAT3, and nuclear factor kappa B (NF-κB) p65. The modulatory effects of STAT3 and NF-κB p65 were investigated using specific inhibitors and activators in wild-type cell lines. High-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) was utilized to identify the chemical constituents present in M-BJJW-medicated serum. The immunomodulatory properties and the anti-tumor activities of M-BJJW were evaluated by co-culturing with peripheral blood mononuclear cells (PBMC) and the CCK-8 assay. Additionally, we assessed M-BJJW's impact on hypoxia-induced alterations in HCC cell lines using immunofluorescence and Western blot assessments. ResultsM-BJJW exhibited substantial therapeutic advantages by effectively alleviating pathological deterioration within the HCC microenvironment. In the DEN-induced rat model, M-BJJW administration notably reduced tumor growth. Flow cytometry analyses revealed an increased proportion of Cytotoxic T lymphocytes (CTLs) accompanied by a simultaneous decrease in regulatory T cells (Tregs). ELISA data supported a marked decrease in pro-inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α). Immunohistochemistry confirmed the suppressive effect of M-BJJW on the expression of HIF-1α and PD-L1. Notably, western blotting unveiled the role of HIF-1α in regulating PD-L1 expression via the STAT3 and NF-κB signaling pathways in HCC cell lines, which was validated using activators and inhibitors of STAT3 and NF-κB. The CCK-8 assay and co-culture techniques demonstrated the anti-tumor activity of M-BJJW. Immunofluorescence and western blotting further confirmed that M-BJJW-containing serum dose-dependently inhibited HIF-1α, PD-L1, p-STAT3, and p-p65 in hypoxic HCC cell lines. ConclusionsM-BJJW demonstrates significant therapeutic potential against HCC by influencing the hypoxic microenvironment, thereby regulating the immunosuppressive milieu. Specifically, M-BJJW modulates the HIF-1α/STAT3/NF-κB signaling pathway, leading to reduced PD-L1 expression and an elevated ratio of cytotoxic T lymphocytes (CTLs), while concurrently decreasing T regulatory cells (Tregs) and immunosuppressive factors. These synergistic effects aid in countering PD-L1-mediated immune evasion, presenting compelling pharmacological evidence supporting the clinical application of M-BJJW as a therapeutic approach for HCC.

Abstract C033: Preliminary pharmacodynamic evaluation of AU-007 in phase 1 dose escalation trial in patients with advanced solid tumors

Abstract AU-007 is a monoclonal antibody currently being evaluated in a Phase 1/2 clinical trial. In conjunction with evaluation of clinical activity, a pharmacodynamic program has been established to assess the biological activity of AU-007 alone or with low dose subcutaneous aldesleukin in patients with advanced solid cancers. AU-007 binds IL-2 on its CD25-binding epitope, thus inhibiting IL-2’s interaction with CD25 in the trimeric IL-2 receptor (IL-2R) but allowing for IL-2’s binding to the dimeric IL-2R. This effectively inhibits the trimeric IL-2R expressing Tregs without impacting dimer expressing memory T cells, NK cells, NKT cells and naïve T cells. We evaluated the effect of AU-007 +/- aldesleukin on peripheral blood cell populations, IFN-gamma, IL-2 and sCD25. Here we report the interim pharmacodynamic data from the ongoing dose escalation portion of the trial. Peripheral blood samples were collected in Cyto-Chex BCT tubes and phenotyped for T cells, NK cells, B cells, and monocytes. A TruCount system was used to obtain absolute cell numbers. In addition, eosinophils were assessed through standard CBC differentials. Serum collections were done to assess circulating markers using the MSD ECL platform. It was observed that, regardless of AU-007 dose with or without aldesleukin, patients had a reduction in the circulating levels of Treg cells (i.e., based on assay validation, a change greater than +/-10% was considered a real change). This is consistent with the mechanism of action of AU-007, by binding IL-2 such that it prevents the interaction with the trimeric IL-2R, Tregs are affected. There was an increase in the CD8+ to Treg ratio consistent with the overall decrease in Tregs. Trends for increases in NK cells and CD8+ T cells were observed in most patients. Eosinophils express the trimeric IL-2R and are known to increase after IL-2 therapy. To date, no drug related increases in eosinophils with AU-007 +/- aldesleukin were observed, and in most cases the levels of circulating eosinophils decreased. IFN-g is a cytokine released after activation of the inflammatory/cytotoxic arm of the immune system. Twelve of the initial 17 patients for whom data are available demonstrated at least a 2-fold or greater increase in the circulating levels of IFN-g, often occurring within 48 hours after dosing. This was observed regardless of dose given. Higher frequency and amounts of IFN-g expression were observed when subcutaneous low dose aldesleukin was also given (dosed 2 – 24h post AU-007 dosing). A dose dependent trend with single-agent AU-007 was observed, leading to more patients with a &amp;gt;2-fold IFN-g. All patients who received low dose aldesleukin had at least 1 timepoint post dose with a &amp;gt;2-fold increase. Overall, AU-007 has demonstrated pharmacodynamic responses consistent with the mechanism of action, with decreases in the regulatory population of cells and increases in inflammatory cells and markers. The initial pharmacodynamic data support continued dose escalation of AU-007 +/- aldesleukin in patients with advanced cancer. Citation Format: Timothy Wyant, Sophia Frentzas, Elizabeth Ahern, Andrew Weickhardt, Andrew Haydon, John Powderly, Drew Rasco, Meredith McKean, Paul De Souza, Jenny Tang, Lori Richards, Aron Knckerbocker, Yanay Ofran, Inbar Amit, James Vasselli. Preliminary pharmacodynamic evaluation of AU-007 in phase 1 dose escalation trial in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C033.

Inflammatory Response to Intravenously Administered Lipopolysaccharide (LPS) in the Bone Marrow of Healthy Volunteers As a Model for Early Phase Drug Development

Introduction Inflammatory responses in the peripheral and central hematological compartments are important for identification of new biomarkers and drug targets in early phase drug development. There is an increasing interest to investigate the modulation of the inflammatory response upon experimental drug exposure using cellular and molecular analyses, specifically in the central compartment. The lipopolysaccharide (LPS) challenge model allows to study TLR4-mediated local and systemic inflammation and therefore the effects of anti-inflammatory compounds. In addition, there is an interest in the effects of TLR4 agonists on early immune cell populations. The aim of the current study is to investigate the effects of intravenous (i.v.) LPS on the peripheral blood and central bone marrow compartment and to perform a proof-of-methodology study for bone marrow analysis in early phase drug research in healthy volunteers. Methods This was an open-label study in 13 healthy young male subjects, aged 18 - 35 years (inclusive). The study was conducted at the Centre for Human Drug Research, Leiden, The Netherlands. The study protocol was approved by the Medical Ethics Committee. All subjects provided written informed consent prior to any study activity. Subjects were divided into control group (n=3), 1 ng/kg i.v. LPS group (n=5) and 2 ng/kg i.v. LPS group (n=5). Blood and bone marrow samples were collected at baseline and at 4h post-dose timepoints. Primary objective was to evaluate the safety and tolerability of bone marrow sampling in healthy volunteers, by continuous safety monitoring, visual analogue scale (VAS) for pain and subject experience questionnaires. To investigate the pharmacodynamic (PD) effects of i.v. LPS challenge on the bone marrow and whole-blood immune compartments, flow cytometry, cytokine analysis and RNA sequencing was performed. Results All 13 subjects completed the study. Adverse events were mild and reversible, and included puncture site pain related to bone marrow puncture, and flu-like symptoms related to LPS administration. Twelve subjects (92%) indicated they would undergo the procedure again in the future. There was an observable difference in VAS pain score between the baseline bone marrow puncture and LPS treatment groups, but no difference between 1 ng/kg and 2 ng/kg groups. Phenotypic changes of inflammatory signatures in peripheral blood and bone marrow cell populations upon LPS administration were detected by multi-parameter flow cytometry. An LPS-induced inflammatory cytokine profile was present. RNA sequencing revealed differential gene expression in inflammatory gene signatures between peripheral blood mononuclear cells and bone marrow cells. Conclusion We demonstrated that bone marrow analysis is feasible and well-tolerable in early phase studies with healthy volunteers and can be used to assess inflammatory responses in the central hematological compartment upon systemic LPS challenge. Access to bone marrow can provide important novel insights into drug effects on the hematopoiesis and differentiation of immune cells and provides added value to early drug development.

Micronuclei in human peripheral blood and bone marrow as genotoxicity markers: A systematic review and meta-analysis.

Can human peripheral blood cells be used as a surrogate for bone marrow cells, in evaluating the genotoxic effects of stressors? We searched the Pubmed/Medline and PubChem databases to identify publications relevant to this question. Micronucleus formation was the genotoxicity endpoint. Three publications comparing exposed vs. non-exposed individuals are included in this analysis; the exposures were to ethylene oxide or ionising radiation (atomic bomb, thorotrast, or radioiodine therapy). Information was extracted on the types of exposure, the numbers of participants, and the micronucleus frequencies. Relative differences (odds ratios) and absolute differences (risk differences) in the numbers of micronuclei between exposed and non-exposed persons were calculated separately for individual cell types (peripheral blood and bone marrow). Random effects meta-analyses for the relative differences in cell abnormalities were performed. The results showed very small differences in the frequencies of micronuclei between exposed and non-exposed individuals, as measured in either peripheral blood or bone marrow cell populations, on both absolute and relative scales. No definite conclusion concerning the relative sensitivities of bone marrow and peripheral blood cells can be made, based on these publications.

The landscape of innate and adaptive immune cell subsets in patients with adult-onset Still's disease.

Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disorder. The understanding of the changes in adaptive immune cells and the crosstalk between innate and adaptive immune systems in AOSD is limited. This study aimed to examine the peripheral immune cell composition and inflammatory protein levels in AOSD patients. Twenty-nine active AOSD patients were enrolled. Flow cytometry was used to analyze the cell populations in peripheral blood. Antibody chips were utilized to detect the protein expression profile in serum. In active AOSD patients, there was an increase in the percentage of classical and non-classical monocytes among peripheral blood mononuclear cells. The proportion of natural killer (NK) cells decreased, with an increase in CD56dim NK1 cells and a decrease in CD56bright NK2 cells compared with healthy controls (HC). The percentage of naïve central memory T cells was decreased, while the percentage of effector and effector memory T cells was increased among adaptive lymphocytes. The proportion of naïve B and memory B cells was decreased, while plasma cells were increased in AOSD patients, indicating activation of the adaptive immune system. Additionally, the serum levels of 40 proteins were elevated in AOSD patients, primarily involved in cytokine-cytokine receptor interaction, inflammatory response, and regulation of MAPK cascade. Our findings showed the activation of the innate and adaptive immune system in AOSD. The protein-protein interaction analysis suggested potential communication between innate and adaptive cell subsets. These findings provide new insights into the pathogenesis of the disease and the development of targeted therapies.

Interim results from a phase I/II study of duvelisib PI3Kδγ inhibitor and nivolumab in patients with advanced unresectable melanoma who have progressed on anti-PD1 therapy.

9540 Background: The majority of unresectable melanoma patients eventually progress on first-line checkpoint blockade therapy. Duvelisib is a potent phosphoinositide 3-kinase (PI3K) δ and γ isoform inhibitor approved for relapsed/refractory CLL. PI3Kγ inhibition has been shown to restore anti-PD1 activity in preclinical tumor models through conversion of tumor associated macrophages (TAMs) from an immunosuppressive to a pro-inflammatory state. PI3Kδ inactivation has been associated with impaired T regulatory cell (Treg) function and reduced intratumoral Treg numbers. We report study design and results of a phase I study to evaluate the safety and efficacy of duvelisib and nivolumab in anti-PD1 refractory patients. Methods: Utilizing a modified 3+3 design with escalating doses of duvelisib (15 mg daily, 25 mg daily, and 25 mg BID), patients with unresectable melanoma were treated with nivolumab and duvelisib until unacceptable toxicity or disease progression. Primary endpoints for the phase I portion were maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of duvelisib. Secondary endpoints include early and late toxicities and anti-tumor activity based on RECIST v1.1. Exploratory endpoints were to evaluate dose-dependent changes in the immune cell populations of peripheral blood and tumor tissue with duvelisib and nivolumab. Results: Ten patients have been enrolled (7 at dose level I, 3 at dose level II) with mean age 64. Two patients had BRAF V600E mutation and were previously treated with BRAF/MEK inhibitors. No dose limiting toxicities (DLT) were observed. Grade 3 or higher adverse events (AEs) attributable to study treatment occurred in 4 patients (40%), including G3 neutropenia (10%), G3 diarrhea (10%), G3 vomiting (10%) and G4 hepatitis (20%). Both instances of hepatitis resolved rapidly with 1-2mg/kg of prednisone. In one patient, hepatitis and nausea/vomiting led to discontinuation of treatment. Out of 6 patients who had follow-up imaging, 4 exhibited progressive disease, one stable disease, and one had partial response of 11 months duration. Given the published data on the regulation of macrophages and Tregs by PI3Kδγ inhibitors, we are utilizing multispectral imaging to measure the spatial interactions of key immune cells within the tumor microenvironment (T cells, B cells, and macrophages) in all matched pre-/post-treatment paraffin-embedded tissues, for which results will be presented. Conclusions: The combination of duvelisib and nivolumab was well-tolerated in patients with advanced unresectable melanoma refractory to ICI without DLTs to date at 15 and 25mg daily dosages. Significant anti-tumor activity was noted in one patient. Enrollment at dose level II is ongoing. Clinical trial information: NCT04688658 .

Does the upper respiratory tract microbiome change in dairy calves fed milk replacer containing probiotics?

Abstract Our overall hypothesis is that feeding probiotics to dairy calves will alter overall immunity and may additionally change the microbiome in the respiratory tract as well as the gut. A group of 20 dairy calves were split into two treatment groups: Control (N=10, milk replacer), and Probiotic (N=10, milk replacer + 0.5 g/day Bovamine Dairy). On day 0 birth weight was obtained and the calves were provided colostrum as per the dairy SOP. On day 2, probiotics were added to the milk replacer of the treated group. Blood was collected along with nasal and tonsil swabs on days 0, 7, 14, 21, 28, 42, and 48. Calves were monitored daily for fecal, nasal, and ocular discharge scores. Our preliminary data showed no significant differences (P&amp;lt;0.5) between the Control and Probiotic groups in immune cell populations in peripheral blood. DNA was extracted from nasal and tonsil swabs to evaluate the bacterial populations in the upper respiratory microbiome. Hypervariable regions 1 through 3 along the 16S ribosomal RNA gene were amplified by PCR and sequenced by Illumina MiSeq to determine the bacterial taxa present. Evaluation of these samples revealed a greater percentage of operational taxonomy units (OTU) were classified at the genus level in the tonsil compared to the nasal samples. The microbiome of the tonsils was more stable over time points compared to the nasal samples. Evaluation of each timepoint for nasal and tonsil samples identified bacterial taxa that changed in relative abundance with the addition of the probiotic compared to the control diet. Addition of probiotic changed the relative abundance of OTU (P&amp;lt;0.01) in the nasal and tonsil sampling sites and at multiple timepoints. Analyses are ongoing. Project supported by fund from the USDA Agricultural Research Service.

The immune system contributes to the effectiveness of vaccine therapy in patients with metastatic melanoma

The aim of the study was to identify differences in the immune system parameters between metastatic melanoma patients who responded and did not respond to dendritic cell vaccination.Material and Methods. The study group included 20 patients with stage III–IV metastatic melanoma, who received vaccine therapy with dendritic cells (DC) in a prophylactic mode. The control groups included 13 patients who had symptoms of disease progression at the time of starting vaccine therapy, and 5 healthy donors. The DC-vaccine was prepared in the form of a suspension of the patient’s autologous dendritic cells loaded with tumor antigens in vitro. A single dose had 2 million dendritic cells in 1 ml of phosphate buffer solution, which was administered intradermally in the nearest site to the regional lymphatic collectors. The immune system status was assessed before starting vaccination. The immune system status was evaluated according to the indices of 25 peripheral blood cell populations using multicolor flow cytometry and integral characteristic in the form of the visual image generated by the visualization method of multidimensional data (NovoSpark, Canada).Results. The immune status in patients with metastatic melanoma at the start of DC-vaccination differed and was associated with the effectiveness of subsequent vaccine therapy. The response to vaccination was observed in patients whose immune system status was similar to that of healthy individuals. Low efficacy of DC-vaccine therapy was shown in patients whose immune system status corresponded to that of patients with disease progression. Alterations of the immune system in patients with metastatic melanoma were registered both at the level of individual immunological parameters and at the level of visualized integral characteristics. The integral characteristics of the immune system associated with the patient’s immunocompromised status can be considered as a criterion for stratification of patients with metastatic melanoma for the effective DC-vaccine therapy.Conclusion. The effectiveness of vaccine therapy with dendritic cells in patients with metastatic melanoma is associated with the immune system state before starting this therapy.

Combined space stressors induce independent behavioral deficits predicted by early peripheral blood monocytes

Interplanetary space travel poses many hazards to the human body. To protect astronaut health and performance on critical missions, there is first a need to understand the effects of deep space hazards, including ionizing radiation, confinement, and altered gravity. Previous studies of rodents exposed to a single such stressor document significant deficits, but our study is the first to investigate possible cumulative and synergistic impacts of simultaneous ionizing radiation, confinement, and altered gravity on behavior and cognition. Our cohort was divided between 6-month-old female and male mice in group, social isolation, or hindlimb unloading housing, exposed to 0 or 50 cGy of 5 ion simplified simulated galactic cosmic radiation (GCRsim). We report interactions and independent effects of GCRsim exposure and housing conditions on behavioral and cognitive performance. Exposure to GCRsim drove changes in immune cell populations in peripheral blood collected early after irradiation, while housing conditions drove changes in blood collected at a later point. Female mice were largely resilient to deficits observed in male mice. Finally, we used principal component analysis to represent total deficits as principal component scores, which were predicted by general linear models using GCR exposure, housing condition, and early blood biomarkers.

Type 2 and type 17 effector cells are increased in the duodenal mucosa but not peripheral blood of patients with functional dyspepsia.

Functional dyspepsia is characterised by chronic symptoms of post-prandial distress or epigastric pain not associated with defined structural pathology. Increased peripheral gut-homing T cells have been previously identified in patients. To date, it is unknown if these T cells were antigen-experienced, or if a specific phenotype was associated with FD. This study aimed to characterise T cell populations in the blood and duodenal mucosa of FD patients that may be implicated in disease pathophysiology. We identified duodenal T cell populations from 23 controls and 49 Rome III FD patients by flow cytometry using a surface marker antibody panel. We also analysed T cell populations in peripheral blood from 37 controls and 61 patients. Where available, we examined the number of duodenal eosinophils in patients and controls. There was a shift in the duodenal T helper cell balance in FD patients compared to controls. For example, patients had increased duodenal mucosal Th2 populations in the effector (13.03 ± 16.11, 19.84 ± 15.51, p=0.038), central memory (23.75 ± 18.97, 37.52 ± 17.51, p=0.007) and effector memory (9.80±10.50 vs 20.53±14.15, p=0.001) populations. Th17 populations were also increased in the effector (31.74±24.73 vs 45.57±23.75, p=0.03) and effector memory (11.95±8.42 vs 18.44±15.63, p=0.027) subsets. Peripheral T cell populations were unchanged between FD and control. Our findings identify an association between lymphocyte populations and FD, specifically a Th2 and Th17 signature in the duodenal mucosa. The presence of effector and memory cells suggest that the microinflammation in FD is antigen driven.