Cell Populations In Peripheral Blood Research Articles

DNA methylome-based validation of induced sputum as an effective protocol to study lung immunity: construction of a classifier of pulmonary cell types

ABSTRACT Flow cytometry is a classical approach used to define cell types in peripheral blood. While DNA methylation signatures have been extensively employed in recent years as an alternative to flow cytometry to define cell populations in peripheral blood, this approach has not been tested in lung-derived samples. Here, we compared bronchoalveolar lavage with a more cost-effective and less invasive technique based on sputum induction and developed a DNA methylome-based algorithm that can be used to deconvolute the cell types in such samples. We analysed the DNA methylome profiles of alveolar macrophages and lymphocytes cells isolated from the pulmonary compartment. The cells were isolated using two different methods, sputum induction and bronchoalveolar lavage. A strong positive correlation between the DNA methylome profiles of cells obtained with the two isolation methods was found. We observed the best correlation of the DNA methylomes when both isolation methods captured cells from the lower parts of the lungs. We also identified unique patterns of CpG methylation in DNA obtained from the two cell populations, which can be used as a signature to discriminate between the alveolar macrophages and lymphocytes by means of open-source algorithms. We validated our findings with external data and obtained results consistent with the previous findings. Our analysis opens up a new possibility to identify different cell populations from lung samples and promotes sputum induction as a tool to study immune cell populations from the lung.

Submicron particle docetaxel intratumoral injection in combination with anti-mCTLA-4 into 4T1-Luc orthotopic implants reduces primary tumor and metastatic pulmonary lesions

We describe here characterization of the response of local and metastatic disease and immunomodulation following intratumoral (IT) injection of submicron particle docetaxel (SPD) administered alone or in combination with systemic antibody anti-mCTLA-4 (anti-mCTLA-4) in the metastatic 4T1-Luc2-1A4 (4T1) murine breast cancer model. In-life assessments of treatment tolerance, tumor volume (TV), and metastasis were performed (n = 10 animals/group). At study end, immune cell populations in tumor-site tissues and peripheral blood were analyzed using flow cytometry. Signs of distress typical of this aggressive tumor model occurred across all animals except for the combination treated which were asymptomatic and gained weight. TV at study end was significantly reduced in the combination group versus untreated [43% reduced (p < 0.05)] and vehicle controls [54% reduced (p < 0.0001)]. No evidence of thoracic metastasis was found in 40% of combination group animals and thoracic bioluminescence imaging (BLI) was reduced vs. untreated controls (p < 0.01). Significant elevations (p < 0.05) in CD4 + T, CD4 + helper T, Treg, and NKT cells were found in tumor and blood in SPD or combination treatment compared to controls or anti-mCTLA-4. Combination treatment increased tumor-associated CD8 + T cells (p < 0.01), peripheral B cells (p < 0.01), and tumor associated and circulating dendritic cells (DC) (p < 0.05). Tumor-associated NK cells were significantly increased in SPD ± anti-mCTLA-4 treatments (p < 0.01). Myeloid-derived suppressor cells (MDSC) were reduced in bloods in SPD ± anti-mCTLA-4 groups (p < 0.05). These data demonstrate that both SPD and anti-mCTLA-4 produce local anti-tumor effects as well as reductions in metastasis which are significantly enhanced when administered in combination.

Glucocorticoids Reduce the Number and Function of CD19+CD24hiCD38hi Regulatory B Cells in Patients with Immune Thrombocytopenia

IntroductionIL-10-producing regulatory B cells (Bregs) are important modulators of immune regulation, and in humans this population has been shown to be enriched within the CD19+CD24hiCD38hi B cell population in peripheral blood. The production of IL-10 by Bregs can be induced through activation of B cells through toll like receptor (TLR) and B cell receptor (BCR) ligation, or indirectly through T cell-mediated activation via CD40:CD40L interactions. Loss of Breg-mediated suppression, either through numerical deficiency or loss of function, has been implicated in the pathogenesis of several autoimmune and inflammation-mediated diseases, including chronic Immune Thrombocytopenia (ITP; Li et al, 2012, Blood, 120:3318-3325). The purpose of this study was to extend this observation by interrogating the effect of systemic immunosuppressive treatment, in particular with glucocorticoids (GCs), on Bregs from the peripheral blood of patients with ITP.MethodsFollowing informed written consent, peripheral blood was drawn from patients with ITP registered at University Hospitals Bristol, UK, and healthy volunteers. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, and multiparameter flow cytometry performed to quantify the frequency of CD19+CD24hiCD38hi B cells. For functional studies, CD19+ B cells and CD4+ T cells were isolated from the peripheral blood of healthy donors using fluorescence activated cell sorting (FACS) and subsequently co-cultured in a 1:1 ratio. Cells were activated with either anti-CD3 (indirect B cell activation via T cells) or a combination of CpG and IgM (direct B cell activation), in the presence or absence of the synthetic GC Dexamethasone (Dex). After 72 hours, intracellular IL-10 expression in B cells was quantified by flow cytometry.ResultsNo significant difference in the proportion of CD19+CD24hiCD38hi Bregs was seen between newly diagnosed ITP patients (n=7) before treatment had started, compared with healthy donors (n=7), (6.5% vs 8.2%, p=0.3176; Figure 1). However, patients receiving GC treatment (n=8) had a significant reduction in the proportion of Bregs when compared with both healthy donors (2.5% vs 8.2%, p=0.0012), and untreated ITP patients (2.5% vs 6.5%, p=0.0093). Similarly, ITP patients receiving another commonly used immunosuppressive drug, mycophenolate mofetil (MMF, n=10), had a significant reduction in the proportion of Bregs compared with both healthy donors (1.7% vs 8.2%, p=0.0004), and untreated ITP patients (1.7% vs 6.5%, p=0.0042). Patients treated with a thrombopoietin (TPO) mimetic (n=6) (not immunosuppressive) had no reduction in the proportion of Bregs compared with untreated ITP patients (5.37% vs 6.49% p=0.4452). Furthermore, in cultures activated by CpG and IgM, Dex did not significantly affect the number of IL-10-producing B cells (5.4% vs 5.4%, p=0.9232, n=10). However, in anti-CD3 activated cultures, Dex significantly reduced the proportion of IL-10 producing B cells (2.0% vs 0.8%, p=0.0094, n=8).ConclusionsThese observations suggest that a deficiency of Bregs in the peripheral blood is not a result of disease activity in ITP patients, but rather the consequence of immunosuppressive drug treatment. Consistent with this, the effect of Dex on B cell IL-10 production is likely to be an indirect phenomenon through the suppression of T cell-mediated activation of Breg function. Overall, these observations show that immunosuppressive treatments such as GCs can deplete Breg numbers in the peripheral blood and, through a T cell mediated mechanism, reduce their ability to produce the immunoregulatory cytokine IL-10. [Display omitted] DisclosuresBradbury:BMS Pfizer: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Other: Travel support; Bayer: Other: Conference attendence; Amgen: Other: Conference attendence. Lee:University of Bristol: Patents & Royalties: International Patent Application No: PCT/GB2014/053804 / US Patent application No. 61/919,404 (A biomarker and companion therapeutic for steroid refractory diseases); EMD Serono: Consultancy; Nanomerics: Consultancy; Roche: Consultancy, Research Funding.

Novel spontaneous myelodysplastic syndrome mouse model.

BackgroundMyelodysplastic syndrome (MDS) is a group of disorders involving hemopoietic dysfunction leading to leukemia. Although recently progress has been made in identifying underlying genetic mutations, many questions still remain. Animal models of MDS have been produced by introduction of specific mutations. However, there is no spontaneous mouse model of MDS, and an animal model to simulate natural MDS pathogenesis is urgently needed.MethodsIn characterizing the genetically diverse mouse strains of the Collaborative Cross (CC) we observed that one, designated JUN, had abnormal hematological traits. This strain was thus further analyzed for phenotypic and pathological identification, comparing the changes in each cell population in peripheral blood and in bone marrow.ResultsIn a specific‐pathogen free environment, mice of the JUN strain are relatively thin, with healthy appearance. However, in a conventional environment, they become lethargic, develop wrinkled yellow hair, have loose and light stools, and are prone to infections. We found that the mice were cytopenic, which was due to abnormal differentiation of multipotent bone marrow progenitor cells. These are common characteristics of MDS.ConclusionsA mouse strain, JUN, was found displaying spontaneous myelodysplastic syndrome. This strain has the advantage over existing models in that it develops MDS spontaneously and is more similar to human MDS than genetically modified mouse models. JUN mice will be an important tool for pathogenesis research of MDS and for evaluation of new drugs and treatments.

Linalool Inhibits MCF-7 Tumor Growth in a Xenograft Model by Apoptosis Induction and Immune Modulation

In this study, the anti-cancer activity of linalool was investigated in MCF-7 breast cancer-bearing mice. Natural killer (NK) and B cell populations in peripheral blood were studied by flow cytometry. The expressions of proliferating cell nuclear antigen (PCNA) and Ki-67 in xenograft tumors were evaluated by immunohistochemistry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to investigate apoptosis induction in an in vivo model. The results indicated that linalool possesses an inhibitory effect on breast cancer growth in the xenograft model. Linalool reduced B cell counts, but increased NK cell counts in mice peripheral blood. The immunosignals of PCNA and Ki-67 were significantly lower in the linalool treatment group than those of the control group. The TUNEL assay showed that linalool significantly induced apoptosis compared to the control group. The findings of this study provide insight and evidence on the antiproliferative activity of linalool on human breast cancer.

Acute radiation syndrome-related gene expression in irradiated peripheral blood cell populations

Purpose In a nuclear or radiological event, an early diagnostic tool is needed to distinguish the worried well from those individuals who may later develop life-threatenFing hematologic acute radiation syndrome. We examined the contribution of the peripheral blood's cell populations on radiation-induced gene expression (GE) changes. Materials and methods EDTA-whole-blood from six healthy donors was X-irradiated with 0 and 4Gy and T-lymphocytes, B-lymphocytes, NK-cells and granulocytes were separated using immunomagnetic methods. GE were examined in cell populations and whole blood. Results The cell populations contributed to the total RNA amount with a ratio of 11.6 for T-lymphocytes, 1.2 for B-cells, 1.2 for NK-cells, 1.0 for granulocytes. To estimate the contribution of GE per cell population, the baseline (0Gy) and the radiation-induced fold-change in GE relative to unexposed was considered for each gene. The T-lymphocytes (74.8%/80.5%) contributed predominantly to the radiation-induced up-regulation observed for FDXR/DDB2 and the B-lymphocytes (97.1%/83.8%) for down-regulated POU2AF1/WNT3 with a similar effect on whole blood gene expression measurements reflecting a corresponding order of magnitude. Conclusions T- and B-lymphocytes contributed predominantly to the radiation-induced up-regulation of FDXR/DDB2 and down-regulation of POU2AF1/WNT3. This study underlines the use of FDXR/DDB2 for biodosimetry purposes and POU2AF1/WNT3 for effect prediction of acute health effects.

An In Vitro Co-Culture Model of Bone Marrow Mesenchymal Stromal Cells and Peripheral Blood Mononuclear Cells Promotes the Differentiation of Myeloid Angiogenic Cells and Pericyte-Like Cells.

Mesenchymal stromal cells (MSCs) are known to stimulate the survival and growth of endothelial cells (ECs) by producing paracrine signals, as well as to differentiate into pericytes and thereby support blood vessel formation and stability. On the other hand, cells with an EC-like phenotype have been found within the CD14+ and CD34+ cell populations of peripheral blood (PB) mononuclear cells (MNCs). The aim of this study was to investigate the proangiogenic differentiation potential of human MSC-MNC co-cultures. Bone marrow-derived MSCs (2,500 cells/cm2) were co-cultured with MNCs (50,000 cells/cm2), which were isolated from the PB of healthy donors. MSCs and MNCs cultured alone at same cell densities were used as controls. Cells in MNC fraction and in co-cultures were isolated for CD14, CD34, and CD31 surface markers with magnetic-activated cell sorting. Co-cultures were analyzed for cell proliferation and morphology, as well as for the expression of various hematopoietic, endothelial, and pericyte markers by immunocytochemistry, quantitative PCR (qPCR), and flow cytometry. Vascular endothelial growth factor (VEGF) expression and secretion was measured with qPCR and enzyme-linked immunosorbent assay, respectively. Our results show that in co-cultures with MSCs, CD14+CD45+ MNCs differentiated into spindle-shaped, nonproliferative, EC-like, myeloid angiogenic cells (MACs) expressing CD31, but also into pericyte-like cells expressing neural/glial antigen 2 (NG2) and CD146. Functionality of the isolated MACs was demonstrated in co-cultures with human umbilical vein endothelial cells, where they supported the formation of tube-like structures. NG2+ cells of MNC-origin were found among both CD34-CD14+ and CD34-CD14- cell populations, indicating the existence of different subtypes of pericyte-like cells. In addition, VEGF was shown to be secreted in MSC-MNC co-cultures, mainly by MSCs. In conclusion, MSCs were shown to possess proangiogenic capacity in MSC-MNC co-cultures as they supported the differentiation of functional MACs, as well as the differentiation of pericyte-like cells of MNC origin. This phenomenon was mediated at least partially via secreted VEGF.

Abstract PS17-37: Immune cell populations in peripheral blood of metastatic breast cancer patients under CDK4/6 inhibitors

Abstract BackgroundCyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have changed the paradigm of Estrogen-receptor positive (ER+) / human epidermal growth factor receptor 2 negative (HER2-) breast cancer (BC) treatment and represent a new standard of care in metastatic setting. Neutropenia is a well-established adverse event associated with CDK4/6i treatment, but the impact of these agents in BC patients’ immune profile is unknown. This study aimed to characterize changes in host circulating immune cell subsets in BC patients undergoing CDK4/6i therapy and investigate how these changes associate with clinical benefit, by assessing progression-free survival (PFS). MethodsA prospective cohort of metastatic ER+/HER2- BC patients treated with CDK4/6i (palbociclib or ribociclib) was included. Baseline and every 12−14-week peripheral blood samples were collected and objective tumor status evaluated by RECIST criteria at the same time points. Cohort was categorized in CDK4/6i therapy non-responders (CDK4/6i NResp, progression &amp;lt; 6.0 months) and responders (CDK4/6i Resp, PFS ≥6.0 months) using primary resistance to endocrine therapy definition of metastatic BC. A total of 66 different immune populations were assessed by flow cytometry, mainly: B cells, natural killer T (NKT), natural killer (NK), effector (Eff; CD4+, CD8+ and γδ T), effector memory (EM; CD4+, CD8+ and γδ T), central memory (CM; CD4+, CD8+ and γδ T) and naive (CD4+, CD8+ and γδ T), and regulatory T (Treg subtypes I, II and III) cells. Statistical analysis of longitudinal data was performed using Linear Mixed Effects models (LME) and Generalized estimation equations models (GEE) in R (version 3.6.1) and RStudio (version 1.2.5019). Models allowed to estimate and compare variation rates of each cell population in time. ResultsA total of 26 patients were included, with a median age of 58 years (min-max, 27−79). All patients received letrozole or fulvestrant, together with LHRH agonist if premenopausal. Fifteen patients received palbociclib and 11 ribociclib, with 17 (65.4%) and 9 (34.6%) patients treated in 1st and 2nd line, respectively.With a median follow-up of 17.18 months (CI 95% 12.84 - 21.52), 6 (23.1%) patients were CDK4/6i NResp, with a median PFS (mPFS) of 3.61 months (IQR 2.28 - 4.42); only two patients in 1st line setting had PFS &amp;lt;6.0 months. Twenty (76.9%) patients were CDK4/6i Resp, with mPFS of 11.97 months (IQR 8.86 - 19.15), 16 (61.5%) of which treated in 1st line.In CDK4/6i NResp subgroup, an increase was identified in CD4/CD8 ratio (1.01%/month, p=0.001), Treg subtype III (2.74%/month, p=0.0008), CD8EM T cells (5.23%/month, p=0.029), and γδ1Eff subset of γδ T cells (6.30%/month, p=0.007). Interestingly, in CDK4/6i Resp subgroup no significant changes were observed in the same cell populations. Additionally, a decrease in total T (-2.38%/month, p=0.002), Treg subtype II (-2.52%/month, p=0.0076), CD8CM T (-3.30%/month, p=0.006), CD4CM T (-3.97%/month, p=0.002), γδ1CM subset of γδ T (-2.73%/month, p=0.047), NK (-0.99%/month, p=0.011), and CD8Eff T (-1.72%/month, p&amp;lt;0.001) cells was observed in CDK4/6i NResp subgroup, with no significant changes in Resp subgroup. Conclusions Results suggest that significant immune profile changes occur in metastatic ER+/HER2- BC patients with early progression to CDK4/6i and endocrine therapy. Citation Format: Soraia Lobo-Martins, Patrícia Corredeira, Patrícia Borges Alves, Marília Antunes, Ângela Rodrigues, António Quintela, André Mansinho, Pedro Filipe, Leonor Ribeiro, Rita Teixeira de Sousa, Conceição Pinto, Catarina Abreu, Gisela Gordino, Julie Ribot, Karine Serre, Marta Martins, Ana Cavaco, Sandra Casimiro, Bruno Silva-Santos, Luís Costa. Immune cell populations in peripheral blood of metastatic breast cancer patients under CDK4/6 inhibitors [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-37.

Biological sex, not reproductive cycle, influences peripheral blood immune cell prevalence in mice.

Traditionally the female sex, compared with the male sex, has been perceived as having greater variability in many physiological traits, including within the immune system. We investigated effects of biological sex and the female reproductive cycle on numbers of circulating leukocytes in C57BL/6J mice. We show that biological sex, but not female reproductive cyclicity, has a significant effect on peripheral blood immune cell prevalence and variability, and that sex differences were not consistent amongst common inbred laboratory mouse strains. We found that male C57BL/6J mice, compared with female mice, have greater variability in peripheral blood immunophenotype, and that this was influenced by body weight. We created summary tables for researchers to facilitate experiment planning and sample size calculations for peripheral immune cells that consider the effects of biological sex. Immunophenotyping (i.e. quantifying the number and types of circulating leukocytes) is used to characterize immune changes during health and disease, and in response to pharmacological and other interventions. Despite the importance of biological sex in immune function, there is considerable uncertainty amongst researchers as to the extent to which biological sex or the female reproductive cycle influence blood immunophenotype. We quantified circulating leukocytes by multicolour flow cytometry in young C57BL/6J mice and assessed the effects of the reproductive cycle, biological sex, and other experimental and biological factors on data variability. We found that there are no significant effects of the female reproductive cycle on the prevalence of peripheral blood B cells, NK cells, CD4+ T cells, CD8+ T cells, monocytes, or neutrophils. Immunophenotype composition and variability do not significantly change between stages of the female reproductive cycle. There are, however, sex-specific differences in immune cell prevalence, with fewer monocytes, neutrophils, and NK cells in female mice. Surprisingly, immunophenotype is more variable in male mice, and weight is a significant contributing factor. We provide tools for researchers to perform a priori sample size calculations for two-group and factorial analyses. We show that immunophenotype varies between inbred mouse strains, and that using equal sample sizes of male and female mice is not always appropriate for within-sex evaluations of immune cell populations in peripheral blood.

Frequency and perturbations of various peripheral blood cell populations before and after eculizumab treatment in paroxysmal nocturnal hemoglobinuria

While red blood cells (RBCs) and granulocytes have been more studied, platelets and reticulocytes are not commonly used in paroxysmal nocturnal hemoglobinuria (PNH) flow-cytometry and less is known about susceptibility to complement-mediated destruction and effects of anti-complement therapy on these populations.We performed flow-cytometry of RBCs and granulocytes in 90 PNH patients and of platelets and reticulocytes in a subgroup (N = 36), to unveil perturbations of these populations during PNH disease course before and after anti-complement treatment.We found that platelets and reticulocytes were less sensitive to complement-mediated lysis than RBCs but not as resistant as granulocytes, as shown by mean sensitive fraction (difference in a given PNH population vs. PNH granulocyte clone size). In treated patients, reticulocytes, platelets, RBCs (with differences between type II and III) and granulocytes significantly increased post-treatment, confirming the role of PNH hematopoiesis within the context of anti-complement therapy. Moreover, we found that PNH platelet clone size reflects PNH granulocyte clone size. Finally, we established correlations between sensitive fraction of PNH cell-types and thrombosis.In sum, we applied a flow-cytometry panel for investigation of PNH peripheral blood populations' perturbations before and after eculizumab treatment to explore complement-sensitivity and kinetics of these cells during the disease course.

Alcoholic hepatitis and metabolic disturbance in female mice: a more tractable model than Nrf2-/- animals.

ABSTRACTAlcoholic hepatitis (AH) is the dramatic acute presentation of alcoholic liver disease, with a 15% mortality rate within 28 days in severe cases. Research into AH has been hampered by the lack of effective and reproducible murine models that can be operated under different regulatory frameworks internationally. The liquid Lieber-deCarli (LdC) diet has been used as a means of ad libitum delivery of alcohol but without any additional insult, and is associated with relatively mild liver injury. The transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) protects against oxidative stress, and mice deficient in this molecule are suggested to be more sensitive to alcohol-induced injury. We have established a novel model of AH in mice and compared the nature of liver injury in C57/BL6 wild-type (WT) versus Nrf2−/− mice. Our data showed that both WT and Nrf2−/− mice demonstrate robust weight loss, and an increase in serum transaminase, steatosis and hepatic inflammation when exposed to diet and ethanol. This is accompanied by an increase in peripheral blood and hepatic myeloid cell populations, fibrogenic response and compensatory hepatocyte regeneration. We also noted characteristic disturbances in hepatic carbohydrate and lipid metabolism. Importantly, use of Nrf2−/− mice did not increase hepatic injury responses in our hands, and female WT mice exhibited a more-reproducible response. Thus, we have demonstrated that this simple murine model of AH can be used to induce an injury that recreates many of the key human features of AH – without the need for challenging surgical procedures to administer ethanol. This will be valuable for understanding of the pathogenesis of AH, for testing new therapeutic treatments or devising metabolic approaches to manage patients whilst in medical care.This article has an associated First Person interview with the joint first authors of the paper.

High-Fat Diet and Feeding Regime Impairs Number, Phenotype, and Cytotoxicity of Natural Killer Cells in C57BL/6 Mice.

Overweight and obesity are major public health challenges worldwide. Obesity is associated with a higher risk for the development of several cancer types, but specific mechanisms underlying the link of obesity and cancer are still unclear. Natural killer (NK) cells are circulating lymphoid cells promoting the elimination of virus-infected and tumor cells. Previous investigations demonstrated conflicting results concerning the influence of obesity on functional NK cell parameters in small animal models. The aim of the present study was to clarify potential obesity-associated alterations of murine NK cells in vivo, implementing different feeding regimes. Therefore, C57BL/6 mice were fed a normal-fat diet (NFD) or high-fat diet (HFD) under restrictive and ad libitum feeding regimes. Results showed diet and feeding-regime dependent differences in body weight, visceral fat mass and plasma cytokine concentrations. Flow cytometry analyses demonstrated significant changes in total cell counts as well as frequencies of immune cell populations in peripheral blood comparing mice fed NFD or HFD in an ad libitum or restrictive manner. Mice fed the HFD showed significantly decreased frequencies of total NK cells and the mature CD11b+CD27+ NK cell subset compared to mice fed the NFD. Feeding HFD resulted in significant changes in the expression of the maturation markers KLRG1 and CD127 in NK cells. Furthermore, real-time PCR analyses of NK-cell related functional parameters in adipose tissue revealed significant diet and feeding-regime dependent differences. Most notable, real-time cytotoxicity assays demonstrated an impaired cytolytic activity of splenic NK cells toward murine colon cancer cells in HFD-fed mice compared to NFD-fed mice. In conclusion, our data demonstrate that feeding a high-fat diet influences the frequency, phenotype and function of NK cells in C57BL/6 mice. Interestingly, restricted feeding of HFD compared to ad libitum feeding resulted in a partial prevention of the obesity-associated alterations on immune cells and especially on NK cells, nicely fitting with the current concept of an advantage for interval fasting for improved health.

Role of fibrocytes and endothelial progenitor cells among low-differentiated CD34+ cells in the progression of lung sarcoidosis

BackgroundSarcoidosis is a multisystemic granulomatous disease with still unknown etiology. Our previous studies showed a significantly higher percentage of CD34 + cells in the peripheral blood in patients with sarcoidosis (SA) compared to the control group. The objective of the present study was to characterized of the CD34 + cell population in peripheral blood in patients with SA with reference to the control group. Moreover in patients with SA, fibrocytes and endothelial cells were analysed and their relationship to the fibrosis process based on assessment of diffusing capacity for carbon monoxide (DLCO).MethodsData from patients diagnosed with SA at Military Institute of Medicine (Warsaw, Poland) between January 2018 and December 2019 were collected and analysed ongoing basis. Peripheral blood was collected from 26 patients with newly diagnosed pulmonary SA and 16 healthy subjects. The immunomagnetic method and flow cytometry were used. Among the CD34+ progenitor cells were assessed: low-differentiated cells, hematopoietic progenitor cells and endothelial progenitor cells. The Statistica 12.0 software was used for a statistical analysis.ResultsWe observed a significantly higher percentage of low-differentiated cells (13.8 vs. 2.3, P = 0.001) and endothelial cells (0.3 vs. 0.0, P = 0.001) in patients with SA compared to the control group. In the study group the median proportion of fibrocytes was 1.877% (0.983–2.340) in patients with DLCO< 80%, while in patients with DLCO> 80% was 0.795% (0.139–1.951) (P = 0.72). The median proportion of endothelial progenitor cells was higher in patients with DLCO< 80%: 0.889% (0.391–1.741), than in patients with DLCO> 80%: 0.451% (0.177–0.857) (P = 0.44).ConclusionsIn conclusion we demonstrated for the first time the immunophenotype of peripheral CD34 + cells with the degree of their differentiation. The study confirmed the involvement of low differentiated cells and endothelial cells in patients with SA.

Normal or reactive minor cell populations in bone marrow and peripheral blood mimic minimal residual leukemia by flow cytometry.

Measurable residual disease (MRD) is a strong independent poor prognostic factor for acute leukemia. Multiparameter flow cytometry (FCM) is a commonly used MRD detection method. However, FCM MRD detection is not well standardized, and the interpretation is subjective. There are normal/reactive minor cell populations in bone marrow (BM) and peripheral blood (PB), which could be confused with MRD. The FCM data of 231 BM and 44 PB pediatric samples performed in a recent 15-month period were retrospectively reviewed. These samples were from 56 B-lymphoblastic leukemia (B-ALL) patients, 11 T-lymphoblastic leukemia (T-ALL) patients, 28 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) patients, 44 cytopenia/leukocytosis patients, and five patients with mycosis fungoides. There were over 10 normal or reactive minor cell populations identified with certain phenotypes mimicking MRD of acute leukemia. These mimickers included CD19+ NK cells, CD22+ basophils, CD22+ dendritic cells (DCs), and plasma cells for B-ALL MRD; CD4/8 double-negative T cells, CD4/8 double-positive T cells, cytoplasmic CD3+ NK cells, CD2- T cells, CD7- T cells, CD5- gamma delta T cells, CD56+ NKT cells for T-ALL MRD; CD33+ NK cells, CD117+ NK cells, basophils, plasmacytoid DCs, non-classical monocytes, CD56+ and/or CD61+ monocytes for AML MRD. These data confirm the presence of a variety of normal/reactive minor cell populations that could mimic MRD of acute leukemia by FCM. Recognizing these MRD mimickers is important for correct FCM MRD interpretation.

Bioimprint Mediated Label-Free Isolation of Pancreatic Tumor Cells from a Healthy Peripheral Blood Cell Population.

New techniques are required for earlier diagnosis and response to treatment of pancreatic cancer. Here, a label-free approach is reported in which circulating pancreatic tumor cells are isolated from healthy peripheral blood cells via cell bioimprinting technology. The method involves pre-fabrication of pancreatic cell layers and sequential casting of cell surfaces with a series of custom-made resins to produce negative cell imprints. The imprint is functionalized with a combination of polymers to engineer weak attraction to the cells which is further amplified by the increased area of contact with the matching cells. A flow-through bioimprint chip is designed and tested for selectivity toward two pancreatic tumor cell lines, ASPC-1 and Mia-PaCa-2. Healthy human peripheral blood mononuclear cells (PBMCs) are spiked with pancreatic tumor cells at various concentrations. Bioimprints are designed for preferential retention of the matching pancreatic tumor cells and with respect to PBMCs. Tumor bioimprints are capable of capturing and concentrating pancreatic tumor cells from a mixed cell population with increased retention observed with the number of seedings. ASPC-1 bioimprints preferentially retain both types of pancreatic tumor cells. This technology could be relevant for the collection and interrogation of liquid biopsies, early detection, and relapse monitoring of pancreatic cancer patients.

Abstract 5590: Treatment of systemic inflammation significantly improves survival in a murine model that recapitulates the rapid health deterioration of advanced cancer

Abstract Cancer immunology research has been centered on how to generate anti-tumor immunity. However, unambiguous clinical evidence has shown that in advanced cancer patients, the tumors induce extensive immune system disorder, i.e., systemic inflammation. In this study, we investigated the impact of systemic inflammation on survival and efficacy of immunotherapy. We found that systemic inflammation drives mice health deterioration during disease progression to late stage and treating the systemic inflammation, with no need to inhibit tumor growth, improves health condition and extends survival. These findings suggest that systemic inflammation in advanced cancer might be an important therapeutic target. Methods 7-week old BALB/c mice were inoculated with 1 x106 C26 tumor cells subcutaneously. Mice body weight and health condition were measured starting 7 days after tumor inoculation. At various time points, peripheral blood was drawn, and immune cells was analyzed with flow cytometry; serum cytokines were measured with mouse cytokine array. Tumor bearing mice were treated with anti-PD1 antibody and anti-inflammation drugs and the treatment effect was monitored. Results Localized tumor growth progressively induces host immune system disorder with increase of Gr1+ cell population and decrease of CD3+ cell population in peripheral blood. When mice began to lose body weight, the cell percentage imbalance was enlarged (Gr1+ %: Naïve 7.90 ± 0.727 vs tumor 72.15 ± 4.143, p&amp;lt;0.0001; CD3+%: Naïve 41.18±2.023 vs tumor 9.18 ±0.249, p&amp;lt;0.0001). The immune cell imbalance along health deterioration (&amp;gt;15% body weight loss) further exacerbates (Gr1+ %: early 72.15 ± 4.143 vs late 93.83 ± 1.971, p=0.0032; CD3+ %: early 9.18±0.249 vs late 6.25±1.085, p=0.0392). In addition, pro-inflammatory cytokines such as IL-1, IL-6 and TNF-a increase expression in mouse serum accompanying health condition deterioration. More importantly, therapeutic interventions with anti-inflammation drug Ketorolac or by depletion of Gr1+ inflammatory cells, could reverse the deterioration process and significantly increase mice survival. Interestingly, when combined with anti-PD-1 antibody, treating systemic inflammation enhances the anti-tumor efficacy of the antibody. Conclusions Progressive systemic inflammation characterized by severe immune cell imbalance and overexpression of multiple inflammatory cytokines drives the rapid overall health deterioration of late stage cancer. Treating the systemic inflammation not only reverses health deterioration but also significantly increases survival. Therefore, targeting systemic inflammation might be a promising approach to the treatment of late stage cancer. Citation Format: Lingbing Zhang, Gennie Liu. Treatment of systemic inflammation significantly improves survival in a murine model that recapitulates the rapid health deterioration of advanced cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5590.

Abstract 951: The effect of neo-adjuvant chemotherapy on immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients

Abstract Introduction: The phase II neoadjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy (docetaxel, carboplatin) in combination with trastuzumab, trastuzumab and lapatinib or lapatinib alone in HER2+ breast cancer patients. Lapatinib is a dual HER2/EGFR tyrosine kinase inhibitor. Trastuzumab is a monoclonal antibody which targets HER2 and is capable of eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by immune effector cells. Studies have shown that tumour infiltrating lymphocyte (TIL) levels and the cytotoxic capacity of circulating immune cells can correlate with response to trastuzumab. Less is known regarding the effect of chemotherapy on the immune response to trastuzumab. This study examines the effects of neo-adjuvant chemotherapy on the phenotype and cytotoxic capacity of peripheral blood mononuclear cells (PBMCs) of HER2+ breast cancer patients. Methods: Matched blood samples were taken pre- and post-neoadjuvant treatment. PBMCs were isolated by density centrifugation and frozen. Direct PBMC-mediated cytotoxicity and trastuzumab-ADCC levels were assessed against the HER2+ breast cancer cell line (SKBR3) and non-MHC class I-restricted leukemic cell line (K562) using a FACS based assay (n=19 matched pre-and post-treatment samples). The immunophenotype of 17 pre-treatment and 13 post-treatment samples was also determined using the DURAClone IM antibody panel (CD16, CD56, CD19, CD14, CD4, CD8, CD3, CD45) on the CytoFLEX platform. Analysis of both data sets was performed using FCS Express and MedCalc. Results: The cytotoxic capacity of PBMCs was reduced following neo-adjuvant chemotherapy. Direct cytotoxicity elicited against the K562 cell line was reduced post-treatment (p=0.04). ADCC elicited against the SKBR3 cell line was also decreased in post-treatment samples (p=0.009). A decrease in cytotoxic capacity was associated with alterations in the immunophenotype of the post-treatment PBMC samples. The proportion of CD56+ NK cells (p=0.001), CD19+ B cells (p=0.001) and CD14+ monocytes (p=0.03) decreased significantly post-treatment. CD56+ NK cells are hypothesised to be the main ADCC effector cell population in peripheral blood. Interestingly, the proportion of CD3+ T cells (p=0.002), including CD4+ (p=0.007) and CD8+ (p=0.035) T cell populations, were increased post-treatment. Conclusion: These results indicate that neo-adjuvant chemotherapy reduces the cytotoxic capacity of circulating immune cells and alters the proportion of adaptive and innate immune cell subsets. These effects warrant further investigation particularly with the emergence of immunotherapies in HER2+ breast cancer. Citation Format: Nicola Gaynor, Alfonso Blanco, Alexandra Canonici, Alexander J. Eustace, Martina McDermott, Barry Moran, Jean M. Fletcher, Norma O'Donovan, John Crown, Denis M. Collins. The effect of neo-adjuvant chemotherapy on immune cell phenotype and cytotoxic capacity in HER2+ breast cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 951.

Correlative studies investigating effects of PI3K inhibition on peripheral leukocytes in metastatic breast cancer: potential implications for immunotherapy.

Patients with localized breast cancer have a 5-year survival rate > 99% compared to patients with metastatic breast cancer (MBC) that have a 5-year survival rate of ~ 27%. Unregulated PI3K/AKT signaling is a common characteristic of MBC, making it a desirable therapeutic target for tumors with activating mutations in this pathway. Interestingly, inhibition of the PI3K/AKT pathway can affect signaling in immune cells, which could potentially alter the immune phenotype of patients undergoing therapy with these drugs. The purpose of this study is to evaluate how PI3K inhibition affects the immune cells of MBC patients during treatment. We investigated the effects of PI3K inhibition on the immune cell populations in peripheral blood of MBC patients enrolled in 4 different clinical trials utilizing PI3K inhibitors. Peripheral blood was drawn at different points in patient treatment cycles to record immune cell fluctuations in response to therapy. MBC patients who responded to treatment with a positive fold-change in cytotoxic T cell population, had an average duration of treatment response of 31.4months. In contrast, MBC patients who responded to treatment with a negative fold-change in cytotoxic T-cell population, had an average duration of therapeutic response of 5months. These data suggest that patients with a more robust, initial anti-tumor T cell response may have a longer therapeutic response compared to patients who do not have a robust, initial anti-tumor T cell response. These results highlight the potential for PI3K inhibition to sensitize tumors to immune checkpoint inhibitors, thus providing additional therapeutic options for patients with MBC.

A novel transgenic mouse strain expressing PKC\u03b2II demonstrates expansion of B1 and marginal zone B cell populations

Protein kinase Cβ (PKCβ) expressed in mammalian cells as two splice variants, PKCβI and PKCβII, functions in the B cell receptor (BCR) signaling pathway and contributes to B cell development. We investigated the relative role of PKCβII in B cells by generating transgenic mice where expression of the transgene is directed to these cells using the Eµ promoter (Eµ-PKCβIItg). Our findings demonstrate that homozygous Eµ-PKCβIItg mice displayed a shift from IgD+IgMdim toward IgDdimIgM+ B cell populations in spleen, peritoneum and peripheral blood. Closer examination of these tissues revealed respective expansion of marginal zone (MZ)-like B cells (IgD+IgM+CD43negCD21+CD24+), increased populations of B-1 cells (B220+IgDdimIgM+CD43+CD24+CD5+), and higher numbers of immature B cells (IgDdimIgMdimCD21neg) at the expense of mature B cells (IgD+IgM+CD21+). Therefore, the overexpression of PKCβII, which is a phenotypic feature of chronic lymphocytic leukaemia cells, can skew B cell development in mice, most likely as a result of a regulatory influence on BCR signaling.

Omalizumab response correlates with reduced IFN-γ-, IL-10- and IL-31-secreting cells in chronic spontaneous urticaria.

The anti-IgE antibody omalizumab has been approved for the treatment of chronic spontaneous urticaria (CSU) in patients insufficiently responding to antihistamines. However, its mode of action in CSU is not clearly understood. The aim of this study was to get a better insight in the immune mechanisms involved in clinical improvement of CSU patients treated with omalizumab. Chronic spontaneous urticaria patients (n=15) were followed for 5months after initiation of omalizumab treatment. Clinical symptoms were assessed by UCT and CU-Q2 oL. Cell-bound IgE was quantified on both FcεRI- and FcεRII-expressing cell populations in peripheral blood. In addition, IgE and IgG as well as their receptors were measured on basophils, and basophil activation was assessed with different concentrations of anti-FcεRI and fMLP. Furthermore, the frequencies of different T-cell subsets secreting IL-5, IL-10, IL-31 or IFN-γ were analysed by ELISpot assay. Seven patients showed a full, five a partial and three no clinical response to omalizumab. Cell-bound IgE was reduced on FcεRI-bearing cells, but not on FcεRII-expressing cells. Likewise, the expression of FcεRI declined. Basophil activation increased upon FcεRI-stimulation while their sensitivity was not affected. Both basophil and T-cell frequencies remained unchanged. However, when comparing the individual response to omalizumab treatment with distinct T-cell subsets, a significant correlation was found between improved UCT and decreased frequencies of IL-10-, IL-31- and IFN-γ-secreting T cells. We here show that besides addressing IgE-dependent immune mechanisms, omalizumab treatment of CSU patients has effects on distinct T-cell subsets, which correlate with clinical improvement.