Flow cytometry analysis of immune cell populations isolated from cervicovaginal secretions of cynomolgus monkeys

ObjectiveCongenital heart block (CHB) with immune cell infiltration develops in the fetus after exposure to maternal Ro/La autoantibodies. CHB-related serology has been extensively studied, but reports on immune-cell profiles of anti-Ro/La-exposed neonates are lacking. In the current study, we characterised circulating immune-cell populations in anti-Ro/La+mothers and newborns, and explored potential downstream effects of skewed neonatal cell populations.MethodsIn total, blood from mothers (n=43) and neonates (n=66) was sampled at birth from anti-Ro/La+ (n=36) and control (n=30) pregnancies with or without rheumatic disease and CHB. Flow cytometry, microarrays and ELISA were used for characterising cells and plasma.ResultsSimilar to non-pregnant systemic lupus erythematosus and Sjögren-patients, anti-Ro/La+mothers had altered B-cell subset frequencies, relative T-cell lymphopenia and lower natural killer (NK)-cell frequencies. Surprisingly, their anti-Ro/La exposed neonates presented higher frequencies of CD56dimCD16hi NK cells (p<0.01), but no other cell frequency differences compared with controls. Type I and II interferon (IFN) gene-signatures were revealed in neonates of anti-Ro/La+ pregnancy, and exposure of fetal cardiomyocytes to type I IFN induced upregulation of several NK-cell chemoattractants and activating ligands. Intracellular flow cytometry revealed IFNγ production by NK cells, CD8+ and CD4+ T cells in anti-Ro/La exposed neonates. IFNγ was also detectable in their plasma.ConclusionOur study demonstrates an increased frequency of NK cells in anti-Ro/La exposed neonates, footprints of type I and II IFN and an upregulation of ligands activating NK cells in fetal cardiac cells after type I IFN exposure. These novel observations demonstrate innate immune activation in neonates of anti-Ro/La+pregnancy, which could contribute to the risk of CHB.

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Antibiotic Therapy Does Not Alter the Humoral Response to Vaccination for Porcine Circovirus 2 in Weaned Pigs

Veterinary Sciences ◽

10.3390/vetsci6020051 ◽

2019 ◽

Vol 6 (2) ◽

pp. 51

Author(s):

Jonathan E. Fogle ◽

Jenna A. Scott ◽

Glen W. Almond

Keyword(s):

Flow Cytometry ◽

Immune Response ◽

Antibiotic Therapy ◽

Humoral Immunity ◽

Immune Cell ◽

Humoral Response ◽

Porcine Circovirus ◽

Cell Populations ◽

Antibiotic Administration ◽

Weaned Pigs

Recent reports suggest that antibiotic therapy may either reduce or enhance the immune response to various porcine vaccines. Based upon these findings, we asked if antibiotic therapy alters immune cell populations, as measured by flow cytometry and/or vaccine-specific humoral immunity, as measured by sample to positive (S/P) antibody ratios. Here, we investigated the immuno-modulatory effects of enrofloxacin, ceftiofur, and tulathromycin on the immune response to a Mycoplasma hyopneumoniae (M. hyopneumoniae) and porcine circovirus type 2 (PCV-2) combination vaccine in weaned pigs. Maternal antibody likely interfered with the induction of immunity to M. hyopneumoniae. Antibiotic administration did not affect immune cell populations, as assessed by flow cytometry and did not affect the induction of humoral immunity to PCV-2.

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Intracerebral Hemorrhage Leads to Infiltration of Several Leukocyte Populations with Concomitant Pathophysiological Changes

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2008.114 ◽

2008 ◽

Vol 29 (1) ◽

pp. 137-143 ◽

Cited By ~ 40

Author(s):

Matthew C Loftspring ◽

Jeremiah McDole ◽

Aigang Lu ◽

Joseph F Clark ◽

Aaron J Johnson

Keyword(s):

Flow Cytometry ◽

Immune System ◽

Intracerebral Hemorrhage ◽

Immune Cell ◽

Adaptive Immune System ◽

Fold Increase ◽

Cytokine Signaling ◽

Cell Populations ◽

Stroke Subtype ◽

Mortality And Morbidity

Intracerebral hemorrhage (ICH) is a stroke subtype with high rates of mortality and morbidity. The immune system, particularly complement and cytokine signaling, has been implicated in brain injury after ICH. However, the cellular immunology associated with ICH has been understudied. In this report, we use flow cytometry to quantitatively profile immune cell populations that infiltrate the brain 1 and 4 days post-ICH. At 1 day CD45hi GR-1+ cells were increased 2.0-fold compared with saline controls ( P ≤ 0.05); however, we did not observe changes in any other cell populations analyzed. At 4 days ICH mice presented with a 2.4-fold increase in CD45hi cells, a 1.9-fold increase in CD45hi GR-1 cells, a 3.4-fold increase in CD45hi GR-1+ cells, and most notably, a 1.7-fold increase in CD4+ cells ( P ≤ 0.05 for all groups), compared with control mice. We did not observe changes in the numbers of CD8+ cells or CD45lo cells ( P = 0.43 and 0.49, respectively). Thus, we have shown the first use of flow cytometry to analyze leukocyte infiltration in response to ICH. Our finding of a CD4 T-cell infiltrate is novel and suggests a role for the adaptive immune system in the response to ICH.

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IMMU-06. DISCERNING THE DIFFERENT BONE MARROW IMMUNE LANDSCAPES

Neuro-Oncology ◽

10.1093/neuonc/noaa215.437 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii105-ii106

Author(s):

Michael Strong ◽

Aqila Ahmed ◽

Anda-Alexandra Calinescu ◽

Xiaofeng Zhou ◽

Tyler Robinson ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Bone Metastasis ◽

Immune Cell ◽

Suppressor Cells ◽

Surface Protein ◽

Long Bone ◽

Cell Populations ◽

Protein Marker ◽

Human T Cell

Abstract Roughly 400,000 people have bone metastases in the U.S. with the vast majority of these occurring in the spine. The etiology of bone metastasis still remains to be fully elucidated. This study explored the differences in immune landscape between long bone and spine that may contribute to higher rates of bone metastasis to the spine. Spines and femurs from male C57BL6/J mice (N=10) were processed for flow cytometry and immunophenotyping using Mass Cytometry by Time-Of-Flight (CyTOF). The cells were analyzed with CyTOF using a 33-surface protein marker mouse antibody panel. Spines (N=3) and femurs (N=2) from patients were analyzed with CyTOF using the Maxpar Complete Human T cell Immuno-Oncology Panel Set. There are global differences in the immune cell composition between the long bone and spine microenvironment. Flow cytometry revealed slight increases in the CD45+ and Cd11b+ cell populations in the bone marrow of murine spines compared to murine long bone, which are markers for myeloid-derived suppressor cells (MDSCs). Using CyTOF, significant differences in the immune cell landscape between long bone and spine were observed. In the murine long bone, an increase in monocytes/macrophages, myeloid progenitors, granulocytic MDSCs, granulocytes, and mast cells was observed compared to the spine. In the murine spine an elevation of CD8a+ DC cells, classical monocytes, MDSCs, pDCs, memory T helper cells, and NK T cells was seen. Evaluation of human long bone and spine revealed similar trends with a predominance of myeloid progenitor cells and monocytes in the human vertebra compared to the human long bone marrow. Significant differences in the immune microenvironment exist between the spine and long bone marrow in both murine and human samples. This is the first report of significant differences in immune cell populations between different skeletal locations. However, the functional significance of these differences has yet to be determined.

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Humanized mice (humice) carrying patient-derived xenograft (PDX) as a platform to develop immunotherapy in bladder cancer (BCa).

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.6_suppl.381 ◽

2017 ◽

Vol 35 (6_suppl) ◽

pp. 381-381 ◽

Cited By ~ 3

Author(s):

Chong-xian Pan ◽

Wei Shi ◽

Ai-Hong Ma ◽

Hongyong Zhang ◽

Primo Lara ◽

...

Keyword(s):

Flow Cytometry ◽

Tumor Growth ◽

Immune Cell ◽

Tumor Regression ◽

Genetic Alterations ◽

Therapeutic Modality ◽

Whole Body ◽

Flow Cytometry Analysis ◽

Nsg Mice ◽

Human Immune

381 Background: Immunotherapy with anti-programmed cell death 1 (PD1) or PD ligand 1 (PD-L1) antibody has emerged as a promising therapeutic modality, but has a response rate of approximately 20% in BCa. There are various drawbacks associated with current animal models. The objective of this study is to establish and characterize humice carrying PDXs in which both the immune cells and BCa cells are derived from humans. Methods: NOD-scid IL2Rgammanull or NSG, mice received CD34+ hematopoietic progenitor cells (HPC) cells i.v. after whole body radiation. PDXs were established through direct implantation of human BCa clinical specimens into NSG mice. Immune cell subpopulations were analyzed through flow cytometry analysis. Humice carrying HLA-unmatched PDXs were treated with an anti-PD1 antibody pemborlizumab (pembro) or in combination with a BKT/ITK inhibitor ibrutinib to determine the anti-tumor efficacy and toxicity. Results: PDXs retained the morphology fidelity and 92-97% of genetic alterations of parental patient cancers. Of the first 8 PDXs tested, 3 had high PD-L1 ( > 10 FPKM) as determined by RNA-seq which was further confirmed with flow cytometry analysis. Major human immune cell sub-populations were reconstituted in humice. No xenograft versus host disease was observed before pembro treatment. In humice with HPC donor 6466, pembro significantly inhibited tumor growth (p = 0.0016 at Day 29) of PDX BL293, but had no effect in another PDX BL440 with the same HPC donor 6466, or with the same PDX BL293 but with a different HPC donor 912. In another set of humice (HPC donor 710) carrying PDX BL293, pembro alone inhibited tumor growth. However, addition of ibrutinib did not potentiate the efficacy of pembro, but increased toxicity. Tumor regression with pembro treatment was associated with decrease of CD4+PD1+, CD8+PD1+ cells at peripheral blood and increased CD45+ and CD8+ cells in PDXs. Conclusions: Humice carrying PDXs reconstitute with human immune system, and can potentially be used to screen for effective immunotherapeutic agents or combinations, and to study resistant mechanisms.

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Abstract P5-04-03: Deconvoluting immune cell populations using ‘in silico flow cytometry’ with CIBERSORT: Association with neoadjuvant therapy response and genomic instability in TNBC

10.1158/1538-7445.sabcs14-p5-04-03 ◽

2015 ◽

Cited By ~ 2

Author(s):

Shaveta Vinayak ◽

Aaron Newman ◽

Sylvia Adams ◽

Anosheh Afghahi ◽

Kristin C Jensen ◽

...

Keyword(s):

Flow Cytometry ◽

Neoadjuvant Therapy ◽

Genomic Instability ◽

In Silico ◽

Immune Cell ◽

Therapy Response ◽

Cell Populations

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Standardization of Functional Immune Cell-Based Assays: An Integral Aspect to Vaccine and Biologic Development and Validation.

Blood ◽

10.1182/blood.v106.11.3933.3933 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3933-3933

Author(s):

Julie Wilkinson ◽

Cecilia Smith ◽

Sybil D’Costa ◽

Enrique Rabellino

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Sample Preparation ◽

Fluorescence Intensity ◽

Mean Fluorescence Intensity ◽

Immune Cell ◽

Cell Populations ◽

Surrogate Markers ◽

Functional Assays ◽

Variable Nature

Abstract The utility of the ex-vivo evaluation of immune cell functionality in the context of a) Determining an efficacious vaccine strategy for infectious diseases/cancer, b) Determining a tolerance profile in autoimmunity and transplantation, and c) understanding the basic mechanisms of immune cell responses in disease pathogenesis is well recognized. However, the benefit of these assays as surrogate markers of immune cell activity in vivo has not been fully realized due to the variable nature of these in vitro assays which is particularly pronounced in T cell functional assays. This variability arises from a variety of factors ranging from choice of assay, source of the cells, the sample processing methodology (isolation, freezing, thawing, and culturing), sample staining protocol for the chosen assay and ultimately data analysis, and data reduction. With a view to reducing variability and standardizing targeted steps of T cell functional assays, an automated methodology for simultaneous staining and analysis of multiple intracellular cytokines and cytotoxicity markers via flow cytometry was developed and validated. A 5-color flow cytometry assay (2–3 surface markers; 2 intracellular markers) was developed to characterize the restricted polyclonal (SEB/CD28) and antigen specific (CEF peptide pool) cytokine and cytotoxic profile response in human PBMCs. A modification to available sample preparation instruments was performed that enabled the automated pipetting, incubation, and staining of intracellular and surface molecules of stimulated human peripheral blood mononuclear cell populations (PBMC) for flow cytometric analysis. Statistically significant reductions in both inter and intra assay variability was observed in the automated methodology as compared to the manual assay with improvements in CVs for positive cell numbers and mean fluorescence intensity. For example, the inter assay CVs for IFNg cytokine producing CD4+ T cell populations improved from approximately 15 to 5, while the mean fluorescence intensity improved nearly 5 fold with automation. Importantly, the automated methodology furnished comparable responses in percent positive cytokine/cytotoxicity profiles as compared to the manual method while reducing the “handson” sample preparation and analysis time from 2 hours to 20 minutes. With the standardization of functional assays, other sources of variability in assays result can now be addressed specifically e.g. specimen handling, freezing, thawing, culturing, or biological. Standardized multiparametric functional profiling of the cells thus reveals the complex nature of the immune response and lends credence to their use as surrogate markers of efficacy and functionality.

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Immunosuppressive Microenvironment Revealed by Immune Cell Landscape in Pre-metastatic Liver of Colorectal Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.620688 ◽

2021 ◽

Vol 11 ◽

Author(s):

Dongqiang Zeng ◽

Miaohong Wang ◽

Jiani Wu ◽

Siheng Lin ◽

Zilan Ye ◽

...

Keyword(s):

Colorectal Cancer ◽

Flow Cytometry ◽

Immune Cells ◽

Immune Cell ◽

Gene Set Enrichment Analysis ◽

Metabolic Reprogramming ◽

Flow Cytometry Analysis ◽

Cell Type ◽

Metastatic Niche ◽

Metastatic Liver

Background: Colorectal cancer, the fourth leading cause of cancer mortality, is prone to metastasis, especially to the liver. The pre-metastatic microenvironment comprising various resident stromal cells and immune cells is essential for metastasis. However, how the dynamic evolution of immune components facilitates pre-metastatic niche formation remains unclear.Methods: Utilizing RNA-seq data from our orthotopic colorectal cancer mouse model, we applied single sample gene set enrichment analysis and Cell type Identification By Estimating Relative Subsets Of RNA Transcripts to investigate the tumor microenvironment landscape of pre-metastatic liver, and define the exact role of myeloid-derived suppressor cells (MDSCs) acting in the regulation of infiltrating immune cells and gene pathways activation. Flow cytometry analysis was conducted to quantify the MDSCs levels in human and mice samples.Results: In the current work, based on the high-throughput transcriptome data, we depicted the immune cell infiltration pattern of pre-metastatic liver and highlighted MDSCs as the dominant altered cell type. Notably, flow cytometry analysis showed that high frequencies of MDSCs, was detected in the pre-metastatic liver of orthotopic colorectal cancer tumor-bearing mice, and in the peripheral blood of patients with stage I–III colorectal cancer. MDSCs accumulation in the liver drove immunosuppressive factors secretion and immune checkpoint score upregulation, consequently shaping the pre-metastatic niche with sustained immune suppression. Metabolic reprogramming such as upregulated glycolysis/gluconeogenesis and HIF-1 signaling pathways in the primary tumor was also demonstrated to correlate with MDSCs infiltration in the pre-metastatic liver. Some chemokines were identified as a potential mechanism for MDSCs recruitment.Conclusion: Collectively, our study elucidates the alterations of MDSCs during pre-metastatic niche transformation, and illuminates the latent biological mechanism by which primary tumors impact MDSC aggregation in the targeted liver.

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Changes in immune Cell Types in Breast Suggest a Decline in Immune Surveillance and Increased Immunosuppression with Age

10.21203/rs.3.rs-430931/v1 ◽

2021 ◽

Author(s):

Arrianna Zirbes ◽

Jesuchristopher Joseph ◽

Jennifer C Lopez ◽

Rosalyn W Sayaman ◽

Mudaser Basam ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Epithelial Cell ◽

Peripheral Blood ◽

Breast Tissue ◽

Immune Cell ◽

Normal Breast Tissue ◽

Normal Breast ◽

Cell Populations

Abstract Background : A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. Methods : We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary organoid preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Results : Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm 2 ) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2:M1 macrophage ratio increased with age and was accompanied by an increased density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. Conclusions : T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age .

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