Multiparameter immunoprofiling for the diagnosis and differentiation of progressive versus nonprogressive nontuberculous mycobacterial lung disease-A pilot study.

Clinical prediction of nontuberculous mycobacteria lung disease (NTM-LD) progression remains challenging. We aimed to evaluate antigen-specific immunoprofiling utilizing flow cytometry (FC) of activation-induced markers (AIM) and IFN-γ enzyme-linked immune absorbent spot assay (ELISpot) accurately identifies patients with NTM-LD, and differentiate those with progressive from nonprogressive NTM-LD. A Prospective, single-center, and laboratory technician-blinded pilot study was conducted to evaluate the FC and ELISpot based immunoprofiling in patients with NTM-LD (n = 18) and controls (n = 22). Among 18 NTM-LD patients, 10 NTM-LD patients were classified into nonprogressive, and 8 as progressive NTM-LD based on clinical and radiological features. Peripheral blood mononuclear cells were collected from patients with NTM-LD and control subjects with negative QuantiFERON results. After stimulation with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides), and control antigens, we performed IFN-γ ELISpot and FC AIM assays to access their diagnostic accuracies by receiver operating curve (ROC) analysis across study groups. Patients with NTM-LD had significantly higher percentage of CD4+/CD8+ T-cells co-expressing CD25+CD134+ in response to PPD stimulation, differentiating between NTM-LD and controls. Among patients with NTM-LD, there was a significant difference in CD25+CD134+ co-expression in MTB300-stimulated CD8+ T-cells (p <0.05; AUC-ROC = 0.831; Sensitivity = 75% [95% CI: 34.9-96.8]; Specificity = 90% [95% CI: 55.5-99.7]) between progressors and nonprogressors. Significant differences in the ratios of antigen-specific IFN-γ ELISpot responses were also seen for RD1-nil/PPD-nil and RD1-nil/anti-CD3-nil between patients with nonprogressive vs. progressive NTM-LD. Our results suggest that multiparameter immunoprofiling can accurately identify patients with NTM-LD and may identify patients at risk of disease progression. A larger longitudinal study is needed to further evaluate this novel immunoprofiling approach.

Convalescent Adaptive Immunity Is Highly Heterogenous after SARS-CoV-2 Infection.

The optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, we recruited 30 unvaccinated convalescent donors who had previously been infected with COVID-19 and 7 unexposed asymptomatic controls. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG via semiquantitative ELISA, and T-cell immunity against S1 and S2 subunits were studied via IFN-γ enzyme-linked immunosorbent spot (ELISpot) and flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2-positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and the determination of optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Comparison of replicating and nonreplicating vaccines against SARS-CoV-2.

Most gene-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are nonreplicating vectors. They deliver the gene or messenger RNA to the cell to express the spike protein but do not replicate to amplify antigen production. This study tested the utility of replication in a vaccine by comparing replication-defective adenovirus (RD-Ad) and replicating single-cycle adenovirus (SC-Ad) vaccines that express the SARS-CoV-2 spike protein. SC-Ad produced 100 times more spike protein than RD-Ad and generated significantly higher antibodies against the spike protein than RD-Ad after single immunization of Ad-permissive hamsters. SC-Ad-generated antibodies climbed over 14 weeks after single immunization and persisted for more than 10 months. When the hamsters were challenged 10.5 months after single immunization, a single intranasal or intramuscular immunization with SC-Ad-Spike reduced SARS-CoV-2 viral loads and damage in the lungs and preserved body weight better than vaccination with RD-Ad-Spike. This demonstrates the utility of harnessing replication in vaccines to amplify protection against infectious diseases.

Antigen Specific Humoral and Cellular Immunity Following SARS-CoV-2 Vaccination in ANCA-Associated Vasculitis Patients Receiving B-Cell Depleting Therapy.

Humoral vaccine responses are known to be suboptimal in patients receiving B-cell targeted therapy, and little is known about vaccine induced T-cell immunity in these patients. In this study, we characterized humoral and cellular antigen-specific anti-SARS-CoV2 responses following COVID-19 vaccination in patients with ANCA-associated vasculitis (AAV) receiving anti-CD20 therapy, who were either B-cell depleted, or B-cell recovered at the time of vaccination and in normal control subjects. SARS-CoV-2 anti-spike (S) and anti-nucleocapsid (NC) antibodies were measured using electrochemiluminescence immunoassays, while SARS-CoV-2 specific T-cell responses to S glycoprotein subunits 1 (S1) and 2 (S2) and receptor binding domain peptide pools were measured using interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assays. In total, 26 recently vaccinated subjects were studied. Despite the lack of a measurable humoral immune response, B-cell depleted patients mounted a similar vaccine induced antigen-specific T-cell response compared to B-cell recovered patients and normal controls. Our data indicate that to assure a humoral response in patients receiving anti-CD20 therapy, SARS-CoV-2 vaccination should ideally be delayed until B-cell recovery (CD-20 positive B-cells > 10/µl). Nevertheless, SARS-CoV-2 vaccination elicits robust, potentially protective cellular immune responses in these subjects. Further research to characterize the durability and protective effect of vaccine-induced anti-SARS-CoV-2 specific T-cell immunity are needed.

Antifibrotics Modify B-Cell-induced Fibroblast Migration and Activation in Patients with Idiopathic Pulmonary Fibrosis.

B-cell activation is increasingly linked to numerous fibrotic lung diseases, and it is well known that aggregates of lymphocytes form in the lung of many of these patients. Activation of B-cells by pattern recognition receptors (PRRs) drives the release of inflammatory cytokines, chemokines, and metalloproteases important in the pathophysiology of pulmonary fibrosis. However, the specific mechanisms of B-cell activation in patients with idiopathic pulmonary fibrosis (IPF) are poorly understood. Herein, we have demonstrated that B-cell activation by microbial antigens contributes to the inflammatory and profibrotic milieu seen in patients with IPF. B-cell stimulation by CpG and ß-glucan via PRRs resulted in activation of mTOR-dependent and independent pathways. Moreover, we showed that the B-cell-secreted inflammatory milieu is specific to the inducing antigen and causes differential fibroblast migration and activation. B-cell responses to infectious agents and subsequent B-cell-mediated fibroblast activation are modifiable by antifibrotics, but each seems to exert a specific and different effect. These results suggest that, upon PRR activation by microbial antigens, B-cells can contribute to the inflammatory and fibrotic changes seen in patients with IPF, and antifibrotics are able to at least partially reverse these responses.

Stochastic changes in gene expression promote chaotic dysregulation of homeostasis in clonal breast tumors.

Cells within tumors vary in phenotype as a result of changes in gene expression caused by a variety of mechanisms, permitting cancers to evolve under selective pressures from immune and other homeostatic processes. Earlier, we traced apparent losses in heterozygosity (LOH) of spontaneous breast tumors from first generation (F1) intercrossed mice to atypical epigenetic modifications in the structure of DNA across the tumor genomes. Here, we describe a parallel pattern of LOH in gene expression, revealed through quantitation of parental alleles across a population of clonal tumors. We found variegated patterns of LOH, based on allelic ratio outliers in hundreds of genes, enriched in regulatory pathways typically co-opted by tumors. The frequency of outliers was correlated with transcriptional repression of a large set of homozygous genes. These findings suggest stochastic losses in gene expression across the genome of tumors generate phenotypic variation among cells, allowing clonal selection during tumor development.

Breaking tolerance with engineered class I antigen-presenting molecules.

Successful efforts to activate T cells capable of recognizing weak cancer-associated self-antigens have employed altered peptide antigens to activate T cell responses capable of cross-reacting on native tumor-associated self. A limitation of this approach is the requirement for detailed knowledge about the altered self-peptide ligands used in these vaccines. In the current study we considered allorecognition as an approach for activating CTL capable of recognizing weak or self-antigens in the context of self-MHC. Nonself antigen-presenting molecules typically contain polymorphisms that influence interactions with the bound peptide and TCR interface. Recognition of these nonself structures results in peptide-dependent alloimmunity. Alloreactive T cells target their inducing alloantigens as well as third-party alloantigens but generally fail to target self-antigens. Certain residues located on the alpha-1/2 domains of class I antigen-presenting molecules primarily interface with TCR. These residues are more conserved within and across species than are residues that determine peptide antigen binding properties. Class I variants designed with amino acid substitutions at key positions within the conserved helical structures are shown to provide strong activating signals to alloreactive CD8 T cells while avoiding changes in naturally bound peptide ligands. Importantly, CTL activated in this manner can break self-tolerance by reacting to self-peptides presented by native MHC. The ability to activate self-tolerant T cells capable of cross-reacting on self-peptide-MHC in vivo represents an approach for inducing autoimmunity, with possible application in cancer vaccines.

Neoantigenic Potential of Complex Chromosomal Rearrangements in Mesothelioma.

INTRODUCTION: Malignant pleural mesothelioma is a disease primarily associated with exposure to the carcinogen asbestos. Whereas other carcinogen-related tumors are associated with a high tumor mutation burden, mesothelioma is not. We sought to resolve this discrepancy. METHODS: We used mate-pair (n = 22), RNA (n = 28), and T cell receptor sequencing along with in silico predictions and immunologic assays to understand how structural variants of chromosomes affect the transcriptome. RESULTS: We observed that inter- or intrachromosomal rearrangements were present in every specimen and were frequently in a pattern of chromoanagenesis such as chromoplexy or chromothripsis. Transcription of rearrangement-related junctions was predicted to result in many potential neoantigens, some of which were proven to bind patient-specific major histocompatibility complex molecules and to expand intratumoral T cell clones. T cells responsive to these predicted neoantigens were also present in a patient's circulating T cell repertoire. Analysis of genomic array data from the mesothelioma cohort in The Cancer Genome Atlas suggested that multiple chromothriptic-like events negatively impact survival. CONCLUSIONS: Our findings represent the discovery of potential neoantigen expression driven by structural chromosomal rearrangements. These results may have implications for the development of novel immunotherapeutic strategies and the selection of patients to receive immunotherapies.

Exploring the effect of library preparation on RNA sequencing experiments.

RNA sequencing (RNA-seq) has become the widely preferred choice for surveying the genome-wide transcriptome complexity in many organisms. However, the broad adaptation of this methodology into the clinic still needs further evaluation of potential effect of sample preparation factors on its analytical reliability using patient samples. In this study, we examined the impact of three major sample preparation factors (i.e., cDNA library storage time, the quantity of input RNA, and cryopreservation of cell samples) on sequence biases, gene expression profiles, and enriched biological functions using RNAs isolated from primary B cell and CD4+ cell blood samples of healthy subjects. Our comprehensive comparison results suggested that different cDNA library storage time, quantity of input RNA, and cryopreservation of cell samples did not significantly alter gene transcriptional expression profiles generated by RNA-seq experiments. These findings shed new lights on the potential applications of RNA-seq technique to patient samples in a regular clinical setting.

Integrative Genome-Wide Analysis of Long Noncoding RNAs in Diverse Immune Cell Types of Melanoma Patients.

Genome-wide identification and characterization of long noncoding RNAs (lncRNA) in individual immune cell lineages helps us better understand the driving mechanisms behind melanoma and advance personalized patient treatment. To elucidate the transcriptional landscape in diverse immune cell types of peripheral blood cells (PBC) in stage IV melanoma, we used whole transcriptome RNA sequencing to profile lncRNAs in CD4+, CD8+, and CD14+ PBC from 132 patient samples. Our integrative computational approach identified 27,625 expressed lncRNAs, 2,744 of which were novel. Both T cells (i.e., CD4+ and CD8+ PBC) and monocytes (i.e., CD14+ PBC) exhibited differential transcriptional expression profiles between patients with melanoma and healthy subjects. Cis- and trans-level coexpression analysis suggested that lncRNAs are potentially involved in many important immune-related pathways and the programmed cell death receptor 1 checkpoint pathways. We also identified nine gene coexpression modules significantly associated with melanoma status, all of which were significantly enriched for three mRNA translation processes. Age and melanoma traits closely correlated with each other, implying that melanoma contains age-associated immune changes. Our computational prediction analysis suggests that many cis- and trans-regulatory lncRNAs could interact with multiple transcriptional and posttranscriptional regulatory elements in CD4+, CD8+, and CD14+ PBC, respectively. These results provide novel insights into the regulatory mechanisms involving lncRNAs in individual immune cell types in melanoma and can help expedite cell type-specific immunotherapy treatments for such diseases.Significance: These findings elucidate melanoma-associated changes to the noncoding transcriptional landscape of distinct immune cell classes, thus providing cell type-specific guidance to targeted immunotherapy regimens. Cancer Res; 78(15); 4411-23. ©2018 AACR.

Gene Expression Signatures Characterized by Longitudinal Stability and Interindividual Variability Delineate Baseline Phenotypic Groups with Distinct Responses to Immune Stimulation.

Human immunity exhibits remarkable heterogeneity among individuals, which engenders variable responses to immune perturbations in human populations. Population studies reveal that, in addition to interindividual heterogeneity, systemic immune signatures display longitudinal stability within individuals, and these signatures may reliably dictate how given individuals respond to immune perturbations. We hypothesize that analyzing relationships among these signatures at the population level may uncover baseline immune phenotypes that correspond with response outcomes to immune stimuli. To test this, we quantified global gene expression in peripheral blood CD4+ cells from healthy individuals at baseline and following CD3/CD28 stimulation at two time points 1 mo apart. Systemic CD4+ cell baseline and poststimulation molecular immune response signatures (MIRS) were defined by identifying genes expressed at levels that were stable between time points within individuals and differential among individuals in each state. Iterative differential gene expression analyses between all possible phenotypic groupings of at least three individuals using the baseline and stimulated MIRS gene sets revealed shared baseline and response phenotypic groupings, indicating the baseline MIRS contained determinants of immune responsiveness. Furthermore, significant numbers of shared phenotype-defining sets of determinants were identified in baseline data across independent healthy cohorts. Combining the cohorts and repeating the analyses resulted in identification of over 6000 baseline immune phenotypic groups, implying that the MIRS concept may be useful in many immune perturbation contexts. These findings demonstrate that patterns in complex gene expression variability can be used to define immune phenotypes and discover determinants of immune responsiveness.

Canonical Stimulation of the NLRP3 Inflammasome by Fungal Antigens Links Innate and Adaptive B-Lymphocyte Responses by Modulating IL-1ß and IgM Production.

The NLRP3 inflammasome is activated in response to different bacterial, viral, and fungal pathogens and serves as modulator of different pattern recognition receptors signaling pathways. One of the main functions of NLRP3 is to participate in IL-1ß maturation which is important in the host defense against Pneumocystis and other fungal infections. However, dysregulation of NLRP3 and IL-1ß secretion are also implicated in the pathophysiology of many auto-inflammatory disorders. Often time's inflammatory flares are preceded by infectious illnesses questioning the role of infection in autoimmune exacerbations. However, we still do not fully understand the exact role that infection or even colonization plays as a trigger of inflammation. Herein, we investigated the role of NLRP3 in circulating B-lymphocytes following activation with two major microbial antigens (ß-glucan and CpG). NLRP3 was determined essential in two independent B-lymphocytes processes: pro-inflammatory cytokine secretion and antibody regulation. Our results show that the ß-glucan fungal cell wall carbohydrate stimulated B-lymphocytes to secrete IL-1ß in a process partially mediated by Dectin-1 activation via SYK and the transcription factors NF-κB and AP-1. This IL-1ß secretion was regulated by the NLRP3 inflammasome and was dependent on potassium efflux and Caspase-1. Interestingly, B-lymphocytes activated by unmethylated CpG motifs, found in bacterial and fungal DNA, failed to induce IL-1ß. However, B-lymphocyte stimulation by CpG resulted in NLRP3 and Caspase-1 activation and the production and secretion of IgM antibodies. Furthermore, CpG-stimulated IgM secretion, unlike ß-glucan-mediated IL-1ß production, was mediated by the mammalian target of rapamycin (mTOR). Inhibition of NLRP3 and the mTOR pathway in CpG activated B-lymphocytes resulted in impaired IgM secretion suggesting their participation in antibody regulation. In conclusion, this study describes a differential response of NLRP3 to ß-glucan and CpG antigens and identifies the NLRP3 inflammasome of human circulating B-lymphocytes as a modulator of the innate and adaptive immune systems.

Flow Cytometric Immune Profiling in Infliximab-Associated Tuberculosis.

Tumor necrosis factor α antagonists are increasingly used to treat inflammatory and autoimmune disorders and are associated with increased risk of active tuberculosis. Diagnosis of active tuberculosis in patients taking tumor necrosis factor α antagonists can be challenging owing to increased incidence of extrapulmonary manifestations and false-negative results on current available diagnostic tests. We present a case of a young woman on infliximab for ulcerative colitis who presented with disseminated tuberculosis. As part of a research study, we performed flow cytometric immune profiling, which has previously not been reported in patients with active tuberculosis taking tumor necrosis α antagonists. The flow cytometry results were within the positive thresholds for tuberculosis infection. Flow cytometric immune profiling may be a valid diagnostic tool for patients taking tumor necrosis factor α antagonists.

Microbial Antigens Stimulate Metalloprotease-7 Secretion in Human B-Lymphocytes Using mTOR-Dependent and Independent Pathways.

Metalloproteinases (MMPs) contribute to tissue remodeling and acute inflammation not only by degrading extracellular matrix proteins but also by controlling the influx of chemokines through the regulation and shedding of syndecans. B-lymphocytes, in addition to their well-known function as antibody producing cells, participate in the innate immune response by secreting inflammatory cytokines and chemokines. However, there is little information about the role of B-lymphocytes in the regulation of MMPs; consequently, herein we investigated whether activated human circulating B-lymphocytes contributed to the secretion of MMPs. We demonstrate that B-lymphocytes activated by un-methylated CpG motifs, found in bacterial DNA, and ß-glucans, found in the cell wall of fungi, both induced MMP-7. Interestingly, while CpG-stimulated cells activated the mTOR pathway via TLR9 receptor to induced MMP-7, ß-glucan-stimulated cells were mTOR-independent and used Dectin-1 receptor. B-lymphocytes did not seem to have a major role in the secretion of tissue inhibitors of metalloproteinases (TIMPs). However, secreted MMP-7 participated in the shedding of Syndecan-4 from the surface of B-lymphocytes. In conclusion, circulating human B-lymphocytes contribute to the regulation of the innate immune system by participating in the secretion of MMP-7 which in turn is important for the shedding of Syndecan-4 in response to infectious stimuli.

Gene expression patterns in CD4+ peripheral blood cells in healthy subjects and stage IV melanoma patients.

Melanoma patients exhibit changes in immune responsiveness in the local tumor environment, draining lymph nodes, and peripheral blood. Immune-targeting therapies are revolutionizing melanoma patient care increasingly, and studies show that patients derive clinical benefit from these newer agents. Nonetheless, predicting which patients will benefit from these costly therapies remains a challenge. In an effort to capture individual differences in immune responsiveness, we are analyzing patterns of gene expression in human peripheral blood cells using RNAseq. Focusing on CD4+ peripheral blood cells, we describe multiple categories of immune regulating genes, which are expressed in highly ordered patterns shared by cohorts of healthy subjects and stage IV melanoma patients. Despite displaying conservation in overall transcriptome structure, CD4+ peripheral blood cells from melanoma patients differ quantitatively from healthy subjects in the expression of more than 2000 genes. Moreover, 1300 differentially expressed genes are found in transcript response patterns following activation of CD4+ cells ex vivo, suggesting that widespread functional discrepancies differentiate the immune systems of healthy subjects and melanoma patients. While our analysis reveals that the transcriptome architecture characteristic of healthy subjects is maintained in cancer patients, the genes expressed differentially among individuals and across cohorts provide opportunities for understanding variable immune states as well as response potentials, thus establishing a foundation for predicting individual responses to stimuli such as immunotherapeutic agents.

Combinatorial Immunoprofiling in Latent Tuberculosis Infection. Toward Better Risk Stratification.

RATIONALE: Most immunocompetent patients diagnosed with latent tuberculosis infection (LTBI) will not progress to tuberculosis (TB) reactivation. However, current diagnostic tools cannot reliably distinguish nonprogressing from progressing patients a priori, and thus LTBI therapy must be prescribed with suboptimal patient specificity. We hypothesized that LTBI diagnostics could be improved by generating immunomarker profiles capable of categorizing distinct patient subsets by a combinatorial immunoassay approach. OBJECTIVES: A combinatorial immunoassay analysis was applied to identify potential immunomarker combinations that distinguish among unexposed subjects, untreated patients with LTBI, and treated patients with LTBI and to differentiate risk of reactivation. METHODS: IFN-γ release assay (IGRA) was combined with a flow cytometric assay that detects induction of CD25(+)CD134(+) coexpression on TB antigen-stimulated T cells from peripheral blood. The combinatorial immunoassay analysis was based on receiver operating characteristic curves, technical cut-offs, 95% bivariate normal density ellipse prediction, and statistical analysis. Risk of reactivation was estimated with a prediction formula. MEASUREMENTS AND MAIN RESULTS: Sixty-five out of 150 subjects were included. The combinatorial immunoassay approach identified at least four different T-cell subsets. The representation of these immune phenotypes was more heterogeneous in untreated patients with LTBI than in treated patients with LTBI or unexposed groups. Patients with IGRA(+) CD4(+)CD25(+)CD134(+) T-cell phenotypes had the highest estimated reactivation risk (4.11 ± 2.11%). CONCLUSIONS: These findings suggest that immune phenotypes defined by combinatorial assays may potentially have a role in identifying those at risk of developing TB; this potential role is supported by risk of reactivation modeling. Prospective studies will be needed to test this novel approach.

Widespread Non-Canonical Epigenetic Modifications in MMTV-NeuT Breast Cancer.

Breast tumors in (FVB × BALB-NeuT) F1 mice have characteristic loss of chromosome 4 and sporadic loss or gain of other chromosomes. We employed the Illumina GoldenGate genotyping platform to quantitate loss of heterozygosity (LOH) across the genome of primary tumors, revealing strong biases favoring chromosome 4 alleles from the FVB parent. While allelic bias was not observed on other chromosomes, many tumors showed concerted LOH (C-LOH) of all alleles of one or the other parent on sporadic chromosomes, a pattern consistent with cytogenetic observations. Surprisingly, comparison of LOH in tumor samples relative to normal unaffected tissues from these animals revealed significant variegated (stochastic) deviations from heterozygosity (V-LOH) in every tumor genome. Sequence analysis showed expected changes in the allelic frequency of single nucleotide polymorphisms (SNPs) in cases of C-LOH. However, no evidence of LOH due to mutations, small deletions, or gene conversion at the affected SNPs or surrounding DNA was found at loci with V-LOH. Postulating an epigenetic mechanism contributing to V-LOH, we tested whether methylation of template DNA impacts allele detection efficiency using synthetic oligonucleotide templates in an assay mimicking the GoldenGate genotyping format. Methylated templates were systematically over-scored, suggesting that the observed patterns of V-LOH may represent extensive epigenetic DNA modifications across the tumor genomes. As most of the SNPs queried do not contain standard (CpG) methylation targets, we propose that widespread, non-canonical DNA modifications occur during Her2/neuT-driven tumorigenesis.

A method for identification and analysis of non-overlapping myeloid immunophenotypes in humans.

The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN-CD33+HLA-DR- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies.

Dopamine D2 receptor agonists inhibit lung cancer progression by reducing angiogenesis and tumor infiltrating myeloid derived suppressor cells.

We sought to determine whether Dopamine D2 Receptor (D2R) agonists inhibit lung tumor progression and identify subpopulations of lung cancer patients that benefit most from D2R agonist therapy. We demonstrate D2R agonists abrogate lung tumor progression in syngeneic (LLC1) and human xenograft (A549) orthotopic murine models through inhibition of tumor angiogenesis and reduction of tumor infiltrating myeloid derived suppressor cells. Pathological examination of human lung cancer tissue revealed a positive correlation between endothelial D2R expression and tumor stage. Lung cancer patients with a smoking history exhibited greater levels of D2R in lung endothelium. Our results suggest D2R agonists may represent a promising individualized therapy for lung cancer patients with high levels of endothelial D2R expression and a smoking history.