Lymphoid Cell Populations Research Articles

Extramedullar central nervous system blast crisis – rare occurrence in chronic myeloid leukemia

Chronic myelogenous leukemia (CML) is a myelo­pro­li­fe­rative neoplasm characterized by the presence of the Philadelphia chromosome. CML is classified into chronic, accelerated and blast phases. The blast crisis is characterized by the rapid expansion of a population of myeloid or lymphoid differentiation-arrested blast cells. Extramedullary blast crisis is defined as blast infiltration in other sites regardless of bone marrow blast proliferation. We report the case of a 59-year-old woman diagnosed with blast phase CML. Initially, the patient had an optimal response to tyrosine kinase inhibitors, but later she developed central nervous system blast crisis.

Natural killer cells in inflammatory autoimmune diseases.

Natural killer (NK) cells are a specialised population of innate lymphoid cells (ILCs) that help control local immune responses. Through natural cytotoxicity, production of cytokines and chemokines, and migratory capacity, NK cells play a vital immunoregulatory role in the initiation and chronicity of inflammatory and autoimmune responses. Our understanding of their functional differences and contributions in disease settings is evolving owing to new genetic and functional murine proof‐of‐concept studies. Here, we summarise current understanding of NK cells in several classic autoimmune disorders, particularly in rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE) and type 1 diabetes mellitus (T1DM), but also less understood diseases such as idiopathic inflammatory myopathies (IIMs). A better understanding of how NK cells contribute to these autoimmune disorders may pave the way for NK cell‐targeted therapeutics.

Regulation of the human NK cell compartment by pathogens and vaccines.

Natural killer cells constitute a phenotypically diverse population of innate lymphoid cells with a broad functional spectrum. Classically defined as cytotoxic lymphocytes with the capacity to eliminate cells lacking self‐MHC or expressing markers of stress or neoplastic transformation, critical roles for NK cells in immunity to infection in the regulation of immune responses and as vaccine‐induced effector cells have also emerged. A crucial feature of NK cell biology is their capacity to integrate signals from pathogen‐, tumor‐ or stress‐induced innate pathways and from antigen‐specific immune responses. The extent to which innate and acquired immune mediators influence NK cell effector function is influenced by the maturation and differentiation state of the NK cell compartment; moreover, NK cell differentiation is driven in part by exposure to infection. Pathogens can thus mould the NK cell response to maximise their own success and/or minimise the damage they cause. Here, we review recent evidence that pathogen‐ and vaccine‐derived signals influence the differentiation, adaptation and subsequent effector function of human NK cells.

Identification of ILC2 in the Lung Using Flow Cytometry.

Group 2 innate lymphoid cells (ILC2), despite their scarcity, are the dominant innate lymphoid cell population in the lung, orchestrating innate immunity and adaptive immunity (Germain and Huang. Curr Opin Immunol 56:76-81, 2019; Krabbendam et al. Immunol Rev 286:74-85, 2018; Mindt et al. Front Immunol 9:840, 2018) . Recent studies reveal that ILC2 play critical roles in inflammation-associated lung injury during sepsis (Lai et al. Cell Death Dis 9:369, 2018; Xu et al. Immunol Cell Biol 96:935-947). Therefore, studies aiming to understand the pathobiology of ILC2 may reveal new therapeutic strategies for sepsis. However, the identification of ILC2 requires multiple surface and intracellular markers. This makes the detection of ILC2 in the lung challenging. Here we describe a method to detect ILC2 in the mouse lung using flow cytometry.

Aging of the immune system and impaired muscle regeneration: A failure of immunomodulation of adult myogenesis.

Skeletal muscle regeneration that follows acute injury is strongly influenced by interactions with immune cells that invade and proliferate in the damaged tissue. Discoveries over the past 20years have identified many of the key mechanisms through which myeloid cells, especially macrophages, regulate muscle regeneration. In addition, lymphoid cells that include CD8+ T-cells and regulatory T-cells also significantly affect the course of muscle regeneration. During aging, the regenerative capacity of skeletal muscle declines, which can contribute to progressive loss of muscle mass and function. Those age-related reductions in muscle regeneration are accompanied by systemic, age-related changes in the immune system, that affect many of the myeloid and lymphoid cell populations that can influence muscle regeneration. In this review, we present recent discoveries that indicate that aging of the immune system contributes to the diminished regenerative capacity of aging muscle. Intrinsic, age-related changes in immune cells modify their expression of factors that affect the function of a population of muscle stem cells, called satellite cells, that are necessary for normal muscle regeneration. For example, age-related reductions in the expression of growth differentiation factor-3 (GDF3) or CXCL10 by macrophages negatively affect adult myogenesis, by disrupting regulatory interactions between macrophages and satellite cells. Those changes contribute to a reduction in the numbers and myogenic capacity of satellite cells in old muscle, which reduces their ability to restore damaged muscle. In addition, aging produces changes in the expression of molecules that regulate the inflammatory response to injured muscle, which also contributes to age-related defects in muscle regeneration. For example, age-related increases in the production of osteopontin by macrophages disrupts the normal inflammatory response to muscle injury, resulting in regenerative defects. These nascent findings represent the beginning of a newly-developing field of investigation into mechanisms through which aging of the immune system affects muscle regeneration.

Established and emergent roles for Ikaros transcription factors in lymphoid cell development and function

Ikaros zinc finger transcription factors are important regulators of the gene programs underlying the development of hematopoietic cell lineages. The family consists of five members: Ikaros, Helios, Aiolos, Eos, and Pegasus, which engage in both homo- and heterotypic intrafamilial interactions to exert diverse functional effects. Pioneering studies focused on the role of these factors in early lymphoid development, as their absence resulted in severe defects in lymphocyte populations. More recent work has now begun to define nuanced, stage-specific roles for Ikaros family members in the differentiation and function of mature T, B, and innate lymphoid cell populations including natural killer (NK) cells. The precise transcriptional mechanisms by which these factors function, both independently and collaboratively, is an area of active investigation. However, several key themes appear to be emerging regarding the pathways influenced by Ikaros family members, including the end-to-end regulation of cytokine signaling. Here, we review roles for Ikaros factors in lymphoid cell development, differentiation, and function, including a discussion of the current understanding of the transcriptional mechanisms they employ and considerations for the future study of this important transcription factor family.

Development and survival of MYC-driven lymphomas require the MYC antagonist MNT to curb MYC-induced apoptosis

AbstractDeregulated overexpression of MYC is implicated in the development and malignant progression of most (∼70%) human tumors. MYC drives cell growth and proliferation, but also, at high levels, promotes apoptosis. Here, we report that the proliferative capacity of MYC-driven normal and neoplastic B lymphoid cells depends on MNT, a MYC-related transcriptional repressor. Our genetic data establish that MNT synergizes with MYC by suppressing MYC-driven apoptosis, and that it does so primarily by reducing the level of pro-apoptotic BIM. In Eμ-Myc mice, which model the MYC/IGH chromosome translocation in Burkitt's lymphoma, homozygous Mnt deletion greatly reduced lymphoma incidence by enhancing apoptosis and markedly decreasing premalignant B lymphoid cell populations. Strikingly, by inducing Mnt deletion within transplanted fully malignant Eμ-Myc lymphoma cells, we significantly extended transplant recipient survival. The dependency of lymphomas on MNT for survival suggests that drugs inhibiting MNT could significantly boost therapy of MYC-driven tumors by enhancing intrinsic MYC-driven apoptosis.

Cellular interactions in aspirin-exacerbated respiratory disease.

The purpose of this review is to summarize the complex cellular interactions of aspirin-exacerbated respiratory disease (AERD) and how these interactions promote pathogenic mechanisms of AERD. In addition to characteristic changes in eicosanoid levels, recent studies have identified increases in alarmin cytokines (IL-33, thymic stromal lymphopoietin) as well as activated innate lymphoid and plasma cell populations in samples from AERD patients. Patients with AERD typically demonstrate high levels of proinflammatory eicosanoids including cysteinyl leukotrienes (CysLTs) and prostaglandin D2 (PGD2) and hyporesponsiveness to prostaglandin E2 (PGE2). CysLTs are released by mast cells, eosinophils, and adherent platelets and promote epithelial release of IL-33, which activates mast cells and group 2 innate lymphoid cells (ILC2s) in concert with CysLTs. TSLP induces PGD2 release from mast cells which activates and recruits eosinophils, basophils, Th2 cells, and ILC2s via CRTH2. In turn, ILC2s and other cell types produce Th2 cytokines IL-4, IL-5, and IL-13 that, along with CysLTs and PGD2, promote bronchoconstriction, eosinophilic tissue inflammation, and mucus production.

HtrA2 is required for inflammatory responses in BMDMs via controlling TRAF2 stability in collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by the destruction of cartilage and bone. The present study aims to investigate the role of HtrA serine peptidase 2 (HtrA2) in the collagen-induced arthritis. The expressions of HtrA2 were determined in the database BioGPS and bone marrow-derived macrophages (BMDMs). The populations of myeloid and lymphoid cells were determined in wild type and HtrA2 knockout (HtrA2MKO) mice using flow cytometry. In addition, the expressions of pro-inflammatory cytokines (Il6, Tnf, and Il1β) were determined in the activated BMDMs from wild type (WT) and HtrA2MKO mice. STRING database was used to predict the interactive proteins of HtrA2 and Co-Immunoprecipitation was used to confirm these interactions. A collagen-induced arthritis model was established to investigate the effects of HtrA2 on the arthritis symptoms. It was found that HtrA2 reduction was associated with the activation of myeloid cells. Interestingly, HtrA2 deficiency did not affect the development of myeloid and lymphoid cells. Further studies demonstrated that HtrA2 deficiency suppressed the production of pro-inflammatory cytokines in BMDMs induced by lipopolysaccharide or CpG. Co-Immunoprecipitation results demonstrated that HtrA2 enhanced the stability of TNF receptor-associated factor 2 (TRAF2). HtrA2 participated in the activation of the inflammatory response in a collagen-induced arthritis model. In summary, HtrA2 modulates inflammatory responses in BMDMs by controlling TRAF2 stability in a collagen-induced arthritis mouse model.

IMMU-34. LANDSCAPE OF GLIOBLASTOMA-ASSOCIATED IMMUNITY BY INTEGRATION OF scRNA-SEQ AND SPATIAL TRANSCRIPTOMICS

Abstract The diversity of molecular states and cellular plasticity of immune cells within the glioblastoma (GBM) environment remain poorly investigated. Here, we conduct deep transcriptional profiling of lymphoid and myeloid cell populations by scRNA-sequencing, map potential cellular interactions and cytokine responses that lead to the dysfunctional and exhausted phenotype of T cells. We identified Interleukin 10 (IL-10) response during T cell activation, which lead to a dysfunctional state of T cells. By the use of a novel method: The nearest functionally connected neighbor (NFCN), an in-silico model to explore cell-cell interaction, the dysfunctional/exhausted phenotype was found to be driven by subset of myeloid cells defined by high expression of HMOX1. By using spatial transcriptomic RNA-sequencing, we identified a correlation between T cell exhaustion and colocalized mesenchymal gene expression. We found that HMOX1 expressing myeloid cells occupying regions marked by T cell exhaustion. Using a human neocortical slice model with myeloid cell depletion we confirmed the functional interaction of myeloid and lymphoid cell leading to the dysfunctional state of T cells. A comprehensive understanding of cellular states and plasticity of lymphoid cells in GBM aids in providing successful immunotherapeutic approaches.

BIOM-30. DECREASED B CELL AND ALTERED CD4+ T CELL SUBSET RATIO PREDICT FAVORABLE RESPONSE TO RUXOLINITIB IN HIGH-GRADE GLIOMAS

Abstract Altered cellular growth and suppression of anti-tumor immune response collectively drive progression and therapeutic resistance of high-grade gliomas (HGGs), suggesting that successful treatments will likely target these key mechanisms. Janus-associated kinase (JAK)-STAT pathway comprises one such therapeutic node, as STAT3 signaling is upregulated in proliferating glioma cells and immunosuppressive cell types. We performed retrospective analysis on 13 HGG patients treated with the JAK1/2 inhibitor ruxolinitib in combination with radiation or radiation plus temozolomide in a Phase I setting (NCT03514069). Disease progression and treatment response was determined based on the Response Assessment in Neuro-Oncology (RANO) criteria. Peripheral blood mononuclear cells (PBMCs) cells were collected from patients prior to treatment, and 2- 4- and 6-week time points to characterize early immunological changes that are indicative of treatment response with flow cytometry. 6 patients responded favorably, define as progression-free survival >6 months. Investigation of myeloid and lymphoid cell populations revealed no differences in dendritic cell, myeloid-derived suppressor cell and CD8+ T cell subset frequencies in blood. Similarly, expression of checkpoint inhibitors PD-1, TIGIT, CTLA-4 and TIM3 were not altered with ruxolinitib treatment. However, there was a ~2-fold reduction in the frequency of peripheral B cells in favorable responders at 4-week time point, whereas the levels remained stable in non-responders (p<0.05). In addition, while 5 out of 7 non-responders had increased CD4+ T cells by 4-weeks, 5 out of 6 responders had stable or reduced CD4+ T cells. This effect was driven by reduction in immunosuppressive regulatory T cells (Tregs) as the relative ratio of immunostimulatory T helper 1 (Th1)-to-Treg ratio was 3-fold higher in responders compared to non-responders. These results support evaluation of B and CD4+ T cells in the peripheral blood as biomarkers to identify HGG patients likely to benefit from such approach.

Exosomes as mediators of immune regulation and immunotherapy in cancer

Exosomes are nanosized extracellular vesicles of endosomal origin that enclose a multitude of functional biomolecules. Exosomes have emerged as key players of intercellular communication in physiological and pathological conditions. In cancer, depending on the context, exosomes can oppose or potentiate the development of an aggressive tumor microenvironment, thereby impacting tumor progression and clinical outcome. Increasing evidence has established exosomes as important mediators of immune regulation in cancer, as they deliver a plethora of signals that can either support or restrain immunosuppression of lymphoid and myeloid cell populations in tumors. Here, we review the current knowledge related to exosome-mediated regulation of lymphoid (T lymphocytes, B lymphocytes, and NK cells) and myeloid (macrophages, dendritic cells, monocytes, myeloid-derived suppressor cells, and neutrophils) cell populations in cancer. We also discuss the translational potential of engineered exosomes as immunomodulatory agents for cancer therapy.

IL-10 production by ILC2s requires Blimp-1 and cMaf, modulates cellular metabolism, and ameliorates airway hyperreactivity

Group 2 innate lymphoid cells (ILC2s) are the dominant innate lymphoid cell population in the lungs at steady state, and their release of type 2 cytokines is a central driver in responding eosinophil infiltration and increased airway hyperreactivity. Our laboratory has identified a unique subset of ILC2s in the lungs that actively produce IL-10 (ILC210s). Our aim was to characterize the effector functions of ILC210s in the development and pathology of allergic asthma. IL-4-stimulated ILC210s were isolated to evaluate cytokine secretion, transcription factor signaling, metabolic dependence, and effector functions invitro. ILC210s were also adoptively transferred into Rag2-/-γc-/- mice, which were then challenged with IL-33 and assessed for airway hyperreactivity and lung inflammation. We have determined that the transcription factors cMaf and Blimp-1 regulate IL-10 expression in ILC210s. Strikingly, our results demonstrate that ILC210s can utilize both autocrine and paracrine signaling to suppress proinflammatory ILC2 effector functions invitro. Further, this subset dampens airway hyperreactivity and significantly reduces lung inflammation invivo. Interestingly, ILC210s demonstrated a metabolic dependency on the glycolytic pathway for IL-10 production, shifting from the fatty acid oxidation pathway conventionally utilized for proinflammatory effector functions. These findings provide an important and previously unrecognized role of ILC210s in diseases associated with ILC2s such as allergic lung inflammation and asthma. They also provide new insights into the metabolism dependency of proinflammatory and anti-inflammatory ILC2 phenotypes.

NK1.1+ innate lymphoid cells in salivary glands inhibit establishment of tissue-resident memory CD8+ Tcells in mice.

NK1.1+ cells found in salivary glands (SG) represent a unique cell population of innate lymphoid cells (ILC) with characteristics of both conventional NKcells and ILC1. Here, we demonstrate that these NK1.1+ cells limit the accumulation and differentiation of virus-specific tissue-resident memory CD8+ Tcells (TRM cells) in SG of mice infected with lymphocytic choriomeningitis virus (LCMV). The negative regulation of LCMV-specific CD8+ TRM cells by NK1.1+ cells in SG is independent of NKG2D, NKp46, TRAIL, and perforin. Moreover, analysis of NKp46iCre+ Eomesfl/fl mice revealed that Eomes-dependent conventional NKcells are dispensable for negative regulation. Since the SG are prone to autoimmune reactions, regulation of TRM cells by tissue-resident ILC may be particularly important to prevent immunopathology in this organ.

Myelomonocytic Skewing In Vitro Discriminates Subgroups of Patients with Myelofibrosis with A Different Phenotype, A Different Mutational Profile and Different Prognosis.

Normal hematopoietic function is maintained by a well-controlled balance of myelomonocytic, megaerythroid and lymphoid progenitor cell populations which may be skewed during pathologic conditions. Using semisolid in vitro cultures supporting the growth of myelomonocytic (CFU-GM) and erythroid (BFU-E) colonies, we investigated skewed differentiation towards the myelomonocytic over erythroid commitment in 81 patients with myelofibrosis (MF). MF patients had significantly increased numbers of circulating CFU-GM and BFU-E. Myelomonocytic skewing as indicated by a CFU-GM/BFU-E ratio ≥ 1 was found in 26/81 (32%) MF patients as compared to 1/98 (1%) in normal individuals. Patients with myelomonocytic skewing as compared to patients without skewing had higher white blood cell and blast cell counts, more frequent leukoerythroblastic features, but lower hemoglobin levels and platelet counts. The presence of myelomonocytic skewing was associated with a higher frequency of additional mutations, particularly in genes of the epigenetic and/or splicing machinery, and a significantly shorter survival (46 vs. 138 mo, p < 0.001). The results of this study show that the in vitro detection of myelomonocytic skewing can discriminate subgroups of patients with MF with a different phenotype, a different mutational profile and a different prognosis. Our findings may be important for the understanding and management of MF.

Abstract 3144: Using artificial intelligence and flow cytometry to confirm the need for a prostate biopsy in at-risk men

Abstract The goal of this study was to create a blood based, liquid biopsy test for confirming the need for a prostate biopsy by objectively analyzing flow cytometry-based immunophenotyping data using machine learning (ML) by evaluating whether a patient is at-risk for having intermediate-/high-grade prostate cancer PCa [Gleason score (GS) ≥ 7]. Currently, there is a lack of blood-based biomarkers that can accurately predict high-risk PCa in at-risk men making it difficult for clinicians to assess disease status. Furthermore, a large percentage of men undergo unnecessary prostate biopsy procedures each year since the majority of biopsies return either benign pathologies, such as benign prostatic hyperplasia (BPH), or low-grade PCa [Gleason score (GS) = 6). Flow cytometry-based immunophenotyping of peripheral blood is an accessible and non-invasive technology, but as more biomarkers are included, new methods must be developed for the efficient analysis and utilization of these large datasets for clinical applications. Here, we used ML to create pattern recognition neural networks (PRNNs) to identify patients with intermediate-/high-grade PCa from those that have either low-grade PCa or BPH based upon the immunophenotyping of 27 different myeloid and lymphoid cell populations. A key population of interest are myeloid-derived suppressor cells (MDSCs) since they are known to be key contributors in supporting tumor progression and escape through their ability to suppress anti-tumor responses mediated through T cell and natural killer (NK) cell activity. We used standard multiparametric flow cytometry to measure MDSCs and other immune cell populations found in the peripheral blood of 114 biopsy-confirmed PCa and 58 biopsy-confirmed BPH patients that were collected in Streck Cyto-Chex® BCT. PRNNs were trained with raw flow cytometry from two data sets: 37 patients with PCa GS ≥ 7 and 40 patients with BPH/PCa GS6. Predictions were evaluated using the performance of the trained PRNNs on known PCa GS ≥ 7 and BPH/PCa GS6 not used in the PRNN training set (holdout samples). In the holdout samples, we identified 21 out of 23 patients who were identified as PCa GS ≥ 7 (Sens = 91.3%, 95%CI = 72.0% to 98.9%; PPV = 33.3%, 95%CI = 28.4% to 38.7%) while 30 out of 72 patients were identified as BPH/PCa GS6 (Spec = 41.7%, 95%CI = 30.2% to 53.9%; NPV = 93.8%, 95%CI = 79.5% to 98.3%) for a combined accuracy of 53.7% (95%CI = 43.2% to 64.0%). The PRNN classified 17 out of 33 BPH, 13 out of 26 GS6 PCa, 13 out of 14 GS7 PCa, and 8 out of 9 &amp;gt; GS7 PCa samples correctly. For this study, out of the 112 patients who were ultimately diagnosed with BPH or low-grade PCa, approximately 47 procedures could have been avoided or delayed. In a clinical setting, we believe that this technology, in use with other known clinical factors, would allow for clinicians to have a more informed decision when recommending their patients for a prostate biopsy procedure while reducing the number of unnecessary biopsy procedures performed each year. Citation Format: George A. Dominguez, John Roop, Anthony J. Campisi, Thomas Schlumpberger, Amit Kumar. Using artificial intelligence and flow cytometry to confirm the need for a prostate biopsy in at-risk men [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 3144.

A Population of Injury-Responsive Lymphoid Cells Expresses mpeg1.1 in the Adult Zebrafish Heart.

Transgenic zebrafish that express fluorophores under the control of mpeg1.1 (mpeg1) and csf1ra (c-fms) promoters have been widely used to study the dynamics and functions of mononuclear phagocytes (MNPs) in larval zebrafish, unveiling crucial roles for these innate immune cells in many processes, including tissue repair. Adult zebrafish are also being increasingly used as a model organism for such studies because of their regenerative capacity and presence of innate and adaptive immune cells. For example, recent investigations highlight roles of MNPs in the regulation of diverse cellular processes during heart regeneration, including scarring, cardiomyocyte proliferation, and neovascularization. However, transgenic lines that stratify MNP subpopulations (monocytes, macrophages, and dendritic cells) are not yet available, preventing functional analysis of these populations. In an attempt to better segregate cardiac MNPs, we assessed the coexpression of mpeg1.1 and csf1ra reporter transgenes in adult zebrafish hearts. Unexpectedly, this also identified a discrete population of mpeg1.1 + csf1ra - lymphoid-like cells, which respond to cardiac cryoinjury in a different temporal pattern to mpeg1.1 + MNPs. mpeg1.1 + lymphoid cells were also abundant in the skin, spleen, and blood, and their frequency was unaffected in the hearts of csf1raj4e1/j4e1 mutant zebrafish, which display deficiencies in MNP populations. Flow cytometry, imaging, and cytological and gene expression analyses collectively indicate that these cells comprise a mixed population of B cells and NK-like cells. Our study therefore highlights the need to identify novel MNP lineage markers but also suggests undetermined roles of B cells and NK-like cells in cardiac homeostasis and repair in adult zebrafish.

Inflammation-Induced Lactate Leads to Rapid Loss of Hepatic Tissue-Resident NK Cells

The liver harbors two main innate lymphoid cell (ILC) populations: conventional NK (cNK) cells and tissue-resident NK (trNK) cells. Using the MCMV model of infection, we find that, in contrast to liver cNK cells, trNK cells initially undergo a contraction phase followed by a recovery phase to homeostatic levels. The contraction is MCMV independent because a similar phenotype is observed following poly(I:C)/CpG or α-GalCer injection. The rapid contraction phase is due to apoptosis, whereas the recovery phase occurs via proliferation in situ. Interestingly, trNK cell apoptosis is not mediated by fratricide and not induced by liver lymphocytes or inflammatory cytokines. Instead, we find that trNK cell apoptosis is the consequence of an increased sensitivity to lactic acid. Mechanistic analysis indicates that trNK cell sensitivity to lactate is linked to impaired mitochondrial function. These findings underscore the distinctive properties of the liver-resident NK cell compartment.

4578 Early life stress promotes chronicity of experimental colitis

OBJECTIVES/GOALS: The overall goal of this study was to determine the effect of early life stress (ELS) on the intestinal CD4+ T cell immune compartment, at homeostasis and after induction of experimental Inflammatory Bowel Disease (IBD). METHODS/STUDY POPULATION: We used a mouse model of ELS, maternal separation with early weaning (MSEW). We used IL-10 reporter mice to enable analysis of IL-10-producing cells. Mice were examined on postnatal day 28 to determine the impact of ELS on gut regulatory T cells. Plasma levels of corticosterone (rodent stress response hormone) was determined by ELISA. Colitis was induced in MSEW and normal rear (NR) mice via intraperitoneal injection of α-IL-10R every 5 days until day 15. Mice were euthanized on days 20 and 30. Colonic tissue sections were stained for histological analysis. Remaining tissue was further processed for flow cytometric analysis of CD4+ T cells and innate lymphoid cells. RESULTS/ANTICIPATED RESULTS: Plasma corticosterone was elevated in MSEW mice compared to their NR counterparts at 4 weeks of age. We observed that the MSEW stress protocol does not affect the baseline colonic CD4+ T cell or innate lymphoid cell populations. There was a reduction in the intestinal CD4+ T cells and regulatory T cells on day 20 in α-IL-10R MSEW mice compared to NR counterparts. This difference disappeared by day 30. Histological scoring showed no difference in disease severity between α-IL-10R treated MSEW and NR mice on day 20. However, on day 30, when α-IL-10R NR mice are recovering from colitis, MSEW mice showed persistent histological inflammation, mainly attributable to sustained epithelial damage. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that ELS prolongs intestinal inflammation and impairs epithelial repair. Future studies will focus on elucidating the mechanisms responsible for ELS-dependent impairment of mucosal repair in experimental colitis.

Abstract B50: Using pattern recognition neural networks to detect prostate cancer: A new method to analyze flow cytometry-based immunophenotyping using machine learning

Abstract Prostate cancer (PCa) screening and detection relies heavily upon prostate-specific antigen (PSA) testing, but PSA testing has a high rate of false positives, leading to increased risks for overdiagnosis and overtreatment; thus, additional blood-based biomarkers for PCa detection are needed. Flow cytometry-based immunophenotyping of peripheral blood is an accessible and noninvasive technology, but as more parameters are included, new computational methods must be developed for the efficient analysis and utilization of these large datasets for clinical applications. Machine learning algorithms, specifically pattern recognition neural networks (PRNNs), have the potential to assist in these types of analyses, but the flow cytometry data need to be transformed into a usable input format. The goal of this study was to use our newly developed “hypervoxelation of cytometry events” computational technique, or HyperVOX, to transform flow cytometry data into a useable format for input into a series of PRNNs to detect PCa of all Gleason scores (GS) from circulating immune cells. We used standard multiparametric flow cytometry techniques to measure 16 different myeloid and lymphoid cell populations found in the peripheral blood of 156 biopsy-confirmed PCa (GS6 n = 59, GS7 n = 68, GS8 n = 12, GS9 n = 16, and GS10 n = 1; median age = 68 ± 8.7 years) along with 99 male healthy donors (HD) (median age = 53 ± 8.5 years). Flow cytometry data were then transformed using HyperVOX in order to create hypervoxels that can be used as the common feature across all samples. Briefly, each channel was used as an axis in a multidimensional space and divided into four segments, with each event being defined by its location within each segment of each axis. The resulting count of events that fall within each hypervoxel for each sample is then used as the input for the PRNN. With this, a screening-type assay was developed to detect PCa compared against HD. PRNNs were trained using raw flow cytometry data processed using HyperVOX from 97 PCa patients and 67 HD controls. Predictions were evaluated using the performance of the trained PRNNs on 59 PCa patients and 32 HD that were not used for PRNN training (holdout test set). The PRNN classified 28 out of 32 HD and 57 out of 59 PCa samples correctly, resulting in a sensitivity of 96.6% (95% CI, 88.3–99.6), specificity of 87.5% (95% CI, 71.0–96.5), positive predictive value (PPV) of 93.4% (95% CI, 85.1–98.2), negative predictive value (NPV) of 93.3% (95% CI, 78.1–98.2), and an AUC of 0.9656 (95% CI, 0.9202–1). Upon Gleason score stratification, the NN classified 27 out of 28 GS6, 18 out of 19 GS7, and 11 out of 11 &amp;gt;GS7 samples correctly. In a clinical setting, this technology would improve PCa detection and allow clinicians to have a more informed decision when recommending their patients for a prostate biopsy procedure and subsequent medical interventions to help reduce overdiagnosis and overtreatment. Citation Format: George A. Dominguez, John Roop, Alexander Polo, Anthony Campisi, Dmitry I. Gabrilovich, Amit Kumar. Using pattern recognition neural networks to detect prostate cancer: A new method to analyze flow cytometry-based immunophenotyping using machine learning [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B50.