Subset Of Patterns Research Articles

MrgprA3 neurons drive cutaneous immunity against helminths through selective control of myeloid-derived IL-33.

Skin uses interdependent cellular networks for barrier integrity and host immunity, but most underlying mechanisms remain obscure. Herein, we demonstrate that the human parasitic helminth Schistosoma mansoni inhibited pruritus evoked by itch-sensing afferents bearing the Mas-related G-protein-coupled receptor A3 (MrgprA3) in mice. MrgprA3 neurons controlled interleukin (IL)-17+ γδ T cell expansion, epidermal hyperplasia and host resistance against S. mansoni through shaping cytokine expression in cutaneous antigen-presenting cells. MrgprA3 neuron activation downregulated IL-33 but induced IL-1β and tumor necrosis factor in macrophages and type 2 conventional dendritic cells partially through the neuropeptide calcitonin gene-related peptide. Macrophages exposed to MrgprA3-derived secretions or bearing cell-intrinsic IL-33 deletion showed increased chromatin accessibility at multiple inflammatory cytokine loci, promoting IL-17/IL-23-dependent changes to the epidermis and anti-helminth resistance. This study reveals a previously unrecognized intercellular communication mechanism wherein itch-inducing MrgprA3 neurons initiate host immunity against skin-invasive parasites by directing cytokine expression patterns in myeloid antigen-presenting cell subsets.

Harnessing Immune Cell Metabolism to Modulate Alloresponse in Transplantation.

Immune cell metabolism plays a pivotal role in shaping and modulating immune responses. The metabolic state of immune cells influences their development, activation, differentiation, and overall function, impacting both innate and adaptive immunity. While glycolysis is crucial for activation and effector function of CD8 T cells, regulatory T cells mainly use oxidative phosphorylation and fatty acid oxidation, highlighting how different metabolic programs shape immune cells. Modification of cell metabolism may provide new therapeutic approaches to prevent rejection and avoid immunosuppressive toxicities. In particular, the distinct metabolic patterns of effector and suppressive cell subsets offer promising opportunities to target metabolic pathways that influence immune responses and graft outcomes. Herein, we review the main metabolic pathways used by immune cells, the techniques available to assay immune metabolism, and evidence supporting the possibility of shifting the immune response towards a tolerogenic profile by modifying energetic metabolism.

Regulation of the programmed cell death protein 1/programmed cell death ligand 1 axis in relapsing-remitting multiple sclerosis.

The programmed cell death protein 1/programmed cell death ligand 1 axis plays an important role in the adaptive immune system and has influence on neoplastic and inflammatory diseases, while its role in multiple sclerosis is unclear. Here, we aimed to analyse expression patterns of programmed cell death protein 1 and programmed cell death ligand 1 on peripheral blood mononuclear cells and their soluble variants in multiple sclerosis patients and controls, to determine their correlation with clinical disability and disease activity. In a cross-sectional study, we performed in-depth flow cytometric immunophenotyping of peripheral blood mononuclear cells and analysed soluble programmed cell death protein 1 and programmed cell death ligand 1 serum levels in patients with relapsing-remitting multiple sclerosis and controls. In comparison to control subjects, relapsing-remitting multiple sclerosis patients displayed distinct cellular programmed cell death protein 1/programmed cell death ligand 1 expression patterns in immune cell subsets and increased soluble programmed cell death ligand 1 levels, which correlated with clinical measures of disability and MRI activity over time. This study extends our knowledge of how programmed cell death protein 1 and programmed cell death ligand 1 are expressed in the membranes of patients with relapsing-remitting multiple sclerosis and describes for the first time the elevation of soluble programmed cell death ligand 1 in the blood of multiple sclerosis patients. The distinct expression pattern of membrane-bound programmed cell death protein 1 and programmed cell death ligand 1 and the correlation between soluble programmed cell death ligand 1, membrane-bound programmed cell death ligand 1, disease and clinical factors may offer therapeutic potential in the setting of multiple sclerosis and might improve future diagnosis and clinical decision-making.

Adenoid lymphocyte heterogeneity in pediatric adenoid hypertrophy and obstructive sleep apnea.

Adenoid hypertrophy is the main cause of obstructive sleep apnea in children. Previous studies have suggested that pathogenic infections and local immune system disorders in the adenoids are associated with adenoid hypertrophy. The abnormalities in the number and function of various lymphocyte subsets in the adenoids may play a role in this association. However, changes in the proportion of lymphocyte subsets in hypertrophic adenoids remain unclear. To identify patterns of lymphocyte subsets in hypertrophic adenoids, we used multicolor flow cytometry to analyze the lymphocyte subset composition in two groups of children: the mild to moderate hypertrophy group (n = 10) and the severe hypertrophy group (n = 5). A significant increase in naïve lymphocytes and a decrease in effector lymphocytes were found in severe hypertrophic adenoids. This finding suggests that abnormal lymphocyte differentiation or migration may contribute to the development of adenoid hypertrophy. Our study provides valuable insights and clues into the immunological mechanism underlying adenoid hypertrophy.

Abstract 5673: Characterization of AB598, a therapeutic anti-human CD39 antibody for the treatment of cancer

Abstract Background: CD39 catalyzes the conversion of extracellular adenosine triphosphate (ATP) into adenosine monophosphate (AMP), resulting in decreased levels of immunostimulatory ATP and increased levels of immunosuppressive adenosine in the tumor microenvironment (TME). By blocking CD39 in the TME, particularly in combination with immunogenic cell death (ICD)-inducing chemotherapies, local levels of ATP can increase, leading to myeloid cell activation and improved tumor control. AB598 potently binds and inhibits enzymatic activity of human and cynomolgus CD39 but not murine CD39. Human CD39 knock-in (hCD39KI) mice have been employed to determine anti-tumor efficacy of AB598 in animals with competent immune systems. Methods: Binding affinity and enzymatic inhibition of AB598 were determined in primary human immune cells. ATP-mediated activation of monocyte-derived dendritic cells (moDCs) and macrophages were assayed in vitro with and without AB598. Whole blood and tissue-based receptor occupancy (RO) assays were developed using AB598-competitive and non-competitive anti-CD39 reagents. In vitro binding of AB598 was determined in samples from human and cynomolgus donors. CD39 protein expression, target engagement, and enzymatic inhibition were assessed in whole blood and tissue samples collected from AB598-dosed animals. CD39 expression patterns in peripheral immune subsets were confirmed in samples from healthy human donors. CD39 enzymatic activity was evaluated in blood-derived cells and serum from healthy human donors and cancer patients. Results: AB598 binds to CD39-expressing monocytes, B cells, and other immune cell populations in human and cynomolgus whole blood with sub-nanomolar affinity. Following AB598 dosing in preclinical species, decreases in CD39 surface expression on peripheral immune cells were observed, consistent with other anti-CD39 therapeutic antibodies. Target engagement and enzymatic inhibition were evident in tissues collected from AB598-dosed animals. Treatment with AB598/chemotherapy combinations resulted in significant syngeneic tumor control in hCD39KI mouse models. Conclusion: When used in combination with an ICD-inducing chemotherapy agent, AB598 can promote anti-tumor immunity by activation of myeloid cells due to increased local ATP levels. Preclinical characterization of the pharmacodynamic effects of AB598 support our therapeutic rationale and demonstrate target engagement and inhibition both peripherally and intratumorally. Citation Format: Amy E. Anderson, Angelo Kaplan, Urvi Vani, Enzo Stagnaro, Kaustubh Parashar, Julie Clor, Nigel P. Walker, Steve W. Young, Matthew J. Walters, Ester Fernandez-Salas, Christine E. Bowman, Lisa Seitz. Characterization of AB598, a therapeutic anti-human CD39 antibody for the treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5673.

Aberrant Expression and Prognostic Potential of IL-37 in Human Lung Adenocarcinoma.

Interleukin-37 (IL-37) is a relatively new IL-1 family cytokine that, due to its immunoregulatory properties, has lately gained increasing attention in basic and translational biomedical research. Emerging evidence supports the implication of this protein in any human disorder in which immune homeostasis is compromised, including cancer. The aim of this study was to explore the prognostic and/or diagnostic potential of IL-37 and its receptor SIGIRR (single immunoglobulin IL-1-related receptor) in human tumors. We utilized a series of bioinformatics tools and -omics datasets to unravel possible associations of IL-37 and SIGIRR expression levels and genetic aberrations with tumor development, histopathological parameters, distribution of tumor-infiltrating immune cells, and survival rates of patients. Our data revealed that amongst the 17 human malignancies investigated, IL-37 exhibits higher expression levels in tumors of lung adenocarcinoma (LUAD). Moreover, the expression profiles of IL-37 and SIGIRR are associated with LUAD development and tumor stage, whereas their high mRNA levels are favorable prognostic factors for the overall survival of patients. What is more, IL-37 correlates positively with a LUAD-associated transcriptomic signature, and its nucleotide changes and expression levels are linked with distinct infiltration patterns of certain cell subsets known to control LUAD anti-tumor immune responses. Our data indicate the potential value of IL-37 and its receptor SIGIRR to serve as biomarkers and/or immune-checkpoint therapeutic targets for LUAD patients. Further, the data highlight the urgent need for further exploration of this cytokine and the underlying pathogenetic mechanisms to fully elucidate its implication in LUAD development and progression.

Host biology, ecology and the environment influence microbial biomass and diversity in 101 marine fish species

Fish are the most diverse and widely distributed vertebrates, yet little is known about the microbial ecology of fishes nor the biological and environmental factors that influence fish microbiota. To identify factors that explain microbial diversity patterns in a geographical subset of marine fish, we analyzed the microbiota (gill tissue, skin mucus, midgut digesta and hindgut digesta) from 101 species of Southern California marine fishes, spanning 22 orders, 55 families and 83 genera, representing ~25% of local marine fish diversity. We compare alpha, beta and gamma diversity while establishing a method to estimate microbial biomass associated with these host surfaces. We show that body site is the strongest driver of microbial diversity while microbial biomass and diversity is lowest in the gill of larger, pelagic fishes. Patterns of phylosymbiosis are observed across the gill, skin and hindgut. In a quantitative synthesis of vertebrate hindguts (569 species), we also show that mammals have the highest gamma diversity when controlling for host species number while fishes have the highest percent of unique microbial taxa. The composite dataset will be useful to vertebrate microbiota researchers and fish biologists interested in microbial ecology, with applications in aquaculture and fisheries management.

Ozone therapy restores immune dysfunction in refractory idiopathic granulomatous mastitis as a novel potential therapeutic approach.

Immunological dysfunction has been suggested to play a major role in the pathogenesis of idiopathic granulomatous mastitis (IGM). We recently showed that ozone therapy was effective in patients with steroid-resistant IGM. This study assessed alterations in intracellular cytokine expression patterns in different T-lymphocyte subsets after ozone therapy in refractory IGM. Peripheral blood T lymphocyte subsets (CD8+ , CD4+ , CD4+ CD25+ CD127- ) were analyzed via flow-cytometry for intracellular cytokine expressions IFN-γ, TNF-α, IL-10, and TGF-β before and after completion of 4-month systemic ozone therapy.Ozone therapy significantly increased the CD4+ IFN-γ+ (p = 0.032), CD4+ TNF-α+ (p = 0.028), and the CD8+ TNF-α+ (p = 0.012) T cells. In contrast, significant decreases in CD4+ IL-10+ (p = 0.047) and CD8+ IL-10+ T cells (p = 0.022) and CD4+ CD25+ CD127-//low Treg cells secreting TGF-β (p = 0.005) were found after ozone therapy. When patients were analyzed according to the response to ozone therapy, patients with a complete remission were more likely to have increased CD3- CD16+ CD56+ natural killer cells (p = 0.0027) and decreased CD19+ B lymphocytes (p = 0.046) following ozone therapy. Our results suggest that ozone therapy stimulated a T-helper-1 response associated with IFN-γ production and downregulation of TGF-β expression in CD4+ CD25+ CD127- Treg cells. These alterations in the immune system following ozone therapy can improve wound healing and restore immune dysfunction in patients with refractory IGM.

Developmental history modulates adult olfactory behavioral preferences via regulation of chemoreceptor expression in Caenorhabditiselegans.

Developmental experiences play critical roles in shaping adult physiology and behavior. We and others previously showed that adult Caenorhabditiselegans which transiently experienced dauer arrest during development (postdauer) exhibit distinct gene expression profiles as compared to control adults which bypassed the dauer stage. In particular, the expression patterns of subsets of chemoreceptor genes are markedly altered in postdauer adults. Whether altered chemoreceptor levels drive behavioral plasticity in postdauer adults is unknown. Here, we show that postdauer adults exhibit enhanced attraction to a panel of food-related attractive volatile odorants including the bacterially produced chemical diacetyl. Diacetyl-evoked responses in the AWA olfactory neuron pair are increased in both dauer larvae and postdauer adults, and we find that these increased responses are correlated with upregulation of the diacetyl receptor ODR-10 in AWA likely via both transcriptional and posttranscriptional mechanisms. We show that transcriptional upregulation of odr-10 expression in dauer larvae is in part mediated by the DAF-16 FOXO transcription factor. Via transcriptional profiling of sorted populations of AWA neurons from control and postdauer animals, we further show that the expression of a subset of additional chemoreceptor genes in AWA is regulated similarly to odr-10 in postdauer animals. Our results suggest that developmental experiences may be encoded at the level of olfactory receptor regulation, and provide a simple mechanism by which C. elegans is able to precisely modulate its behavioral preferences as a function of its current and past experiences.

In This Issue

Inflammatory toxicities resulting from CAR T-cell therapy are common, but clinical features contributing to risk of these toxicities are not well understood. Saini et al. profiled peripheral blood cells of B-cell lymphoma patients prior to CD19-targeting CAR T-cell treatment and found that patients with clonal hematopoiesis (CH) were more likely to experience severe toxicities and therapy-related myeloid neoplasms compared to patients without CH. CH driven by mutations in DNMT3A, TET2, or ASXL1 conferred an especially greater risk of toxicity. This study highlights a high-risk segment of the CAR T recipient patient population and adds to the body of work associating CH with various inflammatory conditions.See article, p. 385.Relapse of acute myeloid leukemia (AML) after allogeneic bone marrow transplantation has been linked to immune evasion due to reduced expression of major histocompatibility complex class II (MHCII) proteins. Here, Eagle et al. developed a transcription network decomposition algorithm that identified a tetrad of transcription factors (TF) responsible for reduced expression of MHCII at relapse. Blasts with altered TF abundance and silenced MHCII expression were detectable at the time of the initial leukemia diagnosis. The results point to an adaptive transcriptional mechanism of immune escape through combinatorial TF fluctuations resulting in emergence of cells with a silenced T-cell stimulation program.See article, p. 394.Acute erythroid leukemia (AEL) is a unique subtype of acute myeloid leukemia with prominent erythroid differentiation and a poorly understood molecular basis. Takeda et al. performed a comprehensive genetic study of 124 AEL cases and revealed that a subset showing extreme (>80%) erythroid differentiation is characterized by multi-hit TP53 alterations, gains/amplifications of EPOR/JAK2, and is associated with a very poor prognosis. Cells from these AEL cases exhibited elevated STAT5 phosphorylation with enhanced proliferation and heme metabolism, and frequently showed sensitivity to ruxolitinib treatment in vitro and in xenograft models, suggesting a potential role of JAK2 inhibition in AEL therapeutics.See article, p. 410.Han and Deng et al. show that mutation-associated low expression of MHC class II (MHCII) on follicular lymphoma (FL) tumor B cells is linked to significant differences in the abundance and gene expression patterns of tumor microenvironment T-cell subsets, including cytotoxic CD4+ T cells. Inferred relative abundance of T-cell subsets in FL defined major microenvironment subtypes, including a “cold” subtype with inferior outcome. Low tumor B-cell MHCII expression was also associated with a reduction in immunotherapeutic target expression. This study provides a framework to consider FL microenvironment characteristics and subtypes in the design and assessment of immune- and cellular-therapy studies.See article, p. 428.Inactivating mutations in TET2 are common in acute myeloid leukemia (AML) and in preneoplastic blood progenitors, suggesting TET2 function is an early barrier to leukemogenesis. To understand which of the TET2-dependent gene circuits are relevant to leukemogenesis, Aivalioti et al. interrogated Tet2 contributions to myeloid differentiation orchestrated by transcriptional master regulator PU.1. Decreased PU.1 activity is commonly observed in AML. Mice with decreased dosages of PU.1 and Tet2 maintain healthy blood formation but develop AML with age. Similar to human AML, progenitor cells from the leukemic mice fail to activate myeloid lineage genes. Hypermethylation at PU.1 binding sites in the enhancer regions of these genes in leukemia points to functional cooperation between PU.1 and Tet2 in sustained activation of myeloid gene program as a critical barrier to leukemogenesis.See article, p. 444.

Mapping of the Sensory Innervation of the Mouse Lung by Specific Vagal and Dorsal Root Ganglion Neuronal Subsets.

The airways are densely innervated by sensory afferent nerves, whose activation regulates respiration and triggers defensive reflexes (e.g., cough, bronchospasm). Airway innervation is heterogeneous, and distinct afferent subsets have distinct functional responses. However, little is known of the innervation patterns of subsets within the lung. A neuroanatomical map is critical for understanding afferent activation under physiological and pathophysiological conditions. Here, we quantified the innervation of the mouse lung by vagal and dorsal root ganglion (DRG) sensory subsets defined by the expression of Pirt (all afferents), 5HT3 (vagal nodose afferents), Tac1 (tachykinergic afferents), and transient receptor potential vanilloid 1 channel (TRPV1; defensive/nociceptive afferents) using Cre-mediated reporter expression. We found that vagal afferents innervate almost all conducting airways and project into the alveolar region, whereas DRG afferents only innervate large airways. Of the two vagal ganglia, only nodose afferents project into the alveolar region, but both nodose and jugular afferents innervate conducting airways throughout the lung. Many afferents that project into the alveolar region express TRPV1. Few DRG afferents expressed TRPV1. Approximately 25% of blood vessels were innervated by vagal afferents (many were Tac1+). Approximately 10% of blood vessels had DRG afferents (some were Tac1+), but this was restricted to large vessels. Lastly, innervation of neuroepithelial bodies (NEBs) correlated with the cell number within the bodies. In conclusion, functionally distinct sensory subsets have distinct innervation patterns within the conducting airways, alveoli and blood vessels. Physiologic (e.g., stretch) and pathophysiological (e.g., inflammation, edema) stimuli likely vary throughout these regions. Our data provide a neuroanatomical basis for understanding afferent responses in vivo.

A Novel Hybrid Approach for Multi-Objective Bi-Clustering in Microarray Data

Background: Today bi-clustering technique plays a vital role to analyze gene expression data in microarray technology. This technique performs clustering on both rows and columns of expression data simultaneously. It determines the expression level of genes set under the subset of several conditions or samples. Basically, obtained information is collected in the form of a sub matrix comprising of microarray data that satisfy coherent expression patterns of subsets of genes with respect to subsets of conditions. These sub matrices are represented as bi-clusters and overall process is called bi-clustering. In this paper, we proposed a new meta-heuristics hybrid ABC-MWOA-CC which is based on artificial bee colony (ABC), modified whale optimization algorithm (MWOA) and Cheng and Church (CC) algorithm to optimize the extracted bi-clusters. In order to validate this algorithm, we also delve into finding the statistical and biological relevancy of extracted genes with respect to various conditions. However, most of the bi-clustering techniques do not address the biological significance of genes belonging to extracted bi-clusters Objective: The major aim of the proposed work is to design and develop a novel hybrid multi-objective bi-clustering approach for in microarray data to produce desired number of valid bi-clusters. Further, these extracted bi-clusters are to be optimized to obtain optimal solution. Method: In the proposed approach, a hybrid multi-objective bi-clustering algorithm which is based on ABC along with MWOA is recommended to group the data into desired number of bi-clusters. Further, ABC with MWOA multi-objective optimization algorithm is applied in order to optimize the solutions using variety of the fitness functions. Results: In the analysis of the result, the multi-objective functions which are employed to judge the fitness calculation like Volume Mean (VM), Mean of Genes (GM), Mean of Conditions (CM) and Mean of MSR (MMSR) leads to improve the performance analysis of the CC bi-clustering algorithm on real life data set such as Yeast Saccharomyces cerevisiae cell cycle gene Expression datasets. Conclusion: The effectiveness of the ABC-MWOA-CC algorithm is comprehensively demonstrated by comparing it with well-known traditional ABC-CC, OPSM and CC algorithm in terms of VM, GM, CM and MMSR.

Correlation of Myeloid Derived Suppressor Cells and Response to Standard Chemoimmunotherapy in Diffuse Large B Cell Lymphoma

INTRODUCTION: Immature myeloid (IM) cells have been shown to induce immune dysfunction(s) in non-Hodgkin's lymphoma (NHL) patients. Myeloid derived suppressor cells (MDSCs) are a subset of IM cells that play a key role in immune suppression associated with malignancies. In solid tumors, MDSCs have been implicated in promoting angiogenesis, tumor cell invasion and metastasis. The presence of MDSCs may correlate with decreased efficacy of immunotherapies. MDSCs have been associated with reduced survival in solid tumors. We aimed at identifying the patterns of immature myeloid cell subsets including MDSCs in DLBCL patients, pre and post-standard R-CHOP or R-EPOCH therapy.METHODS: Peripheral blood was collected on 31 patients with de novo diffuse large B cell lymphoma on an IRB approved study before and after frontline chemo-immunotherapy and at the time of relapse. Clinical variables at diagnosis (age, performance status, stage of the disease, number of extra nodal lesions) were obtained to calculate prognostic indices (IPI and R-IPI). Cell of origin was determined by IHC using the Hans algorithm. Response to therapy was assessed as per the Lugano criteria for response and assessment in lymphoma. Peripheral distribution of myeloid cells (CD11b+ CD33+), IM cells (HLA-DR-/low IL-4R+) and immunosuppressive IM cell subsets [CD14+ monocytic (m) and CD15+ granulocytic (g) myeloid-derived suppressor cells (MDSCs)] in fresh whole blood samples were established by flow cytometry.RESULTS: Median age was 65 years (range 24-88). Twenty-two (70.9%) patients had stage III/IV disease. Thirteen patients (41.9%) had GCB subtype, 16 patients (51.6%) had non-GCB subtype and cell of origin could not be determined in 2 patients (6.5%). Nineteen patients (61.3%) had IPI scores 0-2 and 12 (38.7%) patients had IPI scores of 3-5. Twenty-two patients (70.9%) received R-CHOP or R-mini CHOP, and 9 patients (29%) received R-EPOCH. The end of therapy ORR and CR were 100% and 96.7% respectively. One patient had a PR. Median follow up was 17.5 months. Two patients (6.4%) subsequently progressed. Both are alive after subsequent therapy. The overall survival is 100%. Prior to treatment initiation, patients presented with 0.23±0.05% circulating IM cells, of which 51.6±5.6% were mMDSC and 11.2±4.4% were gMDSC. No correlation between IM cell distribution (including MDSCs) and IPI or disease stage was observed. A trend was observed towards higher mMDSCs in GCB subtype compared to non-GCB subtype (3.49±1.4% vs. 1.44±0.7% of total myeloid cells; p=0.086). Following treatment with chemo-immunotherapy, overall distribution of circulating IM cells decreased to 0.05±0.04% of total WBC (p=0.012 compared with baseline), 31.2±5.6% of which were mMDSC and 21.3±3.7% were gMDSC. Normalization of IM cell distribution was independent of chemo-immunotherapy regimen or IPI. Compared to early stage disease (I/II), patients with advanced stage disease (stage III/IV) tended to retain more IM cells at the end of therapy (0.06±0.03 vs. 0.03±0.03% of total WBC; p=0.069), a significant proportion of which are mMDSC (0.36±0.25 vs. 0.25±0.07% of total myeloid cells; p=0.032). The two patients who relapsed after initial therapy displayed high proportions of MDSCs among total IM cells throughout the course of their disease compared with non-relapsing patients: at baseline (84.8-97.7% vs. 62.2±4.9% of total IM cells), post treatment (87.7-95.7% vs. 52.5±4.4% of total IM cells). This trend was conserved upon relapse with MDCSs representing 91-93% of total IM cells.CONCLUSIONS: To the best of our knowledge, this is the first study to prospectively examine immature myeloid cells and MDSCs in DLBCL at diagnosis, end of therapy and at the time of relapse. Circulating MDSCs, are associated with an active state of DLBCL. Response to chemo-immunotherapy was associated with a dramatic decrease of circulating IM cells, including MDSCs. Patients with advanced stage disease retained a higher titer of circulating mMDSC at end of therapy. For patients who relapsed, a disproportionate abundance of MDSCs (>85% of total IM cells) were seen at diagnosis, post treatment and at the time of relapse. Taken together, MDSCs may represent a potential biomarker and therapeutic target for relapsed and refractory DLBCL patients. [Display omitted] DisclosuresPark:Teva: Consultancy, Research Funding; Takeda: Research Funding; Cornerstone Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead: Speakers Bureau; Seattle Genetics: Research Funding. Ghosh:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau; Jassen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding, Speakers Bureau; TG Therapeutics: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Kite Pharma: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Speakers Bureau.

Patterns of dendritic cell and monocyte subsets are associated with disease severity and mortality in liver cirrhosis patients

Liver cirrhosis is often complicated by an immunological imbalance known as cirrhosis-associated immune dysfunction. This study aimed to investigate disturbances in circulating monocytes and dendritic cells in patients with acute decompensation (AD) of cirrhosis. The sample included 39 adult cirrhotic patients hospitalized for AD, 29 patients with stable cirrhosis (SC), and 30 healthy controls (CTR). Flow cytometry was used to analyze monocyte and dendritic cell subsets in whole blood and quantify cytokines in plasma samples. Cirrhotic groups showed higher frequencies of intermediate monocytes (iMo) than CTR. AD patients had lower percentages of nonclassical monocytes than CTR and SC. Cirrhotic patients had a profound reduction in absolute and relative dendritic cell numbers compared with CTR and showed higher plasmacytoid/classical dendritic cell ratios. Increased plasma levels of IL-6, IL-10, and IL-17A, elevated percentages of CD62L+ monocytes, and reduced HLA-DR expression on classical monocytes (cMo) were also observed in cirrhotic patients. Patients with more advanced liver disease showed increased cMo and reduced tissue macrophages (TiMas) frequencies. It was found that cMo percentages greater than 90.0% within the monocyte compartment and iMo and TiMas percentages lower than 5.7% and 8.6%, respectively, were associated with increased 90-day mortality. Monocytes and dendritic cells are deeply altered in cirrhotic patients, and subset profiles differ between stable and advanced liver disease. High cMo and low TiMas frequencies may be useful biomarkers of disease severity and mortality in liver cirrhosis.

Abnormal glycosphingolipid patterns in human cholangiocarcinoma stem-like subsets

Abnormal glycosphingolipid patterns in human cholangiocarcinoma stem-like subsets

Innate-like Chemokine Receptor Profile and Migratory Behaviour By Terminally Differentiated and Educated NK Cells

Natural Killer (NK) cells play an essential role in cancer surveillance and have a unique capability of spontaneous cytotoxicity against cancer cells. The human NK cell repertoire is functionally diversified through a tightly regulated differentiation process characterized by an early transition from CD56bright to CD56dim NK cells, followed by coordinated changes in expression of inhibitory receptors, including NKG2A and killer cell immunoglobulin-like receptors (KIR). The acquisition of self HLA class I binding KIRs during NK cell differentiation tunes the cytotoxic potential of NK cells in a process termed education, characterized by increased loading of granzyme B in dense core granules. Although NK cell differentiation and education are critical determinants of the functional potential of the cell, little is known about how these events shape the migratory behavior of NK cells. To mediate appropriate and directed immune response against cancer, NK cells must be capable of migration to the tumor site. This process is mediated by chemokines, which guide cell migration by binding to their specific receptors. For example, in multiple myeloma, CXCR3 and CCR5 ligands (MIG, IP-10, and MIP-1a) are significantly upregulated in the bone marrow compared to healthy controls, affecting the composition of immune cells in the tumor microenvironment. In order to delineate the homing patterns of distinct NK cell subsets, we used high-dimensional flow cytometry combined with functional assays to map the NK cell chemokine receptor expression and migratory behavior. We screened resting and cytokine/feeder cell stimulated peripheral blood NK cells for the expression of a panel of 20 chemokine receptors (A). Based on CD56, CD57, NKG2A, and KIR expression, NK cells were divided into 6 phenotypically and functionally distinct subsets that were ordered according to their differentiation status (B). We found that the expression of CX3CR1, CXCR1, CXCR2, and CMKLR1 gradually increased during differentiation, whereas the expression of CXCR3, CCR7, and CCR5 was lower in more differentiated NK cells. CXCR4, CCR4, and CCR2 expression was relatively uniform across all subsets. Interestingly, CCR1 and CXCR6 were expressed mainly on less differentiated NKG2A+ CD56dim NK cells (B). Next, we stratified the chemokine receptor expression on mature KIR+ NK cells based on the expression of self (educated) or non-self KIR (uneducated). Educated NK cells expressed CXCR1, CX3CR1, CCR5, and CMKLR1 at higher levels than the uneducated NK cells. Conversely, CXCR3 was expressed at lower levels on educated NK cells (C). No difference was observed for CXCR2 expression. To determine whether the observed differences in chemokine receptor expression translate into altered chemokine responsiveness between the subsets, we combined the transwell system with multicolor flow cytometry. We found that the chemokine-induced migration capability of NK cells correlated closely with the expression level of corresponding chemokine receptor, leading to subset specific responses to various chemokine gradients (D). The present results show that peripheral blood NK cell chemokine receptor profile changes in a coordinated fashion during NK cell differentiation and is further influenced by the expression of self-specific KIR. Interestingly, receptors which expression declines during NK cell differentiation (CCR5, CCR7, and CXCR3) are commonly associated with adaptive T cell responses to viruses, whereas receptors that are upregulated along the differentiation axis (CXCR1, CXCR2, CX3CR1, CMKLR1) are typical for neutrophils and macrophages as a part of the innate immune response. Thus, our results suggest that NK cell differentiation and education processes together shape the NK cell migratory capabilities to promote homing of the most functional NK cell subsets to the site of inflammation and serve as the first line of defense in the immune response to pathogens and tumors. Figure Disclosures Malmberg: Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.

In vitro immune responses of human PBMCs against Candida albicans reveals fungal and leucocyte phenotypes associated with fungal persistence

Although there is a growing understanding of immunity against Candida albicans, efforts need to be pursued in order to decipher the cellular mechanisms leading to an uncontrolled immune response that eventually oppose disease eradication. We describe here significant intra- and inter-subject variations in immune response patterns of major human leucocyte subsets following an in vitro challenge with C. albicans clinical isolates. We also observed that there are Candida isolate-dependent changes in leucocyte phenotypes. Through a combination of multiple fungal growth and flow cytometric measurements, coupled to the tSNE algorithm, we showed that significant proliferation differences exist among C. albicans isolates, leading to the calculation of a strain specific persistent index. Despite substantial inter-subject differences in T cells and stability of myeloid cells at baseline, our experimental approach highlights substantial immune cell composition changes and cytokine secretion profiles after C. albicans challenge. The significant secretion of IL-17 by CD66+ cells, IFN-γ and IL-10 by CD4+ T cells 2 days after C. albicans challenge was associated with fungal control. Fungal persistence was associated with delayed secretion of IFN-γ, IL-17, IL-4, TNF-α and IL-10 by myeloid cells and IL-4 and TNF-α secretion by CD4+ and CD8+ T cells. Overall, this experimental and analytical approach is available for the monitoring of such fungal and human immune responses.

CD8αα+ T cells show amoeboid shape and frequent morphological change in vitro, and localize to small intestinal intraepithelial region in vivo

Intraepithelial lymphocytes (IELs) are very unique in the intestinal immune system. They include γδT cells and CD4–CD8–TCRαβ+T cells (double negative: DNT), both of which are specific for the intestine, in addition to CD4+ and CD8+ T cells. IELs exist within the monolayer of the intestinal epithelial cells and dynamically move between lamina propria (LP) and intraepithelial (IE) region. The localization and movement patterns of IEL subsets and the regulatory factors have been unknown. Here, we developed a novel in vitro live imaging system and quantified the motility and morphological changes among subsets of IELs. We identified CD8αα as the key regulatory factor. IELs, especially γδ and DNT cells, showed amoeboid shape and frequent morphological change, while most T cells in MLN or SP showed round shape in vitro. TCR signal, IL-15, gut microbes, CCL25, and integrin αEβ7 expression were non-essential for IEL movement in vitro. CD8αα+ cells showed higher motility and larger morphological changes than CD8αα– cells. Adoptive transferred CD8αα+CD4-IELs localized to IE region of recipient NSG mice, while CD8αα–CD4-IELs localized to the LP. Our results showed that the CD8αα/TL signal is essential for the localization of IELs to IE region in vivo. CD8αα/TL may be an effective target to increase the number of IELs, which protects against intestinal infection, allergy, tumorigenesis or inflammation.

Migratory and Dancing Macrophage Subsets in Atherosclerotic Lesions.

Macrophages are essential regulators of atherosclerosis. They secrete cytokines, process lipoproteins and cholesterol, and take up apoptotic cells. Multiple subsets of plaque macrophages exist and their differential roles are emerging. Here, we explore macrophage heterogeneity in atherosclerosis plaques using transgenic fluorescent mice in which subsets of macrophages are labeled by GFP (green fluorescent protein), YFP (yellow fluorescent protein), neither, or both. The objective was to define migration patterns of the visible subsets and relate them to their phenotypes and transcriptomes. Apoe-/-Cx3cr1GFPCd11cYFP mice have 4 groups of macrophages in their aortas. The 3 visible subsets show varying movement characteristics. GFP and GFP+YFP+ macrophages extend and retract dendritic processes, dancing on the spot with little net movement while YFP macrophages have a more rounded shape and migrate along the arteries. RNA sequencing of sorted cells revealed significant differences in the gene expression patterns of the 4 subsets defined by GFP and YFP expression, especially concerning chemokine and cytokine expression, matrix remodeling, and cell shape dynamics. Gene set enrichment analysis showed that GFP+ cells have similar transcriptomes to cells found in arteries with tertiary lymphoid organs and regressing plaques while YFP+ cells were associated with progressing and stable plaques. The combination of quantitative intravital imaging with deep transcriptomes identified 4 subsets of vascular macrophages in atherosclerosis that have unique transcriptomic profiles. Our data link vascular macrophage transcriptomes to their in vivo migratory function. Future work on the functional significance of the change in gene expression and motility characteristics will be needed to fully understand how these subsets contribute to disease progression.

A prospective observational study of immune reconstitution following transplantation with post-transplant reduced-dose cyclophosphamide from HLA-haploidentical donors.

Allogeneic hematopoietic cell transplantation (HCT) from HLA-haploidentical donors with post-transplantation high-dose cyclophosphamide (PT/Cy-haplo) now predominates worldwide. However, to our knowledge, no prospective study has compared immune reconstitution after PT/Cy-haplo with that after conventional HCT. The mechanism by which chronic graft-versus-host disease (GVHD) is inhibited by PT/Cy-haplo also remains unknown. We prospectively compared immune recovery patterns of lymphocyte subsets among four groups of adult patients with hematological disease who received HCT from either HLA-matched related or HLA-matched unrelated donors, cord blood transplantation, or reduced-dose PT/Cy-haplo. Counts of CD4+ T-cell subsets, CD8+ T-cell subsets, and NK cells on days 30 and 60 were often lower in PT/Cy-haplo than those in HLA-matched related HCT. The immune recovery pace in PT/Cy-haplo subsequently caught up with that of the other grafts. The regulatory T cells (Tregs) to conventional CD4+ T-cell (Tcon) ratio was significantly higher until day 90 in PT/Cy-haplo. In multivariate analysis, a higher Tregs-to-Tcon ratio on day 60 was significantly associated with a lower incidence of chronic GVHD (P<0.01). The preservation of Tregs by PT/Cy in the early phase might have resulted in a lower incidence of chronic GVHD.