Cytokine Receptor Expression Research Articles

Involvement of Macrophages in Proliferation of Prostate Cancer Cells Infected with Trichomonas vaginalis.

Macrophages play a key role in chronic inflammation, and are the most abundant immune cells in the tumor microenvironment. We investigated whether an interaction between inflamed prostate cancer cells stimulated with Trichomonas vaginalis and macrophages stimulates the proliferation of the cancer cells. Conditioned medium was prepared from T. vaginalis-infected (TCM) and uninfected (CM) mouse prostate cancer (PCa) cell line (TRAMP-C2 cells). Thereafter conditioned medium was prepared from macrophages (J774A.1 cell line) after incubation with CM (MCM) or TCM (MTCM). When TRAMP-C2 cells were stimulated with T. vaginalis, protein and mRNA levels of CXCL1 and CCL2 increased, and migration of macrophages toward TCM was more extensive than towards CM. Macrophages stimulated with TCM produced higher levels of CCL2, IL-6, TNF-α, their mRNAs than macrophages stimulated with CM. MTCM stimulated the proliferation and invasiveness of TRAMP-C2 cells as well as the expression of cytokine receptors (CCR2, GP130, CXCR2). Importantly, blocking of each cytokine receptors with anti-cytokine receptor antibody significantly reduced the proliferation and invasiveness of TRAMP-C2 cells. We conclude that inflammatory mediators released by TRAMP-C2 cells in response to infection by T. vaginalis stimulate the migration and activation of macrophages and the activated macrophages stimulate the proliferation and invasiveness of the TRAMP-C2 cells via cytokine-cytokine receptor binding. Our results therefore suggested that macrophages contribute to the exacerbation of PCa due to inflammation of prostate cancer cells reacted with T. vaginalis.

35P PD-1+ and TIM-3+ T-cells differ in the expression of common γ-chain cytokine receptors after AHSCT in multiple myeloma patients

Checkpoint pathways appear to involve in multiple myeloma (MM) progression, while targeted therapy (e.g., anti-PD-1/anti-PD-L1) has shown inadequate results. PD-1+ and TIM-3+ T cells following high-dose chemotherapy (HDC) with autologous hematopoietic stem cell transplantation (AHSCT) were significantly higher comparing with the pre-transplant levels, and the early post-transplant period seems to be promising for immunotherapy approaches. The objective of the study was to investigate functional features of T cells expressing PD-1 and TIM-3 under lymphopenic conditions after HDC with AHSCT in MM patients.

The CBFβ-SMMHC/NRP1 Axis Regulates FLT3 and TGF-Beta Pathways in Inv(16) Acute Myeloid Leukemia

Signal transduction pathways regulate the proliferation and viability of acute myeloid leukemia (AML) blasts. The regulation in the expression of cytokine receptors in AML is not well understood. In this study, we investigated how the CBFβ-SMMHC fusion protein regulates expression of cytokine receptors in inv(16) AML, with focus on the co-receptor Neuropilin-1 (NRP1).Knock-down of CBFβ-SMMHC expression, utilizing shRNA transduction, induced G1 phase of cell cycle arrest and reduced the viability of inv(16) ME-1 cells in culture. Expression profile analysis of CBFβ-SMMHC knock-down cells revealed a significant repression of genes associated with transmembrane receptor protein kinase pathways, including NRP1 (-5 fold), FGFR1 (-4.2 fold) , FLT3 (-2 fold) and TGFBR2 (-1.2 fold). The expression of NRP1 was significantly upregulated in inv(16) AML cases when compared to other AML sub-types and to hematopoietic stem and progenitor cells. Functionally, NRP1 knock-down reduced the viability of ME-1 cells with a similar dynamics as when using CBFβ-SMMHC shRNAs. In addition, the proliferation of inv(16) AML cells was reduced 4.1-fold when treated with a function-blocking antibody for the FV/VIII extracellular NRP1 domain, while having no effect in non-inv(16) AML cells or when using blocking antibody for the CUB extracellular domain. Furthermore, deletion of Nrp1 by gene editing reduced the colony-forming unit capacity of primary mouse Cbfb +/MYH11 leukemic cells and extended the median leukemia latency in vivo. To identify the genes regulated by NRP1 in inv(16) AML, we analysed the transcription profile of NRP1 knock-down in ME-1 cells. Gene Set Enrichment and Pathway Analysis revealed a repression in STAT5 pathway, and in signalling receptor activity, including FLT3 (-1.8 fold) and TGFBR2 (-1.8 fold) expression, indicating that NRP1 mediates transcriptional regulation of FLT3 and TGFBR2 expression in inv(16) AML. Furthermore, the regulation of FLT3 and TGFB2 expression by CBFβ-SMMHC and by NRP1 was validated by gene editing in inv(16) AML blasts. Accordingly, NRP1 knock-down in AML cells reduced SMAD2/3 phosphorylation. The repression of RUNX1/CBFβ function, using small molecule inhibitors, in inv(16) AML cells with CBFβ-SMMHC knockdown restored NRP1 expression, suggesting that RUNX1 may repress NRP1 expression in AML cells. To evaluate if RUNX1 directly regulates NRP1 expression, we tested RUNX1 binding in the NRP1 locus of AML cells with CBFβ-SMMHC knockdown. RUNX1 binding at one of six sites with RUNX1 occupancy identified by chromatin immunoprecipitation followed by sequencing (RE5, regulatory element 5) was increased in the CBFβ-SMMHC knock-down cells. The RE5 is located 178kb upstream of the NRP1 transcription start site and it is evolutionarily conserved in vertebrates. The deletion of RE5 by gene editing (~50% editing efficiency) increased NRP1 expression 1.8-fold, suggesting that RUNX1 may repress NRP1 expression at by binding to the RE5 enhancer.Taken together, these studies demonstrate that CBFβ-SMMHC regulates expression of cytokine receptors in inv(16) AML. Specifically, it directly regulates expression of the co-receptor NRP1, which is essential for AML survival, acting (at least in part) by regulating FLT3 and TGFB pathways. DisclosuresGuzman: SeqRx: Consultancy; BridgeMedicines: Consultancy; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

T‐cell subsets in the skin and their role in inflammatory skin disorders

T lymphocytes (T cells) are major players of the adaptive immune response. Naive T cells are primed in the presence of cytokines, leading to polarization into distinct T-cell subsets with specific functions. These subsets are classified based on their T-cell receptor profile, expression of transcription factors, surface cytokine and chemokine receptors, and their cytokine production, which together determine their specific function. This review provides an overview of the various T-cell subsets and their function in several inflammatory skin disorders ranging from allergic inflammation to skin tumors. Moreover, we highlight similarities of T-cell responses across different skin disorders, demonstrating the presence of similar and opposing functions for the different T-cell subsets. Finally, we discuss the effects of currently available and promising therapeutic approaches to harness T cells in inflammatory skin diseases for which efficacy next to unwanted side effects provide new insights into the pathophysiology of skin disorders.

Cytokines, Genetic Lesions and Signaling Pathways in Anaplastic Large Cell Lymphomas

Simple SummaryThis review summarizes the main features of anaplastic large cell lymphoma (ALCL) subtypes focusing on activated biological pathways that may have clinical significance for diagnosis, prognosis and personalized therapy.ALCL is a tumor of activated T cells and possibly innate lymphoid cells with several subtypes according to clinical presentation and genetic lesions. On one hand, the expression of transcription factors and cytokine receptors triggers signaling pathways. On the other hand, ALCL tumor cells also produce many proteins including chemokines, cytokines and growth factors that affect patient symptoms. Examples are accumulation of granulocytes stimulated by IL-8, IL-17, IL-9 and IL-13; epidermal hyperplasia and psoriasis-like skin lesions due to IL-22; and fever and weight loss in response to IL-6 and IFN-γ. In this review, we focus on the biology of the main ALCL subtypes as the identification of signaling pathways and ALCL-derived cytokines offers opportunities for targeted therapies.

Hearing restoration and the stria vascularis: evidence for the role of the immune system in hearing restoration.

This article reviews the current literature regarding the pathogenesis of immune-mediated sensorineural hearing loss, utilizes previously published single-nucleus transcriptional profiles to characterize cytokine and cytokine receptor expression in the adult stria vascularis cell types to support immune system interaction with the stria vascularis and reviews the current literature on immunomodulatory agents currently being used for hearing-restoration treatment. The literature review highlights recent studies that elucidate many cytokines and immune markers, which have been linked to various immune-mediated disease processes that have been observed with sensorineural hearing loss within the stria vascularis and highlights recent publications studying therapeutic targets for these pathways. This review highlights the current literature regarding the pathogenesis of immune-mediated hearing loss. The role of cochlear structures in human temporal bones from patients with immune-mediated sensorineural hearing loss are highlighted, and we review cytokine signalling pathways relevant to immune-mediated sensorineural hearing loss and localize genes encoding both cytokine and cytokine receptors involved in these pathways. Finally, we review immunomodulatory therapeutics in light of these findings and point to opportunities for the application of novel therapeutics by targeting these signalling pathways.

MyD88‐mediated signaling intercedes in neurogenic muscle atrophy through multiple mechanisms

Skeletal muscle atrophy is a debilitating complication of many chronic disease states and disuse conditions including denervation. However, molecular and signaling mechanisms of muscle wasting remain less understood. Here, we demonstrate that the levels of several toll-like receptors (TLRs) and their downstream signaling adaptor, myeloid differentiation primary response 88 (MyD88), are induced in skeletal muscle of mice in response to sciatic nerve denervation. Muscle-specific ablation of MyD88 mitigates denervation-induced skeletal muscle atrophy in mice. Targeted ablation of MyD88 suppresses the components of ubiquitin-proteasome system, autophagy, and FOXO transcription factors in skeletal muscle during denervation. We also found that specific inhibition of MyD88 reduces the activation of canonical nuclear factor-kappa (NF-κB) pathway and expression of receptors for inflammatory cytokines in denervated muscle. In contrast, inhibition of MyD88 stimulates the activation of non-canonical NF-κB signaling in denervated skeletal muscle. Ablation of MyD88 also inhibits the denervation-induced increase in phosphorylation of AMPK without having any effect on the phosphorylation of mTOR. Moreover, targeted ablation of MyD88 inhibits the activation of a few components of the unfolded protein response (UPR) pathways, especially X-box protein 1 (XBP1). Importantly, myofiber-specific ablation of XBP1 mitigates denervation-induced skeletal muscle atrophy in mice. Collectively, our experiments suggest that TLR-MyD88 signaling mediates skeletal muscle wasting during denervation potentially through the activation of canonical NF-κB signaling, AMPK and UPR pathways.

Blockade of IL-2 and IL-15 Gamma Chain Receptor Signaling Decreases Leukemic Cell Viability in T-Cell Large Granular Lymphocyte Leukemia and Adult T-Cell Leukemia

IntroductionInterleukin-15 (IL-15) and IL-2 are important cytokines that drive the progression of many T-cell malignancies such as T-cell Large Granular Lymphocyte Leukemia (T-LGLL) and HTLV-1 derived Adult T-cell Leukemia (ATL).T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with a constitutively active JAK/STAT pathway controlled by cytokines including IL-15 and IL-2. A computational systems approach identified IL-15 as a master regulator of T-LGLL survival and patients have increased IL-15/IL-15Ra serum levels. Cytokine signaling leads to increased phosphorylation and activation of STAT proteins. T-LGLL cells exhibit STAT3 or STAT5 mutational activation in some cases and constitutive activation in all patients. T-LGLL treatments are not selective or curative, and the commonly utilized immunosuppressive agents are not effective in all patients.ATL is an aggressive T-cell disorder that also exhibits aberrant IL-2 and IL-15 cytokine signaling. Tax, identified as an essential oncogene in the disease, is associated with increased cytokine and cytokine receptor expression. ATL patients have high IL-2Ra gene expression and PBMCs from chronic/smoldering ATL patients can depend on autocrine production of cytokines for proliferation. Patients with ATL have poor prognosis and current cytokine-targeting therapies have not proven to be efficacious.The gc receptor is shared by the gc-family cytokines including IL-2, -4, -7, -9, -15 and -21. BNZ-1 (formerly known as BNZ132-1-40) is a pegylated peptide designed to specifically bind the gc receptor to block IL-2, IL-15 and, to a lesser extent, IL-9 cytokine signaling. Because these cytokines are vital to the survival of T cells in T-LGLL and ATL, these diseases represent ideal models to test the hypothesis that treatment of leukemic cells with BNZ-1 will result in reduced proliferation and viability.MethodsThese studies utilized a human T-LGL cell line (TL1), PBMCs from T-LGLL patients and ATL patients, and human IL-15 transgenic NSG mice injected with the ED40515(+) ATL cell line expressing luciferase. In vitro assays were performed after an overnight cytokine withdrawal and 20-minute pretreatment with BNZ-1 prior to addition of cytokine. Leukemic mice were divided into 3 treatment groups; BNZ-1 (40 mg/kg i.v. 2x per week for 4 weeks), JAK1/2 inhibitor Ruxolitinib (Ruxo) (50 mg/kg/day s.c. by pump for 28 days) and PBS control. Tumor burden was measured through bioluminescence imaging (Xenogen IVIS).ResultsOur results demonstrate that in vitro treatment of TL1 cells with BNZ-1 inhibits IL-2 and IL-15 mediated expansion and viability. BNZ-1 impaired the growth of these cytokine-dependent cells to the level observed in cells without exogenous cytokine supplementation. In addition, BNZ-1 was able to drastically block IL-15 and IL-2 induced phosphorylation events, specifically STAT1, STAT3, STAT5, ERK and Akt. The compound blocked IL-15 mediated viability and signaling more effectively than that mediated by IL-2.The BNZ-1 mediated decreased viability of PBMCs from T-LGLL patients was observed in the presence of either IL-2 or IL-15 stimulation and regardless of STAT3 mutational status. A potent reduction of IL-2 and IL-15 mediated STAT1, STAT3, STAT5 and ERK phosphorylation was also observed in T-LGLL PBMCs with BNZ-1 treatment. Proliferation of PBMCs from ATL patients was inhibited by 58 to 80 percent by the addition of either the antiIL-2Ra antibody daclizumab or and the combination treatment of daclizumab, anti-IL-15 and anti-IL-9. BNZ-1 at 10 µM inhibitedproliferation by 60 to 80 percent.Leukemic burden in the ATL tumor bearing mice was reduced by both BNZ-1 and Ruxo when compared to controls. The BNZ-1 treatment group showed comparable efficacy to that observed in the Ruxo group. Furthermore, BNZ-1 therapy was associated with lower levels of IL-2Ra, which reflects reduced tumor burden at 4 and 7 weeks when compared to that observed with the PBS group.In summary, BNZ-1 shows promise as a potential therapy for T-LGLL and ATL. Results from this study support its advancement in clinical trials in the treatment of T-LGLL and ATL patients. DisclosuresTagaya:Bioniz Therapeutics: Employment. Azimi:Bioniz Therapeutics: Employment.

High-Efficiency Lentiviral Genetic Modification of Primary Human Natural Killer Cells

Natural killer (NK) cells have the unique capacity to lyse tumor cells without the need for antigen specific receptors. Modulation of NK cell cytokine, chemokine, and activating and inhibitory receptor expression using gene regimens represents an attractive strategy to further bolster NK cell anti-tumor activity for the purpose of NK cell immunotherapy. Current efforts in clinical-grade genetic modification of patient-derived cells, used in immunotherapy protocols, rely on stable transduction mediated by viral vectors. However, primary NK cells are notoriously difficult to transduce using viral vectors. Here we describe a lentiviral vector-based approach that results in an efficient, robust, and highly reproducible method of stable gene transfer into primary human peripheral blood-derived NK cells, which can subsequently be expanded ex vivo for adoptive infusion into humans. In vitro experiments established highly efficient transduction of ex vivo expanded NK cells could be achieved by activation of primary NK cells in vitro for 2-4 days in cytokine containing media followed by transduction of activated NK cells using LV vectors 2-3 days before integration into our existing ex vivo expansion protocol using irradiated EBV-LCL feeder cells. Unless NK cells were first activated in cytokine-containing media, LV transduction was unsuccessful. Cytokine activation in media containing IL-2 for 2-4 days was found to be sufficient to achieve high transduction efficiency, while additional supplementation of the media with IL-15 or IL-21 had negligible effects. NK cell transduction efficiency and viability with the use of RetroNectin® was uniformly superior to polycation-based transduction protocols. In experiments where NK cells were transduced with identical lentiviral constructs with 8 different promoter sequences driving expression of EGFP, PGK, EFS, and SV40 promoters consistently demonstrated the highest transduction efficiencies reproducibly in the range of 25-60%. Remarkably, when transduced NK cells were stimulated ex vivo using a clinical grade irradiated EBV-LCL feeder cell line, 100-1000 fold expansions of transduced NK cells could be achieved with sustained transgene expression over two weeks. Transduced and expanded primary NK cells remained highly cytotoxic against K562 tumor targets without developing functional deficiencies in degranulation, IFNγ, or TNFα production. These data establish a simple method to produce large numbers (>1011) of genetically modified clinical grade NK cells suitable for infusion. The protocol described herein overcomes previous difficulties described with common transduction techniques opening new avenues to investigate the impact of adoptive infusion of primary NK cells stably transduced to express a variety of different transgenes of interest. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proteomic analyses reveal that immune integrins are major targets for regulation by Membrane-Associated Ring-CH (MARCH) proteins MARCH2, 3, 4 and 9.

MARCH proteins are membrane-associated Ring-CH E3 ubiquitin ligases that dampen immune responses by downregulating cell surface expression of major histocompatibility complexes I and II as well as immune co-stimulatory receptors. We recently showed that MARCH2,3,4 and 9 also downregulate cell surface expression of the inflammatory cytokine receptor for interleukin-6 (IL6Rα). Here we use over-expression of these MARCH proteins in the M1 myeloid leukaemia cell line and cell surface proteomic analyses to globally analyse other potential targets of these proteins. A large range of cell surface proteins regulated by more than one MARCH protein in addition to several MARCH protein-specific cell surface targets were identified most of which were downregulated by MARCH expression. Prominent among these were several integrin complexes associated with immune cell homing, adhesion and migration. Integrin α4β1 (VLA4 or VCAM-1 receptor) was downregulated only by MARCH2 and we showed that in MARCH2 knockout mice, Integrin α4 was upregulated specifically in mature B-lymphocytes and this was accompanied by decreased numbers of B-cells in the spleen.

The Timing and Abundance of IL-2Rβ (CD122) Expression Control Thymic iNKT Cell Generation and NKT1 Subset Differentiation.

Invariant NKT (iNKT) cells are thymus-generated innate-like T cells, comprised of three distinct subsets with divergent effector functions. The molecular mechanism that drives the lineage trifurcation of immature iNKT cells into the NKT1, NKT2, and NKT17 subsets remains a controversial issue that remains to be resolved. Because cytokine receptor signaling is necessary for iNKT cell generation, cytokines are proposed to contribute to iNKT subset differentiation also. However, the precise roles and requirements of cytokines in these processes are not fully understood. Here, we show that IL-2Rβ, a nonredundant component of the IL-15 receptor complex, plays a critical role in both the development and differentiation of thymic iNKT cells. While the induction of IL-2Rβ expression on postselection thymocytes is necessary to drive the generation of iNKT cells, surprisingly, premature IL-2Rβ expression on immature iNKT cells was detrimental to their development. Moreover, while IL-2Rβ is necessary for NKT1 generation, paradoxically, we found that the increased abundance of IL-2Rβ suppressed NKT1 generation without affecting NKT2 and NKT17 cell differentiation. Thus, the timing and abundance of IL-2Rβ expression control iNKT lineage fate and development, thereby establishing cytokine receptor expression as a critical regulator of thymic iNKT cell differentiation.

MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells

Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.

Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy.

Chimeric antigen receptor (CAR) T-cell therapy engineers T-cells to express a synthetic receptor which redirects effector function to the tumor, to improve efficacy and reduce toxicities associated with conventional treatments, such as radiotherapy and chemotherapy. This approach has proved effective in treating hematological malignancies; however, the same effects have not been observed in solid tumors. The immunosuppressive tumor microenvironment (TME) creates a significant barrier to solid tumor efficacy and reduces the anti-cancer activity of endogenous tumor-resident immune cells, enabling cancer progression. In recent years, researchers have attempted to enhance CAR T-cell function in the TME by engineering the cells to express various proteins alongside the CAR. Examples of this engineering include inducing CAR T-cells to secrete cytokines or express cytokine receptors to modulate the cytokine milieu of the TME. Alternatively, the CAR T-cell may secrete antibody-like proteins to target a range of tumor antigens. Collectively, these methods are termed armored CAR T-cell therapy, and in this review, we will discuss the range of armored CAR T-cell approaches which have been investigated to date.

Expression of extracellular matrix components and cytokine receptors in human fibrocytes during rheumatoid arthritis

ABSTRACT Purpose: Fibroblast-like synoviocytes (FLS) represent one of the principal effectors of joint damage in rheumatoid arthritis (RA). Recent discovery of the circulating fibrocyte, a potential precursor of FLS, has raised issues regarding the characterization of fibrocytes with respect to their morphology and their biological role. In this study, we evaluated the morphology of fibrocytes in vitro and their ability to produce different extracellular matrix (ECM) components in comparison with two populations of RA FLS: synovial fluid FLS (fd-FLS) and intimal lining FLS (td-FLS). We also studied the expression of ECM regulators and a set of cytokine receptors involved in the pathogenesis of RA. Materials and Methods: Fibrocytes were cultured from peripheral blood of patients with RA. FLS were cultured from synovial fluids and tissues. ECM proteins (collagen I (col I) and fibronectin), Matrix metalloproteinases (MMP) (MMP3, and MMP9), ECM regulators (β catenin, TCF4, and c-fos), and cytokine receptors (CXCR1, CXCR2, CXCR3, IL1RI, IL1RII, and IL6Rα) were analyzed using qRT-PCR and/or western blot. Results: Our results demonstrated that fibronectin and MMP3 levels were higher in FLS compared to fibrocytes. Although MMP9 was expressed in the three cell types, its level was greater in fibrocytes than in td/fd FLS. The three cell types expressed CXCR3, IL1RI, IL1RII, and IL6Rα, while the expression of CXCR1 and CXCR2 was restricted to fibrocytes. Conclusion: Our results demonstrated that fibrocytes express ECM molecules and cytokines receptors. The observed differences between fibrocytes and FLS may be due to their distinct functions or differentiation state during RA. Abbreviations: RA: Rheumatoid ArthritisFLS: fibroblast-like synoviocytestd: tissue derivedfd: fluid derivedSF: Synovial FluidWnt: WinglessMMP: Matrix MetalloproteinaseCIA: murine collagen induced arthritisECM: Extracellular matrixcol I: Collagen ITCF/LEF: T-cell factor/lymphoid enhancer-binding factorAP1: Activator Protein 1

Newborn BCG Vaccination Induces Robust Infant IFN

Infants are highly susceptible to disease caused by infection with intracellular pathogens, such as Mycobacterium tuberculosis. The Bacille Calmette-Guerin (BCG) vaccine is usually administered at birth to protect against severe forms of tuberculosis in children. BCG also has non-specific protective effects against heterologous infections, thought to be mediated by enhanced innate cell functions. We aimed to determine whether newborn BCG vaccination modulates myeloid and NK cell responses to mycobacteria. We enrolled 130 South African infants in whom BCG vaccination was either administered at birth or delayed to 6 or 10 weeks of age. CD4 T, myeloid and NK cell responses to whole cell mycobacteria were measured by flow cytometry. Levels of cytokines secreted upon whole blood stimulation with BCG were measured by multiplex bead array. Newborn BCG vaccination was associated with significantly higher frequencies of BCG-reactive, cytokine-expressing CD4+ T cells and IFNγ-expressing NK cells, compared with unvaccinated infants at 5-weeks and 9-weeks of age. However, cytokine-expressing CD33+ myeloid cells were not different between vaccinated and unvaccinated infants. Induction of BCG-reactive IFNγ-expressing NK cells was not associated with enhanced NK cell maturation, differentiation or cytokine receptor expression. Although levels of secreted cytokines were not different in vaccinated and unvaccinated infants, BCG-reactive NK cell responses correlated directly with levels of IL-2 and IFNγ, and the innate pro-inflammatory cytokines IL-6, IL-1β and TNF in BCG vaccinated infants only. Our data show that BCG-reactive IFNγ-expressing NK cells are strongly induced by BCG vaccination in infants and are likely amplified through by-stander cytokine-mediated activation. Further studies in infants are needed to improve our understanding of the possible role BCG-induced NK cell responses may play in mediating protection against tuberculosis and other infectious diseases. Funding Statement: NIH (R01AI087915), European and Developing Countries Clinical Trials Partnership (EDCTP, TA.2011.40200.010), South African National Research Foundation (MSc and PhD fellowships to MM). Declaration of Interests: No competing interests. Ethics Approval Statement: Parents or legal guardians provided written, informed consent before enrolment in the study. The Human Research Ethics Committee at the University of Cape Town approved protocols for infants vaccinated at birth (REF: 126/2006) and infants who had delayed BCG vaccination (REF: 177/2011). Good Clinical Practice procedures and the World Medical Association Declaration of Helsinki guidelines were adhered to.

Reconstruction of Intercellular Signaling Network by Cytokine-Receptor Interactions.

Background: The immune system function depends on the coordination activity of the components of system and communications between them which leads to the formation of a complex communication network between immune cells. In this network, cytokines have an important role in the communication between immune cells through the interaction to their specific receptors. These molecules cause to cellular communications and normal function of a tissue. Reconstruction of such a complex network can be a way to provide a better understanding of cytokines’ function.Objective: Our main goal from reconstructing such a network was investigation of expressed cytokines and cytokines receptors in various lineage and tissues of immune cells and identifying the lineage and tissue with the highest expression of cytokines and their receptors.Materials and Methods: In this study, gene expression data related to part of the Immunological Genome Project (ImmGen) and receptor-ligand interactions dataset were used to reconstruct the immune network in mouse. In next step, the topological properties of reconstructed network, expression specificity of cytokines and their receptors and interactions specificity were analyzed.Results: The results of the network analysis were indicated that non- hematopoietic stromal cells have the highest expression of cytokines and cytokine receptors and interactions specificity is very high. Our results show that chemokine receptor of Ccr1 receives the largest number of signals between receptors and only expressed in three hematopoietic lineages.Conclusions: The most of the network communications belonged to non-hematopoietic stromal and macrophage cells. The relationships between stromal cells and macrophages are necessary to create an appropriate environment for differentiation of immune cells. Studying the cellular expression specificity of receptor and ligand genes reveal the high degree of specificity of these genes that indicate non-random transfer of information between cells in multicellular organisms.

Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry.

Although many flow cytometers can analyze 30-50 parameters, it is still challenging to develop a 40+ color panel for the phenotyping of immune cells using fluorochrome conjugated antibodies due to limitations in the availability of spectrally unique fluorochromes that can be excited by the commonly used laser lines (UV, Violet, Blue, Green/Yellow-green, and Red). Spectral flowcytometry is capable of differentiating fluorochromes with significant overlap in the emission spectra, enabling the use of spectrally similar fluorochrome pairs such as Brilliant Blue 515 and FITC in a single panel. We have developed a 43 color panel to characterize most of the immune subsets within the peripheral immune system, including conventional T cells, unconventional T cells such as invariant natural killer T cells (iNKT), Gamma delta (γδ) T-cell subsets (TCR Vδ2, TCR Vγ9) and mucosal-associated invariant T cells (MAIT), B-cell subsets, natural killer (NK) cells, plasmacytoid dendritic cells, dendritic cell subsets, hematopoietic progenitor cells, basophils, and innate lymphoid cell (ILC) subsets (CD117, CRTH2). The panel includes surface markers to analyze activation (CD38, HLA-DR, ICOS/CD278), differentiation (CD45RA, CD27, CD28, CD57), expression of cytokine and chemokine receptors (CD25, CD127, CCR10, CCR6, CCR4, CXCR3, CXCR5, CRTH2/CD294), and co-inhibitory molecules and exhaustion (PD-1, CD223/LAG-3, TIGIT), which enables a deep characterization of PBMCs from peripheral blood. Cells were analyzed on a 5-laser Cytek Aurora and data analysis was done using FlowJo. This panel can help to make a thorough interpretation of immune system, specifically when specimen quantity is low. The panel has not been completely optimized but would rather act as a guide toward the development of a workflow for optimized multicolor immunophenotyping panel.

Phenotyping of Disease-Initiating CD34+/CD38─ Stem Cells in BCR-ABL1─ MPN Reveals Expression of Multiple Cytokine Receptors and Resistance-Related Antigens

The classical BCR-ABL1-negative myeloproliferative neoplasms (MPN) are characterized by over-production of myeloid cells, disease-related mutations in certain driver-genes (JAK2, CALR, MPL) and an increased risk to transform to secondary acute myeloid leukemia (sAML). Although considered stem cell-derived neoplasms, little is known about the phenotype and functional properties of disease-initiating neoplastic stem cells (NSC) in MPN and sAML. Recent data suggest that MPN NSC reside in a CD34+ fraction of the malignant clone. Therefore, these cells are considered most critical target populations to be examined for expression of molecular and immunological targets with the aim to develop improved or even curative NSC-eliminating therapies, such as antibody-based or CAR-T cell approaches. Using a panel of monoclonal antibodies (n=40) and multicolor flow cytometry, we established the immunological phenotype and target expression profiles of putative CD34+/CD38─ NSC and CD34+/CD38+ progenitor cells in patients with polycythemia vera (PV, n=18), essential thrombocythemia (ET, n=29), primary myelofibrosis (PMF, n=38) and post-MPN sAML (n=11). In almost all patients, the putative MPN stem cells expressed the stem cell invasion receptors Hermes (CD44) and ADGRE5 (CD97), C1qR1 (CD93), the migration/adhesion receptor MIC2 (CD99), and the stem cell antigen AC133 (CD133). Contrasting normal stem cells, MPN NCS and sAML stem cells failed to express Thy-1 (CD90). Among the cytokine receptors tested, MPN NSC invariably displayed the TGFßR-related antigen endoglin (CD105), TPOR (CD110), SCFR KIT (CD117), IL-3RA (CD123), CXCR4 (CD184) and IGF-1R (CD221). NSC expressed particularly high levels of KIT and low levels of TPOR and IGF-1R. The IL-2RA (CD25) was identified on NSC in most patients with PMF and sAML, and in a few with ET, but not in patients with PV. Similarly, the GM-CSFR (CD116) was found to be expressed on NSC in most patients with PMF, a few with ET and no with PV. MPN NSC did not exhibit substantial amounts of M-CSFR (CD115), IL-3RB (CD131), FLT3 (CD135), NGFR (CD271) VEGFR-2 KDR (CD309), EPOR, MET or OSMRB. The CD34+/CD38+ MPN progenitor cells displayed a similar profile of cytokine receptors. In addition, MPN and sAML progenitor cells expressed IL-1RAP and CLL-1 in most donors examined. We next examined the expression of various immunological targets and resistance-mediating immune checkpoint antigens on NSC and MPN progenitor cells. In all MPN patients and all sAML patients tested, NSC were found to express substantial amounts of Siglec-3 (CD33) and low levels of Campath-1 (CD52) and MDR-1 (CD243). In addition, MPN NSC and sAML stem cells invariably displayed the "don't eat" me checkpoint IAP (CD47) and the classical checkpoint PD-L1 (CD274). Exposure to interferon-gamma (200 U/ml, 24 hours) resulted in an upregulation of PD-L1 on NSC. In a subset of patients, MPN NSC expressed low levels of HB15 (CD83). In contrast, MPN NSC and sAML stem cells failed to express B7-1 (CD80), B7-2 (CD86), PD-L2 (CD273) and PD1 (CD279). MPN progenitor cells and sAML progenitors expressed an identical profile of cell surface targets and checkpoint antigens. Finally, we confirmed the disease-initiating capacity of MPN stem- and progenitor cells (CD34+ cells) using primary PMF cells in xenotransplantation experiments employing NSGS mice expressing human interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF). After 28 weeks post injection, engraftment of human CD45+ cells in the bone marrow of NSGS mice was found in 15/15 mice injected with bulk mononuclear cells (MNC) containing CD34+ cells and in 0/15 NSGS mice injected with MNC depleted of CD34+ cells. Together, MPN NSC reside in a CD34+ fraction of the malignant clone and display a unique phenotype, including cytokine receptors, immune checkpoint molecules and other target antigens. The phenotypic characterization of neoplastic stem cells should facilitate their enrichment and the development of NSC-eradicating treatment concepts in MPN. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

T helper cells in synovial fluid of patients with rheumatoid arthritis primarily have a Th1 and a CXCR3+Th2 phenotype

BackgroundThe majority of CD4+ T helper (Th) cells found in the synovial fluid (SF) of patients with rheumatoid arthritis (RA) express CXCR3, a receptor associated with Th1 cells. In blood, subsets of Th2 and Th17 cells also express CXCR3, but it is unknown if these cells are present in RA SF or how cytokines from these subsets affect cytokine/chemokine secretion by fibroblast-like synoviocytes (FLS) from patients with RA.MethodsWe examined the proportions of Th1, Th2, CXCR3+Th2, Th17, CXCR3+Th17, Th1Th17, peripheral T helper (TPh) and T follicular helper (TFh) cells in paired SF and blood, as well as the phenotype of TPh and TFh cells in RA SF (n = 8), by the use of flow cytometry. We also examined the cytokine/chemokine profile in paired SF and plasma (n = 8) and in culture supernatants of FLS from patients with chronic RA (n = 7) stimulated with Th-associated cytokines, by the use of cytometric bead arrays and ELISA. Cytokine receptor expression in FLS (n = 3) were assessed by the use of RNA sequencing and qPCR.ResultsThe proportions of Th1 and CXCR3+Th2 cells were higher in SF than in blood (P < 0.05). TPh and PD-1highTFh in RA SF were primarily of a Th1 and a CXCR3+Th2 phenotype. Moreover, the levels of CXCL9, CXCL10, CCL20, CCL2, CXCL8, IL-6 and IL-10 were higher in SF than in plasma (P < 0.05). Lastly, IL-4, IL-13 and IL-17A induced RA FLS to secrete proinflammatory IL-6, CCL2, CXCL1 and CXCL8, while IFNγ mainly induced CXCL10.ConclusionThese findings indicate that not only Th1 but also CXCR3+Th2 cells may have a pathogenic role in RA synovial inflammation.

An Inverse Relationship Between c-Kit/CD117 and mTOR Confers NK Cell Dysregulation Late After Severe Injury

Major trauma-induced tissue injury causes a dysregulation of the immune system. Severe systemic inflammation occurs early after the insult. Later on, an enhanced risk for life-threatening opportunistic infections develops that culminates at the end of the first week after trauma. CD56bright Natural killer (NK) cells play a key role in the defense against infection due to their rapid release of Interferon (IFN) γ in response to Interleukin (IL) 12. NK cells are impaired in IFN-γ synthesis after severe injury due to a disturbed IL-12/IFN-γ axis. Thereby, a circulating factor mediates extrinsic suppression of NK cells. Yet unknown cell-intrinsic mechanisms manifest by day 8 after trauma and render NK cells unresponsive to stimulatory cytokines. In the present study, we investigated the origin of such late NK cell-intrinsic suppression after major trauma. Peripheral blood mononuclear cells (PBMC) were isolated from patients 8 day after severe injury and from healthy control subjects and were stimulated with inactivated Staphylococcus aureus. The expression of diverse cytokine receptors, intracellular signaling molecules, and the secretion of IFN-γ by CD56bright NK cells were examined. After stimulation with S. aureus, NK cells from patients expressed enhanced levels of c-kit/CD117 that inversely correlated with IFN-γ synthesis and IL-12 receptor (IL-12R) β2 expression. Supplementation with IL-15 and inhibition of the transforming growth factor receptor (TGF-βR) I reduced CD117 expression and increased the level of IL-12Rβ2 and IFN-γ. NK cells from patients showed reduced phosphorylation of mammalian target of rapamycin (mTOR). Addition of IL-15 at least partly restored mTOR phosphorylation and increased IL-12Rβ2 expression. The reduced mTOR phosphorylation after severe injury was cell-intrinsic as it was not induced by serum factors. Inhibition of mTOR in purified NK cells from healthy donors by rapamycin decreased the synthesis of IFN-γ. Thus, impaired mTOR phosphorylation in response to a microbial challenge contributes to the cell-intrinsic mechanisms that underlie NK cell dysregulation after trauma. Restoration of the mTOR phosphorylation capacity along with inhibition of the TGF-βRI signaling in NK cells after severe injury might improve the immune defense against opportunistic infections.