Immune Cell Crosstalk Research Articles

Identification of an activated neutrophil phenotype in polymyalgia rheumatica during steroid treatment: a potential involvement of immune cell cross-talk.

We have reported the existence of a distinct neutrophil phenotype in giant cell arteritis (GCA) patients arising at week 24 of steroid treatment. In the present study, we investigated whether longitudinal analysis of neutrophil phenotype in patients with polymyalgia rheumatica (PMR) could reveal a novel association with disease status and immune cell cross-talk. Thus, we monitored PMR patient neutrophil phenotype and plasma microvesicle (MV) profiles in blood aliquots collected pre-steroid, and then at weeks 1, 4, 12 and 24 post-steroid treatment.Using flow cytometric and flow chamber analyses, we identified 12-week post-steroid as a pivotal time-point for a marked degree of neutrophil activation, correlating with disease activity. Analyses of plasma MVs indicated elevated AnxA1+ neutrophil-derived vesicles which, in vitro, modulated T-cell reactivity, suggesting distinct neutrophil phenotypic and cross-talk changes at 24 weeks, but not at 12-week post-steroid.Together, these data indicate a clear distinction from GCA patient neutrophil and MV signatures, and provide an opportunity for further investigations on how to ‘stratify’ PMR patients and monitor their clinical responses through novel use of blood biomarkers.

Toll-like receptors and immune cell crosstalk in the intestinal epithelium

The intestinal epithelium consists of a barrier one cell thick found along the length of the gastrointestinal tract composed of many cell subtypes such as absorptive enterocytes and secretory Paneth cells, Goblet cells and enteroendocrine cells. Primarily known as a cell layer used to absorb nutrients from the products of digestion and as a protective barrier from infection, this has changed in recent years with numerous discoveries indicating its importance in priming and tolerising immune cells. Toll-like receptors are a family of pathogen recognition receptors that are widely expressed in human cells including the intestinal epithelium and are known primarily as initiators of inflammatory responses. However, recent evidence suggest that they may have a variety of roles and are involved in cross-talk with a variety of cell types. This review discusses TLR signalling pathways in the context of the intestinal epithelial microenvironment, namely innate and adaptive immune cells as well as microorganisms that resident in the lumen of the gut. TLR signalling is not only involved in defence against such microorganisms but also in communicating with the underlying immune cells. This review describes the many mechanisms by which such communication is executed. It also highlights potential sources of variation in such signalling in the general population in particular the effects of genetic variation, diversity of the microbiota, concomitant disease, diet and age.

First Insights in NK-DC Cross-Talk and the Importance of Soluble Factors During Infection With Aspergillus fumigatus.

Invasive aspergillosis (IA) is an infectious disease caused by the fungal pathogen Aspergillus fumigatus that mainly affects immunocompromised hosts. To investigate immune cell cross-talk during infection with A. fumigatus, we co-cultured natural killer (NK) cells and dendritic cells (DC) after stimulation with whole fungal structures, components of the fungal cell wall, fungal lysate or ligands for distinct fungal receptors. Both cell types showed activation after stimulation with fungal components and were able to transfer activation signals to the counterpart not stimulated cell type. Interestingly, DCs recognized a broader spectrum of fungal components and thereby initiated NK cell activation when those did not recognize fungal structures. These experiments highlighted the supportive function of DCs in NK cell activation. Furthermore, we focused on soluble DC mediated NK cell activation and showed that DCs stimulated with the TLR2/Dectin-1 ligand zymosan could maximally stimulate the expression of CD69 on NK cells. Thus, we investigated the influence of both receptors for zymosan, Dectin-1 and TLR2, which are highly expressed on DCs but show only minimal expression on NK cells. Specific focus was laid on the question whether Dectin-1 or TLR2 signaling in DCs is important for the secretion of soluble factors leading to NK cell activation. Our results show that Dectin-1 and TLR2 are negligible for NK cell activation. We conclude that besides Dectin-1 and TLR2 other receptors on DCs are able to compensate for the missing signal.

Abstract 703: The anti-cancer drug BPM 31510 elicits immune-modulatory effects through regulation of IL-10

Abstract Mitochondria have emerged as important immune-regulatory organelles, supporting biosynthetic and bioenergetic needs of immune cells and controlling cytokine production. BPM31510 is a clinical stage, nanodispersion of the mitochondrial electron transfer molecule ubidecarenone (CoQ10). Given that cytokine secretion orchestrates immune cell effector function in the tumor microenvironment and importance of mitochondria in this process, the ability of BPM31510 to modulate cytokine secretion was investigated. An ex vivo model using phytohemagglutinin-activated healthy donor peripheral blood mononuclear cells (PBMCs) treated with increasing concentrations of BPM31510 was used to assess cytokine secretion profiles. Analysis of supernatants by multi-analyte ELISA platform revealed effector cytokines IL-2 and IFN-γ to be dose-dependently increased with BPM31510 treatment while IL-10, a key immune-regulatory cytokine, was decreased. Analysis of monocyte depleted PBMCs compared to monocytes alone demonstrated that the observed changes in IL-2 and IFN-γ secretion occur only when both lymphocytes and monocytes are present; however, monocytes alone were identified as the significant source for IL-10. To define the mechanism of BPM31510-induced IL-10 attenuation, two monocytic lineage models were employed in vitro, LPS-stimulated THP-1 monocytes and PMA-differentiated U937 macrophages. Similar to monocytes isolated from PBMCs, treatment of LPS-stimulated THP-1 monocytes with increasing doses of BPM 31510, resulted in a decrease in IL-10 secretion as did treatment of U937 macrophages. The contribution of canonical IL-10 regulatory pathways, ERK, PI3K and STAT3, was assessed by co-treating U937 macrophages with BPM31510 and inhibitors of these pathways (U0126, Wortmannin, and Stattic, respectively). Co-treatment resulted in a further decrease in IL-10 secretion than any inhibitor treatment alone, suggesting that regulation of IL-10 secretion by BPM31510 is not exclusively through canonical signaling pathways. Nevertheless, further investigation into the activation status of these pathways revealed a decrease in STAT3 phosphorylation with BPM31510 treatment, but not of ERK or Akt. Consistent with decreased STAT3 activation, levels of IL-10 transcript, a STAT3 target gene, were also decreased in these samples, indicating that BPM31510 partially regulates IL-10 secretion via inhibition of the STAT3 signaling cascade. Taken together, these data demonstrate BPM31510 attenuates IL-10 secretion from the monocytic lineage through both canonical and non-canonical IL-10 regulatory pathways; this may have important implications for macrophage-mediated immunosuppression and immune cell crosstalk in the tumor microenvironment more broadly upon treatment with BPM31510. Citation Format: Louisa Dowal, Maria D. Nastke, Nidhi Gaur, Richa Singh, Samantha Fowler, Shyamali Jayashankar, Anne R. Diers, Stephane Gesta, Vivek Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan. The anti-cancer drug BPM 31510 elicits immune-modulatory effects through regulation of IL-10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 703.

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System.

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix.Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro, and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model.Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA.Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45+ leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures.Conclusion: Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth.

Immunomodulatory Therapy of Visceral Leishmaniasis in Human Immunodeficiency Virus-Coinfected Patients

Patients with visceral leishmaniasis (VL)–human immunodeficiency virus (HIV) coinfection experience increased drug toxicity and treatment failure rates compared to VL patients, with more frequent VL relapse and death. In the era of VL elimination strategies, HIV coinfection is progressively becoming a key challenge, because HIV-coinfected patients respond poorly to conventional VL treatment and play an important role in parasite transmission. With limited chemotherapeutic options and a paucity of novel anti-parasitic drugs, new interventions that target host immunity may offer an effective alternative. In this review, we first summarize current views on how VL immunopathology is significantly affected by HIV coinfection. We then review current clinical and promising preclinical immunomodulatory interventions in the field of VL and discuss how these may operate in the context of a concurrent HIV infection. Caveats are formulated as these interventions may unpredictably impact the delicate balance between boosting of beneficial VL-specific responses and deleterious immune activation/hyperinflammation, activation of latent provirus or increased HIV-susceptibility of target cells. Evidence is lacking to prioritize a target molecule and a more detailed account of the immunological status induced by the coinfection as well as surrogate markers of cure and protection are still required. We do, however, argue that virologically suppressed VL patients with a recovered immune system, in whom effective antiretroviral therapy alone is not able to restore protective immunity, can be considered a relevant target group for an immunomodulatory intervention. Finally, we provide perspectives on the translation of novel theories on synergistic immune cell cross-talk into an effective treatment strategy for VL–HIV-coinfected patients.

Dominant immunosuppression of dendritic cell function by prostate-cancer-derived exosomes

ABSTRACTExosomes are a distinct population of extracellular vesicles of endocytic origin with a protein repertoire similar to the parent cell. Although tumour-derived exosomes harbour immunosuppressive characteristics, they also carry tumour antigens and thus potentially contribute to immune activation. The aim of this study was to examine the impact of prostate cancer exosomes on tumour antigen cross-presentation. DU145 cells, transduced with shRNA to knockdown Rab27a (DU145KD) that inhibits exosome secretion, triggered significantly stronger tumour-antigen-specific T cell responses when loaded onto dendritic cells (DC) than control DU145 cells. Enhanced T cell response was prevented by adding purified exogenous DU145 exosomes to DU145KD cells, demonstrating that the dominant effect of tumour exosomes is immunosuppression and not antigen delivery. CD8+ T cell responses were impaired via exosomal regulation of DC function; exosomes triggered the expression of CD73, an ecto-5-nucleotidase responsible for AMP to adenosine hydrolysis, on DC. CD73 induction on DC that constitutively express CD39 resulted in an ATP-dependent inhibition of TNFα- and IL-12-production. We identified exosomal prostaglandin E2 (PGE2) as a potential driver of CD73 induction, as inhibition of PGE2 receptors significantly reduced exosome-dependent CD73 induction. The results reveal a hitherto unknown suppression of DC function via exosomal PGE2, adding a new element to tumour exosome–immune cell cross-talk.Abbreviations: AMP: adenosine monophosphate; ATP: adenosine triphosphate; BLCL: B lymphoblastoid cell line; CME: exosomes enriched from cell line conditioned media; DC: dendritic cell; DMSO: dimethyl-sulfoxide; DU145C: DU145 cells with irrelevant knockdown control; DU145KD: DU145 cells with Rab27a knockdown; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; GM-CSF: granulocyte-monocyte colony stimulating factor; HLA: human lymphocyte antigen; IL: interleukin; LPS: lipopolysaccharide; mfi: mean fluorescence intensity; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffer solution; PGE2: prostaglandin E2; TRF: time-resolved fluorescence.

Lung cancer-associated pulmonary hypertension: Role of microenvironmental inflammation based on tumor cell-immune cell cross-talk.

Dyspnea is a frequent, devastating, and poorly understood symptom of advanced lung cancer. In our cohort, among 519 patients who underwent a computed tomography scan for the diagnosis of lung cancer, 250 had a mean pulmonary artery diameter of >28 mm, indicating pulmonary hypertension (PH). In human lung cancer tissue, we consistently observed increased vascular remodeling and perivascular inflammatory cell accumulation (macrophages/lymphocytes). Vascular remodeling, PH, and perivascular inflammatory cell accumulation were mimicked in three mouse models of lung cancer (LLC1, KRasLA2 , and cRaf-BxB). In contrast, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ immunodeficient xenograft and dominant-negative IKK2 mutant triple transgenic (Sftpc-rtTA/Tet-O-Ikk2DN) mice did not develop PH. Coculturing human lung cancer cells with macrophages and lymphocytes strongly up-regulated cytokine release, provoking enhanced migration, apoptosis resistance, and phosphodiesterase 5 (PDE5)-mediated up-regulation of human lung vascular cells, which are typical features of PH. The PDE5 inhibitor sildenafil largely suppressed PH in the LLC1 model. We conclude that lung cancer-associated PH represents a distinct PH category; targeting inflammation in the microenvironment and PDE5 offers a potential therapeutic option.

The Role of Mast Cells in Tuberculosis: Orchestrating Innate Immune Crosstalk?

Tuberculosis causes more annual deaths globally than any other infectious disease. However, progress in developing novel vaccines, diagnostics, and therapies has been hampered by an incomplete understanding of the immune response to Mycobacterium tuberculosis (Mtb). While the role of many immune cells has been extensively explored, mast cells (MCs) have been relatively ignored. MCs are tissue resident cells involved in defense against bacterial infections playing an important role mediating immune cell crosstalk. This review discusses specific interactions between MCs and Mtb, their contribution to both immunity and disease pathogenesis, and explores their role in orchestrating other immune cells against infections.

Parenteral Nutrition-Associated Liver Disease: The Role of the Gut Microbiota.

Parenteral nutrition (PN) provides life-saving nutritional support in situations where caloric supply via the enteral route cannot cover the necessary needs of the organism. However, it does have serious adverse effects, including parenteral nutrition-associated liver disease (PNALD). The development of liver injury associated with PN is multifactorial, including non-specific intestine inflammation, compromised intestinal permeability, and barrier function associated with increased bacterial translocation, primary and secondary cholangitis, cholelithiasis, short bowel syndrome, disturbance of hepatobiliary circulation, lack of enteral nutrition, shortage of some nutrients (proteins, essential fatty acids, choline, glycine, taurine, carnitine, etc.), and toxicity of components within the nutrition mixture itself (glucose, phytosterols, manganese, aluminium, etc.). Recently, an increasing number of studies have provided evidence that some of these factors are directly or indirectly associated with microbial dysbiosis in the intestine. In this review, we focus on PN-induced changes in the taxonomic and functional composition of the microbiome. We also discuss immune cell and microbial crosstalk during parenteral nutrition, and the implications for the onset and progression of PNALD. Finally, we provide an overview of recent advances in the therapeutic utilisation of pro- and prebiotics for the mitigation of PN-associated liver complications.

Hierarchical signaling transduction of the immune and muscle cell crosstalk in muscle regeneration

The muscle regeneration is a complicated bioprocess that involved in many cell types, including necrotic muscle cells, satellite cells, mesenchymal cells, pericytes, immune cells, and other cell types present at the injury site. Immune cells involved in both innate and adaptive immune responses regulate the progress of muscle regeneration. In this review, we discussed the roles of different immune cells in muscle regeneration. The immune cells regulate muscle regeneration through cytokine production, cell-cell contacts, and general immune environment regulation. We also describe the current known mechanism of how immune cells regulating muscle regeneration.

Mycobacterium bovis Bacillus Calmette-Guérin Alters Melanoma Microenvironment Favoring Antitumor T Cell Responses and Improving M2 Macrophage Function.

Intralesional Mycobacterium bovis bacillus Calmette–Guérin (BCG) has long been a relatively inexpensive therapy for inoperable cutaneous metastatic melanoma (CMM), although intralesional BCG skin mechanisms remain understudied. We analyzed intralesional BCG-treated CMM lesions combined with in vitro studies to further investigate BCG-altered pathways. Since macrophages play a pivotal role against both cancer and mycobacterial infections, we hypothesized BCG regulates macrophages to promote antitumor immunity. Tumor-associated macrophages (M2) infiltrate melanomas and impair antitumor immunity. BCG-treated, in vitro-polarized M2 (M2-BCG) showed transcriptional changes involving inflammation, immune cell recruitment, cross talk, and activation pathways. Mechanistic network analysis indicated M2-BCG potential to improve interferon gamma (IFN-γ) responses. Accordingly, frequency of IFN-γ-producing CD4+ T cells responding to M2-BCG vs. mock-treated M2 increased (p < 0.05). Moreover, conditioned media from M2-BCG vs. M2 elevated the frequency of granzyme B-producing CD8+ tumor-infiltrating lymphocytes (TILs) facing autologous melanoma cell lines (p < 0.01). Furthermore, transcriptome analysis of intralesional BCG-injected CMM relative to uninjected lesions showed immune function prevalence, with the most enriched pathways representing T cell activation mechanisms. In vitro-infected MM-derived cell lines stimulated higher frequency of IFN-γ-producing TIL from the same melanoma (p < 0.05). Our data suggest BCG favors antitumor responses in CMM through direct/indirect effects on tumor microenvironment cell types including macrophages, T cells, and tumor itself.

Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells.

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-β and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations.

IL6 Mediates Immune and Colorectal Cancer Cell Cross-talk via miR-21 and miR-29b.

Tumors are surrounded and infiltrated by a variety of stromal cell types, including fibroblasts, immune cells, and vascular endothelial cells, which interact with malignant cells to generate the tumor microenvironment (TME). This complex environment is thought to be regulated by the tumor in order to promote its survival and progression and thus constitutes a potential target for cancer therapy. However, intercellular communication within the microenvironment is not yet well understood. The current study investigates the mechanism by which cancer and immune cells communicate using an in vitro coculture model. It is demonstrated that IL6, a proinflammatory cytokine, secreted by immune cells promotes colorectal cancer cell invasiveness. In addition, in the presence of IL6, the cancer cells were able to secrete circulating miRNAs miR-21 and miR-29b to further induce immune cell IL6 production. Activated immune cells were also found to release miR-21 into the TME. Taken together, these mechanistic findings provide a better understanding of intercellular communication between immune and cancer cells in the TME and offer insight into some of the key players that mediate this cross-talk. This study demonstrates that cocultured cancer and immune cells communicate via IL6 and circulating miRNAs to sustain chronic inflammation and promote prometastatic cancer cell behavior. In addition, critical players are identified that mediate intercellular communication in the TME and suggest possible therapeutic approaches that target the microenvironment.

Innate immune cells in the pathogenesis of primary systemic vasculitis.

Innate immune system forms the first line of defense against foreign substances. Neutrophils, eosinophils, erythrocytes, platelets, monocytes, macrophages, dendritic cells, γδ T cells, natural killer and natural killer T cells comprise the innate immune system. Genetic polymorphisms influencing the activation of innate immune cells predispose to development of vasculitis and influence its severity. Abnormally activated innate immune cells cross-talk with other cells of the innate immune system, present antigens more efficiently and activate T and B lymphocytes and cause tissue destruction via cell-mediated cytotoxicity and release of pro-inflammatory cytokines. These secreted cytokines further recruit other cells to the sites of vascular injury. They are involved in both the initiation as well as the perpetuation of vasculitis. Evidences suggest reversal of aberrant activation of immune cells in response to therapy. Understanding the role of innate immune cells in vasculitis helps understand the potential of therapeutic modulation of their activation to treat vasculitis.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) reprograms M2 macrophages to promote antitumor T cell responses (TUM6P.1007)

Abstract M2 Macrophages (MΦs), also known as tumor-associated MΦs, infiltrate solid tumors and impair antitumor immunity. M. bovis Bacille Calmette-Guérin (BCG) is an intralesional (IL) therapy for cutaneous metastatic melanoma (NCCN Guidelines), and induces regression of injected, but also non-injected lesions. Since MΦs play a pivotal role in tumors and mycobacterial infections, we hypothesized that IL-BCG affects tumor regression by altering M2 MΦs. Thus, we investigated the effects of BCG on in vitro polarized M2 MΦs. BCG induced dramatic transcriptional changes in M2 MΦs on genes involving inflammation, immune cell recruitment, cross-talk and activation. To determine whether BCG could alter the classical M2 phenotype, we compared BCG vs mock-treated M2-MΦ cell surface phenotype and response to LPS. BCG-treated M2-MΦ expressed lower CD163 (p&amp;lt;0.01) and secreted less IL-10 (p&amp;lt;0.05), with no effect on IL-12p40, vs mock-treated M2-MΦ. We also investigated the ability of BCG-treated M2-MΦ to activate T cells, and found an increased frequency of IFN-γ producing CD4+ T cells responding to BCG vs mock-treated M2-MΦ (p&amp;lt;0.05), suggesting an adjuvant effect of BCG. Moreover, supernatant from BCG-treated M2-MΦ increased the frequency of granzyme B-producing CD8+ T cells responding to autologous melanoma cell lines (p&amp;lt;0.01). Together, our data indicates IL-BCG therapy could contribute to a tumor-rejecting environment by inducing a reprogramming of M2 MΦs towards a proinflammatory phenotype.

Abstract P1-07-05: Triple negative breast cancer metastases: Protein signal transduction networks within the tumor-stromal microenvironment complement genomic analysis and stratify local versus distant metastasis

Abstract Background: Genomic profiling of primary TNBC may not reflect the lethal metastatic (mTNBC) lesions that constitute a selected subclone of the primary tumor. Moreover, while the stroma microenvironment of primary tumors has been the subject of study, almost nothing is known about the stromal microenvironment of metastasis. We conducted proteomic signal pathway network analysis of microdissected tumor cells and stroma from mTNBC samples for which whole genome sequencing was known. We addressed the following questions: 1) Can potentially actionable driver mutations in the metastasis be inferred from the activated/ suppressed protein signaling pathways in the tumor cells? 2) Are the tumor cell signaling pathways different between local and distant metastasis? 3) Does the proteomic signaling profile of the stroma provide strategies for stromal therapy of mTNBC? Methods: Enriched populations of mTNBC cells and adjacent stromal cells were collected by Laser Capture Microdissection from 13 fresh frozen mTNBC samples. Signal pathway profiling of 130 protein and/or phosphoprotein endpoints were quantified by reverse phase protein arrays (RPPA). The samples represented local (regional lymph node and chest wall) (n=7) and distant (n=6) metastasis. Spearman rho correlation analysis identified pair-wise protein linakges (rho=0.8, p&amp;lt;0.01). Whole genome sequencing was also performed on the mTNBC samples (Craig DW. Mol Cancer Ther, 2012). Results: We compared the protein signaling network of the tumor and matched stroma to investigate tumor-stroma crosstalk, and also to the genomic alterations (GAs) in the same tumor. GAs in mTNBC fell into two major categories: DNA repair or cell cycle/growth factor signaling. Proteomic analysis confirmed the down-regulation of endocrine (ER, AR, PR) and HER2 pathways. PhosphoJAK1/2 and phosphoHer3 were markedly down-regulated in mTNBCs, while EGFR and Her4 signaling were not. Spearman rho pairwise correlations revealed differences between local and distant metastases as well as between local and distant stroma. Cyclin D1 in locally metastatic tumor cells was linked with Autotaxin, PLCgamma and RUNX1, whereas in distant metastatic tumor cells, Cyclin D1 was linked with HIF-1alpha, phosphoJAK1, and HSP70. Growth factor signaling via phosphoEFGR was more prevalent in distant mTNBC tumor samples (322 linkages) compared to local mTNBC tumor samples (146 linkages). Distant stromal cell linkages were dominated by growth factor (phospho mTOR) and immune cell crosstalk, e.g. 21 linkages between CD63 in distant stromal samples compared to 1 CD63 linkage in local stromal cells. Discussion: Proteomic signal pathway data can stratify metastatic lesions into functionally important groups based on a) clinical phenotype (distant versus local metastasis) and b) GA subtype (DNA repair vs growth factor signaling), thereby providing new actionable strategies for novel therapies, e.g. anti-JAK1 and anti-HSP70, and providing a means to prioritize potential driver mutations. Profiling the signal pathway network of mTNBC stroma, for the first time, provides novel strategies for targeting immune cell and mTOR pathways, for new classes of stromal therapy. Citation Format: Virginia Espina, Maren K Levin, John D Carpten, David W Craig, Daniel Von Hoff, Lance A Liotta, Joyce O'Shaughnessy. Triple negative breast cancer metastases: Protein signal transduction networks within the tumor-stromal microenvironment complement genomic analysis and stratify local versus distant metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-05.

Immune cell crosstalk in obesity: a key role for costimulation?

In the past two decades, numerous experimental and clinical studies have established the importance of inflammation and immunity in the development of obesity and its metabolic complications, including insulin resistance and type 2 diabetes mellitus. In this context, T cells orchestrate inflammatory processes in metabolic organs, such as the adipose tissue (AT) and liver, thereby mediating obesity-related metabolic deterioration. Costimulatory molecules, which are present on antigen-presenting cells and naïve T cells in the AT, are known to mediate the crosstalk between the adaptive and innate immune system and to direct T-cell responses in inflammation. In this Perspectives in Diabetes article, we highlight the newest insights in immune cell interactions in obesity and discuss the role of costimulatory dyads in its pathogenesis. Moreover, the potential of therapeutic strategies that target costimulatory molecules in the metabolic syndrome is explored.

Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs) and NK cells (dNKs), the major decidual immune cell populations. We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3, and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8, and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10, and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.

Genetic Alterations In Immune Cell Crosstalk Genes In Diffuse Large B-Cell Lymphoma Predict Survival

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype accounting for ∼40% of all non-Hodgkin lymphomas. Although R-CHOP has significantly improved outcome in DLBCL, 35% of patients still experience relapsed/refractory disease. Further investigation into the genomic architecture of DLBCL is needed to determine the biological correlates that underlie treatment failure. Immune cell signatures have been shown to impact survival in DLBCL (Lenz et al, NEJM 2008). Recurrent mutations and/or copy number alterations (CNAs) in genes that impact the tumor's immune-cell crosstalk have recently been described, but their clinical impact is unknown. Moreover, mutations and copy-number loss involving genes that foster immune escape has been implicated as a dominant oncogenic mechanism in DLBCL (Challa-Malladi et al, Can Cell 2011). We sought to determine the relationship between genetic alterations involving immune-cell crosstalk pathways and survival in a cohort of DLBCL patients treated with R-CHOP. MethodsFollowing our initial analysis of 91 whole transcriptome sequencing (RNA-seq) including 40 with whole genome sequencing, we validated SNVs in the genes of interest by Sanger sequencing in the tumor and matched germline DNAs if available. RNA-seq-derived gene expression data were also used to determine the cell of origin (COO) distinction. Affymetrix SNP 6.0 microarrays were used to ascertain the CNAs in all 91 DLBCL samples. Clinical outcome correlations were analyzed by Cox regression and the log-rank test in the 82 patients treated with R-CHOP (median follow-up; 82 months). ResultsUsing next-generation sequencing, we identified 97 SNVs in 16 genes (B2M, CD274, CD276, CD58, CD83, CIITA, NLRC4, NLRC5, RFXAP, RFXANK, TNFRSF14, TNFRSF25, TNFSF9, TAP1, TAP2 and TAPBP) that were selected based on an extensive literature review implicating these genes in immune-cell crosstalk pathways in DLBCL. Among them, eight genes were mutated in more than one case including B2M (7%), CD58 (7%), TNFRSF14 (7%), CIITA (4%), TNFSF9 (2%), NLRC5 (3%), CD83 (11%), and RFXAP (2%). These eight loci were further analyzed for CNAs, gene expression and outcome correlations. SNP 6.0 microarrays revealed either focal heterozygous or homozygous deletions in B2M (22%), CD58 (14%), TNFSF9 (10%), and heterozygous deletions in TNFRSF14 (12%), CIITA (3%), NLRC5 (7%), CD83 (2%), and RFXAP (7%). Amongst the 91 cases, 35 cases (38%) had no genetic aberration. 56 cases (62%) had at least one aberration, while 29 (32%) had multiple genetic aberrations indicating that genetic aberrations associated with immune-cell crosstalk are not mutually exclusive in DLBCL. Mutations and CNAs were not COO specific, with the exception of TNFRSF14, which was significantly restricted to the GCB-subtype (p=0.001).We evaluated outcome correlations for each genetic aberrations in the 82 cases with uniform therapy, including two recognized major histocompatibility class (MHC) groups; MHC class I (B2M and NLRC5) and MHC class II (CIITA, RFXAP and CD83). In univariate analysis, the presence of genetic aberrations in the MHCI group and NLRC5 individually were prognostic (5yr PFS, 59% vs 78%, p=0.025, 44% vs 77%, p=0.005, respectively), but not the MHCII group or other individual loci. In subgroup analyses according to COO, both MHCI and MHCII genetic aberrations were associated with inferior 5yr PFS in the ABC subtype (MHCI, 27% vs 79%, p<0.0001) (MHCII, 40% vs 68%, p=0.018), but not in GCB. We next assessed the prognostic impact of the total number of genetic hits and revealed that patients with at least one genetic hit had a 5yr OS and PFS of 65% and 57%, respectively, while those with no genetic hit had a 5yr OS and PFS of 100% and 97%, respectively (both comparisons, p<0.0001). In a Cox model of PFS including IPI and COO, any genetic hit in immune-cell crosstalk pathways remained strongly predictive (HR=15.1 [2.0-113.2], p= 0.008) and was independent of both IPI (HR= 2.1 [1.1-4.0], p= 0.030) and COO (HR= 0.6 [0.3-1.4], p=0.26). ConclusionsRecurrent genetic alterations involving genes related to immune-cell crosstalk in DLBCL are frequent (56%), co-occur and independently contribute to outcome in patients treated with R-CHOP. MHCI and MHCII perturbations are only prognostic in the ABC subtype. [Display omitted] Disclosures:No relevant conflicts of interest to declare.