Cell-cell Contact-dependent Manner Research Articles

Tumor-Associated Monocytes Reprogram CD8+ T Cells into Central Memory-Like Cells with Potent Antitumor Effects.

CD8+ T cells are critical for host antitumor responses, whereas persistent antigenic stimulation and excessive inflammatory signals lead to T cell dysfunction or exhaustion. Increasing early memory T cells can improve T cell persistence and empower T cell-mediated tumor eradication, especially for adoptive cancer immunotherapy. Here, it is reported that tumor-associated monocytes (TAMos) are highly correlated with the accumulation of CD8+ memory T cells in human cancers. Further analysis identifies that TAMos selectively reprogram CD8+ T cells into T central memory-like (TCM-like) cells with enhanced recall responses. L-NMMA, a pan nitric oxide synthase inhibitor, can mitigate TAMo-mediated inhibition of T cell proliferation without affecting TCM-like cell generation. Moreover, the modified T cells by TAMo exposure and L-NMMA treatment exhibit long-term persistence and elicit superior antitumor effects in vivo. Mechanistically, the transmembrane protein CD300LG is involved in TAMo-mediated TCM-like cell polarization in a cell-cell contact-dependent manner. Thus, the terminally differentiated TAMo subset (CD300LGhighACElow) mainly contributes to TCM-like cell development. Taken together, these findings establish the significance of TAMos in boosting T-cell antitumor immunity.

Anti-Malignant Effect of Tensile Loading to Adherens Junctions in Cutaneous Squamous Cell Carcinoma Cells.

Actomyosin contractility regulates various cellular processes including proliferation and differentiation while dysregulation of actomyosin activity contributes to cancer development and progression. Previously, we have reported that actomyosin-generated tension at adherens junctions is required for cell density-dependent inhibition of proliferation of normal skin keratinocytes. However, it remains unclear how actomyosin contractility affects the hyperproliferation ability of cutaneous squamous cell carcinoma (cSCC) cells. In this study, we find that actomyosin activity is impaired in cSCC cells both in vitro and in vivo. External application of tensile loads to adherens junctions by sustained mechanical stretch attenuates the proliferation of cSCC cells, which depends on intact adherens junctions. Forced activation of actomyosin of cSCC cells also inhibits their proliferation in a cell-cell contact-dependent manner. Furthermore, the cell cycle arrest induced by tensile loading to adherens junctions is accompanied by epidermal differentiation in cSCC cells. Our results show that the degree of malignant properties of cSCC cells can be reduced by applying tensile loads to adherens junctions, which implies that the mechanical status of adherens junctions may serve as a novel therapeutic target for cSCC.

Shaping of T Cell Functions by Trogocytosis.

Trogocytosis is an active process whereby plasma membrane proteins are transferred from one cell to the other cell in a cell-cell contact-dependent manner. Since the discovery of the intercellular transfer of major histocompatibility complex (MHC) molecules in the 1970s, trogocytosis of MHC molecules between various immune cells has been frequently observed. For instance, antigen-presenting cells (APCs) acquire MHC class I (MHCI) from allografts, tumors, and virally infected cells, and these APCs are subsequently able to prime CD8+ T cells without antigen processing via the preformed antigen-MHCI complexes, in a process called cross-dressing. T cells also acquire MHC molecules from APCs or other target cells via the immunological synapse formed at the cell-cell contact area, and this phenomenon impacts T cell activation. Compared with naïve and effector T cells, T regulatory cells have increased trogocytosis activity in order to remove MHC class II and costimulatory molecules from APCs, resulting in the induction of tolerance. Accumulating evidence suggests that trogocytosis shapes T cell functions in cancer, transplantation, and during microbial infections. In this review, we focus on T cell trogocytosis and the related inflammatory diseases.

Bone Marrow Mesenchymal Stromal Cells Can Render Multiple Myeloma Cells Resistant to Cytotoxic Machinery of CAR T Cells through Inhibition of Apoptosis.

The microenvironment of multiple myeloma (MM) can critically impair therapy outcome, including immunotherapies. In this context, we have earlier demonstrated that bone marrow mesenchymal stromal cells (BMMSC) protect MM cells against the lytic machinery of MM-reactive cytotoxic T cells (CTL) and daratumumab-redirected natural killer (NK) cells through the upregulation of antiapoptotic proteins Survivin and Mcl-1 in MM cells. Here, we investigated the significance of this mode of immune escape on T cells engineered to express chimeric antigen receptors (CAR T cells). We tested the cytolytic ability of a panel of 10 BCMA-, CD38-, and CD138-specific CAR T cells with different affinities against a model MM cell line and against patient-derived MM cells in the presence versus absence of BMMSCs. Although BMMSCs hardly protected MM cells from lysis by high-affinity, strongly lytic BCMA- and CD38-CAR T cells, they significantly protected against lower affinity, moderately lytic BCMA-, CD38-, and CD138-specific CAR T cells in a cell-cell contact-dependent manner. Overall, there was a remarkable inverse correlation between the protective ability of BMMSCs and the lytic activity of all CAR T cells, which was dependent on CAR affinity and type of costimulation. Furthermore, BMMSC-mediated resistance against CAR T cells was effectively modulated by FL118, an inhibitor of antiapoptotic proteins Survivin, Mcl-1, and XIAP. These results extend our findings on the negative impact of the microenvironment against immunotherapies and suggest that outcome of CAR T cell or conventional CTL therapies could benefit from inhibition of antiapoptotic proteins upregulated in MM cells through BMMSC interactions.

Mesenchymal Stromal Cells Rapidly Suppress TCR Signaling-Mediated Cytokine Transcription in Activated T Cells Through the ICAM-1/CD43 Interaction.

Cell-cell contact participates in the process of mesenchymal stromal cell (MSC)-mediated T cell modulation and thus contributes to MSC-based therapies for various inflammatory diseases, especially T cell-mediated diseases. However, the mechanisms underlying the adhesion interactions between MSCs and T cells are still poorly understood. In this study, we explored the interaction between MSCs and T cells and found that activated T cells could rapidly adhere to MSCs, leading to significant reduction of TNF-α and IFN-γ mRNA expression. Furthermore, TCR-proximal signaling in activated T cells was also dramatically suppressed in the MSC co-culture, resulting in weakened Ca2+ signaling. MSCs rapidly suppressed TCR signaling and its downstream signaling in a cell-cell contact-dependent manner, partially through the ICAM-1/CD43 adhesion interaction. Blockade of either ICAM-1 on MSCs or CD43 on T cells significantly reversed this rapid suppression of proinflammatory cytokine expression in T cells. Mechanistically, MSC-derived ICAM-1 likely disrupts CD43-mediated TCR microcluster formation to limit T cell activation. Taken together, our results reveal a fast mechanism of activated T cell inhibition by MSCs, which provides new clues to unravel the MSC-mediated immunoregulatory mechanism for aGVHD and other severe acute T cell-related diseases.

Multiple Myeloma Bone Marrow Mesenchymal Stromal Cells Inhibit CD8+ T Cell Function in a Process that May Implicate Fibroblast Activation Protein α.

Multiple myeloma (MM) is a malignant plasma cell proliferative disorder with limited immunotherapy treatment because of T cell dysfunction. To investigate the immunomodulatory function of bone marrow mesenchymal stromal cells (MM-BMSCs) on CD8+ T cells. Proliferation and cytotoxicity were detected by cell counting kit-8 assay. Cell cycle was detected by flow cytometry, and p16 expression was detected by PCR. The expression of fibroblast activation protein α (FAPα) was evaluated by immunohistochemistry. Co-culture of CD8+ T cells with MM-BMSCs decreased the cell survival rate and increased the killing rate (p=0.03, p=0.001, respectively), the percentage of cells in G0/G1 phase and p16 expression (p<0.001). FAPα was mainly in the mesenchymal matrix of the MM microenvironment and elevated in MM derived bone marrow compared to healthy donors (p<0.001). The FAPα inhibitor PT-100, increased survival and the killing rate (p<0.001, p=0.043, respectively), and decreased the percentage of cells in G0/G1 phase and p16 expression (p=0.024, p=0.004, respectively). Therefore, MM-BMSCs inhibit the proliferation and cytotoxicity of CD8+ T cells, significantly block the cell cycle and increase p16 expression in co-cultured CD8+ T cells in a cell-cell contact-dependent manner.

Adoptive transfer of regulatory T cells stimulated by Allogeneic Hepatic Stellate Cells mitigates liver injury in mice with concanavalin A-induced autoimmune hepatitis

Regulatory T cell (Treg)-based therapy can effectively control autoimmune hepatitis (AIH). Hepatic stellate cells (HSC) can selectively stimulate allogeneic Treg proliferation following liver transplantation. This study tested the therapeutic effect and potential mechanisms underlying the action of HSC-stimulated Tregs on AIH in a mouse model of Concanavalin A (ConA)-induced AIH. HSC were isolated from BALB/c mice and characterized. Splenic CD4+CD25+ Tregs were isolated from C57BL/6 mice by immunomagnetic beads. The cells were co-cultured with primary (HSC-0), the second generation of HSC (HSC-2) for 72 h. The proliferation of Tregs was determined by flow cytometry. Similarly, the Tregs were co-cultured with HSC in transwell plates to determine the potential cell-cell contact dependent. The CD4+CD25− effector T cells (CFSE-Teffs) were co-cultured with Teff or Tregs in the presence or absence of HSC to determine the suppressive capacity of Tregs. The effects of Tregs or HSC-stimulated Tregs on AIH severity and the frequency of splenic Tregs and Th17 cells were examined in mice. Co-culture with HSC-2 significantly promoted Treg proliferation in a dose- and cell-cell contact-dependent manner, and allogeneic HSC enhanced the suppressive activity of Tregs to inhibit the proliferation of Teff in vitro. Adoptive transfer of Tregs, particularly of HSC-stimulated Tregs, significantly reduced liver injury, inflammation and Ishak modified histology activity index in AIH mice, which were associated with improving the balance of Treg and Th17 cell responses. Our data indicated that mature HSC stimulated allogeneic Treg proliferation in a dose and cell-cell contact-dependent manner, and HSC enhanced the suppressive activity of Tregs to inhibit the proliferation of Teff. Adoptive transfer of HSC-stimulated Tregs significantly reduced liver injury in AIH mice by modulating the balance of Treg and Th17 cell responses.

Expansion of Islet-Resident Macrophages Leads to Inflammation Affecting β Cell Proliferation and Function in Obesity

The nature of obesity-associated islet inflammation and its impact on β cell abnormalities remains poorly defined. Here, we explore immune cell components of islet inflammation and define their roles in regulating β cell function and proliferation. Islet inflammation in obese mice is dominated by macrophages. We identify two islet-resident macrophage populations, characterized by their anatomical distributions, distinct phenotypes, and functional properties. Obesity induces the local expansion of resident intra-islet macrophages, independent of recruitment from circulating monocytes. Functionally, intra-islet macrophages impair β cell function in a cell-cell contact-dependent manner. Increased engulfment of βcell insulin secretory granules by intra-islet macrophages in obese mice may contribute to restricting insulin secretion. In contrast, both intra- and peri-islet macrophage populations from obese mice promote β cell proliferation in a PDGFR signaling-dependent manner. Together, these data define distinct roles and mechanisms for islet macrophages in the regulation of islet β cells.

What Goes Around, Comes Around - HSV-1 Replication in Monocyte-Derived Dendritic Cells.

HSV-1 is a very successful human pathogen, known for its high sero-prevalence and the ability to infect a wide range of different cell types, including dendritic cells (DCs). As very potent antigen-presenting cells DCs play an important role in the induction of antiviral immune responses and therefore represent a strategic target for viral-mediated immune escape mechanisms. It is known that HSV-1 completes its gene expression profile in immature as well as in mature DCs, while lytic infection is only found in immature DCs (iDCs). Notably, HSV-1 infected mature DCs (mDCs) fail to release infectious progeny virions into the supernatant. Apart from HSV-1 dissemination via extracellular routes cell-to-cell spread counteracts a yet unknown mechanism by which the virus is trapped in mDCs and not released into the supernatant. The dissemination in a cell–cell contact-dependent manner enables HSV-1 to infect bystander cells without the exposure toward the extracellular environment. This supports the virus to successfully infect the host and establish latency. In this review the mechanism of HSV-1 replication in iDCs and mDCs and its immunological as well as virological implications, will be discussed.

Characterization of c-Maf+Foxp3\u2212 Regulatory T Cells Induced by Repeated Stimulation of Antigen-Presenting B Cells

The role of B cells in the development of CD4+ regulatory T cells has been emphasized recently. Our previous studies have demonstrated that the antigen-presenting splenic B cells converted naïve CD4+CD25− T cells into CD4+CD25+Foxp3− T cells without additional cytokines or chemicals with regulatory activity and that referred to as Treg-of-B cells. The present study further showed that Treg-of-B cells increased the IL-10-producing population, and the expression of c-Maf, inducible T-cell co-stimulator (ICOS) as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) after repeated stimulation of B cells in a cell-cell contact-dependent manner. Long-term cultured Treg-of-B cells exerted IL-10 and CTLA4-mediated antigen-specific suppressive activity; moreover, the single antigen-specific Treg-of-B cells inhibited in a non-antigen-specific fashion. In conclusion, these results suggest that repeated stimulation of B cells induced IL-10-producing CD4+Foxp3− regulatory T cells in a contact-dependent manner and these Treg-of-B cells possess IL-10 and CTLA4-dependent suppressive function.

Pathogenic conversion of regulatory B10 cells into osteoclast-priming cells in rheumatoid arthritis

Regulatory B10 cells were functionally impaired in rheumatoid arthritis (RA), yet the mechanisms were unclear. B cells are recently recognized as important participants in osteoclastogenesis by producing RANKL. In this study, we investigated whether regulatory B10 cells could convert into RANKL-producing cells, thus impairing their immunosuppressive functions in RA and exacerbating the disease progression. Our results showed that human regulatory B10 cells could ectopically express RANKL. Under RA circumstance, RANKL-producing B10 cells expanded dramatically, partially induced by TNF-α. The frequencies of these cells were positively correlated with RA patient disease activities and tender joint counts, but negatively correlated with the frequencies of regulatory B10 cells. Strikingly, RANKL-producing B10 cells from RA patients, but not healthy individuals significantly promoted osteoclast differentiation and bone erosion in a paracrine and cell-cell contact-dependent manner. Moreover, these pathogenic RANKL-producing B10 cells declined while regulatory IL-10-producing B10 cells increased in RA patients with disease remission after therapy. Collectively, these results showed that in RA, regulatory B10 cells demonstrated the potential of converting into RANKL-producing cells, thus exacerbating osteoclast formation, bone destruction and disease progression. Modulating the status of B10 cells might provide novel therapeutic strategies for RA.

Abstract 3739: An in vitro fluorescence-based co-culture model identifies tumour microenvironment-mediated chemoresistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with poor responses to cytotoxic or targeted agents. PDAC is characterised by abundant desmoplasia and this tumor microenvironment is a major determinant of intrinsic resistance to anticancer therapies. In particular, pancreatic stellate cells/cancer-associated fibroblasts (PSCs/CAFs) are periacinar stromal cells which promote epithelial-mesenchymal transition (EMT) and radioresistance of PDAC. However, this unique tumour microenvironment is not recapitulated in conventional cell monoculture or xenografts, which might attribute to the poor predictivity of these preclinical drug screening models. To address this, we have developed a PDA tumor cell-specific in vitro Fluorescence-Based Co-culture (PDA-FBC) platform to investigate potential mechanisms of stroma-mediated chemoresistance and to evaluate novel agents and combinations. Using K8484 (PDA cell line derived from the LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-Cre (KPC) mouse) stably expressing GFP, we selectively measured tumor cell viability in response to gemcitabine, with and without the presence of stromal cells. Monolayers of CAFs (primary fibroblast-like cells isolated from PDA tumour in KPC mice) or NFs (primary fibroblast-like cells from the normal pancreas of paired PC mice) were grown in 96 well plates and K8484 cells were seeded on top, prior to 72 hour drug exposure. When co-cultured with CAFs, K8484-GFP cells were significantly less sensitive to gemcitabine than cells grown in absence of CAFs: gemcitabine GI50 (50% growth inhibition concentration) is 19+/-3.2nM (n=4) in K8484 cell monolayer, 690+/-250nM (n=4) in the presence of CAFs (p = 0.03). Transwell assays revealed that this CAF-mediated chemoresistance was only induced in a cell-cell direct contact-dependent manner. Moreover, similar stroma-mediated gemcitabine resistance was not detected in co-culture of K8484 with NFs from normal pancreas (GI50=21+/-3.3nM). We are now investigating whether the chemoresistance phenotype extends to other cytotoxic agents. To evaluate potential mechanisms of resistance, RNA sequencing is being conducted to compare molecular profiles of K8484-GFP cells co-cultured with CAFs or NFs (with/without gemcitabine), to identify the genes modulated by the tumor-stroma interaction and potentially responsible for the decreased gemcitabine sensitivity in co-culture. In conclusion, we have identified that cancer-associated fibroblasts induce resistance to gemcitabine in KPC PDAC tumour cells, in contrast to fibroblasts from normal pancreas. This platform will have utility in the assessment of novel drug combinations and a mechanistic understanding of the tumor-stroma interactions may identify new candidate drug combinations. Citation Format: Ruiling Xu, Frances M. Richards, Yao Lin, Duncan I. Jodrell. An in vitro fluorescence-based co-culture model identifies tumour microenvironment-mediated chemoresistance in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3739. doi:10.1158/1538-7445.AM2014-3739

Ex vivo converted double negative T cells suppress activated B cells

Although the ability of endogenous CD4(-)CD8(-) double negative (DN) T cells to suppress B cells has been documented, the extent to which ex vivo converted DN T cells suppress B cells activity is still being explored. The aim of this study was to determine whether and what extent ex vivo converted CD4(-)CD8(-) DN T cells suppress B cell activation and antibody production. We found that ex vivo converted DN T cells suppressed proliferation of activated B cells in a perforin and cell-cell contact dependent manner. In addition, ex vivo converted DN T cells significantly inhibited the production of IgG by stimulated B cells. This study provides evidence that ex vivo converted CD4(-)CD8(-) double negative T cells can down-regulate immune responses by suppressing B cell proliferation and IgG production, and supports efforts to develop ex vivo DN T cell therapies.

Chemokine receptor CCR6-dependent accumulation of γδ T cells in injured liver restricts hepatic inflammation and fibrosis

Chronic liver injury promotes hepatic inflammation, representing a prerequisite for organ fibrosis. We hypothesized a contribution of chemokine receptor CCR6 and its ligand, CCL20, which may regulate migration of T-helper (Th)17, regulatory, and gamma-delta (γδ) T cells. CCR6 and CCL20 expression was intrahepatically up-regulated in patients with chronic liver diseases (n = 50), compared to control liver (n = 5). Immunohistochemistry revealed the periportal accumulation of CCR6(+) mononuclear cells and CCL20 induction by hepatic parenchymal cells in liver disease patients. Similarly, in murine livers, CCR6 was expressed by macrophages, CD4 and γδ T-cells, and up-regulated in fibrosis, whereas primary hepatocytes induced CCL20 upon experimental injury. In two murine models of chronic liver injury (CCl4 and methionine-choline-deficient diet), Ccr6(-/-) mice developed more severe fibrosis with strongly enhanced hepatic immune cell infiltration, compared to wild-type (WT) mice. Although CCR6 did not affect hepatic Th-cell subtype composition, CCR6 was explicitly required by the subset of interleukin (IL)-17- and IL-22-expressing γδ T cells for accumulation in injured liver. The adoptive transfer of WT γδ, but not CD4 T cells, into Ccr6(-/-) mice reduced hepatic inflammation and fibrosis in chronic injury to WT level. The anti-inflammatory function of hepatic γδ T cells was independent of IL-17, as evidenced by transfer of Il-17(-/-) cells. Instead, hepatic γδ T cells colocalized with hepatic stellate cells (HSCs) in vivo and promoted apoptosis of primary murine HSCs in a cell-cell contact-dependent manner, involving Fas-ligand (CD95L). Consistent with γδ T-cell-induced HSC apoptosis, activated myofibroblasts were more frequent in fibrotic livers of Ccr6(-/-) than in WT mice. γδ T cells are recruited to the liver by CCR6 upon chronic injury and protect the liver from excessive inflammation and fibrosis by inhibiting HSCs.

The Survivin Suppressant YM155 Overcomes Microenvironment-Mediated Immune Resistance In a Humanized Multiple Myeloma Mouse Model

BackgroundDurable tumor regression can be achieved in Multiple Myeloma (MM) patients by immunotherapeutic approaches, such as allogeneic stem cell transplantation and donor lymphocyte infusion. However, the low rate of patients, who reach a sustained remission, impedes the success of this therapeutic approach. Currently, much emphasis is placed on the role of the tumor microenvironment in tumor cell immune escape. We previously described that different accessory cells of the microenvironment significantly inhibited the lysis of MM cell lines by T cells in a cell-cell contact dependent manner. Further analysis revealed that this immune escape could be attributed to the induction of a cell adhesion-mediated immune resistance (CAM-IR) of MM against T cell lysis in vitro. Further in vitro studies identified up-regulation of survivin in the MM cells as one of the potential mechanisms of immune resistance. Moreover, co-culture with YM155, a small molecule survivin suppressant, could abrogate accessory cell induced resistance in vitro. Methods and ResultsWe investigated the influence of a human bone marrow microenvironment on the anti tumor activity of T cell therapy in a human-mouse hybrid model (Groen et al. Blood 2012). Immune-deficient RAG2-/- γc-/- -mice were implanted subcutaneously either with uncoated scaffolds or scaffolds coated with human mesenchymal stromal cells (MSC), which generates a human bone marrow environment. Luciferase transduced MM cell line UM9 injected directly in both types of scaffolds grew into MM tumors and were treated with Myeloma reactive minor Histocompatibility antigen specific T cell clones. The T cells induced effective anti-myeloma responses against tumors developed in the uncoated scaffolds. In contrast, and consistent with the in vitro results, no anti-tumor effect was observed in the MSC coated scaffolds. Thus confirming in vivo a microenvironment induced resistance of MM cells to T cell kill. We next investigated if the resistance could be abrogated by combination therapy with T cells and YM155.To this end, mice with tumors in human MSC coated scaffolds were treated with T cells alone, YM155 alone, or the combination of both. YM155- nor T cell-monotherapy induced tumor regression on evaluation of tumor growth based on bioluminescent signal. Moreover, the combination of YM155 with T cells demonstrated a substantial anti-tumor effect. ConclusionYM155 treatment can sensitize myeloma cells to T cell mediated anti-tumor effects and more importantly can overcome microenvironment-mediated resistance of MM cells to T cell treatment. These findings support further development of CAM-IR modulating agents in combination with immune therapy. Disclosures:Minnema:Janssen Cilag: Consultancy, Honoraria. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

Natural Killer Cell Death Mediated By NKG2D-Trogocytosis

IntroductionThe activating receptor, NKG2D, plays crucial role in natural killer (NK) cell-mediated effector function. NKG2D is involved not only in host defense against tumor and viral infection, but also in autoimmune diseases by recognizing stress-induced self-ligands (NKG2DLs). However, the negative feedback regulation of NKG2D has not been fully understood. It has been reported that NK cells undergo rapid apoptosis upon interaction with NK-sensitive tumor cells, suggesting that activation-induced NK cell death can be triggered in certain situations. In this study, we aimed to elucidate underlying mechanism of NK cell death, especially focused on NKG2D-NKG2DLs interaction. MethodsNK cells were purified from splenocytes of C57BL/6, perforin-/-, and DAP10-/-/12-/- mice, and cultured with rhIL-2 (1000 U/ml) for 5 days. We used these IL-2-activated NK cells as effector cells and three target cell lines: mouse T cell lymphoma RMA cells (RMA), RMA lacking MHCI expression (RMA-S), and RMA stably expressing an NKG2DL, Rae-1δ (RMA/Rae-1δ). CFSE-labeled NK cells were co-cultured with target cells for 2 hours, and stained with anti-NK1.1 mAb propidium iodide (PI). The percentage of PI-positive cells within CFSE+ NK1.1+ population was measured by flowcytometry. The cell surface expression of Rae-1 on NK cells after co-culture with target cells was evaluated by flowcytometry and confocal microscopy. ResultsNK cells from WT mice rapidly underwent cell death when co-cultured with Rae-1δ, but not with RMA or RMA-S, suggesting that NKG2D-Rae-1 interaction is involved in NK cell death. NK cells from perforin-/-, and DAP10-/-/12-/- mice did not undergo cell death, highlighting the importance of the NKG2D pathway for NK cell death. However, cross-linking of NKG2D receptor failed to induce NK cell death. To understand underlying the mechanism of NK cell death, we evaluated the cell surface expression of NKG2DLs on NK cells after co-culture with tumor cells. We found that cell surface expression of Rae-1 on NK cells was remarkably induced after co-culture with RMA/Rae-1δ, but no with RMA or RMA-S, implying that these Rae-1-positive NK cells may be lysed by NK cells through NKG2D-induced perforin pathway. The cell surface induction of Rae-1 on NK cells was very rapid (within 5min), and it occurred cell-cell contact dependent manner. Interestingly, NK cells from C57/BL6 mice rapidly became BALB/c allele Rae-1γ-positive after co-culture with RMA/Rae-1γ, indicating that NK cells acquire tumor-derived Rae-1. Consistently, acquisition of Rae-1 by NK cells was confirmed by confocal microscopy. Therefore, NK cells rapidly dress tumor-derived Rae-1 after interaction with tumor cells through intercellular membrane transfer, namely trogocytosis. Trogocytosis of Rae-1 was significantly inhibited in NK cells from DAP10-/-/12-/- mice and by chemical inhibitors of PI3K and Syk, indicating that it requires NKG2D-signaling. To confirm whether Rae-1-dressed NK cells can be recognized and lysed by other NK cells, we used sort-purified Rae-1-dressed NK cells as target cells. Rae-1-dressed NK cells were lysed by WT NK cell in an E/T-ratio dependent manner through NKG2D-induced perforin pathway. Furthermore, adoptively transferred Rae-1-dressed NK cells were significantly eliminated in Rag-1-deficient mice, indicating that Rae-1-dressed NK cells are also recognized and lysed in vivo. ConclusionUpon interaction with NKG2DLs-expressing tumor cells NK cells rapidly dress tumor-derived NKG2DLs, and subsequently undergo fratricide. Our results provide novel insights into activation-induced NK cell death via dynamic intercellular communications. Disclosures:No relevant conflicts of interest to declare.

Bifidobacterium longum Alleviates Dextran Sulfate Sodium-Induced Colitis by Suppressing IL-17A Response: Involvement of Intestinal Epithelial Costimulatory Molecules

Although some bacterial strains show potential to prevent colitis, their mechanisms are not fully understood. Here, we investigated the anti-colitic mechanisms of Bifidobacterium longum subsp. infantis JCM 1222T, focusing on the relationship between interleukin (IL)-17A secreting CD4+ T cells and intestinal epithelial costimulatory molecules in mice. Oral administration of JCM 1222T to mice alleviated dextran sulfate sodium (DSS)-induced acute colitis. The expression of type 1 helper T (Th1)- and IL-17 producing helper T (Th17)-specific cytokines and transcriptional factors was suppressed by JCM 1222T treatment. Intestinal epithelial cells (IECs) from colitic mice induced IL-17A production from CD4+ T cells in a cell-cell contact-dependent manner, and this was suppressed by oral treatment with JCM 1222T. Using blocking antibodies for costimulatory molecules, we revealed that epithelial costimulatory molecules including CD80 and CD40, which were highly expressed in IECs from colitic mice, were involved in IEC-induced IL-17A response. Treatment of mice and intestinal epithelial cell line Colon-26 cells with JCM 1222T decreased the expression of CD80 and CD40. Collectively, these data indicate that JCM 1222T negatively regulate epithelial costimulatory molecules, and this effect might be attributed, at least in part, to suppression of IL-17A in DSS-induced colitis.

Monocytes and the 38kDa-antigen of mycobacterium tuberculosis modulate natural killer cell activity and their cytolysis directed against ovarian cancer cell lines

BackgroundDespite strong efforts to improve clinical outcome of ovarian cancer patients by conventional and targeted immuno-based therapies, the prognosis of advanced ovarian cancer is still poor. Natural killer (NK) cells mediate antibody-dependent cellular cytotoxicity (ADCC), release immunostimulatory cytokines and thus function as potent anti-tumour effector cells. However, tumour cells developed mechanisms to escape from an effective immune response. So highly immunogenic substances, like the 38 kDa-preparation of M. tuberculosis, PstS-1, are explored for their potential to enhance cancer-targeted immune responses. In this study we examined the modulation of different NK cell functions by accessory monocytes and PstS-1. We focussed on NK cell activation as well as natural and antibody-dependent cellular cytotoxicity directed against epidermal-growth-factor-receptor (EGFR)-positive ovarian cancer cell lines.MethodsActivation, cytokine release and cytotoxicity of NK cells stimulated by monocytes and PstS-1 were determined by FACS-analysis, ELISA, Bioplex assay and quantitative polymerase-chain reaction (qPCR). Transwell assays were used to discriminate cell-cell contact-dependent from contact-independent mechanisms. Five ovarian cancer cell lines (A2780, IGROV-1, OVCAR-3, OVCAR-4 and SKOV-3) with different EGFR-expression were used as target cells for natural and antibody-dependent cellular cytotoxicity assays. Cetuximab (anti-EGFR-antibody) was used for ADCC studies.ResultsOur data show that monocytes effectively enhance activation as well natural and antibody-dependent cytolytic activity of NK cells. PstS-1 directly stimulated monocytes and further activated monocyte-NK-co-cultures. However, PstS-1 did not directly influence purified NK cells and did also not affect natural and antibody-dependent cellular cytotoxicity directed against EGFR-positive ovarian cancer cells, even in presence of monocytes. Direct cell-cell contact between NK cells and monocytes was required for NK activation, while released cytokines seemed to play a minor role.ConclusionsOur data suggest that monocytes enhance natural and antibody-dependent cytotoxic activity of NK cells in a cell-cell contact dependent manner. The TLR-agonist PstS-1 provides additional monocyte activation and induces NK activation markers, while NK cytotoxicity remains unaffected. We conclude that monocytes provide accessory function for ADCC exerted by NK during antibody-based cancer immunotherapy directed against EGFR-positive ovarian cancer cells.

Role of class IA PI3K in regulatory T cell function (179.5)

Abstract PI3K (phosphoinositide 3-kinase) family members control variety of cellular responses, such as cell growth, survival, cytoskeletal remodelling and the trafficking of intracellular organelles, in many cell types, including T lymphocytes. The PI3K pathway has emerged as a key regulator of regulatory T cell (Treg) development and homeostasis and may be required for Treg-mediated suppression. To identify the role of regulatory subunits of class IA PI3K in Treg suppressive function, we compared WT and p85α KO Tregs in their function. WT but not p85α KO Tregs showed suppressive capacity in co-cultures with CD4+CD25- T cells stimulated with anti-CD3-coated APCs in vitro. To determine whether p85α KO Tregs were functional in vivo, we conducted experiments in which Tregs offer protection from colitis caused by the transfer of CD4+CD62L high T cells into RAG2-/- recipients. WT Tregs suppressed the capacity of CD4+CD62L high T cells to cause colitis 2 weeks after transfer. In contrast, mice co-injected with p85α KO Tregs developed colitis 2 weeks after transfer that was indistinguishable from that in mice injected with CD4+CD62L high cells alone. Although the precise mechanism of Treg-mediated suppression is unclear, we suppose that p85α functions through cell-cell contact-dependent manner rather than cytokine-dependent manner. S.K. is a consultant for Medical and Biological Laboratories, Co. Ltd. The authors otherwise have no financial conflicts of interest.

EphrinA1-EphA2 Signal Induces Compaction and Polarization of Madin-Darby Canine Kidney Cells by Inactivating Ezrin through Negative Regulation of RhoA

The epithelial cells exhibit either a columnar or a flat shape dependent on extracellular stimuli or the cell-cell adhesion. Membrane-anchored ephrinA stimulates EphA receptor tyrosine kinases as a ligand in a cell-cell contact-dependent manner. The mechanism through which ephrinA1/EphA2 signal regulates the cell morphology remains elusive. We demonstrate here that ephrinA1/EphA2 signal induces compaction and enhanced polarization (columnar change) of Madin-Darby canine kidney epithelial cells by regulating Ezrin, a linker that connects plasma membrane and actin cytoskeleton. Activation of EphA2 resulted in RhoA inactivation through p190RhoGAP-A and subsequent dephosphorylation of Ezrin on Thr-567 phosphorylated by Rho kinase. Consistently, the cells expressing an active mutant of Ezrin in which Thr-567 was replaced with Asp did not change their shape in response to ephrinA1. Furthermore, depletion of Ezrin led to compaction and enhanced polarization without ephrinA1 stimulation, suggesting the role for active Ezrin in keeping the flat cell shape. Ezrin localized to apical domain irrespective of ephrinA1 stimulation, whereas phosphorylated Ezrin on the apical domain was reduced by ephrinA1 stimulation. Collectively, ephrinA1/EphA2 signal negatively regulates Ezrin and promotes the alteration of cell shape, from flat to columnar shape.