Circulating tumour cell enumeration, biomarker analyses, and kinetics in patients with colorectal cancer and other GI malignancies.

Objective: Most of the work in terms of liquid biopsies in patients with solid tumors is focused on circulating tumor DNA (ctDNA). Our aim was to evaluate the feasibility of using circulating tumor cells (CTCs) in peripheral blood samples from patients with advanced or metastatic gastrointestinal (GI) cancers. Methods: In this prospective study, blood samples were collected from each patient in 2 AccuCyte® blood collection tubes and each tube underwent CTC analysis performed utilizing the RareCyte® platform. The results from both tubes were averaged and a total of 150 draws were done, with 281 unique reported results. The cadence of sampling was based on convenience sampling and piggybacked onto days of actual clinical follow-ups and treatment visits. The CTC results were correlated with patient- and tumor-related variables. Results: Data from a total of 59 unique patients were included in this study. Patients had a median age of 58 years, with males representing 69% of the study population. More than 57% had received treatment prior to taking blood samples. The type of GI malignancy varied, with more than half the patients having colorectal cancer (CRC, 54%) followed by esophageal/gastric cancer (17%). The least common cancer was cholangiocarcinoma (9%). The greatest number of CTCs were found in patients with colorectal cancer (Mean: 15.8 per 7.5 ml; Median: 7.5 per 7.5 ml). In comparison, patients with pancreatic cancer (PC) had considerably fewer CTCs (Mean: 4.2 per 7.5 ml; Median: 3 per 7.5 ml). Additionally, we found that patients receiving treatment had significantly fewer CTCs than patients who were not receiving treatment (Median 2.7 versus 0.7). CTC numbers showed noteworthy disparities between patients with responding/stable disease in comparison to those with untreated/progressive disease (Median of 2.7 versus 0). When CTCs were present, biomarker analyses of the four markers human epidermal growth factor receptor 2 (HER2)/programmed death-ligand 1 (PD-L1)/Kiel 67 (Ki-67)/epidermal growth factor receptor (EGFR) was feasible. Single cell sequencing confirmed the tumor of origin. Conclusion: Our study is one of the first prospective real-time studies evaluating CTCs in patients with GI malignancies. While ctDNA-based analyses are more common in clinical trials and practice, CTC analysis provides complementary information from a liquid biopsy perspective that is of value and worthy of continued research.

Beyond Circulating Tumor Cell Enumeration: Cell-Based Liquid Biopsy to Assess Protein Biomarkers and Cancer Genomics Using the RareCyte® Platform.

The practice of medicine has steadily employed less invasive methods to obtain information derived from the tumor to guide clinical management of patients. Liquid biopsy-the sampling of blood-is a non-invasive method for generating information previously only available from tissue biopsies of the tumor mass. Analysis of fragmented circulating tumor DNA in the plasma is clinically used to identify actionable mutations and detect residual or recurrent disease. Plasma analysis cannot, however, assess cancer phenotypes, including the expression of drug targets and protein biomarkers. Circulating tumor cells (CTCs) are intact cancer cells that have entered the blood that have the potential for distant metastasis. While enumeration of CTCs is prognostic of outcome, recently developed technology allows for the interrogation of protein biomarkers on CTCs that could be predictive of response. Furthermore, since CTCs contain intact whole cancer genomes, isolating viable CTCs detected during therapy could provide a rational approach to assessing mutational profiles of resistance. Identification, characterization and molecular analysis of CTCs together will advance the capacity of liquid biopsy to meet the requirements of twenty-first century medicine.

Human ovarian tumor ascites fluids rapidly and reversibly inhibit T cell receptor-induced NF-κB and NFAT signaling in tumor-associated T cells.

Human memory T cells present in ovarian tumor ascites fluids fail to respond normally to stimulation via the T cell receptor (TCR). This immunosuppression is manifested by decreases in NF-κB and NFAT activation, IFN-γ production, and cell proliferation in response to TCR stimulation with immobilized antibodies to CD3 and CD28. The anergy of the tumor-associated T cells (TATs) is mediated by soluble factors present in ovarian tumor ascites fluids. The non-responsiveness of the T cells is quickly reversed when the cells are assayed in the absence of the ascites fluid, and is rapidly reestablished when a cell-free ascites fluid is added back to the T cells. Based upon the observed normal phosphorylation patterns of the TCR proximal signaling molecules, the inhibition of NF-κB, and NFAT activation in response to TCR stimulation, as well as the ability of the diacylglycerol analog PMA and the ionophore ionomycin to bypass the ascites fluid-induced TCR signaling arrest, the site of the arrest in the activation cascade appears to be at or just upstream of PLC-γ. An identical TCR signaling arrest pattern was observed when T cells derived from normal donor peripheral blood were incubated with either malignant or nonmalignant (cirrhotic) ascites fluids. The immunosuppressive activity of ascites fluids reported here suggests that soluble factors acting directly or indirectly upon T cells present within tumors contribute to the anergy that has previously been observed in T cells derived from malignant and nonmalignant inflammatory microenvironments. The soluble immunosuppressive factors represent potential therapeutic targets for ovarian cancer.

An introduction to mass cytometry: fundamentals and applications.

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

Langerhans cells favor skin flora tolerance through limited presentation of bacterial antigens and induction of regulatory T cells.

The mechanisms preventing detrimental T-cell responses against commensal skin bacteria remain elusive. Using monocyte-derived and skin-derived dendritic cells (DCs), we demonstrate that epidermal Langerhans cells (LCs), the DCs in the most superficial layer of the skin, have a poor capacity to internalize bacteria because of low expression of FcγRIIa. Furthermore, LCs show deficiency in processing and major histocompatibility complex II (MHC-II)-restricted presentation of bacterial antigens, as a result of a decreased expression of molecules involved in these functionalities. The reduced capacity to take up, process, and present bacterial antigens cannot be restored by LC activation by ectopically expressed Toll-like receptors or by cytokines. Consequently, bacteria-primed LCs poorly restimulate antibacterial memory CD4(+) T cells and inefficiently induce bacteria-specific effector CD4(+) T cells from naive T cells; however, they initiate the development of regulatory Foxp3(+)CD4(+) T cells, which are able to suppress the proliferation of autologous bystander T cells specific for the same bacteria. In contrast, dermal DCs that reside in the deeper dermal layer of the skin efficiently present bacterial antigens and provoke robust antibacterial naive and memory CD4(+) T-cell responses. In conclusion, LCs form a unique DC subset that is adapted at multiple levels for the maintenance of tolerance to bacterial skin flora.

Image cytometry-based detection of aneuploidy by fluorescence in situ hybridization in suspension.

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects.

ImageStream promyelocytic leukemia protein immunolocalization: in search of promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is a hematological emergency in which a rapid diagnosis is essential for early administration of appropriate therapy, including all-trans retinoic acid before the onset of fatal coagulopathy. Currently, the following methodologies are widely used for rapid initial diagnosis of APL: 1) identification of hypergranular leukemic promyelocytes by using classical morphology; 2) identification of cells with diffuse promyelocytic leukemia (PML) protein distribution by immunofluorescence microscopy; 3) evidence of aberrant promyelocyte surface immunophenotype by conventional flow cytometry (FCM). Here, we show a method for immunofluorescent detection of PML localization using ImageStream FCM. This technique provides objective per-cell quantitative image analysis for statistically large sample sizes, enabling precise and operator-independent PML pattern recognition even in electronic and real dilution experiments up to 10% of APL cellular presence. Therefore, we evidence that this method could be helpful for rapid and objective initial diagnosis and the prompt initiation of APL treatment.

CD81 is essential for the re-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the Akt pathway.

The regulatory mechanisms governing the cell cycle progression of hematopoietic stem cells (HSCs) are well characterized, but those responsible for the return of proliferating HSCs to a quiescent state remain largely unknown. Here, we present evidence that CD81, a tetraspanin molecule acutely responsive to proliferative stress, is essential for the maintenance of long-term repopulating HSCs. Cd81(-/-) HSCs showed a marked engraftment defect when transplanted into secondary recipient mice and a significantly delayed return to quiescence when stimulated to proliferate with 5-fluorouracil (5FU). In addition, we found that CD81 proteins form a polarized patch when HSCs are returning to quiescence. Thus, we propose that the spatial distribution of CD81 during the HSC recovery phase drives proliferative HSC to quiescence, and is important to preserve the self-renewal properties. Here, we show that lack of CD81 leads to loss of HSC self-renewal, and the clustering of CD81 on HSC membrane results in deactivation of Akt, which subsequently leads to nuclear translocation of FoxO1a. Thus, CD81 functions as part of a previously undefined mechanism that prohibits excessive proliferation of HSCs exposed to environmental stress.

ImageStream cytometry extends the analysis of phagocytosis and oxidative burst.

AIM: Phagocytosis is often measured using conventional microscopy and flow cytometry. ImageStream cytometry is a new technology that combines the advantages of both methods, enabling statistically robust microscopic applications. We compared ImageStream cytometry to flow cytometry in a whole blood model of phagocytosis with viable, fluorescence-marked Staphylococcus aureus. We furthermore measured the co-localization of intracellular bacteria to sites of oxidative burst, as well as changes in cell size and actin levels as a result of phagocytosis. EXPERIMENTAL DESIGN: Fluorescence-labeled S. aureus in a ratio of 5:1 bacteria per leukocyte were added to whole blood. Phagocytosis was stopped at different time points. After staining of neutrophils and lysis of erythrocytes, samples were analysed by ImageStream cytometry and flow cytometry. RESULTS: Phagocytosis and oxidative burst determined by flow cytometry and ImageStream cytometry showed strong correlation. In contrast to flow cytometry, ImageStream cytometry easily detected and excluded extracellular adherent bacteria from the measurement of phagocytosis, and enumerated the bacteria within each neutrophil. Using the Bright Detail Similarity score, we identified a subset of neutrophils with intracellular bacteria co-localized to sites of oxidative burst activity. Phagocytosis resulted in an increase in cell size and actin polymerization as determined by an increase in phalloidin fluorescence intensity. CONCLUSIONS: We describe a simple whole blood image-based method for measuring bacterial phagocytosis and oxidative burst. ImageStream cytometry provides the spatial resolution to determine the number of bacteria ingested and the sub-cellular localization and trafficking patterns that enables a more complete evaluation of the phagocytic process.

Visualization and quantification of cytotoxicity mediated by antibodies using imaging flow cytometry.

Conventional approaches for the detection of antibody dependent cell-mediated cytotoxicity (ADCC) activity rely on quantification of the release of traceable compounds from target cells or flow cytometry analysis of population-wide phenomena. We report a new method for the direct imaging and quantification of ADCC of cancer cells. The proposed method using imaging flow cytometry combines the statistical power of flow cytometry with the analytical advantages of cell imaging, providing a novel and more comprehensive perspective of effector/target cell interactions during ADCC events. With this method we can quantify and show in detail the morphological changes in target and effector cells, their apoptotic index, the physical interaction between effector and target cells, and a directional transfer of cytosolic contents from effector to target cells. As a model system we used the therapeutic anti-CD20 antibody rituximab to target CFSE labeled Ramos human Burkitt's lymphoma cells, to CMTPX-labeled human monocytic U-937 effector cells. We expect that similar studies using different effector and target cell populations may contribute to the pre-clinical evaluation of therapeutic antibodies and help to identify mechanisms that could be beneficial in the immunotherapy of cancer.

Exosomes containing glycoprotein 350 released by EBV-transformed B cells selectively target B cells through CD21 and block EBV infection in vitro.

Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV(-) B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350-CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression.

Flow cytometric analysis of microparticles.

Cell-derived microparticles (MPs) are increasingly recognized as important cell-to-cell signaling mechanisms and may exhibit important functions in homeostasis but also in pathogenesis. Indeed, MPs are associated with a number of diseases inhibiting their production that protects against pathogenesis. MPs are distinct from exosomes and apoptotic bodies, often exhibiting the membrane proteins of the activated or apoptotic cell from which they are derived. Electron microscopic analyses have shown that MPs are produced by all cell types tested to date, and ELISA-based assays have established that increased numbers of MPs are produced following cell activation. These approaches do not, however, determine the exact number of MPs and distribution of functional proteins on their surface. Flow cytometry represents an obvious approach to analyze MPs, and we present here a method to assess the number and phenotype of MPs by using a conventional flow cytometer. We also present the caveats with this method and describe a new imaging flow cytometry approach that overcomes these limitations.

C1q deficiency leads to the defective suppression of IFN-alpha in response to nucleoprotein containing immune complexes.

Almost all humans with homozygous deficiency of C1q develop systemic lupus erythematosus (SLE). The precise cellular mechanism(s) by which C1q prevents the development of SLE remains unclear. In this study, we tested the role of C1q in the regulation of IFN-α induced by immune complexes (ICs) in vitro, as well as the consequences of lack of C1q in vivo. Our experiments revealed that C1q preferentially promotes the binding of SLE ICs to monocytes rather than plasmacytoid dendritic cells, but this inhibition was not due to the induction of inhibitory soluble factors. The presence of C1q also altered the trafficking of ICs within monocytes such that ICs persisted in early endosomes. In patients with C1q deficiency, serum and cerebrospinal fluid levels of IFN-α and IFN-γ-inducible protein-10 levels were elevated and strongly correlated with Ro autoantibodies, demonstrating the clinical significance of these observations. These studies therefore associate C1q deficiency with defective regulation of IFN-α and provide a better understanding of the cellular mechanisms by which C1q prevents the development of IC-stimulated autoimmunity.

Immune modulation with sulfasalazine attenuates immunopathogenesis but enhances macrophage-mediated fungal clearance during Pneumocystis pneumonia.

Although T cells are critical for host defense against respiratory fungal infections, they also contribute to the immunopathogenesis of Pneumocystis pneumonia (PcP). However, the precise downstream effector mechanisms by which T cells mediate these diverse processes are undefined. In the current study the effects of immune modulation with sulfasalazine were evaluated in a mouse model of PcP-related Immune Reconstitution Inflammatory Syndrome (PcP-IRIS). Recovery of T cell-mediated immunity in Pneumocystis-infected immunodeficient mice restored host defense, but also initiated the marked pulmonary inflammation and severe pulmonary function deficits characteristic of IRIS. Sulfasalazine produced a profound attenuation of IRIS, with the unexpected consequence of accelerated fungal clearance. To determine whether macrophage phagocytosis is an effector mechanism of T cell-mediated Pneumocystis clearance and whether sulfasalazine enhances clearance by altering alveolar macrophage phagocytic activity, a novel multispectral imaging flow cytometer-based method was developed to quantify the phagocytosis of Pneumocystis in vivo. Following immune reconstitution, alveolar macrophages from PcP-IRIS mice exhibited a dramatic increase in their ability to actively phagocytose Pneumocystis. Increased phagocytosis correlated temporally with fungal clearance, and required the presence of CD4(+) T cells. Sulfasalazine accelerated the onset of the CD4(+) T cell-dependent alveolar macrophage phagocytic response in PcP-IRIS mice, resulting in enhanced fungal clearance. Furthermore, sulfasalazine promoted a TH2-polarized cytokine environment in the lung, and sulfasalazine-enhanced phagocytosis of Pneumocystis was associated with an alternatively activated alveolar macrophage phenotype. These results provide evidence that macrophage phagocytosis is an important in vivo effector mechanism for T cell-mediated Pneumocystis clearance, and that macrophage phenotype can be altered to enhance phagocytosis without exacerbating inflammation. Immune modulation can diminish pulmonary inflammation while preserving host defense, and has therapeutic potential for the treatment of PcP-related immunopathogenesis.

High throughput, parallel imaging and biomarker quantification of human spermatozoa by ImageStream flow cytometry.

Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.

Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow cytometry.

Activation of T lymphocytes by antigen-presenting cells (APC) results in the formation of an immunological synapse. Following contact with the target cell, key signaling and adhesion molecules polarize within minutes to hours to the T cell-APC interface. Multispectral imaging flow cytometry, a new technology which combines flow cytometry with imaging, was used to visualize and quantify the recruitment of the CD3epsilon and Lck signaling molecules during the evolution of an immune synapse. Using this technology, thousands of T cell/macrophage conjugates could be analyzed for each experimental time point. Following Ca++ triggered T cell activation, the dynamics of Lck and CD3epsilon recruitment to the synapse, analyzed by two independent methods, were comparable. However, CD3epsilon exhibited longer residence times (>8 min) at the synapse than Lck.

Measurement of cytoplasmic to nuclear translocation.

A method is described for the quantitative assessment of the translocation of signaling molecules from the cytoplasm to the nucleus in cells. This method utilizes fluorochrome-conjugated antibodies to the signaling molecule and a nuclear dye, and it is based on imagery acquired rapidly in flow with the use of a multispectral imaging cytometer. The analysis correlates the spatial distribution of the stained translocating signaling molecule with nuclear staining, and it generates a quantitative score for each cell using Pearson's correlation coefficient. Examples described in this section use reagents that detect NFkappaB and IRF-7 and measure the translocation of these molecules under stimulating conditions. A protocol for combining cell surface phenotype with cytoplasm to nuclear translocation is also included.

Oncogenic CARD11 mutations in human diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma. In the least curable (ABC) subtype of DLBCL, survival of the malignant cells is dependent on constitutive activation of the nuclear factor-kappaB (NF-kappaB) signaling pathway. In normal B cells, antigen receptor-induced NF-kappaB activation requires CARD11, a cytoplasmic scaffolding protein. To determine whether CARD11 contributes to tumorigenesis, we sequenced the CARD11 gene in human DLBCL tumors. We detected missense mutations in 7 of 73 ABC DLBCL biopsies (9.6%), all within exons encoding the coiled-coil domain. Experimental introduction of CARD11 coiled-coil domain mutants into lymphoma cell lines resulted in constitutive NF-kappaB activation and enhanced NF-kappaB activity upon antigen receptor stimulation. These results demonstrate that CARD11 is a bona fide oncogenein DLBCL, providing a genetic rationale for the development of pharmacological inhibitors of the CARD11 pathway for DLBCL therapy.

Interferon regulatory factor 7-mediated responses are defective in cord blood plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDC) are specialized in massive production of type I interferons (IFN) upon viral infections. Activation of IFN regulatory factor (IRF)-7 is critically required for the synthesis of type I IFN in pDC. IRF-7 is highly expressed by resting pDC and translocates into the nucleus to initiate type I IFN transcription. In a previous work, we observed an impaired IFN-alpha production in enriched cord blood pDC following a TLR9 stimulation using CpG oligonucleotides. Herein, we show that highly purified pDC from cord blood exhibit a profound defect in their capacity to produce IFN-alpha/beta in response to TLR9 as well as to TLR7 ligation or human CMV or HSV-1 exposure. Microarray experiments indicate that expression of the majority of type I IFN subtypes induced by a TLR7 agonist is reduced in cord blood pDC. We next demonstrated a reduced nuclear translocation of IRF-7 in cord blood pDC following CpG and HSV stimulation as compared to adult pDC. We conclude that impaired IRF-7 translocation in cord blood pDC is associated with defective expression of type I IFN genes. Our data provide a molecular understanding for the decreased ability of cord blood pDC to produce type I IFN upon viral stimulation.

Autophosphorylation docking site Tyr-867 in Mer receptor tyrosine kinase allows for dissociation of multiple signaling pathways for phagocytosis of apoptotic cells and down-modulation of lipopolysaccharide-inducible NF-kappaB transcriptional activation.

Efficient clearance of apoptotic cells is essential for tissue homeostasis, allowing for cellular turnover without inflammatory consequences. The Mer (Nyk and c-Eyk) receptor tyrosine kinase (Mertk) is involved in two aspects of apoptotic cell clearance by acting as a receptor for Gas6, a gamma-carboxylated phosphatidylserine-binding protein that bridges apoptotic and viable cells. First, Mertk acts in a bona fide engulfment pathway in concert with alphavbeta5 integrin by regulating cytoskeletal assemblages, and second, it acts as a negative regulator for inflammation by down-modulating pro-inflammatory signals mediated from bacterial lipopolysaccharide-Toll-like receptor 4 (TLR4) signaling, and hence recapitulating anti-inflammatory immune modulation by apoptotic cells. Here we describe Mertk post-receptor events that govern phagocytosis and cytoskeletal signaling are principally mediated by autophosphorylation site Tyr-867. Using the Mertk Y867F mutant and pharmacological inhibitors, we show that Tyr-867 is required for phosphatidylinositol 3-kinase and phospholipase Cgamma2 activation; their activation in turn elicits protein kinase C-dependent signals that act on the actin cytoskeleton. Although Mertk(Y867F) blocked the tyrosine phosphorylation of FAK on Tyr-861 and p130(cas) and also abrogated the phagocytosis of apoptotic cells, this mutant did not suppress lipopolysaccharide-inducible NF-kappaB transcription, nor was NF-kappaB activation dependent on the protein kinase C inhibitor, calphostin C. Finally, unlike the cytoskeletal events associated with Tyr-867 autophosphorylation, the trans-inhibition of NF-kappaB occurred in a postnuclear-dependent fashion independent of cytosolic IkappaB phosphorylation and p65/RelA sequestration. Taken together, these data suggest that Mertk has distinct and separable effects for phagocytosis and for resolving inflammation, providing a molecular rationale for how immune licensing and inflammation can be dissociated from phagocytosis in a single phagocytic receptor.