Cancer stem cells and their niche in the progression of squamous cell carcinoma.

Most cancers harbor a small population of highly tumorigenic cells known as cancer stem cells (CSCs). Because of their stem cell-like properties and resistance to conventional therapies, CSCs are considered to be a rational target for curable cancer treatment. However, despite recent advances in CSC research, CSC-targeted therapies are not as successful as was initially hoped. The proliferative, invasive, and drug-resistant properties of CSCs are regulated by the tumor microenvironment associated with them, the so-called CSC niche. Thus, targeting tumor-promoting cellular crosstalk between CSCs and their niches is an attractive avenue for developing durable therapies. Using mouse models of squamous cell carcinoma (SCC), we have demonstrated that tumor cells responding to transforming growth factor ß (TGF-ß) function as drug-resistant CSCs. The gene expression signature of TGF-ß-responding tumor cells has accelerated the identification of novel pathways that drive invasive tumor progression. Moreover, by focusing on the cytokine milieu and macrophages in the proximity of TGF-ß-responding tumor cells, we recently uncovered the molecular basis of a CSC-niche interaction that emerges during early tumor development. This review article summarizes the specialized tumor microenvironment associated with CSCs and discusses mechanisms by which malignant properties of CSCs are maintained and promoted.

ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters.

Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.

Genomic profiles of colorectal carcinoma with liver metastases and newly identified fusion genes.

Every year, approximately 1.2 million cases of colorectal carcinoma (CRC) are newly diagnosed worldwide. Although metastases to distant organs are often fatal complications of CRC, little information is known as to how such metastatic lesions are formed. To reveal the genetic profiles for CRC metastasis, we conducted whole-exome RNA sequencing on CRC tumors with liver metastasis (LM) (group A, n = 12) and clinical stage-matched larger tumors without LM (group B, n = 16). While the somatic mutation profiles were similar among the primary tumors and LM lesions in group A and the tumors in group B, the A-to-C nucleotide change in the context of "AAG" was only enriched in the LM regions in group A, suggesting the presence of a DNA damage process specific to metastasis. Genes already known to be associated with CRC were mutated in all groups at a similar frequency, but we detected somatic nonsynonymous mutations in a total of 707 genes in the LM regions, but not in the tumors without LM. Signaling pathways linked to such "LM-associated" genes were overrepresented for extracellular matrix-receptor interaction or focal adhesion. Further, fusions of the ADAP1 (ArfGAP with dual PH domain 1) were newly identified in our cohort (3 out of 28 patients), which activated ARF6, an ADAP1-substrate. Infrequently, mutated genes may play an important role in metastasis formation of CRC. Additionally, recurrent ADAP1 fusion genes were unexpectedly discovered. As these fusions activate small GTPase, further experiments are warranted to examine their contribution to CRC carcinogenesis.

Effects of the 2-Substituted Adenosine-1,3-diazaphenoxazine 5'-Triphosphate Derivatives on the Single Nucleotide Primer Extension Reaction by DNA Polymerase.

The adenosine triphosphate derivatives of 2-oxo-1,3-diazaphenoxazine (dAdapTP) showed a significant discrimination ability for the template strand including that between 8-oxo-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) by the single nucleotide primer extension reaction using the Klenow Fragment. In this study, we synthesized new dAdapTP derivatives, i.e., 2-amino-dAdapTP, 2-chloro-dAdapTP and 2-iodo-dAdapTP, to investigate the effect on the selectivity and efficiency of incorporation for the primer extension reaction using a variety of DNA polymerases. In contrast to the previously tested dAdapTP, the selectivity and efficiency of the 2-halo-dAdapTP incorporation were dramatically decreased using the Klenow Fragment. Moreover, the efficiency of the 2-amino-dAdapTP incorporation into the T-containing template was almost the same with that of dAdapTP. In the case of the Bsu DNA polymerase, the efficiency of all the dAdapTP derivatives decreased compared to that using the Klenow Fragment. However, the incorporation selectivity of dAdapTP had improved against the oxodG-containing template for all the template sequences including the T-containing template. Moreover, 2-amino-dAdapTP showed a better efficiency than dAdapTP using the Bsu DNA polymerase. The 2-amino group of the adenosine unit may interact with syn-oxodG at the active site of the Bsu DNA polymerase during the single primer extension reaction.

A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice.

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.

A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbß3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbß3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbß3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to integrin αIIbß3-dependent aggregation in human platelets.

Elevated Expression of ADAP2 is Associated With Aggressive Behavior of Human Clear-Cell Renal Cell Carcinoma and Poor Patient Survival.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common and lethal cancer of the adult kidney. ADAP2 is a GTPase-activating protein was upregulated in clear cell renal cell carcinoma. The role of ADAP2 in ccRCC progression is unknown. METHODS: ADAP2 expression in ccRCC cell lines and tissues was examined via real-time PCR, Western blot and IHC. MTS, colony formation and transwell assay to explore the role of ADAP2 in ccRCC. ADAP2 in growth and metastasis of ccRCC were evaluated in vivo through ccRCC xenograft tumor growth, lung metastatic mice model. The prognostic role of ADAP2 was evaluated by survival analysis. RESULTS: ADAP2 mRNA was expressed at significantly higher levels in 23 pairs of ccRCC tissues than in normal kidney tissues (P < 0.01). Immunohistochemical analysis of 298 ccRCC tissues revealed elevated ADAP2 expression as an independent unfavorable prognostic factor for the overall survival (P = 0.0042) and progression-free survival (P = 0.0232) of patients. The KaplanMeier survival curve showed that patients with a higher expression of ADAP2 showed a significantly lower overall survival rate and disease-free survival rate. Moreover, high expression of ADAP2 at the mRNA level was associated with a worse prognosis for overall survival (P = 0.0083) in The Cancer Genome Atlas (TCGA) cohort. In vivo and in vitro functional study showed that overexpression of ADAP2 promotes ccRCC cell proliferation and metastasis ability, whereas knockdown of ADAP2 inhibited cell proliferation, colony formation, migration and invasion. CONCLUSION: ADAP2 is a novel prognostic marker and could promotes tumor progression in ccRCC.

Identification, Characterization, and Computer-Aided Rational Design of a Novel Thermophilic Esterase from Geobacillus subterraneus, and Application in the Synthesis of Cinnamyl Acetate.

Investigation of a novel thermophilic esterase gene from Geobacillus subterraneus DSMZ 13552 indicated a high amino acid sequence similarity of 25.9% to a reported esterase from Geobacillus sp. A strategy that integrated computer-aided rational design tools was developed to select mutation sites. Six mutants were selected from four criteria based on the simulated saturation mutation (including 19 amino acid residues) results. Of these, the mutants Q78Y and G119A were found to retain 87% and 27% activity after incubation at 70 °C for 20 min, compared with the 19% activity for the wild type. Subsequently, a double-point mutant (Q78Y/G119A) was obtained and identified with optimal temperature increase from 65 to 70 °C and a 41.51% decrease in Km. The obtained T1/2 values of 42.2 min (70 °C) and 16.9 min (75 °C) for Q78Y/G119A showed increases of 340% and 412% compared with that in the wild type. Q78Y/G119A was then employed as a biocatalyst to synthesize cinnamyl acetate, for which the conversion rate reached 99.40% with 0.3 M cinnamyl alcohol at 60 °C. The results validated the enhanced enzymatic properties of the mutant and indicated better prospects for industrial application as compared to that in the wild type. This study reported a method by which an enzyme could evolve to achieve enhanced thermostability, thereby increasing its potential for industrial applications, which could also be expanded to other esterases.

ADAP restraint of STAT1 signaling regulates macrophage phagocytosis in immune thrombocytopenia.

Heightened platelet phagocytosis by macrophages accompanied by an increase in IFN-γ play key roles in the etiology of immune thrombocytopenia (ITP); however, it remains elusive how macrophage-mediated platelet clearance is regulated in ITP. Here, we report that adhesion and degranulation-protein adaptor protein (ADAP) restrains platelet phagocytosis by macrophages in ITP via modulation of signal transducer and activator of transcription 1 (STAT1)-FcγR signaling. We show that ITP was associated with the underexpression of ADAP in splenic macrophages. Furthermore, macrophages from Adap-/- mice exhibited elevated platelet phagocytosis and upregulated proinflammatory signaling, and thrombocytopenia in Adap-/- mice was mitigated by the depletion of macrophages. Mechanistically, ADAP interacted and competed with STAT1 binding to importin α5. ADAP deficiency potentiated STAT1 nuclear entry, leading to a selective enhancement of FcγRI/IV transcription in macrophages. Moreover, pharmacological inhibition of STAT1 or disruption of the STAT1-importin α5 interaction relieved thrombocytopenia in Adap-/- mice. Thus, our findings not only reveal a critical role for ADAP as an intracellular immune checkpoint for shaping macrophage phagocytosis in ITP but also identify the ADAP-STAT1-importin α5 module as a promising therapeutic target in the treatment of ITP.

Enhanced Susceptibility of ADAP-Deficient Mice to Listeria monocytogenes Infection Is Associated With an Altered Phagocyte Phenotype and Function.

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date, only limited data exist regarding the role of ADAP in pathogen-specific immunity during in vivo infection, and its contribution in phagocyte-mediated antibacterial immunity remains elusive. Here, we show that mice lacking ADAP (ADAPko) are highly susceptible to the infection with the intracellular pathogen Listeria monocytogenes (Lm) by showing enhanced immunopathology in infected tissues together with increased morbidity, mortality, and excessive infiltration of neutrophils and monocytes. Despite high phagocyte numbers in the spleen and liver, ADAPko mice only inefficiently controlled pathogen growth, hinting at a functional impairment of infection-primed phagocytes in the ADAP-deficient host. Flow cytometric analysis of hallmark pro-inflammatory mediators and unbiased whole genome transcriptional profiling of neutrophils and inflammatory monocytes uncovered broad molecular alterations in the inflammatory program in both phagocyte subsets following their activation in the ADAP-deficient host. Strikingly, ex vivo phagocytosis assay revealed impaired phagocytic capacity of neutrophils derived from Lm-infected ADAPko mice. Together, our data suggest that an alternative priming of phagocytes in ADAP-deficient mice during Lm infection induces marked alterations in the inflammatory profile of neutrophils and inflammatory monocytes that contribute to enhanced immunopathology while limiting their capacity to eliminate the pathogen and to prevent the fatal outcome of the infection.

The Clinical Aspect of Adaptor Molecules in T Cell Signaling: Lessons Learnt From Inborn Errors of Immunity.

Adaptor molecules lack enzymatic and transcriptional activities. Instead, they exert their function by linking multiple proteins into intricate complexes, allowing for transmitting and fine-tuning of signals. Many adaptor molecules play a crucial role in T-cell signaling, following engagement of the T-cell receptor (TCR). In this review, we focus on Linker of Activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 KDa (SLP-76). Monogenic defects in these adaptor proteins, with known roles in T-cell signaling, have been described as the cause of human inborn errors of immunity (IEI). We describe the current knowledge based on defects in cell lines, murine models and human patients. Germline mutations in Adhesion and degranulation adaptor protein (ADAP), have not resulted in a T-cell defect.

The Multiple Roles of the Cytosolic Adapter Proteins ADAP, SKAP1 and SKAP2 for TCR/CD3 -Mediated Signaling Events.

T cells are the key players of the adaptive immune response. They coordinate the activation of other immune cells and kill malignant and virus-infected cells. For full activation T cells require at least two signals. Signal 1 is induced after recognition of MHC/peptide complexes presented on antigen presenting cells (APCs) by the clonotypic TCR (T-cell receptor)/CD3 complex whereas Signal 2 is mediated via the co-stimulatory receptor CD28, which binds to CD80/CD86 molecules that are present on APCs. These signaling events control the activation, proliferation and differentiation of T cells. In addition, triggering of the TCR/CD3 complex induces the activation of the integrin LFA-1 (leukocyte function associated antigen 1) leading to increased ligand binding (affinity regulation) and LFA-1 clustering (avidity regulation). This process is termed "inside-out signaling". Subsequently, ligand bound LFA-1 transmits a signal into the T cells ("outside-in signaling") which enhances T-cell interaction with APCs (adhesion), T-cell activation and T-cell proliferation. After triggering of signal transducing receptors, adapter proteins organize the proper processing of membrane proximal and intracellular signals as well as the activation of downstream effector molecules. Adapter proteins are molecules that lack enzymatic or transcriptional activity and are composed of protein-protein and protein-lipid interacting domains/motifs. They organize and assemble macromolecular complexes (signalosomes) in space and time. Here, we review recent findings regarding three cytosolic adapter proteins, ADAP (Adhesion and Degranulation-promoting Adapter Protein), SKAP1 and SKAP2 (Src Kinase Associated Protein 1 and 2) with respect to their role in TCR/CD3-mediated activation, proliferation and integrin regulation.

Patients, Social Workers, and Pharmacists' Perceptions of Barriers to Providing HIV Care in Community Pharmacies in the United States.

Retaining people living with HIV (PLWH) in clinical care is a global priority to end the HIV epidemic. Community pharmacies in the United States have structural influences on the success or failure of retention in HIV care by supporting patients' complex needs. However, to date, barriers to retention in care in the community pharmacy setting have not been examined beyond pharmacy services of medication therapy management. We utilized the patient-centered medical home model to examine the barriers to HIV care in the community pharmacy setting. We utilized semi-structured interviews to collect data from 15 participants: five PLWH, five community pharmacists, and five social workers from a midwestern state. Interview data were transcribed and analyzed using directed content analysis. Four key themes emerged regarding the barriers that impact utilization of community pharmacy services by PLWH: the perception of the role of community pharmacists in HIV care, perceptions of pharmacists' HIV knowledge, perceptions of pharmacy operation and services, and negative experiences within the community pharmacy space. Participants' perceptions of solutions for improving HIV care in the community pharmacy focused on improving the relationship between pharmacists and patients, ensuring that the community pharmacy is a private and safe space for patients, and having a diverse pharmacy staff that is equipped to take care of the diverse and marginalized HIV population, such as transgender people.

ABAP1 Plays a Role in the Differentiation of Male and Female Gametes in Arabidopsis thaliana.

The correct development of a diploid sporophyte body and a haploid gametophyte relies on a strict coordination between cell divisions in space and time. During plant reproduction, these divisions have to be temporally and spatially coordinated with cell differentiation processes, to ensure a successful fertilization. Armadillo BTB Arabidopsis protein 1 (ABAP1) is a plant exclusive protein that has been previously reported to control proliferative cell divisions during leaf growth in Arabidopsis. Here, we show that ABAP1 binds to different transcription factors that regulate male and female gametophyte differentiation, repressing their target genes expression. During male gametogenesis, the ABAP1-TCP16 complex represses CDT1b transcription, and consequently regulates microspore first asymmetric mitosis. In the female gametogenesis, the ABAP1-ADAP complex represses EDA24-like transcription, regulating polar nuclei fusion to form the central cell. Therefore, besides its function during vegetative development, this work shows that ABAP1 is also involved in differentiation processes during plant reproduction, by having a dual role in regulating both the first asymmetric cell division of male gametophyte and the cell differentiation (or cell fusion) of female gametophyte.

ADAP1/Centaurin-α1 Negatively Regulates Dendritic Spine Function and Memory Formation in the Hippocampus.

ADAP1/Centaurin-α1 (CentA1) functions as an Arf6 GTPase-activating protein highly enriched in the brain. Previous studies demonstrated the involvement of CentA1 in brain function as a regulator of dendritic differentiation and a potential mediator of Alzheimer's disease (AD) pathogenesis. To better understand the neurobiological functions of CentA1 signaling in the brain, we developed Centa1 knock-out (KO) mice. The KO animals showed neither brain development nor synaptic ultrastructure deficits in the hippocampus. However, they exhibited significantly higher density and enhanced structural plasticity of dendritic spines in the CA1 region of the hippocampus compared with non-transgenic (NTG) littermates. Moreover, the deletion of Centa1 improved performance in the object-in-place (OIP) spatial memory task. These results suggest that CentA1 functions as a negative regulator of spine density and plasticity, and of hippocampus-dependent memory formation. Thus, CentA1 and its downstream signaling may serve as a potential therapeutic target to prevent memory decline associated with aging and brain disorders.

Metabolomics Data Preprocessing Using ADAP and MZmine 2.

The informatics pipeline for making sense of untargeted LC-MS or GC-MS data starts with preprocessing the raw data. Results from data preprocessing undergo statistical analysis and subsequently mapped to metabolic pathways for placing untargeted metabolomics data in the biological context. ADAP is a suite of computational algorithms that has been developed specifically for preprocessing LC-MS and GC-MS data. It consists of two separate computational workflows that extract compound-relevant information from raw LC-MS and GC-MS data, respectively. Computational steps include construction of extracted ion chromatograms, detection of chromatographic peaks, spectral deconvolution, and alignment. The two workflows have been incorporated into the cross-platform and graphical MZmine 2 framework and ADAP-specific graphical user interfaces have been developed for using ADAP with ease. This chapter summarizes the algorithmic principles underlying key steps in the two workflows and illustrates how to apply ADAP to preprocess LC-MS and GC-MS data.

Immune Cell-Type Specific Ablation of Adapter Protein ADAP Differentially Modulates EAE.

The cytosolic adhesion and degranulation-promoting adapter protein ADAP is expressed in various hematopoietic cells including T cells, NK cells, myeloid cells, and platelets but absent in mature B cells. The role of ADAP in T cell activation, proliferation and integrin activation is well-accepted. We previously demonstrated that conventional ADAP knockout mice show a significantly attenuated course of experimental autoimmune encephalomyelitis (EAE). To dissect the impact of different ADAP expressing cell populations on the reduced EAE severity, here, we generated lineage-specific conditional knockout mice. ADAP was deleted in T cells, myeloid cells, NK cells and platelets, respectively. Specific loss of ADAP was confirmed on the protein level. Detailed immunophenotyping was performed to assess the consequence of deletion of ADAP with regard to the maturation and distribution of immune cells in primary and secondary lymphoid organs. The analysis showed equivalent results as for conventional ADAP knockout mice: impaired thymocyte development in ADAPfl/fl Lck-Cre mice, normal NK cell and myeloid cell distribution in ADAPfl/fl NKp46-Cre mice and ADAPfl/fl LysM-Cre mice, respectively as well as thrombocytopenia in ADAPfl/fl PF4-Cre mice. Active EAE was induced in these animals by immunization with the myelin oligodendrocyte glycoprotein35-55 peptide. The clinical course of EAE was significantly milder in mice with loss of ADAP in T cells, myeloid cells and NK cells compared to ADAP-sufficient control littermates. Surprisingly, specific deletion of ADAP in platelets resulted in a more exacerbated disease. These data show that T cell-independent as well as T cell-dependent mechanisms are responsible for the complex phenotype observed in conventional ADAP knockout mice.

Controlling Cytokine Release Syndrome to Harness the Full Potential of CAR-Based Cellular Therapy.

Chimeric Antigen Receptor (CAR)-based therapies offer a promising, targeted approach to effectively treat relapsed or refractory B cell malignancies. However, the treatment-related toxicity defined as cytokine-release syndrome (CRS) often develops in patients, and if uncontrolled, can be fatal. Grading systems have now been developed to further characterize and objectify clinical findings in order to provide algorithm-based guidance on CRS-related treatment decisions. The pharmacological treatments associated with these algorithms suppress inflammation through a variety of mechanisms and are paramount to improving the safety profile of CAR-based therapies. However, fatalities are still occurring, and there are downsides to these treatments, including the possibility of disrupting CAR-T cell persistence. This review article will describe the clinical presentation and current management of CRS, and through our now deeper understanding of downstream signaling pathways, will provide a molecular framework to formulate new hypotheses regarding clinical applications to contain proinflammatory cytokines responsible for CRS.

ADAP Promotes Degranulation and Migration of NK Cells Primed During in vivo Listeria monocytogenes Infection in Mice.

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date only limited and moreover conflicting data exist regarding the role of ADAP in NK cells. To extend existing knowledge we investigated ADAP-dependency of NK cells in the context of in vivo infection with the intracellular pathogen Listeria monocytogenes (Lm). Ex vivo analysis of infection-primed NK cells revealed impaired cytotoxic capacity in NK cells lacking ADAP as indicated by reduced CD107a surface expression and inefficient perforin production. However, ADAP-deficiency had no global effect on NK cell morphology or intracellular distribution of CD107a-containing vesicles. Proteomic definition of ADAPko and wild type NK cells did not uncover obvious differences in protein composition during the steady state and moreover, similar early response patterns were induced in NK cells upon infection independent of the genotype. In line with protein network analyses that suggested an altered migration phenotype in naïve ADAPko NK cells, in vitro migration assays uncovered significantly reduced migration of both naïve as well as infection-primed ADAPko NK cells compared to wild type NK cells. Notably, this migration defect was associated with a significantly reduced expression of the integrin CD11a on the surface of splenic ADAP-deficient NK cells 1 day post-Lm infection. We propose that ADAP-dependent alterations in integrin expression might account at least in part for the fact that during in vivo infection significantly lower numbers of ADAPko NK cells accumulate in the spleen i.e., the site of infection. In conclusion, we show here that during systemic Lm infection in mice ADAP is essential for efficient cytotoxic capacity and migration of NK cells.

The Fyn-ADAP Axis: Cytotoxicity Versus Cytokine Production in Killer Cells.

Lymphocyte signaling cascades responsible for anti-tumor cytotoxicity and inflammatory cytokine production must be tightly regulated in order to control an immune response. Disruption of these cascades can cause immune suppression as seen in a tumor microenvironment, and loss of signaling integrity can lead to autoimmunity and other forms of host-tissue damage. Therefore, understanding the distinct signaling events that exclusively control specific effector functions of "killer" lymphocytes (T and NK cells) is critical for understanding disease progression and formulating successful immunotherapy. Elucidation of divergent signaling pathways involved in receptor-mediated activation has provided insights into the independent regulation of cytotoxicity and cytokine production in lymphocytes. Specifically, the Fyn signaling axis represents a branch point for killer cell effector functions and provides a model for how cytotoxicity and cytokine production are differentially regulated. While the Fyn-PI(3)K pathway controls multiple functions, including cytotoxicity, cell development, and cytokine production, the Fyn-ADAP pathway preferentially regulates cytokine production in NK and T cells. In this review, we discuss how the structure of Fyn controls its function in lymphocytes and the role this plays in mediating two facets of lymphocyte effector function, cytotoxicity and production of inflammatory cytokines. This offers a model for using mechanistic and structural approaches to understand clinically relevant lymphocyte signaling.