Citations

Abstract The two isoforms of phospholipase C (PLC)-γ couple immune recognition receptors to important calcium- and protein kinase C-dependent cellular functions. It has been assumed that PLC-γ1 and PLC-γ2 have redundant functions and that the receptors can use whichever PLC-γ isoform is preferentially expressed in a cell of a given hemopoietic lineage. In this study, we demonstrate that ITAM-containing immune recognition receptors can use either PLC-γ1 or PLC-γ2, whereas the novel NK cell-activating receptor NKG2D preferentially couples to PLC-γ2. Experimental models evaluating signals from either endogenous receptors (FcR vs NKG2D-DAP10) or ectopically expressed chimeric receptors (with ITAM-containing cytoplasmic tails vs DAP10-containing cytoplasmic tails) demonstrate that PLC-γ1 and PLC-γ2 both regulate the functions of ITAM-containing receptors, whereas only PLC-γ2 regulates the function of DAP10-coupled receptors. These data suggest that specific immune recognition receptors can differentially couple to the two isoforms of PLC-γ. More broadly, these observations reveal a basis for selectively targeting the functions initiated by distinct immune recognition receptors.

Abstract Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95vav and phospholipase C-γ isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.

Abstract Invariant chain (Ii) is a non-MHC-encoded molecule, which plays an accessory role in the proper assembly/expression of functional MHC class II molecules and there by plays an important role in Ag processing/presentation. The phenotype of mice lacking Ii depends on the allotype of the MHC class II molecule. In some mice strains, Ii deficiency results in reduction in expression of class II molecules accompanied by defective CD4+ T cell development. Responses to conventional Ags/superantigens are also compromised. In this study, we describe for the first time the functionality of human class II molecules, HLA-DQ6 and HLA-DQ8, in transgenic mice lacking Ii. HLA transgenic Ii−/− mice expressed very low levels of surface DQ6 and DQ8 accompanied by severe reduction in CD4+ T cells both in the thymus and periphery. In vitro proliferation and cytokine production to an exogenous superantigen, staphylococcal enterotoxin B (SEB) was diminished in HLA-transgenic Ii−/− mice. However, SEB-induced in vivo expansion of CD8+ T cells expressing TCR Vβ8 family in DQ8.Ii−/− mice was comparable with that of DQ8.Ii+/+ mice. Systemic IFN-γ production following in vivo challenge with SEB was reduced in DQ8.Ii−/− mice and were also protected from SEB-induced toxic shock. Although the T cell response to a known peptide Ag was diminished in DQ8.Ii−/− mice, DQ8.Ii−/− APCs were capable of presenting that peptide to primed T cells from wild-type DQ8 mice as well as to a specific T cell hybridoma. Differentiation of mature B cells was also affected to a certain extent in DQ8.Ii−/− mice.

Abstract Pancreatic cancer is a highly aggressive, treatment refractory cancer and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas overexpress altered forms of a tumor-specific Ag, mucin 1 (MUC1; an epithelial mucin glycoprotein), which is a potential target for immunotherapy. We have established a clinically relevant animal model for pancreatic cancer by developing a double transgenic mouse model (called MET) that expresses human MUC1 as self molecule and develops spontaneous tumors of the pancreas. These mice exhibit acinar cell dysplasia at birth, which progresses to microadenomas and acinar cell carcinomas. The tumors express large amounts of underglycosylated MUC1 similar to humans. Tumor-bearing MET mice develop low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor vaccine strategies.

The heterotopic thoracic pig‐to‐baboon heart transplantation has been established by our group as a safe preclinical model. Since the recipient's own heart remains in place, it is possible to evaluate immunological reactions of various types and the symptoms of the thrombotic microangiopathy under working heart conditions. However, these experiments are complex and costly. In order to limit the number of transplants, we tested our multi‐transgenic porcine donor hearts in an ex‐vivo perfusion model first.Ex‐vivo model: Both beating ventricles were perfused with heparinized freshly drawn human whole blood using a centrifugal pump and a membrane oxygenator. During 3 h of observation, cardiac function parameters were obtained continuously; specimens from the perfusate, coronary sinus blood, and myocardial biopsies were assessed. Various genetically modified porcine donor hearts were tested; we are currently evaluating the impact of human thrombomodulin on the porcine microcirculation; the efficacy of complement regulation is another work package.Pig‐to‐baboon heterotopic cardiac xenotransplantation: In our latest group, seven baboons received five double (Gal‐KO/hCD46), and two triple (Gal‐KO/hCD46/hTM resp./HLA‐E) transgenic pig hearts. Our immunosuppressive regimen included preoperative anti‐CD20‐antibody, bortezomib, dexamethasone and cyclophosphamide; postoperatively, ATG, tacrolimus, MMF, anti‐CD20‐antibody, bortezomib and dexamethasone were administered. Total lymph node irradiation (6 Gy) was applied on postoperative day five.The triple transgenic hearts survived 35 and 37 days respectively. Both hearts maintained function throughout the experiment. Both recipients succumbed to fungal infections. Humoral rejection was seen only once. The next barrier appears to be the complex event of thrombotic microangiopathy that needs to be addressed with the additional expression of human thrombomodulin strategies.

Abstract Little is known about the regulatory roles of specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in cytotoxic lymphocytes. Recent information suggests that mutations in the SNARE protein syntaxin 11 result in a form of familial hemophagocytic lymphohistiocytosis (FHL). Because genetic abnormalities in key granule components (e.g., perforin) or in regulators of secretion (e.g., Munc13–4) underlie the other identified forms of FHL, we assessed whether syntaxin 11 might also serve a related regulatory role. We determined that syntaxin 11 is expressed in NK cells and activated CTLs and is located in discrete membrane-associated structures in the cytoplasm. Enhanced expression of syntaxin 11 augments the secretion and killing of tumor targets, and suppression of syntaxin 11 expression inhibits these functions. Our data identify and characterize a role for syntaxin 11 in granule exocytosis and in the generation of cell-mediated killing. These results also provide new insights on the mechanisms of hemopoietic dysregulation in FHL.

Abstract NK cells are effector lymphocytes that can recognize and eliminate virally infected and transformed cells. NK cells express distinct activating receptors, including an ITAM-containing FcR complex that recognizes Ab-coated targets, and the DNAX-activating protein of 10 kDa-containing NKG2D receptor complex that recognizes stress-induced ligands. The regulatory role of specific tyrosine kinases in these pathways is incompletely understood. In this study, we show that, in activated human NK cells, the tyrosine kinase IL-2-inducible T cell kinase (Itk), differentially regulates distinct NK-activating receptors. Enhanced expression of Itk leads to increases in calcium mobilization, granule release, and cytotoxicity upon stimulation of the ITAM-containing FcR, suggesting that Itk positively regulates FcR-initiated cytotoxicity. In contrast, enhanced Itk expression decreases cytotoxicity and granule release downstream of the DNAX-activating protein of 10 kDa-containing NKG2D receptor, suggesting that Itk is involved in a pathway of negative regulation of NKG2D-initiated granule-mediated killing. Using a kinase mutant, we show that the catalytic activity of Itk is required for both the positive and negative regulation of these pathways. Complementary experiments where Itk expression was suppressed also showed differential regulation of the two pathways. These findings suggest that Itk plays a complex role in regulating the functions initiated by distinct NK cell-activating receptors. Moreover, understanding how these pathways may be differentially regulated has relevance in the setting of autoimmune diseases and antitumor immune responses where NK cells play key regulatory roles.

Abstract Eosinophils produce and release various proinflammatory mediators and also show immunomodulatory and tissue remodeling functions; thus, eosinophils may be involved in the pathophysiology of asthma and other eosinophilic disorders as well as host defense. Several major questions still remain. For example, how do human eosinophils become activated in diseased tissues or at the site of an immune response? What types of host immunity might potentially involve eosinophils? Herein, we found that human eosinophils react vigorously to a common environmental fungus, Alternaria alternata, which is implicated in the development and/or exacerbation of human asthma. Eosinophils release their cytotoxic granule proteins, such as eosinophil-derived neurotoxin and major basic protein, into the extracellular milieu and onto the surface of fungal organisms and kill the fungus in a contact-dependent manner. Eosinophils use their versatile β2 integrin molecule, CD11b, to adhere to a major cell wall component, β-glucan, but eosinophils do not express other common fungal receptors, such as dectin-1 and lactosylceramide. The I-domain of CD11b is distinctively involved in the eosinophils’ interaction with β-glucan. Eosinophils do not react with another fungal cell wall component, chitin. Because human eosinophils respond to and kill certain fungal organisms, our findings identify a previously unrecognized innate immune function for eosinophils. This immune response by eosinophils may benefit the host, but, in turn, it may also play a role in the development and/or exacerbation of eosinophil-related allergic human diseases, such as asthma.

Abstract NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell–target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.