TCRα-TCRß pairing controls recognition of CD1d and directs the development of adipose NKT cells.

The interaction between the T cell antigen receptor (TCR) expressed by natural killer T cells (NKT cells) and the antigen-presenting molecule CD1d is distinct from interactions between the TCR and major histocompatibility complex (MHC). Our molecular modeling suggested that a hydrophobic patch created after TCRα-TCRß pairing has a role in maintaining the conformation of the NKT cell TCR. Disruption of this patch ablated recognition of CD1d by the NKT cell TCR but not interactions of the TCR with MHC. Partial disruption of the patch, while permissive to the recognition of CD1d, significantly altered NKT cell development, which resulted in the selective accumulation of adipose-tissue-resident NKT cells. These results indicate that a key component of the TCR is essential for the development of a distinct population of NKT cells.

A phase 1b single-arm trial of intratumoral oncolytic virus V937 in combination with pembrolizumab in patients with advanced melanoma: results from the CAPRA study.

BACKGROUND: CAPRA (NCT02565992) evaluated Coxsackievirus A21 (V937) + pembrolizumab for metastatic/unresectable stage IIIB-IV melanoma. METHODS: Patients received intratumoral V937 on days 1, 3, 5, and 8 (then every 3 weeks [Q3W]) and intravenous pembrolizumab 2 mg/kg Q3W from day 8. Primary endpoint was safety. RESULTS: Median time from first dose to data cutoff was 32.0 months. No dose-limiting toxicities occurred; 14% (5/36) of patients experienced grade 3‒5 treatment-related adverse events. Objective response rate was 47% (complete response, 22%). Among 17 responders, 14 (82%) had responses ≥ 6 months. Among 8 patients previously treated with immunotherapy, 3 responded (1 complete, 2 partial). Responses were associated with increased serum CXCL10 and CCL22, suggesting viral replication contributes to antitumor immunity. For responders versus nonresponders, there was no difference in baseline tumor PD-L1 expression, ICAM1 expression, or CD3+ infiltrates. Surprisingly, the baseline cell density of CD3+CD8- T cells in the tumor microenvironment was significantly lower in responders compared with nonresponders (P = 0.0179). CONCLUSIONS: These findings suggest responses to this combination may be seen even in patients without a typical "immune-active" microenvironment. TRIAL REGISTRATION NUMBER: NCT02565992.

The Transcription Factor PLZF Is Necessary for the Development and Function of Mouse Basophils.

Basophils are innate immune cells associated with type 2 immunity, allergic reactions, and host defense against parasite infections. In this study, we show that the transcription factor PLZF, which is known for its essential role in the function and development of several innate lymphocyte subsets, is also important for the myeloid-derived basophil lineage. PLZF-deficient mice had decreased numbers of basophil progenitors in the bone marrow and mature basophils in multiple peripheral tissues. Functionally, PLZF-deficient basophils were less responsive to IgE activation and produced reduced amounts of IL-4. The altered function of basophils resulted in a blunted Th2 T cell response to a protein allergen. Additionally, PLZF-deficient basophils had reduced expression of the IL-18 receptor, which impacted migration to lungs. PLZF, therefore, is a major player in controlling type 2 immune responses mediated not only by innate lymphocytes but also by myeloid-derived cells.

FcγRIIa ligation induces platelet hypersensitivity to thrombotic stimuli.

Platelets are known for their important role in hemostasis, however their significance in other functions, including inflammation and infection, are becoming more apparent. Patients with systemic lupus erythematosus (SLE) are known to have circulating IgG complexes in their blood and are highly susceptible to thrombotic events. Because platelets express a single receptor for IgG, we tested the hypothesis that ligation of this receptor (FcγRIIa) induces platelet hypersensitivity to thrombotic stimuli. Platelets from SLE patients were considerably more sensitive to thrombin compared to healthy volunteers, and this correlated with elevated levels of surface IgG on SLE platelets. To test whether FcγRIIa ligation stimulated thrombin hypersensitivity, platelets from healthy volunteers were incubated with buffer or heat-aggregated IgG, then stimulated with increasing concentrations of thrombin. Interestingly, heat-aggregated IgG-stimulated platelets, but not buffer-treated platelets, were hypersensitive to thrombin, and hypersensitivity was blocked by an anti-FcγRIIa monoclonal antibody (mAb). Thrombin hypersensitivity was not due to changes in thrombin receptor expression (GPIbα or PAR1) but is dependent on activation of shared signaling molecules. These observations suggest that ligation of platelet FcγRIIa by IgG complexes induces a hypersensitive state whereby small changes in thrombotic stimuli may result in platelet activation and subsequent vascular complications such as transient ischemic attacks or stroke.

FcγRIIa requires lipid rafts, but not co-localization into rafts, for effector function.

OBJECTIVE: To determine if receptor localization into lipid rafts, or the lipid rafts themselves, are important for FcγRIIa effector functions. MATERIAL: Wild-type FcγRIIa or mutant FcγRIIa(C208A) that does not translocate to lipid rafts were transfected into Chinese hamster ovary (CHO) cells which have been shown to be reliable cells for studying FcγR function. TREATMENT: Cells were treated with buffer or methyl-ß-cyclodextrin (MßCD) to deplete cholesterol and dissolve the structure of lipid rafts. METHODS: To evaluate lipid raft association, transfected CHO cells were lysed and centrifuged over a sucrose gradient. Fractions were run on SDS-PAGE and blotted for FcγRIIa or sphingolipid GM1 to illustrate the lipid raft fractions. Lateral mobility of GFP-tagged wild-type or mutant FcγRIIa was assessed using fluorescence recovery after photobleaching (FRAP) microscopy. Internalization of IgG-opsonized erythrocytes was assessed by fluorescence microscopy and uptake of heat-aggregated IgG (haIgG) was measured using flow cytometry. RESULTS: We observed that FcγRIIa(C208A) did not localize into lipid rafts. However, the mutant FcγRIIa retained lateral mobility and effector function similar to wild-type FcγRIIa. However, mutant FcγRIIa function was abolished upon treatment with MßCD. CONCLUSIONS: Lipid rafts provide an essential component required for effector activities independent of receptor localization.

Differential requirement of lipid rafts for FcγRIIA mediated effector activities.

Immunoglobulin G (IgG) dependent activities are important in host defense and autoimmune diseases. Various cell types including macrophages and neutrophils contribute to pathogen destruction and tissue damage through binding of IgG to Fcγ receptors (FcγR). One member of this family, FcγRIIA, is a transmembrane glycoprotein known to mediate binding and internalization of IgG-containing targets. FcγRIIA has been observed to translocate into lipids rafts upon binding IgG-containing targets. We hypothesize that lipid rafts participate to different extents in binding and internalizing targets of different sizes. We demonstrate that disruption of lipid rafts with 8mM methyl-ß-cyclodextrin (MßCD) nearly abolishes binding (91% reduction) and phagocytosis (60% reduction) of large IgG-coated targets. Conversely, binding and internalization of small IgG-complexes is less dependent on lipid rafts (49% and 17% inhibition at 8mM MßCD, respectively). These observations suggest that differences between phagocytosis and endocytosis may arise as early as the initial stages of ligand recognition.

Substitution mapping in dahl rats identifies two distinct blood pressure quantitative trait loci within 1.12- and 1.25-mb intervals on chromosome 3.

Substitution mapping was used to refine the localization of blood pressure (BP) quantitative trait loci (QTL) within the congenic region of S.R-Edn3 rats located at the q terminus of rat chromosome 3 (RNO3). An F2(SxS.R-Edn3) population (n=173) was screened to identify rats having crossovers within the congenic region of RNO3 and six congenic substrains were developed that carry shorter segments of R-rat-derived RNO3. Five of the six congenic substrains had significantly lower BP compared to the parental S rat. The lack of BP lowering effect demonstrated by the S.R(ET3x5) substrain and the BP lowering effect retained by the S.R(ET3x2) substrain together define the RNO3 BP QTL-containing region as approximately 4.64 Mb. Two nonoverlapping substrains, S.R(ET3x1) and S.R(ET3x6), had significantly lower BP compared to the S strain, indicating the presence of two distinct BP QTL in the RNO3 q terminus. The RNO3 q terminus was fine mapped with newly developed polymorphic markers to characterize the extent of the congenic regions. The two RNO3 BP QTL regions were thus defined as within intervals of 0.05-1.12 and 0.72-1.25 Mb, respectively. Also important was our difficulty in fine mapping and marker placement in this portion of the rat genome (and thus candidate gene identification) using the available genomic data, including the rat genome sequence.

Internalization of IgG-coated targets results in activation and secretion of soluble CD40 ligand and RANTES by human platelets.

Platelets are crucial elements for maintenance of hemostasis. Other functions attributable to platelets are now being appreciated, such as their role in inflammatory reactions and host defense. Platelets have been reported to bind immunological stimuli like IgG complexes, and for nearly 50 years it has been speculated that platelets may participate in immunological reactions. Platelets have been reported to bind and internalize various substances, similar to other leukocytes, such as macrophages and dendritic cells. In the present study, we tested the hypothesis that human platelets can bind and internalize IgG-coated particles, similar to leukocytes. To this end, we observed that interaction with IgG-coated beads resulted in platelet activation (as measured by CD62P expression), internalization of targets, and significant soluble CD40 ligand (sCD40L) and RANTES (regulated upon activation, normal T cell expresses and secreted) secretion. Blocking FcγRIIA with monoclonal antibody (MAb) IV.3 or inhibiting actin remodeling with cytochalasin D inhibited platelet activation, internalization, and cytokine production. These data suggest that platelets are capable of mediating internalization of IgG-coated particles, resulting in platelet activation and release of both sCD40L and RANTES.