Peter W. Chen

Flash pyrolysis nozzle for generation of radicals in a supersonic jet expansion

A method for the high‐temperature generation of reactive species in supersonic molecular beams has been developed. Pyrolysis temperatures (25–1800u2009°C) can be maintained for longer than 100 h for extended experiments. Short contact time (≊10 μs) of the seeded (<1 Torr partial pressure in 2 atm inert carrier gas) precursor molecules suppresses recombination of the product radicals. Number densities for radicals are estimated to be ≊1014 cm−3 at the sonic orifice with radical fluxes of ≊1016 s−1.

Natural killer cell activation enhances immune pathology and promotes chronic infection by limiting CD8+ T-cell immunity

Infections with HIV, hepatitis B virus, and hepatitis C virus can turn into chronic infections, which currently affect more than 500 million patients worldwide. It is generally thought that virus-mediated T-cell exhaustion limits T-cell function, thus promoting chronic disease. Here we demonstrate that natural killer (NK) cells have a negative impact on the development of T-cell immunity by using the murine lymphocytic choriomeningitis virus. NK cell-deficient (Nfil3−/−, E4BP4−/−) mice exhibited a higher virus-specific T-cell response. In addition, NK cell depletion caused enhanced T-cell immunity in WT mice, which led to rapid virus control and prevented chronic infection in lymphocytic choriomeningitis virus clone 13- and reduced viral load in DOCILE-infected animals. Further experiments showed that NKG2D triggered regulatory NK cell functions, which were mediated by perforin, and limited T-cell responses. Therefore, we identified an important role of regulatory NK cells in limiting T-cell immunity during virus infection.

Rotationally resolved threshold photoelectron spectrum of the methyl radical

We report the rotationally resolved, one‐photon threshold photoelectron spectrum of the methyl radical, CH3, produced by supersonic‐jet, flash pyrolysis. Only rotational transitions with ΔK=0, ±2 are observed and this result is shown to be consistent with photoionization selection rules in D3h symmetry. Assignment of the threshold photoelectron spectrum results in an adiabatic ionization potential of 79u2009349±3 cm−1.

Transgenic mice expressing variants of complement factor H develop AMD-like retinal findings.

PURPOSEnComplement factor H (Cfh) is a key regulator of the alternative complement pathway. A Cfh variant (Y402H) increases the risk for AMD. The purpose of this study was to develop a pathophysiologically relevant animal model of AMD based on this genetic risk factor.nnnMETHODSnThe authors generated chimeric Cfh transgenic mouse lines using two constructs consisting of the human CFH sequence for SCR6-8 (with either 402Y or 402H), flanked by the mouse sequence for SCR1-5 and SCR9-20. They tested the expression of the transgenic mRNA and protein molecules and examined the mice at 12 to 14 months of age for clinical and histologic retinal changes.nnnRESULTSnNuclease protection assay and qRT-PCR analysis demonstrated transgenic mRNA expression in the liver and in the posterior segment of the eye. Western blot analysis showed that the transgenic proteins are present in the circulation at levels comparable to those of mouse Cfh. The chimeric proteins were found to be functional, as demonstrated by their ability to restore physiological serum levels of complement component C3 in Cfh KO mice. Clinical examination showed subretinal drusen-like deposits. Histology demonstrated an accumulation of subretinal cells that stained with a macrophage/microglia marker. Basal laminar deposits, long-spaced collagen, and increased numbers of lipofuscin granules were seen on electron microscopy. Immunohistochemistry showed a thicker sub-RPE band of C3d staining.nnnCONCLUSIONSnChimeric Cfh proteins led to AMD-like characteristics in mice. This may represent a good model for studying the role of complement and other components of the immune system in early AMD.

PD-L1: PD-1 Interaction Contributes to the Functional Suppression of T-Cell Responses to Human Uveal Melanoma Cells In Vitro

PURPOSEnTo assess the expression of PD-L1 on human uveal melanomas and its potential to suppress T-cell function.nnnMETHODSnA panel of primary and metastatic uveal melanoma cell lines was evaluated for PD-L1 expression by RT-PCR and flow cytometric analysis. Uveal melanoma-containing eyes were examined for PD-L1 expression by immunohistochemistry. PD-L1 function was tested by coculturing IFN-gamma-pretreated uveal melanoma cells with activated Jurkat T cells for 48 hours and assessing T-cell production of IL-2 by ELISA.nnnRESULTSnFive of the nine primary and one of the five metastatic uveal melanoma cell lines tested constitutively expressed PD-L1 protein at various levels. However, all primary and metastatic uveal melanoma cell lines upregulated PD-L1 expression after stimulation with IFN-gamma. Immunohistochemistry demonstrated that PD-L1 was not expressed by primary uveal melanomas in situ. IL-2 production by activated Jurkat T cells was decreased significantly when the cells were cocultured with IFN-gamma-pretreated uveal melanoma cells. More than 70% of IL-2 production was restored by addition of either anti-PD-L1 or anti-PD-1 antibody to the coculture assays (P < 0.01).nnnCONCLUSIONSnExpression of PD-L1 by uveal melanoma cells regulates T-cell function by suppressing IL-2 production. The results imply that the presence of IFN-gamma in the tumor local microenvironment promotes upregulation of PD-L1 expression by uveal melanoma, which may, in part, promote immune escape by impairing T-cell function. The selective blockade of PD-L1 is a potential strategy in T-cell-based immunotherapy for uveal melanoma.

Chemotherapy-Induced Genotoxic Stress Promotes Sensitivity to Natural Killer Cell Cytotoxicity by Enabling Missing-Self Recognition

Natural killer (NK) cells can recognize and kill tumor cells lacking self markers, such as class I MHC, but the basis for this recognition is not completely understood. NKR-P1 receptors are members of the C-type lectin-related NK receptor superfamily that are conserved from rodents to humans. Identification of Clr ligands for the NKR-P1 receptors has facilitated functional analysis of MHC-independent target cell recognition by NK cells. One receptor-ligand pair, NKR-P1B:Clr-b, can mediate missing-self recognition of tumor and infected cells, but the role of this axis in sensing stressed cells remains unknown. Here, we show that Clr-b is rapidly downregulated in cells undergoing genotoxic and cellular stress at the level of both RNA and surface protein. Stress-mediated loss of Clr-b on leukemia cells enhanced cytotoxicity mediated by NKR-P1B(+) NK cells. Notably, Clr-b downregulation was coordinated functionally with stress-mediated upregulation of NKG2D ligands (but not class I MHC). Our findings highlight a unique role for the MHC-independent NKR-P1B:Clr-b missing-self axis in recognition of stressed cells, and provide evidence of two independent levels of Clr-b regulation in stressed cells.

Activation of Tumor-specific CD4+ T Lymphocytes by Major Histocompatibility Complex Class II Tumor Cell Vaccines A Novel Cell-based Immunotherapy

Mouse tumor cells transfected with syngeneic MHC class II and costimulatory molecule genes are therapeutic vaccines in mice, provided they do not coexpress the class II-associated invariant chain (Ii). We demonstrated previously that the vaccine cells present tumor peptides via the endogenous antigen presentation pathway to activate CD4+ and CD8+ T cells. Because of their efficacy in mice, we are translating this vaccine strategy for clinical use. To obtain MHC class II+CD80+Ii− human tumor cells, we developed retroviruses encoding HLA-DR and CD80. The HLA-DR virus encodes the DRα and DRβ0101 chains using an internal ribosomal entry site to coordinate expression. SUM159PT mammary carcinoma and Mel 202 ocular melanoma cells transduced with the retroviruses DRB1/CD80 express high levels of DRB0101 and CD80 on the cell surface in the absence of Ii. Irradiated SUM159PT/DR1/CD80 vaccines stimulate proliferation of non-HLA-DRB0101 peripheral blood mononuclear cells and present an exogenous DR1-restricted tetanus toxoid (TT) peptide, indicating that the transduced DRB0101 is functional. SUM159PT/DR1/CD80 vaccines were further transduced with a retrovirus encoding the TT fragment C gene, as a model tumor antigen. These cells stimulate IFN-γ release from TT-primed human DRB0101 peripheral blood mononuclear cells, demonstrating their ability to present “endogenous” tumor antigen. Depletion and antibody blocking experiments confirm that MHC class II-restricted, endogenously synthesized epitopes are presented to CD4+ T cells. Therefore, the MHC class II vaccines are efficient antigen-presenting cells that activate tumor-specific MHC class II-restricted, CD4+ T lymphocytes, and they are a novel and potential immunotherapeutic for metastatic cancers.

Growth of diamond from atomic hydrogen and a supersonic free jet of methyl radicals

The growth of small (∼10-micrometer) diamond particles (on 0.1-or 0.25-micrometer seed crystals) using an effusive glow discharge nozzle for H�and a separate supersonic pyrolysis jet for �CH3 is reported. Laser micro-Raman, scanning electron microscopy, and x-ray photoelectron spectroscopy data are presented as evidence that well-crystallized diamond is indeed formed. Resonant multiphoton ionization spectroscopy is used as a diagnostic for the gas-phase chemistry indicating that the radical sources are clean and quantitative and that there is no detectable interconversion of �CH3 to C2H2 under the conditions of the experiment. Diamond growth is found at substrate temperatures greater than or equal to 650�C with no marked increase in the rate of growth up to 850�C. Acetylene does not give good quality diamond under similar conditions.

Expression of MAGE genes in ocular melanoma during progression from primary to metastatic disease

Primary melanomas that form within the eye have a unique pattern of disease progression as compared with melanomas that form within the skin. A high percentage of patients (approximately 50%) develop metastatic tumors that occur predominately in the liver. An unusual characteristic of ocular melanomas is the prolonged disease-free interval that extends for many years between the development of primary and metastatic tumors. It is estimated that the shortest interval between dissemination of tumor cells from the eye and the appearance of clinically detectable metastases is 6 years. A recent report indicated that fresh uveal melanoma tissue and metastatic tumor biopsies failed to express melanoma antigen gene (MAGE)-1, MAGE-2, or MAGE-3. In the present study, we examined the expression of MAGE genes on fresh and cultured tumor cells obtained from an ocular melanoma patient during different stages of progressive disease. MAGE gene expression was determined by reverse transcription-polymerase chain reaction using MAGE-1, MAGE-2 and MAGE-3 specific primers. Our results demonstrate that primary ocular tumor tissue and cultured tumor cells both express significant levels of MAGE-1, 2, and 3 at the time of enucleation. A high percentage of tumor cells within the primary tumor appear to express MAGE as demonstrated by consistent MAGE expression in 16 tumor cell clones. Metastatic liver tumors that developed 3 years after enucleation and 18 years after the initial formation of the primary tumor also expressed high levels of MAGE-1, -2, and -3. MAGE was expressed on fresh tumor tissue from a single biopsy and cultured tumor cells obtained from three of four different metastatic tumor nodules. When the MAGE-negative metastatic tumor cells were treated with the demethylating agent 5-Aza-2-Deoxycytidine (5-Aza-dC), transcription of MAGE-1 was restored, indicating the MAGE genes were not deleted. Our results demonstrate that in some patients, MAGE genes are expressed on primary and metastatic ocular melanomas.

IL-17A–Dependent CD4+CD25+ Regulatory T Cells Promote Immune Privilege of Corneal Allografts

IL-17A is a proinflammatory cytokine that has received attention for its role in the pathogenesis of several autoimmune diseases. IL-17A has also been implicated in cardiac and renal allograft rejection. Accordingly, we hypothesized that depletion of IL-17A would enhance corneal allograft survival. Instead, our results demonstrate that blocking IL-17A in a mouse model of keratoplasty accelerated the tempo and increased the incidence of allograft rejection from 50 to 90%. We describe a novel mechanism by which CD4+CD25+ regulatory T cells (Tregs) respond to IL-17A and enhance corneal allograft survival. Our findings suggest the following: 1) IL-17A is necessary for ocular immune privilege; 2) IL-17A is not required for the induction of anterior chamber-associated immune deviation; 3) Tregs require IL-17A to mediate a contact-dependent suppression; 4) corneal allograft Tregs suppress the efferent arm of the immune response and are Ag specific; 5) Tregs are not required for corneal allograft survival beyond day 30; and 6) corneal allograft-induced Treg-mediated suppression is transient. Our findings identify IL-17A as a cytokine essential for the maintenance of corneal immune privilege and establish a new paradigm whereby interplay between IL-17A and CD4+CD25+ Tregs is necessary for survival of corneal allografts.