Capacitively coupled nonthermal plasma synthesis of aluminum nanocrystals for enhanced yield and size control.

Uniform-size, non-native oxide-passivated metallic aluminum nanoparticles (Al NPs) have desirable properties for fuel applications, battery components, plasmonics, and hydrogen catalysis. Nonthermal plasma-assisted synthesis of Al NPs was previously achieved with an inductively coupled plasma (ICP) reactor, but the low production rate and limited tunability of particle size were key barriers to the applications of this material. This work focuses on the application of capacitively coupled plasma (CCP) to achieve improved control over Al NP size and a ten-fold increase in yield. In contrast with many other materials, where NP size is controlled via the gas residence time in the reactor, the Al NP size appeared to depend on the power input to the CCP system. The results indicate that the CCP reactor assembly, with a hydrogen-rich argon/hydrogen plasma, was able to produce Al NPs with diameters that were tunable between 8 and 21 nm at a rate up ∼ 100 mg h-1. X-ray diffraction indicates that a hydrogen-rich environment results in crystalline metal Al particles. The improved synthesis control of the CCP system compared to the ICP system is interpreted in terms of the CCP's lower plasma density, as determined by double Langmuir probe measurements, leading to reduced NP heating in the CCP that is more amenable to NP nucleation and growth.

Preclinical Analysis of Candidate Anti-Human CD79 Therapeutic Antibodies Using a Humanized CD79 Mouse Model.

The BCR comprises a membrane-bound Ig that is noncovalently associated with a heterodimer of CD79A and CD79B. While the BCR Ig component functions to sense extracellular Ag, CD79 subunits contain cytoplasmic ITAMs that mediate intracellular propagation of BCR signals critical for B cell development, survival, and Ag-induced activation. CD79 is therefore an attractive target for Ab and chimeric Ag receptor T cell therapies for autoimmunity and B cell neoplasia. Although the mouse is an attractive model for preclinical testing, due to its well-defined immune system, an obstacle is the lack of cross-reactivity of candidate therapeutic anti-human mAbs with mouse CD79. To overcome this problem, we generated knockin mice in which the extracellular Ig-like domains of CD79A and CD79B were replaced with human equivalents. In this study, we describe the generation and characterization of mice expressing chimeric CD79 and report studies that demonstrate their utility in preclinical analysis of anti-human CD79 therapy. We demonstrate that human and mouse CD79 extracellular domains are functionally interchangeable, and that anti-human CD79 lacking Fc region effector function does not cause significant B cell depletion, but induces 1) decreased expression of plasma membrane-associated IgM and IgD, 2) uncoupling of BCR-induced tyrosine phosphorylation and calcium mobilization, and 3) increased expression of PTEN, consistent with the levels observed in anergic B cells. Finally, anti-human CD79 treatment prevents disease development in two mouse models of autoimmunity. We also present evidence that anti-human CD79 treatment may inhibit Ab secretion by terminally differentiated plasmablasts and plasma cells in vitro.

A Prodomain Fragment from the Proteolytic Activation of Growth Differentiation Factor 11 Remains Associated with the Mature Growth Factor and Keeps It Soluble.

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor ß (TGF-ß) family, plays diverse roles in mammalian development. It is synthesized as a large, inactive precursor protein containing a prodomain, pro-GDF11, and exists as a homodimer. Activation requires two proteolytic processing steps that release the prodomains and transform latent pro-GDF11 into active mature GDF11. In studying proteolytic activation in vitro, we discovered that a 6-kDa prodomain peptide containing residues 60-114, PDP60-114, remained associated with the mature growth factor. Whereas the full-length prodomain of GDF11 is a functional antagonist, PDP60-114 had no impact on activity. The specific activity of the GDF11/PDP60-114 complex (EC50 = 1 nM) in a SMAD2/3 reporter assay was identical to that of mature GDF11 alone. PDP60-114 improved the solubility of mature GDF11 at neutral pH. As the growth factor normally aggregates/precipitates at neutral pH, PDP60-114 can be used as a solubility-enhancing formulation. Expression of two engineered constructs with PDP60-114 genetically fused to the mature domain of GDF11 through a 2x or 3x G4S linker produced soluble monomeric products that could be dimerized through redox reactions. The construct with a 3x G4S linker retained 10% activity (EC50 = 10 nM), whereas the construct connected with a 2x G4S linker could only be activated (EC50 = 2 nM) by protease treatment. Complex formation with PDP60-114 represents a new strategy for stabilizing GDF11 in an active state that may translate to other members of the TGF-ß family that form latent pro/mature domain complexes.

Interference with Ca(2+) release activated Ca(2+) (CRAC) channel function delays T-cell arrest in vivo.

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.

Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.

Cytolytic granules mediate killing of virus-infected cells by cytotoxic T lymphocytes. We show here that the granules can take long or short paths to the secretory domain. Both paths utilized the same intracellular molecular events, which have different spatial and temporal arrangements and are regulated by the kinetics of Ca(2+)-mediated signaling. Rapid signaling caused swift granule concentration near the microtubule-organizing center (MTOC) and subsequent delivery by the polarized MTOC directly to the secretory domain-the shortest path. Indolent signaling led to late recruitment of granules that moved along microtubules to the periphery of the synapse and then moved tangentially to fuse at the outer edge of the secretory domain-a longer path. The short pathway is associated with faster granule release and more efficient killing than the long pathway. Thus, the kinetics of early signaling regulates the quality of the T cell cytolytic response.

Radiation-induced CXCL16 release by breast cancer cells attracts effector T cells.

Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8(+)CXCR6(+) activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into "inflamed" peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy.

Peptide-MHC potency governs dynamic interactions between T cells and dendritic cells in lymph nodes.

T cells survey antigen-presenting dendritic cells (DCs) by migrating through DC networks, arresting and maintaining contact with DCs for several hours after encountering high-potency complexes of peptide and major histocompatibility complex (pMHC), leading to T cell activation. The effects of low-potency pMHC complexes on T cells in vivo, however, are unknown, as is the mechanism controlling T cell arrest. Here we evaluated T cell responses in vivo to high-, medium- and low-potency pMHC complexes and found that regardless of potency, pMHC complexes induced upregulation of CD69, anergy and retention of T cells in lymph nodes. However, only high-potency pMHC complexes expressed by DCs induced calcium-dependent T cell deceleration and calcineurin-dependent anergy. The pMHC complexes of lower potency instead induced T cell anergy by a biochemically distinct process that did not affect T cell dynamics.

The duration of exposure to HIV modulates the breadth and the magnitude of HIV-specific memory CD4+ T cells.

The impact of exposure to Ag on the development and maintenance of human CD4(+) memory T cells in general and HIV infection in particular is partially understood. In this study, we measured HIV-specific CD4(+) T cell proliferative responses against HIV proteins and derived peptides one year after highly active antiretroviral therapy initiation in 39 HIV-infected patients who initiated therapy at different times following infection. We show that a brief exposure to HIV of <1 month does not allow the generation of significant detectable frequencies of HIV-specific CD4(+) memory T cells. Patients having prolonged cumulative exposure to high viral load due to therapy failures also demonstrated limited HIV-specific CD4(+) T cell responses. In contrast, patients exposed to significant levels of virus for periods ranging from 3 to 18 mo showed brisk and broad HIV-specific CD4(+) T cell responses 1 year following the onset of therapy intervention. We also demonstrate that the nadir CD4(+) T cell count before therapy initiation correlated positively with the breadth and magnitude of these responses. Our findings indicate that the loss of proliferative HIV-specific CD4(+) T cell responses is associated with the systemic progression of the disease and that a brief exposure to HIV does not allow the establishment of detectable frequencies of HIV-specific memory CD4(+) T cells.

Clonotype tracking of TCR repertoires during chronic virus infections.

Human viral infections such as HIV and EBV typically evoke a strong and diverse CD8(+) T cell response. Relatively little is known about the extent to which TCR repertoire evolution occurs during viral infection or how repertoire evolution affects the efficacy of the CD8(+) T cell response. In this study we describe a general approach for tracking TCR repertoire evolution during viral infection. IFNgamma surface capture and MHC class I tetramer staining were independently used to isolate EBV-specific CD8(+) T cells from peripheral blood. Anchored RT-PCR and clonotype TCR repertoire analysis were performed immediately after isolating the cells. We find that the TCR repertoires of the IFNgamma-secreting and MHC class I tetramer staining populations were similar. In one subject a detailed analysis of the TCR repertoire during the first year of EBV infection was performed and over 600 TCR sequences targeting an EBV-immunodominant epitope were analyzed. Although some repertoire evolution occurred during the year, in general, the degree of repertoire drift was small. TCR repertoire analysis for an HIV-immunodominant epitope revealed a highly conserved amino acid motif in the Dbeta region of TCR that recognizes the epitope and suggested that T cell precursor frequency influences which epitopes are targeted early in HIV infection. This methodology, which allows one to sort antigen-specific T cells based on different functional assays and to obtain a snapshot of their TCR repertoire with relative ease, should lead to a richer understanding of the rules underlying antigen recognition and T cell evolution during viral infection.

Labeling antigen-specific CD4(+) T cells with class II MHC oligomers.

Class I MHC-peptide oligomers (MHC tetramers) have become popular reagents for the detection and characterization of antigen-specific CD8(+) T cells. Class II MHC proteins can be produced by expression in Escherichia coli followed by in vitro folding, or by native expression in insect cells; biotin can be introduced by site-specific chemical modification of cysteine, or by enzymatic modification of a peptide tag; and a variety of fluorescent streptavidin preparations can be used for oligomerization. Here we review methodologies for production of fluorescent oligomers of soluble class II MHC proteins and discuss their use in analysis of antigen-specific CD4(+) T cells. We explore the experimental conditions necessary for efficient staining of CD4(+) T cells using oligomers of class II MHC proteins, and we establish a standard protocol. Finally, we consider complications and challenges associated with these reagents, discuss the interpretation of staining results, and suggest future directions for investigation, in particular the use of MHC oligomers for the study of T cell avidity modulation.