Sensitive Detection and Analysis of Neoantigen-Specific T Cell Populations from Tumors and Blood.

Neoantigen-specific T cells are increasingly viewed as important immunotherapy effectors, but physically isolating these rare cell populations is challenging. Here, we describe a sensitive method for the enumeration and isolation of neoantigen-specific CD8+ T cells from small samples of patient tumor or blood. The method relies on magnetic nanoparticles that present neoantigen-loaded major histocompatibility complex (MHC) tetramers at high avidity by barcoded DNA linkers. The magnetic particles provide a convenient handle to isolate the desired cell populations, and the barcoded DNA enables multiplexed analysis. The method exhibits superior recovery of antigen-specific T cell populations relative to literature approaches. We applied the method to profile neoantigen-specific T cell populations in the tumor and blood of patients with metastatic melanoma over the course of anti-PD1 checkpoint inhibitor therapy. We show that the method has value for monitoring clinical responses to cancer immunotherapy and might help guide the development of personalized mutational neoantigen-specific T cell therapies and cancer vaccines.

MATE-Seq: microfluidic antigen-TCR engagement sequencing.

Adaptive immunity is based on peptide antigen recognition. Our ability to harness the immune system for therapeutic gain relies on the discovery of the T cell receptor (TCR) genes that selectively target antigens from infections, mutated proteins, and foreign agents. Here we present a method that selectively labels peptide antigen-specific CD8+ T cells using magnetic nanoparticles functionalized with peptide-MHC tetramers, isolates these specific cells within an integrated microfluidic device, and directly amplifies the TCR genes for sequencing. Critically, the identity of the peptide recognized by the TCR is preserved, providing the link between peptide and gene. The platform requires inputs on the order of just 100 000 CD8+ T cells, can be multiplexed for simultaneous analysis of multiple peptides, and performs sorting and isolation on chip. We demonstrate 1000-fold sensitivity enhancement of detecting antigen-specific TCRs relative to bulk analysis and simultaneous capture of two virus antigen-specific TCRs from a population of T cells.

T cell receptors for the HIV KK10 epitope from patients with differential immunologic control are functionally indistinguishable.

HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses. Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs). We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells. T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties. Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice. We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity. These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors.

Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2-carboxyethyl)phosphine as a reducing agent by LC-MS/MS.

A simple, robust, and rapid LC-MS/MS method has been developed and validated for the simultaneous quantitation of clopidogrel and its active metabolite (AM) in human plasma. Tris(2-carboxyethyl)phosphine (TCEP) was used as a reducing agent to detect the AM as a disulfide-bonded complex with plasma proteins. Mixtures of TCEP and human plasma were deproteinized with acetonitrile containing 10 ng/mL of clopidogrel-d4 as an internal standard (IS). The mixtures were separated on a C18 RP column with an isocratic mobile phase consisting of 0.1% formic acid in acetonitrile and water (90:10, v/v) at a flow rate of 0.3 mL/min. Detection and quantification were performed using ESI-MS. The detector was operated in selected reaction-monitoring mode at m/z 322.0→211.9 for clopidogrel, m/z 356.1→155.2 for the AM, and m/z 326.0→216.0 for the IS. The linear dynamic range for clopidogrel and its AM were 0.05-20 and 0.5-200 ng/mL, respectively, with correlation coefficients (r) greater than 0.9976. Precision, both intra- and interday, was less than 8.26% with an accuracy of 87.6-106%. The validated method was successfully applied to simultaneously analyze clinical samples for clopidogrel and its AM.

Generation of reactive oxygen species contributes to the development of carbon black cytotoxicity to vascular cells.

Carbon black, a particulate form of pure elemental carbon, is an industrial chemical with the high potential of occupational exposure. Although the relationship between exposure to particulate matters (PM) and cardiovascular diseases is well established, the cardiovascular risk of carbon black has not been characterized clearly. In this study, the cytotoxicity of carbon black to vascular smooth muscle and endothelial cells were examined to investigate the potential vascular toxicity of carbon black. Carbon black with distinct particle size, N330 (primary size, 28~36 nm) and N990 (250~350 nm) were treated to A-10, rat aortic smooth muscle cells and human umbilical vein endothelial cell line, ECV304, and cell viability was assessed by lactate dehydrogenase (LDH) leakage assay. Treatment of carbon black N990 resulted in the significant reduction of viability in A-10 cells at 100 µg/ml, the highest concentration tested, while N330 failed to cause cell death. Cytotoxicity to ECV304 cells was induced only by N330 at higher concentration, 200 µg/ml, suggesting that ECV304 cells were relatively resistant to carbon black. Treatment of 100 µg/ml N990 led to the elevation of reactive oxygen species (ROS) detected by dichlorodihydrofluorescein (DCF) in A-10 cells. Pretreatment of antioxidants, N-acetylcysteine (NAC) and sulforaphane restored decreased viability of N990-treated A-10 cells, and N-acetylcysteine, but not sulforaphane, attenuated N990-induced ROS generation in A-10 cells. Taken together, present study shows that carbon black is cytotoxic to vascular cells, and the generation of reactive oxygen contributes to the development of cytotoxicity. ROS scavenging antioxidant could be a potential strategy to attenuate the toxicity induced by carbon black exposure.