The interplay between membrane topology and mechanical forces in regulating T cell receptor activity

T cells are critically important for host defense against infections. T cell activation is specific because signal initiation requires T cell receptor (TCR) recognition of foreign antigen peptides presented by major histocompatibility complexes (pMHC) on antigen presenting cells (APCs). Recent advances reveal that the TCR acts as a mechanoreceptor, but it remains unclear how pMHC/TCR engagement generates mechanical forces that are converted to intracellular signals. Here we propose a TCR Bending Mechanosignal (TBM) model, in which local bending of the T cell membrane on the nanometer scale allows sustained contact of relatively small pMHC/TCR complexes interspersed among large surface receptors and adhesion molecules on the opposing surfaces of T cells and APCs. Localized T cell membrane bending is suggested to increase accessibility of TCR signaling domains to phosphorylation, facilitate selective recognition of agonists that form catch bonds, and reduce noise signals associated with slip bonds.

A mineralization strategy based on T-cell membrane coated CaCO3 nanoparticles against breast cancer and metastasis

Chemotherapy is always ineffective against cancer metastasis due to the limited diffusion ability of passive agents into the internal tumor. Herein, we designed a mineralization strategy based on the multifunctional...

T cell membrane cloaking tumor microenvironment-responsive nanoparticle with smart “membrane escape mechanism” for enhanced immune-chemotherapy of melanoma

The application of combination immune-chemotherapy makes up for the limitation of monotherapy and achieves superior antitumor activity against cancer. However, combinational therapy is always restricted by poor tumor targeted drug...

84 Quantifying pharmacodynamic biomarker changes in immuno-oncology by mass spectrometry

BackgroundQuantifying pharmacodynamic biomarker changes enables decision making and clinical trials in drug development. Pharmacodynamic biomarkers are used to determine the effects of treatment on disease. Mass spectrometry offers a quantitative, selective, and multiplex platform for pharmacodynamic protein biomarker analysis in clinical samples (e.g. blood and tumor) that is feasible across multiple sample conditions (e.g. fresh, frozen and formalin-fixed paraffin-embedded (FFPE)). To date, however, methodologies for targeted protein analysis by mass spectrometry (i.e. quantitative proteomics) are underdeveloped for application in immuno-oncology.MethodsTo address this, we sought to extract the immuno-oncology-associated T cell membrane proteins CD3, CD4 and CD8 from peripheral blood mononucleate cells (PBMC) and develop a multiplexed mass spectrometry method to quantify their expression. PBMC were isolated from whole blood and using detergent-based lysis buffers fractionated into a cytosolic and membrane protein lysate (figure 1). Analytical methods were then developed to detect proteotypic peptides of all three proteins (table 1 and figure 6) from the lysates by mass spectrometry.ResultsCD3, CD4 and CD8 were detected in the membrane protein fraction but not in the cytosolic protein fraction after whole-proteome tryptic digestion using a filter-aided sample preparation (or FASP) technique but with a signal-to-noise ratio of ≤ 2.0 (figure 2). Applying an additional immunoaffinity (IA) enrichment step with antibody-conjugated magnetic beads, prior to digestion, dramatically improved the analyte signal-to-noise ratios to > 100 (figure 3). Reverse-phase nanoflow liquid chromatography (LC) was used to separate all three analytes in multiplex over a 12-minute run prior to tandem mass analysis (MS/MS) (figure 4). Together, this IA-LC-MS/MS method resulted in detection of endogenous CD3, CD4 and CD8 proteins from small volumes of whole blood (< 0.1 mL) and the analyte responses were linear over at least two orders of magnitude (figure 5).Abstract 84 Figure 1Detergent-based protein extraction and fractionation of PBMCAbstract 84 Table 1Surrogate peptides for selective protein analysis by MS/MS after tryptic digestionAbstract 84 Figure 2Filter-aided sample preparation (FASP) for whole-proteome analysisAbstract 84 Figure 3Immunoaffinity enrichment of proteins from PBMC lysatesAbstract 84 Figure 4Representative multiplex analysis from 1mL of whole bloodAbstract 84 Figure 5Multiplex analysis of endogenous CD3, CD4, and CD8Abstract 84 Figure 6Optimization of tryptic digestion conditionsConclusionsThis method was developed specifically to quantitate pharmacodynamic changes in CD4 and CD8 T cell membrane expressions from clinically feasible samples (i.e. PBMC). This work, however, provides a foundation for developing methodologies to conduct quantitative proteomics applicable to immuno-oncology, which may be used to interrogate additional pharmacodynamic biomarkers.

CD4+ T Cell-Mimicking Nanoparticles Broadly Neutralize HIV-1 and Suppress Viral Replication through Autophagy

ABSTRACT Therapeutic strategies that provide effective and broad‐spectrum neutralization against HIV-1 infection are highly desirable. Here, we investigate the potential of nanoengineered CD4+ T cell membrane-coated nanoparticles (TNP) to neutralize a broad range of HIV-1 strains. TNP displayed outstanding neutralizing breadth and potency; they neutralized all 125 HIV-1-pseudotyped viruses tested, including global subtypes/recombinant forms, and transmitted/founder viruses, with a geometric mean 80% inhibitory concentration (IC80) of 819 μg ml−1 (range, 72 to 8,570 μg ml−1). TNP also selectively bound to and induced autophagy in HIV-1-infected CD4+ T cells and macrophages, while having no effect on uninfected cells. This TNP-mediated autophagy inhibited viral release and reduced cell-associated HIV-1 in a dose- and phospholipase D1-dependent manner. Genetic or pharmacological inhibition of autophagy ablated this effect. Thus, we can use TNP as therapeutic agents to neutralize cell-free HIV-1 and to target HIV-1 gp120-expressing cells to decrease the HIV-1 reservoir. IMPORTANCE HIV-1 is a major global health challenge. The development of an effective vaccine and/or a therapeutic cure is a top priority. The creation of vaccines that focus an antibody response toward a particular epitope of a protein has shown promise, but the genetic diversity of HIV-1 hinders this progress. Here we developed an approach using nanoengineered CD4+ T cell membrane-coated nanoparticles (TNP). Not only do TNP effectively neutralize all strains of HIV-1, but they also selectively bind to infected cells and decrease the release of HIV-1 particles through an autophagy-dependent mechanism with no drug-induced off-target or cytotoxic effects on bystander cells.