Both protein and solid-state nanopores are under intense investigation for the analysis of nucleic acids. A crucial advantage of protein nanopores is that site-directed mutagenesis permits precise tuning of their properties. Here, by augmenting the internal positive charge within the ␣-hemolysin pore and varying its distribution, we increase the frequency of translocation of a 92-nt single-stranded DNA through the pore at ؉120 mV by Ϸ10-fold over the wild-type protein and dramatically lower the voltage threshold at which translocation occurs, e.g., by 50 mV for 1 event⅐s ؊1 ⅐ M ؊1 . Further, events in which DNA enters the pore, but is not immediately translocated, are almost eliminated. These experiments provide a basis for improved nucleic acid analysis with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification.DNA sequencing ͉ electroosmosis ͉ nanopore ͉ protein engineering ͉ single-molecule detection P ores with diameters of a few to hundreds of nanometers, ''nanopores,'' are being developed for a wide variety of analytical applications (1-5). Nanopores can be fabricated by using particle beams and etchants to treat various substrates, including silicon nitride (3, 6) and plastics, for instance poly(ethylene terephthalate) (4). Alternatively, protein pores such as ␣-hemolysin (␣HL) can be used (1, 5). In both cases, to be detected, an analyte must travel into the pore by electrodiffusion, but few systematic attempts have been made to optimize this process. In the present work, we show how the manipulation of charge on the internal surface of the ␣HL pore can be used to improve the rate of capture of an important analyte, DNA.The ␣HL protein nanopore is advantageous in sensing applications because it can be engineered with subnanometer precision with reference to the high resolution crystal structure (7). Further, the ␣HL pore is far more stable than commonly held, operating as normal at temperatures approaching 100°C (8). In stochastic sensing, a binding site for a family of analytes is formed within the pore by site-directed mutagenesis or targeted chemical modification (1). The concentration of an analyte is estimated from the number of binding events per unit time to a single pore. An analyte is identified through the signature provided by the amplitude and mean duration of the individual binding events. In this way, a great variety of analytes have been examined including: cations, anions, organic molecules, and various polymers (1). For example, all 4 DNA bases can be detected as nucleoside monophosphates by an ␣HL pore equipped with an aminocyclodextrin adapter (9). In addition, individual covalent chemical reactions occurring within the lumen of the pore can be observed (5), offering a basis for the detection of reactive molecules (10).Polymers can also be analyzed from the characteristics of transit events through the ␣HL pore (11-15). Studies of nucleic acids have been especially intensive, following the demonstration of the transit of single str...

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Until recently, the known roles of lymphatic endothelial cells (LECs) in immune modulation were limited to directing immune cell trafficking and passively transporting peripheral Ags to lymph nodes. Recent studies demonstrated that LECs can directly suppress dendritic cell maturation and present peripheral tissue and tumor Ags for autoreactive T cell deletion. We asked whether LECs play a constitutive role in T cell deletion under homeostatic conditions. In this study, we demonstrate that murine LECs under noninflamed conditions actively scavenge and cross-present foreign exogenous Ags to cognate CD8+ T cells. This cross-presentation was sensitive to inhibitors of lysosomal acidification and endoplasmic reticulum–golgi transport and was TAP1 dependent. Furthermore, LECs upregulated MHC class I and the PD-1 ligand PD-L1, but not the costimulatory molecules CD40, CD80, or CD86, upon Ag-specific interactions with CD8+ T cells. Finally, Ag-specific CD8+ T cells that were activated by LECs underwent proliferation, with early-generation apoptosis and dysfunctionally activated phenotypes that could not be reversed by exogenous IL-2. These findings help to establish LECs as APCs that are capable of scavenging and cross-presenting exogenous Ags, in turn causing dysfunctional activation of CD8+ T cells under homeostatic conditions. Thus, we suggest that steady-state lymphatic drainage may contribute to peripheral tolerance by delivering self-Ags to lymph node–resident leukocytes, as well as by providing constant exposure of draining peripheral Ags to LECs, which maintain tolerogenic cross-presentation of such Ags.

In melanoma, vascular endothelial growth factor-C (VEGF-C) expression and consequent lymphangiogenesis correlate with metastasis and poor prognosis. VEGF-C also promotes tumor immunosuppression, suggesting that lymphangiogenesis inhibitors may be clinically useful in combination with immunotherapy. We addressed this concept in mouse melanoma models with VEGF receptor-3 (VEGFR-3)-blocking antibodies and unexpectedly found that VEGF-C signaling enhanced rather than suppressed the response to immunotherapy. We further found that this effect was mediated by VEGF-C-induced CCL21 and tumor infiltration of naïve T cells before immunotherapy because CCR7 blockade reversed the potentiating effects of VEGF-C. In human metastatic melanoma, gene expression of VEGF-C strongly correlated with CCL21 and T cell inflammation, and serum VEGF-C concentrations associated with both T cell activation and expansion after peptide vaccination and clinical response to checkpoint blockade. We propose that VEGF-C potentiates immunotherapy by attracting naïve T cells, which are locally activated upon immunotherapy-induced tumor cell killing, and that serum VEGF-C may serve as a predictive biomarker for immunotherapy response.

Detection of chemical processes on a single molecule scale is the ultimate goal of sensitive analytical assays. We recently reported the possibility to detect chemical modifications on individual molecules by monitoring a change in the single ion channel conductance of derivatives of gramicidin A (gA) upon reaction with analytes in solution. These peptide-based nanosensors detect reaction-induced changes in the charge of gA derivatives that were engineered to carry specific functional groups near their C-terminus.1 Here, we discuss five key design parameters to optimize the performance of such chemomodulated ion channel sensors. In order to realize an effective sensor that measures changes in charge of groups attached to the C-terminus of a gA pore, the following conditions should be fulfilled: (1) the change in charge should occur as close to the entrance of the pore as possible; (2) the charge before and after reaction should be well-defined within the operational pH range; (3) the ionic strength of the recording buffer should be as low as possible while maintaining a detectable flow of ions through the pore; (4) the applied transmembrane voltage should be as high as possible while maintaining a stable membrane; (5) the lipids in the supporting membrane should either be zwitterionic or charged differently than the derivative of gA. We show that under the condition of high applied transmembrane potential (>100 mV) and low ionic strength of the recording buffer (< or =0.10 M), a change in charge at the entrance of the pore is the dominant requirement to distinguish between two differently charged derivatives of gA; the conductance of the heterodimeric gA pore reported here does not depend on a difference in charge at the exit of the pore. We provide a simple explanation for this asymmetric characteristic based on charge-induced local changes in the concentration of cations near the lipid bilayer membrane. Charge-based ion channel sensors offer tremendous potential for ultrasensitive functional detection since a single chemical modification of each individual sensing element can lead to readily detectable changes in channel conductance.

Protein nanopores may provide a cheap and fast technology to sequence individual DNA molecules. However, the electrophoretic translocation of ssDNA molecules through protein nanopores has been too rapid for base identification. Here, we show that the translocation of DNA molecules through the α-hemolysin protein nanopore can be slowed controllably by introducing positive charges into the lumen of the pore by site directed mutagenesis. Although the residual ionic current during DNA translocation is insufficient for direct base identification, we propose that the engineered pores might be used to slow down DNA in hybrid systems, e.g. in combination with solid-state nanopores.

Nanoparticle delivery systems are known to enhance the immune response to soluble antigens (Ags) and are thus a promising tool for the development of new vaccines. Our laboratory has engineered two different nanoparticulate systems in which Ag is either encapsulated within the core of polymersomes (PSs) or decorated onto the surface of nanoparticles (NPs). Previous studies showed that PSs are better at enhancing CD4 T cells and antibody titers, while NPs preferentially augment cytotoxic CD8 T cells. Herein, we demonstrate that the differential activation of T cell immunity reflects differences in the modes of intracellular trafficking and distinct biodistribution of the Ag in lymphoid organs, which are both driven by the properties of each nanocarrier. Furthermore, we found that Ags within PSs promoted better CD4 T cell activation and induced a higher frequency of CD4 T follicular helper (Tfh) cells. These differences correlated with changes in the frequency of germinal center B cells and plasma cell formation, which reflects the previously observed antibody titers. Our results show that PSs are a promising vector for the delivery of Ags for B cell vaccine development. This study demonstrates that nanocarrier design has a large impact on the quality of the induced adaptive immune response.

Lymphatic endothelial cells (LECs) chemoattract naïve T cells and promote their survival in the lymph nodes, and can cross-present antigens to naïve CD8 + T cells to drive their proliferation despite lacking key costimulatory molecules. However, the functional consequence of LEC priming of CD8 + T cells is unknown. Here, we show that while many proliferating LECeducated T cells enter early apoptosis, the remainders comprise a long-lived memory subset, with transcriptional, metabolic, and phenotypic features of central memory and stem cell-like memory T cells. In vivo, these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall, and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigenexperienced CD8 + T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.