TEM Cell Research Articles

Wideband transverse electromagnetic cell design and its application in frequency selective surface measurement

An open transverse electromagnetic cell (TEM cell) that covers L-band, S-band, and C-band is proposed in this paper. According to impedance matching theory analysis, the characteristic impedance of coaxial connector and microstrip with air as substrate are considered, which are combined with a taper gradient section to realize the function of TEM cell. The proposed TEM cell was modeled and simulated in CST Microwave Studio. Through full-wave simulation, the proposed TEM cell demonstrates a wideband transmission characteristic, S11 less than −10 dB from 1 to 8 GHz, which is relatively rare among the designs that have been reported. The test flow and data post-processing method are given. To verify the effectiveness of the test method, an example of measuring a classical square slot frequency selective surface that their resonance frequency is 3.7 GHz is designed and simulated in the TEM cell. To verify the simulated results, a prototype is fabricated and measured with the proposed TEM cell. The experimental result demonstrates a good agreement with the simulated result, which indicates that the designed TEM cell has good test performance.

Simultaneous Deep Tracking of Stem Cells by Surface Enhanced Raman Imaging Combined with Single‐Cell Tracking by NIR‐II Imaging in Myocardial Infarction

AbstractStem cell therapy has been used as a potential approach for the treatment of myocardial infarction (MI) over the last two decades. Imaging cellular behaviors of the transplanted stem cells with deep tissue penetration and high precision imaging modalities is crucial for the clinical translation of stem cell therapy approaches for MI. Herein, a gold nanostar (Au‐Star) based second near‐infrared window (NIR‐II) fluorescence/surface enhanced Raman scattering dual‐modal imaging probe (gold nanostar‐3.3′‐diethylthiatricarbocyanine iodide‐silver sulfide nanoparticles, Au‐Star‐DTTC‐Ag2S NPs, GDS NPs) is designed for labeling and precise tracking of the stem cells. The Ag2S compartment generates strong NIR‐II emission, which compensates for the deficiencies of bioluminescent imaging and enables the dynamic observation of in vivo cellular behavior. Subsequently, the specific Raman signal of Au‐Star‐DTTC compartment enables high‐resolution imaging, which could effectively delineate stem cells from the surrounding normal tissues, even at a single‐cell resolution. Using this imaging and tracking approach, it is able to track stem cells in hypodermic and MI models, with high resolution and depth‐independent imaging capabilities, which have not been reported in any other cell tracking platform. This two‐armed imaging toolkit offers new opportunities for a wide range of mechanistic stem cell therapy investigations in different organs.

Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis

Treatment of advanced melanoma with combined PD-1/CTLA-4 blockade commonly causes serious immune-mediated complications. Here, we identify a subset of patients predisposed to immune checkpoint blockade-related hepatitis who are distinguished by chronic expansion of effector memory CD4+ T cells (TEM cells). Pre-therapy CD4+ TEM cell expansion occurs primarily during autumn or winter in patients with metastatic disease and high cytomegalovirus (CMV)-specific serum antibody titres. These clinical features implicate metastasis-dependent, compartmentalised CMV reactivation as the cause of CD4+ TEM expansion. Pre-therapy CD4+ TEM expansion predicts hepatitis in CMV-seropositive patients, opening possibilities for avoidance or prevention. 3 of 4 patients with pre-treatment CD4+ TEM expansion who received αPD-1 monotherapy instead of αPD-1/αCTLA-4 therapy remained hepatitis-free. 4 of 4 patients with baseline CD4+ TEM expansion given prophylactic valganciclovir and αPD-1/αCTLA-4 therapy remained hepatitis-free. Our findings exemplify how pathogen exposure can shape clinical reactions after cancer therapy and how this insight leads to therapeutic innovations.

Electromagnetic Interference assessment of small electronic devices with a TEM cell

Electromagnetic Compatibility deserves an increasing interest because as modern industry 4.0 searches for better results in productivity and efficiency, it is expected the presence of an increasingly number of more complex electronic devices, including portable devices. This paper shows how a TEM cell allows analyzing the effects of Electromagnetic Interference, in an easy way and at small cost, during the development phase of the product.

Tracking Peripheral Memory T Cell Subsets in Advanced Non-Small Cell Lung Cancer Treated with Hypofractionated Radiotherapy and PD-1 Blockade in the ABSCOPAL-1 Clinical Trial

Background: Hypofractionated radiotherapy (HFRT) or chemotherapy combined with programmed death-1 (PD-1) blockade has achieved good clinical control in advanced non-small cell lung cancer (NSCLC). However, the effects of the distinct cell-killing mechanisms induced by radiotherapy and chemotherapy on immune memory remain unclear. We compared T memory subset differentiation in responders and non-responders treated with HFRT combined with PD-1 blockade to those in responders treated with chemo-immunotherapy. Methods: Thirty-eight patients with advanced NSCLC were enrolled in the trial. The frequencies of naive (Tn; CD45RA+CCR7+ ), central memory (Tcm; CD45RA–CCR7+), effector memory (Tem; CD45RA–CCR7–), and effector memory RA (TemRA; CD45RA+ CCR7–) T cell subsets and PD-1 expression were analyzed in CD4+ and CD8+ T cells using flow cytometry on single-cell suspensions from peripheral blood samples. Subsets were compared between healthy control and untreated NSCLC groups; among partial response, stable disease, and progressive disease groups treated using HFRT+PD-1 blockade; and between those treated with chemo-immunotherapy pre- and post-treatment. The correlations of memory T cell subsets and PD-1 expression with overall survival were examined using logistic regression. Findings: NSCLC patients showed expansion of the Tcm and Tem cell subsets among both CD4+ and CD8+ T cells compared with healthy controls. Patients with partial response to HFRT+PD-1 blockade showed reduction in Tn and expansion in TemRA cell subpopulations among CD8+ T cells and reduced PD-1+ CD4+ and PD-1+ CD8+ T cell counts, all of which were significantly correlated with overall survival. The responders to chemo-immunotherapy showed expansion of the TemRA CD4+ T cell subpopulation, decrease in Tcm CD4+ T and CD8+ T cell subpopulation. Conclusion: HFRT+PD-1 blockade and chemo-immunotherapy combination therapies induce differential memory T cell subset differentiation, offering predictive markers for treatment response. Trial Registration: ChiCTR-1900027768 Funding: This research was entirely funded by Functional study of Mir-183 in initiating cells of CD133+/CD326+ lung adenocarcinoma, 2013/01-2015/12, Project leader, Youth Fund project of National Natural Science Foundation of China； Immune mechanism of 3D guided Hypofractionation Radiotherapy in the treatment of lung adenocarcinoma， 2017/01-2019/12， Project leader， Luzhou People's Government - Southwest Medical University science and technology strategic cooperation fund；Luzhou Municipal Government and Southwest Medical University (2019LZXNYDC04);the joint project of People’s Government of Luxian County and Southwest Medical University (2019LXXNYKD-06);Joint Project of Sichuan University and Luzhou Industrial Research Institute (2020CDLZ-24). Declaration of Interest: None to declare. Ethical Approval: This prospective trial was approved by our ethics committee (No. KY2019276)

Signaling via Src Family Kinases Is Required for Normal Internalization of the Receptor c-Kit

AbstractStem cell factor (SCF) exerts its biological effects by binding to a specific receptor, the tyrosine kinase c-Kit, which is expressed on the cell surface. Although normal cellular trafficking of growth factor receptors may play a critical role in the modulation of receptor function, the mechanisms that regulate the distribution of c-Kit on the cell surface and the internalization of c-Kit have not been fully defined. We investigated whether signal transduction via Src family kinases is required for normal c-Kit trafficking. Treatment of the SCF-responsive human hematopoietic cell line MO7e with the inhibitor of Src family kinases PP1 blocked SCF-induced capping of c-Kit and internalization of c-Kit. c-Kit was able to associate with clathrin in the presence of PP1, suggesting that entry of c-Kit into clathrin-coated pits occurs independently of Src family kinases. SCF-induced internalization of c-Kit was also diminished in the D33-3 lymphoid cell line in which expression of Lyn kinase was disrupted by homologous recombination. These results indicate that Src family kinases play a role in ligand-induced trafficking of c-Kit.

Peripheral memory T-cell profile is modified in patients undergoing periodontal management.

T-cells are known to have a role in periodontitis, however, the effect of periodontal therapy on peripheral memory T-cells is unclear. This study evaluated variation in peripheral memory T-cells and red complex bacteria in sub-gingival plaque in patients undergoing periodontal management. Peripheral blood mononuclear cells and sub-gingival plaque were collected from 54 periodontitis patients at baseline, 3-, 6- and 12-months post-therapy and 40 healthy controls. Periodontitis patients were divided into treatment outcome (TxO) groups based on prevalence of sites with probing depth ≥5mm as good (<10% of sites), moderate (10-20%) or poor (>20%) at study conclusion. Naïve (TN -CCR7+ CD45RA+ ), central memory (TCM -CCR7+ CD45RA- ), effector memory (TEM -CCR7- CD45RA- ) and effector memory T-cells re-expressing CD45RA (TEMRA -CCR7- CD45RA+ ) were phenotyped using flow cytometry in CD4+ , CD8+ , CD4+ CD8+ and CD4- CD8- T-cells and red complex bacteria were quantified using qPCR. At baseline, periodontitis subjects had significantly greater mean probing depths and Porphyromonas gingivalis proportions, lower TN but higher CD4+ TCM , CD8+ TCM , CD4+ CD8+ TEM and CD4- CD8- TEM cell proportions compared to health. Periodontal therapy decreased mean probing depths, P.gingivalis proportions, TEM and CD4+ and CD8+ TCM cells, but increased TN and CD4+ and CD8+ TEMRA cells. The T-cell profile in the good TxO group showed therapy-related changes in CD4+ TEM , and CD8+ TN and TEM cells, whereas, no changes were observed in the poor TxO group. Management and the reduction in red complex bacteria were associated with changes in peripheral memory T-cells in periodontitis.

Novel approach for neuronal stem cell differentiation using aqueous two‐phase systems in 3D cultures

AbstractStem cell therapy has emerged as a promising alternative for replacing lost cells involved in neurodegenerative diseases. High efficiency of differentiation and full cell viability are actual challenges to achieve the translation of cell therapies to the clinic. To address this, the construction of aqueous two‐phase systems in three‐dimensional (ATPS‐3D) cultures has been proposed. This technique involves the combination of two polymers in which cells are confined in dextran droplets immersed over a substrate located in a poly(ethylene glycol) phase. The controlled placement of cells in a defined pattern promotes intercellular communication. This review aims to provide insight into the techniques used to enhance neural differentiation and current challenges to achieve the implementation of cell therapies. Cell density, colony size, interconnectivity and an appropriate substrate to modulate paracrine signaling are factors that determine neural differentiation efficiency during the construction of ATPS‐3D cultures. Hence, this contact‐free technique enables the design of neural niches to recapitulate in vivo environments more accurately. © 2020 Society of Chemical Industry (SCI)

Conductive textile structures and their contribution to electromagnetic shielding effectiveness

Fabrics for electromagnetic shielding are especially relevant in nowadays context, contributing to human’s protection and wellbeing and to proper functioning of electronic equipment, in relation to electromagnetic compatibility. Fabrics with electromagnetic shielding properties employ two main technologies, namely insertion of conductive yarns and application of conductive coatings. Magnetron sputtering is a modern technology to enable conductive coatings with thickness in the range of nanometers onto fabrics. This paper aims to analyze contribution of various conductive textile structures out of both fabrics with inserted conductive yarns and coatings to Electromagnetic shielding effectiveness (EMSE). EMSE was measured in the frequency range of 0.1–1000 MHz by using a TEM cell according to standard ASTM ES-07. Results show a gain of 10–25 dB when introducing silver yarns in warp/ weft direction, a variation of 5–35 dB between conductive yarns out of silver and stainless steel and an up to 12 dB gain out of thin copper coating by magnetron plasma onto the fabrics with inserted conductive yarns

External feedback assisted reduction of the lasing threshold of a continuous wave random laser in a dye doped polymer film and demonstration of speckle free imaging

The applications of random laser (RL) are still restricted, due to its complexity, high lasing threshold, unstable nature, difficulty in tuning and modulating its light emission characteristics. However, here a low threshold, continuous wave (CW) RL system has been designed in a fluoresecent laser dye (namely DCM) -doped in a polymer film with ZnO micro-cabbages as scatterers. Interestingly, by putting the active medium inside an external feedback (ExFB) system, a narrowed and stable RL emission at ~598 nm is observed with the subsequent reduction of CW lasing threshold (CWITh) by ~2.4 times. The effective cavity length (Leff) has been varied by an ingenious approach to demonstrate the role of Leff over the RL emission characteristics and the experimental results are supported by a theoretical explanation. The statistical fluctuation in the position of generated modes over time and the role of pump volume amplification on the emission characteristics of the developed RL has been studied in detailed. Thus, it is found here that due to the presence of ExFB, reinjection of pump light into scattering-formed cavities has taken place which has led to the generation of stable RL modes with the reduction of CWITh. The temporal coherency of the ExFB based RL light is found to be ~3.2 times less than that of a conventional He–Ne laser light. As a result, we could demonstrate here speckle free imaging with much lowered value of speckle contrast (C) on two different model systems, such as commercially available carbon film-coated TEM grid and cell membrane of Allium cepa. It is expected that this report will open up new possibilities for the development of stable and tunable RLs with other laser dyes for various optoelectronic applications including in bio-imaging.

Platelet factor 4 enhances CD4+ T effector memory cell responses via Akt‐PGC1α‐TFAM signaling‐mediated mitochondrial biogenesis

BackgroundCell metabolism drives T cell functions, while platelets regulate overall CD4+ T cell immune responses. ObjectiveTo investigate if platelets influence cell metabolism and thus regulate CD4+ T effector memory cell (Tem) responses. MethodsHuman CD4+ Tem cells were activated with αCD3/αCD28 and cultured without or with platelets or platelet‐derived mediators. ResultsPolyclonal stimulation induced rapid and marked Th1 and Treg cell activation of CD4+ Tem cells. Platelet co‐culture enhanced Th1 response transiently, while it persistently enhanced Treg cell activation of Tem cells, with an enhancement that plateaued by day 3. Platelet factor 4 (PF4) was the key platelet‐derived mediator regulating CD4+ Tem cell responses, which involved cellular metabolisms as indicated by mass spectrometric analyses. PF4 exerted its effects via its receptor CXCR3, attenuated Akt activity, and reduced PGC1α phosphorylation, and resulted in elevations of PGC1α function and mitochondrial transcription factor A (TFAM) synthesis. The latter increased mitochondrial biogenesis, and subsequently enhanced Th1 and Treg responses. Consistent with these observations, inhibition of mitochondrial function by rotenone counteracted the enhancements by recombinant PF4, and TFAM overexpression by TFAM‐adenovirus infection mimicked PF4 effects. Furthermore, increased mitochondrial mass elevated oxygen consumption, and enhanced adenosine triphosphate and reactive oxygen species production, which, in turn, stimulated Th1 (T‐bet) and Treg (FoxP3) transcription factor expression and corresponding CD4+ T effector cell responses. ConclusionsPlatelets enhance CD4+ T cell responses of Tem cells through PF4‐dependent and Akt‐PGC1α‐TFAM signaling‐mediated mitochondrial biogenesis. Hence, PF4 may be a promising intervention target of platelet‐regulated immune responses.

Luteolin-mediated Kv1.3 K+ channel inhibition augments BCG vaccine efficacy against tuberculosis by promoting central memory T cell responses in mice.

Despite the availability of multiple antibiotics, tuberculosis (TB) remains a major health problem worldwide, with one third of the population latently infected and ~2 million deaths annually. The only available vaccine for TB, Bacillus Calmette Guérin (BCG), is ineffective against adult pulmonary TB. Therefore, alternate strategies that enhance vaccine efficacy are urgently needed. Vaccine efficacy and long-term immune memory are critically dependent on central memory T (TCM) cells, whereas effector memory T (TEM) cells are important for clearing acute infections. Recently, it has been shown that inhibition of the Kv1.3 K+ ion channel, which is predominantly expressed on TEM but not TCM cells, profoundly enhances TCM cell differentiation. We exploited this phenomenon to improve TCM:TEM cell ratios and protective immunity against Mycobacterium tuberculosis infection in response to BCG vaccination of mice. We demonstrate that luteolin, a plant-derived Kv1.3 K+ channel inhibitor, profoundly promotes TCM cells by selectively inhibiting TEM cells, and significantly enhances BCG vaccine efficacy. Thus, addition of luteolin to BCG vaccination may provide a sustainable means to improve vaccine efficacy by boosting host immunity via modulation of memory T cell differentiation.

Quantifying the Effects of Beam Overlap on Radiation Damage via Radiolysis Products in the In-situ Liquid (S)TEM Cell

Quantifying the Effects of Beam Overlap on Radiation Damage via Radiolysis Products in the <i>In-situ</i> Liquid (S)TEM Cell

Complement-activated interferon-γ-primed human endothelium transpresents interleukin-15 to CD8+ T cells.

Alloantibodies in presensitized transplant candidates deposit complement membrane attack complexes (MACs) on graft endothelial cells (ECs), increasing risk of CD8+ T cell-mediated acute rejection. We recently showed that human ECs endocytose MACs into Rab5+ endosomes, creating a signaling platform that stabilizes NF-κB-inducing kinase (NIK) protein. Endosomal NIK activates both noncanonical NF-κB signaling to synthesize pro-IL-1β and an NLRP3 inflammasome to process and secrete active IL-1β. IL-1β activates ECs, increasing recruitment and activation of alloreactive effector memory CD4+ T (Tem) cells. Here, we report that IFN-γ priming induced nuclear expression of IL-15/IL-15Rα complexes in cultured human ECs and that MAC-induced IL-1β stimulated translocation of IL-15/IL-15Rα complexes to the EC surface in a canonical NF-κB-dependent process in which IL-15/IL-15Rα transpresentation increased activation and maturation of alloreactive CD8+ Tem cells. Blocking NLRP3 inflammasome assembly, IL-1 receptor, or IL-15 on ECs inhibited the augmented CD8+ Tem cell responses, indicating that this pathway is not redundant. Adoptively transferred alloantibody and mouse complement deposition induced IL-15/IL-15Rα expression by human ECs lining human coronary artery grafts in immunodeficient mice, and enhanced intimal CD8+ T cell infiltration, which was markedly reduced by inflammasome inhibition, linking alloantibody to acute rejection. Inhibiting MAC signaling may similarly limit other complement-mediated pathologies.

Operando Liquid-Electrochemical TEM for Monitoring Growth and Dissolution Steps of NaO2 Cubes in Na-O2 Battery

Compared to conventional Li/Na-ion batteries, in which reversible energy storage relies on the use of redox active transition metal oxides as positive electrodes, the metal-O2 battery systems would theoretically offer greater energy density owing to the use of the redox of gaseous oxygen using conductive and light carbon electrodes. Although in sodium−oxygen (Na−O2) batteries show promise as high-energy storage systems, this technology is still the subject of intense fundamental research, owing to the complex reaction by which it operates. To understand the formation mechanism of the discharge product, sodium superoxide (NaO2), liquid- electrochemical TEM holder has been used (Poseidon 510) [7].Here we present for the first time the use of a "Na-O2 microbattery" using a liquid aprotic electrolyte (liquid cell) coupled with fast imaging TEM (Oneview camera) and HAADF-STEM to visualize, in real time, the mechanism of NaO2 nucleation/growth. The electrolyte (NaPF6/DME) was saturated with ultrapure O2, prior to inject into the liquid TEM cell, to induce the reaction Na+ + O2 + e- -> NaO2 using electrochemical echips with a glassy carbon electrode (in the observation window) as working electrode (with two others Pt electrodes).Indeed, the recent development of liquid cell for TEM [1,2] enables an exciting opportunity to explore [3,4] and even quantify [5,6] complex electrochemical reactions occurring inside batteries during operation. Indeed, the miniaturization of new electrochemistry echips (Protochips) [7] with complex electrode designs (3-electrodes cell) adds the capability to perform true in situ/operando quantitative electrochemical reactions, which can be followed using TEM multi-techniques.We observe that the formation of NaO2 cubes [8] onto the glassy carbon electrode during reduction occurs by a solution-mediated nucleation process. The cyclic voltammetry response obtained during discharge and charge of the Na-O2 microbattery exhibits two peaks corresponding to the reduction and oxydation processes. We visualized the solution-mediated growth of NaO2 in real-time and identified that the 3D growth process is governed by the equilibrium between NaO2(solv) ↔ NaO2(solid). Based on the fast imaging acquisition (30 frames/s) of this phenomenon, we quantified the kinetics of growth of NaO2 cubes and demonstrated the strong impact of O2 diffusion in the liquid electrolyte on the NaO2 cube growth. By HAADF-STEM imaging the charge process, we demonstrated that the same solvation−desolvation equilibrium is responsible for the dissolution of the NaO2 discharge product, which consumes the NaO2 cubes from the NaO2−electrolyte interface toward the electrode.Finally, we observed the formation of parasitic shell around NaO2 cubes during the cycling of a Na-O2 battery, in which the fast imaging TEM revealed the chemical reactivity of NaO2 at the interface with the electrolyte. Parasitic products continuously accumulate on the cube surface to form a thick shell surrounding the NaO2 cubes, which passivate the electrode surface. This information is vital for optimization of the battery, since this parasitic shell is responsible for its poor cyclability by preventing crucial O2 redox and further nucleation of NaO2. The assessment of the discharge−charge mechanistic in Na-O2 batteries through operando liquid-electrochemical TEM visualization should facilitate the development of this battery technology [8].

Fractal Hilbert sensor to detect partial discharge on transformer

The design of fractal Hilbert sensor is presented in this paper. The sensor is intended to detect partial discharge (PD) in transformer insulation. The fractal Hilbert sensor designed using 4 order fractal and etching on single layer PCB board. The Hilbert fractal type is chosen as this type of sensor can be built on smaller area compare to other types. The sensor overall dimension is limited to 5x5 cm as it is proposed to be use to detect the PD presence in transformer. The sensor sensitivity is tested using a closed type TEM cell. After the sensitivity of the sensor is tested the sensor then applied to detect the PD signals emitted by void PD defect model. The results show the sensor has sensitivity as high as 10 dB. The sensor also has capability to detect the PD signals generated by the PD defect source. The waveforms captured by the sensor show the sensors can capture high frequency pulse generated by the PD source. Keywords: Partial Discharge (PD), Fractal Hilbert Sensor UHF, Sensor Sensitivity

Research regarding electromagnetic shielding achieved by the fabrics support

Electromagnetic shielding achieved by flexible materials has gained an increased significance in today’s radiation polluted environment. One of the modern approaches for obtaining electromagnetic textile shields with good surface electrical conductivity is the deposition of metallic thin films onto the fabrics. The use of the magnetron sputtering technique ensures some advantages, e.g. improved reflections of the fabric once metallic coatings are deposited on both sides of the fabric, as well as lightweight and excellent flexibility due to nanoscale thickness of layers. In the present work, cotton and polyester fabrics with various structural parameters, such as yarns count, fabric density, and specific mass were used as samples to be coated. PVD magnetron sputtering technique was utilized for the deposition of metallic layers with various thicknesses, either on one side or on both sides of the fabrics, in order to ensure good conductivity of the fabric. Besides, fabric samples with inserted conductive yarns of silver and stainless steel were considered as substrates, as well. The electromagnetic shielding tests using TEM cell evidenced good shielding effectiveness of 10-34 dB within 0.1-1000 MHz.

Real-Time In Situ Observations Reveal a Double Role for Ascorbic Acid in the Anisotropic Growth of Silver on Gold.

Rational nanoparticle design is one of the main goals of materials science, but it can only be achieved via a thorough understanding of the growth process and of the respective roles of the molecular species involved. We demonstrate that a combination of complementary techniques can yield novel information with respect to their individual contributions. We monitored the growth of long aspect ratio silver rods from gold pentatwinned seeds by three in situ techniques (small-angle X-ray scattering, optical extinction spectroscopy and liquid-cell transmission electron microscopy). Exploiting the difference in reaction speed between the bulk synthesis and the nanoparticle formation in the TEM cell, we show that the anisotropic growth is thermodynamically controlled (rather than kinetically) and that ascorbic acid, widely used for its mild reductive properties, plays a shape-directing role, by stabilizing the {100} facets of the silver cubic lattice, in synergy with the halide ions. This approach can easily be applied to a wide variety of synthesis strategies.

Human Epitopes Identified from Herpes Simplex Virus Tegument Protein VP11/12 (UL46) Recall Multifunctional Effector Memory CD4+ TEM Cells in Asymptomatic Individuals and Protect from Ocular Herpes Infection and Disease in "Humanized" HLA-DR Transgenic Mice.

While the role of CD8+ T cells in the control of herpes simplex virus 1 (HSV-1) infection and disease is gaining wider acceptance, a direct involvement of effector CD4+ T cells in this protection and the phenotype and function of HSV-specific human CD4+ T cell epitopes remain to be fully elucidated. In the present study, we report that several epitopes from the HSV-1 virion tegument protein (VP11/12) encoded by UL46 are targeted by CD4+ T cells from HSV-seropositive asymptomatic individuals (who, despite being infected, never develop any recurrent herpetic disease). Among these, we identified two immunodominant effector memory CD4+ TEM cell epitopes, amino acids (aa) 129 to 143 of VP11/12 (VP11/12129-143) and VP11/12483-497, using in silico, in vitro, and in vivo approaches based on the following: (i) a combination of the TEPITOPE algorithm and PepScan library scanning of the entire 718 aa of HSV-1 VP11/12 sequence; (ii) an in silico peptide-protein docking analysis and in vitro binding assay that identify epitopes with high affinity to soluble HLA-DRB1 molecules; and (iii) an ELISpot assay and intracellular detection of gamma interferon (IFN-γ), CD107a/b degranulation, and CD4+ T cell carboxyfluorescein succinimidyl ester (CFSE) proliferation assays. We demonstrated that native VP11/12129-143 and VP11/12483-497 epitopes presented by HSV-1-infected HLA-DR-positive target cells were recognized mainly by effector memory CD4+ TEM cells while being less targeted by FOXP3+ CD4+ CD25+ regulatory T cells. Furthermore, immunization of HLA-DR transgenic mice with a mixture of the two immunodominant human VP11/12 CD4+ TEM cell epitopes, but not with cryptic epitopes, induced HSV-specific polyfunctional IFN-γ-producing CD107ab+ CD4+ T cells associated with protective immunity against ocular herpes infection and disease.IMPORTANCE We report that naturally protected HSV-1-seropositive asymptomatic individuals develop a higher frequency of antiviral effector memory CD4+ TEM cells specific to two immunodominant epitopes derived from the HSV-1 tegument protein VP11/12. Immunization of HLA-DR transgenic mice with a mixture of these two immunodominant CD4+ T cell epitopes induced a robust antiviral CD4+ T cell response in the cornea that was associated with protective immunity against ocular herpes. The emerging concept of developing an asymptomatic herpes vaccine that would boost effector memory CD4+ and CD8+ TEM cell responses is discussed.

Sympathetic Enhancement of Memory T-Cell Homing and Hypertension Sensitization.

Effector memory T lymphocytes (TEM cells) exacerbate hypertension in response to repeated hypertensive stimuli. These cells reside in the bone marrow for prolonged periods and can be reactivated on reexposure to the hypertensive stimulus. Because hypertension is associated with increased sympathetic outflow to the bone marrow, we hypothesized that sympathetic nerves regulate accumulation and reactivation of bone marrow-residing hypertension-specific TEM cells. Using unilateral superior cervical ganglionectomy in wild-type C57BL/6 mice, we showed that sympathetic nerves create a bone marrow environment that supports residence of hypertension-specific CD8+ T cells. These cells, defined by their proliferative response on coculture with dendritic cells from Ang (angiotensin) II-infused mice, were reduced in denervated compared with innervated bone of Ang II-infused mice. Adoptively transferred CD8+ T cells from Ang II-infused mice preferentially homed to innervated compared with denervated bone. In contrast, ovalbumin responsive T cells from OT-I mice did not exhibit this preferential homing. Increasing superior cervical ganglion activity by activating Gq-coupled designer receptor exclusively activated by designer drug augmented CD8+ TEM bone marrow accumulation. Adoptive transfer studies using mice lacking β2AR (β2 adrenergic receptors) indicate that β2AR in the bone marrow niche, rather than T-cell β2AR is critical for TEM cell homing. Inhibition of global sympathetic outflow using Gi-coupled DREADD (designer receptor exclusively activated by designer drug) injected into the rostral ventrolateral medulla or treatment with a β2AR antagonist reduced hypertension-specific CD8+ TEM cells in the bone marrow and reduced the hypertensive response to a subsequent response to low dose Ang II. Sympathetic nerves contribute to the homing and survival of hypertension-specific TEM cells in the bone marrow after they are formed in hypertension. Inhibition of sympathetic nerve activity and β2AR blockade reduces these cells and prevents the blood pressure elevation and renal inflammation on reexposure to hypertension stimuli.