Expression of T-Cell Receptor β-Chain mRNA and Protein in γ/δ T-Cells from Euthymic and Athymic Rats: Implications for T-Cell Lineage Divergence

The relationship between α/β and γ/δ T-cell lineages was studied in rats using RT-PCR analysis of TCRβ transcripts in γ/δ T-cell hybridomas and an intracellular staining technique to detect TCRβ protein in primary γ/δ T-cells. We report the presence of functional TCRβ transcripts in 2/9 γ/δ T-cell hybridomas. About 15 % of peripheral γ/δ T-cells and thymocytes also express TCRβ protein, giving a minimum estimate for successful Tcrb rearrangement based on ex vivo single cell analysis. In athymic rats, γ/δ T-cells expressing intracellular β protein are present but at a lower frequency than in euthymic controls, suggesting that in the thymus, more γ/δ T-cell precursors pass through a stage where functional β rearrangement has occurred than in extrathymic sites. Analysis of TCR expression in purified transitory immature CD4-8+ (iCD8SP) thymocytes and their spontaneously developing CD4+8+ (DP) progeny showed that TCRγ mRNA is expressed in iCD8SP cells but not in their immediate DP progeny that reinitiate RAG-1 transcription and commence α/βTCR expression. We conclude that rat γ/δ T cells can separate from the α/β lineage after TCRβ expression, but not after entry into the DP compartment

Sequential induction of effector function, tissue migration and cell death during polyclonal activation of mouse regulatory T-cells

The ability of CD4+Foxp3+ regulatory T-cells (Treg) to produce interleukin (IL)-10 is important for the limitation of inflammation at environmental interfaces like colon or lung. Under steady state conditions, however, few Tregs produce IL-10 ex vivo. To investigate the origin and fate of IL-10 producing Tregs we used a superagonistic mouse anti-mouse CD28 mAb (CD28SA) for polyclonal in vivo stimulation of Tregs, which not only led to their numeric expansion but also to a dramatic increase in IL-10 production. IL-10 secreting Tregs strongly upregulated surface receptors associated with suppressive function as compared to non-producing Tregs. Furthermore, polyclonally expanding Tregs shifted their migration receptor pattern after activation from a CCR7+CCR52 lymph node-seeking to a CCR72CCR5+ inflammationseeking phenotype, explaining the preferential recruitment of IL-10 producers to sites of ongoing immune responses. Finally, we observed that IL-10 producing Tregs from CD28SA stimulated mice were more apoptosis-prone in vitro than their IL-10 negative counterparts. These findings support a model where prolonged activation of Tregs results in terminal differentiation towards an IL-10 producing effector phenotype associated with a limited lifespan, implicating built-in termination of immunosuppression

Topological Requirements and Signaling Properties of T Cell–activating, Anti-CD28 Antibody Superagonists

Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): “conventional,” TCR signaling–dependent costimulatory mAbs and “superagonistic” mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C′′D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C′′D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C′′D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor κB pathway without inducing phosphorylation of either TCRζ or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs

Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

CD4+CD25+ regulatory T cells (T reg cells) play a key role in controlling autoimmunity and inflammation. Therefore, therapeutic agents that are capable of elevating numbers or increasing effector functions of this T cell subset are highly desirable. In a previous report we showed that a superagonistic monoclonal antibody specific for rat CD28 (JJ316) expands and activates T reg cells in vivo and upon short-term in vitro culture. Here we demonstrate that application of very low dosages of the CD28 superagonist into normal Lewis rats is sufficient to induce T reg cell expansion in vivo without the generalized lymphocytosis observed with high dosages of JJ316. Single i.v. administration of a low dose of the CD28 superagonist into Dark Agouti (DA) rats or Lewis rats that suffered from experimental autoimmune encephalomyelitis (EAE) proved to be highly and equally efficacious as high-dose treatment. Finally, we show that T reg cells that were isolated from CD28-treated animals displayed enhanced suppressive activity toward myelin basic protein–specific T cells in vitro, and, upon adoptive transfer, protected recipients from EAE. Our data indicate that this class of CD28-specific monoclonal antibodies targets CD4+CD25+ T reg cells and provides a novel means for the effective treatment of multiple sclerosis and other autoimmune diseases

The realization of autonomous, aircraft-based, real-time aerosol mass spectrometry in the upper troposphere and lower stratosphere

We report on the developments that enabled the field deployment of a fully automated aerosol mass spectrometer, especially designed for high-altitude measurements on unpressurized aircraft. The merits of the two main categories of real-time aerosol mass spectrometry, i.e. (a) single-particle laser desorption and ionization and (b) continuous thermal desorption and electron impact ionization of aerosols, have been integrated into one compact apparatus with the aim to perform in situ real-time analysis of aerosol chemical composition. The demonstrated instrument, named the ERICA (European Research Council Instrument for Chemical composition of Aerosols), operated successfully aboard the high-altitude research aircraft M-55 Geophysica at altitudes up to 20 km while being exposed to ambient conditions of very low atmospheric pressure and temperature. A primary goal of those field deployments was the in situ study of the Asian tropopause aerosol layer (ATAL). During 11 research flights, the instrument operated for more than 49 h and collected chemical composition information of more than 150 000 single particles combined with quantitative chemical composition analysis of aerosol particle ensembles. This paper presents in detail the technical characteristics of the main constituent parts of the instrument, as well as the design considerations for its integration into the aircraft and its autonomous operation in the upper troposphere and lower stratosphere (UTLS). Additionally, system performance data from the first field deployments of the instrument are presented and discussed, together with exemplary mass spectrometry data collected during those flights

Direct binding of the pH-regulated protein 1 (Pra1) from Candida albicans inhibits cytokine secretion by mouse CD4+ T cells

Opportunistic infections with the saprophytic yeast Candida albicans are a major cause of morbidity in immunocompromised patients. While the interaction of cells and molecules of innate immunity with C. albicans has been studied to great depth, comparatively little is known about the modulation of adaptive immunity by C. albicans. In particular, direct interaction of proteins secreted by C. albicans with CD4+ T cells has not been studied in detail. In a first screening approach, we identified the pH-regulated antigen 1 (Pra1) as a molecule capable of directly binding to mouse CD4+ T cells in vitro. Binding of Pra1 to the T cell surface was enhanced by extracellular Zn2+ ions which Pra1 is known to scavenge from the host in order to supply the fungus with Zn2+. In vitro stimulation assays using highly purified mouse CD4+ T cells showed that Pra1 increased proliferation of CD4+ T cells in the presence of plate-bound anti-CD3 monoclonal antibody. In contrast, secretion of effector cytokines such as IFNγ and TNF by CD4+ T cells upon anti-CD3/ anti-CD28 mAb as well as cognate antigen stimulation was reduced in the presence of Pra1. By secreting Pra1 C. albicans, thus, directly modulates and partially controls CD4+ T cell responses as shown in our in vitro assays

Application of an O-ring pinch device as a constant-pressure inlet (CPI) for airborne sampling

We present a novel and compact design of a constant-pressure inlet (CPI) developed for use in airborne aerosol mass spectrometry. In particular, the inlet system is optimized for aerodynamic lenses commonly used in aerosol mass spectrometers, in which efficient focusing of aerosol particles into a vacuum chamber requires a precisely controlled lens pressure, typically of a few hectopascals. The CPI device can also be used in condensation particle counters (CPCs), cloud condensation nucleus counters (CCNCs), and gas-phase sampling instruments across a wide range of altitudes and inlet pressures. The constant pressure is achieved by changing the inner diameter of a properly scaled O-ring that acts as a critical orifice. The CPI control keeps air pressure and thereby mass flow rate (≈0.1 L min-1) upstream of an aerodynamic lens constant, deviating at most by only ±2 % from a preset value. In our setup, a pressure sensor downstream of the O-ring maintains control of the pinch mechanism via a feedback loop and setpoint conditions are reached within seconds. The device was implemented in a few instruments, which were successfully operated on different research aircraft covering a wide range of ambient pressures, from sea level up to about 55 hPa. Details of operation and the quality of aerosol particle transmission were evaluated by laboratory experiments and in-flight data with a single-particle mass spectrometer. © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License

Optimizing the detection, ablation, and ion extraction efficiency of a single-particle laser ablation mass spectrometer for application in environments with low aerosol particle concentrations

The aim of this study is to show how a newly developed aerodynamic lens system (ALS), a delayed ion extraction (DIE), and better electric shielding improve the efficiency of the Aircraft-based Laser ABlation Aerosol MAss spectrometer (ALABAMA). These improvements are applicable to single-particle laser ablation mass spectrometers in general. To characterize the modifications, extensive sizeresolved measurements with spherical polystyrene latex particles (PSL; 150-6000 nm) and cubic sodium chloride particles (NaCl; 400-1700 nm) were performed. Measurements at a fixed ALS position show an improved detectable particle size range of the new ALS compared to the previously used Liu-type ALS, especially for supermicron particles. At a lens pressure of 2.4 hPa, the new ALS achieves a PSL particle size range from 230 to 3240 nm with 50% detection efficiency and between 350 and 2000 nm with 95% detection efficiency. The particle beam divergence was determined by measuring the detection efficiency at variable ALS positions along the laser cross sections and found to be minimal for PSL at about 800 nm. Compared to measurements by singleparticle mass spectrometry (SPMS) instruments using Liutype ALSs, the minimum particle beam divergence is shifted towards larger particle sizes. However, there are no disadvantages compared to the Liu-type lenses for particle sizes down to 200 nm. Improvements achieved by using the DIE and an additional electric shielding could be evaluated by size-resolved measurements of the hit rate, which is the ratio of laser pulses yielding a detectable amount of ions to the total number of emitted laser pulses. In particular, the hit rate for multiply charged particles smaller than 500 nm is significantly improved by preventing an undesired deflection of these particles in the ion extraction field. Moreover, it was found that by using the DIE the ion yield of the ablation, ionization, and ion extraction process could be increased, resulting in up to 7 times higher signal intensities of the cation spectra. The enhanced ion yield results in a larger effective width of the ablation laser beam, which in turn leads to a hit rate of almost 100% for PSL particles in the size range from 350 to 2000 nm. Regarding cubic NaCl particles the modifications of the ALABAMA result in an up to 2 times increased detection efficiency and an up to 5 times increased hit rate. The need for such instrument modifications arises in particular for measurements of particles that are present in low number concentrations such as ice-nucleating particles (INPs) in general, but also aerosol particles at high altitudes or in pristine environments. Especially for these low particle number concentrations, improved efficiencies help to overcome the statistical limitations of single-particle mass spectrometer measurements. As an example, laboratory INP measurements carried out in this study show that the appli- cation of the DIE alone increases the number of INP mass spectra per time unit by a factor of 2 to 3 for the sampled substances. Overall, the combination of instrument modifications presented here resulted in an increased measurement efficiency of the ALABAMA for different particle types and particles shape as well as for highly charged particles. © 2020 Copernicus GmbH. All rights reserved

Professor of Physiology; M. Castell, PhD, Professor of Physiology; C. Pelegrí, PhD, Professor of Physiology

ABSTRACT. Objective. To determine the immunomodulatory effects of the anti-rat CD28 monoclonal antibody (Mab) JJ316 on the onset of rat adjuvant arthritis (AA). JJ316 is a superagonistic Mab that induces polyclonal T cell proliferation in the absence of T cell receptor (TCR) ligation and promotes the expansion of regulatory T cells. Methods. Female Wistar rats in which AA was induced were treated with JJ316 on Day 0 and Day 9 postinduction. A parallel treatment with JJ319, a "conventional" CD28-specific Mab that costimulates anti-TCR triggered proliferation, was performed. Severity of arthritis was monitored by means of an arthritic score, and by recording hindpaw volume and body weight increases. Serum antibodies against the AA-inducing mycobacteria were also determined by ELISA. To ascertain the effect of JJ316 on T lymphocytes in vivo, blood CD4+CD45RC high (Th1-like) and CD4+CD45RC low (Th2-like) cells were analyzed by flow cytometry, and the relative levels of interleukin 2 (IL-2), IL-10, and interferon-γ (IFN-γ) mRNA in synovial tissue were measured by real-time reverse transcriptionpolymerase chain reaction. Results. JJ316 efficiently prevented the inflammatory process of AA. This effect was associated with a specific decrease in the blood CD4+CD45RC high /CD4+CD45RC low T cell ratio and high IL-10 mRNA expression in the synovia. In addition, anti-mycobacteria antibody levels decreased in JJ316 treated animals. In contrast, administration of the conventional anti-CD28 Mab JJ319 did not improve inflammation. Conclusion. JJ316, a stimulatory CD28-specific Mab known to promote Th2 function and the expansion of regulatory T cells, provides effective protection from AA. (J Rheumatol 2006;33:110-8