Reagent Tracker™ Platform Verifies and Provides Audit Trails for the Error-Free Implementation of T-Cell ImmunoSpot® Assays.

ELISPOT and FluoroSpot assays, collectively called ImmunoSpot assays, permit to reliable detection of rare antigen-specific T cells in freshly isolated cell material, such as peripheral blood mononuclear cells (PBMC). Establishing their frequency within all PBMC permits to assess the magnitude of antigen-specific T-cell immunity; the simultaneous measurement of their cytokine signatures reveals these T-cells' lineage and effector functions, that is, the quality of T-cell-mediated immunity. Because of their unparalleled sensitivity, ease of implementation, robustness, and frugality in PBMC utilization, T-cell ImmunoSpot assays are increasingly becoming part of the standard immune monitoring repertoire. For regulated workflows, stringent audit trails of the data generated are a requirement. While this has been fully accomplished for the analysis of T-cell ImmunoSpot assay results, such are missing for the wet laboratory implementation of the actual test performed. Here we introduce a solution for enhancing and verifying the error-free implementation of T-cell ImmunoSpot assays.

Unbiased, High-Throughput Identification of T Cell Epitopes by ELISPOT.

Recent systematic immune monitoring efforts suggest that, in humans, epitope recognition by T cells is far more complex than has been assumed based on minimalistic murine models. The increased complexity is due to the higher number of HLA loci in humans, the typical heterozygosity for these loci in the outbred population, and the high number of peptides that each HLA restriction element can bind with an affinity that suffices for antigen presentation. The sizable array of potential epitopes on any given antigen is due to each individual's unique HLA allele makeup. Of this individualized potential epitope space, chance events occurring in the course of the T cell response determine which epitopes induce dominant T cell expansions. Establishing the actually-engaged T cell repertoire in each human subject, including the individualized peptides targeted, therefore requires the systematic testing of all peptides that constitute the potential epitope space in that person. The goal of comprehensive, high-throughput epitope mapping can be readily established by the methods described in this chapter.

A push-pull strategy to suppress stable fly (Diptera: Muscidae) attacks on pasture cattle via a coconut oil fatty acid repellent formulation and traps with m-cresol lures.

BACKGROUND: Stable flies [Stomoxys calcitrans (L.)] are economically important pests of cattle and other livestock. As an alternative to conventional insecticides, we tested a push-pull management strategy using a coconut oil fatty acid repellent formulation and an attractant-added stable fly trap. RESULTS: In our field trials we found that weekly applications of a push-pull strategy can reduce stable fly populations on cattle as well as a standard insecticide (permethrin). We also found that the efficacy periods of the push-pull and permethrin treatments following on-animal application were equivalent. Traps with an attractant lure used as the pull component of the push-pull strategy captured sufficient numbers of stable flies to reduce on-animal numbers by an estimated 17-21%. CONCLUSIONS: This is the first proof-of-concept field trial demonstrating the effectiveness of a push-pull strategy using a coconut oil fatty acid-based repellent formulation and traps with an attractant lure to manage stable flies on pasture cattle. Also notable is that the push-pull strategy had an efficacy period equivalent to that of a standard, conventional insecticide under field conditions. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Prediction of B cell epitopes in proteins using a novel sequence similarity-based method.

Prediction of B cell epitopes that can replace the antigen for antibody production and detection is of great interest for research and the biotech industry. Here, we developed a novel BLAST-based method to predict linear B cell epitopes. To that end, we generated a BLAST-formatted database upon a dataset of 62,730 known linear B cell epitope sequences and considered as a B cell epitope any peptide sequence producing ungapped BLAST hits to this database with identity ≥ 80% and length ≥ 8. We examined B cell epitope predictions by this method in tenfold cross-validations in which we considered various types of non-B cell epitopes, including 62,730 peptide sequences with verified negative B cell assays. As a result, we obtained values of accuracy, specificity and sensitivity of 72.54 ± 0.27%, 81.59 ± 0.37% and 63.49 ± 0.43%, respectively. In an independent dataset incorporating 503 B cell epitopes, this method reached accuracy, specificity and sensitivity of 74.85%, 99.20% and 50.50%, respectively, outperforming state-of-the-art methods to predict linear B cell epitopes. We implemented this BLAST-based approach to predict B cell epitopes at http://imath.med.ucm.es/bepiblast .

Development and first evaluation of an attractant impregnated adhesive tape against blood-sucking flies.

Stable flies are one of the most important arthropod pests of livestock that reduce cattle weight gain and milk production leading to annual economic losses in excess of $2 billion to the US cattle industry. The host-seeking behavior is primarily mediated by associated odors from stable fly larval development environments and host animals. The present paper reports the development and evaluation of attractant-impregnated adhesive tapes to reduce stable fly attacks on cattle. Laboratory bioassays showed that only m-cresol impregnated adhesive tapes caught significantly more stable flies (16 ± 1) than the control tape without attractant added (7 ± 1), with a 77% fly recapture rate. Attractant-impregnated adhesive tapes deployed in cattle feedlots showed significant impacts in reducing fly population, with a total of one million stable flies captured over a period of three weeks (mean catches from 57 596 to 102 088 stable flies per trap per week). It further relieved cattle stress with a significant reduction of biting fly avoidance behavior, (6 ± 0.4 cows observed with tail wagging in control vs. 3 ± 0.4 from the trap-deployed). The efficacy of the developed tapes lasted up to 1-week longevity, although 70% of m-cresol was released starting from the second day. The m-cresol impregnated adhesive tape provided an 80% reduction in cattle stress due to stable fly attack. This is the first report of a technology developed by integrating an attractant compound into an adhesive material on a plastic film with demonstrated effectiveness in trapping biting flies that attack livestock animals.

Deconvoluting the T Cell Response to SARS-CoV-2: Specificity Versus Chance and Cognate Cross-Reactivity.

SARS-CoV-2 infection takes a mild or clinically inapparent course in the majority of humans who contract this virus. After such individuals have cleared the virus, only the detection of SARS-CoV-2-specific immunological memory can reveal the exposure, and hopefully the establishment of immune protection. With most viral infections, the presence of specific serum antibodies has provided a reliable biomarker for the exposure to the virus of interest. SARS-CoV-2 infection, however, does not reliably induce a durable antibody response, especially in sub-clinically infected individuals. Consequently, it is plausible for a recently infected individual to yield a false negative result within only a few months after exposure. Immunodiagnostic attention has therefore shifted to studies of specific T cell memory to SARS-CoV-2. Most reports published so far agree that a T cell response is engaged during SARS-CoV-2 infection, but they also state that in 20-81% of SARS-CoV-2-unexposed individuals, T cells respond to SARS-CoV-2 antigens (mega peptide pools), allegedly due to T cell cross-reactivity with Common Cold coronaviruses (CCC), or other antigens. Here we show that, by introducing irrelevant mega peptide pools as negative controls to account for chance cross-reactivity, and by establishing the antigen dose-response characteristic of the T cells, one can clearly discern between cognate T cell memory induced by SARS-CoV-2 infection vs. cross-reactive T cell responses in individuals who have not been infected with SARS-CoV-2.

Functional T Cell Reactivity to Melanocyte Antigens Is Lost during the Progression of Malignant Melanoma, but Is Restored by Immunization.

Healthy human subjects develop spontaneous CD8+ T cell responses to melanoma associated antigens (MA) expressed by normal melanocytes, such as Tyrosinase, MAGE-A3, Melan/Mart-1, gp100, and NY-ESO-1. This natural autoimmunity directed against melanocytes might confer protection against the development of malignant melanoma (MM), where MA are present as overexpressed tumor-associated antigens. Consistent with this notion we report here that functional T cell reactivity to MA was found to be significantly diminished to MAGE-A3, Melan-A/Mart-1, and gp100 in untreated MM patients. Three lines of evidence suggest that the MA-reactive T cells present in healthy subjects undergo exhaustion once MM establishes itself. First, only the MA-specific T cell reactivity was affected in the MM patients; that to third party recall antigens was not. Second, in these patients, the residual MA-specific T cells, unlike third party antigen reactive T cells, were functionally impaired, showing a diminished per cell IFN-γ productivity. Third, we show that immunization with MA restored natural CD8+ T cell autoimmunity to MA in 85% of the MM patients. The role of natural T cell autoimmunity to tumor-associated MA is discussed based on discrete levels of T cell activation thresholds.

CERI, CEFX, and CPI: Largely Improved Positive Controls for Testing Antigen-Specific T Cell Function in PBMC Compared to CEF.

Monitoring antigen-specific T cell immunity relies on functional tests that require T cells and antigen presenting cells to be uncompromised. Drawing of blood, its storage and shipment from the clinical site to the test laboratory, and the subsequent isolation, cryopreservation and thawing of peripheral blood mononuclear cells (PBMCs) before the actual test is performed can introduce numerous variables that may jeopardize the results. Therefore, no T cell test is valid without assessing the functional fitness of the PBMC being utilized. This can only be accomplished through the inclusion of positive controls that actually evaluate the performance of the antigen-specific T cell and antigen presenting cell (APC) compartments. For Caucasians, CEF peptides have been commonly used to this extent. Moreover, CEF peptides only measure CD8 cell functionality. We introduce here universal CD8+ T cell positive controls without any racial bias, as well as positive controls for the CD4+ T cell and APC compartments. In summary, we offer new tools and strategies for the assessment of PBMC functional fitness required for reliable T cell immune monitoring.

Discordance Between the Predicted Versus the Actually Recognized CD8+ T Cell Epitopes of HCMV pp65 Antigen and Aleatory Epitope Dominance.

CD8+ T cell immune monitoring aims at measuring the size and functions of antigen-specific CD8+ T cell populations, thereby providing insights into cell-mediated immunity operational in a test subject. The selection of peptides for ex vivo CD8+ T cell detection is critical because within a complex antigen exists a multitude of potential epitopes that can be presented by HLA class I molecules. Further complicating this task, there is HLA class I polygenism and polymorphism which predisposes CD8+ T cell responses towards individualized epitope recognition profiles. In this study, we compare the actual CD8+ T cell recognition of a well-characterized model antigen, human cytomegalovirus (HCMV) pp65 protein, with its anticipated epitope coverage. Due to the abundance of experimentally defined HLA-A*02:01-restricted pp65 epitopes, and because in silico epitope predictions are most advanced for HLA-A*02:01, we elected to focus on subjects expressing this allele. In each test subject, every possible CD8+ T cell epitope was systematically covered testing 553 individual peptides that walk the sequence of pp65 in steps of single amino acids. Highly individualized CD8+ T cell response profiles with aleatory epitope recognition patterns were observed. No correlation was found between epitopes' ranking on the prediction scale and their actual immune dominance. Collectively, these data suggest that accurate CD8+ T cell immune monitoring may necessitate reliance on agnostic mega peptide pools, or brute force mapping, rather than electing individual peptides as representative epitopes for tetramer and other multimer labeling of surface antigen receptors.

Aleatory Epitope Recognition Prevails in Human T Cell Responses?

Eli Sercarz pioneered epitope recognition by T cells. Studying mice, he made the seminal observation decades ago that epitope dominance is so unpredictable with mixed MHC haplotypes that he coined it aleatory, for dice-like. Accordingly, for every individual there is a unique potential epitope space that is defined by the polymorphic and polygenic MHC molecules (restriction elements) expressed. Of this potential epitope space, some peptides will elicit stronger T cell responses than others, bringing about the actually realized epitope space. The selection of the actually recognized peptides from the potential epitope space is random, however, resulting in unique epitope dominance and hierarchy patterns in individuals. Engaging in brute-force epitope scans, which permit the assessment of the entire potential epitope space at the highest possible resolution, we observe aleatory epitope recognition in human CD8 cell responses to viruses. Because the selection of peptide has fundamental implications for successful T cell immune monitoring, we dedicate this article to Eli Sercarz in a special issue of Critical Reviews™ in Immunology in his honor.

Spatial repellency, antifeedant activity and toxicity of three medium chain fatty acids and their methyl esters of coconut fatty acid against stable flies.

BACKGROUND: Stable flies are one of the most detrimental arthropod pests to livestock. With changing climates and agronomic practices, they expand their roles as pests and disease vectors as well. Their painful bites reduce livestock productivity, annoy companion animals, and interfere with human recreational activities. Current management technologies are unable to effectively control stable flies. The present study reports new results concerning the contact, spatial repellency, and toxicity of a bio-based product, coconut fatty acid and their methyl ester derivatives of free fatty acids of C8:0 , C10:0 and C12:0 to stable flies. RESULTS: Three medium chain fatty acid methyl esters (C8:0 , C10:0 and C12:0 ) showed strong antifeedant activity against stable flies and their strengths were dose-dependent. Only the C8:0 acid, C8:0 - and C10:0 methyl esters elicited significant antennal responses. Laboratory single cage olfactometer bioassays revealed that coconut fatty acid and C8:0 methyl ester displayed active spatial repellency. All three methyl esters showed strong toxicity against stable flies. CONCLUSION: Antifeedant activity is the main method through which coconut fatty acid deters stable fly blood-feeding. The C8:0 , C10:0 and C12:0 methyl esters act not only as strong antifeedants, but also possess strong toxicity against stable fly adults. Limited spatial repellency was observed from coconut fatty acid and C8:0 methyl ester. © 2019 Society of Chemical Industry.

Comprehensive Evaluation of the Expressed CD8+ T Cell Epitope Space Using High-Throughput Epitope Mapping.

T cell immunity is traditionally assessed through functional recall assays, which detect the consequences of the T cells' antigen encounter, or via fluorescently labeled multimers that selectively bind peptide-specific T cell receptors. Using either approach, if the wrong antigen or peptide of a complex antigenic system, such as a virus, is used for immune monitoring, either false negative data will be obtained, or the magnitude of the antigen-specific T cell compartment will go largely underestimated. In this work, we show how selection of the "right" antigen or antigenic peptides is critical for successful T cell immune monitoring against human cytomegalovirus (HCMV). Specifically, we demonstrate that individual HCMV antigens, along with previously reported epitopes, frequently failed to detect CD8+ T cell immunity in test subjects. Through systematic assessment of T cell reactivity against individual nonamer peptides derived from the HCMVpp65 protein, our data clearly establish that (i) systematic testing against all potential epitopes encoded by the genome of the antigen of interest is required to reliably detect CD8+ T cell immunity, and (ii) genome-wide, large scale systematic testing of peptides has become feasible through high-throughput ELISPOT-based "brute force" epitope mapping.

Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify.

It is essential to identify donors who have not been infected with human cytomegalovirus (HCMV) in order to avoid transmission of HCMV to recipients of blood transfusions or organ transplants. In the present study, we tested the reliability of seronegativity as an indicator for the lack of HCMV exposure in healthy human blood donors. Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes. Eighty-two percent (67 of 82) of these HCMV seronegative individuals featured at least one memory cell that was lineage specific for HCMV, with the majority of these subjects possessing CD4+ and CD8+ T cells, as well as B cells, providing three independent lines of evidence for having developed immunity to HCMV. Only 15 of these 82 donors (18%) showed neither T- nor B-cell memory to HCMV, consistent with immunological naïveté to the virus. The data suggest that measurements of serum antibodies frequently fail to reveal HCMV exposure in humans, which may be better identified by direct detection of HCMV-specific memory lymphocytes.

High-Throughput GLP-Capable Target Cell Visualization Assay for Measuring Cell-Mediated Cytotoxicity.

One of the primary effector functions of immune cells is the killing of virus-infected or malignant cells in the body. Natural killer (NK) and CD8 effector T cells are specialized for this function. The gold standard for measuring such cell-mediated cytolysis has been the chromium release assay, in which the leakage of the radioactive isotope from damaged target cells is being detected. Flow cytometry-based single cell analysis of target cells has recently been established as a non-radioactive alternative. Here we introduce a target cell visualization assay (TVA) that applies similar target cell staining approaches as used in flow cytometry but based on single cell computer image analysis. Two versions of TVA are described here. In one, the decrease in numbers of calcein-stained, i.e., viable, target cells is assessed. In the other, the CFSE/PI TVA, the increase in numbers of dead target cells is established in addition. TVA assays are shown to operate with the same sensitivity as standard chromium release assays, and, leaving data audit trails in form of scanned (raw), analyzed, and quality-controlled images, thus meeting requirements for measuring cell-mediated cytolysis in a regulated environment.

Reagent Tracker Dyes Permit Quality Control for Verifying Plating Accuracy in ELISPOT Tests.

ELISPOT assays enable the detection of the frequency of antigen-specific T cells in the blood by measuring the secretion of cytokines, or combinations of cytokines, in response to antigenic challenges of a defined population of PBMC. As such, these assays are suited to establish the magnitude and quality of T cell immunity in infectious, allergic, autoimmune and transplant settings, as well as for measurements of anti-tumor immunity. The simplicity, robustness, cost-effectiveness and scalability of ELISPOT renders it suitable for regulated immune monitoring. In response to the regulatory requirements of clinical and pre-clinical immune monitoring trials, tamper-proof audit trails have been introduced to all steps of ELISPOT analysis: from capturing the raw images of assay wells and counting of spots, to all subsequent quality control steps involved in count verification. A major shortcoming of ELISPOT and other related cellular assays is presently the lack of audit trails for the wet laboratory part of the assay, in particular, the assurance that no pipetting errors have occurred during the plating of antigens and cells. Here, we introduce a dye-based reagent tracking platform that fills this gap, thereby increasing the transparency and documentation of ELISPOT test results.

Spatio-Temporal Characteristics of Inhibition Mapped by Optical Stimulation in Mouse Olfactory Bulb.

Mitral and tufted cells (MTCs) of the mammalian olfactory bulb are connected via dendrodendritic synapses with inhibitory interneurons in the external plexiform layer. The range, spatial layout, and temporal properties of inhibitory interactions between MTCs mediated by inhibitory interneurons remain unclear. Therefore, we tested for inhibitory interactions using an optogenetic approach. We optically stimulated MTCs expressing channelrhodopsin-2 in transgenic mice, while recording from individual MTCs in juxtacellular or whole-cell configuration in vivo. We used a spatial noise stimulus for mapping interactions between MTCs belonging to different glomeruli in the dorsal bulb. Analyzing firing responses of MTCs to the stimulus, we did not find robust lateral inhibitory effects that were spatially specific. However, analysis of sub-threshold changes in the membrane potential revealed evidence for inhibitory interactions between MTCs that belong to different glomerular units. These lateral inhibitory effects were short-lived and spatially specific. MTC response maps showed hyperpolarizing effects radially extending over more than five glomerular diameters. The inhibitory maps exhibited non-symmetrical yet distance-dependent characteristics.

Amyloid beta oligomers (A beta(1-42) globulomer) suppress spontaneous synaptic activity by inhibition of P/Q-type calcium currents.

Abnormal accumulation of soluble oligomers of amyloid beta (Abeta) is believed to cause malfunctioning of neurons in Alzheimer's disease. It has been shown that Abeta oligomers impair synaptic plasticity, thereby altering the ability of the neuron to store information. We examined the underlying cellular mechanism of Abeta oligomer-induced synaptic modifications by using a recently described stable oligomeric Abeta preparation called "Abeta(1-42) globulomer." Synthetically prepared Abeta(1-42) globulomer has been shown to localize to neurons and impairs long-term potentiation (Barghorn et al., 2005). Here, we demonstrate that Abeta(1-42) globulomer does not affect intrinsic neuronal properties, as assessed by measuring input resistance and discharge characteristics, excluding an unspecific alteration of membrane properties. We provide evidence that Abeta(1-42) globulomer, at concentrations as low as 8 nM, specifically suppresses spontaneous synaptic activity resulting from a reduction of vesicular release at terminals of both GABAergic and glutamatergic synapses. EPSCs and IPSCs were primarily unaffected. A detailed search for the precise molecular target of Abeta(1-42) globulomer revealed a specific inhibition of presynaptic P/Q calcium currents, whereas other voltage-activated calcium currents remained unaltered. Because intact P/Q calcium currents are needed for synaptic plasticity, the disruption of such currents by Abeta(1-42) globulomer may cause deficits in cellular mechanisms of information storage in brains of Alzheimer's disease patients. The inhibitory effect of Abeta(1-42) globulomer on synaptic vesicle release could be reversed by roscovitine, a specific enhancer of P/Q currents. Selective enhancement of the P/Q calcium current may provide a promising strategy in the treatment of Alzheimer's disease.