Four-Color ImmunoSpot Assays Requiring Only 1-3 mL of Blood Permit Precise Frequency Measurements of Antigen-Specific B Cells-Secreting Immunoglobulins of All Four Classes and Subclasses.

The B lymphocyte response can encompass four immunoglobulin (Ig) classes and four IgG subclasses, each contributing fundamentally different effector functions. Production of the appropriate Ig class/subclass is critical for both successful host defense and avoidance of immunopathology. The assessment of an antigen-specific B cell response, including its magnitude and Ig class/subclass composition, is most often confined to the antibodies present in serum and other biological fluids and neglects monitoring of the memory B cell (B) compartment capable of mounting a faster and more efficient antibody response following antigen reencounter.

Assessing the Affinity Spectrum of the Antigen-Specific B Cell Repertoire via ImmunoSpot.

The affinity distribution of the antigen-specific memory B cell (B) repertoire in the body is a critical variable that defines an individual's ability to rapidly generate high-affinity protective antibody specificities. Detailed measurement of antibody affinity so far has largely been confined to studies of monoclonal antibodies (mAbs) and are laborious since each individual mAb needs to be evaluated in isolation. Here, we introduce two variants of the B cell ImmunoSpot assay that are suitable for simultaneously assessing the affinity distribution of hundreds of individual B cells within a test sample at single-cell resolution using relatively little labor and with high-throughput capacity.

Monitoring Memory B Cells by Next-Generation ImmunoSpot Provides Insights into Humoral Immunity that Measurements of Circulating Antibodies Do Not Reveal.

Memory B cells (B) provide the second wall of adaptive humoral host defense upon specific antigen rechallenge when the first wall, consisting of preformed antibodies originating from a preceding antibody response, fails. This is the case, as recently experienced with SARS-CoV-2 infections and previously with seasonal influenza, when levels of neutralizing antibodies decline or when variant viruses arise that evade such. While in these instances, reinfection can occur, in both scenarios, the rapid engagement of preexisting B into the recall response can still confer immune protection.

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.

Artificial Intelligence-Based Counting Algorithm Enables Accurate and Detailed Analysis of the Broad Spectrum of Spot Morphologies Observed in Antigen-Specific B-Cell ELISPOT and FluoroSpot Assays.

Antigen-specific B-cell ELISPOT and multicolor FluoroSpot assays, in which the membrane-bound antigen itself serves as the capture reagent for the antibodies that B cells secrete, inherently result in a broad range of spot sizes and intensities. The diversity of secretory footprint morphologies reflects the polyclonal nature of the antigen-specific B cell repertoire, with individual antibody-secreting B cells in the test sample differing in their affinity for the antigen, fine epitope specificity, and activation/secretion kinetics. To account for these heterogeneous spot morphologies, and to eliminate the need for setting up subjective counting parameters well-by-well, CTL introduces here its cutting-edge deep learning-based IntelliCount algorithm within the ImmunoSpot Studio Software Suite, which integrates CTL's proprietary deep neural network.

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.

Antibody Levels Poorly Reflect on the Frequency of Memory B Cells Generated following SARS-CoV-2, Seasonal Influenza, or EBV Infection.

The scope of immune monitoring is to define the existence, magnitude, and quality of immune mechanisms operational in a host. In clinical trials and praxis, the assessment of humoral immunity is commonly confined to measurements of serum antibody reactivity without accounting for the memory B cell potential. Relying on fundamentally different mechanisms, however, passive immunity conveyed by pre-existing antibodies needs to be distinguished from active B cell memory.

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.

Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays.

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity.

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.

B-Cell Activity Predicts Response to Glatiramer Acetate and Interferon in Relapsing-Remitting Multiple Sclerosis.

Objective: We investigated the predictive value of the enzyme-linked immunospot technique (ELISPOT) in identifying patients with relapsing-remitting multiple sclerosis (RRMS) who will respond to treatment with glatiramer acetate (GA) or interferon-β (IFN-β), based on the brain-reactive B-cell activity of peripheral blood cells.

Methods: In this retrospective, cross-sectional, real-world multicenter study, we identified patients with RRMS in the NeuroTransData MS registry and stratified them based on their documented treatment response (relapse-free in the first 12 months of treatment) to GA or IFN-β. The GA group comprised 73 patients who responded to GA and 35 nonresponders.

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 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.

AT-RvD1 Mitigates Secondhand Smoke-Exacerbated Pulmonary Inflammation and Restores Secondhand Smoke-Suppressed Antibacterial Immunity.

Cigarette smoke is a potent proinflammatory trigger contributing to acute lung injury and the development of chronic lung diseases via mechanisms that include the impairment of inflammation resolution. We have previously demonstrated that secondhand smoke (SHS) exposure exacerbates bacterial infection-induced pulmonary inflammation and suppresses immune responses. It is now recognized that resolution of inflammation is a bioactive process mediated by lipid-derived specialized proresolving mediators that counterregulate proinflammatory signaling and promote resolution pathways.

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.

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.

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.

IL-21 in Conjunction with Anti-CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring Antigen-Specific Memory B Cells.

Detection of antigen-specific memory B cells for immune monitoring requires their activation, and is commonly accomplished through stimulation with the TLR7/8 agonist R848 and IL-2. To this end, we evaluated whether addition of IL-21 would further enhance this TLR-driven stimulation approach; which it did not. More importantly, as most antigen-specific B cell responses are T cell-driven, we sought to devise a polyclonal B cell stimulation protocol that closely mimics T cell help.

Quantitative HLA-class-II/factor VIII (FVIII) peptidomic variation in dendritic cells correlates with the immunogenic potential of therapeutic FVIII proteins in hemophilia A.

Background: Plasma-derived (pd) or recombinant (r) therapeutic factor VIII proteins (FVIIIs) are infused to arrest/prevent bleeding in patients with hemophilia A (PWHA). However, FVIIIs are neutralized if anti-FVIII-antibodies (inhibitors) develop. Accumulating evidence suggests that pdFVIIIs with von Willebrand factor (VWF) are less immunogenic than rFVIIIs and that distinct rFVIIIs are differentially immunogenic.

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).

Natural T cell autoreactivity to melanoma antigens: clonally expanded melanoma-antigen specific CD8 + memory T cells can be detected in healthy humans.

We used four-color ImmunoSpot® assays, in conjunction with peptide pools that cover the sequence of tyrosinase (Tyr), melanoma-associated antigen A3 (MAGE-A3), melanocyte antigen/melanoma antigen recognized by T cells 1 (Melan-A/MART-1), glycoprotein 100 (gp100), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) to characterize the melanoma antigen (MA)-specific CD8 + cell repertoire in PBMC of 40 healthy human donors (HD). Tyr triggered interferon gamma (IFN-γ)-secreting CD8 + T cells in 25% of HD within 24 h of antigen stimulation ex vivo. MAGE-A3, Melan-A/MART-1, and gp100 also induced recall responses in 10%, 7.

Multi-Color FLUOROSPOT Counting Using ImmunoSpot Fluoro-X™ Suite.

Multi-color FLUOROSPOT assays for simultaneous detection of several T-cell cytokines and/or classes/sub-classes of immunoglobulins secreted by B cells have recently become a major new avenue of development of ELISPOT technology. Advances in assay techniques and the availability of commercial test kits stimulated development of multi-color FLUOROSPOT data analysis platforms. The ImmunoSpot Fluoro-X™ Software Suite was developed by CTL as an integrated data acquisition, analysis, and management solution for automated high-throughput processing of multi-color T- and B-cell FLUOROSPOT assay plates.

Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental Validation of the Multi-Color ImmunoSpot Software Based on Center of Mass Distance Algorithm.

Over the past decade, ELISPOT has become a highly implemented mainstream assay in immunological research, immune monitoring, and vaccine development. Unique single cell resolution along with high throughput potential sets ELISPOT apart from flow cytometry, ELISA, microarray- and bead-based multiplex assays. The necessity to unambiguously identify individual T and B cells that do, or do not co-express certain analytes, including polyfunctional cytokine producing T cells has stimulated the development of multi-color ELISPOT assays.

Detecting all Immunoglobulin Classes and Subclasses in a Multiplex 7 Color ImmunoSpot Assay.

Antibody molecules in peripheral blood have a relatively short half-life of roughly 20 days, and therefore their persistence in the serum depends on continuous replenishment by plasma cells. Serum antibody titers are thus indirect and unreliable indicators of immunological memory. In contrast, memory B cells persist in peripheral blood for decades, and enumerating these cells provides direct evidence of having developed an immune response to a given antigen.

Multiplex ImmunoSpot Assays for the Study of Functional B Cell Subpopulations.

B cells mediate humoral immunity by producing antibody molecules, but they also participate in innate and acquired immune functions via the secretion of effector molecules such as cytokines, chemokines, and granzyme. B cell subpopulations releasing such effector molecules have been implicated in immunobiology and a number of diseases.Unlike antigen-specific T cells that can be identified by multimer staining, and then counter-stained to define T cell subpopulations, antigen-specific B cells cannot be detected by flow cytometry.