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.

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.

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 .

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.

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.

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.