Lehmann, A. A. | Search bioRxiv

bioRxiv2020

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 (PBMC) 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 inclusion of positive controls that actually evaluate the performance of the antigen-specific T cell and antigen presenting cell (APC) compartments. CEF peptides have been commonly used to this extent. Here we show that CEF peptides fail as a positive control in nearly half of test subjects. Moreover, CEF peptides only measure CD8+ T cell functionality. More reliable alternatives for the assessment of CD8+ T cells are introduced here, as well as positive controls for the CD4+ T cell and APC compartments. In sum, we offer new tools and strategies for the assessment of PBMC functional fitness required for reliable T cell immune monitoring.

immunology↗

bioRxiv2020

Discordance between the predicted vs. the actually recognized CD8+ T cell epitopes of HCMV pp65 antigen and aleatory epitope dominance

CD8+ T cell immune monitoring aims at measuring the numbers and functions of antigen-specific CD8+ T cell populations engaged during immune responses, providing insights into the magnitude and quality of cell-mediated immunity operational in a test subject. The selection of peptides for ex vivo CD8+ T cell detection is critical, however, because for each restricting HLA class I molecule present in a human individual there is a multitude of potential epitopes within complex antigens, and HLA diversity between the test subjects predisposes CD8+ T cell responses to individualized epitope recognition profiles. We report here on a brute force CD8+ T cell epitope mapping approach for the human cytomegalovirus (HCMV) pp65 antigen on ten HLA-A*02:01-matched HCMV infected human subjects. In this approach, in each test subject, every possible CD8+ T cell epitope was systematically tested; that is 553 individual peptides that walk the sequence of the HCMV pp65 protein in steps of single amino acids. Highly individualized CD8+ T cell response profiles with aleatory epitope recognition patterns were observed. We compared the actually detected epitope utilization in each individual with epitope prediction ranking for the shared HLA-A*02:01 allele, and for additional HLA class I alleles expressed by each individual. No correlation was found between epitopes ranking on the prediction scale and their actual immune dominance. The data suggest that accurate CD8+ T cell immune monitoring might depend on the agnostic reliance on mega peptide pools, or brute force mapping, rather than individualized epitope predictions.

immunology↗