The MegaPool Approach to Characterize Adaptive CD4+ and CD8+ T Cell Responses

Abstract

Epitopes recognized by T cells are a collection of short peptide fragments derived from specific antigens or proteins. Immunological research to study T cell responses is hindered by the extreme degree of heterogeneity of epitope targets, which are usually derived from multiple antigens; within a given antigen, hundreds of different T cell epitopes can be recognized, differing from one individual to the next because T cell epitope recognition is restricted by the epitopes’ ability to bind to MHC molecules, which are extremely polymorphic in different individuals. Testing large pools encompassing hundreds of peptides is technically challenging because of logistical considerations regarding solvent‐induced toxicity. To address this issue, we developed the MegaPool (MP) approach based on sequential lyophilization of large numbers of peptides that can be used in a variety of assays to measure T cell responses, including ELISPOT, intracellular cytokine staining, and activation‐induced marker assays, and that has been validated in the study of infectious diseases, allergies, and autoimmunity. Here, we describe the procedures for generating and testing MPs, starting with peptide synthesis and lyophilization, as well as a step‐by‐step guide and recommendations for their handling and experimental usage. Overall, the MP approach is a powerful strategy for studying T cell responses and understanding the immune system's role in health and disease. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Generation of peptide pools (“MegaPools”)Basic Protocol 2: MegaPool testing and quantitation of antigen‐specific T cell responses

Frequency of Dengue Virus-Specific T Cells is related to Infection Outcome in Endemic Settings

“…DENV specific CD4 + T cell memory responses were evaluated following a 24h stimulation of peripheral blood mononuclear cells (PBMCs) with DENV CD4 megapools (MP) and using the Activation Induced Marker Assay (AIM Assay) as readout. Our laboratory has developed the MP approach to study the breadth of T cell responses to diverse pathogens [17]. In previous studies, HLA class I and HLA class II-restricted DENV epitopes spanning the entire sequence of the DENV viruses were predicted and subsequently experimentally tested in different human cohorts from endemic regions [10, 18, 19].…”

“…The MegaPool approach was previously established by Sidney et al [34] and recently detailed protocols were made public R. da Silva Antunes et al [17] allowing the simultaneous assessment of numerous T cells from diverse specificity. Briefly, this method consists of the dissolution of a diverse array of epitopes, their consolidation into a combined pool, and subsequent joint lyophilization to avoid cell cytotoxicity by a larger amount of DMSO.…”

“…We analyzed T cell responses using flow cytometry, evaluating the phenotypes and frequencies of CD4 + and CD8 + T cells individually, and examining relationships with the outcome of subsequent DENV infection. Importantly, we were able to test our question using a substantial sample size (n=71), together with accurate characterization of infection history, information about the HLA type of the donors, and our established DENV peptide MegaPool approach (MP) to asses DENV specific T cell responses [17]. We hereby provide a comprehensive assessment of T cell memory in multiple DENV infections in an endemic setting.…”

PINK1 is a target of T cell responses in Parkinson’s disease

“…Overlapping and predicted class II peptides were combined to form antigen pools for all the respective antigens tested. Our lab routinely identifies CD4 and CD8 T cell epitopes, as well as uses existing data in the Immune Epitope Database and Analysis Resource (IEDB(66)) to develop peptide “megapools” (67). A detailed protocol for making megapools is found in the open-access publication by da Silva Antunes et al (67) The utilization of these megapools allows the ability to test a large number of epitopes spanning multiple HLA-types.…”

“…Our lab routinely identifies CD4 and CD8 T cell epitopes, as well as uses existing data in the Immune Epitope Database and Analysis Resource (IEDB(66)) to develop peptide “megapools” (67). A detailed protocol for making megapools is found in the open-access publication by da Silva Antunes et al (67) The utilization of these megapools allows the ability to test a large number of epitopes spanning multiple HLA-types. Specific sequence identities and reference numbers making up the various antigen pools used in this study can be found in Supplemental Table 2.…”