Adenosine-related small molecules show utility of recall antigen assay to screen compounds for off-target effects on memory T cells.

Extracellular adenosine suppresses T cell immunity in the tumor microenvironment and in vitro treatment of memory T cells with adenosine can suppress antigen-mediated memory T cell expansion. We describe utilizing the recall antigen assay platform to screen small molecule drug off-target effects on memory T cell expansion/function using a dosing regimen based on adenosine treatment. As a proof of principle, we show low dose GS-5734, a monophosphoramidate prodrug of an adenosine analog, does not alter memory T cell recall at lower doses whereas toxicity observed at high dose favors antigen-specific memory T cell survival/proliferation over non-specific CD8+ T cells. Conversely, parent nucleoside GS-441524 at high dosage does not result in cellular toxicity and reduces antigen-specific T cell recall in most donors. Despite similar chemical structure, these drugs displayed opposing effects on memory T cell expansion and viability highlighting the sensitivity of this assay setup in screening compounds for off-target effects.

Assessment of SARS-CoV-2 specific CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers.

The success of SARS-CoV-2 (CoV-2) vaccines is measured by their ability to mount immune memory responses that are long-lasting. To achieve this goal, it is important to identify surrogates of immune protection, namely, CoV-2 MHC Class I and II immunodominant pieces/epitopes and methodologies to measure them. Here, we present results of flow cytometry-based MHC Class I and II QuickSwitchTM platforms for assessing SARS-CoV-2 peptide binding affinities to various human alleles as well as the H-2 Kb mouse allele. Multiple SARS-CoV-2 potential MHC binders were screened and validated by QuickSwitch testing. The screen included 31 MHC Class I and 19 MHC Class II peptides predicted to be good binders by the IEDB web resource provided by NIAID. While several predicted peptides with acceptable theoretical Kd showed poor MHC occupancies, fourteen MHC class II and three MHC class I peptides showed promiscuity in that they bind to multiple MHC molecule types. In addition to providing important data towards the study of the SARS-CoV-2 virus and its presented antigenic epitopes, the peptides identified in this study can be used in the QuickSwitch platform to generate MHC tetramers. With those tetramers, scientists can assess CD4 + and CD8 + immune responses to these different MHC/peptide complexes.