SARS-CoV-2 infection and mRNA vaccination both elicit spike (S)-specific T cell responses. To analyze how T cell memory from prior infection influences T cell responses to vaccination, we evaluated functional T cell responses in naive and previously infected vaccine recipients. Pre-vaccine S-specific responses are predictive of subsequent CD8+ T cell vaccine-response magnitudes. Comparing baseline with post-vaccination TCRβ repertoires, we observed large clonotypic expansions correlated with the frequency of spike-specific T cells. Epitope mapping the largest CD8+ T cell responses confirms that an HLA-A∗03:01 epitope was highly immunodominant. Peptide-MHC tetramer staining together with mass cytometry and single-cell sequencing permit detailed phenotyping and clonotypic tracking of these S-specific CD8+ T cells. Our results demonstrate that infection-induced S-specific CD8+ T cell memory plays a significant role in shaping the magnitude and clonal composition of the circulating T cell repertoire after vaccination, with mRNA vaccination promoting CD8+ memory T cells to a TEMRA-like phenotype.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021).
© 2021 The Authors.

Ending the COVID-19 pandemic will require long-lived immunity to SARS-CoV-2. Here, we evaluate 254 COVID-19 patients longitudinally up to 8 months and find durable broad-based immune responses. SARS-CoV-2 spike binding and neutralizing antibodies exhibit a bi-phasic decay with an extended half-life of >200 days suggesting the generation of longer-lived plasma cells. SARS-CoV-2 infection also boosts antibody titers to SARS-CoV-1 and common betacoronaviruses. In addition, spike-specific IgG+ memory B cells persist, which bodes well for a rapid antibody response upon virus re-exposure or vaccination. Virus-specific CD4+ and CD8+ T cells are polyfunctional and maintained with an estimated half-life of 200 days. Interestingly, CD4+ T cell responses equally target several SARS-CoV-2 proteins, whereas the CD8+ T cell responses preferentially target the nucleoprotein, highlighting the potential importance of including the nucleoprotein in future vaccines. Taken together, these results suggest that broad and effective immunity may persist long-term in recovered COVID-19 patients.
© 2021 The Authors.

Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMC up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states.
© 2021 The Author(s).

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.
© 2021, Swanson et al.