The recent unexpected emergence of the COVID-19 pandemic has spurred significant interest in an improved
understanding of immunological memory to SARS-CoV-2, which consists of humoral (neutralizing antibodies)
and cellular (T and B cells) memory. The generation of immunological memory to the SARS-CoV-2 virus critically
depends on T cell responses. During a viral infection, CD4 helper T cells differentiate largely into either Th1 cells
that orchestrate a type I antiviral immune response or follicular helper (Tfh) cells that enhance antibody
production. CD8 T cells clonally expand and acquire effector function to directly kill virus-infected cells. Despite
the heterogeneity and clonal diversity of virus-specific T cells, the majority of effector T cells die after viral
clearance and only a small portion of them develop into memory T cells that provide long-lasting protection for
the host. Similarly, B cells develop into memory B cells and long-lived plasma cells that produce neutralizing
antibodies. Snapshot observations with multi-parameter flow cytometry-based assays along the course of
infection has yielded abundant knowledge of T cell phenotypic and functional diversity. However, approaches as
such fail to address the developmental trajectory of virus-specific T cells. This becomes more obvious in human
studies given that lineage tracing by genetic alterations or adoptive transfer experiments, done easily in mice,
are inherently difficult or impossible in humans. In this proposal, we will first combine newly developed single-
cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (TCR-seq) techniques on the same cells and
use TCR sequences as natural barcodes to directly “lineage trace” each patient’s SARS-CoV-2-specific CD4
and CD8 T cell effector response and memory formation at the single-cell level throughout the course of natural
infection. Furthermore, we will perform gene regulatory network (GRN) analysis to delineate which transcription
factors collaboratively regulate virus-specific CD4 and CD8 T cell differentiation trajectories. Next, we will
measure T cell clonal diversity and the quality of T cell memory from COVID-19 patients as well as healthy
human controls. The latter will be used to gauge the possible presence of pre-existing immunity (PEI) in the form
of memory T cells derived from cross-reactivity to common coronaviruses. These measurements will use high-
throughput RNA-seq of TCR amplicons and scRNA-seq of memory T cells from recall cultures as inputs for
computational TCR motif analysis. Lastly, successful vaccine development relies on an advanced understanding
of the types of Tfh cells that are generated during natural infection and how they interact with B cells as well as
T regulatory cells for anti-SARS-CoV-2 antibody production in humans. To this end, we propose to monitor
circulating Tfh cells, including three major populations (Th1-, Th2- and Th17-like subsets), and T follicular
regulatory (Tfr) cells in both SARS-Cov-2-infected and healthy human control subjects. In addition, we will
perform T cell-B cell coculture assays to dissect the functional contributions of each subset of Tfh cells and how
they interact with Tfr cells in regulating anti-SARS-CoV-2 neutralizing antibody responses.