Project summary:
Sepsis is prevalent, costly, and deadly. In the U.S, sepsis accounts for 4% of hospitalizations, 13% of in-hospital
healthcare expenditures, and 35% of in-hospital deaths. Although common, sepsis is often difficult to diagnose
and treat effectively, since it is a syndrome defined generically by organ dysfunction resulting from a dysregulated
immune response to infection. This broad definition leads to heterogeneity of disease and misdiagnosis. Clinical
trials thus enroll ill-characterized populations of sepsis patients with variable underlying immune responses to
infection, diluting the effects of novel and otherwise promising therapies that might benefit a defined subset of
patients. Precision immune cell-specific characterization of the dysregulated host immune response in sepsis is
clearly needed. Our group was the first to elucidate an immunosuppressive monocyte substate (MS1) expanded
in patients with urosepsis (Reyes et al. Nat Med 2020) using single cell RNA sequencing (scRNA-seq), which
resolves cellular heterogeneity, revealing rich signatures of immune cell-specific gene expression not evident
from standard immune profiling techniques. Clinical investigation in sepsis would greatly benefit from the scalable
deployment of scRNA-seq across multicenter studies and clinical trials to enable robust characterization of the
host immune response in sepsis, and to explain the effect of disease heterogeneity on clinical course, outcomes,
and treatment effects. Yet in order to facilitate subsequent scRNA-seq, immune cells must be isolated from whole
blood samples, which currently involves complex real-time processing of fresh blood at clinical sites, a process
neither practical nor reproducible enough to allow deployment across multiple clinical centers.
To address this critical need, we piloted a simple method of onsite whole blood cryopreservation that uses only
2 mL of blood and is easily deployable at clinical sites, followed by storage for scRNA-seq at a centralized center
of expertise. In the R21 phase, we will optimize and validate our method of whole blood cryopreservation on
enrolled subjects at 2 local clinical sites versus gold standard methods for immune cell isolation. We will compare
scRNA-seq technical quality metrics and biologically-relevant measures including the MS1 monocyte subtype
and other immune cell states. Importantly, we will test the viability and function of cryopreserved cells in
mechanistic studies. In the R33 phase, we will scale our whole blood cryopreservation method to 5 enrolling
clinical sites around the U.S. to demonstrate feasibility of a multicenter collection with centralized scRNA-seq
and analysis. In this expanded sepsis cohort, we will perform deep immune profiling and derive scRNA-seq-
based endotypes to characterize underlying heterogeneity of host immune responses. We will compare these
endotypes to those derived from bulk RNA sequencing and apply scRNA-seq-derived signatures to our own
clinical trials data that have obtained bulk RNA sequencing. The proposed studies are highly likely to make
available a simple method to facilitate single-cell immune profiling in multicenter studies to greatly enhance our
understanding of the host immune response in sepsis and guide the development of targeted therapies.
