Flow cytometry is a traditional method used by researchers to detect and measure the physical and chemical characteristics of individual cells. By labeling cells with fluorescent molecules designed to bind to cell surface proteins, intracellular molecules, or even nucleic acids, flow cytometers funnel single cells past lasers to detect fluorescent labels and classify cells. While this method enables researchers to measure surface phenotypes of cells, it is limited in identifying critical cell attributes such as cell function and the ability to highly multiplex the proteins that are carrying out these functions.
IsoPlexis’ functional proteomics can help overcome these limitations by allowing for the simultaneous identification of up to 30+ functional proteins (eg. Cytokines, chemokines, and growth factors), giving researchers a more detailed understanding of how cells are orchestrating immune response. This additional data layer can be used alongside flow cytometry to characterize immune cells and responses.
A Broader View of Cytokine Secretion Profiles
While SARS-CoV-2 vaccines have been able to mitigate COVID-19 over time, the emergence of breakthrough infections in vaccinated individuals indicate that individual immune responses to vaccines may differ. Furthermore, researchers are unsure of how effective vaccines targeting COVID-19 spike proteins are against variants that have mutated spike protein sequences. With these limitations in traditional methods, recent studies have shown that multi-parametric platforms alone are insufficient to identify key variants driving COVID-19 response. With IsoPlexis’ functional multi-omic platform, researchers are able to identify the drivers of response through the isolation of cytokine secretion profiles, functionally allowing them to overcome these challenges and develop a broader view of COVID-19 immune response.
In a recently published study in JCI Insights, researchers assessed vaccine-induced SARS-CoV-2 T cell response in 21 healthy, vaccinated individuals with no history of prior COVID-19 infections. All participants had T cells specific to SARS-CoV-2, but the range of response varied between individuals. To determine why people had variable responses, the team looked at differentiation states and cytokine profiles to identify immune features associated with superior recall function and longevity. They were able to identify SARS-CoV-2 specific CD4+ T cells that were polyfunctional (able to secrete multiple cytokines simultaneously) and produced high levels of IL-2, which could be associated with superior longevity.
“Using multiparametric flow cytometry, we identified TNF-α and IL-2 producing CD4+ T cells as the hallmark of vaccine-induced SARS-CoV-2-specific T cell response. However, we were limited to interrogate the expression of three cytokines only per single cell using flow cytometry,” the authors wrote.
To overcome these constraints and better understand cytokine secretion, researchers used IsoPlexis’ highly multiplexed proteomic platform to measure cytokines secreted by SARS-CoV-2 specific T cells. Cell culture supernatant of PBMCs from donors whose T cells had high and low responses when stimulated by spike protein peptides were probed for 22 different cytokines. The team found that IL-2 secretion was only detected in the high responder group, which was consistent with the flow cytometry data. These T cells primarily secreted Th1 cytokines and not Th2 or Th17-prone cytokines, demonstrating that vaccine-induced CD4+ T cells are predominantly Th1 cells. The researchers also measured high levels of cytokines associated with monocytes (including IP-10, MCP-1, IL-8, MIP-1a, and MIP1b) after stimulation with spike protein peptides, revealing a role of monocytes in immune response to COVID-19 as well. The researchers calculated cytokine scores that showed that T cells from high responders secreted more cytokines and a more diverse array of cytokines, correlating with a higher number of vaccine-induced SARS-CoV-2-specific T cells observed with flow cytometry.
Studies Reveal Data Identifying Multiple Facets of COVID-19 Response
IsoPlexis’ functional proteomics has been used in multiple other COVID-19 studies, including two studies published in Cell, allowing researchers to clarify the mechanisms of disease pathogenesis and immune response in SARS-CoV-2.
In the first study, IsoPlexis’ functional immune landscaping demonstrated a marked upregulation of polyfunctional cells with a variety of inflammatory cytokine secretions in both CD4+ and CD8+ T cells as well as in monocytes from patients with COVID-19 infection compared to healthy subjects. IsoPlexis’ single-cell proteomics showed that the polyfunctional strength index, or PSI, of CD4+ T cells, CD8+ T cells and monocytes varied with disease severity and progression. Furthermore, only monocyte PSI was correlated with disease severity in moderate to severe COVID-19 across all time points. These findings suggest that these highly polyfunctional “supervillain” monocytes may contribute to inflammatory processes in moderate to severe COVID-19 cases, improving on current understanding of COVID-19 progression and response.
In the second paper published in Cell, researchers performed a multi-omic study of COVID-19 patients from initial diagnosis to early recovery to better understand post-acute sequelae of COVID-19 (PASC), also known as Long Covid, and its causes. Using IsoPlexis’ technology, the team was able to clarify the immune drivers of PASC.
IsoPlexis’ single-cell proteomics revealed a correlation between PASC endotypes and the increased frequency of “supervillain” T cell subsets, “supervillain” monocytes, or “resting” NK cells. These cells were also associated with disease severity in convalescent patients, indicating the impact of T cells and monocytes on sustained inflammation at convalescence. Functional proteomics at a single-cell level was able to provide a unique assessment dissecting the functional impacts of different cell types across multiple timepoints and the interplay between innate and adaptive immune responses that contributed to effector functions or inflammation in Long Covid.
Single-Cell and Bulk Analyses Play Key Role in COVID-19 Research
IsoPlexis’ multiplexed proteomics has helped researchers better understand multiple facets of COVID-19 response, from risk factors to immunity. Together, these studies identify cells and pathways driving the immune response to COVID-19, uncovering mechanisms of infection and immunity. IsoPlexis’ platform can help accelerate the discovery of immune biomarkers that are critical in the fight against COVID-19 and other infectious diseases, helping researchers develop effective vaccines and therapeutics faster.