Novel cell types implicated in COVID-19 cytokine storm: Findings from the Single Cell Consortium for COVID-19 in China
Researchers from 39 institutes and hospitals across China have come together to deeply profile the immune response to SARS-CoV-2 infection. A testament to the potential of scientific collaboration in enabling large-cohort studies, they collectively performed single cell RNA-sequencing on 284 samples from 196 COVID-19 patients and controls, totaling 1.46 million cells.
Explore their findings regarding the influence of population demographics on the immune landscape, surprising evidence of immune cell susceptibility to SARS-CoV-2 infection, and new cellular culprits of the cytokine storm. Plus, find out how 10x Genomics scientists played a part in enabling the success of this team effort, from connecting labs in the midst of a global pandemic to providing guidance and reagents.
Scaling single cell studies for a big-picture view of COVID-19 immunity
Though scientists have been making strides to understand the biological features that distinguish disease severity in COVID-19 patients, there are certain variables, including age or sex, that must be taken into account for a more systematic understanding of how the immune system responds across all patients. Informing clinical observations with detailed cellular and molecular findings from the bench can drive more personalized, effective treatments. But to do so requires scale—larger patient cohorts, more samples and cells, and pooled data from which significant insights can be derived.
With this challenge in mind, a team of researchers from China set out on an incredible experimental adventure to profile the immune landscape in the lung and peripheral blood of hundreds of COVID-19 patients, which has recently culminated in a publication in Cell, “COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.” Hospitals, medical universities, laboratories, and research institutions across China compose this diverse group, called the Single Cell Consortium for COVID-19 in China, 39-strong and led by primary investigator, Dr. Zemin Zhang of Peking University, Beijing.
The breadth of this study necessitated that all hands were on deck, including the 10x Genomics field scientists and regional marketers involved in supporting the study. In this blog, we share the unique, insider perspective from one 10x-er, Tony Jiang, PhD, Country Manager, who partnered with the Single Cell Consortium for COVID-19 in China. Hear his take on the possibilities of multi-site collaboration and what it takes to get started, and explore the team’s novel insights into the immunobiology of SARS-CoV-2 infection.
Setting up the consortium: From sample collection to single cell data
As Country Manager for China, Tony Jiang works closely with 10x Genomics customers to support their research. “One of my favorite things about my job is getting to share our product portfolio with customers in China and seeing the great publications they produce using our products.”
He tells us that establishing the Single Cell Consortium for COVID-19 in China was a fast-moving project. “I worked with Professor Zemin Zhang to initiate this consortium project in April 2020, in the midst of the global pandemic. In one month’s time, we connected all single cell research labs, and worked with them to set up this consortium.” Part of this 10x-led coordination effort entailed organizing project meetings, establishing action plans, preparing answers to address collaborators’ concerns, and providing safety guidance for working with infectious agents.
Then came the sample collection. 171 patients, ages 6–92, ranging from mild symptoms, to hospitalized with severe disease, to convalescent. 284 samples, including peripheral blood mononuclear cells (PBMCs), bronchoalveolar lavage fluid (BALF), and sputum. 1,462,702 single cells.
“When the scientists needed consumables for their urgent samples and data collection, we provided the support just in time,” said Jiang, who works with distributor teams in China to supply reagents and other components of 10x Genomics kits to researchers. For this project, the consortium leveraged Chromium Single Cell Immune Profiling, an assay that allowed them to analyze whole transcriptome gene expression from individual cells, as well as paired, full-length B-cell and T-cell receptor (BCR/TCR) sequences for a subset of their samples.
Leveraging this single cell RNA-sequencing platform, the team derived an immune mega-dataset, which was processed in a computing center at Peking University. This step in the experimental process, according to Jiang, represents one of the major possibilities of multi-site collaboration, as well as an essential factor for large-scale studies that integrate a breadth of samples—final data consolidation for streamlined processing and analysis. As Jiang notes,
“This is the first COVID-19 single cell mega-data paper from China. It demonstrates incredible leadership and team work by these scientists and ultimately represents China, not just an individual lab.”
Immune changes associated with demographics and disease severity
The discoveries that the research team made about the immune features of SARS-CoV-2 infection also speak to the potential of large-scale single cell studies. With access to a breadth of patient samples across demographic factors and disease stages, and a comprehensive readout of cellular phenotypes, researchers noted different peripheral immune subtype changes associated with distinct clinical features. For example, they observed an increase in plasma B cells in severe disease, as well as an increase in megakaryocytes and CD14+ monocytes in PBMCs from severe COVID-19 patients during the disease progression stage. In contrast, T cells and dendritic cells decreased.
They also observed that age and sex were associated with key compositional changes in peripheral immune populations. For example, they noted a greater diversity of T-cell subsets in females than males, and in younger COVID-19 patients than older (1). These findings may have important clinical implications as scientists seek to understand the wide spectrum of patient responses to infection.
Unexpectedly infected: Immune cells show presence of viral RNA
Transcriptional profiling of immune populations from a subset of severe, disease-progressing COVID-19 patient BALF and sputum samples also revealed unexpected patterns of cellular infection. Specifically, they detected viral RNA in cells from ciliated, secretory, and squamous epithelial cells and a diverse set of immune cells, including neutrophils, macrophages, plasma B cells, T cells, and NK cells. Surprisingly, immune cells hosted more viral RNA sequences than epithelial cells, though immune cells do not express typical infection-mediating surface molecules like ACE2 and TMPRSS2. The team noted a correlation between BSG and TFRC and the abundance of viral RNA in various cell types, however the mystery of how immune cells are infected by SARS-CoV-2 is still unfolding.
And this is one mystery that needs to be solved, as single cell data from this study shows that viral infection of immune cells leads to significant transcriptomic changes in those populations when compared to matched, uninfected cells. For example, they observed elevated expression of interferon-stimulated genes, which are typically associated with viral RNA sensing, in SARS-CoV-2-positive immune cells (1).
New culprits of the cytokine storm
With a big-picture view of immune activity across COVID-19 disease stages, the team next looked to uncover possible culprits of the cytokine storm, a dangerous inflammatory feature of severe disease. To assess cellular sources of cytokine production, they first assigned a cytokine score and inflammatory score to each cell based on its expression of cytokine genes and reported inflammatory response genes, respectively. This narrowed their search to seven cellular subtypes, including three subtypes of monocytes and one subtype of megakaryocytes, with higher detected scores in the scRNA-seq data.
Next, looking at the proportion of these hyper-inflammatory subtypes across patients and disease stages, they found that one monocyte subtype and megakaryocytes had heightened cell ratios in severe COVID-19 patients, indicating they may be major sources of the cytokine inflammatory storm in PBMCs. Moreover, single cell data showed a diverse spread of cytokine gene expression in each cell subtype, including TNF, CCL3, IL1B, CXCL8, IL6, TGFB1, LTB, and IFNG (1). This may suggest a number of diverse mechanisms are in play to drive the inflammation characteristic of the cytokine storm.
As the scientific community continues to decipher the complex, multicellular immune response to COVID-19—including the underlying cellular mechanisms of SARS-CoV-2 infection and systemic inflammation—this rich data source and experimental model represent clear paths toward deeper insights and potential therapeutic interventions.
To explore more details of this study, review the publication here →
Supporting collaboration to advance large-scale single cell studies
With his experience working behind the scenes to ensure the success of this large-scale COVID-19 study, 10x-er Tony Jiang offered some tips for researchers interested in starting their own single cell collaborations. Preparation early on seems to be the key to success: “Before your alliance, standardize and optimize sample collection. It would also be crucial to decide on the technology in advance, whether you want to leverage a 3’ or 5’ single cell RNA-sequencing assay.” Next, Jiang says, you’ll need to set action plans with your collaborators. “Establish how you will do final data consolidation and how to participate in the final paper writing together.”
We’re privileged at 10x Genomics to be a scientific partner to your research efforts and can provide support throughout the process, as exemplified by the Single Cell Consortium for COVID-19 in China. With more crucial work needed to understand the immunobiology of SARS-CoV-2 infection, not to mention many other areas of health and disease, the potential of large-scale single cell atlasing studies is just beginning to be explored.
Find more 10x Genomics applications for infectious disease and COVID-19 research with these resources, and please contact us with any questions about your experiments or initiating a collaborative project.
References:
- Ren X, et al. COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas. Cell 184:1895–1913, 2021.