Researchers at the La Jolla Institute for Immunology cite using Excellos sourced healthy donor starting material to carry out single-cell immune profiling for their research on severe COVID-19. 
Great strides have been made in fighting the SARS-CoV-2 virus, the most noteworthy being the development and distribution of the COVID-19 vaccine. However, we must keep in mind that many people around the world still suffer and die from COVID, particularly when new variants arise. Therefore, it is important that scientists gain a clearer understanding of immune cell function during infection and learn exactly which immune cells or functional subtypes are associated with critical disease and hospitalization.
La Jolla Institute researchers were specifically interested in examining changes in monocyte and dendritic cell populations because these immune cells have been shown to exhibit defective behavior in patients with severe COVID-19.  To put things in perspective, 8 distinct monocyte subsets have been identified in healthy humans, and each type can contribute to disease progression. And while monocytes and dendritic cells are known to be associated with COVID-19 disease progression, scientists are currently lacking any detailed analyses of the specific function of these immune cells in COVID-19 patients.
To conduct their research, the scientists obtained immune cell starting material from healthy volunteers at the San Diego Blood Bank/Excellos, as well as volunteer donations from both hospitalized and non-hospitalized COVID-19 patients. Excellos provides cellular starting material from donors who are extensively characterized and annotated in the donor network database. PBMCs were isolated from donor starting material via density centrifugation.
Following PBMC isolation, leukocytes were labeled using CD45-based barcoding, in preparation for CyTOF analysis. CyTOF is a technique that combines mass spectrometry with single-cell labeling. It allows researchers to quantify labeled target proteins on the surface or interior of single cells, and by extension, to identify differentially expressed protein markers between cell samples. Live CD45-barcoded cells were stained with an established monocyte/T cell panel. Healthy CD45-barcoded PBMCs were then spiked into each COVID-19 sample as an internal control.
To perform single-cell immune profiling on cell populations in COVID-19 patient groups, the scientists used CyTOF data to look for changes in protein expression and in immune cell population frequencies. Lineage markers were used to identify major cell types, while other markers were used to link cells to their primary function, for example, CD9 and CD11 for adhesion, CD73 for migration, and so forth.
Changes in immune cell surface marker cluster frequency and expression were analyzed to look for statistically significant shifts between healthy, non-hospitalized COVID-19 positive, and hospitalized COVID-19 positive subjects.
Single-cell immune profiling reveals remarkable differences in cell surface expression.
The scientists discovered some remarkable differences in cell surface protein expression. Expression of CD9 and CD45RA significantly increased in hospitalized subjects. CD9 mediates cellular adhesion, is associated with cellular senescence, and is known to aggravate atherosclerotic plaque formation. CD45RA is considered a marker for monocyte activation. Hospitalized subjects also displayed significantly increased expression of various monocyte subtypes, CD3+ cells, CD56+ T cells, and CD14+ immature monocyte clusters compared with non-hospitalized subjects and a decrease in the frequency of non-classical monocytes compared to healthy subjects.
While a complete analysis of shifts in cell subtype and function is beyond the bounds of this discussion, one finding does bear mention. CD9-expressing monocytes remained elevated in the blood of hospitalized patients, even 3-4 months post SARS-CoV-2 infection. The CD9+ monocyte subtype strongly correlates with disease severity and progression. This and other data confirm that SARS-CoV-2 results in long-term changes in single-cell immune profiles. The data gathered in this study thus provides a new perspective from which to examine the symptoms experienced by patients with long-COVID-19.
Single-cell immune profiling and other advanced characterization techniques provide unique insight into predicting cellular functionality, disease impact, and therapeutic potential. While this study demonstrates the utility of downstream immune cell profiling techniques, one could easily imagine the benefits of upstream profiling, where highly characterized donors and cellular starting material are used to guide and optimize therapeutic development.
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- Pandori, W. J., et al. (2022). Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9+ monocytes associated with COVID-19 hospitalization. Journal of leukocyte biology, 10.1002/JLB.4COVA0122-076R. Advanced online publication. https://doi.org/10.1002/JLB.4COVA0122-076R
- Schulte-Schrepping, J., et al. (2020). Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment. Cell, 182(6), 1419–1440.e23. https://doi.org/10.1016/j.cell.2020.08.001