Immunotyping of natural killer (NK) cells in COVID-19 patients using high-parameter flow cytometry

SARS-CoV-2 infection elicits a spectrum of immune responses, activating both the innate and adaptive immune systems. Whether these immune responses are targeted and controlled or misdirected and uncontrolled could determine the outcome of the disease. Recent studies using deep immunoprofiling have indicated that the nature of immune responses can also determine the severity of the disease. For example, the failure to launch appropriate interferon responses is correlated with increased levels of IL-6 and the subsequent occurrence of cytokine storm and the severity of COVID-19 disease. Unlike acquired immune responses, the innate immune response toward SARS-CoV-2 infection has not been studied in detail.

NK cells and their role in acute viral infections

As part of the innate immune system, NK cells have the ability to attack and destroy infected cells and can be involved in influencing T cell-mediated adaptive immune responses. Two main NK cell populations are recognized: CD56^bright NK cells (cytokine producing) and CD56^dim NK cells (cytotoxic). Based on a critical threshold level of NK cells, the immune response could be protective or trigger immunopathology. NK cells have been shown to respond to various types of viral infections swiftly. Compared to other immune cells, the involvement and nature of NK cells in COVID-19 disease has not been addressed in detail.

Maucourant et al., in their publication “Natural killer cell immunophenotypes related to COVID-19 disease severity,” use high-parameter flow cytometry and unsupervised analysis to understand the involvement of NK cells in SARS-CoV-2 infection response and if there is a correlation between NK cell activity and COVID-19 disease severity. They analyzed the peripheral blood mononuclear cells of two groups of patients—one with moderate (n = 10) and the other with severe (n = 17) COVID-19 disease—and one control group of SARS-CoV-2 IgG seronegative and symptom-free individuals (n = 17).

Their study revealed several key patterns and details of NK cell activation in COVID-19 disease:

• An analysis using a 28-color NK cell-focused flow cytometry panel revealed that NK cells were highly activated in COVID-19 patients compared to healthy controls. However, the level of activation was not directly correlated with disease severity. Analysis of CD56^bright and CD56^dim NK cells showed that the absolute counts of these cells and of the total NK cells was reduced in COVID-19 patients compared to controls.
• Principal component analysis showed distinct clusters of patient populations compared to controls, marked by changes in expression of several markers associated with NK cell populations (such as CD98, Ki-67, Ksp37 in CD56^bright NK cells and Tim-3, CD98, CD38, CD69 and Ksp37 in CD56^dim NK cells).
• There was a difference in the expression of markers between responding CD56^bright and non-responding CD56^bright cells, as well as between responding and non-responding CD56^dim NK cells.
• Single-cell RNA sequencing and gene ontology enrichment analysis of differentially expressed genes showed distinct gene modules between patient and control populations with several parameters, such as effector functions, activation, proliferation, and interferon response, exhibiting increased expression in patient populations
  compared to controls.
• Analysis of the bronchoalveolar lavage fluid showed an activated and inflamed profile with respect to interferon response, activation/proliferation signatures
  and differential expression.
• Inhibitory receptor expression and NK cell education analysis revealed that NK cell activation seen in patients was independent of inhibitory KIR expression and
  NK cell education.
• Higher frequencies of adaptive NK cells (e.g., NKG2C+CD57+CD56^dim NK cells) were found in severe COVID-19 cases compared to moderate patients. These were proliferation and activation markers, and this adaptive NK cell expansion did not seem to be associated with any CMV reactivation in patients.
• The paper shows an increased adaptive NK cell expansion in COVID-19, for the first time.
• Flow cytometry analysis using an unsupervised approach with FlowJo™ v10.6.2 Software and FlowAI, DownSample, Uniform Manifold Approximation and Projection (UMAP) and PhenoGraph plugins showed different clusters of NK cell populations. PhenoGraph clustering and stratification based on distinct clinical categories revealed 36 clusters with distinct relative abundance of markers that can be associated with disease phenotypes.
• The relative abundance of PhenoGraph clusters revealed two distinct immunotypes—moderate and severe immunotypes.
• Analysis of moderate versus severe immunotypes showed that markers such as MIP-1β, CD98 and TIGIT were upregulated in moderate immunotypes while others such as perforin, NKG2C and Ksp37 were elevated in the severe immunotypes.
• A more detailed analysis of NK cell responses revealed that the expression levels of perforin and granzyme B expression in CD56^bright cells positively correlated with more severe COVID-19 patient phenotype, including sequential organ failure assessment and ongoing SARS-CoV-2 viremia in serum. In addition, upregulation of these markers in CD56^bright cells was also positively correlated with increased levels of several effector molecules and negatively correlated with inhibitory check-point molecules
  such as TIGIT.
• Analysis of soluble serum proteins from COVID-19 patients revealed that several of these were associated with perforin and granzyme B expression in CD56^bright cells. Several soluble factors were associated with other viral infections as well, indicating that NK cell activation pathways may be shared among various viral infection response pathways.
  

These results provide a detailed landscape of early NK cell responses in COVID-19 patients and an understanding of how immune responses change with disease progression.

The significance of the correlation of adaptive NK cell expansion and arming of CD56^bright cells with disease severity should be further investigated with respect to increased levels of pro-inflammatory cytokines observed in patients. The relationship with other innate immune responses involving monocytes and neutrophils also should be examined further to get a detailed understanding of innate immune responses during SARS-CoV-2 infection.

Read the paper for further details.