ハイパラメーターフローサイトメトリーを用いたCOVID-19患者のナチュラルキラー（NK）細胞のイムノタイピング

SARS-CoV-2感染は、さまざまな免疫応答を誘発し、自然免疫系と獲得免疫系の両方を活性化します。 これらの免疫反応が標的にされて制御されているのか、誤った方向に向けられて制御されていないのかによって、病気の結果が決まる可能性があります。免疫プロファイリングを使用した最近の研究では、免疫反応の性質が病気の重症度を決定する可能性があることが示されています。 たとえば、適切なインターフェロン応答を開始できないことは、IL-6のレベルの上昇と、それに続くサイトカインストームの発生およびCOVID-19疾患の重症度と相関しています。 後天性免疫反応とは異なり、SARS-CoV-2感染に対する自然免疫反応は詳細に研究されていません。

NK細胞と急性ウイルス感染におけるそれらの役割

自然免疫系の一部として、NK細胞は感染細胞を攻撃して破壊する能力があり、T細胞を介した適応免疫応答への影響に関与する可能性があります。CD56^bright NK cells（サイトカイン産生）とCD56^dim NK cells（細胞毒性）の2つの主要なNK細胞集団が認識されています。NK細胞の臨界閾値レベルに基づいて、免疫反応は防御的であるか、免疫病理学を引き起こす可能性があります。NK細胞は、さまざまな種類のウイルス感染に迅速に反応することが示されています。他の免疫細胞と比較して、COVID-19疾患におけるNK細胞の関与と性質は詳細に扱われていません。

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 CD56bright and CD56dim 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 CD56bright NK cells and Tim-3, CD98, CD38, CD69 and Ksp37 in CD56dim NK cells).
• There was a difference in the expression of markers between responding CD56bright and non-responding CD56bright cells, as well as between responding and non-responding CD56dim 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+CD56dim 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 CD56bright 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 CD56bright 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 CD56bright 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 CD56bright 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.