Scientists are slowly gaining insights into how respiratory viruses can provoke an inflammatory immune response in the lungs, which could lead to lung damage. A new bioRxiv* preprint paper reveals in detail the changes that occur in peripheral blood natural killer (NK) and T cells in coronavirus disease 2019 (COVID-19). This could help develop new treatment strategies in such infections.
Acute respiratory viral infections cause an increase in chemokines, such as CCL2, CCL3, CXCL1, CXCL3, and IL8, which cause leukocyte recruitment into the lungs. These chemicals attract more cells that have receptors like CCR2, CCR5 and CXCR6 on their surfaces.
Severe lung parenchymal damage and epithelial cell death can result from the hyper-inflammatory reaction occurring in severe COVID-19. The researchers in the current paper examined the involvement of cytotoxic lymphocytes homing to the lung in acute infection. They used flow cytometry as well as transcript analysis using single cell RNA sequencing datasets of cells from peripheral blood and bronchoalveolar lavage fluid.
They concluded that in influenza and in COVID-19, CXCR3 mediates the homing of NK and T cells to the lungs, aided by CXCR6 and CCR5.
Increased NK and T cells with chemokine receptors
The researchers found that all NK and T cells in peripheral blood had chemokine receptors involved in lung homing, whether in healthy individuals or COVID-19 patients. These were less frequent in COVID-19 patients, however. The specific changes were an increase in CCR5+ NK cells and T cells, with a sharp reduction in CXCR2+, CXCR3+, CXCR6+, and CCR2+ cells.
The changes in NK cell chemokine receptor expression were non-significant in both diseases, unlike the large changes in CD8 T cells in both. CXCR3+ and CXCR6+CD8+ T cells were steeply reduced in both conditions, and in influenza, respectively. The fall in CXCR3+ was reflected in NK cells as well, but the change did not attain significance.
The pattern of expression of these chemokine receptors was altered in naïve and exposed T cells of both CD4 and CD8 subtypes, as well as in TEM, TCM and TEMRA CD8 T cells. However, NK cells showed distinct differences in CXCR3 and CCR5 expression between healthy individuals and influenza patients. With influenza vs COVID-19, T cells showed differences primarily in CCR5 (for CD4 cells) and CCR2, CXCR3, and CXCR6 on CD8 cells.
Comparison with other scRNA-seq datasets showed a difference in mRNA expression of chemokine receptors between the circulating NK and T cells. CXCR2 was found mostly on NK cells, but CCR2 and CCR5 in T cells, whether in COVID-19 or in healthy individuals. CXCR3 was lower in NK and T cells in COVID-19 patients, however.
At receptor level, strangely, T cells showed a robust increase in CXCR6, CCR2, and CCR5 expression in COVID-19 patients, perhaps due to post-transcriptional regulation. Influenza patients showed a greater fall in CXCR2, CXCR3 and CXCR6 expression on NK cells than COVID-19 patients did, and CCR5 levels appeared to be higher. Thus, homing factors may be differentially active in these two viral respiratory infections.
Overall, CXCR3 appears to be a common receptor recruiting both NK and T cells to the lungs during COVID-19 or influenza.
Activation markers in NK cells
The researchers also showed that circulating NK cells in moderate COVID-19 had higher activation markers such as CD69, CD38, Ki67, and NKG2D. Among NK cells, CD56brightCD16- and CD56dimCD16+NK cells that expressed lung homing receptors also showed differential induction of CD69 and Ki67, respectively.
The former subset of chemokine receptor-bearing NK cells showed induction of effector enzymes, granzymes and perforin, but perforin expression was threefold higher in patients with influenza compared to those with COVID-19. Conversely, the latter subset of NK cells in influenza patients showed the greatest increase in CD69 expression, whether or not lung homing receptors were also present.
CXCR2 expression was higher on CD56dimCD16+ NK cells that did not have other lung-homing receptors, but since these cells showed a pronounced decrease in COVID-19 patients, relative to healthy individuals, there may be another mechanism of lung homing in these patients.
Activation profiles of NK cells are thus quite different for COVID-19 and influenza, and are stronger in the former, but there were no other phenotypic differences.
Activation markers in T cells
Cytotoxic effector molecules in T cells occurred mostly in cytotoxic CD8 T cells, and some on CD4 T cells. As with NK cells, healthy individuals, as well as COVID-19 and influenza patients, all had the highest levels of granzyme B and perforin in chemokine receptor-negative CD8+ T cells.
CD69 was more often increased in both CD4 and CD8 T cells, in both conditions. However, it was more robustly increased in CD8 T cells bearing lung-homing receptors in COVID-19, but more uniformly, though to a higher level, in influenza, affecting all T cells. CD38 expression was more strongly increased on CD8 T cells in influenza but not COVID-19.
Ki67 was most strongly increased in chemokine receptor-positive CD8+ T cells, as with NK cells bearing CD56dimCD16+ chemokine receptors. This suggests that cytotoxic lymphocytes are especially activated to express lung-homing receptors in COVID-19.
Overall, therefore, both NK and T cells are more strongly activated in influenza vs moderate COVID-19, with cytotoxic lymphocytes expressing lung homing receptors.
BALF shows build-up of activated NK and T cells
In the BALF, activated NK and T cells bearing both CXCR3 and CXCR6 were found to accumulate. Patients with moderate COVID-19 showed very high expression of CXCR3, CXCR6, and CCR5, in both NK and T cells. The researchers attribute the loss of NK cells and T cells expressing lung-homing receptors such as the above, from the circulation, to their migration into the infected lung. Of course, some of these BALF cells may be tissue-resident memory T cells and NK cells.
When cells with high effector molecule concentrations are accumulated in the lung, severe damage is likely. In contrast, the blood NK cells are actually less active in patients with moderate COVID-19. Further research will show if the lung-homing NK cells are still functional, causing infected cells to be destroyed, and further destruction of adjacent tissue. This may contribute to the lung lesions of moderate to severe COVID-19.
What are the implications?
The study thus shows that in both influenza and COVID-19 patients, CXCR3 and CXCR6 are overexpressed in CD8+ T cells, while both NK cells and T cells are activated. In addition, the level of mRNA encoding these lung-homing receptors is lower in influenza patients compared to moderate COVID-19.
The activated effector cells are more likely to express lung homing receptors as well. The BALF in COVID-19 patients have higher levels of leukocyte-attracting chemokines, such as IL-8, CCL5, and CXCL1. Monocytes that bind CXCR3 are expanded in the lung tissue of such patients
The common recruitment of NK and T cells by these lung-homing receptors supports a similar mechanism operating in both influenza and COVID-19, which may help unravel the underlying single pathogenesis of both these conditions. “In addition to CCR5 and CXCR3, CXCR6 has been suggested to be of importance for recruitment of resident memory T cells to the airways both in mice and in moderate COVID-19.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.