The coronavirus disease 2019 (COVID-19) pandemic has claimed over 2.67 million lives in less than a year and a half. However, it is uncommon in children, with less than 2% of the cases being in individuals below the age of 19. Most of these are of mild severity, with the clinical features of severe COVID-19 being rare in this age group.

A new and timely study, which was released on the medRxiv* preprint server, explores the differences in the response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in adults vs. children to help understand how the disease develops and progresses.

The virus binds to the host cell surface protein, angiotensin-converting enzyme (ACE) 2, to accomplish cell entry. Several, but not all, studies indicate an age- and tissue-dependent expression of ACE2, which drives disease severity in children.

The current study aims to delineate the immune landscape in the blood and upper airway from early life through to adulthood. During this period, immune development occurs from the immune tolerant state in fetal life to a pro-inflammatory state later on, as the individual is exposed to antigens and pathogens.

The researchers used multi-omics data from single-cell data sets in 30 healthy children, derived from nasal and tracheal brushings and peripheral blood mononuclear cells (PBMCs), to define normal developmental changes in multiple cell types. The cells came from the airway (42) and blood (31), and the population covered ages from childhood to adolescence.

They also profiled cells from severe pediatric and adult COVID-19 patients and compared these results with the reference data.

Airway epithelial cells in healthy children

The researchers found 15 different cell types, mostly in two groups. One was the pathway of differentiation from basal to secretory cells, including basal, secretory, goblet 1 and 2, and squamous cells. The second was the ciliated cluster.

Epithelial cell types remained constant in composition with age. However, goblet 2 and secretory cells were considerably more frequent in the nasal epithelium compared to the trachea or bronchi.

Novel cell type

The scientists also found a new type of transitional epithelial cell in between secretory and ciliated phenotypes, in children with and without COVID-19. This is among the cell types that show marked antiviral profile, along with neutrophil recruitment. These were termed inflammatory epithelial transit cells (IETCs), being high in inflammatory markers, but also expressing ciliated cell markers and secretory genes.

These cells were found mostly in COVID-19 patients but also in healthy children, which may indicate their emergence both during development and also during wound healing.

The IETCs are probably a separate pathway for differentiation to replace dying ciliated cells. This accounts for the reduction in the main stem cell population, namely, basal I cells. These have been observed to recover their numbers in convalescent patients.

A neutrophil recruitment signature was observed in IETCs, including markers of serum amyloid protein and calprotectin, both of which are expressed in injury and inflammation. The latter is associated with mild COVID-19 as against severe disease.

Calprotectin is seen mostly in myeloid cells, but in the current study, it was found to be expressed in epithelial cells from the nose of an adult COVID-19 patient. This emphasizes how epithelial cells are essential in initiating the innate immune response.

Airway cells in COVID-19

In COVID-19 patients, IETCs, goblet 2 and ciliated cells were the most highly enriched and showed the highest viral reads.

This corresponds to the highest levels of ACE2 and TMPRSS2 expression in these cells in healthy individuals. The researchers postulate that these cells are increased because of a higher rate of proliferation to replace their dying predecessors as a result of infection. This phenomenon has been seen in the lower airways following infection.

Basal cells were reduced. In adults, but not in children or convalescents, acute COVID-19 was associated with higher ACE2 expression.

Other possible viral entry receptors include NRP1, BSG and TFRC, but these failed to show association with actively infected cells from individuals with high viral reads.

Other cells with viral reads included lymphocytes and macrophages, but it is unclear if this reflects true infection or phagocytosis. No viral reads were seen in PBMCs in the COVID-19 patients.

Immune blood cells in healthy children

Across age groups, the researchers found that the profile of naïve lymphocytes showed a development in their gene expression profile. A number of genes expressed indicated increased proliferation capacity, and specialized immune functions, perhaps with increased cytotoxicity.

Both naïve T and B cells have therefore already entered the bloodstream by the first year of life, and continue to develop to their mature state.

Dendritic cells, Tregs and naïve B cells increased in proportion from newborns to infants, but reduced thereafter. Adaptive immune cells like CD8+ cytotoxic T lymphocytes (CTL), plasma cells and gamma delta (g/d) T cells were all high in preschoolers, perhaps because they are exposed to higher antigen exposure.

Non-switched and switched B cells, as well as exhausted cells, were most frequent in the youngest age group, but towards the other extreme, in adolescence, mucosal-associated invariant T (MAIT) cells were increased.

Naïve cells began to decrease with age and were very low in those over 65 years, reflecting ongoing stimulation over a lifetime of exposure.

Expanded lymphocyte clones in adults

In the adult, T and B cell clones, including different T cell subtypes, were expanded, but not in children, and especially in COVID-19 patients. This clonal expansion indicates a memory cell adaptive immune response.

Immune blood cells with COVID-19

The largest change in the blood in children with COVID-19 was an increase in naïve B and T cells, regulatory T cells (Tregs) and innate lymphoid cells (ILCs). This occurred despite a constant mean lymphocyte count. This confirms earlier reports that lymphopenia is less common in pediatric COVID-19.

It could be due to the release of large numbers of naïve and immature cells from the thymus and bone marrow or the migration of mature cells to the site of infection. These trends were reversed in convalescent children.

At the same time, natural killer (NK) cells and monocytes were lost in children, again, perhaps, because of migration to the infection site.

Airway cells in COVID-19 and with age

The scientists again saw several changes in healthy children with age. This included a rapid decrease in neutrophils, activated macrophages and monocytes, from the neonatal period onwards. CD8 T effector cells, γ/δ cells and MAIT cells were high throughout childhood.

Adolescents did not show the same degree of variation in airway cells with age.

With COVID-19, children with extensive immune infiltration had markedly increased B cell subtypes, follicular dendritic cells and naïve T cells, all of which belong to an acute immune response.

Lack of correlation between airway and blood immune cells

The researchers also found that most cell types in blood and the airway closely reflected each other in healthy children showed a strong correlation. However, monocytes and NK cells were poorly correlated at higher lymphocyte concentrations.

In adults with acute COVID-19, lymphocyte frequencies and the correlation between T cells in the blood and airways varied considerably.

Lymphocytes also showed increased expansion of memory and effector cells in adults but not children with COVID-19.

What are the implications?

The response to SARS-CoV-2 in adults versus children strongly reflects the underlying changes in the immune landscape that we observe over the life span.”

The most significant difference between adults and children with SARS-CoV-2 infection was the dominant naïve immune status in the first year of life, but adaptive memory cell recall responses in adults.

In adults, lymphopenia with increased CD8 T effector cells is typical of COVID-19, while CD4 and CD8 cells continue to proliferate in more severe disease. Plasma cells and plasmablasts are high in severe disease, mostly the IgG-producing subtype.

These differences are due to the different structures of the immune repertoire in children and adults.

The study also showed changes in the correlation of airway immune cells and those in the blood with active COVID-19. Thus, both immune cell subsets deserve a separate analysis.

The clonal lymphocyte expansion in adults implies lower immune diversity than in children. This has been reported to be a marker of severe COVID-19 in adults, while partly explaining the less severe presentation in children.

Children and adults with severe COVID-19 failed to show strong interferon (IFN) response in the airway cells, in contrast to the age-dependent increase in healthy children. This is also unlike the high IFN response in adult COVID-19 patients with moderate or mild disease. The strongest IFN response was in secretory and goblet cells.

Further studies will be required to identify whether this lack of IFN response is common to all children with COVID-19 or only severe cases. If the former, it may explain why ACE2 is not upregulated in children with COVID-19.

In view of the neutrophil recruitment markers, more research is also necessary to understand how neutrophils change with age and with COVID-19 in children and in adults. These findings may provide a glimpse of how children develop their epithelial and immune response in health and in COVID-19.

*Important Notice

medRxiv 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.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.