Cytokines are small secreted proteins that are released by various cells and have a specific effect on the interactions and communication between cells (Zhang and An, 2009).

IL-17A is a pro-inflammatory cytokine that causes epithelial cells to secrete neutrophil chemoattractant chemokines (Geha et al., 2017). Cells that produce IL-17A include TCRα/β T cells, TCRγ/δ T cells, and CD45+CD4+TCR−IL-7R+ type 3 innate lymphoid cells (ILC3s), which reside at mucosal surfaces (Geha et al., 2017). Other sources of IL-17A include dendritic cells (DCs), macrophages, neutrophils, and NK cells (Geha et al., 2017).

Macrophages’ response to IFN‐γ induces antimicrobial and antitumor mechanisms as well as the up‐regulation of antigen processing and presentation pathways (Schroder et al., 2004). IFN‐γ controls leukocyte attraction, directs growth, maturation, and differentiation of many cell types, enhances NK cell activity, and regulates B cell functions (Schroder et al., 2004).

TNF (or TNFα) is a key mediator of both acute and chronic systemic inflammatory reactions that result in tumour cell necrosis and apoptosis (Chu, 2013). TNF can induce its own secretion, as well as stimulate the production of other inflammatory cytokines and chemokines (Chu, 2013). TNFα is mainly produced by macrophages (Chu, 2013). CD4 T cells can exert direct antiviral effects by producing cytokines like interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) (Singh et al., 2007).

Interleukin 6 (IL-6) is another pro-inflammatory cytokine produced in response to infections and injuries of the tissue (Tanaka et al., 2014). Defence mechanisms include the stimulation of acute phase responses, hematopoiesis, and immune reactions (Tanaka et al., 2014).

Table 1. Summary of cytokines and their function. Table taken from Chen et al., 2018.

Figure 1. CD4+ T cell production. Image adapted from Bettke, 2020 using BioRender.

There are two subsets of T cells: CD4+ T cells (T helper cells) and CD8+ T cells (cytotoxic T cells). T helper (Th) cells have dual functionality. These cells are responsible for producing cytokines, some of which are necessary for CD8+ T cell activation, and stimulating B cells to generate antibodies for the humoral immune response (Cano & Lopera, 2013). Th cells are especially important for regulating the deletion and amplification of specific immune cells (Monsalvo et al., 2011). There are a number of Th cells: Th1, Th2, Th9, Th17, Th22, T follicular helpers (Tfh), and T regulatory cells (Tregs). The Th cells are identified based on the type of surface markers and cytokines they produce (Cano & Lopera, 2013). Tregs express cell surface markers CD4 and CD25. Due to this, Tregs can also be referred to as CD4+ CD25+ T cells. The most important property of Tregs is preventing immune cells from responding to self-antigen, which helps prevent autoimmune diseases (Cano & Lopera, 2013). Th1 cells produce IFN-γ and elicit a strong immune response to intracellular pathogens (Cano & Lopera, 2013).

On the other hand, CD8+ T cells are cytotoxic T cells that directly destroy malignant or virally infected cells. They do this by inducing apoptosis through the release of cytotoxic granules or by the expression of death ligands (Cano & Lopera, 2013). CD8+ T cells also release important mediators such as IFN-γ, TNF-α, and IL-2 (Cano & Lopera, 2013). The production of IFN-γ is crucial for recovering from an infection, and T cells are responsible for this prominent IFN response (Chen et al., 2020).

B lymphocytes are cells that induce a humoral adaptive immune response. The B cell receptor (BcR) undergoes many phases of gene rearrangement and selection before releasing immunoglobulins. Interactions with antigen-presenting T cells allow for the production of specific antibodies that aid in pathogen neutralization or destruction (Stampfli, 2019).

The extracellular spaces of the body are protected by the humoral immune response, in which antibodies produced by B cells destroy extracellular microorganisms and prevent the spread of intracellular infections (Janeway et al., 2001). Antigens trigger the activation of B cells and their differentiation into antibody-secreting plasma cells and usually require Th cells (Janeway et al., 2001).

Figure 2. The humoral response in three ways. Antibodies will bind to the pathogen (antigen) on the surface of the B cell. The antigen is processed into peptides which activates Th cells. Signals between the Th cell and the B cell allow for the differentiation of the B cell into a plasma cell, secreting a specific antibody. These antibodies protect the host from infection in three ways. 1) Neutralization, 2) Opsonization, 3) Complement activation (Janeway et al., 2001).

There are three main ways in which antibodies can contribute to immunity (Figure 1). Neutralization is when the antibodies bind to the pathogen, preventing the binding of viruses and intracellular bacteria to the target cell surface (Janeway et al., 2001). Neutralization also prevents bacterial toxins from entering cells. This depends on the interaction of the Fv domain of the antibody with the antigen (Tay et al., 2019). Next, antibodies can defend the host through phagocytosis in two ways. Opsonization refers to antibodies coating the pathogen, and these antibodies are then recognized by Fc receptors on phagocytic cells (Janeway et al., 2001). The receptors will bind to the constant C region of the antibody and phagocytosis will occur. Alternatively, complement activation is another way phagocytosis can occur. Through the binding of complement proteins to the pathogen, complement receptors will bind to phagocytes, opsonizing the pathogen further (Janeway et al., 2001). Lysis can also occur with certain microorganisms by pore formation in their membranes. The isotype or class of antibodies produced determines which mechanism is utilized (Janeway et al., 2001).

Antibody interactions may be between: 1) antibodies and the cognate antigens 2) antibody-antigen immune complexes and immunoproteins or 3) antibody-antigen immune complexes and effector cells expressing Fc receptors (Tay et al., 2019). The Fc receptor-dependent function of antibody-dependent cellular phagocytosis (ADCP) provides mechanisms for clearing the virus and virus-infected cells. In addition, ADCP stimulates downstream adaptive immune responses via antigen presentation or via secretion of inflammatory mediators (Tay et al., 2019). The Fc receptor-dependent antibody function links the innate and adaptive immune systems (Tay, Wiehe, & Pollara, 2019). To further develop immune responses, pathogen-associated molecular patterns (PAMPs) derived from virus antigens can be released upon phagocytosis and digestion. The released PAMPs are recognized by PAMP recognition receptors, which stimulate immune cells and activate immune responses (Tay et al., 2019).

The goal of the study was to compare and contrast immune responses presented in children and adults to better understand the mechanisms and clinical course of SARS-CoV-2 infections. Patient samples and data collection was performed at the Montefiore Medical Center located in Bronx, New York City. Further clinical laboratory processing was also performed at the Montefiore Medical Center. A total of 19 authors are affiliated with this study and come from various institutions (Albert Einstein College of Medicine, Yale University, and Montefiore Medical Center). The research paper was accepted for publication on September 16, 2020 and published on October 7, 2020 by the American Association for the Advancement of Science.

The study by Pierce et al. (2020) aimed to compare clinical characteristics as well as cellular and humoral immune responses in hospitalized pediatric and adult patients (Pierce et al., 2020). A total of 125 patients who were admitted to the Montefiore Medical Center between March 13th and May 31st, 2020 were observed. The researchers collected their blood serum levels and their peripheral blood mononuclear cells (PBMCs) to confirm that SARS-CoV-2 infection was present by conducting a polymerase chain reaction (PCR) assay or through positive serology (Pierce et al., 2020). Patients who had pre-existing health conditions that might affect immune responses were eliminated from the study group (Pierce et al., 2020). These conditions include, but are not limited to, cancer, HIV, patients who were receiving chronic immunosuppressive therapy for transplantation, or other conditions (Pierce et al., 2020). Further, PBMCs were segregated from a patient subgroup and the clinical data was elicited from the electronic medical record (EMR) (Pierce et al., 2020). The condition of multisystem inflammatory syndrome in children (MIS-C) was based on the criteria of the CDC (Pierce et al., 2020). According to the CDC, MIS-C is: “a condition in which inflammation occurs in different parts of the body, including the heart, lungs, kidneys, brain, skin, eyes, or gastrointestinal organs,” (Centers for Disease Control and Prevention, n.d.). The inflammation evidence includes, but is not limited to, one or more conditions such as an elevated C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), fibrinogen, procalcitonin, d-dimer, ferritin, lactic acid dehydrogenase (LDH), interleukin 6 (IL-6), elevated neutrophils, reduced lymphocytes, and low albumin (Centers for Disease Control and Prevention, n.d.). Other common symptoms in children and youth (individuals aged less than 24) with MIS-C include fever (greater than or equal to 38.0°C), abdominal pain, vomiting, diarrhea, neck pain, rashes, bloodshot eyes, or drowsiness (Centers for Disease Control and Prevention, n.d.). Some samples that were used as healthy controls were anonymously obtained before the SARS-CoV-2 pandemic and were available from a biorepository of HIV seronegative sera (Pierce et al., 2020). The patients were then divided into five groups based on their age and clinical outcome. The five groups are as follows:

• Group 1: Pediatric patients (children and youth, age <24 years) who did not require ventilation
• Group 2: Pediatric patients with MIS-C
• Group 3: Adults (age >24 years) who did not require ventilation
• Group 4: Adults who required mechanical ventilation or died
• Group 5: Pediatric patients who required mechanical ventilation

(Pierce et al., 2020)

Cytokine levels were measured in the remnant serum samples of the patients after one week of admission (Pierce et al., 2020). The levels were examined due to limited data in the pediatric groups prior to comparison between patients of different age groups and varying clinical outcomes (Pierce et al., 2020). 11-plex Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel was used to measure the cytokine concentrations in sera and supernatants. The corresponding data was collected on a Luminex Magpix and analyzed in the Milliplex Analyst program (Pierce et al., 2020). The process of automation and high throughput screening is performed more efficiently by using Magnetic Beads with features such as walk-away washing (MilliporeSigma, n.d.). This assay required 25 µl of serum or 25 μL of cell culture supernatant per well (MilliporeSigma, n.d.). Any results that were less than the lower limit of detection (LLOD) were grouped together with and set at the LLOD value (Pierce et al., 2020).

Figure 3. How Enzyme-Linked Immunosorbent Assays Work. Image adapted from British Society for Immunology, n.d. Image created using BioRender.

The enzyme-linked immunosorbent assay (ELISA) is an immunological assay that is generally used to measure antibodies, antigens, proteins and glycoproteins in biological samples (British Society for Immunology, n.d.). Indirect ELISA assay was used to measure the anti–SARS-CoV-2 spike protein immunoglobulin A (IgA) as well as total and subclass Immunoglobulin G (IgG) antibodies in the collected sera (Pierce et al., 2020). Unlike the direct ELISA assay where the primary antibody is labelled and detected, the indirect ELISA assay involves a two-step binding process where the labelled secondary antibody is bound to the primary antibody (Enzo Life Sciences, 2017). In this method, the primary antibody is initially incubated with the antigen-coated plates and following incubation, the labelled secondary antibody, which is usually a polyclonal anti-species antibody, is added (Enzo Life Sciences, 2017). Finally, a variable substance is added to produce signal amplification and detect the secondary antibody conjugate (Enzo Life Sciences, 2017). Recombinant SARS-CoV-2 spike protein and either HKU1 S1 protein, 229E S1 protein, or NL63 S1 protein were added as the antigens to the 96-well MaxiSorp plates in a solution of phosphate-buffered saline (PBS) and were then incubated overnight (Pierce et al., 2020). Following, the plates were washed three times with PBS-T solution and incubated with a blocking solution at room temperature (Pierce et al., 2020). A 1:50 dilution of individual serum samples was added to each well and after binding, the wells were washed another three times with PBS-T solution (Pierce et al., 2020). Next, the wells were incubated with the secondary antibody-conjugates, horseradish peroxidase-conjugated anti-human IgG, anti-human IgG isotype-specific, or anti-human IgA antibody at room temperature, and following incubation, the wells were washed six times with PBS-T solution. The reaction was stopped using sulfuric acid and the plates were stained using the tetramethylbenzidine (TMB) substrate (Pierce et al., 2020). Ultimately, the optical density units (ODU) were measured and ODUs from seronegative controls were subtracted from patient samples (Pierce et al., 2020).

In Figure 3, recombinant SARS-CoV-2 spike protein, HKU1 S1 protein, 229E S1 protein, or NL63 S1 protein were added as the antigens to the 96-well MaxiSorp plate. Next, serum samples were added to each well providing the primary antibodies and were able to bind. The wells were then incubated with the secondary antibody-conjugates, horseradish peroxidase-conjugated anti-human IgG, anti-human IgG isotype-specific, or anti-human IgA antibody. Finally, the wells were stained using the tetramethylbenzidine (TMB) substrate.

The neutralization assay is another method used to measure the anti-spike protein IgG antibodies in patient serum samples (Pierce et al., 2020). This method is a serological test that is utilized to detect the presence and magnitude of functional systemic antibodies, responsible for the prevention of infection by a virus (Gauger and Vincent, 2014). The assay is a replication of the infection and subsequent measurement of antibody activity, based on how effectively they neutralize the entry of the recombinant virus into the cells (Nie et al., 2020). The antibody levels are then measured following the staining of the cell culture infected with the virus (Nie et al., 2020). In the study conducted by Pierce et al. (2020) recombinant Vesicular Stomatitis Virus-S was used to incubate the serial twofold dilutions of heat-inactivated serum (1:50 to 1:12800). Duplicated amounts of inoculum were added to 24-well plates containing single layers of African green monkey kidney cells, also known as Vero cells (Pierce et al., 2020). After incubation, the inoculum was extracted, cells were loaded with methylcellulose, further incubated, fixed, and finally stained with crystal violet (Pierce et al., 2020).

Spike protein-coated microspheres were used to measure the activity of non-neutralizing antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in the same serum samples (Pierce et al., 2020). The purification of the extracellular domain of SARS-CoV-2 spike protein, which comprises the amino acids 1 to 1208, was done using nickel affinity chromatography from ExpiCHO-S and biotinylated using the EZ-Link Micro Sulfo-NHS-SS-Biotinylation Kit (Pierce et al., 2020). ExpiCHO-S cells are derived from a non-engineered subclone that has been screened and isolated from Chinese hamster ovary (CHO-S) cells (Thermo Fisher Scientific, n.d.). Sulfo-NHS-SS-Biotin is a thiol-cleavable amine-reactive biotinylation water-soluble reagent that enables biotinylation to be conducted in the absence of organic solvents for applications that cannot tolerate solvents or are complex by their composition (Thermo Fisher Scientific, n.d.). This compound is especially beneficial for labelling and purifying cell surface proteins, since the sulfonate group blocks penetration of cell membranes and its cleavable spacer arm allows initially biotinylated proteins to be released from streptavidin affinity columns (Thermo Fisher Scientific, n.d.).

A FcγRIIIa bioreporter assay was used to measure the activity of ADCC in the patients’ serum samples (Pierce et al., 2020). FcγRIIIa or CD16 is a transmembrane medium affinity receptor that is expressed on natural killer (NK) cells, macrophages, and subsets of monocytes and γδ T cells and interacts with monomeric and complex IgG (Sugita et al., 1999). FcγRIIIa mediates ADCC by NK cells and T cells, phagocytosis by macrophages, cytokine production by NK cells and lymphocytes, and regulation of immunoglobulin production (Sugita et al., 1999). Spike protein-coated microspheres were incubated with sera that were heat-inactivated at a 1:5 dilution ratio at room temperature in flat-bottomed 96-well plates (Pierce et al., 2020). Reporter cells expressing the human FcγRIIIa V variant were added and FcγRIIIa activation was identified by adding luciferin substrate (Pierce et al., 2020). The plates were then read in a SpectraMax M5e and the fold induction was calculated corresponding to the activity of luciferase, an enzyme that produces bioluminescence in the absence of serum (Pierce et al., 2020 & Luker and Luker, 2008).

Figure 4 illustrates antibody-dependent cellular phagocytosis (ADCP). Antibody-dependent cellular phagocytosis (ADCP) was performed to confirm the role of Fc receptors (green) in ADCP and it was measured through the use of spike-protein bead internalization by THP-1 cells, in the presence of patient serum. THP-1 cells are immortalized monocyte-like cells. In the presence of spike protein specific antibodies (yellow) protein beads (red) formed immune complexes with the antibodies leading to the internalization of spike-protein beads by THP-1 cells.

Figure 4. Antibody-dependent cellular phagocytosis (ADCP). Image adapted from Biosciences, 2018 and created using BioRender.

Fc receptors are a group of cell surface proteins that recognize the Fc region of an antibody. They are found on the membrane of certain immune cells, including macrophages and neutrophils (Kohler et al., 2016). Fc receptors bind to antibodies that are attached to pathogens and lead to the protective functions of the immune system (Kohler et al., 2016). Activation of the Fc receptors stimulates phagocytic or cytotoxic cells to destroy pathogens by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (Mellor et al., 2013). Following the formation of the nascent phagosomes, they undergo a process of maturation and subsequently fuse with lysosomes. Ultimately, the pathogen engulfed in the phagolysosome is digested by the lysosomal proteases and reactive oxygen compounds (Mellor et al., 2013).

In this study, diluted heat-inactivated patient serum samples were incubated with spike protein-coated fluorescent beads and THP-1 cells. THP-1 cell line is a valuable in vitro cell model for studying the modulation of monocyte and macrophage functions as it helps support the ultimate in vivo bio-activities of pharmacological activities of drugs (Chanput et al., 2014). Following the incubation, 100 microliters of supernatant were extracted from each well, substituted with an equal volume of 4% paraformaldehyde, and incubated at room temperature (Pierce et al., 2020). Cells were pelleted and rinsed once in a fluorescence-activated cell sorting buffer. The percentage of THP-1 cells with internalized spike protein beads was quantified using a Cytek Aurora flow cytometer and analyzed in FlowJo (Pierce et al., 2020). Human Fc receptor-blocking agent and anti-human ACE-2 R antibody were added to THP-1 cells as further controls (Pierce et al., 2020).

PBMCs obtained from 6 healthy donors before 2020 and 22 patients with SARS-CoV-2 who recovered were fused and resuspended in supplemented 5% human serum (Pierce et al., 2020). The cell suspension was transferred to wells of a 96-well U-bottom plate containing purified SARS-CoV-2 spike protein or media alone (Pierce et al., 2020). After incubation, GolgiStop was added and the cells were harvested, fixed, permeabilized, and stained with antibodies to CD3, CD4, CD8, CD25 APC-CD25, IL-17A, and IFN-γ (Pierce et al., 2020). Electronic gates were put on live CD4+ or CD8+ cells and the proportions of CD25+ or IFN-γ+ cells were measured. The percentage positive was determined by comparison to an isotype control antibody (Pierce et al., 2020).

All of the cytokine data were log-transformed for analysis (Pierce et al., 2020). Other groups were compared using Fisher Exact test or χ2 test, ANOVA with Tukey’s post hoc correction, or multiple regression analysis with a mixed model for the repeated measurements. A P value of less than 0.05 was regarded as significant (Pierce et al., 2020). The absence of any data is attributable to missing laboratory data, insufficient serum/peripheral blood samples (Pierce et al., 2020).

Table 2 showcases clinical manifestation in adults versus pediatric patients who have tested positive for COVID-19. In terms of underlying health conditions, diabetes and hypertension were more likely to occur in adult patients compared to pediatric patients. Additionally, pediatric patients were usually treated by receiving intravenous immunoglobulin (IVIG) while adult patients were usually treated by receiving hydroxychloride. The length of stay (LOS) was also shorter in pediatric patients compared to adult patients and approximately 37% of adult patients required mechanical ventilation whereas only 8% of pediatric patients did. Mortality rates were also more common among adult patients compared to pediatric patients, as 28% of adult patients ended up passing away, while only 3% of pediatric patients passed away.

Table 2. Demographics and clinical features of children and adults with COVID-19. Table taken from Pierce et al., 2020.

*Includes 20 patients with MIS-C.

Figure 5. A comparison of area under the curve (AUC) for neutralizing antibody in pediatric and adult patients (Pierce et al., 2020).

Figure 5 showcases the differences between pediatric and adult patient groups regarding the neutralizing ability of serum in response to the recombinant vesicular stomatitis virus expressing the SARS-CoV-2 spike protein (VSV-S). It shows differences between the area under the dose-response curve for each group. A positive correlation is seen between age and the neutralizing ability of serum. Combined data for group 1 and 2 depicted a significantly lower neutralization activity compared to combined data of groups 3 and 4.

Figure 6. Assessment of ADCP was performed by measuring the percent of THP-1 cells that internalize the spike protein–coated beads in the presence of COVID-19 patient serum after 16 hours of culture (Pierce et al., 2020).

Figure 6 analyzes antibody-dependent cellular phagocytosis (ADCP) in patient serum samples. Spike protein–coated microspheres were used as targets. In Figure 6A pediatric patients were combined into one group and adult patients were combined into another. The phagocytic activity was significantly higher in combined adult patient groups (groups 3 and 4), compared to combined pediatric patient groups (groups 1 and 2). Figure 6B shows the measured ADCP specifically in each of the 5 groups where HC is the healthy control.

Figure 7. Ratio of anti–SARS-CoV-2 spike protein–specific IgG1 to IgG3 in patients with detectable IgG (Pierce et al., 2020).

In Figure 7, different classes of anti-SARS-Co-V-2 spike protein immunoglobulin (IgG) antibodies were measured in patient serum samples. The ratio of IgG1 to IgG3 antibodies in 71 patients were assessed. There were significant differences between groups. Group 2 had a higher proportion of IgG1 versus IgG3 than group 1, group 3 and group 4.

Figure 8. CD4 T Cell Responses. CD4+ T cells were analyzed for (A) intracellular IFN-γ and (B) CD25 (Pierce et al., 2020).

Figure 8A displays representative flow cytometry plots of intracellular IFN-γ staining in CD4+ T cells before and after stimulation with SARS-CoV-2 spike protein. This was done through incubating peripheral blood mononuclear cells (PBMCs) from groups 1 and 4 with SARS-CoV-2 spike protein for 24 hours. As displayed on the graph, Group 1 has both CD4+ T cells and IFN-γ expression where as group 4 has IFN-γ expression but no CD4+ T cell presence. In the no stimulation quadrants, the top graphs reflects the presence of CD4+ T cells without IFN-γ expression and the bottom graphs reflects no presence of CD4+ T cells and no IFN-γ expression. Figure 8B shows the CD25+ staining in CD4+ T cells with and without spike protein stimulation, for groups 1, 2, 3, and 4, as well as healthy control (HC). Groups 1, 2, 3, and 4 had increased CD25+ staining following spike-protein stimulation. Adult groups 3 and 4 showed a robust increase in percent of CD25+ (CD4+) following stimulation, compared to pediatric groups 1 and 2.

Figure 9. ADCP after (A) an Fc-blocking antibody or (B) an ACE2-blocking antibody was added to serum samples to assess the role of Fc receptors in bead internalization by THP-1 cells (Pierce et al., 2020).

Figure 9A displays the changes in ADCP when Fc-blocking antibodies were added to each of the serum samples. This demonstrates the role of Fc-receptors in the internalization of beads by THP-1 cells. It was found that bead uptake was significantly reduced Fc-blocking antibodies were present, which suggests uptake of beads requires an Fc receptor.

Figure 9B shows the changes in ADCP when Angiotensin-converting enzyme 2 (ACE2) blocking antibody were added to each of the serum samples. This was to assess the role of ACE2 receptors in the internalization of beads by THP-1 cells. The uptake of beads was not significantly or consistently reduced.

Figure 10. Cytokine Concentrations in Serum (Pierce et al., 2020).

Figure 10 displays the cytokine concentration in remnant serum samples from both adult and pediatric patients. The serum samples were obtained 7 days after admission. Group 1 pediatric patients were found to have lower concentrations of IL-6, TNF-α, and IP-10 when compared other groups. Moreover, group 4 adult patients, who required ventilation or died, had increased concentrations of IL-6 and TNF-α compared to other groups. The concentration of IL-17A was lowest in group 3 recovered adult patients compared to other groups. Additionally, the concentration of IL-17A and interferon-γ (IFN-γ) was highest in group 2 MIS-C patients compared to other groups.

Figure 11. Correlation between serum concentrations and age for IL-17A and IFN-γ (Pierce et al., 2020).

Figure 11 further looks at cytokine concentration among the pediatric and adult patients, but more specifically examines the relationship between cytokine concentration and age. As age increased, the concentration of IL-17A and IFN-γ decreased, showing a negative correlation. This trend was not consistent with the cytokines IL-6, TNF, or IP-10. The data excludes patients presenting with MIS-C, as they may have a delay in time between infection of SARS-CoV-2 and clinical presentation.

The patients examined in this study helped to identify age-related differences in clinical manifestations of COVID-19. Clinical differences in terms of length of hospitalization, ventilation requirements and rate of mortality were highlighted. Shorter length of stay, decreased ventilation requirement, and decreased mortality rates were characteristic of pediatric patients, in contrast to adults. To provide further insight into age-related differences in the immune response to SARS-CoV-2, the researchers then examined cytokine, humoral, and cellular responses in patients.

One area of immunology that was studied in the paper by Pierce et al., was the ability to generate an adaptive immune response to SARS-CoV-2. Spike protein antibody titres, neutralizing antibody titres, CD4+ T cell responses, and antibody-dependent cell phagocytosis (ADCP) were measured for comparison between pediatric and adult patients. Resulting neutralizing antibody titres and ADCP were found to be higher in adult patients where risk of lung disease and death was higher (Figure 5 & 6A). Based on this, it was concluded that enhanced adaptive responses may lead to worsened pathology. This conclusion is emphasized when comparing different groups within the pediatric age range. Pediatric patients that developed MIS-C (group 2) showed greater ADCP activity than pediatric patients that required no medical ventilation (group 1) (Figure 6B). As such, MIS-C pediatric patients experienced worsened pathology. In previous studies on SARS-CoV-1, it was also found that high neutralizing antibody titres and ADCP activity were found in patients who died compared to those who recovered (Liu et al., 2019). The results of the study by Pierce et al., along with previous coronavirus studies, disprove the hypothesis that poor outcomes may be the result of a failure to generate adaptive immune responses. Instead, it is hypothesized that enhanced adaptive immunity worsens pathology. In terms of pre-existing coronavirus antibodies, this study also suggests that pre-existing antibodies do not modulate clinical responses to SARS-CoV-2 in either pediatric or adult patients (Pierce et al., 2020).

Additionally, pediatric patients that developed MIS-C (group 2) had a greater proportion of IgG1 versus IgG3 than pediatric patients that required no medical ventilation (group 1) (Figure 7). IgG1 is normally most abundant compared to other IgG subclasses (Vidarsson, Dekkers, & Rispens, 2014). On the other hand, IgG3 is the pro-inflammatory antibody responsible for inducing effector functions. In the presence of a viral infection, IgG3 is among the first IgG subclasses to appear. Thus, in pediatric patients that required no ventilation (group 1), higher levels of IgG3 may be more beneficial for viral clearance. This can be explained by immunoglobulin (Ig) subclass binding affinity to specific Fc receptors on effector cell surfaces. IgG3 has the highest affinity for type I Fcγ receptors (i.e. FcγRIIIa) (Tay et al., 2019). In pediatric patients that required no ventilation (group 1), it is hypothesized that the higher proportion of IgG3 antibodies allowed for beneficial effector functions that assisted viral clearance early on; thus, adaptive immune responses are still required. Overall, differences in subclass proportions seen in different groups may result in differences in immune responses to viral proteins, such as spike, nucleocapsid, and membrane proteins (Tay et al., 2019).

In terms of cellular responses, the researchers looked at CD4+ T cell responses to SARS-CoV-2 spike-protein. When analyzing CD4+ T cells for INF-γ expression in pediatric (group 1) versus adult (group 4) patients, the researchers did not find significant intracellular cytokine staining in CD4+ T cells, suggesting that CD4+ T cells are not the source of cytokine expression in patients (Figure 8A). Additionally, the researchers concluded that adults experienced a robust CDT+ T cell response, in contrast to pediatric patients (Figure 8B). This further highlights the reliance on adaptive responses to SARS-CoV-2 in adult patients.

The role of the Fc-receptors was confirmed in this study through the use of Fc-blocking antibodies and ACE2-blocking antibodies. The type I IgG Fc receptor present on THP-1 cells, FcγRIIIa, is an activator receptor that induces phagocytosis (Tay et al., 2019). It is a low-affinity Fc receptor and therefore, can only trigger downstream events in the presence of multivalent antibody-antigen immune complexes (Tay et al., 2019). In the presence of spike-protein coated beads, phagocytic activity was detected. However, in the presence of Fc gamma receptor (FcγR)-blocking antibodies, the phagocytic activity of THP-1 cells was reduced (Figure 9A). Notably, when running the same experiment with angiotensin-converting enzyme 2 (ACE2) polyclonal antibodies, the uptake of spike-protein beads was not reduced significantly in the presence of ACE2 blockage (Figure 9B) (Pierce et al., 2020). This experiment highlights the dependency on Fc receptor-antibody binding for macrophage activation. Fc receptor-dependent antibody activation directly links innate and adaptive immunity by combining the antiviral activity of innate effector cells, such as macrophages and neutrophils, with the adaptive humoral antibody response (Tay et al., 2019). The Fc receptor-dependent nature ADCP provides mechanisms for viral clearance. However, the activation of Fc-dependent pathways also stimulates downstream adaptive immune responses and the secretion of inflammatory mediators which may contribute to worsened pathology (Tay et al., 2019).

What part of the immune response in pediatric patients can further explain the difference in clinical manifestations in hospitalized pediatric and adult patients?

Another area of immunity that was examined in this study is the innate immune response; particularly early cytokine and cellular responses during SARS-CoV-2 infection. In regard to cytokine expression, pediatric patients expressed higher IL-17A and IFN-γ cytokines shortly after the presentation (Figure 10). These cytokines are typically expressed by CD4+ T cells, however at high IFN-γ expression, a robust CD4+ T cell response was not found in group 1 pediatric patients (Figure 8A). Thus, CD4+ T cells are likely not responsible for high IL-17A or IFN-γ expression in pediatric patients. Instead, the researchers propose that resident cells in the respiratory tract are responsible for producing IL-17A and IFN-γ. More importantly, the release of these local cytokines may be protecting patients from progressive respiratory disease (Pierce et al., 2020). This hypothesis is supported by previous studies on bronchoalveolar lavage that have identified tissue-resident immune cells that produce both IL-17A and IFN-γ (Gonzalez et al., 2015). Furthermore, dual cytokine-producing resident memory cells, found in the lung, and pulmonary epithelial cells are known to be a source of IFN-γ during other lung infections (Gonzalez et al., 2015 & Sharma et al., 2007). IL-17A producing cells in particular, may be a crucial contributing factor to immune protection as it may prevent severe respiratory manifestations. This is highlighted in pediatric patient groups 1 and 2 where, in both groups, IL-17A expression is high and lung disease is uncommon. A major finding of this paper is early IL-17A and IFN-γ cytokine expression by tissue-resident cells may help contribute to rapid clearance of SARS-CoV-2.

The age-related difference in IL-17A and IFN-γ expression can be seen in Figure 11, where there is a significant negative correlation between IL-17A and IFN-γ expression, and age. This age-related difference is consistent with the dysfunction of innate immunity in old age. Furthermore, it has been found that old age is characteristic of decreased expression of pattern recognition receptors, such as retinoic-acid inducible gene-1 (RIG-1), and a decrease in number and function of invariant natural killer T (iNKT) cells (Shaw, Goldstein, & Montgomery, 2013). In order to detect viral infection, innate immune cells rely on germline-encoded pattern recognition receptors (PRRs) present on immune cell surfaces or within distinct intracellular compartments of the cell. These PPRs include retinoic acid-inducible gene I-like receptors (RLRs), toll-like receptors (TLRs), nucleotide oligomerization domain-like receptors (NLRs), and cytosolic DNA sensors (Thompson et al., 2011). RIG-1 is a cytosolic PRR that recognizes specific pattern-associated molecular patterns, leading to a signalling cascade that results in the production of inflammatory and/or interferon cytokines. RIG-1 is especially important for recognizing cells that have been infected with RNA viruses, such as SARS-CoV-2 (Thompson et al., 2011). Another important part of the immune system is iNKT cells. iNKT cells are a subset of NKT cells that have both innate and adaptive capabilities. Within hours of infection, these cells secrete a variety of cytokines important for T cell differentiation and regulate other adaptive immune responses (Qin et al., 2019). Previous studies have suggested that iNKT cells enhance the elimination of pathogenic cells and infections (Qin et al., 2019). With these functions in mind, RIG-1 and NKT are important immune properties that, when dysregulated, dampen the innate and/or adaptive immune response.

In contrast to IL-17A and IFN-γ expression, the researchers also examined IL-6 and TNF-α cytokine expression (Figure 10). Pediatric and adult patients that did not require ventilation and did not develop MIS-C or ARDS displayed low levels of IL-6. Based on this, the expression of IL-6 and TNF-α cytokines (by Fc receptor-bearing cells during phagocytosis) may be responsible for inducing the proinflammatory response linked to MIS-C and ARDS in adults (Pierce et al., 2020). Macrophages, neutrophils, mast cells, NKT cells, and B cells are all Fc receptor-bearing cells. Macrophages, in particular, play a big role as Fc-receptor-bearing cells. Once activated, macrophages secrete inflammatory cytokines IL-6 and TNF-α, which induce the fever response (Tanaka et al., 2014 and Chu, 2013). More specifically, IL-6 stimulates hematopoiesis and acts on the liver to induce acute-phase protein responses, leading to inflammation (Tanaka et al., 2014). TNF-α is a key mediator of acute and chronic systemic inflammation, which can have severe effects on the body (Chu, 2013). Additionally, TNF-α stimulates the release of various other cytokines, chemokines and it also induces its own expression (Chu, 2013). As a pro-inflammatory cytokine that induces the adaptive immune response, it is expected that low levels of TNF-α are present in individuals who present mild manifestations of COVID-19.

Pierce et al. concluded that pediatric patients experience a robust innate immune response to SARS-CoV-2 infection, whereas adults experienced a robust adaptive immune response. As such, the robust innate response in pediatric patients may diminish the adaptive immune response thus, preventing progressive responses in children. The hypothesis of a diminished adaptive immune response in pediatric patients is supported by the results of the study. Pediatric patients displayed lower frequencies of antigen-specific CD25+ and IFN-producing CD4+ T cells, lower neutralizing antibody responses, and reduced ADCP - all of which are adaptive responses.

Limitations of the study generally include a small sample size, which would not be an accurate representation of the whole population. Some specific limitations are related to the methods of the study. Patients and their respective immunologic measurements do not reflect all aspects of the immune response. In particular, only responses to the spike protein of SARS-CoV-2 were analyzed, which excludes all other viral protein interactions (membrane, nucleocapsid, accessory proteins, etc). Additionally, some patients were undergoing treatments with drugs such as “hydroxychloroquine, remdesivir, methylprednisolone, [and] IVIG,” which may have significant effects on their immune responses (Pierce et al., 2020). Drugs used to treat SARS-CoV-2 (and the effect of drugs in general) must generate some kind of immune response or antibody production that may be reflected in the quantitative analyses of this study and are misattributed to SARS-CoV-2. Various drug interactions may also further contribute to these immune responses, which may be inaccurately reflected in the study. Another limitation includes the lack of access to bronchoalveolar lavage (BAL) and tissue samples from patients. This study involves analysis of blood samples (and serum) which excludes any effects that could have been present in lung or lymphatic tissues (Pierce et al., 2020). Another significant limitation is the lack of access to serial samples or a time-sensitive analysis of the immune responses. PCR or longitudinal tracking data would be helpful in determining and providing deeper understanding of viral kinetics. This reflects a lack of technology/method to precisely determine the exact time exposure and progression of SARS-CoV-2. This study only assessed outcomes to SARS-CoV-2 clinically, without much quantitative data. In this case, many differences that may have presented themselves during the clinical course remained undetected (Pierce et al., 2020).

The study performed by Pierce et al., examined cytokine, humoral and cellular responses to SARS-CoV-2 in hospitalized pediatric and adult patients. By identifying differences in these responses, the researchers determined age-related immune differences that were associated with differences in clinical outcome. In summary, their findings suggest that the early innate immune response is crucial for viral clearance and patient recovery. Most importantly, cells that produce IL-17A and IFN-γ cytokines allowed for rapid resolution of SARS-CoV-2 infection; whereas, greater reliance on adaptive immune responses, such as antibody neutralization and ADCP, was evident in patients who required ventilation and/or succumbed to the virus (Pierce et al., 2020). The innate immune response, which is especially high in pediatric patients, is concluded to help avoid the progressive cytokine response (or cytokine storm) seen in patients with ARDS and a worsened tissue pathology that occurs as a result of a heightened adaptive immune response (Pierce et al., 2020).

The findings of this study give great insight into the immune responses that are responsible for age-related differences in the clinical manifestation of COVID-19. Moreover, these findings can lead to the focused development of therapeutic interventions against SARS-CoV-2 which target specific immune pathways (Pierce et al., 2020). Due to the conclusion that adaptive responses such as neutralizing antibodies, antibody-induced phagocytosis and T cell responses lead to worsened pathology, therapeutic measures that induced an adaptive response to the spike protein would not be beneficial in patients. Instead, treatments that induce the early innate response to SARS-CoV-2 are hypothesized to lessen symptoms and promote recovery, based on this study’s findings (Pierce et al., 2020).

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