Bracing NK cell based therapy to relegate pulmonary inflammation in COVID-19.

The contagiosity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has startled mankind and has brought our lives to a standstill. The treatment focused mainly on repurposed immunomodulatory and antiviral agents along with the availability of a few vaccines for prophylaxis to vanquish COVID-19. This seemingly mandates a deeper understanding of the disease pathogenesis. This necessitates a plausible extrapolation of cell-based therapy to COVID-19 and is regarded equivalently significant. Recently, correlative pieces of clinical evidence reported a robust decline in lymphocyte count in severe COVID-19 patients that suggest dysregulated immune responses as a key element contributing to the pathophysiological alterations. The large granular lymphocytes also known as natural killer (NK) cells play a heterogeneous role in biological functioning wherein their frontline action defends the body against a wide array of infections and tumors. They prominently play a critical role in viral clearance and executing immuno-modulatory activities. Accumulated clinical evidence demonstrate a decrease in the number of NK cells in circulation with or without phenotypical exhaustion. These plausibly contribute to the progression of pulmonary inflammation in COVID-19 pneumonia and result in acute lung injury. In this review, we have outlined the present understanding of the immunological response of NK cells in COVID-19 infection. We have also discussed the possible use of these powerful biological cells as a therapeutic agent in view of preventing immunological harms of SARS-CoV-2 and the current challenges in advocating NK cell therapy for the same.

Introduction

Humanity is witnessing the devastating effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since late December 2019. The global statistical tally confirms 177,842,616 COVID-19 cases with 3,849,768 tolls on human lives (as of 17th June, 2021) [1]. This pandemic has challenged solidarity across the globe. To date, a few vaccines are licensed to quell COVID-19. Seemingly this warrants a deeper understanding of its pathogenesis. At present, containment strategy, supportive care, and principles of regenerative medicine and immunotherapy form the mainstay of treatment. Ongoing research and development have outlined the pivotal role of biologics in the optimal treatment of COVID-19. In this connotation, evidence has rendered a potential insight into the immunological dysfunction based on Natural Killer (NK) cells which warrants equivalent investigatory focus in view of therapeutic application to relegate the immunological harms caused by SARS-CoV-2.

Among circulating lymphocytes in humans, NK cells contribute to about 5–20 % of the lymphocytic population, which are also called large granular lymphocytes (LGL) [2]. NK cells form a subset of lymphocytes that are cytotoxic and uncharacterized without a clonal-specific receptor [3]. Though lymphocytes belong to the innate immune system, NK cells lack antigen specificity, unlike T cells or B cells. NK cells recognize virus-infected or tumorigenic cells without the presence of antibodies or major histocompatibility complexes (MHC) and through the innate ability of anti-viral and anti-tumorigenic activities [4]. Though the activity of NK cells were documented in human peripheral blood mononuclear cells and rodent splenocytes, these NK cells were found in both lymphoid and non-lymphoid tissues (bone marrow, lymph nodes, skin, gut, tonsils, liver, and lungs) [5, 6]. These cells differentiate from lymphoid progenitors and undergo maturation at specific sites. These include bone marrow, lymph nodes, thymus, tonsils and spleen and thereafter enter into the circulation [7]. The functions of NK Cells include cytolytic granule mediated cell apoptosis (via granzymes like perforin, serine esterase, chondroitin sulfate, phospholipases), antibody-dependent cell-mediated cytotoxicity (via FcyIIIA), cytokine-induced NK cell, and cytotoxic T lymphocyte (CTL) activation (via IL-2, 12, 15, 18, and CCL5), missing self-hypothesis (via MHC-I allele recognition by inhibitory receptors), tumor cell surveillance (via humoral response) and generation of memory NK cells (via CD94/NKG2) respectively.

NK cells express plasticity in differentiation. They acquire CD16+, 2B4+, CD56, CD94/NKG2A+, FasL+, CD158a+, CD158b+, CD161+ surface molecules [8]. The accession of activating receptors (LFA-1, NKp46, NKp30, NKG2D, and DNAM-1) is correlated to the cytotoxicity of NK cells. CD16 and killer immunoglobulin-like receptors (KIR) are expressed in a later phase of development [9]. NK cells do not express T-cell antigen receptor (TCR), pan T marker CD3, or surface immunoglobulins (Ig) B cell receptors [10]. It is worthwhile to understand the balancing relationship between the inhibitory and activating receptor stimulation as it, in turn, determines the activation status of the NK cells. The activating receptors of NK cells are Ly49 (C type lectin receptor), natural cytotoxicity receptor (NCR), and CD16 (FcyIIIA) whereas KIR, CD94/NKG2, ILT, or LIR (immunoglobulin-like receptor) and Ly49 (homodimers) represent inhibitory receptors [7]. Taking advantage of NK cell plasticity, improvement could be observed from NK cell therapy through production of anti-inflammatory cytokine such as IL-10.

This review article represents the fundamental role of NK Cells in the immuno-pathogenesis of COVID-19 and addresses the dire need for investigating the same rapidly through the lens of prospective clinical trials in purview for solidly adducing its therapeutic application in terms of efficacy and safety in COVID-19 patients respectively.

Anti-viral immunology of NK cells

NK cells produce and respond to inflammatory stimuli and play a role in anti-viral and tumor immunology [11, 12]. The starring facets of these cells include the ability to sense RNA viruses, critically responding to those viral invaders via optimal bridging of the innate and adaptive immune system, and execute effector functions to escalate the process of viral clearance. Upon stimulation, NK cells can produce antimicrobial and immuno-regulatory cytokines. After an encounter of microbial challenges, innate cytokines (cells of the innate immune system) elicit responses mediated by NK cell populations. Along with these innate responses, NK cells promote immuno-regulatory functions by the down-streaming adaptive response for defense against microbial organisms [13].

NK cells attack intracellular pathogens by cellular lysis of pathogen-infected cells and expose them to adaptive cell-mediated immunity. The virus inhibits MHC-I expression and upregulates the expression of activating ligands for NK cells. Once the ligand in the virus-infected cells attaches to the NKG2D receptor in NK cells, the NK cells get activated and secrete INF-γ, GM-CSF, and TNF-α and finally kill the virus-infected cell [14, 15]. Notably, the generation of type I IFNs has a pivotal role in facilitating the effector function of NK cells. The effector mechanisms of NK cells are direct mechanisms (virus-infected cells result in the production of type I/III interferon and these NK cells execute antiviral role via degranulation, receptor-mediated killing, and production of antiviral cytokine IFN-γ respectively) and indirect mechanisms (NK cells prime the response of adaptive immune system via promotion of dendritic cell maturation, differentiation of immature helper T cells (Th0) into inflammatory phenotype (Th1) and produces chemokines for attracting other immune cells at the site of inflammation) as shown in Figure 1.

Figure 1

Schematic representation of effector mechanism of NK cells.

Cross-talks between NK cells and viruses

The interaction and cross-talk between viruses and NK cells have been well documented in the literature. Viruses enter the host cells through a specific receptor binding mechanism (direct fusion at the plasma membrane, or clathrin- or caveolin-dependent endocytosis of the viral proteins) [16]. Viral proteins enhance immunogenicity through cell-cell interaction and produce the host viral response. In absence of any specific viral receptors, non-specific viral binding leads to internalization [17]. Though NK cells possess various receptors and ligands for the viral protein entry, NK cells do acquire entry mechanisms through their direct contact (involves immunological synapse) or by exosomal transfer from the virus-infected cells [18]. The virus entry mechanism and NK cell modulation have been tabularized in Table 1 [19-29].

Table 1

Mechanism of viral entry and NK cell modulation.

Viruses	Entry Mechanism	NK Cell Modulation
Receptor-mediated entry
Epstein Barr virus [19]	Receptor – CD21	Morphological changes;NK cell malignancies
Influenza A virus [20]	Clathrin- or caveolin-dependent endocytosis;Receptor – Sialic acids	↑ Apoptosis;↓ Cytotoxicity receptors, cytokines and chemokines
Respiratory syncytial virus [21]	Macropinocytosis;Receptor – FcγRIIIA	↑ INF-γ production;↓ Cytotoxicity receptors, cytokines chemokines
Human immunodeficiency virus – 1 [22]	Receptor – CD4;Co-receptor – CXC4/CCR5	↑ Apoptosis
Cell-cell interaction
Herpes simplex virus [23]	HSV infected fibroblasts	????
Varicella zoster virus [24]	VZV infected epithelial cells	↑ CD57 expression;↓ FcγRIIIA expression
Human T-lymphotropic virus [25]	Interaction with T cells	↑ Proliferation and Survival
Unknown
Cytomegalovirus [26]	? Internalization	????
Human herpes virus 6 [27]	? Internalization	↑ CD4 expression
Measles [28]	? Internalization	↓ Cytotoxicity
Vesicular stomatitis virus [29]	? Internalization	????

Interplay between COVID-19 and NK cells

A good insight into the pathogenesis of novel coronavirus disease (COVID-19) is necessary to command over its management. The correlated evidence from severe patients with lower lymphocytic counts highlighted how dysregulated immune system augments the pathophysiological dynamics in COVID-19 patients [30]. The interaction of NK cells with SARS-CoV-2 is shown in Figure 2.

Figure 2

Schematic representation of interplay of NK cells in SARS-CoV-2 infection (Hypothesized the potential role of NK Cells as a double-edged-sword in the pathogenesis of the COVID-19).

The deterioration of the respiratory system in COVID-19 is caused by a particular exemplary dysfunction of the immune system. This is clearly evident from an unanticipated deterioration of the patient just within 7–8 days after the symptoms start showing up. A study included 54 COVID-19 patients, out of which 28 had a severe respiratory compromise, all patients with respiratory compromise showed very low expression of HLA-DR and macrophage activation syndrome (MAS). A heavy downfall in the count of CD8+ lymphocytes, NK cells as well as CD19 lymphocytes was seen which clearly indicates dysregulation of the immune system among these patients [30]. The decrease in the CD8+ cell count and the drop in the number of NK cells are marked as the distinctive feature of disease by SARS-CoV-2 [30].

In most of the COVID-19 patients, the symptoms ranged from mild to moderate, but around 15% exhibited a progression to severe pneumonia and adding to the severity, a 5% advanced to acute respiratory distress syndrome, MODS (multiple organ dysfunction syndrome), and septic shock [31, 32]. Among patients associated with severe COVID-19 disease, a reduced number of CD8+ T cells, CD4+ T cells as well as NK cells and B cells were seen, thus making lymphopenia a common finding [31, 32, 33, 34, 35]. Also, there was a drop in the fraction of basophils, monocytes, and eosinophils [33, 36]. It has been reported that the COVID-19 was more likely to occur in older men with comorbidities [31, 37, 38, 39].

As depicted in Figure 2, NK cells act as a virus responder in COVID-19 patients without any co-morbidity as well as low-risk individuals but in high-risk individuals, NK cell dysfunction supervenes and hence cytokine storm occurs, which may lead to ARDS and acute lung injury. In high-risk individuals, the evasion of viral load fails as NK cells are dysfunctional due to increased mononuclear cell recruitment besides the production of inflammatory cytokines and chemokines as shown in Figure 3.

Figure 3

Pathogenesis of functionally exhaustive phenotypic expression of NK Cell inhibition by SARS-CoV-2 infection and resultant lung injury.

The two main factors believed to cause disease severity are viral elution of immune responses of the host organism and direct cytopathic effects induced by the virus [39, 40]. The first-line defense against a viral infection is an appropriately functioning innate immune response, but when the same immune response is dysfunctional, it can lead to exaggerated inflammation to which a patient may succumb to death [41].

The two types of immunity in our body i.e. innate and adaptive; both these work inconjunct to counteract the invasion of the pathogen in our body. Physical and epithelial barriers, dendritic cells, phagocytes, and natural killer cells form the main constituents of the innate immune system [42]. Notably, NK cells constitute one of the most important parts of the innate immune system wherein their effector function does not need any pre-stimulation [43].

NK cells are the front players in defencing immunologically against viral infections and cancer through their cytolytic activity and production of cytokines [44, 45, 46, 47]. NK cells can respond to inflammation and recognize these molecular cues on certain target cells, thereby facilitating the production of IFN-γ or the direct cytolysis of those target cells to suppress the virus replication activity [13].

To understand the pathophysiology of severe coronavirus illness, most physicians around the world use sepsis as a prototype because of the alarmingly high level of cytokines associated with the disease [31, 48]. Cytotoxic T cells and NK cells get more and more functionally exhausted as the disease progresses. These cells are important to keep the viral infection in check [49].

The immunobiology of COVID-19 and NK cells is poorly understood. Varchetta et al. analyzed immunological profiles in 32 patients with severe SARS-CoV-2 infection. They reported varied counts of lymphocytes with a raised proportion of mature NK cells and low T cell counts. The patients with poor clinical outcomes exhibited reduced counts of immature CD56 bright and increased counts of CD57+ FcεRIγ neg adaptive NK cells compared to survivors [50]. Evidence showed the emergence of adaptive NK cell expansions and arming of CD56 bright NK cells in severe COVID-19 patients with an increase of pro-inflammatory cytokines. This warrants a longitudinal assessment of NK cell responses in the early phase of COVID-19 infection [51, 52]. Maucourant et al., reported high expression of perforin, NKG2C, and Ksp37, reflecting a high presence of adaptive NK cell expansions in the circulation of patients with severe disease. Engaging the CD56 bright NK cells in the course of COVID-19 disease shows a defined protein-protein interaction network of inflammatory soluble factors [53].

It has been seen that there is a significant spike in the expression of NKG2A on NK cells as well as CD8 cytotoxic lymphocytes in patients with COVID-19 disease which may be related to the functional exhaustion and decreased activity and number of these cells at a primal stage leading to progression of the severity [54]. NKG2A has been demonstrated as an inhibitory receptor and the causative factor for the exhaustion of NK cells in chronic viral infections as shown in Figure 3 [55, 56].

Isolation of NK cells

The potential source to isolate NK cells includes peripheral blood and secondary lymphoid organs (lymph node, tonsil, spleen, and lymph) respectively. Notably, the peripheral blood results in more quantities of NK cells in comparison to other lymphoid organs [57]. The method of NK cell isolation from peripheral blood is described here.

By density centrifugation, lymphocytes are isolated from peripheral blood over a step gradient consisting of a mixture of the carbohydrate polymer Ficoll and the dense iodine-containing compound metrizamide. The resultant comprises lymphocytes and monocytes. Since these re-circulating lymphocytes are being isolated from blood, they never are a representation of the lymphoid system [58]. The procedure of isolating the NK cells has been discussed in Table 2 [58,59].

Table 2

Protocol for isolation of NK cells from peripheral blood.

Collection of peripheral blood in an anticoagulant-containing tube and diluted with an equivalent volume of phosphate buffer saline (PBS).

↓

Diluted blood (2/3rd portion) is layered over 1/3rd of Ficoll™ via pipette which results in the formation of interface distinctly

↓

Centrifugation for 30 min at 800 g at room temperature resulting in the formation of a well-defined layer of lymphocyte at the interface

↓

Pipetting out the layer of lymphocytes from the interface into a fresh centrifuging tube with PBS dilution

↓

Centrifugation for 10 min at 800 g at room temperature resulting in the formation of lymphocyte pellet (Refer Note)

↓

Resuspend the cell pellet in 40 μl of buffer per 107 total cells and add 10 μl of Biotin-Antibody Cocktail (human antibodies against antigens not expressed by NK cells) per 107 total cells

↓

Incubate for 10 min at 4 °C and then wash with buffer by adding 10–20× labeling volume and subjected to centrifugation for 10 min at 300 g

↓

Completely pipette off the supernatant and add 80 μl of buffer per 107 total cells, 20 μl of Anti-Biotin Microbeads per 107 total cells, and 50 μl of anti-CD3 Microbeads per 108 total cells

↓

Magnetic separation of NK cells performed

Note: After centrifugation, platelets may be present among PBMNC of the interface and excessive platelets may interfere with the functional assay or during culture (if needed) of NK cells. Notably, it is better to remove these platelets at this point of time by subjecting it to two-three times of slow centrifugation (3 min at 1 g) followed by centrifugation of yielded supernatant (1 min at 60g) to pellet the cells with each the resulting supernatant will turn clearer as the platelets will be removed. The first centrifugation will clump the platelets to settle down with ease and yield supernatant containing the desired cells. However, the supernatant obtained on second centrifugation is discarded and pelleted cells are re-suspended in PBS respectively. Identification of isolated NK cell relies on an accurate assessment of the frequency of CD56+ CD3− lymphocytes present in peripheral blood as well as the distribution of various CD56 NK cell subsets such as CD56brightCD16- NK cells which produce abundant cytokines such as interferon-gamma and its derivatives such as CD56dimCD16+ NK cells which play their role in the antibody-mediated cellular cytotoxicity [59].

Risk stratified NK cell activity against COVID-19

NK cells play a pivotal role in viral and tumor immune biology. They hypothesized the potential role of NK cells as a double-edged-sword in the pathogenesis of the COVID-19 infection are explained in the following categories (a) COVID-19 Infection in individuals without other co-morbidity: The SARS-CoV-2 infected cells express viral proteins and release cytokines and chemokines which in turn is recognized by the harboring healthy NK cells. These cells execute antiviral response (via direct and indirect effector mechanism) by bridging innate and adaptive immune responses effectively. The generated counter potential immune response shall clear this viral infection and salvage lungs from damage, (b) COVID-19 infection in low-risk individuals: The healthy NK cells shall effectively execute direct and indirect effector mechanisms to counteract the invaded SARS-CoV-2 and salvage lungs by clearing off infection. However, in case of the hyporesponsive immune system of an individual (any co-morbidity slowing down the immune system), it shall result in dwindling of NK Cell response and thereby result in damaging lungs and (c) COVID-19 infection in high-risk individuals: These individuals may have dysfunctional NK Cells which may fail to identify the SARS-CoV-2 infected cells due to the viral immune evasion pattern deployed by the virus [34]. Here, it is hypothesized that the deposited infected epithelial cells along with other immune cells (monocyte, macrophages, neutrophils) produce cytokines and chemokines resulting in the further recruitment of mononuclear cells along with NK cells to the infected site of lungs [60]. It may mark the onset of cytokine storm led by the interferon-gamma. This hyperinflammatory response may pave the way to ARDS and ALI, accounting for significant morbidity and mortality respectively. A decrease in NK cell number and exhausted phenotype has been found in association with SARS-CoV-2 infection apart from severe damage of the lungs as shown in Figure 3.

Moreover, COVID-19 patients develop a hyper-inflammatory response syndrome with increased IL-6 levels, altered coagulation, DIC, septic shock, etc., resembling hemophagocytic lymphohistiocytosis (HLH) [61, 62] and macrophage activation syndrome (MAS) [63, 64]. In children, such clinical presentation resembles Kawasaki disease [65, 66] or even multisystem inflammatory syndrome in children (MIS-C) [67]. Having known that NK cells play a significant role in the management of HLH, MAS, and MIS-C, the usage of NK cells to combat COVID-19 is plausible.

The significance of human NK cells to battle against certain infections of viral etiology has been discussed in numerous studies including human cells and/or in humans. In view of enhancing the functionality of NK cells, humans have been treated with bioactive molecules such as INF-α, IL-2 and 12 and are interestingly reported to be efficacious in disorders associated with depressed NK cell function. There has been considerable evidence implicating NK cells to mediate host defence for fighting infections in humans such as Varicella Zoster Virus (NK cell function reported to rise towards phase of healing) [68, 69, 70], Herpes Simplex Virus (HSV-1 infected target cells lysed by activated human NK cells and limited progression of HSV-1 infection in vitro due to the presence of human NK cells) [71, 72], Cytomegalovirus (CMV infected target cells lysed by activated human NK cells and limited progression of CMV infection in vitro due to the presence of human NK cells) [73, 74, 75], Epstein Barr Virus (patients with EBV infected mononucleosis demonstrated high NK cell activity as LAK cell) [76, 77], Hepatitis B (studies demonstrated surge in NK cell activity in HBV patients in comparison to controls; improvement following interferon therapy) [78, 79, 80, 81, 82], Hepatitis C (2 studies on HCV infected patients treated with INF- α, adduced indirectly for NK cells to limit chronic HCV infection) [83, 84, 85], HIV (numerous aspects of immune function, in fact NK cell activity was increased in HIV positive patients when treated with INF- α) [86, 87, 88, 89, 90, 91, 92] and COVID-19 [34,54].

It is astounding that molecular mechanism is endowed with defective antigen presentation and lymphopenia in combination whereby subjecting lymphoid cells to function in a defective manner. At the same time, it is important to note that these monocytes serve as potent cells for the assembly of TNF-α and IL-6 in severe respiratory failure (SRF) exacerbated by SARS-CoV-2. The analysis of patients infected by SARS-CoV-2 showed circulating concentrations of TNF-α, INF-γ, IL-6, and CRP respectively. INF-γ was below the limit of detection, which indicates that the Th1 response does not involve inflammation. There was a difference in the concentration of circulating levels of TNF-α among COVID-19 patients. In contrast, the concentrations of IL-6 and CRP were significantly elevated in patients with dysregulation of the immune system when compared to patients with an intermediate state of immune activation.

A few patients with immune dysregulation had low detection levels of IL-6, which lead to the understanding of how IL-6 inhibits HLA-DR expression. Notably, it has been hypothesized that low HLA-DR expression on CD14 monocytes is mediated by IL-6 overproduction in COVID-19 patients. The HLA-DR expression on CD14 monocytes was inhibited strongly from the plasma of COVID-19 patients with immune dysregulation, but not from the plasma of patients with an intermediate state of immune activation. The addition of tocilizumab, a precise blocker of the IL-6 pathway facilitated in partial restoration of the HLA-DR expression on the monocytes of all patients with immune dysregulation [30].

NK cells kill the virus-infected cell by secreting several cytokines including INF-γ and α, IL-1 and 3, and GM-CSF [93]. Also, NK cell function is enhanced by IL-2, 7 and 12, and all three classes of interferons (α, β, and γ) [94]. Interestingly IL-6 and other multiple pro-inflammatory cytokines including IFN α and γ and IL-1β also play a role in the cytokines storm [95]. The interplay between the protective function of immune mediators and the striking storm caused by the same mediators will decide the outcome.

Future directives of NK cell therapy for COVID-19

The current consensus on NK cells outlines their potential role in immuno-pathological alterations and virus clearance. However, it mandates rigorous probing of these cells which shall be harnessed and deployed in several worthy ways in near future. These include a multitude of arrays invoking improvisation in donor selection for an allogeneic transplant and NK cell augmented tumor killing as a novel way to prevent tumor escape respectively [96]. Most importantly, genetic engineering of NK cells may serve as a method of utmost significance for manipulation in view of enhancing therapeutic potentiality likewise case of T-cells [97]. Indeed, several clinical trials are afoot across the world for exploring the combination of adoptive infusion of ex-vivo activated autologous or allogeneic NK cells with conventional chemotherapy or with novel immunomodulatory agents with due ethical concerns.

On a positive note, the search result on Clinicaltrials.gov and the World Health Organization's International Clinical Trials Registry Platform (WHO ICTRP) (on 15th June 2021) listed 11 ongoing clinical trials as shown in Table 3 that aim to study the activity of NK Cells in COVID-19 [98]. As per the data, there is a mixture of proof-of-concept studies to understand the role of monocyte and NK cell activity in COVID-19 patients (NCT04375176) and interventional studies to evaluate the efficacy and safety of the proposed treatment with NK cells. Among the interventional studies, four studies were being conducted in China of which two of them (NCT04280224, NCT04324996) evaluated the role of NK cells in severe COVID-19 induced pneumonia or lymphopenia while the other two were open-labeled multicentric exploratory clinical studies (ChiCTR2000030944, ChiCTR2000031735) on the use of cord-blood derived NK cells for COVID-19 induced severe pneumonia. They utilized these studies to evaluate the effectiveness of the various cell lines of NK cells such as NK cells, IL15-NK cells, NKG2D CAR-NK cells, ACE2 CAR-NK cells, NKG2D-ACE2 CAR-NK cells as described in our study to validate the concept on NKG2D and ACE2 receptors in COVID-19. Three studies were from the USA utilizing cell lines like KDS-1000, DVX201, CYNK-001 to study the effectiveness of NK Cells in COVID-19.

Table 3

List of ongoing clinical trials on NK cell therapy for COVID-19.

S.No.	Trial Registration	Title	Interventions	Phase	Location
1	NCT04634370	Phase I clinical trial on NK cells for COVID-19	Biological: NK cells infusion	Phase 1	Brazil
2	NCT04324996	A phase I/II study of universal off-the-shelf NKG2D-ACE2 CAR-NK cells for therapy of COVID-19	Biological: NK cells, IL15-NK cells, NKG2D CAR-NK cells, ACE2 CAR-NK cells, NKG2D-ACE2 CAR-NK cells	Phase 1 Phase 2	China
3	NCT04280224	NK cells treatment for COVID-19	Biological: NK cells	Phase 1	China
4	ChiCTR2000030944	An open, multi-center, control, exploratory clinical study of human NK cells and UC-MSCs transplantation for severe novel coronavirus pneumonia	Biological: MSCs and NK cells	Phase 1	China
5	ChiCTR2000031735	Clinical study for NK cells from umbilical cord blood in the treatment of viral pneumonia include novel coronavirus pneumonia (COVID-19)	Biological: Cord blood NK cells	Phase 1	China
6	IRCT20200417047113N1	Evaluating the safety and efficacy of allogeneic NK cells on COVID-19 induced pneumonia, double-blind, randomized clinical trial	Biological: Allogeneic NK cells	Phase 1	Iran
7	NCT04375176	Monocytes and NK cells activity in Covid-19 patients	Diagnostic Test: Study of immune-mediated mechanisms in patients tested positive for SARS-CoV-2	Phase 1	Italy
8	NCT04578210	Safety Infusion of NK cells or memory T cells as adoptive therapy in COVID-19 pneumonia or lymphopenia	Biological: T memory cells and NK cells	Phase 1 Phase 2	Spain
9	NCT04797975	Off-the-shelf NK Cells (KDS-1000) as immunotherapy for COVID-19	Biological: KDS-1000| Other: Placebo	Phase 1 Phase 2	USA
10	NCT04900454	Allogeneic NK cell therapy in subjects hospitalized for COVID-19	Biological: DVX201	Phase 1	USA
11	NCT04365101	NK Cell (CYNK-001) infusions in adults with COVID-19	Biological: CYNK-001	Phase 1 Phase 2	USA

A paradigm shift has been observed in improving antigenic specificity of NK cells through chimeric antigen receptors (CARs) expression against refractory tumors [99]. The sources of CAR-expressed NK cells has been cultured in an NK-92 homogeneous cell line which is genetically modified to enhance antigenic specificity [100]. Engineered NK cells retain a full array of native cell surface receptors to exert anticancer and antiviral properties [99]. Various clinical trials on engineered NK cells with CAR specificity are ongoing against various leukemia and lymphoma. The incorporation of the CAR principle dictates the specificity of the targeted antigen. These engineered NK cells with CAR specificity can be redirected towards COVID-19. CAR-NK cells prove an off-the-shelf allogeneic product to treat patients with various tumors and viral pathologies.

Similar to therapies utilizing CAR NK cells, memory-like NK cell-based therapy could also be used as a potential targeted anti-viral therapy given their increased function when it is instituted after appropriate activation with pro-inflammatory cytokines [101, 102]. Since, the activation of memory-like NK memory cells needs enhanced autophagy of the viral products, whether COVID-19 would also evade this memory pathway by affecting their autophagy has not been determined yet [103, 104]. Apart from memory-like NK cell therapy, the induced pluripotent stem cell-derived NK cell therapy is another domain that proves to be promising in the perspective of the CAR NK cell therapy [105].

In an individual with an upregulated innate immunity, being an immune cell, the administration of NK cell evades the viral load and establishes a strong immunocompetent environment, and eliminates the viral pathogen. But when there is a dysregulated immunological status of the individual, NK cell may elicit a harmful immunological response and hence the infectious status of the individual may worsen. It is recommended to use NK cells as a therapeutic option in infection when there is an upregulated immune status of the individual.

Conclusion

Research to date provides us with a potential understanding of the biology of NK cells concerning its function and its diversified interactions with receptors. Its role is extremely well-substantiated in neoplastic conditions but warrants a clearer picture in the case of autoimmune conditions and viral infections. The race of finding a definitive cure for COVID-19 has bought in striking efforts from the medical fraternity and researchers. The emerging evidence in COVID-19 hints towards the involvement of NK cells in immune dysregulation especially in severely ill patients. Still, there is a lack of understanding regarding the role of NK cells in asymptomatic or early cases due to the inability of establishing the diagnosis in clinics, and thereby the opportunity to collect their sample for research purposes is undoubtedly skipped. Moreover, it is imperative to decide upon NK cell therapy will perquisite by boosting (as in early presentation) or tuning (late presentation) respectively. NK cell-based therapy may emerge as a major player provided investigations are accelerated in this regard by overcoming this paucity. The current focus should be on establishing this novel therapy wherein techniques for isolation and expansion of these cells in the required count needs to be further addressed.

Declarations

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

No data was used for the research described in the article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.