Skip to main content
NCBI home page
Elsevier - PMC COVID-19 Collection logoLink to Elsevier - PMC COVID-19 Collection
. 2022 Nov 23;228(1):152304. doi: 10.1016/j.imbio.2022.152304

CD4 and IL-2 mediated NK cell responses after COVID-19 infection and mRNA vaccination in adults

Amir M Alhajjat 1,⁎, Catherine R Redden 1, Morgan Langereis 1, Steven T Papastefan 1, Joy AS Ito 1, Katherine C Ott 1, Lucas E Turner 1, HeeKap K Kang 1, Aimen F Shaaban 1
PMCID: PMC9683520  PMID: 36508885

Abstract

A detailed understanding of protective immunity against SARS-CoV-2 is incredibly important in fighting the pandemic. Central to protective immunity is the ability of the immune system to recall previous exposures. Although antibody and T cell immunity have gained considerable attention, the contribution of the NK cell compartment to immune recall and protection from SARS-CoV-2 has not been explored. In this study, we investigate the NK cell responses to stimulation with SARS-CoV-2 in previously exposed and non-exposed individuals. We show that NK cells demonstrate an enhanced CD4+ T cell dependent response when re-exposed to SARS-CoV-2 antigen. The enhanced response is dependent on T cells and correlates with the number of SARS-CoV-2 specific CD4 T cells. We find that IL-2 is a critical mediator of NK cell function. These findings suggest that NK cells contribute to the protective responses against SARS-CoV-2 through a cooperation with antigen-specific CD4 T cells and have significant implications on our understanding of protective immunity in SARS-CoV-2.

Keywords: COVID-19, BNT162b2, NK cells, Memory, Adaptive immunity, CD4, IL-2

1. Introduction

Since January 2020, SARS-CoV-2 has spread throughout the world’s population causing widespread morbidity and mortality (WHO Coronoavirus Disease Covid-19 Dashboard, 2021). Although social distancing, mask use and medical therapies have lessened the impact of the pandemic, control of the pandemic is ultimately achieved by protective immunity. Despite vaccines considerably decreasing the impact of SARS-CoV-2 in the world, our understanding of the protective immunological responses generated by vaccines remains in evolution.

Protective immunity results from the cooperative interaction between multiple components of the immune system to effectively protect against infection (Hoebe et al., 2004). After vaccination, protective immunity is frequently gauged by humoral responses (Plotkin, 2010). Neutralizing antibodies provide some protection against secondary SARS-CoV-2 infection (Zost et al., 2020, Rogers et al., 2020, Khoury et al., 2021). However, neutralizing antibodies have not generally correlated with decreased disease severity. As such, transfer of antibodies after the infection has commenced provides limited benefit (Weinreich et al., 2021). In addition, SARS-CoV-2 antibody titers decay over time (Seow et al., 2020, Jo et al., 2021, Campo et al., 2021). Nonetheless, cellular responses are capable of controlling viral infections even in the absence of antibodies. After hepatitis B infection and hepatitis B vaccination, immune protection persists despite antibody decline (Van Damme and Van Herck, 2007). Similarly, even in the absence of a humoral response, cellular responses are capable of controlling SARS-CoV-2 infection (Bange et al., 2021, Soresina et al., 2020). Fortunately, convalescent COVID-19 patients and BNT162b2 mRNA vaccine recipients develop robust T cell immunity lasting months (Dan et al., 2021, Zuo et al., 2021, Painter et al., 2021, Castellino et al., 2009). These memory T cells have an instrumental role in immune protection from COVID-19 in addition to control, reduction of severity and resolution of COVID-19 infection (Rydyznski Moderbacher et al., 2020).

Although memory has been generally considered the domain of T cells and B cells, NK cells have also been shown to contribute to memory responses both directly and indirectly. In the context of viral infections, NK cells can be activated by cytokines from antigen-specific CD4 T cells (Horowitz et al., 2010, Vargas-Inchaustegui et al., 2012, He et al., 2004), or display distinct antigen-specific memory features akin to T cells (Vivier et al., 2011, Marcus and Raulet, 2013). In either scenario, NK cells are capable of an enhanced recall response and serve protective roles against viral disease. The NK cell recall response to Influenza A vaccination and the NK cell role in control of Simian Immunodeficiency Virus (SIV) are some examples (Vargas-Inchaustegui et al., 2012, Dou et al., 2015). Similar protective responses against SARS-CoV-2 would be valuable in COVID-19 immunity. However, despite numerous reports elucidating an important role for NK cells in both recovery and immunopathology of COVID-19, their role in protective immunity remains unknown.

In this study, we evaluate the NK cell responses to SARS-CoV-2 exposure in naïve and experienced settings. We examine their relationship with T cells and explore the role of IL-2 in the cooperative relationship between SARS-CoV-2 specific CD4 T cells and NK cells. We find that NK cells demonstrate a response to subsequent antigenic exposure that is dependent on CD3 T cells and proportionate to the Ag-specific CD4 T cell response and IL-2 production. We conclude that NK cells are effective mediators of the SARS-CoV-2 memory response elicited through a cooperation with antigen specific CD4+ T cells.

2. Materials and methods

2.1. Study individuals

Institutional review board approval was obtained from Ann & Robert Lurie Children’s hospital and Northwestern University IRB. Seventy-nine subjects were enrolled into the study with a total of 88 blood samples. All subjects gave written informed consent before being enrolled in the study. Participation was voluntary and compensated. Convalescent subjects (n = 18) were those who had recovered from confirmed SARS-CoV-2 infection documented by clinical RT-PCR results in addition to clinical symptoms consistent with COVID-19. None of the subjects had active disease at time of collection and were symptom free for at least 3 weeks. Thirty-one control subjects who have never been diagnosed with COVID-19 or vaccinated. Individuals who have had any suspicious symptoms at any time after January 2020 were asked for documentation of a negative clinical COVID-19 test. Those not able to provide a negative clinical RT-PCR were excluded. Furthermore, presumed control subjects whom were found to have anti-SARS-CoV-2 specific antibodies were excluded due to the possibility of having an asymptomatic infection. Control individuals who were subsequently voluntarily vaccinated were brought back for interval evaluation. Vaccinated individuals were vaccinated with the Pfizer-BioNTech mRNA BNT162b2 vaccine. At each blood draw, a questionnaire was completed and 20 ml of blood was obtained.

2.2. Blood sampling, preparation of PBMCs, cell culture and stimulation with recombinant protein

Blood was collected into EDTA coated vacutainers (BD). Samples were centrifuged at 1500g for 10 min to separate plasma from the cells. Plasma was stored at −80 °C for serological assessment as outlined below. PBMCs were then isolated using Ficoll gradient separation (Sigma-Aldrich). After washing with PBS, PBMCs were counted with a Countess automated cell counter (Thermo Fisher). In a 96 well plate (Thermo Fisher), 106 PBMCs were cultured in RPMI with 10 % fetal bovine serum, 1 % penicillin/streptomycin at 37 °C with 5 % CO2. Stimulation was achieved with addition of recombinant spike protein (1µg/well) (Sino Biological), or recombinant nucleocapsid protein at 1µg/well (Sino biological). Where indicated, blockade of IL-2 was done through addition of 10µg/ml of anti-human IL-2 neutralizing antibody (BD). After 22 h, culture plates were centrifuged, and the resultant supernatant was stored at −80 °C for cytokine analysis as detailed below. The cells were then washed twice and stained for flow cytometry. Data was acquired on a BD LSR II flow cytometer. Flow cytometry data was acquired on BD FACSDiva and analyzed using FlowJo v10.0. Staining was with the following antibodies: CD3 Pe-Cy5 (FN50), CD56 BV510 (NCAM), CD4 FITC (RPA-T4), CD8a Pe-Cy7 (RPA-T8), CD134 BV421 (Ber-ACT35), CD69 PerCPCy5.5 (FN50) (Biolegend). NK cell activation was measured as percent NK cells (CD3- CD56dim) expressing CD69+ above background levels measured in paired unstimulated control samples. CD56 bright NK cells in culture were prohibitively low in the culture and thus not analyzed. CD4 T cell activation was measured by measuring the percentage of CD4 T cells that are double positive for CD69 and OX40 above background levels measured in paired unstimulated control samples. All cultures were done in duplicate.

2.3. Analysis of anti-SARS-CoV-2 antibodies

ELISA for analysis of SARS-CoV-2 antibodies was performed as previously described (Amanat et al., 2020). 96 well plates (Nunc) were coated with 2 µg/ml of either S1 or Nucleocapsid (Sino) overnight at 4 °C. Plates were blocked with 3 % milk in PBS at room temperature for two hours and then washed with PBS with 0.1 % tween 20 (PBST). Plasma samples were prediluted diluted 1:5 with PBS in a separate plate then transferred into the test plate (1:100). Samples were then diluted further (1:300, 1:900) and incubated at room temperature for 2 h. The plate was then washed 3× with PBST. 50 μl of anti-human IgG FAB HRP (Sigma) was added to each well and the plate was incubated for 1 h. Plates were washed 3× with PBST. ELISA Substrate (Thermo Fisher) was added, and plates were developed for 10 min. The reaction was stopped using H2S04 (Thermo Fisher). The plate was read at 450 nm absorbance using a BioTek Epoch 2 microplate reader (Biotek Instruments).

2.4. T cell depletion

Depletion of CD3 T cells was done using magnetic bead separation using manufacturer’s instructions. Briefly, PBMCs were washed and resuspended with Magnetic depletion buffer (Miltenyi Biotech). CD3 microbeads (Miltenyi Biotech) were then added for 15 min at 4 °C. The cells were then washed and resuspended in the buffer. Separation was then achieved by an Auto-MACS (Miltenyi Biotech) using depletes program. Samples were regarded as successfully depleted if the CD3 fraction was <5 % of total lymphocytes.

2.5. Cytokine measurement with flow cytometry-based bead assay

Culture supernatant from the stimulation cultures were stored in 96 well plates at −80 °C. When ready for analysis, the supernatant was thawed and cytokines in the culture supernatant were measured using a flow cytometry bead-based assay (Human CD8/NK Kit, Biolegend) according to the manufacturer’s instructions. Briefly, the samples and mixed beads were incubated together in a 96 well filter plate (Biolegend) on a plate shaker for 2 h at 500 RPM. After two washes, biotin-labeled detection antibodies were added to each well and incubated on the plate shaker for 1 h at 500 RPM. Afterwards, Streptavidin-PE was added to each well for final incubation on the shaker for 30 mins at 500 rpm. Data was acquired on a Cytek Aurora (Cytek Biosciences). Analysis was then performed using the manufacturer’s LEGENDplex software (Biolegend).

2.6. Statistical analysis

Statistical analysis was done in GraphPad Prism (Version 9.1) and Microsoft Excel (Version 16). Statistical details are provided in the respective figure legend. Groups were compared using Mann-Whitney u-test. Correlation analysis was done using Pearson’s coefficient. NK cell and CD4 stimulation were calculated as background subtracted data which was derived by subtracting the stimulation in the no stimulation wells from the stimulation in the corresponding recombinant protein well. Experiments were always done in duplicate. Cytokine data was also measured as background subtracted data which was derived by subtracting the cytokine concentration in the no stimulation wells from the stimulation in the corresponding recombinant protein well.

3. Results

3.1. Clinical characteristics and SARS-COV-2 antibody response of study cohort

Seventy-nine individuals participated in the study. One peripheral blood sample was obtained from most although (9/81) were sampled more than once for longitudinal assessment as outlined in each experiment. Clinical characteristics of study participants can be found in Fig. 1 , Table 1 and supplemental Tables I-III. Thirty-one individuals without a known previous history of COVID-19 or vaccination were included. These were considered control subjects. Nine of the control individuals also voluntarily received the BNT162b2 vaccine and were evaluated again after receiving the vaccine. Eighteen individuals were drawn after recovery from COVID-19 and were symptom free for at least 3 weeks. Median time between positive PCR and blood draw for convalescent individuals was 65 days (range 32–210 days). None of the convalescent donors experienced severe disease and none were hospitalized. One patient was asymptomatic, 8/18 were mild infections while 9/18 had moderate severity as defined by NIH COVID criteria (Spectrum, 2021). Blood was obtained from 39 vaccinated individuals after receiving the of the BNT162b2 mRNA vaccine. Nine blood draws were done 5–14 days (Median: 12 days) after the first dose of the vaccine (Time point 1 (T1)). Sixteen blood draws were 5–22 days (Median: 15.5 days) after receiving the second dose (Time point 2 (T2)). Four blood draws were done 62–82 days (Median 63 days) after the second dose of the vaccine (Time point 3 (T3)) and ten blood draws 106–205 days (Median: 188 days) after the second dose of the vaccine (Time point 4 (T4)). anti-SARS-CoV-2 (Receptor Binding Domain (RBD) specific IgG, Spike (SP) specific IgG and Nucleocapsid (NC) specific IgG) antibodies were measured at every blood sampling and demonstrated findings consistent with current literature. SP and RBD-specific IgG were significantly higher in convalescent and vaccinated individuals when compared to controls. Vaccinated individuals demonstrated levels of NC-specific IgG similar to controls whereas convalescent individuals had higher levels of NC-specific IgG. A slight decay in RBD and Spike specific IgG was seen in T4 individuals highlighting the reduction in humoral immunity months after vaccination consistent with current literature (Seow et al., 2020, Jo et al., 2021, Campo et al., 2021). Seroconversion was seen in all control participants who were additionally tested after vaccination (supplemental Fig. 1).

Fig. 1.

Fig. 1

Open in a new tab

Diagram depicting subjects in the study. T1: 5–14 (median: 12) days after first dose of BNT162b2 vaccine; T2: 5–22 (median:15.5) days after completion of two doses of the vaccine; T3: 62–82 (median:63) days after completion of two doses of the vaccine; T4: 106–205 (median:188) days after completion of the two doses of the vaccine. 9 donors were drawn twice and represented in both the control and vaccinated group.

Table 1.

Characteristics of study individuals.

Control Convalescent Vaccinated
T1 T2 T3 T4
Age Median (Range) 37 (23–67 years) 34.5 (23–62 years) 43 (26–56 years) 39.5 (23–58 years) 34 (26–43 years) 37 (23–67 years)



Sex
Female 30 (96.77 %) 15 (83.33 %) 4 (44.44 %) 9 (56.25 %) 4 (100 %) 8 (80.00 %)
Male 1 (3.23 %) 3 (16.66 %) 5 (55.55 %) 7 (43.75 %) 0 (0 %) 2 (20.00 %)
Median days to Blood Draw (Range) from PCR or vaccination (as applicable) N/A 65 (32–210) days 12 (5–14) days 15.5 (5–22) days 63 (62–82) days 188 (106–205) days
Number of Participants 31 18 9 16 4 10



COVID-19 symptom Severity
Asymptomatic N/A 1 (5.55 %) N/A N/A N/A N/A
Mild N/A 8 (44.44 %) N/A N/A N/A N/A
Moderate N/A 9 (50.00 %) N/A N/A N/A N/A



Race
Caucasian 26 (83.80 %) 16 (88.88 %) 5 (55.55 %) 14 (87.50 %) 4 (100.00 %) 10 (100.00 %)
Non-Caucasian 5 (16.13 %) 2 (11.11 %) 3 (33.33 %) 2 (12.50 %) 0 (0 %) 0 (0 %)
Other 0 (0 %) 0 (0 %) 1 (11.11 %) 0 (0 %) 0 (0 %) 0 (0 %)



Report having long-term health condition(s)
Yes 11 (35.48 %) 11 (61.11 %) 2 (22.22 %) 3 (18.75 %) 1 (25.00 %) 3 (30.00 %)
No 20 (64.52 %) 7 (38.89 %) 7 (77.77 %) 13 (81.25 %) 3 (75.00 %) 7 (70.00 %)
Open in a new tab

3.2. NK cells from convalescent and vaccinated individuals demonstrate antigen-specific recall-type responses

The NK cell response to secondary antigen exposure in convalescent and vaccinated individuals was compared to controls using an in vitro antigen stimulation assay to stimulate peripheral blood mononuclear cells (PBMCs) with SARS-CoV-2 recombinant SP. After overnight stimulation, the frequency of activated NK cells (CD3-CD56dimCD69+) was measured using multi-parameter flow cytometry as illustrated in Fig. 2 . CD69 expression has been shown to correlate with cytotoxic potential of NK cells (Dons'koi et al., 2011, Borrego et al., 1999, Clausen et al., 2003). As shown in Fig. 3 , convalescent (13.59 % ± 3.73) and vaccinated (T1: 13.38 % ± 2.61, T2: 13.90 % ± 3.55, T3: 18.45 % ± 7.56, T4: 22.75 % ± 5.78) individuals demonstrated a significant increase in the frequency of activated NK cells after SP exposure when compared to control individuals (0.90 % ± 0.18). There were no significant differences in the frequency of activated NK cells between convalescent and vaccinated individuals. When measured prior to and after vaccination, all vaccinated individuals exhibited a significantly higher NK cell response to SP after vaccination (1.39 % ± 0.44 pre-vaccine versus 20.37 % ± 5.83 post-vaccine, p < 0.0001) (Fig. 3b). Additionally, the durability of the recall response in NK cells from convalescent and vaccinated individuals was surprisingly evident >6 months after primary antigen exposure even after antibody levels had started to fall (Fig. 3a and supplemental Fig. 2).

Fig. 2.

Fig. 2

Open in a new tab

Flow cytometric analysis. Gating strategy and representative histograms and dot plots used for analysis after in vitro stimulation of PBMCs with SARS-CoV-2 antigens Spike (SP) and Nucleocapsid (NC) in control, convalescent and vaccinated individual.

Fig. 3.

Fig. 3

Open in a new tab

Experienced NK cells demonstrate recall to stimulation with SARS-CoV-2 antigen. PBMCs from controls, convalescent and vaccinated participants were left unstimulated (media alone) or stimulated with recombinant Spike protein (SP). After overnight culture, cells were stained for surface markers and analyzed using flow cytometry. Expression of CD69 on CD56dim CD3- NK cells was measured. (A) NK cell response after stimulation with the SP in controls, convalescent and vaccinated individuals was measured as frequency of CD69 positive cells among CD56dim CD3- NK cells (Control, n = 31; Convalescent, n = 18; T1, n = 9; T2, n = 16; T3, n = 4; T4, n = 10). (B) Frequency of CD69 positive cells among CD56dim CD3- NK cells measured after SP stimulation in individuals prior to and after receiving the vaccine. Post vaccine timepoints are combined (n = 9). (C) NK cell response after stimulation with the NC in controls, convalescent and vaccinated individuals was measured as frequency of CD69 positive cells among CD56dim CD3- NK cells (Control, n = 31; Convalescent, n = 18; T1, n = 9; T2, n = 16; T3, n = 4; T4, n = 10). (D) Frequency of CD69 positive cells among CD56dim CD3- NK cells measured after NC stimulation in individuals prior to and after receiving the vaccine. Post vaccine timepoints are combined (n = 9). T1: 5–14 (median:12) days after first dose of BNT162b2 vaccine; T2: 5–22 (median:15.5) days after completion of two doses of the vaccine; T3: 62–82 (median:63) days after completion of two doses of the vaccine; T4: 106–205 (median:188) days after completion of the two doses of the vaccine. Stimulation was calculated as background subtracted data which was derived by subtracting the stimulation in the no stimulation wells from the stimulation in the corresponding recombinant protein well. Cultures were always done in duplicate. Significance was determined by two-tailed Mann-Whitney u-test. Error bars represent SEM.

Unlike convalescent individuals, vaccinated individuals have not been exposed to SARS-CoV-2 NC as the BNT162b2 mRNA vaccine only encodes the SP (Walsh et al., 2020). Therefore, if secondary NK cell responses are linked to previous exposure to SARS-CoV-2 antigens, vaccinated individuals would not be expected to demonstrate secondary responses to NC. To determine if the observed recall responses were antigen-specific, the NK cell responses to NC was compared between the groups. As demonstrated in Fig. 3c, convalescent individuals (21.03 % ± 4.52) demonstrated a significant increase in activated NK cells following NC stimulation when compared to vaccinated (T1: 1.00 % ± 0.59, T2: 0.52 %±0.18, T3: 5.57 % ± 3.44, T4: 4.16 % ± 1.21) or control individuals (4.69 % ± 1.28). As expected, the frequency of activated NK cells responding to NC stimulation was unchanged after vaccination (6.29 % ± 2.64 pre-vaccine versus 5.26 % ± 1.84 post-vaccine, p > 0.99) (Fig. 3d). Notably, some control and vaccinated individuals demonstrated a slight increase in the frequency of activated NK cells in response to NC likely due to cross-reactivity from prior infection with other coronaviruses (Weiskopf et al., 2020, Ogbe et al., 2021). Collectively, these findings indicate that NK cells previously exposed to the SARS-CoV-2 virus or mRNA vaccine demonstrate durable antigen-specific recall-type responses to subsequent SARS-CoV-2 antigen exposure.

3.3. SARS-CoV-2 NK cell recall response is T cell-dependent

NK cell memory has been shown to occur via direct NK cell receptor binding to cognate antigens and through indirect activation following antigen-specific humoral or T cell responses (Horowitz et al., 2010, Vargas-Inchaustegui et al., 2012, He et al., 2004, Vivier et al., 2011, Marcus and Raulet, 2013). To better understand the mechanisms regulating NK cell recall responses to SARS-CoV-2 antigens, CD4 T cell responses to secondary viral antigen exposure were measured. Following in vitro stimulation with recombinant SP, cytotoxic CD4 T cells were identified by co-expression of both CD69 and OX40 (Porichis et al., 2014, Reiss et al., 2017). The gating scheme and representative samples of the expression are demonstrated in Fig. 2. As demonstrated in Fig. 4 a, significantly more SP-reactive cytotoxic CD4 T cells were identified in convalescent (0.636 % ± 0.05) and vaccinated (T1: 1.173 % ± 0.308, T2: 0.707 % ± 0.181, T3: 0.929 % ± 0.282, T4: 1.400 % ± 0.336) individuals when compared to controls (0.146 % ± 0.054). There was also no significant difference in the frequency of cytotoxic CD4 cells between convalescent and vaccinated individuals. As expected, NC-reactive CD4 T cells were only detected in convalescent (0.448 % ± 0.137) individuals but not controls (0.114 % ± 0.037) or vaccinated (T1: 0.126 % ± 0.046, T2: 0.062 % ± 0.048, T3: 0.128 % ± 0.103, T4: 0.173 % ± 0.067) individuals (Fig. 4b). These findings are consistent with the current understanding of the CD4 T cell responses to SARS-CoV-2 antigens in convalescent and vaccinated individuals (Dan et al., 2021, Zuo et al., 2021, Painter et al., 2021).

Fig. 4.

Fig. 4

Open in a new tab

Frequency of SARS-CoV-2 antigen specific CD4 T cells in control, convalescent and vaccinated individuals. Expression of CD69 and OX40 on CD4 T cells was measured after culture with SARS-CoV-2 SP or NC antigen. Antigen-specific CD4 T cells were defined as those positive for both CD69 and OX40. (A) Antigen specific CD4 T cells after stimulation with SP in controls, convalescent, and vaccinated individuals. (B) Antigen specific CD4 T cells after stimulation with the NC in controls, convalescent individuals measured as CD69 and OX40 positive CD4 T cells. (Control, n = 31; Convalescent, n = 18; T1, n = 9; T2, n = 16; T3, n = 4; T4, n = 10) T1: 5–14 (median:12) days after first dose of BNT162b2 vaccine; T2: 5–22 (median:15.5) days after completion of two doses of the vaccine; T3: 62–82 (median:63) days after completion of two doses of the vaccine; T4: 106–205 (median:188) days after completion of the two doses of the vaccine. Stimulation was calculated as background subtracted data which was derived by subtracting the stimulation in the no stimulation wells from the stimulation in the corresponding recombinant protein well. Cultures were always done in duplicates. Significance was determined by two-tailed Mann-Whitney u-test. Error bars represent SEM.

The relationship between the frequency of SP-reactive cytotoxic CD4 T cells and NK cell recall responses to SARS-CoV-2 antigens in convalescent and vaccinated individuals was examined. As shown in Fig. 5 a, the NK cell recall response correlated with the CD4 T cell response to SP in both convalescent and vaccinated groups when stimulated with SP for both groups (r = 0.52, p < 0.0001, n = 61). To uncouple NK cell responses from T cell responses, CD3+ T cells were depleted from the cytotoxic assays prior to stimulation with SP and compared to non-depleted cytotoxic assays for the convalescent, vaccinated and control groups (Supplemental Fig. 3). As shown in Fig. 5b, the NK cell recall response was strikingly diminished by the loss of T cells in the convalescent (pre-depletion 9.45 % ± 2.85 versus post-depletion 0.56 % ± 0.34, p = 0.029) and vaccinated groups (pre-depletion 30.59 % ± 9.53 versus post-depletion 0.58 % ± 0.21, p = 0.029). Conversely, T cell depletion did not alter the naïve NK cell response seen in control individuals (pre-depletion 0.93 % ± 0.36 versus post-depletion 0.89 % ± 0.84, p = ns) (Fig. 5b). Taken together, these findings suggest that NK cell recall responses to SARS-COV-2 antigens are dependent on T cell activation.

Fig. 5.

Fig. 5

Open in a new tab

NK cell recall response is T cell dependent. (A) Correlation between frequency of CD69+ OX40 + CD4 T cells and CD69+ NK cells when stimulated with Spike (SP) in convalescent and vaccinated individuals (r = 0.52; p < 0.0001; n = 61). (B) NK cell response measured as frequency of CD69 positive cells among CD56dim CD3- NK cells with and without depletion of CD3 cells in controls (n = 4), convalescent (n = 4), and vaccinated subjects (n = 4). Non-parametric Spearman test was used for correlation analysis. For group comparison, significance was determined by two-tailed Mann-Whitney u-test.

3.4. SARS-CoV-2 NK cell recall response is mediated by IL-2

Rapid secretion of IL-2 by activated antigen-specific CD4 T cells is a known pathway for indirect activation of NK cells (Horowitz et al., 2010, He et al., 2004). We thus assessed the importance of IL-2 in the NK cell response to SARS-CoV-2. First, the levels of IL-2 were measured in culture supernatants from control, convalescent and vaccinated individuals after SP stimulation. As shown in Fig. 6 a, IL-2 levels from SP-stimulation assays were significantly higher in convalescent (143.9±66.4 pg/ml) and vaccinated (T1: 157.9±68.2 pg/ml, T2: 87.92±25.19 pg/ml, T3: 56.56±31.46 pg/ml, T4: 256.9±128.0 pg/ml) individuals when compared to control individuals (1.21±1.19 pg/ml). Furthermore, the NK cell response in both convalescent and vaccinated individuals correlated directly with the level of secreted IL-2 in the culture supernatants (Fig. 6b). IL-2 secretion was nearly undetectable in culture supernatants from controls and vaccinated participants when stimulated with NC (Supplemental Fig. 4). An analysis of multiple proinflammatory cytokines revealed a broad elevation in the secreted levels in convalescent (with SP and NC stimulation) and vaccinated individuals (only with SP stimulation) when compared to controls (Supplemental Fig. 5).

Fig. 6.

Fig. 6

Open in a new tab

IL-2 correlates with NK cell response but only partially mediates the response. (A) IL-2 concentration was measured in supernatants from PBMC cultures stimulated with Spike (SP) in control (n = 13), convalescent (n = 14) and vaccinated individuals (T1: n = 4, T2: n = 16, T3: n = 4, T4: n = 7). IL-2 production was significantly increase in convalescent and vaccinated individuals when compared to controls. (B) Correlation of IL-2 and frequency of CD69+ among CD56dimCD3- NK cells in convalescent and vaccinated individuals (n = 45). IL-2 concentrations in supernatant from PBMC cultures with SP correlates with and frequency of CD69+ among CD56dimCD3- NK cell when stimulated with SP. (C) Frequency of CD69+ among CD56dimCD3- NK cell when PBMCs are stimulate with SP with and without neutralizing IL-2 antibodies. (D) Percent reduction in and frequency of CD69+ among CD56dimCD3- NK cell with and without neutralizing IL-2 antibodies and stimulation with SP (n = 10). Post vaccine timepoints are combined (n = 11). T1: 5–14 (median: 12) days after first dose of BNT162b2 vaccine; T2: 5–22 (median: 15.5) days after completion of two doses of the vaccine; T3: 62–82 (median: 63) days after completion of two doses of the vaccine; T4: 106–205 (median: 188) days after completion of the two doses of the vaccine. IL-2 concentration was calculated as background subtracted data which was derived by subtracting the stimulation in the no stimulation wells from the stimulation in the corresponding recombinant protein well. Experiments were always done in duplicates. Significance was determined by two-tailed Mann-Whitney u-test. Error bars represent SEM. Non-parametric Spearman test was used for correlation analysis. LOD: Limit of detection.

To further examine the role of IL-2 in the NK cell response, we cultured fresh PBMCs from vaccinated individuals with SP in presence of IL-2 neutralizing antibody and measured the resultant NK cell activation. Neutralization of IL-2 was confirmed by flow cytometric bead assay. As shown in Fig. 6c and d, there was a significant reduction in the NK cell response to SP stimulation, although this was not statistically significant. This data suggests a significant role for IL-2 as a critical mediator for the NK cell response to SARS-CoV-2. Alternatively, a neutralization of IL-2 may have inhibited CD4 T cell responses leading to further decrease in NK cell responses.

4. Discussion

In this study, we demonstrate an enhanced NK cell response following secondary exposure to the SARS-CoV-2 virus that is dependent on antigen-specific T-cells. Secreted levels of IL-2 appeared to be an important mediator in the NK cell response. These findings reveal a role for NK cells in immunologic memory against SARS-CoV-2 and offer a new method to track sophisticated mechanisms of cellular immunity after vaccination.

NK cells as effectors of innate immunity play a fundamental role in clearance of viral infections (Jost and Altfeld, 2013). An increased susceptibility to viral infections is evident in conditions of diminished NK cell frequency or function (Jawahar et al., 1996). Similarly, normal NK cell function is an important component of the primary response to SARS-CoV-2 whereas defects in NK cell immunity contribute to the disease spectrum (Maucourant et al., 2020, Kramer et al., 2021, Littera et al., 2021, Witkowski et al., 2021). Early evidence for a critical role for NK cells stems from findings in which diminishing numbers of NK cells correlated with increasing severity of SARS-CoV-2 disease (Masselli et al., 2020, Vabret et al., 2020, Ni et al., 2021), a phenomenon also reported in respiratory disease caused by closely related coronaviruses (He et al., 2005, National Research Project for SARS, Beijing Group, 2004). More recent findings have demonstrated a correlation in severity of SARS-CoV-2 disease with the expression of particular subsets of NK cells and receptors (Zheng et al., 2020).

Given the importance of NK cells in acute SARS-CoV-2 infection, a protective NK cell role in SARS-CoV-2 immunity after infection or vaccination is possible and may be mediated by several mechanisms. Notably, NK cells have been shown to harbor explicit antigen-specific memory characteristics in contexts such as hapten-induced contact hypersensitivity (O'Leary et al., 2006), murine cytomegalovirus (Sun et al., 2011), and cytokine induced memory NK cells (Cooper et al., 2009). In humans, NK cell memory has been recognized in human CMV, herpes simplex virus, influenza A and hantavirus although the exact mechanisms remain elusive (Abdul-Careem et al., 2012, Lopez-Vergès et al., 2011, Bjorkstrom et al., 2011, Wijaya et al., 2021, Nikzad et al., 2019, Stary et al., 2020). The results of the current study reveal enhanced NK cell responses in subjects previously exposed to SARS-CoV-2 through either illness or vaccination. The finding that NK cells from vaccinated individuals responded to SP, but not NC, supports that the cooperative CD4 NK cell response is specific and requires prior CD4 T cell antigen exposure to antigen. This was corroborated by the interval assessment of control subjects before and after vaccination; as the NK cell response to SP but not NC increased in these subjects. Expectedly, the responses seemed as durable as the CD4 T cell responses as we identified these NK cell responses in subjects up to 6 months after recovery from COVID-19 and up to 7 months after vaccination.

NK cells are key mediators of antibody-dependent cellular cytotoxicity (ADCC). ADCC is a key component of vaccine-mediated protection against several human pathogens (Haynes et al., 2012, Arora et al., 2018, Kavian et al., 2020, Ferrari et al., 2011). ADCC also likely plays a role in anti-SARS-CoV-2 immunity. Yu et al demonstrated anti-SARS-CoV-2 antibody-dependent NK cell responses to the S protein RBD in vaccinated macaques revealing a role for interplay between neutralizing antibody and antibody-dependent cellular responses in SARS-CoV-2 memory (Yu et al., 2020). However, similar to our findings, antibody-mediated NK cell activation did not correlate with antibody titers. Although that may be a mechanism through which NK cells are activated in our cultures, this is unlikely given the cells were washed prior to cultures. However, the presence of antibody dependent NK cell memory responses remains plausible. Future studies are required to evaluate ADCC in SARS-CoV-2 immunity.

NK cells may also respond indirectly through NK cell cooperation with antigen specific CD4 T cells in control of viral infections. Such CD4 T cell-mediated NK cell functions have been exhibited in the context of hepatitis B virus, influenza A vaccination, rabies and simian immunodeficiency virus (Horowitz et al., 2010, Vargas-Inchaustegui et al., 2012, He et al., 2004). Following exposure to these antigens through infection or vaccination, antigen-specific IL-2 expression by memory CD4 T cells enhances NK cell activation and facilitates clearance of the virus. In rabies vaccinated individuals and SIV, NK cell memory functions have been found to completely depend on T cell-derived IL-2 whereas NK cell memory is elicited in HBV vaccinated individuals despite absence of T cell derived IL-2 (Vargas-Inchaustegui et al., 2012, Wijaya et al., 2021). The findings of the current study revealed that enhanced NK cell responses in SARS-CoV-2 are also mediated by IL-2 expression by memory CD4 T cells. Neutralizing IL-2 antibody only reduced the response indicating a partial but significant role for IL-2 among SARS-CoV-2 vaccinated individuals. This interplay enhances the effectiveness of the antigen-specific adaptive immune response in protection from SARS-CoV-2 infection. Indeed, links between the adaptive and innate immune responses are vital in host defense and allow for amalgamation of the specificity of the adaptive immune response with the rapidity of the innate immune response.

Another potential advantage of memory NK cell responses in viral disease is the broadened response to SARS-CoV-2 variants. Previous studies in subjects that received seasonal influenza vaccine demonstrate an increased and sustained NK cell recall response to multiple influenza virus subtypes despite antibody-mediated responses that were not as broad (Dou et al., 2015). Interestingly, these memory NK cells demonstrated a dynamic increase in intracellular NKp46 which correlated with enhanced IFN-γ response (Dou et al., 2015). Although none of the vaccinated volunteers in the current study reported breakthrough infections prior to the emergence of the omicron variant, future studies are needed to examine NK cell responses to SARS-CoV-2 variants.

Alternatively, NK cells have also been found to have a suppressive effect on adaptive immune responses after vaccination. Mechanisms involved include NK cell clearance of infected and antigen bearing cells, perforin-dependent NK cell killing of activated CD4 and CD8 T cells and production of immunosuppressive cytokines (Rydyznski et al., 2015, Cox et al., 2021). These immunomodulatory effects of NK cells can be protective from immunopathology related to viral infections or detrimental to vaccination efforts.

Collectively, these findings reveal a critical role for NK cells in protective immunity against SARS-COV-2. Weaknesses of the current study include that intracellular cytokines were not measured and the cytotoxic effect of NK cells was not directly measured. Rather we chose the activation status of NK cells demonstrated by the CD69+ expression as a surrogate for enhanced activity. Therefore, an exact contribution of the activated NK cells to anti-SARS-CoV-2 immunity is not well defined. As such, further studies are needed to define whether these activated NK cells contribute to enhanced viral killing versus immunomodulation of the adaptive immune response. Additionally, further studies are also needed to characterize the mechanisms that guide NK cell responses in COVID-19. On a practical level, the NK cell response to SP antigen may provide a method for evaluation of vaccine efficiency to provide broad immunity against different viral subtypes and potentially guide booster intervals.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.imbio.2022.152304.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.pdf (1MB, pdf)

Data availability

Data will be made available on request.

References

  1. Abdul-Careem MF, Lee AJ, Pek EA, Gill N, Gillgrass AE, Chew MV, et al. Genital Hsv-2 infection induces short-term Nk Cell memory. PLoS One (2012) 7(3):e32821. Epub 2012/03/30. doi: 10.1371/journal.pone.0032821. [DOI] [PMC free article] [PubMed]
  2. Amanat F., Stadlbauer D., Strohmeier S., Nguyen T.H.O., Chromikova V., McMahon M., Jiang K., Arunkumar G.A., Jurczyszak D., Polanco J., Bermudez-Gonzalez M., Kleiner G., Aydillo T., Miorin L., Fierer D.S., Lugo L.A., Kojic E.M., Stoever J., Liu S.T.H., Cunningham-Rundles C., Felgner P.L., Moran T., García-Sastre A., Caplivski D., Cheng A.C., Kedzierska K., Vapalahti O., Hepojoki J.M., Simon V., Krammer F. A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat. Med. 2020;26(7):1033–1036. doi: 10.1038/s41591-020-0913-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arora G, Hart GT, Manzella-Lapeira J, Doritchamou JY, Narum DL, Thomas LM, et al. Nk cells inhibit plasmodium falciparum growth in red blood cells via antibody-dependent cellular cytotoxicity. Elife (2018) 7. Epub 2018/06/27. doi: 10.7554/eLife.36806. [DOI] [PMC free article] [PubMed]
  4. Bange E.M., Han N.A., Wileyto P., Kim J.Y., Gouma S., Robinson J., Greenplate A.R., Hwee M.A., Porterfield F., Owoyemi O., Naik K., Zheng C., Galantino M., Weisman A.R., Ittner C.A.G., Kugler E.M., Baxter A.E., Oniyide O., Agyekum R.S., Dunn T.G., Jones T.K., Giannini H.M., Weirick M.E., McAllister C.M., Babady N.E., Kumar A., Widman A.J., DeWolf S., Boutemine S.R., Roberts C., Budzik K.R., Tollett S., Wright C., Perloff T., Sun L., Mathew D., Giles J.R., Oldridge D.A., Wu J.E., Alanio C., Adamski S., Garfall A.L., Vella L.A., Kerr S.J., Cohen J.V., Oyer R.A., Massa R., Maillard I.P., Maxwell K.N., Reilly J.P., Maslak P.G., Vonderheide R.H., Wolchok J.D., Hensley S.E., Wherry E.J., Meyer N.J., DeMichele A.M., Vardhana S.A., Mamtani R., Huang A.C. CD8+ T cells contribute to survival in patients with COVID-19 and hematologic cancer. Nat. Med. 2021;27(7):1280–1289. doi: 10.1038/s41591-021-01386-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bjorkstrom NK, Lindgren T, Stoltz M, Fauriat C, Braun M, Evander M, et al. Rapid Expansion and Long-Term Persistence of Elevated Nk Cell Numbers in Humans Infected with Hantavirus. J Exp Med (2011) 208(1):13-21. Epub 2010/12/22. doi: 10.1084/jem.20100762. [DOI] [PMC free article] [PubMed]
  6. Borrego F, Robertson MJ, Ritz J, Pena J, Solana R. Cd69 Is a Stimulatory Receptor for Natural Killer Cell and Its Cytotoxic Effect Is Blocked by Cd94 Inhibitory Receptor. Immunology (1999) 97(1):159-65. Epub 1999/08/14. doi: 10.1046/j.1365-2567.1999.00738.x. [DOI] [PMC free article] [PubMed]
  7. Campo F, Venuti A, Pimpinelli F, Abril E, Blandino G, Conti L, et al. Antibody Persistence 6 Months Post-Vaccination with Bnt162b2 among Health Care Workers. Vaccines (Basel) (2021) 9(10). Epub 2021/10/27. doi: 10.3390/vaccines9101125. [DOI] [PMC free article] [PubMed]
  8. Castellino F., Galli G., Del Giudice G., Rappuoli R. Generating memory with vaccination: highlights. Eur. J. Immunol. 2009;39(8):2100–2105. doi: 10.1002/eji.200939550. [DOI] [PubMed] [Google Scholar]
  9. Clausen J., Vergeiner B., Enk M., Petzer A.L., Gastl G., Gunsilius E. Functional significance of the activation-associated receptors CD25 and CD69 on human NK-cells and NK-like T-cells. Immunobiology. 2003;207(2):85–93. doi: 10.1078/0171-2985-00219. [DOI] [PubMed] [Google Scholar]
  10. Cooper M.A., Elliott J.M., Keyel P.A., Yang L., Carrero J.A., Yokoyama W.M. Cytokine-induced memory-like natural killer cells. PNAS. 2009;106(6):1915–1919. doi: 10.1073/pnas.0813192106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cox A., Cevik H., Feldman H.A., Canaday L.M., Lakes N., Waggoner S.N. Targeting natural killer cells to enhance vaccine responses. Trends Pharmacol. Sci. 2021;42(9):789–801. doi: 10.1016/j.tips.2021.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dan J.M., Mateus J., Kato Y.u., Hastie K.M., Yu E.D., Faliti C.E., Grifoni A., Ramirez S.I., Haupt S., Frazier A., Nakao C., Rayaprolu V., Rawlings S.A., Peters B., Krammer F., Simon V., Saphire E.O., Smith D.M., Weiskopf D., Sette A., Crotty S. Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science. 2021;371(6529) doi: 10.1126/science.abf4063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dons'koi B.V., Chernyshov V.P., Osypchuk D.V. Measurement of NK activity in whole blood by the CD69 up-regulation after co-incubation with K562, comparison with NK cytotoxicity assays and CD107a degranulation assay. J. Immunol. Methods. 2011;372(1-2):187–195. doi: 10.1016/j.jim.2011.07.016. [DOI] [PubMed] [Google Scholar]
  14. WHO Coronoavirus Disease (Covid-19) Dashboard. (2021) [cited 2021]. Available from: https://covid19.who.int/.
  15. Dou Y, Fu B, Sun R, Li W, Hu W, Tian Z, et al. Influenza Vaccine Induces Intracellular Immune Memory of Human Nk Cells. PLoS One (2015) 10(3):e0121258. Epub 2015/03/18. doi: 10.1371/journal.pone.0121258. [DOI] [PMC free article] [PubMed]
  16. Ferrari G., Pollara J., Kozink D., Harms T., Drinker M., Freel S., Moody M.A., Alam S.M., Tomaras G.D., Ochsenbauer C., Kappes J.C., Shaw G.M., Hoxie J.A., Robinson J.E., Haynes B.F. An HIV-1 gp120 envelope human monoclonal antibody that recognizes a C1 conformational epitope mediates potent antibody-dependent cellular cytotoxicity (ADCC) activity and defines a common ADCC epitope in human HIV-1 serum. J. Virol. 2011;85(14):7029–7036. doi: 10.1128/JVI.00171-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haynes B.F., Gilbert P.B., McElrath M.J., Zolla-Pazner S., Tomaras G.D., Alam S.M., Evans D.T., Montefiori D.C., Karnasuta C., Sutthent R., Liao H.-X., DeVico A.L., Lewis G.K., Williams C., Pinter A., Fong Y., Janes H., DeCamp A., Huang Y., Rao M., Billings E., Karasavvas N., Robb M.L., Ngauy V., de Souza M.S., Paris R., Ferrari G., Bailer R.T., Soderberg K.A., Andrews C., Berman P.W., Frahm N., De Rosa S.C., Alpert M.D., Yates N.L., Shen X., Koup R.A., Pitisuttithum P., Kaewkungwal J., Nitayaphan S., Rerks-Ngarm S., Michael N.L., Kim J.H. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N. Engl. J. Med. 2012;366(14):1275–1286. doi: 10.1056/NEJMoa1113425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. He XS, Draghi M, Mahmood K, Holmes TH, Kemble GW, Dekker CL, et al. T Cell-Dependent Production of Ifn-Gamma by Nk Cells in Response to Influenza a Virus. J Clin Invest (2004) 114(12):1812-9. Epub 2004/12/16. doi: 10.1172/JCI22797. [DOI] [PMC free article] [PubMed]
  19. He Z., Zhao C., Dong Q., Zhuang H., Song S., Peng G., Dwyer D.E. Effects of severe acute respiratory syndrome (SARS) coronavirus infection on peripheral blood lymphocytes and their subsets. Int. J. Infect. Dis. 2005;9(6):323–330. doi: 10.1016/j.ijid.2004.07.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hoebe K., Janssen E., Beutler B. The interface between innate and adaptive immunity. Nat. Immunol. 2004;5(10):971–974. doi: 10.1038/ni1004-971. [DOI] [PubMed] [Google Scholar]
  21. Horowitz A., Behrens R.H., Okell L., Fooks A.R., Riley E.M. NK Cells as Effectors of acquired immune responses: effector CD4 + T cell-dependent activation of NK Cells following vaccination. J. Iummol. 2010;185(5):2808–2818. doi: 10.4049/jimmunol.1000844. [DOI] [PubMed] [Google Scholar]
  22. Jawahar S, Moody C, Chan M, Finberg R, Geha R, Chatila T. Natural Killer (Nk) Cell Deficiency Associated with an Epitope-Deficient Fc Receptor Type Iiia (Cd16-Ii). Clin Exp Immunol (1996) 103(3):408-13. Epub 1996/03/01. doi: 10.1111/j.1365-2249.1996.tb08295.x. [DOI] [PMC free article] [PubMed]
  23. Jo DH, Minn D, Lim J, Lee KD, Kang YM, Choe KW, et al. Rapidly Declining Sars-Cov-2 Antibody Titers within 4 Months after Bnt162b2 Vaccination. Vaccines (Basel) (2021) 9(10). Epub 2021/10/27. doi: 10.3390/vaccines9101145. [DOI] [PMC free article] [PubMed]
  24. Jost S., Altfeld M. Control of Human Viral Infections by Natural Killer Cells. Annu. Rev. Immunol. 2013;31(1):163–194. doi: 10.1146/annurev-immunol-032712-100001. [DOI] [PubMed] [Google Scholar]
  25. Kavian N., Hachim A., Poon L.L.M., Valkenburg S.A. Vaccination with ADCC activating HA peptide epitopes provides partial protection from influenza infection. Vaccine. 2020;38(37):5885–5890. doi: 10.1016/j.vaccine.2020.07.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Khoury D.S., Cromer D., Reynaldi A., Schlub T.E., Wheatley A.K., Juno J.A., Subbarao K., Kent S.J., Triccas J.A., Davenport M.P. Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection. Nat. Med. 2021;27(7):1205–1211. doi: 10.1038/s41591-021-01377-8. [DOI] [PubMed] [Google Scholar]
  27. Kramer B, Knoll R, Bonaguro L, ToVinh M, Raabe J, Astaburuaga-Garcia R, et al. Early Ifn-Alpha Signatures and Persistent Dysfunction Are Distinguishing Features of Nk Cells in Severe Covid-19. Immunity (2021). Epub 2021/10/01. doi: 10.1016/j.immuni.2021.09.002. [DOI] [PMC free article] [PubMed]
  28. Littera R, Chessa L, Deidda S, Angioni G, Campagna M, Lai S, et al. Natural Killer-Cell Immunoglobulin-Like Receptors Trigger Differences in Immune Response to Sars-Cov-2 Infection. PLoS One (2021) 16(8):e0255608. Epub 2021/08/06. doi: 10.1371/journal.pone.0255608. [DOI] [PMC free article] [PubMed]
  29. Lopez-Vergès S., Milush J.M., Schwartz B.S., Pando M.J., Jarjoura J., York V.A., Houchins J.P., Miller S., Kang S.-M., Norris P.J., Nixon D.F., Lanier L.L. Expansion of a unique CD57 + NKG2C hi natural killer cell subset during acute human cytomegalovirus infection. PNAS. 2011;108(36):14725–14732. doi: 10.1073/pnas.1110900108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Marcus A., Raulet D. Evidence for natural killer cell memory. Curr. Biol. 2013;23(17):R817–R820. doi: 10.1016/j.cub.2013.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Masselli E., Vaccarezza M., Carubbi C., Pozzi G., Presta V., Mirandola P., Vitale M. NK cells: A double edge sword against SARS-CoV-2. Adv. Biol. Regul. 2020;77:100737. doi: 10.1016/j.jbior.2020.100737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Maucourant C., Filipovic I., Ponzetta A., Aleman S., Cornillet M., Hertwig L., Strunz B., Lentini A., Reinius B., Brownlie D., Cuapio A., Ask E.H., Hull R.M., Haroun-Izquierdo A., Schaffer M., Klingström J., Folkesson E., Buggert M., Sandberg J.K., Eriksson L.I., Rooyackers O., Ljunggren H.-G., Malmberg K.-J., Michaëlsson J., Marquardt N., Hammer Q., Strålin K., Björkström N.K. Natural killer cell immunotypes related to COVID-19 disease severity. Sci. Immunol. 2020;5(50) doi: 10.1126/sciimmunol.abd6832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ni L, Cheng ML, Feng Y, Zhao H, Liu J, Ye F, et al. Impaired Cellular Immunity to Sars-Cov-2 in Severe Covid-19 Patients. Front Immunol (2021) 12:603563. Epub 2021/02/20. doi: 10.3389/fimmu.2021.603563. [DOI] [PMC free article] [PubMed]
  34. National Research Project for SARS, Beijing Group The involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome. Am. J. Clin. Path. 2004;121(4):507–511. doi: 10.1309/WPK7Y2XKNF4CBF3R. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nikzad R., Angelo L.S., Aviles-Padilla K., Le D.T., Singh V.K., Bimler L., Vukmanovic-Stejic M., Vendrame E., Ranganath T., Simpson L., Haigwood N.L., Blish C.A., Akbar A.N., Paust S. Human natural killer cells mediate adaptive immunity to viral antigens. Sci. Immunol. 2019;4(35) doi: 10.1126/sciimmunol.aat8116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ogbe A., Kronsteiner B., Skelly D.T., Pace M., Brown A., Adland E., Adair K., Akhter H.D., Ali M., Ali S.-E., Angyal A., Ansari M.A., Arancibia-Cárcamo C.V., Brown H., Chinnakannan S., Conlon C., de Lara C., de Silva T., Dold C., Dong T., Donnison T., Eyre D., Flaxman A., Fletcher H., Gardner J., Grist J.T., Hackstein C.-P., Jaruthamsophon K., Jeffery K., Lambe T., Lee L., Li W., Lim N., Matthews P.C., Mentzer A.J., Moore S.C., Naisbitt D.J., Ogese M., Ogg G., Openshaw P., Pirmohamed M., Pollard A.J., Ramamurthy N., Rongkard P., Rowland-Jones S., Sampson O., Screaton G., Sette A., Stafford L., Thompson C., Thomson P.J., Thwaites R., Vieira V., Weiskopf D., Zacharopoulou P., Turtle L., Klenerman P., Goulder P., Frater J., Barnes E., Dunachie S. T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses. Nat. Commun. 2021;12(1) doi: 10.1038/s41467-021-21856-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. O'Leary J.G., Goodarzi M., Drayton D.L., von Andrian U.H. T cell– and B cell–independent adaptive immunity mediated by natural killer cells. Nat. Immunol. 2006;7(5):507–516. doi: 10.1038/ni1332. [DOI] [PubMed] [Google Scholar]
  38. Painter MM, Mathew D, Goel RR, Apostolidis SA, Pattekar A, Kuthuru O, et al. Rapid Induction of Antigen-Specific Cd4(+) T Cells Is Associated with Coordinated Humoral and Cellular Immunity to Sars-Cov-2 Mrna Vaccination. Immunity (2021) 54(9):2133-42 e3. Epub 2021/08/29. doi: 10.1016/j.immuni.2021.08.001. [DOI] [PMC free article] [PubMed]
  39. Plotkin S.A. Correlates of protection induced by vaccination. Clin. Vaccine Immunol. 2010;17(7):1055–1065. doi: 10.1128/CVI.00131-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Porichis F., Hart M.G., Zupkosky J., Barblu L., Kwon D.S., McMullen A., Brennan T., Ahmed R., Freeman G.J., Kavanagh D.G., Kaufmann D.E., Doms R.W. Differential Impact of PD-1 and/or Interleukin-10 Blockade on HIV-1-Specific CD4 T cell and antigen-presenting cell functions. J. Virol. 2014;88(5):2508–2518. doi: 10.1128/JVI.02034-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Reiss S, Baxter AE, Cirelli KM, Dan JM, Morou A, Daigneault A, et al. Comparative Analysis of Activation Induced Marker (Aim) Assays for Sensitive Identification of Antigen-Specific Cd4 T Cells. PLoS One (2017) 12(10):e0186998. Epub 2017/10/25. doi: 10.1371/journal.pone.0186998. [DOI] [PMC free article] [PubMed]
  42. Rogers T.F., Zhao F., Huang D., Beutler N., Burns A., He W.-T., Limbo O., Smith C., Song G.e., Woehl J., Yang L., Abbott R.K., Callaghan S., Garcia E., Hurtado J., Parren M., Peng L., Ramirez S., Ricketts J., Ricciardi M.J., Rawlings S.A., Wu N.C., Yuan M., Smith D.M., Nemazee D., Teijaro J.R., Voss J.E., Wilson I.A., Andrabi R., Briney B., Landais E., Sok D., Jardine J.G., Burton D.R. Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science. 2020;369(6506):956–963. doi: 10.1126/science.abc7520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rydyznski C., Daniels K.A., Karmele E.P., Brooks T.R., Mahl S.E., Moran M.T., Li C., Sutiwisesak R., Welsh R.M., Waggoner S.N. Generation of cellular immune memory and B-cell immunity is impaired by natural killer cells. Nat. Commun. 2015;6(1) doi: 10.1038/ncomms7375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rydyznski Moderbacher C., Ramirez S.I., Dan J.M., Grifoni A., Hastie K.M., Weiskopf D., Belanger S., Abbott R.K., Kim C., Choi J., Kato Y.u., Crotty E.G., Kim C., Rawlings S.A., Mateus J., Tse L.P.V., Frazier A., Baric R., Peters B., Greenbaum J., Ollmann Saphire E., Smith D.M., Sette A., Crotty S. Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity. Cell. 2020;183(4):996–1012.e19. doi: 10.1016/j.cell.2020.09.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Seow J., Graham C., Merrick B., Acors S., Pickering S., Steel K.J.A., Hemmings O., O’Byrne A., Kouphou N., Galao R.P., Betancor G., Wilson H.D., Signell A.W., Winstone H., Kerridge C., Huettner I., Jimenez-Guardeño J.M., Lista M.J., Temperton N., Snell L.B., Bisnauthsing K., Moore A., Green A., Martinez L., Stokes B., Honey J., Izquierdo-Barras A., Arbane G., Patel A., Tan M.K.I., O’Connell L., O’Hara G., MacMahon E., Douthwaite S., Nebbia G., Batra R., Martinez-Nunez R., Shankar-Hari M., Edgeworth J.D., Neil S.J.D., Malim M.H., Doores K.J. Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans. Nat. Microbiol. 2020;5(12):1598–1607. doi: 10.1038/s41564-020-00813-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Soresina A., Moratto D., Chiarini M., Paolillo C., Baresi G., Focà E., Bezzi M., Baronio B., Giacomelli M., Badolato R., Eigenmann P. Two X‐linked agammaglobulinemia patients develop pneumonia as COVID‐19 manifestation but recover. Pediatr. Allergy Immunol. 2020;31(5):565–569. doi: 10.1111/pai.13263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Clinical Spectrum of Sars-Cov-2 Infection (2021). Available from: https://www.covid19treatmentguidelines.nih.gov/overview/clinical-spectrum/.
  48. Stary V., Pandey R.V., Strobl J., Kleissl L., Starlinger P., Pereyra D., Weninger W., Fischer G.F., Bock C., Farlik M., Stary G. A discrete subset of epigenetically primed human NK cells mediates antigen-specific immune responses. Sci. Immunol. 2020;5(52) doi: 10.1126/sciimmunol.aba6232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sun JC, Beilke JN, Bezman NA, Lanier LL. Homeostatic Proliferation Generates Long-Lived Natural Killer Cells That Respond against Viral Infection. J Exp Med (2011) 208(2):357-68. Epub 2011/01/26. doi: 10.1084/jem.20100479. [DOI] [PMC free article] [PubMed]
  50. Vabret N., Britton G.J., Gruber C., Hegde S., Kim J., Kuksin M., Levantovsky R., Malle L., Moreira A., Park M.D., Pia L., Risson E., Saffern M., Salomé B., Esai Selvan M., Spindler M.P., Tan J., van der Heide V., Gregory J.K., Alexandropoulos K., Bhardwaj N., Brown B.D., Greenbaum B., Gümüş Z.H., Homann D., Horowitz A., Kamphorst A.O., Curotto de Lafaille M.A., Mehandru S., Merad M., Samstein R.M., Agrawal M., Aleynick M., Belabed M., Brown M., Casanova-Acebes M., Catalan J., Centa M., Charap A., Chan A., Chen S.T., Chung J., Bozkus C.C., Cody E., Cossarini F., Dalla E., Fernandez N., Grout J., Ruan D.F., Hamon P., Humblin E., Jha D., Kodysh J., Leader A., Lin M., Lindblad K., Lozano-Ojalvo D., Lubitz G., Magen A., Mahmood Z., Martinez-Delgado G., Mateus-Tique J., Meritt E., Moon C., Noel J., O’Donnell T., Ota M., Plitt T., Pothula V., Redes J., Reyes Torres I., Roberto M., Sanchez-Paulete A.R., Shang J., Schanoski A.S., Suprun M., Tran M., Vaninov N., Wilk C.M., Aguirre-Ghiso J., Bogunovic D., Cho J., Faith J., Grasset E., Heeger P., Kenigsberg E., Krammer F., Laserson U. Immunology of COVID-19: Current State of the Science. Immunity. 2020;52(6):910–941. doi: 10.1016/j.immuni.2020.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Van Damme P., Van Herck K. A review of the long-term protection after hepatitis A and B vaccination. Travel Med. Infect. Dis. 2007;5(2):79–84. doi: 10.1016/j.tmaid.2006.04.004. [DOI] [PubMed] [Google Scholar]
  52. Vargas-Inchaustegui D.A., Xiao P., Tuero I., Patterson L.J., Robert-Guroff M. NK and CD4 + T Cell Cooperative Immune Responses Correlate with Control of Disease in a Macaque Simian Immunodeficiency Virus Infection Model. J.I. 2012;189(4):1878–1885. doi: 10.4049/jimmunol.1201026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vivier E., Raulet D.H., Moretta A., Caligiuri M.A., Zitvogel L., Lanier L.L., Yokoyama W.M., Ugolini S. Innate or adaptive immunity? The example of natural killer cells. Science. 2011;331(6013):44–49. doi: 10.1126/science.1198687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Walsh E.E., Frenck R.W., Falsey A.R., Kitchin N., Absalon J., Gurtman A., Lockhart S., Neuzil K., Mulligan M.J., Bailey R., Swanson K.A., Li P., Koury K., Kalina W., Cooper D., Fontes-Garfias C., Shi P.-Y., Türeci Ö., Tompkins K.R., Lyke K.E., Raabe V., Dormitzer P.R., Jansen K.U., Şahin U., Gruber W.C. Safety and immunogenicity of two RNA-based covid-19 vaccine candidates. N. Engl. J. Med. 2020;383(25):2439–2450. doi: 10.1056/NEJMoa2027906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Weinreich D.M., Sivapalasingam S., Norton T., Ali S., Gao H., Bhore R., Musser B.J., Soo Y., Rofail D., Im J., Perry C., Pan C., Hosain R., Mahmood A., Davis J.D., Turner K.C., Hooper A.T., Hamilton J.D., Baum A., Kyratsous C.A., Kim Y., Cook A., Kampman W., Kohli A., Sachdeva Y., Graber X., Kowal B., DiCioccio T., Stahl N., Lipsich L., Braunstein N., Herman G., Yancopoulos G.D. REGN-COV2, a neutralizing antibody cocktail, in outpatients with Covid-19. N. Engl. J. Med. 2021;384(3):238–251. doi: 10.1056/NEJMoa2035002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weiskopf S.R., Rubenstein M.A., Crozier L.G., Gaichas S., Griffis R., Halofsky J.E., Hyde K.J.W., Morelli T.L., Morisette J.T., Muñoz R.C., Pershing A.J., Peterson D.L., Poudel R., Staudinger M.D., Sutton-Grier A.E., Thompson L., Vose J., Weltzin J.F., Whyte K.P. Climate change effects on biodiversity, ecosystems, ecosystem services, and natural resource management in the United States. Sci. Total Environ. 2020;733:137782. doi: 10.1016/j.scitotenv.2020.137782. [DOI] [PubMed] [Google Scholar]
  57. Wijaya RS, Read SA, Truong NR, Han S, Chen D, Shahidipour H, et al. Hbv Vaccination and Hbv Infection Induces Hbv-Specific Natural Killer Cell Memory. Gut (2021) 70(2):357-69. Epub 2020/04/02. doi: 10.1136/gutjnl-2019-319252. [DOI] [PubMed]
  58. Witkowski M, Tizian C, Ferreira-Gomes M, Niemeyer D, Jones TC, Heinrich F, et al. Untimely Tgfbeta Responses in Covid-19 Limit Antiviral Functions of Nk Cells. Nature (2021). Epub 2021/10/26. doi: 10.1038/s41586-021-04142-6. [DOI] [PubMed]
  59. Yu J., Tostanoski L.H., Peter L., Mercado N.B., McMahan K., Mahrokhian S.H., Nkolola J.P., Liu J., Li Z., Chandrashekar A., Martinez D.R., Loos C., Atyeo C., Fischinger S., Burke J.S., Slein M.D., Chen Y., Zuiani A., Lelis F.J.N., Travers M., Habibi S., Pessaint L., Van Ry A., Blade K., Brown R., Cook A., Finneyfrock B., Dodson A., Teow E., Velasco J., Zahn R., Wegmann F., Bondzie E.A., Dagotto G., Gebre M.S., He X., Jacob-Dolan C., Kirilova M., Kordana N., Lin Z., Maxfield L.F., Nampanya F., Nityanandam R., Ventura J.D., Wan H., Cai Y., Chen B., Schmidt A.G., Wesemann D.R., Baric R.S., Alter G., Andersen H., Lewis M.G., Barouch D.H. DNA vaccine protection against SARS-CoV-2 in rhesus macaques. Science. 2020;369(6505):806–811. doi: 10.1126/science.abc6284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zheng M., Gao Y., Wang G., Song G., Liu S., Sun D., Xu Y., Tian Z. Functional exhaustion of antiviral lymphocytes in COVID-19 patients. Cell. Mol. Immunol. 2020;17(5):533–535. doi: 10.1038/s41423-020-0402-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zost S.J., Gilchuk P., Case J.B., Binshtein E., Chen R.E., Nkolola J.P., Schäfer A., Reidy J.X., Trivette A., Nargi R.S., Sutton R.E., Suryadevara N., Martinez D.R., Williamson L.E., Chen E.C., Jones T., Day S., Myers L., Hassan A.O., Kafai N.M., Winkler E.S., Fox J.M., Shrihari S., Mueller B.K., Meiler J., Chandrashekar A., Mercado N.B., Steinhardt J.J., Ren K., Loo Y.-M., Kallewaard N.L., McCune B.T., Keeler S.P., Holtzman M.J., Barouch D.H., Gralinski L.E., Baric R.S., Thackray L.B., Diamond M.S., Carnahan R.H., Crowe J.E. Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature. 2020;584(7821):443–449. doi: 10.1038/s41586-020-2548-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zuo J., Dowell A.C., Pearce H., Verma K., Long H.M., Begum J., Aiano F., Amin-Chowdhury Z., Hoschler K., Brooks T., Taylor S., Hewson J., Hallis B., Stapley L., Borrow R., Linley E., Ahmad S., Parker B., Horsley A., Amirthalingam G., Brown K., Ramsay M.E., Ladhani S., Moss P. Robust SARS-CoV-2-specific T cell immunity is maintained at 6 months following primary infection. Nat. Immunol. 2021;22(5):620–626. doi: 10.1038/s41590-021-00902-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data 1
mmc1.pdf (1MB, pdf)

Data Availability Statement

Data will be made available on request.

Articles from Immunobiology are provided here courtesy of Elsevier

RESOURCES