Identifying SARS-CoV-2 'memory' NK cells from COVID-19 convalescent donors for adoptive cell therapy

Abstract

COVID-19 disease is the manifestation of syndrome coronavirus 2 (SARS-CoV-2) infection, which is causing a worldwide pandemic. This disease can lead to multiple and different symptoms, being lymphopenia associated with severity one of the most persistent. Natural killer cells (NK cells) are part of the innate immune system, being fighting against virus-infected cells one of their key roles. In this study, we determined the phenotype of NK cells after COVID-19 and the main characteristic of SARS-CoV-2-specific-like NK population in the blood of convalescent donors. CD57+ NKG2C+ phenotype in SARS-CoV-2 convalescent donors indicates the presence of 'memory'/activated NK cells as it has been shown for cytomegalovirus infections. Although the existence of this population is donor dependent, its expression may be crucial for the specific response against SARS-CoV-2, so that, it gives us a tool for selecting the best donors to produce off-the-shelf living drug for cell therapy to treat COVID-19 patients under the RELEASE clinical trial (NCT04578210).

Keywords: COVID-19; HLA; KIR; NK cells; cell therapy.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.

Figures

FIGURE 1

Comparison of the expression levels of different surface markers in NK cells of COVID‐19 convalescent donors of different severities. (a) Expression of CD57, NKG2C and CD16 markers in asymptomatic, mild, moderate, severe convalescent donors and healthy donors. (b) Expression of NKp30, NKG2D and NKp46 markers in asymptomatic, mild, moderate, severe convalescent donors and healthy donors. (c) Expression of CD158b, CD158a, CD158e and NKG2A markers in asymptomatic, mild, moderate, severe convalescent donors and healthy donors. (d) Expression of CTLA4, TIGIT, TIM3 and PD1 markers in asymptomatic, mild, moderate, severe convalescent donors and healthy donors. Asymptomatic (n = 11), mild (=22), moderate (n = 6), severe (n = 1) convalescent donors and healthy (n = 6) donors. Each dot corresponds to an individual, and the mean with the standard error of the mean (SEM) is shown. Student's t‐test was used to analyse the data. p‐value: *p  <  0·05, **p  <  0·005, ***p  <  0·001. ****p  <  0·0001

FIGURE 2

Response of convalescent donor NK cells to SARS‐CoV‐2‐specific peptides measured by IFN‐γ production. (a) Percentage of NK cells recovered from PBMCs of blood samples, in purple from convalescent donors (n = 6), in yellow from healthy donors (n = 3). Each dot corresponds to an individual, and the mean with the standard error of the mean (SEM) is shown. (b) Representative figure of gating strategy of the different cell subsets within de CD3‐ CD56+ population (NK cells). On the right below, figure expression of IFN‐γ production by healthy donors’ NK cells. (c) Representative figure of CD16+ population analysis within the CD57+/NKG2C+ population (top), and the CD57+/NKG2C+/IFN‐γ+ population (bottom). (d) Percentage of CD16+ population expression within CD56+ subset, CD57+/NKG2C+ subset and IFN‐γ+ subset of convalescent donors. Each dot corresponds to an individual, and the mean with the standard error of the mean (SEM) is shown. (e) Percentage of IFN‐γ production done by convalescent donors’ NK cells in response to the presence of three different SARS‐CoV‐2 peptides (M, N and S)

FIGURE 3

Expression of the activated population CD57 + NKG2C+ and the naive NK cells population CD62L+ NKG2A+ in the convalescent donors. (a) Representative figure of the expression of CD57 + NKG2C+ and the naive NK cell population CD62L+ NKG2A+ of the best responder (D2) and the worst responder (D4). On the left column, these populations are contained within the CD3‐ CD56+ population, and on the right column, these populations are contained within the CD3‐ CD56+ IFN‐γ + population. (b) Table summarizing the percentage of CD57+ NKG2C+ and CD62L+ in each convalescent donor of IFN‐γ production assay

FIGURE 4

Flow cytometry analysis of NK cells from leukapheresis donors before and after the purification of NK cells. (a) Representative figure of CD57+ NKG2C+ activated population expression on NK cells. On the left, before the purification, and on the right, after purification. (b) Representative figure of CD16+ NK cell maturation marker expression. On the left, before the purification, and on the right, after purification. (c) Comparison of the expression levels of different surface markers in NK cells before and after CliniMACS NK cell purification. (n = 2). Each dot corresponds to an individual, and the mean with the standard error of the mean (SEM) is shown

FIGURE 5

Comparison of the condition of NK cells without freezing and after thawing with different freezing media (FM). (a) Viability of the NK cells in the fresh, FM1, FM2 and FM3 condition (n = 2). (b) Representative figure of the functionality measured by degranulation assay against K562 target cells in the four different conditions. (c) Representative figure of CD57+ NKG2C+ activated population expression on NK cells in the four different conditions. (d) Representative figure of CD16+ marker expression in the four different conditions. The bars represent the mean and error bars represent SEM

FIGURE 6

Potential mechanism of adaptive/‘memory’ NK cells with CD57+/NKG2C+ phenotype activation in SARS‐CoV‐2 infection. (a) Pro‐inflammatory environment with NK cells secretes IFN‐γ that leads to an increase in HLA−E expression. (b) NK cells expressing NKG2C are activated through this receptor by HLA−E presentation. This could lead to an adaptive NK cell. (c) Adaptive NK cells could be activated by the recognition of SARS‐CoV‐2‐specific soluble peptides via NKG2C, which could enter a pro‐inflammatory status secreting IFN‐γ. (d) Activated adaptive/‘memory’ NK cells secrete IFN‐γ in response to SARS‐CoV‐2 soluble peptide‐specific activation