Dynamics of NK, CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID‐19

Abstract Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8+ T, CD4+ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8+ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3+ Tfh‐like cell and CD226+ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS+ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8+ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4+ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.


| INTRODUC TI ON
The outbreak of coronavirus disease 2019 (COVID- 19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an urgent threat to global health. As it is a pandemic, research on its clinical characteristics, treatment, immune response and vaccine development is urged to be conducted.
Natural killer (NK) cells, CD8 + T, CD4 + T and B lymphocytes are essential in antiviral immune response. In a study, the number of NK, T and B cells in various types of viral infection has been reported to be different, indicating a potential correlation between these differences and viral pathogenic mechanisms. 1 In other studies, the dysregulation of immune response was observed in patients with severe COVID-19. 2,3 Moreover, a multicenter retrospective study revealed that lower lymphocyte count was an independent high-risk factor associated with COVID-19 progression. 4 Furthermore, an increase in the number of peripheral lymphocyte subset in patients with COVID-19 was associated with improved clinical symptoms and treatment efficacy. 3,5 In patients with severe COVID-19, lymphopenia resulted from drastically reduced numbers of NK, CD8 + T, CD4 + T and B cells, but not in patients with mild or moderate disease 2,6-8 ; these studies mainly focused on the correlation between lympho- Infection with SARS-CoV-2 can activate innate and adaptive immune responses. NK cells participate in the elimination of virus-infected cells without viral antigen presentation. 9 Cytotoxic T lymphocyte (CTL), an activated subset of CD8 + T cells, can kill virus-infected cells by releasing perforin. 10 CD4 + T cells, particularly T follicular help (Tfh) cells, can promote the production of virus-specific antibodies by activating Tfh-dependent B cells. 11 Moreover, peripheral Tfh-like cells were defined as CXCR5 + CD4 + T cells. 12 After viral infection, the activated immune system would not only directly induce antiviral cellular and humoural immune responses but also develop memory CD4 + and CD8 + T cell subsets as a preparation for secondary infection. 13 B cells differentiate from naïve or transitional to mature cells and finally differentiate into plasma cells, which produce virus-specific antibodies. In addition to the detection of SARS-CoV-2-specific antibodies for the diagnosis of COVID- 19,14 these specific antibodies may neutralize SARS-CoV-2. Furthermore, we analysed other biomolecules, including inhibitory cell surface molecules: T-cell immunoglobulin and mucin 3 (TIM-3), programmed cell death-1 (PD-1), T-cell immunoglobulin and ITIM domain (TIGIT), and active cell surface molecules: inducible costimulatory molecule (ICOS) and CD226, which could bind to CD155 on antigen-presenting cells (APC) and compete with TIGIT. 15,16 The serum inflammatory cytokine levels in patients with severe COVID-19 were observed to be abnormally increased and were used to predict the risk of cytokine storm. 17,18 Furthermore, serum interleukin (IL)-6, IL-1β, IL-8, IL-10, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) levels were analysed to determine the variability of lymphocyte subsets in the present study.
Although patients with severe COVID-19 have high mortality, most patients with the disease exhibit only mild to moderate symptoms. 6,7 Given that SARS-CoV-2 is not a well-known virus, the host resistance of virus-infected patients is a key factor in determining the success or failure of host recovery. The analyses of multiple NK, CD8 + T, CD4 + T and B cell subsets, the production of virus-specific antibodies, the expression of associated-activation and exhaustion molecules, the secretion of inflammatory cytokines in patients with moderate COVID-19 during convalescent period, and the identification of the differences in the cell subsets between SARS-CoV-2 and other pathogenic microorganism (non-SARS-CoV-2) infection would provide useful information on immune response against SARS-CoV-2.

| Patients
A total of 11 patients with COVID-19 (admitted from 31 January to 9 February 2020) and 11 patients with non-SARS-CoV-2 pneumonia received a second phenotyping test on 25 February 2020, as one patient had been discharged at the time. All patients with COVID-19 were administered Kaletra as an antiviral therapy and given the corresponding symptomatic treatment upon the diagnosis of COVID- 19. Arbidol (or ribavirin) was administered in patients with persistent SARS-CoV-2 RNA. The patients whose nasopharyngeal swab tested positive for SARS-CoV-2 were administered interferon-α through atomization. Antibiotics were used in patients with COVID-19, when a combination of bacterial infections was identified. Patients with non-SARS-CoV-2 pneumonia were administered moxifloxacin or levofloxacin. The design of this study was approved by the Ethics Committee of West China Hospital, and all participating patients provided written informed consent prior to enrolment.

| Data collection
Epidemiological history, clinical symptoms, laboratory data, chest CT results and treatment regimen records were collected from the electronic hospital and laboratory information system. To verify data accuracy, the medical records of the patients were independently reviewed by two researchers (XH and GXD). The severity of COVID-19 was de-

| Phenotyping
To determine the NK cells and lymphocyte subsets in patients, hep-

| Statistics
Continuous and categorical variables are presented as median

| Demographic data and clinical characteristics of patients with moderate COVID-19 and control patients
The demographic data and clinical characteristics of patients with moderate COVID-19 who were enrolled are listed in Table 1 and  Table 1.

| NK cells and lymphocyte subsets in patients with COVID-19 and control patients
No significant difference in the total number of NK cells and lymphocyte subsets, the absolute number (or percentage) of CD8 + T, CD4 + T or B cells was found between patients with moderate COVID-19 and control patients ( Figure 2B). The percentage of Tfh-like cells was significantly lower in patients with COVID-19 than in the control patients ( Figure 2F). No significant differences in the absolute number or percentage of naïve CD4 + or CD8 + , memory CD4 + or CD8 + , cells, TIM-3 + , TIGIT + , PD-1 + , ICOS + , CD226 + Tfh-like cells and B-cell subsets at different differentiation stages were observed between the two groups (Data S1,S2). In summary, only NK cells and Tfh-like cells were observed to be different between COVID-19-infected patients and the control patients.

| Changes in the number of NK cells and lymphocyte subsets in patients with COVID-19 during convalescent period
The absolute number of the total NK cells and lymphocyte subsets in patients with COVID-19 increased during the convalescent period  Figure 3A). This increase mainly resulted from the increase in NK, CD8 + T and CD4 + T cells ( Figure 3B). A decrease in B cells ( Figure 3B) was observed in some cases. In summary, the number of NK cells and lymphocyte subsets increased in most patients with COVID-19 during the convalescent period.

| Changes in the number of CD8 + T, CD4 + T and B cell subsets in patients with COVID-19 during convalescent period
The number of effector memory CD8 + (CD45RO + CCR7 − CD8 + ) T cells in patients with COVID-19 increased during the convales-

| D ISCUSS I ON
The outbreak of the highly contagious SARS-CoV-2 was sudden, and the various pathological damages and related pathogenesis COVID- 19 have not yet been fully understood. Many researchers have confirmed in recent studies that the immune response is a doubleedged sword in killing viruses or exacerbating immune damage. 17,18 The present study is the first to report a dynamic analysis of NK  A recent study reported that respiratory SARS-CoV-2 viral load in mild patients decreased after reaching a peak during the second week after the onset of disease. 33 In the present study, we found that

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.