Naturally activated adaptive immunity in COVID‐19 patients

Abstract Coronavirus disease‐2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) has rapidly spread worldwide, threatening the health and lives of many people. Unfortunately, information regarding the immunological characteristics of COVID‐19 patients remains limited. Herein, we collected blood samples from 18 healthy donors (HDs) and 38 COVID‐19 patients to analyse changes in the adaptive immune cell populations and their phenotypes. We observed that the lymphocyte percentage moderately decreased, CD4 and CD8 T cell percentage among lymphocytes were similar, and B cell percentage was increased in COVID‐19 patients in comparison to that in HDs. T cells, especially CD8 T cells, showed an enhanced expression of late activation marker CD25 and exhaustion marker PD‐1. Importantly, SARS‐CoV‐2 infection increased the percentage of T follicular helper– and germinal centre B–like cells in the blood. The parameters in COVID‐19 patients remained unchanged across various age groups. Therefore, we demonstrated that the T and B cells are activated naturally and are functional during SARS‐CoV‐2 infection. These data provide evidence that the adaptive immunity in most patients could be primed to induce a significant immune response against SARS‐CoV‐2 infection upon receiving standard medical care.


| INTRODUC TI ON
A severe pneumonia-associated respiratory syndrome began in Wuhan, China, in December 2019, which was subsequently declared as a public health emergency of international concern by WHO. The novel coronavirus strain was officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1,2 Coronavirus infections, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), can cause severe respiratory diseases. 3,4 SARS-CoV-2 is an enveloped positive-strand RNA virus, which belongs to the same family as SARS-CoV and MERS-CoV based on genome similarity, Coronaviridae. 2,[5][6][7] A number of studies have demonstrated that the adaptive immunity responds to coronavirus infections and is required for efficient clearance of the virus. In patients infected with SARS-CoV, the acute phase of infection is associated with a severe reduction in T cell numbers in the blood, involving a dramatic loss of CD4 and CD8 T cells in comparison to healthy control individuals. 8,9 This suggests that SARS-CoV infection impairs cellular immunity in the early stages of the disease. With the prolonged recovery time of SARS-infected patients, expression of activated T cell markers, such as CD69 and CD25, decreases, 10,11 indicating that T cell activation in response to the virus is impaired. 12 With the improvement of the disease, the ratio of CD4 to CD8 T cells increases, indicating that CD4 T cells recover faster than CD8 T cells. 13 In addition, out of the 92% of cured SARS patients whose B cells initially declined and then increased or continued to increase during the course of the disease, only 8% had a constant or decreasing cell count. 14 Similar to SARS patients, leucopenia and lymphopenia are also observed in MERS patients, albeit to a lesser degree than that observed in SARS patients. A clinical study showed that 14% of MERS patients had leucopenia, while 34% of the patients had lymphopenia. 15 MERS-CoV-infected patients that exhibited distinctively high frequencies of MERS coronavirus-reactive CD8 T cells were associated with severe/moderate illness, whereas CD4 T cell response was minimally detected at this stage. In the convalescent phase, a moderate increase in CD4 T cells was detected. 15 Currently, very few studies have reported that COVID-19 patients develop lymphopenia and exhibit an increase in pro-inflammatory cytokines in severe condition. [16][17][18] The information on changes in immune cells and their functions in response to SARS-CoV-2 infection is still very limited. Based on the fact that T and B cells respond to infections and play critical roles in defending against virus infections, a systematic study on the changes in T and B cells in COVID-19 patients will help uncover the immune response against SARS-CoV-2 infection and will also provide insights for COVID-19 diagnosis and treatment.
A new study showed that people infected with betacoronaviruses including SARS-CoV and MERS could establish T cell immunity to nucleocapsid protein (NP). 19 The analysis of blood samples of 14 COVID-19 patients displayed a strong correlation between neutralization antibody titres and the numbers of virus-specific T cells. 20 Wen et al reported that during the recovery stage of COVID-19, plasma cells underwent a significant increase, whereas naïve B cells decreased remarkably. 21 Several studies reported that SARS-CoV-2 elicits a robust B cell response, as evidenced by the detection of virus-specific IgM, IgA and neutralizing IgG antibodies (nAbs) in the days following infection. 20,22 Importantly, Zost and colleagues identified several human monoclonal antibodies (mAbs) targeting the spike (S) glycoprotein, which exhibited potent neutralizing activity and fully blocked the receptor-binding domain of S (SRBD) from interacting with human ACE2 (hACE2). 23 In addition, two of the most potently ACE2 blocking mAbs have been proven to protect rhesus macaques from SARS-CoV-2 infection. 23 However, further studies will be required to identify, design and synthesize antibodies and drugs targeting SARS-CoV-2.
In this study, we analysed the blood samples from 18 healthy    (Table 1).

| Flow cytometry analysis
The Abs used in the flow cytometry analysis were as follows: and FITC antimouse/human GL7 Antigen (GL7). They were purchased from BioLegend. Blood cells were stained with Abs in the dark at room temperature for 15 minutes and analysed on a FACSCanto II flow cytometer (BD Biosciences). FlowJo 8 was used for data analysis.

| Statistical analysis
The continuous variable of normal distribution is represented by mean ± standard deviation, the non-normal distribution is represented by median [IQR], and the classified variable is represented by count (percentage). Student's t test was performed for two group analysis using SPSS 22.0 software. * and ** stand for P<0.05 and P<0.01, respectively.

| The percentage analysis of T and B cells in COVID-19 patients
To determine the change in the composition of adaptive immune cells, we compared the percentage of T and B cells in the blood samples from HDs and COVID-19 patients using flow cytometry.
Lymphocyte percentage in the whole blood was not significantly different between HDs and COVID-19 patients, though it exhibited a decreasing trend in the patients ( Figure 1A). Within the lymphocyte population, the percentages of CD4 + and CD8 + T cells were comparable ( Figure 1B and C), whereas B cell percentage was significantly increased ( Figure 1D) in COVID-19 patients.

| An activated phenotype of T cells in COVID-19 patients
To evaluate the T cell status in response to SARS-CoV-2 infection, we analysed the expression of CD69, CD25, PD-1, CD45RA, CD45RO and CXCR3 in both CD4 + and CD8 + T cells. In CD4 + T cells of the COVID-19 patients, the expression of CD69 and CD25 (Figure 2A

| An increase in germinal centre-like cells in COVID-19 patients
T follicular helper (Tfh) cells help in activation of B cells and differentiation into effector cells, production of high-affinity antibodies and formation of germinal centres. 24 To study whether COVID-19 patients produced efficient adaptive immune response, we analysed the expression of PD-1 and CXCR5 in CD4 + T cells and the expression of Fas and GL7 in B cells. As shown in Figure 4A

| Correlation analysis between activation signature and patient age
To study whether age affects adaptive immune cell populations and effector features, we performed correlation analysis between T cell activation markers and age. No dramatic change was observed with increase in age of the patients ( Figure 5). The results indicate that there was no defect in CD8 + T cell activation and Tfh-and GCB-like cell differentiation in the aged individuals infected by SARS-CoV-2. Collectively, these studies provide a first glimpse into the phenotypes of T and B cell subsets associated with COVID-19. However, the relevant conclusions need to be interpreted with caution due to the limited number of patients enrolled in these studies. 28 Therefore, further investigation is needed to better determine the phenotype and function of T and B cell subsets in COVID-19
Lymphopenia was observed in COVID patients in previous studies, 25 Epidemiological investigation of coronavirus infection showed that lymphopenia is present in more than 80% of the patients, and serious decline in lymphocytes is correlated with a poor prognosis. 29 However, we did not observe a significant decrease in lymphocyte populations in the COVID-19 patients. This finding could be attributed to the fact that most of the patients in this study showed mild symptoms besides fever. Interestingly, following the division of patients into symptomatic and asymptomatic, we observed a decrease in lymphocytes in the symptomatic patients (data not shown).
PD-1 is a marker of exhausted T cells during chronic and acute infections. 30 Elderly individuals typically exhibit a reduction in the lymphocyte populations and a weaker ability to defend against viral infections. 34 In our correlation analysis, we did not observe a significant correlation between lymphocyte proportions, effector features and age. Our data suggest that the specific populations of T and B cells for SARS-CoV-2 are reserved in aged individuals; this needs to be proven by repertoire sequencing analysis of T cell and B cell receptors in elderly patients.
In summary, our study shows that SARS-CoV-2 could induce relatively normal adaptive immune response in patients. Most people across different age groups are capable of mobilizing the adaptive immune cells, and activating cellular and humoral immunity to defend against the virus with sufficient medical care and anti-viral treatment.

CO N FLI C T O F I NTE R E S T
We declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data, models and code generated or used during the study are available in the submitted article.