Flow cytometric analysis of lymphocyte subsets, monocytes, and HLA-DR expressions on these cells in patients with COVID-19

Introduction

The novel coronavirus (SARS-CoV-2) infection (COVID-19) was reported to have emerged in Wuhan, China, in December 2019. Since then, it has spread first in China and then all over the world, including our country [1]. The main symptoms have been reported as fever, dry cough, myalgia, and fatigue [2]. Some patients may additionally exhibit headaches, hemoptysis, and diarrhea. The disease may progress from asymptomatic and mild form to pneumonia and acute respiratory distress syndrome (ARDS), resulting in secondary infections and complications such as pneumothorax [3]. Five variants of SARS-CoV-2 designated by the World Health Organization, including Omicron, have been reported, and the disease still leads to deaths despite effective vaccination programs [4].

Monocytic phagocytic cells, lymphocytes, and their subsets play a critical role in maintaining the immune response to COVID-19. Lymphocyte subsets were previously analyzed in patients with SARS-CoV from the same viral family, suggesting that the lymphocyte count may be associated with the severity of the disease [5]. However, the immune response to the disease has not yet been fully elucidated. Previous publications have shown leukopenia and lymphopenia in COVID-19, especially a decrease in T, B, and NK lymphocytes [5, 6]. Studies also investigate alterations in T lymphocyte subsets and the CD4/CD8 ratio, but the results are inconsistent. Some publications have reported decreased CD4 and CD8 subset counts but no change in the CD4/CD8 ratio, while others showed no change in CD4 counts but decreased CD8 counts [5, 7]. However, studies investigating lymphocyte and monocyte activation in COVID-19 have been gaining importance in the literature. Today, multiparametric flow cytometry is considered the gold standard for the detection of lymphocyte subsets and activation of cells using percentages of HLA-DR expression on lymphocytes [8, 9]. The intensity of antigen expression (median fluorescent intensity, MFI) on lymphocytes and the internal complexity of cells (side scatter, SSC) can also be used to evaluate the activation of lymphocytes. However, there are a limited number of studies investigating MFI values of HLA-DR expression but no study about neutrophil SSC/lymphocyte SSC ratio in patients with COVID-19 [10, 11].

In this study, we aimed to investigate the lymphocyte subsets, monocytes, MFI of HLA-DR expression, and SSC of lymphocytes by flow cytometry and to detect any correlations between antigen expressions and inflammatory markers in patients with COVID-19.

Materials and methods

Flow cytometry analysis

Flow cytometric analysis of lymphocyte subsets was carried out using the same samples used for the automated blood count autoanalyzer. The lysis no-wash method was performed in accordance with the product manual. In brief, the study tube was labeled with the patient’s name and antibodies inside. After pipetting 10 µL of antibodies (CD3 Allophycocyanin-Alexa Fluor 750 (APC-A750), CD4 Phycoerythrin-Texas Red-X (ECD), CD8 Phycoerythrin-Cyanin 7 (PC7), CD19 Phycoerythrin-Cyanin 5 (PC5), CD56 Allophycocyanin-Alexa Fluor 700, CD16 Phycoerythrin (PE), HLA-DR Pacific Blue (PB), CD45 Krome Orange (KO) Beckman Coulter) according to the manufacturer’s instructions, 100 µL of patient blood sample was added to the labeled tubes. They were incubated in the dark for 15 min at room temperature. At the end of the incubation period, the tubes were vortexed. Then, 2 mL of lysing solution (VersaLyse Lysing Solution, Beckman Coulter) was added to the samples, vortexed, and incubated in the dark for 10 min at room temperature. After vortexing again, data collection was performed on a Beckman Coulter Navios flow cytometer (Brea, CA, USA). At least 10,000 events were collected for each sample, and the results were analyzed using Kaluza software (Treestar, Ashland, OR, USA). The percentage of antigen-expressing cells and MFI values for CD45 and HLA-DR on lymphocytes were recorded. Monocytes were analyzed by gating cells using forward scatter (FSC) and SSC, which were then applied to a CD45 × SSC dot plot. The CD45⁺ events were gated and applied to a CD4 × SSC dot plot. The CD4 dim+ cell population with low SSC to define the monocytes was gated and applied to a CD16 × CD4 dot plot for the characterization of distinct monocyte subsets and MFI of CD16 (Figure 1).

Figure 1:

Lymphocyte and monocyte subgroups gating strategy. SS INT/time (A) fluidic system control; SS INT/SS PEAK (B) singlet cells; FS INT/SS INT (C) viable cells; SSC/CD45 (D) debris exclusion; gated lymphocytes (red), monocytes (green) and granulocytes (blue) in SSC/CD45 (E); CD3⁻ CD19⁺ B lymphocytes (pink) and CD3⁺ CD19⁻ T lymphocytes (blue) (F); CD4⁺ and CD8⁺ T cells at lymphocyte gate (G); CD3⁻ CD19⁻ CD16/56⁺ NK cells (H); CD3⁺ 56⁺ NKT cells (I); CD3⁻ CD4⁺ monocytes (J, K); CD4⁺CD16⁺ monocytes and CD4⁺CD16⁻ monocytes (L).

Absolute counts of lymphocyte subsets were calculated using the “double platform” method [13]. Accordingly, absolute lymphocyte subset counts were calculated by obtaining the percentage of cells from flow cytometry and the total leukocyte count from the whole blood count analyzer. Absolute values of lymphocyte subsets were calculated by the formula: Absolute number/µL=leucocyte count × % lymphocyte × % antibody positivity/10,000.

Daily flow checks (Beckman Coulter) were used to evaluate the device’s performance. The intra-day and inter-day coefficients of variation were <3 %. Utilizing the UK NEQAS external quality evaluation method, the system’s accuracy was evaluated monthly.

Results

Peripheral lymphocyte subset alteration in COVID-19

The flow cytometric analysis results of lymphocyte subsets are shown in Table 3. Both percentages (%) and absolute counts (#) of total lymphocytes, CD3⁺ T, and CD19⁺ B lymphocytes were significantly lower in the patient group compared to the controls. The analysis of T lymphocyte subsets showed significantly lower CD4⁺ T helper and CD8⁺ T cytotoxic lymphocyte counts in the patient group (p=0.007, p<0.05) but no change in their percentages or CD4/CD8 ratios. There was no significant difference between the two groups in terms of the count and percentage of NK cells, but a significantly higher percentage of CD3⁺CD56⁺ NK-T cells was found in the patient group.

Table 3:

Comparison of immunophenotypic features of lymphocyte subsets by flow cytometry in patients with COVID-19 and healthy controls.

Parameters	COVID 19 patients			Healthy controls			p-Value
	Median	Percentile		Median	Percentile
		25	75		25	75
LYM, %	25.5	19.5	33.4	29.4	26.4	37.1	0.033
LYMa	1,468.1	1,122.4	1,868.7	2,261.4	1,670.2	2,622.6	<0.001
CD3+%, T cell	75.0	69.2	80.0	70.6	60.8	76.4	0.019
CD3+ a	1,088.4	830.7	1,384.5	1,377.1	1,042.7	1,848.9	0.008
CD3+CD4+% (T helper)	44.7	37.0	48.8	39.7	32.8	47.3	NS
CD3+CD4+ a	602.7	374.7	878.9	818.6	607.0	1,148.8	0.007
CD3+CD8+% (T cytotoxic)	26.6	20.5	35.4	25.4	19.8	31.1	NS
CD3+CD8+ a	395.4	217.3	644.0	481.1	381.6	783.4	0.05
CD4/CD8 ratio	1.7	1.1	2.3	1.6	1.1	2.6	NS
CD3−CD16+/CD56+%, NK cell	14.0	9.0	22.6	13.5	8.4	19.4	NS
CD3−CD16+/CD56+ a	199.2	110.5	337.1	234.4	179.6	389.3	NS
CD19+%, B cell	9.4	6.7	11.9	12.3	9.7	15.3	0.004
CD19+ a	139.2	88.8	204.5	275.4	181.4	385.6	<0.001
CD3+CD56+ % (NK-T cell)	1.2	0.5	2.2	0.6	0.2	1.2	0.045
CD3+CD56+ a	0.01	0.00	0.03	0.01	0.00	0.02	NS
GRAN CD45 MFI	22,764	20,493	27,552	26,334	20,600	30,359	NS
LYM CD45 MFI	25,446	23,331	27,853	34,848	33,360	36,324	<0.001
GRAN CD45 MFI/LYM CD45 MFI	0.9	0.8	1.1	0.7	0.6	0.9	<0.001
GRAN SSC value	617,472	532,480	712,704	635,904	580,608	668,672	NS
LYM SSC value	72,704	64,512	76,800	62,464	60,416	64,512	<0.001
SSC GRAN/SSC LYM	8.9	7.9	9.9	10.0	8.9	10.9	0.001

1. ^aAbsolute number of related parameters calculated by double platform; LYM, lymphocyte; GRAN, granulocyte; MFI, median fluorescence intensity; SSC, side scatter; NS, non significant.

We also found significantly lower CD45 MFI values and higher SCC values for lymphocytes in the patient group compared with controls (p<0.001, p=0.001, respectively). We also detected significantly increased CD45 MFI values of granulocyte/CD45 MFI values of lymphocytes (p<0.001) and decreased SSC of granulocytes/SSC of lymphocytes (p=0.001) (Table 3).

The percentages of HLA-DR expression on T lymphocytes, T helper, and T cytotoxic subsets were found to be significantly higher in the patient group compared with healthy controls (p=0.001, p<0.001, p<0.001, respectively) (Figure 2A). The MFI values of HLA-DR expression on T lymphocytes and the T cytotoxic subset were significantly increased (Figure 2B).

Figure 2:

Comparison of HLA-DR expression levels in CD3⁺/CD4⁺ T helper and CD3⁺/CD8⁺ T cytotoxic subsets of the patient and control groups as a percentage (A) and MFI value (B).

Discussion

This study examined the complete blood count parameters of patients with mild and moderate COVID-19. T lymphocytes and their subsets, B lymphocytes, and NK cells were analyzed by flow cytometry in patients and compared with the values of healthy controls. Moreover, SSC characteristics and the HLA-DR expressions of lymphocytes and monocytes were measured as percentages and MFI values. In addition, the correlations between antigen expressions and serum CRP, ferritin, and D-dimer values were assessed.

COVID-19 is an acute inflammatory infectious disease. Studies conducted so far have shown alterations in leukocyte and lymphocyte distribution in COVID-19 patients, similar to viral infections such as HIV and SARS. With regard to this, the NLR has been demonstrated to play an important role in the diagnosis and prognosis of viral infections as an inflammatory marker [14, 15]. Similarly, it has been reported that increased NLR is a critical predictor for the assessment of disease severity in COVID-19 patients [16], [17], [18]. While the COVID-19 patients included in the present study had lower absolute leukocyte and lymphocyte counts compared to the healthy controls, there was no significant change in neutrophil counts (Table 1). On the other hand, NLR values were higher in the patient group, which supports the results of other studies. The increased NLR in this study is due to the significant decrease in lymphocyte count. However, no significant correlation was found between NLR, CRP, and ferritin levels. Many publications on COVID-19 have suggested that the progressive reduction of lymphocyte counts in peripheral blood may be a clinical early warning indicator for severe and critical cases in adults [5, 19]. The suppressive effect of cytokines on bone marrow lymphopoiesis has been implicated as a cause of lymphopenia in COVID-19 [20]. In contrast to NLR, the results of this study showed that there was a significant negative correlation between absolute lymphocyte counts and inflammatory markers like CRP and ferritin levels in the patient group (Table 4). Accordingly, it can be suggested that decreasing lymphocyte count may be more useful than NLR because of its negative correlations with inflammatory markers.

In addition to a drop in the number of lymphocytes, changes in the subsets of lymphocytes have been linked to the prognosis of viral diseases and the effectiveness of treatments [1, 5]. A retrospective study showed low CD4⁺T and CD8⁺T lymphocyte counts in SARS patients, associated with a poor prognosis [21]. However, the characteristics of lymphocyte subsets in COVID-19 patients are largely unclear. A study showed decreased T, B, and NK counts in COVID-19 patients. The same study reported a simultaneous decrease in CD4⁺ and CD8⁺ T lymphocytes but no significant change in the CD4/CD8 ratio [5]. Another study showed a decrease in total T, CD8⁺T, and NK cell counts in COVID-19 patients compared to healthy controls but no significant difference in CD4⁺ T and B cell counts between COVID-19 patients and healthy controls [7]. In another study, researchers found decreased T and B cells, monocytes, NK, and NKT cells [22]. The present study analyzed lymphocyte subsets by flow cytometry. The CD3⁺ T lymphocyte and CD19⁺ B lymphocyte counts were significantly lower in the patient group compared to the controls. However, there was no significant difference between the groups in terms of the count and percentage of NK cells. Analysis of T lymphocyte subsets showed that the number of CD4⁺ T helper lymphocytes and CD8⁺ T cytotoxic lymphocytes was significantly lower in the patient group. However, the decrease in the number of cytotoxic T lymphocytes (17.8 %) was less than the decrease in the number of T helper lymphocytes (26.3 %). There was no significant difference in the CD4/CD8 ratio. In other words, despite no change in ratios of both T helper and cytotoxic cells, it can be stated that both parameters decrease numerically, but cytotoxic T lymphocytes are affected relatively less when compared as a percentage. It is well known that CD4⁺ and CD8⁺ T lymphocytes are the basic components of the immune system. Particularly in acute viral infections involving the lung, CD8⁺ T lymphocytes play a vital role in virus clearance [23]. Activation of CD8⁺ T cells results in the expression of cytotoxic molecules, which are responsible for killing the target cell [24]. Therefore, in addition to the numerical changes in lymphocytes, activation states should be considered in viral diseases. The HLA-DR antigen is a well-known activation marker found on T lymphocytes [25]. It has also been reported to be associated with acute viral diseases [26]. Kratzer et al. did a study on people with COVID-19 and found that HLA-DR was increasingly expressed on CD3⁺/CD8⁺ T cells 10 weeks after the infection [27]. Another study found that COVID-19 patients with mild symptoms had a higher rate of polyfunctional CD8⁺ T lymphocyte response than those with severe symptoms. This suggests that CD8⁺ T cells may help reduce the severity of the disease [28]. We measured the expression of HLA-DR on CD8⁺ T and CD4⁺ T lymphocytes to understand their activity status. Both T helper lymphocytes and T cytotoxic lymphocytes in the patient group had significantly increased the percentage of HLA-DR expression (Figure 2). In other words, despite the numerical decrease in lymphocytes, there was a significant increase in the expression of HLA-DR, which is used as an activation marker, on both lymphocyte types. We also found a weak but positive correlation between serum CRP levels and HLA-DR expression on T lymphocytes. These results suggest that both T helper and cytotoxic T lymphocytes play a role in the immune response to COVID-19.

The most commonly used statistical criterion for demonstrating antigen expression in the analysis of lymphocyte subsets is the percentage of positive cells. MFI measurements, however, have the potential to demonstrate the average number of antigen molecules per cell approximately. In this respect, it has been said that showing the staining intensities on a lymphocyte population as MFI values may be more informative than showing them as percentages unless there is no fluctuation in measurement [29]. In the present study, HLA-DR MFI values were calculated in addition to the percentage of HLA-DR expressed on the cell surface. In the patient group, HLA-DR MFI values of CD3⁺/CD4⁺ T lymphocytes were not changed, but their values in CD3⁺/CD8⁺ T cytotoxic cells were significantly higher (Figure 2B). The increase in HLA-DR MFI on the cytotoxic T cell surface indicates that the HLA-DR expression not only increases in percentage but also the amount of protein per cell. This supports the idea that cytotoxic T cells are activated more strongly than T helper cells.

Another molecule associated with lymphocyte activation is CD45 (lymphocyte common antigen) [30]. CD45 is a receptor-linked protein tyrosine phosphatase normally expressed on all leukocytes. It is believed to be essential for maturation, signal transduction, and activation of lymphocytes [30]. CD45 is known to play a role in autoimmune diseases and viral infections, especially HIV [30], [31], [32]. Furthermore, CD45 deficiency results in B and T lymphocyte dysfunction in severe combined immunodeficiency [33]. A previous study showed decreased CD45 expression in patients with COVID-19, suggesting an association with disease severity [33]. The present study supports earlier evidence by showing no difference in CD45 expression of granulocytes but significantly lower CD45 expression of lymphocytes in the patient group compared to the healthy subjects (Table 3). Accordingly, the decrease in CD45 expression in COVID-19 may be another indicator of the deterioration in lymphocyte activity and decrease in maturation.

Side scatter (SSC), which provides information about the internal complexity of cells, such as granularity, is thought to be another indicator of lymphocyte activation [34]. In the present study, the analysis of the SSC characteristics of lymphocytes revealed significantly higher SSC values and a lower ratio of neutrophil SSC to lymphocyte SSC in the patient group compared to the controls. These changes in the light scatter characteristics of lymphocytes indicate increased granularity in their cytoplasm, supporting the increase in cellular activation.

In the event of infection, monocytes are responsible for pathogen recognition, the initiation and resolution of inflammation, and the repair of tissue damage [35]. Three main monocyte types have been identified in peripheral blood based on their CD14 and CD16 (FcR III) expression [36, 37]. These monocyte types are known to exhibit different phenotypes and functions. CD16⁺ monocytes have been shown to express more proinflammatory cytokines with a higher potential for antigen presentation but a reduced ability to perform Fc receptor-mediated phagocytosis [36, 37]. Previous studies have reported an increase in the amount of CD14⁺/CD16⁺ (intermediate and non-classical) monocytes in sepsis, tuberculosis, and HIV infection [38, 39]. To date, there have been few reports of peripheral blood monocyte abnormalities in patients with COVID-19 [40], [41], [42], [43]. The results of the present study, similar to previous studies, showed an increased percentage of CD16⁺ monocytes in COVID-19 patients (Table 5). However, so far, there has been only one study investigating CD16 MFI values in COVID-19 [44]. Measuring CD16 MFI values for the first time in COVID-19, the present study demonstrated significantly lower values compared with healthy controls. In other words, while the percentage of CD16⁺ monocytes was increased in the patient group, CD16 MFI values were found to be decreased. This may be related to decreased ability to perform Fc receptor-mediated phagocytosis. In other words, it can be suggested that CD16⁺ monocytes, which have important monocytic functions such as antigen presentation and proinflammatory cytokine production, increase in COVID-19, but the phagocytic capacity of these monocytes decreases.

HLA-DR expression on monocytes plays a major role in the presentation of antigens to immune cells [45]. This study investigated HLA-DR expression on monocytes. Although there was no significant difference in the percentage of monocytes expressing HLA-DR between the patient and healthy control groups, the patient group had significantly higher HLA-DR MFI values. A previous study suggested that CD16⁺ monocytes express higher levels of HLA-DR in newborns with sepsis and may have higher antigen-presenting cell (APC) activity in these cells [38]. In the present study, COVID-19 patients had significantly increased HLA-DR. MFI values of CD16⁺ monocytes compared to the control group. It can be concluded that the phagocytic properties of CD16⁺ monocytes decreased, whereas their APC properties increased. As a matter of fact, a study reported increased HLA-DR bright positive monocytes in patients with mild COVID-19. However, the same study showed decreased HLA-DR expression in severe cases [35, 46]. Eventually, the severity of HLA-DR expression on monocytes seems to be related to the clinical course of the disease.

In conclusion, both lymphocytes count and CD45 expression on lymphocytes were decreased in COVID-19. However, the increase in granularity of lymphocytes, along with the increased HLA-DR expression on T lymphocytes, suggests that lymphocyte activation has an important role in the immune response to the disease. High HLA-DR MFI values of cytotoxic T lymphocytes indicate that these cells remain at the forefront. The detection of both increased counts and increased APC-related HLA-DR MFI of CD16⁺ monocytes, unlike lymphocytes, supports the hypothesis that these cells play a role in the immune response together with lymphocytes. On the other hand, it can be suggested that the decrease in CD16 MFI values associated with Fc receptor-mediated phagocytosis limits the phagocytic activity of monocytes.