INTRODUCTION:

SARS-CoV-2 is an etiological agen for coronavirus disease-19 (COVID-19) and also included in the group of RNA virus which was also caused SARS outbreak and Middle East Respiratory Syndrome (MERS). WHO stated COVID-19 as a pandemic disease in March 2020¹. Clinical symptoms of SARS-CoV-2 vary from mild to severe-critical symptom with pneumonia as one of the most common characteristic.

Moreover, it also showed a fever, non-productive cough, fatigue to respiratory distress². Coronavirus enter the host cell through spike of transmembrane (S) glycoprotein that is protrunding from the surface of the virus and bind with angiotensin-converting enzyme 2 (ACE2) receptor expressed in the human respiratory tract such as mucosal epithelial cells and lung alveolar type 2 pneumocytes, arterial and venous endothelial cells, and also in other tissue such as gastrointestinal tract, hence mild enteritis is sometimes found in COVID-19 patient^1,3,4.

Innate and adaptive immune responses are very important in controlling viral infection. Innate immunity gives initial response for detection and clearance of viral infection by producing proinflammatory cytokines to inhibit viral replication, stimulates adaptive immune response and recruits other immune cells to the site of infection^5,6. Innate immune cells consisted of phagocyte (macrophage & neutrophils), dendritic cells, mast cells, basophil, eosinophil and natural killer (NK) cells⁷. NK cells are part of innate immune cells whose function is important for primary defense system against viral infection, bacteria, and parasite also tumour surveillance. NK cells also play an important role in integating innate and adaptive immune system⁵. Lymphocyte is an adaptive immune cell which has a role in viral elimination. In classical viral infection, proportion of lymphocyte usually increases, but in influenza viral infection such in H1N1 pandemic and severe acute respiratory syndrome (SARS) lymphopenia occurs. Lymphocyte T play a vital role in adaptive immune system against Influenza type A viral infection. Decreased lymphocyte count or T cell subset (CD4 and CD8) indicate worsening of the patient’s condition^4,5. Several studies has established reference value for lymphocyte subsets in normal population (Table 1) which can be used as a comparison for decreament of lymphocyte subset in current infection. The aim of this research is to analyze the differences in the subset of T lymphocytes and NK cells at various degrees of disease severity in order to be used in stratification of patient management and to predict outcomes for optimal treatment.

Table 1. Normal value of lymphocyte subsets based on previous studies.

Parameters	*Uppal et al⁸*	*Wong et al⁹*	*Yaman et al¹⁰*
CD3
Absolute (cell/ul)	1115–4009	723 - 2271	1152-2208
%		56.09–84.32	64,26- 81,14
CD4
Absolute (cell/ul)	430–1740	396 - 1309	704- 1486
%	30.75–49.60	28.06–53.39	38,27-56,47
CD8
Absolute (cell/ul)	218–1396	224 - 1014	430-908
%	20.06–42.52	16.37–42.65	23-34,99
NK Cell
Absolute (cell/ul)		61 - 607	69-253
%		3.66–26.74	3,77-10,29

METHODS:

Sample:

Samples of 123 confirmed positive COVID-19 patients were taken in July to October 2020 in Dr. Soetomo General Academic Hospital and also from KOGABWILHAN II Forefront Hospital Indrapura Surabaya. The latter was forefront hospital for mild COVID-19 patients. Severity of COVID-19 patients were classified by clinicians to be mild, moderate, and severe to critical according to clinical practice guidelines in Dr. Soetomo regional hospital as follows:

Mild symptoms are characterized by the presence of one or more symptoms and signs of COVID-19 such as fever, cough, runny nose, swallowing pain, headache, muscle pain malaise, nausea, vomiting, diarrhea;without tightness, feeling heavy to breathe and without lung abnormalities on the chest X-ray.

Moderate symptoms are characterized by the presence of one or more symptoms and signs of COVID-19 such as fever, cough, runny nose, swallowing pain, headache, muscle pain malaise, nausea, vomiting, diarrhea;completed with tightness, feeling light/ heavy to breath and/or the presence of an infiltrate on the chest X-ray but the SpO is ≥94% with free air.

Severe symptoms are characterized by the presence of one or more symptoms and signs of COVID-19 such as fever, sough, runny nose, swallowing pain, headache, malaise, muscle pain, nausea, vomiting, diarrhea;Accompanied by RR signs ≥30 times/minute, SpO₂ ≤93% with free air, PaO₂/FiO₂ ≤300 mmHg, thorax photo of an infiltrate ≥50%.

Critical ill: respiratory failure, septic shock or multi-organ failure.

Patients were classified according to severity into 50 patients with mild severity, 34 patients with moderate severity and 39 patients with severe-critical severity. This study has received ethical acceptance from the Ethics Committee of Dr. Soetomo General Academic Hospital.

Laboratory Examination:

Blood sample EDTA 3 ml is taken from patient with COVID-19 conducted full bloodcheck up by using Sysmex XN 1000 hematology analyzer (Sysmex Corporation, Kobe, japan). Examination of CD3+/CD4+/CD8+ T-cell lymphocytes, and CD16+ CD56+ NK cells were examined by flow cytometry using Multitest human monoclonal anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), anti CD8-allophycocyanin (APC) and anti-CD16-APC and anti-CD56-PE antibodies (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Cells were analyzed using BD FACS Calibure flow cytometry system (BD Biosciences). Blood draws are carried out when the patient is initially admitted to the isolation room. Patients with HIV and lysis samples were excluded from this study.

Statistical Analysis:

Statistical analysis was conducted using SPSS software 17.0 (SPSS Inc., Chicago, IL, USA). The differences of multiple parameter between the three groups of disease severity were tested using the comparative nonparametric Kruskal Wallis test test and Man Whitney Test for patient’s outcome. Correlation of T Cell subsets and NK cell with disease severity was evaluated using spearman’s correlation. Cut off value was obtained based on the ROC Curve analysis. P value <0.05 was considered statistically significant.

RESULT AND DISCUSSION:

Patient Characteristics:

A hundred and twenty-three patients for COVID-19 were included in this study, ranging in age from 18 years to 75 years. The average age of patients in the severe, moderate, and mild group was 47 years, 46 years, and 36 years. There was a significant difference in the ages of the three patient groups with p = 0.000. A total of 38 patients (70.3%) were male. There was leukocytosis in 29.6% of cases, lymphopenia in 68.5% of cases. As many as 24% of patients died (Table 2).

Table 2. Demographic and laboratory data.

Variable	Severe-Critical Group (n: 39)	Moderate Group (n: 34)	Mild Group (n: 50)	p-Value
Age (years)	47 ± 12.8	46 ± 11.9	36 ± 10.9	0.000*
Sex
Male	26	17	37
Female	13	17	13
WBC	11.20 ± 5.12	8.79 ± 4.21	7.46 ± 1.87	0.000*
%Lymph	12.84 ±12.89	16.54 ± 7.35	30.85 ±7.91	0.000*
% Neut	79.87 ± 14.09	72.86 ± 9.73	58.5 ±9.45	0.000*
CD3
Absolute (Cells/uL)	673.9 ± 278.82	796.82 ± 270.5	1436.28 ± 406.1	0.000*
%	64.67 ± 12.47	65.16 ±9.08	64,65 ± 9.33	0.778
CD4
Absolute (Cells/uL)	365.0± 192.94	447.71± 199.1	706.44 ±208	0.000*
%	35.04 ± 12.05	35.57 ±8.13	31.79 ±7.05	0.145
CD8
Absolute (Cells/uL)	278.21 ±144.76	309.35 ± 121.72	594.66 ± 257.234	0.000*
%	25.97 ± 8.61	25.09 ±8.52	26.74 ±9.19	0.704
CD16/56 (NK cell)
Absolute (Cells/uL)	164.28 ± 147	192.94±131.5	475.7±267.4	0.000*
%	15.79 ±11.3	15.45 ± 8.57	21.03±9.19	0.003*

*There was significant difference (p value <0.05) for WBC (white blood cell), %Lymph (percentage of lymphocyte),%Neut (percentage of neutrophil), and absolute count of CD3 (Cluster of differentiation 3 or T lymphocyte cell), CD4 (Cluster of differentiation 4 or T helper lymphocyte cell), CD8 (Cluster of differentiation 3 or T Cytotoxic lymphocyte cell), CD16/56 (Cluster of differentiation 16 and 56 or NK cell).

Changes in lymphocyte and NK cell subsets at various clinical degrees of COVID-19:

The number of lymphocyte subsets was compared between severe, moderate, and mild groups (table 2). In COVID-19, there was significant differences in the number of CD 3 cells (p =0.000), CD4 (p =0.000), CD8 (p =0.000), and CD16/56 NK cells (p =0.000) and WBC in the three groups (Figures 1a, 1b, 1c, 1d). In the severe group there was a decrease in the number of CD3, CD4, and CD8 cells below the normal reference value (Table 1).

Figure 1. Comparison of the average counts of a).CD3; b). CD4; c). CD8; d). NK cells; and e) WBC in the severe to critical, moderate and mild degrees COVID-19 groups.

Based on the haemological data, the average of WBC count (in 1000cells/µL) there is a significant difference beween three groups (p=0.030). The percentage increased in neutrophils and the decreases in the lymphocyte’s percentage were significant in the severe and moderate groups compared to the mild group (Table 2). There is a negative correlation between the severeity of COVID-19 and the percentage of lymphocytes, the number of CD3, CD4, CD8 cells and NK cells.

Relationship between lymphocyte subsets and NK cells with patient outcomes:

Further analysis was performed to assess the association of lymphocyte subsets with mortality, resulting in 43.5% mortality in the severe-critical group (17/39). There were difference in CD3, CD4, CD8 and NK cell lymphocyte subsets between the clinical outcomes of recovery (Discharge patients) and death (Table 3).

Table 3. Comparison of lymphocyte subsets based on patient outcomes.

Parameter	Outcome	N	Mean	p value
CD3 Absolute Counts (Cell/uL)	Recovery	106	1084.51±471.9	0.000*
CD3 Absolute Counts (Cell/uL)	Death	17	601.88±324.4	0.000*
CD4 Absolute Counts (Cell/uL)	Recovery	106	554.14±241.26	0.001*
CD4 Absolute Counts (Cell/uL)	Death	17	355.29±249.89	0.001*
CD8 Absolute Counts (Cell/uL)	Recovery	106	443.71±243.73	0.000*
CD8 Absolute Counts (Cell/uL)	Death	17	239.29±158.28	0.000*
CD16/56 (NK cell) Absolute Counts (Cell/uL)	Recovery	106	324.79±255.05	0.001*
CD16/56 (NK cell) Absolute Counts (Cell/uL)	Death	17	136.71±112.42	0.001*
WBC (x1000 cell/uL)	Recovery	106	8.42±3.44	0.000*
WBC (x1000 cell/uL)	Death	17	12.71±5.87	0.000*
Lymph %	Recovery	106	22.47±10.91	0.000*
Lymph %	Death	17	13.12±18.58	0.000*
Neut%	Recovery	106	67.356±12.56	0.000*
Neut%	Death	17	81.124±19.81	0.000*
Age	Recovery	106	41.64±12.58	0.035*
Age	Death	17	48.76±14.05	0.035*

*) Significant p Value < 0.05

ROC analysis was performed to assess the mortality predictors from the various parameters studied (Fig 2). There was an optimal cut off for a decrease in the absolute count of CD4 429 cells/uL, CD8 301 cells/uL and 155 cells/uL NK cells associated with worse severity and mortality.

Figure 2. ROC Curve analysis and Area under curve of the parameters according to the severity of the disease.

Innate and adaptive immune system work together in defense mechanism against virus infection. Virus infection can cause T cell lymphocyte disregulation^11,12. Helper T lymphocyte (CD3⁺CD4⁺), Cytotoxic T cell (CD3⁺CD8⁺), and NK cell (CD16⁺CD56⁺) play roles in cytotoxic and humoral immunity against virus¹³. This study focuses in the change of NK cell and T lymphocyte subsets consist of T helper cells (CD4) and T cytotoxic cell (CD8) in patient with COVID-19 and its relationship with the patient’s outcome. This study has not been yet conducted in Indonesia to help stratificate severity of the disease.

In this study, about 65% of the total patient infected by COVID-19 is male and 35% is female individual. It is different from the previous study conducted that patients who got a risk of infection is higher in female than male group^14,15. The average age in the group with severe-critical ill is higher than in moderate-mild group. This is linear with the previous study which showed in COVID-19 infection, the less higher the age, the bigger the rate of fataility and mortality^14,16. From the result of this study, patient with severe to critical illness and moderate degrees of COVID-19 showed a significant increase in the average of leucocyte count and percentage of neutrophil. It was also followed by the decrease in number of CD3 cells, CD4 cells, CD8 and NK cells. In line with previous studies, there was a significant decrease in CD4 and CD8 in the severe group compared to the mild group. This showed a relationship between changes in lymphocyte subsets with the pathogenesis of SARS-CoV-2, namely immune system disorders such in SARS infection^17,18,19,20.

After the pathogen enters to the respiratory tract, SARS-CoV-2 activates immune system against pathogen, immune cells, and cytokine. NK cells have an important role in natural defense against cancer and virus infection also regulates adaptive immune system²¹. NK cells activation caused killing of the infected cell through cytotoxic degranulation and cytokines generated mainly by interferon gamma^21,22,23. In SARS infection, there was also decrease in the number of peripheral NK cells, as well as a reduction in immunoglobulin-like receptor CD158b+ NK cells which indicate a decrease in cytotoxicity^21,24,25. In COVID-19 case degrees of severe to critical illness and moderate is decreased in the number of NK cells compare with the group of mild degrees accompanied with peripheral NK cells functional exhaustion, increased in in NKG2A expression that indicated a decrease in the cytotoxicity^5,²¹^,²⁶^,,2⁷. SARS-CoV-2 may break down antiviral immunity mediated by NK cells at an early stage of infection, with putative consequences for the development of an efficient adaptive immunity²⁸. In viral infection, defective cytotoxicity leads to the accumulation of antigenic stimuli, perpetuating inflammation and in that triggering tissue damage²¹.

Lymphopenia also occurs with SARS-CoV-2 infection. In SARS-CoV and MERS lymphopenia infection also occurs, this is caused as a direct result of infection in the lymphocyte cell death; viruse that damage multiple target organs such as bone marrow, and thymus or atrophy of lymphoid and spleen tissue; inderect result of inflammatory mediators resulting in apoptosis of lymphocytes including the T cell population and lymphocyte trapped in the infective tissue^28-32. In line with the previous study, it was found lymphophenia in most of the patients impacts CD4 cells³³.

Lymphocyte T cell subsets are an important part from the overall population of immune cells and used as an indicator to detect function of cellular immune function. CD8 T cell has roles in eliminating virus particle with killing of the virus-infected cells mechanism through active biological molecules such as granzyme, perforin and generate cytokine to recruit immune cells to the site of infection^2,³⁴. The decrease in CD8 T cells caused a progress in the severity to more severe level and lost control of COVID-19 virus³⁴. From a previous study it was found that CD8+ T in the severe degree of COVID-19 produce less cytokine upon stimulation^2,¹⁸. CD4+ cells are activated immediately after infection with the SARS-CoV-2 virus to become pathogenic T helper (Th) 1 cells and secrete proinflammatory cytokine such as IL-6, activate monocytes and proliferate in order for the immune cells enter the pulmonary circulation. CD4 T cells also help B cells produce antibodies and prime the response of CD 8+ T cells and other immune cells².

Functional impairment and increase in the activation marker expression and CD4+ cell exhaustion^3,3⁵ which mark T cell overactivation and exhaustion happens at the same time in the patient with COVID-19 and also decrease in CD3+ T and CD4+ cells, accumulation in CD 3+ CD8+ cells and NK cells with decreased cytolytic potency³. Persistant stimulation by the virus induces T cell exhaustion caused decrease in function³⁶^,3⁷. In T cell exhaustion, disfunctional T cell was characterized by poor effector function, as well as expression of inhibitor receptors. Diao et al found that CD8+ and CD4+ T cells had higher levels of T cell exhaustion markers, namely PD-1 and TIM-3. Cytokine storm, especially IL-10, IL-6 and TNF trigger apoptosis and necrosis of T cells, resulting in a decrease of T cell³⁵.

The decrease in CD4 and CD8 T cells in patients who died from COVID-19 suggests more severe patients tended to have lower lymphocyte counts than those who were not severe^38,39. Low CD4 and CD8 counts are associated with poor patient outcomes in SARS infection⁴⁰. In line with the results of this study where in the severe group outcome, 50% died, there was a significant decrease in CD4 and CD8 T cells from this study, an increase in the number of leukocytes could be used to predict mortality in COVID-19 patient.

There are several limitations in this study, including the influence of therapies such as steroids and comorbids which can be a disruptive factor in the number of lymphocyte subsets that have not been analyzed in this study, and the effect of time since symptom onset cannot be equated.

CONCLUSSION:

In summary, there is a correlation between T lymphocyte subset and NK cell with disease severity. The absolute number of CD3, CD4, CD8 and NK cells in moderate and severe-critical COVID-19 patients group were decrease significantly compare to the mild group. This study showed that the decrease in T lymphocyte subset and NK cell was correlate with disease severity and could help in the stratification of patient with poor outcome.

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Received on 19.06.2021 Modified on 24.07.2021

Research J. Pharm. and Tech. 2022; 15(5):2198-2203.

DOI: 10.52711/0974-360X.2022.00365