Determination of the relationship between disease severity and circulating T and B lymphocyte subgroups in the follow-up of COVID-19 patients by means of multiparametric immune phenotyping

Objective: In this study, we aimed to examine the changes detected by flow cytometry in T and B cell subtypes during diagnosis and during follow-up in adult patients with COVID-19 infection with SARS-CoV-2 PCR positivity and its relationship with disease severity. Materials and Methods: The study was conducted prospectively in Çukurova University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology. Patients over the age of 18 who were diagnosed with COVID-19 between 1.10.2021 and 9.9.2022 were included in the study after the approval of the ethics committee and the written consent of the participants. According to the clinical features of the diagnosed patients at the time of admission, they were classified as mild, severe and critical according to the WHO's severity definitions for COVID-19 and were divided into 3 groups according to the follow-up status: outpatient, followed-up in the service and followed in the ICU. 30 patients were included in each group. In outpatients, on the day of diagnosis and on the 7th day of follow-up, the first day of hospitalization and 5-10th day of follow-up among inpatients. On the day of discharge or on the day of discharge, the first day of hospitalization from the patients in the ICU and the 7-14th day of the follow-up. Blood samples were taken on the day of the service or the day of discharge. Complete blood count and flow cytometry studies were performed before the samples were taken. For each patient group, 20 cell types were studied by flow cytometry. In order to determine the subgroups of T lymphocytes to be used in flow cytometry in our study, frequently used T lymphocyte markers such as CD3, CD4, CD8 and CCR7, CD45RA, CD62L, Markers such as CD31 were used. For B lymphocytes, together with CD19, markers such as CD21, CD24, CD27, CD38, IgM were used for the determination of subgroups such as transitional cells, naive cells, memory cells, declassified cells, plasmablast cells. In the analyzes performed, lymphocytes were gated on all leukocytes in CD45/SSC (Side scatter channel) dot plot, and the percentage and number of lymphocytes; For T cells, CD3+ cells were selected from the CD3/SSC graph over lymphocytes, and the percentage and number of T lymphocytes; For B lymphocytes, CD19+ cells were selected from the CD19/SSC graph on lymphocytes and the percentage and number of B lymphocytes were determined. In the next step, separate flow cytometry studies were performed for T and B lymphocytes. SPSS (Statistical Package for the Social Sciences) 25.0 package program was used for statistical analysis of the data. Results: 90 patients with a diagnosis of COVID-19 were included in the study. While the mean age of the patients was 56.7±17.9 (minimum: 18, maximum: 95) years, 46 (51.1%) were male and 44 (48.9%) were female. Smoking was detected in 15 (16.7%) of the patients. 21 (23.3%) were former users. Comorbidity was detected in 66 (73.3%) of the patients. The most common comorbidities were HT in 33 (50%), DM in 22 (33.3%), heart disease in 22 (33.3%), malignancy in 17 (25.8%), 8 ( 12.1% had COPD-asthma, 7 (10.6%) kidney disease, 6 (9.1%) immunosuppression (patient receiving active chemotherapy, neutropenic patient), 5 (7.6%) Autoimmunity was found in 1, chronic liver disease in 1 (1.5%) and other comorbidity findings in 13 (19.7%) patients. According to the WHO scale, 39 (43.3%) patients had mild disease, 33 (36.7%) severe disease, and 18 (20.0%) critical disease. While the rate of critical illness was higher in men (26.1%; n:12) than in women (13.6%; n:6); Although the rate of severe disease was higher in female (21.1%; n:47.7) patients than in male (26.1%; n:12) patients, the difference was not statistically significant. Considering the outcome findings of the relationship between survival and death by gender, 72 (80%) of the patients had healing findings, while 18 (20%) had theAccording to the WHO scale, the comorbidity rate was found to be lower in non-severe patients. At admission and at the time of follow-up, naive B cell rates were found to be lower in patients followed in the ICU. IgM+ memory B cells were found to be significantly lower in outpatients than those followed in the service and ICU. Increase in CD4+ central, memory B cell and plasmablast ratios in outpatients over time; only IgD+ memory B cells and naive B cells were significantly decreased over time. In the patients followed up in the service, CD4+ central, CD4+ effector and class-changed memory B cell ratios increased over time, while only IgM+ memory B cell ratios decreased over time. Increase over time in CD4 neo-immigration, CD4+effector, transitional and class-changed memory B cell ratios in patients followed up in the ICU; only IgM+ memory B cells and plasmablast ratios decreased over time. Conclusion: A wide variety of studies have demonstrated that T and B cell responses are a critical component of immune protection against SARS-CoV-2. In our study, significant differences were found in T and B cell subpopulations in all 3 groups, being outpatient, ward and ICU. Our study shows that SARS-CoV-2 can induce normal adaptive immune response. Collectively, these data shed light on potential variations in T-cell responses as a function of disease severity; We think that this is a key topic for understanding the potential role of immunopathology in the disease, as well as informing vaccine design and evaluation, and will contribute to the literature due to the limited number of studies on temporal changes of T and B lymphocytes, although there are similar studies. presence of death. It was found that the death findings in the ratio of women and men were similar.