key: cord-0808734-meq20a3w authors: Wu, D. X.; Wu, X. P.; Huang, J. S.; Rao, Q. F.; Zhang, Q.; Zhang, W. F. title: Lymphocyte subset alterations with disease severity, imaging manifestation, and delayed hospitalization in COVID-19 patients date: 2020-08-31 journal: nan DOI: 10.1101/2020.08.25.20181321 sha: 943dd2fd0a5a65201279597704dd1180054ded69 doc_id: 808734 cord_uid: meq20a3w Background: COVID-19 continuously threated public health heavily. Present study aimed to investigate the lymphocyte subset alterations with disease severity, imaging manifestation, and delayed hospitalization in COVID-19 patients. Methods: Lymphocyte subsets was classified using flow cytometry with peripheral blood collected from 106 patients. Results: Multivariate logistic regression showed that chest tightness, lymphocyte count, and {gamma}-glutamyl transpeptidase were the independent predictors for severe COVID-19. The T cell, CD4+ T cell and B cell counts in severe patients were significantly lower than that in mild patients (p = 0.004, 0.003 and 0.046, respectively). Only the T cell count was gradually decreased with the increase of infiltrated quadrants of lesions in computed tomography (CT) (p = 0.043). The T cell, CD4+ T cell, and CD8+ T cell counts were gradually decreased with the increase of infiltrated area of the maximum lesion in CT (p = 0.002, 0.003, 0.028; respectively). The T cell count, as well as CD4+ T cell, CD8+ T cell, and NK cell counts were gradually decreased with the increased delayed hospitalization (p = 0.003, 0.002, 0.013, and 0.012; respectively). The proportion of T cell was gradually decreased but B cell was gradually increased with the increased delayed hospitalization (p = 0.031 and 0.003, respectively). Conclusion: T cell and CD4+ T cell counts negatively correlated with disease severity, CT manifestation and delayed hospitalization. CD4+ T cell was mainstay of immunity response in COVID-19 patients. Coronavirus disease 2019 (COVID-19) is a newly emerged viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1] . COVID-19 is highly contagious and has become pandemic quickly. Innate and adaptive immune responses were activated in COVID-19 patients. However, uncontrolled innate and adaptive immune responses may lead to locally and systemically tissue damage, which leads to severe COVID-19 with more frequent intubation and mechanical ventilation, and high mortality. Exhausted lymphocytes were a feature of severe COVID-19 [2] [3] [4] . Recently, the alterations of lymphocyte subsets in COVID-19 patients had attracted the attention of researchers. An overall decline of lymphocyte subsets including CD4 + T cells, CD8 + T cells, B cells, and NK cells has been reported in severe and deceased COVID-19 patients [5, 6] . However, vary patterns of lymphocyte subsets abnormality in severe COVID-19 patients also have been demonstrated by other studies [7] [8] [9] [10] . Reports involving the change of CD4 + to CD8 + T cells ratio were also inconsistent [9, 11, 12] . Thus, the reported patterns of lymphocyte subsets in patients with were diverse and controversial, and necessitated to clear more. Additionally, the correlations of lymphocyte subsets with the lesion manifestation in computed tomography (CT) and with the time from onset to hospitalization (TOH) of patients were also not be well documented. Here, we first investigated alterations of lymphocyte subsets in severe COVID-19 patients. Then, we All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20181321 doi: medRxiv preprint 6 Cytometer (BD Biosciences, Shanghai, China). Anti-CD3 was conjugated by fluorescein isothiocyanate (FITC), anti-CD4, anti-CD8, anti-CD19 were conjugated by allophycocyanin (APC), phycoerythrin (PE), and APC respectively, anti-CD16 and anti-CD56 conjugated by PE (340499 and 340500, BD Biosciences). All tests were performed according to the manufacturer's instructions. Statistical analysis was performed with SPSS 25.0 (SPSS, Inc., Chicago, USA) and MedCalc (MedCalc Software Ltd, Ostend, Belgium). Continuous data were expressed as the mean ± standard deviations or medians with quartile (P25-P75) and categorical data were expressed as numbers (%). Student's t-test or the Mann-Whitney U test as appropriate were used for continuous data, and the χ 2 or Fisher's tests were used for categorical data. Rank correlation was analysed using the Spearman method. A p values of less than 0.05 was considered statistically significant. Independent risk factors were identified using multivariate logistic regression according to the forward Wald method, with entry and removal probabilities of 0.05 and 0.10, respectively. The age of patients with COVID-19 was 46.17 ± 14.39, and 60.4% patients were male. The mean of TOH was 5 days. Among the 106 COVID-19 patients, 83 (78.3%) patients had a clear exposure history, and 33 (31.1%) patients had one or more comorbidities. The most frequent comorbidity was bacterial infection (11.3%), followed by diabetes (9.4%) and hypertension (7.5%). As expected, fever (91.5%), dry cough (43.4%), and chest tightness (32.1%) were the top three frequent symptoms. Chills (20.8%), fatigue (19.8%), and sore throat (18.9%) were also common, but rhinorrhea or rhinobyon (5.7%), All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20181321 doi: medRxiv preprint diarrhea (6.6%), and myalgia (7.5%) were relatively rare in COVID-19 patients (Table 1) . As shown in Table 1 , univariate logistic regression indicated that the TOH of severe patients was significantly longer than of mild patients [7 (4 -10) vs. 4 (2 -7) days; p = 0.011]. The frequency of comorbidities in severe patients was higher than mild patients (45.5% vs. 24.7%; p = 0.035). The frequency of sputum production, chest tightness, and polypnea was more higher in severe patients than mild patients (all p < 0.05). The level of C-reactive protein, as well as levels of direct bilirubin, γ -glutamyl transpeptidase, lactate dehydrogenase, and D-dimer were significantly higher in severe patients than mild patients (all p < 0.05). However, the levels of lymphocyte count and albumin were significantly lower in severe cases compared to mild cases. Multivariate logistic regression showed that chest tightness, lymphocyte count, and γ -glutamyl transpeptidase were the independent indictors to predict severe COVID-19. Giving the lymphocyte count was an important indicator to predict severe COVID-19, we further investigated the alteration of lymphocyte subsets in patients with severe COVID-19. As shown in Figure 1A , the T cell count in severe patients was significantly lower than that in mild patients [ ]. There is no significant difference for CD8 + T cell or NK cell count between severe and mild patients. The difference of CD4 + to CD8 + ratio between severe and mild patients were not All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20181321 doi: medRxiv preprint significant. No significant difference for proportion of lymphocyte subset was observed between severe and mild patients ( Figure 1B ). In order to assess the manifestation of lesions in CT, present study simply scored the number, quadrant, and area of lesions. For the number of lesions, patients were classified to 3 subgroups named patients with no lesion, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20181321 doi: medRxiv preprint The lymphocyte counts were gradually decreased with the increased TOH (p < 0.001) ( Figure 2D ). Lymphocyte subsets analysis showed that the T cell count, as well as CD4 + T cell, and CD8 + T cell Figure 4B ). COVID-19 continuously threated public health heavily, which required more bench and clinic studies to more profoundly profile this disease. The common symptoms in COVID-19 patients, in accordance with previous reports [2, 14, 15] , were fever, and followed by dry cough, and chest tightness. Similar with studies investigated in other area [16, 17] , the age of patients in this study was dramatically younger than those in Wuhan. Notably, the frequencies of hypertension and diabetes for COVID-19 patients in this study were also much lower than patients in Wuhan [2, 14, 18] . The underlying reason may interpret by the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . Lymphocytes and their subsets play a key role in the maintenance of immune function. Complex immune dysregulation has been found in COVID-19 patients [19] . Currently, the change patterns of lymphocyte subsets were not conclusive. It is reported [5, 6] that an whole decline of lymphocyte subsets including CD4 + and CD8 + T cells, B cells, and NK cells were presented in severe and deceased COVID-19 patients. Liu et al. [10] suggested CD8 + T cell count was significant decreased in severe COVID-19 patients than mild patients at the time point of disease onset and 7-9 days later, but their difference in CD4 + T cell count was not significant at any time point. studies [7] [8] [9] also indicated decreased CD4 + and CD8 + T cells were correlated with disease severity of COVID-19, but there is no difference for the level of B or NK cell between severe and mild COVID-19 patients. Additionally, reports involving the change of CD4 + to CD8 + T cells ratio were also inconsistent [9, 11, 12] . In this study, we found the significant percentage change of lymphocyte subsets is rare no matter in patients with severer clinical type, more extensive CT manifestation, or with longer delayed hospitalization. These findings were agreement with previous studies [4, 7] . It is noteworthy that the T cell and CD4 + T cell but not CD8 + T cell were significantly decreased in severe COVID-19 patents, which suggested that CD4 + T cell but not CD8 + T cell play more important role in immunity response to SARS-CoV-2 infection. pneumonitis [20, 21] . Similarly, high-level CD4 + but not the CD8 + T cell response was also observed in All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . SARS patients [22] . What is more, the significantly decreased B cell in severe COVID-19 patents indicated that humoral immunity has been attenuated in antiviral response of SARS-CoV-2. It has reported [23] [24] [25] that T-helper type 1 (Th1), T-helper type 2 (Th2), and regulatory T cells were varying degrees of activated in peripheral blood from critical COVID-19 patients after stimulation with specific antigen of SARS-CoV-2. It can be speculated all the CD4+ T cell subgroups were exhaust in blood of critical COVID-19 patients for the severely damaged lymphoid organs and/or exudation of circulating lymphocytes into lung [9] , although the alteration of CD4+ T cell subsets warrants further investigation. With regard to lymphocyte subset changes with CT manifestation, present study found that the total lymphocyte counts were gradually decreased with the increased number, infiltrated quadrants of lesions, and the area of the maximum lesion. T cell counts were gradually decreased with the increase of infiltrated quadrants of lesions and the area of the maximum lesion rather than the increased lesion number. Gradually decreased CD4 + and CD8 + T cell counts were only observed with increased area of the maximum lesion. Those results revealed that the area of the maximum lesion was closer correlated with the count of lymphocyte subsets and was more appropriate to estimate the severity of COVID-19. The alteration of lymphocyte subsets with the delayed hospitalization has not been reported before, present study firstly observed their correlation and found that the total lymphocyte, T cell, CD4 + and CD8 + T cell counts were gradually decreased with the the increased TOH. This result indicated that the lymphatic organs will continue to be damaged as long as there is no intervention. Liver was a predominantly vulnerable extrapulmonary organ in patients with COVID-19, hepatic dysfunction was seen in 14-53% of cases and particularly in those with severe condition [26] . Similar with the trends of All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20181321 doi: medRxiv preprint lymphocyte subsets, present study also found the level of ALT was gradually elevated with the TOH ( Figure 5) , suggested that delayed hospitalization may cause more liver injure. Therefore, early hospitalization could avoid disease aggravation, more organ damage and the unnecessary use of scarce medical resources. There were several limitations in this study. First, the alteration of CD4 + T cell subsets was not investigated, although CD4 + T cell was demonstrated to be mainstay of immunity response to SARS-CoV-2 infection. Second, only 3 cases with TOH more than 14 days (TOH was 15, 15, and 16 days, respectively), the lymphocyte subset alterations of convalescence of COVID-19 patients was not seen in this study. More studies including patients with TOH more than 14 days need to investigate to observe lymphocyte subset alterations in whole natural history of the disease. In conclusion, present study revealed independent predictors for severe COVID-19 and found CD4 + T cell was mainstay of immunity response to SARS-CoV-2 infection. Total lymphocyte, T cell and CD4 + T cell counts negatively correlated with disease severity, CT manifestation and delayed hospitalization. We believe these findings would provide some new insights in management of COVID-19. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20181321 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . https://doi.org/10.1101/2020.08.25.20181321 doi: medRxiv preprint hospitalization Fig. 3 . The lymphocyte subset alterations with aggravated CT manifestation All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. . 1 8 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted August 31, 2020. Abbreviations: CRP, C-reactive protein; WBC, white blood cell count; RBC, red blood cell count; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ -glutamyl transpeptidase; APTT, activated partial thromboplastin time. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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