In this study, we aimed to analyze the role of lymphocyte subsets in the immunopathogenesis of COVID-19 and severe influenza A, and examined the clinical significance of their alterations, especially in determining the prognosis and recovery duration. Our analyses revealed significant dynamic variations in total lymphocytes and lymphocyte subsets, which get activated in the early stages of COVID-19 and severe influenza A infections, and further demonstrated that severe immune injury tended to be more prominent in patients with severe form of the disease. The recovery rate of patients with severe COVID-19 was comparatively longer than those who received immediate antiviral treatment for severe influenza A and those with non-severe COVID-19.
We retrospectively reviewed the clinical data of 99 patients who were confirmed to have COVID-19, 43 patients with severe influenza A and 110 healthy blood donors who were previously recruited in 2018. All the patients with COVID-19 were divided into two groups, according to the abovementioned diagnostic criteria, including 80 non-severe cases (80.8%) and 19 severe cases (19.2%).Several reports and studies have clearly indicated that older or elderly people are more prone to COVID-19 as their immune systems are likely to get overwhelmed by infections due to their advanced age. Similarly, the elderly who are 65 years or older are particularly at risk for influenza infection, hospitalization, and death due to influenza-related complications, such as pneumonia. In our study, the median age of patients with non-severe COVID-19 was 37 years, while that of those with severe COVID-19 was 67 years. Consistent with the previous studies, our data indicate that the ages of the severe patient group are higher than those of the non-severe COVID-19 group. Further, our study showed that the median age of patients with severe influenza A infection was higher (70 years) than that of those with non-severe COVID-19 (37 years) and healthy controls (45 years). Notably, this difference in age was found to be statistically significant (P < 0.05) among the groups, which suggests that the elderly people represent a large at-risk population.
The WHO-China joint report on COVID-19 provided a comprehensive symptomatology of COVID-19 (n = 55,924).A previous study showed that patients with COVID-19 present with pyrexia in 85% of cases during their illness course, but only 45% are febrile on early presentation. In addition to cough (67.7%) and sputum (33.4%), respiratory symptoms, such as dyspnea, sore throat, and nasal congestion were reported to be present in 18.6%, 13.9%, and 4.8% of cases, respectively. Both COVID-19 and influenza present with common clinical manifestations including fever, cough, rhinitis, sore throat, headache, dyspnea, and myalgia. [7, 14, 15]In our study, all the patients with severe COVID-19 presented with fever, cough, and dyspnea, whereas only 73% and 48% of patients with non-severe COVID-19 had fever and cough, respectively. Similar to the severe COVID-19 group, all patients with severe influenza A infection presented with fever and cough. However, only 77% of the patients had dyspnea. These clinical manifestations of COVID-19 and influenza infections were consistent with other studies. As previously reported the mortality rate also increases in patients with additional comorbidities. Specifically, 63% and 84% patients with severe COVID-19 and severe influenza A, respectively, presented with one or more pre-existing chronic medical conditions. While the mortality rate was 5% due to severe COVID-19, it was even higher (9%) for severe influenza A.
Lymphocytes and their subsets play a crucial role in maintaining immune homeostasis and inflammatory response in the host. As in case of immune disorders and other infections, a viral infection impairs the host’s immune defenses and results in decreased levels of lymphocytes and their subsets.[16, 17] Lymphocyte subsets, namely CD4+ T cells, CD8+ T cells, B cells, and NK cells are primarily involved in the humoral and cytotoxic immunity against viral infection. Therefore, this necessitates the need to understand the mechanism of reduced blood lymphocyte levels and characterize the dynamic alterations of lymphocyte subsets to provide novel insights for an effective treatment and prognosis of COVID-19.As lymphopenia is frequently observed during the initial stages of respiratory viral infection.[3, 18] We identified and analyzed different variations in leukocytes, lymphocytes, and lymphocyte subsets in patients with non-severe and severe COVID-19 and severe influenza A during the first week of illness. Our results showed that the absolute counts of total WBCs and lymphocytes of patients with non-severe (5.34 × 109/L, P < 0.001 and 1.51 × 109/L, P = 0.027, respectively) and severe COVID-19 (3.92 × 109/L, P < 0.0001 and 0.93 × 109/L, P < 0.0001, respectively) were significantly lower than those of healthy donors (6.01 × 109/L and 1.78 × 109/L, respectively). Further, the total lymphocyte counts of patients with severe influenza A were comparatively lower than those of healthy donors, (0.84 × 109/L, P < 0.0001). These findings are corroborated by a previous study which hypothesized that the virus might directly infect lymphocytes resulting in their apoptosis, thus leading to causing a sharp decline in total lymphocyte population and subsequent lymphopenia. Moreover, lymphocytes express the coronavirus receptor angiotensin-convertingenzyme 2(ACE-2), and therefore are a direct target for the virus. Another retrospective study suggested that lymphopenia might be one of the predictive factors for progression to respiratory failure during early stages following Middle East Respiratory Syndrome coronavirus (MERS-Cov) infection. Giving further strength to our study, a study by Geng et al. demonstrated the decline in the populations of T lymphocytes and their subsets, after influenza A virus infection, to be positively correlated with prognosis.We further performed flow cytometric analysis to enumerate total T cell population, CD4+ and CD8+ T cell subsets, B cells and NK cells in patients with non-severe COVID-19, severe COVID-19, and severe influenza A to determine significant changes in different lymphocyte subsets during their first week of illness. Our study demonstrated that patients with severe COVID-19 and severe influenza A had a significantly lower number of total T cells (P = 0.001 and P < 0.0001, respectively), CD4+ T cell subsets (P = 0.001 and P < 0.0001, respectively), and CD8+ T cell subsets (P = 0.001 and P < 0.0001, respectively) than healthy controls. Further, significant differences in total WBCs (P = 0.049), total lymphocytes (P < 0.0001) total T cell population (P < 0.0001), CD4+ T cell subsets (P = 0.001), and CD8 + T cell subsets (P = 0.001) were observed between patients with non-severe and severe COVID-19. Notably, there was a significant reduction in CD8+ T cell subsets (P = 0.036) in patients with non-severe COVID-19 compared with healthy controls. This indicates a more obvious change in CD8+ T cell subsets than in other lymphocyte subsets following SARS-CoV-2 infection. Therefore, our results reiterate the fact that CD8 + T cell responses play a major role in antiviral immunity. Taken together, lymphopenia was common in the patients with COVID-19, indicating a significant impairment in the host’s immune system following SARS-CoV-2 and influenza A infections. Our findings are in line with other studies which also detected these alterations in patients with pneumonia caused by MERS-CoV and Severe acute respiratory syndrome coronavirus (SARS-CoV).[20, 23] In addition, a significant reduction in both CD4+ T cells and CD8 + T cells were specifically observed in patients with severe COVID-19 and severe influenza A. Therefore, this indicates a more severe immune insult in patients with the severe form of the disease. Consequently, the alteration would be more profound, and leads to adverse clinical outcome in these patients. Thus, lymphocytes and their subsets, especially CD8+ T cells, might be a potential predictor for disease severity and clinical efficacy in COVID-19.
Next, we focused on the dynamics of T lymphocytes and their subsets, which played a vital role in cellular immune responses. We compared the absolute counts of leukocytes, total lymphocytes, and lymphocyte subsets of the non-severe and severe COVID-19 patient groups at weeks 2, 3, and 4 with that of those observed during the initial stages of infection. The total leukocyte, lymphocyte, and T cell counts significantly improved at week 3 in patients with non-severe COVID-19. Lymphocyte also recovered markedly at week 3 in severe COVID-19 patients. However, T cell and CD4+ T cell subset population significantly increased at week 4 in patients with severe COVID-19. Our results are consistent with a previous study by He et al. that showed a sharp decline (below normal) in the cell counts of CD45+, CD3+ T cell subsets, CD4+ T cell subsets, and CD8+ T cell subsets during the first week of SARS-Cov infection; their values further declined during the second week before increasing during the third week and returning to normal by the fifth week. Moreover, CD4+ T and CD8+ T cell counts were found to be extremely low in critically ill and deceased patients. Taken together, the alterations in lymphocytes and their subsets gradually improved at later time points in patients with COVID-19. Collectively, our results indicate that the recovery duration of patients with severe COVID-19 is longer than those with the mild form of the disease.
Our study results further revealed a noticeable difference in the time taken for the cell counts to improve among the severe and non-severe COVID-19 and the severe influenza groups. The cell counts of total lymphocytes and their subsets recovered only around week 4 in severe COVID-19; the recovery time was almost delayed by a week compared with those having non-severe COVID-19. On the contrary, the cell counts of total lymphocytes and their subsets in patients with severe influenza A increased and improved drastically at week2; this rapid recovery rate could be attributed to the early initiation of treatment with the neuraminidase inhibitor, oseltamivir, or peramivir, which interfere with virus release from host cells by blocking the viral nucleic acid function, thus preventing infection of new host cells.
NK cells are cytotoxic innate lymphocytes that play an important role in controlling the viral burden. NK cell responses can be specific, and they interact with both innate and adaptive immune cells to coordinate appropriate antiviral responses. Although we observed a reduction in NK cell counts during the initial stages of infection in patients with severe and non-severe COVID-19, their recovery rate (cell counts improved at week 3) showed a similar trend as that of the lymphocytes in both the groups. One plausible reason for this observation could be that the NK cell cross-talk might have got suppressed following the virus attack, which might have further led to impairment of CD4+ T cell responses and their effects on CD8+ T cells. This is further supported by a finding from this study which demonstrated a significant reduction in CD4+ T cell subsets, especially in patients with severe COVID-19. Given the role of B cells in adaptive immune response, we did not observe any significant changes in total B cell population in all patients with COVID-19 during the course of illness. This could possibly be attributed to the poor activation of the adaptive immune response against the virus. Moreover, the lack of detectable virus during stage I (asymptomatic incubation period)of the SARS-CoV-2 infection might have failed to elicit protective B cell immunity.Therefore, future research focusing on strategies to enhance the specific adaptive immune responses during the incubation and non-severe stages of the SARS-CoV-2 infection could significantly boost the B cell population, preventing the progression to the stage III severe respiratory symptomatic stage with high viral load. Furthermore, studies identifying specific immune components or functions that limit protective immunity to virus infection are warranted.
Our study has several limitations. This study was retrospective, small single-center study and only included a small sample of 99 patients with COVID-19 admitted to Beijing Ditan Hospital, which may confound the results and potentially introduce selection bias. This may limit the generalizability of the study. In addition, inconsistencies in time periods between illness onset and admission might have led to missing data which could result in observation biases in the dynamic variations in immune cells.