1. Introduction
For more than four years, a global struggle was caused by the effects of the pandemic due to infections with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Following the initial outbreak in 2019 in Wuhan, China, the virus rapidly spread into a pandemic with about 770 million confirmed cases and resulting in over 6.9 million deaths worldwide (until April 2024) [
1].
Numerous studies have already shown that patients with a clinically severe course of COVID-19 present with abnormalities in several laboratory parameters, including lactate dehydrogenase (LDH), D-dimer, C-reactive protein (CRP), neutrophil counts, and pro-inflammatory cytokines, such as interleukin-6 and others [
2]. Another common feature in patients with severe COVID-19 is lymphopenia with dramatically reduced numbers of T-, B-, and natural killer (NK) cells [
3]. Lower proportions of these cells have been linked to disease severity, as reported in 96% of severe COVID-19 patients [
4]. Lymphocyte subset counts may, therefore, provide prognostic information for COVID-19 disease severity and convalescence when considered in conjunction with other clinical information [
5,
6]. Furthermore, simultaneous recruitment of neutrophil granulocytes by number and function (NETosis) has been proposed to be relevant in COVID-19 outcomes.
Currently, the pathogenesis of reduced lymphocytes in COVID-19 patients is only partially understood. Some of the most common potential mechanisms reported to lead to lymphocyte deficiency are as follows:
- (i)
Increased cellular death caused by the direct infection of SARS-CoV-2 Ribonucleic acid (RNA) in immune cells [
7];
- (ii)
Upregulation of the p53-mediated apoptosis signaling pathway in peripheral blood mononuclear cells (PBMC) [
8];
- (iii)
T cell extravasation and migration to inflamed tissue sites [
4];
- (iv)
Cytokine-storm-induced cellular apoptosis [
9]; and
- (v)
T lymphocyte exhaustion upon repeated activation [
9].
Moreover, a relevant fraction of patients is prone to prolonged multi-organ complaints after the initial time of acute infection and illness. Under these long-term health consequences originating from COVID-19, the World Health Organization (WHO) proposed a clinical definition and the name “Post-COVID-19 Condition” (PCC) to unify various existing terms [
1].
Of hospitalized patients, 87% have at least one persistent symptom at a mean of 60 days after symptom onset [
10]. PCC has also been mentioned in a large number of studies, with a prevalence of up to 43% [
11].
In this study, we aimed to evaluate changes in lymphocyte subpopulations with a particular focus on B cell subsets following SARS-CoV-2 infection in convalescent individuals and patients with PCC. Our evaluation may create new insights into the long-term consequences of SARS-CoV-2 infection and may provide a benefit for patients with persistent symptoms due to the lack of therapeutic alternatives.
4. Discussion
Recovery from SARS-CoV-2 infection is often associated with persistent symptoms months after infection, including fatigue, weakness, and shortness of breath [
10,
16]. Several recent studies suggest ongoing immune dysregulation in COVID-19 convalescents while profiling the immune system using multi-parameter flow cytometry, bulk and single-cell transcriptomics with a focus on T cells [
17,
18]. Using flow cytometry and immunological and serological assays, we provide a comprehensive look at additional immune cell subsets, especially B cell subsets, after COVID-19. To capture the complete range of immune recovery, we included the full spectrum of disease, with severity ranging from asymptomatic infections to mild disease managed in the community to requirements for ICU care. Moreover, we observed changes in symptom characteristics and clinical parameters over time and drew our results in comparison to UHC to point out potential abnormalities.
First, compared to previously published data, we observed a normalization of clinical parameters, e.g., acute-inflammatory biomarkers (CRP, LDH, neutrophils, lymphocytes), which are known to be elevated or decreased in the acute phase [
2,
19]. The NLR, which is an established biomarker for the prediction of progression or severity for COVID-19 [
20], exceeded the proposed threshold for disease severity of ≥4.5 in 44.7% of the COVID-19 cases in the acute phase. Otherwise, the NLR normalized in the follow-up independently to time or present post-COVID syndrome. Another known alteration during acute COVID-19 is the depletion of T cells and their subsets, as well as NK and NKT cells [
17,
21]. With regard to our data, the examined innate immune cell subsets returned to baseline in convalescents within about three months, except for NK cells. Herein, normalization of these cells has only been observed in the latest follow-up. This elevation of NK cells led to the assumption that there could be increased cell proliferation after acute COVID-19. Otherwise, viral infections are often associated with high levels of NK cells, which may indicate a persistent elevation. In general, the main impact of NK and NKT cells in COVID-19 pneumonia has been discussed [
22]; hence, these alterations are indicative of prolonged dysregulation in immune cells.
The number and function of neutrophil granulocytes have furthermore been implicated in the course and outcome of COVID-19 infection. In our current investigation, which mainly focused on lymphocytes, we were, however, not able to delineate a specific predictive value of the neutrophil-to-lymphocyte ratio on either the course or the outcome of the studied cohorts.
Additionally, altered frequencies of adaptive immune cell populations, including activation of cytotoxic T (CD8+) and T helper (CD4+) cells in recovered patients (G3), were significantly changed compared to UHC. These results agree with another study that showed increased cell proliferation of CD4+ and CD8+ T cells after SARS-CoV-2 infection [
23]. In contrast, we noticed a normalization of these cells in G1 and G2, which could be explained by the mild disease severity of our study population, as well as the late start of our observations (85 days after symptom onset).
Another notable driving factor in the heterogeneous immunological responses observed in COVID-19 could be immunosenescence and inflammaging. Immunosenescence refers to the aging of the immune system, which is mainly characterized by a decrease in naïve T cell counts together with an accumulation of CD4+ and CD8+ memory and terminal effector T cells. This may lead to increased vulnerability to infections and an impaired response to vaccination [
24]. Recent publications have presumed that SARS-CoV-2 infection could lead to accelerated immunosenescence in distinct populations [
25].
With regard to age-related alterations in B cells and the significant distribution of the mean age in our cohort and UHC, we have to point out a relatively newly defined subpopulation: CD21low B cells (sometimes referred to as age-associated B cells). CD21low B cells are a naturally occurring population of antigen-experienced B cells that expand continuously with age in healthy individuals but accumulate prematurely in patients with autoimmunity, inborn errors of immunity, and/or infectious diseases [
14]. Wildner et al. also observed an expansion of CD21–/lowCD27– cells in critically ill COVID-19 patients, wherein the number and proportion decreased in patients who recovered [
26]. Although there was no significant difference between our study population and UHC in CD21low B cells, there is a considerable trend in G1 and G2, the two groups with the highest mean age.
Moreover, there is ample evidence that CD21low B cells contribute to the production of both disease-specific antibodies and pathogenic autoantibodies. In several conditions, they, or subsets thereof, are associated with key disease manifestations, e.g., in systemic lupus erythematosus where the frequency of these cells correlates with autoantibody levels and disease activity score as well as rheumatoid arthritis with joint destruction [
27]. Here, we made no distinction between the subsets of CD21low B cells. In order to better understand the role and nature of these cells after the acute phase of COVID-19, further investigations are needed.
In addition, Ryan et al. reported that B cell activation/exhaustion markers remain elevated following SARS-CoV-2 infection [
28]. Our observations showed entirely recovering B cell counts, which is consistent with other data [
17,
22]. Regarding the antiviral immune response, we focused on switched memory B cells that result from the maturation of B cells. Compared to UHC, we observed significant exhaustion of switched B cells in two groups (G1 and G3) (
Figure 3). Contrasting publications have marked a significant increase in memory B cells after SARS-CoV-2 infection, which exhibited protective antiviral functions [
28,
29,
30]. Remarkable other studies have been pointing out that antigen-specific responses to SARS-CoV-2 can persist for several months after infection [
17,
22]. The reasons for these unexpected results might be diverse. SARS-CoV-2 can induce host cell death via different pathways, i.e., apoptosis in response to viral infection or over-activation, necroptosis, pyroptosis, and PANoptosis [
31].
As outlined in another study, different levels of COVID-19 severity could be associated with different levels of immune memory and subsequent immune protection [
32]. In addition, the dysfunction of switched memory B cells after infection can be discussed. Generally, it is well recognized that heterogeneity is a central future of immune memory in SARS-CoV-2 [
29].
Further classification of B cell subsets into transitional (CD38 + IgM++) and IgA+/IgG+ CD27+ and IgM + CD27− B cells revealed a significant expansion of transitional B cells in G1 and G2. This could be caused by the significant reduction in transitional B cells in acute COVID-19, which is characterized by a temporary increase in convalescence. At present, related observations revealed an impairment of immune-regulatory functions of transitional B cells in various immune diseases like autoimmune rheumatic diseases and neuro-immunological diseases [
33]. An expansion of transitional B cells is also frequently reported in patients with systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SS) which strengthens the hypothesis that autoimmunity has an impact on PCC.
In addition, current studies have observed that virus-specific antibodies are detectable for several months after recovery [
22,
29,
32]. As expected, our findings demonstrated detectable IgG and IgA antibody responses against SARS-CoV-2 after infection. Although we found no remarkable difference in the amount of IgA + CD27+ B cells in convalescents and UHC in G1 and G2, the reduction in G3 was significant and in accord with other findings [
18]. In addition, sustained production of neutralizing IgG+ virus-specific antibodies has been consistently correlated with protection from virus infection [
34]. The drop of IgG + CD27+ B cells in our latest follow-up could lead to the assumption that the concentration of IgG+ antibodies decreases within 7–11 months after infection.
In agreement with a previously published study, the B cell profiles of convalescent plasma donors after COVID-19 disease frequently differs [
30]. Examining B cell phenotypes in convalescent patients highlights that alterations in B cell subsets during severe acute COVID-19 are largely restored upon convalescence. These viral-neutralizing antibodies are secreted by plasma cells to provide durable protection after infection. Further studies revealed that circulating antibody-secreting cells (ASC) defined as CD19 + CD27hiCD38hiCD138+ were expanded in severe SARS-CoV-2 infections, although their occurrence was not associated with virus-specific IgM [
35]. In convalescents, we detected a normalization of these cell subsets. Hence, the rapid antibody decay is a manifestation of apoptosis of the nascent blood ASC.
Individuals suffering from PCC demonstrated subtle differences in immune responses compared with those without persistent symptoms. Risk factors for PCC may include age, more severe acute infection, socioeconomic factors, and female sex [
36]. Given the high prevalence (51%) of persistent symptoms among our study population, we evaluated immune responses in individuals with or without PCC and identified no significant differences but rather trends that should be mentioned. Indeed, B cells are known to be reduced in the acute phase of SARS-CoV-2 infection [
3,
37]. Moreover, Hu F. et al. have reported a correlation between decreased numbers of B and T cells and persistent SARS-CoV-2 shedding, which may further perpetuate chronic immune activation in PCC [
38]. Although there was no significant difference between NPCC and PCC, we found reduced amounts of B cells in PCC patients in comparison to NPCC patients (G1:
p < 0.126, G2:
p < 0.596, G3:
p < 0.087). Whether reduced numbers of B cells could influence the pathophysiology of PCC has not yet been proven, although their impact on COVID-19 disease has been discussed [
39]. Another trend is noticeable in switched memory B cells (CD21 + CD27+); here, we observed a reduction of these cells in PCC compared to NPCC, especially in G3 (with no significant value). Additionally, switched memory B cells were significantly decreased in PCC compared to UHC. Nevertheless, the variability in the penetrance of this phenotype has already been mentioned by others and may be explained by the influence of gender and pre-existing autoimmunity [
30].
Interestingly, many of our PCC patients met the diagnostic criteria for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a neuroinflammation-linked condition characterized by a range of debilitating chronic symptoms [
40,
41]. Overlap between PCC and ME/CFS diagnoses is not surprising since numerous cases of ME/CFS begin with a viral infection, e.g., Epstein–Barr Virus (EBV) [
42,
43] and Human Herpesvirus-6 (HHV6) [
44]. Consistent abnormal findings in ME/CFS include T cell exhaustion and other T cell abnormalities, diminished NK cell function, mitochondrial dysfunction, and vascular and endothelial abnormalities [
45], which have been observed in PCC patients as well [
46].
Furthermore, the presence and reactivation of chronic viral infections, such as cytomegalovirus (CMV), EBV, and human immunodeficiency virus (HIV), have been proposed as potential contributors to PCC [
47]. Serological evidence in a prior study has already suggested that recent EBV reactivation is associated with a higher likelihood of develo** PCC [
47]. Notably, in this study, we did not screen for coincidental viral infections after SARS-CoV-2 infection.
Historically suggested, viral infections have had a complex relationship with a variety of autoimmune diseases, e.g., rheumatoid arthritis (RA), systemic sclerosis (SS), and systemic lupus erythematosus (SLE). Several autoimmunity phenomena were observed during acute COVID-19, e.g., develo** new IgG- autoantibodies in hospitalized patients [
48] producing autoantibodies against immunomodulatory proteins, including cytokines, chemokines, and cell surface proteins [
49]. These features raise questions about whether they result from an immune inflammatory or autoimmune process triggered by COVID-19, and whether they are truly novel syndromes or are characteristics of previously described post-viral inflammatory syndromes. This autoimmune hypothesis could justify the higher incidence of this syndrome in women, which mostly conforms to our findings.
Currently, the SARS-CoV-2 pandemic seems to be under control in most countries, and the consequences of SARS-CoV-2 infection have come to the fore. Indeed, research into PCC has accelerated, but existing research is not enough to improve the outcomes for people who are suffering from PCC.
Compared to other studies, the sample size of our study is limited, particularly in the case of patients with more severe diseases. This is important given the apparently highly heterogeneous recovery in immune dysregulation over time. In addition, the mean age of our control cohort was much lower than in our study population, which could have an impact on lymphocyte counts. Another limiting factor might be the lack of inclusion of patients with non-European ancestry. It is also noteworthy to consider the lack of differentiation in circulating virus variants, which could have an influence on the pathogenesis. While our flow cytometry analyses enabled the assessment of multiple parameters, they did not include markers for neutrophils, monocytes, lymphokines, or dendritic cells (DC), which are altered in COVID-19 convalescents, as shown in a previous study [
49]. Further larger studies are needed to more fully assess the differences due to disease severity, comorbidities, treatment, and other confounders.