Introduction

The global COVID-19 pandemic, which was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has left an indelible mark, it poses a grave threat to both public health and daily routines, leading to a substantial surge in infections and fatalities on a global scale [1]. Since the discovery of SARS-CoV-2, the number of patients experiencing reinfection has been continuously rising. There is evidence to suggest that reinfection is associated with an increase in risks of death and hospitalization during the acute phase and after, as well as the occurrence of multiple organ sequelae [38]. DNT cells, constituting 1–3% of peripheral T cells, demonstrate immunoregulatory potential by eliminating B cells during chronic graft-versus-host disease [39]. In addition, DNT cells with some CD4 + T cell functions were shown to be associated with a nonpathogenic outcome following HIV infection. The role of DNT cell shifts in COVID-19 and their implications for the disease warrant further investigation. In the context of B cells, De Biasi et al. reported a decrease in the numbers of total and naïve B cells, as well as reduced proportions and numbers of memory switched and unswitched B cells in COVID-19 patients [40]. Interestingly, our study revealed a unique pattern in the behavior of B cell subsets. Within the initial 7 days after viral RNA clearance, both naïve and memory B cell numbers were notably higher. However, by the 3-month mark, these numbers exhibited a decline. This phenomenon might be attributed to the activation of B cells during the virus clearance phase, prompting proliferation, and subsequently, a gradual return to baseline levels during the recovery period. This nuanced dynamic of B cell populations sheds light on their role in the immune response during and after COVID-19 recovery. Additionally, Maucourant et al. have reported on distinct NK cell immunotypes characterized by the expression of perforin, NKG2C, and Ksp37. These immunotypes have demonstrated a correlation with COVID-19 disease severity [41]. The results indicated that although NK cells decreased in number, they were activated and functioned against several viral infections. CD14 + CD16 − monocytes are a classical monocyte subset, which was reported to be decreased in severe COVID-19 cases [42,43,44]. However, as patients transition toward recovery, the number of these cells gradually rises, indicating rebuilding of the immune system and stronger innate immunity and pathogen clearance capacity.

It is established that antibody levels targeting the SARS-CoV-2 S antigen wane post-viral RNA clearance or vaccination [13, 14, 45]. Similarly, we observed a decrease in both anti-S IgM and IgG antibody levels within 6 months after viral RNA clearance. We found the antibody levels correlated with CD4 + T cells, gdT cells, DNT cells, and NK cells. After SARS-CoV-2 infection, SARS-CoV-2 spike-specific memory B cells can rely on CD4 + T cells to produce high-affinity antibodies [46], the positive correlation between CD4 + T cells and anti-S IgG levels is understandable in our study. NK cells can secrete various cytokines, such as IFN-γ, TNF-α, etc. Kaneko et al. found that excessive TNF-α inhibited the formation of germinal center reactions, suppressed the generation of long-lasting antibody responses originating from germinal centers, resulting in the low and transient antibody responses in COVID-19 patients [47]. This may be one of the reasons for the negative correlation between NK cells and IgG antibody levels. Notably, DNT cells exhibited positive correlations with both IgM and IgG levels, which had not been reported previously. We speculated that it might be related to the limited long-term persistence of antibodies in COVID-19 patients. DNT cells have the ability of promoting B cell apoptosis, inhibiting B cell proliferatxion and plasma cell formation [48]. We hypothesized that after SARS-CoV-2 infection, when B cells produced antibodies, DNT cells might also continuously promote the apoptosis of B cells. This resulted in the situation that antibodies could not exist for a long time and could not provide long-term immune protection. However, this assumption still requires subsequent large-sample, dynamic exploration.

In T cells, except for naïve CD4 + T cells, which showed a decrease at 6 months after recovery compared to 7 days, other CD4 + T cells and CD8 + T cells maintained higher levels at 6 months after COVID-19 recovery. Similarly, NK cells also maintained higher levels at 6 months compared to 7 days after recovery. This is consistent with the widely recognized notion that there is relatively strong immune protection against COVID-19 during the first 3–6 months after the initial infection.While higher levels of T cells and NK cells provide protection against reinfection with the SARS-CoV-2 virus, it’s worth noting that currently, SARS-CoV-2 subvariants BQ.1 (a subvariant of BA.5) and XBB (a subvariant of BA.2) have globally replaced the previously dominant Omicron variant strains (including BA.5) [49]. Viral mutations resulting in immune escape increase the probability of reinfection. After infection, the increase in the number of naïve and memory B cells is sustained for a relatively short period before declining, followed by a gradual return to levels close to those in uninfected individuals by 6 months after viral RNA clearance, suggests that their protective role against reinfection in the event of a new COVID-19 exposure may be relatively limited.

This is the first study to examine the changes in PBMCs in patients with COVID-19 within 6 months after viral RNA clearance. Although, the small number of patients enrolled in this study partially limits the reliability of the experimental results, longitudinal dynamic observations revealed consistent changes across different samples. Another limitation of this study is that the biological functions of the most altered subsets of PBMCs were not investigated in detail, and further studies are required to determine the significance of these changes to the host immune status.Furthermore, selecting 6 months as the observation endpoint, longer-term changes in immune cell profiles after clearance of COVID-19 RNA are also worth investigating.

In conclusion, we described longitudinal dynamic changes in the PBMCs of COVID-19 patients within 6 months after viral RNA clearance. Our results identified distinct changes in the subpopulations of T cells, B cells, NK cells, and monocytes. In addition, we described possible relationships between anti-S antibodies and several subsets of immune cells, which could help elucidate the immune changes that occur during COVID-19 recovery. Our study provides a strategy for guiding prevention against reinfection in the current scenario of continuously changing COVID-19 virus strains.