Recovery Of Innate Immune Cells And Persisting Alterations in Adaptive Immunity in The Peripheral Blood Of Convalescent Plasma Donors At Eight Months Post SARS-CoV-2 Infection Part 2

3.4. Alterations in Adaptive Immunity Persist at Eight Months Post Infection

In contrast to the innate arm of immunity, our data provide evidence of some persisting activation of the adaptive arm even at eight months post-SARS-CoV-2 infection. It is noticeable that the percentage of total B cells did not restore to normal levels even after eight months (mean percentage—18.7% in controls vs. 13.1% and 12.7% in CP donors at two months and eight months post-infection, p < 0.001), even though lymphopenia was fully reversed already at two months post-infection. 

The innate immune system is the body's most basic immune system. It works from birth to protect us from germs and microorganisms. This is in contrast to the acquired immune system, which only kicks in when exposed to germs or viruses and takes time to develop an immune response.

The innate immune system includes the skin, mucous membranes, macrophages, and platelets, among others, which help protect the body from infection and external aggressors. This is why we feel that in some cases the body will overcome the disease on its own without any external intervention. People with strong innate immunity are usually better able to cope with various illnesses.

However, the innate immune system is not perfect and cannot protect against all viruses and bacteria. At this time, the acquired immune system plays a role, which helps the body produce specific antibodies and fight against various bad pathogens. The stronger the acquired immunity, the more resistant the body will be and be able to get rid of pathogens and toxins faster and more effectively. To have a healthy immune system, in addition to having enough exercise, a healthy diet, and adequate sleep, we also need to pay attention to our mood. Stress and tension lower immunity and make the body prone to disease. Balancing the mood and maintaining a happy attitude can improve the body's immunity and contribute to the long-term health of the body.

Proper eating habits are also very important in optimizing our immune system. Diet can provide the body with sufficient nutrients and remove toxins from the body. Especially sugar and refined foods, these foods tend to destroy the body's health. Eating more natural foods, such as vegetables, fruits, whole grains, nuts, fish, etc., can provide the body with various essential nutrients and help strengthen the immune system.

In general, the cooperation of innate immunity and acquired immunity can greatly improve our body's resistance. In the long run, it can help you maintain your health and allow you to have a higher quality of life. Remember, everyone can improve their immunity and maintain good health by adopting the right lifestyle, let us encourage each other. From this point of view, we need to improve immunity. Cistanche can significantly improve immunity. Cistanche is rich in a variety of antioxidant substances, such as vitamin C, vitamins, carotenoids, etc. These ingredients can scavenge free radicals, reduce oxidative stress, and improve immunity. immune system resistance.

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Regarding the CD4+ T cell compartment, our previous analysis highlighted an overall restoration of the COVID-19-related skewed CD4 polarization at two months post-infection [13], except for some individual cases with a higher prevalence of the Th9- and Th17-type subsets. CP donors’ Th cells tested at eight months retained the recovery of the Th1/Th2 ratio, whereas percentages of Th17- and Th9-type cells decreased, sensibly due to the limited need for further activation of this type of polarized immune response. 

On the contrary, our analyses showed significant persisting changes at eight months post-infection towards a decreased CD4/CD8 ratio (mean CD4/CD8 ratio—4.5 in controls vs. 2.2 at both two and eight months, post-infection, p < 0.01) and a marked reduction of CD8+ Tregs that remained steadily low, since the initial infection. Furthermore, NKT cells and especially their CD56+CD16- compartment were found substantially increased even at eight months post-COVID-19 infection. 

Of note, the percentages of the CD56+CD16- NKT compartment showed a significantly increased rate between the two- and the eight-month evaluation points (mean percentage—6% vs. 7.1% of total CD3+ cells at two and eight months, respectively, p = 0.002).

4. Discussion

COVID-19 is strongly associated with unique immune cascades, and several studies have reported alterations in immune profiling following the SARS-CoV-2 immune response [4,9,10,15]. However, the vast majority of these studies have focused on alterations during the active COVID-19 disease and/or within a short-term follow-up. In the present study, we report our results on the long-term evaluation of the immune profile of CP donors, who were previously infected with SARS-CoV-2 and mounted a humoral response, as confirmed by the production of specific antibodies against the S1-protein of the virus.

Our results show that some alterations, particularly in specific lymphocyte subsets of the adaptive arm of immunity, persist even at eight months post-infection. We also noticed that at the same time point, the percentages of innate immunity major populations (monocytes, granulocytes, and NK cells), mostly of phenotypically distinct subsets thereof, were the same or very similar to the levels of healthy controls, suggesting that the resolution of the inflammatory profile is likely linked to the altered presence of these innate soluble mediator (chemokine and cytokine)-producing cells.

Firstly, although the percentage of total monocytes in CP donors at eight months was increased compared to the two-month and control values, our data highlighted particular kinetics of the different monocytic subsets following COVID-19 infection. 

In more detail, we have previously reported a marginal increase in the total monocyte population in the PB of active COVID-19 patients, sensibly due to the generic recruitment of macrophages to the lung or other infected areas [13]. In agreement with other studies, we also noticed the apparent increase in the intermediate (CD14+CD16+) monocyte subset and a less significant elevation in the non-classical compartment (CD14-CD16+) at the expense of classical monocytes (CD14+CD16-) during the active COVID-19 phase [13,16,17]. 

The role of intermediate monocytes is not fully elucidated, though they are known to be antigen-presenting cells that participate in the proliferation and stimulation of T cells with established involvement in anti-viral responses [18–20]. They are capable of secreting GM-CSF, TNF-α, and IL-6, which are critical for the inflammatory cytokine storms suffered by COVID-19 patients; hence, higher percentages of this subset have been associated with severe pulmonary complications, ICU admission, and increased time to discharge from the hospital [16,21]. The non-classical monocytes are mobile with a patrolling behavior in the endothelium, which may also be implicated in anti-viral responses [22]. 

Their unique phenotypic signature converges towards an anti-inflammatory mode of action, while new evidence supports their ability to undergo local conversion into alternatively activated macrophages [22,23]. In our cohort, we noticed that the percentages of intermediate monocytes recovered shortly (at two months) post-infection, implying their critical role during the acute phase of COVID-19. 

On the contrary, the recovery of non-classical monocytes was delayed and was evident at eight months, suggesting a prolonged immune activation lasting for at least two months post-infection, further supporting their contribution to the resolution of inflammation. 

Our results are in agreement with Neeland et al., who most recently reported a similar monocyte signature in CP adults at seven weeks post-infection, with increased percentages in total monocytes and a non-statistically significant increase in the same monocyte subsets as analyzed herein [12]. It should also be noted that in our analysis, total monocytes were gated on WBCs, and thus, may comprise phenotypes that do not necessarily express the markers used for identifying the aforementioned subsets (e.g., CD14- immature monocytes).

Interesting kinetics was also observed in granulocyte subsets which showed a gradual restoration over time, although the percentage of total granulocytes at eight months was slightly reduced compared to two-month values. 

As previously shown [13], the percentage of total granulocytes was increased during active COVID-19 with a substantial rise of their CD11b+ activated subset and a pronounced reduction of the CD11b- fraction. After two months post-infection, the percentages of CD11b+ granulocytes remained high in CP donors (mean—72.3% of total granulocytes in active COVID-19 vs. 64,5% in CP donors two months post-infection vs. 46.1% in healthy donors), thus providing additional evidence of an ongoing immune response. 

At eight months post-infection, the prevalence of the CD11b+ subset has significantly decreased, resulting in a CD11b+/CD11b- granulocyte ratio similar to that of normal healthy donors. CD11b, a protein subunit of the heterodimeric amb2, is a surface integrin present on several leukocytes that mediate among others cell migration, phagocytosis, chemotaxis, and cell-mediated cytotoxicity. CD11b immunophenotyping has been used to discriminate distinct immune profiles in lung inflammatory processes [24], whereas, in COVID-19, we and others have highlighted the direct correlation between the increased CD11b+/CD11b- ratio to more adverse symptoms [13,25]. 

Moreover, we previously showed that these CP donors who did not develop SARS-CoV-2 antibodies despite their PCR-positive testing had intermediate levels of CD11b+ granulocytes, lower compared to CP donors with detectable antibodies and comparable to unaffected controls, an observation that further associates the severity of the disease with hyperinflammation and stronger immune responses [26]. 

As our antibody panel did not include specific markers that would distinguish neutrophils from other granulocytes (eosinophils, basophils, mast cells), the reduction in their total percentage at eight months compared to two months could be due to lower percentages of these subsets in PB. In support, the number of eosinophils was shown to reduce during the CP phase [12], whereas activated mast cells that are implicated in the development of fibrotic conditions during active COVID-19, would also be expected to decrease after the resolution of the infection [27].

The percentages of NK cells at eight months were also similar to the levels of healthy controls. This is of importance since NK cells are crucial for anti-viral immunity and their effector functions are practically the first line of defense that limits viral replication. Thus, restoration of their levels to normal suggests a complete resolution of the infection at eight months. The marginal decrease in the CD56-CD16+ NK cell subset, a phenotype characterizing memory-like cells, additionally conforms with the resolution of the viral infection [28].

The aforementioned recovery of monocytes, granulocytes, and NK cells in the PB of COVID-19-recovered CP donors eight months post-infection could be likely predicted. Viral clearance is largely a function of the innate immune system, being confronted with the rapid kinetics of virus elimination.

Nevertheless, profiling of the adaptive immune cells did not seem to follow the same pattern. We have previously reported significant alterations in adaptive cell subsets during the active COVID-19 phase, which quantitatively affected the percentages of B cells, T cells, and NKT cells due to the apparent lymphopenia that characterizes COVID-19, and accordingly affected the relevant components of their various immune subsets. 

Firstly, the total number of B cells was found to be reduced throughout the whole study period and detected even at eight months post-infection. Although our panel did not include specific B cell markers for an in-depth B cell subset analysis, the statistically significantly increased Th2-type cells during active COVID-19 combined with the reduced total numbers of B cells may imply a skewed ratio towards antibody-secreting effector B cells and maybe terminally differentiated and highly secreting plasma cells [10]. 

The persisting deregulated B cell deficiency at eight months post COVID-19 infection may indicate B cell exhaustion in the periphery that needs more time to fully restore; it could also be a result of the hyperinflammatory (IFN-γ high) environment during the active phase of COVID-19 reported to negatively impact on B cell development [29]. However, since CP donors included in the study developed detectable levels of anti-SARS-CoV-2 antibodies, a more detailed B cell panel is needed to clarify the reasons(s) for B cell impairment.

Convalescent individuals show a strong and probably durable memory immune response mediated by both CD4+ and CD8+ T cells [10,30,31]. In our study, the vast majority of CP donors showed increased percentages of CD8+ T cells both at two and eight months post-infection, which, combined with the persisting low levels of CD8+ Tregs, probably indicates a durable cytotoxic immune reaction against possible SARS-CoV2 residuals. 

Further support of this notion derives from the fact that NKT cells and especially the CD56+CD16- compartment were found substantially increased both at two months [13] and eight months post-infection. CD3+CD56+ NKT cells share NK and T cell characteristics and reportedly have been considered intermediate mediators of innate and adaptive immunity with an important role in anti-viral responses [32,33]. NKT cells, although found in very low numbers in PB, produce high levels of IFN-γ, regulate the equilibrium of Th1/Th2 cells, and enhance CD4+ and CD8+ T cell functions, whereas when depleted in vivo, viral replication is greatly enhanced. 

In COVID-19, increased levels of NKT cells were noticed mostly in mild cases and were proposed to promote rapid control of the infection, being either directly cytotoxic or mediating antibody-dependent cell-mediated cytotoxic effects [34]. Therefore, the presence of notable percentages of NKT cells up to eight months post-infection may, on the one hand, support the evidence that the inflammatory trigger (i.e., likely some inactive viral remnants) is not fully cleared, but on the other hand, may also indicate an NKT cell-mediated cytotoxic activation that is still alert and probably compensates the role of the restored to-nearly-normal levels NK and CD8+ T cells.
Regarding the limitations of our study, we should note that the evaluation of the components of the immune response against SARS-CoV-2 was among the secondary study endpoints in the context of the phase 2 clinical trial of CP. Thus, it may be underpowered in terms of highlighting significant associations in subgroup analyses. Furthermore, all the analyses were performed in PB samples from the participants. Since COVID-19 is primarily a pulmonary disease, alveolar samples could provide a more accurate overview of the immune responses generated.

5. Conclusions

In conclusion, CP donors present with a unique immune landscape at eight months post-COVID-19 infection, which is characterized by the restoration of the components of innate immunity along with a persisting imprint of SARS-CoV-2 on the adaptive immunity. Elucidating the short- and long-term immune cell alterations to SARS-CoV-2 is essential in terms of therapeutics, prevention, and for formulating effective vaccination strategies. 

Although the long-term immune response following vaccination remains to be determined, the sustained alterations of adaptive immunity following COVID-19 seem to reinforce the value of vaccination. Vaccines aim to prime the immune system against SARS-CoV-2 more rapidly than natural infection. Neutralizing antibodies and SARS-CoV-2-specific memory B and T cell responses will subsequently prevent the infection or at least control the infection promptly to prevent severe disease.

Author Contributions:

Conceptualization, O.E.T. and E.T.; methodology, N.O.-S., P.R., I.V.K., I.P.T., O.E.T.; software, P.R., I.V.K.; validation, I.N.-S., E.K., M.G., I.C., I.P.T., E.T.; formal analysis, I.V.K., N.O.-S.; investigation, N.O.-S., E.K., O.E.T., C.P., I.N.-S., I.C., E.K., M.G., E.T.; resources, M.-A.D., O.E.T., E.T.; data curation, P.R., E.K., E.T.; writing—original draft preparation, I.V.K., N.O.-S.; writing— review and editing, I.N.-S., E.T., I.C., O.E.T., E.K., E.K., M.G., I.P.T., M.-A.D.; visualization, N.O.-S., I.V.K.; supervision, M.-A.D., O.E.T., E.T.; project administration, P.R., C.P., E.K., O.E.T., E.T.; funding acquisition, E.T., M.-A.D. All authors have read and agreed to the published version of the manuscript.

Funding:

This research was partially funded by SYN-ENOSIS (17315/S.A.R.G.-N.K.U.A).

Institutional Review Board Statement:

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee) of “Alexandra” General Hospital, Athens, Greece (protocol code 245, 16 April 2020).

Informed Consent Statement: 

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:

Data is available upon request from the authors.

Conflicts of Interest:

The authors declare no conflict of interest.

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