The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

Abstract

Among numerous studies on coronavirus 2019 (COVID-19), we noted that the infection and mortality rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) increased with age and that fetuses known to be particularly susceptible to infection were better protected despite various mutations. Hence, we established the hypothesis that a new immune system exists that forms before birth and decreases with aging. Methods: To prove this hypothesis, we established new ex-vivo culture conditions simulating the critical environmental factors of fetal stem cells (FSCs) in early pregnancy. Then, we analyzed the components from FSCs cultivated newly developed ex-vivo culture conditions and compared them from FSCs cultured in a normal condition. Results: We demonstrated that immunoglobulin M (IgM), a natural antibody (NAb) produced only in early B-1 cells, immunoglobulins (Igs) including IgG3, which has a wide range of antigen-binding capacity and affinity, complement proteins, and antiviral proteins are induced in FSCs only cultured in newly developed ex-vivo culture conditions. Particularly we confirmed that their extracellular vesicles (EVs) contained NAbs, Igs, various complement proteins, and antiviral proteins, as well as human leukocyte antigen G (HLA-G), responsible for immune tolerance. Conclusion: Our results suggest that FSCs in early pregnancy can form an independent immune system responding to unlearned antigens as a self-defense mechanism before establishing mature immune systems. Moreover, we propose the possibility of new solutions to cope with various infectious diseases based on the factors in NAbs-containing EVs, especially not causing unnecessary immune reaction due to HLA-G.

Keywords: IgG3; SARS-CoV-2; extracellular vesicles; fetal immune system; natural antibody.

Figure 1

A schematic diagram of the process of verifying hypotheses to identify a new immune system built up by FSCs in early pregnancy. (A) The developing progress of the fetal immune system. Igs-secreting B cell progenitors and pre pro-B cells were detected only in the fetal liver around 9 weeks of gestation, and mature B cells expressing B cell receptors were found in various fetal tissues after 18 weeks of gestation. In addition, maternal IgG can be transferred to the fetus through the umbilical cord after 15 weeks of gestation. However, it is not known whether the fetus produces Igs before the innate and adaptive immune system are formed. Adapted from with permission from AAAS. (B) To investigate the mechanism of self-protection by FSCs before establishing the innate and adaptive immune system by immune cells in early pregnancy, we established hypotheses and prepared a flow chart to verify them. (1) We confirmed whether Igs, particularly NAbs, including IgG3, which act on the innate immune system, were secreted from FSCs under a typical 2D culture condition. (2) We established an ex-vivo culture condition that simulates in vivo environment of FSCs to induce the secretion of NAbs from them. We confirmed the secretions of Igs and NAbs from FSCs^Ex-vivo (NAbs-FSCs) cultured in this culture condition and compared them with FSCs^Control cultured in a typical 2D culture condition. (3) We confirmed NAbs, proteins related to the promotion of Ig secretion, and complement proteins in NAbs-FSC-EVs secreted from NAbs-FSCs cultured in a newly established ex-vivo culture condition to induce the secretion of NAbs and compared them with FSCs^Control-EVs. In addition, we confirmed the difference in induction aspects according to the culture conditions by comparing the content of HLA-G proteins inducing immune tolerance and various anti-viral proteins induced by IFN in NAbs-FSC-EVs with FSCs^Control-EVs. Moreover, we compared and analyzed the induction aspects of NAbs such as IgG3 and IgM in NAbs-FSC-EVs and FSCs^Control-EVs through Exoview®. This verification process confirmed that FSCs cultivated in a newly established ex-vivo culture condition could secrete NAbs, complement proteins, and various anti-viral proteins, which could construct the new immune system to protect themselves from external infectious agents at the cellular level.

Figure 2

Establishment of new ex-vivo culture conditions to induce the secretion of NAbs in FSCs in early pregnancy. (A) In vivo environment of FSCs in the first trimester of pregnancy and factors inducing the secretion of NAbs in FSCs. (B) A schematic diagram of novel in vivo environment-like ex-vivo culture conditions inducing NAbs secretion in FSCs. (C) An ex-vivo cell culture temperature profile with a 5-6 day-cycle simulating the 28-day cycle of female hormones and body temperature changes applied to new ex-vivo culture conditions to induce hormone secretion in TBCs and FSCs replacing the internal nervous system . (D) A graph of 24-hour-cycle vibration condition applied to new ex-vivo culture conditions for inducing pregnancy-related hormone secretion in TBCs and FSCs .

Figure 3

Analysis of NAbs expression and secretion characteristics of FSCs cultured in the novel ex-vivo culture conditions for inducing the secretion of Nabs. (A) A schematic diagram to verify NAbs expression and secretion potential in FSCs (FSCs^Ex-vivo) cultured under new ex-vivo culture conditions. (B) Flow cytometry results comparing the expression patterns of stem cell-specific markers in FSCs cultured in FSCs^Control and FSCs^Ex-vivo. The results showed that the expression aspects of MSC markers (CD73, CD90, and CD105) and ESC marker (SSEA4) were very similar in FSCs^Control and FSCs^Ex-vivo. (C) Flow cytometry results comparing the cell membrane and intracellular expression patterns of Igs and NAbs in FSCs^Control and FSCs^Ex-vivo. The expression of membrane-bound IgM is rather higher in FSCs^Control than FSCs^Ex-vivo, but the expressions of intracellular IgM and IgG3 (NAbs) are much higher in FSCs^Ex-vivo than FSCs^Control. The expression pattern of IgG is similar in FSCs^Control and FSCs^Ex-vivo. (D) Results of protein antibody microarray analysis comparing the relative expression levels of Igs in the culture supernatants of FSCs^Control and FSCs^Ex-vivo. Compared to FSCs^Control, FSCs^Ex-vivo secreted higher levels of Igs. Error bars represent standard deviation. *, P <0.05; **, P<0.01; ***, P<0.001; Student's t-test.

Figure 4

Characterization of NAb-secreting FSCs-derived EVs (NAbs-FSC-EVs). (A) A schematic diagram verifying whether NAbs are contained in NAbs-FSC-EVs secreted from NAbs-FSCs cultured under new ex-vivo culture conditions. (B) The results of NTA on NAbs-FSC-EVs and FSC^Control-EVs secreted from FSCs^Control cultured on a typical 2D plate. The concentrations, average sizes, and distribution of FSC^Control-EVs and NAbs-FSC-EVs are almost similar. There is no significant difference in EV secretion characteristics according to the culture conditions. (C) SEM (left) and TEM (right) images of FSC^Control-EVs and NAbs-FSC-EVs, respectively. There are no significant differences in shapes and size distributions in both EVs according to the culture conditions. (D) The results of flow cytometry analysis to compare the expression patterns of exosome markers (CD9, CD63, and CD81) in FSC^Control-EVs and NAbs-FSC-EVs. The expression aspects that CD63 and CD81 are expressed at higher levels than CD9 are similar in both EVs. (E) The results of co-expression analysis of exosome markers (CD9, CD63, CD81, and syntenin) in FSC^Control-EVs and NAbs-FSC-EVs using ExoView®. They show expression patterns very similar to the flow cytometry results in Figure 4D, and these common expression patterns appear to be a unique characteristic of FSCs-derived EVs. (F) The results of flow cytometry analysis comparing the presence of IgG and NAbs (IgG3 and IgM) in FSC^Control-EVs and NAbs-FSC-EVs. (G) The results of exosomal cargo analysis to compare the content of NAbs in FSC^Control-EVs and NAbs-FSC-EVs using ExoView®. Similar to the flow cytometry results in Figure 4F, they show that NAbs-FSC-EVs contain higher levels of NAbs than FSC^Control-EVs. (H) The results of analyzing the patterns of NAbs content in both EVs captured by each exosomal marker using Exoview®. (I) The results of protein antibody microarray analysis comparing relative expression levels of B cell-specific proteins related to the promotion of Ig transcription induced in FSC^Control-EVs and NAbs-FSC-EVs. They show that B cell-specific proteins related to the promotion of Ig transcription are induced in NAbs-FSC-EVs at higher levels than FSC^Control-EVs. (J) The results of analyzing the concentrations of IgG3 and IgM contained in both EVs obtained from the subculture of FSCs^Control and NAbs-FSCs using each ELISA kit. IgG3 and IgM were not detected in all FSC^Control-EVs, but the contents of IgG3 and IgM in NAbs-FSC-EVs gradually increased as the subculture progressed. Error bars represent standard deviation. *, P <0.05; **, P<0.01; ***, P<0.001; Student's t-test; N.D., Not Detected.

Figure 5

Analysis results of various complement proteins contained in NAbs-FSC-Evs. The complement system is activated by the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). C1 subcomponents such as C1q, C1r, and C1s form the C1 complex in the CP, and the C5, C6, C7, C8, and C9 complement proteins form the membrane attack complex. C3a, C4a, and C5a proteins, generated in this process, recruit various immune cells to spread the inflammatory response . Comparisons of relative expression levels of complement proteins contained in NAbs-FSC-EVs and FSC^Control-EVs using SET100 protein antibody microarray (A) and human L493 array (B). (C) Comparisons of absolute quantification of complement proteins contained in NAbs-FSC-EVs and FSC^Control-EVs using ESI-Q-TOF MS/MS. We confirm that NAbs-FSC-EVs contain higher levels of various complement proteins compared to FSC^Control-EVs in all analysis results. Error bars represent standard deviation. *, P <0.05; **, P <0.01; ***, P<0.001; ****, P<0.0001; Student's t-test.

Figure 6

Comparison of secretion characteristics of cytokines, HLA-G, and anti-viral proteins in FSCs established in various culture conditions. (A) Various FSCs established in different culture conditions to characterize the self-defense mechanism formed by NAbs-FSCs. (B) Cytokine analysis results of EVs from FSCs obtained under each culture condition using human cytokine array. The secretion levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in FSC^Control-EVs, itFSC-EVs, and NAbs-FSC-EVs were similar, but the secretion levels of Th1 cytokines (IFN-γ and IL-2), showing increased expression in asymptomatic infected person to COVID-19, were the highest in NAbs-FSC-EVs. (C) The results of flow cytometry analysis to compare the expression aspects of membrane-bound HLA-G1 and intracellular soluble HLA-G5/G6 in each FSC using 87G (detecting β2m of HLA-G1 and HLA-G5 isoforms) and 2A12 (detecting intron 4 of soluble HLA-G5 and HLA-G6 isoforms) Abs. Similar to the previous study results , all HLA-G expressions were increased in NAbs-FSCs and itFSCs compared to FSCs^Control. In addition, the expression of HLA-G in NAbs-FSCs was also higher than in itFSCs due to the effect of increased IFN-γ stimulation. (D) sHLA-G (shedding HLA-G1 and HLA-G5) and HLA-G5/6 concentration analysis results of EVs from FSCs obtained under each culture condition through ELISA analysis using MEM-G/9 (detecting β2m of HLA-G1 and HLA-G5 isoforms) and 5A6G7 (detecting intron 4 of soluble HLA-G5 and HLA-G6 isoforms) Abs, respectively. Similar to the previous study results , all HLA-G concentrations were higher in itFSC-EVs and NAbs-FSC-EVs than in FSC^Control-EVs. However, unlike HLA-G5/6 concentrations, sHLA-G concentration was higher in NAbs-FSC-EVs than itFSC-EVs. (E) A comparison of the concentrations of interferon-induced proteins with anti-viral functions in NAbs-FSC-EVs and itFSC-EVs. IFITM3 protein was expressed at a higher level in NAbs-FSC-EVs than itFSC-EVs using SET100 protein antibody microarray, and LY6E protein was contained at a higher level in NAbs-FSC-EVs than itFSC-EVs according to ESI-Q-TOF MS/MS analysis. (F) A comparison of absolute quantification of another anti-viral protein (TRPML2) in NAbs-FSC-EVs and itFSC-EVs using ESI-Q-TOF MS/MS. Error bars represent standard deviation. *, P <0.05; **, P <0.01;***, P<0.001; ****, P<0.0001; Student's t-test.

Figure 7

Overview of HLA-G-based immune tolerance environment and NAbs-based self-defense mechanism induced by hormones secreted during pregnancy. During pregnancy, the fetus must protect itself from various foreign pathogens and the maternal immune system. Protection from the maternal immune system is achieved by an immune tolerance environment induced by various HLA-G isoforms secreted mainly by EVTs, which continuously infiltrate the maternal decidua. The transcription, alternative splicing, and secretion of soluble HLA-G isoforms, which can induce extensive immune tolerance, are induced by the pregnancy-related hormones hCG and PR , and the transcription and expression of membrane-bound HLA-G, which induces local immune tolerance, is also upregulated by IFN stimulation (the left panel). On the other hand, we suggest that before the immune system establishment, the fetal self-defense mechanism against foreign pathogens can be induced by various factors, including IL-10, Pax-5, NF-κB, and Oct-1/2, that promote the transcription of Igs, especially NAbs, such as IgG3 and IgM, which have the most outstanding anti-viral effector functions, through the pathway triggered by ER, another pregnancy-related hormone secreted from TBCs (the right panel). Therefore, our results demonstrate that hormones secreted during pregnancy may induce the different immune systems that fully protect the fetus from maternal immune system and foreign antigens.

Figure 8

Suggested Primal Immune System as a Self Defense Mechanism of Fetal Stem Cells before the establishment of innate and adaptive immune systems. Since virus invasion and infection into host cells are affected by the host's innate and adaptive immune responses, it was recognized that the fetus was very susceptible to viral infection before forming innate and adaptive immune systems. However, our results for FSCs in early pregnancy suggest that the fetus possesses very complicated and sophisticated self-defense mechanisms (primal immune system) at the cellular level exist in addition to the protective function of the placental TBC, which was considered as the only barrier to protect the fetus from external infections so far. (A) Extracellular defense mechanism by secretion of NAbs and complement proteins. Our results show that FSCs in early pregnancy can produce and secrete various NAbs, including IgP (fetal IgG3) polyreactive to unexperienced antigens, along with complement proteins to eliminate external pathogens immediately by CDC and ADCC, before umbilical cord generation, the main delivery route of maternal IgG, and before the complete development of B cells. In particular, IgP secreted from early pregnancy FSCs was proved to have the primal Ig repertoire that can instantly recognize various antigenic epitopes pathogens. Furthermore, IgP has the most outstanding Fc effector function to induce ADCC by binding with the highest affinity to Fcγ receptor of NK cells. These functions of IgP may contribute to much effective removal of the infectious agents. Also, we demonstrated that itPG-FSCs could secrete various NAbs such as IgP, IgM, and IgA as they contained factors, including Igll1, Pax5, and Cstf, that promote the secretion of soluble Igs. This finding may indicate that the primal immune system can establish the protective mechanism against various infection routes such as the upper respiratory tract in addition to the placenta and maternal blood. (B) The defense mechanism in the cellular and endosomal membranes to inhibit virus entry and replication by IFN-inducing proteins. Over the past decade, several IFN-inducible proteins, including interferon-inducible transmembrane family (IFITMs), ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and LY6E, have been known to regulate the infectious entry of various viruses . Still, few studies have been performed on the fetus during pregnancy. However, we proved through new ex-vivo culture conditions containing HSC/UCB-MSC^CO-EVs that the expressions of IFN-inducible proteins such as IFITM3, LY6E, and HLA-G were increased in fetal stem cells by increased IFN stimulation. Our results that itPG-FSC-EVs contained a higher level of LY6E and IFITM3 than FSC-EVs suggest that FSCs also have a sophisticated self-protection mechanism that suppresses the viral infection by LY6E mainly expressed in the plasma membrane at the beginning of virus entry and by IFITM3 expressed in the endosomal membrane after cell entry. In addition, membrane-bound HLA-G, whose expression is increased by IFN stimulation, may inhibit the relative expression of receptors that can be used as entry pathways of viruses and protect infected host cells by inducing delayed immune response through trogocytosis by contact with immune cells . (C) Intercellular defense mechanism to inhibit virus transmission by activating anti-viral autophagy. Transient Receptor Potential Mucolipin Subfamily (TRPMLs) are proteins constituting endosome cation channels and perform various physiological functions. TRPML1 is receiving attention as a target molecule that inhibits the fusion of the SARS-CoV-2 envelopes and endosomes , and a novel role of TRPML2, only expressed in the recycled endosome, in the innate immune response was recently revealed , but there is still little study on fetal stem cells during pregnancy. From our results, the expression of TRPML2 was increased in itPG-FSC EVs. We intend to suggest a new mechanism that the viral antigen information degraded by IFITMs upregulated by interferon stimulation acts as a pattern recognition receptor (PRR), thereby increasing TRPML2 expression , . In addition, upregulated TRPML2 may contribute to the intracellular protective mechanism that can prevent the replication of infected inhibits virus by binding to the endosomes containing the virus entering the cell, thereby inhibiting the entry of the virus into the cell nucleus and by inducing the degradation through anti-viral autophagy (lysosomal degradation).

Figure 9

Suggested Immune System for the whole life. To date, the antibody-based humoral immune system against foreign pathogen infection is divided into three categories according to the infection stage . The first is the immediate innate immune response by NAbs already present in the body before antigen exposure, and it acts as the first line of defense against infectious agents. The second is an extrafollicular "innate-like" antibody response induced within the early few days after antigen exposure, which serves to eliminate the infection temporarily until the specialized antibody response matures. Lastly, it is an acquired antibody response that appears delayed 1-2 weeks after infection by highly assembled antibodies through somatic cell hypermutation and class switch recombination processes. However, we found that the primal immune system by IgP existed before the fetal immune system establishment during pregnancy, and the adaptabilities of innate and adaptive immune systems against foreign antigens depend on the primal repertoire that was experienced and formed during this period. (A) Primal Immunity. According to the study for healthy fetuses in early pregnancy , circulating B lymphocytes in fetal blood at 12 weeks of gestation have a diverse BCR repertoire, and immunoglobulin heave chain variable region gene (IGHV)-containing clones analyzed at 26 weeks of gestation are similar to those of healthy infants. It was confirmed that it exists in proportion, but this can be called the primary repertoire. On the contrary, the repertoire of NAbs, including IgG3 secreted from FSCs obtained around 10 weeks of pregnancy, which we confirmed through this study, has not experienced any antigens except for themselves, and in other words, can respond to any antigens. Therefore, it can be a primal repertoire, and it will be gradually diversified to the primary repertoires with antigen-specific diversities due to modifying factors such as Abs transmitted from the mother, food and environmental factors, vaccinations, infections, or diseases . (B) Innate Immunity. The primary repertoire formed from the Primal repertoire in early pregnancy plays a critical role in manifesting symptoms after infection in the later innate and adaptive immune systems. Younger children who have a relatively similar repertoire to the Primal repertoire can react immediately to new antigens and eliminate them before the adaptive immune system acts. However, since birth, the number of naïve B-1 cells that secrete natural antibodies acting on the innate immune system, the ability to secrete antibodies, and the adaptability of the repertoire to new antigens gradually decrease with age. The ability to cope with new viruses, such as SARS-CoV-2, also decreases with age . For this reason, many children and younger generations have asymptomatic infections of COVID-19, and even young children are well adapted to the mutant virus , while the older, the more symptomatic and severe the proportion of patients increases. (C) Adaptive Immunity. The evasion mechanisms to the initial innate immunity of all viruses, including SARS-CoV-2, and the resulting delayed priming of T cell responses and adaptive immunity are receiving attention as the main factors that increase the severity and fatality of COVID-19 with age . In other words, as the naïve T cell pool gradually decreases with age, it is difficult to generate a T cell response capable of recognizing a new antigen. This assumption can be supported by the findings that systemic excessive immune pathology, including cytokine storms, is caused by an explosive innate immune response activated to replace the delayed adaptive immune system . In particular, the delayed adaptive immune response can cause taste and olfactory abnormalities in asymptomatic and mild patients accounting for more than half of SARS-CoV-2 infections , so the most effective prevention and treatment for various infections including SARS-CoV-2 It can be said that the strategy lies in an effective innate immune system capable of triggering a normal adaptive immune response. From this point of view, it is expected that our research results, which first proposed the concept of the primal immune system before the formation of the innate immune system, will contribute to finding a fundamental solution to protect humanity against unknown infectious agents in the future. Another important finding in our results is that IgA is also contained in itPG-FSC-EVs secreted from FSCs in early pregnancy. It can be expected to provide an expanded variety of applicability to various infection pathways, such as the upper respiratory tract in the existing IgG-centered Ab therapeutics.