Abbreviations: ADEM: acute disseminated encephalomyelitis; AIHA: autoimmune hemolytic anemia; AIP: autoimmune pancreatitis; ANA: antinuclear antibody; CCR6: C-C Motif Chemokine Receptor 6; DCs: dendritic cells; EGFR: epidermal growth factor receptor; Foxp3: forkhead box P3 ; GBS: Guillain Barré syndrome; HLH: Hemophagocytic lymphohistiocytosis; IFN: interferon; IL-10: interleukin-10; IgG4-RD: IgG4-related disease; IgG: immunoglobulin G ; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; NIK: NF-κB Inducible Kinase; PD-1: programmed cell death 1; PD-L1: programmed cell death-ligand 1; RBD: receptor binding domain; RGD: arginine-glycine-aspartate tripeptide motif; SOCS3: suppressor of cytokine signalling 3; STAT3: signal transducer and activator of transcription 3; TB-IRIS: tuberculosis-immune reconstitution inflammatory syndrome; TGF-β: transforming growth factor-β; TLR: toll-like receptor; TME: tumor microenvironment; Teff: T effector cell ; Treg: T regulatory cell; VARs: vaccine adverse reactions; iTregs: inducible Treg cells; MST1: Mammalian Sterile 20-like Kinase 1; mTECs: medullary thymic epithelial cells; mTreg: memory Treg Cell; miR: microRNA; nTregs: naïve Treg cells; scRNA-seq: single-cell mRNA sequencing.

Keywords: Treg cells; SARS-CoV-2 mRNA vaccination; immunosenescence; thymic involution; cancer; autoimmunity; TGF-β; IL-6; NF-κB; IgG4.

Upon stimulation from a specific antigen, the immune homeostasis of T regulatory cell responses preserves self-tolerance and halts exaggerated T cell immune responses to protect from tissue damage [1,2] The discovery of Treg cells (either of thymic or peripheral origin) in mammals, including humans, has offered considerable insights into the regulation of the adaptive immune response [1]. Both CD4+ and CD8+ regulatory T cells offer a homeostatic balance in the immune system to avoid both autoimmunity and cancer [3]. Treg cells release cytokines such as interleukin-10 (IL-10) that suppress the activity of Teff cells. When the immune cells lose self-tolerance, Treg cells play a role in preventing an excessive inflammatory response that could injure tissues. On the other hand, a large population of Treg cells resident in the tumor microenvironment maladaptively protects cancer cells from immune attack, leading to accelerated tumor growth [4].

During aging, T cells develop increased affinity to self-antigens, which is concurrent with and offset by a clonal expansion of peripheral (inducible) Tregs (iTregs), In parallel, thymic T cell capacity shrinks, impairing the ability to generate new T cells. The increase in iTregs can help to suppress autoimmunity, but it comes with a high cost of increased risk to cancer and sepsis [5].

The thymus gland plays a central role in the development of the immune system in mammals. Beginning in utero, stem cells migrate from the bone marrow into the thymus, where they first mature into thymocytes. These thymocytes undergo a transformation involving a complex process of negative and positive selection that ultimately yields a pool of CD4+ and CD8+ T cells, as well as a naïve Treg (nTreg) cell population.

The selection process involves exposing the cells to diverse human proteins, and those thymocytes that bind strongly to human proteins are eliminated via apoptosis. Those that bind weakly are retained and become the dominant source of CD4+ and CD8+ Teff cells. For cells that show intermediate binding, the situation is more complicated. Many of them evolve into nTreg cells, that, when activated, are able to suppress clonal expansion and activation of Teff cells. A unique marker for Treg cells is the forkhead box P3 (Foxp3) transcription factor. Some Teff cells still survive in this pool of intermediate-binding cells, and they play a significant role in autoimmune disease, especially in association with immunosenescence and inflammation linked to aging [6,7]. Besides nTreg cells that emerge from the thymus, peripheral CD4+ Teff cells can also transform into Treg cells in response to the cytokines IL-2 and transforming growth factor-β (TGF-β), which are overexpressed in association with cellular stress [8,9].

Ionizable cationic lipids are key components of the lipid nanoparticles used for delivery of mRNA in the mRNA vaccines [10]. While one important feature of these lipids is that they can release the mRNA by endosomal rupture to support protein synthesis, they can also delay release until the lysosomal stage, which activates the NLRP3 inflammasome [11,12]. This can be beneficial as an adjuvant to induce an immune response, but it may cause unintended negative consequences through oxidative stress leading to mitochondrial damage and inducing necrosis, syncytia formation, and pyroptosis [13].

Activation of the NLRP3 inflammasome induces caspase-1 release from mitochondria due to excessive reactive oxygen species and mitochondrial DNA damage [14]. Damage response signalling results in the formation of membrane pores and the initiation of a necrotic form of cell death called pyroptosis. The NLRP3 inflammasome and caspase-1 together lead to secretion of the pro-inflammatory cytokine IL-1β [14]. These activities are essential for launching the immune response to the vaccine antigens that will ultimately lead to a strong antibody response, the desired outcome.

There is another lesser known but equally important member of the interleukin-1 family that is also activated by the DNA damage response and caspase-1 signalling, IL-18 [15,16]. IL-18 plays several roles both in immune activation and in autoimmune disease. On the positive side, it promotes the proliferation of cytotoxic CD8+ T cells [17]. However, through an unusual mechanism that we will describe in detail later on, it induces self-reactive innate antibody responses that play an essential role in autoimmune disease [18]. It also promotes inflammation-induced carcinogenesis in squamous cell carcinoma [19]. For our purposes, the most interesting aspect of IL-18 is its ability to induce peripheral activated mTreg cells to migrate back to the thymus, particularly in younger persons before thymic involution, where they play a powerful role in disrupting innate nTreg development and release into the periphery [20]. We hypothesize that this effect is the primary mechanism by which IL-18 leads to excessive activation of self-reactive antibodies, through a reduction in the naïve Treg pool in the periphery.

IL-18 signalling upregulates C-C Motif Chemokine Receptor 6 (CCR6) expression in peripheral activated mTreg cells, and this results in their migration to and homing in the thymus. These recirculating thymic Tregs then inhibit the production of new nTreg cells in the thymus, by consuming IL-2, resulting in its depletion [21].

A multi-author study has shown through a whole blood test quantifying the Th1 cytokines – interferon- γ (IFN-γ), tumor necrosis factor- α (TNF-α), and IL-2 – that spike-specific T cells produced all these cytokines in abundance at two weeks after the second dose [22]. Prior infection with COVID-19 leads to an increased production of IL-2 in response to the mRNA vaccines [23], inducing the transformation of peripheral Teff cells into Treg cells. A published case study involved a patient who developed severe myocarditis following a single dose of the mRNA vaccine. He had had a mild case of COVID-19 three months earlier, which primed a powerful NLRP3 inflammasome reaction to the vaccine. This patient’s monocytes expressed increased levels of IL-18 compared to others who had been vaccinated for COVID-19, likely leading to homing of induced mTregs to the thymus and increasing the risk for an autoimmune attack on the heart [24].

The thymus plays a central role in shaping the immune system during childhood. With in- creasing age, the thymus shrinks over time, a process known as thymic involution, a property common to all vertebrates. Increasingly, it is becoming clear that thymic involution may be the most important factor in immunosenescence and the associated chronic smoldering inflammatory state known as “inflammaging” [25,26]. The NLRP3 inflammasome has a direct effect on the thymus, accelerating thymic demise [27]. IL-18 has been shown to suppress regeneration in the thymus, by activating the IL-18 receptor on natural killer cells [28]. It is widely accepted that immunosenescence leads to increased risk to infection, autoimmune disease, and impaired cancer immunosurvelliance [29].

Treg cells play an important role in thymic involution [30]. Remarkably, as thymic involution progresses, the homing mTreg cells maintain their numbers, while the counts of all the other cell types in the thymus decrease. In fact, these mature Tregs constitute the majority of the Treg pool in the aged thymus [20]. The elderly population generally has a high Treg/Teff ratio in the periphery, but the Treg population is predominantly composed of long-lived mTregs that have already committed to the specific antigen that they were originally exposed to. These mTregs will suppress the T cell response to new exposures to the same antigen, but they have little ability to react to novel threats. Naïve Tregs able to respond to a new insult are in short supply, and this results in poorly controlled autoimmune attack by self-reactive T cells [31].

Those who suffer from conditions associated with immunosenescence, e.g., cancer, cardiovascular disease, rheumatoid arthritis, metabolic diseases, neurodegenerative diseases, etc., are at increased risk for suffering from severe and sometimes fatal COVID-19 infection [32]. When people with high-risk preconditions are vaccinated with the mRNA vaccines, there is an increased production of both TGF-β and IL-2, likely leading to the production of a large mTreg cell population, poor response to the vaccine, and further acceleration of thymic involution [33].

A recent analysis by Chouerini TK et al. [34], although concluding in support of vaccinating patients with cancer (cancer(+) patients) with mRNA vaccines, reveals important findings for considering immunological disorders of SARS-CoV-2 vaccinees. In this study, it was found that the mRNA vaccinated cancer(+) patients, and especially those who had received 2 or 3 booster doses prior to SARS-CoV-2 infection, develop breakthrough SARS-CoV-2 infections more frequently than the unvaccinated cancer(+) control group, suggesting a Treg-suppressed immune system after repeated mRNA exposure. Importantly, within the vaccinated cancer(+) population in this study, the development of hematologic malignancies was encountered more frequently than in the unvaccinated cancer(+) control group. Also, the vaccinated cancer(+) group required more anti-neoplastic drugs to treat their malignant conditions.

The authors of this study concluded that the use of further mRNA vaccination [34], in addition to the initial two vaccines in cancer(+) patients, would help to prevent increased mortality rates from COVID-19 in this population group. However, their findings also imply immunological irregularities in the cancer(+) vaccinees after mRNA exposure. Importantly, they described an ill-defined abnormally enhanced Treg response that suppressed anti-spike-protein Teff cell immunity in the cancer(+) patients, brought about by the mRNA injections. According to the aforementioned studies on immunosenescence and Treg responses, [31-33], it is therefore likely that further injections would lead to even greater immune suppression, and further accelerate cancer progression [35].

These findings led us to review the literature and provide further analysis of the immune system responses developed upon mRNA vaccination in the cancer(+) and cancer(-) populations, with a focus on the Treg cell population. In general, high immunogenicity is associated with more severe side effects, and, depending on the initial state of the immune system, vaccines can, in the extreme cases, either fail to produce an effective immune response or produce such a strong immune response that it induces severe and even life-threatening adverse reactions. Sophisticated machine learning methods have been developed to evaluate vaccine-induced immunity and reactogenicity [36].

Tregs behave differently in healthy and in malignant tissues [37]. A propensity toward autoimmunity is induced by mRNA vaccination in both cancer(+) and cancer(-) individuals. The clinical course in these two scenarios, though, is quite different. Insufficient suppression by an inadequate Treg pool in the cancer(-) scenario creates conditions favoring development of “classical” autoimmunity (autoimmune thyroiditis, rheumatoid arthritis, etc.). In the cancer(+) individual, though, enhanced suppression of the immune response by a resident abundant Treg pool is most relevant for its impairment of anti-cancer immunity and consequent risk of accelerated cancer progression [38,39].

Cancer and autoimmunity are in juxtaposition from a deregulated Treg response [40], which we argue is happening after mRNA vaccination. The autoimmunity occurring in cancer(+) patients under immunotherapy following primary and especially booster mRNA shots is considered to be a downstream effect of a dysregulated T cell response [41]. Moreover, the development of autoimmunity is closely linked to primary immunodeficiency syndromes that manifest with recurrent infections [42]. In this case, the breakthrough infections encountered in the cancer(+) patients after the mRNA vaccination is a sign that the mRNAs produce an exacerbation of their preexisting immunodeficiency [34]. Breakthrough infections occur also in the cancer(+) patients that have not received immunotherapy treatments (although in lower numbers).

Therefore, with the mRNA vaccinations against COVID-19, important questions arise that concern immune competence in both the cancer(-) and cancer(+) populations. These are: 1) in the cancer(-) population, could the immune system be provoked toward more frequent development of any particular types of malignancy by the mRNA vaccines [39]? and 2) what is the absolute increased risk of new cancer (in cancer(-)) or enhanced growth/spread of cancer (in cancer(+)) for individuals receiving one or multiple mRNA injections? In this regard, the Treg responses after the mRNA vaccinations could potentially be of prognostic value [43]. The functioning Treg cells have on the one hand a suppressor function that allows malignant cells to survive, but, on the other hand, when the Treg cells are inhibited, this lets autoimmunity develop, as a consequence of the intense inflammatory response induced by the spike protein [44]. With these questions in mind, we review the available literature on immune responses after the mRNA vaccinations. We then examine the similar but distinct implications of the dysregulation of Treg cells in the cancer(-) and cancer(+) populations. In doing so, we offer clear and concerning answers to the questions posed.

Autoimmunity involves an impairment of Treg homeostatic balance [45]. Conceptually, when a Treg response is raised upon a specific antigen stimulus, T cells are prevented from becoming activated into functional effector cells. During autoimmunity, the Treg cells lose their suppressive function and Teff cells that have lost self-tolerance cause disease. Concerning the mRNA vaccinations for COVID-19, a thorough review by Diani S et al. determined that the natural immunity conferred by a previous SARS-CoV-2 infection, both cellular and humoral, is robust and long lasting compared to more rapidly waning protection afforded by vaccines. Vaccination carries greater risk of adverse reactions in previously infected individuals, with a higher risk of inducing autoimmune disease with repeated vaccination [46].

A study of Tormo N et al. evaluated T cell responses after the mRNA vaccinations according to a) the age (before and after 60 years of age) and b) whether they have been previously infected or not with SARS-CoV-2. They noted substantial differences in the immune response to the administered vaccines over time based on both age and previous infection status [47]. We will describe their results in more detail later in this paper, as they nicely illustrate the concepts we are proposing. Two papers that set the stage for our arguments are Lourenço EV et al., which provides a review of the role of dysregulated natural Treg cells in autoimmunity [48], and Sanchez et al., which describes their important role during infection [49].

We have searched the PubMed and ScienceDirect databases for papers describing the immune response to the mRNA vaccines, as well as a large number of papers that review the complex mechanisms of the immune system and the processes by which it ages. In the below, we begin with a section specifically focusing on the unique aspects of the immune response to the vaccines compared to SARS-CoV-2 infection. After depicting the observed Teff and Treg responses, inferred from the Tormo et al. study [47], we examine the criteria of autoimmunity development in both cancer(+) and cancer(-) populations. These observations led us to further predict the development of immunosenescence as a consequence of the return of activated dendritic cells and Treg cells to the thymus, accelerating thymic involution. Based in part on the study of Pellerin et al. [50], which discusses immune loss of regulation due to an altered function of FOXP3+ Treg cells, we predict a subsequent Treg/Teff imbalance in the mRNA vaccinated individuals. The Treg/Teff imbalance involves either an enhancement or a reduction of the Teff cell response in these population groups under differing initial immune states, leading to differing pathological outcomes. Finally, the immune senescence pathogenic mechanisms that are underlying and complicate the final effect of repeated mRNA vaccinations led us to investigate the deleterious outcomes from an altered Treg/Teff balance in the immune systems of vaccinees, particularly after repeated booster shots [3,51].

mRNA viruses induce expression of type I interferons (IFN-α and IFN-β) by infected cells, due to the detection of double-stranded RNA during replication [52]. A major distinction between the immune response to the mRNA vaccines and that provoked by a viral infection is that, in the case of the vaccine, the type I IFN response is not induced due to the absence of replicating viruses. Not only are the enzymes needed for replication lacking, but also the mRNA has been disguised to resemble a human mRNA molecule [53].

Type I IFNs play a major role in the initial immune response to a viral infection. They cause the activation of naïve CD4+ and CD8+ T cells in the early stages of the infection, inducing clonal expansion and differentiation into a pool of Teff cells as well as a pool of iTreg cells [50]. Type I IFNs maintain the Foxp3+ expression that characterizes Treg cells under inflammatory conditions [54]. However, type I IFNs actually suppress the activity of Treg cells, holding them in check until the viral load has dissipated [55]. Over time, the level of type I IFNs decreases, due to the fact that cytotoxic immune cells, also induced by the IFN, have cleared the virus-infected cells and halted viral replication. Once the type I IFN expression is sufficiently reduced, the iTreg cells that had been standing by are now free to release the immune-suppressing cytokines, including interleukin-10 (Il-10) and TGF-β, which are effective in shutting down the inflammatory response after the virus has been successfully cleared [56].

The SARS-CoV-2 spike protein has been demonstrated experimentally to inhibit and damage the ACE2 receptor protein expression in epithelial cells. This induced a hyperinflammatory signalling cascade that led to activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and increased release of TNF-α and IL-6 [57]. A study involving 50 COVID-19 patients revealed that those with severe disease were characterized by a persistent viral load and high levels of TNF-α and IL-6 expression, associated with a highly impaired type I IFN response, in some cases due to the presence of anti-type-I-IFN autoantibodies. The lack of type I IFN delayed the immune response to the virus, allowing the virus to replicate freely, and inducing severe disease. Furthermore, an insufficient pool of mTreg cells caused sustained immune activation, and the overactive immune response was the major source of severe symptoms [58].

A study of the immune response to the vaccines compared to the response to infection revealed that the vaccine induces a response pattern comparable to that of severe disease [59]. These authors wrote: “We find that BNT162b2 vaccination produces IgG responses to spike and RBD [receptor binding domain] at concentrations as high as those of severely ill COVID-19 patients and follows a similar time course.” [59] This result aligns with the concept that the vaccine simulates an impaired type I IFN response. A detailed study on the mRNA vaccines revealed that there was a refractory period immediately following vaccination prior to the induction of a specific immune response, and the authors proposed that this delay could explain the higher risk of infection during this early period [60]. This delay may be a manifestation of a missing type I IFN response.

The mRNA vaccines create a mosaic of cells that synthesize spike protein, inducing a response in transfected cells that results in the abundant release of exosomes containing not only the spike protein but also microRNAs (miR-148a and miR-590) that specifically suppress the response to type I IFN. When these exosomes are taken up by microglia (immune cells in the brain), they induce a potent inflammatory response [61]. Exosomes presenting the spike protein on their surface are still present in the circulation four months after vaccination [62]. Large-scale single-cell mRNA sequencing (scRNA-seq) technology revealed dramatic alterations in gene expression of almost all immune cells after vaccination. Increased NF-κB signalling and a reduced type I IFN response were most notable, and there was a marked deficiency in CD8+ T cells [33]. Type I IFNs induce a massive expansion of antigen specific CD8+ T cells, both effector and memory, in response to viral infection [63]. Type I IFNs also protect CD8+ T cells from destruction by natural killer cells [64]. A study on mice with impaired type I IFN receptors in Tregs found that these Tregs had enhanced suppressor activity during both acute and chronic infection, resulting in CD8+ T cell anergy, defective generation of memory T cells, and viral persistence [55].

With mRNA vaccines, immune cells would be expected to respond to the situation as an unnatural circumstance in which human cells are producing a toxic foreign protein. The detection of antigen on the surface of transfected cells activates CD4+ immune cells and launches the cascade that eventually leads to a strong antibody response to the spike protein. This response is heavily skewed towards immunoglobulin G (IgG), with little or no IgM or IgA antibody production [59]. IgG is the primary antibody type that induces autoimmune disease, and this effect is enhanced in the absence of secreted IgM antibodies [65,66]. The replacement of every uridine in the vaccine mRNA molecules with methylpseudouridine assures that the mRNA will survive for a long time and continue to be translated into spike protein, resulting in sustained immune activation [67].

There is extensive homology between heptapeptides from immunoreactive epitopes in SARS-CoV-2 and human proteins that can lead to autoimmune disease via molecular mimicry. Cross-reactive IgG antibodies could mistakenly attack human proteins with similar peptide sequences, and a constellation of diseases, including neurological disorders, cardiovascular alterations, coagulopathies, pregnancy dysfunctions, multiple cancers and anosmia, among others, could ensue [68].

The spike protein can induce an intense inflammatory response in endothelial cells via integrin binding. The arginine-glycine-aspartate (RGD) tripeptide motif exposed on the surface of the receptor binding domain (RBD) of the spike protein binds to integrin 51 expressed by endothelial cells. This activates the NLRP3 inflammasome through the NF-κB signalling pathway. NF-κB signalling also induces vascular leakage and leukocyte adhesion. As a result of NF-κB activation, proinflammatory cytokines, chemokines, and coagulation factors are upregulated in endothelial cells [69]. Treg cells dramatically increase their suppressive function in response to inflammation, releasing high levels of the immunosuppressive cytokines Il-10 and TGF-β [70].

iTregs, but not nTregs, interact with endothelial selectins and transmigrate past the endothelial barrier. In response to antigen presentation (e.g., spike), they suppress TNF-α and Il-1β, as well as Teff cell adhesion to the endothelium, which is critical for T cell influx into inflamed tissues [71]. This fast-acting suppression is mediated by TGF-β released by the iTregs [72]. The anti-idiotype antibodies become quite relevant in this regard. They can be structurally identical to the original antigen, i.e., spike proteins, and thus push forward this suppression [73].

Cancer is associated with an imbalance in Teff and Treg cells where the Tregs far outnumber the Teffs in the tumor microenvironment [35,74]. The NLRP3 inflammasome promotes carcinogenesis in squamous cell carcinoma. Huang et al. found that Foxp3 was highly overexpressed in the tumor, and Treg cells comprised 45% of the CD4+ T cells there [19]. Induction of high levels of TNF-α and IL-6 by the spike protein through activation of NLRP3 will lead to increased production of Il-10 and TGF-β by the pre-existing Treg pool. This can be expected to cause excessive immune suppression in the tumor microenvironment, leading to accelerated tumor progression. Autoimmune disease has the opposite problem [45,75]. The increased activation of Teff cells by the vaccine in the context of an insufficient Treg pool will exacerbate autoimmune disease.

Under normal conditions, immunosenescence occurs as the immune system ages [76]. As aging progresses, the peripheral Treg population increases in number, but most of those Tregs are mTregs already committed to specific antigens, and the ability to induce an iTreg response to a novel exposure is lowered [77]. As these cell populations continue to shift with time, the cumulative loss of Treg activation in response to self-reactive antibodies results in an increased risk of autoimmune disease developing with increasing age.

IFN-γ, a Th1 cytokine and the only type II IFN, is produced by hyperactivated CD4+ and CD8+ T cells in response to a virus infection. T cell hyperactivation has been associated with severe cases of COVID-19 [78]. During Mycobacterium tuberculosis infection in the lungs, IFN-γ-producing CD4+ T cells are essential for controlling the pathogen, but overproduction of this cytokine causes lung injury, leading to tuberculosis-immune reconstitution inflammatory syndrome (TB-IRIS) [79]. CD25-expressing hyperactivated Teff cells produce the protease furin, which cleaves the spike protein, facilitating viral entry [78].

Type I IFN induces proliferation of Foxp3+ Treg cells, which, when activated, suppress the expression of IFN-γ [80]. A seminal paper comparing the immune response in cases of severe COVID-19 with milder disease revealed many aspects of immune dysfunction that were associated with an impaired type I IFN response. The T cells of severe cases highly expressed CD25 (the IL-2 receptor), but they were deficient in Foxp3. Foxp3-CD25+CD4+ T cells were very effective as Teff cells, producing high, even toxic, levels of IFN-γ, as well as furin. The authors hypothesized that these cells were very short-lived and died off before being able to transform into Foxp3+ Treg cells. They concluded that tissue damage in the lungs associated with severe disease was mainly due to an overactive immune response, leading to excessive and prolonged inflammation. Thus, an impaired Foxp3-mediated negative feedback loop characterized severe disease [78].

The aforementioned study by Tormo et al. provides an opportunity to compare vaccine responses among young and old and to assess the effect of previous exposure to SARS-CoV-2 [47]. The authors looked specifically at 50 individuals who were either nursing home residents (old) or nursing home employees (young). Thus, they provided cohorts for both a <60 and a >60 population, as well as a distinction between those who had previously recovered and those whose first exposure to the spike protein was through the vaccine.

The first thing to note from this study is that a prior infection resulted in a very modest IgG antibody response to the spike protein. All of the cases had been mild, and this is reflected in the fact that the CoV2+ <60 group had a median IgG level of 5 RU/ml, and the median for the CoV2+ >60 group was only 36 RU/ml, just prior to vaccination. This is to be contrasted with peak values greater than 800 RU/ml for all four cohorts following vaccination. So, one can conclude that the dramatic response to the vaccine more closely emulates severe disease.

However, prior infection clearly had a powerful effect on the reaction to the vaccine. The antibody response to the first vaccine was far greater in the CoV2+ cohort than in the CoV2- cohort. This was likely due to memory Teff cells ready to respond immediately to the spike protein being produced by the transfected cells.

The CoV2+ >60 population achieved a median IgG response of 2882 RU/ml in response to the first vaccine. This was the highest titer achieved in this group – the second vaccine added no further benefit. The authors proposed that a single vaccine would be more than adequate for those already infected, and that the second vaccine might even do harm.

The CoV2- cohort showed a slower and lower increase in both humoral (anti-spike IgG antibodies) and cellular (IFN-γ) response markers, compared to the CoV2+ cohort. This was especially true for the CoV2- >60 group. CD4+ IFN-γ responses for this population remained low the entire time, reaching a maximum level of just 0.07 IU/ml four weeks after the second vaccine. It was not until two weeks after the second vaccine that any of them achieved a level above the proposed cutoff threshold. Since these individuals were all nursing home residents, it is likely that immunosenescence was a cause of their poor response. While the >60 CoV2- group had the poorest immune response, by contrast the >60 CoV2+ group acquired more than twice the serum antibody titers and IFN-γ levels even compared to the <60 CoV2+ group. So, the contrast between CoV2- and CoV2+ was especially dramatic for the 60+ population.

The precipitous fall in IFN-γ during the two-week period following the second vaccine for the CoV2+ population was perhaps the most remarkable result of these experiments, and this was especially pronounced in the >60 group, where CD4+ IFN-γ levels fell from 1.61 just before the second vaccine to only 0.89 two weeks later. The authors hypothesized that Treg cells may have suppressed the response to control exacerbated inflammatory damage, but this would also of course limit the effectiveness of the vaccine, and potentially accelerate inflammaging. These Treg cells were likely induced by simultaneous excessive production of IL-2 and TGF-β in response to the first vaccine [9,22,33]. This sharp downregulation appeared to be transient, however, as a level comparable to that following the first vaccine was restored one month after the second dose, perhaps due to persistent production of the spike protein by transfected cells showing a continued Treg/Teff cell imbalance.

Lozano-Ojalvo et al. compared vaccine reactions in CoV2- and CoV2+ populations with similar findings as those of the Tormo et al. study. These authors showed that CoV2+ individuals produced very high levels of both IL-2 and IFN-γ just ten days after the first vaccine. Furthermore, the second vaccine actually set them back by causing a reduction in cellular immunity [81].

The natural immunity of unvaccinated CoV2+ individuals, both cell-mediated and humoral, is superior to the mRNA vaccine-induced immunity, which decays more rapidly over time [82]. Natural SARS-CoV-2 antigens are superior to the mRNA-derived spike protein for inducing long-lasting immunity [83]. A bigger concern is that the vaccine may be inducing immunosenescence, increasing risk to infections with other pathogens. A study based in Israel found a significant increase in non-COVID respiratory infections from April to June 2021, immediately following an aggressive nationwide vaccination campaign [84]. While the authors suggested easing of social distancing as a likely cause, the induction of immunosenescence by the vaccine might also have contributed to this result.

It had long been believed that the thymus is immune privileged (i.e., is insensitive to foreign protein exposure), but more recent research has shown that this is not true. In fact, chronic infection of the thymus by viruses that are highly pathogenic can drive the immune system to immune tolerance towards that pathogen. This could happen through at least three distinct mechanisms: (1) negative selection of pathogen-reactive T cells, (2) excessive generation of pathogen-specific Tregs, or (3) T cell anergy. They may all be at play [85].

SARS-CoV-2 can infect the thymus, particularly in the youth, and this induces a loss of function that correlates with disease severity [86]. ACE2 is expressed by the thymic epithelium, particularly the medullary thymic epithelial cells (mTECs), which are mainly responsible for negative selection, and so they should be susceptible to SARS-CoV-2 infection. The SARS-CoV-2 virus can target TECs and downregulate critical genes involved with epithelial cell adhesion and survival [86]. Rosichini et al. verified in an in vitro study that cultured TECs from the thymus of children expressed ACE2 and were able to be infected with SARS-CoV-2. Spike-positive human TECs were identified at both 24 and 48 hours after infection. There was increased mortality among the mTECs compared to cortical TECs, reflecting their higher ACE2 expression [86]. The spike protein induces IL-6 and TNF-α in epithelial cells [57]. Both of these cytokines have been implicated in acute thymic involution [87]. Defects in thymus epithelial cells are associated with the aged thymus [88].

An experiment involving mice with a genetic defect that interfered with the induction of T-cell tolerance in the thymus resulted in a strong mouse model for autoimmune hepatitis. The mutation led to depletion of mTECs that would normally cause autoreactive T-cells to be eliminated before they exit the thymus. This resulted in a reduction in the release of naive Tregs from the thymus and an increase in the release of self-reactive CD4+ and CD8+ T cells [89]. Autoimmune hepatitis has been associated with the mRNA vaccines [90].

The thymus is easily accessible via the lymphatic system, so this implies that the mRNA vaccines could enable the delivery of the spike protein and even the spike mRNA and the ionizable cationic lipids through the lymph system, beginning with the axillary lymph nodes. Swelling of the axillary and chest lymph nodes is one of the more common side effects of the vaccine, clearly indicating that the dendritic cells (DCs) reacting to the injection in the deltoid muscle are migrating to the lymph node [91]. The DCs would almost certainly endocytose the mRNA nanoparticles while resident in the muscle tissue.

A case study involved a 64-year-old woman with breast calcification who was assessed for breast cancer via ultrasonography six months before her first SARS-CoV-2 vaccine, and again 7 days after the vaccine due to obvious lymph node enlargement in the vaccinated arm. Six months later, a follow-up examination revealed that the lymph node was still swollen, although somewhat reduced, even though there was no evidence of breast cancer [92].

Dendritic cells play an essential role in controlling the transformation of thymocytes into new antigen-specific T cells in the thymus. As many as half of the DCs in the thymus are of peripheral origin, rather than recently emerging from the bone marrow. Some of the circulating DCs return home to the thymus and carry antigens from the periphery to the thymus. Ominously, this implies that DCs could directly deliver vaccine mRNA and synthetic cationic lipids to the thymus. Once in the thymus, these cells proliferate, likely distributing the vaccine mRNA among their offspring. They not only present antigen to T cells, but also induce antigen-specific Foxp3+CD25+CD4+ Tregs from Foxp3-CD25-CD4+ thymocytes. By contrast, Tregs were not induced by similar DCs in the spleen [93].

Thus, these activated antigen-expressing DCs that migrate back to the thymus induce both negative selection of antigen-specific T cells and an antigen-specific Treg pool to further control any self-reactive antibodies that escape selection. These returning DCs are the major hemopoietic cells that serve in this capacity in the thymus. While these activities can serve well to protect from autoimmune disease due to molecular mimicry, they could also induce tolerance to a virus, jeopardizing a memory response.

The S1 segment of the spike protein could be cleaved by furin from spike protein exposed on the membrane of DCs and released freely into the external milieu [94]. Since S1 contains the receptor binding domain of the spike protein, it could bind to the ACE2 receptors on mTECs, inducing a damaging inflammatory effect, as has been demonstrated for endothelial cells [69]. Even independently of the spike protein, returning DCs have been shown to directly inhibit TEC proliferation and induce their apoptosis by activating the Jagged1/Notch3 signalling pathway [95].

An impaired type I IFN response may play a critical role in the pathological overshooting of immune activation associated with the vaccines. In response to a viral infection, type I IFN induces a massive clonal expansion of antigen specific CD8+ T cells. It has been shown that antigen-specific CD8+ T cells expand nearly 10,000-fold during the first week after mice are infected with lymphocytic choriomeningitis virus [63]. As we have stated, the vaccines do not elicit a type I IFN response, due to the lack of double-stranded mRNA associated with viral replication [53,96].

Severe COVID-19 has been linked to a deficiency in the glycoprotein perforin, resulting in a pathogenic auto-inflammatory feedback loop [97]. Perforin, released by cytotoxic CD8+ T cells, generates pores in the target cell membrane, resulting in cell death. The number of perforin-positive lymphocytes declines precipitously above the age of 70, and this could help explain the increased susceptibility to severe COVID-19 in the elderly [98]. Furthermore, the S1 subunit of the spike protein has been shown experimentally to suppress perforin expression in CD8+ T cells [99].

Cytotoxic CD8+ T cells are essential for eliminating hyperactivated antigen presenting DCs in the thymus, a process that critically depends on perforin [100]. Hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome, is a life-threatening, hyperinflammatory disorder, characterized by malignant inflammation and multi-organ failure. A study on perforin-deficient mice provided a compelling demonstration that these mice were susceptible to HLH, due to an impaired ability of CD8+ T cells to prune off hyperactivated antigen-presenting DCs in the thymus [100]. HLH has been reported as an adverse reaction to the mRNA vaccines in multiple publications [101-104].

In summary, the mRNA vaccines prime the thymus to produce spike-specific autoreactive T cells that fail to be transformed into Treg cells due to a deficiency in activated CD8+ T cells. These T cells can be a source of autoimmune disease and HLH, a hyperinflammatory attack on the organs. At the same time, transfected DCs in the thymus may continue to produce spike protein for weeks if not months after vaccination, causing damage to mTECs, accelerating thymic involution, and driving the immune system towards anergy. These ideas are schematized graphically in Figure 1.