Figure 1. The presentation of SARS-CoV-2 spike protein by DCs to the thymus leads to thymic involution and inflammatory disease. The spike presentation by DCs causes mTEC apoptosis that leads to thymic atrophy and failure of Treg transformation, eventually resulting in thymic involution and inflammatory diseases [96-104].
7. The Molecular Reasons for Treg Irregularities in the Cancer(-) Population after the mRNA Doses
Kasper et al. (2016) have detailed the complex molecular networks that control Treg induction and function beyond IL-2 and TGF- β, including transcription factors, kinases, phosphatases, Notch family receptors, mTOR signalling, etc. [105]. The Treg cells, when functioning well, have a protective effect against cancer, autoimmune reactivity, and transplant rejection. A key aspect of their protective role is through conferring mTreg immunity; that is, they respond efficiently to re-exposure to an antigen they were primed with earlier [70,106]. In general, the main role of Treg CD4+ T cell subpopulations is influenced by a complex cascade of genetic, molecular and T cell interactions (for more details, as this description is outside the scope of this review, see [70]) ultimately to provide an efficient mTreg response. The final outcome of Treg cellular interactions is for differentiated Treg cells to release IL-10, TGF-β and other suppressive chemokines that will negatively control the pro-inflammatory responses and thus limit prolonged and chronic inflammation. As the Treg cells are subdivided into CD4+ Treg and CD8+ Treg cells [107,108], from each Treg subpopulation an antigen-specific mTreg cell subset is created that will keep the immune system in check, preventing an overwhelming future immune stimulation by the same virus re-infection and/or viral antigen vaccine boosters [109].
However, in the case of SARS-CoV-2 spike protein, an extensive robust NF-κB activation occurs. This causes an upregulation of genes involved in a) TNF-α signalling, b) the pro-inflammatory response, and c) cytokine-to-cytokine receptor interactions [110]. Overall, the activation of NF-κB signalling upon the stimulation of a specific viral antigen (for a detailed review see [111]), on its own, initiates the formation of a Treg response. The NF-κB-mediated Treg response specific to the stimulating antigen thereafter leads to the formation of specific subpopulations of mTreg cells which have the role to become activated upon later stimulations from the specific viral antigen [112]. NF-kB has two branches (pathways) that are simultaneously activated by viral antigens, a) the canonical pathway which leads to inflammation, and b) the non-canonical or alternative pathway which is involved in immune cell differentiation, maturation, and organogenesis.
The stimulation of NF-κB has been mainly considered as an optimal activator of CD4+ mTreg cells through the activation of the NF-κB canonical pathway. The mTreg cells, as we have stated previously, are needed for the organism to avoid autoimmunity [113], but their activation promotes cancer progression [114]. The role of the alternative pathway activation in the formation of Treg cells has remained obscure until recently. The SARS-CoV-2 spike protein stimulation of the T lymphocyte toll-like receptor (TLR) system releases excessive TNF cytokines [115]. Hence, the stimulation of TNF receptor family members (such as OX40, CD40 and LT-βR) by the spike protein will result also in the activation of the alternative NF-κB pathway by the stabilisation and energisation of NF-κB Inducible Kinase (NIK) [111].
In experiments that investigated the role of NIK overexpression in relation to Treg development, it has been shown that the overstimulation and constitutive expression of NIK leads to aggressive and lethal autoimmunity. The Treg cells produced under the overwhelming stimulation of NIK in these experiments were defective in inducing immune suppression [116]. In these experiments, the tested mice were engineered to constitutively overexpress NIK and the phenotype of the T cell response was characterized by OX40+ hyper-reactive T cells and Tregs that were deficient in Foxp3.
The expression of Foxp3 by T cells is catalytic for an optimum Treg suppressive activity. Under the influence of NIK overstimulation, there is a loss of the capacity to distinguish between self and non-self-antigens by the immune system that leads to a disturbed self-tolerance, a hallmark for autoimmunity initiation and progression [45]. CD4+ T cells at inflammatory sites in rheumatoid arthritis are known to be resistant to suppression by Treg cells [45]. Overall, this leads to a state of hyper-inflammation in the organism.
A study on the immune response to SARS-CoV-2 mRNA vaccines found that IFN-γ and Il-2 were highly expressed following vaccination, with a statistically significant increased expression in those who were vaccinated following infection with COVID-19. The level of these cytokines was highly correlated with the IgG response [23]. Il-2 plays an important role in Treg induction and persistence [30]. Interestingly, Treg cells accumulate with age, but the reason for this is surprising. It is not through clonal expansion from either the thymic or the peripheral pool, but rather simply because aging Treg cells show reduced expression of the protein Bim, a pro-apoptotic signalling molecule. As a consequence, they survive much longer than Tregs expressing high levels of Bim. Chronic stimulation by Il-2 leads to preferential expansion of Tregs with low expression of Bim, allowing them to accumulate, and increasing the size of the overall Treg pool through lack of attrition [117]. As we have already discussed, some of these long-lived Treg cells migrate to the thymus and facilitate accelerated thymic involution.
The extensive study of Świerkot, J et al., investigated the emergence of an autoimmune response after SARS-CoV-2 mRNA vaccination in a cancer(-) population [118]. In this study, the individuals who had completed their mRNA vaccination (2 mRNA injections) and had presented with more severe vaccine adverse reactions (VARs,), had significantly higher antinuclear antibody (ANA) titers when compared to the individuals with less severe VARs [119]. The authors did not find a correlation between prior SARS-CoV-2 infection status and severity of VARs. However, another study found that more severe VARs was most strongly associated with individuals who had COVID-19 and were subsequently mRNA vaccinated [120]. Furthermore, many studies show that autoimmunity can arise after COVID-19 vaccinations. One study describes 27 cases of autoimmune reactions following SARS-CoV-2 vaccination (17 flares and 10 new) [121]. In a case report of systemic lupus erythematosus, symptom onset occurred just two days after immunization with the first mRNA injection [122]. A 63-year-old man experienced acute severe autoimmune-like hepatitis just one week after his first dose of an mRNA vaccine [123]. A review article described 27 cases of autoimmune hepatitis following COVID-19 vaccines, ranging in age from 27 to 82, 20 of which were due to mRNA vaccines. None of them used any hepatotoxic drugs that could explain their disease [90]. Cases of autoimmune hemolytic anemia are described as a serious adverse reaction of mRNA vaccination [124,125]. A single-center study based in Saudi Arabia identified 31 cases of autoimmune disease following mRNA vaccination, including vasculitis, systemic lupus erythematosus and neurological diseases. All but four of them were new-onset disease, where symptoms first appeared on average just seven days after the vaccine [126]. A comprehensive review article found considerable evidence of new-onset autoimmune disease following mRNA vaccination, including autoimmune glomerulonephritis, autoimmune rheumatic diseases, and autoimmune hepatitis [127].
8. The Immune Response of Cancer(+) Patients after Receiving the mRNA Injections: the Influence of Vaccination on the Treg Responses
In general, the activation of dendritic cells, through the stimulation of Toll like receptors (TLRs), pro-inflammatory cytokines, and CD40, is naturally designed to produce a subpopulation of Treg cells (for review see [70]). The generation of Treg cells promotes cancer development and exerts immunosuppression in the tumor microenvironment (TME), lowering the natural cellular anti-tumor activity and enhancing the growth of tumors [43]. The mechanisms of tumor enhancement by Treg cells are several, and the generation of Tregs has a prognosis favouring the development of many cancers while at the same time inhibiting the development of autoimmune diseases [128,129].
Treg cells inhibit anti-tumor immunity, and enhanced Treg responses are associated with cancers of poor prognosis. The elimination of Treg cells in cancer is a hallmark for successful treatment results during immunotherapy [130]. Basic research on Treg inhibition in the past has provided fundamental insights on tumor regression and, moreover, has revealed correlations between the inhibition of a Treg cell response and the development of autoimmunity. When the research group of Shimizu et al. [131] specifically blocked the CD25+ CD4+ suppressive Treg cells, the peripheral CD4+ T cells were able to eliminate syngeneic tumors in normal naïve mice. The results of another research group, that of Takahashi et al. [132], showed that the elimination of CD25+ CD4+ Treg cells in naïve mice led to spontaneous development of autoimmune diseases. The CD25+ CD4+ Treg cells are naturally anergic, and when activated exert immune suppression. Moreover, the antigen concentration that is required to make the Treg cells become suppressive is lower than the antigen concentration required to make the CD25-CD4+ T cells, i.e., Teff cells, become activated and proliferate. The expression of CD25 (also known as the IL-2 receptor α chain) facilitates distinguishing between the true Treg cells, characterized by being responsive to IL-2 and immunosuppressive, and cells that are non-responsive to IL-2 (CD25-), which are not true Treg cells and are non-suppressive.
Only a few subsets of CD25- cells can evolve, regain their CD25 expression, and function as regulatory (suppressive) cells during a specific antigen’s repetitive activation of the immune response [133]. A thorough analysis of the T cell responses elicited after the full dose (two injections) of the mRNA vaccination in cancer(+) patients highlights that their T cell responses are very low 6 months after vaccination as compared to their T cell responses that were developed three weeks after their mRNA full (two dose) vaccinations [134]. Although this can be attributed to the overall immunodeficiency caused by cancer in these patients, this can also mean that the immune system of these patients develops a sufficient Treg subclass of cells, specific for spike protein, which remains responsive in time and eventually suppresses the T cell response against the spike protein.
Cancer(+) patients being treated with immune-suppressing therapy face a difficult situation where they are likely to experience severe disease from a viral infection, but they are also not likely to respond as well as cancer(-) patients to the vaccine. A careful investigation of the immune response of cancer(+) patients to repeated mRNA vaccination revealed an ominous sign that such patients could reach a point where further vaccination against COVID-19 is counterproductive [135]. Eleven out of 36 patients being studied showed an optimal response after the second vaccine, but then suffered from T cell exhaustion following the booster shot, due to repeated exposure to the spike antigen. A marked fall-off of IFN-γ production was associated with a marked upregulation of programmed cell death 1 (PD-1) on CD4+ and CD8+ T cells [135]. PD-1 is a known marker for T cell exhaustion [136]. Several studies have shown that PD-1 is upregulated in CD8+ and CD4+ T cells during COVID-19 disease, and that PD-1 levels are higher in association with severe disease [137] (and references therein). This suggests that the booster shot may have actually made these patients more susceptible to severe disease from COVID-19. Furthermore, PD-1 expressing exhausted T cells are less able to suppress tumor growth [138].
A study on mice clearly demonstrated that repeated booster shots immunizing against the spike RBD domain led to increased PD-1 expression in T cells, which was associated with profoundly impaired CD4+ and CD8+ T cell activation and a poor antibody response [139].
Severely immunosuppressed cancer patients suffering from multiple myeloma generate a specific memory Teff subpopulation against spike protein which increases after two to five weeks from the second mRNA vaccination dose [140]. A specific mTreg cell subpopulation was also generated after the mRNA vaccines, and was sustained over time, in the immune system of the mRNA vaccinated multiple myeloma patients [84]. The Treg and mTreg cells are generally CD25+, CD27+, FOXP3+, and CD127+. As a reminder, the general rule is that the CD25+ (true Treg) T cells will become activated with less antigen concentration than the CD25- (not true Treg) T cells [132].
Furthermore, the immune suppression conferred by the CD25+ Treg cells is independent of the humoral response developed by the B cells encountering the antigen, as this kind of T cell response relies purely on the antigen-presenting cell interactions. Therefore, the increased activation of B cells upon the third booster dose of mRNA in patients with solid cancer shown in the study of Scroff FT et al. [141] is not related to the true Treg response developed in these patients. The finding of this study, which illustrates a poor effector T cell response after the third booster mRNA, is alarming and prognoses for further deterioration of the overall health of the solid cancer patients. This is due to the development of the Treg response which suppresses T cell clonal activation.
Because the researchers were unable to detect any presence of antigen presenting cells, specific subtyping of T cells was not performed. Also, after the third (booster) dose of mRNA, the humoral B cell response lacked coordination between various immune aspects which are normally linked, suggesting a diminished T cell effector response. Regarding T cell adaptive immunity, this means that the Treg and subsequently the mTreg responses which have been developed in these cancer(+) patients were feasibly robust, and their suppressive activities outweighed any beneficial Teff cell response against the mRNA coded spike protein after the booster (third) dose of mRNA vaccination [3]. Also, a disorganised B regulatory cell activity leads to a downregulated Teff cell response [142].
9. PD-L1 Upregulation Following mRNA Vaccination
Programmed cell death ligand 1 (PD-L1) is a regulatory molecule expressed on many types of immune cells and cancer cells, and, by binding to its receptor PD-1, expressed on the surface of activated T cells, it leads to T cell dysfunction and apoptosis [143]. At least two studies have shown that PD-L1 is overexpressed in circulating immune cells following the second vaccine. Loacker et al. (2022) found significant upregulation of PD-L1 expression levels on monocytes and granulocytes two days after the second mRNA vaccine in 62 vaccinated individuals, compared to unvaccinated controls. They suggested that this indicated a regulatory response to avoid autoimmune collateral damage [144]. Özbay et al. (2022) examined expression of PD-L1 in antigen-presenting monocytes at 6 different time points starting before the first vaccine and ending 12 weeks after the booster shot. They found particularly high expression levels two weeks after the second vaccine. The level subsided somewhat but remained elevated at all the subsequent measurement times, up to 12 weeks after the booster shot [145]. They too suggested that this reaction could be a protective mechanism suppressing overactivated T cells induced by vaccines. However, there is concern that sustained upregulation of PD-L1 could accelerate tumor growth, because PD-L1 expressing monocytes in circulation could infiltrate the tumor environment. PD-L1 ligating to PD-1 on activated CD8+ T cells in the tumor microenvironment would suppress their activity, preventing them from killing the tumor cells. PD-L1 also causes PD-1-expressing activated CD4+ T cells to transform into Tregs [146].
PD-L1 is expressed on many types of cancer cells, and, by binding to its receptor PD-1, expressed on the surface of activated T cells, it leads to T cell dysfunction and apoptosis [143]. Furthermore, the PD-L1 upregulation depends on sensing IFN-γ secreted by activated CD8+ T cells [147]. PD-1/PD-L1 inhibitors are a group of immune checkpoint inhibitors that are becoming an attractive choice for cancer immunotherapy in multiple types of cancer [148]. These work by blocking PD-1/PD-L1 signalling, enabling tumor-resident immune cells to kill the tumor cells. However, these drugs often come with severe and even fatal side effects that limit their usefulness. Treatment is associated with increased risk of severe immune-mediated inflammation in the lungs, the colon, the liver and the kidneys, as well as autoimmune diabetes [148]. This is likely due to the fact that the tissue resident immune cells are now able to launch an inflammatory response.
Impaired PD-1/PD-L1 function plays an important role in many autoimmune diseases [149]. The fact that the activation of PD-1 is essential for the prevention of autoimmunity caused by the mRNA vaccines is shown in a study involving cancer(+) patients [150]. These patients were under immunotherapy treatment with checkpoint signalling inhibitors that block PD-L1 expression and therefore PD-1 activation [41]. The patients developed autoimmune antibodies after the mRNA booster doses, likely due to the antigenic over-stimulation of Teff cells by spike protein, in the absence of a protective response normally induced by PD-L1. It is reasonable to assume that the CD25- effector T cells are protagonists in this pathway [1]. These T cells permit the development of autoimmunity while offering protection against cancer [3,35].
A characteristic of the aged immune system is inflexibility and an inability to adapt to new challenges. As the system ages, an imbalance sets in between Tregs and Teffs. Age-related loss of Treg function renders the host susceptible to a syndrome of chronic smoldering inflammation, whereas age-related gain of Treg function leads to increased risk to cancer and infection. It appears that the aged immune system has reached a steady-state condition that often errs in one direction or the other, regarding Treg function, which dictates a trade-off between autoimmune disease and cancer [77]. Thus, autoimmunity and cancer are two sides of the same coin [151].
10. Impaired mTOR-mediated Treg Cell Function
As we mentioned earlier, an important role for type I IFNs is to stimulate the synthesis of a pool of Tregs ready to “spring into action” once the viral load subsides. This process depends on activation of the PI3K/Akt/mTOR pathway [152]. In 2013, Zeng et al. demonstrated an essential role for mTORC1 as a positive regulator of Treg function, via experiments on mice with a disruption of mTORC1 function through Treg-specific deletion of the essential component raptor [153]. These mice developed a fatal early onset hyperinflammatory disorder due to ineffective Treg suppressor function. Mechanistically, Raptor-dependent mammalian target of rapamycin complex 1 (mTORC1) signalling in Tregs coordinates Treg proliferation and upregulation of suppressive molecules to establish Treg functional competency.
The NLRP3 inflammasome recruits macrophages and neutrophils, which in turn cause reactive oxygen species (ROS) production [28] [154]. Excessive production of ROS is known to inhibit the phosphoinositide 3 kinase (PI3K)/Akt pathway [155]. The spike protein has been demonstrated to induce an intense inflammatory response that may be initiated even prior to cellular infection. Even spike pseudovirions and recombinant SARS-CoV-2 spike protein treatment induce apoptosis and phagocytosis in ACE2-expressing cells, as a consequence of ROS inactivation of the PI3K/Akt/mTOR pathway [156]. The authors of this work proposed that this effect could account for multi-organ failure associated with severe cases of COVID-19 [156].
Interferon regulatory factor 3 (IRF3) is a transcription factor that plays an essential role in detecting double strand viral RNA and then launching the type I IFN response and activating the PI3K/Akt pathway [157]. It has been demonstrated experimentally that the spike protein interacts with IRF3 and mediates its proteasomal degradation, thus terminating IFN-I activation [158]. Thus, the vaccines not only do not induce an IFN-I response, but also facilitate the production of a large pool of spike proteins that would directly interfere with IFN-I activation by other pathogens. This specific effect of the spike protein could partially explain the occasional reactivation of latent viruses such as Herpes and Varicella following mRNA vaccination [159].
The sustained hyperinflammatory state induced by the mRNA vaccines may be primarily due to the impaired ability of the immune system to provide an adequate pool of activated effector Tregs to suppress the cytokine response by effector T cells. As we will see in subsequent sections, repeated mRNA vaccination eventually induces a response that suggests the development of immune tolerance to the spike protein, likely to prevent tissue damage due to excessive cytokine production. But this also means that the vaccine will lose its effectiveness to protect from COVID-19 after repeated booster shots. A relevant study on cancer(-) immunosuppressed individuals shows that only after selective drug-induced mTOR inhibition do CD4+ and some CD8+ hyperactive T cells develop after the mRNA vaccination [160]. When mTOR is active, the differentiation of T effector cells is favored, while the formation of memory T cells is inhibited. Likewise, the inhibition of mTOR promotes the generation of memory immunity [161]. As already described, the favoring of memory Teff responses will further promote immunosenescence and inflammaging, and this requires a thorough evaluation of mRNA vaccines safety, especially in the elderly population [162]. Figure 2 shows the mechanisms by which the SARS-CoV-2 spike protein could induce an inflammatory response.