Figure 4. Potential activation of STAT3 by SARS CoV-2 spike protein stimulatory effects. Under normal conditions, IL-6R remains dormant as a) it is inhibited by SOCS3 and b) it does not synergise with unstimulated EGFRs to produce STAT3. This condition can be bypassed and reversed by (A) the activation of NF-κB and subsequent expression of IL-6 through TLR2 and TLR4 stimulation by spike protein [174,176] and (B) the direct stimulation of EGFR by spike protein and synergy with IL-6R [177]. The final effect between IL-6R, EGFR and IL-6 would be the continuous production of STAT3, although the SOCS3 will still be present [179].
Elevated levels of Th17 cells are implicated in the etiopathogenesis of numerous inflammatory and autoimmune diseases [181]. Furthermore, in some instances, Th17 cells can promote cancer [182]. Th17 cells are shown to be strongly implicated in spike protein induced immunopathology. In a recent study that concluded that the spike protein aggravates rheumatoid arthritis, the Th17 cell population was markedly increased, whilst the Treg cell population was decreased [183].
Th17 cells produce the cytokine IL-17, which promotes inflammation. Th17 cells are believed to be involved in the pathogenesis of myocarditis, which has been identified as a sometimes-fatal complication of mRNA vaccination [184,185]. They recruit other immune cells, such as neutrophils, to the heart, and they release pro-inflammatory molecules such as IL-17. The levels of Th17 cells are elevated in patients with myocarditis. Blocking Th17 cell activity via drugs such as Bazedoxifene ameliorates myocarditis in experimental models [186]. Furthermore, a connection to macrophage activation syndrome is suggested by a study that confirmed that macrophages infiltrated the heart muscle and became activated, releasing toxic cytokines, in association with vaccine-induced myocarditis [187].
Th17 cells also play an important role in autoimmune hemolytic anemia (AIHA). A study by Xu et al. found that patients with AIHA had elevated levels of Th17 cells, which were closely correlated not only with disease severity but also with the levels of IL-17 and anti-RBC IgG antibodies [188]. IgG antibodies are the most common class of autoantibodies against RBCs, often acting through molecular mimicry. CD8+ T cells bind to IgG antibodies and become activated to release cytokines that destroy RBCs. Abnormalities of immunoregulatory cytokines associated with AIHA include elevated levels of IL-6, IL-2, and IL-17, and increased secretion of TGF-β [189]. Reduced numbers of circulating CD4+ nTregs are also linked to the disease [190]. As we have seen, all of these are consistent with known effects of the spike protein.
Yonker et al, (2023) found that the concentration of free unbound circulating spike protein is elevated in the blood of vaccinated individuals who suffer from post-vaccine myocarditis [191]. Whereas in a control group without myocarditis, circulating spike protein was appropriately bound by antibodies. Some cases of myocarditis due to the mRNA vaccination are considered to be the result of autoimmune activation [192]. Additionally, it is worrisome that the DCs and monocytes increase their TGF-β and IL-2 signalling upon engagement with the spike protein synthesized by human cells from the vaccine mRNA (Table 1) [33]. The spike protein on its own has been shown to activate TGF-β signalling [163]. The monocytes and macrophages are mainly DC-derived antigen presenting cells [193]. The intense TGF-β signalling can also be attributed to spike protein induced inflammation via TLR2-mediated NF-κB hyperactivation [174].
Proper Treg-DC cellular interactions are crucial for the well-controlled suppression of the effector CD4+ T lymphocytes [1]. Impairments during Treg-DC cellular interactions will produce autoimmune disease [1]. TGF-β signalling inhibits DC functions in general, and latent TGF-β signalling by the DCs will contribute in favour of Th17 cell differentiation, and, hence, to the development of autoimmune disease.
This immune impairment seems to be tightly connected with the mRNA vaccines. An autoimmune origin of disease is sufficiently described in a relevant case of encephalomyelitis due to mRNA vaccination [194]. Moreover, the several pathological neurological outcomes that follow COVID-19 mRNA vaccines, including Guillain Barré syndrome (GBS), transverse myelitis, and acute disseminated encephalomyelitis (ADEM) (amongst several others), also have an autoimmune origin [195]. Again, in relation to the spike protein expressed by the mRNAs, autoimmune encephalitis was the diagnosis of disease after three doses of mRNA (Pfizer) vaccination in a case study, and the mRNA vaccines were found to be the only factor causing the disease in this patient [196].
12. Th17, PD-L1 and IgG4
As we have said, the mRNA vaccines induce a strong IgG antibody response to the spike protein. There are four subtypes of IgG antibodies, labelled as IgG1, IgG2, IgG3, and IgG4. IgG3 is very effective at protecting from infection, whereas IgG4 is uniquely unable to protect from infection, and, in fact, actively blocks access to the spike protein by effector antibodies [197]. IgG4 is normally the least common variant in human serum. However, elevated levels of IgG4 are triggered by repeated exposure to inflammation-inducing antigens. A seminal paper tracked the evolution of IgG variant distribution over time following the initial two shots and subsequent booster shots of mRNA SARS-CoV-2 vaccines [198]. Remarkably, they found that class switching towards IgG4 increased over time in the months following vaccination. IgG4, which normally represents no more than 5% of the total pool, was sharply elevated upon administration of the booster shot. Furthermore, the level continued to rise after the booster, reaching nearly 20% of the IgG pool five months after the booster shot. A subsequent article proposed that IgG4 induced by the booster shot constitutes an immune tolerance mechanism that would suppress the natural antiviral responses to the SARS-CoV-2 virus [199]. Another publication confirmed that IgG4 is highly expressed several months after mRNA vaccination, and that this phenomenon does not occur for the DNA vector-based vaccines [200].
IgG4-related disease (IgG4-RD) is a newly recognized disease that is characterized by elevated serum levels of IgG4 and excess fibrosis in multiple organs [201]. PD-L1 plays a role in IgG4-RD. Concentrations of soluble PD-1 and PD-L1 are significantly elevated in patients with IgG4-RD, and the expression of PD-1 on Treg cells is upregulated. Furthermore, stimulation of naïve T cells from IgG4-RD patients with PD-L1 caused them to transform into CD4+CD25+ iTreg cells. The authors concluded that the PD-1/PD-L1 pathway could promote Treg cell differentiation into iTregs, and that this may play an important role in the observed elevation of Treg cells in IgG4-RD patients [202]. Most target organs of IgG4-RD have Treg cell infiltration, and Treg cells are also abundant in the blood [203].
Type I autoimmune pancreatitis (AIP) is commonly found in association with IgG4-RD. An increased number of circulating iTregs, particularly those releasing Il-10, was found in association with IgG4-RD-related pancreatitis [204]. On the other hand, circulating nTreg levels are low, a pattern consistent with immunosenescence. These abundant iTregs appear to be ineffective at controlling the inflammation, and a likely explanation for this is a decreased expression of Mammalian Sterile 20-like Kinase 1 (MST1), which is essential for allowing the cell-to-cell contact needed for iTregs to act on Teff cells [205]. Patients with IgG4-RD are at increased risk to both pancreatic cancer and lymphoma [206]. Several case reports of acute pancreatitis have been reported in association with mRNA vaccines [207-209]. A case study described a patient who experienced rapid progression of lymphoma following an mRNA booster vaccine [210]. Several other case reports involving lymphoma following mRNA vaccination have been published [211-214]. One of 14 mice involved in a study of the BNT162b2 mRNA vaccine developed a fatal aggressive multi-organ malignant B-cell lymphoblastic lymphoma shortly after the second shot [215].
13. Conclusion
In this paper, we have provided an extensive review of the role of Treg cells in the immune system, with a particular focus on the apparent disruption of their behavior caused by the mRNA vaccines. It appears that the vaccines typically induce an intense IgG antibody response due to the toxicity of the spike protein, along with an extreme inflammatory response through cytokine release by T cells, and, ultimately, the potential for autoantibodies to attack the tissues through recognition of non-self spike protein on the cell surface. Because a natural infection is replaced by an abnormal situation in which human cells are producing large quantities of a toxic viral protein, the type I IFN response is suppressed. Normally, this response to double-stranded viral RNA induces the clonal expansion of a pool of Treg cells, but also keeps them suppressed until the viral load has sufficiently subsided. The mRNA in the vaccines is resistant to breakdown and concealed from the immune system due to its humanized code. This causes an unnatural and often inappropriate immune response, where the consequences are highly dependent on the prior immune state of the vaccinated individual, particularly with respect to their Treg cell population. Some of the activated DCs return to the thymus and induce a response that damages the thymic epithelium and accelerates thymic involution, leading to inflammaging and immunosenescence. This can also induce a life-threatening macrophage activation syndrome (HLH), as was observed in several case studies on the mRNA vaccines. Repeated booster vaccination can lead to the development of self-tolerance to the spike protein, which may make the person less resistant to the virus than a fully unvaccinated person.
We have analyzed the response to the mRNA vaccines against COVID-19 differentially depending on a distinction between cancer(-) and cancer(+) populations. The mRNA vaccines cause a Treg dysregulation in both populations. The Treg dysregulation in the cancer(-) population predominantly causes immune senescence and promotes autoimmunity, in part due to homing of mTreg cells to the thymus and accelerated thymic involution. In cancer(+) cases, depending in part upon whether they receive PD-1/PD-L1 inhibitors, the patients develop a hyperimmune response and also have a tendency to develop autoimmunity. Moreover, the cancer(+) patients who do not receive PD-1/PD-L1 blockers are prone to cancer progression by the mRNA vaccines. Furthermore, the development of a high Th17 response may also result in tumorigenesis, and, therefore, further studies are needed to evaluate the potential of the mRNA vaccines to induce cancer. The inhibition of mTOR may accelerate immunosenescence due to enhancement of the memory Teff response, and this is especially dangerous for the elderly population receiving the mRNA vaccines, who are at risk for both autoimmune and neoplastic disease.
Acknowledgements: Special thanks to Mrs. Trinity Brami for her assistance in creating the figures of this paper.
Funding: This research was funded in part by Quanta Computers, Taiwan, under the auspices of the Qmulus project. The funder played no role in the preparation of the manuscript.
Authors’ contributions: AK wrote the first draft. AK, SS and GN participated in multiple rounds of edits and expansions. AK, SS, GN, and PMc all participated in further refinements and verification of factual content.
Competing Interests: The authors declare no competing interests.