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.