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.
1. Introduction
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].
2. mRNA Vaccine Responses in Patients with and without Cancer
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.
3. The Criteria for Assessing Treg Dysregulation after mRNA Vaccinations
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].
4. Delayed but Enhanced Immune Response to mRNA Vaccines
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.
5. The Treg Response after mRNA Vaccination: Potential Role for Immune
Senescence
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.
6. Potential for Damage to the Thymic Epithelium and Accelerated Thymic
Involution
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.