Crohn’s cell expressing the lectin receptor, CD161, as well

Crohn’s
disease (CD) is an Inflammatory Bowel Disease (IBD) distinguished by
discontinuous and excessive inflammation along the gastrointestinal tract (GI).
Advanced or untreated CD, leads to scar formation or ulcers, further resulting
in intestinal stricture and bowel obstruction. 
In these cases, surgery is required to excise the affected region of
either the small or the large intestine. 
Currently, the most prevalent treatments against CD include
anti-inflammatory drugs (corticosteroids) and immunomodulators (anti-TNF),
however, efficient outcomes appear only to a proportion of patients (NHS, 2015).  Additionally, CD approximately affects 2.5
million individuals of any age in the Western world and has an accelerating
increase in incidence in the developing countries.  The above evidence illustrates the necessity
of discovering novel therapies in order to bear a spectrum of alternative treatments
that can be implemented according to the individual (Boyapati, Satsang and Ho, 2015). 

The
aetiology as well as pathogenesis of CD, and more generally of IBD is ambiguous
and complicated.  The most accepted
hypothesis suggests that the synergy between genetic and environmental factors
trigger disease onset by stimulating continuous expression of pro-inflammatory
cytokines.  Under normal conditions, the
intestinal mucosal barrier is essential for preventing invasion of commensal bacteria
in the epithelial cells.  This is
achieved through maintaining immune self-tolerance. However, the aforementioned
risk factors of CD cause imbalances of the gut microenvironment, leading to
increase of intestinal permeability which results in dysfunction of mucosal
barrier and subsequent breakdown of tolerance (Wang et al, 2016).  Therefore,
the breakdown of tolerance elicits excessive immune responses.  According to this, GWAS-studies indicated
that genetically susceptible individuals may carry specific genetic
polymorphisms related to bacteria recognition, further promoting tolerance
impairment. CD is considered a cell-mediated disease, as Th1 and Th17 responses
are predominantly associated with its pathogenesis, illustrated in Figure 1. 

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The
balance between Treg/Th17 cells is essential for mucosal homeostasis, through
sustaining the equilibrium between pro- and anti-inflammatory cytokines.  Depending on the cytokine microenvironment,
the pro-inflammatory responses of Th17 cells are restricted by the activity of regulatory
T cells (Tregs), and vice versa (Ziegler
and Buckner, 2009).  This is achieved
by the presence of TGF?, a
common mediator for Th17 and Treg differentiation.  Th17 cells are thought to be originated from
a subset of naïve T cell expressing the lectin receptor, CD161, as well as the
transcription factor ROR?t (Cosmi et al, 2008).  Their differentiation depends on the presence
of the pro-inflammatory cytokines, IL-1?,
IL-6, IL-21 and TGF?. Following
activation, Th17 cells express high levels of IL-17, IL-21, and IL-22
pro-inflammatory cytokines which have an essential role in host defences
against extracellular pathogens by triggering further recruitment of innate
immune cells (Galvez, 2014).  In contrast, Tregs are divided into two
categories; natural and induced.  Natural
Tregs (nTreg) arise from the thymus already expressing FOXP3, while induced
Treg (iTreg) develop from mature CD4+ effector T cells in the periphery.  Treg differentiation in the periphery is
achieved upon exposure to high concentrations of TGF?. 
The main function of FOXP3+ Treg’s involves modulation of excessive immune
responses for maintening self-tolerance and thus prevention of autoimmune
diseases.  This is achieved through TGF? and IL-10 anti-inflammatory cytokine secretion
at the site of inflammation (Yadav,
Stephan and Bluestone, 2013).

 

During
CD, mucosal barrier dysfunction alters the Th17/Treg balance towards Th17-induced
inflammatory response, and thus significantly eliminating iTreg within the
intestinal lamina propria.  Although these
subtypes exhibit contradictory functions, TGF? is required for their
differentiation.  At low concentrations,
TGF? synergizes with IL-6 and IL-6-dependent IL-21 to promote IL-23 receptor
expression, and thus stimulating Th17 differentiation.  Conversely, high concentrations of TGF? and
absence of IL-6 or IL-21, expression of IL-23R is supressed resulting in
inhibition of ROR?t and induction of iTregs (Omenetti and Pizzaro, 2015). 
This suggests, that Th17 cells can inhibit Tregs by the expression of
IL-21, while Tregs inhibit Th17 through IL-10 production (Raza et al, 2012).  However,
in CD elevated levels of TGF? are
ineffective, as the downstream signalling pathway is disrupted by the blockage
of smad3 phosphorylation.  This is
achieved by the up-regulation of the intracellular inhibitor of smad
signalling, known as smad7 which acts as a negative feedback on smad3.  Interestingly, both iTreg and Th17 cells have
been illustrated of being unstable with a degree of trans-differentiation into
other CD4+ subtypes.  This was illustrated,
as a fraction of FOXP3+ Tregs showed high-degree of plasticity towards an IL-17-producing
phenotype along with loss of FOXP3 expression. 
This is caused due to the accumulation of Th17-generated cytokines, as
plasticity is thought to be an adaptive mechanism caused by the loss of immune
tolerance.   (Ueno et al, 2015).  Oppositely,
a research indicated that under homeostatic conditions, Th17 cells can be
converted into CD4+ Foxp3- type1 Tregs (Tr1) in the
presence of TGF?
via aryl hydrocarbon
receptor (AhR) activation (Gagliani et
al, 2015).  As Tregs expressing IL-17
were found up-regulated in IBD patients, trans-differentiation can be exploited
as a potential therapeutic target against pathogenesis. 

IL-35
is a novel anti-inflammatory cytokine, member of IL-12 family.  It is a heterodimer containing p35 and
Epstein-Bar virus-induced gene 3 (EBI3) subunits. This cytokine activates IL-35
receptor by inducing the dimerization of gp130 and IL1R?2 domains.  A recent study, illustrated that this
cytokine is essential for Tregs to achieve their maximum regulatory activity,
both in vitro and in vivo. Moreover, IL-35 was found to be
mainly produced by Tregs and B regulatory cells (Bregs) but not from Th17 effector
cells.  Activation of the IL-35R, induces
STAT1 and, STAT4 to form a heterodimer which results in a positive feedback-loop,
promoting continuous IL-35 expression.  Researches
have also observed expression of IL-35 in a population of IL-35-only induced
CD4+ Tregs, referred as iTr35. These cells were indicated of being able to
supress both CD4+ and CD8+ T cells proliferation, as well as their
differentiation into Th17 cells (Collison
et al, 2010).  Ebi3 deficient mice have a significant
increase in the production of IL-17. 
This is achieved through Treg expansion and increased production of
IL-10. (Oslon, Sullivan and Burlingham,
2013).  In models of inflammatory
bowel disease, IL-35 gene therapy and the adoptive transfer of IL-35-expressing
Tregs have been shown to cure colitis symptoms. 
According to the ability of IL-35 of inducing the autologous Breg, IL-35+ Breg
cells, as well as, iTr35 can be utilised to eliminate excessive immune
responses and allow mucosal healing (Choi,
Leung and Bowlus, 2016).

All
of the above indicate that cytokines are a key element of CD pathogenesis. A treatment
based on elevating TGF?
levels in the inflamed mucosa will be inefficient, as smad7 will interrupt its
signalling.  For this reason, an
alternative aspect must be identified to increase targeted-immunosuppression.  T cell redirected for universal-mediated
killing (TRUCKs) can be used to direct IL-35, specifically to the inflamed area
and expand regulatory cells without TGF?
signalling requirement.   Additionally,
introduction of anti-IL-6R monoclonal antibody can prevent Th17 differentiation
and plasticity, permitting further production of anti-inflammatory
cytokines.  

 

 

 

TRUCKs

TRUCK
is a fourth-generation chimeric antigen receptor (CAR)-redirected T cell which causes
the release of any transgenic product such as cytokines to promote accumulation
in a targeted tissue, following T cell activation.  Generally, CARs are consisted of an
ectodomain, a transmembrane domain, and an endodomain as shown in Figure 2.  The ectodomain is a variable portion of both
heavy and light chains of an immunoglobulin known as scFv.  The transmembrane domain, mostly CD28 is connected
to the artificial binding domain in order to provide stability to the
receptor.  Lastly, the endodomain is the
functional end of the receptor which is the CD3? component along with three
immunoreceptor tyrosine-based activation motifs (ITAMs), essential for T cell
activation.  In contrast to CARs, TRUCKs
in this case require the transfer of two trans-genes; CAR and IL-35.  To avoid transactivation of the cytokine
cassette by the CAR promoter, the two genes must be integrated at different
genomic sites.  Additionally, the nuclear
factor of the activated T cell (NFAT)-responsive expression cassette must be utilised
for inducing the expression of the cytokine (Zhang et al, 2017). 

 

 

 

Leukapheresis
can be used to collect leukocytes from the patient by which CD4/CD8 composition
naïve T cells are separated through antibody bead markers.  T cell activation and expansion can be
achieved in vitro by either purifying
patient’s antigen presenting cells (APC) or using beads coated with
anti-CD3/anti-CD28 monoclonal antibodies. 
The combination with IL-2 growth factors, will further induce increase T
cell growth (Smith et al, 2015).  The delivery of foreign genes into naive T
cells can be achieved either using a viral or a non-viral vector system.  The most commonly used vectors are the
genetically engineered retroviruses such as lentivirus.  Lentivirus vector system has the safer
integration site profile, as it has the lower-risk for insertional mutagenesis
caused by the integration of vector DNA into host cells.  Following T cell activation, the cells must
be infected by the recombinant virus vector encoding both the IL-35 and CARs.
As IL-35 is consisted of two subunits, the cytokine will be expressed in the
single chain format composed of covalently linked (p35-EBI3) chains.  Upon infection, the RNA will be
reverse-transcribed into DNA and permanently integrated into the genome of the
patient’s cells.  For TRUCK-T cell
culture, the CliniMACS bioreactor system can be used.   It is
a single device that can effectively enrich, activate, and expand the cells to
reach a clinically-approved threshold, significant process for transfusion into
the patient.  Transfusion can be carried
out through an intravenous infusion to the beforehand (Chmielewski, Hamback and Abken, 2014). 

The
main aim of TRUCKs is the accumulation of any cytokine to promote a second
immune response in a locally restricted manner, as the ideal outcome is to
cause the dampening of excessive inflammation by avoiding any systemic
cytotoxicity.  Several clinical trials
illustrated that direct administration of cytokines, usually result in cytokine
release syndrome accompanied by other adverse side effects. In this case, TRUCKs can be efficient even at low
levels. In comparison with tumour-infiltrating lymphocytes (TIL) IL-12 therapy,
the cell doses used for TRUCK-induced IL-12 were 50- to 100- fold lower.  Another advantage of TRUCKs, is the fact that
re-administration may not be required, since the targeted antigen will be
continuously released, further promoting T cell activation and differentiation (Chmielewski and Abken, 2015).  Integrin a4?7 and CCR9 are key homing receptors for
lymphocyte migration to gut mucosa.  The
induction of Vitamin A metabolite, known as Retinoic Acid (RA) can cause the
expression of these receptors, as a safety measure to prevent any ‘on-target
off-tumour’ TRUCK activation (Calisto et
al, 2011). 

IL-6 Inhibitor

Overexpression
of the pro-inflammatory cytokine IL-6 was found to be highly associated with CD
pathology, as it’s a major mediator involved in Th17 differentiation as well as
plasticity.  The IL-6 receptor (IL-6R) is
consisted by two chains; the IL-6 binding subunit and the membrane glycoprotein
gp130.  Additionally, IL-6 can achieve trans-signalling
through the presence of a naturally occurring soluble IL-6R (sIL-6R), even in
the absence of membrane IL-6R. 
Tocilizumab is a humanised anti-IL6R monoclonal antibody of lgG1 class
which inhibits signal transduction through blocking IL-6 attachment to both
membrane and soluble IL-6R (Tanaka,
Narazaki and Kishimoto, 2011).  Favourably,
the function of sgp130 is not altered by the inhibitory effects of tocilizumab,
as it is an essential subunit for IL-35R signalling (Hashizume and Mihara, 2009). A randomised trial of the drug was
executed which demonstrated ideal clinical responses to approximately 80% of
patients, suggesting its effectiveness.  The
half life of the particular drug is estimated to be approximately 240 hours
after the third dose of 8mg and last longer in humans than in mice.  However, in case of increase dosage, the
clearance rate decreases which can be a limitation during an emergency that the
treatment must be interrupted. 
Additionally, endoscopic and histologic healing was not observed during
clinical trials, indicating the necessity for improvement (Ito et al, 2004).

 

 

 

Animal Model

To
investigate the possible effects of the drug, a suitable animal model is
required which predominantly represent characteristics of CD pathogenesis.  The chemically-induced colitis hapten reagent
2,4,6-trinitrobenzene sulfonic acid (TNBS) mouse model is commonly manipulated
for investigating immunologic aspects of the disease such as cytokine secretion
and potential immunotherapeutic effects. 
However, the pathogenic mechanism towards CD-like disease, remains
unclear.  The Th1 phenotype is the major
response which further promotes secretion of potent pro-inflammatory cytokines
to the colonic tissue, mostly TNF? and
IL-12.  Ideally, IL-17 was shown to be
essential for the development of acute colitis in TNBS-treated mice, as IL-17R-/-
mice treated with TNBS indicated mild inflammation, in contrast to the wild-type.  Thus, Th17 cells also demonstrated an
effector function in TNBS-inducing colitis. 
A research group observed that p19 (IL-23R subunit) deficient mice,
developed exacerbate colitis and suggested that Th17 can carry out both pro-
and anti-inflammatory responses (Antoniou
et al, 2016).  However, IL-23 was
shown to be significant for activation of Group 3 Innate Lymphoid Cell (ILC3) which
further secrete the protective cytokine, IL-22. 
This provides a possible explanation regarding the advantageous role of
Th17 against CD (Longman et al, 2014).
 

Recently,
a study showed that TNBS-induced colitis mouse model with high salt diet (HSD),
established increased Th17 responses, Treg dysfunction, and thus intensify
immune responses (Wei, et al, 2017).  This can be used as a method to
strengthen mouse’s inflammatory responses, generating a more ultimate animal
model for drug administration.  Following
drug application into the TNBS mouse, the TRUCK must recognise a
tissue-specific antigen to prohibit ‘on-target off-antigen’ activation in
healthy tissues.  The regenerating
islet-derived type 4 (REG-4) is a protein constitutively produced by the
enteroendocrine cells in colon epithelial (Granlund,
et al, 2013).  Studies indicated that
REG-4 is highly expressed during colon inflammation and its associated with
poor prognosis in colorectal cancer (Que
et al, 2007).  The particular protein
can be found in the colon of both mice and humans, thus it can be considered a
suitable candidate for TRUCK-recognition. 

Discussion:

The
expected aftermath of this therapeutic concept is the continuous expression of
anti-inflammatory cytokines which may lead to targeted immunosuppression and
gradual mucosal healing.  Continuous
secretion of IL-35 by the activated TRUCK T cells will promote the expansion of
CD4+ CD25+ Treg, iTr35, as well as Bregs and at the same time inhibiting Th17
production.  According to Bregs, IL-35
will induce the production of a unique Breg subtype, expressing IL-35 and
IL-10.  A research group, indicated that
IL-35-producing B cells have a crucial role in controlling immune responses
during infection and autoimmune disease. 
For example, during Uveitis it was suggested that the production of
IL-35 have a positive feedback loop that further expand Treg and Breg
production, associated to the suppression of uveitis.  This illustrates that the TRUCK-induced IL-35
can be used as a treatment for human uveitis as well as other autoimmune
disease such as multiple sclerosis (Wang
et al, 2014).    IL-10 is another important mediator in
preserving intestinal mucosal homeostasis in both mice and humans.  This was observed, as IL-10 or
IL10R-deficient mice developed spontaneous colitis.  IL-10 suppression allows the up-regulation of
the anti-inflammatory cytokines, IFN?,
??F?, and IL-17, thus triggering excessive
inflammation.  Regarding the innate
immune cells, IL-10 can regulate immune responses mediated by the activation of
pattern-recognition receptors (PRRs), as in its absence there is exaggerated
inflammation in innate cells (Shouval et
al, 2014). These observations propose that both IL-10 and IL-35 can execute
immunosuppression without the support of TGF? signalling.  

The
pro-inflammatory cytokine IL-6 has been associated with CD progression mostly
through the stimulation of Th17 cell differentiation.  Except form Th17 production, IL-6 is crucial
for the development of other T cell subtypes, such as Th17, Th22, and TFh (Hunter and Jones, 2015).  Th22 activity is associated with the presence
of IL-17 or TNF and is also essential for the production of IL-22.  This cytokine is an IL-10 member and recently
several studies focused on a possible protective role during IBD through
promoting mucus production, and thus epithelial regeneration.  For this reason, the administration of anti-IL6R
may remarkably decrease IL-22, preventing barrier regeneration (Zhang, Pan and Ye, 2011).  However, ILC3s are tightly associated with
intestinal homeostasis through IL-22 expression in response to the presence of
IL-23 and IL-1?.  Although, Th17 and Th22 cell will be blocked
by anti-IL-6R administration, IL-22 will not be severely affected as ILC3s
function will be intact.  Regulation of
IL-22 production can also be beneficial, as excessive production of this
cytokine was linked with colon cancer development (Jianq et al, 2013).  Furthermore,
Tfh cells produce high levels of IL-21 which impact B cell maturation and thus
antibody production.  During CD,
auto-antibodies such as the IgG Anti-Saccharomyces
cerevisiae antibody (ASCA) was reported in 60%-70% of patients with the
disease (Carbone et al, 2013).  Tocilizumab may lessen auto-antibody
production, as it causes decrease of IL-21 production.  Lastly, IL-6 stimulate the production of the
C-reactive protein (CRP) which is used as serum marker for the identification
of immune responses in IBD.  High levels
of CRP during inflammation was found of inducing a threefold increase of sIL-6R
expression.  Thus, CRP levels can be
utilised for the determination of drug efficacy against inflammation (Jones et al, 1999).  Mucosal healing identification can be
observed mainly through colonoscopy in humans while in mice sacrifice is
required. 

Conclusion:

The
TRUCK T cell prospective can be a promising treatment against autoimmune
diseases through reduction of excessive inflammation in a tissue-targeted
manner.  Combination of TRUCK with other
immunomodulators can further assist in its activity and allow the restoration
of immune homeostasis.  CD is a
complicated disease, as several factors are associated with its development,
making the identification of novel therapies relatively difficult.  Cytokine manipulation can be considered a
target, as are considered key players in CD pathogenesis as well as
progression.  

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