Crohn’s the thymus already expressing FOXP3, while induced Treg

Crohn’sdisease (CD) is an Inflammatory Bowel Disease (IBD) distinguished bydiscontinuous excessive inflammation along the gastrointestinal tract (GI). Advancedor untreated CD, commonly leads to scar formation or ulcers, further resultingin intestinal stricture and bowel obstruction. In such cases, surgery is required to remove the affected region ofeither small or large intestine.  Currently,the most prevalent treatments against CD include anti-inflammatory drugs(corticosteroids) and immunomodulators (anti-TNF), however efficient outcomesappear to a proportion of patients. Additionally, CD approximately affects 2.

5 million individuals of anyage in the Western world and has an accelerating increase in incidence in thedeveloping world.  The above evidence illustratesthe necessity of discovering novel therapies in order to bear a spectrum ofalternative treatments that can be implemented according to the individual.  Theaetiology as well as pathogenesis of CD, and more generally of IBD is ambiguousand complicated.

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  The most acceptedhypothesis suggests that the synergy between genetic and environmental factorstrigger disease onset by stimulating continuous expression of pro-inflammatorycytokines.  Under normal conditions, theintestinal mucosal barrier is essential for preventing invasion of commensal bacteriain the epithelial cells.  This isachieved through maintaining immune self-tolerance. However, the aforementionedrisk factors of CD cause imbalances of the gut microenvironment, leading toincrease of intestinal permeability which results in dysfunction of mucosalbarrier and subsequent breakdown of tolerance (Wang et al, 2016).

  Therefore,the breakdown of tolerance elicits excessive immune responses.  According to this, GWAS-studies indicatedthat genetically susceptible individuals may carry specific geneticpolymorphisms related to bacteria recognition, further promoting toleranceimpairment. CD is considered a cell-mediated disease, as Th1 and Th17 responsesare predominantly associated with its pathogenesis, illustrated in Figure 1.  Thebalance between Treg/Th17 cells is significant for mucosal homeostasis, throughsustaining the equilibrium among pro- and anti-inflammatory cytokines.  Dependent on the microenvironment, the pro-inflammatoryresponses of Th17 cells are restricted by the activity of Tregs, and vice versa(Ziegler and Buckner, 2009).

  This is achieved in the presence of TGF?, a common mediator significant for Th17and Treg differentiation.  Th17 cells arethought to be originated from a subset of naïve T cell expressing the lectinreceptor, CD161 as well as the transcription factor ROR?t (Cosmiet al, 2008).  Their differentiation dependson the presence of the pro-inflammatory cytokines, IL-1?, IL-6, IL-21 and TGF?. Following activation, Th17 cells expresshigh levels pro-inflammatory of IL-17, IL-21, and IL-22 which have an essentialrole in host defences against extracellular pathogens by triggering furtherrecruitment of innate immune cells (Galvez,2014).  In contrast, regulatory Tcells (Tregs) are divided into two categories; natural and induced.  Natural Tregs (nTreg) arise from the thymusalready expressing FOXP3, while induced Treg (iTreg) develop from mature CD4+effector T cells in the periphery.  Tregdifferentiation in the periphery is achieved upon exposure to highconcentrations of TGF?.   FOXP3+ Treg’s main function involves regulationof excessive immune responses as well as maintenance of self-tolerance toprevent autoimmune disease development.

 This is achieved through TGF?and IL-10 anti-inflammatory cytokine secretion at the site of inflammation (Yadav, Stephan and Bluestone, 2013). DuringCD, mucosal barrier dysfunction alters the Th17/Treg commitment towards Th17-inducedinflammatory response, and thus significantly eliminating iTreg within theintestinal lamina propria.  As mentionedbefore, even if these both subtypes exhibit contradictory functions, TGF? is requiredfor their differentiation.

  At lowconcentrations, TGF? synergizes with IL-6 and IL-6-dependent IL-21 to promoteIL-23 receptor expression, and thus Th17 differentiation.  On the other hand, at high concentrations ofTGF? and absence of IL-6 or IL-21, IL-23R is supressed resulting in inhibitionof ROR?t and induction of iTregs (Omenettiand Pizzaro, 2015).  This suggests,that Th17 cells can inhibit Tregs by the expression of IL-21, while Tregs throughIL-10 production (Raza et al, 2012).  However, in CD even if TGF? levels are elevated, the downstream signallingpathway is destructed by the blockage of smad3 phosphorylation.  This is achieved by the up-regulation of a naturalintracellular inhibitor of smad signalling, known as smad7 which acts as a negativefeedback.  Interestingly, both iTreg andTh17 cells illustrated of being instable with a degree of trans-differentiationinto other CD4+ subtypes.  To illustrate this,a fraction of FOXP3+ Tregs showed high-degree of plasticity towards IL-17-producingcells as well as loss of FOXP3 expression.

 This is caused due to the accumulation of Th17-generating cytokines, asplasticity may be an adaptive mechanism (Uenoet al, 2015).  Oppositely, a researchindicated that under homeostatic conditions, Th17 cells can be converted into CD4+Foxp3- type1 Tregs (Tr1) in the presence of TGF? and aryl hydrocarbon receptor (AhR) (Gagliani et al, 2015).  As Tregs expressing IL-17 were foundup-regulated in IBD patients, trans-differentiation can be utilised as apotential therapeutic target. IL-35is a novel anti-inflammatory cytokine, member of IL-12 family.

  It is a heterodimer containing p35 andEpstein-Bar virus-induced gene 3 (EBI3) subunits while the IL-35 receptorrequires the dimerization of gp130 and IL1R?2 domains.  A recent study, illustrated that thiscytokine is essential for Tregs to achieve their maximum regulatory activity,both in vitro and in vivo. IL-35 was found to be mainlyproduced by Tregs and B regulatory cells (Bregs) but not from effector cells.  Once IL-35 engagement to the receptor, STAT1and STAT4 form a unique heterodimer which results in a continuousfeedback-loop, promoting IL-35 expression. Research has also observed expression of IL-35 in a population of IL-35-onlyinduced CD4+ Tregs, referred as iTr35 cells (Collison et al, 2010).  IL-35-induced cells were indicated of beingable to supress both CD4+ and CD8+ T cells proliferation, as well as theirdifferentiation into Th17 cells.  Ebi3deficient mice have a significant increase in the production of IL-17.  This is achieved through Treg expansion andincreased production of IL-10.

(Oslon,Sullivan and Burlingham, 2013).  Inmodels of inflammatory bowel disease, IL-35 gene therapy and the adoptivetransfer of IL-35-expressing Tregs have been shown to cure colitissymptoms.  According to the ability ofIL-35 of inducing the autologous Breg, IL-35+ Breg cells, aswell as, iTr35 can be utilised to eliminate excessive immune responses andallow mucosal healing (Choi, Leung andBowlus, 2016). Allthe above indicate that cytokines are a key element underlying CD pathogenesis.A treatment based on elevating TGF?levels in the inflamed mucosa will be inefficient, as smad7 will interrupt itssignalling.  For this reason, analternative aspect must be identified to increase targeted-immunosuppression.  T cell redirected for universal-mediatedkilling (TRUCKs) can be used to direct IL-35, specifically to the inflamed areaand expand regulatory cells without TGF?signalling requirement.

   Additionally,introduction of anti-IL-6R monoclonal antibody can prevent Th17 differentiationand plasticity, permitting further production of anti-inflammatorycytokines.     TRUCKs TRUCKis a fourth-generation chimeric antigen receptor (CAR)-redirected T cell which causesthe release of any transgenic product such as cytokines to promote accumulationin a targeted tissue, following T cell activation.  Generally, CARs are consisted of anectodomain, a transmembrane domain, and an endodomain as shown in Figure 2.  The ectodomain is a variable portion of bothheavy and light chains of an immunoglobulin known as scFv.  The transmembrane domain, mostly CD28 is connectedto the artificial binding domain in order to provide stability to thereceptor.

  Lastly, the endodomain is thefunctional end of the receptor which is the CD3? component along with threeimmunoreceptor tyrosine-based activation motifs (ITAMs), essential for T cellactivation.  In contrast to CARs, TRUCKsin this case require the transfer of two trans-genes; CAR and IL-35.  To avoid transactivation of the cytokinecassette by the CAR promoter, the two genes must be integrated at differentgenomic sites.  Additionally, the nuclearfactor of the activated T cell (NFAT)-responsive expression cassette must be utilisedfor inducing the expression of the cytokine.  Leukapheresiscan be used to collect leukocytes from the patient by which CD4/CD8 compositionnaïve T cells are separated through antibody bead markers.  T cell activation and expansion can beachieved in vitro by either purifyingpatient’s antigen presenting cells (APC) or using beads coated withanti-CD3/anti-CD28 monoclonal antibodies. The combination with IL-2 growth factors, will further induce increase Tcell growth.

  The delivery of foreigngenes into naive T cells can be achieved either using a viral or a non-viralvector system.  The most commonly usedvectors are the genetically engineered retroviruses such as lentivirus.  Lentivirus vector system has the saferintegration site profile, as it has the lower-risk for insertional mutagenesiscaused by the integration of vector DNA into host cells.  Following T cell activation, the cells mustbe 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 thesingle chain format composed of covalently linked (p35-EBI3) chains.

  Upon infection, the RNA will bereverse-transcribed into DNA and permanently integrated into the genome of thepatient’s cells.  For TRUCK-T cellculture, the CliniMACS bioreactor system can be used.   It isa single device that can effectively enrich, activate, and expand the cells toreach a clinically-approved threshold, significant process for transfusion intothe patient.  Transfusion can be carriedout through an intravenous infusion to the beforehand.  Themain aim of TRUCKs is the accumulation of any cytokine to promote a secondimmune response in a locally restricted manner, as the ideal outcome is tocause the dampening of excessive inflammation by avoiding any systemiccytotoxicity.  Several clinical trialsillustrated that direct administration of cytokines, usually result in cytokinerelease syndrome accompanied by other adverse side effects. In this case, TRUCKs can be efficient even at lowlevels. 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 thatre-administration may not be required, since the targeted antigen will be continuouslyreleased, further promoting T cell activation and differentiation (Chmielewski and Abken, 2015).  Integrin a4?7 and CCR9 are key homing receptors forlymphocyte migration to gut mucosa.  Theinduction of Vitamin A metabolite, known as Retinoic Acid (RA) can cause theexpression of these receptors, as a safety measure to prevent any ‘on-targetoff-tumour’ TRUCK activation (Calisto etal, 2011).      IL-6 Inhibitor Overexpressionof the pro-inflammatory cytokine IL-6 was found to be highly associated with CDpathology, as it’s a major mediator involved in Th17 differentiation as well asplasticity.  The IL-6 receptor (IL-6R) isconsisted by two chains; the IL-6 binding subunit and the membrane glycoproteingp130.  Additionally, IL-6 can achieve trans-signallingthrough the presence of a naturally occurring soluble IL-6R (sIL-6R), even inthe absence of membrane IL-6R.

 Tocilizumab is a humanised anti-IL6R monoclonal antibody of lgG1 classwhich inhibits signal transduction through blocking IL-6 attachment to bothmembrane 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 wasexecuted which demonstrated ideal clinical responses to approximately 80% ofpatients, suggesting its effectiveness.   However, endoscopic and histologic healingwas not observed, indicating the necessity for improvement (Ito et al, 2004). Animal ModelToinvestigate the possible effects of the drug, a suitable animal model isrequired which predominantly represent characteristics of CD pathogenesis.  The chemically-induced colitis hapten reagent2,4,6-trinitrobenzene sulfonic acid (TNBS) mouse model is commonly manipulatedfor investigating immunologic aspects of the disease such as cytokine secretionand potential immunotherapeutic effects.

 However, the pathogenic mechanism towards CD-like disease, remainsunclear.  The Th1 phenotype is the majorresponse which further promotes secretion of potent pro-inflammatory cytokinesto the colonic tissue, mostly TNF? andIL-12.  Ideally, IL-17 was shown to beessential 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 aneffector 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.  However,IL-23 was shown to be significant for activation of Group 3 Innate LymphoidCell (ILC3) which further secrete the protective cytokine, IL-22.  This provides a possible explanationregarding the   Recently,a study showed that TNBS-induced colitis mouse model with high salt diet (HSD),established increased Th17 responses, Treg dysfunction, and thus intensifyimmune responses (Wei, et al, 2017).  This can be used as a method tostrengthen mouse’s inflammatory responses, generating a more ultimate animalmodel for drug administration.

  Followingdrug application into the TNBS mouse, the TRUCK must recognise atissue-specific antigen to prohibit ‘on-target off-antigen’ activation inhealthy tissues.  The regeneratingislet-derived type 4 (REG-4) is a protein constitutively produced by theenteroendocrine cells in colon epithelial (Granlund,et al, 2013).  Studies indicated thatREG-4 is highly expressed during colon inflammation and its associated withpoor prognosis in colorectal cancer (Queet al, 2007).

  The particular proteincan be found in the colon of both mice and humans, thus it can be considered asuitable candidate for TRUCK-recognition.