Treg cell-based therapies challenges and persp.pdf

The therapeutic potential of a unique FOXP3+immunosuppressive subset of regulatory T(Treg)cells has been demonstrated in various preclinical models of graftversushost disease(GVHD)13,solid organ transplantation4,5,type 1 diabetes mellitus(T1D)6,7,systemic lupus erythematosus(SLE)8,inflammatory bowel disease9,10 and multiple sclerosis(MS)11.This has stimulated advances in the clinical development of adoptive cell therapy(ACT)of Tregcells in the clinic.There are now more than 50 active and completed clinical trials testing the safety and efficacy of Tregcell therapy for indications such as kidney or liver transplantation,pemphigus vulgaris,SLE,inflammatory bowel disease,autoimmune hepatitis,allergy and asthma12.Although separation and expansion protocols for the Tregcells vary,early clinical trial results report manufacturing success and excellent safety profiles1217.Broadening the therapeutic potential of Tregcell ACT will depend on the use of novel technologies to alter the genome of the cells to enhance functional activity,stability,persistence and antigen specificity.Numerous preclinical studies have demonstrated that antigenspecific Tregcells are more potent than polyclonal Tregcells in models of T1D6,7,autoimmune central nervous system disease18 and transplantation1922.Moreover,antigenspecific Tregcells predominantly localize at the site of antigen presentation,decreasing the risk of generalized immunosuppression.Thus,antigenspecific Tregcells may be both safer and more efficient than unselected polyclonal Tregcells for ACT.Because of their high precursor frequency23,24,antigenselected human Tregcells with direct allospecificity,derived from the transplant recipient,can be efficiently expanded invitro using allogeneic antigenpresenting cells(APCs)from the transplant donor25.This is currently being evaluated as a cellbased therapy in several clinical trials26,27.However,Tregcells with indirect allospecificity as well as selfantigenreactive Tregcells have a precursor frequency predicted to be 1,000fold to 10,000fold lower than the frequency of direct alloreactive Tregcells and have not been effectively expanded to date23,28.Hence,alternative strategies such as genetic engineering are needed to produce Tregcells specific for a particular nominal antigen.In this Review,we focus on the therapeutic potential of Tregcells to treat and reverse immunemediated diseases.We provide an overview of adoptive cell therapy with Tregcells and discuss the specificity,function and activity of this small immunosuppressive cell subset,the accumulating evidence that these cells can protect patients from GVHD,autoimmunity and organ transplant rejection,as well as practical aspects of the implementation of Tregcell therapy in the clinic.Finally,we describe current efforts to enhance Tregcell specificity,survival and function as a nextgeneration therapeutic,using new techniques such as CRISPRbased gene editing and the use of bone marrow,induced pluripotent stem cells and embryonic stem cells to generate Tregcells,in order to advance the use of this essential regu lator of immune homeostasis for various immune and nonimmunerelated diseases.Tregcell backgroundThe concept of immune regulation by specialized lymphocytes has been a doctrine of immunology for more than four decades.However,the absence of clear molecular markers challenged the dogma and for a long while relegated the notion of suppressor cells to the backburner of immunology.Then,a series of studies by Sakaguchi etal.29 revitalized the field by demonstrating that a small subset of cells,identified by the expression of CD4 and CD25,could be used to transfer tolerance in animals that develop autoimmunity as a consequence of neonatal thymectomy.A major breakthrough came when three groups independently identified the Xlinked gene FOXP3 in mouse and human,Direct allospecificityA process by which donor-derived antigen-presenting cells present allogeneic major histocompatibility complexpeptide complexes.Indirect allospecificityA process by which host-derived antigen-presenting cells present self-major histocompatibility complexallogeneic peptide complexes.Tregcell-based therapies:challenges and perspectivesCarolineRaffin1,2,LindaT.Vo1,2 and JeffreyA.Bluestone 1*Abstract|Cellular therapies using regulatory T(Treg)cells are currently undergoing clinical trials for the treatment of autoimmune diseases,transplant rejection and graft-versus-host disease.Inthis Review,we discuss the biology of Tregcells and describe new efforts in Tregcell engineering to enhance specificity,stability,functional activity and delivery.Finally,we envision that the success of Tregcell therapy in autoimmunity and transplantation will encourage the clinical useofadoptive Tregcell therapy for non-immune diseases,such as neurological disorders and tissuerepair.1Sean N.Parker Autoimmune Research Laboratory,Diabetes Center,University of California,San Francisco,San Francisco,CA,USA.2These authors should be considered joint first authors:Caroline Raffin,Linda T.Vo.*e-mail:Jeff.bluestone ucsf.eduhttps:/doi.org/10.1038/s41577-019-0232-6ReviewSNature reviews|Immunologyas the transcription factor that determines the Tregcell functional programme by inducing a specific gene expression programme and epigenetic signature during Tregcell development3032.The critical role for FOXP3 and,as a consequence,for Tregcells in immune tolerance was clearly established with the discovery that earlyonset fatal multiorgan inflammation and autoimmune disorders were observed in patients and mice with a disrupted FOXP3 gene.In humans,males with deleterious mutations develop immunodysregulation polyendocrinopathy enteropathy Xlinked(IPEX)syndrome and mice with mutant FOXP3(scurfy mice)develop lethal multiorgan autoimmunity.Both syndromes are a direct consequence of a systemic defect in Tregcells3335.In fact,the first demonstrated utility for adoptive transfer of Tregcells as a therapy was performed in FOXP3 mutant mice and provides a key rationale for the use of these cells both as a therapy for IPEX patients and in humans with various immune diseases.Tregcells,which comprise 57%of CD4+Tcells,develop both directly in the thymus(tTreg cells)and in the periphery(pTreg cells).pTreg cells develop,especially in the gut,from CD4+conventional Tcells under conditions of high levels of transforming growth factor(TGF)and retinoic acid in the environment or in response to metabolites produced by microbiota36,37.The Tcell receptor(TCR)repertoire of the pTreg cell and conventional Tcell populations is largely nonoverlapping3840.Indeed,tTreg cells mainly recognize selfantigens,whereas the pTreg cell TCR repertoire also includes TCRs specific for nonself infectious antigens or innocuous commensal microbiotaderived antigens,which are important for the maintenance of mucosal tolerance41.Unlike in the mouse,where neuropilin 1(NRP1)has been defined as a marker of pTreg cells42,43,there is currently no marker able to distinguish tTreg cells from pTreg cells in humans.Thus,Tregcells isolated from peripheral blood are likely a combination of tTreg cells and pTreg cells.Importantly,Tregcells can be found locally in tissues as well as systemically during an inflammatory response.They migrate via afferent lymphatics from the inflamed tissue to the draining lymph node,where they can also function to control antigen presentation.Thus,Tregcells exert their suppressive function both at the tissue site of inflammation and in local secondary lymphoid tissues4447.Tregcells confer immune tolerance via multiple mechanisms48.Through the expression of antiinflammatory soluble mediators,such as IL10,TGF and IL35,the consumption of IL2,and the expression of negative regulatory cell surface receptors such as cytotoxic T lymphocyte antigen 4(CTLA4),CD39 and CD73(ref.49),they can target T cells directly or indirectly by modulating APCs.For example,CTLA4 binding to CD80/CD86 on APCs can lead to the induction of indolamine 2,3dioxygenase(IDO)50,51.In addition,Treg binding to APCs can result in stripping of the cell surface molecules (trogocytosis)altering costimulation and antigen presentation52,53.In fact,some data have sugges ted that Tregcells,expressing perforin and granzyme B,may directly kill APCs expressing the target antigen53.Importantly,many of these suppressive activities of Tregcells can function in an antigen nonspecific manner,called dominant bystander suppression,allowing them to suppress effector Tcells of diverse specificities54.Tregcells can also modulate the tissue micro environment through the production of some of these same immunosuppressive molecules,promoting the emergence of other immunosuppressive cell populations such as Tregcells with different specificities and T regulatory 1(Tr1)cells5558.This phenomenon,called infectious tolerance,supports the idea that,in a therapeutic setting,adoptively transferred Tregcells may not need to persist indefinitely but for long enough to confer suppressive capacity to other immune cells located in the affected tissue59.It is important to highlight that the mechanisms of action of Tregcells and effector Tcells differ,with the latter functioning in a largely cell contactdependent manner directed against antigenbearing cells(fig.1).These differences need to be taken into consideration when designing Tregcellbased therapies.Finally,a detailed biological and biochemical understanding of these immunoregulatory Tcells,combined with recent advances in Tregcell subset identification(Box1),the ability to engineer antigen specificity through TCR and chimeric antigen receptor(CAR)transduction,genome editing to generate more potent and more stable Tregcell populations,and strategies to produce Tregcells denovo from stem cell populations,provides an unparalleled opportunity for ACT.Ultimately,these advances will enable the development of allogeneic Tregcell products for more general use in various therapeutic settings.Engineering antigen-specific TregcellsThere are currently several approaches to generate and/or expand antigenspecific Tregcells invitro(fig.2).As mentioned,endogenous antigenspecific Tregcells are more potent than polyclonal Tregcells but their expansion is challenging owing to low precursor frequencies.Another approach to generate antigenspecific Tregcells is to redirect polyclonal Tregcells by introducing synthetic receptors.These receptors can take the form of CARs,enabling direct antigen recognition,or engineered TCRs,which target antigens in the context of an antigenMHCpeptide complex.A third approach is to convert antigenspecific effector Tcells into Tregcells through overexpression of FOXP3.Tregcells with engineered TCRs.Animal studies have shown that engineered tissue antigenspecific Treg cells are significantly more efficient than polyclonal Tregcells in preclinical models of T1D6,7,colitis60,rheumatoid arthritis(RA)61,62,MS18 and transplantation63.Importantly,TCRtransduced Tregcells accumulate in tissue targeted during autoimmunity in an antigendriven manner,where they exert both antigenspecific repression and nonspecific bystander suppression following activation18,61,62.Given that,in many cases,Tregcell populations with a single antigenspecific TCR can suppress polyspecific pathogenic Tcell populations,autoantigenreactive engineered Tregcells may be as effective as polyclonal Treg cells to shut down pathogenic T cells independent of their specificity.However,in some settings,oligoclonal or pauciclonal Tregcells may be more efficacious.In this regard,it was shown that expanded Tregcells with TrogocytosisA process whereby lymphocytes physically extract surface molecules from antigen-presenting cells.Infectious toleranceA phenomenon in which a tolerance-inducing state is transferred from one cell population to another.PauciclonalThe presentation of limited clonal allogeneic specificity were more efficient in promoting heart graft tolerance when also transduced with a TCR with specificity for donor antigens presented by host APCs(indirect alloantigen specificity)63.Thus,antigenspecific expansion and genetic engineering with recombinant TCRs may be combined to gene rate dualspecific Tregcells that have higher efficiency in promoting graft tolerance.These results encouraged the development of TCR transgenic human Tregcells specific for antigens that are abundantly present in affected tissues.For example,human Tregcells with a transgenic TCR specific for the antihaemophilic factor VIII(FVIII)efficiently suppressed proliferation and cytokine production of FVIIIspecific effector Tcells and reduced FVIII antibody production in splenocytes of FVIIIimmunized HLADR1 transgenic haemophilic mice invitro64.Moreover,Tregcells with a transgenic TCR specific for myelin basic protein(MBP)were able to suppress both MBPspecific Tcells and Tcells with other specificities in a preclinical mouse model of MS65.The ability of human Tregcells transduced with TCRs to achieve bystander suppression was also observed in other preclinical studies using TCRs with diverse antigen specificities6668.Interestingly,the specificity of human Tregcells was also efficiently redirected by class Irestricted transgenic TCRs,which may optimize the effectiveness of the Tregcells in the autoimmune tissue66,69.It should be noted that the majority of current efforts have relied on antigenspecific TCRs isolated from effector Tcells and transduced into Tregcells.It is possible that the intrinsic affinity and specificity of TCRs isolated from Tregcells can be distinct from effector Tcells,affecting migration into specific niches and functional activity.Thus,an approach that utilizes TCRs isolated from Tregcells to redirect antigen specificities in Tregcells may more closely replicate the intrinsic functionality of antigenspecific Tregcells.Chimeric antigen receptors.CARs are artificial receptors comprising an antigen binding site of a monoclonal antibody(mAb)in their extracellular domain and Tcell stimulatory and costimulatory intracellular domains(fig.3).CARengineered effector Tcells(CAR Tcells)CD8CD4+T cellCD4+T cellCD4+T cellCD8+T cellTCRAPCTreg cellTreg cellCD4a Effector T cellsb Treg cellsCTLPerforins andgranzymesTarget tissuedestructionInfectioustoleranceMHCclass IIMHCclass IIL-2APCBystandersuppressionEffector T cellsof distinct antigenspecificityIFN IL-2 consumption IL-10,TGF Inhibitory receptors Granzymes Metabolic control(IDO,adenosine)Fig.1|Mechanisms of action of effector Tcells versus Tregcells.a|The recognition of specific antigens presented or cross-presented by antigen-presenting cells(APCs)activates CD4+and CD8+Tcells,respectively.Following activation,CD4+Tcells produce cytokines that help CD8+Tcells to differentiate into cytotoxic T lymphocytes(CTLs).CTLs then kill target cells by performing their cytotoxic activity in an antigen-specific and cell contact-dependent manner.b|Like effector Tcells,regulatory T(Treg)cell activation is antigen sp