REVIEW published: 22 February 2019 doi: 10.3389/fimmu.2019.00181 Ways Forward for Tolerance-Inducing Cellular Therapies- an AFACTT Perspective Anja ten Brinke 1,2*, Marc Martinez-Llordella 3 , Nathalie Cools 4 , Catharien M. U. Hilkens 5 , S. Marieke van Ham 1,2,6 , Birgit Sawitzki 7 , Edward K. Geissler 8 , Giovanna Lombardi 9 , Piotr Trzonkowski 10 and Eva Martinez-Caceres 11,12 1 Department of Immunopathology, Sanquin Research, Amsterdam, Netherlands, 2 Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands, 3 Department of Inflammation Biology, MRC Centre for Transplantation, School of Immunology and Microbial Sciences, Institute of Liver Studies, King’s College London, London, United Kingdom, 4 Laboratory of Experimental Hematology, Faculty of Medicine and Health Sciences, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp, Belgium, 5 Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom, 6 Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands, 7 Charité-Universitaetsmedizin Berlin, Berlin Institute of Health, Institute for Medical Immunology, Humboldt-Universitaet zu Berlin, Berlin, Germany, 8 Section of Experimental Surgery, Department of Surgery, University Hospital Regensburg, University of Regensburg, Regensburg, Germany, 9 Division of Transplantation Immunology and Mucosal Biology, MRC Centre for Transplantation, Guy’s Hospital, King’s College London, London, United Kingdom, Edited by: 10 Department of Clinical Immunology and Transplantology, Medical University of Gdansk, Gdansk, Poland, 11 Division of Duncan Howie, Immunology, Germans Trias i Pujol University Hospital, LCMN, IGTP, Badalona, Spain, 12 Department of Cellular Biology, University of Oxford, United Kingdom Physiology and Immunology, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain Reviewed by: Sylvaine You, Institut National de la Santé et de la Clinical studies with cellular therapies using tolerance-inducing cells, such as tolerogenic Recherche Médicale (INSERM), antigen-presenting cells (tolAPC) and regulatory T cells (Treg) for the prevention of France transplant rejection and the treatment of autoimmune diseases have been expanding Ciriana Orabona, University of Perugia, Italy the last decade. In this perspective, we will summarize the current perspectives of the *Correspondence: clinical application of both tolAPC and Treg, and will address future directions and the Anja ten Brinke importance of immunomonitoring in clinical studies that will result in progress in the field. a.tenbrinke@sanquin.nl Keywords: tolerance, dendritic cells, regulatory T cells, autoimmunity, transplantation, cell therapy, clinic, Specialty section: immunomonitoring This article was submitted to Immunological Tolerance and Regulation, INTRODUCTION a section of the journal Frontiers in Immunology The number of patients with autoimmune diseases, severe allergies and recipients of organ or Received: 24 October 2018 stem cell transplants is increasing worldwide. Currently, many of these patients require lifelong Accepted: 21 January 2019 administration of immunomodulatory drugs, which cause generalized immunosuppression and Published: 22 February 2019 hereby only partially alleviate the symptoms but do not cure the disease. Besides these drugs Citation: are inevitably associated with a risk of immediate or late-occurring severe adverse effects ten Brinke A, Martinez-Llordella M, (e.g., life-threatening infections, cancer). Targeting the fundamental cause of autoimmunity, Cools N, Hilkens CMU, van Ham SM, i.e., loss of tolerance to self-antigens, or inhibiting induction or execution of undesired Sawitzki B, Geissler EK, Lombardi G, immunity in transplantation and allergy will provide the next steps forward to avoid general Trzonkowski P and immunosuppression. Accumulating knowledge on mechanisms of immune activation and Martinez-Caceres E (2019) Ways Forward for Tolerance-Inducing tolerance has led to the development of tolerance-inducing cellular therapies with regulatory T Cellular Therapies- an AFACTT cells (Treg) and tolerogenic antigen presenting cells (tolAPC), such as tolerogenic dendritic cells Perspective. Front. Immunol. 10:181. (tolDC) and regulatory macrophages (Mreg) [as reviewed in (1, 2)], with the specific objective to doi: 10.3389/fimmu.2019.00181 restrain unwanted immune reactions long-term. Frontiers in Immunology | www.frontiersin.org 1 February 2019 | Volume 10 | Article 181 ten Brinke et al. Ways Forward for Tolerance-Inducing Cellular Therapies The development of cell-based therapies is clinically attractive This invalidates their putative use as therapeutic products for for many reasons, not in the least through their potential of tolerance induction. Therefore, different strategies to generate being of low-toxicity, to simultaneously control many different stable tolAPC have been explored, including treatment with inflammatory cells and induction of antigen-specific immunity. pharmacological agents or cocktails of immunomodulatory Since immunological tolerance is a self-reinforcing state (3), cytokines, genetic engineering, and exposure to apoptotic cells the therapeutic effects of cell therapy are expected to outlast (9, 27, 28). Most of these in vitro conditioning regimens aim the lifespan of the therapeutic cells themselves, opening the at stabilizing a semi-mature state of tolDC, maintaining the possibility of curative treatments. Production costs for these capacity to induce immune hyporesponsiveness of T cells, even tolerogenic cell products range from 10,000 to 40,000 Euro in presence of powerful pro-inflammatory signals. depending on the therapeutic cell product and production site, Importantly, tolAPC inhibit T cell proliferation, albeit which is relatively low considering that few injections of cells may through different immunosuppressive mechanisms depending be sufficient to induce long-lasting tolerance. on the approach used to generate tolAPC in vitro. Induction In recognition of the potential of tolerance inducing cell- of peripheral T cell anergy and apoptosis (29), attenuation based therapies and to join forces in the ongoing efforts in the of effector and memory T cell responses and the generation field, A FACTT (Action to Focus and Accelerate Cell Based and activation of Treg populations (30, 31) result in part Tolerance-inducing Therapies (CTT) was initiated through EU from presentation of low levels of antigen in the absence COST Action Funding. Our goal was to initiate a network that of costimulation; these are typical mechanisms attributed to would coordinate European CTT efforts to minimize overlap and a variety of tolerogenic subtypes (10, 32, 33). Additionally, maximize comparison of the diverse approaches across Europe. tolerogenic DC may express various inhibitory receptors Now, looking back at 4 years of very active network interactions, such as programmed death-ligand (PD-L)1, PD-L2 (34), we have evaluated our combined current stance in the field and immunoglobulin-like transcripts (ILT) (35), FasL (36, 37), and defined avenues to support future directions. TRAIL (38). Secretion of anti-inflammatory cytokines such as IL-10 (39, 40) and TGF-β (41, 42), as well as reduced expression of pro-inflammatory cytokines, also may contribute TOLEROGENIC THERAPY WITH ANTIGEN to tolerance induction. A study comparing tolDC generated PRESENTING CELLS IN CLINICAL in presence of dexamethasone and rapamycin demonstrated PRACTICE that while both tolDC subsets were able to impair T cell proliferation, rapamycin-treated tolDC have a mature phenotype Over the past 20 years extensive experimental research has and are not able to produce IL-10 upon stimulation with LPS, been invested in the generation and characterization of tolAPC, as opposed to vitamin D3 - or dexamethasone-treated tolDC including tolDC and Mreg, with the aim to restore tolerance in (27). Whereas, it was demonstrated that rapamycin-treated autoimmune diseases (4–10) and transplant rejection (5, 11–14). tolDC induce Treg (27), vitD3 -treated tolDC induce T-cell To date, clinical trials exploring the safety, feasibility and efficacy hyporesponsiveness and antigen-specific Treg (7, 43). Moreover, of different types of tolAPC are a reality [reviewed in (1) and DC-10 induce Tr1 cells (44), while autologous tolDC have a Table 1], and have confirmed so far that tolAPC therapy is safe, weak capacity to stimulate allogeneic T cells and suppress T- with no relevant side effects, and is well-tolerated by patients. cell proliferation and IFN-γ production (45). Mreg have been Hence, to advance tolerogenic therapy with antigen-presenting shown to convert allogeneic CD4+ T cells to IL-10-producing, cells (APC), we should stand on the shoulders of these pioneers TIGIT+ , FoxP3+ -induced Treg (46). Variations in the process and address remaining challenges, such as the optimal dose, to generate tolAPC may initiate regulation through distinctive injection route, frequency of administration, antigen-specificity, mechanisms, making it difficult to compare these different types and the related issue of suitable biomarkers of cell therapy- of tolAPC. Therefore, efforts have been made to find common induced reduction of general inflammatory state and induction features unique for tolerance-inducing cells (47). For example, of tolerance, in the design of the next-generation clinical trials. since tolDC conditioned using vitamin D3 and dexamethasone exhibit high cell surface expression of TLR2 (48) or CD52 (49), Phenotypic and Functional Identification of such markers might be considered to assess the quality and in vitro Generated TolAPC stability of tolDC in future cell-based clinical trial protocols. Both tolDC and Mreg can be generated in vitro starting In addition, it was demonstrated that the expression of single from CD14+ monocytes and share some phenotypic and immunoglobulin IL-1-related receptor has a role in maintaining functional characteristics. Indeed, both tolAPC types express low low levels of costimulatory molecules and in regulation of Treg to intermediate levels of T-cell costimulatory molecules, and expansion (50). Others demonstrated that C-lectin receptor secrete low amounts of pro-inflammatory cytokines, indicative CLEC-2 upregulation by DC is associated with Treg induction of a partially matured APC. Similarly, immature DC (iDC) (51). So far, however, gene expression studies comparing different display minimal expression of costimulatory molecules and little tolDC and Mreg protocols have not been able to identify secretion of inflammatory cytokines, demonstrating potential common biomarkers of tolerance induction [reviewed by (52, 53) optimal requirements for tolerance induction in vivo (23, and (54)]. 24). However, iDC are unstable and may differentiate into The difficulty in comparing characteristics of different clinical immunogenic DC under inflammatory conditions (25, 26). tolAPC suggests the need for a uniform set of metrics for Frontiers in Immunology | www.frontiersin.org 2 February 2019 | Volume 10 | Article 181 TABLE 1 | Completed or ongoing trials with tolAPC in autoimmunity and transplantation. Study ID Phase Cell product Indication Dosing scheme tolDC per dose Route of Outcome Center References administration ten Brinke et al. NCT00445913 I Antisense ODN targeting T1D 4 injections, 1 × 107 Intradermal -No adverse effects Pittsburgh/USA (15) CD40/CD80/CD86 tolDC bi-weekly (AE) -Increase of B220+CD11c+ B cells -Evidence for C-peptide reactivation NCT02354911 II Antisense ODN targeting Recent onset T1D 4 injections, 1 × 107 Intradermal Not Recruiting DiaVacs Inc. Frontiers in Immunology | www.frontiersin.org CD40/CD80/CD86 tolDC bi-weekly Pittsburgh/USA NTR5542 I VitD3 and Dex tolDC pulsed T1D 2 injections, 4 weeks Dose-escalation: 5, 10, or Intradermal Finished Leiden/NL with proinsulin peptide apart 20 × 106 Rheumavax I NF-kB inhibitor Bay HLA-risk positive RA Single injection Low dose 0.5-1 × 106 Intradermal -Grade I AE. Brisbane/AUS (16) 11-7082 tolDC pulsed with (minimal disease High dose 2-4.5 × 106 -Decrease DAS28 4 citrullinated peptides activity) -Decrease eff T cells, ratio Teff/Treg -Decrease pro-inflammatory cytokines and chemokines NCT01352858 I Dex and VitD3 tolDC pulsed Inflammatory Arthritis Single injection Dose-escalation: Intraarticular -Safe, feasible, Newcastle upon (17) 3 (AutoDECRA) with autologous synovial 1 × 106 acceptable Tyne/UK fluid 3 × 106 -Knee symptomse 10 × 106 stabilized in 2 patients receiving the highest doses I tolDC pulsed with RA 5 injections Low dose 0.5 × 107 Not indicated -Treatment well Seoul/KOR (18) CreaVax-RA recombinant PAD4, RA33, High dose 1.5 × 107 tolerated CRiS KCT0000035 citrullinated, fillagrin and -Antigen –specific vimentin autoantibodies decreased in 5/9 positive patients NCT03337165 I Dex and IFN-α tolDC RA Single injection Dose-escalation: 1, 3, 5, 8, Intraarticular Recruiting Novosibirk/RUS (TolDCfoRA) 10 × 106 NCT02283671 I Dex tolDC pulsed with MS and 3 injections, Not indicated Intravenous Recruiting Barcelona/ES relevant disease peptides Neuro-myelitis optica bi-weekly NCT02618902 I/IIa VitD3 tolDC pulsed with Active MS patients 6 injections, 4 Dose-escalation: Intradermal Recruiting Antwerp/BE myelin-derived peptides bi-weekly and 2 5 × 106 monthly 10 × 106 15 × 106 NCT02903537 I/IIa VitD3 tolDC pulsed with Active MS patients 6 injections, 4 Dose-escalation: Intranodal Recruiting Badalona, (Tolervit-MS) myelin-derived peptides bi-weekly and 2 5 × 106 Pamplona/ES monthly 10 × 106 15 × 106 (Continued) Ways Forward for Tolerance-Inducing Cellular Therapies February 2019 | Volume 10 | Article 181 TABLE 1 | Continued Study ID Phase Cell product Indication Dosing scheme tolDC per dose Route of Outcome Center References administration ten Brinke et al. I Dex and VitA tolDC Refractory Crohn’s Single injection or 3 Dose-escalation: Intra-peritoneal -No AE (3 patients Barcelona/ES (19) disease injections bi-weekly 2 × 106 withdrew due to 5 × 106 worsening of 10 × 106 symptoms) -Clinical improvement in 3 patients -Increase of Treg and decrease of IFN-g Frontiers in Immunology | www.frontiersin.org levels NCT02622763 I Dex tolDC Crohn’s disease Not listed 10 × 106 Intralesional Recruiting Barcelona/ES 100 × 106 NCT02252055 I/II Low-dose Kidney Tx from living Single injection, 1 day 1 × 106 /kg bw Intravenous Nantes/FR (20) (ONEatDC) GM-CSF-recipient tolDC donor before Tx NCT03726307 I VitD3 and IL-10 donor Kidney Tx from living Single injection, 7 Dose - escalation: Intravenous Not yet recruiting Pittsburgh/USA tolDC (Dcreg) donor days before Tx 0,5 ± 0,1 × 106 /kg bw, 1,2 ± 0,2 × 106 /kg bw, 2,5 to 5 × 106 /kg bw NCT03164265 I VitD3 and IL-10 donor Liver Tx Single injection, 7 Not described Intravenous Enrolling by invitation Pittsburgh/USA tolDC (Dcreg) days before Tx TAIC-I I/II Cell product containing Kidney Tx from Single injection, 5 0.5–7.5 × 106 /kg bw Intravenous Safe Regensburg/DE (21) 4 donor Mreg deceased donor days after Tx TAIC-II I Cell product containing Kidney Tx from living Single injection, 5 1.7–10.4 × 107 /kg bw Intravenous -Safe Regensburg/DE (22) donor Mreg donor days before Tx -No acute or delayed AE were associated with the infusion. −3 of the 5 patients were able to tolerate low-dose tacrolimus monothera- py and one patient was withdrawn from all immunosuppression for over 8 months NCT02085629 I/II Donor derived Mreg Kidney Tx from living Single injection, 7 2.5–7.5 × 106 /kg bw Intravenous Completed, no results Regensburg/DE (20) donor days before Tx yet This table is based on information deposited on www.clinicaltrials.gov, www.clinicaltrialsregister.eu and/or indicated references. AE, adverse event; RA, rheumatoid arthritis; T1D, type 1 diabetes; Tx, transplantation; Dex, dexamethasone; MS, multiple sclerosis; /kg bw, per kg body weight; vitD3, vitamin D3. Ways Forward for Tolerance-Inducing Cellular Therapies February 2019 | Volume 10 | Article 181 ten Brinke et al. Ways Forward for Tolerance-Inducing Cellular Therapies their description, including full characterization of (at least) pool of distinct, candidate disease-related peptides (63–65) the immune phenotype and their functional activity (potency). (NCT02283671, NCT02618902, and NCT02903537). Hence, a better identification of the characteristics that identify In contrast to autoimmunity, transplant rejection is mediated the tolerance-inducing properties of tolAPC, irrespective of the by an undesired immune response against epitopes that differ conditioning regimen, would be valuable for safe cell therapy between the transplanted donor graft and the recipient host, delivery into patients. Joint efforts in translating tolAPC into the so-called allorecognition (66). Specifically, recipient T cells may clinic by harmonizing protocols and defining functional quality initiate a strong immune response leading to transplant rejection parameters have been initiated (1, 55). A Minimum Information in the absence of adequate immunosuppression. To avoid Model on Antigen-presenting cells (MITAP) has been defined transplant rejection, the induction of tolerance to donor-specific to harmonize reporting on tolAPC therapy to ultimately allow antigens has been coined as a therapeutic target for decades. the uncovering of commonalities between tolAPC and to define For this, both donor tolAPC and recipient tolAPC loaded with common quality control biomarkers and potency assays for the donor-specific antigens are being considered for development of various tolAPC products for clinical use (55). Likewise, using cell-based immunotherapeutic protocols in the transplantation similar immunomonitoring protocols in different clinical trials setting (67). However, whereas the clinical use of donor- could help to better understand the in vivo mechanism of action derived tolAPC is only feasible in the context of living donor of these cells (56). transplantation and entails a risk of sensitization (including development of allo-antibodies), the use of autologous, i.e., recipient-derived, tolAPC is a less risky approach to begin with. Antigen Specificity of TolAPC-Based Indeed, the use of recipient autologous tolAPC is likely to be Immunomodulation more feasible than that of donor-derived tolAPC, since cell Targeted regulation of antigen-specific T cell responses would products can be prepared from the peripheral blood of the avoid generalized immunosuppression as observed with recipient before transplantation, stored while the patient is on immunosuppressive drugs and monoclonal antibodies currently the waiting list, and loaded with donor-derived antigens (such as in use in the clinics and may thus overcome occurrence HLA peptides or donor cell lysates) at the time of transplantation. of impaired immune-surveillance leading to infections or In the context of the ONE study, two trials using tolDC and development of malignancies. Ex vivo generated tolAPC have Mreg are being performed in living-donor kidney transplant the potential to therapeutically induce, enhance, or restore recipients (Table 1). antigen-specific tolerance. Indeed, after loading these cells with exogenous or endogenous antigens, one major advantage is their Route of Administration of TolAPC capability to act in an antigen-specific manner. Although it is generally accepted that the route of administration A number of in vivo studies demonstrate that antigen is important for optimal tolAPC effector function, the best route loading of tolAPC is indispensable to reach efficient clinical of tolAPC administration is not known. To date, a variety of responsiveness following tolAPC therapy. For instance, a routes of administration have been used (see Table 1), including beneficial effect of vitamin D3 -tolDC loaded with MOG40−55 intradermal, intraperitoneal (19), intravenous and intra-articular peptide was demonstrated in experimental autoimmune (17). Different routes of administration may be required to allow encephalomyelitis (EAE), whereas no clear beneficial effect on tolDC to reach the relevant draining lymphoid tissue for T cell the clinical score of EAE mice was found when mice were treated encounter or to end up at the site of inflammation. Especially with vitamin D3 - tolDC not loaded with myelin peptides (57, 58). since tolDC demonstrate a reduced expression of CCR7 and Similar findings have been demonstrated in other animal models consequently a reduced (but not absent) ability to migrate in of autoimmune diseases, including collagen-induced arthritis response to the CCR7 ligand CCL19 (68), the capacity of tolDC to and autoimmune thyroiditis (59–61). Altogether, these findings reach the lymph nodes is a critical concern. While the migration suggest that selection of the target self-antigen is critical for of DC toward the lymph nodes increases following intradermal disease-specific tolerance induction in vivo. Suitable disease- as compared to subcutaneous administration, only 2–4% of associated self-antigens responsible for T cell priming have been DC migrate to the draining lymph nodes after intradermal identified for T1D and multiple sclerosis (MS). However, this is administration, but the situation may be different in patients not the case for other autoimmune diseases, such as rheumatoid with autoimmune diseases where monocyte-derived DC from arthritis or Crohn’s disease, for which specific disease-associated MS patients have shown a significantly higher proportion of antigens are unknown or not tissue specific. Moreover, not all CCR7-expressing cells compared to healthy controls (69). Given patients uniformly display the same set of self-antigens for a these observations, and that in the setting of cancer vaccine given disease. MS, for example, is associated with a range of development, DC injected into a lymphatic vessel showed a self-antigens and auto-antibodies that are differentially expressed prolonged half-life as compared to DC injected intravenously among patients and at different points during the disease (62). (70, 71), direct intranodal injection of tolDC is being evaluated While the targetable autoreactive T cell populations may be in a clinical setting (see Table 1). limited to a select number of antigens at the onset of clinical As an alternative to lymph node targeting of tolAPC, tolAPC disease, other “late antigen” or spread epitope autoreactive T may also be introduced directly into the site of inflammation. cell populations may drive autoimmunity during progression of Indeed, injection into the affected disease site (i.e., an inflamed the disease. To overcome this hurdle, some groups have chosen joint) where the tolAPC could suppress auto-reactive effector T to load the tolerance-inducing therapeutic cells with a broad cell responses is logical. In this context, intra-articular injection Frontiers in Immunology | www.frontiersin.org 5 February 2019 | Volume 10 | Article 181 ten Brinke et al. Ways Forward for Tolerance-Inducing Cellular Therapies of tolDC differentiated using dexamethasone and vitamin D3 antigen-specific FoxP3+ regulatory T cells in an animal model and loaded with autologous synovial fluid in patients with of arthritis (79) and that administration of phosphatidylserine- rheumatoid arthritis was demonstrated to be safe and feasible. rich liposomes loaded with disease-specific autoantigens lead Hence, despite the fact that tolDC were directly injected at the to a beneficial effect in experimental models of T1D and site of inflammation, no adverse events were observed in most MS (80, 81). Nevertheless, DC represent a heterogeneous cell patients and hypertrophy, vascularity and synovitis were stable population arising from bone marrow-restricted precursors in all treated cohorts. Moreover, two patients receiving 10 million identified in humans. While multiple subsets of DC have cells showed a decrease in synovitis score (17). Similarly, a phase been found in the peripheral blood, lymphoid organs and I randomized clinical study currently evaluates the safety and tissues, most of the hallmark DC markers are promiscuously efficacy of tolDC injected into the intestinal lesions in patients expressed making it difficult to unambiguously discriminate with refractory Crohn’s disease (Table 1). In some conditions between DC subpopulations and specifically target those DC such as T1D, direct injection of cells in the inflammatory subpopulations that induce tolerance. Extensive phenotypic site, e.g., pancreas, might not be possible and require tolDC screening combined with gene expression profiling allows the administration adjacent to the inflammation site. For the identification of tolerance-inducing DC counterparts present treatment of inflammatory diseases of the brain, the blood–brain in vivo. For instance, Gregori and co-workers identified a barrier (BBB) may represent a major hurdle. Considering this DC subset expressing HLA-DR+ CD14+ CD16+ that exhibits potential problem, it was demonstrated that enhancing CCR5 potent tolerogenic activity (44). In targeting only such tolerance- expression in tolDC using mRNA electroporation endowed these inducing DC cell type-specific targeting may emerge as another cells with CCR5-driven migratory capacity. This enabled the promising approach in DC-based immunotherapy. cells to migrate to inflammatory sites, even when it required crossing of functional barriers such as the BBB (72). Similarly, Combination Therapy introducing CCR7 expression in tolDC using the proposed Since a variety of often complementary mechanisms are involved approach of chemokine receptor mRNA electroporation could in the maintenance of immune tolerance, a more complex overcome the limited lymphoid homing capacity of tolDC. therapeutic approach using combinations that target different Indeed, DC transduced with lentiviral vectors coding for CCR7 pathways that contribute to induction and maintenance of and IL-10 genes were able to migrate to the lymph nodes tolerance may be required to fully control autoimmunity. and spleen, prolonging cardiac allograft survival in mice (73). For instance, combinations of tolAPC with disease-modifying However, there are still many unknowns and there is a clear treatments that reduce the frequency of disease-causing cells, clinical need to characterize the pharmacodynamics of tolDC e.g., alemtuzumab, should be explored as the latter therapy in humans and relate this to clinical efficacy. Advances in cell reduces the disease-causing cells to a number that may imaging techniques, for example magnetic resonance imaging of be more effectively controlled by antigen-specific tolerance- 19F-labeled cells, have made it possible to address this question inducing strategies, such as tolDCs. Alternatively, therapies in future studies. like fingolimod, an antagonist of sphingosine−1-phosphate receptor which retains naïve and central memory T cells in the lymph nodes, are promising as co-medication with tolAPC TolAPC Therapy: What Does the Future as it could increase the number of tolDC-T cell interactions Hold? in the lymph node thereby facilitating Treg priming and In vivo Targeting consequently the efficacy of tolDC-based strategies. Also in While our knowledge of tolAPC biology has expanded greatly, the context of solid organ transplantation, tolDC therapy and in vitro generated tolDC and Mreg are currently being used could be improved by the addition of a complementary in various clinical trials (Table 1), clinical-grade manufacturing treatment. For instance, the combination of adoptive transfer of tolAPC is still a time-consuming and expensive process. of tolDC and CTLA-4 Ig, a fusion protein that blocks B7- It requires cell precursors that need to be isolated from the CD28 costimulation, resulted in extended survival of MHC- patient’s blood, modulated ex vivo and reintroduced into the mismatched heart allografts in mice (82, 83), while the pancreatic patient. Direct antigen delivery to tolAPC in vivo may limit islet allograft survival improved by combination of autologous the workload and costs. Indeed, specific antigen-targeting of tolDC and CD3 targeting antibodies (84). With the recent DC-restricted endocytic receptors (DEC-205) with monoclonal data on the important role of other coinhibitory molecules antibodies has been shown to induce antigen-specific T cell for T cell-mediated inflammation such as CD96 the portfolio hyporesponsiveness in experimental models (74). Interestingly, of combination therapies might increase in the next years a phase I clinical trial demonstrated that in vivo targeting of (85). However, since antigens can easily trigger immunity human DC could be achieved by antibodies against DEC205 with instead of tolerance, a primary concern remains the safety subsequent antigen presentation and robust humoral and cellular of combining two immune-modulatory vaccination strategies responses (75). In vivo targeting of DC with biomaterials such as in autoimmune diseases and in the prevention of transplant liposomes, microparticles and nanoparticles is also a promising rejection. Although one can envisage that concomitant use of approach [as reviewed in (76–78)]. This is exemplified by the immunosuppressive therapies might synergistically reduce the fact that liposomes loaded with NFkB inhibitors targeting APC risk to unexpectedly worsen antigen-specific reactions (86), in situ, suppress the cellular responsiveness to NF-kB and induce any novel manipulation of the immune system may involve Frontiers in Immunology | www.frontiersin.org 6 February 2019 | Volume 10 | Article 181 ten Brinke et al. Ways Forward for Tolerance-Inducing Cellular Therapies an unpredictable risk. Furthermore, combination therapy may been reported in only one trial to date, however it was concluded introduce confounding factors inducing additive, synergistic or that the tumor was present before the therapy with Treg was antagonistic effects complicating the evaluation of the precise applied (94). The safety and feasibility of adoptive transfer of mode of action. ex vivo expanded Treg was further confirmed in T1D patients (2), which has driven the application of Treg therapy to clinical Conclusion trials in other autoimmune conditions such as MS, autoimmune Several protocols to generate and administer tolAPC have hepatitis, systemic lupus erythematosus, Crohn’s disease, and been tested in phase I clinical trials with highly encouraging autoimmune uveitis (102) (Table 2). Another clinical trial was results from a safety point of view and in terms of adverse recently published where polyclonal Treg were injected into T1D effects (Table 1). Further phase I/II studies are under way in patients; results from this trial confirm the safety of this type of Crohn’s disease, T1D, rheumatoid arthritis, MS and kidney therapy and also show for the first time, by deuterium labeling of transplantation (Table 1). However, for the success of future the Treg, that some of the injected Treg can be detected for up to tolAPC trials, there is great need to define the optimal vaccination 1 year after infusion (103). protocol; to ensure optimal in vivo-acting of the tolAPC, future Treg therapy is now being applied as a “tolerogenic” therapy trials may require changes in administration route, dose or to reduce dependency on immunosuppressant drugs in patients could demand repeated tolAPC administration. Furthermore, receiving solid organ transplants. The idea behind this strategy is the identification of a common set of tolerogenic markers very similar to the application of Treg in autoimmune diseases, would enable optimized comparison of tolAPC products and namely to tilt the balance toward Treg dominance over rejection- their tolerance-inducing potential and provide an improved causing Teff cells. The first reports using adoptive transfer of understanding of how these cells modulate the T cell response Treg in kidney transplant patients have been recently published both locally and systemically. It would be of great help to analyze demonstrating the safety of this strategy in the context of solid critical pathways contributing to programming and function of organ transplantation (103, 104). Recently, clinical trials are tolAPC. Ultimately, this may set the stage for new approaches being completed using different variations of Treg products improving the therapeutic potential of tolAPC for the future. (The ONE Study and ThRIL) (Table 2). The ONE Study includes Phase I clinical trials comparing the safety of different types of CD4+ REGULATORY T CELLS (TREG) regulatory cells, including polyclonal and donor-reactive Treg in patients receiving kidney transplants (www.onestudy.org) (20). CD4+ Treg are recognized as a dominant cell population The ThRIL trial is a Phase I/IIa dose-escalation clinical trial in responsible for induction and maintenance of immune tolerance. the setting of liver transplantation. Results from the ThRIL and They may be generated either in the thymus as natural regulatory the various ONE Study trials are currently being prepared for T cells (nTreg or tTreg) or in the periphery as induced regulatory publication. The impact of Treg on the recipient immune system T cells (iTreg). Both subsets can induce tolerance toward auto- will be revealed only when the very detailed immunomonitoring and alloantigens utilizing a variety of mechanisms including is completed, which is a major objective of the described clinical cell-to-cell contacts, secretion of immunosuppressive cytokines trials (105–107). and inhibitory molecules (e.g., adenosine or prostaglandin E), Altogether, from the outcomes of the completed clinical local depletion of IL-2, or through killing of other cells (87). trials so far, it can be concluded that adoptive transfer of Treg Treg actively traffic to inflammatory sites and the suppressive is safe and technically feasible (Table 2). Therefore, increasing activity is usually localized without a significant impact on the efforts are currently focusing on clinical trials to test their general immunity. Since their more precise identification 2–3 therapeutic efficacy. Importantly, several lessons have been decades ago in the mouse, and more recently in the human, learned from recent experiences with Treg to improve future steady advances in understanding Treg biology have eventually trial designs. For example, the clinical state of the patients has provided sufficient knowledge to culture, manipulate and expand been shown to influence the function and properties of Treg, the cells in vitro under Good Manufacturing Practice (GMP) and therefore can condition the success of ex vivo cell product conditions for therapeutic purposes. Indeed, Treg have become expansion (108). Furthermore, the specific expansion protocol a promising cellular drug that can potentially be used to control can affect Treg function and specificity, and can improve tissue disease-causing immune responses. targeting and suppression capacity (108). Finally, the immune modulatory therapies received by patients at the time of Treg Treg in Clinical Practice adoptive transfer can positively or negatively impact therapeutic While the application of Treg for the treatment of autoimmune outcome (109, 110). diseases is currently under intense investigation, Treg were first used in the clinic to treat patients with graft vs. host disease Treg Therapy: What Does the Future Hold? (GvHD) after hematopoietic stem cell transplantation (HSCT) Antigen-Specificity of Treg Therapy (88) (Table 2). Results from the clinical trials in GvHD with Studies in preclinical models using murine Treg have polyclonal expanded Treg have suggested that altogether these demonstrated that specificity for either the auto or allo cells are safe, but there is some concern about the occurrence of (transplantation) -antigens may offer an advantage for Treg mild to moderate infections, and it still is unclear whether Treg function compared to polyclonal Treg (111). Adoptively treatment could promote cancer (92, 94). The latter problem has transferred allospecific murine Treg generated by using either Frontiers in Immunology | www.frontiersin.org 7 February 2019 | Volume 10 | Article 181 TABLE 2 | Completed or ongoing trials with Treg in autoimmunity and transplantation. Study ID Phase Cell product Indication Dosing scheme Tregs per dose Outcome Center References HSCT ten Brinke et al. NKEBN/458- I Expanded polytTregs GvHD treatment Single injection or 1 × 105 /kg bw -Safe Gdansk/PL (88) 310/2008 3 injections 3 × 106 cells/kg bw -Reduced immunosuppression in chronic GVHD -Only transient improvement in acute GVHD NCT00602693 I Expanded CB GvHD prophylaxis Single injection Dose-escalation: 1, 3, -Safe Minnesota/USA (89, 90) polytTregs 10, 30, 30+30, 100, -Increased incidence of 300, 1,000 and 3,000 infections Frontiers in Immunology | www.frontiersin.org × 105 /kg bw -Reduced incidence of acute GvHD/GvL effect 01/08 I Fresh polytTregs GvHD prophylaxis Single injection Dose-escalation: -Safe Perugia/IT (91, 92) 0.5 × 106 Tcons/kg bw -Reduced number of with 2 × 106 Tregs/kg leukemia relapses bw -Reduced incidence of 1 × 106 Tcons/kg bw GVHD with 2 × 106 /Tregs kg bw −2 × 106 Tcons/kg bw with 4 × 106 Tregs/kg bw 8 I Fresh polytTregs GvHD prophylaxis Single injection up to 5 × 106 /kg bw Safe Regensburg/DE (93) Treg002 EudraCT: 2012-002685-12 EK 206082008 I Expanded polytTregs GvHD treatment Single or 2 0.97–4.45 × 106 /kg -Two cases of tumors Dresden/DE (94) injections bw -Stable chronic GvHD ALT-TEN I Tr1 (IL-10 DLI or DC-10 GvHD prophylaxis Single injection Dose-escalation: 1 × -Safe Milan/IT (95) DLI) 105 , 3 × 105 and 1 × -Long-term free-disease 106 , 3 × 106 CD3+ T survival in 4 patients cells/kg bw NCT02749084 I/II Multiple Treg DLI Severe Refractory Chronic 3 injections Dose-escalation: 1.7 × Recruiting Bologna/IT GvHD prophylaxis 105 , 3.3 × 105 and 6.6 × 105 /kg bw per injection NCT02991898 II Fresh CB polyTregs aGvHD prophylaxis after CB Single injection No data Suspended Minnesota/USA with IL-2 Tx NCT01911039 I polyTregs Steroid Dependent/ Single injection Dose-escalation: 1 × Unknown Stanford/USA Refractory Chronic GvHD 105 , 5 × 105 , 1.5 × treatment 106 /kg bw NCT02385019 I/II Fresh donor polyTregs Steroid-Refractory Chronic Single injection Dose-escalation: 0.5 × Recruiting Lisboa/PT GvHD treatment 106 , 1.0 × 106 and 2.0–3.0 × 106 /kg bw NCT01937468 I Fresh polyTregs with Steroid -Refractory Chronic Unknown Unknown Active/not recruiting Boston/USA IL-2 GvHD treatment Ways Forward for Tolerance-Inducing Cellular Therapies February 2019 | Volume 10 | Article 181 (Continued) TABLE 2 | Continued Study ID Phase Cell product Indication Dosing scheme Tregs per dose Outcome Center References ten Brinke et al. NCT01903473 I Fresh polyTregs with aGvHD and cGvHD Single injection ≥ 0.5 × 106 /kg bw Unknown Liege/BE rapamycin treatment EudraCT: I Fresh polyTregs with Steroid -Refractory Chronic Single injection ≥ 0.5 × 106 /kg bw Recruiting Liege/BE 2012-000301-71 rapamycin GvHD treatment NCT01795573 I Donor polyTregs aGvHD prophylaxis Unknown Unknown Recruiting Tampa/USA expanded with recipient DC Frontiers in Immunology | www.frontiersin.org NCT01660607 I/II Fresh polyTregs with aGvHD prophylaxis Single injection initial doses will be 1 × Recruiting Stanford/USA Tconv 106 Treg/kg bw to 3 × 106 Tcon/kg bw (ratio 1:3) NCT02423915 I Fucosylated fresh CB GvHD prophylaxis Single injection Dose-escalation: Active/Not Recruiting Houston/USA polyTregs 1 × 106 /kg bw 1 × 107 /kg bw I Fresh allogeneic GvHD prophylaxis Single injection Dose-escalation: 1 × Terminated, GvHD was Stanford/USA BMT Protocol 204 polyTregs with Tconv 105 Treg and 3 × 105 within parameters to NCT01050764 T con/kg bw or 3 × continue, but the study was 105 Treg and 1 × 106 terminated due to poor Tcon/kg bw or 1 × 106 outcomes prior to sufficient 9 Treg and 3 × 106 Tcon accrual to set the MTD, kg/bw or 3 × 106 Treg even at the only dose tested and 1 × 107 Tcon/kg (1 × 105 Treg and 3 × 105 bw T con/kg bw). Primary outcome result is null. NCT01634217 I iTregs GvHD prophylaxis Unknown Unknown Active, not recruiting Minnesota/USA TRANSPLANTATION NCT02129881 I/II Expanded polytTregs Living donor kidney Tx Single injection 1–10 × 106 /kg bw Completed, no results yet London, (20) Oxford/UK I/II Expanded polytTregs Living donor kidney Tx Single injection Dose-escalation: 0.5 × Completed, no results yet Berlin/DE (20) ONEnTreg13 106 , 1 × 106 , and 3 × NCT02371434 106 /kg bw EudraCT: 2013-001294-24 I/II Donor-Alloantigen- Living donor kidney Tx Single injection Dose-escalation: 300 No longer recruiting San (20) DART Reactive (dar) × 106 darTreg or 900 Francisco/USA NCT02244801 Tregs × 106 darTreg NCT02091232 I/II Belatacept-conditioned Living donor kidney Tx Unknown Unknown No longer recruiting Boston/USA (20) Tregs ThRIL I Expanded polytTregs Liver Tx Single injection low dose and high Completed, no results yet London/UK NCT02166177 dose (Continued) Ways Forward for Tolerance-Inducing Cellular Therapies February 2019 | Volume 10 | Article 181 TABLE 2 | Continued Study ID Phase Cell product Indication Dosing scheme Tregs per dose Outcome Center References ten Brinke et al. NCT02188719 I Donor-Alloantigen- Liver Tx Single injection Dose-escalation: 0 × Recruiting San Reactive 106 darTreg or 50 × Francisco/USA Tregs 106 darTreg or 200 × 106 darTreg or 800 × 106 darTreg NCT02088931 I Expanded polytTregs Subclinical rejection in Single injection 200 × 106 Open/Not recruiting San kidney Tx Francisco/USA NCT02474199 I Donor-Alloantigen- CNI reduction in liver Tx Single injection 300–500 × 106 /kg bw Recruiting San Frontiers in Immunology | www.frontiersin.org Reactive Francisco/USA Tregs NCT01624077 I Donor-antigen Liver Tx Multiple injections 1 × 106 /kg bw per Unknown Nanjing/CHN expanded Tregs at several intervals injection NCT01446484 I polytTregs Living donor kidney Tx Single injection 2 × 108 s.c. Unknown Moscow/RUS sub-cuntaneous I Expanded polytTregs Living donor kidney Tx Unknown Unknown Active, Not recruiting Chicago/USA TRACT NCT02145325 NCT02711826 I/II Donor-Alloantigen- Subclinical rejection in Single injection 400 ± 100 × 106 Recruiting Birmingham, Reactive Tregs vs. kidney Tx darTregs Los Angeles, polytTregs San Francisco, 10 Ann Arbor, Cleveland/USA AUTOIMMUNITY I Expanded polytTregs Recent T1D Single or 2 Dose-escalation: 10 × -Completed Gdansk/PL (96–98) TregVAC injections 106 , 20 × 106 , or 30 × -Safe ISRCTN06128462 106 /kg bw -Reduced insulin consumption (insulin independence in 2 out of 12 patients) -Better stimulated C-peptide secretion profiles NCT01210664 I Expanded polytTregs T1D Single injection Dose-escalation: 0.05 Completed/Safe San (99) × 108 , 0.4 × 108 , 3.2 Francisco/USA × 108 , 26 × 108 CATS1 I/II Ovalbumin-specific Tr1 Refractory Crohn’s disease Single injection Dose-escalation: 1 × -Safe Lille/FR (100) 106 , 107 , 108 , or 109 -Clinical response in 40% of patients II Ovalbumin-specific Tr1 Refractory Crohn’s disease Single injection 1 × 106 /kg bw Terminated/completed Valbonne/FR CATS29 Multicenter: AT, EudraCT: BE, FR, GE, IT, UK 2014-001295-65 NCT02327221 (Continued) Ways Forward for Tolerance-Inducing Cellular Therapies February 2019 | Volume 10 | Article 181 TABLE 2 | Continued Study ID Phase Cell product Indication Dosing scheme Tregs per dose Outcome Center References ten Brinke et al. II Expanded polytTregs Recent T1D 2 injections, 3 30 × 10/kg bw per Recruitment closed/Follow Gdansk/PL TregVAC2.0 combined with months apart injection up in progress EudraCT: antiCD20 antibody 2014-004319-35 I Expanded polytTregs MS Single injection: Up to 40 × 106 /kg bw Recruiting Gdansk/PL TregSM Cohort I – EudraCT: intravenous 2014-004320-22 Cohort II - intrathecal Frontiers in Immunology | www.frontiersin.org NCT02704338 I Expanded polytTregs Autoimmune hepatitis Single injection 10-20 × 106 /kg bw Not yet recruiting Nanjing/CHN NCT02772679 II Expanded polytTregs Recent T1D Single injection 3 or 20 × 106 /kg bw Suspended San with IL2 Francisco/USA NCT02428309 II Expanded polytTregs Systemic lupus Single injection Dose-escalation: 1 × Active/Not Recruiting San erythematosus 108 or 4 × 108 or 16 × Francisco/USA 108 NCT02932826 I Expanded third-party Recent T1D Single injection 1–5 × 106 /kg bw Recruiting Hunan/CHN CB polyTregs NCT03011021 I Expanded third-party Recent T1D Single injection 1–5 × 106 /kg bw Recruiting Hunan/CHN CB polyTregs and Liraglutide II Expanded polytTregs Recent T1D Single injection low dose and high Active/Not Recruiting San Francisco, 11 T-Rex Study dose Aurora, NCT02691247 New Haven, Gainesville, Miami, Indianapolis, Boston, Fargo, Kansas City, Portland, Sioux Falls, Nashville/USA OTHER NCT03101423 I Donor polyTregs DLI Beta Thalassemia Major Unknown Unknown Active/Not Recruiting Nanning/CHN NCT03241784 I Donor polyTregs DLI Amyotrophic Lateral 8 injections iv in 1 × 106 /kg bw per completed Houston/USA (101) Sclerosis (ALS) total concomitant injection with IL2 sc.: 4 injections over 2 months at early stage and 4 injections over 4 months at later stages This table is an updated version of the table in (102) and is based on information deposited on www.clinicaltrials.gov, www.clinicaltrialsregister.eu and/or indicated references. Route of administration of the Tregs is intravenous, except where otherwise noted. AE, adverse event; T1D, type 1 diabetes; Tx, transplantation; MS, multiple sclerosis; /kg bw, per kg body weight; HSCT, hematopoetic stem cell transplantation; CB, cord blood; GvHD, graft vs. host disease. Ways Forward for Tolerance-Inducing Cellular Therapies February 2019 | Volume 10 | Article 181 ten Brinke et al. Ways Forward for Tolerance-Inducing Cellular Therapies donor-derived APC or TCR transduction promote indefinite the in vivo treatment of autoimmune syndromes. For example, heart allograft survival, even in completely mismatched mouse while rapamycin administered to T1D patients preferentially strains (111). This strategy was subsequently applied to human increased Treg levels, pancreas function deteriorated due to Treg in a humanized mouse model, where Treg were generated islet toxicity (125). Nonetheless, the complex and specific in the presence of donor APC /DCs or B cells) and shown to be pathogenesis of autoimmune syndromes may provide hints superior to polyclonal Treg in protecting from human skin graft toward the design of new combined therapies. Tandemly rejection (112). More recently, by conferring specificity using targeting different effector mechanisms involved in particular a chimeric antigen receptor (CAR), human Treg transduced syndromes with Treg therapy may improve outcomes. For with a lentivirus encoding for HLA-A2-CAR were superior example, an ongoing trial in early phase of T1D (EudraCT: to polyclonal Treg in protecting HLA-A2+ human skin grafts 2014-004319-35) supports the idea of a synergistic approach by (113–115). CAR constructs are now being developed to increase combining Treg administration with B cell depletion. Treg stability and function. Based on promising results with antigen-specific Treg New Technical Advancements in pre-clinical models, the use of alloantigen-specific Treg It is now accepted that the optimal way to manufacture generated by culturing recipient Treg with donor-specific Treg for clinical use requires an efficient ex vivo expansion cells, either using activated donor-derived B cells (112) or rate while maintaining purity and suppression potency before donor-derived DCs is being tested in clinical trials (Table 2). GMP product release. Two main Treg isolation strategies are Compared to the transplantation field where the antigens are currently being used in clinical trials to purify the starting known, generation of antigen-specific Treg in autoimmunity is Treg, immunomagnetic selection and flow cytometry cell sorting. more challenging because the inciting antigens are often not The magnetic platform (CliniMACS CD4+ CD25+ selection) known (see also paragraph Antigen Specificity of tolAPC-Based provides a highly automated GMP-grade approach which is Immunomodulation). New data suggest that regulatory and easy to standardize across centers. However, the resultant Treg effector cell subsets are driven by different epitopes (116, 117). In product does generally contain a minor population of CD127+ addition, the auto-antigen triggering the autoimmune condition cells that could jeopardize product purity after expansion. can change during disease progression due to epitope spreading Addition of rapamycin to the culture media has helped to and antigen-specific Treg may thus need to be tuned toward maintain Treg purity and function, without reducing the specific stages of disease. expansion rate too much (126). The second method, which uses a flow cytometry approach, results in a highly pure Treg Combination Therapy population due to CD127+ cell depletion ability. However, Since adoptive transfer with Treg alone, particularly with a one- this cell-sorting strategy entails more complex protocols and time infusion, may not be sufficient to control the immune challenges to maintain GMP grade. With the appearance of response, combined or successive therapies are being tested. new GMP-compatible cell sorters (Tyto MacQuant, Miltenyi or One approach is based on evidence that low doses of IL-2 can Influx, BD Biosciences), this sorting approach is likely to become preferentially increase the endogenous pool of Treg; so far, low- the preferential Treg isolation method. dose IL-2 treatment has been safe in inflammatory conditions Although cell-sorting of Treg increases their initial purity, such as GvHD after HSCT (118). In a preclinical model it was a search for an optimally stable final Treg products is critical demonstrated that IL-2/anti-IL-2 complexes not only promote therapeutically. It is well known that prolonged in vitro culture Treg proliferation, they increase Treg survival and function results in epigenetic changes in Treg—methylation of TSDR while synergizing with calcineurin inhibitors to prolong graft region of foxp3 gene—which in-turn reduces suppressive ability survival (119). Recently, in a murine model of transplantation (127). The use of anti-methylation agents in cultures to prevent it was shown that by combining donor-specific Treg with the epigenetic changes has failed due to culture viability issues. The IL-2/anti-IL-2 complexes, a synergistic effect in extending skin addition of rapamycin to the expansion culture, as used in both transplant survival is observed (Ratnasothy et al., unpublished The ONE Study and ThRIL, was proposed as a remedy to such data). These results pave the way for the first clinical trial in liver changes as the drug preferentially expands Treg both in vitro transplant patients to combine Treg and low-dose IL-2 therapy and in vivo (122). However, proper choice of media, addition (NCT02949492). More caution is being exercised regarding of autologous serum, limited time frame for the expansion and ◦ similar trials using low-dose IL-2 in autoimmune diseases, temperature decreases during in vitro culture to ≈33 C, all since in contrast to HSCT or solid organ transplantation, impact the suppressive capacity of final Treg products (128–130). autoimmune patients are not lymphopenic and are likely to produce IL-2 themselves (98). However, in some conditions Conclusion such as systemic lupus erythematosus an intrinsic defect in Treg therapies are currently undergoing intense testing. An Tregs contributes to disease progression and here low-dose IL-2 interest in therapeutic Treg preparations has resulted in therapy was shown to correct the defect (120, 121). Furthermore, several ongoing clinical trials in the transplantation setting, in T1D settings low dose IL-2 treatment is currently being in autoimmune diseases, but now also in conditions such as investigated (NCT02411253). beta thalassemia major and amyotrophic lateral sclerosis (101) The positive effects of rapamycin on Treg in vitro or in a (Table 2). We await new testing in the setting of hypersensitivity transplant setting (122–124) could not be effectively translated to and cardiovascular disease (102), and anticipate that Treg therapy Frontiers in Immunology | www.frontiersin.org 12 February 2019 | Volume 10 | Article 181 ten Brinke et al. Ways Forward for Tolerance-Inducing Cellular Therapies will be considered for any condition where there is evidence for must keep in mind that ATMP must be affordable for patients and an immune regulation imbalance. society. Interestingly, scientists are largely responsible for current guidelines, and should revisit those recommendations based on factors such as cost (55, 132). TIMING OF TOLERANCE-INDUCING CELL THERAPY IMMUNOMONITORING OF Timing of tolerance-inducing cell therapy in relation to the TOLERANCE-INDUCING CELLULAR transplant or in the course of autoimmune disease development THERAPIES needs specific consideration. Clinical trials using Treg for GvHD indicate that Treg should be injected as early as possible, Tackling Immunomonitoring in Tolerogenic preferentially before disease onset (89, 91, 131). Early treatment Therapies is particularly important to achieve a high ratio between Treg Since cell-based therapies are becoming more common, it and detrimental effector T cells, and thus prevent development is important to reliably monitor the immune system for of acute rejection or GvHD. In both HSCT and solid organ both desired and undesired immunological effects. Rigorous transplantation, tolerance-inducing cells are therefore mainly immunomonitoring will therefore provide information about given around, just before or shortly after, the transplantation the safety of these treatments, ideally at early time points (see Tables 1, 2). Although early treatment is likely more after CTT administration. In addition it will give insight in effective, this approach encounters limitations such as increased the therapy-related mechanisms of tolerance-induction and immunosuppression doses and anti-CD25 treatment, which can maintenance and may aid in patient-tailoring of therapy. interfere with the activity of infused cells. Thus, Tregs have also To accurately measure the effects, especially across different been infused at later time points e.g., 6 months after kidney trials, it will require introduction of harmonized and validated transplantation in patients with biopsies showing evidence of immunomonitoring assays. inflammatory infiltrates to treat ongoing chronic rejection (103). There are a number of possible assays that can determine It must also be considered that disease diagnosis timing is cell therapy effects in humans. For instance, measurement of a factor in this respect. Although it is likely best to give circulating cytokines, C-reactive protein or changes in antibody the tolerance-inducing cell treatment as early as possible in titer can determine immune status. Similarly, measuring CD4+ disease development, it is currently not feasible in treating T cell responses after viral-antigen stimulation is possible by autoimmunity since a majority of autoimmune diseases start flow cytometry via CD40L expression or cytokine production long before clinical symptoms and diagnosis, this brings in the in a functional assay; these assays could potentially identify added factor that the functional capacity of the attacked organ non-specific immunosuppressive effects (133). Although the may already be irreparably damaged by the ongoing autoimmune completed clinical trials have shown so far that cell-based process. Future insight into autoimmune disease development tolerogenic therapies are safe and do not cause serious and early biomarkers will hopefully allow for earlier treatment undesirable immune responses (96, 99, 103, 104), the extent with tolerance-inducing cell products. to which these treatments achieve therapeutic efficacy remains largely undetermined. Current cell-based tolerogenic trials in REGULATIONS autoimmunity and transplantation include clinical outcome measures such as C-peptide response, insulin consumption The perspectives of tolerance-inducing cellular therapy depend or reduction of immunosuppressive doses. However, clinical also on recently introduced regulations. The majority of tested endpoints may not necessary reflect the efficacy of cell-based preparations in Europe are now classified as drugs under therapies, since the tolerogenic effect of the transferred cells the 1394/2007 EU directive on advanced therapy medicinal may not directly lead to immediate changes in systemic products (ATMP). This significantly changes the legal path parameters such as inflammation, potentially underestimating a for their registration requirements, for manufacturing license longer term effect. It is therefore important that future clinical and marketing authorization. While the idea of an ATMP in trials incorporate suitable monitoring methods to assess the Europe is relatively nascent, and it is continuously evolving immunomodulatory effects of cellular therapies. through public consultations with interested parties, cellular drugs have a distinct central paneuropean path for registration. General vs. Specific Monitoring Assays While the Committee for Advanced Therapies (CAT) steers this To assess therapeutic effectiveness, different methods have been process in Europe to optimize safety for patients, it would be proposed to identify tolerogenic responses. The assessment of useful to introduce wider rules allowing for introduction of the in vitro autoreactive or donor-specific T cells responses prior cells as a routine treatment. To accelerate the whole process, to and after treatment could provide a precise evaluation of acknowledgment of flexible new types of manufacturing cGMP therapeutic efficacy. Antigen-specific assays allow discrimination equipment and reagents could open the way to more widespread between targeted tolerance to the induction of general immune ATMP use. Furthermore, measures to reduce manufacturing suppression and loss of responses to pathogens. In addition, costs would lessen this major limitation to new trials. When these methods provide an efficacy readout for antigen-specific considering cell therapy, scientists, physicians and regulators therapies such as tolerogenic APC loaded with antigens or the Frontiers in Immunology | www.frontiersin.org 13 February 2019 | Volume 10 | Article 181 ten Brinke et al. Ways Forward for Tolerance-Inducing Cellular Therapies generation of donor-specific Treg. The identification of antigen- clones and identifying them among the T cell compartment specific immune responses by ELISPOT (134) or through repertoire through TCR sequencing analysis (147). flow cytometry detection of CD40L upregulation (135) have shown promising results in predicting kidney and liver allograft rejection, suggesting potential applicability for the evaluation Harmonization to Allow Comparison of tolerogenic therapies. Monitoring targets will be different Current tolerogenic cell-based trials are highly heterogeneous, depending on the main immune population involved in the comprising different cell types with variable manufacturing disease (e.g., CD4, CD8 T cells or antibodies). Unfortunately, approaches, and targeting various autoimmune diseases and the antigens mediating the immune responses in autoimmunity transplantation settings. Furthermore, by their very nature such are not always available or identified (as discussed in paragraph trials tend to involve low numbers of patients, often participating Antigen Specificity of tolAPC-Based Immunomodulation); HLA in different centers or countries. It is therefore essential to antigens in the case of transplantation are known and can establish common immunomonitoring strategies in the research be used, or stimulation with donor or donor-matched cells is community to achieve robust and reliable data which can possible. Though less specific, identification of phenotypic or be compared and combined between trials. First, not all the functional changes by flow cytometry on the total pool of cells immunomonitoring methods are similarly standardisable, and targeted (e.g., Treg) by the tolerogenic treatment may reveal second, not all the centers have the technical expertise or therapeutic effectiveness. Indeed, the acquisition of tolerance in infrastructure to perform certain assays. Sample collection and animal models and in the clinic is associated with an increased storage of whole blood or tissue biopsies for transcriptional number of regulatory cells and decreased pro-inflammatory analysis does not involve extensive sample manipulation, making function of innate and adaptive immune cells (5, 136, 137). standardization of this method achievable. On the contrary, while Therefore, flow cytometry analysis to delineate the distribution flow cytometry analysis of circulating blood is an accessible and activation status of different cell types, or in vitro assays technology, instrumentation must be calibrated appropriately to to evaluate the suppressive and inflammatory function of accurately define cell subsets, and analysis of the data needs to circulating cells can provide a non-specific approach to assess the be strictly regulated. Although centralized phenotypic analysis development of tolerogenic properties (138). Other non-specific in multicenter trials is feasible (105), this involves significant strategies such as gene expression profiling of circulating immune logistical challenges, including the decision to either test fresh cells or tissue biopsies can add to the functional assessment of or frozen samples; frozen samples sacrifice accuracy due to immune responses. Furthermore, there is a growing body of loss of certain cell populations during separation and freezing work focusing on the validation of transcriptional signatures to procedures. In general, flow cytometry standardization requires predict transplantation tolerance in liver and kidney transplant extensive cooperation between centers and precise planning. recipients (139–141), which may prove to be a valuable tool in To further harmonize flow cytometry data implementation of assessing the efficacy of tolerogenic therapies. automated gating approaches will be of outmost importance in the future (148–150). Finally, functional in vitro assays Tracking of Cellular Product also represent a challenging method to standardize, involving The evaluation of homeostatic characteristics of infused cells different approaches depending of the cell type and function. such as survival, stability or tissue migration, constitutes another Nevertheless, several efforts have been made to establish a monitoring objective to assess therapeutic efficacy. Being able minimal harmonization of antigen-specific functional assays to track infused cells will help to determine the best site for (56). While these assays are likely to be the most informative in administration/application of tolerogenic cell products. While the assessment of therapeutic efficacy, it is unlikely that current simple phenotypic detection (flow cytometry) of the transferred assay results will be directly comparable between independent cell subset after treatment suggests the presence of infused trials. Therefore, the inclusion of adequate control cohorts cells, it does not distinguish transferred from endogenous and reference groups in the assays, considering the specific cells. Therefore, current techniques for tracking infused cells cell therapy approaches and disease characteristics, remains an depend on direct cell labeling strategies, such as indium objective to achieve feasible comparisons (107). labeling or deuterium introduction during ex vivo expansion, with subsequent isotope detection in the different tissues or compartments (99, 103, 142). Unfortunately, this method is FINAL CONCLUSION only semi-quantitative, since individual cells are not detected. Individual cells can be labeled with rare earth metals and Tolerance-inducing cellular therapies have great potential. detected with precision in vivo using laser ablation-inductively Several cell types are now in early-stage clinical trials, including coupled plasma-mass spectrometry, but so far this has only been various types of Treg and tolAPC (including tolDC and Mreg) tested in mouse models (143, 144). Other emerging therapeutic (Tables 1, 2). At the present time it is unclear which of these approaches such as CAR-Treg could take advantage of genetic cell types will prove most suitable as a cell-based therapy; each modifications to adapt reported gene imaging strategies to detect likely has particular advantages that may be suitable for one the transferred cells by non-invasive methods (e.g., MRI, PET, particular disease or another. In Table 3, the main specific SPECT) (145, 146). Additionally, the use of T cell receptor (TCR) limitations to be considered for the treatment of autoimmunity engineered Treg or ex vivo expanded antigen-specific Treg create or transplantation with tolerance-inducing cell therapies are the opportunity to track infused cells by predefining the TCR summarized. It becomes clear that although we might treat these Frontiers in Immunology | www.frontiersin.org 14 February 2019 | Volume 10 | Article 181 ten Brinke et al. Ways Forward for Tolerance-Inducing Cellular Therapies TABLE 3 | Main specific differences in tolerance-inducing cell treatment between transplantation and autoimmune disease setting. Transplantation Autoimmunity Antigen - Alloantigens (MHC alleles and other disparities) - Autoantigen not always known - Autoantigens in case of underlying or de novo developed - Epitope spreading might occur during disease progression autoimmune diseases Pathogenic immune response - Normal, but undesired, immune response against foreign - Loss of tolerance to self-antigen antigen Timing - Time point of antigen contact is known, treatment can be - Disease already develops before clinical symptoms given around time point of transplantation - Better diagnosis and biomarkers are needed to be able to intervene at earlier time point Route of administration - Intravenous - Intradermal - Local injection in affected tissue or draining lymph node of the tissue is to be considered Co-medication - High dose of conventional immune suppression (steroids, - Varies and is disease specific CNI, MMF) +/- antibody-based induction therapy at the moment of transplantation Clinical efficacy evaluation - Prevention of acute rejections - Disease-dependent. E.g. In T1D C-peptide response or insulin - Reduction conventional immune suppression. It is difficult to consumption can be determined. In other AID disease-specific lower co-medication without good markers to predict scores can be used. transplant tolerance - Depending on disease progression on moment of application. Irreversible tissues destruction will not improve. New relapses/lesions can be scored. Immunomonitoring - Donor antigen-specific T cells - Autoantigen not always known. - Donor specific antibodies When known, T cell responses are difficult to detect, since they are very low frequent and of low affinity diseases with the same manufactured cell product, the optimal is a funding agency for research and innovation networks. treatment regime could be quite different. COST Actions help connect research initiatives across Europe Immunomonitoring is an indispensable aspect of current and enable scientists to grow their ideas by sharing them with and future tolerogenic cell-based therapies that will provide their peers. This boosts their research, career and innovation. fundamental information to understand and optimize cell COST is supported by the EU Framework Programme Horizon therapies. Monitoring will also aid in identifying biomarkers with 2020. Part of the work discussed is funded by The ONE the capacity for early identification of therapy responders and Study, EU FP7 Funding Program, BIO-DrIM, EU FP7 Funding non-responders and patient-tailoring of therapy. As part of the Program and ReSToRe, EU H2020 Funding Program. PT is overall strategy to increase implementation of ATMPs, it will supported by the National Center for Research and Development, be critical to harmonize GMP manufacturing protocols, product PL (grant no: STRATEGMED1/233368/1/NCBR/2014). EM-C characterization and immunomonitoring. Minimal information acknowledges the support by projects PI14/01175, PI16/01737 models such as MITAP and MiTREG (132, 151) will serve as and PI17/01521, integrated in the Plan Nacional de I+D+I and important tools in this respect. co-supported by the ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER). NC AUTHOR CONTRIBUTIONS and EM-C acknowledge the support by project 140191 from the Institute for the Promotion of Innovation by Science and All authors listed have made a substantial, direct and intellectual Technology (IWT-TMB) in Flanders (Belgium). contribution to the work, and approved it for publication. 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