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Tolerogenic Cell-Based Therapy in Transplantation

Organ transplantation is currently the preferred treatment for patients with end stage organ failure.  However, it has been limited by the morbidity and mortality caused by the immunosuppressive drugs (IS) that are the mainstay of graft survival.  Patients treated with immunosuppressive drugs suffer from various side effects including opportunistic infections, hypertension, diabetes, malignancy, end organ damage and failure, and chronic rejection(3).  Approximately only half of kidney transplant recipients are alive at 10 years as a result.  Recently, tolerogenic cell-based therapies have emerged as a promising approach to induce immunological tolerance to solid organ allografts.  The primary benefit of cell-based therapy is the elimination of the life-long need for immunosuppressive drugs, thereby avoiding their associated toxicity.  Cell-based therapies are bringing transplant recipients one step closer to achieving the “Holy Grail” of transplantation: donor-specific tolerance.  For the first time the concept of one graft for life may be a clinical reality.  In this chapter, we review the role of regulatory T cells (Treg), tolerogenic dendritic cells (DC), graft facilitating cells (FC), regulatory B cells (Breg), and mesenchymal stem cells (MSC) in transplantation tolerance and their potential clinical application.
Mechanisms of Immunological Tolerance
Central Tolerance To induce tolerance, natural tolerogenic mechanisms must be utilized.  During maturation in the thymus, T cells gain an enormous diversity of T cell receptors (TCR) which can act as a double-edged weapon.  Not only do these receptors allow T cells to recognize most harmful non-self antigens, but they also can recognize self-antigens. For this reason, the role of central tolerance in preventing the escape of harmful self-reactive T cells into the circulation is of utmost importance to prevent autoimmunity. The two processes by which central tolerance is achieved are positive and negative selection, which take place in the cortex and medulla of the thymus respectively (4).  A series of complex developmental steps result in the mature Tcell repertoire.
Positive selection Immature T cells migrate from the bone marrow to the thymus where they acquire their TCR, CD4 and CD8 co-receptors; they are now called double positive (DP) thymocytes.  For double positive cells to continue to function they must receive a rescue signal from cortical thymic epithelial cells that express self-peptide or major histocompatibility complex (MHC). DP cells which don’t receive this rescue signal undergo apoptosis. As such the T cell repertoire is generated.  Positive selection allows antigen-presenting cells to present antigen to the TCR.
Negative selection  The rescued clones will now transform into single positive (SP) cells that express either the CD4 or CD8 co-receptor (5,6).  They then enter the thymic medulla where they encounter tissue specific antigens.  Self-reactive T cells undergo apoptosis.  The final result is the emergence of T cells which are tolerant to self-antigens but reactive to foreign antigens.
Peripheral Tolerance  Despite the efficiency of central tolerance in eliminating self-reactive T cells, some self-reactive T cells escape into the periphery due to the paucity of surface antigens expressed in the thymus (7). Peripheral tolerance compensates by eliminating these cells, inducing functional inactivation (anergy) and deletion of self-reactive T cells.
Anergy For T cells to be activated, two signals must be received.  The first signal occurs through the TCR engraftment and the second through co-stimulatory molecule (CD28) ligation. Co-stimulation will result in secretion of IL-2. The absence of one or both of the activating signals leads to a state of hyporesponsiveness or anergy in T cells encountering an antigen (8). This group of cells will remain alive, but they are rendered unresponsive.  Tolerogenic DCs can also induce cell anergy by mechanisms to be described later. Finally, regulatory T cells (Tregs) can transform into adaptive Tregs under certain conditions which can induce anergy in order to establish tolerance.
Peripheral deletion Deletion of self-reactive lymphocytes in the periphery is achieved by induction of apoptosis (programmed cell death) through two main pathways:  Fas ligand (Fas)-mediated and Bcl-2-like protein 11 (Bim)-mediated apoptosis. Fas activation leads to activation of Caspase and promotion of apoptosis (9).  Bim activation increases mitochondrial membrane permeability which releases Cytochrome C  inducing  apoptosis (10).  Both anergy and peripheral deletion are imperative to the maintenance of our natural immune systems, but they can also be manipulated for various therapies to induce tolerance.
Tolerogenic Strategies and Outcomes
Current standard of practice dictates that after an organ transplant, patients are maintained on immunosuppression for the remainder of their lives.  General immunosuppression leads to an increased incidence of infection, cancer, and ultimately a shortened lifespan of both the transplanted organ and the patient.  Strategies to minimize immunosuppression (IS) have been suboptimal as there is a fine balance to avoid excess immunosuppression leading to complications while avoiding rejection.  A study by Hoshino et al. describes the consequences of insufficient immunosuppression.  Seventy-two patients received living-donor kidney transplants with a clonal deletion protocol (total lymphoid irradiation or bortezomib) involving gradual tapering from IS starting at two months.  Development of donor specific antibodies (DSA) was inversely proportional to the level of IS, with 80% of the low IS group positive for DSA by 12 months and 74% by 6 months.  IS withdrawal or minimization without the development of DSA, rejection, or impaired kidney function, is extremely rare. A study by Shapiro et al between August 1991 and December 1996 at the University of Pittsburgh was performed to study the outcome of steroid withdrawal on patients who receive Tacrolimus-based immunosuppression.  Despite the acceptable outcomes in steroids withdrawal group, patients were not completely weaned off IS  (11)Shapiro REF.  Cell based therapies with the potential to target more specific immune processes could reduce the need for IS and improve quality of life for patients who may otherwise find themselves falling into these statistics (12).
Operational tolerance (OT) refers to non-chimeric transplant patients off all immunosuppression with stable organ function for one year or more. Most cases of operational tolerance have been reported in liver transplant patients.  At the University of California subjects who had stable hepatic function on immunosuppressive monotherapy were tapered off their IS over a minimum of 36 weeks. Twelve out of 20 pediatric liver transplant patients were successfully withdrawn from IS over a minimum of 36 weeks and remained tolerant with stable graft function and histology for at least one year.  A follow-up more than two years after complete IS withdrawal showed no significant change in protocol liver biopsy (13). The higher incidence of tolerance in liver transplant patients is hypothesized to be due to the unique venous endothelium of the liver which can be replaced by bone marrow-derived cells (14).
In 2010, Orlando et al. divided operationally tolerant patients into three categories after kidney or liver transplantation.  The first is a group of patients who were noncompliant with their immunosuppression treatments.  The second category includes patients who developed toxicity or intolerable side effects from IS.  The final group includes patients for whom tolerogenic protocols were applied and weaning under careful observation was conducted.  The majority of this final group consists of liver transplant patients who generally have a higher chance of operational tolerance.(15,16) (17).
An early venture into cell based therapies in conjunction with renal transplantation was conducted by Shapiro et al. who used a leucocyte depleting strategy followed by low-dose tacrolimus prior to transplantation. While this group successfully minimized immunosuppression, operational tolerance was not achieved. The study was halted due to the development of acute rejection in 20% of the patients and the appearance of DSA in 15% of the patients during weaning (18). The investigators had no way to predict which subjects would develop rejection or DSA.  The negative effects were possibly due to the persistence of effector memory cells after lymphoid depletion. Calcineurin inhibitor (CNI)-sensitive effector memory cells are not pro-tolerant, which would explain the difficulty in achieving operational tolerance (19).
The Role of Regulatory T cells in Tolerance Induction
The major population of regulatory T cells (Treg) are CD4+CD25+ FoxP3+ Treg. They are a key population responsible for controlling immune responses to alloantigens and preventing rejection in vivo.  Tregs exert their immunoregulatory functions through naturally-occurring Treg (nTreg) which arise from the thymus and regulate autoimmunity now called Thymus-derived Treg (tTreg).  When CD4+ T cells are subjected to tolerogenic stimuli, including contact with DCs or immunosuppressive cytokines such as IL-10, TGF-β, and IL-35, they differentiate into adaptive Treg (aTreg) or induced Treg (iTreg) now called Peripherally derived Treg (pTreg) (Abbas AK, Benoist C, Bluestone JA, Campbell DJ, Ghosh S, Hori S, et al. Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol (2013) 14:307–8. doi:10.1038/ni.2554).  This is effectively a form of clonal anergy with failure to produce IL-2, IL-5, IL-10, interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor as occurs in activation of conventional T cells (20,21).  The molecular mechanisms involved in Treg suppressive activity are summarized in Figure [1].
Regulatory T cells type 1 (Tr1), a subpopulation of iTregs, develop in the presence of IL-10.  They were originally defined in severe combined immune disorder (SCID) patients who had undergone bone marrow transplantation (22). The population is defined as IL-10 and IFN-γ-secreting cells which are CD25FoxP3(23). Tr1 cells inhibit APCs, effector T cells, graft versus host disease (GVHD) and allograft rejection by producing pro-inflammatory cytokines, reducing expression of co-stimulatory molecules, and suppressing memory and naïve T-cell activity (24), lending them as a promising avenue for future research and development.
Mechanisms of Immunosuppression by Treg
Tregs exert their immunoregulatory functions by modulation of four important immunological pathways.  The first is by metabolic regulation of Teffector cell (Teff) via Treg surface ectoenzymes CD39 and CD73.  These surface proteins convert ATP to AMP which increases production of the immune regulator adenosine. Transfer of cAMP to effector T cells through gap junctions leads to decreased production of IL-2 which in turn leads to apoptosis (25,26).  A second pathway aims at manipulating antigen presenting cells (APC) by hindering the ability of APC to express co-stimulatory molecules, such as CD80(B7-1) and CD86 (B7-2), thereby crippling their stimulatory role on T cells (27), Tregs exert this by overexpressing Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). When B7 molecules on APC bind CD28 surface receptor on CD4+T cells effective immune response will be initiated (Raab, M., Cai, Y.C., Bunnell, S.C., Heyeck, S.D., Berg, L.J., and for T cell costimulation. Proc. Natl. Acad. Sci. USA 92, 8891–8895, however when B7 molecules bind CTLA-4, which is the same receptor family as CD28, an immunoregulatory response will be activated   (Cohen, P.T. (1997). Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem. Sci. 22, 245–251.
A third route operates through secretion of inhibitory cytokines including IL-10, IL-35 and TGF-β which reduce effector T cell activation (28-30).  The final mode of operation for Treg revolves around suppression of CD8+ cells through PD-1 receptor (Programmed cell death-1), which also belongs to the CD28 family. Binding of PD-1 receptor to its ligands (PD-L1 and PDL-2) on APCs hinders CD8+ cells activation. Targeted immunotherapy for PD-l and PD-1 ligands is a promising treatment for many types of cancer. (The PD-1 Pathway in Tolerance and Autoimmunity Loise M. FranciscoPeter T. Sage, and Arlene H. Sharpedoi:  10.1111/j.1600-065X.2010.00923.x , Immunol Rev. 2010 Jul; 236: 219–242.)  By increasing levels of Treg in the circulation, a combination of these pathways can be induced to decrease inflammation, thereby increasing the chance of successful engraftment, favoring tolerance over immune activation.
Regulatory T cells in experimental animal models in transplantation
The role of Treg in prevention of graft rejection was studied in mice that received allogeneic islet-cell transplantation combined with Treg transplantation.  Different expansion protocols for Treg generation were utilized including expansion in vivo (31), expansion ex vivo with rapamycin (32), and induction of Tr1 Treg cells in vivo (31,33). The outcome of prevention of allograft rejection was promising, resulting in transplantation into the clinic.
Regulatory T cells in clinical application
The presence of naturally occurring populations of FoxP3+ Treg cells in renal transplant biopsies (34), and peripheral blood samples (35)correlate with increased graft success.  Accordingly, several groups have conducted preliminary clinical trials using Tregs as a cellular therapy in conjunction with organ transplantation.  The outcomes have been mixed (36-40).  The ONE study is a multicenter phase I/IIa clinical trial utilizing Treg in Europe which began in the summer of 2014.  Cellular therapy is being investigated in renal transplantation using nTreg, T regulatory type 1 cells, tolerogenic dendritic cells, and/or regulatory macrophages. The studies are underway at two UK centers and a center in Berlin.  Researchers are administering autologous ex vivo expanded nTreg to recipients of living-donor renal transplants (41).
In the United States, Bluestone et al. are executing a similar clinical trial utilizing a different approach for Treg expansion.  They have used the same Treg selection criteria as the European group based on CD4+CD25+CD127lo/- markers and CD45RA expression, however, two methods of expansion are used: polyclonal and alloantigen-reactive Treg expansion.   In polyclonal expansion, anti-CD3 and anti-CD28-coated beads supplemented with IL-2 and Rapamycin are used (42,43).  Alloantigen-reactive Treg are expanded using donor antigen-presenting cells such as dendritic cells, B cells, and unfractionated peripheral blood mononuclear cells. Alloantigen-reactive Treg are more potent than polyclonally expanded Treg.  Alloantigen-reactive Tregs are also highly donor-reactive and can be produced in a short time frame compared to polyclonal and autologous ex vivo expanded Tregs.   These studies will provide valuable data and insight into the role of Treg therapy in renal transplantation in the coming years.
Regulatory Dendritic Cells (DCreg) in Transplantation
DCs have a wide range of functions; depending upon their maturation and T-cell responses, they can induce either inflammatory activation or tolerance (44,45). Mature DCs act as APC and are distributed in all organs and in the circulation.  These cells express high levels of co-stimulatory molecules (mainly CD40, CD80, and CD86) and adhesion molecules for interaction with naive T cells.  These characteristics help them to be the most powerful line of activators to transform naive T cells into T effector cells (46). DCreg are characterized by low expression of co-stimulatory molecules which induces anergy in memory T cells.  These regulatory cells reduce production of pro-inflammatory IL-12 and increase secretion of the immune regulator IL-10 which induces Treg proliferation (47), and facilitates deletion of Teff cells (48).
Pharmacological modulation of DC with anti-inflammatory cytokines and agents such as rapamycin, dexamethasone, and Vitamin D3 has been used in animal models to induce tolerogenic DCs, which suppress T cell function in transplantation and autoimmune disease (49-51,51-53). In animal studies DCreg can be transfused before transplant, leaving unresponsive T cells and healthy grafts. Positive outcomes are not dependent on DCreg persistence in vivo unlike Treg therapy which requires repeated infusions to ensure the persistence of Treg (54).
Clinical application of tolerogenic (DCreg).
Giannoukakis et al. were the first to conduct a human trial using DCreg in adult patients with type-1 diabetes (55).  The regimen included ex vivo conditioning of autologous DCs with anti-sense oligonucleotides targeting CD40, CD80, and CD86 to render them tolerogenic. This phase I trial demonstrated the safety of DCreg transplantation; however, there was no clinical improvement.
A phase I/II study at the University of Pittsburgh is examining the safety of donor-derived DCreg combined with conventional immunosuppression on rejection and host alloimmune responses (56).  The investigators developed a GMP protocol for induction of maturation-resistant DCreg from peripheral blood monocytes. The University of Nantes, France is also using a distinct monocyte-derived DCreg paired with live-donor renal transplantation as a part of The ONE Study (41,54).  Although no trials have taken place yet, many groups will be looking towards these proposed phase I and phase II trials. In conclusion, DCreg infusion combined with immunosuppression has the potential to help prevent rejection, allowing for potential reduced or CNI- free immunosuppression.  The outcomes of several clinical trials are awaited with much anticipation.
The Role of Regulatory B cells in Transplantation
Regulatory B cells (Breg) are also termed “suppressive” cells due to their role in terminating the immune response by producing IL-10 (57).  They secrete transforming growth factor β (TGF-β) which induces apoptosis of CD4+ (58) and anergy in CD8+ effector T cells (59).  Breg manipulation in mouse models has given insight to the role of Bregs and B cells in rejection; however, it has not yet been tested as a novel therapy for renal transplant. The genetic modification of mice to deplete Breg results in the development of chronic inflammation, indicating that induction of Breg can be a potential therapeutic modality in immune-mediated inflammatory conditions (60). Deletion of IL-10-producing genes from Bregs in mice is also associated with increasing inflammation which decreases graft health (61,62).  In addition, IL-35-stimulated B cells produce IL-35 which inhibit autoimmune diseases (63), while mice lacking expression of IL-35 develop an exacerbation of experimental autoimmune encephalitis (64).  Bregs can suppress the immune response by inducing the proliferation  of Treg and suppressing pro-inflammatory lymphocytes such as tumor necrosis factor α (TNF-α)-producing monocytes, IL-12-producing dendritic cells, Th17 cells, Th1 cells, and cytotoxic CD8+ T cells through the production of IL-10, TGF-β, and IL-35 (65).  These results highlight the importance of Breg in maintaining stable graft function and controlling inflammation.
The role of B cells as a biomarker of transplant tolerance
In operationally tolerant patients a B cell signature has been identified.  Several groups report increased expression of B cell phenotypic profiles (35,66,67), genotypic profiles (68-70) (71,72), and miRNA profiles (73) which correlate with OT.  Patients who are OT have a greater number of naïve and transitional B cells compared with patients with chronic rejection.  Increased expression of IGKV1D-13 appears to be the most stable and discriminating gene as a marker of OT of all of the B cell associated gene expression markers (70).
In addition, the ratio of TNF-a and IL-10 production by Breg has been found to depend upon the stability of the Treg population (74).  This observation suggests that the ratio between IL-10 and TNF-α may correlate with the state of tolerance. A study conducted by Cherukuri et al. concluded that the IL-10/TNF-α ratio in transitional B cells (TrB) from recipients with chronic rejection was low compared with those with stable graft function (74).  These biomarkers could be used to predict early graft rejection, as well as ability to minimize immunosuppression and may guide future research in order to increase the rate of tolerance.
The role of Mesenchymal Stromal Cells (MSCs) in Transplantation
The interaction between MSCs, DCs, and natural killer (NK) cells is critical for each of  their immunomodulatory functions. MSCs halt antigen presenting potential of mature DCs by down regulating cell-surface expression of MHC class II molecules, CD11c, CD83, co-stimulatory molecules, and cytokines including interleukin-12, a mechanism which plays an indirect role in cellular anergy (75). MSCs decrease the pro-inflammatory potential of DCs by inhibiting their production of tumor-necrosis factor, and stimulating plasmacytoid DCs (pDCs) to produce the immunoregulatory cytokine IL-10 (76).  In addition, MSCs increase Tregs through secretion of pGE2, IL-10 (by pDCs), and TGF-β (77).  In combination, these signals have the potential to enhance tolerance. NK cells oppose the effects of MSCs and the mutual interaction between MSCs and NK cells leads to a delicate balance of immune response and tolerance.  Expression of NKp30 and natural-killer group 2, member D (NKG2D) activating receptors inhibits NK cell cytotoxic activity and consequently, target-cell killing (78). Because of the versatility and malleability of these cells based on the microenvironment, MSCs may impact future transplant therapies.
MSCs Move to Clinic
A pilot study by Remuzzi et al. utilizedMSCs to attempt to induce tolerance in two recipients of kidney allografts from living-related donors.  The protocol included induction by T cell depletion therapy and maintenance with CNI and mycophenolate mofetil (MMF).  MSCs were infused on day 7 post-transplant. There was an early increase in creatinine 7 to 14 days post infusion, but after one year the number of Tregs had started to rise. Both patients are in good health and have stable grafts (79). The Tan et altrial used autologous BM-derived MSC to spare patients from lymphodepletion with antithymocyte globulin (ATG) or alemtuzumab in 159 renal transplant recipients.  Autologous MSC (1−2 × 106/kg) were infused at kidney reperfusion and two weeks later. Patients were divided into three groups, 53 patients received standard IS plus MSC, 52 patients received low dose CNIs plus MSC and control group of 51 patients received anti-IL-2 receptor antibody plus standard dose CNIs.  Patients who received MSCs had a lower incidence of acute rejection, opportunistic infection, and better eGFR at 1 year compared with those who received anti-IL-2 receptor antibody induction (80).
Facilitating Cells
CD8+/TCR Facilitating cells (FC) from mouse bone marrow significantly enhance hematopoietic stem cell (HSC) engraftment and induce transplantation tolerance in vivo (81). The effects of FC on HSC include enhancing clonogenicity of HSC, preventing apoptosis of HSC, and improving the bone marrow stromal microenvironment to support cobblestone area formation by HSC (82).  FC is not a CD4+/CD25+/FoxP3+ Treg and does not express the genes linked to Treg (83).  However, FC have been shown to induce the formation of antigen-specific Treg and interleukin-10-producing type 1 regulatory T cells in vitro from naive CD4+/CD25– T cells (84,85).
A comprehensive assessment of FC surface markers using flow cytometric analysis of sorted FC populations by Fugier-Vivier et al. revealed a broad heterogeneity in subpopulations with facilitating potential (86).  The majority of mouse CD8+/TCR FC were CD11c+ (65% to 70%) and B220+ (75% to 88%). While only 15% of the B220+ population was composed of B cells, 55% co-expressed CD11c (CD11c+/B220+), suggesting a dendritic cell component.  Other studies by Taylor et al. demonstrated that FC express CD3ε  on FC is associated with the TCRβ chain and a novel 33-kDa protein in the complex termed FCp33 is critical for the HSC engraftment-enhancing properties of FC (85).  Further analysis of the CD11c+ dendritic subset revealed that a plasmacytoid precursor dendritic cell (p-preDC) phenotype (CD11dim/B220+/CD11b) is the predominant cell type (93% to 95%) within this CD11c+ subset(86,87).
CD8a+ p-preDC FC are critical to the generation of antigen-specific Treg in vivo. This was revealed by the removal of the CD8a+ p-preDC FC subpopulation from total FC which abrogated the generation of Treg in vivo (88). The co-transplantation of p-preDC rescues allogeneic mouse recipients from radiation-induced aplasia, significantly enhancing alloengraftment and survival compared with ablated recipients of HSC alone.  However, facilitation by this p-preDC component alone was inferior to that observed when FC total was administered.  This observation suggests that the p-pre DC subset is necessary, but not sufficient, for HSC facilitation and cannot replace effect of the total FC population.
Taylor and colleagues demonstrated that FCs induce CD4+/CD25+ /FoxP3+ Treg cells (85,89). The researchers proposed a mechanism of activation of FC similar to that of p-preDC, to generate Treg, a mechanism that requires direct cell-to-cell contact (85). Recent studies have suggested that the differentiation of CD4+/CD25 T cells into CD4+/CD25+ T cells requires stimulation from various sources such as TGFβ, pDC, or rapamycin.  The B7 receptors (CD80 and CD86) play an important role in the maintenance or development of Treg (90,90,91). B7-deficient mice have a significantly lower number of CD4+/CD25+  regulatory T cells (85).  CD86 is thought to be another important component in assisting FC to produce CD4+/CD25+/FoxP3+ Treg because CD86 expression on FC is upregulated after CpG stimulation.  More recently, CD8α+ p-preDC FC were shown to induce antigen-specific Treg in vivo (88).  Notably, FC from bone marrow from DOCK2-/- mice exhibited impaired ability to induce Treg and IL-10 producing Trl cells in vitro (84).
The characterization of human FC
Two distinct and complementary FC subpopulations are present in human CD8+/TCR FC from G-CSF mobilized PBMC: CD8+/TCR/CD56neg (CD56neg) FC and CD8+/TCRCD56bright (CD56bright) FC (92) (Figure).  The CD56neg FC subpopulation promotes rapid reconstitution of human HSPC in NOD/SCID/IL2rgnull (NSG) mice without GVHD. Co-culture of CD56bright FC with HSPC upregulates cathelicidin and beta-defensin 2, factors that prime responsiveness of HSPC to SDF-1. Both CD56bright FC and CD56neg FC subpopulations significantly upregulate mRNA expression of Flt3 ligand (>3500 fold and > 10 fold, respectively). Human FC total significantly prevented apoptosis of HSPC in vitro.  These data suggested that the two predominant FC subpopulations are phenotypically distinct and exert complementary functions to HSPCs to promote homing, migration, clonogenicity and engraftment of HSC (93).  Bridenbaugh et al. also reported that human CD8+/TCR/CD3+ FC from G-CSF mPBMC enhance human HSPC engraftment in NOD/SCID mice in vivo (94).
Induction of Tolerance through Mixed Chimerism
Mixed chimerism refers to the co-existence of donor and recipient bone marrow cells in the recipient’s body.  HSCs from the organ donor are transplanted into the recipient in the hope that these cells will become fully functional, and the recipient’s immune system will recognize any transplanted organ as self.  Chimerism has some of the most promising data, leaving several patients IS-free after renal transplant.  As such, cell-based therapies inch closer and closer to becoming standard of care in transplantation.  Three main groups in the United States and several other groups worldwide have been working to induce tolerance through chimerism using distinct approaches. The safety and feasibility of the approaches have already been established.
The Massachusetts General Hospital (MGHgroup
HLA-matched subjects with renal failure and multiple myeloma (MM) were transplanted with bone marrow and a kidney from a single living donor (Table). Preparative therapy for the transplant consisted of high-dose cyclophosphamide (60 mg/kg/day on days −5 and −4), equine ATG  (15 to 20 mg/kg on transplant days −1,+1, +3, and +5) and thymic irradiation (700 cGy on day −1). Cyclosporine (CyA) was utilized for immunosuppression and then tapered and discontinued as early as day 73 post-transplant (95).
IS was successfully removed from 5 out 10 patients with maintained antitumor response in 30 % (96).
One patient only received low-dose total body irradiation and ATG before transplant as the original regimen was aborted due to development of sepsis and multi-organ failure. Ten patients received the same therapy with a slight modification.  The development of cyclophosphamide related toxicities led to its replacement with 400 cGy TBI.  Researchers followed these patients for more than 17 years. Six of the ten patients are alive. Two of these six are in complete remission for five and thirteen years respectively. Two of the remaining four patients relapsed to  progressive MM and underwent a second HSCT from their original donors. Two patients developed acute GVHD and four patients developed chronic GVHD. Five of the ten patients developed transient chimerism. Studies at this site recently resumed and have expanded to include patients with other hematologic malignancies and blood disorders paired with end stage renal disease.  The protocol continues to be refined, and at its current stage, involves no cyclophosphamide, but instead TBI (400 cGy) with the previously described regimen (96).
The MGH group further conducted a study in HLA-mismatched patients with malignancy.  This protocol is modeled off of the most successful protocols previously described in literature for HLA-mismatched transplants using umbilical cord blood. The conditioning regimen includes ATG, low-dose cyclophosphamide, and TBI (200 cGy) before simultaneous kidney and bone marrow transplant.  Post-transplant, subjects receive high-dose cyclophosphamide, tacrolimus, and MMF.
The first patient to undergo this therapy experienced graft rejection within two weeks.  She has since recovered and has had over three years of successful graft function. Because the first patient developed rejection, ATG was replaced by fludarabine in the conditioning. Researchers adjusted the dose of fludarabine appropriately to prevent toxicity in patients with renal dysfunction. The second patient stopped IS after eight months and lived for two years post-transplant with no GVHD, no evidence of recurrent MM, and normal kidney function.  The third patient expired six months after transplant from presumed neurotoxicity due to fludarabine.
As a result, the number of doses of fludarabine was reduced and the duration of hemodialysis lengthened to reduce the fludarabine toxicity as it is eliminated by the kidneys.  At the time of publication, the fourth patient had reached six months after transplant with no GVHD, a full taper off IS, and normal kidney function.  This trial has included six patients total, each receiving a more refined treatment plan.  Presently, the trial continues and the current treatment includes a reduced intensity fludarabine paired with hemodialysis which will most likely undergo more revision (96).
The MGH group also conducted an HSCT study with 10 HLA-mismatched subjects without malignancy using three protocols, each an improvement on the previous based on results (Table). The first was the NKD03 regimen, which consisted of pre-transplant cyclophosphamide (60mg/kg i.v. on days -5 and -4 before transplant), humanized anti-CD2 mAb (0.6 mg/kg/dose on days -2, -1, 0 and +1), CyA (5mg/kg) and TI (700cGy on day -1, day 0). The kidney and unprocessed BM (2-3 x108 mononuclear cells/kg) were transplanted on day 0.  The first three patients received oral CyA (8-12 mg/kg/day) post-operatively (target trough blood levels of 250-350 ng/mL tapered and discontinued over 9-14 months) to prevent antibody-mediated rejection (97). The modified protocol included the addition of Rituximab (375 mg/m2/dose on days -7 and -2), and prednisone (2 mg/kg/dose day 0 taper to day +10). Two patients who were treated with that regimen developed biopsy proven chronic humoral rejection after immunosuppression stoppage despite the addition of these agents (98).  Consequently, the protocol was modified once more (ITN036) for the last five patients.  This modified protocol adds two more Rituximab doses (days 5 and 12), extension of the prednisone course until day 20, and Tacrolimus instead of CyA to be tapered at 8 months after confirming no rejection by a 6-month protocol biopsy.
In summary, seven of ten patients tapered completely off IS, four out of those seven patients have had a good graft function without rejection episodes for 4 to 11 years. The remaining three patients returned to IS after 6 to 8 years due to chronic humoral rejection in two of them, and recurrence of original disease (membranoproliferative GN) in one patient. The remaining three patients lost their grafts within 18 months of transplantation before weaning due to kidney rejection in two patients and thrombotic microangiopathy (TMA) in one.  The net result is that four patients out of ten were withdrawn completely off IS and achieved transient mixed chimerism, but nine out the ten patients developed “engraftment syndrome”  (99). Each of these protocols is currently being modified in order to achieve better results.  This study confirmed the safety of the approach and though this protocol found limited success, there is still a need for a better line of treatment.
The Stanford group
Twenty-two HLA-matched kidney transplant recipients have been enrolled to date on a combined kidney/HSCT protocol.  Conditioning consisted of total lymphoid irradiation (TLI) and rabbit ATG. Patients received CD34+ cells obtained from G-CSF-mobilized peripheral blood mononuclear cells with an add-back of 1 x 106 T cells/kg (100) (Table?).  Eighteen of the 22 patients had at least 12 months of mixed chimerism, and completely withdrew from IS drugs. Withdrawal occurred after one month of MMF treatment and six to twelve months of CyA treatment (100). Patients were observed for up to seven years after IS drug withdrawal (median, 29 months), and none had subsequent rejection episodes or GVHD.  All 22 patients had good kidney graft function at the last observation point (100).
Northwestern University (NU)/Institute for Cellular Therapeutics (ICT)
A Northwestern University group utilized FC and HSC combined therapy with renal transplantation.  FC, a bone marrow derived CD8+TCR cell population, enables engraftment of HSC of HLA-matched and HLA-mismatched donors without causing GVHD (81).  The NU/ICT group has developed a nonmyeloablative reduced-intensity conditioning approach to establish high levels of donor chimerism without GVHD or engraftment syndrome in allogeneic recipients of combined kidney/HSC/FC transplants (101).  A tolerance-promoting FC-based HSC graft, named FCRx product consists of a G-CSF mobilized PBMC that were apheresed from the donor and processed to remove GVHD producing cells yet retain HSC and FC, and cryopreserved until administration the day after the kidney transplantation (101).  Recipients received fludarabine (30mg/kg/dose, days -5, -4, and -3), cyclophosphamide (50mg/kg/dose, days -3 and +3)  and TBI (200 cGy day -1) followed by the living donor kidney transplantation and FCRx infusion.  A total of 31 subjects were transplanted during this phase II trial with follow up ranging from one month to 72 months. These subjects range from 18-65 years of age and from 5/6 HLA-matched related to 0/6 HLA-matched unrelated to their donors.  Nineteen subjects with durable donor chimerism were successfully weaned off all immunosuppression (time off IS ranging from 3-65 months). Sixteen subjects exhibited full chimerism (>98% donor cells), and three subject with mixed chimerism. Five subjects with transient chimerism are maintained on low dose IS with normal kidney function. Two subjects have lost their renal allografts.  Subjects with durable donor chimerism showed immunocompetence and donor-specific tolerance in an in vitro proliferative assay.  All chimeric subjects retained chimerism after removal of IS, with stable normal renal functions (ATC abstract, Ildstad et al. 2016).   FCRx offers a novel cell-based therapeutic approach to induce transplantation tolerance, treat a number of autoimmune diseases, and improve outcomes in inherited metabolic diseases (Figure).
Induction of transplantation tolerance avoids long-term use of immunosuppressive drugs which is associated with better quality of life for kidney transplant recipients.  Clinical trials using cell based therapies in order to create safer organ transplantation are promising.   While research in this area has just begun in the past couple of decades, there is sound ground work using animal models on which to build.  Eventually, these treatment regimens will go beyond the current disease models.  While focus is now on end stage renal failure and diabetes mellitus, it may soon shift to anything from sickle cell anemia, to inherited metabolic disorders, and a variety of autoimmune disorders.
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