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Kidney transplantation is the preferred mode of renal replacement therapy for most patients with end-stage renal disease. Despite the increasing success of transplantation over the years, allograft rejection remains a major problem. Recently, there has been considerable improvement in understanding the role of the immune system in rejection. In the setting of transplantation, T cells have proved to be crucial players in the immune response, and their activation has been shown to be a very tightly regulated process involving numerous interactions of receptors including the T-cell receptor (TCR):CD3 complex, co-stimulatory receptors, and appropriate signaling molecules, resulting in production of cytokines as well as clonal expansion and differentiation of effector T lymphocytes. In this review, current knowledge of the mechanisms of lymphocyte activation as well as potential targets for various immunosuppressive agents are discussed.
Allorecognition, the recognition of transplantation antigens by T cells, which is determined by the inheritance of co-dominant genes of the major histocompatibility complex (MHC), is the main event that triggers rejection. T-cell activation is a very tightly regulated process involving numerous interactions of receptors including the T-cell receptor (TCR):CD3 complex, co-stimulatory receptors, and appropriate signaling molecules, resulting in the production of cytokines as well as clonal expansion and differentiation of effector T lymphocytes. When T cells interact with antigen-presenting cells (APCs), they undergo complex structural and cytoskeletal changes leading to cell cycle progression.1 The end result is the activation and expansion of a pool of alloreactive effector cells that participate in graft injury and destruction.
In order to be fully activated, the T cell must be presented with distinct activation signals. To explicate the mechanisms for T-cell activation a three-signal hypothesis has been constructed. The initial interaction between the antigen-speciﬁc T cells and APCs provides the ﬁrst signal (signal 1) for T-cell activation. The most proximal event is the tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR/CD3 complex, followed by the activation of various distinct signaling pathways, including the Ras/ERK MAPK cascade, the Ca/calcineurin/NFAT pathway, and the PKC/NFχB pathway.2
Several novel immunosuppressive agents affecting signal 1 have been in development. ISA247, also called voclosporin, is a semisynthetic structural analog of cyclosporine A (CsA) that inhibits the calcineurin signal transduction pathway. Statistically longer allograft survival has been reported in animal studies for voclosporin compared with CsA. Another agent, alefacept, a dimeric fusion protein consisting of the CD2-binding portion of the human lymphocyte function-associated antigen-3 (LFA-3) linked to the Fc portion of human IgG1, is being tested in kidney transplantation. LFA-3-Ig is thought to neutralize the effect of CD2-expressing cells through several mechanisms including complement-mediated lysis and interruption of CD2's interactions with LFA-3, limiting CD4+ T-cell adhesion to APCs and disrupting the engagement of effector TCR with antigen and MHC molecules.
In addition to MHC-peptide complexes, a co-stimulatory signal, signal 2, is required for T-cell activation. T cells become anergic when presented with signal 1 in the absence of co-stimulatory signals. One of the most important co-stimulatory receptors is CD28 on naïve T cells; it has been previously hypothesized that the CD28 pathway would be important for T-cell activation when CD28-deﬁcient mice had severely diminished CD4+ T-cell pro-liferation.3 The CD28 receptor binds to two co-stimulatory molecules, B7-1 (CD80) and B7-2 (CD86). CD28 is constitutively expressed on all T cells in mice and on 95% of CD4+ T cells and 50% of CD8+ T cells in humans.4 In addition to CD28, inducible co-stimulator (ICOS), which is a member of the CD28 family and is induced after T-cell activation, also plays a signiﬁcant role in the co-stimulation of T cells. Its ligand B7H/B7RP-1 is expressed on B cells and in non-immune tissues. It has been demonstrated that in mice deﬁcient in ICOS or its ligand, B7H, CD4+ T-cell activation and production of effector cytokines, especially interleukin-4 (IL-4), are defective.5
Many inhibitory co-stimulatory molecules have also been identiﬁed such as the cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), a homologue of the CD28 molecule, which is induced on activated T cells and serves as a negative regulator of T-cell activation and proliferation. CTLA4 also binds to B7-1 and B7-2 and inhibits IL-2 production and cell cycle progression. Belatacept, a selective co-stimulation blocker, is a human fusion protein combining a modiﬁed extracellular portion of CTLA-4 with the constant-region fragment (Fc) of human IgG1 CTLA4-Ig, which binds to CD80/CD86 surface ligands of APCs. Belatacept is currently being tested for prevention of allograft rejection and preservation of kidney function in kidney transplant recipients as a potential replacement for calcineurin inhibitors (CNIs). It differs from abatacept by only two amino acids and has greater binding avidity to CD80 and CD86.
Belatacept provides more potent inhibition of T-cell activation than abatacept. In a partially blinded, randomized, multicenter Phase 2 study in 218 patients, Vincenti and colleagues demonstrated that the incidence of acute rejection was similar among three groups of patients: 7% for intensive belatacept, 6% for less intensive belatacept, and 8% for CsA.6 At 12 months, both renal function and histology was much better in patients treated with belatacept, raising the possibility of eliminating CNIs without compromising transplant outcomes. In a 3-year, randomized, active-controlled, parallel-group, multicenter Phase 3 study conducted at 100 centers worldwide, belatacept has been tested in 444 transplant recipients of standard kidneys (using a more [MI] or less intensive [LI] regimen of belatacept versus CsA).7 In this particular study, the incidence of acute rejection at 12 months was higher in the belatacept groups compared with CsA (22% MI; 17% LI; 7% CsA). In another Phase 3 study that assessed belatacept use versus CsA in adult expanded-criteria donor (ECD) kidney transplant recipients, the incidence of acute rejection was similar across groups (18% MI; 18% LI; 14% CsA).8
These studies have demonstrated fewer cases of diabetes, better blood pressure control, and better lipid proﬁles after transplantation with belatacept compared with CsA. Interestingly, few patients developed donor-speciﬁc antibodies on belatacept. Patients on belatacept also had better glomerular ﬁltration rates at 12 months compared with patients on CsA. An important issue is that belatacept has to be given intravenously, although might be helpful in patient compliance with immunosuppressive regimens by assuring the delivery of the medication. One concern is that more patients have developed post-transplant lymphoproliferative disease (PTLD) in the belatacept arm; most of these patients were Epstein-Barr virus (EBV) negative. Belatacept seems to be a promising agent with a different mechanism of action in immunosuppression. The long-term results of these two Phase 3 trials, as well as other studies such as conversion from CNIs to belatacept, pairing belatacept and sirolimus, and using alemtuzumab along with belatacept in kidney transplant recipients, are eagerly awaited.
We now have considerable evidence that CD8+ T cells also require cytokine signals, produced by macrophages and/or dendritic cells, at distinct stages of the T-cell response for optimal generation of effector and memory population, and for survival.9, 10 CD8+ T cells that do not receive a third signal in addition to signal 1 and signal 2 fail to develop cytolytic function, and become unresponsive. Signals provided by inﬂammatory cytokines, pre-dominantly IL-12 and type I interferons (IFNα/β), and possibly IL-21, are now considered to be required for a productive T-cell response and are termed signal 3. The requirement for a third signal was initially demonstrated in vitro using highly puriﬁed naive CD8+ T cells from TCR transgenic mice and artiﬁcial MHC protein/peptide antigen complexes immobilized on inert microspheres.11 With this technique, the potential effects of inﬂammatory cytokines on APCs were eliminated. In the absence of IL-12, a low level of proliferation occurred in vitro in response to antigen and IL-2. Addition of IL-12 in vitro resulted in a strong proliferative response, and in vivo administration of IL-12 along with peptide resulted in extensive clonal expansion.
In a newly developed murine heart transplant model, Filatenkov and colleagues studied the coordinated efforts between CD4+ and CD8+ T cells in the development of an effective CTL response.12 They have shown that CD4+ T cells conditioned dendritic cells to produce IL-12 and that IL-12 was necessary to support development of CD8+ T-cell effector functions and graft rejection. In a recent study, by using oligonucleotide microarray analysis of naive cells, Agarwal and colleagues studied the extent and molecular nature of the differentiation program induced by IL-12 and IFNα/β along with TCR and CD28 signals.13 When they stimulated the naïve cells for 3 days with artiﬁcial APCs and B7-1, they demonstrated altered expression of about 2,300 genes. When compared with expression in cells stimulated with only antigen-B7, addition of IL-12 or IFNα/β was found to further regulate expression of a modest number of genes/expressed sequence tags (730 genes and 350 genes, respectively). It appears that signals provided by antigen-B7 initiate changes in expression levels within 24 hours, but the changes are transient and expression reverts to naive levels by 72 hours. It has also been shown that in the presence of either IL-12 or IFNα/β, the changes in gene expression increase and persist at 72 hours, and additional changes occur, consistent with these cytokines inducing a critical sustained differentiation program in CD8+ T-cell function and memory.
Many of the set of genes regulated by IL-12 and IFNα/β, such as granzymes, IFNγ, CD25, Ox-40, and Bcl-3, are involved in effector functions, proliferation and co-stimulation, survival, trafﬁcking, and migration of T cells. Furthermore, it has been demonstrated that CD4+ T-cell responses in vitro are also enhanced by inﬂammatory cytokines. Interestingly, while IL-12 is required along with antigen and IL-2 to stimulate a signiﬁcant CD8+T-cell response, IL-1 has been shown to enhance the response to antigen and IL-2 in CD4+ T cells. Recently, in animal studies, Ben-Sasson and colleagues have demonstrated that IL-1 causes a marked increase in the degree of expansion of naïve and memory CD4+ T cells in response to challenge with their cognate antigen.14 The response was not induced by a series of other cytokines and does not depend on IL-6 or CD-28, indicating that parallel to the CD8+ T cells, CD4+ T cells might also need a third signal to proliferate. CP-690550 is a synthetic orally available inhibitor of janus kinase-3 (JAK3), which mediates signal transduction of receptors of the common γ-chain cytokines, inhibiting signal 3. Studies are under way in transplantation using different molecules, with high selectivity for JAK3.
The signal 3 cytokines (IL12 and IFNα/β for CD8+ T cells and IL-1 for CD4+ T cells) play a crucial role not only in transplantation, but also in tumor biology and vaccine development. They promote transcription of numerous genes needed for T-cell differentiation and effector functions, which could be developed into targets for various therapies in the future and allow us to pick and choose the best individual immunosuppressive regimen for the individual patient.