Approaches to the Induction of Tolerance

Historical Perspective

In 1951 Billingham and Medawar published a landmark article entitled “The Technique of Free Skin Grafting in Mammals.” These classic experiments provide the foundation for what would become the field of transplantation immunology and the groundwork for many concepts in modern immunology, including immunologic memory. Further work by Medawar and his team, based upon earlier writings of Ray D. Owen, involved skin grafting dizygotic mammalian twin calves. The observation that these grafts were accepted by both hosts led to the hypothesis that a phenomenon of immunologic tolerance to the skin grafts was achieved secondary to “foreign” blood cells that persisted in each twin as a consequence of placental fusion.

These breakthroughs in research translated to the clinic in 1954, when Joseph Murray and colleagues performed the first successful kidney transplant between monozygotic twins at the Peter Bent Brigham Hospital in Boston, Massachusetts. The success of this procedure was in part because of the lack of immunosuppression needed when an organ was transplanted between monozygotic twins. Allografts that were attempted subsequently failed because of uncontrolled acute rejection responses mounted by the immune system. The quest to identify methods of both immunosuppression and tolerance induction in transplantation began.

Definition of “Tolerance”

Generally, the concept of tolerance (operational) refers to the persistent survival of a transplanted allograft in the absence of continuing immunosuppressive therapy and an ongoing destructive immune response targeting the graft. This functional definition is appropriate, because multiple immunologic mechanisms together with donor and recipient factors are involved in both inducing and maintaining tolerance to a defined set of donor antigens in vivo . Achieving functional tolerance in transplant recipients will mandate that specific allograft-destructive responses are “switched off” while the global immune response to pathogens and carcinogens remains intact. The most robust form of transplantation tolerance thus has to be donor-specific, as opposed to mere immuno-incompetence, a requirement that can be tested experimentally by grafting third-party transplants and by challenging tolerant recipients to respond to virus infections and tumors. The concept of graft-specific tolerance is essential both to maintain long-term survival of graft and host, and to eliminate the adverse events associated with lifelong nonspecific immunosuppression.

Need for Tolerance in Clinical Transplantation

The immune response to an allograft is an ongoing dialogue between the innate and adaptive immune system that if left unchecked will lead to the rejection of transplanted cells, tissues, or organs (see Chapter 32 ). Elements of the innate immune system, including macrophages, neutrophils, and complement, are activated as a consequence of tissue injury sustained during cell isolation or organ retrieval and ischemia reperfusion. Activation of the innate immune system inevitably leads to the initiation and amplification the adaptive response that involves T cells, B cells, and antibodies. T cells require a minimum of two signals for activation, antigen recognition (often referred to signal 1) and costimulation (referred to as signal 2). The majority of B cells require help from T cells to initiate antibody production. Antibodies reactive to donor antigens, including major and minor histocompatibility antigens and blood group antigens, can trigger or contribute to rejection early, and late, after transplantation.

Multiple factors determine the decision as to how the immune response to a transplant will be triggered and evolve, including where the antigen is “seen” and the conditions that are present at the time—in particular, the presence or absence of inflammation associated with activation of the innate response. In general, the innate response is neither specific nor is it altered significantly with multiple antigenic challenges. In contrast, the adaptive response is specific for a particular antigen or combination of antigens and “remembers” when it encounters the same antigen again, augmenting its activity and the rapidity of the response at each encounter. When the immune system encounters an antigen, it has to decide which type of response to make. In most cases, even though one component of the immune system may dominate and lead to rejection, the process is usually multifactorial, resulting from the integration of multiple mechanisms.

Understanding the molecular and cellular mechanisms that lead to allograft rejection has provided insights leading to the development of therapeutics that suppress this unwanted immune response after transplantation. A diverse collection of small-molecule and biologic immunosuppressive agents are approved and available for use in the clinic that have the potential to control or inhibit allograft rejection. In the context of solid-organ transplantation, the drugs that currently are available for clinical use include azathioprine, cyclosporine, tacrolimus, mycophenolate mofetil, rapamycin, antithymocyte globulin, anti-CD25 monoclonal antibodies, belatacept, and steroids ( Table 21.1 ). Each immunosuppressive agent acts on a different aspect of the immune response to an allograft and can therefore be used effectively in combination. Unfortunately, all of these agents are globally nonspecific in their suppressive activity, and each has some deleterious side effects.

These immunosuppressive drugs can be used with good success to prevent or control acute allograft rejection; however, they are less effective at controlling the long-term response to injury and activation of the immune system. They also appear to be unable to induce the development of unresponsiveness or tolerance to the donor alloantigens consistently, at least in the way they are used clinically at present. For nearly all transplant recipients, continued survival of the allograft depends on life-long administration of several immunosuppressive drugs. The exception to this statement is liver transplantation where in a proportion of pediatric and adult recipients it is possible to wean a small proportion of selected patients (<10%–20%) treated with immunosuppressive drugs off their immunosuppression, especially in patients with stable graft function over the first 4 to 5 years.

The inability of current immunosuppressive drug regimens to induce tolerance to donor antigens in the majority of patients may be due in part to the nonspecific nature of the immunosuppression resulting from their inability to distinguish between the potentially harmful immune response mounted against the organ graft and immune responses that could be beneficial, protecting the recipient from infectious pathogens and providing mechanisms to control the development of malignant cells. In general, current immunosuppressive drugs act by interfering with lymphocyte activation and/or proliferation irrespective of the antigen specificity of the responding cells ( Fig. 21.1 ). These mechanisms do not discriminate between effector cells that could be damaging to the transplant and immune regulatory cells that have the potential to control allograft rejection. This lack of immunologic specificity means that the immune system of a patient treated with one or more of these therapeutic agents is compromised not only in its ability to respond to the transplant, but also in its ability to respond to any other antigenic stimuli that may be encountered after transplantation. Therefore patients are more susceptible to infections (see Chapter 15, Chapter 16, Chapter 17, Chapter 18, Chapter 19 ) and are at a higher risk for developing cancer (see Chapters 34 and 35).

The development of immunologic tolerance or specific unresponsiveness to donor alloantigens in the short term or the long term after transplantation appears to offer the best possibility of achieving effectiveness and specificity in the control of the immune system after transplantation in either the absence or at least reduced loads of nonspecific immunosuppressive agents. If tolerance to donor alloantigens could be achieved reliably, it would ensure that only lymphocytes in the patient’s immune repertoire responding to donor antigens were suppressed or controlled, leaving the majority of lymphocytes immune competent and able to perform their normal functions after transplantation, including protecting the body from infection and cancer after transplantation. This chapter is therefore dedicated to discussion of the mechanisms underlying tolerance induction and strategies used to induce unresponsiveness in transplanted allografts.

Overview of T Cell Activation

Understanding the cellular and molecular mechanisms of activation and immune system regulation is important for the development of novel tolerance induction approaches in the context of transplantation and autoimmunity. The next section of the chapter sets the scene for discussing the different approaches to tolerance induction being explored most actively at present.

Hematopoietic stem cells (HSC) present in the bone marrow give rise to all of the leukocyte populations that participate in innate and adaptive immune responses. The thymus is the key organ that shapes the T cell repertoire. T cell precursors leave the bone marrow and migrate to the thymus where they rearrange the genes that encode the antigen recognition structure, the T cell receptor (TCR). Thymocytes that express TCRs with low affinity for self-antigen presented by self major histocompatibility complexes (MHC) molecules are “neglected” and die as they will be of no use to the host. In contrast, thymocytes expressing TCRs with a high affinity for self-antigen undergo programmed cell death and are “deleted” from the repertoire as they could respond to self-antigens in the periphery and therefore be harmful to the host. This leaves the T cells with receptors that have an intermediate affinity to enter the bloodstream where they recirculate between blood and peripheral lymphoid tissue. A subpopulation of T cells that will be discussed later, so-called thymus-derived or naturally occurring regulatory T cells (Treg), are also selected in the thymus and migrate to the periphery. A mature T cell repertoire is developed through this thymic selection process that is not only diverse, but can also react to foreign antigen while still remaining tolerant to self-antigens.

Naïve T cells encounter antigen in the form of a peptide MHC complex on the surface of antigen-presenting cells (APCs). Antigen presentation to T cells can be performed by a variety of APCs, including dendritic cells (DCs), macrophages, and B cells, although dendritic cells are the most immunostimulatory of all the APCs and the most potent at stimulating naïve T cells to respond.

As a direct consequence of organ retrieval and implantation, the tissue within the transplant is injured and stressed. Cells of the innate immune system express invariant pathogen-associated pattern recognition receptors (PRRs) that enable them to detect not only repeating structural units expressed by pathogens, referred to as pathogen-associated molecular patterns (PAMPs), but also markers of tissue injury or damage-associated molecular patterns (DAMPs). Local tissue damage and ischemia reperfusion injury generates many potential DAMPS, including reactive oxygen species, heat shock proteins, heparin sulfate, and high mobility group box 1 (HMBG1), after capture by the receptor for advanced glycation end products (RAGE) complex and fibrinogen, that can bind to PRRs. There are several families of PRRs, including transmembrane proteins present at the cell surface, such as toll-like receptors (TLRs), and inside the cell, including the NOD-like receptors (NODR).

The sensing of DAMPS by PRRs results in potent activation of the inflammasome, upregulating the transcription of genes and production of microRNAs involved in inflammatory responses setting up amplification and feedback loops that augment the response and trigger adaptive immunity. The end result is production of inflammatory mediators including the proinflammatory cytokines, interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF), type I interferons, chemokines (chemoattractant cytokines), and the rapid expression of P-selectin (CD62P) by endothelial cells. These events identify the transplant as a site of injury and inflammation, modifying the activation status, permeability, and viability of endothelial cells lining the vessels, triggering the release of soluble molecules, including antigens from the graft, inducing the production of acute phase proteins, including complement factors systemically and in some cases by the organ itself, stimulating the maturation and migration of donor-derived DCs from the transplant to recipient lymphoid tissue. This process results in upregulation of donor MHC, costimulatory and adhesion molecules by the donor-derived APC, enabling them to become potent stimulators of naïve T cells. The presentation of donor alloantigens to recipient T cells by donor-derived APCs results in T cell activation via the direct pathway of allorecognition.

Activation of the innate immune system within the allograft also triggers the recruitment of inflammatory leukocytes of recipient origin into the graft. These recipient APCs have the capacity to acquire donor histocompatibility antigens from the graft tissue and process them into peptides that can be presented to T cells via the indirect pathway of allorecognition. A third pathway of presentation of alloantigen to T cells has also been described, the so-called semidirect pathway of allorecognition whereby as a result of close contact between recipient APCs and donor cells, sections of membrane containing histocompatibility molecules are transferred from one cell to the other for presentation to T cells.

The interaction between APCs of donor or recipient origin and T lymphocytes is pivotal to the adaptive arm of the immune response. Immunostimulatory APCs are brought into close proximity to naïve T cells that may have TCRs capable of recognizing either intact donor alloantigens via the direct or semidirect pathways of allorecognition or donor peptides presented by recipient MHC molecules via the indirect pathway. An immunologic synapse (IS) is formed by the close interaction between the APC and the T cell that is dependent on the successful dynamic rearrangement and polarization of the filamentous actin in the DCs cytoskeletal membrane to bring the MHC-peptide complex in close relation to the TCR, thereby initiating an activation response. Specific T cell membrane compartments termed “lipid rafts” serve as recruitment centers for costimulatory molecules to concentrate in the cytoskeleton allowing for closer interactions with molecules on the APC ( Fig. 21.2 ).

For a T cell to become fully activated, a threshold number of TCRs need to be engaged. T cell receptor recognition of a donor MHC-peptide complex present on an APC, often referred to as signal 1, results in signal transduction through the cluster of differentiation (CD3) proteins that associate with the TCR at the T cell surface. This signal transduction initiates a cascade of biochemical signaling pathways that are contributed to by interactions between accessory, costimulatory, and adhesion molecules and ultimately culminates in cytokine production and proliferation of the triggered T cell and its differentiation into an effector cell ( Fig. 21.3 ).

Accessory and costimulatory molecules that have been shown to be important in triggering T cell activation on the T cell side include CD4, CD11b/CD18 (LFA-1), CD28, and CD154 (CD40 ligand). These molecules must engage their ligands on APCs, MHC class II, intracellular adhesion molecule (ICAM), CD86/80 (B7-1/B7-2), and CD40 respectively to ensure that once antigen recognition by TCR (signal 1) has occurred, the threshold for activation of a naïve T cell is overcome by delivering signal 2.

The two-signal model of T cell activation is well accepted, but it is important to note that this is a simplification. The cytokine and chemokine milieu present at the time these molecular engagements occur affects the differentiation pathway a T cell takes and the course of the response. Cytokines and chemokines can modulate expression of the cell surface molecules mentioned previously in addition to the expression of cytokine and chemokine receptors themselves. This modulation can result in differential signaling in the T cell and APC, tipping the balance of the response from full to partial activation or, in some circumstances, inactivation of the cells involved, modifying dramatically the downstream events (i.e., cell migration patterns and the generation of effector cells). Activation signals in the form of cytokines propagate the responses initiated by signals 1 and 2 and are often referred to as the third signal in T cell activation.

Mechanisms of Tolerance to Donor Antigens

The human immune system has evolved naturally to respond to challenges in a precise and controlled way. A constant balance exists to ensure an effective but not excessive response to unwanted stimuli. Many mechanisms of tolerance are, in fact, continuously used by the body to prevent reactions against self-antigens that would ultimately lead to autoimmune pathologies. It is when this balance in the immune system is disrupted that immune pathology leading to disease can occur. Many of these same mechanisms and regulatory cell populations can be harnessed to induce and maintain tolerance to alloantigens, at least in animal models.

The mechanisms identified as responsible for inducing or maintaining tolerance to donor antigens include the following:

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  Deletion of donor-reactive cells centrally in the thymus and in the periphery

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  T cell ignorance, or a state of T cell unresponsiveness that is relevant to grafts placed at “immunologically privileged” sites such as the cornea or brain

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  Exhaustion, in which the ability of donor-reactive cells to harm the allograft is eliminated as a result of overstimulation and cell death

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  Anergy, defined as a state of unresponsiveness that is refractory to further stimulation despite the continuing presence of antigen after transplantation

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  Immunoregulation—an active process whereby the immune response to an allograft is controlled by populations of regulatory immune cells

To exploit regulation of the immune response to an organ graft for therapeutic purposes, a clearer understanding of the mechanisms by which this phenomenon operates is required. Although theoretically regulation could function exclusively through a single mechanism, such as deletion of donor-reactive T cells and B cells from the repertoire (as will be discussed next), at present there is little evidence to support this as the only or even the dominant mechanism for inducing and maintaining unresponsiveness to cell and organ transplants. The more likely scenario is that different mechanisms work in concert and that distinct combinations of mechanisms are brought into play depending on donor and recipient characteristics, immunosuppression, infection, and so on, as the immune response to the transplant evolves.

Mechanisms of Tolerance Induction and Maintenance