Liver and pancreas transplantation immunobiology

Innate immunity

The cellular components of the innate immune system include dendritic cells, macrophages, neutrophils, and natural killer cells. These cells are activated by binding of either microbial by-products (pathogen-associated molecular patterns [PAMPs]) or products of sterile tissue injury (damage-associated molecular patterns [DAMPs]) to pattern-recognition receptors located in both intracellular and cell surface compartments. Perhaps the most well-described pattern-recognition receptor is the Toll-like receptor (TLR). This highly conserved family of receptors responds to PAMPs associated with microbial infection, including LPS as well as DAMPs generated by cell death, including high mobility group box-1 (HMGB1). Interestingly, the TLRs expressed in the liver differ from those expressed in the periphery and tend to be less responsive to ambient LPS. This is likely an adaptation to portal bacteremia and is thought to make the liver more tolerant of minor perturbations that would evoke an innate response in other organs. It is important to note that innate immune receptors are conserved between individuals and do not demonstrate the polymorphic nature of MHC as described previously.

Binding of PAMPs and DAMPs to innate immune receptors initiate signaling cascades resulting in generalized inflammation and activation of complement. ^{, ,} Of particular relevance in transplantation, ischemia-reperfusion injury and associated cell death results in the release of multiple DAMPs derived from different subcellular compartments including the nucleus (extracellular DNA, HMGB1) and mitochondria (mitochondrial DNA, reactive oxygen species). Recently, greater attention has been paid to the effects of DAMP-mediated inflammation on both early graft injury and function and the connection with the subsequent adaptive immune response. ^,

The complement system acts as the primary mediator of cytolysis, and the by-products of complement help link the innate and acquired immune responses. ^, Platelets have also been increasingly recognized as serving an innate immune role through the release of chemotactic proteins and other immunostimulatory molecules.

Acquired immunity

Specific recognition is the hallmark of the acquired immune system. The immense structural diversity of the lymphocyte receptors (T-cell and B-cell receptors) facilitates recognition of a vast array of antigens. Furthermore, initial antigen recognition induces physiologic change in the lymphocyte, lowering the threshold for activation in response to subsequent encounters. This phenomenon underlies the concept of immunologic memory, which results in a more rapid (anamnestic) response to subsequent antigen encounters. T-cell receptors (TCRs) bind peptide antigens that have been processed and presented in combination with MHC. B-cell Igs bind antigens in their native conformation and can be secreted in soluble form as circulating antibodies that act remotely.

Cellular immunity

Understanding the unique nature of the TCR is fundamental to understanding its function. ^, T cells, formed in the bone marrow and fetal liver, migrate to the thymus during development, where they undergo rearrangement of the DNA that encodes the TCR. Each gene rearrangement results in the generation of a TCR with specificity generally restricted to one epitope, although some cross-reactivity can occur with similar epitopes. The sum of all random TCR gene rearrangements generates TCRs with approximately 10 ⁹ unique specificities. If such a vast array of T cells was to be released immediately into the periphery, they would mediate widespread and fatal autoimmune processes. Accordingly, the process of thymic selection eliminates those T cells likely to induce autoimmunity. ^,

The initial phase of thymic selection occurs through the interaction of cortical thymic epithelial cells with the cohort of developing T cells. These T cells express the accessory cell surface markers CD4 or CD8, which facilitate stronger binding interactions between the TCR and MHC. If T-cell binding does not occur to the self MHC molecules presented by the thymic epithelium, the T cells are eliminated because they are unable to interact with self MHC molecules, which is required for normal immune surveillance and function. This process is known as positive selection.

Positive selection produces a repertoire of T cells capable of recognizing and binding to self MHC, which is critical for normal functioning in the periphery. However, this step does not distinguish T cells with normal affinity from those with inappropriately strong affinity to self MHC, which would predispose to autoimmunity. To eliminate cells with a high risk of autoimmune recognition, the developing T cells then move into the thymic medulla, where either CD4 or CD8 expression is lost. If binding to self MHC in the medulla results in a high-affinity interaction, these T cells are also eliminated, a process known as negative selection. Therefore, after this step the majority of T cells released from the thymus bind to self MHC but fail to become activated in the absence of additional costimulatory molecules. This process is imperfect, and autoreactive cells occasionally escape thymic selection, contributing to disease processes such as primary sclerosing cholangitis (see Chapters 10 and 41 ), autoimmune hepatitis (see Chapters 10 and 68 ), and type 1 diabetes. However, the bar for activation remains high and thus limits the risk of autoimmunity. For example, a single interaction of TCR and antigen-bearing MHC is inadequate to trigger T-cell activation; rather, approximately 8000 TCR-MHC interactions over the course of several hours are needed to initiate activation, which further limits the likelihood of autoimmunity. Costimulatory molecules greatly alter this need for redundancy, as discussed subsequently.

T cells are further distinguished by the accessory cell surface molecules CD8 and CD4 that influence the cell types that they are capable of interacting with and their roles in the immune response. ^, CD8 ⁺ T cells, known as cytotoxic T cells , bind to cells expressing class I MHC molecules. This binding interaction is facilitated by CD8 stabilization of TCR-MHC class I ligation. All parenchymal cells express class I MHC and display internal cellular peptides within the binding groove of this molecule. Cytotoxic T cells recognize diseased or infected parenchymal cells and activate cytolytic mechanisms. T-cell killing can occur through either calcium ion (Ca ²⁺ )-dependent secretory mechanisms or Ca ²⁺ -independent direct cell contact mechanisms.

CD4 ⁺ T cells, also known as helper T cells, interact with APCs expressing MHC class II including dendritic cells, macrophages, and activated endothelial cells. This interaction is mediated by CD4 stabilization of TCR-MHC class II ligation. MHC class II displays peptide fragments that have been phagocytized from the extracellular space. ^, Interestingly, resting sinusoidal endothelial cells of the liver are capable of presenting antigen to T cells, making the liver an organ with considerable ability to evoke or suppress an immune response. The interaction between CD4 ⁺ T cells and APCs influences subsequent activation of CD8 ⁺ T cells. ^, This process is mediated through upregulation of cell surface molecules on APCs known as costimulation receptors. Thus while APCs act as initiators of an immune response, they require CD4 ⁺ T cells to activate the primary effector arm of the acquired immune system mediated by CD8 ⁺ T cells.

An additional subset of T cells, regulatory T cells (Treg), further limit promiscuous immune responses. Treg cells have the ability to suppress cytokine secretion, adhesion molecule expression, and costimulatory signaling. The most extensively studied population of Treg cells express CD4 and CD25, the high-affinity α-chain of the interleukin (IL)-2 receptor. Animal models suggest that these cells play a critical role in controlling immune activation. ^, The prevailing evidence suggests that Treg cells are responsive to established inflammation, rather than serving a prophylactic role in preventing inflammation. However, harnessing the power of Treg cells to quell counteradaptive immune responses such as rejection is an ongoing area of research in autoimmunity and alloimmunity.

Humoral immunity

B cells recognize antigen in its native, unprocessed form. When antigen binds to two cell surface antibody receptors, the antibodies are brought together in a process known as cross-linking, stimulating B-cell proliferation and differentiation into an antibody-secreting plasma cell. The activation threshold for a resting B cell is relatively high, and similar to TCR recognition, costimulation can lower this threshold substantially. B cells also have the ability to internalize antigen bound to surface immunoglobulins and process them for presentation to T cells along with costimulation molecules.

Antibody structure is determined in the bone marrow through mechanisms similar to those that govern the generation of TCR diversity in the thymus. ^, Five different heavy-chain loci (μ, γ, α, ε, and δ) on chromosome 14 and two different light-chain loci (κ and λ) on chromosome 2, each with V, D, and/or J, and C regions, are brought together randomly by the RAG1 and RAG2 apparatus to form a functional antigen receptor. The basic antibody structure consists of two identical heavy chains and two identical light chains. The type of heavy chain dictates the Ig class: IgM, IgG, IgA, IgE, or IgD. The overall structure of the antibody results in two identical antigen-binding sites and a common region, the Fc portion. Bound antibody triggers activation of the complement cascade. In addition, most phagocytic cells have receptors for the Fc portion of IgG, allowing them to actively engulf antibody-coated cells.

Unlike the TCR, B-cell immunoglobulin loci undergo several forms of alteration after stimulation to improve the functionality of the secreted antibody. Isotype switching is the process of shifting from the initial heavy-chain IgM to one of four types to improve function and specialization of the secreted antibody. IgG is the most significant soluble mediator of opsonization and is the dominant antibody produced in response to alloantigen. IgA is important in mucosal immunity, IgE is involved in mast cell–mediated immunity, and IgD is primarily cell bound. After a B cell is activated, the specific D and J regions of the heavy- and light-chain genes undergo random alterations of the antigen-binding site. The resultant B-cell clones have altered antigen affinity, and this process is termed affinity maturation. Clones that have higher affinity for the target antigen have a selective survival advantage and form the basis for a more vigorous response on reexposure to the antigen.

Evolving cellular composition with age

A hallmark of immunity is the ability to adapt based on prior immune experience, such that initial immune responses are less robust than anamnestic responses. As humans age, immune experience grows, manifesting in an observed loss of naive T and B cells and a commensurate accumulation of cells expressing a memory phenotype. This transition is accentuated by physiologic thymic atrophy, which slows the production of naive T cells. In fact, the pool of available naive T cells is a function of thymic involution and the ongoing conversion to memory and senescent CD8 T cells. This transition with aging imparts a significant effect on the immune system that is particularly evident in elderly persons but measurable in most people by the fourth decade of life.

By using established markers of resting naive T cells and memory cell activation, four T-cell subpopulations have been described that appear to differ in terms of their degree of antigen experience, prior activation history, and migratory capabilities. These can be defined as naive T cells, effector memory T cells, central memory T cells, and terminally differentiated effector cells. During the last decade, numerous surface markers have been used to assess the myriad differentiation pathways seen after antigen exposure, and it is now clear that the lymphocyte repertoire is dynamic over time. Although it is beyond the scope of this chapter to define the specifics of this migration from naiveté to experience, it should be recognized that each individual’s immune repertoire is a product of both their inherited molecular makeup and their prior immune exposures. This is increasingly important because many of the molecules targeted in transplantation, particularly costimulatory molecules, are altered with immune experience such that optimal immune management is likely to require cognizance of these changes.

Mediators of context: Costimulation and cytokines

Isolated TCR binding with an MHC-peptide complex or antibody ligation with an antigen is not usually sufficient for naive lymphocyte activation. Receptor-ligand pairs on T and B cells and APCs, known as costimulation receptors, determine the character of the T-cell response ( Fig. 104.1 ). The type of costimulatory signal received by the lymphocyte determines whether the cell will become activated, remain quiescent, die, or become resistant to subsequent immune stimulation.

The biology of T-cell costimulation is substantially more developed than that of B-cell costimulation. Examples of T-cell costimulation receptors include CD28 and CTLA-4 (CD152). CD28 promotes T-cell activation and leads to the expression of CTLA-4, which then promotes downregulation of the T-cell response; thus activation typically begets deactivation. The B7 molecules CD80 and CD86, found on APCs, are the ligands for CD28 and CTLA-4. Although B7 molecules can bind to either receptor, their affinity for CTLA-4 is much greater; when B7 is in limited supply, the higher-affinity interaction with CTLA-4 predominates. Because B7 molecules are not expressed by normal tissues, CD8 ⁺ T-cell interaction with class I self MHC does not elicit a proliferative response; instead it reinforces quiescence of autoreactive T-cell clones.

Although the mechanisms of costimulation have not been completely elucidated, it is known that binding of CD28 allows more efficient T-cell signal transduction. Through CD28-B7 interactions, the number of binding events required to trigger activation of a T cell decrease from 8000 to 1500. ^, In contrast, when CTLA-4 binds B7, the T cell becomes incapable of producing IL-2 during the encounter and even in subsequent interactions. The CD19-CD21 complex provides comparable control of antigen receptor binding for B cells.

Additionally, costimulation is mediated through another pair of receptors: CD40, found on dendritic cells, endothelium, B cells and other APCs, and CD154 on T cells and platelets. The ability of APCs to stimulate a cytotoxic T-cell response is greatly augmented by the effects of CD40 binding. After CD40 ligation, activating cytokines are released and B7 molecules are upregulated. ^, CD154 is upregulated after TCR ligation and provides positive feedback to the APC. In addition, CD154 is found in and released by activated platelets at sites of endothelial injury. Therefore sites of trauma that recruit platelets create an environment of activating costimulatory molecules, thereby bridging the innate and acquired immune systems.

Direct cell-to-cell contact is not the only means by which immune cells communicate. Soluble mediators of communication known as cytokines, or interleukins, are polypeptides that are released from many cells; they can either activate or suppress adjacent cells, and the pattern of cytokine expression is thought to influence the resultant type of T-cell response. ^, Once activated, T cells have been described by one of several cytokine-secretion phenotypes. T cells that promote cytotoxic responses are characterized by expression of IL-2, IL-12, IL-15, and interferon (IFN)-γ and are called helper T type 1 (Th1) cells; T cells that promote humoral or eosinophilic responses are characterized by secretion of IL-4, IL-5, IL-10, and IL-13 and are called helper T type 2 (Th2) cells. More recently, additional cytokine lineages have emerged; the most relevant to this chapter is the Th17 lineage, involved in gut immunity and driven by IL-6, IL-23, and transforming growth factor (TGF)-β.

In addition to cytokines, other soluble mediators of inflammation are capable of promoting increased blood flow and improved exposure of an area of injury to innate and acquired immune elements. Some suggest that the APCs of the liver are more efficient in generating Th2 responses, which may be a mechanism by which liver allografts avoid late cellular rejection.

Clinical rejection syndromes