Chapter 63. Immunobiology of Heart and Heart-Lung Transplantation

This chapter on the immunology of transplantation is a refreshed effort designed to promote a foundation for understanding the basic transplant immunologic fundamentals necessary for competency in caring for heart and lung transplant recipients. Although surgeons must acquire the technical expertise to perform these often demanding surgeries safely, the recipients' well-being additionally benefits from a surgical team well versed in basic transplant immunology. Comfort in treating patients who receive immunosuppressive therapies, both conventional and innovative, requires familiarity with the nonsurgical language of transplantation. The goal of this chapter is to squeeze the essentials1 into an understandable short text. The knowledge gained should permit surgeons to read immunology-tilted manuscripts with better understanding and thus make better decisions in treating their own patients. The core features of this alloresponse and new science are presented with some specific references to hearts and lungs so that the surgeon participating in the care of thoracic organ recipients can more easily build an understanding of the complex and evolving science. The material is organized to reflect a unifying theory of the immune response that determines the fate of an allograft and most often the heart or lung recipient as well. This includes: (1) histocompatibility; (2) activation of alloresponse T lymphocytes and T-cell–mediated rejection (TMR); (3) antibody-mediated rejection (AMR); (4) the underappreciated immune pathways, natural killer (NK) cells, and memory lymphocytes; and (5) immune plus gene monitoring. The text will clarify acute, cell-mediated, hyperacute, and chronic rejection pathways (Fig. 63-1).

Major histocompatibility complex (MHC) molecules are a family of proteins that vary quite a lot between individuals (genetic polymorphism) and represent the molecular basis for how immune systems distinguish self from nonself with respect to infections and transplants. MHC molecules on donor cells or MHC fragments, shed from thoracic organ transplants, are determined to be foreign by the immune regulatory system of the host. Intact MHC molecules expressed on the surface of cells serve two key functions in the context of transplantation. Fragments of foreign proteins, including fragments of MHC molecules, are presented in the binding groove of MHC and are recognized by T-cell receptors of the recipient that happen to have high affinity for that protein fragment (indirect donor antigen presentation). In addition, recipient T cells directly recognize donor MHC as "foreign" (direct donor antigen presentation).

Human MHC molecules are known as human leukocyte antigens (HLA) because they are expressed at high levels on leukocytes and were first measured on peripheral blood lymphocytes. HLA are heterodimeric glycoproteins expressed on the surface of almost every cell in the human body. These proteins are immunologically active and can trigger a proliferative or cytotoxic T-cell response in vivo and in vitro. For these reasons, they are thought to play a critical role in graft rejection in solid-organ transplantation.

The genes coding for these antigens are located on the short arm of chromosome 6 (6p21.3). This region spans over 4 million base pairs in length and encodes for over 200 genes involved with host immune surveillance and immune regulation.

The HLA is divided into three regions or Classes: Class I, II, and III (Fig. 63-2).

Classic Class I HLA proteins/antigens are HLA-A, HLA-B, and, more recently, HLA-C. These proteins consist of an α-heavy chain and a β-light chain (Fig. 63-3). The α-light chain is encoded in the MHC; however, the β-chain is β2-microglobulin that is encoded on chromosome 15. When folded, the α-heavy chain only contains the peptide-binding region which presents peptide antigen to the T cell. Class I antigens are constitutively expressed on nearly all cells. B-cell lymphocytes may express Class I antigen at a higher density than the T-cell lymphocytes. Class I antigen expression is upregulated on the endothelium and parenchymal cells in many organs and tissues in association with inflammation, including after ischemia and reperfusion.

Classic Class II HLA proteins/antigens are HLA-DR, HLA-DRw, HLA-DQ, and more recently HLA-DP. These proteins consist of an α-heavy chain and a β-light chain, both of which are encoded in the MHC. When folded, the α-heavy chain and β-light chain come together with each contributing part of the peptide-binding region (Fig. 63-3). Class II antigens are expressed mainly on B-cell lymphocytes, activated T cells, and dendritic cells. Other cell types such as endothelial cells, may express Class II antigen expression after activation.

Each person has two different HLA genes at each HLA locus (HLA-A, B, D, etc). HLA antigens are codominantly expressed on the cell surface so that each cell has two different HLA proteins for each HLA locus. HLA haplotypes (the sequence of A, B, D genes on one chromosome, see Fig. 63-2) are usually inherited as a group from each parent in a Mendelian fashion. Thus, two offspring of the same parents have a 50% chance of inheriting one identical haplotype, 25% chance of inheriting two identical haplotypes, and 25% chance of having no haplotypes in common. In addition, because of the distance between these genes in the MHC, there are hot spots of gene recombination. Recombination in offspring occurs approximately 1 to 2% between HLA-A and C, or between HLA-B and HLA-DR, and approximately 30% between HLA-DP and HLA-DQ. This recombination accounts for most of the occasional exceptions to faithful transmission of intact parental haplotypes to their children. HLA haplotypes are found in varying frequencies across the major population groups (white, Hispanic, black, Asian, and American Indian).

Hyperacute Rejection

In the early days of organ transplantation, hyperacute rejection occurred because of preexisting IgM alloantibodies. Best known examples of these are the ABO blood group IgM antibodies. Although still a barrier to xenotransplantation, blood group typing has virtually eliminated this particular cause of immediate organ destruction. Of interest is the success of blood group-mismatched organs in neonatal recipients who have not yet developed IgM antibodies to blood group antigens. Antibodies against blood group antigens arise between 6 and 18 months postnatal because of the presence of related carbohydrate antigens on intestinal bacteria. Today, when hyperacute rejection is seen, it is normally caused by preexisting IgG antibodies usually directed against donor HLA proteins (see Fig. 63-1). These antibodies are often the result of previous blood cell transfusions (most particularly multidonor platelets owing to the high HLA load) or previous pregnancies or transplants. After wide adoption of the first crossmatch technique by Patel and Terasaki in 1969, hyperacute rejection based on HLA antibodies became rare.2 Further improvements in screening for HLA antibodies have nearly eliminated it. New flow cytometric crossmatch techniques occasionally reveal previously unsuspected anti-HLA antibodies (see Flow Cytometric Assays) at titers below the threshold detected by the crossmatch test; the importance of these low-titer antibodies is uncertain, but at least in some circumstances they are associated with an accelerated failure of the organ transplant.

Success in heart and lung transplantation requires control over the adaptive immune response to donor MHC antigens expressed by the allograft. Rejection of allografts is initiated by T lymphocytes primed to donor antigens in the peripheral lymphoid tissues and recruited into the donor organ where those antigens are expressed. Much of the recent advance in clinical transplantation has been because of a better understanding of T-cell–mediated responses. Transplant immunologists are closing in on the critical steps that contribute to different effector mechanisms of tissue injury. The nature of allograft rejection is influenced by alloantigen specificity, frequencies (measured as absolute number or proportion), and cytokine profiles of naïve and "memory" donor-specific T cells. Currently, it is held that alloreactive T lymphocytes are primed by alloantigens presented to them on donor (direct) and/or host (indirect) antigen-presenting cells (APCs). The T cells are the primary actors in the pathologic reaction to the transplanted organ. They participate in cytotoxicity and cytokine-mediated inflammation. B cells, antibodies, and macrophages contribute to destruction of the graft via a variety of effector pathways. Ischemia/reperfusion injury of the allograft triggers innate immunity that can amplify adaptive immunity. Molecular modulators of innate immunity have recently been an expanding area of interest. These include toll-like receptors (TLR), cytokines, chemokines and complement. Although APCs are emphasized, B cells and natural killer (NK) cells exert influence on a host's decision to reject an allograft.3

T-Lymphocyte Alloactivation

A T cell can only become triggered to proliferate and differentiate if it links to a specialized cell that presents fragments of donor peptides bound to HLA molecules on its surface (Fig. 63-4). These HLA-peptide displaying cells are called antigen-presenting cells (APCs). "Professional" APCs differ from other HLA-expressing cells in that they also express costimulatory molecules that efficiently stimulate reciprocal activation of both the APCs and the responding T cell. Professional APCs include dendritic cells, B lymphocytes, and macrophages. The peptide HLA antigen complex may be presented by resident donor passenger APCs found in the transplanted heart or lung (direct presentation) or host APCs that endocytose HLA protein shed from the allograft (indirect presentation) (Fig. 63-5). HLA-peptide antigens become the specific key that fits into the T-cell receptor. T cells react against alloantigens because of the cross-reactivity of the T-cell receptor (TCR) with self and foreign HLA molecules. Host and passenger donor dendritic cells are thought to be the most efficient type of APCs for activating naïve T cells.

Donor Antigen Presentation to T Cells by APCs

Donor passenger APCs transplanted with the heart or lung directly process endogenous HLA-derived peptides from within the graft or themselves and present them associated with a groove on Class I HLA complexes on their surfaces (Fig. 63-6A). CD8+ T lymphocytes have Class I HLA T-cell receptor (TCR) molecules on their surfaces into which the Class I protein MHC complexes presented on the donor-derived APCs fit (lock and keys). This pathway is responsible for the majority of initial T cells that react to alloantigens. It is known as the direct pathway of peptide presentation and is responsible for most early events of acute cell-mediated cytotoxicity.

Later alloresponses are directed more by CD4+ T cells because they recognize Class II HLA donor-specific peptide-HLA surface molecules presented on recipient APCs. These recipient APCs gradually replace those donor APCs transplanted with the heart or lungs of the donor. Host APCs trafficking through the allograft phagocytose interstitial and nodal HLA protein antigen shed from the donor graft (Fig. 63-6B). This shedding is induced when the graft is stressed by ischemia (preservation), inflammation (bacterial and viral infection), and rejection. The donor protein undergoes endosmic proteolysis and is combined with a Class II HLA molecule. The donor peptide Class II HLA complex is transported to the surface of the host APC and presented indirectly as the key to the TCR complex on CD4+ T cells. Because CD4+ T cells provide help to donor-specific B cells, later alloresponses tend to be associated with appearance of alloantibody.

Activated dendritic cells also provide costimulators to the naïve T cells that are required for a full T-cell response. Macrophages present antigens to differentiated CD4+ cells that activate the macrophage to promote cell-mediated immunity. B cells also serve an APC function by presenting antigens to helper T cells. These then activate the B cell to become part of the effector humoral immune response through production of antibodies (Fig. 63-7). Recently it has been learned that recipient APCs can acquire intact HLA molecules directly from contact with a donor APC bearing the HLA molecule. It can also receive the HLA molecules by fusing with exosomes from donor APCs containing the protein. These APCs can stimulate CD4+ T cells by direct or indirect pathways.4

APC-Induced T-Cell Activation: Signal 1

T lymphocytes are able to respond to the peptide HLA complexes expressed on APCs because they express membrane receptors that: (1) keep the cells together via the APC-adherence ligands; (2) include the TCR that specifically recognize (cognate) the peptide–HLA complexes displayed in the APC; and (3) costimulatory receptor molecules accessory to the TCR that either facilitate (CD28) or inhibit (cytotoxic T lymphocyte-associated antigen 4 [CTLA-4]) the transduction of activation signals to the nucleus or provide second signals that strengthen the TCR activation. The TCR complex includes variable antigen-binding TCR heterodimer and invariant CD3, ξ, ε, and γ proteins, and ξ chains that enable activation signal transduction to the nucleus (Fig. 63-8).

CD8 and CD4 surface proteins act as coreceptors that bind to the Class I or II HLA molecule, respectively, displayed on the APC. As discussed, the CD8 and CD4 interact with Class I and II HLA, respectively, when a donor peptide–HLA complex is recognized. CD8 and CD4 strengthen the binding of the TCR to the HLA molecule and participate in the intracellular signaling process.

Costimulation Pathways: Signal 2

In addition to activation of the T cell by engagement of foreign peptides and its TCR, several other surface molecules participate in a T-cell activation. Those that enhance TCR signaling (CD4 in CD4+ T cell and CD8 in CD8+ T cell) are called coreceptors because they bind to part of the same HLA molecules that engage the TCR. Another group of T-cell activating surface molecules is called costimulatory receptors (Fig. 63-9). Blockade of costimulatory pathways provides important new possible therapy to prolong organ transplant survival and even reach a tolerant state. Costimulatory receptors or ligands on T cells recognize respective cognitive ligands or receptors presented on APCs or the graft tissue itself. The best defined costimulatory receptor and ligand pairs are the constitutively expressed CD28 T-cell costimulatory receptor and its APC ligands B7-1 (CD80) and B7-2 (CD86). This pathway is particularly important in activation of naïve T cells. When engaged, these pairs deliver antiapoptotic signals and trigger the expression of and growth factors, including IL-2 that encourage proliferation of the alloantigen-specific CD4+ or CD8+ cells. A second inducible costimulatory receptor for B7 molecules has been identified and termed CTLA-4 (CD152). Unlike the structurally homologous CD28, CTLA-4 appears on recently activated T cells and is a negative regulator that promotes pathways that limit proliferation. It is efficient that the same costimulator (B7) can prompt initial proliferative signals via constitutive CD28 engagement and later when CTLA-4 is induced subsequently limiting signals to the T cell. Therapeutic targeting of this pathway with CD80 and CD86 blockade with CTLA-4 Ig–like molecules has been effective in primates and is currently in clinical trials in kidney transplantation.5 Inducible costimulator (ICOS) and Programmed Death-1 (PD-1) are more recent discoveries in the T-cell CD28 family. When ICOS joins its ligand ICOS-L on an activated APC, it stimulates the T-cell effector response by promoting IL4 and IL10 production. PD-1 is the coreceptor for costimulator PD-ligand and, like CTLA-4, represses T-cell responses.

A second family of costimulatory molecules belong to the TNF superfamily. The prototypic pathway is represented by the costimulatory receptor CD40 on APC binding to CD40L on T cells. Transplantation immunobiology is influenced by the innate immunity directors against substances from microbes, including bacteria, viruses, and fungi. These substances, often nucleic acids, are called pathogen-associated molecular patterns (PAMPs) (see Fig. 63-9). PAMPs can bind to PAMP-receptors on APCs called toll-like receptors (TLR). This engagement has the effect of strengthening the APC response. This begins by increasing the expression of costimulatory molecules and proinflammatory cytokines. The CD40 signaling promotes additional B7 expression and elaboration of T-cell proliferation IC12. By reciprocal activation, CD40L on T cells makes the APC better and is thought to license additional APCs to participate in the T-cell activation process. A clinical trial of anti-CD154 was unexpectedly associated with thromboembolic complications. This led to consideration of blocking CD40, and a human trial is currently underway in transplant.

In severe rejection, B cells join the alloresponse when they receive help from CD4+ T cells (see Fig. 63-7). CD4+ T-cells are activated by APCs to express CD40L (CD154). Ligand B cells expressing CD40 and MHC Class II receptors engage helper CD4+ T cells and proliferate. B cells participate in the rejection of allografts by efficiently providing APC function to helper CD4+ T cells and by elaboration of antibodies that: (1) recognize and destroy the donor endothelial targets; (2) participate in antibody dependent cytotoxicity (ADCC); or (3) stimulate the proliferation and migration of activated endothelial cells.6 In the former, the endothelial antigen–antibody complexes activate the classic complement pathway, causing C3 and C5 to cleave, inciting inflammation (C5a) and formation of the membrane attack complex (MAC), which is composed of C5b complexed with C6, 7, 8, and 9 (C5b-9). The MAC causes vascular cell death by inducing pores to form in the cell membrane. When endothelial cells are activated or killed by complement, this in turn results in microvascular thrombosis and inflammation. ADCC describes the process by which antidonor IgG antibodies coating donor cells bind to the Fcγ RIII receptor of natural killer lymphocytes (NK cell). Cells of the innate immune system such as natural killer cells are also present in allografts during rejection. These recognize alloantigens because they constitutively express inhibiting receptors specific for self HLA Class I protein. The NK cell produces proinflammatory cytokines like IFN-γ and directly kills the antibody-engaged target cell by injecting its proteolytic enzymes into the donor cell.

Cell-Mediated Rejection

Despite use of immunosuppressive therapy, transplanted heart and lungs often acutely reject. Both CD4+ and CD8+ cells participate, but the response is primarily mediated by the CD4 T cells. The early cell-mediated response begins when specific clones of CD8+ cells that bear TCR molecules which fit and engage donor-specific peptide HLA Class I on donor APCs are stimulated to proliferate. In the presence of help from CD4+ T cells, they become cytolytic lymphocytes CTLs and directly attach to and kill graft endothelial and parenchymal cells bearing the identical Class I donor protein HLA presented to them by the APC activators. This is known as the direct pathway because the CD8 cell directly kills the allotarget. It is responsible for most acute cell-mediated rejection early (in the first few months) after transplant. In the presence of calcium, the protein perforin polymerizes onto the Class I bearing target cell and causes 16- to 20-nm pores to open in the cell membrane, resulting in osmotic collapse. CTL granular content, including granzyme B and other cytotoxic molecules, is directly injected into the target cell, causing necrosis. Alternatively, apoptosis (programmed cell death) can also be stimulated in the target cell by interaction of the lymphocyte Fas ligand with the APO-1/Fas receptor of the target cell. Second messengers are elicited that activate endonucleases and proteases to cause fragmentation of DNA and thus organize the dissolution of the Class I bearing donor target T cell (Fig. 63-10).

The CD4+ T cells primarily follow an indirect alloresponsive pathway by secreting various cytokines that drive inflammation and graft loss (see Fig. 63-10). IL-2 increases the expression of its own receptors (IL-2R) on CD4+ cells, driving the proliferation and further differentiation of CD4+ cells. The activated CD4 cells secrete additional lymphokines, including interferon-γ (IFN-γ), which along with IL-2 stimulates CD8+ CTLs to bind to the allograft cells presenting donor MHC protein molecules. CD4+ T cells are very heterogeneous. The four major categories are now recognized: Th1, Th2, Th17, and regulatory T cells (Tregs). Th1 cells are the main mediators of cell-mediated rejection. They produce IFN-γ and favor IL-12 production by macrophages. Th2 cells make IL-4, 5, and 13 and promote humoral responses. Th17 produce IL-17, a cytokine recently discovered and involved in inflammation. In conditions in which the immune response is partially inhibited, Tregs develop with the ability to inhibit immune responses.

Memory T Cells

With improvement in immunosuppression that limits activation of naïve T cells, donor-reactive memory cells have been recognized as a previously underappreciated risk to the allograft. This group of donor-specific cells has an effector-memory phenotype. This is defined by an immediate and strong recall that is no more or less sensitive to costimulation blockade. Memory cells are thought to arise from heterologous immunity. This occurs when there is a resemblance between microbial antigens and self-protein HLA complex antigens. Some CD4+ and CD8+ cells exposed to Epstein-Barr, herpes simplex, and cytomegalovirus (CMV) become primed to recognize allogenic HLA molecules. Memory T-cell numbers are proportionately increased following lymphoablative treatment with rabbit ATG or alemtuzumab (anti-CD52 ab). It is unclear whether these memory cells are more resistant to depletion or whether they represent a conversion from naïve T cells during repopulation. Memory CD4+ cells provide help for the alloresponse. They provide growth and proinflammatory factors that affect CD8+ T cells and B-cell antibody production. They can elicit the proinflammatory response from nodal-bearing tissue remote from the allograft. Memory CD4+ cells recruit CD8+ cells that infiltrate the allograft across the endothelium. The memory CD8+ cells proliferate and recruit macrophages, neutrophils, and additional activated T effector cells into the donor organ.7 To date, memory T cells continue to evade efforts of therapeutic targeting. LFA-1 is one target that is under study for this purpose. They are very heterogeneous and have multiple functions.

Immunologic Tolerance

Peripheral tolerance mechanisms are responsible for tolerance to transplant-specific HLA protein not present in the thymus. Immunomechanisms to achieve immunologic tolerance of an allograft include considerations of cell death by apoptosis (deletion) of alloreactive T cells or induction of functional unresponsiveness (anergy or ignorance) and active regulation of alloimmunity by either donor-antigen–specific or nondonor-antigen–specific mechanisms (regulation). Anergy has been induced by exposing CD4+ T cells to MHC antigen in the absence of costimulation. In addition, much of the current interest has been in better understanding of a subset of CD4+ T cells called regulatory T cells (Tregs) whose function is to suppress immune responses and maintain self-tolerance.8 Tregs may occur naturally (nTreg) and are important in autoimmunity. Those that are inducible (iTregs) in response to an allograft are distinct. Most Tregs express the IL-2 receptor α chain (CD25). TGF-β, IL2, and B7, CTLA-4 costimulation are required for nTreg production and survival. It remains to be fully elucidated how these molecules direct naïve T cells to differentiate into effector, memory, or regulatory phenotypes under various clinically relevant circumstances. FoxP3 is a forkhead family transcription factor important for Treg-suppressive function, but is also found in activated T cells in humans, and thus is not specific to identify human Tregs.

There has been limited success in translation of laboratory rodent-based tolerance protocols in clinical practice. Freedom from immunosuppression rarely is accomplished universally in strictly controlled large animal models. Management of preexisting memory cells appears to be a major hurdle. Finally, redundant effector cell mechanisms, cytokines, and costimulatory pathways make single or even dual approaches seem underpowered to establish tolerance in the clinic. There has been a limited number of patients in whom tolerance was induced by a regimen, inducing a mixed allogeneic chimerism (coexistence of recipient and donor cells) for kidney allografts. However, work in the nonhuman primate model showed that even this highly tolerogenic regimen did not induce tolerance to cardiac allografts.9 Heart and lung allografts are less easily amendable to tolerance than kidney; in other words, they are more immunogenic.10

Chronic rejection of thoracic organs takes the form of induced obliterative fibrosis of small airways in the lung (obliterative bronchiolitis [OB]) or bronchiolitis obliterans syndrome (BOS) and diffuse obliterative transplant vasculopathy (TV) in heart (also termed cardiac allograft vasculopathy [CAV]). OB and CAV are likely caused by a response of the vessels and airways to a combination of peritransplant injury from preservation-related ischemia, acute rejection, and a chronic delayed-type hypersensitivity immune reaction (DTH) (see Fig. 63-1). DTH response is driven primarily by CD4+ T cells reacting to Class II alloantigens on the endothelium and smooth muscle on the coronary arteries or the epithelium and bronchial smooth muscle of the donor airway. The responding CD4+ T cells elaborate tumor necrosis factor (TNF) and interferon-γ (IFN-γ), which promotes release of growth factors and chemokines from the endothelial, epithelial, and smooth muscle cells and recruit regional macrophages. Smooth muscle cells in the vessel and airway walls proliferate and move into the intima (subendothelial) or subepithelial compartment. The result is proliferation followed by fibrosis with resultant progressive luminal occlusion. As this process evolves, the transplanted organ parenchyma becomes progressively more fibrotic. CAV has been associated with systemic soluble markers of general inflammation. These include C-reactive protein, a marker of systemic inflammation, vascular cell adhesion molecule (VCAM-1), a marker of endothelial cell activation and neopterin, a marker of macrophage activation.11 Further, these inflammatory mediators correlated with necrotic core and dense calcified components of the plaque. The necrotic core component is composed of lipid cells, necrotic and lymphocyte remnants together with tissue microcalcification.12 At 5 years posttransplantation, CAV was responsible for death or retransplantation in 7%, and 50% patients with severe disease experienced these outcomes.12 Its course more often is an indolent one, yet if present before 2 years, it acts like an inflammatory vasculitis and is associated with a poor survival.13 The International Society for Heart and Lung Transplantation has recently published a consensus on the diagnosis and nomenclature for CAV.14

Circulating HLA antibodies may form in a sensitization process through prior exposures to blood transfusions, pregnancy, organ transplantation, or use of a ventricular assist device. Transplant candidates with preexisting HLA antibodies not only are at risk of hyperacute and acute antibody-mediated rejection very early after transplantation, but also have lower rates of longer-term survival as well. Sensitive new bead methods now allow detection of circulatory antibodies and use of those antibody specificities to define "unacceptable" donor HLA antigens. The role of non-HLA antibodies in acute and chronic rejection and treatment strategies before transplant (desensitization), or when antibodies are detected after transplantation, is becoming better understood but remains in evolution.

HLA Antibody Analysis

Serologic Testing

The techniques used for identification of HLA antibodies have evolved significantly in recent years. The serological lymphocytotoxic assay for antibody screening is based on mixing patient serum (unknown) with a panel of cells whose HLA typing is known and adding complement (complement-dependent cytotoxicity or CDC assay). If antibodies are present in the patient serum that react with the cell's HLA molecules, cell death occurs because of complement activation. This method is limited to detection of antibodies represented by the panel of antigens tested and by issues with sensitivity and accuracy. The specificity of the antigen to which the antibody is reacting (HLA or non-HLA antibody) is sometimes not clear because of the presence of other proteins in the patient serum, or the antibody isotype (IgG or IgM, etc; some antibody isotypes do not fix complement), and the titer (low, high) is not determined. Several enhancements of this assay were developed to address these issues: (1) heat or chemical treatment of the patient serum to inactivate IgM antibodies and identify IgG antibodies, which were felt to be more important to outcomes; (2) additional washes of the target cells following incubation of the patients serum to "wash off" the nonspecific reactants; and (3) addition of antihuman globulin (AHG) to the reaction to increase the detection of low titer IgG antibody (CDC-AHG method). There are other subvariations of these serologic enhancements.

The antibody-screening assay consists of a panel of known HLA-typed cells, with each cell representing a unique set of antigen targets for the serum to react. The assay generates the panel reactive antibody titer (PRA), which is reported out as a percent of the prospective donor pool that would likely be killed by the patient's serum. The result of this analysis is termed percent calculated PRA or %cPRA. The %cPRA gives a better indication of the patient's likelihood of having a compatible offer of a UNOS deceased donor organ because it is based on actual UNOS-typed donors. The panel may consist of any number of cells; however, minimally, cells from at least 30 carefully selected individuals are needed to cover the most common HLA antigen targets. In addition to the %PRA, the specificity of the antigen targets could be identified based on the individual cell reactions. The more different HLA antigens to which the patient is sensitized, the higher the %PRA and the less likely the patient is to have a compatible donor identified. Using conventional matching criteria, %PRA greater than 10% and greater than 25% have been associated with incrementally lower survival large registry reports.

Despite the use of enhancing techniques, serological antibody screening and methods of identification are not able reliably to detect low levels of HLA antibody, and are relatively poor for characterizing Class II antibody.

Solid-Phase Assay

An ELISA plate method was the first solid-phase assay developed. In this assay, solubilized known HLA antigen protein targets are captured onto the surface of the wells of a microtiter plate. Patient serum is reacted with these proteins in each respective well. A secondary antihuman IgG antibody conjugated to a colorimetric reporter molecule is then added to the wells. This allows quantitative spectrophotometric determinations with a threshold absorbance used to distinguish a positive versus negative antigen–antibody reaction. Two additional hallmarks of the ELISA assay are the ability to: (1) objectively evaluate and quantify HLA antibody reactivity; and (2) eliminate the need for dependence on complement fixing for antibody detection, making ELISA a complement-independent assay. The colorimetric ELISA assay was able to evaluate better the presence of HLA Classes I and II antibody than the CDC-AHG method. Key faults of the ELISA assay are: (1) sensitivity of the colorimetric detection methods; and (2) purity and reliability of the solubilized HLA antigen captured in the well, because HLA can change its shape and thus antibody reactivity when bound to the plate.

Flow Cytometric Assays

The development of solid-phase microbead-based flow cytometry assays represents a monumental improvement from the initial solid-phase ELISA antibody screening and identification techniques. The use of fluorescent dyes is foremost as report molecules, allowing detection of the antigen–antibody reactions using a flow cytometer. Fluorescent dye light emissions are severalfold more sensitive than colorimetric dyes. As with the ELISA assay, the detection of the antibody–antigen reaction is made using beads coated with HLA molecules. Each HLA molecule-specific bead is identified by its unique mixture of two fluorescent dye colors incorporated into the bead. A secondary antihuman antibody is conjugated to a reporter molecule, in this case, a fluorescent dye of another color. The higher the titer of the antibody in the patient's serum, the more antibody is available to react to the antigen conjugated to the bead, and the more intense is binding of the secondary flouresceinated antibody. The more reporter dye becomes bound to the bead complex, the more fluorescent emission is produced from the bead when analyzed by the flow cytometer. The fluorescent emission signal of each bead type is then averaged and the normalized value is reported as the mean fluorescence intensity (MFI) of the bead. For most laboratories, the MFI value greater than or equal to 1000 is considered to be a positive reaction for the presence of the HLA antibody. This cutoff was derived in a similar fashion to that for the ELISA assay, namely, twice the MFI value for the negative control serum.

This resultant value is called the shift median channel value (sMCV). The sMCV cutoff for a positive versus negative flow crossmatch is statistically determined by each HLA laboratory initially and periodically by crossmatch of up to 100 individuals with serum that does not contain HLA antibody (negative). Shift values of less than or equal to two standard deviations from the mean of these histograms median channel value is considered to be negative, between two and three standard deviations to be equivocal, and three or more standard deviations to be positive.

The HLA antigen is conjugated directly to the microbead, not simply captured, and the source of which is recombinant cell lines. This allowed manufacturing of beads coated not only with the antigens of a single individual mimicking a cell (multi-antigen beads), but also with a single HLA antigen (single antigen beads). These single antigens can be further described to the exact HLA allele of that antigen. Because individuals become sensitized to the amino acid epitopes encoded by the antigen allele, microbead-based antibody analysis opened a completely new level of insight into characterizing antibodies contained in patient serum. This process is called epitope mapping. In combination with allele level typing of patients and potential donors, epitope mapping of antibodies can better predict outcomes of transplants in highly sensitized patients. The flow cytometer instrument may be either a larger instrument, which can acquire data from cells or beads, or a mini-flow cytometer, which only acquires data from beads, such as a Luminexx instrument (Fig. 63-11).

Using solid-phase HLA assays (SPA) or flow cytometric assays, the laboratory can easily and reliably test for patient antibodies to HLA-A, -B, C, DRB1, DRB3, DRB4, DRB5, DQB1, DQA1, and DPB1.

The results of the serum antibody test and the donor crossmatch are interpreted together as a final assessment of recipient and donor compatibility. Composite MFI values of 4000 or greater are generally predictive of a positive flow crossmatch. This composite MFI value is derived from the sum of MFI values and the single antigen–antibody analysis for each donor target antigen present, either Class I or II. This is a rule of thumb, as cellular expression of these target antigens varies. Most centers post unacceptable antigens in UNOS for those single antigens with MFI values of 4000 or greater, because these single antigens alone may result in a positive flow crossmatch with donors. Based on these MFI values, a virtual or paper crossmatch can be reliable in predicting the actual cellular crossmatch, especially with high MFI values. In addition, unacceptable antigens can be identified by virtual crossmatch (discussed in the following), excluding a particular donor that expresses certain donor antigens from consideration for a recipient who is sensitized against one or more of those antigens.

HLA Crossmatch

Crossmatch of patient serum with potential donors is no different than performing antibody testing.

Virtual Crossmatch

Practical realities of matching sensitized candidates with nonlocal donors resulted in the acceptance of the virtual crossmatch (VXM). The VXM is a comparison of the donor HLA genotype determined by organ procurement organizations routinely as part of the donor (heart, lung) organ distribution process to the gene families represented by beads that bound antibody from the sensitized candidate recipient. If gene families are shared between donor and the bead, the VXM is positive. For example, a candidate with antibodies against A1, A11, and B7 by Luminexx single antigen beads would be incompatible with a donor typed as A11, A25, B55, and B57.15 The anti-A1 antibody determined by SPA corresponds to the donor typed A1. Although currently there is no method to determine the functional characteristics of the antibodies, recently it was estimated that the positive predictive value of an incompatible VXM compared with cytotoxic crossmatch was nearly 80%. Most centers refuse nonlocal donors based on an incompatible VXM and insist on a prospective CDC-AHG crossmatch for sensitized patients when possible.

Non-HLA Antibodies

It has become increasingly clear that non-HLA antibodies that can cause injury in thoracic organ transplants.16,17 About 16% of HLA antibody-negative heart recipients may lose their graft to the mixed diagnosis of primary failure within 30 days of transplant.18 SPA does not detect non-HLA antibodies, but flow cytometry methods can detect MICA/B. Antibodies-to-donor endothelial antigens are the largest poorly discussed group of clinically important non-HLA antibodies. These include endothelial, autoantibodies and those to MHC Class I chain A (MICA) and B (MICB). Endothelial antigen targets may exist, constitutively or as induced autoantigens because of activation of the endothelium. MICA and MICB are polymorphic antigens expressed on epithelial and, to an unknown extent, on endothelium. MICA antibodies may occur in up to 20% of candidates and have been associated with poorer survival but not increased rejection.18

Autoantibodies against conserved (non-polymorphic) proteins are frequently found in the blood of thoracic transplant recipients in association with chronic rejection. Vimentin (an intracellular cytoskeletal protein found in vessel walls and activated lymphocytes), cardiac proteins (cardiac myosin), and collagen type 5 (expressed mainly in the lung) are among the antigens against which autoantibodies have been described. Antivimentin antibodies form earlier than anti-HLA after transplantation and are a response in up to 30% of heart recipients to exposure of antigens mounted on the surface of damaged and activated cells.19 Antivimentin antibodies reflect tissue injury but might also activate platelets and neutrophils.20 Antiheart antibodies exist preoperatively in some because of their primary cardiac disease. At present, it is difficult to know their true significance. Finally, IgM non-HLA antibodies are cytotoxic and can react to all leukocytes, even the patient's own. Antigen specificity is unknown, as is the clinical relevance.

Desensitization Therapy

Once unacceptable antigens are determined from SPA, they can be entered into the UNOS website (http://optn.transplant.hrsa.gov/resources/professionalResources.asp?index=10). This will provide the cPRA. If the percentage chance of any donor will not be acceptable is greater than 50%, then it is reasonable to initiate a desensitization protocol. An optimal protocol has not been established. High-dose IVIG (2 g/kg/over 2 days q 2-4 weeks), plasmapheresis (1.5 volume exchange ×5 days), monoclonal anti-CD20 B-cell therapy (rituximab 1 g IV weekly ×4), and in the past cyclophosphamide (1 mg/kg/day) have been used in various combinations.

At UCLA, plasmapheresis, IVIG, and rituximab reduced circulatory antibody levels from 70.5 to 30.2%. Heart transplantation of these candidates after negative CDC resulted in similar 5-year outcomes to control patients and untreated but high PRA patients (81.1%, 75.7%, 71.4%). Of interest, the freedom from CAV was 74.3, 72.7, and 76.2%, respectively.21

It has been agreed that patients waiting for transplantation with circulating HLA antibodies should be studied every 3 months, and those desensitized every 2 weeks after therapy. Patients on VADs or who receive blood transfusions or with infections should be closely monitored as well. After transplantation donor-specific antibody monitoring is recommended at regular intervals and when a humoral rejection event is suspected. Donor-specific titers should be measured daily for 1 to 2 weeks and frequently thereafter in desensitized patients and those considered high risk for antibody response. This higher-risk group of recipients is generally treated with thymoglobulin plus IVIG, plasmapheresis and/or rituximab. Maintenance immunosuppressive therapy that has best controlled cellular and antibody-mediated rejection includes tacrolimus, mycophenolate mofetil, and prednisone.22

Antibody-Mediated Rejection

Hyperacute antibody-mediated rejection (AMR) became extremely rare with improving crossmatch techniques, but clinical interest in AMR was renewed in 1990 by Halloran and associates who described features of pure AMR in a few renal transplant recipients.23 Later, C4d became established as an important histologic associate.24 The incidence of AMR after cardiac transplantation is uncertain because of a lack of universal screening in asymptomatic recipients. Symptomatic pure AMR without a component of acute cellular rejection occurs in 10 to 15% of cardiac transplant recipients, but AMR features have been reported in up to 40% of patients with acute cellular rejection (mixed rejection).25 Clinical symptoms of cardiac AMR are those common in heart failure using echo. A greater than 25% decrease in ejection fraction and an increase in left ventricular mass distinguished antibody-mediated rejection from cell-mediated rejection.26 Reduced R-wave voltage-conduction abnormalities, including bundle branch block, are electrocardiographic associates. As discussed, presensitization to HLA Class I or II antibodies predisposes to AMR.27 Those recipients who develop presensitized antibodies, especially donor-specific, risk AMR.28 Non- HLA antibodies, including those against cardiac myosin, vimentin, and endothelial cells have been less often associated with AMR.29–31 Cardiac AMR occurs early (weeks to months) after transplant and, if avoided early, rarely occurs later.32 Classically, histologic diagnosis of cardiac AMR has included evidence of endothelial swelling and activation macrophages in the graft plus confirming immunofluorescence or immunoperoxidase staining for immunoglobulin (IgG or IgM) (Figs. 63-12 and 63-13). Although absence of immunofluorescence seems reasonable to rule out AMR, capillary swelling (63%) and macrophage vascular adherence (30%) do not33 and complement (C3d and C4d). Non-AMR causes of C4d staining include organ reperfusion injury, immunosuppression, treatment with monoclonal antibodies, and viral infections.34 To date, the distribution (diffuse versus localized) and intensity of the stains has not found a standardization for grading. The suggested algorithm for AMR includes: (1) Present clinical evidence of cardiac graft dysfunction and histologic evidence with or without interstitial edema and hemorrhage; and (2) positive immunofluorescence or immunoperoxidase for donor-specific antibodies. This creates further questions about the importance of asymptomatic (allograft function conserved) AMR-associated histopathology. It is likely that AMR is a pathologic continuum with clinical involvement in later or more aggressive forms. Asymptomatic AMR has been related to worse cardiovascular outcomes and the presence of cardiac allograft vasculopathy (CAV).35,36

Although a clinical phenotype for AMR after lung transplantation and its response to anti-antibody regimens has been discussed, unfortunately, no strong consensus exists regarding its diagnostic characteristics.37 Recently, the Washington University program evaluated the effect on donor-specific HLA antibodies (DSA) and bronchiolitis obliterans syndrome (BOS) in 65/116 recipients developed DSA. Antibody-depleting therapy with IVIG, with or without rituximab, resulted in no differences in the incidence of acute rejection, lymphocytic bronchiolitis, and BOS between those with and those without antibody.38

Gene Expression Profiling

A DNA microarray-based real-time polymerase chain reaction–derived bio signature for cardiac rejection has been developed by XDx, the maker of allomap molecular expression testing (XDx, Brisbane, CA).39 Peripheral blood mononuclear cells provided a panel of 11 informative genes that were associated with acute cellular rejection.40 Several pathways were identified that participate in regulation of effector cell activation, trafficking and morphology, platelet activation, plus corticosteroid sensitivity. These included PDCD1 and ITGA4 for T-cell activation and migration, ILIR2 steroid-responsive gene, the decoy for IL-2, and WDR40A plus CMIR of the micro-RNA gene family. Peripheral blood samples are assigned a score with higher numbers associated with progressive risk of lack of immunologic quiescence. This approach was recently randomized against surveillance endomyocardial biopsy in a multicenter trial in low-risk rejection cardiac recipients, 6 months to 5 years after transplantation.41 The evaluation excluded recipients with a significant history of rejection, CAV, or allograft dysfunction. Results indicated 14.5% of patients profiled versus 15.3% of those surveilled by endomyocardial biopsy reached a composite endpoint of rejection with hemodynamic compromise, graft dysfunction owing to other causes, death, or retransplantation. The low risk for rejection of the enrolled patients made interpretation to earlier and higher-risk recipients problematic.42 Other reports found that the profiled genes regulatory T-cell homeostasis and corticosteroid sensitivity can distinguish mild from moderate and severe rejection and are evident before histologic, detectable rejection.43,44

Functional Activity of Immune System

A recipient's individual immune responses are affected by their susceptibility to immunosuppression, their clinical state, genetic background, age, gender, and diet. An assay of cell function would be useful to quantitate the dynamic positive and negative regulatory responses of the immune system. Clinicians have struggled, often in relative ignorance, to titrate doses of immunosuppressive drugs to achieve levels that deter the alloresponse while also minimizing the risk of infection. A long-imagined goal of selective treatment based on a quantitative assessment of the net state of immunosuppression has been approached clinically with some success. An assay has been developed (Cylex, ImmuKnow, Columbia, MD) to measure the intracellular concentration of adenosine triphosphate (ATP) of CD4+ T cells. ImmuKnow measures T-cell responses by quantifying ATP activity45 to phytohemagglutinin, a T-cell mitogen. In general, the collective studies suggest that ATP levels less than 100 ng/mL correlate with an increased risk of infection.46 In a study in 296 heart recipients spanning 2 weeks to 10 years posttransplant, infection in 39 recipients occurred with an average 187 + 126 ng/ATP/mL versus a steady state of 280 + 126 ng/ATP/mL (Fig. 63-14). Rejection scores in eight recipients averaged 328 ng/ATP/mL and did not differ from baseline. However, 3 of 8 with antibody-mediated rejection scored 49/I-12/ng/ATP.46 This assay opens the field to personalized immunosuppression that might help balance risks of infection and rejection.

The author wishes to thank Agnes Azimzadeh and Richard N. Pierson III for their critical review of this chapter, and Debra KuKuruga for her assistance and expertise on the HLA portion of this chapter.