The authors would like to acknowledge Norman Barker and Dr. Peter Illei for their help with the images in this chapter.

After the technical challenge of lung or heart transplantation has been successfully achieved, the process of maintaining a healthy graft begins. For this, a general understanding of the immunobiology of thoracic transplantation is needed. This chapter will provide a background on the immune system and allorecognition. It will describe how the immune system mediates rejection of lung and heart transplants, types of organ rejection, and the histologic appearance of rejection. It will further describe methods to monitor allograft rejection and the criteria upon which rejection is characterized. Finally, the chapter will cover basics of immunomodulation therapy.

History of Primary Graft Failure and an Understanding of the Role of Allorecognition

Experimental heart transplantation developed slowly through the 1940s and 1950s, but became established in the laboratory of Dr. Norman Shumway by the mid-1960s. Human heart transplantation was first performed by Dr. Christian Bernard in 1967. Heart transplant outcomes were initially poor due to quick rejection of the organ. Experimental lung transplantation in mammals occurred throughout the first half of the 20th century. The first human lung transplantation occurred in 1963, although “successful” transplantation did not occur until the early 1980s. For both organs, early survivability was dependent on a number of factors, but key among them was an understanding and control of organ rejection. These early transplants failed because there was no effective way to control organ rejection without overly suppressing the immune system leading to fatal infections. The revolution in solid organ transplant was made possible by the immunosuppressive drug cyclosporine, discovered in the mid 1970s and introduced clinically in 1983. It prevented acute rejection of the organ, but allowed the body to ward off infectious agents. Thus successful organ transplantation is dependent upon a keen understanding of the interaction of the immune system with the transplanted organ. Our understanding of this interaction is focused on the role of the major histocompatibility complex (MHC) and the innate immune system in solid organ rejection.

The MHC is one of the essential parts of the immunoregulatory process. The MHC genomic region encodes a large and diverse group of proteins that are expressed on all cells. These MHC proteins present antigen (polypeptide fragments of cell proteins) to immune cells, predominantly T cells. There are two major classes of MHC molecules. Class I molecules are found on all nucleated cells and present antigen to CD8 positive (CD8⁺) “cytotoxic” T cells and natural killer (NK) cells. Class II MHC proteins occur on antigen presenting cells (APCs) and present antigen to CD4 positive (CD4⁺) “helper” T cells. In the lung and heart, native dendritic cells are the primary MHC class II positive APCs.

With all human cells constantly presenting antigen to T cells, one might wonder why our bodies are not under constant immunologic attack. The answer is that through our first year of life, our body learns to differentiate between self and nonself antigens. This process occurs primarily in the thymus in which an enormous diversity of T cells each displaying a different T-cell receptor complex interacts with thymic epithelial cells. Only those T cells that have an intermediate affinity for self-antigen MHC complexes (an ability to recognize, but not react) are allowed to survive. T cells with either no affinity or high affinity to self peptide MHC complexes are removed. Thus, after the first year of life, our T cells have developed the capability to perform immunosurveillance and differentiate between self and nonself antigens presented by MHC complexes.¹

APCs are the most potent initiators of alloimmune responses because they express high concentrations of both MHC class I and II antigens (Fig. 49-1). The MHC class II antigens are required to activate CD4 helper T cells. APCs also express numerous costimulatory molecules that provide secondary signals necessary for expansion of the T cells’ response to the target antigens. APCs can participate in three pathways of allorecognition. All three pathways are involved in lung and heart rejection. The direct pathway is the mechanism by which recipient T cells recognize nonself antigen present on donor APCs. Transplants contain large numbers of donor APCs at the time of transplantation that are progressively replaced by recipient APCs. The indirect pathway occurs when necrotic or apoptotic cellular debris from donor cells are taken up by recipient APCs. These nonself antigens are then displayed via MHC class II molecules resulting in a CD4⁺ cell-driven process. A third potential pathway is the semidirect pathway. Here, small cellular vesicles (exosomes) containing MHC move from a donor APC to a recipient APC and stimulate both CD4⁺ and CD8⁺ T cells.² In addition to the described T cell MHC interaction, a costimulatory event must occur to optimally activate the T-cell response. The best studied costimulation interaction is between the CD28 protein on the T cell and a B7 molecule (B7-1 and B7-2; CD 80 and 86, respectively) on the APC.³ A new immunosuppressive agent, LEA29Y (belatacept), blocks this interaction, although it is not yet used in thoracic transplantation.

Secondary Stimulation of T-cell Response

Once a T cell has become activated, a number of inflammatory pathways become upregulated. The pathways described here are all targeted by immunosuppressive drugs that will be covered later in this chapter. T-cell receptor activation by MHC activates calcineurin. Calcineurin, in turn, activates a transcription factor (NF-AT) which increases the production of interleukin-2 (IL-2) and additional cytokines (Fig. 49-1). Secreted IL-2 binds an IL-2 receptor (IL-2R) on the cell surface stimulating expansion of the local immune cell population (predominantly T cells). This cell proliferation is partly mediated by a direct stimulatory effect of IL-2R on mTOR which regulates the cell cycle.³ The result of this activation pathway is increased inflammatory cells in the donor organ and injury seen as epithelial cell or myocyte death.

In distinction to the adaptive immune response that is defined by antigen specific receptors on lymphocytes (T cells and B cells), the innate immune system encompasses mediators and cells with invariant receptors (Table 49-1). These include proteins of the complement system as well as an array of cells, notably platelets, granulocytes, monocytes, and macrophages. Components of the innate immune system evolved to survive pathogens and tissue injury (Fig. 49-2). Toll-like receptors (TLR) and mannose binding lectin (MBL) are good examples of what Janeway⁴ termed pattern recognition receptors that bind to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PAMPs include structural motifs unique to prokaryotes, such as lipopolysaccharide (LPS), bacterial lipopeptides, flagellin, and unmethylated DNA. DAMPs are intracellular or cryptic molecules that are exposed by tissue disruption or produced by cells in response to stress. Among the best-defined DAMPs are heat shock proteins (HSP) and fragmented hyaluronan. Other DAMPs such as high-mobility group box 1 (HMGB1) and IL-33 are located in the nucleus of live cells and released by necrotic cells.

Toll receptors were discovered in fruit flies because genetically deficient flies had morphogenic defects and were susceptible to fungal infections. On the basis of sequence homologies more than 10 TLRs have been identified in mammals. In humans, TLR 1, 2, and 6 can form heterodimers and TLR4 forms homodimers on the surface of leukocytes and parenchymal cells where they bind signature PAMPs and DAMPs (Fig. 49-3). TLR4, for example, binds LPS and fragmented hyaluronan.⁵ Other TLRs, such as TLR3, are located on intracellular endosomes and recognize viral and bacterial RNA or DNA. Cell surface TLRs signal through MyD88 to upregulate a group of proinflammatory cytokines, including TNFα, IL-1, IL-6, and IL-12 as well as the chemokines IL-8 and MCP-1 that attract neutrophils and monocytes. TLR4 also signals through TRIF as does the intracellular TLR3 to upregulate IFNα and β (Fig. 49-3).

MBL is integrated into the complement system of mammals as one of three initiators of the complement cascade. MBL is a member of the collectin family of proteins that are characterized by collagen-like tails with globular lectin heads, hence “col-lectin.”⁶ The lectin heads can bind to carbohydrates terminating in mannose, fucrose and N-acetylglucosamine (GlcNac) that typify bacteria. The affinity of these reactions is low, but MBL forms oligomers of two, three, four, or six molecules that bind arrays of repeated carbohydrate motifs with relatively high avidity. Homologues of collectins and MBL have been identified in invertebrate animals such as sea squirts. These animals lack most of the remainder of complement components. Therefore, collectins arose as primitive pattern recognition molecules that opsonized bacteria for phagocytes. They also opsonize subcellular debris from injured or senescent cells for removal by phagocytes. In mammals, innate immunity functions both independently and in conjunction with the adaptive immune response.⁷

Unlike adaptive immune responses, which require days to generate, the invariant receptors of the innate immune system provide immediate responses in the postoperative period. Of particular relevance to transplantation are the innate immune responses to ischemia imposed by organ procurement followed by reperfusion when the transplant operation is completed (Table 49-2). Unmitigated ischemia–reperfusion injury is characterized by activation of the complement cascade, platelets, and granulocytes. The ensuing tissue injury results in macrophage infiltration. Even with current preservation solutions, which are designed to diminish this injury, early protocol biopsies often contain some evidence of ischemic injury histologically identifiable by macrophage infiltrates and deposits of complement split products (C4d and C3d) on damaged myocytes.

Organs from deceased donors are subject to multiple insults that can augment innate immune responses. These include sequella of traumatic injury, brain death, and various emergency interventions (Fig. 49-2). Surgical procedures cause tissue injury resulting in exposure of DAMPs and initiation of wound healing responses. In addition, incisions break mechanical and chemical barriers, providing portals for infectious agents and stimulation of innate immunity through PAMPs.

Thoracic organ transplants that require cardiopulmonary bypass have additional inciting stimuli for the innate immune system. These include the bioincompatible surfaces of the bypass equipment that cause complement activation. Any platelet transfusions also introduce partially activated platelets that release chemokines and cytokines. The inflammatory effects of the innate immune responses can be decreased by administration of steroids postoperatively.

Components of the innate immune system facilitate de novo adaptive immune responses and augment preexisting or ongoing adaptive immune responses. Macrophages in ischemic sites, for example, can upregulate MHC class II expression and present antigen to T cells. Preexisting or newly induced antibodies can activate complement and generate biologically active split products that chemoattract and activate neutrophils and macrophages. As a result, these are the most common leukocytes in antibody-mediated rejection (AMR). C5 is a pivotal component of complement because it is cleaved into two key split products when it is activated: C5a, which is a powerful chemoattractant and activator of neutrophils and macrophages; and C5b that initiates the formation of the membrane attack complex (C5b–C9) that can lyse endothelial cells. This has led to the development of therapeutic agents to block C5 including monoclonal antibodies to C5, small-molecule antagonists for C5a, and soluble receptors for C5a.