Immunotherapy in Transplantation: Principles and Practice

By Bruce Kaplan

This comprehensive reference source will benefit all transplant specialists working with pharmacologic and biologic agents that modulate the immune system. Compiled by a team of world-renowned editors and contributors covering the fields of transplantation, nephrology, pharmacology, and immunology, the book covers all anti-rejection drugs according to a set template and includes the efficacy of each for specific diseases.

• Language: English
• Publisher: Wiley
• Release date: Feb 23, 2012
• ISBN: 9781444355604

Book preview

CHAPTER 1

The Immune Response to a Transplanted Organ: An Overview

Fadi G. Lakkis

Thomas E. Starzl Transplantation Institute, Departments of Surgery, Immunology, and Medicine, University of Pittsburgh, Pittsburgh, PA, USA

Basic definitions

Organs transplanted between two members of the same species are rejected unless the donor and recipient are genetically indistinguishable (identical twins in the case of humans). Rejection is caused by the recipient’s immune response to foreign elements present on the transplanted organ. These elements are usually proteins that differ between the donor and recipient and are called alloantigens. The transplanted organ itself is referred to as the allograft and the immune response mounted against it as the alloimmune response or alloimmunity. The prefix xeno, on the other hand, is used to denote the transplantation of organs between members of different species, as in the terms xeno-antigens, xenografts, and xenotransplantation.

The principal players

The T lymphocyte is the principal mediator of the alloimmune response [1, 2]. Experimental animals devoid of T cells do not reject tissue or organ allografts [3, 4]. Similarly, T cell depletion in humans prevents rejection effectively until T cells return to the circulation [5]. T cells cause direct injury to the allograft through a variety of cytotoxic molecules or cause damage indirectly by activating macrophages and other inflammatory cells (Chapter 3). T cells also provide help to B lymphocytes to produce a host of antibodies that recognize alloantigens (alloantibodies). Alloantibodies inflict injury on the transplanted organ by activating the complement cascade or by activating macrophages and natural killer cells (Chapter 4). An exception to the T cell requirement for allograft rejection is the rapid rejection of organs transplanted between ABO blood-group-incompatible individuals. In this case, allograft destruction is mediated by preformed anti-ABO antibodies that are produced by B-1 lymphocytes, a subset of B cells that are activated independent of help from T cells. Another potential mechanism of T-cell-independent rejection is graft dysfunction mediated by monocytes. This has been observed in renal transplant recipients after profound T cell depletion [5], but it is unlikely that monocytes lead to full-blown rejection in the absence of T cells or preformed antibodies.

The principal alloantigens recognized by T cells, B cells, and antibodies are the human leukocyte antigens (HLAs). These are cell-surface proteins that are highly variable (polymorphic) between unrelated individuals. Two main classes of HLA proteins have been identified. Class I molecules (HLA-A, -B, and -C) are expressed on all nucleated cells, whereas class II molecules (HLA-DP, -DQ, and -DR) are present on cells of the immune system that process and present foreign proteins to T cells; these are referred to as antigen-presenting cells (APCs) and include B cells, dendritic cells, macrophages, and other phagocytic cells (Chapter 2). In humans, activated T cells and inflamed endothelial cells also express class II molecules. Since HLA inheritance is codominant, any given individual shares one haplotype (one set of alleles) with either biological parent and has a 25 % chance of being HLA-identical (sharing both haplotypes) with a sibling. The chance that two unrelated individuals are HLA-identical is less than 5 %, because of the highly polymorphic nature of the HLA. Although HLA matching between donor and recipient confers long-term survival advantage on grafts [6], it does not in any way obviate the need for immunosuppression. The immune system is, in fact, capable of recognizing any non-HLA protein that differs between the donor and recipient as foreign and of mounting an alloimmune response to it that is sufficient to cause rejection. Non-HLA proteins that trigger an alloimmune response and are targeted during allograft rejection are referred to as minor histocompatibility antigens (Chapter 2). It is likely that a large number of minor antigens exist, making it very difficult to match for them.

Types of rejection

Pathologists have traditionally divided allograft rejection into three groups based on the tempo of allograft injury: hyperacute, acute, and chronic. Hyperacute rejection is a very rapid form of rejection that occurs within minutes to hours after transplantation and destroys the allograft in an equally short period of time. It is triggered by preformed anti-ABO or anti-HLA antibodies present in the recipient [7, 8]. Blood typing and clinical cross-matching, whereby preformed anti-HLA antibodies are screened for by mixing recipient serum with donor cells, or more commonly nowadays by sensitive flow-cytometric methods, has virtually eliminated hyperacute rejection. Acute rejection, in contrast, leads to allograft failure over a period of several days rather than minutes or hours. It usually occurs within a few days or weeks after transplantation, but it could happen at much later time points if the immune system is awakened by infection or by significant reduction in immunosuppression. Chronic rejection is a slow form of rejection that primarily affects the graft vasculature (or the bronchioles and bile ducts in the case of lung and liver transplants respectively) and causes graft fibrosis. Chronic rejection may become manifest during the first year after transplantation, but more often progresses gradually over several years, eventually leading to the demise of the majority of transplanted organs, with the exception perhaps of liver allografts. Since acute and chronic rejections are caused by T cells, antibodies, or both, it is increasingly common to label rejection by its predominant immunological mechanism, cellular or antibody mediated, in addition to its temporal classification (Chapters 3 and 4). Rejection is also graded according to agreed-upon criteria known collectively as the Banff classification [9]. These are important advances in transplantation pathology, as they often guide the choice of anti-rejection treatment and are used as prognosticators of long-term allograft outcome.

Distinguishing features of the alloimmune response

Although alloimmune responses resemble antimicrobial immune responses in many ways, they are distinguishable by several salient features. These features are highlighted here, as they have direct implications for the development of anti-rejection therapies.

Alloimmune responses are vigorous responses that involve a relatively large proportion of the T cell repertoire

Humans carry a large repertoire of T lymphocytes that recognize and react to virtually any foreign protein with a high degree of specificity. The diversity of T cell reactivity is attributed to the random rearrangement during T cell ontogeny of genes that code for components of the T cell receptor (TCR) for antigen (Chapter 3). The same applies to B cells, leading to an immense variety of antibodies that detect almost any conceivable foreign antigen (Chapter 4). The high specificity of T cells is explained by the fact that TCRs do not recognize whole antigens; instead, they recognize small peptides derived from foreign proteins and presented in the context of HLA molecules on antigen-presenting or infected cells (Chapter 2). This leads to fine molecular specificity in which only a very small proportion of T cells react to a non-self peptide. It is estimated that only 1 in 10 000 or less of all T cells in a human being recognize peptides derived from any given microbe. The small proportion (or precursor frequency) of microbe-specific T cells is nevertheless sufficient to eliminate the infection because of the ability of T lymphocytes to proliferate exponentially (a phenomenon referred to as clonal expansion) before differentiating into effector cells. In sharp contrast, the immune response to an allograft involves anywhere between 1 and 10 % of the T cell repertoire [10, 11] – essentially 10–100 times more than an antimicrobial response. The large-scale participation of T cells in the alloimmune response can be readily demonstrated in the mixed lymphocyte reaction (MLR), a laboratory test in which coculturing recipient peripheral blood mononuclear cells (PBMCs) with donor PBMCs results in conspicuous proliferation of recipient T lymphocytes. Detecting T cell proliferation against microbial antigens, on the other hand, is a much more difficult feat because of the low precursor frequency of microbe-specific lymphocytes. Alloimmune responses, therefore, are especially vigorous responses because of the participation of a significant proportion of T cells with a wide range of specificities. The reasons for this phenomenon, perhaps the dominant obstacle to improving allograft survival without unduly compromising the recipient’s immune system, are explained next.

T cell alloreactivity is cross-reactivity

The immune system has evolved to protect animals against infection. It is not surprising, therefore, that humans and most other vertebrate species are armed with T cells that recognize microbial antigens. Why is it, then, that we also carry a disproportionately large proportion of T cells that react to alloantigens? Based on cellular and molecular studies in humans and experimental animals, it has become evident that TCRs specific for a microbial peptide (presented in the context of self-HLA) are also capable of recognizing allogeneic, non-self HLA [11]. This phenomenon is known as cross-reactivity or heterologous immunity and has been best demonstrated for T cells specific to Epstein–Barr virus (EBV) antigens [12]. The same is likely to be true of T cells specific to other viruses. The inherent ability of developing T cells to bind to HLA molecules also contributes to the high precursor frequency of alloreactive T cells in the mature T cell repertoire [13]. The inherent bias to generate TCRs that see HLA is attributed to the fact that T cell education in the thymus and the ultimate development of a mature cellular immune system are dependent on recognition of peptides bound to HLA (Chapter 3). Therefore, alloreactivity is an unintended side effect of an immune system that has evolved to effectively fend off foreign, generally microbial, antigens.

T cell alloreactivity is in large part a memory response, even in naive individuals not previously exposed to alloantigens

The primary immune response to a foreign antigen not previously encountered by the host is mediated by naive T lymphocytes (Chapter 3). Naive T cells specific to the foreign antigen are present at a low precursor frequency, have a relatively high stimulation threshold (e.g., stringent dependence on costimulatory molecules), can only be activated within secondary lymphoid tissues (e.g., the spleen and lymph nodes) [14], and are, therefore, slow to respond. In contrast, the secondary immune response to an antigen previously encountered by an individual (e.g., after vaccination or infection) is mediated by memory T cells and is significantly stronger and faster than a primary response. Antigen-specific memory T cells are long-lived lymphocytes that exist at a greater precursor frequency than their naive counterparts, have a low stimulation threshold and high proliferative capacity, and can be activated within secondary lymphoid tissues or at non-lymphoid sites – for example, the site of infection or in the allograft itself [15]. Memory B cells and plasma cells share some of the properties of memory T cells thus, endowing vaccinated individuals with the ability to rapidly produce high titers of antigen-specific antibodies upon reinfection (Chapter 4). Immunological memory, therefore, provides humans with optimal protection against microbes.

Humans for the most part are not exposed to alloantigens, with the exception of mothers who may have been sensitized to paternal antigens during pregnancy or individuals who had prior transfusions or organ transplants. Yet all humans, including those presumably never exposed to allogeneic cells or tissues, harbor alloreactive memory T cells. Accurate quantitation of alloreactive T cells has demonstrated that approximately 50 % of the alloreactive T cell repertoire in humans is made up of memory T lymphocytes [11, 16, 17]. This finding can again be explained by the phenomenon of cross-reactivity, whereby memory T cells specific to microbial antigens also recognize alloantigens and contribute to the high precursor frequency of alloreactive T cells. Therefore, the extent of one’s alloreactivity is intimately shaped by one’s immunological memory to foreign antigens not necessarily related to the graft.

The distinguishing features of alloimmunity summarized above have important implications for both the immunological monitoring of transplant recipients and the development of anti-rejection therapies. It is becoming increasingly clear that measuring anti-donor memory T cells or donor-specific antibodies either before or after transplantation could predict rejection incidence and graft outcomes [18]. Moreover, T-lymphocyte-depleting agents used to prevent rejection invariably skew T cells that repopulate the host towards memory [19, 20]. These memory T cells arise from antigen-independent, homeostatic proliferation of undepleted naive or memory T cells – a phenomenon known as lymphopenia-induced proliferation [21]. Lymphopenia-induced T cell proliferation is responsible for early and late acute rejection episodes in lymphocyte-depleted transplant recipients and creates an obstacle to minimizing immunosuppression [22]. Another clinical implication of alloreactive memory T cells is that anti-rejection agents that inhibit naive lymphocyte activation or migration are not expected to be as effective as those that suppress both naive and memory lymphocytes. Targeting memory T or B cells, therefore, is desirable but leads to the important conundrum of how to inhibit alloreactivity without compromising beneficial antimicrobial memory. Overcoming this challenge could pave the path towards developing the next generation of immunotherapeutic agents in transplantation.

Immune regulation

The alloimmune response is subject to regulatory mechanisms common to all immune responses. Four principal regulatory mechanisms have been described: activation-induced cell death (AICD), regulation by specialized lymphocyte subsets known as TREG and BREG, anergy, and exhaustion. These mechanisms ensure that collateral damage to the host is kept to a minimum during or after a productive immune response.

Primary and secondary T cell responses are characterized by exponential proliferation of antigen-specific T cells followed by a crash phase in which the majority of activated or effector T cells die by apoptosis (Plate 1.1). This process prevents unnecessary immunopathology while allowing T cells that escape apoptosis to become memory lymphocytes. The same is true for B cells, where the process of expansion followed by death allows for the selection of B lymphocytes with the highest affinity to their target antigens (affinity maturation) (Chapter 4). Most immunusuppressive drugs available for clinical use target lymphocyte proliferation and in some cases (e.g., calcineurin inhibitors) prevent AICD [23], leaving the possibility of developing agents that selectively enhance the apoptosis of activated T cells open. Such a strategy would be more specific than pan-T-cell depletion, as only T cells that have been activated by alloantigens are killed.

The isolation of T and B cell subpopulations that downregulate immune responses in vitro and in vivo has led to a resurgence of studies on regulatory lymphocytes (Chapter 6). TREG and BREG populations have been identified in rodents and, in the case of the former, in humans as well. Regulatory lymphocytes suppress mixed lymphocyte reactions in vitro and prolong allograft survival in rodent transplantation models. The mechanisms by which TREG suppress immune responses are varied. They include cytokines (e.g., IL-10 and TGFβ), inhibitory membrane molecules (e.g., CTLA-4), and possibly direct cytotoxicity to naive or effector lymphocytes. In addition to interest in isolating and expanding TREG for adoptive cell therapy in transplant recipients, there has been an important focus on developing or exploiting existing immunosuppressive drugs that spare or enhance regulatory lymphocytes. One example is the mTOR inhibitor rapamycin, which in mice generates a favorable TREG to effector T cell ratio that may contribute to long-term allograft survival. It is not certain, however, whether the salutary effects of rapamycin on TREG in rodents will translate to longer allograft survival in humans because of the pleiotropic functions of mTOR signaling in different cells of the immune system.

Anergy and exhaustion refer to the state in which T cells or B cells become unresponsive to restimulation with antigen. Anergy occurs when naive lymphocytes encounter antigen in the absence of critical costimulatory signals necessary for their full activation. A prime example of costimulation is the B7–CD28 pathway (Chapter 3). B7 molecules expressed on antigen-presenting cells engage CD28 on T cells concurrent with T cell stimulation through the TCR. Blocking B7-CD28 interaction renders T cells anergic and/or induces their apoptosis [24, 25]. CTLA4-Ig, a fusion protein that binds B7 molecules and prevents them from engaging CD28, is currently approved for use in renal transplant recipients. Published data suggest that CTLA4-Ig may be an effective substitute for calcineurin inhibitors. Finally, exhaustion occurs when effector or memory T cells repeatedly encounter a persistent antigen, as would occur during chronic viral infection or in the case of an allograft. Repeated antigenic stimulation induces the expression of inhibitory molecules that keep T cells hypo- or un-responsive. One example of such inhibitory molecules is PD-1, shown in rodents to suppress alloreactive effector T cells [26]. These regulatory pathways provide interesting opportunities for developing novel strategies to inhibit T cells that have been activated by alloantigens. By targeting activated but not naive T cells, these strategies may prove more selective than currently available immunosuppressive therapies.

The innate immune system in transplantation

The mammalian immune system consists of two integrated arms: the innate and adaptive.

The adaptive immune system (the subject of discussion of this chapter so far) consists of T and B lymphocytes which express diverse and highly specific antigen receptors brought about by gene rearrangement, expand clonally, and generate immunological memory. Unlike the adaptive system, the innate immune system is made up of inflammatory cells (dendritic cells, monocytes, macrophages, neutrophils, eosinophils, basophils, and other cells) that do not express rearranging receptors, have limited proliferative capacity, and, for the most part, do not generate memory. Cells of the innate immune system instead express nonrearranging, germ-line-encoded receptors that detect conserved molecular patterns present in microbes but not shared by mammalian cells [27]. A representative example of innate receptors is toll-like receptor (TLR)-4, which recognizes lipopolysaccharide on Gram-negative bacteria (Chapter 5). It should be noted that the innate immune system also encompasses noncellular mediators capable of microbial recognition – for example, complement proteins. Activation of the innate immune system by microbial ligands causes inflammation, the first line of defense against infection, but more importantly induces the maturation and migration of antigen-presenting cells to secondary lymphoid tissues where they trigger primary T cell and B cell responses. The latter function of the innate immune system is critical for initiating adaptive immunity to infection and vaccines in the naive host. The innate immune system, therefore, is responsible for the first self–non-self recognition event that ultimately leads to productive T and B cell immunity.

Although the innate recognition pathways required for establishing antimicrobial immunity have been uncovered for many infectious diseases, how the innate immune system triggers the adaptive alloimmune response is not as straightforward. Several endogenous ligands released by dying cells in the graft participate in ischemia–reperfusion injury (Chapter 6), but it is not clear whether any single ligand has a dominant role or whether any are critical for triggering either naive or memory T cell activation. These uncertainties could be due to the release of myriads of redundant activators of the innate immune system by the graft at the time of transplantation or due to the possibility that memory T cell activation, an important component of the alloimmune response, could occur independent of innate immune activation. Nevertheless, it is generally accepted that inflammation influences the migration of effector and memory T cells to the transplanted organ and increases the intensity of rejection [28]. Prolonged cold or warm ischemia not only predisposes allografts to delayed function after transplantation, but also to increased risk of acute and chronic rejection [29]. Recent studies have suggested that the innate immune system may be capable of distinguishing between self and allogeneic non-self [30, 31], akin to its role in detecting microbial non-self. This intriguing possibility could imply that an innate allorecognition system that precedes allorecognition of HLA by the adaptive immune system maintains immunity against allografts long after the early inflammatory phase has subsided. The nature of such innate allorecognition and whether it contributes to either acute or chronic rejection remains to be determined.

Concluding remarks

The immune system is composed of rich layers of cellular and humoral mediators that work in concert to protect humans against potentially fatal infections. One price that humans pay for this highly developed defense system is the rejection of life-saving organ transplants. Better understanding of the regulatory mechanisms embedded in the immune system and of the subtle distinctions between antimicrobial and alloimmunity should pave the path towards selective immunotherapies that prevent rejection but preserve beneficial immunity against infection. Studying the immune system is like peeling an onion: beneath each layer we find another; chopping the onion will bring tears … only during peeling does it speak the truth [32].

References

1 Hall BM, Dorsch S, Roser B. The cellular basis of allograft rejection in vivo. II. The nature of memory cells mediating second set heart graft rejection. J Exp Med 1978;148:890–902.

2 Hall BM, Dorsch S, Roser B. The cellular basis of allograft rejection in vivo. I. The cellular requirements for first-set rejection of heart grafts. J Exp Med 1978;148:878–889.

3 Miller JFAP. Effect of neonatal thymectomy on the immunological responsiveness of the mouse. Proc R Soc Lond B 1962;156:415–428.

4 Bingaman AW, Ha J, Waitze S-Y et al. Vigorous allograft rejection in the absence of danger. J Immunol 2000;164:3065–3071.

5 Kirk AD, Hale DA, Mannon RB et al. Results from a human renal allograft tolerance trial evaluating the humanized CD52-specific monoclonal antibody alemtuzumab (CAMPATH-1 H). Transplantation 2003;76:120–129.

6 Takemoto S, Terasaki PI, Cecka JM et al. Survival of nationally shared, HLA-matched kidney transplants from cadaveric donors. The UNOS Scientific Renal Transplant Registry. N Engl J Med 1992;327:834–839.

7 Starzl TE, Lerner RA, Dixon FJ et al. Shwartzman reaction after human renal homotransplantation. N Engl J Med 1968;278:642–648.

8 Patel R, Terasaki PI. Significance of the positive crossmatch test in kidney transplantation. N Engl J Med 1969;280:735–739.

9 Solez K, Colvin RB, Racusen LC et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant 2008;8:753–760.

10 Suchin EJ, Langmuir PB, Palmer E et al. Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question. J Immunol 2001;166:973–981.

11 Macedo C, Orkis EA, Popescu I et al. Contribution of naïve and memory T-cell populations to the human alloimmune response. Am J Transplant 2009;9:2057–2066.

12 Burrows S, Khanna R, Burrows J, Moss D. An alloresponse iln humans is dominated by cytotoxic T lymphocytes (CTL) cross-reactive with a single Epstein–Barr virus CTL epitope: implications for graft-versus-host disease. J Exp Med 1994;179:1155–1161.

13 Zerrahn J, Held W, Raulet DH. The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell 1997;88:627–636.

14 Lakkis FG, Arakelov A, Konieczny BT, Inoue Y. Immunologic ignorance of vascularized organ transplants in the absence of secondary lymphoid tissue. Nature Med. 2000;6:686–688.

15 Chalasani G, Dai Z, Konieczny BT et al. Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs. Proc Natl Acad Sci U S A 2002;99:6175–6180.

16 Merkenschlager M, Terry L, Edwards R, Beverley PC. Limiting dilution analysis of proliferative responses in human lymphocyte populations defined by the monoclonal antibody UCHL1: implications for differential CD45 expression in T cell memory formation. Eur J Immunol 1988;18:1653–1661.

17 Lombardi G, Sidhu S, Daly M et al. Are primary alloresponses truly primary? Int Immunol 1990;2:9–13.

18 Dinavahi R, Heeger PS. T-cell immune monitoring in organ transplantation. Curr Opin Organ Transplant 2008;13:419–424.

19 Pearl J, Parris J, Hale D et al. Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T-cell depletion. Am J Transplant 2005;5:465–474.

20 Toso C, Edgar R, Pawlick R et al. Effect of different induction strategies on effector, regulatory and memory lymphocyte sub-populations in clinical islet transplantation. Transpl Int 2009;22:182–191.

21 Surh CD, Sprent J. Homeostasis of naive and memory T cells. Immunity 2008;29:848–862.

22 Wu Z, Bensinger SJ, Zhang J et al. Homeostatic proliferation is a barrier to transplantation tolerance. Nat Med 2004;10:87–92.

23 Li Y, Li XC, Zheng XX et al. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat Med 1999;5:1298–1302.

24 Dai Z, Konieczny BT, Baddoura FK, Lakkis FG. Impaired alloantigen-mediated T cell apoptosis and failure to induce long-term allograft survival in IL-2-deficient mice. J Immunol 1998;161:1659–1663.

25 Wells AD, Li XC, Li Y et al. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat Med 1999;5:1303–1307.

26 Habicht A, Kewalaramani R, Vu MD et al. Striking dichotomy of PD-L1 and PD-L2 pathways in regulating alloreactive CD4+ and CD8+ T cells in vivo. Am J Transplant 2007;7:2683–2692.

27 Palm NW, Medzhitov R. Pattern recognition receptors and control of adaptive immunity. Immunol Rev 2009;227:221–233.

28 Chalasani G, Li Q, Konieczny BT et al. The allograft defines the type of rejection (acute versus chronic) in the face of an established effector immune response. J Immunol 2004;172:7813–7820.

29 Murphy SP, Porrett PM, Turka LA. Innate immunity in transplant tolerance and rejection. Immunol Rev 2011;241:39–48.

30 Fox A, Mountford J, Braakhuis A, Harrison LC. Innate and adaptive immune responses to nonvascular xenografts: evidence that macrophages are direct effectors of xenograft rejection. J Immunol 2001;166:2133–2140.

31 Zecher D, van Rooijen N, Rothstein D et al. An innate response to allogeneic nonself mediated by monocytes. J Immunol 2009;183:7810–7816.

32 Grass G. Peeling the Onion. New York: Harcourt; 2007.

CHAPTER 2

Antigen Presentation in Transplantation

Martin H. Oberbarnscheidt and Fadi G. Lakkis

Thomas E. Starzl Transplantation Institute, Departments of Surgery, Immunology, and Medicine, University of Pittsburgh, Pittsburgh, PA, USA

Transplantation antigens

Transplantation of organs between genetically disparate individuals of the same species (allogeneic individuals) leads to recognition and rejection of the allogeneic tissue by the recipient’s immune system. The immune process of discriminating between self- and non-self-tissues is called allorecognition. The principal transplantation antigens, or alloantigens, recognized during this process are genetically encoded polymorphic proteins that are expressed on tissues of individuals. These polymorphic determinants are the major histocompatibility complex (MHC) antigens (in humans known as human leukocyte antigens or HLAs) and the minor histocompatibility antigens (mHAgs). MHC antigens are glycoproteins expressed by polymorphic multigene clusters located on chromosome 17 in mice and chromosome 6 in humans (Figure 2.1), whereas mHAgs can be any other polymorphic protein that is encoded virtually anywhere in the genome.

Figure 2.1 HLAs. Organization of the genes coding for the HLA on human chromosome 6.

MHC molecules

The MHC is polygenic, polymorphic and codominantly expressed. All of these features contribute to the large inter-individual variability – at least 300 HLA alleles have been identified in the human population. There are two classes of MHC molecules, class I and II, that differ in their structure, function, and tissue distribution. MHC class I proteins are expressed on all nucleated cells, whereas MHC class II expression is restricted to antigen-presenting cells (APCs), activated T cells, and endothelial cells. APCs include B cells, dendritic cells (DCs), macrophages, and other phagocytic cells described in more detail later in this chapter. An individual can express at least three different MHC class I (HLA-A, -B and -C) and class II (HLA-DR, -DP and -DQ) proteins (Figure 2.1). Typically, most individuals will be heterozygous at the MHC locus, and the MHC class II HLA-DR cluster will contain an additional β chain (HLA-DRB), resulting in the expression of six different MHC class I and eight different MHC class II molecules on cells.

The primary function of MHC molecules is to bind short peptides that are derived from proteins processed by the cell. The cellular and biochemical mechanisms of antigen processing are reviewed elsewhere [1]. Briefly, exogenous antigens are processed into small peptides in endosomes and lysosomes, whereas endogenous (cytosolic) antigens are processed by proteosomes. Peptide loading onto MHC molecules occurs in the Golgi apparatus with the help of specialized transporter proteins. Peptide:MHC complexes then translocate to the cell membrane where they can be surveyed by the immune system. Non-self-peptide:MHC complexes expressed by APCs trigger T cell activation by binding the T cell receptor (TCR) for antigen (Chapter 3). In the case of non-APCs, non-self-peptide:MHC complexes serve as a target for T cells specific for the presented antigenic peptide. The MHC, therefore, ensures that foreign antigens, especially those derived from infecting organisms, are detected and eliminated by the immune system with great specificity. The high degree of polymorphism (diversity) of the MHC guarantees that virtually any microbial-derived peptide can be presented to the immune system.

Peptides presented by MHC class I molecules are usually derived from endogenous, cytosolic proteins (either self or foreign), although exogenous antigens may also be presented. The presentation of exogenous proteins in the context of MHC class I is referred to as cross-presentation or cross-priming [2, 3]. MHC class I complexes interact with the TCR on CD8 T cells. They are comprised of a heavier glycosylated α chain and a lighter β2-microglobulin chain. The α chain contains a transmembrane domain and three extracellular domains: α-1, α-2, and α-3. The variable α-1 and α-2 helices form a groove, which is closed at the ends, that serves as the binding site for processed peptides. The specificity of the peptide-binding groove is determined by the different alleles encoding the α-1 and α-2 helices. The peptide and surrounding amino acids of the α-1 and α-2 helices are recognized by the TCR. α-3 is not variable and interacts with CD8 to increase the binding of the MHC:peptide complex to the TCR. β2-Microglobulin is also not variable and is encoded on a different chromosome (chromosome 15) than the α chain.

MHC class II molecules generally present peptides derived from exogenous proteins, which are taken up by APCs and processed in the vesicular compartment (endosomes and lysomes), but MHC class II molecules can also present endogenous, cytosolic peptides [4–6]. MHC class II complexes interact with the TCR on CD4 T cells. They are heterodimers composed of an α and a β chain – both of which are polymorphic. The variable α-1 and β-1 domains of each of these chains form the peptide-binding pocket, which is open so that longer peptides can be presented than those of MHC class I. The bound peptide in conjunction with surrounding amino acids is then recognized by the TCR. CD4 interacts with the nonvariable α-2 domain of MHC class II molecules to strengthen the binding of the MHC:peptide complex to the TCR.

Although the principal function of MHC molecules is to present microbial peptides to T cells, they are also the most prominent transplantation antigens. Non-self-MHC is recognized by 1–10 % of the T cells of an individual (Chapter 1). Since T cells are selected to recognize foreign peptides in the context of self-MHC, it is thought that cross-reactivity of TCRs allows allorecognition by T cells. Allorecognition by T cells, therefore, includes the recognition of self-peptide:non-self-MHC, non-self-peptide:self-MHC or non-self-peptide:non-self-MHC complexes. The different models proposed to explain TCR cross-reactivity are reviewed by Yin & Mariuzza [7].

mHAgs

The mHAgs play an important role in allorecognition and account for the rejection of HLA-matched organs and graft versus host disease (GvHD) after hematopoietic stem cell (bone marrow) transplantation. An example of a clinically relevant mHAg is the H-Y antigen present in male mice and humans. In mice, transplantation of skin from a male to an otherwise syngeneic female recipient will result in rejection of the graft in most strains [8]. Recent reports reviewed the role of H-Y and other mHAgs in solid organ transplantation [9, 10]. In humans, Gratwohl et al. [9] showed in a retrospective multivariate analysis that female recipients of male kidney transplants have a higher risk of graft failure, suggesting an immunological effect of H-Y in kidney transplantation. The effect of sex-mismatched hematopoietic stem cell transplantation (female donor to male recipient) leading to GvHD has been well described [11, 12]. Antigen-specific T cells and antibodies against H-Y-encoded gene products could be detected in these recipients [13–15]. So far, more than 50 mHAgs in mice and humans have been described, with differences between donor and recipient ranging from single amino acid residues to extensive variation, but it is estimated that there may be hundreds more. mHAgs are presented in the context of recipient MHC class I and II. How subtle differences in mHAgs influence their presentation by MHC molecules and lead to TCR recognition is an area of active investigation.

Other transplantation antigens

No discussion of transplantation antigen is complete without underscoring the fact that the first alloantigens to be discovered were those that define blood groups in humans, the ABO system being the most prominent. ABO antigens are carbohydrate determinants expressed on red blood cells, as well as in other cells and tissues. Since they do not evoke T cell responses, they are not classified as mHAgs. Matching for ABO blood types made both blood transfusion and organ transplantation possible – as transplantation across ABO-incompatibility without prior desensitization led to the hyperacute rejection of organ allografts. Hyperacute rejection is caused by preformed IgM antibodies produced during infancy by B-1 lymphocytes, so-called innate B cells, independent of T cell help (Chapter 4).

APCs

An important link between the innate and adaptive immune system are professional APCs. APCs take up, process, and present antigens in the context of MHC molecules and, in addition, possess the necessary molecules to activate or inhibit the adaptive immune system through a variety of cell-surface proteins; these include adhesion molecules (e.g., integrins) and costimulatory molecules (e.g., B7 family members CD80 and CD86) (Chapter 3). DCs, macrophages, and B cells are the main populations of professional APCs. Other APCs include subtypes of phagocytic cells, such as Kupfer and stellate cells in the liver.

DCs

DCs were first described morphologically by Steinman and colleagues and were found to be potent stimulators of the mixed leukocyte reaction (MLR) [16, 17], an in vitro test that measures T cell alloreactivity. All DCs originate from myeloid precursors in the bone marrow and are distributed throughout most tissues of the body. DCs are specialized in antigen uptake through a variety of different mechanisms, which include phagocytosis, macropinocytosis, and receptor-mediated endocytosis (e.g., via Fc, C-type lectin, scavenger, and complement receptors). They also process antigens into small peptides that are presented in the context of MHC molecules. DCs are highly migratory. Activated DCs that have taken up antigen and have received innate activation signals (e.g., from microbes) mature and migrate to secondary lymphoid tissue where they present antigen to T cells.

DCs are often divided into lymphoid tissue DCs (CD8+ and CD8− DC) and non-lymphoid tissue DCs (tissue-specific subsets, like Langerhans cells in the skin or intestinal CD103+ and CD103− DC) [18, 19]. Non-lymphoid tissue DC subsets differ in their migratory properties, although in general they migrate to secondary lymphoid tissue after antigen uptake and innate activation. While most DCs originate from pre-DCs in the bone marrow (these DCs are increasingly referred to as conventional DCs or cDCs), there is increasing evidence that monocytes are the precursors of certain non-lymphoid tissue DC subsets under steady-state conditions and, more importantly, upon inflammation. DCs play a twofold role in transplantation: donor DCs that accompany the allograft can be directly recognized by the recipient’s T cells (direct allorecognition), whereas recipient’s DCs take up donor alloantigen, process it, and present it to recipient T cells (indirect allorecognition) (see next section).

DCs also include a distinct but small subset known as plasmacytoid DCs (pDCs) because of their plasma cell-like morphology [20]. Despite their name, pDCs derive from a myeloid precursor in the bone marrow; but unlike other DCs, they express CD4 and produce large amounts of type I interferons in response to viral infection. They are capable of presenting alloantigen to T cells, but recent evidence suggests that pDCs may play a role in tolerance induction in transplantation [21].

Macrophages and monocytes

Macrophages are phagocytic cells that engulf pathogens and clear dying cells. They are equipped with a variety of receptors, such as mannose, scavenger, and complement receptors. Macrophages, like DCs, process antigen and present it in the context of MHC molecules, but they are not as potent activators of the MLR as DCs. Because macrophages scavenge dead cells, expression of costimulatory molecules is tightly regulated and thought to be only upregulated in the context of non-self recognition. Monocytes are rapidly recruited to sites of inflammation and are thus also a prominent cell type in allograft rejection. Although their main role has been seen as pro-inflammatory effector cells that differentiate into macrophages and contribute to graft destruction, there is increasing evidence that they differentiate into either DCs or immunregulatory cells sometimes referred to as myeloid-derived suppressor cells [22]. The role of host monocyte-derived DCs in transplantation is currently under investigation.

B lymphocytes

B cells can bind specific soluble antigens through their cell surface immunoglobulin, internalize them, and present peptides in the context of MHC class II. B cells constitutively express high levels of MHC class II, but require induction of costimulatory molecules for efficient T cell priming upon activation. Antigen presentation by B cells has been shown to be relevant in autoimmunity [23–25] and microbial immunity [26], as well as in transplantation [27, 28]. In the latter case, antigen presentation by B cells has been shown to potentiate the alloimmune response, leading to increased generation of memory T cells.

Antigen presentation pathways

Three pathways of antigen presentation have been described in the context of transplantation: direct, indirect, and semi-direct (Figure 2.2). In direct presentation, recipient T cells recognize self- or non-self-peptides presented in the context of allogeneic, donor MHC molecules expressed on donor APCs [29, 30]. In indirect presentation, alloantigens are taken up and processed by recipient APCs and presented in the context of recipient MHC molecules to recipient T cells. Semi-direct presentation refers to the acquisition of intact donor MHC:peptide complexes by recipient APCs and their presentation to recipient T cells.

Figure 2.2 Pathways of alloantigen presentation. The three pathways of alloantigen presentation (direct, indirect, and semi-direct) are shown. The frequency (%) of T cells that react to a specific pathway is indicated where known. APC: antigen-presenting cell; MHC: major histocompatibility complex molecule; TCR: T cell receptor for antigen.

Direct alloantigen presentation

Direct presentation has historically been viewed as the major pathway involved in acute allograft rejection. This view is supported by the strong T cell proliferation observed in the MLR, as well as by the original studies of Lafferty and coworkers showing that depletion of APCs (so-called passenger leukocytes) from thyroid allografts by culturing prior to transplantation results in prolonged graft survival in the absence of immunosuppression [31, 32]. Another early study by Lechler et al. showed that retransplantation of renal allografts parked in an intermediate, immunosuppressed host (causing depletion of donor APCs) leads to graft acceptance in rats [33]. Pietra et al. showed that CD4 T cells adoptively transferred to severe combined immunodeficiency (SCID) or recombination-activating-gene-deficient (Rag−/−) mice were not able to reject MHC class II deficient cardiac allografts [34]. Conversely, Rag−/− MHC class II−/− recipients were able to reject the cardiac allografts after the adoptive transfer of CD4 T cells, indicating that CD4 T cells with direct allorecognition are sufficient to mediate allograft rejection. The high precursor frequency of directly alloreactive T cells is most likely due to cross-reactivity [35]. Supportive of this hypothesis are the findings that in humans a large number of alloreactive T cells have a memory phenotype [36, 37]. Human memory T cell populations contributing to the alloresponse contain virus-specific T cells (e.g., reactivity against EBV) that cross-react with specific alloantigens (e.g., HLA-B44) [38]. Since direct recognition is dependent on the presence of donor APCs in the allograft, it is thought to dominate T cell priming in the immediate post-transplantation period. After donor APC frequency declines by means of apoptosis and rejection by the recipient’s immune response, the number of direct recognition recipient T cells also declines. Therefore, it is presumed that direct antigen presentation is particularly relevant to acute rejection.

Indirect alloantigen presentation

Indirect antigen presentation is in fact the default pathway of T cell priming outside of transplantation, as T cells are selected to recognize antigens in the context of self-MHC. In transplantation, it was suggested as a pathway of allograft rejection after the observation that donor-leukocyte-depleted renal allografts in rats were still rejected, although much more slowly than nondepleted allografts [39]. Accelerated allograft rejection was demonstrated by Fabre and coworkers by immunizing recipients with peptides of allogeneic donor MHC antigens [40]. More evidence came from experiments using skin grafts from MHC class II−/− mice, which fail to initiate direct recognition by CD4 T cells and were still rejected in a CD4 T-cell-dependent manner by MHC class I−/− recipients (these recipients lack CD8 T cells) [41]. Indirect allorecognition, therefore, is largely host CD4 T cell dependent and MHC class II dependent. This could be further demonstrated in murine cardiac allograft experiments using combinations of MHC class I, class II, and double knock-out mice as donors or recipients [42]. Here, MHC class II deficiency of the cardiac allograft was necessary to obtain indefinite graft survival, and a corresponding experiment resulted in prolonged graft survival only if the recipient lacked CD4 T cells (MHC class II−/− recipient). Because donor-derived APCs (passenger leukocytes) are eliminated within a short period after transplantation, it is thought that indirect allorecognition is the principal alloantigen presentation pathway that leads to chronic rejection. There is ample evidence to support this in mice and humans, but it is also increasingly recognized that indirect allorecognition is sufficient and perhaps critical for acute rejection.

Like CD4 T cells, CD8 T cells are also capable of indirect recognition, a phenomenon known as cross-priming. Cross-priming refers to the process of presenting peptides derived from exogenous donor alloantigens in the context of recipient MHC class I molecules on recipient APCs. One study provided evidence that recipient endothelium can indirectly cross-present donor skin graft antigens in the context of MHC class I to TCR-transgenic CD8 T cells, which resulted in skin graft rejection [43]. However, the same group showed that, in a cardiac allograft model, indirectly primed CD8 T cells are nonpathogenic bystanders [44]. The significance of the indirect CD8 T cell pathway (cross-priming) in organ transplantation is, therefore, not completely understood.

Semi-direct alloantigen presentation

Semi-direct presentation has been described as a novel pathway of alloantigen presentation by APCs [45]. Herrera et al. reported that mouse DCs acquire allogeneic MHC:peptide complexes after in vitro incubation with allogeneic DCs but also in vivo upon migration of donor APCs to regional lymph nodes. The semi-direct antigen presentation model proposed would link T cells with direct and indirect allospecificity. The traditional model of CD4 T cell help for generating cytotoxic CD8 T cells requires priming by the same APCs. In transplantation, this could unlink the direct from the indirect presentation pathway. The semi-direct presentation model allows a recipient APC to present processed allopeptide:self-MHC complexes as well as acquired allopeptide:donor MHC complexes, linking both the direct and indirect presentation pathway (Figure 2.2). This model also helps explain the finding that embryonic thymic epithelium, which lacks APCs, is rejected in the absence of indirect allorecognition [46] and that costimulation-deficient cardiac allografts (CD80−/−CD86−/− donors) are acutely rejected by MHC class II−/− recipients lacking the indirect pathway [47]. The significance of the semi-direct pathway in vivo has not yet been fully determined.

Migration of APCs and sites of interaction with T cells

APC trafficking in organ transplantation encompasses the migration of donor DCs from the graft to the secondary lymphoid tissues (e.g., spleen and lymph nodes) where they interact with host naive and memory T cells, and the migration of host monocytes and other DC precursors to the graft. The latter migration pattern leads to repopulation of the graft with host-derived DCs, which interact with effector and memory T cells in the graft or migrate back to the secondary lymphoid tissues after picking up donor alloantigens. It is important to note that naive T cell activation by APCs occurs within the organized structures of secondary lymphoid tissues, whereas memory T cells can be activated in lymphoid and non-lymphoid tissues (e.g., within the graft itself) [48–50]. One exception to this rule is the activation of naive T cells within the graft if ectopic lymphoid tissues, also known as tertiary lymphoid tissues, are present, as is the case in organs undergoing chronic inflammation/rejection [51–54]. Naive T cell activation by resident APCs also occurs in lung allografts, which contain broncho-alveolar lymphoid tissues (BALT), and in small-bowel allografts, which contain Peyer’s patches [55, 56]. Since naive and memory T cells are both involved in initiating and propagating the alloimmune response in humans (Chapter 1), all three sites of T cell activation (the graft, secondary lymphoid tissues, and tertiary lymphoid tissues) are important in the rejection process.

DC migration from sites of inflammation to secondary lymphoid tissues occurs via the blood or afferent lymphatic channels [57]. The principal mechanism responsible for DC migration to lymph nodes is the chemokine receptor CCR7. CCR7 is a G protein-coupled receptor that binds CCL19 and CCL21, chemokines present in high concentration on high endothelial venules in lymph nodes. The adhesion molecules involved in adhesion and transendothelial migration of DCs into lymph nodes are L-selectin and possibly other molecules, such as the C-type lectin receptor DC-SIGN (CD209). Monocyte migration from the bone marrow, spleen, and blood into sites of inflammation, such as the graft, is dependent on the chemokine receptors CCR2 and/or CX3CR1 (fractalkine receptor) [58].

Relevance to immunotherapy

Although immunosuppressive agents currently in clinical use for solid organ transplantation are not viewed as immunotherapies that target antigen presentation, some do influence APC function. Prime examples are corticosteroids and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. There is evidence that corticosteroids suppress the maturation of and MHC class II and inflammatory cytokine expression by monocytes, macrophages, and DCs, making them less effective at presenting antigen and activating T cells. Rapamycin, on the other hand, has contrasting effects on APCs [59]. It inhibits the maturation of in-vitro-generated DCs but surprisingly also increases antigen presentation by enhancing autophagy in monocytes, macrophages, and DCs. The complex effects of rapamycin on the immune system could explain its pleiotropic effects in humans and may underlie its less than predictable effects on long-term allograft survival.

Further elucidation of the types of APC and antigen-presentation pathways involved in allograft rejection promises to yield novel immunotherapies in transplantation. Potential strategies include (a) specific targeting of APC subsets by monoclonal antibodies or pharmacological agents, (b) inhibition of APC migration from or to the graft, and (c) manipulating DCs in vitro to render them tolerogenic. Tolerogenic DCs can then be administered to humans at the time of transplantation to downmodulate the alloimmune response by a process known as negative vaccination [60]. APCs, therefore, provide an attractive target for immunotherapy in transplantation.

References

1 Cresswell P. Antigen processing and presentation. Immunol Rev 2005;207:5–7.

2 Bevan MJ. Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay. J Exp Med 1976;143(5):1283–1288.

3 Bevan MJ. Cross-priming. Nat Immunol 2006;7(4):363–365.

4 Dissanayake SK, Tuera N, Ostrand-Rosenberg S. Presentation of endogenously synthesized MHC class II-restricted epitopes by MHC class II cancer vaccines is independent of transporter associated with Ag processing and the proteasome. J Immunol 2005;174(4):1811–1819.

5 Nimmerjahn F, Milosevic S, Behrends U et al. Major histocompatibility complex class II-restricted presentation of a cytosolic antigen by autophagy. Eur J Immunol 2003;33(5):1250–1259.

6 Paludan C, Schmid D, Landthaler M et al. Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 2005;307(5709):593–596.

7 Yin Y, Mariuzza RA. The multiple mechanisms of T cell receptor cross-reactivity. Immunity 2009;31(6):849–851.

8 Eichwald EJ, Silmser CR. Skin. Transplant Bull 1955;2:148–149.

9 Gratwohl A, Dohler B, Stern M, Opelz G. H-Y as a minor histocompatibility antigen in kidney transplantation: a retrospective cohort study. Lancet 2008;372(9632):49–53.

10 Dierselhuis M, Goulmy E. The relevance of minor histocompatibility antigens in solid organ transplantation. Curr Opin Organ Transplant 2009;14(4):419–425.

11 Miklos DB, Kim HT, Miller KH et al. Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission. Blood 2005;105(7):2973–2978.

12 Stern M, Passweg JR, Locasciulli A et al. Influence of donor/recipient sex matching on outcome of allogeneic hematopoietic stem cell transplantation for aplastic anemia. Transplantation 2006;82(2):218–226.

13 Goulmy E, van Leeuwen A, Blokland E et al. Major histocompatibility complex-restricted H-Y-specific antibodies and cytotoxic T lymphocytes may recognize different self determinants. J Exp Med 1982;155(5):1567–1572.

14 Voogt PJ, Fibbe WE, Marijt WA et al. Rejection of bone-marrow graft by recipient-derived cytotoxic T lymphocytes against minor histocompatibility antigens. Lancet 1990;335(8682):131–134.

15 Spierings E, Vermeulen CJ, Vogt MH et al. Identification of HLA class II-restricted H-Y-specific T-helper epitope evoking CD4+ T-helper cells in H-Y-mismatched transplantation. Lancet 2003;362(9384):610–615.

16 Steinman RM, Witmer MD. Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proc Natl Acad Sci U S A 1978;75(10):5132–5136.

17 Steinman RM, Cohn ZA. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med 1973;137(5):1142–1162.

18 Liu K, Nussenzweig MC. Origin and development of dendritic cells. Immunol Rev 2010;234(1):45–54.

19 Helft J, Ginhoux F, Bogunovic M, Merad M. Origin and functional heterogeneity of non-lymphoid tissue dendritic cells in mice. Immunol Rev 2010;234(1):55–75.

20 Swiecki M, Colonna M. Unraveling the functions of plasmacytoid dendritic cells during viral infections, autoimmunity, and tolerance. Immunol Rev 2010;234(1):142–162.

21 Ochando JC, Homma C, Yang Y et al. Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat Immunol 2006;7(6):652–662.

22 Geissmann F, Manz MG, Jung S et al. Development of monocytes, macrophages, and dendritic cells. Science 2010;327(5966):656–661.

23 O’Neill SK, Shlomchik MJ, Glant TT et al. Antigen-specific B cells are required as APCs and autoantibody-producing cells for induction of severe autoimmune arthritis. J Immunol 2005;174(6):3781–3788.

24 Chan OT, Hannum LG, Haberman AM et al. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus. J Exp Med 1999;189(10):1639–1648.

25 Noorchashm H, Lieu YK, Noorchashm N et al. I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice. J Immunol 1999;163(2):743–750.

26 Lund FE, Hollifield M, Schuer K et al. B cells are required for generation of protective effector and memory CD4 cells in response to Pneumocystis lung infection. J Immunol 2006;176(10):6147–6154.

27 Noorchashm H, Reed AJ, Rostami SY et al. B cell-mediated antigen presentation is required for the pathogenesis of acute cardiac allograft rejection. J Immunol 2006;177(11):7715–7722.

28 Ng YH, Oberbarnscheidt MH, Chandramoorthy HC et al. B cells help alloreactive T cells differentiate into memory T cells. Am J Transplant 2010;10(9):1970–1980.

29 Warrens AN, Lombardi G, Lechler RI. Presentation and recognition of major and minor histocompatibility antigens. Transpl Immunol 1994;2(2):103–107.

30 Whitelegg A, Barber LD. The structural basis of T-cell allorecognition. Tissue Antigens 2004;63(2):101–108.

31 Talmage DW, Dart G, Radovich J, Lafferty KJ. Activation of transplant immunity: effect of donor leukocytes on thyroid allograft rejection. Science 1976;191(4225):385–388.

32 Lafferty KJ, Cooley MA, Woolnough J, Walker KZ. Thyroid allograft immunogenicity is reduced after a period in organ culture. Science 1975;188(4185):259–261.

33 Batchelor JR, Welsh KI, Maynard A, Burgos H. Failure of long surviving, passively enhanced kidney allografts to provoke T-dependent alloimmunity. I. Retransplantation of (AS X AUG)F1 kidneys into secondary AS recipients. J Exp Med 1979;150(3):455–464.

34 Pietra BA, Wiseman A, Bolwerk A et al. CD4 T cell-mediated cardiac allograft rejection requires donor but not host MHC class II. J Clin Invest 2000;106(8):1003–1010.

35 Gras S, Kjer-Nielsen L, Chen Z et al. The structural bases of direct T-cell allorecognition: implications for T-cell-mediated transplant rejection. Immunol Cell Biol 2011;89(3):388–395.

36 Merkenschlager M, Ikeda H, Wilkinson D et al. Allorecognition of HLA-DR and-DQ transfectants by human CD45RA and CD45R0 CD4 T cells: repertoire analysis and activation requirements. Eur J Immunol 1991;21(1):79–88.

37 Lombardi G, Sidhu S, Daly M et al. Are primary alloresponses truly primary? Int Immunol 1990;2(1):9–13.

38 Macedo C, Orkis EA, Popescu I et al. Contribution of naive and memory T-cell populations to the human alloimmune response. Am J Transplant 2009;9(9):2057–2066.

39 Lechler RI, Batchelor JR. Immunogenicity of retransplanted rat kidney allografts. Effect of inducing chimerism in the first recipient and quantitative studies on immunosuppression of the second recipient. J Exp Med 1982;156(6):1835–1841.

40 Benham AM, Sawyer GJ, Fabre JW. Indirect T cell allorecognition of donor antigens contributes to the rejection of vascularized kidney allografts. Transplantation 1995;59(7):1028–1032.

41 Auchincloss H, Jr, Lee R, Shea S et al. The role of indirect recognition in initiating rejection of skin grafts from major histocompatibility complex class II-deficient mice. Proc Natl Acad Sci U S A 1993;90(8):3373–3377.

42 Campos L, Naji A, Deli BC et al. Survival of MHC-deficient mouse heterotopic cardiac allografts. Transplantation 1995;59(2):187–191.

43 Valujskikh A, Lantz O, Celli S et al. Cross-primed CD8+ T cells mediate graft rejection via a distinct effector pathway. Nat Immunol 2002;3(9):844–851.

44 Valujskikh A, Zhang Q, Heeger PS. CD8 T cells specific for a donor-derived, self-restricted transplant antigen are nonpathogenic bystanders after vascularized heart transplantation in mice. J Immunol 2006;176(4):2190–2196.

45 Herrera OB, Golshayan D, Tibbott R et al. A novel pathway of alloantigen presentation by dendritic cells. J Immunol 2004;173(8):4828–4837.

46 Pimenta-Araujo R, Mascarell L, Huesca M et al. Embryonic thymic epithelium naturally devoid of APCs is acutely rejected in the absence of indirect recognition. J Immunol 2001;167(9):5034–5041.

47 Mandelbrot DA, Kishimoto K, Auchincloss H, Jr et al. Rejection of mouse cardiac allografts by costimulation in trans. J Immunol 2001;167(3):1174–1178.

48 Lakkis FG, Arakelov A, Konieczny BT, Inoue Y. Immunologic ‘ignorance’ of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat Med 2000;6:686–688.

49 Chalasani G, Dai Z, Konieczny BT et al. Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs. Proc Natl Acad Sci U S A 2002;99:6175–61780.

50 Lakkis F. Where is the alloimmune response initiated? Am J Transplant 2003;3:241–242.

51 Baddoura FK, Nasr IW, Wrobel B et al. Lymphoid neogenesis in murine cardiac allografts undergoing chronic rejection. Am J Transplant 2005;5(3):510–516.

52 Nasr IW, Reel M, Oberbarnscheidt MH et al. Tertiary lymphoid tissues generate effector and memory T cells that lead to allograft rejection. Am J Transplant 2007;7(5):1071–1079.

53 Kerjaschki D, Regele HM, Moosberger I et al. Lymphatic neoangiogenesis in human kidney transplants is associated with immunologically active lymphocytic infiltrates. J Am Soc Nephrol 2004;15(3):603–612.

54 Thaunat O, Field A-C, Dai J et al. Lymphoid neogenesis in chronic rejection: evidence for a local humoral alloimmune response. Proc Natl Acad Sci U S A 2005;102:14723–14728.

55 Gelman AE, Li W, Richardson SB et al. Cutting edge: acute lung allograft rejection is independent of secondary lymphoid organs. J Immunol 2009;182(7):3969–3973.

56 Wang J, Dong Y, Sun JZ et al. Donor lymphoid organs are a major site of alloreactive T-cell priming following intestinal transplantation. Am J Transplant 2006;6(11):2563–2571.

57 Randolph GJ, Ochando J, Partida-Sanchez S. Migration of dendritic cell subsets and their precursors. Annu Rev Immunol 2008;26:293–316.

58 Gelman AE, Okazaki M, Sugimoto S et al. CCR2 regulates monocyte recruitment as well as CD4 T1 allorecognition after lung transplantation. Am J Transplant 2010;10(5):1189–1199.

59 Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nat Rev Immunol 2009;9(5):324–337.

60 Thomson AW. Tolerogenic dendritic cells: all present and correct? Am J Transplant 2010;10(2):214–219.

CHAPTER 3

The T Cell Response to Transplantation Antigens

Didier A. Mandelbrot¹, Bryna Burrell², Mohamed H. Sayegh³, and Peter S. Heeger⁴

¹The Transplant Institute and Harvard Medical School, Boston, MA, USA

²Mount Sinai School of Medicine, Recanati/Miller Transplantation Institute, New York, NY, USA

³Brigham and Women’s Hospital, Boston, MA, USA

⁴Mount Sinai School of Medicine, Recanati/Miller Transplantation Institute, New York, NY, USA

Basic T cell lexicon

T lymphocytes are a critical component of immune responses. Using their T cell receptors (TCRs), they recognize specific peptide antigens presented in the context of major histocompatibility (MHC) molecules expressed on antigen-presenting cells (APCs). The two main types of mature T cells are CD4-expressing T helper (Th) lymphocytes, which recognize peptides expressed on class II MHC, and the CD8-expressing cytolytic T lymphocytes (CTLs), which recognize peptides expressed on class I MHC. CD4 T cells regulate immune responses both by directly engaging signaling receptors on APCs and by secreting cytokines, which act locally to regulate additional T cells, B cells and other leukocytes. CD4 T cells are important in both stimulating and downregulating various specific immune responses. CD8 T lymphocytes can kill cells expressing foreign antigens, such as cells infected with microbes or transplanted cells that express alloantigens, but also produce cytokines. While circulating T cells express either CD4 or CD8, during thymic development T cell precursors can express one, neither (double negative, DN), or both (double positive, DP) of these molecules.

T cell activation requires two signals. The first signal is provided by cognate interactions between the Ag/MHC complex on APCs and the TCRs on T cells. The second, or costimulatory, signal is provided by ligands on APCs such as B7-1 and B7-2 to receptors on T cells, such as CD28. Further activation signals are provided by cytokines such as IL-2 and by numerous intracellular signaling pathways. For example, the calcineurin and target of rapamycin (TOR) pathways transmit and integrate signals from cell-surface receptors, resulting in T cell proliferation and differentiation. Activation of naive T cells, which occurs predominantly in secondary lymphoid organs (LN and spleen), induces proliferation, differentiation, and an ability to migrate to peripheral organs where, upon re-encounter of their specific antigen, they mediate effector functions. Thus, CD4 T cells serve to regulate both the afferent and efferent limbs of the immune response: they both recognize the transplanted graft antigens and mediate the effector mechanisms that reject grafts. CD4 cells can differentiate into Th1, Th2, and Th17 cells, which have distinct phenotypes and functions in immune responses. CD4 cells can also differentiate into regulatory T cells (Tregs), which are important for downregulating immune responses, and into memory cells. In the effector phase of the alloresponse, helper CD4 cells provide direct cellular signals as well as cytokine signals to other CD4 cells, CD8 cells, B cells, and macrophages. Interactions between APCs and T cells, as well as leukocyte trafficking in general, are regulated by a number of adhesion molecules, such as leukocyte factor antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1.

Anti-rejection therapies in clinical use target both cell-surface molecules, such as CD3 and CD25, and intracellular signaling pathways. Therapies that are likely to be used in the future target B7, CD28, CD40, and other costimulatory molecules, as well as adhesion molecules [1].

T cell development and maintenance

Origins of T cell precursors

T cells originate in the fetal liver prenatally and in the bone marrow as hematopietic stem cells (HSCs) postnatally [2]. HSC lymphoid progenitors were initially described as Thy-1.1lo, Lineage (Lin)− and stem cell antigen-1 (Sca-1)+ [3]. Although only 0.02–0.06 % of mouse bone marrow cells fit this phenotype, reconstitution by as few as 30 of these cells results in the rescue and viability of half of syngeneic irradiated mice [3]. Further characterization shows these cells to fall into one of three groups: long-term HSCs possessing unlimited self-renewal capacity, short-term HSCs with some degree of self-renewal capacity, and multipotent progenitors that cannot self-renew [4]. These groups can be further differentiated based on low versus negative expression of Mac-1 and CD4. In irradiated recipients, HSCs negative for these markers provide long-term reconstitution, whereas low expression of these markers on HSCs correlates with transient reconstitution [5]. Additionally, HSCs are delineated by CD34 expression in humans [6] and are CD117 (c-kit)+ and CD34lo/− in mice [7]. Lymphoid progenitors are thought to be Lin−c-kitloIL-7Rα+ [4].

T cell maturation

T cells mature in the thymus, and at least six T cell populations are exported from the thymus: γ/δ T cells, naive CD4+ and CD8+ α/β T cells, NKT cells, regulatory T cells, and intraepithelial lymphocytes [2]. Thymocyte precursors have only a limited self-renewal capacity [2], and primitive lymphoid cells are continually imported into the thymic medullary cortex [8] from the circulation to varying degrees throughout life [9]. As these cells progressively develop through stages of maturation, their progeny lose their differentiation potential. Once in the thymus, this CD4 and CD8 negative (DN) precursor population migrates through the thymus and progresses through the DN stages [8], DN2 (CD25+CD44+), DN3 (CD25+CD44−), and DN4 (CD25−CD44−), as cells migrate across the cortex to the subcapsular zone (SCZ) [8] (Figure 3.1). Rearrangement of the TCR β-chain locus occurs during the DN2 to DN3 progression. DN3