Immune Biology of Allogeneic Hematopoietic Stem Cell Transplantation: Models in Discovery and Translation

antigens

CH 1

Overview of the immune biology of allogeneic hematopoietic stem cell transplantation

Gérard Socié

Service d’Hématologie-Greffe de Moelle, Hôpital Saint-Louis, AP-HP, Paris, Université Paris VII Denis-Diderot, and Unité INSERM U940, Paris, France

Bruce R. Blazar

Cancer Center and Department of Pediatrics, Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, Minnesota, USA

Introduction

Much of our understanding of the biology of graft-versus-host disease (GVHD) has developed from two preclinical animal models, the mouse and the dog (reviewed in references [1–6]). Since there are significant species differences between humans and mice, five points are important to consider when drawing conclusions from studies with animal models and before correlation to the clinical allogeneic hematopoietic stem cell transplantation (HSCT) scenario (Box 1.1).

BOX 1.1 Caveats in directly translating results from animal models into human studies

1. Conditioning regimen

• In murine studies, usually irradiation alone (without chemotherapy)

• Irradiation often is used with large fraction doses and high dose rates not commonly used in patients

2. Immunological disparity between donor and recipient

• Inbred strain combinations are used, resulting in a variety of MHC- and/or minor histocompatibility antigen (MiHA)-disparate models

• These different strain combinations have different Th1/Th2/Th17 as well as Treg content and can sway the dominance of CD4+ or CD8+ T-cell effectors in GVHD

• Thus, conclusions in one model may not translate into other immunologically distinct models or into the clinic

3. Source of donor cells

• Typically spleen cells and/or lymph node T cells are added to the bone marrow graft, in contrast to peripheral blood or bone marrow grafts (contaminated with peripheral blood cells) in human studies

4. Microbial baseline

• Whereas in rodents, mice are housed under specific pathogen free conditions since birth, humans are not. Therefore, extrapolation of murine data between laboratories may be difficult and clinical translation of such findings into HSCT recipients may be even more challenging, especially for those innate and adaptive immune responses most readily influenced by the microenvironment

5. Age of the donors and recipients

• The majority of murine HSCT studies use primarily young adult mice and only infrequently older mice will be used. Older age in mice is known to alter antigen-presenting cell (APC) capacity, thymopoiesis and peripheral T-cell recovery, and sensitivity to radiation

6. GVHD prophylaxis

• Generally not included systematically in the experimental setting

7. GVL

• Experimental studies are limited by the use of a limited repertoire of cell lines and only infrequently transformed or mutated primary hematopoietic cells given to the host

In this overview we introduce the main concepts and experimental results concerning the major areas developed in the book including the GVHD, the graft-versus-leukemia (GVL) effect, rejection and immune deficiency.

We will focus on recent advances and their translation into clinical knowledge or therapies. In each section we summarize key experimental data and then provide a perspective as to how these data succeeded or failed to be translated to the bedside. The main differences between experimental systems and human beings, as well the tools used to study GVHD and GVL, are illustrated in Figure 1.1.

FIGURE 1.1 (a) Tools in experimental GVHD. (b) Tools and challenges in studying human GVHD. KO = knock out; pbmnc = peripheral blood mononuclear cells.

Immune rejection

Preclinical studies show that allogeneic hematopoietic stem cell (HSC) graft rejection can be mediated by host natural killer (NK) cells, NK T cells, γδ T cells, and/or CD4+ and CD8+ T cells that recognize histocompatibility antigens (MiHA) on the donor cells (reviewed in chapter 5, and in references [6] and [7]). In clinical practice, graft rejection of related HLA-identical bone marrow (BM) or mobilized peripheral blood (PB) after myeloablative conditioning is rare. Graft rejection or graft failure occurs primarily following transplant of cells from related HLA-mismatched or matched unrelated donors (MUDs) and/or use of T-cell-depleted grafts. Rejection or graft failure can be assessed by the extent of donor chimerism measuring the proportion of the recipient cells. However, early elimination of myeloid precursors and their progeny does not always correlate with long-term engraftment, and researchers should take care to avoid over-interpretation of results. Early studies to characterize murine NK cells suggested that host NK cells could reject donor BM in a non-MHC-restricted manner, as evidenced by a phenomenon called hybrid resistance in which parental BM cells are rejected by F1 hybrid recipients. Investigators now recognize that NK cells bear inhibitory and activating receptors directed to MHC and other cellular determinants that are critical to target cell identification and subsequent NK cell-mediated killing (reviewed in chapter 15). During graft rejection, the effector pathways used by recipient T cells differ on the basis of prior sensitization of the host to alloantigen. In naïve, un-sensitized recipient mice, perforin, granzyme B and Fas/FasL can mediate rejection of MHC- and/or MiHA mismatched BM by CD8+ T cells. CD4+ T cells mediate allogeneic BM destruction. However, CD8+ T cells from sensitized recipients with alloantigen can reject BM by an unknown mechanism that appears independent of the numerous pathways, as determined using gene knockout mice and neutralizing antibodies. Prior exposure to histocompatibility antigens, which can occur by blood product transfusions, pregnancies or immunization in experimental models, is attributed to cytotoxic T cells, which can be identified in alloantigen-sensitized recipients. However, antibodies capable of recognizing MHC or MiHA on donor cells can induce graft rejection or lineage-specific aplasia.

Immune deficiency

A major problem limiting the efficacy of allogeneic HSCT is the issue of promoting immune reconstitution without increasing GVHD (reviewed in chapter 6 and in reference [6]). Patients are profoundly immunosuppressed following transplant as a result of the cytoreductive conditioning, immunosuppressive drugs to prevent GVHD, and the paucity of transplanted T cells compared with the size of the T-cell compartment in an immune competent person. In addition, acute GVHD induces lymphoid hypoplasia, thus tying GVHD to immune impairment. This leaves the patient susceptible to a number of opportunistic infections. Infectious complications associated with neutropenia early post-transplant are no longer as prominent in clinical practice. However, cytomegalovirus, Epstein–Barr virus (EBV) and fungal infections, predominantly Candida species and Aspergillus fumigatus that arise after neutrophil recovery, are now major contributors to morbidity and mortality following allogeneic HSCT. Unfortunately, there are few preclinical models that have been developed to study these opportunistic infections and the complicating effects of GVHD on their occurrence.

There are two sources for T cells in the recovering recipient: peripheral expansion of mature T cells and de novo production of naïve T cells derived from transplanted stem cells and produced in the recipient thymus. However, the thymus begins to involute at puberty, and the capacity for thymic-derived T-cell production is greatly diminished in adulthood. In addition, the cytoreductive conditioning can induce tissue damage to the epithelial cells of the thymus and a decreased ability to produce IL-7. Thus, a reduced ability to generate new T cells is a function both of increasing age and of conditioning dose intensity. An older HSCT recipient is especially prone to limited recovery of the CD4+ T-cell repertoire following allogeneic HSCT. A slow recovery is associated with an increased risk of opportunistic infections and a decreased ability to generate a response to vaccination. The benefit of de novo generation of T cells post-transplant is the production of donor-derived T cells that are tolerant of both the graft and the recipient and generation of a broad T-cell receptor (TCR) repertoire.

Enhancing immune reconstitution is an area of intensive research. An increasing variety of approaches has been explored pre-clinically and clinically: infusion of IL-7, keratinocyte growth factor, growth hormone, mature cytotoxic lymphocytes with defined immunological properties against pathogens or tumor antigens and blockade of sex hormones. New developments of allogeneic HSCT, e.g. umbilical cord blood or haploidentical graft preparations leading to prolonged immunodeficiency, have further increased the need to improve immune reconstitution. While slow T-cell reconstitution is regarded as primarily responsible for susceptibility to infections with viruses and fungi, GVHD and propensity for post-HSCT relapse, the importance of innate immune cells for disease and infection control is currently being re-evaluated. In the future, individualized therapy partially based on genetic features of the underlying disease will likely come of age (reviewed in reference [8]).

GVHD pathophysiology

GVHD is a complex disease resulting from donor T-cell recognition of a genetically disparate recipient that is unable to reject donor cells following allogeneic HSCT. The classical scheme of GVHD [2,6,9] development includes five basic steps:

Step 1: Priming of the immune response. Cytoreductive conditioning induces tissue damage and the release of a storm of proinflammatory cytokines that promote the activation and maturation of antigen-presenting cells (APCs) and the rapid amplification of donor T cells [10–12].

Step 2: T-cell activation and costimulation. Activation occurs as the result of the recognition and interaction of the TCR and costimulatory molecules with their cognate ligands expressed on the surface of the APC.

Step 3: Alloreactive T-cell expansion and differentiation.

Step 4: Activated T-cell trafficking. Activated T-cell migration to GVHD target tissues (gut, liver, skin and lung) is followed by the recruitment of other effector leukocytes [13].

Step 5: Destruction of the target tissues by effector T cells. Destruction occurs via exposure to cell surface and release of soluble immune effector molecules. Tissue damage then leads to increased inflammatory signals, perpetuating and augmenting the disease process by contributing to the cytokine storm that fuels GVHD.

Previous reviews [2,6,9,10,13–15] and chapters in this book have detailed these phases of GVHD initiation and tissue destruction.

Priming of the immune response

The earliest phase of acute GVHD is set into motion by the damage caused by the underlying disease and exacerbated by conditioning regimens (reviewed in chapter 8). Damaged host tissues secrete proinflammatory cytokines, such as TNF-α and IL-1, which contribute to the cytokine storm increasing the expression of adhesion molecules, costimulatory molecules, MHC antigens and chemokine gradients that alert the residual host and the infused donor immune cells. These danger signals activate host tissue cells including APCs. Damage to the GI tract from the conditioning is particularly important in this process, because it allows for systemic translocation of lipopolysaccharide that further enhances host APC activation [9,16]. This scenario is in accord with the increased GVHD risk associated with intensive conditioning regimens in some human randomized trials [17–19]. However, preclinical studies in dogs [1,3,20–22] and clinical studies have indicated that reduced intensity conditioning is associated with less morbidity and less early acute GVHD [23].

It is noticeable that IL-1 blockade [24] or protection of epithelial tissue damage by infusion of keratinocyte growth factor, although partially efficacious in some experimental GVHD models [25,26], thus far have proved ineffective in preventing acute GVHD in randomized human trials performed in matched sibling donors [27] (including both randomized IL-1 and KGF trials). Because the mechanisms associated with acute (late onset) GVHD after reduced (eventually minimal) conditioning have not been well elucidated, additional studies are warranted that go back to the bench to develop the so-called mini transplant in the mouse setting that may complement the aforementioned canine investigations.

T-cell activation and costimulation

The core of the graft-versus-host immune reaction lies within the second step, in which donor T cells proliferate and differentiate in response to host APCs [28,29] (reviewed in chapter 9). Recent advances have indicated the presence of a subset of post-mitotic, self-renewing CD44 (lo)/CD62L (hi)/CD8+ T cells that can generate and sustain all allogeneic T-cell subsets in GVHD reactions, including central memory, effector memory and effector CD8+ T cells [30]. The danger signals generated in the first phase augment this activation, at least in part, by increasing expression of costimulatory molecules. In mouse models, in which genetic differences between donor and recipient strains can be tightly controlled, CD4+ T cells induce acute GVHD to MHC class II differences and CD8+ T cells induce acute disease to MHC class I differences [29,31–38]. Under typical bone marrow transplantation (BMT) conditions, murine studies with MiHA-disparate models have demonstrated that GVHD initiation requires donor T-cell recognition of host antigen in the context of host APCs [29,31–38]. Donor-derived APCs are then able to augment CD8+ T-cell-mediated GVHD by acquiring and presenting host antigens [34].

In humans, the incidence of acute GVHD is directly related to the degree of mismatch between HLA determinants [39], mapped by high-resolution DNA typing of HLA genes with PCR-based techniques, largely replacing earlier cellular methods (reviewed in chapter 2). However, recipients of HLA-identical grafts can still develop systemic acute GVHD due to genetic differences that lie outside the MHC loci and that encode proteins referred to as MiHAs (reviewed in chapter 3). Thus, there is strong evidence for MiHA-mismatch mediated GVHD in humans [40–42]. Although individual human MiHA antigens associated with GVHD have been identified, the relative contribution of diverse MiHA and the existence (if any) of single, dominant MiHAs in humans (such as B6dom and H60 that have been well characterized in rodents [43,44]) is unknown. With respect to the donor-versus-host origin of APCs initiating GVHD in humans, little data are available. However, recent studies on the fate of human Langerhans cells, dermal dendritic cell and macrophages in patients suggest that host-derived APCs at least participate to the early stage of the disease [45–47]. Donor and recipient polymorphisms of cytokine genes ascribed to the cytokine storm in rodents and humans have also been implicated as risk factors for the disorder. For example, TNF-α, IL-10 and INF-γ variants have correlated with GVHD in some, but not all, studies (reviewed in reference [48] and in chapter 16). Genetic polymorphisms of proteins connected with innate immunity, such as NOD2, have been associated with acute GVHD in patients [49]. Lastly, in some experimental models, polymorphisms in members of the Toll-like receptor family have been linked to GVHD risk [50].

Costimulatory molecules play pivotal roles in experimental GVHD

A major role for GVHD initiation in rodent models has been ascribed to CD28/CTLA-4 (CD152):B7 interactions which consists of both a positive (CD28/B7) and an inhibitory (CTLA-4:B7) pathway (reviewed in chapter 10). Another B7 supergene family member, ICOS (inducible costimulator) (CD278), binds the ligand B7h (CD275) expressed on host APCs and thereby promotes T-effector responses. Blockade or absence of ICOS on donor T cells diminishes gut and liver GVHD [51,52]. Other costimulatory molecules with potent implication in GVHD include OX40 (CD134), CD40L (CD154), 4–1BB (CD137) and glucocorticoid-induced tumor necrosis factor receptor (GITR).

Inhibitory pathways that downregulate GVHD

In response to tissue injury and activated T cells, inhibitory pathways are upregulated in an attempt to protect the host against injury. CTLA-4 and programmed death-1 (PD-1; CD279) are upregulated on donor T cells during acute GVHD and serve to dampen the immune response. Both also are primarily expressed in the cytoplasm of activated T cells and CD4+CD25+Treg cells (reviewed in references [6,15]). In rodents, selective blockade of CTLA-4:B7 interactions accelerated acute GVHD lethality. Thus, an ideal reagent for inhibiting GVHD would be one that selectively blocks CD28/B7 blockade or absence of PD-1 on donor cells accelerates GVHD and is associated with increased IFN-γ production [53]. Conversely the future development of molecules that signal via PD-1 or its downstream pathways may prove useful in inhibiting GVHD. In addition to surface molecules, the intracellular tryptophan catabolic pathway, indoleamine 2,3-dioxygenase, induced by IFN-γ in GVHD target organs especially in the GI tract, diminishes T effector cell destruction via local mechanisms that result in both an increased donor T-cell apoptosis and decreased proliferation [54–56]. Activation of indoleamine 2,3-dioxygenase or provision of tryptophan catabolites has been shown in rodent models to reduce GVHD.

Translating data on costimulatory molecules for GVHD prevention into the clinic turns out to be much more difficult. Data on a limited number of patients suggest that costimulatory blockade accomplished by adding CTLA4-Ig to an in vitro mixed lymphocyte reaction culture resulted in donor anti-host hypo-responsive T cells that supported relatively rapid T-cell immune recovery and a seemingly low propensity to cause acute GVHD when added to a haploidentical stem cell graft [57,58]. More broadly directed in vitro methodologies have been recently devised to depleted alloreactive T cells and such methodologies have been applied to studies in a limited number of patients [59]. The new CTLA4-Ig derivative of Abatacept, Belatacept, which preferentially blocks CD28/B7 interactions, is highly efficient in the treatment of rheumatoid arthritis and psoriasis and in preventing acute solid organ graft rejection, but has not been tested to date for acute GVHD prophylaxis [60].

Acute GVHD and T-cell subpopulations

Conventional T cells

Using new methods such as green fluorescent protein marking or bioluminescence technology, it has been reported that T cells can undergo a massive and much earlier than previously thought expansion in lymph nodes and Peyer patches (reviewed in chapter 11 and in reference [61]). In mice, naive CD44(lo)CD62L(hi) CD8+ T cells generate and sustain allogeneic CD8+ T cells in GVHD reactions [62,63]. Murine memory T cells isolated from non-allosensitized donors fail to induce GVHD in experimental models [62]. In contrast, alloantigen-sensitized effector memory CD44(hi)CD62L(lo) as well as naïve phenotype CD44(lo)CD62L(hi), but not central memory CD44(hi)CD62L(hi) CD8+ T cells, cause GVHD following adoptive transfer into secondary recipients [30]. Both alloantigen-sensitized effector memory CD4+ and CD8+ T cells are involved in the transfer of GVHD under these conditions.

In the clinic, quantification of the degree and location of early T-cell expansion is not readily possible given the limitations of current technology that can be applied to HSCT recipients. Nonetheless, clinical studies currently evaluate transferring enriched memory T cells rather than naïve T cells to the recipient at the time of HSCT. Such studies will provide important proof-of-concept as to whether the removal of naïve T cells from the donor graft is sufficient to reduce or prevent acute GVHD.

Regulatory T cells

CD4+CD25+Foxp3+ regulatory T cells (Tregs) have potent suppressor activity both in vitro and in vivo (reviewed in chapter 12). Donor Treg cell infusion blocks acute GVHD. Murine L-selectin (CD62L) expressing Treg cells preferentially home to secondary lymphoid organs, and in particular lymph nodes, resulting in GVHD prevention [64]. Conversely, depletion of CD25+ T cells from the donor graft or in the recipient immediately following allogeneic HSCT promotes acute and chronic GVHD in various mouse models while still maintaining a graft-versus-hematopoietic cell malignancy response in most but not all studies [65–69]. Because of the relatively low frequency of Tregs in lymphoid organs, ex vivo expansion of Tregs has often been used to increase the number and to activate Tregs prior to in vivo adoptive transfer. Immunosuppressive drugs given to prevent or control GVHD also affect Treg cell expansion and function. Calcineurin inhibitors such as cyclosporin decrease IL-2 production, leading to a reduction in Treg proliferation and function. In contrast, rapamycin preferentially spares Tregs as opposed to effector T cells and induces or functionally increases murine and human Tregs in ex vivo culture systems, albeit at the expense of overall cell yield [70].

Some challenges have arisen in the manipulation of human Tregs during allogeneic HSCT. A combination of CD4, CD25 and CD127 (IL-7R) has permitted the isolation of a highly purified Treg population that included both CD4+CD25+ and CD4+CD25− T-cell subsets both of which were as suppressive as the classic CD4+CD25(hi) Treg cell subset [71,72]. However, it is unknown whether the expansion of this Treg subpopulation will permit retention of as high a level of suppressor function as the CD4+25+ population [73]. Furthermore experimental data both in mice and in vitro human studies has demonstrated the extraordinary potential of T helper cell subsets (Th1, Th2 and Th17) and of Tregs to exhibit plasticity, shifting from one phenotype to another one (reviewed in references [74,75]). This aspect of plasticity may also be of concern when administering Tregs to patients with inflammatory diseases. However, early phase I–II clinical trials have demonstrated the feasibility of using Treg in the clinical setting [76,77] without significant GVHD or toxicities. New techniques of Treg expansion [78] now allow the production of sufficient functional Treg for clinical use.

NKT cells

A second inhibitory population shown to inhibit acute GVHD lethality is the NKT subset that co-expresses NK and T-cell surface determinants [79]. In rodents, total lymphoid irradiation combined with anti-thymocyte globulin has been shown to induce host NKT cells that also promote the generation of Tregs and the production and release of anti-inflammatory cytokines [80]. In HSCT human recipients, studies indicate that the reduced acute GVHD lethality seen despite the infusion of high numbers of T cells contained in a G-CSF mobilized PB stem cell graft is associated with increased donor NKT cells [81,82].

Th17 cells

Th17 cells (reviewed in chapter 13 and in reference [83]) have recently emerged as a new player in GVHD. Although the role of this new T-cell subset has been dissected in certain experimental models including inflammatory bowel disease, lung and skin GVHD, experimental GVHD studies have led to seemingly discordant GVHD lethality results that may be ascribed to distinct differences in experimental GVHD conditions [84–86]. As yet the role of Th17 cells in humans is uncertain [87].

T-cell trafficking

How T cells are recruited into tissues could be pivotal for understanding the stereotypical involvement of skin, liver and bowel in GVHD. While the migration of T cells into secondary lymphoid organs during GVHD and other inflammatory responses has been well characterized, the migration of leukocytes into parenchymal organs is less well understood. This process may involve changes in vascular permeability and, in certain systems, has been shown to require specific selectin–ligand, chemokine–receptor and integrin–ligand interactions (reviewed in chapter 16 and in reference [13]). During a GVHD reaction, donor T cells initially migrate to spleen and peripheral lymphoid tissues within hours [88]. Naïve donor T cells traffic to lymphoid tissues, where the subset of alloreactive T cells receive activation signals by APCs, and then subsequently migrate to specific GVHD target organ sites, essential for the induction and pathogenesis of acute GVHD [89]. Almost all tissues express transplantation antigens; however, acute GVHD pathology is primarily limited to only a few locations rich in epithelial cells and expressing high levels of MHC antigens – gut, skin, liver, lung, secondary lymphoid organs and thymus. The ability of alloreactive donor T cells to home to specific organs is regulated by a unique combination of signals that bind to corresponding receptors on host tissues and counter-receptors expressed on donor T cells, including members of the chemokine family. MIP1a and other chemokines (such as CCL2–CCL5, CXCL2, CXCL9, CXCL10, CXCL11, CCL17 and CCL27) are over-expressed during GVHD generation and can enhance the homing of cellular effectors to GVHD target organs.

These results suggest that strategies that influence T-cell migration, particularly to GVHD target organs, may offer promise for reducing GVHD target organ specific injury, although the redundancy of chemokines and their receptors may hinder clinical efficacy in the context of GVHD prevention or therapy. As such, targeting lymphocyte/integrin interaction may be a more promising way to explore this issue. Indeed the research of targeting lymphocyte trafficking has been taken into the clinic in diseases related to GVHD, such as rheumatoid arthritis and colitis.

Effector stage; T cells and others

After migration of alloreactive effector T cells to the target tissues of GVHD, these cells can mediate tissue destruction through both direct cytotoxic activity and the recruitment of other leukocytes. Targeting these effector pathways has been studied as a strategy to prevent or reduce GVHD severity. Researchers have considered acute GVHD to be a Th1/T cytotoxic-type (IL-12, IL-2 and IFN-γ) disease on the basis of the predominance of cytotoxic T-cell-mediated pathology and of increased production of Th1-type cytokines. However, several recent studies have suggested that the influence of Th1 and Th2 cytokines in acute and chronic GVHD is not so simply explained (reviewed in chapter 11).

The concentration and timing of cytokine release into the circulation and relevant target organs appear to be critical for GVHD (reviewed in chapter 16). For example, IL-10 promotes Th2 and type 1 regulatory T-cell responses, which can be important in the induction of tolerance to allografts (reviewed in reference [6]). Higher production of IL-10, as demonstrated in human recipients with an IL-10 polymorphism, is associated with reduced occurrence and severity of GVHD [90]. Paradoxically, high dose of IL-10 administration can accelerate GVHD in a murine model, and high-serum IL-10 levels in patients after HSCT are associated with a fatal outcome. However, conversely, low-dose IL-10 administration can inhibit acute GVHD in mice (reviewed in reference [6]). These findings highlight the pleiotropic, sometimes opposing, nature of cytokines during the different phases of GVHD pathogenesis and on various effector and regulatory cell populations.

T cells mediate the final effector pathway in GVHD by multiple pathways [30,91,92]. The expression of both Fas and FasL is increased on CD8+ and CD4+ donor T cells during acute GVHD in patients and mice, and serum levels of soluble FasL and Fas were found to correlate with GVHD severity or the response to GVHD therapy. Several studies in experimental mouse models have analyzed the role of the Fas–FasL and perforin–granzymes pathways in the development of GVHD by using mice that are deficient for FasL (gld mice), perforin or granzyme B as donors, or by the in vivo administration of neutralizing anti-FasL antibodies. Although these differences in experimental design affect the opportunity to draw a uniform conclusion, most studies have shown a role for the Fas–FasL pathway in GVHD mortality. With respect to the perforin–granzyme pathway, approximately two-thirds of studies demonstrated the importance of this pathway in GVHD pathogenesis (reviewed in reference [5]).

In studies of transplant patients, polymorphisms in the TNF-α gene of HSCT recipients are associated with higher levels of production of the cytokine and are correlated with a higher incidence of severe acute GVHD [93] (and reviewed in reference [48]), which suggests that, in humans, induction of TNF-α from recipient cells may make an important contribution to disease. Regardless of the source of TNF-α, its importance in GVHD is borne out with the demonstration that treatment of steroid-resistant GVHD with a TNF-α blocker has shown efficacy, especially against gastrointestinal disease, in some studies [94–96] or when administrated for GVHD prophylaxis [97]. Although preliminary clinical studies also suggested that an anti-TNF-α antibody (Etanercept) in addition to steroids was superior to steroids alone as initial treatment for acute GVHD [96], a recent multicenter four-arm BMT-CTN randomized trial designed to identify the most promising agent(s) for initial therapy for AGVHD indicated that Etanercept was not the most effective agent to be combined with steroids for GVHD therapy [98].

Promise from molecular biology and proteomics?

New molecular tools including proteomic and gene profiling (reviewed in chapters 19 and 21) have already begun to pave the way for such a response, allowing for a more precise definition of acute GVHD or the construction of a predictive model for acute GVHD severity both in humans and in the experimental setting. In this regard, a recent paper measured the gene-expression profiles of CD4+ and CD8+ T cells from 50 donors with microarray technology. Using quantitative PCR, established statistical tests, and analysis of multiple independent training-test datasets, Perreault and colleagues found that dangerous donor trait (occurrence of GVHD in the recipient) is under polygenic control and is shaped by the activity of genes that regulate transforming growth factor-beta signaling and cell proliferation [99]. These findings strongly suggest that the donor gene-expression profile can have a dominant influence on the occurrence of chronic GVHD in the recipient. However, it should be stressed that currently no gene-expression profiling data are available on acute GVHD. The application of proteomic tools that allows screening for differentially expressed or excreted proteins in body fluids has generated considerable interest in the field. Using proteomics, authors have screened for plasma proteins specific for GVHD. Paczesny et al. [100] aimed to isolate candidate proteins using high throughput assays on a large number of patient samples, and to determine their significance with respect to patient outcome (reviewed in chapter 19).

The graft-versus-leukemia effect

The graft-versus-leukemia (GVL) reaction refers to the ability of donor immune cells to eliminate host leukemic cells after allogeneic HSCT. In 1956, Barnes et al. were the first to report cure of leukemia in mice after total body irradiation and HSCT. Key insights into its mechanisms were reported in a landmark study from the International Bone Marrow Transplant Registry in 1990 [101]. Strikingly, the latter study, that was based on data from over 2000 subjects, showed that GVL was abrogated if T cells were depleted from the graft or if the HSCT donor was an identical twin. On the basis of these data, it was therefore inferred that GVL depended on donor T cells and on the existence of histocompatibility differences between the donor and its recipient (reviewed in reference [102]).

The GVL effect has been extensively reviewed elsewhere [102–104] and almost every chapter in this book aims to review the relative contribution of the different cell subsets implicated in its genesis. Although the evidence for a GVL effect after allogeneic HSCT is now well accepted, the mechanisms involved in the effect are not completely known. However, because GVHD is intimately associated with GVL, it can be assumed that similar mechanisms control GVHD and GVL. GVHD requires the recognition by donor T cells of antigens presented by the MHC molecules on the recipient cells initiating clonal expansion of responders and an effector response involving lymphocytes and cytokines. In GVHD, this leads to the clinical features of acute and chronic GVHD. In GVL reactions, the allogeneic response suppresses residual leukemia. GVHD reactions are directed against a broad spectrum of tissues, including BM. The dominant antigens on leukemia cells driving the GVL response are not known: major or minor histocompatibility antigens co-expressed on GVHD targets (such as normal skin and gut cells) and leukemic cells could induce a non-specific GVH/GVL allogeneic response. The response against either normal or malignant bone marrow-derived cells may also overlap. Thus GVL may in part be a graft-versus-hematopoietic effect involving lymphoid or myeloid lineages or both. Additionally, leukemia cells could induce a more specific allogeneic response if they express antigens, either not present or underexpressed in cells of other tissues (reviewed in chapter 7).

Separating GVHD from GVL has been successfully accomplished in mouse models using various strategies, including depletion of alloreactive T cells; inhibition of inflammatory cytokines; interfering with T-cell cytolytic pathways, co-stimulatory pathways and trafficking; purifying T cells of certain activation states; and using immunosuppressive cell populations, including regulatory T cells and NK T cells [102–104]. NK cells have been found to substantially contribute to GVL responses, which were previously thought to be largely mediated by T cells alone. T-cell therapies continue to be a major area of interest. Researchers have identified several types of antigens that are recognized by allogeneic T cells, including various MiHAs (reviewed in chapter 3), as well as tumor-specific antigens (reviewed in chapter 7), including proteinase 3 (Pr3; also known as myeloblastin), Wilms’ tumor 1 (WT1) and BCR-ABL. This has allowed the expansion of antigen-specific T cells using culture techniques, in which T cells are grown ex vivo in the presence of APCs, the target antigen and supportive cytokines. Alternatively, genetic engineering techniques can endow T cells with tumor-specificity by introducing previously cloned antigen-specific T-cell receptor genes, or modified T-cell receptor-like genes called chimeric-antigen receptors, which recognize extracellular proteins that are expressed by tumors.

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CH 2

The HLA system in hematopoietic stem cell transplantation

Dominique Charron

Laboratoire Jean Dausset, Immunology-Immunogenetics-Histocompatibility, Université Paris-Diderot, Hôpital Saint-Louis AP-HP, Paris, France

Effie Petersdorf

Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

Introduction

Since the discovery of the HLA system [1,2], there has been unprecedented discovery of the gene number, structure and sequences, polymorphism, haplotype composition and linkage disequilibrium (LD) within the MHC [3]. More than 300 genes reside within the MHC and of these, approximately 15–20% have immune-related function including antigen processing and presentation, immune regulation, inflammation, complement, maternal-fetal immunology, stress response, leukocyte maturation and the immunoglobulin superfamily [3]. New information on the regulatory polymorphisms is emerging from efforts of the Human Epigenome Project (HEP) and provides insight into methylation and histone acetylation profiles within the MHC [4,5].

Classical HLA

Organization

The MHC is composed of three regions termed class I, class III and class II (Figure 2.1). The HLA system includes the classical loci HLA-A, C and B, and the non-classical genes HLA-E, HLA-F, HLA-G, MICA and MICB that reside within the class I region. HLA-DR, HLA-DQ and DP reside within the class II region. The class III region comprises genes of importance to the stress response (TNF, HSP, LTA) and the complement cascade.

FIGURE 2.1 Map of the human major histocompatibility complex (MHC).

The MHC is encoded on chromosome 6p21 and is organized into three major regions: class I, class III and class II. The figure shows the location of each of the classical and non-classical HLA genes that have been described as playing a role in hematopoietic cell transplantation.

Class I

Class I HLA-A, -B and -C genes are each composed of a series of eight exons delineated by intervening introns. Each class I exon has a unique function: exon 1 encodes the leader sequence; exons 2, 3 and 4 encode the α1, 2 and 3 domains, respectively; exon 5 encodes the trans-membrane portion, and exons 6, 7 and 8 encode the cytoplasmic tail. These products give rise to the heavy α chain of the mature HLA class I molecule, and define the HLA phenotype (e.g. HLA-A1 or A2). The heavy chain is non-covalently bound to a β2-microglobulin light chain whose gene resides on chromosome 15. The delineation of the crystallographic structure of HLA-A2 in 1987 was pivotal to understanding the structure–function relationship of MHC molecules. Those landmark studies revealed that class I molecules are composed of two α-helical regions that overlay an eight-strand anti-parallel β-pleated sheet; together these form the functional groove of class I molecules for peptide binding [6].

Sequence variation in exons 2, 3 and 4 define the allospecificity of HLA-A, HLA-B and HLA-C antigens. As of March 2012, over 1757 HLA-A, 2338 HLA-B and 1304 HLA-C alleles are recognized by the World Health Organization Nomenclature Committee for Factors of the HLA System [7,8]. The nucleotide substitutions in exons 2, 3 and 4 are commonly observed at hypervariable positions that define the peptide binding groove and T-cell receptor (TCR) contact residues of the α1 and α2 domains [9]. In addition to interactions with the TCR, HLA-C and some HLA-B and A molecules serve as ligands for natural killer inhibitory receptors (KIR). The specificity of ligand–receptor interactions is defined by residues 77–80 for HLA-C and by residues 77–83 (the Bw4 epitope) for HLA-B.

Non-classical MHC genes: HLA-E, F, G and MIC

In addition to the classical class I HLA loci, the class I region of the MHC encodes a series of genes that are termed the non-classical class I genes, HLA-E, HLA-F, HLA-G and the MHC class I-related chain A and B genes (MICA and MICB, respectively). Known as the class 1b family [10], these genes share homology, but differ with respect to their specific regulation, expression patterns and epigenetic profiles. Their role in transplantation is emerging (Table 2.1).

Table 2.1 The Role of MHC Genes in Hematopoietic Cell Transplantation

∗Genotype models: HLA-E∗01:01/01:03; MICA val129met; HLA-G ins/del.

HLA-E

HLA-E is an extensively studied MHC class Ib antigen. In contrast to the classical MHC class I HLA-A, B, C genes, HLA-E is minimally polymorphic with only two non-synonymous functional HLA-E alleles, HLA-E∗01:01 and HLA-E∗01:03. HLA-E∗01:03 differs from HLA-E∗01:01 by a single amino acid substitution (gly to arg) at position 107 of the α2 heavy chain domain [8]. Although the two alleles appear to be evenly distributed in the human population (approximately 50% each), they differ with respect to their quantitative cell surface expression. HLA-E∗01:03 is expressed at higher concentration on transfected cells compared to HLA-E∗01:01 [11]. The HLA-E molecules preferentially bind nonameric self-peptides derived from the leader sequences of the various HLA class I molecules. HLA-E also presents non-canonical peptides derived from pathogens or stress-related proteins. Overall, the HLA-E/peptide complexes differ in thermal stability [12].

As a ligand for the CD94/NKG2 receptors on NK cells [13] and for the TCR on NKT cells, HLA-E molecules are involved in both innate and adaptive immunity. Recent evidence supports a role for the involvement of HLA-E in presentation of peptides to the αβ TCR expressed on CD8+ T cells. The HLA-E CD94/NKG2 A/C system modulates either inhibition or activation of the NK cell-mediated cytotoxicity and cytokine production; at the same time, HLA-E can present microbial-derived peptides from human viruses or bacteria, thereby inducing T-cell responses. These diverse roles highlight the importance of HLA-E molecules as restriction elements for the specific T-cell responses against pathogens such as CMV, Epstein–Barr virus (EBV) or mycobacteria (Mycobacterium tuberculosis) [14–16]. In the same way, the Qa-1 molecule, a murine counterpart of the human HLA-E, has been shown to bind and present an immunodominant peptide recognized by salmonella-specific CD8+ T lymphocytes (CTL) and also participates in the host response against Listeria monocytogenes.

HLA-G

The human leucocyte antigen G (HLA-G) is a non-classical HLA class Ib locus whose products have distinct immunomodulatory, anti-inflammatory and tolerogenic properties. HLA-G maps telomeric to HLA-A (Figure 1). Although HLA-G molecules are structurally similar to their classical counterparts, they are distinguished by their limited tissue distribution in physiological conditions, the diversity of isoforms generated by alternative splicing (four membrane-bound [HLA-G1 to G4] and three soluble [HA-G5 to G7] isoforms [sHLA-G]) [17] and their unique pattern of polymorphisms in the non-coding regions, especially within the promoter and the 3′-untranslated region (3′UTR; [18]). To date, HLA-G alleles encode a limited number of exonic mutations; these polymorphisms are often silent mutations that result in 47 unique alleles that give rise to only 15 protein variants [8]. HLA-G allele frequencies vary extensively between Caucasian, Asian and African populations.

HLA-G mediates immune responses of NK and T lymphocytes by interacting directly with a series of inhibitory receptors: KIR 2DL4 (CD158d) on NK and a subpopulation of T cells; ILT2 (CD85j) on T, B, NK, dendritic cell (DC) and monocytes, and ILT4 (CD85d) expressed exclusively on antigen presenting cells (APC). Each HLA-G allele bears either a 14-base pair (bp) insertion (ins) or deletion (del) polymorphism in the 3′UTR which influences HLA-G expression. Such indels have functional consequences. The insertion allele (+14 bp), albeit initially described to increase HLA-G mRNA stability, was subsequently correlated with lower levels of HLA-G mRNA and serum sHLA-G isoforms [19,20] with likely functional consequences on the properties of HLA-G molecules. Cell surface HLA-G was first found on cytotrophoblasts and shown to maintain fetal–maternal tolerance. HLA-G is also physiologically expressed on CD14+ monocytes and thymic epithelium. Overall HLA-G molecules are involved in the inhibition of NK cell activity, CD4+ T lymphocyte and DC maturation, apoptosis of CD8+ cytotoxic T cells (CTL) and development of regulatory T cells (Tregs).

MIC genes

The MIC gene family comprises two expressed genes, MICA and MICB, and five pseudo genes (MICC, MICD, MICE, MICF and MICG) [21]. Located at the centromeric end of HLA classical class I region, MICA maps approximately 46 kb from HLA-B. MICA is the most polymorphic non-classical class I gene with over 70 alleles reported so far [8].

A distinguishing feature of MICA sequence variation is the presence of polymorphism in both the α2 and the α3 domains. In fact, none of the polymorphic residues of MICA correlate with those of the α1 and α2 domains of classical class I molecules, the latter of which contact the antigenic peptide and/or TCR. Overall the significance of most MICA/B polymorphisms remains to be elucidated in terms of their ancestral origins and evolution. It is suggested that, based on the α3 domain polymorphisms, MICA alleles can be divided into two large families which might have evolved from two different ancestral lineages.

From a functional point of view, a methionine (met) to valine (val) change at position 129 of the α2-heavy domain categorizes MICA alleles into strong (MICA-129 met) and weak (MICA-129 val) binders of the NKG2D receptor involved in activation and co-stimulation of NK and T cells [22]. Another level of diversity has been identified in the trans-membrane region (TM) of MICA with the insertion of short tandem repeats (STR) that result in a series of alleles (A4–A10). In addition, a nucleotide insertion (GCT → GGCT) in the TM region of the A5.1STR allele results in a premature stop codon. This sequence is present in MICA∗008, the most frequent MICA allele described in various populations.

Although similar in structure to an HLA class I heavy chain, MICA does not bind β2 microglobulin nor any specific peptide. Therefore MICA molecules are not involved in TCR-mediated immunity but rather engage NKG2D, a C-type lectin expressed on effector cells, including NK and αβ- and γδ-T cells. Such engagement triggers NK cells and co-stimulates T lymphocytes to mount subsequent immune responses. Further, a soluble isoform of MICA (sMICA), resulting from the proteolytic shedding of the membrane-bound molecules, was shown to result in NKG2D receptor downregulation. The ensuing immune modulation highlights the functional duality of the membrane-bound and soluble MICA isoforms. Preferentially expressed on epithelial and endothelial cells, MICA and MICB appear more ubiquitous at the mRNA level. Expression has also been reported in activated immune cells including DCs and T lymphocytes. The specific patterns of expression of MICA/B are related to the regulatory sequences which are devoid of interferon response elements, while a stress response element has been identified in the promoter region [23].

Class II

Historically the class II region has been known as the HLA-D region following the characterization of HLA-A, HLA-B and HLA-C. The class II region has since been extensively sequenced, and its organization has been well-defined in many populations [3]. Not only does the class II region encode genes that define the classical HLA-DR, HLA-DQ and HLA-DP alloantigens, but it also contains genes that play a critical role in antigen loading and presentation (TAP and LMP); the concentration of genes with related function make class II a truly unique region of the human