GVHD PREVENTION: AN OUNCE IS BETTER THAN A POUND

The pathophysiology of acute graft-versus-host disease (aGVHD) is known to involve donor T cells responding to host histoincompatible allo-antigens presented by the host antigen presenting cells (APCs) and the subsequent induction of pro-inflammatory cytokines and cellular effectors that cause target organ damage. In a more general sense, GVHD can be considered as an immune response against foreign antigens that has gone awry. Similar to all immune responses, GVHD, can be understood as a process that consists of (A) triggers, (B) sensors, (C) mediators, and (D) effectors of GVHD.

Triggers for Induction of GVHD

Like all immune responses, certain triggers are critical for induction of acute graft-versus-host disease (aGVHD). These include: (1) Disparities between histocompatibility antigens: antigen disparity can be at the level of major histocompatibility complex (MHC), that is, MHC mismatched or at the level of minor histocompatibility antigens (miHA), that is, MHC matched but miHA mismatched [1]. The severity of aGVHD is directly related to the degree of MHC mismatch [2]. In bone marrow transplants (BMT) that are MHC matched but miHA disparate, donor T cells still recognize MHC-peptide derived from the products of recipient polymorphic genes, the miHAs. The expression of miHAs is wide and variable. Some miHAs such are primarily found on hematopoietic cells, whereas some others such as the H-Y antigens, are ubiquitously expressed [3]. It is now being increasingly appreciated that the extent of the allo-antigens can impact the degree of allo-specific T cell responses [4]. (2) Damage induced by conditioning regimens and underlying diseases: under most circumstances, the initiation of an adaptive immune response is triggered by the innate immune response. The innate immune system is triggered by certain exogenous and endogenous molecules. This is likely the case in the induction of aGVHD. Pattern recognition receptors such as Toll-like receptors (TLR), nucleotide-binding oligomerization domain containing 2 (NOD2) [5] play an essential role in innate immunity and in initiating the cellular signaling pathways that activate cytokine secretion, such as NF-kB. Some of their ligands, such as lipopolysaccharide, CpG, and MDP2, which is recognized by TLR-4, TLR-9, and NOD2, respectively, are released by the preparative regimens and contribute to the induction and enhancement of allo-T cell responses [6–10]. Inthisway, the conditioning regimens amplifythe secretion of proinflammatory cytokines like interleukin (IL)-1, tumor necrosis factor (TNF)-α [7,11,12], IL-6, and other interferon family members in a process described as a “cytokine storm.” In addition to the exogenous microbial-associated molecules, endogenous noninfectious triggers as a consequence of damage, called damage-associated molecular patterns (DAMPs) such as adenosine-5′-triphosphate also play a critical role in GVHD [13,14]. In fact, the proinflammatory cytokines themselves might serve as DAMPs. Other infectious and sterile stimuli, as yet not reported, might also play a role in triggering an allo-T cell cells to host antigens.

Sensors of GVHD

The triggers that initiate an immune response have to be sensed and presented. APCs might be considered the sensors for aGVHD. The APCs sense the DAMPs, present the MHC disparate or miHA disparate protein, and provide the critical secondary (costimulatory) and tertiary (cytokine) signals for activation of the alloreactive T cells, the mediators of aGVHD. APCs sense allo-disparity through MHC and peptide complexes. Dendritic cells (DCs) are the most potent APCs and the primary sensors of allo-disparity [15]. Recipient DCs that have been primed by the conditioning regimen will process and present MHC and peptide complexes to donor T cells at the time of transplant [16]. In the case of hematopoietic cell transplants (HCTs), recipient APCs present the endogenous and the exogenous antigens to donor CD8⁺ and CD4⁺ T cells, respectively. As such, they are crucial for the induction of donor regulatory T cell responses and in suppressing GVHD [17]. DCs are important initiators and regulators of immune responses. The role of DC subsets in GVHD is just beginning to be understood. The role of other hematopoietic derived APCs, such as Langerhan cells, macrophages, B cells, and basophils has been recently investigated. In the presence of other hematopoietic-derived APCs, these APCs either play no role or have a regulatory effect on the severity of GVHD induction [18–22]. The role of nonhematopoietic-derived APCs, in the absence of radiosensitive hematopoietic-derived APCs, such as endothelial and epithelial cell subsets in this process sensing and inducing an allo-T cell response is as yet not well explored. Furthermore, the kinetics of the switch from recipient to donor APCs, the contributions of different APCs subsets, the importance of direct alloantigen presentation, and the magnitude of indirect alloantigen presentation in GVHD remains to be largely determined. An intriguing and potentially novel aspect of the role of APCs is whether they can be modulated to enhance the presentation of tumor specific antigens while not concomitantly enhancing allo-antigen presentation in order to accentuate graft-versus-tumor effects without aggravating GVHD.

APCs provide the critical costimulation signals for turning on the aGVHD process. The interaction between the MHC/allopeptide complex on APCs and the T cell receptor of donor T cells along with the signal via T cell costimulatory molecules and their ligands on APCs is required to achieve T cell activation, proliferation, differentiation, and survival and the in vivo blockade of positive costimulatory molecules (such as CD28, ICOS, CD40, CD30, etc.), or inhibitory signals (such as PD-1 and CTLA-4) mitigate or exacerbate aGVHD, respectively [23].

Mediators of GVHD

These include primarily the donor T cell subsets. Evidence suggests that alloreactive donor T cells consist of several subsets with different stimuli responsiveness, activation thresholds, and effector functions. The allo-antigen composition of the host determines which donor T cells subsets differentiate and proliferate. As mentioned previously, in the majority of HLA-matched HCT, aGVHD may be induced by either or both CD4⁺ and CD8⁺ subsets responses to miHAs. The repertoire and immunodominance of the GVHD-associated peptides presented by MHC class I and class II molecules has not been defined [24]. Donor naive CD62L⁺ T cells are the primary alloreactive T cells that drive the GVHD reaction while the donor effector memory CD62L− T cells do not [25,26]. Interestingly, donor regulatory T cells (Tregs) expressing CD62L are also critical to the regulation of GVHD [27,28]. We now know that it is possible to modulate the alloreactivity of naive T cells by inducing anergy with costimulation blockade, deletion via cytokine modulation, or mixed chimerism. Donor effector memory T cells that are nonalloreactive do not induce GVHD, yet are able to transfer functional memory [25]. In contrast, memory T cells that are alloreactive can cause severe GVHD [22,29,30]. GVHD is negatively regulated by Tregs. In mouse BMT models, naturally occurring donor-derived Tregs suppress the proliferation of conventional T cells, prevent GVHD, and preserve graft-versus-leukemia (GVL) effects depending upon the ratio of effector T cells to Tregs [31–36]. In addition, based on the dominant cytokines that are produced upon activation, T cells can be distinguished into various subsets such as Th1, Th2, and Th17 cells. The Th1 cytokines (interferon [IFN]-γ, IL-2 and TNF-α) have been implicated in the pathophysiology of aGVHD. IL-2 production by donor T cells remains the main target of many current clinical therapeutic and prophylactic approaches, such as cyclosporine, tacrolimus, and monoclonal antibodies (mAbs) against the IL-2 and its receptor to control aGVHD [37,38]. But emerging data indicate an important role for IL-2 in the generation and maintenance of CD4⁺CD25⁺Foxp3⁺Tregs, suggesting that prolonged interference with IL-2 may have an unintended consequence in the prevention of the development of long-term tolerance after allogeneic HCT [39–42]. Furthermore, the role of Th1/Th2 and Th17 cytokines is complex and might be model dependent [43–55]. Moreover, these cells are required for the GVL effects.

Effectors and Amplifiers of GVHD

The effector phase that leads to GVHD target organ damage is a complex cascade that involves cytolytic cellular effectors such as CD8 cytotoxic T lymphocytes (CTLs), CD4 T cells, natural killer cells, and inflammatory molecules such as IL-1b, TNF-α, IFN-γ, IL-6, and reactive oxygen species [56]. The cellular effectors require cell-cell contact to kill the cells of the target tissues via activation of perforingranzyme, Fas-FasL (CD95-CD95L), or TNFR-TRAIL pathways. Other CTLs killing mechanisms such as TWEAK, and LTβ/LIGHT pathways have also been implicated in GVHD [57–64]. It is important to note that CTL pathways are essential for GVL effects as well. Inflammatory pathways, by contrast, based on animal models, do not require cell-cell contact to kill target cells and are not particularly critical of GVL. GVHD damage by the cellular effectors is amplified by these inflammatory mediators including IFN-γ produced by T cells, TNF-α, and IL-1 produced by T cells and monocytes/macrophages.

All of the above aspects of the biology of aGVHD have been summarized in the mold of a normal immune response. Although this allows for accessing the biology of GVHD, it is important to note that GVHD is a complicated systemic process with as yet still many unknowns and is not a simplified, linear, or cyclical process.