Pathophysiology of Acute Graft-vs-Host Disease: Recent Advances (V体育安卓版)

1. KIR polymorphisms

KIR receptors on NK cells that bind to the HLA class I gene products are encoded on chromosome 19. Polymorphisms in the trans-membrane and cytoplasmic domains of KIR receptors governs whether the receptor has inhibitory potential (such as KIR2DL1, -2DL2, -2DL3 and 3DL1 and their HLA class I ligands (HLA-C and HLA-Bw4) or activating potential; at this time, there is limited information on the clinical significance of activating KIR genes. Two competing models have been proposed for HLA-KIR allo-recognition by donor NK cells following HSCT: “mismatched ligand” and the “missing ligand” models(39–43). The former posits that NK alloreactivity occurs when donor NK cells recognize recipient target cells that lack the class I allele of the donor (HLA mismatching between the donor and recipient in the GVH direction); the latter hypothesizes that donor NK alloreactivity occurs when the host lacks the correct HLA class I ligand(s) to provide the inhibitory signal for donor KIR(42, 43). Both models are supported by some clinical observations, albeit in patients receiving very different transplant and immunosuppressive regimens(40, 44–46). Clearly, further validation is warranted and it is likely that the immuno-biology of the interface between HLA and KIR genetics will be an area of intense future investigation.

2. Cytokine gene polymorphisms

Pro-inflammatory cytokines, involved in the classical ‘cytokine storm’ of GVHD (discussed below), cause pathological damage of target organs such as skin, gut and liver(47). Several cytokine gene polymorphisms, both in hosts and donors, have been implicated. Specifically, TNF polymorphisms (TNFd3/d3 in the recipient, TNF-863 and -857 in donors and/or recipients and TNFd4, TNF-α-1031C and TNFRII-196R- in the donors) have been associated with an increased risk of acute GVHD and transplant-related mortality(48, 49). The three common haplotypes of the IL-10 gene promoter region in recipients representing high, intermediate and low production of IL-10 have been associated with the severity of acute GVHD following allo-BMT after HLA-matched sibling donors(50). By contrast, smaller studies have found neither IL-10 nor TNF-α polymorphisms to be associated with GVHD after HLA-mismatched cord blood transplants(49, 51). Furthermore, it is important to note that not all of the polymorphism analyses so far support the paradigm that excess of pro-inflammatory and Th1 cytokines are always associated with greater GVHD or mortality. For example IFN-γ polymorphisms of the 2/2 genotype (high IFN-γ production) and 3/3 genotype (low IFN-γ) have been associated with decreased or increased acute GVHD, respectively(49, 52). This is consistent with accelerated acute GVHD in IFN-γ knockout mouse models suggesting therefore that IFN-γ may be involved in the down-regulation of GVHD via a negative feed-back loop (discussed below)(53, 54). Similarly, possession of IL-6-174 polymorphism in the recipients (high production of IL-6) associated with both acute and chronic GVHD after MRD sibling HCT(52), which is in contrast to the results of IL-6 genetics in other Th1 mediated disease such as juvenile onset chronic arthritis (55).

By contrast, genotype (high production) of another Th1 cytokine, IL-2 (-330 allele G) was noted to increase the risk of acute GVHD in MUD transplants (56) while a different study showed that Th2 cytokine, IL-13, production by donor T cells is also predictive of acute GVHD in unrelated donor stem cell cohorts (57). Thus data from cytokine polymorphism studies so far do not seem to suggest a simple paradigm for GVHD. Nonetheless, the fact that most of the studies are small, not properly stratified must be considered while interpreting these data.

A small study in pediatric recipients of unrelated HSCT suggested that the presence of the IL-1α-889 allele in either donor or recipient decreased transplant related mortality but did not decrease GVHD(49). NOD2/CARD15 gene polymorphisms in both the donors and recipients were recently shown to have a striking association with GI GVHD and overall mortality after both related and unrelated allogeneic HSCT(58). It is likely non-HLA gene polymorphisms might play differing roles depending on the donor source (related vs. unrelated), HLA disparity (matched vs. mismatched), source of the graft (CB vs. PBSC vs. BM), and the intensity of the conditioning.

1. Co-stimulation

The interaction of donor lymphocyte TCR with the host allo-peptide presented on the MHC of APCs alone is insufficient to induce T cell activation(93). Both TCR ligation and co-stimulation via a ‘second’ signal through interaction between the T cell co-stimulatory molecules and their ligands on APCs are required to achieve T proliferation, differentiation and survival(94). The danger signals generated in phase 1 augment these interactions and significant progress has been made on the nature and impact of these ‘second’ signals(95, 96). Costimulatory pathways are now known to deliver both positive and negative signals and molecules from two major families, the B7 family and the TNF receptor (TNFR) family play pivotal roles in GVHD(97). Interruption of the second signal by blockade of various positive co-stimulatory molecules (CD28, ICOS, CD40, CD30, 4-1BB and OX40) reduces acute GVHD in several murine models while antagonism of the inhibitory signals (PD-1 and CTLA-4) exacerbates the severity of acute GVHD(2, 98–103). The various T cell and APC co-stimulatory molecules and the impact on acute GVHD are summarized in Table 2. The specific context and the hierarchy in which each of these signals play a dominant role in the modulation of GVHD remain to be determined.

2. T cell subsets

T cells consist of several subsets whose responses differ based on antigenic stimuli, activation thresholds and effector functions. The alloantigen composition of the host determines which donor T-cell subsets proliferate and differentiate.

3. Cytokines and T cell differentiation

APC and T cell activation result in rapid intracellular biochemical cascades that induce transcription of many genes including cytokines and their receptors. The Th1 cytokines (IFN-γ, IL-2 and TNF-α) have been implicated in the pathophysiology of acute GVHD(158–160). 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 acute GVHD(161, 162). But emerging data indicate an important role for IL-2 in the generation and maintenance of CD4⁺CD25⁺ Foxp3+ T regs, 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(163–166).

Studies by Sykes and colleagues and others have demonstrated that the role of Th1 cytokines is complex. For example exogenous administration of IFN-γ or T cells from IFN-γ deficient donors have demonstrated a reduction and enhancement of GVHD respectively(53, 54, 167, 168). These data are consistent with the clinical IFN-γ polymorphism data (discussed above). A recent study suggests that IFN- γ might play a differential role in the severity of distinct GVHD target organs(169). Likewise, early injection of IL-2 has also been shown to reduce GVHD(170, 171). Thus whether the Th1 cytokines are the regulators or inducers of GVHD severity depends on the degree of allo-mismatch, the intensity of conditioning and the T cell subsets that are involved after BMT(172–174). Thus although the “cytokine storm” initiated in phase 1 and amplified by the Th1 cytokines correlates with the development of acute GVHD, early Th1 polarization of donor T cells to HCT recipients can attenuate acute GVHD suggesting that physiological and adequate amounts of Th1 cytokines are critical for GVHD induction, while inadequate production (extremely low or high) could modulate acute GVHD through a breakdown of negative feedback mechanisms for activated donor T cells(160, 174–177). Several different cytokines that polarize donor T cells to Th2 such as IL-4, G-CSF, IL-18, IL-11, rapamycin and the secretion of IL-4 by NK1.1⁺ T cells can reduce acute GVHD(178–185). But Th1 and Th2 subsets cause injury of distinct acute GVHD target tissues and some studies failed to show a beneficial effect of Th2 polarization on acute GVHD(186). Thus the Th1/Th2 paradigm of donor T cells in the immuno-pathogenesis of acute GVHD has evolved over the last few years and its causal role in acute GVHD is complex and incompletely understood.

IL-10 plays a key role in suppression of immune responses and its role in regulating experimental acute GVHD is unclear(187). Recent clinical data demonstrate an unequivocal association of IL-10 polymorphisms with the severity of acute GVHD(50). TGF-β, another suppressive cytokine was shown to suppress acute GVHD but to exacerbate chronic GVHD(188). The roles of some other cytokines, such as IL-7 (that promotes immune reconstitution) and IL-13 remain unclear(189–192). The role for Th17 cells, a recently described novel T cell differentiation in many immunological processes, is not yet known(193). In any case, all of the experimental data so far collectively suggest that the timing of administration, the production of any given cytokine, the intensity of the conditioning regimen and the donor-recipient combination may all be critical to the eventual outcome of acute GVHD.

4. Leukocyte migration

Donor T cells migrate to lymphoid tissues, recognize alloantigens on either host or donor APCs and become activated. They then exit the lymphoid tissues and traffic to the target organs and cause tissue damage(194). The molecular interactions necessary for T cell migration and the role of lymphoid organs during acute GVHD have recently become the focus of a growing body of research. Chemokines play a critical role in the migration of immune cells to secondary lymphoid organs and target tissues(195). T-lymphocyte production of macrophage inflammatory protein-1alpha (MIP-1α) is critical to the recruitment of CD8⁺ but not CD4⁺ T cells cells to the liver, lung, and spleen during acute GVHD(196). Several chemokines such as CCL2-5, CXCL2, CXCL9-11, CCL17 and CCL27 are over-expressed and might play a critical role in the migration of leukocyte subsets to target organs liver, spleen, skin and lungs during acute GVHD(194, 197). CXCR3+ T and CCR5+ T cells cause acute GVHD in the liver and intestine(194, 198–200). CCR5 expression has also been found to be critical for Treg migration in GVHD(201). In addition to chemokines and their receptors, expression of selectins and integrins and their ligands also regulate the migration of inflammatory cells to target organs(195). For example, interaction between α4β7 integrin and its ligand MadCAM-1 are important for homing of donor T cells to Peyer’s patches and in the initiation of intestinal GVHD(68, 202). αLβ2/ICAM1, 2, 3 and α4β1/VCAM-2 interactions are important for homing to the lung and liver after experimental HCT(194). The expression of CD62L on donor Tregs is critical for their regulation of acute GVHD suggesting that their migration in secondary tissues is critical for their regulatory effects(88). The migratory requirement of donor T cells to specific lymph nodes (e.g. Peyer’s patches) for the induction of GVHD might depend on other factors such as the conditioning regimen, inflammatory milieu etc(68, 203). Furthermore, FTY720, a pharmacologic sphingosine-1-phosphate receptor agonist, inhibited GVHD in murine but not in canine models of HCT(204, 205). Thus, there might also be significant species differences in the ability of these molecules to regulate GVHD.

1. Cellular Effectors

Cytotoxic T cells (CTLs) are the major cellular effectors of GVHD(208, 209). The Fas-Fas ligand (FasL), the perforin-grazyme (or granule exocytosis) and TNFR-like death receptors (DR), such as TNF-related apoptosis-inducing ligand (TRAIL: DR4, 5 ligand) and TNF-like weak inducers of apoptosis (TWEAK: DR3 ligand) are the principle CTL effector pathways that have been evaluated after allogeneic BMT(209–214). The involvement of each of these molecules in GVHD has been testing by utilizing donor cells that are unable to mediate each pathway. Perforin is stored in cytotoxic granules of CTLs and NK cells, together with granzymes and other proteins. Although the exact mechanisms remain unclear, following the recognition of a target cell through the TCR-MHC interaction, perforin is secreted and inserted into the cell-membrane, forming “perforin pores” that allow granzymes to enter the target cells and induce apoptosis through various downstream effector pathways such as caspases(215). Ligation of Fas results in the formation of the death-inducing signaling complex (DISC) and also activates caspases(216, 217).

Transplantation of perforin deficient T cells results in a marked delay in the onset of GVHD in transplants across MiHA disparities only, both MHC and MiHA disparities (126), and across isolated MHC I or II disparities(209, 218–222). However, mortality and clinical and histological signs of GVHD were still induced even in the absence of perforin-dependent killing in these studies, demonstrating that the perforin-granzyme pathways plays little role in target organ damage. A role for the perforin-granzyme pathway for GVHD induction is also evident in studies employing donor-T-cell subsets. Perforin- or granzyme B-deficient CD8+ T cells caused less mortality than wild-type T cells in experimental transplants across a single MHC class I mismatch. This pathway, however, seems to be less important compared to Fas/FasL pathway in CD4-mediated GVHD(221–223). Thus, it seems that CD4+ CTLs preferentially use the Fas-FasL pathway, whereas CD8+CTLs primarily use the perforin-granzyme pathway.

Fas, a TNF-receptor family member, is expressed by many tissues, including GVHD target organs(224). Its expression can be upregulated by inflammatory cytokines such as IFN-γ and TNF-α during GVHD, and the expression of FasL is also increased on donor T cells, indicating that FasL-mediated cytotoxicity may e a particularly important effector pathway in GVHD(209, 225). FasL-defective T cells cause less GVHD in the liver, skin and lymphoid organs(220, 223, 225). The Fas-FasL pathway is particularly important in hepatic GVHD, consistent with the keen sensitivity of hepatocytes to Fas-mediated cytotoxicity in experimental models of murine hepatitis(209). Fas-deficient recipients are protected from hepatic GVHD, but not from other organ GVHD, and administration of anti-FasL (but not anti-TNF) MAbs significantly blocked hepatic GVHD damage occurring in murine models(209, 226, 227). Although the use of FasL-deficient donor T cells or the administration of neutralizing FasL MAbs had no effect on the development of intestinal GVHD in several studies, the Fas-FasL pathway may play a role in this target organ, because intestinal epithelial lymphocytes exhibit increased FasL-mediated killing potential(228). Elevated serum levels of soluble FasL and Fas have also been observed in at least some patients with acute GVHD(229, 230).

The utilization of a perforin-granzyme and FasL cytotoxic double-deficient (cdd) mouse provides an opportunity to address whether other effector pathways are capable of inducing GVHD target organ pathology. An initial study demonstrated that cdd T cells were unable to induce lethal GVHD across MHC class I and class II disparities after sublethal irradiation(219). However, subsequent studies demonstrated that cytotoxic effector mechanisms of donor T ells are critical in preventing host resistance to GVHD(213, 231). Thus when recipients were conditioned with lethal dose of irradiation, cdd CD4+ T cells produced similar mortality to wild type CD4+ T cells(213). These results were confirmed by a recent study demonstrating that GVHD target damage can occur in mice that lack alloantigen expression on the epithelium, preventing direct interaction between CTLs and target cells(214).

The participation of another death ligand receptor signaling pathway, TNF/TNFRs, has also been evaluated. Experimental data suggests that this pathway is crucial for GI GVHD (discussed more below). Recently, several additional TNF family apoptosis-inducing receptors/ligands have been identified, including TWEAK, TRAIL and LTβ/LIGHT have all been proposed to play a role in GVHD and GVL responses(2, 232–238). However whether these distinct pathways play a more specific role for GVHD mediated by distinct T cell subsets in certain situations remains unknown. Intriguingly, recent data suggest that none of these pathways might be critical for mediating the rejection of donor grafts(232, 239). Thus it is likely that their role in GVHD might be modulated by the intensity of conditioning and by the recipient T cell subsets. Existing experimental data suggest that perforin and TRAIL cytotoxic pathways are associated with CD8+ T cell–mediated GVL(209). The available experimental data are strongly skewed toward CD8+ T cell–mediated GVL based on the dominant role of this effector population in most murine GVT models; however, CD4+ T cells can mediate GVL and might be crucial in clinical BMT depending on the type of malignancy and the expression of immuno-dominant antigens.

Taken together, although experimental data suggest that might be some distinction between the use of different lytic pathways for the specific GVHD target organs and GVL, but the clinical applicability of these observations is as yet largely unknown

2. Inflammatory Effectors

Inflammatory cytokines synergize with CTLs resulting in the amplification of local tissue injury and further promotion of an inflammation, which ultimately leads to the observed target tissue destruction in the transplant recipient(47). Macrophages, which had been primed with IFN-γ during step 2, produce inflammatory cytokines TNF-α and IL-1 when stimulated by a secondary triggering signal(240). This stimulus may be provided through Toll-like receptors (TLRs) by microbial products such as LPS and other microbial particles, which can leak through the intestinal mucosa damaged by the conditioning regimen and gut GVHD(241, 242). It is now apparent that immune recognition through both TLR and non-TLRs (such as NOD) by the innate immune system also controls activation of adaptive immune responses(241, 243). Recent clinical studies of GVHD suggested the possible association with TLR/NOD polymorphisms and severity of GVHD(58, 244, 245). LPS and other innate stimuli may stimulate gut-associated lymphocytes, keratinocytes, dermal fibroblasts, and macrophages to produce pro-inflammatory effectors that play a direct role in causing target organ damage. Indeed experimental data with MHC mismatched BMT suggest that under certain circumstances these inflammatory mediators are sufficient in causing GVHD damage even in the absence of direct CTL induced damage(71). The severity of GVHD appears to be directly related to the level of innate and adaptive immune cell priming and release of pro-inflammatory cytokines such as TNF- α, IL-1 and nitric oxide (NO)(71, 242, 246–248).

The cytokines TNF- α and IL-1 are produced by an abundance of cell types during processes of both innate and adaptive immunity; they often have synergistic, pleiotrophic, and redundant effects on both activation and effector phases of GVHD(160). A critical role for TNF- α in the pathophysiology of acute GVHD was first suggested over 20 years ago because mice transplanted with mixtures of allogeneic BM and T cells developed severe skin, gut, and lung lesions that were associated with high levels of TNF- α mRNA in these tissues(249). Target organ damage could be inhibited by infusion of anti-TNF- α MAbs, and mortality could be reduced from 100% to 50% by the administration of the soluble form of the TNF- α receptor (sTNFR), an antagonist of TNF- α(62, 65, 247). Accumulating experimental data further suggest that TNF -α is involved in a multistep process of GVHD pathophysiology. TNF-α can (1) cause cachexia, a characteristic feature of GVHD, (2) induce maturation of DCs, thus enhancing alloantigen presentation, (3) recruit effector T cells, neutrophilis, and monocytes into target organs through the induction of inflammatory chemokines, and (4) cause direct tissue damage by inducing apoptosis and necrosis. TNF-α also involves in donor-T-cell activation directly trough it’s signaling via TNFR1 and TNFR2 on T cells. TNF-TNF1 interactions on donor T cells promote alloreactive T-cell responses and TNF-TNFR2 interactions are critical for intestinal GVHD(235, 250). TNF-α also seems to be important effector molecules in GVHD in skin and lymphoid tissue(249, 251). Additionally, TNF-α might also be involved in hepatic GVHD, probably by enhancing effector cell migration to the liver via the induction of inflammatory chemokines(252). An important role for TNF-α in clinical acute GVHD has been suggested by studies demonstrating elevated serum levels or TNF- α or elevated TNF- α mRNA expression in peripheral blood mononuclear cells in patients with acute GVHD and other endothelial complications, such as hepatic veno-occlusive disease (VOD)(252–255). Phase I–II trials using TNF-α antagonists reduced the severity of GVHD suggesting that it is a relevant effector in causing target organ damage(256, 257).

The second major pro-inflammatory cytokine that appears to play an important role in the effector phase of acute GVHD is IL-1(258). Secretion of IL-1 appears to occur predominantly during the effector phase of GVHD of the spleen and skin, two major GVHD target organs(259). A similar increase in mononuclear cell IL-1 mRNA has been shown during clinical acute GVHD. Indirect evidence of a role for IL-1 in GVHD was obtained with administration of this cytokine to recipients in an allogeneic murine BMT model. Mice receiving IL-1 displayed a wasting syndrome and increased mortality that appeared to be an accelerated form of disease. By contrast, intra-peritoneal administration of IL-1ra starting on d 10 post-transplant was able to reverse the development of GVHD in the majority of animals, providing a significant survival advantage to treated animals(260). However, the attempt to use IL-1ra to prevent acute GBHD in a randomized trial was not successful(261).

As a result of activation during GVHD, macrophages also produce NO, which contributes to the deleterious effects on GVHD target tissues, particularly immunosuppression(248, 262). NO also inhibits the repair mechanisms of target tissue destruction by inhibiting proliferation of epithelial stem cells in the gut and skin(263). In humans and rats, the development of GVHD is preceded by an increase in serum levels of NO oxidation products(264–267).

Existing data demonstrate important role for various inflammatory effectors in GVHD. The relevance of currently studied or as yet unknown specific effectors might however be determined by other factors, including the intensity of preparatory regimens, the type of allograft, the T cell subsets and the duration of BMT. In any event, both experimental and clinical data suggest an important role for both the cellular and inflammatory mediators in GVHD induced target organ damage.