Increased expression of Fas and FasL is observed in both CD8⁺ and CD4⁺ T cells during GVHD (46–48) and is associated with the severity of GVHD (48, 49). Blockade of Fas/FasL pathway led to decreased overall mortality in GVHD (50, 51) and reduced tissue specific organ damage (52). Meanwhile, single-nucleotide polymorphism (SNP) analysis showed that SNP of Fas in recipients can be used to improve prognostic stratification of GVHD (53, 54). Furthermore, selective depletion of host-sensitized donor lymphocytes by pre-treatment of soluble FasL can prevent GVHD (54–56). These results indicate that Fas/FasL is a key molecule in the pathogenesis of GVHD. Mizrahi et al. (57) found that short-term mobilization of peripheral blood by FasL reduced GVHD and improved survival following lipopolysaccharide stimulation, while retaining GVT activity. Likewise, engineered T cells displaying novel form of FasL (streptavidin-FasL) eliminated alloreactive T cells without significantly affecting GVT effect (58). However, the expression level of Fas failed to serve as a sensitive and specific marker for GVHD (59).

Variable mechanisms have been proposed for the function of Fas/FasL pathway in GVHD. Using murine parent to F1 models, it was reported that FasL pathway was important for both CD4⁺ and CD8⁺ T cell-mediated GVHD. Host mice receiving FasL-deficient donor T cells developed significantly less GVHD compared with WT donor T cells (60). FasL-deficiency in donor T cell did not affect T cell proliferation, homing, activation, cytokine production, and anti-tumor activity, but decreased mature T cell expansion after allo-HCT (50, 60). However, allo-HCT of FasL-deficient T cells led to decreased donor cell engraftment and subsequent chimerism (61). On the recipient side, both Fas-deficient and FasL-deficient mice had higher GVHD mortality compared with WT mice (62, 63). Together, these findings show that Fas/FasL pathway in the host is vital to resist donor cell engraftment and subsequent GVHD, while important for donor cell engraftment in allogeneic host to form stable chimerism after non-myeloablative conditioning. Therefore, how to attenuate Fas-mediated GVHD, while not affecting donor cell engraftment is a great challenge. Further study showed brief exposure of unstimulated naïve donor lymphocytes to FasL in vitro preferentially depleted FasL-sensitive cells, and attenuated GVHD without impairing engraftment or GVT activity (64). In addition, FasL had been found to enhance the killing activity of CD25⁺ regulatory T cells (killer Treg) and abrogate autoimmunity. Infusion of killer Treg cells increased apoptosis of effector lymphocytes and ameliorated GVHD severity (65).

Previously, it was believed that CD4⁺ T cells cause cytotoxicity mainly through Fas/FasL pathway while CD8⁺ T cells prefer the perforin/granzyme pathway (66). However, reports afterwards demonstrated that the perforn/granzyme pathway was involved in cytotoxic function of CD4⁺ T cells and Fas/FasL is important for that of CD8⁺ T cells as well, though the potency was variable (60, 67). Maeda et al. (68) reported that deficiency in either perforin or FasL in CD8⁺ T cells decreased the development of GVHD, indicating that both were required for the function of alloreactive CD8⁺ T cells. However, another study showed that donor T cell cytotoxicity via Fas/FasL or perforin was not prerequisite for induction of GVHD (69). T cells lacking perforin and FasL function can still cause lethal GVHD after bone marrow transplantation (69). Furthermore, it was reported that memory CD8⁺ T cells in the host mediated resistance to donor cell engraftment through a mechanism that was independent of FasL and perforin pathways (70). Sleater et al. (71) demonstrated that the absence of either perforin or Fas had little impact on rejection of pancreatic islet. However, simultaneous disruption of both pathways prevented allograft rejection despite T cell infiltration. These findings painted a complicated picture about how Fas/FasL in the host and donor cells affect GVHD. We postulate that the perforin/granzyme and Fas/FasL pathways comprise alternative and required mechanisms for T cell-mediated cytotoxic function in the context of allo-HCT. In addition, FasL is the critical for NK cell-mediated cytotoxicity. Donor NK cells have been found to suppress GVHD while inducing GVT effect after allo-HCT (72, 73). Olson et al. also showed that co-injection of donor NK cells with alloreactive T cells decreased host GVHD severity by reducing cytokine production, T cell activation, and proliferation, via a mechanism that involved T cell apoptosis induced by NK cells through the FasL and perforin pathways (74).

Skin, liver, and intestines are typical target organs in acute GVHD, while primary and second lymphoid organs are also susceptible. In a human skin explant model, higher GVHD score was associated with Fas expression in epithelium and blockade of Fas-mediated apoptosis decreased severity of cutaneous GVHD damage (75). Likewise, in oral mucosa lesions, allogeneic lymphocytes from FasL-defective mice did not induce vascular damage, or epithelial cell death in recipients, suggesting a major role of FasL by allogeneic lymphocyte-mediated mucosal GVHD (76). It was found that radiation conditioning prior to allo-HCT upregulated Fas expression on thymic stromal cells and donor alloreactive T cells used FasL to medicate thymic GVHD (77). In addition, bone marrow atrophy is mediated by p53-dependent up-regulation of Fas (78). Ceramide-rich macrodomains are sites where Fas is concentrated on cells. Sphingomyelinase-deficient mice, which cannot generate ceramide, revealed reduced GVHD-related organ damage, attenuated cytokine storm, and CD8⁺ T cell proliferation. These results indicate that GVHD-mediated cutaneous damage is associated with Fas expression in recipients (79). However, studies of GVHD in liver and intestines are controversial. Hepatotoxicity is more likely through FasL-Fas pathway (80), while intestinal GVHD is associated with FasL-dependent TNFα level (81). Specifically, hepatic lesions were improved by administration of anti-FasL antibody whereas intestinal lesions were protected by anti-TNFα antibody but not by anti-FasL antibody (82). This result indicates that FasL and TNFα differentially contribute to GVHD pathogenesis. Contradictory results were also reported that administration of anti-FasL and anti-TNFα antibodies or using FasL-deficient donor T cells was not able to prevent intestinal GVHD (52, 82, 83). Furthermore, hematopoietic stem cells are also susceptible to FasL-induced cell apoptosis. A recent murine model study indicated that bone marrow cells pretreated with IFNγ increased expression of Fas and related caspases and proapoptotic genes which cause engraftment failure after allo-HCT (84). Therefore, it becomes evident that multiple pathways are involved in this sophisticated network and further investigations need to evaluate the role of Fas/FasL pathway in crosstalk with other molecules during GVHD target organ damage.

Initially, the FasL/Fas system was believed to engage in GVHD only (32). Depletion of FasL led to decreased lethal GVHD while GVT activity remained intact, suggesting that other molecular pathways are responsible for GVT effect (85). However, another report showed that CD8⁺ T cell-mediated GVT activity depended on IFNγ and FasL but did not require TNFα, perforin or TRAIL (35). Other studies showed that FasL and perforin were both required for CD8⁺ T cell-mediated GVT effect (86), while the perforin/granzyme pathway may be more dominant in GVT activity mediated by CD8⁺ T cells (60). It is also possible that CD4⁺ T cell-mediated GVT effect relies on both the Fas/FasL and the perforin/granzyme pathways (60). These studies used different donor-host combinations for allo-HCT and various tumor models, which may explain the discrepancy on the contributions of these pathways to GVT effect. While there is not a comprehensive all-in-one model to quantify the contributions of various pathways to GVT activity in different cancers that are enormously heterogenous, it is reasonable to conclude that the FasL/Fas system is important for GVT effect against certain tumors.

Grossman et al. first showed that human adaptive Treg cells (converted from CD4⁺ conventional T cells) preferentially express GzmB and can kill allogeneic target cells in a perforin-dependent manner (99). On the other hand, human CD4⁺CD25⁺ natural Treg cells express GzmA but very little GzmB. Both Treg subtypes display perforin-dependent cytotoxicity against autologous target cells, including activated CD4⁺ and CD8⁺ T cells, CD14⁺ monocytes, and both immature and mature dendritic cells (100). Based on in vitro activation of human T cells, these findings suggest that the perforin/granzyme pathway is one of the mechanisms that human Treg cells use to control immune responses. A recent study from Choi et al. (101) reported that the hypomethylating agent azacytidine could drive Foxp3 expression in non-Treg cells and convert them into Tregs that could suppress GVHD without decreasing GVT effect in a murine model. And the suppressive function in those converted Tregs was partially dependent on perforin, but not GzmB. However, our studies with in vivo mouse tumor models showed that GzmB is important for natural Treg cell-mediated suppression of anti-tumor response (102). For natural Treg cell-mediated allogeneic T cell response, it was learned that GzmB was not required for donor natural Treg cell-mediated suppression of murine GVHD (103). Furthermore, our recent work has proven that GzmB is not required for natural Treg cell-mediated suppression of GVT effect either (104). Therefore, it seems that inhibiting GzmB will cause minimal influence on natural Treg-mediated suppression of murine GVHD and GVT effect. However, GzmA has recently been reported to be required for Treg-mediated suppression of murine GVHD, providing protection against GI tract damage (105). In a recent clinical study, Ukena S et al. analyzed CD4⁺CD25^hiCD127^lo Treg population from patients with and without GVHD after allo-HCT and found that higher GzmA expression in Treg cells had better tolerance to allo-graft (106).

Using MHC I-mismatched and MHC-fully mismatched murine models, Graubert et al. reported in 1996 that GzmB was important for CD8⁺ T cells to cause lethal GVHD. GzmB deficiency in CD8⁺ T cells significantly decreased the lethality and severity of GVHD after transplantation (96). Recent studies by our lab added to two new discoveries. First, while GzmB^−/− CD8⁺ T cells exhibit reduced ability to cause GVHD, which was expected, surprisingly GzmB^−/− CD8⁺ T cells showed significantly enhanced GVT activity with several tumor models (107). GzmB-mediated activation-induced T cell death may account for the different anti-tumor immune responses between WT and GzmB^−/− CD8⁺ T cells. Secondly, we have found that a TLR5 agonist, could not only enhance GVT activity via activating antigen presenting cells (APCs) (108), but also stimulate up-regulation of endogenous GzmB inhibitor, Spi6, in accessory immune cells including APCs. In addition, our new report showed that Spi6 protects alloreactive T cells from GzmB-mediated mitochondrial damage, preserving their ability to cause GVHD without affecting GVT effect (109). Yet our study also suggests a novel function for Spi6, which contributes to GzmB-independent protection of intestinal epithelial cells in murine GVHD (110).

Initially, it was thought that GzmB was not important for CD4⁺ T cell-mediated GVHD. However, from our study published recently, we found that GzmB expression was upregulated in CD4⁺CD25⁻ conventional T cells after allo-HCT (111). GzmB^−/− CD4⁺CD25⁻ T cells exhibited enhanced expansion which was due to decreased activation-induced cell death (AICD). More GI tract damage and more cytokine production were observed in the hosts mice receiving GzmB^−/− CD4⁺CD25⁻ T cells. Using both MHC-mismatched (B6 to BALB/c) and minor antigen mismatched (129/SvJ to B6) models, we confirmed that GzmB^−/− CD4⁺CD25⁻ T cells caused more severe GVHD compared to WT counterparts (111). On the flip side, GzmB^−/− CD4⁺CD25⁻ conventional T cells partially lost GVT effect compared with WT T cells (104).

These new results reveal a more complicated paradigm for this pathway in allo-HCT as GzmB function in different T cell subsets (CD4⁺ vs. CD8⁺) unexpectedly leads to opposite outcomes in GVHD and GVT effect. Therefore, simply targeting GzmB in total T cell population is probably not beneficial for improving allo-HCT. Instead, disabling GzmB function in selected CD8⁺ donor T cells but not in CD4⁺CD25⁻ donor T cells may lead to favorable outcomes desired for allo-HCT patients.

TNFα is a type II transmembrane protein which can be cleaved to a soluble form. It has been well-studied and known as a pro-inflammatory cytokine (114, 115). Holler et al. first reported that increased blood level of TNFα was observed in acute GVHD (116). Soluble TNF receptors (TNFRs) were also associated with GVHD related complications (117). Choi et al. demonstrated the dynamical change of TNFR1 level before and after allo-HCT and its correlation with high grade II-IV GVHD (118). Clinical trial investigating the combination of TNFα monoclonal antibody (Etanercept) plus methylprednisolone as initial therapy for GVHD found substantial majority of remission, delayed onset of acute GVHD and reduced organ damage (112). Subsequent phase II trial revealed that lower TNFR1 level was linked with better prognosis. However, adding Etanercept to standard prophylaxis did not affect the overall rate of GVHD (119). In a multicenter prospective study, Etanercept arm had lower rate of GVHD initially, but achieved similar response in the end (120). Infliximab, a murine-human chimerized monoclonal antibody against TNFα, failed to lower the risk of GVHD in a small prospective study (121). In addition, TNFα has been a promising target in GVHD prevention particularly in gastrointestinal system (122). It should be noted that none of the studies stratify the patients based on their TNFα or TNFRs levels and these ambiguous findings should prompt us to revamp the conventional concept of TNFα. On the flip side, TNFα performs critical regulatory function in Treg cells after allo-HCT (123). In a murine allo-HCT study, donor Treg cells primed by TNFα can decrease GVHD, prolong animal survival and maintain GVT effect (124). Overall speaking, TNFα plays a fundamental role in allo-HCT, including GVHD initiation and progression, affecting clinical outcome and response to therapy, yet it functions much more like a pro-inflammatory cytokine than a cytotoxic molecule.

IFNγ plays a central role in host defense by regulating both innate and adaptive immunity, including specific effects on T cell differentiation and proliferation (125). IFNγ exerts paradoxical effect in GVHD. Exogenous IL-12 treatment stimulates IFNγ-mediated protection against GVHD after lethal irradiation conditioning on the day of allo-HCT (126). However, using IFNγ knockout mice, two groups independently reported that neither donor nor host derived IFNγ is required for the development of GVHD (127, 128). Further studies confirm that the protective effect of IFNγ may depend on IL-12, IL-18, or Fas (129–131). Although the exact mechanism of IFNγ in GVHD remains unclear, it may implicate that IFNγ signaling in recipient non-hematopoietic cells is more important in the process of GVHD development (125). In a recent study, Kim et al. (132) showed that human MSCs, primed with IFNγ before infusion, displayed stronger suppression of GVHD in vivo in an indoleamine 2,3-dioxygenase (IDO)-dependent manner. On the other aspect, IFNγ production is essential for tumor eradication as well (133). GVT effect was diminished in the hosts receiving IFNγ-deficient donor cells as IFNγ was also shown to promote FasL-dependent GVT activity of CD8⁺ T cells (35). Furthermore, lack of IFNγ led to impaired Treg function and exacerbated GVHD (134). Among these studies, we note that IFNγ may function as a cytotoxic molecule as well as a proinflammatory cytokine. While both functions are involved in GVHD and GVT effect, a better mechanistic understanding of the INFγ signaling is still required for dissociating the GVT effect from GVHD.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to TNF superfamily. TRAIL can induce target cell apoptosis though binding to death receptor (DR) 4 or 5 (135). TRAIL is upregulated after allogeneic stimulation and does not affect donor T cell proliferation and cytokine production. It has been reported that TRAIL contributes to optimal GVT effect since TRAIL^−/− donor T cells exhibit decreased anti-tumor activity (37). NK cell-derived TRAIL was also shown to kill acute lymphoblastic leukemia cells after HCT (136). A study using over-expression system revealed that TRAIL⁺ T cells induced less GVHD but augmented GVT effect (137), while another study reported that the level of soluble TRAIL in peripheral blood after allo-HCT was corelated with better prognosis with less GVHD (138), suggesting that TRAIL may be a feasible target for GVHD and GVT management.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.