Anti-thymocyte globulin: To reduce acute and chronic GvHD incidence by in vivo T-cell depletion (TCD), anti-thymocyte globulin (ATG), a polyclonal IgG purified from immunized horses or rabbit serum by human thymocytes or T-cell lines, has been extensively evaluated. The results demonstrated that ATG could reduce the severity of chronic graft versus host disease (cGvHD) without having a detrimental effect on overall survival rate; however, the effects of aGvHD were not reduced. Further studies have shown that excessive duration or dosing of ATG can have adverse effects, such as increasing non-rebreathing masks and relapse rates or causing immunosuppressive toxicity. Therefore, the dosing of this treatment is customized based on the total number of lymphocytes per unit of body weight, and can be used as an approach to control GvHD [7].

Post-transplant cyclophosphamide: Post-transplant cyclophosphamide (PTCy) is a game changer for GvHD prophylaxis; this procedure allows the safe and effective use of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) [16]. Unlike the highly immunosuppressive regimens that can be utilized for haplo-HSCT, which lead to transplant-associated toxicity, the innovative use of PTCy treatment following non-myeloablative haplo-HSCT reduces GvHD severity [7]. T-effector cell exhaustion and Treg preservation can play an important role in the decrease in GvHD in mice [16]. PTCy can act by causing the depletion of alloreactive T-cells by eliminating their proliferation and intrathymic clonal deletion of alloreactive T-cell precursors; numerous studies have replicated these results, causing PTCy to become the most widely used haplo-HSCT regimen [17].

Sirolimus: Sirolimus is an attractive immunologic profile operator for GvHD prevention that inhibits CD8⁺ cells and promotes Treg proliferation in vitro. Sirolimus is a mammalian target of rapamycin (mTOR) inhibitor that synergizes with CNI to reduce Teff proliferation and activity without causing nephrotoxicity that may otherwise be caused by CNI treatment alone [18]. Although less nephrotoxic, this treatment is avoided in patients with a higher risk of veno-occlusive disease (VOD) because of its association with higher rates of VOD [19]. Additionally, in combination with CNI, sirolimus has been associated with increased rates of TMA; additionally, TMA can be resolved by discontinuation of this treatment regimen [20]. Eventually, the sirolimus/PTCy combination as a CNI-free, less nephrotoxic treatment regimen with acceptable rates of engraftment and aGvHD was assessed; consequently, it is currently reserved for conditions precluding CNI use, such as sickle cell HSCT and renal dysfunction [21].

A clinical-grade cell separation technology has been introduced in graft manipulation for both pan and selective TCD.

Pan-TCD: Donor graft ex vivo TCD using experimental methods, such as monoclonal antibodies with or without complement, immunotoxins, or counter-flow elutriation, has been utilized to prevent GvHD due to the competing risks of relapse and non-relapse mortality. It has been has suggested that increasing rates of graft failure have been observed in TCD grafts [22, 23]. Therefore, with the availability of clinical-scale cell separation techniques, more selective methods of TCD are being explored to prevent GvHD while preserving graft-versus-leukemia (GvL) [16].

Selective TCD: For selective TCD, various solutions have been implemented; however, most of these techniques have not exhibited lasting success. Depletion of CD5⁺ and CD8⁺ T-cells was attempted for the first time in the 1990s. Although the rates of GvHD were an incentive to continue this strategy, relapse rates were high, leading to its subsequent discontinuation [16]. Using specific antibodies to deplete and modulate subset-selective T-cells has become an interesting strategy that can ameliorate GvHD without compromising the GvL effect. In contrast to α/β T-cells, γ/δ T-cells have important innate immune functions and, through α/β TCD, these γ/δ T-cells can release cytokines rapidly, thereby killing tumor and virally-infected cells without inducing GvHD [24]. Currently, the ex vivo TCD method has been established and accepted for transplant recipients. Nonetheless, all procedures, from pan to selective TCD, are associated with different limitations, advantages, and disadvantages; disadvantages include the potential for life-threatening infections but these can be blocked by the use of adoptive specific T-cells [25]. Therefore, it is hoped that, with further clinical trials, these limitations can be overcome, and this strategy can be used as an effective treatment method.

Following antigen–T-cell receptor interaction and T-cell activation, we can observe the initiation of activity in co-stimulatory and co-inhibitory signaling pathways, the modulation of which can be an approach used to prevent GvHD.

Abatacept, which contains the extracellular and immunoglobulin heavy chain portions of the cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4), can block CD28 and CTLA-4 as co-stimulatory and co-inhibitory T-cell receptors; this prevents CD28 and CTLA-4 from binding to the APC ligands B7-1/CD80 and B7-2/CD86 ligands, respectively, and ultimately leads to an inhibitory signal. Therefore, abatacept could be considered the most promising GvHD treatment strategy that has FDA breakthrough designation.

The OX40L blockade is another co-stimulator modifying pathway. Specifically, OX40L is a ligand for OX40 (CD134), a co-stimulatory receptor on T-cells, and a strong negative regulator of Foxp3(⁺) Tregs; therefore, using an antagonistic anti OX40L monoclonal antibody (moAb) may have a beneficial effect on GvHD. Consequently, this effective strategy has been gradually introduced in clinical practice [16].

Having a significant role in immune homeostasis, Tregs (CD4⁺CD25⁺Foxp3⁺) perform their suppressive function by producing TGF-β, IL-10, and IL-35. TGF-β plays a key role in inducing stem cell quiescence and promoting osteoblastogenesis, which is essential for the conservation of the HSC niche [26]. Several studies have shown that the infusion of ex vivo expanded Tregs, or their in vivo upregulation and functional enhancement after transplantation, can be beneficial for tolerance induction or GvHD prevention [16].

In ECP, a cell-based immunomodulatory treatment, the buffy coat of peripheral blood is separated and subsequently treated with a photosensitizing agent (8-methoxy psoralen). This sample, which contains leukocytes and platelets, is re-infused back into the patient after exposure to Ultraviolet A light. ECP has been shown to have various effects on different parts of the immune system. Therefore, ECP can be an effective and safe treatment for patients with GvHD without increasing the risk of relapse or infection after HSCT [27].

Fibrosis, defined as excess connective tissue formation as a reparative or reactive process, is developed in the final stage of inflammatory processes like GvHD by inflammatory Th17 cells. A sphingosine-1-phosphate (S1P) analog, fingolimod (FTY720), is an effective immunosuppressive factor that accumulates T-cells in lymph nodes and blocks lymphocyte migration throughout the body via S1P1 internalization. By evaluating this treatment in an animal model, it was demonstrated that FTY720 could decrease the number of splenocytes but increase the number of T lymphocytes in mesenteric lymph nodes. Thus, FTY720 can reduce inflammation by suppressing the migration of T-cells to transplanted organs and preventing GvHD [8].

TGF-β is an immunosuppressive cytokine that reduces IFN-γ production and degranulation alongside decreasing the expression of activating NK cell receptors, such as NKG2D and NKp30, and decreasing total cytotoxic functions; overall, these processes can inhibit NK cells. Host Tregs are an important source of TGF-β and their depletion induces stronger NK cell–dependent allograft rejection, resulting in the suppression of NK cell activity. In contrast, exogenous administration of TGF-β prevents IFN-γ production by altering the balance between inhibitory and activating receptors and resulting in protection against GvHD [26, 39, 40]. Therefore, there is a direct relationship between increased IFN-γ levels and the incidence of GvHD [38].

Granulocyte-colony-stimulating factor (G-CSF) accelerates engraftment and increases neutrophil numbers for umbilical cord blood (UCB) transplantation, affecting group 3 innate lymphoid cells (ILC3) and NK cell differentiation [41]; additionally, before collection of HSC from donor peripheral blood, G-CSF can also be used as a potent HSC mobilizing agent [41, 42]. NKG2D expression differs among different NK cell subtypes [43]. Accordingly, several studies have indicated a postponed and reduced ILC3 expression and NK cell differentiation from HSCs that were recovered after G-CSF-induced mobilization in vitro compared to HSCs that were isolated from bone marrow (BM) or UCB. Therefore, G-CSF may affect ILC3 production [33, 41] and GvHD occurrence [33, 41, 44].

Notably, immunosuppressive drugs, such as calcineurin inhibitors, are used for the treatment of GvHD and may affect NK cell generation and differentiation. However, cyclosporine or corticosteroids may not affect helper-ILC reconstitution [41].

Many conditioning regimens for stem cell transplantation contain ATG, a polyclonal antibody that is derived from rabbits, horses, or pigs and targets fresh human thymocytes [45]. ATG can decrease T-cell levels following stem cell transplantation; additionally, this treatment results in functional NK cell reconstitution [45, 46] and death of targeted immune cells by induction of apoptosis, complement-mediated lysis, or NK-cell-mediated lysis [47]. As a result, adding ATG to pretreatment protocols can limit the incidence of GvHD by increasing the proportion of NK cells. Subsequently, an increase in NK cells leads to KIR-ligand mismatching, which ultimately reduces mortality rates after transplantation [45, 46, 48].

With respect to specific genotypes, the presence of KIR haplotype B in donors has been observed to significantly decrease the incidence of GvHD [33, 49, 50]. In particular, donor NK cells expressing KIR2DS1 are effective in haploidentical allograft settings because they kill allogenic DCs and protect against GvHD [33].

DCs are APCs that recognize foreign and self-antigens and stimulate adaptive immune cells, such as effector or memory T-cells. Plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) are characterized by their remarkable capacity to generate type I IFNs upon viral infection [53]. pDCs are important effectors of the innate immune system [54]. Further, cDCs detect bacterially-derived molecules and generate pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-12p70, to stimulate T-cell subsets (Th1 and Th17) that attract and activate cytotoxic T lymphocytes [55] (Fig. 2).

Tolerogenic, or regulatory, DCs express high levels of T-cell co-inhibitory ligands, such as programmed death ligand-1 [52]. The production of DC subsets is regulated by inflammatory stimulation. These DC-stimulating cells can selectively produce specific cytokines, such as IL-12 and IL-23, and ligands, such as delta-like 1 (DLL1) and DLL4 [55]. Immune tolerance induced by immature DCs is dependent on their low expression of CD40 and capacity to produce high levels of anti-inflammatory cytokines, such as IL-10 [56].

Anderson et al. [57] demonstrated that host-derived DCs are important for donor T cells and are essential for the induction of disease and initiation of GvHD in MHC-mismatched transplants. DC depletion appears to be an effective way to prevent organ transplant rejection as DCs play an important role in allograft tolerance. DCs that modulate and suppress immune responses are called Tol-DCs. These Tol-DCs are characterized by a decreased expression of IL-12 and other pro-inflammatory cytokines, and an increased expression of anti-inflammatory molecules, such as IL-10 and TGF-β. Additionally, Tol-DCs can decrease T-cell proliferation and lead to T-cell apoptosis, anergy, and reduced responsiveness. This DC subset can also promote Treg induction to induce immune tolerance in transplanted cells. After allo-HSCT occurs, pattern recognition receptors induce active DCs, leading to maturation of DCs, stimulation of co-stimulatory molecules, and production of proinflammatory cytokines [58].

Selective removal of both host- and donor-type APCs can reduce GvHD. Additionally, DCs can induce allogeneic T cell responses. Yu et al. [9] examined numerous donor DCs from the peripheral blood of 50 patients who underwent allo-HSCT. A small number of circulating DCs were found to decrease the risk of relapse and acute GvHD and could predict patient death after allo-HSCT; nonetheless, Perkey and Maillard [6] showed that DLL4 blockade can eliminate this effect, thereby preventing GvHD while preserving antitumor activity. Additionally, CTLA-4-Ig modulated DCs have exhibited positive results in suppressing immune response [56]. In vitro induction of Tol-DCs requires various stimulators and clinically approved drugs, including rapamycin, dexamethasone, vitamin D3, IL-10, and TGF-β [58]. Stenger et al. [59] evaluated a polyclonal ATG antibody with various immunologic effects, including several impacts on DCs, such as inhibiting antigen uptake and maturation, decreasing antigen-presenting capacity, and inducing complement-mediated lysis of these cells.

Histone deacetylase inhibitors reduce DC numbers through Toll-like receptor–induced co-stimulatory molecule presentation, pro-inflammatory cytokine release, and T-cell allo-stimulatory activity. There are numerous open clinical trials surrounding the efficacy of cellular therapies and biological interventions for the treatment or prevention of GvHD [59].

Immature DCs have been genetically modified to express soluble TNF-α receptor I and C-C chemokine receptor type 7 on GvHD and GVL in allogeneic BM transplantation mice. Studies have shown that pDCs inhibit proliferation and activate autologous T-cells via a type I IFN signaling–dependent mechanism. Alternatively, GvHD prevents the regeneration of tolerogenic donor pDCs by depleting DC progenitors, rather than inhibiting pDC maturation. The uncontrolled production of functional donor DCs is a challenge for GvHD [60]. DCs can be targeted to reduce the effects of GvHD and the reactive T-cell response. For example, the co-transfer of tolerogenic DCs and Tregs may be more useful in reducing GvHD reactions in vivo. However, one major challenge for this treatment is the need to produce a large number of donor-type tolerogenic DCs with sufficient time to exert their function [9]. Tol-DCs are a small group of DCs that are distinguished by low expression levels of co-stimulatory agents and pro-inflammatory factors. Tol-DCs stimulate immune tolerance by inducing Treg proliferation and inhibiting T-cell activation, resulting in a reduced effect of GvHD.

MSC-derived exosomes have streamlined regulatory approval and commercialized products for the treatment of GvHD, alongside a diverse range of inflammatory conditions [63].

MSCs can facilitate HSC engraftment via paracrine mechanisms, including secreting mediators and hematopoietic cytokines and suppressing the remaining host immunity [64]. The first patient with steroid-refractory aGvHD was treated with allogeneic MSCs in 2004 [65]. Giebel et al. [54] showed that MSC-derived extracellular vesicles (EVs) indirectly block the inhibitory effects of T lymphocytes and control the improvement of GvHD symptoms. Nonetheless, it is unclear whether the anti-inflammatory abilities of EVs are associated with high levels of TGF-β, IL-10, and HLA-G.

EV therapy of UCB stem cells has been observed to induce an increase in expression of C–X–C chemokine receptor type 4, a key part of HSC niche, and induce the completion of CD34⁺ cell migration from the peripheral blood to BM niche. Winer et al. [66] indicated that BM-MSC-EVs alleviated symptoms in a treatment-resistant, grade IV aGvHD patient. Consequently, many researchers have designed strategies to modify and improve MSC therapy in GvHD [64].

Other studies in mice have shown that MSCs inhibit GvHD via IL-10, or IFN-γ with nitric oxide [40]. MSCs, which can be extracted using various methods, have features that are not found in other cells. Several years of research has demonstrated that MSCs are important cells that interact with their immediate surroundings and adjacent cells, thereby supplying cell-based reactions that can be utilized in therapeutics [67]. Several studies have been conducted on the co-transplantation and co-culture of MSCs with HSCs to improve HSC expansion and differentiation. These studies evaluated the clinical capacity, inhibition, and potential treatment of GvHD in HSCT; however, the corresponding results of these studies revealed different effects in this treatment. In addition, there are several disadvantages to the clinical usage of MSCs, such as pneumonia-related death in allo-HSCT patients, increased tumor expansion, pulmonary embolism due to unregulated differentiation of MSCs, and ectopic tissue formation [68]. Nonetheless, MSCs are observed to be immunosuppressive in vitro, in preclinical animal models, and in clinical studies. An advantage of MSC use in GvHD treatment is their high safety with few side effects [68]. Ringdén et al. [62] found that DSCs have immunosuppressive activity and in vitro alloreactivity when used in combination with lymphocyte culture. DSCs are also more effective than BM-MSCs in treating acute GvHD; however, an advantage of MSCs, compared to DSCs, is their toxicity profile. Some recent studies have used MSC-EVs, because of their additional benefits, for cellular therapy in allo-HSC grafts, engraftment, and inhibition of GvHD. Application of MSC-EVs improved the effects of HSCs, including enhancing self-renewal, inhibiting differentiation, promoting homing for HSC expansion, and preventing GvHD following HSCT [62].