Azathioprine (AZA) was the first anti-proliferative agent available for clinical use and is commonly used in SOT patients in combination with other drugs, mainly Cyclosporine and Prednisone. AZA is converted to 6-mercaptopurine (6-MP) in vivo, which in turn is converted into 6-thiouric acid, 6-methyl-MP, and 6-thioguanine. These metabolites are incorporated into the DNA, block the de novo pathway of purine synthesis and inhibit cell proliferation. The main toxic effects induced by AZA are leukopenia, thrombocytopenia, anemia, and hepatotoxicity (22). Immunosuppressive function of AZA is due to its ability to negatively interfere with the function and proliferation of T and B lymphocytes, with a relatively higher selectivity for T cells. In addition, AZA was shown to photosensitize the skin (23), promoting the accumulation of 6-thioguanine in DNA, which results in enhanced production of mutagenic reactive oxygen species after exposure to ultraviolet A (UVA) (24). These effects were suggested to have an impact on the development of squamous cell carcinomas (SCC) and other skin cancers in transplanted patients. A meta-analysis showed that treatment with AZA was associated with a significantly increased risk of SCC in SOT patients, whereas no significant association was observed between exposure to AZA and basal cell carcinoma (BCC) (25). A more recent study reported a significantly elevated risk of both BCC and SCC in SOT patients treated with AZA (26). Notably, multivariate analysis disclosed that AZA was associated with significantly higher risk than mycophenolate mofetil, sirolimus, cyclosporine, or tacrolimus (26). A recent whole exome sequencing study (WES) on cutaneous SCC from immunosuppressed patients has revealed a high mutation rate with an average of 50 mutations per megabase pair DNA. In particular, mutational signature analysis reveals the presence of a novel signature, whose presence correlates with chronic exposure to AZA (27). This signature is probably the result of combined action of UV exposure and incorporation of AZA metabolites into DNA, ultimately promoting tumor progression. These findings have clinical relevance for patients under treatment with AZA, who should be counseled about their skin cancer risk and UV photosensitivity.

Mycophenolic acid (MPA), isolated in 1896 from Penicillium brevicompactum by Bartolomeo Gosio, is the active metabolite of mycophenolate mofetil (MMF) (28). MMF is a potent immunosuppressive drug used especially in combination with Calcineurin inhibitors (CNIs). MPA is the reversible uncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH), the enzyme that synthesizes de novo guanosine nucleotides, thereby inhibiting DNA synthesis. In 2000, MMF was approved by FDA for use in liver transplantation, and in 2005 it has been shown (29) that treatment with MMF improved graft and survival of adult liver transplanted patients. Common side effects are nausea, vomiting, diarrhea (30) and high risk of opportunistic infection, in particular due to Cytomegalovirus (CMV) (31, 32). As compared to the use of AZA, MPA was associated with significantly lower risk of cutaneous SCC in cohorts of kidney, kidney–pancreas, heart and lung transplant patients (33, 34), an effect probably related to a decreased ability of MMF to induce ultraviolet light-related transforming effects (35).

Myeloid-derived suppressor cells (MDSCs) are innate cells that play a pivotal role in inhibiting T-cell dependent responses. MDSCs are not a terminally differentiated cell population and are characterized by CD33⁺ expression, whereas CD3, CD14, CD19, CD56, and HLA-DR are usually negative. In mice, MDSCs consist of two large groups of cells termed granulocytic or polymorphonuclear (PMN-MDSCs) characterized by CD11b⁺Ly6G⁺Ly6C^lo expression, and monocytic (M-MDSCs) characterized by CD11b⁺Ly6G⁻Ly6C^hi expression (112). Also in human, two main groups of MDSCs have been identified: granulocyte MDSCs (G-MDSCs, CD33⁺CD11b⁺CD14⁻CD15⁺) and monocytic MDSCs (M-MDSCs, CD33⁺CD11b⁺CD14⁺CD15^−/low) (112, 113). M-MDCSs contributes to production of inflammatory cytokines and growth factors, which may have a strong immunosuppressive effect, including the inhibition of T-cell proliferation (112, 114), and impaired maturation and development of DCs. In fact, DCs generated in the presence of MDSCs were found to be less effective in antigen uptake, migration and induction of IFN-γ production by T cells (115). Furthermore, MDSCs are able to down-regulate the production of IL-12 by macrophages while increasing IL-10 production in response to cell-cell contact (116). Experiments carried out in mouse models demonstrated that MDSCs identified with phenotypic biomarkers were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Interestingly, CD4⁺CD25^highFoxP3⁺ Treg cells were insensitive in vitro to MDSC-mediated suppression. These results suggest that increasing numbers of MDSCs can inhibit alloreactive T-cell proliferation in vivo and that these cells may participate in the maintenance phase of tolerance (114). In an analysis of a cohort of 50 kidney transplant patients, Meng et al confirmed that the number of MDSCs was associated with long-term graft survival and showed that this population regulates the imbalance between Tregs and Th17 cells (117). Moreover, in an experimental mouse model of transplantation, Garcia et al. showed that MDSC were associated with an increased accumulation of Foxp3-expressing Tregs in the allografts of transplant recipient mice following tolerogenic treatment (118). The impact of CsA on the myeloid population was investigated in a skin transplantation study showing that CsA can stimulate the accumulation of CD11b⁺Gr1⁺ cells (MDSCs in mice) by improving the immune responses through the NFAT pathway, also promoting the differentiation into CD4⁺ and CD8⁺ T cells. In a murine cardiac transplantation model, RAPA treatment led the recruitment of MDSCs, which also expanded as a consequence of the effects of the treatment on the mTOR pathway (119). In addition, inhibition of the mTOR signaling was shown to promote a shift of MDSCs toward the G-MDSCs subtype (120). A recent study in human kidney transplantation demonstrated that CD33⁺CD11b⁺HLA-DR⁻ MDSCs were able to expand Tregs in vitro, through the release of TGF-β and IL-10 (121). RAPA was also shown to decrease M-MDSCs differentiation from myeloid progenitors by blocking the glycolytic metabolic pathway (76). An excessive expansion of MDSCs promoted by immunosuppressive drugs may contribute to increase the risk of cancer in SOT patients. In fact, MDCSs constitute a critical component of the immune suppressive niche characterizing tumor microenvironment, where these cells may promote immune escape and malignant progression by affecting both the innate and adaptive immune responses (122). Studies carried out so far in the SOT setting focused mainly on the role of MDSCs in the induction of tolerance and allograft control and no data are currently available with regard to the possible correlation between expansion/overactivity of MDSCs and risk of the novo malignancy in these patients. Some indirect clues derived from a study carried out in a mouse neuroblastoma-bearing chimeras, which showed that adoptive recipient leukocyte infusion enhanced anti-tumor responses of allogeneic bone marrow transplantation. These anti-tumor effects, however, were counteracted by expansion of host MDSCs pointing to a relevant role of these immunosuppressive cells in limiting the efficacy of anti-tumor immunity in the transplant setting (123).

Macrophages were identified by Elie Metchnikoff as an essential component of innate immune system that forms the first line of defense against pathogens (124). According to their differentiation status and functional role in the immune system, macrophages are conventionally classified into M1 and M2 subtypes, although these cells has the ability to differentiate into a variety of phenotypes in response to different stimuli from the microenvironment (125). The M1 phenotype can be identified by the overexpression of surface molecules such as MHC-II and CD86, and increased ability to present antigens and kill intracellular pathogens. In vitro, classical activation can be induced by stimulating macrophages with IFN-γ and LPS, causing TNFα production, associated with microbicide activity, production of pro-inflammatory cytokines and cellular immunity. Macrophages then undergo a process of activation toward a pro-inflammatory phenotype, increasing their ability to kill intracellular pathogens and contributing to the progression of the inflammatory process. M2 macrophages are generated in the presence of the anti-inflammatory cytokines IL-4, IL-10, and IL-13 and participate in tissue remodeling and long term repair (126). In addition to the host defense role, macrophages play an important role in homeostasis, and pathological processes such as obesity and malignancy (127). In particular, they show a more immunosuppressive phenotype characterized by impaired antigen presentation to T cells and production of cytokines that stimulate a Th2 response (128). Evidence accumulated so far however indicates that the binary classification of macrophages in M1 and M2 subtypes is an oversimplification that does not account for the marked plasticity of these cells that may acquire a broad and continuous spectrum of different phenotypes, with M1 and M2 representing the two extremes (128). Examples of non-M1/M2 cells are: CD169⁺ macrophages, detected in bone marrow, lymph node, liver, and spleen, and mainly involved in erythropoiesis and immune regulation; macrophages expressing TCRαβ or TCRγδ identified in inflammatory and infectious diseases; a novel subtype of tumor-associated macrophages characterized by an M2-like immunosuppressive gene profile and expressing a novel receptor “macrophage receptor with collagenous structure” (MARCO), detected in mouse tumor models of mammary carcinoma, colon cancer and B16 melanoma (128). The function of these non-M1/M2 macrophages however remain far from being fully characterized. In the SOT setting, macrophages play a controversial role: the presence of CD86⁺ macrophages is associated with acute rejection in kidney transplants (129) and in mouse models of heart transplant (130). Likewise, macrophages may exert anti-inflammatory responses and immunosuppressive effects that help maintain the peripheral tolerance, being able to produce IL-10 and TGF-β, involved in the resolution of graft inflammation (131). The use of immunosuppressive drugs has also an impact on the macrophage compartment: the use of CsA and TAC was associated with a decrease in the production of inflammatory mediators, such as IL-2 and TNF-α (69). Moreover, CNIs were shown to promote the differentiation of macrophages to the M2 phenotype (132). CsA was shown to impair the phagocytosis activity of macrophages and enhance the severity of infectious diseases (133). mTORi were shown to significantly decrease the production of chemokines induced by LPS, such as MCP-1, IL-8, RANTES, MIP-1α, and MIP-1β and their combination with glucocorticoids increased the production of the anti-inflammatory cytokine IL-10 through the STAT3 transcription factor (134). mTORi could also induce macrophage apoptosis, mainly affecting the M2 rather than M1 subset (77). It has also been documented that the administration of MMF reduces the synthesis of nucleotides by inhibiting the inosine monophosphate dehydrogenase pathway (135), decreases the production of IL-1β, IL-10, TNF-α (134) and the expression of adhesion molecules (136) by macrophages. Studies aiming at assessing the role of macrophages in SOT patients with cancer are scarce. No significant difference in the density of tumor-associated macrophages was observed in SCCs from SOT recipients as compared to SCC of non-transplant patients. These findings seem to rule out that the density of these cells may contribute to a worse prognosis of invasive SCC in transplant patients. Intriguingly, however, the density of tumor-associated macrophages infiltrating SCC in situ was markedly lower than that detected in non-transplant patients (137). It remains to be elucidated whether this is the result of direct effects of immunosuppressive drugs and whether this decreased infiltration of macrophage contributes to the enhanced invasiveness of SCC in SOT patients.

DCs constitute a heterogeneous population of APCs that mediates critical connections between innate and adaptive immunity (138). These cells play a pivotal role in antigen processing and presentation resulting in the induction of effective immune responses against pathogens and tumor cells. Notably, these cells are the most powerful APCs, being able to orchestrate a primary immune response but also to induce immune tolerance (138). In particular, DCs play a central role in priming naive T and B cells, the first critical steps in the induction of an antigen-specific immune response. DCs develop from CD34⁺ bone-marrow progenitor cells or from CD14⁺ monocytes and differentiate into immature DCs (iDCs), which are functionally specialized to take up exogenous antigens. After recognition, exogenous antigens are internalized and the activated DCs migrate to the draining lymph node, where they can induce an adaptive immune response (139). This successful outcome requires the processing of antigens and their loading in the form of small peptides on the main histocompatibility complex (MHC) molecules. Peptides loaded on MHC-II molecules are recognized by antigen-specific CD4⁺ T helper cells, while peptides loaded on MHC-I molecules are recognized by antigen-specific CD8⁺ T lymphocytes. The presentation of internalized antigens on MHC-I molecules is defined as cross-presentation, a crucial process in the induction of effective adaptive immune responses against tumors and viruses that do not infect DC directly and that may induce peripheral tolerance (140). The MHC-I pathway is normally used to present endogenous antigens and cross-presentation is particularly important because it allows DCs to present through the MHC-I pathway also exogenous antigens, which are usually mainly presented by MHC-II molecules (141). Antigen cross-presentation involves two main pathways: (1) the vacuolar pathway, in which the processing/loading takes place within the endo/lysosomal compartment and (2) the endosome pathway, in which the internalized antigens are transported from the endosome to the cytosol where they are degraded by the proteasome (142). The co-stimulatory CD40/CD40L axis along with the danger signal provided by an exogenous antigen are catalysts for DC licensing. Therefore, exogenous antigen cross presentation and the consequent activation of naive CD8⁺ cytotoxic T cells, provide the immune system with an important mechanism for generating immunity to viruses while preserving tolerance to self (143).

DCs exist in two differentiation states, each of which has distinct phenotypic, morphological and functional characteristics: immature dendritic cells (iDCs) and mature dendritic cells (mDCs). iDCs phenotypically show high expression of CCR1, CCR5, CCR6, and CD68, while the expression of CCR7, CD86, CD80, CD40, and CD83 is low. These cells are able to capture and process the antigens they encounter and subsequently evolve to mDCs, which are characterized by high expression levels of CD83, CD86, CD49, CD80, CCR7, MHC-II, CD1a, and CD11c (144).

Another classification of DCs is based on different DC subpopulations: classical DCs (cDCs), plasmacytoid DCs (pDCs), monocyte-derived DCs (moDCs), and Langherans cells (LCs). cDCs are professional APCs that play a key role in shaping appropriate adaptive immune responses (145). There are two major subsets of cDC: cDC1, which are HLADR⁺CD11c⁺CD9α⁺ and CD24⁺ or CD103⁺ and require the transcription factors IRF8 (146), Batf3 (147), Nfil3 (148), and Bcl6 (149) for their development. These cells also express the CD141⁺ or blood DC antigen 3 (BDCA3). This population was found in spleen, tonsil, liver, lung and skin. Similar to mouse DCs, human cDC1 CD141⁺ express TLR3, but unlike the mouse subset, they lack the TLR8 and TLR9. This population is particularly efficient at cross-presentation of cellular antigens and are highly competent to stimulating allogenic or autologous CD4⁺ T cell immune responses. The second major cDC subset, cDC2, expresses HLADR⁺ CD11c^hi and the IRF4 transcription factor. This subset is present in lymphoid tissues and CD141 molecule is expressed by splenic cCD2. Human blood cDC2 express high levels TLR2 and TLR8 and very low levels of TLR4. They are able, without TRL activation, to cross-present soluble antigens, a process that may be enhanced bafilomycin-mediated inhibition of endosomal acidification (145, 150). From an ontogenetic point of view, available data are consistent with the existence of common DC progenitors that give rise to pDCs and intermediate precursors of cDCs (pre-cDCs) that may differentiate into CD1c⁺ (BDCA-1) or CD141⁺ (BDCA-3) cDCs. In human peripheral blood mononuclear cells, the HLA-DR⁺CD14⁻CD11b⁻ subpopulation includes the CD1c⁺ DC subset (CD172α⁺ and IRF4⁺), the CD141^high DC subset (Clec9A⁺, XCR1⁺, IRF8⁺, and TLR3⁺), and the pDC subset (BDCA-2⁺, BDCA-4⁺, and CD123^bright).

pDCs correspond to a subset of immature CD11c⁻ population distinct from classical myeloid CD11c⁺ DCs. Pre-pDCs express CD4 but lack T-cell receptor alpha (TCRα), TCRβ, TCRγ, TCRδ, or CD3 chains and are negative for B-cell lineage (CD19, CD21) or myeloid (CD13, CD14, CD33) markers. This population is characterized by the production of type I interferon during viral infection as they express constitutively IRF7. pDCs circulate in the blood and in peripheral organs and are strongly dependent on Fltl3 ligand (Flt3L), a potent endogenous DC growth factor, for their development and function. After maturation in response to TLR-ligation or CD40-engagement, pDCs became capable to present antigens to T cells and preferentially, induce generation of a unique type of CD8⁺ Treg cells (151).

LCs are the DCs of the epidermidis, although they may be also present in other stratified epithelia, such as the mucosal oral and vaginal epithelium. LCs share with cDCs common myeloid or monocytic progenitors. LCs are stellate cells expressing MHC-II molecules and the integrin αX chain CD11c. They express also CD1a and CD1c, two MHC-I-related molecules involved in the presentation of lipid antigens. This population is considered sentinel tissue-resident DCs and their development is independent of the Flt3/Flt3L axis stimulation (152).

moDCs are phenotypically difficult to differentiate from cDCs because they share the expression of CD11c, CD11b, and MHC-II, but they express the Fc-gamma receptor 1 (FcγRI) (153). These cells also express CD1α, which is characteristic of the DCs derived from monocytes. Their mature phenotype is characterized by the loss of CD14, a marker of differentiated monocytes and by an increase in the expression of CD1α, MHC-II, CD83, and CD86. moDCs can be successfully grown in the presence of GMC-SF and IL-4 and are capable of induce effective antigen-specific immune responses (154).

Immunosuppressive drugs employed in the management of allograft rejection in SOT patients can influence the phenotypic and functional characteristics of DCs, suggesting that their impaired function may also play a role in the development of tumors or in promoting the reactivation of oncogenic viral infections in immunosuppressed patients. MMF was shown to down-regulate co-stimulatory and adhesion molecules, such as CD40, CD54, CD80, and CD86, in human monocyte-derived DC in vitro, a decreased production of TNF-α, IL-10, IL-12, and IL-18, as well as less efficient stimulation of alloreactive T cells (65). Similarly, the 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), i.e., the active form of vitamin D3, inhibits the differentiation and maturation of human DCs, leading to down-regulated expression of CD40, CD80, and CD86 costimulatory molecules and inhibition of alloreactive T cell activation (155). Interestingly, Gregori et al. showed that short-term treatment of transplant recipient mice with 1,25(OH)2D3 in combination with MMF induced tolerance to islet allografts and expanded CD4⁺CD25⁺ Treg cells that were shown to adoptively transfer transplantation tolerance. Moreover, these DCs displayed a tolerogenic phenotype characterized by down-regulation of CD40, CD80, and CD86 and reduced IL-12 production (156).

The effects of CNIs on DCs are well-recognized and several groups have investigated the impact of these drugs on mouse and human DCs. TAC and CsA were found to inhibit the allostimulatory capacity of in vitro-generated myeloid DCs without affecting DC maturation (70). On the contrary, the pioneering work of Lee et al has shown how the treatment with CsA impaired the allostimulatory capacity of in vitro generated mouse bone marrow-derived DCs by downregulating CD40, CD80, and CD86 expression associated with reduced nuclear translocation of NF-κB, a transcription factor promoting DC maturation (157, 158). In addition, CsA inhibited DC-dependent production of INF-γ, IL-2, and IL- 4 by T cells, and IL-6, IL-12p40, and IL-12p70 by DCs (72). Recently, it was demonstrated that DCs generated in the presence of CsA lose their ability to induce Treg proliferation, with a strong reduction of IL-12 secretion and particularly of IL-2, which is necessary for the proliferation of CD4⁺CD25⁺Foxp3⁺ T cells (159), thus having a negative impact on this population (160). Moreover, TAC was shown to inhibit the ability of DCs to stimulate T cells and to decrease the production CXCL10 and IL-12. Although IL-12 production by DC was impaired by TAC, these cells did not promote Th2 development as T cells stimulated by TAC-treated DCs produced less interferon IFN-γ, IL-4, and IL-10 (161) (Figure 1).

In mouse models, RAPA strongly impacts DCs maturation and function resulting in the induction of well-defined phenotypic characteristics of these cells. Data accumulated so far indicate that RAPA is able to impair maturation of DCs, reduce DCs costimulatory molecule upregulation, decrease the production of pro-inflammatory cytokines, inhibit T cells stimulatory capacity (78) and promote the selectively development of Treg cells in animal models of solid organ transplantation (162) (Figure 1). Mouse and human DCs treated with RAPA have an immature phenotype with low levels of CD80 and CD40 receptors and with a decreased expression of B7-H1, the PD-L ligand, a negative regulator of T cells activation and inducer of peripheral tolerance (163).

During the maturation process, DCs downregulate the CCR1 and CCR5 receptors and upregulate the expression of CCR7, which promotes the DCs migration from the peripheral tissues to the sites where they will encounter the naive T cells for their priming (144). In both mouse and human DCs, immunosuppressive drugs have divergent effects on the modulation of chemokine receptors (CKR) in maturing DC, important in regulating DCs localization and homing (164). In an elegant study, Sordi et al. showed how the use of immunosuppressive drugs may interfere with the generation of effective immune responses by affecting DC function. In particular, these authors investigated the functional relevance of CKR in the process of DC maturation both in vitro and in vivo. CsA and TAC were shown to slightly modulate the expression of CCR7 but without affecting the function of this CKR. In contrast, RAPA increased the expression of CCR7 at the mRNA and protein level and enhanced the in vitro migration of human DCs to CCL19 and of mouse DCs to lymph nodes in vivo, suggesting that it could be due to the inhibition of endogenous IL-10 production (165), which has an inhibitory activity on DCs maturation (166).

RAPA-treated alloantigen-pulsed DCs were shown to induce antigen-specific regulation and prolong experimental heart allograft survival. Taner et al. demonstrated that RAPA-exposed DC loaded with donor cell lysates and their adoptive infusion prior to experimental heart transplantation prolonged fully MHC mismatched murine heart allograft survival (167). Notably, RAPA-treated DCs were also shown to stimulate the generation of antigen-specific Foxp3⁺ Treg cells thereby promoting organ transplant tolerance (168). The induction of tolerance by RAPA is further enhanced by the combination with Flt3L, which promotes the generation of tolerogenic, immunosuppressive DCs along with the production of CD25⁺Foxp3⁺ Treg cells and IL-10 secretion (169).

T cell responses are modulated by cytokines secreted by mDCs including pro-inflammatory cytokines, such as IL-12 and INF-α, and anti-inflammatory cytokines, such as IL-10. DCs treated with RAPA have a distinct cytokine secretion profile at different stages of maturation. In iDCs, RAPA reduced IL-10 and IL-12 production after LPS stimulation and increased apoptosis, while mDCs are resistant to RAPA-induced apoptosis, but they also show decreased production of IL-10 and TNF-α (79). A recent study demonstrated that relevant concentrations of RAPA (20 ng/ml) inhibit the ability of both TLR7- and TLR9-activated pDCs to stimulate the production of IFN-γ and IL-10 by allogeneic T cells. On the contrary, RAPA-treated TLR7-activated pDCs were capable of stimulating the activation of naive and memory T cells, while also stimulating the generation and proliferation of CD4⁺ FoxP3⁺ Treg cells (80).

In recent years, tolerogenic DCs (t-DCs) attracted a growing interest for the development of new strategies to prevent and control allograft rejection after SOT. Enhanced generation of t-DCs, however, may negatively impact the induction of antitumor immune responses in this setting. There are two main critical factors that could shape DC functions into immunologic or tolerogenic: the stage of differentiation and the environmental factors that DCs encounter. Indeed, t-DCs comprise most immature DCs and DCs with different maturation states. One of the major hallmarks of t-DCs is their ability to induce tolerogenic responses to a much greater extent and in a shorter time as compared with immature DCs. t-DCs exert a unique immune surveillance function and normally express low levels of MHC and costimulatory molecules (CD80, CD86, CD40, OX40L) on their surface, and display a low capacity of activating T cells, which is potentially associated with T cell anergy and increased Treg cell generation. Moreover, t-DC can express high levels of inhibitory molecules such as programmed death ligand (PD-L)-1 and PD-L2, leukocyte Ig-like transcripts (ILTs) 2,34, HLA-G and galectins that may contribute to their tolerogenic potential (170–172).

Another subpopulation of t-DC was shown to have a semi-mature phenotype characterized by the high expression of MHC class II and B7 costimulatory molecules and a low production of proinflammatory cytokines, such as IL-1β, IL-6, IL-12p40, and IL-12p70 or TNF-α. However, mature DCs can also became tolerogenic. Indeed, in a subset of both mature and immature monocyte-derived DCs, Munn DH et al., demonstrated the expression of indoleamine 2,3-dioxygenase (IDO), a negative regulator of T-cell proliferation. The authors hypothesized that the presence of IL-10 during DC maturation (a regulatory cytokine associated with the development of tolerogenic DCs) prevented IFN-γ-induced down-regulation of IDO, resulting in sustained expression of functional IDO even in mature, IFN-γ-activated DCs (173). IDO and t-DC induction were also found to be associated with spontaneous renal allograft acceptance (174).

Gregori et al identified a novel subset of t-DC, named DC-10, which secretes high levels of IL-10, express ILT4 and HLA-G, and have the specific function to induce Type 1 T regulatory (Tr1) cells which are critical in maintaining tolerance to self- and non-self-antigens (172). There is also a unique subset of human DC, characterized by high expression of CD11b and low expression of MHC class II, which can negatively regulate immune responses. These DC have high expression of Fas that can inhibit CD4⁺ T-cell proliferation and produce IL-10 and IP-10 via ERK-mediated inactivation of GSK-3 and subsequent up-regulation of β-catenin (175). pDC were also found to exert tolerogenic functions. In particular, these cells display heterogeneous properties with regard to antigen presentation, maturation and expression of different costimulatory molecules. Indeed, pDCs do not induce strong T-cell responses, but they were found to induce IL-10-producing regulatory Tr1 cells in vitro (176). Moreover, Abe M et al. reported the ability of pre-pDC to prolong vascularized organ graft survival promoting tolerance (177). A subset of pDCs expressing CCR9 was identified by Hadeiba et al. in resting secondary lymphoid tissues. This population was a potent enhancer of Treg cell function and effectively prevented acute graft-vs.-host disease induced by allogeneic CD4+ donor T cells. In support of the role of pDC in tolerance induction, some other studies suggested a possible functional correlation between the enhanced presence of pDC and the elevated frequency of Treg cells (178) with successful withdrawal of immunosuppression in liver transplant tolerance (179).

Immunosuppressive factors and molecules expressed by T cells can also shape DC into a more tolerogenic state. Indeed, IL-10-producing Treg cells suppressed DC maturation and prevented Th1 cell differentiation. Moreover, interaction between t-DCs and Tregs is essential for the establishment of immune tolerance induced by apoptotic cell administration. Indeed, Wu et al. demonstrated that t-DCs promote the expansion of Tregs via PD-L1 on their surface, and Tregs facilitate t-DCs to sustain tolerogenic state via IL-10 and TGF-β (180). Another example of DC-tolerance induction by T cells occurs via Foxp3⁺ Tregs that inhibit the expression of MHC-II molecules in DC that were not able to make cognate interactions with CD4+ T cells (181). In addition, regulatory DCs can selectively recruit Th1 cells and inhibit Th1 proliferation, and promote the generation of IL-4-producing alternative memory CD4 T cells with suppressive activity (182).

The possible direct role of immunosuppressive drugs on DC function in SOT patients with or without cancer is difficult to assess. The extent and quality of T-cell responses specific for epitopes of tumor-associated antigens or oncogenic viruses may constitute a surrogate marker of the function of DC in various transplant settings. This possibility is best exemplified by the clinical relevance of the monitoring of EBV-specific T-cell responses in transplanted patients at risk to develop EBV-associated PTLD (183).