Solid organ transplantation is the optimal treatment for patients with terminal organ failure [1]. Transplantations conducted from September 1, 1987 to December 31, 2012, saved more than 2 million life-years for patients who underwent transplantation of the kidney, liver, heart, lung, pancreas, or intestines [2]. However, the survival benefit is hampered by allograft rejection. To prevent this, patients with transplants must take immunosuppressives for the rest of their lives. These regimens aim to prevent the proliferation and cytotoxic activity of T cells [3].

CD4 T helper (Th) cells include type 1 Th (Th1), type 2 Th (Th2), Th17, and regulatory T cell (Treg) populations. Th1 cells produce interferon (IFN)-γ and are implicated not only in host defense against pathogens such as protozoa, bacteria, and viruses but also in the development of autoimmune diseases. Th2 cells secrete interleukin (IL)-4, IL-5, and IL-13, and defend against the invasion of pathogens, including helminths. They also participate in the pathogenesis of allergic diseases such as asthma and atopic dermatitis [4]. Th17 cells produce the cytokines IL-17A, IL-17F, IL-21, IL-22, and granulocyte-macrophage colony-stimulating factor. They protect against bacterial infection and are involved in the development of diseases such as experimental autoimmune encephalomyelitis, multiple sclerosis, psoriasis, and inflammatory bowel disease [5]. Tregs are important in the maintenance of immunological self-tolerance and homeostasis. They express a high level of CD25 and express the transcription factor, forkhead box P3 (FOXP3). Treg-mediated suppression is associated with cell surface factors (cytotoxic T lymphocyte antigen-4 [CTLA-4], CD25, TIGIT, CD39, and CD73), cytokines (IL-2, IL-10, transforming growth factor [TGF]-β, and IL-35), and secreted or intracellular factors (granzyme, cyclic adenosine monophosphate [cAMP], and indoleamine 2,3-dioxygenase [IDO]) [6]. Cytotoxic effector CD8⁺ T cells eliminate cells infected with intracellular pathogens or cancer cells. CD8⁺ T cells cause pore formation in the target cell membrane and secrete death-inducing granules containing granzymes, perforin, cathepsin C and granulysin [7,8].

This review primarily focuses on the roles of Th17 cells in the rejection of transplanted solid organs, as well as their interactions with other T cell types. We also examine the roles of other T cells, including CD4⁺ T cells (Th1, Th2, and Tregs) and CD8⁺ T cells, in the rejection process. Additionally, we discuss strategies to suppress the functions of Th17 and other T cell types to mitigate rejection, such as the use of immunosuppressive drugs and the adoptive transfer of regulatory cells.

Th17 cells affect the activity of other T cell subsets, contributing to allograft rejection. Additionally, the activity of Th17 cells is regulated by Tregs. T cells can either induce or suppress allograft rejection through various mechanisms (Fig. 1) [9–22].

Figure 1. Roles of T cell subtypes in allograft rejection. Th17 cells promote the infiltration of neutrophils into a graft in an interleukin (IL)-17A-independent pathway [9]. Neutrophils undergo NETosis to promote allograft rejection [10,11]. Th17 cells also activate endothelial cells and fibroblasts. Endothelial cells release chemokines and increase their expression of adhesion molecules. In turn, immune cells are recruited and move from the bloodstream across the endothelial monolayer into the blood vessel wall. This immune cell infiltrate is a hallmark of transplant vasculopathy [12]. Activated fibroblasts contribute to fibrosis [13]. Activated endothelial cells and fibroblasts secrete IL-6, promoting the differentiation of Th17 cells [14]. IL-17 modulates allograft rejection by promoting the maturation of dendritic cells (DCs) [15]. Compared to immature DCs, which are specialized in endocytosis, mature DCs express higher levels of MHC and costimulatory molecules on their surface for efficient antigen presentation [16]. Mature DCs present peptides with MHC molecules to activate CD4⁺ and CD8⁺ T cells. The peptides are produced by the processing of donor MHC molecules. CD8⁺ and CD4⁺ T cells recognize the peptides via the direct, indirect, and semidirect pathways. In the direct pathway, recipient CD8⁺ T cells and CD4⁺ T cells engage complexes of MHC molecules and peptides derived from donor MHC molecules on the surface of donor antigen-presenting cells (APCs), and CD8⁺ T cells receive assistance from recipient CD4⁺ T cells. In the indirect pathway, CD4⁺ T cells engage complexes composed of recipient MHC molecules and peptides produced by the processing of donor MHC molecules, thereby forming recipient APC/CD4 T cell couplets. Recipient CD8⁺ T cells recognize MHC class Ⅰ: peptide complexes on donor APCs and obtain help from CD4⁺ T cells. In the semidirect pathway, CD8⁺ T cells recognize intact donor MHC class Ⅰ molecules on recipient APCs presenting donor MHC molecule-derived peptides with recipient MHC Ⅱ molecules to CD4⁺ T cells. CD8⁺ T cells obtain help from CD4⁺ T cells [17]. Activated CD4⁺ T cells differentiate into Th1, Th2, or Th17 cells, depending on the local cytokine environment [18]. Th1 cells damage allografts via Fas/Fas ligand (FasL)-mediated cytotoxicity and produce interferon (IFN)-γ and the growth factor IL-2, thereby triggering alloreactive CD8⁺ cytotoxicity. Th1 cells induce delayed-type hypersensitivity (DTH) by macrophages, which release nitric oxide (NO), tumor necrosis factor (TNF)-α, and oxygen species, leading to allograft damage. Th2 cells secrete IL-4 and IL-5 to activate eosinophils, which release harmful enzymes causing graft destruction. Th2 cells induce the production of alloreactive antibodies by B cells. Th2 cells express IL-4 and IL-10, which inhibit Th1 responses. Activated CD8⁺ T cells produce perforin and granzyme. Perforin forms channels in the allogeneic cell membrane, through which granzyme moves into cytoplasm where it induces apoptosis [19,20]. In addition to cytotoxicity, CD8⁺ T cells regulate effector T cells. CCR7⁺CD8⁺ T cells reduced the proportion of IFN-γ⁺ (Th1) and IL-17⁺CD4⁺ (Th17) T cells [21]. An increased Th17-to-regulatory T cell (Treg) ratio contributes to transplant rejection [22].

T Helper 17 Cells

Allograft rejection after organ transplantation is accompanied by increased IL-17 and Th17 levels in organ allografts, draining lymph nodes (LNs), spleen, and peripheral blood. After renal allograft transplantation, IL-17 expression in the renal allograft was elevated in rats with acute rejection [23]. The levels of Th17 cells and IL-17 in peripheral blood, serum, and plasma were increased in renal transplant patients with renal rejection [24,25]. Haouami et al. [26] reported increased IL-23 levels in the plasma of patients with renal acute rejection. IL-23 maintains and promotes the expansion of Th17 cells [27]. After liver transplantation, the number of Th17 cells was elevated in the liver and peripheral blood of rats that experienced acute rejection after liver allograft transplant. IL-17 levels in liver homogenate and serum were also significantly increased [28]. After liver transplantation, circulating Th17 levels were higher in patients with acute liver rejection than in those who were stable, and demonstrated a positive correlation with the degree of liver transplant rejection [29,30]. Serum levels of IL-17 were higher in liver transplant patients with acute rejection than in those with nonacute rejection [31]. Fábrega et al. [32] also reported increased serum levels of IL-17 and IL-23 in hepatic transplant patients with acute rejection. After cardiac allograft transplant, the proportion of CD4⁺IL-17⁺ (Th17) cells among splenocytes peaked at day 5 in allogeneic mice and was higher in allogeneic individuals than in syngeneic individuals. In addition to Th17 cells, IL-17 was produced by CD8⁺ cells and neutrophils [33]. Wang et al. [34] reported elevated levels of Th17, Th1 and Treg in the peripheral blood of cardiac transplant patients with acute rejection. The percentage of CD4⁺ IL-17⁺ T cells increased in the cervical draining LNs of mice 6 days after cornea allograft transplantation, compared to mice with isografts [35]. The frequency of Th17 cells was positively correlated with the severity of acute rejection after intestine transplantation in rats [36]. In one study on rats, the number of Th17 cells in the spleen peaked 7 days after skin transplantation, while IL-17 levels in peripheral blood peaked at day 5 [37]. After lung transplantation, IL-17 mRNA levels in allografts increased in mice with acute rejection [38]. In lung transplant patients with acute rejection, IL-17 levels in bronchoalveolar lavage increased at 28 days after transplantation but decreased at 90 days [39]. Th17 cell levels were related to sensitization to donor human leukocyte antigen (HLA) molecules, which delay access to transplantation. Some patients with end-stage renal disease are highly sensitized (HS) or nonsensitized (NS) to the donor HLA molecule. In one study, peripheral blood mononuclear cells (PBMCs) from HS patients before transplant were stimulated with PMA/ionomycin and showed higher frequencies of CD4⁺ T cells expressing IL-17A, IFN-γ, IL-4, and IL-6 than NS patients. Allogeneic stimulation of PBMCs from HS patients with donor antigen-presenting cells (APCs) resulted in higher percentages of CD4⁺ T cells expressing IL-17A, IL-4, and IL-6 than in NS patients [40].

Interruption of the action of IL-17 or inhibition of Th17 differentiation attenuates allograft rejection, implicating IL-17 therein. Treatment of mice with an IL-17R:Fc fusion protein after cardiac transplant prolonged the survival of non-vascularized and vascularized cardiac allografts [15]. IL-17^–/– mice that underwent cardiac allograft transplant had weaker chronic rejection than wild-type mice. Interestingly, γ:δ T cells, rather than IL-17-producing CD4⁺ and CD8⁺ cells, were the main source of IL-17 in the allografts of wild-type recipients [41]. In one study, transplantation of lung allografts deficient in IL-17A into mice who were repeatedly administered lipopolysaccharide into the airway resulted in alleviated periairway and pulmonary fibrosis, acute rejection, and epithelial hyperplasia compared to wild-type mice [42]. Chen et al. [38] showed that neutralization of IL-17A using anti-IL-17A attenuated acute rejection after lung transplantation in mice. In another study on mice after heart transplantation, suppression of Th17 differentiation by inhibition of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway decreased Th17 infiltration and IL-17 expression in myocardial tissue, thereby inhibiting acute rejection [43].

Th17-mediated allograft rejection is associated with Th1 cells. T-box transcription factor (T-bet) is necessary in Th1 cell development. In T-bet^–/– mice, allograft rejection was accelerated after cardiac transplantation, which was accompanied by decreased production of a Th1 cytokine (IFN-γ) and increased production of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13). Th17 cells predominated among the graft-infiltrating lymphocytes. Splenocytes from the T-bet^–/– recipients produced significantly higher amounts of IL-17, as well as other inflammatory cytokines, IL-6 and IL-12p40. Neutralization of IL-17 inhibited rejection in the T-bet^–/– recipients. This suggests that, in the absence of Th1 cells, Th17 cells mediated rejection, and that Th17-mediated allograft rejection can be downregulated by Th1 cells [44]. Another study also showed that T-bet deficiency promoted IL-17 production. T-bet^–/– mice developed severe experimental autoimmune myocarditis after immunization with MyHC-α emulsified in Complete Freund's Adjuvant. Heart-infiltrating CD3⁺ T cells from the immunized T-bet^–/– mice produced more IL-17 than those from T-bet^+/+controls [45].

IL-17 affects allograft rejection by promoting the maturation of dendritic cells (DCs). DCs are APCs that activate T cells, and their maturation is important for T cell activation [16,46]. Immature DCs are highly endocytic and take up antigens via macropinocytosis, receptor-mediated endocytosis, and phagocytosis. However, they express low levels of major histocompatibility complex (MHC) molecules and costimulatory molecules (CD80 and CD86) on their surface and are unable to process and present them to T cells. By contrast, mature DCs have an increased antigen-processing ability and express higher levels of MHC and costimulatory molecules on their surface, although their ability to engage in endocytosis decreases. Therefore, they can efficiently present antigens to T cells [16]. IL-17 promotes the maturation of DCs, the activation of T cells, and the rejection of allografts. It promotes the phenotypic differentiation of DC progenitors, as indicated by increased expression of CD11c, CD40, MHC class II (I-Ab), CD80, and CD86 [15]. Duan et al. [47] showed that inhibition of endogenous IL-17 suppressed DC maturation and the Th1 immune response and decreased the levels of inflammatory cytokines during acute heart-allograft rejection in mice. Adoptive transfer of DCs from mice treated with anti-IL-17 prolonged allograft survival and decreased the level of the Th1 cytokine IFN-γ. Th17 cells promote neutrophil infiltration into skin grafts, resulting in graft rejection. In one study, female C57BL/6.RAG1^–/– mice were infused with Marilyn-derived long-term Th17 cells and grafted with male C57BL/6.RAG1^–/– skin. The Th17-injected mice showed slow skin graft rejection and dense infiltration of neutrophils in skin grafts 48 days after transplantation, suggesting that neutrophils mediate Th17 cell-induced rejection. However, neutrophil infiltration was not dependent on IL-17A or IFN-γ; therefore, other IL-17A-independent pathways may be involved. Neuralization of IL-17A and IFN-γ did not affect neutrophil infiltration [9]. Neutrophil extracellular traps (NETs) participate in the rejection of transplanted solid organs, including the liver and kidney. Neutrophils release NETs while undergoing a type of cell death termed NETosis. NETs consist of chromatin, DNA fibers, and granule proteins. They are abundant in bioactive molecules that promote thrombosis, inflammation, and fibrosis [10,11,48]. In addition, Th17 cells activate endothelial cells and fibroblasts, which contribute to rejection [14]. Activated endothelial cells release chemokines and exhibit increased expression levels of adhesion molecules. In turn, immune cells are recruited and translocate from the bloodstream across the endothelial monolayer into the blood vessel wall. This immune cell infiltrate is a hallmark of transplant vasculopathy [12]. In cardiac allograft vasculopathy, the intima thickens and fills with macrophages and T cells, while the tunica adventitia is infiltrated by B cells, T cells, and myeloid cells [13]. Chronic lung allograft dysfunction is characterized by subepithelial and interstitial fibrosis, extracellular matrix deposition, epithelial activation, and fibroblast hyperproliferation [13]. Activated endothelial cells and fibroblasts secrete IL-6, promoting the differentiation of Th17 cells [14]. IL-17 contributes to rejection by regulating gene expression. In vitro, IL-17 increases the expression of profibrotic genes, including ACTA-2 and CTGF in human renal proximal tubular epithelial cells; these genes are linked to renal cell injury [24]. Research on the roles or levels of Th17 and IL-17 in allograft rejection is listed in Table 1.

Table 1. The roles of Th17 and IL-17 in allograft rejection

Study	Type of allograft	Subject	Findings
Wang et al. (2022) [23]	Kidney	Rats	• IL-17 levels increased in kidney grafts
Chung et al. (2015) [24]	Kidney	Humans	• Th17 cell and IL-17 levels increased in peripheral blood and serum, respectively • IL-17 increased the expression of profibrotic genes, including ACTA-2 and CTGF in the renal tubular cell line HPRTEpiC, which underwent chronic allograft injury
Crispim et al. (2009) [25]	Kidney	Humans	• IL-17 levels increased in serum
Haouami et al. (2018) [26]	Kidney	Humans	• IL-17 and IL-23 levels increased in plasma
Deteix et al. (2010) [54]	Kidney	Humans	• Th17 cell levels increased in kidney grafts
Negi et al. (2024) [40]	Kidney	Humans	• Allogeneic stimulation of PBMCs from highly sensitized patients with donor APCs showed higher percentages of CD4+ T cells expressing IL-17A than nonsensitized patients
Xie et al. (2010) [28]	Liver	Rats	• Th17 cell levels increased in the liver and peripheral blood • IL-17 levels in liver homogenate and serum increased
Assadiasl et al. (2022) [29]	Liver	Humans	• Th17 cell levels increased in peripheral blood • Th17 cell frequency was negatively associated with liver allograft functions
Fan et al. (2012) [30]	Liver	Humans	• Th17 cell levels increased in peripheral blood. • Th17 cell frequency was negatively correlated with the rejection activity index
Afshari et al. (2014) [31]	Liver	Humans	• IL-17 levels increased in serum
Fábrega et al. (2009) [32]	Liver	Humans	• IL-17 and IL-23 levels increased in serum
Wang et al. (2019) [55]	Liver	Humans	• Th17 cell levels increased in peripheral blood
Antonysamy et al. (1999) [15]	Heart	Mice	• Prolonged nonvascularized or vascularized cardiac allograft survival and inhibition of T cell proliferation were observed after treatment of recombinant mIL-17R: Fc fusion protein to inhibit IL-17 action • Phenotypic differentiation of DC progenitors after treatment of recombinant IL-17 to bone marrow-derived cells
Min et al. (2009) [33]	Heart	Mice	• Th17 cell frequencies increased among splenocytes • IL-17 levels increased in serum
Wang et al. (2011) [34]	Heart	Humans	• Th17 cell levels increased in peripheral blood
Itoh et al. (2010) [41]	Heart	Mice	• Weaker chronic rejection was observed after transplant to IL-17 deficient recipient mice • γ: δ T cells was as a main source of IL-17 after transplant to wild-type recipient mice
Zhang et al. (2021) [43]	Heart	Mice	• Weaker acute rejection, and decrease in Th17 cell infiltration and IL-17 expression in myocardial tissue were observed after treatment of JAK2/STAT3 signaling inhibitor AG490
Yuan et al. (2008) [44]	Heart	Mice	• Infiltration of Th17 cells into allografts was promoted after transplant to T-bet-deficient recipient mice • Neutralization of IL-17 with anti-IL17 weakened allograft rejection
Chen et al. (2016) [38]	Lungs	Mice	• Increase in IL-17 levels in lung graft • Weaker acute rejection was observed after neutralization of IL-17A with anti-IL-17A
Vanaudenaerde et al. (2006) [39]	Lungs	Humans	• Increase in IL-17 levels in bronchoalveolar lavage
Watanabe et al. (2023) [42]	Lungs	Mice	• Weaker acute rejection was observed after transplant to donor mice deficient in IL-17A receptor and administration of LPS
Chen et al. (2009) [35]	Cornea	Mice	• Increase in Th17 cell levels in cervical draining lymph nodes
Yang et al. (2013) [36]	Intestine	Rats	• Increase in Th17 cell levels in intestine grafts
Zheng et al. (2014) [37]	Skin	Rats	• Increase in Th17 cell percentages among splenocytes • Increase in IL-17 in serum
Agorogiannis et al. (2012) [9]	Skin	Mice	• Infusion of recipient mice with Marilyn-derived “long-term” Th17 cells caused dense infiltration of neutrophils in skin grafts

Th17, T helper 17; IL, interleukin; PBMC, peripheral blood mononuclear cell; APC, antigen-presenting cell; DC, dendritic cell; JAK2/STAT3, Janus kinase 2/signal transducer and activator of transcription 3; LPS, lipopolysaccharide.

T Helper 1 and T Helper 2 Cells

The transplantation of allogeneic organs elicits a complex immune response, which differentiates between self and nonself. CD4⁺ T cells are activated upon recognizing alloantigens presented by APCs via direct or indirect pathways. In the direct allorecognition pathway, recipient CD4⁺ T cells recognize a complex of a MHC molecule and a peptide produced by processing donor MHC molecules on the surface of donor APCs. The indirect pathway involves the recognition by recipient CD4⁺ T cells of complexes on recipient APCs; these complexes consist of a recipient MHC molecule and a peptide [49]. Activated CD4⁺ T cells differentiate into Th1, Th2, or Th17 cells, depending on the local cytokine environment [18].

Th1 cells contribute to organ allograft rejection, a finding supported by numerous studies. In rats with liver allografts, serum levels of IFN-γ and IL-10, which are Th1 and Th2 cytokines respectively, were higher than in those with isografts [50]. In patients with renal allograft rejection, a high plasma level of IFN-γ before renal transplantation and a decrease posttransplantation were associated with acute rejection. The increase in IFN-γ suggests that ongoing inflammatory responses promoted rejection [51,52]. T cell clones from renal biopsies of patients with acute interstitial grade I/II rejection (AIR) and borderline rejection (BLR) had a higher proportion of T cells producing IFN-γ (Th1) than of T cells producing IL-4 and IL-5 (Th2). This implicates Th1 cells in AIR and BLR [53]. The frequency of T-bet⁺ cells (Th1) was significantly higher in biopsies from patients with kidney grafts with delayed graft function (DGF) than in pretransplant biopsies. The frequency of GATA-3⁺ cells (Th2) increased nonsignificantly. The T-bet⁺/GATA-3⁺ cell ratio (Th1/Th2) was significantly higher in patients with DGF [56]. In patients with acute cardiac graft rejection, the percentages of circulating Th1 significantly increased, suggesting that Th1 cells are associated with rejection [34]. In cornea transplants, the levels of CD4⁺ IFNγ⁺ T cells (Th1) increased in cervical draining LNs of mice with allografts 12 and 18 days after cornea allograft transplantation, compared to mice with isografts [35]. A Th1 response in BALB/c-dm2 (dm2) mice with no MHC class I L^d molecules was induced by the MHC I determinant, L^d 61–80 peptide, emulsified in complete Freund’s adjuvant. The dm2 mice were transplanted with L^d+ skin from BALB/c mice and showed accelerated acute rejection [57].

Th1 cells participate in allograft rejection by producing IFN-γ and the growth factor IL-2, which activate cytotoxicity in CD8⁺ T cells. Th1 responses are amplified by the IFN-γ produced by CD8⁺ T cells. Th1 cells induce delayed-type hypersensitivity (DTH) by macrophages, which release toxic factors such as nitric oxide (NO), tumor necrosis factor-α, and oxygen species. In addition, they promote the synthesis of complement-fixing immunoglobulin IgG2a by B cells and damage allografts via Fas/Fas ligand (FasL)-mediated cytotoxicity [19,20].

Th2 cells can induce or suppress allograft rejection. The level of IL-4 was higher before transplantation and shortly afterward in patients who did not experience rejection, suggesting that IL-4 may suppress acute rejection. In patients who experienced rejection, IL-4 levels increased after transplantation, suggesting that IL-4 influences acute rejection [52]. Following the polarization of the T cell response to Th2 and skin allograft transplantation, dm2 mice did not show a CD8⁺ T cell cytotoxic response, and rejection was delayed to 60–80 days. After cardiac allograft transplant, an indirect Th2-mediated alloimmune response, but not a Th1-mediated alloimmune response, caused chronic rejection [57].

Unlike Th1 cells, Th2 cells lack FasL and are not directly cytotoxic. However, Th2 cells express IL-4 and IL-5, which activate eosinophils. Eosinophils release granules containing harmful enzymes, leading to graft destruction. Th2 cells also induce the production of alloreactive antibodies by B cells [20] and prevent rejection by producing IL-4 and IL-10, which inhibit Th1 responses [19].

CD8⁺ T Cells

The cytotoxic effect of CD8⁺ T cells on allografts is implicated in rejection. Activated CD8⁺ T cells express perforin and granzyme; the former forms channels in the allogeneic cell membrane, through which the latter enters the cytoplasm, resulting in apoptosis [20]. The ratio of CD8⁺ T cells to all lymphocytes infiltrating the portal tract of the liver of patients with rejection was positively correlated with the RAI score, the standard grading system for liver allograft rejection [58]. CD69⁺ CD8⁺ T cells in the peripheral blood of patients with rejection correlated with acute renal graft rejection [59]. The induction of a Th1 response in mice followed by transplantation of allogeneic skin significantly increased the antidonor cytotoxic response of CD8⁺ T cells, thereby accelerating acute rejection [57].

The proper development of CD8⁺ T cells requires help from CD4⁺ T cells, including the supply of IL-2 [18]. Ekkens et al. [60] showed that Th1 and Th2 cells promoted primary and memory CD8⁺ T cell responses. CD8⁺ T cells obtain help via the direct, indirect, and semidirect pathways of allorecognition. In the direct pathway, recipient CD8⁺ T cells and recipient CD4⁺ T cells engage complexes of an MHC molecule and a peptide derived from donor MHC molecules on the surface of donor APCs, and the latter provide assistance to the former [17]. In one study, CD4⁺ T cells from B6 mice were transferred into B6CII^–/– mice, which are deficient in CD4⁺ T cells and have APCs lacking MHC class II molecules. The CD4⁺ T cell-reconstituted B6CII^–/– recipients rejected the BALB/c heart allografts as did wild-type B6 recipients. The CD4⁺ T cell-reconstituted B6CII^–/– recipients showed stronger CD8⁺ T cell responses than wild-type recipients. Therefore, the help of CD4⁺ T cells via the direct pathway generated a strong cytotoxic CD8⁺ T cell response, resulting in rejection [61]. In the indirect pathway, recipient CD8⁺ T cells recognize MHC class I molecule–peptide complexes on donor APCs and obtain help from recipient CD4⁺ T cells forming recipient APC/CD4⁺ T cell couplets. The CD4⁺ T cells engage complexes consisting of a recipient MHC molecule and a peptide produced by processing donor MHC molecules [17]. Lymphocytes from renal allograft recipients cocultured with allopeptides corresponding to the hypervariable regions of HLA class II on CD4⁺ T cells show heightened proliferation [62]. In the semidirect pathway, CD8⁺ T cells recognize intact donor MHC class I molecules on the recipient APCs presenting donor MHC molecule-derived peptides with recipient MHC molecules to CD4⁺ T cells, and they obtain help from CD4⁺ T cells. Harper et al. [17] showed that host secondary lymphoid tissues (SLTs) were required for CD8⁺ T cell-mediated rejection (TCMR) of murine cardiac allograft. In SLTs, recipient APCs obtained MHC class I molecules from graft parenchymal cells and presented them intact to CD8⁺ T cells. They also processed MHC class I molecules and presented them to CD4⁺ T cells, enabling CD8⁺ T cells to receive assistance from CD4⁺ T cells [17].

CD8⁺ T cells can mediate allograft rejection without help from CD4⁺ T cells. In one study, CD8⁺ T cells with specificity for the MHC class I molecule H2K^b were transferred to thymectomized and CD8⁺ and CD4⁺ T cell-depleted CBA (H2^k) mice, followed by transplantation with cardiac allografts with H2K^b. The CD8⁺ T cells proliferated and caused cardiac rejection. Fifty days after transplantation, the levels of CD8⁺ T cells with the memory phenotype (CD44^hi) and of cells producing IL-2 and IFN-γ were higher in the transplanted than in the nontransplanted group [63].

In addition to cytotoxicity, CD8⁺ T cells regulate effector T cells. In one study, CCR7⁺CD8⁺ T cells from kidney transplant patients regulated effector T cells participating in TCMR. They inhibit the proliferation of CD4⁺ T cells and reduce the proportions of IFN-γ⁺ (Th1) and IL-17⁺CD4⁺ T (Th17) cells and the production of inflammatory cytokines (IL-2 and IL-17) [21].

Regulatory T Cells

Tregs are classified into thymus-derived and peripherally induced Tregs (iTregs), also known as natural Tregs (nTregs) and iTregs, respectively. nTregs are generated in the thymus during T cell development, and iTregs are produced from peripheral naïve conventional T cells during an immune response [64]. Treg levels are diminished in patients with transplant rejection. Treg levels were found to be lower in peripheral blood from patients with hepatic acute rejection than in patients who had stable graft function [65]. Demirkiran et al. [66] showed that Treg levels in peripheral blood decreased after liver transplantation and were lowest 3 months posttransplantation. Tregs induce transplant tolerance. Some transplant patients are naturally tolerant, which is known as operational tolerance (OT) and is associated with Tregs. Such patients show normal allograft function without immunosuppressants [64]. FOXP3 expression was found to be higher in OT patients than in those with chronic rejection [67]. The percentage of CD4⁺ T cells with demethylation in the FOXP3 Treg-specific demethylated region was higher in OT patients than in those with stable graft function under immunosuppression, with symptoms of chronic rejection, and healthy volunteers [68]. Tregs are immunosuppressive and inhibit the activation of other immune cells, including CD4⁺ T cells and CD8⁺ T cells, via various mechanisms [64]; for further details, see the section on the use of cell-based therapy to induce transplant tolerance.

Imbalance between T Helper 17 and Regulatory T Cells

The balance between Th17 and Treg cells is important in the suppression of allograft rejection. An increased Th17/Treg ratio (i.e., an imbalance) promotes organ allograft rejection. In liver transplantation, Th17 cells, their related cytokines (IL-17, IL-6 and IL-23), and transcription factor (RORγt) levels increased in mice with acute rejection after liver transplantation. By contrast, Tregs, cytokine (IL-10 and TGF-β1), and transcription factor (FOXP3) levels decreased. This was accompanied by impaired liver function and morphological changes. The mean ratio of Th17 to Tregs in peripheral blood was higher in patients with acute rejection 6 months after liver transplantation than in stable recipients [29]. In one study, Th17 and Treg levels in the peripheral blood of patients with acute rejection were higher and lower, respectively, 2 and 4 weeks after liver transplantation than in healthy controls [55]. In another study, after kidney transplantation, the ratio of Th17 to Tregs infiltrating allograft tissues was positively correlated with the severity of allograft tissue lesions [69]. Deteix et al. [54] analyzed intragraft infiltrate from patients with chronic renal rejection. Lymphocytes in the interstitium of kidney grafts that survived for <8 years consisted mainly of Th17, while Tregs were scarce. The opposite was true for kidney grafts that survived for >8 years [54].

Tregs modulate the quantity of Th17 cells. The transfer of exogenous iTregs decreased the proportion of Th17 and IL-17⁺ γ:β T cells among splenocytes in a murine model of orthotopic lung transplantation and reduced the severity of acute lung allograft rejection [70]. IL-17 also affects Tregs. Neutralization of IL-17 increased the number of Tregs, prolonged survival, and improved liver function in individuals who experienced rejection [70,71].

Acute and chronic graft rejection are major obstacles to long-term graft survival in recipients of solid organ transplants. Immunosuppressive drugs are used to prevent rejection and to induce tolerance. Long-term administration of immunosuppressive drugs causes adverse effects, which can be attenuated using multiple drugs at lower doses. A variety of immunosuppressive drugs regulate the signals that activate T cells. T cells may be activated by signal 1 (T cell receptor [TCR] signaling pathway), signal 2 (costimulation), and signal 3 (IL-2 signaling pathway) [72]. When the TCR and coreceptors bind MHC–peptide complexes on APCs, immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tails of the CD3 chains are phosphorylated. The protein kinase zeta-chain-associated protein kinase 70 (Zap70) binds the ITAM and phosphorylates two adaptor molecules, SLP-76 and linker for activated T cells (LAT). Activated phospholipase C (PLC)-γ binds to the adaptor molecules and is phosphorylated by IL-2 inducible T cell kinase (Itk). PLC-γ cleaves the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP₂) to diacylglycerol (DAG) and inositol trisphosphate (IP₃). IP₃ induces Ca²⁺ release from the endoplasmic reticulum. Ca²⁺ activates the calmodulin/calcineurin pathway, leading to activation of the transcription factor, nuclear factor of activated T cells (NFAT). DAG activates protein kinase C (PKC)-θ, thereby inducing activation of the transcription factor, nuclear factor kappa B (NF-κB). When the costimulatory molecules B7.1 (CD80) and B7.2 (CD86) on the APC surface bind to CD28 on T cells, the cytoplasmic tail of CD28 binds to phosphoinositide 3-kinase (PI3K). PI3K converts the membrane lipid PIP₂ into PIP₃. PIP₃ activates protein kinase B (Akt), which causes the activation of mammalian target of rapamycin (mTOR) [73,74]. The binding of IL-2 to IL-2R causes phosphorylation of JAK 1 and JAK3. JAKs are cytoplasmic tyrosine kinases associated with cell surface signaling receptors, particularly members of the cytokine receptor common gamma chain family. The JAKs include JAK1, 2, and 3 and tyrosine kinase 2, which bind to receptors after the binding of cytokines. Activated JAK phosphorylates STAT, leading to STAT dimerization and nuclear transport to regulate gene expression. Their interaction also activates the PI3/AKT pathway [75,76]. Immunosuppressive drugs are classified into pharmacological and biological drugs, such as polyclonal and monoclonal antilymphocyte antibodies [77]. The effects of immunosuppressive drugs on T cell signaling associated with T cell activation are shown in Fig. 2.

Figure 2. Immunosuppressive drugs and their sites of action that inhibit the activation of T cells. Some of the sites are linked to signals 1, 2, and 3. Rectangles with red lines, biological drugs; rectangles with blue lines, pharmacological drugs. APC, antigen-presenting cell; IL, interleukin; MHC, major histocompatibility complex; TCR, T cell receptor; JAK, Janus kinase; STAT, signal transducer and activator of transcription; PI3K, phosphoinositide 3-kinase; PIP₂, phosphatidylinositol 4,5-bisphosphate; PIP₃, inositol trisphosphate; Akt, protein kinase B; mTOR, mammalian target of rapamycin; Itk, inducible T cell kinase; Zap70, zeta-chain-associated protein kinase 70; ITAM, immunoreceptor tyrosine-based activation motif; LAT, linker for activated T cell; PLC, phospholipase C; DAG, diacylglycerol; PKC, protein kinase C; MPA, mycophenolic acid; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor kappa B.

Biological Drugs

Muromonab CD3 is an anti-CD3 monoclonal antibody that suppresses signal 1 [78]. APCs present antigens to the TCR and activate T cells via the CD3–TCR complex [72]. Belatacept and abatacept block signal 2. Both contain the extracellular region of human CTLA-4 and a part of the modified Fc region of human IgG1 [79]. These agents bind to CD80/86 ligands on APCs, preventing them from binding to CD28 [80]. Lulizumab is an anti-CD28 monoclonal antibody that blocks T cell activation [72].

Basiliximab and daclizumab are chimeric and humanized antibodies against the IL-2 receptor α-chain, respectively, and they block signal 3. They inhibit the binding of IL2 to the α-chain and the proliferation of activated T cells [81]. Ruxolitinib and tofacitinib inhibit JAK1/2 and JAK3, respectively [72]. Siplizumab is an anti-CD2 monoclonal antibody. By binding to LFA-3, CD2 mediates the adhesion of T cells to APCs [72].

Pharmacological Drugs

Cyclosporine and tacrolimus inhibit calcineurin activation [77]. Sotrastaurin strongly inhibits PKC-θ, which is required for signal transduction in CD3/CD28-induced activation of T cells [82]. Sirolimus and everolimus form a complex with the FK binding protein (FKBP-12), which binds to the kinase mTOR, thereby suppressing the mTOR intracellular signaling pathway [83]. Azathioprine is converted into 6-thioguanine by the hypoxanthine-guanine phosphoribosyltransferase pathway. The product is a purine analogue that inhibits the synthesis of DNA and RNA and the proliferation of immune cells [77,84]. Mycophenolic acid (MPA) is an inhibitor of inosine-59 monophosphate dehydrogenase, which is necessary for the de novo synthesis of guanosine nucleotides. FK778 is an inhibitor of pyrimidine synthesis [85]. FTY720 is a high-affinity agonist of G protein-coupled S1P₁ receptors on lymphocytes, preventing their translocation to grafts from secondary lymphoid organs. The binding of FTY720 to S1P₁ receptor triggers the internalization and degradation of the receptors. As a result, cells can not react to the egress signal provided by S1P and exit lymphoid organs [86,87]. Glucocorticoids (GCs) are steroid hormones that decrease the number of circulating monocytes and inhibit the synthesis of proinflammatory cytokines by macrophages. They also inhibit MHC class I antigen presentation by APCs such as DCs. The number of circulating T cells, and their IL-2 synthesis and action, are inhibited by GCs; the number of eosinophils and basophils decreases, and the number of neutrophils increases [88,89]. Glucocorticoids also inhibit NF-κB activation by interacting with one of NF-κB subunits, NF-κB1, or increasing the expression of an inhibitor protein of NF-κB, IκB. NF-κB is a dimeric transcription factor known to play critical roles in various cellular processes including adaptive and innate immunity, cell differentiation, proliferation, and apoptosis. It is also involved in allograft rejection [90]. NF-κB regulates the expression of proteins involved in alloresponses including cytokines, chemokines, adhesion molecules, MHC antigens, and proinflammatory enzymes. Csizmadia et al. [91] showed the nuclear translocation of NF-κB subunits, p50, p52 and p65, as well as an increase in the expression of the genes encoding NF-κB and IκB in transplanted hearts during cardiac allograft rejection.

Multiple studies have shown the effects of immunosuppressive agents on Th17 cells. MPA and tacrolimus significantly decreased Th17-related transcripts including IL-17A, and IL-21 after in vitro T cell activation. MPA had stronger inhibitory effects on IL-17 transcript levels than tacrolimus. Treatment of MPA in combination with tacrolimus decreased serum IL-17 levels, compared to treatment with tacrolimus alone [92]. The plasma concentrations of IL-17 in renal transplant recipients treated with sirolimus were significantly lower than those in the normal control group and the group treated with tacrolimus [93]. In the murine skin transplant model, treatment with tacrolimus and resveratrol increased skin graft survival, significantly decreasing the percentage of Th17 cells among splenocytes. Meanwhile, tacrolimus alone prolonged graft survival, but did not affect Th17 levels. Resveratrol is a natural polyphenol compound that has therapeutic effects in metabolic disorders, including diabetic nephropathy [94].

Immunosuppressive agents have several side effects, including nephrotoxicity, increased cardiovascular risk, and systemic overimmunosuppression, which can lead to opportunistic infection and malignancy. Therefore, strategies that minimize the use of immunosuppressive drugs to induce graft tolerance are needed. Extensive research has focused on cell-based therapy for graft tolerance. Such therapy involves regulatory cell types including Tregs, myeloid-derived suppressor cells (MDSCs) and IL-10-producing B cells. The field is continuously growing and shows promise. Numerous studies on the adoptive transfer of Tregs and MDSCs have been carried out, whereas information on IL-10-producing B cells is limited compared to them [22]. Herein, we review the adoptive transfer of Tregs and MDSCs. The mechanisms by which immune cells regulate T cells and APCs are shown in Fig. 3.

Figure 3. Cell-based therapies for the induction of graft tolerance. Regulatory T cells (Tregs) inhibit the activation of effector T cells and antigen-presenting cells (APCs). A high level of high-affinity interleukin (IL)-2 receptor on Tregs prevents the use of IL-2 by effector T cells. The interaction of cytotoxic T lymphocyte antigen-4 (CTLA-4) on Tregs with CD80/86 on effector T cells and APCs inactivates the latter. CD39 and CD73 on Tregs produce the anti-inflammatory factor adenosine from ATP and 5'-adenosine monophosphate (5’-AMP). Tregs secrete granzyme B to induce the apoptosis of effector T cells [22,95]. Tregs produce transforming growth factor (TGF)-β and IL-10. TGF-β induces apoptosis of CD4⁺ T cells and suppresses the function of CD8⁺ T cells; IL-10 suppresses the activity of T helper 17 cells. Tregs interact with PD-L1 and PD-L2 on the surface of T cells via PD-1 to inhibit T cell responses. The transfer of cyclic adenosine monophosphate (cAMP) to T cells through intercellular gap junctions inhibits their activation. Tregs transfer miRNAs into T cells via exosomes [64]. Lymphocyte activation gene-3 (LAG-3) on Tregs binds to major histocompatibility complex (MHC) Ⅱ and inhibits antigen presentation by APCs. IL-10 downregulates their expression of MHC Ⅱ and costimulatory molecules. Granzyme B from Tregs induces the apoptosis of APCs. Inducible nitric oxide synthase (iNOS) in myeloid-derived suppressor cells (MDSCs) consumes L-arginine to produce nitric oxide (NO), preventing the use of the former by T cells to proliferate. In addition, arginase 1 (ARG1) produced by MDSCs cleaves L-arginine to ornithine and urea, preventing the use of the former by T cells. The production of a large quantity of heme oxygenase-1 (HO-1) by MDSCs contributes to the inactivation of T cells. CCL5 produced by MDSCs recruits Tregs from secondary lymphoid organs to grafts, where they induce tolerance. The interaction of B7-H1 (PD-L1) on MDSCs with PD-1 on Tregs promotes the migration, proliferation, and function of the latter. In the presence of interferon-γ, MDSCs produce IL-10 and TGF-β, which trigger the activation of Tregs [22]. Indoleamine 2,3-dioxygenase (IDO) on MDSCs consumes tryptophan and promotes kynurenine production. Tryptophan deficiency and excessive kynurenines suppress lymphocyte responses [96,97].

Regulatory T Cell

Clinical trials have evaluated the effect of adoptive transfer of Tregs on allograft rejection of solid organs, including the liver and kidney [22]. In the phase 1/2 trial NCT02474199, five subjects with liver transplants were infused with autologous donor alloantigen-reactive Tregs (darTregs). Two darTreg-infused participants reached the primary end point, a 75% decrease in calcineurin inhibitor dose with stable liver function for at least 12 weeks [98]. In the phase 1 trial NCT02088931, a subset of infused autologous Tregs was detected in the circulation of three patients with kidney transplants for at least 1 month after infusion. Graft inflammation improved in two of the three patients [99]. Tregs inhibit the activation of effector T cells and APCs. A high level of high-affinity IL-2 receptor on Tregs deprives effector T cells of IL-2, which is important for their survival [22]. The interaction of CTLA-4 on Tregs with CD80/86 on effector T cells and APCs suppresses the action of effector T cells [22,95]. CD39 and CD73 on Tregs suppress effector T cells by converting ATP and 5’-AMP into adenosine, an anti-inflammatory factor. Tregs secrete granzyme B to induce the apoptosis of effector T cells [22]. They produce TGF-β and IL-10. The former induces apoptosis of CD4⁺ T cells and suppresses the function of CD8⁺ T cells. The latter suppresses the activity of Th17 cells. Tregs interact with PD-L1 and PD-L2 on the surface of T cells via their PD-1, thereby inhibiting T cell responses. cAMP may be transferred to T cells through intercellular gap junctions, inhibiting their activation. Tregs transfer miRNAs into T cells via exosomes [57] and suppress the function of APCs. Lymphocyte activation gene-3 (LAG-3) on Tregs binds to MHC II and inhibits antigen presentation. IL-10 produced by Tregs downregulates MHC II and costimulatory molecules. Granzyme B from Tregs induces apoptosis in APCs [22].

Myeloid-Derived Suppressor Cells

MDSCs are a heterogeneous group of myeloid cells that suppress acute inflammatory responses by inhibiting immune responses and inflammation [100]. They are subdivided into granulocyte-like MDSCs (G-MDSCs) and mononuclear phagocyte-like MDSCs (M-MDSCs). Their phenotypes are unclear, but M-MDSCs are typically defined as CD11b⁺CD14⁺CD33⁺HLA^–DR^low/neg, and G-MDSCs as CD11b⁺ CD15⁺HLA^–DR^lowCD66b⁺. In mice, these cell populations have been defined as CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ Ly6C^low, respectively [101]. MDSCs suppress the proliferation of T cells by producing NO via inducible nitric oxide synthase (iNOS). To produce NO, iNOS consumes L-arginine, which is necessary for T cell proliferation. The arginase 1 produced by MDSCs cleaves L-arginine to ornithine and urea, preventing the use of the former by T cells. The production of a large amount of heme oxygenase-1 by MDSCs contributes to the inactivation of T cells. The interactions between B7-H1 (PD-L1) on MDSCs and PD-1 on Tregs promote the migration, proliferation, and function of the latter. CCL5 produced by MDSCs recruits Tregs from secondary lymphoid organs to the graft, where they induce tolerance. In the presence of IFN-γ, MDSCs produce IL-10 and TGF-β, which stimulate the activation of Tregs [22]. MDSCs contain IDO, which consumes tryptophan and produces N-formylkynurenine, and promotes kynurenine production. Tryptophan deficiency and excessive kynurenines suppress lymphocyte responses. G-MDSCs and M-MDSCs induce immunosuppression in tumor models with high levels of reactive oxygen species and iNOS, respectively. In organ transplants, M-MDSCs play more critical roles in transplant tolerance induction. M-MDSCs promote tolerance by mechanisms involving iNOS or IDO [96,97].

This review has summarized the roles of Th17 cells in allograft rejection and their relationship with other T cell subsets. Numerous studies have shown that Th17 cells play critical roles in rejection. IL-17 and Th17 cell levels increased in patients with allograft rejection, and inactivation of IL-17 or inhibition of Th17 differentiation mitigated the rejection. This threat posed by Th17 cells to organ transplantation has led to research on ways to inactivate Th17 cells to induce tolerance. Multiple studies have investigated which immunosuppressive drugs effectively suppress Th17 cells or decrease IL-17 levels. However, further research on a wider range of drugs is needed to more effectively regulate Th17 cells.

Th17 cells affect other T cell subset activity to contribute to allograft rejection. Th17 activity is also controlled by regulatory cells. Tregs and MDSCs can downregulate the contribution of T cell types to allograft rejection. The adoptive transfer of regulatory cells to induce tolerance is a field of active research. It induces allograft tolerance and minimizes the use of immunosuppressive agents, which come with side effects. Numerous studies involving adoptive transfer of Tregs or MDSCs have been conducted to induce tolerance. In comparison, studies on IL-10-producing B cells are limited, and further research is needed. Oberholtzer et al. [22] suggested the adoptive transfer of multiple regulatory cell types, considering their synergistic effects. In addition to Tregs, MDSCs, and IL-10-producing B cells, regulatory cells include tolerogenic DCs, regulatory NK cells and regulatory macrophages [22]. To effectively administer combinations of regulatory cells, studies on the adoptive transfer of these three cells are also required.

Identifying the immune profile of allograft recipients with allograft tolerance is crucial for defining the optimal ranges of T cells and their cytokines required to establish tolerance. Understanding the optimal immune profile can guide the adjustment of immunosuppressive drug doses to minimize immunosuppression, which is commonly defined as the administration of the lowest amount of immunosuppressive drugs to induce a rejection-free state [29,102]. Th17 cells and other T cell subsets mutually influence each other during allograft rejection. A comprehensive understanding of the interaction between different T cell types during allograft rejection can deepen our knowledge of the immune profile associated with graft tolerance. Thus, further research on the roles of Th17 cells in allograft rejection, particularly in the context of other T cell subsets, is required.

The levels of Th17 cells and IL-17 are elevated in patients with allograft rejection. Th17 cells promote rejection by accelerating neutrophil infiltration into grafts, activating endothelial cells, recruiting immune cells, and activating fibroblasts. Specifically, they can activate other CD4⁺ T and CD8⁺ T cells by promoting the maturation of DCs. Th1 cells damage allografts by triggering Fas/FasL-mediated cytotoxicity, alloreactive CD8⁺ cytotoxicity, and DTH by macrophages. Th2 cells modulate rejection by activating eosinophils, inducing alloreactive antibody production by B cells, and inhibiting Th1 responses to allografts. CD8⁺ T cells cause apoptosis of allograft cells. A high Th17-to-Treg ratio is associated with rejection. Immunosuppressive drugs are used to inactivate T cells, inhibit rejection, and induce tolerance, but they have side effects; this can be overcome by the adoptive transfer of regulatory cells. Th17 cells promote transplant rejection by interacting with a variety of cell types. Further research on the interaction will provide insight into the pathogenesis of transplant rejection and lay the foundation for the development of strategies to induce tolerance.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Funding/Support

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2024-00454685).

Author Contributions

All the work was done by Young Joon Lee and Mi-La Cho. Both authors read and approved the final manuscript.