Secondary Logo

Journal Logo

Wnt Signaling as Master Regulator of T-Lymphocyte Responses

Implications for Transplant Therapy

Staal, Frank J.T. PhD; Arens, Ramon PhD

doi: 10.1097/TP.0000000000001393

T cell-mediated immune responses to the grafted tissues are the major reason for failed organ transplantation. The regulation of T cell responses is complex and involves major histocompatibility complex molecules on transplanted organs, cytokines, regulatory cells, and antigen-presenting cells. The evolutionary conserved Wnt signal transduction pathway has long been known for its importance in development of stem cells and immature T cells in the thymus. Recent evidence indicates the Wnt pathway as a master regulator of T cell immune responses via governing the balance between T helper 17/regulatory T cells and by regulating the formation of effector and memory cytotoxic CD8 T cell responses. In doing so, Wnt signals influence the outcome of immune responses in transplantation settings.

Staal and Arens review the Wnt pathway influence on the outcome of allotransplantation, by serving as a master regulator governing the balance between Th17/Treg and the formation of effector and memory cytotoxic CD8 T cell responses.

1 Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.

Received 4 March 2016. Revision received 26 May 2016.

Accepted 10 June 2016.

F.J.T.S. is supported in part by a TOP grant from The Netherlands Organization for Health Research and Development, ZonMw Project 40-00812-98-09050 and ZonMW E-RARE (grant 40-41900-98-020). R.A. is supported by a KWF grant (UL2015-7817).

The authors declare no conflicts of interest.

F.J.T.S. and R.A. wrote, discussed, and edited the article.

Correspondence: Frank J.T. Staal, PhD, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, PO Box 9600, 2300 RC/Leiden, The Netherlands. (

Back to Top | Article Outline

Immune Responses in Transplant Therapy

The transplantation of cells, tissues, or complete organs to replace damaged or dysfunctional organs is a well-established medical therapy. The success of the transplantation is largely determined by the level of acceptance of the graft by the host. Three types of rejection are commonly discerned: (1) hyperacute rejection occurring minutes to hours after transplantation due to preexisting antibodies, (2) acute rejection happening within the first 6 months after transplantation due to adaptive immune responses mediated by T cells, and (3) chronic rejection after 6 months occurring due to a combination of immunological mechanisms and other factors.1,2 In most cases, adaptive immune responses to the grafted tissues are the major impediment to successful transplantation. Acute rejection is caused by immune responses to alloantigens on the graft, which are proteins that vary from individual to individual within a species, and are thus perceived as foreign by the recipient.

Highly polymorphic MHC molecules almost always trigger a T cell response against the grafted organ. Mature, naive T cells that have not yet encountered antigen express CD45RA in humans and are CD44low in mice. Upon encounter of foreign antigens presented by antigen-presenting cells (APC) in peripheral lymphoid organs, naive T cells are activated by interactions between the T cell receptor (TCR) and a foreign-peptide/MHC-complex, costimulatory signals such as interactions of CD28 on T cells and CD80/CD86 on APCs, and the availability of cytokines, such as IL-2, IL-12, and IL-15.3 In case of transplantation, the foreign MHC/HLA molecules are powerful triggers of T cell responses directed against the transplant.

CD4+ T cells differentiate into T-helper (Th) subpopulations, of which several different types have been described: Th1, Th2, Th17, and T follicular helper (Tfh) cells.4 Differentiation into one of the Th subpopulations is highly dependent on the cytokine milieu present during T cell activation. For instance, IL-12 enhances the expression of the transcription factor (TF) T-bet (Tbx21), which commits cells to the Th type 1 (Th1) lineage. Over the last decade, the proinflammatory Th17 lineage has been described. Commitment of this lineage is enhanced by the TF retinoic acid-related orphan receptor-γt (RORγt) and requires the presence of IL-6 or other proinflammatory cytokines, such as IL-1, TNF, and TGF-β.4 In addition, antigen-stimulated dendritic cells (DCs) and macrophages produce IL-23, which promotes the development of Th17 cells. Th17 cells subsequently produce IL-17 and IL-22 and are thought to be involved in immunity against extracellular pathogens particularly at mucosal surfaces and are involved in inflammatory reactions mainly through the IL23-IL17 axis.

CD8+ T cells differentiate into cytotoxic T cells (CTLs) that are involved in the destruction of virus-infected cells and tumor cells.3 Recognition of a target cell will result in contact-mediated cytotoxicity, a process in which cytolytic molecules, such as perforin and granzyme B, released by CTLs are involved in inducing apoptosis of the target cell.5 Additionally, binding of Fas-ligand present on CTLs to Fas (CD95) molecules present on target cells induces the formation of a death-inducing signaling complex, which also initiates apoptosis pathways in the target cell. Activated CD8 T cells also secrete cytokines, such as IFN-γ, TNF, and IL-2.3

Effector T cells are short-lived cells, and approximately 90% will die during resolution of inflammation, thereby restoring the resting state of the immune system.3 Only approximately 5% to 10% of the effector cells survive the first inflammatory state and become long-lived memory cells that can rapidly respond to reinfection with the same pathogen. These memory T cell populations depend on IL-7 and IL-15 signals for survival and proliferation.6,7

The memory T cell compartment consists mainly of central and effector memory cells, which both express CD45RO in humans and are CD44high in mice.7,8 Central-memory T cells (Tcm) express the lymph node homing receptors CCR7 and CD62L (L-selectin) and recirculate through lymph nodes and the spleen. Central-memory T cells largely lack effector functions but readily proliferate and differentiate upon antigenic reencounter, providing reactive memory.7-9 Effector-memory T (Tem) cells lack expression of certain homing receptors and reside also in nonlymphoid tissues. Effector-memory T cells are capable of producing effector cytokines within hours after TCR stimulation, thereby providing rapid protective memory.7,9 A special type of T cells is able to persist in nonlymphoid tissues without recirculating to lymphoid organs. These so-called tissue-resident memory T cells (Trm) cells are characterized by expression of the activation marker CD69 and CD103.10

Additionally, several subpopulations of regulatory T cells (Treg) cells with immunosuppressive function exist that control immune responses.11 Thymus-derived Tregs are CD4+ T cells generated as a separate subpopulation in the thymus (historically sometimes still referred to as natural Treg).12 In humans and mice, these cells express CD25 and the TF FoxP3. Naive T cells can also acquire FoxP3 expression in the presence of TGF-β, IL-2, and retinoic acid; these cells are called peripherally derived Treg (previously referred to as induced Treg.11,12 Next to FoxP3+ Treg, several other types of Treg, such as Tr1 and Tr3, can be induced from naive CD4+ T cells. Tr1 T cells secrete IL-10 and TGF-β, whereas Tr3 T cells mainly secrete TGF-β.13

Back to Top | Article Outline

Wnt Signaling

The evolutionary conserved Wnt pathway plays essential roles in the development of almost every organ in the body.14 In adults, Wnt signaling is mostly important in organ systems with active stem cells, such as skin, hair, intestine, and the hematopoietic system. Over the last few years, several laboratories including ours have shown that Wnt signaling may also regulate T cell–mediated immune responses, making this pathway of relevance for transplantation, autoimmunity, infectious disease, and cancer immunotherapy. We will here first describe the pathway's components and its well-established role in immune and blood cell development before discussing its role in regulating Th cells, Treg, and CTLs.

The Wnt signaling pathway is subdivided into canonical (β-catenin–dependent) and noncanonical (β-catenin–independent) pathways (illustrated schematically in Figures 1 and 2, respectively). Binding of different Wnt proteins to frizzled (Frz) receptors can trigger different Wnt pathways. So far, 19 Wnt ligands, 10 Frz receptors and several coreceptors (most importantly, low-density lipoprotein receptor-related proteins [Lrp], eg, Lrp5/Lrp6) have been discovered in mammals.16 In addition, the leucine-rich repeat-containing G-protein–coupled receptor proteins 4, 5, and 6 function as receptors for R-spondin, which dramatically increases Wnt signaling responses.17 Wnt proteins function as proliferation-inducing growth factors but may also affect cell-fate decisions, apoptosis, and quiescence.17,18 Canonical Wnt proteins bind to their receptors (Figure 1), thereby preventing proteosomal degradation of the Wnt mediator β-catenin. Subsequently, β-catenin is translocated to the nucleus where it will form an active transcription complex with 1 of the 4 TFs downstream of the Wnt pathway: T cell factor (Tcf) 1, 3, or 4 or lymphocyte-enhancer binding factor (Lef)1. In the hematopoietic system Tcf1 and Lef1 are most abundantly expressed Wnt-responsive TFs. Upon transcriptional activation, several target genes will be activated (including Axin-2, c-Myc, etc.) that are important for proliferation and/or cell fate decisions. Non-canonical Wnt signaling (Figure 2) involves recognition of distinct Wnt ligands by cognate Frz-ROR/RYK receptor complexes and activation of RHO, and RAC small GTPases as well as JUN kinase that phosphorylates and regulates the TF JUN. An additional noncanonical pathway involves heterotrimeric G protein activation of phospholipase C and turnover of phospholipid membranes in the endoplasmic reticulum, and the release of intracellular Ca2+ ions. In some contexts increased intracellular Ca2+ activates protein kinase C, as well as other calcium sensitive enzymes, such as calmodulin-dependent kinase II, and the calcineurin-dependent TF nuclear factor of activated T cells (NFAT).19 This might be particularly important in lymphocytes, where NFAT plays crucial roles. Interestingly, crosstalk between the noncanonical calcium pathway and the canonical Wnt pathway via glycogen synthase kinase (GSK)3β, which regulates nuclear egress of NFAT,20 has been proposed. Noncanonical Wnt signaling pathway also regulates cellular polarization and migration (the so-called planar-cell-polarity pathway).21,22





Back to Top | Article Outline

Wnt Signaling During Hematopoiesis and Lymphopoiesis

In the hematopoietic system, a role for Wnt signaling was first demonstrated during T cell development in the thymus where it provides proliferation signals to immature thymocytes (reviewed in Staal et al and Rothenberg et al16,23). These studies involved among others, the Tcf1 DNA binding protein, which is encoded by the Tcf7 gene. Indeed, Tcf7 deficiency affects the highly proliferative stages double-negative (DN) 2 and DN4,24,25 and conditional deletion of β-catenin inhibits T cell development at β-selection checkpoint (DN3).26 Furthermore, activation of the pathway by in vivo stabilization of β-catenin resulted in thymocyte development without the requirement of pre-TCR signaling and impaired transition from DN to double-positive (DP) stages of T cell development.27

Studies on mice deficient for the Wnt-responsive TFs revealed crucial roles for Tcf1 in T cell development25 and Lef1 in B-cell development.28,29Tcf7−/− mutant mice had a severe reduction of thymic cellularity and a partial block in thymocyte differentiation at the transition from the CD8+ immature single positive (ISP) stage to the CD4+CD8+ DP stage.25 The ISP and DN thymocytes of Tcf7−/− mice did not proliferate as strong as their wild-type counterparts.30 These data indicated that lack of Tcf1 mainly results in lack of proliferation and therefore expansion of the thymocytes. Although Lef1−/− mice have normal T cell development, mice deficient in both Lef1 and Tcf1 have a complete block in T cell differentiation at the ISP stage,31 which indicates redundancy between these 2 factors. Indeed, important recent work has underscored this point and showed that deficiency of both Tcf1 and Lef1 in DP thymocytes results in diminished output of CD4+ T cells and redirected these cells to a CD8+ T cell fate.32 This is mediated by intrinsic histone deacetylase activity of Tcf1 and Lef-133 and by balancing the expression of the TFs Th-POK (Zbtb7b) and Runx3d. Moreover, when both Lef1 and Tcf1 are deficient in adult mice, a very small thymus with severe developmental blocks remains.34 Of interest, recent work including from our laboratory indicated that Tcf1 has another essential function in the thymus besides acting as the nuclear effector of Wnt signaling in thymocytes, namely a role as a critical tumor suppressor gene for the development of thymic lymphomas, the murine counterpart of human T cell acute lymphoblastic leukemia.17,34,35 That is, mice deficient for Tcf1 develop thymic lymphomas with high frequency due to ectopic upregulation of Lef1 and paradoxically extremely high Wnt signaling levels, which form the initialing step for leukemia development, often followed by additional oncogenic hits, such as Notch1 mutations.35

Besides regulating T cell development in the thymus, Wnt signaling was also shown to play a role in the regulation of hematopoietic stem cell (HSC) function. We and others recently showed that Wnt is necessary for normal HSC function by using either Wnt3a-deficient mice,36 or by overexpressing the Wnt-negative regulator Dickkopf1 (DKK1) in the osteoblastic stem cell niche,37 or by Vav-Cre–mediated conditional deletion of β-catenin.38 However, Mx-Cre–mediated deletion of β-catenin39 or β- and γ-catenin simultaneously40,41 did not affect hematopoiesis, probably due to the fact that Wnt signaling was not completely abolished in these models.40 Besides these approaches to inhibit Wnt signaling, gain-of-function approaches to activate the pathway in HSCs were performed with conflicting results. Stabilized forms of β-catenin resulted in either enhancement of HSC function and maintenance of an immature phenotype,42-45 or exhaustion of the HSC pool followed by failure of repopulation in transplantation assays.46,47 These differences may be explained by the different levels of Wnt pathway activation,48,49 resulting from the different approaches used and/or the interference of other signals in the context of Wnt activation.50-52 The controversies surrounding Wnt signaling in hematopoiesis and lymphopoiesis have recently been discussed in detail elsewhere.53 It also needs to be noted that results obtained with Tcf7−/− mice need to be interpreted with caution. Tcf1 may work as a suppressor of Wnt signaling in absence of ligand by recruitment of corepressors and paradoxically in Tcf7−/− cells, the levels of Lef1 can increase, thereby actually promoting Wnt signaling.

Back to Top | Article Outline

Wnt Signaling as Regulator of Mature T Cell Responses

Wnt signaling has traditionally been studied in the context of thymocyte development and stem cell biology (reviewed in Staal et al and Luis et al16,54). However, by using reporter mice, it was found that T cells are the only mature blood cells actively undergoing Wnt signaling, hinting to a potential important role in regulating T cell responses.55

Back to Top | Article Outline

CD8 T Cells

The Wnt-responsive TFs, Tcf1, and Lef1 are highly expressed by naive mouse and human CD8+ T cells. After a productive encounter with antigen and subsequent expansion and differentiation into cytotoxic effector T cells, the expression levels rapidly drop however, and increase again during memory formation.56-58 Moreover, within the memory CD8 T cell subsets, differential expression exist: cells with high levels of active Wnt signaling and concomitant high Tcf1 expression is found in Tcm, whereas lower levels of Tcf1 are found in Tem cells.58,59 The Trm cells have strongly downregulated Tcf1, which is concomitant with their elevated activation status60 Of interest, Tcf1 functions as a repressor of CD8+ effector T cell formation in a β-catenin/Wnt-independent manner,61 which may be related to its requirement for efficient memory formation.57,61-63 Lef1 is also implicated in the stimulation and formation of effector and memory CD8+ T cells.64

Important work by the laboratory of Restifo indicates that canonical Wnt signaling induced by activated β-catenin, Wnt3a, or GSK3β inhibitors arrested CD8 T cell differentiation and favored CD8+ T cell memory formation by suppressing their maturation into terminally differentiated effector T cells.58 These investigators have proposed a role for WNT signaling in the generation of a novel CD8+ T cell memory population named T memory stem cells that possessed superior self-renewal capability in serial transplant experiments and had the multipotent capacity to generate Tcm, Tem, and effector T cells.58,65,66 Consistently, Tcf1 deficiency was shown to limit the proliferation of CD8+ effector T cells and impair differentiation toward a central-memory phenotype.57 Taken together, canonical Wnt signaling inhibits effector CTL responses but enhances memory CD8+ T cell maturation.

Back to Top | Article Outline

CD4 T Cells

The role of Wnt signaling in CD4+ T cells is controversial, especially regarding the role of Wnt signaling in formation and function of Th1 and Th2 cells, but the emerging picture is somewhat clearer with respect to Treg and Th17 cells. The most convincing studies, combining biochemistry, molecular biology, genetics, loss-of-function and gain-of-function studies and clinical samples have led to the insight that proinflammatory Th17 cells are favored in their function by Wnt signaling and that Treg are inhibited by canonical Wnt signaling (described in detail below). Of note, Treg can interfere with T cell priming in organ-draining lymph nodes, which is important when considering their role in transplant rejection or acceptance. Treg cells can constrain T cell activation by, for example, limiting the ability of effector T cells to form stable contacts with APCs.11,13 Consistently, Treg inhibited the alloantigen-driven proliferation of T cells in graft-draining lymph nodes in models of skin allograft rejection.67 The use of Treg in adoptive transfer is subject of intense study, including the famous ONE study in solid organ transplantation (

A seminal study by Gounari and coworkers68 showed that Wnt-β-catenin signaling induced expression of RORγT resulting in high amounts of IL-17 and predisposition to inflammation, colitis, and intestinal tumors.68 This is consistent with earlier reports showing that Tcf1 binds directly to the IL17a gene69,70 and reports showing that at least some Th17 cells are long-lived, express high levels of Tcf1, and β-catenin for their self-renewal–like proliferation.71 These studies are however in contrast with a report in which activation of Wnt-β-catenin by GSK3β inhibitors repressed Th17 differentiation in in vitro polarization studies.72 It might be that timing and dosage of the Wnt signals given is crucial, as early administration of not yet fully committed/polarized cells might favor survival of naive T cells.35 A similar cautionary remark might be made on the role of Wnt signaling in Treg. An initial study reported that retroviral transduction of a stabilized β-catenin mutant resulted in increased Treg survival73 and would favor Treg suppression. However, continuous high amounts of β-catenin are not favorable, and it has been shown that the expression of stabilized β-catenin in thymocytes can result in the development of thymic lymphomas.74,75 Therefore, interpreting experiments with the overexpression of stabilized β-catenin mutants can be difficult and the effects of stabilized catenin may also have resulted in increased survival of Treg precursors. Coffer and coworkers76 in contrast showed that Wnt signaling directly modulates Foxp3 activity and thereby Treg function. Tcf1 directly binds to FoxP3 and β-catenin-Tcf inhibits Foxp3 transcriptional activity. These investigators used a variety of approaches, including recombinant Wnt3a, a Wnt production inhibitor, multiple distinct GSK3 inhibitors (Wnt mimetics), and Apcmin/+ and Tcf7−/− mice to manipulate Wnt signaling in vitro and in vivo and thereby robustly studied the mechanism underlying these observations.76 The emerging picture is that Wnt signaling reduces Treg-mediated suppression in vitro and in vivo. Interestingly, in autoimmune diseases, such as rheumatoid arthritis, Wnt3a levels were increased in synovial fluids leading to suppressed Treg activity.76 Together with the reports on Th17 cells, we propose that canonical Wnt signals, generated by DCs and other APCs although perhaps also by T cells themselves favor the development of proinflammatory Th17 cells and inhibit Treg development and function. Preliminary work from our laboratory indicates that in polarization assays a Th17/Treg bipotential precursor can be skewed toward Th17 or Treg lineages by modulating Wnt signaling. Taken together with the work discussed above, canonical Wnt signaling is likely a master regulatory pathway in governing the balance between Th17/Treg and thereby influences the outcome of immune responses (Figure 3).



Recent work from research on experimental models for allergy indicated that Th1/Th2 CD4+ T cell responses are also controlled by canonical Wnt signals.77 That is the Wnt inhibitor DKK1 stimulated strong Th2 responses through induction of the Th2 TFs GATA3 and c-Maf and inhibited Th1 responses.77 Conversely, one could extrapolate that Wnt signaling favors Th1 responses while inhibiting Th2 responses. However, previous work by the Misra-Sen laboratory showed that Tcf1 and β-catenin stimulate rather than inhibit Th2 responses. These investigators showed direct binding of Tcf1 to a GATA3 transcriptional regulatory element and subsequent increased IL-4 production.78 Moreover, deletion of Tcf1 impaired Th2 responses in vivo,78 suggesting that Tcf1 promoted Th2 differentiation via stimulation of GATA-3. It is currently unclear why such disparate results were obtained. It is likely that the different Th1/Th2 models used for the experiments can cause different outcomes. Clearly, there is a need for further careful analysis, first at the molecular level, to understand if and how Tcf1 and β-catenin regulate expression of the lineage promoting TFs T-bet and GATA3, followed by in vitro polarization studies and robust in vivo models.

Tcf1 and Lef1 also orchestrate Tfh cell differentiation by regulating the responsiveness of CD4+ T cells to IL-6 signaling and by influencing the levels of the transcriptional repressor Bcl-6.79 Interestingly, Tcf1 functions in Tfh cells by negatively regulating IL-2 and Blimp.80 Indeed, Tcf1-deficient Tfh cells are severely compromised and fail to efficiently clear viral infections.80

Back to Top | Article Outline

Wnt Signaling in DCs

DCs process antigens and microenvironmental signals to control innate and adaptive immunities. DC-specific deletion of β-catenin increased proinflammatory cytokine production and intestinal inflammation in mice.81 In contrast, Wnt signals have also been reported to shift DCs from promoting immune responses into a tolerogenic state.82,83 However, in all these situations, it is unclear whether Wnt signaling also plays such roles physiologically, because only skin DCs (Langerhans cells) express appreciable levels of the ubiquitous Wnt target gene Axin2 in vivo.

Back to Top | Article Outline

Wnt Signaling in Organ Transplantation

As discussed above, Wnt signaling can significantly affect T lymphocyte responses and therefore also the outcome of T cell-mediated acute and chronic rejection of solid organs after transplantation. In transplanted organs, regeneration of tissue function is crucial. The Wnt pathway is one of the major pathways activated during regeneration of cells and tissues.84-88 However, this needs to be done in a controlled fashion and in interplay with many other signals, inflammatory factors, and growth factors. Recent evidence links inflammation and Wnt signalling to tissue regeneration. In a recent overview article,89 inflammatory cytokines were proposed to trigger pathways interacting with the Wnt pathway in adult stem cells in liver, kidney, or intestine. This leads to activation of TFs (NF-kB, intracellular-Notch, transcriptional enhancer associate domain in the Hippo pathway) that directly interact with components of the Wnt pathway, which provoke a regenerative response by inducing genes that promote dedifferentiation (such as Wnts can do42,90), stemness, and proliferation; all activities are integrally linked to the Wnt pathway.

In the kidney, the Wnt pathway is active in normal renal tissue in establishing tissue homeostasis.91 Wnt proteins control cell polarity, proliferation, and other processes, especially in the cilia which are located in proximal and distal tubules, the macula densa, and the collecting duct.92 Chronic renal allograft damage is manifested by an inflammatory process that leads to transplant glomerulopathy, diffuse interstitial fibrosis, and tubular atrophy with loss of tubular structures. Reactivation and dysregulation of Wnt pathways underlies this chronic fibrosis.93 Whether this dysregulated Wnt activity also influences T cell responses is unclear. On the other hand, it may be that infiltrating T cells and innate inflammatory cells secrete Wnt proteins that influence renal tissue. Nevertheless, several studies point to inappropriate Wnt activation in chronic renal transplant failure. Interestingly, modulation of the Wnt pathway using retinoic acid may alleviate some of the inappropriate Wnt activation, at least in rat models of chronic renal allograft damage.94

A recent report directly links Wnt signaling in immune cells with transplant outcome via the often occurring re-activation of cytomegalovirus infection following solid organ transplantation. Ueland et al95 showed that high levels of the Wnt signaling inhibitor DKK1 were lower in CMV+ patients. This could be due to lower Th17 or Th1 responses that are normally favored by higher Wnt levels, but in CMV+ patients decreased due to the high DKK1 levels.

Back to Top | Article Outline

Special Cases: HSC Transplantation and Adoptive T Cell Transfer: A Potential Role for Wnt Signaling

Two special types of transplantation, namely, HSC transplantation (as for instance in bone marrow transplantation) and adoptive T cell transfer requires special attention. As indicated above, Wnt signaling can affect the self-renewal of HSCs. Thus, provided the correct dosage is given, Wnt signaling may be important for expansion of HSCs ex vivo before transplantation. Support from this notion comes from many laboratories.96-98 Clearly, a too high dosage of Wnt signaling can have detrimental effects.46,99 Examination of Wnt signaling in young and aged HSCs revealed an association of reduced Wnt signaling with impaired T cell differentiation in aged cells.100 In addition, noncanonical Wnt signaling can affect the properties of HSCs leading to an aged phenotype with more myeloid offspring than lymphoid and less self-renewal. As reported by Geiger and coworkers101,102 elevated expression of Wnt5a in aged HSCs caused stem cell ageing. Wnt5a treatment of young HSCs induces ageing-associated stem cell polarity, reduction of regenerative capacity, and an ageing-like myeloid-lymphoid differentiation skewing.103 Conversely, we recently showed that in vivo treatment with Wnt3a in HSCs leads to the opposite effects, namely, production of more lymphocytes and fewer myeloid cells, suggesting that a “rejuvenated” HSC phenotype had arisen.90 Collectively, these data show that Wnt signaling not only affects self-renewal and therefore ex vivo expansion of HSCs,104 but also directly impacts cell fate decisions toward lymphoid or myeloid lineages. This leads to the exciting notion that modulating Wnt levels could help to produce desired types of cells upon transplantation.

Adoptive T cell transfer (adoptive cellular therapy [ACT]) is an important type of transplantation that especially is used in cancer immunotherapy and in combatting viral reactivation after transplantation (eg, CMV reactivation).105 Traditionally, patient-derived specific T cells are expanded in vitro using IL-2 and other agents to transfer large quantities of, for example, tumor-specific CTLs back to the patient. Recently, the use of engineered T cells with desired specifies has come to great fruition.106 Instead of transducing T cells with additional αβTCRs, it is possible to transfer chimeric TCRs, which may be generated by joining the light and heavy chain variable regions of a monoclonal antibody expressed as a single chain Fv molecule with the transmembrane and cytoplasmic signaling domains derived from CD3ζ chain or Fc receptor γ chain through a flexible spacer. These T cells that thus combine the antigen specificity of an antibody and the cytotoxic properties of a T cell in a single-fusion molecule are referred to as chimeric antigen receptor T cells.106 However, the off-targets effects and sometimes serious side effects make efforts of using memory CD8 T cells into transplantation still worthwhile. As discussed above, such memory cells are highly dependent on Wnt signaling and therefore ex vivo treatment with Wnt modifying agents may be useful in clinical use of ACT.

Back to Top | Article Outline

Future Perspectives

For the outcome of transplantation, the balance between Treg, Th1/Th17, and CTL responses is crucial. The current state-of-the-art in regulation of immune responses points to a potentially critical role for Wnt signaling in this process. As low Wnt signaling would inhibit Th17 responses and memory CTL responses and enhance Treg activity, Wnt signaling inhibitors could be of interest to stimulate graft acceptance in vivo. Several compounds, such as 2,4-diamino-quinazoline, quercetin, ICG-001, PKF115-584, and BC2059, which all inhibit the interaction between β-catenin and TCFs, or drugs that inhibit sections of Wnts (porcupine inhibitors), such as inhibitor of Wnt production or axin activators (inhibitor of Wnt response), are available to this end.107-110 Activation of Wnt signaling in CD8 T memory stem cells and likely also in other memory CD8 T cell subsets would allow for better proliferation and/or survival of these cells in adoptive transfer settings. Given the current clinical interest in Treg transfers, as exemplified by the ONE study, ex vivo manipulation of Treg with Wnt inhibitors could well be beneficial to enhance Treg activity and lead to higher graft acceptance upon transplantation. On the other hand, activation of Wnt signals by natural ligands or GSK3β inhibitors may improve ACT and HSC transplantation applications. A cautionary remark here is that many of the studies done thus far have only been done in mice. We would therefore applaud more studies with Wnt reporters in human cells in vitro and in vivo in xenograft models such as the Nod-severe combined immunodeficiency-Gamma mice.111,112 Together such studies yield another level of manipulating T cell responses in vivo besides the current immune suppressive drugs and may eventually lead to improved outcome after organ transplantation.

Back to Top | Article Outline


1. Claas FH. Predictive parameters for in vivo alloreactivity. Transpl Immunol. 2002;10:137–142.
2. Claas FH. Transplantation: changing dogmas in clinical transplantation immunology. Curr Opin Immunol. 2005;17:533–535.
3. Arens R, Schoenberger SP. Plasticity in programming of effector and memory CD8 T cell formation. Immunol Rev. 2010;235:190–205.
4. Zhu J, Paul WE. Peripheral CD4+ T cell differentiation regulated by networks of cytokines and transcription factors. Immunol Rev. 2010;238:247–262.
5. Schaniel C, Sallusto F, Ruedl C, et al. Three chemokines with potential functions in T lymphocyte-independent and -dependent B lymphocyte stimulation. Eur J Immunol. 1999;29:2934–2947.
6. Boyman O, Létourneau S, Krieg C, et al. Homeostatic proliferation and survival of naïve and memory T cells. Eur J Immunol. 2009;39:2088–2094.
7. Surh CD, Sprent J. Homeostasis of naive and memory T cells. Immunity. 2008;29:848–862.
8. Pepper M, Jenkins MK. Origins of CD4(+) effector and central memory T cells. Nat Immunol. 2011;12:467–471.
9. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol. 2004;22:745–763.
10. Gebhardt T, Mueller SN, Heath WR, et al. Peripheral tissue surveillance and residency by memory T cells. Trends Immunol. 2013;34:27–32.
11. Waldmann H, Hilbrands R, Howie D, et al. Harnessing FOXP3+ regulatory T cells for transplantation tolerance. J Clin Invest. 2014;124:1439–1445.
12. Abbas AK, Benoist C, Bluestone JA, et al. Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol. 2013;14:307–308.
13. Devaud C, Darcy PK, Kershaw MH. Foxp3 expression in T regulatory cells and other cell lineages. Cancer Immunol Immunother. 2014;63:869–876.
14. Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature. 2005;434:843–850.
15. Staal FJ, Noort Mv M, Strous GJ, et al. Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin. EMBO Rep. 2002;3:63–68.
16. Staal FJ, Luis TC, Tiemessen MM. WNT signalling in the immune system: WNT is spreading its wings. Nat Rev Immunol. 2008;8:581–593.
17. Staal FJ, Clevers H. Tales of the unexpected: Tcf1 functions as a tumor suppressor for leukemias. Immunity. 2012;37:761–763.
18. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127:469–480.
19. Clipstone NA, Crabtree GR. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992;357:695–697.
20. Beals CR, Sheridan CM, Turck CW, et al. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science. 1997;275:1930–1934.
21. Kokolus K, Nemeth MJ. Non-canonical Wnt signaling pathways in hematopoiesis. Immunol Res. 2010;46:155–164.
22. Korswagen HC. Canonical and non-canonical Wnt signaling pathways in Caenorhabditis elegans: variations on a common signaling theme. Bioessays. 2002;24:801–810.
23. Rothenberg EV, Moore JE, Yui MA. Launching the T-cell-lineage developmental programme. Nat Rev Immunol. 2008;8:9–21.
24. Schilham MW, Wilson A, Moerer P, et al. Critical involvement of Tcf-1 in expansion of thymocytes. J Immunol. 1998;161:3984–3991.
25. Verbeek S, Izon D, Hofhuis F, et al. An HMG-box-containing T cell factor required for thymocyte differentiation. Nature. 1995;374:70–74.
26. Xu Y, Banerjee D, Huelsken J, et al. Deletion of beta-catenin impairs T cell development. Nat Immunol. 2003;4:1177–1182.
27. Gounari F, Aifantis I, Khazaie K, et al. Somatic activation of beta-catenin bypasses pre-TCR signaling and TCR selection in thymocyte development. Nat Immunol. 2001;2:863–869.
28. Galceran J, Hsu SC, Grosschedl R. Rescue of a Wnt mutation by an activated form of LEF-1: regulation of maintenance but not initiation of Brachyury expression. Proc Natl Acad Sci U S A. 2001;98:8668–8673.
29. Reya T, O'Riordan M, Okamura R, et al. Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism. Immunity. 2000;13:15–24.
30. Schilham MW, Oosterwegel MA, Moerer P, et al. Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4. Nature. 1996;380:711–714.
31. Okamura RM, Sigvardsson M, Galceran J, et al. Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1. Immunity. 1998;8:11–20.
32. Steinke FC, Yu S, Zhou X, et al. TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4(+) T cell fate and interact with Runx3 to silence Cd4 in CD8(+) T cells. Nat Immunol. 2014;15:646–656.
33. Xing S, Li F, Zeng Z, et al. Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity. Nat Immunol. 2016;17:695–703.
34. Yu S, Zhou X, Steinke FC, et al. The TCF-1 and LEF-1 transcription factors have cooperative and opposing roles in T cell development and malignancy. Immunity. 2012;37:813–826.
35. Tiemessen MM, Baert MR, Schonewille T, et al. The nuclear effector of Wnt-signaling, Tcf1, functions as a T-cell-specific tumor suppressor for development of lymphomas. PLoS Biol. 2012;10:e1001430.
36. Luis TC, Weerkamp F, Naber BA, et al. Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation. Blood. 2009;113:546–554.
37. Fleming HE, Janzen V, Lo Celso C, et al. Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell Stem Cell. 2008;2:274–283.
38. Zhao C, Blum J, Chen A, et al. Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo. Cancer Cell. 2007;12:528–541.
39. Cobas M, Wilson A, Ernst B, et al. Beta-catenin is dispensable for hematopoiesis and lymphopoiesis. J Exp Med. 2004;199:221–229.
40. Jeannet G, Scheller M, Scarpellino L, et al. Long-term, multilineage hematopoiesis occurs in the combined absence of beta-catenin and gamma-catenin. Blood. 2008;111:142–149.
41. Koch U, Wilson A, Cobas M, et al. Simultaneous loss of beta- and gamma-catenin does not perturb hematopoiesis or lymphopoiesis. Blood. 2008;111:160–164.
42. Baba Y, Garrett KP, Kincade PW. Constitutively active beta-catenin confers multilineage differentiation potential on lymphoid and myeloid progenitors. Immunity. 2005;23:599–609.
43. Malhotra S, Baba Y, Garrett KP, et al. Contrasting responses of lymphoid progenitors to canonical and noncanonical Wnt signals. J Immunol. 2008;181:3955–3964.
44. Reya T, Duncan AW, Ailles L, et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature. 2003;423:409–414.
45. Willert K, Brown JD, Danenberg E, et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature. 2003;423:448–452.
46. Kirstetter P, Anderson K, Porse BT, et al. Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block. Nat Immunol. 2006;7:1048–1056.
47. Scheller M, Huelsken J, Rosenbauer F, et al. Hematopoietic stem cell and multilineage defects generated by constitutive beta-catenin activation. Nat Immunol. 2006;7:1037–1047.
48. Suda T, Arai F. Wnt signaling in the niche. Cell. 2008;132:729–730.
49. Trowbridge JJ, Moon RT, Bhatia M. Hematopoietic stem cell biology: too much of a Wnt thing. Nat Immunol. 2006;7:1021–1023.
50. Goessling W, North TE, Loewer S, et al. Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration. Cell. 2009;136:1136–1147.
51. Huang J, Zhang Y, Bersenev A, et al. Pivotal role for glycogen synthase kinase-3 in hematopoietic stem cell homeostasis in mice. J Clin Invest. 2009;119:3519–3529.
52. Nemeth MJ, Topol L, Anderson SM, et al. Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation. Proc Natl Acad Sci U S A. 2007;104:15436–15441.
53. Staal FJ, Chhatta A, Mikkers H. Caught in a Wnt storm: complexities of Wnt signaling in hematopoiesis. Exp Hematol. 2016;44:451–457.
54. Luis TC, Ichii M, Brugman MH, et al. Wnt signaling strength regulates normal hematopoiesis and its deregulation is involved in leukemia development. Leukemia. 2012;26:414–421.
55. Luis TC, Naber BA, Roozen PP, et al. Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion. Cell Stem Cell. 2011;9:345–356.
56. Willinger T, Freeman T, Herbert M, et al. Human naive CD8 T cells down-regulate expression of the WNT pathway transcription factors lymphoid enhancer binding factor 1 and transcription factor 7 (T cell factor-1) following antigen encounter in vitro and in vivo. J Immunol. 2006;176:1439–1446.
57. Zhao DM, Yu S, Zhou X, et al. Constitutive activation of Wnt signaling favors generation of memory CD8 T cells. J Immunol. 2010;184:1191–1199.
58. Gattinoni L, Zhong XS, Palmer DC, et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat Med. 2009;15:808–813.
59. Boudousquie C, Danilo M, Pousse L, et al. Differences in the transduction of canonical Wnt signals demarcate effector and memory CD8 T cells with distinct recall proliferation capacity. J Immunol. 2014;193:2784–2791.
60. Mackay LK, Minnich M, Kragten NAM, et al. Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes. Science. 2016;352:459–463.
61. Tiemessen MM, Baert MR, Kok L, et al. T cell factor 1 represses CD8+ effector T cell formation and function. J Immunol. 2014;193:5480–5487.
62. Jeannet G, Boudousquie C, Gardiol N, et al. Essential role of the Wnt pathway effector Tcf-1 for the establishment of functional CD8 T cell memory. Proc Natl Acad Sci U S A. 2010;107:9777–9782.
63. Zhou X, Yu S, Zhao DM, et al. Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1. Immunity. 2010;33:229–240.
64. Zhou X, Xue HH. Cutting edge: generation of memory precursors and functional memory CD8+ T cells depends on T cell factor-1 and lymphoid enhancer-binding factor-1. J Immunol. 2012;189:2722–2726.
65. Gattinoni L, Lugli E, Ji Y, et al. A human memory T cell subset with stem cell-like properties. Nat Med. 2011;17:1290–1297.
66. Lugli E, Dominguez MH, Gattinoni L, et al. Superior T memory stem cell persistence supports long-lived T cell memory. J Clin Invest. 2013;123:594–599.
67. Joffre O, Santolaria T, Calise D, et al. Prevention of acute and chronic allograft rejection with CD4+CD25+Foxp3+ regulatory T lymphocytes. Nat Med. 2008;14:88–92.
68. Keerthivasan S, Aghajani K, Dose M, et al. beta-Catenin promotes colitis and colon cancer through imprinting of proinflammatory properties in T cells. Sci Transl Med. 2014;6:225ra28.
69. Yu Q, Sharma A, Ghosh A, et al. T cell factor-1 negatively regulates expression of IL-17 family of cytokines and protects mice from experimental autoimmune encephalomyelitis. J Immunol. 2011;186:3946–3952.
70. Yu Q, Sharma A, Sen JM. TCF1 and beta-catenin regulate T cell development and function. Immunol Res. 2010;47:45–55.
71. Muranski P, Borman ZA, Kerkar SP, et al. Th17 cells are long lived and retain a stem cell-like molecular signature. Immunity. 2011;35:972–985.
72. Lee YS, Lee KA, Yoon HB, et al. The Wnt inhibitor secreted Frizzled-Related Protein 1 (sFRP1) promotes human Th17 differentiation. Eur J Immunol. 2012;42:2564–2573.
73. Ding Y, Shen S, Lino AC, et al. Beta-catenin stabilization extends regulatory T cell survival and induces anergy in nonregulatory T cells. Nat Med. 2008;14:162–169.
74. Guo Z, Dose M, Kovalovsky D, et al. Beta-catenin stabilization stalls the transition from double-positive to single-positive stage and predisposes thymocytes to malignant transformation. Blood. 2007;109:5463–5472.
75. Sharma A, Sen JM. Molecular basis for the tissue specificity of beta-catenin oncogenesis. Oncogene. 2013;32:1901–1909.
76. van Loosdregt J, Fleskens V, Tiemessen MM, et al. Canonical Wnt signaling negatively modulates regulatory T cell function. Immunity. 2013;39:298–310.
77. Chae WJ, Ehrlich AK, Chan PY, et al. The Wnt antagonist Dickkopf-1 promotes pathological type 2 cell-mediated inflammation. Immunity. 2016;44:246–258.
78. Yu Q, Sharma A, Oh SY, et al. T cell factor 1 initiates the T helper type 2 fate by inducing the transcription factor GATA-3 and repressing interferon-gamma. Nat Immunol. 2009;10:992–999.
79. Choi YS, Gullicksrud JA, Xing S, et al. LEF-1 and TCF-1 orchestrate T(FH) differentiation by regulating differentiation circuits upstream of the transcriptional repressor Bcl6. Nat Immunol. 2015;16:980–990.
80. Wu T, Shin HM, Moseman EA, et al. TCF1 is required for the T follicular helper cell response to viral infection. Cell Rep. 2015;12:2099–2110.
81. Manicassamy S, Reizis B, Ravindran R, et al. Activation of beta-catenin in dendritic cells regulates immunity versus tolerance in the intestine. Science. 2010;329:849–853.
82. Oderup C, LaJevic M, Butcher EC. Canonical and noncanonical Wnt proteins program dendritic cell responses for tolerance. J Immunol. 2013;190:6126–6134.
83. Suryawanshi A, Manoharan I, Hong Y, et al. Canonical wnt signaling in dendritic cells regulates Th1/Th17 responses and suppresses autoimmune neuroinflammation. J Immunol. 2015;194:3295–3304.
84. Cadigan KM, Nusse R. Wnt signaling: a common theme in animal development. Genes Dev. 1998;11:3286–3305.
85. Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004;20:781–810.
86. Rattis FM, Voermans C, Reya T. Wnt signaling in the stem cell niche. Curr Opin Hematol. 2004;11:88–94.
87. Trompouki E, Bowman TV, Lawton LN, et al. Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration. Cell. 2011;147:577–589.
88. Zamurovic N, Cappellen D, Rohner D, et al. Coordinated activation of notch, Wnt, and transforming growth factor-beta signaling pathways in bone morphogenic protein 2-induced osteogenesis. Notch target gene Hey1 inhibits mineralization and Runx2 transcriptional activity. J Biol Chem. 2004;279:37704–37715.
89. Karin M, Clevers H. Reparative inflammation takes charge of tissue regeneration. Nature. 2016;529:307–315.
90. Famili F, Naber BA, Vloemans S, et al. Discrete roles of canonical and non-canonical Wnt signaling in hematopoiesis and lymphopoiesis. Cell Death Dis. 2015;6:e1981.
91. Lin SL, Li B, Rao S, et al. Macrophage Wnt7b is critical for kidney repair and regeneration. Proc Natl Acad Sci U S A. 2010;107:4194–4199.
92. Christensen ST, Pedersen SF, Satir P, et al. The primary cilium coordinates signaling pathways in cell cycle control and migration during development and tissue repair. Curr Top Dev Biol. 2008;85:261–301.
93. von Toerne C, Schmidt C, Adams J, et al. Wnt pathway regulation in chronic renal allograft damage. Am J Transplant. 2009;9:2223–2239.
94. von Toerne C, Bedke J, Safi S, et al. Modulation of Wnt and Hedgehog signaling pathways is linked to retinoic acid-induced amelioration of chronic allograft dysfunction. Am J Transplant. 2012;12:55–68.
95. Ueland T, Rollag H, Hartmann A, et al. Secreted Wnt antagonists during eradication of cytomegalovirus infection in solid organ transplant recipients. Am J Transplant. 2014;14:210–215.
96. Huang J, Nguyen-McCarty M, Hexner EO, et al. Maintenance of hematopoietic stem cells through regulation of Wnt and mTOR pathways. Nat Med. 2012;18:1778–1785.
97. Luis TC, Staal FJ. WNT proteins: environmental factors regulating HSC fate in the niche. Ann N Y Acad Sci. 2009;1176:70–76.
98. North TE, Goessling W, Walkley CR, et al. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature. 2007;447:1007–1011.
99. Duinhouwer LE, Tüysüz N, Rombouts EW, et al. Wnt3a protein reduces growth factor-driven expansion of human hematopoietic stem and progenitor cells in serum-free cultures. PLoS One. 2015;10:e0119086.
100. Khoo ML, Carlin SM, Lutherborrow MA, et al. Gene profiling reveals association between altered Wnt signaling and loss of T cell potential with age in human hematopoietic stem cells. Aging Cell. 2014;13:744–754.
101. Artandi SE, Blau HM, de Haan G, et al. Stem cells and aging: what's next? Cell Stem Cell. 2015;16:578–581.
102. Geiger H, de Haan G, Florian MC. The ageing haematopoietic stem cell compartment. Nat Rev Immunol. 2013;13:376–389.
103. Florian MC, Nattamai KJ, Dorr K, et al. A canonical to non-canonical Wnt signalling switch in haematopoietic stem-cell ageing. Nature. 2013;503:392–396.
104. Staal FJ, Luis TC. Wnt signaling in hematopoiesis: crucial factors for self-renewal, proliferation, and cell fate decisions. J Cell Biochem. 2010;109:844–849.
105. Rosenberg SA, Restifo NP, Yang JC, et al. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer. 2008;8:299–308.
106. Kalos M, June CH. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity. 2013;39:49–60.
107. Kaniwa K, Arai MA, Li X, et al. Synthesis, determination of stereochemistry, and evaluation of new bisindole alkaloids from the myxomycete Arcyria ferruginea: an approach for Wnt signal inhibitor. Bioorg Med Chem Lett. 2007;17:4254–4257.
108. Kuhl M. The WNT/calcium pathway: biochemical mediators, tools and future requirements. Front Biosci. 2004;9:967–974.
109. Li X, Zhang Y, Kang H, et al. Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem. 2005;280:19883–19887.
110. Zhang Q, Major MB, Takanashi S, et al. Small-molecule synergist of the Wnt/beta-catenin signaling pathway. Proc Natl Acad Sci U S A. 2007;104:7444–7448.
111. Wiekmeijer AS, Pike-Overzet K, Brugman MH, et al. Sustained engraftment of cryopreserved human bone marrow CD34(+) cells in young adult NSG mice. Biores Open Access. 2014;3:110–116.
112. Shultz LD, Lyons BL, Burzenski LM, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174:6477–6489.
Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.