Role of T cell-NF-κB in Transplantation

Abstract

NF-κB is a pleiotropic transcription factor that is ubiquitously expressed. Following transplantation of solid organs, NF-κB in the graft is activated within a few hours as a consequence of ischemia/reperfusion, and then again after a few days in intra-graft infiltrating cells during the process of acute allograft rejection. In the present article, we review the components of the NF-κB pathway, their mechanisms of activation, their role in T cell and antigen-presenting cell (APC) activation and differentiation, and in solid organ allograft rejection. Targeted inhibition of NF-κB in selected cell types may promote graft survival with fewer side effects than global immunosuppressive therapies.

Introduction

A quarter of a century ago, Ranjan Sen and David Baltimore discovered a transcription factor that bound to the enhancer of the immunoglobulin κ light chain in B cells. Its DNA-binding activity was inducible by posttranslational modifications upon LPS or phorbol esters stimulation. They named it Nuclear Factor of the κ light chain in B cells, most commonly known as NF-κB (1). Later studies showed that NF-κB is present in almost all mammalian cells. This transcription factor is extremely conserved through evolution, as it is not only present in animals from insects to mammals, but there is also evidence of NF-κB homologs in plants (2). NF-κB is a family of pleiotropic transcription factors involved in embryonic development of various organs (3), inflammation, immune response, cell survival, proliferation and differentiation (4). NF-κB activation is achieved with diverse stimuli, such as bacterial or viral infections, inflammatory cytokines, UV and oxidative stress, engagement of antigen receptors and ischemia/reperfusion, and the pattern of transcribed genes that it regulates is stimulus- and cell type-dependent (4).

The family of NF-κB transcription factors

The family of NF-κB transcription factors shares homology with the avian oncogene v-Rel; therefore the family is called NF-κB/Rel. The five members of the NF-κB/Rel family are RelA, RelB, c-Rel, p105/p50 (NF-κB1) and p100/p52 (NF-κB2) (5). All the members contain a Rel homology domain in their amino terminus, but only RelA, RelB and c-Rel contain transactivation domains, composed of abundant serine, acidic and hydrophobic amino acids, that are essential for the transcriptional capacity of NF-κB. In contrast, p50 and p52 need to associate with other family members to acquire transcriptional activity. The most common dimers are composed of p50 bound to RelA or c-Rel, and p52 bound to RelB (6). NF-κB recognizes the decameric DNA sequence GGGRNNYYCC, (R=purine Y=pyrimidine). In addition, dimeric complexes of p65/p65, p65/c-Rel, p65/p52, c-Rel/c-Rel, p52/c-Rel, p50/c-Rel, p50/p50, RelB/p50, and RelB/p52 have been described, some of them only in limited cell subsets (3). Interestingly, the subunits NF-κB1/p50 and NF-κB2/p52 are generated as larger precursors, p105 and p100 respectively, that are processed before being part of the active dimers. As will be described in more detail below, both p100 and p105 contain domains that can serve as NF-κB inhibitors themselves, while their processing permits NF-κB activation (6). Post-translational modifications of NF-κB subunits further regulate the activity of these factors, where usually phosphorylation and acetylation are associated with an increase in function (7).

Regulation of the NF-κB pathway: IκB molecules

In resting cells, the NF-κB dimers are sequestered in the cytoplasm by their inhibitors, IκB (IκBα, IκBβ and IκBε). A signal that activates the NF-κB pathway promotes the phosphorylation of the IκB inhibitors, which in turn leads to their K48-ubiquitination and further degradation by the 26S proteasome. The enzymatic complex that phosphorylates the IκB molecules is the IκB kinase (IKK) complex, composed of the kinases IKKα, IKKβ and the regulatory subunit NEMO (IKKγ).

There is a common structural pattern among the IκB inhibitors, consisting of a group of ankyrin motifs that bind to the NF-κB dimers. Despite their structural similarities, the different IκBs have preferential binding for different NF-κB dimers: the complex RelA/p50 is preferentially targeted by IκBα and to a lesser extent by IκBβ, while the dimers composed only by the transactivating subunits RelA/c-Rel bind both IκBβ and IκBε, and RelA/RelA binds to IκBε (8, 9). Transcriptional regulation of the various IκBs is strikingly different, as IκBα transcription is rapidly upregulated by NF-κB activation, thus quickly negatively controlling NF-κB activation, whereas IκBβ and IκBε are constitutively produced (6, 10). Binding of hypophosphorylated IκBβ to RelA/c-Rel has recently been shown to be critical for transcription of TNF upon LPS stimulation, and IκBβ-deficient mice are resistant to septic shock (11). Therefore, inhibition of IκBβ could be a promising therapy in diseases involving TNF production, such as ischemia/reperfusion injury (see below).

More recently, NF-κB2/p100 was shown to function as a fourth IκB protein, independent of its p52 domain, as its ankyrin domain (IκBδ) sequesters RelA and RelB complexes in the cytoplasm (12). Though NF-κB/p105 contains an ankyrin motif, there is as of yet no evidence that this domain has inhibitory functions.

Bcl3, which shares homology with the IκB molecules, interacts with the p50 and p52 homodimers either to repress (13, 14) or to induce gene expression, if it is complexed to p50/p50 dimers (15, 16). Expression of another IκB-like inducible protein, IκBζ (NF-κBiζ), is triggered by exposure to LPS and IL-1α/IL-1β, but not TNF, in a NF-κB-dependent manner (17). Similar to Bcl3-containing complexes, IκBζ/p50/p50 complexes are transcriptionally active (18).

Activation of NF-κB

The IKK complex is a hexamer composed of two subunits each of the kinases IKKα and IKKβ and of the regulatory subunit NEMO/IKKγ. IKKβ and NEMO, but not IKKα, are pivotal factors for activation of the NF-κB canonical pathway. Mice lacking IKKβ die at an embryonic stage, and tissue-specific deletions were required for analysis of this kinase in different cell types. The role of IKKα in the IKK complex is partially redundant, but this enzyme is absolutely required for the activation of the alternative NF-κB pathway (see below). Activation of the IKK complex requires K63-ubiquitination of the regulatory subunit NEMO/IKKγ (19). Expression of a myristoylated form of NEMO in Jurkat cells, which results in its accumulation at the plasma membrane, was sufficient to render it constitutively active (20). As with IKKβ, mice deficient in NEMO die at an embryonic stage (4).

Canonical pathway of NF-κB activation

The most abundant form of NF-κB dimers, RelA/p50, is present in the cytoplasm sequestered by its inhibitor IκBα. Several stimuli, such as IL-1 and TLR agonists can promote its activation, with the most paradigmatic being TNF via engagement of the TNF receptor (TNFR) (Figure 1). Briefly, ligation of the TNFR promotes the recruitment of TRAF2/5 (TNF Receptor-Associated Factor 2/5) and RIP1 (Receptor-Interacting Protein 1) via TRADD (TNFR1-Associated Death Domain Protein). TRAF2 causes the K63 non-degradable ubiquitination of RIP1 and also recruits and stabilizes the IKK complex towards the TNFR. TAK1 (TGF-β Activated Kinase 1) and TRAF2 interact with TAB2/3 (TAK1-Binding Protein 2/3), inducing TAK1 activation, which in turn phosphorylates IKKβ. The IKK complex can also be autophosphorylated, enhancing its activation. This active IKK complex is then ready to phosphorylate IκBα, therefore targeting it for K48 ubquitination and 26S proteasomal degradation. This event liberates RelA/p50 to translocate into the nucleus to exert its transcriptional activity (6).

Figure 1. Classical and alternative NF-κB activation pathways.

The classical pathway activates IKKβ and IKKγ in the IKK complex. IKKβ phoshorylates IκBα, targeting it for K48-ubiquitination and subsequent degradation by the 26S proteasome, releasing the dimers RelA/p50 or cRel/p50 for nuclear translocation. The alternative pathway involves NIK stabilization, activation of IKKα, which phoshorylates NF-κB2/p100 and directs it for K48-ubiquitiniation and partial degradation by the 26S proteasome, liberating the newly generated RelB/p52 for nuclear translocation.

Alternative pathway of NF-κB activation

Receptors belonging to the TNFR superfamily, such as CD40, LTβR and RANKL, activate NF-κB by IKKα-dependent processing of the NF-κB2/p100 subunit bound to RelB independently from IKKβ or NEMO (Figure 1). The pathway leading to this activation depends on the stabilization of the NF-κB-Inducing Kinase (NIK). In resting cells, NIK is constitutively produced, but also constantly degraded in a cIAP (cellular Inhibitor of APoptosis) 1/2- and TRAF3-mediated polyubiquitination-dependent manner (21, 22). Upon stimulation, NIK is stabilized and phosphorylates IKKα, which in turn directs processing of p100, such that RelB/p52 dimers are free to translocate into the nucleus (23). . Mice deficient in NIK (aly/aly mice) have a phenotype very similar to mice expressing a non-phosphorylatable form of IKKα (IKKα^AA), as both lack lymph nodes (23, 24).

Interestingly, components of the NF-κB alternative pathway can activate the canonical branch: IKKβ can be activated by stabilized NIK, and processing of p100 can also lead to assembly of RelA/p52, as well as RelB/p52 dimers (12, 25).

NF-κB and cell survival

Apoptosis is a mechanism of programmed cell death. This process usually involves an increased ratio of pro-apoptotic (i.e., Bax, Bim) versus anti-apoptotic (i.e., Bcl-2, Bcl-x_L) molecules. TNF/TNFR1 and Fas/FasL pathways can directly initiate this type of apoptosis. NF-κB antagonizes apoptosis and promotes cell survival by inducing expression of pro-survival genes (Bcl-2, Bcl-x_L, IEX1) and by repressing the pro-apoptotic genes Bax and Bim (26–29)(30). While Bcl-2 remains constant during mature T cell activation, Bcl-x_L is strongly upregulated upon TCR triggering.

NF-κB activation in T cells

Full activation of naïve T cells requires stimulation through their TCR and the costimulatory molecule CD28. These events trigger the activation of several intracellular signaling pathways that lead to T cell proliferation/clonal expansion, cytokine secretion, cell survival, differentiation and generation of memory cells. Signaling through the canonical pathway of NF-κB is critical for proper T cell activation (Figure 2).

Figure 2. Activation of NF-κB by the TCR.

Upon stimulation, the TCR/CD3 complex activates Lck, which promotes recruitment and activationof ZAP70 to the CD3 complex and PI3K to CD28. ZAP70 activates PLCγ, which generates dyacylglycerol (DAG), which in combination with PI3K-activated PDK1 promotes activity of PKCθ. This kinase phosphorylates CARMA1, which in turn recruits Bcl10 and MALT1 (CBM complex), that promotes the activation of IKK complex via the K63 ubiquitination of IKKγ, resulting in the canonical activation of NF-κB.

When the TCR/CD3 and CD28 respectively bind to peptide-MHC and B7 molecules in APCs, the signaling cascade that triggers NF-κB activation begins with the dephosphorylation of the membrane kinase Lck (Lymphocyte-Specific Protein-Tyrosine Kinase) by CD45, which results in activation of two pathways. First, Lck activates Zap-70, which in turn phosphorylates and recruits the scaffold protein LAT (Linker for Activation of T Cells), promoting PLCγ1 (Phospholipase C γ1) activity that cleaves phosphatidylinositol 4,5-biphosphate (PIP2) into the second messengers diacylgylcerol (DAG) and inositol triphosphate (IP3). At the same time, Lck activates PI3K (Phosphatidyl Inositol 3-Kinase) upon CD28 stimulation. Active PI3K produces phosphatidylinositol 1,4,5-triphosphate (PIP3), which activates PDK1 (3-Phosphoinositide-Dependent Protein Kinase 1), an enzyme that in turn activates both Akt and PKCθ (Protein Kinase C θ), the latter being dependent on DAG. PKCθ phosphorylates the lipid raft-associated CARMA1 [CAspase Recruitment Domain (CARD) Membrane Associated GUanylate Kinase (MAGUK) protein 1], recruiting it to the immunological synapse. Phosphorylated CARMA1 associates with Bcl10 (B-cell CLL/lymphoma 10) and MALT1 (Mucosa-Associated Lymphoid Tissue 1), driving their localization to the plasma membrane. Bcl10, now as a complex with CARMA1 and MALT1, promotes K63-ubiquitination and activation of NEMO, therefore activating the IKK complex and the NF-κB canonical pathway (31). Interestingly, phosphorylation of Bcl10 by IKKβ has been shown to dissociate it from MALT1, working as a negative feedback loop for TCR-NF-κB signaling (32). In T cells, the formation of the CARMA1-Bcl10-MALT1 (CBM) adaptosome activates JNK2 (c-Jun Kinase 2) and promotes c-Jun accumulation, without affecting AP-1 signaling (33, 34). The adaptor protein ADAP (Adhesion and Degranulation Adaptor Protein), required for T cell adhesion upon LFA-1 (Lymphocyte Function-associated Antigen, Type 1) engagement, has been shown to be necessary for the formation of the CBM complex, by recruiting it to PKCθ (35). Upon TCR stimulation, activation of PKCθ results in activation of the transcription factors NF-κB, NFAT (Nuclear Factor of Activated T cells) and AP-1 (Activator Protein-1) (36), thus controlling T cell survival mostly via induction of Bcl-x_L and IL-2 (37)(38)(39). Though both RelA/p50 and c-Rel/p50 dimers bind to the IL-2 promoter, only c-Rel/p50 is important for chromatin remodeling and IL-2 transcription (40, 41).

Despite almost undetectable levels of RelB in unstimulated T cells, the alternative pathway of NF-κB activation plays a role in T cell activation, although it seems to bear opposite effects on naïve and memory CD4 populations (42). Activation of unfractionated CD4⁺ T cells has been shown to be strongly impaired in NIK- and RelB-deficient cells. However, when separating naïve from memory cells, NIK-deficient naïve CD4⁺ T cells were found to be hyperresponsive, produced higher levels of IL-2, expressed more IL-2Rα, displayed increased homeostatic proliferation and promoted autoimmunity. In contrast, memory CD4⁺ T cells were hyporresponsive and strongly suppressive, thus regulating the activation of the naïve population (42).

Several of the molecules required for NF-κB activation in T cells are restricted to either T cells themselves or to cells of leukocytic origin (CARMA1, PKCθ, ADAP). The development of drugs that specifically target these molecules could potentially inhibit lymphocyte activation selectively, thus being of interest in autoimmunity or transplantation settings.

NF-κB and T cell development

T cell precursors are generated in the bone marrow and migrate to the thymus where they differentiate into CD4/CD8 double negative (DN)2, DN3, DN4, CD4/CD8 double positive (DP), CD4 and CD8 single positive TCRαβ T cells as well as TCRγδ and NKT cells. Using a super-repressor form of IκBα expressed in T cells under control of the Lck promoter (IκBαΔN-Tg), Boothby and colleagues demonstrated that early activation of the canonical pathway of NF-κB was strongly required for thymic CD8⁺ T cell development and to a lesser extent for that of CD4⁺ T cells (43). The same model showed that canonical NF-κB was critical for early generation of NKT cells, mostly by expression of pro-survival factors (44). Inhibition of the canonical pathway of NF-κB in T cells, either by specific deletion of either IKKβ or NEMO/IKKγ also strongly impaired CD8 development, and only modestly affected the CD4 lineage (43, 45). Surprisingly, although lack of CARMA1, Bcl10 and MALT1 altered the ratio of DN4 to DN3 thymocyte precursors, positive and negative selection during thymic development remained largely unimpaired (46–49), suggesting that TCR-mediated NF-κB activation is not required for conventional T cell development.

Given their capacity to suppress allograft immune responses and prolong allograft survival, regulatory CD4⁺CD25⁺Foxp3⁺ T cells (Tregs), are of particular interest in transplantation, and a lot of effort has been devoted to developing methods for expansion of thymic or peripherally generated Tregs to improve allograft survival (50). The canonical pathway of NF-κB activation is very important for the thymic generation of natural Tregs (nTregs), as mice lacking IKKβ from the DP stage on (CD4-Cre × IKKβ^FL/FL) have drastically reduced Treg numbers (45). Interestingly, while the signaling of NF-κB downstream of the TCR did not affect the thymic development of conventional CD4⁺ and CD8⁺ T cells, lack of PKCθ, Bcl10, or CARMA1 strongly impaired Treg development (51–54). Most recently, the subunit c-Rel was shown to be particularly important for thymic expression of Foxp3 (55), by binding to an intronic enhancer of the Foxp3 gene (56). Conversely, transgenic expression of constitutively active IKKβ in T cells (Lck promoter) resulted in increased numbers of Foxp3⁺ cells and restored nTreg development in CARMA1-deficient mice. However, these cells lost their suppressor capacity, suggesting that NF-κB is important for Treg development but may be counterproductive for Treg function (57). It is worth mentioning that NF-κB and NFAT signaling was blunted in wild type nTregs upon TCR stimulation (58), and that the pharmacological inhibition of PKCθ resulted in enhanced Treg suppressor function (59) thus making this inhibitor an interesting candidate to improve graft survival.

NF-κB and T cell differentiation

Upon TCR stimulation, T cells differentiate into particular effector phenotypes depending on the cytokine milieu. The ratio of Th1 and Th17 versus Th2 and iTreg subsets has been linked to allograft rejection (60).

Solid organ transplantation is strongly associated with increased IFNγ production by alloreactive Th1 cells. Differentiation into Th1 effector cells requires TCR stimulation in combination with IL-12 and IL-18, in a process that involves upregulation of the transcription factors T-bet and STAT4. Both canonical and alternative pathways of NF-κB activation are important in Th1 differentiation. Inhibition of the canonical pathway by either expression of a super-repressor form of IκBα, or by lack of RelA specifically in T cells was shown to result in strongly reduced IFNγ production (61, 62). The alternative pathway of NF-κB activation also modulated Th1 differentiation, in that lack of RelB prevented expression of both T-bet and STAT4 (63). Similarly, a deficiency in CARMA1 strongly impaired recruitment of IKKβ to the immunological synapse, T cell proliferation and IL-2 and IFNγ production (33, 46). Bcl3-deficient mice were also incapable of mounting a Th1 response, and they succumbed to Toxoplasma gondii infection (64). Interestingly, not all murine models with impaired NF-κB activity in T cells had the same defects in Th1 development, as mice deficient in PKCθ generated normal Th1 and cytotoxic T cell (CTL) responses against Leishmania major (65).

Though Th2 polarization has been mostly associated with allograft survival due to the ability of IL-4 to inhibit Th1 differentiation, there is clear evidence that the Th2 cytokines IL-4 and IL-13 can also be deleterious to graft function (66). The transcription factor GATA3 is the master regulator for Th2 differentiation, leading to the expression of IL-4, IL-5 and IL-13. The p50/p50 NF-κB homodimer, but not RelA, is thought to control GATA3 expression and the priming for Th2 differentiation (67), possibly due to the Bcl3/p50/p50 complex binding to the GATA3 proximal promoter (63). PKCθ, the kinase that links the TCR to NF-κB signaling controls Th2 differentiation (68), as mice deficient in PKCθ had blunted Th2-mediated responses to helminths and allergic stimuli (69), and did not develop eosinophilia or produce Th2 cytokines in a model of airway inflammation (70). In a murine model of asthma, CARMA1 was also required for IL-4 production and allergic airway inflammation (71). Interestingly, the transcriptional repressor Schnurri-2 has been shown to compete with RelA/NF-κB for the κB sites (72). Mice deficient in Schnurri-2 had constitutively active NF-κB in their T cells, and enhanced Th2 differentiation, maybe due to an enhanced effect of NF-κB1/p50 on GATA3 expression (67).

Th17 responses, characterized by production of IL-17A and F, are mounted against extracellular bacteria and predominate in several types of autoimmune disorders. Although not yet examined directly, NF-κB may play a role in Th17 differentiation. Indeed, mice with IKKβ-deficient T cells failed to develop experimental autoimmune encephalomyelitis, a Th17-dependent disease (73). Similarly, PKCθ-deficient mice did not develop IL-17-producing T cells in a mouse model of autoimmune myocarditis, though viral infection and TLR ligands could restore IL-17 production (74). In addition, the IκBζ/p50/p50 complex has been shown to interact with RORγt to promote IL-17A expression, and mice deficient in IκBζ had Th17 differentiation defects and were also resistant to experimental autoimmune encephalomyelitis (18). Recently, either RelA, RelB or cRel were shown to be dispensable for the generation of IL-17-producing CD4 T cells, although RelA and RelB were required for IL-17 production by γδ T cells (75, 76), suggesting that mature CD4 T cells compensate for the lack of these subunits to enable the generation of Th17 cells.

Although c-Rel/NF-κB is critical for thymic nTreg development (as mentioned above), peripheral conversion of naïve T cells into inducible Tregs (iTregs) may take place in the absence of TCR-NF-κB signaling. TGFβ-mediated iTreg differentiation requires IL-2, a cytokine heavily controlled by NF-κB. Accordingly, the inability by NF-κB-impaired T cells to secrete normal amounts of IL-2 prevents naïve T cells to upregulate Foxp3 under iTreg differentiating conditions. However, if exogenous IL-2 is provided, T cells impaired in NF-κB signaling via lack of NF-κB1, Bcl10, CARMA1 or c-Rel, have all been shown to differentiate successfully into functional iTregs (54, 77, 78). Indeed, our own results indicate that NF-κB signaling is detrimental for iTreg differentiation when cells are stimulated with high doses of TCR stimuli (79). Considering the information above, inhibition of NF-κB during an allograft response could both block differentiation of proinflammatory cytokines while promoting suppressive cells.

NF-κB and cytotoxic T cell responses

Allogeneic cytotoxic CD8⁺ T cells are potent mediators of allograft rejection (80). Upon antigen encounter, CD8⁺ T cells become effector cytotoxic cells and gain the ability to kill target cells through secretion of granzyme B and perforin or through induction of apoptosis via Fas-FasL signaling. The role of NF-κB during the CTL response is still a matter of debate. Evidence suggesting that NF-κB may play a role in CTL differentiation is supported by the impaired CD8 and CTL responses to reovirus reported in IκBαΔN-Tg mice (81). However, CTL differentiation was normal in PKCθ-deficient mice infected with Leishmania major (65), in ADAP-deficient mice (82, 83), and in c-Rel-deficient T cells if exogenous IL-2 was provided (84). Considering the importance of IL-2 for CTL differentiation in c-Rel-deficient T cells, it may be of interest to test whether lack of CTL response in infected IκBαΔN-Tg mice can be corrected with exogenous IL-2.

NF-κB and T cell memory responses

Upon TCR stimulation, T cells are activated and become effector and memory cells. Memory T cells are characterized by a lower threshold of activation and a faster response upon antigen re-encounter, as well as by increased expression of CD44, and of chemokine receptors that direct the cells more efficiently towards inflamed tissues (85). The presence of antigen-experienced memory T cells strongly decreases the ability of costimulation blockade-based therapies to induce transplantation tolerance (86). Several lines of evidence suggest that NF-κB is important for the generation of memory T cells. Mice bearing IKKβ-deficient T cells (CD4-Cre × IKKβ^Fl/Fl) (45), as well as Bcl10- and CARMA1-deficient mice, have very few CD44^hi memory cells suggesting that TCR-NF-κB signaling is required for the generation of memory cells (46, 87). In support of this hypothesis, Teixeiro and colleagues recently showed that a mutation in the TCRβ chain of the OTI CD8⁺ T cells (βTMDmut cells) selectively abrogated NF-κB signaling upon antigen stimulation. Whereas IFNγ, TNF, IL-2 and granzyme B effector responses of βTMDmut cells were normal upon in vivo priming with ovalbumin-expressing-Listeria monocytogenes, recall responses were completely abrogated, suggesting that the domain of the TCR that controls NF-κB activation is critical for the generation of a memory response (88). Once memory T cells are generated, TCR-NF-κB may be dispensable for their function, as Bcl10-deficient memory CD44^hi cells displayed normal in vitro proliferation, as well as IFNγ and IL-4 production (87). However, the NF-κB inhibitor deoxyspergualin, in combination with anti-CD40L and CTLA4-Ig, enabled tolerance to skin transplants in small animals with memory T cells generated through heterologous immunity to viral antigens (89), suggesting that the role of T cell-NF-κB in memory T cells needs to be addressed in more detail.

Activating the T cells: NF- κB in dendritic cells

Antigen presentation for T cell priming is mediated by dendritic cells (DCs), B cells and macrophages, regarded as professional APCs. TLR signaling and pro-inflammatory cytokines (TNF and IL-1) promote maturation of the APCs that upregulate MHC class I and II, costimulatory ligands such as CD80, CD86 and CD40, and cytokines that help activate and differentiate T cells. In contrast, interaction of T cells with immature DCs can drive T cells towards a suppressor or anergic phenotype. Signaling via CD40, IL-1R, TNFR and TLR2,-4,-7 and -9 triggers NF-κB activation and plays a central role in DC maturation (90, 91). DC development from bone marrow precursors has been shown to proceed normally in the absence of either RelA or p50, but not when both subunits are absent (92). Interestingly, neither single deficiency of RelA, p50 or c-Rel, nor combined cRel/p50 double deficiency affected DC-mediated capacity to induce normal T cell proliferation, but the low levels of IL-12 produced by p50-cRel double knockout DCs may be insufficient to sustain Th1 differentiation (92).

The alternative NF-κB activation pathway is very important in DC development and function. NIK promotes dendritic cell activation, as an adenovirus encoding NIK was shown to strongly upregulate pro-inflammatory cytokines (TNF, IL-6, IL-12, IL-15, and IL-18), MHC class I/II and CD80 and CD86, that in turn promoted a Th1 response (93). Conversely, mice deficient in NIK had DCs with lower levels of MHC II, CD80, CD86 and reduced capacity to stimulate T cell activation (94).

NF-κB and transplantation

NF-κB is activated in transplanted tissue at several stages leading to solid organ rejection (Figure 3), first during ischemia/reperfusion, and second in activated allogeneic T cells that infiltrate the organ. The first evidence associating NF-κB with allograft rejection was described by Csizmadia and colleagues, where murine cardiac allografts undergoing rejection on day 7 post-transplantation were shown to have higher mRNA expression levels of p50, p52, RelA, c-Rel, RelB and Bcl3, and enhanced NF-κB activity compared to native hearts and hearts treated with a tolerizing anti-CD154-based regimen (95). More recently, Ma and collaborators used mice expressing a luciferase reporter controlled by NF-κB enhancers to analyze kinetics of NF-κB activation following cardiac transplantation. In this system, NF-κB in the graft was activated at 4–24h (due to ischemia), peaked at 4d (initiation of allograft rejection) and returned to basal levels upon cardiac rejection on day 10 (96). Interestingly, immunosuppression with anti-CD154 strongly reduced activation of NF-κB, which was restored in the presence of CpG, a TLR9 ligand that prevents the induction of transplantation tolerance (96, 97).

Figure 3. Kinetics of NF-κB activation during allograft rejection.

NF-κB is activated at least twice in transplanted organs. The first induction of NF-κB, a product of ischemia reperfusion, occurs very early (30 min–24h) after transplantation in donor cells (parenchymal and passenger leukocytes), as a result of signaling through TLR2/4, IL-1R and TNFR. The recruitment of inflammatory neutrophils releasing ROS leads to organ injury and increased susceptibility to chronic organ rejection. The second peak of NF-κB activity occurs in recipient hematopoietic cells that infiltrate the graft during the process of acute allograft rejection (day 4–7 post-transplantation in mice). Activated alloreactive T cells with active NF-κB produce effector cytokines and exert cytotoxicity. In the presence of a tolerizing regimen such as anti-CD154 mAb (gray line), the level of activation of NF-κB in the graft is strongly diminished.

NF-κB in transplantation: ischemia and reperfusion

Damage due to ischemia/reperfusion (I/R) can harm the function of solid organ transplants. During ischemia, tissues experience damage due to lack of oxygen and nutrients. During reperfusion, when the blood flow is restored into the ischemic organ, reoxygenation generates free radicals and production of the pro-inflammatory cytokines TNF, IL-1β, IL-6 and IFNγ, leading to tissue damage mediated by neutrophils and monocytes (98). The proinflammatory cytokine TNF is known to mediate multiorgan dysfunction, and ablation of TNF/TNFR signaling in a gut model of I/R prevented cytokine production, NF-κB signaling and intestinal damage (99). The pro-inflammatory cytokines TNF, IL-1β, IL-6 and the neutrophil chemoattractants MCP-1 (Monocyte Chemotactic Protein 1) and MIP-1α (Macrophage Inflammatory Protein 1−α) are gene targets of NF-κB, thus making inhibition of this transcription factor of special interest to prevent or attenuate I/R injury. Indeed, preconditioning of hearts, kidneys and lungs of small animals with various NF-κB pharmacological inhibitors (IMD-0354, NF-κB ODNs or pyrrolidine dithiocarbamate, among others) decreased infiltration of neutrophils into ischemic organs and enhanced animal survival upon I/R (100–102). During I/R injury, NF-κB activation is important both in leukocytes and in the parenchymal cells of the transplanted organ. In fact, decreased NF-κB activity by specific abrogation of IKKβ in the enterocytes (Villin-Cre × IKKβ^FL/FL) prevented the expression of pro-inflammatory mediators (TNF, IL-1β, IL-6, eotaxin, MCP-1 and MIP-1α and iNOS) and subsequent injury (99). Nevertheless, reduced NF-κB activity in these tissues made them especially susceptible to TNF cytotoxicity (99), throwing a cautionary note for the use of global NF-κB inhibitors to prevent I/R injury. Another model of decreased NF-κB activity in the liver (Alfp-Cre × IKKβ^FL/FL) demonstrated partial inhibition of I/R-mediated NF-κB activation and protection of mice from liver I/R injury (103). Similarly, preconditioning with the IKKβ pharmacological inhibitor AS602868 protected mice from liver injury after I/R (103). Production of TNF during I/R may be due to activation of NF-κB downstream of TLR2 and TLR4 crosslinking by endogenous ligands, DAMPs (Damage-Associated Molecular Patterns), released from necrotic cells (104, 105). Ablation of the TLR2/4 signaling molecule MyD88 or TLR4, decreased the production of the inflammatory mediators TNF, IL-6 and iNOS and enhanced survival of mice upon I/R. In lungs of mice subjected to I/R, pharmacological blockade of TLR4 or adenovirus-mediated NF-κB inhibition prevented ischemia-induced apoptosis and edema and improved oxygenation of transplanted lungs (106, 107)(108).

In summary, NF-κB is activated repeatedly during I/R injury. First, release of endogenous DAMPs results in TLR2- and TLR4-dependent activation of NF-κB, that induces expression of IL-1 and TNF. Signaling downstream of these cytokines elicits a second wave of NF-κB activity that upregulates the expression of other effector molecules (MCP-1, IL-8) thus attracting leukocytes to the inflamed site and promoting injury mediated by the release of reactive oxygen species (ROS).

NF-κB and transplantation: NF-κB in alloimmunity

Considering the importance of NF-κB in T cell activation and differentiation, it is not surprising that mice bearing NF-κB-impaired T cells have a reduced ability to reject allografts. Interestingly, the nature of the transplanted organ determines whether T cell-NF-κB impairment is sufficient or not to induce long-term allograft acceptance.

The role of individual NF-κB subunits in allograft rejection has been assessed by genetic ablation. Deficiency of p50 and p52 not only did not prevent cardiac allograft rejection, but also in fact resulted in resistance to anti-CD154-mediated tolerization, possibly due to the requirement of NF-κB for TCR-mediated CD154 upregulation (95, 109). In contrast, mice lacking the NF-κB subunit c-Rel fully accepted cardiac allografts (110) and 40% of allogeneic islets (111). The role of T cell-NF-κB was further probed using mice expressing a super-repressor form of IκBα selectively in T cells (IκBαΔN-Tg). IκBαΔN-Tg mice were shown to permanently accept cardiac allografts, with almost no leukocyte infiltration in the allografts and with reduced expression of the proinflammatory cytokines IL-1β, IL-6 and IFNγ (109). Interestingly, though IκBαΔN-Tg mice accepted allogeneic hearts, our group showed that they readily rejected skin allografts (112). Forced expression of Bcl-x_L or blockade of Fas restored the capacity of IκBαΔN-Tg mice to reject cardiac allografts, thus suggesting that NF-κB is required for the survival of alloreactive T cells (113)(114). Similar to IκBαΔN-Tg mice, we have shown that CARMA1-deficient mice accept cardiac allografts long-term, while they reject allogeneic skin, suggesting that TCR-driven NF-κB signaling is critical for cardiac allograft rejection (115). PKCθ-deficient T cells transferred into lymphopenic mice failed to promote cardiac allograft rejection, whereas the overexpression of the pro-survival factor Bcl-x_L in these T cells also restored rejection, further supporting a role for T-cell/PKCθ/NF-κB in the survival of alloreactive T cells (116). Consistent with the requirement of ADAP for NF-κB-activation downstream of the TCR, genetic deficiency in ADAP also enhanced the survival of cardiac and intestinal allografts, although acceptance of the latter required adjunctive treatment with CD154 costimulation blockade (82, 83).

The kinase RIP2 mediates NF-κB activation in innate and adaptive immune cells upon CD40, LPS and TCR engagement (117, 118). Ruefli-Brasse showed that RIP2 phosphorylates Bcl10, and that RIP2-deficiency resulted in reduced NF-κB activation and T cell proliferation in vitro, as well as delayed rejection of neonatal cardiac allografts (119). In contrast to these results, however, Fairhead and collaborators reported that RIP2 deficiency had no effect on either TCR-mediated NF-κB activity, in vitro T cell proliferation, Th1 differentiation or cardiac allograft survival (120). A possible explanation for these discrepancies may be that the various RIP2-deficient strains of mice were generated with different targeting strategies, therefore possibly affecting RIP2 gene expression in different manners.

The alternative pathway of NF-κB activation is important at least in models of bone marrow transplantation, as NIK-deficient T cells did not develop graft versus host disease (GVHD) when transferred into lymphopenic allogeneic recipients (121).

Considering the role of NF-κB in DC maturation, an approach tested for its potential to achieve tolerance has been the generation of immature immunosuppressive DCs by blocking NF-κB activation with NF-κB decoy oligonucleotides (NF-κB-ODN). In vitro, these cells displayed low levels of MHC II, CD80, CD86, and a reduced capacity to stimulate allogeneic T cells, but their adoptive transfer was not sufficient to induce long-term cardiac allograft acceptance, also requiring the ectopic expression of CTLA4-Ig to control cardiac allograft rejection (122).

Importantly, certain DC subsets such as epidermal Langerhans cells, can overcome the reduced function of NF-κB-impaired T cells. As mentioned above, IκBαΔN-Tg mice accept cardiac allografts long-term but reject skin allografts with similar kinetics as wild type mice (112). Interestingly, this difference was due at least in part to the presence of epithelial-derived Langerhans cells in the skin allografts, as systemic injection of donor Langerhans cells, but not splenic DCs, restored cardiac allograft rejection in IκBαΔN-Tg mice (115). These results indicate that Langerhans cells can stimulate NF-κB-impaired T cells more potently than splenic DCs, partially restoring their function. Whether this is due to induction of transcription factors that can compensate for lack of NF-κB, remains to be investigated.

Taken together, these results suggest that specific targeting of T cell-NF-κB may be a promising therapy for preventing rejection of allografts with moderate immunogenicity such as heart and kidney, while keeping in mind that very immunogenic allografts, such as skin and intestine, may overcome NF-κB deficiency and trigger allograft rejection. Although T-cell specific NF-κB inhibitors may impair thymic Treg development, they may also enhance Treg suppressive function and conversion of conventional T cells into induced Tregs, therefore being overall favorable in transplant settings. Targeting the NF-κB alternative pathway may also have beneficial effects in transplantation via inhibition of DC function and memory T cell activation. However, unlike PKCθ and the CBM complex that selectively link the TCR to the canonical NF-κB pathway, a molecule essential for activation of the alternative pathway that would be selectively expressed in leukocytes and could serve as a therapeutic target remains to be identified.

Pharmacological inhibition of NF-κB in the clinic

Bortezomib, a proteasome inhibitor, was originally developed for the treatment of relapsed multiple myeloma and mantle cell lymphoma (123). The capacity to inhibit the proteasome prevents the degradation of IκB, therefore reducing NF-κB activation. Similarly to the fate of alloreactive NF-κB-impaired T cells in mice, Bortezomib selectively induced apoptosis of human activated T cells while preserving viability of resting T cells as the latter display minimal NF-κB activity (124). Bortezomib also inhibited the function of conventional T cells while preserving Treg suppressive capacity (125). Although this drug appears promising for the treatment of GVHD, its positive effects in transplantation are not yet clear and its use has been mostly intended to inhibit antibody production by B cells. In small clinical trials in patients with kidney transplants, bortezomib reduced the titers of anti-HLA Abs in patients undergoing acute rejection, but did not improve serum creatinine levels (126).

Prednisone is a glucocorticoid (GC) widely used to prevent rejection of transplanted organs (127). The immunosuppressive function of GCs is due to their ability to bind the GC receptor (GR), thus inhibiting the expression of inflammatory cytokines. Although the precise mechanism is still not completely understood, an important part of the anti-inflammatory action of GCs may be their inhibitory effects on NF-κB activation, either through interactions between the GC-GR complex and the RelA/p50 dimer bound to DNA, or by inducing the expression of IκBα (128).

15-Deoxyspergualin (DSG) and its analogs are immunosuppressive drugs that inactivate NF-κB possibly via their specific binding to the nuclear transport protein HSC70 and subsequent inhibition of nuclear translocation of proteins (129). Treatment of mice with DSG resulted in reduced antigen processing and presentation by APCs, impaired B cell development, decreased antibody production and diminished T cell activation (130, 131). Treatment with DSG in small animal models also prolonged the survival of transplanted cardiac allografts (132) and induced long-term acceptance of allogeneic pancreatic islets in 75% of mice (133), but did not prevent heart allograft rejection in rats presensitized with donor blood (134). Of note, the therapeutic window of DSG seems narrow, as doses of 0.3 mg/kg were ineffective and doses beyond 10 mg/kg were toxic in a rat model of aortic allograft atherosclerosis (chronic rejection) (135). In combination with anti-CD45RB, treatment with the DSG analog LF 15-0195 (LF), which is less toxic and more potent than DSG, induced donor-specific tolerance to mouse cardiac allografts, seemingly by generation of tolerogenic DCs, CD4⁺CD25⁺ Tregs and B cells, as these populations could transfer donor-specific tolerance (136)(137, 138).

In non-human primates, LF significantly prolonged renal allograft survival in monkeys, but renal rejection occurred two weeks after drug withdrawal (139). In Japan, DSG has been used as a recovery treatment in liver transplant patients undergoing acute rejection that is steroid-resistant, with 6 out of 10 patients recovering when treated with 3–5 mg/kg DSG for 4–14 days (140).

Thus, drugs that inhibit NF-κB ubiquitously show potent immunosuppressive effects, but have a narrow pharmaceutical window limited by side effects. New drugs that could target NF-κB only in immune cells may hold promise in transplantation.

Conclusions

The current literature implicates NF-κB in the harmful effects of ischemia/reperfusion, in the survival of activated T cells, the differentiation of Th1, Th2 and Th17 effector T cells, the generation of memory T cells and the maturation of DCs whereas it may antagonize the peripheral development of Tregs (Figure 4). Thus, specific NF-κB inhibitors that target the canonical pathway of NF-κB activation downstream of the TCR may be less toxic than pan-NF-κB inhibitors and useful to prevent rejection of moderately immunogenic organs, as they may reduce generation of effector and memory T cells and facilitate iTreg differentiation. However, more immunogenic organs, such as skin and intestine, can trigger allograft rejection in the face of NF-κB-impaired T cells and may require additional or altogether different therapies. It should also be kept in mind that whereas inhibition of NF-κB may promote peripheral iTreg differentiation, it might also block thymic development of nTregs. In addition, although naïve T cells encountering antigen for the first time are very susceptible to NF-κB inhibition, pre-existing memory T cells may not be as dependent on NF-κB activation. The development of animal models in which the canonical versus the alternative NF-κB pathways can be turned off in an inducible manner in peripheral cells of different origins at different time points after transplantation, or the availability of cell-specific pharmacological inhibitors of NF-κB, will be necessary to dissect these questions in more depth and assess global outcomes in vivo. Finally, inhibition of NF-κB in the donor organ may also be of interest for cytoprotection of the graft.

Figure 4. Transplant-related NF-κB activating stimuli and NF-κB-mediated functions.

Several stimuli activate NF-κB, that plays a major role in development, activation and differentiation of alloreactive T cells and DC, as well as cell survival and TNF-mediated cytotoxicity.