NF-κB is a key regulator of transcription after TCR and costimulatory receptor ligation. To determine the role of T cell-intrinsic NF-κB activation in acute allograft rejection, we used IκBαΔN-Tg mice (H-2b) that express an inhibitor of NF-κB restricted to the T cell compartment. We have previously shown that these mice permanently accept fully allogeneic (H-2d) cardiac grafts and secondary donor skin grafts, and that splenocytes from these tolerant mice have reduced alloreactivity when restimulated in vitro. These results were compatible with either deletion or suppression of allospecific T cells as possible mechanisms of tolerance. The aim of this study was to investigate the mechanism of transplant tolerance in these mice. IκBαΔN-Tg mice did not have increased numbers or function of CD4+CD25+ regulatory T cells either before or after cardiac transplantation. In addition, tolerance could not be transferred to fresh NF-κB-competent T cells and was not permissive for linked suppression to skin grafts sharing donor and third-party alloantigens, suggesting that dominant suppression is not the mechanism by which IκBαΔN-Tg mice achieve tolerance. In contrast, overexpression of the antiapoptotic protein Bcl-xL in T cells from IκBαΔN-Tg mice resulted in effective rejection of cardiac allografts and correlated with an increased frequency of splenocytes producing IFN-γ in response to alloantigen. Together, these results suggest that the death of alloreactive T cells may be partly responsible for the transplantation tolerance observed in mice with defective T cell-intrinsic NF-κB activation.

Solid organ transplantation is often the only cure for end-stage organ failure. However, unless tissues are donated by identical twins, transplantation is limited by the occurrence of acute allograft rejection. Acute allograft rejection is mediated by T lymphocytes, and its prevention requires transplant recipients to take life-long immunosuppressive therapies, with the potential complications of infections and virus-induced tumors that are common with chronic immunosuppression. Therefore, the goal of transplantation research is to develop immunosuppressive regimens that inactivate only alloreactive T cells, leaving the rest of the immune system competent to react against pathogens and tumor Ags, a state termed donor-specific tolerance. Thus, understanding the biochemical pathways in T cells that are necessary to mount acute allograft rejection episodes is essential for the design of such novel therapies.

T cell activation follows engagement by the TCR of specific peptide/MHC complexes displayed on the surface of APCs, concurrently to the ligation of coreceptors (CD4 or CD8) and costimulatory receptors on T cells. An essential costimulatory receptor for naive T cells is CD28, which binds B7-1 (CD80) and B7-2 (CD86) on APCs (1). Engagement of TCR in the absence of CD28 ligation results in T cell death or T cell inactivation (2, 3). Biochemical signals transduced upon TCR/CD28 engagement include activation of the Src kinase Lck, phosphorylation of ITAM motifs in TCR ζ-chains, and recruitment and activation of the tyrosine kinase ZAP70 and subsequently of the scaffolding adaptors Lat and SLP76 that are essential for the formation of signalosomes localized to lipid rafts at the plasma membrane (4). These signalosomes promote activation of downstream transcription factors that in naive T cells include NFAT, AP-1, and NF of the B cell κ-chain (NF-κB). Although these events have been well established in vitro, it is not clear whether all these pathways take place after T cell activation in vivo or what steps are necessary for mounting a productive immune response in the context of transplant rejection. Several groups have begun investigating the role of transcription factors during acute allograft rejection. For instance, inhibition of NFAT using cell-permeable inhibitory peptides resulted in prolongation of islet allograft survival in mice (5).

Our laboratory has chosen to concentrate on the role of NF-κB activation in T cells during acute allograft rejection, because NF-κB had been implicated in cell survival, proliferation, and cytokine production in several model systems in vitro (6) and therefore was likely to play an important role in immune responses in vivo. The Rel/NF-κB family of proteins is composed of five members that each contain a Rel homology domain (RHD)5 important for protein and DNA binding. p50 and p52 bind DNA well, but have weak transactivating capacity, whereas RelA (p65), RelB, and c-Rel are poor DNA binders, but contain a DNA transactivating domain. Typical NF-κB molecules are composed of a heterodimeric combination of p50 or p52, bound with p65, RelB, or c-Rel. T cells contain all NF-κB family subunits. In resting cells, NF-κB dimers are sequestered in the cytoplasm by IκB family members. IκB molecules bind to and cover the nuclear localization signals on NF-κB heterodimers, thus preventing their translocation to the nucleus (6). Engagement of TCR and CD28 results in activation of IκB kinases (IKK), which phosphorylate IκB. The phosphorylated IκB is then ubiquitinated and subsequently degraded by the proteasome. This exposes the nuclear localization signal on the NF-κB dimer, which can move into the nucleus and activate gene transcription. Nuclear translocation of NF-κB subunits has been reported in cardiac allografts at the time of T cell infiltration and initiation of rejection (7).

Many immunosuppressive drugs commonly used to prevent or treat allograft rejection inhibit NF-κB as part of their mechanism of action, although they may also exert many other effects. Corticosteroids, for instance, induce de novo transcription of IκBα molecules and therefore inhibit further NF-κB activation (8, 9). Several nonsteroidal anti-inflammatory drugs reduce the phosphorylation of IκB by the IKK complex (10). Even cyclosporin A, a compound known for its inhibitory activity of NFAT cells, has been shown to reduce proteasomal degradation of phosphorylated IκBs (10). The immunosuppressive activity of these drugs is probably due at least in part to their NF-κB inhibitory effects.

Transplantation experiments have been performed on mice genetically deficient in single NF-κB subunits, but prolongation of graft survival in these mice has been slight, presumably due to redundancy and compensation by other subunits. Thus, p50- or p52-deficient mice effectively reject cardiac allografts (11, 12). The role of T cell-intrinsic NF-κB activation during allograft rejection has been reinvestigated using mice that have reduced NF-κB activity selectively in T cells. These mice express a super-repressor transgenic form of IκBα driven by the Lck proximal promoter and CD2 locus control region (13). This transgene bears a deletion of the region containing the serines that serve as targets for the IKK complex. Thus, this mutant IκBα cannot be phosphorylated by the IKK complex and therefore retains the NF-κB subunits to which it is bound in the cytoplasm. We and others have shown that the majority of IκΒαΔN-Tg mice permanently accept heart transplants (14, 15), but reject skin allografts (15). However, IκΒαΔN-Tg mice tolerant to cardiac allografts permanently accept secondary donor, but not third-party, skin allografts, indicative of donor-specific tolerance (15). Splenocytes from these mice had reduced reactivity against donor, but not third-party, Ags (15), suggesting that alloreactive T cells had been deleted or were hyporesponsive either because they had become anergic or because they were being suppressed by regulatory cells. Several transplantation models in which long-term graft survival has been achieved have identified either T cell apoptosis or T cell regulation as a major mechanism to attenuate rejection (16). In this study we have investigated whether graft acceptance in IκBαΔN-Tg mice is due to regulation or deletion of alloreactive T cells. Tolerance in this model was not transferable to fresh wild-type T cells. In contrast, overexpression in T cells of the antiapoptotic protein Bcl-xL resulted in cardiac allograft rejection by IκΒαΔN-Tg mice, suggesting that apoptosis, but not regulation, may be the main mechanistic pathway of tolerance in these mice.

IκΒαΔN-Tg mice (H-2b) have been previously described (13) and were a gift from M. Boothby (Vanderbilt University, Nashville, TE). Bcl-xL-Tg mice (H-2b) have been previously described (17) and were provided by C. Thompson (University of Pennsylvania, Philadelphia, PA). IκBαΔN-Tg and Bcl-xL-Tg mice were intercrossed to generate double-transgenic mice (Bcl-xL/IκBαΔN-Tg). C57BL/6 (B6; H-2b) and BALB/c (H-2d) were purchased from Frederick Cancer Research Facilities. B6/RAG1-deficient (RAG1-KO; H-2b) mice were bred in our animal facility. Animals were housed in ventilated racks in a specific pathogen-free animal facility. Experiments were performed in agreement with our Institutional animal care and use committee and according to the National Institutes of Health guidelines for animal use.

Abdominal heterotopic cardiac transplantation was performed using a technique adapted from that originally described by Corry et al. (18). Cardiac allografts were transplanted in the abdominal cavity by anastomosing the aorta and pulmonary artery of the graft end-to-side to the recipient’s aorta and vena cava, respectively. The day of rejection was defined as the last day of a detectable heartbeat in the graft. Graft rejection was verified in selected cases by necropsy and pathological examination of H&E-stained graft sections.

Cervical cardiac transplantation was performed using a technique adapted from Chen (19). The cardiac graft was placed under the skin of the front of the neck. The innominate artery of the donor heart was anastomosed end-to-end to the right common carotid artery of the recipient. The pulmonary artery of the donor heart was anastomosed end-to-end to the external jugular vein of the recipient.

Some B6 mice were transplanted with BALB/c hearts and treated with a combination of anti-CD40L (MR1, 1 mg i.p. on days 0, 7, and 14 posttransplantation; Ligocyte Pharmaceuticals) and donor-specific transfusion (DST; 5 × 106 BALB/c splenocytes i.v. on day 0).

Splenocytes were isolated and processed into single cell suspensions. Cells were stained with anti-CD4-FITC- and anti-CD25-PE-coupled Abs (BD Pharmingen) and analyzed by flow cytometry (FACScan I or II; BD Biosciences).

To assess the regulatory capacity of CD4+CD25+ T cells, splenocytes from unmanipulated B6 and IκBαΔN-Tg mice either unmanipulated or transplanted >50 days previously with a BALB/c heart were isolated and stained with dialyzed anti-CD4-FITC and anti-CD25-PE Abs. Cell populations were sorted using a MOFLO-HTS cell sorter (DakoCytomation) into CD4+CD25 (responding cells) or CD4+CD25+ (regulatory cells). Responding cells were resuspended in DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES, 2-ME (50 μM), and additional amino acids and seeded in 96-well, round-bottom plates (3 × 104 cells/well) in the presence of soluble anti-CD3 (10 μg/ml) and irradiated (2000 rad) syngeneic splenocytes (15 × 104 cells/well). Increasing numbers of CD4+CD25+ T cells were added to the wells. Supernatants were collected at 24 h, and the concentration of IL-2 was measured by ELISA using Ab pairs (BD Pharmingen). Parallel plates were pulsed with [3H]thymidine for the last 8 h of a 72-h culture (1 μCi/well).

Spleens were harvested from unmanipulated B6 mice or anti-CD40L- plus DST-treated, long-term BALB/c cardiac graft-accepting B6 mice or IκBαΔN-Tg mice that had accepted a BALB/c heart transplanted >50 days previously. Indicated numbers of splenocytes were resuspended in 150 μl of PBS and injected i.v. into the retro-orbital plexus of recipient mice. Recipient mice in some experiments were syngeneic RAG1-KO mice that had been transplanted 1 day previously with BALB/c cardiac allografts. In other experiments, recipient mice were anti-CD40L- plus DST-treated, long-term BALB/c cardiac graft-accepting B6 mice or IκBαΔN-Tg mice that had accepted a cervical BALB/c heart for >50 days. These recipients received a second abdominal BALB/c cardiac transplant 1 day before the adoptive transfer.

Splenocytes from untransplanted mice or from mice transplanted 25 days previously with a BALB/c heart (106/well) were restimulated in vitro with irradiated B6 or BALB/c splenocytes (4 × 105/well) and incubated for 24 h in a 5% CO2 incubator. The ELISPOT assay was conducted according to the instructions of the manufacturer (BD Biosciences), and the numbers of spots per well were calculated using the ImmunoSpot Analyzer (CTL Analyzers LLC).

CD4+CD25+ T cells are naturally occurring regulatory cells that develop in the thymus. It was possible that reduced NF-κB activation during thymic maturation promoted the development of Treg at the expense of effector T cells, so that the ratio of CD4+CD25+ to CD4+CD25 T cells be altered and favor tolerance induction. To test this hypothesis, we compared the number of CD4+CD25+ T cells in wild-type and IκBαΔN-Tg littermates. Consistent with the known reduction in the number of total CD4+ T cells in IκΒαΔN-Tg mice compared with wild-type mice, the total number of CD4+CD25+ was also reduced in the spleens of these mice (Table I). However, the percentage of CD4+CD25+ within the CD4+ population was similar to that in wild-type mice in the spleen of both unmanipulated (Table I) and tolerant (data not shown) IκΒαΔN-Tg mice. Similar results were obtained in lymph nodes of these animals (data not shown). This result indicates that reduced NF-κB activation in T cells does not favor the development of a greater proportion of CD4+CD25+ T cells either spontaneously or after alloantigenic encounter.

Table I.

IκBαΔN-Tg mice do not have an increased number of CD4+CD25+ cellsa

C57BL/6IκBαΔN-Tg
Number of CD4+CD25+ 2 ± 0.5 × 106 (10.6%) 1.1 ± 0.2 × 106 (11.3%) 
Number of CD4+CD25 16.6 ± 2.3 × 106 10.3 ± 2 × 106 
C57BL/6IκBαΔN-Tg
Number of CD4+CD25+ 2 ± 0.5 × 106 (10.6%) 1.1 ± 0.2 × 106 (11.3%) 
Number of CD4+CD25 16.6 ± 2.3 × 106 10.3 ± 2 × 106 
a

Splenocytes from unmanipulated B6 and IκBαΔN-Tg mice were stained with FITC-coupled anti-CD4 and PE-coupled anti-CD25 and analyzed by flow cytometry. The results represent the mean and SD of three control mice and five IκBαΔN-Tg mice from three independent experiments. Numbers in parentheses represent the percentage of CD4+CD25+ T cells of all CD4+ T cells.

Although the number of CD4+CD25+ T cells was not increased in IκΒαΔN-Tg mice when compared with wild-type mice, it was possible that IκΒαΔN-Tg CD4+CD25+ T cells had increased suppressor function on a per cell basis or that IκΒαΔN-Tg CD4+CD25 cells were more susceptible to suppression than wild-type T cells. To explore these possibilities, CD4+ cells from unmanipulated IκΒαΔN-Tg and control littermates mice were sorted into populations of CD4+CD25+ and CD4+CD25 cells, and IL-2 production or [3H]thymidine incorporation by CD4+CD25 cells was measured in the presence of anti-CD3 mAb and increasing numbers of CD4+CD25+ T cells. As shown in Fig. 1, 50% inhibition of the maximum level of IL-2 secreted by both B6 and IκΒαΔN-Tg conventional cells occurred at a ratio of 0.25 regulatory cells to 1 responding cell regardless of the type (wild-type or IκΒαΔN-Tg) of CD4+CD25+ or CD4+CD25 cell used. Similar results were observed when proliferation was assayed in anti-CD3-stimulated cultures (Fig. 2) as well as in cultures stimulated with irradiated allogeneic BALB/c splenocytes (data not shown). These results indicate that IκBαΔN-Tg Tregs have similar suppressor ability as wild-type CD4+CD25+ cells, and that IκBαΔN-Tg CD4+CD25 T cells have similar susceptibility to suppression as wild-type cells. Finally, similar results were found when T cells from tolerant, rather than unmanipulated, IκΒαΔN-Tg mice were used as the source of CD4+CD25+ and CD4+CD25 T cells (Figs. 3 and 4), suggesting that the state of tolerance does not correlate with a global increase in the total number or function of CD4+CD25+ IκBαΔN-Tg T cells. Taken together, these results indicate that reduced NF-κB activation in T cells does not result in increased intrinsic or induced suppressor function by CD4+CD25+ cells or increased susceptibility to suppression of CD4+CD25 T cells.

FIGURE 1.

T cells from naive IκBαΔN-Tg mice do not have increased capacity to suppress IL-2 or augmented susceptibility to suppression. CD4+CD25 and CD4+CD25 cells were sorted from the spleen of B6 or unmanipulated IκBαΔN-Tg mice. CD4+CD25 T cells were stimulated with soluble anti-CD3 and irradiated syngeneic B6 splenocytes in the presence of increasing numbers of CD4+CD25+ cells. Supernatants were collected at 24 h and analyzed for IL-2 content by ELISA. The plot represents the mean and SD of triplicate determinations. This result is representative of three independent experiments.

FIGURE 1.

T cells from naive IκBαΔN-Tg mice do not have increased capacity to suppress IL-2 or augmented susceptibility to suppression. CD4+CD25 and CD4+CD25 cells were sorted from the spleen of B6 or unmanipulated IκBαΔN-Tg mice. CD4+CD25 T cells were stimulated with soluble anti-CD3 and irradiated syngeneic B6 splenocytes in the presence of increasing numbers of CD4+CD25+ cells. Supernatants were collected at 24 h and analyzed for IL-2 content by ELISA. The plot represents the mean and SD of triplicate determinations. This result is representative of three independent experiments.

Close modal
FIGURE 2.

T cells from naive IκBαΔN-Tg mice do not have increased capacity to suppress proliferation. The experiment was performed as described in Fig. 1, but plates were incubated for 72 h and pulsed with [3H]thymidine for the last 8 h of culture.

FIGURE 2.

T cells from naive IκBαΔN-Tg mice do not have increased capacity to suppress proliferation. The experiment was performed as described in Fig. 1, but plates were incubated for 72 h and pulsed with [3H]thymidine for the last 8 h of culture.

Close modal
FIGURE 3.

T cells from tolerant IκBαΔN-Tg mice do not have increased capacity to suppress IL-2 or augmented susceptibility to suppression. CD4+CD25 and CD4+CD25 cells were sorted from the spleen of unmanipulated B6 or of IκBαΔN-Tg mice that had received a BALB/c heart >50 days previously. Cells were assayed as described in Fig. 1.

FIGURE 3.

T cells from tolerant IκBαΔN-Tg mice do not have increased capacity to suppress IL-2 or augmented susceptibility to suppression. CD4+CD25 and CD4+CD25 cells were sorted from the spleen of unmanipulated B6 or of IκBαΔN-Tg mice that had received a BALB/c heart >50 days previously. Cells were assayed as described in Fig. 1.

Close modal
FIGURE 4.

T cells from tolerant IκBαΔN-Tg mice do not have increased capacity to suppress proliferation. The experiment was performed as described in Fig. 3, but plates were incubated for 72 h and pulsed with [3H]thymidine for the last 8 h of the culture.

FIGURE 4.

T cells from tolerant IκBαΔN-Tg mice do not have increased capacity to suppress proliferation. The experiment was performed as described in Fig. 3, but plates were incubated for 72 h and pulsed with [3H]thymidine for the last 8 h of the culture.

Close modal

Although the proportion and function of CD4+CD25+ T cells were normal in IκBαΔN-Tg mice, it was possible that the tolerance observed in these mice was due to a different subset of spontaneously arising or induced regulatory cells. Thus, to assess whether these transplanted mice had developed regulatory cells capable of suppressing the function of conventional T cells, we investigated whether splenocytes from tolerant mice could suppress the rejection mediated by fresh wild-type splenocytes in an adoptive transfer model. As shown in Fig. 5, left panel, splenocytes from wild-type, but not from tolerant, IκBαΔN-Tg mice were capable of inducing rejection of BALB/c hearts when transferred into syngeneic RAG1-KO recipients. Notably, the rejection capacity of wild-type splenocytes was not suppressed by addition of splenocytes from tolerant mice at a 1:1 ratio, suggesting the lack of strong regulation in the spleens of tolerant IκBαΔN-Tg mice. As a positive control for these experiments, we used B6 mice transplanted with BALB/c hearts and immunosuppressed with a combination of anti-CD40L mAb and perioperative injection of donor splenocytes (anti-CD40L+DST). This treatment resulted in long-term cardiac allograft acceptance (data not shown). In contrast to the lack of suppression of fresh B6 splenocytes by splenocytes from tolerant IκBαΔN-Tg mice, splenocytes from anti-CD40L+DST mice effectively reduced the capacity of B6 splenocytes to reject a donor heart in RAG1-KO recipient mice (Fig. 5, right panel).

FIGURE 5.

Fresh, wild-type splenocytes are not suppressed by tolerant IκBαΔN-Tg splenocytes in RAG1-KO recipients. Splenocytes from unmanipulated B6 (n = 4, left panel; n = 3, right panel), from IκBαΔN-Tg mice that had accepted a BALB/c heart for >50 days (left panel; alone, n = 3; with B6 splenocytes at a 1:1 ratio, n = 6), or from B6 mice treated with anti-CD40L and DST that had accepted a BALB/c heart for >43 days (right panel; alone, n = 3; with B6 splenocytes at a 1:1 ratio, n = 7) were adoptively transferred into syngeneic RAG1-deficient mice 1 day after the transplantation of a BALB/c heart. Graft survival in the transferred mice was assessed over time.

FIGURE 5.

Fresh, wild-type splenocytes are not suppressed by tolerant IκBαΔN-Tg splenocytes in RAG1-KO recipients. Splenocytes from unmanipulated B6 (n = 4, left panel; n = 3, right panel), from IκBαΔN-Tg mice that had accepted a BALB/c heart for >50 days (left panel; alone, n = 3; with B6 splenocytes at a 1:1 ratio, n = 6), or from B6 mice treated with anti-CD40L and DST that had accepted a BALB/c heart for >43 days (right panel; alone, n = 3; with B6 splenocytes at a 1:1 ratio, n = 7) were adoptively transferred into syngeneic RAG1-deficient mice 1 day after the transplantation of a BALB/c heart. Graft survival in the transferred mice was assessed over time.

Close modal

It remained possible that the regulation, if any, was not contained in the spleen of tolerant mice. Therefore, to address whether tolerant IκBαΔN-Tg mice could suppress the function of wild-type cells, a different adoptive transfer model was designed. Unmanipulated IκBαΔN-Tg mice or IκBαΔN-Tg mice that had been transplanted with BALB/c hearts >50 days previously were transferred with wild-type splenocytes at the time of transplantation of a second fresh BALB/c heart. The second heart was transplanted to avoid concerns that putative lack of rejection of the original heart may be due to graft adaptation over time. A dose of 80 × 106 splenocytes was chosen, because this was the minimum number of cells that consistently promoted cardiac allograft rejection when transferred into naive, freshly transplanted IκBαΔN-Tg mice. As shown in Table II, wild-type splenocytes effectively promoted rejection of fresh BALB/c hearts whether transferred into naive or tolerant IκBαΔN-Tg mice, indicating that regulation, if it exists in these mice, is not strong enough to suppress wild-type splenocytes. In contrast to the fresh second set of heart transplants that was rejected, some first-set heart grafts were retained after the adoptive transfer of fresh splenocytes, suggesting that attrition of passenger leukocytes or graft adaptation may have indeed occurred in those long-term accepted transplants. In contrast, B6 mice having accepted an initial BALB/c heart after anti-CD40L+DST treatment retained a second fresh donor heart transplant after transfer of fresh B6 splenocytes, indicating that in some models of long-term graft acceptance, dominant suppression does develop.

Table II.

Fresh wild-type splenocytes are not suppressed in tolerant IκBαΔN-Tg micea

Donors of Splenocytes (80 × 106)RecipientsBALB/c Cardiac Allograft Survival (Days)
None IκBαΔN-Tg >50 × 3 
B6 Naive IκBαΔN-Tg First graft: 14, 21, 33 
B6 Tolerant IκBαΔN-Tg Second graft: 23, 37, 42 
B6 Naive B6 First graft: 13, 13, 16 
B6 Long-term graft-accepting B6 (anti-CD40L + DST) Second graft: >50 × 6 
Donors of Splenocytes (80 × 106)RecipientsBALB/c Cardiac Allograft Survival (Days)
None IκBαΔN-Tg >50 × 3 
B6 Naive IκBαΔN-Tg First graft: 14, 21, 33 
B6 Tolerant IκBαΔN-Tg Second graft: 23, 37, 42 
B6 Naive B6 First graft: 13, 13, 16 
B6 Long-term graft-accepting B6 (anti-CD40L + DST) Second graft: >50 × 6 
a

Splenocytes (80 × 106) from unmanipulated B6 mice were transferred into naive IκBαΔN-Tg or B6 mice, or into tolerant IκBαΔN-Tg mice or anti-CD40L + DST-treated B6 mice that had accepted a BALB/c heart for > 50 days. Recipients had also received a first or second BALB/c heart, as indicated, 1 day prior to the adoptive transfer of B6 splenocytes and survival of this fresh transplant was assessed over time.

One hallmark of dominant tolerance is that mice tolerant to Ag A will accept skin grafts from A×B mice, which coexpress the initial Ag to which tolerance was induced and a new Ag to which the recipient mice are naive (20). To test whether tolerant IκBαΔN-Tg mice can develop this linked suppression, IκBαΔN-Tg mice that had accepted a BALB/c heart for >50 days were transplanted with BALB/c × A/J F1 skin grafts. All transplanted animals rapidly rejected these skin grafts, whereas they retained their BALB/c cardiac allografts (data not shown). Taken together with the adoptive transfer experiments, these results suggest that dominant suppression is not the major mechanism of tolerance in IκBαΔN-Tg mice.

Aside from regulation, another major mechanism of allograft tolerance that has been uncovered in certain transplantation models is that of deletion of allospecific T cells (21). Because NF-κB activation results in the transcription of several genes involved in cell survival, including A1, A20, inhibitor of apoptosis, and Bcl-xL, it was possible that NF-κB-deficient mice would achieve transplantation tolerance because allospecific T cells would die upon antigenic stimulation secondary to the absence of prosurvival proteins up-regulation. Overexpression of Bcl-xL in T cells has been shown to protect T cells from apoptosis induced by TCR stimulation (22). Therefore, we crossed Bcl-xL-Tg (H-2b) with IκBαΔN-Tg (H-2b) mice and transplanted the resulting single- and double-transgenic animals with BALB/c hearts. T cells from double-transgenic mice have also been shown to have reduced susceptibility to apoptosis (23). As shown in Fig. 6 A, although the majority of IκBαΔN-Tg littermates accepted BALB/c hearts long-term, all Bcl-xL/IκBαΔN-Tg mice rejected cardiac allografts, albeit with delayed kinetics compared with Bcl-xL-Tg control littermates.

FIGURE 6.

Expression of a Bcl-xL transgene in T cells prevents tolerance induction in IκBαΔN-Tg mice. BALB/c hearts were transplanted into littermates of the different strains. A, Graft survival was assessed over time (wild-type, n = 7; Bcl-xL-Tg, n = 3; IκBαΔN-Tg, n = 8; IκBαΔN/Bcl-xL-Tg littermates, n = 9). B, Splenocytes were stimulated with irradiated B6 or BALB/c splenocytes on day 25 posttransplant, and the precursor frequency of IFN-γ-producing cells was determined using an ELISPOT assay. The plot represents the means and SDs of at least triplicate determinations. ∗∗∗, p < 0.001, as determined by Student’s t test.

FIGURE 6.

Expression of a Bcl-xL transgene in T cells prevents tolerance induction in IκBαΔN-Tg mice. BALB/c hearts were transplanted into littermates of the different strains. A, Graft survival was assessed over time (wild-type, n = 7; Bcl-xL-Tg, n = 3; IκBαΔN-Tg, n = 8; IκBαΔN/Bcl-xL-Tg littermates, n = 9). B, Splenocytes were stimulated with irradiated B6 or BALB/c splenocytes on day 25 posttransplant, and the precursor frequency of IFN-γ-producing cells was determined using an ELISPOT assay. The plot represents the means and SDs of at least triplicate determinations. ∗∗∗, p < 0.001, as determined by Student’s t test.

Close modal

To address whether expression of the Bcl-xL transgene promoted survival of allospecific T cells, an IFN-γ-specific ELISPOT assay was performed using splenocytes from mice transplanted with a BALB/c heart 25 days previously. IκBαΔN-Tg mice that had accepted BALB/c hearts had few IFN-γ-producing cells. In addition to promoting rejection, expression of the Bcl-xL transgene in IκBαΔN-Tg mice resulted in markedly enhanced numbers of IFN-γ-producing cells, almost as high as in NF-κB-sufficient mice that had also rejected their grafts (Fig. 6 B). This was not due to an increased number of T cells in wells assaying the responsiveness of Bcl-x/IκBαΔN-Tg splenocytes, because the percentage of T cells was similar in all IκBαΔN-Tg and Bcl-x/IκBαΔN-Tg spleens, as determined by flow cytometry (data not shown). This result implies that BALB/c-specific cells are alive and functional in Bcl-x/IκBαΔN-Tg mice. Together, these data indicate that overexpression of Bcl-xL in T cells results in rejection of cardiac allografts by mice with impaired T cell-intrinsic NF-κB activation and suggest that T cell deletion plays an important role in the tolerance observed in IκBαΔN-Tg mice.

We have previously reported that mice with reduced T cell-intrinsic NF-κB activation permanently accept primary fully allogeneic heart transplants and secondary donor, but not third-party, skin grafts (15), indicating robust donor-specific tolerance. These results identified NF-κB in T cells as a possible target for immune modulation in the clinic, but also called for vigorous investigation of the mechanisms by which this tolerance was achieved to evaluate possible clinical applicability. In this study we demonstrate that T cell regulation is not the major mechanism leading to tolerance in this model. Rather, transgenic expression of Bcl-xL in T cells restores cardiac allograft rejection in T cell-intrinsic, NF-κB-impaired mice.

Several immunosuppressive regimens have been associated with long-term cardiac allograft acceptance in mice. These include treatment with Abs or fusion proteins thought to block engagement of costimulatory receptors such as CD28/CD80/CD86 (24) or CD40/CD154 (25), administration of nondepleting anti-CD4 and anti-CD8 Abs (26), treatment with gallium nitrate (27), or combination therapies that include donor-specific transfusions (28, 29). The mechanisms by which these treatments induce graft acceptance can be divided into two broad nonexclusive categories: T cell depletion and regulation of T cell responses. For instance, treatment with CTLA-4-Ig or anti-CD154 mAb has been reported to promote T cell depletion (22, 30), but administration of anti-CD154 mAb has also been associated with the development of regulation in a skin graft model (31, 32). Nondepleting anti-CD4 and anti-CD8 mAbs have been shown to promote dominant suppressive tolerance in skin graft models (33), whereas transfusion of donor cells has been reported to promote either regulation (34) or deletion (29) of alloreactive T cells in cardiac or skin transplant models.

Reduced NF-κB activation in T cells also results in long-term cardiac allograft acceptance (14, 15). However, we were unable to uncover the existence of a regulatory mechanism in IκBαΔN-Tg mice. It was clear that IκBαΔN-Tg mice did not have an increased percentage or number of native CD4+CD25+ T cells. In fact, NF-κB activation has recently been reported to be necessary for the thymic development of regulatory cells, because mice with either conditional deletion of IKK subunits in T cells or p50/cRel double-deficient mice fail to develop CD4+CD25+ T cells (35, 36). It would appear that the residual level of NF-κB activation in T cells from IκBαΔN-Tg mice is sufficient for the development of innate regulatory T cells in these mice. Although rejection of cardiac allografts is mainly dependent on the presence of CD4+ and not CD8+ T cells (37, 38, 39, 40, 41), immunohistochemistry of graft tissue at the time of rejection usually reveals a majority of CD8+ T cells, suggesting that this subset is an active, albeit dispensable, participant in the effector phase of the rejection process (42). CD4+CD25+ T cells can suppress the function of CD8+ T cells (43, 44, 45). IκBαΔN-Tg mice have markedly reduced numbers of CD8+ T cells (13). Thus, although the proportion of CD4+CD25+ T cells relative to the number of CD4+ T cells appears normal in IκBαΔN-Tg mice, the ratio of Treg to CD8+ T cells is indeed increased. Therefore, although the suppressor capacity of IκBαΔN-Tg CD4+CD25+ cells is not increased on a per cell basis, we cannot exclude the possibility that these mice have a greater capacity to regulate their CD8 effector T cell responses in vivo.

Several groups have now shown that CD4+CD25+ T cells that suppress allogeneic responses in vivo can arise from CD4+CD25 T cells after transplantation (46, 47). However, we did not find increased numbers of CD4+CD25+ T cells in tolerant IκBαΔN-Tg mice (data not shown). Aside from CD4+CD25+ cells, other subsets of T cells, such as NKT cells, some subpopulations of CD8+ T cells, and induced T regulatory 1 or Th3 cells, can also suppress T cell responses in some settings (48). Thus, it was possible that reduced NF-κB activation in T cells would affect the development of other regulatory T cell subsets in IκBαΔN-Tg mice or prompt the development of induced regulatory T cells upon encounter of T cells with allogeneic Ags in vivo. Although it was unlikely that regulation would be supported by NKT cells in this model, because NF-κB activation in T cells is necessary for the thymic development of this cell type (49, 50), other cell subsets could have been involved. However, we could not find evidence for dominant regulation in tolerant IκBαΔN-Tg mice. The lack of strong regulation was concluded because of the concordant results of three lines of experiments: fresh wild-type splenocytes were not suppressed by splenocytes from tolerant IκBαΔN-Tg mice in RAG1-deficient mice; fresh wild-type splenocytes were not suppressed in tolerant IκBαΔN-Tg mice transplanted with fresh second donor hearts; tolerant IκBαΔN-Tg mice accepted donor skin, but rejected F1 skin grafts that expressed donor Ags. We cannot exclude that tolerant IκBαΔN-Tg mice have developed a low level of regulation that we are unable to detect with our assays. For instance, although a 1:1 ratio of tolerant to wild-type fresh splenocytes did not prevent wild-type splenocyte-mediated cardiac allograft rejection in RAG1-KO mice, it is possible that a greater ratio would be successful. Nevertheless, we conclude that if regulation exists in this model, it is not dominant, not easily transferable, and of lower magnitude than that induced in B6 mice by anti-CD40L + DST treatment.

In contrast with the lack of evidence for dominant suppression, it was clear that Bcl-xL/IκBαΔN-Tg mice were capable of rejecting cardiac allografts. Although NF-κB promotes the transcription of multiple antiapoptotic genes, including Bcl-xL, overexpression of just Bcl-xL has been shown to be sufficient to reduce the death of IκBαΔN-Tg T cells (23). However, it is also possible that transgenic expression of Bcl-xL in T cells modifies aspects of T cell biology other than survival, and that cardiac allograft rejection in Bcl-xL/IκBαΔN-Tg mice is not due to prevention of death of IκBαΔN-Tg T cells, but, rather, to increased T cell effector function, for instance. Similar to our model, overexpression of Bcl-xL in T cells has previously been reported to prevent tolerance induction in mice treated with a combination of donor-specific transfusion with CTLA-4-Ig or anti-CD154 (22). These results support the idea that reducing T cell death can prevent transplantation tolerance in models in which apoptosis is the major mechanism of tolerance, although in neither case can it be excluded that Bcl-xL is operating through alternative mechanisms than prevention of apoptosis. Future experiments using TCR transgenic/IκBαΔN-Tg mice to be able to follow the fate of alloreactive T cells after transplantation will help determine whether the effect of Bcl-xL depends on prevention of T cell death.

The importance of the precursor frequency of graft-specific T cells in the success or failure to reject a transplant is becoming increasingly apparent (51, 52). Because regulation of alloresponses can coexist with a certain level of deletion of alloreactive T cells, it has been suggested that it may be possible to tip the balance of these two mechanisms in favor of transplantation tolerance (53). In fact, in the absence of regulation or constant depletion of new emerging T cells, T cell deletion may impede transplantation tolerance as new developing T cells may undergo homeostatic expansion to reconstitute the T cell repertoire and thus acquire memory characteristics that confer resistance to certain regimens of tolerance induction, such as costimulatory blockade (54). It is possible that IκBαΔN-Tg mice remain tolerant indefinitely without histological evidence of chronic graft rejection (14) because of the continuous deletion of new emerging alloreactive T cells as they encounter alloantigen in the presence of blunted NF-κB activation.

The pathway that leads to NF-κB activation in T cells downstream of TCR/CD28 engagement is unique. It involves recruitment and activation of protein kinase Cθ, the adaptors CARMA1, Bcl10, and MALT1 and the recruitment of a ubiquitinating complex that includes TRAF6 and results in activation of TAK1, followed by activation of the IKK complex (55). Protein kinase Cθ is mostly restricted to T cells, whereas CARMA1, Bcl10, and MALT1 appear lymphocyte-restricted. Therefore, it may be possible to develop small molecule inhibitors that are cell-permeable and inhibit the binding or activation of these lymphocyte-restricted proteins. These therapies should only inhibit TCR- or BCR-mediated NF-κB activation and may reproduce in normal mice or patients the susceptibility to tolerance observed in IκBαΔN-Tg mice. However, whether transient deletion of alloreactive T cells will be sufficient to induce and maintain tolerance and whether these therapies will be effective to induce deletion of pre-existing cross-reactive memory cells remain to be established.

The authors have no financial conflict of interest.

We thank James Marvin and Ryan Duggan for expert help with cell sorting.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by American Heart Association Grant 0350261N, Juvenile Diabetes Research Foundation Grant 1-2003-188, and National Institutes of Health Grant RO1AI052352-01.

5

Abbreviations used in this paper: RHD, Rel homology domain; DST, donor-specific transfusion; IKK, IκB kinase; RAG1-KO, RAG1-deficient; Treg, regulatory T cell.

1
Lenschow, D. J., T. L. Walunas, J. A. Bluestone.
1996
. CD28/B7 system of T cell costimulation.
Annu. Rev. Immunol.
14
:
233
.
2
Jenkins, M. K., R. H. Schwartz.
1987
. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo.
J. Exp. Med.
165
:
302
.
3
Schwartz, R. H..
1990
. A cell culture model for T lymphocyte clonal anergy.
Science
248
:
1349
.
4
Wange, R. L., Y. Huang.
2004
. T cell receptor signaling: beyond complex complexes.
J. Biol. Chem.
14
:
14
.
5
Noguchi, H., M. Matsushita, T. Okitsu, A. Moriwaki, K. Tomizawa, S. Kang, S. T. Li, N. Kobayashi, S. Matsumoto, K. Tanaka, et al
2004
. A new cell-permeable peptide allows successful allogeneic islet transplantation in mice.
Nat. Med.
10
:
305
.
6
Karin, M., A. Lin.
2002
. NF-κB at the crossroads of life and death.
Nat. Immunol.
3
:
221
.
7
Ruland, J., T. W. Mak.
2003
. From antigen to activation: specific signal transduction pathways linking antigen receptors to NF-κB.
Semin. Immunol.
15
:
177
.
8
Auphan, N., J. A. DiDonato, C. Rosette, A. Helmberg, M. Karin.
1995
. Immunosuppression by glucocorticoids: inhibition of NF-κB activity through induction of IκB synthesis.
Science
270
:
286
.
9
Marx, J..
1995
. How the glucocorticoids suppress immunity.
Science
270
:
232
.
10
Karin, M., Y. Yamamoto, Q. M. Wang.
2004
. The IKK NF-κB system: a treasure trove for drug development.
Nat. Rev. Drug Discov.
3
:
17
.
11
Smiley, S. T., V. Csizmadia, W. Gao, L. A. Turka, W. W. Hancock.
2000
. Differential effects of cyclosporine A, methylprednisolone, mycophenolate, and rapamycin on CD154 induction and requirement for NFκB: implications for tolerance induction.
Transplantation
70
:
415
.
12
Csizmadia, V., W. Gao, S. A. Hancock, J. B. Rottman, Z. Wu, L. A. Turka, U. Siebenlist, W. W. Hancock.
2001
. Differential NF-κB and IκB gene expression during development of cardiac allograft rejection versus CD154 monoclonal antibody-induced tolerance.
Transplantation
71
:
835
.
13
Boothby, M. R., A. L. Mora, D. C. Scherer, J. A. Brockman, D. W. Ballard.
1997
. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-κB.
J. Exp. Med.
185
:
1897
.
14
Finn, P. W., J. R. Stone, M. R. Boothby, D. L. Perkins.
2001
. Inhibition of NF-κB-dependent T cell activation abrogates acute allograft rejection.
J. Immunol.
167
:
5994
.
15
Zhou, P., K. W. Hwang, D. A. Palucki, Z. Guo, M. Boothby, K. A. Newell, M. L. Alegre.
2003
. Impaired NF-κB activation in T cells permits tolerance to primary heart allografts and to secondary donor skin grafts.
Am. J. Transplant.
3
:
139
.
16
Lechler, R. I., O. A. Garden, L. A. Turka.
2003
. The complementary roles of deletion and regulation in transplantation tolerance.
Nat. Rev. Immunol.
3
:
147
.
17
Chao, D. T., G. P. Linette, L. H. Boise, L. S. White, C. B. Thompson, S. J. Korsmeyer.
1995
. Bcl-xL and Bcl-2 repress a common pathway of cell death.
J. Exp. Med.
182
:
821
.
18
Corry, R. J., H. J. Winn, P. S. Russell.
1973
. Primarily vascularized allografts of hearts in mice: the role of H-2D, H-2K, and non-H-2 antigens in rejection.
Transplantation
16
:
343
.
19
Chen, Z. H..
1991
. A technique of cervical heterotopic heart transplantation in mice.
Transplantation
52
:
1099
.
20
Graca, L., A. Le Moine, S. P. Cobbold, H. Waldmann.
2003
. Antibody-induced transplantation tolerance: the role of dominant regulation.
Immunol. Res.
28
:
181
.
21
Chiffoleau, E., P. T. Walsh, L. Turka.
2003
. Apoptosis and transplantation tolerance.
Immunol. Rev.
193
:
124
.
22
Wells, A. D., X. C. Li, Y. Li, M. C. Walsh, X. X. Zheng, Z. Wu, G. Nunez, A. Tang, M. Sayegh, W. W. Hancock, et al
1999
. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance.
Nat. Med.
5
:
1303
.
23
Mora, A. L., R. A. Corn, A. K. Stanic, S. Goenka, M. Aronica, S. Stanley, D. W. Ballard, S. Joyce, M. Boothby.
2003
. Antiapoptotic function of NF-κB in T lymphocytes is influenced by their differentiation status: roles of Fas, c-FLIP, and Bcl-xL.
Cell Death Differ.
10
:
1032
.
24
Pearson, T. C., D. Z. Alexander, K. J. Winn, P. S. Linsley, R. P. Lowry, C. P. Larsen.
1994
. Transplantation tolerance induced by CTLA4-Ig.
Transplantation
57
:
1701
.
25
Larsen, C. P., D. Z. Alexander, D. Hollenbaugh, E. T. Elwood, S. C. Ritchie, A. Aruffo, R. Hendrix, T. C. Pearson.
1996
. CD40-gp39 interactions play a critical role during allograft rejection: suppression of allograft rejection by blockade of the CD40-gp39 pathway.
Transplantation
61
:
4
.
26
Chen, Z., S. Cobbold, S. Metcalfe, H. Waldmann.
1992
. Tolerance in the mouse to major histocompatibility complex-mismatched heart allografts, and to rat heart xenografts, using monoclonal antibodies to CD4 and CD8.
Eur. J. Immunol.
22
:
805
.
27
Orosz, C. G., E. Wakely, S. D. Bergese, A. M. VanBuskirk, R. M. Ferguson, D. Mullet, G. Apseloff, N. Gerber.
1996
. Prevention of murine cardiac allograft rejection with gallium nitrate: comparison with anti-CD4 monoclonal antibody.
Transplantation
61
:
783
.
28
Bushell, A., P. J. Morris, K. J. Wood.
1994
. Induction of operational tolerance by random blood transfusion combined with anti-CD4 antibody therapy: a protocol with significant clinical potential.
Transplantation
58
:
133
.
29
Adams, A. B., M. M. Durham, L. Kean, N. Shirasugi, J. Ha, M. A. Williams, P. A. Rees, M. C. Cheung, S. Mittelstaedt, A. W. Bingaman, et al
2001
. Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression.
J. Immunol.
167
:
1103
.
30
Monk, N. J., R. E. Hargreaves, J. E. Marsh, C. A. Farrar, S. H. Sacks, M. Millrain, E. Simpson, J. Dyson, S. Jurcevic.
2003
. Fc-dependent depletion of activated T cells occurs through CD40L-specific antibody rather than costimulation blockade.
Nat. Med.
9
:
1275
.
31
Honey, K., S. P. Cobbold, H. Waldmann.
1999
. CD40 ligand blockade induces CD4+ T cell tolerance and linked suppression.
J. Immunol.
163
:
4805
.
32
Graca, L., K. Honey, E. Adams, S. P. Cobbold, H. Waldmann.
2000
. Cutting edge: anti-CD154 therapeutic antibodies induce infectious transplantation tolerance.
J. Immunol.
165
:
4783
.
33
Qin, S., S. P. Cobbold, H. Pope, J. Elliott, D. Kioussis, J. Davies, H. Waldmann.
1993
. ‘Infectious’ transplantation tolerance.
Science
259
:
974
.
34
Bushell, A., M. Karim, C. I. Kingsley, K. J. Wood.
2003
. Pretransplant blood transfusion without additional immunotherapy generates CD25+CD4+ regulatory T cells: a potential explanation for the blood-transfusion effect.
Transplantation
76
:
449
.
35
Schmidt-Supprian, M., G. Courtois, J. Tian, A. J. Coyle, A. Israel, K. Rajewsky, M. Pasparakis.
2003
. Mature T cells depend on signaling through the IKK complex.
Immunity
19
:
377
.
36
Zheng, Y., M. Vig, J. Lyons, L. Van Parijs, A. A. Beg.
2003
. Combined deficiency of p50 and cRel in CD4+ T cells reveals an essential requirement for nuclear factor κB in regulating mature T cell survival and in vivo function.
J. Exp. Med.
197
:
861
.
37
Campos, L., A. Naji, B. C. Deli, J. H. Kern, J. I. Kim, C. F. Barker, J. F. Markmann.
1995
. Survival of MHC-deficient mouse heterotopic cardiac allografts.
Transplantation
59
:
187
.
38
Krieger, N. R., D. P. Yin, C. G. Fathman.
1996
. CD4+ but not CD8+ cells are essential for allorejection.
J. Exp. Med.
184
:
2013
.
39
Han, W. R., L. J. Murray-Segal, P. L. Mottram.
1999
. Assessment of peripheral tolerance in anti-CD4 treated C57BL/6 mouse heart transplants recipients.
Transplant. Immunol.
7
:
37
.
40
Mottram, P. L., A. Raisanen-Sokolowski, T. Glysing-Jensen, A. N. Stein-Oakley, M. E. Russell.
1998
. Redefining peripheral tolerance in the BALB/c to CBA mouse cardiac allograft model: vascular and cytokine analysis after transient CD4 T cell depletion.
Transplantation
66
:
1510
.
41
He, G., O. S. Kim, J. R. Thistlethwaite, J. Hart, C. T. Siegel, G. L. Szot, K. A. Newell.
1999
. Differential effect of an anti-CD8 monoclonal antibody on rejection of murine intestine and cardiac allografts.
Transplant. Proc.
31
:
1239
.
42
Szot, G. L., P. Zhou, I. Rulifson, J. Wang, Z. Guo, O. Kim, K. A. Newel, J. R. Thistlethwaite, J. A. Bluestone, M. L. Alegre.
2001
. Different mechanisms of cardiac allograft rejection in wildtype and CD28-deficient mice.
Am. J. Transplant.
1
:
38
.
43
Piccirillo, C. A., E. M. Shevach.
2001
. Cutting edge: control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells.
J. Immunol.
167
:
1137
.
44
Green, E. A., L. Gorelik, C. M. McGregor, E. H. Tran, R. A. Flavell.
2003
. CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells through TGF-β-TGF-β receptor interactions in type 1 diabetes.
Proc. Natl. Acad. Sci. USA
100
:
10878
.
45
Dai, Z., Q. Li, Y. Wang, G. Gao, L. S. Diggs, G. Tellides, F. G. Lakkis.
2004
. CD4+CD25+ regulatory T cells suppress allograft rejection mediated by memory CD8+ T cells via a CD30-dependent mechanism.
J. Clin. Invest.
113
:
310
.
46
Karim, M., C. I. Kingsley, A. R. Bushell, B. S. Sawitzki, K. J. Wood.
2004
. Alloantigen-induced CD25+CD4+ regulatory T cells can develop in vivo from CD25-CD4+ precursors in a thymus-independent process.
J. Immunol.
172
:
923
.
47
Cobbold, S. P., R. Castejon, E. Adams, D. Zelenika, L. Graca, S. Humm, H. Waldmann.
2004
. Induction of foxP3+ regulatory T cells in the periphery of T cell receptor transgenic mice tolerized to transplants.
J. Immunol.
172
:
6003
.
48
Francois Bach, J..
2003
. Regulatory T cells under scrutiny.
Nat. Rev. Immunol.
3
:
189
.
49
Sivakumar, V., K. J. Hammond, N. Howells, K. Pfeffer, F. Weih.
2003
. Differential requirement for Rel/nuclear factor κB family members in natural killer T cell development.
J. Exp. Med.
197
:
1613
.
50
Stanic, A. K., J. S. Bezbradica, J. J. Park, N. Matsuki, A. L. Mora, L. Van Kaer, M. R. Boothby, S. Joyce.
2004
. NF-κB controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes.
J. Immunol.
172
:
2265
.
51
Li, Y., X. C. Li, X. X. Zheng, A. D. Wells, L. A. Turka, T. B. Strom.
1999
. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance.
Nat. Med.
5
:
1298
.
52
He, C., S. Schenk, Q. Zhang, A. Valujskikh, J. Bayer, R. L. Fairchild, P. S. Heeger.
2004
. Effects of T cell frequency and graft size on transplant outcome in mice.
J. Immunol.
172
:
240
.
53
Zheng, X. X., A. Sanchez-Fueyo, M. Sho, C. Domenig, M. H. Sayegh, T. B. Strom.
2003
. Favorably tipping the balance between cytopathic and regulatory T cells to create transplantation tolerance.
Immunity
19
:
503
.
54
Wu, Z., S. J. Bensinger, J. Zhang, C. Chen, X. Yuan, X. Huang, J. F. Markmann, A. Kassaee, B. R. Rosengard, W. W. Hancock, et al
2004
. Homeostatic proliferation is a barrier to transplantation tolerance.
Nat. Med.
10
:
87
.
55
Sun, L., L. Deng, C. K. Ea, Z. P. Xia, Z. J. Chen.
2004
. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes.
Mol. Cell
14
:
289
.