A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy

T-bet plays a crucial role in Th1 development. We investigated the role of T-bet in the development of allograft rejection in an established MHC class II–mismatched (bm12 into B6) model of chronic allograft vasculopathy (CAV). Intriguingly, and in contrast to IFN-γ−/− mice that are protected from CAV, T-bet−/− recipients develop markedly accelerated allograft rejection accompanied by early severe vascular inflammation and vasculopathy, and infiltration by predominantly IL-17–producing CD4 T cells. Concurrently, T-bet−/− mice exhibit a T helper type 1 (Th1)–deficient environment characterized by profound IFN-γ deficiency, a Th2 switch characterized by increased production of interleukin (IL) 4, IL-5, IL-10, and IL-13 cytokines, as well as increased production of the proinflammatory cytokines IL-6, IL-12p40, and IL-17. Neutralization of IL-17 inhibits accelerated allograft rejection and vasculopathy in T-bet−/− mice. Interestingly, CD4 but not CD8 T cell deficiency in T-bet−/− mice affords dramatic protection from vasculopathy and facilitates long-term graft acceptance. This is the first study establishing that in the absence of Th1-mediated alloimmune responses, CD4 Th17 cells mediate an aggressive proinflammatory response culminating in severe accelerated allograft rejection and vasculopathy. These results have important implications for the development of novel therapies to target this intractable problem in clinical solid organ transplantation.

Until recently, primed CD4 Th cells were considered to segregate into two distinct populations, each producing its own set of cytokines and mediating separate eff ector functions ( 1 ). Th1 cells produce IFN-␥ , and mediate activation of macrophages and induction of delayedtype hypersensitivity responses. Th2 cells produce IL-4, IL-5, IL-10, and IL-13, which provide help for B cell function and are important in IgG switching, IgE production, and eosinophilic infl ammation ( 2 ).
T-bet is a Th1-specifi c T-box transcription factor that plays a crucial role in Th1 development, in part via the induction of IFN-␥ . T-bet not only induces Th1 development but also actively suppresses Th2 diff erentiation, and more remarkably, introduction of T-bet into fully Th2-diff erentiated CD4 T cells triggers conversion to a Th1 phenotype. In the absence of T-bet, CD4 T cells fail to diff erentiate into the Th1 lineage and default to a Th2 fate. Mice with homozygous deletion of T-bet show normal lymphoid development but fail to generate functional Th1 responses, have impaired regulation of antibody isotype class switching, and are protected from several T cell -mediated autoimmune diseases but show evidence of heightened allergic (Th2) responses ( 3 ).
It has been proposed that a Th1 phenotype promotes allograft rejection via IFN-␥ -induced activation of macrophage function and up-regulation of MHC class II antigen expression that favors T cell allosensitization. In contrast, the Th2 phenotype has been proposed to favor longterm graft survival in some models ( 4 ). Thus, in groups; Fig. 1 A ). Further, grafts harvested from T-bet Ϫ / Ϫ recipients 2 wk after transplantation were notable for histological signs of severe vascular infl ammation and vasculopathy, characterized by perivascular and intraluminal leukocytic infi ltration, that were entirely absent in the case of IFN-␥ Ϫ / Ϫ recipients ( Fig. 1 B ). Interestingly, grafts from the T-bet Ϫ / Ϫ recipients were heavily infi ltrated with PMNs (including eosinophils and neutrophils) compared with a predominantly mononuclear infi ltration in the case of the WT recipients ( Fig. 1 C and Table I ). Because the PMN-dominant infi ltrate is also consistent with antibody-mediated rejection, we performed bm12 heart transplants in T-bet/Ig double KO (DKO) recipients that lacked both T-bet and mature B cells. Interestingly, T-bet/Ig DKO recipients also rejected the bm12 heart grafts with the same tempo as T-bet Ϫ / Ϫ recipients (MST of 15 d in the T-bet Ϫ / Ϫ group vs. 14 d in the T-bet/Ig DKO group; n = 6 in each group; P = 0.8676; Fig.  1 A ), and the pathology, including PMN infi ltration, of the grafts harvested from T-bet/Ig DKO mice was very similar to those harvested from T-bet Ϫ / Ϫ recipients, indicating no role of antibodies or B cells in this model (not depicted). 8 wk after transplantation, grafts from the IFN-␥ Ϫ / Ϫ recipients demonstrated an intermediate pattern of infi ltration, with more PMNs and fewer mononuclear cells compared with WT recipients. As previously reported ( 11 ), we also noted that IFN-␥ Ϫ / Ϫ mice demonstrated no evidence of allograft vasculopathy up to 8 wk after engraftment, in contrast to WT recipients in which occlusive CAV was noted in 50% of the vessels examined ( Fig. 1 B ).
These observations were at fi rst somewhat surprising in that T-bet Ϫ / Ϫ mice rejected the grafts despite exhibiting a Th1defi cient environment, characterized by profound IFN-␥ defi ciency and increased Th2 cytokines, whereas many Th1dominated diseases are repressed in T-bet defi ciency ( 13 -15 ). Further, STAT4 Ϫ / Ϫ mice, which like T-bet Ϫ / Ϫ mice exhibit a Th1-defi cient environment, are in contrast protected from CAV ( 12 ). However, paradoxical results have been obtained involving the STAT1 -T-bet -STAT4 -IFN-␥ axis in various models of auto-and alloimmunity. For example, STAT1 Ϫ / Ϫ and IFN-␥ Ϫ / Ϫ mice are susceptible to EAE, whereas T-bet Ϫ / Ϫ and STAT4 Ϫ / Ϫ mice are resistant ( 15,18 ). Some of these inconsistencies are now beginning to be unraveled by the appreciation of a novel, IL-17 -secreting Th cell lineage termed Th17 as a mediator of tissue infl ammation. In view of this and the peculiar pattern of graft-infi ltrating leukocytes consisting of predominantly PMNs, we examined the grafts showing severe vascular infl ammation for the presence of IL-17 -producing cells. 2 wk after transplantation, when the grafts from T-bet Ϫ / Ϫ recipients had undergone rejection, there was infi ltration of PMNs along with CD4 and CD8 T cells with severe vascular infl ammation and vasculopathy that were entirely absent in IFN-␥ Ϫ / Ϫ recipients. Besides intense PMN infi ltration observed by light microscopy ( Fig. 1 C ), immuno fl uorescence staining of the grafts indicated that both CD4 and CD8 T cells were present, but the majority of the graft-infi ltrating lymphocytes in T-bet Ϫ / Ϫ recipients were the transplant setting, it was widely believed that allograft rejection is predominantly a Th1-mediated immune response, although this conclusion was challenged by data in cytokinedefi cient mice that indicated that rejection could proceed in the absence of IL-2 or IFN-␥ . Mice defi cient in the hallmark Th1 cytokine IFN-␥ rapidly reject fully mismatched cardiac allografts, as do mice lacking STAT4, a transcription factor associated with the development of Th1 responses ( 5 -7 ). Th2 cytokines, on the other hand, have also been noted in grafts undergoing acute vascular rejection ( 8,9 ). As a result, the validity of the Th1/Th2 paradigm in transplantation has been questioned ( 10 ). Nevertheless, in MHC class II -mismatched cardiac allograft models, transplanted hearts are protected from chronic allograft vasculopathy (CAV) in both STAT4-and IFN-␥ -defi cient mice, suggesting a key role for IFN-␥ and/or Th1 CD4 T cells in the development of subacute immunemediated vascular injury and CAV ( 11,12 ).
We hypothesized that in the absence of T-bet, a Th2predominant environment in combination with impaired CD8 eff ector function would result in inhibition of destructive alloimmune responses and could promote transplantation tolerance and prevent the development of CAV. This hypothesis is supported by studies in Th1-mediated autoimmune disease models in which T-bet defi ciency confers protection ( 13 -15 ), as well as earlier studies in chronic rejection models in which STAT4-and IFN-␥ -defi cient mice are protected from CAV ( 11,12 ). However, in contrast to our a priori hypothesis, we now report for the fi rst time the role of T-bet in alloimmunity, and show that T-bet defi ciency contributes to the development of aggressive CD4 Th17 cell -mediated infl ammatory alloimmune responses that ultimately culminate in accelerated rejection and vasculopathy in an established model of chronic rejection.

T-bet defi ciency accelerates cardiac allograft rejection in a model of CAV
T-bet expression appears to be critical for the development of a variety of infl ammatory autoimmune diseases classically considered related to Th1 immunity, such as experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis, Crohn ' s disease, and type 1 diabetes ( 13 -15 ). Allograft rejection is also considered a predominantly Th1 immune response. Moreover, IFN-␥ is a key regulatory cytokine associated with chronic allograft rejection and is thought to play a critical role in the pathogenesis of CAV ( 11 ). Therefore, we investigated the role of T-bet in alloimmune responses and the development of CAV in an established MHC class IImismatched model (bm12 into C57BL/6) of CAV, where vascularized cardiac allografts survive long term without immunosuppression but develop CAV ( 16,17 ). Unexpectedly, T-bet Ϫ / Ϫ mice ( n = 6) rejected their grafts with a median survival time (MST) of 15 d compared with WT ( n = 8) and IFN-␥ Ϫ / Ϫ ( n = 6) mice in which 75% (two out of eight grafts were rejected at days 39 and 41) and 100% of grafts survived for > 60 d, respectively (P < 0.002 compared with both IL-17 -producing CD4 T cells ( Fig. 2 ). There were hardly any and no IL-17 -producing cells in grafts from WT and IFN-␥ Ϫ / Ϫ recipients, respectively. Further, there were no NK cells and all of the T cells expressed the ␣ ␤ TCR, indicating that there were no ␥ ␦ T cells infi ltrating the grafts from T-bet Ϫ / Ϫ recipients (not depicted). Collectively these results suggest that T-bet negatively regulates the development of Th17 cells that mediate the vascular infl ammation and acute rejection in a model of CAV.

T-bet defi ciency promotes CD4 and CD8 T cell effector memory differentiation and proliferation during an alloimmune response
To investigate whether T-bet defi ciency, in addition to promoting Th17 cells, had any eff ect on CD4 and CD8 eff ector memory T cell (T EM ) populations and proliferation in response to alloantigens, we fi rst evaluated the frequency of T cells bearing the T EM phenotype (CD44 hi CD62L low ) in the peripheral blood of bm12 graft recipients 2 wk after transplantation. We found that the activation of CD4 T cells in Tbet Ϫ / Ϫ and IFN-␥ Ϫ / Ϫ recipients (11.8 ± 0.2% and 13.3 ± 3.4%, respectively) was slightly but signifi cantly higher than in WT recipients (9 ± 0.2%; P < 0.05). Further, the percentage of CD8 T cells with T EM phenotype in T-bet Ϫ / Ϫ recipients (32.3 ± 2.4%) was markedly higher than in IFN-␥ Ϫ / Ϫ and WT recipients (9.56 ± 3.1% and 4.9 ± 1.5%, respectively; P < 0.001), with the majority of the CD8 T cells in T-bet Ϫ / Ϫ recipients expressing low levels of CD62L ( Fig. 3 A ). These fi ndings are consistent with recent reports of increased CD8central/memory T cell generation in T-bet Ϫ / Ϫ mice ( 19 ).
Up-regulation of Th2 and Th17 cytokine production by T-bet ؊ / ؊ recipients T-bet controls Th cell polarization toward a Th1 phenotype ( 3 ). In EAE studies, clinical improvement correlated with reduced T-bet expression, reduced IFN-␥ production, and a reciprocal increase in the Th2 transcription factor GATA3 in splenocytes ( 15 ). Recently, it has been noted that both IFN-␥ and T-bet repress the diff erentiation of Th17 cells ( 18,22 ). Further, T-bet expression has been reported to be a critical determinant of Th1 versus Th17 cell fate ( 23 ). Moreover, Tbet appears to negatively regulate certain autoimmune diseases by suppressing IL-17 production ( 21,24 ). As shown below, we noted that the majority of the graft-infi ltrating lympho- Five high-power fi elds were counted per slide, with one slide each from fi ve to six heart grafts per group. Results are presented as mean numbers of cells ± SD. the CD4 cell population and are consistent with the observation that CD4 but not CD8 T cell defi ciency or depletion in T-bet Ϫ / Ϫ recipients aff ords dramatic protection from vasculopathy and facilitates long-term acceptance of heart grafts ( Fig. 5 C ). Collectively, these data indicate that T-bet in CD4 cells plays a critical role in determining the fate and type of the alloimmune response.
Anti -IL-17 antibody inhibits accelerated cardiac allograft rejection in a model of CAV in T-bet ؊ / ؊ recipients From the foregoing, it is reasonable to consider that IL-17producing Th17 cells play a critical role in mediating alloimmune responses. Further, blocking IL-17 with anti -IL-17 antibody resulted in markedly reduced skin infl ammation in a model of systemic autoimmunity ( 21 ). We therefore administered anti -IL-17 mAb or control Ig to T-bet Ϫ / Ϫ recipients of bm12 heart grafts and observed for graft survival to directly determine whether IL-17 neutralization can inhibit accelerated cardiac allograft rejection in this model of CAV. Neutralization of IL-17 with anti -IL-17 mAb indeed resulted in signifi cant prolongation of allograft survival in T-bet Ϫ / Ϫ recipients compared with the group that received control IgG (MST of 38 vs. 15 d, respectively; P < 0.01; Fig. 6 A ). The protection from acute rejection, although signifi cant, was not complete and may have been caused by the relatively brief duration of anti -IL-17 mAb administration. We then, as above, examined alloantigen-specifi c production of Th1, Th2, indicating that the diff erences in levels of IL-12p40 could be caused by diff erences in IL-23 (a heterodimer of IL-12p40 and IL-23p19) production ( 25 ), consistent with previous reports of increased IL-17 and IL-23 production in autoimmune disease models in T-bet Ϫ / Ϫ mice ( 21,24 ). However, on direct measurement of IL-23 by ELISA we noted that IL-23 production by splenocytes harvested from bm12 heart allograft recipients was generally low and not diff erent between Tbet Ϫ / Ϫ and IFN-␥ Ϫ / Ϫ recipients (2.67 ± 2.31, 5.47 ± 7.49, and 5.7 ± 7.35 pg/ml in WT, IFN-␥ Ϫ / Ϫ , and T-bet Ϫ / Ϫ recipients, respectively; P = NS between all groups). Collectively, in the presence of increased IL-6 and in the absence of Th1-polarizing conditions and the loss of direct negative regulatory eff ect of T-bet on Th17 cells in the T-bet Ϫ / Ϫ recipients, a positive feedback loop for the generation of Th17 cells is established, ultimately resulting in a heavily skewed immune response toward the Th17 phenotype.

Proinfl ammatory cytokine production and accelerated cardiac allograft rejection in a model of CAV in T-bet ؊ / ؊ mice is dependent on CD4 T cells
Both CD4 and CD8 lymphocytes contribute to CAV development via secretion of IFN-␥ , a known mediator of CAV in this model ( 26 ). Recently, it has been suggested that T-betexpressing CD8 T cells mediate suppression of experimental autoimmune myocarditis ( 24 ). To evaluate the relative importance of T-bet defi ciency in CD4 or CD8 T cell compartments in mediating the accelerated rejection, we injected CD4 or CD8 depleting mAb into T-bet Ϫ / Ϫ recipients of bm12 hearts. CD8 depletion resulted in a slight prolongation of MST from 15 to 21 d (P < 0.001; Fig. 5 A ). In contrast, CD4 depletion resulted in a dramatic prolongation of allograft survival ( > 60 d) in T-bet Ϫ / Ϫ recipients ( Fig. 5 A ). In addition, we transplanted bm12 hearts into CD4/T-bet DKO and CD8/Tbet DKO recipients. CD4/T-bet DKO recipients, similar to the CD4-depleted T-bet Ϫ / Ϫ recipients above, also accepted the grafts for > 60 d, whereas CD8/T-bet DKO mice rejected the heart grafts with an MST of 19 d ( Fig. 5 A ). Further, pathological examination of the grafts from CD4-depleted Tbet Ϫ / Ϫ and CD4/T-bet DKO recipients revealed only mild vasculopathy in 20 and 12.5% of the vessels, respectively, 8 wk after transplantation, whereas grafts from CD8-depleted Tbet Ϫ / Ϫ or CD8/T-bet DKO recipients showed severe vasculopathy 3 wk after transplantation ( Fig. 5 B ). These data are in keeping with the observation that the majority of the IL-17producing graft-infi ltrating lymphocytes in T-bet Ϫ / Ϫ recipients were CD4 T cells ( Fig. 2 ) and indicate that T-bet expressed in the CD4 rather than CD8 T cell compartment prevents accelerated rejection in this model of CAV. Further, splenocytes from T-bet/CD4 DKO recipients compared with those from T-bet Ϫ / Ϫ and T-bet/CD8 DKO recipients produced signifi cantly less IL-6 (12.9 ± 3.8 vs. 940.7 ± 176.3 and 1,240.5 ± 215.6 pg/ml, respectively; P < 0.01) and IL-17 (7.8 ± 4.6 vs. 210.2 ± 73.4 and 113.1 ± 61.2 pg/ml, respectively; P < 0.01) cytokines. These data indicate that both of these cytokines are predominantly produced by IL-17 cytokine production in T-bet Ϫ / Ϫ recipients and infi ltration of vascular lesions with IL-17 -producing CD4 T cells, we conclude that CD4 Th17 cells are the main cells responsible for mediating the accelerated rejection and CAV in Tbet Ϫ / Ϫ recipients.

DISCUSSION
Since the original description of Th lymphocyte subsets (Th1 and Th2) and their divergent eff ector functions, each has been linked with the development of specifi c diseases or immune responses, including transplant rejection or tolerance ( 28,29 ). It has been postulated that Th1 cells are critically involved in the development of allograft rejection, whereas Th2 cells are involved in promoting transplantation tolerance ( 29 ). T-bet is believed to be crucial for the development of a variety of infl ammatory diseases by virtue of being the master regulator of Th1 diff erentiation ( 30 ). T-bet Ϫ / Ϫ mice are resistant to EAE ( 13 ), and silencing T-bet by small interfering RNA specifi c for T-bet at the time of EAE induction reduced the incidence and severity of disease, indicating that T-bet is critical for the diff erentiation of autoreactive T cells. Further, silencing T-bet after disease onset signifi cantly improves the clinical course of established EAE ( 15 ). Intriguingly, we found that T-bet defi ciency in cardiac allograft recipients, on the contrary, accelerates rejection in a model of CAV despite exhibiting a Th1-defi cient environment characterized by a profound deficiency of IFN-␥ and increased Th2 cytokines. This was particularly surprising because IFN-␥ is thought to play a critical role in the pathogenesis of CAV, and either STAT4 or IFN-␥ defi ciency confers protection from CAV in this model ( 11,12 ), raising questions about the notion that IFN-␥ is the key regulatory cytokine associated with chronic allograft rejection. However, intriguing and paradoxical results have been obtained involving the STAT1 -T-bet -STAT4 -IFN-␥ axis in various models of auto-and alloimmunity. For example, STAT1 Ϫ / Ϫ mice and IFN-␥ Ϫ / Ϫ mice are susceptible to EAE, whereas T-bet Ϫ / Ϫ and STAT4 Ϫ / Ϫ mice are resistant ( 15,18 ). IFN-␥ has been implicated in the acute rejection process in and proinfl ammatory Th17 cytokines in the anti -IL-17 mAb or control Ig -treated T-bet Ϫ / Ϫ recipients of bm12 cardiac allografts 14 or 40 d after transplantation. Consistent with our earlier observations, splenocytes from T-bet Ϫ / Ϫ recipients at each time point produced very little Th1 cytokine IFN-␥ ( Fig. 6 B ). Further, at days 14 and 40, splenocytes from T-bet Ϫ / Ϫ recipients treated with anti -IL-17 mAb in comparison to those from control Ig -treated T-bet Ϫ / Ϫ recipients at day 14 produced signifi cantly less of the proinfl ammatory cytokines IL-17 (13.6 ± 0.43 and 72.3 ± 81.12 vs. 337 ± 111.85 pg/ml, respectively; P < 0.01) and IL-12p40 (3.42 ± and 1.12 ± 0.43 vs. 18,433 ± 152.75 pg/ml, respectively; P < 0.001). Notably again, IL-12p70 (representing IL-12, a heterodimer of IL-12p35 and IL-12p40) levels were not signifi cantly diff erent between the three groups (unpublished data). Interestingly, splenocytes from T-bet Ϫ / Ϫ recipients treated with anti -IL-17 mAb in comparison to those from control Ig -treated T-bet Ϫ / Ϫ recipients produced signifi cantly higher amounts of the Th2 cytokines IL-4 (174.83 ± 22.12 and 200.8 ± vs. 22.2 ± 33.5 pg/ml, respectively; P < 0.01) and IL-5 (2,448.33 ± 675.79 and 641 ± 36.11 vs. 204.7 ± 19 pg/ml, respectively; P < 0.001). IL-10 levels were not signifi cantly diff erent between the three groups; however, IL-13 production was initially increased at day 14 and then diminished at day 40 in anti -IL-17 -treated groups ( Fig. 6 B ). IL-6 is upstream of IL-17 in the Th17 diff erentiation pathway ( 27 ); consequently, IL-6 production is markedly elevated in anti -IL-17 -treated groups (1,405.2 ± 203.44 and 726 ± 116.9 vs. 262 ± 12.12 pg/ml, respectively; P < 0.001). Pathological examination of heart allografts from T-bet Ϫ / Ϫ recipients treated with anti -IL-17 mAb at day 14 revealed minimal vascular infl ammation and no changes of CAV ( Fig. 6 C and Table II ). However, examination at day 40, when the anti -IL-17 -treated T-bet Ϫ / Ϫ recipients had rejected the heart allografts, revealed considerable leukocytic infi ltration in the parenchyma but minimal CAV ( Fig. 6 C and Table II ). These data are consistent with previous reports of reduced skin infl ammation with IL-17 neutralization in a model of systemic autoimmunity ( 21 ). Collectively, with the increased allograft dysfunction independent of IL-12p70 or IL-23 ( 38 ). Collectively, in the presence of increased IL-6 and in the absence of Th1-polarizing conditions and the loss of a direct negative regulatory eff ect of T-bet on Th17 cells in the T-bet Ϫ / Ϫ recipients, a positive feedback loop for the generation of Th17 cells is established, ultimately resulting in a heavily skewed immune response toward the Th17 phenotype. Interestingly, IL-17 neutralization in T-bet Ϫ / Ϫ recipients inhibited accelerated cardiac allograft rejection and was associated with markedly reduced IL-17 and IL-12p40 production ( Fig. 6,  A and B ). However, production of IL-6 and Th2 cytokines, including IL-13, was actually increased with IL-17 neutralization, indicating that IL-17 is downstream of IL-6 and that IL-17 is critical for mediating cardiac allograft rejection and vasculopathy. In an antigen-induced allergic airway infl ammation recipients of allografts, whereas IFN-␥ defi ciency has also been reported to result in severe vascular infl ammation leading to acute rejection and to interfere with tolerance induction ( 31 ). The diff erences seen in Th17 response after targeting T-bet with small interfering RNA in the EAE model ( 15 ) versus the CAV model in our studies are likely related to the diff erences in the model, mode of priming, precursor T cell frequency, strength of TCR stimulation, and partial versus complete T-bet defi ciency. Interestingly, recent studies have shown that T-bet acts as a negative regulator of Th17-mediated immune responses in two diff erent models of autoimmunity ( 21,24 ). In a model of experimental autoimmune myocarditis, heightened disease severity in T-bet Ϫ / Ϫ mice was suggested to be caused by increased CD4 T cell -mediated IL-17 production in the heart ( 24 ). Lohr et al., in another model of systemic autoimmune disease, reported worsening of disease in T-bet Ϫ / Ϫ mice, suggesting a reciprocal relationship between Th1 responses and IL-17 production indicating that IL-17 is an important cytokine in tissue infl ammation ( 21 ). Our observation of accelerated rejection in T-bet Ϫ / Ϫ recipients associated with severe vascular infl ammation characterized by PMNs and IL-17 -producing T cell infi ltration are in keeping with these previous reports of T-bet as a negative regulator of Th17-mediated immune responses in vivo. These results suggest that T-bet negatively regulates the development of Th17 cells that mediate the vascular infl ammation and acute rejection in a model of CAV.
Interestingly, the accelerated allograft rejection and vasculopathy are associated with increased production of the proinfl ammatory cytokines IL-6, IL-17, and IL-12p40. These observations are consistent with previous reports of increased IL-17 production in autoimmune disease models in T-bet Ϫ / Ϫ mice ( 21,24 ). T-bet decreases antigen receptor -induced IL-17 production ( 23 ). IL-23, on the other hand, promotes IL-17 expression and the proliferation of IL-17 -producing cells from a pool of memory T cells, thus playing an important role in maintaining Th17 eff ector function ( 32,33 ). It has been proposed that in an infl ammatory milieu, IL-6 and TGF-␤ together induce Th17 cells and up-regulate IL-23R, thereby allowing IL-23 to strengthen and sustain the Th17 phenotype ( 34 ). However, in this model of CAV, although IL-12p40 levels were elevated, IL-23 production in vitro by splenocytes from allograft recipients was minimal, indicating that IL-23 -independent mechanisms may be responsible for the maintenance of the Th17 response. IL-12p40 is believed to have opposing immunomodulatory eff ects in diff erent experimental conditions; for example, IL-12p40 specifi cally inhibited the eff ects of IL-12 and IL-23 heterodimers ( 35,36 ), whereas IL-12p40 homodimers exacerbated cardiac allograft rejection by enhancing alloantigen-specifi c Th2 development ( 37 ). However the latter study predated the discovery of Th17 cells, and the aggressive alloimmune responses were attributed to skewing of the immune response toward a Th2 phenotype and Th17 responses were not examined. Further, in lung transplantation, enhanced epithelial production of IL-12p40 homodimers was associated with progressive chronic immune response, appears to be an attractive strategy for the inhibition of Th17 diff erentiation.
IL-17 is a potent proinfl ammatory cytokine that induces chemokine expression and leukocyte infi ltration and mediates tissue infl ammation ( 18 ). IL-17 induces granulopoiesis via G-CSF production and regulates neutrophil recruitment via CXC chemokine induction by epithelial cells ( 43 ). Homozygous deletion of IL-17RA abrogates the increase in splenic neutrophil progenitors induced by overexpression of IL-17A ( 43 ). Further, in a model of acute trinitrobenzenesulfonic acid -induced colitis, mice defi cient in IL-17RA expression were signifi cantly protected from trinitrobenzenesulfonic acid -induced weight loss and colonic infl ammation and substantially reduced PMN immigration into the colon ( 44,45 ). Neutralization of IL-17A homodimers strongly inhibits neutrophilic airway infl ammation mediated by adoptive transfer of ovalbumin-specifi c polarized Th17 cells and airway challenge with antigen ( 46 ). In keeping with these, we noted intense PMN infi ltration within the lesions of vascular infl ammation in grafts from T-bet Ϫ / Ϫ recipients; interestingly, however, the PMN infi ltration consisted of both neutrophils and eosinophils, perhaps as a result of markedly increased Th2 cytokines along with a Th17 immune response. Eosinophilic infi ltration is well described in cardiac allografts associated with a Th2 immune response ( 47 ). Further, neutralization of IL-17 in T-bet Ϫ / Ϫ recipients markedly reduced the vascular infl ammation, particularly neutrophil infi ltration, suggesting a link between IL-17 and neutrophil recruitment to the graft ( Fig. 6 C and Table II ).
Despite the predominantly PMN infi ltration, CD4 cells appear to be critical for the development of CAV, because CD4 depletion or defi ciency in T-bet Ϫ / Ϫ recipients aff ords complete protection from CAV and long-term allograft survival ( Fig. 5, A and B ). Further, IL-6 and IL-17 production is markedly diminished in CD4/T-bet DKO recipients ( Fig. 5 C ), indicating the CD4 cells are critical for the initiation of the aggressive Th17 alloimmune response. Moreover, there is an increase in proliferation of T cells and an expansion of CD4 and CD8 T EM cells in T-bet Ϫ / Ϫ recipients ( Fig. 3 A ). These fi ndings are consistent with recent reports of increased CD8central/memory T cell generation in T-bet Ϫ / Ϫ mice ( 19 ). Further, we examined whether this net increase in T cells of the T EM phenotype is caused by a defi ciency in Foxp3 + T reg cells, particularly because a reciprocal relationship between the development of pathogenic Th17 cells and Foxp3 + T reg cells orchestrated by IL-6 and TGF-␤ has been recently demonstrated ( 20 ). In addition, IL-6, together with IL-1 and STAT3, induces genetic reprogramming of Foxp3 + T reg cells ( 48 ). However, we did not fi nd a decrease in Foxp3 + T reg cells in T-bet Ϫ / Ϫ recipients ( Fig. 3 B ). These observations are consistent with a previous report that there was no defect in numbers of T reg cells in the absence of IFN-␥ or T-bet ( 21 ). Whether the function of CD4 + CD25 + Foxp3 + T reg cells in T-bet Ϫ / Ϫ mice is also unaltered remains to be investigated. Further, the infl ammatory milieu within the grafts in T-bet Ϫ / Ϫ recipients may promote the conversion of T reg model, T-bet inhibited diff erentiation of both Th17 and IL-13 -producing Th2 cells ( 39 ). In addition, IL-13 appears to down-regulate Th17 diff erentiation in models of pulmonary and CNS infl ammation ( 40,41 ). Therefore, targeting IL-13 may not be benefi cial in the prevention of Th17 immunity. On the other hand, IL-6 blockade inhibited the development of EAE, indicating that IL-6 targeting prevents diff erentiation of Th17 cells but does not inhibit committed Th17 cells ( 42 ). Therefore, IL-6 blockade, particularly early in the allo- seemingly inconsistent fi ndings in models of Th1-mediated autoimmune infl ammation such as EAE and collagen-induced arthritis in IFN-␥ Ϫ / Ϫ or STAT1 Ϫ / Ϫ mice ( 13 ). Th17 immunity has been implicated in various models of immune-mediated tissue injury, particularly in the absence of a Th1 environment ( 21,53,54 ). Our studies have extended this novel paradigm to the transplant setting, enabling us to explain transplant rejection by infl ammatory processes mediated by Th17 cells in the absence of Th1-dominant immune responses. Indeed, IL-17 has been implicated in both experimental and human renal and lung allograft rejection ( 51, 55 -57 ). Further, IL-17 promotes secretion of infl ammatory cytokines by stromal cells and macrophages and promotes DC maturation. IL-17R: Fc administration promoted cardiac allograft survival by inhibiting maturation of DC progenitors ( 58 ), and local expression of soluble IL-17R by gene therapy prolonged cardiac allograft survival ( 59 ). From the data presented in this paper, it is clear that CD4 Th17 cells are the major mediators of vascular infl ammation, thereby setting the stage for the progression of these lesions to CAV. We have shown that T-bet negatively regulates this vascular infl ammation and suppresses the development of CAV. Further, neutralization of IL-17 prevents vascular infl ammation and suppresses CAV. In conclusion, IL-17 -producing CD4 Th17 cells are important in allograft rejection and may serve as a novel target of strategies for the prevention of transplant rejection and graft vasculopathy.

MATERIALS AND METHODS
Animals. WT C57BL/6, B6.C-H2 bm12 (bm12), IFN-␥ Ϫ / Ϫ , CD4 Ϫ / Ϫ , Ig Ϫ / Ϫ (B-less), and T-bet Ϫ / Ϫ mice, all on a C57BL/6 background, were purchased from the Jackson Laboratory. Mice defi cient in both T-bet and CD4, CD8, or B cells were generated by crossbreeding. Genotyping was used to confi rm homozygous deletion of the T-bet and CD4, CD8, or Ig genes. Experiments were approved by the Institutional Animal Care and Use Committee at the Harvard Medical School Center for Animal Resources and Comparative Medicine.
Heart transplantation and antibody treatment. In the MHC class II mismatch model, bm12 mice were used as donors, and C57BL/6 WT, IFN- all on C57BL/6 background, were used as recipients. Vascularized heart grafts were transplanted using a microsurgical technique as previously described ( 16 ). Depleting anti-CD4 (GK1.5) and -CD8 (2.43; both from Bio-Express) were administered i.p. at a dose of 0.1 mg on days Ϫ 6, Ϫ 3, and Ϫ 1. Neutralizing IL-17 mAb (MAB421; R & D Systems) was administered i.p. at a dose of 100 μ g daily on days 0 -3, followed by every other day until day 13 after transplantation. Graft function was assessed by daily palpation of the abdomen. Rejection was defi ned as complete cessation of cardiac contractility as determined by direct visualization. cells to Th17 phenotype in an IL-6 -dependent manner, as has been recently demonstrated in the context of priming with activated DCs ( 49 ). Whether this is the case in our model needs further investigation. CD4 Th17 cells have been implicated in many autoimmune diseases ( 50 ), and collagen type V -specifi c Th17 cells contribute to obliterative bronchiolitis in human lung transplants ( 51 ). Therefore, it is conceivable that the cardiac allograft rejection in T-bet Ϫ / Ϫ recipients is mediated by Th17 cells reacting against self-antigens exposed in the ischemically injured heart allografts and needs further investigation. However, we believe that this is unlikely because the strong Th17 recall response of the splenocytes from T-bet Ϫ / Ϫ recipients is in response to alloantigen, and in general, the frequency of alloreactive T cells is signifi cantly higher than that of autoreactive T cells.
In another model of vascular rejection, however, IL-17 antagonism attenuated acute rather than chronic rejection ( 52 ). It has been reported that an initial vascular infl ammation, characterized by perivascular and intraluminal lymphocytic infi ltration, precedes development of the classical fi broproliferative arteriosclerotic lesions of CAV ( 11 ). This initial vascular infl ammation likely represents a counterpart of acute rejection in clinical transplantation and is a harbinger of pathological changes of impending CAV or clinical chronic rejection. In our experiments with T-bet Ϫ / Ϫ recipients, this early vascular infl ammation is so severe that acute rejection ensues, precluding assessment of the impact of T-bet deficiency on CAV. However, in WT recipients, vascular infl ammation of the grafts becomes apparent by 2 wk after transplantation and progresses to typical lesions of CAV by 8 wk. Indeed, IL-17 neutralization in our model of CAV in T-bet Ϫ / Ϫ recipients resulted in a marked decrease in initial vascular infl ammation ( Fig. 6 C and Table II ) and, consequently, prolongation of graft survival ( Fig. 6 A ). Interestingly, IL-17 neutralization converts the phenotype (in relation to CAV) of the T-bet Ϫ / Ϫ recipients to that of IFN-␥ Ϫ / Ϫ recipients, and the grafts undergo delayed parenchymal rejection with minimal changes of CAV ( Fig. 6 C ), similar to a previous report in IFN-␥ Ϫ / Ϫ recipients in this model ( 11 ). This suggests that early vascular infl ammation is mediated by CD4 Th17 cells and contributes to the eventual development of CAV, and that even a brief antagonism of CD4 Th17 cells by IL-17 neutralization aff ords protection from CAV.
In summary, recent elucidation of a novel lineage of Thelper Th17 cells ( 18,22,25 ) has shed light on some of the Histology. Grafts harvested at 2 or 8 wk or at the time of rejection were sectioned transversely, fi xed in 10% buff ered formalin, and stained by hematoxylin and eosin (H & E), and elastin stains and the severity of parenchymal rejection and CAV were scored as previously described ( 16 ). For the phenotype of graft-infi ltrating leukocytes, fi ve high-power fi elds were counted per slide, with one slide each from fi ve to six heart grafts per group. Results are presented as mean numbers of cells ± SD. The remaining portions of the graft were used for frozen sections for immunofl uorescence staining using goat anti -mouse IL-17 (R & D Systems) and rat anti -mouse CD4 and CD8 (both from BioExpress) as primary antibodies. Secondary detection was performed using Cy2-conjugated donkey anti -rat IgG and Cy3-conjugated donkey anti -goat IgG (Jackson ImmunoResearch Laboratories). Images were captured using a confocal laser scanning microscope (C1 Plus; Nikon).
Cytokine analysis by Luminex and ELISA assay. For cytokine analysis, splenocytes harvested at 14 d after transplantation from recipients of bm12 heart allografts were restimulated by irradiated donor spleen cells. The cellfree supernatants of individual wells were removed after 48 h and analyzed by a multiplexed cytokine bead -based immunoassay using a preconfi gured 21-plex mouse cytokine detection kit (Millipore) according to the manufacturer ' s instructions. In brief, the assay is based on conventional sandwich assay technology. The antibody specifi c to each cytokine was covalently coupled to Luminex microspheres uniquely labeled with a fl uorescent dye. The 25-μ l microspheres were incubated overnight at 4 ° C with standards, controls, and samples (50 μ l) in a 96-well microtiter fi lter plate. The plate was washed to remove excess reagent after incubation, and a detection antibody (25 μ l biotin; Beadlyte Mouse 21-plex Multi-Cytokine kit; Millipore) was added in the form of a mixture containing each of the antibodies. After 30 min of incubation at room temperature, streptavidin-phycoerythrin was added for an additional 30 min. After a fi nal wash step, the beads were resuspended in buff er and analyzed on the Luminex 100 instrument to determine the concentration of the cytokine of interest. IL-23 was measured by a specifi c ELISA kit from eBioscience according to the manufacturer ' s instructions. All samples were tested in triplicate wells.
Adoptive transfer model. 40 million CFSE-labeled lymphocytes from WT, T-bet Ϫ / Ϫ , or IFN-␥ Ϫ / Ϫ mice were injected i.v. into lethally irradiated (1,000 rads) bm12 mice in 0.25 ml of sterile PBS. Recipients were killed 3 d later, and spleen and lymph nodes were harvested. The proliferation of CFSE-labeled donor cells in the host spleen and lymph nodes was examined by fl ow cytometry based on the dilution profi le of the CFSE dye. Apoptosis was evaluated by Annexin V and 7-amino-actinomycin D staining.

Statistics.
For the graft survival, Kaplan-Meier graphs were constructed and log-rank comparison of the groups was used to calculate p-values. For the cytokine levels by Luminex, data are presented as means ± SD, and comparisons between the values were performed using the two-tailed Student ' s t test. For all statistical analyses, the level of signifi cance was set at a probability of P < 0.05. All experiments were repeated at least three to fi ve times.