The Notch signaling pathway has been recently shown to contribute to T cell differentiation in vitro. However, the in vivo function of Notch signaling in transplantation remains unknown. In this study, we investigated the importance of Delta1 in regulating the alloimmune response in vivo. Delta1 expression was upregulated on dendritic cells and monocytes/macrophages upon transplantation in a BALB/c into B6 vascularized cardiac transplant model. Whereas administration of anti-Delta1 mAb only slightly delayed survival of cardiac allografts in this fully MHC-mismatched model, it significantly prolonged graft survival in combination with single-dose CTLA4-Ig or in CD28 knockout recipients. The prolongation of allograft survival was associated with Th2 polarization and a decrease in Th1 and granzyme B-producing cytotoxic T cells. The survival benefit of Delta1 blockade was abrogated after IL-4 neutralization and in STAT6KO recipients, but was maintained in STAT4KO recipients, reinforcing the key role of Th2 cell development in its graft-prolonging effects. To our knowledge, these data demonstrate for the first time an important role of Delta1 in alloimmunity, identifying Delta1 ligand as a potential novel target for immunomodulation in transplantation.
The adaptive immune system has permitted the development of specific defense mechanisms against a large variety of pathogens and abnormal cells. In transplantation, both CD4+ and CD8+ T cells play a dominant role in cell-mediated immunity against alloantigens and have been shown to be of key importance in allograft rejection (1–3). After Ag recognition, naive CD4+ T cells can differentiate into Th1, Th2, Th17, or regulatory T cells, whereas naive CD8+ T cells can become CTLs. The type of differentiation is determined by signals delivered by APCs and the cytokine microenvironment (4). Accumulating evidence indicates that Notch and its ligands on APCs might be important mediators of T cell differentiation (5–11).
The Notch pathway is an important intercellular signaling pathway that plays a major role in controlling cell fate (5, 7). The Notch family consists of four highly conserved receptors (Notch1, 2, 3, and 4), which can interact with at least five Notch ligands (Jagged1 and 2; Delta1, 3, and 4). Within the immune system, the ligands are mainly expressed on APCs, whereas the Notch receptors are expressed on T cells (6, 7, 12). At the molecular level, Notch is a heterodimeric surface receptor consisting of an extracellular ligand-binding region noncovalently associated with a transmembrane polypeptide with a long intracellular tail, which is cleaved during activation. This intracellular domain then translocates into the nucleus, where it forms a complex with CSL/RBP-Jκ/MAML/p300, which activates the expression of target genes (7, 13).
Depending on the predominant Notch ligand expressed on APCs, different target genes are activated by Notch signaling. As an example, Delta1-expressing dendritic cells (DCs) have increased ability to activate naive CD4+ T cells and promote Th1 cell development in vitro via upregulation of T-bet and IFN-γ, and inhibition of IL-4R signaling (6, 11). In contrast, Jagged-expressing APCs lead to Th2 cell differentiation via enhancement of GATA3 transcription (6). Finally, transfected Delta1 on DCs are also able to direct differentiation of naive CD8+ T cells into CTLs by promoting granzyme B and eomesodermin (Eomes) transcription (10, 14).
In transplantation, a Th1 response is the dominant phenotype in allograft rejection, whereas a Th2 response favors long-term survival in some transplant models (15–17). Therefore, we decided to study whether blockade of Delta1 could potentially tip the balance toward a favorable T cell subtype and improve allograft survival. In this study, we demonstrate that an anti-Delta1 blocking mAb is able to prolong allograft survival in a fully MHC-mismatched model of cardiac transplantation, in particular with blockade of the B7:CD28 costimulation. This effect was characterized by an increase in Th2 polarization and a decrease in Th1 and cytotoxic T cells. Finally, the prolongation in allograft survival was shown to require the presence of Th2 cytokines, in particular IL-4. These data lead us to conclude that Delta1 signaling is important in alloimmunity in vivo, and blockade of this pathway could be a potential additional target to improve graft outcome.
Materials and Methods
C57BL/6 (H-2b, B6), BALB/c (H-2d), CD28-deficient mice on B6 background (B6.129S2-Cd28tm1Mak/J), and STAT4-deficient and STAT6-deficient mice on BALB/c background (C.129S2-Stat4tm1Gru/J and C.129S2-Stat6tm1Gru/J, respectively) were purchased from The Jackson Laboratory. All mice were 8–12 wk of age and housed in accordance with institutional and National Institutes of Health guidelines.
Heterotopic heart transplantation
Vascularized heart grafts were placed in an intra-abdominal location using microsurgical techniques, as described by Corry et al. (18). Graft function was assessed by palpation of the heartbeat. Rejection was determined by complete cessation of palpable heartbeat and was confirmed by direct visualization after laparotomy. Graft survival is shown as the median survival time (MST) in days.
Abs and in vivo treatment protocol
The anti-Delta1 (HMD1-5) mAb was generated, as previously described (19). This mAb was manufactured and purified from the original hybridoma by a commercial source, BioXCell (West Lebanon, NH). Control hamster IgG was also obtained from the same source. Cardiac allograft recipients were treated with mAbs i.p. at 500 μg at day 0, and 250 μg at days 2, 4, 6, 8, and 10 after transplantation. CTLA4-Ig is a fusion protein composed of a human IgG1 Fc fused to the extracellular domain of CTLA4, which was purchased from Bristol-Myers-Squibb. CTLA4-Ig was given i.p. at day 2 after transplantation (250 μg). For IL-4 neutralization, 1 mg anti–IL-4 mAb (11B11) was administered on days 0, 1, 3, 5, and 8 after transplantation (BioXCell).
Measurement of cytokines by ELISPOT and Luminex assay
20). The frequencies of cytokine-secreting alloreactive cells were expressed as the number of cytokine-producing cells per 0.5 × 106 responder cells. For Luminex assay, cell-free supernatants of individual wells were removed after 48 h of incubation and analyzed by a multiplexed cytokine bead–based immunoassay using a preconfigured 21-plex mouse cytokine detection kit (Millipore), as described previously (21). All samples were tested in triplicate wells.
Splenocytes from recipients at 10–14 d after transplantation were stained with fluorochrome-labeled mAbs against CD4, CD8, CD11b, CD11c, B220, CD62 ligand (CD62L), CD44, CD25, CD80, CD86, PDL-1, CD1d, CD5, CD21, and FoxP3 (BD Biosciences, San Jose, CA). Intracellular FoxP3 staining was performed using the Cytofix/Cytoperm intracellular staining kit. Flow cytometry was performed with a FACSCalibur system (BD Biosciences) and analyzed using FlowJo software. To characterize Delta1 expression, we first incubated splenocytes from naive and transplanted animals 7 d after transplantation with anti-mouse CD16/CD32 purified (BD Biosciences) to block nonspecific binding to FcγR. Then cells were incubated with biotinylated anti-mouse Delta1 (HMD1-5) and Delta4 (HMD4-2) Abs, generated as previously described (1919).
Intracellular cytokine staining
To isolate cells from the allografts, hearts were excised, minced, and digested with ∼500 U/ml collagenase (Worthington Biochemical) for 30 min at 37°C. Cells were then mashed through 70-μm filters, and RBCs were lysed. Cells (0.5 × 106) were resuspended in HL-1 medium (BioWhittaker); supplemented with 1% l-glutamine (BioWhittaker), 1% penicillin (BioWhittaker), and 10% FCS (BioWhittaker); and restimulated with PMA (5 ng/ml; Sigma-Aldrich) plus ionomycin (500 ng/ml; Sigma-Aldrich); and brefeldin A (10 μg/ml; Sigma-Aldrich) was added. Cells were incubated for 4 h at 37°C. After staining for the surface markers (CD4 and CD8), cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences), according to the manufacturer’s instructions, and incubated with PE-conjugated anti–IFN-γ (XMG1.2) and allophycocyanin-conjugated anti–IL-17 (eBio17B7) for 30 min at 4°C. A gate was set on CD4+ and CD8+ cells, and the percentage of IFN-γ and IL-17 cells was determined by flow cytometric analysis.
Ex vivo cytotoxicity assay
Splenocytes from control and anti-Delta1–treated recipients were harvested after transplantation, and CD8+ T cells were isolated by MACS (CD8a; Ly-2 microbeads from Miltenyi Biotec). B cells from allogeneic BALB/c mice were used as target cells after cell sorting with CD19 microbeads (Miltenyi Biotec) with >95% purity. For the cytotoxicity assay, CD8+22). The percentage of calcein AM negative (apoptotic cells) and ethidium positive (dead cells) was determined by flow cytometry to differentiate apoptotic/dead target cells from viable ones. The specific cytotoxicity of CD8+ cells was calculated as follows: percentage of specific cytotoxicity = (percentage of dead/apoptotic target cells in the presence of effector cells) − (percentage of dead/apoptotic target cells alone) (23).
Cardiac graft samples from transplanted mice were harvested from rejecting (cessation of heartbeat by palpation) mice and at 7–14 d after transplantation when some of the grafts started to reject. Grafts were then fixed in 10% formalin, embedded in paraffin, transversely sectioned, and stained with H&E stain and Elastin Van Gieson stain. Immunoperoxidase staining using CD4 and CD8 Abs was also performed on smaller pieces of frozen sections following air drying and fixation with acetone. For each immunoperoxidase Ab, the number of stained leukocytes per high power field was recorded by averaging the number of positively stained leukocytes in the most affected five high power fields. Acute cellular rejection was semiquantitatively graded (0R-3R) using the revised International Society of Heart and Lung Transplantation classification (24). Because the revised International Society of Heart and Lung Transplantation histologic schema was proposed to grade small endomyocardial biopsies rather than resection specimens, additional parameters were used to further assess the severity and extent of the histologic changes. The extent of the cellular infiltration (percentage of inflammation) and myocyte loss was assessed and expressed as the percentage of surface areas involved by the aforementioned processes (25, 26). A transplant pathologist (I. Batal) blinded to the groups read all the samples.
Graft survival was expressed graphically using the Kaplan-Meier method, and statistical differences in survival between the groups were assessed by the log-rank test. Student t test was used for comparison of means. A p value <0.05 was considered statistically significant.
Delta1 expression is upregulated on APCs in an acute rejection model
First, we decided to characterize Delta1 and Delta4 ligand expression in the naive and transplant setting. We analyzed their expression on hematopoietic cells from lymph nodes and spleens of naive B6 wild-type (WT) animals by flow cytometry. Interestingly, Delta1 was predominantly expressed on DCs (CD11c+) and monocytes/macrophages (CD11b+CD11c−), whereas it was minimally present on B cells and T cells (Fig. 1). In contrast, Delta4 expression was low in all hematopoietic cells (e.g., CD11c+, 0.41 ± 0.08; Supplemental Fig. 1). Next, we performed cardiac transplants in a fully allogeneic model of cardiac transplantation and noted that Delta1 expression was significantly upregulated upon transplantation, especially close to the time of allograft rejection (7 d) (Fig. 1), whereas Delta4 did not significantly vary (Supplemental Fig. 1). This observation suggests a potential role of Delta1 in allograft rejection; however, it does not prove a direct contribution of this Notch ligand to the alloimmune response. Because Delta1 ligand signals through Notch receptors, we also evaluated the expression of these receptors on naive and transplanted T cells. Notch2 was the predominant receptor on CD4+ cells (18.96% ± 0.33), whereas CD8+ T cells expressed similar amounts of both Notch2 and Notch3 receptors (∼19%) (Supplemental Fig. 2). Both Notch2 and Notch3 receptors were upregulated upon transplantation on T cells, whereas Notch1 and Notch4 remained low (<1%) in both naive and transplant settings (Supplemental Fig. 2).
Delta1 blockade prolongs cardiac graft survival
To evaluate whether Delta1 plays a role in allograft rejection, we decided to use a Delta1 blocking mAb (HMD1-5) in vivo. We transplanted BALB/c hearts into B6 recipients and administered either hamster IgG control or anti-Delta1 mAb for 10 d (500 μg on day 0; 250 μg on days 2, 4, 6, 8, and 10). Although control animals had a MST of 7 d, anti-Delta1–treated group had a slight delayed graft survival (MST = 9 d, n = 6, p = 0.0005). However, when CD28 knockout (KO) mice were used as recipients, Delta1 blockade significantly delayed allograft rejection (MST = 52 d versus 12 d in controls, n = 8, p < 0.0001) (Fig. 2A). A similar finding was also observed when B6 WT recipients were treated with anti-Delta1 in combination with a single dose of CTLA4-Ig (250 μg on day 2) (MST = 58 versus 29 d in sCTLA4-Ig alone, n = 7, p < 0.0001) (Fig. 3A). These data suggest that brief Delta1 blockade is able to significantly delay allograft rejection, especially in the absence of strong B7:CD28 costimulation.
Anti-Delta1 decreases cellular infiltration and myocyte loss of cardiac allografts
To elucidate the delayed rejection in anti-Delta1–treated group, we started by analyzing cardiac grafts at 2 wk after transplantation, when control grafts started to be rejected. Although control grafts had intense inflammatory infiltrates and myocyte losses, Delta1 blockade on CD28KO recipients led to significantly less myocyte loss and inflammatory infiltrates (Fig. 2B, 2C), with lower rejection scores by International Society of Heart and Lung Transplantation classification (1.8 ± 0.1 versus 2.4 ± 0.24, p < 0.05). Similar picture was also observed in B6 WT recipients treated with sCTLA4-Ig and anti-Delta1 (Fig. 3B, 3C). Furthermore, the latter grafts had less CD4+ and CD8+ infiltrating cells when analyzed by immunohistochemistry (Fig. 3D, 3E). These findings suggest that Delta1 blockade leads to a decrease in the intensity of the alloimmune response.
Anti-Delta1 reduced the frequency of CD4 and CD8 effector/memory cells
To investigate the mechanisms of reduced alloimmunity in response to Delta1 blockade, we first measured the frequency of lymphocyte subtypes in transplanted groups 14 d after transplantation. Anti-Delta1 administration resulted in a significant decrease in both CD4 and CD8 effector/memory T cells (CD44highCD62Llow) in CD28KO recipients (p = 0.001) (Supplemental Fig. 3) as well as in B6 WT recipients cotreated with sCTLA4-Ig (p < 0.05) when compared with controls (Fig. 4). Regulatory T cells were not significantly different between groups (Fig. 4). Furthermore, the percentage of APC subtypes was similar between groups as well as the activation status of DCs, as assessed by CD80, CD86, and PDL-1 expression (Supplemental Fig. 4A). Because Delta1 has been shown to be important in the development of marginal zone B (MZB) cells, we also checked the frequency of this subpopulation in the spleens of treated and control animals. Despite a lower trend in percentage of total B cells as well as subtypes (regulatory B cells and MZB cells), there was no statistically significant difference between anti-Delta1 group and controls (Supplemental Fig. 4B). In summary, anti-Delta1 lowered the frequency of effector/memory CD4 and CD8 cells, whereas it did not affect the activation status of DCs or other APC subtypes.
Anti-Delta1 mAb tipped the balance toward Th2 cells
Next, we sought to characterize the influence of Delta1 blockade on Th cell phenotype in lymph nodes and spleens of CD28KO recipients 2 wk after transplantation. The anti-Delta1–treated group showed increased production of IL-4 and IL-5, whereas IFN-γ and IL-17 levels were decreased, as measured by Luminex bead-based assay (Fig. 5A). Similar observation was also noted in B6 WT recipients cotreated with sCTLA4-Ig and anti-Delta1 (Fig. 6A); however, the decrease in IFN-γ was more dramatic in this model. Moreover, Delta1 blockade also led to lower frequency of granzyme B-producing cells, as demonstrated by ELISPOT (Figs. 5B, 6B). This was confirmed by an in vitro cytotoxicity assay, in which CD8+ T cells from anti-Delta1–treated group demonstrated lower cytotoxicity activity against allogeneic target cells when compared with controls (8% apoptotic/dead cells versus 16% on controls, p = 0.0001) (Fig. 7).
Finally, when lymphocytes infiltrating the allografts were isolated and cultured with irradiated donor cells, the frequency of both CD4+ and CD8+ IFN-γ+ T cells was significantly lower in the anti-Delta1 group (Fig. 8). These results indicate that Delta1 blockade leads to polarization into Th2 cells and decreases Th1 and cytotoxic T cells, potentially explaining the delay in allograft rejection.
Anti-Delta1 prolongation of allograft survival is dependent on Th2 cytokines
Th1 and Th2 development is dependent on STAT4 and STAT6 signaling, respectively (27). To evaluate whether Delta1 signaling may induce Th1 differentiation indirectly via suppression of Th2 cells through IL-4 signaling blockade, we decided to dissect what was the dominant beneficial consequence of Delta1 blockade. Because STAT4KO mice were only commercially available on BALB/c background, we inverted the strains for this experiment. First, we confirmed the effect of anti-Delta1 mAb in this strain combination by demonstrating a slight prolongation of graft survival on BALB/c recipients of B6 hearts treated with anti-Delta1 mAb (MST = 11) when compared with controls (MST = 9, n = 5/group, p = 0.0025), similarly to what was observed on the BALB/c into B6 model. By transplanting B6 hearts into STAT4KO or STAT6KO recipients on BALB/c background, we were able to observe that prolongation of allograft survival was only maintained in STAT4KO treated with anti-Delta1 (Fig. 9A), whereas STAT6KO recipients had similar accelerated rejection as controls (Fig. 9B). Furthermore, neutralization of IL-4 abrogated the prolonging effects of Delta1 blockade in CD28KO recipients of BALB/c hearts (MST = 52 versus 13 in the cotreated group with anti-Delta1 and anti–IL-4 mAb, n = 5, p = 0.0002) (Fig. 9C). These findings suggest that the improved survival by Delta1 blockade is mainly dependent on Th2 polarization.
The role of the Notch pathway in T cell differentiation in vitro has been increasingly explored lately. In particular, Delta1 ligands expressed on DCs have been shown to promote Th1 cell development and CTL differentiation (6, 10, 11, 14), whereas Delta4 promotes IL-17 production (28). Nevertheless, little is known about the contribution of Notch ligands in the transplant setting in vivo, especially of Delta1, because both Th1 and CTLs are known to play a major role in allograft rejection (15–17, 29). In this study, we demonstrated that Delta1 is strongly upregulated upon transplantation in APCs in an acute cardiac rejection model (Fig. 1), whereas Delta4 remains mainly unchanged. The expression of Delta1 on DCs is induced by several stimuli, including infection with virus and bacteria, exposure to TLR ligands, and CpG-containing DNA (6, 11, 30). In alloimmunity, multiple stimuli might act in synergy in the upregulation of Delta1, especially TLR ligands released after surgical trauma and ischemic injury (31, 32). Based on observations of Delta1 expression after syngeneic cardiac transplants in which Delta1 was not as significantly upregulated as in allotransplants (data not shown), other unknown stimuli must be present to contribute to the dominant Delta1 expression upon transplantation.
Delta1 deficiency in mice is an embryonically lethal condition because Delta1 plays a major role in the compartmentalization of somites during embriogenesis (33). Cre-transgenic mice with selective deletion of Delta1 after birth demonstrated that the proportion of T and B cells was normal in both spleens and lymph nodes when compared with age-matched controls (34), suggesting that Delta1 is dispensable for postnatal T cell development in the thymus. However, a subpopulation of B cells, splenic MZB cells, was significantly decreased in these Delta1-null mice (34). Later, it was shown that despite the essential role of Delta1 for the maintenance of MZB cells in normal mice, anti-Delta1 blocking Ab did not significantly affect MZB cells in lupus-prone (New Zealand Black × New Zealand White)F1 mice (19), indicating that other signals might contribute to MZB cell maintenance. Similarly to these findings, we noticed that MZB cell subpopulation was not significantly decreased in the transplant setting at both 7 and 14 d after transplantation (Supplemental Fig. 4B), whereas it was decreased in the naive B6 mice that received anti-Delta1 without a transplant (data not shown). These findings suggest that Delta1 blockade does not reduce the alloimmune response through MZB cell depletion or by affecting T cell development; however, it could be affecting T cell activation and/or differentiation.
T cell activation requires the TCR engagement and costimulatory signals. Adding to this complexity, Notch proteins have emerged as potentiators of TCR signaling (5). Notch signaling has been shown to promote the nuclear retention of the NF-κB proteins p50 and p65 (35, 36) and to be involved in a positive feedback loop that regulates IL-2 production and CD25 expression (37). Furthermore, chemical γ-secretase inhibitors that block Notch signaling decrease T cell proliferation significantly (36). Corroborating with these findings, blockade of Delta1 in our in vivo model led to a decrease in the frequency of both CD4 and CD8 effector/memory cells, suggesting that Delta1 signaling might contribute to T cell proliferation. On the contrary, the transfection of small interfering RNAs into DCs against different Notch ligands led to enhanced IFN-γ production in MLR experiments with CD4+ T cells (38). These discrepancies seem to depend on the experimental system used and will only be resolved by performing experiments with conditionally gene-targeted mice or perhaps further development of in vivo small interfering RNA technologies.
CD8+ T cells have been reported to be major contributors of chronic rejection (29, 39), and its different requirements for costimulation (3) might pose a challenge to the development of tolerogenic strategies. The differentiation of naive CD8+ T cells into a functional CTL requires the induction of the two following key genes: T-bet and Eomes (40). CTLs can then mediate direct cell killing via granzyme B, perforin, or Fas ligand expression. Delta1-expressing DCs have increased capacity to promote CTL differentiation in vitro via interaction with Notch2 on naive CD8+ T cells (10). After this interaction, Notch2 intracellular domain migrates to the nucleus in combination with CREB1 to regulate granzyme B expression (10). Furthermore, Cho et al. (14) have shown that Notch1 is also able to directly regulate the expression of Eomes, perforin, and granzyme B via binding of the promoters of these crucial effector molecules. Reinforcing these findings, Delta1 blockade decreased the frequency of granzyme B-producing cells in our transplant models (Figs. 5B, 6B) and led to lower cytotoxicity activity of CD8+ T cells ex vivo against alloantigen target cells (Fig. 7), suggesting an important role of Delta1–Notch interaction in the regulation of cytolytic effector function of CD8+ T cells. Nonetheless, we realize that our Ab approach is unable to define whether Notch signaling is truly required to mount an efficient CTL response in vivo.
In sharp contrast to our findings, Delta1-transfected L cells (carrying both MHC class I and II molecules) injected into recipients 14 d before transplantation were able to prolong cardiac allograft survival in another study (41). This inhibition of rejection was CD8 dependent and presumably due to the emergence of regulatory IL-10–producing CD8+ cells. However, this conclusion was based on in vitro findings using Delta1-Fc, and not Delta1-transfected L cells like the in vivo model, raising concern about their conclusion. Moreover, the experimental approach undertaken with preinjection of cells in combination with MHC molecules and the properties of Delta1 on L cells could account for the differences observed. Nonetheless, we did not observe any effect of Delta1 blockade on IL-10 production in our experiment in vivo.
Different approaches have been undertaken to prove the role of Delta1 in Th1 cell differentiation, including transfection of Delta1 on APCs (6, 11) and use of Delta1-Fc proteins (30, 42). Furthermore, a neutralization Ab specific for Delta1 ligand (HMD1-5), the same clone used in this study, was able to decrease the frequency of Th1 cells and reduce the severity of disease in a model of experimental autoimmune encephalomyelitis (42). In agreement with these reports, our anti-Delta mAb was able to decrease Th1 and increase Th2 cells in murine cardiac transplantation, in particular when B7:CD28 pathway was blocked (Figs. 5, 6), suggesting an important role of Delta1 signaling in the determination of Th cell polarization in vivo.
It is believed that once Delta1 ligand interacts with the Notch receptor on CD4+ T cells, it activates the transcription of Tbx21 via a RBP-J–dependent mechanism, which encodes the Th1 cell-specifying transcription factor T-bet (43). Moreover, Sun et al. (11) have shown that Delta1 signaling could also induce a preferential Th1 response by inhibiting IL-4R signaling, suggesting that it is actually the suppression of Th2 development that favors Th1 polarization. Interestingly, our results with IL-4 neutralization and STAT4- and STAT6-deficient recipients go in support of this hypothesis, in which the dominant effect of Delta1 signaling is possibly through its suppressive effect on Th2 cell development. In neutral conditions, Th cells seem to favor Th2 polarization through GATA3 expression (44), and Delta1 affects IL-4 responsiveness to allow Th1 cell development (11). In the transplant setting, Th2 response favors long-term survival in some transplant models (15–17, 45), and tipping the balance from a Th1 to a Th2 response via Delta1 blockade demonstrated to be effective in reducing the alloimmune response. Among the Th2 cytokines, IL-4 was demonstrated in our model to be specifically important for the protective effect of Delta1 blockade, because neutralization of IL-4 abrogated the prolonging graft effect of anti-Delta1. However, the mechanism by which Delta1-initiated signaling blocks Th2 cell development remains to be established.
Taken together, our findings support a key role of Delta1 in the regulation of Th cell polarization and CTL differentiation in transplantation (Fig. 10). We were able to show that Delta1 is significantly upregulated on APCs upon transplantation, and, using an Ab approach, a brief blockade of Delta1 was able to decrease CTLs and tip the balance toward a Th2 response, in synergy with B7:CD28 blockade. This effect was shown to be dependent on Th2 cytokines, in particular IL-4, suggesting a protective role of Th2 polarization in delaying rejection. In face of the poor long-term outcomes of current immunosuppressive drugs in organ transplantation, the identification of beneficial effects of Delta1 blockade might help in the development of potential novel targets for tolerogenic strategies in alloimmunity.
The authors have no financial conflicts of interest.
We thank Mollie Jurewicz and Olaf Boenisch for invaluable technical assistance and Mohamed H. Sayegh for thoughtful discussions.
This work was supported by a research grant from the American Society of Transplantation (to L.V.R.) and National Institutes of Health Grants RO1 AI51559, 2PO1056229, and AI70820. T.U. is a recipient of an American Heart Association Scientist Development grant.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- dendritic cell
- median survival time
- marginal zone B
- single-dose CTLA4-Ig
- Received December 16, 2010.
- Accepted August 25, 2011.
- Copyright © 2011 by The American Association of Immunologists, Inc.