MerTK is required for apoptotic cell–induced T cell tolerance

Self-antigens expressed by apoptotic cells (ACs) may become targets for autoimmunity. Tolerance to these antigens is partly established by an ill-defined capacity of ACs to inhibit antigen-presenting cells such as dendritic cells (DCs). We present evidence that the receptor tyrosine kinase Mer (MerTK) has a key role in mediating AC-induced inhibition of DC activation/maturation. Pretreatment of DCs prepared from nonobese diabetic (NOD) mice with AC blocked secretion of proinflammatory cytokines, up-regulation of costimulatory molecule expression, and T cell activation. The effect of ACs on DCs was dependent on Gas6, which is a MerTK ligand. NOD DCs lacking MerTK expression (NOD.MerTKKD/KD) were resistant to AC-induced inhibition. Notably, autoimmune diabetes was exacerbated in NOD.MerTKKD/KD versus NOD mice expressing the transgenic BDC T cell receptor. In addition, β cell–specific CD4+ T cells adoptively transferred into NOD.MerTKKD/KD mice in which β cell apoptosis was induced with streptozotocin exhibited increased expansion and differentiation into type 1 T cell effectors. In both models, the lack of MerTK expression was associated with an increased frequency of activated pancreatic CD11c+CD8α+ DCs, which exhibited an enhanced T cell stimulatory capacity. These findings demonstrate that MerTK plays a critical role in regulating self-tolerance mediated between ACs, DCs, and T cells.

The processes of tissue remodeling, wound healing, and removal of damaged cells rely on cell death by apoptosis. Apoptosis entails a highly ordered process characterized by cell shrinkage, retention of organelles, nuclear chromatin condensation, and fragmentation, as well as redistribution of phosphatidylserine (PS) on the plasma membrane ( 1,2 ). Under homeostatic conditions, APCs such as macrophages and DCs bind and ingest apoptotic cells (ACs) using a variety of receptors, including Mer tyrosine kinase (MerTK), the PS receptor, scavenger receptor-A (SR-A), CD36, and ␣ v ␤ 5 integrin ( 3 -5 ). In this way, the breakdown and release of potentially immunogenic self-antigens by ACs is avoided ( 6 ). Indeed, ineffi cient clearance of ACs has been linked to autoimmunity ( 7 ).
The binding and ingestion of ACs also results in immunoregulation of APCs to further promote self-tolerance. For instance, activation of macrophages is blocked and TGF-␤ secretion is induced by ACs ( 8,9 ). Similarly, immature DCs become refractory to subsequent stimulation upon encounter with ACs, and maintain a " tolerogenic " phenotype characterized by low levels of MHC, CD40, CD80, and CD86 expression and the lack of proinfl ammatory cytokine secretion ( 10 -12 ). Consequently, these DCs are unable to eff ectively stimulate naive T cells or promote type 1 CD4 + T cell diff erentiation. In the nonobese diabetic (NOD) mouse model of autoimmune diabetes for example, induction of limited apoptosis of pancreatic ␤ cells prevents progression of the diabetogenic response. Lack of diabetes correlates with the establishment of tolerogenic DCs, and subsequent induction of immunoregulatory T cells ( 13 ).
The Tyro3, Axl, and MerTK (TAM) family of receptor tyrosine kinases (RTKs) has been reported to regulate homeostatic activation of macrophages and DCs. Mice lacking all three RTKs exhibit systemic autoimmunity marked by highly activated APCs in vivo ( 14 ). MerTK, which and, in addition to binding MerTK, it also serves as a PS opsonin, mediating phagocytosis of ACs ( 21 ).
Recently, we demonstrated that AC-induced immunoregulation of DCs is mediated by selective inhibition of the NF-B pathway, which regulates expression of several genes involved in DC activation and maturation ( 22 ). Importantly, upon binding of ACs, MerTK was shown to transduce signals necessary for inhibition of NF-B activation in BM-derived DCs (BMDCs) and splenic DCs (sDCs) ( 22 ). With this in mind, the current study was performed to determine how MerTK regulates DC eff ector function in response to ACs in vitro and in vivo, and in turn the eff ect this has on T cell self-tolerance. also is expressed by NKT cells, certain epithelial cell types, and reproductive tissues, is required for effi cient phagocytosis of ACs by peritoneal macrophages ( 5,15 ). Lupus-like symptoms detected in aging C57BL/6 mice in which MerTK expression is blocked because of insertion of a neomycin cassette in the kinase domain of the Mertk gene (MerTK KD ), are believed to be, in part, the result of ineffi cient clearance of ACs by macrophages ( 5,16 ). Investigation of retinal pigment epithelial cells (RPEs) has also shown that engulfment of apoptotic outer segments shed from the photoreceptor is mediated by MerTK via recognition of growth arrest -specifi c protein-6 (Gas6) and protein S ( 5, 17 -20 ). Gas6 is ubiquitously expressed (B) BMDCs from NOD or NOD.MerTK KD/KD mice were untreated or co-cultured with ACs, stimulated with varying concentrations of LPS for 72 h, and IL-12p70 secretion was measured by ELISA. (C) Ex vivo -derived sDCs from NOD and NOD.MerTK KD/KD mice were untreated or co-cultured with ACs, and then treated with 1 g/ml LPS for 72 h and IL-12p70 measured. (D) NOD (open histograms) and NOD.MerTK KD/KD (fi lled histograms) BMDCs were untreated or co-cultured with ACs, stimulated with 100 ng/ml LPS for 24 h, and assessed for costimulatory molecule expression on CD11c + gated cells. (E) NOD and NOD.MerTK KD/KD BMDCs were untreated or co-cultured with ACs, stimulated by cross-linking CD40 for 72 h, and IL-12p70 secretion measured. All DCs were collected from 10 -12-wk-old female mice (4 -8 mice per group). Data are representative of two independent experiments. *, P < 0.001; † , P = 0.011 (Student ' s t test). Error bars represent the mean ± the SEM.
CD4 + and CD8 + T cells were isolated from the spleens of NOD.BDC ( 23,24 ) and NOD.CL4 ( 25 ) TCR transgenic mice, respectively. In the absence of ACs, both peptide-pulsed NOD and NOD.MerTK KD/KD BMDCs stimulated high IL-2 secretion by BDC CD4 + ( Fig. 2 A ) and CL4 CD8 + ( Fig. 2 B ) T cells. AC pretreatment, however, signifi cantly inhibited the Our data demonstrate that ACs no longer prevent proinfl ammatory cytokine secretion, costimulatory molecule expression, and T cell stimulation by MerTK-defi cient DCs. Additionally, the number and activation status of pancreatic CD11c + CD8 ␣ + DCs are increased, and T cell -mediated ␤ cell autoimmunity is exacerbated in NOD mice lacking MerTK expression in both TCR-transgenic and T cell adoptive transfer models.

MerTK is necessary for AC-induced inhibition of DC activation, maturation, and effector function in vitro
Studies have demonstrated that ACs inhibit activation and maturation of DCs. Indeed, AC pretreatment for 3 h blocked LPS-stimulated intracellular IL-12p70 expression in CD11c + CD11b + and CD11c + CD8 ␣ + sDCs prepared from NOD mice ( Fig. 1 A ). MerTK expression is readily detected on CD11c + CD11b + and CD11c + CD8 ␣ + sDCs, but is reduced on plasmacytoid DCs (Fig. S1, available at http://www.jem.org/cgi/ content/full/jem.20062293/DC1). To investigate the role of MerTK in AC-induced inhibition of DC eff ector function, NOD.MerTK KD/KD mice were used as a source of DCs lacking MerTK expression. A 17-cM segment of 129/Ola Chromosome 2 harboring the MerTK KD mutation was introgressed onto the NOD genotype for 11 backcross generations ( 22 ). Pretreatment of NOD BMDCs with ACs for 3 h completely inhibited LPS-stimulated IL-12p70 secretion that otherwise was detected in cultures of NOD BMDCs treated with LPS-alone ( Fig. 1 B ). In contrast, NOD.MerTK KD/KD BMDCs secreted similar levels of IL-12p70 in response to LPS, regardless of AC pretreatment ( Fig. 1 B ). ACs also blocked LPS-stimulated IL-12p70 secretion by sDCs prepared from NOD but not NOD.MerTK KD/KD mice ( Fig. 1 C ). Similarly, AC pretreatment inhibited secretion of other proinfl ammatory cytokines such as TNF ␣ ( 22 ) and IL-1 ␣ / ␤ (unpublished data) in NOD and not MerTK KD/KD DCs. LPS-stimulated up-regulation of CD40, CD80, and CD86 expression was also inhibited by AC pretreatment of NOD BMDCs ( To rule out the possibility that AC-induced inhibition was specifi c for LPS stimulation, NOD versus NOD.MerTK KD/KD BMDCs treated with anti-CD40 mAb were compared. Crosslinking with anti-CD40 mAb induced IL-12p70 secretion by NOD BMDCs that was signifi cantly reduced by AC pretreatment (P < 10 Ϫ 3 ; Fig. 1 E ).Conversely, anti-CD40 mAb-stimulated IL-12p70 secretion by NOD.MerTK KD/KD BMDCs was not aff ected by AC pretreatment ( Fig. 1 E ). These results indicate that in the absence of MerTK expression, DCs are resistant to the inhibitory eff ect of ACs on secretion of proinfl ammatory cytokines and up-regulation of costimulatory molecule expression.
Next, the eff ect of AC pretreatment on the capacity of NOD and NOD.MerTK KD/KD BMDCs to stimulate CD4 + and CD8 + T cells in vitro was investigated. For this purpose,  capacity of peptide-pulsed NOD BMDCs to stimulate BDC CD4 + (P < 10 Ϫ 3 ) and CL4 CD8 + (P < 10 Ϫ 3 ) T cells ( Fig. 2, A and B ). In contrast, AC pretreatment of peptide-pulsed NOD.MerTK KD/KD BMDCs had no eff ect on IL-2 secretion by BMDCs were stimulated with 100 ng/ml LPS and IL-12p70 secretion measured by ELISA 72 h later (B) or costimulatory molecule surface expression measured via FACS 24 h later (C). Open histograms represent untreated DCs and fi lled histograms represent the indicated treatment group plus LPS. BMDCs were cultured with (D) BDC peptide and BDC CD4 + T cells or (E) HA peptide and CL4 CD8 + T cells for 72 h, and IL-2 secretion measured by ELISA. BMDCs were isolated from 10 -12 wk-old female mice (4 -8 per group). Data are representative of two independent experiments *, P < 0.001 (Student ' s t test). Error bars represent the mean ± the SEM. present on ACs even under serum-free culture conditions, a direct role for Gas6 in AC-mediated inhibition of DCs was established by using a blocking anti-Gas6 polyclonal antibody. Pretreatment of ACs with anti-Gas6, but not an isotype control antibody, blocked the capacity of ACs to inhibit LPSstimulated IL-12p70 secretion by NOD BMDCs ( Fig. 4 C ).
The lack of sensitivity of NOD.MerTK KD/KD DCs to AC pretreatment could be caused by an artifact of DCs developing in the absence of MerTK expression, and/or the genetic background of the mice. To rule out these possibilities, the eff ect of a blocking anti-MerTK polyclonal antibody on NOD and BALB/c BMDCs was assessed. The inhibitory activity of the polyclonal antibody was verifi ed by demonstrating that phosphorylation of MerTK induced by ACs was inhibited by treating NOD DCs with anti-MerTK, but not an isotype control antibody ( Fig. 3 A ). Despite AC pretreatment, LPS-stimulated IL-12p70 secretion ( Fig. 3 B ) and upregulation of costimulatory molecule expression ( Fig. 3 C ) were readily detected in NOD BMDCs incubated with anti-MerTK antibody. Furthermore, ACs failed to inhibit stimulation of BDC CD4 + ( Fig. 3 D ) and CL4 CD8 + ( Fig. 3 E ) T cells by NOD BMDCs incubated with the anti-MerTK, but not the isotype control antibody.
Next, BALB/c DCs were treated with blocking anti-MerTK or isotype control antibody before AC treatment. Similar to NOD DCs, ACs prevented LPS-stimulated up-regulation of costimulatory molecule expression ( Fig. S2 A, available at http://www.jem.org/cgi/content/full/jem.20062293/DC1) and IL-12p70 secretion ( Fig. S2 B), and inhibited T cell stimulation ( Fig. S2 C) by BALB/c BMDCs. Anti-MerTK, but not isotype control antibody, however, blocked AC-induced inhibition of IL-12p70 and T cell stimulation by BALB/c DCs (Fig. S2, A -C). Together, these fi ndings demonstrate that ACinduced inhibition of activation, maturation, and eff ector function of BMDCs and sDCs in vitro is dependent on MerTK.

AC-induced inhibition of DCs is Gas6 dependent
Gas6 binds MerTK, and some evidence indicates that protein S is also a MerTK ligand ( 26 -29 ). Other yet to be identifi ed ligands have also been proposed. Initially, protein S, which is found in serum, was investigated. Protein S mRNA expression by AC was not detected via RT-PCR ( Fig. S3 A, available at http://www.jem.org/cgi/content/full/jem.20062293/DC1), and LPS-stimulated IL-12p70 secretion by NOD BMDCs was inhibited by ACs despite serum-free culture conditions (Fig. S3 B). These results indicate that protein S is not necessary for MerTK binding of ACs, and they are consistent with recent observations by our group that Tyro3, and not MerTK, binds protein S ( 30 ). Whether Gas6 functioned as a MerTK ligand for ACs was then tested. Gas6 has been reported to bind PS ( 31 ), which would be exposed on the inverted plasma membrane of ACs. Accordingly, Gas6 surface expression on apoptotic thymocytes induced in vitro via ␥ irradiation or prepared directly from the thymus was measured by FACS. Apoptotic thymocytes were identifi ed by costaining with a VAD-FMK-FITC conjugate. Gas6 was detected on the surface of apoptotic thymocytes regardless of whether apoptosis was induced in vitro or in vivo ( Fig. 4 A ). Furthermore, Gas6 protein expression as determined by Western blot was induced in a temporal manner in irradiated thymocytes cultured under serum-free conditions ( Fig. 4 B ). Because protein S may bind to thymocytes in vivo and therefore be Western blot in whole-cell lysates prepared from untreated (U/T) thymocytes or apoptotic thymocytes at various times after ␥ irradiation. 10 ng of recombinant murine Gas6 protein (rmGas6) was loaded as a positive control (note that a truncation causes rmGas6 to have a reduced molecular weight compared with endogenous Gas6; see Materials and methods). (C) NOD BMDCs were pretreated with ACs incubated with anti-Gas6 or isotype control antibody, and LPS-induced (100 ng/ml) IL-12p70 secretion was measured via ELISA 72 h later. Thymocytes were isolated from 3 -4 wk-old female NOD mice. BMDCs were isolated from 10 -12-wk-old female NOD mice (4 per group). Data are representative of two independent experiments. *, P = 0.005 (Student ' s t test). Error bars represent the mean ± the SEM. animals (Fig. S4, available at http://www.jem.org/cgi/content/ full/jem.20062293/DC1). Furthermore, a kinetic analysis of apoptotic ␤ cell clearance/persistence showed no diff erence between STZ-treated NOD and NOD.MerTK KD/KD mice (Fig. S5). CFSE-labeled BDC CD4 + T cells were adoptively transferred, and 72 h later, proliferation was measured in the draining PLNs. Only limited proliferation of BDC CD4 + T cells was detected in NOD recipients treated with either STZ or citrate saline ( Fig. 6 A ). Similarly, the majority CFSE + BDC CD4 + T cells (71%) had not proliferated in citrate saline -treated NOD.MerTK KD/KD recipients ( Fig. 6 A ). In contrast, BDC CD4 + T cells exhibited signifi cant proliferation in NOD.MerTK KD/KD mice treated with STZ ( Fig. 6 A ). Additionally, the frequency of CFSE + IFN ␥ -producing cells was These results demonstrate that Gas6 expression is induced on the surface of ACs, and that it is required for MerTKmediated inhibition of DCs by ACs.

MerTK is required to maintain T cell tolerance to apoptotic ␤ cells
The lack of sensitivity of MerTK KD/KD DCs to AC-induced inhibition in vitro would be expected to promote autoimmunity in vivo. To test this hypothesis, TCR transgenic NOD.BDC mice were bred with NOD.MerTK KD/KD animals, and the development of diabetes was compared in female mice lacking MerTK expression (NOD.MerTK KD .BDC) or expressing wild-type levels of MerTK (NOD.BDC). Although only 10% (1/10) of NOD.BDC female mice developed diabetes over 52 wk ( Fig. 5 A ), which is consistent with the diabetes incidence reported for other NOD.BDC colonies, diabetes onset was accelerated, and the incidence of diabetes signifi cantly increased (7/11; P = 0.038, 2 ) in NOD.MerTK KD .BDC female mice ( Fig. 5 A ). Furthermore, ELISPOT demonstrated a signifi cantly (P = 0.002, Student ' s t test) increased frequency of IFN ␥ -secreting CD4 + T cells in the pancreatic lymph nodes (PLNs) of 4 wk-old NOD.MerTK KD .BDC versus NOD.BDC female mice ( Fig. 5 B ).
One possible explanation for exacerbated ␤ cell autoimmunity in NOD.MerTK KD .BDC mice is that apoptotic ␤ cells failed to inhibit DC activation and/or maturation, leading to enhanced activation of BDC CD4 + T cells. Analysis of DCs infi ltrating the pancreas of 4-wk-old mice demonstrated that the number of CD11c + CD8 ␣ + DCs was increased in NOD.MerTK KD .BDC mice ( Fig. 5 C ). Furthermore, an increased frequency of CD86 HI and CD80 HI CD8 ␣ + DCs was found among pancreatic CD11c + CD8 ␣ + DCs in NOD. MerTK KD .BDC when compared with NOD.BDC female mice ( Fig. 5 C ). No diff erence, however, was detected in the activation status of pancreatic CD11c + CD11b + DCs ( Fig. 5 C ) in the two groups of mice. These fi ndings demonstrate that in the absence of MerTK expression, enhanced ␤ cell autoimmunity correlated with increased activation and numbers of pancreatic CD11c + CD8 ␣ + DCs.
To better defi ne events going on between apoptotic ␤ cells, DCs, and CD4 + T cells, a model system was established in which naive BDC CD4 + T cells were transferred into either 4 wk-old NOD or NOD.MerTK KD/KD female recipients and ␤ cell apoptosis synchronized by treatment with a single dose of streptozotocin (STZ). This approach was necessary because analysis of NOD.BDC and NOD.MerTK KD .BDC mice is limited by heavy islet infi ltration at an early age (i.e., by 3 wk of age), and the observation that ␤ cell apoptosis is not synchronized. At 4 wk of age, both NOD and NOD.MerTK KD/KD mice lack islet infi ltration. NOD and NOD.MerTK KD/KD mice received a single i.p. injection of 120 mg/kg of STZ or citrate saline as a vehicle control. STZ at 120 mg/kg induces only moderate ␤ cell apoptosis, and the treated NOD mice remain diabetes free ( 13 ). Equivalent levels of Gas6 were detected on the surface of the few apoptotic ␤ cells detected in untreated mice, and on apoptotic ␤ cells in the STZ-treated BDC CD4 + T cells continued to increase in STZ-treated NOD.MerTK KD/KD recipients ( Fig. 6, C and D ). Notably, the increased T cell reactivity of transferred CFSE + BDC CD4 + T cells in STZ-treated NOD.MerTK KD/KD recipients was DCdependent, based on experiments using NOD.MerTK KD/KD mice expressing the diphtheria toxin (DT) receptor (DTR) transgene driven by the CD11c promoter as recipients. The CD11c-DTR transgenic model has been used to transiently deplete DCs in vivo upon DT injection ( 32 ). NOD.MerTK. DTR mice were treated with DT or left untreated, and 24 h later, mice received STZ and CFSE + BDC CD4 + T cells. As demonstrated in Fig. 6 E , DT treatment, which induces a > 10-fold reduction of PLN DCs, signifi cantly reduced proliferation and the frequency of CFSE + IFN ␥ + BDC CD4 + T cells compared with the non-DT treated group of mice. markedly increased in STZ-treated NOD.MerTK KD/KD recipients (36%) compared with STZ-treated NOD or citrate salinetreated NOD and NOD.MerTK KD/KD recipients ( ‫ف‬ 2 to 3%; Fig. 6 B ). Consistent with these latter fi ndings, the total number of CFSE + IFN ␥ + BDC CD4 + T cells in the PLN of STZ-treated NOD.MerTK KD/KD recipients increased with time relative to the other groups. At 72 h after transfer, no signifi cant diff erence in the number of CFSE + BDC CD4 + T cells among the four treatment groups was detected ( Fig. 6 C ), although the number of CFSE + IFN ␥ + T cells was increased in STZ-treated NOD.MerTK KD/KD recipients ( Fig. 6 D ). At 96 h after transfer, the number of CFSE + BDC CD4 + T cells signifi cantly declined among STZ-and citrate saline -treated NOD mice, and citrate saline -treated NOD.MerTK KD/KD mice. In contrast, both the number of CFSE + and CFSE + IFN ␥ +

CD11c + CD8 ␣ + DCs from STZ-treated MerTK-defi cient NOD mice have an increased capacity to stimulate T cells
Analysis of pancreatic DCs showed no signifi cant diff erence in the frequency of CD11c + CD11b + and CD11c + CD8 ␣ + DCs in citrate saline -treated NOD and NOD.MerTK KD/KD recipients, or STZ-treated NOD recipients ( Fig. 7 A ). In contrast, an increase in the frequency of pancreatic CD11c + CD8 ␣ + DCs was detected in STZ-treated NOD.MerTK KD/KD recipients ( Fig. 7 A ). The frequency of CD11c + CD8 ␣ + DCs expressing elevated surface CD80 and CD86 in the pancreas ( Fig. 7 A ), and intracellular IL-12 in the PLN ( Fig. 7 B ) was also increased in STZ-treated NOD.MerTK KD/KD recipients relative to the other three groups. These fi ndings indicate that enhanced proliferation and IFN ␥ expression by CFSE + BDC CD4 + T cells transferred into STZ-treated NOD.MerTK KD/KD mice is DC dependent and correlates with an increased frequency of activated CD11c + CD8 ␣ + DCs.
To determine whether acquisition of ␤ cell antigens in vivo by NOD.MerTK KD/KD DCs promoted ␤ cell -specifi c T cell expansion, mice were treated with STZ as above, DCs were sorted via FACS from the pancreas, and the sorted DCs were cultured with CFSE + BDC CD4 + T cells (without exogenous peptide). As demonstrated in Fig. 8 A , a signifi cant increase in proliferation was detected in cultures prepared with DCs isolated from STZ-treated NOD.MerTK KD/KD as compared with NOD mice (P = 0.025, Student ' s t test). Next, pancreatic CD11c + CD11b + and CD11c + CD8 ␣ + DCs were sorted, and the capacity to stimulate BDC CD4 + T cells in vitro was tested, again, in the absence of exogenous antigen. Similar to results obtained with a pool of sorted pancreatic DCs ( Fig. 8 A ), no increase in CFSE + BDC CD4 + T cell proliferation was seen for either CD11c + CD11b + or CD11c + CD8 ␣ + DCs prepared from STZ-versus citrate saline -treated NOD mice ( Fig. 8 B ). Strikingly, a 9.7-fold increase in BDC CD4 + T cell proliferation was observed for CD11c + CD8 ␣ + DCs sorted from STZ-treated compared with citrate saline -treated NOD.MerTK KD/KD mice ( Fig. 8 B ). On the other hand, only a marginal increase (1.8-fold) in CD4 + T cell proliferation was detected for CD11c + CD11b + DCs sorted from the pancreas of NOD.MerTK KD/KD mice treated with STZ versus citrate saline ( Fig. 8 B ). These data demonstrate that DCs lacking MerTK expression, upon encounter with apoptotic ␤ cells, drive expansion of ␤ cell -specifi c CD4 + T cells. Furthermore, ␤ cell -specifi c CD4 + T cell stimulation is predominately mediated by CD11c + CD8 ␣ + DCs in NOD.MerTK KD/KD mice.

DISCUSSION
ACs provide a continuous source of self-antigen, which can be ingested, processed, and presented by DCs ( 4 ). Autoimmune reactivity to these self-antigens, however, is blocked by Figure 7. An increased frequency of activated CD11c + CD8 ␣ + DCs is detected in STZ-treated MerTK-defi cient recipient mice. (A and B) 5 × 10 6 BDC CD4 + T cells were transferred, and after 72 h, CD11c + CD11b + and CD11c + CD8 ␣ + DCs were isolated from the pancreas and stained for CD11b, CD8 ␣ , CD80 and CD86 (A), or PLN CD11c + CD8 ␣ + DCs stained for intracellular IL-12 p40/p70, and the total number of IL-12 expressing CD11c + CD8 ␣ + DCs determined per PLN (B). *, P = 0.026 (Student ' s t test). Error bars represent the mean ± the SEM. Furthermore, Gas6 expression was detected on the surface of apoptotic thymocytes and ␤ cells ( Figs. 4 A and S4), and a direct role for this PS opsonin was shown by blocking the inhibitory eff ect of ACs via anti-Gas6 antibody ( Fig. 4 C ). Importantly, in the absence of AC pretreatment, NOD and NOD.MerTK KD/KD DC activation and APC function did not signifi cantly diff er ( Figs. 1 and 2 ). Based on these data, we conclude that MerTK primarily serves to bind ACs and transduce signals that block subsequent activation and maturation in DCs. In contrast to RPE and certain subsets of macrophages, however, DCs do not require MerTK for effi cient phagocytosis of ACs (Fig. S6, available at http://www.jem.org/cgi/ content/full/jem.20062293/DC1) ( 16,30 ).
The roles of and mechanisms by which TAM RTKs regulate innate cell activation and/or function are complex, and clearly cell-type dependent. Recent work by Ivashkiv et al. demonstrated that IFN ␣ -mediated inhibition of TNF ␣ secretion in macrophages is caused by Axl binding of Gas6 or the capacity of ACs to inhibit subsequent DC activation and maturation ( 10 -12, 30, 33 ). MerTK is involved in phagocytosis of ACs by certain subsets of macrophages ( 5,15,34 ) and RPEs ( 35 ). In addition, MerTK and TAM family members Axl and Tyro3 have been shown to regulate homeostatic activation of innate eff ector cells ( 14,16 ). More recently, we have demonstrated that ACs immunoregulate DCs in part by preventing activation of the NF-B pathway via MerTKmediated signaling events ( 22 ). Accordingly, the role for MerTK in AC-induced immunoregulation of DCs and self-tolerance was investigated.
In vitro analyses of BMDCs and ex vivo sDCs demonstrate an essential role for MerTK in AC-induced inhibition of DC activation, maturation, and eff ector function. AC pretreatment of NOD, but not NOD.MerTK KD/KD DCs or DCs incubated with anti-MerTK antibody inhibited proinfl ammatory cytokine secretion, up-regulation of costimulatory molecule expression, and T cell stimulation ( Figs. 1 -3 and S2). NOD.MerTK KD/KD mice (Fig. S5) ruling out the possibility that " antigen-load " infl uenced BDC CD4 + T cell expansion and IFN ␥ expression in a disparate manner among the respective groups. Therefore, the lack of MerTK expression results in the development of pathogenic ␤ cell -specifi c type 1 T eff ector cells once ␤ cells are undergoing apoptosis.
Several observations indicate that MerTK-defi cient CD11c + CD8 ␣ + DCs contribute to ␤ cell autoimmunity in the two model systems studied. For instance, in both NOD.MerTK KD . BDC mice and STZ-treated NOD.MerTK KD/KD mice, the frequency and activation status of CD11c + CD8 ␣ + DCs was increased relative to CD11c + CD11b + DCs ( Fig. 5 C and  Fig. 7 A ). Furthermore, pancreatic CD11c + CD8 ␣ + DCs from STZ-treated NOD.MerTK KD/KD mice exhibited an increased T cell stimulatory capacity in vitro compared with CD11c + CD11b + DCs from the same mice, or CD11c + CD8 ␣ + and CD11c + CD11b + DCs from the other experimental groups ( Fig. 8 B ). Importantly, T cell stimulation was directly dependent on the capacity of the respective DCs to endocytose, process, and present antigen derived from STZ-induced apoptotic ␤ cells in vivo. These results are similar to reports indicating that CD11c + CD8 ␣ + DCs are highly effi cient at presenting antigens derived from ACs ( 39,40 ), and are potent producers of IL-12p70 ( 41,42 ). Furthermore, CD11c + CD8 ␣ + DCs have been implicated in driving ␤ cell autoimmunity ( 43 ).
Although MerTK-expressing macrophages and NKT cells were not directly examined, it is unlikely that these innate eff ectors play a signifi cant role in promoting ␤ cell autoimmunity in the two model systems. For instance, no difference in the number and activation status of CD11b + F4/80 + macrophages was detected in the PLN or pancreas of NOD.BDC versus NOD.MerTK KD .BDC mice or NOD versus NOD.MerTK KD/KD recipients (unpublished data). The observation that clearance of apoptotic ␤ cells over a 24-h period was similar in STZ-treated NOD or NOD.MerTK KD/KD recipients (Fig. S5) further suggests no signifi cant defect in MerTK-defi cient macrophages; macrophages are generally considered to be the most effi cient cell type for removal of ACs ( 6,44,45 ). NKT cells have been shown to have an immunoregulatory role in T1D, and reduced numbers of NKT cells in NOD mice has been linked to diabetes progression ( 43 ). Using CD1d tetramers complexed with an ␣ -galactosylceramide peptide, no diff erence in the number of NKT cells was detected under the various conditions tested, regardless of MerTK expression (unpublished data). Although work by Behrens et al. ( 46 ) suggests that MerTK is required for activation of NKT cells in vitro, the lack of MerTK expression appears to have no signifi cant eff ect on an already diminished pool of NKT cells in NOD mice. Finally, results obtained with NOD.MerTK.DTR mice in which proliferation and IFN ␥ expression of adoptively transferred BDC CD4 + T cells in the PLNs were significantly reduced after DT injection and in vivo DC depletion ( Fig. 6 E ), further demonstrate a primary role for DCs in mediating enhanced ␤ cell -specifi c T cell reactivity in MerTK-defi cient NOD mice. Importantly, the frequency of ACs, even though MerTK and Tyro3 are expressed and bind these ligands ( 36 ). Here, IFN ␣ was found to up-regulate Axl, which in turn diff erentially induced the transcriptional suppressor Twist ( 36 ). Interestingly, Axl-induced Twist blocked TNF ␣ expression by preventing NF-B binding to the TNF ␣ gene promoter. As noted above, MerTK binding of ACs in DCs also regulates NF-B -dependent transcription, but in this case via inhibition of upstream I B kinase activation in a phosphatidylinositol 3-kinase -dependent manner ( 22 ). These fi ndings suggest that NF-B is a common target of the TAM family in macrophages and DCs and that the mechanism by which NF-B activity is regulated is dependent on the particular RTK, the context of the signaling events, and cell type.
The inability of ACs to block activation and maturation of DCs lacking MerTK expression would be expected to break T-cell tolerance in vivo. In fact, this was observed in the two in vivo model systems studied. In NOD.MerTK KD .BDC mice, overt diabetes developed at an enhanced frequency and accelerated onset, which corresponded with an increased frequency of IFN ␥ -secreting CD4 + T cells in the PLN compared with NOD.BDC mice ( Fig. 5, A and B ). Furthermore, BDC CD4 + T cells transferred into STZ-treated NOD.MerTK KD/KD exhibited enhanced expansion and IFN ␥ expression, resulting in a signifi cantly increased number of type 1 eff ectors at 96 h after transfer compared with the other groups of recipients ( Fig. 6 ). Importantly, this eff ect was dependent on the induction of ␤ cell apoptosis because expansion and IFN ␥ expression by BDC CD4 + T cells in citrate saline -treated NOD.MerTK KD/KD recipients was limited, and mimicked that of STZ-or citrate saline -treated NOD recipient mice ( Fig. 6 ). Surprisingly, the total number of CFSE + BDC CD4 + T cells in the PLN of STZ-treated NOD.MerTK KD/KD recipients at 72 h after transfer was similar to the other 3 groups ( Fig. 6 C ), despite increased proliferation ( Fig. 6 A ). This discrepancy was caused by enhanced traffi cking of BDC CD4 + T cells from the PLN into the islets of the STZ-treated NOD.MerTK KD/KD recipients (unpublished data). The marked decrease in the number of BDC CD4 + T cells observed between 72 and 96 h after transfer for STZ-or citrate saline -treated NOD recipients ( Fig. 6, C and D ) is typical of antigen-induced tolerance, in which T cells undergo expansion and then contraction. This result is consistent with a report by the Glaichenhaus group demonstrating that STZ-induced ␤ cell apoptosis in young NOD mice induces tolerogenic DCs that prevent the expansion and development of pathogenic T eff ector cells ( 13 ). Indeed, pancreatic DCs from STZ-treated NOD.MerTK KD/KD recipients exhibited a typical " proinfl ammatory " phenotype based on increased expression of costimulatory molecules and IL-12 relative to STZ-treated NOD recipients ( Figs. 7 and 8 ). Furthermore, real-time PCR analysis showed that TNF ␣ and IL-1 ␤ mRNA expression was increased 9.5-and 9.9-fold, respectively, in DCs sorted from the pancreas of STZ-treated NOD.MerTK KD/KD versus NOD recipients (unpublished data). The latter is noteworthy in view of the cytotoxic eff ect TNF ␣ and IL-1 ␤ have on ␤ cells ( 37,38 ). Importantly, the frequency of apoptotic ␤ cells was similar in STZ-treated NOD and onset and frequency as wild-type NOD mice. Use of mice was approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.

Preparation of BMDCs and sDCs.
BMDCs and sDCs were prepared from male or female mice between 8 -12 wk of age as described ( 52 ). In brief, BM cells were fl ushed from tibias and femurs, and RBCs were lysed. BM cells were cultured for 8 d in RPMI 1640 complete media (10% FBS, penicillin/streptomycin, l-glutamine, sodium pyruvate, nonessential amino acids, and 2-mercaptoethanol) with 10 ng/ml GM-CSF (PeproTech) and 10 ng/ml IL-4 (PeproTech). On day 8, BMDCs were washed and cultured in RPMI complete media (without cytokines) for experiments.

Pretreatment of DCs with ACs.
Thymocytes prepared from 4 -6-wk-old mice were adhered to plastic for 2 h to remove DCs and macrophages, ␥ irradiated at 600 R, and cultured in base medium for 12 h. FACS demonstrated > 90% apoptotic and < 10% necrotic thymocytes based on Annexin V and propidium iodide staining. Apoptosis was confi rmed via DNA fragmentation analysis. DCs were co-cultured with ACs at a ratio of 1:10 (DC:AC) for 3 h. In some experiments, DCs were treated with anti-MerTK antibody before AC incubation. In brief, DCs (5 × 10 6 /well) were incubated with anti -mouse Fc ␥ III/II (BD Biosciences) in 6-well ultra-low cluster plates for 0.5 h at 37 ° C to block Fc receptor binding. DCs were treated for 1 h at 37 ° C with 20 μ g of goat anti-MerTK antibody or goat IgG, which is an isotype control. Alternatively, some AC samples were pretreated with 20 μ g polyclonal anti-Gas6 or goat IgG for 30 min before co-culture with DCs. In other experiments, DCs were co-cultured with ACs in serum-free HL1 media (Cambrex) to eliminate serum-derived protein S. Measurement of IL-12 production from DCs. DCs (9 × 10 5 /well) were pretreated with or without ACs for 3 h, washed, and stimulated with LPS for 72 h. Supernatants were collected and assayed for IL-12p70 in triplicate using an ELISA kit (BD Biosciences) following the manufacturer ' s instructions. Intracellular IL-12 staining was performed using a Cytofi x/Cytoperm cell permeabilization kit (BD Biosciences) and PE-labeled anti -mouse IL-12p40/70. Alternatively, DCs were stimulated by cross-linking CD40 with 10 μ g/ml of anti-CD40 IgM (HM40-3; BD Biosciences) and anti-IgM antibody (G188-2; BD Biosciences) for 30 min.
Our results suggest that pancreatic CD11c + CD8 ␣ + DCs lacking MerTK expression are no longer sensitive to the inhibitory eff ects of ACs. These DCs, upon encountering apoptotic ␤ cells, process and present antigens, but maintain the capacity to be activated and in turn promote ␤ cell -specifi c T cell reactivity. A reduced frequency of CD11c + CD11b + DCs, which may have a protective role in NOD mice under certain conditions, may also indirectly promote ␤ cell autoimmunity in the respective models. Interestingly, specifi c polymorphisms within the MERTK gene have been identifi ed in patients with systemic lupus erythematosus ( 47 ). Furthermore, MERTK and Mertk are located within the T1D-susceptibility intervals IDDM12/13 and Idd13 both in human populations and mice, respectively ( 48 -51 ). Sequence analysis of cDNAs encoding MerTK from a variety of mouse strains have identifi ed at least three allelic variants (unpublished data). Therefore, it is possible that allelic variants of MERTK and Mertk encode proteins that alter the response of DCs or other innate eff ectors (i.e., macrophages and NKT cells) to ACs, leading to the promotion or prevention of autoimmunity.
Noteworthy is that, despite a periphery that is " proinfl ammatory " ( Figs. 6 -8 ), NOD.MerTK KD/KD female (and male) mice remain diabetes-free. This is caused by increased efficiency of thymic negative selection in NOD.MerTK KD/KD mice and eff ective purging of the ␤ cell -specifi c T cell repertoire (unpublished data). Consequently, insulitis is significantly reduced, and NOD.MerTK KD/KD mice fail to develop diabetes. In marked contrast, BDC clonotypic thymocytes in NOD.MerTK KD/KD .BDC mice are not deleted, and the high frequency of mature T cells that enter the periphery readily diff erentiate into pathogenic eff ectors and drive ␤ cell autoimmunity ( Fig. 5 A ). Similarly, transferred BDC CD4 + T cells proliferate and diff erentiate into type 1 eff ectors in STZtreated NOD.MerTK KD/KD , but not NOD recipients ( Fig. 6 ). Together, these fi ndings suggest that MerTK infl uences the " normal " progression of ␤ cell autoimmunity in at least two mutually nonexclusive ways: (a) in the thymus, by regulating the effi ciency of negative selection by thymic DCs, and (b) in the periphery, by eff ecting DC responses to ACs and in turn infl uencing the general proinfl ammatory milieu. Eff orts are ongoing to further defi ne the roles of MerTK in T1D. In summary, our fi ndings demonstrate that MerTK is necessary for establishing T cell tolerance to AC-expressed proteins, and that altered function of this RTK may, in addition to T1D, contribute to other T cell -mediated autoimmune diseases.