Tolerance induction by exosomes from immature dendritic cells and rapamycin in a mouse cardiac allograft model

Background

Dendritic cells (DCs) release bioactive exosomes that play an important role in immune regulation. Because they express low levels of class I major histocompatibility complex (MHC) and co-stimulatory molecules, exosomes derived from donor immature DCs (imDex) prolong allograft survival by inhibiting T-cell activation. However, this effect is limited and does not induce immunological tolerance when imDex are administered alone. Thus, we tested the effect of combined treatment with donor imDex and low-dose rapamycin on inducing tolerance in a mouse cardiac transplantation model.

Methods

ImDex were obtained from the culture supernatant of immature DCs derived from donor mouse (C57BL/6) bone marrow and were injected with suboptimal doses of rapamycin into recipient mouse (BALB/c) before and after transplantation. The capacity of this treatment to induce immune tolerance was analyzed in vitro and in vivo using the mouse cardiac transplantation model.

Results

Donor imDex expressed moderate levels of MHC class II and low levels of MHC class I and co-stimulatory molecules, but neither imDex nor subtherapeutic rapamycin dose alone induced cardiac allograft tolerance. Combined treatment with imDex and rapamycin, however, led to donor specific cardiac allograft tolerance. This effect was accompanied by decreased anti-donor antigen cellular response and an increased percentage of spleen CD4⁺CD25⁺ T cells in recipients. Furthermore, this donor specific tolerance could be further transferred to naïve allograft recipients through injection of splenocytes, but not serum, from tolerant recipients.

Conclusion

Combined with immunosuppressive treatment, donor imDex can prolong cardiac allograft survival and induce donor specific allograft tolerance.     

Thioredoxin Priming Prolongs Lung Allograft Survival by Promoting Immune Tolerance

Tolerance to allograft antigen is the major challenge and final goal of transplant medicine. Our previous study demonstrated that thioredoxin-1 (Trx) priming of donor lung significantly protected allogeneic lung graft. To determine whether Trx priming of donor lung inhibits allograft rejection, extends allograft survival and induces immune tolerance, orthotopic left lung transplantation was performed from Lewis to Sprague-Dawley rats without immunosuppression. Donor lungs were primed with Trx at 4°C for 4 hr prior to transplantation. After up to 37 days post-transplantation, allograft lung morphology, recipient T cell and humoral alloantigen-specific immune responses were examined. We found that Trx-primed lungs exhibited much reduced acute rejection and associated lung injuries resulting in loss of graft functional area at 5-37 days post-transplant in contrast to the control groups. CD4⁺ T cells from the recipients with Trx-primed grafts responded to the stimulation of dendritic cells (DCs) of donor origin, in contrast to DCs from the third party, with significantly reduced proliferation. Consistent with above findings, we observed that CD4⁺Foxp3⁺ regulatory T cells in spleen cells from the recipients with Trx-primed grafts were significantly increased compared to controls, and CD4⁺ T cells from the recipients with Trx-primed grafts produced much higher levels of immunosuppressive cytokine, IL-10 when stimulated with allogeneic donor DCs. In addition, humoral immune tolerance was also induced as there was no significant increase levels of serum antibodies against donor antigens in Trx-lung recipients when re-challenged with allogeneic donor antigens. Our results demonstrate that one-time Trx-priming of donor lung grafts prior to transplantation significantly prolongs the survival of the grafts through inducing or promoting cellular and humoral alloantigen-specific immune tolerance, which might be associated with the induction of immunosuppressive regulatory T cells.     

Transfusion induces blood donor-specific suppressor cells

Transfusion with blood from the organ donor before transplantation can prolong the survival of renal allografts in the rat. To determine if the beneficial effect of preoperative blood transfusion was due to the generation of donor-specific suppressor cells, in vivo and in vitro adoptive transfer experiments were performed. Lymphoid cells were harvested from transfused and untreated rats. These cells were then either (1) transferred to lightly irradiated (200 R) syngeneic hosts which were subsequently challenged with a kidney allograft (in vivo assay) or (2) titrated as regulator cells into naive unidirectional MLC such that the regulator and responder populations were syngeneic. In the LEW-RT1 to DA-RT1av1 strain combination, the adoptive transfer of thoracic duct lymph (TDL) or lymph node (LN) cells (5 x 10(7) to 7.5 x 10(7) cells) from DA animals transfused with LEW blood, 7 days previously into syngeneic (DA), lightly irradiated (200 R) hosts resulted in the indefinite survival of LEW kidney allografts. The phenomenon was blood donor-specific and dose-dependent. In contrast the adoptive transfer of spleen cells (10(7) to 10(8] from blood transfused hosts 7 days after transfusion had no effect on renal allograft survival. In vitro the addition of LN or TDL regulator cells, harvested from DA rats transfused with LEW blood, to a unidirectional MLC (DA responders, LEW stimulators) resulted in a significant depression of the proliferative response when compared with the proliferation of these same cells without the addition of these regulator cells or with the addition of LN or TDL regulator cells from a DA rat transfused with third party (PVG-RT1c) blood. The depression of the proliferative response observed in vitro, was blood donor specific. When LN or TDL regulator cells from a DA rat transfused with PVG-RT1c blood were added to a unidirectional MLC between DA responders and PVG stimulators, a significant depression in the proliferative response was observed. These in vitro findings were confirmed in two other strain combinations (LEW-PVG, and DA-PVG). Thus a single blood transfusion results in the induction of donor-specific suppressor cells detectable both in vivo and in vitro 7 days after transfusion in some but not all lymphoid compartments.     

Induction of allospecific tolerance by immature dendritic cells genetically modified to express soluble TNF receptor

The ability of dendritic cells (DC) to initiate immune responses or induce immune tolerance is strictly dependent on their maturation state. TNF-alpha plays a pivotal role in the differentiation and maturation of DC. Blockade of TNF-alpha action may arrest DC in an immature state, prolonging their window of tolerogenic opportunity. Immature DC (imDC) were transfected with recombinant adenovirus to express soluble TNF-alpha receptor type I (sTNFRI), a specific inhibitor of TNF-alpha. The capacity of sTNFRI gene-modified imDC (DC-sTNFRI) to induce immune tolerance was analyzed. sTNFRI expression renders imDC resistant to maturation induction and impairs their capacity to migrate or present Ag. This process leads to induction of allogeneic T cell hyporesponsiveness and the generation of IL-10-producing T regulatory-like cells. In vivo pretreatment of transplant recipients with DC-sTNFRI induces long-term survival of cardiac allografts in 50% of cases, and leads to a substantial increase in the generation of microchimerism and T regulatory cell numbers. Thus, blockade of TNF-alpha action by sTNFRI genetic modification can inhibit the maturation of DC and potentiate the in vivo capacity of imDC to induce donor-specific immune tolerance and prolong allograft survival.     

负载供者抗原的第三方树突状细胞延长大鼠移植心脏存活

目的观察负载供者抗原第三方未成熟DC对大鼠同种异体移植心脏存活期的影响。方法培养骨髓来源第三方大鼠未成熟DC,负载供者抗原,CTLA-4 Ig致耐受处理。建立大鼠心脏腹部移植模型,A组：心脏移植;B组：心脏移植,术前输注供者源未成熟DC;C组：心脏移植,术前输注第三方未成熟DC;D组：心脏移植,术前输注负载供者抗原致耐受第三方未成熟DC。分别行移植心病理、IFNγmRNA表达和移植心存活时间观察。结果D组的移植心病理改变轻,IFNγmRNA表达下调,移植心脏存活时间显著延长。结论负载供者抗原第三方致耐受未成熟DC能够诱导供者特异性免疫耐受,明显延长大鼠移植心脏存活时间。     

Role of the passive apoptotic pathway in graft-versus-host disease

Donor T cells have been shown to undergo apoptosis during graft-vs-host disease (GVHD). Although active apoptosis mediated through Fas/Fas ligand interactions has been implicated in GVHD, little is known about the role of the passive apoptotic pathway. To examine this question, we compared the ability of normal donor T cells and T cells overexpressing the antiapoptotic protein, Bcl-x(L), to mediate alloreactive responses in vitro and lethal GVHD in vivo. In standard MLCs, T cells that overexpressed Bcl-x(L) had significantly higher proliferative responses but no difference in cytokine phenotype. Overexpression of Bcl-x(L) prolonged survival of both resting and alloactivated CD4(+) and CD8(+) T cells as assessed by quantitative flow cytometry, accounting for the higher proliferative responses. Analysis of engraftment in murine transplantation experiments demonstrated an increase in donor T cell chimerism in animals transplanted with Bcl-x(L) T cells, suggesting that overexpression of Bcl-x(L) prolonged T cell survival in vivo as well. Notably, transplantation of Bcl-x(L) T cells into nonirradiated F(1) recipients also significantly exacerbated GVHD as assessed by mortality and pathological damage in the gastrointestinal tract. However, when mice were irradiated no difference in GVHD mortality was observed between animals transplanted with wild-type and Bcl-x(L) T cells. These data demonstrate that the passive apoptotic pathway plays a role in the homeostatic survival of transplanted donor T cells. Moreover, the susceptibility of donor T cells to undergo passive apoptosis is a significant factor in determining GVHD severity under noninflammatory but not inflammatory conditions.     

Testicular immune privilege promotes transplantation tolerance by altering the balance between memory and regulatory T cells

Immune responses are suppressed in immunologically privileged sites, which may provide a unique opportunity to prolong allograft survival. However, it is unknown whether testicular immune privilege promotes transplantation tolerance. Mechanisms underlying immune privilege are also not well understood. Here we found that islet transplantation in the testis, an immunologically privileged site, generates much less memory CD8(+) T cells but induces more Ag-specific CD4(+)CD25(+) regulatory T cells than in a conventional site. These CD4(+)CD25(+) cells exhibited the suppression of alloimmune responses in vivo and in vitro. Despite the immune regulation, intratesticular islet allografts all were rejected within 42 days after transplantation although they survived longer than renal subcapsular islet allografts. However, blocking CD40/CD40L costimulation induced the tolerance of intratesticular, but not renal subcapsular, islet allografts. Tolerance to intratesticular islet allografts spread to skin allografts in the non-privileged sites. Either transfer of memory CD8(+) T cells or deletion of CD25(+) T cells in vivo broke islet allograft tolerance. Thus, transplantation tolerance requires both costimulatory blockade, which suppresses acute allograft rejection, and a favorable balance between memory and regulatory T cells that could favorably prevent late allograft failure. These findings reveal novel mechanisms of immune privilege and provide direct evidence that testicular immune privilege fosters the induction of transplantation tolerance to allografts in both immunologically privileged and non-privileged sites.     

PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20

It is well known that adoptive transfer of donor-derived tolerogenic dendritic cells (DCs) helps to induce immune tolerance. RelB, one of NF-κB subunits, is a critical element involved in DC maturation. In the present study, our results showed tolerogenic DCs could be acquired via silencing RelB using small interfering RNA. Compared with imDCs, the tolerogenic DCs had more potent ability to inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines. They both mediated immune tolerance via the increased of T cell apoptosis and generation of regulatory T cells. Administration of donor-derived tolerogenic DCs significantly prevented the allograft rejection and prolonged the survival time in a murine heart transplantation model. Our results demonstrate donor-derived, RelB-shRNA induced tolerogenic DCs can significantly induce immune tolerance in vitro and in vivo.     

Immature and mature CD8alpha+ dendritic cells prolong the survival of vascularized heart allografts.

CD8alpha+ and CD8alpha- dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8alpha-(myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8alpha+ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2(b)) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2(k)) recipients for Th1 responses. Paradoxically and in contrast to their CD8alpha- counterparts, mature CD8alpha+ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8alpha+ and CD8alpha- DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8alpha+ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.     

Cutting edge: the Wiskott-Aldrich syndrome protein is required for efficient phagocytosis of apoptotic cells

Phagocytosis of apoptotic cells by macrophages and dendritic cells is necessary for clearance of proinflammatory debris and for presentation of viral, tumor, and self Ags. While a number of receptors involved in the cognate recognition of apoptotic cells by phagocytes have been identified, the signaling events that result in internalization remain poorly understood. Here we demonstrate that clearance of apoptotic cells is accompanied by recruitment of the Wiskott-Aldrich syndrome (WAS) protein to the phagocytic cup and that it's absence results in delayed phagocytosis both in vitro and in vivo. Therefore, we propose that WAS protein plays an important and nonredundant role in the safe removal of apoptotic cells and that deficiency contributes significantly to the immune dysregulation of WAS. The efficiency of apoptotic cell clearance may be a key determinant in the suppression of tissue inflammation and prevention of autoimmunity.     

Different programs of activation-induced cell death are triggered in mature activated CTL by immunogenic and partially agonistic peptide ligands

Activation-induced cell death (AICD) of mature T cells plays an important role in the control of immune homeostasis and peripheral tolerance. TNFRs and Fas have been implicated in the induction of AICD. However, these molecules were shown to be dispensable, at least in some experimental systems, for downsizing of Ag-induced T cell expansions and development of tolerance in vivo. The conditions of T cell activation leading to T cell deletion in a death receptor-independent manner are not well characterized. Here we show that human CTLs die through a death receptor-independent apoptotic program upon triggering with a partially agonistic peptide ligand. This apoptotic process exhibits some features of T cell death due to lymphokine deprivation and is blocked by exogenous IL-2. Our data demonstrate that engagement of TCR by MHC-peptide complexes can trigger diverse apoptotic programs of AICD and that the choice between these programs is determined by the agonistic potency of MHC-peptide ligand.     

B cell-mediated antigen presentation is required for the pathogenesis of acute cardiac allograft rejection

Acute allograft rejection requires the activation of alloreactive CD4 T cells. Despite the capacity of B cells to act as potent APCs capable of activating CD4 T cells in vivo, their role in the progression of acute allograft rejection was unclear. To determine the contribution of B cell APC function in alloimmunity, we engineered mice with a targeted deficiency of MHC class II-mediated Ag presentation confined to the B cell compartment. Cardiac allograft survival was markedly prolonged in these mice as compared to control counterparts (median survival time, >70 vs 9.5 days). Mechanistically, deficient B cell-mediated Ag presentation disrupted both alloantibody production and the progression of CD4 T cell activation following heart transplantation. These findings demonstrate that indirect alloantigen presentation by recipients' B cells plays an important role in the efficient progression of acute vascularized allograft rejection.     

Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 +/- 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-gamma, inducible NO synthase, and TGF-beta; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.     

Targeted lymphoid homing of dendritic cells is required for prolongation of allograft survival

Accumulating evidence that dendritic cells (DC) are important regulators of peripheral immune tolerance has led to the concept that donor-derived DC may be useful for inducing donor-specific transplantation tolerance. Although in vitro studies in this field have been encouraging, in vivo results have been inconsistent. Recent evidence has suggested a critical role of lymphoid organs in tolerance induction. In this study, we use a novel gene transduction technique to show that engineered expression of CCR7 on immature DC can markedly increase DC homing to lymphoid organs, leading to increased interaction with Ag-specific T cells. Moreover, we show that a single infusion of DC coexpressing CCR7 and the immunomodulatory molecule viral IL-10 (vIL-10) markedly prolongs cardiac allograft survival (mean survival time >100 days); importantly, DC expressing either vIL-10 alone or CCR7 alone was not effective. These results demonstrate an important paradigm for immune modulation using DC.     

Tolerance might be stably induced by both mesenchymal stem cell and hepatocyte growth factor in heart transplantation

Heart transplantation still remains the best choice of treatment for many kinds of end-stage heart diseases, but the side-effects of immunosuppressive agents and cardiac allograft vasculopathy (CAV) remain the main two obstacles in improving the long outcome of cardiac allografts. Mesenchymal stem cells (MSCs) are nonhematopoietic pluripotent cells that retain the ability to undergo differentiation into cells of different lineages. What's more, MSCs have been demonstrated to exert many profound inhibitory effects on many lymphocytic subpopulations such as T cells, regulatory T cells, and dendritic cells in vitro. In vivo studies also suggested that MSCs could be used to attenuate immune-mediated disorders such as transplant rejection and autoimmune diseases including rheumatoid arthritis, multiple sclerosis and GVHD. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in protecting infarcted myocardial by its antiapoptotic, antifibrotic and angiogenic effects. And also it was suggested that the administration of HGF could effectively suppress acute and chronic cardiac allograft rejection. We based our hypothesis on that HGF-modified MSCs would engraft stably in recipient and interact with important immune cells such as T cells, B cells and dendritic cells, then stable immune tolerance be induced possibly by both immunomodulation of MSCs and cardioprotective and immunomodulative effects of HGF. Further work is necessary to highlight the specific underlying mechanisms.     

Phagocytosis of apoptotic cells and the resolution of inflammation

Clearance of apoptotic cells by phagocytic cells plays a significant role in the resolution of inflammation, protecting tissue from harmful exposure to the inflammatory and immunogenic contents of dying cells. Apoptosis induces cell surface changes that are important for recognition and engulfment of cells by phagocytes. These changes include alterations in surface sugars, externalization of phosphatidylserine and qualitative changes in the adhesion molecule ICAM-3. Several studies have contributed to clarify the role of the receptors on the surface of phagocytes that are involved in apoptotic cell clearance. The phagocytic removal of apoptotic cells does not elicit pro-inflammatory responses; in contrast, apoptotic cell engulfment appears to activate signals that suppress release of pro-inflammatory cytokines. Therefore, clearance of apoptotic leucocytes is implicated in the resolution of inflammation and mounting evidence suggests that defective clearance of apoptotic cells contributes to inflammatory and autoimmune diseases. Defining the ligands on apoptotic cells and the corresponding receptors on phagocytes with which they engage, is likely to lead to the development of novel anti-inflammatory pro-resolution drugs. In this article, we will review the recognition and signaling mechanisms involved in the phagocytosis of apoptotic cells as well as the role of endogenous compounds that play a relevant role in the modulation of inflammation. We will also discuss what is currently known about diseases that may reflect impaired phagocytosis and the consequences on inflammation and immune responses.     

Identification of TLT2 as an engulfment receptor for apoptotic cells

Clearance of apoptotic cells (efferocytosis) is critical to the homeostasis of the immune system by restraining inflammation and autoimmune response to intracellular Ags released from dying cells. TLRs-mediated innate immunity plays an important role in pathogen clearance and in regulation of the adaptive immune response. However, the regulation of efferocytosis by activation of TLRs has not been well characterized. In this study, we found that activation of TLR3 or TLR9, but not of TLR2, enhances engulfment of apoptotic cells by macrophages. We found that the activation of TLR3 upregulates the expression of triggering receptor expressed on myeloid cells (TREM)-like protein 2 (TLT2), a member of the TREM receptor family, on the surface of macrophages. Blocking TLT2 on the macrophage surface by either specific anti-TLT2 Ab or soluble TLT2 extracellular domain attenuated the enhanced ability of macrophages with TLR3 activation to engulf apoptotic cells. To the contrary, overexpression of TLT2 increased the phagocytosis of apoptotic cells. We found that TLT2 specifically binds to phosphatidylserine, a major "eat me" signal that is exposed on the surface of apoptotic cells. Furthermore, we found that TLT2 mediates phagocytosis of apoptotic cells in vivo. Thus, our studies identified TLT2 as an engulfment receptor for apoptotic cells. Our data also suggest a novel mechanism by which TREM receptors regulate inflammation and autoimmune response.     

Induction of allograft tolerance in the absence of Fas-mediated apoptosis.

Using certain immunosuppressive regimens, IL-2 knockout (KO) mice, in contrast to wild-type (wt) controls, are resistant to the induction of allograft tolerance. The mechanism by which IL-2 regulates allograft tolerance is uncertain. As IL-2 KO mice have a profound defect in Fas-mediated apoptosis, we hypothesized that Fas-mediated apoptosis of alloreactive T cells may be critical in the acquisition of allograft tolerance. To definitively study the role of Fas in the induction of transplantation tolerance, we used Fas mutant B6.MRL-lpr mice as allograft recipients of islet and vascularized cardiac transplants. Alloantigen-stimulated proliferation and apoptosis of Fas-deficient cells were also studied in vivo. Fas mutant B6.MRL-lpr (H-2b) mice rapidly rejected fully MHC-mismatched DBA/2 (H-2d) islet allografts and vascularized cardiac allografts with a tempo that is comparable to wt control mice. Both wt and B6.MRL-lpr mice transplanted with fully MHC-mismatched islet allografts or cardiac allografts can be readily tolerized by either rapamycin or combined costimulation blockade (CTLA-4Ig plus anti-CD40L mAb). Despite the profound defect of Fas-mediated apoptosis, Fas-deficient T cells can still undergo apoptotic cell death in vivo in response to alloantigen stimulation. Our study suggests that: 1) Fas is not necessarily essential for allograft tolerance, and 2) Fas-mediated apoptosis is not central to the IL-2-dependent mechanism governing the acquisition of allograft tolerance.     

Macrophages and dendritic cells use different Axl/Mertk/Tyro3 receptors in clearance of apoptotic cells

The clearance of apoptotic cells is important for regulating tissue homeostasis, inflammation, and autoimmune responses. The absence of receptor tyrosine kinases (Axl, Mertk, and Tyro3) results in widespread accumulation of apoptotic cells and autoantibody production in mice. In this report, we examine the function of the three family members in apoptotic cell clearance by different phagocytic cell types. Mertk elimination nearly abolished macrophage apoptotic cell phagocytosis; elimination of Axl, Tyro3, or both, reduced macrophage phagocytosis by approximately half, indicating that these also play a role. In contrast, apoptotic cell clearance in splenic and bone marrow-derived dendritic cells (DCs) is prolonged compared with macrophages and relied primarily on Axl and Tyro3. The slower ingestion may be due to lower DC expression of Axl and Tyro3 or absence of GAS6 expression, a known ligand for this receptor family. In vivo, phagocytosis of apoptotic material by retinal epithelial cells required Mertk. Unlike macrophages, there did not appear to be any role for Axl or Tyro3 in retinal homeostasis. Likewise, clearance of apoptotic thymocytes in vivo was dramatically reduced in mertk(kd) mice, but was normal in axl/tyro3(-/-) mice. Thus, cell and organ type specificity is clearly delineated, with DCs relying on Axl and Tyro3, retina and thymus requiring Mertk, and macrophages exhibiting an interaction that involves all three family members. Surprisingly, in macrophages, tyrosine phosphorylation of Mertk in response to apoptotic cells is markedly diminished from axl/tyro3(-/-) mice, suggesting that the interactions of these receptors by heterodimerization may be important in some cells.