Apoptotic cell-based therapies against transplant rejection: role of recipient’s dendritic cells

One of the ultimate goals in transplantation is to develop novel therapeutic methods for induction of donor-specific tolerance to reduce the side effects caused by the generalized immunosuppression associated to the currently used pharmacologic regimens. Interaction or phagocytosis of cells in early apoptosis exerts potent anti-inflammatory and immunosuppressive effects on antigen (Ag)-presenting cells (APC) like dendritic cells (DC) and macrophages. This observation led to the idea that apoptotic cell-based therapies could be employed to deliver donor-Ag in combination with regulatory signals to recipient’s APC as therapeutic approach to restrain the anti-donor response. This review describes the multiple mechanisms by which apoptotic cells down-modulate the immuno-stimulatory and pro-inflammatory functions of DC and macrophages, and the role of the interaction between apoptotic cells and APC in self-tolerance and in apoptotic cell-based therapies to prevent/treat allograft rejection and graft-versus-host disease in murine experimental systems and in humans. It also explores the role that in vivo-generated apoptotic cells could have in the beneficial effects of extracorporeal photopheresis, donor-specific transfusion, and tolerogenic DC-based therapies in transplantation.     

Cytokine expanded myeloid precursors function as regulatory antigen-presenting cells and promote tolerance through IL-10-producing regulatory T cells

The initiation of graft-vs-host disease (GVHD) after stem cell transplantation is dependent on direct Ag presentation by host APCs, whereas the effect of donor APC populations is unclear. We studied the role of indirect Ag presentation in allogenic T cell responses by adding populations of cytokine-expanded donor APC to hemopoietic grafts that would otherwise induce lethal GVHD. Progenipoietin-1 (a synthetic G-CSF/Flt-3 ligand molecule) and G-CSF expanded myeloid dendritic cells (DC), plasmacytoid DC, and a novel granulocyte-monocyte precursor population (GM) that differentiate into class II+,CD80/CD86+,CD40- APC during GVHD. Whereas addition of plasmacytoid and myeloid donor DC augmented GVHD, GM cells promoted transplant tolerance by MHC class II-restricted generation of IL-10-secreting, Ag-specific regulatory T cells. Importantly, although GM cells abrogated GVHD, graft-vs-leukemia effects were preserved. Thus, a population of cytokine-expanded GM precursors function as regulatory APCs, suggesting that G-CSF derivatives may have application in disorders characterized by a loss of self-tolerance.     

Dendritic cells: limited potential in immunotherapy

Dendritic cells (DC) represent the most potent antigen-presenting cells (APC) of the immune system for their unique capability of presenting antigen to T-cells. Their use as cellular vaccines after charging with antigen ex vivo has been shown to induce protective and therapeutic anti-tumor immunity with regression of tumor manifestations in animal models of experimental cancer therapy. Human monocyte-derived DC (MoDC) generated in vitro in the presence of GM-CSF and IL-4 are regarded equivalent to immature DC. They can be induced to mature under various experimental conditions. MoDC, in their immature as well as mature state have been widely used for experimental as well as for clinical purposes. However, unequivocal proof for the clinical efficiency of MoDC-based anti-tumor vaccinations is still missing. There is now increasing experimental evidence demonstrating that MoDC may be hampered in their ability to migrate in response to inflammatory as well as homeostatic chemataxins. We therefore suggest that MoDC may not represent the equivalent of migratory DC in vivo limiting their use as magic bullets in tumor immunotherapy.     

Impact of mammalian target of rapamycin inhibition on lymphoid homing and tolerogenic function of nanoparticle-labeled dendritic cells following allogeneic hematopoietic cell transplantation

Dendritic cells (DC) play a major role in the pathogenesis of graft-vs-host disease (GvHD). Directed modification of surface molecules on DC that provide instructive signals for T cells may create a tolerogenic DC phenotype that affects GvHD severity. To investigate the impact of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on in vivo migratory capacities, tolerogenic function, and B7 superfamily surface expression on DC following allogeneic hematopoietic cell transplantation (aHCT), we generated a platform for magnetic resonance imaging and bioluminescence imaging based cell trafficking studies. Luciferase transgenic DC were labeled with superparamagnetic iron oxide nanoparticles bound to a murine IgG Ab that allowed for Fc-gammaR-mediated endocytosis. Locally injected luc(+) DC could be tracked within their anatomical context by bioluminescence imaging and magnetic resonance imaging after aHCT, based on stable intracellular localization of superparamagnetic iron oxide-IgG complexes. RAPA preconditioned DC (DC-R) displayed reduced expression of MHC class II, B7-1 (CD80), and B7-2 (CD86) but not B7-H4 whose ligation of T cells has a profound inhibitory effect on their proliferation and cytokine secretion. DC-R of recipient genotype reduced GvHD severity that is compatible with their tolerogenic phenotype. CCR5, CCR7, and CD62L expression was not affected by mTOR inhibition, which allowed for DC-R in vivo trafficking to secondary lymphoid compartments where immunregulation is required. This study is the first to delineate the impact of RAPA on DC migration and tolerogenic function after aHCT. Modification of the DC phenotype by mTOR inhibition may have therapeutic potential in an attempt to reduce GvHD following aHCT.     

CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells

Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14- Langerhans' cells (LC), CD1a-CD14- dermal DC (DDC), and CD1a-CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-beta1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a-CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.     

Fascin is involved in the antigen presentation activity of mature dendritic cells

Maturation of dendritic cells (DC) is critical to their development into potent APCs. Upon maturation, DC up-regulate the expression of MHC class II as well as costimulatory and adhesion molecules, all of which are important in Ag presentation. In addition, they undergo structural changes characterized by the expression of numerous long dendrites. Fascin is an actin-bundling protein that has been reported to be important for the development of dendrites. In this study, we evaluated fascin expression and function during DC maturation into potent APC. In vitro, treatment of bone marrow-derived DC (BM-DC) with GM-CSF resulted in increased levels of fascin expression. This increase correlated directly with an increase in MHC class II and B7-2 expression. Fascin expression was decreased by the addition of TGF-ss and increased by the addition TNF-alpha to the culture. These cytokines suppress or enhance DC maturation, respectively. Increased levels of fascin expression were found to correlate with increased APC activity in a one-way MLR. Specific inhibition of fascin expression, using antisense oligonucleotides, markedly reduced this APC allostimulatory activity. These data demonstrate that fascin expression correlates with DC maturation into APC, and it plays a significant role in the ability of DC to function as APC. This observation is the first evidence linking fascin-mediated dendrite formation with the APC activity of DC.     

HLA-DR-mediated apoptosis susceptibility discriminates differentiation stages of dendritic/monocytic APC

Professional APC are characterized by their ability to present peptide via HLA class II in the presence of costimulatory molecules (CD40, CD80, and CD86). The efficiency of Ag presentation can be classed as follows: mature dendritic cells (DC) are most efficient, immature DC and macrophages are intermediate, and monocytes are considered poor APC. There is a large body of evidence demonstrating that HLA-DR transmits signals in the APC. In this study, we have addressed the question of the outcome of HLA-DR signals on APC of the monocyte/DC lineages throughout their differentiation from immature to mature APC. DC were generated from both monocytes and CD34+ cells of the same individual, macrophages were differentiated from monocytes. Immunophenotypical analysis clearly distinguished these populations. HLA-DR-mediated signals led to marked apoptosis in mature DC of either CD34 or monocytic origin. Significantly less apoptosis was observed in immature DC of either origin. Nonetheless, even immature DC were more susceptible to HLA-DR-mediated apoptosis than macrophages, whereas monocytes were resistant to HLA-DR-mediated apoptosis. The mechanism of HLA-DR-mediated apoptosis was independent of caspase activation. Taken together, these data lead to the notion that signals generated via HLA-DR lead to the demise of mature professional APC, thereby providing a means of limiting the immune response.     

Human antigen-presenting cells respond differently to gut-derived probiotic bacteria but mediate similar strain-dependent NK and T cell activation

The intestinal microbiota is essential for homeostasis of the local and systemic immune system, and particularly strains of lactic acid bacteria and Escherichia coli have been shown to have balancing effects on inflammatory conditions such as allergy and inflammatory bowel disease. However, in vitro assessment of the immunomodulatory effects of distinct strains may depend strongly on the cell type used as a model. To select the most appropriate model for screening of beneficial bacteria in human cells, the response to strains of intestinal bacteria of three types of antigen-presenting cells (APC) was compared; blood myeloid dendritic cells (DC), monocyte-derived DC and monocytes, and the effector response of natural killer cells and na?ve T cells was characterized. Maturation induced by gut-derived bacteria differed between APC, with blood DC and monocytes responding with the production of IL-6 and tumour necrosis factor-alpha to bacteria, which elicited mainly IL-10 in monocyte-derived DC. In contrast, comparable IFN-gamma production patterns were found in both natural killer cells and T cells induced by all bacteria-matured APC. An inhibitory effect of certain strains on this IFN-gamma production was also mediated by all types of APC. The most potent responses were induced by monocyte-derived DC, which thus constitute a sensitive screening model.     

The presence of GM-CSF and IL-4 interferes with effect of TGF-beta1 on antigen presenting cells in patients with multiple sclerosis and in rats with experimental autoimmune encephalomyelitis

The possibility to generate and expand tolerogenic dendritic cells (DC) with TGF-β1 in vitro opens new therapeutic perspectives for the treatment of autoimmune diseases. In the present study, GM-CSF+IL-4 induced the differentiation of DC from adherent peripheral blood mononuclear cells, which had a higher expression of HLA-DR, CD86 and CD1a and the capacity to stimulate T cells. TGF-β1 alone slightly promoted the generation of antigen presenting cells (APC) with higher expression of CD14, but did not differentiate them into E-cadherin + Langerhans cell (LC)-like DC. TGF-β1-driven APC exhibited the morphology, phenotypes and functions of tolerogenic immature DC, and had lower capacity to stimulate T cells. In vivo experiment demonstrates that TGF-β1-treated APC exhibited the therapeutic potential in Lewis rats with experimental autoimmune encephalomyelitis (EAE), followed by increase of IL-10 production in lymph nodes and decrease of inflammatory cells in spinal cords. Most importantly, GM-CSF/IL-4 used in DC preparation abolished the effect of TGF-β1 to induce tolerogenic APC in vitro and in vivo. The results reveal that the usage of GM-CSF for the generation of tolerogenic DC should not be copied from DC preparation for anti-tumor therapy.     

Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats

Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals.     

IFN-gamma inhibits presentation of a tumor/self peptide by CD8 alpha- dendritic cells via potentiation of the CD8 alpha+ subset

Using an in vivo model of tumor/self peptide presentation for induction of class I-restricted skin test reactivity, we have previously shown that a minority population of CD8+ dendritic cells (DC) negatively regulates the induction of T cell reactivity by peptide-loaded CD8- DC in DBA/2 mice. However, the CD8- fraction can be primed by IL-12 to overcome inhibition by the CD8+ subset when the two types of DC are cotransferred into recipient hosts. We report here that exposure of CD8+ DC to IFN-gamma greatly enhances their inhibitory activity on Ag presentation by the other subset, blocking the ability of IL-12-treated CD8- DC to overcome suppression. In contrast, IFN-gamma has no direct effects on the APC function of the latter cells and does not interfere with IL-12 signaling. The negative regulatory effect triggered by IFN-gamma in CD8+ DC appears to involve interference with tryptophan metabolism in vivo. Through tryptophan depletion affecting T cell responses, IFN-gamma acting on CD8+ DC may thus contribute to regulation of immunity to tumor/self peptides presented by the CD8- subset.     

Efficient priming and expansion of antigen-specific CD8+ T cells by a novel cell-based artificial APC

The ex vivo priming and expansion of human CTL by APC, such as autologous monocyte-derived dendritic cells (DC), has the potential for use in immunotherapy for infectious diseases and cancer. To overcome the difficulty of obtaining sufficient number of autologous DC from patients, we have developed cell-based artificial APC (aAPC), designated Med-APC. These aAPC rapidly activate and expand the corresponding Ag-specific CD8+ T cells when pulsed with CTL epitope peptide(s) as efficiently as mature DC (mDC). We have also shown that Med-APC possess an innate cellular machinery that is sufficient to support the processing of complete Ag into immunodominant peptides, which considerably extends the usefulness of this technology. In addition, we have developed a novel expression vector system that expresses ubiquitinated Ag, resulting in an enhanced APC function of this system. Genetically encoded Ag can be easily introduced into Med-APC by transfection with this vector. Med-APC transfected with ubiquitinated Ag can efficiently expand the corresponding Ag-specific CTL without exogenous peptides. Therefore, Med-APC may have important therapeutic implications for adoptive immunotherapy and can be used for the detection of Ag-specific CTL for immunomonitoring.     

The selective estrogen receptor modulators, tamoxifen and raloxifene, impair dendritic cell differentiation and activation

Most immune cells, including myeloid progenitors and terminally differentiated dendritic cells (DC), express estrogen receptors (ER) making these cells sensitive to estrogens. Our laboratory recently demonstrated that 17-beta-estradiol (E2) promotes the GM-CSF-mediated development of CD11c+ CD11b(int) DC from murine bone marrow precursors. We tested whether the therapeutic selective estrogen receptor modulators (SERM), raloxifene and tamoxifen, can perturb DC development and activation. SERM, used in treatment of breast cancer and osteoporosis, bind to ER and mediate tissue-specific agonistic or antagonistic effects. Raloxifene and tamoxifen inhibited the differentiation of estrogen-dependent DC from bone marrow precursors ex vivo in competition experiments with physiological levels of E2. DC differentiated in the presence of SERM were assessed for their capacity to internalize fluoresceinated Ags as well as respond to inflammatory stimuli by increasing surface expression of molecules important for APC function. Although SERM-exposed DC exhibited increased ability to internalize Ags, they were hyporesponsive to bacterial LPS: relative to control DC, they less efficiently up-regulated the expression of MHC class II, CD86, and to a lesser extent, CD80 and CD40. This phenotype indicates that these SERM act to maintain DC in an immature state by inhibiting DC responsiveness to inflammatory stimuli. Thus, raloxifene and tamoxifen impair E2-promoted DC differentiation and reduce the immunostimulatory capacity of DC. These observations suggest that SERM may depress immunity when given to healthy individuals for the prevention of osteoporosis and breast cancer and may interfere with immunotherapeutic strategies to improve antitumor immunity in breast cancer patients.     

Unopposed production of granulocyte-macrophage colony-stimulating factor by tumors inhibits CD8+ T cell responses by dysregulating antigen-presenting cell maturation

Tumor cells gene-modified to produce GM-CSF potently stimulate antitumor immune responses, in part, by causing the growth and differentiation of dendritic cells (DC). However, GM-CSF-modified tumor cells must be gamma-irradiated or they will grow progressively, killing the host. We observed that 23 of 75 (31%) human tumor lines and two commonly used mouse tumor lines spontaneously produced GM-CSF. In mice, chronic GM-CSF production by tumors suppressed Ag-specific CD8+ T cell responses. Interestingly, an inhibitory population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused the observed impairment of CD8+ T cell function upon direct cell-to-cell contact. The inhibitory cells were positive for some markers associated with Ag presenting cells, like F4/80, but were negative for markers associated with fully mature DC like DEC205, B7. 2, and MHC class II. We have previously reported that a similar or identical population of inhibitory "immature" APC was elicited after immunization with powerful recombinant immunogens. We show here that these inhibitory cells can be elicited by the administration of recombinant GM-CSF alone, and, furthermore, that they can be differentiated ex vivo into "mature" APC by the addition of IL-4 and GM-CSF. Thus, tumors may be able to escape from immune detection by producing "unopposed" GM-CSF, thereby disrupting the balance of cytokines needed for the maturation of fully functional DC. Further, CD11b/Gr-1 double-positive cells may function as "inhibitory" APC under the influence of GM-CSF alone.     

Dendritic cells,chemokine receptors and autoimmune inflammatory diseases

Dendritic cells (DC) have been implicated in the induction of autoimmune diseases and have been identified in lesions associated with several autoimmune inflammatory diseases. Since DC are regarded as the professional antigen-presenting cell (APC) of the immune system and the only APC capable of activating na?ve T cells, they are likely to play a significant role in breaking tolerance of self-reactive lymphocytes and in supporting autoimmune responses in these diseases. A number of studies have revealed that small molecular weight chemotactic proteins known as chemokines are present within the autoimmune lesions and may contribute to the recruitment not only of DC populations, but also of immune cells such as T cells, B cells, neutrophils and monocytes into the site, and to the formation of organized lymphoid tissue structures within the target organ. The focus of this review will be a discussion of the role of chemokines in the recruitment of DC in human autoimmune inflammatory disorders, specifically the trafficking of DC into the inflammatory sites and the subsequent migration of differentiated DC from the inflammatory sites into the draining lymph nodes. Once DC are properly positioned within the lymph nodes, circulating antigen specific na?ve T cells can interact with DC and become activated, clonally expanded and stimulated to undergo differentiation into antigen-experienced memory T cells. Subsequent reactivation of memory T cells that enter the autoimmune lesions by DC present in the inflammatory lesion is thought to play a central role in tissue inflammation.     

IL-10-producing B220+CD11c- APC in mouse spleen

APC acting at the early stages of an immune response can shape the nature of that response. Such APC will include dendritic cells (DCs) but may also include populations of B cells such as marginal zone B cells in the spleen. In this study, we analyze APC populations in mouse spleen and compare the phenotype and function of B220(+)CD11c(-) populations with those of CD11c(+) spleen DC subsets. Low-density B220(+) cells had morphology similar to DCs and, like DCs, they could stimulate naive T cells, and expressed high levels of MHC and costimulatory molecules. However, the majority of the B220(+) cells appeared to be of B cell lineage as demonstrated by coexpression of CD19 and surface Ig, and by their absence from RAG-2(-/-) mice. The phenotype of these DC-like B cells was consistent with that of B cells in the marginal zone of the spleen. On bacterial stimulation, they preferentially produced IL-10 in contrast to the DCs, which produced IL-12. Conventional B cells did not produce IL-10. The DC-like B cells could be induced to express low levels of the DC marker CD11c with maturational stimuli. A minority of the B220(+)CD11c(-) low-density cells did not express CD19 and surface Ig and may be a DC subset; this population also produced IL-10 on bacterial stimulation. B220(+) APC in mouse spleen that stimulate naive T cells and preferentially produce IL-10 may be involved in activating regulatory immune responses.     

HIV-1-specific CTL responses primed in vitro by blood-derived dendritic cells and Th1-biasing cytokines

Vaccine strategies designed to elicit strong cell-mediated immune responses to HIV Ags are likely to lead to protective immunity against HIV infection. Dendritic cells (DC) are the most potent APCs capable of priming both MHC class I- and II-restricted, Ag-specific T cell responses. Utilizing a system in which cultured DC from HIV-seronegative donors were used as APC to present HIV-1 Ags to autologous T cells in vitro, the strength and specificity of primary HIV-specific CTL responses generated to exogenous HIV-1 Nef protein as well as intracellularly expressed nef transgene product were investigated. DC expressing the nef gene were able to stimulate Nef-specific CTL, with T cells from several donors recognizing more than one epitope restricted by a single HLA molecule. Primary Nef-specific CTL responses were also generated in vitro using DC pulsed with Nef protein. T cells primed with Nef-expressing DC (via protein or transgene) were able to lyse MHC class I-matched target cells pulsed with defined Nef epitope peptides as well as newly identified peptide epitopes. The addition of Th1-biasing cytokines IL-12 or IFN-alpha, during priming with Nef-expressing DC, enhanced the Nef-specific CTL responses generated using either Ag-loading approach. These results suggest that this in vitro vaccine model may be useful in identifying immunogenic epitopes as vaccine targets and in evaluating the effects of cytokines and other adjuvants on Ag-specific T cell induction. Successful approaches may provide information important to the development of prophylactic HIV vaccines and are envisioned to be readily translated into clinical DC-based therapeutic vaccines for HIV-1.     

Establishment of recipient model for spermatogonial stem cells transplantation in Kunming mice

The objective of this study was to establish a recipient model for spermatogonial stem cells (SSCs) transplantation in the Kunming mice after different doses busulfan treatment. The results showed that the most optimal dose of busulfan was 20 mg/kg and the most appropriate time for transplantation was 5–7 wk after busulfan treatment. Then, the cloned fragments existed in the testis of recipient mice after 20 mg/kg busulfan treatment and the offspring with enhanced green fluorescent protein (EGFP) were produced by the transplanting SSCs. Hence, we established the effective recipient model for donor-derived SSCs transplantation in Kunming mice.     

Conceiving wholeness: women, motherhood, and ovarian transplantation, 1902 and 2004

Scholars have shown that organ transplantation may transform ideas about one's body, with recipients feeling that they are receiving not just a body part but also a part of the donor's identity. This article focuses on a different way in which organ transplantation shapes recipient identity: the idea of becoming whole. We present the case studies of two women separated by a century (one in 1902 and the other in 2004) who sought ovarian transplantation, and examine how ovarian transplantation can engender a sense of wholeness on the individual, the familial, and the cultural levels, due to its ability to enable a recipient to naturally conceive and experience pregnancy.     

The dendritic cell cytoskeleton is critical for the formation of the immunological synapse

The binding of a T cell to an APC results in T cell actin cytoskeletal rearrangement leading to the formation of an immunological synapse. The APC cytoskeleton has been thought to play a passive role in this process. In this study, we demonstrate that dendritic cells (DC), unlike other APC, actively polarize their actin cytoskeleton during interaction with T cells. DC cytoskeletal rearrangement was critical for both the clustering and the activation of resting T cells. This study provides compelling evidence that the APC cytoskeleton plays an active role in the immunological synapse and may explain the unique ability of DC to activate resting T cells.