RESUMO
We have previously shown that pancreatic islets engineered to transiently display a modified form of FasL protein (SA-FasL) on their surface survive indefinitely in allogeneic recipients without a need for chronic immunosuppression. Mechanisms that confer long-term protection to allograft are yet to be elucidated. We herein demonstrated that immune protection evolves in two distinct phases; induction and maintenance. SA-FasL-engineered allogeneic islets survived indefinitely and conferred protection to a second set of donor-matched, but not third-party, unmanipulated islet grafts simultaneously transplanted under the contralateral kidney capsule. Protection at the induction phase involved a reduction in the frequency of proliferating alloreactive T cells in the graft-draining lymph nodes, and required phagocytes and TGF-ß. At the maintenance phase, immune protection evolved into graft site-restricted immune privilege as the destruction of long-surviving SA-FasL-islet grafts by streptozotocin followed by the transplantation of a second set of unmanipulated islet grafts into the same site from the donor, but not third party, resulted in indefinite survival. The induced immune privilege required both CD4+ CD25+ Foxp3+ Treg cells and persistent presence of donor antigens. Engineering cell and tissue surfaces with SA-FasL protein provides a practical, efficient, and safe means of localized immunomodulation with important implications for autoimmunity and transplantation.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Proteína Ligante Fas , Sobrevivência de Enxerto , Privilégio Imunológico , Tolerância ImunológicaRESUMO
Immune imbalance in autoimmune disorders such as type 1 diabetes may originate from aberrant activities of effector cells or dysfunction of suppressor cells. All possible defective mechanisms have been proposed for diabetes-prone species: (i) quantitative dominance of diabetogenic cells and decreased numbers of regulatory T cells, (ii) excessive aggression of effectors and defective function of suppressors, (iii) perturbed interaction between effector and suppressor cells, and (iv) variations in sensitivity to negative regulation. The experimental evidence available to date presents conflicting information on these mechanisms, with identification of perturbed equilibrium on the one hand and negation of critical role of each mechanism in propagation of diabetic autoimmunity on the other hand. In our analysis, there is no evidence that inherent abnormalities in numbers and function of effector and suppressor T cells are responsible for the immune imbalance responsible for propagation of type 1 diabetes as a chronic inflammatory process. Possibly, the experimental tools for investigation of these features of immune activity are still underdeveloped and lack sufficient resolution, in the presence of the extensive biological viability and functional versatility of effector and suppressor elements.
Assuntos
Autoimunidade , Diabetes Mellitus Tipo 1/imunologia , Camundongos Endogâmicos NOD , Animais , Apoptose/imunologia , Comunicação Celular/imunologia , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/metabolismo , Progressão da Doença , Inflamação/imunologia , Inflamação/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Secretion of ligands of the tumor necrosis factor (TNF) superfamily is a conserved response of parenchymal tissues to injury and inflammation that commonly perpetuates elimination of dysfunctional cellular components by apoptosis. The same signals of tissue injury that induce apoptosis in somatic cells activate stem cells and initiate the process of tissue regeneration as a coupling mechanism of injury and recovery. Hematopoietic stem and progenitor cells upregulate the TNF family receptors under stress conditions and are transduced with trophic signals. The progeny gradually acquires sensitivity to receptor-mediated apoptosis along the differentiation process, which becomes the major mechanism of negative regulation of mature proliferating hematopoietic lineages and immune homeostasis. Receptor/ligand interactions of the TNF family are physiological mechanisms transducing the need for repair, which may be harnessed in pathological conditions and transplantation. Because these interactions are physiological mechanisms of injury, neutralization of these pathways has to be carefully considered in disorders that do not involve intrinsic aberrations of excessive susceptibility to apoptosis.
Assuntos
Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/genética , Proliferação de Células , Células-Tronco Hematopoéticas/fisiologia , Humanos , Ligantes , Família Multigênica , Ligação Proteica , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais/genéticaRESUMO
The non-obese diabetic (NOD) mouse is a prevalent disease model of type 1 diabetes. Immune aberrations that cause and propagate autoimmune insulitis in these mice are being continually debated, with evidence supporting both dominance of effector cells and insufficiency of suppressor mechanisms. In this study we assessed the behaviour of NOD lymphocytes under extreme expansion conditions using adoptive transfer into immunocompromised NOD.SCID (severe combined immunodeficiency) mice. CD4(+) CD25(+) T cells do not cause islet inflammation, whereas splenocytes and CD4(+) CD25(-) T cells induce pancreatic inflammation and hyperglycaemia in 80-100% of the NOD.SCID recipients. Adoptively transferred effector T cells migrate to the lymphoid organs and pancreas, proliferate, are activated in the target organ in situ and initiate inflammatory insulitis. Reconstitution of all components of the CD4(+) subset emphasizes the plastic capacity of different cell types to adopt effector and suppressor phenotypes. Furthermore, similar immune profiles of diabetic and euglycaemic NOD.SCID recipients demonstrate dissociation between fractional expression of CD25 and FoxP3 and the severity of insulitis. There were no evident and consistent differences in diabetogenic activity and immune reconstituting activity of T cells from pre-diabetic (11 weeks) and new onset diabetic NOD females. Similarities in immune phenotypes and variable distribution of effector and suppressor subsets in various stages of inflammation commend caution in interpretation of quantitative and qualitative aberrations as markers of disease severity in adoptive transfer experiments.
Assuntos
Transferência Adotiva , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Hospedeiro Imunocomprometido , Fatores Etários , Animais , Imunofenotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Tumor necrosis factor-α (TNF-α) has been suggested to exert detrimental effects on hematopoietic progenitor function that might limit the success of transplants. In this study, we assessed the influences of TNF-α and its two cognate receptors on the function of fresh umbilical cord blood (UCB) and cryopreserved mobilized peripheral blood (mPB). CD34(+) progenitors from both sources are less susceptible to spontaneous apoptosis than lineage-committed cells and are not induced into apoptosis by TNF-α. Consequently, the activity of UCB-derived severe combined immune deficiency (SCID) reconstituting cells and long-term culture-initiating cells is unaffected by this cytokine. On the contrary, transient exposure of cells from both sources to TNF-α stimulates the activity of myeloid progenitors, which persists in vivo in UCB cell transplants. Progenitor stimulation is selectively mediated by TNF-R1 and involves activation of caspase-8, without redundant activity of TNF-R2. Despite significant differences between fresh UCB cells and cryopreserved mPB cells in susceptibility to apoptosis and time to activation, TNF-α is primarily involved in tropic signaling in hematopoietic progenitors from both sources. Cytokine-mediated tropism cautions against TNF-α neutralization under conditions of stress hematopoiesis and may be particularly beneficial in overcoming the limitations of UCB cell transplants.
Assuntos
Caspase 8/metabolismo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Antígenos CD34/metabolismo , Apoptose , Células Cultivadas , Ativação Enzimática , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos SCID , Receptores Tipo II do Fator de Necrose Tumoral , Transdução de SinaisRESUMO
Treg cells endowed with enhanced killing activity through decoration with Fas-ligand (FasL) protein (killer Treg) have been effective in delay of hyperglycemia in prediabetic non-obese diabetic (NOD) mice. In this study, we assessed the therapeutic efficacy of these cells, harvested from age-matched euglycemic NOD donors, on the course of disease in new-onset diabetics. One dose of 4 × 10(6) killer Treg cells stabilized blood glucose associated with increased insulin levels in 5 of 9 mice and partially reversed the severity of islet inflammation, whereas naive Treg cells did not modulate the course of disease significantly. Killer Treg cells were shown to operate through induction of cell apoptosis within the pancreatic lymph nodes, resulting in reduced efficiency of adoptive disease transfer to NOD/SCID recipients. A second mechanism of action consisted of increased fractions of CD4(+)CD25(-)FoxP3(+) T cells in the pancreas and all lymphoid organs. Immunomodulation with FasL rather than Treg cells enhanced the expression of CD25 and FoxP3 in the thymus, suggesting a possible contribution of thymic output to prolonged stabilization of the glucose levels. Autologous Treg cells evolve as excellent vehicles for targeted delivery of FasL as an immunomodulatory protein, which delete pathogenic cells at the site of inflammation and induce systemic dominance of suppressor subsets.
Assuntos
Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Imunomodulação , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Linfócitos T Reguladores/imunologia , Animais , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Graft-versus-host disease (GVHD) can be prevented by Fas-mediated selective depletion of host-sensitized donor lymphocytes ex vivo. We tested the hypothesis that Fas-mediated depletion of lymphocytes in the absence of host-specific antigenic stimulation can alleviate GVHD. Brief exposure (24 hours) of unstimulated donor lymphocytes to Fas ligand (FasL) ex vivo results in balanced apoptosis of CD8(+) and CD4(+) subsets with preferential depletion of CD62L and CD69, increased T regulatory fractions, and sustained responses to stimulation. This procedure ameliorates weight loss and improves the clinical and histologic score of skin and gastrointestinal GVHD with and without concurrent transplantation of hematopoietic progenitors and irrespective of conditioning-induced tissue injury. Although FasL-resistant donor T cells are less potent effectors of GVHD, they facilitate hematopoietic progenitor engraftment when infused with or after the graft and retain the potential to elaborate graft-versus-tumor reactions. These findings in a preclinical model together with the known trophic effects of FasL on primitive hematopoietic progenitors suggest that brief ex vivo incubation of hematopoietic grafts with FasL may improve the outcome and safety of clinical T cell-replete allogeneic and haploidentical transplants.
Assuntos
Proteína Ligante Fas/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/métodos , Linfócitos/imunologia , Linfócitos T/imunologia , Animais , Apoptose/imunologia , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/métodos , Processos de Crescimento Celular/imunologia , Doença Enxerto-Hospedeiro/imunologia , Efeito Enxerto vs Tumor/imunologia , Humanos , Ativação Linfocitária , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos TransgênicosRESUMO
Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.
Assuntos
Proteína Ligante Fas/imunologia , Tolerância Imunológica/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/cirurgia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Imuno-Histoquímica , Indicadores e Reagentes , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estreptavidina/imunologia , Transplante HomólogoRESUMO
Neuroblastoma represents a relatively common childhood tumor that imposes therapeutic difficulties. High risk neuroblastoma patients have poor prognosis, display limited response to radiochemotherapy and may be treated by hematopoietic cell transplantation. Allogeneic and haploidentical transplants have the distinct advantage of reinstitution of immune surveillance, reinforced by antigenic barriers. The key factors favorable to ignition of potent anti-tumor reactions are transition to adaptive immunity, recovery from lymphopenia and removal of inhibitory signals that inactivate immune cells at the local and systemic levels. Post-transplant immunomodulation may further foster anti-tumor reactivity, with positive but transient impact of infusions of lymphocytes and natural killer cells both from the donor, the recipient or third party. The most promising approaches include introduction of antigen-presenting cells in early post-transplant stages and neutralization of inhibitory signals. Further studies will likely shed light on the nature and actions of suppressor factors within tumor stroma and at the systemic level.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Neuroblastoma , Humanos , Criança , Transplante Homólogo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Imunoterapia , Neuroblastoma/terapia , Neuroblastoma/patologiaRESUMO
Alloreactive T-effector cells (Teffs) are the major culprit of acute graft-versus-host disease (aGVHD) associated with hematopoietic stem cell transplantation. Ex vivo nonspecific depletion of T cells from the donor graft impedes stem cell engraftment and posttransplant immune reconstitution. Teffs upregulate Fas after activation and undergo Fas ligand (FasL)-mediated restimulation-induced cell death (RICD), an important mechanism of immune homeostasis. We targeted RICD as a means to eliminate host-reactive Teffs in vivo for the prevention of aGVHD. A novel form of FasL protein chimeric with streptavidin (SA-FasL) was transiently displayed on the surface of biotinylated lymphocytes, taking advantage of the high-affinity interaction between biotin and streptavidin. SA-FasL-engineered mouse and human T cells underwent apoptosis after activation in response to alloantigens in vitro and in vivo. SA-FasL on splenocytes was effective in preventing aGVHD in >70% of lethally irradiated haploidentical mouse recipients after cotransplantation with bone marrow cells, whereas all controls that underwent transplantation with nonengineered splenocytes developed aGVHD. Prevention of aGVHD was associated with an increased ratio of CD4+CD25+FoxP3+ T regulatory (Tregs) to Teffs and significantly reduced transcripts for proinflammatory cytokines in the lymphoid organs and target tissues. Depletion of Tregs from the donor graft abrogated the protection conferred by SA-FasL. This approach was also effective in a xenogeneic aGVHD setting where SA-FasL-engineered human PBMCs were transplanted into NSG mice. Direct display of SA-FasL protein on donor cells as an effective means of eliminating alloreactive Teffs in the host represents a practical approach with significant translation potential for the prevention of aGVHD.
Assuntos
Doença Enxerto-Hospedeiro , Camundongos , Humanos , Animais , Proteína Ligante Fas , Estreptavidina , Doença Enxerto-Hospedeiro/prevenção & controle , Linfócitos T , LinfócitosRESUMO
T cell depletion prevents graft-versus-host disease (GVHD) but also removes T cell-mediated support of hematopoietic cell engraftment. A chimeric molecule composed of IL-2 and caspase-3 (IL2-cas) has been evaluated as a therapeutic modality for GVHD and selective ex vivo depletion of host-reactive T cells. IL2-cas does not affect hematopoietic cell engraftment and significantly reduces the clinical and histological severity of GVHD. Early administration of IL2-cas reduced the lethal outcome of haploidentical transplants, and survivor mice displayed markedly elevated levels of X-linked forkhead/winged helix (FoxP3(+); 50%) and CD25(+)FoxP3(+) T cells (35%) in the lymph nodes. The chimeric molecule induces in vitro apoptosis in both CD4(+)CD25(-) and CD4(+)CD25(+) subsets of lymphocytes from alloimmunized mice, and stimulates proliferation of cells with highest levels of CD25 expression. Adoptive transfer of IL2-cas-pretreated viable splenocytes into sublethally irradiated haploidentical recipients resulted in 60% survival after a lethal challenge with lipopolysaccharide, which is associated with elevated fractions of CD25(high)FoxP3(+) T cells in the lymph nodes of survivors. These data demonstrate that ex vivo purging of host-presensitized lymphocytes is effectively achieved with IL2-cas, and that IL-2-targeted apoptotic therapy reduces GVHD severity in vivo. Both approaches promote survival in lethal models of haploidentical GVHD. The mechanism of protection includes direct killing of GVHD effectors, prevention of transition to effector/memory T cells, and induction of regulatory T cell proliferation, which becomes the dominant subset under conditions of homeostatic expansion.
Assuntos
Transplante de Medula Óssea/imunologia , Doença Enxerto-Hospedeiro , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T/transplante , Transferência Adotiva , Animais , Antígenos CD/biossíntese , Antígenos CD/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Caspase 3/genética , Caspase 3/imunologia , Células Cultivadas , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/terapia , Interleucina-2/genética , Interleucina-2/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos , Terapia de Alvo Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Índice de Gravidade de Doença , Baço/citologia , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Transplante Isogênico , Irradiação Corporal TotalRESUMO
We hypothesized that regulatory T cells (Treg) effectively target diabetogenic cells, and reinforcing their killing capacity will attenuate the course of disease. For proof of concept, Fas-ligand (FasL) protein was conjugated to CD25(+) Treg (killer Treg) to simulate the physiological mechanism of activation-induced cell death. Cytotoxic and suppressive activity of killer Treg was superior to naïve Treg in vitro. Administration of 3-4 × 10(6) Treg prevented hyperglycemia in 65% prediabetic NOD females, however only killer Treg postponed disease onset by 14 weeks. CD25(+) Treg homed to the pancreas and regional lymph nodes of prediabetic NOD females, proliferated and ectopic FasL protein induced apoptosis in CD25(-) T cells in situ. This mechanism of pathogenic cell debulking is specific to killer Treg, as FasL-coated splenocytes have no immunomodulatory effect, and only killer Treg prevent the disease in 80% of NOD.SCID recipients of effector:suppressor T cells (10:1 ratio). All immunomodulated mice displayed increased fractional expression of FoxP3 in the pancreas and draining lymph nodes, which was accompanied by CD25 only in recipients of killer Treg. A therapeutic intervention that uses the affinity of Treg to reduce the pathogenic load has long-term consequences: arrest of destructive insulitis in mice with established disease prior to ß-cell extinction.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Homeostase/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Proteína Ligante Fas/imunologia , Feminino , Imunomodulação , Inflamação/imunologia , Inflamação/patologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Tumor necrosis factor (TNF) family receptors/ligands are important participants in hematopoietic homeostasis, in particular as essential negative expansion regulators of differentiated clones. As a prominent injury cytokine, TNF-alpha has been traditionally considered to suppress donor hematopoietic stem and progenitor cell function after transplantation. We monitored the involvement of TNF receptors (TNF-R) 1 and 2 in murine hematopoietic cell engraftment and their inter-relationship with Fas. Transplantation of lineage-negative (lin(-)) bone marrow cells (BMC) from TNF receptor-deficient mice into wild-type recipients showed defective early engraftment and loss of durable hematopoietic contribution upon recovery of host hematopoiesis. Consistently, cells deficient in TNF receptors had reduced competitive capacity as compared to wild-type progenitors. The TNF receptors were acutely upregulated in bone marrow (BM)-homed donor cells (wild-type) early after transplantation, being expressed in 60%-75% of the donor cells after 6 days. Both TNF receptors were detected in fast cycling, early differentiating progenitors, and were ubiquitously expressed in the most primitive progenitors with long-term reconstituting potential (lin(-)c-kit(+) stem cell antigen (SCA)-1(+)). BM-homed donor cells were insensitive to apoptosis induced by TNF-alpha and Fas-ligand and their combination, despite reciprocal inductive cross talk between the TNF and Fas receptors. The engraftment supporting effect of TNF-alpha is attributed to stimulation of progenitors through TNF-R1, which involves activation of the caspase cascade. This stimulatory effect was not observed for TNF-R2, and this receptor did not assume redundant stimulatory function in TNFR1-deficient cells. It is concluded that TNF-alpha plays a tropic role early after transplantation, which is essential to successful progenitor engraftment.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Apoptose , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/deficiênciaRESUMO
Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD4(+)CD25(+)Foxp3(+) T cells in mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25(+) T cells in vitro, with lower toxicity of IL2-cas to Foxp3(+) T cells. These data infer that targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25(+) Treg. The IL2-cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.
Assuntos
Caspase 3/uso terapêutico , Toxina Diftérica/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Peso Corporal/efeitos dos fármacos , Caspase 3/administração & dosagem , Caspase 3/genética , Caspase 3/farmacologia , Proliferação de Células/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/patologia , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/farmacologia , Toxina Diftérica/administração & dosagem , Toxina Diftérica/genética , Toxina Diftérica/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Interleucina-2/genética , Interleucina-2/metabolismo , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Análise de Sobrevida , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Ácido Trinitrobenzenossulfônico/administração & dosagem , Ácido Trinitrobenzenossulfônico/farmacologiaRESUMO
The feasibility and safety of immunotherapy mediated by intentionally mismatched rIL-2 activated killer lymphocytes (IMAK) with no prior stem cell engraftment was investigated in patients with advanced chemotherapy-resistant hematological malignancies and metastatic solid tumors. Our goals were to maximize anti-cancer activity by using intentionally mismatched donor lymphocytes; amplify killing of target cancer cells by rIL-2 activation of killer cells in vitro and in vivo, and avoid the risk of graft-versus-host disease (GVHD) by anticipated rejection of alloreactive donor lymphocytes. Conditioning consisted of 5 days of fludarabine 25 mg/m(2) or a single dose of cyclophosphamide 1,000 mg/m(2), 2 subcutaneous injections of alpha interferon (IFN) 3 x 10(6) and COX2 inhibitors, followed by administration of IMAK (65 +/- 5 CD3(+)CD56(-); 17 +/- 5 CD3(-)CD56(+)) in conjunction with low dose subcutaneous rIL-2 (6 x 10(6) IU/m(2)/day) for 5 days for continuous activation of alloreactive donor lymphocytes prior to their anticipated rejection. Here, we present our phase 1 clinical study data in a cohort of 40 high-risk patients with metastatic solid tumors and hematological malignancies. Treatment was accompanied by some malaise and occasional self-limited fever but otherwise well tolerated on an outpatient basis. Transient engraftment of donor cells was documented in two patients and only one developed self-limited grade 1 GVHD. Among patients with chemotherapy-resistant disease, long-term progression-free survival was recorded in 5 of 21 evaluable patients with metastatic solid tumors and in four of five patients with hematological malignancies. We conclude that the proposed procedure is feasible, safe, and potentially effective, with some otherwise resistant cancer patients long-term disease-free, thus justifying larger Phase II studies in patients with hematological malignancies and metastatic solid tumors, preferably at a stage of minimal residual disease with the goal in mind to eradicate all malignant cells at an early stage of the disease.
Assuntos
Células Matadoras Induzidas por Citocinas/transplante , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hematológicas/terapia , Imunoterapia , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Neoplasias/terapia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Interleucina-2/uso terapêutico , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia , Fatores de Risco , Linfócitos T/efeitos dos fármacos , Linfócitos T/transplante , Adulto JovemRESUMO
Mounting evidence indicates that immunosuppressive therapy and autologous bone marrow transplantation are relatively inefficient approaches to treat autoimmune diabetes. In this study we assessed the impact of immunosuppression on inflammatory insulitis in NOD mice, and the effect of radiation on immunomodulation mediated by adoptive transfer of various cell subsets. Sublethal radiation of NOD females at the age of 14 weeks (onset of hyperglycemia) delayed the onset of hyperglycemia, however two thirds of the mice became diabetic. Adoptive transfer of splenocytes into irradiated NON and NOD mice precipitated disease onset despite increased contents of CD25(+)FoxP3(+) T cells in the pancreas and regional lymphatics. Similar phenotypic changes were observed when CD25(+) T cells were infused after radiation, which also delayed disease onset without affecting its incidence. Importantly, irradiation increased the susceptibility to diabetes in NOD and NON mice (71-84%) as compared to immunomodulation with splenocytes and CD25(+) T cells in naïve recipients (44-50%). Although irradiation had significant and durable influence on pancreatic infiltrates and the fractions of functional CD25(+)FoxP3(+) Treg cells were elevated by adoptive cell transfer, this approach conferred no protection from disease progression. Irradiation was ineffective both in debulking of pathogenic clones and in restoring immune homeostasis, and the consequent homeostatic expansion evolves as an unfavorable factor in attempts to restore self-tolerance and might even provoke uncontrolled proliferation of pathogenic clones. The obstacles imposed by immunosuppression on abrogation of autoimmune insulitis require replacement of non-specific immunosuppressive therapy by selective immunomodulation that does not cause lymphopenia.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/terapia , Imunoterapia , Radioterapia , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Animais , Autoimunidade/efeitos da radiação , Contagem de Células , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Suscetibilidade a Doenças/etiologia , Feminino , Fatores de Transcrição Forkhead/biossíntese , Hiperglicemia , Imunomodulação/efeitos da radiação , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Pâncreas/patologia , Radioterapia/efeitos adversos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Linfócitos T Reguladores/efeitos da radiaçãoRESUMO
The critical role played by Fas ligand (FasL) in immune homeostasis renders this molecule an attractive target for immunomodulation to achieve tolerance to auto- and transplantation Ags. Immunomodulation with genetically modified cells expressing FasL was shown to induce tolerance to alloantigens. However, genetic modification of primary cells in a rapid, efficient, and clinically applicable manner proved challenging. Therefore, we tested the efficacy of donor splenocytes rapidly and efficiently engineered to display on their surface a chimeric form of FasL protein (SA-FasL) for tolerance induction to cardiac allografts. The i.p. injection of ACI rats with Wistar-Furth rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor, but not F344 third-party cardiac allografts. Tolerance was associated with apoptosis of donor reactive T effector cells and induction/expansion of CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells. Treg cells played a critical role in the observed tolerance as adoptive transfer of sorted Treg cells from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite survival of secondary Wistar-Furth grafts. Immunomodulation with allogeneic cells rapidly and efficiently engineered to display on their surface SA-FasL protein provides an effective and clinically applicable means of cell-based therapy with potential application to regenerative medicine, transplantation, and autoimmunity.
Assuntos
Proteína Ligante Fas/metabolismo , Transplante de Coração/imunologia , Baço/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T/imunologia , Tolerância ao Transplante , Transferência Adotiva , Animais , Proliferação de Células , Proteína Ligante Fas/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Masculino , Ratos , Ratos Endogâmicos ACI , Ratos Endogâmicos F344 , Ratos Endogâmicos WF , Proteínas Recombinantes de Fusão/metabolismo , Baço/citologia , Baço/metabolismo , Linfócitos T Reguladores/metabolismo , Transplante Homólogo/imunologiaRESUMO
A large body of evidence on the activity of regulatory T (Treg) cells was gathered during the last decade, and a similar number of reviews and opinion papers attempted to integrate the experimental findings. The abundant literature clearly delineates an exciting area of research but also underlines some major controversies. A linear cause-result interpretation of experimental maneuvers often ignores the fact that the activity of Treg cells is orchestrated with the effector T (Teff) cells within an intricate network of physiological immune homeostasis. Every modulation of the activity of the effector (cytotoxic) immune system revolves to affect the activity of regulatory (suppressive) cells through elaborate feedback loops of negative and positive regulation. The lack of IL-2 production by innate Treg cells makes this cytokine a prime coupler of the effector and suppressive mechanisms. Here we attempt to integrate evidence that delineates the involvement of IL-2 in primary and secondary feedback loops that regulate the activity of suppressive cells within the elaborate network of physiological immune homeostasis.
Assuntos
Homeostase/imunologia , Interleucina-2/imunologia , Linfócitos T Reguladores/imunologia , Animais , Humanos , Interleucina-10/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Modelos Imunológicos , Receptores de Interleucina-2/imunologia , Subpopulações de Linfócitos T/imunologia , Fator de Crescimento Transformador beta/imunologiaRESUMO
Continuous efforts are dedicated to develop immunotherapeutic approaches to neuroblastoma (NB), a tumor that relapses at high rates following high-dose conventional cytotoxic therapy and autologous bone marrow cell (BMC) reconstitution. This study presents a series of transplant experiments aiming to evaluate the efficacy of allogeneic BMC transplantation. Neuro-2a cells were found to express low levels of class I major histocompatibility complex (MHC) antigens. While radiation and syngeneic bone marrow transplantation (BMT) reduced tumor growth (P < 0.001), allogeneic BMT further impaired subcutaneous development of Neuro-2a cells (P < 0.001). Allogeneic donor-derived T cells displayed direct cytotoxic activity against Neuro-2a in vitro, a mechanism of immune-mediated suppression of tumor growth. The proliferation of lymphocytes from congenic mice bearing subcutaneous tumors was inhibited by tumor lysate, suggesting that a soluble factor suppresses cytotoxic activity of syngeneic lymphocytes. However, the growth of Neuro-2a cells was impaired when implanted into chimeric mice at various times after syngeneic and allogeneic BMT. F1 (donor-host) splenocytes were infused attempting to foster immune reconstitution, however they engrafted transiently and had no effect on tumor growth. Taken together, these data indicate: (1) Neuro-2a cells express MHC antigens and immunogenic tumor associated antigens. (2) Allogeneic BMT is a significantly better platform to develop graft versus tumor (GVT) immunotherapy to NB as compared to syngeneic (autologous) immuno-hematopoietic reconstitution. (3) An effective GVT reaction in tumor bearing mice is primed by MHC disparity and targets tumor associated antigens.
Assuntos
Transplante de Medula Óssea/imunologia , Efeito Enxerto vs Tumor/imunologia , Imunoterapia/métodos , Neuroblastoma/imunologia , Neuroblastoma/terapia , Animais , Processos de Crescimento Celular/imunologia , Epitopos , Antígenos de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/patologia , Linfócitos T/imunologia , Linfócitos T/transplante , Condicionamento Pré-TransplanteRESUMO
Bone marrow transplantation has been adoptively transferred from oncology to the treatment of autoimmune disorders. Along with extension of prevalent transplant-related concepts, the assumed mechanism that arrests autoimmunity involves elimination of pathogenic cells and resetting of immune homeostasis. Similar to graft versus tumor (GVT) reactivity, allogeneic transplants are considered to provide a better platform of immunomodulation to induce a graft versus autoimmunity reaction (GVA). It is yet unclear whether recurrence of autoimmunity in both autologous and allogeneic settings reflects relapse of the disease, transplant-associated immune dysfunction or insufficient immune modulation. Possible causes of disease recurrence include reactivation of residual host pathogenic cells and persistence of memory cells, genetic predisposition to autoimmunity and pro-inflammatory characteristics of the target tissues. Most important, there is little evidence that autoimmune disorders are indeed abrogated by current transplant procedures, despite reinstitution of both peripheral and thymic immune homeostasis. It is postulated that non-specific immunosuppressive therapy that precedes and accompanies current bone marrow transplant strategies is detrimental to the active immune process that restores self-tolerance. This proposition refocuses the need to develop strategies of immunomodulation without immunosuppression.