RESUMO
Hematopoietic chimerism after allogeneic bone marrow transplantation may establish a state of donor antigen-specific tolerance. However, current allotransplantation protocols involve genotoxic conditioning which has harmful side-effects and predisposes to infection and cancer. Here we describe a non-genotoxic conditioning protocol for fully MHC-mismatched bone marrow allotransplantation in mice involving transient immunosuppression and selective depletion of recipient hematopoietic stem cells with a CD117-antibody-drug-conjugate (ADC). This protocol resulted in multilineage, high level (up to 50%), durable, donor-derived hematopoietic chimerism after transplantation of 20 million total bone marrow cells, compared with ≤ 2.1% hematopoietic chimerism from 50 million total bone marrow cells without conditioning. Moreover, long-term survival of bone marrow donor-type but not third party skin allografts is achieved in CD117-ADC-conditioned chimeric mice without chronic immunosuppression. The only observed adverse event is transient elevation of liver enzymes in the first week after conditioning. These results provide proof-of-principle for CD117-ADC as a non-genotoxic, highly-targeted conditioning agent in allotransplantation and tolerance protocols.
Assuntos
Transplante de Medula Óssea/métodos , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas/métodos , Imunoconjugados/farmacologia , Proteínas Proto-Oncogênicas c-kit/imunologia , Tolerância ao Transplante/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Células-Tronco Hematopoéticas , Tolerância Imunológica , Terapia de Imunossupressão/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Pele/patologia , Transplante de Pele/métodos , Quimeras de Transplante , Transplante HomólogoRESUMO
WHIM-09 is the first patient described with WHIM syndrome, an autosomal dominant form of neutropenia related to bone marrow retention of neutrophils. Originally diagnosed incorrectly with autoimmune neutropenia, the patient underwent splenectomy at age 9, but the absolute neutrophil count (ANC) did not rise. Subsequently, she was spontaneously cured by chromothripsis (chromosome shattering), which deleted the disease allele CXCR4 R334X , and 163 other genes, on chromosome 2 in a single hematopoietic stem cell (HSC). Chromothriptic CXCR4 +/o HSCs replaced CXCR4 +/R334X WHIM HSCs, and the ANC rose to a new sustained and benign baseline ~ 2-3-fold above normal that had remained unexplained. Here, we show that splenectomized Cxcr4 +/o mice had sustained and benign neutrophilia, phenocopying neutrophilia in WHIM-09. In addition, WHIM-09's granulocyte-macrophage precursor cells possessed increased granulocyte colony-forming activity ex vivo. Thus, WHIM-09's neutrophilia may be multifactorial, involving neutrophil-extrinsic factors (splenectomy), as well as CXCR4 haploinsufficiency-dependent neutrophil-intrinsic factors (increased myeloid precursor cell differentiation). The strong bone marrow retention signal for neutrophils conferred by the WHIM mutation may have prevented neutrophilia after splenectomy until the mutation was deleted by chromothripsis.
Assuntos
Cromotripsia , Células-Tronco Hematopoéticas/fisiologia , Síndromes de Imunodeficiência/diagnóstico , Mutação/genética , Neutrófilos/fisiologia , Receptores CXCR4/genética , Verrugas/diagnóstico , Alelos , Animais , Diferenciação Celular/genética , Criança , Modelos Animais de Doenças , Feminino , Haploinsuficiência , Humanos , Síndromes de Imunodeficiência/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças da Imunodeficiência Primária , Esplenectomia , Verrugas/genéticaRESUMO
Hematopoietic chimerism is known to promote donor-specific organ allograft tolerance; however, clinical translation has been impeded by the requirement for toxic immunosuppression and large doses of donor bone marrow (BM) cells. Here, we investigated in mice whether durable chimerism might be enhanced by pre-treatment of the recipient with liposomal clodronate, a macrophage depleting agent, with the goal of vacating BM niches for preferential reoccupation by donor hematopoietic stem cells (HSC). We found that liposomal clodronate pretreatment of C57BL/6 mice permitted establishment of durable hematopoietic chimerism when the mice were given a low dose of donor BM cells and transient immunosuppression. Moreover, clodronate pre-treatment increased durable donor-specific BALB/c skin allograft tolerance. These results provide proof-of-principle that clodronate is effective at sparing the number of donor BM cells required to achieve durable hematopoietic chimerism and donor-specific skin allograft tolerance and justify further development of a tolerance protocol based on this principle.
Assuntos
Quimerismo/efeitos dos fármacos , Ácido Clodrônico/farmacologia , Macrófagos/efeitos dos fármacos , Transplante de Pele/métodos , Tolerância ao Transplante/efeitos dos fármacos , Aloenxertos , Animais , Transplante de Medula Óssea/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBARESUMO
Hematopoietic chimerism established by allogeneic bone marrow transplantation is known to promote donor-specific organ allograft tolerance; however, clinical application is limited by the need for toxic host conditioning and "megadoses" of donor bone marrow cells. A potential solution to this problem has been suggested by the observation that recipient bone marrow mobilization by the CXCR4 antagonist AMD3100 promotes chimerism in congenic bone marrow transplantation experiments in mice. Here we report that a single subcutaneous dose of 10 mg/kg AMD3100 in recipient C57BL/6 mice was able to enhance hematopoietic chimerism when complete MHC-mismatched BALB/c donor bone marrow cells were transplanted 1h after drug dosing. However, levels of chimerism measured 30 days post-transplantation were not sustained when mice were reexamined on day 90 post-transplantation. Moreover, transient chimerism induced by this protocol did not support robust donor-specific skin allograft tolerance. Using the same transient immunosuppression protocol, we confirmed that "megadoses" of donor bone marrow cells could induce durable chimerism associated with donor-specific skin allograft tolerance without AMD3100 pre-treatment. We conclude that in this protocol AMD3100 pretreatment may empty bone marrow niches that become reoccupied by allogeneic donor hematopoietic progenitor cells but not by true long-lived donor hematopoietic stem cells, resulting in short-lived chimerism and failure to support durable donor-specific allograft tolerance.
Assuntos
Transplante de Medula Óssea , Células-Tronco Hematopoéticas/efeitos dos fármacos , Compostos Heterocíclicos/administração & dosagem , Receptores CXCR4/antagonistas & inibidores , Transplante de Pele , Animais , Benzilaminas , Quimerismo , Ciclamos , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Compostos Heterocíclicos/efeitos adversos , Antígenos de Histocompatibilidade/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Nicho de Células-Tronco/efeitos dos fármacos , Condicionamento Pré-Transplante , Tolerância ao Transplante/efeitos dos fármacosRESUMO
AMD3100 (plerixafor), is a specific CXCR4 antagonist approved by the FDA for mobilizing hematopoietic stem cells from bone marrow to blood for transplantation in cancer. AMD3100 also mobilizes most mature leukocyte subsets to blood; however, their source and trafficking potential have not been fully delineated. Here, we show that a single injection of AMD3100 10 mg/kg into C57Bl/6 mice rapidly mobilizes (peak â¼ 2.5 h) the same leukocyte subsets to blood as in humans. Using this model, we found that AMD3100 mobilization of neutrophils, lymphocytes, and monocytes to blood is not reduced by splenectomy or by blockade of lymphocyte egress from lymph node with FTY720, but is coupled to (i) reduced content of each of these cell types in the bone marrow; (ii) reduced T-cell numbers in thymuses; (iii) increased lymphocytes in lymph nodes; and (iv) increased neutrophil and monocyte content in the lung. Direct intrathymic labeling showed that AMD3100 selectively mobilizes naïve thymic CD4(+) and CD8(+) T cells to blood. Finally, AMD3100-induced neutrophil mobilization to blood did not reduce neutrophil trafficking to thioglycollate-inflamed peritoneum. Thus, AMD3100 redistributes lymphocytes, monocytes, and neutrophils from primary immune organs to secondary immune organs, peripheral tissues, and blood, without compromising neutrophil trafficking to inflamed sites.
Assuntos
Movimento Celular/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Receptores CXCR4/antagonistas & inibidores , Animais , Benzilaminas , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Movimento Celular/imunologia , Ciclamos , Feminino , Imunofenotipagem , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Contagem de Leucócitos , Leucócitos/imunologia , Pulmão/imunologia , Pulmão/patologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos , Fenótipo , Baço/efeitos dos fármacos , Baço/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/efeitos dos fármacos , Timo/imunologiaRESUMO
We developed a mouse model of heterotopic limb transplantation in which we took advantage of Thy1.1 and Thy1.2 congenic strains to track and characterize donor T cells, to determine the role of recipient's thymus in mixed T-cell chimerism induction as well as transplant immunocompetence. The vascularized Thy1.1 limb graft composed of femur, muscle, and skin (VBT) survived long-term in more than 87.5% of Thy1.2 recipients. Percentages of donor-type Thy1.1 T cells increased from day 30 to 90 in thymus and spleen of recipients. Most peripheral donor T cells displayed a naïve phenotype and a few others were regulatory T cells. Thymectomy prevented peripheral T-cell chimerism. Congenic VBT in immunodeficient RAG mice restored their ability to reject skin allografts. These observations suggest that donor T cells differentiated in host thymus might contribute to maintenance of mixed chimerism after transplantation of tissue composite grafts that include vascularized bone.
Assuntos
Extremidades/transplante , Linfócitos T/citologia , Timo/citologia , Transplante Heterotópico/imunologia , Animais , Transplante de Medula Óssea/imunologia , Proliferação de Células , Rejeição de Enxerto/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T/imunologia , Antígenos Thy-1/metabolismo , Timo/imunologia , Quimeras de Transplante/imunologiaRESUMO
PURPOSE: We studied the gene expression in rat cardiac allografts retransplanted into (donor x recipient) F1 animals to identify unknown or unexpected genes whose expression might contribute to the progression of transplant vasculopathy. METHODS: Gene expression was first studied using a mRNA differential display, then it was further investigated using quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. RESULTS: We found that the rat immunoglobulin kappa chain gene was preferentially induced in retransplanted cardiac allografts in which transplant vasculopathy was developing. The diseased vessels in the same allografts were heavily infiltrated with CD45R-positive B cells. The expression of two genes related to B-cell responses, B-lymphocyte chemoattractant, and CD40 ligand, showed a similar time course to that of the immunoglobulin kappa chain gene. We observed a heavy deposit of both IgM and IgG on the pathological neointima late in the development of transplant vasculopathy (i.e., 30 days after retransplantation) that was absent from the allografts immediately after retransplantation. CONCLUSION: During the development of transplant vasculopathy in a (donor x recipient) F1 environment, B cells were selectively recruited into the allografts and stimulated; meanwhile, antibodies against the pathological neointima were formed. These antibodies may be involved in the pathogenesis of transplant vasculopathy.