RESUMO
Immune responses to HLA and development of anti-donor HLA (DSA) were shown to play a role in chronic rejection following transplantation. We hypothesized that Abs to MHC change microRNAs (miRNAs), leading to chronic lung allograft rejection. Microarray analysis was performed in a murine model of anti-MHC-induced obliterative airway disease (OAD), a correlate of obliterative bronchiolitis. A unique profile of dysregulated miRNAs was detected in OAD mice on days 7 and 15 after Ab administration compared with control. Sixty-seven miRNAs were increased and 42 miRNAs were decreased in OAD mice on day 7. In addition, 15 miRNAs were overexpressed and 16 miRNAs were underexpressed in OAD mice on day 15. The expression of miR-16 and miR-195 was significantly decreased in lungs of OAD mice, as assessed by quantitative RT-PCR and in situ hybridization, with increases in H-2 Aa and H-2 Dma mRNA levels. Significant reductions in miR-16 and miR-195 levels were also noted in lung transplant (LTx) patients with DSA compared with LTx patients without DSA. Bioinformatic TargetScan and reporter assays identified the binding of miR-16 and miR-195 to the 3'-untranslated region of regulatory factor X 5. Quantitative PCR and immunohistochemistry indicated posttranscriptional increases in regulatory factor X 5 mRNA and protein expression in OAD mice, as well as in LTx recipients with DSA, which was associated with increased expression of HLA-DPA1, HLA-DQA1, and HLA-DRA mRNA. Therefore, our results demonstrated that miRNAs induced by alloimmunity may play important roles in chronic rejection after LTx.
Assuntos
Regulação da Expressão Gênica , Genes MHC da Classe II , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade/imunologia , Isoanticorpos/imunologia , MicroRNAs/genética , Animais , Análise por Conglomerados , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Transplante de Pulmão , Camundongos , Modelos Animais , Interferência de RNA , RNA Mensageiro/genética , Fatores de Transcrição de Fator Regulador X , Reprodutibilidade dos Testes , Doadores de Tecidos , Fatores de Transcrição/genética , TransplantadosRESUMO
The role of interferons, either pathogenic or protective, during autoimmune diabetes remains controversial. Herein, we examine the progression of diabetes in NOD mice lacking the type I (IFNAR) or type II (IFNGR) interferon receptor and, for the first time, in mice deficient in both receptors (double knockout [DKO]). All mice were bred, maintained, and monitored in a single specific pathogen-free facility with high female and low male diabetes incidence. Our expectation was that removal of interferon signaling would reduce autoimmune destruction. However, examination of diabetes incidence in the IFNAR- and IFNGR-deficient NOD mice showed a reduction in females and an increase in males. In DKO mice, diabetes occurred only in female mice, at decreased incidence and with delayed kinetics. These results show that interferons act as both positive and negative modulators of type 1 diabetes disease risk dependent on sex.
Assuntos
Envelhecimento , Doenças Autoimunes/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Ilhotas Pancreáticas/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/fisiopatologia , Progressão da Doença , Intervalo Livre de Doença , Feminino , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/fisiopatologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Caracteres Sexuais , Organismos Livres de Patógenos EspecíficosRESUMO
BACKGROUND: Intra-islet endothelial cells (IECs) express high levels of major histocompatibility complex (MHC) and are pivotal for posttransplant islet revascularization. We postulated that donor-specific sensitization would result in hyperacute rejection of IECs and prevent islet engraftment. Furthermore, ligation of endothelial cells with subsaturating concentrations of anti-MHC class I antibody (Ab) results in "accommodation" conferring protection against Ab/complement-mediated lysis. Therefore, we investigated whether accommodation of IECs would prevent hyperacute rejection of islets in sensitized recipients. METHODS: Islets were transplanted beneath the kidney capsule and allograft survival monitored using daily blood glucose (diabetes >300 mg/dL, normoglycemia <150 mg/dL). Recipients were presensitized with donor islets, splenocytes, or skin. Accommodation was induced by incubating human or murine islets with varying concentrations of anti-MHC class I Ab ex vivo. RESULTS: Isografts remained functional for >100 days, whereas allografts were rejected by day 14. Islet allo-transplantation induced donor-specific but not third-party anti-MHC Abs. Donor-specific, but not third-party, sensitization induced hyperacute rejection of subsequent islet allografts (median survival 1 day) associated with complement deposition. Preincubation of islets with subsaturating concentrations of anti-MHC-I Abs (1-100 ng/mL) up-regulated Bcl-2, Bcl-xl, and HO-1 within CD31+ IEC. These accommodated islets were resistant against hyperacute rejection when transplanted into donor-(splenocyte) sensitized recipients without any immunosuppression (median survival 6 days). CONCLUSIONS: Pretransplant sensitization against donor antigens results in hyperacute rejection of murine islets. IECs may play a crucial role in development of donor-specific immunity after islet transplantation. Significantly, accommodation of IEC may confer resistance to hyperacute rejection in sensitized recipients.
Assuntos
Células Endoteliais/imunologia , Sobrevivência de Enxerto/fisiologia , Transplante das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/citologia , Animais , Proteínas do Sistema Complemento/imunologia , Rejeição de Enxerto/prevenção & controle , Antígenos de Histocompatibilidade Classe I/imunologia , Ilhotas Pancreáticas/imunologia , Complexo Principal de Histocompatibilidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Homólogo/imunologiaRESUMO
BACKGROUND: In recent years, transplantation of islets and pancreas has become a viable option for patients debilitated with type I diabetes. The success of islet transplantation has been attributed to the ability to isolate high quality islets for transplantation and capacity to maintain the recipient's immunosuppressive levels within a specific target range following transplantation. The purpose of this study was to determine the role of pretransplant sensitization to human leukocyte antigen (HLA) in islet transplantation. METHODS: We retrospectively analyzed seven patients that were transplanted with islets under the auspices of the Juvenile Diabetes Research Foundation and Islet Cell Resource Center/National Institutes of Health. Humoral sensitization towards donor antigens both prior to and following islet transplantation was detected by FLOW panel reactive antibodies (PRA) and donor-specific cellular sensitization was detected by performing enzyme-linked immunospot assay analysis for cytokines interferon-gamma and interleukin-2. RESULTS: Our analysis demonstrates that humoral and cellular sensitization to histocompatibility antigens prior to and after islet transplantation are associated with the failure of transplanted islets CONCLUSION: Patient selection based on sensitization to donor HLA may be one of the factors crucial for the success of islet transplant. Further, in some patients, rejection of islets can be associated with sensitization to mismatched donor histocompatibility antigens.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Histocompatibilidade/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Diabetes Mellitus Tipo 1/sangue , Antígenos HLA/imunologia , Teste de Histocompatibilidade , Humanos , Insulina/sangue , Transplante Homólogo , Resultado do TratamentoRESUMO
Recently it was demonstrated that exposure of the developing brain during the period of synaptogenesis to drugs that block NMDA glutamate receptors or drugs that potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. All currently used general anesthetic agents have either NMDA receptor-blocking or GABA(A) receptor-enhancing properties. To induce or maintain a surgical plane of anesthesia, it is common practice in pediatric or obstetrical medicine to use agents from these two classes in combination. Therefore, the question arises whether this practice entails significant risk of inducing apoptotic neurodegeneration in the developing human brain. To begin to address this problem, we have administered to 7-d-old infant rats a combination of drugs commonly used in pediatric anesthesia (midazolam, nitrous oxide, and isoflurane) in doses sufficient to maintain a surgical plane of anesthesia for 6 hr, and have observed that this causes widespread apoptotic neurodegeneration in the developing brain, deficits in hippocampal synaptic function, and persistent memory/learning impairments.
Assuntos
Anestésicos/toxicidade , Encéfalo/efeitos dos fármacos , Deficiências da Aprendizagem/induzido quimicamente , Transtornos da Memória/induzido quimicamente , Doenças Neurodegenerativas/induzido quimicamente , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Doença Crônica , Combinação de Medicamentos , Antagonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Agonistas GABAérgicos/toxicidade , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Técnicas In Vitro , Isoflurano/toxicidade , Deficiências da Aprendizagem/complicações , Deficiências da Aprendizagem/patologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/complicações , Transtornos da Memória/patologia , Midazolam/toxicidade , Doenças Neurodegenerativas/complicações , Doenças Neurodegenerativas/patologia , Óxido Nitroso/toxicidade , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacosRESUMO
Standard islet potency testing uses transplantation of islets under the kidney capsule in diabetic severe combined immunodeficient (d-SCID) mice. Even though it is possible to achieve normoglycemia in the majority of recipients by this method, the surgical procedure, by itself, is technically difficult and associated with an appreciable mortality of animals. In addition, the spatially limited renal subcapsular site restricts the mass of islet tissue that can be transplanted. Matrigel basement membrane matrix (MATRIGEL), extracted from a mouse sarcoma, is rich in angiogenic growth factors and has been shown to support the growth of mammalian cells using murine models. In this report we demonstrate that subcutaneous islet transplantation with MATRIGEL can effectively achieve normoglycemia and that this is a simple and reproducible model for in vivo islet potency testing in d-SCID mice that overcomes many drawbacks of the conventional method of kidney subcapsular islet transplantation.
Assuntos
Transplante das Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/citologia , Animais , Glicemia/metabolismo , Colágeno , Combinação de Medicamentos , Transplante das Ilhotas Pancreáticas/métodos , Transplante das Ilhotas Pancreáticas/fisiologia , Laminina , Camundongos , Camundongos SCID , Modelos Animais , Proteoglicanas , Valores de ReferênciaRESUMO
BACKGROUND: Oleanolic acid (OA) is a natural plant-derived triterpenoid with potent anti-inflammatory properties. Since inflammatory cytokines released following islet transplantation hinders engraftment and long-term function, we determined the synergistic ability of OA to with Cyclosporine-A (CSA), a calcineurin inhibitor in improving islet allograft's function and survival. METHODS: C57BL/6 mice were rendered diabetic using streptozotocin (200mg/kg). BALB/c islets were transplanted under the kidney capsule alone (control) or along with administration of OA alone, CSA alone or a combination of both OA and CSA (OA+CSA). T-cell infiltration was analyzed by immunohistochemistry; cytokine concentration was analyzed by Luminex; T-cell cytokine phenotype was analyzed by ELISpot; and alloimmune response was analyzed by flow cytometry. RESULTS: OA+CSA markedly prolonged islet allograft survival compared to controls (37 ± 5 days vs. 8 ± 3 days). A significant decrease of CD4+ (34 ± 9 vs. 154 ± 42 cells/hpf) and CD8+ T-cellular (46 ± 22 vs. 224 ± 51 cells/hpf, p<0.0001) infiltration into the graft in OA+CSA treated mice compared to controls. A significant decrease in T cell infiltration was demonstrated in the OA+CSA cohort over either compound application individually. An increase in anti-inflammatory molecules, IL-10 (2.4-fold) and vascular endothelial growth factor (1.6-fold), along with decreased pro-inflammatory cytokines IFN-γ, IL-1ß (1.3-2.4-fold) and IL-17 (3.2-fold) was demonstrated. OA+CSA also significantly decreased the frequency of allo-specific T-cell responses. Development of antibodies against donor antigens was also delayed (39 vs 22 days; p<0.05) in the OA+CSA cohort over administration of either agent individually. CONCLUSIONS: OA and CSA exert synergistic effect towards enhancing islet allograft survival and function. This synergistic effect resulted in markedly reduced graft infiltrating cells with attenuation of inflammatory cytokine milieu leading to impairment of both cellular and humoral alloimmune responses. Therefore, novel therapeutic approaches involving combination of OA with calcineurin-inhibitor based immunosuppressant CSA will produce potential beneficial outcomes in clinical islet transplantation.
Assuntos
Inibidores de Calcineurina , Ciclosporina/farmacologia , Diabetes Mellitus Experimental/terapia , Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressores/farmacologia , Transplante das Ilhotas Pancreáticas , Ácido Oleanólico/farmacologia , Aloenxertos , Animais , Calcineurina/imunologia , Ciclosporina/agonistas , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Sinergismo Farmacológico , Imunossupressores/agonistas , Camundongos , Camundongos Endogâmicos BALB C , Ácido Oleanólico/agonistas , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
BACKGROUND: The IL-17 axis is implicated in pathogenesis of chronic rejection after human lung transplantation. Using a murine model of obliterative airway disease (OAD), we recently demonstrated that Abs to MHC class I antigens can induce immune responses to self-antigens that contributes to immunopathogenesis of chronic rejection. Using a murine model of OAD, we determined the role of IL-17 family members in induction of autoimmunity leading to OAD after ligation of MHC class I. METHODS: Anti-MHC class I or control antibodies (Abs) were administered intrabronchially to wild-type (WT) and IL-17a knock out (IL-17A-/-) C57BL/6. RESULTS: By day 30, anti-MHC I administered endobronchially in IL-17A-/- mice demonstrated significant reduction in cellular infiltration, a 36.8% reduction in CD4 T cells, 62.7% in CD11b macrophages, 37.5% in degree of fibrosis, 1.94 fold and 2.17 fold decrease in anti-KAT and anti-Col-V, respectively, when compared with wild-type mice. Analysis of lung infiltrating cells in anti-MHC I WT revealed increase in IL-17A (KAT:92+21,Col-V:103+19spm) and IL-17F (KAT:5.03%,Col-V:2.75%) secreting CD4+ T cells. However, administration of anti-MHC I in IL-17A-/- demonstrated increase only in IL-17F for KAT (13.70%) and Col-V (7.08%). Anti-IL-17(A-F) mAb administration after anti-MHC I abrogated OAD in both WT and IL-17A-/-. CONCLUSION: Our findings indicate that IL-17A and IL-17F secreted by CD4+Th17 cells specific to lung self-antigens are critical mediators of autoimmunity leading to the pathogenesis of OAD.
Assuntos
Autoanticorpos/imunologia , Autoimunidade , Bronquíolos/imunologia , Bronquiolite Obliterante/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Células Th17/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Biomarcadores/metabolismo , Bronquíolos/patologia , Bronquiolite Obliterante/genética , Bronquiolite Obliterante/patologia , Bronquiolite Obliterante/prevenção & controle , Antígeno CD11b/metabolismo , Colágeno Tipo V/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Interleucina-17/deficiência , Interleucina-17/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Fibrose Pulmonar/prevenção & controle , Transdução de Sinais , Fatores de Tempo , Tubulina (Proteína)/imunologiaRESUMO
BACKGROUND: Delayed angiogenesis remains a significant challenge to the survival of transplanted islets. In this study, using a murine model of subcutaneous islet transplantation with matrigel basement membrane matrix, we determined the role of the proangiogenic growth factors in enhancing the islet engraftment. METHODS: BALB/c islets were transplanted subcutaneously in growth factor reduced (GFR) or growth factor supplemented (GFS) matrigel into diabetic severe combined immunodeficient mice. GFS matrigel was prepared by supplementing GFR with proangiogenic factors, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The functioning grafts were harvested at 15 days and vessel formation was analyzed histopathologically. RESULTS: Our results demonstrate that suboptimal (250) islet equivalents in GFS-VEGF+HGF were able to restore normoglycemia, whereas those transplanted in GFR failed to reverse diabetes. Histopathology of the GFS-VEGF+HGF graft revealed 12±3 blood vessels per field, whereas GFR, GFS-VEGF, and GFS-HGF grafts had only 3±1, 6±2, and 4±1 blood vessels, respectively. Insulin staining demonstrated increased number of islets in matrigel supplemented with VEGF and HGF. Protein and mRNA analysis demonstrated enhanced intercellular adhesion molecule and vascular cell adhesion molecule within the islets when supplemented with both VEGF+HGF suggesting stable blood vessel formation. Transcription factors focal adhesion kinase phosphorylation and extracellular signal-regulated kinase1/2 phosphorylation were also increased (8-fold and 4.6-fold, respectively) when both the growth factors were present. There was weak expression of transcription factors when VEGF or HGF were supplemented alone. CONCLUSION: We conclude that proangiogenic growth factors, VEGF and HGF, synergistically enhance angiogenesis after islet transplantation leading to stable engraftment.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Transplante das Ilhotas Pancreáticas/métodos , Transplante das Ilhotas Pancreáticas/fisiologia , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Imunodeficiência Combinada Severa/cirurgia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Colágeno , Combinação de Medicamentos , Fator de Crescimento de Hepatócito/uso terapêutico , Hipoglicemia/prevenção & controle , Insulina/metabolismo , Laminina , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/efeitos dos fármacos , Proteoglicanas , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
BACKGROUND.: Oleanolic acid (OA) is a ubiquitous triterpenoid, with potent antioxidant and anti-inflammatory properties. Here, we tested whether these combined properties of OA can prevent nonimmunologic primary nonfunctioning and immunologic phenomena ascribed to graft rejection hence prolong islet allograft survival. METHODS.: Islet transplants were performed under kidney capsule of streptozotocin-induced diabetic C57BL/6 mice with BALB/c islets. Recipients were treated with 0.5 mg/day of OA intraperitoneally, and serum samples were collected once in 2 days and used for luminex, ELISA, and donor-specific antibody screening. Transplanted mice were killed at different time intervals to obtain splenocytes and kidney samples for ELISPOT, mixed leukocyte reaction, and immunohistochemical studies. RESULTS.: After transplantation, the decrement of blood glucose was significantly faster in mice receiving OA less than 2+/-1 days compared with untreated (4+/-2 days). OA prolonged survival of transplanted islets up to 23+/-3 days and reversed diabetes even with 250 islets. Treatment group showed increased serum interleukin (IL)-10 (twofold) and decreased inducible protein-10 and IL-4 (threefold) in luminex. Significantly reduced frequency of interferon-gamma (4.5-fold), IL-4 (3.5-fold), IL-2 (2.3-fold), and IL-17 (fourfold) producing T-cell populations were found in ELISPOT. OA-treated grafts had significant reduced and delayed infiltration of CD4+ and CD8+ T cells. OA also delayed donor-specific antibody generation up to 19 days after transplantation. Combined treatment with cyclosporine A, OA further prolonged the islet allograft survival to 34+/-3 days. CONCLUSIONS.: In conclusion, OA is an attractive, dietary nontoxic plant triterpenoid, which suppresses the production of proinflammatory cytokines and delays graft-specific immune responses to prolong islet allograft survival.
Assuntos
Sobrevivência de Enxerto/fisiologia , Transplante das Ilhotas Pancreáticas/fisiologia , Ácido Oleanólico/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Diabetes Mellitus Experimental , Sobrevivência de Enxerto/efeitos dos fármacos , Insulina/sangue , Transplante das Ilhotas Pancreáticas/imunologia , Transplante das Ilhotas Pancreáticas/métodos , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Ensaio de Cápsula Sub-Renal , Transplante Homólogo/imunologia , Transplante Homólogo/fisiologiaRESUMO
INTRODUCTION: Mammaglobin-A(mam-A) is expressed in over 80% of human breast tumors. We recently reported that mam-A DNA vaccination resulted in breast cancer immunity in a preclinical model. Here we investigated whether mam-A HLA-class-I tetramers could be used to monitor and define the role of CD8(+)cytotoxic T-lymphocytes(CTL) in mediating breast cancer immunity following mam-A DNA vaccination. STUDY DESIGN: Mam-A DNA vaccination was performed in HLA-A2(+)huCD8(+ )transgenic mice. HLA-A2 tetramers carrying the immunodominant mamA2.1 peptide were used to monitor CD8(+)CTL. Human breast cancer colonies were developed in immunodeficient SCID-beige mice. ELISPOT was used to correlate frequency of mamA2.1 tetramer(+)CD8(+)T cells and IFN-gamma production [spots per million cells (spm)] in human subjects. RESULTS: Vaccination of HLA-A2(+)huCD8(+) mice with mam-A DNA vaccine, but not empty vector, led to the expansion of mamA2.1 tetramer(+)CD8(+)T-cells in peripheral blood (<0.5% pre-vaccination compared to >2.0% post-vaccination). CD8(+)T cells from vaccinated mice specifically lysed UACC-812(HLA-A2(+)/mam-A(+), 25% lysis) but not MDA-MB-415(HLA-A2(-)/mam-A(+)) or MCF-7(HLA-A2(+)/mam-A(-)) breast cancer cells. Adoptive transfer of purified CD8(+)T cells from vaccinated mice into immunodeficient SCID-beige mice with established human breast cancer colonies led to tetramer(+)CD8(+ )T-cell infiltration with regression of UACC-812 but not MCF-7 tumors. HLA-A2(+) breast cancer patients revealed increased frequency of mamA2.1 tetramer(+)CD8(+ )T-cells compared to normal controls (2.86 +/- 0.8% vs. 0.71 +/- 0.1%, P = 0.01) that correlated with the IFN-gamma response to mamA2.1 peptide (48.1 +/- 20.9 vs. 2.9 +/- 0.8 spm, P = 0.03). CONCLUSIONS: CD8(+ )T-cells are crucial in mediating breast cancer immunity following mam-A DNA vaccination. Mam-A HLA-class-I tetramers can be effectively used to monitor development of CD8(+ )T-cells following mam-A vaccination.
Assuntos
Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Antígeno HLA-A2/imunologia , Proteínas de Neoplasias/imunologia , Uteroglobina/imunologia , Vacinas de DNA/imunologia , Animais , Citotoxicidade Imunológica , Feminino , Humanos , Mamoglobina A , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: A novel breast cancer-associated antigen, mammaglobin-A, is expressed in 80% of primary breast tumors. The characterization of immune responses against this highly expressed breast cancer-specific antigen would be of value in the development of new therapeutic strategies for breast cancer. METHODS: We developed an in vivo model using human leukocyte antigen-A*0201/human CD8+ (HLA-A2+/hCD8+) double-transgenic mice to define the epitopes and to study the level of protection acquired by mammaglobin-A cDNA vaccination toward mammaglobin-A+/HLA-A2+ breast cancer cell lines. Mammaglobin-A epitopes were identified using an HLA class I peptide binding prediction computer program, and their activity was verified using gamma interferon ELISPOT and cytotoxicity assays. RESULTS: We identified seven mammaglobin-A-derived candidate epitopes that bind the HLA-A*0201 molecule (Mam-A2.1-7). CD8+ cytotoxic T lymphocytes (CTLs) from HLA-A2+/hCD8+ mice reacted to the Mam-A2.1 (amino acids [aa] 83-92, LIYDSSLCDL), Mam-A2.2 (aa 2-10, KLLMVLMLA), Mam-A2.4 (aa 66-74, FLNQTDETL), and Mam-A2.6 (aa 32-40, MQLIYDSSL) epitopes. CD8+ CTLs from breast cancer patients also recognized a similar epitope pattern as did those in the HLA-A2+/hCD8 mice and reacted to the Mam-A2.1, Mam-A2.2, Mam-A2.3, Mam-A2.4, and Mam-A2.7 epitopes. Passive transfer of mammaglobin-A-reactive CTLs into SCID (severe combined immunodeficient) beige mice with actively growing mammaglobin-A+ tumors resulted in statistically significant regression (P<.001) in the growth of the tumors. CONCLUSIONS: The HLA-A2+/hCD8+ mouse represents a valuable animal model to characterize the HLA-A*0201-restricted CD8+ CTL immune response to mammaglobin-A in vivo, and the data reported here demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and/or prevention of breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Vacinas Anticâncer/farmacologia , Imunoterapia/métodos , Proteínas de Neoplasias/farmacologia , Uteroglobina/farmacologia , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , DNA Complementar/uso terapêutico , Modelos Animais de Doenças , Epitopos , Feminino , Humanos , Mamoglobina A , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/uso terapêutico , Linfócitos T Citotóxicos/imunologia , Uteroglobina/genética , Uteroglobina/uso terapêuticoRESUMO
We have previously demonstrated that human T cells responding to porcine islets are primarily CD4+ and recognized porcine major histocompatibility complex class I molecules through the indirect pathway of antigen presentation. To determine whether this mechanism is responsible for rejection of adult porcine islets xenografts, porcine islets from adult pigs were transplanted under the kidney capsule of streptozotocin-treated CD4-knockout (KO), CD8-KO, Ig-KO and normal C57BL/6 mice. Islet xenografts were acutely rejected with similar kinetics when transplanted into normal C57BL/6 (MST=17.6 +/- 3.5 days) and Ig-KO (MST=19.0 +/- 1.7 days) mice. Interestingly, islet xenografts were rejected significantly earlier when transplanted into CD8-KO mice as compared with normal C57BL/6 (MST=7.0 +/- 0.01 days, P=2 x 10-4). Histopathological analysis revealed classical acute cellular rejection with severe diffuse interstitial cellular infiltrates in all rejected islet xenografts. In contrast, islet xenografts were not rejected when transplanted into CD4-KO mice (MST >/= 100 days, P=1 x 10-9). Histopathological analysis revealed no cellular infiltrates and intact islet xenografts. CD4+ T cells from both normal C57BL/6 and CD8-KO xenograft recipients showed detectable proliferative responses to porcine islets in the presence but not in the absence of syngeneic antigen-presenting cells. In addition, the anti-islet proliferative responses observed in normal C57BL/6 mice were significantly lower than those observed in CD8-KO mice. IgG anti-porcine antibodies were readily detected in C57BL/6 and CD8-KO xenograft recipients but not in Ig-KO or CD4-KO recipients. These results indicate that indirectly activated CD4+ T cells mediate acute rejection of adult porcine islet xenografts and that xenoreactive CD8+ T cells and antibodies are not necessary in this process.
Assuntos
Reações Antígeno-Anticorpo/imunologia , Linfócitos T CD4-Positivos/imunologia , Rejeição de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Animais , Anticorpos Heterófilos/imunologia , Apresentação de Antígeno/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Suínos , Transplante HeterólogoRESUMO
A breast cancer-associated antigen, mammaglobin-A, is specifically expressed in 80% of primary breast tumors. The definition of immune responses against this highly expressed breast cancer-specific antigen should be of great value in the development of new therapeutic strategies for breast cancer. Thus, the purpose of this study was to identify HLA-A2-restricted mammaglobin-A-derived epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL). We identified seven mammaglobin-A-derived candidate epitopes that bind the HLA-A2 molecule (Mam-A2.1-7) by means of a HLA class I-peptide binding computer algorithm from the Bioinformatics & Molecular Analysis Section of the National Institutes of Health. Subsequently, we determined that CD8+ CTLs from breast cancer patients reacted to the Mam-A2.1 (83-92, LIYDSSLCDL), Mam-A2.2 (2-10, KLLMVLMLA), Mam-A2.3 (4-12, LMVLMLAAL), Mam-A2.4 (66-74, FLNQTDETL), and Mam-A2.7 (32-40, TINPQVSKT) epitopes using an IFN-gamma ELISPOT assay. Interestingly, healthy individuals also showed high reactivity to the Mam-A2.2 epitope. Two CD8+ CTL lines generated in vitro against TAP-deficient T2 cells loaded with the candidate epitopes showed significant cytotoxic activity against the Mam-A2.1-4 epitopes. These CD8+CTL lines recognized a HLA-A2+breast cancer cell line expressing the Mam-A2.1 epitope. In addition, DNA vaccination of HLA-A2+/human CD8+ double-transgenic mice with a DNA construct encoding the Mam-A2.1 epitope and the HLA-A2 molecule induced a significant expansion of epitope-specific CD8+ CTLs that recognize the same HLA- A2+/Mam-A2.1+ breast cancer cell line. In conclusion, these results demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and prevention of breast cancer.