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1.
Proc Natl Acad Sci U S A ; 116(47): 23682-23690, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31685610

RESUMO

Following antigen stimulation, naïve T cells differentiate into memory cells that mediate antigen clearance more efficiently upon repeat encounter. Donor-specific tolerance can be achieved in a subset of transplant recipients, but some of these grafts are rejected after years of stability, often following infections. Whether T cell memory can develop from a tolerant state and whether these formerly tolerant patients develop antidonor memory is not known. Using a mouse model of cardiac transplantation in which donor-specific tolerance is induced with costimulation blockade (CoB) plus donor-specific transfusion (DST), we have previously shown that systemic infection with Listeria monocytogenes (Lm) months after transplantation can erode or transiently abrogate established tolerance. In this study, we tracked donor-reactive T cells to investigate whether memory can be induced when alloreactive T cells are activated in the setting of tolerance. We show alloreactive T cells persist after induction of cardiac transplantation tolerance, but fail to acquire a memory phenotype despite becoming antigen experienced. Instead, donor-reactive T cells develop T cell-intrinsic dysfunction evidenced when removed from the tolerant environment. Notably, Lm infection after tolerance did not rescue alloreactive T cell memory differentiation or functionality. CoB and antigen persistence were sufficient together but not separately to achieve alloreactive T cell dysfunction, and conventional immunosuppression could substitute for CoB. Antigen persistence was required, as early but not late surgical allograft removal precluded the acquisition of T cell dysfunction. Our results demonstrate transplant tolerance-associated T cell-intrinsic dysfunction that is resistant to memory development even after Lm-mediated disruption of tolerance.


Assuntos
Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/imunologia , Subpopulações de Linfócitos T/imunologia , Imunologia de Transplantes , Aloenxertos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Fatores de Transcrição Forkhead/análise , Genes Reporter , Rejeição de Enxerto/imunologia , Antígenos H-2/imunologia , Transplante de Coração , Antígenos de Histocompatibilidade Classe II/imunologia , Memória Imunológica , Isoantígenos/imunologia , Listeria monocytogenes , Listeriose/imunologia , Transfusão de Linfócitos , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Complicações Pós-Operatórias/imunologia , Linfócitos T Reguladores/imunologia , Doadores de Tecidos
2.
In Vivo ; 33(5): 1477-1484, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31471395

RESUMO

BACKGROUND/AIM: Leukocyte activation is thought to be a major step in sepsis-induced pulmonary edema. We attempted to confirm whether pulmonary edema can be reproduced under intravital microscopy in a model of transfusion-related acute lung injury (TRALI) using MHC class I-specific antibody. MATERIALS AND METHODS: The surface pulmonary microcirculation was observed using an epi-fluorescence microscope through a thoracic window in 50 male mice. Monoclonal MHC class I-specific antibody (Ab) was administered to the animals, while the control group received saline. The leukocytes and macro-molecular leakage in the pulmonary circulation were analyzed. RESULTS: Leukocytes accumulated in the capillaries (52.5±12.7 leukocytes per designated area in Ab group vs. 20.8±3.1 in control). The air-containing alveolus area significantly shrank from 2,224.9±934.9 µm2 to 509.7±380.8 µm2 in the Ab group. CONCLUSION: Pulmonary edema develops rapidly following leukocyte accumulation in the lung. We confirmed that leukocyte accumulation without an underlining condition is sufficient to induce pulmonary edema.


Assuntos
Anticorpos Monoclonais/efeitos adversos , Antígenos H-2/imunologia , Edema Pulmonar/etiologia , Edema Pulmonar/patologia , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/patologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Biomarcadores , Biópsia , Contagem de Células Sanguíneas , Gasometria , Modelos Animais de Doenças , Injeções Intravenosas , Masculino , Camundongos , Imagem Óptica , Edema Pulmonar/diagnóstico por imagem
3.
Nature ; 572(7770): 481-487, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391585

RESUMO

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Doença Celíaca , Células Clonais/citologia , Células Clonais/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Antígenos H-2/imunologia , Humanos , Imunização , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Glicoproteína Associada a Mielina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Adulto Jovem
4.
Transpl Immunol ; 55: 101202, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30904624

RESUMO

Graft-versus-host disease (GVHD) and transplant rejection as a result of host-versus-graft (HVG) response have remained two major complications of allogeneic hematopoietic stem cell transplantation (allo-HSCT). When donors are partially HLA-mismatched unrelated or haploidentical related, their severity correlates with the degree of HLA disparity. Specific elimination of alloreactive donor or recipient T cells targeting the mismatched HLA products could markedly alleviate both complications while only minimally affecting graft-versus-tumor (GVT) response or engraftment. To redirect human CD8 T cells against alloreactive CD8 T cells we electroporate these cells with in-vitro-transcribed mRNA encoding MHC-I heavy chains fused with the signaling portion of CD3ζ. Here we show that peripheral blood human CD8 T cells expressing H-2Kb/CD3ζ or H-2Kd/CD3ζ respond to anti-MHC-I stimuli in a strictly specific manner. This study paves the way for further advancing this approach as a means to dampen GVHD and HVG that are caused by HLA disparity in allo-HSCT.


Assuntos
Complexo CD3/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Antígenos H-2/imunologia , Complexo CD3/genética , Linfócitos T CD8-Positivos/patologia , Eletroporação , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Antígenos H-2/genética , Humanos , Células Jurkat
5.
Immunol Cell Biol ; 97(6): 552-562, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30768806

RESUMO

The NOD-like receptor (NLR) family plays an important role in innate immunity. Class II transactivator and NOD-like receptor caspase activation and recruitment domain CARD containing 5 (NLRC5) are unusual members of the NLR family that instead of recognizing pathogen-associated or damage-associated molecular patterns, form enhanceosomes with adaptor molecules and modulate major histocompatibility complex (MHC) class II and MHC class I expression, respectively. While NLRC5 has been shown to play a role during intracellular pathogen infection and tumor cell immune evasion, its role in regulating antigen-specific CD8+ T-cell responses at the intestinal mucosa has not been investigated. Here, we take advantage of the rotavirus model in adult mice to dissect the impact of NLRC5 on CD8+ T-cell responses to this viral infection at the gut mucosa. We show that while Nlrc5-/- mice exhibited normal proportions of T-cell subpopulations in the intraepithelial and lamina propria compartments, these mice had decreased baseline MHC class I expression on various immune cells in the lamina propria. Upon rotavirus infection, Nlrc5 deficiency resulted in impaired H2-Kb -restricted antigen-specific CD8+ T-cell responses, which were recapitulated in mice deficient for Nlrc5 within the dendritic cell compartment. The impaired CD8+ T-cell response in Nlrc5-/- mice was not significant enough to impact viral titers, suggesting compensation in Nlrc5-/- mice, perhaps as a result of higher numbers of activated B cells in the mesenteric lymph nodes and normal rotavirus-specific immunoglobulin A responses. Collectively, our results demonstrate a minor role for NLRC5 in modulating H2-Kb -restricted antigen-specific CD8+ T-cell responses in the small intestine during rotavirus infection in adult mice.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Mucosa Intestinal/imunologia , Infecções por Rotavirus/imunologia , Rotavirus/fisiologia , Animais , Apresentação do Antígeno , Antígenos Virais/imunologia , Células Cultivadas , Antígenos H-2/metabolismo , Epitopos Imunodominantes/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Carga Viral
6.
Immunol Cell Biol ; 97(3): 326-339, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30537346

RESUMO

Class Ib major histocompatibility complex (MHC) is an extended family of molecules, which demonstrate tissue-specific expression and presentation of monomorphic antigens. These characteristics tend to imbue class Ib MHC with unique functions. H2-Q10 is potentially one such molecule that is overexpressed in the liver but its immunological function is not known. We have previously shown that H2-Q10 is a ligand for the natural killer cell receptor Ly49C and now, using H2-Q10-deficient mice, we demonstrate that H2-Q10 can also stabilize the expression of Qa-1b. In the absence of H2-Q10, the development and maturation of conventional hepatic natural killer cells is disrupted. We also provide evidence that H2-Q10 is a new high affinity ligand for CD8αα and controls the development of liver-resident CD8αα γδT cells. These data demonstrate that H2-Q10 has multiple roles in the development of immune subsets and identify an overlap of recognition within the class Ib MHC that is likely to be relevant to the regulation of immunity.


Assuntos
Antígenos H-2/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores Imunológicos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Biomarcadores , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Antígenos H-2/genética , Antígenos H-2/metabolismo , Imunomodulação/genética , Imunofenotipagem , Células Matadoras Naturais/citologia , Ligantes , Fígado/imunologia , Fígado/metabolismo , Camundongos , Ligação Proteica , Subpopulações de Linfócitos T/citologia
7.
Front Immunol ; 9: 2348, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30374353

RESUMO

The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.


Assuntos
Antígenos Virais/imunologia , Glicoproteínas/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Proteínas Virais/imunologia , Transferência Adotiva , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos CD8/metabolismo , Citocinas/metabolismo , Epitopos/química , Epitopos/imunologia , Glicoproteínas/química , Glicoproteínas/genética , Antígenos H-2/imunologia , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/virologia , Camundongos , Camundongos Transgênicos , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Transdução de Sinais , Proteínas Virais/química , Proteínas Virais/genética
8.
Biochem Biophys Res Commun ; 502(2): 226-231, 2018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29792863

RESUMO

Human infections by type B influenza virus constitute about 25% of all influenza cases. The viral hemagglutinin is comprised of two subunits, HA1 and HA2. While HA1 is constantly evolving in an unpredictable fashion, the HA2 subunit is highly conserved, making it a potential candidate for a universal vaccine. However, immunodominant epitopes in the HA2 subunit remain largely unknown. To delineate MHC Class I epitopes, we first identified 9-mer H-2Kd-restricted CD8 T cell epitopes in the HA2 domain by in silico analyses, followed by evaluating the immunodominance of these peptides in mice challenged with the virus. Of three peptides selected through in silico analysis, the universally conserved peptide, YYSTAASSL (B/HA2-190), possessed the highest predicted binding affinity to MHC Class I and was most effective in inducing IL-2 and TNF-α in mouse splenocytes. Importantly, the peptide demonstrated best capability of stimulating peptide-specific ex-vivo cytotoxicity against target cells. Taken together, this finding would be of value for assessment of cell-mediated immune responses elicited by vaccines based on the highly conserved HA2 stalk domain.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza B/imunologia , Animais , Antígenos CD8/química , Simulação por Computador , Feminino , Antígenos H-2/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Imunidade Celular , Epitopos Imunodominantes/química , Vírus da Influenza B/química , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos DBA , Modelos Imunológicos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Subunidades Proteicas , Linfócitos T Citotóxicos/imunologia , Fator de Necrose Tumoral alfa/biossíntese
9.
FEBS J ; 285(15): 2728-2745, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29673068

RESUMO

In the first half of the 20th century, the major histocompatibility complex (MHC) of the laboratory mouse, the H-2 complex, was defined by a combination of serology and genetics. In the second half of the 20th century, its human counterpart, the human leukocyte antigen (HLA) complex was similarly defined and shown to mediate rejection of allogeneic kidney grafts. The clinical relevance of the transplantation antigens created the field of transplant immunology, which aimed to reduce graft rejection by HLA matching of transplant donors and recipients, and to use immunosuppressive drugs to prevent and treat rejection. Because tissue transplantation is not a natural phenomenon, the relevance of the MHC for immunology and immune defense against microbial pathogens was frequently questioned. In the 1970s, the general observation that cytotoxic T-cell responses to viral infection required recognition of both a viral antigen and a transplantation antigen argued for the immunological importance of the MHC. Proving this point was not achieved until close to the end of the 20th century. This required detailed biochemical and structural analysis of the transplantation antigens, the viral antigens, and the T-cell receptors that recognized them. This century of research culminated in 1996 with the three-dimensional crystallographic structure of the complex of these three components. In this complex is MAC, the very first HLA antigen to be detected and now more formally known as HLA-A*02:01.


Assuntos
Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/fisiologia , Transplante de Tecidos , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Feminino , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Antígenos H-2/química , Antígenos de Histocompatibilidade/história , História do Século XX , Humanos , Camundongos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/imunologia , Gravidez , Transplante de Pele , Solubilidade , Linfócitos T Citotóxicos/imunologia , Microglobulina beta-2/imunologia
10.
Cancer Immunol Res ; 6(6): 636-644, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29615400

RESUMO

With the advancement of personalized cancer immunotherapies, new tools are needed to identify tumor antigens and evaluate T-cell responses in model systems, specifically those that exhibit clinically relevant tumor progression. Key transgenic mouse models of breast cancer are generated and maintained on the FVB genetic background, and one such model is the mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) mouse-an immunocompetent transgenic mouse that exhibits spontaneous mammary tumor development and metastasis with high penetrance. Backcrossing the MMTV-PyMT mouse from the FVB strain onto a C57BL/6 genetic background, in order to leverage well-developed C57BL/6 immunologic tools, results in delayed tumor development and variable metastatic phenotypes. Therefore, we initiated characterization of the FVB MHC class I H-2q haplotype to establish useful immunologic tools for evaluating antigen specificity in the murine FVB strain. Our study provides the first detailed molecular and immunoproteomic characterization of the FVB H-2q MHC class I alleles, including >8,500 unique peptide ligands, a multiallele murine MHC peptide prediction tool, and in vivo validation of these data using MMTV-PyMT primary tumors. This work allows researchers to rapidly predict H-2 peptide ligands for immune testing, including, but not limited to, the MMTV-PyMT model for metastatic breast cancer. Cancer Immunol Res; 6(6); 636-44. ©2018 AACR.


Assuntos
Biologia Computacional/métodos , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Antígenos de Histocompatibilidade/imunologia , Neoplasias/imunologia , Peptídeos/imunologia , Software , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatografia Líquida , Modelos Animais de Doenças , Feminino , Antígenos H-2/química , Antígenos H-2/genética , Antígenos H-2/imunologia , Haplótipos , Humanos , Ligantes , Neoplasias Mamárias Animais , Neoplasias Mamárias Experimentais , Espectrometria de Massas , Camundongos , Ligação Proteica
11.
Front Immunol ; 9: 3173, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30693005

RESUMO

Natural killer (NK) cell function is regulated by a balance between activating and inhibitory receptors, but the details of this receptor interplay are not extensively understood. We developed a flow cytometry-based assay system in which Ca2+ flux downstream of antibody-mediated activating receptor triggering was studied in the presence or absence of inhibitory receptor co-crosslinking. We show that the inhibitory influence on activating receptor-induced Ca2+ flux is quantitatively regulated, both on murine and human NK cells. Furthermore, both activating and inhibitory receptors operate in an additive way, suggesting that a fine-tuned balance between activating and inhibitory receptors regulate proximal NK cell signaling. We also demonstrate that murine NK cell expression of H2Dd lowered the capacity of Ly49A to deliver inhibitory signals after antibody crosslinking, suggesting that the cis interaction between H2Dd and Ly49A reduce the signaling capacity of Ly49A in this setting. Finally, we show that priming of NK cells by IL-15 rapidly augments Ca2+ flux after activating receptor signaling without attenuating the potential of inhibitory receptors to reduce Ca2+ flux. Our data shed new light on NK cell inhibition and raises new questions for further studies.


Assuntos
Cálcio/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Receptores KIR/metabolismo , Receptores de Células Matadoras Naturais/metabolismo , Animais , Membrana Celular/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Expressão Gênica , Antígenos H-2/genética , Antígenos H-2/imunologia , Humanos , Ativação Linfocitária/genética , Camundongos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligação Proteica , Transdução de Sinais
12.
J Immunol Res ; 2018: 2942679, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30596107

RESUMO

Overexpression of metastasis-associated protein 1 (MTA1) has been observed in many human malignancies and is significantly related to tumor invasion and metastasis, therapeutic resistance to radiation and chemotherapy, making MTA1 an ideal candidate tumor antigen. We identified several human leukocyte antigen- (HLA-) A2-restricted epitopes in MTA1 and evaluated their binding ability to HLA-A∗0201 molecules. Subsequently, a recombinant fragment encompassing the dominant epitopes, MTA1(1-283), was expressed, and the abilities of the selected epitopes of MTA1 and the MTA1(1-283) fragment to induce cytotoxic T lymphocytes (CTLs) were examined. Our results indicated that the epitopes and MTA1(1-283) fragment elicited HLA-A2-restricted and antigen-specific CTL responses both in vitro and in vivo. The new epitopes identified here may help promote the development of new therapeutic vaccines for HLA-A2+ patients expressing MTA1.


Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Vacinas Anticâncer/imunologia , Neoplasias Colorretais/imunologia , Epitopos de Linfócito T/imunologia , Histona Desacetilases/imunologia , Proteínas Repressoras/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Apresentação do Antígeno , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Mapeamento de Epitopos , Antígenos H-2/genética , Antígeno HLA-A2/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Ligação Proteica , Especificidade do Receptor de Antígeno de Linfócitos T
13.
Mol Immunol ; 93: 115-124, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29175591

RESUMO

Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti-cancer immunity. However, a viable approach to use antibody-complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof-of-concept we investigated if anti-tetanus IgG could induce potent cross-presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA-specific CD8+ T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide-peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high-titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MTTE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti-MTTE antibodies with MTTE-containing conjugates are able to induce DC and T cell activation using model antigens.


Assuntos
Apresentação Cruzada/imunologia , Ovalbumina/imunologia , Toxoide Tetânico/imunologia , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito B , Epitopos de Linfócito T/imunologia , Antígenos H-2/imunologia , Humanos , Hibridomas , Imunoconjugados/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Toxoide Tetânico/química , Vacinação
14.
Circulation ; 137(5): 488-503, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-28775077

RESUMO

BACKGROUND: Cardiac transplantation is an excellent treatment for end-stage heart disease. However, rejection of the donor graft, in particular, by chronic rejection leading to cardiac allograft vasculopathy, remains a major cause of graft loss. The lymphatic system plays a crucial role in the alloimmune response, facilitating trafficking of antigen-presenting cells to draining lymph nodes. The encounter of antigen-presenting cells with T lymphocytes in secondary lymphoid organs is essential for the initiation of alloimmunity. Donor lymphatic vessels are not anastomosed to that of the recipient during transplantation. The pathophysiology of lymphatic disruption is unknown, and whether this disruption enhances or hinders the alloimmune responses is unclear. Although histological analysis of lymphatic vessels in donor grafts can yield information on the structure of the lymphatics, the function following cardiac transplantation is poorly understood. METHODS: Using single-photon emission computed tomography/computed tomography lymphoscintigraphy, we quantified the lymphatic flow index following heterotrophic cardiac transplantation in a murine model of chronic rejection. RESULTS: Ten weeks following transplantation of a minor antigen (HY) sex-mismatched heart graft, the lymphatic flow index was significantly increased in comparison with sex-matched controls. Furthermore, the enhanced lymphatic flow index correlated with an increase in donor cells in the mediastinal draining lymph nodes; increased lymphatic vessel area; and graft infiltration of CD4+, CD8+ T cells, and CD68+ macrophages. CONCLUSIONS: Chronic rejection results in increased lymphatic flow from the donor graft to draining lymph nodes, which may be a factor in promoting cellular trafficking, alloimmunity, and cardiac allograft vasculopathy.


Assuntos
Movimento Celular , Rejeição de Enxerto/imunologia , Transplante de Coração , Linfa/imunologia , Vasos Linfáticos/imunologia , Aloenxertos , Animais , Doença Crônica , Modelos Animais de Doenças , Feminino , Rejeição de Enxerto/diagnóstico por imagem , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Antígenos H-2/genética , Antígenos H-2/imunologia , Histocompatibilidade , Linfangiogênese , Vasos Linfáticos/diagnóstico por imagem , Vasos Linfáticos/patologia , Linfocintigrafia/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Fatores de Tempo
15.
PLoS One ; 12(12): e0189584, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29253009

RESUMO

Post-translational modifications significantly broaden the epitope repertoire for major histocompatibility class I complexes (MHC-I) and may allow viruses to escape immune recognition. Lymphocytic choriomeningitis virus (LCMV) infection of H-2b mice generates CD8+ CTL responses directed towards several MHC-I-restricted epitopes including the peptides GP92 (CSANNSHHYI) and GP392 (WLVTNGSYL), both with a N-glycosylation site. Interestingly, glycosylation has different effects on the immunogenicity and association capacity of these two epitopes to H-2Db. To assess the structural bases underlying these functional results, we determined the crystal structures of H-2Db in complex with GP92 (CSANNSHHYI) and GP392 (WLVTNGSYL) to 2.4 and 2.5 Å resolution, respectively. The structures reveal that while glycosylation of GP392 most probably impairs binding, the glycosylation of the asparagine residue in GP92, which protrudes towards the solvent, possibly allows for immune escape and/or forms a neo-epitope that may select for a different set of CD8 T cells. Altogether, the presented results provide a structural platform underlying the effects of post-translational modifications on epitope binding and/or immunogenicity, resulting in viral immune escape.


Assuntos
Apresentação do Antígeno , Antígenos Virais/imunologia , Antígenos H-2/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Asparagina/química , Linfócitos T CD8-Positivos/metabolismo , Cristalografia por Raios X , Epitopos/imunologia , Glicosilação , Camundongos , Peptídeos/imunologia , Processamento de Proteína Pós-Traducional , Solventes , Linfócitos T Citotóxicos/imunologia
16.
Cancer Immunol Res ; 5(10): 908-919, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28851693

RESUMO

To understand why vaccine-activated tumor-specific T cells often fail to generate antitumor effects, we studied two α-fetoprotein-specific CD8+ T cells (Tet499 and Tet212) that had different antitumor effects. We found that Tet499 required high antigen doses for reactivation, but could survive persistent antigen stimulation and maintain their effector functions. In contrast, Tet212 had a low threshold of reactivation, but underwent exhaustion and apoptosis in the presence of persistent antigen. In vivo, Tet499 cells expanded more than Tet212 upon reencountering antigen and generated stronger antitumor effects. The different antigen responsiveness and antitumor effects of Tet212 and Tet499 cells correlated with their activation and differentiation states. Compared with Tet212, the population of Tet499 cells was less activated and contained more stem-like memory T cells (Tscm) that could undergo expansion in vivo The TCR signaling strength on Tet499 was weaker than Tet212, correlating with more severe Tet499 TCR downregulation. Weak TCR signaling may halt T-cell differentiation at the Tscm stage during immune priming and also explains why Tet499 reactivation requires a high antigen dose. Weak TCR signaling of Tet499 cells in the effector stage will also protect them from exhaustion and apoptosis when they reencounter persistent antigen in tumor lesion, which generates antitumor effects. Further investigation of TCR downregulation and manipulation of TCR signaling strength may help design cancer vaccines to elicit a mix of tumor-specific CD8+ T cells, including Tscm, capable of surviving antigen restimulation to generate antitumor effects. Cancer Immunol Res; 5(10); 908-19. ©2017 AACR.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/imunologia , Memória Imunológica , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Animais , Antígenos de Neoplasias/imunologia , Apoptose/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Antígenos H-2/imunologia , Humanos , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Peptídeos/imunologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , alfa-Fetoproteínas/química , alfa-Fetoproteínas/imunologia
17.
J Am Heart Assoc ; 6(7)2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28711866

RESUMO

BACKGROUND: T cells are found in atherosclerotic plaques, with evidence supporting a potential role for CD8+ T cells in atherogenesis. Prior studies provide evidence of low-density lipoprotein and apoB-100 reactive T cells, yet specific epitopes relevant to the disease remain to be defined. The current study was undertaken to identify and characterize endogenous, antigen-specific CD8+ T cells in atherosclerosis. METHODS AND RESULTS: A peptide fragment of apoB-100 that tested positive for binding to the mouse MHC-I allele H2Kb was used to generate a fluorescent-labeled H2Kb pentamer and tested in apoE-/- mice. H2Kb pentamer(+)CD8+ T cells were higher in apoE-/- mice fed an atherogenic diet compared with those fed a normal chow. H2Kb pentamer (+)CD8+ T cells in atherogenic diet-fed mice had significantly increased effector memory phenotype with a shift in Vß profile. H2Kb pentamer blocked lytic activity of CD8+ T cells from atherogenic diet-fed mice. Immunization of age-matched apoE-/- mice with the apoB-100 peptide altered the immune-dominant epitope of CD8+ T cells and reduced atherosclerosis. CONCLUSIONS: Our study provides evidence of a self-reactive, antigen-specific CD8+ T-cell population in apoE-/- mice. Immune modulation using the peptide antigen reduced atherosclerosis in apoE-/- mice.


Assuntos
Doenças da Aorta/prevenção & controle , Apolipoproteína B-100/administração & dosagem , Apolipoproteína B-100/imunologia , Aterosclerose/prevenção & controle , Linfócitos T CD8-Positivos/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Vacinas/administração & dosagem , Vacinas/imunologia , Animais , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica , Modelos Animais de Doenças , Predisposição Genética para Doença , Antígenos H-2/imunologia , Imunização , Epitopos Imunodominantes , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Fenótipo , Placa Aterosclerótica , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia
18.
Sci Rep ; 7: 45775, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28374766

RESUMO

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation.


Assuntos
Sistemas CRISPR-Cas , Complexo Principal de Histocompatibilidade/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Fibroblastos , Citometria de Fluxo , Antígenos H-2/genética , Antígenos H-2/imunologia , Macrófagos/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos BALB C
19.
Proc Natl Acad Sci U S A ; 114(5): 1099-1104, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28096390

RESUMO

Maternal microchimerism (MMc) has been associated with development of allospecific transplant tolerance, antitumor immunity, and cross-generational reproductive fitness, but its mode of action is unknown. We found in a murine model that MMc caused exposure to the noninherited maternal antigens in all offspring, but in some, MMc magnitude was enough to cause membrane alloantigen acquisition (mAAQ; "cross-dressing") of host dendritic cells (DCs). Extracellular vesicle (EV)-enriched serum fractions from mAAQ+, but not from non-mAAQ, mice reproduced the DC cross-dressing phenomenon in vitro. In vivo, mAAQ was associated with increased expression of immune modulators PD-L1 (programmed death-ligand 1) and CD86 by myeloid DCs (mDCs) and decreased presentation of allopeptide+self-MHC complexes, along with increased PD-L1, on plasmacytoid DCs (pDCs). Remarkably, both serum EV-enriched fractions and membrane microdomains containing the acquired MHC alloantigens included CD86, but completely excluded PD-L1. In contrast, EV-enriched fractions and microdomains containing allopeptide+self-MHC did not exclude PD-L1. Adoptive transfer of allospecific transgenic CD4 T cells revealed a "split tolerance" status in mAAQ+ mice: T cells recognizing intact acquired MHC alloantigens proliferated, whereas those responding to allopeptide+self-MHC did not. Using isolated pDCs and mDCs for in vitro culture with allopeptide+self-MHC-specific CD4 T cells, we could replicate their normal activation in non-mAAQ mice, and PD-L1-dependent anergy in mAAQ+ hosts. We propose that EVs provide a physiologic link between microchimerism and split tolerance, with implications for tumor immunity, transplantation, autoimmunity, and reproductive success.


Assuntos
Quimerismo , Células Dendríticas/imunologia , Vesículas Extracelulares/imunologia , Tolerância Imunológica , Transferência Adotiva , Animais , Antígeno B7-2/biossíntese , Antígeno B7-2/imunologia , Antígeno B7-H1/biossíntese , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Transfusão Feto-Materna/imunologia , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígeno de Histocompatibilidade H-2D/genética , Antígeno de Histocompatibilidade H-2D/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Isoantígenos/imunologia , Masculino , Troca Materno-Fetal/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Imunológicos , Gravidez , Especificidade do Receptor de Antígeno de Linfócitos T
20.
J Hepatol ; 66(4): 765-777, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27914923

RESUMO

BACKGROUND & AIMS: Induction of donor-specific immune tolerance is a good alternative to chronic life-long immunosuppression for transplant patients. Donor major histocompatibility complex (MHC) molecules represent the main targets of the allogeneic immune response of transplant recipients. Liver targeted gene transfer with viral vectors induces tolerance toward the encoded antigen. The aim of this work was to determine whether alloantigen gene transfer to hepatocytes induces tolerance and promotes graft acceptance. METHODS: C57BL/6 (H-2b) mice were treated with adeno-associated viral (AAV) vector targeting the expression of the MHC class I molecule H-2Kd to hepatocytes, before transplantation with fully allogeneic pancreatic islet from BALB/c mice (H-2d). RESULTS: AAV H-2Kd treated mice were tolerant to the alloantigen, as demonstrated by its long-term expression by the hepatocytes, even after a highly immunogenic challenge with an adenoviral vector. After chemical induction of diabetes, the AAV treated mice had significantly delayed rejection of fully allogeneic pancreatic islet grafts, with more than 40% of recipients tolerant (>100days). AAV-mediated expression of H-2Kd in the liver induced the local expansion of CD8+ T lymphocytes with allo-specific suppressive properties. The adoptive transfer of these liver-generated CD8+ Tregs into naive diabetic mice promoted the long-term survival of allogeneic pancreatic islet grafts. CONCLUSION: AAV-mediated long-term expression of a single MHC class I molecule in the liver induces the generation of a subset of allo-specific CD8+ Treg cells, which promote tolerance toward fully allogeneic graft. Liver gene transfer represents a promising strategy for in vivo induction of donor-specific tolerance. LAY SUMMARY: The liver has a special immune system, biased toward tolerance. In this study, we investigated the possibility of harnessing this property of the liver to induce tolerance to an allogeneic transplantation. We demonstrate for the first time that the in vivo gene transfer of an allogeneic antigen with an adeno-associated viral vector to mouse hepatocytes induces the expansion of a population of CD8+ regulatory T lymphocytes. These Tregs are then instrumental in preventing the rejection of allogeneic pancreatic islets transplanted in these animals. Allogeneic transplantation is the main treatment for the end-stage diseases of a number of organs. Life-long immunosuppressive treatments are still required to limit graft rejection, and these treatments exhibit serious side effects. Our present findings open a new avenue for promoting allo-specific tolerance via in vivo induction of CD8+ Treg expansion.


Assuntos
Hepatócitos/imunologia , Imunossupressão/métodos , Transplante das Ilhotas Pancreáticas/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/terapia , Técnicas de Transferência de Genes , Vetores Genéticos , Sobrevivência de Enxerto/imunologia , Antígenos H-2/genética , Isoantígenos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Parvovirinae/genética , Doadores de Tecidos , Transplante Homólogo
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