RESUMO
Little is known about whether presentation of endogenous and exogenous hepatitis B virus (HBV) surface antigens on APCs targeted by vaccination and/or virus-harboring hepatocytes influences de novo priming of CD8(+) T cells. We showed that surface antigen-expressing transfectants exclusively display a K(b) /S190 epitope, whereas cells pulsed with recombinant surface particles (rSPs) exclusively present a K(b) /S208 epitope to CD8(+) T cells. The differential presentation of these epitopes largely reflects the selective, but not exclusive, priming of K(b) /S190- and K(b) /S208-specific T cells in C57BL/6 mice by endogenous/DNA- or exogenous/protein-based vaccines, respectively. Silencing the K(b) /S190 epitope (K(b) /S190V194F ) in antigen-expressing vectors rescued the presentation of the K(b) /S208 epitope in stable transfectants and significantly enhanced priming of K(b) /S208-specific T cells in C57BL/6 mice. A K(b) /S190-mediated immunodominance operating in surface antigen-expressing cells, but not in rSP-pulsed cells, led to an efficient suppression in the presentation of the K(b) /S208 epitope and a consequent decrease in the priming of K(b) /S208-specific T cells. This K(b) /S190-mediated immunodominance also operated in 1.4HBV-S(mut) transgenic (tg) hepatocytes selectively expressing endogenous surface antigens and allowed priming of K(b) /S208- but not K(b) /S190-specific T cells in 1.4HBV-S(mut) tg mice. However, IFN-γ(+) K(b) /S208-specific T cells could not inhibit HBV replication in the liver of 1.4HBV-S(mut) tg mice. These results have practical implications for the design of T-cell-stimulating therapeutic vaccines.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular Tumoral , Feminino , Antígenos H-2/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Tumor dormancy is a clinical phenomenon related to immune equilibrium during cancer immunoediting. The mechanisms involved in dormant metastases are poorly understood due to the lack of preclinical models. Here, we present a nontransgenic mouse model in which spontaneous metastases remain in permanent immunomediated dormancy with no additional antitumor treatment. After the injection of a GR9-B11 mouse fibrosarcoma clone into syngeneic BALB/c mice, all animals remained free of spontaneous metastases at the experimental endpoints (3-8 months) but also as long as 24 months after tumor cell injection. Strikingly, when tumor-bearing mice were immunodepleted of T lymphocytes or asialo GM1-positive cells, the restraint on dormant disseminated metastatic cells was relieved and lung metastases progressed. Immunostimulation was documented at both local and systemic levels, with results supporting the evidence that the immune system was able to restrain spontaneous metastases in permanent dormancy. Notably, the GR9-B11 tumor clone did not express MHC class I molecules on the cell surface, yet all metastases in immunodepleted mice were MHC class I-positive. This model system may be valuable for more in-depth analyses of metastatic dormancy, offering new opportunities for immunotherapeutic management of metastatic disease.
Assuntos
Metástase Neoplásica/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Fibrossarcoma/imunologia , Fibrossarcoma/patologia , Gangliosídeo G(M1)/fisiologia , Antígenos H-2/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Skin or organ allograft rejection is dependent on noncytotoxic CD4(+) T cells, but the mechanisms of recognition and rejection remain elusive. Previously, we demonstrated C57BL/6 (H-2D(b)K(b)) macrophage-mediated, cell-to-cell contact-dependent, d haplotype-specific lysis of allografts (e.g., BALB/c skin and Meth A cells; H-2D(d)K(d)) in the rejection site and isolated two cDNA clones encoding receptors on macrophages for H-2D(d) and H-2K(d), macrophage major histocompatibility complex receptor (MMR) 1 and 2, respectively. METHODS: To elucidate the role of MMR2 and T-cell receptors (TCRs) in graft rejection, we generated MMR2 knockout (KO) mice on a C57BL/6 background and transplanted D(d), K(d), or D(d)K(d) transgenic C57BL/6 skin or EL-4 lymphoma cells onto or into these KO mice. RESULTS: MMR2 KO mice lacking MMR2 mRNA or protein expression in their monocytes had no obvious abnormalities in terms of cell number in or composition of their lymphoid tissues or in T lymphocyte responses to alloantigen or nonalloantigen, whereas they failed to reject K(d) transgenic skin grafts. Surprisingly, they also lacked MMR1 mRNA and protein expression in their monocytes and failed to reject D(d) or D(d)K(d) transgenic skin grafts. However, they did reject skin grafts from mice expressing H-2I(d), minor H(d), or third-party major histocompatibility complex. On the contrary, D(d)-, K(d)-, or D(d)K(d)-EL-4 cells injected intradermally or intraperitoneally into MMR2 KO mice were rejected by TCR(αß)(+)/CD8(+) T cells in a transgene number-dependent and MMR-independent manner. CONCLUSIONS: These results demonstrate that MMRs on monocytes/macrophages and TCRs on cytotoxic T lymphocytes in mice were essential for recognition and rejection of allografted skin and lymphoma, respectively.
Assuntos
Rejeição de Enxerto/etiologia , Linfoma/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Imunológicos/fisiologia , Transplante de Pele/efeitos adversos , Animais , Antígenos H-2/fisiologia , Células HEK293 , Antígeno de Histocompatibilidade H-2D/fisiologia , Humanos , Hipersensibilidade Tardia/etiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
Innate memory phenotype (IMP) CD8(+) T cells are nonconventional αß T cells exhibiting features of innate immune cells and are significantly increased in the absence of ITK. Their developmental path and function are not clear. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8(+) T cells using bone marrow chimeras. Wild-type bone marrow gives rise to IMP CD8(+) T cells in MHCI(-/-) recipients, resembling those in Itk(-/-) mice, but distinct from those derived via homeostatic proliferation, and independent of recipient thymus. In contrast, MHCI(-/-) bone marrow does not lead to IMP CD8(+) T cells in wild-type recipients. OTI IMP CD8(+) T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific "naive" CD8(+) IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Antígenos H-2/fisiologia , Homeostase/imunologia , Imunidade Inata , Memória Imunológica , Ativação Linfocitária/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Antígenos H-2/genética , Hematopoese/genética , Hematopoese/imunologia , Imunofenotipagem , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Quimera por Radiação/imunologia , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
Cellular peptides generated by proteasomal degradation of proteins in the cytosol and destined for presentation by MHC class I (MHC-I) are associated with several chaperones. Heat shock proteins 70, 90, and the TCP-1 ring complex have been implicated as important cytosolic players for chaperoning these peptides. In this study, we report that gp96 and calreticulin are essential for chaperoning peptides in the endoplasmic reticulum. Importantly, we demonstrate that cellular peptides are transferred sequentially from gp96 to calreticulin and then to MHC-I forming a relay line. Disruption of this relay line by removal of gp96 or calreticulin prevents the binding of peptides by MHC-I and hence presentation of the MHC-I-peptide complex on the cell surface. Our results are important for understanding how peptides are processed and trafficked within the endoplasmic reticulum before exiting in association with MHC-I H chains and beta2-microglobulin as a trimolecular complex.
Assuntos
Calreticulina/metabolismo , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Antígenos H-2/metabolismo , Glicoproteínas de Membrana/metabolismo , Ovalbumina/metabolismo , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos H-2/fisiologia , Antígeno de Histocompatibilidade H-2D , Camundongos , Chaperonas Moleculares/metabolismo , Transporte Proteico/imunologia , Microglobulina beta-2/metabolismoRESUMO
Previously, we showed that 2B4 is a dominant inhibitory receptor in SHIP-deficient NK cells that prevents efficient cytolysis of complex targets. We show in this study that 2B4 deficiency restores homeostatic control and cytolytic function to SHIP-deficient NK cells. However, 2B4(-/-)SHIP(-/-) NK cells still exhibit a profound disruption of their NK receptor repertoire and are compromised for induction of IFN-gamma by several NK-activating receptors, including NKp46, NK.1.1, and NKG2D. In addition, we find that 2B4(-/-) NK cells have an extensively disrupted repertoire, including a supernormal frequency of NKp46(+) NK cells. Consequently IFN-gamma is induced on a much higher percentage of 2B4(-/-) NK cells following engagement of NKp46. We also find that both SHIP and 2B4 are required to prevent expression of Ly49B, a myeloid lineage MHC class I receptor not normally expressed by the NK lineage. Finally, when SHIP-deficient NK cells are on an H-2(d) background, they exhibit supernormal levels of Ly49A and possess normal cytolytic function against MHC-matched tumor targets and enhanced cytolysis of MHC mismatched tumor targets. However, despite normal or elevated cytolytic function, H2d SHIP(-/-) NK cells exhibit poor induction of IFN-gamma like their H2b(+) or 2B4(-/-) counterparts, demonstrating a uniform requirement for SHIP in induction of IFN-gamma downstream of key NK activating receptors. These findings reveal a complex interplay of SHIP, 2B4, and MHC in the regulation of homeostasis, effector function, and repertoire formation in the NK cell lineage.
Assuntos
Antígenos CD/fisiologia , Citotoxicidade Imunológica , Antígenos H-2/metabolismo , Homeostase/imunologia , Células Matadoras Naturais/imunologia , Monoéster Fosfórico Hidrolases/fisiologia , Receptores de Células Matadoras Naturais/biossíntese , Transdução de Sinais/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígeno CD48 , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citotoxicidade Imunológica/genética , Feminino , Antígenos H-2/fisiologia , Homeostase/genética , Inositol Polifosfato 5-Fosfatases , Interferon gama/biossíntese , Interferon gama/fisiologia , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Knockout , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Imunológicos/fisiologia , Receptores de Células Matadoras Naturais/metabolismo , Receptores de Células Matadoras Naturais/fisiologia , Transdução de Sinais/genética , Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
In mice and humans, the immunologic effects of developmental exposure to noninherited maternal antigens (NIMAs) are quite variable. This heterogeneity likely reflects differences in the relative levels of NIMA-specific T regulatory (T(R)) versus T effector (T(E)) cells. We hypothesized that maintenance of NIMA-specific T(R) cells in the adult requires continuous exposure to maternal cells and antigens (eg, maternal microchimerism [MMc]). To test this idea, we used 2 sensitive quantitative polymerase chain reaction (qPCR) tests to detect MMc in different organs of NIMA(d)-exposed H2(b) mice. MMc was detected in 100% of neonates and a majority (61%) of adults; nursing by a NIMA+ mother was essential for preserving MMc into adulthood. MMc was most prevalent in heart, lungs, liver, and blood, but was rarely detected in unfractionated lymphoid tissues. However, MMc was detectable in isolated CD4+, CD11b+, and CD11c+ cell subsets of spleen, and in lineage-positive cells in heart. Suppression of delayed type hypersensitivity (DTH) and in vivo lymphoproliferation correlated with MMc levels, suggesting a link between T(R) and maternal cell engraftment. In the absence of neonatal exposure to NIMA via breastfeeding, MMc was lost, which was accompanied by sensitization to NIMA in some offspring, indicating a role of oral exposure in maintaining a favorable T(R) > T(E) balance.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Quimerismo , Antígenos H-2/fisiologia , Hipersensibilidade Tardia/imunologia , Tolerância Imunológica/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Cruzamentos Genéticos , Feminino , Citometria de Fluxo , Hipersensibilidade Tardia/metabolismo , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Mães , Reação em Cadeia da Polimerase , Linfócitos T Reguladores/metabolismoRESUMO
The identification of CTL epitopes from tumor antigens is very important for the development of peptide-based, cancer-specific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumor-associated antigen. Although several epitopes of heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. The present study was designed to predict and identify the CTL epitopes in the mouse heparanase protein. For this purpose, H-2K(b)-restricted CTL epitopes were identified by using the following four-step procedure: (a) a computer-based epitope prediction from the amino acid sequence of mouse heparanase, (b) a peptide-binding assay to determine the affinity of the predicted epitopes with the H-2K(b) molecule, (c) the testing of the induction of CTLs toward various carcinoma cells expressing heparanase antigens and H-2K(b), and (d) the induction of immunoprotection and immunotherapy in vivo. The results showed that, of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398 to 405 (LSLLFKKL; mHpa398) and 519 to 526 (FSYGFFVI; mHpa519) lysed three kinds of carcinoma cells expressing both heparanase and H-2K(b) (B16 melanoma cells, EL-4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also treating tumor-bearing hosts successfully. Our results suggest that the mHpa398 and mHpa519 peptides are novel H-2K(b)-restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo. These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies.
Assuntos
Glucuronidase/biossíntese , Antígenos H-2/fisiologia , Linfócitos T Citotóxicos/metabolismo , Animais , Autoimunidade , Linhagem Celular Tumoral , Células Dendríticas/citologia , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Antígenos H-2/metabolismo , Sistema Imunitário , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/químicaRESUMO
The interaction between T cell receptors (TCR) and peptide-major histocompatibility complex (pMHC) antigens can lead to varying degrees of agonism (T cell activation), or antagonism. The P14 TCR recognises the lymphocytic choriomeningitis virus (LCMV)-derived peptide, gp33 residues 33-41 (KAVYNFATC), presented in the context of H-2D(b). The cellular responses to various related H-2D(b) peptide ligands are very well characterised, and P14 TCR-transgenic mice have been used extensively in models of virus infection, autoimmunity and tumour rejection. Here, we analyse the binding of the P14 soluble TCR to a broad panel of related H-2D(b)-peptide complexes by surface plasmon resonance, and compare this with their diverse cellular responses. P14 TCR binds H-2D(b)-gp33 with a KD of 3 microM (+/-0.5 microM), typical of an immunodominant antiviral TCR, but with unusually fast kinetics (k(off) = 1 s(-1)), corresponding to a half-life of 0.7 s at 25 degrees C, outside the range previously observed for murine agonist TCR/pMHC interactions. The most striking feature of these data is that a very short half-life does not preclude the ability of a TCR/pMHC interaction to induce antiviral immunity, autoimmune disease and tumour rejection.
Assuntos
Antígenos H-2/metabolismo , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos Virais/metabolismo , Antígenos Virais/fisiologia , Glicoproteínas/metabolismo , Glicoproteínas/fisiologia , Antígenos H-2/fisiologia , Antígeno de Histocompatibilidade H-2D , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Peptídeos/genética , Peptídeos/fisiologia , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Proteínas Virais/metabolismo , Proteínas Virais/fisiologiaRESUMO
The mouse multimember family of Qa-2 oligomorphic class I MHC genes is continuously undergoing duplications and deletions that alter the number of the two "prototype" Qa-2 sequences, Q8 and Q9. The frequent recombination events within the Q region lead to strain-specific modulation of the cumulative Qa-2 expression levels. Q9 protects C57BL/6 hosts from multiple disparate tumors and functions as a major CTL restriction element for shared tumor-associated Ags. We have now analyzed functional and structural properties of Q8, a class I MHC that differs significantly from Q9 in the peptide-binding, CTL-interacting alpha(1) and alpha(2) regions. Unexpectedly, we find that the extracellular domains of Q8 and Q9 act similarly during primary and secondary rejection of tumors, are recognized by cross-reactive antitumor CTL, have overlapping peptide-binding motifs, and are both assembled via the transporter associated with the Ag processing pathway. These findings suggest that shared Ag-presenting functions of the "odd" and "even" Qa-2 loci may contribute to the selective pressures shaping the haplotype-dependent quantitative variation of Qa-2 protein expression.
Assuntos
Sequência Conservada , Rejeição de Enxerto/imunologia , Antígenos H-2/química , Antígenos H-2/genética , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Apresentação Cruzada/genética , Apresentação Cruzada/imunologia , Líquido Extracelular/imunologia , Marcadores Genéticos/imunologia , Rejeição de Enxerto/genética , Antígenos H-2/fisiologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/metabolismo , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Transplante de Neoplasias , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , TransfecçãoRESUMO
Genetic modification of hematopoietic stem cells (HSCs) resulting in a state of molecular chimerism can be used to induce donor-specific tolerance to allografts. However, the requirements for maintaining tolerance in molecular chimeras remain unknown. Here, we examined whether long-term expression of a retrovirally encoded alloantigen in hematopoietic cells is required to maintain donor-specific tolerance in molecular chimeras. To this end, mice were reconstituted with syngeneic bone marrow transduced with retroviruses carrying the gene encoding the allogeneic MHC class I molecule Kb. Following induction of molecular chimerism, mice were depleted of cells expressing Kb by administration of the anti-Kb monoclonal antibody Y-3. Mice that were effectively depleted of cells expressing the retrovirally encoded MHC class I antigen rejected Kb disparate skin allografts. In contrast, control molecular chimeras accepted Kb disparate skin allografts indefinitely. These data suggest maintenance of tolerance in molecular chimeras requires long-term expression of retrovirally transduced alloantigen on the progeny of retrovirally transduced HSCs.
Assuntos
Transplante de Medula Óssea , Terapia Genética/métodos , Antígenos H-2/fisiologia , Células-Tronco Hematopoéticas/imunologia , Quimeras de Transplante/imunologia , Tolerância ao Transplante , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/virologia , Camundongos , Quimera por Radiação/genética , Quimera por Radiação/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Pele/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução Genética , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
Peptide-MHC tetramers have been engineered to allow accurate detection of antigen-specific cytotoxic C lymphocytes (CTL) by flow cytometry. Here, we propose a novel use for peptide-MHC tetramers in the specific and sensitive analysis of the cytotoxic function of antigen-specific CTL by blocking MHC-restricted antigen-specific cytotoxicity. We found that pretreatment of ovalbumin (OVA)-specific CD8(+) CTL (OT-1 CTL), derived from OT-1 T-cell receptor (TCR)-transgenic mice, with OVA(257-264) peptide-H-2K(b) tetramer caused a marked inhibition of the cytotoxicity against OVA-expressing EG-7 tumor cells. OVA(257-264) peptide-H-2K(b) tetramer did not block the cytotoxicity mediated by 2C mouse (H-2(b))-derived CD8(+) CTL, which recognize allo (H-2L(d)) antigens. Moreover, OT-I CTL activity was not inhibited by an irrelevant HBV(208-216) peptide-H-2K(b) tetramer. These results indicate that the blocking of CTL activity with peptide-MHC tetramer was caused by interference with the interaction between the TCR and H-2K(b)-OVA(257-264) peptide complex, but not with the CD8-MHC class I interaction. The blocking activity of OVA(257-264) peptide-H-2K(b) tetramer was reversible because OT-I CTL pretreated with the tetramer recovered their cytotoxicity after culturing with interleukin-2 for 24 h. The same results were also demonstrated in freshly isolated, in vivo-primed OT-1 CTL sorted by the tetramer. These results demonstrate that peptide-MHC tetramer is a useful tool for defining MHC-restricted antigen-specific CTL function. Moreover, our finding implies that the measurement of CTL activity immediately after tetramer-guided sorting is not a suitable method for evaluating the function of in vivo-induced tetramer-positive CTL. We believe that the tetramer-blocking assay presented here will be useful for functionally monitor the induction of MHC-restricted antigen-specific CTL during vaccination therapy against tumor and infectious diseases.
Assuntos
Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/imunologia , Ovalbumina/metabolismo , Linfócitos T Citotóxicos/imunologia , Timoma/imunologia , Animais , Antígenos H-2/genética , Antígenos H-2/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia , Timoma/genética , Timoma/metabolismo , Células Tumorais CultivadasRESUMO
The immunoreceptor NKG2D stimulates activation of cytotoxic lymphocytes upon engagement with MHC class I-related NKG2D ligands of which at least some are expressed inducibly upon exposure to carcinogens, cell stress, or viruses. In this study, we investigated consequences of a persistent NKG2D ligand expression in vivo by using transgenic mice expressing MHC class I chain-related protein A (MICA) under control of the H2-K(b) promoter. Although MICA functions as a potent activating ligand of mouse NKG2D, H2-K(b)-MICA mice appear healthy without aberrations in lymphocyte subsets. However, NKG2D-mediated cytotoxicity of H2-K(b)-MICA NK cells is severely impaired in vitro and in vivo. This deficiency concurs with a pronounced down-regulation of surface NKG2D that is also seen on activated CD8 T cells. As a consequence, H2-K(b)-MICA mice fail to reject MICA-expressing tumors and to mount normal CD8 T cell responses upon Listeria infection emphasizing the importance of NKG2D in immunity against tumors and intracellular infectious agents.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação para Baixo/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Células Matadoras Naturais/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/biossíntese , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/microbiologia , Células CHO , Linhagem Celular Tumoral , Técnicas de Cocultura , Cricetinae , Citotoxicidade Imunológica/genética , Regulação para Baixo/genética , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Antígenos H-2/fisiologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/fisiologia , Imunidade Inata/genética , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/microbiologia , Listeriose/genética , Listeriose/imunologia , Listeriose/patologia , Ativação Linfocitária/genética , Linfoma de Células T/genética , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores Imunológicos/fisiologia , Receptores de Células Matadoras NaturaisRESUMO
Defective antitumor immune responses are frequent consequences of defects in the expression of major histocompatibility complex (MHC) class I and costimulatory molecules. We demonstrated that statins, inhibitors of HMGCoA reductase, enhance mIFN-gamma induced expression of MHC class I antigens on murine B16F10 melanoma. GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor, mimics this effect of statins. This effect is related to peptide transporter protein TAP1 up-regulation. Simultaneously, GGTI-298 induces the expression of CD80 and CD86 costimulatory molecules. C3 exoenzyme, which selectively inactivates Rho proteins, phenocopies the effects of GGTI-298, indicating a role for Rho proteins in these events. Furthermore, the treatment of B16F10 cells with GGTI-298 or C3 exoenzyme associated with mIFN-gamma induces in vivo tumor growth slowing down in immunocompetent but not in nu/nu syngeneic mice. Both in vivo injections and in vitro restimulation of splenocytes with GGTI-298- and mIFN-gamma-treated B16F10 cells induces an enhancement of specific CD8 T lymphocytes labeled by TRP-2/H-2K(b) tetramers. Finally, these effects are not limited to mouse models since they were also reproduced in two human melanoma cell lines. These observations indicate that protein geranylgeranylation as well as Rho protein are critical for costimulatory and IFN-gamma-dependent MHC class I molecule expression in melanoma.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antígeno B7-1/fisiologia , Antígeno B7-2/fisiologia , Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Antígenos H-2/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Melanoma Experimental/imunologia , Animais , Antígeno B7-1/análise , Antígeno B7-2/análise , Linfócitos T CD8-Positivos/fisiologia , Linhagem Celular Tumoral , Feminino , Interferon gama/farmacologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.
Assuntos
Apoptose/imunologia , Citotoxicidade Imunológica/imunologia , Inibidores do Crescimento/fisiologia , Antígenos H-2/fisiologia , Oligopeptídeos/fisiologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologia , beta-Alanina/análogos & derivados , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Clonais , Ciclosporina/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Dimerização , Relação Dose-Resposta Imunológica , Cinética , Potenciais da Membrana/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Necrose , Espécies Reativas de Oxigênio/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/imunologia , Solubilidade , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , beta-Alanina/farmacologiaRESUMO
We used fibrosarcoma from an H2-K/V-JUN transgenic mouse to derive a series of three immortal cell lines (JUN-1, -2, and -3). The cell lines exhibit strikingly different behavior regarding phenotype transformation. Features examined include contact inhibition and density limitation of growth, proliferation, invasiveness, motility, and organization of the microfilament system. Overall, JUN-2 and JUN-3 represent extreme phenotypes, with JUN-2 having a phenotype indicative of low-level cellular transformation and JUN-3 meeting all the criteria of the transformed phenotype. JUN-1 cells can also be regarded as transformed, but to a lesser extent than JUN-3. Their phenotype is in the majority of characteristics intermediary between JUN-2 and JUN-3. The transformation status is inversely related to the expression of the V-JUN transgene, which is the highest in JUN-2, lower in JUN-1 and very low in JUN-3. This might be related to the MHC class I promoter driving its expression and to the general observation of repression of MHC class I genes coupled with cellular transformation. Based on this premise, we present a model of H2-K/V-JUN-mediated tumorigenesis, in which v-jun-conditioned transformation represents merely an initial phase of tumorigenesis. Later during tumor progression, additional oncogenes are activated and/or tumor suppressor genes inactivated, leading on the one hand to further exacerbation of the transformed phenotype, and on the other hand to the repression of the H2-K/V-JUN transgene (fixed in JUN-3). We believe that the system of JUN cell lines can be valuable for further molecular analysis of transformation-related traits.
Assuntos
Transformação Celular Neoplásica , Fibrossarcoma/patologia , Genes jun , Antígenos H-2/genética , Actinas/química , Animais , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Genes jun/fisiologia , Antígenos H-2/fisiologia , Camundongos , Camundongos Transgênicos , Invasividade NeoplásicaRESUMO
Leishmania infection inhibits the capacity of macrophages (MPhi) to present antigens to CD4(+) T cells. Relocation of MHC class II and H-2DM to the parasitophorous vacuole (PV) and their subsequent degradation by the parasite may contribute to this defect. Dendritic cells (DC) are critical for initiation of primary T cell responses. DC can process Leishmania antigen and elicit Leishmania-specific T cells, but it is unknown whether exposure to Leishmania impairs this capacity. In particular, it is not clear whether DC containing live parasites efficiently process and present antigens. We investigated the ability of mouse bone marrow-derived DC infected with L. mexicana to generate pigeon cytochrome c (PCC) peptide-MHC class II complexes, using the mAb D4, which recognizes PCC(89-104) H-2E(k), and the PCC-specific T cell hybridoma 2B4. We show that H-2DM-dependent complex generation is not compromised by infection and that complexes are fully recognized by specific T cells. We further show that in contrast to infected MPhi, in infected DC cytoplasmic H-2DM is not down-regulated and not relocated to the parasite-containing vacuole. This observation may explain the continued ability of infected DC to present PCC, and also indicates differences in the habitat of these intracellular parasites in DC compared to MPhi.
Assuntos
Citocromos c/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Antígenos H-2/metabolismo , Antígenos H-2/fisiologia , Leishmania mexicana/imunologia , Animais , Apresentação de Antígeno , Columbidae , Feminino , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos CBARESUMO
Low levels of IFN-gamma secreted by immature B cells prevent their own migration and homing to the lymph nodes and premature encounter with Ag. In this study we followed the mechanism regulating IFN-gamma secretion by immature B cells. We show that the MHC class I receptor, Ly49D, is expressed on immature B cells and is down-regulated during maturation. Activation of this receptor leads to increase in IFN-gamma transcription and translation and results in the altered ability of B cells to polymerize actin in response to chemokine stimulation. Moreover, we show that H2-D blockage inhibits the ability of immature B cells to transcribe the IFN-gamma gene and results in rescue of cytoskeletal rearrangement. Thus, Ly49D that is expressed on immature B cells recognizes MHC class I on the peripheral tissues, inducing the secretion of low levels of IFN-gamma and thereby down-regulating immature B cell homing to the lymph nodes or to sites of inflammation.
Assuntos
Actinas/metabolismo , Antígenos Ly/fisiologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Movimento Celular/imunologia , Interferon gama/metabolismo , Receptores de Retorno de Linfócitos/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Antígenos Ly/biossíntese , Subpopulações de Linfócitos B/citologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Movimento Celular/genética , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos H-2/fisiologia , Antígeno de Histocompatibilidade H-2D , Interferon gama/antagonistas & inibidores , Interferon gama/biossíntese , Lectinas Tipo C , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília A de Receptores Semelhantes a Lectina de Células NK , Biossíntese de Proteínas/imunologia , Receptores de Retorno de Linfócitos/biossíntese , Receptores Semelhantes a Lectina de Células NK , Transcrição Gênica/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
In a variety of malignancies, the immune-escape phenotype is associated, in part, with the inability of tumor cells to properly present their Ags to CTLs due to a deranged expression of MHC class I glycoproteins. However, these molecules were found to possess broader nonimmune functions, including participation in signal transduction and regulation of proliferation, differentiation, and sensitivity to apoptosis-inducing factors; processes, which are characteristically impaired during malignant transformation. We investigated whether the deranged expression of MHC class I expression by tumor cells could affect proper receptor-mediated signal transduction and accentuate their malignant phenotype. The malignant and H-2K murine MHC class I-deficient B16BL6 melanoma cells were characterized by an attenuated capacity to bind insulin due to the retention of corresponding receptor in intracellular stores. The restoration of H-2K expression in these cells, which abrogated their capacity to form tumors in mice, enhanced membrane translocation of the receptor, presumably, by modulating its glycosylation. The addition of insulin to H-2K-expressing melanoma cells cultured in serum-free conditions precluded apoptotic death by up-regulating the activity of protein kinase B (PKB)/Akt. In contrast, the deficiency for H-2K characteristic to the malignant clones was associated with a constitutive high activity of PKB/Akt, which rendered them resistant to apoptosis, induced by deprivation of serum-derived growth factors. The possibility to correct the regulation of PKB/Akt activity by restoration of H-2K expression in B16BL6 melanoma cells may be considered as an attractive approach for cancer therapy, since an aberrant activation of this enzyme is characteristic to resistant malignancies.
Assuntos
Adjuvantes Imunológicos/fisiologia , Apoptose/imunologia , Glicoproteínas/biossíntese , Antígenos H-2/biossíntese , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Proteínas Serina-Treonina Quinases , Receptor de Insulina/fisiologia , Transdução de Sinais/imunologia , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Animais , Linhagem Celular Tumoral , Células Clonais , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas/fisiologia , Glicosilação , Substâncias de Crescimento/deficiência , Antígenos H-2/genética , Antígenos H-2/metabolismo , Antígenos H-2/fisiologia , Imunidade Inata , Insulina/metabolismo , Melanoma Experimental/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptor de Insulina/metabolismoRESUMO
Productive immune responses require an appropriate environment to support peripheral CD8(+) T cell survival. Although host MHC class I molecules appear to be required for this process, the cellular and molecular requirements have not been comprehensively studied. Using adoptive transfer of 2C/recombinase-activating gene-2 (RAG-2)(-/-) TCR-transgenic T cells, we found that the survival of both naive and effector CD8(+) T cells was dependent upon host expression of the same MHC class I alleles that supported thymic selection. Expression of appropriate MHC class Iby either bone marrow- or non-bone-marrow-derived cells was sufficient, suggesting that professional antigen-presenting cells were not mandatory. In contrast to MHC class I, neither T cell expression of CD28 nor host expression of ICAM-1 was required for peripheral T cell survival. Finally, T cell death in the absence of appropriate host MHC class I was overcome by elimination of Fas signaling but not by overexpression of Bcl-x(L) by CD8(+) T cells. These results suggest that, in the absence of a survival signal provided by engagement of host MHC/self peptide complexes, CD8(+) T cells die via a Fas-dependent, mitochondria-independent pathway.