RESUMO
The parasite Toxoplasma gondii replicates in a specialized intracellular vacuole and causes disease in many species. Protection from toxoplasmosis is mediated by CD8(+) T cells, but the T. gondii antigens and host genes required for eliciting protective immunity are poorly defined. Here we identified GRA6, a polymorphic protein secreted in the parasitophorous vacuole, as the source of the immunodominant and protective decapeptide HF10 presented by the H-2L(d) major histocompatibility complex class I molecule. Presentation of the HF10-H-2L(d) ligand required proteolysis by ERAAP, the endoplasmic reticulum aminopeptidase associated with antigen processing. Consequently, expansion of protective CD8(+) T cell populations was impaired in T. gondii-infected ERAAP-deficient mice, which were more susceptible to toxoplasmosis. Thus, endoplasmic reticulum proteolysis is critical for eliciting protective immunity to a vacuolar parasite.
Assuntos
Antígenos de Protozoários/metabolismo , Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Leucil Aminopeptidase/deficiência , Proteínas de Protozoários/metabolismo , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Apresentação de Antígeno , Leucil Aminopeptidase/imunologia , Leucil Aminopeptidase/metabolismo , Camundongos , Toxoplasma/fisiologia , Vacúolos/imunologiaRESUMO
Many NK cells express inhibitory receptors that bind self-MHC class I (MHC I) molecules and prevent killing of self-cells, while enabling killing of MHC I-deficient cells. But tolerance also occurs for NK cells that lack inhibitory receptors for self-MHC I, and for all NK cells in MHC I-deficient animals. In both cases, NK cells are unresponsive to MHC I-deficient cells and hyporesponsive when stimulated through activating receptors, suggesting that hyporesponsiveness is responsible for self-tolerance. We generated irradiation chimeras, or carried out adoptive transfers, with wild-type (WT) and/or MHC I-deficient hematopoietic cells in WT or MHC I-deficient C57BL/6 host mice. Unexpectedly, in WT hosts, donor MHC I-deficient hematopoietic cells failed to induce hyporesponsiveness to activating receptor stimulation, but did induce tolerance to MHC I-deficient grafts. Therefore, these two properties of NK cells are separable. Both tolerance and hyporesponsiveness occurred when the host was MHC I deficient. Interestingly, infections of mice or exposure to inflammatory cytokines reversed the tolerance of NK cells that was induced by MHC I-deficient hematopoietic cells, but not the tolerance induced by MHC I-deficient nonhematopoietic cells. These data have implications for successful bone marrow transplantation, and suggest that tolerance induced by hematopoietic cells versus nonhematopoietic cells may be imposed by distinct mechanisms.
Assuntos
Tolerância Imunológica , Células Matadoras Naturais/imunologia , Tolerância a Antígenos Próprios , Transferência Adotiva , Animais , Transplante de Medula Óssea , Citocinas , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/classificação , Células Matadoras Naturais/fisiologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Quimera por RadiaçãoRESUMO
The elimination of solid tumors largely depends on effective T-cell priming by dendritic cells (DCs). For decades, studies focusing on antitumoral immune responses have been performed with tumors transplanted subcutaneously (s.c.). These studies however do not take into account the heterogeneous tissue distribution and functionality of the different DC subsets. Given the crucial role of DCs in inducing protective immune response, we postulated that the anatomic location of tumor development may greatly impact tumor immunogenicity. We therefore implanted tumor cells either in the DC-rich dermis environment or in the s.c. tissue that mainly contains macrophages and monocytes. We showed that intradermal (i.d.), but not s.c. tumors are rapidly rejected in a T-cell-dependent manner and induce protective T-cell responses. The rejection of i.d. tumors correlates with rapid recruitment of dermal DCs presenting the tumor antigen to both CD4 and CD8 T cells in the draining lymph nodes (dLNs). The same DC subsets were mobilized upon s.c. tumor transplantation but with delayed kinetics. Altogether, our results show that the anatomical site of tumor development influences tumor immunogenicity, notably by controlling the kinetics of DC mobilization in the draining LNs.
Assuntos
Células Dendríticas/imunologia , Linfonodos/citologia , Neoplasias/imunologia , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/fisiologia , Derme/imunologia , Células de Langerhans/imunologia , Ativação Linfocitária , Camundongos , Neoplasias/fisiopatologia , Tela Subcutânea/imunologiaRESUMO
Ligands for the NKG2D stimulatory receptor are frequently upregulated on tumor lines, rendering them sensitive to natural killer (NK) cells, but the role of NKG2D in tumor surveillance has not been addressed in spontaneous cancer models. Here, we provided the first characterization of NKG2D-deficient mice, including evidence that NKG2D was not necessary for NK cell development but was critical for immunosurveillance of epithelial and lymphoid malignancies in two transgenic models of de novo tumorigenesis. In both models, we detected NKG2D ligands on the tumor cell surface ex vivo, providing needed evidence for ligand expression by primary tumors. In a prostate cancer model, aggressive tumors arising in NKG2D-deficient mice expressed higher amounts of NKG2D ligands than did similar tumors in wild-type mice, suggesting an NKG2D-dependent immunoediting of tumors in this model. These findings provide important genetic evidence for surveillance of primary tumors by an NK receptor.
Assuntos
Adenocarcinoma/imunologia , Fibrossarcoma/imunologia , Síndromes de Imunodeficiência/imunologia , Vigilância Imunológica , Linfoma de Células B/imunologia , Neoplasias da Próstata/imunologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Adenocarcinoma/genética , Animais , Benzo(a)Antracenos/toxicidade , Modelos Animais de Doenças , Feminino , Fibrossarcoma/induzido quimicamente , Fibrossarcoma/genética , Síndromes de Imunodeficiência/genética , Vigilância Imunológica/genética , Linfoma de Células B/genética , Masculino , Metilcolantreno , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Neoplasias da Próstata/genética , Receptores Imunológicos/fisiologia , Receptores de Células Matadoras NaturaisRESUMO
The antitumor activity of CD4(+) T cells is increasingly acknowledged in both humans and mice. The involved mechanisms have been mostly studied using transplanted tumor mouse systems. In these models, many tumor cells die at the time of implantation leading to the release of Ag in an inflammatory context contrasting with the slow and nondestructive growth of early-stage human tumors. In this study, we show that the presentation of a MHC class II-restricted model Ag (male, DBY) released by dying tumor cells may last more than 4 wk. The duration of Ag presentation varies according to the way the cells are killed before implantation. To avoid this artifactual early priming of the host precluding the study of the interactions between the immune system and tumors at the steady state, we generated a cell line expressing the DBY Ag in an inducible manner. Ag expression can be efficiently induced in vivo several days after tumor implantation. We show that the Ag reaches the lymph node and activates naive CD4(+) T cells to proliferate and recirculate. We did not observe de novo induction of tumor-specific regulatory T cells. However, we observed Th1/Th17 effector cells in the tumor draining lymph node and tumors. Thus, when a neoantigen appears in established tumors, the immune system is not ignorant and naive CD4(+) T cells are not tolerized. This opens up the possibility of therapeutic vaccines improving the immune response toward tumor-specific neoantigens.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Neoplasias/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Neoplasias/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , FenótipoRESUMO
Inhibitory receptors specific for major histocompatibility complex (MHC) class I molecules govern the capacity of natural killer (NK) cells to attack class I-deficient cells ('missing-self recognition'). These receptors are expressed stochastically, such that the panel of expressed receptors varies between NK cells. This review addresses how the activity of NK cells is coordinated in the face of this variation to achieve a repertoire that is self-tolerant and optimally reactive with diseased cells. Recent studies show that NK cells arise in normal animals or humans that lack any known inhibitory receptors specific for self-MHC class I. These NK cells exhibit self-tolerance and exhibit functional hyporesponsiveness to stimulation through various activating receptors. Evidence suggests that hyporesponsiveness is induced because these NK cells cannot engage inhibitory MHC class I molecules and are therefore persistently over-stimulated by normal cells in the environment. Finally, we discuss evidence that hyporesponsiveness is a quantitative trait that varies depending on the balance of signals encountered by developing NK cells. Thus, a tuning process determines the functional set-point of NK cells, providing a basis for discriminating self from missing-self, and at the same time endowing each NK cell with the highest inherent responsiveness compatible with self-tolerance.
Assuntos
Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Receptores KIR/imunologia , Tolerância a Antígenos Próprios , Animais , Antígenos CD/imunologia , Antígenos de Superfície/imunologia , Moléculas de Adesão Celular/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/imunologia , Camundongos , Camundongos Transgênicos , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Receptores Imunológicos/imunologia , Receptores KIR/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transativadores/imunologiaRESUMO
Inhibitory receptors that engage self-MHC class I molecules enable NK cells to detect disease-associated loss of MHC class I on surrounding cells. Previous studies showed that some NK cells lack all receptors for self-MHC class I, yet fail to exhibit autoimmunity because they are generally hyporesponsive to stimulation. We asked whether NK cells exist in only two states, responsive and hyporesponsive, corresponding to cells that express or fail to express inhibitory receptors for self-MHC class I. The alternative model is that NK cells vary continuously in their responsiveness, based on variations in the number of different inhibitory and stimulatory receptors they express, which is known to vary. In this study, we show in the murine system that NK cell responsiveness increases quantitatively with each added self-MHC-specific inhibitory receptor. Genetic analysis demonstrated that interactions of each of the receptors with self-MHC class I were necessary to observe augmented responsiveness. These findings suggest that NK cell responsiveness is comparable to a rheostat: it is tuned to an optimal set point depending on the inhibitory and stimulatory interactions encountered in the normal environment, so as to ensure self-tolerance and yet optimize sensitivity to changes in normal cells.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Modelos Imunológicos , Animais , Linhagem Celular Tumoral , Interferon gama/biossíntese , Interferon gama/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Especificidade por SubstratoRESUMO
The regulation of CD4 T-cell numbers during an immune response should take account of the amount of antigen (Ag), the initial frequency of Ag-specific T cells, the mix of naive versus experienced cells, and (ideally) the diversity of the repertoire. Here we describe a novel mechanism of T-cell regulation that potentially deals with all of these parameters. We found that CD4 T cells establish a negative feedback loop by capturing their cognate major histocompatibility class (MHC)/peptide complexes from Ag-presenting cells and presenting them to Ag-experienced CD4 T cells, thereby inhibiting their recruitment into the response while allowing recruitment of naive T cells. The inhibition is Ag specific, begins at day 2 (long before Ag disappearance), and cannot be overcome by providing new Ag-loaded dendritic cells. In this way, CD4 T-cell proliferation is regulated in a functional relationship to the amount of Ag, while allowing naive T cells to generate repertoire variety.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular/imunologia , Retroalimentação Fisiológica/imunologia , Animais , Células Apresentadoras de Antígenos , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/citologia , Feminino , Antígenos de Histocompatibilidade , Imunidade Celular , Memória Imunológica , Ativação Linfocitária , Masculino , Camundongos , Camundongos Transgênicos , Subpopulações de Linfócitos TRESUMO
T cells need to migrate to and accumulate inside tumors before mediating rejection of the tumor. The number of specific T cells inside tumors may depend on the efficiency of priming in the draining lymph node (DLN), intratumor deletion, suppressive phenomena, or both. We used monoclonal anti-male antigen CD4 (Marilyn) T cells and tumor cell lines expressing or not the corresponding antigen (Dby) to analyze CD4 T-cell accumulation in tumors. Priming by MHC II(+) or MHC II(-) male splenocytes or Dby(+) tumor cells induced similar Marilyn T-cell expansion in the DLN and recirculation in other lymph nodes and capacity to produce IFN-gamma. However, intratumor accumulation was different for each priming condition. In mice with Dby(-) tumors, MHC II(+) male splenocyte priming induced greater, although not statistically significant, Marilyn T-cell accumulation in the tumors than MHC II(-) male splenocyte priming. In mice with Dby(+) tumors, priming in the tumor DLN induced less Marilyn T-cell intratumor accumulation than priming by MHC II(+) male splenocytes. We saw comparable differences for Marilyn T-cell accumulation in gut lamina propria, suggesting that priming affects effector T-cell accumulation in inflamed tissues. Mature dendritic cells were loaded with graded doses of Dby peptide to control for antigen-presenting cell characteristics during priming. We observed similar proliferation, with higher concentrations inducing higher intratumor accumulation. Thus, intratumor accumulation requires stronger stimulation than for proliferation or the capacity to secrete lymphokines. In this system, priming intensity alone can explain the number of intratumor T cells without having to call for intratumor deletion or suppression phenomena.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Fibrossarcoma/imunologia , Linfocinas/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , RNA Helicases DEAD-box , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfocinas/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Proteínas/genética , Proteínas/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Some mature natural killer (NK) cells cannot be inhibited by major histocompatibility complex (MHC) I molecules, either because they lack corresponding inhibitory receptors or because the host lacks the corresponding MHC I ligands for the receptors. Such NK cells nevertheless remain self-tolerant and exhibit a generalized hyporesponsiveness to stimulation through activating receptors. To address whether NK cell responsiveness is set only during the NK cell differentiation process, we transferred mature NK cells from wild-type (WT) to MHC I-deficient hosts or vice versa. Remarkably, mature responsive NK cells from WT mice became hyporesponsive after transfer to MHC I-deficient mice, whereas mature hyporesponsive NK cells from MHC I-deficient mice became responsive after transfer to WT mice. Altered responsiveness was evident among mature NK cells that had not divided in the recipient animals, indicating that the cells were mature before transfer and that alterations in activity did not require cell division. Furthermore, the percentages of NK cells expressing KLRG1, CD11b, CD27, and Ly49 receptors specific for H-2(b) were not markedly altered after transfer. Thus, the functional activity of mature NK cells can be reset when the cells are exposed to a changed MHC environment. These findings have important implications for how NK cell functions may be curtailed or enhanced in the context of disease.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Animais , Antígeno CD11b/biossíntese , Antígeno CD11b/imunologia , Diferenciação Celular , Divisão Celular , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Células Matadoras Naturais/transplante , Lectinas Tipo C , Camundongos , Subfamília A de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptores Imunológicos/biossíntese , Receptores Imunológicos/imunologia , Tolerância a Antígenos Próprios/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/biossíntese , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di-guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor kappaB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid-sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.
Assuntos
GMP Cíclico/análogos & derivados , Citosol/metabolismo , Regulação da Expressão Gênica/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/metabolismo , Sistemas do Segundo Mensageiro/imunologia , Animais , Linhagem Celular Tumoral , GMP Cíclico/imunologia , GMP Cíclico/metabolismo , Citosol/imunologia , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Interferon Tipo I/imunologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
Researchers designing antitumor treatments have long focused on eliciting tumor-specific CD8 cytotoxic T lymphocytes (CTL) because of their potent killing activity and their ability to reject transplanted organs. The resulting treatments, however, have generally been surprisingly poor at inducing complete tumor rejection, both in experimental models and in the clinic. Although a few scattered studies suggested that CD4 T "helper" cells might also serve as antitumor effectors, they have generally been studied mostly for their ability to enhance the activity of CTL. In this mouse study, we compared monoclonal populations of tumor-specific CD4 and CD8 T cells as effectors against several different tumors, and found that CD4 T cells eliminated tumors that were resistant to CD8-mediated rejection, even in cases where the tumors expressed major histocompatibility complex (MHC) class I molecules but not MHC class II. MHC class II expression on host tissues was critical, suggesting that the CD4 T cells act indirectly. Indeed, the CD4 T cells partnered with NK cells to obtain the maximal antitumor effect. These findings suggest that CD4 T cells can be powerful antitumor effector cells that can, in some cases, outperform CD8 T cells, which are the current "gold standard" effector cell in tumor immunotherapy.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias/imunologia , Animais , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Imunoterapia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Transgênicos , Linfócitos T Citotóxicos/imunologiaRESUMO
Tumor infiltrating lymphocytes (TIL) display activation markers and their presence is often associated with a favorable outcome. The role of tumor antigens in T cell recruitment into tumors is unclear. In an attempt to address this issue, we purified lymphocytes from breast tumor or nontumor, mammary tissue from patients, and normal mammary tissue from healthy individuals. In all groups, including healthy individuals, the majority of cells displayed an effector/memory (CD45RA(lo)/CD27(+/-)) phenotype and quite surprisingly the early and transient activation marker CD69, thus, questioning the tumor antigen specificity of TIL. Because the human repertoire is diverse, the T cells found in the tumors could recognize both self/tumor and environmental antigens through cross-reactivity. To test this hypothesis, we used two anti-male HY monospecific TCR transgenic mouse models. We found an infiltration of HY negative tumors by the CD4(+) and CD8(+) monoclonal T cells after priming with HY positive cells in the periphery. Thus, the presence of activated effector/memory T lymphocytes in tumors can be independent of reactivity against tumor antigens. These results suggest that to find activated effector T cells in a tissue does not always mean that a specific immune response is taking place.