RESUMO
We generated a CD8 T-cell receptor (TCR) transnuclear (TN) mouse specific to the Ld -restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T-cell-mediated response against this pathogen. The TN T cells bound Ld -Gra6 tetramer and proliferated upon unspecific and peptide-specific stimulation. The TCR beta sequence of the Gra6-specific TN CD8 T cells is identical in its V- and J-region to the TCR-ß harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma-infected mice carried Gra6 TN cells already at day 8 post-infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T-cell response to Toxoplasma in both the acute and the chronic phases of infection.
Assuntos
Antígenos de Protozoários/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Epitopos Imunodominantes/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Toxoplasmose/imunologia , Doença Aguda , Transferência Adotiva , Animais , Antígenos de Protozoários/imunologia , Modelos Animais de Doenças , Progressão da Doença , Antígenos de Histocompatibilidade Classe I/metabolismo , Hibridomas , Epitopos Imunodominantes/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas de Protozoários/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
BACKGROUND: Dendritic cells (DCs) are the highly specialized antigen presenting cells of the immune system that play a key role in regulating immune responses. DCs can efficiently initiate immune responses or induce tolerance. Due to this dual function, DCs are studied in the context of immunotherapy for both cancer and autoimmune diseases. Characterization of DC-specific genes, leading to better understanding of DC immunobiology, will help to guide their use in clinical settings. We previously identified DC-STAMP, a multi-membrane spanning protein preferentially expressed by DCs. DC-STAMP resides in the endoplasmic reticulum (ER) of immature DCs and translocates towards the Golgi compartment upon maturation. In this study we knocked down DC-STAMP in mouse bone marrow-derived DCs (mBMDCs) to determine its function. RESULTS: We demonstrate that DC-STAMP knock-down mBMDCs secrete less IL-6, IL-12, TNF-α and IL-10 while IL-1 production is enhanced. Moreover, LPS-matured DC-STAMP knock-down mBMDCs show impaired T cell activation potential and induction of Th1 responses in an alloreaction. CONCLUSIONS: We show that DC-STAMP plays an important role in cytokine production by mBMDCs following LPS exposure. Our results reveal a novel function of DC-STAMP in regulating DC-initiated immune responses.
Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Linfócitos T/metabolismo , Animais , Medula Óssea/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Ativação Linfocitária/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , RNA Interferente Pequeno/genética , Linfócitos T/patologiaRESUMO
Dendritic cell-specific transmembrane protein (DC-STAMP) has been first identified as an EST in a cDNA library of human monocyte-derived dendritic cells (DC). DC-STAMP is a multimembrane spanning protein that has been implicated in skewing haematopoietic differentiation of bone marrow cells towards the myeloid lineage, and in cell fusion during osteoclastogenesis and giant cell formation. To gain molecular insight in how DC-STAMP exerts its function, DC-STAMP interacting proteins were identified in a yeast-2-hybrid analysis. Herein, we report that amplified in osteosarcoma 9 (OS9) physically interacts with DC-STAMP, and that both proteins colocalize in the endoplasmic reticulum in various cell lines, including immature DC. OS9 has previously been implicated in ER-to-Golgi transport and transcription factor turnover. Interestingly, we now demonstrate that toll-like receptor (TLR)-induced maturation of DC leads to the translocation of DC-STAMP from the ER to the Golgi while OS9 localization is unaffected. Applying TLR-expressing CHO cells we could confirm ER-to-Golgi translocation of DC-STAMP following TLR stimulation and demonstrated that the DC-STAMP/OS9 interaction is involved in this process. Collectively, the data indicate that OS9 is critically involved in the modulation of ER-to-Golgi transport of DC-STAMP in response to TLR triggering, suggesting a novel role for OS9 in myeloid differentiation and cell fusion.
Assuntos
Células Dendríticas/metabolismo , Retículo Endoplasmático/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células CHO , Cricetinae , Cricetulus , Células Dendríticas/citologia , Células Dendríticas/imunologia , Retículo Endoplasmático/metabolismo , Humanos , Lectinas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Deleção de Sequência/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismoRESUMO
Rac1 is a small GTPase that regulates the actin cytoskeleton but also other cellular processes. To investigate the function of Rac1 in skin, we generated mice with a keratinocyte-restricted deletion of the rac1 gene. Rac1-deficient mice lost nearly all of their hair within a few weeks after birth. The nonpermanent part of mutant hair follicles developed constrictions; lost expression of hair follicle-specific keratins, E-cadherin, and alpha6 integrin; and was eventually removed by macrophages. The permanent part of hair follicles and the sebaceous glands were maintained, but no regrowth of full-length hair follicles was observed. In the skin of mutant mice, epidermal keratinocytes showed normal differentiation, proliferation, cell-cell contacts, and basement membrane deposition, demonstrating no obvious defects of Rac1-deficient epidermis in vivo. In vitro, Rac1-null keratinocytes displayed a strong spreading defect and slightly impaired adhesion. These data show that Rac1 plays an important role in sustaining the integrity of the lower part of hair follicles but not in maintenance of the epidermis.
Assuntos
Folículo Piloso/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Deleção de Genes , Folículo Piloso/citologia , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/crescimento & desenvolvimento , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Morfogênese/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rac1 de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
T cell receptor-major histocompatibility complex (TCR-MHC) affinities span a wide range in a polyclonal T cell response, yet it is undefined how affinity shapes long-term properties of CD8 T cells during chronic infection with persistent antigen. Here, we investigate how the affinity of the TCR-MHC interaction shapes the phenotype of memory CD8 T cells in the chronically Toxoplasma gondii-infected brain. We employed CD8 T cells from three lines of transnuclear (TN) mice that harbor in their endogenous loci different T cell receptors specific for the same Toxoplasma antigenic epitope ROP7. The three TN CD8 T cell clones span a wide range of affinities to MHCI-ROP7. These three CD8 T cell clones have a distinct and fixed hierarchy in terms of effector function in response to the antigen measured as proliferation capacity, trafficking, T cell maintenance, and memory formation. In particular, the T cell clone of lowest affinity does not home to the brain. The two higher affinity T cell clones show differences in establishing resident-like memory populations (CD103+) in the brain with the higher affinity clone persisting longer in the host during chronic infection. Transcriptional profiling of naïve and activated ROP7-specific CD8 T cells revealed that Klf2 encoding a transcription factor that is known to be a negative marker for T cell trafficking is upregulated in the activated lowest affinity ROP7 clone. Our data thus suggest that TCR-MHC affinity dictates memory CD8 T cell fate at the site of infection.
Assuntos
Encéfalo/imunologia , Encéfalo/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Memória Imunológica , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transferência Adotiva , Animais , Antígenos CD/metabolismo , Antígenos de Protozoários/imunologia , Biomarcadores , Cálcio/metabolismo , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Imunofenotipagem , Cadeias alfa de Integrinas/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Complexo Principal de Histocompatibilidade/genética , Camundongos , Proteínas de Protozoários/imunologiaRESUMO
T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and ß genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity-hence reactivity to self-and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 Ld-restricted epitope, derived from the Rop7 protein of Toxoplasma gondii We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds.
Assuntos
Linfócitos T CD8-Positivos/citologia , Receptores de Antígenos de Linfócitos T/genética , Toxoplasmose/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Divisão Celular , Perfilação da Expressão Gênica , Rearranjo Gênico do Linfócito T , Camundongos , Proteínas de Protozoários/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Toxoplasmose/imunologiaRESUMO
The ubiquitous apicomplexan parasite Toxoplasma gondii stimulates its host's immune response to achieve quiescent chronic infection. Central to this goal are host dendritic cells. The parasite exploits dendritic cells to disseminate through the body, produce pro-inflammatory cytokines, present its antigens to the immune system and yet at the same time subvert their signaling pathways in order to evade detection. This carefully struck balance by Toxoplasma makes it the most successful parasite on this planet. Recent progress has highlighted specific parasite and host molecules that mediate some of these processes particularly in dendritic cells and in other cells of the innate immune system. Critically, there are several important factors that need to be taken into consideration when concluding how the dendritic cells and the immune system deal with a Toxoplasma infection, including the route of administration, parasite strain and host genotype.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Interações Hospedeiro-Patógeno , Fatores Imunológicos/metabolismo , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Fatores de Virulência/metabolismo , Animais , Humanos , Evasão da Resposta Imune , Fatores Imunológicos/imunologia , Toxoplasmose/imunologia , Toxoplasmose/parasitologia , Fatores de Virulência/imunologiaRESUMO
Dendritic cells (DCs) are professional antigen presenting cells of the immune system that play a crucial role in initiating immune responses and maintaining self tolerance. Better understanding of the molecular basis of DC immunobiology is required to improve DC-based immunotherapies. We previously described the interaction of transcription factor LUMAN (also known as CREB3 or LZIP) with the DC-specific transmembrane protein DC-STAMP in DCs. Target genes of LUMAN and its role in DCs are currently unknown. In this study we set out to identify genes regulated by LUMAN in DCs using microarray analysis. Expression of a constitutively active form of LUMAN in mouse DC cell line D2SC/1 identified Apolipoprotein A4 (ApoA4) as its target gene. Subsequent validation experiments, bioinformatics-based promoter analysis, and silencing studies confirmed that ApoA4 is a true target gene of LUMAN in bone marrow-derived DCs (BMDCs).
Assuntos
Apolipoproteínas A/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Brefeldina A/farmacologia , Linhagem Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores da Síntese de Proteínas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Dendritic cells (DCs) are the professional antigen-presenting cells (APC) which efficiently prime the immune response or induce tolerance. We recently identified Dendritic Cell Specific TrAnsMembrane Protein (DC-STAMP), a novel 470 amino acid protein preferentially expressed by dendritic cells. Previously we demonstrated that DC-STAMP re-localizes towards the Golgi upon DC maturation. To identify proteins that interact with DC-STAMP, a yeast-2-hybrid analysis was performed. Here, we report a physically interacting partner of DC-STAMP in the endoplasmic reticulum (ER), called LUMAN (also known as CREB3 or LZIP). LUMAN was previously described as an ER-resident transcription factor with unknown function. It is activated in a process called regulated intramembrane proteolysis (RIP), which involves translocation to the Golgi and subsequent proteolytic cleavage. The proteolytically activated form of the protein then translocates to the nucleus. Our data indicate that DC-STAMP plays an important role in the modulation of LUMAN activation. Moreover, we demonstrate that LUMAN is endogenously expressed by DC and becomes activated by RIP upon DC maturation induced by various different stimuli. These data define LUMAN/DC-STAMP as a novel regulatory circuit in DC.