No description available.

No description available.

Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

Interleukin (IL) -7 is an essential non-redundant cytokine that influences T-cell differentiation, proliferation, homeostasis and T-cell functions. In T-cells, IL-7 signals are transduced via IL-7's heterodimeric receptor composed of a common, γ chain (CD132) and an IL-7 specific, α chain (CD127). In light of the many roles that IL-7 plays in T-cell biology, it is no surprise that CD127 expression is tightly regulated in T-cells. In this study, I explore the effects that disease specific mutations in CD127 have on CD127 expression, regulation and signal transduction using an in vitro T-cell model. Here I specifically examined four disease associated mutations of CD127: P132S associated with severe combined immunodeficiency; L242_L243insNPC associated with T-cell acute lymphoblastic leukemia; I356V & T244I associated with autoimmune diseases like multiple sclerosis, rheumatoid arthritis and type 1 diabetes. In developing my model, I decided to use Jurkat cells because they expressed high endogenous surface levels of CD132, low endogenous surface levels of CD127 and endogenous STAT5. Jurkat cells were transduced with lentiviruses that induced expression of either WT or one of the four mutant CD127. I found that transduced Jurkat cells produced the WT and all four mutant CD127 proteins. I also found that wild type CD127, I356V, L242_L243insNPC and T244I mutant CD127 proteins were all expressed at the same level on the cell surface. However, I could not detect P132S mutated CD127 protein in its native state on the surface or intracellularly. I also found no differences between the mutant CD127 and wild type CD127 with regards to the level of soluble CD127 transcripts. I found that cell lines expressing L242_L243insNPC, I356V and T244I mutant CD127 protein, down-regulated surface CD127 at high IL-7 doses (25ng/mL) to the same extent as in the cell line expressing wild type CD127 protein. Interestingly, at the low IL-7 dose (1ng/mL) these mutant CD127 cell lines down-regulated surface CD127 to a lesser degree the wild type CD127 cell line. Further studies are required to elucidate whether P132S mutated CD127 is expressed on the surface and if T224I and I356V mutations in CD127 enhance signaling. By understanding CD127 dysregulation and dysfunction in disease states, we can potentially develop therapeutics that can return the function of CD127 to normalcy.

Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

HIV Tat protein downregulates surface expression of the interleukin-7 receptor alpha-chain (CD127) on CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. Once taken up by CD8 T cells, Tat binds directly to the cytoplasmic tail of CD127 inducing receptor internalization and degradation. Given the important roles of CD127 in proper immune function, the Tat/CD127 interactions were characterized and the mechanisms required to induce receptor loss from the surface of CD8 T cells were investigated. Tat deletion mutants were generated each sequentially lacking a region of the protein. CD8 T cells isolated from HIV negative volunteers were exposed to exogenous or intracellular Tat proteins before surface CD127 expression was analyzed by flow cytometry. To characterize Tat/CD127 physical interactions, wild type Tat and Tat mutants were incubated with lysates from a CD127+ Jurkat cell line followed by CD127/Tat co-immunoprecipitation. The effect of Tat on CD127 post-translational modifications was also investigated. Removal of the N-terminus of Tat (aa 1-10 or aa 17-21) prevented Tat from downregulating CD127 and prevented Tat from binding CD127 as assessed by co-immunoprecipitation. Deletion of the basic region (aa 48-59) also prevented Tat from downregulating CD127 but did not prevent Tat from interacting physically as demonstrated by co-immunoprecipitation. Strikingly, endogenously expressed Basic Tat acted as a dominant negative mutant, causing an accumulation of CD127 at the cell surface. These observations suggest that Tat may bind CD127 via its N-terminus to disrupt the normal recycling of the receptor, and then recruit cellular endocytic machinery to the receptor via it’s basic region, to remove the receptor from the cell surface and target it for degradation. Furthermore, Tat encourages the ubiquitination of CD127 by recruiting the cytokine-inducible SH2 containing (CIS) protein to the receptor, possibly leading to accelerated CD127 internalization and proteasomal degradation. I propose a model whereby Tat binds CD127 via its N-terminal region then recruits CIS via its basic region. CIS in turn recruits a cellular E3 ubiquitin ligase to ubiquitin tag the receptor for internalization and proteasome degradation. This research may lead to novel treatments designed to maintain IL-7 signalling and strengthen CD8 T cell function in HIV+ persons.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-11T16:05:45Z No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-05-11T16:05:58Z (GMT) No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) / Made available in DSpace on 2016-05-11T16:05:58Z (GMT). No. of bitstreams: 1 Barbara Maria Nascimento de Oliveira Produção... 2016.pdf: 2363764 bytes, checksum: ec48ade14eccf109aecb436eec7cd66a (MD5) Previous issue date: 2016 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO. A interleucina 7 (IL-7) é uma citocina pleiotrópica produzida principalmente por células estromais da medula óssea, células epiteliais tímicas e intestinais. IL-7 e que promove o desenvolvimento de linfócitos T e B e diferenciação de células T memória, especialmente CD4+. Por isso, diversos autores têm estudado a capacidade de IL-7 promover a restauração da população de linfócitos em situações de linfopenia e resposta imune de memória. Cães que desenvolvem leishmaniose visceral e recebem tratamento especfico apresentam cura clínica. No entanto, após a interrupção do tratamento, a maioria dos animais exibe recaída da doença, mesmo na ausência de reinfecção. É possível que esse fenômeno esteja associado a uma dificuldade de estabelecimento de linfócitos T de memória específicos para Leishmania em cães. Em nosso laboratório, tentativas prévias foram realizadas para produzir a IL-7 canina em Escherichia coli, no entanto, o rendimento da proteína biologicamente ativa foi pequeno. OBJETIVO. Por isso, resolveu-se avaliar a viabilidade de produzir IL-7 do sistema baculovírus-células de inseto. MATERIAL E MÉTODOS. No presente trabalho, uma construção de DNA foi concebida contendo uma sequência de nucleotídeos que codificam o peptídeo sinal da proteína GP64 do baculovírus da Autographa californica (AcMNPV), a IL-7 canina (proteína madura) com códons otimizados para a tradução em Trichoplusia ni, um espaçador com 23 aminoácidos e uma cauda de seis histidinas. A construção de DNA elaborada foi sintetizada quimicamente, inserida em um plasmídeo de clonagem (pUC57-GP64caIL-7), subclonada em um plasmídeo carreador (pFastBac1- GP64caIL-7), adequado para o sistema baculovirus-célula de inseto, e transferida para o cromossoma artificial do baculovírus recombinante (bacmídeo). RESULTADOS. A produção de IL-7 recombinante canina (rcaIL-7) em células de inseto infectadas com baculovírus recombinante foi otimizada. Em seguida, a rcaIL-7 foi produzida em células High-five cultivadas em suspensão utilizando multiplicidade de infecção (MOI) de 5 e tempo de infecção (TOI) de 48 h. A proteína recombinantes foi purificada por cromatografia de afinidade em Sepharose-níquel, obtendo-se um rendimento médio de 5,5 mg por litro de cultivo. CONCLUSÃO. RcaIL-7 purificada, avaliada a 40 ng/mL, mostrou-se capaz de promover a proliferação de células mononucleares de sangue periférico de cães sadios, sem estímulo prévio ou concomitante, indicando que a proteína foi produzida biologicamente ativa. Futuros estudos serão realizados para avaliar a capacidade imunomoduladora da rcaIL-7 em cães e determinar a utilidade dessa citocinas no tratamento de enfermidades caninas, como por exemplo, na leishmaniose visceral canina. / INTRODUCTION. The interleukin 7 is a pleiotropic cytokine produced mainly by bone marrow stromal cells, thymus and intestinal epithelial cells. IL-7 promotes the development of T and B limphocytes and differentiation T cells to memory cells, especially T CD4+ cells. For these reasons, several authors have studied the ability of IL- 7 to promote restoration of the lymphocyte population in cases of lymphopenia and memory imune responses. Dogs which develop visceral leishmaniasis and are treated with specific drugs develop only transient clinical cure. After treatment withdrawal, most animals show disease relapse, even in the absence of reinfection. One possible explanation for the lack of control of the infection after treatment in dogs is a difficulty to develop/maintain specific memory T cells. Therefore, it is possible that effective treatments for canine visceral leishmaniasis be developed by manipulating the immune system. Previously, in our laboratory, it was attemped to produce recombinant canine IL- 7 (rcaIL-7) in Escherichia coli, however there was a low yield of biologically active protein. OBJECTIVE. In the current project, the baculovírus-insect cell system was evaluated to produce rcaIL-7. MATERIAL AND METHODS. In this study, a construction of DNA was designed to encode nucleotides of Autographa californica GP64 signal peptide, canine IL-7 (mature protein) with optimized codons for Trichoplusia ni, a 23-amino acid spacer and a six histidine tail. The DNA construct was chemically synthesized, inserted into a cloning plasmid (pUC57-GP64caIL-7), subcloned into a carrier plasmid (pFastBac1-GP64caIL-7), suited to the baculovirus-insect cell expression system, and transfered to an artificial chromosome of the recombinant baculovirus (bacmid). RESULTS. Recombinant protein expression was optimizatized, then, the rcaIL-7 was produced in a cell culture in suspension using multiplicity of infection (MOI) 5 and time of infection (TOI) 48 hours. Subsequently rcaIL-7 was purified by Nickel Sepharose-Affinity chromatography, obtaining an average yield of 5.5 mg per liter of culture. CONCLUSION. The purified recombinant rcaIL-7 at 40 ng/mL was able to induce prolifereation of peripheral blood mononuclear cells of heath adult dog, suggesting it was biologically active. Given the results, the rcaIL-7 may be used in further studies to assess its ability to modulate canine immune system, that would be useful for the development immunotherapy

HIV Tat protein downregulates surface expression of the interleukin-7 receptor alpha-chain (CD127) on CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. Once taken up by CD8 T cells, Tat binds directly to the cytoplasmic tail of CD127 inducing receptor internalization and degradation. Given the important roles of CD127 in proper immune function, the Tat/CD127 interactions were characterized and the mechanisms required to induce receptor loss from the surface of CD8 T cells were investigated. Tat deletion mutants were generated each sequentially lacking a region of the protein. CD8 T cells isolated from HIV negative volunteers were exposed to exogenous or intracellular Tat proteins before surface CD127 expression was analyzed by flow cytometry. To characterize Tat/CD127 physical interactions, wild type Tat and Tat mutants were incubated with lysates from a CD127+ Jurkat cell line followed by CD127/Tat co-immunoprecipitation. The effect of Tat on CD127 post-translational modifications was also investigated. Removal of the N-terminus of Tat (aa 1-10 or aa 17-21) prevented Tat from downregulating CD127 and prevented Tat from binding CD127 as assessed by co-immunoprecipitation. Deletion of the basic region (aa 48-59) also prevented Tat from downregulating CD127 but did not prevent Tat from interacting physically as demonstrated by co-immunoprecipitation. Strikingly, endogenously expressed Basic Tat acted as a dominant negative mutant, causing an accumulation of CD127 at the cell surface. These observations suggest that Tat may bind CD127 via its N-terminus to disrupt the normal recycling of the receptor, and then recruit cellular endocytic machinery to the receptor via it’s basic region, to remove the receptor from the cell surface and target it for degradation. Furthermore, Tat encourages the ubiquitination of CD127 by recruiting the cytokine-inducible SH2 containing (CIS) protein to the receptor, possibly leading to accelerated CD127 internalization and proteasomal degradation. I propose a model whereby Tat binds CD127 via its N-terminal region then recruits CIS via its basic region. CIS in turn recruits a cellular E3 ubiquitin ligase to ubiquitin tag the receptor for internalization and proteasome degradation. This research may lead to novel treatments designed to maintain IL-7 signalling and strengthen CD8 T cell function in HIV+ persons.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21027号 / 医科博第88号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 杉田 昌彦, 教授 濵﨑 洋子, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM