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Littérature scientifique sur le sujet « T cell transfer »

Auteur : Grafiati

Publié le 9 mars 2023

Mis à jour le 10 mars 2023

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Articles de revues sur le sujet "T cell transfer"

Chapman, Paul B. « Programming T cells for adoptive T cell transfer therapy ». Pigment Cell & ; Melanoma Research 23, n^o 2 (1 février 2010) : 155–56. http://dx.doi.org/10.1111/j.1755-148x.2010.00681.x.

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Sharma, Preeti, et David M. Kranz. « T Cell Receptors for Gene Transfer in Adoptive T Cell Therapy ». Critical Reviews in Immunology 39, n^o 2 (2019) : 105–22. http://dx.doi.org/10.1615/critrevimmunol.2019030788.

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Berger, Carolina, Michael C. Jensen et Stanley R. Riddell. « Establishing T Cell Memory by Adoptive Transfer of T Cell Clones. » Blood 108, n^o 11 (16 novembre 2006) : 866. http://dx.doi.org/10.1182/blood.v108.11.866.866.

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Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for >30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

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Itzhaki, Orit, Daphna Levy, Dragoslav Zikich, Avraham J. Treves, Gal Markel, Jacob Schachter et Michal J. Besser. « Adoptive T-cell transfer in melanoma ». Immunotherapy 5, n^o 1 (janvier 2013) : 79–90. http://dx.doi.org/10.2217/imt.12.143.

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Kessels, Helmut W. H. G., Monika C. Wolkers et Ton N. M. Schumacher. « Adoptive transfer of T-cell immunity ». Trends in Immunology 23, n^o 5 (mai 2002) : 264–69. http://dx.doi.org/10.1016/s1471-4906(02)02219-6.

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Conrad, Heinke, Peter Meyerhuber, Barbara Kast, Julia Mueller, Christian Peschel, Wolfgang Uckert et Helga Bernhard. « Redirection of human T lymphocytes towards HER2 by T cell receptor gene transfer for adoptive T cell transfer (41.6) ». Journal of Immunology 182, n^o 1_Supplement (1 avril 2009) : 41.6. http://dx.doi.org/10.4049/jimmunol.182.supp.41.6.

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Abstract The clinical goal of our studies is the adoptive transfer of primary T cells transduced with a HER2-specific T cell receptor (TCR) for patients with HER2-overexpressing breast cancer. HLA-A2-, CD8 T cells were stimulated with allogeneic HLA-A2+ dendritic cells (DC) pulsed with the peptide HER2369-377. HER2369-377-reactive T cells were screened, cloned and further tested in functional assays. The TCR from the HER2-reactive CTL clone KU1 was cloned into a retrovirus. Primary T lymphocytes were transduced with this construct and functionally compared with the parental CTL clone KU1. The TCR-transduced primary T cells recognized the peptide HER2369-377 exogenously loaded onto T2 cells or endogenously expressed by tumor cells and transfectants. Similar to the parental CTL clone KU1, the transgenic T cells were not only able to recognize HER2369-377, but also the corresponding peptides from HER3 and HER4. Following TCR optimization (codon optimization, murinization), the TCR-transduced T cells displayed an enhanced tumor cell recognition. In conclusion, the fine specificity of a HER2-reactive TCR is conserved following transduction into primary T lymphocytes. These results facilitate the design of HER2-directed immunotherapies based on TCR-transduced T cells. The observed cross-recognition especially with HER3 may be beneficial as HER2 and HER3 overexpressing tumors are particularly aggressive. DFG

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Katsura, Y., T. Kina, T. Amagai, T. Tsubata, K. Hirayoshi, Y. Takaoki, T. Sado et S. I. Nishikawa. « Limiting dilution analysis of the stem cells for T cell lineage. » Journal of Immunology 137, n^o 8 (15 octobre 1986) : 2434–39. http://dx.doi.org/10.4049/jimmunol.137.8.2434.

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Abstract Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.

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Heemskerk, Mirjam H. M., Manja Hoogeboom, Renate Hagedoorn, Michel G. D. Kester, Roel Willemze et J. H. Frederik Falkenburg. « Reprogramming of Virus-specific T Cells into Leukemia-reactive T Cells Using T Cell Receptor Gene Transfer ». Journal of Experimental Medicine 199, n^o 7 (29 mars 2004) : 885–94. http://dx.doi.org/10.1084/jem.20031110.

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T cells directed against minor histocompatibility antigens (mHags) might be responsible for eradication of hematological malignancies after allogeneic stem cell transplantation. We investigated whether transfer of T cell receptors (TCRs) directed against mHags, exclusively expressed on hematopoietic cells, could redirect virus-specific T cells toward antileukemic reactivity, without the loss of their original specificity. Generation of T cells with dual specificity may lead to survival of these TCR-transferred T cells for prolonged periods of time in vivo due to transactivation of the endogenous TCR of the tumor-reactive T cells by the latent presence of viral antigens. Furthermore, TCR transfer into restricted T cell populations, which are nonself reactive, will minimize the risk of autoimmunity. We demonstrate that cytomegalovirus (CMV)-specific T cells can be efficiently reprogrammed into leukemia-reactive T cells by transfer of TCRs directed against the mHag HA-2. HA-2-TCR–transferred CMV-specific T cells derived from human histocompatibility leukocyte antigen (HLA)-A2+ or HLA-A2− individuals exerted potent antileukemic as well as CMV reactivity, without signs of anti–HLA-A2 alloreactivity. The dual specificity of these mHag-specific, TCR-redirected virus-specific T cells opens new possibilities for the treatment of hematological malignancies of HLA-A2+ HA-2–expressing patients transplanted with HLA-A2–matched or –mismatched donors.

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Koste, L., T. Beissert, H. Hoff, L. Pretsch, Ö. Türeci et U. Sahin. « T-cell receptor transfer into human T cells with ecotropic retroviral vectors ». Gene Therapy 21, n^o 5 (3 avril 2014) : 533–38. http://dx.doi.org/10.1038/gt.2014.25.

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Vanham, Guido, Lieve Penne, Heidi Allemeersch, Luc Kestens, Betty Willems, Guido van der Groen, Kuan-Teh Jeang, Zahra Toossi et Elizabeth Rich. « Modeling HIV transfer between dendritic cells and T cells : importance of HIV phenotype, dendritic cell– T cell contact and T-cell activation ». AIDS 14, n^o 15 (octobre 2000) : 2299–311. http://dx.doi.org/10.1097/00002030-200010200-00011.

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Thèses sur le sujet "T cell transfer"

Sommermeyer, Daniel. « Generation of dual T cell receptor (TCR) T cells by TCR gene transfer for adoptive T cell therapy ». Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16051.

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Die Herstellung von T-Zellen mit definierten Spezifitäten durch den Transfer von T-Zellrezeptor (TCR) Genen ist eine effiziente Methode, um Zellen für eine Immuntherapie bereitzustellen. Eine besondere Herausforderung ist dabei, ein ausreichend hohes Expressionsniveau des therapeutischen TCR zu erreichen. Da T-Zellen mit einem zusätzlichen TCR ausgestattet werden, entsteht eine Konkurrenzsituation zwischen dem therapeutischen und dem endogenen TCR. Bevor diese Arbeit begonnen wurde war nicht bekannt, welche TCR nach einem Gen-Transfer exprimiert werden. Daher haben wir Modelle etabliert, in denen TCR Gene in Maus und humane T-Zellen mit definierten endogenen TCR transferiert wurden. Die Expression beider TCR wurde mithilfe von Antikörpern und MHC-Multimeren analysiert. Diese Modelle haben gezeigt, dass bestimmte TCR andere TCR von der Zelloberfläche verdrängen können. Dies führte in einem Fall zu einer vollständigen Umkehr der Antigenspezifität. Aufgrund dieser Ergebnisse haben wir das Konzept von „starken“ (gut exprimierten) und „schwachen“ (schlecht exprimierten) TCR vorgeschlagen. Zusätzlich wurde die Verdrängung „schwacher“ und „starker“ humaner TCR durch Maus TCR beobachtet. Parallel dazu wurde berichtet, dass die konstanten (C) Regionen von Maus TCR für die erhöhte Expression auf humanen Zellen verantwortlich sind. Dies führte zu einer Strategie zur Verbesserung der Expression humaner TCR, die auf dem Austausch der humanen C-Regionen durch die von Maus TCR basiert (Murinisierung). Ein Problem ist dabei die mögliche Immunogenität dieser hybriden Konstrukte. Deshalb haben wir jene Bereiche der Maus C-Regionen identifiziert, die für die erhöhte Expression verantwortlich sind. In der TCRalpha Kette wurden vier und in der TCRbeta Kette fünf Aminosäuren gefunden, die ausreichend für diesen Effekt waren. Primäre humane T-Zellen mit TCR, die diese neun „Maus“ Aminosäuren enthielten, zeigten eine bessere Funktionalität als T-Zellen mit Wildtyp TCR.
The in vitro generation of T cells with a defined antigen specificity by T cell receptor (TCR) gene transfer is an efficient method to create cells for immunotherapy. One major challenge of this strategy is to achieve sufficiently high expression levels of the therapeutic TCR. As T cells expressing an endogenous TCR are equipped with an additional TCR, there is a competition between therapeutic and endogenous TCR. Before this work was started, it was not known which TCR is present on the cell surface after TCR gene transfer. Therefore, we transferred TCR genes into murine and human T cells and analyzed TCR expression of endogenous and transferred TCR by staining with antibodies and MHC-multimers. We found that some TCR have the capability to replace other TCR on the cell surface, which led to a complete conversion of antigen specificity in one model. Based on these findings we proposed the concept of ‘‘strong’’ (well expressed) and “weak” (poorly expressed) TCR. In addition, we found that a mouse TCR is able to replace both “weak” and “strong” human TCR on human cells. In parallel to this result, it was reported that the constant (C)-regions of mouse TCR were responsible for the improved expression of murine TCR on human cells. This led to a strategy to improve human TCR by exchanging the C-regions by their murine counterparts (murinization). However, a problem of these hybrid constructs is the probable immunogenicity. Therefore, we identified the specific parts of the mouse C-regions which are essential to improve human TCR. In the TCRalpha C-region four and in the TCRbeta C-region five amino acids were identified. Primary human T cells modified with TCR containing these nine “murine” amino acids showed an increased function compared to cells modified with wild type TCR. For TCR gene therapy the utilization of these new C-regions will reduce the amount of foreign sequences and thus the risk of immunogenicity of the therapeutic TCR.

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Wright, G. P. « Generation of antigen-specific regulatory T cells by T cell receptor gene transfer ». Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18952/.

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Regulatory T cells (Tregs) have shown considerable potential in the treatment of murine models of immuno-pathology. Whilst poly-clonal Tregs are able to suppress immuno-pathology in a number of models, the superiority of Ag-specific Treg treatment has been demonstrated using Tregs from T cell receptor (TCR)- transgenic animals. Translation of these promising results to the clinic has been hampered by difficulties in isolating or enriching the rare Ag-specific Tregs from the polyclonal population. Here I describe two distinct approaches to generate Ag-specific T cells with regulatory ability: firstly, TCR gene transfer into purified CD4+CD25+ T cells was used to redirect the specificity of naturally occurring Tregs. Secondly, co-transfer of FoxP3 and TCR genes served to convert conventional CD4+ T cells into Ag-specific ‘Treg-like’ cells. Both approaches generated T cells that suppressed in vitro and engrafted efficiently, retaining TCR and FoxP3 expression, when adoptively transferred into recipient mice. Using an established arthritis model, I demonstrate Ag-driven accumulation of the gene modified T cells at the site of joint inflammation, which resulted in a reduction of joint swelling. In animals treated with TCR-transferred natural Tregs this was accompanied by a local reduction in the number of inflammatory Th17 cells and a significant decrease in arthritic bone destruction. Together, I have described a strategy to rapidly generate Ag-specific Tregs capable of antigen-dependent amelioration of autoimmune damage in the absence of general immune suppression. These approaches could practicably be translated into the clinic in order to treat numerous different immuno-pathologies.

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Bracq, Lucie. « Analysis of HIV-1 cell-to-cell transfer to macrophages ». Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB063/document.

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Les macrophages sont une cible particulièrement importante de l’infection par le VIH-1 et jouent un rôle crucial dans la physiopathologie de l’infection. Lorsqu’ils sont infectés, leur capacité de survie dans les tissus leur permet de jouer un rôle essentiel dans la dissémination virale et l’établissement de réservoirs viraux au niveau des différents territoires tissulaires. In vitro, les étapes précoces et tardives du cycle de réplication virale dans les macrophages ont été analysées dans le cadre de l’infection par des virus libres. Cependant, les modalités d’infection des macrophages lors d’une transmission intercellulaire reste largement inexplorées. Les travaux présentés ici ont permis d’établir un modèle de transmission intercellulaire du VIH-1 de lymphocytes T infectés vers les macrophages. Nous avons montré que les lymphocytes T infectés sont capables d’interagir étroitement avec les macrophages, conduisant ainsi à la fusion cellulaire de ces deux cellules et permettant le transfert de matériel viral dans les macrophages cibles. Ce transfert viral par fusion cellulaire, rapide et efficace, est restreint aux virus utilisant le corécepteur CCR5 et dépend de l’interaction entre l’enveloppe virale et le récepteur CD4. Les virus transférés sont alors stockés au sein de compartiment cytoplasmique des cellules fusionnées mais nous observons également des évènements précoces d’assemblage et de bourgeonnement du VIH-1 à la membrane plasmique des cellules fusionnées résultant de la fusion des membranes des lymphocytes T infectés et des macrophages cibles. Ces cellules fusionnées acquièrent alors la capacité de fusionner avec les macrophages non infectés environnants permettant la dissémination du VIH-1. L’ensemble de ces résultats met en évidence un nouveau mécanisme de transmission intercellulaire entre lymphocytes T et macrophages via un mécanisme de double fusion cellulaire dépendant de l’enveloppe virale et des récepteurs CD4 et CCR5. Ces évènements successifs de fusion entre lymphocytes T et macrophages puis entre macrophages permettent la formation de cellules géantes multinucléés capables de produire de grande quantité de virus infectieux. Ces cellules multinculées pourraient correspondre aux macrophages multinuclées observés in vivo dans les organes lymphoïdes et le système nerveux central de patients infectés par le VIH-1 ou de singes infectés par le SIV. Ce mécanisme représente donc un modèle de transmission intercellulaire original permettant la dissémination virale et la formation de macrophages réservoirs durant l’infection par le VIH-1
Macrophages are important targets of HIV-1 and play crucial roles in physiopathology of infection. Because of their long time survival capacity, infected macrophages participate in virus dissemination and establishment of persistent virus reservoirs in numerous tissues. In vitro, macrophages infection and analysis of the different steps of the virus cycle have been largely documented using cell-free virus infection. However, there is a paucity in knowledge of the mechanisms that control infection and dissemination to macrophages by cell-to-cell transfer. In the work presented here, we establish a model of HIV-1 cell-to-cell transfer from infected T cells to macrophages. We observed that infected T cells are able to interact with macrophages leading to cell fusion for transfer of viral material to macrophages targets. This cell-to-cell fusion transfer, very fast and efficient, is restricted to CCR5-tropic viruses, and mediated by viral envelope-receptor interactions. Transferred viruses can then accumulate in cytoplasmic compartments of newly lymphocyte/macrophages fused cells but we also observed early viral assembly and budding events at the plasma membrane of these fused cells, resulting from the merge of viral material between infected T cells and macrophages. These cells then acquire the ability to fuse with neighboring non-infected macrophages for virus dissemination. Together, these two-sequential envelope-dependent cell fusion process lead to the formation of highly virus-productive multinucleated giant cells reminiscent of the infected multinucleated giant macrophages detected in vivo in lymphoid organs and the central nervous system of HIV-1 infected patients and simian immunodeficiency virus-infected macaques. These mechanisms may represent an original mode of virus transmission for viral spreading and formation of macrophage virus reservoirs during HIV-1 infection

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Böhm, Stefanie [Verfasser], et Lars [Akademischer Betreuer] Nitschke. « Adoptive T-cell-receptor transfer to examine human T-cell immunology in vitro / Stefanie Böhm. Betreuer : Lars Nitschke ». Erlangen : Universitätsbibliothek der Universität Erlangen-Nürnberg, 2013. http://d-nb.info/1033688193/34.

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Chakupurakal, Geothy. « Preclinical studies of adenovirus-specific T-cells for adoptive transfer to haemopoietic stem cell transplant recipients ». Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/2883/.

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Allogeneic stem cell transplantation (SCT) is the only curative treatment option for many haematological malignancies. Adenovirus (Ad) infections are a significant cause of morbidity and mortality post SCT. Lack of effective anti-viral treatment for Ad disease has led to the development of adoptive immunotherapy of Ad-specific T-cells as a promising therapeutic option for patients in this setting. The aim of this project was to establish preclinical criteria for the development of a clinical trial comparing two T-cell enrichment methods- multimer selection and cytokine secretion selection to enrich Ad-specific T-cells for the purposes of adoptive transfer directly without the need for in vitro culture. Eight pHLA tetramers containing HLA class I restricted Ad epitopes were generated and their ability to identify and enrich Ad-specific T-cells investigated. HLA A*01 TDL tetramer consistently detected T-cells in all (13/13) healthy adult donors screened. Frequency and enrichement of Ad-specific T-cells by cytokine secretion and selection was also investigated. Despite the low frequency of Ad-specific T-cells, clinical grade enrichment was feasible by both methods. T-cells selected by both methods were then characterised for homing and proliferative potential. Ad-specific T-cells identified by either method had a high proliferative potential, possessed a novel minimally differentiated memory phenotype, were cytotoxic towards Ad species responsible for infections in SCT recipients and capable of limiting virus replication. In conclusion, Ad-specific T-cells enriched by multimer selection or cytokine secretion selection are suitable for adoptive transfer to patients with Ad infection following HSCT. Both methods also allow the monitoring of Ad-specific immune reconstitution after adoptive transfer.

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Carluccio, S. « GENERATION OF TUMOR-SPECIFIC CYTOTOXIC T-LYMPHOCYTES FROM PEROPHERAL BLOOD OF COLORECTAL CANCER PATIENTS FOR ADOPTIVE T-CELL TRANSFER ». Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/231155.

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Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth most common cause of death in the developed Western countries. Adoptive T-cell transfer (ACT) refers to an immunotherapeutic approach in which anti-tumor T lymphocytes, usually the tumor infiltrating lymphocytes (TIL), are identified, grown ex vivo and then re-infused into the cancer patient. ACT of EBV-specific T-cell lines and T Cytotoxic Lymphocytes (CTLs) for the therapy of EBV-induced lymphomas is the best demonstration of clinically efficacious ACT, but there are many evidences also for leukemia and multiple myeloma. As regards to the solid tumors, ACT using autologous TIL, grown ex-vivo and then re-infused into the cancer patient, has emerged as an effective treatment for metastatic melanoma and renal cell carcinoma (RCC), that are the most immunogenic tumors in humans. Randomized clinical trials are ongoing for gastric cancer, hepatocellular carcinoma and lung cancer. These approaches mainly use the TIL and the definition of tumor associated antigen (TAA), tumor specific antigen (TSA) or cancer testis antigen (CTA), that are generally correlated with tumor progression and immunogenicity in various types of cancer. However these antigens are often found to be poorly expressed in CRC, and few is known about their relationship with this type of neoplasia. In addition, although a clear association between TIL and clinical outcome of CRC has been documented, active and adoptive immunotherapy do not play yet an important role in the treatment of advanced CRC. In order to develop an ACT protocol for CRC treatment, we designed an experimental approach that does not require neither the definition of molecular defined tumor antigens, nor the availability of TIL. Our strategy was based on the in vitro stimulation of patient’s CD8+-enriched T-cells from peripheral blood mononuclear cells (PBMCs) with dendritic cells (DCs), pulsed with apoptotic tumor cells as a source of tumor antigens, in order to generate autologous CTLs with strong anti-tumor activity. In this study, 78 CRC patients were enrolled. Tumor biopsies were obtained at surgery, together with 100 ml of heparinized peripheral blood (PB). Tumors were mechanically dissociated to a single-cell suspension and cultured to obtain tumor cell line from each patient. DCs were generated from previously separated PBMCs, using a magnetic positive selection of CD14+ monocytes, cultured in presence of recombinant human Interleukin-4 (rh IL-4) and recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rh GM-CSF). Anti-tumor CTLs were elicited in co/micro-culture using DCs as antigen-presenting cells, autologous apoptotic tumor cells as source of antigens and T CD8+ lymphocytes enriched effectors, with weekly stimulation. CTLs Interferon-γ (IFN-γ) secretion was assessed by ELISpot assay to evaluate their activation in response to autologous tumor. Tumor cell lines were obtained from 20 out of 78 patients (25,6%), because gut intestinal flora had adversly affected the establishment of primary tumor cell line and a loss of expansion of tumor cells was observed. DCs were generated from 26 patients, but only 6 patients had the corresponding tumor cell line, indispensable for the co-culture setting up. This was the reason why co/micro-cultures were set up only for 6 patients. ELISpot assay was performed at the end of co/micro-culture stimulations to evaluate effectors IFN-γ secretion. ELISpot results showed that strong and significant IFN-γ secretion was detected at the third, fourth and fifth stimulations for one patient and at the second for another patient, whereas for three patients a weak secretion was detected during the second and third stimulations. Although our immunological study must be performed on an increased number of CRC patients, and the CTLs expansion, together with CTLs lytic ability against autologous tumor cells, must be still performed, our results suggested that the generation of tumor-specific CTLs could be useful for supporting an ACT approach in CRC.

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Gräf, Patricia [Verfasser]. « Serial transfer of single cell-derived immunocompetence reveals stemness of CD8+ central memory T cells / Patricia Gräf ». München : Verlag Dr. Hut, 2015. http://d-nb.info/1070124389/34.

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Akhter, Waseem. « Le rôle du transfert de mitochondries des cellules stromales mésenchymateuses (CSM) dans la suppression des réponses des cellules T CD4+ et CD8+ ». Thesis, Université de Montpellier (2022-….), 2022. http://www.theses.fr/2022UMONT011.

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Les lymphocytes T cytotoxiques CD8+ (LTC) et les cellules T helper CD4+ sont des effecteurs clés dans les maladies auto-immunes, les maladies du greffon contre l'hôte et le rejet de greffe. Les cellules stromales mésenchymateuses (CSM) sont des cellules multipotentes auto-renouvelables qui possèdent des propriétés de réparation tissulaire et d'immunomodulation. En raison de leur capacité à réprimer les réponses immunitaires pathogènes, elles présentent un potentiel thérapeutique pour le traitement des maladies à médiation immunitaire. Les CSMs ont la capacité unique d'exporter leurs propres mitochondries vers les cellules voisines en réponse à une blessure ou une inflammation. Cependant, on ne sait pas si le transfert de mitochondries a lieu et s'il joue un rôle dans la répression des réponses des lymphocytes T CD4+ Th1 et CD8+. Nous avons abordé cette question en utilisant un modèle de souris transgénique de la maladie et des CSMs allogéniques dérivées de la moelle osseuse in vitro et in vivo. Nos résultats ont montré :1) Les CSMs ont inhibé l'expansion, le gain du phénotype effecteur et la production de la cytokine effectrice IFNγ in vitro et le potentiel diabétogène in vivo des cellules CD4+ Th1 après activation. De façon remarquable, les cellules T CD4+ ont absorbé les mitochondries des CSMs pendant la suppression. Le transfert de mitochondries de CSMs aux cellules Th1 CD4+ a entraîné une diminution de la prolifération et de la production d'IFNγ. Enfin, nous avons démontré que les CSMs et les mitochondries de CSMs empêchent la régulation à la hausse du facteur de transcription maître Th1 sur les cellules T CD4+ activées.2) Les CSMs ont diminué l'expansion, le gain du phénotype effecteur et la production de la cytokine effectrice IFNγ dans les cellules T CD8+ après activation in vitro. Notamment, nous avons constaté que pendant leur interaction conduisant à la suppression, les CSMs ont également transféré des mitochondries aux LTC. Le transfert des mitochondries des CSM aux cellules T CD8+ a entraîné une diminution de la prolifération et de la production d'IFNγ lors de l'activation, contribuant à l'effet suppressif global des CSMs. Enfin, nous avons démontré que les mitochondries de CSMs et de CSMs régulent de manière différentielle l'équilibre de deux facteurs de transcription clés pour la différenciation des LTC, T-bet et Eomes, sur les cellules T CD8+ activées
CD8+ Cytotoxic T lymphocytes (CTL) and CD4+ T helper cells are key effectors in autoimmune, graft versus host diseases and graft rejection. Mesenchymal Stromal Cells (MSCs) are self-renewing multipotent cells with tissue repair and immunomodulatory properties. Due to their ability to repress pathogenic immune responses they show therapeutic potential for the treatment of immune mediated diseases. MSCs have the unique ability to export their own mitochondria to neighboring cells in response to injury and inflammation. However, whether mitochondrial transfer occurs and has any role in the repression of CD4+ Th1 and CD8+ T cell responses is unknown. We have addressed this issue utilizing a transgenic mouse model of disease and allogeneic bone marrow derived MSCs in vitro and in vivo. Our results showed:1) MSCs inhibited expansion, gain of effector phenotype and the production of the effector cytokine IFNγ in vitro and the diabetogenic potential in vivo of CD4+ Th1 cells after activation. Remarkably, CD4+ T cells took up mitochondria from MSCs during suppression. The transfer of MSC mitochondria to CD4+ Th1 cells resulted in decreased proliferation and production of IFNγ. Finally, we demonstrated that both MSCs and MSC mitochondria prevent the upregulation of the master Th1 transcription factor on activated CD4+ T cells.2) MSCs decreased the expansion, gain of effector phenotype and the production of the effector cytokine IFNγ in CD8+ T cells after activation in vitro. Notably, we found that during their interaction conducting to suppression, MSCs also transferred mitochondria to CTL. MSC mitochondrial transfer to CD8+ T cells resulted in decreased proliferation and production of IFNγ upon activation contributing to the global suppressive effect of MSCs. Finally, we demonstrated that both MSCs and MSC mitochondria differentially regulate the balance of two transcription factors key for CTL differentiation, T-bet and Eomes, on activated CD8+ T cells

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Uhlig, Holm H. « Intestinal bacterial flora and the inflammatory immune response in the T cell transfer model of colitis ». Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403771.

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Petschenka, Jutta [Verfasser]. « Safety and therapeutic efficacy of adoptive p53-specific T cell antigen receptor (TCR) gene transfer / Jutta Petschenka ». Mainz : Universitätsbibliothek Mainz, 2014. http://d-nb.info/1052080685/34.

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Livres sur le sujet "T cell transfer"

Lee, Paul H. Effects of adoptive transfer of OSP A-specific TH2 T cells on the evolution of lyme borreliosis in mice. [New Haven, Conn : s.n.], 1995.

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Kang, Joonsoo. Analysis of T cell receptor-antigen interaction using retrovirus-mediated gene transfer. 1994.

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Chapitres de livres sur le sujet "T cell transfer"

Wang, Mingjun. « Adoptive T Cell Transfer ». Dans Encyclopedia of Cancer, 1–4. Berlin, Heidelberg : Springer Berlin Heidelberg, 2014. http://dx.doi.org/10.1007/978-3-642-27841-9_7111-1.

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Shaffer, Donald R., Conrad Russell Y. Cruz et Cliona M. Rooney. « Adoptive T Cell Transfer ». Dans Cancer Immunotherapy, 47–70. New York, NY : Springer New York, 2012. http://dx.doi.org/10.1007/978-1-4614-4732-0_3.

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Wang, Mingjun. « Adoptive T-Cell Transfer ». Dans Encyclopedia of Cancer, 114–17. Berlin, Heidelberg : Springer Berlin Heidelberg, 2014. http://dx.doi.org/10.1007/978-3-662-46875-3_7111.

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Newrzela, Sebastian, Brandenburg Gunda et Dorothee von Laer. « T Cell Culture for Gammaretroviral Transfer ». Dans Genetic Modification of Hematopoietic Stem Cells, 71–82. Totowa, NJ : Humana Press, 2009. http://dx.doi.org/10.1007/978-1-59745-409-4_6.

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Holtick, Udo, et Elie Azoulay. « ICU ». Dans The EBMT/EHA CAR-T Cell Handbook, 161–63. Cham : Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-030-94353-0_31.

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AbstractCAR-T cell treatment comes with significant side effects that challenge the structure and capacity of haematology wards and will regularly necessitate intermittent patient transfer to the ICU. Life-threatening adverse events include cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, which can occur within hours or days after administration. Sepsis might also require ICU admission within the days that follow CAR-T infusion in these high-risk immunocompromised patients.

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Miyazaki, Yumi, et Shunichi Shiozawa. « In Vivo Cell Transfer Assay to Detect Autoreactive T Cell Subsets ». Dans Methods in Molecular Biology, 49–53. New York, NY : Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0404-4_6.

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Banerjee, Daliya, et Motomu Shimaoka. « Lentiviral Gene Transfer Method to Study Integrin Function in T Lymphocytes ». Dans Integrin and Cell Adhesion Molecules, 47–54. Totowa, NJ : Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-166-6_4.

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Slater, S., U. Kreuzburg-duffy, R. Jaggar et C. Macdonald. « Transfer of SV40 - T Expression Vectors into Human Monocytes ». Dans Animal Cell Technology : Developments Towards the 21st Century, 85–90. Dordrecht : Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-011-0437-1_14.

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Weigmann, Benno. « Induction of Colitis in Mice (T-Cell Transfer Model) ». Dans Methods in Molecular Biology, 143–51. New York, NY : Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1212-4_14.

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Lugli, Enrico, et Luca Gattinoni. « Harnessing Stem Cell-Like Memory T Cells for Adoptive Cell Transfer Therapy of Cancer ». Dans Cancer Drug Discovery and Development, 183–209. Cham : Springer International Publishing, 2015. http://dx.doi.org/10.1007/978-3-319-21167-1_8.

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Actes de conférences sur le sujet "T cell transfer"

Wilking, Alice, Lili Wang, Benjamin K. Chen, Thomas Huser et Wolfgang Hubner. « Resolving T cell — T cell transfer of HIV-1 by optical nanoscopy ». Dans 2017 Conference on Lasers and Electro-Optics Europe (CLEO/Europe) & European Quantum Electronics Conference (EQEC). IEEE, 2017. http://dx.doi.org/10.1109/cleoe-eqec.2017.8087773.

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Kristensen, Nikolaj Pagh, Christina Heeke, Siri A. Tvingsholm, Anne-Mette Bjerregaard, Arianna Draghi, Amalie Kai Bentzen, Rikke Andersen, Marco Donia, Inge Marie Svane et Sine Reker Hadrup. « Abstract A14 : Neoepitope-specific CD8+ T cells in adoptive T-cell transfer ». Dans Abstracts : AACR Special Conference on Tumor Immunology and Immunotherapy ; November 17-20, 2019 ; Boston, MA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/2326-6074.tumimm19-a14.

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Kephart, Jacob, et G. F. Jones. « Material Distribution Optimization in a Metal Matrix Heat Sink Using a Constructal-Design Inspired Unit T-Cell ». Dans ASME 2017 Heat Transfer Summer Conference. American Society of Mechanical Engineers, 2017. http://dx.doi.org/10.1115/ht2017-4950.

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Constructal principles are used to investigate the optimization of material utilization in a metal matrix heat sink that maximizes the total heat transfer rate through the base of heat sink. This approach utilizes a two-dimensional geometry to examine spatial heat flow and optimal material distribution in a metal matrix in the plane perpendicular to the coolant flow direction. The matrix is composed of multiple layers of conductive tees built up from the smallest constituent, the unit T-cell. The unit cell consists of a conductive tee-shaped geometry with the two rectangular void regions each making up half of a cooling channel. The horizontal boundaries must match the temperature and heat flux at the boundaries of the neighboring unit cells as this is a conjugate conduction/convection problem. The geometry of the unit cell is characterized by aspect ratios of channel width to height, overall cell width to height, and channel height to cell height. The matrix structure is assembled by stacking unit cells into multiple layers where the number of cells in each layer is an integer multiple of the cells contained in the lower layer. The solution is obtained for optimal T-cell geometric parameters under a set of predetermined constraints including overall volume, solid fill fraction, and number of layers. When a large number of stacked unit cells are considered, the results describe the ideal spatial distribution of porosity and pore sizes for two dimensional functionally graded metal-matrix heat sink. These results will lead to a better understanding of the role played by the porosity distribution in a metal-matrix heat sink and may be applied to the analysis, optimization, and design of more effective heat sinks.

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Inderberg, Else M., Sébastien Wälchli, Marit R. Myhre, Kari Lislerud, Gunnar Kvalheim et Gustav Gaudernack. « Abstract 2310 : With a little help from CD4 T cells in adoptive T-cell transfer ». Dans Proceedings : AACR 107th Annual Meeting 2016 ; April 16-20, 2016 ; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-2310.

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Heeke, Christina, Anne-Mette Bjerregaard, Amalie Kai Bentzen, Marco Donia, Rikke Andersen, Marie Stentoft Svane et Sine Reker Hadrup. « Abstract B015 : T-cell recognition profiling of CD8+ T-cells in tumor-infiltrating lymphocytes expanded for adoptive cell transfer ». Dans Abstracts : Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference : Translating Science into Survival ; September 30 - October 3, 2018 ; New York, NY. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/2326-6074.cricimteatiaacr18-b015.

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Howsmon, Daniel, Juergen Hahn, Shelby Steinmeyer, Arul Jayaraman et Robert Alaniz. « Neural networks elucidate T cell priming conditions for adoptive transfer ». Dans 2015 41st Annual Northeast Biomedical Engineering Conference (NEBEC). IEEE, 2015. http://dx.doi.org/10.1109/nebec.2015.7117044.

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Cooper, Laurence. « Abstract IA47 : Nonviral gene transfer to redirect T cell specificity ». Dans Abstracts : CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference : Translating Science into Survival ; September 16-19, 2015 ; New York, NY. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/2326-6074.cricimteatiaacr15-ia47.

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Rylander, M. Nichole, Kenneth R. Diller, Sihong Wang et Shanti J. Aggarwal. « Correlation of HSP70 Expression and Cell Viability Following Thermal Stimulation of Aortic Endothelial Cells ». Dans ASME 2004 Heat Transfer/Fluids Engineering Summer Conference. ASMEDC, 2004. http://dx.doi.org/10.1115/ht-fed2004-56383.

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The goal of this study was to determine the kinetics of HSP70 expression and variations in cell viability resulting from thermal preconditioning of the heart. Experiments were conducted to determine combinations of elevated temperatures and brief heating durations that could produce enhanced HSP70 levels while maintaining high cell viability. Bovine aortic endothelial cells were heated with a water bath at temperatures ranging from 44–50°C and periods of 1–30 min consistent with anticipated protocols for cardiac preconditioning prior to surgery. Western blot and cell adhesion analysis in culture following heating were employed to determine HSP70 level and cell viability respectively. The results demonstrate that HSP70 expression and cell viability are not maximized simultaneously. To ensure cell viability greater than 90% required a heating protocol for which HSP70 expression was less than its maximum level. HSP70 expression increments from 2.3 to 3.6 times the value of the control can be achieved for thermal stimulation protocols varying from T = 46°C for 10 min to T = 50°C for 1 min while maintaining a cell viability of 90% or greater. An Arrhenius injury model was fit to the cell damage data yielding values for scaling and activation energy terms of A = 1.4×1066 s−1 and Ea = 4.1×105 J/mole.

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Kakimi, Kazuhiro, Tomohiro Murakawa, Takeshi Fukami, Shigenori Goto, Toru Kaneko, Yukihiro Yoshida, Shin-ichi Takamoto et Jun Nakajima. « Abstract 1926 : Adoptive γδ T cell transfer therapy for non-small cell lung cancer ». Dans Proceedings : AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010 ; Washington, DC. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/1538-7445.am10-1926.

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Moon, Edmund K., Carmine Carpenito, Carl June et Steven M. Albelda. « Optimizing Adoptive T Cell Transfer (ACT) Immunotherapy For Malignant Pleural Mesothelioma (MPM) ». Dans American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado. American Thoracic Society, 2011. http://dx.doi.org/10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a2365.

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Rapports d'organisations sur le sujet "T cell transfer"

Tzfira, Tzvi, Michael Elbaum et Sharon Wolf. DNA transfer by Agrobacterium : a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, décembre 2005. http://dx.doi.org/10.32747/2005.7695881.bard.

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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.

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Ron, Eliora, et Eugene Eugene Nester. Global functional genomics of plant cell transformation by agrobacterium. United States Department of Agriculture, mars 2009. http://dx.doi.org/10.32747/2009.7695860.bard.

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The aim of this study was to carry out a global functional genomics analysis of plant cell transformation by Agrobacterium in order to define and characterize the physiology of Agrobacterium in the acidic environment of a wounded plant. We planed to study the proteome and transcriptome of Agrobacterium in response to a change in pH, from 7.2 to 5.5 and identify genes and circuits directly involved in this change. Bacteria-plant interactions involve a large number of global regulatory systems, which are essential for protection against new stressful conditions. The interaction of bacteria with their hosts has been previously studied by genetic-physiological methods. We wanted to make use of the new capabilities to study these interactions on a global scale, using transcription analysis (transcriptomics, microarrays) and proteomics (2D gel electrophoresis and mass spectrometry). The results provided extensive data on the functional genomics under conditions that partially mimic plant infection and – in addition - revealed some surprising and significant data. Thus, we identified the genes whose expression is modulated when Agrobacterium is grown under the acidic conditions found in the rhizosphere (pH 5.5), an essential environmental factor in Agrobacterium – plant interactions essential for induction of the virulence program by plant signal molecules. Among the 45 genes whose expression was significantly elevated, of special interest is the two-component chromosomally encoded system, ChvG/I which is involved in regulating acid inducible genes. A second exciting system under acid and ChvG/Icontrol is a secretion system for proteins, T6SS, encoded by 14 genes which appears to be important for Rhizobium leguminosarum nodule formation and nitrogen fixation and for virulence of Agrobacterium. The proteome analysis revealed that gamma aminobutyric acid (GABA), a metabolite secreted by wounded plants, induces the synthesis of an Agrobacterium lactonase which degrades the quorum sensing signal, N-acyl homoserine lactone (AHL), resulting in attenuation of virulence. In addition, through a transcriptomic analysis of Agrobacterium growing at the pH of the rhizosphere (pH=5.5), we demonstrated that salicylic acid (SA) a well-studied plant signal molecule important in plant defense, attenuates Agrobacterium virulence in two distinct ways - by down regulating the synthesis of the virulence (vir) genes required for the processing and transfer of the T-DNA and by inducing the same lactonase, which in turn degrades the AHL. Thus, GABA and SA with different molecular structures, induce the expression of these same genes. The identification of genes whose expression is modulated by conditions that mimic plant infection, as well as the identification of regulatory molecules that help control the early stages of infection, advance our understanding of this complex bacterial-plant interaction and has immediate potential applications to modify it. We expect that the data generated by our research will be used to develop novel strategies for the control of crown gall disease. Moreover, these results will also provide the basis for future biotechnological approaches that will use genetic manipulations to improve bacterial-plant interactions, leading to more efficient DNA transfer to recalcitrant plants and robust symbiosis. These advances will, in turn, contribute to plant protection by introducing genes for resistance against other bacteria, pests and environmental stress.

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Lee, Chung, Timothy Kuzel, Richard Meagher, Ximing Yang, Norm Smith et Qiang Zhang. Preparation for a Clinical Trial Using Adoptive Transfer of Tumor-Reactive TGF_Beta-Insensitive CD8+ T Cells for Treatment of Prostate Cancer. Fort Belvoir, VA : Defense Technical Information Center, juillet 2006. http://dx.doi.org/10.21236/ada462885.

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Lee, Chung. Preparation for a Clinical Trial Using Adoptive Transfer of Tumor-Reactive TGF_Beta-Insensitive CD8+ T Cells for Treatment of Prostate Cancer. Fort Belvoir, VA : Defense Technical Information Center, juillet 2006. http://dx.doi.org/10.21236/ada463479.

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Firon, Nurit, Prem Chourey, Etan Pressman, Allen Hartwell et Kenneth J. Boote. Molecular Identification and Characterization of Heat-Stress-Responsive Microgametogenesis Genes in Tomato and Sorghum - A Feasibility Study. United States Department of Agriculture, octobre 2007. http://dx.doi.org/10.32747/2007.7591741.bard.

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Exposure to higher than optimal temperatures - heat-stress (HS) - is becoming increasingly common to all crop plants worldwide. Heat stress coinciding with microgametogenesis, especially during the post-meiotic phase that is marked by starch biosynthesis, is often associated with starch-deficient pollen and male sterility and ultimately, greatly reduced crop yields. The molecular basis for the high sensitivity of developing pollen grains, on one hand, and factors involved in pollen heat-tolerance, on the other, is poorly understood. The long-term goal of this project is to provide a better understanding of the genes that control pollen quality under heat-stress conditions. The specific objectives of this project were: (1) Determination of the threshold heat stress temperature(s) that affects tomato and sorghum pollen quality whether: a) Chronic mild heat stress conditions (CMHS), or b) Acute heat stress (AHS). (2) Isolation of heat-responsive, microgametogenesis-specific sequences. During our one-year feasibility project, we have accomplished the proposed objectives as follows: Objectrive 1: We have determined the threshold HS conditions in tomato and sorghum. This was essential for achieving the 2nd objective, since our accumulated experience (both Israeli and US labs) indicate that when temperature is raised too high above "threshold HS levels" it may cause massive death of the developing pollen grains. Above-threshold conditions have additional major disadvantages including the "noise" caused by induced expression of genes involved in cell death and masking of the differences between heatsensitive and heat-tolerant pollen grains. Two different types of HS conditions were determined: a) Season-long CMHS conditions: 32/26°C day/night temperatures confirmed in tomato and 36/26°C day maximum/night minimum temperatures in sorghum. b) Short-term AHS: In tomato, 2 hour exposure to 42-45°C (at 7 to 3 days before anthesis) followed by transfer to 28/22±2oC day/night temperatures until flower opening and pollen maturation, caused 50% reduced germinating pollen in the heat-sensitive 3017 cv.. In sorghum, 36/26°C day/night temperatures 10 to 5 days prior to panicle emergence, occurring at 35 days after sowing (DAS) in cv. DeKalb28E, produced starch-deficient and sterile pollen. Objective 2: We have established protocols for the high throughput transcriptomic approach, cDNA-AFLP, for identifying and isolating genes exhibiting differential expression in developing microspores exposed to either ambient or HS conditions and created a databank of HS-responsivemicrogametogenesis-expressed genes. A subset of differentially displayed Transcript-Derived Fragments (TDFs) that were cloned and sequenced (35 & 23 TDFs in tomato and sorghum, respectively) show close sequence similarities with metabolic genes, genes involved in regulation of carbohydrate metabolism, genes implicated in thermotolerance (heat shock proteins), genes involved in long chain fatty acids elongation, genes involved in proteolysis, in oxidation-reduction, vesicle-mediated transport, cell division and transcription factors. T-DNA-tagged Arabidopsis mutants for part of these genes were obtained to be used for their functional analysis. These studies are planned for a continuation project. Following functional analyses of these genes under HS – a valuable resource of genes, engaged in the HS-response of developing pollen grains, that could be modulated for the improvement of pollen quality under HS in both dicots and monocots and/or used to look for natural variability of such genes for selecting heat-tolerant germplasm - is expected.

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Ficht, Thomas, Gary Splitter, Menachem Banai et Menachem Davidson. Characterization of B. Melinensis REV 1 Attenuated Mutants. United States Department of Agriculture, décembre 2000. http://dx.doi.org/10.32747/2000.7580667.bard.

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Brucella Mutagenesis (TAMU) The working hypothesis for this study was that survival of Brucella vaccines was directly related to their persistence in the host. This premise is based on previously published work detailing the survival of the currently employed vaccine strains S19 and Rev 1. The approach employed signature-tagged mutagenesis to construct mutants interrupted in individual genes, and the mouse model to identify mutants with attenuated virulence/survival. Intracellular survival in macrophages is the key to both reproductive disease in ruminants and reticuloendothelial disease observed in most other species. Therefore, the mouse model permitted selection of mutants of reduced intracellular survival that would limit their ability to cause reproductive disease in ruminants. Several classes of mutants were expected. Colonization/invasion requires gene products that enhance host-agent interaction or increase resistance to antibacterial activity in macrophages. The establishment of chronic infection requires gene products necessary for intracellular bacterial growth. Maintenance of chronic infection requires gene products that sustain a low-level metabolism during periods characterized little or no growth (1, 2). Of these mutants, the latter group was of greatest interest with regard to our originally stated premise. However, the results obtained do not necessarily support a simplistic model of vaccine efficacy, i.e., long-survival of vaccine strains provides better immunity. Our conclusion can only be that optimal vaccines will only be developed with a thorough understanding of host agent interaction, and will be preferable to the use of fortuitous isolates of unknown genetic background. Each mutant could be distinguished from among a group of mutants by PCR amplification of the signature tag (5). This approach permitted infection of mice with pools of different mutants (including the parental wild-type as a control) and identified 40 mutants with apparently defective survival characteristics that were tentatively assigned to three distinct classes or groups. Group I (n=13) contained organisms that exhibited reduced survival at two weeks post-infection. Organisms in this group were recovered at normal levels by eight weeks and were not studied further, since they may persist in the host. Group II (n=11) contained organisms that were reduced by 2 weeks post infection and remained at reduced levels at eight weeks post-infection. Group III (n=16) contained mutants that were normal at two weeks, but recovered at reduced levels at eight weeks. A subset of these mutants (n= 15) was confirmed to be attenuated in mixed infections (1:1) with the parental wild-type. One of these mutants was eliminated from consideration due to a reduced growth rate in vitro that may account for its apparent growth defect in the mouse model. Although the original plan involved construction of the mutant bank in B. melitensis Rev 1 the low transformability of this strain, prevented accumulation of the necessary number of mutants. In addition, the probability that Rev 1 already carries one genetic defect increases the likelihood that a second defect will severely compromise the survival of this organism. Once key genes have been identified, it is relatively easy to prepare the appropriate genetic constructs (knockouts) lacking these genes in B. melitensis Rev 1 or any other genetic background. The construction of "designer" vaccines is expected to improve immune protection resulting from minor sequence variation corresponding to geographically distinct isolates or to design vaccines for use in specific hosts. A.2 Mouse Model of Brucella Infection (UWISC) Interferon regulatory factor-1-deficient (IRF-1-/- mice have diverse immunodeficient phenotypes that are necessary for conferring proper immune protection to intracellular bacterial infection, such as a 90% reduction of CD8+ T cells, functionally impaired NK cells, as well as a deficiency in iNOS and IL-12p40 induction. Interestingly, IRF-1-/- mice infected with diverse Brucella abortus strains reacted differently in a death and survival manner depending on the dose of injection and the level of virulence. Notably, 50% of IRF-1-/- mice intraperitoneally infected with a sublethal dose in C57BL/6 mice, i.e., 5 x 105 CFU of virulent S2308 or the attenuated vaccine S19, died at 10 and 20 days post-infection, respectively. Interestingly, the same dose of RB51, an attenuated new vaccine strain, did not induce the death of IRF-1-/- mice for the 4 weeks of infection. IRF-1-/- mice infected with four more other genetically manipulated S2308 mutants at 5 x 105 CFU also reacted in a death or survival manner depending on the level of virulence. Splenic CFU from C57BL/6 mice infected with 5 x 105 CFU of S2308, S19, or RB51, as well as four different S2308 mutants supports the finding that reduced virulence correlates with survival Of IRF-1-/- mice. Therefore, these results suggest that IRF-1 regulation of multi-gene transcription plays a crucial role in controlling B. abortus infection, and IRF-1 mice could be used as an animal model to determine the degree of B. abortus virulence by examining death or survival. A3 Diagnostic Tests for Detection of B. melitensis Rev 1 (Kimron) In this project we developed an effective PCR tool that can distinguish between Rev1 field isolates and B. melitensis virulent field strains. This has allowed, for the first time, to monitor epidemiological outbreaks of Rev1 infection in vaccinated flocks and to clearly demonstrate horizontal transfer of the strain from vaccinated ewes to unvaccinated ones. Moreover, two human isolates were characterized as Rev1 isolates implying the risk of use of improperly controlled lots of the vaccine in the national campaign. Since atypical B. melitensis biotype 1 strains have been characterized in Israel, the PCR technique has unequivocally demonstrated that strain Rev1 has not diverted into a virulent mutant. In addition, we could demonstrate that very likely a new prototype biotype 1 strain has evolved in the Middle East compared to the classical strain 16M. All the Israeli field strains have been shown to differ from strain 16M in the PstI digestion profile of the omp2a gene sequence suggesting that the local strains were possibly developed as a separate branch of B. melitensis. Should this be confirmed these data suggest that the Rev1 vaccine may not be an optimal vaccine strain for the Israeli flocks as it shares the same omp2 PstI digestion profile as strain 16M.

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