Introduction: Virus-specific CD8+ T cells are crucial to suppress the viral replication in HIV infection. Their functional status is important as well. Also, the chronic nonspecific immune activation of T and B lymphocytes plays an important role in the immunopathogenesis of HIV infection. Aim of the study: To analyze the frequency and functional status of HIV-specific CD8+ T cells and the expression of nonspecific activation markers on B and T cells in HIV+ patients and to assess the effect of combined antiretroviral therapy (cART) on these parameters. Patients and methods: Our cohort included 80 HIV+ patients: 36 HIV+ patients on cART, 18 patients without therapy, in whom cART was introduced during our study, 9 patients without therapy, 10 patients with primary HIV infection, 3 long-term non-progressors and 4 patients initially on cART, in whom the therapy was discontinued. Control group consisted of 34 HIV- healthy individuals. We examined CD4+ a CD8+ T cell counts, viral load, expression of nonspecific activation markers on T cells and the frequency of HIV-specific CD8+ T cells by ELISpot method and flow cytometry using MHC tetramers and intracellular cytokine detection. Results: No significant differences in HIV-specific CD8+ T cells were found between treated and untreated HIV+ patients. The frequency...

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Final publication is available at http://www.bloodjournal.org/ / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19614号 / 医博第4121号 / 新制||医||1015(附属図書館) / 32650 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 山田 亮, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Le rhabdomyosarcome (RMS) est le cancer des tissus mous le plus courant chez l'enfant, et moins de 30 % des patients à haut risque obtiennent une rémission. Par conséquent, il existe un besoin pour une immunothérapie nouvelle et efficace. Les cellules tueuses naturelles (NK), avec leur capacité intrinsèque à tuer les cellules cancéreuses, représentent un outil thérapeutique prometteur. Cependant, leur efficacité clinique est limitée. Ainsi, nous proposons de concevoir ces cellules avec un récepteur antigénique chimérique (CAR) qui permettra aux cellules NK de cibler plus efficacement les cellules de RMS. De plus, nous proposons aussi de concevoir grâce à la technologie CRISPR-Cas9, des NK n’exprimant pas NKG2A, un récepteur impliqué dans l'inhibition des cellules NK par le microenvironnement tumoral. Nous avons développé un vecteur lentiviral codant pour une construction CAR reconnaissant B7-H3 et FGFR4, deux protéines surexprimées à la surface des cellules RMS, associées à une queue intracellulaire optimisée pour l'activation des NK. Les cellules NK primaires expandues ont été transduites et triées en fonction de l'expression du CAR, conduisant à une population de cellules CAR+- NK enrichie. L'efficacité des deux CAR a été évaluée par des tests cytotoxiques et de dégranulation contre les lignées cellulaires de RMS, RH-30 et RD, toutes deux exprimant B7-H3 et FGFR4. Les résultats préliminaires ont montré une augmentation de la cytotoxicité de 20 % par rapport aux NK de type sauvage pour les CAR anti-B7-H3. Les cellules NK ont également été transduites pour éliminer l’expression du gène KLRC1, codant pour NKG2A, en utilisant CRISPR Cas9. Ceci a permis d’augmenter la cytotoxicité des NK de 20% à 25% comparativement aux NK qui expriment NKG2A. Nous avons aussi combiné les deux modifications génétiques, obtenant ainsi des NK qui expriment un CAR contre les cellules du RMS et n’exprimant pas NKG2A. Des résultats préliminaires nous ont permis d’observer que les NK doublement modifiées étaient 60% plus cytotoxiques que les NK non-transduites et 20% plus efficaces que les CAR-NK ou les NK n’exprimant pas NKG2A. Ce projet sera une preuve de principe qu'une thérapie hautement innovante basée sur l'ingénierie des cellules NK est efficace et applicable au cancer solide. / Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in childhood, and less than 30% of high-risk patients achieve remission. Therefore, there is a need for new and efficient immunotherapy. Natural killer (NK) cells, with their intrinsic ability to kill cancer cells, represent a promising therapeutic tool. However, their clinical efficacy is limited. Thus, we propose to engineer these cells with a Chimeric Antigen Receptor (CAR) that will allow NK cells to target RMS cells more efficiently and though the knock-out of NKG2A, a receptor involved in the inhibition of NK cells by the tumor microenvironment. We developed a lentiviral vector coding for a CAR construct recognizing B7-H3 and FGFR4, two proteins overexpressed at the surface of RMS cells, combined to an intracellular tail optimized for NK activation. Expanded primary NK cells were transduced and sorted based on CAR expression, leading to an enriched CAR+ -NK cells population. Efficacy of both CARs was evaluated by cytotoxic and degranulation assays against RH-30 and RD RMS cell lines, both expressing B7-H3 and FGFR4. Preliminary results showed an increase in cytotoxicity of 20% compared to wild type NK for CAR anti-B7-H3. NK cells were also knocked-out for the gene coding for NKG2A, using CRISPR Cas9, thereby increasing cytotoxicity by 20% to 25%. The combination of both genetic modifications should significantly increase the efficacy of NK-cell based therapy in RMS. Indeed, preliminary results allowed us to observe that doubly modified NKs were more than 60% more cytotoxic than non-transduced NKs and 20% more effective than CAR-NKs or NKs not expressing NKG2A. This project will be a proof of principle that a highly innovative therapy based on NK-cell engineering is efficient and applicable to solid cancer.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24885号 / 医科博第152号 / 新制||医科||10(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 河本 宏, 教授 濵﨑 洋子, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Syfte: Målet med denna studie var att undersöka ett möjligt samband mellan de olika HLA-DRB1*-allelerna med mängden Mutans Streptococci (MS) i saliven, med fokus på HLA DRB1*04, i en grupp förskolebarn i södra Sverige, Skåne.Material och metod: Salivprover från 318 HLA-DRB1*-typade barn odlades på MSB-agarplattor och antalet MS CFU räknades. Resultaten analyserades statistiskt med chi-två-test.Resultat: Inget signifikant samband kunde fastställas mellan någon av DRB1*04-allelerna och mängden MS. Dock var höga MS-värden ungefär dubbelt så vanligt för homozygota DRB1*04 alleler (p = 0,354). Höga MS-värden var vanligare för DRB1*04:01 allelen (p = 0,717). Däremot var höga MS-värden procentuellt mycket mindre förekommande för DRB1*04:04 allelen (p = 0,098). Ett statistisk signifikant samband (p = 0,003) kunde ses för pojkar positiva för DRB1*07:01-allelen. Höga MS-värden var mycket vanligare för DRB1*07:01-allelen.Slutsats: Ett samband mellan HLA-DRB1-alleler och mängden MS i saliven kan finnas. Resultaten indikerar att barn positiva för DRB1*04:01-allelen och homozygota DRB1*04-alleler har en tendens att uppvisa ett högre MS-värde i saliven jämfört med barn negativa för allelen. HLA-typen är antagligen bara en av många faktorer som påverkar mängden MS. Resultaten från denna studie är delvis i linje med tidigare studier och ytterligare studier behövs. / Objective: The aim of the study was to investigate a possible relationship of the different HLA-DRB1* alleles, with focus on HLA-DRB1*04, and the amount of Mutans Streptococci (MS) in saliva from a group of preschool children in the Southern part of Sweden, in the County of Skåne.Material and method: Saliva samples from 318 HLA-DRB1* typed children were cultivated on MSB agar and CFU of MS was counted. The results were statistically analyzed using chi-square tests.Results: No statistical significant relationship could be established between any DRB1*04 allele and the amount of MS. However, high numbers of MS was found to be about twice as common for homozygote DRB1*04 alleles (p = 0.364). High numbers of MS was more common for DRB1*04:01 alleles (p = 0.717). On the contrary, high levels of MS was much less common for DRB1*04:04 alleles (p = 0.098). A statistical significant relationship (p = 0.003) could be seen for boys positive for the DRB1*07:01 allele. High MS count was much more common for DRB1*07:01 alleles.Conclusion: A relationship between HLA-DRB1 alleles and the amount MS in the saliva might exist. The results indicate that children positive for the DRB1*04:01 allele and homozygote DRB1*04 alleles have a tendency to display more MS in their saliva compared to children negative to the alleles. The HLA type might just be one of many factors affecting the amount of MS. The results are partly in line with earlier studies and further studies are needed.

<p>AMR as a cause of graft rejection has been long recognized and the presence of pre formed antibodies against donor HLA is a risk factor for increased graft rejection. FlowXM is the current clinical gold standard for detecting harmful DSA in the recipients and a positive FlowXM is considered a strong contraindication to transplantation. However, newer techniques such as SAB provide with a highly sensitive and specific method for DSA detection that is unattainable by FlowXM. But due to the intrinsic limitations associated with SAB assays, the clinical relevance of DSA detected on SAB has been highly disputable. Therefore, the overall aim of this study was to investigate the utility of SAB in predicting harmful DSA levels, by establishing a fluorescence range on SAB that correlated to positive FlowXM. This was done by retrospectively testing the highest serum dilutions on FlowPRA SAB that produced positive B or T cell FlowXM from 15 variably sensitized patients. Thus, a very narrow MFI range on SAB was established, for B and T cells separately, that correlated to positive FlowXM. On B cells this correlate ranged from 2780-7772 MFI (Mean MFI =5641), whereas T cell range was 1089-6731 (Mean MFI= 3226). In order to test these ranges for prediction of positive FlowXM, B and T cell FlowXM tests were carried out using various serum/cell combinations. DSA MFI of >3000 on SAB resulted in a significantly higher T cell positive FlowXM (p</p> / Master of Science (MSc)