The aim of this study was to investigate the impact of thymic dysplasia on the phenotypic and functional characteristics of T cells in patients with 22q11.2 deletion syndrome, including T-cell phenotype, transcriptional profile, cytokine production, as well as the possibility of utilizing IL-7 to recover their numbers and function. We found a strong bias towards Th1 response in pediatric and young adult 22q11.2DS patients, expansion of CXCR5+ follicular helper cells and CXCR3+CCR6- Th1 cells, increased production of cytokines IFN-γ, IL-10, IL-2, IL-21 and TNF-α. This Th1 skew was primarily driven by expanded terminally differentiated T cells. IL-7 further reduced naive T cells, increased cytokine production and caused an upregulation of exhaustion markers. Thus, Th1 bias in T cell populations persists from infancy into adolescence and is accompanied by accelerated maturation of T cells into memory stages. This phenotype is exacerbated by IL-7 which causes further decrease in naïve T cells in vitro.

• MeSH
• cytokiny MeSH
• DiGeorgeův syndrom * MeSH
• dítě MeSH
• interferon gama * MeSH
• interleukin-7 MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• paměťové T-buňky MeSH
• Th1 buňky MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While TALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALL is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here we used single-cell mass cytometry to study signal transduction pathways such as JAK-STAT, PI3K-AKT-mTOR and MEK-ERK in 16 diagnostic and five relapsed T-ALL primary samples, and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKT-mTOR and MEK-ERK pathways and increased expression of proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy.

• MeSH
• dítě MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• interleukin-7 * farmakologie   MeSH
• lidé MeSH
• lymfoblastická leukemie-lymfom z prekurzorových T-buněk * farmakoterapie   metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• recidiva MeSH
• T-lymfocyty metabolismus   MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Supraphysiological levels of IL-7 induce increase counts of pre-B cells, naive T cells and memory phenotype CD8+ T cells. Immunocomplexes of IL-7 and αIL-7 mAb M25 (IL-7/M25) were described as IL-7 superagonist in vivo. Thus, treatment of mice with IL-7/M25 remarkably increases the size of the T cell pool. We decided to use IL-7/M25 in order to expand the T cell population prior to the administration of αCTLA-4 and αPD-1 mAbs in tumor-bearing mice and in turn boost the immunotherapy based on a combination of CTLA-4 and PD-1 blockage. We found that just four doses of IL-7/M25 increased the absolute numbers of splenocytes approximately fivefold and significantly shifted the CD4+:CD8+ T cell ratio in favor of CD8+ T cells. There was also a substantive increase in relative counts of memory phenotype CD8+ T cells (approximately threefold) within CD8+ T cells but a significant decrease (approximately 30%) in relative counts of Treg cells within CD4+ T cells. All these data suggest that IL-7/M25 offer a suitable approach to potentiate tumor immunotherapy through CTLA-4 and PD-1 blockage. Unexpectedly, IL-7/M25 significantly abrogated the antitumor activity of αCTLA-4 plus αPD-1 mAbs in the following mouse tumor models: MC-38 and CT26 colon carcinoma and B16F10 melanoma. This paradoxical effect of IL-7/M25 on the antitumor activity of CTLA-4 and PD-1 blockage was not mediated via either increased levels of IL-10 or TGF-β in the sera or increased counts of IL-10-producing B or T cells in the spleen of mice injected with IL-7/M25. Thus, our work shows that caution should be exercised when combining two immunotherapy approaches together.

• MeSH
• antigen CTLA-4 imunologie   MeSH
• antigeny CD279 imunologie   MeSH
• CD4-pozitivní T-lymfocyty imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• imunoterapie metody   MeSH
• interleukin-10 imunologie   MeSH
• interleukin-7 imunologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• melanom imunologie   MeSH
• modely nemocí na zvířatech MeSH
• monoklonální protilátky imunologie   MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory tračníku imunologie   MeSH
• protinádorové látky imunologie   MeSH
• regulační T-lymfocyty imunologie   MeSH
• transformující růstový faktor beta imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND AIMS: Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. METHODS: We present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. RESULTS: We found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. CONCLUSIONS: Our protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.

• MeSH
• aktivace lymfocytů účinky léků   genetika   MeSH
• buněčné kultury metody   MeSH
• buňky PC-3 MeSH
• chimerické antigenní receptory genetika   metabolismus   MeSH
• elektroporace MeSH
• genetické vektory MeSH
• HEK293 buňky MeSH
• imunoterapie adoptivní metody   MeSH
• interleukin-4 farmakologie   MeSH
• interleukin-7 farmakologie   MeSH
• interleukiny farmakologie   MeSH
• kultivované buňky MeSH
• Lentivirus genetika   MeSH
• lidé MeSH
• proteinové inženýrství metody   MeSH
• protinádorové vakcíny genetika   imunologie   MeSH
• receptory antigenů T-buněk genetika   metabolismus   MeSH
• T-lymfocyty * cytologie   účinky léků   imunologie   metabolismus   MeSH
• transdukce genetická metody   MeSH
• transpozibilní elementy DNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Sepse a septický šok představují celosvětově významný medicínský i socioekonomický problém. V současnosti je obecně přijímán dvoufázový koncept významného porušení imunitní homeostázy v sepsi s enormní aktivací imunitních mechanismů v jejím úvodu s následnou kompenzatorní reakcí, při níž je kompromitována většina funkcí na nejrůznějších úrovních vrozené i získané imunity. Aktuální poznatky však ukazují na simultánní aktivaci prozánětlivých a protizánětlivých pochodů již v samotném úvodu systémového zánětu. Tato sekundární alterace obranných mechanismů, vedoucí ke snížené rezistenci k primárně málo virulentním nozokomiálním a oportunním patogenům a ke „chronické“ multiorgánové dysfunkci, může být příčinou zvýšené morbiditya mortality kriticky nemocných. Tento přehledový článek se stručnou formou zabývá právě dysregulací imunitní odpovědi nemocných v sepsi/septickém šoku, jejími konsekvencemi, diagnostickými možnostmi a možnými terapeutickými intervencemi.

Sepsis and septic shock represent an important medical and socio-economic burden worldwide. The double-phased concept of significant immune homeostasis impairment in sepsis has generally been accepted. In this theory, the initial phase is characterized by enormous activation of immune system followed by the compen-satory phase resulting in profound immunosuppression. However, this paradigm has recently been challenged and the concept of simultaneous pro-inflammatory, anti-inflammatory and adaptive immunity suppressing response occurring early in sepsis has been introduced. These immune alterations leading to the failure to combat relatively avirulent, nosocomial and opportune pathogens, and prolonged multiorgan dysfunction seem to be a major cause of increased morbidity and mortality in critically ill patients. This review briefly summarizes the current concept of sepsis-induced immune deregulation and discusses diagnostic tools and emerging immune-based therapeutic interventions.

• MeSH
• adaptivní imunita MeSH
• B-lymfocyty MeSH
• bakteriální proteiny fyziologie   MeSH
• buněčná imunita fyziologie   MeSH
• buňky NK MeSH
• CD4-pozitivní T-lymfocyty MeSH
• cytokiny MeSH
• dendritické buňky fyziologie   MeSH
• homeostáza * fyziologie   MeSH
• imunitní systém - jevy * fyziologie   MeSH
• interleukin-7 MeSH
• lidé MeSH
• mediátory zánětu MeSH
• membránové proteiny MeSH
• monocyty fyziologie   MeSH
• neutrofily fyziologie   MeSH
• přirozená imunita MeSH
• průtoková cytometrie MeSH
• receptory imunologické * fyziologie   klasifikace   MeSH
• sepse * imunologie   MeSH
• septický šok * imunologie   MeSH
• T-lymfocyty fyziologie   MeSH
• virové proteiny MeSH
• Check Tag
• lidé MeSH