Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory\ud T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-c chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.\ud Methodology/Principal Findings: CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21)\ud using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/ mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p,0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg6RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/ mTOR pathway.\ud Conclusions/Significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation

Salvage surgery for head and neck squamous cell carcinoma

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Reduction of Radiation Dosage in Visualization of Paranasal Sinuses in Daily Routine

Background. Preoperative imaging of the nose and paranasal sinus is standard in otorhinolaryngology. Previous studies on phantoms demonstrated the potential for dose reduction of cone beam computed tomography (CBCT) by varying the application parameters. Methodology. Based on previous studies, the standard protocol of paranasal sinus imaging by CBCT was altered. One hundred and fifty examinations using the old protocol (01/2010–01/2011, high dosage) and 150 examinations using the new protocol (09/2012–09/2013, low dosage) were evaluated and compared for the visibility of 17 anatomical structures, the Lund-Mackay Score, and technical parameters. Results. Alteration of the protocol resulted in a significant reduction in dosage (6.64 mGy versus 2.88 mGy). Both groups showed the same amount of pathology (Lund-Mackay Score: 4.95±3.79 versus 5.26±5.77; p=0.558). There was a significant better visibility of the anatomical structures (all visible = 1, nothing visible = 4) (results: 1.25 versus 1.17; p=0.001) in the low-dosage group. Conclusion. Despite a significant reduction in the applied dosage, reliable visualization of the bony anatomy of the anterior skull base is possible by CBCT. This demonstrates the need for the discussion of the required clinical imaging quality

Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells

Introduction: Adaptive regulatory T cells (Tr1) are induced in the periphery by environmental stimuli. CD73 expression and adenosine (ADO) production by tumor cells may influence Tr1 generation and their immunosuppressive activity. Material and Methods: Tr1 were generated in co-cultures of CD4+CD25neg T cells, autologous immature dendritic cells (iDC), and irradiated ADO-producing CD73+ or non-producing CD73neg breast cancer (BrCa) cell lines (TU). The expression of ectonucleotidases and other surface markers on Tr1 was determined by flow cytometry. Tr1-mediated suppression of proliferation was evaluated in CFSE-based assays. Luciferase-based ATP detection assays and mass spectrometry were used to measure ATP hydrolysis and ADO levels. Cytokine levels were measured by ELISA or Luminex. CD73 expression on tumor cells or T cells in TU tissues was assessed by immunofluorescence. Results: CD73+ TU induced higher numbers of Tr1 cells (p < 0.01) than CD73neg TU. Tr1TU73+ hydrolyzed more exogenous ATP, produced more ADO, and mediated higher suppression than Tr1TU73neg (p < 0.05 for all). ARL67156, an ectonucleotidase inhibitor, and ZM241385, A2A receptor antagonist, reduced suppression of proliferation mediated by Tr1TU73+ cells (p < 0.01). Basal-like primary BrCa cells expressed higher levels of ectonucleotidases and induced more Tr1 than less aggressive primary luminal-like BrCa. Conclusion: BrCa producing ADO (CD73+ TU) favor the induction of Tr1, which expresses CD39 and CD73, hydrolyzes ATP to ADO, and effectively suppresses anti-tumor immunity

Transoral surgery for oropharyngeal tumors using the Medrobotics(®) Flex(®) System - a case report.

Transoral resection of pharyngeal tumors with acceptable oncological and functional results can be challenging due to their location in a narrow anatomic space

TMV promote expansion of human Treg in culture.

<p>The fold expansion of fresh (<i>left panel</i>) or rapamycin-expanded (<i>right panel</i>) CD4⁺CD25^high T cells to which TMV or DC-derived MV were added on day 0. Cells were stimulated with OKT3, anti-CD28 Abs and IL-2 (500 IU/mL) and cultured for 14–21 d. The data are means ± SD of six independent co-cultures. Asterisks indicate significant differences (p<0.05) between the cultures ± TMV.</p

TMV increase suppressor activity of Treg.

<p>The FLOCA was used to simultaneously measure suppression proliferation of CFSE-labeled autologous CD4⁺CD25^neg RC and their apoptosis upon co-incubation with CFSE-negative Treg. RC cells stimulated with OKT3, anti-CD28 mAb and IL-2 (150 IU/mL) were co-cultured for 5 d with Treg pre-incubated or not with TMV. At harvest, cells were stained with 7-AAD and examined by flow cytometry. The suppressor assays were performed at the S:RC ratio of 1∶1. Treg pre-incubated with TMV induced higher levels of apoptosis (<b><u>A</u></b>) and greater inhibition of RC proliferation (<b><u>B</u></b>). The data are from one experiment of five performed. When Treg were pretreated with Concanamycin A or GrB inhibitor I and then incubated with TMV, the frequency of 7-AAD⁺ RC was lower (<b><u>C</u></b>) as was RC proliferation inhibition (<b><u>D</u></b>). Treg pretreated with FasL Ab and then incubated with TMV induced RC death (<b><u>C</u></b>) and inhibited RC proliferation (<b><u>D</u></b>).</p