Invasive aspergillosis remains a serious complication in patients undergoing allogeneic stem cell transplantation (SCT). Since it became clear that lymphocytes provide a critical secondary defense against fungi, adoptive transfer of functionally active anti-Aspergillus T cells might be an option to restore adaptive immune effector mechanisms. Using the interferon (IFN)-␥ secretion assay, we isolated human activated T cells upon stimulation with a cellular extract of Aspergillus fumigatus. Culturing this cell population for 14 days, we obtained an average of 1.1 ؋ 10 7 cells from a single 100-mL blood draw in 7 of 7 healthy individuals. Within another 14 days, these cells were expanded to an average number of 2.0 ؋ 10 8 T-helper 1 (T H 1) cells secreting IFN-␥ on stimulation with Aspergillus antigens. Testing various fungal antigen extracts, similar proportions of IFN-␥-producing CD3 ؉ /CD4 ؉ cells were obtained upon activation with antigen extracts of A fumigatus, A flavus, A niger, and Penicillium chrysogenum, whereas no significant IFN-␥ production was observed upon activation with antigen extracts of Alternaria alternata and Candida albicans. In addition, generated T cells were able to induce damage to A fumigatus hyphae, and significantly increased hyphal damage induced by human neutrophils. CD4 ؉ T-cell-mediated alloreactivity of generated anti-Aspergillus T cells was clearly reduced compared with that of the original cell population. In conclusion, we present a simple and feasible strategy for rapid generation of a high number of functional active T cells against Aspergillus from a single blood draw. Our data suggest that functionally active T cells against Aspergillus could be a promising treatment option for patients undergoing allogeneic SCT. IntroductionInvasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients with hematologic malignancies, particularly in patients who have undergone allogeneic stem cell transplantation (SCT). 1 Important risk factors for IA are neutropenia and defects in the phagocyte cell function. 2 On the other hand, there is an increasing body of evidence that adaptive immune response also plays a critical role in the host defense against Aspergillus fumigatus, the most frequent cause of IA. For example, IA has been reported with increasing frequency in patients with advanced AIDS, 3 and up to two-thirds of patients with IA diagnosed after allogeneic SCT are not neutropenic. 1 In contrast to neutrophil recovery, which generally occurs within the first 2 to 3 weeks after SCT, the number of functional T cells and T-cell function increase slowly over the first few months after transplantation. 4 Prolonged immune suppression due either to transplant conditioning and graft-versus-host disease (GVHD) prophylaxis or to treatment of GVHD further increase the risk of IA in SCT patients. 5,6 It has recently been shown that a significant antigen-specific proliferation of interferon (IFN)-␥-producing T cells occurred in healthy individuals and in patients survi...

Chimeric antigen receptor (CAR) T cell therapy is a potent new treatment option for relapsed or refractory hematologic malignancies. As the monitoring of CAR T cell kinetics can provide insights into the activity of the therapy, appropriate CAR T cell detection methods are essential. Here, we report on the comprehensive validation of a flow cytometric assay for peripheral blood CD19 CAR T cell detection. Further, a retrospective analysis (n = 30) of CAR T cell and B cell levels over time has been performed, and CAR T cell phenotypes have been characterized. Serial dilution experiments demonstrated precise and linear quantification down to 0.05% of T cells or 22 CAR T cell events. The calculated detection limit at 13 events was confirmed with CAR T cell negative control samples. Inter-method comparison with real-time PCR showed appreciable correlation. Stability testing revealed diminished CAR T cell values already one day after sample collection. While we found long-term CAR T cell detectability and B cell aplasia in most patients (12/17), some patients (5/17) experienced B cell recovery. In three of these patients the coexistence of CAR T cells and regenerating B cells was observed. Repeat CAR T cell infusions led to detectable but limited re-expansions. Comparison of CAR T cell subsets with their counterparts among all T cells showed a significantly higher percentage of effector memory T cells and a significantly lower percentage of naïve T cells and T EMRA cells among CAR T cells. In conclusion, flow cytometric CAR T cell detection is a reliable method to monitor CAR T cells if measurements start without delay and sufficient T cell counts are given.

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Background: Central venous access devices (CVAD) provide important benefits in the management of oncological pediatric patients. However, these catheters are responsible for severe complications. Methods: In this context, we aimed to analyze all patients receiving a CVAD in the Department of Pediatric Hematology and Oncology of the University hospital of Mainz over a period of 9 years, focused on CVAD related complications. Data on demographics, as well as intraoperative and postoperative complications were extracted. Results: A total of 296 patients with a mean age 93.2 ± 62.4 months were analyzed. The majority suffered from leukemia ( n = 91, 30.7%), lymphomas ( n = 50, 16.9%), and brain tumors ( n = 48, 16.2%). In 63 (21.3) patients, complications were observed. No death caused by complications of CVADs was found in our series. Catheter-related blood stream infections (BSI) (7.4%) were most prevalent, followed by dislodgements (5.4%), occlusions (2.7%), thrombosis (2.4%), and catheter leakage (2.4%). Insertion site infections were observed in three patients (1.0%). Fifty-nine percent of all patients with catheter-related BSI suffered from Leukemia. In patients with Catheter-related BSIs we detected the condition leukemia as the underlying disease as a risk factor compared to solid tumors as the underlying disease. Overall, totally implanted devices (ports) have a lower complication rate than tunneled catheter. Conclusion: Implantation of CVADs seems to be safe and reliable in this large pediatric patient cohort. Even if complications occur in the long-term management of CVADs, they can be treated successfully and long-term catheter survival rates are excellent.

Because lymphocytes play a major role in the host response to Candida infection, adoptive transfer of anti-Candida T cells might be a therapeutic option in patients undergoing allogeneic hematopoietic stem-cell transplant (alloHSCT) who have invasive Candida infection. Using the interferon (IFN)- gamma secretion assay, we isolated human anti-Candida T cells after stimulation with a cellular extract of C. albicans. These cells were expanded within 4 weeks to an average number of 2.6x107 T helper 1 type lymphocytes and significantly lost their alloreactive potential, compared with the original cell population. The generated cells were also stimulated by antigens of C. tropicalis but not by antigens of C. glabrata or various molds. In addition, generated anti-Candida T cells were able to induce damage to C. albicans hyphae and significantly increased hyphal damage induced by human neutrophils. Our data suggest that the generation of functionally active anti-Candida T cells is feasible and may be a promising treatment option for patients undergoing alloHSCT.

High-grade neuroepithelial tumor of the central nervous system with BCOR alteration (HGNET-BCOR) is a rare, highly malignant tumor. At the time of this publication, no standard protocol exists to treat this tumor entity. In this work, we tested the responsiveness of the primary culture PhKh1 derived from tumor tissue from a pediatric HGNET-BCOR patient (P1) to inhibitors of the Sonic hedgehog pathway combined with radiation. The SMO inhibitors vismodegib and itraconazole had low effect on the proliferation of the PhKh1 cells. However, the GLI inhibitor arsenic trioxide reduced the expression of GLI target genes in the PhKh1 cells and in combination with radiotherapy significantly decreased their clonogenic potential. PhKh1 cells resistant to arsenic trioxide were characterized by the overexpression of molecular chaperones. We combined arsenic trioxide and radiation in the relapse therapy protocol of P1, achieving complete remission after seven weeks. Clinical remission lasted for six months, when P1 developed systemic metastases. Meanwhile, an increase in the concentration of circulating tumor DNA carrying a BCOR internal tandem duplication was observed. Molecular characterization of a second patient (P2) was also performed. In P2, we detected a larger tandem duplication and greater activation of the Sonic hedgehog pathway than in P1. These findings suggest that combining arsenic trioxide with radiotherapy may represent a new therapeutic approach. Moreover, peripheral blood analysis for circulating tumor DNA could help in the early detection of systemic metastases.