Flow cytometry data mining by cytoChain identifies determinants of exhaustion and stemness in TCR-engineered T cells

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (T_SCM) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-T_SCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.     

The influence of small field output factors simulated uncertainties on the calculated dose in VMAT plans for brain metastases: a multicentre study

Objectives:This multicentric study was carried out to investigate the impact of small field output factors (OFs) inaccuracies on the calculated dose in volumetric arctherapy (VMAT) radiosurgery brain plans.Methods:Nine centres, realised the same five VMAT plans with common planning rules and their specific clinical equipment Linac/treatment planning system commissioned with their OFs measured values (OFbaseline). In order to simulate OFs errors, two new OFs sets were generated for each centre by changing only the OFs values of the smallest field sizes (from 3.2 × 3.2 cm² to 1 × 1 cm²) with well-defined amounts (positive and negative). Consequently, two virtual machines for each centre were recommissioned using the new OFs and the percentage dose differences ΔD (%) between the baseline plans and the same plans recalculated using the incremented (OFup) and decremented (OFdown) values were evaluated. The ΔD (%) were analysed in terms of planning target volume (PTV) coverage and organs at risk (OARs) sparing at selected dose/volume points.Results:The plans recalculated with OFdown sets resulted in higher variation of doses than baseline within 1.6 and 3.4% to PTVs and OARs respectively; while the plans with OFup sets resulted in lower variation within 1.3% to both PTVs and OARs. Our analysis highlights that OFs variations affect calculated dose depending on the algorithm and on the delivery mode (field jaw/MLC‐defined). The Monte Carlo (MC) algorithm resulted significantly more sensitive to OFs variations than all of the other algorithms.Conclusion:The aim of our study was to evaluate how small fields OFs inaccuracies can affect the dose calculation in VMAT brain radiosurgery treatments plans. It was observed that simulated OFs errors, return dosimetric calculation accuracies within the 3% between concurrent plans analysed in terms of percentage dose differences at selected dose/volume points of the PTV coverage and OARs sparing.Advances in knowledge:First multicentre study involving different Planning/Linacs about undetectable errors in commissioning output factor for small fields.     

Development of large numbers of mast cells at sites of idiopathic chronic dermatitis in genetically mast cell-deficient WBB6F1-W/Wv mice

The normal skin and other tissues of adult mast cell-deficient WBB6F1- W/Wv or WCB6F1-Sl/Sld mice contain less than 1.0% the number of mast cells present in the corresponding tissues of the congenic normal (+/+) mice. As a result, genetically mast cell-deficient WBB6F1-W/Wv or WCB6F1-Sl/Sld mice are widely used for studies of mast cell differentiation and function. We found that mast cells developed at sites of idiopathic chronic dermatitis in WBB6F1-W/Wv mice and that the number of mast cells present in the skin of WBB6F1-W/Wv mice was proportional to the severity of the dermatitis (in ear skin, there were 33 +/- 4 mast cells/mm2 of dermis at sites of severe dermatitis v 9 +/- 3 at sites of mild dermatitis, 0.8 +/- 0.3 in skin without dermatitis, and 100 +/- 7 in the normal skin of congenic WBB6F1-+/+ mice; in back skin, the corresponding values were 2.0 +/- 0.6, 1.1 +/- 0.9, 0.025 +/- 0.025, and 26.2 +/- 3.2). The development of mast cells was a local, not systemic, consequence of the dermatitis. Thus, WBB6F1-W/Wv mice with severe dermatitis lacked mast cells in skin not showing signs of dermatitis and also in the peritoneal cavity, stomach, cecum, and tongue. Idiopathic chronic dermatitis was not associated with the local development of mast cells in WCB6F1-Sl/Sld mice, a mutant whose mast cell deficiency is due to a mechanism distinct from that of WBB6F1-W/Wv mice. These findings may have implications for understanding the nature of the mast cell deficiency in WBB6F1-W/Wv and WCB6F1-Sl/Sld mice and for the use of these mutants to analyze mast cell differentiation and function.