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Severe combined immunodeficiency (SCID) has many phenotypes, including those which result in the formation of T cells but render them incapable of an optimal level of proliferation following stimulation by an antigen. Phytohaemagglutinin (PHA) is a mitogen commonly used to assess T-cell proliferation, although anti-CD2/CD3/CD28-coated beads may provide a more physiological trigger. Some patients and healthy controls display poor T-cell proliferation (<45%) in response to PHA, but may be capable of an increased percentage proliferation when stimulated by another mitogen. This study aimed to develop an anti-CD2/CD3/CD28 bead-based T-cell proliferation assay and to compare T-cell proliferation generated by two mitogens both in healthy controls and in poor responders to PHA. A bead-based T-cell proliferation assay was optimized and validated. Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood samples of 10 healthy controls and 3 poor PHA responders. PBMCs were incubated with carboxyfluorescin diacetate succinimidyl ester and three separate stimulants: PHA, Beads only and Beads plus IL-2. Flow cytometry was performed on Day 6, following fluorochrome staining with CD3 PerCP and CD4 APC, to identify CD4+ T cells. No significant difference was found between the results generated by PHA- or Bead stimulation in 10 healthy controls. A strong positive correlation was observed between the use of the Beads alone and the addition of IL-2. A varied response to the Beads was produced among the poor PHA responders. It has been concluded that the bead-based assay will be run alongside every PHA test in the Oxford Immunology diagnostic laboratory. The step to add IL-2 on Day 3 will not be included as this did not significantly impact T-cell proliferation. The Bead assay provides a more physiological addition to the current PHA test and may be useful in assessing T-cell proliferation in those patients who respond poorly to PHA. However, some individuals who respond poorly to PHA may also respond poorly to the anti-CD2/CD3/CD28 Beads. [ABSTRACT FROM PUBLISHER]

Background: AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets., Methods: VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale., Results: AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP., Conclusion: Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse., Competing Interests: Competing interests: MAP owns stock in and is employed Autolus Therapeutics; an inventor on patents licensed to Autolus Therapeutics to which he receives a share of revenues. KSP share holder and consultant of Autolus Therapeutics. CR speaker fees and advisory boards for Novartis, Kite/Gilead, Amgen and BMS/Celgene., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: It has been suggested that dietary freedom in functional insulin therapy may be detrimental to glycemic control in type 1 diabetes. This study evaluates the effect of carbohydrate intake on glycemic control and postprandial blood glucose concentrations., Methods: Insulin pump data from 148 adults with type 1 diabetes, trained in functional insulin therapy, using pumps for ≥6 months, with ≥2 weeks of consecutive downloaded data, ≥80% use of a bolus calculator, ≥3 capillary blood glucose tests/day, and a concurrent HbA1C, were analyzed. More detailed periprandial data (pre- and postmeal glucose, carbohydrate intake, insulin bolus) were collected from a subset of 105 downloads (3495 meals)., Results: Mean (± SD) age of contributors was 43 ± 13 years, HbA1C 7.84% ± 0.93 (62.19 mmol/mol); daily carbohydrate intake 166 ± 71 g. HbA1C reduced with increased meals/day (r = -.370, P < .0005) and increased with mean carbohydrate content/meal (r = .198, P = .043). However, total daily carbohydrate intake had a weak but significant negative association with HbA1C (r = -.181, P = .027). There was no association between standard deviation of carbohydrate intake and HbA1C (r = .021, P = .802) or between meal carbohydrate content and postprandial change in blood glucose (r = -.004, P = .939) for meals with early postprandial (1-3 hours; n = 390) readings. There was a weak positive correlation (r = .184, P = .008) between meal carbohydrate content and late (4-7 hours; n = 390) postprandial readings., Discussion: With appropriate training, patients using insulin pumps can accommodate a flexible diet with variable carbohydrate intake, without detriment to glycemic control. However, large carbohydrate meals may contribute to poorer outcomes, through impact on late postprandial glycemia., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article:PC has received speaker fees and travel support from Medtronic Ltd, Roche Ltd, Johnson and Johnson and Abbott and is a member of their advisory boards., (© 2016 Diabetes Technology Society.)