Poster Z3 - Rapid Isolation of CD4+ T Cells Using a Functionally Closed Microfluidic System for Generating Induced Regulatory T Cells for Adoptive Therapy

Introduction: Regulatory T cells (Tregs), a small subset of CD4+ T cells, are crucial in maintaining self-tolerance and preventing autoimmune diseases. Adoptive Treg therapies have shown promise in alleviating autoimmune symptoms, but the current manufacturing approaches face challenges. Isolating low-prevalence natural Tregs (nTregs, < 5% of circulating CD4+ T cells) requires large blood volumes, and the lack of specific surface markers results in a highly variable separation purity. Additionally, existing magnetic-/fluorescence-activated cell sorting approaches are complex, expensive, and time-consuming. This study aimed to develop a simple and efficient process for manufacturing clinically relevant numbers of Tregs from small blood volumes in a short timeframe.

Materials and

Methods: An alternative approach was investigated where instead of directly isolating low prevalence nTregs, much more prevalent CD4+ T cells were isolated to generate induced Tregs (iTregs) in cell culture. Rosette-Enabled SIZE-based separation (RESIZE) (Abhishek et al., Lab Chip 23(7):1804-1815, 2023) was used to separate CD4+ T cells from blood using a controlled incremental filtration (CIF) microfluidic device. CD4+ T cells were stimulated with anti-CD3/anti-CD28, IL-2, TGF-β, ATRA, and rapamycin to generate iTregs in 2-week cultures. Flow cytometry was used to assess iTreg percentages using viability dye, anti-CD45, anti-CD4, anti-CD25, and anti-Foxp3 staining. The purity was calculated as 100×([Foxp3+ cells]/[CD4+CD25+ cells]).

Results, Conclusions, and Discussions: The RESIZE separation performed using CIF microfluidic devices recovered 92.4 ± 1.2% of CD4+ T cells with a purity of 94.1 ± 4% from 8 mL of healthy whole blood in < 90 min. For the optimized conditions of Treg induction, the total yield of iTregs was (81.3 ± 47.2)×106 with a purity of 61.1 ± 7.2%. Notably, the mean fluorescence intensity expression of Foxp3, a functional marker of Tregs, was significantly higher in iTregs compared to nTregs from the same subject, suggesting a higher suppressive ability of the Tregs generated in culture. This proof-of-concept study developed an efficient process for manufacturing clinically relevant numbers of high-FoxP3-expressing iTregs from a small volume of blood. The RESIZE separation using a CIF device enabled rapid isolation of CD4+ T cells with >90% recovery and purity. Within 2 weeks of culture, the optimized induction protocol generated >80 million iTregs with >60% purity, meeting the reported requirements of current adoptive Treg therapies. By streamlining the generation of large numbers of potent autologous iTregs, this protocol could enable wider clinical implementation of Treg adoptive therapies.

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