Background: Regulatory T cells (Tregs) are a subset of CD4+ T lymphocytes, endowed with suppressive capacities. Tregs are greatly dependent on IL-2 signaling for their survival and functionality. In autoimmune diseases Tregs are impaired and unable to control the disease. In our lab, we recently demonstrated the safety and efficacy of anti-CD19 CAR-Tregs in controlling autoimmune manifestations in a humanized mouse model of SLE. However, engineered cells showed a limited persistence in the peripheral blood. To increase CAR-Treg persistence we evaluated the engineered cell fitness after the manufacturing, due to the influence of the cell source and phenotype on the survival of engineered cells. In literature, several Treg expansion protocols have been reported but a direct comparison is still lacking.

Objectives: In this project we aim to better understand the persistence of CAR-Tregs and to compare different expansion protocols with the final goal to find the best one to obtain a high yield of functional cells.

Methods: To track CAR-Tregs in vivo we generated Tregs transduced with two lentiviral vectors encoding for the CAR construct and for the Luciferase protein (LucCAR-Tregs) and only with the luciferase as control (Luc-Tregs). We injected the cells in humanized mice and tracked them both trough in vivo imaging system (IVIS) and flow cytometry. For the comparison of CAR-Treg manufacturing protocols we decided to test different intensity of stimulation, different concentrations of IL2 and the addition of a stimulation step. We decided to combine these variables on CD4+CD25+ sorted Tregs as cell source and rapamycin in culture. We compared them with our standard manufacturing process: a single stimulation with anti-CD3/CD28 beads with a bead-to-cell ratio of 3:1, rapamycin and IL2 500 IU/ml. For each tested protocol we evaluated the expansion, transduction, purity and memory phenotype of the resulting cellular products. Moreover, we assessed the expression of inhibitory markers and the functional abilities of engineered cells.

Results: In our humanized mouse model by combining flow cytometry and bioluminescence data, Luc+ CAR-Tregs showed an initial expansion with the peak at day +5 after their infusion and a subsequent contraction till their disappearance from the peripheral blood after 39 days, thus confirming the kinetic already observed in our previous experiments. Overall, CAR-Treg injection proved safe, without signs of Cytokine Release Syndrome or B cell aplasia. For the expansion protocols comparison we firstly tested the influence of IL2 concentration. Cells cultured with beads 3:1 and increased (1000 IU/ml) or decreased (250 IU/ml) IL2 didn’t show differences in expansion, transduction, phenotype or inhibitory markers expression. Differences in the suppressive abilities of CAR-Tregs have been detected and resulted to be negatively correlated to the amount of IL2.A, reduced quantity of the cytokine generated a superior suppression contrary to high IL2 that decreased the suppressive ability of CAR-Treg. Not seeing any increased expansion of the cells, we introduced a different stimulation to the protocol reducing the amount of beads used to a 1:1 ratio. We combined the 1:1 stimulation with the different IL2 concentrations but we couldn’t observe any change in the analyzed parameters. We also decided to culture the cells by employing two rounds of stimulation, at day +0 and +9, with a bead-to-cell ratio of 1:1 in the presence of the different concentrations of IL2. Compared to the standard, CAR-Tregs expanded with the restimulation step showed a significant increased expansion without differences in the phenotype, transduction or suppressive abilities. Moreover, we could observe a significant decreased CTLA-4 expression, worth of further studies.

Conclusion: We were able to track CAR-Tregs in vivo confirming our previous results on cells persistence. We generated and compared different expansion protocols and were able to find the best conditions for an increased expansion of CAR-Tregs.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.