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Biomaterials

Biomaterials - Poster Session D

Poster B6 - Metabolite Based Reprogramming of Human CD4+ T Cells

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Methylmalonic Acid (MMA) is a known immuno-suppressant that has been found to induce CD8+ T cell exhaustion (Joanne D. Tejero, et. al.). We are investigating the influence of MMA on CD3+ T cell populations, and seek to establish the effect of long-term MMA release from MMA-based polymers. These polymers could act as therapeutics for inflammatory and autoimmune disease via injection or implant, ensuring a gradual release of MMA from the designed polymer to avoid repetitive treatments.

Materials and

Methods:  For this study, blood was obtained from 2 healthy donors and T cells were isolated using a CD3+ isolation kit (biolegend). The T cells were then plated in a 96 well plate and treated with MMA polymer (named as 1,4 MMA), free MMA, and 1,4 butanediol. For each drug, three serial dilutions were tested. Free MMA treatments consisted of 0.62, 0.062, and 0.0062 mg/ml, the 1,4 butanediol treatments were, 3.79, 0.379, and 0.0379 mg/ml, and the MMA polymer 0.1, 0.01, and 0.001 mg/ml concentrations. After a 40 minute drug-only incubation period, the cells were all treated with phorbol 12-myristate-13-acetate (PMA) and ionomycin, which are potent T cell activation agents. In addition to the treatment groups, proper control groups were incubated simultaneously. The total treatment time was 36 hours. The cells were then stained for CD4 and CD69 for flow cytometry. CD69, an early T cell activation marker, was the marker analyzed across CD4 cell populations to determine the effect of the MMA polymer on T cell activation.

Results, Conclusions, and Discussions: The cells treated with PMA/ionomycin alone showed high CD69 expression levels as compared to the no treatment group. Cells treated with free MMA showed high CD69 expression levels among CD4 cells at low treatment concentrations, 0.0062 and 0.062 mg/ml, as compared to no treatment. The cell groups treated with the highest concentration of free MMA, 0.62 mg/ml, showed low CD69 expression levels in CD4 cells as compared to PMA/ionomycin alone. Cells treated with 1,4 butanediol overall showed a similar level of CD69 expression as the PMA/ionomycin alone. Cells treated with the MMA polymer treatments at 0.001 and 0.01 mg/ml showed similar CD69 expression levels as compared to PMA/ionomycin alone, but showed a sharp decrease at 0.1 mg/ml.

Based on control groups we can conclude that the PMA/ionomycin treatment was effective in inducing early activation, because the CD69 expression levels of the PMA/ionomycin cell groups was high compared to the no treatment cell groups. We can also conclude that MMA is effective in inhibiting the expression of the CD69 early activation marker due to the decrease in CD69 expression levels in helper T cells as the concentration of the free MMA treatment increases. The 1,4 butanediol treated helper T cells showed CD69 expression levels that were lower than the PMA/ionomycin treated helper T cells but higher than the free MMA treated helper T cells. From this we can conclude that free MMA is more potent than 1,4 butanediol in inhibiting CD69 expression. The lowest level of CD69 expression was achieved by the highest concentration MMA polymer treatment (0.1 mg/ml).

These studies confirm that MMA released from the polymer is effective in reducing CD69 expression on CD4+ T cells in vitro. However, other subtypes of CD4 T cells and CD8 cell populations remain to be investigated. Future tests will look at CD8 T cell and other CD4 T cell responses to the same treatment conditions. Furthermore, the effects of lower MMA polymer concentrations are yet to be investigated by determining the release profile of MMA from the polymer using release kinetic studies.

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