Improving regulatory T cell production through mechanosensing

Abstract

Induced Tregs (iTregs) have great promise in adoptive immunotherapy for treatment of autoimmune diseases. This report investigates the impacts of substrate stiffness on human Treg induction, providing a powerful yet simple approach to improving production of these cells. Conventional CD4⁺ human T cells were activated on materials of different elastic modulus and cultured under suppressive conditions. Enhanced Treg induction was observed on softer materials as early as 3 days following activation and persisted for multiple weeks. Substrate stiffness also affected epigenetic modification of Treg specific genes and Treg suppressive capacity. Tregs induced on substrates of an optimal stiffness balance quantity and suppressive quality.

Keywords: PDMS; T cells; Treg induction; Treg statbility; Treg suppressive capacity.

Figure 1.. PDMS substrates for Treg induction.

(A) Indentation study of PDMS with different ratios of cross-liner and monomer (Data are mean, whiskers are min and max, n=2 individual experiments for 1:59, n=4 for 1:10, and n = 5 for 1:25, and 2 replicates each, One-way ANOVA). (B) Fluorescence intensity of PDMS surface coating imaged with an epifluorescence microscope in 40X (Data are mean, whiskers are min and max, n=3 individual experiments and 2 replicates each, One-way ANOVA). (C) Treg induction experimental setup (D) Representative gating setup for all samples. Unstained control was used for gating FOXP3⁺ T cells.

Figure 2.. Impact of substrate stiffness on Treg induction.

(A & B) Comparison across 3 different PDMS stiffnesses, based on (A) percent and (B) total count of FOXP3⁺ cells. (C & D) Comparison between softest PDMS (17kPa) surface and stiff controls of plate-bound OKT3 and 9.3 and Dynabeads (2 beads per cell) based on (C) percent and (D) total count of FOXP3⁺ cells. For all panels, Data are mean ± SEM, n=5 individual experiments and 2 replicates each. Two-way ANOVA with Tukey multiple comparisons; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 3.. Mechanosensing in Treg induction relies on cell contractility.

Effect of Y-27623 inhibition of actomyosin contractility based on (A) percent and (B) total count of FOXP3⁺ cells. Data are mean ± SEM, n=5 individual experiments and 2 replicates each; Two-way ANOVA with Tukey multiple comparisons; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 4.. Suppressive capacities of Treg on different PDMS substrates.

(A) Percent suppression with 1:1, 1:2, and 1:4 ratios of Treg and Tconvs on 3 PDMS substrates, and gating was created based on unstimulated control. (B) Percent suppression with 1:1, 1:2, and 1:4 ratios of Treg and Tconvs comparing 870kPa to plate-bound control and Dynabeads. One-way ANOVA was conducted with GraphPad Prism, and 870kPa is the control group for comparison with Dynabeads and Plate-bound control for (B) Data are mean, whiskers are min and max, n=3 individual experiments; * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5.. Substrate stiffness impacts induced Treg epigenetic modification.

(A) PCA analysis of samples epigenetic modification profilers. (B) Volcano plot of differentially methylation analysis comparing tTregs vs Tconvs. Cutoffs are 5% methylation difference, q-value <0.05. (C) Volcano plot of differentially methylation analysis comparing 2600kPa vs 17kPa. Cutoffs are 1% methylation difference, q-value <0.05. (D, E, F) Percent DNA methylation level for all samples on ctla-4, cd254, FoxP3.