The immune system maintains a balance between protection and tolerance. Regulatory T cells (Tregs) act as a tolerance mechanism to suppress effector immune cells. Recent studies suggest that the induction of Tregs from conventional T cells (Tconvs) has many practical and therapeutic advantages. Previously, both human and mouse T cells were reported to exhibit different responses to activating substrates of different rigidities as indicated in IL-2 secretion levels. In this work, we explore the previously unknown effect of substrate rigidity on the induction of Tregs from Tconvs. We used Sylgard 184 poly(dimethylsiloxane) (PDMS) to obtain rigidity ranging a few hundred kilopascals to megapascals. Mouse CD4+ T cells (Foxp3-GFP linked B6 mouse) were obtained from spleen and further isolated to CD25+ and CD25− cells using magnetic bead-based isolation kits. CD4+CD25− T cells (>99% Foxp3−) were then seeded onto the surfaces coated with antibodies to CD3 and CD28 in IL-2 and TGF-b-enriched media. Surprisingly, there was a significant increase in Treg induction rate at lower substrate rigidities (i.e., Young’s modulus, E ~ 100 kPa) compared to high rigidity (i.e., E ~ 3 MPa). To confirm that this significant difference in induction rate is truly related to T cell mechanosensing, we administered compound Y-27632 (cY) to inhibit myosin contractility. In the presence of cY, the difference in induction rate at varying rigidities was significantly reduced. This study furthers our understanding of mechanosensing properties of immune cells and raises questions about the underlying molecular mechanisms involved in this process of T cells choosing to proliferate or differentiate.

Immunotherapy using regulatory T cells (Tregs) has shown recent successes in the treatment of autoimmune and inflammatory diseases such as type 1 diabetes. While natural Tregs are unstable and dysfunctional in the inflammatory milieu, induced Tregs are more potent and durable. Tregs can be induced from CD4+CD25− T cells in vitro with TGF-β and IL-2 during activation. Chemical pathways in Treg induction have been heavily investigated, but the impact of mechanical cues on Treg induction has not been thoroughly explored. As T cell activation has been shown to be sensitive to the rigidity of the activating substrate, Treg induction may also be modulated by substrate rigidity. To test this hypothesis, Tregs were induced with 10 ng/ml TGF-β and IL-2 on different rigidities of polyacrylamide gels (5 to 110 kPa) coated with anti-CD3 and anti-CD28. The hydrogel stiffness was controlled by adjusting the acrylamide monomer and crosslinker content. The density of activating antibodies was varied by altering the concentration of streptavidin acrylamide that allows the binding of biotinylated antibodies to the gels. High ligand density on the stiffer substrate significantly upregulated the rate of Treg induction, measured by percent Foxp3 high T cells. Decreasing the ligand density shifted the optimal rigidity to softer substrates. This preliminary data has shown that Treg induction is mechanosensitive and dependent on ligand density, which can contribute to the understanding of mechanosensing in Treg induction and the improvement of biomaterial design for generating functional and stable Tregs to advance Treg adoptive therapy.

Regulatory T cells (Tregs) play a pivotal role in modulating immune response hence supporting immune tolerance. Recently, CD45 ligation with anti-CD45RB mAb MB23G2 (anti-CD45RB) was reported to enhance graft-survival; evidence shows it was by homeostatic Treg proliferation in vivo. While many in vivo data support the notion that anti-CD45RB supports Tregs expansion, it is still unclear if the ligation ex vivo is sufficient to enhance Tregs’ avidity for activation and further promote their expansion and function. Here, we were to see if anti-CD45RB ligation can enhance Treg’s sensitivity to perceive activating signals in varying strength in vitro. To test this hypothesis, mouse CD4+ T cells (Foxp3-GFP linked B6 mouse, both Foxp3+ and Foxp3−, or Tregs and Tconvs, respectively) were seeded onto surfaces micropatterned with an antibody targeting the CD3 on T cell (anti-CD3) with and without the presence of anti-CD45RB. These surfaces were created by first microcontact printing of 2-μm diameter features of anti-CD3 at various concentrations (i.e., 1, 5, and 20 μg/mL) on glass coverslips and back-filled with ICAM-1. The effect of anti-CD45RB on Treg and Tconv activation was compared by the ratios of phosphorylation of Lck at Tyr 394 to 505 sites, cell registration on anti-CD3 dot array, and motility of each cell type on varying concentration of activation signals. Overall results show that anti-CD45RB specifically slow-down Treg motility and, therefore, enhance the probability to contact to activating signals (cell registration) and improve T cell activation indicated by ratios of phospho-Lck at active over inactive sites. This study assures the importance of further investigation on the role of anti-CD45RB in Treg activation mechanism.