The immune system maintains a balance between protection and tolerance. Regulatory T cells (Tregs) function as a vital tolerance mechanism in the immune system to suppress effector immune cells. Additionally, Tregs can be utilized as a form of immunotherapy for autoimmune disorders. As T cells have previously been shown to exhibit sensitivity to the rigidity of an activating substrate upon activation via IL-2 secretion, we herein explore the previously unknown effect of substrate rigidity on the induction of Tregs from conventional naïve mouse CD4+ T cells. Substrates with modulatable rigidities ranging from a hundred kilopascals to a few megapascals were fabricated via poly(dimethylsiloxane). We found that there was a significant increase in Treg induction at lower substrate rigidities (i.e., E ~ 100 kPa) compared to higher rigidity levels (i.e., E ~ 3 MPa). To confirm that this significant difference in induction rate was truly related to T cell mechanosensing, we administered compound Y-27632 to inhibit myosin contractility. In the presence of Y-27632, the myosin-based contractility was disrupted and, as a result, the difference in Treg induction caused by the substrate rigidity was abrogated. This study demonstrates that mechanosensing is involved in Treg induction and raises questions about the underlying molecular mechanisms involved in this process.

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.

Regulated cell death in response to microbial infection plays an important role in immune defense and is triggered by pathogen disruption of essential cellular pathways. Gram-negative bacterial pathogens in the Yersinia genus disrupt NF-κB signaling via translocated effectors injected by a type III secretion system (T3SS), thereby preventing induction of cytokine production and antimicrobial defense. In murine models of infection, Yersinia blockade of NF-κB signaling triggers cell-extrinsic apoptosis through Receptor Interacting Serine-Threonine Protein Kinase 1 (RIPK1) and caspase-8, which is required for bacterial clearance and host survival. Unexpectedly, we find that human macrophages undergo apoptosis independently of RIPK1 in response to Yersinia or chemical blockade of IKKα/β. Instead, IKK blockade led to decreased cFLIP expression, and overexpression of cFLIP contributed to protection from IKK blockade-induced apoptosis in human macrophages. Importantly, IKK blockade also induces RIPK1 kinase-independent apoptosis in human T cells and human pancreatic cells. Altogether, our data indicate that, in contrast to murine cells, blockade of IKK activity in human cells triggers a distinct apoptosis pathway that is independent of RIPK1. These findings have implications for the contribution of RIPK1 to cell death in humans and the efficacy of RIPK1 inhibition in human diseases.