Mechanical regulation of lymphocyte activation and function.

Mechanosensing, or how cells sense and respond to the physical environment, is crucial for many aspects of biological function, ranging from cell movement during development to cancer metastasis, the immune response and gene expression driving cell fate determination. Relevant physical stimuli include the stiffness of the extracellular matrix, contractile forces, shear flows in blood vessels, complex topography of the cellular microenvironment and membrane protein mobility. Although mechanosensing has been more widely studied in non-immune cells, it has become increasingly clear that physical cues profoundly affect the signaling function of cells of the immune system. In this Review, we summarize recent studies on mechanical regulation of immune cells, specifically lymphocytes, and explore how the force-generating cytoskeletal machinery might mediate mechanosensing. We discuss general principles governing mechanical regulation of lymphocyte function, spanning from the molecular scale of receptor activation to cellular responses to mechanical stimuli.

Cathepsin B causes trogocytosis-mediated CAR T cell dysfunction.

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacy in cancer treatment. Still, most patients receiving CAR T cells relapse within 5 years of treatment. CAR-mediated trogocytosis (CMT) is a potential tumor escape mechanism in which cell surface proteins transfer from tumor cells to CAR T cells. CMT results in the emergence of antigen-negative tumor cells, which can evade future CAR detection, and antigen-positive CAR T cells, which has been suggested to cause CAR T cell fratricide and exhaustion. Whether CMT indeed causes CAR T cell dysfunction and the molecular mechanisms conferring CMT remain unknown. Using a selective degrader of trogocytosed antigen in CAR T cells, we show that the presence of trogocytosed antigen on the CAR T cell surface directly causes CAR T cell fratricide and exhaustion. By performing a small molecule screening using a custom high throughput CMT-screening assay, we found that the cysteine protease cathepsin B (CTSB) is essential for CMT and that inhibition of CTSB is sufficient to prevent CAR T cell fratricide and exhaustion. Our data demonstrate that it is feasible to separate CMT from cytotoxic activity and that CAR T cell persistence, a key factor associated with clinical CAR T cell efficacy, is directly linked to CTSB activity in CAR T cells.

A tug of war between filament treadmilling and myosin induced contractility generates actin rings.

In most eukaryotic cells, actin filaments assemble into a shell-like actin cortex under the plasma membrane, controlling cellular morphology, mechanics, and signaling. The actin cortex is highly polymorphic, adopting diverse forms such as the ring-like structures found in podosomes, axonal rings, and immune synapses. The biophysical principles that underlie the formation of actin rings and cortices remain unknown. Using a molecular simulation platform called MEDYAN, we discovered that varying the filament treadmilling rate and myosin concentration induces a finite size phase transition in actomyosin network structures. We found that actomyosin networks condense into clusters at low treadmilling rates or high myosin concentrations but form ring-like or cortex-like structures at high treadmilling rates and low myosin concentrations. This mechanism is supported by our corroborating experiments on live T cells, which exhibit ring-like actin networks upon activation by stimulatory antibody. Upon disruption of filament treadmilling or enhancement of myosin activity, the pre-existing actin rings are disrupted into actin clusters or collapse towards the network center respectively. Our analyses suggest that the ring-like actin structure is a preferred state of low mechanical energy, which is, importantly, only reachable at sufficiently high treadmilling rates.

Cytotoxic T Lymphocyte Activation Signals Modulate Cytoskeletal Dynamics and Mechanical Force Generation.

Cytotoxic T lymphocytes (CTLs) play an integral role in the adaptive immune response by killing infected cells. Antigen presenting cells (APCs), such as dendritic cells, present pathogenic peptides to the T cell receptor on the CTL surface and co-stimulatory signals required for complete activation. Activated CTLs secrete lytic granules containing enzymes that trigger target cell death at the CTL-target contact, also known as the immune synapse (IS). The actin and microtubule cytoskeletons are instrumental in the killing of CTL targets. Lytic granules are transported along microtubules to the IS, where granule secretion is facilitated by actin depletion and recovery. Furthermore, actomyosin contractility promotes target cell death by mediating mechanical force exertion at the IS. Recent studies have shown that inflammatory cytokines produced by APCs, such as interleukin-12 (IL-12), act as a third signal for CTL activation and enhance CTL proliferation and effector function. However, the biophysical mechanisms mediating such enhanced effector function remain unclear. We hypothesized that the third signal for CTL activation, IL-12, modulates cytoskeletal dynamics and force exertion at the IS, thus potentiating CTL effector function. Here, we used live cell total internal reflection fluorescence (TIRF) microscopy to study actomyosin and microtubule dynamics at the IS of murine primary CTLs activated in the presence of peptide-MHC and co-stimulation alone (two signals), or additionally with IL-12 (three signals). We found that three signal-activated CTLs have altered actin flows, myosin dynamics and microtubule growth rates as compared to two signal-activated CTLs. We further showed that lytic granules in three-signal activated CTLs are less clustered and have lower velocities than in two-signal activated CTLs. Finally, we used traction force microscopy to show that three signal-activated CTLs exert greater traction forces than two signal-activated CTLs. Our results demonstrate that activation of CTLs in the presence of IL-12 leads to differential modulation of the cytoskeleton, thereby augmenting the mechanical response of CTLs to their targets. This indicates a potential physical mechanism via which the third signal can enhance the CTL response.