Chemokine receptor distribution on the surface of repolarizing T cells.

T cells migrate constitutively with a polarized morphology, underpinned by signaling compartmentalization and discrete cytoskeletal organizations, giving rise to a dynamic and expansive leading edge, distinct from the stable and constricted uropod at the rear. In vivo, the motion and function of T cells at various stages of differentiation is highly directed by chemokine gradients. When cognate ligands bind chemokine receptors on their surface, T cells respond by reorientating their polarity axis and migrating toward the source of the chemokine signal. Despite the significance of such chemotactic repolarization to the accurate navigation and function of T cells, the precise signaling mechanisms that underlie it remain elusive. Notably, it remained unclear whether the distribution of chemokine receptors on the T cell surface is altered during repolarization. Here, we developed parallel cell-secreted and microfluidics-based chemokine gradient delivery methods and employed both fixed imaging and live lattice light-sheet microscopy to investigate the dynamics of chemokine receptor CCR5 on the surface of primary murine CD8+ T cells. Our findings show that, during constitutive migration, chemokine receptor distribution is largely isotropic on the T cell surface. However, upon exposure to a CCL3 gradient, surface chemokine receptor distributions exhibit a transient bias toward the uropod. The chemokine receptors then progressively redistribute from the uropod to cover the T cell surface uniformly. This study sheds new light on the dynamics of surface chemokine receptor distribution during T cell repolarization, advancing our understanding of the signaling of immune cells in the complex chemokine landscapes they navigate.

T cell cytoskeletal forces shape synapse topography for targeted lysis via membrane curvature bias of perforin.

Cytotoxic T lymphocytes (CTLs) lyse target cells by delivering lytic granules that contain the pore former perforin to the cytotoxic immunological synapse. Here, we establish that opposing cytoskeletal forces drive lytic granule polarization and simultaneously shape T cell synapse topography to enhance target perforation. At the cell rear, actomyosin contractility drives the anterograde movement of lytic granules toward the nucleus. At the synapse, dynein-derived forces induce negatively curved membrane pockets to which granules are transported around the nucleus. These highly concave degranulation pockets are located directly opposite positively curved bulges on the target cell membrane. We identify a curvature bias in the action of perforin, which preferentially perforates positively curved tumor cell membrane. Together, these findings demonstrate murine and human T cell-mediated cytotoxicity to be a highly tuned mechano-biochemical system, in which the forces that polarize lytic granules locally bend the synaptic membrane to favor the unidirectional perforation of the target cell.

T cell morphodynamics reveal periodic shape oscillations in three-dimensional migration.

T cells use sophisticated shape dynamics (morphodynamics) to migrate towards and neutralize infected and cancerous cells. However, there is limited quantitative understanding of the migration process in three-dimensional extracellular matrices (ECMs) and across timescales. Here, we leveraged recent advances in lattice light-sheet microscopy to quantitatively explore the three-dimensional morphodynamics of migrating T cells at high spatio-temporal resolution. We first developed a new shape descriptor based on spherical harmonics, incorporating key polarization information of the uropod. We found that the shape space of T cells is low-dimensional. At the behavioural level, run-and-stop migration modes emerge at approximately 150 s, and we mapped the morphodynamic composition of each mode using multiscale wavelet analysis, finding 'stereotyped' motifs. Focusing on the run mode, we found morphodynamics oscillating periodically (every approx. 100 s) that can be broken down into a biphasic process: front-widening with retraction of the uropod, followed by a rearward surface motion and forward extension, where intercalation with the ECM in both of these steps likely facilitates forward motion. Further application of these methods may enable the comparison of T cell migration across different conditions (e.g. differentiation, activation, tissues and drug treatments) and improve the precision of immunotherapeutic development.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation.

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8+ T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activation-induced gains in fluorescence were also observed in naïve and CD4+ T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.

Insulin-like growth factor binding protein-7: A marker of conjunctivalization in an animal model of limbal stem cell deficiency.

PURPOSE: Limbal stem cell deficiency (LSCD) is characterized by the loss of limbal epithelial stem cells, resulting in a pathological process termed 'conjunctivalization' which compromises corneal transparency, leading to blindness. Current diagnosis for LSCD is limited because reliable conjunctiva-specific biomarkers are lacking. This study sought to address this shortcoming through the serendipitous discovery of insulin-like growth factor binding protein (IGFBP)-7. METHODS: IGFBP-7 expression was determined in normal (n=83) and conjunctivalized (n=52) mouse corneas with experimentally-induced LSCD, and in cadaveric normal human corneas (n=7) and human pterygia (n=15); a disease characterized by the invasion of a conjunctivalized, fibrovascular pannus. Clinical assessments including slit-lamp microscopy, fluorescein staining and impression cytology, and biochemical, molecular and immunological assays were also conducted. RESULTS: Mass spectrometry of conditioned media from mouse limbal explant-derived cells revealed the presence of IGFBP-7. This factor was expressed in normal limbal and conjunctival epithelium and conjunctivalized corneas from mice with LSCD, and in human pterygium epithelium but not in normal mouse or human corneal epithelium. Four weeks after inducing LSCD, IGFBP-7 staining was increased by 2.9-fold in mouse corneas compared to steady-state, and by 1.6-fold in impression cytology specimens derived from the same mice. Notably, IGFBP-7 was detected approximately 2-weeks earlier than Muc5AC. CONCLUSIONS: This study provides novel insights into the specificity of IGFBP-7 for the mammalian conjunctival epithelium in health and disease. A point-of-care test for IGFBP-7 could be developed to assist clinicians in early diagnosis, and in monitoring disease progression, severity and therapeutic outcomes in patients with LSCD.