Exocrine gland-resident memory CD8+ T cells use mechanosensing for tissue surveillance.

Tissue-resident CD8+ T cells (TRM) continuously scan peptide-MHC (pMHC) complexes in their organ of residence to intercept microbial invaders. Recent data showed that TRM lodged in exocrine glands scan tissue in the absence of any chemoattractant or adhesion receptor signaling, thus bypassing the requirement for canonical migration-promoting factors. The signals eliciting this noncanonical motility and its relevance for organ surveillance have remained unknown. Using mouse models of viral infections, we report that exocrine gland TRM autonomously generated front-to-back F-actin flow for locomotion, accompanied by high cortical actomyosin contractility, and leading-edge bleb formation. The distinctive mode of exocrine gland TRM locomotion was triggered by sensing physical confinement and was closely correlated with nuclear deformation, which acts as a mechanosensor via an arachidonic acid and Ca2+ signaling pathway. By contrast, naïve CD8+ T cells or TRM surveilling microbe-exposed epithelial barriers did not show mechanosensing capacity. Inhibition of nuclear mechanosensing disrupted exocrine gland TRM scanning and impaired their ability to intercept target cells. These findings indicate that confinement is sufficient to elicit autonomous T cell surveillance in glands with restricted chemokine expression and constitutes a scanning strategy that complements chemosensing-dependent migration.

Dasatinib-induced spleen contraction leads to transient lymphocytosis.

The tyrosine kinase inhibitor dasatinib is approved for Philadelphia chromosome-positive leukemia, including chronic myeloid leukemia (CML). Although effective and well tolerated, patients typically exhibit a transient lymphocytosis after dasatinib uptake. To date, the underlying physiological process linking dasatinib to lymphocytosis remains unknown. Here, we used a small rodent model to examine the mechanism of dasatinib-induced lymphocytosis, focusing on lymphocyte trafficking into and out of secondary lymphoid organs. Our data indicate that lymphocyte homing to lymph nodes and spleen remained unaffected by dasatinib treatment. In contrast, dasatinib promoted lymphocyte egress from spleen with kinetics consistent with the observed lymphocytosis. Unexpectedly, dasatinib-induced lymphocyte egress occurred independently of canonical sphingosine-1-phosphate-mediated egress signals; instead, dasatinib treatment led to a decrease in spleen size, concomitant with increased splenic stromal cell contractility, as measured by myosin light chain phosphorylation. Accordingly, dasatinib-induced lymphocytosis was partially reversed by pharmacological inhibition of the contraction-promoting factor Rho-rho associated kinase. Finally, we uncovered a decrease in spleen size in patients with CML who showed lymphocytosis immediately after dasatinib treatment, and this reduction was proportional to the magnitude of lymphocytosis and dasatinib plasma levels. In summary, our work provides evidence that dasatinib-induced lymphocytosis is a consequence of drug-induced contractility of splenic stromal cells.

The Tec Kinase Itk Integrates Naïve T Cell Migration and In Vivo Homeostasis.

Naïve T cells (TN) constitutively recirculate through secondary lymphatic organs (SLOs), where they scan dendritic cells (DCs) for cognate peptide-loaded major histocompatibility complexes (pMHC). Continuous trafficking between SLOs not only enables rapid clonal selection but also ensures TN homeostasis by providing access to prosurvival signals from TCR, IL-7R, and the chemokine receptor CCR7. Inside the lymphoid tissue, CCR7-mediated TN motility is mainly driven by the Rac activator DOCK2, with a separate contribution by a phosphoinositide-3-kinase γ (PI3Kγ)-dependent pathway. Tec tyrosine kinases and the Rac activator Tiam1 constitute prominent downstream effectors of PI3K signaling. Yet, the precise role of Tec kinase versus Tiam1 signaling during CCR7-mediated TN migration and homeostasis remains incompletely understood. Here, we examined the function of the Tec family member interleukin-2-inducible T-cell kinase (Itk) and Tiam1 during TN migration in vitro and in vivo using intravital microscopy. Itk deficiency caused a mild decrease in CCR7-triggered TN migration, mirroring observations made with PI3Kγ;-/- T cells, while lack of Tiam1 did not affect TN motility. In silico modeling suggested that reduced migration in the absence of Itk does not result in a substantial decrease in the frequency of TN encounters with DCs within the lymphoid tissue. In contrast, Itk was important to maintain in vivo homeostasis of CD4+ TN, also in MHCII-deficient hosts. Taken together, our data suggest that Itk contributes to TN migration and survival by integrating chemokine receptor and TCR signaling pathways.

Organ-Specific Surveillance and Long-Term Residency Strategies Adapted by Tissue-Resident Memory CD8+ T Cells.

Tissue-resident CD8+ T cells (CD8+ TRM) populate lymphoid and non-lymphoid tissues after infections as first line of defense against re-emerging pathogens. To achieve host protection, CD8+ TRM have developed surveillance strategies that combine dynamic interrogation of pMHC complexes on local stromal and hematopoietic cells with long-term residency. Factors mediating CD8+ TRM residency include CD69, a surface receptor opposing the egress-promoting S1P1, CD49a, a collagen-binding integrin, and CD103, which binds E-cadherin on epithelial cells. Moreover, the topography of the tissues of residency may influence TRM retention and surveillance strategies. Here, we provide a brief summary of these factors to examine how CD8+ TRM reconcile constant migratory behavior with their long-term commitment to local microenvironments, with a focus on epithelial barrier organs and exocrine glands with mixed connective-epithelial tissue composition.

Immune synapse instructs epigenomic and transcriptomic functional reprogramming in dendritic cells.

Understanding the fate of dendritic cells (DCs) after productive immune synapses (postsynaptic DCs) with T cells during antigen presentation has been largely neglected in favor of deciphering the nuances of T cell activation and memory generation. Here, we describe that postsynaptic DCs switch their transcriptomic signature, correlating with epigenomic changes including DNA accessibility and histone methylation. We focus on the chemokine receptor Ccr7 as a proof-of-concept gene that is increased in postsynaptic DCs. Consistent with our epigenomic observations, postsynaptic DCs migrate more efficiently toward CCL19 in vitro and display enhanced homing to draining lymph nodes in vivo. This work describes a previously unknown DC population whose transcriptomics, epigenomics, and migratory capacity change in response to their cognate contact with T cells.

Salivary gland macrophages and tissue-resident CD8+ T cells cooperate for homeostatic organ surveillance.

It is well established that tissue macrophages and tissue-resident memory CD8+ T cells (TRM) play important roles for pathogen sensing and rapid protection of barrier tissues. In contrast, the mechanisms by which these two cell types cooperate for homeostatic organ surveillance after clearance of infections is poorly understood. Here, we used intravital imaging to show that TRM dynamically followed tissue macrophage topology in noninflamed murine submandibular salivary glands (SMGs). Depletion of tissue macrophages interfered with SMG TRM motility and caused a reduction of interepithelial T cell crossing. In the absence of macrophages, SMG TRM failed to cluster in response to local inflammatory chemokines. A detailed analysis of the SMG microarchitecture uncovered discontinuous attachment of tissue macrophages to neighboring epithelial cells, with occasional macrophage protrusions bridging adjacent acini and ducts. When dissecting the molecular mechanisms that drive homeostatic SMG TRM motility, we found that these cells exhibit a wide range of migration modes: In addition to chemokine- and adhesion receptor-driven motility, resting SMG TRM displayed a remarkable capacity for autonomous motility in the absence of chemoattractants and adhesive ligands. Autonomous SMG TRM motility was mediated by friction and insertion of protrusions into gaps offered by the surrounding microenvironment. In sum, SMG TRM display a unique continuum of migration modes, which are supported in vivo by tissue macrophages to allow homeostatic patrolling of the complex SMG architecture.

Dynamic spherical harmonics approach for shape classification of migrating cells.

Cell migration involves dynamic changes in cell shape. Intricate patterns of cell shape can be analyzed and classified using advanced shape descriptors, including spherical harmonics (SPHARM). Though SPHARM have been used to analyze and classify migrating cells, such classification did not exploit SPHARM spectra in their dynamics. Here, we examine whether additional information from dynamic SPHARM improves classification of cell migration patterns. We combine the static and dynamic SPHARM approach with a support-vector-machine classifier and compare their classification accuracies. We demonstrate that the dynamic SPHARM analysis classifies cell migration patterns more accurately than the static one for both synthetic and experimental data. Furthermore, by comparing the computed accuracies with that of a naive classifier, we can identify the experimental conditions and model parameters that significantly affect cell shape. This capability should - in the future - help to pinpoint factors that play an essential role in cell migration.

In Vivo Function of the Lipid Raft Protein Flotillin-1 during CD8+ T Cell-Mediated Host Surveillance.

Flotillin-1 (Flot1) is an evolutionary conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1-deficient neutrophils is impaired because of a decrease in myosin II-mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. In this study, we analyzed morphology and migration parameters of murine wild-type and Flot1-/- CD8+ T cells using in vitro assays and intravital two-photon microscopy of lymphoid and nonlymphoid tissues. Flot1-/- CD8+ T cells displayed significant alterations in cell shape and motility parameters in vivo but showed comparable homing to lymphoid organs and intact in vitro migration to chemokines. Furthermore, their clonal expansion and infiltration into nonlymphoid tissues during primary and secondary antiviral immune responses was comparable to wild-type CD8+ T cells. Taken together, Flot1 plays a detectable but unexpectedly minor role for CD8+ T cell behavior under physiological conditions.

Regulation of global CD8+ T-cell positioning by the actomyosin cytoskeleton.

CD8+ T cells have evolved as one of the most motile mammalian cell types, designed to continuously scan peptide-major histocompatibility complexes class I on the surfaces of other cells. Chemoattractants and adhesion molecules direct CD8+ T-cell homing to and migration within secondary lymphoid organs, where these cells colocalize with antigen-presenting dendritic cells in confined tissue volumes. CD8+ T-cell activation induces a switch to infiltration of non-lymphoid tissue (NLT), which differ in their topology and biophysical properties from lymphoid tissue. Here, we provide a short overview on regulation of organism-wide trafficking patterns during naive T-cell recirculation and their switch to non-lymphoid tissue homing during activation. The migratory lifestyle of CD8+ T cells is regulated by their actomyosin cytoskeleton, which translates chemical signals from surface receptors into mechanical work. We explore how properties of the actomyosin cytoskeleton and its regulators affect CD8+ T cell function in lymphoid and non-lymphoid tissue, combining recent findings in the field of cell migration and actin network regulation with tissue anatomy. Finally, we hypothesize that under certain conditions, intrinsic regulation of actomyosin dynamics may render NLT CD8+ T-cell populations less dependent on input from extrinsic signals during tissue scanning.

Fam65b Phosphorylation Relieves Tonic RhoA Inhibition During T Cell Migration.

We previously identified Fam65b as an atypical inhibitor of the small G protein RhoA. Using a conditional model of a Fam65b-deficient mouse, we first show that Fam65b restricts spontaneous RhoA activation in resting T lymphocytes and regulates intranodal T cell migration in vivo. We next aimed at understanding, at the molecular level, how the brake that Fam65b exerts on RhoA can be relieved upon signaling to allow RhoA activation. Here, we show that chemokine stimulation phosphorylates Fam65b in T lymphocytes. This post-translational modification decreases the affinity of Fam65b for RhoA and favors Fam65b shuttling from the plasma membrane to the cytosol. Functionally, we show that the degree of Fam65b phosphorylation controls some cytoskeletal alterations downstream active RhoA such as actin polymerization, as well as T cell migration in vitro. Altogether, our results show that Fam65b expression and phosphorylation can finely tune the amount of active RhoA in order to favor optimal T lymphocyte motility.

Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies.

With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches.