Lymphocyte migration and retention properties affected by ibrutinib in chronic lymphocytic leukemia.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is widely used for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). A prominent effect of ibrutinib is to disrupt the retention of CLL cells from supportive lymphoid tissues, by altering BTK-dependent adhesion and migration. To further explore the mechanism of action of ibrutinib and its potential impact on non-leukemic cells, we quantified multiple motility and adhesion parameters of human primary CLL cells and non-leukemic lymphoid cells. In vitro, ibrutinib affected CCL19-, CXCL12- and CXCL13-evoked migration behavior of CLL cells and non-neoplastic lymphocytes, by reducing both motility speed and directionality. De-phosphorylation of BTK induced by ibrutinib in CLL cells was associated with defective polarization over fibronectin and inability to assemble the immunological synapse upon B-cell receptor engagement. In patients' samples collected during a 6-month monitoring of therapy, chemokine-evoked migration was repressed in CLL cells and marginally reduced in T cells. This was accompanied by profound modulation of the expression of chemokine receptors and adhesion molecules. Remarkably, the relative expression of the receptors governing lymph node entry (CCR7) versus exit (S1PR1) stood out as a reliable predictive marker of the clinically relevant treatment-induced lymphocytosis. Together, our data reveal a multifaceted modulation of motility and adhesive properties of ibrutinib on both CLL leukemic cell and T-cell populations and point to intrinsic differences in CLL recirculation properties as an underlying cause for variability in treatment response.

Mutations affecting the actin regulator WD repeat-containing protein 1 lead to aberrant lymphoid immunity.

BACKGROUND: The actin-interacting protein WD repeat-containing protein 1 (WDR1) promotes cofilin-dependent actin filament turnover. Biallelic WDR1 mutations have been identified recently in an immunodeficiency/autoinflammatory syndrome with aberrant morphology and function of myeloid cells. OBJECTIVE: Given the pleiotropic expression of WDR1, here we investigated to what extent it might control the lymphoid arm of the immune system in human subjects. METHODS: Histologic and detailed immunologic analyses were performed to elucidate the role of WDR1 in the development and function of B and T lymphocytes. RESULTS: Here we identified novel homozygous and compound heterozygous WDR1 missense mutations in 6 patients belonging to 3 kindreds who presented with respiratory tract infections, skin ulceration, and stomatitis. In addition to defective adhesion and motility of neutrophils and monocytes, WDR1 deficiency was associated with aberrant T-cell activation and B-cell development. T lymphocytes appeared to develop normally in the patients, except for the follicular helper T-cell subset. However, peripheral T cells from the patients accumulated atypical actin structures at the immunologic synapse and displayed reduced calcium flux and mildly impaired proliferation on T-cell receptor stimulation. WDR1 deficiency was associated with even more severe abnormalities of the B-cell compartment, including peripheral B-cell lymphopenia, paucity of B-cell progenitors in the bone marrow, lack of switched memory B cells, reduced clonal diversity, abnormal B-cell spreading, and increased apoptosis on B-cell receptor/Toll-like receptor stimulation. CONCLUSION: Our study identifies a novel role for WDR1 in adaptive immunity, highlighting WDR1 as a central regulator of actin turnover during formation of the B-cell and T-cell immunologic synapses.

Switching between individual and collective motility in B lymphocytes is controlled by cell-matrix adhesion and inter-cellular interactions.

Lymphocytes alternate between phases of individual migration across tissues and phases of clustering during activation and function. The range of lymphocyte motility behaviors and the identity of the factors that govern them remain elusive. To explore this point, we here collected unprecedented statistics pertaining to cell displacements, cell:matrix and cell:cell interactions using a model B cell line as well as primary human B lymphocytes. At low cell density, individual B lymphocytes displayed a high heterogeneity in their speed and diffusivity. Beyond this intrinsic variability, B lymphocytes adapted their motility to the composition of extra-cellular matrix, adopting slow persistent walks over collagen IV and quick Brownian walks over fibronectin. At high cell density, collagen IV favored the self-assembly of B lymphocytes into clusters endowed with collective coordination, while fibronectin stimulated individual motility. We show that this behavioral plasticity is controlled by acto-myosin dependent adhesive and Arp2/3-dependent protrusive actin pools, respectively. Our study reveals the adaptive nature of B lymphocyte motility and group dynamics, which are shaped by an interplay between and cell:matrix and cell:cell interactions.

The Wiskott-Aldrich Syndrome Protein Contributes to the Assembly of the LFA-1 Nanocluster Belt at the Lytic Synapse.

T lymphocyte cytotoxicity relies on a synaptic ring of lymphocyte function-associated antigen 1 (LFA-1), which permits polarized delivery of lytic granules. How LFA-1 organization is controlled by underlying actin cytoskeleton dynamics is poorly understood. Here, we explored the contribution of the actin cytoskeleton regulator WASP to the topography of LFA-1 using a combination of microscopy modalities. We uncover that the reduced cytotoxicity of Wiskott-Aldrich syndrome patient-derived CD8+ T lymphocytes lacking WASP is associated with reduced LFA-1 activation, unstable synapse, and delayed lethal hit. At the nanometric scale, WASP constrains high-affinity LFA-1 into dense nanoclusters located in actin meshwork interstices. At the cellular scale, WASP is required for the assembly of a radial belt composed of hundreds of LFA-1 nanoclusters and for lytic granule docking within this belt. Our study unravels the nanoscale topography of LFA-1 at the lytic synapse and identifies WASP as a molecule controlling individual LFA-1 cluster density and LFA-1 nanocluster belt integrity.

T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors.

The actin cytoskeleton is composed of a dynamic filament meshwork that builds the architecture of the cell to sustain its fundamental properties. This physical structure is characterized by a continuous remodeling, which allows cells to accomplish complex motility steps such as directed migration, crossing of biological barriers, and interaction with other cells. T lymphocytes excel in these motility steps to ensure their immune surveillance duties. In particular, actin cytoskeleton remodeling is a key to facilitate the journey of T lymphocytes through distinct tissue environments and to tune their stop and go behavior during the scanning of antigen-presenting cells. The molecular mechanisms controlling actin cytoskeleton remodeling during T lymphocyte motility have been only partially unraveled, since the function of many actin regulators has not yet been assessed in these cells. Our review aims to integrate the current knowledge into a comprehensive picture of how the actin cytoskeleton drives T lymphocyte migration. We will present the molecular actors that control actin cytoskeleton remodeling, as well as their role in the different T lymphocyte motile steps. We will also highlight which challenges remain to be addressed experimentally and which approaches appear promising to tackle them.

Dioxin receptor regulates aldehyde dehydrogenase to block melanoma tumorigenesis and metastasis.

BACKGROUND: The dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell. We have shown that AhR knockdown promotes melanoma primary tumorigenesis and lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi. METHODS: Mouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both. They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro. Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging. RESULTS: Depletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR). Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice. However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo. Aldh1a1 knockdown reduced the high levels of CD133(+)/CD29(+)/CD44(+) cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells. Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells. In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells. CONCLUSIONS: Aldh1a1 overactivation in an AhR-deficient background enhances melanoma progression. Since AhR may antagonize the protumoral effects of Aldh1a1, the AhR(low)-Aldh1a1(high) phenotype could be indicative of bad outcome in melanoma.

Collective cell motility promotes chemotactic prowess and resistance to chemorepulsion.

Collective cell migration is a widespread biological phenomenon, whereby groups of highly coordinated, adherent cells move in a polarized fashion. This migration mode is a hallmark of tissue morphogenesis during development and repair and of solid tumor dissemination. In addition to circulating as solitary cells, lymphoid malignancies can assemble into tissues as multicellular aggregates. Whether malignant lymphocytes are capable of coordinating their motility in the context of chemokine gradients is, however, unknown. Here, we show that, upon exposure to CCL19 or CXCL12 gradients, malignant B and T lymphocytes assemble into clusters that migrate directionally and display a wider chemotactic sensitivity than individual cells. Physical modeling recapitulates cluster motility statistics and shows that intracluster cell cohesion results in noise reduction and enhanced directionality. Quantitative image analysis reveals that cluster migration runs are periodically interrupted by transitory rotation and random phases that favor leader cell turnover. Additionally, internalization of CCR7 in leader cells is accompanied by protrusion retraction, loss of polarity, and the ensuing replacement by new leader cells. These mechanisms ensure sustained forward migration and resistance to chemorepulsion, a behavior of individual cells exposed to steep CCL19 gradients that depends on CCR7 endocytosis. Thus, coordinated cluster dynamics confer distinct chemotactic properties, highlighting unexpected features of lymphoid cell migration.

The Dioxin receptor modulates Caveolin-1 mobilization during directional migration: role of cholesterol.

BACKGROUND: Adhesion and migration are relevant physiological functions that must be regulated by the cell under both normal and pathological conditions. The dioxin receptor (AhR) has emerged as a transcription factor regulating both processes in mesenchymal, epithelial and endothelial cells. Indirect results suggest that AhR could cooperate not only with additional transcription factors but also with membrane-associated proteins to drive such processes. RESULTS: In this study, we have used immortalized and primary dermal fibroblasts from wild type (AhR+/+) and AhR-null (AhR-/-) mice to show that AhR modulates membrane distribution and mobilization of caveolin-1 (Cav-1) during directional cell migration. AhR co-immunoprecipitated with Cav-1 and a fraction of both proteins co-localized to detergent-resistant membrane microdomains (DRM). Consistent with a role of AhR in the process, AhR-/- cells had a significant reduction in Cav-1 in DRMs. Moreover, high cell density reduced AhR nuclear levels and moved Cav-1 from DRMs to the soluble membrane in AhR+/+ but not in AhR-/- cells. Tyrosine-14 phosphorylation had a complex role in the mechanism since its upregulation reduced Cav-1 in DRMs in both AhR+/+ and AhR-/-cells, despite the lower basal levels of Y14-Cav-1 in the null cells. Fluorescence recovery after photobleaching revealed that AhR knock-down blocked Cav-1 transport to the plasma membrane, a deficit possibly influencing its depleted levels in DRMs. Membrane distribution of Cav-1 in AhR-null fibroblasts correlated with higher levels of cholesterol and with disrupted membrane microdomains, whereas addition of exogenous cholesterol changed the Cav-1 distribution of AhR+/+ cells to the null phenotype. Consistently, higher cholesterol levels enhanced caveolae-dependent endocytosis in AhR-null cells. CONCLUSIONS: These results suggest that AhR modulates Cav-1 distribution in migrating cells through the control of cholesterol-enriched membrane microdomains. Our study also supports the likely possibility of membrane-related, transcription factor independent, functions of AhR.

The dioxin receptor controls ß1 integrin activation in fibroblasts through a Cbp-Csk-Src pathway.

Recent studies have suggested a regulatory role for the dioxin receptor (AhR) in cell adhesion and migration. Following our previous work, we report here that the C-terminal Src kinase-binding protein (Cbp) signaling pathway controls ß1 integrin activation and that this mechanism is AhR dependent. T-FGM AhR-/- fibroblasts displayed higher integrin ß1 activation, revealed by the increased binding of the activation reporter 9EG7 anti-ß1 mAb and of a soluble fibronectin fragment, as well as by enhanced talin-ß1 association. AhR-/- fibroblasts also showed increased fibronectin secretion and impaired directional migration. Notably, interfering Cbp expression in AhR-/- fibroblasts reduced ß1 integrin activation, improved cell migration and rescued wild-type cell morphology. Cbp over-expression in T-FGM AhR-/- cells enhanced the formation of inhibitory Csk-Cbp complexes which in turn reduced c-Src p-Tyr(416) activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsive residues p-Tyr(576) and p-Tyr(577). The c-Src target and migration-related protein Cav1 was also hypophosphorylated at p-Tyr(14) in AhR-/- cells, and such effect was rescued by down-modulating Cbp levels. Thus, AhR regulates fibroblast migration by modulating ß1 integrin activation via Cbp-dependent, Src-mediated signaling.