RESUMEN
T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin-related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F-actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3-knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon-like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three-dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3-dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Movimiento Celular/genética , Linfocitos T Citotóxicos/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteína 3 Relacionada con la Actina/genética , Actinas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Interferente Pequeño , Análisis de la Célula Individual , Linfocitos T Citotóxicos/citología , Pez CebraRESUMEN
Immunotherapies based on the autologous adoptive transfer of ex vivo-manipulated T cells are rapidly evolving for the treatment of both metastatic and primary malignancies. However, extended ex vivo culturing reduces the functionality of isolated T cells. Cryopreservation of rapidly expanded T cells for subsequent use throughout an immunotherapeutic regimen is a highly desirable recourse, thus far encumbered by a lack of studies investigating its effects on effector T-cell functionality. Here we directly compare murine tumour-reactive CD8(+) T cells cryopreserved during ex vivo expansion to freshly isolated populations. We show that cryopreservation fully conserves the differentiation potential of effector T cells, secretion of pro-inflammatory cytokines, cytotoxic function and does not impair the three-dimensional scanning motility of T cells or their capacity to infiltrate and reject tumours.