Visualizing the dynamics of HIV-specific cytotoxic T-cells in extracellular matrix

Abstract

Cytotoxic lymphocytes (CTLs) traffic through tissues in search of antigen and mount protective immune responses against viral infections and cancer. While molecular mechanisms of CTL antiviral effector functions have been established in vitro, they have been defined in the absence of physiological dynamics and migration. Furthermore, longterm dynamics of single cells have been inaccessible in vivo, where brief imaging durations have been achieved (-30-60 min). Presently, several key aspects of CTL dynamics and function remain unknown: whether individual CTLs migrating within tissues kill multiple targets, if CTLs exhibit spatiotemporal coordination of effector functions, or if migrating CTLs effect these functions in different compartments. Thus, a mechanistic understanding of multidimensional CTL function might directly inform therapeutic strategies. In this thesis, we first developed an approach for long-term high-speed optical imaging of cellular dynamics for continuous periods of 24 hours. HIV-specific CTLs were visualized as they encountered CD4+ target cells within a three-dimensional extracellular matrix tissue model supporting migration of both CTLs and targets. Using this approach, we found that high-avidity CTLs engaged, arrested, and killed the first target encountered with near-perfect efficiency. These CTLs remained in contact with dead targets for hours, accumulating TCR signals and upregulating antiviral cytokine and chemokine secretion for >12 hours, but were refractory to killing additional targets. By contrast, lower-avidity CTLs exhibited poor efficiency and target migration directly impeded CTL killing. Thus, high-avidity CTLs coordinate multiple antiviral functions in four dimensions (3D space and time): effectively destroying the first detected infected cell during an initial "commitment phase", but rapidly transitioning to a prolonged "secretory phase." In vivo, coordination of lytic and non-lytic effector functions will direct the local inflammatory milieu and recruit additional effectors to the tissue. We conclude that the efficiency of antigen recognition by individual migrating CTLs is a critical, but previously undefined, parameter of CTL function. Furthermore, TCR avidity and initial CTL efficiency are prerequisites for sustained antiviral polyfunctionality; together these parameters define a highly effective, multidimensional CTL response, which may inform the design of increasingly effective vaccines.