| Two-photon imaging to investigate fundamental immune processes as they occur in real-time has revealed the choreography of cell movement and interactions that take place hidden from view inside lymphoid organs. The goal of this dissertation was to characterize, at single-cell resolution, the behavior and cellular interaction dynamics of intranodal natural killer (NK) cells using two-photon microscopy. The results from this study demonstrate that intranodal NK cells exhibit remarkably dynamic behavior, actively patrolling lymphoid organs and preferentially forming stable conjugates to eliminate cognate MHC-mismatched targets. In addition, the study demonstrates that a technique commonly used to isolate NK cells modifies effector function, resulting in augmented cytokine secretion with concomitant diminished cytotoxicity and decreased motility.;With the feasibility of immuno-imaging established, the physiological significance of NK cells in alloimmunity was investigated. Traditionally, it has been believed that dendritic cells (DC) that are passenger leukocytes and are present within the transplanted organ are responsible for activating the recipient's immune system to reject the transplanted tissue. This direct presentation of alloantigens by donor-derived DC has been thought to be the dominant pathway of alloreactivity. However, the indirect pathway, by which recipient DC present alloantigen, has been gaining support and mounting evidence proposes a role for the indirect pathway in chronic allograft rejection. Both pathways focus on the mechanism of generating T cell-mediated alloimmunity, but fail to account for a potential role of NK cells during the initial stages of the response. Using a combination of functional assays as well as two-photon microscopy, this study demonstrates that host NK cells eliminate donor-derived DC before they are able to stimulate T cells with direct allospecificity, thus limiting a functional role for direct presentation in vivo and restricting alloantigen recognition to the indirect presentation pathway. Taken together, these studies serve to refine our understanding of the interaction between innate and adaptive immunity in alloresponses, and begin to define the mechanisms underlying the importance of the indirect pathway in acute rejection. |
