A novel method to allow noninvasive, longitudinal imaging of the murine immune system in vivo

Abstract

In vivo imaging has revolutionized understanding of the spatiotemporal complexity that subserves the generation of successful effector and regulatory immune responses. Until now, invasive surgery has been required for microscopic access to lymph nodes (LNs), making repeated imaging of the same animal impractical and potentially affecting lymphocyte behavior. To allow longitudinal in vivo imaging, we conceived the novel approach of transplanting LNs into the mouse ear pinna. Transplanted LNs maintain the structural and cellular organization of conventional secondary lymphoid organs. They participate in lymphocyte recirculation and exhibit the capacity to receive and respond to local antigenic challenge. The same LN could be repeatedly imaged through time without the requirement for surgical exposure, and the dynamic behavior of the cells within the transplanted LN could be characterized. Crucially, the use of blood vessels as fiducial markers also allowed precise re-registration of the same regions for longitudinal imaging. Thus, we provide the first demonstration of a method for repeated, noninvasive, in vivo imaging of lymphocyte behavior. (Blood. 2012; 119(11):2545-2551) IntroductionMultiphoton laser scanning microscopy (MPLSM) has provided key insights into the kinetics and dynamics of the cellular interactions that govern the initiation of adaptive immune responses. [1][2][3][4][5] These studies have been performed ex vivo on excised lymph nodes (LNs) or in situ with surgically exposed LNs. 6 However, long-term imaging is limited by the effect of these procedures on the physiologic integrity of the LN, and longitudinal studies have been impractical. [1][2][3][4][5]7 We have therefore developed the novel approach of transplanting a LN into the murine ear pinna to facilitate in vivo MPLSM directly through the skin, allowing noninvasive, longitudinal imaging of cellular behavior in defined regions of the same LN.Although previous studies have established the ear pinna as a site for transplantation of tissue such as spleen and heart, [8][9][10][11][12] the concept of transplanting a LN into the ear pinna of a mouse to facilitate imaging is entirely novel. Lymphoid tissue transfer has also long been accepted as a suitable method to study the development of lymphoid structures in vivo. 8,9,13 For example, the kidney capsule as a site of engraftment has proven invaluable for studying the development and organization of lymphoid tissue, 9,14 although relatively inaccessible for in vivo imaging. In this regard, LN transplantation into the ear pinna has a clear attraction. Here, we demonstrate that these transplanted LNs (tLNs) maintain the structural features, cellular organization, and functional capabilities of conventional LNs. Significantly, we show that tLNs have fully functional vascular and lymphatic supplies, allowing lymphocyte recirculation, Ag drainage by the afferent lymphatics, and levels of T-cell activation equivalent to those of normal LNs. Thus, our novel model provides a fully functional LN in an ac...

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