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The epithelial component of the thymic environment is organized into discrete cortical and medullary compartments that mediate different aspects of thymocyte differentiation. The processes controlling the growth and organization of these epithelial compartments are poorly defined. In this study we have used a novel approach to define the three-dimensional organization of thymic epithelial (TE) compartments to demonstrate that the organization of the medullary TE compartment is very complex. A spatial relationship of medullary thymic epithelium with vascular elements of the thymus was demonstrated by simultaneous immunohistochemical labeling of vascular elements and medullary TE. Medullary TE was often arranged as perivascular cuffs surrounding intermediate-sized vessels, but was not associated with either the capillary network or large centrally located vessels. Similar analyses of RAG-2^-/- thymi revealed a striking physical association of medullary TE with vascular elements. Ultrastructural analysis of the RAG-2^-/- thymus indicated a preferential association of focal accumulations of medullary TE with post-capillary venules. These data suggest that discrete segments of the thymic vasculature provide cues that act in concert with thymocyte-derived stimuli to effect normal development of the thymic environment.

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I–bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of FcγR+ target cells in a reverse antibody-dependent cellular cytotoxicity–type assay and induces apoptosis in Ly49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.