C57BL/6 mice genetically deficient in interleukin 15 (IL-15−/− mice) were generated by gene targeting. IL-15−/− mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8+ T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15−/− mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15−/− mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15−/− mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15−/− mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15−/− mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.

SummaryInterleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.

The physiological role of the TNF receptor (TNFR) family member, RANK, was investigated by generating RANK-deficient mice. RANK −/− mice were characterized by profound osteopetrosis resulting from an apparent block in osteoclast differentiation. RANK expression was not required for the commitment, differentiation, and functional maturation of macrophages and dendritic cells from their myeloid precursors but provided a necessary and specific signal for the differentiation of myeloid-derived osteoclasts. RANK −/− mice also exhibited a marked deficiency of B cells in the spleen. RANK −/− mice retained mucosal-associated lymphoid tissues including Peyer's patches but completely lacked all other peripheral lymph nodes, highlighting an additional major role for RANK in lymph node formation. These experiments reveal that RANK provides critical signals necessary for lymph node organogenesis and osteoclast differentiation.

SynopsisImaging flow cytometry combines the statistical power and fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. The technique is a good fit for clinical applications by providing a convenient means for imaging and analyzing cells directly in bodily fluids. Examples are provided of the discrimination of cancerous from normal mammary epithelial cells and the high throughput quantitation of FISH probes in human peripheral blood mononuclear cells. The FISH application will be further enhanced by the integration of extended depth of field imaging technology with the current optical system. Keywordsimaging; flow; cytometry; fluorescence; brightfield; darkfield; multispectral Quantifying Cellular Structure in Health and DiseaseThe eukaryotic cell is a highly structured, three-dimensional object containing a wide range of molecular species. The size, shape, and structure of the cell, as well as the abundance, location, and co-location of any of these constituent biomolecules may be of significance in any given clinical situation or research application. For instance, in hematopoiesis, as cells differentiate and mature, different subsets of molecules are expressed that reflect a specialized functional capacity for that unique cell type (e.g., granulocytes vs. lymphocytes). In general the characterization of this array of constituent molecules by imaging or flow cytometry provides insight into the physiological function of any particular cell or alternatively, pathological changes that may have occurred or accrued. In clinical practice and in research settings, cellular evaluation by imaging technologies and flow cytometry provides significant information reflecting the particular cellular phenotype, both normal and pathological. Microscopy provides a wealth of information, but data acquisition rates are slow and analysis is generally subjective. In flow cytometry, data acquisition is rapid and better suited for the evaluation of pathologies present in low frequency, but the data are only intensity-based, thus lacking the morphology that truly lends credence to the analysis.In addition, the assessment and evaluation of cell samples by imaging and flow cytometric techniques is complicated by a number of factors. For instance, changes in a cell type or aCorresponding author for proofs and reprints:

SummaryPurified CD4 + lymph node T cells were sorted into two populations on the basis of their expression of CD45RB (CD45RB hi and CD45RB 1~ and injected into congenic severe combined immunodeficient (SCID) mice. After a period of time that was dependent on the number of cells injected, the SCID mice that received CD45RBhi/CD4 + T cells developed a wasting disease that was not seen in SCID mice that received the CD4+/CD45RB 1~ cells or whole lymph node cells. At death, SCID mice that received the CD4 +/CD45RB hi cells had increased spleen and lymph node cellularity compared with normal SCID mice and SCID mice that received the CD4+/CD45RB 1~ T cells. The spleen and lymph node contained CD4 + cells and neither CD8 + nor surface immunoglobulin M-positive cells, plus a population of cells that did not express any of those markers. At necropsy, the SCID mice that received the CD4 +/CD45RB hi cells had significant hyperplasia of the intestinal mucosa with significant lymphoid cell accumulation in the lamina propria. Interestingly, mice that received mixtures of whole lymph node or purified CD4 * cells with CD4 +/CD45RB ~ cells did not develop weight loss, indicating that the unseparated CD4 + population contained cells that were capable of regulating the reactivity of the CD4 +/CD45RB hi cells. utoaggressive immunological reactivity can be driven by CD4 § thymus-derived lymphocytes. For instance, it has been demonstrated that experimental autoimmune encephalomyelitis and diabetes can be induced in normal animals by injecting them with CD4 + T cell clones or lines derived from animals with autoimmune disease of that particular tissue (1-5). Also, T cell reactivity to self-antigens can be demonstrated in normal animals by immunizing them with a closely related antigen in a unique way or by depleting them of a regulatory population (6-8). These data indicate that T cells with specificity for self-antigens exist normally, but that their reactivity is controlled by immunoregulatory mechanisms.It is well appreciated that thymus-derived lymphocytes can be categorized according to the cell surface antigens they express. This has allowed the classification ofdass I or II MHCrecognizing T cells based on their expression of CD8 or CD4 (9). Also, recent data indicate that virgin and memory T cells can be distinguished by their expression of other cell surface markers such as CD44 or CD45 (10-12). The ability to associate T cell function with the expression of a unique array of cell surface determinants is useful in studying the function of these populations in isolation as well as in defined combinations. For instance, Powrie and Mason (13) have separated CD4 + T cells based on their expression of CD45R and injected the resultant subpopulations into congenic, athymic (nude) animals. They found that nude rats injected with congenic CD45Rhi/CD4 + T cells developed wasting disease characterized by inflammatory infiltrates in many organs. Rats injected with unfractionated CD4 + cells (a mixture of CD45R hi and CD45R l~ cells) did n...