White pulp compartments in the spleen of the turtle Mauremys caspica

Summary The aim of the present study was to analyze the nature of lymphoid and non-lymphoid cellular components occurring in distinct histological compartments of the splenic white pulp of the turtle, Mauremys caspica, in order to define their possible correlations with those of the spleen of higher vertebrates, principally mammals. The white pulp of M.caspica consisted of 3 clearly distinguishable regions: (1) the periateriolar lymphoid sheath, and (2) the inner and (3) the outer zones of the periellipsoidal lymphoid sheath. Reticular cells intimately associated with reticular fibres constituted an extensive meshwork in the periarteriolar lymphoid sheath which housed principally Ig-negative lyphoid cells, mature and immature plasma cells, and interdigitating cells. A few Ig-positive cells were also present in the peripheral region of the periarteriolar lymphoid sheath. The inner and outer zones of the periellipsoidal lymphoid sheath were separated by a discontinuous layer of reticular cell processes. In the inner zone, surface Ig-positive lymphoid cells predominated as well as dendritic cells, resembling ultrastructurally the mammalian follicular dendritic cells, although no germinal centres were found in the turtle spleen. Macrophages, some cytoplasmic Ig-positive cells, and Ig-negative lymphoid cells appeared in the outer zone of the periellipsoidal lymphoid sheath. These results allow us to speculate on a phylogenetic relationship between the periarteriolar lymphoid sheath and the inner and the outer zones of the periellipsoidal lymphoid sheath of the spleen of M. caspica and the periarteriolar lymphoid tissue, the lymphoid follicles and the marginal zone, respectively, of the mammalian splenic white pulp.     

Variation of lymphoid activity in the spleen of a migratory bird,the pied flycatcher (Ficedula hypoleuca; Aves,passeriformes)

The size and microscopic structure of the spleen of the migratory pied flycatcher (Ficedula hypoleuca) show marked changes during the reproductive cycle. Upon the spring return to their northern breeding sites, the birds have a small spleen with little lymphoid activity and a poorly developed red pulp. During the breeding period the volume of red and white pulp increases, the number and distinctness of lymphoid follicles (germinal centres) in the white pulp increase, and groups of cells with intensely basophilic cytoplasm, probably B cells (plasma cells), appear. The findings suggest that the immune system of the adult pied flycatcher is activated during periods when it is bound to the nest. Young flycatchers beginning their autumn migration also show a marked increase of lymphoid activity in the spleen.     

The spleen and skin wound healing in Xenopus adults

In most vertebrates, the regenerative capacity to restore lost/damage tissues to original structure and functionality decreases at some time during ontogenesis. To evaluate the role of the acquired immunity in the decline of regenerative potential, we examined the cellular responses elicited in the spleen during skin repair in Xenopus adults. Modifications in the architecture were found to be induced and were remarkable 14 days postinjury when the spleen increased significantly in size. In white pulp, the periarteriolar lymphoid sheaths were associated with follicles having central light zones, morphologically similar to germinal centers. With the progress of healing, pigment‐containing cells were seen to accumulate in both white and red pulp regions. Moreover, compared to controls, the cells immunoreactive to anti‐cytokines (TNF‐α, TGF‐β1) and ‐iNOS increased from the first days after wounding. The 14th day, the positive cells formed a dense network of reticular cells in central regions of lymphoid follicles and more frequent reactive leukocytes were detected within the red pulp. A higher number of lymphoid cells immunostained with anti‐CD3ε were also observed in the perifollicular zone. The results suggest that the spleen of adult frogs is involved in skin wound healing with the expansion of lymphoid compartments. J. Morphol. 277:888–895, 2016. © 2016 Wiley Periodicals, Inc.     

Macrophage colony-stimulating factor is indispensable for repopulation and differentiation of Kupffer cells but not for splenic red pulp macrophages in osteopetrotic (<Emphasis Type="Italic">op</Emphasis>/<Emphasis Type="Italic">op</Emphasis>) mice after macrophage depletion

We previously reported that macrophage colony-stimulating factor (M-CSF, CSF-1) played important roles in the process of the repopulation of Kupffer cells after their elimination by administration of liposome-entrapped dichloromethylene diphosphonate (lipo-MDP). In this study, we examined the repopulation of Kupffer cells and splenic red pulp macrophages in osteopetrotic (op/op) mice defective in the production of functional M-CSF and their littermate mice by using the lipo-MDP model. In untreated op/op mice, numbers of F4/80-positive Kupffer cells in the liver and F4/80-positive splenic red pulp macrophages were reduced. Repopulation of Kupffer cells and splenic macrophages was observed in littermate (op/+) mice liver by 14 days after depletion. However, in op/op mice, repopulation of Kupffer cells was not observed in Kupffer-cell-depleted op/op mice until 56 days after depletion, whereas splenic red pulp macrophages repopulated and recovered to the level of control op/op mice by 10 days after depletion. Single injection of M-CSF was effective for the induction of the repopulation of Kupffer cells, and daily administration of M-CSF induced remarkable repopulation and maturation of Kupffer cells and proliferation of macrophage precursor cells in the liver of Kupffer-cell-depleted op/op mice. These results suggest that Kupffer cells are completely M-CSF-dependent tissue macrophages, whereas splenic red pulp macrophages are composed of M-CSF-dependent macrophages and M-CSF-independent macrophages. This mouse model provides a useful tool for the study of effects of growth factor on Kupffer cell differentiation in vivo. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, NIH grant CA20408, and a Tsukada Memorial Grant (2000).     

Splenic white pulp as a thymus-independent area in the African clawed toad,Xenopus laevis

Summary An indirect immunofluorescence study of the frozen sections of the spleen of an anuran amphibian, Xenopus laevis, showed that lymphocytes bearing a small amount of immunoglobulin (Ig) were localized mostly in the white pulp of non-immunized toads. There were fewer fluorescent cells in the red pulp. In the toads hyperimmunized with human gamma globulin (HGG), cells with strong cytoplasmic fluorescence increased significantly in the outer part of the white pulp. Electron microscopy of spleens from these toads showed that plasma cells at different stages of maturation were abundant in the white pulp, whereas in the red pulp, a smaller number of maturer plasma cells were observed. These results indicate that, in contrast with its mammalian counterpart, the splenic white pulp of this anuran is the site where thymusin-dependent lymphocytes commence blast formation and transformation into plasma cells.     

Lymphoid organ development in Xenopus thymectomized at eight days of age

This paper examines the effect of early thymectomy on the subsequent development of lymphoid tissues in the toad, Xenopus laevis. At the time of thymic removal (8 days post-fertilization) all the lymphoid organ anlagen are at a rudimentary state of differentiation and contain few, if any, small lymphocytes. Despite the absence of any thymic tissue all thymectomized animals grew normally. Thymectomized larvae developed relatively normal lymphoid organs. However, lymphoid depletion was apparent in the splenic red pulp and in the pharyngeal ventral cavity bodies. Examination of the lymphoid organs of post-metamorphic Xenopus revealed reduction in spleen size following thymectomy. Lymphoid depletion was evident in the splenic red pulp of many thymectomized toadlets and reduction in proportion of white to red pulp was also noted in a few of these animals. Absence of the thymus had no apparent effect on the histology of the other lymphoid organs examined.     

Ultrastructure of splenic white pulp of the turtle,Mauremys caspica

Summary The ultrastructure of splenic tissue of non-immunized turtles, Mauremys caspica, shows two areas, namely, the white pulp which is lymphoid in nature, and the red pulp which is formed by cell cords and sinusoids. Between both areas there is always a marginal zone with gaps through which cells leak. In the white pulp, there are two blood vessel types; one with muscled walls, and the other showing thinner walls sheathed by reticular cells. Reticular cells constitute a network where there occur dendritic macrophages, lymphoblasts and small and medium lymphocytes. Mature plasma cells are scarce in the white pulp.     

Cellular events during the primary immune response in the spleen

Summary The cellular events during the primary immune response in T and B cell compartments in the splenic white pulp were analysed in germfree mice immunized with sheep erythrocytes. Light, fluorescence and electronmicroscopic studies revealed that the initial formation of lymphoid blast cells occurs in the thymus-dependent area, i.e. the central periarteriolar lymphatic sheath (central PALS), 2 days after immunization. Lymphoblasts were found in close relation with erythrocyte-containing macrophages and with interdigitating cells. With fluorescence microscopy these blast cells were Ig negative. Lymphoblasts in the central PALS showed many polyribosomes in the cytoplasm, but were virtually devoid of endoplasmic reticulum. The ultrastructure of lymphoblasts in the central PALS, and their relation with interdigitating cells, suggests that these cells are the progeny of antigen-activated T cells.Cells with a positive cytoplasmic fluorescence, plasmablasts, appeared 3 days after immunization in the peripheral part of the PALS. During the progress of the immune response these cells accumulated around branches of the central arteriole, and moved along marginal zone bridging channels towards the red pulp. In the electron microscope plasmablasts showed many polyribosomes, short strands of rough endoplasmic reticulum close to mitochondria, and a few electron-dense bodies. The cell organelles of plasmablasts were frequently gathered in a so called uropod, which is a morphological sign of active cell movement.Germinal center formation started within primary follicles, 4 days after immunization. Blast cells in germinal centers did not show cytoplasmic fluorescence. During the course of the immune response, germinal centers extended in diameter, and fluorescent dendritic cells appeared at the periphery of the germinal center.From the present observations we conclude that: (1) cellular cooperation between different lymphoid and non-lymphoid cell types during the immune response against SRBC takes place in the PALS, (2) the cellular cooperation in the PALS results in the differentiation of B cells into immunoglobulin-producing plasmablasts, (3) the cellular cooperation in the PALS preceeds the formation of germinal centers in primary follicles, hence germinal centers are not involved in early T-B cell cooperation.     

Postnatal development of the splenic white pulp in the golden hamster Mesocricetus auratus. I. The periarterial lymphoid sheath (PALS)

The histological organization of the periarterial lymphoid sheath (PALS) was studied during the postnatal life of the golden hamster Mesocricetus auratus with special interest in the cell components occurring in each of their regions. Our results suggest a role of the cell components defining the splenic microenvironment at each developmental stage in governing the developmental process. This process can be temporarily and histologically divide into three stages: 1. At birth, a few lymphocytes and lymphoblasts surrounding the central artery define primitive PALS. 2. A second period is determined on the 2nd day by the appearance of a marginal sinus which bounds the two splenic compartments, white and red pulp. The PALS increases circumferentially around the central artery defined by the pattern of reticular cells and fibres. 3. Between the 4th and 10th days, the PALS reaches its definitive organization, except for the absence of primary and secondary lymphoid follicles, defining an inner and outer region. The marginal zone appears on the 6th day.     

Macrophages and Reticulum Cells in the Spleen of the Dogfish,Scyliorhinus canicula

The presence and ultrastructural features of reticulum cells and macrophages were studied in the spleen of the dogfish Scyliorhinus canicula. Three morphologically distinguishable regions of the spleen were identified: the white pulp, the red pulp and the ellipsoids. In all three, the splenic parenchyma was a meshwork supported by reticulum cells and fibres. Reticulum cells in both the white and the red pulp are irregular elements, the processes of which are joined by cell junctions and embrace developing reticular fibres. The ellipsoids of the dogfish spleen are terminal branches of the splenic arteries of the white pulp, with a sheath consisting of reticulum cells, reticular fibres, ground substance, macrophages and occasional lymphocytes. Isolated melanomacrophages also occur in the ellipsoid walls as well as in the red pulp. In both the white and the red pulp phagocytic reticulum cells, and macrophages appear frequently forming cell associations with surrounding blood cells, mainly lymphocytes. The functional significance of the ellipsoids and the cell-cell clusters of the white and the red pulp is discussed in relation to the immune capacities demonstrated in elasmobranchs.     

Appendix and γM-antibody formation: VII. Rosette-forming cells in rabbits immunized with sheep erythrocytes

After intravenous immunization with sheep red blood cells the rabbit spleen shows a sharp rise in the number of plaque-forming cells but there is no detectable rise in PFC in the appendix or mesenteric lymph node of the same animals. Repeated immunization via an appendicostomy blind loop results in virtually no local PFC and only a small rise in splenic PFC.In lethally irradiated animals neither thymocytes nor appendix cells alone restore the splenic PFC response. Simultaneous injection of the two cell types restores both direct and indirect plaque formation. The injected cells were labeled with tritiated adenosine and a standard rosette assay for specific antigen-binding cells applied to recipients' spleen cells following immunization. Rosettes appeared by 3 days after immunization whether thymocytes or appendix cells were labeled. Labeled rosettes were observed only in animals receiving labeled appendix cells.This result demonstrates the presence of rosette forming cell precursors in rabbit appendix cell populations and suggests that the cells of gut-associated lymphoid tissues include antibody-forming cell precursors which are normally seeded to the spleen and draining lymph nodes.     

The argentophil reticular cells of the mammalian spleen. II. The argentophil reticular cell arrangement inside the red pulp and its relationship with the endothelial lining cells of the splenic sinuses

The red pulp's argentophil reticular cell network of the spleen is composed by 3 types of fixed cells: 1. the primitive reticular cell, slightly argentophil; 2. the small reticular cell; 3. the larger reticular cell, strongly argentophil and phagocytic. This latter shows the classical morphological characteristics attributed to the reticular cells of the spleen. The large argentophil reticular cell may become free, constituting a 4th cell type, the free macrophage. A 5th reticular cell type is the dendritic cell found into the lymphatic follicles of the white pulp. The argentophil reticular cells of the red pulp assemble together to form the reticular cells' network, that occurs inside the red pulp cords. The primitive and the small reticular cell form the fundamental network on which the large cells are apposed. The reticular cells of this network maitain relationship with the arterial terminal vessels of the red pulp, being responsible by the ellipsoid structure. In those arteriolar segments without ellipsoid and in those mammalian species devoid of ellipsoid, the white pulp reticular cells, that surround the blood vessel as a part of the lymphoid periarteriolar sheath, mix with the red pulp's reticular cells and both can hardly be discriminated. The ellipsoids are formed by large argentophil cells arranged in concentrical layers around its lumen that sometimes appear devoid of endothelial lining cells. The red pulp's argentophil reticular cells, either the small or the large ones, contributed to the structure of the splenic sinuses' wall; its thin processes surround the sinus wall outside the endothelial lining cell as fibrillar structures that cross the back side of the lining cells. Two or more argentophil reticular cells send fibrillar processes to a single sinus. The perisinusal reticular cells may send a process between adjacent endothelial lining, cells that insinuate and attain the sinus lumen; this process becomes thick and eventually, the reticular cell enter the sinus lumen as a free macrophage. The argentophil reticular cells of the red pulp make connection between the capsule or the trabeculae and the reticular cell network. The endothelial lining cells of the splenic sinuses are not argentophil.     

Specific localization of epidermal-type fatty acid binding protein in dendritic cells of splenic white pulp

Dendritic cells in the splenic white pulp of mice were intensely immunoreactive for epidermal-type fatty acid binding protein (E-FABP). This specific immunostaining revealed a clear difference in morphology between the dendritic cells in the periarterial lymphoid sheath (PALS) and follicular dendritic cells in the follicles in terms of cell sizes and process branching. No immunoreactivity was detected in dendritic cells in the marginal zones and the red pulp, although endothelial cells of almost all capillaries in the red pulp were immunoreactive for E-FABP. After peritoneal injection of lipopolysaccharide, the immunoreactive cells in PALS progressively enlarged and became rounded in shape with a peak in size at 24 h postinjection and they eventually resumed the dendritic form at 48 h postinjection. Within each of the enlarged immunoreactive cell perikarya were included small immunonegative apoptotic cells, presumptive lymphocytes. Taken together, E-FABP is useful as a marker for dendritic cells in the splenic white pulp, and may be involved through combination with fatty acids in antigen presentation and retention as well as in cytokine production.     

Pro-enkephalin opioid peptides are abundant in porcine and bovine splenic nerves,but absent from nerves of rat,mouse, hamster,and guinea-pig spleen

The opioidergic innervation of the mammalian spleen and possible species differences were investigated. Light-microscopic immunohistochemistry revealed that splenic nerves of bovine and porcine spleen, but not of rat, mouse, hamster and guinea-pig spleen contained proenkephalin-derived opioidergic innervation. Immunoreactivity to both prodynorphin and pro-opiomelanocortin was absent from splenic nerves. In bovine and porcine spleen, fibers immunoreactive for met-enkephalin, met-enkephalin-Arg-Phe, met-enkephalin-Arg-Gly-Leu, leu-enkephalin and peptide F formed perivascular plexus, traveled in trabecular connective tissue, and extended into the capsule. Spatial relationships with immune cells were apparent in the white and red pulp, excluding lymphoid follicles. Colocalization of enkephalin immunoreactivity with immunoreactivities for tyrosin hydroxylase, dopamin--hydroxylase, and neuropeptide Y, but not for substance P or calcitonin gene-related peptide were found. Our results provide evidence that opioid expression in splenic innervation is strongly species-dependent and exclusively proenkephalin-derived. Colocalization with marker enzymes of noradrenergic neurons indicates a mainly postganglionic sympathetic origin of proenkephalinergic splenic innervation. Opioidergic perivascular nerves probably control the splenic blood flow. A close interrelationship of opioidergic fibers with immune cells provides the anatomical basis for direct effects of neurally released opioids on splenic immune functions.     

Comparative evaluation of the immune status of congenitally athymic and euthymic rat strains bred and maintained at different institutes: 2. Athymic rats.

We performed a comparative evaluation of the immune status, focused on the T-cell system, in congenitally athymic rat strains. From 11 institutes around the world, we sampled 15 groups of animals at ages of 1 1/2-2 months and 1/2 year. The analysis included weight of body and spleen; antibody response and delayed-type hypersensitivity response after immunization with ovalbumin; and (immuno)histology of spleen, lymph nodes and lymphoid tissue along the gastrointestinal tract. Morphometric analysis was done for alpha beta-T-cell receptor-bearing cells in spleen tissue as a measure of the periarteriolar lymphocyte sheath; it was also done for splenic red pulp using the antibody ED2 recognizing red pulp macrophages. For almost all variables analyzed, statistically significant differences between the groups were observed. The extent of alpha beta-T-cell receptor-bearing cells in the spleen increased with age. The functioning of these cells in immunological responses can be questioned, because an immune response to ovalbumin was invariably absent. But secondary follicles with germinal centers, reflecting T-cell-dependent B-cell reactivity, were observed in lymph nodes and Peyer's patches (up to 40% and 75%, respectively, depending on the group), with a higher prevalence in older animals. A cluster analysis on the basis of body and spleen weight and composition of spleen compartments did not yield clusters with a different profile in regard to the animals' group of origin. The data presented are useful when comparing studies performed with various athymic rat strains at different institutes.     

Low CXCL13 expression, splenic lymphoid tissue atrophy and germinal center disruption in severe canine visceral leishmaniasis

Visceral leishmaniasis is associated with atrophy and histological disorganization of splenic compartments. In this paper, we compared organized and disorganized splenic lymphoid tissue from dogs naturally infected with Leishmania infantum assessing the size of the white pulp compartments, the distribution of T, B and S100⁺ dendritic cells, using immunohistochemistry and morphometry and the expression of CCR7 and the cytokines, CXCL13, lymphotoxin (LT)-α, LT-β, CCL19, CCL21, TNF-α, IL-10, IFN-γ and TGF-β, using by real time RT-PCR. The lymphoid follicles and marginal zones were smaller (3.2 and 1.9 times, respectively; Mann-Whitney, P<0.02) in animals with disorganized splenic tissue in comparison to those with organized splenic lymphoid tissue. In spleens with disorganized lymphoid tissue, the numbers of T cells and S100⁺ dendritic cells were decreased in the follicles, and the numbers of B cells were reduced in both the follicles and marginal zones. CXCL13 mRNA expression was lower in animals with disorganized lymphoid tissue (0.5±0.4) compared to those with organized lymphoid tissue (2.7±2.9, both relative to 18S expression, P = 0.01). These changes in the spleen were associated with higher frequency of severe disease (7/12) in the animals with disorganized than in animals with organized (2/13, Chi-square, P = 0.01) splenic lymphoid tissue. The data presented herein suggest that natural infection with Leishmania infantum is associated with the impairment of follicular dendritic cells, CXCL13 expression, B cell migration and germinal center formation and associates these changes with severe clinical forms of visceral leishmaniasis. Furthermore the fact that this work uses dogs naturally infected with Leishmania infantum emphasizes the relevance of the data presented herein for the knowledge on the canine and human visceral leishmaniasis.     

Changes in spleen histology in response to antigenic stimulation in the snapping turtle,Chelydra serpentine

Proliferative and migratory changes of lymphoid cells in the spleen were observed in turtles stimulated with KLH and maintained at 30°C. Small foci of pyroninophilic lymphoblasts were first seen in the white pulp at about day 5. Progressive enlargement of these centers continued and peaked by days 8-12. By days 15-20 the white pulp returned to a normal (unimmunized) state, while the number of pyroninophilic cells, primarily plasma cells, increased markedly in the red pulp. At days 22-25, the number of plasma cells returned to normal levels and the spleen appeared normal for the remainder of the 60 day observation period. These events suggest that at 30°C,-the turtle is capable of a strong and prompt proliferative response in the white pulp sheaths, followed by migration and differentiation of lymphoblasts into plasma cells, n the red pulp., Observations of pyroninophilic cells in sinuses, venules and veins of the spleen and a concomitant depletion of cells in red pulp, further suggest a migration from the spleen to other sites. Following a second antigenic challenge, at day 60, no significant histological changes were observed at 30°C. Nor were any changes observed following primary or secondary antigenic challenge, in animals maintained at 10°C. These findings are discussed with regard to immunological memory and low temperature immunosuppression in ectothermic vertebrates.     

The architecture of the spleen of the yellow-bellied toad, Bombina variegata

The spleen of the yellow-bellied toad, Bombina variegata , consists of distinct white and red pulps. The well-developed white pulp is formed by a large central lymphocytic region around the numerous blood vessels and by its smaller peripheral ramifications, both surrounded by the more or less developed connective tissue boundary layer. Large peripheral sinuses of the white pulp, filled mostly with lymphocytes, are usually present at the inner side of this boundary. At the outer side of the boundary layer, the lymphocytic marginal zone is often observed. This zone merges into the erythrocyte-rich red pulp formed by cellular cords and small venous sinusoids.
The structure of the spleen of Bombina variegata differs considerably from the spleens of other anuran species studied so far. The highly developed white pulp and its distinct separation from the red pulp may be connected with the important role of the spleen as the main secondary lymphoid organ of B. variegata. The splenic compartmentalization makes the yellow-bellied toads a useful model for experimental immunobiological studies.     

Leishmania donovani: evolution and architecture of the splenic cellular immune response related to control of infection.

Infection with the protozoan Leishmania donovani in humans is usually subclinical. Parasites probably persist for the life of the host and the low-level infection is controlled by the cellular immune response. To better understand the mechanisms related to the control of infection, we studied the evolution and architecture of the splenic cellular immune response in a murine model that is most representative of human subclinical infection. Following systemic inoculation with L. donovani, the parasites were primarily localized to the macrophage-rich splenic red pulp. There was an initial increase in the numbers of T cells and dendritic cells in the periarteriolar lymphoid sheath and marginal zone, but the red pulp (where parasitized macrophages were prominent) remained free of these cells until later in the course of infection. Thus, T cells did not colocalize with parasitized red pulp macrophages until later in the course of infection. Early in the course of infection, IL-10 production within the marginal zone and TGF-beta production by cells in the red pulp were prominent. These macrophage-inhibitory cytokines may contribute to the establishment of the infection and early parasite replication. By day 28 of infection, when the visceral parasite burden began to decline, the number of IL-10-producing spleen cells was back to the baseline level, but IFN-gamma production was higher and the number of IL-12-producing cells was increased dramatically. At this time T cells and dendritic cells had moved out of the lymphoid follicle and marginal zone into the red pulp where the parasites were located. These findings therefore suggest that control of infection is associated with IFN-gamma and IL-12 production and migration of T cells and dendritic cells to the site of chronic parasitism.     

Analysis of in situ NK cell responses during viral infection.

NK cells are required for early control of murine CMV (MCMV) infection, but the distribution of murine NK cells in situ has not been clearly defined. We tested the reactivity of all available NK cell receptor-specific mAbs by immunohistochemistry. Only one mAb, 4D11 (anti-Ly-49G2), was reactive with C57BL/6 tissue sections. mAb 4D11-reactive cells expressed the nuclear morphology and flow cytometric profile of NK cells. In lymphoid organs, NK cells were distributed primarily in the splenic red pulp, between adjacent lobes in lymph node and randomly in the cortex and medulla of the thymus. No NK cells were detected in normal liver sections. Two days following MCMV infection, most splenic NK cells were associated with the lymphoid follicles and marginal zone. By day 3 following infection, the number of liver NK cells had increased significantly and the cells were detected within inflammatory foci. These changes were independent of IL-12, IFN-gamma, and TNF-alpha, as assessed in mice with targeted mutations. Concurrent immunostaining for NK cells and viral Ags revealed close association of NK cells and MCMV-infected cells in the spleen and liver. Similar results were obtained in CD1(-/-) and recombination activation gene-1(-/-) mice lacking NK T or T and B cells, respectively, indicating specificity of staining for NK cells. Thus, following MCMV infection, NK cells accumulate at sites of viral replication in an IL-12-, IFN-gamma-, and TNF-alpha-independent manner.