Occurrence of nonlymphoid leukocytes that are not derived from blood islands in Xenopus laevis larvae

Previous immunohistochemical observations using the monoclonal antibody (XL-1) which recognizes all types of leukocytes in Xenopus laevis revealed the occurrence of XL-1+ cells in the mesenchyme throughout the early larval body, before the appearance of any lymphocytes. The present experiments were performed to determine whether these leukocytes originate, like lymphocytes and red blood cells (RBCs), in the ventral blood islands (VBI) or the dorsolateral plate (DLP). For tracing the derivation of cells, a specific staining by quinacrine to nuclei of X. laevis and Xenopus borealis hybrid (LB) cells was used to distinguish them from X. laevis (LL) cells. Orthotopic graftings of VBI tissue from st.22-23 LB embryos to the stage-matched LL embryos and examinations at st.44-45 before differentiation of the lymphocytes showed that the proportion of XL-1+ LB cells was always significantly lower than that of RBCs with the same marker in all experimental larvae. The head (LB)-body (LL) chimeras from st.22-23 embryos and culture of the head-portions as VBI- and DLP-free explants from st.14-23 embryos both demonstrated that a significant number of XL-1+ cells which had originated in the head portions had begun to differentiate by st.42-43. These results indicate that there is a significant population of larval nonlymphoid leukocytes (mostly macrophages) that do not originate from either the VBI or DLP region, and are distributed in the mesenchyme throughout the body.     

Induction of blood cells in Xenopus embryo explants

 A Xenopus-specific anti-leukocyte monoclonal antibody designated XL-2 was isolated and used to identify leukocytes in tailbud embryos and activin A-treated explants of blastula animal cap. XL-2 bound to a 135-kDa polypeptide in western blots of protein extracts from adult thymocytes, tailbud embryos, tadpoles, and explants. In cell suspensions, it immunostained the cell surface of all types of adult leukocytes including lymphocytes, monocyte/macrophages, thrombocytes, and granulocytes. At embryonic stage 24, immunocytochemistry revealed XL-2-positive leukocytes, the earliest time at which such cells have been recognized. Whole-mount staining of tailbud embryos and tadpoles showed a widely dispersed population of XL-2-reactive leukocytes, many of which had elongated shapes and ameboid pseudopodia. In activin A-treated animal caps, XL-2 recognized a subpopulation of cells within the lumen of the central fluid-filled cavity as well as cells in the interstitium of mesenchymal and mesothelial components of the explant. Together, activin A and human interleukin-11 induced 100% of explants to form lumenal blood cells. Compared to activin A alone, murine stem cell factor plus activin A significantly increased the numbers of XL-2-reactive leukocytes and erythrocytes. These results support the view that activin A induces leukocyte and erythrocyte progenitors during Xenopus embryogenesis. Received: 29 August 1997 / Accepted: 28 October 1997     

C-type virus-lymphocyte interactions in developing mouse thymus.

The appearance of C-type virus particles in thymus cells of Swiss mouse embryos, 11.5 to 15.5 days post-conception age (PCA), was studied with the electron microscope. In thymic rudiments of all specimens examined, virus particles were seen in epithelial cytoplasm, budding from epithelial cell surfaces and in extracellular spaces. Lymphoid cells were first seen in thymic rudiments of 13.5 days PCA, and did not display virus particles at this stage. At 14.5 days PCA, thymic lymphocytes had localized plasmalemmal thickenings of high electron-density which were adjacent to extracellular virus particles. Viruses appeared to be penetrating thymic lymphocytes by viropexis in embryos of 15.5 days PCA. At this stage, many lymphocytes also had cytoplasmic virus-containing vesicles and viral buds at their surfaces. These observations suggest the possibility that, in embryos, C-type viruses are transmitted horizontally from thymic epithelium to early populations of thymic lymphocytes.     

Ontogeny of the immune system inSebastiscus marmoratus: Histogenesis of the lymphoid organs and effects of thymectomy

Synopsis The ontogenetic development of the immune system in a marine teleostSebastiscus marmoratus was studied by histological examination and removal of the thymus. The pronephros and the spleen had been differentiated at the time of birth and contained small numbers of haemopoietic cells. In contrast to most vertebrates, the rudiments of the thymus were first visible 1 week post-birth in the dorsoposterior part of the pharynx, the same location as in the adults. However, small lymphocytes first appeared in the thymus of fish at 3 weeks of age, followed by the pronephros at 4 weeks and the spleen at 6 weeks. Complete or partial suppression of the antibody response to sheep red blood cells (SRBC) occurred in fish that were thymectomized at 1.5 months of age and immunized 2 weeks later, and a marked decrease in lymphocytes was observed in the pronephros and spleen. The thymectomy of adult fish also caused reduced serum antibody titres in fish immunized 1 month after the operation. These results suggest that the thymus plays an essential role in the development of the immune system and its functions continue into adult life.     

Effect of cadmium on lymphocyte subsets distribution in thymus and spleen

This work was designed to analyze the possible dose dependent effects of cadmium on the distribution of lymphocyte subsets within the thymus and spleen. Cadmium accumulation was also evaluated in these tissues. For this purpose, adult male rats were exposed for one month to 0, 5, 10, 25, 50 or 100 ppm of cadmium chloride (CdCl2) in the drinking water. In both spleen and thymus, the B lymphocytes increased with the doses of 5 and 10 ppm of CdCl2, and decreased with the doses of 25-100 ppm. In spleen, the doses of 25 and 50 ppm decreased CD4+ cells and the doses of 5 and 10 ppm increased CD8+ cells, while the percentage of thymus T, CD4+, CD8+ and CD4(+)-CD8+ cells was not modified by cadmium treatment at any dose used in this study. After cadmium exposure, the metal was accumulated in the spleen only from the dose of 50 ppm on, and in the thymus, from the dose of 10 ppm on. In conclusion, although the accumulation of the metal is higher in thymus than in spleen, the metal affected CD4+ and CD8+ lymphocytes at the spleen but not at the thymus.     

The cellular composition of the blood and haematopoietic organs of turbot Scophthalmus maximus L.

The cellular composition of the blood, anterior kidney, spleen and thymus of turbot Scophrhalmus maximus L., aged 1 + was determined. Ninety-four per cent of blood cells belonged to the erythrocyte lineage of which 82% were mature erythrocytes. The leucocytes, which represented 4.5% of the blood cells, were mainly lymphocytes (50%). The presence of crythroblasts in the anterior kidney and the spleen demonstrated an erythropoietic activity in both organs. However, this activity appeared to be prevalent in the spleen which also appeared to act as a storage zone for erythrocytes and as the centre point for thrombopoiesis. Although 96% of the anterior kidney cells were leucocytes, the number of white cells per gram of organ was higher in the spleen.     

Differential participation of ventral and dorsolateral mesoderms in the hemopoiesis of Xenopus, as revealed in diploid-triploid or interspecific chimeras

The thymocytes in the early larvae of Xenopus laevis have been shown to be derived from precursor cells immigrating interstitially through the mesenchyme into the organ rudiments at 3-4 days of age (Nieuwkoop and Faber stages 42-45). Orthotopic grafting of diploid tissues onto triploid stage 22 embryos followed by ploidy analyses of their hemopoietic cells revealed that both thymocytes and erythrocytes in early larvae are derived from the ventral blood islands (VBI), whereas those in late larvae and adults come mainly from the dorsolateral plate (DLP). To study how the VBI cells of embryos at stage 22 participate in hemopoiesis, a number of interspecific chimeras were produced in X. laevis and X. borealis embryos. Sections of the chimeras at various developmental stages were examined by employing the unique stainability of X. borealis nuclei to quinacrine as a marker; the results show that the VBI-derived cells enter into the circulation around stage 35/36, and that some of them leave the blood vessels to migrate interstitially through the mesenchyme toward the thymic rudiment during stages 43-45. A minor population of the VBI-derived cells was also found extravascularly in the mesonephric primordia. In contrast to the VBI, the DLP-derived cells contributed to the hemopoietic cell population not in early larvae, but in late ones as a major constituent in the mesonephros, thymus, liver, and peripheral blood.     

Analysis of hemopoietic lineage of accessory cells in the developing thymus of Xenopus laevis

The developmental history of accessory cells in the thymus was studied by grafting hemopoietic stem cells into cytogenetically distinct frog embryos (diploid-2N or triploid-3N) before the establishment of circulation and overt differentiation and colonization of the thymus. The DNA content of cortical thymocytes and circulating erythrocytes was quantified by staining with propidium iodide and measuring the amount of red fluorescence emitted by individual nuclei with the use of flow cytometry. Accessory cells from thymic medulla were separated by incubating for 2 hr on glass slides. For comparison, the developmental history of peritoneal macrophages was examined as representative, myeloid-derived phagocytic cells. DNA content of adherent cells was quantified by staining with the DNA-specific Feulgen reaction and measuring light absorption of individual nuclei by microdensitometry. Thymic accessory cells were subdivided into phagocytic and nonphagocytic phenotypes on the basis of latex bead ingestion. Phagocytic cells in the thymus were usually nonspecific esterase positive and phenotypically resembled peritoneal macrophages. Nonphagocytic cells from the thymus were usually esterase negative and had a dendritic morphology characterized by branched cytoplasmic extensions. Nonphagocytic cells were positive for cytoplasmic RNA based on staining with methyl green-pyronin Y. Phagocytic cells from both the thymus and the peritoneal cavity had no levels of cytoplasmic RNA detectable by this method. Analysis of the embryonic derivation of thymic accessory cells, based on the proportion of cells carrying the cytogenetic marker, demonstrated that thymic lymphocytes and thymic accessory cells were a concordant pair of cells, distinct from myeloid-derived erythrocytes and possibly macrophages. These experiments provide circumstantial evidence suggesting thymocytes and thymic accessory cells could arise from a bipotential precursor that diverges into these separate lineages after colonization of the epithelial thymic rudiment during early development.     

Immunolocalization of Phostensin in Lymphatic Cells and Tissues

Phostensin binds to the pointed ends of actin filaments and modulates actin dynamics. The genomic location of phostensin is between the HLA-C and HLA-E gene clusters on human chromosome 6, and the mRNA of this protein is predominantly distributed in the spleen, thymus, and peripheral leukocytes. However, the distribution of phostensin in leukocyte cell populations and the subcellular localization have not yet been determined. In this study, an anti-phostensin monoclonal antibody (PT2) that recognizes residues 89–124 of phostensin was prepared and used to examine the subcellular localization and distribution of phostensin in white blood cell populations and in lymphatic tissues. It was found that phostensin is mainly concentrated at the cell periphery and co-localizes with actin filaments. In addition, phostensin was abundant in helper T-lymphocytes, cytotoxic T-lymphocytes, mature monocytes, macrophages, B-lymphocytes, natural killer cells, and granulocytes as well as in the lymphatic tissues, such as the thymus, lymph nodes, and spleen. Phostensin is expressed in the mature lymphocytes of the thymic medulla but not in the immature lymphocytes of the thymic cortex. Taken together, phostensin is a ubiquitous protein in leukocytes, and it may play an important role in modulating the cellular functions of leukocytes.     

The 46,000-dalton tyrosine protein kinase substrate is widespread, whereas the 36,000-dalton substrate is only expressed at high levels in certain rodent tissues

Proteins of molecular mass 46,000 (p46) and 34,000-39,000 (p36) daltons are phosphorylated at tyrosine in Rous sarcoma virus-transformed chicken and mouse fibroblasts. p46 has recently been identified as an isozyme of enolase but the function of p36 is unknown. The expression of these proteins in various mouse and rat tissues has been examined. In most tissues, except muscle, p46 is found at relatively constant levels. In muscle, a more basic, related protein is present. In contrast, the abundance of p36 varies more widely from tissue to tissue, suggesting that it has a function in some but not all differentiated cells. By SDS gel electrophoresis and immunoblotting, high levels of p36 (60-120% of its relative abundance in fibroblasts) were found in small intestine, lung, and thymus, and intermediate levels (20-50%) were found in spleen, lymph nodes, and testes. No p36 was detectable in brain and muscle. Where studied, p36 mRNA expression paralleled protein levels. The cell types within each tissue expressing p36 were identified by immunofluorescence and immunoperoxidase staining. These cell types include all endothelial cells and fibroblastic cells examined, as well as various epithelial cells, cardiac muscle cells, macrophages, and testicular interstitial cells. We were unable to detect p36 in skeletal or smooth muscle cells, erythrocytes, nerve cells, or lymphocytes in any of the examined tissues. p36 appears to be concentrated in the terminal web region of intestinal columnar epithelial cells.     

Evidence for a cyclic renewal of lymphocyte precursor cells in the embryonic chick thymus

Experiments involving sequential transplantations of the chick embryonic thymus at E9 to E12 into a first 3-day host quail embryo and then into a second chick host allowed demonstration of the cyclic periodicity of hemopoietic cell seeding of the embryonic thymus. After a first wave of colonization occurring between E6.5 and E8, the thymus becomes refractory to hemopoietic cell entry for about 4 days. It resumes its capacity to be seeded by a second wave of blood-borne stem cells at E12. After a second period of non receptivity starting at E14, a third wave of incoming cells reaches the thymus around E18. Therefore, with a slightly different periodicity, the same cyclic mechanism regulates the renewal of lymphocytes in chick and quail embryos. Quail hemopoietic cells were immunostained in the chimeric thymuses, with a species specific monoclonal antibody (anti-MB1) which recognizes a common surface antigenic determinant on all endothelial and blood cells of the quail (except erythrocytes). Two steps could thus be distinguished in the seeding process. When the thymus becomes receptive for hemopoietic cells, the latter first accumulate in the intrathymic blood vessels before penetrating massively in the thymic parenchyma. The quail chick-chimera system combined with the use of a species- and cell-type-specific antibody provides a unique tool for studying thymic colonization by lymphocyte precursors.     

Lipid peroxidation of rat spleen and thymus lymphocytes treated with x-rays (0-5-1.0 Gy)

It is established, that low doses of X-ray irradiation have affected activation of lipid peroxidation (LPO) in immunocompetent cells of the spleen and thymus. The amount of malonic dialdehyde (MDA) in lymphocytes of spleen and thymocytes increases 2 times twenty-four hours after animals' irradiation by X-rays in a dose of 0.5 Gy; when a dose grows to 1.0 Gy, the MDA content in the spleen lymphocytes increases from the 1st to the 6th days and in thymocytes from the 1st to the 3d days reaching its maximum at the 3d day. MDA accumulation in the immunocompetent cells of irradiated animals varies depending on the method of lipid peroxidation initiation.     

ACTH receptor distribution and modulation among murine mononuclear leukocyte populations

Murine mononuclear leukocytes express adrenocorticotropin (ACTH) receptors that were recognized by a monospecific antiserum to the ACTH receptor on Y-1 adrenal cells. The antiserum was utilized in an immunofluorescence (IF) assay to characterize the distribution of ACTH receptors on resting murine mononuclear leukocyte populations. Forty-seven percent of spleen cells, 32% of lymph node cells, and 1% of thymocytes constitutively expressed ACTH receptors. Separation of lymphocytes into purified B cell and T cell populations, followed by IF analysis revealed that 47% of B cells and 23% of T cells possessed ACTH receptors. Helper T cells (CD4+ T cells) constituted the majority of ACTH receptor-positive T lymphocytes. Furthermore, 47% of resident peritoneal macrophages, purified by adherence to plastic, expressed ACTH receptors. The T-lymphocyte mitogen, concanavalin A, interferon gamma, and ACTH enhanced ACTH receptor expression. The differential distribution of ACTH receptor-positive cells among specific leukocyte populations explains in part why differential cellular responses are observed and implies important regulatory functions for these receptors in the generation or regulation of immune responses.     

The latex particle adherence (LPA) assay for detection of leukocytes with adhesive surface properties.

A new, simple, and rapid in vitro assay has been developed for identification of adherent and nonadherent leukocytes. The assay is based on adherence of latex (polystyrene) particles to the cell surface. Using the latex particle adherence (LPA) assay, the percentage of adhesive leukocytes has been determined in human peripheral blood mononuclear preparations and in the lymph nodes, thymus, bursa of Fabricius, spleen, and bone marrow of mouse, chicken, and rat origin. The highest proportion of LPA-positive cells was found in peritoneal exudate, bone marrow, and spleen, the lowest proportion, in thymus and bursa of Fabricius. LPA-Positive cells in human peripheral blood mononuclear preparations were identified as surface immunoglobulin-positive lymphocytes nonrosetting with sheep red blood cells. LPA-Positive cells in peritoneal exudate were identified as macrophages. Incubation of leukocyte suspensions on polystyrene petri dishes or nylon wool columns reduces substantially the percentage of LPA-positive cells in the nonadherent fraction. The LPA assay seems to be a method of choice for establishing the relationship between adhesiveness of the cell surface and other cell membrane markers on a single-cell level.     

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.     

A monoclonal antibody to the p220 component of sheep LCA identifies B cells and a unique lymphocyte subset

The leukocyte common antigen (LCA, CD45) of humans and rodents is expressed exclusively by leukocytes, and has been implicated in a number of immune functions (1-4), although its precise function is still unknown. Three monoclonal antibodies (mAbs) were produced which identified different epitopes on the LCA of sheep. mAbs 1-11-32 and 38-42 reacted with determinants of LCA expressed on all leukocytes, but showed differential reactivity with thymocytes. Another antibody, 20-96, identified an epitope of LCA expressed mainly on B cells, but also on a unique lymphocyte subset contained mostly in peripheral blood, which was 20-96high, sIg-, CD4-, CD8-, SBU-T19-, but CD5+. These cells constituted only 5-6% of PBL. The cellular lineage of this latter subset is uncertain since these cells appeared to be unrelated to B cells, and were absent from the thymus. Unlike the other two mAbs to LCA, 20-96 was not reactive with macrophages and granulocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of material immunoprecipitated by the "pan" LCA-specific mAbs revealed lymphocyte forms with molecular weights (MW) of 220, 210, and 190K, whereas 20-96 immunoprecipitated only a 220K MW form. The expression and MW of LCA on thymocytes or ileal Peyer's patch (IPP) cells differed from those on peripheral lymphocytes. B cells in IPP, which constitute 98% of cells, expressed the 20-96 determinant at low density, in contrast to its high expression on peripheral B cells. LCA from IPP existed in two forms of 220 and 190K MW, whereas LCA from peripheral B cells was entirely 220K MW, and thymus 210 and 190K MW.     

Reutilization by maternal and embryonal cells of nuclear materials from H3-thymidine labeled lymphocytes introduced into the lumen of intestine of rats

Summary H³-thymidine labeled lymphocytes from thymus and lymph nodes of donor rats were washed and injected in to the intestine of recipient rats on the 11th and 19th day of gestation; subsequent labeling of maternal and embryonal cells was studied autoradiographically 24 hours after injection. In 12-day embryos, numerous stem cells or hemocytoblasts were labeled frequently intensely. In 20-day embryos, stem cells or hemocytoblasts scattered throughout the liver were often labeled. In other fetal tissues at this stage, cells in thymus, spleen, mesenteric lymph node and intestine were labeled but scarcely and weakly. In mothers, labeling in lymphoid tissues was scarce but definite, in thymus, mesenteric lymph node and spleen. These results suggest that nuclear materials from lymphocytes emigrated into the intestinal canal of the mother could be reutilized by maternal and embryonal cells.     

Further Studies on Guinea Pig Z-1 Antigen That Is Involved in Phagocytosis of Zymosan by Macrophages: Cell Type Distribution of the Antigen and Cross-Reactivity of Anti-Z-1 with Human CR3

We have previously identified a heterodimer molecule, Z-1, on guinea pig peritoneal macrophages (Møs) by the newly prepared monoclonal antibody, anti-Z-1, and Z-1 has been assumed to be the complement receptor type three (CR3) in this species. To clarify this assumption, the cell type distribution of the antigen in guinea pig and the cross-reactivity of anti-Z-1 with other species were analyzed. It was demonstrated that Z-1 was expressed on peritoneal Møs, pulmonary Møs, peritoneal polymorphonuclear leukocytes (PMN), peripheral neutrophils, and some subpopulations of spleen cells and of bone marrow cells, but not on erythrocytes, circulating lymphocytes, and lymphocytes in both spleen and bone marrow in detectable amounts. Thus the expression of Z-1 seems to be restricted to phagocytes as is CR3 of other species. Furthermore, it was found that anti-Z-1 bound with peripheral neutrophils from human, horse and goat and HL-60 cells differentiated into monocytes. Any cross-reactivity of the antibody was not detected with neutrophils from rabbit, cow, sheep and dog and nondifferentiated HL-60 cells. Human Z-1 was indistinguishable from human CR3, since both were the heterodimer consisting of α chain of 170 kDa (pI = 6.6-7.2) noncovalently associated with β chain of 100 kDa (pI = 5.6-6.7). In addition, human CR3 in detergent-lysate of neutrophils was completely adsorbed with anti-Z-1 F(ab')2-Sepharose. These findings indicate that guinea pig Z-1 shares an antigenic determinant with human CR3. It thus seems to be possible that Z-1 may function as CR3 in guinea pigs.     

Development of T lymphocytes in Xenopus laevis: appearance of the antigen recognized by an anti-thymocyte mouse monoclonal antibody

Development of T lymphocytes in Xenopus laevis was studied using a mouse monoclonal antibody (mAb), XT-1, that was produced against surface determinants on thymocytes of J strain frog. Ontogenic studies, employing immunofluorescence, showed that cells positive for the determinant recognized by XT-1 mAb (XT-1⁺ cells) were first detected in the thymus of J strain Xenopus by Nieuwkoop and Faber stage 48 (7 days postfertilization) and then in the spleen, liver and kidney by stage 52 (20 days postfertilization). Percentages of XT-1⁺ cells in the thymus increased rapidly by stage 49 (10 days postfertilization) and reached adult levels by stage 52, and those in the spleen, liver, and kidney reached adult levels by stage 56 (40 days postfertilization). Electron microscopic immunohistochemistry revealed that most XT-1⁺ cells in thymuses from stage 56 larvae were typical small lymphocytes (4–7 μm in diameter). In contrast, many XT-1⁺ cells in larval thymuses at stage 49 are large (8–10 μm in diameter) lymphoblastoid cells. Thymectomy at stage 46 (5 days postfertilization) depleted XT-1⁺ cells in larval and adult lymphoid organs to background levels. These results suggest that the XT-1⁺ cells are differentiated from the lymphoid precursor cells in the thymus before the appearance of small lymphocytes and migrate into peripheral lymphoid organs. The cell surface determinant recognized by the XT-1 mAb may provide an important marker for the differentiation of T lymphocytes in Xenopus.     

Properties and kinetics of a population of B-lymphocytes during rat ontogenesis]

Cells bearing surface immunoglobulins (Ig+-cells) detected by the indirect immunofluorescent method and cells forming rosettes (RFC) with sheep erythrocytes coated with antibody and complement (EAC rosettes) were found in the liver and the spleen on the 15th and the 20th days of prenatal life of rats. The percentage of Ig+-cells and RFC in the liver was high and remained unchanged and at about the same level during the whole postnatal life. The spleen and the bone marrow displayed an increase of the Ig+-cells and RFC increased throughout the 1st month of postnatal life with the maximum at the 30th day after birth; a sharp decrease occurred in old animals. In the thymus the Ig+-cells and the RFC were either absent or present in very small amounts only at some periods of study. Ig+-cells with "capping" were discovered in the spleen and bone marrow on the 5th--10th days of postnatal life; their count increased considerably in 30-day and adult rats. Such cells were absent in the lymphoid tissues of old 40-month rats.