B lymphocyte differentiation in lethally irradiated and reconstituted mice

The recovery of the B lymphocyte compartments was investigated in lethally irradiated mice reconstituted with fetal liver cells. This was done by means of immunofluorescence on frozen sections of spleen, lymph nodes and Peyer's patches. The first B lymphocyte recovery in the spleen was observed on day 8, a few days earlier than in lymph nodes and Peyer's patches (day 13). These early B cells in the spleen were found in the central part of the periarteriolar lymphatic sheath (PALS). Later on, while increasing in number, the B cells formed growing follicles at the periphery of the PALS. Subsequently, brightly fluorescent B cells appeared in the marginal zone, which surrounded the follicles. Another two weeks later, around day 30, also germinal center formation was observed in the follicles of the spleen. B cell development in lymph nodes and Peyer's patches started somewhat later than in the spleen, but once started, the recovery of the different compartments was completed very fast. Germinal center reactions were found in lymph nodes and Peyer's patches already on day 25, and thus earlier than in the spleen, but later than the first occurrence of the strongly fluorescent cells in the marginal zone. Apparently, germinalcenter formation is not essential for the recovery of the population of brightly fluorescent B cells in the marginal zone after irradiation and reconstitution.     

The effect of neonatal rat graft-vs-host disease (GVHD) on Fc receptor lymphocytes.

The level of Fc receptor rosette-forming lymphocytes (Fc-RFL) was examined in spleen and lymph node cell suspension from neonatal DA and BN rats inoculated within 24 hr of birth with either allogeneic L (experimental) or syngeneic (control) lymphoid cells. In addition, these levels were compared to fetal and neonatal animals that received no injection. The indicator cells (EA) were sheep erythrocytes sensitized with one-half concentration of the highest dilution of rabbit anti-sheep erythrocyte IgG(A) which agglutinated an equal amount of 1% suspension of E. Care was taken to exclude scoring macrophages by injecting colloidal carbon at least 1 hr before killing the test animals. The spleen of 19-day DA fetal rats exhibited a level of 19.3% Fc-RFL, similar to that of animals having received adult syngeneic cells at birth (40.0%) by day 7. Thereafter the level of Fc-RFL did not vary appreciably between these two groups. However, as early as 2 days after inoculation there was a significantly greater number of Fc-RFL in the spleen of experimental DA neonates compared to controls. The lymph nodes of experimental animals did not exhibit a significantly greater number of Fc-RFL until day 6 with both tissue compartments peaking at day 10 and remaining significantly higher than controls until death. In neonatal BN animals significantly higher levels of Fc-RFL in experimental animals were not evident as early and peaked later (day 12) in both tissue compartments but again these differences remained until death. Cytotoxic alloantisera demonstrated that on days 6, 10, and 12 most, if not all, of the Fc-RFL were host in origion in both DA and BN GVHD, with a very significant host plasma cell response in such GVHD animals. One-micron tissue section revealed the presence of a great number of plasma cell especially prominent in the medulla of lymph nodes with the cortex of lymph nodes and white pulp of the spleen markedly depleted of lymphocytes indicative of cytotoxicity.     

Ontogeny of human fetal lymph nodes.

Developing lymph nodes from 30 human embryos and fetuses with crown-rump lengths (CRL) of 18 mm (5.6 wk) to 245 mm (26 wk) were examined by light microscopy. The nodes were embedded in araldite, and the sections examined were approximately 1 mu in thickness. The development of nodes was divided into three stages: 1. the lymphatic plexus and connective tissue invagination (30 mm to 67 mm CRL); 2. the early fetal lymph node (43 mm to ,5 mm CRL); and 3. the late fetal lymph node (CRL greater than 75 mm). The lymphatic plexus was formed by connective tissue invaginations and bridges which divided a lymph sac into a meshwork of channels and spaces. Connective tissue invaginations were endothelially-lined and were surrounded by lymphatic space. Reticular cells, macrophages, and blood vessels were found in these invaginations. Early fetal lymph nodes were formed from invaginations when the cellular density and lymphocyte content increased. The lymphatic space surrounding the early node was the developing subcapsular sinus. With further development the early node became packed with lymphocytes, increasing the cellular density and size of the node. The connective tissue surrounding the subcapsular sinus condensed to form the capsule. Afferent lymphatic vessels pierced the capsule. Capillaries, veins, postcapillary venules, and occasional arteries were found in early and late nodes.     

Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1.

We have examined the generation of CTL immunity immediately after localized footpad infection with herpes simplex virus 1 (HSV-1) using three coordinated in vivo T cell tracking methodologies. Tetrameric MHC class I containing the immunodominant peptide from HSV-1 glycoprotein B (gB) showed that after infection the proportion of Ag-specific T cells peaked at day 5 within draining popliteal lymph nodes and 2 days later in the spleen. Preferential expression of the activation marker CD25 by tetramer-positive cells in draining popliteal nodes but not spleen suggested that gB-specific T cells were initially activated within the lymph node. In vivo cytotoxicity assays showed that Ag-specific effector cells were present within the draining lymph nodes as early as day 2 after infection, with a further 2-day lag before detection in the spleen. Consistent with the very early arming of effector CTL in the draining lymph node, adoptive transfer of CFSE-labeled gB-specific transgenic T cells showed that they had undergone one to four rounds of cell division by day 2 after infection. In contrast, proliferating T cells were first detected in appreciable numbers in the spleen on day 4, at which time they had undergone extensive cell division. These data demonstrate that HSV-1-specific T cells are rapidly activated and armed within draining lymph nodes shortly after localized HSV-1 infection. This is followed by their dissemination to other compartments such as the spleen, where they further proliferate in an Ag-independent fashion.     

Absence of Peyer's patches and abnormal lymphoid architecture in chronic proliferative dermatitis (cpdm/cpdm) mice

The chronic proliferative dermatitis (cpdm) mutation causes inflammation in multiple organs, most prominently in the skin. Examination of the immune system revealed severe abnormalities in the architecture of lymphoid tissues. Peyer's patches were absent. In contrast, the spleen, lymph nodes, and nasal-associated lymphoid tissues were present. The spleen had normal numbers of T and B cells, but the spleen, lymph nodes, and nasal-associated lymphoid tissues had poorly defined follicles and lacked germinal centers and follicular dendritic cells. The marginal zone in the spleen was absent. The total concentration of serum IgG, IgA, and IgE in cpdm/cpdm mice was significantly decreased, whereas serum IgM was normal. Fecal IgA was low to undetectable in mutant mice, and the concentration of fecal IgM was increased. The titer of DNP-specific Abs following immunization with DNP-keyhole limpet hemocyanin was significantly decreased for all IgG subclasses. In contrast, T cell function appeared normal as assessed by evaluation of the contact hypersensitivity response in cpdm/cpdm mice. The cpdm mutation causes a complex phenotype that is characterized by multiorgan inflammation and the defective development of lymphoid tissues. The cpdm/cpdm mouse may be a useful model to study the factors that control the development of lymphoid tissues, in particular the Peyer's patches, and the mechanisms that control the humoral immune response.     

Detection and quantification of IgM(+) lymphocytes in fetal lamb spleen, liver and lymph nodes by flow cytometry

The first stage in Peyer's patch development in the fetal lamb is characterized by the colonization of the rudimentary Peyer's patches by precursor cells expressing the IgM surface receptor. In the fetal lamb, the spleen has been implicated as the source of gene-rearranged IgM(+) B lymphocytes. This study was intended to quantitate IgM(+) lymphocytes in the spleen, lymph nodes and liver of fetal lambs at various gestational ages between 63 and 110 days using flow cytometry. Flow-cytometric analysis revealed that IgM(+) lymphocytes were rare in the liver being consistently less than 1% at every gestational age examined. IgM(+) lymphocytes were detected in the spleen (mean 9.18%) and prescapular lymph nodes (mean 11.89%) as early as 63 days. In both spleen and lymph nodes, the highest representation of IgM(+) lymphocytes occurred between 70 and 86 days gestation. The highest mean percentage of IgM(+) lymphocytes was observed in the spleen (22.63%) and lymph nodes (17.02%) at 75 days gestation. From 98 days onwards, B-lymphocyte density gradually decreased in both spleen and prescapular lymph nodes. This study indicates that substantial populations of IgM(+) lymphocytes were present in both the spleen and prescapular lymph nodes from 70 days gestation and implies that both of these locations could be potential sources for the normal colonization of the ileal Peyer's patches.     

Ontogeny of murine T lymphocytes: II. Postnatal appearance and organ distribution of lymphocytes bearing Qa-4 and Qa-5 surface antigens

Cells from the lymphoid organs of C57BL/6 mice (from birth to 20 weeks) were monitored by the cytotoxicity assay for the presence of Qa-4 and Qa-5 surface antigens. Qa-4- and Qa-5-bearing cells are detectable in spleen, lymph nodes, and Peyer's patches, but not in thymus, liver, or bone marrow. Both antigens are present on small fractions of cells in each of these organs during the first week after birth. At 4–6 weeks of age, the fractions of Qa-4- and Qa-5-bearing cells rise to maximal levels which are then maintained throughout the ages studied (4–20 weeks). The relative proportion of these cell populations is greatest in the lymph nodes and smallest in the Peyer's patches, and in all three organs, more Qa-4- than Qa-5-positive cells are detected. The majority of Qa-4- and Qa-5-positive cells are Thy-1 positive, however, not all Thy-1- positive cells are Qa-4, Qa-5 positive. During postnatal development the ratio of Qa-4 or Qa-5-positive cells to Thy-1-positive cells increases in spleen, lymph nodes, and Peyer's patches indicating that cells bearing these antigens become a larger fraction of the T-cell population with age.     

3H-deoxythymidine incorporation in graft-versus-host disease in the Norway rat. I. Liquid scintillation studies.

Graft-versus-host-disease was produced in newborn Brown Norway (BN) rats with an intravenous (iv) injection of adult allogeneic Lewis (L) lymph node cells (experimental) and the response was compared to littermates injected with adult syngeneic BN cells (control). By 4 days the reaction in the spleen of experimental animals was such that the spleen index was 1.70 and 2.58 on day 7, and continued to increase until death. A one hour iv pulse of tritiated deoxythymidine (3HdT) administered to experimental and control animals revealed a whole organ peak incorporation of 3HdT on day 6 in experimental spleens. A second larger peak occurred on day 10 in the experimental spleen as compared to a single peak at days 6 or 7, respectively, in the experimental mesenteric and combined superficial lymph nodes. However, analysis of the incorporation of 3HdT with respect to organ weight revealed a peak incorporation in animals receiving L cells on day 4--6 with a second smaller peak on day 10 in the experimental spleen and again a single peak on day 5 or 6 in the lymph nodes. Total 3HdT incorporation within both experimental lymph node compartments became less than controls by day 15 even though experimental nodes had a larger mass. 3HdT incorporation per milligram tissue weight decreased in all tissue compartments of experimental animals by day 13--14. The contribution of donor and host cell proliferation to the various peaks observed is discussed.     

Evidence of extensive lymphocyte death in sheep Peyer's patches. II. The number and fate of newly-formed lymphocytes that emigrate from Peyer's patches

The emigration of newly produced lymphocytes from Peyer's patches (PP) of lambs was studied. Mesenteric lymph nodes (MLN) were excised from most animals a few weeks after birth, and then at 8 to 10 wk of age, the dividing cells in 3 to 4 m of the small intestine were labeled in situ with [3H]thymidine. An extracorporeal perfusion system was used to restrict the 15-min period of labeling to the perfused lengths of intestine, which included either the large continuous ileal PP or a number of smaller jejunal PP. One or 3 days later, the number of labeled cells in the perfused tissue and in other lymphoid organs was studied by autoradiography. In the perfused tissues, labeled lymphocytes accounted for 63.7% of ileal PP cells by 1 day and for 86.7% by 3 days compared with only 9.6% of lymphocytes in the perfused MLN. Labeled lymphoid cells in the perfused PP were nearly all in the follicles. Labeled lymphocytes that must have been produced in the segments of ileum or jejunum at the time of the perfusion, subsequently emigrated via the lymphatics, and were identified in the spleen, MLN, other lymph nodes, blood, jejunal PP, and at a lower frequency in the thymus, nonperfused ileal PP, and bone marrow. In lymph nodes, spleen, and nonperfused PP, more than 80% of the immigrant newly formed PP-derived cells were small- and medium-sized lymphocytes, and about 15% were large lymphocytes. The nature of the labeled cells in the lamina propria of the nonperfused small intestine was quite different in that approximately 50% were plasma cells as early as 24 hr after the cells were born in the perfused gut. It is proposed that terminal B cell differentiation was most likely initiated within the PP in response to the entry of antigen. It was estimated that at both 1 and 3 days after perfusion there were about 100 times more labeled cells in the perfused ileal PP than could be accounted for by emigration to other organs. It was concluded that these results provide additional support for the view that PP in lambs produce a tremendous number of lymphocytes, but relatively few leave their site of production; most apparently die in situ.     

Lymphangiogenesis and its role in cancer

In many tumour types, lymphatic vasculature serves as a major route for tumour metastasis. The dissemination of malignant cells to the regional lymph nodes is an early step in the progression of many solid tumours and is an important determinant of prognosis. Lymphangiogenesis (formation of new lymphatic vessels) is thought to be crucial for cancer cells to metastasise to the regional lymph nodes. However research in this important process has been neglected largely due to the lack of molecular markers specific to the lymphatic endothelium. Recently, several specific markers have been identified including LYVE-1, podoplanin and prox-1. Although the biology of lymphangiogeneis, particularly its regulation, is still far from clear, it is now well established that tumours are lymphangiogenic i.e. they could induce the generation of their own lymphatics and metastasise to the regional lymph nodes. It is thought that the interruption of the main signalling pathways involved in this process could help to prevent lymphatic spread of many tumours. Furthermore, understanding the molecular mechanisms in lymphangiogenesis might help to develop new therapeutic strategies against cancer lymphatic spread. Here, we reviewed the literature in regards to the biology of lymphangiogenesis, its molecular regulation, lymphatic markers and the significance in human solid tumours.     

Transdiaphragmatic lymphatic transport of intraperitoneally administered marker in hamsters.

OBJECTIVE: To determine whether transdiaphragmatic transport in hamsters is similar to that described in other animals by examining transport of an intraperitoneally administered marker. METHODS: Monastral blue B suspension was administered intraperitoneally to 28 male Syrian hamsters (Mesocricetus auratus). Four hamsters each were euthanized 7, 15, and 30 min, and 1, 2, 3, and 24 h later. Specimens were examined microscopically for presence of marker. RESULTS: Marker was present in intrathoracic lymphatic vessels and cranial and caudal mediastinal lymph nodes by 7 min after its administration. The amount of marker in lymph nodes increased with time. The subcapsular distribution of marker was consistent with lymphatic transport. By 1 h after its administration, marker was present in the liver, spleen, bone marrow, and mesenteric and mandibular lymph nodes. Patterns of marker distribution in these tissues were consistent with hematogenous transport, but the amount of marker was considerably less than that in the intrathoracic lymph nodes at corresponding times. CONCLUSIONS: Particulates were most likely translocated from the hamster peritoneal cavity to intrathoracic lymph nodes via transdiaphragmatic lymphatic vessels. A portion of the translocated particulates entered the blood, where they were distributed to a variety of tissues within a short time.     

The histophysiology of antibody-forming sites in the marine toad

Summary The normal histologioal features of the lymphatic organs — pericardial nodes, jugular bodies, spleen, and kidney — of the marine toad, Bufo marinus, are decribed. The lymph nodes and spleen of the marine toad lack the compartmental organization of corresponding mammalian organs and contain relatively less internal connective tissue. The cellular stroma composed of reticulum cells and fixed macrophages plays a more important role in maintaining structural organization than do the connective tissue.Changes in the cellular composition of the lymphatic parenchyma were observed in animals immunized with bovine serum albumin suspended in Freund's complete adjuvant. In addition to an increase in the number of lymphocytes and the presence of lymphoid hemocytoblasts, cells occurred which possessed many of the morphological characteristics of mammalian plasma cells. These plasma cells, which exhibited positive fluorescent antibody reaction, were more abundant in the kidney than in the lymph nodes or spleen of an immunized animal.Granulomas developed at the site (gastrocnemius muscle) of injection of antigen in complete adjuvant, and similar cystic lesions arose in the kidney. Apparently, the antigen-adjuvant mixture found its way from the site of injection (gastrocnemius muscle) into the kidney, probably via the renal portal system, and established lesions in the kidney. Appreciable numbers of antibody-forming cells, or plasma cells, were found in the muscle granulomas and in the kidney lesions.The lymphoid tissue of the kidney is considered the principal site of antibody formation in the marine toad, Bufo marinus.This investigation was supported by grants HD-2614-1 and GM-11782 from the United States Public Health Service administered by Dr. Ronald R. Cowden and Dr. E. Peter Volpe, respectively.     

Vitamin A Supplementation in Early Life Enhances the Intestinal Immune Response of Rats with Gestational Vitamin A Deficiency by Increasing the Number of Immune Cells

Vitamin A is a critical micronutrient for regulating immunity in many organisms. Our previous study demonstrated that gestational or early-life vitamin A deficiency decreases the number of immune cells in offspring. The present study aims to test whether vitamin A supplementation can restore lymphocyte pools in vitamin A-deficient rats and thereby improve the function of their intestinal mucosa; furthermore, the study aimed to identify the best time frame for vitamin A supplementation. Vitamin A-deficient pregnant rats or their offspring were administered a low-dose of vitamin A daily for 7 days starting on gestational day 14 or postnatal day 1, day 14 or day 28. Serum retinol concentrations increased significantly in all four groups that received vitamin A supplementation, as determined by high-performance liquid chromatography. The intestinal levels of secretory immunoglobulin A and polymeric immunoglobulin receptor increased significantly with lipopolysaccharide challenge in the rats that received vitamin A supplementation starting on postnatal day 1. The rats in this group had higher numbers of CD8⁺ intestinal intraepithelial lymphocytes, CD11_C ⁺ dendritic cells in the Peyer''s patches and CD4⁺CD25⁺ T cells in the spleen compared with the vitamin A-deficient rats; flow cytometric analysis also demonstrated that vitamin A supplementation decreased the number of B cells in the mesenteric lymph nodes. Additionally, vitamin A supplementation during late gestation increased the numbers of CD8⁺ intestinal intraepithelial lymphocytes and decreased the numbers of B lymphocytes in the mesenteric lymph nodes. However, no significant differences in lymphocyte levels were found between the rats in the other two vitamin A supplement groups and the vitamin A-deficient group. In conclusion, the best recovery of a subset of lymphocytes in the offspring of gestational vitamin A-deficient rats and the greatest improvement in the intestinal mucosal immune response are achieved when vitamin A supplementation occurs during the early postnatal period.     

Rapid anti-helminthic response of B lymphocytes in the intestinal mucosal tissues of rats

B cell response to Trichinella spiralis (Ts) adult antigen (Ag) was studied in rats 1-20 days postinfection. B cell recoveries from the mesenteric lymph node (MLN), Peyer's patches (PP), thoracic duct lymph (TDL), and the spleen were determined by FACS analysis and Ag-specific antibody-producing cells (Ab-pc) in these tissues were enumerated using the immunoplaque assay. Total B cell numbers increased 2-70 times from day 3 postinfection in the MLN and TDL obtained from MLN-resected rats (MX) and such proliferation was not found in the PP or the spleen. Ab-pc of all isotypes increased from day 3 in the MLN and from day 2 in the MX-TDL. Among all isotypes, IgE- and IgG1-pc showed the strongest response. Immunofluorescence study revealed that these B cells were activated in the non-PP region of the small intestine. These results indicate an early isotype switch to IgG1 and IgE production in Ts-infected small intestine.     

3H-deoxythymidine incorporation in graft-versus-host disease in the Norway rat. II. Autoradiographic studies.

A sequential analysis was made of various areas within the lymph nodes and spleen of newborn Brown Norway (BN) rats suffering from graft-versus-host disease (GVHD) subsequent to an allogeneic injection of adult Lewis (L) lymph node cells (experimental). One micron thick autoradiographs were compared between such experimental and control littermates having received the same number of syngeneic adult BN cells. Both experimental and control animals received tritiated deoxythymidine (3HdT) one hour before killing. The autoradiographs revealed a 2.25 and 2.50 times higher thymidine labeling index of lymphocytes in the deep cortex of mesenteric lymph nodes and white pulp of the spleen, respectively, for experimental animals. The experimental effect occurred within one day. The majority of the labeled cells in experimental animals were large lymphoblasts with prominent nucleoli. The labeling index within these areas remained significantly higher than control values until day 8 in the spleen and through day 14 within the lymph nodes. However, differences in labeled cells present in high powered microscopic fields reached a peak on day 3 within compartments in experimental animals but fell significantly below control values by day 9 owing to a pronounced disappearance of both small and large lymphocytes from these areas, and a decreased intensity of individual cell labeling as the reaction progressed. In contradistinction the concentration of labeled cells present in high powered microscopic fields of lymph nodes' medulla became 3.13 times controls by day 4. Most of these labeled cells contained a more basophilic cytoplasm than those found in the deep cortex and some were distinctly plasma cell precursors. In contrast to the deep cortex their concentration remained approximately three times control values until death. The data indicates that the major proliferative events within the spleen and lymph nodes in neonatal rat GVHD are initially restricted to donor cell localization areas of these tissue compartments. Subsequently the GVHD-related events may be attributed to other areas and possibly cell types. Thus any proliferation contributing to splenomegaly in the latter stages of GVHD appears to occur in the red pulp and that contributing to lymph node enlargement a medullary response.     

AN ELECTRON MICROSCOPE STUDY OF LYMPHATIC TISSUE IN RUNT DISEASE

The thymus, spleen, and lymph nodes were studied in runt disease induced by a graft of intravenously injected homologous splenic cells into newborn rats and mice. Adult Long-Evans cells (70 x 10⁶) were injected into Sprague-Dawley rats. Adult DBA cells (7 x 10⁶) were injected into C57BL/6 mice. Runted rats were sacrificed at 14 to 28 days of age; mice at 10 to 20 days. The thymic cortex is depleted of small lymphocytes. Those remaining are severely damaged and phagocytized. Evidence of damage includes swelling of mitochondria, myelin figure formation, margination of chromatin, and sharp angulation in nuclear contour. Large numbers of macrophages are present. Epithelial-reticular cells which envelop small cortical blood vessels are often retracted, with the result that the most peripheral layer in the thymic-blood barrier suffers abnormally large gaps. Lymphocytes of the periarterial lymphatic sheaths of spleen and of the cortex of lymph nodes are reduced in number and damaged. Vast numbers of plasma cells and many lymphocytes are evident throughout lymph nodes, in the periarterial lymphatic sheaths, and in the marginal zone and red pulp of the spleen. Plasma cells are of different sizes, the larger having dilated sacs of endoplasmic reticulum. Lymphocytes are small to medium in size. They contain, in varying quantity, ribosomes and smooth membrane-bounded cytoplasmic vesicles approximately 350 to 500 A in diameter. Most plasma cells and lymphocytes are damaged and many of these are phagocytized. Many lymphocytes in lymph nodes, however, show no evidence of damage. Reticular cells and other fixed cells of the connective tissues seldom appear affected. Thus, the major cell types reacting in runt disease are lymphocytes, plasma cells, and histiocytes or macrophages. It appears, therefore, that both the delayed and immediate types of sensitivity play a part in this disease.     

IgE formation in the rat following infection with Nippostrongylus brasiliensis: I. Proliferation and differentiation of IgE-bearing cells

Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis-infected animals.     

Peyer's patches export lymphocytes throughout the lymphoid system in sheep

The lymphocyte output from small intestine containing either the long continuous ileal Peyer's patch (PP) or several smaller jejunal PP was examined in young lambs. Most studies were done in 2-mo-old lambs, 1 mo after removal of mesenteric lymph nodes (MLN). Extracorporeal perfusion of part of the intestine and addition of fluorescein isothiocyanate to the perfusate led to the labeling, in their normal microenvironment, of a regionally defined population of cells. One day later considerable numbers of emigrant lymphocytes were identified by fluorescence microscopy in the spleen, MLN and peripheral lymph nodes, jejunal PP, and bone marrow. In nonperfused ileal PP and thymus the labeling indexes were low. The highest labeling index was in the blood where 3.7% of the lymphocytes were labeled. A similar organ distribution of emigrant cells was found on day 3. When MLN were included in the perfused region more emigrants were identified. In some animals the intestinal lymphatic draining the perfused ileum was cannulated. Continual lymph drainage caused a dramatic decrease in the labeling indexes in other lymphoid organs. A substantial number of lymphocytes leave both ileal PP and jejunal PP via lymphatics and travel to all other lymphoid organs. However, the number of emigrant lymphocytes compared with the total number of labeled lymphocytes in the perfused tissue was about 10 times greater after perfusing gut with the jejunal PP than after ileal PP perfusion. We conclude that relatively more lymphocytes emigrate from the jejunal PP than from the ileal PP.     

Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriformis: tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions, and in vitro lymphocyte transformations during primary infections

The kinetics of the lymphocyte responses of Trichostrongylus colubriformis-infected and normal guinea pigs were measured by the in vivo uptake of tritiated thymidine either as dpm ³H/mg tissue or as the percentage change in [³H] -labeled lymphoblasts in autoradiographs of tissue impression smears and sections. The lymphoid response was predominantly a local one centering on the infected area of the small intestine. The greatest lymphocyte reactions as assessed by counts of labeled lymphoblasts occurred in the Peyer's patches and mesenteric lymph nodes where the peak responses took place 11 and 6 days after infection, respectively. The local nature of the responses was exemplified by the fact that the mesenteric lymph nodes of the anterior small intestine showed a peak response on the sixth day but the response from the posterior small intestine peaked 7 days later. A similar but less dramatic relationship existed among the Peyer's patches. In addition no labeled lymphoblast response was elicited in the inguinal lymph nodes or cecal lymphoid patches throughout the infection and the first increased responsiveness of the spleen did not take place until after Day 13, by which time the lymphoid proliferations associated with the infected intestine had subsided. Initially, the spleen showed a marked depletion of labeled blast cells during the first 7 days of the infection. This was taken as indicating at the time the infection was being established the export of cells capable of transformation in response to parasite antigen. This was supported by the observation that large numbers of phytohemagglutinin responsive lymphocytes were found in the peripheral circulation at this time. The in vitro responsiveness of peripheral lymphocytes to T. colubriformis antigen was also studied. Positive lymphocyte transformations first occurred 6 days after infection but thereafter declined to the normal level by Day 13; the peak transformation ratio was found 25 days after infection but by Day 38 it had declined to a low but persistently positive level. There was a correlation between the circulation of specifically sensitized cells, probably of thymic origin, IgE antibody titers, and the development of positive dermal delayed hypersensitivity reactions in infected guinea pigs, suggesting a close relationship among these three immunological phenomena.All lymphoblast responses in Peyer's patches, mesenteric lymph nodes, and lamina propria of the intestine were completed before the immune elimination of the parasite commenced 10 days after infection. During the first 10 days of infection specifically sensitized lymphocytes appeared and disappeared from the circulation. The loss of circulating sensitized lymphocytes at the time immune elimination of the parasite was taking place in the gut suggested that the sensitized cells were “homing-in” on the local area of infection. After the immune elimination of the parasite had commenced, the level of sensitized lymphocytes and IgE antibodies then increased rapidly in the blood. Evidence from the kinetics of the hemagglutinating antibodies indicated that stage specific antigens occur in T. colubriformis.     

Lymphatic tracing and T cell responses following oral vaccination with live Mycobacterium bovis (BCG)

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis bacille Calmette-Guérin (BCG) expands a subset of interferon-gamma (IFN-gamma)-secreting T cells and mediates protection against aerosol mycobacterial challenge. We have traced the movement of the live vaccine through the regional lymphatics of mice and monitored the resultant immune response. Six hours after oral vaccination BCG was detected in low numbers systemically and in draining lymphatic tissue. However, after 48 h, BCG was predominantly associated with alimentary tract lymphatic tissues, such as the cervical and mesenteric lymph nodes and Peyer's patches. Lymphocytes that produced IFN-gamma in response to PPD-B or BCG-pulsed dendritic cells predominated in the spleen and were almost exclusively CD4(+), CD44(+) and CD62L(-), thus resembling an effector memory T cell population. Despite the fact that an oral route was used for immunization, splenic IFN-gamma-secreting T cells in vaccinated mice did not express the mucosal homing antigens alpha(4)beta(7) integrin or alphaIEL (CD103). However, a proportion of BCG-specific CD4(+) T cells expressed the CD29 integrin (beta(1)) chain, potentially involved in lung homing function. Thus, oral priming with M. bovis BCG appears to induce a subset of spleen-resident CD4(+) T cells with the potential to provide protective immunity in the lung.