Antigen-bearing dendritic cells from the sublingual mucosa recirculate to distant systemic lymphoid organs to prime mucosal CD8 T cells

Effector T cells are described to be primed in the lymph nodes draining the site of immunization and to recirculate to effector sites. Sublingual immunization generates effector T cells able to disseminate to the genital tract. Herein, we report an alternative mechanism that involves the recirculation of antigen-bearing dendritic cells (DCs) in remote lymphoid organs to prime T cells. Sublingual immunization with a muco-adhesive model antigen unable to diffuse through lymphatic or blood vessels induced genital CD8 T cells. The sublingual draining lymph nodes were not mandatory to generate these lymphocytes, and antigen-bearing DCs from distant lymph nodes and spleen were able to prime specific CD8 T cells in a time- and dose-dependent manner. This study demonstrates, for the first time, that antigen-bearing DCs originating from the site of immunization recirculate to distant lymphoid organs and provides insights into the mechanism of distant CD8 T-cell generation by sublingual immunization.     

Different reticular elements in rat lymphoid tissue identified by localization of Ia, Thy-1 and MRC OX 2 antigens.

An indirect immunoperoxidase method was used to localize the Ia, Thy-1 and MRC OX 2 antigens in rat lymphoid tissues. The antigens were detected using mouse monoclonal antibodies and all were notable since they were found to be expressed on different dendritic or reticular elements in lymphoid tissue. Ia antigen was present on bone marrow-derived dendritic cells in the T-dependent areas of spleen and lymph nodes in addition to B cells. MRC OX 2 antibody labelled dendritic cells in the follicles of spleen and lymph nodes but not the Ia positive cells in the T-dependent areas. Cells associated with blood vessels including the endothelium of the post capillary venules were also labelled with MRC OX 2 antibody. In contrast, anti-Thy-1 antibody labelled the pericyte sheath surrounding the post capillary venules and connective tissue elements in lymphoid tissues. The odd patterns of distribution of these antigens are discussed with respect to possible functions of the antigens and the cells they label.     

Cells involved in the immune response: XXVI. The demonstration of bone-marrow specific antigens in the rabbit

Horse anti-rabbit bone marrow cell antiserum was tested for its cytotoxic activity with respect to the lymphocytes of the various lymphoid organs. The unabsorbed antiserum was highly cytotoxic with respect to the circulating WBC and cells of the bone marrow and thymus but demonstrated low cytotoxic activity with respect to spleen, lymph node and SAPP cells (sacculus rotundus, appendix and Peyer's patches). However, following absorption with thymocytes, lymph node cells or SAPP cells, cytotoxic activity directed toward any of these cell types disappeared without affecting the cytotoxic activity with respect to bone marrow and circulating lymphocytes. On the other hand, bone marrow and spleen cells and circulating white blood cells were capable of absorbing out completely the cytotoxic activity directed toward these cells. On the basis of a comparison of efficiency of absorption of anti-bone marrow cell activity by cells of the different lymphoid organs and cytotoxicity assays of the absorbed antiserum, it is concluded that approximately 15–25 per cent of the spleen lymphocytes and 20–40 per cent of the circulating lymphocytes in the rabbit are bone marrow-derived cells. The other lymphoid organs do not normally appear to possess these cells.     

Immunohistochemical study of S-100 protein in the bovine lymph node and spleen

Immunohistochemistry using anti-bovine S-100 protein serum was examined in the bovine lymph node and spleen. In the lymph node, immunoreactivity was found in endothelial cells of lymph vessels and in endothelial and reticular cells of the sinuses. In the spleen, immunoreactivity was observed in endothelial cells of the trabecular artery, central artery, penicillar artery, sheathed artery, terminal capillary, trabecular vein and lymph vessel. In addition, the follicular dendritic cells in germinal centers both of the lymph node and spleen were stained with S-100 protein. These findings suggest that S-100 protein of the vascular systems may be related to the flow of lymph and blood.     

The role of lymphoid organs in the formation of antitoxic immunity

Summary Experiments carried out on 110 rabbits were devoted to the comparative study of the relationship between immunological reactivity of lymphoid organs and the character of the antigen administered (crude and adsorbed) as well as the immunization scheme. Toxoid was injected subcutaneously into the interior part of the posterior extremity. Antitoxin was determined in dynamics in the regional and distant lymph nodes, as well as in the spleen and blood. As shown by the investigations, primary injection of the antigen, irrespective of its character, is accompanied by a weak regional lymph node antitoxin production and complete absence of antitoxin accumulation in the distant lymphoid organs.With repeated antigen administration the immunological activity of the regional lymph node showed a marked rise. Immunological reactivity of lymphoid organs is directly proportional to the intensity of the primary immunization. Crude toxoid provokes a high revaccination effect limited to the regional lymph node. Administration of the same antigen to the animals which received adsorbed toxoid in primary immunization is accompanied by an intense antitoxin synthesis by the regional lymph node with a brief involvement into this process of distant lymph nodes and spleen. The same antigen administered with a 1% alum causes a diffuse type of reaction.(Presented by Active Member of the Akad. Med. Nauk SSSR P. F. Zdrodovskii) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 51, No. 3, pp. 80–84, March, 1961     

Monoclonal antibodies against mouse splenic stromal cells

A panel of rat monoclonal antibodies directed against mouse splenic stromal cells were isolated. These monoclonal antibodies were Immunohistochemically divided into four groups which reacted with non-lymphoid cells of the murine spleen; (1) in the white pulp, (11) at the marginal zone, (111) in the red pulp, and (IV) on the endothelium of splenic blood vessels. These monoclonal antibodies were studied Immunohistochemically In lymphoid organs by means of light and electron microscopy. Monoclonal antibodies SS-4 (group I) reacted with fibroblastic reticulum cells that were distributed only in the white pulp of the spleen and In the follicular areas of lymph nodes. The SS-4 staining cell, In clustered splenic stromal cells, formed colonies which Included a small number of Thy-1 positive lymphocytes. Therefore, we concluded that SS4 staining stromal cells comprise the lymphoid cornpartment. In contrast, monoclonal antibodies SS-1, SS-3 and SS-5 (group II) reacted with dendritic shaped cells in the marginal zone of the spleen. Examination of splenic extra-medullary hematopolesis in mice rescued by bone marrow transplantation after lethal irradiation revealed that SS-3 and SS-5 reacted with dendritic shaped stromal cells in clonal nodules of engrafted marrow in the red pulp. SS-3 and SS-5 staining cells could not be observed in physiologic hematopoiesis of non-transplanted mice. It was suggested that SS3 and SS-5 staining stromal cells are Involved in primitive hematopoiesls. Monoclonal antibodies SS2, SS-6 and SS-7 (group 111) mainly reacted with dendritic cells and macro-phages in the red pulp. Monoclonal antibodies SS-8 and SS-9 (group IV) reacted with endothelial cells of blood vessels and sinuses. These findings of heterogeneity in mouse splenic stromal cells are further evidence that specific micro-envlronments are composed by speclalired stromal cells.     

In-vivo modulation of thymus-derived lymphocytes with monoclonal antibodies in mice. I. Effect of anti-Thy-1 antibody on the tissue distribution of lymphocytes.

A procedure is described for the in vivo removal of all detectable T lymphocytes from spleen and lymph nodes in mice. A single intraperitoneal injection of monoclonal anti-Thy-1 antibody into mice leads to rapid depletion of functional T cells from peripheral lymphoid organs, but not thymus. The extent of T-cell depletion is dependent on the cytotoxic titre of the anti-Thy-1 antibody used. Antibody with a median cytotoxic titre greater than 10(6) causes the complete removal of cells bearing Thy-1, Lyt-1 and Lyt-2 surface antigens from peripheral lymphoid populations in 3 days. Eight days after treatment Thy-1+, Lyt-1+ and Lyt-2+ cells begin to reappear in these organs. Splenic B cells, assayed by the expression of surface immunoglobulin (sIg) and by mitogenic responsiveness to bacterial lipopolysaccharide (LPS), are not affected by this treatment. The monoclonal anti-Thy-1 antibody does not appear to penetrate thymus tissue and bind to thymocytes. Anti-Thy-1 antibody, but not F(ab')2 is required for in-vivo T-cell depletion. These findings indicate that anti-Thy-1 antibody causes the removal of Thy-1+ cells from peripheral lymphoid tissue, and as the circulating levels of anti-Thy-1 antibody decrease, cells from the thymus repopulate the thymus-dependent areas of the depleted lymphoid organs.     

Two closely related antigens expressed on granulocytes, macrophages and some reticular elements in rat lymphoid tissues: characterization by monoclonal antibodies

Two distinct novel antigen systems preferentially expressed in rat granulocytes and macrophages were detected using two different monoclonal antibodies (R2-1A6 and R2-2B1). These two antibodies reacted with approximately 50% of rat bone marrow cells, most granulocytes, blood monocytes, alveolar macrophages and peptone-elicited peritoneal macrophages, but not with red blood cells, platelets, thymocytes and T lymphocytes. In addition, R2-2B1 but not R2-1A6 antibody cross-reacted weakly with rat B cells. These two monoclonals also reacted with some reticular elements in rat lymphoid organs including epithelial reticular cells in the thymic medulla and follicular dendritic cells in the lymphoid germinal centre, as well as with the specialized endothelium in the marginal sinuses of the spleen and the post-capillary venules of the lymph node, where lymphocyte recirculation takes place. These antibodies, however, did not label so-called 'dendritic cells' bearing Ia antigens on their cell surfaces, which were found to be located in the thymic medulla, thymus-dependent areas of rat lymphoid tissues and the interstitium of various non-lymphoid organs, suggesting that these dendritic cells, presumably ascribed to those associated with accesory cell function, are separable from the mononuclear phagocyte system in rats by their different reactivities with R2-1A6 and R2-2B1 antibodies.     

Immunobiological studies on experimental visceral leishmaniasis. I. Changes in lymphoid organs and their possible role in pathogenesis

Studies were carried out to determine changes in lymphoid organs i.e. spleen, lymph node and bone marrow (BM) in progressive experimental visceral leishmaniasis. Mononuclear phagocytes in the BM were increased; spleens showed a hypercellularity coupled with a rise in parasite burden while secondary follicles with no apparent depletion of paracortex were seen in the lymph node. This enhanced proliferation of mononuclear phagocytes in the BM and probably their subsequent recruitment in the spleen could be induced in naive recipients by injecting nylon wool-purified lymph node cells derived from infected mice together with sonicated leishmanial antigen(s). Similar changes could also be induced in the BM of naive recipients by injecting serum of infected mice. In control experiments where both donor and recipients were uninfected such changes were not apparent. A working hypothesis is proposed to delineate the role of lymphoid organs in the pathogenesis of experimental visceral leishmaniasis.     

The Recirculation of T and B Lymphocytes in the Athymic, Nude Rat

The recirculation of lymphocytes through the tissues and their return to the blood were compared in nude and euthymic rats. Three approaches were used: the organ distribution of 15Cr-labelled lymphocytes from nude or euthymic donors at 24 h after injection; the compartmental distribution of B and T lymphocytes as assessed by autoradiography of the spleen, lymph nodes, and Peyer's patches; and the tempo of recirculation from blood to thoracic duct lymph as estimated by counting timed fractions of lymph from a recipient of labelled lymphocytes. The following conclusions were drawn: (1) The distribution of lymphocytes between organs and within organs is very similar in nude and euthymic recipients. In particular, B lymphocytes proceed normally to the follicular areas in the absence of T cells. (2) The recirculation from blood to lymph is delayed in nude rats. (3) For equal numbers of B and T cells injected intravenously about half as many B cells as T cells enter mesenteric and cervical lymph nodes, but approximately equal numbers of B and T cells enter the spleen and Peyer's patches.     

Expression of asialo GM1 by both Thy-1-positive and Thy-1-negative lymphocytes: evidence for modification of asialo GM1 by sialic acid

In order to determine the extent to which Thy-1-positive and Thy-1-negative lymphocytes express the asialo GM1 antigen, lymphocytes in normal spleen and lymph node were examined for simultaneous expression of the asialo GM1 and Thy-1.2 determinants. The results presented herein demonstrate that 55-57% of Thy-1.2-positive cells in spleen and 61-70% of Thy-1.2-positive cells in lymph node express asialo GM1. Furthermore, a significant frequency of Thy-1-negative cells in spleen (12-19%) and in lymph node (28-32%) also express asialo GM1. Since asialo GM1 has previously been shown to be absent on Ig-positive lymphocytes [9], these results establish that asialo GM1 is a marker shared by lymphocytes belonging to the T-cell lineage and lymphocytes apparently not committed to the T-cell to the T-cell lineage, most probably including natural killer (NK) cells. The implication of this finding as to the controversy regarding the possible relation of NK cells to T cells is discussed. Pretreatment of lymphocytes from spleen, thymus and lymph node with neuraminidase resulted in subsequent reactivity of 80-90% of these cells with anti-asialo GM1 anti-bodies. A smaller increase in asialo GM1 detection after neuraminidase treatment was seen with bone-marrow cells (65%). Protease treatment did not affect the subsequent reactivity of lymphocytes with anti-asialo GM1 antibodies. It is concluded that in situ enzymatic modification of asialo GM1 by the addition of sialic acid may be an important regulatory event in lymphocyte differentiation.     

Cells involved in the immune response. XXVIII. the cellular composition of the lymphoid organs in the normal outbred rabbit.

Horse anti-rabbit spleen cell antiserum was obtained by the intravenous immunization of horses with rabbit spleen cells. The antisera obtained were analysed for their lymphocytotoxic activity with respect to the lymphoid cells of the different lymphoid organs prior to and following absorption with the different lymphoid cells. The results have been integrated with those obtained in previous investigations and an all-embracing concept of the interrelationship of the lymphoid cells in the different lymphoid organs in the rabbit has emerged. The distinction between the heretofore considered central lymphoid organs (bone marrow and thymus) and peripheral lymphoid organs (spleen, lymph node and appendix) has been blurred by the finding that, at least in the rabbit, appendix cells possess specific antigen markers and participate in cell-mediated immune reactions in vitro and the observation that the spleen also has a central lymphoid function in the generation of cells in vivo capable of carrying out a cell-mediated immune reaction in vitro. It is concluded that the distinction of central and peripheral lymphoid function may be an artificial one in that it may reflect a function of the particular organ in only one of the many different types of immune reactions and that it should not influence the investigator in the elucidation of the cellular mechanisms participating in the mediation of the different immune responses.     

Distribution of interferon-gamma receptor in human tissues.

Interferon-gamma (IFN-gamma) is produced by activated T lymphocytes and plays a regulatory role in immune responses. The nature and location of cells that express the IFN-gamma receptor (R) and respond to this lymphokine are not well documented. The distribution of human IFN-gamma-R (HuIFN-gamma-R) was, therefore, investigated in situ by immunohistochemistry, using affinity-purified rabbit polyclonal antibodies directed against the extracellular domain of the receptor. In lymphoid organs, IFN-gamma-R expression is restricted to the B cell areas of lymph nodes, adult and fetal spleen, tonsils, appendix, and mucosa-associated lymphoid tissue of the small bowel. Macrophages and other reticular cells in lymphoid tissues and other organs are strongly positive for IFN-gamma-R, whereas its expression was consistently negative in the cortical and medullary thymocytes. Two-color flow cytofluorometric analysis of blood, lymph node, tonsil, spleen and thymus cells confirms that most B lymphocytes are IFN-gamma-R positive, whereas T lymphocytes are negative. However, after in vitro activation, peripheral blood T cells become IFN-gamma-R+. In non-lymphoid organs, IFN-gamma-R is expressed on endothelial cells of the medium- and small-size vessels. In epithelial tissues, high expression of IFN-gamma-R is detected on trophoblastic epithelium, glandular cells of stomach, ileum and colon, lung alveolar cells, salivary duct cells, renal tubular cells, and endometrial mucosa cells. Hepatocytes are weakly positive, while squamous epithelial cells are negative. The distribution of the HuIFN-gamma-R is discussed in view of the known functions of IFN-gamma.     

Quantitative analysis of the chimeric state in mice: III. Comparison of the colonizing capacities of syngeneic cell suspensions from different sources

Cytological analysis of the lymphoid organs of mice neonatally injected with syngeneic cell suspensions derived from different sources showed that the proportions of donor cells in the host spleen, thymus and bone marrow were low, irrespective of the type of suspension administered at birth. In contrast, the proportions of donor cells found in the host lymph nodes were for all inocula demonstrably higher than those found in other lymphoid organs. Various types of inocula, however, displayed different lymph node colonizing capacities depending upon the tissue from which the inoculum was prepared. The order of the lymph node colonizing properties showed a fair correlation with the known order of tolerance-conferring capacities of these inocula. Both phenomena are in accord with the hypothesis that the higher the content in recirculating small lymphocytes in a given tissue, the higher the capacity of the inoculum prepared from that tissue to colonize the host lymph node system and to induce tolerance.     

Cells involved in the immune response: XXII. The demonstration of thymus-specific antigens in the rabbit

Horse anti-rabbit thymus cell serum (HARTS) was obtained by immunizing a horse with rabbit thymocytes intravenously at weekly intervals for 3 weeks. The horse was bled 2 weeks later and the antiserum was analysed for its cytotoxic activity with respect to the lymphocytes of the various lymphoid organs. It was demonstrated that the cytotoxic activity of the antiserum was several orders of magnitude greater for thymus cells than for cells of the other organs tested. Only thymus and lymph node cells were capable of absorbing the thymocytotoxic activity of the antiserum; however, ten to fifteen times as many lymph node cells as thymus cells were required to neutralize the thymocytotoxic activity of the serum. Absorption of the antiserum with the cells of the other lymphoid organs (spleen, bone marrow, appendix, sacculus rotundus, Peyer's patches and circulating leucocytes) resulted in a slight but significant decrease in the thymocytotoxic activity. At no time was the thymocytotoxic activity completely absorbed with cells of these organs. The cytotoxic activity of the antiserum with respect to the cells of the different lymphoid organs other than the thymus could be abolished following absorption of the antiserum with the cells of any of the lymphoid organs. On the basis of our data, it is concluded that (a) the thymocytes possess two groups of antigens—one thymocyte specific and one common to all rabbit lymphocytes and (b) only the lymph nodes of all the lymphoid organs other than the thymus possess significant numbers of thymus-derived or T-cells. However, the proportion of these cells in the lymph node does not exceed 7–10 per cent, a figure much lower than that found in the lymph nodes of the mouse. Less than 1 per cent of the circulating lymphocytes in the rabbit are T-cells.     

Long term administration of cyclophosphamide in MRL/1 mice. I. The effects on the development of immunological abnormalities and lupus nephritis

Weekly injection of cyclophosphamide (Cy) into MRL/Mp-lpr/lpr (MRL/l) mice, at a dose of 10-20 mg/kg, from 1 month of age prevented the development of generalized lymph node enlargement, decreased serum levels of anti-DNA antibodies and immune complexes, and markedly prolonged their life span. Cy effectively suppressed enhanced differentiation of B cells, as evidenced by decreased number of immunoglobulin secreting cells in the spleen. Cy was also shown to suppress abnormal expansion of Thy-1 positive cells in lymphoid organs of MRL/1 mice. These results suggested that Cy prevented the development of murine lupus like syndrome in MRL/1 mice through suppression of spontaneous polyclonal B cell activation and also by reducing the number of T cells to exert excessive helper activity on B cells.     

Significance of lymphatic invasion on regional lymph node metastasis in early gastric cancer using LYVE-1 immunohistochemical analysis

It has been reported that lymphatic invasion is a predictor for lymph node metastasis in early gastric cancer (EGC); however, it has been impossible to differentiate between lymphatic invasion and blood vessel invasion using current staining techniques. We studied the significance of lymphatic invasion on regional lymph node metastasis in EGC by using human lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) antibody, specific to lymphatic vessels, and von Willebrand factor (vWF) antibody, specific to the blood vessels, to clearly distinguish these vascular tissues.EGC tissues were obtained from 66 node-positive and 66 node-negative subjects and were matched by age and sex. These tissues were immunostained with antibodies against LYVE-1 and vWF. Multivariate logistic regression analysis demonstrated that lymphatic invasion was a significant independent predictor for regional lymph node metastasis (odds ratio, 4.667; P = .0094), whereas blood vessel invasion was not. Thus, lymphatic invasion identified by LYVE-1 antibody could predict the existence of regional lymph node metastasis in EGC.     

In vitro synthesis of some complement components (C1q, C3 and C4) by lymphoid tissues and circulating leucocytes in man.

Human lymphoid tissues and peripheral blood leucocytes and monocytes were studies with respect to the synthesis of complement components (C1q, C3 and C4) using an in vitro culture technique. All of the lymphoid tissues investigated (bone marrow, thymus, lymph node, spleen, tonsil, adenoid) synthesize complement components in different patterns. C3 was produced by all lymphoid tissues except the spleen, which was the only lymphoid tissue in which C4 production was regularly found. C1q synthesis was demonstrated in the spleen and adenoid cultures, and occasionally also in those of lymph node tissue. Lymphocytes in peripheral blood from normal individuals and in thoracic duct lymph, and also from patients suffering from chronic lymphatic leukaemia, do not synthesize any of these complement components. Peripheral blood leucocyte samples from normal individuals, containing 60 per cent lymphocytes and 40 per cent monocytes, do synthesize C3, however. Separation of the monocytes from these samples showed that it was in these cells that the synthesis of C3 occurred. Production of C3 by mononuclear phagocytes is also supported by the finding that peripheral blood leucocytes from patients suffering from acute monocytic leukaemia synthesize C3. C1q and C4 synthesis could not be demonstrated in any of the cultures of circulating leucocytes.     

Age-decrease of cells sensitive to an autoantibody-specific for thymocytes and thymus-dependent lymphocytes in NZB mice

NZB mice naturally produce an autoantibody which in the presence of complement is specifically cytotoxic for thymocytes and thymus-dependent lymphocytes (T-cells) in the peripheral lymphoid tissues (lymph nodes and spleen) and the circulation of mice. Using a direct cytotoxicity test with a NZB mouse serum pool which contained the high titred autoantibody, a progressive decrease was observed with age in the proportion of the autoantibody-sensitive cells in mesenteric lymph node, spleen, and blood of NZB mice in comparison with mice of other strains (C57BL/6J and NZW). The numerical decrease in the population of autoantibody-sensitive cells was evident at younger age and greater degree in the peripheral blood than in the lymph node and spleen. The age-decrease in the number of autoantibody-sensitive cells in lymph node and spleen contrasted with the numerical increase in nucleated cells in these organs. The age-decrease in the proportion and number of the autoantibody-sensitive cells in the blood exceeded the decrease in the blood lymphocyte count. This finding indicated that T-cells in the blood are selectively depleted with the ageing of NZB mice. A similar observation was made on the blood lymphocytes of (NZB × NZW)F₁ hybrid mice. The depletion of T-cells in the blood in association with the production of natural thymocytotoxic autoantibody is termed autoimmune thymus-dependent lymphocytopenia.     

Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood

Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2⁺ CD25^lo NK cells were the predominant subset of NK cells within the blood. In contrast, CD2⁻ CD25^hi NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2⁻ NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2⁻ subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection.