Mouse monoclonal antibodies against rat major histocompatibility antigens. Two Ia antigens and expression of Ia and class I antigens in rat thymus

A rat Ia (RT-1B) antigen (called Ia-A) equivalent to the mouse I-A product has been defined with mouse monoclonal antibodies (W. R. McMaster and A. F. Williams, Eur. J. Immunol. 1979. 9: 426). To identify other Ia antigens mouse monoclonal antibodies were raised against rat spleen glycoproteins depleted of the Ia-A antigen. An IgG antibody (called MRC OX17) was obtained and used to purify a molecule which had a similar structure to the Ia-A antigen and reacted with anti-Ia alloantibodies. There was no cross-reaction between the two Ia glycoproteins in assays with mouse monoclonal antibodies, alloantibodies or rabbit antibodies. In one alloantiserum almost all the detectable anti-Ia antibodies reacted with a mixture of the two Ia glycoproteins. The MRC OX17 antibody did not bind to mouse cells, but rabbit antibodies to the pure rat glycoprotein cross-reacted and recognized determinants mapping to the mouse I-E region. In the thymus the rat Ia-E antigen was on cortical epithelial and medullary reticular cells. An IgG monoclonal antibody (MRC OX18) to isotypic determinants of rat histocompatibility RT-1A antigens was also produced and used to analyze these antigens on thymus cells. The heavily labeled thymocytes were those with characteristics of mature T lymphocytes. Cortical epithelial cells and medullary dendritic-like cells were also RT-1A positive.     

Electrophoretic separation of lymphocytes from rat spleen and thymus and their response to mitogens in vitro

Electrophoretic mobility (EPM) of lymphocytes from the thymus and spleen of Wistar and August rats was investigated by free flow electrophoresis and the ability of lymphocytes with different surface electric charges to undergo mitogenic transformation under the influence of phytohemagglutinin and concanavalin A was compared. An in vitro culture showed that splenic lymphocytes can be divided into two principal groups depending on their surface charge: cells with high and low mobility respectively. Separation of the thymocytes showed them to be a group of cells consisting of several (8 to 10) fractions differeing in EPM. Ability of the lymphocytes to be stimulated by mitogens was shown to depend on their surface charge. Stimulation of [³H]thymidine incorporation was observed during exposure of splenic lymphocytes with high mobility in an electric field to mitogens. Lymphocytes with low mobility were not stimulated by mitogens. The subpopulation of thymocytes with low EPM was not stimulated by concanavalin A. Rat thymocytes were shown virtually not to react to phytohemagglutinin irrespective of their EPM.Laboratory for Organ and Tissue Transplantation, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kovanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 9, pp. 322–324, September, 1979.     

Characterization of lymphoid cell lines from murine spleen which can localize in thymus.

Several murine lymphoid cell lines have been tested for specific capacity to localize in thymus. These are all continuous, cloned Radiation leukemia virus-induced cell lines which have a common phenotype resembling lymphoid stem cells or immature T cells. Since each of these cell lines has a cloning efficiency approaching 100%, the number of cells which enters thymus during a 3 h homing assay has been estimated by limit dilution cloning analysis taking into account extra-binomial variation caused by individual mice. Only two out of seven of these cell lines have been found to have this specific property. These two cell lines, 16C1 and 5C2B, have been characterized as immature lymphoid cells, bearing no rearrangement at the TCR gamma and beta loci, and having the phenotype of CD3+CD4-CD8-, immature T cells. A maximum number of 2000 16C1 cells and 2500 5C2B cells can enter thymus during a 3 h homing assay, suggesting a limited number of sites in thymus to which these cells can bind. The capacity of 16C1 to enter thymus in low frequency has been found to be a stable property and was not increased by repetitive passage through mouse thymus. Using this assay, we have also been able to confirm that entry of 16C1 cells into thymus can be inhibited by antibody specific for the Ly24 (Pgp-1) molecule.     

Cells bearing Ia antigens in the murine thymus. An ultrastructural study

Light- and electron-microscopic immunohistochemical examination of the murine thymus revealed that epithelial cells throughout the thymus expressed Ia antigens, as did interdigitating cells, monocytes, and macrophages located in perivascular "cuffs" associated with corticomedullary and medullary blood vessels. Thymic lymphocytes also expressed Ia antigens, but only at areas of contact with Ia-positive nonlymphoid cells. We observed evidence for synthesis of Ia antigens by epithelial cells, but not by thymic lymphocytes. These data indicate that several morphologically distinct populations of cells in the thymus express Ia antigens and thus may be involved in cellular interactions regulating I-region-restricted T-lymphocyte differentiation.     

Induction of early alloantigen tolerance in thymus and spleen

Both thymocytes and splenocytes from B6 (H-2b) mice injected as neonates with C3H(H-2k) or (B6 X C3H)F1 cells exhibited specific nonresponsiveness to the H-2KD alloantigens in question. Thus, tolerance could be induced in strain combinations which did not share H-2 antigens. Induction of specific allotolerance did not require the presence of Thy-1.2+, Ia+ or G10-Sepharose-adherent cells in the donor inoculum, however injection of irradiated (1200 rad) cells was ineffective. Evidence of specific unresponsiveness was found as early as 5 days after birth and was not abrogated by adding either irradiated normal spleen cells or exogenous interleukin-2 in vitro. In fact, in some experiments there was evidence for the presence of stimulated helper T cells within the population of 'tolerant' cytotoxic T cells exposed to alloantigen. However, this could reflect the operation of a syngeneic mixed lymphocyte reaction directed at, for instance, fetal calf serum components expressed in the context of the tolerated Ia determinants.     

Rh glycoproteins in epithelial cells: lessons from rat and mice studies.

Rhesus glycoproteins are a recently discovered family of ammonium transporters and a new branch of the Mep/AMT proteins superfamily that was identified more than 15 years ago in lower organisms and plants. Despite many ex vivo studies showing evidences that Rh glycoproteins can accelerate transmembrane NH3 or NH4+ transfer, their role in normal and disease physiology remains unknown. This review focuses on some of the different studies carried out in animal models to gain insight into Rh glycoprotein function. Immunolocalization studies have added new evidence that this protein family is related to ammonium transport or metabolism in epithelial cells. However, the absence of distal tubular acidosis or hyperammonemia in Rhbg KO mice have raised new questions about the physiological significance of these proteins.     

Immortalization of rat spleen and thymus T cells by human T-cell leukemia virus type I

Co-cultivation of thymus and spleen cells of Fisher and Lewis rats with lethally irradiated MT-2 cells harboring human T-cell leukemia virus type I (HTLV-I) resulted in the establishment of lymphoid cell lines, FIRT-1, FIRS-1, LERT-1, and LERS-1, respectively. Cells of these cell lines had rat T-cell characters as demonstrated by the positive reaction to monoclonal antibodies (MAbs) to rat T cell antigens (Thy 1 and pan T). They lacked surface immunoglobulins and strongly expressed rat interleukin-2 receptor antigen (Tac) and Ia antigen. Karyotypic analysis revealed that they had the normal rat karyotype in early cultures, but showed marked aneuploidy after long cultivation. None of them expressed HTLV gag proteins (p19 and p24) or virus particles, but they contained HTLV-I proviral DNA monoclonally and weakly expressed pX gene products (p40x). They were not transplantable into syngeneic newborn rats.     

Immune regulation by mesenchymal stem cells derived from adult spleen and thymus

We show here that human and mouse mesenchymal stem cells (MSCs) can be obtained not only from bone marrow (BM), but also from adult spleen and thymus. In vitro, both human and mouse spleen- and thymus-derived MSCs exhibit immunophenotypic characteristics and differentiation potential completely comparable to BM-MSCs. In addition, they can inhibit immune responses mediated by activated T lymphocytes with efficiency comparable to BM-MSCs. In vivo, mouse MSCs from BM, spleen, and thymus, if injected together with a genetically modified tumor cell vaccine, can equally prevent the onset of an anti-tumor memory immune response, thus leading to tumor growth in normally resistant mice. Our data suggest that not only do spleen and thymus have a stem cell reservoir to build up their stromal architecture, but also contain microenviromental immunoregulatory cells with the same properties of BM-MSCs.     

Suppression in Xenopus laevis: thymus inducer, spleen effector cells.

Studies were carried out on suppressor function in the amphibian Xenopus laevis, the South African clawed toad. Suppression by the thymus of haemagglutinin (HA) production by spleen is antigen-dependent, partially specific and not MHC-restricted in this species (Ruben, Buenafe & Seivert, 1983). Three questions were considered in this study. Does the thymus effect suppression by stimulating peripheralized spleen effector cells, or do effector cells reside within the thymus? Do macrophages participate in the induction and/or expression of thymus-dependent suppressor function? Can thymus suppressor and helper functions be distinguished by using irradiation treatment? The capacity of immunized thymus to suppress HA when co-cultured with spleen fragments from immunized, cyclophosphamide (CyP)-injected animals was tested. Immunized thymus failed to suppress the high levels of HA production by spleen fragments from CyP-treated, immunized donors. Colloidal carbon injection resulted in blockade of macrophage function, and both the capacity of thymuses to suppress and of spleens to be suppressed in co-cultures. Finally, the effect of thymus exposure to gamma-irradiation in vitro was tested using autogeneic thymus/spleen combinations. This enabled the visualization of thymic helper function, which is MHC-restricted in Xenopus (Bernard et al., 1981). Four dosages of irradiation were tested after antigen challenge. The highest HA titres were produced by spleen co-cultures with thymuses which had received 1000 rads. We conclude that suppression of HA production in spleen is not the result of thymus suppressor effector cells, but that suppressor function is mediated by thymus inducer cells which stimulate suppressor effectors in spleen. Both the thymic inducers and effectors in the spleen are sensitive to CyP and macrophage blockade. Our studies further suggest that we are able to distinguish between the thymic functions of help and suppression in Xenopus by taking advantage of their differential sensitivities to irradiation. While it has been postulated, on other grounds, that suppression was one of the earliest thymic regulatory functions to have evolved (L.N. Ruben & R.H. Clothier, submitted), here we suggest the presence of sequential activities of more than one cellular subset, as early in evolution as the primitive anuran (tail-less) amphibia.     

Immunoglobulin-positive human spleen and thymus cells during embryogenesis

The relative percentages and types of luminescence of immunoglobulin-positive cells were investigated by the indirect immunofluorescence method in the spleen and thymus of 32 human fetuses between the 11th and 32nd weeks of development. The number of Ig-positive cells in the spleen increased during embryogenesis from 13 to 33.7%, but in the thymus it remained at 0–2% regardless of the stage of development of the fetus. For the first time a differential count of Ig-positive cells based on types of luminescence was carried out in the course of embryogenesis. Thymus cells had solitary points of luminescence on their surface. Lymphocytes with single, and later with multiple points, progressing to solid luminescence over the whole surface and, finally, cells with caps of luminescence appeared successively in the spleen during development of the fetus. The differences in the density of immunoglobulin receptors on the surface reflect the degree of differentiation of the lymphocyte.Laboratory of Embryonic Histogenesis, Research Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 1, pp. 38–40, January, 1980.     

Identification and purification of arginine deiminase that originated from Mycoplasma arginini.

A lymphocyte blastogenesis inhibitory factor, (LBIF), was purified from the culture supernatant of human histiocytic lymphoma U937 by fast protein liquid chromatography. In this study, we demonstrated, first, that LBIF originated from a mycoplasma, Mycoplasma arginini, infecting U937 cells, and second, that LBIF bore the arginine deiminase activity. The implication of in vivo immunosuppression induced by arginine-utilizing mycoplasma species is discussed.     

Distribution of Ia antigens and T lymphocyte subpopulations in rat lungs.

In order to examine the mechanism of specific immunity in the lung, the distribution of Ia antigens and T lymphocyte populations was determined using immunoperoxidase-staining of cryostat sections of lungs from specific pathogen-free rats. BALT was found to be divided into three regions of lymphoid tissue. The central region was primarily composed of B cells, and was surrounded by a peripheral region of T cells (MRC OX-19+) which included both T helper (W3/25+) and T suppressor/cytotoxic (MRC OX-8+) cells. The subepithelial region contained a dense network of W3/25+, non-T cells. A majority of BALT cells, including the lymphoepithelial cells, were Ia+. The alveolar walls were found to contain numerous Ia+ dendritic-shaped cells. Alveolar macrophages found in sections, as well as those collected using bronchoalveolar lavage, were Ia- and W3/25-. Mechanisms for the induction of immunity within both BALT and the alveolar region are proposed.     

Synthesis and migration of glycoproteins in cells of the rat thymus,as shown by radioautography after 3H-fucose injection

Young male rats received a single intravenous injection of ³H-fucose and were killed after various time-intervals. Light- and electron-microscopic radioautographic studies of the thymus in animals killed shortly after injection showed that all of the different cell types present incorporated ³H-fucose label. The heaviest uptake occurred in macrophages and in hypertrophic epithelial cells located near the cortico-medullary border. Somewhat lighter incorporation was observed in medullary and cortical stellate epithelial cells and in cells designated as special cells, while the lightest reaction appeared over lymphocytes. In all cells the label was localized initially to the Golgi apparatus, where, presumably, it was incorporated into glycoproteins. With time, some of the labeled putative glycoproteins in all cell types migrated to the plasma membrane. In macrophages, much of the label migrated to lysosomal bodies, while in the special cells the label migrated to dense bodies which may also be of lysosomal nature. In stellate and hypertrophic epithelial cells much of the label migrated to characteristic vacuoles. The possible relationship between the observed glycoprotein synthesis in these cells and hormone production is discussed.     

The fine structure of dense lysosomes isolated from rat spleen.

A dense fraction from rat spleen was shown to consist of membrane bound organelles of varying shape and size which were packed with electron dense particles with the appearance of ferritin. Electron microscope microanalysis confirmed that iron was present in the organelles and the amount correlated with the density as noted in transmission electron microscopy. The organelles also stained, positively for acid phosphatase and, therefore, confirmed the biochemical findings that dense lysosomes (L30) had been isolated. The role lysosomes play in the degradation of red blood cells, and as possible sites of iron storage, are briefly discussed.     

Development of lymphocyte populations in the human foetal thymus and spleen

T and B lymphocytes in the human foetal thymus and spleen were studied to determine the distribution and degree of development which takes place before exposure to environmental antigens occurs. Tests applied were spontaneous and complement-dependent rosette formation and immunofluorescence to detect surface immunoglobulins.Most thymus lymphocytes were spontaneous rosette-forming cells: the percentage of these cells in the spleen was lower. Complement receptor lymphocytes (CRL) were found in the spleen but not the thymus, suggesting that these tissues contain lymphocytes of different origin. Lymphocytes with surface immuno-globulin (SIg lymphocytes) were more numerous in the spleen than the thymus. Analysis of class-specific heavy chain and light chain determinants suggests that some foetal B cells carry heavy chains of more than one class. A possible model for foetal B-cell development and its relationship to antigen drive is discussed.