Identification of Ia glycoproteins in rat thymus and purification from rat spleen.

A fraction of la-like glycoproteins was prepared from rat thymocytes by lentil lectin affinity chromatography and gel filtration in deoxycholate. Spleen cells from mice immunized with this preparation were fused with myeloma cells to produce antibody-secreting hybrid cell lines. Antibody from four lines called MRC OX, 3, 4, 5, 6 reacted with the la-like glycoproteins, and MRC OX 3 antibody recognized an antigenie determinant polymorphic in the rat. All four antibodies also bound to mouse spleen cells and all detected polymorphisms. Studies on recombinant mouse strains suggest that the determinants are coded by the I-A subregion of the H-2 complex. MRC OX 3 correlates with Ia specificity 9, while MRC OX 4, 5, 6 correlate with specificity 17 or 18. MRC OX 4 monoclonal antibody was used for affinity chromatography to purify Ia glycoproteins from rat spleen. The rat Ia glycoprotein complex was composed of two noncovalently linked polypeptide chains of apparent mol. wt. (unreduced) 30 000 and 24 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The purified Ia glycoprotein partially inhibited the binding to thoracic duct lymphocytes of an alloantiserum which detects Ia antigens linked to the major histocompatibility complex. The monoclonal anti-la antibodies bound to the majority of peripheral B lymphocytes and 18% of thymocytes, but did not significantly bind to peripheral T lymphocytes. There were on average 150000 molecules of Ia glycoprotein per la-positive B lymphocyte, and 45 000 molecules per la-positive thymocyte. From the same fusion, another cell line was prepared called MRC OX 2 which secretes monoclonal antibody to a previously undefined thymus glycoprotein of apparent mol. wt. 60000. Preliminary studies showed that the antigen was expressed on all thymocytes and on peripheral B lymphocytes in smaller amounts. It was also present in brain, but not liver or kidney homogenate.     

The localization of Thy-1.1, MRC OX 2 and Ia antigens in the rat ovary and fallopian tube.

The localization of Thy-1, MRC OX 2 and Ia antigens as defined by monoclonal antibodies MRC OX 7, MRC OX 2 and MRC OX 6 was determined by an indirect immunoperoxidase technique on cryostat sections of rat ovaries. Thy-1 antigen was present significantly in the theca interna of growing antral follicles. Developing corpora lutea exhibited an increasing presence of Thy-1 antigen and it was still present in degenerating ones. Thy-1 antigen was constantly present in fallopian tube tunica propria. The MRC OX 2 antigen was expressed most on ovarian structures that do not develop further, i.e. granulosa of degenerating antral follicles and third generation of corpora lutea. MRC OX 2 antibody stained the capillaries of the fallopian tube; the most heavily MRC OX 2+ were the cells of ovarian germinal epithelium. The Ia+ cells were occasionally found within the growing ovarian structures but they were more frequent in degenerating ones. Rare or no la+ cells within the ovary and heavily Ia-depleted thymus medulla and Ia areas in the spleen were, however, observed in some rats. The role of these antigens with respect to the structures they label is discussed.     

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.     

The origin of Ia antigen-expressing cells in the rat kidney.

The lineage of Ia antigen expressing (Ia+) cells that have been detected in the parenchyma and interstitium of the rat kidney has not been defined. The authors have studied the origins of Ia+ cells in chimeric rats using monoclonal antibodies to define cells of bone marrow and parenchymal origin. PVGc RTI rats (recipients) received intravenously 2 X 10(6) bone marrow cells from F1 hybrid PVG RTIc/RTIu rats (donors) 1 day after 1000 rads whole body irradiation. Ia chimerism was monitored in blood and isolated glomeruli by immunofluorescence and in frozen sections by immunoperoxidase, using monoclonal antibodies MRC OX3 (anti-Ia RTIu), MRC OX4 (anti-RTIc and u), and MRC OXI (anti-rat leukocyte common antigen). In normal F1 hybrid kidneys, glomerular cell counts were as follows: OXI+, 7.19 +/- 0.23/gl; OX4+, 3.03 +/- 0.14; OX3+, 2.34 +/- 0.1 (76% detectable expression of RTIu). OXI+, OX4+, and OX3+ cells were codistributed in cells in the interstitium between renal tubules. Proximal tubules were weakly OX4+, OX3+. In chimeric rats 5 days after irradiation, blood leukocytes, and renal OX1+ and OX4+ cells were depleted; OX3+ cells were not detected; by 4 weeks blood leukocytes were restored to normal numbers, and 85% of Ia+ cells were OX3+. By 6 weeks OXI+ and OX4+ cells were restored in glomeruli and interstitium, with increasing expression of OX3+ cells; at 10 weeks 75% of glomerular Ia+ cells were OX3+ (equivalent to detectable level of OX3+ cells in normal F1 hybrids) and OX1+, OX4+, and OX3+ cells appeared in equivalent numbers in the interstitium. Groups of proximal tubules were OX4+ and OX3-. These results in established bone marrow chimeras show that in the normal rat kidney bone marrow derived leukocytes expressing Ia antigen are present in the glomerulus and interstitium. Ia antigen is also expressed on some proximal tubular cells. There is no evidence for endothelial Ia positivity.     

A monoclonal mouse anti-rat Ia antibody which cross reacts with a human HLA-DRw determinant

A mouse monoclonal antibody, called MRC OX 3, which detects a polymorphic Ia determinant in the rat and cross reacts with an Ia determinant coded for by the I-A subregion in the mouse, detects a polymorphic determinant on human B cell lines. MRC OX 3 antibody binds to human B cell lines which express HLA-DRw specificities DRw 1, DRw2 and DRw6 but does not bind to those cells which only express other HLA-DRw specificities. No significant binding of MRC OX 3 antibody to either normal or mitogen stimulated human peripheral blood leukocytes, some of which are typed as HLA-DRw1, DRw2 or DRw6, could be detected. The binding of MRC OX 3 antibody to a human B cell line could be completely inhibited by pre-absorbing the antibody with purified rat Ia antigen.     

In vivo induction of Ia antigen in resident leukocytes in the normal rat renal glomerulus

The normal rat glomerulus contains a mesangial population of mononuclear leukocytes, 35 to 70% of which express class II major histocompatibility complex antigen (equivalent to mouse Ia) Schreiner GF, Unanue ER: Lab Invest 51:515, 1984. Inducing factors for cellular expression of class II antigens in normal leukocytes are unknown. In this population, glomerular leukocyte origin and Ia expression have been studied in kidneys from bone marrow-irradiated Lewis rats transplanted into normal syngeneic recipients. Leukocytes (LC+) and Ia positive (Ia+) cells were enumerated in isolated glomeruli by immunofluorescence using monoclonal antibodies to rat common leukocyte antigen (MRC OX1) and rat Ia antigen (MRC OX3, and OX4). Irradiation to donors caused extreme depletion of glomerular LC+ cells. Twenty-four hours after renal transplantation into normal recipients the glomerular LC+ population was restored to 65% of normal, and by 3 days was complete. In contrast Ia+ cells which were also depleted by irradiation, remained low 24 hours post-transplantation and only reached normal levels after 4 days. Thus, Ia antigen expression is induced in a population of leukocytes derived from circulating mononuclear leukocytes after a residence time of 3 to 4 days in the glomerulus. While the glomerular factors responsible are unknown, these experiments provide the first evidence for in vivo basal Ia induction in a normal resident mononuclear leukocyte population.     

Non-activated guinea-pig T cells and thymocytes express Ia antigens: FACS analysis with alloantibodies and monoclonal antibodies.

Conventional alloantisera and monoclonal antibodies to guinea-pig Ia antigens were used for analysis of Ia expression by guinea-pig T cells and thymocytes. Indirect immunofluorescent staining was performed with alloantisera or with ascitic fluid as a source of monoclonal antibody followed by flow microfluorometry analysis on the fluorescence activated cell sorter. About 80% of normal, non-activated peritoneal exudate T cells, lymph node T cells and thymocytes expressed Ia antigens. These data are therefore in contrast to studies with human or murine T cells where Ia antigens were shown to be expressed predominantly on activated but not on non-activated T cells. All the reactivity of the anti-Ia alloantisera for strain 2 T cells could be removed by absorption with an Ia-bearing B cell leukaemia, EN-L2C, but not by its Ia-negative variant, BZ-L2C. Thus, the Ia determinants identified on T and B cells are probably identical. One monoclonal antibody, 25E3, which had previously been shown by serologic analysis to react exclusively with an alloantigenic determinant of strain 2 Ia antigens displayed an unusual pattern of reactivity in that it clearly stained strain 13 thymocytes, but not mature strain 13 T or B lymphocytes. The significance of this possible expression of inappropriate Ia determinants by thymocytes remains unclear. This phenomenon might be associated with differentiation processes in the thymus.     

Reactivity of monoclonal antibodies against human leucocyte antigens with lymphocytes of non-human primate origin

The phylogenetic distribution of antigens present on human lymphocytes was investigated by incubating human or simian cells with murine anti-human monoclonal antibodies and then determining the level of reactivity with a radiolabelled anti-murine IgG reagent. The monoclonal antibodies used were specific for a T-cell antigen, lymphoid and lymphoid:myeloid antigens, Ia antigens, and beta 2 microglobulin. The cells examined included B- and T-lymphoblastoid cell lines and fresh peripheral blood lymphocytes separated by sheep erythrocyte rosetting into T-cell and non T-cell fractions. Results of these studies showed that the antibodies gave complete cross-reactivity with gorilla and chimpanzee cells while B-cell lines of orangutan origin had lost lymphoid and beta 2 microglobulin markers. Gibbon cells and cells of Old World and New World monkeys reacted strongly only with monoclonal antibodies against Ia antigenic determinants. These Ia antigens were found on the non T-cell fraction of fresh peripheral lymphocytes, on B-cell lines and on some virus induced T-cell tumour lines. Immunoprecipitation analysis using the anti-Ia antibodies showed a degree of molecular diversity on owl monkey and marmoset cells compared to the Ia antigens associated with human cells.     

Differential expression and serologically distinct subpopulations of human Ia antigens detected with monoclonal antibodies to Ia alpha and beta chains

Studies with two monoclonal antibodies (DA6.147 and DA6.231) which react, respectively, with isolated human Ia alpha and beta chains are reported. Both antibodies detect epitopes expressed on all DR-heterozygous and DR-homozygous cell lines tested (n = 17) and bind to the Epstein-Barr virus-negative cell line Ramos. Ia subunit specificity of the antibodies was determined by an adaptation of an electroblot technique which transfers separated Ia chains from polyacrylamide gels to nitrocellulose paper. In radioimmunobinding assays, peripheral blood B cell-enriched fractions, phytohemagglutinin-activated T cells and pokeweed mitogen-activated cells gave strong reactions with DA6.231 (anti-Ia beta). In contrast, DA6.147 (anti-Ia alpha) reacted only weakly, if at all, with peripheral B cells, pokeweed mitogen blasts and activated T cells. However, both antibodies bound to isolated Ia from activated T cells and peripheral B cells after Nonidet-P40 solubilization of the cells and DA6.147+ antigens could be found in the cytoplasm of activated T cells by indirect immunofluorescence techniques. Results of serological inhibition procedures following fractionation of lymphoblastoid cell lysates on monoclonal antibody affinity columns showed that the DA6.147 alpha chain epitope is carried on only a minor subpopulation of human Ia.     

The molecular basis of T helper cell function--I. Allotype- and MHC-linked determinants on antigen-specific, H-2-restricted T cell lines, hybridomas and lymphomas

A T-cell hybridoma clone, which produces antigen-specific helper factors and a T-cell lymphoma clone which produces non-specific helper factors was used to study the expression of T-cell allotypes and Ia antigens. Use was made of rabbit antisera against isolated T-cell receptor material and of monoclonal mouse antibodies against isolated rat Ia antigen. The rabbit antisera detected endogenously produced determinants both on the membrane and on intracellular polypeptides of these cells. The monoclonal mouse anti-rat-Ia antibodies detected polymorphic determinants on mouse Ia antigens and reacted with endogenously produced molecules on the membrane and on intracellular molecules of the hybridoma and lymphoma cells. The molecules carrying Tcr allotypes were single-chain polypeptides with mol. wts of 60,000-70,000 and the molecules carrying Ia-like antigenic determinants were single-chain polypeptides with mol. wts of 40,000-50,000. Thus T-cell allotypes and Ia antigens were found on separate polypeptide chains. The role and genetic localization of allotype-like and Ia-like molecules in T-cell products is discussed.     

Biochemical variation of human Ia like antigens detected with monoclonal antibodies

Four mouse monoclonal antibodies to human B cell surface determinants previously described as being directed against Ia like (MHC class II) antigens, have been shown to precipitate Ia alpha and beta chains. Electrophoretic transfer experiments showed one antibody to be directed against Ia alpha chains and two others to be against Ia beta chains. The antibodies were then used to analyse a range of cell types and a large number of lymphoblastoid and lymphoma cell lines. Ia antigens could not be detected on peripheral blood T cells, cord endothelium or T cell lines but their presence was confirmed on activated T cells and peripheral blood non-T cells. There was both qualitative and quantitative variation of Ia like antigen expression on B cell lines, including an apparent genetic polymorphism in alpha chain structure unrelated to DR allotypes and a single instance of a beta chain of abnormally high molecular weight.     

Antigenic phenotyping of isolated and in situ rodent follicular dendritic cells (FDC) with emphasis on the ultrastructural demonstration of Ia antigens

The antigenic phenotype of mouse lymph node follicular dendritic cells (FDCs) was studied by immunocytochemical techniques. Indirect fluorescence was used in conjunction with monoclonal antibodies to localize FDC surface antigens on FDC-enriched cell preparations and in cryostat sections. Lymph nodes from rats and mice were also labeled directly for Ia antigens with fluorescein- or peroxidase-conjugated Ia-specific monoclonal antibodies (i.e., MRC Ox4 and 10-2.16, respectively). Lymphoid tissue was also prepared for electron microscopy to allow clear distinction between Ia antigens of B lymphocytes and FDCs in situ. In these experiments, gold-labeled antigen was used to clearly identify FDCs and their processes among the Ia-positive cells of lymph node follicles. The labeling observed by light and electron microscopy showed that FDCs expressed Ia in situ and in vitro. Additional surface determinants shown to be expressed by FDCs included H2-K, common leukocyte antigen, and the receptor for the Fc portion of IgG1 and IgG2b. Neither macrophage antigens, such as Mac-1, Mac-2, Mac-3, and F4/80, nor the lymphocyte markers Ly-1, Ly-2, and Thy-1 were expressed by FDCs. Thus, the antigenic phenotype of FDCs, along with their distinctive dendritic morphology, their nonphagocytic and nonadherent nature, and their ability to trap and retain immune complexes on their plasma membrane, identifies them as a unique cell population.     

Species cross-reactive Ia determinants: detection with a monoclonal antibody to human Ia of a murine Ia. 7 epitope

A.TH anti-A.TL alloserum has previously been shown to react with monomorphic determinants of human Ia molecules. In an attempt to produce monoclonal antibodies detecting human-mouse cross-reacting epitopes, A.TH mice (Is) which lack I-E antigens were immunized with human Ia glycoproteins. Five hybridoma lines were established which recognize monomorphic human Ia determinants. Only one of the lines, 21w4, is reactive with murine cells and also with pig and sheep cells but not with rat cells. Binding studies to murine target cells show a strain distribution analogous to that of an Ia. 7-like specificity. The binding of 21w4 antibody to human and murine cells was compared to that of two other monoclonal antibodies directed to the murine Ia. 7-like specificity. Although all three antibodies had a similar pattern of reactivity with cells of different mouse strains, only 21w4 bound to the human cells tested. Competitive inhibition studies on murine cells have revealed that the epitopes were spatially related but not identical. Our results support the view that Ia. 7, the putative species cross-reacting specificity, might be composed of clusters of epitopes with variable degrees of conservation throughout evolution.     

Heterogeneity of human peripheral blood mononuclear cells detected by monoclonal antibodies to monomorphic determinants of human Ia antigens

The reactivity patterns of several monoclonal antibodies specific for monomorphic determinants of human Ia antigens were studied using flow cytometric techniques. We observed differential reactivity of these antibodies with human lymphoid cell lines, normal fresh human mononuclear cells, and lymphoblasts from PHA-activated cultures. The molecular heterogeneity of Ia antigens previously identified with immunochemical techniques was accompanied by heterogeneity of cell surface expression as identified by an immunofluorescent probe. The determinants identified by these anti-Ia monoclonal antibodies may provide useful markers in the isolation of cellular subpopulations responsive in the immune system.     

Two subsets of rat T lymphocytes defined with monoclonal antibodies

A new monoclonal mouse antibody that recognizes a subset of rat peripheral T cells has been prepared by immunizing mice with rat thymocyte glycoprotein. This antibody, designated MRC OX 8, labels all peripheral T cells that are unlabeled by the previously described W3/25 monoclonal antibody. No peripheral T cells were found that bound both antibodies, but, in contrast, 90% of thymocytes were doubly labeled. Thoracic duct lymphocytes of congenitally athymic nude rats were not labeled by either antibody, but the spleens of such animals contained both W3/25⁺ cells and MRC OX 8⁺ cells. These splenocyte subpopulations did not overlap. Using the fluorescence-activated cell sorter to isolate cells binding MRC OX 8 antibody, the phenotype of T cells mediating various T cell functions was established. Combining the present results with those published previously, it is shown that the cells providing help for antibody responses and those mediating graft-vs. -host reactions are phenotypically W3/25⁺ MRC OX 8^?. On the other hand, parental T cells that suppress antibody formation in F₁ hosts were identified as W3/25^? MRC OX 8⁺. The relationship between the rat T cell subsets defined by these antibodies and those in the mouse identified by the Ly series of alloantibodies is discussed and a comparison made between the rat W3/25⁺ subset and a recently identified human T cell subset.     

Cytoplasmic as opposed to surface Ia antigens expressed on human peripheral blood lymphocytes and monocytes

Peripheral blood leucocytes were examined for the presence of HLA-Dr or Ia-like antigen on the cell surface and in the cytoplasm. Surface staining of viable cell suspensions, using monoclonal anti-Ia antibody in both immunofluorescence and immunoperoxidase methods gave similar results. Using a newly developed immunoperoxidase double staining procedure, which stains the cells when either viable or fixed, both the surface and cytoplasm of the individual cells were labelled and examined. Twenty to thirty percent of the mononuclear cells were positive for Ia staining on the cell surface and in the cytoplasm. Morphologically these positive cells were identified as both lymphocytes and monocytes. A small percentage (5-9%) of these monocytes and lymphocytes consistently labelled only cytoplasmic Ia determinants. In contrast, only surface binding was observed with monoclonal antibodies recognizing T cell subpopulations. The ability to detect intracellular Ia antigenic determinants is of value in distinguishing stages of lymphocyte and monocyte development.     

Role of Ia antigens in the human autologous mixed lymphocyte reaction

The role of Ia antigens in the human autologous mixed lymphocyte reaction (MLR) was analyzed. To do this, different mononuclear cell populations were fractionated according to their Ia antigen expression and used as stimulating cells in autologous MLR. Also examined was the effect of two hybridoma monoclonal antibodies specific for human Ia molecules on the autologous MLR. The results show that the stimulating capacity of peripheral blood mononuclear cells is restricted to Ia-bearing cells. While peripheral blood T cells are unable to function as stimulating cells, highly purified Ia+ mixed lymphocyte culture-activated T cells are very effective. The possibility that Ia molecules are directly involved in the autologous MLR is further substantiated by the strong inhibitory effect of monoclonal anti-Ia antibodies.     

Expression of Ia like (HLA-DR) antigens on human alveolar macrophages.

The distribution of Ia like (HLA-DR) antigens on human alveolar macrophages (HAM phi) has been investigated by indirect immunofluorescence staining of viable macrophages with a panel of monoclonal antibodies (MoAb) to common determinants of these antigens. HAM phi were characterized by non-specific esterase stain, plastic adherence, phagocytosis and IgG-Fc receptor expression. Ia like antigens were expressed in approximately 45-80% of HAM phi, being localized as patchy and lineal fluorescence along the membrane. Ia like expression was higher in macrophages from non-smoker subjects (P less than 0.025). No difference in Ia like antigen expression was found between adherent and non-adherent HAM phi subsets. Ia like positive HAM phi from both smoker and non-smoker subjects consisted of a large subpopulation of phagocytic cells (60-70%) and a smaller non-phagocytic subpopulation (20-25%). These subpopulations were also present in the Ia like negative HAM phi. The percentage of Ia like positive macrophages showed variable results depending on the MoAb used, suggesting that not all anti-Ia like antibodies recognize the same antigenic determinants. Moreover, lack of staining of one macrophage subset occurred with all MoAb tested, over a large range of concentrations.     

Relationship between histocompatibility antigens, other surface determinants and the IgE receptor on rat mast cells.

Rat alloantibodies recognizing classical transplantation antigens (CTA) or non-H-1 determinants were able to compete effectively with monomeric IgE or IgG-coated sheep erythrocytes for receptor sites on the rat mast cell surface. Inhibitory capacity, however, was entirely confined to anti-CTA antibodies of the IgG2a subclass, whereas IgG1 antibodies lacked this ability. Analogously, F(ab')2 fragments of anti-CTA antibody consistently failed to affect IgE binding, but exposure of cell-bound F(ab')2 to anti-rat IgG restored its inhibitory capacity. From these results it was concluded that receptor sites recognizing the Fc portion of the anti-CTA molecule are involved in the inhibition process. Based on a cytotoxicity assay and on comparative absorption studies on alloantisera, the existence and relative amount of CTA and I region-associated antigens on purified rat mast cells and lymph node cells were analyzed. Whereas the CTA concentration per unit surface area on both cell types was very similar, rat mast cells consistently lacked Ia antigens.     

Role of Ia antigens and interleukin 1 in T-cell proliferation to phytohemagglutinin

Highly purified human T cells were obtained by a four-step purification procedure which included: removal of plastic adherent cells, rosetting with sheep red cells, passage over nylon-wool columns, and treatment with mouse monoclonal antibodies to human Ia antigens and complement. The resulting T cells did not proliferate to phytohemagglutinin (PHA). Purified human interleukin 1 (IL-1) could not substitute for accessory cells in supporting a PHA response. Reconstitution with as little as 0.03% adherent cells resulted in a proliferative response to PHA. T-Cell proliferation to PHA was supported by monocytes, by Ia+ Epstein-Barr virus-transformed lymphoblastoid B-cells lines, and by Ia- cultured human dermal fibroblasts but not by Ia-containing liposomes. Addition of anti-Ia antibodies to monocyte-containing cultures did not inhibit the T-cell proliferative response to PHA. These results suggest that Ia antigen expression by accessory cells is neither necessary nor sufficient to support T-cell proliferation to PHA and that IL-1 is not sufficient to support the proliferation of T cells to PHA.