Production of Specific Antisera to Human B Lymphocytes

Antisera have been prepared, in mice and rabbits, to membrane and sub-membrane fractions of human B lymphocyte derived lymphoid lines. Antisera to a protein subfraction were, after only minimal absorption, specific for human peripheral B lymphocytes, monocytes and B cell derived lymphoid lines. The antigen(s) recognised by these antisera were not the same as the previously described B-cell markers; immunoglobulin, Fc receptor, complement receptor and Ia antigens. The antigen(s) could not be removed from cells by lysostrip with anti- β₂ microglobulin.     

Pharmacological modification of immunoregulatory T lymphocytes . II. Modulation of T lymphocyte cell surface characteristics.

Human peripheral blood lymphocytes were fractionated into non-T lymphocyte, T lymphocyte, theophylline resistant (TR) and theophylline sensitive (Ts) T lymphocyte subpopulations. The proportion of cells bearing surface membrane immunoglobulin (sIg), Clq, Ia antigen, beta 2 microglobulin and T lymphocyte specific antigens detected by monoclonal antibodies OKT3, OKT4, OKT5 and OKT8 was studied using immunofluorescent techniques. Incubation of T lymphocytes or TR lymphocytes with adenosine or impromidine, an H2 histamine agonist, under conditions previously shown to increase Fc gamma receptors and radioresistant suppressor cell activity, was found to increase the proportion of cells expressing readily detectable surface beta 2 microglobulin and the antigen detected by OKT8. Cells expressing OKT4 antigen declined and there was no change in OKT3, OKT5, Ia, Clq, sIg or Es receptor expression. These data indicate that the expression of T lymphocyte Fc gamma receptors, beta 2 microglobulin and the antigens detected by the monoclonal antibodies OKT4 and OKT8 are, at least in part, regulated by agents acting upon adenosine and H2 histamine receptors.     

Surface markers of primate B and T lymphoid cell lines identified by antibodies to human and simian lymphocyte antigens

Simian B and T lymphoid cell lines were shown to maintain surface markers found on mature lymphocytes in vivo. The T lymphoid cell lines expressed Ia-like antigens on their surfaces, further suggesting that they represent mature, activated T cells. These Ia antigens show a structural similarity to Ia on human cells although some diversity exists. The Ia antigen expressed on T lymphoid cell lines was shown to be very similar to those on B lymphoid cell lines. Owl monkey and marmoset T lymphoid cell lines were also shown to express a VH immunoglobulin-related determinant, a marker which is thought to be associated with T cell antigen receptor. Owl monkey and marmoset T cell lines express a surface antigen which identifies the sheep erythrocyte receptor on human T cells and some of these lines express an antigen found on human helper T cells. It is noteworthy that substantial conservation of surface components has occurred within primate evolution such that monoclonal antibodies to human Ia, OKT-11a and Leu 3a markers can be used to type lymphocytes of lower primates.     

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.     

Allorecognition of HLA DR2 and DR5 Molecules by V-Beta-8-Positive T-Cell Clones

V-beta-8-positive T cells were isolated from human peripheral blood lymphocytes using a monoclonal antibody specific for the V beta 8 family. Alloreactive T-cell lines were generated by stimulation with mononuclear cells from individuals homozygous for HLA DR1-DR9. Cloned V beta 8-positive T cells were then assayed for alloreactivity based on a proliferative assay using irradiated B-cell lines. V beta 8-positive T-cell clones alloreactive to DR2 and DR5 molecules were chosen for further study based on the association of these MHC antigens with autoimmune disease. DNA sequence analysis confirmed the use of the V beta 8 gene family as well as providing information on the use of the V, D, J and N segments in these alloreactive T-cell clones.     

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.     

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.     

Thyroglobulin-induced T-cell in vitro proliferation in Hashimoto's thyroiditis: identification of the responsive subset and effect of monoclonal antibodies directed to Ia antigens

Recently it was reported that the peripheral blood and thyroid gland of patients with Hashimoto's thyroiditis contain activated (Ia+ and/or MLR4+) T cells and high levels of 5/9+ ("helper") T lymphocytes. In normal individuals the 5/9 monoclonal antibody recognizes a T-cell fraction that includes all T lymphocytes with inducer activities. Here, circulating 5/9+ and 5/9- T lymphocytes were isolated from patients with Hashimoto's disease, and the proliferative response induced by human thyroglobulin was investigated. The results show that the total thyroglobulin-induced lymphocyte DNA synthesis is confined to the 5/9+ T-cell fraction. Further subfractionation of 5/9+ into MLR4+ and MLR4- cells clearly indicates that no substantial differences exist in their proliferative capacities. Whether 5/9, MLR4, and Ia antigens, all expressed on the thyroglobulin-responsive T-cell subset, are involved in thyroglobulin-induced cell proliferation, was also analyzed. Although both 5/9 and MLR4 monoclonal antibodies had no effect, complete inhibition of antigen-induced blastogenesis was observed upon addition of monoclonal antibodies (D1/12 and BT2/9) directed to common determinants of Ia antigens. This inhibitory effect was also observed when T or non-T fractions were separately incubated with the monoclonal antibodies before culture. These results indicate that in humans, as in animals, the major histocompatibility complex may play a role in autoimmune thyroiditis. The data show that (a) the thyroglobulin-induced proliferative response is confined to a subset (5/9+) of T lymphocytes and (b) Ia antigens are involved in thyroglobulin-induced lymphocyte DNA synthesis in Hashimoto's disease.     

Recovery of Peripheral Lymph Cells from Congenitally Athymic Nude Rats

Thoracic-duct cannulation of mesenteric lymphadenectomized (MLNx) congenitally athymic nude rats was studied as a method of obtaining peripheral lymph cells. A higher recovery of non-lymphoid cells (NLC) was obtained from nude than from euthmyic littermates. Both a higher percentage and a greater number of NLC were found in nude animals. Most of these cells resembled dendritic or veiled cells and were strongly positive for Ia antigens. This population could further be enriched by irradiation of the animal, but with a risk of cell damage. Splenectomy had no effect on early output of Ia+ NLC. A substantial population of lymphoid cells from MLNx nude rats expressed T-cell antigens defined by the monoclonal antibodies OX 19, OX 8 or W3/25. These cells were more radiosensitive than were mature T cells. In addition, a large population of cells in the peripheral lymph from nude rats did not display surface antigens recognized by monoclonal antibodies directed either against B cells or against T cells. This cell fraction increased after irradiation. These cells resembled small lymphocytes but had a more irregular nucleus and multiple large granules.     

Monoclonal anti-T-cell antibodies react with circulating myeloid leukemia cells and normal tissue macrophages

Rabbit and monoclonal antibodies to human myeloid leukemia cells, monocytic leukemia cells and human thymocytes have shown the existence of common T-cell/myeloid/monocyte antigens. For this reason, the specificity of a series of monoclonal antibodies to human T-cells (OKT 1, 3, 4, 5, 6, 8, 9, 10; and NA1/34) was tested by immunofluorescence (cytofluorograph) and complement-mediated cytotoxicity against human myeloid leukemia and normal blood cells and leukemic cell lines. In addition, an immunohistological analysis of the specificity of OKT4, 9.3, Leu 3a, OKT3 and NA1/34 antibodies was performed using normal lymphoid tissues and a sensitive immunoperoxidase technique. Normal human peripheral blood mononuclear cells reacted with OKT3 ("pan T-cell", mean 54%), OKT4 ("helper T-cell", mean 35%) and OKT 5/8 ("suppressor T-cell", mean 18%) as previously reported. However, OKT3 reacted with the cell lines K562 (myeloid), RC2a and THP-1 (monocytoid) and U937 (macrophage) as well as with cells from 9/65 myeloid leukemia patients. OKT4 reacted with the cell lines HL60 (promyelocyte), RC2a and U937 and also with cells from 6/60 myeloid leukemia patients. OKT5 reacted with the cell lines K562 and THP-1. OKT1 ("pan T-cell") reacted with THP-1 and with myeloid and monocytic leukemia samples (5/32) as did OKT6 ("cortical thymocyte") (3/32). OKT10 ("common thymocyte") reacted with a range of leukemia cell lines (B-cell, pre- B-cell and macrophage) as well as 7/21 myeloid leukemia samples. In tissue sections Leu 3a, (9.3 and OKT4 to a lesser extent), stained paracortical lymphocytes, plus subcapsular and medullary macrophages, and dendritic cells present within the paracortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical and ultrastructural characterization of human T-lymphotropic virus type I (HTLV-I)-producing rabbit lymphoid cell lines

Summary Fine structural and immunocytochemical characterization of rabbit lymphoid cell lines transformed by human T-lymphotropic virus type I (HTLV-I) was carried out. All nine cell lines tested were reactive with anti-HTLV-I-positive human, monkey, and rabbit sera and monoclonal antibody to HTLV-Ip 19, but not with anti-HTLV-I-negative sera and monoclonal antibodies to human Ia and pan-T antigens. All cell lines were strongly positive for monoclonal antibodies to rabbit Ia and pan-T antigens. Ultrastructurally, each cell line contained C-type virus particles in varying numbers in the extracellular space. These particles showed replication patterns similar to those in HTLV-I or simian T-lymphotropic virus type I (STLV-I)-producing human or monkey cells. In addition, anti-HTLV-I-positive rabbit serum gave positive immunoreactivity to HTLV-I or STLV-I by indirect immunoferritin method.These results indicate that the ultramorphology and replication patterns of HTLV-I in rabbit cell lines are indistinguishable from those of HTLV-I in human and monkey cell lines, HTLV-I in rabbit cells shares the common surface antigenic determinants with HTLV-I or STLV-I in human or monkey cells, and that these cells are definitely rabbit T cells bearing their own Ia antigens.     

Monoclonal antibodies define the characteristics and cellular distribution of an Ia-associated protein

In a recent report we described the identification of physical associations between Major Histocompatibility Complex (MHC) Class II (Ia) antigens and other structures of Mr 67,000, which were significantly enhanced following brief T-B cell co-culture (1). To further investigate this 67K Ia-associated product, monoclonal antibodies (MAb) were produced against isolated 67K material and their reactivity examined. Cell surface binding by these MAb was detected only after perturbation of the membrane by cellular adherence or following aldehyde fixation, which indicates that the determinant recognized by these mAb is retained in the plasma membrane in a covert fashion. All lymphoid cells tested showed reactivity with the MAb as determined by immunofluorescence and by ELISA, but no binding was detected on bone marrow or peritoneal macrophages. Expression of the antigen reactive with these antibodies followed a similar pattern with established murine cell lines, with T and B cell lines and a pre-B cell line showing reactivity, while no antigen was detected on macrophage-like and fibroblast cell lines. The intensity of antigen expression by normal lymphoid cells was ordered: thymocytes greater than splenic T cells greater than or equal to bone marrow lymphocytes greater than splenic B cells. No correlation was observed between expression of Ia antigens by non-lymphoid cells and expression of the 67K molecule. These observations suggest that this antigen is primarily a marker of lymphoid cells, with the highest expression on cells of the T lymphocyte lineage. Finally, inhibition of antigen-specific, MHC-restricted T-cell activation by the MAb directed against the 67K structure suggests an important functional role for this interesting molecule originally identified by its physical association with Ia following T-B cell interactions.     

Detection and Quantitation of Human Ia-Type Antigens with Iodinated Protein A and Specific Purification of Antibodies Against Ia-Type Alloantigens

Antibodies directed against specific human Ia-type antigens can easily be detected and quantitated by an improved radioimmunoassay using iodinated protein A bound to a specific antibody-Ia-antigen complex on the surface of freshly drawn peripheral human leukocytes, cultured human cell lines, or lymphoid cells fixed with glutardialdehyde or formaldehyde. The same principle can also be used for the detection of Ia alloantigens on human lymphocytes when testing them with specific antisera known to contain antibodies against transplantation antigens. These anti-Ia-alloantigen antibodies had been purified by a two-step procedure involving ion-exchange chromatography on DEAE-cellulose at pH 6.3 and the specific absorption on formaldehyde-fixed Ia-alloantigen-carrying homozygous cell lines, followed by elution of these antibodies with isotonic citrate buffer at pH 3.0. In this way an about 90-fold purification could be achieved. After such a purification the highly enriched antibody fraction still reacted selectively with one specificity of the Ia antigen system.     

Immunoultrastructural and morphometric analysis of B lymphocytes in human germinal centers. Evidence for alternate pathways of follicular transformation.

Monoclonal antibodies to B-cell differentiation antigens B1, B2, C3b, and Ia were used for ultrastructural characterization of B lymphocytes undergoing follicular transformation in human germinal centers. Morphologic alterations and morphometric parameters including form factor (FF) and nuclear contour index (NCI) were evaluated. Antibodies to B1, Ia, and C3b revealed uninterrupted linear surface membrane staining in B cells at various stages of transformation, while staining for B2 appeared as aggregates of gold particles localized to sites of antigen expression along the cell membrane. B cells with highly irregular or convoluted nuclei (NCI greater than 6.5) formed 3% of follicular lymphocytes and may explain the derivation of rare follicular center cell lymphomas with marked nuclear irregularity which mimic T-cell lymphomas histologically. Cleaved cells (NCI greater than or equal to 4.5) comprised 48% of the cellular population and were present at all stages of transformation. Results of morphometric studies suggest that small cleaved cells (centrocytes) and noncleaved cells transform to large lymphoid cells (centroblasts) along parallel lines, and without following the sequential differentiation pathway suggested by Lukes and Collins.     

Characterization of rabbit cells by monoclonal and polyclonal antibodies.

Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a T-cell antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed.     

Immunoultrastructural localization of Ia antigens in human endometrium

The distribution of Ia antigens was studied at the light and ultrastructural levels in 34 proliferative and 16 secretory endometria with two monoclonal antibodies using an avidin-biotin-peroxidase complex method. The endothelial cells, many lymphocytes, and various monocytic-macrophagic cells in the endometrial stroma were Ia positive. Furthermore, Ia antigens were localized to normal endometrial epithelium in the proliferative phase, and focally in epithelium adjacent to stromal lymphoid aggregates throughout the cycle. Expression of Ia antigens in the secretory epithelium was focal or absent. At the ultrastructural level, Ia-positive epithelial cells exhibited staining on the plasma membrane, in free and membrane-bound ribosomes, rough endoplasmic reticulum, and occasional perinuclear cisternae. In the endothelial cells and lymphocytes, Ia antigens were also localized to the plasma membrane and rough endoplasmic reticulum. These findings indicate that endometrial epithelium expresses Ia antigens which may be regulated by endometrial lymphoid cells and/or hormones. The plasma membrane expression of Ia antigens by the epithelial cells of endometrium appears to result from active synthesis, and not merely from passive absorption of Ia antigens.     

Anti-Ia reactivity in sera of untreated patients with active Hodgkin's disease

The effect of sera from eight patients with Hodgkin's disease on the autologous and allogeneic mixed lymphocyte response of normal individuals was examined. Sera from three patients with active disease caused marked inhibition of both autologous and allogeneic mixed lymphocyte reaction without inducing significant reduction of the phytohemagglutinin-induced proliferative response. The inhibitory activity of Hodgkin's disease sera on the autologous mixed lymphocyte reaction was removed by adsorption with non-T, but not T, lymphocytes and it was correlated with the ability of such sera to block the binding of monoclonal anti-Ia antibody to Ia-positive target cells. Anti-Ia antibodies were detected in the same sera by double antibody radioimmunoassay and analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using 125I-labeled, partially purified, Ia antigens from two different human B-cell lines. This anti-Ia reactivity was strongly reduced or absent in sera taken from the same patients at the completion of multidrug chemotherapy.     

Diffuse malignant lymphoma with cerebriform nuclei: A B-cell lymphoma studied with monoclonal antibodies

A lymph node biopsy performed on a 55-year-old woman with asymptomatic generalized lymphadenopathy revealed a diffuse, malignant lymphoma composed of small to intermediate-sized lymphocytes with cerebriform-shaped nuclei; electron microscopy confirmed the nuclear complexity. The cerebriform nuclear configuration, coupled with an interfollicular pattern of nodal involvement with encroachment upon residual germinal centers, was presumptive of either mycosis fungoides or a peripheral T-cell lymphoma. Immunologic evaluation, however, indicated that the cerebriform lymphocytes represented a monoclonal B-cell population (IgM-IgD, lambda). Staining with monoclonal antibodies disclosed a phenotype of Ia+, B1+, BA-1+, BA-2+, Leu-1+; the neoplastic cells were unreactive with T-cell, lineage-specific antibodies (anti-Leu-2a, -3a, -4, -5) and with J5 (CALLA). In light of the immunophenotype and the distributional pattern, the cerebriform-shaped lymphocytes may represent an extreme morphologic variant of intermediate lymphocytic lymphoma.     

Transformation of human fetal thymus and spleen lymphocytes by human T-cell leukemia virus type I

Co-cultivation of human thymus and spleen lymphocytes, which were obtained from 26-week and 27-week fetuses, with a lethally-irradiated human cord T-cell line harboring human T-cell leukemia virus type I (HTLV-I) resulted in the establishment of T-cell lines positive for adult T-cell leukemia-associated antigens and producing HTLV-I. These cell lines had the phenotype of a helper/inducer subset of peripheral T-cells as evidenced by the reactivity with monoclonal antibodies to human T-cells.     

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.