Observations on rabbit thymocytes and peripheral T cells. I. Anomalous results in the mixed antiglobulin reaction caused by non-specific adsorption of sheep immunoglobulin.

The "single-stage" mixed antiglobulin reaction (MAR) was carried out with rabbit thymocytes. This test involved treating the cells with either sheep or goat anti-rabbit globulin sera, and subsequently reacting them with indicator erythrocytes coated with rabbit immunoglobulin (Ig) so as to form rosettes. An unexpectedly high number (up to 38%) of thymocytes reacted, although the rosettes were weaker than those given by peripheral B lymphocytes. When blood and lymph node lymphocytes or thymus cells which had already been treated with sheep anti-rabbit globulin serum were subsequently exposed to rabbit anti-sheep Ig serum and then rosetted with indicator cells coated with ox Ig (cross-reacts with sheep Ig) almost 100% reaction was obtained in each of the cell suspensions. This was designated the "two-stage" MAR. The anomalous results, both in the one-stage and two-stage MAR, were abolished by pepsin-treating the sheep anti-rabbit globulin serum; thus indicating that sheep Ig is adsorbed non-specifically via the Fc part of the molecules to the surface of rabbit thymocytes and peripheral T lymphocytes.     

Analysis of T-lymphocyte subpopulations in juvenile-onset diabetics.

The antigenic properties of leukaemic cells from five patients with adult T cell leukaemia were studied with rabbit anti-MOLT-4 and anti-human thymocyte antisera using indirect membrane immunofluorescent staining. The E rosette-positive, surface immunoglobulin (sIg) negative leukaemic cells from these patients gave a positive reaction with the appropriately absorbed antisera, which reacted specifically with thymocytes, cells from T cell acute lymphoblastic leukaemia (T-ALL) and T-ALL-derived lymphoblastoid cell lines (T-LCLs) and normal peripheral blood T cells. Nevertheless, the antisera further absorbed with fresh normal peripheral blood lymphocytes (FN-PBL) lost almost all the reactivities with the leukaemic cells as well as with normal peripheral blood T cells but still retained the reactivities with thymocytes, T-LCLs and T-ALL cells. The results suggest that adult T cell leukaemia cells possess a peripheral blood T cell antigen but not a thymocyte-specific antigen.     

Identification of canine lymphocyte populations by immunofluorescence surface marker analysis

A T-cell-specific rabbit anti-dog thymocyte antiserum was prepared and used in an indirect fluorescent antibody technique to identify T lymphocytes. The antiserum was rendered specific by absorptions with normal dog red blood cells, lyophilized serum, and bone marrow cells. By the indirect immunofluorescence assay, 73.6 +/- 1.2 (SEM)% of the peripheral blood lymphocytes were identified as T lymphocytes. By direct immunofluorescence assay of surface membrane immunoglobulin-positive lymphocytes, 18.2 +/- 0.8% of the peripheral blood lymphocytes were identified as B lymphocytes. The two assays used in conjunction can thus identify over 90% of the peripheral blood lymphocytes as either T or B lymphocytes, with the remaining 8.2 +/- 1.0% as null cells.     

Characterization of a novel bovine leukocyte protein involved in cell-cell adhesion

Abstract: Preliminary characterization of an apparently novel bovine leukocyte adhesion protein is described. Two IgG₁ monoclonal antibodies, UC-C1 and UC-H5, raised against established cultures of IL-2-dependent bovine peripheral blood lymphocytes (PBL) were found to react with an antigen expressed by the majority of bovine peripheral blood leukocytes. Immunoprecipitation and polyacrylamide gel electrophoresis of the antigen produced a distinct protein band of molecular weight 160 000, and additional diffuse protein bands of approximate molecular weight 180 000, 175 000 and 150 000. Two-color flow cytometric analyses showed that the antigen was expressed at low density on a small proportion of circulating B lymphocytes, but was highly expressed on all circulating T lymphocytes. The majority of monocytes and all granulocytes expressed the antigen at a density lower than that of T lymphocytes. Peripheral blood lymphocytes stimulated with concanavalin A had an approximately 3-fold increased expression of the antigen, which was apparent within 18 h and remained stable in long-term cultures. Expression of the antigen in thymus, analyzed by the immunoperoxidase technique, was predominantly restricted to thymocytes in the immediate subcapsular cortex and medulla; expression in lymph nodes and spleen was predominantly confined to lymphocytes in T-cell areas. Flow-cytometric analysis demonstrated that thymocytes and the majority of peripheral and mesenteric lymph node-derived T cells had relatively low surface density of antigen compared to circulating T cells. Binding of UC-C1 or UC-H5 to the antigen on lymphocytes induced homotypic aggregation. UC-C1 completely blocked binding of FITC-conjugated UC-H5 to blood mononuclear cells, suggesting that the antibodies recognize the same epitope or proximal epitopes.     

抗人淋巴细胞T8抗原样McAb的研制和初步鉴定

用杂交瘤技术,将经人外周血E花环阳性细胞免疫小鼠脾细胞与NS-1骨髓瘤细胞融合后,产生一分泌IgG_1亚型McAb杂交瘤株。其所分泌抗体经微量放射免疫测定及间接免疫荧光法分析,表明它只能与T细胞系、76％的胸腺细胞及22％的外周血T淋巴细胞反应,不与其它各种不同细胞反应。将此抗体所识别入外周血T淋巴细胞亚群与抗Leu-2a识别T8~+细胞、抗Leu-3a识别T4~+细胞比较,发现此抗体与抗Leu-2a识别同一群细胞。此抗体能从T细胞表面沉淀出30KD(还原条件)或78KD(非还原条件)分子,并完全阻断FITC标记抗Leu-2a与T细胞的结合,说明此抗体是识别T8抗原样的McAb。     

Analysis with monoclonal antibodies of human lymphoid cells forming rosettes with rabbit red blood cells.

Rabbit red blood cells have previously been shown to rosette with a subpopulation of thymocytes and with mitogen activated peripheral lymphocytes but not with unstimulated lymphocytes. Using monoclonal antibodies and double marker assays we studied the phenotype of these cells. In thymus, over 90% of rosetting cells express antigens of immature thymocytes (HTA1, OKT6). A proportion of the rosetting cells shows in addition antigens of mature thymocytes (OKT3, UCHT1). These cells probably correspond to a stage of intrathymic maturation between common and mature thymocytes. Virtually all rosetting cells are T cells and express an antigen related to T cell activation (TAC) when lymphocytes are activated by mitogens like PHA or Con A. Few rosetting cells are Ia positive. Two other antigens (OKT9, OKT10) known to be associated with proliferating and immature cells, are found in variable proportions on rosetting cells. After stimulation with allogeneic lymphocytes, fewer rosettes are detected than after stimulation by mitogens. Cells activated by a soluble antigen (PPD) and forming rosettes with rabbit red blood cells have a helper phenotype (Leu3a positive). Screening of leukaemia cell samples revealed that only cells from patients with T-ALL form rosettes with rabbit red blood cells. Rosette formation is almost totally inhibited by a polyclonal anti-thymocyte serum and two monoclonal antibodies (OKT11A,Lyt3) which have been shown to block rosettes with sheep erythrocytes.     

Rabbit leukocyte surface antigens defined by monoclonal antibodies

Several monoclonal antibodies (mAb) against rabbit leukocytes were characterized in binding and functional studies. mAb 1.24 stains thymocytes, bone marrow cells, peripheral T and B cells and blood monocytes. T cells express more 1.24 antigen than B cells. In the absence of added complement (C), mAb 1.24 inhibits alloantigen-, concanavalin A (Con A)-, and phytohemagglutinin (PHA)-, but not pokeweed mitogen (PWM)- or anti-immunoglobulin (Ig)-induced cell proliferation. It also strongly blocks anti-sheep erythrocyte plaque-forming cell responses. A second mAb, designated 4.B9, binds to 20% of thymocytes and to most, if not all, peripheral T cells and in vitro-activated T cell blasts. A third one, 10.B3, is reactive with the nearly entire thymocyte and a major peripheral T cell population. Two-color membrane immunofluorescence reveals the presence of a small population of peripheral blood leukocytes which bear surface Ig and are weakly stained by mAb 4.B9 and 10.B3. Without C, both 4.B9 and 10.B3 inhibit Con A- and PHA-induced mitogenesis, but have no effect on PWM-, antigen-, or alloantigen-induced cell proliferation. Depletion of 4.B9+ cells by panning or complement lysis completely abrogates proliferative responsiveness to antigen and alloantigen, significantly reduces responsiveness to the T cell mitogens Con A and PHA, but enhances that to the B cell mitogen anti-Ig. A fourth mAb, 12.C7, binds to 60% of thymocytes and to 10-30% of peripheral T lymphocytes at high-level fluorescence. T cell blasts obtained in mixed leukocyte reactions are partially stained by mAb 12.C7, while those obtained after Con A or PHA activation are not. In addition, mAb 12.C7 is completely unreactive with B cells or monocytes. Without complement, it does not seem to interfere with any of the in vitro functions tested. All antigens studied here do not appear to be expressed in nonleukon tissues, as they do not bind to erythrocytes and are absent from brain, heart, liver and kidney as shown by quantitative absorption analysis.     

Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens

We have prepared 2 mouse monoclonal antibodies which react with differentiation antigens on guinea pig lymphoid cells. Monoclone 5AB2 recognizes an antigen expressed on both T and B lymphocytes and absent on macrophages. It has proven useful in the preparation of populations of antigen presenting cells which are free of T and B lymphocytes. The second monoclonal, 8BE6, is specific for peripheral T cells and 10% of thymocytes. It reacts with a 68,000 dalton molecule which is also expressed on the guinea pig B cell leukemia, EN-L2C. 8BE6 has proven to be lytic for peripheral T cells in the presence of rabbit complement and has been used to deplete T cells from heterogenous cell populations.     

Characterization of two sheep lymphocyte differentiation antigens, SBU-T1 and SBU-T6

The monoclonal antibodies 25.91 and 20.27 define two lymphocyte cell surface antigens of sheep. 25.91 is reactive with 60-80% of lymphocytes and 98% of thymocytes, and only stains surface immunoglobulin-negative peripheral lymphocytes. 25.91 immuno-precipitates a 67,000 MW protein from lymphocyte lysates under both reducing and non-reducing conditions, whereas immunoprecipitation of thymocyte lysates reveals a 67,000, 62,000 MW complex. The tissue distribution and molecular weight analysis reported here for the antigen recognized by 25.91 indicate that this antigen is the sheep homologue of the human T1 and mouse Ly 1 antigens. The monoclonal antibody 20.27 is reactive with 80% of thymocytes and the majority of cell surface immunoglobulin-positive peripheral blood lymphocytes (B cells), but is unreactive with peripheral blood T cells. 20-27 also stains Langerhans cells in skin tissue sections and large dendritic-like cells in the paracortex sections and large dendritic-like cells in the paracortex of lymph node tissue sections. Immunoperoxidase staining of thymus tissue sections with 20.27 shows intense staining of cortical thymocytes and an absence of staining within the medulla. Molecular weight analysis of the 20.27 antigen reveals two major bands of 46,000 and 12,000 MW under both reducing and non-reducing conditions. The 20.27 antigen has properties resembling MHC class I-like antigens such as T6 in the human and TL in the mouse.     

Enumeration of T cells, B cells and monocytes in the peripheral blood of normal and lymphocytotic cattle.

The rosette-forming capacity of bovine peripheral blood lymphocytes (PBL) was determined with dextran and 2-aminoethylisothiouronium bromide (AET)-treated sheep erythrocytes (SRBC). Both dextran and AET-enhanced rosette formation; however, AET-treated SRBC detected a larger percentage of rosette-forming cells and thus was used in this study. The specificity of rosette formation by bovine thymus-derived (T) lymphocytes was shown by (1) demonstration of rosettes and surface-membrane immunoglobulins sIg) on different cells in PBL and nylon-wool fractionated lymphocyte populations and (2) rosette formation by a large percentage (83--90%) of thymocytes from three bovine foetuses and two 14-month-old heifers. A procedure was also developed to identify bovine monocytes by latex phagocytosis and 10--30% latex-ingesting cells were detected in PBL preparations isolated by Ficoll-Hypaque flotation. The frequency of sIg-bearing latex-ingesting, and sIg-bearing latex non-ingesting cells in bovine peripheral blood was also determined. These procedures were utilized to determine the distribution of T and bone-marrow derived (B) lymphocytes in peripheral blood of normal and lymphocytotic cattle. PBL from twenty normal cattle contained approximately 63% T and 11% B (sIg+ latex non-ingesting) lymphocytes. In peripheral blood of three cattle with persistent lymphocytosis, a prodromal stage of bovine leukaemia, the percentage of B cells was elevated approximately to 59% whereas T lymphocytes decreased to 35%, thus providing additional evidence that persistent lymphocytosis is a B-cell disease.     

Separation of lymphocyte subpopulations in carp Cyprinus carpio L. by monoclonal antibodies: immunohistochemical studies.

Lymphoid cell populations in various organs of the carp Cyprinus carpio L. were investigated using a series of mouse monoclonal antibodies raised against carp thymocytes and carp serum Ig. Clones have been designated as Ig+T+, Ig+T- or Ig-T+ on the basis of the reactivity with thymocytes and serum Ig in the enzyme-linked immunosorbent assay (ELISA) screening. Their reaction to the lymphoid organs of carp was investigated on cryostat sections and cytocentrifuge slides using immunoperoxidase techniques. IG+T+ clones could be subdivided into those that stained reticular fibres around blood vessels in various organs (R+) and those that did not (R-). The former stained most thymocytes and most peripheral lymphocytes as well as plasma cells whereas the latter did not stain cortical thymocytes and some peripheral lymphocytes. IG+T- clones were negative for thymocytes but positive for plasma cells and a certain population of peripheral lymphocytes. Ig-T+ clones reacted similarly to Ig+T+R- clones. It is concluded that fish lymphoid cell populations can be distinguished based upon differences in cell surface and/or cytoplasmic determinants. The monoclonal antibodies described can be used for further structural analysis of the determinants and for functional separation of T- and B-like cells in the 'lower' vertebrates.     

Effect of hydrocortisone on immune system of the lizard, II. Differential action on T and B lymphocytes

Lymphocytes of thymus, spleen, peripheral blood (PB) and bone marrow (BM) collected from adult lizards, were cultured for 24 hr in the presence of 10⁻³M hydrocortisone acetate (HC) in order to assess the effect of in vitro HC on lizard T and B cell viability. The results indicated that HC induced stepwise, time-dependent mortality of the majority of thymocytes carrying T cell specific antigen(s) (TSA), 30–50% of T cells of spleen, PB and BM, and of a proportion of splenic B lymphocytes. Administration of 1 mg/g body weight HC to adult . lead to depletion of all TSA⁺ thymocytes. In contrast, T lymphocytes in the peripheral lymphoid compartments revealed both sensitivity and resistance to HC; similarly, B lymphocytes constituted susceptible and resistant subpopulations.     

Heterologous specific antiserum for identification of human T lymphocytes*

Peripheral blood lymphocytes (PBL) from two patients with Bruton-type agammaglobulinaemia were used for the preparation of heterologous anti-human T cell-sera which were absorbed with cultured B lymphoblast cells, peripheral blood lymphocytes from a patient with chronic lymphatic leukaemia and `adherent' cells. Using multiple criteria, one antiserum (ATCS) was shown to be specific for T lymphocytes. This antiserum asserts the existence of human-specific T-lymphocyte antigen(s) (HTLA) and provides another method for identifying human T cells. In the presence of rabbit complement, ATCS was cytotoxic for 65·5% (range 49–78) of normal PBL and 97% of thymocytes (the latter cells having also a higher surface density of HTLA than PBL). The study of PBL from a variety of patients showed that the percentage of ATCS-sensitive cells was high in Bruton-type agammaglobulinaemia, variable from patient to patient and from time to time in common variable hypogammaglobulinaemia and generally low in active lepromatous leprosy, in patients under antilymphocyte globulin therapy and in chronic lymphatic leukaemia. Cultured lymphoblasts from various B cell lines or from a Burkitt lymphoma cell line were resistant to ATCS.     

Bovine pan T-cell monoclonal antibodies reactive with a molecule similar to CD2.

Monoclonal antibodies (mAb) CH128A and CH61A react with molecules of 50,000-60,000 MW. They are expressed by all T cells in cattle, comprising 44-69% of peripheral blood mononuclear leucocytes (PBM), the majority of lymphocytes in T-dependent areas of lymph node, and 75-80% of cells derived from the thymus including both cortical and medullary thymocytes. The molecule recognized by these mAbs is not expressed on B lymphocytes, monocytes/macrophages, or granulocytes. Both mAb inhibit spontaneous rosette formation by sheep erythrocytes and bovine lymphocytes. We postulate that these mAb see the bovine homologue of the human sheep red blood cell receptor CD2 and has been named BoT2.     

Restricted expression of a human T cell lineage membrane antigen

An antiserum raised against human fetal and childhood thymocytes (anti-Thy) and absorbed with peripheral lymphocytes (tonsil) detected an antigen(s) shared by thymocytes, T cell-acute leukemias, activated peripheral T cells and a subset of peripheral T cells. The antigen was expressed by the negative circulating T cell subset after mitogen activation of that separated population. The antigen was shown to be separate from the E rosette receptor, another anti-T cell serum detected antigen, and β₂-microglobulin; its expression was not related to a particular phase of the cell cycle. The results suggest that the antigen is expressed by T cells only under certain maturational and proliferative conditions.     

Two molecularly independent surface receptors identify bovine T lymphocytes

E rosette formation is a commonly used technique to identify T lymphoytes in many species; however, treatment of bovine E rosettes with rhodamine-conjugated F(ab′)₂ anti-immunoglobulin resulted in 10% (range 3–18%) of these cells exhibiting fluoresncence. The failure of E rosettes to identify only non-immunoglobulin bearing cells suggested that an alternative method for T lymphocyte identification was essential. Using a labeled heterologous T cell antiserum and peanut agglutinin (PNA), identical populations of bovine T lymphocytes were identified. Unique and molecularly independent cell surface receptors were detected by dual fluorescent staining experiments, differential inhibition of cellular binding of PNA by its carbohydrate ligand, and capping experiments. Moreover, both reagents bound to approximately 62% of peripheral blood lymphocytes (PBLs) and virtually all thymocytes. Use of either T cell reagent in combination with rhodamine-conjugated F(ab′)₂ anti-bovine immunoglobulin provided a rapid, simple and reliable method for simultaneous enumeration of bovine T and B cells and permitted the detection of rare cells (<1%) expressing both T and B cell markers. Approximately 90% of all PBLs were identified as either T or B cells.     

T lymphocytes modifying factors in sera of patients with Myasthenia gravis

The examination of patients with Myasthenia gravis revealed that before thymectomy the number of T and B cells in the peripheral blood did not deviate from the norm. It was also observed that their sera contained factor stimulating T lymphocytes to the spontaneous rosette formation, theta antigen generation in xynogeneic system and hydrocortisone-resistant thymocytes. No factor inhibiting the spontaneous rosette formation by T lymphocytes or causing the transmission from Thy-1.2+ cells to Thy-1.2-cells was detected in these sera. Thymectomy resulted in the immediate fall of T lymphocytes number in the peripheral blood of the patients examined. Besides, serum of most patients almost totally lost the stimulating factor. The fall of T cells number did not involve the appearance of the inhibitory factor in the patients' serum. Different than in others results gave blood and serum analysis in the patient tymectomized 10 years earlier. Her blood contained the normal number of T and B lymphocytes and the stimulatory factor of a "limited" activity. It displayed only the ability of generating hydrocortisone-resistant cells, it appeared nonactive in generating theta antigen or restoring the ability of rosette formation by T cells.     

Staphylococcus Aureus and Protein A as Mitogens for Rabbit T Lymphocytes

The thymidine uptake by rabbit lymph nodes, spleen and peripheral blood lymphocytes was stimulated by Protein A (SpA) and SpA containing staphylococci. The cell fraction enriched in T lymphocytes showed a higher degree of stimulation with SpA than the fraction enriched in B cells. Rabbit thymocytes showed a marked thymidine uptake by treatment with SpA but not with staphylococci. The monovalent fragment B of SpA (reacting with IgG) neither stimulated nor inhibited the incorporation of thymidine in rabbit lymph node lymphocytes indicating that the Fc reacting site of SpA cannot be responsible for the mitogenicity.     

Four distinct antigen systems on human thymus and T cells defined by monoclonal antibodies: immunohistological and immunochemical studies.

Human thymus and T cell antigens were identified by using four distinct monoclonal antibodies (MoAb), designated 2D5, 5B3, 7A5 and 9D4. 2D5 antibody reacted with most human thymocytes and a few peripheral lymphocytes as well as with a subpopulation (20%) of bone marrow cells, and precipitated a 45K molecular weight (mol. wt.) component from 125I-labelled thymus cell lysate. 7A5 antibody also reacted with the majority (80%) of thymocytes but neither with peripheral lymphocytes nor with bone marrow cells. The antigen detected by 7A5 was a glycoprotein consisting of 48K and 12K mol. wt. components, which were non-covalently associated with each other. 5B3 reacted with virtually all of human thymus and T cells but not with the majority of B cells and bone marrow cells. This reagent precipitated a 72K mol. wt. glycoprotein from thymus and T cells. An additional 65K mol. wt. glycoprotein was precipitated by 5B3 together with the 72K mol. wt. component, but with poor reproducibility. 9D4 antibody, on the other hand, reacted with a 200K mol. wt. component from thymus and T cells as well as 220K and 210K components from the non-T cell fraction of tonsil lymphocytes. Whereas antigens detected by 2D5 and 7A5 appeared to be highly expressed on cortical thymocytes, the antigen defined by 5B3 occurred much more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Based on the data described above, it is suggested that 7A5, 5B3 and 9D4 MoAb recognize human homologues of mouse TL, Ly-1 and Ly-5 antigens, respectively, whereas 2D5 antibody seems to resemble OKT10, as described by others.     

Quantitative analysis of tissue distribution of Ly10-like murine alloantigen(s) with antisera and monoclonal antibodies

Tissue distribution of non-Lyt1.1 ("Ly10-like") antigen or antigens encoded by short chromosomal segment differentiating B6-Ly-1a congenic strain from B6 strain of mice was studied by quantitative absorption of (BALB/c X B6)F1 anti B6-Ly-1a antiserum and by direct cytotoxicity of Ly-10-132-12-26 monoclonal antibody on lymphoid cell populations. Identical strain but not tissue distribution pattern does not allow to conclude whether antiserum and monoclonal antibody detect the same or closely linked antigens. Absorption experiments revealed the highest antigen content in the brain tissue, lower in testis and kidney, still lower in lymphoid organs and the lowest in liver and lung. Among lymphoid cells, bone marrow cells had highest absorbing capacity, followed by thymus, spleen and lymph nodes. Monoclonal antibody lysed almost 100% of thymocytes, 30% bone marrow cells and 10-20% of spleen and lymph node cells (both T-cell and B-cell enriched populations contained the same proportion of positive cells). Cortisone resistant thymocytes showed the same sensitivity as cortical thymocytes to Ly-10-132-12-26 antibody which is distinguishable characteristics of medullary thymocytes from peripheral T cells. Mitogen activated lymphocytes exhibited significantly higher expression of Ly10-like antigen than resting peripheral lymphocytes.