Detection of ecto-5'-nucleotidase on rat B and T lymphocyte subpopulations using a monoclonal antibody in rosetting reactions

A mouse monoclonal antibody to rat 5'-nucleotidase (5N 4-2 McAb) was used in the direct anti-determinant rosetting reaction (DARR) to demonstrate the ecto-5'-nucleotidase molecule in preparations of rat lymphocytes. Results indicated that 35.5 +/- 7.5% of peripheral blood lymphocytes (PBL), 37.3 +/- 4.8% of lymph node cells (LN) and 37.0 +/- 8.5% of spleen lymphocytes expressed the 5N 4-2 antigen. Depletion studies and mixed rosetting reactions (MRR) showed that the 5N 4-2 antigen was mainly expressed on rat T lymphocytes rather than on B lymphocytes: In fact 59.6 +/- 3.2% (in PBL), 76.5 +/- 0.6% (in LN) and 67.1 +/- 1.3% (in spleen) of T lymphocytes exhibited the 5N 4-2 antigen compared to only 26.5 +/- 2.6% (in PBL), 34.0 +/- 2.1% (in LN) and 46.1 +/- 12.0% (in spleen) of B lymphocytes. As expected a strong association was found between the expression of 5N 4-2 antigen and 5'-nucleotidase enzyme activity on lymphocytes. Both 5N 4-2 positive cells and enzyme activity were preferentially exhibited in the T lymphocyte subpopulation, and 92% of the enzyme activity was observed in a 5N 4-2 antigen positive subpopulation.     

Use of a monoclonal antibody specifically non-reactive with T cells to delineate lymphocyte subpopulations.

Rat monoclonal antibody M1/69.16 reacts with a heat stable antigen of mouse commonly expressed in the majority of cell types in blood, spleen, bone marrow and thymus, including cells of erythroid, myeloid and lymphoid series. However, subpopulations of cells in lymphoid tissues can be identified which are non-reactive with this antibody using the fluorescence-activated cell sorter. All surface Ig positive cells seem to react with M1/69.16 while more than 96% of Ig negative cells in spleen and lymph nodes are M1/69.16 negative. Most cells (80%-90%) in the M1/69.16 negative populations in spleen lymph nodes and bone marrow express Thy-l. Thus, peripheral T cells are specifically non-reactive with this antibody. In contrast, approximately 95% of thymocytes react with M1/69.16, leaving a minor population which is negative. The negative population (5%) is enriched in cells expressing high amounts of H-2 antigen and those bearing H9/25 antigen which is specific for lymphocyte subsets, indicating that M1/69.16 negative thymocytes represent a specific subpopulation, possibly "mature' thymocytes.     

Determination of T lymphocyte subpopulations by monoclonal antibodies in rheumatoid arthritis. Influence of immunomodulating agents

The pathogenesis of rheumatoid arthritis is unknown, but clear abnormalities of the immune system are well documented in this disease. We therefore evaluated T cell subpopulations in patients with rheumatoid arthritis using monoclonal antibodies previously shown to react with all T cells (OKT3), with inducer/helper T cells (OKT4) and with suppressor/cytotoxic T cells (OKT8). These investigations disclosed evidence of a significant decrease in the number of OKT8⁺ cells/mm³ and a high inducer-helper/suppressor-cytotoxic (OKT4⁺/OKT8⁺) ratio in active rheumatoid arthritis. A modest number of patients with active arthritis were treated with levamisole or with synthetic thymopoietin 32–36 (thymopoietin pentapeptide or TP-5). These individuals responded with ratio decreases to more normal levels.Our data support the hypothesis that monoclonal T cell antibodies may offer an important tool for the further evaluation of patients with rheumatoid arthritis and their individual response to treatment.     

Immunoregulatory T cell subpopulations in patients with scleroderma using monoclonal antibodies.

Twenty-eight patients with scleroderma were compared with 22 healthy age-matched subjects. Monoclonal antibodies were used to detect the whole T cell population (OKT3), T helper cells (OKT4), and T suppressor/cytotoxic cells (OKT8) by indirect immunofluorescence on isolated peripheral blood mononuclear cells. A subset of scleroderma patients (i.e. 30% or eight of 28 patients) exhibited an elevated ratio of OKT4/OKT8 cells which could be accounted for, mainly by a reduction in OKT8 cells compared with controls. The scleroderma patients with an elevated OKT4/OKT8 ratio tended to be younger, have a shorter disease duration and more extensive skin involvement than patients with a normal OKT4/OKT8 ratio. There was no correlation with the presence of autoantibodies, drug therapy, or HLA-DR type. In order to further determine whether this imbalance in immunoregulatory cell subpopulations was specific for scleroderma, we further studied 16 patients with psoriatic arthritis but without manifest autoimmunity and delineated a similar subset of patients with an elevated OKT4/OKT8 cell ratio (i.e. 38% or six of 16 patients). The results demonstrate similar immunoregulatory T cell imbalances in patients with scleroderma and psoriatic arthritis. These findings suggest that numerical imbalances in lymphocyte subpopulations may not be specific for autoimmune disorders.     

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.     

An immunohistological analysis of lymphocyte subpopulations and their microenvironment in the synovial membranes of patients with rheumatoid arthritis using monoclonal antibodies.

We have used monoclonal antibodies of the orthoclone (OKT) series to identify T cell subsets in an immunohistological analysis of the synovial membranes obtained from normal individuals and patients with osteoarthritis or rheumatoid arthritis. T cells of the inducer and the suppressor/cytotoxic subsets were identified by the OKT4 and OKT8 antibodies respectively while HLA-DR (Ia-like) antigens were recognized by a conventional antiserum. In the normal and osteoarthritic synovial membranes, virtually no lymphocytes were identified whereas the mononuclear cell infiltrates of the rheumatoid synovial membranes were composed predominantly of T cells expressing the OKT4 inducer phenotype with few OKT8+ suppressor/cytotoxic cells. The OKT4+ cells were found to be intimately related to B cells and strongly HLA-DR+ cells which morphologically resembled the interdigitating cells of lymph nodes. The micro-anatomical arrangement of these different cell types in the mononuclear infiltrates of the rheumatoid synovial membranes closely resembled that of the paracortical or T cell dependent area of normal lymph nodes except few OKT8+ lymphocytes were present. These findings are explained in terms of rheumatoid arthritis as a disease of altered T lymphocyte/macrophage immunoregulation.     

Phenotype of rat natural killer cells defined by monoclonal antibodies marking rat lymphocyte subsets.

The fluorescence-activated cell sorter and a rosette-depletion technique were used to separate rat spleen lymphocytes and BCG-activated lymphocytes into subpopulations with and without the antigens defined by W3/13, W3/23 and OX8 monoclonal antibodies. The resultant populations were then tested for natural killer (NK) activity in a quantifiable 6 hr 51Cr release assay. The data establish that rat natural killer cells are heterogeneous with respect to their surface antigen expression and that subpopulations of rat NK cells express the OX8 and W3/13 defined antigens. However, rat NK cells do not express the antigen defined by W3/24 monoclonal antibody.     

Distribution pattern of lymphocyte subpopulations in human tonsils as analysed by monoclonal antibodies with double immunoenzymatic labeling

Summary The distribution of B- and T-lymphocytes, T-cell subpopulations, including natural killer cells and monocytes/macrophages, was studied in cryostat sections of human tonsils by the avidin-biotin-peroxidase technique using monoclonal antibodies. A double immunostaining procedure was also developed to detect two types of lymphocytes on one single section simultaneously using horseradish peroxidase and alkaline phophatase as labeling enzymes.The primary follicles and the germinal centers of the secondary follicles were mainly found to be positive for B-cells. T-cells were predominantly localized in the follicular caps and in the interfollicular areas. The ratio of helper/inducer cells to suppressor/cytotoxic cells was in favour of helper T-cells. Both subpopulations were also predominant in follicular caps and interfollicular areas.The quantity of natural killer cells was very variable and nearly all were localized exclusively in the germinal centers.Monocytes/macrophages were only seen occasionally in the interfollicular areas. The double-immunoenzymatic labeling was useful for the visualization of combinations of antigens, however, without demonstrating the presence of two different surface antigens on one single cell.     

T lymphocyte subpopulations defined by two sets of monoclonal antibodies in chronic active hepatitis and systemic lupus erythematosus.

Lymphocyte subpopulations were enumerated in human peripheral blood using murine monoclonal antibodies with specificity for all peripheral blood T lymphocytes (OKT3, alpha-Leu 1) and for the helper subset (OKT4, alpha Leu 3a) and suppressor/cytotoxic subset (OKT8, alpha Leu 2a). Patients with chronic active hepatitis (CAH) (23) or systemic lupus erythematosus (SLE) (10), compared with healthy subjects (20), had a lower mean T lymphocyte count. Patients with CAH had normal numbers of suppressor/cytotoxic (TSC) cells, but fewer helper (TH) cells than healthy subjects (0 . 96 +/- 0 . 11 X 10(9)/1 versus 1 . 45 +/- 0 . 15 X 10(9)/1), and those with SLE also had fewer TH cells (0 . 93 +/- 0 . 11 X 10(9)/1). Patients with CAH receiving azathioprine (n = 8) had significantly fewer TSC cells, and a higher TH/TSC ratio (2 . 69 +/- 0 . 35) than those (n = 15) not on this therapy (1 . 85 +/- 0 . 15). When patients taking azathioprine were excluded, no correlation was found between disease activity and the TH/TSC ratio for either disease.     

Immunoaffinity chromatography of lymphocyte subpopulations using tert-amine derived matrices with adsorbed antibodies

New polymeric solid-phase matrices for cell affinity chromatography were prepared and their advantageous characteristics compared with conventional matrices were highlighted. These new matrices are derivatives of poly(2-hydroxyethyl methacrylate) (PHEMA) containing a slight quantity of amino compounds as a co-monomer. They were applied to immunoaffinity selection between IgG+ and IgG- lymphocytes of the rat mesenteric lymph node. Simple physical adsorption was sufficient for anti-rat IgG antibodies to be immobilized on these matrices, allowing us to omit the laborious procedure of covalent-linking of antibodies on a matrix. As these matrices themselves showed extremely low non-specific adsorption of lymphocytes, a very dilute solution of antibody (0.02-0.08 mg/ml) was enough for column conditioning. This separation method gave IgG- lymphocytes of more than 90% purity and almost 95% yield within as short a time as 7 min. Further, IgG+ lymphocytes were obtained in good yield (80-90% of loaded number) by recovering the adsorbing cell fraction from the column by gentle pipetting of the matrix.     

Annual stability in the levels of lymphocyte subpopulations identified by monoclonal antibodies in blood of healthy individuals

The absolute numbers and percentages of lymphocytes, monocytes, and lymphocyte subpopulations in the blood mononuclear cells were examined monthly in two healthy individuals over a 22-month period. The object of this study was to determine whether the levels of lymphocyte subpopulations identified by monoclonal antibodies, Leu4⁺ (T), Leu3⁺ (helper T), Leu2⁺ (suppressor/cytotoxic T), and Leu7⁺ (natural killer) cells, were stable during the year for healthy donors. The results were analyzed by the cosiner method to estimate the rhythmicity of these subpopulations. The number of lymphocytes varied, showing a moderate circannual rhythm with a peak in early summer, whereas the number of monocytes also varied but its variation did not show a specific rhythm. The absolute numbers of T-lymphocyte subpopulations and Leu3⁺ and Leu2⁺ cells showed a covariation, with a peak in early summer in parallel with the circannual rhythm of total lymphocyte counts. A subpopulation of granular lymphocytes with natural killer function, Leu7⁺ cells, also showed a significant variation during the year. Of particular interest is that Leu3/Leu2 ratios were considerably stable during the year. The two-time point examination of these lymphocyte markers including HB-2⁺ B cells in August and January in 15 normal donors did not show any significant differences, although the mean values were slightly higher in summer. The stability and variability of these lymphocyte markers are displayed graphically and the details of these variations are listed.     

Characterization of human adenoid cells using surface and functional markers for lymphocyte subpopulations.

Adenoid lymphocytes from children undergoing adenoidectomy were compared with blood cells from the same children using techniques for identifying T cells and B cells. A high proportion of adenoid lymphocytes were immunoglobulin positive cells. Of these only a minor fraction carried receptors for the Fc part of IgG. Adenoid B lymphocytes respond poorly if at all to polyclonal B-cell activators, such as LPS or PPD, which show a different reactivity compared to human splenic cells. The response to anti B2-microglobulin was also different; blood cells responded better than adenoid cells. Thus distinct subpopulations of B lymphocytes reside in different lymphoid organs. The adenoid lymphocyte reactivity might reflect their function in the defence mechanism against infections.     

Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies.

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.     

The effect of cyclophosphamide in vivo on the expression of lymphocyte markers, detected by monoclonal antibodies, in the rat

Surface markers on lymphocytes from the thymus, lymph node and spleen of the rat were examined in single cell suspensions using a panel of monoclonal antibodies. These were used particularly to investigate Pan T, TH (T-helper), TS/C (T-suppressor-cytotoxic) and Ia antigens. The expression of these markers in rats treated with a single dose of cyclophosphamide (100 mg/kg body weight) was followed for 3 weeks after treatment. Maximum changes were detected at 3 days and recovery took up to 3 weeks to near completion. Comparison was made with histological observations of the effect of CY on these organs. At 3 days, the thymus showed maximum weight loss and gross cortical depletion. This associated with significant drop in the expression of the TS/C marker on small and large lymphocytes. Regeneration of the cortex, beginning at 7 days, was associated with the presence of many large pyroninophilic cells. This was accompanied by an increase in the expression of the TS/C marker on both small and large lymphocytes. The mesenteric lymph node showed marked depletion of the B-cell areas at 3 days. There was also a significant drop in TS/C and Ia expression and a marked rise in Pan T and TH. TS/C expression recovered rapidly with a rebound at 7 days. However, the expression of Pan T and TH did not return to normal until 21 days. In the spleen there was a similar decrease in the lymphocytes populating the B-cell areas at 3 and 7 days with an increase in the expression of Pan T and TH, and a decrease in the expression of Ia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two distinct antigenic markers for rat thymus and T cells defined by monoclonal antibodies.

Rat thymus and T-cell antigens were defined by using two distinct monoclonal antibodies (R-1-3B3 and R-1-12C5). R-1-3B3 antibody, when tested for its reactivity with rat lymphoid cells by immunofluorescence, labelled virtually all thymus and T cells but not B cells and bone marrow cells. The antigen defined by this R-1-3B3 antibody occurred more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Immunochemical data showed that R-1-3B3 antibody recognized a single glycoprotein with a molecular weight of 67,000, which were able to interact with Lens culinaris haemagglutinin. R-1-12C5 antibody, on the other hand, reacted with all of thymus and T cells as well as with a subpopulation (approximately 20%) of bone marrow cells. In contrast to the antigen defined by R-1-3B3, that detected by R-1-12C5 antibody existed largely on cortical thymocytes and to a much lesser extent on medullary thymocytes and peripheral T cells. R-1-12C5 antibody detected a single glycoprotein with a 95,000 molecular weight, which could also interact with Lens culinaris haemagglutinin. Based on these data described above and since both antigens defined by R-1-3B3 and R-1-12C5 antibodies were absent from rat brain tissue, we concluded that they were distinct from brain-associated thymic antigens in rats including Thy-1 and W3/13 antigen systems.     

Reactivity of normal rat epiphyseal chondrocytes with monoclonal antibodies recognizing different leucocyte markers.

The aim of the study was to test whether normal rat epiphyseal chondrocytes react with monoclonal antibodies (MoAbs) detecting different markers of lymphoid cells. For this purpose, isolated chondrocyte and, for comparison, splenocyte cytosmears were exposed to a battery of different MoAbs followed by indirect immunoperoxidase staining. As we were able to show, all chondrocytes reacted with OX17 MoAb detecting Class II (Ia) antigen encoded by the RT1.D subregion of rat major histocompatibility complex (MHC). However, unlike splenocytes they did not react with OX6 MoAb detecting the RT1.B encoded Class II molecule. Moreover, all chondrocytes were stained with W3/25 MoAb specific for the rat equivalent of human CD4 (T4) antigen and with W3/13 MoAb specific for rat leucocyte sialoglycoprotein. A positive reaction was also obtained with the antibody against the S-100 protein. By contrast, chondrocytes did not react with antibodies specific for all T (OX19) or B (HIS14) cells, rat CD8 (T8) equivalent, monocytes/macrophages (ED1, ED2), factor VIII (M616), or glial fibrillary acidic protein (Z334).     

Measurement of IgG-blocking antibodies by ELISA using monoclonal antibody and fluorogenic substrate

An enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antihuman gamma-chain antibody and a fluorogenic substrate has been developed for quantitation of IgG-blocking antibodies in human serum. Generation of fluorescent product was linear with time to 60 min. Using optimal conditions the ELISA was sensitive to less than 1 ng/ml of specific IgG to short ragweed pollen. The assay demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation = 8.8%). Reproducibility was determined by constructing precision profiles for intra and interassay variation for the entire working range of the assay. Intraassay CVs ranged from a mean of 13% at threshold to less than 5% at higher antibody concentration. Interassay reproducibility similarly ranged from 18 to 10%. In this assay the effect of serum dilution on nonspecific binding was minimal and specific binding of 4-10 ng IgG antibody to the antigen-adsorbed wells was largely complete (75.8 +/- 4.8%) and highly specific (greater than 98%). This application of ELISA for ragweed IgG antibody measurement has performance specifications equal or superior to previously developed radioimmunoassay and ELISA systems.     

Cross reaction of monoclonal antibodies to human MHC class I and class II products with bovine lymphocyte subpopulations

Peripheral blood lymphocytes (PBL) from cattle have been separated into T & B cell subpopulations using a panning technique. These T and B cell preparations have then been tested by direct and indirect complement mediated cytotoxicity tests with a series of monoclonal antibodies (Moabs) reacting with HLA class I and class II products.
The anti-class I monoclonals were monomorphic or non-reactive and where reactive killed both B and T cells.
The anti-class II monoclonals reacted only with B lymphocytes. There was no relationship between their reactivity pattern and any BoLA specificity.
This work was supported by the National Institutes of Health Grants AI21384 and CA38469.