Mouse lymphocytes with and without surface immunoglobulin: preparative scale separation in polystyrene tissue culture dishes coated with specifically purified anti-immunoglobulin.

Mouse spleen cells could be preparatively separated into immunoglobulin positive (Ig+) and immunoglobulin-netative (Ig-)populations by incubating as many as 2 X 10(8) cells per 100 mm diameter petri plate coated with specifically purified goat anti-mouse immunoglobulin. The non-adherent population was 95% or more Ig-, and possessed graft versus host and cytotoxic effector activities, as would be expected for T cells. They could also give a mixed lymphocyte reaction and generate cytotoxic effector activity on culture in vitro. The adherent cells could not be released undamaged from plates coated with undiluted anti-Ig, but they could be released from plates coated with a 1/4 or 1/10 dilution of anti-Ig in an irrelevant antibody. The released cells were over 90% viable by trypan-blue staining, and 94% or more of the viable cells were Ig+.     

Concanavalin A Induction of Suppressor Activity in the T-Helper Subset Defined by Monoclonal Antibodies

Human monocyte-depleted peripheral blood mononuclear cells were separated in T4+ and T8+ populations by a panning technique. Petri dishes coated with goat anti-mouse antibodies were plated by peripheral blood mononuclear cells coated by monoclonal antibodies, either T4 or T8. The cell populations were separated into adherent and non-adherent populations based on binding to the goat anti-mouse-coated plastic dishes. The purity of the adherent populations was 96%. T4+ and T8+ populations were used as effector cells in the concanavalin A-induced suppressor test. The T4+ population revealed a pronounced suppressor activity similar to that exhibited by the T8+ population. This finding was independent of two different sources of monoclonal antibodies, T4/T8 and OKT4/OKT8. The registered suppressor activity in the monoclonal antibody-defined helper population could not be explained either by a switch of the membrane phenotype from T4+ to T8+ cells or by an increased interleukin 2 consumption of the concanavalin A-treated cells.     

Effects of anti-Ig reagents on T cell functions I. Activation of rabbit T cells by anti-allotype antibodies

Rabbit lymphocytes were analyzed by flow microfluorometry, using anti-T cell and anti-Ig reagents. Rabbit T cells and cells expressing surface Ig (B cells) appeared to belong to distinct subpopulations which could be separated on the basis of their selective adherence to nylon wool columns or to anti-Ig-coated dishes. Using flow microfluorometry, no evidence was obtained for the expression of a allotypes (VH framework) on T cells. Separated lymphocyte populations were functionally characterized using an in vitro proliferation assay. B and T cells from rabbit spleen or peripheral blood responded in a differential fashion to B and T cell-specific mitogens and to anti-Ig antibodies. Although such T cells did not respond upon stimulation with anti-Ig antibodies alone, significant proliferation could be induced by simultaneous addition of anti-Ig and T cell growth factor. In addition, activated T cells, derived from lymph nodes of immunized rabbits, generated a proliferative response upon stimulation with anti-Ig reagents alone. The above-mentioned effects on T cells could be obtained using heterologous anti-Ig antibodies or isologous anti-allotype antibodies, directed either against a allotypes (VH framework) or against b allotypes (kappa light chain). Antibodies against the Fc portion of rabbit Ig or against irrelevant allotypic specificities were ineffective in triggering T cells. Fab fragments from anti-allotype antibodies were equally stimulatory for T cells as compared to intact IgG, indicating that cross-linking of Ig-like molecules is not a necessary requirement for anti-Ig-induced T cell activation.     

The role of a human antigen specific T8+ cell subset in antigen presentation, helper function and contrasuppression

Regulation of the human immune response was studied by sequential separation of subsets of T cells, followed by assessment of their helper and suppressor functions in a series of reconstitution experiments. T8+ lymphocytes were separated by panning on streptococcal antigen (SA) coated plates into T8+ SA-adherent cells (T8+SA+) and T8+ SA-non-adherent cells (T8+SA-). The helper and suppressor functions of the T8+SA+ and T8+SA- cells, reconstituted with T4+ helper cells were then studied by a direct antibody forming cell assay. T4+ cells will not induce helper activity by 1000 ng SA alone but require the accessory function of monocytes (Mo). However, replacing Mo by T8+SA+ cells will elicit a similar helper activity by T4+ cells and SA as that induced by Mo. In addition to the antigen-specific presentation and induction of helper activity, the T8+SA+ subset displays the properties of antigen-specific contrasuppressor cells. Thus, reconstitution of T4+ cells and T8+SA- (suppressor cells) with T8+SA+ and 1000 ng SA induces helper and no suppressor activity. Substitution of Mo for the T8+SA+ cells converts the helper to a predominantly suppressor-cell function. T8+SA- cells elicit suppression with 1 ng SA in the absence of accessory cells and reconstitution with Mo, T8+SA+ or T4+ cells failed to affect the suppressor activity. Total reconstitution of the four principle subsets of T4+, T8+SA+, T8+SA- cells and Mo elicited similar antigen dose-dependent responses as those of the unseparated mononuclear cells. It seems that all four cell subsets are required for optimal immunoregulation. We suggest that the T8+SA+ can present antigen to T4+ helper cells and induce helper activity, but in addition these cells can prevent the suppressor subset of T8+ cells from inhibiting T4+ helper cells and function as contrasuppressor cells. The mechanism of these functions is not known but HLA class II antigens might play an essential role in antigen binding, presentation and contrasuppression of the T8+SA+ cells, as the latter are significantly enriched in Ia+ cells.     

T lymphocyte subsets in the peripheral blood in patients with primary biliary cirrhosis

T lymphocyte subsets and natural killer (NK) cells in the peripheral blood of 10 patients with primary biliary cirrhosis (PBC) were examined by using various kinds of monoclonal antibodies directed to the surface markers of the lymphocytes. We found that the population of Leu 2a+, Leu 15- cytotoxic T cells was decreased in PBC patients compared to normal controls, especially in those with a high serum titer of anti-mitochondria antibodies. However, there was no significant difference in the population of Leu 2a+, Leu 15+ suppressor T cells between PBC patients and normal controls. Although the population of Leu 3a+, Leu 8- helper T cells was not significantly different, the population of Leu 3a+, Leu 8+ suppressor-inducer T cells was remarkably increased in PBC patients compared to normal controls. On the other hand, no such difference was seen for the NK cells. Furthermore, the Leu 3a+/Leu 2a+ ratio was significantly higher in PBC patients especially in those with a high serum anti-mitochondria antibody titer, which was thought to be due to the decreased population of cytotoxic T cells.     

Monoclonal antibody analysis of T-lymphocyte subsets in young and aged adults

Eleven monoclonal antibodies identifying surface antigens present on human T lymphocytes were utilized to investigate the effects of advanced age on peripheral blood lymphocyte subsets. Both the proportion and number of lymphocytes recognized by five antibodies reactive with 'pan' T cell antigens (OKT3, OKT11, Leu4, T101 and Lyt3) decreased with age. The percentage of helper/inducer (OKT4+, Leu3a+) cells remained constant; however the proportion of Leu2a+, suppressor/cytotoxic cells declined. There was no change with age in the percent representation of OKT9+ or OKT10+ cells, nor in the ratio of helper/inducer to suppressor/cytotoxic cells (OKT4+/OKT8+ or Leu3a+/Leu2a+). Absolute numbers of helper/inducer (OKT4+, Leu3a+), suppressor/cytotoxic (OKT8+, Leu2a+), OKT9+ and OKT10+ cells were lower in elderly individuals as the result of lymphocytes constituting a lower percentage of the peripheral blood white cell population in this age group. While only small differences existed between the lymphocyte populations of young and aged men; aged women, compared to young women, had more dramatic shifts in their T cell populations. Comparison of antibodies putatively identifying similar (the same) functional groups of T cells demonstrated excellent correlation between the percentage of cells enumerated with the antibodies OKT3+: Leu4+ (r = .951), OKT4+: Leu3a+ (r = .914), OKT8+: Leu2a+ (r = .896), and in the ratio of helper/inducer to cytotoxic/suppressor OKT4+/OKT8+: Leu3a+/Leu2a+ (r = .926) cells.     

Unique Immunological Behaviour of CD8+ (Suppressor T Cell) Leukaemia Cells

A patient with chronic lymphocytic leukaemia and elevated serum IgM antibody is described. A multiparameter surface marker analysis of his peripheral blood lymphocytes demonstrated a unique phenotype of OKT3-, OKT11+, E-rosetting+, OKT4-, OKT8+, OKIa1+, OKCLL+, OKDR+, Tac+, characterizing the disease as suppressor (CD8+) T cell chronic lymphocytic leukaemia. Because of the uncommon phenotype and the abnormal serum immunoglobulin pattern, in vitro functional assays were performed that showed decreased mitogenic response to the phytohaemagglutinin, but increased response to concanavalin A. However, these leukaemic T cells demonstrated phytohaemagglutinin-specific suppressor cell activity on normal blood lymphocytes. In vitro functional studies indicated that a defect in the patient's T cells may cause IgM hypergammaglobulinaemia. The regulatory function of the patient's T cells on immunoglobulin synthesis by normal B cells was found to be mediated by a soluble factor secreted from neoplastic T cells.     

Soluble anti-mu monoclonal antibodies prime resting B cells to secrete immunoglobulins in response to interleukins-4 and -5.

Soluble anti-immunoglobulin (Ig) antibodies have been generally found to inhibit Ig secretion in B cells, via largely unknown mechanisms. To investigate this phenomenon further a two-step culture system was used in which B cells are primed for 24-72 h with various soluble monoclonal or polyclonal anti-Ig antibodies: after washing the cells were placed in readout cultures with a combination of interleukin (IL)-5 and IL-4. Using this protocol B cells primed with (mitogenic or nonmitogenic) anti-mu monoclonal antibodies differentiated into large numbers of IgM-secreting cells, comparable to responses to lipopolysaccharide. In contrast, priming with polyclonal rabbit anti-Ig or monoclonal anti-kappa antibodies, markedly inhibited Ig secretion induced by IL-4 + IL-5. In addition, anti-mu was markedly inhibitory if left in the readout cultures with the two lymphokines. These results, therefore, indicate that appropriate cross-linking of surface IgM receptors on B cells can prime the cells to secrete Ig when they are restimulated by T cell-derived lymphokines in the absence of anti-mu. In contrast co-ligation of both surface IgM and surface IgD receptors apparently results in powerful inhibition of Ig secretion, which is not reversed by stimulation with IL-4 plus IL-5.     

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.     

Surface immunoglobulins on the lymphocytes of the skate Raja naevus

Living lymphocytes of the Elasmobranch fish, Raja naevus, have been examined for surface immunoglobulin (Ig) by treatment with a fluorescent anti-Ig system. Large numbers of Ig-positive cells (60-80%) were found in peripheral blood, spleen and thymus. Following modulation of the surface Ig with anti-Ig, resynthesis occurred, showing that the surface Ig is a product of the individual lymphocytes rather than material passively absorbed from the serum. Formation of caps was independent of temperature, occurring as readily at 4 degrees C as at 20 degrees C, a finding which presumably reflects the environmental conditions normally experienced by the skate. The presence of Ig-bearing lymphocytes in the adult skate thymus suggests a similarity to the amphibian larval thymus, which may be a primary lymphoid organ for the production of both T and B lymphocyte analogues.     

Contents to volume 70 (1984)

Bovine red blood cells linked to polyclonal or monoclonal anti-immunoglobulin antibodies are used in the direct antiglobulin rosetting reaction to detect surface-Ig on human lymphocytes. The sensitivity of this test is markedly increased by pretreating the red cells with trypsin. Enzyme-treated red cells, coupled to anti-human Fab or anti-light chain antibodies, react not only with innate Ig on B lymphocytes but also with smaller amounts of passively adsorbed, cytophilic Ig on up to 25% of freshly prepared peripheral blood (non-B) lymphocytes. In contrast, trypsinized red cells carrying anti-Ig isotype-specific antibodies react exclusively with B cell surface-Ig. Cytophilic Ig is abnormally firmly bound to lymphocytes separated on Ficoll-Hypaque at 20°C or below, and is released very slowly during 3 h or more at 37°C in vitro.Lymphocytes are free of detectable cytophilic Ig when isolated on Ficoll-Hypaque at 37°C, and very little Ig is retained by non-B cells in suspensions purified on Percoll which, unlike Ficoll, does not increase Ig binding affinity. These lymphocyte separation procedures are recommended as a preliminary to B cell assays by sensitive antiglobulin techniques.     

人外周血T细胞主要功能亚群的分离及某些性质的研究

用本实验室自制CD_4类抗体T_(4a)T_(4b)和CD_3类抗体T_(3b)及流式细胞荧光激活分类仪(FACS440)将正常人外周血分离成T_(4a)(T_(4b))~+、T_(4a)(T_(4b))~-、T_(8b)~+、T_(8b)~-细胞。对所分离的细胞纯度分析表明.其纯度达95%以上.用CD_4、CD_(?)类对所分离细胞进行双染分析,发现T_(4a)(T_(4b)~+、T_(4a)(T_(4b))~-,T_(8b)~+中不含T_(4a)(T_(ab))~-、T_(8b)细胞.同样在阴性细胞中,阳性细胞的含量不超过2.2%.用此高度纯化的T_(4a)(T_(4b))~+、T_(8b)细胞,观察PHA诱导分泌IL-2的情况.发现仅T_(4a)(T_(4b)_~+细胞分泌IL-2.在本实验条件下,T_(8b)细胞不分泌IL-2.     

Enumeration and isolation of human T and B lymphocytes by rosette formation with antibody-coated erythrocytes.

Rosette techniques are presented for the enumeration and separation of both Ig+ T- and Ig- T+ human lymphocytes. In order to enumerate Ig+ cells, the direct immunocytoadhesion technique was employed using human erythrocytes (E) coated with purified anti-kappa or anti-lambda light chain antibodies. Specificity of these rosettes was shown with chronic lymphocytic leukaemias of either the kappa or lambda type. T+ cells were enumerated by a new indirect rosette technique in which the lymphocytes were initially treated with rabbit anti-human thymus cell antiserum followed by direct rosetting with human E coated with purified anti-rabbit light chain antibody. For normal individuals, 24-32% Ig+ T- cells and 65-71% Ig- T+ cells were found among the lymphocytes of peripheral blood as well as tonsils with these rosette methods. The Ficoll-Hypaque method was used to obtain purified Ig- T+ and Ig+ T- cells by removing rosetted Ig+ cells or T+ cells, respectively. The purity of the Ig- T+ cells was indicated by greater than 99% indirect rosetting of cells sensitized with anti-human thymus cell antibody (Ab) and by less than 1% direct rosetting with anti-kappa Ab-E+ anti-lambda Ab-E. The purity of the Ig+ T- cells obtained was indicated by 92-96% direct rosetting with anti-kappa Ab-E+anti-lambda Ab-E and by less than 1% indirect rosetting with anti-human thymus cell antibody. A small percentage of Ig- T- 'null' cells could not be identified by either reagent. Thus, essentially pure Ig- T+ and Ig+ T- cells were readily and efficiently isolated by 'negative selection' thereby lessening the possibility of functional changes that may develop by more extensive manipulation of lymphocytes.     

Functional characterization of a regulatory human T-cell subpopulation increasing during autologous MLR.

The present study was undertaken to investigate the heterogeneity of helper T cells in humans using two different monoclonal antibodies: 5/9 and MLR4. The former identifies 15-20% of resting T lymphocytes from peripheral blood and corresponds to an anti-helper/inducer T cell. The second antibody, MLR4, recognizes 5% of total T lymphocytes and partially overlaps with the 5/9+ T cells. In order to investigate functional differences within the 5/9+ cells, we separated two different subsets (5/9+ MLR+ and 5/9+ MLR4-) by a rosetting technique. Although both subsets provide help for Ig synthesis in a PWM-stimulated culture, only the 5/9+ MLR4- fraction gave a proliferative response in both autologous and allogeneic MLR and to soluble protein antigens. The effect of radiation on the ability of the two subsets to provide help for Ig synthesis showed that the 5/9+ MLR4+ subset is highly radiation-sensitive, while 5/9+ MLR- is relatively radiation-resistant. In a further series of experiments, 5/9+ MLR4+ cells isolated after activation in an autologous MLR but not by Con A, were no longer able to induce T-cell differentiation but now showed a strong suppressor effect. The 5/9+ MLR4- subset separated from the same cultures did not display any suppressor function. These data demonstrate in fresh PBL the existence of a radiation-sensitive regulatory subset exerting a helper activity, and which acquires suppressor activity after activation in autologous MLR.     

Phenotypic Convergence and Divergence of Surface Immunoglobulin and CD40 Signals

Both anti-CD40 antibodies and anti-immunoglobulin (Ig) coupled to Sepharose induced proliferation of resting B cells and suppressed lipopolysaccharide (LPS)-induced B-cell differentiation to immunoglobulin secretion at comparable levels determined with the plaque-forming assay and Ig RNA steady state levels. Anti-CD40 antibodies also increased the proliferation of B cells stimulated by T helper cells in vitro while suppressing their differentiation to Ig secretion. Further, B cells preactivated by anti-Ig, anti-CD40 or a combination of the two mitogens could be restimulated by anti-CD40 but not by anti-Ig antibodies. Phenotypic divergence of Ig and CD40 signals regarding surface expression of activation markers was observed. Restimulation of anti-Ig- or anti-CD40-prestimulated cells with anti-Ig induced apoptosis whereas apoptosis could be inhibited when cells were recultivated with anti-CD40.     

Human T cell differentiation antigens characterizing a cytotoxic/suppressor T cell subset

Monoclonal antibodies were raised against the leukemic T cells from a patient with chronic lymphocytic leukemia. Two antibodies, termed T411 and T811, were obtained which were reactive by indirect immunofluorescence only with cells of the T cell lineage. The T411 antibody recognized a polypeptide chain of 100,000 dalton apparent molecular weight which was present on the surface of 94 +/- 4% of peripheral blood T lymphocytes, but only on 20 +/- 8% of thymus cells. The antibody T811 reacted with a surface molecule composed of 2 poly-peptide chains of 32,000 and 34,000 dalton apparent molecular weight, which was expressed only on 25 +/- 10% of blood T lymphocytes and on 90 +/- 4% of thymus cells. Functional analysis of the T811+ and T811- T cell subsets isolated by rosetting with anti-mouse Ig coated ox erythrocytes revealed that both subpopulations were able to mount a proliferative response to allo-antigens, whereas allo-antigen induced cytotoxic cells and their precursors were only found in the T811+ subset. The pokeweed mitogen induced in vitro differentiation of B lymphocytes into immunoglobulin secreting cells was dependent on the presence of the T811- subset, whereas the T811+ T cells efficiently suppressed this differentiation.     

Surface immunoglobulin-positive T lymphocytes in HIV-1 infection: relationship to CD4+ lymphocyte depletion

T lymphocytes bound to autologous immunoglobulin (surface Ig + T cells) and serum antibodies that bind to allogeneic lymphocytes have been detected in HIV-1-infected individuals, but their significance in the immunopathogenesis of HIV-1 infection is uncertain. We tested peripheral blood from HIV-1-infected individuals to determine if surface Ig+ T cells are specific for HIV-1 infection and are associated with CD4+ lymphocyte depletion. The majority of HIV-1-infected individuals contained substantial numbers of circulating surface Ig+ T cells. The presence of such cells was restricted to seropositive individuals and not related to risk factors associated with the acquisition of HIV-1 infection. Autologous immunoglobulin was detected on both CD4+ and CD8+ cells in all patients tested. Most individuals with surface Ig+ T lymphocytes also had serum anti-T-lymphocyte antibodies. The presence of surface Ig+ T lymphocytes correlated significantly with lower absolute CD4+ lymphocyte counts only in asymptomatic, HIV-1-infected individuals.     

Rapid recovery of antigen-binding receptors on chicken B cells following anti-Ig serum treatment.

Rosette-forming cells (RFC) from the peripheral blood of sheep red blood cell (SRBC)-immunized chickens were characterized as B cells which manifest antigen-binding receptors but no antibody secretion. Lymphocytes were pretreated at 0 degrees C with anti-Ig serum, washed, incubated further at various temperatures and harvested for the rosette-forming cells (RFC) assay. Anti-Ig treatment blocked all RFC formation and providing that the cells were maintained at less than 10 degrees C regeneration did not occur up to 6 h. Complete recovery of RFC formation occurred within 10 min at 37 degrees C following treatment with low but not with high concentrations of anti-Ig serum. However, recovery from inhibition by high-dose anti-Ig treatment was achieved by including chicken IgG in the incubation medium. The recovery of receptors was reduced following "sandwich" treatment of lymphocytes. When cells were treated with anti-Ig antibody, washed and exposed to various drug inhibitors of "capping" the regeneration of RFC was prevented; in contrast, inhibitors of protein synthesis were ineffective. The results were interpreted in terms of dissociation of anti-Ig antibodies from the Ig receptor at an early stage of the ligand-induced receptor redistribution process.     

Regulation of delayed-type hypersensitivity IV. Antigen-specific suppressor cells for delayed-type hypersensitivity induced by lipopolysaccharide and sheep erythrocytes in mice.

Mice injected subcutaneously with 1 x 10(8) sheep red blood cells (SRBC) developed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after injection. Such DTH was suppressed when 100 microgram lipopolysaccharide (LPS) was injected intravenously 1-2 days before or at the time of SRBC injection. This suppression of DTH was transferable by spleen, lymph node, thymus and bone marrow cells to sensitized or normal syngeneic recipients, but could not be transferred by serum. Suppressor cells were not induced by LPS alone or SRBC alone, and they were antigen-specific since DTH to chicken red blood cells was not affected. The suppressor cells appeared in the spleen in optimum number 3-4 days after induction. They were theta-negative and Ig-positive as judged by antiserum plus complement treatment and by Ig rosette separation. Attempts to obtain soluble suppressor factor from the suppressor cells by sonication or in vitro incubation were unsuccessful. Mitomycin C treatment of the suppressor cells completely abolished the suppressor activity. Thus, LPS, in conjunction with antigen, appears to induce a population of specific suppressor B cells which are capable of regulating T cell function.     

Intrathyroidal HLA-DR-positive lymphocytes in Hashimoto's disease: increases in CD8 and Leu7 cells

The peripheral and intrathyroidal HLA-DR-positive (DR+) lymphocyte subsets that were activated in vivo in patients with Hashimoto's disease (HD) were examined by two-color flow cytometry with monoclonal antibodies against CD3, CD4, CD8, Leu7, CD19, and HLA-DR antigens. The proportions of total DR+ cells in peripheral lymphocytes and the proportions of DR+ cells in the CD3+, CD4+, and Leu7+ lymphocytes were higher in patients with HD than in normal controls. Furthermore, the proportions of total DR+ cells among intrathyroidal lymphocytes isolated from thyroid tissue of individuals with HD were higher than those in their peripheral lymphocytes. Interestingly, the proportions of DR+ cells among the CD3+, CD8+, and Leu7+ lymphocytes in the thyroid were greatly increased. These data indicate that (i) CD3+ T, especially CD4+ T helper/inducer, lymphocytes and Leu7+ NK/K cells are activated in peripheral blood in Hashimoto's disease and that (ii) CD3+ T, especially CD8+ T suppressor/cytotoxic, lymphocytes and Leu7+ NK/K cells are predominantly activated in Hashimoto's goiter, suggesting an increase of cell-mediated cytotoxicity in the thyroid in Hashimoto's disease.