Spontaneous Reverse Haemolytic Plaque Formation

A sensitive reverse haemolytic plaque assay was used on freshly isolated blood cells from normal human subjects to show that T lymphocytes and monocytes were both necessary for immunoglobulin production by unstimulated B cells cultured only for the time necessary to form plaques. When lymphocyte preparations were fully depleted of T cells by E-rosette formation overnight on ice followed by Ficoll-Hypaque centrifugation, the number of plaque-forming cells was reduced by up to 94%; this reduction was reversed by the replacement of T cells, although excess T cells suppressed plaque formation. Moreover, when T-cell function was blocked by 10-1000 ng of two monoclonal anti-T-cell antibodies, OKT3 or UCHT1, this significantly reduced or abolished spontaneous IgG plaques, and higher concentrations of either OKT3 or UCHT1 reduced the numbers of IgA and IgM plaques formed by B cells. The role of monocytes in spontaneous plaque formation was investigated. The removal of plastic-adherent cells from mononuclear cell preparations did not consistently result in a reduction in the numbers of plaques, but complement-mediated lysis of monocytes with either of two monoclonal antibodies with specificity for monocytes, OKM1 and FMC17, reduced by 50% the number of IgG, IgA and IgM plaques. This effect was reversed by addition of as few as 1% plastic-adherent cells. Decreased plaque formation by B cells, resulting from either blocking of T-cell function with monoclonal antibody or complement-mediated lysis of monocytes, or both, was fully reversed by soluble factors present in cell-free conditioned medium from lectin-activated T cells. Thus spontaneous plaque formation by human peripheral blood B cells requires T cells and a small number of monocytes, and the major function of these cells is to help B cells by the production of soluble factors.     

A study on IgE antibody-producing cells from the peripheral blood lymphocytes of atopic patients

In the present study, the peripheral blood lymphocytes (PBL) of six rye-grass and 22 ragweed atopic patients demonstrated secretion of IgE antibody from 7-day cultures as measured by a sensitive ultra-low RAST. The RAST binding ranged from 1.5% to 21% whereas the cell supernatants from the PBL of eight non-atopic individuals showed little or no response (1.2%). The addition of antigens (rye grass 1 or AgE) or interleukin (IL-2) to the cultures on day 0 failed to cause an increase in response. But examination of PBL from four of these patients by a reverse hemolytic plaque assay showed that challenge of these cells by antigen or IL-2 caused an increase in the number of antigen-specific IgE plaque forming cells. The bulk of the IgE antibody secreting cells were located in a sheep red blood cell rosetted fraction (cell fraction 2) whereas most of IgE antibody PFC were found in the non-rosetting fraction (cell fraction 1). It appears that the reverse hemolytic plaque assay detects IgE antibody-producing cells which can still undergo immune regulation and may represent an earlier stage of B cell differentiation whereas the ultra-low RAST appears to measure spontaneous plasmablast cell IgE antibody response.     

Distinctive functional characteristics of human "T" lymphocytes defined by E rosetting or a monoclonal anti-T cell antibody

The properties of human lymphocyte fractions isolated either by sheep red cell(E) rosetting or by fluorescence-activated cell sorting after staining with UCHT1 monoclonal anti-T cell antibody have been compared. Two populations of E+ cells with very different phenotype and function have been identified. E+/UCHT1+ cells respond well to the T cell mitogens phytohemagglutinin and concanavalin A and provide help for an in vitro specific antibody response. They can also suppress the antibody response of allegeneic peripheral blood mononuclear cells. In contrast, the E+/UCHT1- population, which has no other markers characteristic of T cells, fails to respond to mitogens or to provide help or suppression for an antibody response. These cells, however, are highly active natural killers. They possess Fc gamma receptors and have a characteristic staining pattern of nonspecific esterase enzyme activity. It is concluded that not all cells capable of forming E rosettes are thymus-processed cells and that this heterogeneity can be revealed by staining with the monoclonal anti-T cell reagent UCHT1.     

Cellular mechanisms of restricted immunoglobulin formation in the human neonate

The functional capacity of human neonatal B lymphocytes has been investigated by in vitro methods using T lymphocyte-dependent (pokeweek mitogen, PWM) and -independent (Epstein-Barr virus, EBV) polyclonal B cell activators. B cell activation of single cells was detected by class-specific immunoglobulin (Ig) secretion using a reversed hemolytic plaque assay. It was found that neonatal B cells were triggered to secretion of IgM by EBV, with a magnitude comparable to adult levels, but that, in contrast to B cells from adults, they did not secret IgG. Cord lymphocytes did not secret Ig although they displayed a sizable DNA synthetic response to PWM. Using cell separation and culture experiments, it was shown that (allogeneic) adult T lymphocytes could restore cord B cell responsiveness to PWM and that cord T lymphocytes could not cooperate with adult B cells. In addition to this immaturity of cord T helper function for antibody synthesis, we found cells in the cord T cell-enriched fraction which inhibited the polyclonal response of adult lymphocytes to both PWM and EBV. These lymphocytes suppressed adult B lymphocytes directly but appeared ineffective against neonatal B lymphocytes themselves. The nature of these suppressing cells and their possible role in the fetal/maternal relationship are a matter of speculation.     

T cell activation by anti-T3 antibodies: comparison of IgG1 and IgG2b switch variants and direct evidence for accessory function of macrophage Fc receptors

The human T3 antigen is closely associated with the T cell receptor. Some anti-T3 antibodies cause T cell proliferation in the presence of monocytes which have Fc receptors (FcR) that bind particular antibody subclasses. Such an interaction is thought to determine whether or not an anti-T3 antibody is mitogenic. We examined the mitogenicity of an IgG1 antibody, UCHT1, and an IgG2b switch variant of identical specificity, UCHT1B. With autologous monocytes, 76% of individuals responded to UCHT1 and 9% to UCHT1B, falling into three patterns of responsiveness. Both antibodies in the absence of monocytes induced responsiveness to recombinant interleukin 2, even for UCHT1B nonresponder T cells. The proliferation induced by UCHT1B, however, was always less than that induced by UCHT1. These findings demonstrate the critical role played by the Fc region for mitogenesis, and suggest a possible role for the hinge region. We then obtained direct evidence that mitogenicity can be mediated exclusively via FcR. Mouse macrophages have distinct FcR: FcRI binds IgG2a but FcRII binds IgG1 and IgG2b and its function can be inhibited by the specific antibody 2.4G2. Because UCHT1 and UCHT1B were of the correct subclass to interact with FcRII we examined the accessory function of mouse peritoneal macrophages. Without exception, human T cells now responded to both antibodies. Proliferation was drastically inhibited by 2.4G2 but not by an irrelevant anti-macrophage antibody, F4/80, nor by an anti-human neutrophil FcR antibody, 3G8. Furthermore, 2.4G2 did not inhibit the accessory function of mouse macrophages for OKT3, an IgG2a antibody that presumably interacts with FcRI, and did not inhibit the function of human monocytes for UCHT1 and UCHT1B. Mouse B cells, in contrast to macrophages, have an FcR which binds all three subclasses, but which can be inhibited by 2.4G2. B cells, however, were not accessory cells for mitogenesis with UCHT1, UCHT1B or OKT3. These findings are discussed in relation to other requirements for T cell activation by anti-T3 antibodies.     

Investigation of early T cell activation: analysis of the effect of specific antigen, interleukin 2 and monoclonal antibodies on intracellular free calcium concentration

The three mitogenic anti-T3 antibodies, UCHT1, anti-Leu-4 and WT-32, all produce a rapid increase in T cell intracellular Ca2+ ( [Ca2+]i) in all individuals, as measured by quin 2 tetra-acetoxymethyl ester fluorescence. This indicates that the lack of responsiveness of approximately 30% of individuals to UCHT1 in proliferation assays is not due to failure of the antibody to elicit Ca2+ mobilization and that a rise in [Ca2+]i is per se not adequate to induce cell division. Another mitogenic antibody, WT-31, which is directed against the constant portion of the T cell receptor, did not, however, produce a rapid calcium rise in peripheral blood T cells. The clone HA1.7 gave a similar Ca2+ response to UCHT1. WT-31 did not induce a rise in [Ca2+]i, nor did the specific antigen to which the clone responded. Accessory cells may be required to induce Ca2+ mobilization with these ligands. There was no response to IL2, or an antibody (anti-Tac) to the IL2 receptor. In contrast to peripheral blood T cells treatment of HA1.7 with WT-31 led to an enhancement of the calcium response to subsequent UCHT1 addition. Furthermore, cross-linking of WT-31 on the surface of HA1.7 cells did produce a small rise in [Ca2+]i. The IL2-independent malignant T cell line, HUT78, exhibited a calcium response to both UCHT1 and WT-32. Both of these responses occurred without cross-linking. The T cell receptor is closely associated with cell-surface proteins, including the T3 antigen and these studies confirm the importance of the T3 antigen in T cell activation. They also suggest that the relationship between the T cell receptor and the T3 antigen may vary in T cells in different proliferative states.     

Effect of lobenzarit disodium (CCA) on B cell differentiation to antibody secreting cells

We investigated the effect of the immunomodulator CCA on B cells, using the system of Staphylococcus aureus Cowan 1-stimulated B cells developing into antibody secreting cells in the presence of T cell factors. Results were expressed as the number of plaque-forming cells (PFC), as measured by reverse hemolytic plaque assay. Serially diluted CCA was added to the culture system to evaluate its effect (500-0.005 micrograms/ml). Different results were obtained by adding CCA at a high dose or a low dose. High-dose CCA showed an inhibitory effect on PFC (p less than 0.01). On the other hand, low-dose CCA showed an inhibitory effect on high PFC responses of B cells and a stimulatory effect on low responses. Our data suggest that CCA has a direct effect on B cells and that low-dose CCA has immunomodulatory action on normal variations in immune responses.     

Separation of human suppressor and helper T cells by concanavalin A-coated sheep erythrocytes

Normal human peripheral blood mononuclear leukocytes (PBML) were activated by concanavalin A (Con A). Con A-activated and non-activated T cells were separated by E (AET) rosettes (2-aminoethylisothiouronium hydrobromide treated sheep erythrocyte rosettes). Purified T cells were rosetted with Con A-coated sheep red blood cells (Con A-SRBC) at 37 degrees C resulting in Con A-SRBC rosetted and non-rosetted T cells. The Con A-SRBC rosetted T lymphocytes in the T lymphocytes from Con A-activated and non-activated PBML were 44.4 +/- 5.4 percent and 16.0 +/- 7.5 percent (Mean +/- S.D.) while the Con A-SRBC non-rosetted T lymphocytes were 55.6 +/- 5.4 percent and 84.0 +/- 7.5 percent respectively. The Con A-SRBC rosetted and non-rosetted T cells were separated by Ficoll-Hypaque gradient centrifugation. Functional studies of Con A-SRBC rosetted and non-rosetted T cells were performed by in vitro tests using pre-amplified reverse hemolytic plaque assay for measuring numbers of immunoglobulin G (IgG) secreting cells and ELISA quantitation of IgG concentration. Both techniques were used to assess the suppressor and helper functions of the Con A-SRBC rosetted and non-rosetted T cells. The Con A-SRBC rosetted cells obtained from T cells of Con A-activated PBML showed strong suppressor activities to normal PBML in both pre-amplified reverse hemolytic plaque assay and sandwidh ELISA of IgG concentration, while the Con A-SRBC non-rosetted T cells demonstrated strong helper activities to normal PBML in both assay systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

T cell activation by anti-CD3 antibodies: function of Fc receptors on B cell blasts, but not resting B cells, and CD18 on the responding T cells

Mouse anti-human CD3 (T3) antibodies can induce T cell proliferation in the presence of Fc receptor (FcR)-bearing accessory cells. Depending on whether the particular antibody can interact with the FcR, it can be mitogenic or otherwise. Previously, some of us (Smith, K. G. C. et al., Eur. J. Immunol. 1986. 16:478) examined human T cell responses to the murine anti-CD3 antibody switch variants UCHT1 (IgG1) and UCHT1B (IgG2b). Using a novel xenogeneic system with mouse macrophages (M phi) and an anti-FcR antibody, 2.4G2, we obtained direct evidence for accessory function of FcR in these responses. However, mouse B cells which also possess FcR were not accessory cells. Here we show that resting B cells do not inhibit anti-CD3 responses in the presence of other accessory cells, and they do not synergize with them. They appear to be inert in these responses but this is not simply because of their radiosensitivity. In contrast, B cell blasts proved to be potent stimulators of responses with UCHT1, UCHT1B and OKT3 (IgG2a). All three responses were inhibited by 2.4G2, whereas we have shown previously that the OKT3 response with M phi was not, in keeping with the known specificities of B cell and M phi FcR. These findings are discussed in relation to the molecular cloning of FcR, and we consider the possibility that distinct FcR could be expressed on resting and activated B cells. A report that anti-CD18 (LFA-1) antibodies blocked the UCHT1 response with human monocytes raised the possibility that this molecule might also be involved in accessory function. However, we show that this inhibition is in fact at the level of the T cell, since anti-human, but not anti-mouse CD18 antibodies, inhibited proliferative responses and clustering with both human and mouse accessory cells. Our results demonstrate that the principal contribution of accessory cells to anti-CD3 responses may be the provision of an FcR, and that CD18 is most probably required at the level of the T cell.     

Interferon-gamma inhibits the proliferation but not the differentiation of murine B cells in response to IL-5

Interferon-gamma (IFN-gamma) is supposed to be produced by type 1 helper T cells (TH1) and inhibits IL-4-dependent B cell growth and differentiation. IL-5 (T cell-replacing factor, TRF), is a T cell-derived lymphokine which is predominantly produced by type 2 helper T cells (TH2) and regulates proliferation and differentiation of activated B cells. In this study, the effect of IFN-gamma on IL-5-dependent B cell growth and differentiation has been studied using murine chronic B cell leukemic cells (BCL1), normal splenic B cells, and cloned early B cell line. IFN-gamma selectively inhibits the IL-5-mediated proliferation of activated B cells as well as cloned early B cell lines at a low concentration (2 U/ml) in which polyclonal IgM production was not affected. This inhibitory effect of IFN-gamma occurs within 24 h after the onset of culture, as demonstrated by the inability of antibody to IFN-gamma to reverse totally the IFN-gamma-mediated suppressive effects if it was added later than 24 h after the onset of the culture. On the contrary, IL-5-mediated IgM secretion of BCL1 and IgA formation of LPS-stimulated normal B cells were relatively resistant to the suppressive effect of IFN-gamma. IFN-gamma does not affect the receptor expression for IL-5. Interestingly, IL-4-mediated IgG1 formation of LPS-stimulated B cells was markedly suppressed by IFN-gamma at 10 U/ml. These results strongly suggest that IFN-gamma may have differential effects on IL-5-mediated B cell triggering.     

A pre-amplified reverse hemolytic plaque assay

A pre-amplified reverse hemolytic plaque assay has been developed. Sheep red blood cells (SRBC) were coated with staphylococcal protein A (SPA) by the chromic chloride method. The protein A-coated SRBC (SPA-SRBC) was then pre-amplified with an appropriate amount of human class-specific Ig. The pre-amplified Ig-SPA-SRBC was used to detect class-specific Ig-producing cells. It was found that this pre-amplified reverse hemolytic plaque assay gave clearer, larger and more numerous hemolytic plaques which were easy to count and thus gave more accurate results.     

Defect in the tissue cellular immune response: experimental visceral leishmaniasis in euthymic C57BL/6 ep/ep mice.

In BALB/c mice, successful defense against visceral leishmaniasis is T cell dependent, expressed by tissue granuloma formation, and probably mediated by macrophages activated by cytokines, including gamma interferon (IFN-gamma). C57BL/6 ep/ep (pale ear) mice, which reportedly exhibit impaired IFN-gamma production, were challenged with Leishmania donovani to determine the outcome of infection in a euthymic host with an apparent defect in lymphokine secretion. In BALB/c and normal C57BL/6 mice, L. donovani liver burdens peaked at 2 weeks and were largely eliminated by 4 weeks. In contrast, in pale ear mice, infection progressed until after 4 weeks and persisted at high levels at 8 weeks. The failure to resolve hepatic infections was not related to deficiencies in (i) Thy-1+, L3T4+, or Lyt-2+ T cells; (ii) IFN-gamma secretion; (iii) liver tissue Ia expression; (iv) macrophage antimicrobial capacity; or (v) antileishmanial antibody production. However, despite the anticipated influx of mononuclear cells into livers, these cells were not properly focused on the parasitized Kupffer cells, the inflammatory infiltrate receded prematurely, and mature granulomas failed to develop. These results suggest that there is a cellular immune defect at the tissue level and emphasize the critical role of granuloma formation in successful resolution of systemic intracellular infections.     

Enumeration of cytokine-secreting cells at the single-cell level

A sensitive assay utilizing enzyme-linked immunosorbent assay methodology has been developed for the quantitation of single cells secreting interferon (IFN)-gamma or tumor necrosis factor (TNF). Cloned T cells or cells from lymphoid organs were stimulated with antigen, concanavalin A, or phorbol myristate acetate and ionomycin in microwells coated with antibodies specific for IFN-gamma. Discrete "spots" overlying areas where cells secrete IFN-gamma were then developed by incubation with a second antibody to IFN-gamma, followed by an enzyme-labeled antibody conjugate and substrate. Similarly, using TNF-specific antibody reagents, TNF-secreting cells were detected and quantitated in cell populations obtained from normal lymphoid tissues, bone marrow and peripheral blood, following activation with phorbol myristate acetate and ionomycin. Provided specific antibodies are available, this method has the potential to measure the frequency of cells secreting any cytokine.     

Induction of T-cell activation or anergy determined by the combination of intensity and duration of T-cell receptor stimulation, and sequential induction in an individual cell

It has been shown that anergic T cells have important roles in peripheral tolerance, although the precise mechanism for inducing anergy is still unclear. We analysed the kinetics of anergy induction at an individual cell level by flow cytometry. We first successfully obtained T helper type 1 (Th1) cells that had been made uniform with the level of interferon-gamma (IFN-gamma) production induced by antigen stimulation. We then used these Th1 cells to evaluate the degree of anergy for each Th1 cell treated with an anti-CD3 monoclonal antibody according to the level of IFN-gamma secretion. Our results demonstrate that anergic stimulation could induce both activation and anergy, depending on the duration and intensity of stimulation at the level of an individual cell. Each Th1 cell was first activated and then gradually became anergic depending on the duration of stimulation. The duration of the stimulus required for inducing anergy became shorter as the intensity of stimulation became stronger. We also show that the calcineurin signal controlled the induction of activation or anergy depending on the activity. This study contributes to better understanding of the precise mechanism for inducing T-cell anergy.     

Analysis of mammosomatotropic cells in normal and neoplastic human pituitaries

The reverse hemolytic plaque assay (RHPA) was used to analyze hormone secretion in normal and neoplastic human pituitary cells. Immunocytochemical (ICC) staining for PRL and GH with the peroxidase method and ultrastructural ICC with colloidal gold labeling were used along with the RHPA to analyze for mammosomatotropic (MS) cells in these dissociated and cultured pituitary cells. MS cells were identified in both normal and neoplastic pituitary tissues. Quantitation of plaque areas, showed that PRL cells from normal pituitaries had significantly larger plaque areas than PRL cells from PRL-producing adenomas or from mixed PRL-GH producing adenomas.     

Generation of immunoglobulin secreting cells in mixed lymphocyte culture

The nature of the cellular interactions and role of the HLA system in the generation of immunoglobulin secreting cells in primary and secondary mixed lymphocyte cultures were investigated. The B lymphocyte response to alloantigen stimulation as measured by a Protein A reverse hemolytic plaque assay, consisted of polyclonal activation with production of IgG, IgM, IgA secreting cells detectable as early as day 4 in a primary and by 24 hr in a secondary mixed lymphocyte culture. B cell activation was shown to be dependent upon collaboration with T helper cells. A disparity at the HLA D/DR region between responding and stimulating cell populations was required for the induction of T helper cells. However, once activated, T helper cells could collaborate with autologous or allogeneic B lymphocytes and, without additional antigen, trigger immunoglobulin production. The mixed lymphocyte culture may now be considered a model of B cell as well as T cell activation.     

Detection of intracellular expression and secretion of interferon-gamma at the single-cell level after activation of human T cells with tetanus toxoid in vitro

Activation of T cells results in intracellular expression and secretion of cytokines such as interferon (IFN)-gamma. Here we have used three different assays for determination of IFN-gamma in tetanus toxoid- or mitogen-activated human T cell cultures. Two of these assays [intracytoplasmic immunofluorescence and enzyme-linked immuno spot assay (ELISPOT)] determined the expression and secretion of IFN-gamma at the single-cell level while the third assay enzyme-linked immunosorbent assay (ELISA) measured IFN-gamma secreted into the culture supernatant. Comparison of all three tests revealed a good correlation between the ELISPOT assay and the ELISA, whereas expression of intracellular IFN-gamma showed a qualitative but not a quantitative correlation with the latter. Both the immunospot assay and the immunofluorescence may be used to detect approximate numbers of specific T cells even when present at low frequencies. With the use of the immunospot assay antigen-specific T cells could be detected even in the absence of detectable IFN-gamma in the culture supernatants. However, the ELISA assay should be more convenient for screening large clinical material.     

Effects of monoclonal antibodies to the alpha and beta chains of the human lymphocyte function-associated (H-LFA-1) antigen on T lymphocyte functions

The ability of two antibodies, one specific for the alpha chain, p180, and the other for the beta chain, p95, of the human lymphocyte function-associated (LFA-1) antigens, to inhibit T cell function was measured. Both antibodies inhibited T cell-mediated lysis of virus-infected target cells and of K562 cells. Only the anti-beta chain antibody inhibited natural killer cell lysis of K562. The antibodies inhibited cytotoxic T lymphocyte cell (CTL) lysis of HLA-mismatched target cells in the presence of concanavalin A at 6.25-12.5 micrograms/ml, but at higher doses of Con A no inhibition was seen. When the lytic process was divided into calcium-independent (adherence) and -dependent (lysis) steps the antibodies were found to block at the initial step of conjugate formation. The effects of these antibodies on T cell proliferative responses showed that responses to antigens, alloantigens, mitogens and anti-CD3 (UCHT1) antibody were greatly inhibited. All of these responses are adherent cell dependent and proliferation of adherent cell-depleted mononuclear cells to Sepharose-coupled UCHT1 was not inhibited by anti-LFA-1 antibodies. Proliferation to paired anti-CD2 (T11) antibodies was also only weakly inhibited. Release of interferon-gamma by CTL on contact with target cells was also inhibited by anti-LFA antibody. These results are evidence that the LFA antigen is necessary for a nonspecific interaction with antigen-presenting cells that is essential for activation of T cells through the CD3-T cell receptor complex.