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.     

Cellular composition of suppurative granulomas: an immunohistochemical study of suppurative granulomatous lymphadenitis

The immunohistochemical findings from an investigation of suppurative granulomatous lymphadenitis (SGL) are presented. With a broad panel of monoclonal and polyclonal antibodies directed against B cells, T cells, monocytes/macrophages, HLA-DR antigens, and the transferrin receptor, early, nonsuppurative granulomas were found to consist of OKM1+ OKIa1+ OKT9+ epithelioid histiocytes and multinucleated giant cells, admixed with variable numbers of OKT4+ Leu-3a+ helper/inducer T cells. These nonsuppurative lesions were surrounded by distinctive cuffs of BA1+ B1+ sIgM+ sIgD+ OKIa1+ lymphocytes. In contrast, suppurative granulomas were bordered by palisades of OKM1+ OKIa1+ OKT9+ epithelioid histiocytes, admixed with some OKT8+ suppressor/cytotoxic T cells. These suppurative lesions lacked distinctive cuffs of B lymphocytes, but half of the lesions were surrounded by numerous plasma cells that expressed cytoplasmic IgA and IgG. Based on these immunohistochemical findings, it is concluded that a shift in the nature of the predominant intragranulomatous T-cell subset occurs during the successive phases of the immune response in SGL. The cause of the central necrosis and suppuration may be related to the excessive numbers of intragranulomatous OKT8+ T cells or to the formation of immune complexes by the surrounding plasma cells.     

OKT4+ T cell deficiency and an association of immunoglobulin deficiency in systemic lupus erythematosus.

A patient with systemic lupus erythematosus and selective serum IgA and IgM deficiency also had a deficiency of OKT4+, OKT4A+, OKT4B+, and OKT4E+ T cells in circulating blood. This deficiency of helper/inducer T cell subsets in blood was associated with impaired T cell responses to phytohaemagglutinin in vitro, and an impaired ability to produce IgA and IgM by the patient's B cells. The patient's OKT4+ (CD4+) T cell deficient population suppressed IgM synthesis by normal B and T cells.     

Differences in the stimulating capacity of immobilized anti-CD3 monoclonal antibodies: variable dependence on interleukin-1 as a helper signal for T-cell activation.

Monoclonal antibodies to the CD3 antigen on human T lymphocytes have been shown to induce accessory cell-dependent T-cell activation. One function of the accessory cells is cross-linking of CD3 by Fc receptor-binding of the anti-CD3 antibodies. Whether additional accessory signals are still required when anti-CD3 is presented in immobilized form is controversial. In the present study we stimulated purified human T cells with several anti-CD3 monoclonal antibodies, which were immobilized by coating the culture wells with goat anti-mouse IgG. A first group of immobilized anti-CD3 antibodies (anti-Leu-4, UCHT1, anti-T3, WT32 and 64.1) induced vigorous T-cell proliferation in the complete absence of monocytes, even when anti-interleukin-1 beta antiserum was added to the cultures. Other immobilized anti-CD3 antibodies (OKT3, WT31) required interleukin-1 beta in order to induce T-cell proliferation. However, when OKT3 was immobilized by direct coating of the culture wells with OKT3, it was also able to induce accessory cell-independent production of interleukin-2 and T-cell proliferation. Interleukin-1 beta further enhanced the interleukin-2-dependent proliferative response and it could provide help to induce proliferation at doses of immobilized OKT3 which, by themselves, were insufficient for full T-cell activation. We conclude that the requirement for interleukin-1 beta to induce interleukin-2-dependent proliferation of T cells when stimulated with anti-CD3 antibodies is not absolute, but depends on the CD3 epitope recognized, on the way of antibody presentation, on the antibody concentration and on other, still undefined, characteristics of the monoclonal antibodies used.     

T-cell subsets in multiple sclerosis: a comparative study between cell surface antigens and function

Pokeweed mitogen (PWM)-driven immunoglobulin synthesis (IgG, IgA, IgM) and concanavalin A (Con A)-stimulated suppression of allogeneic mixed leukocyte reaction (MLR) were studied and compared to T-cell subsets defined by monoclonal antibodies OKT4 and OKT8 in patients with multiple sclerosis (MS). The group of patients with active progressive MS showed diminished suppressor activity as measured by T-cell functional tests and also an elevated OKT4/OKT8 ratio. The group of MS patients in remission did not show these abnormalities. However, this correlation between functional tests and T-cell phenotypes was not found when separate individuals were considered within the subgroups of MS. Since neither OKT4 nor OKT8-reactive cells represent homogeneous functional subsets of T cells, the OKT4/OKT8 ratio does not account for the functional immunological status of separate individuals but rather provides a global evaluation of T-cell subset disturbances in different groups of diseases.     

T-cell influence on the B-cell differentiation process induced by Klebsiella

Klebsiella pneumoniae K43 cell membrane preparations (Klebs M) have been characterized previously as a human polyclonal B cell activator (PBA) that stimulates purified B cells to differentiate into immunoglobulin (Ig) secreting cells with negligible prior or parallel proliferation and in the absence of T cells. The aim of the present study was to define the cellular interactions in the regulation of Klebs M induced B-cell differentiation. For this purpose OKT4+ and OKT8+ cell populations were negatively selected with reasonable purity by means of a panning technique or by complement-mediated cytolysis using monoclonal OKT4 and OKT8 antibodies. The resulting cell populations were added to purified autologous B cells exposed to Klebs M or, as a control, pokeweed mitogen (PWM). In the Klebs M system both the OKT4+ and the OKT8+ cell subsets markedly enhanced IgM production; however, the helper effect of the OKT4+ cell subset was much more intense than that of the OKT8+ subset. In the PWM system only the OKT4+ cells provided help for B-cell differentiation. The OKT8+ subset demonstrated suppressor activity in the presence of an adequate helper cell (OKT4+ subset) function. These results indicate that Klebs M behaves like a "relatively T cell-independent PBA".     

Selective partial IgM deficiency: Functional assessment of T and B lymphocytesin vitro

Seven patients with selective IgM deficiency (SIgMD) were studied for cell surface immunoglobulin (SmIg), T-cell subpopulations, and immunoglobulin (Ig) synthesisin vitro by peripheral blood lymphocytes (PBL). Serum IgM levels were less than 25 mg/dl, while IgA, IgG, and IgD were within normal levels. The patients had respiratory or urinary tract infections and two were diagnosed as having systemic lupus erythematosus (SLE). T/B-cell ratios in PBL were within normal ranges. Percentage ratios of B cells bearing SmIg were normal in five patients and decreased in two; however, normal values were seen after 7 days of culture in the presence of PWM. OKT4/OKT8 ratios decreased in five of seven patients, in whom two were due to a decrease in OKT4 and two to an increase in OKT8 cells. One showed a decrease in OKT4 and an increase in OKT8. Analysis of lymphocyte function for Ig synthesisin vitro, using a coculture of counterpart T and B cells from healthy individuals and patients with SIgMD, revealed that the increased function of IgM isospecific suppressor T cells (Ts) was responsible for the IgM deficiency in all seven patients.     

Ganglioside GT1b suppresses immunoglobulin production by human peripheral blood mononuclear cells

Gangliosides are sialic acid-containing glycolipids and have various immunomodulatory effects. We previously reported that various gangliosides in vitro either inhibited or enhanced spontaneous immunoglobulin production by human peripheral blood mononuclear cells (PBMC). Among them, GT1b was the most inhibitory. In this study, we further examined the mechanism for the inhibitory effect of GT1b. The inhibitory effect of GT1b was apparent at 0.1 micrometers, increased dose dependently, and was maximal at 10 micrometers. In the presence of 10 micrometers GT1b, spontaneous production of immunoglobulin (Ig)G, IgM and IgA in human PBMC was reduced by 60%, 59.5% and 58%, respectively, compared with controls. GT1b did not affect the proliferation and viability of PBMC, and did not enhance their apoptosis. GT1b did not alter immunoglobulin production of B cells alone. Interleukin (IL)-6 and IL-10 each partially reversed the GT1b-induced inhibition of immunoglobulin production by PBMC, and the presence of both cytokines completely reversed the inhibition. GT1b inhibited IL-6 and IL-10 production in monocytes, without affecting that in T or B cells. When monocytes were preincubated with GT1b, washed and then cultured with B and T cells, the immunoglobulin production was also suppressed. These results suggest that GT1b may indirectly suppress immunoglobulin production of B cells in whole PBMC via reducing the production of IL-6 and IL-10 in monocytes. It is thus indicated that GT1b may act as an important inhibitor for human humoral immune responses.     

Production of human immune interferon (Hu IFN-gamma) studied at the single cell level. Origin, evidence for spontaneous secretion and effect of cyclosporin A

A reverse hemolytic plaque assay has been developed which specifically detects secretion of human immune interferon (Hu IFN-gamma) at the single cell level. Unstimulated peripheral blood lymphocytes from healthy adult volunteers spontaneously secreted IFN-gamma. Stimulation of these cells with concanavalin A, phytohemagglutinin, or the UCHT1 monoclonal anti-human T cell antibody significantly increased the number of IFN-gamma-secreting cells. The cell producing IFN-gamma, both spontaneously and after UCHT1 antibody stimulation, is an OKT3+,4+,8-,HLA-DR-T lymphocyte as determined at the single cell level. Finally, cyclosporin A, a potent and selective immunosuppressive drug for T cells, strongly inhibited the secretion of IFN-gamma as assayed at the cell level. This IFN-gamma reverse hemolytic plaque assay has great potential for the further study of IFN-gamma both in physiological and pathological conditions.     

Immunological parameters in the aged and in Alzheimer's disease.

Peripheral blood from patients with Alzheimer's disease, elderly normal subjects and young (normal subjects) was examined with respect to leucocyte phenotypes and proliferative responses to lectins. Whole blood cell analysis showed that the neutrophil count was similar in all three groups. However, the lymphocyte count was depressed in the Alzheimer group. The monocyte count was reduced in the healthy aged and further reduced in the Alzheimer group. Analysis of peripheral blood mononuclear cells showed no change in the proportion of E+ cells, total T determined with the monoclonal antibody UCHT1 (similar to OKT3) and the T cell subsets: active E and T dot (discrete esterase staining). However, the proportion of T cells bearing the antigen detected by UCHT3 monoclonal antibody (similar to Leu 1 and OKT1) was reduced in the healthy aged and further reduced in the Alzheimer group. Proliferative responses to PHA, Con A, PA, and PWM were similarly depressed in both the aged and Alzheimer groups.     

Induction of Human T Colony Formation by Phorbol Myristate Acetate

Phorbol myristate acetate (PMA) is a potent inducer of T colony formation by peripheral blood lymphocytes. A mean cloning efficiency of 0.3% (0.05-0.5%) is obtained with PMA concentrations of 100-1000 ng/ml. PMA-induced T colony formation does not require the presence of monocytes and therefore differs from other mitogens in this respect. Purified T-colony-promoting activity (TCPA) (devoid of phytohaemagglutinin (PHA)) increases PMA-induced T colony numbers and induces T colony formation at low PMA doses (0.01 to 1 ng), concentrations at which no T colonies are detected in the absence of added TCPA. PMA-induced colonies are mainly composed of cells bearing Fc receptors for IgM (54%), which is not the case for colonies obtained with PHA (11%). PMA-induced colony cells do not bind OKT3 and OKT4 monoclonal antibodies, whereas 23% are able to bind OKT8 antibody. These results demonstrate that PMA is a potent inducer of T colony formation and may therefore serve as a useful tool for the study of T-cell differentiation.     

Antibodies to the T3 antigen complex on human T cells render thymocytes unresponsive to mitogen.

Human thymocytes differed from peripheral blood T cells, in that they did not proliferate in response to mouse monoclonal antibodies specific for the T3 antigen complex (UCHT1 and OKT3), even in the presence of T-cell growth factor (IL-2) or with monocytes plus IL-2. The failure to respond was not the result of inhibition by cortical thymocytes, since OKT6-negative cells also did not respond when tested under conditions of limiting dilution. Of more importance, these antibodies rendered human thymocytes unresponsive to the lectin phytohaemagglutinin when added before, during, or immediately after addition of the lectin, an effect which was much more profound than the decrease in mitogenesis caused with blood mononuclear cells. These results illustrate a clear difference between medullary thymocytes and peripheral T cells in their ability to respond to signals transduced through the T3 antigen complex.     

A monoclonal antibody with specificity for murine mu heavy chain which inhibits the formation of antigen-specific direct IgM plaques

A panel of monoclonal rat antibodies binding to mouse mu heavy chain were tested for their ability to inhibit the formation of antigen-specific plaques in the hemolytic plaque assay. Nine antibodies inhibited SRC-specific direct IgM plaques at high concentrations (greater than 20 micrograms/ml). In contrast to all others, however, one antibody inhibited these plaques at much lower concentrations (down to 0.4 microgram/ml) when added to the assay. This antibody also inhibited plaques formed by cells secreting antibodies against trinitrophenyl or phosphorylcholine determinants. IgG plaques with any of the above specificities were not inhibited. IgM secretion was unaffected by the monoclonal anti-mu antibody. Its inhibitory effect on plaque formation rather appears to be a consequence of its ability to inhibit complement dependent, IgM mediated lysis of erythrocytes. This monoclonal anti-IgM antibody therefore provides a convenient reagent to distinguish specific direct IgM plaques from indirect IgG plaques.     

Heterogeneity of immunological abnormalities in ataxia-telangiectasia

Peripheral blood mononuclear cells from five patients with ataxia-telangiectasia were evaluated for their reactivity with a panel of monoclonal antibodies directed against T-cell subsets and for theirin vitro functions in a pokeweed mitogen-induced immunoglobulin biosynthesis assay. All the patients had significantly reduced proportions of cells identified by monoclonal antibodies to subpopulations of T lymphocytes with helper activity (OKT4 and 5/9) and produced low amounts or no IgA and IgGin vitro. Immunoglobulin biosynthesis was increased by the addition of normal x-irradiated peripheral blood mononuclear cells in one of three patients, suggesting a helper T-cell deficiency in this patient and intrinsic B-cell defects in the other two. Two patients had increased proportions of cells identified by a monoclonal antibody to a subpopulation of T lymphocytes which includes suppressor T cells (OKT8), and their cells were able to suppress immunoglobulin biosynthesis by peripheral blood mononuclear cells from normal donors. These findings indicate heterogeneous disturbances of immunoregulatory mechanisms in ataxia-telangiectasia.     

The cellular composition of human lymph nodes after allogenic bone marrow transplantation: an immunohistological study

Using immunohistological techniques, the cellular composition of lymph nodes was assessed in 18 patients who had died 15 to 326 days after allogeneic bone marrow transplantation for leukaemia. The lymph nodes showed reduced cellularity of the cortex and paracortex, dilated sinuses and no lymphoid follicles. The majority of leucocytes were T lymphocytes with an inversion of the normal T4:T8 ratio. No cells were detected expressing immature cortical thymocyte antigens, using NA1/34 and OKT10, but an excess of T11 (E rosette receptor)+ cells over the sum of T4+, T8+ and HNK1+ cells raised the possibility of the presence of immature cells. B lymphocytes were extremely rare and present as clusters in only two patients. Despite this, plasma cells were prominent in many cases and their number increased with time post transplant. The predominant immunoglobulin heavy chain class was IgA in seven cases, IgG in three cases, IgM in two cases and IgE in one case with no relationship between dominant class and days post transplant. In patients with graft-versus-host disease (GvHD), there was a significantly lower T4:T8 ratio but no increase in expression of lymphocyte activation markers. Pyknotic leucocytes were present in half of the cases with GvHD and none of the other cases. No differences were detected in patients who had received marrow purged with monoclonal antibodies (Campath-I or UCHT1). Chimeric studies on three recipients of one haplotype matched marrow, using a monoclonal antibody specific for HLA-A2 and A28 antigens, showed a significant influx of donor cells by 56 days but this did not appear to be an immediate prelude to full morphological reconstitution.     

In vitro immunoglobulin production by mononuclear cells in rheumatoid arthritis

Since patients with rheumatoid arthritis (RA) exhibit serum hypergammaglobulinemia and autoantibody (rheumatoid factor) production, we compared elaboration and control of in vitro RA mononuclear cell (MNC), Ig assayed by enzyme-linked immunoassays or by hemolytic plaque formation, in 37 RA patients and 17 normal subjects. We found (1) RA spontaneous plaque-forming cells were significantly reduced (RA 344 vs normal 627 PFC/10(6) MNC, P less than 0.002); (2) RA spontaneous IgG and IgM (but not IgA) elaboration was significantly diminished (IgG RA 339, normal 776; IgM RA 255, normal 869 ng/ml, P less than 0.001; IgA RA 87, normal 124); (3) RA stimulated IgG and IgM production (but not IgA) was also decreased (IgG RA 2434, normal 3862, P less than 0.06; IgM RA, 1676, normal 3323, P less than 0.005; IgA RA 1859, normal 2315); (4) reduced RA Ig elaboration was not clearly due to altered numbers of T or non-T cells, age, medications, clinical features of disease, or response kinetics; (5) relative improvement of RA in vitro IgG, but not usually IgM, secretion followed removal of adherent cells, addition of indomethacin or addition of mitomycin C-treated T cells; (6) MNC from synovial fluids, but not bone marrows, exhibited spontaneous Ig production in excess of stimulated synovial fluid cellular or peripheral blood Ig elaboration. These observations indicate selective impairment of peripheral blood MNC IgG and, particularly, IgM secretion in RA. This defect appears to reflect accessory cell influences which differ from normal as well as the sequestration of primed or activated cells in the synovial fluid.     

B cell activation by pokeweed mitogen in cultures of normal peripheral blood lymphocytes depleted of T regulator subsets by treatment with OKT4 and OKT8 monoclonal antibodies.

OKT4+ (T helper/inducer) and OKT8+ (T cytotoxic/suppressor) subsets were depleted from peripheral blood lymphocytes (PBL) by complement-mediated lysis and residual cells examined for responsiveness to pokeweed mitogen (PWM) using a protein A haemolytic plaque assay for immunoglobulin secreting B cells. It was shown that: (1) three cycles of cell killing were required to totally abolish T helper function; (2) OKT4- PBL did not respond to PWM, but in a co-culture system, an equal number of unfractionated normal PBL could entirely reconstitute responsiveness of the residual B cells; (3) OKT8- PBL gave enhanced numbers of PWM-induced plaque forming cells (PFC); (4) addition of 4 micrograms/ml concanavalin A (con A) to PWM stimulated OKT8- PBL failed to suppress PFC generation, but suppression was induced by 12.5 and 25 micrograms/ml con A and (5) kinetics of PWM-induced PFC development were similar in the presence or absence of OKT8+ cells.     

Characterization of the suppressor activity in lymphocytes from patients with common variable hypogammaglobulinemia: evidence for an associated primary B-cell defect

The pathogenetic mechanisms responsible for the impaired immunoglobulin production in common variable hypogammaglobulinemia (CVH) are diverse with abnormalities in both B cells and immunoregulatory T cells. Production of IgG, IgM, and IgM-rheumatoid factor (IgM-RF) was measured in pokeweed mitogen (PWM) or Epstein-Barr virus (EBV)-stimulated cultures using various combinations of CVH, cord blood mononuclear cells (CBMC), and normal adult control B and T cells. The following results were obtained. First, the proportion of OKT3+ and OKT8+ cells were increased in CVH patients. Second, the T cells from four CVH patients and CBMC suppressed PWM-induced IgG, IgM, and IgM-RF production by normal B cells. Furthermore, major suppressor activity was found in the OKT8+ T-cell subpopulations in CBMC and three out of four CVH patients. There was no significant difference in relative suppression by OKT8+ cells from normal adults, CVH patients, or CBMC. However, in one CVH patient suppressor T cells were found in both OKT4+ as well as OKT8+ fractions. In the CVH patient with OKT4+ suppressor cells, X irradiation (1250 rads) abrogated suppressor activity and restored helper activity in the OKT4+ T-cell fraction. Irradiation of normal OKT4+ cells did not increase helper activity. When non-E-rosetting cells from normal subjects, CVH, and CBMC were stimulated with EBV it was observed that normal adult B cells could be induced to secrete IgG, IgM, and Ig-RF whereas CVH and CBMC could only produce IgM and IgM-RF but not IgG. The present study demonstrates for the first time that a radiosensitive OKT4+ suppressor cell is present in some CVH patients.     

Uptake of free adenosine and adenosine from adenosine monophosphate by human peripheral blood lymphocytes: possible kinetic role for ecto-5''-nucleotidase in the regulation of intracellular adenosine.

Human gut-associated immunoregulatory events were studied in a pokeweed mitogen (PWM)-stimulated culture system using lymphocytes obtained from the mesenteric lymph nodes (MLN) of female subjects undergoing gastroplasty for obesity. Compared with peripheral blood lymphocytes, lymphocytes obtained from MLN secreted IgG, IgA and IgM isotypes that differ in pattern and distribution despite similar proportions of T cells and B cells expressing isotype-specific surface membrane immunoglobulin (SmIg). Among the isotypes secreted, IgA appeared to be increased relatively to other isotypes in MLN cultures. Crossover coculture experiments using T and B cells isolated from both MLN and blood by E-rosetting and cell panning procedures demonstrated that IgA was particularly sensitive to help and suppression exerted by MLN T cells and T cell subsets defined by monoclonal antibodies OKT4 and OKT8 respectively, when compared with similar subsets isolated from blood. The results presented provide a basis for study of gut handling of ingested antigen in man, and of disturbed immunoregulatory events in inflammatory and neoplastic disease of the human gut.     

Immunological studies of ageing. X. Impaired T lymphocytes and normal monocyte response from elderly humans to the mitogenic antibodies OKT3 and Leu 4.

Lymphocytes from old and young humans were cultured with PHA or the monoclonal antibodies OKT3 or Leu 4. The incorporation of [3H]TdR was significantly lower in cultures from old as compared to young donors, and the response of lymphocytes stimulated with OKT3 was the best discriminator of donor age. The mitogenic response of lymphocytes to these monoclonal antibodies requires monocytes. The response of T cells containing less than 5% adherent cells was diminished and the difference between old and young donors was not seen. The age-associated response was recovered when autologous or allogeneic monocytes were added to T cells. The age-associated response of T cells was the same, whether cultured with monocytes from young or old donors. Thus, monocytes from elderly subjects are not impaired with respect to their capacity to facilitate the proliferative response of T cells stimulated with monoclonal antibodies. Although lymphocytes from elderly donors were more sensitive to the inhibitory effect of prostaglandin E2, this did not account for the age-associated defect as indomethacin did not eliminate this defect. We conclude that the proliferative response of lymphocytes to OKT3 and Leu 4 is a more sensitive discriminator of lymphocyte donor age than is response to plant lectins, and that the age-associated defect in this response appears to reside within the T-cell population and not the monocyte population.