Heterogeneity of concanavalin A-induced suppressor T cells in man defined with monoclonal antibodies.

Human peripheral blood T lymphocytes were enriched for OKT4+ or OKT8+ subpopulations using complement mediated lysis with OKT8 or OKT4 monoclonal antibodies. These subpopulations and unfractionated T cells were separately stimulated with concanavalin A (Con A) for a period of 48 hr and were then examined for their suppressive influence on proliferative response of autologous T cells to phytohaemagglutinin (PHA) or allogeneic non-T cells. Con A-activated unfractionated T cells, OKT4+ and OKT8+ T cell subsets markedly suppressed both these responses. Both OKT4+ and OKT8+ T cell subsets when enriched following Con A-activation of unfractionated T cells also caused significant suppression of proliferative responses of autologous T cells to PHA and allogeneic non-T cells in mixed lymphocyte cultures. The suppressive influence of Con A-activated T subsets was abolished by irradiation (2,000 rad) of activated cells. These studies indicate that Con A-induced suppressor T cells are heterogeneous. Precursors of Con A-induced suppressor T cells appear to reside in both OKT4+ and OKT8+ T cell populations.     

Immunological studies in patients with isolated growth hormone deficiency.

The proportion of total T cells, OKT4+ (helper/inducer phenotype) T cells and activated T cells (Tac+) in four growth hormone deficient children were compared to simultaneously studied age and sex matched healthy controls. Proportions of OKT8+ T cells (suppressor/cytotoxic phenotype), and B lymphocytes (surface immunoglobulin positive) were increased when compared to healthy controls. Increased proportion of OKT8+ T cells resulted in abnormally low ratios of OKT4+/OKT8+ cells. Proliferative response to PHA and in the AMLR were comparable to the control group. In allogeneic MLR, T cells from three of four patients responded poorly and three of four patients non-T cells stimulated poorly in MLR. Con A activated suppressor cell activity was comparable to healthy control group. This study supports the role of growth hormone in certain immune response.     

Differences in sensitivity to melphalan between con A-activated and nonactivated human T-cell subsets

In vitro treatment with melphalan (L-PAM, L-phenylalanine mustard), 2 micrograms/2 X 10(6) cells, significantly decreased the total number of E-rosette-positive (E+) T lymphocytes from peripheral blood (PBL) of healthy human donors as well as those of the OKT4 (precursor suppressor/helper/inducer T cells) and OKT17 populations (suppressor cells within the OKT4 subset). The OKT8 population (cytotoxic/mature suppressor cells) was not affected by a similar L-PAM treatment. The sensitivity of concanavalin A (Con A)-activated E+ T-cell populations to subsequent L-PAM treatment in vitro was different from that of Con A-untreated T cells: Thus, L-PAM treatment did not affect the expression of OKT3 and OKT4 antigens, increased the percentage of OKT17 cells, and inhibited the expression of OKT8 antigen. Depletion of OKT8 from Con A-activated E+ T cells (OKT4+-OKT8(-)-OKT17+) did not affect their suppressive activity on PHA stimulation in L-PAM-treated as well as untreated cells. Further depletion of OKT17+ cells from the OKT4+-OKT8(-)-OKT17+ subset (OKT4+-OKT8(-)-OKT17-) abolished the suppressive effect on PHA stimulation. Suppressive activity of the OKT4+-OKT8(-)-OKT17- subset was again evident after treatment of this population with L-PAM. The results obtained indicate that the sensitivity to L-PAM treatment of various T-cell phenotypes is changed by Con A activation and that after depletion of specific T suppressor cells L-PAM seems affect the immunoregulatory circuit within the Con A-activated OKT4 subset.     

Regulatory Influences on the Response of Rabbit T Cells to Concanavalin A and Phytohaemagglutinin

The proliferative response, induced in rabbit spleen cells by concanavalin A (Con A) and phytohaemagglutinin (PHA), is abolished when T cells are killed with antibody against rabbit thymus lymphocyte antigen (RTLA) in the presence of complement. The response was examined with purified spleen T cells, to which various helper cell fractions were added; it could be shown that B cells help the responding T cells. The helper effect in the response to PHA is abolished and the response to Con A is reduced by any manoeuvre which destroys or removes B cells. Help by B cells is given when helper cells have lost proliferative capacity as a consequence of mitomycin-C treatment. Spleen cells adhering to the walls of culture tubes help suspended T spleen cells in their response to Con A. This help could be abolished by complement mediated cell kill with antibody to rabbit bursal equivalent lymphocyte antigen (RABELA). On the other hand, the helper effect in Con A response was increased when T cells were removed. Thus the response of T cells to Con A is regulated by helper B cells and suppressor T cells.     

In vitro immune cell function in six cases of immunoproliferative small intestinal disease after long term remission

We have studied several parameters of in vitro immune cell function in peripheral blood mononuclear cells from six patients with immunoproliferative small intestinal disease after long term remission. We have observed two groups of patients with different patterns of response. (a) After stimulation with pokeweed mitogen (PWM) and Staphylococcus aureus, three patients showed a significant reduction of the Ig synthesis (A, G and M) and the proliferative response. In two of them, we found increased spontaneous suppressor T cell activity. In the third case, the diminished response could not be attributed, according to our assays, either to suppressor T cells, lack of T helper activity (although the number of OKT4+ cells was diminished) or an intrinsic B cell defect. The three patients showed normal or augmented NK activity and an inversion of the OKT4+/OKT8+ ratio was detected in two of them. (b) The remaining three patients showed a normal Ig synthesis after stimulation with PWM and a slightly depressed IgM synthesis in response to S. aureus. They expressed a normal T helper cell function and did not show increased spontaneous suppressor T cell activity. They had low levels of natural killer cytotoxicity and the OKT4+/OKT8+ ratio was not significantly altered. Taken together, our data indicate that significant alterations of the in vitro immune response can be found in peripheral blood mononuclear cells of some immunoproliferative small intestinal disease patients after long term remission.     

Studies on immunoregulatory mechanisms in acute and chronic hepatitis B

Patients with acute hepatitis B and HBV-induced chronic hepatitis as well as normal control persons participated in the study. Hepatitis patients of both groups have decreased OKT4+/OKT8+T cell ratios due to an percental increase of OKT8+T cells in peripheral blood compared to the data of controls. Lymphocyte cultures of chronic hepatitis patients show reduced DNA synthesis after stimulation by allogeneic non-T cells, PHA, Con A and PWM. PWM-induced immunoglobulin secretion by B cells, determined by means of a reverse haemolytic plaque assay (RHPA) and a solid phase ELISA, showed comparable results in hepatitis B patients and controls. The AMLR, which is thought to reflect an autologous immunoregulatory phenomenon, is slightly impaired in cultures of hepatitis B patients in comparison to controls. Con A-induced suppressor cell activity on T cell reactions is decreased in hepatitis, whereas suppressor cell activity on B cell activation is within the same range as in cultures of controls. It is concluded from these data, that suppressor cell activity on T cell function is impaired in hepatitis B, whereas B cell functions and suppressor cell activity on B cell function are in the normal range. The results with the functional assays and the finding of increased proportions of OKT8+T cells in hepatitis B are considered to reflect properties of different T cell subpopulations, responsible for different immunoregulatory functions.     

Peripheral T Cell Lymphomas: An Immunological Study of Seven Unusual Cases

A multiparameter study of malignant lymph node cells and peripheral blood lymphocytes of seven patients with peripheral T cell lymphoma is presented. The results of monoclonal marker studies showed three cases of helper-suppressor T cell lymphoma (OKT4+, OKT8+), one case of suppressor T cell lymphoma (OKT8+), and three cases of helper T cell lymphoma (OKT4+). Immunophenotypic heterogeneity of neoplastic T cells with expression of pan-T antigens, OKT3+, and OKT11+ (erythrocyte rosetting+) was observed in most patients. Six of the seven cases tested showed Ia and DR antigens. No relationship was detected between patterns of reactivity with T cell reagents and histological types. When tested, the in-vitro malignant T cells of five patients proliferated in response to concanavalin A (Con A), but had poor response to phytohaemagglutinin. The interleukin 2 receptors showed maximum expression on Con A-activated T cells of five patients, and phytohaemagglutinin-activated T cells of one patient. The neoplastic T cells (OKT4+, OKT8+) of one patient studied had suppressor activity for IgG and IgA, and helper activity for IgM synthesis on pokeweed mitogen-induced normal B cell differentiations.     

Reconstitution of in vitro humoral immune function in bone marrow transplant recipients

The process by which humoral immune function is re-established following bone marrow transplantation was investigated in 50 transplant recipients. The recovery of in vitro-specific antibody production directed at sheep red blood cells was found to proceed through three phases. B cell function and helper T cell function were undetectable in the first phase (encompassing the first 5 months after transplantation), in which only suppressor cells reached functional maturity. Suppressor cells controlled responsiveness in the second phase (encompassing the period 5 to 15 months postgrafting). During this period, B cells and helper T cells became fully responsive; their activity became measurable, however, only after removal of Sephadex G-10-adherent suppressor cells. Normal responsiveness (associated with the loss of excessive suppressor cell activity) was characteristic of the third phase (over 15 months posttransplantation). Particular attention was paid to the role of suppressor cells in the recovery of humoral immune function. Their activity was positively correlated with graft-versus-host disease (GVHD), particularly of the chronic type. Suppressor cell activity in the early posttransplant period was predominantly mediated by OKT8-positive T lymphocytes, whereas suppressor cell activity in chronic GVHD patients was predominantly mediated by peripheral blood mononuclear cells that were retained on Sephadex G-10 columns, but did not express OKT8 antigen.     

A suppressor cell in the response of rabbit T cells to Con A.

The response to Concanavalin A is regulated by a helper cell, described elsewhere (4), and by a suppressor cell, described in this paper. This suppressor cell is an adherent T cell with Fc receptors. Evidence for properties of the suppressor cell was obtained by two types of experiments: 1) regulatory cells gave more help if Fc-bearing subpopulations were removed from them, i.e. the suppressor cell could not be removed with Degalan beads, coated with anti-allotype antibody, but not with beads, coated with F(ab')2 fragments of the antibody; 2) help for T cells, in their response to Con A, was augmented when T cells were eliminated from the regulating adherent cell preparation. Thus, the response of spleen T cells to Con A is regulated by two adherent cells, a B helper cell and a T suppressor cell. We have previously shown that the response to phytohemagglutinin (PHA) is regulated by an adherent helper cell (4). We have found no evidence, in the present study for an adherent suppressor cell which participates in the T cell response to PHA.     

Immunoregulation in glomerulonephritis, Henoch--Schonlein purpura and lupus nephritis.

Immunoregulation was examined in normal controls and in patients with immune complex glomerulonephritis and lupus nephritis (SLE) using OKT monoclonal anti-bodies against helper (OKT4) and suppressor (OKT8) T cell subsets. Functional studies assessed T cell control of in vitro immunoglobulin synthesis by cultured peripheral blood mononuclear cells (PBMC). IgG and IgA synthesis was measured in unstimulated, pokeweed mitogen (PWM) stimulated and PWM + concanavalin A (Con A) stimulated cultures. Patients with primary membranous nephropathy (MN) and mesangial IgA nephropathy (IgA GN) were found to have elevated T4/T8 ratios secondary to a deficiency of the T8+ subset. Patients with SLE had low T4/T8 ratios. B cell activation with high spontaneous immunoglobulin synthesis was present in cell cultures from patients with SLE, IgA GN and Henoch-Schonlein purpura (HSP). Defective Con A inducible suppression of in vitro immunoglobulin synthesis was found in SLE, HSP and to a lesser extent, primary MN. Functional Con A inducible suppressor defects correlated with elevated T4/T8 ratios only in patients with MN. All four disorders appear to share disturbances of cellular immune response with various degrees of defective immune suppression; however, it is not clear from these studies whether the defects are primary or secondary phenomena.     

A human autoantibody to renal collecting duct cells associated with thyroid and gastric autoimmunity and possibly renal tubular acidosis

Peripheral blood mononuclear cells (PBMC), obtained from BCG vaccinated healthy donors, were induced to proliferation by BCG for five days in vitro. When re-exposed to BCG, they failed to proliferate. However, they partially retained the ability to respond to Con A and allogeneic cells. The addition of graded numbers of such cultured cells to fresh autologous PBMC suppressed their proliferative response to BCG. These suppressor cells could also inhibit the proliferation of fresh cells to other mycobacterial antigens, both in particulate form, i.e. Mycobacterium leprae, or in soluble form, i.e. PPD and SPA30. However, these pre-cultured cells did not inhibit the response of fresh cells to non-specific mitogens, i.e. Con A and alloantigens. The inhibition of the response to non-mycobacterial soluble antigens, i.e. tetanus toxoid (TT) and diphtheria toxoid (DT) varied with little suppression in some individuals and stronger suppression in others. The suppression to BCG was found to be mediated by T cells. Subfractionation of T cells by monoclonal antibodies OKT4 and OKT8 allocated the suppressor cells to the OKT4+ class of T cells. The suppression in the autologous system was quite strong, whereas it was much weaker in allogeneic systems.     

Phenotype of human T helper and suppressor cells in an in vitro specific antibody response

Monoclonal anti-human T cell antibodies have been used to identify cells which provide help for an in vitro specific antibody response to influenza virus and also those which suppress the response of allogeneic peripheral blood mononuclear cells. The helper cells carry antigens defined by the Leu 3a and OKT4-antisera, whereas the suppressor cells are predominantly Leu 2a- and OKT8-positive. Help and suppression in this secondary in vitro antibody response are therefore provided by antigenically distinct T cell subsets.     

Absence of the OKT4 epitope on blood T cells and thymus cells in a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia

Human helper/inducer T-lymphocytes that express the T4 antigen are important in the regulation of B and T cell functions. Several epitopes of the T4 molecule have now been recognized; however, the precise role of these molecules in the function of helper/inducer T cells is unclear. We studied a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia whose blood lymphocytes and thymus cells did not express the epitope recognized by OKT4 monoclonal antibody but did display the T4 epitopes recognized by OKT4A and Leu3A monoclonal antibodies. The absence of the OKT4 epitope on the patient's thymus cells suggested that the abnormality occurred during early T cell differentiation. The patient had intact delayed hypersensitivity to 4/4 antigens, and his blood lymphocytes proliferated normally to phytohemagglutinin, concanavalin A, pokeweed mitogen, tetanus toxoid, and allogeneic cells. The patient's T cells demonstrated augmented suppressor activity that was localized to the OKT8+ population rather than to the unusual T4 subset. Irradiation abrogated suppressor activity and rendered his T cells capable of providing help for polyclonal B cell differentiation. The data emphasize the limitations of OKT4 as the sole reagent for characterizing the subset of human helper/inducer cells and demonstrate that the expression of the T4 epitope recognized by OKT4 monoclonal antibody is not required for certain helper/inducer T cell functions in vitro and in vivo.     

T-cell dysfunctions in IgA nephropathy: specific abnormalities in the regulation of IgA synthesis

This work was undertaken to determine the cellular abnormalities that could explain the high levels of serum IgA frequently found in patients with IgA nephropathy. Seventeen control subjects and twenty-seven patients who had received no therapy were studied. After in vitro pokeweed mitogen (PWM) stimulation, significantly higher amounts of IgA were produced by peripheral blood mononuclear cells (PBM) of patients when compared with those of the control group (560 +/- 97 vs 231 +/- 57 ng/ml, P less than 0.0025). No differences were observed in the synthesis of IgG and IgM. Twenty out of twenty-seven patients presented an increase in the percentages of OKT4+ cells (mean + 2 SD), in relation to the control group, with normal or elevated percentages of OKT8+ cells. The OKT4+/OKT8+ cell ratio was elevated in 12 out of 27 patients. All patients presented some abnormality in the generation of IgA-specific suppressor cells at variable doses of concanavalin A (Con A) on in vitro PWM-stimulated culture of PBM. In both assays low doses of Con A (2.5 micrograms/ml) induced a certain suppression of IgA synthesis in patients that was not observed in the majority of the control group. At these doses some patients also showed an enhancement in the synthesis of IgG and IgM. On the contrary, higher doses of Con A (50 micrograms/ml) produced significantly less IgA suppression than the controls. Normal IgA-suppression values were found at 10 micrograms/ml of Con A. T cells obtained from patients were significantly more efficient than T cells from controls in providing IgA-helper activity for normal allogeneic enriched B cells (P less than 0.025) in PWM-stimulated cocultures. These results show that patients with IgA nephropathy present, after mitogen stimulation in vitro, a specifically increased production of IgA as well as an augmentation in the activity of IgA-helper T cell and a deregulation on IgA-suppressor T-cell function. According to these data, it is suggested that the alteration observed in helper T cells might precede that of suppressor T cells. These immunoregulatory abnormalities might contribute to the pathogenesis of the disease.     

T cells which proliferate in response to concanavalin A include cells which proliferate in mixed leucocyte reactions.

Selection in long-term culture of alloreactive T cells, by successive in vitro restimulation with semi-allogeneic cells, results in primed responder cell populations which maintain full proliferative reactivity to allogeneic cells as well as to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) but are depleted of cells which can effect target cell destruction in either a specific or nonspecific manner. Con A-induced T cell blasts (selected by velocity sedimentation) can revert to small resting lymphocytes in the presence of inert "filler" cells. Con A blasts which have reverted, readily proliferate in response to Con A or allogeneic stimulator cells but are largely depleted of effector killer cells and PHA-responsive cells.     

T cell malignancy in Richter's syndrome presenting as hyper IgM. Induction and characterization of a novel CD3+, CD4-, CD8+ T cell subset from phytohemagglutinin-stimulated patient's CD3+, CD4+, CD8+ leukemic T cells

A patient is described, having Richter's syndrome and immunodeficiency with hyper IgM, who developed suppressor T cell lymphoma (CD3+, CD4-, CD8+) following untreated helper-suppressor T cell chronic lymphocytic leukemia (CD3+, CD4+, CD8+). The neoplastic T cells in both malignancies expressed interleukin (IL) 2 receptors but were deficient in typical CD2+ and CD5+ pan T antigens. Additionally, a large percentage of malignant lymph node T cells expressed HLA-DR+ activation antigens. In vitro immunoglobulin-production experiments demonstrated that the patient's leukemic blood T cells had an excess helper function for IgM synthesis but a suppressor function for IgG and IgA synthesis by normal B and T cells. The leukemic blood T cells demonstrated a poor response to phytohemagglutinin (PHA). A defect in IL 2 receptor expression was evident in PHA-stimulated leukemic blood T cells. Of interest was the observation that PHA stimulated the induction of a novel CD3+, CD4-, CD8+ T cell subset from patient's CD3+, CD4+, CD8+ leukemic blood T cells. These PHA-induced CD3+, CD4-, CD8+ T cell subsets produced an elevated proliferative response to PHA and concanavalin A, had a helper cell function for IgM synthesis and produced highly elevated amounts of IL 2.     

Lymphocyte subpopulations and suppressor cell activity in acute polyradiculoneuritis (Guillain-Barré syndrome).

Ficoll-Triosil separated peripheral blood mononuclear (PBM) cells were analysed by fluorescent microscopy with Ortho monoclonal antisera to T cells (OKT3+) helper cells (OKT4+) and suppressor cytotoxic cells (OKT8+) and with polyclonal antiserum to surface immunoglobulin. Twenty-five normal subjects, 16 patients with acute polyradiculoneuritis (Guillain-Barré syndrome, AP) and 10 patients with other neurological diseases were studied. The percentages of OKT3+, OKT4+ and immunoglobulin bearing cells were similar in the three patient groups but the percentage of OKT8+ cells was reduced to 13.5 +/- 4.1 (mean +/- s.d.) compared with 19.4 +/- 4.9 in the normal subjects and 18.5 +/- 3.1 in the patients with other neurological diseases. The ratio of OKT4+/OKT8+ cells was correspondingly increased to 3.5 +/- 2.1 compared with 2.1 +/- 0.5 in the normal subjects and 2.1 +/- 0.4 in the patients with other neurological diseases. In one test of suppressor cell function Con A incubated mitomycin treated PBM cells were added to autologous PBM cells stimulated with Con A to compare the degree of suppression with that produced by control incubated mitomycin treated cells (Con A suppression test). In a second test of suppressor cell function short lived suppressor cell (SLS) activity was assayed by comparing PBM stimulation by Con A added at the start of culture with that produced by Con A added after 24 hr. Neither test revealed any significant differences between AP patients and control subjects.     

Immunological reconstitution after bone marrow transplant with Campath-1 treated bone marrow.

Immunological reconstitution after allogeneic bone marrow transplant (BMT) was studied in 20 patients who received Campath-1 treated bone marrow. The peripheral blood lymphocyte phenotype was analysed with a panel of monoclonal antibodies at 3, 6 and 12 months. T cell proliferative capacity was evaluated by stimulation with PHA and Con A and in the mixed lymphocyte reaction (MLR). Natural killer (NK) cell activity was analysed against the K562 cell line at specified times after BMT in nine patients. Absolute numbers of T lymphocytes were reduced in all patients at 3 and 6 months. A marked decrease in the number of CD4+ cells persisted beyond 12 months. CD8+ cells regenerated more rapidly and reached normal at 6 months. No correlation was found between changes in lymphocyte subpopulations and the presence of graft-versus-host disease or cytomegalovirus infection. B cells recovered rapidly and maintained normal numbers throughout the study. A moderate increase in HNK1+ (Leu7) cells was observed at 3 and 6 months simultaneously with a low expression of NK15 (Leu11) and OKM1 antigens at 3 and 6 months, suggesting the presence of immature NK cells early after the transplant. A profound decrease of T cell proliferative capacity was observed both after mitogen stimulation and in the mixed lymphocyte reaction. NK cell activity was raised during the first month after transplant in all but one patient but no correlation was found with the presence of GVHD or cell marker analysis.     

Altered T-Cell Subsets and Defective T-Cell Function in Young Children with Down Syndrome (Trisomy-21)

Children with Down syndrome (DS) suffer an increased incidence of severe viral and bacterial infections particularly during the first 5 years of life. Unfortunately, few studies have been performed on the immune systems of young children with Down syndrome. Peripheral blood mononuclear cells (PBMC) from a group of non-institutionalized children less than 6 years of age were studied and compared with PBMC from age-matched controls. The children with DS had reduced numbers of circulating OKT4+ (helper/inducer) T cells and a significantly depressed ratio of OKT4+ to OKT8+ (suppressor/cytotoxic) T cells. PBMC from the DS subjects exhibited reduced proliferative responses to phytohemagglutinin and to an optimal concentration of concanavalin A (Con A), but normal responses to suboptimal doses of Con A and pokeweek mitogen. PBMC from young children with DS appeared to produce normal levels of interleukin-2 (IL-2). These findings provide evidence that the primary immune defect in DS is in part a depressed number and function of helper T cells. They also indicate that IL-2 production may not be defective in DS, but rather that the mechanism for response to IL-2 may be faulty.     

Altered T-cell subsets and defective T-cell function in young children with Down syndrome (trisomy-21)

Children with Down syndrome (DS) suffer an increased incidence of severe viral and bacterial infections particularly during the first 5 years of life. Unfortunately, few studies have been performed on the immune systems of young children with Down syndrome. Peripheral blood mononuclear cells (PBMC) from a group of non-institutionalized children less than 6 years of age were studied and compared with PBMC from age-matched controls. The children with DS had reduced numbers of circulating OKT4+ (helper/inducer) T cells and a significantly depressed ratio of OKT4+ to OKT8+ (suppressor/cytotoxic) T cells. PBMC from the DS subjects exhibited reduced proliferative responses to phytohemagglutinin and to an optimal concentration of concanavalin A (Con A), but normal responses to suboptimal doses of Con A and pokeweek mitogen. PBMC from young children with DS appeared to produce normal levels of interleukin-2 (IL-2). These findings provide evidence that the primary immune defect in DS is in part a depressed number and function of helper T cells. They also indicate that IL-2 production may not be defective in DS, but rather that the mechanism for response to IL-2 may be faulty.