Recombinant human interleukin-2 reverses in vitro-deficient cell-mediated immune responses to tuberculin purified protein derivative by lymphocytes of tuberculous patients.

In vitro lymphocyte proliferative response to purified protein derivative of tuberculin (PPD) was investigated in patients with tuberculosis. Peripheral blood lymphocytes (PBL) from patients with advanced, refractory tuberculosis showed a significantly depressed response compared with the response of PBL from patients with newly diagnosed tuberculosis (P less than 0.01). A further characterization of this low responsiveness to PPD revealed that PBL from these advanced tuberculous patients failed to generate interleukin-2 (IL-2) in response to PPD stimulation. IL-2 receptor (Tac antigen) expression on the surface of T cells after PPD stimulation was also impaired, although to a lesser extent, in the patients with advanced, refractory tuberculosis. We attempted to overcome the depressed in vitro response observed in PBL from patients with advanced, refractory tuberculosis and found that the addition of exogenous, recombinant IL-2 returned the depressed PPD-induced PBL proliferation in these patients to the level of response observed in PBL from patients with newly diagnosed tuberculosis. The addition of recombinant IL-2 also had a restorative effect (up regulation) in vitro on the partly impaired PPD-induced IL-2 receptor expression by PBL from the patients with advanced, refractory tuberculosis. Our results suggest that recombinant IL-2 may offer a novel approach to the therapy of advanced, drug-resistant tuberculosis.     

Immunotherapy of patients with terminal-stage malignant tumors with an immunopotentiator OK-432--a comparison of SU-PS test-responding and -nonresponding patients

Terminal malignant tumor cases were treated with immunopotentiator OK-432. The absolute numbers of neutrophils and lymphocytes and different lymphocyte subsets were determined, and SU-PS and PPD skin tests were also performed to monitor the immunologic status of each patient. The SU-PS test changed to positive in one-third of the patients 2-6 weeks after the start of therapy. The SU-PS responding patients showed an increase in lymphocytes and Leu II+ cells, and an elevation of the OK T4/T8 ratio 2 weeks later. In all patients from the responding group, the (OKIal+ - Bl+) cells decreased in the peripheral blood immediately after the positive change in the SU-PS test. In the SU-PS nonresponding group, the OKT4+ cells tended to decline with time, while the OKT8+ cells increased. That is, the OKT4/T8 ratio remained low throughout the test period. In the SU-PS responding group, OK-432 therapy prolonged the survival time.     

The fine structure of normal lymphocyte subpopulations--a study with monoclonal antibodies and the immunogold technique.

The ultrastructural characteristics of normal lymphocyte subpopulations, identified by monoclonal antibodies and visualized by a colloidal gold labelled anti-mouse IgG were analysed. Our study demonstrates: (1) the major T lymphocyte subsets (OKT4+ and OKT8+) have distinct ultrastructural morphology. The majority of OKT4+ cells have a high nuclear/cytoplasmic ratio (N/C) and few cytoplasmic organelles whilst most OKT8+ cells have a low N/C ratio and numerous organelles, namely a well developed Golgi apparatus, lysosomal granules and parallel tubular arrays (PTA); (2) a unique subtype with irregular nuclear outline that resembles Sézary cells was seen in 5-10% of OKT4+ lymphocytes; (3) OKM1, a reagent that reacts with monocytes and granulocytes, is positive in a small lymphocyte subset which appears to be negative with the OKT reagents and is morphologically identical to OKT8+ cells; (4) 'hand-mirror' cells were only seen labelled with OKT8 and OKM1; (5) B lymphocytes labelled with FMC4 (anti-IA) could be distinguished from OKT3+ lymphocytes by having numerous profiles of endoplasmic reticulum (ER) and ribosomes; these were particularly prominent in lymphoplasmacytoid cells. Morphological similarities between normal T lymphocyte subsets and T neoplasias of the same membrane phenotype suggest that these disorders arise from specific T cell types present in normal peripheral blood or from common precursors.     

Increase of gamma/delta T cells in hospital workers who are in close contact with tuberculosis patients.

gamma/delta T cells are likely to participate in the immune response to tuberculous infection in humans. In this study, we carried out an investigation to characterize the responsiveness of gamma/delta T cells from tuberculous patients and healthy individuals to mycobacterial stimulation in vitro. Healthy subjects were assigned to the following two groups: those who had been exposed to tuberculosis (contacts) and those who had not been exposed (noncontacts). The percent gamma/delta T cells in fresh peripheral blood obtained from health care workers who were tuberculin skin test positive and who had constant contact with patients with active tuberculosis (healthy contacts) was significantly higher, whereas healthy noncontacts showed the normal range of gamma/delta T cells. Patients with active pulmonary tuberculosis also had low levels of gamma/delta T cells. HLA-DR antigen-bearing activated gamma/delta T cells were observed in higher percentages among healthy contacts than among healthy noncontacts or patients with pulmonary tuberculosis. In healthy contacts, gamma/delta T cells increased as a percentage of peripheral blood mononuclear cells after in vitro stimulation with purified protein derivative (PPD) tuberculin compared with the percentage of fresh peripheral blood mononuclear cells that they made up, whereas no such increase was observed in patients with tuberculosis or in healthy noncontacts. Phenotypic analysis of the gamma/delta T cells in healthy contacts, which increased in number in vitro in response to PPD, revealed the preferential outgrowth of CD4+ V gamma 2+ gamma/delta T cells. This expansion of gamma/delta T cells by PPD required accessory cells, and it was inhibited by the addition of an antibody against HLA-DR in culture. Proteolytic digestion of PPD showed that gamma/delta T cells increased in number in response to peptide, but not nonpeptide, components of PPD. These findings suggest that gamma/delta T cells, especially CD4+ V gamma 2+ gamma/delta T cells, may participate in the immune surveillance of tuberculous infections in humans.     

Nonspecific recruitment of lymphocytes in purified protein derivative-induced lymphocyte proliferative response of patients with tuberculosis.

Peripheral blood lymphocytes (PBL) from tuberculosis patients were studied for their in vitro proliferative response stimulated with purified protein derivative (PPD)-tuberculin. Studies were designed to characterize the lymphocytes involved in the PPD-induced proliferation. PPD-responsive lymphocytes were eliminated in PBL by the procedure of cultivation of PBL with PPD in the presence of 5-bromodeoxyuridine and light illumination of the cultured cells. These PBL which lost PPD reactivity were no longer able to proliferate to PPD stimulation but were still capable of proliferation in the presence of both PPD and X-irradiated, autologous fresh PBL or upon addition of culture fluids from PPD-stimulated PBL. In addition, these nonspecifically activated lymphocytes released a soluble factor into the culture fluids which inhibited the migration of leukocytes. It was likely that large numbers of nonspecific T cells were induced to proliferate as a result of the presentation of specific T cells with the antigen PPD. It is suggested that a similar recruitment of lymphocytes by PPD-stimulated T cells takes place in vivo during the establishment of tuberculosis or antituberculous immunity or both.     

Lymphocyte Subpopulations in Gastric Disease; Effect of Histamine and Cimetidine on Immunoregulatory T Cell Subsets

The numbers of T lymphocytes, helper and suppressor: T lymphocytes, were measured in peripheral blood of 10 patients with duodenal ulcer, 8 patients with chronic gastritis, 13 patients with gastric cancer and 20 normal controls. T lymphocyte subpopulations were enumerated b the use of monoclonal antibodies OKT3 (pan-T lymphocytes7, OKT4(helper/inducer lymphocytes) and OKT8(cytotoxic/suppressor lymphocytes)in an indirect immunofluorescence technique. Furthermore, the possible phrmacological modulation of 10-4 histamine and 10-4, 10-8 M cimetidine of T lymphocyte subsets was investigated. Lymphocyte subpopulations were found to range in normal values in patients with ulcer and chronic gastritis. 8 marked decrease of OKT3 and OKT8 positive lymphocytes was noted in patients with gastric cancer, whereas OKT4 lymphocytes were normal. Exposure of normal lymphocytes and lymphocytes from the three groups of patients to histamine and cimetidine resulted in no significant changes of lymphocyte subsets.     

Tuberculous Meningitis: Immune Reactions within the Central Nervous System

Cerebrospinal fluid (CSF) lymphocytes from two patients with tuberculous meningitis proliferated stronger than the corresponding peripheral blood lymphocytes (PBL) when stimulated with tuberculin purified protein derivative (PPD) in the lymphocyte transformation test after 3 days of culture. This might indicate an accumulation of specifically primed lymphocytes within the central nervous system. CSF lymphocytes and PBL from nine of ten patients with acute aseptic meningitis investigated as controls showed no or low responses when stimulated with. PPD, whereas the remaining patient displayed a significant proliferation of CSF lymphocytes, which was more pronounced than that of PBL. Stimulation with the mitogens phytohaemagglutinin, concanavalin A, and pokeweed mitogen gave lower proliferation of CSF lymphocytes compared with PBL in tuberculous and aseptic meningitis. Evaluation of the proliferative response of CSF lymphocytes compared with PBL on stimulation with PPD might be a useful complement in the diagnosis of tuberculous meningitis.     

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.     

Defective gamma-interferon production in peripheral blood leukocytes of patients with acute tuberculosis

Production of interferon (IFN)-gamma by peripheral blood leukocytes (PBL) was examined in cultures of unseparated fresh whole blood exposed to phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). The yield of IFN-gamma was measured by a newly developed immunoradiometric assay. Nine of 14 patients with acute pulmonary tuberculosis (TB) showed a depressed IFN-gamma response to Con A and/or PWM. Only four of these TB patients also showed a depressed IFN-gamma response to PHA. Stimulation of the patients' PBL cultures with PHA in the presence of exogenous interleukin 2 (IL 2) produced normal IFN-gamma yields in all but the most severely depressed patients. PBL cultures of TB patients with defective IFN-gamma production in response to mitogenic lectins also produced less IFN-gamma after stimulation with tuberculin PPD. Although some patients showed a moderate degree of lymphopenia, their OKT4/T8 lymphocyte ratios were mostly normal or close to normal, with the notable exception of one TB patient who has been diagnosed to have the acquired immune deficiency syndrome (AIDS).     

结核性胸膜炎合并慢性阻塞性肺疾病患者免疫功能的变化及意义

目的 通过观察T淋巴细胞亚群、B细胞、NK细胞活性来探讨结核性胸膜炎合并慢性阻塞性肺疾病(COPD)患者免疫功能变化.方法 采用流式细胞术分别测定45例结核性胸膜炎合并COPD患者(研究组)、45例COPD患者(COPD对照组)和45例健康体检者(健康对照组)外周血T淋巴细胞亚群、B细胞和NK细胞水平.结果 研究组CD3+、CD4+、CD4+/CD8+、B细胞和NK细胞表达率降低,与健康对照组差异有统计学意义(P ＜0.05);COPD对照组CD3+、CD4+、CD4+/CD8+、B细胞和NK细胞表达率与健康对照组差异有统计学意义(P＜0.05);研究组与COPD对照组的差异无统计学意义.结论 结核性胸膜炎合并COPD患者免疫功能明显低于正常健康人群,增强免疫治疗非常必要.     

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.     

T-cell subsets in the hyporesponsiveness to hepatitis B surface antigen (HBsAg) and antigen-specific suppressor lymphocytes in chronic hepatitis B virus (HBV) infection

In contrast to convalescent hepatitis B patients, who exhibit the ability to elicit a specific immune response to HBsAg, patients with chronic hepatitis B virus (HBV) infection are markedly hyporesponsive to HBsAg and show a decrease in the normal ratio of OKT4-positive (helper/inducer) to OKT8-positive (suppressor/cytotoxic) lymphocytes. In this study the role of OKT4-positive and OKT8-positive cells on cellular immune response to HBsAg was evaluated in patients with chronic HBV infection and the ability of such patients to develop antigen-specific suppressor lymphocytes after in vitro sensitization to HBsAg. Elimination of OKT8-positive cells markedly improved the in vitro lymphocyte proliferative response to HBsAg without altering the reactivity of cells from the same donor to PPD or Candida. In contrast, the degree of responsiveness to HBsAg was not affected by the depletion of OKT4-positive cells. In vitro co-culture experiments, performed in the seven chronically HBV-infected patients who showed a proliferative response when their PBM were cultured with purified HBsAg or PPD, have demonstrated that lymphocytes from chronic HBV carriers, stimulated with HBsAg, inhibit the response of autologous PBM to HBsAg but not to the unrelated antigen PPD.     

Phenotype study with monoclonal antibodies of T lymphocyte colonies in normal individuals and in patients with chronic OKT8+ lymphocytic leukaemia.

The lymphocyte colony forming capacity of peripheral blood mononuclear cells from normal controls and from two patients with chronic OKT8+ lymphocytic leukaemia was determined in agar culture under PHA stimulation. The number and size of the colonies in patients were reduced compared to normal. The lymphocytic phenotype of colony cells was studied with monoclonal antibodies in colonies harvested from agar culture and in colonies expanded in liquid culture in the presence of TCGF. This study was performed in individual colonies and in pooled colonies. Colonies from normal controls contained a mixture of the OKT4+ and OKT8+ lymphocyte subsets. In contrast, colonies from the two patients contained essentially OKT4+ lymphocytes. The data indicate that, in the patients, progenitors of the OKT8+ subset are unresponsive to normal proliferative and/or differentiative stimuli under the present culture conditions.     

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.     

Evaluation of T lymphocyte subpopulations in children with nephrotic syndrome.

We have evaluated lymphocyte subsets both quantitatively and functionally in children with nephrotic syndrome. Ten children had minimal change nephrotic syndrome (MCNS), seven had focal segmental glomerular sclerosis (FSGS) and four had recovered from MCNS. Two patients with FSGS had decreased OKT4+ cells and a decreased T4/T8 ratio. Patients with active MCNS synthesized significantly less IgM in vitro than did normal subjects (P less than 0.005). In vitro IgG synthesis was significantly decreased in patients with active nephrotic syndrome whether it was due to MCNS or FSGS. Co-culture studies involving lymphocyte subsets suggested that both patients with decreased percentages of T4+ cells had decreased T helper cell activity as well. The results of this study support previous speculation about abnormal immune function in some patients with nephrotic syndrome.     

Alteration of T cell subsets and immunoglobulin synthesis in vitro during high dose gamma-globulin therapy in patients with idiopathic thrombocytopenic purpura.

T cell subsets of peripheral lymphocytes were studied using monoclonal antibodies (OKT3, OKT4, OKT8) and immunoglobulin (IgG, IgM, IgA) synthesis in vitro by peripheral mononuclear cells stimulated with pokeweed mitogen was studied in adult patients with idiopathic thrombocytopenic purpura before and after high-dose intravenous intact gamma(gamma-)globulin therapy, at a daily dose of 0.4g/kg body weight for 5 consecutive days. A transient increase in platelet count which reached a peak on the 1st to 2nd days after the end of the therapy was observed in four of five patients. Immunoglobulin synthesis in vitro was suppressed remarkably following therapy in all cases: the mean reduction of IgG, IgM and IgA was 78, 66 and 48%, respectively. Titres of various autoantibodies, TGHA, MCHA, ANF and anti-DNA antibody, also decreased following therapy. In correspondence to this, phenotypic analysis in T cell subsets showed a decrease of OKT4+:OKT8+ ratio following therapy, without a change in the proportion of OKT3+ cells. These data indicate that the intravenous gamma-globulin preparations suppressed synthesis of antibodies non-specifically and caused a relative increase of OKT8+ suppressor T cells.     

Cellular immunity in tuberculous pleural effusions: evidence of spontaneous lymphocyte proliferation and antigen-specific accelerated responses to purified protein derivative (PPD).

The kinetics of in vitro cellular proliferation against a PPD of Mycobacterium tuberculosis or streptococcal antigen (streptokinase-streptodornase) was evaluated in pleural fluid and peripheral blood mononuclear cells (PBMC) from patients with tuberculous and non-tuberculous pleuritis. The peak proliferative response to PPD by mononuclear cells from pleural fluid occurred earlier (day 3) in 65% of patients with tuberculosis, a finding not seen in non-tuberculous effusions. Spontaneous lymphocyte proliferation of both peripheral blood lymphocytes and pleural effusion lymphocytes was frequently observed, irrespective of etiology. However, 20 of 21 tuberculous patients manifesting spontaneous lymphocyte proliferation had accelerated kinetics of proliferation to PPD, which was antigen-specific. These results suggest that spontaneous lymphocyte proliferation occurs as a response to antigen stimulation at the site of disease, and is not a non-specific response to inflammation. Furthermore, enhanced reactivity against mycobacterial antigen, manifested by accelerated kinetics of proliferation, has diagnostic potential in patients with pleural effusions.     

T-cell differentiation antigens and antigenic lymphocyte reactivity in pleural effusions

Blood and pleural effusion mononuclear cells from thirteen patients were examined for the expression of T lymphocyte differentiation antigens as well as in vitro thymidine incorporation. The ratio of T4 to T8 cells was significantly greater among pleural effusion lymphocytes than among blood lymphocytes. Effusion lymphocyte responses to phytohaemagglutinin were less than those of blood lymphocytes. Unstimulated thymidine incorporation was greater in pleural effusion lymphocytes. Antigen-stimulated lymphocyte reactivity was not consistently greater in either blood or effusion lymphocytes. Lymphocytes from tuberculous effusions all reacted to tuberculin. Pleural effusion lymphocytes, regardless of the etiology of the effusion, possessed the same range of antigenic specificities as did blood lymphocytes. Therefore, effusion lymphocyte responsiveness to tuberculin does not prove the presence of tuberculous pleurisy but does indicate sensitisation to tuberculin.     

Studies on the immunoregulation of thyroid autoantibody production in vitro.

The spontaneous production (without mitogen or antigen) of antithyroglobulin and antimicrosomal antibodies by peripheral (PBL) and thyroid-derived lymphocytes from patients with Hashimoto's thyroiditis (HT) has been studied with particular emphasis on the regulation of this phenomenon. Based on studies of DNA and protein synthesis, kinetic studies and B/T reconstitution experiments, in most HT patients, spontaneous production by PBL is accounted for by secretion of preformed antithyroglobulin (termed Type 1 patients), whereas active production is observed in a small minority (termed Type 2). In none of 24 HT patients could active antimicrosomal antibody production by PBL be detected. Conversely, thyroid-derived lymphocytes produced both autoantibodies by an active process. Pokeweed mitogen (PWM) stimulation enhanced antibody production by PBL in the Type 1 group but not in Type 2 or thyroid-derived lymphocytes. T lymphocytes were required for antibody synthesis in both thyroid antigen-driven and peripheral PWM-driven cultures. By separating T lymphocytes into T4+ (helper) and T8+ (suppressor) subsets with monoclonal antibodies, T-cell modulation of autoantibody production in both systems was studied. In a PWM-induced system, both thyroid and peripheral T-cell subsets were capable of modulating peripheral antibody production. In the thyroid lymphocyte antigen-specific system, further addition of thyroid derived T8+ cells alone caused partial suppression of antibody production but not with peripheral T8+ cells. Of interest was the partial decrease of antibody production by the thyroid lymphocytes by added peripheral T4+ cells. The fact that the production of thyroid autoantibodies by thyroid-derived mononuclear cells (which included T suppressor, T helper and B lymphocytes) could be reduced by the addition of more suppressor T lymphocytes suggests that an antigen-specific defect in the T4+/T8+ thyroid cell balance may account for the in vivo production of these antibodies in patients with Hashimoto's thyroiditis.     

Immunoregulatory T cells in men with a new acquired immunodeficiency syndrome

We have evaluated the functional properties of the OK-T8+/OKT4+ T-cell subpopulations in nine patients with a new syndrome of acquired immune deficiency (AIDS). Despite polyclonal hypergammaglobulinemia in the sera of these patients, their peripheral blood lymphocytes (PBL) produced negligible quantities of immunoglobulin (Ig) when cultured in vitro for 8 days in the presence of pokeweed mitogen (PWM). Patient B cells, however, synthesized normal quantities of immunoglobulin when cocultured with T cells from healthy donors, indicating preservation of B-cell function. Unfractionated PBL or T cells of patient origin mediated marked suppression of pokeweed mitogen-driven immunoglobulin production by T and B cells from healthy donors. The suppressive activity was contained within the population of T cells bearing the OKT8 antigen and was sensitive to in vitro irradiation. On a per-cell basis, patient OKT8+ cells appeared to have greater suppressive activity than normal control OKT8+ cells. In addition, OKT4+ cells from patients had less helper activity for induction of immunoglobulin synthesis than control OKT4+ cells. Increased T suppression and reduced T help are probably a consequence of one or more viral infections and may contribute to progressive immune deficiency and susceptibility to malignancy in patients with the acquired immuno deficiency syndrome.