Immunological evaluation in children with juvenile chronic arthritis treated with auranofin.

An immunological evaluation was performed before therapy and every four months during the first year of treatment with auranofin in 6 children with juvenile chronic arthritis. The immunological tests included: IgG, IgA, IgM, IgE and "natural" antibody serum levels, CH50 of the classical and alternative complement pathways, PWM-induced IgM production in vitro, and polymorphonuclear neutrophil functions. A reduction of the in vitro IgM synthesis and in the CH50 of the classical pathway of complement, and a normalization of impaired chemotaxis, occurred in patients who presented a clinically significant improvement during auranofin treatment.

Analysis with OKT monoclonal antibodies of T-lymphocyte subsets present in blood and liver of patients with chronic active hepatitis.

The relative distribution of T lymphocyte subsets, as defined by the monoclonal antibodies OKT, was determined by cytofluorimetric analysis in peripheral blood and in cells isolated from liver biopsies of 31 patients with chronic active hepatitis (CAH). The percentage of peripheral blood lymphocytes binding OKT8 (directed against cytotoxic/suppressor T cells) was found to be elevated in patients with HBsAg and HBeAg positive chronic active hepatitis. Patients with CAH who had seroconverted to anti-HBe, had an increased number of OKT3-positive cells in their blood, which was directed against a common T cell surface antigen, associated with a decreased number of OKT8 positive cells. Lymphocytes isolated from liver biopsies of patients with CAH presented a general increase of OKT8-positive cells associated with a decreased number of OKT4-positive (helper/inducer) T cells. It is likely that OKT8-positive cells found in liver biopsies represent cytotoxic T cells directed against either viral or liver cell determinants.

Low beta-adrenergic receptor concentration on human thymocytes.

Several agents are known that can elevate cyclic AMP levels in lymphoid cells, e.g. isoproterenol, PGE1 and adenosine. We have studied the cyclic AMP increasing effect of these agents on thymocytes from mouse and man and on human peripheral T lymphocytes. In contrast to mouse thymocytes and human peripheral T lymphocytes, human thymocytes appeared to be insensitive to isoproterenol, but did respond to PGE1 and adenosine. Furthermore, the density of beta-adrenergic receptors on the cells was determined by measuring the specific binding of 3H-dihydroalprenolol. A correlation was found between the receptor density on the cells and the rise in intracellular cyclic AMP induced by isoproterenol: human thymocytes appeared to have very few beta-adrenergic receptors, in contrast to thymocytes from mouse or to T lymphocytes from human blood. We conclude that the development of beta-adrenergic receptors in T cell ontogeny is different for mice and human beings. Comparison of animal models with the situation in man should be made with caution.

Isolation and partial characterization of a novel subset of human T lymphocytes defined by monoclonal antibodies.

A novel subset of human blood lymphocytes was isolated by means of labelling with monoclonal antibodies and fluorescence-activated cell sorting. In normal individuals, the new subset accounts for about 2% of the blood T lymphocytes. The cells of this subset bind monoclonal antibodies specific for T lymphocytes in general [e.g. OKT3, Hu-Lyt 3(9 . 6) and Leu-22] and they also form E rosettes. However, no binding is seen with monoclonal antibodies to T-lymphocyte subsets (OKT4, OKT8, Leu-2A and Leu-3A). Moreover, the lymphocytes of this new subset express neither Ia antigens nor membrane immunoglobulins. They do not bind OKM1, an antibody against cells of the myelomonocytic lineage that also reacts with natural killer cells, nor do they bind OKT6 or OKT10, specific for thymocyte antigens. The cells have a high specific gravity, a thymocyte-like pattern of lactate dehydrogenase isoenzymes and do not contain terminal deoxynucleotidyl transferase. Although these lymphocytes are viable, also after culture in vitro, and can be stored in liquid nitrogen, they are inert in all functional systems tested: they neither proliferate upon stimulation with mitogens or allogeneic cells, nor do they display suppressor or natural killer cell activity. A patient who was successfully reconstituted by bone marrow transplantation for severe combined immunodeficiency, was found to contain an abnormally high (25%--30%) fraction of these OKT3 positive, OKT4 and OKT8 negative cells among his circulating T lymphocytes.

Changes in human T lymphocytes after thymectomy and during senescence.

Peripheral lymphocytes from individuals who had been thymectomized in adult life for myasthenia gravis (MG) or for other, nonimmunological reasons showed a moderate decrease in proliferative response capacity to several T-cell mitogens as compared to lymphocytes from normal individuals. The decrease of the response to mitogens and allogeneic lymphocytes was 20-30% within 5 years after thymectomy and about 50% more than 15 years after thymectomy. A comparable decrease in lymphocyte proliferative response capacity was found in healthy aged humans (68-97 years old). Analysis of T lymphocytes from both aged and thymectomized individuals with monoclonal (OKT) antibodies showed a similar pattern: the proportion of T lymphocytes binding OKT6, OKT10, or OKI1 were found. A biochemical parameter for human T-cell differentiation, the lactate dehydrogenase (LDH) isoenzyme pattern, showed a significantly lower H/M ratio in the group of elderly people compared to young individuals. Furthermore, among patients thymectomized for MG, a significant correlation was observed between the LDH isoenzyme pattern of the T lymphocytes and the proliferative response to mitogens of these cells. In contrast, in healthy thymectomized individuals the LDH isoenzyme pattern appeared to be normal. These findings indicate that, after thymectomy or involution of the thymus, at least part of the peripheral blood T lymphocytes have properties different from those of the cells of young individuals. These cells might represent immature and/or not fully differentiated lymphocytes.

The cell-lineage of T gamma cells.

In patients with severe combined immunodeficiency, who have been successfully treated by bone-marrow transplantation, the occurrence of split take has been well documented: whereas myelomonocytic hemopoietic cell lines remain of host origin, the T-lymphoid compartment is of donor origin, while the B-lymphoid compartment may be either of host or of donor origin. We have studied the T gamma cells of two patients, successfully treated by bone-marrow transplantation from donors of the opposite sex, with respect to the sex-chromosome pattern, the binding of OKM1 and OKT3 monoclonal antibody and their K and NK activity. All T gamma cells of both patients were found to be of donor origin. These T gamma cells contained two serologically distinct subpopulations, one OKM1+ OKT3+, the other OKM1+ OKT3-, as was also found in normals. However, the ratio between these two subpopulations is 0.6 in normals, whereas these patients revealed a ratio of 3.0. Furthermore, the patients' T gamma cells displayed a strongly reduced K and NK activity (+/- 30% of normal). We concluded that at least part of the OKM1+ OKT3+ and of the OKM1+ OKT3- T gamma cells are derived from other than the myelomonocytic lineage, presumably from the lymphocytic lineage. The origin of the K and NK active T gamma cells, however, cannot be conclusively determined from these experiments. These findings also imply that the antigen detected by OKM1 should obviously no longer be regarded as exclusively present on myelomonocytic cells.

Characterization of two subsets of human T gamma cells.

Normal human E rosette-forming, Fc-IgG receptor-bearing cells (so-called T gamma cells) were separated into two functionally different subpopulations. Both subpopulations bind the monoclonal antibody OKM1 (directed against an antigen present also on monocytes and granulocytes). The first subpopulation accounts for about 70% of the total T gamma cell population, does not bind OKT3 (a monoclonal antibody directed against an antigen present on most T lymphocytes), and displays strong killer (K) cell and natural killer (NK) cell activity. The second subpopulation accounts for about 30% of the total T gamma population, binds both OKT3 and OKM1 (confirmed with a double-labeling assay), and displays low K and NK cell activity. Each subset contained less than 10% null cells. Comparison of T gamma cell populations obtained by adherence to a monolayer of IgG-coated human erythrocytes or by rosette formation with these cells revealed that pure T gamma cells with normal killer cell and natural killer cell activity were best obtained with the monolayer technique. Comparison of the enzymic and functional profile of T gamma cells, monocytes and granulocytes, as well as changes during culture of these cells in vitro, failed to indicate a relationship between T gamma cells and cells of the myelomonocytic lineage.

Non-histone chromatin proteins in human thymocytes and T lymphocytes from blood.

The composition of nuclear proteins from human thymocytes and T lymphocytes from peripheral blood was analyzed. Total thymocytes and total peripheral blood T lymphocytes differed markedly in non-histone chromatin proteins (both phosphorylated and non-phosphorylated), but did not differ in histones. When the cells were separated according to density, T-lymphocyte fractions with a close specific gravity showed restricted differences in non-histone chromatin patterns.

T lymphocyte characteristics in bone marrow-transplanted patients. II. Analysis with monoclonal antibodies.

The relative distribution of T cell subsets, as defined by the monoclonal antibodies OKT, was analyzed in peripheral blood lymphocytes of 8 children after bone marrow transplantation for aplastic anemia. The percentage of peripheral blood lymphocytes binding OKT3 (directed against a common T cell surface antigen and defining most peripheral T cells) reached normal values shortly after transplantation. In 5 patients, lymphocytes binding OKT8 (directed against an antigen present on the suppressor/cytotoxic T cell subset) were found in high proportion, and lymphocytes binding OKT4 (detecting an antigen present on inducer/helper T cells) in low percentage. This resulted in an inverted ratio OKT4/OKT8 compared with that of lymphocytes from normal individuals. In all patients the lymphocytes bound abnormally high amounts of OKT10 (directed against an antigen present on all thymocytes but not on mature peripheral T cells). In the course of time, a trend towards normalization was observed for all parameters investigated; however, the kinetics of the recovery showed a marked heterogeneity. From the analysis of this phenomenon, it is likely that, among other conditions yet unknown, a minimum of 20% of OKT4-positive lymphocytes is required for a normal proliferative response to T cell mitogens in vitro. No other correlation was found between any lymphocyte phenotype, as defined by the OKT antibodies, and proliferative response in vitro. Furthermore, lymphocytes from the patient with chronic graft-vs-host disease (greater than 50% OKT8 positive) failed to suppress the proliferative response to mitogens and antigens of the lymphocytes of the histo-identical bone marrow donor.

Effect of ascorbate on abnormal neutrophil, platelet and lymphocytic function in a patient with the Chediak-Higashi syndrome.

A diminished chemotactic response was observed with the neutrophils of a patient with the Chediak-Higashi syndrome, who was not in the accelerated phase of the disease. An abnormally low release of myeloperoxidase from these cells during phagocytosis was also noted; this resulted in a decreased iodination capacity and probably also caused the defect in the intracellular killing of bacteria by the neutrophils. The level of cyclic AMP in these cells was elevated, but decreased after treatment with ascorbate either in vitro or in vivo. During ascorbate therapy, the bactericidal activity of the neutrophils normalized, whereas the chemotactic response remained low. Nevertheless, the patient had significantly less infections during ascorbate therapy. The bleeding tendency, due to a storage-pool disorder of the Chediak-Higashi platelets, was unaffected by treatment with ascorbate. The patient's lymphocytes did not display any activity in antibody-dependent lymphocytotoxicity. This defect was not affected by treatment with ascorbate either.

Influence of adult thymectomy on immunocompetence in patients with myasthenia gravis.

The influence of adult thymectomy on several parameters of immunocompetence in patients with myasthenia gravis (MG) was investigated. Since incomplete thymectomy may lead to the presence of thymic remnants, we determined the activity of a thymus-dependent factor in the sera of the MG patients. As measured by several parameters, MG patients showed a normal immunocompetence compared with healthy controls, except in the response to DNCB sensitization in vivo. When tested at least 5 years after thymectomy, MG patients were found to have decreased response to mitogens, and a decreased cytotoxic T cell response in cell-mediated lympholysis. The response to challenge with DNCB in vivo was decreased both in thymectomized and nonthymectomized MG patients. No difference was found in a) the percentage of circulating T, B, non-B/non-T cells; b) the response to allogeneic cells (MLR); c) the antibody-dependent lymphocytotoxicity; d) the production of immunoglobulins in vitro by pokeweed mitogen-stimulated cells; and e) the anamnestic response to antigens in vitro. We conclude that adult thymectomy results in a decrease in the function of some subpopulations of lymphocytes.

Thymocyte maturation induced by a thymus-dependent serum factor.

By means of direct measurement of intracellular cyclic AMP in thymocytes in vitro we demonstrated the existence in human serum of a thymus-dependent factor (SF). This activity of SF appeared to be due to a very low M.W. (less than 500) peptide distinct from other circulating thymic peptides. SF was found to act selectively on immunologically immature, hydrocortisone (HC)-sensitive peanut lectin-agglutinating thymocytes. In a period of 240 min SF sequentially induces in thymocyte synthesis of cyclic AMP, protein synthesis and synthesis of phosphorylated non-histone chromatin proteins of high molecular weight. Because SF does not induce a change in DNA synthesis, it seems likely that the events induced by SF are associated with a process requiring DNA translation, but not DNA replication. The biochemical events induced by SF are accompanied by the acquisition of immunological maturation by thymocytes. This has been shown by the acquisition of HC resistance, by the expression of FC receptors for IgM, by the decrease of terminal transferase activity and by the induction of the capacity to elicit a graft-versus-host reaction. We conclude that, most likely, SF acts on immature thymocytes inducing a part of them to acquire some properties of mature T cells.