Depressed lymphocyte function after measles-mumps-rubella vaccination.

Healthy children receiving routine measles-mumps-rubella vaccine developed an impaired in vitro lymphocyte response to stimulation with antigen (Candida) but not with mitogen (phytohemagglutinin and pokeweed mitogen). Response of lymphocytes was determined by measurement of the amount of [14C]thymidine incorporated by the cells. The impaired response to antigen lasted from one to five weeks after vaccination. There was no alteration in the number of either total or thymus-derived lymphocytes in the peripheral blood after vaccination; These results suggest that viral vaccination causes a depression of lymphocyte function rather than a depletion of functional lymphocytes.     

Hypogammaglobulinaemia in systemic lupus erythematosus: report of a case with evidence for spontaneously activated T suppressor cells

A patient with hypogammaglobulinaemia associated with systemic lupus erythematosus (SLE), a healthy HLA-A, B, C, D-identical and mixed lymphocyte culture (MLC)-negative sibling, and two mutually HLA-A, B, C, D-identical siblings were investigated. Blood mononuclear cells from the patient contained a high proportion of T lymphocytes with the suppressor/cytotoxic phenotype, and in vitro no development of Ig-secreting cells was observed in response to pokeweed mitogen (PWM) or to Epstein-Barr virus (EBV), as opposed to cell cultures from the siblings. In cell cultures from the two healthy HLA-identical siblings, T-lymphocytes as well as monocytes/macrophages (M phi's) could be replaced with corresponding cells from the sibling without major alterations of the pokeweed mitogen (PWM)-induced B cell response. In PWM-stimulated co-cultures of B cells from the patient with healthy HLA-identical T cells, moderate numbers of IgM-secreting cells developed, but not IgG- or IgA-secreting cells. T cells from the patient co-cultured with healthy HLA-identical B cells suppressed their Ig-secretion; this effect was abolished by irradiation of the T cells. The in vitro generation of T suppressor cells by concanavalin A (ConA) was normal. No evidence for abnormal suppressor function of monocytes/macrophages was obtained. Thus in this patient, spontaneously activated T suppressor cells as well as defective B cells were associated with hypogammaglobulinaemia.     

Immune reactions in patients with Graves' disease.

Although Graves' disease exhibits many features of an autoimmune disturbance there is uncertainty regarding, in particular, the role of cell-mediated immunity. Therefore, we undertook a sequential study of peripheral lymphocyte responsiveness to phytomitogens, concentrations and ratios of T and B lymphocytes, and skin hypersensitivity to multiple antigens, throughout periods of antithyroid drug therapy in patients with hyperthyroidism of Graves' disease. Careful attention was paid to obtaining control lymphocyte data with blood from normal subjects matched for age and sex. A micromethod using whole blood was applied to measurement of peripheral lymphocyte responses (incorporation of tritiated [³H] thymidine) to phytohemagglutinin (PHA) and to pokeweed mitogen (PWM).Total lymphocyte concentrations and the proportions of T and B cells were normal in 26 patients studied; there was a mild excess of total lymphocytes and T cells in 12 patients with fresh disease versus that in six patients with persisting or recurrent hyperthyroidism. Responses of lymphocytes to pokeweed mitogen were normal but to phytohemagglutinin, significant differences from controls were identified: With one day of culture, cells from the patients were more responsive; with six days of incubation, they were less responsive than were cells from corresponding control subjects. These differences were not found with patients restudied after three to 10 months of treatment with propylthiouracil. The ratio of T:B cells varied with the donor's age, as did responses to pokeweed mitogen. Responses to phytohemagglutinin after one and six days of incubation, and to pokeweed mitogen after three days correlated positively with the concentration of serum thyroxine.Delayed hypersensitivity to four antigens injected intradermally was normal; the degree of skin response to tuberculin purified protein derivative correlated significantly with in vitro lymphocyte responses to that antigen.Our data confirm some abnormalities of lymphocyte function in Graves' disease. It remains unclear to what degree reversion to normal during therapy reflects a change in basic pathogenetic mechanisms or an influence of thyroid hormones per se. The importance of age and sex in studies of this nature is emphasized.     

Immunosuppressive serum factors in viral hepatitis. I. Characterization of serum inhibition factor(s) as lymphocyte antiactivator(s)

Sera from patients with acute viral hepatitis B were found to inhibit the in vitro proliferation of normal lymphocytes induced by different mitogens and antigens. In addition, an effect on concanavalin A-induced T suppressor cell activity and pokeweed mitogen-stimulated IgG and IgM synthesis was demonstrated. Studies concerning the kinetics of serum immunosuppressive effects indicated that serum immunosuppressive factor (SIF) interfered with the intermediate phase of mitogen-induced lymphocyte activation which was defined by protein and RNA synthesis. Thus, when SIF-positive sera were added to lymphocytes, which were already activated by phytohemagglutinin, for 8, 12, or 18 hr, the inhibitory effect decreased in relation to the duration of lymphocyte activation. No inhibition could be demonstrated when SIF-positive sera were added 24 hr after initiation of mitogen stimulation. Furthermore, similar inhibitory effects were found measuring either uptake of [3H]uridine (RNA synthesis) or [3H]leucine (protein synthesis) in a 24 hr culture of phytohemagglutinin-stimulated lymphocytes or [3H]thymidine uptake (DNA synthesis) after 48 hr. These results indicate that SIF act(s) like an antiactivator and may belong to immunoregulatory physiologic serum factors.     

Spontaneous and induced immunoglobulin secretion by synovial fluid B lymphocytes in rheumatoid arthritis.

The functional properties of B lymphocytes in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were analysed by means of a reverse haemolytic plaque forming cell (PFC) assay. SF mononuclear cells spontaneously secreted IgG, but little IgM or IgA. The SF cells failed to respond to the polyclonal B cell activators pokeweed mitogen (PWM) and Epstein-Barr virus. However, SF B cells cocultured with autologous T lymphocytes from the blood and stimulated with PWM secreted IgG but little IgM or IgA. The PFC responses of blood B cells cocultured with autologous SF T cells in the presence of PWM were low; irradiation of the T cells increased the blood B lymphocyte responses, but the differences were not statistically significant. It is concluded that suppressor SF T cells may be partly responsible for the poor response of SF B cells to PWM.     

The Sézary Syndrome Lymphoid Cell: Abnormal Surface Properties and Mitogen Responsiveness

The peripheral blood lymphoid cells of five patients with Sézary syndrome (SS) were examined with respect to their surface membrane characteristics and their response to mitogens. These cells showed markedly defective mitogenic responses to a broad dose range of phytohaemagglutinin (PHA), pokeweed mitogen, concanavalin A, and a rabbit antihuman lymphocyte antiserum (ATS), when compared with normal human lymphocytes. SS lymphoid cells (three patients studied) also displayed diminished or nearly absent capacity to form rosettes with unsensitized sheep erythrocytes (E-rosettes), and lacked surface immunoglobulin determinants. Despite their poor mitogenic response to ATS, they were as susceptible as normal lymphocytes to ATS-induced, complement mediated cytotoxicity. By comparison with lymphocytes from patients with chronic lymphocytic leukaemia, however, SS lymphoid cells showed decreased susceptibility to leukoagglutination by PHA. By way of contrast, three patients with mycosis fungoides having normal-appearing peripheral blood lymphocytes showed normal lymphocyte responses to mitogens, as well as normal proportions of E-rosette forming and surface immunoglobulin-bearing lymphocytes. These studies demonstrate that the SS lymphoid cell may, in some cases, lack surface properties and mitogen response characteristics of both B- and T-lymphocytes.     

In vitro immunoglobulin production, proliferation, and cell markers before and after antithymocyte globulin therapy in patients with aplastic anemia

Peripheral blood lymphocytes from 16 aplastic anemia patients were studied for in vitro biosynthesis of immunoglobulins (Ig), proliferative responses, and cell markers before and after antithymocyte globulin (ATG) treatment in an attempt to identify immune functions that would be useful in predicting responses to ATG therapy. Six of the 16 aplastic anemia patients were complete responders to ATG therapy, two were partial responders, and eight failed to respond to ATG therapy. The proportion of E+, CD4, CD8, and surface Ig-positive cells did not correlate with in vitro lymphocyte functions nor clinical responses before or after ATG therapy. Lymphocyte proliferative responses to phytohemagglutinin, tetanus toxoid, alloantigens, or pokeweed mitogen were generally present before and after ATG therapy. When pokeweed mitogen, herpes simplex type I virus, and tetanus toxoid were used as probes to elicit in vitro Ig production using a hemolytic plaque assay, some patients had 1) B cells that failed to produce Ig, 2) T cells that failed to provide helper activity, and 3) T cells that exhibited excessive suppressor activity in the various antibody production systems. These measures of immune function, however, did not correlate with clinical responses to ATG therapy.     

Antithyroglobulin (ATG) production by peripheral blood leukocytes in vitro.

Antithyroglobulin is produced in vitro by pokeweed mitogen stimulation of peripheral blood lymphocytes from patients whose serum contains the autoantibody. Antithyroglobulin synthesis requires T lymphocyte help but is suppressed by larger numbers of T lymphocytes. T cells from both patients and normals perform both of these functions.     

Sulfasalazine treatment and lymphocyte function in patients with rheumatoid arthritis

Sulfasalazine is now an established 2nd line agent in the treatment of rheumatoid arthritis (RA) but its mode of action is unknown. Two separate studies have investigated the possibility that it works in RA by influencing lymphocyte function. After 12 weeks of treatment with sulfasalazine, elevated levels of circulating activated lymphocytes and abnormal ex vivo mitogen response to concanavalin A (Con-A) in 11 patients with RA reverted to normal. An in vitro study investigated the effect of sulfasalazine and its metabolites on mitogen response by healthy and RA peripheral blood mononuclear cells (PBMC). Sulfapyridine (SP) and 5-hydroxy SP suppressed the response of RA PBMC to Con-A. Sulfasalazine, SP and N-acetyl SP suppressed the response of healthy PBMC to pokeweed mitogen. 5-aminosalicylic acid also affected mitogen response and cell viability, which may be relevant to actions of this metabolite within the gut.     

Analysis of impaired in vitro immunoglobulin synthesis in rheumatoid arthritis.

Decreased immunoglobulin production in pokeweed mitogen driven lymphocyte cultures has been reported in rheumatoid arthritis (RA). Here various activators and experimental designs have been used to determine the contribution of B cells, T cells, or monocytes to this low response. Sixty patients with RA and paired controls were studied at the onset of disease and again six months later. Concentrations of IgA, IgG, and IgM in cultures of RA peripheral blood mononuclear cells stimulated with thymus dependent activators were already decreased at the onset of the disease. Six months later RA mononuclear cells produced even lower concentrations of immunoglobulin. In contrast, stimulation with a T cell independent activator showed that RA B lymphocytes had retained normal potential to synthesise immunoglobulin. Poor helper function was indicated by costimulation experiments and cultures of mixed mononuclear cells from patients and controls. This notion was supported also by the fact that phytohaemagglutinin induced interleukin-2 production by RA mononuclear cells was less than half of the control values. Nonspecific suppressor activity was similar in RA and controls. Monocyte functions were normal when tested by addition of indomethacin or 2-mercaptoethanol to the mitogen activated cultures. The defect in mitogen stimulated immunoglobulin production in vitro of RA mononuclear cells thus was more pronounced with time and probably reflects impaired mediator associated help in the differentiation of B lymphocytes into immunoglobulin secreting cells.     

Regulation of the induction of colonies in vitro by normal human lymphocytes.

Lymphocytes isolated from normal human peripheral blood can be induced to form colonies in vitro by incubation with the appropriate inducer. Phytohemagglutinin can induce colonies with T (thymus-derived) lymphocytes. Optimun colony formation with about two colonies per 10(2) peripheral blood lymphocytes was obtained by adding, in addition to phytohemagglutinin, autologous plasma, autoologous red blood cells, and fresh L-glutamine or L-cystine. In the absence of these fresh amino acids, no colonies were formed at seeding levels below 10(5) cells per 35 mm petri dish. The addition of either of these amino acids gave a 10-fold decrease in the minimum number of cells that had to be seeded for colony formation. Lipopolysaccharide did not induce the formation of colonies, but enhanced the formation of T cell colonies by phytohemagglutinin. The mixing of lymphocytes from persons with and deficient in glucose-6-phosphate-dehydrogenase (EC 1-1-1-49) has shown that phytohemagglutinin-induced colonies can be derived from single cells and are, therefore, clones. No colonies were formed by lethally irradiated cells. Incubation with pokeweed mitogen also induced the formation of colonies. With autologous plasma and autologous red blood cells, pokeweek mitogen induced about one colony per 5 X 10(3) cells seeded and no colonies at seeding levels below 10(5) cells per petri dish. The minimum number of cells needed for colony formation by pokeweed mitogen was not decreased by fresh L-glutamine or L-cystine. The results indicate that normal human lymphocytes can be cloned in vitro and that induction of these lymphocyte colonies can be regulated by lectins and other specific factors.     

Cytofluorometric analysis of the kinetics of lymphocyte transformation after phytohemagglutinin stimulation: comparison with the kinetics of thymidine incorporation.

The technique of flow cytofluorometry has been employed to assess the response of unfractionated and highly purified human lymphocyte subpopulations to phytohemagglutinin (PHA) and pokeweed mitogen. Normal values for cytofluorometric responses were established and compared to the uptake of tritiated thymidine in simultaneous experiments. Cytofluorometric analysis offered the advantages of increased sensitivity and direct measurement of DNA content per cell, and provided percentages and absolute numbers of responding cells. B-cell responses to pokeweed mitogen were absent, but brisk T-cell responses were noted. Between 4% and 8% of highly purified human B cells were found to respond to PHA by increasing their DNA content; modest but significant uptake of tritiated thymidine by B cells following PHA stimulation was also observed.     

Cell-mediated immunity in rheumatic diseases

Lymphocyte responses to phytohemagglutinin, concanavalin A, and pokeweed mitogen were tested in normal patients and in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma (PSS), other connective tissue disease, and other illnesses. The relationship of lymphocyte response to diagnosis, therapy, and T- and B-lymphocyte populations was analyzed. Additional studies included the determination of proliferative responses of various combinations of purified T and B lymphocytes cultured with plant mitogens. Lymphocytes from patients with RA and SLE incorporated significantly less thymidine in the presence of plant mitogens as compared to normal and comparably ill subjects. Treatment had no effect on mitogen response. Responses to all three mitogens correlated closely in patients with RA, SLE, or PSS; no correlation was noted between the response to mitogen of lymphocytes in culture and the number of T cell ultured.     

Studies of Lymphocyte Proliferation in Hairy Cell Leukaemia: Activity in Mixed Lymphocyte Reaction and Responses to Mitogens

Subpopulations of splenic lymphocytes from patients with hair cell leukaemia (HCL) were compared with similar subpopulations of lymphocytes from reference individuals for their ability to respond to mitogens and to participate in allogenic and autologous mixed lymphocyte reactions. T cell enriched subpopulations were obtained by double passage of mononuclear cells through mylon wool columns. Non-T cell subpopulations were collected by eluting adherent cells from nylon wool columns and by incubating them with sheep erythrocytes followed by density gradient centrifugation. Unfractionated mononuclear cells, T enriched and non-T subpopulations were compared. Enriched T cell subpopulations from HCL and reference patients responded similarly to allogeneic antigens and phytohaemagglutinin. Splenic non-T cells from reference patients produced a stronger stimulus in the allogeneic mixed lymphocyte reaction than did the unfractionated or the T enriched cells. In contrast, the non-T subpopulations from patients with HCL produced a reduced response compared to that of reference splenic cells when mixed with allogeneic lymphocytes. In addition, non-T cells from HCL patients failed to respond to pokeweed mitogen. Neither reference nor HCL splenic cells produced a significant response in the autologous mixed lymphocyte reactions. The data suggest that splenic non-T cells from patients with HCL either suppress the stimulatory capacity of normal B lymphocytes or fail to stimulate allogeneic lymphocytes in the mixed lymphocyte reactions.     

Spontaneous and induced immunoglobulin synthesis and anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis: relation to leukocyte subpopulations in blood and active lesions

Summary Using a reverse plaque forming cell (PFC) assay the production of immunoglobulin (Ig) by peripheral blood mononuclear cells (MNCs) in vitro was studied in 12 patients with Wegener's granulomatosis (WG). Spontaneous IgG production was increased in two of six untreated patients. The IgG response of MNCs from eight untreated patients to pokeweed mitogen (PWM) and Epstein-Barr virus (EBV) stimulation was significantly depressed. The IgM and IgA production followed the individual pattern of IgG. Blood B-cell and T-cell subset concentrations were normal before therapy, whereas the monocyte concentration was increased in four of six patients. Titers of anti-neutrophil cytoplasm autoantibodies (ANCAs) did not correlate with spontaneous or induced Ig production nor with blood leukocyte subset concentrations. Biopsy specimens from upper respiratory tract lesions in seven untreated patients showed numerous macrophages, activated T lymphocytes, and plasma cells, suggesting a pathogenetic role of these cells in the development of lesions and local production of ANCAs.     

Interaction of cytomegalovirus with leukocytes from patients with mononucleosis due to cytomegalovirus.

Cytomegalovirus (CMV) was isolated from hemic cells from seven of seven patients with acute CMV mononucleosis and from one of six patients with mononucleosis during convalescence. CMV was isolated from the mononuclear leukocyte fraction, the polymorphonuclear leukocyte fraction, or both cell fractions. Virus was not detected in washed erythrocytes, plasma, or leukocyte lysates. Mononuclear leukocytes from patients with acute CMV mononucleosis displayed an elevated level of incorporation of [3H]thymidine on the day of donation compared with that in cells from convalescent patients or normal donors. Responses to pokeweed mitogen and concanavalin A were significantly lower in patients with CMV mononucleosis than in normal donors. There were no significant differences among the acute, convalescent, and normal donor groups in response to phytohemagglutinin or in the one-way mixed leukocyte reaction. These findings suggest that in patients with CMV mononucleosis virus infects and may persist within peripheral blood leukocytes and that the lymphocytes of these patients are selectively hyporesponsive to certain mitogens.     

T lymphocyte colonies stimulated by different mitogens require diverse culture conditions

Normal human peripheral blood mononuclear cells form colonies of T lymphocytes in a semi-solid agar matrix when stimulated by a variety of mitogens. In this report, we attempt to determine the optimal conditions for the formation of T lymphocyte colonies by cells stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM), Concanavalin A (Con A), or Staphylococcal protein A (SPA). We conclude that optimal conditions differ for each mitogen used. Cultures stimulated by PWM or Con A showed a significant requirement for feeder layers composed of human peripheral blood mononuclear cells. Two-mercaptoethanol significantly enhanced the number of T-cell colonies when PWM, Con A, or SPA, but not PHA, were added as mitogens. Fetal calf serum (FCS) was required for optimal conditions when Con A or SPA but not PWM or PHA were used to stimulate mononuclear cells. Cells stimulated by PHA or PWM produced more T-cell colonies in a 2-step assay than a 1-step assay, whereas the reverse was true with Con A or SPA. Optimal cell concentrations, mitogen doses, and culture kinetics also differed for each mitogen used in the T-cell colony assay.     

Development of a chemically defined serum- and protein-free medium for growth of human peripheral lymphocytes.

A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foundation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The peripheral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic makeup and biochemical environmental history of the individual at the time the cells were formed. Examination of the role of various factors in lymphocyte activation and subsequent events may be simplified by the utilization of a medium that is protein-free and chemically defined. The CFBI 1000 medium supports the growth response of human peripheral lymphocytes to mitogen as measured by [3H]thymidine incorporation to an extent comparable to other media used widely in assessment of lymphocyte proliferation.     

In vitro regulation of immunoglobulin synthesis after human marrow transplantation. II. Deficient T and non-T lymphocyte function within 3-4 months of allogeneic, syngeneic, or autologous marrow grafting for hematologic malignancy

Immunoglobulin secretion was studied in 37 patients between 19 and 106 days after allogeneic HLA-identical (30 patients), allogeneic one HLA- haplotype-identical (three patients), syngeneic (three patients), or autologous (one patient) marrow grafting. E rosette-positive (T) and E rosette-negative (non-T) peripheral blood mononuclear cells were cocultured with pokeweed mitogen for 6 days. Polyvalent immunoglobulin secretion was determined by counting plaque forming cells in a reverse hemolytic plaque assay. The number of antibody secreting cells in cocultures of autologous T and non-T lymphocytes was low in 40 of 44 tests conducted on samples from the 37 patients. Mononuclear or non-T cells from 38 of 40 tests failed to produce antibody when cultured with normal helper T cells. T cells from 23 of 37 tests failed to help normal non-T cells secrete antibody. T lymphocytes from 23 of 41 tests suppressed antibody production greater than 80% by normal T and non-T cells. The suppressor cells were radiosensitive in 17 of the 25 tests. The abnormal function of lymphocyte subpopulations in patients during the first 3 mo after syngeneic, allogeneic or autologous marrow grafting was similar regardless of the type of graft or the presence of acute graft versus host disease.     

Detection of antinuclear ribonucleoprotein (nRNP) antibody producing cells in peripheral blood lymphocytes from patients with mixed connective tissue disease

Antinuclear ribonucleoprotein (nRNP) antibody forming cells were detected in pokeweed mitogen activated peripheral blood lymphocytes (PBL) from 5 of 6 patients with mixed connective tissue disease (MCTD) in the presence of rabbit antihuman IgG antiserum and guinea pig complement as hemolytic plaque forming cells (indirect PFC). In the absence of rabbit antihuman IgG antiserum, direct PFC were detected only in a case of PBL from the 6 patients. Although we have successfully detected mainly IgG anti-nRNP antibody forming cells in PBL from patients with MCTD, numbers of PFC did not correlate with the serum levels of IgG anti-nRNP determined by the enzyme linked immunosorbent assay.