Helper activity for immunoglobulin synthesis of T helper type 1 (Th1) and Th2 human T cell clones: the help of Th1 clones is limited by their cytolytic capacity.

A large number of CD4+ human T helper type 1 (Th1) clones specific for purified protein derivative and of Th2 clones specific for the excretory/secretory antigen of Toxocara canis, derived from the same individuals, were analyzed for both cytotoxic capacity and helper function for immunoglobulin (Ig) synthesis. The great majority of Th1, but only a minority of Th2 clones exhibited cytolytic activity. All Th2 (noncytolytic) clones induced IgM, IgG, IgA, and IgE synthesis by autologous B cells in the presence of the specific antigen, and the degree of response was proportional to the number of Th2 cells added to B cells. Under the same experimental conditions, Th1 (cytolytic) clones provided helper function for IgM, IgG, and IgA, but not IgE, synthesis with a peak response at 1:1 T/B cell ratio. At higher T/B cell ratios, a strong decrease of Ig synthesis was observed. All Th1 clones lysed Epstein-Barr virus transformed autologous B cells pulsed with the specific antigen. The decrease of Ig production at high T/B cell ratios correlated with the lytic activity of Th1 clones against autologous antigen-presenting B cell targets. These data suggest that Th1 differ from Th2 human T cell clones not only for their profile of cytokine secretion, but also for cytolytic potential and mode of help for B cell Ig synthesis.

Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production.

A large series of T cell clones (TCC) specific for purified protein derivative (PPD) of Mycobacterium tuberculosis (total 60) or Toxocara canis excretory/secretory (TES) antigen (total 69) were established from the peripheral blood of two healthy individuals and analyzed for their profile of cytokine production in response to stimulation with either the specific antigen or the polyclonal activator phorbol myristate acetate plus anti-CD3 antibody. Under both these experimental conditions, the great majority of PPD-specific TCC secreted IL-2 and IFN-gamma but not, or limited amounts of, IL-4 and IL-5. In contrast, most TES-specific TCC secreted IL-4 and IL-5 but not, or limited amounts of, IL-2 and IFN-gamma. PPD-specific TCC that failed to secrete IL-4 and IL-5, and TES-specific TCC that failed to secrete IL-2 and IFN-gamma, were found to lack transcripts for IL-4 and IL-5, or for IL-2 and IFN-gamma, respectively. During the course of the study, over a 6-mo period, the functional phenotype of both TES- and PPD-specific TCC was repeatedly assessed and remained constant. These data demonstrate that T cells with stable Th1 or Th2 functional pattern exist not only in mice but also in humans and suggest that in the course of natural immunization certain infectious agents preferentially expand T cell subsets with stable and definite profile of cytokine production.

Recombinant interferon-alpha selectively inhibits the production of interleukin-5 by human CD4+ T cells.

The effects of recombinant IFN-alpha on the production of IL-5 by human CD4+ T cells were first analyzed on resting CD4+ T cells purified from normal PBMC and stimulated either with a combination of PMA and anti-CD28 mAb or anti-CD3 mAb cross-linked on B7-1/CD32-transfected mouse fibroblasts. We found that IFN-alpha profoundly inhibited in a dose-dependent manner IL-5 production by resting CD4+ T cells whereas IL-10 was upregulated in both systems. The addition of a neutralizing anti-IL-10 mAb to PMA and anti-CD28 mAb upregulated IL-5 production by resting CD4+ T cells but did not prevent IFN-alpha-induced IL-5 inhibition. We then analyzed the effect of IFN-alpha on the production of cytokines by differentiated type 2 helper (Th2) CD4+CD3- cells isolated from peripheral blood of two patients with the hypereosinophilic syndrome. In both cases, IFN-alpha markedly inhibited IL-5 production while it induced mild upregulation of IL-4 and IL-10. Finally, the inhibitory effect of IFN-alpha on IL-5 production was confirmed on a panel of Th2 and Th0 clones generated in vitro. In 2 out of 6 clones, IL-5 inhibition was associated with upregulation of IL-4 and IL-10. We conclude that IFN-alpha selectively downregulates IL-5 synthesis by human CD4+ T cells.

Abnormalities of in vitro immunoglobulin synthesis by peripheral blood lymphocytes from untreated patients with Hodgkin's disease.

The immunoglobulin-synthesizing activities of peripheral blood mononuclear cells from 57 untreated patients with Hodgkin's disease and 47 normal subjects were compared. Cumulative amounts of IgM and IgG synthesized and secreted by unstimulated and pokeweed mitogen-stimulated cells over a 7-d period were determined in a solid-phase radioimmunoassay. Synthesis of IgM in unstimulated cultures and of both IgM and IgG in cultures stimulated with pokeweed mitogen was markedly reduced in patients with Hodgkin's disease, whereas the mean level of the spontaneous IgG synthesis was enhanced. The degree and frequency of in vitro abnormalities were not influenced by disease stage or histology. Depression of pokeweed mitogen-induced immunoglobulin synthesis did not correlate with excessive number of monocytes and it was unaffected by removal of phagocytic cells or addition to the cultures of monocytes from normal individuals. On the other hand, monocytes isolated from blood of patients with Hodgkin's disease were even more effective than normal monocytes in supporting pokeweed mitogen-induced immunoglobulin synthesis by normal phagocyte-depleted mononuclear cells. Synthesis of both IgM and IgG induced by pokeweed mitogen remained subnormal after addition to patient B cell cultures of autologous irradiated T cells or allogeneic normal T lymphocytes. T cells from patients with Hodgkin's disease appeared at least as effective as normal T cells in helping pokeweed mitogen-induced immunoglobulin production by normal B cells. However, when normal T cells were co-cultured with B cells from patients with Hodgkin's disease, spontaneous IgG synthesis declined, whereas the addition of patient T cells to normal B cells resulted in an increase of spontaneous IgG synthesis. In patients showing depression of pokeweed mitogen-induced immunoglobulin synthesis the lymphoproliferative response and immunoglobulin synthesis stimulated by Staphylococcus aureus bacteria of the Cowan first strain, a T cell independent B cell mitogen, were also markedly reduced. These studies demonstrate impairment of immunoglobulin synthesis by cultured lymphocytes from untreated patients with Hodgkin's disease after stimulation with polyclonal B cell activators and suggest that the in vitro abnormalities may be, at least in part, the result of a preexisting in vivo activation of lymphocytes in Hodgkin's disease patients.

In vivo CD30 expression in human diseases with predominant activation of Th2-like T cells.

CD30 is a member of the tumor necrosis factor (TNF) receptor family, originally described as a marker for Hodgkin and Reed-Sternberg cells in Hodgkin's disease, which has been found to be preferentially expressed by T cells producing Th2-type cytokines. The presence of CD30 expression was assessed by both immunohistochemistry and reverse transcriptase-polymerase chain reaction in the target organs of patients with Th1- or Th2-dominated disorders. CD30 expression was found in neither the gut of patients with Crohn's disease nor in the gastric antrum of Helicobacter pylori-infected patients, where there was high interferon-gamma (IFN-gamma) expression. In contrast, high CD30 expression in the apparent absence of IFN-gamma expression was observed in the skin of patients with systemic sclerosis or chronic graft versus host disease (GVHD), which can be considered Th2-dominated disorders. Moreover, high levels of soluble CD30 were found in the serum of both systemic sclerosis and GVHD patients but not in the serum of patients suffering from multiple sclerosis, a Th1-dominated disorder. Thus, CD30 expression appears to be preferentially associated with Th2-type responses not only in vitro but also in vivo.

Surface markers and function of circulating thyroid autoantibody-producing cells.

The in vitro synthesis of antithyroglobulin (anti-Tg) and antithyroid microsomal (anti-M) autoantibodies by peripheral blood mononuclear cells (MNC) from patients with autoimmune thyroid diseases was investigated using sensitive immunoradiometric assays. Cultures were carried out in the presence or in the absence of pokeweed mitogen (PWM). Thyroid autoantibodies were undetectable in supernatants of MNC cultures from 9 normal subjects. Supernatants of MNC cultured without PWM had detectable levels of anti-Tg and anti-M in 5 (19.3%) and in 2 (7.7%) of 26 patients with autoimmune thyroid diseases, respectively. In the presence of PWM, a marked increment in the antibody concentrations occurred in all but 1 of these cultures, and the number of positive cultures increased to 13 (50.1%) for anti-Tg and to 15 (57.7%) for anti-M. Studies of MNC fractions depleted of T lymphocytes (non-T cells) were carried out on selected patients showing antibody synthesis only after PWM stimulation. Autoantibody production was not found with non-T cells, but the effect of the mitogen was restored by readdition of T cells. Irradiation (1000 rad) of T cells before coculturing significantly enhanced autoantibody production. With this model no significant functional difference was found between autologous and allogenic T cells from thyroid autoimmune disease patients or from normal subjects. The cells involved in PWM-driven thyroid autoantibody synthesis, as defined by depletion studies, were lymphocytes bearing DR antigens and surface immunoglobulin G (IgG) without detectable surface immunoglobulin M (IgM). Depletion from MNC suspensions of Tg-binding cells abolished PWM-stimulated anti-Tg production, but did not alter the synthesis of anti-M. Further studies were carried out on MNC from a single patient with Hashimoto's thyroiditis, whose non-T cells consistently produced large amounts of anti-M and total IgG in the absence of PWM. The addition of PWM to these unfractionated MNC slightly increased the production of anti-M, but inhibited antibody synthesis after depletion of T lymphocytes. Interestingly, the addition of autologous T lymphocytes to non-T cells inhibited the spontaneous synthesis of anti-M. These data indicate that in vitro synthesis of anti-Tg and anti-M by MNC may be frequently induced by stimulation with PWM in patients with thyroid autoimmune disorders. PWM-stimulated synthesis of thyroid autoantibodies appears to be T-cell dependent and modulated by radiosensitive T lymphocytes. The cells responsible for PWM-dependent thyroid autoantibody synthesis are B lymphocytes with surface membrane IgG and have receptors specific for the autoantigen.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytolytic T lymphocytes with natural killer activity in thyroid infiltrate of patients with Hashimoto's thyroiditis: analysis at clonal level.

T Lymphocytes from thyroid infiltrates and peripheral blood (PB) of 3 patients with Hashimoto's thyroiditis (HT) were cloned using a microculture system previously shown to allow the clonal expansion of virtually all PB T lymphocytes from normal individuals. The phenotypic and functional features of a total number of 153 clones from thyroid infiltrates and 206 clones from PB were examined and compared with those of 272 clones derived from normal PB and spleens. The majority of clones derived from thyroid infiltrates of patients with HT had the cytotoxic/suppressor (T8+) phenotype, whereas the majority of clones from PB expressed the helper/inducer (T4+) phenotype. In addition, a consistent proportion (25%) of clones derived from PB of one patient had a phenotype (T3+T4-T8-) that was only occasionally found on clones obtained from PB or spleens of normal subjects. Most clones derived from both PB and thyroid infiltrates of the patients with HT had cytolytic activity, assessed by a lectin-dependent cytolytic assay against the murine P815 tumor cell line. The high frequency of cytotoxic T cells in thyroid infiltrates was related to the increased proportion of T8+ cells, whereas enhanced percentages of cytotoxic cell precursors with T4+ and T3+T4-T8- phenotypes primarily accounted for the high frequency of cytolytic T cells in the PB of the same patients. Many cytolytic T cell clones derived from thyroid infiltrates also had natural killer activity against human K562 and MOLT-4 target cells. These data provide the first functional analysis of T lymphocytes infiltrating the thyroid gland in patients with HT and suggest that the high proportions of cytolytic T cell precursors found in both thyroid infiltrates and PB of these patients may be of importance in determining the tissue damage in thyroid autoimmune disease.

A simple solid-phase radioimmunoassay for the measurement of IgG secreted in vitro by human lymphocytes.

A radioimmunoassay is described for measuring IgG, based on the ability of immunoglobulins of this class to inhibit the binding of radioiodinated staphylococcal protein A to IgG linked to a solid phase. The solid phase is represented by ox erythrocytes coated with anti-ox erythrocyte rabbit IgG, a reagent used for detecting cells equipped with receptors for the Fc fragment of IgG. By this assay the IgG secreted in vitro by human peripheral blood lymphocytes stimulated with PWM and those present in samples of very diluted human sera were measured. It was found that the assay is a very rapid, simple and reproducible procedure for the detection of IgG immunoglobulin at the nanogram level.

High potential to tumor necrosis factor alpha (TNF-alpha) production of thyroid infiltrating T lymphocytes in Hashimoto's thyroiditis: a peculiar feature of destructive thyroid autoimmunity.

T lymphocytes present in thyroid infiltrates of 6 patients with Hashimoto's thyroiditis (HT) and of 4 patients with Graves' disease (GD) were analyzed at clonal level and their profiles of mitogen-induced lymphokine secretion were characterized. Production of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-gamma (IFN-gamma) was measured in culture supernatants of a total number of 332 T cell clones (TCC) from HT, of 269 TCC from GD infiltrates and of 266 control TCC derived from normal lymphoid tissues. No significant difference was found in the ability to produce IL-2 between TCC from HT or GD infiltrates and control TCC. The proportion of HT- or GD-derived TCC able to produce IL-4 was extremely low (4 and 5%, respectively) in comparison with controls (19%). In contrast, the proportion of interferon-gamma (IFN-gamma)-producing (IFN-P) TCC derived from either HT (87%) or GD (80%) infiltrates was much higher (p less than 0.0005) than that found in controls (59%). In addition, most of IFN-P TCC from either HT or GD usually released higher amounts (p less than 0.002) of IFN-gamma than did control clones. No significant difference was found between GD infiltrates and controls in the proportions of TCC able to secrete TNF-alpha (39% and 47%, respectively), whereas the proportion of TNF-alpha-producing (TNF-P) TCC derived from HT (78%) was significantly higher (p less than 0.0001). In addition, most of both CD8 and CD4 TCC from HT released higher amounts of TNF-alpha than did TNF-P clones from controls or GD. These data suggest that T cells present in autoimmune thyroid infiltrates share a number of functions, such as high production of IFN-gamma, but differ with regard to their ability to secrete TNF-alpha, which is peculiar of most T cells present in the thyroid of HT patients.

Thyroiditis as a model of organ specific autoimmune disease.

In the last few years a great deal of information on the etiopathogenetic aspects of organ-specific autoimmune diseases has been obtained from the extensive study of both animal models of experimental or spontaneous thyroiditis and of human thyroid autoimmune diseases. It has been clearly shown that genetic factors play a fundamental etiologic role. They are responsible for the dysregulation of the immune system and for the target organ susceptibility which favor the onset of the disease. Environmental factors are presumed to act as initiating or precipitating events, leading genetically predisposed individuals to thyroid autoimmunity. A number of immune mechanisms able to trigger autoimmune responses, such as antigenic cross-reactions and the aberrant expression of HLA class II molecules, have been suggested, but the definition of why and how they become operative requires further investigation. Data obtained from experimental models and from human thyroid diseases clearly indicate that the ongoing expansion of autoreactive T cells with specificity for thyroid autoantigens represents the main immunological event responsible for induction and maintenance of thyroid damage. Such autoreactive T cells can induce tissue lesions through activation of different effector systems and secretion of different combinations of lymphokines. In overt thyroid autoimmune diseases autoantibodies directed against functional molecules or cellular receptors can also be involved in the pathogenesis of tissue lesions. However, the pathogenesis of inflammatory destructive lesions of the thyroid is more complex and not yet fully elucidated. It is worthy of note that a large proportion of T cells present in inflammatory human thyroid infiltrates are apparently not directed against the thyroid autoantigens recognized so far.(ABSTRACT TRUNCATED AT 250 WORDS)

CD8 T-cell clones producing interleukin-5 and interferon-gamma in bronchial mucosa of patients with asthma induced by toluene diisocyanate.

OBJECTIVES: The aims of the present study were to determine whether specific in vivo stimulation of asthmatics sensitized with toluene diisocyanate (TDI) induces the activation of T lymphocytes in bronchial mucosa and to characterize their phenotype and cytokine secretion profile. METHODS: Bronchial biopsies from two subjects with occupational asthma due to TDI were obtained 48 h after an asthmatic reaction induced by an inhalation challenge with TDI and after three months of no exposure to TDI, at the time when the subjects had recovered from their asthma. The fragments of bronchial mucosa were cultured in the presence of interleukin-2 so that the in vivo activated T cells present in the tissue would expand, and T blasts were then cloned under limiting dilution conditions. RESULTS: From the two 48-h specimens, 65 and 63 T-cell clones were obtained. Most of the clones exhibited the CD8 phenotype (82 and 83%). All of the CD8 clones produced interferon-gamma and 44% produced interleukin-5, but only 6% secreted interleukin-4 as well. Three months after the cessation of exposure, growing T cells could not be recovered from bronchial biopsies cultured in interleukin-2. CONCLUSIONS: The results suggest that, in sensitized subjects, exposure to TDI induces the activation of a subset of CD8 lymphocytes producing interferon-gamma and interleukin-5.

Heterologous specificity of reticulin RS antibodies.

Reticulin antibodies were detected by the indirect immunofluorescence technique in 1450 sera from miscellaneous patients. R1 pattern on human and rat tissues was found only in 2 cases with adult coeliac disease. Reticulin Rs staining, involving intrasinusoidal cells of rat liver as well as interstitial and endothelial structures of various rat tissues, was demonstrated in 3.7% of cases, without correlation with gastro-intestinal or skin disorders. Rs pattern, due to IgG antibodies, could be absorbed by rat erythrocytes. Only Rs positive sera showed high titres of anti-rat IgG-type hemoagglutinins. Rat RBC agglutinating activity was demonstrated by a modified indirect Coomb's test. Heterologous Rs antibodies appear unrelated to particular diseases and should not be mistaken for the R1 and R2 patterns, which may be useful for diagnostic procedures.

Isoantibodies to human exocrine pancreas and endothelium.

A cytoplasmic immunofluorescence of some human pancreatic exocrine cells was obtained in 19.2% of 270 patient undiluted sera and confirmed in 20.4% of 98 healthy controls. No relation was found with clinical disorders or autoantibodies. This reaction was always associated with human endothelial antibodies and both were clearly related to high titre "incomplete" IgG anti-A group isoagglutinins.

T-cell independence of immunoglobulin synthesis by human peripheral blood lymphocytes stimulated with SpA-containing staphylococci.

Unfractionated and T-cell depleted human peripheral blood lymphocytes (PBL) were cultured in vitro in the presence of pokeweed mitogen (PWM) and Staphylococcus aureus strain Cowan I (StaCw). After 7 days of culture, the cells were assayed for cytoplasmic immunoglobulins (Cyto-Ig) by direct staining using fluorescein-labelled F(ab')2 fragments prepared from specific antisera against human IgG F(ab')2. The amount of immunoglobulin of the IgM and IgG class released into the cell-free supernatants was also measured by radioimmunoassay. In unfractionated PBL StaCw, like PWM, was able to induce a significant increase of either the number of Cyto-Ig containing cells for the amount of IgM and IgG secreted into the supernatant. In contrast, the amount of IgM and IgG immunoglobulin released into the supernatant of T-cell depleted suspensions stimulated with PWM was significantly reduced in comparison with that of unfractionated populations, whereas it was unchanged in T-cell depleted vs unfractionated suspensions stimulated with StaCw. The addition of a few T lymphocytes restored the ability of T-cell depleted suspensions to produce Ig in the presence of PWM, whereas despite addition of high numbers of T cells no further augmentation of the Ig production induced by StaCw on T-cell depleted suspensions was observed. Cultures of umbilical cord blood lymphocytes (UCBL) stimulated with PWM did not generate Ig-producing cells, whereas UCBL stimulated with StaCw showed significant production of Ig of both IgM and IgG classes. The results indicate that T lymphocytes are probably not involved either with stimulation or with the suppression of Ig production induced by StaCw.

In vitro IgE synthesis induced by human T cell clones in normal B cells and its suppression by heterogenous T cell populations.

Thirty-four out of 119 human T cell clones established from tonsillar or peripheral blood T cell suspensions of 3 nonallergic individuals were able to induce normal B cells to synthesize remarkable amounts of IgE in vitro. The activity of these clones was apparently mediated by triggering of the monomorphic molecular complex CD3 immediately before or during their incubation with the target B cells. The addition to the cultures of mitogen-stimulated autologous unfractionated T cells inhibited in a dose-dependent fashion, the T cell clone induced IgE, but not IgG, synthesis.

New trends on the pathogenetic aspects of allergic bronchial asthma.

The central role of IgE antibodies in allergic bronchial asthma (ABA) is not disputed any longer. Immunological mechanisms other than IgE-mediated reactions in the pathogenesis of ABA have not yet been definitely demonstrated. There is controversy as to whether IgG STS antibodies, probably IgG4, play a role in the disease process. There is no evidence to support the involvement of immune complexes. Anti-beta-2-adrenergic receptor autoimmune responses cannot be considered as responsible for the development of the disease at the present time. In the last few years a great effort has been made to understand the immunological mechanisms underlying the enhanced and long-standing IgE antibody production in atopic diseases. The results obtained in different laboratories, including our own, are in favor of a high responder status to allergen epitopes in a large proportion of atopics and suggest that a preferential expression of IgE isotype in the antibody responses may occur through different mechanisms. Of great interest is the recent demonstration of IgE-binding factors with IgE-potentiating and suppressive activity and of other related regulatory molecules and receptors. Progress has been recently achieved in the characterization of mediators responsible for the different pathological changes of ABA. A linkage between immediate IgE-mediated reactions, bronchial late-phase reactions (LPR) and chronic inflammation (CI) has been reported. It has been demonstrated that a cascade of mediators and cell interactions induce both LPR and CI. There is evidence of a close relationship between LPR-CI and nonspecific bronchial hyperreactivity. A better knowledge of pathogenetic mechanisms of ABA would open new perspectives in the therapy. A modulation of IgE antibody production can be attempted in different ways. At present a control of mediator release and of airway hyperreactivity can be achieved by several pharmacological interventions and by the avoidance of common and/or occupational allergens or pollutants.

IgE synthesis in vitro induced by T cell factors from patients with elevated serum IgE levels.

The effect of unstimulated T cell culture supernatants (TCS) from patients with atopic dermatitis and high serum IgE levels on the IgE production in vitro by B cell rich suspensions from normal individuals or grass pollen sensitive patients with mild atopy was evaluated. TCS from patients with raised IgE enabled B cell suspensions from normal individuals to produce detectable amounts of IgE and potentiated the spontaneous IgE synthesis in vitro by B cell suspensions from grass sensitive patients. In contrast, the addition of TCS from normal subjects with low serum IgE levels did not increase or even reduced IgE synthesis by B cell cultures. When the same B cell cultures were analysed for their ability to produce IgG or IgM protein, no significant differences were observed. These findings indicate that T lymphocytes from patients with high serum IgE levels can release soluble factor(s) possessing isotype (IgE) specific potentiating activity.