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The existence of functionally polarized human T cell responses based on their profile of cytokine secretion in both the CD4+ T helper (Th) and the CD8+ T cytotoxic cell subset has been established. Human Th1 and Th2 cells not only produce a different set of cytokines but also exhibit distinct functional properties and preferential expression of some activation markers, such as LAG-3 and CD30, respectively. Several factors are involved in the Th cell differentiation into the polarized Th1 or Th2 pathway. They include the cytokine profile of 'natural immunity' evoked by different offending agents, the nature of the peptide ligand, as well as the activity of some costimulatory molecules and microenvironmentally secreted hormones, in the context of different host genetic backgrounds. Polarized Th1-type and Th2-type responses play different roles in protection, Th1 being effective in the defense against intracellular pathogens and Th2 against intestinal nematodes. Moreover, they are responsible for different types of immunopathological reactions. Th1 responses predominate in organ-specific autoimmune disorders, acute allograft rejection, unexplained recurrent abortions, and in some chronic inflammatory disorders of unknown etiology. In contrast, Th2 responses predominate in Omenn's syndrome, transplantation tolerance, chronic graft versus host disease, systemic sclerosis; moreover allergen-reactive Th2 cells are involved in the triggering of atopic disorders.

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.

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.

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.

Biopsy specimens were obtained from the bronchial or the nasal mucosa of three patients with grass pollen-induced bronchial asthma or rhinitis 48 h after positive bronchial or nasal provocation test with grass pollen extract. T cell clones (TCC), derived from these and control specimens, were then assessed for their phenotype, allergen-specificity, profile of cytokine secretion and ability to provide B cell help for IgE synthesis. Out of 50 and 61 CD4+ TCC derived from the bronchial mucosa of the two patient with atopic asthma 11 (22%) and 19 (31%), respectively, showed both proliferation and cytokine production in response to grass pollen allergens under major histocompatibility complex-restricted conditions. Of these 21 (70%) exhibited a clear-cut type 2 T helper (Th2) profile and induced IgE synthesis in autologous peripheral blood B cells in the presence of grass allergens. All the other 9 grass-specific clones showed a Th0 pattern of cytokine secretion, but only 1 provided moderate help for IgE synthesis. In contrast, the majority of TCC, derived under the same experimental conditions from the bronchial mucosa of two nonatopic patients with toluene diisocyanate-induced asthma, were CD8+ and most of them produced interferon-gamma or interferon-gamma and interleukin-5, but not interleukin-4, in response to nonspecific stimulation. Of 22 CD4+ TCC3 (14%) derived from the grass-stimulated mucosa of the patient with allergic rhinitis, but none of those derived from the unstimulated nostril of the same patient, exhibited proliferation and cytokine production in response to grass allergens. All had a clear-cut Th2 profile and provided help for IgE synthesis by autologous B cells. These data indicate that inhalation of the relevant allergen results in the activation of allergen-specific Th2 lymphocytes in the airway mucosa of patients with allergic respiratory disorders. These cells may play a central role in determining the nature of the inflammatory response in the airways of atopic patients.

Objective: The serum concentration of soluble interleukin-2 receptor (sIL-2R) is a marker of T-lymphocyte activation. Increased circulating sIL-2R has been reported in untreated Graves' disease. This finding has been interpreted as the consequence of the autoimmune activation, but recent data suggest that sIL-2R is directly correlated to thyroid state. The aim of this study was to elucidate the respective roles of autoimmunity and thyroid function in modulating serum sIL-2R., Design and Patients: sIL-2R was evaluated in 20 normal euthyroid subjects and in a large series of patients with autoimmune and non-autoimmune thyroid disorders in different functional state., Measurements: sIL-2R was assayed by a solid-phase monoclonal antibody assisted ELISA method., Results: Serum sIL-2R in normals was 461 +/- 186 U/ml (mean +/- SD). Increased sIL-2R was found in 61 hyperthyroid patients with Graves' disease (1610 +/- 962 U/ml, P < 0.0001) and in 23 with toxic adenoma (1121 +/- 598 U/ml, P < 0.0001). Restoration of euthyroidism lowered to normal sIL-2R in both groups. Serum sIL-2R was higher in euthyroid Graves' disease patients with active than in those with non-active ophthalmopathy. Decreased serum sIL-2R (228 +/- 93 U/ml, P < 0.0001) was found in 30 patients hypothyroid after total thyroidectomy. Highly variable circulating sIL-2R (range 100-1456 U/ml, mean +/- SD: 379 +/- 301 U/ml) was found in 49 patients with hypothyroid Hashimoto's thyroiditis (P = NS vs normals; P < 0.02 vs post-thyroidectomy hypothyroid patients). Treatment with L-thyroxine increased sIL-2R in all thyroidectomized and in the majority of Hashimoto's thyroiditis patients. In individual Hashimoto's thyroiditis patients (mostly with increased serum sIL-2R), L-thyroxine caused a decrease of circulating sIL-2R, sIL-2R was normal in 29 patients with euthyroid Hashimoto's thyroiditis. Both in Graves' disease and in Hashimoto's thyroiditis, no correlation was found between sIL-2R and anti-thyroglobulin, anti-thyroid peroxidase and anti-thyrotrophin-receptor autoantibodies. Highly significant positive correlation between serum thyroid hormones and sIL-2R was found in all study groups., Conclusions: In thyroid disorders thyroid hormones are the main regulator of serum sIL-2R concentration. The contribution of autoimmune activation may be detected only in some patients with autoimmune hypothyroidism, while in Graves' disease the role of the immune system is masked by the hyperthyroid state.

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.

High serum concentration of soluble interleukin-2 receptor (sIL-2R) is considered a reliable marker of T lymphocyte activation. It has been recently reported that sIL-2R levels are increased in untreated Graves' disease. This finding has been interpreted as the consequence of an active autoimmune state, but the relevance of the thyroid function per se was not investigated. In the present study we assayed sIL-2R by ELISA in 20 normal subjects and in a series of patients with immunogenic (Graves' disease, GD) or nonimmunogenic (toxic adenoma, TA) hyperthyroidism. Significant increased concentrations of sIL-2R were found in 46 patients with untreated hyperthyroid GD (mean +/- SD: 1,683 +/- 1016 U/ml, vs 461 +/- 186 U/ml in normal controls, p less than 0.0001) and in 21 with untreated TA (1,111 +/- 617 U/ml, p less than 0.0001 vs normals). Restoration of the euthyroid state by antithyroid drugs or 131I administration was associated with a normalization of sIL-2R (516 +/- 174 U/ml in 38 patients with GD and 365 +/- 90 U/ml in 12 with TA; p = NS vs normals and p less than 0.001 vs the untreated state for both groups). A highly significant positive correlation between serum sIL-2R and free triiodothyronine (FT3) (r = 0.724, p less than 0.0001) or free thyroxine (FT4) (r = 0.698, p less than 0.0001) concentrations was found in combined sera obtained from all untreated and treated patients, irrespectively of the autoimmune or nonautoimmune nature of the underlying hyperthyroid disease.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The availability of high efficiency T-cell cloning techniques recently allowed the identification and characterization of clones derived from the thyroid infiltrate of patients with autoimmune thyroid diseases. Phenotypical and functional analysis of T-cell clones obtained from thyroid infiltrates of patients with Hashimoto's thyroiditis show that most of them are progenies of CD8+ cytolytic T cells with natural killer activity. This phenomenon, of potential importance in tissue damage, is markedly less pronounced in Basedow's disease glands. In both Hashimoto's thyroiditis and Basedow's disease only a minority of clones appear to be specific for autologous thyroid cells and most of them are potent interferon-gamma producers, while increased secretion of tumor necrosis factor-alpha is observed only in Hashimoto's thyroiditis. In contrast with normal lymphoid tissue, only very few T cell clones derived from both BD and HT infiltrates were able to produce detectable amounts of IL-4, suggesting that most of the thyroid-infiltrating T cells represent quite homogeneous populations of Th1-type "inflammatory" T cells. This peculiar potential of lymphokine secretion could play a role in the expression and/or maintenance of thyroid autoimmunity and thyroid functional damage.

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)

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.

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.

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.

The IgE helper function of human T cell clones or their phytohemagglutinin-induced supernatants was positively correlated with their ability to produce or their content in interleukin 4 (IL-4), whereas it was inversely correlated with production of or content in gamma interferon. The addition to B cell cultures of anti-IL-4 antibody abolished not only the IgE synthesis induced by recombinant human IL-4, but also that induced by IL-4-producing T cell clones or their phytohemagglutinin-induced supernatants. A clonal analysis in nonatopic donors and patients with common atopy showed that atopics possess in their peripheral blood significantly higher numbers of T cells able to secrete IL-4 and to provide helper function for IgE.

The role of T cells and monocytes, as well as that of cytokines, such as IL-1, IL-2 and IL-6, on the IL-4-dependent in vitro human IgE synthesis was investigated. Recombinant IL-4, IL-4-containing T-cell clone supernatants and different combinations of recombinant cytokines failed to induce highly purified B cells to synthesize IgE. IL-4-dependent IgE synthesis was restored by addition to purified B cells of either untreated or mitomycin C-treated autologous T lymphocytes. Addition to purified B cells of autologous monocytes did not restore the IgE response, but usually it exerted a potentiating effect on the synthesis of IgE induced by IL-4 in the presence of suboptimal concentrations of T cells. The activity of T cells apparently preceded that of IL-4 and required a physical contact with B cells. The presence in culture of IL-2 also appeared to be necessary for the T-cell and IL-4-dependent IgE synthesis. Even though not essential, IL-6 was able to potentiate IgE synthesis in most experiments, whereas IL-1 did not display any modulatory effect.

Sera from 46 patients with malignant melanoma (MM) in different clinical stages and from 25 healthy blood donors were studied by an indirect conventional immunofluorescent technique (IIT) and by an immunofluorescent complement-fixation test (ICFT). No specific and reliable serological reactivity could be demonstrated against autologous and homologous MM and other nonmalignant pigmented tissues. An increased incidence of diffused antinuclear (ANA; 41%) and cytoplasmic epidermal cell autoantibodies (A-CYT; 71%) was observed in the patients by IIT. Statistical evaluation of the results gave significant values in the chi 2 test in patients for ANA (chi 2 = 5.2061; p less than 0.05) and for A-CYT (chi 2 = 8.9176; p less than 0.01) versus age-matched controls. Thyroid microsomal, gastric parietal cell, smooth muscle and mitochondria autoantibodies were, respectively, detected in 4.3, 4.3, 4.3 and 0% of patients' sera.

Peripheral blood lymphocytes (PBL) from 15 of 38 patients with chronic lymphocytic leukaemia (CLL) were capable of forming rosettes with human erythrocytes coated with staphylococcal protein A (SpA-HRBC). PBL from seven patients also showed a marked proliferative response after stimulation with Staphylococcus aureus bacteria strain Cowan I (Cowan Staph). The SpA-rosetting of CLL cells was inhibited by incubation with F(ab')2 fragments of anti-immunoglobulin (Ig) antibodies. In addition, incubation with these fragments inhibited the proliferative response of leukaemic B cells to Cowan Staph. The Cowan-Staph induced proliferation of CLL cells was also impaired by the addition of normal human IgG and human IgG F(ab')2 fragments to the cultures, whereas non-immune rabbit IgG was not inhibitory. The inhibitory activity of human IgG F(ab')2 fragments was retained by a SpA-Sepharose column, and it was found in the material recovered from the same column by acid elution. These data indicate that an interaction between SpA and a structure located in the F(ab')2 region of surface Ig of different classes is responsible for either SpA-binding or the Cowan-Staph-induced proliferative response of PBL from some patients with CLL.

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.

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.

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.

Thirty-nine patients (14 non-diabetics, 8 chemical diabetics, and 17 overt diabetics) with circulating islet cell antibodies (ICA) were studied. Insulin and glucagon secretion after oral (100 g) and intravenous glucose loading (200 mg/kg bolus injection followed by an infusion of 20 mg/min over 60 min) and arginine infusion (25 g over 30 minutes) were evaluated in these patients and in non diabetic and diabetic ICA-negative controls. In the non-diabetic groups with or without ICA, insulin and glucagon responses to glucose were similar. Moreover, in ICA positive patients the response of these hormones to arginine infusion was reduced. Similar alterations in insulin and glucagon secretion were observed in the CIA positive and negative patients with chemical or overt diabetes. In particular, fasting hyperglucagonaemia and glucagon hyperresponse to arginine are associated with a lack of insulin secretion in the patients with overt diabetes. Hormonal differences between diabetics with and without ICA could not be detected.

We have studied the surface antigen pattern, enzymatic phenotype, and functional capacity of peripheral blood lymphocytes from a patient with Sézary syndrome (SS). The majority of these cells formed E rosettes but lacked the Fc(mu) receptor. The neoplastic cells were reactive with pan-T cell (OKT3)- and helper T cell (OKT4)-subset monoclonal antibodies; however, they lacked the 5/9 antigen, which identifies a more restricted subset of helper T cells. Most SS cells also reacted with PTF 29.12, a monoclonal antibody which recognizes DR determinants. Only 35% of the cells expressed single, focal accumulations of alpha-naphthyl-acid esterase activity, which is a characteristic of T.M cells, but 85% of them showed this focal staining pattern with acid phosphatase or beta-glucuronidase. Mononuclear cells from the SS patient showed poor or no proliferative response to phytohemagglutinin, pokeweed mitogen, concanavalin A, purified protein derivative, Candida, and allogeneic cells and lacked both helper and suppressor activity for pokeweed mitogen driven production of IgM and IgG immunoglobulins by normal B cells, but they were able to stimulate a marked proliferative response in mixed-lymphocyte culture. The defective expression of enzymatic and surface membrane characteristics, together with the lack of some T-cell functions, suggests that the patient cells may be immature T.M 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.

Factor I (fibrinogen) and factor VIII were shown, by an indirect immunofluorescence technique, to be present in human washed platelets. In the case of fibrinogen, the immunofluorescent pattern had a 'clod distribution' up to a 1:128 dilution of the antiserum. Factor VIII showed a 'speckled' pattern up to the 1:64 dilution. Cross-absorption studies confirmed the presence of such factors. Factor II, VII, IX, X and XIII, however, were not found in washed platelets. A diffuse fluorescence was noted using normal rabbit sera, but this was due to a cross-reaction, since it disappeared using a 1:32 diluted sera.

The in vitro effects of radiation, diterpine forskolin (FK), and hydrocortisone (HC) on the in vitro spontaneous IgE synthesis by peripheral blood B-lymphocytes from atopic patients were investigated. Without affecting cell viability, in vitro irradiation inhibited in a dose-dependent fashion de novo IgE synthesis in vitro by B cells from all patients examined with a mean 40% reduction of in vitro IgE product after treatment with 100 rads. In contrast, the in vitro IgE production by the U266 myeloma cell line was unaffected, even by irradiation with 1600 rads. The addition to B cell cultures from atopic patients of FK consistently resulted in a dose-dependent inhibition of the spontaneous IgE production in vitro. The addition to cultures of 10(-5) and 10(-6) molar concentrations of HC was also usually inhibitory, whereas lower HC concentrations were uneffective or even enhanced the spontaneous in vitro IgE synthesis. When 10(-6) molar concentrations of both HC and FK were combined in culture, a summation inhibitory effect on the spontaneous IgE synthesis was observed. In contrast, neither FK nor HC had inhibitory effect on the in vitro spontaneous IgE synthesis by the U266 myeloma cell line. The spontaneous in vitro IgE synthesis by B cells from patients with Hodgkin's disease, demonstrating high levels of serum IgE, was strongly reduced or virtually abolished after patients underwent total nodal irradiation to prevent the spread of the disease. In addition, the in vitro spontaneous IgE synthesis by B cells from atopic patients was markedly decreased or abolished by in vivo administration of betamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously demonstrated that a proportion of human T cell clones (TCC) derived from tonsil or peripheral blood (PB) of non-allergic donors, upon triggering with phytohaemagglutinin (PHA) or anti-CD3 monoclonal antibody (MoAb), were able to provide help for IgE synthesis in B cells from both allergic and non-allergic individuals. In this study we show that, upon PHA stimulation, culture supernatants from 10 selected TCC active on IgE synthesis also provided helper activity for IgE, whereas supernatants from unstimulated cultures of the same TCC were ineffective. In contrast, culture supernatants derived from five PHA-stimulated TCC, unable to provide helper function for IgE synthesis, consistently failed to elicit production of IgE. While the induction of IgE synthesis by TCC occurred in B cells from virtually all allergic and non-allergic donors, their soluble factor(s) were found to be able to provide substantial help for IgE production only in B cells from a proportion of donors tested. In addition, B cells from non-atopic donors usually appeared to be less responsive than atopic B cells to the activity of such factor(s). In contrast, synthesis of both IgG and IgM was induced in every B cell donor by both TCC and their supernatants. Partial characterization of the factor(s) providing helper function for IgE synthesis in B cells showed that it apparently had a mol. wt between 10 and 50 kD and did not bind to immobilized IgE. Such an activity appeared to be associated with the presence of interleukin 4 (IL-4) in supernatants and it was inhibited by adding both gamma-interferon and anti-human IL-4 antibody in culture.

High proportions of T8+ cells with inverted T4/T8 ratio were found in freshly isolated thyroid lymphocytes from patients with Hashimoto's thyroiditis. In addition, about one third of thyroid infiltrating cells expressed the TAC antigen, whereas in patient peripheral blood (PB) or normal lymphocytes from PB or lymphoid organs the percentage of TAC-positive cells was consistently lower than 10%. Following negative selection with OKT4 or OKT8 monoclonal antibodies and complement, TAC+ T cells were enriched in the T8+ cell population. Thyroid infiltrating T cells from two patients underwent two different cloning procedures. In the first, single T cells were initially activated with phytohaemagglutinin (PHA) and interleukin 2 (IL-2), in the other with recombinant IL-2 (rIL-2) alone. The majority of T cell clones obtained by initial PHA-stimulation (55-65%) had the T8+ phenotype, but the frequency of T8+ clones obtained by stimulating T cells with rIL-2 alone was even higher (78 & 71%, respectively). The majority of T8+ clones elicited by PHA (35/37 & 36/38) and all the T8+ clones (36/36 & 22/22) obtained from thyroid infiltrates with initial stimulation by rIL-2 displayed cytolytic activity. Most of cytolytic T8+ clones obtained from thyroid infiltrates with both cloning procedures, displayed NK activity against human K562 and MOLT-4 target cells, but not against a NK-resistant target, such as Raji cells. These data suggest that in Hashimoto's disease a considerable proportion of thyroid infiltrating T cells are in vivo activated T8+ cytolytic T cells with NK activity, which may be of importance in determining or maintaining the tissue damage of the target gland.

Peripheral blood mononuclear cells from atopic donors were stimulated in vitro with allergens (Rye group I or Dermatophagoides pteronyssinus). T cell lines were originated and maintained in long term culture using IL-2 and periodical restimulations with allergen. The lines were antigen specific (i.e. responded to the allergen used to raise them and not to other antigens) and required that the antigen was presented by autologous cells (i.e. they were restricted). The restriction elements were probably at the level of HLA-DR antigens since the proliferative response was specifically blocked by anti-HLA-DR antibodies. Surface marker analysis revealed that the lines comprised mainly cells with an helper/inducer phenotype, although cells with markers of the suppressor/cytotoxic T cells were also present. The lines could be cloned by limiting dilution and clones with the same restriction and specificity as the parental line were isolated. These studies demonstrate the possibility of obtaining a large number of allergen specific human T cells that can be used for further in vitro studies on the regulation of the IgE response.

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.

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.

Supernatants (SN) from 10 phytohemagglutinin (PHA)-stimulated human T cell clones (TCC), selected for their helper function on IgE synthesis, were found to provide IgE helper activity in atopic B cells showing low or undetectable spontaneous in vitro IgE synthesis. In contrast, SN from 5 PHA-stimulated TCC unable to provide helper function for IgE synthesis consistently failed to elicit IgE production. SN active on IgE synthesis contained high concentrations of interleukin-4 (IL-4), whereas inactive SN did not contain detectable amounts of IL-4. Moreover, the IgE helper activity of TCC SN was strongly inhibited by the addition of interferon-gamma (IFN-gamma) to B cell cultures. These data suggest that IL-4 may play a role in the induction of in vitro human IgE synthesis, whereas IFN-gamma displays an inhibitory effect.