Cytotoxic lymphocytes in Hashimoto''s thyroiditis

The ability of peripheral lymphocytes from patients with Hashimoto's disease to destroy target cells coated with thyroid-related-autoantigens has been demonstrated. Direct cytotoxicity was demonstrable against mouse mastocytoma target cells coated with human thyroglobulin or microsomal antigen from thyrotoxic thyroid glands. No correlation was noted between circulating antibody and the ability of these cells to be cytotoxic.     

Intrathyroidal HLA-DR expression and T lymphocyte phenotypes in Graves'' thyrotoxicosis, Hashimoto''s thyroiditis and nodular colloid goitre.

Thyroid surgical biopsies from 21 individuals were examined by a double immunoenzymatic technique with respect to HLA-DR expression and lymphocytic infiltration. HLA-DR positive thyrocytes were observed in two examined Hashimoto goitres and in nine of 11 specimens from patients with Graves' disease. HLA-DR positive thyrocytes were localized to areas harbouring infiltrating lymphocytes, whereas regions with no lymphocytes only rarely expressed HLA-DR antigens. In two specimens of nodular goitre HLA-DR positive thyrocytes were observed in the vicinity of lymphocytic infiltration. Tissues from another three nodular goitres, from one follicular adenoma and from two normal individuals contained no HLA-DR positive thyrocytes and no or only a few lymphocytes. The lymphocytic infiltrates were dominated by cells with the Leu 3a helper/inducer phenotype irrespective of underlying disease, although most pronounced in Hashimoto's thyroiditis. The results indicate that HLA-DR antigens is expressed on thyrocytes in thyroid disorder. The extent of expression correlated with lymphocytic infiltration, which suggests that the two findings are related and of importance for the development of thyroid autoimmunity.     

Spontaneous secretion of thyroid autoantibodies by cultured peripheral blood lymphocytes from patients with Hashimoto''s thyroiditis detected by micro-ELISA techniques

The spontaneous in vitro production of anti-thyroglobulin (aTg) and anti-microsomal (aM) antibodies by mononuclear cells (MNC) from patients with Hashimoto's thyroiditis (HT) was analysed by an ELISA detection system. MNC from 35 HT patients spontaneously produced detectable levels of both autoantibodies in vitro (i.e., without mitogenic or antigenic stimulation). aTg was quantified using a reference aTg IgG standard and ranged from 55 to 9,000 ng aTg. Specificity of aTg by ELISA was assessed using heterologous Tg antigen (Ag). Microsomal Ag obtained by gel filtration was far less contaminated with Tg than the ultracentrifugation pellet. Specificity of aM ELISA was assessed using insulinoma membrane as unrelated Ag and by blocking aM detection only with microsomal Ag. aM levels in the 35 supernatants ranged from 0.1 to 1.12 OD. A direct correlation was found between aM serum titres detected by haemagglutination and in vitro aM spontaneous production, but not for aTg. This lack of correlation for aTg might have biological relevance. Tg restimulation in vitro enhanced aTg production in only four out of 18 cases, of which only one was significant. This system provides a tool for studies of the immunoregulation of thyroid autoantibody formation in vitro.     

T and B lymphocytes in peripheral blood of splenectomized subjects]

The report is a part of more extensive studies on the role of the spleen in immune processes. Assessing the ability of lymphocytes T to non-immunological binding of sheep erythrocytes and the ability of B cells to bind immunological complexes through the receptor for C3 component of complement, and the presence of immunoglobulins on the surface of lymphocytes B the number of these cells in peripheral blood was determined in 14 healthy individuals who had been splenectomized. In all these subjects the degree of blastic transformation of lymphocytes in PHA-stimulated 3 day-old white blood cell cultures was determined. It was found that the number of T and B lymphocytes in the peripheral blood of splenectomized and non-splenectomized patients was similiar. Lymphocytes obtained from splenectomized patients had however, an impairment of transformation ability after PHA stimulation. It is suggested that, apart from determination of T and B cell pool in the peripheral blood, an evaluation of their transformation ability after PHA stimulation is necessary for assessment of the immunological state in vitro investigations.     

Peripheral blood T lymphocytes in patients with duodenal ulcers treated with cimetidine.

The influence of cimetidine on the numbers of circulating T lymphocytes was studied in 24 patients with duodenal ulcer treated with cimetidine. The patients were divided into two groups according to the way of cimetidine administration and T lymphocytes were measured during treatment. No significant difference in the numbers of T lymphocytes was found in both groups of patients suggesting that cimetidine given therapeutically has no effect on circulating T lymphocytes.     

In vivo activated cytotoxic T cells in the thyroid infiltrate of patients with Hashimoto''s thyroiditis.

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.     

Enhanced production of gamma-interferon by thyroid-derived T cell clones from patients with Hashimoto''s thyroiditis.

T lymphocytes from thyroid infiltrate and peripheral blood (PB) of four patients with Hashimoto's thyroiditis (HT) were analysed at clonal level for their ability to secrete interleukin 2 (IL-2) and gamma-interferon (gamma-IFN). As controls, T cell clones from PB of four normal donors and from spleen of two trauma victims were used. While no abnormality was found in the capacity to produce IL-2, the proportion of gamma-IFN-producing (IFN-P) T cell clones derived from HT infiltrates was significantly higher (P less than 0.0005) than that of IFN-P clones derived from normal or patient PB. Most of CD4+ and CD8+ IFN-P clones from thyroid infiltrates, as well as a proportion of CD4+ PB-derived clones of patients with HT, released higher amounts of gamma-IFN than control clones. A relationship could be demonstrated between high gamma-IFN production and natural killer (NK) activity in T cell clones from thyroid and PB of HT patients. In fact, the percentage of IFN-P clones with NK potential (NK+) was remarkably higher (P less than 0.0005) in thyroid infiltrates than in normal spleen or PB. The proportion of IFN-P NK+ clones from patient PB was also significantly increased (P less than 0.02) but, unlike thyroid-derived clones in which the majority of IFN-P NK+ clones were CD8+, most PB-derived IFN-P NK+ clones from the same patients expressed the CD4+ phenotype. Almost all thyroid NK+ clones could be triggered to produce more gamma-IFN, while gamma-IFN synthesis by NK-negative thyroid clones was comparable to that of control clones. In view of the multiple effects ascribed to gamma-IFN in the cascade of events leading to immune responses, the abnormal potential to gamma-IFN secretion shown by intrathyroidal T lymphocytes may be of importance in the pathogenesis of autoimmune thyroiditis.     

Peripheral blood T lymphocytes in seasonal bronchial asthma

Variations in T lymphocytes in asthmatic patients are related to disease severity. However, the effects of natural exposure to pollens on peripheral blood T lymphocytes have not been clarified. In this paper, the effects on peripheral blood CD4 and CD8 lymphocytes from pollen-sensitive subjects and from nonatopic donors were studied during and outside the pollen season. In patients who suffer from seasonal asthma, we found an increase in the CD4/CD8 bright ratio and a decrease in the mean number of CD4 receptors per cell during the pollen season. No variation was observed in healthy subjects. These results suggest that CD4 lymphocytes may be causally linked to the pathogenesis of seasonal bronchial asthma.     

Activated T and B lymphocytes in peripheral blood of patients with Chagas' disease

Whole blood preparations from patients with either the indeterminate(asymptomatic) or cardiac clinical forms of chronic Trypanosomacruzl infection were analyzed by flow cytometry using double-labelingto identify subsets of circulating lymphocytes. Several significantdifferences were demonstrated between the blood lymphocyte profilesof chagaslc patients and non-chagaslc controls. Clear increasein the percentages and actual numbers of double-positive cellsof the phenotype CD3⁺/HLA-DR⁺, as well as decrease in the percentageof CD45RA⁺/CD4⁺ and CD45RA⁺/CD8⁺ T cells, Indicate greater numbersof activated T cells circulating in the blood of infected patients.Consistent parallel increases were seen also in the B lymphocytesubset which stained double-positive for CD19/CD5. There wereno significant differences in the circulation of these chronicchagaslc patients in the CD4:CD8 ratios. Also, no substantivephenotyplc differences were observed in the lymphocyte populationsbetween the two ends of the clinical spectrum (Indeterminateversus cardiac) in chronic human Chagas' disease. These observationsdemonstrate that increased levels of activated T cells and CD5⁺B cells are present in the circulation of people with chronicChagas' disease. These are cell phenotypes that have been associatedin other conditions with autoimmune, polyclonal, and hyperlmmuneresponses. The specificities of these activated cells and theroles they may play in resistance or pathogenesis during chronicChagas' disease need now to be determined.     

Immunohistological phenotyping of thyroid infiltrating lymphocytes in Graves'' disease and Hashimoto''s thyroiditis.

Subsets of lymphocytes in the thyroid were immunophenotyped by their surface antigens in frozen tissues of Hashimoto's thyroiditis and Graves' disease. Using triple layer immunoperoxidase staining (IP), monoclonal antibodies (T3, Leu 3, T8, anti-Tac and Leu 7) were employed to detect markers of T cell subsets, activated T cells, and a natural killer associated antigen. B cells were identified by 2 step IP with anti-IgD antisera. Excluding those cells forming lymphoid follicles, the density of lymphocytes infiltrating between thyroid epithelial cells was much higher in Hashimoto's thyroiditis than in Graves' disease. However, relative proportions of subsets were similar in both diseases. Most of the infiltrating cells were T3 positive T cells (T3+), with more T8+ (suppressor/cytotoxic T) than Leu 3+ (inducer/helper T). Some Leu 7+ were occasionally seen, but surface IgD positive mature B cells (IgD+) were almost absent. In contrast, IgD+ cells were densely aggregated in primary lymphoid follicles and mantle zones of secondary follicles. In these regions, Leu 3+ cells were about twice as frequent as T8+ cells. Some Leu 7+ and scarce Tac+ cells were also found. The present study indicates a major involvement of immunoregulatory T cells in autoimmune thyroid disease, and also suggested intrathyroidal maturation of B cells.     

Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients

The anti-α4 monoclonal antibody natalizumab inhibits lymphocyte extravasation into the central nervous system and increases peripheral T and B lymphocytes in multiple sclerosis patients. To investigate whether the lymphocyte accumulation was due to a higher lymphocyte production, an altered homeostasis, or a differential transmigration of lymphocyte subsets through endothelia, T-cell receptor excision circles and kappa-deleting recombination excision circles were quantified before and after treatment, T-cell receptor repertoire was analyzed by spectratyping, and T- and B-lymphocyte subset migration was studied using transwell coated with vascular and lymphatic endothelial cells. We found that the number of newly produced T and B lymphocytes is increased because of a high release and of a low propensity of naïve subsets to migrate across endothelial cells. In some patients this resulted in an enlargement of T-cell heterogeneity. Because new lymphocyte production ensures the integrity of immune surveillance, its quantification could be used to monitor natalizumab therapy safety.     

T and B lymphocytes in the liver and peripheral blood of subjects with acute and chronic hepatitis.

E and EAC rosette-forming cells in the peripheral blood and in the liver of subjects with acute and chronic hepatitis were studied. We found a highly significant reduction (P less than 0.01) of E rosette percentage in the lymphocytes isolated from the liver patients with chronic persistent, and chronic active, hepatitis. EAC rosette-forming cells were significantly increased in the liver of patients with chronic active hepatis (P less than 0.01). In this condition lymphocytes with Fc receptor were also found.     

Changes of B and T lymphocytes and selected apopotosis markers in Hashimoto's thyroiditis

The aim was to assess changes of B and T lymphocytes and selected apoptotic markers in Hashimoto thyroiditis (HT) cases on the basis of quantitative immunohistochemical studies (CD20, CD43, CD8, Bcl-2, caspase-3). The control group comprised colloid goitres without inflammatory infiltrate taken from 10 female patients. Thyroid specimens were obtained retrospectively from 40 patients. The immunohistochemical reactions were subject to quantitative evaluation performed using image-processing methods, including a spatial visualisation of the markers' expression. The percentage of Bcl-2 reactions in HT (mean 3.65%, SD 2.94%) was significantly lower than in the control group (mean 13.99%, SD 5.04%), while the thyroid follicles in HT samples exhibited a higher degree of staining for caspase-3 (mean 1.10%, SD 1.03%) in contrast to normal control tissues (mean 0.48%, SD 1.02%). The results from this study indicate that apoptosis plays a major role in the patogenesis of autoimmune thyroid diseases containing the main pathogenic events in the lesion of thyroid follicular cells in HT. Moreover, the reactivity of CD43 and CD20 was significantly higher in Hashimoto disease, while CD8 was not significantly different from the control group.     

Functional analysis of T and B cells from blood and thyroid tissue in Hashimoto''s disease

The existence of antibodies against alcohol-treated rabbit hepatocytes in sera of patients with alcoholic liver disease was investigated utilizing a 125I-labelled protein A assay. The sera, after two absorptions with normal rabbit hepatocytes, were incubated with hepatocytes isolated from rabbits which had been treated for 4 days with a daily dose of ethanol (1 g/kg body weight) intravenously. Serum antibodies against alcohol-treated hepatocytes were detected in 21 of 55 patients with alcoholic liver disease; this was associated with a distinct, mixed granular and linear fluorescence staining the surface of alcohol-treated hepatocytes. By contrast, none of 23 patients with other liver diseases who had no history of excessive alcohol intake showed the antibodies in their sera. The values of the antibodies of patients with alcoholic active cirrhosis and alcoholic hepatitis were significantly higher than those of other types of alcoholic liver disease. These results showed that antibodies against alcohol-treated hepatocytes are present in alcoholic liver disease with inflammation. The role of these antibodies on liver cell damage in alcoholic liver disease remains to be clarified.     

T and B lymphocytes in patients with enteric fever.

Estimation of T and B lymphocytes was done in 50 patients of enteric fever, 50 duration matched non enteric fever patients and 50 normal healthy individuals. The difference in both early and late rosette forming T lymphocytes was found to be statistically significant in enteric versus non-enteric patients. Significant difference was also observed in enteric versus normal individuals in case of late rosette forming T lymphocytes.     

Long term spectrotypic and idiotypic stability of thyroglobulin autoantibodies in patients with Hashimoto''s thyroiditis.

Isoelectric focusing of serial bleeds from patients with Hashimoto's thyroiditis was carried out and thyroglobulin (Tg) autoantibodies visualized using 125I-labelled Tg followed by autoradiography. Although the spectrotype of these antibodies was polyclonal and varied from patient to patient, each individual's spectrotype remained constant during the disease. Similar results were obtained if immunoblots were stained with rabbit anti-idiotype (anti-Id) raised to these autoantibodies. Using radioimmunoassay (RIA), it is shown that the levels of Id remain constant over several years whether assayed on crude immunoglobulin (Ig) fractions or affinity-purified anti-Tg. Therefore, once established, the autoimmune disease appears to be stable in terms of autoantibody spectrotype and idiotype in the patients studied.