Blood and thyroid-infiltrating lymphocyte subclasses in juvenile autoimmune thyroiditis.

We have studied the distribution of T and B lymphocytes in the blood and in the thyroid lymphocytic infiltrates (obtained by fine needle aspiration biopsy) in sixteen patients with juvenile autoimmune thyroiditis (JAIT). The same cell populations were also tested for cell-mediated immunity (CMI) to thyroid antigen in the leucocyte migration test (LMT). The relative and absolute numbers of blood T lymphocytes were normal (71-76%), as were the numbers of blood B lymphocytes (19%). The thyroid infiltrate contained 48% B lymphocytes, whereas only 40-44% of the infiltrating lymphocytes were T cells. Half of the JAIT patients showed a positive CMI to thyroid antigen with blood leucocytes, but when thyroid-infiltrating lymphocytes of these patients were tested in the LMT, they were negative. Thus, in contrast to what is generally assumed, we were unable to demonstrate T cell-dominated lymphocytic infiltrates or the accumulation of specifically sensitized T lymphocytes within the thyroid gland in autoimmune thyroiditis.     

T细胞受体Vβ基因在自身免疫性甲状腺病病变组织中的表达及意义

目的研究国人Graves病、桥本甲状腺炎甲状腺内浸润T淋巴细胞的T细胞受体(TCR)Vβ基因家族的利用,发现优势利用基因,为阐明自身免疫性甲状腺病(AITD)的T细胞克隆的分布模式及防治提供依据.方法穿刺或手术取得12例Graves病、15例桥本甲状腺炎病人的甲状腺组织,提取RNA.抽取5例Graves病、5例桥本甲状腺炎和7例正常人之外周静脉血,分离外周血淋巴细胞(PBL),提取RNA.以24个Vβ基因家族特异的寡核苷酸为上游引物,以一个Cβ特异的寡核苷酸为下游引物,进行逆转录-聚合酶链反应,比较各个Vβ基因家族的表达.结果Graves病及桥本甲状腺炎患者甲状腺内TCRVβ基因家族的平均表达阳性数分别为(5.3±1.2)个,(13.4±3.0)个;而且Vβ3、Vβ5和Vβ8的表达在Graves病更常见.以上两组病人及正常对照PBL之TCRVβ基因家族的平均表达分别为(23.0±1.0)个,(22.2±1.3)个和(22.4±1.7)个.结论Graves病甲状腺内淋巴细胞的TCRVβ基因显示明显的限制性利用,某些Vβ家族的使用率更高,提示寡克隆扩增的甲状腺抗原特异性T细胞可能与Graves病的发病有关,这可能有重要的治疗意义.相反,桥本甲状腺炎病变内TCRVβ利用的限制性较差或无限制性,可能与随疾病进展,非特异性免疫机制的介入有关.     

CA 19-9 expression in subacute (de Quervain's) thyroiditis: an immunohistochemical study.

Carcinoma antigen 19-9 (CA 19-9) and carcinoembryonic antigen (CEA) expression was immunohistochemically investigated in 48 cases of subacute granulomatous (de Quervain's) thyroiditis, two of focal lymphocytic thyroiditis, three of Hashimoto's thyroiditis, two of Graves' disease, and seven follicular adenomas, 27 follicular carcinomas, and eight papillary carcinomas of the thyroid. CA 19-9 expression was found in all cases of subacute thyroiditis, lymphocytic thyroiditis, and papillary carcinomas examined and in approximately 50% of follicular adenomas and carcinomas. The strongest CA 19-9 staining was demonstrated in late stage subacute thyroiditis and in papillary carcinomas with marked sclerosis. Occasionally CA 19-9 expression was present in seemingly normal thyroid parenchyma adjacent to the thyroid lesions investigated. CEA was found in the center of the granulomatous lesions in acute stage subacute thyroiditis. All neoplasms were CEA negative. CA 19-9 and CEA could be demonstrated occasionally in multinucleated giant cells of subacute thyroiditis, which may suggest that these giant cells are of either histiocytic or follicular cell origin. Immunohistochemical investigation with antibodies against CA 19-9 and CEA may help to histomorphologically define subacute granulomatous thyroiditis.     

甲状腺激素检测在甲状腺毒症病因鉴别诊断中的临床应用价值

目的 探讨甲状腺激素检测在亚急性甲状腺炎与弥漫性毒性甲状腺肿(Graves病)这两种不同病因的甲状腺毒症中的鉴别诊断价值.方法 采用全自动化学发光免疫法检测157例亚急性甲状腺炎甲亢期患者及162例Graves病患者血清中甲状腺激素的含量,包括游离三碘甲状腺原氨酸(fT3)、三碘甲状腺原氨酸(T3)、甲状腺素(T4)、...     

Marked increase of CD5 + B cells in hyperthyroid Graves' disease

We examined the proportions of B lymphocytes bearing CD5 cell surface antigen (CD5+ B cells), which are capable of making autoantibodies, in peripheral blood from patients with various thyroid diseases. The level of CD5+ B cells was markedly increased (>9.0%) above the normal range (0.5-7.7%) in untreated, hyperthyroid patients with Graves' disease, although about 10% of the patients had no detectable serum thyroid-stimulating hormone (TSH) receptor antibody (TRAb). However, the levels of CD5+ B cells were normal in untreated patients with destructive thyrotoxicosis due to aggravation of Hashimoto's thyroiditis or subacute thyroiditis. In patients with stimulated hyperthyroid Graves' disease the levels of CD5+ B cells were correlated with those of thyroid hormones and TRAb, all significantly increased. However, once hyperthyroidism was controlled by anti-thyroid drugs, CD5+ B cells were decreased, followed in turn by reduction of TRAb. We conclude that the proportion of CD5+ B cells is useful as a therapeutic index and for diagnosis of Graves' disease and its differentiation from destruction-induced thyrotoxicosis.     

Proliferation of T8-positive cytolytic T lymphocytes in response to thyroglobulin in human autoimmune thyroiditis: analysis of cell interactions and culture requirements

These experiments were designed to analyze the involvement of T-lymphocyte subpopulations in autoimmune thyroid disorders such as Graves' Disease (GD) and Hashimoto's Disease (HD). In a first set of experiments, lymphocytes isolated from thyroid infiltrates or from peripheral blood of GD and HD patients were analyzed for the expression of various surface antigens. While HLA-DR + T cells were numerous among thyroid infiltrating T lymphocytes in both groups of patients, the proportions of T8 + cells (as defined by their reactivity with the B 9.4 monoclonal antibody specific for T8 surface molecule) were strikingly different in HD and GD. In the latter group of patients only 19% of infiltrating T cells were T8 +, whereas these cells represented approximately 50% in four HD patients. Given the previous demonstration that all T cells expressing T8 antigen are cytolytic T lymphocytes (CTL) or their precursors (CTL-P) in conjunction with the fact that lymphocytes from HD or GD patients are known to proliferate in vitro in response to human tg (Htg), we further analyzed the T-cell subset(s) responsible for in vitro proliferation to Htg. In these experiments, peripheral blood T lymphocytes purified from patients with GD or HD were cultured with 1 microgram/ml Htg and irradiated autologous T-depleted mononuclear cells as the source of antigen presenting cells (APC). The proportions of T8 + cells declined considerably during culture in GD patients, but at Days 6 to 9, T8 + cells represented as much as 51% of cultured T lymphocytes from patients with HD. Moreover, the majority of T8 + cells were medium-large size lymphoblasts. Removal of Htg at Day 6 resulted in both abrogation of proliferative responsiveness and in decreases of T8 + percentages. Further analysis of the cell interactions leading to T8 + cell proliferation in response to Htg showed that helper/inducer T cells, as defined by 5/9 antigen expression, were strictly required. Collectively, these features are reminiscent of the T-cell involvement in experimental autoimmune thyroiditis of mice and stress for the first time the potential role of CTL in tissue damage occurring in Hashimoto's thyroiditis.     

Participation of a histamine-Sepharose-adherent subpopulation of human mononuclear cells in the production of leucocyte migration inhibition factor (LIF) in healthy children.

The separation of mouse splenic T lymphocytes into distinct subpopulations by fractionation on histamine-rabbit serum albumin Sepharose (H-RSAS) columns has been described. The H-RSAS-adherent T cells have been attributed regulatory functions associated with B cell activity, T cell-mediated cytotoxicity and the secretion of mediators such as immuno-interferon. The possibility that H-RSAS-adherent T cells exert a similar regulatory effect on an in vitro parameter of T cell-mediated immunity was investigated by assaying the production of leucocyte migration inhibition factor (LIF) in human blood samples, using the agarose droplet method. Phytohaemagglutinin (PHA) and BCG-purified protein derivative (PPD) were used as stimulants of LIF secretion which was measured as a percentage of inhibition of linear leucocytic migration. In normal individuals a highly significant (P less than 0.001) decrease was demonstrated in the production of LIF by peripheral blood leucocytes depleted of H-RSAS-adherent cells. Migration inhibition dropped from 36 +/- 11.7% to 21.2 +/- 12.9% in eighteen cases tested with PHA and from 29.3 +/- 11.7% to 17.2 +/- 9.8% in twelve cases tested with PPD. These results suggest the existence of a lymphocytic subpopulation involved in LIF production which expresses histamine receptors.     

Studies on the immunoregulation of thyroid autoantibody production in vitro.

The spontaneous production (without mitogen or antigen) of antithyroglobulin and antimicrosomal antibodies by peripheral (PBL) and thyroid-derived lymphocytes from patients with Hashimoto's thyroiditis (HT) has been studied with particular emphasis on the regulation of this phenomenon. Based on studies of DNA and protein synthesis, kinetic studies and B/T reconstitution experiments, in most HT patients, spontaneous production by PBL is accounted for by secretion of preformed antithyroglobulin (termed Type 1 patients), whereas active production is observed in a small minority (termed Type 2). In none of 24 HT patients could active antimicrosomal antibody production by PBL be detected. Conversely, thyroid-derived lymphocytes produced both autoantibodies by an active process. Pokeweed mitogen (PWM) stimulation enhanced antibody production by PBL in the Type 1 group but not in Type 2 or thyroid-derived lymphocytes. T lymphocytes were required for antibody synthesis in both thyroid antigen-driven and peripheral PWM-driven cultures. By separating T lymphocytes into T4+ (helper) and T8+ (suppressor) subsets with monoclonal antibodies, T-cell modulation of autoantibody production in both systems was studied. In a PWM-induced system, both thyroid and peripheral T-cell subsets were capable of modulating peripheral antibody production. In the thyroid lymphocyte antigen-specific system, further addition of thyroid derived T8+ cells alone caused partial suppression of antibody production but not with peripheral T8+ cells. Of interest was the partial decrease of antibody production by the thyroid lymphocytes by added peripheral T4+ cells. The fact that the production of thyroid autoantibodies by thyroid-derived mononuclear cells (which included T suppressor, T helper and B lymphocytes) could be reduced by the addition of more suppressor T lymphocytes suggests that an antigen-specific defect in the T4+/T8+ thyroid cell balance may account for the in vivo production of these antibodies in patients with Hashimoto's thyroiditis.     

Gamma delta lymphocytes in endocrine autoimmunity: evidence of expansion in Graves' disease but not in type 1 diabetes.

Endocrine autoimmune disorders are mediated by T cell-dependent responses to organ-specific antigens, but the mechanisms initiating the process remain unknown. Lymphocytes which use the gamma delta heterodimer as T cell receptor (TCR) for antigen constitute a distinct subset of T cells whose function remains elusive. In order to investigate their possible involvement in endocrine autoimmunity we have determined the proportion of gamma delta T cells in the peripheral blood of 23 patients with type 1 (insulin-dependent) diabetes mellitus (type-1 DM) and 30 patients with autoimmune thyrotoxicosis (Graves' disease). T lymphocyte TCR expression was assessed by fluorescence-activated flow cytometry on peripheral blood mononuclear cells using MoAbs UCHT1 (CD3), TCR delta 1 (gamma delta TCR), WT31 and beta F1 (alpha beta TCR) and both the percentage of T cells expressing gamma delta and the ratio gamma delta/alpha beta were calculated. In the diabetic patients gamma delta cells were not significantly different from the control group (7.7 +/- 54% versus 8.0 +/- 5.5% of T cells, P NS). There was no relation between the proportion of gamma delta lymphocytes and the presence of islet cell antibodies (ICA) in the sera. The Graves' patients showed a tendency towards a higher proportion of gamma delta T lymphocytes than the controls (gamma delta/alpha beta ratios: 0.095 +/- 0.047 versus 0.063 +/- 0.022, P = 0.03). In 14 Graves' patients the number of gamma delta were measured in paired samples of peripheral and intrathyroidal lymphocytes, demonstrating an expansion of gamma delta within the thyroid glands (0.21 +/- 0.3 versus 0.095 +/- 0.047, P = 0.032). Immunohistochemical studies showed that gamma delta cells were scattered among the predominant alpha beta lymphocytes infiltrating the thyroid gland and that they account for 10% of intraepithelial lymphocytes. No relation was found between the increase of gamma delta lymphocytes and any clinical features.     

Identification of lymphocyte subsets in peripheral blood and thyroid gland in Graves' disease by alpha-naphthyl-acetate-esterase reaction

T lymphocyte subsets were prospectively examined in the peripheral blood and thyroid aspirates of 10 patients with hyperthyroid Graves' disease before and after treatment with methimazole and attainment of euthyroidism. T lymphocyte subsets were identified with monoclonal antibodies and pattern of alpha-naphthyl-acetate esterase (ANAE) staining pattern in the case of peripheral blood and ANAE staining pattern with thyroid aspirate smears. Before treatment, OKT8+ lymphocytes were significantly decreased (18.4% +/- 4.8) (S. D.) in the patients compared to control (28.8 +/- 6.7%, p less than 0.05), the OKT4/OKT8 ratio was increased (2.92 vs 2.11). Percent OKT8+ lymphocytes were not different from the controls when the ten patients had been rendered euthyroid. ANAE mononuclear cells with a diffuse pattern (presumed suppressor cells) were 4.2% +/- 1.8 before treatment and 8.3 +/- 2.4 (p less than 0.05) after treatment and 11.5% +/- 2.2 in controls. ANAE mononuclear cells with diffuse pattern represented 4.2% +/- 1.8 of the mononuclear cells infiltrating the thyroid gland of untreated patients and rose to 8.3% +/- 2.4 after the patients had become euthyroid. ANAE negative cells (B cells and some T cells) were increased in the thyroid of untreated patients. It is concluded that mononuclear cells with presumed suppressor T cell phenotype are decreased in the blood and thyroid glands of patients with active Graves' disease and that this defect is corrected when euthyroidism has been established.     

Clonal analysis of T lymphocytes infiltrating the thyroid gland in Hashimoto's thyroiditis

T cells isolated from thyroid tissue and peripheral blood of 2 patients with Hashimoto's thyroiditis were studied by a high cloning efficiency microculture technique. Clonal efficiencies of 37 and 24% were obtained from thyroid-derived T cell cultures, while 40 and 90% efficiencies resulted from peripheral-blood-derived cultures. A prevalence of T4-/T8+ T cell clones were found in thyroid infiltrates. The functional analysis of the clones demonstrated significantly higher proportions of clones with cytolytic activity in a lectin-dependent assay in thyroid-derived microcultures, as compared to peripheral blood-derived ones. The proportion of clones displaying natural-killer-like activity was increased in 1 patient only. Cytolytic activity was displayed not only by all T4-/T8+, but also by several T4+/T8- intrathyroid clones. Remarkable proportions of cytolytic clones were also able to release interleukin-2 upon phytohemagglutinin stimulation. Finally, the proportion of T cell clones able to release gamma-interferon following mitogen stimulation was significantly higher in thyroid- vs. peripheral-blood-derived microcultures. These results provide further data about the possible pathogenetical role of both regulatory and effector T lymphocytes in human autoimmune thyroiditis.     

Autologous CD8-positive cells suppress T cell proliferation in response to thyroid antigens in Hashimoto's thyroiditis

To assess whether there is a defect in thyroid antigen-specific suppressor cells in Hashimoto's thyroiditis (HT) and whether such cells actively prevent thyroid autoreactivity in control subjects, T cell proliferation was measured before and after removal of CD8-positive cells from the lymphocyte population. CD8 depletion significantly increased the proliferation of HT lymphocytes to soluble thyroglobulin and to thyroid microsomal antigen immunoblotted onto nitrocellulose; some of these cultures also reacted with an unidentified thyroid antigen, mol wt approx 16 kDa. However, CD8 depletion did not permit normal lymphocytes to respond to thyroid antigens. Peripheral blood CD8 cells were also found to suppress proliferation of a thyroid T cell line, derived from a patient with HT, in response to autologous, Ia-positive thyroid follicular cells. These results do not support the existence of a defect in antigen-specific suppressor cells in HT, nor the active suppression of thyroid autoreactivity by such cells in normal subjects. The data suggest that there may be an imbalance in T cell helper and suppressor influences in thyroiditis, but in some patients a new balance is achieved, so that T cell sensitization to thyroid autoantigens can only be identified by removal of CD8-positive cells.     

Induction of suppressor cells by Mycobacterium paratuberculosis antigen in inflammatory bowel disease.

We studied the M. paratuberculosis-induced proliferation and suppressor cell generation by peripheral blood lymphocytes from patients with inflammatory bowel disease. Peripheral blood lymphocytes were separated from 33 patients with Crohn's disease, 18 with ulcerative colitis, nine with other intestinal diseases, and five with autoimmune disorders. Proliferation of peripheral blood lymphocytes from normal individuals in response to 10 micrograms/ml of M. paratuberculosis antigen was reduced by depletion of CD4+ T cells. The ability of M. paratuberculosis antigen to suppress concanavalin A-induced proliferation (expressed as a percentage suppression) was reduced by depletion of CD8+ T cells. This suppression was the same whether peripheral blood lymphocytes were from normal individuals, patients with intestinal diseases other than inflammatory bowel disease, or patients with autoimmune disorders (47 +/- 14%, 44 +/- 24%, and 30 +/- 26%, respectively). In contrast, the suppression induced by M. paratuberculosis for patients with Crohn's disease and ulcerative colitis (66 +/- 22% and 67 +/- 22%) was much greater than that for normal individuals (P less than 0.001). In particular, lymphocytes from patients with active Crohn's disease demonstrated little proliferation in response to this antigen but marked suppressor activity (79 +/- 13%). How the immunomodulatory effects of this antigen relate to the pathogenesis of the inflammatory bowel diseases remains to be determined.     

Pertinence of kappa and lambda recombinant antibodies directed against thyroid peroxidase in thyroid autoimmune disease

Forty-one single-chain variable region fragments (scFvs) directed against thyroid peroxidase (TPO) were obtained by phage display libraries constructed from thyroid-infiltrating B cells of Graves' disease patients. Among these scFvs, 24.4% used a Vkappa light chain whereas 75.6% shows a light chain of Vlamda origin. Study of light chain gene usage in the TPO antibody repertoire demonstrated a dominance of the Vkappa 1-39 and Vlambda 1-51 genes. Thyroid peroxidase probing of overlapping peptides covering the amino acid sequences of anti-TPO T2/kappa and T13/lambda variable regions demonstrated a more restricted antigen recognition on T13/lambda than on T2/kappa. These two recombinant antibodies, expressed as whole IgG1 in the baculovirus/insect cell system, inhibited the binding to TPO of serum TPO autoantibodies whatever the light chain. Our study indicates that lambda as well as kappa light chain usage are found in the TPO antibody repertoire of thyroid-infiltrating B cells and are pertinent in the pathogenesis of autoimmune thyroid disease.     

Antigen specific immune response potential and HLA typing of Israeli patients with thyroid autoimmune diseases (TAD).

The immune response potential to the synthetic polypeptide antigen (T,G)-A--L was studied in 35 patients with thyroid autoimmune diseases (TAD). For this purpose the ability of their antigen activated peripheral blood lymphocytes (PBL) to generate a (T,G)-A--L specific helper factor was tested. In addition, the patients were typed for their HLA determinants. The results of the study have shown that 20/35 (57%) patients responded to (T,G)-A--L, a similar proportion to that found among healthy donors that were tested as control. No significant difference was found in the rate of responses between patients with Graves' disease and Hashimoto's thyroiditis. The responses in these groups of patients were shown to be 13/22 (59%) and 7/13 (54%) respectively. HLA typing of 26 patients with TAD did not demonstrate any association of the disease or the immune response potential with any specific HLA determinant. It is proposed that unlike the general lack of regulation that we have previously observed in patients with systemic lupus erythematosus, the abnormal autoimmune reaction in TAD and probably in other organ-specific autoimmune diseases, is towards a specific organ without affecting other arms and functions of the immune system.     

Particularity of Local Immunity in the Nasopharynx

Immunological functions of the pharyngeal tonsil, palatine tonsils and blood leucocytes of children undergoing tonsillectomy were evaluated by determining T or B lymphocytes, the response to mitogens, and the cell-mediated immunological responses to tuberculin. In all the test systems used similar results were obtained with cells derived from either the palatine or pharyngeal tonsils. The mean percentage of T lymphocytes was significantly higher in the peripheral blood than in tonsils, but the reverse was true of B lymphocytes. The reaction to PHA was lower in tonsillar cell culture than in blood cell culture, but tonsillar cells reacted better to Con A than blood cells. In lymphocyte transformation tests tonsillar cells reacted to specific antigen (tuberculin) and this reaction was significantly higher than that of the parallelly tested blood lymphocytes. Further, in about 50% of the children tested, tuberculin caused migration inhibition of the mixture containing tonsillar cells and guinea pig peritoneal cells. Surprisingly, nearly identical results were obtained if migration inhibition test was performed with tonsillar cells alone. Consequently, poorly migrating tonsillar cells are nevertheless usable for direct migration inhibition testing.     

四聚体技术检测原发性胆汁性肝硬化患者血循环中抗原特异性T细胞

目的定量检测原发性胆汁性肝硬化(PBC)患者体内抗原特异性T淋巴细胞含量,探讨其在PBC发病机制中的作用。方法采用四聚体技术检测15例HLA-A0201阳性(A2 )PBC患者外周血单个核细胞(PBMC)经抗原肽诱导生长的细胞毒性T淋巴细胞(CTL)中PDC-E2159~167aa与PDC-E2165~174aa特异性CD8 T细胞频率,以A0201阴性(A2-)PBC患者与A2 的其他慢性肝病和健康自愿者为对照组。结果在A2 PBC患者PDC-E2159~167aa与PDC-E2165~174aa诱导的CTL中可检测到其相应的四聚体/CD8 细胞,平均频率分别为0.42%±0.24%(0.17%~1.08%)、0.27%±0.17%(0.05%~0.56%),各对照组的四聚体阳性细胞频率均低于0.1%,差异非常显著(P<0.001);A2 PBC组中PDC-E2159~167aa特异性的CTL频率与PDC-E2165~174aa特异性的CTL无显著性差异(P>0.05)。处于临床Ⅰ、Ⅱ期的A2 PBC患者中CD8 特异性CTL频率均较Ⅲ期的要高(P<0.05)。PDC-E2159~167aa特异性CTL与PDC-E2165~174aa特异性CTL频率在抗-PDC阳性PBC组和抗-PDC阴性组之间均无显著性差异(P>0.05)。结论HLA-A0201限制性的PDC-E2159~167aa和PDC-E2165~174aa特异性CD8 CTL在PBC疾病进展中起重要作用,抗线粒体抗体阴性或抗-PDC阴性PBC患者与阳性患者可能有着相似的T细胞介导的免疫发病机制。     

TMA与TGA检测对甲状腺疾病的临床诊断价值

根据174例疑诊患者的TMA、TGA，T3，T4放射免疫测定结果及其临床表现进行诊断，结果表明桥本氏甲状腺炎和甲亢所占的比例(分别为38．51％及31．61％）显著高于甲减和亚急性甲状腺炎(分别为6．32％和4．02％)，P＜0．05。甲减患者TGA TMA的阳性率（81．82％)显著高于甲亢(50．91％)，桥本氏甲状腺炎(50．75％)及亚急性甲状腺炎(57．14％)，P＜0．05。单项抗体阳性者以TMA较TGA为多。本文结果提示在甲状腺疾病中TMA，TGA的阳性结果存在相互重叠现象，与T3、T4联检并结合临床表现进行综合分析有助于甲状腺疾病的鉴别诊断并正确指导治疗。     

Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases

For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.     

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.