Cytokine treatment of human bone marrow activates anti-leukaemia effector cells: monitoring of purging by polymerase chain reaction and DNA analysis.

The aims of this study were to investigate the role of cytokines (tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma) and interleukin-2 (IL-2) in augmenting graft-versus-leukaemia (GVL). We have investigated the effector cells involved in GVL, by studying the role of these cells in purging of the cell line K562 in short-term bone marrow cultures. The effect of the addition in vitro of rGCSF was also studied. Monitoring of purging was achieved by cytotoxicity assays, DNA analysis and the use of the polymerase chain reaction for the detection of bcr/abl transcripts in the Philadelphia positive (Ph+) K562 cell line. Supernatants from IL-2-treated and non-treated bone marrow were tested for cytokine production (TNF alpha and IFN gamma). The results have shown that the main cytotoxic effector cells in the bone marrow generated by IL-2 have the CD56+ CD8+ phenotype. Overnight incubation of bone marrow was sufficient to generate cytotoxic cells as measured by Chromium51 (Cr51) release assays. Measurable levels of TNF alpha but not IFN gamma were also detected in supernatants. Addition of TNF alpha and IFN gamma to the IL-2 in the bone marrow cultures augmented the cytotoxicity but tended to inhibit progenitor cell growth as measured by granulocyte-macrophage colony-forming unit (GM-CFU) and erythroid blast-forming unit (BFU-e) assays. An estimate of the purging of the marrow could also be achieved by DNA analysis of K562 DNA in bone marrow. The bcr/abl transcript could still be detected by PCR analysis in marrow containing 1% K562 and treated with IL-2 for 24 h, but by 6 days of incubation the bcr/abl transcript was weak or undectable. The results suggest that although reduction in the proportion of leukaemia in contaminated marrow can be detected after incubation with IL-2 for 24 h, complete elimination of minimal residual disease requires longer incubation times.

A technique for the isolation and mitogenic activation of thyroglobulin-specific human B lymphocytes.

In previous studies we demonstrated that Hashimoto peripheral blood lymphocytes enriched for thyroglobulin (Tg) binding activity could be activated to secrete increased amounts of Tg antibody by Epstein - Barr virus (EBV) but not by pokeweed mitogen (PWM). We now report an investigation into the requirements for the isolation of Tg receptor positive (TgR+) B cells capable of being stimulated by PWM. The interaction between Hashimoto lymphocytes and Tg coated erythrocytes followed by red cell lysis interfered with the ability of the population to synthesize Tg antibody. However, this could be overcome if the rosettes formed between Tg coated erythrocytes and the Tg receptors on B cells were dissociated by digestion followed by red cell lysis and overnight incubation before the addition of PWM. Using this approach, Hashimoto TgR+ B cells could be stimulated by the mitogen to secrete immunoglobulin with a higher Tg antibody specific activity than unfractionated lymphocytes. Consequently, enriched populations of antigen specific human B cells capable of responding to mitogenic signals can be prepared by a positive selection technique.

TSH receptor antibody synthesis by thyroid lymphocytes.

Several indirect observations have indicated that lymphocyte in the thyroid may be an important site of TSH receptor antibody synthesis in Graves' disease and we now describe an investigation of this possibility using improved lymphocyte isolation and TSH receptor antibody assay procedures. Our studies demonstrate that thyroid lymphocytes spontaneously produce TSH receptor antibody in culture. Furthermore, experiments with mitogen tend to suggest that these cells, in contrast to lymphocytes from lymph nodes draining the thyroid, are part of an active immune response to the TSH receptor.

Subpopulations of thyroid autoantibody secreting lymphocytes in Graves' and Hashimoto thyroid glands.

Lymphocytes isolated from Graves' and Hashimoto thyroid tissue by enzymatic (dispase) digestion or mechanical disaggregation were markedly different in terms of their ability to synthesize thyroid autoantibodies in culture. Dispase digestion, followed by removal of thyroid follicular cells, gave a lymphocyte population with a high T:B cell ratio (6:1). However, the ability of these cell suspensions to synthesize microsomal (Mic) and thyroglobulin (Tg) antibodies spontaneously was significantly increased compared with lymphoid suspensions isolated by mechanical means. Spontaneous synthesis of thyroid autoantibodies was not markedly enhanced in cell suspensions prepared from patients' lymph node tissue by digestion compared with mechanical disaggregation. Further, Mic and Tg antibody production by thyroid lymphocytes prepared using dispase was inhibited by pokeweed mitogen (PWM) whereas in most cases suspensions prepared from the same tissues by mechanical dispersion synthesized low or undetectable levels of autoantibodies whether PWM was present or absent. Digestion of tissue debris remaining after mechanical removal of lymphocytes gave suspensions which had an increased proportion of suppressor/cytotoxic T cells compared with suspensions produced mechanically or by digestion alone; however, in terms of spontaneous autoantibody synthesis and PWM induced inhibition, these suspensions were similar to these obtained by digestion alone. It would therefore seem that enzymatic digestion of thyroid tissue resulted in the isolation of a lymphoid population which was different from that extracted by mechanical disaggregation. The digestion process appears to permit the recovery of lymphocytes closely associated with thyroid follicular cells and our studies suggest that it is this population which makes the major contribution to autoantibody synthesis.

T cell regulation of thyroglobulin autoantibody IgG subclasses in Hashimoto's thyroiditis.

Microsomal and thyroglobulin (Tg) antibodies in patients with autoimmune thyroid disease are usually predominantly of subclasses IgG1 and/or IgG4 and the distribution pattern is characteristic for the serum of an individual. We have studied the role of T cells in synthesis of total IgG and Tg antibody IgG subclasses (measured by ELISA) in cultures of peripheral blood lymphocytes (PBL) from Hashimoto patients. Unfractionated PBL incubated with the T dependent activator pokeweed mitogen (PWM) synthesized IgG of all four IgG subclasses in the proportions 69% IgG1, 20% IgG2, 8% IgG3 and 3% IgG4; these values are similar to the proportions of the subclasses in serum. In contrast, the IgG subclass of Tg antibody was predominantly IgG1 in one patient, approximately equal proportions of IgG1 and IgG4 in four patients, and almost completely restricted to IgG4 in one patient; these patterns were similar to the subclass distribution of the autoantibodies in the individual patients' serum. B cells incubated alone secreted little Tg antibody but the response could be restored to the original levels and proportions of IgG1 and/or IgG4 Tg antibody by the addition of T cells either from the same individual or from another donor. Further, removal of suppressor T cells had little effect on the proportions of IgG1 and IgG4 Tg antibody although the total amounts of Tg antibody of both subclasses were sometimes increased. Our studies indicate that T cells are required in this in vitro system to elicit Tg antibody synthesis and to control the magnitude of the antibody response. However, the characteristic IgG subclass distribution of Tg antibody in an individual is determined at the level of the B cell.

The thyroid microenvironment in autoimmune thyroid disease: effects of TSH and lymphokines on thyroid lymphocytes and thyroid cells.

Thyroid lymphocytes synthesize thyroid autoantibodies in close proximity to thyroid cells and consequently soluble mediators such as TSH and interleukins (IL) 1 and 2 may have unforeseen effects on lymphocytes and thyrocytes, respectively. Investigations of thyroid autoantibody synthesis by thyroid lymphocytes in vitro showed that TSH did not affect microsomal (Mic) antibody production, but thyroglobulin (Tg) antibody synthesis was decreased, probably as a result of complexing between Tg antibody and Tg secreted by small numbers of thyrocytes in the cell suspension. IL-1 and IL-2 partially mimicked the inhibitory effects on spontaneous autoantibody synthesis induced by Pokeweed mitogen (PWM) in cultures of thyroid lymphocytes. This inhibition may require a number of soluble mediators released by T cells in response to the mitogen; however, depletion studies indicated that the cell type responsible for PWM inhibition is unlikely to be a suppressor T cell and may be an NK cell. IL-1 and IL-2 had little effect on the viability of thyrocyte monolayers in an 18 h assay, but antibody dependent cells cytotoxicity (ADCC) using blood lymphocytes and thyroid autoantibody positive sera was demonstrated; further, the cytotoxicity appeared to be due to Mic antibodies. It is possible that IL-1 and/or IL-2 (as well as other cytokines) may affect thyroid cells after longer periods of exposure, either by altering them functionally or by direct damage. However, assuming that NK cells are present in sufficient numbers in the gland, ADCC could play a major role in the development of hypothyroidism in Hashimoto's disease.

Thyroid autoantigens and human T cell responses.

We investigated the ability of T cells from patients with Hashimoto's thyroiditis and with Graves' disease as well as control donors to proliferate in response to thyroid peroxidase (TPO) and thyroglobulin using (i) lymphoid cells from different lymphoid organs; (ii) unfractionated or CD8- depleted lymphoid suspensions or T cells + autologous low density cells (LDC); (iii) 200-microliters well cultures and 20-microliters hanging-drop microcultures; and (iv) intact TPO and thyroglobulin, denatured thyroglobulin and 12 synthetic peptides predicted on the basis of the amino acid sequence of TPO to be T cell epitopes. In 200-microliters well cultures, proliferative responses (assessed in terms of 3H-thymidine uptake) to intact TPO or thyroglobulin, digested thyroglobulin or synthetic TPO peptides were not significantly different in unfractionated or CD8-depleted lymphoid suspensions from blood, thyroid or lymph nodes of TPO/thyroglobulin autoantibody-positive patients, autoantibody-negative patients or control donors. In contrast, blood T cells from some high titre patients with Hashimoto's thyroiditis (but not from healthy individuals) proliferated in response to intact thyroglobulin or TPO presented by autologous LDC in hanging-drop microcultures. Heat denatured thyroglobulin (with which thyroglobulin autoantibodies do not interact) did not stimulate proliferation and this observation, together with the ability of T cells from some patients to respond to intact thyroglobulin in the absence of LDC, indicated that thyroglobulin-specific B cells may be involved in antigen presentation. As we were unable to demonstrate proliferation by blood T cells + LDC from all thyroglobulin antibody-positive patients with Hashimoto's thyroiditis, our studies suggest that the presence of sufficient precursor T cells, as well as the number and type of antigen-presenting cells, are critical for T cell proliferative responses to human TPO and thyroglobulin.

IgG subclass distribution of thyroid autoantibodies: a 'fingerprint' of an individual's response to thyroglobulin and thyroid microsomal antigen.

The IgG subclass distribution of autoantibodies to thyroglobulin and thyroid microsomal antigen was studied in 21 patients with Graves' disease during fluctuations in total IgG class autoantibody levels induced by various forms of therapy. In addition, changes in autoantibody subclass distributions were investigated during the natural course of Hashimoto's disease in seven patients taking thyroxine. The autoantibodies were principally of subclasses IgG1 and/or IgG4 in Graves' patients although IgG2 contributed significantly to thyroglobulin antibodies in 5/7 Hashimoto sera. In Graves' disease the distribution of microsomal and thyroglobulin antibodies among the IgG subclasses remained essentially unchanged over periods of 6 months-2 years whether autoantibody levels decreased during carbimazole therapy or increased transiently following 131Iodine treatment or subtotal thyroidectomy. Similar observations were made for thyroglobulin antibodies in Hashimoto patients studied over 2 1/2-4 years; furthermore, the IgG subclass distribution of microsomal antibodies was usually different from that of thyroglobulin antibodies in the same patient. These observations suggest that the microsomal and/or thyroglobulin antibody subclass distribution is characteristic for a particular individual and may be regarded as the 'fingerprint' of an individual's response to these thyroid autoantigens.