A juxta-membrane epitope on the human acetylcholine receptor recognized by T cells in myasthenia gravis.

T cell proliferative responses to synthetic peptides taken from the human nicotinic acetylcholine receptor (AChR) alpha-chain sequence, or to whole AChR purified from electric fish (Torpedo marmorata), have been studied, using blood, thymus, and lymph node cells, from 34 patients with myasthenia gravis (MG) and 17 controls mostly with other neurological diseases. Peptides were selected because they contained amino acid motifs that recur in most defined T cell epitopes. Peptide 257-269 (from the extracellular loop of the AChR alpha-chain between the second and third trans-membrane domains) stimulated cells from six patients and no controls. Peptides from region 125-143 (from the main extracellular 1-210 stretch), which is thought to be an important T cell epitope in rats, provoked responses in 26% of patients and 41% of controls. Two patients responded both to these peptides and to peptide 257-269, thereby implying some heterogeneity of their reacting T cells. Whereas the initial blood T cell samples sometimes responded both to Torpedo AChR and to the 125-143 peptides, T cell lines selected with either antigen subsequently showed no response to the other. This observation suggests that it may be essential to use human AChR sequences for studying truly autoreactive T cells in MG. Finally, no strong association was found between any of the responses to peptides and the HLA types of the responding individuals.

Immunohistological studies of the thymus in myasthenia gravis. Correlation with clinical state and thymocyte culture responses.

In frozen sections of thymus from 9 out of 12 myasthenia gravis patients, the medulla contained follicles of B lymphocytes with germinal centres showing the same immunofluorescence staining pattern as is seen normally in reactive lymphoid tissues. Only 1 similar follicle was seen in 9 normal thymus samples. There was a positive association between the extent of germinal centres, plasma anti-acetylcholine receptor (AChR) titre, and spontaneous anti-AChR production by thymocytes in vitro. These thymic changes were not universally found, and are thus probably not central to the initiation of myasthenia. Between the follicles, in 9 cases, there was an apparent increase in interdigitating cells with closely associated 'inducer' (OKT4+)T lymphocytes. Thymic antigen presenting cells--either here or in the germinal centres--could be involved in breaking self-tolerance, or in perpetuating the autoimmune response, and it may be their removal that is therapeutic.

Attempted isolation of viruses from myasthenia gravis thymus.

A systematic study of thymus homogenates and cell suspensions from 13 patients with myasthenia gravis (MG) of recent onset, and 6 non-myasthenic controls, has failed to detect or isolate virus by cell culture with 'rescue techniques', electron microscopy, or intracerebral inoculation into neonatal mice. These results do not support the case for persistent viral infection in the thymus, and impose constraints on hypotheses of a viral aetiology of MG.

Thymus dependence of the antibody response to tetanus toxoid in mice.

Both the IgM and IgG antibody responses to tetanus toxoid were greatly reduced in adult thymectomized, irradiated, foetal liver reconstituted mice, in contrast with results published for neonatally thymectomized mice. This evidence lends further support to the notion that all IgG responses to protein antigens are thymus dependent.

Variation in spotting among the close relatives of the butterfly, Maniola jurtina.

A study of spotting in seven related butterfly species in the genera Maniola and Pyronia has been initiated, in the hope of complementing previous work on Maniola jurtina. Marked individual variability has been found in six of the species. Stability of spotting over large areas has not been prominent, but a high degree of apparent geographical variability has been found in four species. Sometimes this has taken the form of clines and sometimes of quantal steps. Some of the quantal changes coincide with the appearance of different "subspecies". Among the incomplete data presented, parallel geographical variation in different species has not been an obvious feature. There have not been enough samples to test for temporal variation in spotting.

Greatly improved recovery of in vitro B-memory cell function after enzymic dispersion of immunized mouse spleens.

We have used the protease dispase to disperse the fine clumps that persist after mechanical disruption of spleens from immunized mice. After 4-8 days in culture, the resulting 'D/C' cells spontaneously generated many more IgG plaque-forming cells (PFC) against sheep erythrocytes (SRBC) than did conventional (CONV) suspensions. The difference averaged 12-fold and was consistently high after a wide range of immunization protocols. The major difference between the two cell preparations proved to be in the B-cell lineage rather than in antigen-presenting cells or T cells and, indeed, the response was largely T-cell independent. Antigen-driven culture responses to SRBC were also more than 10-fold higher with D/C than with CONV suspensions, and again there was apparently an improved recovery of B-memory cells. However, when fresh cell preparations were assayed immediately for PFC, there was no D/C:CONV difference--just as we have previously reported for memory responses on cell transfer to irradiated recipients. One simple interpretation is that germinal centres tend to remain as fine clumps on mechanical disruption, and their constituent B-memory cells are enriched by our procedure. If so, their responses are much more evident in vitro than after cell transfer.

Radioactive antigen suicide of an anti-DNP (2,4-dinitrophenyl) clone. II. Follow-up of clones relatively resistant to radioactive antigen suicide when initially selected.

After suicide of the anti-dinitrophenyl (DNP) clone E21 in a previous experiment (Eur. J. Immunol. 1975. 5:58) several new clones (S clones) appeared promptly in the recipients. There was compelling evidence that they were then resistant to suicide, relative to E21. We now report further studies on these S clones and their antibodies. The antibodies of half of the S clones had affinities ten to one hundred times higher than E21 (3 x 10(-7) M); none was lower. The four tested were also able to bind DNP on the particular conjugate used for the suicide procedure. Three of the S clones were serially propagated, they showed a very great capacity for proliferation, transferring into 510, 85 and 110 recipients each. When their cells were tested at subsequent passages, they recovered early after inhibition by radioactive antigen. Two S clones may still have been somewhat refractory to suicide but, probably in all three, rapid proliferation was largely responsible for the recovery of the few cells escaping suicide, and helped them to appear suicide resistant.

B cell tolerance induced by polymeric antigens. III. Dissociation of antibody formation and memory generation in tolerant mice.

Hapten (2,4-dinitrophenyl (DNP))-substituted type 3 pneumococcal polysaccharide (DNP-lys-S3) effectively suppresses both primary and secondary anti-DNP antibody responses to DNP-proteins. The present experiments show that tolerizing doses of DNP-lys-S3 have much less effect on B cell memory (re)generation in three distinct situations: (i) in serial transfers of DNP-protein-primed cells, (ii) in virgin mice tolerized before priming with DNP-hemocyanin, and (iii) in tolerized, hemocyanin-primed mice boosted with DNP-hemocyanin. Under some conditions tolerized mice developed 10-50% of normal memory in the absence of significant antibody formation. Isoelectric focusing analyses revealed that many B cell clones proliferate in tolerant mice, but produce very little antibody until transferred to further hosts. Clones that escape suppression in partially tolerant mice do not appear to be resistant to DNP-lys-S3, when retested. Since there is independent evidence that the generation of B memory cells is less T cell-dependent than the development of antibody-forming cells, these data are best explained by assuming that DNP-lys-S3 blocks lymphocyte cooperation. This would be expected to preferentially suppress antibody formation, and to spare memory generation.

B cell tolerance induced by polymeric antigens. V. Different avidities of primed and virgin precursor cells for paucivalent antigen.

We have compared the avidity of virgin and primed hapten-specific precursor cells for DNP-lys-S3 (dinitrophenylated type 3 pneumococcal polysaccharide) by measuring its capacity to prevent them from binding highly radioactive DNP-hemocyanin, and thereby protect them from committing hapten-specific "suicide". Moderately substituted conjugates (e.g. DNP-lys2.7S3) protected virgin and memory B cells impartially. In contrast, DNP-lys0.6S3 protected memory cells efficiently, but protected virgin cells only at considerably higher concentrations. These results are consistent with previous evidence - from this and other systems - that there is a higher density of receptors on primed than on unprimed B cells - the significance of which is discussed.

Greatly increased autoantibody production in myasthenia gravis by thymocyte suspensions prepared with proteolytic enzymes.

We have previously reported that cell suspensions prepared by mechanical dispersion of thymic tissue from myasthenia gravis (MG) patients spontaneously synthesise anti-acetyl-choline receptor autoantibodies (anti-AChR) in culture in many cases. We now find a 2-200-fold greater anti-AChR production if the cell suspensions are prepared with the proteolytic enzymes collagenase and dispase (THYC + D). This difference has been seen in seven of the first 10 cases tested. Using these enzymes, we now also find comparable anti-AChR synthesis by MG lymph node cells, confirming the presumed extra-thymic production of much of this autoantibody. The increased anti-AChR synthesis by THYC + D is not simply a mitogenic effect of the enzymes, but apparently depends on several distinct factors: (1) plasma cells are often isolated much more efficiently--particularly by dispase--than by mechanical dispersion of these tissues (or of 'normal' tonsil), and the same may also be true of specific B cells. (2) The greatly increased numbers of fibroblasts, macrophages and other adherent cell types exert 'feeder' effects which enhance anti-AChR and total IgG production non-specifically. (3) Specific antigen presenting cells (APC) may also be recovered more efficiently and selectively stimulate anti-AChR production by B memory cells. These results imply that APC, plasma cells, and perhaps also B memory cells are recovered from germinal centres much more efficiently by enzymic than by conventional dispersion, a conclusion that may be relevant to workers studying antibody forming and memory B cells in other systems or species.

Cell types required for anti-acetylcholine receptor antibody synthesis by cultured thymocytes and blood lymphocytes in myasthenia gravis.

In most young myasthenia gravis patients, the thymic medulla contains germinal centres. Thymocytes from these cases spontaneously synthesize anti-acetylcholine receptor autoantibody (anti-AChR) in culture; after irradiation they may also selectively stimulate anti-AChR antibody production by autologous blood lymphocytes. By depleting cortical or mature thymic T cells by complement killing, we now show that neither of these responses depends on thymic T cells, unlike the total IgG response to pokeweed mitogen which is T cell-dependent and shows T/B cell synergy. The results suggest that much of the spontaneous anti-AChR production is by autonomous thymic plasma cells, which may be HLA-DR-. The ability to stimulate autologous blood lymphocytes does not require viable HLA-DR+ thymic cells but appears to depend on rare antigen presenting cells from the germinal centres. In preliminary experiments, blood T cells were apparently also necessary.

Circulating T cell subsets in the Lambert-Eaton myasthenic syndrome.

Peripheral blood T cell subsets were measured using monoclonal antibodies and a fluorescence activated cell sorter in 15 untreated patients with Lambert-Eaton myasthenic syndrome (nine with small cell carcinoma, one undifferentiated epithelial tumour (ca-LEMS], five with no demonstrable tumour (non-ca-LEMS), 10 age-matched healthy controls and 10 patients with small cell carcinoma without neurological disease. OKT8+ (suppressor/cytotoxic) T cells were significantly decreased in ca-LEMS compared with non-ca LEMS (p less than 0.001) ca-controls (p less than 0.01) and healthy controls (p less than 0.001). In one patient depressed OKT8+ T cells antedated clinically evident tumour by five months. OKT3+ (total) and OKT4+ (helper) T cells were similar in ca-LEMS, non-ca LEMS and controls. The mechanism underlying the loss of circulating OKT8+ T cells in ca-LEMS is unknown, but these changes may help to predict the presence of carcinoma in this disease.

Immunoglobulin M receptors on memory cells of immunoglobulin G antibody-forming cell clones.

The memory cells of two antibody-forming cell clones had receptors of the IgM class, even though the clones had been producing IgG1 or IgG2a anti-2,4-dinitrophenyl antibodies for 9-15 months previously (on exposure to antigen). Thus a phenotypic switch in heavy chain constant region evidently occurred after re-exposure of these memory cells to antigen. To show that, we first removed the clonal cells' surface immunoglobins by "capping" and "stripping", with class- or subclass-specific antisera. Then, to assay their remaining receptor activity, the cells were incubated with antigen in vitro, washed and transferred (together with carrier primed cells) to irradiated recipients, and their antibody responses to this in vitro boost were assayed by iselectric focusing. Pretreatment with anti-mu serum, as well as with anti-Fab(kappa), prevented the responses of the IgG1 and IgG2a clones to an in vitro boost, while anti-gamma1 and anti-gamma2a antisera had no effect. An antiserum to the putative mouse IgD also had no effect. The anti-mu serum failed to react with the IgG1 and IgG2A clonal serum antibodies in the test tube. Some other contaminating clones were suppressed completely only by the anti-Fab serum. This result strongly suggests that switching in class commitment may occur during the differentiation of memory cells to antibody producers, and may therefore be antigen-dependent. It also implies that some apparently naive cells with surface IgM may, in reality, be B memory cells.

In vitro studies on the ontogeny of lymphocyte populations.

We have investigated the ontogency of lymphocyte populations in the mouse embryo using organ culture techniques. Our results indicate that B lymphocytes are generated independently in foetal liver, foetal spleen and foetal bone marrow, while T lymphocytes are generated in thymus. We have characterised the structural and functional properties of developing lymphocytes in the organs. Our results suggest that foetal lymphocytes may differ from adult lymphocytes in a number of important respects which may have a bearing on the generation of tolerance to self antigens.

Potent immunostimulatory dendritic cells can be cultured in bulk from progenitors in normal infant and adult myasthenic human thymus.

Low density cells can readily be enriched from thymus tissue both of children undergoing cardiac surgery and of older patients with myasthenia gravis, and can be cryostored in bulk. When fresh or thawed cells are cultured with granulocyte-macrophage colony-stimulating factor and stem cell factor with or without tumour necrosis factor-alpha (TNF-alpha), they generate numerous cells with the characteristic ultrastructural, phenotypic and functional properties of dendritic cells. These proved to be very potent, both as stimulators of primary mixed leucocyte responses and as costimulators in oxidative mitogenesis. Especially after exposure to TNF-alpha, these dendritic cells also processed a natural epitope from a 437-residue polypeptide and presented it efficiently to an autoimmune T-cell clone (of T helper type 0 phenotype). Thus, immunostimulatory dendritic cells can be cultured in relative abundance from progenitors in infant and adult human thymus. Both are convenient sources of potent antigen-presenting cells of identifiable origins, e.g. for use in selecting human T-cell lines.

Infection of mouse liver by human adenovirus type 5.

CBA mice, inoculated intravenously with large doses of adenovirus type 5, showed raised levels of serum aspartate aminotransferase (SAAT; EC 2.6.I.I) and died within a few days from histologically demonstrable hepatic necrosis. After inoculation of I LD50, virus was rapidly taken up by the tissues where infectivity then declined greatly. Organ titres then increased about 100-fold by 48 h p.i. but, in the liver, which showed intranuclear inclusion bodies, and by electron microscopy, scattered intranuclear and intracytoplasmic adenovirions, the increase was 10000- to 100000-fold. P antigen was detected by single radial diffusion in liver extracts, and by immunofluorescence in 80% of liver cells at 36 h p.i. Hexon, penton base and fibre antigens appeared later and in fewer cells. The maximum amount of hexon, of demonstrable type 5 specificity, was shown by radioimmunoassay to be equivalent to up to 5 x 1011 whole adenovirions/g liver. It is concluded that human adenovirus type 5 undergoes an abortive but lytic infection in most liver cells but that replication may proceed to completion in a few.