- Email: [email protected]
A-4: Mechanisms of Autoimmunity
Workshop A-4 Mechanisms of Autoimmunity
Department of Microbiology and Immunobiology, The Queen's University of Belfast, Belfast, Northern Ireland
63. Macrophage sub-population variation in patients with multiple sclerosis MARILYN A. ARMSTRONG and MARGARET HAIRE
Populations of monocyte-derived macrophages from patients with multiple sclerosis (MS) and from age and sex-matched control subjects were investigated using the ecto-enzyme 5' nucleotidase (5'NT) as a marker for sub-population variation during culture with autologous serum. Immunofluorescent staining with monoclonal antibodies was carried out in parallel with each enzyme assay (performed according to the method described by SMITH et al.) (1). Early indications suggest that 5'NT levels are different in MS and control subjects.
1. SMITH, G. P., T. SHAH, A. D. B. WEBSTER, and T. J. PETERS. 1982. Studies on the kinetic properties and subcellular localization of adenine nucleotide phosphatases in peripheral blood lymphocytes from control subjects and patients with common variable primary hypogammaglobulinaemia. Clin. expo Immunol., 49: 393-400.
Department of Immunology, Mayo Clinic, Rochester, MN 55905, and Department of Microbiology and Immunology, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A.
64. T cell and B cell recognition of human and mouse thyroglobulins CHELLA S. DAVID, C.
J.
KRCO, and Y. M. KONG
Mice of the H-2k and H-2' haplotypes are susceptible to autoimmune thyroiditis induced by immunization with mouse thyroglobulin. By day 28 after immunization with 60 !-Ig of Mouse Thyroglobulin (MTG) in Complete Freunds Adjuvant (FCA), these mice develop severe thyroiditis characterized by mononuclear cell infiltration. On days 8-14, popliteal lymph node cells (LNC) from these mice proliferate in vitro in response to mouse thyroglobulin. On days 12-18, T cells cytotoxic for syngeneic thyroid cells are generated following culture of lymph node cells with mouse thyroglobulin. The proliferative response to MTG in vitro is mediated by Thy-l +, Ly-l +z- cells and can be blocked by polyclonal or monoclonal antibodies to I-Ak. Mouse thyroglobulin primed lymph node cells proliferate in response to human thyroglobulin indicating the presence of T cell clones recognizing shared determinants. To further confirm this, BIO.BR (H-2k) mice were immunized with human thyroglobulin and the
16th International Leucocyte Culture Conference, Cambridge· 41 draining lymph node cells and the immune sera were collected. Immune T cells responded strongly to challenge with human thyroglobulin (.... cpm 37,000) but not to mouse, bovine, or pig thyroglobulin (.... cpm's less than 2,000). In some cases, human thyroglobulin sensitized lymph node cells responded also to mouse thyroglobulin. When human thyroglobulin primed LNC were cultured with mouse thyroglobulin or human thyroglobulin, T cells cytotoxic for mouse thyroid targets were generated. Antigen presentation assays showed that human thyroglobulin sensitized T cells were also restricted by the I-A subregion. In contrast to the T cell response, immune sera revealed extensive crossreaction between human and mouse thyroglobulins in ELISA assays. Monoclonal antibodies prepared by the fusion of immune B cells to Fa/O myeloma cells exhibited two patterns of reactivity, one specific for human thyroglobulin and the other reactive with all four species of thyroglobulin. These results suggest that T cell and B cell repertoire against thyroglobulin are distinct.
Clinical Immunology Research Centre, University of Sydney, Sydney, Australia
65. Suppressor T cells in the induction of self tolerance
J. GIBSON and A. BASTEN Evidence exists to support the contention that suppressor T cells (Ts) playa significant «fail safe» role in the maintenance of tolerance to self as well as foreign antigens (1). In contrast, their contribution to the induction of self tolerance remains controversial. In order to test the latter possibility a murine model was established in which I-J+ cells were eliminated during embryonic life, offspring subsequently being assessed for their susceptibility to develop autoantibody responses. Anti I-Jk antibodies were generated by repeated immunisation of BI0.A(3R)(I-Jb) female mice with spleen and thymus cells from the congenic strain BI0.A(5R)(I-Jk) at weekly intervals for six weeks. The efficacy of this regimen was confirmed by showing that serum from immunised BI0.A(3R) animals contained sufficient anti I-Jk activity to eliminate I-J+Ts in a standard assay for suppression. After the final immunisation the BI0.A(3R) females and a control group were mated with CBA (H-2k) males thereby exposing F J progeny to IgG anti I-Jk antibodies in utero and during lactation. At six to seven weeks of age the two groups of offspring were tested for their ability to generate an autoantibody response to: (a) autologous erythrocytes as measured by direct antiglobulin test (DAGT) after challenge with rat erythrocytes (2) and (b) DNA (by enzyme immunoassay) following in vivo pokeweed mitogen (PWM) stimulation (3). In a representative experiment with the autoimmune haemolytic anaemia model all anti I-J exposed animals (6/6) became DAGT positive with a mean log2 titre (±SEM) of 6.7 ± 0.4 two weeks after a single immunisation of 2 X 108 rat erythrocytes. The response persisted for greater than six weeks. By contrast only 1/6 control animals were DAGT positive at two weeks (mean 1.5 ± 1.1) and thereafter all were negative. Pooled data from two subsequent experiments gave similar results while demonstrating at the same time that the effect of anti-I-J antibodies was specific for autoantibody production since serum anti-rat erythrocyte haemagglutination titres measured concurrently were not significantly different between test and control groups. Comparable results were obtained in the second experimental system. Mice exposed to anti-I-J during ontogeny developed significantly (p < 0.001) more anti-DNA antibodies (OD = 1.260 ± 0.048) than did controls (0.958 ± 0.032) following PWM stimulation. These findings support the concept that I-J+ Ts playa key role in the induction of self tolerance. 1. FAZEKAS DE ST. GROTH, B., B. BASTEN, and R. H. LOBLAY. Eur. J. Immunol. (in press). 2. PLAYFAIR, J. H. L., and S. MARSHALL-CLARKE. 1973. Nature (New BioI.) 243: 213. 3. FISH, F., and M. ZIFF. 1982. J. Immunol. 128: 409.
42 . 16th International Leucocyte Culture Conference, Cambridge Neuroimmunology Laboratory, Dept. of Neurology, Temple University School of Medicine, Philadelphia, Pa., and Neuroimmunology Branch, NINCDS, NIH, Bethesda, MD., U.S.A.
66. Abnormal augmentation of responses to self and non-self antigens in multiple sclerosis
J. I.
GREENSTEIN, B. B. MISHRA, H. F. MCFARLAND, and S. JACOBSON
In normal late-convalescent individuals, the cellular immune response to measles virus (MV) as measured, by lymphocyte proliferation, is considerably lower than the proliferative responses to both mumps and to vaccinia viruses. After natural infection significant responses to MV occur, but they decline rapidly, probably as the result of a failure to maintain cion ally expanded populations of antigen-reactive lymphocytes in the circulation (1). Previous studies in twins with multiple sclerosis (MS) demonstrated that 20 % of affected twins had significantly elevated proliferative responses to MV (2). This was the result of interleukin 2 production by, and proliferation of OKT4+ lymphocytes (3). The responses were not suppressed either by the addition of autologous OKT8+ to OKT4+ cells or by the addition of HLA-identical unaffected twin, low responder (LR) E+ cells to HR E+ cells. The original cohort of MS twins have now been studied longitudinally over a 5 year period. In six twin sets, 3 HR MS twins have had persistently elevated, and 3 twins have had fluctuating proliferative responses to MV. In addition, 6 sporadic MS cases have been studied sequentially during periods of disease activity and quiescence. Elevated proliferative responses to MV occurred in all during acute phases. Because of this, the MV HR were also examined for their ability to generate MV specific cytotoxic T cells (CTL). Significant CTL were generated only in MV HR. During the state of low responsiveness in sporadic MS cases significant levels of specific MV CTL were not generated - a response similar to that seen in normal late convalescents (4). Further, the response to self antigens, measured in the autologous mixed lymphocyte response has been studied in the individuals. Elevated responses occurred in the HR MS twins, or during acute activity in sporadic cases, but not in unaffected LR twins or during disease quiescence. Thus, the cells responsible for producing the elevated measles responses in HR appear to reside in a T cell subset with helper-inducer phenotype and function. These cells are presumably responsible for the clonal expansion of measles specific T cell populations in the MS HR. Analogous responses to self-antigens also appear to occur in these HR individuals. The mechanisms involved may be of potential relevance to the understanding of immunoregulatory abnormalities in MS. 1. 2. 3. 4.
GREENSTEIN, J. I., H. F. McFARLAND. 1983. Infect. Immun. 40: 198. GREENSTEIN, J. I., et al. 1984. Ann. Neurol. 15: 79. GREENSTEIN, J. I., et al. 1984. Ann. N.Y. Acad. Sci. (in press). LUCAS, C. J., et al. 1982. Infect. Immun. 38: 226.
Department of Neurology and Med. Microbiology of the Johannes-Gutenberg-University, 6500 Mainz, FRG
67. Changes in the number of complement-binding leucocytes in the peripheral blood of multiple sclerosis patients. Indication to a reduction of C3bi-binding T cells in the early acute phase of MS K. P. HAMMANN, D. v. STELDERN, M. P. DIERICH, and H. CH. HOPF The aetiology and pathogenesis of multiple sclerosis (MS) are still unknown. Immunologic, pathologic, and epidemiologic findings point to an aberration in the immune system and/or an
16th International Leucocyte Culture Conference, Cambridge· 43 infectious agent such as a virus. Among the numerous aberrations of the immune system in MS patients there are reports on the number of complement (C3) receptor bearing cells in the peripheral blood of MS patients and that the number of T cells is reduced during the active phase of MS. In a recent paper WAHLIN et al. (1) reported that about 60 % of the C3bi-binding cells were T cells and that 30-35 % of K cells bound C3bi. Since it is known that C3bi may augment antibody dependent cytotoxicity (ADCC) (2), which is assumed to play an essential role in demyelination (3, 4), we examined peripheral blood leucocytes (PBL) of MS patients for their reactivity with EAC3b-, EAC3b-~lH-, and EAC3bi-intermediates and C3b- and ~lH coated microspheres (ms). We found a marked decrease in the number of EAC3b-~lH (P = 0.04)- and EAC3bi(P = 0.03)-rosettes accompanied with a decrease in the number of T cells (P = 0.0015), when MS patients were examined within the first fourteen days after occurance of disturbances. On the other hand, when MS patients were examined later than fourteen days after onset of disturbances or those with a progressive course were examined EAC3b-~lH(P = 0.0002)- and EAC3bi(P = 0.0002)-rosettes were markely increased. In our studies where we applied fluorescence-activated cell sorter analysis on C3b- and ~lH-coated ms, we obtained similar results as found with EAC-intermediates. These observations as well as previous findings suggest that the decrease in EAC3b-~lH-, EAC3bi-, and ~lH-ms binding PBL was predominantly due to a loss of ~lH-binding T cells which might be of relevance for the demyelinating mechanism(s). 1. 2. 3. 4.
WAHLIN, B., et al. 1983. J. Immunol. 130: 2831. PERLMANN, H., et al. 1981. J. Exp. Med. 153: 1592. BROSNAN, c., et al. 1977. J. Immunol. 118: 2103. FRICK, E. and STICKL. 1982. Acta neurol. scandinav. 65: 30.
Karolinska Institute, Stockholm, Sweden
68. Activation of cell mediated immunity by absence or deleted expression of normal cellular gene products, i.e. by «no self» rather than «non self»? K. KARRE, H. G. LjUNGGREN, G. PIONTEK, R. KIESSLING, G. KLEIN, K. TANIGUCHI, and A. GRONBERG
Self non self discrimination may in theory operate through two distinct strategies: 1) By detecting the presence of foreign elements; 2) by scanning for the absence of normal self encoded gene products. The latter principle applies in rejection of allogeneic cells as well as in prevention of self fertilization in certain invertebrate species. The mammals have evolved adaptive immune responses based on the first strategy, with the additional requirement for recognition of self MHC simultaneously with detection of «non self». Infected or otherwise aberrant cells might therefore escape detection despite the expression of foreign antigens, provided their expression of MHC is switched off or deleted. It follows that the more primitive defence strategy may have been conserved also in mammals, provided it was modified to specifically eliminate MHC loss variant cells, which would otherwise escape the evolving T-cell system. Several observations on Fl hybrid rejection of parental hematopoetic grafts, resistance against metastasis, and in vitro NK -activity may actually be reinterpreted in support of such an alternate surveillance strategy in mice, geared to detect absence or incomplete expression of class I MHC gene products. In our studies of the murine RBL-5 lymphoma, we found that it is highly malignant in syngeneic H_2b/b hosts, whereas it is rejected by H_2d/b hosts, towards which the lymphoma can be considered to have incomplete MHC expression (lack of H-2 d). If this rejection is actually due to the postulated defence
44 . 16th International Leucocyte Culture Conference, Cambridge against «no/incomplete self»; one would further predict: 1) deletion of H-2b expression in the RBL-5 lymphoma should make it rejected also by syngeneic H_2b/b hosts; 2) introduction of H-2d expression in the lymphoma by DNA mediated gene transfer of class I genes should make it fully competent to grow out efficiently also in H_2d/b hosts. We are currently testing the second postulate, while the first has been tested and confirmed for a RBL-5 variant with no or very low MHC expression. We have also observed a switch from highly malignant phenotype to non-metastatic or non-tumorigenic phenotype accompanying the loss of MHC expression in variants of other murine tumor lines. The hypothesis of NK mediated surveillance against «no (incomplete) self" can not be rejected at this stage, and we conclude that it deserves further critical testing. A model for preferential triggering of NK-cells by targets with incomplete MHC expression will be presented. MHC class I gene products are not implicated in the initial effectorltarget contact, but rather in regulating activation of lysis according to this model. It is also suggested that a part of the interferon induced protection of target cells from NK-lysis is mediated via enhanced expression of MHC class I products.
Corporate Bioscience Group - Cell Biology Section, Imperial Chemical Industries PLC, Alderley Park, Nr Macclesfield, Cheshire SKI0 ETJ, U.K.
69. Induction of auto reactive T cells specific for autologous erythrocyte antigens D. G. KELLY and E.
J.
CULBERT
In an accompanying abstract (PATRICIA E. RIDDELL et al.) we have demonstrated that antierythrocyte (RBC) autoantibodies elicited in mice immunised with cross-reactive rat RBC are controlled by helper T cells reactive with rat, but not mouse RBC. By deliberate immunisation with autologous (mouse) RBC membrane in adjuvant, we have been able to elicit autoreactive T cells which proliferate in vitro in response to mouse RBC, but show no reactivity with rat RBC at the whole population level. We will show the results of clonal analysis of the specificity of these autoreactive T cells for autologous vs. xenogenic (rat) RBC, and compare the regulatory properties of auto reactive and rat RBC-specific T cell lines on in vivo autoantibody responses.
Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A.
70. The crucial role of circulatory thyroglobulin in activation of suppressor mechanisms M. LEWIS, A. A. GIRALDO, and Y. M. KONG
We report here the crucial role of circulatory levels of a self-antigen in activating suppressor mechanisms which prevent autoimmune disease. Previously we have shown that T -cellmediated suppression of experimental autoimmune thyroiditis (EAT) in genetically susceptible haplotypes is evoked by injection of soluble mouse thyroglobulin (MTg) (1). Moreover, injections of thyroid-stimulating hormone (TSH) or TSH-releasing hormone (TRH) can also activate suppression. The treated high responder mice become resistant to subsequent induction of EAT. Preliminary studies suggest that one definite link between the two tolerogenic schemes is the increase in circulatory MTg level, as measured by monoclonal antibodies to
16th International Leucocyte Culture Conference, Cambridge . 45 MTg in an ELISA (2). Calculations of tlh show that tolerogenic doses of MTg (~ 100 flg i.v.) equilibrate at a rate of 50 % reduction every 3 hr with about 1 % remaining after 24 hr (alpha phase). This rapid decline is followed by sustained levels above baseline for ~ 2-3 days with a tlh of about 10 hr (beta phase). Steady infusion of TSH or TRH i.p. via osmotic pumps results in comparable MTg levels, peaking around 72 hr. We have now examined further the importance of sustaining above threshold levels of MTg for a critical interval in activating suppression by using lipopolysaccharide (LPS). LPS has been shown earlier to augment suppression when given after the injection of TSH or TRH (1), and was used in the present study in conjunction with a nontolerogenic dose of MTg or with TSH pump. LPS (20 flg i.v.) administered to female CBA/J (H-2k) mice 24 hr before a nontolerogenic dose of MTg (::5 25 flg i.v.) extended the initial clearance rate of MTg from 3 to 5 hr, thereby sustaining MTg levels above baseline for 2-3 days. After challenge 10--11 days later with MTg + adjuvant, MTg antibody titers were very low and none of the mice developed EAT. In contrast, 100 % of the mice given 20 flg MTg (non-tolerogenic) had high antibody titers and thyroid lesions. LPS given i.v. 6 hr after the implantation of TSH pump (0.25 U/d for 7 days) likewise raised MTg levels above those in mice given TSH pump alone, without altering the overall kinetics. After challenge, antibody titers were low and only about 30 % developed lesions, compared to 50 and 100 % respectively in mice given only TSH or saline pump. These data demonstrate the important role of circulatory MTg in activating suppressor mechanisms to maintain seIftolerance. Supported by grants from the National Institutes of Health, AM 30975 & AM 31827. 1. KONG, Y. M., et al. 1982. New York Acad. Sci. 392: 191. 2. LEWIS, M., A. A. GIRALDO, and Y. M. KONG. 1984. Proc. Fed. Amer. Soc. Exp. BioI. 343: In press.
Tumour Immunology Unit, Department of Zoology, University College London, Gower Street, London WC1E 6BT, and Department of Immunology, Middlesex Hospital Medical School, Tottenham Street, London, U.K.
71. Cloning of autoreactive T lymphocytes from thyroid glands with Graves' disease M. LONDEI, G. F. BOTIAZZO, and M. FELDMANN
Graves' disease, autoimmune thyrotoxicosis, is often treated by surgical removal of the gland. We have dissociated the thyroid epithelial cells (TEC) which unlike normal glands strongly express HLA-DR antigens and the lymphocytes from such glands, and by incubating the lymphocytes in IL-2 were able to clone out T cells which were activated, and expressed receptors for IL-2 in vivo. Many of the T cell clones reacted with autologous TEC, but not mismatched TEe. These cloned T cells bound strongly to TEe. Immunofluorescent analysis of these autoreactive clones indicated that they reacted with Leu 3 (T4 antigen) but not UCHT4 (T8 antigen), indicating that these are cells of the «helper» phenotype. Because these cells were induced to proliferate by autologous DR + TEC, it indicates that DR + thyroid epithelial cells may act as antigen presenting cells, demonstrating that aberrant la/DR acts as in antigen presentation. This observation was confirmed by the capacity of monoclonal antibodies to DR to inhibit the proliferation of auto reactive T cell clones to TEe. As well as autoreactive T cell clones recognizing TEe, there were T cell clones which recognized autologous but not mismatched PBL, thus presumably representing the autologous mixed lymphocyte response cells that have been speculated to be of import once in the genesis of autoimmunity.
46 . 16th International Leucocyte Culture Conference, Cambridge Tumour Immunology Unit, Department of Zoology, University College London, Gower Street, London WC1E 6BT, and Department of Immunology, Middlesex Hospital Medical School, Tottenham Street, London, U.K.
72. Thyroid epithelial cells can present antigen M. LONDEI,
J. R.
LAMB, G. F. BOTIAZZO, and M. FELDMANN
While it is now well established that MHC Class II molecules (Ia or HLA-DR) are essential for antigen presentation in the induction of immune responses, it is not clear whether all cells capable of expressing MHC Class II determinants can function as antigen presenting cells in the appropriate circumstances. It has become apparent that during autoimmune responses, there is aberrant expression of DR antigens, for example in Graves' thyroiditis. We have speculated that this aberrant expression could permit these cells to act as antigen presenting cells, and so by presenting their cell surface autoantigens, initiate and perpetuate the autoimmune process. This hypothesis was tested using Influenza A haemagglutin specific human T cell clones, which can be triggered by either Influenza A virus, or synthetic peptides derived from the haemagglutinin sequence in conjunction with HLA-DC determinants. Thyroids of Graves' disease patients, expressing MHC Class II removed at operation, from donors whose blood cells present to the existing clones, were used as antigen presenting cells in a T cell proliferation assay, using peptide or killed virus. A strong response to the peptide, but none to the whole virus was obtained. This is unlike the results obtained with E- cells (B cells and monocytes) from the same donors, and indicates that the antigen presentation of the thyroid suspension must have been by the thyroid epithelial cells. This was confirmed by the lack of other possible antigen presenting cells, monocytes and B cells, and the role of the response and its abrogation by anti DC but not anti DR monoclonals.
Imperial Cancer Research Fund, Tumour Immunology Unit, University College London, London, U.K.
73. Suppressor T cells to liver F antigen M. L. LUKIC, A. RODRIGEZ, and N. A. MrrCHISON
Liver specific glycoprotein F offers a model to analyse the role of different cellular mechanisms in preveting T cell reactivity to autoantigens. It occurs in mice in two allelic forms: type 1 (F.l) and type 2 (F.2). Mice are unresponsive to their own F type but respond to immunization with allogeneic F by producing antibodies which cross-react with self-type F. Natural tolerance to F is found to be due to a repertoire purging mechanisms at the T cell level. However, the abolition of T suppressor cell induction enhances the response to allo F. Suppressor T cells are also responsible for non-MHC linked low responsiveness to allo F. We therefore attempted to develop an in vitro system in which suppressor cell activity induced by F antigen could be studied. Draining lymph node T cells obtained from CBA mice (F.l type) immunized with CBA or BlO.S (F.2 type) liver homogenate in CFA were stimulated in vitro with 1-20 !lg of F.l or F.2 obtained by single-step immunosorbent purification as described elsewhere (Wedderburn et al., submitted). Spleen adherent cells from untreated CBA mice were used as antigen presenting cells. Lymph node T cells obtained from CBA mice immunized with F.2 type liver extract responded in the dose-dependent fashion to
16th International Leucocyte Culture Conference, Cambridge . 47 immunopurified F.2. T cells from mice primed with self-type, F.1 did not respond to the in vitro stimulation with either F.1 or F.2. We then studied the effect of spleen T cells obtained from CBA mice 7 days after i.v. injection of 2.5-3.5 mg soluble F.1 or F.2 on the responses of primed lymph node T cells to F.2. It was found that spleen cells from mice pretreated with soluble F.2 induced profound suppression of the proliferative response. Spleen cells from mice injected with self-type F (F.1) also exhibited a suppressive effect on the proliferative response to allo F (F.2). However, the degree of suppression was always lower than the one induced by spleen cells treated with allo F. The addition of spleen T cells from mice treated with saline or BGG did not induce any suppressive effect indicating that observed suppression was F antigen specific. The results are compatible with the notion that self F specific suppressor T cells are not eliminated known to be operative at the T cell level.
Louisiana State University and Tulane University Medical Centers, New Orleans, Louisiana, U.S.A.
74. Immune abnormalities associated with HLA-B8 in healthy subjects CANDACE C. MCCOMBS,
J. P. MICHALSKI, R.
D. DESHAZO, and B. BOZELKA
The antigen HLA-B8 occurs with increased frequency in patients with at least nine diseases having a known or suspected autoimmune component, suggesting that a gene in linkage disequilibrium with HLA-B8 predisposes to the development of autoimmunity. We have observed that many healthy young adults with HLA-B8 have alterations of immune functions when compared to sex and age matched controls without HLA-B8. These alterations include decreased T cell proliferation in response to suboptimal concentrations of mitogens, increased pokeweed mitogen driven immunoglobulin synthesis, and decreased Con A induced suppression. With regard to T cell proliferation, 9 out of 15 HLA-B8 positive subjects incorporated less than 2,500 cpm in response to 0.1 !lg/ml of PHA, compared to only lout of 15 subjects without HLA-B8. The mean response of the subjects with HLA-B8 was 1,919 cpm, significantly less than the response (6,384 cpm) of the subjects without HLA-B8 (p < 0.007). Similarly decreased proliferation was also apparent in response to a suboptimally stimulating concentration of Con A, but not to optimally stimulating concentrations of either mitogen. In additional studies, we examined B lymphocyte function in subjects with and without HLA-B8 by measuring pokeweed mitogen driven differentiation of B cells to IgM bearing blasts. We found that nine subjects with HLA-B8 had a mean of 83,426 IgM positive blasts per culture compared to 27,713 for nine subjects without HLA-B8 (p = 0.0013). Con A induced suppression of immunoglobulin synthesis was also examined, using a one-step method in which Con A is added, along with pokeweed mitogen, at the initiation of culture. Subjects with HLA-B8 were found to have decreased Con A induced suppression in this assay (p = 0.007). To further analyze the cellular basis of these immunological abnormalities, lymphocyte subsets were enumerated using monoclonal antibodies and a fluorescence activated cell sorter. B cells staining for surface IgM were increased in twelve subjects with HLA-B8 (9.6 % ± 1.2) compared to twelve subjects without HLA-B8 (6.1 % ± 0.7, p = 0.013). OKT4 positive cells (helper cells) were increased in the group with HLA-B8 (41.2% vs 36.9%), while OKT8 positive cells (suppressor/cytotoxic cells) were decreased (19.8% vs 23.1 %). When the OKT4/0KT8 ratio was calculated for each subject, the mean ratio of the subjects with HLAB8 (2.49) was significantly higher (p = 0.0078) than the ratio of the subjects without this antigen (1.69). These alterations of lymphocyte subsets and immune responsiveness may contribute to the predisposition to autoimmunity associated with HLA-B8.
48 . 16th International Leucocyte Culture Conference, Cambridge Louisiana State University Medical Center, New Orelans, Louisiana, U.S.A.
75. Interleukin 2 production by PHA stimulated lymphocytes from subjects with HLA-B8
J.
P. MICHALSKI and CANDACE C. MCCOMBS
The histocompatibility antigen HLA-B8 is associated with an increased frequency of immune mediated diseases, including myasthenia gravis, Graves' disease, Sjogren's syndrome and systemic lupus erythematosus. Several altered immunologic functions occur in normal subjects with HLA-B8, which may reflect the action of a gene or genes, linked to HLA-B8, which contributes to autoimmunity. These abnormalities include impaired response to suboptimal concentrations of PHA or Con A, increased spontaneous or pokeweed mitogen driven immunoglobulin production and decreased Con A induced suppressor activity. Because cellular interactions are involved in these in-vitro immune functions, we have begun a study of interleukin production and response, to probe the mechanism of altered lymphocyte function in subjects with HLA-B8. In a preliminary study of 6 subjects with HLA-B8 and 6 individuals without this antigen, we cultured peripheral blood mononuclear cells with several concentrations of PHA and harvested supernatants after 24 and 48 hours. Interleukin 2 was assayed by a microassay utilizing an IL-2 dependent murine cytotoxic T cell line. In most subjects, supernatant IL-2 stimulated by an optimal concentration of PHA peaked at 24 hours whereas suboptimally stimulated cultures reached peak levels at 48 hours. IL-2 concentrations in optimally stimulated cultures generally ranged from 5-13 units/ml whereas levels with suboptimal PHA concentrations were 10-20 times lower. At 48 hours of culture,S of 6 subjects with HLA-B8 had lower IL-2 production by sub optimally stimulated mononuclear cells than age matched controls without HLA-B8. IL-2 in optimally stimulated cultures was comparable for the two groups. Exogenous IL-2 was added to PHA stimulated cultures of 2 subjects with HLA-B8 who had very low proliferative responses. The impaired suboptimal PHA response improved significantly compared with relatively little change in two individuals with a normal response and who lacked HLA-B8. Our preliminary data suggest that levels of IL-2 in PHA stimulated cultures correlate with subsequent incorporation of tritiated thymidine. Subjects with HLA-B8 produce smaller amounts of IL-2 after stimulation with a suboptimal concentration of PHA. In two subjects, impaired proliferative responsiveness was at least partially corrected by exogenous IL-2, demonstrating a capacity to respond to this substance. Impaired production of IL-2 may contribute to altered immunity associated with HLA-B8.
University of California, Los Angeles, California, U.S.A.
76. 112 response and production in multiple sclerosis patients
J.
E. MERRILL, C. MOHLSTROM, V. KERMANI, G. W. ELLISON, and L. W. MYERS
In an effort to analyze the defective proliferative response to antigens and mitogens by T lymphocytes in multiple sclerosis (MS) patients, we examined the interleukin 2 (IL2) response and production by peripheral blood lymphocytes (PBL) and cerebrospinal fluid (CSF) mononuclear cells. For IL2 response experiments, nylon wool passed (NWP) PBL were used. Endogenous IL2 production was prevented by 1O- 6 M Dexamethasone and response was measured to exogenous lectin-free IL2. Cells were stimulated with 10 Itg/ml PHA-P and 10 % IL2. Chronic progressive MS patients had an abnormally low response to exogenous IL2 compared to controls. While acute relapse patients' PBL demonstrated a normal IL2 response
16th International Leucocyte Culture Conference, Cambridge· 49 during exacerbation, they showed reduced responsiveness during remission. These abnormalities could not be explained by different dose or kinetic response optima to PHA or III nor could they be explained by abnormal numbers of III or transferrin receptor-bearing lymphocytes. In contrast, CSF cells contained normal numbers of IL2 receptor-bearing lymphocytes but depressed numbers of transferrin receptor-bearing cells which might explain their reduced IL2 response compared to homologous PBL. Production of IL2 was measured from supernatants of preNW and NWP cells. NWP cells made 8-10 fold more III than preNW cells and thus were used in all experiments. Production of IL2 was elevated in chronic progressive patients and acute relapse patients during remission. IL2 levels were diminished during exacerbation. Removal of NW adherent cells and 1200 rad x-irradiation of the NWP lymphocytes augmented IL2 production to a 3 fold greater extent in MS patients than controls indicating the likelihood that both macrophages and suppressor T cells play a role in the regulation of IL2 production in multiple sclerosis. The inability of MS patients' lymphocytes to cion ally expand in response to IL2 may contribute to the pathogenicity of this disease. Supported by grants from the MS Society-JF 2012-A-3 and the NIH-NS08711 and NS 19145.
Corporate Bioscience Group - Cell Biology Section, Imperial Chemical Industries PLC, Central Toxicology Laboratory, Alderley Park, Nr. Macclesfield, Cheshire, SKI0 4TJ, U.S.A.
77. In vivo modulation of anti-erythrocyte autoantibodies by T cell lines PATRICIA E. RIDDELL, D. G. KELLY, A.
J. ALDRIDGE, R. T. ROBSON, and E. J.
CULBERT
CBA mice immunised with rat erythrocytes (RBC) produce an anti-RBC autoantibody response which is preferentially suppressed on rechallenge with rat RBC (1). Monoclonal autoantibodies bind to a cross-reactive determinant(s) shared between rat and mouse RBC, suggesting that the autoantibodies are a normal component of the anti-rat RBC response. To investigate the specificity of helper T cells regulating the autoantibody response, we have produced T cell lines reactive with rat RBC. These lines proliferate in response to rat RBC, but not mouse RBC, and augment autoantibody production in mice immunised with rat RBC. Injection of the rat RBC-reactive lines in the absence of rat RBC does not result in autoantibody production. These results demonstrate that the autoantibody response in this model can be regulated by rat RBC-specific helper T cells, and suggest that helper T cells reactive with autologous RBC do not operate in this model. The implications of these findings for possible mechanisms of specific suppression of autoantibodies will be discussed. 1. COOKE, A., P. R. HUTCHINGS, and J. H. L. PLAYFAIR. 1978. Suppressor T cell in experimental autoimmune haemolytic anaemia. Nature 273: 154.
Wayne State University School of Medicine, Detroit, Michigan, 48201, U.S.A.
78. T cell recognition of unique and shared determinants of auto antigenic mouse thyroglobulin in experimental autoimmune thyroiditis (EAT) L. L. SIMON and Y. M. KONG
Weare studying the antigenic determinants recognized by T and B lymphocytes from mice of a thyroiditis-susceptible strain following immunization and/or tolerization with homolo-
50 . 16th International Leucocyte Culture Conference, Cambridge gous (mouse) and heterologous (human) thyroglobulin (Tg). High responder mice immunized with 100 Itg mouse Tg (MTg) in complete Freund's adjuvant (CFA) develop autoantibodies to MTg and EAT characterized by mononuclear infiltration. On days 8-14 following immunization, their popliteal lymph node cells (LNC) proliferate in vitro in response to MTg, and also to human Tg (HTg) (1). Our current findings indicate that a minor portion of the autoantibodies produced after MTg-immunization reacts with HTg in passive hemagglutination but the major portion is directed against MTg-specific rather than shared determinants. LNC from mice immunized with heterologous HTg proliferate strongly in vitro to HTg and to a much lesser degree to MTg. After HTg-immunization, the antibody response is primarily directed against HTg-specific determinants but antibodies to shared determinants are also produced. To study the contribution of mouse-specific and shared determinants in T cell proliferation, reciprocal Tg's are used for cross tolerization and immunization. Previous studies have shown that suppression of EAT induction and autoantibody production can be achieved in high responder mice by the injection of soluble MTg (200 ltg, i.v.) on days 10 and 3 prior to immunization and their LNC do not proliferate in response to MTg. We have now observed that comparable treatment with soluble HTg before immunization with HTg and CFA dramatically reduces the proliferative response to HTg. MTg-induced tolerance followed by immunization with either MTg or HTg in CFA eliminates the proliferative response to MTg, but only partially reduces the response to HTg after HTg-CFA immunization. Interestingly, treatment with aqueous HTg prior to immunization with MTg-CFA greatly reduces the proliferative response to MTg. These results indicate that the proliferative response to MTg in high responder mice is readily elicited against determinants shared with HTg and suppressed by pretreatment with soluble HTg. Using tolerance induction to functionally delete T cell subsets reactive with epitopes on MTg and HTg will make it possible to study the relative roles of these epitopes and T cell subsets in autoantibody production and EAT induction. In addition, Tg-specific T cell lines and clones reactive with unique and shared determinants on the auto antigen MTg can be developed. Supported by National Institutes of Health AM 31827 and AM 30975.
1. SIMON et aI., 1984. Proc. Fed. Amer. Soc. Exp. BioI. 43: in press.
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, U.S.A.
79. Determinant specificity of the immune response to insulin in man
J. W. THOMAS, VALERIE J. VIRTA, and LAURA J. NELL Insulin has been used as a model antigen in studies of the fine specificity of immune response (Ir) gene function in inbred animals. Those studies revealed that both the appropriate MHClinked gene product and unique amino acid exchanges between the immunizing insulin and the animals' endogenous insulin were critical for initiation of the immune response to this antigen. We have used a similar approach in the outbred human population to identify the determinants on the insulin molecule recognized by T cells from insulin-treated diabetics. Two patterns of antigen recognition were found in human lymphocyte responses to mammalian insulins. In one group, the differential responses to insulins from several species identified the amino acid residues necessary for antigen recognition. These residues are clustered on the surface of the molecule and include the A chain loop of beef insulin and the carboxy-terminus of the B chain of beef and pork insulin. Studies employing insulins that have additional amino acid substitutions in the A chain loop (horse and sheep) indicate that lymphocytes from different individuals may recognize this limited region of the molecule in several ways. Since the cellular
16th International Leucocyte Culture Conference, Cambridge· 51 responses of these subjects are directed at primary amino acid exchanges, they are analogous to the responses seen with inbred animals. However, primary structure alone is not always sufficient for recognition of A loop determinants indicating that conformational integrity of this region of the molecule is also important. This contrast with the animal studies is most notable in a second group of subjects whose lymphocytes do not show a differential response to various insulins. This group's lymphocytes respond to all insulins, including human. Further studies in this «autoimmune» group with isolated A and B chain peptides and with monomeric sodium insulin suggest that primary amino acid exchanges are not critical for the response. Rather, we believe that conformational determinants are created by the molecular interactions of insulin dimers and hexamers and that these determinants are recognized by immune T cells. This conclusion is further supported by the observation that diabetics treated with human insulins develop anti-insulin antibodies. Our studies demonstrate that despite the complexity of an outbred population antigenic determinants that are recognized by man can be identified when a highly defined antigen is employed. In addition, this approach reveals patterns of antigen recognition that were not previously demonstrated when the same antigen was used in inbred animals.
Laboratory of Immunology NIAID, NIH, Bethesda, MD 20205 U.S.A.
80. Monoclonal autoantibodies from normal mice P.
WASSMER
Autoantibodies reacting with different tissues are commonly found in the sera of patients with autoimmune disorders, but are also present at a low frequency in healthy individuals. The mechanisms of appearance and the pathological roles of these self-reactive antibodies are not yet clear. We report here the production of monoclonal autoantibodies from non-immunized healthy mice. Antibody-secreting hybridomas were prepared by fusing spleen cells from normal mice with P3-653Ag8 myeloma cells. Hybridomas producing immunoglobulins as determined by ELISA were screened for reactivity with tissue sections by indirect immunofluorescence. Up to 10 % of hybridomas secreting immunoglobulins produced antibodies reacting with islets of Langerhans, anterior pituitary, gastric mucosa, smooth muscle, salivary gland or cellular nuclei from various tissues. This finding suggests that non autoimmune individuals without detectable serum autoantibodies may possess a number of antibody secreting B-Iymphocytes with affinity for self antigens.
Department of Microbiology and Immunobiology, The Queen's University of Belfast, Belfast, Northern Ireland
63. Macrophage sub-population variation in patients with multiple sclerosis MARILYN A. ARMSTRONG and MARGARET HAIRE
Populations of monocyte-derived macrophages from patients with multiple sclerosis (MS) and from age and sex-matched control subjects were investigated using the ecto-enzyme 5' nucleotidase (5'NT) as a marker for sub-population variation during culture with autologous serum. Immunofluorescent staining with monoclonal antibodies was carried out in parallel with each enzyme assay (performed according to the method described by SMITH et al.) (1). Early indications suggest that 5'NT levels are different in MS and control subjects.
1. SMITH, G. P., T. SHAH, A. D. B. WEBSTER, and T. J. PETERS. 1982. Studies on the kinetic properties and subcellular localization of adenine nucleotide phosphatases in peripheral blood lymphocytes from control subjects and patients with common variable primary hypogammaglobulinaemia. Clin. expo Immunol., 49: 393-400.
Department of Immunology, Mayo Clinic, Rochester, MN 55905, and Department of Microbiology and Immunology, Wayne State University School of Medicine, Detroit, MI 48201, U.S.A.
64. T cell and B cell recognition of human and mouse thyroglobulins CHELLA S. DAVID, C.
J.
KRCO, and Y. M. KONG
Mice of the H-2k and H-2' haplotypes are susceptible to autoimmune thyroiditis induced by immunization with mouse thyroglobulin. By day 28 after immunization with 60 !-Ig of Mouse Thyroglobulin (MTG) in Complete Freunds Adjuvant (FCA), these mice develop severe thyroiditis characterized by mononuclear cell infiltration. On days 8-14, popliteal lymph node cells (LNC) from these mice proliferate in vitro in response to mouse thyroglobulin. On days 12-18, T cells cytotoxic for syngeneic thyroid cells are generated following culture of lymph node cells with mouse thyroglobulin. The proliferative response to MTG in vitro is mediated by Thy-l +, Ly-l +z- cells and can be blocked by polyclonal or monoclonal antibodies to I-Ak. Mouse thyroglobulin primed lymph node cells proliferate in response to human thyroglobulin indicating the presence of T cell clones recognizing shared determinants. To further confirm this, BIO.BR (H-2k) mice were immunized with human thyroglobulin and the
16th International Leucocyte Culture Conference, Cambridge· 41 draining lymph node cells and the immune sera were collected. Immune T cells responded strongly to challenge with human thyroglobulin (.... cpm 37,000) but not to mouse, bovine, or pig thyroglobulin (.... cpm's less than 2,000). In some cases, human thyroglobulin sensitized lymph node cells responded also to mouse thyroglobulin. When human thyroglobulin primed LNC were cultured with mouse thyroglobulin or human thyroglobulin, T cells cytotoxic for mouse thyroid targets were generated. Antigen presentation assays showed that human thyroglobulin sensitized T cells were also restricted by the I-A subregion. In contrast to the T cell response, immune sera revealed extensive crossreaction between human and mouse thyroglobulins in ELISA assays. Monoclonal antibodies prepared by the fusion of immune B cells to Fa/O myeloma cells exhibited two patterns of reactivity, one specific for human thyroglobulin and the other reactive with all four species of thyroglobulin. These results suggest that T cell and B cell repertoire against thyroglobulin are distinct.
Clinical Immunology Research Centre, University of Sydney, Sydney, Australia
65. Suppressor T cells in the induction of self tolerance
J. GIBSON and A. BASTEN Evidence exists to support the contention that suppressor T cells (Ts) playa significant «fail safe» role in the maintenance of tolerance to self as well as foreign antigens (1). In contrast, their contribution to the induction of self tolerance remains controversial. In order to test the latter possibility a murine model was established in which I-J+ cells were eliminated during embryonic life, offspring subsequently being assessed for their susceptibility to develop autoantibody responses. Anti I-Jk antibodies were generated by repeated immunisation of BI0.A(3R)(I-Jb) female mice with spleen and thymus cells from the congenic strain BI0.A(5R)(I-Jk) at weekly intervals for six weeks. The efficacy of this regimen was confirmed by showing that serum from immunised BI0.A(3R) animals contained sufficient anti I-Jk activity to eliminate I-J+Ts in a standard assay for suppression. After the final immunisation the BI0.A(3R) females and a control group were mated with CBA (H-2k) males thereby exposing F J progeny to IgG anti I-Jk antibodies in utero and during lactation. At six to seven weeks of age the two groups of offspring were tested for their ability to generate an autoantibody response to: (a) autologous erythrocytes as measured by direct antiglobulin test (DAGT) after challenge with rat erythrocytes (2) and (b) DNA (by enzyme immunoassay) following in vivo pokeweed mitogen (PWM) stimulation (3). In a representative experiment with the autoimmune haemolytic anaemia model all anti I-J exposed animals (6/6) became DAGT positive with a mean log2 titre (±SEM) of 6.7 ± 0.4 two weeks after a single immunisation of 2 X 108 rat erythrocytes. The response persisted for greater than six weeks. By contrast only 1/6 control animals were DAGT positive at two weeks (mean 1.5 ± 1.1) and thereafter all were negative. Pooled data from two subsequent experiments gave similar results while demonstrating at the same time that the effect of anti-I-J antibodies was specific for autoantibody production since serum anti-rat erythrocyte haemagglutination titres measured concurrently were not significantly different between test and control groups. Comparable results were obtained in the second experimental system. Mice exposed to anti-I-J during ontogeny developed significantly (p < 0.001) more anti-DNA antibodies (OD = 1.260 ± 0.048) than did controls (0.958 ± 0.032) following PWM stimulation. These findings support the concept that I-J+ Ts playa key role in the induction of self tolerance. 1. FAZEKAS DE ST. GROTH, B., B. BASTEN, and R. H. LOBLAY. Eur. J. Immunol. (in press). 2. PLAYFAIR, J. H. L., and S. MARSHALL-CLARKE. 1973. Nature (New BioI.) 243: 213. 3. FISH, F., and M. ZIFF. 1982. J. Immunol. 128: 409.
42 . 16th International Leucocyte Culture Conference, Cambridge Neuroimmunology Laboratory, Dept. of Neurology, Temple University School of Medicine, Philadelphia, Pa., and Neuroimmunology Branch, NINCDS, NIH, Bethesda, MD., U.S.A.
66. Abnormal augmentation of responses to self and non-self antigens in multiple sclerosis
J. I.
GREENSTEIN, B. B. MISHRA, H. F. MCFARLAND, and S. JACOBSON
In normal late-convalescent individuals, the cellular immune response to measles virus (MV) as measured, by lymphocyte proliferation, is considerably lower than the proliferative responses to both mumps and to vaccinia viruses. After natural infection significant responses to MV occur, but they decline rapidly, probably as the result of a failure to maintain cion ally expanded populations of antigen-reactive lymphocytes in the circulation (1). Previous studies in twins with multiple sclerosis (MS) demonstrated that 20 % of affected twins had significantly elevated proliferative responses to MV (2). This was the result of interleukin 2 production by, and proliferation of OKT4+ lymphocytes (3). The responses were not suppressed either by the addition of autologous OKT8+ to OKT4+ cells or by the addition of HLA-identical unaffected twin, low responder (LR) E+ cells to HR E+ cells. The original cohort of MS twins have now been studied longitudinally over a 5 year period. In six twin sets, 3 HR MS twins have had persistently elevated, and 3 twins have had fluctuating proliferative responses to MV. In addition, 6 sporadic MS cases have been studied sequentially during periods of disease activity and quiescence. Elevated proliferative responses to MV occurred in all during acute phases. Because of this, the MV HR were also examined for their ability to generate MV specific cytotoxic T cells (CTL). Significant CTL were generated only in MV HR. During the state of low responsiveness in sporadic MS cases significant levels of specific MV CTL were not generated - a response similar to that seen in normal late convalescents (4). Further, the response to self antigens, measured in the autologous mixed lymphocyte response has been studied in the individuals. Elevated responses occurred in the HR MS twins, or during acute activity in sporadic cases, but not in unaffected LR twins or during disease quiescence. Thus, the cells responsible for producing the elevated measles responses in HR appear to reside in a T cell subset with helper-inducer phenotype and function. These cells are presumably responsible for the clonal expansion of measles specific T cell populations in the MS HR. Analogous responses to self-antigens also appear to occur in these HR individuals. The mechanisms involved may be of potential relevance to the understanding of immunoregulatory abnormalities in MS. 1. 2. 3. 4.
GREENSTEIN, J. I., H. F. McFARLAND. 1983. Infect. Immun. 40: 198. GREENSTEIN, J. I., et al. 1984. Ann. Neurol. 15: 79. GREENSTEIN, J. I., et al. 1984. Ann. N.Y. Acad. Sci. (in press). LUCAS, C. J., et al. 1982. Infect. Immun. 38: 226.
Department of Neurology and Med. Microbiology of the Johannes-Gutenberg-University, 6500 Mainz, FRG
67. Changes in the number of complement-binding leucocytes in the peripheral blood of multiple sclerosis patients. Indication to a reduction of C3bi-binding T cells in the early acute phase of MS K. P. HAMMANN, D. v. STELDERN, M. P. DIERICH, and H. CH. HOPF The aetiology and pathogenesis of multiple sclerosis (MS) are still unknown. Immunologic, pathologic, and epidemiologic findings point to an aberration in the immune system and/or an
16th International Leucocyte Culture Conference, Cambridge· 43 infectious agent such as a virus. Among the numerous aberrations of the immune system in MS patients there are reports on the number of complement (C3) receptor bearing cells in the peripheral blood of MS patients and that the number of T cells is reduced during the active phase of MS. In a recent paper WAHLIN et al. (1) reported that about 60 % of the C3bi-binding cells were T cells and that 30-35 % of K cells bound C3bi. Since it is known that C3bi may augment antibody dependent cytotoxicity (ADCC) (2), which is assumed to play an essential role in demyelination (3, 4), we examined peripheral blood leucocytes (PBL) of MS patients for their reactivity with EAC3b-, EAC3b-~lH-, and EAC3bi-intermediates and C3b- and ~lH coated microspheres (ms). We found a marked decrease in the number of EAC3b-~lH (P = 0.04)- and EAC3bi(P = 0.03)-rosettes accompanied with a decrease in the number of T cells (P = 0.0015), when MS patients were examined within the first fourteen days after occurance of disturbances. On the other hand, when MS patients were examined later than fourteen days after onset of disturbances or those with a progressive course were examined EAC3b-~lH(P = 0.0002)- and EAC3bi(P = 0.0002)-rosettes were markely increased. In our studies where we applied fluorescence-activated cell sorter analysis on C3b- and ~lH-coated ms, we obtained similar results as found with EAC-intermediates. These observations as well as previous findings suggest that the decrease in EAC3b-~lH-, EAC3bi-, and ~lH-ms binding PBL was predominantly due to a loss of ~lH-binding T cells which might be of relevance for the demyelinating mechanism(s). 1. 2. 3. 4.
WAHLIN, B., et al. 1983. J. Immunol. 130: 2831. PERLMANN, H., et al. 1981. J. Exp. Med. 153: 1592. BROSNAN, c., et al. 1977. J. Immunol. 118: 2103. FRICK, E. and STICKL. 1982. Acta neurol. scandinav. 65: 30.
Karolinska Institute, Stockholm, Sweden
68. Activation of cell mediated immunity by absence or deleted expression of normal cellular gene products, i.e. by «no self» rather than «non self»? K. KARRE, H. G. LjUNGGREN, G. PIONTEK, R. KIESSLING, G. KLEIN, K. TANIGUCHI, and A. GRONBERG
Self non self discrimination may in theory operate through two distinct strategies: 1) By detecting the presence of foreign elements; 2) by scanning for the absence of normal self encoded gene products. The latter principle applies in rejection of allogeneic cells as well as in prevention of self fertilization in certain invertebrate species. The mammals have evolved adaptive immune responses based on the first strategy, with the additional requirement for recognition of self MHC simultaneously with detection of «non self». Infected or otherwise aberrant cells might therefore escape detection despite the expression of foreign antigens, provided their expression of MHC is switched off or deleted. It follows that the more primitive defence strategy may have been conserved also in mammals, provided it was modified to specifically eliminate MHC loss variant cells, which would otherwise escape the evolving T-cell system. Several observations on Fl hybrid rejection of parental hematopoetic grafts, resistance against metastasis, and in vitro NK -activity may actually be reinterpreted in support of such an alternate surveillance strategy in mice, geared to detect absence or incomplete expression of class I MHC gene products. In our studies of the murine RBL-5 lymphoma, we found that it is highly malignant in syngeneic H_2b/b hosts, whereas it is rejected by H_2d/b hosts, towards which the lymphoma can be considered to have incomplete MHC expression (lack of H-2 d). If this rejection is actually due to the postulated defence
44 . 16th International Leucocyte Culture Conference, Cambridge against «no/incomplete self»; one would further predict: 1) deletion of H-2b expression in the RBL-5 lymphoma should make it rejected also by syngeneic H_2b/b hosts; 2) introduction of H-2d expression in the lymphoma by DNA mediated gene transfer of class I genes should make it fully competent to grow out efficiently also in H_2d/b hosts. We are currently testing the second postulate, while the first has been tested and confirmed for a RBL-5 variant with no or very low MHC expression. We have also observed a switch from highly malignant phenotype to non-metastatic or non-tumorigenic phenotype accompanying the loss of MHC expression in variants of other murine tumor lines. The hypothesis of NK mediated surveillance against «no (incomplete) self" can not be rejected at this stage, and we conclude that it deserves further critical testing. A model for preferential triggering of NK-cells by targets with incomplete MHC expression will be presented. MHC class I gene products are not implicated in the initial effectorltarget contact, but rather in regulating activation of lysis according to this model. It is also suggested that a part of the interferon induced protection of target cells from NK-lysis is mediated via enhanced expression of MHC class I products.
Corporate Bioscience Group - Cell Biology Section, Imperial Chemical Industries PLC, Alderley Park, Nr Macclesfield, Cheshire SKI0 ETJ, U.K.
69. Induction of auto reactive T cells specific for autologous erythrocyte antigens D. G. KELLY and E.
J.
CULBERT
In an accompanying abstract (PATRICIA E. RIDDELL et al.) we have demonstrated that antierythrocyte (RBC) autoantibodies elicited in mice immunised with cross-reactive rat RBC are controlled by helper T cells reactive with rat, but not mouse RBC. By deliberate immunisation with autologous (mouse) RBC membrane in adjuvant, we have been able to elicit autoreactive T cells which proliferate in vitro in response to mouse RBC, but show no reactivity with rat RBC at the whole population level. We will show the results of clonal analysis of the specificity of these autoreactive T cells for autologous vs. xenogenic (rat) RBC, and compare the regulatory properties of auto reactive and rat RBC-specific T cell lines on in vivo autoantibody responses.
Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A.
70. The crucial role of circulatory thyroglobulin in activation of suppressor mechanisms M. LEWIS, A. A. GIRALDO, and Y. M. KONG
We report here the crucial role of circulatory levels of a self-antigen in activating suppressor mechanisms which prevent autoimmune disease. Previously we have shown that T -cellmediated suppression of experimental autoimmune thyroiditis (EAT) in genetically susceptible haplotypes is evoked by injection of soluble mouse thyroglobulin (MTg) (1). Moreover, injections of thyroid-stimulating hormone (TSH) or TSH-releasing hormone (TRH) can also activate suppression. The treated high responder mice become resistant to subsequent induction of EAT. Preliminary studies suggest that one definite link between the two tolerogenic schemes is the increase in circulatory MTg level, as measured by monoclonal antibodies to
16th International Leucocyte Culture Conference, Cambridge . 45 MTg in an ELISA (2). Calculations of tlh show that tolerogenic doses of MTg (~ 100 flg i.v.) equilibrate at a rate of 50 % reduction every 3 hr with about 1 % remaining after 24 hr (alpha phase). This rapid decline is followed by sustained levels above baseline for ~ 2-3 days with a tlh of about 10 hr (beta phase). Steady infusion of TSH or TRH i.p. via osmotic pumps results in comparable MTg levels, peaking around 72 hr. We have now examined further the importance of sustaining above threshold levels of MTg for a critical interval in activating suppression by using lipopolysaccharide (LPS). LPS has been shown earlier to augment suppression when given after the injection of TSH or TRH (1), and was used in the present study in conjunction with a nontolerogenic dose of MTg or with TSH pump. LPS (20 flg i.v.) administered to female CBA/J (H-2k) mice 24 hr before a nontolerogenic dose of MTg (::5 25 flg i.v.) extended the initial clearance rate of MTg from 3 to 5 hr, thereby sustaining MTg levels above baseline for 2-3 days. After challenge 10--11 days later with MTg + adjuvant, MTg antibody titers were very low and none of the mice developed EAT. In contrast, 100 % of the mice given 20 flg MTg (non-tolerogenic) had high antibody titers and thyroid lesions. LPS given i.v. 6 hr after the implantation of TSH pump (0.25 U/d for 7 days) likewise raised MTg levels above those in mice given TSH pump alone, without altering the overall kinetics. After challenge, antibody titers were low and only about 30 % developed lesions, compared to 50 and 100 % respectively in mice given only TSH or saline pump. These data demonstrate the important role of circulatory MTg in activating suppressor mechanisms to maintain seIftolerance. Supported by grants from the National Institutes of Health, AM 30975 & AM 31827. 1. KONG, Y. M., et al. 1982. New York Acad. Sci. 392: 191. 2. LEWIS, M., A. A. GIRALDO, and Y. M. KONG. 1984. Proc. Fed. Amer. Soc. Exp. BioI. 343: In press.
Tumour Immunology Unit, Department of Zoology, University College London, Gower Street, London WC1E 6BT, and Department of Immunology, Middlesex Hospital Medical School, Tottenham Street, London, U.K.
71. Cloning of autoreactive T lymphocytes from thyroid glands with Graves' disease M. LONDEI, G. F. BOTIAZZO, and M. FELDMANN
Graves' disease, autoimmune thyrotoxicosis, is often treated by surgical removal of the gland. We have dissociated the thyroid epithelial cells (TEC) which unlike normal glands strongly express HLA-DR antigens and the lymphocytes from such glands, and by incubating the lymphocytes in IL-2 were able to clone out T cells which were activated, and expressed receptors for IL-2 in vivo. Many of the T cell clones reacted with autologous TEC, but not mismatched TEe. These cloned T cells bound strongly to TEe. Immunofluorescent analysis of these autoreactive clones indicated that they reacted with Leu 3 (T4 antigen) but not UCHT4 (T8 antigen), indicating that these are cells of the «helper» phenotype. Because these cells were induced to proliferate by autologous DR + TEC, it indicates that DR + thyroid epithelial cells may act as antigen presenting cells, demonstrating that aberrant la/DR acts as in antigen presentation. This observation was confirmed by the capacity of monoclonal antibodies to DR to inhibit the proliferation of auto reactive T cell clones to TEe. As well as autoreactive T cell clones recognizing TEe, there were T cell clones which recognized autologous but not mismatched PBL, thus presumably representing the autologous mixed lymphocyte response cells that have been speculated to be of import once in the genesis of autoimmunity.
46 . 16th International Leucocyte Culture Conference, Cambridge Tumour Immunology Unit, Department of Zoology, University College London, Gower Street, London WC1E 6BT, and Department of Immunology, Middlesex Hospital Medical School, Tottenham Street, London, U.K.
72. Thyroid epithelial cells can present antigen M. LONDEI,
J. R.
LAMB, G. F. BOTIAZZO, and M. FELDMANN
While it is now well established that MHC Class II molecules (Ia or HLA-DR) are essential for antigen presentation in the induction of immune responses, it is not clear whether all cells capable of expressing MHC Class II determinants can function as antigen presenting cells in the appropriate circumstances. It has become apparent that during autoimmune responses, there is aberrant expression of DR antigens, for example in Graves' thyroiditis. We have speculated that this aberrant expression could permit these cells to act as antigen presenting cells, and so by presenting their cell surface autoantigens, initiate and perpetuate the autoimmune process. This hypothesis was tested using Influenza A haemagglutin specific human T cell clones, which can be triggered by either Influenza A virus, or synthetic peptides derived from the haemagglutinin sequence in conjunction with HLA-DC determinants. Thyroids of Graves' disease patients, expressing MHC Class II removed at operation, from donors whose blood cells present to the existing clones, were used as antigen presenting cells in a T cell proliferation assay, using peptide or killed virus. A strong response to the peptide, but none to the whole virus was obtained. This is unlike the results obtained with E- cells (B cells and monocytes) from the same donors, and indicates that the antigen presentation of the thyroid suspension must have been by the thyroid epithelial cells. This was confirmed by the lack of other possible antigen presenting cells, monocytes and B cells, and the role of the response and its abrogation by anti DC but not anti DR monoclonals.
Imperial Cancer Research Fund, Tumour Immunology Unit, University College London, London, U.K.
73. Suppressor T cells to liver F antigen M. L. LUKIC, A. RODRIGEZ, and N. A. MrrCHISON
Liver specific glycoprotein F offers a model to analyse the role of different cellular mechanisms in preveting T cell reactivity to autoantigens. It occurs in mice in two allelic forms: type 1 (F.l) and type 2 (F.2). Mice are unresponsive to their own F type but respond to immunization with allogeneic F by producing antibodies which cross-react with self-type F. Natural tolerance to F is found to be due to a repertoire purging mechanisms at the T cell level. However, the abolition of T suppressor cell induction enhances the response to allo F. Suppressor T cells are also responsible for non-MHC linked low responsiveness to allo F. We therefore attempted to develop an in vitro system in which suppressor cell activity induced by F antigen could be studied. Draining lymph node T cells obtained from CBA mice (F.l type) immunized with CBA or BlO.S (F.2 type) liver homogenate in CFA were stimulated in vitro with 1-20 !lg of F.l or F.2 obtained by single-step immunosorbent purification as described elsewhere (Wedderburn et al., submitted). Spleen adherent cells from untreated CBA mice were used as antigen presenting cells. Lymph node T cells obtained from CBA mice immunized with F.2 type liver extract responded in the dose-dependent fashion to
16th International Leucocyte Culture Conference, Cambridge . 47 immunopurified F.2. T cells from mice primed with self-type, F.1 did not respond to the in vitro stimulation with either F.1 or F.2. We then studied the effect of spleen T cells obtained from CBA mice 7 days after i.v. injection of 2.5-3.5 mg soluble F.1 or F.2 on the responses of primed lymph node T cells to F.2. It was found that spleen cells from mice pretreated with soluble F.2 induced profound suppression of the proliferative response. Spleen cells from mice injected with self-type F (F.1) also exhibited a suppressive effect on the proliferative response to allo F (F.2). However, the degree of suppression was always lower than the one induced by spleen cells treated with allo F. The addition of spleen T cells from mice treated with saline or BGG did not induce any suppressive effect indicating that observed suppression was F antigen specific. The results are compatible with the notion that self F specific suppressor T cells are not eliminated known to be operative at the T cell level.
Louisiana State University and Tulane University Medical Centers, New Orleans, Louisiana, U.S.A.
74. Immune abnormalities associated with HLA-B8 in healthy subjects CANDACE C. MCCOMBS,
J. P. MICHALSKI, R.
D. DESHAZO, and B. BOZELKA
The antigen HLA-B8 occurs with increased frequency in patients with at least nine diseases having a known or suspected autoimmune component, suggesting that a gene in linkage disequilibrium with HLA-B8 predisposes to the development of autoimmunity. We have observed that many healthy young adults with HLA-B8 have alterations of immune functions when compared to sex and age matched controls without HLA-B8. These alterations include decreased T cell proliferation in response to suboptimal concentrations of mitogens, increased pokeweed mitogen driven immunoglobulin synthesis, and decreased Con A induced suppression. With regard to T cell proliferation, 9 out of 15 HLA-B8 positive subjects incorporated less than 2,500 cpm in response to 0.1 !lg/ml of PHA, compared to only lout of 15 subjects without HLA-B8. The mean response of the subjects with HLA-B8 was 1,919 cpm, significantly less than the response (6,384 cpm) of the subjects without HLA-B8 (p < 0.007). Similarly decreased proliferation was also apparent in response to a suboptimally stimulating concentration of Con A, but not to optimally stimulating concentrations of either mitogen. In additional studies, we examined B lymphocyte function in subjects with and without HLA-B8 by measuring pokeweed mitogen driven differentiation of B cells to IgM bearing blasts. We found that nine subjects with HLA-B8 had a mean of 83,426 IgM positive blasts per culture compared to 27,713 for nine subjects without HLA-B8 (p = 0.0013). Con A induced suppression of immunoglobulin synthesis was also examined, using a one-step method in which Con A is added, along with pokeweed mitogen, at the initiation of culture. Subjects with HLA-B8 were found to have decreased Con A induced suppression in this assay (p = 0.007). To further analyze the cellular basis of these immunological abnormalities, lymphocyte subsets were enumerated using monoclonal antibodies and a fluorescence activated cell sorter. B cells staining for surface IgM were increased in twelve subjects with HLA-B8 (9.6 % ± 1.2) compared to twelve subjects without HLA-B8 (6.1 % ± 0.7, p = 0.013). OKT4 positive cells (helper cells) were increased in the group with HLA-B8 (41.2% vs 36.9%), while OKT8 positive cells (suppressor/cytotoxic cells) were decreased (19.8% vs 23.1 %). When the OKT4/0KT8 ratio was calculated for each subject, the mean ratio of the subjects with HLAB8 (2.49) was significantly higher (p = 0.0078) than the ratio of the subjects without this antigen (1.69). These alterations of lymphocyte subsets and immune responsiveness may contribute to the predisposition to autoimmunity associated with HLA-B8.
48 . 16th International Leucocyte Culture Conference, Cambridge Louisiana State University Medical Center, New Orelans, Louisiana, U.S.A.
75. Interleukin 2 production by PHA stimulated lymphocytes from subjects with HLA-B8
J.
P. MICHALSKI and CANDACE C. MCCOMBS
The histocompatibility antigen HLA-B8 is associated with an increased frequency of immune mediated diseases, including myasthenia gravis, Graves' disease, Sjogren's syndrome and systemic lupus erythematosus. Several altered immunologic functions occur in normal subjects with HLA-B8, which may reflect the action of a gene or genes, linked to HLA-B8, which contributes to autoimmunity. These abnormalities include impaired response to suboptimal concentrations of PHA or Con A, increased spontaneous or pokeweed mitogen driven immunoglobulin production and decreased Con A induced suppressor activity. Because cellular interactions are involved in these in-vitro immune functions, we have begun a study of interleukin production and response, to probe the mechanism of altered lymphocyte function in subjects with HLA-B8. In a preliminary study of 6 subjects with HLA-B8 and 6 individuals without this antigen, we cultured peripheral blood mononuclear cells with several concentrations of PHA and harvested supernatants after 24 and 48 hours. Interleukin 2 was assayed by a microassay utilizing an IL-2 dependent murine cytotoxic T cell line. In most subjects, supernatant IL-2 stimulated by an optimal concentration of PHA peaked at 24 hours whereas suboptimally stimulated cultures reached peak levels at 48 hours. IL-2 concentrations in optimally stimulated cultures generally ranged from 5-13 units/ml whereas levels with suboptimal PHA concentrations were 10-20 times lower. At 48 hours of culture,S of 6 subjects with HLA-B8 had lower IL-2 production by sub optimally stimulated mononuclear cells than age matched controls without HLA-B8. IL-2 in optimally stimulated cultures was comparable for the two groups. Exogenous IL-2 was added to PHA stimulated cultures of 2 subjects with HLA-B8 who had very low proliferative responses. The impaired suboptimal PHA response improved significantly compared with relatively little change in two individuals with a normal response and who lacked HLA-B8. Our preliminary data suggest that levels of IL-2 in PHA stimulated cultures correlate with subsequent incorporation of tritiated thymidine. Subjects with HLA-B8 produce smaller amounts of IL-2 after stimulation with a suboptimal concentration of PHA. In two subjects, impaired proliferative responsiveness was at least partially corrected by exogenous IL-2, demonstrating a capacity to respond to this substance. Impaired production of IL-2 may contribute to altered immunity associated with HLA-B8.
University of California, Los Angeles, California, U.S.A.
76. 112 response and production in multiple sclerosis patients
J.
E. MERRILL, C. MOHLSTROM, V. KERMANI, G. W. ELLISON, and L. W. MYERS
In an effort to analyze the defective proliferative response to antigens and mitogens by T lymphocytes in multiple sclerosis (MS) patients, we examined the interleukin 2 (IL2) response and production by peripheral blood lymphocytes (PBL) and cerebrospinal fluid (CSF) mononuclear cells. For IL2 response experiments, nylon wool passed (NWP) PBL were used. Endogenous IL2 production was prevented by 1O- 6 M Dexamethasone and response was measured to exogenous lectin-free IL2. Cells were stimulated with 10 Itg/ml PHA-P and 10 % IL2. Chronic progressive MS patients had an abnormally low response to exogenous IL2 compared to controls. While acute relapse patients' PBL demonstrated a normal IL2 response
16th International Leucocyte Culture Conference, Cambridge· 49 during exacerbation, they showed reduced responsiveness during remission. These abnormalities could not be explained by different dose or kinetic response optima to PHA or III nor could they be explained by abnormal numbers of III or transferrin receptor-bearing lymphocytes. In contrast, CSF cells contained normal numbers of IL2 receptor-bearing lymphocytes but depressed numbers of transferrin receptor-bearing cells which might explain their reduced IL2 response compared to homologous PBL. Production of IL2 was measured from supernatants of preNW and NWP cells. NWP cells made 8-10 fold more III than preNW cells and thus were used in all experiments. Production of IL2 was elevated in chronic progressive patients and acute relapse patients during remission. IL2 levels were diminished during exacerbation. Removal of NW adherent cells and 1200 rad x-irradiation of the NWP lymphocytes augmented IL2 production to a 3 fold greater extent in MS patients than controls indicating the likelihood that both macrophages and suppressor T cells play a role in the regulation of IL2 production in multiple sclerosis. The inability of MS patients' lymphocytes to cion ally expand in response to IL2 may contribute to the pathogenicity of this disease. Supported by grants from the MS Society-JF 2012-A-3 and the NIH-NS08711 and NS 19145.
Corporate Bioscience Group - Cell Biology Section, Imperial Chemical Industries PLC, Central Toxicology Laboratory, Alderley Park, Nr. Macclesfield, Cheshire, SKI0 4TJ, U.S.A.
77. In vivo modulation of anti-erythrocyte autoantibodies by T cell lines PATRICIA E. RIDDELL, D. G. KELLY, A.
J. ALDRIDGE, R. T. ROBSON, and E. J.
CULBERT
CBA mice immunised with rat erythrocytes (RBC) produce an anti-RBC autoantibody response which is preferentially suppressed on rechallenge with rat RBC (1). Monoclonal autoantibodies bind to a cross-reactive determinant(s) shared between rat and mouse RBC, suggesting that the autoantibodies are a normal component of the anti-rat RBC response. To investigate the specificity of helper T cells regulating the autoantibody response, we have produced T cell lines reactive with rat RBC. These lines proliferate in response to rat RBC, but not mouse RBC, and augment autoantibody production in mice immunised with rat RBC. Injection of the rat RBC-reactive lines in the absence of rat RBC does not result in autoantibody production. These results demonstrate that the autoantibody response in this model can be regulated by rat RBC-specific helper T cells, and suggest that helper T cells reactive with autologous RBC do not operate in this model. The implications of these findings for possible mechanisms of specific suppression of autoantibodies will be discussed. 1. COOKE, A., P. R. HUTCHINGS, and J. H. L. PLAYFAIR. 1978. Suppressor T cell in experimental autoimmune haemolytic anaemia. Nature 273: 154.
Wayne State University School of Medicine, Detroit, Michigan, 48201, U.S.A.
78. T cell recognition of unique and shared determinants of auto antigenic mouse thyroglobulin in experimental autoimmune thyroiditis (EAT) L. L. SIMON and Y. M. KONG
Weare studying the antigenic determinants recognized by T and B lymphocytes from mice of a thyroiditis-susceptible strain following immunization and/or tolerization with homolo-
50 . 16th International Leucocyte Culture Conference, Cambridge gous (mouse) and heterologous (human) thyroglobulin (Tg). High responder mice immunized with 100 Itg mouse Tg (MTg) in complete Freund's adjuvant (CFA) develop autoantibodies to MTg and EAT characterized by mononuclear infiltration. On days 8-14 following immunization, their popliteal lymph node cells (LNC) proliferate in vitro in response to MTg, and also to human Tg (HTg) (1). Our current findings indicate that a minor portion of the autoantibodies produced after MTg-immunization reacts with HTg in passive hemagglutination but the major portion is directed against MTg-specific rather than shared determinants. LNC from mice immunized with heterologous HTg proliferate strongly in vitro to HTg and to a much lesser degree to MTg. After HTg-immunization, the antibody response is primarily directed against HTg-specific determinants but antibodies to shared determinants are also produced. To study the contribution of mouse-specific and shared determinants in T cell proliferation, reciprocal Tg's are used for cross tolerization and immunization. Previous studies have shown that suppression of EAT induction and autoantibody production can be achieved in high responder mice by the injection of soluble MTg (200 ltg, i.v.) on days 10 and 3 prior to immunization and their LNC do not proliferate in response to MTg. We have now observed that comparable treatment with soluble HTg before immunization with HTg and CFA dramatically reduces the proliferative response to HTg. MTg-induced tolerance followed by immunization with either MTg or HTg in CFA eliminates the proliferative response to MTg, but only partially reduces the response to HTg after HTg-CFA immunization. Interestingly, treatment with aqueous HTg prior to immunization with MTg-CFA greatly reduces the proliferative response to MTg. These results indicate that the proliferative response to MTg in high responder mice is readily elicited against determinants shared with HTg and suppressed by pretreatment with soluble HTg. Using tolerance induction to functionally delete T cell subsets reactive with epitopes on MTg and HTg will make it possible to study the relative roles of these epitopes and T cell subsets in autoantibody production and EAT induction. In addition, Tg-specific T cell lines and clones reactive with unique and shared determinants on the auto antigen MTg can be developed. Supported by National Institutes of Health AM 31827 and AM 30975.
1. SIMON et aI., 1984. Proc. Fed. Amer. Soc. Exp. BioI. 43: in press.
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, U.S.A.
79. Determinant specificity of the immune response to insulin in man
J. W. THOMAS, VALERIE J. VIRTA, and LAURA J. NELL Insulin has been used as a model antigen in studies of the fine specificity of immune response (Ir) gene function in inbred animals. Those studies revealed that both the appropriate MHClinked gene product and unique amino acid exchanges between the immunizing insulin and the animals' endogenous insulin were critical for initiation of the immune response to this antigen. We have used a similar approach in the outbred human population to identify the determinants on the insulin molecule recognized by T cells from insulin-treated diabetics. Two patterns of antigen recognition were found in human lymphocyte responses to mammalian insulins. In one group, the differential responses to insulins from several species identified the amino acid residues necessary for antigen recognition. These residues are clustered on the surface of the molecule and include the A chain loop of beef insulin and the carboxy-terminus of the B chain of beef and pork insulin. Studies employing insulins that have additional amino acid substitutions in the A chain loop (horse and sheep) indicate that lymphocytes from different individuals may recognize this limited region of the molecule in several ways. Since the cellular
16th International Leucocyte Culture Conference, Cambridge· 51 responses of these subjects are directed at primary amino acid exchanges, they are analogous to the responses seen with inbred animals. However, primary structure alone is not always sufficient for recognition of A loop determinants indicating that conformational integrity of this region of the molecule is also important. This contrast with the animal studies is most notable in a second group of subjects whose lymphocytes do not show a differential response to various insulins. This group's lymphocytes respond to all insulins, including human. Further studies in this «autoimmune» group with isolated A and B chain peptides and with monomeric sodium insulin suggest that primary amino acid exchanges are not critical for the response. Rather, we believe that conformational determinants are created by the molecular interactions of insulin dimers and hexamers and that these determinants are recognized by immune T cells. This conclusion is further supported by the observation that diabetics treated with human insulins develop anti-insulin antibodies. Our studies demonstrate that despite the complexity of an outbred population antigenic determinants that are recognized by man can be identified when a highly defined antigen is employed. In addition, this approach reveals patterns of antigen recognition that were not previously demonstrated when the same antigen was used in inbred animals.
Laboratory of Immunology NIAID, NIH, Bethesda, MD 20205 U.S.A.
80. Monoclonal autoantibodies from normal mice P.
WASSMER
Autoantibodies reacting with different tissues are commonly found in the sera of patients with autoimmune disorders, but are also present at a low frequency in healthy individuals. The mechanisms of appearance and the pathological roles of these self-reactive antibodies are not yet clear. We report here the production of monoclonal autoantibodies from non-immunized healthy mice. Antibody-secreting hybridomas were prepared by fusing spleen cells from normal mice with P3-653Ag8 myeloma cells. Hybridomas producing immunoglobulins as determined by ELISA were screened for reactivity with tissue sections by indirect immunofluorescence. Up to 10 % of hybridomas secreting immunoglobulins produced antibodies reacting with islets of Langerhans, anterior pituitary, gastric mucosa, smooth muscle, salivary gland or cellular nuclei from various tissues. This finding suggests that non autoimmune individuals without detectable serum autoantibodies may possess a number of antibody secreting B-Iymphocytes with affinity for self antigens.