Abstracts
Eur. surg. Res. 11: 205-216 (1979)
10th Round Table Symposium on Applied Immunology1 Axams/Tirol, January 19-23, 1979 Organized by W. Brendel and K. Messmer, Munich
Ultramicrotechmques for the Quantitative Measurement of Enzyme Activities and Membrane-Bound Antigens in Single Cells: Synthesis of Ig Antigenic Determinants in T as well as B Cells
P. Hosli, S. Avrameas, M. Stanislavski, A. Ullmann, M. Rodrigot and E. Vogt
Abstracts are listed in order of presentation.
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The combined use of specially developed cell culture techniques (plastic film dish, PFD) (1) and biochemical ultramicromethods (parafilms micro cuvette, PMC) (2) permits one to routinely and quantitatively measure enzyme activities of single visually selected cells, making use of either fluorogenic or radioactive substrates. Based on these techniques, an enzyme immunoassay for quantitation at the single cell level of immunoglobulin antigenic determinants, present on the membrane of murine lymphocytes (Mlg), has been developed (3). This immunoassay makes use of specific Fab: anti-Ig, anti-K, anti-u and anti-7 conjugated to E. coli (J-galactosidase combined with the micromanipulation of lymphocytes and the measurement of the coupled (3-galactosidase activity in the PMC with the fluorogenic substate 4-methylumbellifcryl-i5-£>-galactopyranoside. With this technique Mlg could be measured in substantial amounts on the surface of almost all splenic and lymph node lymphocytes examined, but a larger proportion of cells with higher amounts of Mlg were found in the spleen than in the lymph nodes. However, when the same Fabs were used to localize Mlg by conventional immunohistochemical staining, only a fraction of cells corresponding to published values could be scored as positive. The fact that Mlg is associated with the vast majority of lymphoid cells implies that T cells carry such antigens. In order to investigate this further, experiments were performed on splenic B- and T-cell fractions obtained using either an anti-lg immunoadsorbent or after treatment with anti-0 serum plus complement. B-cell fractions possessed greater amounts of Mlg (1,500-79,000) than those of T-cell fractions (0-39,000), but on the average B cells carried only about 2.5 times more u-. * and 7-antigenic determinants than T cells. Immunoadsorbent column-fractionated T cells, from which Mlg was removed by anti-mouse Ig stripping, were able to regenerate Mlg after 24 h of culturing. As the
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quantities reexpressed equaled those found on T cells before anti-lg treatment, the present results strongly suggest that T cells synthesize and express MIg. Irrespective of the molecular nature of the T-cell structure detected by the present study, it is hypothesized that the difficulty in detecting MIg by standard immunochemicalstaining methods may reflect differences in the membrane composition of B and T cells. The local concentration of MIg could be higher or the microredistribution of MIg could be achieved more easily on B than on T cells. The credit for the immunological part of these studies belongs to S. Avrameas, M. Stanislawski and M. Y. Rodrigot (cf. 3, 4). References
1 2 3
4
Hôsli, P.: Tissue cultivation on plastic films (Tecnomara, Ziirich 1972). Hôsli, P.: Quantitative assays of enzyme activity in single cells: early prenatal diagnosis of genetic disorders. Clin. Chem. 23: 1476 (1977). Hôsli, P.; Avrameas, S.; Ullmann, A.; Vogt, E., and Rodrigot, M.Y.: Quantitative ultramicro-scalc immunoenzymic method for measuring Ig antigenic determinants in single cells. Clin. Chem. 24: 1325 (1978). Avrameas, S.; Hôsli, P.; Stanislawski, M.; Rodrigot, M.Y., and Vogt, E.: A quantitative study at the single cell level of immunoglobulin antigenic determinants present on the surface of murine B and T lymphocytes. J. Immun, (in press). Address: Institut Pasteur, Département de Biologie moléculaire, Unité de Cytogénétique expérimentale, 25, rue du Docteur-Roux, F -75724 Paris Cédex 15 (France)
T-Cell Receptors and T-Cell Antigens
H. Binz, H. Frischknecht, A. Kimura and H. Wigzell
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Subsets of T cells exist with specific functions and unique surface properties. An analysis of the surface glycoproteins found on functionally distinct T blasts has revealed several unique properties as studied with alloantisera or lectins. In this regard it has been found that a particular glycoprotein appears on the cytotoxic T cells of murine or of human origin which is not found on other T cells or cytotoxic cells of non-T type. A similar uniqueness is found on the antigen-binding receptors of T cells belonging, in the mouse, to the s.c. Lyt subsets. Here it could be shown that Lyt-I*2~ T cells and Lyt-1 ~V T cells display distinct, largely non-overlapping idiotypic receptors when tested in anti-MHC' reac tions. Elimination of the former cells would cause a highly significant reduction of the MLC response (now inducing the elimination by anti-idiotypic antisera against the relevant anti-MHC T blasts with the corresponding Lyt-phenotype) whereas killing the idiotypic T cells with anti-idiotypic sera absorbed with Lyt-1 ' V cells would leave the killing properties but not the proliferative properties intact. In the MHC system it is also clear that anti-idiotypic antibodies may functionally mimic the allo-MHC structures and induce specific cytotoxic T cells with the correct allo-MHC specificity even in the absence of ‘helper’ T cells of the Lyt-1 *2' phenotype. The latter finding may be taken as an indication
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that proliferating helper T cells even in allo-MHC situations can function via auto-anti-idiotypic reactions. Address: Department of Immunology, Uppsala University, Box 782, S 751 23 Uppsala (Sweden) Newer Concepts of T-Lymphocyte Responses to Alloantigens
F.H Bach Responses of T lymphocytes can be studied from two perspectives: first, the nature of the antigens that evoke reactions and second, the classification of the cell types involved. Although much of our understanding of cell-mediated immunity in these respects has come from in vitro studies of T-lymphocy te response to major histocompatibility complex-encoded alloantigens, recent work has extended our concepts to reactions to 'altered-self antigens. The basic dogma, as it developed, stated that there are at least two types of MHC-encoded alloantigens (LD and CD) that preferentially evoke responses in two functionally disparate cell types: Lyt 1* T helper (Th) and Lyt 2* cytotoxic T (Tc) lymphocytes, respectively. Further, that altered-self antigens, such as TNP-modified syngeneic cells or syngeneic tumor cells evoke a response that involves a Lyt 1,2* cell that is not involved in the ordinary response to alloantigens. We and others have obtained results that suggest that this model may be an oversimpli fication in that a Lyt 1,2* cell can also be involved in the response to alloantigens. Our data arc consistent with the possibility that there are two alternative pathways of T-lymphocyte activation: one involving the Lyt 1,2* cell the other not. Which pathway will predominate in any given reaction may depend either on the overall strength of the reaction or on the presence of LD antigens on the stimulating cells. Further, the two pathways may be differentially responsive to augmentation or suppression thus providing an additional mecha nism by which T-lymphocyte reactivity can be regulated. We would hypothesize that these two pathways of T-lymphocyte activation may represent the cell-mediated analogue of the primary and secondary B-lymphocyte response. To the extent that the presence and strength of the LD stimulus controls which pathway will be the strongest in any response, these findings may help to explain the accumulating evidence that matching for LD correlates with improved renal allograft survival. The large number of different T-lymphocyte classes that can be involved in these responses forces careful comparison of results such as those discussed above with our present understanding of T-lymphocyte receptors and control of T-lymphocyte reactivity by anti-idiotypic sera. Address: Immunobiology Research Center, Madison, Wise. (USA) Immunological Reactivity in Patients with Colorectal Cancer
P.J. Morris and P. Grantly Gill
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The survival of patients with colorectal cancer is poor, the crude 5-year survival in Oxford being 25.8% for all pathological stages and 37.3% after a curative resection. Many
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factors have been shown to influence survival in 335 patients treated in Oxford between 1966 and 1971, the most significant of which were the pathological stage of the tumor, the age of the patient, the clinical presentation, and the type of resection performed (1). However, recent studies of smaller groups of patients suggest that the patients’ immunologi cal reactivity might also determine survival and metastasis. Firstly, a significant correlation between lymphocyte and macrophage infiltration (but not plasma cells) at the tumor edge and both survival and metastatic spread was noted, but no such correlation with cellular infiltration within the tumor stroma was seen. Secondly a hemolytic assay of peripheral blood monocyte function (using sensitized A1 red cells as targets) in patients with colorectal cancer showed a significant decrease in activity (2). Thirdly diminished DNCB reactivity has been shown in patients with colorectal cancer by Bolton et al. (3). These findings were felt to justify a controlled trial of immunotherapy with C. parvum, a potent stimulant of macrophage activity, given over a 2-year period. There were 2 deaths and 4 patients with recurrent disease in the treatment group compared to 2 deaths and 8 recurrences in the non-treatment group. A significant increase in monocyte numbers can be demonstrated in blood 7 days after C. parvum infusion. No conclusions can be drawn about the value of this method of immunotherapy for colorectal cancer at this time. References
1 2 3
Gill, P.G. and Morris, P.J.: Br. J. Surg. 65: 17 (1978). Gill, P.G.; Waller, C.A., and Maclennan, I.C.M.: Immunology 33: 873 (1977). Bolton, P.B., et al.: Br. med. J. Hi: 18 (197S). Address: Nuffield Department of Surgery, University of Oxford, Oxford (UK)
H-2 Control of Anti-H-Y T-Cell Responses
Elizabeth Simpson, T. Matsunaga. C. Hetherington, M. Hurme, Mary Brenan and P.R. Chandler
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Females of certain inbred strains of mice can reject syngeneic male skin and can generate H-2 restricted cytotoxic T-cell responses to appropriate male cells (1). The male specific antigen, H-Y, is a weak transplantation antigen coded by a gene(s) on the Y chromosome, and the responses to it have been used as models for responses to tumour antigens, which in many ways act as weak transplantation antigens. Immune response (Ir) genes mapping in the H-21 region determine the ability of mice to make both the anti-H-Y skin graft response and the anti-H-Y cytotoxic T-cell response. However, each of these responses is controlled by genes mapping in different subregions of H-21: in H-2b mice the gene(s) enabling graft rejection are in IBb whilst those allowing cytotoxic responses map in lAb (2, 3); the H-2b genes are dominant (1). In H-2d mice there is a dominant ld gene which allows cytotoxic responses, but not graft rejection (4). A variety of other dependent haplotypes have complementary I-region Ir genes whose activity is shown in appropriate FI mice which make cytotoxic T-cell responses to H-Y, although neither of the parental strains do, at least some of these complementary genes map in the 1C
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subregion (2). Skin graft rejection is not seen in FI hybrids showing complementary anti-H-Y cytotoxic T-cell responses. The separation of the control of skin graft rejection and cytotoxic responses suggest that graft rejection may not be mediated by cytotoxic cells per se. The cytotoxic T-cell responses are also dependent on appropriate K/D associative antigens. The target cell specificity of these responses is such as to suggest that the target antigen consists of two closely associated components, self-H-2K or D plus H-Y. The molecular organisation of this association is not understood but there is clearly a hierarchy of association suggesting that certain K/D molecules ‘fit’ better with H-Y than others, e.g. Kk > Db > Dk > Kd and that certain of them, e.g. Kb, Dd, Ks, Kq, are completely non-associative. Recombinant strains such as B10A (5R) (bbbddd) with permissive Ir genes (lAb) but non-associative K/D antigens (Kb’ Dd) are non-responders, indicating the strict limitation put on responsiveness by appropriate associative self-antigens. It seems likely that K/D antigens are associative for Te cells, whilst 1 antigens are associative for T^ cells. We have recently suggested that Ir genes function by producing associative la antigens such that ‘Ir gene’ products and ‘restrictive’ I-region products are identical and determine activation of T[4 cells necessary for differentiation of T(j cells (4). This novel way of looking at Ir-gene function could, we believe, be equally well applied to Ir-genc control of T-helper and T-suppressor function in antibody responses. The significance of this work in applied immunology is that MHC antigens may well control the extent and effectiveness of responses to all antigens and the manner in which this control is exercised is important to understand, if we are to manipulate the immune response in controlling graft rejection, the growth of tumours, and autoimmune diseases. T-cell responses of mice to H-Y may be excellent models, because all aspects appear to be controlled by MHC genes, and many of these have been mapped both for Tc (cytotoxic cells) and the putative and probably mandatory T^ (T helper) cells. References
1 2 3 4
Simpson, E. and Gordon, R.D.: Immunol. Rev. 35: 59 (1977). Hurme, M.; Hetherington, C.M.; Chandler, P.R., and Simpson, E.: J. exp. Med. 147: 758 (1978). Hurme, M.; Chandler, P.R.; Hetherington, C.M., and Simpson, E.: J. exp. Med. 147: 768 (1978). Matsunaga, T. and Simpson, E.: Proc. natn. Acad. Sci. (in press, 1978). Address: Clinical Research Centre, Division of Transplantation Biology, Watford Road, Harrow, Middlesex HA1 3UJ (UK)
Induction and Effect of Suppressor Cells
H. Wagner
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Mainly due to Ly differentiation antigens, the regulatory influences of interactions amongst murine lymphocyte subpopulations on B-cell-mediated antibody responses has been worked out with precision. For example the obligatory helper T cell expresses the Ly 1
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cell marker, which contrasts to the Ly 2.3 positive T suppressor cell. Recently Tada and associates have described two amplifying systems. In the helper amplification system, activated Ly 1 cells interact with Ly 123 cells to increase the activity of the Ly 1 cell (positive loop). A similar system is described for suppression (negative loop). A T cell with the phenotype of a suppressor cell (Ly 23) makes a product which acts on Ly 123 cell, which then increases the amount of suppression via Ly 2.3 cells. Opposed to the Tada loops, the work of Gershon and Cantor indicates the existence of feedback suppression. In this instance Ly 1 cells act on Ly 123 cells. However, instead of producing more Ly 1 activity, this interaction leads to Ly 23 T-cell-mediated suppression. Observations showing that T cells can suppress the induction of T-cell immune responses have been reported only recently. Accordingly T suppressor cells can suppress in an antigen-specific or nonspecific fashion the in vitro generation of cytotoxic T lympho cytes (1, 2). We have studied the nature and mechanism of action of antigen-specific suppressor T cells obtained either in vivo or in vitro. The results show that suppressor T cells and cytotoxic T cells arc different. References
1 2
Wagner, H.; Starzinski-Powitz, A.; Pfizenmaier, K., and Röllinghoff, M.: Eur. J. Immunol. 6: 873 (1976). Al-Adra, A.R. and Pilarski, L.M.: Eur. J. Immunol. 8: 504 (1978). Address: Institut für Medizinische Mikrobiologie der Universität, Hochhaus Augustusplatz, Mainz (FRG)
Influence of ALG on Suppressor Lymphocytes in vitro (Cultures) and in vivo (Kidney Grafts)
Anti-lymphocyte globulin (ALG) is a potent immunosuppressive drug, widely used to reduce the frequency and severity of graft rejection. Its main effects are the impairment of the antibody response, but also the destruction of lymphocytes, especially those of the T-celt population. However, suppressor cells, supposed to play a beneficial role in protecting a graft from rejection, are found mainly in this T-lymphocyte fraction. It was therefore the aim of the study to monitor the fluctuations of lymphocyte populations with respect to the suppressive activity in vivo and in vitro. 35 kidney recipients received a combination of 5 mg/kg body weight azathioprine, 100 mg prednisolone and 20 mg protein of antilymphocyte globulin/kg body weight over a period of 10 days after transplantation. A steady reduction reached a basic therapy of 2.5 mg azathioprine and 15 mg cortisone after 8 months. White blood cell counts, shifting of T + B cells in the peripheral blood, circulating levels of lymphocytes which bind ALG to their surface were monitored in 3-day intervals. The effect of this immunosuppressive therapy and the action of ALG on suppressor cells was measured in an in vitro assay with patients’ peripheral lymphocytes and cells from healthy donors. The suppressive action of spontaneous and concanavalin-A-activated circulating suppressor cells on mixed lymphocyte
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C. Hammer, W. Bullinger, W. Land and W. Brendel
211
Abstracts
cultures and mitogen-stimulated lymphocytes was recorded preoperatively under immuno suppressive therapy with and without ALG and during rejection episodes. The influence of various concentrations of ALG on lymphocytes of healthy donors was measured with MLC 'specific' suppressor cells in MLC and mitogen stimulation. Suppressive activity of lymphocytes of kidney-transplant patients (%) Pre-op.
No rejection
Rejection no ALG treatment
ALG treatment
ALG treatment
no ALG treatment
MLC/spontaneous Lys MLC/activated Lys
24 41
+ 134 13
+ 130 19
+ 117 4
31 59
PHA/spontaneous Lys PHA/activated Lys
29 39
14 50
+ 132 27
21 30
46 54
Results: After transplantation and the start of immunosuppressive therapy, especially under ALG, lymphocyte counts dropped to 40% of the initial values. As B cells remained relatively constant, the T-cell population reflects the major reduction. ALG-positivc circulat ing lymphocytes decreased from 70% preoperatively to 30% after 7 days of ALG therapy. At the same time values of spontaneous and activated suppressor cells are reduced. In most patients the suppression of MLC and PHA response shifts to stimulation. Patients with rejection crises treated or untreated with ALG show a similar behavior. As in vivo, lymphocytes incubated with ALG and rabbit complement for 1 h in vitro loose their MLC-specific and unspecific suppressor activity with increasing ALG concentrations. The results indicate that immunosuppressive therapy in combination with antilymphocyte globu lin hits cytotoxic lymphocytes as well as suppressor cells in patients. It seems, however, that the suppressive activity recovers faster within hours or Jays than does the cytotoxic cell population. References
2
3 4
Bullinger, W.; Hammer, C.; Land, W., and Welter, H.: Arch. klin. Chir., Chir. Forum, 320, suppl. (1979). Hammer, C.; Jerry, L.M., and Lewis, M.G.: Suppressor cell activity of peripheral blood and lymph node lymphocytes in cancer patients; in Rainer, Immunotherapy of malignat diseases, p. 12 (Schattauer, Stuttgart 1978). Thomas, F.; Lee, H.; Wolf, J.S.; Mendez-Pican, G., and Thomas, J.: Surgery, St Louis 79; 408 (1976). Thomas, J.; Thomas, F.; Mendez-Pican, G., and Lee, H.: Surgery, St Louis 87: 125 (1977). Address: Institut fiir Chirurgische Forschung im Klinikum Grosshadern, Marchioninistrasse 15, D 8000 München 70 (FRG) Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 7:51:28 PM
1
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Role of Suppressor Cells in Autoimmune Diseases
R. Boyd and G. Wick 3 years ago at the Kitzbiihcl meeting (1), we presented evidence fora multigenic basis for the development of spontaneous autoimmune thyroiditis (SAT) which occurs in chick ens of the obese strain (OS) (2). In the meantime our efforts have been aimed at the elucidation of effector and, more recently, suppressor mechanisms involved in the pathogen esis of this disease. Based on the earlier observation that a primary defect exists in the thyroids of OS birds our present working hypothesis postulates that thyroglobulin-autoantibodies (Tg-AAb), vertically transferred from the mother hen via the egg yolk into the embryo, is the first pathogenetic stimulus followed by the influx of Tg-binding B cells. The latter cells invade the thyroid epithelial lining (periopolesis) and differentiate into plasma cells which produce Tg-AAb in situ and lead to further destruction. In addition recent results speak for a participation of antibody-dependent cellular cytotoxicity (ADCC) in the pathogenesis of SAT. In contrast to experimentally induced autoimmune diseases neonatal thymectomy increases the frequency and severity of SAT in the OS and also entails the formation of AAb to non-thyroid antigens. Furthermore, OS chickens generally display a heightened immune response to foreign antigens. These data led to the assumption that the underlying immuno logic defect in OS chickens is a quantitative or qualitative defect of suppressor cells. To investigate this possibility further, the ability of OS thymus suspensions to suppress the in vitro ADCC or direct cytotoxicity of autologous blood leucocytes to thyroglobulin-coated chicken erythrocyte targets, was studied. A decrease in ADCC reactivity was taken as a parameter for the suppressive activity of the added cell suspension. Preliminary results suggest that it is not a defect of suppressor cells, but rather a defect of target cells for suppression which underlies the development of SAT in the OS. This work was supported by a grant of the Austrian Research Council (project No. 3120). References
1 2
Wick, G.: 6th Round Table Symposium on Applied Immunology, Kitzbiihel 1975. Wick, G.; Sundick, R.S., and Albini, B.: Clin. Immunobiol. Immunopathol. 3: 272 300 (1974). Address: Institute for General and Experimental Pathology, University of Innsbruck, Fritz-Pregl-Strasse 3, Innsbruck (Austria)
On the Structure and Function of Transplantation Antigens
The HLA-A, -B and -C antigens are composed of two polypeptide chains. The small invariant chain, dj-microglobulin, is structurally similar to immunoglobulins. The alloantigenic, heavy chain contains two immunoglobulin-like disulfide bridges and amino acid sequence information suggests that the two HLA-A, -B and -C antigen subunits are as similar
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P.A. Peterson
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to immunoglobulin-G constant domains as are the latter to one another. An evolutionary relationship between antibodies and transplantation antigens, therefore, seems likely. That there may exist a functional similarity between the two types of molecules is indicated by the observation that an adenovirus protein, E l9, has been isolated as a physical complex with AgB antigens, the rat equivalents of the HLA-A, -B and -C antigens. Data have been obtained supporting the view that T killer cells recognize the E l 9-transplantation antigen complex. Moreover, some bacteria have been shown to bind to liposome-integrated HLA-A, -B and -C antigens. Thus, transplantation antigens may display the general property to interact with non-self-structures. The HLA-D antigens, whose structure is much less known, have been shown to occur on most if not all epithelial cells. It is particularly noteworthy that these antigens seem to be under hormonal regulation as regards the expression on mammary gland epithelium. The functional significance of the non-lymphoid HLA-B antigens will be discussed. Address: Department of Cell Research, the Wallenberg Laboratory, Box 561, S 751 22 Uppsala (Sweden)
Effects, Significance and Clinical Applicability of Total Lymphoid Irradiation
S. Slavin and S. Strober Total lymphoid irradiation (TL1) given in daily fractions of 200 rads has been used extensively during the past years to treat lymphoid malignancies in humans. It has been well established that TL1 results in long-lasting impairment of the thymus-derived (T) cell-medi ated immune responsiveness in experimental animals as well as in man (1, 2). During the past years we have examined the potential use of TLI for studying a number of clinically relevant problems related to the fields of transplantation immunobio logy and autoimmunity (table I). TLI allows for a permanent survival of bone marrow (BM) allografts (including engraftment of white and red blood cell series) in both inbred mice (1) and rats (3) as well as in outbred unrelated dogs (4) across the major histocompatibility barriers. BM recipients
Immunosuppressive therapy
Immunomanipulation
1 Adjuvant induced polyarthritis in cats(6)
1 Establishment of transplantation tolerance Bone marrow transplantation (1, 3 5) Organ transplantation (1 ,3 -5 ) 2 Tolerance induction for soluble antigens (7)
2 SLE-like disease in NZB/NZWF, mice (unpublished observations) 3 Clinical autoimmune disorders (unpublished observations)
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Table I. Experimental and clinical applicability of TLI
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develop no clinical signs of graft versus host disease ( 1 5 ) . BM recipients develop permanent and specific tolerance to donor-type alloantigens with a permanent survival of skin and heart allografts (1, 3). One-way mixed lymphocyte reactivity of recipient versus donor could be specifically blocked by spleen cells from chimeric mice. Specific tolerance to donor-type skin allografts was also adoptively transferred into normal syngeneic recipients. The data suggest that the tolerance state is due to generation of specific suppressor cells follow ing TL1. Preliminary studies indicate that TLI has a beneficial effect in the treatment of experimental autoimmune disorders including adjuvant-induced polyarthritis in rats (6) as well as rheumatoid arthritis (unpublished observations). The immunosuppressive effects of TLI and the potential generation of suppressor cells following TLI indicate that further investigations should be carried out with regard to applicability of TLI to clinical BM and organ transplantation as well as for the treatment of autoimmune disorders. References
1 2 3 4 5 6 7
Slavin, S., et al.: J. exp. Med. 146: 34 (1977). Funks, Z., et al.: J. clin. Invest. 58: 803 (1976). Slavin. S., et al.: J. exp. Med. 147: 700 (1978). Slavin, S., et al.: Transplantation (in press). Slavin, S„ et al.: J. exp. Med. 147: 963 (1978). Schurman, D., et al.: Transactions Orth. Res. Soc. 3: 38 (1978). Zan-Bar, I., et al.: J. Immunol. 121: 1400 (1978). Address: Department of Medicine A. Hadassah University Hospital, Jerusalem (Israel)
The Beneficial Effect of a Single Blood Transfusion before Kidney Transplantation on Allograft Survival
After much initial scepticism, the findings of Opelz and Terasaki that pretransplant blood transfusion cannot only endanger kidney graft survival but can protect it as well, have been amply confirmed and the graft protecting effect of pretransplant blood transfusion is now generally accepted (1). Animal studies especially those in rhesus monkeys with standard immunosuppression have been essential in this (2). It was generally assumed that blood transfusions induced not only the formation of cytotoxic graft destructive antibodies but also enhancing or graft-protecting antibodies. During a retrospective study van H ooff et al. (3) and Persijn et at. (4) noticed that a single blood transfusion even if given many months before transplantation had a similar graft-protecting effect. This has now been confirmed in a controlled prospective study (5). 19 patients received one blood transfusion each between I and 18 months before transplant ation. 15 out of 19 of the transplants functioned at 6 months posttransplantation. The single blood transfusion had been ‘washed’, i.e. about 90% of the buffyeoat had been removed in one centrifugation step. 8 patients received I 3 blood transfusions which had
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J.J. van Rood. G.G. Persijn, A. van I.eeuwen, E. Goulmy. B.A. Bradley
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been made buffycoat-free by passing them through a cottonwool filter® (Central Laborato ry Red Cross Bloodtransfusion Service. Amsterdam). Only 2 of the 8 transplants functioned at 6 months posttransplantation. It seems improbable that anti-DR-enhancing antibodies are responsible for the graftprotecting effect of a single transfusion. If it is assumed however that antibodies with other specificities could enhance as well, enhancement remains a realistic explanation (6 ). Another possible explanation could be the induction of CML non-responsiveness which can be found after kidney transplantation and which sometimes is caused by suppressor cells (5, 7, 8). References
1 2 3 4 5 6 7 8
Opelz, G., et al.: Transplantn Proc. 5: 253 (1973). Es, A.A. van, et al.: Lancet i: 506 ( 1977). Hooff, J.O. van, et al.: Transplantation 22: 306 (1976). Persijn. G.G., et ah: Transplantn Proc. 9: 503 (1977). Rood, J.J. van, et al.: Transplantn Proc, (in press, 1979). Jonker, M. and Rood, J.J. van: Tissue Antigens 11: 251 (1978). Thomas. J., et al.: Transplantn Proc. 9: 85 (1977). Woningeit, K. and Pichlmayr. R.: Dialysis Transplantn 6: 58 (1977). Address: Department of Immunohaematology, University Medical Center, Leiden (The Netherlands)
The Immunosuppressive Action of Cyclosporin A
R. Y. Caine
Address: Department of Surgery, University of Cambridge, Cambridge (UK)
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An analysis of the results of organ graft experiments in animals treated with Cyclospo rin A (CyA) in the Cambridge University Department of Surgery would suggest that CyA is active by mechanisms quite different from any other immunosuppressant so far studied. It appears to have a highly specific effect on transforming lymphocytes in response to both antigenic and mitogen challenge. The lipotrophic nature of the molecule and its unique amino acid are likely to be involved in its mode of action, which is not yet known but is under study and will be discussed. 7 patients on dialysis with renal failure received transplants from mismatched cadaver donors and were treated with CyA initially as the sole immunosuppressive agent. CyA was effective in inhibiting rejection but there was clear evidence of both nephro- and hepatotoxicity, attributable to the drug. A cyclophosphamide analoque, Cytimun (Astawcrke), was added to the CyA treatment in 6 of the patients. 5 patients are out of hospital with functioning allografts, 2 of these have received no steroids. 1 patient required an allograft nephrectomy because of pyelonephritis developing in the graft. Another patient developed a fatal Aspergillus and Candida infection. Further careful study of this potentially valuable drug will be required before it can be recommended in clinical practice.
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Abstracts Modification of the Immune Response by Oral Antigen Application
J Seifert, K. Hallfeld, G. Figacz, B. Eberle and W. Brendel Former investigations have shown that it is possible to induce tolerance as well as immunization if a soluble protein antigen is cntcrally applied. For clinical use it is of more interest to change a preformed immune response against a certain antigen in unresponsive ness via enteral antigen administration. Therefore rabbits were immunized against human 7-globulin (HGG). Animals with precipitating antibodies against HGG were divided into 3 groups. 1 group was orally fed with 500 mg HGG. a second group was given pepsin-digested HGG in the same dosage and a third group remained untreated as controls. After an absorption period of 4 h the rabbits were anesthetized for blood pressure and blood flow measurements and intravenously challenged with 50 mg HGG. Furthermore, antibodies against HGG were checked before oral treatment during the absorption time and after intravenous challenge with HGG. Control animals without oral pretreatment had a mortality rate of 60% within 90 min. The oral application of HGG improved this to 30%. The best results, however, were achieved with pepsin-digested HGG. After the absorption of HGG fragments the mortality rate was 0. The behavior of blood pressure and blood flow are shown in the table.
Organ flow after 60 min, ml/min and g
Blood pressure, mm Hg n before Pretreatment Oral HGG 7 Digested 8 HGG Controls 14
lowest
105 ± 7.3
58 ± 6.1
112 t 7.7 107 ± 5.7
78 ± 13.0 35 ±4.6
kidney
liver
96 ± 5.3
2.6 ± 0.49
0.17 ± 0.04 0.35 ± 0.05
110 ± 10.8 60 ± 8.8
6.2 ± 0.51 1.2 ± 0.28
0.39 ± 0.02 0.35 ± 0.04 0.05 ± 0.02 0.12 ± 0.01
60 min
gut
In control animals, blood pressure and flow values were decreased after intravenous challenge similar to that found in a shock situation. Animals did not recover within 60 min. Oral pretreatment with HGG results not only in a smaller decrease of blood flow and blood pressure, but also in a recovery to nearly normal values. Rabbits orally pretreated with pepsin-digested HGG, however, did not show a significant decrease in circulatory parameters indicating that disorders in blood pressure and blood flow can be prevented by this kind of treatment. Circulating antibodies tested by agar diffusion technique were not influenced by oral pretreatment. Therefore it must be speculated that mainly the cellular side of the immune response was influenced. But these findings show that the antigen-induced circula tory reaction can be manipulated via the gut. Especially in patients sensitized against protein or even allergens the oral application of pepsin-digested antigens could be of clinical use.
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Address: Institut für Chirurgische Forschung im Klinikum Grosshadern, Marchioninistrasse 15, D-8000 München 70 (FRG)
Eur. surg. Res. 11: 205-216 (1979)
10th Round Table Symposium on Applied Immunology1 Axams/Tirol, January 19-23, 1979 Organized by W. Brendel and K. Messmer, Munich
Ultramicrotechmques for the Quantitative Measurement of Enzyme Activities and Membrane-Bound Antigens in Single Cells: Synthesis of Ig Antigenic Determinants in T as well as B Cells
P. Hosli, S. Avrameas, M. Stanislavski, A. Ullmann, M. Rodrigot and E. Vogt
Abstracts are listed in order of presentation.
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The combined use of specially developed cell culture techniques (plastic film dish, PFD) (1) and biochemical ultramicromethods (parafilms micro cuvette, PMC) (2) permits one to routinely and quantitatively measure enzyme activities of single visually selected cells, making use of either fluorogenic or radioactive substrates. Based on these techniques, an enzyme immunoassay for quantitation at the single cell level of immunoglobulin antigenic determinants, present on the membrane of murine lymphocytes (Mlg), has been developed (3). This immunoassay makes use of specific Fab: anti-Ig, anti-K, anti-u and anti-7 conjugated to E. coli (J-galactosidase combined with the micromanipulation of lymphocytes and the measurement of the coupled (3-galactosidase activity in the PMC with the fluorogenic substate 4-methylumbellifcryl-i5-£>-galactopyranoside. With this technique Mlg could be measured in substantial amounts on the surface of almost all splenic and lymph node lymphocytes examined, but a larger proportion of cells with higher amounts of Mlg were found in the spleen than in the lymph nodes. However, when the same Fabs were used to localize Mlg by conventional immunohistochemical staining, only a fraction of cells corresponding to published values could be scored as positive. The fact that Mlg is associated with the vast majority of lymphoid cells implies that T cells carry such antigens. In order to investigate this further, experiments were performed on splenic B- and T-cell fractions obtained using either an anti-lg immunoadsorbent or after treatment with anti-0 serum plus complement. B-cell fractions possessed greater amounts of Mlg (1,500-79,000) than those of T-cell fractions (0-39,000), but on the average B cells carried only about 2.5 times more u-. * and 7-antigenic determinants than T cells. Immunoadsorbent column-fractionated T cells, from which Mlg was removed by anti-mouse Ig stripping, were able to regenerate Mlg after 24 h of culturing. As the
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quantities reexpressed equaled those found on T cells before anti-lg treatment, the present results strongly suggest that T cells synthesize and express MIg. Irrespective of the molecular nature of the T-cell structure detected by the present study, it is hypothesized that the difficulty in detecting MIg by standard immunochemicalstaining methods may reflect differences in the membrane composition of B and T cells. The local concentration of MIg could be higher or the microredistribution of MIg could be achieved more easily on B than on T cells. The credit for the immunological part of these studies belongs to S. Avrameas, M. Stanislawski and M. Y. Rodrigot (cf. 3, 4). References
1 2 3
4
Hôsli, P.: Tissue cultivation on plastic films (Tecnomara, Ziirich 1972). Hôsli, P.: Quantitative assays of enzyme activity in single cells: early prenatal diagnosis of genetic disorders. Clin. Chem. 23: 1476 (1977). Hôsli, P.; Avrameas, S.; Ullmann, A.; Vogt, E., and Rodrigot, M.Y.: Quantitative ultramicro-scalc immunoenzymic method for measuring Ig antigenic determinants in single cells. Clin. Chem. 24: 1325 (1978). Avrameas, S.; Hôsli, P.; Stanislawski, M.; Rodrigot, M.Y., and Vogt, E.: A quantitative study at the single cell level of immunoglobulin antigenic determinants present on the surface of murine B and T lymphocytes. J. Immun, (in press). Address: Institut Pasteur, Département de Biologie moléculaire, Unité de Cytogénétique expérimentale, 25, rue du Docteur-Roux, F -75724 Paris Cédex 15 (France)
T-Cell Receptors and T-Cell Antigens
H. Binz, H. Frischknecht, A. Kimura and H. Wigzell
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Subsets of T cells exist with specific functions and unique surface properties. An analysis of the surface glycoproteins found on functionally distinct T blasts has revealed several unique properties as studied with alloantisera or lectins. In this regard it has been found that a particular glycoprotein appears on the cytotoxic T cells of murine or of human origin which is not found on other T cells or cytotoxic cells of non-T type. A similar uniqueness is found on the antigen-binding receptors of T cells belonging, in the mouse, to the s.c. Lyt subsets. Here it could be shown that Lyt-I*2~ T cells and Lyt-1 ~V T cells display distinct, largely non-overlapping idiotypic receptors when tested in anti-MHC' reac tions. Elimination of the former cells would cause a highly significant reduction of the MLC response (now inducing the elimination by anti-idiotypic antisera against the relevant anti-MHC T blasts with the corresponding Lyt-phenotype) whereas killing the idiotypic T cells with anti-idiotypic sera absorbed with Lyt-1 ' V cells would leave the killing properties but not the proliferative properties intact. In the MHC system it is also clear that anti-idiotypic antibodies may functionally mimic the allo-MHC structures and induce specific cytotoxic T cells with the correct allo-MHC specificity even in the absence of ‘helper’ T cells of the Lyt-1 *2' phenotype. The latter finding may be taken as an indication
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that proliferating helper T cells even in allo-MHC situations can function via auto-anti-idiotypic reactions. Address: Department of Immunology, Uppsala University, Box 782, S 751 23 Uppsala (Sweden) Newer Concepts of T-Lymphocyte Responses to Alloantigens
F.H Bach Responses of T lymphocytes can be studied from two perspectives: first, the nature of the antigens that evoke reactions and second, the classification of the cell types involved. Although much of our understanding of cell-mediated immunity in these respects has come from in vitro studies of T-lymphocy te response to major histocompatibility complex-encoded alloantigens, recent work has extended our concepts to reactions to 'altered-self antigens. The basic dogma, as it developed, stated that there are at least two types of MHC-encoded alloantigens (LD and CD) that preferentially evoke responses in two functionally disparate cell types: Lyt 1* T helper (Th) and Lyt 2* cytotoxic T (Tc) lymphocytes, respectively. Further, that altered-self antigens, such as TNP-modified syngeneic cells or syngeneic tumor cells evoke a response that involves a Lyt 1,2* cell that is not involved in the ordinary response to alloantigens. We and others have obtained results that suggest that this model may be an oversimpli fication in that a Lyt 1,2* cell can also be involved in the response to alloantigens. Our data arc consistent with the possibility that there are two alternative pathways of T-lymphocyte activation: one involving the Lyt 1,2* cell the other not. Which pathway will predominate in any given reaction may depend either on the overall strength of the reaction or on the presence of LD antigens on the stimulating cells. Further, the two pathways may be differentially responsive to augmentation or suppression thus providing an additional mecha nism by which T-lymphocyte reactivity can be regulated. We would hypothesize that these two pathways of T-lymphocyte activation may represent the cell-mediated analogue of the primary and secondary B-lymphocyte response. To the extent that the presence and strength of the LD stimulus controls which pathway will be the strongest in any response, these findings may help to explain the accumulating evidence that matching for LD correlates with improved renal allograft survival. The large number of different T-lymphocyte classes that can be involved in these responses forces careful comparison of results such as those discussed above with our present understanding of T-lymphocyte receptors and control of T-lymphocyte reactivity by anti-idiotypic sera. Address: Immunobiology Research Center, Madison, Wise. (USA) Immunological Reactivity in Patients with Colorectal Cancer
P.J. Morris and P. Grantly Gill
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The survival of patients with colorectal cancer is poor, the crude 5-year survival in Oxford being 25.8% for all pathological stages and 37.3% after a curative resection. Many
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factors have been shown to influence survival in 335 patients treated in Oxford between 1966 and 1971, the most significant of which were the pathological stage of the tumor, the age of the patient, the clinical presentation, and the type of resection performed (1). However, recent studies of smaller groups of patients suggest that the patients’ immunologi cal reactivity might also determine survival and metastasis. Firstly, a significant correlation between lymphocyte and macrophage infiltration (but not plasma cells) at the tumor edge and both survival and metastatic spread was noted, but no such correlation with cellular infiltration within the tumor stroma was seen. Secondly a hemolytic assay of peripheral blood monocyte function (using sensitized A1 red cells as targets) in patients with colorectal cancer showed a significant decrease in activity (2). Thirdly diminished DNCB reactivity has been shown in patients with colorectal cancer by Bolton et al. (3). These findings were felt to justify a controlled trial of immunotherapy with C. parvum, a potent stimulant of macrophage activity, given over a 2-year period. There were 2 deaths and 4 patients with recurrent disease in the treatment group compared to 2 deaths and 8 recurrences in the non-treatment group. A significant increase in monocyte numbers can be demonstrated in blood 7 days after C. parvum infusion. No conclusions can be drawn about the value of this method of immunotherapy for colorectal cancer at this time. References
1 2 3
Gill, P.G. and Morris, P.J.: Br. J. Surg. 65: 17 (1978). Gill, P.G.; Waller, C.A., and Maclennan, I.C.M.: Immunology 33: 873 (1977). Bolton, P.B., et al.: Br. med. J. Hi: 18 (197S). Address: Nuffield Department of Surgery, University of Oxford, Oxford (UK)
H-2 Control of Anti-H-Y T-Cell Responses
Elizabeth Simpson, T. Matsunaga. C. Hetherington, M. Hurme, Mary Brenan and P.R. Chandler
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Females of certain inbred strains of mice can reject syngeneic male skin and can generate H-2 restricted cytotoxic T-cell responses to appropriate male cells (1). The male specific antigen, H-Y, is a weak transplantation antigen coded by a gene(s) on the Y chromosome, and the responses to it have been used as models for responses to tumour antigens, which in many ways act as weak transplantation antigens. Immune response (Ir) genes mapping in the H-21 region determine the ability of mice to make both the anti-H-Y skin graft response and the anti-H-Y cytotoxic T-cell response. However, each of these responses is controlled by genes mapping in different subregions of H-21: in H-2b mice the gene(s) enabling graft rejection are in IBb whilst those allowing cytotoxic responses map in lAb (2, 3); the H-2b genes are dominant (1). In H-2d mice there is a dominant ld gene which allows cytotoxic responses, but not graft rejection (4). A variety of other dependent haplotypes have complementary I-region Ir genes whose activity is shown in appropriate FI mice which make cytotoxic T-cell responses to H-Y, although neither of the parental strains do, at least some of these complementary genes map in the 1C
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subregion (2). Skin graft rejection is not seen in FI hybrids showing complementary anti-H-Y cytotoxic T-cell responses. The separation of the control of skin graft rejection and cytotoxic responses suggest that graft rejection may not be mediated by cytotoxic cells per se. The cytotoxic T-cell responses are also dependent on appropriate K/D associative antigens. The target cell specificity of these responses is such as to suggest that the target antigen consists of two closely associated components, self-H-2K or D plus H-Y. The molecular organisation of this association is not understood but there is clearly a hierarchy of association suggesting that certain K/D molecules ‘fit’ better with H-Y than others, e.g. Kk > Db > Dk > Kd and that certain of them, e.g. Kb, Dd, Ks, Kq, are completely non-associative. Recombinant strains such as B10A (5R) (bbbddd) with permissive Ir genes (lAb) but non-associative K/D antigens (Kb’ Dd) are non-responders, indicating the strict limitation put on responsiveness by appropriate associative self-antigens. It seems likely that K/D antigens are associative for Te cells, whilst 1 antigens are associative for T^ cells. We have recently suggested that Ir genes function by producing associative la antigens such that ‘Ir gene’ products and ‘restrictive’ I-region products are identical and determine activation of T[4 cells necessary for differentiation of T(j cells (4). This novel way of looking at Ir-gene function could, we believe, be equally well applied to Ir-genc control of T-helper and T-suppressor function in antibody responses. The significance of this work in applied immunology is that MHC antigens may well control the extent and effectiveness of responses to all antigens and the manner in which this control is exercised is important to understand, if we are to manipulate the immune response in controlling graft rejection, the growth of tumours, and autoimmune diseases. T-cell responses of mice to H-Y may be excellent models, because all aspects appear to be controlled by MHC genes, and many of these have been mapped both for Tc (cytotoxic cells) and the putative and probably mandatory T^ (T helper) cells. References
1 2 3 4
Simpson, E. and Gordon, R.D.: Immunol. Rev. 35: 59 (1977). Hurme, M.; Hetherington, C.M.; Chandler, P.R., and Simpson, E.: J. exp. Med. 147: 758 (1978). Hurme, M.; Chandler, P.R.; Hetherington, C.M., and Simpson, E.: J. exp. Med. 147: 768 (1978). Matsunaga, T. and Simpson, E.: Proc. natn. Acad. Sci. (in press, 1978). Address: Clinical Research Centre, Division of Transplantation Biology, Watford Road, Harrow, Middlesex HA1 3UJ (UK)
Induction and Effect of Suppressor Cells
H. Wagner
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Mainly due to Ly differentiation antigens, the regulatory influences of interactions amongst murine lymphocyte subpopulations on B-cell-mediated antibody responses has been worked out with precision. For example the obligatory helper T cell expresses the Ly 1
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cell marker, which contrasts to the Ly 2.3 positive T suppressor cell. Recently Tada and associates have described two amplifying systems. In the helper amplification system, activated Ly 1 cells interact with Ly 123 cells to increase the activity of the Ly 1 cell (positive loop). A similar system is described for suppression (negative loop). A T cell with the phenotype of a suppressor cell (Ly 23) makes a product which acts on Ly 123 cell, which then increases the amount of suppression via Ly 2.3 cells. Opposed to the Tada loops, the work of Gershon and Cantor indicates the existence of feedback suppression. In this instance Ly 1 cells act on Ly 123 cells. However, instead of producing more Ly 1 activity, this interaction leads to Ly 23 T-cell-mediated suppression. Observations showing that T cells can suppress the induction of T-cell immune responses have been reported only recently. Accordingly T suppressor cells can suppress in an antigen-specific or nonspecific fashion the in vitro generation of cytotoxic T lympho cytes (1, 2). We have studied the nature and mechanism of action of antigen-specific suppressor T cells obtained either in vivo or in vitro. The results show that suppressor T cells and cytotoxic T cells arc different. References
1 2
Wagner, H.; Starzinski-Powitz, A.; Pfizenmaier, K., and Röllinghoff, M.: Eur. J. Immunol. 6: 873 (1976). Al-Adra, A.R. and Pilarski, L.M.: Eur. J. Immunol. 8: 504 (1978). Address: Institut für Medizinische Mikrobiologie der Universität, Hochhaus Augustusplatz, Mainz (FRG)
Influence of ALG on Suppressor Lymphocytes in vitro (Cultures) and in vivo (Kidney Grafts)
Anti-lymphocyte globulin (ALG) is a potent immunosuppressive drug, widely used to reduce the frequency and severity of graft rejection. Its main effects are the impairment of the antibody response, but also the destruction of lymphocytes, especially those of the T-celt population. However, suppressor cells, supposed to play a beneficial role in protecting a graft from rejection, are found mainly in this T-lymphocyte fraction. It was therefore the aim of the study to monitor the fluctuations of lymphocyte populations with respect to the suppressive activity in vivo and in vitro. 35 kidney recipients received a combination of 5 mg/kg body weight azathioprine, 100 mg prednisolone and 20 mg protein of antilymphocyte globulin/kg body weight over a period of 10 days after transplantation. A steady reduction reached a basic therapy of 2.5 mg azathioprine and 15 mg cortisone after 8 months. White blood cell counts, shifting of T + B cells in the peripheral blood, circulating levels of lymphocytes which bind ALG to their surface were monitored in 3-day intervals. The effect of this immunosuppressive therapy and the action of ALG on suppressor cells was measured in an in vitro assay with patients’ peripheral lymphocytes and cells from healthy donors. The suppressive action of spontaneous and concanavalin-A-activated circulating suppressor cells on mixed lymphocyte
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C. Hammer, W. Bullinger, W. Land and W. Brendel
211
Abstracts
cultures and mitogen-stimulated lymphocytes was recorded preoperatively under immuno suppressive therapy with and without ALG and during rejection episodes. The influence of various concentrations of ALG on lymphocytes of healthy donors was measured with MLC 'specific' suppressor cells in MLC and mitogen stimulation. Suppressive activity of lymphocytes of kidney-transplant patients (%) Pre-op.
No rejection
Rejection no ALG treatment
ALG treatment
ALG treatment
no ALG treatment
MLC/spontaneous Lys MLC/activated Lys
24 41
+ 134 13
+ 130 19
+ 117 4
31 59
PHA/spontaneous Lys PHA/activated Lys
29 39
14 50
+ 132 27
21 30
46 54
Results: After transplantation and the start of immunosuppressive therapy, especially under ALG, lymphocyte counts dropped to 40% of the initial values. As B cells remained relatively constant, the T-cell population reflects the major reduction. ALG-positivc circulat ing lymphocytes decreased from 70% preoperatively to 30% after 7 days of ALG therapy. At the same time values of spontaneous and activated suppressor cells are reduced. In most patients the suppression of MLC and PHA response shifts to stimulation. Patients with rejection crises treated or untreated with ALG show a similar behavior. As in vivo, lymphocytes incubated with ALG and rabbit complement for 1 h in vitro loose their MLC-specific and unspecific suppressor activity with increasing ALG concentrations. The results indicate that immunosuppressive therapy in combination with antilymphocyte globu lin hits cytotoxic lymphocytes as well as suppressor cells in patients. It seems, however, that the suppressive activity recovers faster within hours or Jays than does the cytotoxic cell population. References
2
3 4
Bullinger, W.; Hammer, C.; Land, W., and Welter, H.: Arch. klin. Chir., Chir. Forum, 320, suppl. (1979). Hammer, C.; Jerry, L.M., and Lewis, M.G.: Suppressor cell activity of peripheral blood and lymph node lymphocytes in cancer patients; in Rainer, Immunotherapy of malignat diseases, p. 12 (Schattauer, Stuttgart 1978). Thomas, F.; Lee, H.; Wolf, J.S.; Mendez-Pican, G., and Thomas, J.: Surgery, St Louis 79; 408 (1976). Thomas, J.; Thomas, F.; Mendez-Pican, G., and Lee, H.: Surgery, St Louis 87: 125 (1977). Address: Institut fiir Chirurgische Forschung im Klinikum Grosshadern, Marchioninistrasse 15, D 8000 München 70 (FRG) Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 7:51:28 PM
1
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Role of Suppressor Cells in Autoimmune Diseases
R. Boyd and G. Wick 3 years ago at the Kitzbiihcl meeting (1), we presented evidence fora multigenic basis for the development of spontaneous autoimmune thyroiditis (SAT) which occurs in chick ens of the obese strain (OS) (2). In the meantime our efforts have been aimed at the elucidation of effector and, more recently, suppressor mechanisms involved in the pathogen esis of this disease. Based on the earlier observation that a primary defect exists in the thyroids of OS birds our present working hypothesis postulates that thyroglobulin-autoantibodies (Tg-AAb), vertically transferred from the mother hen via the egg yolk into the embryo, is the first pathogenetic stimulus followed by the influx of Tg-binding B cells. The latter cells invade the thyroid epithelial lining (periopolesis) and differentiate into plasma cells which produce Tg-AAb in situ and lead to further destruction. In addition recent results speak for a participation of antibody-dependent cellular cytotoxicity (ADCC) in the pathogenesis of SAT. In contrast to experimentally induced autoimmune diseases neonatal thymectomy increases the frequency and severity of SAT in the OS and also entails the formation of AAb to non-thyroid antigens. Furthermore, OS chickens generally display a heightened immune response to foreign antigens. These data led to the assumption that the underlying immuno logic defect in OS chickens is a quantitative or qualitative defect of suppressor cells. To investigate this possibility further, the ability of OS thymus suspensions to suppress the in vitro ADCC or direct cytotoxicity of autologous blood leucocytes to thyroglobulin-coated chicken erythrocyte targets, was studied. A decrease in ADCC reactivity was taken as a parameter for the suppressive activity of the added cell suspension. Preliminary results suggest that it is not a defect of suppressor cells, but rather a defect of target cells for suppression which underlies the development of SAT in the OS. This work was supported by a grant of the Austrian Research Council (project No. 3120). References
1 2
Wick, G.: 6th Round Table Symposium on Applied Immunology, Kitzbiihel 1975. Wick, G.; Sundick, R.S., and Albini, B.: Clin. Immunobiol. Immunopathol. 3: 272 300 (1974). Address: Institute for General and Experimental Pathology, University of Innsbruck, Fritz-Pregl-Strasse 3, Innsbruck (Austria)
On the Structure and Function of Transplantation Antigens
The HLA-A, -B and -C antigens are composed of two polypeptide chains. The small invariant chain, dj-microglobulin, is structurally similar to immunoglobulins. The alloantigenic, heavy chain contains two immunoglobulin-like disulfide bridges and amino acid sequence information suggests that the two HLA-A, -B and -C antigen subunits are as similar
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P.A. Peterson
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to immunoglobulin-G constant domains as are the latter to one another. An evolutionary relationship between antibodies and transplantation antigens, therefore, seems likely. That there may exist a functional similarity between the two types of molecules is indicated by the observation that an adenovirus protein, E l9, has been isolated as a physical complex with AgB antigens, the rat equivalents of the HLA-A, -B and -C antigens. Data have been obtained supporting the view that T killer cells recognize the E l 9-transplantation antigen complex. Moreover, some bacteria have been shown to bind to liposome-integrated HLA-A, -B and -C antigens. Thus, transplantation antigens may display the general property to interact with non-self-structures. The HLA-D antigens, whose structure is much less known, have been shown to occur on most if not all epithelial cells. It is particularly noteworthy that these antigens seem to be under hormonal regulation as regards the expression on mammary gland epithelium. The functional significance of the non-lymphoid HLA-B antigens will be discussed. Address: Department of Cell Research, the Wallenberg Laboratory, Box 561, S 751 22 Uppsala (Sweden)
Effects, Significance and Clinical Applicability of Total Lymphoid Irradiation
S. Slavin and S. Strober Total lymphoid irradiation (TL1) given in daily fractions of 200 rads has been used extensively during the past years to treat lymphoid malignancies in humans. It has been well established that TL1 results in long-lasting impairment of the thymus-derived (T) cell-medi ated immune responsiveness in experimental animals as well as in man (1, 2). During the past years we have examined the potential use of TLI for studying a number of clinically relevant problems related to the fields of transplantation immunobio logy and autoimmunity (table I). TLI allows for a permanent survival of bone marrow (BM) allografts (including engraftment of white and red blood cell series) in both inbred mice (1) and rats (3) as well as in outbred unrelated dogs (4) across the major histocompatibility barriers. BM recipients
Immunosuppressive therapy
Immunomanipulation
1 Adjuvant induced polyarthritis in cats(6)
1 Establishment of transplantation tolerance Bone marrow transplantation (1, 3 5) Organ transplantation (1 ,3 -5 ) 2 Tolerance induction for soluble antigens (7)
2 SLE-like disease in NZB/NZWF, mice (unpublished observations) 3 Clinical autoimmune disorders (unpublished observations)
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Table I. Experimental and clinical applicability of TLI
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develop no clinical signs of graft versus host disease ( 1 5 ) . BM recipients develop permanent and specific tolerance to donor-type alloantigens with a permanent survival of skin and heart allografts (1, 3). One-way mixed lymphocyte reactivity of recipient versus donor could be specifically blocked by spleen cells from chimeric mice. Specific tolerance to donor-type skin allografts was also adoptively transferred into normal syngeneic recipients. The data suggest that the tolerance state is due to generation of specific suppressor cells follow ing TL1. Preliminary studies indicate that TLI has a beneficial effect in the treatment of experimental autoimmune disorders including adjuvant-induced polyarthritis in rats (6) as well as rheumatoid arthritis (unpublished observations). The immunosuppressive effects of TLI and the potential generation of suppressor cells following TLI indicate that further investigations should be carried out with regard to applicability of TLI to clinical BM and organ transplantation as well as for the treatment of autoimmune disorders. References
1 2 3 4 5 6 7
Slavin, S., et al.: J. exp. Med. 146: 34 (1977). Funks, Z., et al.: J. clin. Invest. 58: 803 (1976). Slavin. S., et al.: J. exp. Med. 147: 700 (1978). Slavin, S., et al.: Transplantation (in press). Slavin, S„ et al.: J. exp. Med. 147: 963 (1978). Schurman, D., et al.: Transactions Orth. Res. Soc. 3: 38 (1978). Zan-Bar, I., et al.: J. Immunol. 121: 1400 (1978). Address: Department of Medicine A. Hadassah University Hospital, Jerusalem (Israel)
The Beneficial Effect of a Single Blood Transfusion before Kidney Transplantation on Allograft Survival
After much initial scepticism, the findings of Opelz and Terasaki that pretransplant blood transfusion cannot only endanger kidney graft survival but can protect it as well, have been amply confirmed and the graft protecting effect of pretransplant blood transfusion is now generally accepted (1). Animal studies especially those in rhesus monkeys with standard immunosuppression have been essential in this (2). It was generally assumed that blood transfusions induced not only the formation of cytotoxic graft destructive antibodies but also enhancing or graft-protecting antibodies. During a retrospective study van H ooff et al. (3) and Persijn et at. (4) noticed that a single blood transfusion even if given many months before transplantation had a similar graft-protecting effect. This has now been confirmed in a controlled prospective study (5). 19 patients received one blood transfusion each between I and 18 months before transplant ation. 15 out of 19 of the transplants functioned at 6 months posttransplantation. The single blood transfusion had been ‘washed’, i.e. about 90% of the buffyeoat had been removed in one centrifugation step. 8 patients received I 3 blood transfusions which had
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J.J. van Rood. G.G. Persijn, A. van I.eeuwen, E. Goulmy. B.A. Bradley
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been made buffycoat-free by passing them through a cottonwool filter® (Central Laborato ry Red Cross Bloodtransfusion Service. Amsterdam). Only 2 of the 8 transplants functioned at 6 months posttransplantation. It seems improbable that anti-DR-enhancing antibodies are responsible for the graftprotecting effect of a single transfusion. If it is assumed however that antibodies with other specificities could enhance as well, enhancement remains a realistic explanation (6 ). Another possible explanation could be the induction of CML non-responsiveness which can be found after kidney transplantation and which sometimes is caused by suppressor cells (5, 7, 8). References
1 2 3 4 5 6 7 8
Opelz, G., et al.: Transplantn Proc. 5: 253 (1973). Es, A.A. van, et al.: Lancet i: 506 ( 1977). Hooff, J.O. van, et al.: Transplantation 22: 306 (1976). Persijn. G.G., et ah: Transplantn Proc. 9: 503 (1977). Rood, J.J. van, et al.: Transplantn Proc, (in press, 1979). Jonker, M. and Rood, J.J. van: Tissue Antigens 11: 251 (1978). Thomas. J., et al.: Transplantn Proc. 9: 85 (1977). Woningeit, K. and Pichlmayr. R.: Dialysis Transplantn 6: 58 (1977). Address: Department of Immunohaematology, University Medical Center, Leiden (The Netherlands)
The Immunosuppressive Action of Cyclosporin A
R. Y. Caine
Address: Department of Surgery, University of Cambridge, Cambridge (UK)
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An analysis of the results of organ graft experiments in animals treated with Cyclospo rin A (CyA) in the Cambridge University Department of Surgery would suggest that CyA is active by mechanisms quite different from any other immunosuppressant so far studied. It appears to have a highly specific effect on transforming lymphocytes in response to both antigenic and mitogen challenge. The lipotrophic nature of the molecule and its unique amino acid are likely to be involved in its mode of action, which is not yet known but is under study and will be discussed. 7 patients on dialysis with renal failure received transplants from mismatched cadaver donors and were treated with CyA initially as the sole immunosuppressive agent. CyA was effective in inhibiting rejection but there was clear evidence of both nephro- and hepatotoxicity, attributable to the drug. A cyclophosphamide analoque, Cytimun (Astawcrke), was added to the CyA treatment in 6 of the patients. 5 patients are out of hospital with functioning allografts, 2 of these have received no steroids. 1 patient required an allograft nephrectomy because of pyelonephritis developing in the graft. Another patient developed a fatal Aspergillus and Candida infection. Further careful study of this potentially valuable drug will be required before it can be recommended in clinical practice.
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Abstracts Modification of the Immune Response by Oral Antigen Application
J Seifert, K. Hallfeld, G. Figacz, B. Eberle and W. Brendel Former investigations have shown that it is possible to induce tolerance as well as immunization if a soluble protein antigen is cntcrally applied. For clinical use it is of more interest to change a preformed immune response against a certain antigen in unresponsive ness via enteral antigen administration. Therefore rabbits were immunized against human 7-globulin (HGG). Animals with precipitating antibodies against HGG were divided into 3 groups. 1 group was orally fed with 500 mg HGG. a second group was given pepsin-digested HGG in the same dosage and a third group remained untreated as controls. After an absorption period of 4 h the rabbits were anesthetized for blood pressure and blood flow measurements and intravenously challenged with 50 mg HGG. Furthermore, antibodies against HGG were checked before oral treatment during the absorption time and after intravenous challenge with HGG. Control animals without oral pretreatment had a mortality rate of 60% within 90 min. The oral application of HGG improved this to 30%. The best results, however, were achieved with pepsin-digested HGG. After the absorption of HGG fragments the mortality rate was 0. The behavior of blood pressure and blood flow are shown in the table.
Organ flow after 60 min, ml/min and g
Blood pressure, mm Hg n before Pretreatment Oral HGG 7 Digested 8 HGG Controls 14
lowest
105 ± 7.3
58 ± 6.1
112 t 7.7 107 ± 5.7
78 ± 13.0 35 ±4.6
kidney
liver
96 ± 5.3
2.6 ± 0.49
0.17 ± 0.04 0.35 ± 0.05
110 ± 10.8 60 ± 8.8
6.2 ± 0.51 1.2 ± 0.28
0.39 ± 0.02 0.35 ± 0.04 0.05 ± 0.02 0.12 ± 0.01
60 min
gut
In control animals, blood pressure and flow values were decreased after intravenous challenge similar to that found in a shock situation. Animals did not recover within 60 min. Oral pretreatment with HGG results not only in a smaller decrease of blood flow and blood pressure, but also in a recovery to nearly normal values. Rabbits orally pretreated with pepsin-digested HGG, however, did not show a significant decrease in circulatory parameters indicating that disorders in blood pressure and blood flow can be prevented by this kind of treatment. Circulating antibodies tested by agar diffusion technique were not influenced by oral pretreatment. Therefore it must be speculated that mainly the cellular side of the immune response was influenced. But these findings show that the antigen-induced circula tory reaction can be manipulated via the gut. Especially in patients sensitized against protein or even allergens the oral application of pepsin-digested antigens could be of clinical use.
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