Exp. Cliii. Endocrino!. Vol.. 95, No. 1, 1990, pp. 148-456
J. A. Barth, Leipzig
Central Institute of Diabetes "Gerhardt Katsch" (Director: 0MB Prof. Dr. se. med. H. Bibergeil), Karlsburg/GDR
S. KNOSPE, ERIKA KÖHLER, ILONA RJASANOWSKI and KARIN TrrzE
With 3 Figures
Summary. The autologous mixed lymphocyte reaction (AMLR) represents activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Deteriorations in AMLR have been reported in many autoimmune diseases and in diseases with a derangement in T cell regulatory function. We have studied AMLR in 23 newly diagnosed Type-1 diabetic patients and 32 healthy subjects. T and non-T cells were purified by rosetting mononuclear cells with sheep erythrocytes and 8epa-
rating the rosetted T cells from the nonrosetted non.T cells by density gradient centrifugation. Purity of T-lymphocytes isolated was 90% as determined by indirect immunofluorescent analysis with monoclonal antibodies. Proliferation of lymphocytes was measured in response to phytohaemagglutinin and of concanavalin A in a lymphocyte transformation test. In the present study, a deficient AMLR is demonstrated in patients with newly diagnosed Type-1 diabetes. Our data provide evidence for an aberrant immune regulation at the time of diabetes manifestation. The deficient AMLR may represent the in-vitro expression of an in-vivo process against pancreatic cells.
Key words: Type-1 diabetes - AMLR - Lymphocyte-Purification - Lymphocyte-Transformation - Lymphocyte-Phenotyping
Introduction immunologic self-recognition and autologous regulation of the immune response are fundamental characteristics of healthy organisms (Rosenkranz et al., 1987). The human autologous mixed lymphocyte reaction (AMLR) is a unique phenomenon of cellular communication and constitutes activation, proliferation and differentiation of T cells subsequent to specific recognition of self determinants on non-T cells bearing class-TI major histocompatibility antigens (Romain et al., 1984; Takada et al., 1985). Purified helper/inducer T-lymphocytes from normal persons respond to autologous non-T cells in the AMLR hut not suppressor/cytotoxic T-cells. The latter can proliferate, however, in response to autologous non-T cells in the presence of helper/inducer T-lymphocytes and interleukin 2 (Takada et al., 1985). Failure of T cells to recognize the autologous cell surface antigen on non-T cells results in disruption of normal T cell response (Smith * Dedicated to Professor H. Bibergeil on the occasion of his 65th birthday
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Autologous Mixed Lymphocyte Reaction in Newly Diagnosed Type-1 Diabetes*
S. KNosnF et al., Autologous Mixed Lymphocyte Reaction in Diabetes
149
and Talai, 1982). Deficiency in the AMLR have been reported in many autoimmune diseases, and have been interpreted as reflecting altered immunoregulatory function (Kuntz et al., 1979; Smith and De Horatius, 1982; Gupta et al., 1983; Conte et al., 1985; Takada et aI., 1985; Ahmann and Burman, 1987). There are only a few studies on AMLR in patients with diabetes mellitus which is considered as an auto-immune disease (Gupta et al., 1983; Chandy et al., 1984). Therefore it was of interest to investigate the AMLR at the time of diabetes onset. Material and Methods
diabetes and 32 healthy controls without family history of Type-1 diabetes (aged between 16 and 28 years, 17 females and 15 males) were investigated.
Isolation of Mononuclear Cells All procedures were performed under sterile conditions. Mononuclear cells (MNC) were separated from 50 ml heparinized peripheral venous blood by density gradient centrifugation (Boyum, 1976),
washed 3 times with phosphate buffered saline (PBS) and resuspended in PBS at a concentration of 1.5 X 10 cells/mi. MNC were depleted of phagocytic cells by means of incubation of 3 x 10 cells for 30 min with 100 mg carbonyl iron (Flucka AG, Buchs, Switzerland) in 2.5 ml Eagle medium at 37°C. Cells having incorporated iron particles were separated by magnetic fixation. Remaining cells were decanted and used for separation of T-lymphocytes (E-rosetting cell fraction) from non-T lymphocytes (non.E.rosetting cell fraction) as follows:
Separation of T and non-T Cells T and non-T cells were purified by rosetting MNC with sheep erythrocytes (SRBC) and separating the rosetted T cells from the non-rosetted non-T cells by density gradient centrifugation (see separation scheme of T and non-T lymphocytes, Table 1). Because of the loss of cells during this procedure, at least 1.5 x 10 MNC were required in order to obtain the necessary number of non-rosetted cells for AMLR. 1.2 X 10 MNC and 2.4x 10 SRBC per 15 ml Eagle medium with 40% fetal calf serum (FCS) were incubated for 30 min at 37°C. For better cell contact the tubes were centrifuged (200 g, 5 mm) and then incubated for 12-15 h at 4°C. The bulk of the supematant was removed thereafter and the pellet was gently resuspended in 10 ml Eagle medium and layered onto separation medium. After density gradient centrifugation (400 g, 30 min at 4°C) non-rosetted cells were taken from the interface and rosetted cells from the pellet. In order to block their proliferative capacity non-T cells (5 x 106/ml) were treated with 25 g mitomycin C/mI buffer (Boehringer, Mannheim, FRG) for 30 min at 37°C. The cells were washed 3 times thereafter and resuspended at 2 x 106/ml medium supplemented with 20% heat-inactivated autologous serum. The rosetted T cell pellets were resuspended and re-layered onto fresh separation medium and centrifuged again. The nonrosetted cells were removed effectively from rosetted cells by this repeated purification procedure. SRBC attached to T cells were lysed for 30 min at 4°C with Tris buffer (17 mmol/l, pH 7.2) containing ammonium chloride (0.134 mol/1). The T cells were washed 3 times and resuspended at 1 X 10 cells/ ml medium supplemented with 20% heat-inactivated autologous serum. The viability of the isolated cells was >95% as assessed by trypan blue exclusion test.
Autologous Mixed Lymphocyte Culture AMLR cultures were performed in 96-well round-bottomed Titertek microtiter plates (Flow Laboratories) at 37°C in an atmosphere of 5% CO2 - 95% humidified air. Each well contained 200 l Eagle culture medium supplemented with 20% autologous serum consisting of lx 10 re-
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Patients 23 patients (aged between 16 and 24 years, 11 females and 1-2 males) with a newly diagnosed Type-1
Exp. Clin. Endocrinol. 95 (1990) 1
150
Scheme of the separation procedure of T and non-T lymphocytes 50 ml Heparinized venous blood V
Density gradient centrifugation (400 g, 30 mm) V
Mononuclear cells (6-8x107) (Interface) V
3 Washings V
Resuspension at 1.5>< 10 cells/nil V
Carbonyl iron treatment (3 x ioe cells/2.5 ml plus 200 mg carbonyl iron) 30mm, 37°C V
Magnetic separation of phagocytes V
Phagocytic depleted mononuclear cells (MNC) V
2 Washings V
Separation of T and non-T cells by SRBC-rosetting (1.2>< 10 MNC plus 2.4>< 10 SRBC/15 ml,) 30 mm, 37°C V
Centrifugation (200 g, 5 mm, 4°C) V
Incubation
(12-15h, 4°C) gentle Restispensioti V
(Continued p 151)
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Table 1
S. KNOSPE et al., Autologous Mixed Lymphocyte Reaction in Diabetes
151
Table 1 (Continued)
y Density graçiient centrifugation (400 g, 30 mm, 4°C)
Non-T cells (Interlace)
Pellet re-layered to fresh separation medium
2 Washings
y
Mitomycin C treatment (23 vg/mI)
Density gradient centrifugation (400 g/30 mm)
y (5>< 10° cells/mi, 30 mm, 37°C)
y 3 Washings
y Cell lysis (0.134 mol NH4C1/l Tris buffer, 17 mmol/l, pH 7.2, 30 mm, 4°C)
y Resuspension at 2>< 106 cells/mi
y 3 Washings
y Resuspension at 1 x 106 cells/mi
sponder T cells and 2>< 10 mitomycin C treated non-T lymphocytes as stimulator cells. Control cultures contained only the respective number of responder or stimulator cells. The lymphocytes were cultured for 6 days. Thereafter, they were pulsed with 20 KBq [3H] thymidine for 24h. The cells were harvested onto a glass-fiber filter strip using an automated multiple harvester (Flow Laboratories). [3H] Thymidine incorporation was determined by a liquid scintillation counter (Packard, model 2 650). Mixkl lymphocyte cultures as weil as controls were performed 6-fold. The AMLR stimulation index was calculated according to
(NON + T) - (NON) T
whereby (NON + T) represented the actual autoiogous mixed lymphocyte reaction. NON and T were the stimulator- and the responder cells, respectively.
Mitogen-Induced Transformation In order to verify the responsiveness of lymphocytes to phytohaemagglutinin (PilA Wellcome, Beckenham, England) and concanavalin A (ConA Pharmacia, Uppsala, Sweden) and to control the mitomycin C mediated inhibition of the non-T cell proliferation in 19 each of the diabetic and of the control persons the mitogen mediated proliferation of T-lymphocytes and of mitomyoin C treated non-T cells were investigated. These lymphocyte transformation tests (LTT) were performed in parallel to the AMLE assay using 96-well Titertek microtiter plates at a temperature of 37°C in an atmosphere of 5% CO2 - 95% humidified air. Each weil contained 125 l Eagle culture medium supplemented with the respective mitogen concentration (dilution: PHA 1:8 to 1:128; ConA 1.6 to 50 sg/ml), with 20 mmol/I Hepes and 10% FCS. All other conditions were identical to the AMLR assay.
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y
y Rosetted T cells (Pellet)
152
Exp. Clin. Endocrinol. 95 (1990) 1
The antibodies used were directed either against pan-T cells (OKT 3, Bl-T2), T helper cells (OKT 4), T suppressor/cytotoxic cells (OKT 8, Bl-E12), activated HLA-DR positive cells (Bl.DR/1, Bl. DR/2), activated T cells (4F2) or monocytes/granulocytes (Bl.M/G). OKT monoclonal antibodies were obtained from Ortho Pharmaceutical Corporation (Raritan, New Jersey, USA) and the monoclonal antibodies of the Bl series were kindly supplied by Dr. Fiebig (Karl-Marx-Universität, Leipzig GDR). Their specificity has been well documented (Kung et al., 1979; Reinherz et aL, 1979; Behn et al., 1984; Seliger et al., 1984; Fiebig et al., 1985). After fixation on slides by means of PDDA (Polydimethyl-diallyl-ammonium-chloride), the lymphocytes were incubated for 45 min at 4°C with monoclonal antibody. The samples were then labelled for 30 min at 4°C with a fluoresceinconjugated goat anti-mouse serum (Staatliches Institut für Immunpräparate und Nährmedien, Berlin, GDR). The fluorescent cells were counted on the fluorescent microscope Fluoval (VEB Carl Zeiss Jena, GDR) (Kupper et al., 1983).
Results There was a nearly 90% enrichment of T cells in the rosetted cell fraction as shown by the pan-T cell monoclonal antibodies OKT 3 and B1-T2 (Table 2). There was no differ-
ence between cells of healthy controls and newly diagnosed diabetics, neither in the degree of enrichment of T-lymphocytes nor in the part of subpopulations obtained such as helper/inducer or suppressor/cytotoxic cells. The number of moriocytes/granulocytes was low. Also both lILA-DR expression and the number of activated T cells were mode-
rate only. There was a small but significant difference in activated T cell number as revealed by the antibody Bl-DR/2.
Table 2 Characterization of the T cell enriched subsets by monoclonal antibodies (Percentage of cells positive with monoclonal antibody)
BL-T2 OKT3 OKT4 OKT8 BL-E12 BL-M/G BL-DR/1 BL-DR/2 4F2
Healthy controls Number of phenotyped subjects Mean (%) Standard error
31 88.0 0.5
89.1 1.2
Newly diagnosed diabetics Number of phenotyped patients 24 88.1 Mean (%) Standard error 0.7
6
6
6
87.9 1.4
34.9 3.4
28.9 4.6
*
9
9 30.1 4.1
9
28.0 2.2
14 27.1 1.9
21
15 29.0 1.6
18 6.0 0.6
5.9 0.6
30 11.5 0.7
27
11.0* 0.8
18 11.0 1.2
23
21
1-2
13.2 1.3
14.4* 2.1
12.6 1.8
p < 0.10
Lymphocyte Transformation Test T cells from controls and diabetics proliferated in response to PilA and ConA, whereas the mitomycin C treated non-T cells showed no mitogen-dependent [3H] thymidine incorporation (Fig. 1).
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Analysis of Lymphocytes by Monoclonal Antibodies
5 xiO4
15
Q-
o-
9 z 4
\
10
o o
zo
D
\
\
Con A
C-)
o
ü 1:8
1:16 1:32 1:64
1:128
0
50
dilution
Fig. 1
T NON+T HeaLthy controLs NON
T NON+T NewLy diagnosed NON
Typel diabetics
Fig. 2
6
0
(jigImL)
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î\
153
8. Kwosr et al., Autologous Mixed Lymphocyte Reaction in Diabetes
154
Exp. Clin. Endocrino!. 95 (1990) 1
Autologous Mixed Lymphocyte Reaction The AMLR was significantly decreased in newly diagnosed diabetic patients, when compared with simultaneously studied healthy controls (Fig. 2). [H] Thymidine incorporation in purified T-lymphocytes alone was 16.6
% a)
Newly diagnosed Type 1 diabetics % a) S.J. Range n
4
78.1 9.4 12.4
3.1-6.2 >6.2
32
99.9
n 25 3
0-3.1
2
78.3 13.0 8.6
23
99.9
18 3
Frequency of cases in the respective S.J. range
Discussion
The ability of human T-lymphocytes to proliferate in response to autologous non-T mononuclear cells in an autologous mixed lymphocyte reaction is generally considered necessary for an intact immune surveillance (Romain et al., 1984; Conte et al., 1985; Takada et al., 1985). But there remains an uncertainty concerning the in vivo significance of this in vitro phenomenon (Bretscher, 1986). The AMLR is thought to indicate the capacity of a subset of T cells to be stimulated by class II major histocompatibility complex molecules in the absence of other antigens. AMLR deficiency is associated with clinically active immune diseases. It has been shown significantly impaired in lupus erythematosus and rheumatoid arthritis (Kuntz et al., 1979; Smith and Horatius, 1982; Takada et al., 1985). Abnormalities of immune regulation seem also to contribute to the pathogenesis of insulin-dependent diabetes. In NOD-mice with high incidence of insulin-dependent diabetes, Baxter et al. (1988) demonstrated a defective syngeneic mixed lymphocyte response. The respective diabetesresistant NOD strain, on the other hand, was able to respond. Deficient AMLR was also shown in patients with insulin-dependent diabetes of long duration (Gupta et al., 1983). Chandy et al. (1984) found in a subset of patients with recent-onset insulin-dependent diabetes a diminished AMLR and a reduced allogenic mixed lymphocyte response. In the present study, we have demonstrated deficient AMLR in patients with newly diagnosed Type-1 diabetes. Results of AMLR reported here provide evidence for an aberrant immune regulation at the time of diabetes manifestation. The disturbed AMLR may represent the in vitro expression of an in vivo phenomenon which is associated with the auto-immune process against beta-cells. Acknowledgement. The study is part of the research project HFR M22 of the Ministry of Health of the German Democratic Republic. ReYerenees AimIANN, A. J.; BUEMAN, K. D.: The role of T lymphocytes in autoimmune thyroid disease. Endocrinol. Metab. Clin. N. Amer. 16 (1987) 287-326. BAXTER, A. G.; FAVILLA, M. L.; MANDEL, T. E.: Defective syngeneic mixed lymphocyte response reflects incidence of diabetes in NOD mice. In: The immunology of diabetes, IXth. Intern. Workshop, Melbourn, Australia, November 27-29, 1988. BEHN, I.; KUPPEE, H.; FIEBIG, H.: Testsatz monoklonaler Antikörper zur quantitativen Be-
stimmung von humanen T- und B-Zellen durch Enzymbrückentechnik unter Verwendung monoklonaler Anti-Meerrettichperoxydase-Antikörper. Wiss. Z. Karl-Marx-Univ. Leipzig, Math.-Naturwiss. R. 33 (1984) 668-671. BØYUM, A.: Isolation of lymphocytes, granulocytes and macrophages. Scand. J. Immunol. 5 (1976) 9-15.
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ft)
155
156
Exp. Clin. Endocrinol. 95 (1990) 1 BRETSCIIER, P. A.: How significant are autoreactive T cells? Immunol. Today 7 (1986) 301-305. CBANDY, K. G.; CIinLEs, A. M.; KURSUNAN, A.; BUCKINGIIAM, B.; WALDECK, N.; GUPTA, S.:
GUPTA, S.; FnKING, S. M.; ORTI, E.: Autologous mixed lymphocyte reaction in manS VI. Deficiency of autologous mixed lymphocyte reaction in Type 1 (insulin-dependent) diabetes mellitus. J. Clin. Lab. Immunol. 11 (1983) 59-62. GUPTA, S.; C1íAJLES, M. A.; BUCKrNGIIAM, B.; KI1SHNAR, A.: Immunological profile in newly
diagnosed patients with Type 1 diabetes. Diabetes 32 (1983) 153. KUNo, P. C.; GOLDSTEIN, G.; REINHERZ, E. L.; SCIILOSSMAN, S. F.: Monoclonal antibodies defining distinctive human T cell surface antigens. Science 206 (1979) 347-349. KUNTZ, M. M.; INNES, J. B.; WEKSLER, ME.: The Cellular Basis of the Impaired Autologous Mixed Lymphocyte Reaction in Patients with Systemic Lupus Erythematosus. J. Clin. Invest. 63 (1979) 151-153. KUPPER, H.; TYPLT, H.; GRIamncKE, H.-D.; FIEBIG, H.: Objekttriigertest zur immunfluores-
zenzmikroskopischen und enzymimmunologischen Erfassung von Zeilmembranantigenen. Allergie u. Immunol. 29 (1983) 223-228. REINHzmz, E. L.; KUNO, P. C.; GOLDSTEIN, G.; SCHLOSSMAN, S. F.: Further characterization
of the human inducer T cell subset defined by monoclonal antibody. J. Immunol. 123 (1979) 2894-2896k RoitIN, P. L.; SCHLOSSMAN, S. F.; REINHERz, E. L.: Surface molecules involved in selfrecognition and T cell activation in the autologous mixed lymphocyte reaction. J. Immunol 133 (1984) 1093-1098. ROSENKBANTZ, K.; DUPoNT, B.; WuIL&Ms, D.; FLOMENBERU, N.: Autocytotoxic and auto-
suppressor T-cell lines generated from autologous lymphocyte cultures. Hum. Immunol 19 (1987) 189-203. SEr.IGER, C.; KUPPEn, H.; FIBEW, H.: Rosettentest zur Bestimmung von Lymphozytenpopu-
lationen unter Verwendung an Rindererythrozyten gekoppelter monokionaler Antikörper. Wiss. Z. Karl-Marx-Univ. Leipzig, Math.-Naturwiss. R. 33 (1984) 672-676. SMrrIi, J. B.; rn HOBATIUS. R. J.: Deficient autologous mixed lymphocyte reaction correlates
with disease activity in systemic lupus erythematosus and rheumatoid arthritis. Clin. Exp. Immunol. 48 (1982) 155-162. SMITH, J. B.; TATAL, N.: Significance of Self-Recognition and Interleukin-2 for Immunregula. tion, Autoimmunity and Cancer. Scand. J. Immunol. 16 (1982) 269-278. TAXADA, S.; UEDA, Y.; SuzUKI, N.; MURAKAWA, Y.; Homir«o, T.; GREEN, J.; STEINBERG, A. D.; HORwITz, D. A.; SAKANE, T.: Abnormalities in autologous mixed lymphocyte reaction. activated immunologic processes in systemic lupus erythematosus and their possible correction
by interleukin 2 Eur. J. Immunol. 15 (1985) 262-267. (Accepted 25 May 1989)
Author's address: S. KNospE, Central Institute of Diabetes "Gerhardt Katsch", Greifswalder Str. lia, Karlsburg, DDR-2201
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Autologous mixed lymphocyte reaction in man: XV. Cellular and molecular basis of deficient autologous mixed lymphocyte response in insulin-dependent diabetes mellitus. J. Clin. Immunol. 4 (1984) 424-428. CONTE, R.; TAzzaI, P. L.; FrNELLI, C.: Deficiency of autologous mixed lymphocyte reaction in patients with idiopathic autoimmune hemolytic anemia. Vox Sang. 49 (1985) 285-291. FIEmO, H.; Bmiw, I.; KUPPER, H.; FIEBACH, H.: Charakterisierung von monokionalen Antikörpern gegen Monozyten. Allerg. Immunol. 31 (1985) 313-320. GUPTA, S.: Autologous mixed lymphocyte reaction in health and disease states in man. Vox Sang. 44 (1983) 265-288.
J. A. Barth, Leipzig
Central Institute of Diabetes "Gerhardt Katsch" (Director: 0MB Prof. Dr. se. med. H. Bibergeil), Karlsburg/GDR
S. KNOSPE, ERIKA KÖHLER, ILONA RJASANOWSKI and KARIN TrrzE
With 3 Figures
Summary. The autologous mixed lymphocyte reaction (AMLR) represents activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Deteriorations in AMLR have been reported in many autoimmune diseases and in diseases with a derangement in T cell regulatory function. We have studied AMLR in 23 newly diagnosed Type-1 diabetic patients and 32 healthy subjects. T and non-T cells were purified by rosetting mononuclear cells with sheep erythrocytes and 8epa-
rating the rosetted T cells from the nonrosetted non.T cells by density gradient centrifugation. Purity of T-lymphocytes isolated was 90% as determined by indirect immunofluorescent analysis with monoclonal antibodies. Proliferation of lymphocytes was measured in response to phytohaemagglutinin and of concanavalin A in a lymphocyte transformation test. In the present study, a deficient AMLR is demonstrated in patients with newly diagnosed Type-1 diabetes. Our data provide evidence for an aberrant immune regulation at the time of diabetes manifestation. The deficient AMLR may represent the in-vitro expression of an in-vivo process against pancreatic cells.
Key words: Type-1 diabetes - AMLR - Lymphocyte-Purification - Lymphocyte-Transformation - Lymphocyte-Phenotyping
Introduction immunologic self-recognition and autologous regulation of the immune response are fundamental characteristics of healthy organisms (Rosenkranz et al., 1987). The human autologous mixed lymphocyte reaction (AMLR) is a unique phenomenon of cellular communication and constitutes activation, proliferation and differentiation of T cells subsequent to specific recognition of self determinants on non-T cells bearing class-TI major histocompatibility antigens (Romain et al., 1984; Takada et al., 1985). Purified helper/inducer T-lymphocytes from normal persons respond to autologous non-T cells in the AMLR hut not suppressor/cytotoxic T-cells. The latter can proliferate, however, in response to autologous non-T cells in the presence of helper/inducer T-lymphocytes and interleukin 2 (Takada et al., 1985). Failure of T cells to recognize the autologous cell surface antigen on non-T cells results in disruption of normal T cell response (Smith * Dedicated to Professor H. Bibergeil on the occasion of his 65th birthday
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Autologous Mixed Lymphocyte Reaction in Newly Diagnosed Type-1 Diabetes*
S. KNosnF et al., Autologous Mixed Lymphocyte Reaction in Diabetes
149
and Talai, 1982). Deficiency in the AMLR have been reported in many autoimmune diseases, and have been interpreted as reflecting altered immunoregulatory function (Kuntz et al., 1979; Smith and De Horatius, 1982; Gupta et al., 1983; Conte et al., 1985; Takada et aI., 1985; Ahmann and Burman, 1987). There are only a few studies on AMLR in patients with diabetes mellitus which is considered as an auto-immune disease (Gupta et al., 1983; Chandy et al., 1984). Therefore it was of interest to investigate the AMLR at the time of diabetes onset. Material and Methods
diabetes and 32 healthy controls without family history of Type-1 diabetes (aged between 16 and 28 years, 17 females and 15 males) were investigated.
Isolation of Mononuclear Cells All procedures were performed under sterile conditions. Mononuclear cells (MNC) were separated from 50 ml heparinized peripheral venous blood by density gradient centrifugation (Boyum, 1976),
washed 3 times with phosphate buffered saline (PBS) and resuspended in PBS at a concentration of 1.5 X 10 cells/mi. MNC were depleted of phagocytic cells by means of incubation of 3 x 10 cells for 30 min with 100 mg carbonyl iron (Flucka AG, Buchs, Switzerland) in 2.5 ml Eagle medium at 37°C. Cells having incorporated iron particles were separated by magnetic fixation. Remaining cells were decanted and used for separation of T-lymphocytes (E-rosetting cell fraction) from non-T lymphocytes (non.E.rosetting cell fraction) as follows:
Separation of T and non-T Cells T and non-T cells were purified by rosetting MNC with sheep erythrocytes (SRBC) and separating the rosetted T cells from the non-rosetted non-T cells by density gradient centrifugation (see separation scheme of T and non-T lymphocytes, Table 1). Because of the loss of cells during this procedure, at least 1.5 x 10 MNC were required in order to obtain the necessary number of non-rosetted cells for AMLR. 1.2 X 10 MNC and 2.4x 10 SRBC per 15 ml Eagle medium with 40% fetal calf serum (FCS) were incubated for 30 min at 37°C. For better cell contact the tubes were centrifuged (200 g, 5 mm) and then incubated for 12-15 h at 4°C. The bulk of the supematant was removed thereafter and the pellet was gently resuspended in 10 ml Eagle medium and layered onto separation medium. After density gradient centrifugation (400 g, 30 min at 4°C) non-rosetted cells were taken from the interface and rosetted cells from the pellet. In order to block their proliferative capacity non-T cells (5 x 106/ml) were treated with 25 g mitomycin C/mI buffer (Boehringer, Mannheim, FRG) for 30 min at 37°C. The cells were washed 3 times thereafter and resuspended at 2 x 106/ml medium supplemented with 20% heat-inactivated autologous serum. The rosetted T cell pellets were resuspended and re-layered onto fresh separation medium and centrifuged again. The nonrosetted cells were removed effectively from rosetted cells by this repeated purification procedure. SRBC attached to T cells were lysed for 30 min at 4°C with Tris buffer (17 mmol/l, pH 7.2) containing ammonium chloride (0.134 mol/1). The T cells were washed 3 times and resuspended at 1 X 10 cells/ ml medium supplemented with 20% heat-inactivated autologous serum. The viability of the isolated cells was >95% as assessed by trypan blue exclusion test.
Autologous Mixed Lymphocyte Culture AMLR cultures were performed in 96-well round-bottomed Titertek microtiter plates (Flow Laboratories) at 37°C in an atmosphere of 5% CO2 - 95% humidified air. Each well contained 200 l Eagle culture medium supplemented with 20% autologous serum consisting of lx 10 re-
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Patients 23 patients (aged between 16 and 24 years, 11 females and 1-2 males) with a newly diagnosed Type-1
Exp. Clin. Endocrinol. 95 (1990) 1
150
Scheme of the separation procedure of T and non-T lymphocytes 50 ml Heparinized venous blood V
Density gradient centrifugation (400 g, 30 mm) V
Mononuclear cells (6-8x107) (Interface) V
3 Washings V
Resuspension at 1.5>< 10 cells/nil V
Carbonyl iron treatment (3 x ioe cells/2.5 ml plus 200 mg carbonyl iron) 30mm, 37°C V
Magnetic separation of phagocytes V
Phagocytic depleted mononuclear cells (MNC) V
2 Washings V
Separation of T and non-T cells by SRBC-rosetting (1.2>< 10 MNC plus 2.4>< 10 SRBC/15 ml,) 30 mm, 37°C V
Centrifugation (200 g, 5 mm, 4°C) V
Incubation
(12-15h, 4°C) gentle Restispensioti V
(Continued p 151)
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Table 1
S. KNOSPE et al., Autologous Mixed Lymphocyte Reaction in Diabetes
151
Table 1 (Continued)
y Density graçiient centrifugation (400 g, 30 mm, 4°C)
Non-T cells (Interlace)
Pellet re-layered to fresh separation medium
2 Washings
y
Mitomycin C treatment (23 vg/mI)
Density gradient centrifugation (400 g/30 mm)
y (5>< 10° cells/mi, 30 mm, 37°C)
y 3 Washings
y Cell lysis (0.134 mol NH4C1/l Tris buffer, 17 mmol/l, pH 7.2, 30 mm, 4°C)
y Resuspension at 2>< 106 cells/mi
y 3 Washings
y Resuspension at 1 x 106 cells/mi
sponder T cells and 2>< 10 mitomycin C treated non-T lymphocytes as stimulator cells. Control cultures contained only the respective number of responder or stimulator cells. The lymphocytes were cultured for 6 days. Thereafter, they were pulsed with 20 KBq [3H] thymidine for 24h. The cells were harvested onto a glass-fiber filter strip using an automated multiple harvester (Flow Laboratories). [3H] Thymidine incorporation was determined by a liquid scintillation counter (Packard, model 2 650). Mixkl lymphocyte cultures as weil as controls were performed 6-fold. The AMLR stimulation index was calculated according to
(NON + T) - (NON) T
whereby (NON + T) represented the actual autoiogous mixed lymphocyte reaction. NON and T were the stimulator- and the responder cells, respectively.
Mitogen-Induced Transformation In order to verify the responsiveness of lymphocytes to phytohaemagglutinin (PilA Wellcome, Beckenham, England) and concanavalin A (ConA Pharmacia, Uppsala, Sweden) and to control the mitomycin C mediated inhibition of the non-T cell proliferation in 19 each of the diabetic and of the control persons the mitogen mediated proliferation of T-lymphocytes and of mitomyoin C treated non-T cells were investigated. These lymphocyte transformation tests (LTT) were performed in parallel to the AMLE assay using 96-well Titertek microtiter plates at a temperature of 37°C in an atmosphere of 5% CO2 - 95% humidified air. Each weil contained 125 l Eagle culture medium supplemented with the respective mitogen concentration (dilution: PHA 1:8 to 1:128; ConA 1.6 to 50 sg/ml), with 20 mmol/I Hepes and 10% FCS. All other conditions were identical to the AMLR assay.
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y
y Rosetted T cells (Pellet)
152
Exp. Clin. Endocrinol. 95 (1990) 1
The antibodies used were directed either against pan-T cells (OKT 3, Bl-T2), T helper cells (OKT 4), T suppressor/cytotoxic cells (OKT 8, Bl-E12), activated HLA-DR positive cells (Bl.DR/1, Bl. DR/2), activated T cells (4F2) or monocytes/granulocytes (Bl.M/G). OKT monoclonal antibodies were obtained from Ortho Pharmaceutical Corporation (Raritan, New Jersey, USA) and the monoclonal antibodies of the Bl series were kindly supplied by Dr. Fiebig (Karl-Marx-Universität, Leipzig GDR). Their specificity has been well documented (Kung et al., 1979; Reinherz et aL, 1979; Behn et al., 1984; Seliger et al., 1984; Fiebig et al., 1985). After fixation on slides by means of PDDA (Polydimethyl-diallyl-ammonium-chloride), the lymphocytes were incubated for 45 min at 4°C with monoclonal antibody. The samples were then labelled for 30 min at 4°C with a fluoresceinconjugated goat anti-mouse serum (Staatliches Institut für Immunpräparate und Nährmedien, Berlin, GDR). The fluorescent cells were counted on the fluorescent microscope Fluoval (VEB Carl Zeiss Jena, GDR) (Kupper et al., 1983).
Results There was a nearly 90% enrichment of T cells in the rosetted cell fraction as shown by the pan-T cell monoclonal antibodies OKT 3 and B1-T2 (Table 2). There was no differ-
ence between cells of healthy controls and newly diagnosed diabetics, neither in the degree of enrichment of T-lymphocytes nor in the part of subpopulations obtained such as helper/inducer or suppressor/cytotoxic cells. The number of moriocytes/granulocytes was low. Also both lILA-DR expression and the number of activated T cells were mode-
rate only. There was a small but significant difference in activated T cell number as revealed by the antibody Bl-DR/2.
Table 2 Characterization of the T cell enriched subsets by monoclonal antibodies (Percentage of cells positive with monoclonal antibody)
BL-T2 OKT3 OKT4 OKT8 BL-E12 BL-M/G BL-DR/1 BL-DR/2 4F2
Healthy controls Number of phenotyped subjects Mean (%) Standard error
31 88.0 0.5
89.1 1.2
Newly diagnosed diabetics Number of phenotyped patients 24 88.1 Mean (%) Standard error 0.7
6
6
6
87.9 1.4
34.9 3.4
28.9 4.6
*
9
9 30.1 4.1
9
28.0 2.2
14 27.1 1.9
21
15 29.0 1.6
18 6.0 0.6
5.9 0.6
30 11.5 0.7
27
11.0* 0.8
18 11.0 1.2
23
21
1-2
13.2 1.3
14.4* 2.1
12.6 1.8
p < 0.10
Lymphocyte Transformation Test T cells from controls and diabetics proliferated in response to PilA and ConA, whereas the mitomycin C treated non-T cells showed no mitogen-dependent [3H] thymidine incorporation (Fig. 1).
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Analysis of Lymphocytes by Monoclonal Antibodies
5 xiO4
15
Q-
o-
9 z 4
\
10
o o
zo
D
\
\
Con A
C-)
o
ü 1:8
1:16 1:32 1:64
1:128
0
50
dilution
Fig. 1
T NON+T HeaLthy controLs NON
T NON+T NewLy diagnosed NON
Typel diabetics
Fig. 2
6
0
(jigImL)
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î\
153
8. Kwosr et al., Autologous Mixed Lymphocyte Reaction in Diabetes
154
Exp. Clin. Endocrino!. 95 (1990) 1
Autologous Mixed Lymphocyte Reaction The AMLR was significantly decreased in newly diagnosed diabetic patients, when compared with simultaneously studied healthy controls (Fig. 2). [H] Thymidine incorporation in purified T-lymphocytes alone was 16.6
% a)
Newly diagnosed Type 1 diabetics % a) S.J. Range n
4
78.1 9.4 12.4
3.1-6.2 >6.2
32
99.9
n 25 3
0-3.1
2
78.3 13.0 8.6
23
99.9
18 3
Frequency of cases in the respective S.J. range
Discussion
The ability of human T-lymphocytes to proliferate in response to autologous non-T mononuclear cells in an autologous mixed lymphocyte reaction is generally considered necessary for an intact immune surveillance (Romain et al., 1984; Conte et al., 1985; Takada et al., 1985). But there remains an uncertainty concerning the in vivo significance of this in vitro phenomenon (Bretscher, 1986). The AMLR is thought to indicate the capacity of a subset of T cells to be stimulated by class II major histocompatibility complex molecules in the absence of other antigens. AMLR deficiency is associated with clinically active immune diseases. It has been shown significantly impaired in lupus erythematosus and rheumatoid arthritis (Kuntz et al., 1979; Smith and Horatius, 1982; Takada et al., 1985). Abnormalities of immune regulation seem also to contribute to the pathogenesis of insulin-dependent diabetes. In NOD-mice with high incidence of insulin-dependent diabetes, Baxter et al. (1988) demonstrated a defective syngeneic mixed lymphocyte response. The respective diabetesresistant NOD strain, on the other hand, was able to respond. Deficient AMLR was also shown in patients with insulin-dependent diabetes of long duration (Gupta et al., 1983). Chandy et al. (1984) found in a subset of patients with recent-onset insulin-dependent diabetes a diminished AMLR and a reduced allogenic mixed lymphocyte response. In the present study, we have demonstrated deficient AMLR in patients with newly diagnosed Type-1 diabetes. Results of AMLR reported here provide evidence for an aberrant immune regulation at the time of diabetes manifestation. The disturbed AMLR may represent the in vitro expression of an in vivo phenomenon which is associated with the auto-immune process against beta-cells. Acknowledgement. The study is part of the research project HFR M22 of the Ministry of Health of the German Democratic Republic. ReYerenees AimIANN, A. J.; BUEMAN, K. D.: The role of T lymphocytes in autoimmune thyroid disease. Endocrinol. Metab. Clin. N. Amer. 16 (1987) 287-326. BAXTER, A. G.; FAVILLA, M. L.; MANDEL, T. E.: Defective syngeneic mixed lymphocyte response reflects incidence of diabetes in NOD mice. In: The immunology of diabetes, IXth. Intern. Workshop, Melbourn, Australia, November 27-29, 1988. BEHN, I.; KUPPEE, H.; FIEBIG, H.: Testsatz monoklonaler Antikörper zur quantitativen Be-
stimmung von humanen T- und B-Zellen durch Enzymbrückentechnik unter Verwendung monoklonaler Anti-Meerrettichperoxydase-Antikörper. Wiss. Z. Karl-Marx-Univ. Leipzig, Math.-Naturwiss. R. 33 (1984) 668-671. BØYUM, A.: Isolation of lymphocytes, granulocytes and macrophages. Scand. J. Immunol. 5 (1976) 9-15.
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ft)
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156
Exp. Clin. Endocrinol. 95 (1990) 1 BRETSCIIER, P. A.: How significant are autoreactive T cells? Immunol. Today 7 (1986) 301-305. CBANDY, K. G.; CIinLEs, A. M.; KURSUNAN, A.; BUCKINGIIAM, B.; WALDECK, N.; GUPTA, S.:
GUPTA, S.; FnKING, S. M.; ORTI, E.: Autologous mixed lymphocyte reaction in manS VI. Deficiency of autologous mixed lymphocyte reaction in Type 1 (insulin-dependent) diabetes mellitus. J. Clin. Lab. Immunol. 11 (1983) 59-62. GUPTA, S.; C1íAJLES, M. A.; BUCKrNGIIAM, B.; KI1SHNAR, A.: Immunological profile in newly
diagnosed patients with Type 1 diabetes. Diabetes 32 (1983) 153. KUNo, P. C.; GOLDSTEIN, G.; REINHERZ, E. L.; SCIILOSSMAN, S. F.: Monoclonal antibodies defining distinctive human T cell surface antigens. Science 206 (1979) 347-349. KUNTZ, M. M.; INNES, J. B.; WEKSLER, ME.: The Cellular Basis of the Impaired Autologous Mixed Lymphocyte Reaction in Patients with Systemic Lupus Erythematosus. J. Clin. Invest. 63 (1979) 151-153. KUPPER, H.; TYPLT, H.; GRIamncKE, H.-D.; FIEBIG, H.: Objekttriigertest zur immunfluores-
zenzmikroskopischen und enzymimmunologischen Erfassung von Zeilmembranantigenen. Allergie u. Immunol. 29 (1983) 223-228. REINHzmz, E. L.; KUNO, P. C.; GOLDSTEIN, G.; SCHLOSSMAN, S. F.: Further characterization
of the human inducer T cell subset defined by monoclonal antibody. J. Immunol. 123 (1979) 2894-2896k RoitIN, P. L.; SCHLOSSMAN, S. F.; REINHERz, E. L.: Surface molecules involved in selfrecognition and T cell activation in the autologous mixed lymphocyte reaction. J. Immunol 133 (1984) 1093-1098. ROSENKBANTZ, K.; DUPoNT, B.; WuIL&Ms, D.; FLOMENBERU, N.: Autocytotoxic and auto-
suppressor T-cell lines generated from autologous lymphocyte cultures. Hum. Immunol 19 (1987) 189-203. SEr.IGER, C.; KUPPEn, H.; FIBEW, H.: Rosettentest zur Bestimmung von Lymphozytenpopu-
lationen unter Verwendung an Rindererythrozyten gekoppelter monokionaler Antikörper. Wiss. Z. Karl-Marx-Univ. Leipzig, Math.-Naturwiss. R. 33 (1984) 672-676. SMrrIi, J. B.; rn HOBATIUS. R. J.: Deficient autologous mixed lymphocyte reaction correlates
with disease activity in systemic lupus erythematosus and rheumatoid arthritis. Clin. Exp. Immunol. 48 (1982) 155-162. SMITH, J. B.; TATAL, N.: Significance of Self-Recognition and Interleukin-2 for Immunregula. tion, Autoimmunity and Cancer. Scand. J. Immunol. 16 (1982) 269-278. TAXADA, S.; UEDA, Y.; SuzUKI, N.; MURAKAWA, Y.; Homir«o, T.; GREEN, J.; STEINBERG, A. D.; HORwITz, D. A.; SAKANE, T.: Abnormalities in autologous mixed lymphocyte reaction. activated immunologic processes in systemic lupus erythematosus and their possible correction
by interleukin 2 Eur. J. Immunol. 15 (1985) 262-267. (Accepted 25 May 1989)
Author's address: S. KNospE, Central Institute of Diabetes "Gerhardt Katsch", Greifswalder Str. lia, Karlsburg, DDR-2201
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Autologous mixed lymphocyte reaction in man: XV. Cellular and molecular basis of deficient autologous mixed lymphocyte response in insulin-dependent diabetes mellitus. J. Clin. Immunol. 4 (1984) 424-428. CONTE, R.; TAzzaI, P. L.; FrNELLI, C.: Deficiency of autologous mixed lymphocyte reaction in patients with idiopathic autoimmune hemolytic anemia. Vox Sang. 49 (1985) 285-291. FIEmO, H.; Bmiw, I.; KUPPER, H.; FIEBACH, H.: Charakterisierung von monokionalen Antikörpern gegen Monozyten. Allerg. Immunol. 31 (1985) 313-320. GUPTA, S.: Autologous mixed lymphocyte reaction in health and disease states in man. Vox Sang. 44 (1983) 265-288.
