Immunology Letters, 4 (1982) 289-294 Elsevier Biomedical Press

D E F I C I E N C Y OF S U P P R E S S O R T - C E L L S IN I N S U L I N - D E P E N D E N T D I A B E T E S M E L L I T U S

An analysis wi th m o n o c l o n a l an t ibod ies

Sudhir GUPTA, Senih M. FIKRIG, Srniti KHANNA and Eduardo ORTI Memorial Sloan-Kettering Cancer Center, New York, NY and Downstate Medical Center, Brooklyn, NY, U.S.A.

(Received 14 November 1981) (Modified version received 5 January 1982)

(Accepted 16 February 1982)

1. Summary

Peripheral blood from 25 patients with insulin- dependent diabetes mellitus was examined for any alteration in the proportions and or functions of immunoregulatory T-cell subsets, defined with mono- clonal antibodies. Ten of 25 (40%) patients demon- strated deficiency of OKT8 ÷ (suppressor/cytotoxic) T-cells. Eight of these 10 patients had abnormally high ratios of OKT4*/OKT8 ÷ T-cells. Nine of 10 patients with abnormally low proportion of OKT8 ÷ T-cells had deficient concanavalin A-induced suppressor cell activity against the proliferative response of autologous or allogeneic lymphocytes to phytohemagglutinin. No correlation was observed between the deficiency of suppressor T-cells and the control of diabetes. There- fore, it is likely that the deficiency of suppressor cells is related to insulin-dependent diabetes mellitus itself and not to the metabolic changes that are associated with diabetes mellitus. This study demonstrates both quantitative and qualitative deficiency of suppressor T-cells in at least some patients with insulin-dependent diabetes, that might play an important role in the pathogenesis and autoimmune manifestations of a proportion of patients with insulin-dependent diabetes mellitus.

2. Introduction

Insulin-dependent diabetes mellitus is characterized

Key words: monoclonal antibodies - suppressor cells - dia- betes mellitus

by a lack of glucose-induced insulin secretion and a progressive loss of ~-cells from the pancreatic islets, resulting in a loss of endogenous insulin production. Although the etiology of insulin-dependent diabetes mellitus is not clearly understood, a large body of evidence strongly suggests that the immune system plays an important role in its pathogenesis. Buschard e t al. [1 ] have been able to passively transfer hyper- glycemia by transplanting peripheral blood lympho- cytes from newly diagnosed patients with insulin- dependent diabetes mellitus into athymic nude mice. Recently, Doberson et al. [2] have reported the presence of cytotoxic auto-antibodies to 13-cells of the islets of Langerhans in the sera of patients with insulin- dependent diabetes mellitus. Presence of a variety of organ and tissue-specific autoantibodies and associa- tion of autoimmune diseases with the insulin-depen- dent diabetes mellitus [3-10], suggest an aberration in immune regulation. Recently, distinct subpopula- tions of human T-cell subsets have been defined using murine monoclonal antibodies [11 ]. Helper/inducer T-cell subsets are defined by OKT4 antibody and OKT8 antibody reacts with surface antigens present on suppressor/cytotoxic T-cells [ 11 ]. In this investiga- tion we have quantified the proportions Of OKT4 ÷ and OKT8 ÷ T-cell subsets using monoclonal antibodies and correlated them with concanavalin A-inducible suppressor activity in patients with insulin-dependent diabetes mellitus.

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3. Subjects and methods

Peripheral venous blood was obtained from 25 patients with insulin-dependent diabetes mellitus. The characteristics of these patients are shown in Table 1. Twenty-five young (mean +- S.D.; 18 -+ 4 years) and sex-matched donors with no evidence of any gross inamunodeficiency served as controls.

Mononuclear cells were isolated from fresh hepa- rinized blood on a Ficoll-Hypaque (FH) density gradi- ent. Cells were washed three times in Hank's balanced salt solution (HBSS) and resuspended in medium RPMI-1640 containing 20% heat-inactivated fetal calf serum (Grand Island Biologicals, Grand Island, NY) at a concentration of 4 X 106/ml. A small aliquot of this cell suspension was used to label the monocytes by ingestion of latex polystyrene particles (Dow Chem- icals, IN) for the analysis of total T-cells. Analysis of T-cell subset was performed on purified T-cells.

Aliquots of mononuclear cell suspensions in medium containing fetal calf serum were mixed with carbonyl iron (Lymphocyte Separator Reagent, Technicon, Tarrytown, NY) and incubated at 37°C on a rotator for 30 min. Lymphocytes were separated from phago- cytic cells on FH gradient by a method described ear- lier [12]. Lymphocytes were washed and resuspended in HBSS at a concentration of 4 X 106/ml. T-Cells were separated from non-T-cells by rosetting lympho- cytes with neuramindase-treated sheep red blood cells (SRBC) and subsequent centrifugation on FH gradi- ent [121.

Analysis of T-cell subsets was done on purified T-cells using OKT4 and OKT8 murine monoclonal antibodies that define helper/inducers and suppressor/ cytotoxic T-cells, respectively (Ortho Pharmaceuticals, Raritan, N J). We have used a technique modified from that mentioned in the Ortho technical information. In brief, 5 X 10 s T-lymphocytes were washed twice with phosphate-buffered saline/2% bovine serum albumin (PBS/BSA) and cells resuspended in 25/11 of PBS/BSA. Ten ml of monoclonal antibody was added to cell suspension and incubated on ice for 30 rain. Cells were washed twice with PBA/BSA and incubated with 25/.d of goat anti-mouse fluorescein isothiocynate-conjugated IgG (f(ab')2) antibody (1:2 dilution; Cappel Laboratory, Cochraneville, PA) on ice for another 30 min. Cells were washed three times with PBS/BSA and resuspended in 25/A of PBS/BSA.

Wet mounts were prepared and 200-500 lymphocytes counted with Lietz Orthoplan fluorescence micros- cope equipped with epi-lllumination. Results of T-cell subsets are expressed as percent of T-cells.

Concanavalin-A-induced suppressor cell activity was assayed by a technique described [13]. Data on concanavalin-A-induced suppressor activity in some of these patients have been published [14]. Statistical analysis was performed by the Student's t-test.

4. Results

Data regarding T-cell subsets in insulin-dependent diabetes mellitus are shown in Table 2. The proportion of total T- (mean -+ S.D. ;71.9 +-9.9)and OKT4 ÷ T-cells (mean + S.D.; 52.9 -+ 12.3)in patients were comparable to healthy controls (total T-, 78 -+ 9.6; OKT4*, 54.9 -+ 6.7). Only two patients had a lower propor- tion of T-cells when compared to healthy controls. The proportions of OKT4 ÷ cells were lower in one and higher in 4 patients when compared to the range for normal controls. Proportion of OKT8 ÷ cells in the patient group (mean +-- S.D.; 21.4 + 8.6) was signifi- cantly (P < 0.025) lower than in the control group (mean -+ S.D.; 27.6 -+ 4.0). Ten of 25 patients had a proportion of OKT8 ÷ cells lower than the lowest for the range in normal controls. The ratios of OKT4*/ OKT8 ÷ cells in the diabetes group (mean -+ S.D; 3.0 - 1.8) was significantly (P < 0.025) higher than those in control group (mean -+ S.D.; 2.0 -+ 0.5). Eight of 25 patients had increased ratios of OKT4*/OKT8 ÷ cells as compared to range for healthy controls. No direct correlation was observed between deficiency of OKT8 ÷ T-cells or ratios of OKT4+/OKT8 ÷ T-cells and the control of diabetes as determined by the levels of glycohemoglobin or urinary sugar (Table 2) or with the levels of blood sugar (Table 1).

Correlation between Con A-induced suppressor cell activity and the proportion of OKT8 ÷ T-cells is shown in Fig. 1. Nine of 10 patients with OKT8 ÷ cells of <20% had deficient Con A-induced suppressor activ- ity (<13%) against allogeneic responder cells and 7 had against autologous responder cells. Similarly, 14 of 15 patients with a normal proportion of OKT8 ÷ T-cells (~<20%) had normal Con A-induced suppressor cell activity (~<13%)against allogeneic responders and 13 against autologous responders.

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Table 2 T-Cell subsets in patients with insulin-dependent diabetes mellitus a

Patient E-RFCC b OKT4 + OKT8 ÷ OKT4/OKT8 Control of diabetes e (Total) (Helper/inducer) (Suppressor/cytotoxic)

1 82 36 9 c 4,0 d good 2 61 27 c 24 1,1 ~ir 3 61 53 9 c 5.9 d good 4 65 55 19 c 2.9 good 5 56 c 59 24 2.4 good 6 70 48 32 1.2 poor 7 69 45 24 1.9 poor 8 82 59 30 2.0 good 9 60 50 29 1.7 poor

10 77 45 28 1.6 good 11 92 67 d 14 c 4.8 d poor 12 82 60 30 2.0 good 13 69 70 d 25 2.8 poor 14 83 48 26 1.8 poor 15 78 59 22 2.7 poor 16 74 60 24 2,5 fair 17 86 39 9 c 4.3 d poor 18 79 40 19 c 2.1 good 19 72 40 43 0.9 poor 20 75 40 21 1.9 poor 21 56 c 56 25 2.2 poor 22 59 60 8 c 7.5 d poor 23 68 70 d 10 c 7,0 d poor 24 76 56 16 c 3,5 d good 25 65 80 d 16 c 5.0 d poor

Patients(25) Mean±S.D. 71 .9±9 .9 52 .9±12.3 21 .0±8.6 3 .0+1.8 Range (56-92) (27-80) (8 43) (0.9-7.5) Controls(25) Mean±S.D. 7 8 ± 9 . 6 54 .9+6 .7 27 ,6±4 .9 2 .0±0.5 Range (58-93) (39-69) (20-32) (1 .1-3.0) P-value NS NS <0.025 <0.025

aOKT4 ÷ and OKT8 ÷ T-cell subsets are expressed as % of T-cells. bTotal T-cells as determined by spontaneous rosette-formation with SRBC. CAbnormally low value. dAbnormally high value. eGood control = no sugar or traces of sugar in urine or[and glycohemoglobin <9%.

Fair control = 1+ to 2+ sugar in urine or/and glycohemoglobin 9%-11%, Poor control = 3+ to 4+ sugar with acetone in urine or/and glycohemoglobin > 11%.

5. Discussion

During the last several years , ev idence has accu- m u l a t e d to suggest i m m u n o l o g i c a l p e r t u r b a t i o n s in

pa t i en t s w i th i n s u l i n - d e p e n d e n t d iabe tes meUitus.

Some o f these i m m u n o l o g i c a l abno rma l i t i e s inc luded : the presence o f a va r ie ty o f organ- and t issue-specif ic

( inc lud ing against 13-cells) a u t o a n t i b o d i e s [3 ,4 ,6 ,7 ];

c i rcula t ing i m m u n e complexes [15] ; ce i l -media ted

i m m u n e response to pancrea t i c an t igen [ 16]; depressed

prol i fera t ive response to PHA [ 1 7 - 1 9 ]; and cy to tox i -

c i ty against cell l ines f rom in su l inoma [20] . The role

o f ce l l -media ted i m m u n i t y in i n su l i n -dependen t dia-

be tes mel l i tus is fu r the r d e m o n s t r a t e d b y the presence

o f l y m p h o c y t i c i n f i l t r a t ion in the pancreas dur ing an

early stage o f the disease [21 ]. Data regarding n u m b e r s

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Fig. 1. Relationship between the proportion of OKT8 + T-cell subsets and concanavalin A-induced suppressor cell activity against proliferative response of autologous (~) and allogeneic (-) responder cells to phytohemagglutinin. Interrupted lines are the lower limits of normal values for OKT4 + T-cells (20%) and concanavalin A-induced suppression (13%).

of peripheral blood T-cells have been rather conflicting, reporting normal [22,23] or decreased numbers of total T-cells [24]. Muller et al. [24] have reported a decrease in the absolute numbers of "total" and "active" T-cells in insulin-dependent diabetes mellitus. In the present study we have observed a normal pro- portion of total T-cells in a majority of patients with insulin-dependent diabetes mellitus.

Recently, immunoregulatory T-cell subsets have been defined by murine monoclonal antibodies. OKT4 antibody defines differentiation antigen present on the surface of T-cells containing helper/inducer func- tions, and OKT8 antibody defines surface phenotype of cells containing suppressor/cytotoxic functions [ 11 ]. The presence of wide variety of autoantibodies

and association with autoimmune endocrinopathies, strongly suggests a defect in immune regulation in insulin-dependent diabetes mellitus. In our present study we have demonstrated an imbalance of OKT4+/ OKT8 ÷ cells contributed mostly by abnormally low values of OKT8 ÷ cells in 40% of patients with insulin- dependent diabetes mellitus. This quantitative abnor- mality of low proportions of OKT8 ÷ T-cells correlated well with the deficiency of Con A-inducible suppressor cell function against proliferative response of allogeneic or autologous lymphocytes to PHA. This finding is in agreement with the observation of Reinhertz et al. [25], who demonstrated that OKT8 + T-cells contain precursors of Con A-inducible suppressor function. The lack of Con A-inducible suppression or enhance- ment (Fig. 1), cannot be attributed to allogeneic effects of co-culture because of the background response of co-cultures of control mitomycin-C-treated cells, and responders were similar to those of mito- mycin-C-treated Con A-induced suppressors and responder cells co-cultured in the absence of PHA. Furthermore, similar abnormalities were observed in the autologous system. No correlation was observed between the deficiency of suppressor T-cells and the control of diabetes. Therefore, it appears likely that the abnormality of suppressor cells is related to the primary disorder of insulin-dependent diabetes mellitus in at least a subpopulation of patients with this disorder, and is not a consequence of metabolic abnormalities produced by diabetes. Recently Buschard et al. [26] have also reported lack of Con A-induced suppressor functions in patients with newly diagnosed insulin-dependent diabetes mellitus.

In summary, we have demonstrated a quantitative and qualitative deficiency of suppressor T-cells in some patients with insulin-dependent diabetes mellitus. This deficiency of suppressor T-cells, that is also con- sidered important in the mechanism of tolerance, might play an important role in the pathogenesis and autoimmune manifestations of at least a proportion of patients with insulin-dependent diabetes mellitus.

Acknowledgements

This work was supported by grants from USPH: CA-17404, CA-19267, AC--00541, and the Judith Harris Selig Memorial Fund.

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