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Diabeto logia (1984) 27:102 -105 D i a b e t o l o g i a9 Springer-Verlag1984

A lterations in lym phocyte subpo pulations in T yp e 1 ( insulin-dependent)diabetes m elli tus: explorat ion of po ssible mechanisms and re lat ionshipsto auto imm une phenomenaK . C . H e r o l d , A . H u e n , L . G o u l d , H . T r a i s m a n a n d A . H . R u b e n s t e i nDepartment of Medicine, University of C hicago, Pritzker School of M edicine,and Department o f Pediatrics Northwestern University Medical School Chicago, I llinois, USA

Summary. P r e v io u s s t ud i e s h a v e s h o w n a v a r i e t y o f i m m u n o -logica l abnor mal i t i e s in T ype 1 ( insu l in- depend ent ) d iabe tes ,inc lud ing d i s tur bances in pe r ipher a l lym phocy tes and an t i -l y m p h o c y t e a n t i bo d i e s . W e m e a s u r e d s u b se t s o f T a n d n a t u r a lk i l l er cel ls w i th mo noc lona l an t ibodies in pa t ien t s w i th d iabe-tes , and a l so as sayed f or an t i - lymphocyte an t ibodies us ingd u a l c o l o u r i m m u n o f l u o r e s c e n c e a n d f l o w c y t o m e t ry . W ef oun d a s ign i f ican t decr ease in number s of Leu 3 a ( he lpe r / in -ducer ) ce l ls in Type I d iab e t ic pa t ien t s o f r ecent onse t an d in-te r med ia te l eve ls in pa t ien t s w i th longer dur a t io n o f the d i s-ease . Leu 4 ( pan T ce l l) leve l s w er e r edu ced in Type 1 d iabe t icpa t ien t s o f mor e than 4 mo nths d ur a t ion . Le u 7 ( na tur a l k i ll e rce l ls ) w er e inc r eased in Type 2 ( no n- insu l in- depe ndent ) d ia -be t ic pa t ien t s . I nd iv idua l Typ e 1 d iabe t ic pa t ien t s o f r ecentonse t sh ow ed low leve ls o f Leu 7 an d 11 a ( na tur a l k i l l er ce ll )l eve l s w i th nor mal 3a leve l s . A utoant ibodies aga ins t Leu

3 a + ce ll s w er e pr esen t in h igher t i t r e s in the Type 1 d iabe t icpa t ien t s o f r ecent onse t than in cont r o l sub jec t s . We con c lude :( 1) Leu 3 a ce l ls m ay be decr eased in Type I d iabe t ic pa t ien t so f r e c e n t o n s e t a n d r e t u r n t o n o r m a l w i t h t i m e ; ( 2) lo w n u m -ber s o f Leu 7 and 11 a ce l l s w i th nor m al n um ber s of Leu 3 am a y b e s e e n i n s o m e T y p e 1 d i ab e t i c p a ti e n t s o f r e c e n t o n s e t ,w h i c h m a y h e l p e x p l a i n p r e v i o u s r e p o r t s o f d e c r e a s e d s u p -pr es sor ce l l s ; ( 3 ) Leu 7 leve l s may be inc r eased in Ty pe 2 d ia -be tes ; ( 4) au toant ibo dies aga ins t Leu 3 a+ per iphe r a l lym-p h o c y t e s m a y b e s e e n i n T y p e 1 d i a b e t ic p a t i e n t s o f r e c e n to n s e t. T h e s e a p p e a r t o b e a m a r k e r o f a u t o i m m u n e p h e n o m e -n a r a t h e r t h a n i m m u n o l o g i c a l m e d i at o r s.Key words: L y m p h o c y t e s , a n t i - ly m p h o c y t e a n t i b o d i e s , k i ll e rcel ls , cel l-mediated immunity.

A v a r i e t y o f d i s t u rb a n c e s i n c e l l - m e d i a t e d i m m u n i t yh a v e b e e n f o u n d i n T y p e 1 d i a b e te s , i n c l u d i n g c e l l u l a rh y p e r s e n s i t i v i t y t o p a n c r e a t i c e x t r a c t [ 1], c y t o t o x i c e f -f e c ts o f d i a b e t i c l y m p h o c y t e s a g a i n s t i n s u l i n o m a c e ll s[2 ] a n d a l t e ra t i o n s i n s u b s e ts o f p e r i p h e r a l b l o o d l y m -p h o c y t e s [ 3 -7 ] . S tu d i e s i n T y p e 1 d i a b e t e s a n d o t h e ra u t o i m m u n e d i s e a se s h a v e s u g g e s t e d t h a t a n t i b o d i e s d i -r e c t e d a g a i n s t l y m p h o c y t e s m a y p l a y a p a r t i n th e p a -t h o g e n e s i s o f t h e s e d i s o r d e r s [ 8, 9 ]. W a r m a n d c o l d -r e a c t i n g a u t o a n t i b o d i e s a g a i n s t T a n d B c el ls h a v e b e e nf o u n d i n d i a b e t i c s e r a [1 0], a n d a n I g G a g a i n s t l y m p h o -c y t e s h a s b e e n i d e n t i f i e d i n t h e B B W i s t a r r a t [1 1]. S i m i -l a r a n t i b o d i e s h a v e b e e n d e s c r i b e d i n o t h e r a u t o i m -m u n e d i s e as e s b u t t h e p a t h o g e n i c s i g n i f i c a n c e o f t h e s ea n t i b o d i e s a n d t h e i r r e l a t io n s h i p t o d i s t u r b a n c e s i n p e-r i p h e ra l b l o o d l y m p h o c y t e s u b se ts h a s n o t b e e n d e -

f i n e d . T h e p r e s e n t s t u d y w a s d e s i g n e d t o a d d r e s s t h e s eq u e s t i o n s .

S u b j e c t s a n d m e t h o d sSubjectsPeripheral blood lymphocyte subpopulations were measured in threegroups o f diabetic patients and 14normal control subjects. The pat-ients included: (a) 15 Type 1 d iabetic patients of recent onse t (aged3-23 years; within 4 months of presentation). The mean C-peptidelevel was 0.28_+0.01 nm ol/l [13]; HbA1 was 10.0+0 .5% [14]; sevenhad anti-thyroid antibodies (anti-microsomal haemagglutination andanti-thyroglobu lin haem agglutination ) [15]. (b) Thirteen T ype I dia-betic patients o f more than 4 months duration of the disease (aged9-49 years). Two had anti-thyroid antibodies. Mean C-peptide levelwas 0.08 _+0.3 nmo l/1; H bAa was 10.7 _+0.5 %. (c) Eleven obese Type 2diabetic patients of 4 months - 22 years duration (aged 29-69 years),

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K . C . H e r o l d e t a l . : L y m p h o c y t e s u b p o p u l a t i o n s i n T y p e I d i a b e t e sT a b l e 1 . L y m p h o c y t e s u b s e t s a n d a n t i - l y m p h o c y t e a n t i b o d i e s i n t h e p a t i e n t g r o u p s s t u d i e d

10 3

G r o u p M o n o c l o n a l a n t i b o d i e s f o r p e r i p h e ra l b l o o d l y m p h o c y t e s u b s e t s A n t i - l y m p h o c y t e a n t i b o d i e sL e u - 2 a L e u - 3 a L e u - 4 L e u - 7 L e u -1 1 a N u m b e r o f N u m b e r o f( c y t o t o x i c / ( h e l p e r / ( p a n T ( n a t u r a l ( n a t u r a l L e u - 2 a - L e u - 3 a -su p p re sso r ) i n d u c e r ) c e l l ) k i l l e r c e l l ) k i l l e r c e l l ) p o s i t i v e p o s i t i v e(%) (%) (%) (%) (%) ce l l s c e l l s

C on tr ol sub jec ts 25.1 _+ 1.5 48.3 + 1.5 74.0 _+ 1.4 9.5 _+ 1.4 8.6 _+ 1.0Ty pe 1 di ab eti c 26.4 _+ 1.3 42.6 _+ 1.5 b 72.1 + 1.7 6.8 _+ 1.1 6.9 _+ 1.3p a t i e n t s o fr e c e n t o n s e t( < 4 m o n t hs )T y p e 1 d i a b e t i c 2 5 . 2 + 1 . 4 4 3 . 7 + 1 .3 a 6 9 . 8 + 1 . 3 a 9 . 3 + 1 . 4 8 . 7 + 1 . 8p a t i e n t s o f l o n g e rd u r a t i o n ( > 4 m o n t h s )Typ e 2 d ia be t ic pa t ien ts 24 .5 + 9 .2 43 .2 + 2 .9 69 .9 __. 3 .2 20 .3 + 2 .6~ 12.5 _+ 2.2

84.7 _+ 10. 9 20 .4 _+ 2.68 2 . 1 + 9 . 25 3 5 . 3 + 6 . 9 a

a p < 0 . 0 5 , b p < 0 . 0 2 , C p < 0 . 0 0 1 v e r su s c o n t r o l s u b je c t s

T w o h a d a n t i t h y r o i d a n t i b o d i e s . M e a n C - p e p t i d e l e v e l w a s 0 . 9 9 _ +1 .5 n m o l / l ; H b A ~ w a s 1 0 . 0 + 0 .5 % . W r i t t e n i n f o r m e d c o n s e n t w a s o b -t a i n e d f r o m a l l s u b j e c t s . T h e s t u d y w a s a p p r o v e d b y t h e C l i n i c a l I n -v e s t i g a t i o n C o m m i t t e e o f t h e U n i v e r s i t y o f C h i c a g o .Preparation and staining o fperipheral blood lymph ocytesM o n o n u c l e a r c e ll s w e re i s o l a te d f r o m 2 0 m l o f p e r i p h e r a l b l o o d u s i n gF i c o l l / P a q u e b y s t a n d a r d m e t h o d s [ 1 6] . T h e c e l ls w e r e s t a i n e d e i t h e rp h y c o e r y t h r i n c o n j u g a t e d w i t h a n t i - L e u 2 a o r 3 a o r f l u o r e s c e i n i s o-t h i o c y a n a t e ( F I T C ) c o n j u g a t e d w i t h a n t i - L e u 4 , 7 o r 1 1 a ( B e c t o nD i c k i n s o n , M o n o c t o n a l C e n t e r , M o u n t a i n V i e w , C a l i f o r n i a ) a c c o r d -i n g t o e s t a b l i s h e d p r o c e d u r e s [ 16 ]. T h e s e r e c e n t l y c h a r a c t e r i z e d a n t i -b o d i e s s p e c i f y c y t o t o x i c / s u p p r e s s o r T c e l ls , h e l p e r / i n d u c e r T c e l l s,l a r g e g r a n u l a r l y m p h o c y t e s a n d n a t u r a l k i l le r c el ls ( H N K - 1 a n t i g e n ) ,a n d n e u t r o p h i l a n d n a t u r a l k i l le r c e l l F c I g G r e c e p t o r s r e s p e c t i v e ly[ 1 2 ] . C o n t r o l s i n c l u d e d c e l l s i n c u b a t e d w i t h m e d i a a l o n e . T h e c e l l sw e r e a n a l y s e d o n a F A C S I V .5 ( B e c t o n D i c k i n s o n , F A C S D i v i s i o n ,M o u n t a i n V ie w , C a l i f o r n i a , U S A ) . 2 0 , 0 0 0 c e l ls w e r e a n a l y s e d i n d u -p l i c a t e a n d t h e m e a n d a t a e x p r e s s e d a s a p e r c e n t a g e a r e r e p o r t e d .

Staining of anti-lymphoeyte antibodiesP e r i p h e r a l b l o o d l y m p h o c y t e s w e r e i s o l a t e d f r o m t h r e e n o r m a l d o -n o r s a s d e s c r i b e d a b o v e a n d p o o l e d . H e a t - i n a c t i v a t e d u lt r a c e n t r i -f u g e d s e r a f r o m T y p e 1 d i a b e t i c p a t i e n t s o f r e c e n t o n s e t o r c o n t r o ls u b j e c t s w a s i n c u b a t e d w i t h c e ll s f o r 1 h a t 4 ~ A f t e r w a s h i n g , t h ec e l l s w e r e s t a i n e d w i t h F I T C c o n j u g a t e d g o a t F ( a b ) 2 a n t i -h u m a n I g G( Fc f r a g m e n t s p e c if i c) w h i c h w a s p r e - a b s o r b e d w i t h m o u s e i m m u n o -g l o b u l i n . T h e c e l l s ' we re w a s h e d a n d t h e n s t a i n e d w i t h p h y c o e r y t h r i na n t i - L e u 2 a o r 3 a . N e g a t i v e c o n t r o l c e l l s i n c l u d e d l y m p h o c y t e ss t a i n e d w i th F I T C a n t i - I g G a n d p h y c o e r y t h r i n c o n j u g a t e d a n t i- L e u2 a o r 3 a . A s a p o s i t i v e c o n t r o l , r a b b i t a n t i - t h y m o c y t e g l o b u l i n i n a 1 :5 0 d i l u t i o n w a s u s e d i n t h e p l a c e o f s e r a . 3 0 , 0 00 c e l ls w e r e a n a l y s e df o r t w o c o l o u r i m m u n o f l u o r e s c e n c e o n t h e i n s t r u m e n t n o t e d a b o v ew i t h g a t e s s e t o n l y m p h o c y t e p e a k s o f f o r w a r d a n d w i d e a n g l e s c a tt e r .T h e n u m b e r o f d u a l c o l o u r f l u o r e s c e n t ce l ls w a s c a l c u l a te d u s i n g aT e x t r o n i x c o m p u t e r ( m o d e l 4 0 2 5 , T e x t r o n i x , B e a v e r t o n , O r e g o n ,U S A ) . E a c h s e r u m s a m p l e w a s a s s a y e d o n t h r e e o c c a s i o n s w i t h t h es a m e c e ll d o n o r s a n d t h e m e a n n u m b e r o f d u a l c o l o u r c el ls i s r e p o r t-ed .Statistical analysisT h e s i g n i f i c a n c e o f d i f f e r e n c e s b e t w e e n p a t i e n t g r o u p s a n d c o n t r o ls u b j e c t s w e r e e v a l u a t e d b y u n p a i r e d t - t e s ts o r W i l c o x o n s c o r e s . A l l r e -s u l t s a r e e x p r e s s e d a s m e a n + S E M .

Resul ts

Lympho cyte subpopulationsT h e q u a n t i t a t i v e a n a l y s i s o f l y m p h o c y t e s u b p o p u l a -t ions i s seen in Ta b le 1 . Leu 3a l evel s were s ig n i f i can t lyr e d u c e d i n t h e T y p e 1 d i a b e t i c p a ti e n ts o f r e c e n t o n s e t(p < 0 .0 2) co m p are d w i t h t h e co n t ro l s u b jec t s an d d i a -b e t i c p a ti e n ts o f lo n g e r d u r a t i o n ( p < 0 . 0 5 ) , a n d u n -ch a n g e d i n T y p e 2 d i ab e t i c p a t i en ts . L eu 2 a l ev e ls w e ren o t d i f f e r en t f r o m co n t ro l s u b j ec t s i n an y o f t h e d i ab e t i cg r o u p s . R e d u c e d n u m b e r s o f L e u 4 c e ll s w e r e s e e n i nt h e d i ab e t i c p a t i en ts o f l o n g e r d u ra t i o n (p < 0 .0 5) . A l -t h o u g h , a s a g ro u p , s i g n i f i can t d i f f e r en ces in L e u 7 o r1 l a ce l ls w e re n o t s een i n t h e r ecen t o n s e t T y p e 1 d i ab e t -i c p a t ien t s , t h e r e w as a c l ea r t en d en cy fo r s t r i k in g l y lo wv a l u es i n p a t i en ts w i t h d i ab e t e s o f s h o r t d u ra t i o n . F u r -t h e rm o re , t h e l ev el s o f L eu 7 an d 1 1 a ce l ls w e re s i g n i fi -c a n t l y c o r r e l a t e d ( r = 0 . 7 7 , p < 0 . 0 0 1 ) , w h i c h w a s n o ts een i n t h e co n t ro l g ro u p . A s t ro n g i n v e r s e co r r e l a t i o nw a s s e e n b e t w e e n c h a n g e s i n L e u 3 a a n d 1 1 a l e v e l s( r = 0 .6 7, p < 0 .0 1 ), an d a p o s i ti v e co r r e l a t i o n b e t w e en 2 aan d 1 1 a lev e ls ( r= 0 . 6 8 , p < 0 .0 1) . L e u 7 l ev e ls w e re s ig -n i f i can t l y i n c r eas ed i n t h e T y p e2 d i ab e t i c p a t i en t s ,w h i c h is u n e x p l a i n e d b y d i f f e re n c e s in g l u c o s e c o n t r o la s H b A 1 l ev e l s w e re s i mi l a r i n t h e t h r ee ex p e r i men t a lg ro u p s .Anti-lymphocyte antibodiesT o d e t e r m i n e w h e t h e r a n t i - l y m p h o c y t e a n t i b o d ie s a r ep re s e n t i n t h e T y p e 1 d i ab e t i c p a t i en t s o f r ecen t o n s e tan d d e f i n e t h e i r p o s s i b l e r e l a t i o n s h i p t o p e r i p h e ra lb l o o d l y m p h o c y t e a b n o r m a l i ti e s , w e d e v e l o p e d a m e t h -o d f o r d e t e c ti o n o f l y m p h o c y t e b i nd i n g i m m u n o g l o b u -l in u s i n g d u a l c o l o u r i m m u n o f l u o r e s c e n c e . T h e l e v e l o fb ac k g r o u n d s t a i n i n g i n t h e a s s ay w as < 1% o f cel ls . Ap o s i t iv e co n t ro l , r ab b i t an t i - t h y m o cy t e g l o b u l i n , b o u n d5 16 3 L eu 2 a + ce ll s an d 4 32 9 L eu 3 a + ce ll s. L o w l ev e l s

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10 4o f b i n d i n g w ere s een ag a i n s t L eu 2 a an d 3 a ce l ls w i t h se -r a f ro m b o t h d i ab e t i c an d c o n t ro l s u b jec t s . T h e l ev e l s o fb i n d i n g ag a i n s t t h e 3 a s u b p o p u l a t i o n w as s i g n i f i can t l yg rea t e r i n t h e d i ab e t i c s e r a t h an t h e co n tro l~ (p

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K. C. Herold et al.: Lymphocyte subpopulations in Type 1 diabetes 1053. Horita M, Suzuki H, Onodera F, Ginsberg-Fellner F, Fauci A,Notkins A (1982) Abnormalities of immunoregulatory subsets inpatients with insulin-dependentdiabetes mellitus. J Immunol 129:1426-14294. Pozzilli P, Gorsuch A, Sensi M, Bottazzo G, Cudworth A (1979)Evidence for raised K-cell levels in Type 1 diabetes. Lancet 2:173-1755. Jackson R, Mon'is M, Haynes B, Eisenbarth G (1982) Increased

circulating Ia-antigen-bearing T cel ls in Type 1 diabetes mellitus.New Engl J Med 306:785-7886. Muscart-Lemone F, Delespesse G, Dorchy H, Lemiere B, ServaisG (1982) Characterization of immunoregulatory T lymphocytes ininsulin-dependentdiabetic children by means of monoclonal an-tibodies. C lin Exp Immunol 47:296-3007. Pozzilli P, Zuccar ini O, Iavicoli M, Andreani D, Sensi M, SpencerK, Bottazzo G, Beverley P, Kyner J, Cudworth A (1983) Mono-clonal antibodies defined abnormalities of T-lymphocytes inType 1 (insulin-dependent)diabetes. Diabetes 32: 91-948. DeHoratius R (1980) Lymphocytotoxic antibodies. In: SchwartzRS (ed) Progress in clinical immunology, Vol 4. Grune & Stratton,New York, pp 151-1749. Serjeantson S, Theophilus J, Zimmet P, Court J, Crossley J, ElliottR (1981) Lymphocytotoxic antibodies and histocompatibility anti-gens in juvenile-onsetdiabetes mellitus. Diabetes 30:26-2910. Ozturk G, Terasaki P (1979) Non-HLA lymphocyte cytotoxins invarious diseases. Tissue Antigens 14:52-5811. Dyrberg T, Nakhooda A, Baekkeskov S, Lernmark A, Poussier P,

Marliss E (1982) Islet cell surface and lymphocyte ant ibodies inthe spontaneouslydiabetic BB Wistar rat. Diabetes 31 : 278-28112. Lanier L, Loken M (1984) Human lymphocyte subpopulationsidentified by using three-color immunofluorescence and flow cy-tometry analysis. J Immunol 132:151-15613. Faber O, Binder C, Markussen J, Heding L, Naithani V, KuzuyaH, Blix P, Horwitz D, Rubenstein A (1978) Characterization ofseven C-peptide antisera. Diabetes 27:170-17714. Gabbay K, Sosenko J, Banuchi G, Mininsohn M, Fluckiger R(1979) Glycosylated hemoglobins: increased glycosylation of he-moglobin A in diabetic patients. Diabetes 28:337-34015. Beall G, Soloman D (1973) Immunologic features of thyroid dis-ease. Postgrad Med 54:181-18916. Becton Dickinson Monoc lonal Ant ibody Source Book (1983)B.D. Monoclonal Antibody Center, Sunnyvale, California17. Wall J, Baur R, Schleusener H, Bandy-Dafoe P (1983) Peripheralblood and intrathyroidal mononuclear cell popula tions in patientswith autoimmune thyroid disorders. J Cl in Endocrinol Metab 56:164-169

Dr. Kevan C. HeroldDepartment of Endocrinology/MedicineUniversity of Chicago Hospitals and Clinics5841 South Maryland AvenueChicago, I1. 60637USA