BARUJ BENACERRAF Harvard Medical School, Boston, M a 02115, U

T HE R OLE OF M HC GE NE PR O D UCTS IN I M MUNE REGULATION AND ITS RELEVA NCE T O ALL OREACTIVITY

Nobel Lecture, 8 Dece mber, 1980

BARUJ BENACERRAF Harvard Medical School, Boston, M A 02115, U. S. A.

The i m mune syste m has evolved the capacity to react specifically with a very large n u m b er of f or ei g n m ol e c ul es wit h w hi c h it h a d n o pr e vi o us c o nt a ct, w hil e a v oi di n g r e a cti vit y f or a ut ol o g o us m ol e c ul es, n at ur all y a nti g e ni c in ot h er s p e ci es or in ot h er individuals of the sa me species. I m munological research has been directed to the elucidation of this pheno menon ever since Ehrlich (1) proposed that i m munoco mpetent cells bear r e c e pt ors f or a nti g e n i d e nti c al wit h t h e a nti b o di es t o b e pr o d u c e d. G o w a ns ( 2) identified ly mphocytes as the cells responsible for i m mune pheno mena. Burnet ( 3) pr o p os e d t h e cl o n al s el e cti o n t h e or y of i m m u nit y w hi c h p ost ul at e d t h at: 1) ly mphocytes differentiate as clones bearing antibody receptors of unique s p e cifi cit y, 2) a nti b o d y r es p o ns es r efl e ct t h e s el e cti v e e x p a nsi o n of s p e cifi c l y m p h o c yt es, f oll o wi n g t h e bi n di n g of a nti g e n, a n d t h eir diff er e nti ati o n as s e cr et ors of a nti b o d y, i d e nti c al i n s p e cifi cit y wit h t h e a nti g e n bi n di n g r e c e pt ors o n t h e ori gi n al cl o n es. T h e B ur n et h y p ot h esis w as v erifi e d e x p eri m e nt all y ( 4, 5, 6) and was accepted as a major advance, concerned pri marily with the response of a nti b o d y pr o d u ci n g c ells, lat er i d e ntifi e d as B l y m p h o c yt es ( 7) a n d pl as m a c ells. A c c or di n gl y, st u di es o n t h e s p e cifi cit y of a nti b o di es a n d o n t h e str u ct ur e of i m munoglobulins revealed that these molecules ( 8, 9) a n d t h eir str u ct ur al g e n es ( 1 0,ll) evolved in a way that ensures the enor mous diversity of antibody co mbin- ing sites observed. T h e dis c o v er y b y Mill er ( 1 2) a n d b y G o o d ( 1 3) t h at l y m p h o c yt es diff er e nti at e i nt o t w o se p ar at e cl ass es of c ells ( T a n d B) wit h disti n ct f u n cti o ns, th e id e ntifi c a- tion of cellular i m mune pheno mena mediated by T cells (14) and the de monstration that i m mune responses are regulated by helper (15,16,17) a n d s u p pr ess or ( 1 8, 1 9) c ells a n d b y m a cr o p h a g es ( 2 0) e m p h asi z e d t h e c o m pl e xit y of t h e i m m u n e s yst e m a n d t h e criti c al r ol e pl a y e d b y T ly mphocytes in the regulation of i m m u nit y. It b e c a m e i n cr e asi n gl y a p p ar e nt t h at t h e cl o n al s el e cti o n t h e or y, alt h o u g h correct, did not take into account the co mplex cellular and molecular i nt er a cti o ns ess e nti al t o i m m u n e p h e n o m e n a or t h e r estri cti o ns t h es e i nt er a cti o ns di ct at e i n t h e s p e cifi cit y of T c ells. A n a d diti o n al s yst e m, b esi d e s p e cifi c i m m u n o gl o b uli ns, i n v ol vi n g t h e pr o d u cts of t h e m aj or hist o c o m p ati bilit y c o m- pl e x ( M H C) w as s h o w n t o b e criti c all y i n v ol v e d i n t h e m a n n er b y w hi c h T c ells

5 9 7 5 9 8 Physiology or Medicine 1980 perceive antigens on the surface of cells and therefore in the nature of i m muno- gc nicit y. I propose to give a historical account of ho w our present understanding of’1 cell i m munity and of T cell i m mune regulation has evolved with particular e mphasis on the genes of the M H C and the molecules for which they code that regulate essential i m mune mechanis ms.

Carrier F u nctio n a nd the Specificity of T Ly mphocytes The pioneering experi ments of Landsteiner (21) established that antibodies can be produced against any type of molecule provided it is presented to the i m mune syste m coupled to an i m munogenic carrier molecule. The deter mi- n a n t s a g a i n s t w h i c h a n t i b o d i e s c a n b e m a d e w e r e t e r m e d “ h a p t e n s ” a n d “carriers” the essential i m munogenic molecules required to initiate i m mune responses. Landsteiner’s experi ments i mplied the existence of a co mplex pro- c ess i n v ol vi n g t h e r e c o g niti o n of a “carrier” f u ncti o n b y a n e ntit y disti nct fr o m antibody to initiate i m mune responses. Spurred by Landsteiner’s observations of the “ c arri er ” eff e ct, G ell a n d I ( 2 2) investigated the specificity of cellular i m mune responses to haptenprotein conjugates. We noted a funda mental difference bet ween the specificity of cellular i m mune reactions and of antibodies. I m mune cells displayed classical “carrier” specificity in contrast to antibodies which can be largely hapten specific. This was latter sho wn to be a general property of T cell mediated i m mune responses (23). Moreover, we also de monstrated another critical difference bet ween the type of deter minants reactive with antibodies and with T cells. Extensive denaturation of protein antigens capable of decreasing drastically reactivity with specific antibody had little effect on the ability of such proteins to initiate or elicit delayed type sensitivity ( D T H) to the intact molecules (24). This indicated again that T and B ly mphocytes may not be specific for the sa me deter minants, and that T cells react preferentially with sequential deter minants on proteins. These observations were co nfir me d by Sc hirr mac her a n d Wi gzell (25) a n d b y Is hiza ka et al. (26).

The Discovery of I m mune Response Genes The identification of the genes which deter mine biological pheno mena and the study of the control they exert on these pheno mena has proven to be the most successful approach to a detailed understanding of the mechanis m of biological processes. So me of the most significant advances in molecular biology have relied upon the methodology of genetics. The sa me state ment may be made concerning our understanding of i m munological pheno mena. I m munologists had not infrequently observed that certain individuals are weak responders to selected antigens. The co mplexity of most antigens and the marked heterogeneity of the antibody response did not encourage a genetic analysis of specific i m mune responsiveness. Ho wever, when synthetic polypep- tides with relatively restricted structural heterogeneity were synthtesizcd (27), the appropriate antigens were available to i m munologists to study the genetic require ments for i m munogenicity. The response of outbred guinea pigs to hapten conjugates of the poly- L-lysine ho mopoly mer ( D N P- P L L) was the first specific i m mune response docu mented to be under the control of a single do minant autoso mal gene (28). We introduced the ter ms “responders” and “nonresponders” to distinguish ani mals possessing or not possessing the gene, and the gene responsible was referred to as an i m mune response or Ir gene. Fortunately, t wo inbred strains of guinea pigs developed originally by Se well Wright were available at the National Institute of Allergy and Infectious Diseases, strain 2 and strain 13. Strain 2 ani mals responded to D N P- P L L and s t r a i n 1 3 g u i n e a p i g s d i d n o t , w h e r e a s ( 2 x 1 3 ) F 1 w e r e r e s p o n d e r s . T h e pheno menon was extended to other polypeptide antigens ( Table 1), the rando m copoly mers of L-gluta mic acid and L-lysine ( G L), L-gluta mic acid and L- alanine ( G A) and L-gluta mic acid and L-tyrosine ( G T) (29). The response to conventional antigens, weak isologous antigens (30) or foreign protein antigens, ad ministered at li miting i m munizing doses (31, 32) to ensure response to only the most i m munogenic deter minants, is under si milar c o ntr ol of i n di vi d ual Ir ge nes. The pheno menon was extended to other experi mental species. Mc Devitt and Sela de monstrated the Ir gene control of the response of inbred mice to a very interesting set of branched copoly mers synthesized by Sela, ( T, G)- A- L, ( H, G)- A- L, and ( Phe, G)- A- L which differed only in one of the a mino acids on the side chain (33). The responses to these copoly mers were under the control ofdistinct Ir genes. In collaboration with Maurer we also de monstrated Ir gene control of the response of inbred mice to linear rando m copoly mers of L-a mino acids (34). Genetic control of i m mune responsiveness was also report- c d i n r ats ( 3 5, 3 6), a n d r h es us m o n k e ys ( 3 7), ill ustr ati n g t h e g e n er alit y of t his pheno menon for different antigens and in different species.

Li n k a ge of Ir Ge nes to t he M aj or Hist oc o m p ati bilit y C o m ple x. M a p pi n g of t he Ge nes a n d Gene Co mple mentation The availability of inbred strains of mice and guinea pigs per mitted the rapid mapping of Ir genes. Mc Devitt and Chinitz (38) m a d e t h e e x citi n g fi n di n g t h at responsiveness of inbred mice to ( T, G)- A- L, ( H, G)- A- L, and ( Phe, G)- A- L could be predicted on the basis of their H-2 genotype. The linkage of murine Ir genes with the H-2 co mplex was confir med for nu merous antigens by many laboratories and is appropriately considered one of the distinctive features of s pecific Ir ge nes ( 3 9). A s u m mar y of t he data is s h o w n i n Fi g. 1 ( 4 0). I d e nti c al li n k a g e b et w e e n g ui n e a pi g Ir ge nes a n d M H C s pecificities i n t hat s pecies was doc u me nte d i n o ur laboratory (41). T he strategy e m ploye d i n t hese ex peri me nts is ill ustrate d i n Ta ble 1. T he ge nes f or t he res p o nses t o P L L, G A an d BS A were observe d to be linke d to the loc us controlling the major histoco m pati bilit y co m plex of strai n 2 g ui nea pi gs. Si milarl y t he G T ge ne a n d t he ge nes co ntrolli ng res po nsive ness to li miti ng doses of D N P- G P A were fo u n d to be li n ke d to t he major H loc us of strai n 13 g ui nea pi gs. Li nkage of Ir ge nes to t he M H C of t he rat (35, 36) a n d r hes us mo nkey (37) w as als o est a blis h e d, ill ustr ati n g t h e g e n er al si g nifi c a n c e of t h e fi n di n g. I n c o ntrast, Ir ge nes were s h o w n n ot t o be li n ke d t o t he str uct ural ge nes f or t he H chain of i m m unoglob ulins (39). 6 0 0

a b d f j k n p q r s u v

The availability of congenic resistant mouse strains developed by Snell (42) and of strains with docu mented reco mbinant events within the H-2 co mplex per mitted Mc Devitt, Deak, Shreffler, Klein, Sti mpfling and Snell (43) to map the murine Ir- 1 locus controlling responsiveness to ( T, G)- A- L to a ne w region of t he mo use H-2 co m plex ter me d t he I regio n (Fig. 2). Ma p pi ng of i n di vi d ual m uri ne Ir ge nes by se veral laboratories (re vie we d i n 40) re veale d t hat most Ir ge nes ma p i n I- A, a s maller n u m ber ma p i n I- B whereas responsiveness to so me antigens map in both I- A and I- E. The latter cases deserve to be discussed in so me detail as they provide the genetic basis for the molecular identification of Ir gene products, to be discussed in another section. Whereas most i m mune responses investigated are under the control of 6 0 1

si n gl e l o ci, c o m pl e m e nt ati o n of Ir g e n es f or t h e r es p o ns e t o c ert ai n a nti g e ns is obser ve d i n rare cases. T h us Dorf a n d I s ho we d t hat t he res po nse to t he ter poly mer of L-gl uta mic aci d, L-lysine an d L- phenylalanine ( G L Ø) is deter- mi ne d b y t wo Ir ge nes w hic h co m ple me nt i n bot h t he cis a n d tra ns co nfi g ura-

Fi g ur e 2. G e n etic m a p of t h e H- 2 c o m pl e x s h o wi n g t h e v ari o us l oci a n d t h e s u br e gi o ns of I. N ot e that Ir genes have been ma p pe d in I- A, I- B an d I- E. 6 0 2 Physiology or Medicine 1980 tio n to per mit a res po nse to G L Ø to de velo p (44). T hese ge nes w hic h we ter me d α a n d β m a p i n t h e I- E a n d I- A s u br e gi o n ( Fi g. 2), r es p e cti v el y ( T a bl e 2). P ossessi o n of eit her α or β ge nes alo ne does not co nfer res po nsive ness to G L Ø w hic h req uire t he prese nce of bot h ge nes. Res po nse to se veral ot her a nti g e ns f oll o ws t h e p att er n of t h e G L Ø r es p o ns e ( 4 5). We s hall disc uss later t he evi de nce t hat Ir ge ne co m ple me ntatio n for G L Ø res po nses reflects t he molec ular co m ple me ntatio n of t he α a n d β s u b u nits of t h e Ia glyco protein. This molec ule m ust be ex presse d on the s urface of macro- p hages a n d B ly m p hocytes for t he res po nse to G L Ø. I n t he case of t his Ia m ol e c ul e, t h e a a n d β c h ai ns will b e s h o w n t o b e c o d e d r es p e cti v el y i n I- E a n d I- A. W he n Ir ge nes ma p i n a si n gle re gi o n s uc h as I- A, disti nct a a n d β s u b u nits, t h e a n d c hai ns, are co de d i n t he sa me A s u bre gio n.

Ia Molec ules a nd Histoco mpatibility A ntige ns Ta ki n g a d va nta ge of t he existe nce of mo use a n d g ui nea pi g strai ns w hic h differ s ol el y at t h e I r e gi o n of t h eir M H C s u c h as t h e A T L a n d A T H str ai ns of mi c e a n d t he g ui nea pig strai n 2 a n d strai n 13, atte m pts were ma de to pro d uce antibo dies s pecific for the Ir gene pro d ucts by cross-i m m unization with ly m p hoi d tiss ue. Alloa ntisera pre pare d i n t his ma n ner by S hreffler a n d Davi d (46), Klei n a n d Ha u ptfel d (47) a n d Mc Devitt a n d associates (48) i n mice a n d Sch wartz, Pa ul an d Shcvach (49) i n g ui nea pigs reacte d wit h alloa ntige ns ter me d Ia (i m m une res ponse-associate d) antigens ex presse d on B ly m phocytes a n d a sig nifica nt fractio n of macro p hages (50). A detaile d st u dy by S hreffler a n d Da vi d (46) of t he s pecificities detecte d by a nti-Ia a ntisera re veale d t he consi derable poly mor phis m of these molec ules. C ulle n et al (51, 52) st u die d t he str uct ure of m uri ne Ia a nti ge ns ex presse d on B ly m phocytes, an d analyze d the me mbrane antigens s pecifically reactive wit h a nti-Ia a ntibo dies. S uc h a ntibo dies bo u n d glyco protei ns fro m B cells co m pose d of a n α a n d a β c hai n wit h molec ular weig hts of 33,000 a n d 28,000 dalto ns res pecti vel y. Si milarly, 13 a nti-2 a n d 2 a nti-13 reci procal alloa ntisera detecte d ho molo- go us Ia molec ules with corres pon ding α a n d β c hai n s ub u nits o n g ui nea pig macro phages an d B ly m phocytes (49,53). A gra p hic re prese ntatio n of a n Ia molec ule is s ho w n i n Fi g. 3 a n d co m pare d with a classical trans plantation antigen of the M H C ex presse d on all cells a n d co m prise d of a 45,000 dalto n poly mor p hic c hai n associate d wit h

β 2 , micr o gl o b uli n (54). A n a nalysis of t he i m m u nological pro perties of t he hig hly poly mor p hic Ia molec ules on macrophages an d B ly mphocytes reveale d that these pro d ucts sti m ulate t he alloreacti ve proliferatio n of u n pri me d clo nes of T ly m p hocytes i n a n i n vitr o test ter me d t he mixe d le ukocyte reactio n ( M L R) (55). T he ability of Ia beari ng cells to sti m ulate M L R res po nses is effecti vely blocke d by a nti-Ia a nti b o dies (56). I re gi o n differe nces a n d Ia m olec ules o n cells sti m ulate str o n g graft vers us host reactio ns (57) a n d vi goro us ho mo graft rejectio ns (58). 6 0 4 Physiology or Medicine 1980

Fig ure 3. Gra phic re presentation of the chain str uct ure of an Ia molec ule, co m pare d with the str uct ure of a histoco m patibility a ntige n. Esse ntially co m parable res ults were obtai ne d for t he mo use H-2 an d the h u man H L A an d the g uinea pig G P L A co m plexes.

F u nctio n of Ir Ge nes T he st u d y or Ir ge ne f u ncti o n c o ntri b ute d t o o ur u n dersta n di n g of t he i ntricate reg ulatory mec ha nis ms evolve d by T cells a n d macro p hages to reg ulate s pecific i m m u ne res po nses. Ex peri me nts were i nitially desig ne d to i de ntify t he cells of the i m m une syste m in which Ir genes are ex presse d an d the nat ure of the process t hey co ntrol. H-li nke d Ir ge nes were s ho w n to deter mi ne bot h h u moral a n d c ell ul ar i m m u n e r es p o ns es ( 2 8). A f urt h er a n al ysis r e v e al e d t h at t h e g e n es c o ntr ol t he rec o g niti o n of t he “carrier” molec ule as a n i m m u noge n (59), a pro perty of T ly m phocytes. Th us, res pon der g uinea pigs which make anti- D N P antibo dy upon i m munization with D NP-P L L are equally able to make anti- benzylpenicilloyl (BP O) antibody to BP O-PLL whereas nonresponder guinea pigs to D N P- P L L do not (59). Si milarly, no nres po n der a ni mals w ho faile d to make anti- D NP antibody to D NP-PLL, make anti- D NP antibody when i m munize d wit h D N P co nj ugates of a co nve ntio nal a ntige n. Moreover, Ira Gree n i n my laboratory ma de t he sig nifica nt observatio n t hat t he D N P- P L L ge netic defect co ul d be by passe d an d nonres pon der ani mals in d uce d to for m anti- D N P- P L L antibo dies if D N P- P L L is treate d as a macro molec ular ha pten an d a d mi nistere d co u ple d to a n i m m u noge neic carrier s uc h as ovalb u mi n (60). C o nsist e nt wit h t h e criti c al r ol e of t h e c arri er i n c ell ul ar i m m u nit y, t h e g e n eti c defect for cell ular i m m unity was not by passe d an d the nonres pon der g uinea pigs i m m unize d with D N P- P L L-ovalb u min di d not develo p delaye d ty pe sensitivity to D N P- P L L in s pite of making large a mo unts of anti- D N P- P L L antibo dies. D u n ha m, U na n ue a n d I (61) t he n verifie d t he prese nce of B cells wit h a ntibo dy rece ptors for no ni m m u noge neic poly pe pti des i n t he s plee ns of no nre- s pe n der mice. We co ncl u de d fro m t hese ex peri me nts t hat t he process gover ne d by s pecific H-li nke d Ir ge nes co ntrols T cell i m m u ne res po nses a n d affects a nti b o d y pr o d ucti o n o nl y as a res ult of t he ner d of hel per T cells f or B cell res po nses. I n agree me nt wit h t his co ncl usio n, H-li nke d Ir ge nes were s ho w n to co ntrol o nly t he res po nse to T de pe n de nt a ntige ns (39). ‘I‘ i n de pe n de nt re- s po nses w hic h res ult fro m t he direct acti vatio n of B l y m p hoc ytes b y a nti ge n are not u n der H-li n ke d Ir ge ne co ntrol. T h e i n v ol v e m e nt of Ir g e n es i n ‘I‘ c ell r es p o ns es c o ul d r es ult fr o m eit h er: 1) t h e e x pr essi o n of Ir g e n es i n T c ells a n d t h eir c o di n g f or t h e T c ell r e c e pt or, or 2) t he ex pressio n of Ir ge nes i n macro p ha ge a n d B cells a n d t heir role i n deter mi na nt selectio n, a ntige n prese ntatio n a n d T cell- B cell i nteractio n. T he latter alter nati ve was s ho w n to be correct i n e very res pect. S he vac h a n d Rose nt hal (62), worki ng wit h t he g ui nea pig syste ms we de velo pe d, ma de U S C of . . t h e fi n di n g t h at pri m e d T c ell cl o n es pr olif er at e i n vitr o an d incor porate H 3 thy mi dine when presente d with antigen by antigen p ulse d macro phages. (2 x

1 3) F 1 g uinea pigs were i m m unize d with t wo antigens, D N P- G L, controlle d by a str ai n 2 Ir g e n e, a n d G T c o ntr oll e d b y a str ai n 1 3 Ir g e n e ( T a bl e 1). T h eir T

cells were ex pose d to D N P- G L or G T o n macro p hages of 2, 13 or F 1 o ri gi n.

T he res ults were u neq uivocal. Pri me d (2 x 13)F 1 T cells res pon de d to D N P-

G L o n strai n 2 or F 1 macro p hages b ut not o n strai n 13 macro p hages. I n

co ntrast, t he sa me pri me d cell po p ulatio ns res po n de d to G T o n strai n 13 or F 1 b ut not strai n 2 macro p hages (Fig. 4). T hese ex peri me nts were exte n de d i n mice b y Sre d ni, Matis, Ler ner, Pa ul a n d Sc h wartz (63) usi n g a G L Ø s pecific T cell lint clone d fro m a res pon der B10. A(5 R) mo use. S uch G L Ø s pecific clone li nes o nly proliferate d w he n prese nte d G L Ø by a ntige n prese nti ng cells ( mac-

ro phages) fro m high res pon der mice [ B10. A(5 R) or ( B10. A X B10) F 1 ] e x- pressi ng bot h Ir- G L Ø α a n d β g e n es i n t h e s a m e c ell ( T a bl e 3). T h e n e e d f or I a bearing macro phages for T cell sti m ulation was f urther doc u mente d in o ur laboratory by Ger mai n a n d S pri nger (64). T reat ment of antigen presenting cells wit h mo noclo nal a nti-Ia a ntibo dy a n d co m ple me nt abolis he d t he ability of t h e c ells t o pr es e nt a nti g e n f or pr olif er ati v e r es p o ns es t o pri m e d T c ells ( T a bl e 4). A n ot h er a p pr o a c h t o t h e r ol e of Ir g e n es i n t h e pr es e nt ati o n of a nti g e n t o T cells by macro p hages i nvolve d t he use of a nti-Ia a ntisera wit ho ut co m ple me nt to block a ntige n prese ntatio n. T he origi nal ex peri me nts were carrie d o ut i n g ui nea pigs by S hevac h, Pa ul a n d Gree n (65) a n d later i n mice by Sc h wartz a n d ass ociates i n c olla b orati o n wit h o ur la b orat or y (66). T cells fr o m (2 X

1 3) F 1 guinea pigs pri med to D NP- GL and GT were exposed to D NP- GL or

G T i n vitr o t o g et h er wit h ( 2 X 1 3) F I macro p ha ges a n d alloa ntisera directe d to 2 or 1 3 I a s p e cifi citi es. A nti- 2 a ntis er a bl o c k e d o nl y t h e r es p o ns e t o D N P- G L a n d not to G T w hereas a nti-13 a ntisera bloc ke d t he res po nse to G T b ut not to 6 0 6 Physiology or Medicine 1980

D N P- G L ( Table 5). T hese ex peri me nts le d to t he i m porta nt co ncl usio n t hat: 1) Ir genes are ex presse d on antigen presenting cells with the mor phology of macr o p ha ges, a n d 2) T cells detect a nti ge n o n t he s urface of a nti ge n prese nti n g cells a n d are s pecific f or f orei g n a nti ge ns percei ve d i n t he c o ntext of a ut ol o g o us I a m ol e c ul es. Deter mi na nt selectio n for T cell res po nses clearl y res ults fro m s uc h a process. T h us, e v e n i n t h e c as e w h er e t w o i n br e d str ai ns ar e r es p o n d ers t o t h e s a m e T de pe n de nt a ntige n t he st u dies of Barci nski a n d Rose nt hal (67) o n t he i m m u ne res po nse of g ui nea pig to i ns uli n reveale d t hat strai ns 2 a n d 13 res po n d to disti nct deter mi na nts; strai n 2 T cells res po n d to a deter mi na nt o n t he A c h ai n of i ns uli n ( A R- A 9- Al ”), w h er e as i n str ai n 1 3 g ui n e a pi gs, t h e r es p o ns e is directe d to seq ue ntial deter mi na nts o n t he B c hai n of i ns uli n i nvolvi ng t he histi di ne at positio n 10 (68). Si milar data co ncer ni n g deter mi na nt selectio n i n ot her a ntige ns was re porte d by Berzofsky et al (69) a n d Ki p ps et al i n o ur la b orat or y (70). The Role of M H C Gene Products in I m mune Regulation 6 0 7

T a bl e 3. Sti m ul ati o n of G L Ø S p e cifi c B 1 0. A( 5 R) T C ell Cl o nes Req uires A ntige n Prese nti ng Cells ( Macrophages) Expressing Both α a n d β Ir- G L Ø Gene Pro d ucts in the Sa me Cell

N ot e: Clo nes 6.2 a n d 6.4 were selecte d fro m G L Ø pri me d T cells of B10. A(5 R) res po n der mice. 5 x 1 0 3 cells fro m those cloned lines were sti mulated with 100 µg/ ml GL Ø in the presence of irra diate d antigen presenting s pleen cells. Sti m ulation was assaye d by meas uring the incor po- r ati o n of 3 H-t h y mi di n e. A d a pt e d fr o m Sr e d ni, B., M atis, L. A., L er n er, E. A., P a ul, W. E. a n d S c h w art z, R. H. i n J. E x p. M e d. 1 5 3, 6 7 7, 1 9 8 1.

Table 4 Treat ment with Monoclonal Anti-Ia Antibody and Co mple ment Eli minates Macrophages Req uire d for G A T I n d uce d T Cell Proliferatio n

Res po n di ng cells (a) A d de d antigen presenting cells (b)

a. 4 X 1 0 5 nylon passed ly mph node T cells fro m G AT- CF A pri med B ALB/c mice, treated with C alone, or M5/114 + C, then culture d with or without 100 µg/ ml G A T for 3 days, pulse d for 1 8 hr wit h 3 H-thy mi dine, harveste d, an d counte d. b. 3 X 1 0 5 1 5 0 0 R y-irr a di at e d s y n g e n ei c s pl e e n c ells.

Table 5. A nti-Ia Alloa ntisera Block A ntige n Prese ntatio n to Pri me d T Cells Res po n di ng cells A nti ge n A ntisera a d de d Increase d D N A synthesis

N ot e:

( 2 X 1 3) F 1 guinea pig T cells were pri med to D NP- GL and GT. Their response to D NP- GL is blocke d by 13 %-a nti-2 a ntisera a n d t heir res po nse to G T is blocke d by Z-a nti-13 a ntisera. T hese alloa ntisera are s pecific for Ia a ntige ns o n strai n 2 a n d 13 res pectively. A d a pt e d fr o m S h e v a c h, E. M., P a ul, W. E., a n d Gr e e n, I., J. E x p. M e d. 1 3 6, 1207 (1972). 6 0 8 Physiology or Medicine 1980

T he ge netic restrictio ns dictate d b y I re gio n co ntrolle d a nti ge n prese ntatio n t o T c ells c a n als o b e o bs er v e d w h e n att e m pts ar e m a d e t o tr a nsf er d el a y e d t y p e sensitivity. I ha d ma de the p uzzling observation with Pa ul an d Green that delaye d ty pe sensitivity to D N P- P L L in rando m bred guinea pigs could only be a do pti vely tra nsferre d to reci pie nts t hat were also res po n ders to t his a ntige n (71). Usi n g c o n ge nic resista nt i n bre d strai ns of mice, Miller et al (72) later s h o w e d t h at t h e s u c c essf ul tr a nsf er of d el a y e d t y p e r e a cti vit y r e q uir es I r e gi o n i d e ntit y b et w e e n t h e s e nsiti z e d T c ell a n d t h e r e ci pi e nt mi c e w hi c h pr o vi d e t h e a ntige n prese nti ng macro p hage w he n t he test a ntige n is i njecte d. Moreo ver, as ex pecte d, sensitize d cells fro m (res pon der x nonres pon der)F 1 mice di d n ot tra nsfer D T H to no nres po n der reci pie nts lacki ng t he a ntige n prese nti ng cells ( 7 3). At t h e s a m e ti m e as t h e N.I. H. gr o u p d o c u m e nt e d t h e i m p ort a n c e of Ir g e n es a n d Ia m olec ules i n a nti ge n prese nti n g cells a n d t heir critical r ole i n t he prese ntatio n of a nti ge n to T cells, ex peri me nts were carrie d o ut b y Katz, Ha maoka, Dorf a n d me (74) a n d by Ki n dre d a n d S hreffler (75) de mo nstrati ng t h e r ol e of I r e gi o n g e n es i n t h e c o ntr ol of T c ell- B c ell i nt er a cti o ns i n a nti b o d y res po nses. We devise d a do uble a do ptive tra nsfer protocol w hereby ha pte n s pecific B c ells a n d c arri er s p e cifi c T c ells fr o m eit h er t h e s a m e p ar e nt al str ai n or disti n ct

pare ntal strai ns were tra nsferre d t o irra diate d F 1 reci pie nt mice pri or t o seco n dary c halle nge (74). T he res ults were u neq ui vocal. Carrier s pecific hel per T cells a n d ha pte n pri me d B cells nee d to s hare I re gio n ge nes for a nti bo d y res po nse to develo p to t he ha pte n-carrier co nj ugate. S uccessf ul T cell- B cell

interactions were observe d bet ween F 1 T cells a n d pare ntal B cells, or pare ntal

T c ell s a n d F 1 B cells w hic h o nly nee d to s hare o ne ha ploty pe for s uccessf ul res po nses pro vi de d bot h strai ns are res po nses to t he carrier a ntige n use d ( Ta ble 6).

N ot e:

Carrier pri me d T cells were a d o pti vel y tra nsferre d t o (a X b) F 1 reci pie nts w hic h were irra diate d; t he n a nti- T hy 1 a n d C treate d, ha pte n pri me d s plee n cells ( B cells) were a do ptively tra nsferre d

t o t h e s a m e ( a X b) F 1 reci pie nts. T he a ni mals were c halle nge d wit h t he ha pte n- protei n co nj u- gates a n d t he seco n dary a nti- ha pte n res po nse meas ure d as a n i n dicatio n of T- B cell coo perative i nteracti o ns. A d a pt e d fr o m K at z, D. H., H a m a o k a, T., D orf, M. E. a n d B e n a c err af, B. i n Pr o c. N atl. A c a d. S ci. ( U S A) 7 0, 2 6 2 4 ( 1 9 7 3). The Role of M H C Gene Products in I m mune Regulation 6 0 9

When an antigen un der Ir gene control was use d s uch as the co poly mer

G L T, (res po n der X n o n r e s p o n d e r ) F 1 T cells s pecific f or G L T hel pe d t he res pon der b ut not the nonres pon der ha pten s pecific B cells when challenge d wit h D N P- G L T ( 7 6), i n di c ati n g t h e criti c al r ol e of Ir g e n e e x pr essi o n i n B c ells, i n T c ell- B c ell i nt er a cti o ns ( Fi g. 5). T h e n e e d f or I r e gi o n i d e ntit y f or T c ell- B c ell i nt er a cti o ns w as c o nfir m e d b y S prent (77) an d Ka p pler an d Marrack (78) with different syste ms. More rece ntl y, C hiller, wor ki n g wit h clo nall y deri ve d a nti ge n s pecific hel per T cell li nes, obser ve d t he sa me I regio n re q uire me nt for s uccessf ul T- B cell i nteracti o n. T he data of Si n ger a n d Ho des (79) i n dicate t hat i n certai n ex peri me ntal c o n diti o ns w h er e t h e a nti b o d y r es p o ns e i n v ol v es s ol el y u n pri m e d B c ells of t h e Ly b5 p he noty pe, a ntibo dy res po nses may be hel pe d by T cells, across I diff er e n c es. It is cl e ar, h o w e v er, t h at t h e m aj orit y of t h e r es p o ns es of pri m e d B c ells r e q uir e t h e t y p e of I r e gi o n c o ntr oll e d T c ell- B c ell i nt er a cti o n dis c uss e d e arli er. T h e s p e cifi cit y of t h e i nt er a cti o n f or I a is d et er mi n e d b y t h e s p e cifi cit y

Fr o m K at z, D. H., H a m a o k a, T., D orf, M. E., M a ur er, P. H., a n d B e n a c err af, B. i n J. E x p. M e d. 1 3 8, 7 3 4 (1973). 6 1 0 Physiology or Medicine 1980 of t he T cell cl o nes sti m ulate d w he n a nti ge n is ori gi nall y prese nte d b y t he Ia bearing macrophages. W h at is t h e m e c h a nis m of I r e gi o n c o ntr oll e d T c ell- B c ell i nt er a cti o n a n d Ir ge ne f u ncti o n at t his le vel? T he data is n ot as defi niti ve as i n t he case of macro p ha ge- T cell i nteractio n disc usse d earlier. I feel, ne vert heless, t hat s u b- sta ntial e vi de nce exists for t he vie w t hat m uri ne L y l + T c ells ar e s p e cifi c f or a ntige n percei ve d i n t he co ntext of Ia molec ules o n a ntige n prese nti ng cells. T he cells are sti m ulate d to proliferate a n d differe ntiate i nto D T H or hel per T c ells. T h e h el p er T c ells will i n t ur n i nt er a ct wit h I a b e ari n g B c ells w hi c h bo u n d a ntige n t hro ug h t heir i m m u noglob ui n rece ptors. T he hel per T cells d eli v er t h eir diff er e nti ati n g si g n al b y i nt er a cti n g wit h a nti g e n a n d I a m ol e c ul es o n B c ell s i n a si mil ar m a n n er a s o n a nti g e n pr e s e nti n g c ell s.

T he la M olec ules are t he Ir Ge ne Pr o d ucts T here is no w s ubsta ntial a n d very co nvi nci ng evi de nce for t he vie w t hat Ia molec ules are t he Ir ge ne pro d ucts a n d deter mi ne s pecific i m m u ne res po nsive- ness to t hy m us de pe n de nt a ntige ns. 1) Ia molec ules, t he s urface glyco protei ns co m pose d of a a n d B c hai ns, ex presse d pri marily on a po p ulation of macro phages an d B ly m phocytes, are c o d e d f or i n pr e cis el y t h e s a m e s u br e gi o n of I i n w hi c h Ir g e n es m a p: I- A a n d I- E. 2) A nti-Ia a ntisera a n d partic ularly mo noclo nal a nti-I- A a ntibo dies s pecificall y bl oc k i n vitr o res po nses by i nteracti ng wit h Ia molec ules o n a ntige n pr es e nti n g c ells. 3) T he ma p pi n g of t he str uct ural ge nes co di n g for t he α a n d β c h ai ns of I a molec ules i n t he I re gio n i n dicates a n i nti mate correlatio n bet wee n -c hai n str uct ure a n d t he co ntrol of t he res po nse to G L Ø by co m ple me nti ng α a n d β g e n es i n I- A a n d I- E. T he str uct ural a nal ysis of Ia m olec ules a n d of t heir c o m p o ne nt c hai ns i n t he m o use was carrie d o ut b y se veral la b orat ories usi n g t he tec h ni q ues of 2 di me n- sio nal gel electro p horesis a n d pe pti de ma p pi n g to a nal yse t he basis of pol y mor- p his ms. Jo nes et al (80) ma de t he f u n da me ntal observatio n t hat, i n strai ns beari ng t he a p pro priate H-2 ha ploty pe, a ge ne i n t he I- E s ubregio n, co ntrols t he cell surface expression of an Ia molecule, whose poly morphic deter minants are largely co ntrolle d by t he I- A s ubregio n. Cook, Vitetta, U hr a n d Ca pra (81) a n d later Sil ver et al (82) co nfir me d t hese fi n di n gs a n d de mo nstrate d t hat t he 1- E s u br e gi o n c o ntr ols t h e s y nt h esis of t h e I a E a c h ai n w hi c h o n t h e c ell s urf a c e is no nco vale ntly associate d wit h t he β c hai n deter mi ne d i n t he I- A s ubregio n ( Fi gs. 2 a n d 6). Strai ns wit h t he H-2 b h a pl ot y p e i n t h e I- E s u br e gi o n f ail t o s y nt hesize t his a c hai n a n d as a co nse q ue nce t he corres po n di n g Ia molec ule is not ex presse d o n t he cell me mbra ne, alt ho ug h t he β c h ai n c o d e d i n I- A is s y nt h esi z e d a n d f o u n d i n t h e c yt o pl as m. T h e g e n eti c c o ntr ol of t h e p ol y p e pti d e c h ai ns of t his cl ass of I a m ol e c ul es b y I- E a n d I- A correlates co m pletely wit h t he Ir ge ne co m ple me ntatio n observe d i n t he res po nse to G L Ø disc usse d earlier (44). Moreover t hose strai ns t hat Ge nes

. .

ex hi bit a res p o n der a ge ne at I- E s y nt hesize a n a c hai n c o ntr olle d b y t his l oc us. It is i n d e e d f ort uit o us t h at w e c all e d t h e I- E g e n e a a n d t h e I- A g e n e b at a ti m e w h e n w e di d n ot k n o w t h at t h e y d et er mi n e d, r es p e cti v el y, t h e a a n d b c h ai ns of the corres pon ding Ia molec ule. T he a vaila bilit y of cl o ne d li nes of a nti ge n s pecific ‘I’ cells a n d of m o n ocl o nal a nti-Ia a ntibo dy pro vi de d still more co ncl usi ve e vi de nce for t he i de ntity be- t wee n Ia molec ules a n d Ir ge ne pro d ucts. Sre d ni, Sc h wartz a n d associates (63) clo ne d a G L Ø s pecific T cell li ne derive d fro m a B10. A(5 R) res po n der mo use w hic h was sti m ulate d o nly by G L Ø prese nte d o n B10. A(5 R) or ( B10 X B10. A) F, prese nti n g cells ( Ta ble 3). T he i n vitr o res po nse of t his clo ne to G L Ø was s pecificall y bl oc ke d b y a m o n ocl o nal a nti-Ia a nti b o d y s pecific f or t he co nfor matio nal deter mi na nts (83) on the Ia molec ules res ulting fro m the i nteracti o n of t he I- E c o de d α c h ai n wit h t h e I- A c o d e d β c h ai n. T he ot her major m uri ne Ia molec ule has bot h α a n d β c hai ns co de d for i n I- A. T he possi bilit y still exists for ge netic co m ple me ntatio ns at t he molec ular level corres pon ding with Ir gene co m ple mentation in ani mals heterozygo us at I- A, w hic h is precisely w hat occ urs. S uc h co m ple me ntatio n is more diffic ult to detect a n d de pe n ds u po n clo nal a nalysis of T cells s pecific for a n a ntige n t he res po nse to w hic h is co ntrolle d at I- A, s uc h as t he ter poly mer G A T. Clo ne d T c ell li n es s p e cifi c f or G A T w er e s el e ct e d b y F at h m a n et al ( 8 4) a n d b y Sr e d ni et al ( 6 3) fr o m a ( B 1 0. A X B10) F 1 mouse i m munize d with G A T. So me of these clones res pon de d to G A T when presente d by B10. A macro phages, other clones r es p o n d e d t o G A T o n B 1 0 m a cr o p h a g es a n d a t hir d t y p e of cl o n e r es p o n d e d t o the antigen only when presente d on ( Bl0. A x B10)F 1 macro p hages ( Table 7).

We ca n c o ncl u de t hat t he res p o nse t o G A T i n ( B 10. A X B 1 0 ) F 1 mi c e i s d et er mi n e d b y t hr e e t y p es of g e n eti c all y disti n ct I- A c o d e d I a m ol e c ul es w hi c h, 6 1 2 Physiology or Medicine 1980

to get her wit h a nti ge n, s pecificall y select t he t hree t y pes of clo nes sti m ulate d. T he exte nt to w hic h t hese t hree Ia molec ules i nteract wit h t he sa me deter mi- na nt o n t he G A T a nti ge n or wit h differe nt o nes has not bee n ascertai ne d.

Si g nific a nce of Ir Ge ne S pecificit y We have ma de co nsi derable progress i n o ur u n dersta n di ng: 1) of Ir ge ne f u nctio n i n a nti ge n prese nti n g cells a n d i n B cells, 2) of t he i de ntit y of Ia molec ules a n d Ir ge ne pro d ucts, a n d 3) of t he co m mit me nt of m uri ne T cells wit h t h e L y l + phenoty pe (85) to react with a utologo us Ia molec ules an d antigens. B ut an i m portant iss ue re mains unresolve d which concerns the pr ocess b y w hic h t he s pecificit y of Ir ge ne f u ncti o n is i m parte d, i.e., w h y certai n Ia molec ules o n a ntige n prese nti ng cells deter mi ne T cell res po nse to s o m e a nti g e ns a n d n ot t o ot h ers. T h e iss u e c a n als o b e pr es e nt e d i n ot h er ter ms, i.e. what mechanis m deter mines the develo p ment of T cell clones with co m- bi ne d s pecificit y for a utolo go us Ia molec ules a n d selecte d a nti ge ns. T c ells b e ar r e c e pt ors c o d e d at l e ast i n p art b y t h e i m m u n o gl o b uli n H c h ai n linkage gro u p as sho wn by Binz an d Wigzell (86), Eich mann (87) an d o ur la b orat or y (88-90). Ir ge nes d o n ot nee d t o be ex presse d i n T cells f or res po nses to occ ur. T cells beco me co m mitte d to host M H C s pecificities as t he y differe ntiate. T h us, no nres po n der pare ntal T cells ca n be t ur ne d i nto res po n der T cells by bei ng de velo pe d i n (res po n der X no nres po n der)F, irra diate d reci pi- e nts ( 7 3, 9 1). S u c h T c ells r es p o n d t o t h e p ut ati v e a nti g e n, if it is pr es e nt e d o n res po n der macro p hages beari ng t he a p pro priate Ia molec ules wit h w hic h t he T cells i nteracte d d uri n g differe ntiati o n. The Role of M H C Gene Products in I m mune Regulation... 6 1 3

T wo ty pes of hy pot heses have bee n for m ulate d to acco u nt for Ir ge ne co ntrolle d restrictio ns. vo n Boe h mer, Haas a n d Jer ne (92) pro pose d t hat T cells ge nerate t heir re pert oire f or f orei g n a nti ge ns fr o m t heir rece pt ors f or a utolo go us M H C a nti ge ns a n d t hat Ir ge ne defects reflect t he a bse nce of clo nes beari n g rece pt ors f or certai n a nti ge ns, base d u p o n t he restricti o n place d o n t he re pertoire b y t he co m mit me nt of T cells to a partic ular set of a utolo go us M H C a nti ge ns. As T cells differe ntiate a n d are selecte d t o react wit h differe nt M H C a nti ge ns i n differe nt i n di vi d uals, differe nt H-li n ke d Ir ge ne defects res ult. An alternative hy pothesis was pro pose d in de pen dently by Rosenthal (93) a n d m yself (94). It p ost ulates: 1) t hat Ia molec ules are ca pable of reacti ng selecti vely wit h certai n a mi no aci d seq ue nces o n protei n a ntige ns, 2) t hat s uc h a selecti ve i nteracti o n i n a nti ge n prese nti n g cells res ults i n t he for matio n of a n Ia molec ule-a nti ge n co m plex reacti ve wit h T cell clo nes differ- e ntiate d t o bear rece pt ors f or a ut ol o g o us Ia a n d a nti ge n ( Fi g. 7). A li mite d n u mber of s uc h bi n di n g sites o n a relati vel y s mall n u mber of Ia molec ules ca n ge nerate fro m available a ntige ns a n al most u nli mite d n u mber of deter mi na nts s pecificall y rec o g nize d b y T cells. T he size of t he bi n di n g site o n t he Ia molec ule for t he a ntige n or its frag me nt s ho ul d e nco m pass a li mite d n u m ber of a mi no aci ds i n or der not to i m pose u n d ue restrictio ns o n t he s yste m a n d t o per mit a gi ve n site t o bi n d t o a great variet y of f orei g n pr otei ns. T he li keli hoo d t hat s uc h a se q ue nce is prese nt i n a gi ve n protei n varies i n versel y wit h t h e si z e of t h e s e q u e n c e a n d is c o nsi d er a bl e f or a p ost ul at e d si z e i n v ol vi n g t hr e e or at m ost f o ur a mi n o a ci ds. T h e l o c ati o n of t h e bi n di n g sit es will v ar y i n differe nt protei ns. T his dictates t he a nti ge nic deter mi na nts wit h w hic h T cells react i n co nj u nctio n wit h Ia molec ules. A gi ve n Ia molec ule co ul d t h us react wit h a lar ge n u m ber of a nti ge ns a n d yet i m pose u ni ge nic restrictio n to i m m u ne res ponsiveness. T he i de ntificatio n of t he a mi no aci d seq ue nces critical for i m m u noge nicity m a y pr o vi d e s o m e i n di c ati o n of t h e si z e of t h e p ost ul at e d sit e of i nt er a cti o n wit h t he Ia molec ules. T he ele ga nt st u dies of Sc hloss ma n a n d associates (95, 96) o n the i m m unogenicity of D N P-oligo- L-lysines for strain 2 g uinea pigs is very infor mative. The s mallest oligolysine poly mer which is i m m unogenic has seven lysi ncs. Ho we ver, a pe pti de co ntai ni ng o nly fo ur lysi nes a n d a se q ue nce of L- alanines, ter minate d with a D N P-lysinc, is eq ually i m m unogenic in strain 2 g ui nea pi gs, alt h o u g h t he s pecificit y of t he res p o nse is differe nt. It w o ul d a p pear t heref ore t hat t he critical i nteracti o n site i n t his a nti ge n f or strai n 2 g ui n e a pi gs m a y c o nsist of at m ost 4 l ysi n es. T h e d at a o n t h e i m m u n o g e ni cit y of i ns uli n A c h ai n f or str ai n 2 g ui n e a pi gs a n d of B c h ai n f or str ai n 1 3 g ui n e a pi gs (68) a n d o n t he precise a mi n o aci ds res p o nsi ble s u g gest als o a n i nteracti o n site i n v ol vi n g 3 or at m ost 4 a mi n o aci ds. Alt ho ug h t he seco n d hy pot hesis a p pears more co m patible wit h t he available fi n di ngs, defi nite e vi de nce of Ia molec ule-a ntige n i nteractio n is lacki ng. So me recent ex peri ments of Ne po m an d Ger main in o ur laboratory may also be i nter prete d to i n dicate a necessary i nteractio n bet wee n Ia molec ule a n d a ntige n f or bi n di n g of t h e c o m pl e x t o T c ells. W e h a v e i n d e e d o bs er v e d t h at w h e n F 1 T cells s pecific far a nti ge ns u n der Ir ge ne co ntrol s uc h as G A T or G L Ø are sti m ulate d to proliferate by a ntige n, t hey selecti vely bi n d a utologo us Ia mole- c ules of precisely t he ty pe w hic h deter mi ne d res po nsive ness to t he a ntige n. C o nsi d eri n g t h at s o m e of t h e T c ells i n t h e p o p ul ati o n st u di e d s h o ul d h a v e b e e n s p e cifi c f or t h e all eli c I a m ol e c ul e, t h e s el e cti v e bi n di n g o bs er v e d m a y i n d e e d i m ply a req uisite i nteractio n bet wee n Ia molec ules o n macro p hagcs a n d a nti- g e n.

I Regio n Co ntrol of T Cell S uppressor Respo nses Selecte d a ntige ns s uc h as t he ter poly mer G A T i n d uce prefere ntially s u p pressor T cells i n certai n no nres po n der mo use strai ns, w hic h co ntrib ute to t he u nre- s po nsi ve ness obser ve d (97). T hese T cells, a do pti vely tra nsferre d, s u p press t he anti- G A T antibo dy response to G A T co uple d to an i m m unogenic carrier. Prefere ntial s u p pressor T cell res po nses were also obser ve d for ot her a ntige ns s uch as the co poly mer G T (98) by us an d hen egg lysozy me by Scrcarz an d ass ociates (99). T h e a bilit y t o d e v el o p s p e cifi c s u p pr ess or T c ells als o pr o v e d t o b e c o ntr oll e d i n t h e I r e gi o n of t h e m uri n e H- 2 c o m pl e x ( T a bl e 8). T h e a n al ysis of t h e g e n es res p o nsi ble, of t heir pr o d ucts, a n d of t he pr ocesses i n v ol ve d i n t he ge nerati o n of s pecific s u p pressor T cells has not yet per mitte d a defi niti ve u n dersta n di n g of t h es e c o m pl e x p h e n o m e n a. As i n t h e c as e of I r e gi o n c o ntr ol of T c ell r es p o ns es, i m m unogenicity of a co m plex antigen is deter mine d by the deter minants it bears. A p pare ntly certai n deter mi na nts i n mice of t he a p pro priate H-2 ha ploty pe i n d uce selectively s u p pressor T cell res po nses (99, 100). Moreover, t he prese nce of deter mi na nts w hic h sti m ulate s u p pressor T cells prefere ntially may res ult i n s u p pressio n of res po nses to ot her deter mi na nts o n t he a ntige n w hic h ot her wise wo ul d sti m ulate hel per T cells a n d t hereby a ntibo dy res po nses. I n a d ditio n to t his t y pe of I re gio n co ntrol of s u p pressor T cell res po nses The Role of M H C Gene Products in I m mune Regulation... 6 1 5

N ot e: 100 µg G T was a d mi nistere d i ntra perito neally; 3 days later t he ex peri me ntal a n d matc he d control gro u ps were i m m unize d with G T co u ple d to the i m m unogenic carrier, methylate d b o vi n e s er u m al b u mi n, a n d t h e a nti b o d y r es p o ns es c o m p ar e d t o d et er mi n e t h e s u p pr essi o n eli cit e d b y G T pr ei m m u ni z ati o n. Do minant G T specific suppression can be generate d in mice with H-2 d , H-2’ an d H-2” b ut not t he H-2” or H-2 b ha ploty pes. T he res po nsible ge nes ma p pe d i n t he I regio n. I n tra nsfer experi ments the GT suppression observed was sho wn to be mediated by suppressor T cells. A d a pt e d fr o m D e br é, F., K a p p. J., D orf, M., a n d B e n a c err af, B. i n J. E x p. M e d. 1 4 2, 1447 (1975).

w hic h is s pecific a n d deter mi na nt orie nte d, t he I regio n affects s u p pressor T cell res po nses b y co di n g for s pecificities ex presse d o n all s u p pressor T cells. M ur p h y et al (101), a n d Ta da et al (102, 103) disc o vere d t hat a ne w s ubregio n of I, t he I-J, co ntrols alloa ntige ns ex presse d o nly o n s u p pressor T c ells. All t h e T c ells i n t h e s u p pr ess or T c ell cir c uits b e ar I-J c o d e d s p e cifi c- ities. Moreo ver, a ntige n s pecific s u p pressor factors were extracte d fro m a ntige n s p e cifi c s u p pr ess or T c ells ( 1 0 4, 1 0 5). S u c h f a ct ors w er e s h o w n t o sti m ul at e t h e ge neratio n of s u p pressor T c ells ( 1 0 6) a n d to bear o n t he sa me molec ule deter mi na nts co de d by t he I-J s ubregio n as well as i dioty pic deter mi na nts co de d for by the i m m unologlob ulin heavy chain linkage gro u p (88, 89). The str uct ure of s u p pressor factor a n d t he nat ure of t he cell ular i nteractio ns w hic h res ult i n s u p pressor T cell res po nses are c urre ntly u n der i n vestigatio n i n se veral laboratories.

T he F u ncti o n al S pecificit y of T Cells f or t he A nti ge ns of t he M H C - T he Ori gi n of All ore acti vit y I n a prece di n g sectio n we disc usse d t he co m mit me nt of hel per a n d D T H T cells to react with antigen an d a utologo us Ia molec ules. An analogo us co m mit ment of cytolytic T cells ( C T L) to histoco m patibility a ntige ns of t he M H C has bee n de monstrate d by Zinkernagel an d Doherty (107) by Shearer et al (108) an d b y Be va n (109) ill ustrati n g t he ge neral nat ure of t he co m mit me nt of T l y m p hocytes to react wit h a ntige n o nly o n cell s urfaces a n d i n relatio n wit h ge ne pro d ucts of the M H C. Zi nker nagel a n d Do herty ( 107) d e monstrated that CTL fro m mice i m mune 6 1 6 Physiology or Medicine 1980 to Ly m phocytic chorio meningitis vir us ( L C M) only lyse L C M infecte d target cells w hic h s hare H-2 a ntige ns wit h t he killer cell. T he M H C loci co ncer ne d ma p at eit her K or D of t he H-2 co m plex. T h us, C T L recog nize a ntige n i n t he co ntext of t he K or D histoco m patibilit y a nti ge ns, li ke hel per cells react wit h a nti ge n a n d Ia molec ules. T he e v ol uti o nar y si g nifica nce of t hese restricti o ns a n d of t he r ole pla ye d b y M H C a ntige ns beco mes rea dily a p pare nt w he n we co nsi der t hat T cell i m m u ne res po nses are pri marily res po nsible for mo nitori ng self a n d no nself o n cell s urfaces. T cells nee d to deter mi ne w he n a n a utologo us cell beco mes malig na nt or virall y i nfecte d a n d m ust be destr o ye d. T his s ur veilla nce f u ncti o n is o pti mal- l y perf or me d if a lar ge n u m ber of T l y m p h oc yte cl o nes are s pecialize d t o detect s mall varia nts o n M H C molec ules. Taki ng a dva ntage of t his process, T cells ha ve also e vol ve d t he ca pacity to reg ulate i m m u ne res po nses as a co nseq ue nce of t heir ability to recog nize a ntige n o n cell me mbra nes. Beca use u nreg ulate d i m m une res ponses can be very har mf ul, we have develo pe d highly s pecific T cell me diate d mechanis ms of i m m une reg ulation which req uire the recognition b y T cells of cl o nes of ot her i m m u ne cells beari n g a nti ge n. Beca use of t he t wo ty pes of M H C s pecificities ex hibite d by hel per a n d.. cytolytic T ly m p hocytes, t wo ty pes of Ir ge ne defects ca n be obser ve d i n C T L res p o nses. A maj or t y pe of Ir ge ne defect ma ps i n t he I re gi o n (92, 110) a n d c o ncer ns t he ge nerati o n of hel per cells, as i n t he case of a nti b o d y. T he ot her t y p e m a ps i n K ( 1 1 1) or D a n d r efl e cts t h e a bilit y of C T L cl o n es t o r e a ct wit h a nti ge ns o n cell s urfaces as t he y are prese nte d i n relatio n wit h K or D ge ne pr o d ucts. T h e m aj or t o pi c of t his p a p er h as b e e n t h e s p e cifi cit y of T c ells f or a ut ol o g o us M H C a ntige ns a n d t he ma n ner i n w hic h foreig n a ntige ns are perceive d by T cells i n t he co ntext of M H C ge ne pro d ucts. We post ulate t hat M H C a ntige ns ha ve e vol ve d for precisel y t his f u nctio n. Yet t he y ha ve bee n ori gi nall y disco v- ere d b y Corer et al (112) a n d i de ntifie d i n a differe nt co ntext as t he major a ntige ns, wit hi n a s pecies, res po nsible for alloreacti vity a n d t he rejectio n of allografts, a pheno menon which is of li mite d evol utionary val ue. The iss ue of t he ori gi n of all oreacti vit y ca n n o w be a p pr o priatel y a d dresse d as it a p pears t o be cl osel y relate d t o t he pr ocess w here b y T cells bec o me co m mitte d as a class to reactivity with a utologo us M H C antigens d uring differe ntiati o n. Jerne (113) pro pose d a theory which was f urther elaborate d by o urselves (114, 115) t o ex plai n t he ge nerati o n i n t he t h y m us of T cells s pecific f or a utologo us M H C a ntige ns. Accor di ng to t he t heory, i n t he first stage T cells i nitially s pecific for self M H C ge ne pro d ucts are selecte d i n t he t hy m us to differe ntiate a n d pr oliferate. T he n, i n a sec o n d sta ge, o nl y t h ose T cells w hic h bear lo w affi nity rece ptors for self M H C a ntige ns are allo we d to mat ure a n d l e a v e t h e t h y m us as f u n cti o n al T c ells. S u c h T c ells, h a vi n g l o w r e a cti vit y f or self M H C a ntige ns, have co nco mita ntly hig h affi nity for varia nts of self M H C a ntige ns. T hese varia nts a p pear to be t he sa me or si milar to t he alloge neic M H C a ntige ns ex presse d i n t he sa me s pecies. Weaker affi nity for xe noge neic M H C antigens would thus be expected. Si multaneously and independently 6 1 7

these T cells develo p recognition for deter minants on conventional thy m us de pen dent antigens. T he hig h degree of reactivity to M H C a ntige ns w hic h co nstit ute t he poly- mor p hic po p ulatio n e nco u ntere d i n t he sa me s pecies (i.e. alloa nti ge ns) a n d t he l o w er r e a cti vit y t o x e n o g e n ei c M H C a nti g e ns m a y b e attri b ut e d t o t h e f a ct t h at lo w affi nity rece ptors for self M H C a ntige ns arc ex pecte d to react o pti mally wit h alloge neic M H C a ntige ns, b ut m uc h less so wit h xe noge neic a ntige ns. T his w o ul d acc o u nt f or t he para d ox t hat t he str o n gest T cell res p o nses arc n ot elicite d by a ntige ns f urt her re move d p hyloge netically fro m t he res po n der. T wo pre dicti o ns fr o m t his t he or y are: (a) t hat cl o nes of T cells i n d uce d b y xe n o ge- neic M H C antigens sho ul d be highly cross-reactive with allogeneic M H C a nti ge ns, e ve n to t he exte nt t hat t he y ma y de mo nstrate a heteroclitic s pecificity. T his has i n dee d bee n de mo nstrate d by B urakoff et al. w he n mo use a nti-rat C T L were s ho w n to be co m prise d of clo nes cross-reacti ve wit h allo ge neic tar get c ells ( 1 1 6) ( T a bl e 9). ( b) All or e a cti v e T c ells s h o ul d b e e x p e ct e d t o b e hi g hl y. cr oss-r e a cti v e wit h m o difi e d s y n g e n ei c c ells. T his w as als o s h o w n t o b e t h e c as e w he n we observe d co nsi derable cross-reactivity by alloreactive cells for T N P co nj u gate d tar get cells s y n ge neic to t he res po n der (114) ( Table 10).

N ot e: C57BL/6 ( H-2 b ) s pl e e n c ells w er e s e nsiti z e d wit h r at s pl e e n c ells of t h e L e wis str ai n. T h e C T L were assa ye d f or s pecific 5 1 Cr release o n xe n o ge neic sti m ulat or rat cells or o n all o ge neic B10. B R ( H-2 k ) or B10. D2 ( H-2”) mo use cells. I dentical res ults were obtaine d with A CI an d B N sti m ul at ors. A d a pt e d fr o m B ur a k off, S.J, R at n ofs k y, S. E., a n d B e n a c err af, B. i n Pr o c. N atl. A c a d. S ci. ( U S A) 7 4, 4 5 7 2 ( 1 9 7 7).

Table I O. Cytolysis of Syngeneic T N P- Mo difie d Taracts by Allogenically Sti m ulate d Cytolytic T C ells

Responder Sti m ulator 6 1 8 Physiology or Medicine 1980

Si nce t he T cell re pertoire for M H C s pecificities is nor mally deter mi ne d by t h e s elf M H C a nti g e ns of t h e t h y m us, w e s h o ul d e x p e ct t h e T c ell r e p ert oir e t o v ar y a c c or di n g t o t h e M H C of t h e t h y m us i n w hi c h T c ells diff er e nti at e. R e c e nt ex peri me nts utilizi ng ra diatio n c hi meras by Zi nker nagel a n d associates (117) a n d Be va n (118) ha ve de mo nstrate d t his to be t he case. T he post ulate t hat alloreacti vit y res ults fro m T cells differe ntiati n g i n t he t h y m us t hat are stro ngly reacti ve for varia nts of self M H C a nti n ge ns lea ds to t he ex pectatio n t hat i m m u nizatio n wit h virally i nfecte d sy nge neic cells s ho ul d res ult i n t he sti m ulati o n of T cell cl o nes rescti ve wit h t he virall y i nfecte d sy nge neic cells use d to i m m u nize a n d also reactive wit h u ni nfecte d alloge neic t ar g et c ells. Fi nberg et al i n o ur laboratory have rece ntly s ho w n t hat i m m u nizatio n of B ALB/c ( H-2 d ) mice wit h Se n dai coate d sy nge neic cells sti m ulates C T L w hic h lyse Se n dai coate d B A L B /c target cells b ut also lyse u ncoate d H-2 b , H- 2 q , H- 2 k , H- 2 S a n d H-2’ allo ge neic tar get cells to a n a p precia ble de gree (119) ( Tabell 11). We f urt her de mo nstrate d by t he col d target i n hibitio n tec h niq ue t hat t he sa me clo nes t hat lyse d B A L B / c coate d Se n dai targets also crossreacti- vely lyse d t he alloge neic targets (Fig. 8). F urt her more, it was obser ve d t hat . . se parate C T L cl o nes l yse d eac h of t he differe nt all o ge neic tar gets. I n a d diti o n, t h er e w as si g nifi c a ntl y l ess l ysis of t ar g et c ells b e ari n g t h e H- 2 q ha ploty pe than of t ar g et c ells b e ari n g t h e H- 2 k or H-2” ha plot y pes. T his latter fi n di n g s u g gests t hat t he associatio n of Se n dai vir us a ntige ns wit h t he H-2 d ge ne pro d ucts of B A L B/c mice creates deter minants which are more crossreactive with H-2 k a n d H-2’ t ha n wit h H-2 q ge ne pro d ucts. Usi n g clo ne d T cell li nes, t he all oreacti vit y of C T L s pecific f or virall y i nfecte d s y n ge neic cells was c o nfir me d by vo n Boc h mer et al (120) a n d exte n de d by Sre d ni a n d Sc h wartz (121) to T cells reacti ve wit h a utologo us I regio n pro d ucts a n d foreig n a ntige ns. B10. A T cells specific for D NP-ovalbu min ( D NP-ova) were clone d on D NP-ova pulse d macro p hages. S uc h clo ne d li nes proliferate d s pecifically w he n ex pose d to D N P- o v a o n s y n g e n ei c m a cr o p h a g es. S o m e of t h es e cl o n es c o ul d als o b e sti m ul at e d t o proliferate by H-2” alloge neic macro p hages i n t he abse nce of D N P-ova ( Table 12). T herefore t he sa me clo ne selecte d o n D N P-ova p ulse d B 10. A macro p hages reacte d i dentically to D NP-ova pulse d B10. A macrophages or B10.S macro-

N ot e: A d a pt e d fr o m Fi n b er g, R. S., B ur a k off, S. J., C a nt or, H. a n d B e n a c err af, B. i n Pr o c. N atl. A c a d S ci. ( U S A) 7 5, 5 1 4 5 ( 1 9 7 8). 6 1 9

Fi g ure 8. Mat ure c yt ol ytic T cells s pecific f or Se n dai i nfecte d s y n ge neic tar get cells cr ossreacti vel y l yse no ni nfecte d allo ge neic tar get cells. Usi n g col d tar get i n hi bitio n of l ysis b y C T L, t his ex peri- me nt ill ustrates t hat disti nct p o p ulati o ns of Se n dai s pecific C T L l yse differe nt all o ge neic tar gets. A d a pt e d fr o m Fi n b er g, R. S., B ur a k off, S. J., C a nt or, H.. a n d B e n a c err af. B.. i n Pr o c. N atl. A c a d. S ci. ( U S A) 7 5, 5 1 4 5 ( 1 9 7 8). p hages wit ho ut a ntige n. I n bot h cases t he ability to sti m ulate ma p pe d i n t he I- A s u bre gio n de mo nstrati n g t hat alloreacti vit y to I re gio n a nti ge ns also arises as a c o ns e q u e n c e of t h e c o m mit m e nt of T c ells t o a ut ol o g o us M H C s p e cifi citi es. T he re mai ni n g iss ue co ncer ns t he precise nat ure of t he T cell rece ptor a n d ho w t he s pecificity for foreig n a ntige ns a n d M H C co de d molec ules is co nco mi-

Table 12. Bl0. A Clo nes S pecific for A nti ge n a n d Self Ia Ma y Also Be Selecti vel y Alloreacti ve

So urce of s plee n cells H- 2 Proliferative res ponse ( C P M) 6 2 0 Physiology or Medicine 1980 t a ntl y m ai nt ai n e d. O n t h e b asis of i di ot y pi c a n d g e n eti c e vi d e n c e, t h e v ari a bl e regio ns of i m m u noglob uli n hea vy c hai ns a p pears to be res po nsible for bot h t he s pecificity directe d to M H C a n d t he s pecificity for foreig n deter mi na nts, w he n a nalyze d i n de pe n de ntly. T he proble m still re mai ns w het her T cells have o ne rece ptor or t wo co u ple d rece ptors a n d w het her o ne or t wo V H re gi o ns are i nvolve d. Moreover, t he sig nifica nce of I regio n co de d deter mi na nts o n a ntige n-s pecific, i di ot y pc beari n g re g ulat or y pr o d ucts o n T cells m ust be clarifie d. I a m not te m pte d to g uess at t he a ns wer co nsi deri n g t he prese nt a vailabilit y of cl o n e d li n es of s p e cifi c T c ells, a n d of T c ell h y bri ds. A d efi niti v e a ns w er s h o ul d be fort hco mi ng fro m t he laboratory, a n d t he ge nes co di ng for t he T cell r e c e pt ors will s o o n b e i d e ntifi e d.

Co ncl usio ns T he evol utio nary sig nifica nce of t he co m mit me nt of T cells to M H C a ntige ns s ho ul d be assesse d fro m se veral va nta ge poi nts. Fro m t he poi nt of vie w of t he i n divi d ual co ncer ne d, t he existe nce of s uc h a broa dly poly mor p hic syste m to deter mine s pecific res ponsiveness an d s u p pression will unesca pably res ult in i n divi d uals wit h differe nt i m m u nological pote ntial to a give n c halle nge. So me.. will cl e arl y b e at gr e at er ris k, w h er e as ot h ers will b e b ett er pr e p ar e d t o r esist t o certai n i nfectio us a ge nts, a n d it is not s ur prisi n g t hat i m m u nolo gical diseases are li nke d to t he M H C. As far as t he s pecies is co ncer ne d, t his poly mor p hic defe nse syste m res ults i n a very sig nifica nt s ur vi val a d va ntage to u nforesee n c h all e n g es a n d a b ett er p ossi bilit y f or t h e i m m u n e s yst e m t o a d a pt t o e v ol uti o n- ar y press ures. As biologists we conte m plate with a d miration an d a we the won dro us array of s o p histi c at e d c ell i nt er a cti o ns a n d r e c o g niti o ns e v ol v e d i n t h e T c ell i m m u n e s yst e m w hi c h m ust b e a m o d el f or ot h er si mil arl y c o m pl e x bi ol o gi c al s yst e ms of hig hly differe ntiate d orga nis ms.

A C K N O W L E D G E M E N T S

I wo ul d li ke to ex press m y dee p a p preciatio n a n d m y profo u n d affectio n for m y n u mero us st u de nts a n d associates w ho have s hare d wit h me i n t he toil a n d s ho ul d no w e q ually s hare i n t he ho nor. I a m also dee ply gratef ul to t he Natio nal I nstit ute of Allergy a n d I nfectio us Diseases w hic h s u p porte d my work fait hf ull y a n d ge nero usl y si nce 1957, a n d ho use d me fro m 1968 to 1970, a n d to the National Cancer Instit ute which s u p porte d my progra m for the last ten years. I o we also a debt of gratit u de to Ne w York U ni versity Me dical Sc hool an d to Harvar d Me dical School which provi de d the sti m ulating aca de mic environ ment witho ut which o ur work co ul d not have progresse d. T he R ole of M H C Gene Products in I m mune Regulation... 6 2 1

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8 2. Sil v er, J., R uss ell, W. A., R eis, B. L., a n d Fr eli n g er, J. A. Pr o c. N atl. A c a d. S ci. 7 4, 5 1 3 1, 1 9 7 7. 8 3. L er n er, E. A., M atis, L. A., J a n e w a y, C. A., Jr., J o n es, P. P., S c h w art z, R. H., a n d M ur p h y, D. B. J. E x p. M e d. 1 5 2, 1 0 8 5, 1 9 8 0. 8 4. Ki m ot o, M., a n d F at h m a n, C. G. J. E x p. M e d. 1 5 2, 7 5 9, 1 9 8 0. 8 5. C a nt or, H., a n d B o ys e, E. A. J. E x p. M e d. 1 4 1, 1 3 9 0, 1 9 7 5. 8 6. Bi n z, H., a n d Wi g z ell, H. J. E x p. M e d. 1 4 2, 1 9 7, 1 9 7 5. 8 7. Ei c h m a n n, K. A d v. I m m u n ol. 2 6, 1 9 5, 1 9 7 8. 8 8. Gr e e n e, M. I., B a c h, B. A., a n d B e n a c err af, B. J. E x p. M e d. 1 4 9, 1 0 6 9, 1 9 7 9. 8 9. G er m ai n, R. N., J u, S- T., Ki p ps, T. J., B e n a c err af, B., a n d D orf, M. E. J. E x p. M e d. 1 4 9, 6 1 3, 1 9 7 9. 9 0. W ei n b er g er, J. Z., Gr e e n e, M. I., B e n a c err af, B., a n d D orf, M. E. J. E x p. M e d. 1 4 9, 1 3 3 6, 1 9 7 9. 9 1. L o n g o, D. L., a n d S c h w art z, R. H. F e d. Pr o c. 3 9, 1 1 2 7, 1 9 8 0. 9 2. v o n B o e h m er, H., H a as, W., a n d J er n e, N. K. Pr o c. N atl. A c a d. S ci. 7 5, 2 4 3 9, 1 9 7 8. 9 3. R os e nt h al, A. S. I m m u n ol. R e v. 4 0, 1 3 5, 1 9 7 8. 9 4. B e n a c err af, B. J. I m m u n ol. 1 2 0, 1 8 0 9, 1 9 7 8. 9 5. S c hl oss m a n, S. F. Tr a ns pl a nt. R e v. 1 0, 9 7, 1 9 7 2. 9 6. Y ar o n, A., D u n h a m, E. K., a n d S c hl oss m a n, S. F. Bi o c h e mistr y 1 3, 3 4 7, 1 9 7 4. 9 7. K a p p, J. A., Pi er c e, C. W., a n d B e n a c err af, B. J. E x p. M e d. 1 4 0, 1 7 2, 1 9 7 4. 9 8. D e br é, P., W alt e n b a u g h, C., D orf, M. E., a n d B e n a c err af, B. J. E x p. M e d. 1 4 4, 2 7 2, 1 9 7 6. 9 9. A d ori ni, L., H ar v e y, M. A., Mill er, A., a n d S er c ar z, E. E. J. E x p. M e d. 1 5 0, 2 9 3, 1 9 7 9. . . 1 0 0. S c h w art z, M., W alt e n b a u g h, C., D orf, M., C esl a, R., S el a, M., a n d B e n a c err af, B. Pr o c. N atl. A c a d. S ci. 7 3, 2 8 6 2, 1 9 7 6. 1 0 1. M ur p h y, D. B., H er z e n b er g, L. A., O k u m ur a, K., H er z e n b er g, L. A., a n d M c D e vitt, H. O. J. E x p. M e d. 1 4 4, 6 9 9, 1 9 7 6. 1 0 2. T a d a, T., T a ni g u c hi, M., a n d D a vi d, C. S. J. E x p. M e d. 1 4 4, 7 1 3, 1 9 7 6. 1 0 3. T a d a, T., T a ni g u c hi, M., a n d D a vi d, C. S. C ol d S pri n g H ar b or S y m p. Q u a nt. Bi ol. 4 1, 1 1 9, 1 9 7 6. 1 0 4. T a d a, T., T a ni g u c hi, M., H a y a k a w a, K., a n d Ok u m ura, K. I n: T a n d B Ly m p hocytes: R e c o g niti o n a n d F u n cti o n E. S. Vit ett a a n d C. F. F o x, e ds., A c a d e mi c Pr ess, N e w Y or k p. 2 9 3, 1 9 7 9. 1 0 5. T ht z e, J., K a p p, J. A., a n d B e n a c err af, B. J. E x p. M e d. 1 4 5, 8 3 9, 1 9 7 7. 1 0 6. W alt e n b a u g h, C., T ht z e, J., K a p p,J. A., a n d B e n a c err af, B. J. E x p. M e d. 1 4 6, 9 7 0, 1 9 7 7. 1 0 7. Zi n k er n a g el, R. M., a n d D o h ert y, P. C. J. E x p. M e d. 1 4 1, 1 4 2 7, 1 9 7 5. 1 0 8. S h e ar er, G. M., R e h n, T. G., a n d G ar h ari n o, C. A. J. E x p. M e d. 1 4 1, 1 3 4 8, 1 9 7 5. 1 0 9. B e v a n, M. J. J. E x p. M e d. 1 4 2, 1 3 4 9, 1 9 7 5. 1 1 0. Si m ps o n, E., a n d G or d o n, R. D. I m m u n ol. R e v. 3 5, 5 9, 1 9 7 7. 1 1 1. Zi n k er n a g el, R. M., Alt h a g e, A., C o o p er, S., Kr e e b, G., Kl ei n, P. A., S eft o n, B., Fl a h ert y, L., Sti m pfli n g, J., S hr e m er, D., a n d KI ei n,J. J. E x p. M e d. 1 4 8, 5 9 2, 1 9 7 8. 1 1 2. G or er, P. A., L y m a n, S., a n d S n ell, G. D. Pr o c. R o y. S o c. L o n d. [ Bi ol.] 1 3 5, 4 9 9, 1 9 4 8. ‘ 1 1 3. Jer n e, N. K. E ur. J. I m m u n ol. 1, 1, 1 9 7 1. 1 1 4. L e m o n ni er, F., B ur a k off, S. J., G er m ai n, R. N., a n d B e n a c err af, B. Pr o c. N atl. A c a d. S ci. U S A 7 4, 1 2 2 9, 1 9 7 7. 1 1 5. B ur a k off, S. J., Fi n b er g, R., Gli m c h er, L., L e m o n ni er, F., B e n a c err af, B., a n d C a nt or, H. J. E x p. M e d. 1 4 8, 1 4 1 4, 1 9 7 8. 1 1 6. B ur a k off, S. J., R at n ofs k y, S. E., a n d B e n a c err af, B. Pr o c. N atl. A c a d. S ci. U S A 7 4, 4 5 7 2, 1 9 7 7. 1 1 7. Zi n k er n a g el, R. M., Alt h a g e, A., C o o p er, S., C all a h a n, G., a n d Kl ei n, J. J. E x p. M e d. 1 4 8, 8 0 5, 1 9 7 8. 1 1 8. B e v a n, M. J, N at ur e 2 6 9, 4 1 7, 1 9 7 7. 1 1 9. Fi n b er g, R., B ur a k off, S., C a nt or, H., a n d B e n a c err af, B. Pr o c. N atl. A c a d. S ci. 7 5, 5 1 4 5, 1 9 7 8. 1 2 0. v o n B o e h m er, H., T urt o n, K., a n d H a as, W. E ur. J. I m m u n ol. 9, 5 9 2, 1 9 7 9. 1 2 1. Sr e d ni, B., a n d S c h w art z, R. H. I m m u n ol. R e v. 5 4, 1 8 7, 1 9 8 1.