Targets for killer T cells.

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Human Lymphocyte Differentiation Its Application to Cancer INSERM Symposium No 8

Editors Β Serrou and C Rosenfeld

TARGETS FOR KILLER Τ CELLS

BEN A. BRADLEY, ELS GOULMY, IEKE SCHREUDER and JON J. VAN ROOD

Department of Immunohaematology, University Hospital, Leiden (The Netherlands)

In tumour immunity as in allograft immunity killer Τ cells are thought to play an essential role in the response . Although more is known of the target antigens in the latter case, the gene products involved have never been p r e -cisely defined. The main tool for the investigation of these antigens is the cell mediated lympholysis (CML) assay, in which allogeneic, PHA-induced, blast cells (T cells) were used as targets . Generalizations deduced from this assay can thus be applied to situations involving killer Τ cells and other foreign tissues, only after due consideration of the phenotypic differences existing between these and PHA-blasts.

It is generally agreed that when a CML test is performed between two random-ly selected individuals most of the structures recognized a r e associated with

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gene products in the H L A region . Fmrthermore it is strongly argued that the HLA-A, -B and -C antigens defined by tissue typing sera and complement are the antigens involved . These are hereafter referred to as HLA-SD antigens. Despite these claims the data remains ambiguous in the sense that some foreign HLA-SD antigens elicit weak o r zero killer cell response

In addition to these apparent anomalies w e and others ' ' have found that the association of the target antigen with HLA-SD specificities is often incomplete in that not all target cells which carry the appropriate antigen in an unrelated panel a r e killed and when segregation studies are performed in families the pattern obtained is untypical of HLA-SD antigens which normally segregate in a codominant w a y . Α recessive pattern may, in theory, have account-ed for this, because in the mouse there existaccount-ed a recessive H-2 associataccount-ed antigen, the haemopoietic resistence (Hh) antigen which segregated in a re-cessive manner and which w a s identifiable by CML ' . However, no such pattern has y e t been observed with H L A associated CML antigens in m a n , neither did it f i t o u r data .

Recent new ideas, which had their conceptual origins in certain mouse expe-riments have led us to reexamine the assumption that HLA-SD antigens a r e Τ cell targets. In these mouse experiments foreign minor hιstocompatibi1ity anti-gens w e r e shown to be capable of eliciting killing reactions only if they w e r e accompanied on the target cell by H-2D and Κ (HLA-SD equivalent) antigens of

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232

the ' s e i f type. Such antigens w e r e expressed on the effector and target ce)) and could thus be excluded as the targets in t h e m s e l v e s . Nevertheless both

' s e i f H-2D and/or Κ and the minor 'foreign' antigen was required for killing 22 23

and< neither alone eticited a killing response ' .

Following these observations an opportunity arose in man to study a simllar phenomenon and the results formed the basis of the classification of antigens proposed h e r e .

Initially a patient (Mrs. R e e f . ) , w h o had been suffering from aplastic a n a e m i a , was studied. She had received multiple blood transfusions and a bone m a r r o w allograft from her HLA identical brother which was ultimately rejected. T h e r e a f t e r her periphera] blood lymphocytes w e r e shown to be capable of killing target cells from her HLA identical brother thus suggesting that minor histo-compatibi1ity antigens w e r e recognized. It was subsequently shown that when these effector cells w e r e tested on a panel of targets from unrelated indivi-duals only male H L A - A 2 p o s i t i v e , targets w e r e killed despite the fact that the HLA-A2 antigen was a ' s e i f antigen present on the cells of M r s . R e e f . h e r s e i f . This led to the conclusion that the target was a minor histocompatibi1ity antigen (HY) encoded on the Υ sex chromosome and that killing required the s i m u l -taneous expression of the ' s e i f compatible H L A - A 2 antigen on effector and target. Hitherto three examples of this p h e n o m e n o n have been described and in all cases an H L A - A 2 restricted H-Y killing o c c u r r e d , but in one case this was supplernented by restriction to another ' s e i f compatible antigen, the HLA-B7 antigen .

The methods used for the cell mediated lympholysis (CML) technique have beer described in detail el sewhere2''. Brief ly periphera) blood lymphocytes w e r e sen-sitized in vitro in one-way MLC's w h e r e responder c e l l s , A, w e r e sensen-sitized to irradiated cells B. After 6 days these cells w e r e tested in a k hour killing assay on PHA blast cells obtained at 3 days of c u l t u r e . These had been p r e -viously labelled with sodium chromate so that the degree of killing could be measured by the amount of isotope released into the supernatant. Controls in-cluded PHA blasts of the original sensitizing c e l l , B, as p o s i t i v e control and a u t o l o g o u s , A , PHA blasts as negative control. Since one of the two major sources of Variation in these studies was the ability of PHA blasts to take up and release Cr the results were always corrected for the difference between spontaneous release and maximal release for each individual target cell used. The second major source of Variation was the size of the effector cell clone w h i c h in turn determined the m a x i m u m kill by any effector cell population. In the data shown positive reactions w e r e those which gave percentage kill values

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comparable to the positive control value (see for further discussion refs. 18,28,

In two series of experiments an association between antigens recognized in CML and 'seif HLA-SD was demonstrated. In both cases the latter were excluded as the antigens in themsetves because the same antigens were present on the effector cel1s. HLA-SI) HAM-MINOR HAM-MAJOR SD Serologiedlly Defined

HAM HLA-SI) Associated at Membrane MAJOR (ILA Linked

MINÜR Non-Linked to Hl.A

Fig. 1. Schematic representation of a target cel1 showing two types of targets for Τ ki 1 ler cells. The foreign component of the complex target rnay be encoded outside the major histocompatibi1ity complex (HAM-minor) or in linkage with it

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Panel HLA-B.C

No. type Sera

a b c d e f g h i j k 1 10 20 -30 50 . 55 . W35,Cw4 W35,NonCw4 •w53,Cu4 W35,CwA 56 70 } Non Bw!35/53

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gUB Positive 1 J Negative

I · 1 Not Tested

* HLA-B locus homozygote or potential homozygote

a b,c d e,£ g>h, J > ^ 1 m η anti CwA " w51 " w5l,w52 " w5l,w52,w53 i " w5l,w52,w53,w35 w53,w35 w35 anti a " b F i g . 2 . S u m m a r y of r e s u l t s o b t a i n e d f r o m a panel o f 70 i n d i v i d u a l s w h o had been t y p e d w i t h v a r i o u s a n t i s e r a w h i c h r e c o g n i z e d the B w 3 5 g r o u p of a n t i g e n s , and w i t h t w o CML e f f e c t o r cell p r e p a r a t i o n s (m and n ) w h i c h r e c o g n i z e d B w 3 S a s s o c i a t e d a n t i g e n s , a and b .

In an a t t e m p t to c l a r i f y w h a t at f i r s t a p p e a r e d to be c o n f u s i n g o b s e r v a t i o n s , a h y p o t h e t i c a l c l a s s i f i c a t i o n of a n t i g e n s ij n o w p r o p o s e d . In g e n e r a ) it c o u l d b e said that two t y p e s of a n t i g e n e x i s t bcih being a s s o c i a t e d w i t h 'seif' H L A - S D a n t i g e n s at the cell m e m b r a n e l e v e l . U n d e r no c i r c u m b L a n c e s is the a s s o c i a t i o n r e f e r r e d to m e a n t to imply m o l e c u l a r i n t e r a c t i o n b e t w e e n the H L A - S D d e t e r m i n a n t and the o t h e r d e t e r m i n a n t c o n c e r n e d , this q u e s t i o n r e m a i n s o p e n . T h e term is used s o l e l y to imply that a s i m u l t a n e o u s ( a s s o c i a t e d ) r e c o g n i t i o n of both a n t i -g e n s m u s t o c c u r b e f o r e k i l l i n -g t a k a s p l a c e . A n t i -g e n s w h i c h a r e HLA A s s o c i a t e d at the M e m b r a n e a r e a b b r e v i a t e d to HAM a n t i g e n s . T h e s e can be s u b d i v i d e d into a n t i g e n s w h i c h a r e e n c o d e d o u t s i d e the HLA r e g i o n , as in the c a s e of m i n o r h i s -t o c o m p a -t i b ί 1 i -t y a n -t i g e n s f r o m w h i c h w e d e r i v e -the -t e r m HAM m i n o r a n -t i g e n s , o r , a l t e r n a t i v e l y they can be in l i n k a g e w i t h the HLA c o m p l e x and a r e thus p r o d u c t s of the m a j o r h i s t o c o m p a t i b i 1 i t y c o m p l e x and b e c a u s e of this w e d e r i v e the term H A M m a j o r a n t i g e n s (see F i g . 1 ) .

A n e x a m p l e o f a HAM m i n o r a n t i g e n w a s o n e w h i c h w a s e n c o d e d on the Υ c h r o m o s o m e . It w a s r e c o g n i z e d in a s s o c i a t i o n w i t h the ' s e i f c o m p a t i b l e H L A A 2 a n t i gen and it o c u r r e d in t h r e e i n d e p e n d e n t c a s e s , o n e of w h i c h a l s o involved H L A -B 7 . T h e s e r e s u l t s w e r e d e s c r i b e d e l s e w h e r e

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236

Α p o s s i b l e e x a m p l e of a HAM major antigen was an a n t i g e n w h i c h w a s r e c o g n i z ed by e f f e c t o r cells raised in v i t r o between HLASD i d e n t i c a l , but g e n o t y p i c a l

-ly HLA n o n - i d e n t i c a l , fami-ly m e m b e r s . T h e s e data h a v e been most-ly described 2 7

e l s e w h e r e . Family s e g r e g a t i o n s t u d i e s w e r e c o m p a t i b l e w i t h the view that the a n t i g e n s detected by CML (a and b) w e r e encoded in the HLA region. W h e n tested upon an e x t e n s i v e panel of individuals w h o typed p o s i t i v e l y or n e g a t i v e l y for H L A - B w 3 5 a n d / o r its W 4 a s s o c i a t e d a n t i g e n , B w 5 3 , only individuals w h o carried these HLA-SD a n t i g e n s w e r e k i l l e d . F i f t y - f i v e individuals w h o carried Bw35 a n d / or Bw53 w e r e tested and it was r e m a r k a b l e that cells from all 55 individuals w e r e killed by e i t h e r anti-a or a n t i - b . None of the 17 individuals w h o w e r e n e g a t i v e for these HLA-SD a n t i g e n s w e r e p o s i t i v e and a l t h o u g h this rendered the p o s s i b i l i t y of non B w 3 5 / 5 3 ' s b e i n g killed highly u n l i k e l y , further c o n f i r m a t i o n of this a w a i t e d the testing of a larger panel (Fig. 2 ) . T h e reaction pattern o b t a i n e d w i t h the e f f e c t o r c e l l s , anti-a and a n t i - b , failed to c o r r e l a t e w i t h any of the p a t t e r n s o b t a i n e d w i t h the a v a i l a b l e typing s e r a . T h e s e CML defined a n t i g e n s t h e r e f o r e a p p e a r e d to fulfill the c r i t e r i a for HAM m a j o r a n t i g e n s in that they w e r e e n c o d e d in the m a j o r h i s t o c o m p a t i b i 1 i t y c o m p l e x and w e r e seen only in a s s o c i a t i o n w i t h s p e c i f i c ' s e i f c o m p a t i b l e H L A - S D a n t i g e n s .

One I n t e r p r e t a t i o n of this data is that HAM major a n t i g e n s f o i l o w the same rules of a s s o c i a t i v e r e c o g n i t i o n as h a v e already been d e m o n s t r a t e d for HAM m i n o

a n t i g e n s and that a similar c o m p l e x a n t i g e n is formed by the p r o d u c t s of two s e p a r a t e c i s t r o n s w h i c h are in this case both encoded in the HLA region. In s u g g e s t i n g this it should be stated that w e c a n n o t c o m p l e t e l y e x c l u d e the p o s -sibility that these CML s p e c i f i c i t i e s are in fact represented w i t h i n ehe HLA-SD typing sera used, most of which are clearly m u l t i v a l e n t (Fig. 2 ) . An a l t e r n a t i v Interpretation is that Τ killer cells r e c o g n i z e a c a r r i e r m o l e c u i e plus an a p p r o p r i a t e hapten (Bw35/53) w h e r e a s the a n t i s e r a only r e c o g n i z e the hapten p o r t i o n of the m o l e c u i e .

T h e v i e w that HLA-SD antigens in themselves acted as target antigens in CML

t was lent further support by some recent e x p e r i m e n t s in m a n2-5. In g e n e r a l , h o w

-e v -e r th-e data r-emain i n c o n c l u s i v -e , it was first sugg-est-ed that such antig-ens w e r e a s s o c i a t e d w i t h killing when it was shown in the m o u s e that target cells w h i c h had been previously ineubated w i t h a n t i - H - 2 sera s u b s e q u e n t i y b e c a m e re-s i re-s t e n t to killing . F u r t h e r m o r e m o u re-s e cell 1inere-s w h i c h failed to e x p r e re-s re-s the m o l e c u l e s c a r r y i n g H-2D and Κ a n t i g e n s w e r e neither c a p a b l e of s e n s i t i z i n g nor c a p a b l e of being killed in CML a s s a y s , thus implying a s p e c i f i c role for these m o l e c u l a r s t r u e t u r e s in Τ cell k i l l i n g , d e s p i t e the p o s s i b i l i t y that they may

31 32 not be targets in themselves .

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237

t In man discrepancies have been recorded regarding the behaviour of HLA-SD antigens as targets in so far that not all antigens performed equally well in the generation of effector cells. It was suggested that the antigens HLA-A2, -B7> -B8, -B12, -Bw35 were strong immunogens whereas others were weak immuno-gens e.g. HLA-A1, -A3, -B11, and -Aw19 . Such observations were originally

interpreted to Support a Suggestion that target antigens constituted an inte-gral part of the 'backbone' structure of the HLA-SD molecules which were Seen exclusively by killer Τ c e l l s1 6'1 8.

HLA-D region products have hitherto been considered by most workers to be incapable of behaving as targets for Τ killer cells *ϋ>5 _ However most attempts to demonstrate HLA-D target antigens have used 'conventional' CML assays of the type described here. Three pieces of data suggested that this may have been an inappropriate technique. Firstly, in the mouse it was possible to demonstrate CML to all regions of the H-2 complex, including the H-2 IA region (HLA-D equivalent), providing the appropriate technical modifications were

in-35"37

troduced . Secondly, gene products of the Mls locus in the mouse, which induced strong proliferative responses in allogeneic cel1 mixtures consistently failed to behave as targets in conventional CML assays but were thought to be detectable by the so called 'cytostasis' assay in which macrophage monolayers

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were used as targets . Thirdly, it was recently demonstrated in man that in CML assays in which blood monocyte-macrophage cells were used as target cells,

39 HLA-DR associated gene products could behave as antigens

Finally, with regard to the relevance of this discussion to tumour immunity one should mention phenomena in mouse tumour modeis where, in sonne cases,

40 ^1

changes in the H-2D and Κ structures have been observed ' . In others H-2D and Κ restricted CML reactions to tumour associated antigens have been demon-strable ' . However, exceptions to this latter phenomenon have also been des-cribed . We feel therefore that it is legitimate to suggest that tumour asso-ciated antigens in man may in some cases form antigen complexes in association with 'seif HLA-SD antigens which are recognized by the host's killer Τ cells.

SUMMARY

"Conventional" CML assays in man have been widely used to investigate anti-gens recognized by killer Τ cells. Although the detection of these antianti-gens appears to be dependent on expression of the molecular structures which carry HLA-SD (HLA-A, -B and -C encoded serologically defined) antigens there is no clear evidence that these behave as targets in themselves. Two hypothetical categories of targets detected by killer Τ cells are proposed both of which

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are HLA-SD Associated at the cell Membrane level (HAM). The allogeneic or foreign portion of the HAM antigen may be encoded outside the major histocompa-tibility complex and such a complex is termed a HAM minor antigen, and an example is given of the HLA-A2 associated H-Y antigen. AIternatively it may be encoded in 1inkage with the major histocompatibi1ity complex and is thus termed a HAM major antigen. Α possible example of this is an HLA encoded antigen which is associated with the HLA-Bw35 group of antigens. If such a classification is generally applicable, it may explain some of the apparent ambiguities regarding HLA-SD antigens and targets for ki1ler Τ cells.

ACKNOWLEDGEMENTS

This work was in part supported by the Dutch Foundation for Medical Research (FUNGO) which is subsidized by the Dutch Organization for the Advancement of Pure Research (ZWO), the Dutch Organization for Health Research (TNO) and the J.A. Cohen Institute for Radiopathology and Radiation Protection (IRS). REFERENCES

1. Cantor, H. (1978) in Proc.Int.Symp. on Human Lymphocyte Differentiation, Its Application to Cancer (Supported by INSERM), Serrou.B. and Rosenfeld, C. eds. Elsevier, Amsterdam (in press).

2. Lightbody, J.J., Bernoco, D., Miggiano, V.C. and Ceppellini, R. (1970 G.Batt.Virol.Immunol. 64, 243-254.

3- Trinchieri, G., Bernoco, D., Curtoni, E.S., Miggiano, V.C. and Ceppellini, R. (1973) in Ηistocompatibi1ity Testing 1972, Dausset, J. and Colombani, J. eds. Munksgaard, Copenhagen, pp. 509-519.

4. Miggiano, V . C , Bernoco, D., Lightbody, J., Trinchieri, G. and Ceppellini, R· (1972) Transpi.Proc. 4, 231-237.

5. Sondel, P.M. and Bach, F.H. (1975) J.exp.Med. 142, 1339-1348. 6. Sondel, P.M. and Bach, F.H. (1976) Tissue Antigens 7, 173-180.

7- Grunnet, N., Kristensen, T., Jsirgensen, F. and Kissmeyer-Ni el sen, F. (1974) Tissue Antigens 4, 218-228.

8. Bonnard, G.D., Chappuis, M., Glauser, Α., Mempel, W., ßaumann, P.,

Grosse-Wilde, H. and Albert, E.D. (1973) Transpl.Proc. 5, 1679-1682.

9. Eijsvoogel, V.P., du Bois, M.J.G.J., Melief, C.J.M., de Groot-Kooy, M.L., Koning, C , van Rood, J.J., van Leeuwen, Α., du Toit, E. and Schellekens,

P.Th.A. (1973) in Ηistocompatibi1ity Testing 1972, Dausset, J. and Colombani, J. eds. Munksgaard, Copenhagen, pp. 501-508.

10. Kristensen, T., Grunnet, N. and Kissmeyer-Nielsen, F. (1975) Tissue Antigens 6, 229-236.

11. Willumsen, J. and Heron, I. (1974) Tissue Antigens 4, 172-177.

12. Long, M.A., Handwerger, B.S., Arnos, D. and Yunis, E.J. (1976) J.Immunol. 117, 2092-2099.

13. Geha, R.S., Malakian, Α., Geha, 0. and Yunis, E.J. (1977) J.Immunol. 118, 1286-1291.

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237

In man discrepancies have been recorded regarding the behaviour of HLA-SD antigens as targets in so far that not all antigens performed equally well in the generation of effector cells. It was suggested that the antigens HLA-A2, -B7, -68, -B12, -Bw35 were strong imniunogens whereas others were weak

immuno-1 8

gens e.g. HLA-Al, -A3, -B11, and -Awl9 . Such observations were original ly interpreted to support a Suggestion that target antigens constituted an inte-gral part of the 'backbone' structure of the HLA-SD molecules which were seen

-ι r ι Q exclusively by killer Τ cells ' .

HLA-D region products have hitherto been considered by most workers to be 8 9 12 33 34

incapable of behaving as targets for Τ killer cells ' . However most attempts to demonstrate HLA-D target antigens have used 'conventional' CML assays of the type described here. Three pieces of data suggested that this may have been an inappropriate technique. Firstly, in the mouse it was possible to demonstrate CML to all regions of the H-2 complex, including the H-2 ΓΑ region (HLA-D equivalent), providing the appropriate technical modifications were in-troduced . Secondly, gene products of the Mls locus in the mouse, which induced strong proliferative responses in allogeneic cel1 mixtures consistently failed to behave as targets in conventional CML assays but were thought to be detectable by the so called 'cytostasis' assay in which macrophage monolayers were used as targets . Thirdly, it was recently demonstrated in man that in CML assays in which blood monocyte-macrophage cells were used as target cells,

39 HLA-DR associated gene products could behave as antigens .

Finally, with regard to the relevance of this discussion to tumour immunity one should mention phenomena in mouse tumour modeis wnere, in some cases,

4Q 41

changes in the H-2D and Κ structures have been observed ' . In others H-2D and Κ restricted CML reactions to tumour associated antigens have been

demon-42 43

strable ' . However, exceptions to this latter phenomenon have also been

des-44

cribed . We feel therefore that it is legitimate to suggest that tumour asso-ciated antigens in man may in some cases form antigen complexes in association with 'seif HLA-SD antigens which are recognized by the host's killer Τ cells.

SUMMARY

"Conventional" CML assays in man have been widely used to investigate anti-gens recognized by killer Τ cells. Although the detection of these antianti-gens appears to be dependent on expression of the molecular structures which carry HLA-SD (HLA-A, -B and -C encoded serological ly defined) antigens there is no clear evidence that these behave as targets in themselves. Two hypothetical categories of targets detected by killer Τ cells are proposed both of which

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are HLA-SD Associated at the cel1 Membrane level (HAM). The allogeneic or foreign portion of the HAM antigen may be encoded outside the major histocompa-tibility complex and such a complex is termed a HAM minor antigen, and an example is given of the HLA-A2 associated H-Y antigen. A]ternatively it may be encoded in linkage with the major histocompatibi1ity complex and is thus termec a HAM major antigen. Α possibie example of this is an HLA encoded antigen whier is associated with the HLA-Bw35 group of antigens. If such a classification is generally applicable, it may explain some of the apparent ambiguities regardinc HLA-SD antigens and targets for killer Τ cells.

ACKNOWLEDGEMENTS

This work was in part supported by the Dutch Foundation for Medical Research (FUNGO) which is subsidized by the Dutch Organization for the Advancement of Pure Research (ZWO), the Dutch Organization for Health Research (TNO) and the J.A. Cohen Institute for Radiopathology and Radiation Protection (IRS). REFERENCES

1. Cantor, H. (1978) in Proc.Int.Symp. on Human Lymphocyte Differentiation, Its Application to Cancer (Supported by INSERM), Serrou,B. and Rosenfeld, C. eds. Elsevier, Amsterdam (in press).

2. Lightbody, J.J., Bernoco, D., Miggiano, V.C. and Ceppellini, R. (1971) G.Batt.Virol.Immunol. 64, 243-254.

3. Trinchieri, G., Bernoco, D., Curtoni, E.S., Miggiano, V.C. and Ceppellini, R· (1973) in Histocompatibi1ity Testing 1972, Dausset, J. and Colombani, J. eds. Munksgaard, Copenhagen, pp.

509-519-4. Miggiano, V . C , Bernoco, D., Lightbody, J., Trinchieri, G. and Ceppellini, R. (1972) Transpl.Proc. 4, 231-237.

5. Sondel, P.M. and Bach, F.H. (1975) J.exp.Med. 142, 1339-1348. 6. Sondel, P.M. and Bach, F.H. (1976) Tissue Antigens 7, 173-180.

7- Grunnet, N., Kristensen, T., Jizirgensen, F. and Ki ssmeyer-Ni el sen, F. (1974) Tissue Antigens 4, 218-228.

8. ßonnard, G.D., Chappuis, M., Glauser, Α., Mempel, W., Baumann, P., Grosse-Wilde, H. and Albert, E.D. (1973) Transpl.Proc. 5, 1679-1682.

9. Eijsvoogel, V.P., du Bois, M.J.G.J., Melief, C.J.M., de Groot-Kooy, M.L., Koning, C , van Rood, J.J., van Leeuwen, Α., du Toit, E. and Schellekens, P.Th.A. (1973) in Histocompatibi1ity Testing 1972, Dausset, J. and Colombani, J. eds. Munksgaard, Copenhagen, pp. 501-508.

11. Willumsen, J. and Heron, I. (1974) Tissue Antigens 4, 172-177.

12. Long, M.A., Handwerger, B.S., Arnos, D. and Yunis, E.J. (1976) J.immunol. 117, 2092-2099.

13. Geha, R.S., Malakian, Α., Geha, 0. and Yunis, E.J. (1977) J.Immunol. 118, 1286-1291.

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4. Schendel, O.J., Wank, R., Hansen, J.A. and Dupont, B. (1977) Transpl.Proc. 9, 1777-1780.

6. Kristensen, T. , Grunnet, N., JeSrgensen, F., Lamm, L.U. and KissmeyerNielsen, F. (1976) Tissue Antigens 8, 299316

-17. Grunnet, N., Kristensen, T. and Kissmeyer-Nielsen, F. (1976) Tissue Antigens 7,

301-303-18. Kristensen, T. (1978) in Ηistocompatibi1ity Testing 1977, Bodmer, W.F. ed. Munksgaard, Copenhagen (in press).

19. Long, M.A. and Yunis, E.J. (1977) Transpl.Proc. 9, 455-460.

20. Matossian-Rogers, A. and Festenstein, H. (1977) Cliη.exp.Immunol. 27, 335-340.

21. Shearer, G.M., Cudkowicz, G., Schmitt-Verhulst, Α.Μ., Rehn, T.G., Waksal, H. and Evans, P.D. (1976) Cold Spring Harbor Symp. on Quant.Biol. pp. 511-518.

12. Gordon, R.D., Simpson, E. and Samueison, L.E. (1975) J.exp.Med. 142,

1108-1120.

II. Bevan, M.J. (1975) J.exp.Med. 142, 1349-1364.

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