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Influence of MB compatibility of survival of kidney transplants from related and cadaveric donors
Abstracts
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ISOLATION OF HUNANT CELL SUBSETS ON AFFINITY COLUNNS USING NONOCLONALA,q~IBODIES A . J . S . D u a r t % . C . B . C a r p e n t e r , J . E . Go~uen, B.J.. C a r p e n t e r and N.R. Carovo~, Laboratory of l~munogeneCzcs and T r a n s p l a n t a t i o n , ~rxgham & Women', H o ~ p i t a l , B o s t c n , Hass. 02115. Study of lymphocyte subsets has been f a c i l i t a t e d by the d e t e c t i o n of variou~
sur~ace~arkers which are relate~ to d~stinet functxo.m. ,e have explored the use of a f f i n i t y me~hods for separatzon of large quantities(50 x106) of c e l l s . Antibody-coated Degalan V26 (poly~ethylmethacrylate) bead col,-.ns provide e x c e l l e n t e n r i c h m e n t of c e l l s b e a r i n g t h e r e l e v a n t a n t i g e n . Adherent c e l l s a r e r e c o v e r e d by removing t h e beads and a g i t a t x n g them i n a t e s t t u b e . Normal human p e r i p h e r a l blood lymphocytes were p r e p a r e d by t h e Ftcoll-Rypaque • e t h o d . B c e l l s were f i r s t removed on a rabb~t anti-human Ig column y ~ e l d l n g T c e l l s contaminated w i t h l e s s t h a n 5~ B c e l l s . Honoclonal(mouse) a n t z b o d i e s t o e i t h e r h e l p e r / $ u d u c e r o r s u p p r e s s o r / c y t o t o x i c s u b s e t s (OKT~ and oKTg) were i n c u b a t e d w i t h t h e s e T c e l l s f o r 1 hour a t room t e m p e r a t u r e . A t r e r washing,
the c e l l s were applied to a goat anti-mouse Ig coated Degalan column. In general, the adherent fraction contained O-3~ contamtnatxon v t t h the opposite subset, v h i l e the non-adherent fraction ncc~:!onally had more than IOZ contamination. Total y i e l d of cel.s was 60-100~. Cells were I00~ v~able and could be used for HLA typing, e.g. OKT8+ c e l l s prepared by e i t h e r .egative or p o s i t i v e s e l e c t i o n gave i d e n t i c a l r e a c t i o n s w i t h HLA-A,~,and C a n t ~ s e r a as t h e s t a r t i n g p r e p a r a t i o n , and were completely n e g a t i v e w i t h anti°D~ a l l o and x e n o - a n t i s e r a . A n t i b o d y - c o a t e d Degalan beads can be reused a t t e a s t 30 t i m e s . T h i s economical t e c h n i q u e i s a p p l i c a b l e t o s e p a r a t i o n o f l a r g e q u a n t i t i e s of c e l l s i n a r e l a t i v e l y s h o r t txme.
CYTOTOXIC T CELL LINES" S p e c i f i c i t y ~galnst HLA and non HLA antlgen~. D. Dubey, D. Stauntono H. Peelln, S. Stux, and E.J. Yunls. Harvard Medical School, Boston, ~A Cytotoxlc T cell lines (95% E-RFC) with a11ogene~c s p e c l f l c l t y w e r e qenerated from secondary stimulated responder c e l l s (18 combinations, 8 lndividLals) i n l n t e r l e u k i n 2 and tested a f t e r 2°3 months of continuous culture. CTLs were tested against PHA blasts from HLA panel o f 10 individuals formx~n~r~ HLA (A,B,C and DR) differences. The data s~owed differences of cytotoxtc a c t t v l t y ( l y r i c a c t w 1 t y expressed in LU/IO • ce11~) between 3,746 to 14,000 units against the prlm|ng c e l l s and between I to 2040 of other tarqet c e l l s . Fourteen of the CTL combinations-tested gave higher lyClc values (746-14,000 units) with priming c e l l s as targets against t h i r d party targets with phenotypes of p a r t i c u l a r HLA CREGs (1-2040 units). [n two combinations the s p e c i f i c i t y of the CTLs against the HLA antigens of the or~mlng c e l l s and CRE~ g~oups o f the nanel of tarqets was s i m i l a r (2750-7840 ~n~ts). In ano~er experiment, we tested the response to a co~on st~mulator showing a strong response against the target-prlmng c e i l s (2,273 to 4,820 units) but the respone varied fr0m very 10w (low res~nder) to moderate (h~gh responders}: Z to 19 and 171 to 303 The rema~nlng two co~b~natlons recognized a s p e c i f i c i t y not attributed to known HLA antigens (cytolyt~c units of l i t and 571). The checkerboard method to generate CTLs described can no, be used to study 1) the genetics of responders and non-responders against a110o detemlnants o f lymphoid c e l l s and 2) the genetics of non HLA antigens present on a lymphocyte T c e l l panel (PHA blasts) (Support. NC! G~ant CA 20531, CA 27063 and CA 26158)
INFLUENCE OF MB COMPATIBILITY ON SURVIVAL OF KIDNEY TRANSPLANTS FROM RELATED AND C~J)AVERIC DONORS. R . J . D u q u e s n o y ~ M . M a r r a r i , S A . H s e k b a r t h and ll.M.KauiYmlm. J r . , T h e Blood C e n i e r of S o u t h e a s t e r n Wisconsin a n d t h e D e p t . o f S u r g e r y , T h e Medical College o f W i s c o n s i n . M i l w a u k e e , Wisconsin. ~.IB i8 • m / I t e m of B c e l l a l l o a n t l g e n s s t e o n ~ l y associated w i t h HLA*DR and c o d e d for by • d i s t i n c t l o c u s i n t h e HLA-D rel~ion. We h a v e p r e v i o u s l y r e p o e t e d t h a t MB eompatibfllty s i g n i f i c a n t l y e n h a n c e s l - y e a * s u r v i v a l o f 21 k i d n e y t r a n s p l a n t s from I h s p l o t y p e m i s m a t c h e d r e l a t e d donoes (Duquesnoy e t 81. ~ .
360
Annual AACHT Meeun~ 1981 Eng. J . Med., 302: 821. 1980). Our observatfons FAve been extended to 29 vatients with the following correlations of ~ compatibility v s . I-year survival:
++ 11; +- 1; -+ 1; -- 16 (X 2 = 17.95; p~lO'S). We have also evelu~ted the role of MB compstfbflfty fn 51 eadaverfe Iddney transplants as detemined b y 6re:ruth graft survival and the number of MB fnooranatibfl/t/es i n the donor. The following results were c h a i n e d . 0 MB Ins: 50~ survival (N = 12): I MB ino: 81t auzv/~r-1 ¢R = 36); 2 MB fnc: 67~ 8m'vlval (N = 3). Also, we foun~ no effect of M~ antigen s h a r i n g on csd~verle trsnsphmt s u r v l v a l . We obaerve~ no influence of HLA-DK emd MT eompaUbflity on 6-month Rrsit survival i n *hi~ group of psttents. Although these results should be considered prel~L~az-;, we find it difficult to explain the nut~ked differences i n the influence of ~ .-ompaUbfllW on the outcome of related v s . cadaverie transplants. It is possible that these dffTez~mees are :'elated to r e ~ p f e n t di~erences i n pl~l"ansplant blood trened~usfon history, dialysis duration as well as treatment nf reject/on episodes b y exchange p l u m s p h e r e s f s . (SuppoSed by Grant AI-12507).
DESCRIPTIONOF A NEWHLA-LINKED LOCUS USING CLONED PLT CELLS. D.O. Eckels t R.3. Hartzman~ D. S~oote F. Robtns~ R. Hargrove and T. Ltonettt, Im~.nologtc Oncology D-~;('~ion, Georgetown University School of I%.dtclne, Mashlngton, D.C. 20007 and the Naval Medical Research Institute, 8ethesda, M.D. 21)014. Responder cells from a ;)w3 HTC were stimulated wtth cells from a Dwl,3 heterozygote tn primary ~.C fro" 4 days. Responding blast cells were removed and cloned by limiting dilution at 0.3 celTs per ~ell for 3 weeks in the presence of TCGF. Clones were tested agatnst a panel of 32 cells contatntnq Owl,3 and other spectf|cittes tn a d d t t i ~ to the 10 family members of the ortgtna] sttmulator(FLA~). Two clones, TLC 14-14 and 14o8K, gave postttve responses ~n all family cells expressing the %" haplotype and a Dw5 HTC (JPSU) but failed to respond to al1 other panel members including 7 Dwtposttive cells. Three individuals from the JI~U family who were neaattve for the "c" haplot~pe failed to stimulate 11.C 14-14. Family members expressing the "c a haplotype f e l l ~nto two groups: 3 strong postttve~ and ~ weak positives. There were no HLA-D, GLO recombtnants. These data suggest the presence of restimulattng antigens encoded by ~ . . ~ outside the ~ region. Panel member HaplotyPe TLC ]4o14 TLC TLC 14-12 ~ ~ FLAH a/b 20215 34791 -- 15959- ~ . ~ - - , LLAH b/c 1502(; 26964 11674 S; EL~ a/c 460 :331 299 2075 JPSU a/c 11605 16775 355 3S TESU b/Cd** 909 n.t. n.t. n.% RISU b/c 11272 n.t. n.t. n.t. *Median cpm from t r i p l i c a t e cultures. **P(~q3 recombinant.
DOES HLA-D EXTST~D.D. Eckels t R.J. Hartzmant D. Smoot, F. Rot~tns e R. Harceove and T. Ltonettt, Imunologtc Oncology Dtvlstoo, Georgetown University School--of'Medlctn--~', gash|ngton; D.C. 2000! and the Naval Medical Research Institute, Oethesda, H.D. 20014. Cells from ItLA-Dw3 and I),2 HTCs were stimulated by cells from a I~vI,3 heretozygote (FLNq) tn primary MLC for 4 days. Blast cells were r ~ , L ~ .;,d :::)ned by ltmtttng dilution at 0.3 cells/well tn the presence of T ( ~ aria |rradtated feeders. Clones were expanded for 3 weeks and then tested with a panel of 32 stimulators containing Owl-9 as ~11 as FLk~I's f m t l y . Of 58 "LCs tested, 52~ clearly respo,ded to the postttve control while 17~ segreg&ted wtth cells from stbltngs carrying the "b" haplotvpe; 3Sg demonstrated conplex segregation patterns in the f ~ t l y . TLCS never were restlmulated exclusively hy all Dwl+ cells. Further, most TLCs were resttmulated by subsets of ~ l cells. Generally, the differences between poslttve and negative responses was
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ISOLATION OF HUNANT CELL SUBSETS ON AFFINITY COLUNNS USING NONOCLONALA,q~IBODIES A . J . S . D u a r t % . C . B . C a r p e n t e r , J . E . Go~uen, B.J.. C a r p e n t e r and N.R. Carovo~, Laboratory of l~munogeneCzcs and T r a n s p l a n t a t i o n , ~rxgham & Women', H o ~ p i t a l , B o s t c n , Hass. 02115. Study of lymphocyte subsets has been f a c i l i t a t e d by the d e t e c t i o n of variou~
sur~ace~arkers which are relate~ to d~stinet functxo.m. ,e have explored the use of a f f i n i t y me~hods for separatzon of large quantities(50 x106) of c e l l s . Antibody-coated Degalan V26 (poly~ethylmethacrylate) bead col,-.ns provide e x c e l l e n t e n r i c h m e n t of c e l l s b e a r i n g t h e r e l e v a n t a n t i g e n . Adherent c e l l s a r e r e c o v e r e d by removing t h e beads and a g i t a t x n g them i n a t e s t t u b e . Normal human p e r i p h e r a l blood lymphocytes were p r e p a r e d by t h e Ftcoll-Rypaque • e t h o d . B c e l l s were f i r s t removed on a rabb~t anti-human Ig column y ~ e l d l n g T c e l l s contaminated w i t h l e s s t h a n 5~ B c e l l s . Honoclonal(mouse) a n t z b o d i e s t o e i t h e r h e l p e r / $ u d u c e r o r s u p p r e s s o r / c y t o t o x i c s u b s e t s (OKT~ and oKTg) were i n c u b a t e d w i t h t h e s e T c e l l s f o r 1 hour a t room t e m p e r a t u r e . A t r e r washing,
the c e l l s were applied to a goat anti-mouse Ig coated Degalan column. In general, the adherent fraction contained O-3~ contamtnatxon v t t h the opposite subset, v h i l e the non-adherent fraction ncc~:!onally had more than IOZ contamination. Total y i e l d of cel.s was 60-100~. Cells were I00~ v~able and could be used for HLA typing, e.g. OKT8+ c e l l s prepared by e i t h e r .egative or p o s i t i v e s e l e c t i o n gave i d e n t i c a l r e a c t i o n s w i t h HLA-A,~,and C a n t ~ s e r a as t h e s t a r t i n g p r e p a r a t i o n , and were completely n e g a t i v e w i t h anti°D~ a l l o and x e n o - a n t i s e r a . A n t i b o d y - c o a t e d Degalan beads can be reused a t t e a s t 30 t i m e s . T h i s economical t e c h n i q u e i s a p p l i c a b l e t o s e p a r a t i o n o f l a r g e q u a n t i t i e s of c e l l s i n a r e l a t i v e l y s h o r t txme.
CYTOTOXIC T CELL LINES" S p e c i f i c i t y ~galnst HLA and non HLA antlgen~. D. Dubey, D. Stauntono H. Peelln, S. Stux, and E.J. Yunls. Harvard Medical School, Boston, ~A Cytotoxlc T cell lines (95% E-RFC) with a11ogene~c s p e c l f l c l t y w e r e qenerated from secondary stimulated responder c e l l s (18 combinations, 8 lndividLals) i n l n t e r l e u k i n 2 and tested a f t e r 2°3 months of continuous culture. CTLs were tested against PHA blasts from HLA panel o f 10 individuals formx~n~r~ HLA (A,B,C and DR) differences. The data s~owed differences of cytotoxtc a c t t v l t y ( l y r i c a c t w 1 t y expressed in LU/IO • ce11~) between 3,746 to 14,000 units against the prlm|ng c e l l s and between I to 2040 of other tarqet c e l l s . Fourteen of the CTL combinations-tested gave higher lyClc values (746-14,000 units) with priming c e l l s as targets against t h i r d party targets with phenotypes of p a r t i c u l a r HLA CREGs (1-2040 units). [n two combinations the s p e c i f i c i t y of the CTLs against the HLA antigens of the or~mlng c e l l s and CRE~ g~oups o f the nanel of tarqets was s i m i l a r (2750-7840 ~n~ts). In ano~er experiment, we tested the response to a co~on st~mulator showing a strong response against the target-prlmng c e i l s (2,273 to 4,820 units) but the respone varied fr0m very 10w (low res~nder) to moderate (h~gh responders}: Z to 19 and 171 to 303 The rema~nlng two co~b~natlons recognized a s p e c i f i c i t y not attributed to known HLA antigens (cytolyt~c units of l i t and 571). The checkerboard method to generate CTLs described can no, be used to study 1) the genetics of responders and non-responders against a110o detemlnants o f lymphoid c e l l s and 2) the genetics of non HLA antigens present on a lymphocyte T c e l l panel (PHA blasts) (Support. NC! G~ant CA 20531, CA 27063 and CA 26158)
INFLUENCE OF MB COMPATIBILITY ON SURVIVAL OF KIDNEY TRANSPLANTS FROM RELATED AND C~J)AVERIC DONORS. R . J . D u q u e s n o y ~ M . M a r r a r i , S A . H s e k b a r t h and ll.M.KauiYmlm. J r . , T h e Blood C e n i e r of S o u t h e a s t e r n Wisconsin a n d t h e D e p t . o f S u r g e r y , T h e Medical College o f W i s c o n s i n . M i l w a u k e e , Wisconsin. ~.IB i8 • m / I t e m of B c e l l a l l o a n t l g e n s s t e o n ~ l y associated w i t h HLA*DR and c o d e d for by • d i s t i n c t l o c u s i n t h e HLA-D rel~ion. We h a v e p r e v i o u s l y r e p o e t e d t h a t MB eompatibfllty s i g n i f i c a n t l y e n h a n c e s l - y e a * s u r v i v a l o f 21 k i d n e y t r a n s p l a n t s from I h s p l o t y p e m i s m a t c h e d r e l a t e d donoes (Duquesnoy e t 81. ~ .
360
Annual AACHT Meeun~ 1981 Eng. J . Med., 302: 821. 1980). Our observatfons FAve been extended to 29 vatients with the following correlations of ~ compatibility v s . I-year survival:
++ 11; +- 1; -+ 1; -- 16 (X 2 = 17.95; p~lO'S). We have also evelu~ted the role of MB compstfbflfty fn 51 eadaverfe Iddney transplants as detemined b y 6re:ruth graft survival and the number of MB fnooranatibfl/t/es i n the donor. The following results were c h a i n e d . 0 MB Ins: 50~ survival (N = 12): I MB ino: 81t auzv/~r-1 ¢R = 36); 2 MB fnc: 67~ 8m'vlval (N = 3). Also, we foun~ no effect of M~ antigen s h a r i n g on csd~verle trsnsphmt s u r v l v a l . We obaerve~ no influence of HLA-DK emd MT eompaUbflity on 6-month Rrsit survival i n *hi~ group of psttents. Although these results should be considered prel~L~az-;, we find it difficult to explain the nut~ked differences i n the influence of ~ .-ompaUbfllW on the outcome of related v s . cadaverie transplants. It is possible that these dffTez~mees are :'elated to r e ~ p f e n t di~erences i n pl~l"ansplant blood trened~usfon history, dialysis duration as well as treatment nf reject/on episodes b y exchange p l u m s p h e r e s f s . (SuppoSed by Grant AI-12507).
DESCRIPTIONOF A NEWHLA-LINKED LOCUS USING CLONED PLT CELLS. D.O. Eckels t R.3. Hartzman~ D. S~oote F. Robtns~ R. Hargrove and T. Ltonettt, Im~.nologtc Oncology D-~;('~ion, Georgetown University School of I%.dtclne, Mashlngton, D.C. 20007 and the Naval Medical Research Institute, 8ethesda, M.D. 21)014. Responder cells from a ;)w3 HTC were stimulated wtth cells from a Dwl,3 heterozygote tn primary ~.C fro" 4 days. Responding blast cells were removed and cloned by limiting dilution at 0.3 celTs per ~ell for 3 weeks in the presence of TCGF. Clones were tested agatnst a panel of 32 cells contatntnq Owl,3 and other spectf|cittes tn a d d t t i ~ to the 10 family members of the ortgtna] sttmulator(FLA~). Two clones, TLC 14-14 and 14o8K, gave postttve responses ~n all family cells expressing the %" haplotype and a Dw5 HTC (JPSU) but failed to respond to al1 other panel members including 7 Dwtposttive cells. Three individuals from the JI~U family who were neaattve for the "c" haplot~pe failed to stimulate 11.C 14-14. Family members expressing the "c a haplotype f e l l ~nto two groups: 3 strong postttve~ and ~ weak positives. There were no HLA-D, GLO recombtnants. These data suggest the presence of restimulattng antigens encoded by ~ . . ~ outside the ~ region. Panel member HaplotyPe TLC ]4o14 TLC TLC 14-12 ~ ~ FLAH a/b 20215 34791 -- 15959- ~ . ~ - - , LLAH b/c 1502(; 26964 11674 S; EL~ a/c 460 :331 299 2075 JPSU a/c 11605 16775 355 3S TESU b/Cd** 909 n.t. n.t. n.% RISU b/c 11272 n.t. n.t. n.t. *Median cpm from t r i p l i c a t e cultures. **P(~q3 recombinant.
DOES HLA-D EXTST~D.D. Eckels t R.J. Hartzmant D. Smoot, F. Rot~tns e R. Harceove and T. Ltonettt, Imunologtc Oncology Dtvlstoo, Georgetown University School--of'Medlctn--~', gash|ngton; D.C. 2000! and the Naval Medical Research Institute, Oethesda, H.D. 20014. Cells from ItLA-Dw3 and I),2 HTCs were stimulated by cells from a I~vI,3 heretozygote (FLNq) tn primary MLC for 4 days. Blast cells were r ~ , L ~ .;,d :::)ned by ltmtttng dilution at 0.3 cells/well tn the presence of T ( ~ aria |rradtated feeders. Clones were expanded for 3 weeks and then tested with a panel of 32 stimulators containing Owl-9 as ~11 as FLk~I's f m t l y . Of 58 "LCs tested, 52~ clearly respo,ded to the postttve control while 17~ segreg&ted wtth cells from stbltngs carrying the "b" haplotvpe; 3Sg demonstrated conplex segregation patterns in the f ~ t l y . TLCS never were restlmulated exclusively hy all Dwl+ cells. Further, most TLCs were resttmulated by subsets of ~ l cells. Generally, the differences between poslttve and negative responses was