ANTIGENS
ON
H-2 ALLOGENEIC
CELLS*
By JAMES FORMAN AND J. WAYNE STREILEIN
From the Department of Microbiology and the Department of Cell Biology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 Sensitized murine thymus-derived (T) lymphocytes possess the preferential ability to recognize extrinsic antigens on H-2 syngeneic cells (1). However, the T-cell repertoire also includes the capacity to recognize H-2 alloantigens (2). Because a relatively high proportion of cytotoxic T-cell precursors recognize H-2 alloantigens (3), the possibility exists that a portion o f this alloreactivity includes clones of T cells that recognize extrinsic antigens in the context of H-2 allodeterminants. T o determine whether there exist clones of T cells that can recognize antigens in the context of H2 allodeterminants (antigen H-2 aUospecific recognition) in an animal, we utilized animals rendered neonatally tolerant of H-2 alloantigens in which the degree of chimerism is exceedingly low. By eliminating the H-2 alloreactivity, we are able to demonstrate that cytotoxic T lymphocytes of host origin recognize minor histocompatibility (H) x alloantigens on the tolerated H-2 allogeneic cells. Materials and Methods
Animals. All mice were produced and maintained in our colonies at the University of Texas Health Science Center at Dallas, Dallas, Texas. Neonatally tolerant animals were produced by a method previously described (4). Briefly, 15 × 106 semiallogeneic bone marrow and spleen cells were inoculated i.v. within 24 h of birth. Animals were assayed for tolerance at 8 wk of age by the application of an orthotopic skin graft identical to the donor of the tolerizing inoculum. Immunization Protocols. Mice were sensitized against minor H-antigens by i.p. inoculation of 50 × 108 spleen cells. 3 wk to 3 mon later, the recipients were killed, their spleen cells were placed in culture with 3,000-rad-irradiated stimulator cells, and cytotoxic T-cell activity was assayed 5 d later. Cytotoxicity Assay. This procedure has been described in detail in a previous communication (5). Data is expressed as the net isotope release, which equals the percentage of 51Cr release from target cells in the presence of immune cells minus the percentage of ~lCr release from target cells in the presence of control cells. Control cells represent cultures lacking stimulator cells. Results
Minor H-Antigens Presented on Tolerated Haplotype Induce T-Effector Cells Restricted to Tolerated Haplotype. Strong alloreactivity to an unrelated H-2 haplotype in normal animals precludes identifying reactivity to minor H-antigens in an H-2-allogeneic context. Therefore, adult B10.A (H-2 a) animals, rendered tolerant to H-2 f antigens * Supported by National Institutes of Health grants AI-13111, AI-10678, and CA-09082. Abbreviation used m this paper: H, histocompatibility. J. Exv. MED. © T h e Rockefeller University Press • 0022-1007/79/10/1001/0751.00 Volume 150 October 1979 1001-1007
1001
ANTIGEN H-2 ALLOSPECIFIC RECOGNITION
1002
TABLE I
B I O.A Effector Cells Tolerant of B l O.M Recognize Minor H-Antigens on H-2 Allogeneic Cells
Line
1 2
Immu-
Responder nization
BI0.M
A.CA
Target cells
A.CA(KtDt)$
MinorH-2-reH antigion shargen ining becompat- tweentaribility between get cells and cells reused for sponder immuniand tarzation get cells
100:1
50:1
10:1
31.1 0.1 2.5 28.3
18.9 -0.2 0.7 19.4
1.5
1.2
4.7 -0.2 0.5 4.9 2.5
10.4 0.9 0.9 8.9 0.l -1.7
2.5 -0.1 -0.2 2.9 1.0 -0.6
15.5
4.7
3 4
A(KkD a) A.TFRI(KSD t)
5
B10.M(llR)(KfD a)
yes yes yes yes no
A.CA A.SW A A.TFR l B10.M(llR) B10.M
yes yes yes yes no no
K D
-- D K -K D
18.8 -0.2 3.0 13.8 -0.4 -1.3
no
K D
22.5
A.SW(KSD~)
6 7 8 9 10 11
B10.Atoler- A . C A ant to B10.M
12
B10.A
B10.M B10.M
K D
Net 51Crrelease* (effector-to-target cell ratio)
-- D K --
* SlCr release from target cells in the presence of nonimmune (control) effector cells ranged from 8.9 to 12%. "~H-2K and H-2D alleles. by n e o n a t a l i n o c u l a t i o n of (B 10.A × B10.M)F1 spleen cells, were inoculated i.p. with A.CA (H-2 f) spleen cells. A l t h o u g h B10.M a n d A.CA share the H - 2 [ haplotype, they have different alleles at m u l t i p l e m i n o r H-loci representing the difference between C 5 7 B L / 1 0 a n d A strain b a c k g r o u n d genes. 3 wk to 3 m o n after in vivo i n o c u l a t i o n of the H-2t-tolerant B10.A a n i m a l s with A.CA l y m p h o i d cells, spleen cells from these mice were cocultured with irradiated A.CA spleen cells a n d s u b s e q u e n t l y tested for cytotoxic activity against a panel of target cells. As a positive control, n o r m a l B 10.M a n i m a l s were sensitized with A.CA spleen cells a n d the resultant cytotoxic effector cells were tested against the same panel of targets. B10.M a n i m a l s sensitized against m i n o r H - a n t i g e n disparate spleen cells (A.CA) generate a cytotoxic effect against A.CA target cells (line 1, T a b l e I). This response is 1-1-2 restricted in that H - 2 - i n c o m p a t i b l e target cells b e a r i n g the a p p r o p r i a t e m i n o r Hantigens are not lysed (lines 2 a n d 3); whereas a cytotoxic effect is observed if only the H - 2 D f region is shared between the s t i m u l a t o r a n d target cell (line 4). B10.M(11R) target cells are not lysed because they lack the A strain b a c k g r o u n d m i n o r H - a n t i g e n s (line 5). B 10.A a n i m a l s tolerant to B 10.M, sensitized in vivo a n d boosted in vitro with A.CA cells, display a strong cytotoxic effect against A.CA target cells (line 6). This response is H-2 restricted to the same extent as that observed w h e n testing the B10.M spleen ceils. T h a t is, H - 2 - i n c o m p a t i b l e target cells are not lysed (lines 7 a n d 8), whereas target cells s h a r i n g only the H - 2 D f region with the s t i m u l a t o r cells are lysed (line 9).
JAMES FORMAN AND J. WAYNE STREILEIN
1003
TAnLE II Minor H-Antigens Presentedon the ToleratedHaplotype Induce T-Effector Cells Restricted to Host Haplotype O-2-re-
Line
Responder
Cells Cells used for used for in vitro in vivo chalpriming lenge
Target cells
gion sharing between cells used for in vitro thailenge and
Net SXCrRelease* (eflector-to-target cell ratio) 100:1
50:1
10:1
target cells 1 2 3 4
BI0.A tolerant to B10.M
A.CA
A.CA
A.CA A C3H(/~Dk):~ C3H.SW(KbD b)
K D
28.0 -0.6 2.2 0.5
28.3 1.3 2.9 0.4
12.5 0.9 3.6 0.4
5 6 7 8
B10.A tolerant to B10.M
A.CA
A
A A.CA C3H C3H.SW
K D
35.3 1.0 16.8 0.5
27.2 0.6 13.0 0.7
11.7 3.4 3.8 1.8
K --
* SXCrrelease from target cells in the presence of nonimmune (control) effector cells ranged from 10.3 to 21.6%. H-2K and H-2D alleles. B 1 0 . M ( I I R ) target cells l a c k i n g the m i n o r H - a n t i g e n s o f the A strain b a c k g r o u n d are not lysed (line 10). T h e s e d a t a also d e m o n s t r a t e t h a t the H - 2 t - t o l e r a n t B 10.A a n i m a l s do not generate a n t i - H - 2 killer-cell a c t i v i t y against B10.M target cells (line 11); whereas n o n t o l e r a n t a n i m a l s d o generate such a c t i v i t y (line 12). T h u s , a significant a m o u n t o f specific cytotoxic T-cell a c t i v i t y c a n be g e n e r a t e d in B 10.A a n i m a l s t o l e r a n t to H-2 f antigens w h e n these a n i m a l s are sensitized against m i n o r H - a n t i g e n s in the context o f the t o l e r a t e d H-2-allogeneic h a p l o t y p e . Minor H-Antigens Presented on the Tolerated Haplotype Induce T-Effector Cells Restricted to Host Haplotype. Bevan (6) has d e m o n s t r a t e d t h a t m i n o r H - a n t i g e n s p r e s e n t e d on H 2 semiallogeneic cells m a y be used to cross p r i m e mice in vivo so t h a t the a n t i g e n can be p r e s e n t e d on the allogeneic H-2 h a p l o t y p e . I n these experiments, we wished to d e t e r m i n e w h e t h e r a n i m a l s tolerant to an H - 2 h a p l o t y p e c o u l d be p r e s e n t e d with m i n o r H - a n t i g e n s on the t o l e r a t e d h a p l o t y p e so t h a t the antigens c o u l d cross-prime a n d restrict for the host h a p l o t y p e . B 10.A a n i m a l s t o l e r a n t to B 10.M were i n o c u l a t e d in vivo with A . C A l y m p h o i d cells. W h e n spleen cells from these t o l e r a n t a n i m a l s were later c u l t u r e d in vitro w i t h A . C A cells, a cytotoxic response was g e n e r a t e d against A . C A , b u t not H - 2 - i n c o m p a t i b l e target ceils ( T a b l e II, lines 1-4), in a g r e e m e n t w i t h the d a t a shown in T a b l e I. F u r t h e r m o r e , w h e n freshly h a r v e s t e d spleen cells were c h a l l e n g e d in vitro with A strain spleen cells, the cytotoxic effect g e n e r a t e d was now restricted to K k D a target cells, i n c l u d i n g C 3 H , which indicates t h a t the A a n d C 3 H strains share some m i n o r H-alleles (lines 5 a n d 7). I m p o r t a n t l y , A . C A cells a n d o t h e r H - 2 - i n c o m p a t i b l e target cells b e a r i n g the a p p r o p r i a t e m i n o r H - a n t i g e n s were not lysed (lines 6 a n d 8). Thus, these results i n d i c a t e t h a t m i n o r H - a n t i g e n s p r e s e n t e d on cells o f a n allogeneic H-2 h a p l o t y p e can p r i m e the a n i m a l for m i n o r H - a n t i g e n recognition on the host's H-2 h a p l o t y p e .
ANTIGEN H-2 ALLOSPECIFIC RECOGNITION
1004
TABLE III Minor H-Antigens Presentedon Host Haplotype Fail to Induce T Effector Cells Restricted to Tolerated Haplotype
Line
1
Responder
BI0.A tolerant to B10.M
Cells Cells used for used for in vivo in vitro chalpriming lenge A
A
2 3 4
Target cells
A
H-2-region sharing between cells used for in vitro challenge and target cells K D
A.CA B10.A tolerant to B10.M
A
A.CA
A.CA A
K D
Net 51Cr release* (effector-to-target cell ratio) 100:1
50:1
10:1
15.8
8.5
2.9
--0.4
-3.5
- 1.0
-3.2
-1.1
-0.3
-1.1
-1.7
0
* S~Cr release from A and A.CA target cells in the presence of nonimmune (control) effector cells was 20.0 and 24.4%, respectively.
Minor H-Antigens Presented on Host Haplotype Fail to Induce T-Effector Cells Restricted to Tolerated Haplotype. W e have previously d e t e r m i n e d t h a t the extent o f c h i m e r i s m in a n i m a l s r e n d e r e d tolerant as neonates is e x t r e m e l y low (4, 7). Therefore, it w o u l d be expected t h a t the n u m b e r o f (B 10.A X B 10.M)FI l y m p h o r e t i c u l a r cells a b l e to present a n t i g e n in B 10.A a n i m a l s tolerant o f the B 10.M h a p l o t y p e w o u l d be c o r r e s p o n d i n g l y low. In the following e x p e r i m e n t , we d e t e r m i n e d w h e t h e r the chimeric F1 cells in tolerant a n i m a l s could present m i n o r H - a n t i g e n s to the host's T-cell system. B10.A a n i m a l s tolerant of B10.M were i n o c u l a t e d in vivo w i t h A strain l y m p h o i d cells followed b y reehallenge in vitro with either A or A . C A cells. T h e specificity o f effector ceils g e n e r a t e d following in vitro c u l t u r e is shown in T a b l e III. As expected, B 10.A a n i m a l s tolerant o f B I 0 . M a n d i m m u n i z e d with A strain cells in vivo a n d in vitro g e n e r a t e d cytotoxic effector cells with specificity for A strain a n d not for A . C A target cells (lines 1 a n d 2). However, when the tolerant spleen cells were c h a l l e n g e d in vitro with A . C A spleen cells, no cytotoxic effect was observed against A . C A target cells (line 3). Therefore, m i n o r H - a n t i g e n s p r e s e n t e d on A strain spleen cells c a n n o t be processed in vivo to allow for T-cell recognition o f m i n o r H - a n t i g e n s on the t o l e r a t e d H-2 f h a p l o t y p e . This finding suggests t h a t host reprocessing of m i n o r H - a n t i g e n s is not a requisite for sensitization of cytotoxic T lymphocytes. Minor H-Antigens Presented on Tolerated Haplotype Induce T-Effector Cells Restricted to Tolerated Haplotype. B10.M a n i m a l s were m a d e t o l e r a n t as neonates to B10.A cells, p r i m e d in vivo a n d c h a l l e n g e d in vitro with A strain l y m p h o i d cells. T h e i r spleen cells were then tested against a p a n e l o f target cells ( T a b l e IV). T h e specificity o f the cytotoxic cells g e n e r a t e d was restricted to H - 2 - c o m p a t i b l e target cells (lines 1-3). W h e n the a n i m a l s were c h a l l e n g e d in vitro with A . C A r a t h e r t h a n A strain l y m p h o i d cells, a strong cytotoxic effect was observed against A . C A target cells (lines 4 a n d 5), d e m o n s t r a t i n g cross-priming similar to t h a t shown in T a b l e II. Therefore, B10.M a n i m a l s tolerant to B 10.A also possess T cells t h a t can recognize m i n o r H - a n t i g e n s in the context o f the t o l e r a t e d H-2 h a p l o t y p e .
JAMES FORMAN AND J. WAYNE STREILEIN
1005
TABLE IV B I O.M Effector Cells Tolerant of B l O.A Recognize Minor H-Antigens on H-2 Allogeneic Cells H-Y-re-
gion sharCells
Line
Responder
Cells used for used for in vitro in vivo chalpriming
1
BI0.M tolerant to B10.A
A
lenge
A
2 3 4 5
B 10.M tolerant to BI0.A
A
Target cells
A.CA
ing between cells used for in vitro challenge and target cells
Net 5~Cr Release* (effector-to-target cell ratio) 100:1
50: l
10:1
A
K D
33.4
24.6
9.8
A.CA A.TH (KSDa):]:
-- D
2.3 32.2
0.4 24.0
0.9 10.2
A.CA
K D
39.3
32.1
11.2
6.0
4.7
3.4
A
* 51Cr release from target cells in the presence of nonimmune (control) effector cells ranged from 13.8 to 15.8%. H-2K and H-2D alleles. Discussion T h e d a t a in this report d e m o n s t r a t e t h a t a n i m a l s r e n d e r e d tolerant as neonates possess a significant n u m b e r o f cytotoxic T-cell precursors t h a t c a n recognize m i n o r H - a n t i g e n s in the context o f the allogeneic H - 2 h a p l o t y p e to which t h e y were r e n d e r e d tolerant. Secondly, because the specificity o f the effector cells g e n e r a t e d are restricted b y the H - 2 h a p l o t y p e o f the cells used for the in vitro challenge, it is c o n c l u d e d t h a t two different sets o f cytotoxic effector cells co-exist in t o l e r a n t animals. Because we c a n n o t find cytotoxic T cells (or their precursors) t h a t can recognize the t o l e r a t e d H 2 a l l o a n t i g e n s themselves in these animals, it is surprising t h a t T cells from these a n i m a l s t h a t recognize m i n o r H - a n t i g e n s can be restricted b y the H-2 a l l o a n t i g e n s to w h i c h t h e y are tolerant. This s i t u a t i o a m a y be e x p l a i n e d b y p o s t u l a t i n g t h a t alloreactive T cells b e a r receptors with a s p e c t r u m o f avidities for H-2 antigens. In the tolerance protocol, h i g h - a v i d i t y r e c e p t o r b e a r i n g T cells are deleted so t h a t only those w i t h low-avidity receptors remain. T h e s e p u t a t i v e l y l o w - a v i d i t y - r e c e p t o r clones could b i n d a l l o a n t i g e n with high avidity, however, if the H-2 a l l o a n t i g e n is presented together with m i n o r H-antigens. A l t e r n a t i v e l y , T cells recognizing m i n o r H - a n t i g e n s in the context o f H2 a l l o d e t e r m i n a n t s could represent a subset o f anti-self clones t h a t c o i n c i d e n t a l l y cross-react with m i n o r H - a n t i g e n s a l o n g with the allo-H-2 d e t e r m i n a n t s , a n d thus w o u l d not be e x p e c t e d to be affected b y the tolerance p r o c e d u r e itself. It is very unlikely t h a t FI cells utilized for the tolerance i n d u c t i o n are m e d i a t i n g the cytotoxic effects o b s e r v e d because we have previously shown t h a t (a) the extent o f l y m p h o i d cell c h i m e r i s m in a n i m a l s r e n d e r e d t o l e r a n t b y this protocol is < 5 % (4, 7), a n d (b) t r e a t m e n t o f the effector cells with a n t i s e r u m against the FI cells does not affect the cytotoxic response (7). It is possible t h a t t h e a c t i v i t y of the effector ceils from B10.A mice tolerant to
1006
ANTIGEN H-2 ALLOSPECIFIC RECOGNITION
B 10.M that were sensitized and tested against A.CA is H-2 unrestricted and directed against Qa-Tla antigens (8) rather than A strain minor H-antigens restricted by H2K t or H-2D f. This possibility is unlikely, however, because (a) both B 10.M and A.CA have the same alleles at the Qa-Tla loci that control antigens that are recognized by cytotoxic T cells, and (b) attempts to demonstrate H-2-unrestricted anti-Qa-Tla effector cell activity by cross-sensitization of B 10.M animals with A.CA cells and vice versa has yielded negative results (8; and James Forman, unpublished results). Bevan (6) has demonstrated that cross-priming occurs in vivo when minor Hantigens are presented on an H-2 haplotype that is different than that used for in vitro boosting. Therefore, it is possible that minor H-antigens have to be reprocessed on host cells to generate cytotoxic effector cells. However, our data demonstrate that this is not a requirement. Thus, B10.A animals tolerant of B10.M when primed in vivo and boosted in vitro with A.CA cells generate H-2f-restricted anti-minor-Hantigen effector cells. It could be argued that A strain minor H-antigens are processed on the small number of (B10.A × B10.M)F1 cells residing in the tolerant animals (
ON
H-2 ALLOGENEIC
CELLS*
By JAMES FORMAN AND J. WAYNE STREILEIN
From the Department of Microbiology and the Department of Cell Biology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 Sensitized murine thymus-derived (T) lymphocytes possess the preferential ability to recognize extrinsic antigens on H-2 syngeneic cells (1). However, the T-cell repertoire also includes the capacity to recognize H-2 alloantigens (2). Because a relatively high proportion of cytotoxic T-cell precursors recognize H-2 alloantigens (3), the possibility exists that a portion o f this alloreactivity includes clones of T cells that recognize extrinsic antigens in the context of H-2 allodeterminants. T o determine whether there exist clones of T cells that can recognize antigens in the context of H2 allodeterminants (antigen H-2 aUospecific recognition) in an animal, we utilized animals rendered neonatally tolerant of H-2 alloantigens in which the degree of chimerism is exceedingly low. By eliminating the H-2 alloreactivity, we are able to demonstrate that cytotoxic T lymphocytes of host origin recognize minor histocompatibility (H) x alloantigens on the tolerated H-2 allogeneic cells. Materials and Methods
Animals. All mice were produced and maintained in our colonies at the University of Texas Health Science Center at Dallas, Dallas, Texas. Neonatally tolerant animals were produced by a method previously described (4). Briefly, 15 × 106 semiallogeneic bone marrow and spleen cells were inoculated i.v. within 24 h of birth. Animals were assayed for tolerance at 8 wk of age by the application of an orthotopic skin graft identical to the donor of the tolerizing inoculum. Immunization Protocols. Mice were sensitized against minor H-antigens by i.p. inoculation of 50 × 108 spleen cells. 3 wk to 3 mon later, the recipients were killed, their spleen cells were placed in culture with 3,000-rad-irradiated stimulator cells, and cytotoxic T-cell activity was assayed 5 d later. Cytotoxicity Assay. This procedure has been described in detail in a previous communication (5). Data is expressed as the net isotope release, which equals the percentage of 51Cr release from target cells in the presence of immune cells minus the percentage of ~lCr release from target cells in the presence of control cells. Control cells represent cultures lacking stimulator cells. Results
Minor H-Antigens Presented on Tolerated Haplotype Induce T-Effector Cells Restricted to Tolerated Haplotype. Strong alloreactivity to an unrelated H-2 haplotype in normal animals precludes identifying reactivity to minor H-antigens in an H-2-allogeneic context. Therefore, adult B10.A (H-2 a) animals, rendered tolerant to H-2 f antigens * Supported by National Institutes of Health grants AI-13111, AI-10678, and CA-09082. Abbreviation used m this paper: H, histocompatibility. J. Exv. MED. © T h e Rockefeller University Press • 0022-1007/79/10/1001/0751.00 Volume 150 October 1979 1001-1007
1001
ANTIGEN H-2 ALLOSPECIFIC RECOGNITION
1002
TABLE I
B I O.A Effector Cells Tolerant of B l O.M Recognize Minor H-Antigens on H-2 Allogeneic Cells
Line
1 2
Immu-
Responder nization
BI0.M
A.CA
Target cells
A.CA(KtDt)$
MinorH-2-reH antigion shargen ining becompat- tweentaribility between get cells and cells reused for sponder immuniand tarzation get cells
100:1
50:1
10:1
31.1 0.1 2.5 28.3
18.9 -0.2 0.7 19.4
1.5
1.2
4.7 -0.2 0.5 4.9 2.5
10.4 0.9 0.9 8.9 0.l -1.7
2.5 -0.1 -0.2 2.9 1.0 -0.6
15.5
4.7
3 4
A(KkD a) A.TFRI(KSD t)
5
B10.M(llR)(KfD a)
yes yes yes yes no
A.CA A.SW A A.TFR l B10.M(llR) B10.M
yes yes yes yes no no
K D
-- D K -K D
18.8 -0.2 3.0 13.8 -0.4 -1.3
no
K D
22.5
A.SW(KSD~)
6 7 8 9 10 11
B10.Atoler- A . C A ant to B10.M
12
B10.A
B10.M B10.M
K D
Net 51Crrelease* (effector-to-target cell ratio)
-- D K --
* SlCr release from target cells in the presence of nonimmune (control) effector cells ranged from 8.9 to 12%. "~H-2K and H-2D alleles. by n e o n a t a l i n o c u l a t i o n of (B 10.A × B10.M)F1 spleen cells, were inoculated i.p. with A.CA (H-2 f) spleen cells. A l t h o u g h B10.M a n d A.CA share the H - 2 [ haplotype, they have different alleles at m u l t i p l e m i n o r H-loci representing the difference between C 5 7 B L / 1 0 a n d A strain b a c k g r o u n d genes. 3 wk to 3 m o n after in vivo i n o c u l a t i o n of the H-2t-tolerant B10.A a n i m a l s with A.CA l y m p h o i d cells, spleen cells from these mice were cocultured with irradiated A.CA spleen cells a n d s u b s e q u e n t l y tested for cytotoxic activity against a panel of target cells. As a positive control, n o r m a l B 10.M a n i m a l s were sensitized with A.CA spleen cells a n d the resultant cytotoxic effector cells were tested against the same panel of targets. B10.M a n i m a l s sensitized against m i n o r H - a n t i g e n disparate spleen cells (A.CA) generate a cytotoxic effect against A.CA target cells (line 1, T a b l e I). This response is 1-1-2 restricted in that H - 2 - i n c o m p a t i b l e target cells b e a r i n g the a p p r o p r i a t e m i n o r Hantigens are not lysed (lines 2 a n d 3); whereas a cytotoxic effect is observed if only the H - 2 D f region is shared between the s t i m u l a t o r a n d target cell (line 4). B10.M(11R) target cells are not lysed because they lack the A strain b a c k g r o u n d m i n o r H - a n t i g e n s (line 5). B 10.A a n i m a l s tolerant to B 10.M, sensitized in vivo a n d boosted in vitro with A.CA cells, display a strong cytotoxic effect against A.CA target cells (line 6). This response is H-2 restricted to the same extent as that observed w h e n testing the B10.M spleen ceils. T h a t is, H - 2 - i n c o m p a t i b l e target cells are not lysed (lines 7 a n d 8), whereas target cells s h a r i n g only the H - 2 D f region with the s t i m u l a t o r cells are lysed (line 9).
JAMES FORMAN AND J. WAYNE STREILEIN
1003
TAnLE II Minor H-Antigens Presentedon the ToleratedHaplotype Induce T-Effector Cells Restricted to Host Haplotype O-2-re-
Line
Responder
Cells Cells used for used for in vitro in vivo chalpriming lenge
Target cells
gion sharing between cells used for in vitro thailenge and
Net SXCrRelease* (eflector-to-target cell ratio) 100:1
50:1
10:1
target cells 1 2 3 4
BI0.A tolerant to B10.M
A.CA
A.CA
A.CA A C3H(/~Dk):~ C3H.SW(KbD b)
K D
28.0 -0.6 2.2 0.5
28.3 1.3 2.9 0.4
12.5 0.9 3.6 0.4
5 6 7 8
B10.A tolerant to B10.M
A.CA
A
A A.CA C3H C3H.SW
K D
35.3 1.0 16.8 0.5
27.2 0.6 13.0 0.7
11.7 3.4 3.8 1.8
K --
* SXCrrelease from target cells in the presence of nonimmune (control) effector cells ranged from 10.3 to 21.6%. H-2K and H-2D alleles. B 1 0 . M ( I I R ) target cells l a c k i n g the m i n o r H - a n t i g e n s o f the A strain b a c k g r o u n d are not lysed (line 10). T h e s e d a t a also d e m o n s t r a t e t h a t the H - 2 t - t o l e r a n t B 10.A a n i m a l s do not generate a n t i - H - 2 killer-cell a c t i v i t y against B10.M target cells (line 11); whereas n o n t o l e r a n t a n i m a l s d o generate such a c t i v i t y (line 12). T h u s , a significant a m o u n t o f specific cytotoxic T-cell a c t i v i t y c a n be g e n e r a t e d in B 10.A a n i m a l s t o l e r a n t to H-2 f antigens w h e n these a n i m a l s are sensitized against m i n o r H - a n t i g e n s in the context o f the t o l e r a t e d H-2-allogeneic h a p l o t y p e . Minor H-Antigens Presented on the Tolerated Haplotype Induce T-Effector Cells Restricted to Host Haplotype. Bevan (6) has d e m o n s t r a t e d t h a t m i n o r H - a n t i g e n s p r e s e n t e d on H 2 semiallogeneic cells m a y be used to cross p r i m e mice in vivo so t h a t the a n t i g e n can be p r e s e n t e d on the allogeneic H-2 h a p l o t y p e . I n these experiments, we wished to d e t e r m i n e w h e t h e r a n i m a l s tolerant to an H - 2 h a p l o t y p e c o u l d be p r e s e n t e d with m i n o r H - a n t i g e n s on the t o l e r a t e d h a p l o t y p e so t h a t the antigens c o u l d cross-prime a n d restrict for the host h a p l o t y p e . B 10.A a n i m a l s t o l e r a n t to B 10.M were i n o c u l a t e d in vivo with A . C A l y m p h o i d cells. W h e n spleen cells from these t o l e r a n t a n i m a l s were later c u l t u r e d in vitro w i t h A . C A cells, a cytotoxic response was g e n e r a t e d against A . C A , b u t not H - 2 - i n c o m p a t i b l e target ceils ( T a b l e II, lines 1-4), in a g r e e m e n t w i t h the d a t a shown in T a b l e I. F u r t h e r m o r e , w h e n freshly h a r v e s t e d spleen cells were c h a l l e n g e d in vitro with A strain spleen cells, the cytotoxic effect g e n e r a t e d was now restricted to K k D a target cells, i n c l u d i n g C 3 H , which indicates t h a t the A a n d C 3 H strains share some m i n o r H-alleles (lines 5 a n d 7). I m p o r t a n t l y , A . C A cells a n d o t h e r H - 2 - i n c o m p a t i b l e target cells b e a r i n g the a p p r o p r i a t e m i n o r H - a n t i g e n s were not lysed (lines 6 a n d 8). Thus, these results i n d i c a t e t h a t m i n o r H - a n t i g e n s p r e s e n t e d on cells o f a n allogeneic H-2 h a p l o t y p e can p r i m e the a n i m a l for m i n o r H - a n t i g e n recognition on the host's H-2 h a p l o t y p e .
ANTIGEN H-2 ALLOSPECIFIC RECOGNITION
1004
TABLE III Minor H-Antigens Presentedon Host Haplotype Fail to Induce T Effector Cells Restricted to Tolerated Haplotype
Line
1
Responder
BI0.A tolerant to B10.M
Cells Cells used for used for in vivo in vitro chalpriming lenge A
A
2 3 4
Target cells
A
H-2-region sharing between cells used for in vitro challenge and target cells K D
A.CA B10.A tolerant to B10.M
A
A.CA
A.CA A
K D
Net 51Cr release* (effector-to-target cell ratio) 100:1
50:1
10:1
15.8
8.5
2.9
--0.4
-3.5
- 1.0
-3.2
-1.1
-0.3
-1.1
-1.7
0
* S~Cr release from A and A.CA target cells in the presence of nonimmune (control) effector cells was 20.0 and 24.4%, respectively.
Minor H-Antigens Presented on Host Haplotype Fail to Induce T-Effector Cells Restricted to Tolerated Haplotype. W e have previously d e t e r m i n e d t h a t the extent o f c h i m e r i s m in a n i m a l s r e n d e r e d tolerant as neonates is e x t r e m e l y low (4, 7). Therefore, it w o u l d be expected t h a t the n u m b e r o f (B 10.A X B 10.M)FI l y m p h o r e t i c u l a r cells a b l e to present a n t i g e n in B 10.A a n i m a l s tolerant o f the B 10.M h a p l o t y p e w o u l d be c o r r e s p o n d i n g l y low. In the following e x p e r i m e n t , we d e t e r m i n e d w h e t h e r the chimeric F1 cells in tolerant a n i m a l s could present m i n o r H - a n t i g e n s to the host's T-cell system. B10.A a n i m a l s tolerant of B10.M were i n o c u l a t e d in vivo w i t h A strain l y m p h o i d cells followed b y reehallenge in vitro with either A or A . C A cells. T h e specificity o f effector ceils g e n e r a t e d following in vitro c u l t u r e is shown in T a b l e III. As expected, B 10.A a n i m a l s tolerant o f B I 0 . M a n d i m m u n i z e d with A strain cells in vivo a n d in vitro g e n e r a t e d cytotoxic effector cells with specificity for A strain a n d not for A . C A target cells (lines 1 a n d 2). However, when the tolerant spleen cells were c h a l l e n g e d in vitro with A . C A spleen cells, no cytotoxic effect was observed against A . C A target cells (line 3). Therefore, m i n o r H - a n t i g e n s p r e s e n t e d on A strain spleen cells c a n n o t be processed in vivo to allow for T-cell recognition o f m i n o r H - a n t i g e n s on the t o l e r a t e d H-2 f h a p l o t y p e . This finding suggests t h a t host reprocessing of m i n o r H - a n t i g e n s is not a requisite for sensitization of cytotoxic T lymphocytes. Minor H-Antigens Presented on Tolerated Haplotype Induce T-Effector Cells Restricted to Tolerated Haplotype. B10.M a n i m a l s were m a d e t o l e r a n t as neonates to B10.A cells, p r i m e d in vivo a n d c h a l l e n g e d in vitro with A strain l y m p h o i d cells. T h e i r spleen cells were then tested against a p a n e l o f target cells ( T a b l e IV). T h e specificity o f the cytotoxic cells g e n e r a t e d was restricted to H - 2 - c o m p a t i b l e target cells (lines 1-3). W h e n the a n i m a l s were c h a l l e n g e d in vitro with A . C A r a t h e r t h a n A strain l y m p h o i d cells, a strong cytotoxic effect was observed against A . C A target cells (lines 4 a n d 5), d e m o n s t r a t i n g cross-priming similar to t h a t shown in T a b l e II. Therefore, B10.M a n i m a l s tolerant to B 10.A also possess T cells t h a t can recognize m i n o r H - a n t i g e n s in the context o f the t o l e r a t e d H-2 h a p l o t y p e .
JAMES FORMAN AND J. WAYNE STREILEIN
1005
TABLE IV B I O.M Effector Cells Tolerant of B l O.A Recognize Minor H-Antigens on H-2 Allogeneic Cells H-Y-re-
gion sharCells
Line
Responder
Cells used for used for in vitro in vivo chalpriming
1
BI0.M tolerant to B10.A
A
lenge
A
2 3 4 5
B 10.M tolerant to BI0.A
A
Target cells
A.CA
ing between cells used for in vitro challenge and target cells
Net 5~Cr Release* (effector-to-target cell ratio) 100:1
50: l
10:1
A
K D
33.4
24.6
9.8
A.CA A.TH (KSDa):]:
-- D
2.3 32.2
0.4 24.0
0.9 10.2
A.CA
K D
39.3
32.1
11.2
6.0
4.7
3.4
A
* 51Cr release from target cells in the presence of nonimmune (control) effector cells ranged from 13.8 to 15.8%. H-2K and H-2D alleles. Discussion T h e d a t a in this report d e m o n s t r a t e t h a t a n i m a l s r e n d e r e d tolerant as neonates possess a significant n u m b e r o f cytotoxic T-cell precursors t h a t c a n recognize m i n o r H - a n t i g e n s in the context o f the allogeneic H - 2 h a p l o t y p e to which t h e y were r e n d e r e d tolerant. Secondly, because the specificity o f the effector cells g e n e r a t e d are restricted b y the H - 2 h a p l o t y p e o f the cells used for the in vitro challenge, it is c o n c l u d e d t h a t two different sets o f cytotoxic effector cells co-exist in t o l e r a n t animals. Because we c a n n o t find cytotoxic T cells (or their precursors) t h a t can recognize the t o l e r a t e d H 2 a l l o a n t i g e n s themselves in these animals, it is surprising t h a t T cells from these a n i m a l s t h a t recognize m i n o r H - a n t i g e n s can be restricted b y the H-2 a l l o a n t i g e n s to w h i c h t h e y are tolerant. This s i t u a t i o a m a y be e x p l a i n e d b y p o s t u l a t i n g t h a t alloreactive T cells b e a r receptors with a s p e c t r u m o f avidities for H-2 antigens. In the tolerance protocol, h i g h - a v i d i t y r e c e p t o r b e a r i n g T cells are deleted so t h a t only those w i t h low-avidity receptors remain. T h e s e p u t a t i v e l y l o w - a v i d i t y - r e c e p t o r clones could b i n d a l l o a n t i g e n with high avidity, however, if the H-2 a l l o a n t i g e n is presented together with m i n o r H-antigens. A l t e r n a t i v e l y , T cells recognizing m i n o r H - a n t i g e n s in the context o f H2 a l l o d e t e r m i n a n t s could represent a subset o f anti-self clones t h a t c o i n c i d e n t a l l y cross-react with m i n o r H - a n t i g e n s a l o n g with the allo-H-2 d e t e r m i n a n t s , a n d thus w o u l d not be e x p e c t e d to be affected b y the tolerance p r o c e d u r e itself. It is very unlikely t h a t FI cells utilized for the tolerance i n d u c t i o n are m e d i a t i n g the cytotoxic effects o b s e r v e d because we have previously shown t h a t (a) the extent o f l y m p h o i d cell c h i m e r i s m in a n i m a l s r e n d e r e d t o l e r a n t b y this protocol is < 5 % (4, 7), a n d (b) t r e a t m e n t o f the effector cells with a n t i s e r u m against the FI cells does not affect the cytotoxic response (7). It is possible t h a t t h e a c t i v i t y of the effector ceils from B10.A mice tolerant to
1006
ANTIGEN H-2 ALLOSPECIFIC RECOGNITION
B 10.M that were sensitized and tested against A.CA is H-2 unrestricted and directed against Qa-Tla antigens (8) rather than A strain minor H-antigens restricted by H2K t or H-2D f. This possibility is unlikely, however, because (a) both B 10.M and A.CA have the same alleles at the Qa-Tla loci that control antigens that are recognized by cytotoxic T cells, and (b) attempts to demonstrate H-2-unrestricted anti-Qa-Tla effector cell activity by cross-sensitization of B 10.M animals with A.CA cells and vice versa has yielded negative results (8; and James Forman, unpublished results). Bevan (6) has demonstrated that cross-priming occurs in vivo when minor Hantigens are presented on an H-2 haplotype that is different than that used for in vitro boosting. Therefore, it is possible that minor H-antigens have to be reprocessed on host cells to generate cytotoxic effector cells. However, our data demonstrate that this is not a requirement. Thus, B10.A animals tolerant of B10.M when primed in vivo and boosted in vitro with A.CA cells generate H-2f-restricted anti-minor-Hantigen effector cells. It could be argued that A strain minor H-antigens are processed on the small number of (B10.A × B10.M)F1 cells residing in the tolerant animals (
