Immunological Rev. (1977), Vol. 35

Published by Munksgaard, Copenhagen, DenmarkNo part may be reproduced by any process without written permission from the author(s)

Responsiveness to HY Antigen

Ir Gene Complementation

and Target Cell Specificity

ELIZABETH SIMPSON^ & ROBERT D. GORDON^

INTRODUCTION

From earlier in vivo studies (reviewed by Gasser & Silvers 1972) it wasclear that strains difEered in the ability of females to reject syngeneic maleskin grafts. Females of all strains of the U2^ haplotype, e.g. C57B1/10 (BIO)C3H.SW, 129/J and A.BY, fairly rapidly rejected such grafts and showedclassical second set responses when subsequently challenged with a secondsyngeneic male graft. The influence of non-H-2 genes on the ability to re-ject syngeneic male skin grafts seemed small. Females of other H-2 haplo-types examined, e.g. H2'', H2^, were non-responders, and did not rejecteither first or second grafts in any regular fashion. Certain BIO congenicrecombinant strains showed intermediate responses and since there was acorrelation between responsiveness to HY by skin graft rejection and im-mune responsiveness to the synthetic polypeptides (T,G)-AL and H,G-AL,Stimpfling & Reichert (1971) suggested that reactivity to H-Y was at leastpartially under control of the Ir-1 locus.

Fl mice with one H2^ parent are capable of rejecting male skin of eitherparental strain (Gasser & Silvers 1972). This suggests that the immune re-sponse gene(s) controlling responsiveness are dominant, and that male cellsof non-responder strains bear HY antigen. It has been shown that the tissuedistribution of HY antigen is ubiquitous. Goldberg et al. 1972, demonstratedcytotoxic anti-HY antibody, both in inbred strains which reject and in thosewhich fail to reject syngeneic male skin grafts. Thus, interstrain differences

' Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow,Middlesex HAl 3UJ, England.

^ Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts 02114,USA.

m SIMPSON & GORDON

in immune responses to HY may depend on cell-mediated mechanisms.Goldberg et al. (1973) demonstrated cytotoxic T cells against C57B1/6male lymphocytes in C57B1/6 female mice following rejection of syngeneicmale skin.

In this paper we propose to review the nature of the anti-HY cytotoxicT cell responses generated in vitro following in vivo priming. We have fotindthat all strains capable of rapid rejection of syngeneic male skin grafts cangenerate such cytotoxic cells (Gordon & Simpson 1977), that the targetcell specificity of the response is H-2 restricted in the manner first describedby Zinkemagel & Doherty (1974) for cytotoxic responses to certain viruses,and by Shearer (1974) for such responses to haptens (Gordon et al. 1975),and that Fj mice, of certain parental H-2 haplotypes which themselves arenon-responders, are responders, suggesting H-2 Ir gene complementationfor the anti-HY cytotoxic response, analogous to the Ir gene complemen-tation which has been shown for certain antibody responses e.g. Munro &Taussig 1975 (Gordon & Simpson 1977, Simpson & Gordon 1977).

METHODS

Mice were obtained from the Breeding Unit, C.R.C. The methods used toelicit and assay a cytotoxic T cell response have been previously describedGordon et al. 1975, Simpson et al. 1975). Briefly, spleens were removedfrom female mice primed in vivo by skin grafting with syngeneic male skinor by i.p. injection of 10' syngeneic male spleen cells 14 days to 4 monthspreviously. The spleens were teased apart and the resulting cell suspensionwashed once in a balanced salt solution (BSS), then resuspended at 5XlO«cells/ml in bicarbonate-buffered RPMI medium containing 10 % FCS, with10 mM hepes, glutamine, penicillin and streptomycin, and 5 x 1 0 " ^ 2mercaptoethanol. The responding cells were co-cultured with an equal num-ber of 2000R irradiated male spleen cells in 25 cm^ plastic tissue cultureflasks. After 5 days incubation in a humidified, 10 % CO2 atmosphere, thecultures were harvested, washed once in BSS and then resuspended inEagles minimal essential medium with 10 % FCS and 10 mM hepes. Thecell concentration was adjusted and three doubling dilutions made. Thevarious concentrations of attacking cells were then plated in triplicate inwells of a microtiter plate, and 1 X 10^ ^^Cr-labeled target cells added perwell. TTie target cells were spleen cells which had been cultured for 48-72 h in the presence of 4 //g/ml Con A, labeled for 90 min with ^ Crsodium chromate and then washed twice. The attacker: target cell ratiosusually used were 8:1, 4:1, 2:1 and 1:1. The plates were spun briefly andthen incubated at 37°C in a 10 % CO2 atmosphere for 3 h before harvest-

T CELL RESPONSE TO HY ANTIGEN 61

ing the supernatants for gamma counting. Maximum release was that amountof ^'Cr released from triton-treated target cells; spontaneous release wasthat released by target cells incubated in medium alone.

The response of H-2'' mice

The response of unseparated spleen cells and splenic T cells from C57B1/10(BIO) female mice primed m vivo and boosted in vitro with BIO male cellsis shown in Figure IA. Figure IB shows that the cytotoxic cells are sen-sitive to the effect of anti-Thy 1.2 serum plus complement. From these datait is clear that the HY response is a classical cytotoxic T cell response.However, primary in vitro responses have not been obtainable in this system(Figure 2),

Table I shows the results of four experiments In which the target cellspecificity of the anti-HY cytotoxic cells for primed and boosted BIO fe-males was examined. All male target cells of H2^ haplotype were killed,

1.0 2 .0 3.0 4.D

ATTACKING CELLS X 10-5

1.0 2 .0 3.0 4 .0

AHACKING CELLS X 10-5

Figure 1. Cytotoxicity of unseparated female spleen cells and female splenic T-cellsagainst *'Cr-labeled male spleen target cells. (A) cytotoxic activity of unseparated ( • )and nylon wool filtered (» ) BIO female spleen cells against ''Cr-labeled BIO maletargets. Female spleen cells from mice primed in vivo by i.p. injection of isologousmale spleen cells were cultured far 5 days in vitro with equal numbers of irradiatedmale spleen cells. One aliquot of female cells was filtered through a nylon woolcolumn prior to culture to remove nearly all the B-lymphocytes. (B) Unseparated fe-male spleen cells primed in vivo and cultured in vitro for 5 days with male spleencells were treated with anti-Thy 1.2 serum, preabsorbed with TTiy 1.1 thymocytes,with (# ) and without ( • ) complement just prior to assay against ^^Cr labeled maletarget cells. All responding cell cultures were assayed in quadruplicate for 3 h at fourA:T ratios. Background (spontaneous *'Cr release) was 19.6%. Standard errors wereless than 4 % of mean counts. (From: Gordon et al. 1975).

SIMPSON & GORDON

TABLE IH-2 target cell specificity of celt-mediated responses to H-Y antigen

A

B

C

D

Target cell

BIO ,3BIO9B10.A(4R)(3B10.A(4R)9B10.A(5R) (3

B10(3B10.A(2R) SB10.A(2R)9B10.A(5R)c5B10.C(47N)(5B10.C(47N)9C3H.SW SC3H.SW$C3H5

BIO ,3B10.D2 5Balb/c ,3D2.GD(3BIO.BR 5CBA (5B10JVC3

Ac3

B10(5(B10XBalb/C)Fi(5(B10XBalb/C)Fj9/ [In TV./i ^ V D1 AMI? ^\tja.i I?/ \^ /\ 0 3-Kf}r -i 0

f/^Ti A V Dl ffc/t .^

( B 1 0 X A ) F I J 3

(B10.D2xBalb/C)F,5

K

bbkkb

bkkbbbbbk

bdddkkkk

H-2 haplotypeIA

bbkkb

bkkbbbbbk

bdddkkkk

IB

bb

bbb

bkkbbbbbk

bddbk

kkk

IC

bb

b

CT

"

d

bdddbbbbk

bddbk

kdd

S

b

bbbd

bdddbbbbk

bddbkkdd

Parental haplotypesH-2'' /H-2b

H-2ti / H-2«>H-2'' /H-2d

H-2'' / H-2''H-21' /H-2b

H-2'' / H-2K/1H-2'' / H-2d

D

bb

bbd

bbbdbbbbk

bddbkkdd

Corrected% lysis

22.17 ±0.852.03 ±0.23

20.74 ± 0.725.06 ±1.193.30 ±0.22

33.48 ± 2.9530.56 ±2.102.24±0.173.92 ±0.34

33.26 ±2.452.52±0.21

38.48 ± 3.801.51 ±0.117.30 ±1.25

16.74 ±0.702.78 ±0.451.14 ±0.20

15.41 ±0.541.32 ±0.273.30 ±0.422.80 ±0.292.60 ±0.49

33.48 ±2.9522.49 ±2.33

2.29 ±0.1623.05 ±3.1424.29 ±1.3421.20 ±0.90

3.18 ±0.25

0.990.981.000.880.99

0.970.970.970.970.980.990.960.980.88

0.990.930.911.000.940.880.980.92

0.970.960.990.930.990.990.99

BIO 9 spleen cells from mice primed in vivo and boosted in vitro with BIO ^ spleencells were assayed in quadruplicate for 3 h with ^^Cr labeled target cells at A:T = 4:1,2:1, 1:1, and 0.5:1. Corrected % lysis is the % kill of target cells at A:T = 4:1 asdetermined from a four point linear regression curve ± one standard error, r is ameasure of the goodness of fit. Background ' 'Cr release ranged from 5 % to 20 %.Standard errors were less than 5 % of mean counts. Maternal parents are listed firstin describing the origins of F , animals, e.g., (BlOxA)Fi means (B10$XAc5)Fj. (From:Gordon et al. 1975).

T CELL RESPONSE TO HY ANTIGEN 63

regardless of genetic background (i.e. BIO, C3H.SW, BIO C(47)N) or ofwhether only one parent of an Fi contributed the Iri2^ haplotype (i.e. (BIOxBalb)F,, (BalbxBlO)Fi, (CBAxBlO)Fi, (BlOxA)Fa). Female cellswere not killed to any significant extent, nor were cells bearing H2 haplo-types other than H2'' (i.e. C3R CBA, A, BIO.A, Balb/C, B10.D2). Thus,target cell specificity is H2^-Umited. Of the H2'' recombinant strain targetsexamined, B10.A(2R) male cells were killed, whilst those of B10.A(5R)were not. Therefore the sharing between responder and target of H2D*' isnecessary and sufficient. The nature of the association between the HYantigen, presumably a product of the Y chromosome, and H-2D'', a productof the MHC, which is on chromosome 17, is not indicated in these experi-ments, but from the F: target data shown it is clear that the HY chromo-

50

0.5 1,0 1.5 2,0 ^1,0 8,D

ATTACKING CELLS X 10-5

Figure 2. Cytotoxic response of sensitized BIO female spleen cells against lXlQ^ BIO'^'Cr-labeled male spleen target cells. All responding cells were cultured for 5 dayswith an equal number of irradiated BIO male spleen cells. Each culture of respondingcells was assayed in quadruplicate for 3 h at six attacking cell to target cell (A:T)ratios. Linear regression lines were plotted from the origin through A:T = 2:1 (i.e.2X10* attacking cells). ( • • ) unprimed (normal) BIO female spleen cells (pri-mary in vitro sensitization). (Q D) female responding cells primed in vivo by

grafting with BIO male tail skin 44 days prior to culture. Data fit a linear regressionline with slope 6.44±0.16 and r (the coefficient of determination, a measure of thegoodness of fit) = 1.00. ( • • ) female responding cells primed in vivo by i.p. in-jection of l x l O ' BIO male spleen cells 14 days prior to culture. Data fit a linearregression line with slope 15.2011.95 and r = 0.95. (O O) female respondingcells primed in vivo by two previous male skin grafts applied 5 and 3 months earlierand boosted with lXlO'' BIO male spleen cells injected i.p. 14 days prior to culture.Data fit a linear regression line with slope 19.6214.34 and r — 0.87. Background(spontaneous ^'Cr release) was 8.6 %. Standard errors were less than 4 % of meancounts. (From: Gordon et al. 1975).

SIMPSON & GORDON

TABLE IISpecificity of cell-mediated cytotoxic responses by BIO'^ primed with

B10.BR C5 or B10.BR^ skin

Respondingcell

BIO?

BIO?

BIO?

BIO?

B10$

BIO?

Antigen

In vivo In vitro

BIO.BR (5 BIO.BR t5

B10.BR{3 B10(5

BIO.BR? BIO.BR 5

BIO.BR? BIO 5

BIO (5 BIO.BR (3

Targetcell

BIO^BIO?BIO.BR (5BIO.BR?

BIO ,3BIO?BIO.BR (5BIO.BR?

B10<5BIO?B10,BR(5BIO.BR?

BIO 6BIO?BIO.BR 5BIO.BR?

D1 n .O AU M

BIO?

BIO.BR?IJ ±\J (\

BIO?

Corrected % lysis(A:T= 4:1)

-3.04 ± LOO-3.61 ±0.6117.38 ±0.1416.49 ±0.11

18.42 + 2.85-1.01 ±0.58

0.55 ±0.510,31 ± 1.30

-1.46 ±1.33-1.89 ±0,9324.88 ±1.4425,46 ±2.81

-0.32 ±0.36-1.61 ±0.85-0.76 ±0,55-0.46 ±1.31

3.0611.311.71 ±0.82

44,01 ±2.9347,65 ± 0.41

57.31 ± 1.095.20 ±1.57

BIO female spleen cells from mice primed in vivo with BIO.BR male or female orBIO male skin grafts and challenged in vitro with BIO.BR or BIO male spleen cellswere assayed in triplicate for 3 h with =^Cr-labeled target cells at A:T = 1:1, 2:1, 4:1and 8:1, Corrected % lysis is the % kill of target cells at A:T = 4:1 as determinedfrom a four point linear regression fit ± 1 SE. Background (spontaneous) ^'Cr releasewas less than 13 % and SE were less than 5 % of mean counts. (From: Gordon et al.(1976)).

some can come from one parent (and not necessarily the BIO parent) andthe HZ^-bearing chromosome from the other parent.

Further evidence that, with respect to the cytotoxic response, the HYpart of the antigen on the target and therefore presumably on the priming

T CELL RESPONSE TO HY ANTIGEN 65

TABLE mSpecificity of celt-mediated cytotoxic responses by

primed and challenged with CBA (5 and/or BIO i^ cells

Respondingcell

Antigen

In vivo In vitro

Targetcell

Corrected % lysis(A:T= 4:1)

CBA 5 CBA (5(CBAxBlO)Fi?

(CBAxBlO)Fj?

(CBAxB10)Fj?

(CBA X B10)Fi? BIO (5 CBA ^

tJ 1 A ^

BIO?CBA (3CBA?

D 1 O .^

BIO?CBA (3CBA?

D1 n ^

BIO?CBA 5CBA?

BIO (5BIO?CBAc5CBA9

-0.1110.200.4910.27

21.7311.385.3710.30

1.9110.621.7310,551,10 + 0.673.9810.43

26,3412.342.7811.020,9211.071.8910.20

-0.59 ±0.802.8611.230.5510.041.6510.33

(CBAXBlO)Fj female spleen cells from mice primed in vivo with a CBA or BIO maleskin graft and challe^nged in vitro with CBA or BIO male spleen cells were assayedin triplicate for 3 h with ='Cr-labeIed target cells at A:T = 1:1, 2:1, 4:1 and 0.5:1 or8:1. Corrected % lysis is the % kill of target cells at A:T = 4:1 as determined froma four point linear regression fit 1 1 SE, Background (spontaneous) ='Cr release wasless than 15 % and SE were less than 5 % of mean counts. Maternal parents are listedfirst in describing the origins of F^ mice e.g, CCBAxB10)Fi means (CBA?

i: Gordon et al. (1976)).

and boosting male cell is shared between strains comes from experimentsin which BIO responder females were primed with allogeneic male cells.Such mice were not only primed to give second set skin graft responsesto syngeneic male skin (data not shown: see Gordon et al. 1976) but alsogave HZ^-restricted cytotoxic HY responses (Table II). However, it was notpossible to elicit an HY response when the boosting antigen was an allo-geneic male cell, for in that case an anti-H2 response took over (Table II),

Immunological Rev. (1977), Vol. 35

66 SIMPSON & GORDON

TABLE IVMapping of HY association with H-2 haplotypes d, k and a

ResponderPriming

and boostingantigen

Targetcell

H-2haplotype

Corrected% lysis

H-2association

(B10XBa!b)Fj$

(B10XCBA)Fi9 CBA

B105B109B10.A(2R)(5B10.A(2R)9B10.A(5R)(5Balb(5

Balb^Balb$D2GD5BIO.HTT^HTIc5BIO (5

CBAc5CBA5B10.Ac5C3H.OH c5B10c3ATL<5

A ,3A9B10.A(2R)5Balb/c (5CBAc5CBA9BIO ,5

bbbbbbbbbbbbkkkddbkkkddbbbbddddddddd

ddddddddddddddbbbbssskkdbbbbbdbbbbbb

kkkkkkkkkkkkkkkddddddddkbbbbbbskkkkd

kkkdddkkkdddkkkddbddddddkkkkkkkkkkkkbbbbbb

10.45 ±1.552.00 ±1.26

15.7511.75-1.58 ±1.20

0.78 ±0.332.34 ±0.75

14.89 ±2.90-1.66 ±0.2625.01 ±3.042.63 ±0.292.57 ±1.46

-0.56±0.19

31.09 ±0.832.39±0.514.62 ±0.90

35.11 ±1.861.19±0.311.01 ±0.11

16.99 + 0.962.71 ±0.67

27.14±1.715.21 ±0.68

23.6811.443.5910.221.57 ±1.05

H-2D'*

H-2K''

H-2D!'

H-2K''

Spleen cells were from responder mice primed in vivo and boosted in MLC in vitrowith male cells of the strain indicated. Assay was performed in triplicate at four at-tacker: target cell ratios. Corrected % lysis is the % kill of target cells at A:T 4:1as determined from a four point regression curve.

The response of Fi mice with one H2^ parent

Fl mice which are responders by virtue, prestmiably, of the inheritanceof dominant immune response gene(s) from an H2'' parent can reject maleskin from either parent, and also generate cytotoxic T cells to male cellsof each parent. However, as shown by the data in Table III, there are ap-

T CELL RESPONSE TO HY ANTIGEN 67

TABLE V'Parental preference' of HY association in response of Fj mice with one H-2^ parent

Responder Priming antigen Boosting antigenTarget cell

Balb/c 5

(Balb/c xB10)Fi (3 (Balb/C XB10)F, (5BIO (5Balb/C S

BIO 5

40.91 ±2.3424.60 ±2.110.76 ±0.38

43.19 ±0.321.64 ±0.81

4.68 ±0.350.47 ±0.134.16 ±0.172.50 ±0.30

15.52 ±1.01

(CBAxB10)Fj$

BIO (5CBA 6

-1.24 ±0.331.38 ±0.73

-2.89 ±0.4021.92±1.59-1.62 ±0.37

BIO 320.94 ±2.9343.00±2.79

1.23 ±0.67

19.72 ±1.520.61 ±0.42

15.08 ±0.370.16 ±1.22

33.20 ±0.62

1.69 ±0.043.34 ±0.806.98 ± 0.74

Spleen cells were from responder mice primed in vivo and boosted in MLC in vitro withmale cells of Ihe strains indicated. Assay was performed in triplicate at four attacker :target cell ratios. Corrected % lysis is the % kill of target cells at A:T 4:1 as deter-mined from a four point regression curve.

parently two separate clones of HY-reactive cells in such an Fi, and primingone set with parent 1 (e.g. CBA male) only enabled an HY response whenthe boosting antigen was CBA male, and the target cell specificity of sucha response was H2''-liniited. Likewise, priming and boosting with parent 2(BIO male) gave an H2Mimited or -associated HY response. Such responseshave enabled us to map the H-2 association of HY cytotoxic responses forthree non-responder haplotypes, H-2" (CBA), U-l^ (Balb, B10.D2) andH2" (A). In each case Fi responders were made between BIO and thestrain in question, and then stimulated with male cells of the non-responderhaplotype. The H-2 restriction and mapping of these responses are shownin Table IV, which also indicates that an Fi sueh as (B10xBalb/C)Fi,when stimulated with H2'' male cells, makes an H-2D''-associated HY re-sponse indistinguishable in specificity from a response by a BIO female (seeTable I). In summary, H2^ HY responses in (BlOxBalb)Fi mice are H-2K^-associated, H-2'' responses in (BlOxCBA)Fi mice H-2D''-associated, whilstin (BlOxA)Fi mice the association was with H-2K'', indicating that under

68 SIMPSON & GORDON

TABLEIn vitro cell-mediated cytotoxic

Responder

A C57BL/10$CBA2B10.A$Balb/C$A.SW9

B C57BLyiO$

B10.A(4R)$B10.A(5R)9HTI5HTG$D2.GD9B10.HTT$A.TH$

C C3H.SW9

129/J$

A.BYJ

P (CBAXBaIb)Fi9

(CBAxBaIb)Fi2

Antigen

C57BiyiO ^CBA^B10.A (5Balb/C SA.SW (3

C57BIV10 (3

BIO.A(4R)(5B10.A(5R)5HTISHTG5D2.GD 5BIO.HTT^

A.THS

C3H.SW(5

129/J S

A.BY 5

CBA^

Balb/c^

Target cell

C57BiyiOCBABIO.ABalb/CA.SW

D2.GDB10.A(4R)B10.A(5R)B10.A(4R)B10.A(5R)i f nHTGD2.GDBIO.HTTBIO.HTT

C57B1V10B10.A(2R) 'B10.A(5R)

. C57BLyiOB10.A(2R)B10.A(5R)C57B1710B10.A(2R)B10.A(5R)

CBABalb/CC57BLyiOCBABalb/CC57BLyiO

Spleen cells from female mice, primed in vivo and challenged in MLC with the malecells (antigen) shown, were assayed in triplicate for 3 h against lXlO ' ^^Cr-labeledtarget cells at A:T = 1:1, 2:1, 4:1 and 8:1. Corrected % lysis is the kill of ^'Cr-labeledtarget cells, corrected for background, at A:T = 4:1 as determined from a four pointlinear regression fit ± SE. Background (spontaneous *'Cr release) ranged from 8 %to 2 5 % . (CBAXBalb)Fj means (CBA$xBalb/C(5)Fi. The H-2 haplotype of A.THh s s s s s d. (From: Gordon & Simpson 1977).

T CELL RESPONSE TO HY ANTIGEN 69

VIresponses to H-Y antigen

K

bkk

ds

dk

bkhbddss

bkbbkbbkb

kdbkdb

H-2 haplotype

IA

bkkd

s

dkbkbbddss

bkb

bkbbkb

kdbkdb

IB

bkkd

s

bb

bbbbd

bss

bkbbkbbkb

kdbkdb

IC

bkdd

s

bbdbdbdbkk

bdd

bddbdd

kdbkdb

S

bk

dds

bbdbdbdbkk

bdd

bddbdd

kdbkdb

D

bk

dds

bbdbddbbdd

bbd

bbdbbd

kdbkdb

Corrected %

S target

18.72 ± 0.280.01 ± 0.02

-0.55 ± 1.03-0.73 ± 0.44

0.81 ± 0.27

19.60 ± 0.6021.02 ± 1.68-0.11 ± 0.41

0.95 ± 0.610.11 ± 0.504.26 ± 4.42

-1.19 ± 0.08-1.03 ± 0.290.02 ± 0.660.31 ± 0.58

24.28 ± 0.7525.51 ± 1.93-1.09 ± 0.9615.74 ± 0.7314.23 ± 0.61^ .02 ± 2.3021.34 ± 0.6322.45 ± 1.84^ .81 ± 1.52

25.61 ± 1.862.37 ± 0.431.66 ± 0.751.87 ± 0.472.22 + 0.250.84 ± 0.55

lysis (A:T = 4:1)

$ target

2.75 ± 0.92

-1.46 ± 0.460.87 ± 0.15

-1.64 ± 0.09

-1.46 ± 0.99

-0.93 ± 0.38

2.40 ± 0.22

appropriate conditions the yUK^ antigen(s) can 'associate with' HY presum-ably both at the level of antigen presentation and at the target cell.

There are, however, certain peculiarities about the HY response of Fimice having one H-2'' parent. One is that it has proved very difficult toprime them allogeneically, with male cells differing either just at H-2 orat H-2 plus non H-2. Such allogeneically primed Fi mice, when boosted

70 SIMPSON & GORDON

in vitro with male cells of either parent, fail to produce any cytotoxic re-sponse, in contrast to BIO female responder mice primed allogeneic ally.

Another interesting and unexpected finding was that Fi mice with oatH-l" parent, when primed and boosted with Fi male cells, produced anHY response which was predominantly associated with only one parentalhaplotype. These results, shown in Table V, show that for (BalbxBlO.Fifemales the 'preferred parent' is BIO, whilst for (CBA X B10)Fi it is CBA,and for (AxBlO)Fi it is BIO. The selection of only one of two possibleclones {i.e. one to each HY-associated haplotype) is an event which takesplace during priming, since its effects are fixed after Fj priming, and there-after a response to the non-preferred parent could not be elicited by boost-ing with male cells of that parent. These results might suggest that thereis a hierarchy of association, or antigenicity of HY in association with dif-ferent H-2 products, so that H-2D'' was strongest {CBA the preferred par-ent for {CBAxB10)Fi anti-Fi male responses), followed by H-2D^ {BIOthe preferred parent for (AxBlO)Fi responses), then H-2K' and H-2K^by virtue of A and Balb/C being the 'non-preferred parents' in (AxBlO)Fiand (Balb/C xB10)Fi responders. However, an additional factor whichnow needs consideration is the function or interaction of HY Ir genes whichwe know exist in non-responder haplotypes (Gordon & Simpson 1977,Simpson & Gordon 1977; see also sections below and discussion).

The response of various strains to HY

Table VI details the results of attempting to elicit anti HY responses inthe manner described, in a number of different strains. From experimentsA and C it will be seen that all the H-l^ strains examined, regardless ofnon-H-2 'background' are responders, and share the same target cell spe-cificity. Experiment B shows that none of the H-2'* recombinant strainsexamined show cytotoxic T cell responsiveness to HY, nor do two otherrecombinant strains of different haplotypes. It is of particular interest thatstrain HTI is a non-responder, since it is of the H2'' haplotype from Kthroughout the I region, differing only at D.

There are two possible explanations for this negative finding: firstly, thatone or more Ir gene(s) necessary for the HY response are coded at, or verynear the D end of the MHC complex; or secondly, that H-2D' is an in-appropriate antigen for HY presentation/association. In fact, we have notfound an example of H-2D^ association with HY in any HY responders -(Balb/C xB10)Fi females primed and boosted with Balb/C male cells makean H-2K<'-associated response, and {AxB10)Fi females primed and boostedwith A male cells make an H-2K"-associated response.

T CELL RESPONSE TO HY ANTIGEN

TABLE VIIH-2-associated mapping of HY responses

11

Responder O(haplotype)

Antigen Target

6Target

haplotype

Corrected% lysis

(A:T = 4:1)

Mappingof HY

association

(CBAXBalb)Fikkkkkk/dddddd(CBA X BaIb)Fi(B10.BrxB10.D2)Fikkkkkk/dddddd(CBAXBIO.S)F^kkkkkk/ssssss(CBAxBlO.S)Fj

(CBAXA)Fkkkkkk/kkkddd(CBAXA)F,C3H.OHdddddk(B10.A(4R) X B10.A(5R)Fikkbbbb/bbbddd

CBA

BalbBlO.Br

CBA

BIO.S

CBA

AC3H.0H

BIO

C3H.OH2R

BalbC3H.OH

CBAA

C3H.0HBIO.SATH

AC3H.0H

AC3H.0H

Balb4R5R

dddddkkkkddbdddddddddddkkkkkkkkkkddddddddksssssssssssdkkkddddddddkkkkddddddddkddddddkkbbbbbbbddd

40.720.9

2.1

14.317.122.712.514.1

23.88.5

17.5

±1.2±1.4±1.4±0.4±0,9±0.6±2.3±2.20

±0.9±1.40

±0.70

17.38 ±0.892.88 ±0.48

j

(K'O D'

I KI' ni;j1 ^> K^ nK

1 ^ ""

11 Db

Spleen cells from female mice, primed in vivo and challenged in MLC with the malecells (antigen) shown, were assayed in triplicate for 3 h against 1X10= ^^Cr labeledtarget cells at A:T = 1:1, 2.1, 4:1 and 8:1. Corrected % lysis is the kill of ^'Cr labeledtarget cells corrected for background at 4:1 as determined from a four point linear re-gression fit ± SE. Background (spontaneous ^'Cr release) ra^iged frmn 10 % to 25 %.(From: Simpson &. Gordon 1977).

The responses of Fj mice of non-responder parents

From experiment A {Table VI) it is evident that neither Balb/C nor CBAare responder strains to HY, and yet experiment D shows that the Fi be-tween them is a responder, albeit only to CBA male cells. Some detailsof the mapping of the H-2 association of this response are given in TableVII. This remarkable finding of a responder Fi derived from two non-responder haplotypes H2'' and H2^ immediately suggests Ir gene comple-mentation, and since Ihe effects on HY responsiveness of non-H-2 geneshas been considered minimal, (for skin grafting, see Gasser & Silvers (1972),and for the cytotoxic T cell response, experiment C, Table VI), it seemedmost likely that the Ir gene complementation was between genes in theH-2 region. However, this needed confirmation using H-2 congenic mice,and some pertinent data on (BlO.Br xB10.D2)Fi responders are given inTable VII. In short, these Fi mice, too, are responders, at least to BlO.Br

SIMPSON & GORDON

male cells, whereas neither parent is a responder. Several more examplesof Ir gene complementation, probably involving genes within the MHCcomplex, have now been found and are detailed in Table VII. In the caseof each Fi responder, neither of the parental strains was a responder (CBA,BIO.S, A, B10.A(4R), B10.A(5R)). In some of these cases, an HY re-sponse can be elicited to male cells of both parents, e.g. (CBAxBlO.S)Fi,whereas in others only one male parental cell is 'immunogenic' (e.g. (CBAxBalb)Fi (CBAxA)Fi). This is a little surprising in the latter case, since(CBAxA)Fi responds to CBA male cells, and the association is bothH-2K' and H-2DS whereas male A cells, which also bear H-ZK" antigen(s)cannot elicit the response. It was also very interesting to find an exampleof gene complementation in a recombinant strain involving H2' and H2''haplotypes. However, as the I region in C3H.OH is entirely derived fromthe H2'' haplotype from which this strain differs only by being H-2'' at theD end, this strain may be an example rather of appropriate antigen presen-tation (i.e. the H2D'' association of HY may be immunogenic, whereas H2D^association may not - see also the discussion of HTI non-responsivenessabove). This may also be true of the (B10.A(4R) X B10.A(5R))Fi com-plementation shown, especially in view of the HTI discussion in relationto H2'' Ir genes. It is clear from the data in Table VII that Ir genes forHY responsiveness, wherever they map, are present in non-H2'' 'non-re-sponder' haplotypes. Whether additional genes, coding for antigen(s) at theK and/or D end, and implicated in antigen presentation as well as targetcell specificity, are also involved, is not clear at the moment.

DISCUSSION

The cytotoxic response to HY exemplifies the response to minor i.e. non-H-2 transplantation antigens. Bevan (1975) has found that responses toseveral other non-H-2 antigens, either genetically isolated in co-isogenicstrains (such as H7 in B10.C(47)N and BIO) or several together, as in re-sponses between different strains sharing the H-2 region (e.g. Balb/C andB10.D2) are likewise H-2-restricted in target cell specificity. Thus, in eachcase so far examined the target cell needs to bear both the appropriateminor antigen and K and/or D determinants of the responder strain. Thenature of the association between self H-2 and minor alloantigens is stillnot understood, either at the level of the cell surface or at the biochemicallevel (see Doherty et al. 1976, Shearer et al. 1976 for discussion on thispoint). However, since it seems intrinsically unlikely that either determinantis biochemically 'modified' by the other, some type of dual or associativerecognition system on the part of the receptor(s) of the effector cytotoxic

T CELL RESPONSE TO HY ANTIGEN 73

cell seems likely. Since it has not been possible to elicit a primary cytotoxicresponse in vitro, either to HY or any other minor transplantation antigen,it is difficult to look at the question of the antigenic requirements for prim-ing, or whether more than one type of precursor cell is involved. It mayvery well be that primary in vitro responses are not obtained because thenumber of precursor cells is so small, and only reaches detectable numbersfollowing priming. The in vivo cross-priming of BIO responder females byallogeneic male cells could be via the reprocessing of HY antigen on theallogeneic cells on which they are presented, during the anti-allograft re-sponse which they must elicit, so that subsequently HY is presented to theanti-HY cytotoxic precursor cells as HY on self cells, e.g. macrophages.However, this hypothesis does not explain the apparent failure to prime Fimice allogeneically.

From the results obtained using anti-HY responder Fj mice derived fromnon-responder parents, it is clear that Ir gene interaction occurs, and thateven in 'non-responder' haplotypes, Ir gene(s), an albeit incomplete com-plement exists. This is a separate question from that of antigenicity of malecells of different haplotypes, since all haplotypes examined have been ca-pable of eliciting a response, both in vivo (e.g. the rejection of CBA maleskin grafts by (CBAxBlO)Fi females - but see Gasser & Silvers 1972), andin vitro (e.g. Table IV). Thus the gene complementation observed implies Irgene complementation and suggests that each F] has an 'array' of at leasttwo Ir genes, capable of 'seeing' HY in a different associative context.Thus, (CBAxBlO)Fi females 'see' HY on CBA male cells in associationwith D'', the (AxBlO)Fi female anti-A male response is K* -associated,whilst both (Balb/C xCBA)F, and (B10.D2xB10.Br)Fi mice respond toH2'' male cells in such a way that the association with k is H-2K'' andH-2D''. All the available evidence points to the Ir genes being in the MHC(minimal influence of non-H-2 genes in syngeneic male skin graft rejectionby H2^ females, and in generation of cytotoxic responses in these strains,similar behaviour of (B10.D2xB10.Br)Fi and (Balb/C X CBA)Fi re-sponders).

It seems unlikely that the Ir genes are coded at the K or D ends them-selves, at least for H2' responders, since we have found that H2'' mutantsboth at the K end (B6HZ1, B6HZ170) and at the D end (B6.HZW42) areresponders (Gordon et al. unpublished). This suggests that the Ir genes liebetween K and D, perhaps in the I region where so many Ir genes for anti-body responses to various antigens are situated. Our ability to map theirposition using the available recombinant strains would be increased by find-ing some non-complementing haplotypes for the HY response.

No direct evidence has yet been found for suppressor genes, but they

74 SIMPSON & GORDON

could exist in tioo-responder haplotypes, and even modify the effects ofIr genes in responders, for example in Fi"s which have one H-l" parent.Such gene interaction may be involved in some of the pecuharities of theresponse of these Fi's, viz. the failure to prime allogeneically, and thefinding of 'parental preference' when Fi male cells are used to prime (Ta-ble V).

There is very little evidence of MHC Ir genes limiting the H-2 restrictedcytotoxic responses of different haplotypes to viruses (see Doherty et al.1976), but there is at least one example of MHC Ir gene involvement inthe H-2-restricted cytotoxic response to haptenic determinants (for reviewsee Shearer et al. 1976). It may well be that the evolutionary importanceof these types of T cell responses to viruses have precluded non-respondertypes surviving. The function of minor transplantation antigens, includingHY, in ehdting self MHC restricted responses, is probably different fromthat of viruses, but one which may also have evolutionary significance andmay, as Ohno (1977) has suggested, be the signal for organo-genesis anddifferentiation. Certainly, the MHC-restricted cytotoxic response to HY isnot limited to mice, since it has now also been shown in human beings(Goulmy et al. 1977).

SUMMARY

The H-2 restricted nature of the cytotoxic T cell response to the HY an-tigen in mice has be«n reviewed, together with mapping data for the H-2 Kand/or D association of responses to male cells of the following haplotypes:H-2'', H-Z*", H-2'', H-2^, H-2^ Ir gene complementation is imphcated in theHY response of a number of Fi mice derived from non-responder parentalhaplotypes, and in one recombinant strain. Thus, at least two and possiblymore Ir genes are involved in the anti-HY response, in addition to the Kand/or D gene products with which HY is obligatorily associated, certainlyat the target cell level.

REFERENCES

Bevan, M. J. (1975) The major histocompatibility complex determines susceptibility tocytotoxic T cells directed against minor histocompatibility antigens. / . exp. Med.142, 1349.

Doherty, P. C , Blanden, R. V. & Zinkernagel, R. M. (1976) Specificity of virus-immuneT-cells for H-2K or H-2D compatible interactions: implications for H-antigendiversity. Transplant. Rev. 29, 89.

Gasser, D. L. & Silvers, W. K. (1972) Genetics and immunology of sex-linked antigens.Adv. Immunol. 15, 215.

I T CELL RESPONSE TO HY ANTIGEN 75

Goldberg, E. H., Boyse, E. A., Scheid, M. & Bennett, D. (1972) Production of H-Y anti-body by female mice that fail to reject male skin. Nature New Biol. 238, 55.

Goldberg, E. G.. Shen,F. & Tokuda, S. (1973) Detection of H-Y (male) antigen onmouse lymph node cells by the cell to cell cytotoxicity test. Transplantation (Bal-timore) 15, 334.

Gordon, R. D.. Mathiesan, B. J., Samelson, L. E., Boyse, E. A. & Simpson, E. (1976) Theeffect of allogeneic presensitization on H-Y graft survival and in vitro cell-medi-ated responses to H-Y antigen. /. exp. Med. 144, 810.

Gordon, R.D. & Simpson, E. (1977) Immune response gene control of cytotoxic Tcell responses to HY. Trans. Proc. 9 (in press).

Gordon, R.D., Simpson, E. & Samelson, L. E. (1975) In vitro cell-mediated immuneresponses to the male specific (HY) antigen in mice. / . exp. Med. 142, 1108.

Goulmy,E., Termijtelen, A., Bradley, B. A. & van Rood, J. J. (1977) Y antigen killingby women is restricted by HLA. Nature (in press).

Munro, A. J. & Taussig, M. (1975) Two genes in the major histocompatibility complexcontrol immtine response. Nature 256, 103,

Ohno, S. (1977) The ariginal function of MHC antigens as the general plasma mem-brane anchorage state of organogenesis-directing proteins. Transplant. Rev. 33.

Shearer, G.M. (1974) Cell-mediated cytoloxicity to trinitrophenyl modified syngeneiclymphocytes. Europ. J. Immunol. 4, 527.

Shearer, G. M., Rehn,T. G. & Schmitt-Verhulst, A. (1976) Role of the murine majorhistocompatibility complex in the specificity of in vitro T-ceU mediated lympho-lysis against chemically-modified autologous cells. Transplant. Rev. 29, 222.

Simpson, E. & Gordon, R. D. (1977) Ir gene complementation in the HY response.Nature (submitted for publication).

Simpson, E., Gordon, R. D., Taylor, M., Mertin,J. & Chandler, P. (1975) Micromethodsfor induction and assay of mouse mixed lymphocyte reactions and cytotoxicity.Europ. J. Immunol. 5, 451.

Stimpfling, J. H. & Reichert, A. E. (1971) Male-specific graft rejection and the H-2locus. Transplantation (Baltimore) 12, 527.

Zinkemagel, R. M. & Doherty, P. C. (1974) Immunological surveillance against alteredself components by sensitized T lymphocytes in lymphocytic choriomeningitis.Nature (Lond.) 248, 701.