T-cell and antibody typing of a mouse population segregating for Sxr and H-2 haplotype

CELLULAR

IMMUNOLQGY

98,46-56 (1986)

T-Cell and Antibody

Typing of a Mouse Population for Sxr and H-2 Haplotype

ELIZABETH SIMPSON,*,* PHILLIP CHANDLER,* KWHEI BRUCE LOVELAND,* ANDANNEMCLAREN'~

Segregating

TOMONARI,*

*Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow, Middlesex, HAI 3UJ, England; and TMRC Mammalian Development Unit, Worfsn House, 4 Stephenson Way, London NWl, England Received June 18. 1985; accepted October 15. 1985 During investigation of the frequency of recombination of the testis determining gene, Tdy, and the minor histocompatibility antigen gene Hya on the Sxr segment in an outbred mouse stock, we identified two fertile males, one XY and the other XYSxr, which typed H-2k positive using the H-2b anti-H-2k monoclonal antibody HB50, but whose cells failed either to stimulate H-Y specific H-2k restricted T-cell clones, or to be killed by anti-H-2k or anti-H-2’ restricted H-Y specific cytotoxic T cells. We investigated these two mice and their existing relatives, using H-2 and H-Y typing methods. The progeny of their test matings with H-2b homozygous C57BL/ 6 females were also investigated. The results indicate that the transmission of the Hya gene on the Y chromosomes from both mice, and the additional Hya gene on the Sxr segment of the carrier male, allowed for the expression of the H-Y antigen and its detection in the presence of an H-2 haplotype for which we had H-2 restricted H-Y specific typing cells (H-2b and H-2k). Furthermore, we identified the haplotype of the two original males as expressed in the H-2 homozygous and heterozygous F2 progeny as H-2q and discovered an unexpected cross reactivity of the monoclonal anti Kkw antibody HB13 with half the cells of H-2q homozygotes, but not qb heterozygotes. o 1986 AC&&C RCS, IX.

INTRODUCTION

The male specific minor H antigen, H-Y, can only be recognised by T cells when presented to them in the context of appropriate H-2 molecules. T-cell clones capable of recognising H-Y are available for determining the presence of H-Y on spleen cells of mice of the H-2b and H-2k haplotypes (1, 2). Bulk cultures of cytotoxic T cells capable of specifically lysing target cells carrying H-Y + Db or H-Y + Dk can also be readily generated (1) and used to complement the H-Y typing by T-cell clones, which we assessby a proliferative assay (2). For both types of assays, however, it is essential to know the H-2 type of the cells under investigation for the presence of H-Y, since otherwise false H-Y negatives could occur. During studies of the transmission of Sxr, an X-Y linked dominantly expressed factor causing sex reversal in XXSxr mice, we used a stock of outbred mice carrying the T( 16; X) 16H translocation (T 16/H) (3, 4). Inheritance of the translocated chro’ To whom reprint requests should be addressed. 46

oooa-8749186 $3.00 Copyright 0 1986 by Academic Rrs, Inc. All rights of reproduction in any form -cd.

T-CELL

AND ANTIBODY

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47

mosome T 16/H from the mother and XSxr from an Sxr carrier male often results in the development of a phenotypically normal female who is fertile (4) and positive for the male specific minor histocompatibility (H) antigen, H-Y (1, 5). In the course of study of H-Y expression in Sxr-carrying mice, we typed for H-Y a number of animals of various genotypes. We were aware from the time of our first study of the H-Y antigen in TlBH/XSxr mice (1) that whilst most of the mice in the population under study were of the H-2b and/or H-2k haplotype, there existed in this population at least one other H-2 haplotype, the possession of which (in the absence of H-2b or H-2k) made it impossible to type these mice for H-Y. The present studies were made after finding two fertile male mice, which although KkDk positive with a monoclonal antibody derived from a fusion of spleen cells from a C3H.SW mouse immunised with C3H cells (H-2b anti-H-2k) (6), were not positive for H-2k when tested with H-2b anti-H-2’cytotoxic T cells, and nor did their spleen cells stimulate the proliferation of an H-2k restricted H-Y specific T-cell clone. One of these mice, number 75, was an Sxr carrier male with the karyotype XYSxr whilst the other, number 53, was XY. MATERIALS

AND

METHODS

Mice. Stock carrying the T( 16; X) 16H translocation (T 16/H) and Sxr have already been described together with the breeding programme which gives T 16H/XSxr females (4). C57BL/6 mice used for test mating males 53 and 75 were maintained at the MRC Mammalian Development Unit; C57BL/lO, CBA, BALB.B, BALBK, SWR, (CBA X C57BL/ 10)F1, and (BALB/c X C57BL/ 10)FI were from the Clinical Research Centre. C57BL/lO females and (CBA X C57BL/lO)Fi females were immunised in vivo with C57BL/ 10 male and CBA male cells, respectively, as previously described (1). Partial splenectomy of test mice to be H-2 and H-Y typed was performed under ether anaesthesia. Spleens from control mice were removed aseptically following cervical dislocation. All mice were coded by number and the code was broken after computation of the results. Spleen cell suspensions were made in balanced salt solution containing 5% foetal calf serum (FCS). Red blood cells were removed by water lysis, viable cells were counted and resuspended at 8 X 106/ml in complete culture medium: RPM1 1640 with 10% FCS, 10 mM Hepes, 5 X 1Op5M 2-mercaptoethanol, penicillin, streptomycin, and glutamine. Aliquots of these cells were used after 2000 R irradiation as stimulator cells for the T-cell clones (see below). Aliquots were also placed in culture under appropriate conditions (see below) for use as target cells 24- 120 hr later in the T-cell cytotoxicity assays, and for the antibody plus complement lysis assay (see below). T-cell clones. The following were used: lo-2- 12 (specific for H-Y + Db), 2-l- 1 (H-Y + IAb), C3 and A4 (H-Y + Dk), and Clone 4 (IEk). Their derivation, the mapping, and their restriction specificities and use for H-Y and H-2 typing have been described [( 1,2) and Tomonari, submitted]. They were used in proliferation assays, with 1 X lo4 cloned cells per well (96-well flat-bottom microtitre plate) incubated with 8 X lo5 irradiated stimulator cells per well. All assays had foetal calf serum at a final concentration of 15% in the culture medium. Assays using class I restricted clones (10-2-12, C3, and A4) also included lo-25% IL-2 (rat Con A supernatant) in the medium, and were incubated 4 or 5 days before being pulsed with [3H]thymidine, 1 &i/well for 612 hr before harvesting. Assays using class II restricted clones (2-l-1, clone 4) had no

48

SIMPSON

ET

AL.

exogenous IL-2 added: they were incubated 3 days before pulsing with [3H]thymidine for 6-12 hr. Cytotoxic T cells were generated in 5-day bulk culture as previously described (1). Anti-H-2k were primary BALB.B or C57BL/lO anti-CBA, anti-H-2b were primary BALB.K or CBA anti-C57BL/lO, anti-H-2“ were primary (BALB/c X C57BL/lO)Fi anti-SWR, anti-H-Y cultures were derived from spleens of female mice previously immunised in vivo with syngeneic or semisyngeneic male cells: anti-H-Yb were C57BL/lO P anti-C57BL/lO 8, and anti-H-Yk were (CBA X C57BL/10)F1 9 anti-CBA male. After 5 days in culture, cytotoxic attacker cells were harvested, counted, and resuspended at 3 X 106/ml in culture medium before plating in triplicate in 100 ~1 volumes per round-bottomed microtitre well, then serially diluted in tripling dilutions to give four attacker: target cell ratios, 30: 1, 10: 1,3: 1, and 1: 1 when “Cr labeled target cells were added at 1 X 104/well in 100 ~1 volumes. Following addition of the target cells, the plates were spun briefly at approximately 15Og and then incubated for 3 hr at 37°C in a humidified incubator. One hundred microliters per well was then removed for gamma counting and the percentage specific lysis computed according to the formula: % specific lysis = 100 X

(cpm of experimental - cpm of medium control) (cpm of maximum - cpm of medium control) ’

where experimental = wells containing attacker and target cells, medium control = wells containing target cells in medium, maximum = target cells incubated with 5% Triton. In controls with medium only (medium control) less than 30% of the “Cr was released (spontaneous release). The percentage specific lysis was computed for each A:T ratio and from these data regression analysis of each A/T cell combination made. From this analysis the computed percentage specific lysis at A:T 10: 1 was taken. This figure was taken as positive (underlined in the tables) when the data titrated with a regression line having r* > 0.80, and where the positive values were significantly greater than those on negative control targets (e.g., Table 3, experiment 2 which had high “non-specific” kill: this was unusual). Antibodies. Monoclonal anti-H-2 antibody-producing hybridomas were obtained from the American Tissue Culture Collection [for original references see (6,7)]. Some of these antibodies were known to react with shared (public) determinants on particular, different H-2 haplotypes (Table 1). They were grown in flasks and the supematants collected, aliquoted, and stored at -20°C. Rabbit serum was used as a source of complement. That used in the series of experiments reported here came from a single rabbit whose serum had been stored at -70°C in 2-ml aliquots. It was diluted to a final 1:30 for the one-step complement-mediated lysis assay, into which 50 ~1 antibody (neat hybridoma supematant) and 50 ~1 “Cr labeled target cells ( lo4 or 2X 104) were added followed by 100 ~1 rabbit complement. The plates were incubated at 37°C for 1 hr before being centrifuged briefly, then a 100-~1 aliquot of supematant was removed from each well for gamma counting. Percentage lysis was calculated according to the formula: %lysis=

(E-c) 100X(M-c),

where E = cpm released by antibody plus complement, C = cpm released by complement alone, M = maximum released cpm by targets incubated in 5%. Triton. Gen-

T-CELL

AND ANTIBODY

TYPING

49

TABLE 1 Monoclonal Antibodies Used ATCC number

Original designation

HB13 HB19 HB41 HB50

15-3-18 28-11-5s 28-13-38 12-2-2s

Reactivityjcrossreactivity

Reference

K’/D’/r Db/Dd(?Ld)q, p Kb/f KkDk/Kq p r

Note. All the monoclonal antibodies listed were from C3H antiC3H.SW (HB19, HB41) or C3H.SW antiC3H (HB 13, HB50) immunizations.

erally lysis by complement alone was 5-20% of the total releasable counts, depending on whether 24,48,72, or 120 hr targets were used. 120 hr targets (see below) gave the lowest background counts both in the T-cell-mediated cytotoxicity test and in the antibody test. Each antibody was tested in triplicate wells on each target. The positives (+ in Tables 2, 3, and 4) were clear, with the majority of cells being killed (>70% above complement controls). The only exception to this was the result using HB13, which lysed the majority of H-2 target cells but only 50% (+ in Table 4) of H-2q homozygous cells (see Results and Discussion). Target cells. These were set up from the rbc-free spleen cell suspensions of controls and mice to be tested. They were incubated 24, 48, 72, or 120 hr with appropriate concentrations of Con A (4 pg/ml for 24 or 48 hr targets and 1 &ml for 72 or 120 hr targets), foetal calf serum (FCS) (10% for 24,48, and 120 hr targets, 1% for 72 hr targets), IL2 (25% rat spleen Con A supernatant for 120 hr targets only), and with spleen cells at the appropriate concentration: 5 X 106/ml with 2 ml in 5 ml capacity wells in a 12-well cluster plate for 24, 48, or 72 hr targets, 1 X lo6 in 12.5 ml in 50-cm* flasks incubated upright for 120 hr targets. RESULTS Breeding Data

Following discovery of males 53 and 75, we tested several of their existing relatives (father and brother of 53, sons of 75) (see Figs. l-3 and Table 2) for H-2 and H-Y, using two monoclonal antibodies, anti-KkDk, HB50, and anti-Kb, HB4 1, as well as bulk culture cytotoxic T cells (anti-H-2b, anti-H-2’, anti-H-Yb, and anti-H-Yk) and H-2k and H-2b restricted T-cell clones specific for H-Y. These studies indicated that (i) at least some of the male relatives of 53 and 75 were H-Y positive (H-2b or H-2k restricted) and (ii) amongst the sons of 75 which typed positive with the anti-KkDk antibody, some were H-Y positive and some H-Y negative when tested with H-2k restricted T cells. The H-Y negative mice were also negative with b anti-k cytotoxic T cells. We then test mated 53 and 75 with C57BL/6 females (H-2b homozygotes) and typed their Fi progeny for H-Y in association with H-2k and H-2b (Figs. l-3 and Table 2). All the F, males were negative for H-2k-H-Y and positive for H-24H-Y, indicating that the Y chromosome inherited from their respective fathers coded/controlled the expression of the H-Y antigen. We then brother-sister mated some of the

50

SIMPSON ET AL. /

bb

v

See

propositus

(q@

H-yk-b-

1

W

Heyk+b-

fig. 2

I 63.227.228X/X g 82,83T16H/Y iw

81 T16HIY

bb

b(q) H-y k- b+

@ll

)+yk-b-

w

H-yk-b-

+I H-yk+b-

blq)

Heyk-b+

RG. 1. H-2 and H-Y typing of progeny of male 75 and female 35. Each mouse typed has a number and this appears in a square (males) or circle (females) within which is also given information about the sex chromosome constitution, including whether the mice were known Sxr carriers (Sxr) or might have been (fSxr). Under each square and circle appears the H-2 and H-Y typing summary of the mice, deduced from antibody and T-cell data of the type shown in Tables 2-4. The definition of b, k, q, (q), and [q] is given in the results, under “H-2 and H-Y typing data.” H-Yk+sb+ indicates that the mice typed H-Y positive with both H-2k and H-2b restricted T cells (clones and/or cytotoxic cells), H-pmb- that the mice were negative for H-Y with both H-2’ and H-2b restricted T cells. H-p+& or H-pew indicates that they typed positive with H-2k or H-2b H-Y specific cells, respectively, but not both. Horizontal lines between circles and squares indicate mating pans.

F, generation, thus deriving an F2 generation in which the H-2b and the paternally derived H-2 haplotype was segregating (Figs. 1-3 and Table 3). Since Falconer’s outbred Q strain had contributed genetically to the mouse stock we were using, and we knew from a previous study that the H-2b and H-2q haplotypes were present in sublines derived from the Q strain (8) we surmised that H-2q might be our unidentified haplotype. Known H-2q homozygous mice are positive when typed with HB50 (6). The FZ mice were therefore typed not only for H-2’, H-2b, H-Y’, and H-Yb but also with a larger panel of monoclonal antibodies allowing the discrimination between b, k, and q haplotypes (Tables 1 and 4) and with cytotoxic T cells generated in bulk culture against the H-2q haplotype. Results of typing the FZ generation mice indicated that they were segregating for H-2b and H-2q (Table 4): all males which were bb or bq were positive for H-Yb (with cytotoxic T cells and H-2b restricted clones): all qq males were

T-CELL

AND ANTIBODY /

51

TYPING

prcpositus

1

@-&q-@ bb

b(q)

H-yk-b-

w

H-yk-b-

b(q)

H-y”b+

+ See fig. 1

%I H-yktb-

-

b(q)

Heyk-b+

El bb

Heykb+

IO 251 253

163 247

ba

Heyk-b+

ba H-Y

k-b-

qq H-Y

k-b-

w

H-y’+

FIG. 2. H-2 and H-Y typing of progeny of male 75 and a C57BL/6 female. For explanation of symbols, see caption to Fig. 1.

negative for H-Yb and H-Yk, as would be expected (Table 3). They were identified as H-2q by antibody and by cytotoxic T cells (Tables 3 and 4). H-2q mice have H-2 determinants recognised by antibody in common with H-2k, and other determinants in common with H-2b, but the pattern with the reagents used allows us to distinguish b, k, and q homozygotes and all F1 combinations. Moreover T-cell recognition of each haplotype is so’precise that the presence or absence of a particular H-2 allele can readily be determined. H-2 and H-Y Typing Data

Figures l-3 show the family trees of mice 53 and 75 together with the H-2 and H-Y typing inferred from data including that shown in Tables 2 and 3. Where k and b are written, these haplotypes have been identified by cytotoxic T cells and/or a Tcell clone whose proliferation is specific for H-Yk or H-Yb. The designation (q) in the figures and Table 2 is given when spleen cells have been killed by anti-KkDk monoclonal HB50, in the absence of killing by anti-Kb (HB41), by the failure of anti-H-2k or antiH-Yk restricted cytotoxic effector cells to lyse these targets and by the failure of spleen cells to stimulate H-2k restricted T-cell clones (Table 2, experiments 1, 2, and 3A). The presence of H-2q can only be inferred ([q] in Figs. 1 and 2) in mice that also have

52

SIMPSON

propositus 2

\

ET AL.

1

53 XY w

71 XY lq)b

I

r

149 159

158 If4 176 216

bb H-yk- b+ FIG.

Ed 173 114

XY

bb H-Y’- b-

lb0 167 114 217

XY

b9 H-Y’- b+

I

bq ,,.yk-b-

w Hmyk-b-

XY

w H-yk-b-

3. H-2 and H-Y typing of relatives of male 53. For explanation of symbols, see caption to Fig. 1.

the H-2k haplotype: this inference is made for mouse 35 and the k[q] progeny of 35 and 75 (Fig. 1). The positive H-Y typing of mouse 35 is already published (3). The ?bq haplotype of the mother of 53 and 77 is inferred from the typing of 76, 53, and 77, since she was not available for testing at the time of the discovery of 53. The designation q is given to mice in the Fz generation (Figs. 2 and 3 and Tables 3 and 4) where a more extensive investigation of this non-b haplotype was made using a panel of monoclonal antibodies (Table 4), as well as (BALB/c X C57BL/10)F1 anti-SWR (anti-H-2q) cytotoxic T cells (Table 3, experiments 1 and 2). It is the results of this analysis of the F2 generation that convinces us that the previously unidentified hap lotype is H-2q. From the uniform typing of all F2 mice designated qq from the families of both 53 and 75 it is clear that 53 and 75 both contributed the same H-2 haplotype and that they themselves must have been homozygous for it. The pattern of monoclonal antibody reactivities on the control and experimental mice (Table 4) show that (i) HI341 (anti Kb) distinguishes H-2b (positive) from both H-2’ and H-2q (negative), (ii) HB50 (anti-KkDk) distinguishes H-2q and H-2k (both positive) from H-2b (negative), (iii) HB19 (anti-Db) distinguishes H-2k (negative) from H-2q and H-2b (positive) but

53 55

8

T 16H/Y?Sxr T16H/Y?Sxr T 16HJY?Sxr XY sxr XY

81 a2 83 84 a5

86

88

8 8 8

9

6 8 8 8

8 8

9

8 6

92 93 95

89 90 91

II

xx XYsxr xY?sxr

xx XY XY XY XY

+

XY XY XY

6 8 8

58

15 76

+ + +

+ + +

-

f

XY

Genotype

HB50 KsDk

HB41 Kb

XX unrelated XY 1 controls

9 8

No.

Sex

ceus

Reactivity with MAb

0

16

-14

-3 -1

5

8

7 0 4

TF TF

TF

TF TF TF TF TF

TF TF TF TF TF

12 9 15 24 22 0

7

I

0 19

0

uH-Zb

0

0

-2 5 6

-8 -3

-1

0

30 26 38

6

6 49 7

0 0 29

aH-Yk

cytotoxicity

46

20

11 16

aH-2k

~

80

3 2

23

0 46

303 10

8

5 15 21 21 26

-3

-3

15 1960 10

540

45

81

7 275

4 9 35

22

0

0

aH-Yb

cpm X 10-r

79

7 68

9 82 81 93 19

13 11 11 13 11

ND ND ND

141 52 1295

1

4 6

5

5 6

5 115 285

232 167

90

118

446

32 44

38

16 9

Clone (antigen specificity) C3/A4 10-2-12 2-l-l (@ + H-Y) (Db + H-Y) (IAb + H-Y)

Proliferation

(q)b (q)b (0

(0 &b W (q)b (0

ml) k(q) k(q) k(q) q(q)

(99) k(q) (q)b

kb

(99) (qk or kk

H-2

Summary

H-y’-‘H.Yk-‘+ H-Y-*

H-Y-H-Y*-” H-y-’ H-Y’” H-y-+

H-y’-” H-y k+‘H-Y.“H-yk+‘H-Y’-”

H-Yk-” H-yk*‘H-Y-*

H-Yk”*

H-yk-” H-yk--

H-Y

of typing

’ T-Cell cytotoxicity is the percentage specific lysis at an attackerztarget (A:T) ratio of IO: 1 taken from the regression curves of a titration (see Materials and Methods). Boldface figures indicate positives as defined under Materials and Methods. TF: Technical failure. Attacker cells failed to lyse H-2b male targets. b Proliferation shown is cpm X 10-s. Figures in boldface indicate positive stimulation, significantly greater than control cpm, either of cloned cells in the presence of medium alone (data not shown) or in the presence of female cells and/or male cells of an inappropriate H-2 haplotype (data shown). See also Materials and Methods. ’ Reactivity with antibody. + = ~70% cells killed, - < 10% cells killed. + = -30-501 cells killed in the presence of MAb plus rabbit complement (see Materials and Methods).

(3C)

(3B)

(3N

(2)

(1)

Expt

source of

stimulating

Results of H-2 and H-Y Typing of Propositi 53 and 75 and Their First Degree Relatives and Controls“*

TABLE 2

8 9 6 9 9 9 9 9 8 6

8 9 8 9 9 6 6 8 9 9

1

2

247 248 249 253 254

C57BL/IO C57BL/lO CBA CBA SWR 209 210 211 216 217

No.

I

3 5 z

cc See footnotes to Table 2.

Sex

Expt

source of stimulating cells

XY + Sxr XXSxr xx xx

XY xx XY xx xx XY f Sxr

XY xx XY xx xx xx xx xx XY XY

Genotype

+

+

+

+ -

+ + + + + +

+

-

-

-

+

+ + + -

+ +

+

+

+ +

+ + -

Typing

10

34

19 41 1

46

27

53 26 13 17 14 5 2

55

49 43 3

1

2 1 1 8 8 2 13 1

15

17

CCH-2b

of Mice

0 4

-8 -2 -1

8 2 0

0 11

5

aH-2’

and H-Y

HB50 KQk

Reactivity with MAb

of H-2

HB41 Kb

Results

3

11 a 49 24 19 29 24 16 -8 -1

2 0 16 -1 1 0 -7 1 1 5

aH-Y’

Cytotoxicity

62 27 28 24 27 56 41 50 30 18

1

18

17 1 2 3 3 0 -4 0

aH-Yb

of F2 Generation

TABLE

9 a 13 12 70 9 47 58 41 48

7 3 7 7 50 28 -9 35 2 48

aH-2q

and H-2b,

ND ND ND ND ND ND ND ND ND ND

2 ND 6 2 2 2 3

134

1 2

ControlsH (cpm x lo-*)

42 38

811

26 28 70 27 894 a

9%

a 11 la ND 10 19 6 903 15

914

3 56 1 1 1 2 58 4 165 2 2

134

58 2 I 2 ND 3 4 4

Clone (antigen spccilicity) 10-2-12 2-l-l (Db + H-Y) (IAb + H-Y)

ProIifmtion

and H-2q

C3JA4 (D’ + H-Y)

H-2k,

ND ND ND ND ND ND ND ND ND ND

ND 2 2 3 a 2

1 1 513

No. 4 IEk

bb bb kk kk 99 bb 99 bs w-l h

E WI

bb bb kk kk 99 bs bb

H-2

of

H-y’” H-y-‘H-y’+‘H-YeH-y’-H-yk-‘+ H-Y nH-yk-+ H-yH-y-‘-

H-y-+ H-Y k-bH-y”‘H-Y tiH-yH-Y tiH-ycbH-ye”H-Y-+ H-y--

H-Y

Summary typing

T-CELL

AND ANTIBODY

55

TYPING

TABLE 4 Antibody Plus Complement Typing of F2 Generation of Mice and H-2 9 H-2 ‘, H-2q Homozygous Controls Antibody (specificity) Mouse C57BL/6 CBA SWR 149 159 160 162 167 169 172 173 174 209 210 211 212 216 217 241 248 249 250 251 252 253 254

Sex

Grandfather

HB41(Kb)

+ 8 6 8 6 8 9 9 6 6 9 9 9 9 6 6 8 d 8 8 9 9 9 9

53 53 53 75 53 53 53 53 53 53 53 53 53 53 53 75 75 75 75 75 75 15 75

+ + + + + + + + + + +

HBSO(K’Q’)

+ + + + + + + + + + + + + + +

HB13(KkDk)

+ f* + + + f + + + 2

-

HB19(Db)

H-2 type

+ + + + + + + + + + + + + + + + + + + + + + + + +

Note. Results shown are from three separate experiments: some mice were typed on two occasions. *Variable reaction on five individuals. (FsSee footnotes to Table 2.

cannot separate H-29 and H-2b, (iv) HB 13 (anti-KkDk) reacts in an interesting manner: only with H-2k mice are more than 70% of the cells killed with this antibody. This is true for both H-2k homozygotes and heterozygotes (data on heterozygotes not shown). HB 13 also reacts with about half the number of H-2q homozygous cells that are killed with HB 14 and HB 19. The specificity of this “50% reactivity” has not yet been explored, but it might represent cross reaction with a Qa/Tla molecule consistent with the original typing of a weak reaction on a Dq - recombinant strain (6). It is not invariably found, even in inbred SWR mice: of five separate experiments performed, the SWR control gave 50% reactivity in three but failed to do so in two (data not shown). The experimental qq mice in Table 4 also show some variability with respect to this reaction: of 12 tested, 9 gave it and 3 did not. Exceptionally, 1 bq (out of 8 tested) heterozygote gave 50% reactivity: this could represent an intra-H-2/Qa/Tla recombinant.

SIMPSON ET AL.

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DISCUSSION From the H-Y typing results of the F2 generation (Table 3, experiments 1 and 2) it is clear that the negative H-Y findings with mice 53 and 75 using H-2k restricted T cells specific for H-Y are explained by these mice being H-2q homozygotes. The absence of the H-2k haplotype in the F2 mice and by implication in 53 and 75 is shown not only by the monoclonal antibody analysis (Table 4) but by their failure to stimulate clone No. 4, which reacts specifically with the Ek molecule (Table 3 and Tomonari, submitted). The cross reactivity of anti-H-2 monoclonal antibodies against public specificities makes it necessary to H-2 type in addition with alloreactive and H-2 restricted T cells. For precise identification, cloned T cells should ideally be used, since uncloned bulk culture stimulated populations inevitably contain some clones which cross react with other H-2 or H-2 + X specificities and in some experiments this can give a high “background” making interpretation difficult. The T-cell cytotoxicity results of experiment 2, Table 3, show this type of high cross reactivity. In this experiment, interpretation is facilitated by the clone data (Table 3, experiment 2) and the antibody analysis of the same mice (Table 4). The nature and function of minor H antigens such as H-Y remain unresolved. The interaction of cell surface molecules such as MHC, minor H, and receptor molecules may be essential not only for immunological but also for other physiological functions (9, 10). Identification of each of these components is an important step in assigning their function. The specificity of T-cell recognition already implies association between a variety of cell surface molecules (9, 10). This paper explores the relationship between H-Y and H-2 and pinpoints potential hazards in identifying these molecules, particularly in a non-inbred population. It underlines the capacity of H-2 restricted T-cell clones in particular to discriminate H-2 alleles, and the use of this capacity to type for a minor H antigen in an outbred population. Should typing for minor antigens be undertaken in an outbred species such as man, similar attention to fine specificity of HLA molecules will be necessary: this approach has already shown that A-2, A-3, and B27 variants exist in man, distinguishable by T cells making HLA restricted responses to viruses and minor H antigens (11-13). REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Simpson, E., McLaren, A., Chandler, P., and Tomonari, K., Transplantation 37, 17, 1984. Tomonari, K., J. Immunol. 131, 1641, 1983. Lyon, M. F., Searle, A. G., Ford, C. E., and Ohno, S., Cytogenetics 3, 306, 1964. McLaren, A., and Monk, M., Nature (London) 300,446, 1982. McLaren, A., Simpson, E., Tomonari, K., Chandler, P., and Hogg, H., Nature (London) 312,552, 1984. Ozato, K., Mayer, N., and Sachs, D. H., J. Immurwl. 124,533, 1980. Ozato, K., and Sachs, D. H., J. Immunol. 126, 317, 1981. Simpson, E., Bultield, G., Brenan, M., Fitzpatrick, W., Hetherington, C., and Blann, A., Immunogenetics 15,63,

1982.

9. Simonsen, M., and Olsson, L., Ann. Immunol. MD(l), 85, 1983. 10. Simpson, E., Ann. Immunol. (Inst. Pasteur) 135C, 410, 1984. 11. Biddison, W. E., Krangel, M. S., Strominger, J. L., Ward, D. E., Shearer, G. M., and Shaw, S., Hum. Immunol. 3, 225, 1980. 12. Biddison, W. E., Shearer, G. M., and Shaw, S., J. Immunol. 127, 2231, 1981. 13. Breuning, M. H., Lucas, C. J., Breur, B. S., Engelsma, M. Y., deLange, G. G., Dekker, A. J., Biddison, W. E., and Ivani, P., Hum. Immunol. 5, 259, 1982.