Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene...

Immunogenegcs 33: 54-61, 1991 111111111110- genetics

© Springer-Verlag 1991

Variable spread of X inactivation affecting the expression of different epitopes of the Hya gene product in mouse B-cell clones

Diane Scott ~', Anne McLaren 2, Julian Dyson 1, and Elizabeth Simpson 1

1 Transplantation Biology, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, England 2 MRC Mammalian Development Unit, University College London, 4 Stephenson Way, London NW1 2HE, England

Received August 17, 1990; revised version received September 17, 1990

Abstract. Cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed due to X inactivation and from a male X/XSxr mouse, both of the (kxb)F 1 haplotype, were examined for H-Y expression. This was determined both by their ability to act as targets for H-2 k and H-2b-restricted H-Y-specific cytotoxic T cells and by their ability to stimulate the proliferation of H-2K k, H-2D b (class I) and A b (class II)-restricted T-cell clones. In B-cell clones from the T16H/XSxr mouse, expression of H-Y/D b exhibited partial X inactivation and only a proportion ( = 30%) of the cells were targets for or stimulated H-2Db-restricted H-Y-specific T cells. In contrast, H-Y epitopes restricted by H-2 k (H-Y/K k, H- Y/D k) and A b (H-Y/A b) exhibited no X inactivation. Fur- thermore, no inactivation of H-Y/D b, H-Y/A b, or H-Y k was observed in the male X/XSxr mouse. These results indicate that the T16H/XSxr female is a mosaic, as a result of the variable spread of X inactivation into the Sxr region. They further suggest that the H-Y antigen recognized in association with H-2 k and H-2D b class I molecules and A b class II molecules may be the product of more than one gene.

Introduction

Sex-reversed (Sxr) X/X and X/O male mice were originally described by Cattanach and co-workers (1971). They were subsequently shown to have been derived from car- rier males with a duplication of the region of the Y chromosome which includes the testis-determining gene (Tdy). This region is normally located on the short arm of the Y chromosome (McLaren et al. 1988) and the duplication becomes attached to the pseudoautosomal region at the telomeric end of the Y chromosome

Offprint requests to: E. Simpson.

(McLaren and Monk 1982; Singh and Jones 1982; Cat- tanach et al. 1982). During male meiosis this duplicated region can be transferred to the distal end of the X chromosome and individuals inheriting XSxr develop as phenotypic, although sterile, X/XSxr males. These mice express H-Y, the male-specific transplantation antigen (Simpson et al. 1981), indicating that the Hya gene controlling H-Y expression is also located in the Sxr translocation.

In normal X/X individuals, one of the two X chromosomes is inactivated resulting in dosage compen- sation of X-linked genes (Lyon 1961). When X/YSxr car- rier males are mated with females carrying Searle's translocation T (X; 16)16H (T16H; Lyon et al. 1964), the translocated X chromosome is preferentially expressed in the adult and therefore the paternally derived XSxr chromosome is the inactive one in all cells. However, the expression of Sxr genes including Tdy in these T 16H/XSxr individuals is incompletely suppressed, presumably because X inactivation may not spread completely into the Sxr region. Thus, mice of the genotype T16H/XSxr may develop as females, hermaphrodites, or males (Cattanach et al. 1982; McLaren and Monk 1982). Both female and male T16H/XSxr mice were found, with a single exception, to express the H-Y antigen (Simpson et al. 1984). Thus Hya expression can occur when Tdy expression is suppressed by X inactivation.

Since in T16H/XSxr mice Tdy and Hya were found to be under differential control by X inactivation, we have examined H-Y expression in individual cloned cell lines obtained by viral transformation of immature B cells from the bone marrow of both T16H/XSxr and X/XSxr mice. We have found that in cloned B-cell lines from a female T16H/XSxr mouse in which Tdy expression was suppressed by X inactivation, the expression of the H-Y epitope (determinant) that is presented and restricted by the major histocompatibility complex (MHC) class I molecule H-2D b exhibited partial, and possibly reversible, X inactivation. The H-Y epitope presented and restricted by the

D. Scott et al.: Hya expression in mouse B-cell clones 55

H-2 k class I molecule exhibited no X inactivation. In addition, in X/XSxr (male) mice we could find no evidence of X inactivation of H-Y expression at the clonal level. The implications of these findings with regard to models of Hya gene expression are discussed.

Materials and methods

Mice. CBA/Ca, C57BL/10/ScSn(B10), B10.A(2R), SWR/CRC, (CBA x B 10)F1, and (B 10 × SWR)Ft mice were bred at the Clinical Research Centre (Harrow, UK). X/XSxr and T16H/XSxr mice were bred at the Mammalian Development Unit (London, UK). All mice carrying Sxr in these studies were H-2 k:'b heterozygotes.

Media. Phosphate-buffered balanced salt solution (BSS) containing penicillin, streptomycin, and 2% fetal calf serum (FCS) was used for the preparation of spleen and bone marrow cell suspensions. RPMI- 1640 containing 5 x l 0 -5 M 2-mercaptoethanol, 10 mM hydroxyethyl- piperazine-ethanesulfonic acid (HEPES), glutamine, penicillin, and streptomycin was used for cell culture. This RPMI-1640 medium was further supplemented with 5%, 10%, or 15% FCS as indicated. Rat spleen cell concanavalin A (Con A) supernatant was used as a source of IL2 for proliferative assays. Cytotoxic T-cell assays were performed in Dulbecco's minimal essential medium (DMEM) supplemented with 10 mM HEPES and 5% FCS.

Cytotoxic T-cell assays. H-2 k- and H-2b-restricted H-Y-specific cytotoxic T cells were produced as described previously (Simpson and Gordon 1977). H-2q-restricted H-Y-specific cells were generated by immunizing (B10 x SWR)F 1 females with SWR male spleen cells. An- ti-H-2 b, anti-H-2 k, and anti-H2 q cytotoxic ceils were produced in primary mixed lymphocyte culture (MLC) by 5 days' culture of spleen cells from unimmunized CBA (H-2 k) with irradiated B10 (H-2 b) spleen cells, from B10 with irradiated CBA spleen cells, and from (CBAxB10)F 1 with irradiated SWR (H-2 q) spleen cells. Anti-D b cytotoxic cells were produced by incubating spleen cells from CBA mice immunized with B10.A(2R) [KkAkEkD b] with irradiated B10.A(2R) cells. At the end of the 5-day culture, the anti-H-Y and anti-H-2 cytotoxic cells were harvested, counted, and resuspended to 2-3 x 106 cells/ml. Duplicate 150 gl samples were distributed into 96-well round-bottomed microtiter plates and three serial threefold dilutions were made. Target cells (1 X 106), 2- or 5-day spleen cell Con A blasts (McLaren et al. 1984), or Abelson leukaemia virus-transformed cloned B-cell lines were washed and labeled by incubation for 90 rain at 37 °C in 100 gl BSS/FCS containing 50 gCi 51sodittrn chromate. The cells were washed three times in BSS/FCS and resuspended at 1 x 105 cells/ml in DMEM/FCS. 100 ~1 samples were added to the wells containing the dilutions of cytotoxic T cells. Controls of target cells incubated with 100 ~tl medium alone (C) or 100 gl Triton X-100 (maximum, M) were included. After 3 h incubation at 37 °C the plates were centrifuged and 100 ~i supernatant was harvested and counted. Percent specific lysis at each effector: target (E:T) ratio was computed from (Exp-C/M-C) x 100, where Exp as cpm of supernatant from target cells incubated with effector cells, and C and M are cpm from supernatant of target cells incubated with medium alone and with Triton X-100, respectively. The percent lysis at E:T 10:1 was computed from regression analysis of the percent lysis at each E:T ratio. Positive values are defined as those obtained from regression curves with a correlation coefficient of r2>0.80 and are underlined in the tables.

Proliferation assays. H-Y-specific T-cell clones were obtained by limiting dilution from secondary MLC cells from C57BL/10, B6.C-H-16 ~, and CBA mice previously immunized with syngeneic male cells and were the kind gift of K. Tomonari (Clinical Research

Centre). Clone M1 is Db-restricted, B9 is Ab-restricted, and C6 is K k- restricted (Simpson and Tomonari 1989). Both clones M1 and C6 res- pond to irradiated male spleen cells that express the appropriate MHC restriction element, in the presence of IL2. Clone B9 responds to male spleen cells expressing the A b molecule and, to a lesser extent, to male cells expressing an inappropriate MHC class II molecule provided that irradiated female spleen cells bearing the appropriate restriction element are present to act as antigen-presenting cells. This phenomenon was originally reported by Matzinger (1988). This clone does not require exogenous IL2. Proliferation assays were set up using 1 x 104 indicator T-clone cells/well in a final volume of 200 [xl RPMI-1640 medium containing 15% FCS and IL2 (for clones M1 and C6) or 1 x 105 irradiated (3500 rad) H-2 b female splenic lymphocytes (for clone Bg). Triplicate samples of stimulator cells, either irradiated (3500 rad) splenic lymphocytes (8 x 10S/welI in flat-bottomed microtiter plates) or irradiated (7000 rad) cloned Abelson-transformed B-cell lines (1 x 10S/well in round-bottomed microtiter plates), were added. The cells were incubated at 37 °C for 72 h (clone B9) or 96 h (clones M1 and C6). 3Hthymidine (1 gCi) was added for the last 6-8 h incubation. The wells were harvested and counted using a beta plate counter.

Production of Abelson leukaemia virus. Abelson leukaemia virus was obtained from the "producer" cell line 54C1 z (kindly donated by V. Henson, The Jackson Laboratory, Bar Harbor, Maine). The 54C12 cells were grown in RPMI-1640 with 10% FCS to a concentration of approximately 106/ml. Cells and supernatant were pooled and stored at - 7 0 °C. After thawing, the cell debris was removed by centrifugation (3000 g, 30 rain) and the virus containing supernatant was filtered (0.45 ~t), aliquoted, and stored at - 7 0 °C.

Transformation o f mouse bone marrow B cells with Abelson leukaemia virus. Bone marrow cells were harvested under sterile conditions from the femurs of mice of approximately 12 weeks of age. The cells were filtered through nylon gauze, washed twice in BSS/FCS, and resuspended to 2-5 x 105/ml in RPMI-1640 containing 8 ~tg/ml polybrene (Aldrich Chemical Company, Gillingham, Kent, UK) and 5 % FCS. Bone marrow cells (4 ml) were aliquoted into 50 mm petri dishes and 1 rnl virus-containing supernatant was added. After incubation at 37 °C for 90 rain, 1 ml FCS was added and the cells were incubated overnight. RPMI-1640 (2 ml) containing 20 % FCS was then added. The cells were re-fed as necessary after 6-7 days. When growth was established the cells were transferred to 25 ml flasks and cultured in 10 ml RPMI-20% FCS in an upright position.

Cloning o f Abelson-transformed B cells. Abelson leukaemia virus- transformed B cells were distributed at 1, 5, and 10 cells/well in round- bottomed microtiter plates containing 1 x 104 irradiated (7000 rad) autologous Abelson-transformed B cells as feeder cells, in a final volume of 200 ~tl RPMI-1640 with 20% FCS. Approximately 2 weeks later, wells were screened for growth and positive wells were transferred to 24-well plates in 0.5 ml RPMI-1640 with 20% FCS. Usually between 5 and 15 wells per 96-well microtiter plate gave positive growth even when seeded at 5 and 10 cells/well, reflecting the low cloning efficiency of these cells and indicating that the positive wells obtained were likely to have been derived from a single cell.

Fluorescence-activated cell sorter (FACS) analysis. Cells to be assessed for expression of D b and K k molecules were incubated with monoclonal antibodies HB19 (ATCC, Db-specific) or HB25 (ATCC, Kk-specific), then washed and incubated with anti-mouse immunoglobulin (Ig) coupl- ed to fluorescein, iso-thiocyanate (FITC) followed by washing and analysis on a FACStarPLUS (Becton Dickinson, Mountain View, California). Ten thousand cells of eacb sample were assessed.

Southern analysis. DNA was prepared from B-cell lines as described by Wyke and Quade (1980); DNA from an X/XSxr liver was prepared

56 D. Scott et al.: Hya expression in mouse B-cell clones

as described by Maniatis and co-workers (1982). DNA (8 gg) was digested with the restriction enzyme Eco RI (New England Biolabs, Beverley, Massachusetts), electrophoresed through a 0.8 % agarose gel, and transferred to a nylon membrane (Southern 1975). The membrane was hybridized with a kilobase 2.3 (kb) subfragment ofpDP1122 (Mar- don and Page 1989) labeled by random oligonucleotide priming (Feinberg and Vogelstein 1984). Hybridization and high-stringency washing was performed as described by Maniatis and co-workers (1982). The membrane was then air-dried and exposed to X-ray film at - 7 0 °C for 4 days.

Results

Cytotoxicity. The results of cytotoxicity assays examining the expression of H-Y by a series of cloned B-cell lines derived from T16H/XSxr female and X/XSxr male mice, both with an (H-2kxH-2b)F1 H-2 haplotype, are summarized in Table 1. All cell lines tested expressed the H-Y epitope restricted by the H-2K k and H-2D k class I molecules (H-yk). In contrast, while all cell lines (6

Table 1. Lysis of Abelson-transformed clones and control spleen cells by H-Y- and H-2-specific cytotoxic effector T cells.

Targets Effectors from mice

Anti-H-y k Anti-H-y b Anti-H-2 k Anti-H-2D b Anti-H-2 q (B10 x CBA)F 1 9 c~CBA o" B10 ~ e~B10cy B10 anti-CBA CBA anti-2R (CBA x B10)~SWR

T16H/XSxr clone (kxb) 9 5H2 39* 70 40 98 0 5E12 32 59 31 76 4 5G8 32 71 30 86 - 3 5A8 27 51 33 78 1 1F10 28 49 26 80 - 3 5H7 33 35 33 78 6 5B3 50 22 46 100 - 1 5C3 39 8 34 75 - 4 1A6 32 10 41 79 - 4 1D5 ~ 10 3--~ 7~ 0 5F1 42 13 45 81 2 5C2 31 13 30 76 2 5D4 41 7 45 99 - 2 1D4 34 11 32 76 2 1F3 36 9 36 67 6 1C8 49 9 49 91 - 4

X/XSxr clone (kxb) cy 1F8 35 69 29 72 3 1Fll 37 65 34 88 5 1F12 31 55 31 80 2 1F5 41 74 39 106 2 1A3 34 61 31 75 8 1D1 41 89 47 102 18

x/x 9 (k~b) 1 10 21 48 1

Control spleen cells H-2bmale - 4 17 - 8 69 - 5 H-2bfemale 1 --8 0 5~ 2 H-2kmale 14 3 20 - ~ - 4 H-2kfemale - 7 2 1-8 - 3 2 H-2qmale - 5 - 2 ~ 28 t 20 H-2qfemale 0 1 4 45' 34 B10.A(2R) male (H-2K~D b) 3 * 54 16 65 2

* Percent lysis at E:T of 10:1. Underlined figures represent positive values obtained from regression curves with a correlation coefficient of r 2 > 0.80. See Materials and methods. Cross-reactive lysis on H-2 q targets by CBA anti-B10.A(2R) CTLs.

* Failure to lyse B10.A(2R) male targets by H-2k-restricted H-Y-specific cytotoxic effectors is due to the predominant use of D k as a restriction molecule. Lysis of these targets by H-2b-restricted H-Y CTLs demonstrates the use of D b as a restriction molecule by these effectors.

D. Scott et al. : Hya expression in mouse B-cell clones 57

shown of a total of 12 tested) from the X/XSxr male mice expressed the H-Y molecule restricted by H-2D b (H-yb), only a proportion of the cell lines from the female T16H/XSxr mouse expressed this epitope. Of the 24 cell lines tested (16 of which are shown in Table 1), 5 (clones 5H2-1F10) consistently expressed H-Y b, 17 (represent- ed in Table 1 by clones 5C3-1C8) did not express H-Y b, while 2, clones 5H7 and 5B3, were initially negative (three experiments) but in subsequent experiments, as shown in Table 1, appeared to have acquired H-Y b expression. Controls showed that all cell lines expressed MHC class I molecules of the H-2 k and H-2 b haplotypes (Table 1, columns 1 and 2) including H-2D b, the class I molecule that restricts H-Y recognition in H-2 t' mice (Simpson and Gordon 1977). Further controls including anti H-Y q (data not shown) and anti H-2 q (Table 1, column 3) and the targets of a B-cell line derived by Abelson virus transformation of bone marrow from a normal H-2 ~b female mouse demonstrated the specificity of the cytotoxicity observed in this experiment. The only nonspecific cytotoxicity observed was the ability of the anti-H-2D b effector cells to kill target cells expressing H-2 q. This was presumably because of both the high level of lysis produced by these effectors, which were from CBA mice immunized in vivo with B 10.A(2R) cells before in vitro restimulation, and the expression of an epitope on the H-2 q class I molecule that is cross reactive with D b (Simpson et al. 1978). Primary anti-H-2 b cytotoxic effector cells (from primary cultures of CBA spleen cells incubated with irradiated C57BL/10 spleen cells) did not kill target cells expressing H-2 q (data not shown).

Proliferation. The ability of these transformed B-cell lines to stimulate proliferation of T-cell clones specific for H- Y/D b (clone M1), H-Y/A b (clone B9), and H-Y/K k (clone C6) are summarized in Table 2. H- y/Db-restricted proliferation by these cell lines gave essentially similar results to the cytotoxicity data. Six of the clones derived from the T16H/XSxr mouse that were killed by H-yb-restricted cytotoxic cells also stimulated M1, and these included clone 5H7 that was originally negative in cytotoxic assays. Clone 5B3 that was also initially negative in cytotoxicity assays has to date remained unable to stimulate M1. Clones 5D4 and 1F3 appeared to stimulate M1 in several experiments, however the high background proliferation of these clones (see "no clone control" column) makes the significance of this observation questionable. The other clones that were insensitive to anti-H-Y b killing also failed to stimulate M1. Again, in contrast, all the clones derived from the X/XSxr mouse were able to stimulate M1. Abelson-transformed B cells from the normal X/X female mouse did not stimulate M1.

FACS analysis of the cloned B-cell lines indicated that they did not express significant levels of class II molecules

(results not shown). In order, therefore, to stimulate the Ab-restricted clone B9, irradiated female cells were included in the assay to act as antigen-presenting cells (Mat- zinger 1988). The results show that, in addition to the six clones (5H2-5H7) that stimulated M1, clones 1D5, 5F1, 5C2, and 1C8 also strongly stimulated the proliferation of B9. When compared with the stimulation produced by control normal X/X female B cells or male cells expressing an inappropriate class II molecule, all the clones (with the exception of clone IF3 that had a high background and 5D4), both from T16H/XSxr and X/XSxr, caused significant proliferation of B9. This proliferation was less than that seen with H-2 b male splenic lymphocytes and pro- bably reflects the inefficiency of processing of H-Y by the antigen-presenting H-2 b female spleen cells.

The ability of the cloned B-cell lines to stimulate the H-2Kk-restricted T-cell clone C6 was low compared with the control of H-2 k male spleen cells. This might reflect sub-optimal assay conditions and perhaps the inability of the B-cell lines to produce sufficient accessory molecules that might be required, in addition to the exogenous IL2 that was present in the assay, for optimal proliferation of C6. However, the stimulation produced by the T16H/XSxr clones (with the exception of 5C2 and 1F3) and X/XSxr B-cell clones was again significantly (two- to tenfold) higher than that produced by the Abelson- transformed B cells from a normal X/X female.

FACS analysis. The inability of the T16H/XSxr clones to be recognised by the H-yb-restricted cytotoxic T-cell lines and clones might be due to a low level of surface expression of H-2D b, the relevant restriction molecule, by these cells. The results of FACS analysis of the T16H/XSxr and X/XSxr B-cell lines for expression of H-2K k and H-2D b class I molecules are summarized in Table 3. For each antibody the results are shown as a percentage of cells in the population expressing that molecule and the median distribution of the peak of fluorescence which gives a measure of fluorescence intensity. The results indicate that for both H-2K k and H-2D b there is some variation in the levels of surface expression between the cell lines. However, this variation does not correlate with nor is it sufficiently great to explain the inability of some of the T16H/XSxr-derived lines to be recognized by T cells as expressing H-Y b.

Southern analysis. To investigate the possibility that the loss of the H-Y b antigen by some T16H/XSxr B-cell clones was due to deletion or rearrangement of part of the Sxr region, a Southern analysis was performed using pDPl122, a cDNA clone derived from Zfy-2 which hybridizes to both Zfy-1 and Zfy-2 and weakly to Zfx. Zfy-1 and Zfy-2 are the mouse homologues of human ZFY, a zinc finger protein encoded in the sex-determining region of the Y chromosome. They are located in the Sxr

58 D. Scott et al. : Hya expression in mouse B-cell clones

Table 2. Stimulation of H-Y-specific T-cell clones by Abelson-transformed clones and control spleen cells.

Stimulator cells Responder clones

H-YD b (M1) H-Y-A b (B9) H-Y-K k (C6) No clone control

T16H/XSxr clones (kxb)

5H2 13419* (307) t 10525 (310) 2482 (101) 153

5E12 14549 (155) 10 125 (1310) 2 872 (289) 47

5G8 17213 (132) 9485 (1308) 2419 (235) 149

5A8 20 701 (1313) 5 050 (67) 9 947 (98) 168

1F10 17996 (2 151) 14043 (976) 2046 (229) 35

5H7 14 051 (429) 8 729 (649) 2 747 (480) 35

5B3 2 958 (282) 2 015 (529) 2 053 (171) 30

5C3 4709 (424) 4214 (2 141) 3524 (128) 492

1A6 1 961 (404) 4 098 (849) 3 693 (205) 23

1D5 2719 (647) 9626 (2262) 1708 (587) 80

5F1 4632 (87) 13489 (1787) 1758 (20) 31

5C2 2 402 (202) 8 784 (575) 1046 (91) 35

5D4 7534 (132) 918 (110) 2902 (162) 428

108 2654 (430) 4818 (2866) 2316 (253) 28

1F3 (8 701) (167) (5 067) (1 120) (3 526) (85) 3 533 * 1C8 3 103 (542) 11291 (362) 4 865 (1329) 39

X/XSxr clones (kxb)

1F8 8 923 (883) 1258 (66) 1774 (222) 34

1F11 8 502 (117) 1 269 (476) 1781 (28) 31

1F12 25 299 (244) 6 000 (791) 9 474 (241) 1577 *

1F5 11994 (165) 2 994 (628) 1443 (105) 43

1A3 11 357 (1 216) 2 856 (302) 1677 (129) 35

1D1 7 896 (604) 4 447 (36) 1541 (166) 29

X/X female (kxb) 4 392 (625) 94 (22) 898 (7) 37

Control spleen cells

H-2 b male 17634 (2116) 37610 (2632) 921 (120) ND

H-2 b female 1 866 (261) 164 (27) 2 076 (373) ND

H-2 k male 3 591 (359) 1 018 (102) 20 807 (2705) ND

H-2 k female 3 511 (491) 470 (188) 3 018 (362) ND

Medium 4 615 (291) 39 (4) 381 (84) ND

* Underlined figures represent significant stimulation. t Figures in brackets indicate variance. * Represents radio-resistant growth of the Abelson B-ceil clone: this figure has to be taken into account in the levels of proliferation shown using

these B-cell clones to stimulate the H-Y-specific T cell clones. Brackets indicate these. ND, Not done.

region (Page et al. 1987; Mardon and Page 1989). Zfx, also homologous to ZFY, has been located on both mouse and human X chromosomes. It has been shown in humans to escape X inactivation (Schneider-G~idicke et al. 1989). Figure 1 illustrates that all of the B cell lines derived from Sxr-carrying mice analyzed have both Zfy-1 and Zfy-2.

While this does not exclude the possibility of DNA deletion or rearrangement leading to selective loss of Hya epitopes, any such process has not involved loss of Zfy-1 or Zfy-2. This mechanism is made unlikely by two obser- vations, first the frequency and consistency of outcome (loss of H-Y b) and second the acquisition of H-Y b in two

D. Scott et al.: Hya expression in mouse B-cell clones

Table 3. Levels of expression of H-2K k and H-2D b molecules on Abelson-transformed clones.

59

Cells Antibody

Anti-H-2K k Anti-H-2D b FITC anti-mouse Ig control

Percent* Peak/Mode t Percent Peak/Mode Percent Peak/Mode

T 16H/XSxr clones 5H2 99.4 716 99.0 225 99.6 6 5E12 99.5 597 98.6 260 99.5 5 5G8 99.0 769 99.6 242 99.5 5 5A8 98.7 742 99.5 270 99.5 4 1F10 99.5 347 99.6 251 99.4 4 5H7 99.3 691 98.9 195 99.5 5 5B3 99.5 742 98.5 225 99.8 4 5C3 99.1 742 97.5 175 99.0 5 1A6 99.4 742 99.4 202 99.7 5 1D5 98.1 827 100.0 556 99.3 4 5F1 99.5 556 99.5 195 99.6 5 5C2 99.5 360 99.7 242 99.7 5 5D4 NE NE 98.2 121 99.5 5 ID8 NE NE 99.9 202 99.5 5 1F3 NE NE 99.7 260 99.6 4 1C8 NE NE 99.8 195 99.6 4

X/XSxr clones 1F8 99.9 373 99.7 260 99.3 5 1F11 99.9 432 99.6 290 99.7 6 1F12 99.6 312 96.3 188 99.5 5 1F5 99.9 448 99.6 270 99.5 7 1A3 99.8 402 99.5 202 99.8 4 1D1 99.8 416 99.5 175 99.7 4

* Percent of cells expressing antigen. t Peak/Mode: intensity of fluorescent staining. Higher values represent greater fluorescence. NE, not estimated.

initially negative clones (5H7 and 5B3). X inactivation seems a far more likely possibility than DNA recom- bination.

Fig. 1. All B-cell clones derived from Sxr containing animals retain Zfy-1 and Zfy-2. A 2.3 kb subfragment of the 2.8 kb Zfy-2 cDNA pDP1122 was hybridized to Eco RI-digested genomic DNA. Lane 1, X/X 9 B-cell line; lane 2, A8; lane 3, F l l ; lane 4, D l l ; lane 5, 5A8; lane 6, 5H7; lane 7, 1F10; lane 8, 5G8; lane 9, 5C3; lane 10, 5B3; lane 11, 5E12; lane 12, 1A6; lane 13, X/XSxr liver; 14, xxliver. The bands corresponding to Zfy-1 ( l l and 12 kb) and Zfy-2 (5 kb) are indicated. Size markers were the Hind III fragments of iambda.

Discussion

From the data presented in Tables 1 and 2 it is clear that, whereas all of the Abelson-transformed pre B-cell clones derived from an H-2 k~b X/XSxr male expressed H-Y antigen in association with H-2 k [K k using clone C6 and D k using cytotoxic T lymphocytes (CTLs)], A b (clone B9), and H-2D b (using clone M1 and CTLs), the majority of similar clones from a T16H/XSxr female failed to express H-Y antigen in association with D b, although all expressed H-Y in association with H-2 k and A b. FACS analyses of D b and K k expression by the B-cell clones show that the levels of both molecules are similar in cells derived from T16H/XSxr and X/XSxr mice, making it unlikely that low levels of D b expression account for the failure to present H-Y to Db-restricted indicator T cells.

60 D. Scott et al. : Hya expression in mouse B-cell clones

The simplest hypothesis to account for these observa- tions is that the spread of X inactivation in the Sxr translocation is variable and that at a clonal level one may find cells expressing different numbers of genes within this segment.

The finding that the same clone can express H-Y in association with H-2 k class I molecules and A b class II molecules but not with H-2D b suggests that the presumed H-Y peptide(s) that associate with H-2K k and H-2D k class I molecules and A b class II molecules are encoded in a different part of the Hya gene from the H-Y peptide(s) that bind to H-2D b molecules. The expression of one or more of these peptides in the absence of the other suggests they are under the control of separate promoters and may indeed be the products of more than one gene. An alter- native explanation may be provided by the work of Boon and colleagues, who have shown that antigenic peptides representing tumour-specific transplantation antigens can be directly transcribed as short subgenic fragments presumably controlled by separate cryptic promoters (Lurquin et al. 1989; Boon and Van Pel 1989). Our obser- vations would be consistent with either of these hypotheses.

The findings reported here support the hypothesis of the variable spread of X inactivation in the Sxr region. This was originally suggested by the finding that T16H/XSxr individuals can be females, males, or hermaphrodites (Cattanach et al. 1982; McLaren and Monk 1982). A similar variable spread of X inactivation into X-autosome translocations has been demonstrated by Cat- tanach (1974).

The observation that two of the clones (5B3 and 5H7) that were initially negative for H-Y/D b expression subsequently became positive argues against genetic loss (deletion) of this part of the Hya gene in the H-Y/Db-negative c lones-as was also suggested by the Southern analysis shown in Figure 1. It further indicates that the variable spread of X inactivation in this region is unstable, although it is not clear whether this instability was caused by prolonged in vitro culture or whether it could also be an in vivo feature. Such somatic cell reactivation of inactivated X-autosome translocations has previously been demonstrated (Cattanach 1974). In addition, age-related reactivation of true X-linked genes has been shown (Wareham et al. 1987; Brown and Rastan 1988).

The variable spread of X inactivation into the Sxr segment, as indicated by H-Y/Db gene expression of individual clones, indicates that despite the invariable inactivation of the X chromosome carrying Sxr, this T16H/XSxr female is a mosaic. One would not necessari- ly expect this sort of mosaicism to be confined to T16H/XSxr females. No evidence for mosaicism was found in cells from the X/XSxr male; however, only 12 such clones were examined and only about 50 % of these would have had the Sxr-carrying X inactivated; no more

than five clones would therefore have been expected to show inactivation of the gene controlling recognition of H-Y in association with H-2D b. Thus, the failure to detect such clones could have been due to chance alone. Alternatively, this could be due to an in vitro advantage of (X-inactive) XSxr clones over X (XSxr-inactive) clones, which would not apply to T16H/XSxr clones in which all the Sxr-carrying X chromosomes are inactive.

The data reported here showing a variation in H-Y expression in clones of cells from a T16H/XSxr female establish mosaicism in this individual, presumably as a result of the variable spread of X inactivation into the Sxr region. They further imply that the H-Y antigen recognised in association with H-2 k (K k and D k) and H-2D b class I molecules and A b class II molecules may be products of more than one gene. If there are indeed two genes controlling the recognition of H-Y antigen in association with H-2 k and H-2D b, respectively, our results suggest that the most likely order of the genes involved is: te lomere- recognition of H-Y in association with H-2 k (Hya k) (0% inactivation) - recognition of H-Y in association with H-2D b (Hya b) (19/24=79% inactivation). Tdy is likely to lie near this latter locus, or even closer to the junction of Sxr with the X chromosome, since Tdy must have suf- fered inactivation in some 75 % or more of cells in the gonad primordium for the embryo to develop as a female.

Acknowledgments. We thank Mrs. Vivien Tikerpae for help in preparing the manuscript, Dr. K. Tomonari for use of his T-cell clones and Dr. D. Page for plasmid DPl122.

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