CELLULAR IMMUNOLOGY 120,2 18-229 (I 989)

The C57BL/6 Nude, Beige Mouse: A Model of Combined T Cell and NK Effector Cell lmmunodeficiency

*Laboratory of Immunology, Louis Pasteur University Strasbourg I, BP 24,6 7401 Illkirch-Cedex, France, and tPreclinica1 Research 386/125, Sandoz Ltd, postfach, CH 4002 Basel, Switzerland

Received October I I, 1988; accepted December 30, I988

This article describes the construction and establishment of a double congenic nude, beige C57BL/6 (B6 nu, bg) mouse strain. The mice do not show higher fragility than C57BL/6 nude mice and the double congenic strain can be maintained under conventional mouse housing conditions. Although the B6 nu, bg display a very low natural killer activity which cannot be enhanced by an interferon inducer (poly(I-C)), they lack responsiveness to a T cell mitogen (concanavalin A); and they also show extremely low responsiveness to a B cell mitogen (0 128: B I2 Escherichia coli lipopolysaccharide) probably as a result of combined effects of the beige and nude genes in the C57BL/6 genetic context. o 1989 Academic PWS, IIIC.

INTRODUCTION

Mice with profound deficiencies of immune surveillance are valuable tools both as in vivo hosts for cancer cells in short term studies on tumor growth, metastasis, and drug sensitivity and as recipients for long term studies on the development, the causes, and the prevention of autoimmunity syndromes which are characteristic of the donors of the grafted cells. In the case of tumor grafts (e.g., human tumors grown on outbred nude mice) there is no real need, at least generally, of a same genetic background for the cell donor and the host. In attempts to study autoimmunity disor- ders, the construction of the donor + recipient chimeras must be done in the same genetic background.

Because of their lack of mature T cells, the nude, athymic mice (homozygous for the ..* mutation) could rapidly be used for growing tumor cells from very diverse origins ( l-3). However, it appeared early that nude mice could better resist threshold inocula of certain syngeneic tumor transplants than their euthymic +/nu littermates. The phenomenon was attributed to the elevated levels of natural killer (NK) cell activity in nu/nu mice (4,5).

On the contrary, mice homozygous for the beige (bg) mutation have a blockade in expression of NK function (6-S) and they show a decreased resistance to low dose

’ Abbreviations used: B6, C57BL/6; bg, beige; +, wild; lpr, lymphoproliferation gene/phenotype; nu, nude, athymic gene/phenotype; Con A, concanavalin A; dpm, disintegration per minute; f, female; ip, intraperitoneal; LPS, lipopolysaccharide; m, male; NK, natural killer; poly (I-C), poly(inosinic)-poly(cyti- dilic) acid.

218

OOO8-8749/89$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

NK-, T- NUDE BEIGE C57BL/6 MICE 219

inocula of syngeneic tumors compared to phenotypically normal +/bg littermates (9, 10).

The types of tumor cells which are particular targets for NK cells are not entirely defined, but classical NK targets in the mouse, i.e., EL4 and YAC 1 lymphoma cells, are early thymomas, thus a rather immature T cell subset. Related to this immature characteristic, natural killer and/or natural suppressor activities have been implicated in the control of stem cell growth (11-13). Both tumor and normal targets of NK cells are thus rapidly proliferating cells which belong to an early type of precursor cell. These characteristics are actually shared by an abnormal type of cell which causes lymphadenopathy in the lpr (lymphoproliferation) locus-homozygous mice: al- though these cells bear aberrant epitopes (i.e., non-T cell epitopes), they belong to a rapidly dividing, immature T cell lineage subset ( 14).

Most attempts to transfer the lymphoproliferative syndrome of lpr mice by classical methods have failed: neither congenic normal mice nor their +/fpr FI hybrids, either irradiated or treated with radiomimetics and then grafted with bone marrow or spleen cells, nor congenic nude mice allowed the growth of the abnormal Zpr T cell subset ( 15- 18). Particularly the lack of lymphoproliferation transfer and the profound lym- phoid aplasia found in X-irradiated C57BL/6 (B6) recipients of bone marrow cells from lpr homozygous B6 (B6 lpr) mice (17, 18) could be due to the recognition of lpr locus-encoded epitopes on the grafted lymphoid precursors by radiation-resistant NK cells of the host (18). Similarly, the lack of lymphoproliferation in IZU homozygous B6 (B6 nu) recipients of B6 lpr spleen cells could be due to the elimination of abnor- mal cells of the inoculum by the powerful NK activity of the nude host ( 15).

A T cell-deprived, NK activity-deficient recipient would thus be useful for the study of a variety of single locus-encoded autoimmune disorders. In order to have it avail- able on the C57BL/6 genetic background, we have constructed a double congenic B6 y1u, bg mouse strain. This paper describes the pedigree and some practical problems inherent to the construction of such double immunodeficient mice, as well as evi- dence for their low NK activity, their lack of T cell mitogen responsiveness, and their low B cell mitogen responsiveness.

MATERIALS AND METHODS

Mice. Our (normal) C57BL/6 (B6 +, or B6 +/+, +/+), C57BL/6 beige (B6 bg or B6 +/+, bg/bg), and C57BL/6 nude (B6 nu or B6 nu/nu, +/+) colonies originated from breeding pairs obtained from, respectively, the CSEAL (Orleans, France), the Jackson Laboratory (Bar Harbor, ME), and Dr. Friis (Bomholgard, Ry, Denmark). Our B6 nu colony was maintained by crossing hairy (nu-heterozygous females) with nude (nu-homozygous) males. The double congenic strain (B6 y1u, bg or B6 nu/nu, bg/bg) was constructed as described under Results. All mice were bred in our animal colony and kept in isolators under positive pressure (ESI, Cachan, France). They were provided with autoclaved water and bedding and with sterilized food (X-irradiated, UAR, Epinay/Orge, France). Yet, their maintenance status should be considered conventional rather than specific-pathogen free. To induce a higher NK activity, some mice were injected ip with poly(inosinic)-poly(cytidylic) acid (poly(I-C), Sigma, St. Louis, MO; 100 rg in 50 ~1 of NaClO.9%).

Cell cultures. The murine NK cell target was the YAC 1 cell line, a T cell lymphoma of mouse strain A/Sri origin. It was maintained in RPM1 1640 medium supplemented

220 PFLUMIO ET AL.

with antibiotics (penicillin and streptomycin), glutamin, pyruvate, and 10% heat- inactivated fetal calf serum (all from GIBCO, Paisley, UK). YACl cells were passed in new cultures 1 day before use as target cells in cytotoxicity assays. Spleen cells were prepared from mice euthanized when 6 to 9 weeks old. They were first washed twice in Hanks’ buffered saline solution and then counted for cell viability with the vital staining procedure ( 19). They were adjusted to the appropriate concentration in the RPM1 medium indicated above and cultured at 37°C in a humidified CO* incubator.

Chromium release cytotoxicity assay (20). The YAC 1 target cells were labeled with “Cr (sodium chromate, 1.1 mCi/ml, Amersham, Buckinghamshire, England) and mixed with effector spleen cells at four effector:target ratios (100: 1, 50: 1, 25: 1, and 12.5: 1). The test was performed in 96-well microtiter plates with 2. lo4 target cells per well. Spontaneous lysis was determined by addition of medium only (no spleen cells) to the target cells, while 100% lysis was determined by addition of 1 M HCl. The assays were run for 4 hr at 37°C in a humidified CO2 incubator. The plates were centrifuged before supematant harvesting; 50% of the supernatant fluid was counted for radioactivity in a gamma counter. Spontaneous lysis was generally less than 15% and never exceeded 20%. The percentage specific 5’Cr release was equal to 100 X [(cpm test sample - cpm spontaneous lysis)/(cpm HCl control - cpm spontane- ous lysis)].

Mitogenic activation (21). Samples of 5 X lo5 cells/well of trypan blue-defined via- ble spleen cells were cultured in triplicate in flat-bottom 96-well microtiter plates. Concanavalin A (Con A, Pharmacia, Uppsala, Sweden) or bacterial lipopolysaccha- ride (LPS from Escherichia coli, batch 0 128:B 12. Difco, Detroit, MI) was added to the wells (2.5, 5.0, and 10.0 hg/ml for Con A; 6.25, 12.5, 20, and 25 pg/ml for LPS). Six hours prior to the end of the 3-day culture, [3H]thymidine ([3H]TdR, 1 PCi) was added to each well. The cultures were harvested with a multiple sample harvester (Titertek, Flow, France) and the radioactivity incorporated in DNA was determined by liquid scintillation spectroscopy. To allow comparisons of different experiments performed at very different times during this work, some of the data were expressed as the percentage of the mitogenic responses of control B6 mice.

RESULTS

Pedigree and Viability of 86 nu, bg

Procedures (P -W F2 generations). Our nude, beige B6 mice were produced by a series of crosses starting from B6 bg females (f) and B6 nu males (m). The Fl genera- tion (B6 +/nu, bg/+) had thus the black hair phenotype. The offsprings were inter- crossed in randomly selected breeding pairs. Since the two loci were not linked (nu: chromosome 11; bg: chromosome 13), they segregated independently. Thus, 16 different combinations could be obtained in the F2 progeny, 25% being nude (nu/nu, ?/?), 18.75% being beige (+/?, bg/bg), and 56.25% being black (+/?, +/?). Out of the F2 mice with a nude phenotype, one-fourth should theoretically be +/+, another fourth bg/bg, and one-half +/bg at the bg locus.

The multiple intercrosses of Fl mice actually provided a F2 progeny with a lower proportion of viable nude mice than expected. This may be due to important death between birth and weaning (at 5 weeks for the nudes): 20.5% of the 1175 F2 mice born from Fl intercrosses died early. The mice which survived weaning had (in per-

NK-, T- NUDE BEIGE C57BL/6 MICE 221

TABLE 1

F2 and F3 Pedigrees

F2

Crosses and genotypes (established a posteriori)

Females (n) X Males(n)

F3

Phenotypes/genotypes (n (% obtained; % theory))

Total Early born death Black hair Beige hair Nude (n) (n(W)) (+/?, +/?) (+/?, bglbg) (nu/nu, ?/?) Notes

+/+, bg/bg (2) m/m, +/bg (2) 30 0 13 (43.5; 50) 17 (56.7; 50) 0 (1

+/nu, bglbg (4) nu/nu, +/+ (4) 63 lS(28.6) 28 (44.4; 50) 0 17 (27; 50) h +/nu, bg/bg (I 1) nu/nu, +/bg (10) 183 74 (40.4) 45 (24.6; 25) 49 (26.8; 25) 15 (8.2; 50) c +/nu, bg/bg(l) +lnu. bglbg (I) 14 l(7.2) 0 11 (78.3;75) 2(14.3;25) d +/nu, bglbg (4) nulnu, bglbg (1) 30 12(40) 0 17 (56.7; 50) I (3.3; 50) ’

0 All these F3 beige mice were +/nu, bg/bg and could be used for further crosses. ’ These F2 females could be used for further crosses as well as their F3 nude progeny (since they were

+/bg). Out of the 18 F3 mice dead early, at least 11 were nude. ’ Both these F2 parents could be used further, although not in this type of mating since it provided F3

progeny of unclear genotype. Among these F3 nudes, at birth, 213 should be bg/bg and l/3 should be +/ bg. However, at least 50 out of the 74 F3 mice dead early were nudes, and death might have affected the nu/nu, bg/bg selectively.

d Only one out of five such breeding pairs was informative. Both F2 parents could be used further in this type of mating which could provide nu/nu, bg/bg F3 mice. These beige haired F3 mice were unfortunately ?/? at the nude locus and were discarded.

’ The presence of only beige mice and nude mice in these F3 indicated that the F2 female was heterozy- gous at the nu locus and that this F2 male had at least one bg allele. Among the 30 mice born from this type of mating, 22 were phenotypically identified (17 beige mice and 5 nudes among which 4 died before weaning). The probability of having no black mouse in F3 if this F2 nude male was bg/+ was l/560 (0.7522). The probability was thus good that this F2 nude male, as well as the F3 nudes, was nu/nu, bg/bg, but this needed further test crosses.

centage of born mice) the following phenotype: black hair (58%), beige hair (15%), and nude (6.5%).

Although it has been reported that nu/nu, bg/bg F2 mice issued from B6 X C3H crosses could be identified by their lighter skin and pale ears (23), we could not con- vincingly see a difference for B6 congenics, and we found it necessary to establish a nude, beige colony only after test crosses were performed. All F2 mice with the black hair phenotype were euthanazied as they were +/?, +/?, whereas mice with the beige hair phenotype (theoretical proportions at the nude locus: l/3 +/+ and 2/3 +/nu) and mice with the nude phenotype (theoretical proportions at the beige locus: l/4 +/ +, l/2 +/bg and l/4 bg/bg) were engaged in specific test crosses. These were designed in ways such that the F3 littermates’ phenotypes allowed a formal establishment of the F2 parents’ genotypes. In practice, +/?, bg/bg (36 f) were mated with nulnu, ?/? (25 m) or with +/?, bg/bg (5 m). Out of the 63 F3 litters obtained, only 55 were large enough to provide reliable information on the parents’ genotypes (see Table 1).

Selection of the double nu, bg homozygotes (F3 and F4 generations). The actual numbers of F3 animals which could be used for further intercrosses were limited: 5 1 +/nu, bg/bg (30 f and 2 1 m) and 5 nude males (4 bg/+ and 1 bg/bg; nude females do not produce litters). Intercrosses of beige F3 females with nude F3 males were thus

222 PFLUMIO ET AL.

limited and gave only litters of small size, with expected numbers of nudes, which had to be nu/nu, bg/bg but 75% of them died before weaning.

Most of the F3 intercrosses were thus performed between +/nu, bg/bg littermates giving birth to a progeny of 330 F4 mice in proportions close to the theoretical values of 75% +/?, bg/bg (244 mice, 73.9%) and 25% nulnu, bglbg(76 mice, 23%), 10 mice (3.1%) dying early after birth. However, 63% of the nude, beige mice died before weaning following a weight loss. The critical period for nude, beige survival could be due to selective care by the hairy mother; in further matings the nude mice were identified at birth by the absence of vibrissae (24), allowing the euthanasia of most hairy littermates, and their weaning was postponed as long as they looked fragile.

An additional production of nude, beige mice was obtained by selective back- crosses of F3 males to F2 females, leading to an F4’ progeny. Crosses between 13 +/ nu, bg/bg F2 females and 11 +/nu, bg/bg F3 males gave birth to 307 F4’ mice from which, at 2 weeks of age, 228 (74.3%) were found to be beige and 60 (19.5%) nude (thus nude, beige).

B6 nu, bg strain establishment (FS and further generations). Three nude, beige males with well-established bg/bg genotype and four hairy, beige females with well- established +/nu genotype were selected and intercrossed. The offspring from such matings +/nu, bg/bg (l) X nu/nu, bg/bg (m) was constituted of beige mice (50%, +/ nu, bg/bg) and of nude mice (50%, nujnu, bgjbg).

The B6 nu, bg strain is maintained by performing the same type of intercross at all generations (+/nu, bglbg females X nulnu, bg/bg males). All nulnu, bglbg females are available for experimentation. Intercrosses of +/nu, bg/bg mice produce more nulnu, bg/bg progeny (25%) for experimentation.

Our data on viability of the double immunodeficient C57BL/6 nude, beige are still limited particularly because most of them were euthanized around 8 weeks of age for in vitro analyses of their immune system or were used as recipients for construction of chimeric mice by injection of cells from autoimmune donors. The only values available so far are those of B6 nu, bg males engaged in repeated matings for B6 nu, bg litter production: out of 18 males observed in that way, the following survival times were obtained (weeks [number]: 9 [2]; 10-l 1 [5]; 13 [2]; 14-15 [3]; 17 [2]; 19 [ 11; 26 [I]; 29 [l] and one 12 months old and still alive).

So far no spontaneous tumor development has been observed either on these B6 nu, bg males or on other long term survivor chimeric mice prepared for the study of autoimmunity phenotype expression (B6 nu, bg mice of both sexes grafted with lymphoid cell suspensions from B6 +, B6 bg, B6 lpr, and B6 me” mice).

In Vitro Responses of B6 nu, bg Spleen Cells

NK activity. The tests were performed on healthy looking 6- to 9-week-old mice with NK activity being at its maximum in normal mice. The NK activity in the spleen was studied with individual mice, since work on pooled lymphoid cell suspensions from several animals would not reveal the actual level of variability among individual mice and may hinder the occurrence of possible leaky mutants (as occurs, for in- stance, with the SCID mouse). Six completely independent experiments (i.e., includ- ing mice obtained from different litters) are reported in Table 2. Each of the first four experiments included the comparison of the NK activity in two (or one) B6 nu, bg mice with those of two B6 bg, two B6 +, and two B6 nu, age and sex matched, mice;

Stra

in:

96 +

/+

+ /+

TABL

E 2

NK

Cel

l Act

ivity

in

Nor

mal

and

Mut

ant

C57

BL/

6 M

ice

96 +

/+

bg/b

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Rat

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1 5O

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2.8

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5 0.

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3.3

2 1

11.9

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4 0.

6 0

0.6

2 10

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7

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4.9

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1.7

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2

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4.1

1.4

0.6

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1.0

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1.7

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7 4.

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7 0.

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3.9

3.1

0.1

1.6

0.3

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48.8

26

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16.0

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30.6

18

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17

.7

11.4

19

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11.6

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1

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12

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11.4

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25

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12.5

:1

10.0

11

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1.3

5.8

0.8

0 0

m

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3 2.

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0 2.

2 0.

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9 5.

9 2.

5 1.

5 4.

1 10

.9

8.4

4.1

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2 r k 5

1 1.

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8 0.

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0 1.

0 1.

0 30

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18

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N

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2 1.

4 0.

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9 2.

8 2.

6 1.

0 1.

2 31

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30.7

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5 2.

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21.4

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8.9

5.9

3.0

1.9

1.1

SD

3.4

1.4

0.8

0.5

1.8

1.2

1.4

0.4

17.2

13

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13.2

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5 4.

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4

224 PFLUMIO ET AL.

TABLE 3

Effects of Poly(l-C) on the NK Cell Activity

% of specific cytotoxicity (R = 50: 1)

Mice Without poly(I-C) With poly(I-C) (mean If: SD (n)O) (mean f SD(n))

+I+, +I+ 2.2 f 1.4(12)b 12.3 + 1 (4)’ +I+, bglk 1.2 f 1.1 (12)b 5.3 t- 4.3 (4)’ nu/nu. +/+ 21.4 f 13.2 (12)b 29.1 _+ 4.5 (3)’ nulnu, bglbg 3.0 +- 3.5 (7)d 7.0 f 3.7 (6)’

a (n) = number of animals. ’ Mean of six experiments. ’ Mean of two experiments. d Mean of four experiments.

B6 nu, bg mice were not included in the other two reported comparisons. All mice were from our colony except the B6 nu of experiment 1, which were obtained from another source and had a much higher NK activity. The lower NK activity of B6 nu mice from our colony might be due to better animal housing conditions with lower incidence of viral infections. The comparison of the four congenic strains showed, in each experiment, that the NK activity of B6 nu mice was the highest and that of B6 bg mice was the lowest whereas B6 + mice and B6 nu, bg were intermediate but low. Although the mean percentages of specific NK cytotoxicity at the 50: 1 effector:target ratio were 2.2 for B6 +, 21.4 for B6 nu, 1.2 for B6 bg, and 3.0 for B6 nu, bg, the individual variability was higher for B6 nu, bg than for B6 +.

To evaluate if the higher NK activity of some individual mice might be due to latent viral infections, a series of mice of each congenic strain was injected with poly(I- C) 2 days before euthanasia and their splenic NK cell activity was compared with that of sham-treated mice. The results (Table 3) showed an increase of NK activity in B6 + mice, little effects in B6 nu mice, no effect for 3/4 B6 bg mice, l/4 behaving like B6 + mice, and little or no effect on B6 nu, bg mice: at least not one of the six B6 11u, bg mice treated with poly(I-C) acquired an activity as high as poly(I-C)-treated B6 y1u mice, and they were at best like poly(I-C)-treated B6 + mice.

Con A responsiveness. Although the B6 nu, bg mice, like B6 nu mice, lacked a macroscopically detectable thymus, we found it necessary to control the lack of T cell mitogen responsiveness. The proliferative responses to Con A were compared with spleen cells of all four congenic mouse strains. A sample experiment shows the Con A responses obtained at different mitogen doses (Fig. l), and the mitogenic responses obtained with 5 pg/ml Con A in independent experiments are also shown (Fig. 2). The athymic mouse B6 nu, bg and B6 nu spleen cells did not respond to the T cell mitogen as expected, while the euthymic control mouse B6 + and B6 bg spleen cells gave high incorporation values. There did not seem to be differences either between the two athymic mouse strain or between the two euthymic mouse strains with regard to Con A responsiveness.

LPS responsiveness. We also compared the B cell mitogen responsiveness of spleen cells of individual mice of the four congenic mouse strains. A sample experiment

NK-, T- NUDE BEIGE CS7BL/6 MICE 225

2OOQQO

15ooocl

100000

50000

0 I 0 2.! 5 5 IO

FIG. 1. Responsiveness of normal and mutant B6 mice to various Con A doses. This typical experiment shows the responsiveness of spleen cells (5 X lO’/well) of two B6 +, two B6 bg, one B6 nu, and one B6 nu, bg mouse to various doses of Con A (&ml, Xaxis). Each column represents the mean dpm (*SD, Y axis) of [3H]thymidine incorporated into DNA of triplicate cultures.

shows the LPS responses obtained at different mitogen doses (Fig. 3), and the mito- genie responses obtained with 25 pg/ml LPS in independent experiments are also shown (Fig. 4). The B6 + spleen cells gave a high proliferative response, which was higher than the response of B6 bg spleen cells, a finding which we did not expect although there had been a few preliminary reports of such a fact. The LPS responsive- ness of all tested B6 IZU, bg spleen cell suspensions was very low; the B6 ytu spleen cells were generally less responsive to LPS than B6 + cells. These patterns of respon- siveness fitted with data obtained with larger numbers of B6 bg and B6 71~ mice com- pared with B6 + mice for the independent effects of the 11u gene and of the bg gene on B cell responsiveness, to be reported independently.

DISCUSSION

Pedigree and Viability

During the construction of the B6 nu, bg mouse strain, a high proportion of early dying mice (death when 2 and 3 weeks old) concerned the phenotypically nude litter- mates. On some independent matings, we tried early grafting of spleen cells from

FIG. 2. Comparison of the responsiveness of B6 + mice with those of mutant B6 mice to 5 &ml Con A. Data from four different experiments have been pooled, totalizing 10 B6 +, 10 B6 bg, 4 B6 nu, and 6 B6 nu, bg mice. To allow the comparison, the data are expressed (as percentage Y axis) as the ratios of the dpm obtained for each individual mouse to the mean dpm obtained for the respective control (B6 +) mice.

226 PFLUMIO ET AL.

60000

0 0 6.25 12.5 20 25

FIG. 3. Responsiveness of normal and mutant B6 mice to various LPS doses. This typical experiment shows the responsiveness of spleen cells (5 X lO’/well) of two B6 +, two B6 6g, two B6 au, and two B6 nu, bg mice to various doses of LPS (&ml, X axis). Each column represents the mean dpm (*SD; Yaxis) of [3H]thymidine incorporated into DNA of triplicate cultures.

hairy littermates into the nude ones, but without better success (not shown). Some improvement of the nude littermates’ viability was obtained by euthanasia of hairy ones thus favoring mother’s care for the nudes and by delaying the weaning of the latter. But a more rigorous control of the temperature of the breeding room (and isolators) was later found to be the most essential condition to avoid the critical period of 2-3 weeks of age: it should not drop under 18°C or rise above 23°C. Working at 20-22°C in a >95% humid atmosphere, specific early loss of nude littermates in the matings for strain maintenance has practically been eliminated: 50% of the [+/nu, bg/ bg(f) X nu/nu, bg/bg (m)] littermates who reach weaning at 5 weeks are indeed nude.

It is therefore likely that the lower percentages than expected of nude littermates obtained during the construction of the B6 nu, bg strain were due to accidental death related to inappropriate environment, and indeed the percentages of early dead mice could easily account for the lacking percentages of nude littermates. Our B6 y1u, bg mice do not seem more fragile than B6 nu mice, although we have not specifically performed survival studies since all available B6 TZU, bg mice were either reserved for production of new litters or were used experimentally and euthanized when 2 or 3 months old. We had however a few B6 nu, bg males who were active breeders for more than 6 months.

FIG. 4. Comparison of the responsiveness of B6 + mice with those of mutant B6 mice to 25 &ml LPS. Data from four different experiments have been pooled, totalizing 10 B6 +, 10 B6 bg, 3 B6 nu, and 6 B6 nu. bg mice. To allow the comparison, the data are expressed (as percentage) as the ratios of the dpm obtained for each individual mouse to the mean dpm obtained for the respective control (B6 +) mice.

NK-, T- NUDE BEIGE C57BL/6 MICE 227

Three different groups have reported the construction of double immunodeficient nude, beige mice but no congenic strains were established. Thus, nude, beige mice were identified in F2 littermates of parental B6 X C3H crosses by their lighter skin and pale ears and they remained viable up to 3 months under conventional condi- tions (23). Healthy nude, beige mice, with a life span comparable to that of regular nude mice, were also obtained through crosses of C57BL/6N-beige and N:NIH(S)- nude mice (25). As already stated in the introduction, such “outbred” nude, beige mice may be used as recipients for tumor cells but they should not be considered appropriate recipients for grafts of cells which carry a potential autoimmunity disor- der. Indeed, even if graft-versus-host reactions could be avoided, the genetic context could strongly affect the expression of a given loci; i.e., environmental factors could play a role. Particularly, the phenotypic expression of the lymphoproliferative and autoimmune syndrome, caused by homozygosity at the Ipr locus, differs in MRL, B6, SJL, C3H, and other genetic contexts. Any attempt to dissect the cellular components taking part in the etiopathology of the whole Zpr syndrome observed in a B6 lpr mouse ought to be performed under congenic conditions. Nude, beige mice on the C57BL/ 6 background had been made in the past (26) but no further work was published, perhaps because of problems of maintenance of poorly viable mice or because of limited interest in such mice as tumor graft recipients in comparison to classi- cal nudes.

NK Activity

Nude, beige mice on the B6 genetic background have low NK activity, thus con- firming, with a larger number of animals, data reported by others (23, 25, 26). For the pool of experiments reported in Table 2 and which represent a comparable group since each experiment included mice of the four different genotypes, comparative percentages of spleen NK activity at the 50: 1 effector:target ratio were 2.2% for B6 +, 2 1.4% for B6 nu, 1.2% for B6 bg, and 3% for B6 RU, bg. It is a matter for speculation, whether the slightly higher NK activity found in B6 IZU, bg compared with B6 bg should be taken as evidence that the bg-mediated blockade in NK activity expression is leaky in some individual mice, or if only a part of the enhanced NK activity of nude mice can be controlled by the bg locus, or if still a new cell type is involved. Further work is required for this matter, but the actual fact that B6 IZU, bg mice, although much deprived of NK activity compared with B6 y1u, have slightly more NK activity than B6 bgconfirms preliminary reports (23,25,26). In comparison with them, the NK activity of our mice, particularly B6 +, was rather low. The B6 strain is indeed a “low NK” strain (23) possibly because it carries a number of non-immune system-linked resistance genes to viral infections (22), calling for little contribution of NK cells under normal conditions. Higher NK activity could be induced by an interferon inducer (poly(I-C)) in B6 +, and even to a small extent in B6 bg, but there was no evident enhancement of either the high NK activity of B6 nu or the low NK activity of B6 nu, bg (although again there may be a slight effect of poly(I-C) in both athymic strains).

T and B Cell Mitogen Responsiveness

The results of responsiveness to the T cell mitogen, Con A, were as expected, whereas the study of the responsiveness to the B cell mitogen, LPS, gave some unex-

228 PFLUMIO ET AL.

petted results, although similar observations had already been mentioned by others, but in reserved terms. Thus, there was no response of B6 nu and B6 IZU, bg to Con A and the responses of B6 bg and of B6 + to this mitogen were generally similar both for the dose-response curve and for the maximal tritiated thymidine uptake. This does not necessarily mean that the T cell compartment of immune surveillance in B6 bg mice functions in an entirely normal way. On the contrary, there were both early (26) and recent (27) reports that the generation of functional cytotoxic T lym- phocytes is also affected by the bg mutation. If the basic effect of the bg mutation is at the level of microtubule function (28), alterations of many cells other than NK could be expected, which is indeed the case for several nonlymphoid cells of beige individuals (29).

The rather unexpected results were actually that both B6 y1u and B6 nu, bg mice, and also, although to a lesser extent, B6 bg mice, responded less to the B cell mitogen LPS than B6 +. The lower response to LPS of bg-homozygous mice was briefly men- tioned by two other groups of investigators who indicated that the LPS responses in B6 bg/bg mice were reduced to 60% of those in the B6 +/+ and B6 +/bg mice (25), and that “8 weeks old SB bg/+ mice had somewhat higher” (about twofold) “re- sponses to LPS than bg/bg homozygous littermates” (30). Although the nu locus was initially reported as enhancing LPS responsiveness, decreased, unchanged, and en- hanced responses to LPS have been reported in the literature (25, 31-34). It is un- known whether there are differences due to the different genetic contexts into which the nu locus is expressed, due to different experimental conditions or to different LPS sources. We have repeatedly observed lower responses of B6 ~tu compared with B6 + mice, using this and other sources of LPS, but no systematic comparison has been done.

Given the fact that both the beige mutation and the nude mutation, at least in the C57BL/6 genetic context, had decreasing effects on the LPS responses of spleen cells, it is not surprising that the double congenic B6 nu, bg showed extremely low LPS responsiveness. Whether these mice are as depleted of mature B cells as mice doubly homozygous for the nu and Xid (X-linked immunodeficiency) is not yet known. However, we have evidence that other phenotypic characteristics of the Xid locus are shared by the bg locus.

CONCLUDING REMARKS

Once established and maintained under good conventional housing conditions, the double congenic B6 nu, bg do not show higher fragility than B6 nu mice. They display however a very low NK activity which cannot be enhanced by an interferon inducer; they lack responsiveness to a T cell mitogen and they show an extremely low responsiveness to a B cell mitogen, probably resulting from cumulative effects of the bg gene and of the izu gene expressed in the B6 context. Preliminary results from experiments currently in progress show that B6 nu, bgare very good recipients for the graft of mature lymphoid cells from B6 congenic mice, with particularly prolonged survival with spleen cell grafts from B6 + and B6 bg donors but even from B6 lpr and B6 me” donors, thus providing an interesting recipient model for the cellular dissec- tion of the components of such autoimmunity syndromes.

NK-, T- NUDE BEIGE C57BL/6 MICE 229

ACKNOWLEDGMENTS

This work was supported in part by an INSERM grant. The authors are very grateful to Miss C. Bertin for her help with animal care and to Miss C. Loor for her help with the English writing of this manuscript.

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