The role of splenic colony-forming units in autoimmune disease

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

49, 133-142 (1988)

The Role of Splenic Colony-Forming Autoimmune Disease CURTIS L. SCRIBNERAND Cellular

Immunology

Section,

Units in

ALFRED D. STEINBERG

National Institute of Arthritis, Musculoskeletal, Bethesda, Maryland 20892

and Skin Diseases,

Stem cell activity in murine lupus was investigated by analyzing endogenous splenic colony-forming units in sublethally irradiated inbred, congenic, and consomic mice as well as Fl crosses. Splenic colony-forming units (CFU-s) were elevated (>lOO) in young NZB mice as compared with nonautoimmune-prone mice (
INTRODUCTION A number of inbred mouse strains, Fl crosses, and recombinant inbred lines develop various features of human systemic lupus (l-3) caused by at least three different types of genes. Autoimmunity-inducing genes include lymphoproliferation @r&r), generalized lymphoproliferative disease (gld/gld), and motheaten (me/me) types (2,4-8). The SB/Le- and BXSB-Y chromosome genes are accelerating genes that cannot by themselves induce autoimmunity on a normal (e.g., CBA/J background), but can cause acceleration of disease on autoimmune-prone backgrounds such as BXSB, NZB, and C57BL/64pr/lpr (5, 9, 10). NZB, MRL + / + , and (NZB x NZW) Fl mice have a number of background genes which contribute to the development of disease (11-13). Although considerable progress is being made in the analysis of these various mice, precise mechanisms of disease induction have not yet been elucidated. Polyclonal B cell activation and autoantibody production is the final common pathway of disease induction in all of these murine models of systemic lupus erythematosus (14-16). All have increased numbers of Ig-secreting cells which are reduced when the X-linked immunodeficiency (xid) gene is introduced (17-20). Though the abnormalities underlying polyclonal B cell activation may be somewhat different in the various strains, they have one common feature: their pluripotent hematopoietic stem cells carry the information for the autoimmune phenotype which is ultimately expressed by more mature cells (13, 21,22). NZB and (NZB x NZW) Fl bone marrow stem cells can transfer disease potential to a nonautoimmune strain following lethal irradiation (23, 24). The BXSB male stem 133 0090-1229/88 $1.50 Copyright All rights

0 1988 by Academic Press, Inc. of reproduction in any form reserved.

134

SCRIBNER

AND

STEINBERG

cells can transfer the phenotype of accelerated disease to female littermates (25). MRL-lpr/lpr stem cell transplants can kill +/+ recipients, either by a GvH-like process (26), or, when the transferred newborn lprllpr spleen is placed under the kidney capsule of a +/+ , through the development of lpr disease without the wasting syndrome (27). In order to study a possible common feature of the autoimmune mice, we analyzed endogenous hematopoietic stem cell colony formation in the spleen (CFU-s) as a measure of stem cell function. Such an assay provides the means to assess stem cell function in a large number of autoimmune mice with different genetic mechanisms. We also questioned whether or not (i) the numbers of CFU-s might correlate with autoimmune disease as has been suggeseted in a preliminary report (28), and (ii) increased stem cell activity, as measured by CFU-s, might precede the manifestations of disease in some or all of the autoimmune mice. The data suggest that increased numbers of CFU-s are associated with the development of autoimmune disease in many strains and crosses, and that in some there is a temporal correlation between the number of CFU-s and the development of autoimmune disease. MATERIALS

AND METHODS

Mice. Nonautoimmune BALB/c, C3H/HeJ, CBA/J, CBA/N, C57BL/6, DBA/2, MRL + / + , and NFS mice were obtained from animal production at the National Institutes of Health or commercial breeders (The Jackson Laboratory, Bar Harbor, ME; Charles River, Wilmington, MA). NZB and (NZB x NZW) Fl mice were maintained and bred in our own production facilities as were BXSB and MRL lprllpr which originally came from The Jackson Laboratory (4,9). Xid mice were created by crossing CBA/N males with the desired end strain and the offspring repeatedly backcrossed to the end strain type while testing for xid markers (low IgM and failure to respond to TNP-ficoll) as previously reported (18). The autosomal recessive lymphoproliferation (lpr) gene was transferred from the original MRL background to C3H and C57BL/6 backgrounds using cross and repeated backcross breedings. The SB/Le-Y chromosome was transferred by crossing BXSB or SB/Le males with females of the appropriate background strain with the resulting males backcrossed to females of the same background strain through greater than 5-10 generations before study (29). The SB/Le-Y and lpr transfers have been maintained in our breeding facilities. CFU-s. Endogenous colony-forming units of the spleen were enumerated using a modification of the original Till and McCulloch protocol (30) as previously described (12). Briefly, the animals were given 650R of y radiation via a Cesium137 Gammacel40 (Atomic Energy of Canada, Ltd.). They were kept on standard mouse chow and water ad libitum for 8-10 days in a laminar flow hood before being killed by cervical dislocation and their spleens removed into Bouin’s fixative. Distinctive raised, pale, round colonies were counted using a dissecting microscope at 5x power. Mortality before 8 days was extremely rare. The numbers reported are the average of a minimum of five animals in a set; variability was very small from animal to animal. There was also very good agreement between

SPLENIC

COLONY-FORMING

UNITS

IN AUTOIMMUNE

135

DISEASE

experiments using mice of the same age, sex, and strain on different occasions separated by as much as 18 months. RESULTS CFU-s in “nonautoimmune” mice. CFU-s levels were determined in multiple strains of mice that are not known to express major autoimmune phenomena. These nonautoimmune mice averaged between 0 and 11 CFU-s at all ages (Table 1). There were no major differences in numbers of CFU-s between males and females in this study. CFU-s in the classically autoimmune NZB and (NZB X NZW) Fl strains. NZB mice had consistently elevated CFU-s at all ages averaging more than 10 times that found in nonautoimmune mice (Table 1, Fig. 1). CFU-s in NZB females lagged somewhat behind those in males until 20 weeks when they reached comparable levels (Table 2). CFU-s in (NZB x NZW) Fl males and females on the other hand, while starting at the same elevated level as the NZB females at 4 weeks, fell rapidly to the level of nonautoimmune strains by 6 weeks of age (Table 2, Fig. 1). The (NZB x NZW) Fl males remained there through 26 weeks. In contrast, CFU-s of the females rebounded to ~100 CFU-s at age 20 weeks, the time of expression of their autoimmune disease. The effect of the SBILe-Y chromosome accelerator factor on CFU-s. BXSB males possess the Y chromosome of the parent SB/Le mouse which carries one or more factors that accelerate autoimmune disease. BXSB females, which do not have the Y chromosome accelerator factor, had low CFU-s levels comparable to other nonautoimmune strains at all ages (Table 3, Fig. 2). BXSB males, however, had small numbers of CFU-s early in life; they developed elevated numbers of CFU-s by 18 weeks of age, averaging 5-10 times the CFU-s of BXSB females after that time. The addition of the SB/Le-Y chromosome accelerating factor to nonauTABLE SPLEENIC

COLONY-FORMING

UNITS

1

IN SEVERAL

INBRED

MOUSE

STRAINS

Weeks Strain”

Sex

BALBIC C3H CBA/J CBA/N C57BLl6 DBAl2 MRL i-l+ NFS

M/F M/F M/F M/F M/F M/F M/F M/F

NZB

M

5-15 Ob 2 5 5 3 5 4 2 >lOO

16-25 0 0 5 6 5 5 11 4 >loo

2+lo 0 ND’ 6 5 5 5 3 2 >lOO

u Greater than five mice per group. Means are shown; standard deviations were within two CFU of the mean. b CFU-s were enumerated g-10 days after 650 R. c Not determined.

136

SCRIBNER

AND

STEINBERG

0

0

10 WEEKS

20

NZB

MALE

3

NZB FEMALE

0

(NZB x NZWi

Fl

MALE

a

INZB

Fl

FEMALE

30

x NZWi

40

OF AGE

FIG. 1. Change in splenic colony-forming units (CFU-s) in NZB and (NZB x NZW) Fl mice at several ages. NZB males have uniformly high numbers of CFU-s at all ages while NZB females have an increase in CFU-s number at 20 weeks of age. Both (NZB x NZW) Fl males and females have a rapid drop in CFU-s number between 3 and 5 weeks and stay low in the males. By 25 weeks CFU-s in the females have risen to the level of NZB mice. Each point is the mean of more than two groups of five mice assayed at separate times.

toimmune strains (e.g., CBA/J, C57BL/6) through cross breeding resulted in no increase in CFU-s in the males. Of note, when the SB/Le-Y chromosome was bred into NZB mice, there was a reduction in CFU-s through 15 weeks of age. The effect of the lpr and gld genes on CFU-s. MRL + I + male and female mice averaged 4-8 CFU-s at all ages between 6 and 20 weeks of age (Table 4, Fig. 3). In MRL-lpr/lpr mice, CFU-s were markedly increased to approximately 30 by 11-15 weeks and ~100 by 20-25 weeks (Table 4). The addition of the SB/Le-Y chromosome accelerator gene to MRL-lprltpr resulted in a still higher level of CFU-s (>lOO) at 6 weeks or about three times the level expected in MRL-fprllpr males (Table 4). The addition of lprllpr to nonautoimmune C3H or C57BL/6 mice did not cause an increase in CFU-s through 25 weeks in spite of detectable autoantibodies and lymphadenopathy. There was, however, a late occurring threeTABLE CFU-s

IN NEW

2

ZEALAND

MICE Weeks

___-NZB

Group”

(NZB x NZW)Fl

Sex

4

5-1s

16-25

24-40

M F

NDb ND

>I00 53 k 8

>I00 >I00

>I00 3~100

M F

55 * 7 42 2 5

17 2 4 8?4

422

41-i

>loo

>loO

0 Greater than five mice per group. Means ir standard deviations are shown. b Not determined.

SPLENIC

COLONY-FORMING

UNITS IN AUTOIMMUNE

2100

0

BXSB

MALE

0

BXSB

FEMALE

MALE

BXSB.xld AND FEMALE

0

“: 3 8

137

DISEASE

50 -

25 -

WEEKS

OF AGE

2. Change in splenic colony-forming units of BXSB and BXSB.xid mice at various ages. BXSB females have low numbers of CFU-s at all time points. BXSB males with the SB/Le-Y chromosome accelerator gene(s) display an increase in CFU-s by 18 weeks of age. This rise is blocked by xid. Each point is the mean of more than two groups of five mice assayed at separate times. FIG.

to fivefold rise in CFU-s noted after 30 weeks in C57BL/6-lpr/lpr mice and an even larger increase in C3H/HeJ-lpr/lpr mice after 40 weeks of age. Similar results are seen with C57BL/6-gld/gld mice (Table 4). The (MRL-fpr/lpr x C57BL/6-lpr/lpr) Fl mice showed an increase in CFU-s over time (Table 5). (MRL-lpr/lpr x C3H-lpr/lpr) Fl females had a marked and early increase in CFU-s reminiscent of MRL-lpr/lpr mice. In these crosses, the females had more CFU-s than male littermates (Table 5). Control crosses of (MRL. +/ + x C3H-lpr/lpr) and the reverse (C3H-lpr/lpr x MRL. +/+) gave expectedly low levels of CFU-s in these lprl + mice. The effect of the xid gene orz CFU-s. CBA/N mice with the xid gene did not differ from CBA/J without the xid gene (Table 1). Similarly, xid had no effect on the relatively low CFU-s of BXSB females (Table 6, Fig. 2), though it did prevent the late increase in CFU-s in BXSB males (Table 6). The xid gene did not prevent the early high CFU-s in NZB mice; late in life NZB. xid mice and a major reduction in CFU-s (Table 6). TABLE 3 EFFECT OF THE SBlLe-Y CHROMOSOME ON CFU-s Weeks Strain”

Sex

5-15

16-25

26-40

BXSB

M F

10 2 3 11 ?4

53 f 11 423

58 Z!Y6 a+2

CBA/J.SB/Le-Y CVBLl6.SB/Le-Y

M M

121 4? 1

3+-l 4*3

a Greater than five mice per group. Means f standard deviation are shown. b Not determined.

NDb ND

138

SCRIBNER AND STEINBERG TABLE OF Iprllpr

EFFECT

4 gldlgld

AND

CFU-s

ON

Weeks Strain”

Sex

MRL +I+ MRL-lprllpr

M/F M F M M F M F M F

MRL-lpr/lpr.SB/Le-Y C5lBLl6-lprllpr C3H-lprilpr C57BL/6-gldlgld

5-15

16-25

Xi-40

:..4t

4-+2

11 -+2

3+-2

ht

30 2 3 28 +- 6

>I00 >I00

>lOO

>I00

>lOO

>I00

ND’ ND

ND ND

7t3 423 6k1

421 5i3

17 -+ 3 20 i 5

621

ND ND

211 25 I

4L1

5x2

8z2

,100

521

ND

523

rlO0

1

ND

ND .-IO0 ND

a Greater than five mice per group. Means t standard deviation are shown, b Not determined.

DISCUSSION The pivitol role of the pluripotent hematopoietic stem cell in autoimmune disease is clear: autoimmune disease can be transferred into a nonautoimmune-prone host through bone marrow or stem cell transplants which suggests that the pluripotent stem cell is suffkient to cause the disease. That is, the autoimmune tendencies are encoded in the cellular genome and are not directly dependent on the milieu of the surrounding tissue in which the stem cells expand. However, >I00

-

75 -

Y 0

MRL

lpr

MALE

0

MRL

lpr

FEMALE

0

MRL

+il+

MALE

n

MRL

+ ‘i

FEMALE

A

10

20

WEEKS FIG. 3. Change MRL- +I+ mice modest increases Each point is the

30

i 40

OF AGE

in splenic colony-forming units MRL- + I + and MRL-lprllpr mice at various ages. have essentially normal numbers of CFU-s before 20 weeks of age. Thereafter, are noted. MRL-lpr/lpr mice have a rapid increase in CFU-s by 12 weeks of age. mean of more than two groups of five mice assayed at separate times.

SPLENIC

COLONY-FORMING

UNITS

IN AUTOIMMUNE

139

DISEASE

TABLE 5 CFU-s IN Fl MICE: EFFECT OF SEX Weeks Cross” (MRL.lpr/lpr

C57BLl6.lprllpr)

X

(MRL.lpr/lpr

x C3H.lprIlpr)

(MRL. + / + X C3H,lpr/lpr) (C3H.lpr/lpr

x

MRL. + / + )

Sex

5-15

16-25

2tio

M F M F M F M F

5&l 10 2 102 37 !I 12 !z 13 + 20 f 18 t

18 * 2 20 ? 3 10 f 3 >lOO 14 -+ 2 42-2 12? 1 422

26 + 2 >lOO >lOO >loa 5’1 9-tl 10 k 2 11 t 1

2 1 5 3 1 1 4

a Greater than five mice per group. Means 2 standard deviation are shown.

whether overexuberant stem cell division is the cause or sine qua non of autoimmune disease is not known. A multitude of new murine autoimmune disorders have been described that covers the gamut from multigenic (e.g., NZB) to single gene (e.g., gld) causation, each with a varied expression depending on the propensity of the background strain to autoimmunity. In this paper we have studied the role of the stem cell number and/or division rate, as measured by the ability of the hematopoietic stem cell pool to react to a massive proliferation stimulus caused by sublethal radiation. Nonautoimmune-prone strains have a very predictable low number of CFU-s while NZB mice have many CFU-s from an early age which correlates with the previous findings of increased antibody production and an increased number of B cells in young NZB mice. This suggested that an early hyperplasia of hematopoietic stem cells played a role in NZB autoimmune disease. In order to determine whether this would hold for autoimmune disease in general, other mouse strains and crosses were compared. BXSB males had many more CFU-s than did their female littermates supporting a correlation between numbers of CFU-s and au-

EFFECT

OF THE xid GENE

ON

TABLE 6 CFU-s: STUDIES ON AUTOIMMUNE

BACKGROUNDS

Weeks Straina NZB NZB . xid BXSB BXSB. xid

Sex

5-15

16-25

26-40

M F M F M F M F

>loo 53 -+ 8 >loo 51 -+4 10 IT 3 11 *4 6+2 10 * 1

>loo >loo >lOO 50 ?I 7 59 Ii 5 4*3 13 2 1 821

>loo >loa 421 NDb 58 f 6 822 12 k 2 623

L(Greater than five mice per group. Means + standard deviation are shown. b Not determined.

140

SCRIBNER

AND

STEINBERG

toimmune disease, though the SB/Le-Y chromosome on the nonautoimmune CBA/J and C57BL/6 strains was not sufficient to cause either an increase in CFU-s or autoimmune phenomena. The development of increased numbers of CFU-s in BXSB males at about 16 weeks of age correlated roughly with the onset of disease in our colony. Similarly, MRL-Eprllpr mice with SB/Le-Y chromosome had both accelerated development of autoimmune disease and accelerated elevation in CFU-s. The analysis of multiple strains of lprllpr mice did not provide a single coherent picture; however, it did provide potentially important insights. The data suggest CFU-s hyperexpansion by itself does not play a primary role in their autoimmune disease. There is an 8- to lo-fold rise in CFU-s in MRL-lpr/lpr mice over MRL- + I+ mice between 5 and 15 weeks of age. By approximately 16 weeks the MRL-lpr/lpr levels are at the level of NZB mice, suggesting an induction of increased stem cell numbers or division by the developing disease process in the MRL mice. However, when Ipr is bred into essentially nonautoimmune C57BL/6 or C3H mice, it becomes clear that stem cell hyperplasia, by itself, is not necessary for Ipr-mediated autoimmune disease, but that stem cells are induced to increased activity by some stress late in Ipr-mediated autoimmune disease. This is further exemplified by the findings in (MRL-lpr/lpr x C3H- or C57BLi 6-lpdlpr) Fl mice where intermediate levels of CFU-s are found at different ages with the ultimate increase to high levels of CFU-s retarded when compared to the MRL-lpr/lpr model. This suggests that background gene interactions also contribute importantly to CFU-s number, perhaps by altering the ability of the FI mouse to make and release the cytokines necessary to induce CFU-s hyperplasia. Xid, on the other hand, might not be expected to influence CFU-s number since it acts to block development of late-maturing B cells and thereby prevents autoantibody expression. As expected, it did not prevent high CFU-s number in either male or female NZB.xid mice. However, in BXSB.xid males where both the Y chromosome-linked accelerator gene and the X chromosome-linked immunodeficiency gene are operative, the expected increase in CFU-s after 16 weeks is blocked in concordance with marked decrease in autoimmune manifestations of the BXSB.xid mouse. This would suggest that in BXSB mice the presence of autoantibodies or an autoantibody-mediated disease is able to induce CFU-s hyperplasia either directly or indirectly through cellular damage or cytokine release. Alternatively, it is possible that in BXSB mice, as in NZB mice (12), a single gene underlies the increase in CFU-s and that in the process of breeding the BXSB.xid strain that gene was lost. Of interest, the prototype of murine autoimmune disease, the (NZB x NZW) Fl mouse, is a cross between an intermediate and a high CFU-s strain and has iow levels of CFU-s until 16 weeks when they suddenly increase in the female, but not in the male. This sudden spurt in CFU-s occurs just prior to the accelerated production of IgG antibodies to native DNA in these mice, a process which is maximal between 51/2 and 7% months of age. Therefore, the data are consistent with a large increase in CFU-s in (NZB x NZW) FI mice just prior to major disease. However, the data are also consistent with a process which separately accelerates CFU-s and IgG anti-DNA production. It is even possible that minor

SPLENIC

COLONY-FORMING

UNITS IN AUTOIMMUNE

DISEASE

141

immune complex disease activity at 3% to 4 months of age might lead to the increase in CFU-s before the major disease manifestations appear. This last formulation would put the increase in CFU-s into a positive feedback cycle such that increased antibodies lead to increased factors which lead to increased CFU-s and then a further increase in antibodies. This study would suggest that increased numbers of CFU-s are characteristic of all of the autoimmune mouse strains and that they are an early event in the NZB model of autoimmune disease. Their increase may more frequently be a secondary phenomenon in some of the other strains, perhaps related to factors released by the autoimmune process. The induced stem cell hyperplasia might thereby act to amplify and markedly worsen the ongoing preexisting autoimmune phenomena by supplying increased numbers of lymphocytes and their products. Whether or not stem cell hyperplasia, in and of itself, is necessary for the maximal manifestations of autoimmune disease is still an open question. The strong correlations between increased CFU-s and autoimmune disease suggest that this is a strong possibility. REFERENCES 1. Bumet, F. M., and Holmes, M. C., The natural history of the NZB/NZW Fl hybrid mouse: A laboratory model of systemic lupus erythematosis. Australas. Ann. Med.14, 185, 1965. 2. Andrews, B. S., Eisenberg, R. A., Theofilopoulos, A. N., Izui, S., Wilson, C. B., McConahey, P. .I., Murphy, E. D., Roths, .I. B., and Dixon, F. J.. Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains. J. Exp. Med. 148, 1198-1215, 1978. 3. Scribner, C. L., Seldin, M. F., Santoro, T. J., Smith, H. R., and Steinberg, A. D., Studies of the genetic bases of murine lupus. Mendalian analyses of background genes, accelerating genes, inducing genes, and modifying genes. In “New Horizons in Animal Models of Autoimmune Disease” (M. Kyogoku, and H. Wigzell, Eds.). Academic Press, Tokyo, 1987. 4. Murphy, E. D., and Roths, J. B., Mouse News/. 58, 51, 1978. 5. Roths, J. B., Murphy, E. D., and Either, E. M., A new mutation, gld, that produces lymphoproliferation and autoimmunity in C3H/HeJ mice. J. Exp. Med. 159, l-20, 1984. 6. Davidson, W. F., Dumont, F. J., Bedigan, H. G., Fowlkes. B. J., and Morse, H. C., III: Phenotypic, functional, and molecular genetic comparisons of the abnormal lymphoid cells of C3Hlprllpr and C3H-gld/gld mice. J. Immunol. 136, 4075-4084, 1986. 7. Shultz, L. D., and Green, M. C., Motheaten, an immuno-deficient mutant of the mouse. II. Depressed immune competence and elevated serum immunoglobulins. J. Zmmunol. 116,936946, 1976. 8. Davidson, W. F., Morse, H. C., III, Sharrow, S. 0.. and Chused, T. M., Phenotypic and functional effects of the motheaten gene on murine B and T lymphocytes. J. Immunol. 122, 884-895, 1979. 9. Murphy, E. D., and Roths, J. B., A Y-chromosome associated factor in strain BXSB producing accelerated autoimmunity and lymphoproliferation. Arthritis Rheum. 22, 1188-l 194, 1978. 10. Steinberg, R. T., Miller, M. L., and Steinberg, A. D., Effect of the BXSB Y chromosome accelerating gene on autoantibody production. Clin. Immunol. Immunopathol. 35, 67-72, 1985. 11. Raveche, E. S., Novotny, E. A., Hansen, C. T., Tjio, J. H., and Steinberg, A. D., Genetic studies in NZB mice. V. Recombinant inbred lines demonstrate that separate genes control autoimmune phenotype. J. Exp. Med. 153, 1187-1197, 1981. 12. Miller, M. L.. Raveche, E. S., Laskin, C. A.. Klinman, D. M., and Steinberg, A. D., Genetic Studies in NZB Mice. VI. Association of autoimmune traits in recombinant inbred lines. J. Immunol. 133, 1325-1331, 1984. 13. Raveche, E. S., Chused, T. M., Steinberg, A. D., Laskin, C. A., Edison. L. J., and Tjio, J. H.. Comparison of response to stem cell differentiation signals between normal and autoimmune mouse strains. J. Immunol. 134, 865-871, 1985.

142

SCRIBNER AND STEINBERG

14. Theolilopoulos, A. N., and Dixon, F. J.. Etiopathogenesis of murine SLE. lmmunol. ReLt. 55, 179-216, 1981. 15. Smith, H. R., Cellular abnormalities. Systemic lupus erythematosus: lnsights from animal models. Ann. Intern. Med. 100, 714-727, 1984. 16. Kincade, P. W., Lee, G., Fernandes. G., Moore, M. A. S., Williams, N.. and Good, R., Abnormalities in clonable B lymphocytes and myeloid progenitors in autoimmune NZB mice. Proc. Natl. Acad. Sci. USA 76, 3464-3468. 1979. 17. Amsbaugh, D. F., Hansen, C. T.. Prescott, B., Stashak, P. W., Barthold, D. R., and Baker, P. J., Genetic control of the antibody response to type-111 pneumococcal polysaccharide in mice. 1. Evidence that an X-linked gene plays a decisive role in determining responsiveness. J. Exp. Med. 136, 931-943, 1972. 18. Steinberg, B. J., Smathers, P. A., Frederiksen, K.. and Steinberg, A. D.. Ability of the xid gene to prevent autoimmunity in (NZB x NZW) Fl mice during the course of their natural history, after polyclonal stimulation, or following immunization with DNA. /. C/in. Invest. 70, 587-597, 1982. 19. Scher, I., Steinberg, A. D.. Beming, A. K., and Paul, W. E., X-linked B lymphocyte immune defect in CBA/N mice. II. Studies of the mechanisms underlying the immune defect. J. Exp. Med. 142, 637-650, 1975. 20. Scher, I., Sharrow, S. 0.. Paul. W. E., X-linked B lymphocyte defect in CBA/N mice. 111. Abnormal development of B lymphocyte populations defined by their density of surface immunoglobulins. J. Exp. Med. 144, 507-519, 1976. 21. Raveche, E. S.. Laskin, C., Rubin, C., Tjio, J. H., and Steinberg, A. D., Comparison of stem cell recovery in autoimmune and normal strains. Cell. Immunol. 79, 56-67. 1983. 22. Yoshida, S., Dorshkind, K., and Gershwin, M. C., Hemopoietic cell dysfunction in murine lupus. Clin. Exp. Rheum. 5, 79-87, 1987. 23. Morton. J. L., Siegel, B. V., and Moore, R. D., Transplantation of autoimmune potential. Il. Glomerulonephritis in lethally irradiated DBA/Z recipients of NZB bone marrow cells. Transpkuntation 19, 464-469, 1975. 24. Morton, J. L., and Siegel, B. V., Transplantation of autoimmune potential. IV. Reversal of the NZB autoimmune syndrome by bone marrow transplantation. Transplantation 27, 133-134. 1979. 25. Jyonouclii, H., Kincade, P. W., and Good, R. A., Age-dependent changes in B lymphocyte lineage cell populations of autoimmune-prone BXSB mice. J. Immunol. 134, 858-864, 1985. 26. Theofilopoulos, A. N., Balderas, R. S., Gozes, Y.. Aguado, M. L., Hang, L. M., Morrow, P. R.. and Dixon, F. J.. Association of the /pr gene with a graft-vs-host disease-like syndrome. J. Exp. Med. 162, l-18, 1985. 27. Smathers, P. A., Santoro, T. J.. Chused, T. M., Reeves, J. P., and Steinberg. A. D.. Studies of lymphoproliferation in MRL-lpr/lpr mice. J. Immunol. 133, 1955-1961, 1984. 28. Izui, S., Kelley, V. E., Masuda, K., Yoshida, H., Roths, J. B., and Murphy, E. D.. Induction of various autoantibodies by mutant gene Ipr in several strains of mice. J, fmmunol. 133, 227-233. 1984. 29. Hudgins, C. C.. Steinberg, R. T., Klinman, D. M., and Steinberg, A. D., Studies of consomic mice bearing the Y chromosome of the BXSB mouse. J. Immunoi. 134, 3849-3854. 1985. 30. Till, J. E., and McCulloch, E. A., Early repair processes in marrow cells irradiated and proliferating in vivo. Radiat. Res. 18, 96-105, 1963. Received December 1, 1987; accepted with revision April 4. 1988