Alterations in peripheral B cells and B cell progenitors following ...

International Immunology, Vol. 13, No. 4, pp. 553–558

© 2001 The Japanese Society for Immunology

Alterations in peripheral B cells and B cell progenitors following androgen ablation in mice Thomas M. Ellis, Michael T. Moser, Phong T. Le, Robert C. Flanigan and Eugene D. Kwon Departments of Medicine, Urology, and Cell Biology, Neurobiology and Anatomy, Loyola University School of Medicine, 2160 South First Avenue Maywood, IL 60153, USA Keywords: androgen ablation, B cell, B cell progenitor

Abstract The production of B lymphocytes is regulated in part by physiologic levels of androgens and estrogens. While these sex hormones down-regulate B lymphopoiesis, augmentation of B lymphopoiesis occurs under conditions where androgen or estrogen levels are decreased. In this study we examine the effect of androgen ablation of male mice on B lymphopoiesis and on the phenotypic composition of peripheral B lymphocyte populations. Spleen and thymic weights are significantly increased following castration, as is the total number of peripheral blood lymphocytes. However, the absolute numbers of B cells in the periphery are selectively increased following castration; the numbers of T cells, NK cells and granulocytes remain unchanged. The increase in circulating B cells is due largely to increases in the numbers of recent bone marrow emigrants expressing a B220lo⍣CD24hi⍣ phenotype and these cells remain significantly elevated in castrated mice for up to 54 days post-castration. Similar increases in the percentages of newly emigrated B cells are observed in mice that lack a functional androgen receptor (Tfm). Finally, assessments of B cell progenitors in the bone marrow revealed significant increases in the relative numbers of IL-7-responsive B cell progenitors, including cells in Hardy fractions B (early pro-B cells), C (late pro-B cells), D (pre-B cells) and E (immature B cells). These findings demonstrate that androgen ablation following castration significantly and selectively alters the composition of peripheral B cells in mice. Further, these alterations result from the potentiating effects of androgen ablation on IL-7-responsive pro-B cell progenitors. Introduction Androgens and estrogens exert potent regulatory influences over the immune system, although the full nature of these effects and the mechanisms underlying hormone-induced changes in host immunity are poorly understood. Both humoral and cell-mediated responses are affected, as demonstrated by the ability of estrogen administration to enhance antibody responses and depress cell-mediated responses as well as by the potentiation of cell-mediated functions following castration of male or female mice (1–4). The increased incidence of autoimmune disorders in females further suggests a role for sex hormones in the regulation of lymphocyte function and autoreactivity (5). Several observations indicate that sex hormones serve as important regulators of B and T lymphopoiesis. Thymic

involution that occurs during puberty is associated with the onset of sex hormone production and can be delayed by castration prior to puberty (1,2). Castration of mice after puberty reverses thymic involution and leads to thymic hypertrophy, a process that can be reversed by replacement of androgen or estrogen (1,2). More recent evidence indicates that the expression of progesterone receptors by thymic stromal cells is required for normal fertility and that T cell development during pregnancy is inhibited by a progesterone-dependent blockade in T lymphopoiesis at the early CD3–CD44⫹CD25⫹ stage of development (6). There is considerable evidence that B lymphopoiesis is negatively regulated by steroid sex hormones. Estrogen is a potent inhibitor of stromal cell-dependent B cell lymphopoiesis

Correspondence to: T. M. Ellis Transmitting editor: J. P. Allison

Received 5 September 2000, accepted 8 January 2001

554 Effects of castration on B lymphopoiesis in vitro, and pregnancy is associated with a reduction in B lymphopoiesis and the numbers of IL-7-sensitive precursors (7–9). Likewise, treatment of mice with dihydrotestosterone leads to a reduction in the numbers of IL-7-responsive B cell precursors (10). Conversely, loss of androgen production or function results in significant increases in B lymphopoiesis and in the numbers of peripheral B cells, as shown in castrated mice, mice bearing mutations in the androgen receptor (AR) (Tfm) and in mice possessing genetic deficiencies in gonadal steroidogenesis (hgp) (11,12). Smithson et al. showed that sex hormones target an IL-7-responsive B progenitor cell, corresponding to either the pro- or pre-B cell stage (7,11). However, the exact stage of B cell differentiation that is affected by the loss of sex hormone production or function and the consequences of such on peripheral B cell population are unknown (11,12). These studies were undertaken to identify the stage of B cell development that is affected by androgen ablation of male mice and to assess the effects of androgen ablation on the composition of circulating B cells. We show that castration selectively increases the numbers of peripheral B cells in part by increasing the numbers of newly emigrating immature B cells from the bone marrow. Further, castration affects B lymphopoiesis at the early pro-B cell level, which represents the earliest identifiable IL-7-responsive B cell progenitor.

marrow from each femur was removed by flushing with 1 ml RPMI 1640 (using a 26 gauge needle and a 1 cm3 syringe) into a single well of a 24-well culture plate (Costar, Corning, NY).

Methods

Cell staining and FACS analysis

Mice C57BL/6 males (4–6 weeks of age) were purchased (Taconic, Germantown, NY) for use in this study. Animals were acclimated on site for at least 4 weeks prior to the initiation of the study and were 10–12 weeks of age at the start of each experiment. Male Tfm mice and wild-type controls [Ta(6J)⫹/Y] were purchased from the Jackson Laboratory (Bar Harbor, ME) and were used at 12–16 weeks of age. All studies were conducted according to NIH guidelines for the proper use of animals and with the approval of the IACUC at Loyola University Medical Center. Castration Surgeries were performed under isofluorane anesthesia (IsoFlo; Abbott, IL) using sterile instruments and gloves within a laminar flow bio-cabinet. A transverse scrotal incision was made, testicles exposed, ligated and removed (sham: unligated testicles returned to the scrotum). The scrotal incision was then closed using a single skin clip. Animals were placed in a clean cage and monitored until fully recovered from the effects of anesthesia. Harvesting of tissues Peripheral blood was collected via cardiac puncture using a heparinized 3 cm3 syringe with a 26 gauge needle on mice anesthetized with isofluorane (IsoFlo) on day 7, 14 or 28 postsurgery. The thymus and spleen were dissected, cleaned of facia and adipose tissue, and their weights were recorded. Organ size is represented as an organ index, which is the organ/body weight ratio. The femur was dissected free from the muscle, cut both at the knee and hip, and removed. Bone

Cell isolation Mononuclear cells were collected by centrifugation over Lympholyte-M (Cedarlane, Hornby, Ontario, Canada), and were washed 3 times in HBSS without Ca2⫹ and Mg2⫹ (Life Technologies, Rockville, MD) prior to staining. Splenocytes and thymocytes were isolated by gentle dissection with 19 gauge needles followed by centrifugation over a LympholyteM gradient. Mononuclear cells from bone marrow were isolated by centrifugation over Lympholyte-M and cell preparations were washed 3 times in cold PBS containing 5% newborn calf serum prior to use. Antibodies Fluorochrome-conjugated antibodies for use in immunofluorescence analysis were purchased from BD Biosciences/ PharMingen (San Diego, CA) and included PerCP– or phycoerythrin (PE)–anti-CD3, CyChrome–anti-CD45, FITC–antiCD44, FITC–anti-heat stable antigen (CD24), biotinylatedanti-CD43, PE–anti-BP-1 and allophycocyanin–avidin. PEconjugated anti-mouse IgM was obtained from Jackson ImmunoResearch (West Grove, PA).

For each staining sample, 1–5⫻106 cells were incubated on ice for 30 min with saturating amounts of antibody. Cells were washed with ice-cold PBS containing 5% newborn calf serum. Flow cytometric analyses were performed using a dual-laser FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). The instrument was calibrated daily using bead standards (CaliBRITE; Becton Dickinson) and FACSComp software (Becton Dickinson). For four-color analyses of bone marrow, compensation and PMT settings were optimized manually using bone marrow cells stained singly with each antibody used for analysis. For analysis of spleen and blood cell populations, gates were set to include those cells that were viable by forward and right angle scatter profiles, and were CD45⫹. At least 20,000 gated events were collected and analyzed. For flow cytometric analysis of bone marrow, 50,000 events were collected with gates set to include large and small lymphocytes as defined by forward and side scatter signals. Results were analyzed using CellQuest software (Becton Dickinson) and positive populations were defined as those events that fell beyond a marker set to include 95% of control-stained cells. On those occasions in which markers fell within the negative peak, and where visual resolution of positive and negative peaks was clear-cut, markers were adjusted manually to the natural breakpoint between positive and negative peaks. Bone marrow B cell progenitors were classified according to the scheme of Hardy (13), as follows: Fraction A (pre/pro-B cells; CD45R⫹CD43⫹CD24–IgM–), Fraction B (pro-B cells; CD45R⫹CD43⫹ CD24⫹IgM–), Fraction C (pre-B cells; CD45R⫹CD43⫹ CD24hi⫹IgM–), Fraction D (small pre-B cells; CD45R⫹CD43–CD24⫹IgM–), Fraction E (immature B cells; CD45Rlo⫹CD43–CD24–IgM⫹) and Fraction F (mature B cells; CD45Rhi⫹CD43–CD24–IgM⫹).

Effects of castration on B lymphopoiesis 555 Table 1. Comparison of spleen and thymus organ size and peripheral leukocyte counts in castrated and sham-operated mice

Shamc Castratedc

Organ index (⫻10–3)a

Cells/ml (⫻106)b

Thymus

Spleen

Lymphocytes

Granulocytes

B cells

T cells

NK cells

1.3 ⫾ 0.1 2.8 ⫾ 0.1d

2.8 ⫾ 0.2 3.5 ⫾ 0.1d

3.4 ⫾ 0.3 6.5 ⫾ 1.5e

0.8 ⫾ 0.1 0.8 ⫾ 0.1

2.3 ⫾ 0.6 4.0 ⫾ 0.6e

0.9 ⫾ 0.1 0.8 ⫾ 0.2

0.3 ⫾ 0.0 0.2 ⫾ 0.0

an ⫽ 16 each for sham and control bn ⫽ 10 each for sham and control cAnalyzed 14 days post-surgery. dSignificantly eSignificantly

groups. groups.

different from sham controls at P ⬍ 0.01. different from sham controls at P ⬍ 0.05.

Statistical analysis Differences between mean values were assessed for statistical significance using two-sided Students t-tests. Results Effect of surgical castration on peripheral lymphocytes Ablation of sex hormone production following castration of male mice or ovarectomy of female mice leads to increased T and B lymphopoiesis, and elevated numbers of circulating lymphocytes. Experiments were performed to determine the effects of castration of male mice on the composition of peripheral lymphocyte populations. Consistent with previous findings, spleen and thymus weights were significantly increased in castrated animals 14 days following castration, compared to animals undergoing sham surgery (Table 1) (14,15). Increased organ weights were observed as late as 54 days post-castration surgery, the latest time point examined (not shown). Thus, the increase in thymus and spleen size after castration was stable, and did not simply reflect acute, short-term changes restricted to the early post-castration period. Castration-induced increases in thymus and spleen weights were accompanied by a significant increase in the number of peripheral blood leukocytes compared to sham controls. Total peripheral blood leukocyte counts increased on day 14 following castration to a mean of 7.1 ⫾ 1.9⫻106 compared to 4.1 ⫾ 0.5⫻106 cells/ml for sham controls (P ⬍ 0.05). Significant increases were observed as early as day 7 and as late as day 54 following castration (data not shown). Analysis of specific leukocyte populations showed that the increase is restricted entirely to the B lymphocyte population, as the absolute number of peripheral blood B220⫹ cells increased 75.6 ⫾ 8.1% over levels of B220⫹ cells in sham controls (Table 1). The numbers of circulating T cells (7.8 ⫾ 1.6⫻105 versus 7.5 ⫾ 1.1⫻105 cells/ml), NK cells (1.8 ⫾ 0.3⫻105 versus 1.5 ⫾ 0.2⫻105 cells/ml) and granulocytes (0.6 ⫾ 0.1⫻106 versus 0.7 ⫾ 0.1⫻106 cells/ml) did not differ between shams and castrates respectively. Similar changes in B and T cells seen in peripheral blood were also observed in the spleen (not shown). Thus, castration induces significant and selective increases in the B cell population, but does not affect the numbers of peripheral T cells, NK cells or granulocytes.

Fig. 1. Probability (20%) flow cytometric contour plots depicting elevated numbers of newly emigrated B cells (B220lo⫹CD24hi⫹) in castrated mice, sham-operated control mice, Tfm mice and Tfm-trait bearing Ta(6J)⫹/Y controls. With the exception of Tfm and Ta(6J)⫹/Y mice, these profiles are from animals assayed 14 days post-surgery and are representative of profiles obtained from at least four animals in each group.

This implies that the castration-induced peripheral leukocyte expansion does not reflect a general increase in bone marrow leukopoiesis nor does it involve T and NK cells. Castration induces increased numbers of immature B cells (B220lo⫹CD24hi⫹) in the periphery The increase in peripheral B lymphocytes following castration suggests either an increase in the numbers of mature lymphocytes and/or increased emigration of newly formed B cells from the bone marrow. Newly emigrated B cells exhibit a B220lo⫹CD24hi⫹ phenotype and normally comprise 10–15% of the adult peripheral (splenic) B cell pool (16). Phenotypic analyses of B220⫹ lymphocytes from blood and spleens of mice collected 14 days after castration or mock surgery showed an increase in the fraction of B220⫹ B cells that express the B220lo⫹CD24hi⫹ phenotype of newly emigrated, immature B cells (Figs 1 and 2). The absolute numbers of both newly emigrated (B220lo⫹CD24hi⫹) and mature (B220hi⫹CD24lo⫹) B cells were significantly increased in peripheral blood of castrated mice (Table 2), although the increase in the numbers of newly emigrated B cells in blood

556 Effects of castration on B lymphopoiesis

Fig. 2. Composition of peripheral blood B cells in castrated and sham-operated animals. Bars represent the mean percentage of total B220⫹ B cells in spleen and peripheral blood of castrated (n ⫽ 10) and sham-operated (n ⫽ 10) animals, as well as the relative mean fraction of newly emigrated B cells (B220lo⫹CD24hi⫹) and mature B cells (B220hi⫹CD24lo⫹) within the peripheral B220⫹ population.

Table 2. Comparison of the absolute numbers of mature B cells in blood of castrated and sham-operated mice Cells/ml (⫻10–6)a

Shamb Castratedb

B220lo⫹CD24hi⫹

B220hi⫹CD24lo⫹

0.4 ⫾ 0.1 1.2 ⫾ 0.3c

1.7 ⫾ 0.3 3.8 ⫾ 0.3c

an ⫽ 10 each for sham and castrate groups. bAnalyzed 14 days post-surgery. cSignificantly different from sham controls at P

⬍ 0.001

was proportionally higher than the increase in mature B cells when compared to sham controls (750 versus 131%; Fig. 2). B220lo⫹CD24hi⫹ cell numbers remained significantly elevated even at the latest time point examined (54 days post-castration), thereby suggesting that the effects of castration on this population are somewhat stable (not shown). Thus, although both mature and new emigrants contribute to the increase in peripheral B cells following castration, the increase in new emigrants suggests that the effects of castration on peripheral B cell numbers might be due, at least in part, to increased B cell output from the marrow. Mice with testicular feminization syndrome (Tfm) possess a point mutation in the X-linked AR gene and thus are unresponsive to androgen (10,17). If the effects of castration on B lymphopoiesis reflect the direct activities of androgen on bone marrow, then we expect to see similar changes in Tfm mice. Similar to castrated wild-type animals, Tfm mice exhibit elevated percentages of B220lo⫹CD24hi⫹ cells in the blood (Fig. 1) when compared to trait-carrying male controls [Ta(6J)⫹/Y]. This indicates that the loss of androgen responsiveness alone is sufficient to promote the appearance of new B cell emigrants. Thus, reductions in androgen brought about either by decreases in androgen production or by impaired receptor function both lead to stable increases in the numbers of peripheral B cells by increasing the numbers of newly emigrated immature B cells from the bone marrow.

Fig. 3. Relative abundance of B cell progenitors in bone marrow of castrated and sham-operated mice. Cells were analyzed by flow cytometry on day 14 post-surgery. Values represent the mean ⫾ SEM from six mice from each treatment group and asterisks indicate significant changes from sham-operated controls.

Effects of castration on B lymphopoiesis in bone marrow Either the loss of sex hormone production or AR function lead to increases in B lymphopoietic activity by increasing IL-7responsive, B220⫹CD24⫹ B cell progenitors in the bone marrow (7,10,11). However, the specific progenitor stage of B cell differentiation affected by androgen ablation has not been identified. To determine this, bone marrow was collected from castrated or surgical control animals 14 days after surgery, and the cells were subjected to immunofluorescence and flow cytometry to resolve subpopulations of progenitor B cells at various stages of differentiation using the Hardy classification scheme (13). The percentages of pre-pro B cells (B220⫹CD24lo⫹CD43⫹; Fraction A), the earliest identifiable B cell progenitor, were decreased following castration and the percentages of early pro-B cells (Fraction B) were unchanged (Fig. 3). Although the percentages of pre-pro B cells (Fraction A) were decreased, their absolute numbers were not significantly affected by castration (Table 3). However, significant increases were observed following castration in both the relative and absolute numbers of the early pro-B cell population (Fraction B) and all subsequent stages of B cell development, with the greatest increase occurring in the late pro-B cell stage (Fraction C; Table 3). Thus, castration affects B lymphopoiesis at the early pro-B cell stage, which also represents the earliest fraction of IL-7-responsive cells (13). These findings indicate that androgen sensitivity is directed at the initiation of IL-7-responsive lineages, and leads to significant increases in downstream B cell progenitors and in the numbers of peripheral mature B cells. Discussion In this study we have characterized the effects of castration of male mice on the composition of circulating B lymphocytes and also identified the B cell progenitor populations affected by androgen ablation. Previous studies documented that physiologic levels of sex hormones exert a powerful inhibitory effect on B lymphopoiesis (7–11). However, the mechanism by which sex hormones mediate their effects and

Effects of castration on B lymphopoiesis 557 Table 3. Comparison of the absolute numbers of B cell progenitor populations in bone marrow of castrated and shamoperated mice Bone marrow fraction (no. per bone ⫻10–5)a

Shamb Castratedb

A (pre/pro-B)

B (early pro-B)

C (late pro-B)

D (pre-B)

E (immature B)

1.1 ⫾ 0.2 0.9 ⫾ 0.2d

1.5 ⫾ 0.1 2.4 ⫾ 0.3e

1.5 ⫾ 0.4 4.5 ⫾ 0.7c

12.1 ⫾ 3.1 21.3 ⫾ 5.2c

7.0 ⫾ 2.3 15.0 ⫾ 4.6f

an ⫽ 6 each for sham and castrate bAnalyzed 14 days post-surgery. cSignificantly

groups.

different from sham controls at P ⬍ 0.001.

dNot significantly different from sham controls. eSignificantly different from sham controls at 0.001 fSignificantly

⬍ P ⬍ 0.01. different from sham controls at P ⬍ 0.05.

the consequences on the composition of the peripheral lymphocyte pool are not completely established. Previous studies showed that castration increases lymphocyte numbers in both central (thymus and bone marrow) and peripheral lymphoid organs, leading to significant increases in thymic and splenic mass (14,15). Here we confirm and extend these observations by demonstrating that the increases in splenic cellularity and peripheral blood lymphocytes are due largely to significant increases in newly formed B cells, although the numbers of mature B cells are also increased. Despite an increase in thymic size and cellularity, the numbers of circulating T and NK cells are unaffected by castration. Further, we demonstrate that the numbers of circulating granulocytes are unaffected by androgen ablation. Thus, castration effects are specific for T and B lymphopoiesis; there is no detectable effect on granulopoiesis. Previous investigators have shown that MAC-1 positive cells in bone marrow and spleen are unaltered in genetically hypogonadal female (hpg) mice or in AR-deficient Tfm mice, providing further support for the lymphocyte specificity of the castration effect (7). The increase in circulating B cells following castration reflects the appearance of large numbers of newly emigrated immature (B220lo⫹CD24hi⫹) and mature B cells (B220hi⫹CD24lo⫹) in the periphery, although the relative increase was considerably greater in the newly emigrated population. These new emigrants normally constitute 10–15% of the adult B cell pool, whereas in castrated animals they represent up to 45% of the total circulating B220⫹ B cell pool (16,18). Similar increases in newly emigrated B cells are observed in Tfm mice, which possess a mutation in the AR that ablates responsiveness to this hormone (17). Thus, a loss of androgen production or function can singly increase B lymphopoiesis and peripheral emigration, although it is still unclear whether androgens mediate this effect directly or indirectly. Also, evidence indicates that the immature B cell population undergoes extensive selection in the periphery and that only 30% of these cells that reach the spleen become mature B cells (18,19). Thus, the increase in B220lo⫹CD24hi⫹ cells observed following castration might also reflect effects of androgen ablation on normal peripheral deletion of this subset, although this possibility was not directly addressed in these studies. An increase in B220lo⫹CD24hi⫹ cells of

this magnitude may be functionally significant, given the observation that B220lo⫹CD24hi⫹ cells differ from their more mature counterparts in that they are refractory to stimulation by anti-IgM antibodies, while still maintaining the ability to generate primary antibody responses if provided with T cell help (16,18). The earliest detectable effect of castration on B lymphopoiesis occurs at the early pro-B cell level, with the greatest relative increase appearing in the late pro-B cell population (Fraction C). These populations correspond to the stages of B cell differentiation that are responsive to IL-7, a cytokine produced by bone marrow stromal cells, and required for B cell proliferation and differentiation (20). In vitro B cell differentiation cultures suggest that the inhibitory effects of estrogen on B cell lymphopoiesis are mediated through bone marrow stromal cells. However, given the expression of AR on both bone marrow stromal cell lines and pro-B cell lines, the possibility still exists that androgen ablation exerts direct effects on both stem cells and B progenitors (12). Hormonemediated inhibition of in vitro differentiation of pre-B cells in vitro occurred even in the presence of exogenous IL-7, indicating that although the effect is directed at IL-7responsive B cell progenitors, IL-7 is not the primary target of the hormonal effect. Interestingly, IL-7 is also required for T cell development in the thymus, another organ under the regulation of sex hormones (20). Although IL-7-responsive B cell progenitors increase following estrogen or androgen withdrawal, this effect appears to be indirect since IL-7responsive precursors are unaffected by direct estrogen exposure in vitro (7). Estrogens have been shown to reduce IL-6 production by stromal cells in vitro, suggesting that sex hormones might also affect IL-7 production (21). Although effects on IL-7 production by stromal cells cannot be ruled out, recent observations suggest the inhibitory effects of sex hormones on B lymphopoiesis might be through steroidinduced production of negative regulators of B lymphopoiesis (7). This is further supported by our own observations that dihydrotestosterone does not affect IL-7 production by murine bone marrow stromal cell lines (unpublished observation). It is noteworthy that, despite a significant increase in the cellularity of both bone marrow and thymus after castration, only B cells are significantly increased in the periphery. This difference might reflect the more open architecture of bone

558 Effects of castration on B lymphopoiesis marrow, yet also suggest the existence of tight regulatory controls over T cell emigration from the thymus. Although castration clearly affects B cell output in mice, treatment of mice with progesterone and estrogen does not significantly affect peripheral T or B cell output, despite the ability of this treatment to induce both thymic involution and reduce B lymphopoiesis (6). Thus, the numbers of B cells in male mice are tightly controlled by physiologic levels of androgen. Ablation of sex hormone function or production constitutes a common therapeutic strategy for the treatment of hormone responsive tumors, including prostate and breast cancer. Previous evidence suggests that androgen ablation therapy of prostate cancer patients induces elevations in circulating lymphocytes, a change which is associated with a more favorable prognosis (22). Thus, further understanding of therapy-induced alterations in the underlying composition of circulating lymphocytes may be important to the development and to the optimal application of immunotherapy in these diseases. Studies are in progress to determine whether similar affects of anti-androgen therapy on peripheral lymphocyte composition and function occur in humans. Acknowledgements This work was supported in part by grant CA82185 from the NCI (E. D. K.), grant PC991568 from the Department of Defense (E. D. K.) and a grant from the Potts Foundation (T. M. E.).

Abbreviation PE

phycoerythrin

References 1 Eidinger, D. and Garrett, T. J. 1972. Studies of the regulatory effects of the sex hormones on antibody formation and stem cell differentiation. J. Exp. Med. 136:1098. 2 Tartakovsky, B., De Baetselier, P., Feldman, M. and Segal, S. 1981. Sex-associated differences in the immune response against fetal major histocompatibility antigens. Transplantation 32:395. 3 Ainbender, E., Weisinger, R. B., Hevizy, M. and Hodes, M. L. 1968. Difference in the immunoglobulin class of polio antibody in the serum of men and women. J. Immunol. 101:92. 4 Graff, R. J., Lappe, M. A. and Snell, G. D. 1969. The influence of the gonads and adrenal glands on the immune response to skin grafts. Transplantation 7:105. 5 Smith, H. R. and Steinberg, A. D. 1983. Autoimmunity—a perspective. Annu. Rev. Immunol. 1:175. 6 Tibbetts, T. A., DeMayo, F., Rich, S., Conneely, O. M. and O’Malley, B. W. 1999. Progesterone receptors in the thymus are required for thymic involution during pregnancy and for normal fertility. Proc. Natl Acad. Sci. USA 96:12021.

7 Smithson, G., Beamer, W. G., Ponting, I. and Kincade, P. W. 1995. Estrogen suppresses stromal cell-dependent lymphopoiesis in culture. J. Immunol. 155:3409. 8 Medina, K. L., Smithson, G. and Kincade, P. W. 1993. Suppression of B lymphopoiesis during normal pregnancy. J. Exp. Med. 178:1507. 9 Medina, K. L. and Kincade, P. W. 1994. Pregnancy-related steroids are potential regulators of B lymphopoiesis. Proc. Natl Acad. Sci. USA 91:5382. 10 Smithson, G., Couse, J. F., Lubahn, D. B., Korach, K. S. and Kincade, P. W. 1998. The role of estrogen receptors and androgen receptors in sex steroid regulation of B lymphopoiesis. J. Immunol. 161:27. 11 Smithson, G., Beamer, W. G., Shultz, K. L., Christianson, S. W., Shultz, L. D. and Kincade, P. W. 1994. Increased B lymphopoiesis in genetically sex-steroid-deficient hypogonadal (hpg) mice. J. Exp. Med. 180:717. 12 Viselli, S. M., Reese, K. R., Fan, J., Kovacs, W. J. and Olsen, N. J. 1997. Androgens alter B cell development in normal mice. Cell. Immunol. 182:99. 13 Hardy, R. R., Carmack, C. E., Shinton, S. A., Kemp, J. D. and Hayakawa, K. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213-? 14 Olsen, N. J., Watson, M. B., Henderson, G. S. and Kovacs, W. J. 1991. Androgen deprivation induces phenotypic and functional changes in the thymus of adult male mice. Endocrinology 129:2471. 15 Viselli, S. M., Stanziale, S., Shults, K., Kovacs, W. J. and Olsen, N. J. 1995. Castration alters peripheral immune function in normal male mice. Immunology 84:337. 16 Allman, D. M., Ferguson, S. E. and Cancro, M. P. 1992. Peripheral B cell maturation. I. Immature peripheral B cells in adults are heat-stable antigenhi and exhibit unique signaling characteristics. J. Immunol. 149:2533. 17 Charest, N. J., Zhou, Z. X., Lubahn, D. B., Olsen, K. L., Wilson, E. M. and French, F. S. 1991. A frame shift mutation destabilizes androgen receptor messenger RNA in the Tfm mouse. Mol. Endocrinol. 5:573. 18 Allman, D. M., Ferguson, S. E., Lentz, V. M. and Cancro, M. P. 1993. Peripheral B cell maturation. II. Heat stable antigenhi splenic B cells are an immature developmental intermediate in the production of long-lived marrow-derived B cells. J. Immunol. 151:4431. 19 Levine, M. H., Haberman, A. M., Sant’Angelo, D. B., Hannum, L. G., Cancro, M. P., Janeway, C. A. and Shlomchik, M. J. 2000. A B-cell receptor-specific selection step governs immature to mature B cell differentiation. Proc. Natl Acad. Sci. USA 97:2743. 20 Grabstein, K. H., Waldschmidt, T. J., Finkelman, F. D., Hess, B. W., Alpert, A. R., Boiani, N. E., Namen, A. E. and Morrissey, P. J. 1993. Inhibition of murine B and T lymphopoiesis in vivo by an anti-IL-7 monoclonal antibody. J. Exp. Med. 178:257. 21 Girasole, G., Jilka, R. L., Passeri, G., Boswell, S., Boder, G., Williams, D. C. and Manolagas, S. C. 1992. 17β-estradiol inhibits interleukin-6 production by bone marrow-derived stromal cells and osteoblasts in vitro: a potential mechanism for the antiosteoporotic effect of estrogens. J. Clin. Invest. 89:883. 22 Oliver, R. T. D. and Gallagher, C. J. G. 1998. Intermittent endocrine therapy and its potential for chemoprevention of prostate cancer. Cancer Surv. 23:191.