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Increased Severity of Murine Lupus in Female Mice Is Due to Enhanced Expansion of Pathogenic T Cells ¹

Thomas J. Lang^2,*, Phuong Nguyen^*, John C. Papadimitriou and Charles S. Via^*

^* Research Service, Baltimore Veterans Affairs Medical Center, and Division of Rheumatology and Clinical Immunology, and Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201

	Abstract

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A strong female predominance is a well-recognized feature of human lupus. The mechanism by which sex influences disease expression and severity is not fully understood. To address this question, we used the parent-into-F₁ (pF₁) model of chronic graft-vs-host disease (cGVHD) in which lupus-like humoral autoimmunity and renal disease are induced in normal F₁ mice. An advantage of this model is that the pathogenic T cells driving disease (donor strain) can be studied separately from nonspecifically activated T cells (host strain). We observed that lupus-like disease using female donor and host mice (fF cGVHD) is characterized by more severe long-term disease (glomerulonephritis) than with male donor and host (mM cGVHD). Interestingly, differences in disease parameters could be seen at 2 wk after parental cell transfer, as evidenced by a 2- to 3-fold greater engraftment of donor CD4⁺ T cells in fF cGVHD mice, which persisted throughout disease course. Enhanced engraftment of donor CD4⁺ T cells in fF cGVHD mice was not due to differences in splenic homing, alloreactive precursor frequency, initial proliferation rates, or apoptotic rates, but rather to sustained high proliferation rates during wk 2 of disease compared with mM cGVHD mice. Crossover studies (mF, fM) demonstrated that enhanced donor CD4⁺ T cell proliferation and engraftment segregate with the sex of the host. These results demonstrate that the sex of the recipient can influence the expansion of pathogenic T cells, thus increasing long-term the burden of autoreactive T cells and resulting in greater disease severity.

	Introduction

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Systemic lupus erythematosus is an autoimmune disease characterized by T cell-dependent B cell hyperactivity, resulting in autoantibody formation and immune complex glomerulonephritis (1, 2, 3). A striking and unexplained feature of human lupus is a female predominance approaching 90% of cases, especially within the 15- to 64-year-old age group (4). These data strongly suggest a role for female sex hormones in lupus pathogenesis. Because sex hormone manipulation can alter the severity of murine lupus (reviewed in Refs.5, 6, 7), it has been thought that hormones may act as modulators of disease expression in systemic lupus erythematosus (SLE) ³ (8). Studies in murine lupus, particularly the New Zealand Black/White (NZB/W) model, have supported this hypothesis. For example, female NZB/W mice exhibit earlier onset of disease, more rapid progression, and earlier mortality compared with males (9, 10, 11). Additionally, disease is accelerated with administration of estrogenic compounds in both castrated males and females (11, 12). In contrast, androgenic hormones are protective in NZB/W mice (11, 13, 14). Treatment with flutamide, an androgen receptor antagonist, accelerates mortality in females (15) and castration accelerates disease in males (12).

The mechanisms by which sex influences disease expression in murine lupus are not fully understood. A major difficulty in the study of spontaneous murine lupus models such as NZB/W mice is the inability to identify and study the pathogenic T cells that drive disease expression. To this end, we have used an induced murine model of lupus, the parent-into-F₁ (pF₁) model of chronic graft-vs-host disease (cGVHD) in which the pathogenic T cells driving disease expression can be identified and studied separately from nonspecifically activated T cells. In this model, normal F₁ mice develop a lupus-like disease following the i.v. transfer of homozygous parental strain CD4⁺ T cells (16). Previously, it has been reported that multiple transfers of parental strain donor cells result in lupus-like renal disease that is more severe in females (17). In the present study, we demonstrate that a single transfer of parental DBA splenocytes into BDF₁ mice results in lupus-like renal disease that is more severe in females, and that the increased disease severity is mediated by enhanced donor CD4⁺ T cell expansion in female hosts during the second week of disease.

	Materials and Methods

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Mice

Six- to 8-wk-old female and male DBA/2J (DBA) (H-2^d) and B6D2F₁ (BDF₁) (H-2^b/d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

Induction of cGVHD

Single cell suspensions of DBA splenocytes were prepared, and the number of CD4⁺ T cells was quantitated by flow cytometry before injection. For all experiments, cGVHD was induced by injecting unfractionated splenocytes containing 10–12 x 10⁶ DBA CD4⁺ T cells into the tail vein of recipient BDF₁ mice. Control mice were age- and sex-matched uninjected F₁ mice. These controls are phenotypically identical with syngeneic controls (F₁F₁), as neither develops features of cGVHD, such as renal disease (18).

Assessment of glomerulonephritis in cGVHD

Mice were sacrificed 10 wk after parental cell transfer, and kidneys were fixed in phosphate-buffered Formalin, paraffin embedded, and stained routinely with H&E. All slides were scored blindly by a renal pathologist (J.C.P.). Glomerular capillary membrane thickness and crescents were semiquantitatively graded using the following scale: 0 = normal; 1⁺ = mild; 2⁺ = moderate; 3⁺ = severe. A glomerular cellular score was also determined in which perivascular cell infiltrates and mesangial cell numbers for each group were quantitated by counting these features in 10 fields per section and calculating the group mean ± SEM (19). Proteinuria was measured by urine dipstick and reported on a scale of 0–4⁺ (Albustix; Bayer, Elkhart, IN).

Flow cytometric analysis

Single cell suspensions of splenocytes were first incubated with anti-murine FcR mAb 2.4G2 (20), then stained with saturating concentration of either FITC-conjugated, PE-conjugated, or biotin-conjugated anti-CD4, anti-CD8, anti-B220, and anti-H-2K^b (BD PharMingen, San Diego, CA). Three-color flow cytometry was performed using a FACScan flow cytometer (BD Immunocytometry Division, San Jose, CA.). Lymphocytes were gated based on forward and side scatter and analyzed using CellQuest acquisition and analysis software. Ten thousand events were collected per sample. Donor CD4⁺ and CD8⁺ T cells were identified as staining positively for CD4 or CD8 and negatively for H-2K^b, whereas host T cells stain positively for H-2K^b.

Serologic assays

Serum was tested by ELISA for the presence of anti-ssDNA IgG Abs, as described (21). Briefly, microtiter plates were coated with heat-denatured salmon sperm DNA, blocked with 2% BSA-PBS, and incubated with serial dilutions of mouse serum beginning at a dilution of 1/40. Wells were washed and incubated with anti-mouse IgG alkaline phosphatase conjugate (Southern Biotechnology Associates, Birmingham, AL). OD was determined at 405 nm. MRL/lpr sera was assayed as a standard, and arbitrary units were calculated assuming that serum diluted 1/40 contains 100 U anti-ssDNA Ab/ml.

Splenic homing, proliferation, and alloreactive precursor frequency of donor CD4⁺ T cells

CFSE (Molecular Probes, Eugene, OR) labeling of donor splenocytes and analysis of donor cell proliferation by flow cytometry were performed, as previously described (22, 23). For homing studies, mice were sacrificed at 24 h after transfer of CFSE-labeled donor cells. For donor CD4⁺ T cell proliferation and alloreactive precursor frequency studies, mice were sacrificed at 72 h after parental cell transfer.

5-bromo-2'-bromodeoxyuridine (BrdU) incorporation

On days 7, 10, or 14 after donor cell transfer, mice received two doses of 0.8 mg BrdU i.p. separated by a 2-h interval. Two hours after the second dose, mice were sacrificed and splenocytes were stained with anti-CD4 PE and anti-H-2K^b CyChrome (BD PharMingen). BrdU-positive cells were identified using BrdU Flow Kit (BD PharMingen). Proliferating donor T cells were defined as staining positively for BrdU and CD4, but negatively for H-2K^b.

Determination of apoptotic donor CD4⁺ T cells

Splenocytes from cGVHD mice at day 7 or 10 were stained for CD4 and H-2K^b, as described above, then stained by either TUNEL (Apo-Direct), annexin V (Apo-annexin V), or anti-activated caspase 3 Ab (all from BD PharMingen), according to the manufacturer’s instructions.

In vitro MLR

MLRs were performed in round-bottom 96-well microtiter plates. Normal male and female DBA responders were plated at 4 x 10⁵ splenocytes per well. Stimulators consisted of day 7 mM or fF cGVHD splenocytes irradiated with 3000 rad and plated at 1 x 10⁵ or 2 x 10⁵ cells/well. Cells were pulsed with [³H]thymidine 12 h before harvesting at either 48 or 60 h.

Statistical analysis

Data were examined for normality and equal variance (Kolmogorov-Smirnov). If satisfactory, groups were compared by two-tailed Student’s t test.

	Results

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A single injection of 80 x 10⁶ DBA splenocytes results in more severe lupus-like disease in fF than mM cGVHD

Previous work has reported that four sequential injections of 30 x 10⁶ DBA splenocytes administered every 3 days into BDF₁ mice resulted in lupus-like renal disease that is more severe in females than in males (17). Lupus-like renal disease can also be induced in BDF₁ mice following a single injection of 80–100 x 10⁶ DBA splenocytes (19, 24). To determine whether lupus-like cGVHD induced by a single dose of parental spleen cells is more severe in females, we compared disease outcome seen when both donors and hosts were female (fF cGVHD) with that when both donors and hosts were male (mM cGVHD). Renal histology performed at 10 wk after donor cell transfer (Fig. 1) demonstrates that fF cGVHD mice exhibit more severe glomerulonephritis (Fig. 1, C and D) compared with either mM cGHVD mice (Fig. 1B), normal female F₁ mice (Fig. 1A), or normal male F₁ mice (data not shown). Specifically, fF cGVHD mice exhibited thickened capillary loops, mesangial proliferation, sclerosis, and glomerular enlargement. Severity of hyalinosis and crescent formation in fF cGVHD ranged from 1⁺ to 3⁺, while in mM cGVHD, it was 0 to 1⁺. Moreover, fF cGVHD kidneys exhibited greater glomerular cellularity (normal male or female F₁ = 25 ± 3 cells/glomerulus; fF = 40 ± 3.7 cells/glomerulus; mM: 28 ± 1 cells/glomerulus; p < 0.05, fF vs mM). Only a female glomerulus is shown in Fig. 1, as histologic scoring of normal male and female glomeruli did not differ.

View larger version (192K): [in this window] [in a new window]   FIGURE 1. Glomerulonephritis in fF cGVHD is more severe than in mM cGVHD mice. cGVHD was induced, as described in Materials and Methods, and animals were sacrificed at 10 wk of disease (n = 5 mice/group). Representative glomeruli are shown in the following panels at x40 magnification: A, normal female F1; B, mM cGVHD; C and D, fF cGVHD (two different animals). Shown in C and D are capillary loop thickening (thin arrowhead), mesangial hyperproliferation (thick arrowhead), and mesangial matrix deposition (*). Similar results were seen in a separate independent experiment.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. FF cGVHD mice exhibit increased levels of anti-ssDNA Ab and increased proteinuria. fF or mM cGVHD was induced as described, and controls consisted of age-matched uninjected male or female F1 mice (n = 5 mice/group). A, Proteinuria was determined as described, and results are shown for individual mice at the indicated time points after GVHD induction. Similar results were seen in a separate independent experiment (*, p < 0.05). B, Mice were bled at the indicated times, and anti-ssDNA IgG Ab levels were determined by ELISA. Results are shown as mean arbitrary U/ml ± SEM; *, p < 0.05.

Increased disease severity in fF cGVHD mice is associated with increased engraftment of donor CD4⁺ T cells and expansion of host B cells at 2 wk after donor cell transfer

FF cGVHD exhibit an increase in serum anti-ssDNA Ab compared with mM cGVHD mice as early as 4 wk after disease induction (Fig. 2B). Although the presence of anti-dsDNA Ab is more specific for SLE renal disease in both humans and mice, anti-dsDNA Ab in cGVHD mice are typically not detected until 4 wk of cGVHD and occur in as few as 40% of cGVHD mice (25). The explanation for this delay is not clear and may reflect more complex processes such as epitope spreading or stochastic events. By contrast, increased anti-ssDNA Ab is a reliable marker of alloantigen-driven, polyclonal B cell hyperactivity and is observed in nearly 100% of cGVHD mice as early as 10 days after disease induction (26). Moreover, for donor inocula 10⁸ cells, the level of anti-ssDNA Ab correlates with the magnitude of the allogeneic T cell response in that the injection of greater numbers of donor T cells results in higher levels of IgG anti-ssDNA Ab at 2 wk (24). To determine whether the elevated anti-ssDNA in fF cGVHD mice reflect greater T and/or B cell expansion, flow cytometric analysis of donor and host lymphocyte subsets was performed 2 and 10 wk after disease induction. At 2 wk, both mM and fF cGVHD mice exhibit the characteristic increase in host B cells compared with control F₁ mice (Table I); however, the increase in splenic B cell numbers is significantly greater for females (140% increase over control F₁) than for males (60% increase over control F₁) (p < 0.01, fF vs mM). A more striking difference was seen for donor CD4⁺ T cell engraftment with fF cGVHD mice exhibiting 2- to 3-fold greater engraftment of donor CD4⁺ T cells compared with mM cGVHD mice, despite the injection of equal numbers of CD4⁺ T cells. Because cGVHD is a CD4⁺ T cell-driven disease (16, 26, 27), these results suggest that enhanced expansion of B cells and autoantibody production in fF cGVHD mice is a consequence of enhanced CD4⁺ T cell engraftment and expansion (24, 28). Moreover, the increased numbers of donor CD4⁺ T cells and host B cells seen for fF cGVHD relative to mM cGVHD are still observed at 10 wk of disease, indicating that early differences in engraftment and expansion of donor cells are long lasting.

View this table: [in this window] [in a new window]   Table I. Expansion of donor CD4+ T cell and host B cells is greater in fF vs mM cGVHD mice at both 2 and 10 wk of disease

Enhanced engraftment of donor CD4⁺ T cells in fF cGVHD is not due to increased splenic homing

Sex-dependent differences in T cell homing have been previously reported in an animal model of drug-induced SLE (29). To determine whether differential splenic homing of donor cells in cGVHD contributed to enhanced donor CD4⁺ T cell engraftment in fF cGVHD, the presence of donor T cells in host spleens was determined within 24 h after the injection of unfractionated CFSE-labeled splenocytes containing equivalent numbers of donor CD4⁺ T cells (12 x 10⁶/mouse). CFSE-stained female and male donor CD4⁺ T cells display a single peak of fluorescence, indicating that donor cells have not undergone cell division at 24 h after cell transfer (Fig. 3, A and B). Thus, the number of donor cells in both groups at 24 h postinjection reflects splenic homing alone and does not differ significantly between fF (1.96 ± 0.15 x 10⁶) and mM (2.06 ± 0.10 x 10⁶) cGVHD mice (Fig. 3C).

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Splenic homing of donor CD4+ T cells is equivalent in mM and fF cGVHD. cGVHD was induced as described using DBA male or female splenocytes labeled with CFSE. Host spleens were evaluated by flow cytometry 24 h postinjection. Representative tracings are shown for A, fF, or B, mM cGVHD mice. Shown in C are the total number of engrafted donor CD4+ T cells at 24 h for fF or mM cGVHD (p = NS, n = 8 mice/group).

Enhanced engraftment of donor CD4⁺ T cells in fF cGVHD is not due to increased frequency of alloreactive precursors

To address whether sex differences in alloreactive precursor frequency contribute to differences in donor CD4⁺ T cell engraftment, CFSE-labeled donor splenocytes were injected into F₁ mice, and the donor CD4⁺ T cells were examined at 3–4 days postinjection by flow cytometry (23). Representative donor CD4⁺ T cell CFSE-staining profiles at 72 h postinjection are shown in Fig. 4, A and B, for a single fF and mM cGVHD mouse, respectively. There was no significant difference in the calculated DBA CD4⁺ anti-F₁ precursor frequency between males and females (Fig. 4C) (males = 3.1 ± 0.3%; female = 3.7 ± 0.5%; p = 0.11; n = 6/group).

View larger version (55K): [in this window] [in a new window]   FIGURE 4. fF and mM cGVHD mice exhibit similar donor CD4+ T cell alloreactive precursor frequencies. cGVHD was induced using CFSE-labeled DBA splenocytes, as described for Fig. 3, and mice were assessed at 72 h. Representative CFSE staining of donor CD4+ T cells is shown for A, fF cGVHD, and B, mM cGVHD mice. C, Donor CD4+ alloreactive precursor frequencies were calculated individually as described and shown as the group means ± SEM. D, Cells that have undergone n cell divisions are shown as a percentage of total donor CD4+ T cells.

Enhanced engraftment of donor CD4⁺ T cells in fF cGVHD is due to greater proliferation during the second week of disease

CFSE-labeled donor cells were used to determine whether differential proliferation rates could account for the enhanced donor CD4⁺ T cell engraftment in fF cGVHD (23). At 72 h, donor CD4⁺ T cells in both mM and fF cGVHD mice have undergone a maximum of five cell divisions, indicating similar rates of division during this time period (Fig. 4, A and B). Moreover, the rate of cell division, as reflected in the percentage of total donor CD4⁺ T cells that have undergone one, two, three, four, or five cell divisions, did not differ significantly between mM and fF cGVHD (Fig. 4D).

Because CFSE staining is not useful for determining proliferation rates beyond 4–5 days of disease, BrdU pulse labeling was used to estimate proliferative rates at days 7, 10, and 14 after parental cell transfer. Increased donor CD4⁺ T cell engraftment in fF cGVHD is not detected at day 7, but by day 10 a >2-fold increase is seen compared with mM cGVHD (Table II). Additionally, both the percentage and absolute number of donor CD4⁺ T cells incorporating BrdU were significantly greater at days 7 and 10 in fF compared with mM cGVHD. However, by day 14, BrdU incorporation in fF cGVHD declines to a level comparable to mM cGVHD. Thus, the increased engraftment of donor CD4⁺ T cells observed at day 14 for fF cGVHD (Tables I and II) can be seen as early as day 10 after parental cell transfer and reflects the prolonged female donor CD4⁺ T cell proliferation during wk 2 of disease.

View this table: [in this window] [in a new window]   Table II. Increased donor CD4+ T cell proliferation in fF cGVHD mice during week 2 of GVHDa

View larger version (41K): [in this window] [in a new window]   FIGURE 5. Apoptotic rates of donor CD4+ T cells are similar in fF and mM cGVHD. cGVHD was induced as described, and apoptosis of donor CD4+ T cells was assessed ex vivo by A, Annexin V at day 7, or B, TUNEL staining at day 10. Results represent the group mean of the percentage of apoptotic donor CD4+ T cells ± SEM (n = 5/group). Control F1 mice in A are pooled male and female F1, which individually were not significantly different.

To determine whether enhanced donor CD4⁺ T cell expansion segregates with the sex of the donor or the host, female and male donor splenocytes were injected into either the same or the opposite sex BDF₁ hosts. Donor CD4⁺ T cell engraftment in fF cGVHD is significantly greater at 2 wk of disease compared with mM cGVHD (Fig. 6), consistent with the data in Tables I and II. Of note, engraftment of donor CD4⁺ T cells in mF cGVHD mice is significantly increased compared with mM cGVHD (p < 0.001), and did not differ significantly from that of fF cGVHD (p = NS). In contrast, donor CD4⁺ T cell engraftment in fM cGVHD was significantly reduced compared with fF cGVHD (p < 0.001), and was comparable to that seen in mM cGVHD (p = NS). In a separate experiment, BrdU incorporation into female donor CD4⁺ T cells at day 10 was significantly increased when injected into female hosts as compared with male hosts (fF = 11.1% ± 1.1 vs fM = 6.7 ± 1.0; p < 0.05). These results indicate that it is the sex of the host that influences donor CD4⁺ T cell proliferation and engraftment in cGVHD.

View larger version (47K): [in this window] [in a new window]   FIGURE 6. Enhanced engraftment of donor CD4+ T cells in cGVHD mice correlates with the sex of the host. cGVHD was induced as described, and donor CD4+ T cell engraftment was determined by flow cytometry at day 14. Female and male DBA donor splenocytes were injected into either male or female hosts. Results represent the group mean ± SEM x 10-6 (n = 5 mice/group). *, p < 0.001, fF vs fM cGVHD, and mM vs mF cGVHD.

It is possible that the foregoing sex-dependent differences in T cell proliferation reflect differences in the ability of host APCs (including B cells) to drive donor CD4⁺ T cell proliferation. Based on the data presented in Table II demonstrating that differential donor T cell proliferation is not observed until day 7 of disease, we tested the APC function of day 7 cGVHD splenocytes in a MLR. Splenocytes from either fF or mM cGVHD mice were obtained at day 7 of disease and used as stimulators for naive parental male or female DBA responder cells in an in vitro MLR. There was no significant difference in the incorporation of [³H]thymidine by responder DBA T cells (male or female) stimulated with either male or female cGVHD splenocytes (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrate that lupus-like disease induced using female donor and host mice is more severe than that seen using male donor and host mice. Long-term outcome variables such as proteinuria and renal pathology are worse in fF than in mM cGVHD. Importantly, differences in disease severity can be observed as early as 2 wk after parental cell transfer. Serum anti-ssDNA Ab titers, donor CD4⁺ T cell engraftment, and host B cell expansion are significantly increased in fF cGVHD mice compared with mM cGVHD mice and reflect greater donor CD4⁺ T cell proliferation during the second week of disease in fF cGVHD. Specifically, donor CD4⁺ T cell homing, alloreactive precursor frequency, and proliferation during the first week after cell transfer do not differ between mM and fF cGVHD mice. However, during the second week of disease, donor CD4⁺ T cell proliferation declines in mM cGVHD mice, but is sustained in fF cGVHD mice. The relatively longer period of proliferation in fF cGVHD mice results in a 2- to 3-fold greater engraftment of donor CD4⁺ T cells by the end of the second week of disease compared with mM cGVHD mice. This difference in the absolute number of donor CD4⁺ T cells is maintained throughout the observed disease course. Thus, relative differences in the duration of donor CD4⁺ T cell proliferation early in disease have long-term effects on the total burden of persisting pathogenic T cells, which in turn influence long-term disease severity.

Crossover experiments (mF and fM) clearly demonstrate that enhanced donor CD4⁺ T cell proliferation and engraftment are determined by the sex of the host. The exact nature of the host mechanism(s) that promotes donor CD4⁺ T cell expansion in female hosts is not understood at present and is most likely complex. Our results demonstrating that differential donor CD4⁺ T cell expansion is not seen until after day 7 of disease argue that the initial host APC function (DC and macrophages) is equivalent. However, it has been previously shown that by day 7 of disease, host B cells are significantly activated in cGVHD mice (30). The APC function of activated host B cells could contribute to the differential expansion of donor CD4⁺ T cells during wk 2 of disease. However, in experiments designed to compare the relative capacity of day 7 cGVHD splenocytes to drive proliferation of naive T cells, we were unable to demonstrate a difference between fF and mM cGVHD day 7 splenocytes in vitro.

The dependence of enhanced donor CD4⁺ T cell proliferation on the sex of the host suggests a role for sex-specific host-derived factors, such as sex hormones (i.e., estrogen, androgen, and prolactin). Sex hormones have been previously studied for their ability to modulate not only normal immune responses, but also autoimmune responses (6, 7). Specifically, lupus-like disease in NZB/W mice is significantly worse in females as measured by autoantibody titers, glomerulonephritis, and mortality (9, 10, 11). Castration causes acceleration of disease (13), while treatment of ovarectomized females with androgen reduced disease severity (11). Additionally, estrogen treatment of mice transgenic for an anti-DNA IgH inhibits development of tolerance and enhances bcl-2 expression in naive B cells (31). Prolactin also accelerates disease in NZB/W mice (32) and promotes expansion of Ag-specific T cells (33). Similarly, sex hormones are most likely important in the cGVHD model of lupus, as ovarectomy has been reported to reduce both autoantibody levels and proteinuria (34). The exact mechanisms by which sex hormones influence disease expression in murine lupus are not fully explained. The potential immunomodulatory effects of sex hormones in cGVHD are complex, and further studies are required to understand their role in modulating donor CD4⁺ T cell expansion in the cGVHD model.

In conclusion, we have demonstrated that long-term lupus-like disease in the pF₁ model is significantly influenced by the sex of the host. Moreover, increased disease severity in female cGVHD mice is due to a greater long-term burden of pathogenic CD4⁺ T cells that becomes apparent as early as 7–10 days of disease. These results suggest that strategies to reduce T cell expansion early in the course of human lupus may have long-term therapeutic effects.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI47466 and Department of Veterans Affairs Merit Review Grant (to C.S.V.). T.J.L. is supported by a Department of Veterans Affairs Career Development Award.

² Address correspondence and reprint requests to Dr. Thomas J. Lang, Division of Rheumatology and Clinical Immunology, MSTF 8-34, 10 S. Pine Street, University of Maryland School of Medicine, Baltimore, MD 21201. E-mail address: tlang001{at}umaryland.edu

³ Abbreviations used in this paper: SLE, systemic lupus erythematosus; 5-bromo-2'-deoxyuridine; cGVHD, chronic graft-vs-host disease.

Received for publication April 1, 2003. Accepted for publication September 17, 2003.

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