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The Journal of Immunology, 2008, 181, 2399 -2406
Copyright © 2008 by The American Association of Immunologists, Inc.
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Homeostatic Proliferation in the Mice with Germline FoxP3 Mutation and its Contribution to Fatal Autoimmunity1

Xing Chang*, Pan Zheng*,{dagger} and Yang Liu2,*,{ddagger}

* Division of Immunotherapy, Section of General Surgery, Department of Surgery, {dagger} Department of Pathology, and {ddagger} Division of Molecular Medicine and Genetics, Department of Internal Medicine and Pathology, Comprehensive Cancer Center and Program of Molecular Mechanisms of Disease, University of Michigan Medical Center, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
FoxP3 has emerged as a critical regulator for the development and function of regulatory T cells. Recent studies by several groups have demonstrated that FoxP3 is expressed outside T cell lineages. In this context, we have reported that germline mutation of FoxP3 caused defective thymopoiesis, although its potential contribution to autoimmune diseases has not been analyzed. In this study, we report that, during perinatal period, germline mutation of FoxP3 in scurfy mice caused lymphopenia in the spleen and massive homeostatic proliferation, characterized by the independence from cognate Ags and expression of bona fide markers for homeostatic proliferation. The homeostatic proliferation is suppressed by increases in T cell numbers but not by adoptive transfer of regulatory T cells (Treg). Adoptive transfer of Treg-containing bulk T cells was dramatically more effective than transfer of either Treg alone or Treg-depleted CD4 T cells in curing the scurfy mice. Our data demonstrated that FoxP3 mutation not only ablates Treg, but also dramatically increased homeostatic proliferation during the perinatal period. Homeostatic proliferation acts in concert with Treg defects in causing acute and fatal autoimmune diseases in the FoxP3 mutant mice. These results demonstrated that germline mutation of FoxP3 caused two defects that work in concert to cause lethal autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Paradoxically, autoimmune disease and immune deficiency, which are usually regarded as two opposite extremes of immune response, can coexist in one patient (1, 2). This paradox can be resolved by the homeostatic proliferation associated with lymphopenia, as best illustrated by the spontaneous proliferation and expansion of naive T cells after the transfer into a T cell-deficient host (3, 4). With less intensity, homeostatic proliferation is also observed in newborns as a physiological response (5). Compared with other mechanisms of T cell proliferation, homeostatic proliferation has some unique attributes which could facilitate destructive autoimmunity. During this process, T cells are stimulated by low affinity self Ags rather than cognate Ags (4, 6) and gain features of memory T cells (7, 8). As such, homeostatic proliferation may prime naive T cells of broad specificities, possibly including the otherwise quiescent autoreactive T cells. More recent studies suggest that homeostatic proliferation overcomes T cell tolerance (9, 10, 11). A causal connection between homeostatic proliferation and autoimmunity in the NOD mice has also been proposed (12).

Regulatory T cells (Tregs)3 have recently emerged as a major regulator of autoimmune disease. Defective development and function of Tregs have been observed in various animal models and human patients (13). FoxP3 mutations result in lethal autoimmune diseases in mouse and man (14, 15, 16, 17). Because the FoxP3 gene is essential for the development and/or function of Tregs (18, 19, 20), and because the deletion of the gene by the CD4-Cre has led to lethal autoimmune disease (21), it has been suggested that the absence of Tregs in the scurfy mice is solely responsible for deadly autoimmune diseases (18, 21). More recently, in support of the notion of essential role for Treg throughout the lifespan, it was reported that deletion of FoxP3-expressing cells cause acute lethal inflammation in the immune competent adult mice (22), although this finding has been disputed by another group (23). However, given our observation of FoxP3 expression in epithelial cells in mammary gland, thymus (24, 25), and lung (26), it is unclear whether the cellular ablation is restricted to the Treg lineage. Expression of the FoxP3 gene in lung epithelial cells may well contribute to the lethal inflammation when high doses of diphtheria toxin was used to treat the mice in which the diphtheria toxin receptor were knocked into the FoxP3 locus. In fact, a careful examination of the immunohistochemical analysis of published data (23) indicated that in both mouse thymus and spleen, expression of neither FoxP3 nor the knocked in diphtheria toxin receptor is restricted to mature T cells. Meanwhile, expression of FoxP3 in TCR-negative cells was reported in human thymus (27).

The attribution of all phenotypes in the scurfy mice to a Treg defect is not supported by early studies that showed that bone marrow from the FoxP3 mutant mice failed to reconstitute autoimmune diseases in lethally irradiated syngeneic hosts (28). Likewise, we showed that T-depleted scurfy bone marrow did not transfer lethal autoimmune diseases into RAG-1-deficient hosts (24). However, Komatsu and Hori (29) reported that chimera consisting of bone marrow from the scurfy mice were short lived. The difference between these analyses remains to be resolved.

Given the reported synergy between lymphopenia and Treg defects in the development of autoimmune diseases (30), it is intriguing whether the severe autoimmune diseases in the scurfy mice and immune dysregulation, polyendocrinopathy, X-linked syndrome (IPEX) patients are due to combined abnormalities in Treg and homeostatic proliferation. In support of this notion, we observed massive but transient homeostatic proliferation in the scurfy mice with a germline mutation of FoxP3 and lethal autoimmune diseases. Restoration of cellularity using T cells from wild type (WT) mice is more effective than transfer of Treg alone in curing the scurfy mice. Our data demonstrate that, in addition to the Treg defects, FoxP3 mutation causes homeostatic proliferation, and that the two defects likely act in concert to cause acute and fatal autoimmune diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Thy1.1 BALB/c mice with a mutation of FoxP3 (FoxP3sf) were produced after more than 12 generations of backcrossing at the University of North Carolina. B6 FoxP3sf/+ mice were purchased from The Jackson Laboratory and were bred with WT B6. The genotype of the scurfy mice was determined through allele-specific PCR as described (24). B6 OT-1 (31), BALB/c DO11.10, and B6 Rag–/– mice were purchased from The Jackson Laboratory. B6. OT-1 Rag–/– mice were purchased from Taconic Farms. All studies involving experimental animals have been approved by Laboratory Animal Use and Care Committee of The Ohio State University and the University of Michigan.

BrdU incorporation and measurements

Mice were injected i.p. with nucleotide analog BrdU (1 mg/mouse in 100 µl PBS) 3 h before being sacrificed. Splenocytes and lymph node cells were prepared and BrdU incorporation was detected by flow cytometry with a BrdU Flow Kit in conjunction with other cell surface markers, as described by the manufacturer (BD Biosciences).

Abs and flow cytometry

Single cell suspensions of thymuses, spleens, or lymph nodes were prepared and first blocked with anti-FcR (2.4G2) to eliminate Fc-mediated nonspecific bindings. For cell surface staining, the samples were stained with Abs on ice for 30 min in staining buffer and were fixed by 1% paraformaldehyde. Intracellular staining of FoxP3 was performed as described by the manufacturer (eBioscience). The following Abs were used: PerCp cy5.5 conjugated anti-CD4 and anti-CD8 (BD Biosciences), allophycocyanin-conjugated anti-CD4, anti-CD8, anti-Thy1.1, and anti-Thy1.2, PE-conjugated anti-CD25 (PC61), anti-CD69, anti-CD44, anti-Foxp3 (FJK-16), allophycocyanin-Alexa Fluor 750 conjugated anti-CD4, and anti-CD8 (eBioscience).

Cell purification and adoptive transfer

To purify the OT-1 T cells, the spleens and lymph node cells from 6- to 8-wk-old B6 OT-1 mice were first incubated with anti-FcR (2.4 G2), anti-CD4 (GK1.5), anti-CD11b (MAC-1), anti-B220, and N418 (anti-CD11c) Abs. The Ab-coated cells were then depleted with anti-Rat IgG-coated magnetic beads (Dynal, Invitrogen). The purified cells were then labeled with 2.5 µM CFSE at 37°C in the presence of 0.1% FBS. Unlabeled CFSE was then washed away using 5% RPMI 1640 three times. Similar procedure was also used to purify DO11.10 CD4 T cells using anti-CD8 (T1B210).

To purify the CD4+CD25+ cells, CD4+ T cells were first purified using the Dynal beads to remove non-CD4 cells and then the CD25+CD4+ T cells were further purified using the MACS beads. In brief, the spleens and lymph node cells from 6 to 8 wk old BALB/C or B6 Thy1.1 mice were first incubated with anti-FcR (2.4 G2), anti-CD8 (2.4.3), anti-CD11b (MAC-1), anti-B220, and N418 (anti-CD11c) Abs. The Ab-coated cells were then depleted with anti-Rat IgG-coated magnetic beads (Dynal, Invitrogen). Purified CD4 T cells were stained with anti-CD25 PE followed by anti-PE MACS beads (Miltenyi Biotec), and the CD4+CD25+ cells were then positively selected using MACS LS columns. The purity of the CD4+CD25+ cells was routinely around 92 to 95%. One million purified CD4+CD25+ cells were resuspended in serum-free RPMI 1640 and i.v injected into 2–3 days old syngeneic scurfy mice and their WT littermates.

Bone marrow chimera

Bone marrow cells after T cell depletion were isolated from either 15-day-old CD45.1+Foxp3sf mice or CD45.1+Foxp3wt mice and mixed with the bone marrow of 10-wk-old WT B6 mice (CD45.2+) at a 1:1 ratio. The mixed bone marrow was then i.v. transferred into B6.Rag-1–/– mice, which received 500 rad {gamma}-irradiation 1 day before the transplantation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Reduced cellularity in splenic T cells correlates with T cell hyperproliferation in newborn scurfy mice

We systematically investigated the possibility that the severe autoimmune diseases observed in scurfy mice and IPEX patients may be due to homeostatic proliferation that works in concert with Treg defect. Because homeostatic proliferation is normally driven by lymphopenia, we first compared the percentages and absolute numbers of both CD4 and CD8 T cells in the spleens of scurfy mice to those in their WT littermates (Fig. 1, a and b). In the 2- to 3-day-old mice, the number and percentage of CD4 and CD8 T cells were similar between the two groups. However, from days 6 to 16, the scurfy mice had fewer CD4 T cells in the spleen than the WT mice in regards to both percentage and absolute number. The maximum difference was reached at day 10 after birth, when the number of CD4 T cells in the scurfy spleen was less than 1/2 of their WT counterpart. By the third week, the number of CD4 cells in the scurfy spleen caught up with their WT counterpart. The number and frequency of CD8 T cells were similar in the two groups in the first 10 days. By day 16, the number of CD8 T cells was substantially higher in the scurfy spleen.


Figure 1
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  		FIGURE 1. Reduced T cell numbers in the scurfy mice during perinatal period. Scurfy mutant mice or their WT littermates in the C57BL/C background were analyzed for the percentages (a) and absolute numbers (b) of CD4 and CD8 T cells in the spleen at different ages. The percentages of CD4 and CD8 T cells were determined with CD4 and CD8 staining, and the absolute numbers were calculated based on the total number of splenocytes x CD4 or CD8 percentage. Data are summary of at least three mice of each age and error bars indicate SEM. a, Upper panel, R2 = 0.97 (scurfy) and 0.94 (WT); lower panel, R2 = 0.92 (scurfy) and 0.97 (WT). b, Upper panel, R2 = 0.99 (scurfy) and 0.97 (WT); lower panel, R2 = 0.96 (scurfy) and 0.97(WT).

 
Mathematical modeling of T cell numbers revealed distinct patterns of the expansion of the T cell compartment between scurfy and WT mice. In the WT mice, the increase of splenic T cells during the first 3 wk can be best modeled by linear regression (CD4: y = 118962x – 390557; CD8: y = 62256x – 214431; y, number of cells; x, age), which suggests that T cell expansion is primarily due to continuous emigration from the thymus. However, in the scurfy mice, the increases in T cell numbers best fit with an exponential regression (CD4: y = 22567e0.2701x; CD8: y = 8727.9e0.3733x), which suggests a major role for T cell division in the periphery with the increase of T cell numbers.

To test the contributions of T cell proliferation to the expansion of the T cell compartment, mice at different ages were pulsed with BrdU for 3 h and their splenocytes were harvested for flow cytometry. On day 3, the T cells had the same division rate in the scurfy mice as in the WT mice. Thereafter, the rate of T cell division in the scurfy mice was substantially higher than in the WT mice (Fig. 2, a and b). A peak in T cell division was found on day 10 in the scurfy mice, and after day 10, the division rate, especially of CD4 T cells, gradually decreased (Fig. 2b).


Figure 2
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  		FIGURE 2. Hyperproliferation of T cells in the newborn scurfy mice. B6 scurfy mice and their WT littermates at different ages were pulsed with 1 mg BrdU/mouse for 3 h and dividing cells were accessed based on the BrdU+ cells. a, Representative FACS profiles (a) and summary of BrdU+ percent cells (b) among CD4 or CD8 T cells at different ages. Data are a summary of at least three mice for each age and error bars indicate SEM.

 
Homeostatic proliferation expands T cells in newborn scurfy mice

Data in Fig. 2 also revealed that, after 3 days, the difference in T cell numbers correlated strongly with the difference in the division rate among WT and scurfy T cells, which raised the possibility that the initial phase of proliferation observed may be predominantly homeostatic in nature. We then tested all known criteria for homeostatic proliferation in the scurfy mice.

First, we conducted the phenotypic analysis of the dividing T cells in newborn scurfy mice. In the 10-day-old scurfy mice, when proliferation is at its peak in the scurfy mice, the majority of the dividing T cells expressed CD44, but not early activation markers such as CD69 or CD25 (Fig. 3). Such phenotypes are consistent with homeostatic proliferation in normal neonates (5).


Figure 3
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  		FIGURE 3. Increased homeostatic proliferation in the scurfy mice during perinatal period. Dividing T cells in the newborn scurfy mice exhibited markers of homeostatic proliferation. CD4 or CD8 T cells in the spleen of 10-day-old B6 scurfy mice or their WT littermates were analyzed for BrdU incorporation and CD69 (upper panel), CD25 (middle panel), or CD44 (lower panel) expression. Data are representative of at least five mice of each genotype from three independent experiments.

 
Second, we tested whether naive T cells could divide in newborn scurfy mice in the absence of their cognate Ags. We labeled naive RAG-1+/+ (Fig. 4a) and RAG-1–/– (Fig. 4b) OT-1 transgenic T cells with CFSE and transferred them into 3-day-old syngeneic scurfy mice. Seven days later, the spleens and lymph nodes were harvested and analyzed for their division, although most of the injected cells were found in the lymph nodes. Whereas few cells divided in the WT recipients in the 7-days period, most of those injected into the scurfy mice had undergone 2–4 divisions (Fig. 4, a and b). As expected, the majority of the divided T cells expressed CD44 but not CD69 (Fig. 4a, lower panel), further indicating the nature of homeostatic proliferation. Similar results were obtained when DO11.10 CD4 T cells were transferred into the 3-day-old scurfy mice in the BALB/c background (Fig. 4c). Therefore, newborn scurfy mice can support naive T cell proliferation in the absence of their cognate Ags.


Figure 4
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  		FIGURE 4. Neonatal scurfy mice support naive T cell proliferation independent of their specificities. Purified CD8 T cells (3 x 106/mouse) from B6 RAG-1+/+ (a) and RAG-1–/– (b) OT-1 Tg mice were labeled with CFSE and transferred into 3- or 4-day-old B6 scurfy mice. Seven days after the transfer, donor CD8+ T cells in the lymph nodes were gated and analyzed for CFSE dilution. Donor cells were also analyzed for the expression of CD69 (upper panel) and CD44 (lower panel). Data are representative at least four scurfy mice and three WT mice from two independent experiments. c, Neonatal scurfy mice support naive CD4 T cells proliferation independent of their specificities. As in b, purified CD4 T cells (3 x 106/mouse) from DO11.10 mice were transferred into 4-day-old BALB/c scurfy mice, and the donor cells (CD4+KJ126+) were analyzed for their division.

 
Third, we tested whether proliferation of naive T cells could be suppressed by bulk naive T cells. We cotransferred 2 x 106 CFSE-labeled OT-1 T cells together with 4 x 107 CD25– polyclonal Thy1.1+ T cells into 3-day-old scurfy mice. Seven days after the transfer, we examined the CFSE dilution of donor OT-1 cells. As shown in Fig. 5a, when transferred with polyclonal naive T cells, the OT-1 cells divided much slower than when transferred alone. In support of the role for total T cell numbers in the intensity of homeostatic proliferation, the suppression of OT-1 division inversely correlated with the graft efficiency of Thy1.1 T cells (Fig. 5b). To rule out a potential contribution of Treg-mediated suppression, we also used unlabeled OT-1 CD8 T cells, which are incapable of expressing FoxP3 in vivo, as competitors. As shown in Fig. 5, unlabeled OT-1 cells also effectively inhibited division of the CFSE-labeled OT-1 cells.


Figure 5
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  		FIGURE 5. Bulk T cell transfer inhibits the naive T cell proliferation in the scurfy mice. CFSE-labeled OT-1 CD8 T cells were transferred into 3-day-old B6 Scurfy mice together with either 4 x 107 CD25– total Thy1.1 T cells or 3 x 107 unlabelled OT-1 CD8 T cells. Seven days after the transfer, division of the CFSE labeled OT-1 cells was determined by CFSE dilution. Numbers in each plot represent the percentage of donor cells undergoing more than two cell cycles. Representative FACS profiles are shown in a and the summary is shown in b. Note when transferred with Thy1.1 T cells, more donor cells in the host led to less divisions of OT-1 cells (a, left panel).

 
Finally, to determine whether homeostatic proliferation decreased as T cell cellularity increased, we compared the rate of homeostatic proliferation in 1- or 2-wk-old mice. As shown in Fig. 6a, a majority of the dividing T cells in the 7-day-old mice were CD69–CD44+, which is consistent with the phenotype of homeostatic proliferation. By day 15, the majority of the BrdU+ T cells acquired CD69 expression with a reduced rate of proliferation (Fig. 6a). To determine whether the lack of homeostatic proliferation in the 2-wk-old mice was due to a change of host environment, but not endogenous T cells per se, purified OT-1 cells or naive Thy1.1 CD8 T cells were purified and, after CFSE labeling, transferred into 5- or 16-day-old scurfy mice. Four days later, the spleens and lymph nodes were harvested and the number of T cells undergoing proliferation was determined by the dilution of CFSE. As shown in Fig. 6b, consistent with the homeostatic proliferation in newborn scurfy mice, the substantial proportion of T cells injected on day 5 divided in the four days period. When transferred into 16 days old scurfy mice, however, few T cells divided. Thus, restoration of cellularity by the expansion of T cells in the scurfy mice inhibited homeostatic proliferation.


Figure 6
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  		FIGURE 6. Cessation of homeostatic proliferation after reconstitution T cell cellularity by the expansion of endogenous T cells. a, Phenotypic change of the dividing T cells between 7- and 15-day-old scurfy mice. Thy1.1 BALB/c scurfy mice at either 7 or 15 days old were pulsed with BrdU for 3 h, and lymph node cells were stained with anti-BrdU and anti-CD69 (upper panel) or anti-CD44 (lower panel). Data are repeated two times with similar results. Similar results were also obtained from the splenocytes (data not shown). b, Only 1-wk-old mice support Ag-independent proliferation. In brief, 3–4 x 106 purified OT-1 CD8 T cells or polyclonal Thy1.1 CD8 T cells were labeled with CFSE and transferred into B6 scurfy mice or their WT littermates at different ages. Four days after the transfer, donor cells were analyzed for CFSE dilution. Numbers in each plot depict the percentage of donor cells that divided at least once. The data in b have been repeated at least twice involving three mice at each time points. Similar results were obtained using transgenic mice expressing TCR specific for tumor Ag P1A (data not shown).

 
FoxP3 mutation in T cells does not affect homeostatic proliferation in response to lymphopenia

Besides being constitutively expressed in Tregs, FoxP3 can also be induced in nonregulatory T cells during homeostatic proliferation in the periphery (32). Therefore, enhanced T cell homeostatic proliferation in the scurfy mice might be due to the T cells’ intrinsic defects caused by the FoxP3 mutation. To test this possibility, we compared the homeostatic proliferation of scurfy T cells and WT T cells in response to the lymphopenic environment. As shown in Fig. 7a, the rate of BrdU incorporation was comparable between injected WT T cells and the endogenous FoxP3-deficient T cells.


Figure 7
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  		FIGURE 7. No intrinsic role of Foxp3 in inhibiting homeostatic proliferation. a, WT T cells divided as equally as FoxP3 mutant T cells in new born Scurfy mice. Five million purified total T cells from WT BALB/c mice were transferred into 2-day-old Thy1.1 BALB/c scurfy mutant mice. Seven to eight days after the transfer, the mice were pulsed with BrdU for 3 h and the donor CD4 cells were compared with the host CD4 T cells for the BrdU incorporation. Similar results were obtained for CD8 T cells. b, FoxP3 mutant T cells responded equally to lymphopenic cue. Bone marrow cells from FoxP3sfCD45.1+ and FoxP3wtCD45.2+ mice were mixed at a 1:1 ratio and used to reconstitute Rag1–/– mice. Total T cells purified from chimera mice were labeled with CFSE and transferred into new Rag1–/– mice. At either day 4 or 7 after the transfer, donor cells were analyzed for their division based on CFSE dilution. Left panel, Ratio of two genotypes in either o the CD4 or CD8 compartments before transfer. Right panel, Dividing of donor cells in Rag1–/– mice. Note CD45.1+ represents FoxP3sf T cells, and CD45.1– represents Foxp3wt T cells. Data are representative of two independent experiments.

 
To use T cells with the same activation status, we constructed the mixed bone marrow chimera using scurfy bone marrow (CD45.1+Foxp3sf) and congenic WT bone marrow (CD45.2+Foxp3wt) to reconstitute Rag1–/– mice. The chimera mice were healthy with no sign of autoimmune diseases (18). In addition, the phenotypes of the two sources of T cells are essentially the same (18). Interestingly, although the same number of bone marrow were used to generate chimera, the number of T cells from the scurfy bone marrow was somewhat reduced. It is possible that the stem cell function is somehow reduced as a result of autoimmune response in the donors.

T cells from the chimera mice were then purified and transferred into new Rag1–/– mice after CFSE labeling. CFSE dilution of T cells with two genotypes was compared at 4 and 7 days after the transfer. As shown in Fig. 7b, T cells with the scurfy mutation (CD45.1+) had the same pace of division as WT T cells (CD45.2+). Additionally, in the CD4 compartment, the ratio between CD45.1+ and CD45.2+ T cells after the homeostatic proliferation remained the same on both days 4 and 7 as to what was observed before transfer. Although the CD8 T cells ratio remained the same at day 4, there appear to a preferential accumulation of CD45.2+ (FoxP3wt) CD8 T cells on day 7. Because FoxP3 is not expressed in CD8 T cells, this is likely due to a fluctuation of immune T cell response in the different recipients. These data demonstrate that the FoxP3 defect in the CD4 T cells had no effect on their response to homeostatic cue.

Efficient rescue of the scurfy mice requires suppression of homeostatic proliferation and reconstitution of Treg

A critical prediction of the notion that homeostatic proliferation is an essential part of the pathogenesis is that suppression of homeostatic proliferation is sufficient to rescue the scurfy mice. To test this prediction, we adoptively transferred bulk or CD4+CD25– T cells from WT into the scurfy mice, and tested the effect of the bulk T cells on homeostatic T cell proliferation and life span of the recipients. Because the bulk T cells also contained ~5% of the FoxP3+ T cells, we also transferred comparable number of Treg into the scurfy recipients. As shown in Fig. 8, a and b, transfer of bulk or CD4+CD25– T cells, but not Treg alone significantly reduced homeostatic proliferation. The Treg reconstitution reached 2–3% of the CD4 T cells on average in the scurfy host when either bulk or Treg are transferred (Fig. 8c). Importantly, the bulk T cells, but neither Treg alone nor CD4+CD25– T cells, rescued the majority of the Scurfy mice (Fig. 8d). Thus, restoration of Treg and suppression of homeostatic proliferation are both important for the control of lethal autoimmune disease.


Figure 8
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  		FIGURE 8. Homeostatic proliferation and Treg defects must both be corrected to prevent autoimmune disease in the Scurfy mice. a and b, Suppression of homeostatic proliferation by either bulk T cells or CD4+CD25– T cells (a) but not Treg (b). The 2- to 3-day-old Scurfy mice received either 20 x 106 bulk or CD4+CD25– T cells, or 1 x 106 Treg or left untreated. Seven to ten days after the transfer, the mice were pulsed with BrdU for 3 h and sacrificed for analysis. Summary of the BrdU incorporation of host T cells in the spleen (a) or the lymph nodes (b) are presented. c, Successful reconstitution of Treg compartment by adoptive transfer of 106 Treg into the new born FoxP3sf/y mice. Data shown are percentage of donor Treg cells in the lymph nodes of the recipients of bulk T cells or CD4+CD25+ T cells. d, Lifespan of the mice that received either Treg or bulk T cells, in comparison to untreated scurfy mice. Although no extension of lifespan was achieved by Treg transfer or CD4+CD25– T cell transfer, bulk T cells substantially increased the survival of the scurfy mice (p < 0.001 in comparison to all other groups). Error bars in a–c depict SE of mean.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
A major tenant for the hypothesis that Tregs are both necessary and sufficient to protect against fatal and acute autoimmunity is the phenotype of the mice with mutations in the FoxP3 gene. However, recent studies have demonstrated expression of the FoxP3 protein in other normal tissues, including thymic (24), mammary (25), and lung epithelia (26). These data call for an investigation to determine whether other mechanisms may act in concert with Treg defect in causing lethal autoimmune diseases.

In searching for a critical cofactor that synergize with Treg defects in causing lethal autoimmune diseases, we observed that proliferating T cells in mice with germline mutation of FoxP3 exhibit markers of homeostatic proliferation (BrdU+CD44+CD69–). Given the extensive data linking homeostatic proliferation to autoimmune diseases (33), it is possible that homeostatic proliferation may be an underlying cause for the pathogenesis of autoimmune diseases in the scurfy mice.

We found that during the perinatal period, splenic T cells in the scurfy mice were significantly fewer than their WT littermate. Coinciding with lymphopenia, we observed a much stronger homeostatic proliferation in the scurfy mice in comparison to their WT littermates, characterized by the independence from cognate Ags, expression of bona fide markers for homeostatic proliferation. The homeostatic proliferation was prevented by increases in T cell numbers but not restoration of Treg. Interestingly, the homeostatic proliferation was quickly overtaken by what is optionally defined as Ag-driven proliferation. This kinetic suggests that homeostatic proliferation may prime the potentially autoreactive T cells in mice with germline mutation of FoxP3.

More importantly, the data presented in this study demonstrate that homeostatic proliferation is an important provision for the lethal autoimmune disease in scurfy mice. Thus, adoptive transfer of bulk T cells suppressed homeostatic proliferation and rescued the majority of the scurfy mice. This suppression cannot be explained by cotransferred Treg, as equal aliquots of Treg alone failed to suppress autoimmune disease. However, we would like to stress that the poor activity of adoptively transferred Treg as observed by us (none) and others (short-term amelioration) (18, 21, 34) does not argue for a lack of role for Treg in the pathogenesis of autoimmune disease. In fact, removing Treg from the bulk T cells ablated the rescue. Thus, although neither suppression of homeostatic proliferation nor restoration of Treg number is sufficient to suppress autoimmune disease, both are necessary. It should noted that suppression of homeostatic proliferation was heterogenous, which may explain incomplete rescue of some mice even when both Treg and bulk T cells were used.

In the scurfy mice with germline mutation of FoxP3, we have reported defective thymopoeisis associated with mutation of the FoxP3 gene in thymic epithelial cells. As a result, we have observed a reduced thymic cellularity before the development of overt of autoimmune diseases (24). Correspondingly, the number of new thymic emigrants were reduced in the scurfy mice (our unpublished observation). Although the lymphopenia is moderate in the scurfy mice, it must be emphasized that lymphopenia was observed despite the obscuring effect of massive ongoing lympho proliferation. Because the homeostatic proliferation is suppressed by bulk T cells but not Treg, we suggest that the homeostatic proliferation in the scurfy mice is not a consequence of Treg defect.

A hallmark of homeostatic proliferation is the lacking of CD69 in dividing T cells. In this regard, it is of interest to note that CD69 inhibits S1P1 chemotactic function and thus suppresses the emigration of activated T cells out of the lymphoid organ (35). As such, a lack of CD69 may not merely be a marker of homeostatic proliferation, but rather serves as an important function to promote autoimmune diseases. Therefore, it is plausible that T cells undergoing homeostatic proliferation, by virtue of lacking CD69, more readily emigrate into target tissues to cause autoimmune damage.

Taken together, our data revealed that a single gene defect can affect two important factors implicated in autoimmune diseases: Treg defects and homeostatic proliferation, whose interaction is responsible for what appears to be the most severe autoimmune diseases occurring in both mice and humans. This binary model provided an immunological basis to explain the superior therapeutic effect using polyclonal T cells in comparison to purified Treg. As such, our data offer valuable insights for the treatment of IPEX patients.


   Acknowledgments
 
We thank Drs. Weiping Zou and Philip King for critical reading of the manuscript and Lynde Shaw for editorial assistance. Part of the work was conducted when the authors were at The Ohio State University.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by grants from the U.S. Department of Defense and National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Yang Liu, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109. E-mail address: yangl{at}umich.edu Back

3 Abbreviations used in this paper: Treg, regulatory T cell; WT, wild type; IPEX, immune dysregulation, polyendocrinopathy, X-linked syndrome. Back

Received for publication April 7, 2008. Accepted for publication June 10, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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