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The Journal of Immunology, 2007, 179, 7439-7447
Copyright © 2007 by The American Association of Immunologists, Inc.
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Murine Lupus Susceptibility Locus Sle1a Controls Regulatory T Cell Number and Function through Multiple Mechanisms1

Carla M. Cuda*, Suigui Wan2,*, Eric S. Sobel{dagger}, Byron P. Croker*,{ddagger} and Laurence Morel3,*

* Department of Pathology, Immunology, and Laboratory Medicine and {dagger} Department of Medicine, Division of Rheumatology and Clinical Medicine, University of Florida, Gainesville, FL 32610; and {ddagger} Pathology and Laboratory Medicine Service, Malcolm Randall Veterans Affairs Medical Center, Gainesville, FL 32608


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Sle1 locus is a key determinant of lupus susceptibility in the NZM2410 mouse model. Within Sle1, we have previously shown that Sle1a expression enhances activation levels and effector functions of CD4+ T cells and reduces the size of the CD4+CD25+Foxp3+ regulatory T cell subset, leading to the production of autoreactive T cells that provide help to chromatin-specific B cells. In this study, we show that Sle1a CD4+ T cells express high levels of ICOS, which is consistent with their increased ability to help autoreactive B cells. Furthermore, Sle1a CD4+CD25+ T cells express low levels of Foxp3. Mixed bone marrow chimeras demonstrated that these phenotypes require Sle1a to be expressed in the affected CD4+ T cells. Expression of other markers generally associated with regulatory T cells (Tregs) was similar regardless of Sle1a expression in Foxp3+ cells. This result, along with in vitro and in vivo suppression studies, suggests that Sle1a controls the number of Tregs rather than their function on a per cell basis. Both in vitro and in vivo suppression assays also showed that Sle1a expression induced effector T cells to be resistant to Treg suppression, as well as dendritic cells to overproduce IL-6, which inhibits Treg suppression. Overall, these results show that Sle1a controls both Treg number and function by multiple mechanisms, directly on the Tregs themselves and indirectly through the response of effector T cells and the regulatory role of dendritic cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dominant suppression through regulatory T cells (Tregs),4 and specifically CD4+CD25+ T cells expressing the Foxp3 transcription factor, has now been accepted as a major mechanism by which self-tolerance is maintained. A decrease in Treg numbers or function has been directly associated with autoimmune pathogenesis in multiple diseases and their associated mouse models (1, 2). Reduced numbers of Tregs (3, 4) as well as defective Treg function (5) have more recently been described in lupus patients. Tregs play a key role in maintaining tolerance to DNA in a transgenic mouse model (6). In spontaneous models of lupus, tolerance induction was dependent on the generation of Foxp3+ Tregs in (NZB x SWR)F1 (7) and (NZB x NZW)F1 (BWF1) mice (8), and Treg transfers showed a significantly protective effect in BWF1 mice (9). Other murine studies have documented a Treg protective effect for autoantibody production, but not for end-organ pathology (10, 11). Although these studies have documented a role for Tregs in controlling at least some aspects of lupus pathogenesis, they did not determine the mechanisms responsible for the observed Treg deficiency in either number or function.

We have used NZM2410-derived congenic strains to address these questions. The major lupus susceptibility locus Sle1 controls tolerance to nuclear Ags (12, 13) and intrinsically affects both B and T cells (14, 15). Multiple loci contribute to the Sle1 phenotype (16) and we have shown that Sle1a and Sle1c are largely responsible for the generation of autoreactive T cells, with Sle1a alone accounting for CD4+ T cell phenotypes equivalent to that of the entire Sle1 locus (17). CD4+ T cells expressing Sle1a show significantly increased levels of activation and proliferation, as well as increased production of cytokines. Furthermore, purified Sle1a CD4+ T cells are able to induce in vivo the production of anti-nuclear Abs from either Sle1 or normal B cells (17). Finally, Sle1a is associated with a reduction of CD4+CD25+CD62L+Foxp3+ Treg numbers (17). Conversely, the B6.Sle1.Sle2.Sle3 (B6.TC) strain, which reconstitutes the full autoimmune pathogenesis with the three major NZM2410 susceptibility loci (18), produces dendritic cells (DCs) that prevent Treg inhibitory functions on effector T cells (Teffs) (19). Production of high amounts of IL-6 by B6.TC DCs is a major mechanism by which this interference occurred, and we have shown that this phenotype maps to Sle1 (19).

In this study, we examined the functional consequences of Sle1a expression on Tregs and cells directly interacting with them. Treg function can be affected by multiple factors, including their number and intrinsic function. Many studies have reported a critical role of accessory cells, especially DCs, for optimal Treg development and function (20), and imaging studies have clearly shown that Tregs exert their regulatory function through direct contact with DCs (21, 22). Teffs can also be resistant to suppression, as was shown in the MRL/lpr model of lupus (23). The complexity of a regulatory system in which these three cellular compartments play a critical role requires a model in which each compartment can be assayed independently in a syngeneic/autologous fashion (24). The NZM2410-congenic strains, which share >96% of their C57BL/6 (B6) genome, offer such a model. By comparing the B6.Sle1a congenics to B6 controls, we first confirmed that Sle1a results in a reduced subset of CD4+CD25+CD62L+Foxp3+ cells. Sle1a Tregs, however, appeared normal regarding expression of markers commonly associated with the regulatory phenotype and were capable of normal regulatory activity at high Treg:Teff ratios. Sle1a also induced an increased level of activation in CD4+ T cells and DCs, and both of these compartments significantly interfered with Treg regulatory function. Finally, we showed that the activated CD4+ T cell phenotypes and reduced Treg numbers required Sle1a expression in these T cells, suggesting that the generation of autoreactive T cells results from additive intrinsic defects in both Sle1a-expressing CD4+ T cells and DCs. Overall, these results identify Sle1a as a locus playing a major role in T cell tolerance through Treg regulation by multiple mechanisms.


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

C57BL/6J (B6), C57BL/6J-Cg-IghaThy1aGpila/J (B6.Thy1a), and B6.129S7-Rag1tm1Mom/J (B6.Rag) mice were originally obtained from The Jackson Laboratory. The B6.NZM2410-Sle1 (B6.Sle1)-congenic strain contains a 37-cM NZM2410-derived interval defined by the D1MIT101 and D1MIT155 markers (25). The B6.NZM2410-Sle1a (B6.Sle1a) subcongenic line represents a 2.96-Mb interval between and excluding D1MIT370 and D1MIT147 (16, 17). Unless specified, experiments were conducted with 8- to 12-mo-old female congenic mice and B6- matched controls. This age is past the induction of anti-nuclear Abs and autoreactive cells in most B6.Sle1 and B6.Sle1a mice (12, 16). All mice were bred and maintained at the University of Florida in specific pathogen-free conditions. All experiments were conducted according to protocols approved by the University of Florida Institutional Animal Care and Use Committee.

Flow cytometry

Briefly, cells were first blocked on ice with staining buffer (PBS, 5% horse serum, and 0.09% sodium azide) supplemented with 10% rabbit serum and pretreated with anti-CD16/CD32 (2.4G2) to block FcR-mediated binding. Cells were then stained with pretitrated amounts of the following FITC-, PE-, allophycocyanin-, or biotin-conjugated Abs: CD4 (RM4-5), CD69 (H1.2F3), CD25 (7D4), CD62L (MEL-14), glucocorticoid-induced TNF receptor (GITR) (DTA-1), CD103, ICOS (CD278 clone 7E.17G9), B220 (RA3-6B2), CD3 (145-2C11), CD11b (M1/70), CD11c (HL3), CD19 (1D3), CD40 (HM40-3), CD62L, CD80 (16-10A1), CD86 (GL1), I-Ab (AF6-120.1), NK1.1 (PK126), TER119, and mPDCA-1 (Miltenyi Biotec) or isotype controls. All Abs were obtained from BD Biosciences unless otherwise specified. A combination of PE-conjugated anti-CD3, CD19, NK1.1, and TER119 Abs were used to exclude CD11clow T cells, B cells, NK cells, and erythroblasts, respectively. Biotin-conjugated Abs were revealed using streptavidin-PerCP-Cy5.5 (BD Biosciences). Intracellular expression of CD152 (CTLA-4) and IL-10 was analyzed in fixed permeabilized cells with a Cytofix/Cytoperm Plus kit (BD Pharmingen). For IL-10 expression, splenocytes were cultured in the presence of anti-CD3 and anti-CD28 (1 µg/ml) for 3 days and intracellular IL-10 levels in CD4+ICOS+ cells were assessed by flow cytometry. IL-10 was also measured in the culture supernatant using an OptEIA Mouse IL-10 ELISA kit (BD Pharmingen) according to the manufacturer’s instructions. Foxp3 expression was determined using an intracellular Foxp3-PE staining kit (eBioscience). Cell staining was analyzed using a FACSCalibur (BD Biosciences). At least 50,000 events were acquired per sample, and dead cells were excluded based on scatter characteristics. Positive staining was determined as equal to or greater than the top 5% of the isotype control.

Suppression assays

CD4– (APC), CD4+CD25– Teff, and CD4+CD25+ Treg populations were purified from splenocytes with magnetic beads using the CD4+CD25+ Treg cell kit according to the manufacturer’s instructions (Miltenyi Biotec) and cultured in 96-well flat-bottom plates in the presence of 1 µg/ml anti-CD3 to assess in vitro suppression levels of Tregs. Teffs and Tregs FACS analysis consistently showed >90% purity. The number of Teffs was kept constant at 5 x 105 cells/well, whereas the number of Tregs was titrated using 4-fold dilutions. Cultures were maintained for 54 h before pulsing with 1 µCi/well [3H]thymidine for an additional 18 h. Cells were then collected onto fiber filter mats with a PHD cell harvester (Cambridge Technology) and counted using a beta scintillation counter. To assess the suppressive function of Tregs in vivo, CD4+CD25– Teff and CD4+CD25+ Treg populations from 2-mo-old female donor mice were purified with magnetic beads and transferred into age-matched female B6.Rag mice by injection into the tail vein. Recipients received 4 x 105 B6 or B6.Sle1a Teffs in the presence or absence of B6 or 1 x 105 B6.Sle1a Tregs. After injection, mice were monitored for clinical signs of colitis for up to 8 wk and body weight was monitored weekly. Mice that lost 15% or more of body weight or showed overt clinical signs of disease were sacrificed. Routine colon, stomach and kidney histology was performed to compare B6 and B6.Sle1a Teff and Treg functions and scored blindly in a semiquantitative fashion. The colon multiplicative score (0–81) was calculated by multiplying the thickness score by the infiltrate score in both the mucosa and the muscularis. The kidney additive score (0–4) was computed by adding 1 to the infiltrate score for the presence of granulomas.

Generation of DCs and DC phenotyping

DCs were generated from bone marrow (BM) with GM-CSF and IL-4 (R&D Systems) as previously described (19). To assess activation levels and cytokine production, BM-derived CD11c+ DCs were cultured for 24 h with LPS (Sigma-Aldrich) at 1 µg/ml. The supernatants were harvested and stored at –80°C until assayed with commercial ELISA kits (BD Pharmingen).

BM chimeras

Chimeras were prepared as previously described (15). In brief, 6- to 8-wk-old female B6 mice were lethally irradiated with two doses of 525 rad gamma irradiation (4 h apart) in a Gammacell 40 137Cs apparatus (MDS Nordion). Donor BM cells were depleted of mature T cells using CD5 Microbeads (Miltenyi Biotec). Production of mixed BM chimeras was performed at a 1:1 ratio for the B6.Thy1a and B6.Sle1a strains. Ten million cells were given to each mouse by tail vein injection. Chimeric mice were maintained for 8 wk, and lymphocytes were analyzed by flow cytometry to evaluate proliferation, activation, and Treg levels. The B6.Thy1a and B6.Sle1a origin of the T cells was determined with CD90.1 (Thy1a) and CD90.2 (Thy1b). CD4+ cellular proliferation was measured by staining splenocytes with 2.5 µM CFSE (Molecular Probes) before stimulation with anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) and cultured for 48 h in a 37°C/5% CO2 incubator. Activation was measured by staining lymphocytes with CD4 and CD69 after 12 h of anti-CD3 and anti-CD28 stimulation. Treg levels were measured by staining lymphocytes with CD4, CD25, and CD62L before culture.

Statistical analysis

Unpaired t test statistics (two- or one-tailed as indicated) were used to compare the phenotypes of the B6.Sle1 and B6.Sle1a strains with that of B6. Comparisons for BM chimeras were made with paired two-tailed Student’s t tests after verification that the data were normally distributed with GraphPad Prism 4. Nonparametric Mann-Whitney U tests were used when the data were not normally distributed. Comparisons for colon and kidney pathology were made with one-way ANOVA tests. Each in vitro experiment was performed at least twice with reproducible results.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Sle1 and Sle1a are associated with increased levels of activated T cells and decreased levels of Tregs

Previous results indicated that Sle1 is associated with a significantly increased number of activated CD4+ T cells (13, 14) as well as a decreased number of CD4+CD25+ Tregs, and that this phenotype mapped to Sle1a and to a lesser extent to Sle1c (17, 26). We confirmed these results by showing that congenic mice expressing Sle1 or Sle1a showed a significant increase in activated CD4+CD69+ or CD4+CD44+ T cells and a significant decrease in naive CD4+CD62L+CD44– T cells as compared with B6 (data not shown). In addition, we show here that both Sle1 and Sle1a CD4+ T cells showed a significantly increased expression of ICOS (Fig. 1A), which is a costimulatory molecule that is pivotal for T-B interactions and highly expressed on follicular helper T cells (27). We further analyzed CD4+ICOS+ cells by culturing total splenocytes in the presence of anti-CD3 and anti-CD28 to assess intracellular levels and secreted IL-10. We observed a trend of increased levels of CD4+ICOS+IL-10+ cells as well as production of IL-10 in the culture supernatant associated with Sle1a, but not to a statistically significant degree (data not shown). B6.Sle1a- congenic mice also showed significantly decreased percentages of CD4+CD25+CD62Lhigh (Fig. 1B), with significantly fewer CD4+CD25+ cells expressing CD62L, indicating that this locus induced a higher proportion of recently activated cells CD4+CD25+ cells as opposed to Tregs. These findings were confirmed by intracellular expression of Foxp3 (Fig. 1, C and D). It is of note that CD4+CD25+CD62Lhigh cells have lost the Foxp3high peak in the B6.Sle1 and B6.Sle1a mice, suggesting that this population contains less functional Foxp3+ Tregs in these mice than in B6. Similar results were obtained for younger mice ranging from 5 to 7 mo of age (data not shown). Overall, these results confirm that Sle1a expression increases the number of activated T cells and diminishes the Foxp3+ Treg compartment. However, the expression of markers commonly associated with Tregs, namely, GITR, CD103, and CTLA-4, was not affected by Sle1a expression in either CD4+CD25+ (Fig. 2A) or CD4+CD25+CD62L+ populations (Fig. 2B), suggesting that Sle1a Tregs may be functional, although reduced in numbers.


Figure 1
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  		FIGURE 1. Sle1a results in increased CD4+ T cell activation and a diminished Treg compartment. Splenocytes from B6, B6.Sle1, and B6.Sle1a mice were labeled for surface CD4, ICOS, CD25, and CD62L and intracellular Foxp3 expression and analyzed by FACS. Each point represents an individual animal. Representative gating on a B6 mouse is shown in the left-hand column (marker for A and rectangular gate for B–D) and representative expression levels in all three strains are shown in the right-hand column. The light gray filled histograms show isotype controls, dark gray filled histograms show B6 values, while thick and thin black lines represent B6.Sle1 and B6.Sle1a, respectively. All comparisons were performed with B6 values. Two-tailed t tests: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 

Figure 2
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  		FIGURE 2. Sle1a does not affect expression of markers associated with the regulatory phenotype. CD4+CD25+ (A) or CD4+CD25+CD62L+ (B) splenocytes from B6 and B6.Sle1a mice were compared for GITR, CD103, or intracellular CTLA-4 expression. Representative histograms of at least five mice per strain are shown. The percentage of positive cells (based on the isotype control shown by the gray histograms) is indicated above each marker.

 
Sle1a Tregs require a higher Treg:Teff ratio to support inhibition to a level equivalent to B6 Tregs

We assessed Sle1a Treg function using standard suppression assays in which the proliferation of CD4+CD25– Teffs was measured in response to anti-CD3 stimulation in the presence of APCs and graded ratios of Tregs. In these assays, the only variable was the Treg origin, B6 or B6.Sle1a, while all other cells were of B6 origin. As shown in Fig. 3, there was no difference between the inhibitory capability of Sle1a and B6 Tregs at a 1:1 Treg:Teff ratio. A significantly diminished inhibitory function appeared however at 1:4 and 1:16. At this latter ratio, inhibition by Sle1a Tregs was no longer observed and in some cases increased proliferation was observed with Sle1a Treg addition, as we have previously reported for B6.TC Tregs (19). This titration result is consistent with the CD4+CD25+ population containing a smaller proportion of functional Tregs in B6.Sle1a mice.


Figure 3
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  		FIGURE 3. Sle1a Tregs are deficient in inhibiting proliferation at low Treg:Teff ratios. Inhibition of proliferation assays were set up with B6-derived APCs and Teffs and either B6 ({blacksquare}) or B6.Sle1a ({square}) Tregs at the indicated ratio. A, Representative assay comparing proliferation in the presence of B6 or Sle1a Tregs (six mice per strain). Means and SEs and results of one-tailed t tests between the 0:1 assays and the various Treg:Teff ratios for each strain. B, Normalized percentage inhibition of proliferation of the 0:1 assays at the various Treg:Teff ratios for each strain combined from three different assays (15 mice per strain). Means and SEs and results of one-tailed t tests between the two strains for each Treg:Teff ratio. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
Sle1a expression increases DC activation

We have recently shown that B6.TC DCs display an abnormal phenotype and hinder Treg function in an IL-6- dependent manner (19). Furthermore, elevated IL-6 production coupled with Treg inhibition mapped to Sle1. We show here that the Sle1a mediates an expansion of CD11c+CD11b+B220– myeloid DCs in the spleen (Fig. 4A) and lymph nodes (data not shown). Plasmacytoid DCs gated as CD11c+B220+ (Fig. 4A), but not as B220+PDCA-1+ (data not shown), were also modestly expanded in B6.Sle1a spleens. In addition, Sle1a DCs displayed a significantly increased expression of activation markers as shown for CD86 (Fig. 4C) and CD80 (Fig. 4D) that is similar to that of Sle1 DCs. These ex vivo phenotypes were age dependent as they reached statistical significance only in old mice. Increased levels of activation markers such as CD40 and CD86 (Fig. 4B), or CD80 and class II MHC (data not shown), and increased production of IL-6 (Fig. 4E) and IL-12 (Fig. 4F) were readily obtained by LPS stimulation of DCs derived from either young (2–3 mo old) or old B6.Sle1a BM. These levels were similar to what we have previously described for B6.Sle1. Overall, these results show that Sle1a induces an age-dependent DC accumulation in secondary lymphoid organs and that these DCs produce more inflammatory cytokines than those of the B6 controls. We have previously reported that Sle1 increases activation of peripheral B cells (13, 15). In this study, we show that Sle1a splenic B cells also expressed a significantly higher level of CD19, CD80, and CD86 in old mice (data not shown). Overall, these results show that Sle1a increases activation not only in CD4+ T cells but also in DCs and B cells.


Figure 4
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  		FIGURE 4. Sle1a expression activates DCs. A, Plasmacytoid DCs (pDC) and myeloid DCs (mDC) were significantly expanded in the spleen of B6.Sle1a mice as compared with B6 mice. B, CD40 and CD86 were significantly up-regulated in B6.Sle1a BM-derived DCs in response to LPS. Splenic DCs were significantly more activated in B6.Sle1 and B6.Sle1a than in B6 mice, as shown by CD86 (C) and CD80 (D) expression. DCs derived from B6.Sle1 or B6.Sle1a BM produced significantly more IL-6 (E) and IL-12 (F) than B6 after LPS exposure. Each point represents an individual animal. All comparisons were performed with B6 values. Two-tailed t tests: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
Sle1a expression affects the ability of both DCs to support Treg suppression and Teffs to be inhibited

Given that Sle1a expression affects all cellular compartments in a suppression assay, namely, Tregs, Teffs, and APCs, we investigated the consequences of Sle1a expression independently in each of these cellular compartments on the ability of Tregs to suppress Teff proliferation (Fig. 5). As seen earlier, expression of Sle1a in Tregs had a significant effect on Treg function at a low Treg:Teff ratio. Interestingly, Sle1a expression in Teffs significantly hindered the action of Tregs, although this effect was no longer significant at the 1:16 Treg:Teff ratio. Expression of Sle1a in APCs significantly prevented inhibition at all three ratios and even induced enhanced proliferation at the 1:16 Treg:Teff ratio. This latter effect was observed with DCs from B6.TC mice (19), suggesting that the Sle1a locus plays a major role in the DC defective functions in this model. In conclusion, Sle1a expression in either one of the three members of the suppression assay significantly impacts the ability of Tregs to suppress Teff proliferation.


Figure 5
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  		FIGURE 5. Sle1a expression in Tregs, Teffs, or APCs affects the extent of the inhibition of Teff proliferation. The inhibition of CD4+CD25+ Teff proliferation in the presence of 1:1, 1:4, or 1:16 Treg:Teff ratios is expressed as a percentage of the proliferation induced in the absence of Tregs for each condition. The origin, B6 or B6.Sle1a, of Tregs, Teffs, and APCs is indicated under each column. The graphs show the means and SEs of three independent assays with three to four mice per strain at 6 mo of age in each assay. Results of one-tailed t tests between each condition and the "all B6 condition" are indicated for each Treg:Teff ratio. +, #, *: p < 0.05; ++, ##, **: p < 0.01; +++, ###, ***: p < 0.001.

 
We also assessed in vivo the effect of Sle1a expression on effector and regulatory CD4+ T cell function in a rapid model of disease adapted from the experimental colitis model (28). B6.Rag mice received 4 x 105 CD4+CD25– Teffs from either 2-mo-old B6 or B6.Sle1a in the presence or absence of 1 x 105 CD4+CD25+ Tregs from B6 or B6.Sle1a mice. As expected, B6 Teffs induced weight loss and colitis, which was abrogated by the presence of B6 Tregs (Figs. 6 and 7, A and B). Sle1a-expressing Tregs did not suppress B6 Teff function as well as B6 Tregs, possibly due to a lower ratio of functional Tregs within the injected CD4+CD25+ population. We also observed that the Sle1a-expressing Teffs are resistant to suppression by either B6 or Sle1a Tregs. In addition to lymphocytic infiltrates, Sle1a Teffs resulted in the presence of elevated numbers of polymorphonuclear neutrophils (PMN) in the lesions, leading to cryptitis (Fig. 6C2). Finally, the combination of Sle1a Teffs and Tregs led to the most severe pathology with the presence of giant cells (Fig. 6D2). Interestingly, similar results were observed in the kidneys (Fig. 7C and data not shown), but not in the stomach. Teffs induced interstitial inflammation and granulomas in B6.Rag kidneys, which were suppressed by B6 Tregs only when the Teffs were of B6 origin. As in the colon, Sle1a Teffs were also associated with giant cells in the kidneys, indicating a greater level of inflammation. Overall, this in vivo experiment confirmed that the Sle1a CD4+CD25+ population is less effective at suppressing Teff functions and that Sle1a Teffs are resistant to Treg suppression.


Figure 6
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  		FIGURE 6. Sle1a expression in either Tregs or Teffs affects the extent of disease in an adoptive transfer model. CD4+CD25– Teff and CD4+CD25+ Treg populations from 2-m- old female donor mice were transferred into age-matched female B6.Rag1–/– recipient mice. Representative colon histology: All parts of the composite labeled 1 are the same magnification (0.5 mm) and all parts labeled 2 (0.1 mm) are x4 those labeled 1. Each two subfigures with the same letter (e.g., A1 and A2) are from the same animal. A1 and A2, B6 Teffs and B6 Tregs. This figure is representative of the control group with normal thickness and minimal lymphocytic infiltrate in the lamina propria (A2, circled area, center). B1 and B2, B6 Teffs and Sle1a Tregs. This figure is representative of ~2-fold increase in thickness (B1). There is a notable increase in lamina propria thickness and mononuclear cell infiltrate. There are also increases in epithelial infiltrating lymphocytes, epithelial apoptosis, and mitosis (B2). C1 and C2, Sle1a Teffs and B6 Tregs. This figure is representative of an additional increase in thickness (C1). In addition to the lymphocytes and other findings noted above, increased PMN were present in the lamina propria and glands (cryptitis and crypt abscess, C2, center). D1 and D2, Sle1a Teffs and Sle1a Tregs. This is the greatest overall thickness with inflammation extending into the muscularis propria (D1, bottom). There is an increase in PMN and lymphocytes throughout the mucosa. Occasional multinucleated giant cells are present (D2, circled area).

 

Figure 7
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  		FIGURE 7. Quantification of the effects of CD4+CD25– Teff and CD4+CD25+ Treg transfers into B6.Rag1–/– mice. A, Maximum weekly percentage of body weight loss up to 8 wk after transfer. The box and whisker plot shows the medians, 25th and 75th percentiles, and minimal and maximum for each group. B, Multiplicative colitis pathology score. C, Additive kidney pathology score (infiltrate score + giant cell presence). B and C, The strain of origin of Teff and Tregs is indicated on the x-axes, with 0 indicating the absence of Tregs. ANOVA: *, p < 0.05.

 
Sle1a expression intrinsically affects CD4+ T cell phenotypes

The results presented above showed that Sle1a expression affects the function of multiple hemopoietic cell compartments, which prompted us to examine whether Sle1a expression was required for CD4+ T cells to show the functional defects reported above. To address this question, we produced mixed BM chimeras by injecting T cell-depleted BM cells from either normal B6.Thy1a or B6.Sle1a (Thy1b) donor mice into lethally irradiated B6 hosts. As shown in Fig. 6, the increased proliferation and activation of CD4+ T cells, as well as the decreased percentage of Tregs were completely reproduced by Sle1a BM-derived cells (cf B6.Thy1a->B6 and B6.Sle1a->B6). More interestingly, in mixed chimeras containing both Sle1a-expressing and normal CD4+ T cells, only those T cells expressing Sle1a displayed enhanced proliferation, as measured by in vitro CFSE dilution (Fig. 8A), and activation, as measured by CD69 expression (Fig. 8B). Corresponding histograms show CFSE levels on gated CD4+ T cells (Fig. 8, A and B, respectively). Conversely, the percentage of CD62L+ Treg was significantly lower in Sle1a-expressing T cells than in B6 and can be visualized in the corresponding histogram depicting CD62L levels on CD4+CD25+ gated cells (Fig. 8C). Taken together, these results show unambiguously that Sle1a results in intrinsically activated CD4+ T cells. Sle1a expression in nonhemopoietic cells is not required for induction of these phenotypes. The abnormal phenotypes are not transferable to bystander normal T cells, excluding Sle1a being mediated through a soluble factor.


Figure 8
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  		FIGURE 8. Sle1a expression affects CD4+ function in a cell-intrinsic manner. B6 hosts were reconstituted with B6.Thy1a and/or B6.Sle1a BM. Connected samples indicate values for CD4+ T cells expressing the Thy1a (CD90.1-gated) or Thy1b in B6.Sle1a mice (CD90.2-gated) alleles within the same mouse. Controls are represented by B6.Thy1a->B6 and B6.Sle1a->B6 single-strain BM transfers. A, In vitro anti-CD3-induced proliferation measured as the percentage of CD4+CFSElow lymphocytes with representative histogram showing CFSE expression on gated CD4+ T cells. B, Activation measured as the percentage of CD4+CD69+ lymphocytes with representative histograms showing CD69 expression on gated CD4+ T cells. C, Treg levels, measured as the percentage of CD4+CD25+ splenocytes expressing CD62L with representative histograms, showing CD62L expression. Each point represents an individual animal. Two-tailed t tests: *, p < 0.05; **, p < 0.01. Data representative of two independent sets of BM chimeras with five mixed chimeras in each.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have shown here that expression of Sle1a is sufficient to induce increased activation levels of CD4+ T cells, DCs, and B cells, as well as to down-regulate Treg levels. We also show that Sle1a CD4+ T cells express increased levels of the costimulation marker ICOS, which has been shown to play a critical role in T cell help to B cells, especially in germinal centers (29, 30). Elevated ICOS expression on T cells from lupus patients has now been reported in three independent studies (31, 32, 33). These last two studies reported that ICOS stimulation of lupus T cells significantly enhanced anti-dsDNA Ab production from autologous B cells, which is equivalent to what we have shown for Sle1a T cells, which were able to induce antichromatin production in both autologous Sle1a-expressing B cells and normal B cells (17). These results also suggest that Sle1a confers a T cell phenotype that is found in lupus patients, which further validates the need to determine the identity of the Sle1a gene(s). Future experiments should address the specific role of ICOS in this process. High levels of ICOS have been associated with IL-10 secretion by CD4+ T cells (34) and IL-10 production by CD4+ T cells is significantly increased in the NZM2410 model (35). There was however no consistent increase of IL-10 production by Sle1a CD4+ T cells, suggesting that another mechanism may be involved.

Sle1a induces a reduction in the size of the Treg compartment, but these cells express normal levels of CTLA-4, CD103, and GITR, molecules which have been commonly associated with the regulatory phenotype. In addition, at the higher ratios of Treg:Teff, Sle1a-expressing Tregs are fully capable of suppressing the proliferation of B6 Teffs on a per-cell basis in the presence of B6 APCs. However, at lower ratios of Treg:Teff, this suppressive capability is decreased, consistent with a reduced proportion of functional Tregs within the CD4+CD25+ T cell population of the B6.Sle1a mice. In addition to in vitro suppression assays, we also performed adoptive transfers adapted from the experimental model of colitis to test the in vivo effect of Sle1a on Treg and Teff functions in a rapid model of disease. Results from the in vivo study confirmed our in vitro data. We cannot exclude, however, that Sle1a also affects Treg inhibitory functions. Indeed, a recent construct with a nonfunctional Foxp3 has demonstrated that the expression of Treg signature makers can develop normally in cells that completely lack inhibitory functions (36). A definitive answer to that question will require breeding of Sle1a to a Foxp3 reporter construct, which we are currently pursuing.

Although we have shown that the Sle1a-expressing Tregs are capable of suppression, in situations where either the Teffs or the APCs express Sle1a, the suppressive capability of normal B6 Tregs is significantly reduced, suggesting that the Sle1a locus confers a resistance to suppression of Teff proliferation and that the APCs are playing a role in this phenomenon. It is of note that the APC population used in our in vitro suppression assays contains not only DCs but B cells as well. We have previously shown the effects of Sle1a DCs on Treg suppression (19); however, Sle1a affects both of these cell types. This indicates a potential role of activated B cells on Treg function and is an avenue to be studied further. A similar Teff resistance has been previously reported in another model of lupus (23), but it is not clear at this point whether this resistance is the mere consequence of hyperactivation or a result of involvement with a specific mechanism. Cbl-b deficiency results in a resistance to Treg regulation involving TGF-β, and this mutation also induces an increased level of activation in effector T cells (37). To our knowledge, no other mechanisms of resistance to Tregs have been reported and additional experiments will be necessary to determine how Sle1a confers this resistance in CD4+ T cells. We have previously shown that DCs from the NZM2410 triple congenic strain B6.TC prevent Tregs from performing their inhibitory functions, primarily through the production of IL-6 (19). We report here that Sle1a-expressing DCs present the same phenotype of high IL-6 production and Treg inhibition, indicating that this locus plays a major role in the overall DC phenotype of lupus-prone mice. Interestingly, the type 1 diabetes-prone NOD mice, which have a reduced number of Tregs (38, 39), also produce APCs that fail to fully support Treg functions (39). These results suggest that defective support or active inhibition of Treg functions by DCs may be a common mechanism affecting autoimmune pathogenesis.

Mixed BM chimeras have shown here that the increased proliferation and activation of Sle1a-expressing T cells, as well as the reduced Sle1a Treg level require that Sle1a be expressed in these T cells. These results differ from what might have been predicted from the in vitro reconstitution experiments shown in Fig. 5, where B6.Sle1a-derived APCs inhibited Treg function. The BM chimera results do not mean that Sle1a exclusively affects CD4+ T cells. In an analogous set of experiments, BM chimeras showed that T cell activation and autoreactivity mediated by Sle3 were the indirect result of Sle3 expression within the myeloid compartment (40, 41). It is therefore possible that the Sle1a-induced intrinsic defects in CD4+ T cells are indirectly responsible for the DC and B cell abnormalities. Alternatively, the Sle1a gene(s) may control a pathway present in all three cellular compartments. In any event, indirect or direct activation of DCs by Sle1a was not sufficient to convey extrinsic changes to B6-derived CD4+ T cells in vivo. The exact cause for these differences is unclear and highlights the need to confirm in vitro findings with in vivo studies. Additional mixed BM chimeras will be necessary to address whether Sle1a expression in these DCs and B cells is necessary for production of the activated phenotypes.

Autoreactive T cells are a feature common to many autoimmune diseases for which a genetic basis has been demonstrated, yet only very few genes have been identified as responsible for this phenotype (42). In addition to Cbl-b discussed above (37), null alleles of Gadd45a (43) or E2f2 (44) result in a lower threshold for T cell activation culminating in autoimmune pathogenesis, while null alleles in Ctla4 (45) and Foxp3 (46) result in massive inflammatory and autoimmune responses through the disruption of the Treg compartment. More recently, a natural polymorphism in the Il2 gene has been identified as responsible for the diabetes susceptibility locus Idd3 in the NOD mouse, also through an impairment of Treg function (47). The Sle1a interval does not contain any gene with obvious function in T cells. Our vitro results showed that Sle1a confers an autoimmune phenotype to CD4+ T cells in the colon, which is not typically associated with lupus pathogenesis. This indicates that Sle1a affects a genetic pathway regulating production of Tregs and responses to Tregs in a manner that is not restricted to tolerance to nuclear Ags. The identification of the Sle1a gene(s) will therefore uncover a novel and broad pathway by which autoreactive T cells are regulated by Tregs.


   Acknowledgments
 
We thank Leilani Zeumer and Xuekun Su for excellent animal care, Pui Lee for his advice with DC phenotyping, and the members of the Morel laboratory for stimulating discussions.


   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 work was supported by National Institutes of Health Grants R01 AI 45050 (to L.M.) and T32 AR007603 (to C.M.). Back

2 Current address: Xuanwu Hospital, Capital University of Medical Science, Beijing, China. Back

3 Address correspondence and reprint requests to Dr. Laurence Morel, Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610-0275. E-mail address: morel{at}ufl.edu Back

4 Abbreviations used in this paper: Treg, regulatory T cell; DC, dendritic cell; Teff, effector T cell; BM, bone marrow; PMN, polymorphonuclear neutrophil; GITR, glucocorticoid-induced TNF receptor. Back

Received for publication March 20, 2007. Accepted for publication September 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sakaguchi, S., M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, T. Takahashi, T. Nomura. 2006. Foxp3+CD25+CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease. Immunol. Rev. 212: 8-27. [Medline]
  2. Paust, S., H. Cantor. 2005. Regulatory T cells and autoimmune disease. Immunol. Rev. 204: 195-207. [Medline]
  3. Miyara, M., Z. Amoura, C. Parizot, C. Badoual, K. Dorgham, S. Trad, D. Nochy, P. Debre, J. C. Piette, G. Gorochov. 2005. Global natural regulatory T cell depletion in active systemic lupus erythematosus. J. Immunol. 175: 8392-8400. [Abstract/Free Full Text]
  4. Lee, J. H., L. C. Wang, Y. T. Lin, Y. H. Yang, D. T. Lin, B. L. Chiang. 2006. Inverse correlation between CD4+ regulatory T-cell population and autoantibody levels in paediatric patients with systemic lupus erythematosus. Immunology 117: 280-286. [Medline]
  5. Valencia, X., C. Yarboro, G. Illei, P. E. Lipsky. 2007. Deficient CD4+CD25high T regulatory cell function in patients with active systemic lupus erythematosus. J. Immunol. 178: 2579-2588. [Abstract/Free Full Text]
  6. Seo, S. J., M. L. Fields, J. L. Buckler, A. J. Reed, L. Mandik-Nayak, S. A. Nish, R. J. Noelle, L. A. Turka, F. D. Finkelman, A. J. Caton, J. Erikson. 2002. The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells. Immunity 16: 535-546. [Medline]
  7. Kang, H. K., M. A. Michaels, B. R. Berner, S. K. Datta. 2005. Very low-dose tolerance with nucleosomal peptides controls lupus and induces potent regulatory T cell subsets. J. Immunol. 174: 3247-3255. [Abstract/Free Full Text]
  8. La Cava, A., F. M. Ebling, B. H. Hahn. 2004. Ig-reactive CD4+CD25+ T cells from tolerized (New Zealand Black x New Zealand White)F1 mice suppress in vitro production of antibodies to DNA. J. Immunol. 173: 3542-3548. [Abstract/Free Full Text]
  9. Scalapino, K. J., Q. Tang, J. A. Bluestone, M. L. Bonyhadi, D. I. Daikh. 2006. Suppression of disease in New Zealand Black/New Zealand White lupus-prone mice by adoptive transfer of ex vivo expanded regulatory T cells. J. Immunol. 177: 1451-1459. [Abstract/Free Full Text]
  10. Bagavant, H., C. Thompson, K. Ohno, Y. Setiady, K. S. K. Tung. 2002. Differential effect of neonatal thymectomy on systemic and organ-specific autoimmune disease. Int. Immunol. 14: 1397-1406. [Abstract/Free Full Text]
  11. Bagavant, H., K. S. K. Tung. 2005. Failure of CD25+ T cells from lupus-prone mice to suppress lupus glomerulonephritis and sialoadenitis. J. Immunol. 175: 944-950. [Abstract/Free Full Text]
  12. Morel, L., C. Mohan, Y. Yu, B. P. Croker, N. Tian, A. Deng, E. K. Wakeland. 1997. Functional dissection of systemic lupus erythematosus using congenic mouse strains. J. Immunol. 158: 6019-6028. [Abstract]
  13. Mohan, C., E. Alas, L. Morel, P. Yang, E. K. Wakeland. 1998. Genetic dissection of SLE pathogenesis: Sle1 on murine chromosome 1 leads to a selective loss of tolerance to H2A/H2B/DNA subnucleosomes. J. Clin. Invest. 101: 1362-1372. [Medline]
  14. Sobel, E. S., M. Satoh, W. F. Chen, E. K. Wakeland, L. Morel. 2002. The major murine systemic lupus erythematosus susceptibility locus Sle1 results in abnormal functions of both B and T cells. J. Immunol. 169: 2694-2700. [Abstract/Free Full Text]
  15. Sobel, E. S., C. Mohan, L. Morel, J. Schiffenbauer, E. K. Wakeland. 1999. Genetic dissection of SLE pathogenesis: adoptive transfer of Sle1 mediates the loss of tolerance by bone marrow-derived B cells. J. Immunol. 162: 2415-2421. [Abstract/Free Full Text]
  16. Morel, L., K. R. Blenman, B. P. Croker, E. K. Wakeland. 2001. The major murine systemic lupus erythematosus susceptibility locus, Sle1, is a cluster of functionally related genes. Proc. Natl. Acad. Sci. USA 98: 1787-1792. [Abstract/Free Full Text]
  17. Chen, Y., C. Cuda, L. Morel. 2005. Genetic determination of T cell help in loss of tolerance to nuclear antigens. J. Immunol. 174: 7692-7702. [Abstract/Free Full Text]
  18. Morel, L., B. P. Croker, K. R. Blenman, C. Mohan, G. Huang, G. Gilkeson, E. K. Wakeland. 2000. Genetic reconstitution of systemic lupus erythematosus immunopathology with polycongenic murine strains. Proc. Natl. Acad. Sci. USA 97: 6670-6675. [Abstract/Free Full Text]
  19. Wan, S., C. Xia, L. Morel. 2007. IL-6 produced by dendritic cells from lupus-prone mice inhibits CD4+CD25+ T cell regulatory functions. J. Immunol. 178: 271-279. [Abstract/Free Full Text]
  20. Tarbell, K. V., S. Yamazaki, R. M. Steinman. 2006. The interactions of dendritic cells with antigen-specific, regulatory T cells that suppress autoimmunity. Semin. Immunol. 18: 93-102. [Medline]
  21. Tang, Q., J. Y. Adams, A. J. Tooley, M. Bi, B. T. Fife, P. Serra, P. Santamaria, R. M. Locksley, M. F. Krummel, J. A. Bluestone. 2006. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat. Med. 7: 83-92.
  22. Tadokoro, C. E., G. Shakhar, S. Shen, Y. Ding, A. C. Lino, A. Maraver, J. J. Lafaille, M. L. Dustin. 2006. Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo. J. Exp. Med. 203: 505-511. [Abstract/Free Full Text]
  23. Monk, C. R., M. Spachidou, F. Rovis, E. Leung, M. Botto, R. I. Lechler, O. A. Garden. 2005. MRL/Mp CD4+,CD25– T cells show reduced sensitivity to suppression by CD4+,CD25+ regulatory T cells in vitro: a novel defect of T cell regulation in systemic lupus erythematosus. Arthritis Rheum. 52: 1180-1184. [Medline]
  24. Mudd, P. A., B. N. Teague, A. D. Farris. 2006. Regulatory T cells and systemic lupus erythematosus. Scand. J. Immunol. 64: 211-218. [Medline]
  25. Morel, L., Y. Yu, K. R. Blenman, R. A. Caldwell, E. K. Wakeland. 1996. Production of congenic mouse strains carrying genomic intervals containing SLE-susceptibility genes derived from the SLE-prone NZM2410 strain. Mamm. Genome 7: 335-339. [Medline]
  26. Chen, Y., D. Perry, S. A. Boackle, E. S. Sobel, H. Molina, B. P. Croker, L. Morel. 2005. Several genes contribute to the production of autoreactive B and T cells in the murine lupus susceptibility locus Sle1c. J. Immunol. 175: 1080-1089. [Abstract/Free Full Text]
  27. Vinuesa, C. G., M. C. Cook, C. Angelucci, V. Athanasopoulos, L. Rui, K. M. Hill, D. Yu, H. Domaschenz, B. Whittle, T. Lambe, et al 2005. A RING-type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 435: 452-458. [Medline]
  28. Maloy, K. J., L. Salaun, R. Cahill, G. Dougan, N. J. Saunders, F. Powrie. 2003. CD4+CD25+ T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms. J. Exp. Med. 197: 111-119. [Abstract/Free Full Text]
  29. Mcadam, A. J., R. J. Greenwald, M. A. Levin, T. Chernova, N. Malenkovich, V. Ling, G. J. Freeman, A. H. Sharpe. 2001. ICOS is critical for CD40-mediated antibody class switching. Nature 409: 102-105. [Medline]
  30. Tafuri, A., A. Shahinian, F. Bladt, S. K. Yoshinaga, M. Jordana, A. Wakeham, L. M. Boucher, D. Bouchard, V. S. Chan, G. Duncan, et al 2001. ICOS is essential for effective T-helper-cell responses. Nature 409: 105-109. [Medline]
  31. Hutloff, A., K. Buchner, K. Reiter, H. J. Baelde, M. Odendahl, A. Jacobi, T. Dorner, R. A. Kroczek. 2004. Involvement of inducible costimulator in the exaggerated memory B cell and plasma cell generation in systemic lupus erythematosus. Arthritis Rheum. 50: 3211-3220. [Medline]
  32. Yang, J. H., J. Zhang, Q. Cai, D. B. Zhao, J. Wang, P. E. Guo, L. Liu, X. H. Han, Q. Shen. 2005. Expression and function of inducible costimulator on peripheral blood T cells in patients with systemic lupus erythematosus. Rheumatology 44: 1245-1254. [Abstract/Free Full Text]
  33. Kawamoto, M., M. Harigai, M. Hara, Y. Kawaguchi, K. Tezuka, M. Tanaka, T. Sugiura, Y. Katsumata, C. Fukasawa, H. Ichida, et al 2006. Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-{gamma} and anti-double-stranded DNA antibody production. Arthritis Res. Ther. 8: R62[Medline]
  34. Lohning, M., A. Hutloff, T. Kallinich, H. W. Mages, K. Bonhagen, A. Radbruch, E. Hamelmann, R. A. Kroczek. 2003. Expression of ICOS in vivo defines CD4+ effector T cells with high inflammatory potential and a strong bias for secretion of interleukin 10. J. Exp. Med. 197: 181-193. [Abstract/Free Full Text]
  35. Blenman, K. R., B. Duan, Z. Xu, S. Wan, M. A. Atkinson, T. R. Flotte, B. P. Croker, L. Morel. 2006. IL-10 regulation of lupus in the NZM2410 murine model. Lab. Invest. 86: 1136-1148. [Medline]
  36. Lin, W., D. Haribhai, L. M. Relland, N. Truong, M. R. Carlson, C. B. Williams, T. A. Chatila. 2007. Regulatory T cell development in the absence of functional Foxp3. Nat. Immunol. 8: 359-368. [Medline]
  37. Wohlfert, E. A., M. K. Callahan, R. B. Clark. 2004. Resistance to CD4+CD25+ regulatory T cells and TGF-β in Cbl-b–/– mice. J. Immunol. 173: 1059-1065. [Abstract/Free Full Text]
  38. Wu, A. J., H. Hua, S. H. Munson, H. O. McDevitt. 2002. Tumor necrosis factor-{alpha} regulation of CD4+C25+ T cell levels in NOD mice. Proc. Natl. Acad. Sci. USA 99: 12287-12292. [Abstract/Free Full Text]
  39. Alard, P., J. N. Manirarora, S. A. Parnell, J. L. Hudkins, S. L. Clark, M. M. Kosiewicz. 2006. Deficiency in NOD antigen-presenting cell function may be responsible for suboptimal CD4+CD25+ T-cell-mediated regulation and type 1 diabetes development in NOD mice. Diabetes 55: 2098-2105. [Abstract/Free Full Text]
  40. Sobel, E. S., L. Morel, R. Baert, C. Mohan, J. Schiffenbauer, E. K. Wakeland. 2002. Genetic dissection of systemic lupus erythematosus pathogenesis: evidence for functional expression of Sle3/5 by non-T cells. J. Immunol. 169: 4025-4032. [Abstract/Free Full Text]
  41. Zhu, J. K., X. B. Liu, C. Xie, M. Yan, Y. Yu, E. S. Sobel, E. K. Wakeland, C. Mohan. 2005. T cell hyperactivity in lupus as a consequence of hyperstimulatory antigen-presenting cells. J. Clin. Invest. 115: 1869-1878. [Medline]
  42. Chen, Y. F., L. Morel. 2005. Genetics of T cell defects in lupus. Cell. Mol. Immunol. 2: 403-409. [Medline]
  43. Salvador, J. M., M. C. Hollander, A. T. Nguyen, J. B. Kopp, L. Barisoni, J. K. Moore, J. D. Ashwell, A. J. J. Fornace. 2002. Mice lacking the p53-effector gene Gadd45a develop a lupus-like syndrome. Immunity 16: 499-508. [Medline]
  44. Murga, M., O. Fernandez-Capetillo, S. J. Field, B. Moreno, L. Borlado, Y. Fujiwara, D. Balomenos, A. Vicario, A. C. Carrera, S. H. Orkin. 2001. Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation, leading to the development of autoimmunity. Immunity 15: 959-970. [Medline]
  45. Tivol, E. A., F. Borriello, A. N. Schweitzer, W. P. Lynch, J. A. Bluestone, A. H. Sharpe. 1995. Loss of Ctla-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of Ctla-4. Immunity 3: 541-547. [Medline]
  46. Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, S. A. Yasayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, F. Ramsdell. 2001. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 27: 68-73. [Medline]
  47. Yamanouchi, J., D. Rainbow, P. Serra, S. Howlett, K. Hunter, V. E. S. Garner, A. Gonzalez-Munoz, J. Clark, R. Veijola, R. Cubbon, et al 2007. Interleukin-2 gene variation impairs regulatory T cell function and causes autoimmunity. Nat. Genet. 39: 329-337. [Medline]



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