The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2009, 182, 274 -282
Copyright © 2009 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schmidt, E. M.
Right arrow Articles by Walker, L. S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schmidt, E. M.
Right arrow Articles by Walker, L. S. K.

CTLA-4 Controls Regulatory T Cell Peripheral Homeostasis and Is Required for Suppression of Pancreatic Islet Autoimmunity1

Emily M. Schmidt2,*, Chun Jing Wang2,*, Gemma A. Ryan*, Louise E. Clough*, Omar S. Qureshi*, Margaret Goodall*, Abul K. Abbas{dagger}, Arlene H. Sharpe{ddagger}, David M. Sansom* and Lucy S. K. Walker3,*

* Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, United Kingdom; {dagger} Department of Pathology, University of California, San Francisco, CA 94143; and {ddagger} Department of Pathology, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CTLA-4 pathway is recognized as a major immune inhibitory axis and is a key therapeutic target for augmenting antitumor immunity or curbing autoimmunity. CTLA-4-deficient mice provide the archetypal example of dysregulated immune homeostasis, developing lethal lymphoproliferation with multiorgan inflammation. In this study, we show that surprisingly these mice have an enlarged population of Foxp3+ regulatory T cells (Treg). The increase in Treg is associated with normal thymic output but enhanced proliferation of Foxp3+ cells in the periphery. We confirmed the effect of CTLA-4 deficiency on the Treg population using OVA-specific Treg which develop normally in the absence of CTLA-4, but show increased proliferation in response to peripheral self-Ag. Functional analysis revealed that Ag-specific Treg lacking CTLA-4 were unable to regulate disease in an adoptive transfer model of diabetes. Collectively, these data suggest that the proliferation of Treg in the periphery is tuned by CTLA-4 signals and that Treg expression of CTLA-4 is required for regulation of pancreas autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Polymorphisms in the CTLA-4 gene are linked with several autoimmune diseases including type 1 diabetes, thyroiditis, systemic lupus erythematosus, and rheumatoid arthritis (reviewed in Refs. 1 and 2). Attention was first drawn to the importance of CTLA-4 by the description of CTLA-4-deficient mice over a decade ago (3, 4); however, the basis for the severe disease observed in these animals remains the subject of debate. Potential explanations include the ability of CTLA-4 to raise the threshold of T cell activation (5), modulate T cell motility (6), alter T cell thymic selection (7), or control the function of regulatory T cells (Treg)4 (8, 9). A role for CTLA-4 in Treg function was first suggested on the basis that this population constitutively expresses CTLA-4 (8, 9) and is consistent with the ability of CTLA-4-expressing cells to exert dominant tolerance over CTLA-4-deficient cells in mixed bone marrow chimeras (10). The first demonstration that anti-CTLA-4 Abs were capable of abrogating disease protection by Treg was provided by Powrie and colleagues (8) and Sakaguchi and colleagues (9), although these studies did not formally rule out the potential for effector cells to also be altered by such treatments. This work prompted further investigation of the role of CTLA-4 specifically in the Treg population. Although CTLA-4-deficient Treg appeared to retain suppressive function (11, 12), generation of such cells relied on the simultaneous abrogation of CD28 signaling (by CD80/86 blockade or deficiency). It is not clear whether the residual population of Treg that develops in the absence of CD28 is equivalent to wild-type Treg, both in efficiency and mechanism of action. Ab-mediated targeting of CTLA-4 has provided similarly controversial results: such an approach either does (9, 11, 12) or does not (13, 14) abrogate Treg suppression, with different batches of mAb preparations sometimes yielding opposite results (15).

To analyze the role of CTLA-4 in Treg, we examined Foxp3 expression in CTLA-4–/– mice. In addition, to examine CTLA-4 deficiency in a setting not complicated by lymphoproliferation, we used a TCR-transgenic system to generate Ag-specific Treg lacking CTLA-4. Our results indicate that Treg selection is not impaired in the absence of CTLA-4 but that CTLA-4 deficiency dysregulates Treg proliferation and abrogates Treg function in vivo. These data provide new insight into the mechanisms by which CTLA-4 preserves immune homeostasis and are relevant to the therapeutic targeting of this pathway.


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

DO11.10 and BALB/c mice were purchased from The Jackson Laboratory and Rag2–/– mice from Taconic Farms. CTLA-4–/– mice were provided by A. Sharpe. (Harvard, Boston, MA) rat insulin promoter (RIP)-mOVA mice expressing membrane OVA under the control of the insulin promoter (from line 296-1B) were a gift from W. Heath (Walter and Eliza Hall Institute). Mice were housed at the Birmingham Biomedical Services Unit and used according to institutional guidelines.

Flow cytometry

Cells were stained with mAb against Foxp3 (FJK-16s; eBioscience), CD25 (PC61), CD4 (LT34), CD8 (53-6.7), and Ki67 (B56) or CD69 (H1.2F3). All Ab were purchased from BD Biosciences unless otherwise indicated. For Foxp3 and Ki67 staining, cells were fixed and permeabilized according to the manufacturer’s instructions. Lymphocytes from the pancreas were obtained by digesting with liberase (Roche) at 37°C and centrifugation over Lympholyte-M (Cedarlane Laboratories). For measurement of IL-10 and surface TGFβ levels, cells were stimulated overnight with anti-CD3/CD28 beads (Dynal), then TGFβ was detected by biotinylated Ab (R&D Systems) followed by streptavidin-PE and IL-10 expression was measured by capture assay (Miltenyi Biotec) according to the manufacturer’s instructions.

Immunofluorescence

Five-micrometer spleen sections were acetone fixed and stained with rabbit anti-Ki67, biotinylated rat anti-mouse Foxp3 and Alexa Fluor 647 (Invitrogen)-conjugated rat anti-mouse CD4. Rabbit Ab were detected with Alexa Fluor 488-conjugated donkey anti-rabbit IgG and biotinylated Ab by Alexa Fluor 555-conjugated streptavidin (both Invitrogen). Images were obtained by confocal microscopy (Zeiss LSM 510).

In vivo Ab treatment

Six- to 8-wk-old BALB/c mice were injected with 500 µg of blocking anti-CTLA-4 mAb (4F10, gift from J. Bluestone, University of California, San Francisco) or control hamster IgG i.p. as indicated. 4F10 was grown in Miniperm bioreactors (Grenier Bio-One) using IgG-depleted FCS and purified by passing over staphylococcal protein A (GE Healthcare).

Adoptive transfer

Whole lymph nodes (LN), CD4+CD25– cells or CD4+CD25high cells from CTLA-4–/– mice were injected i.v. into rag–/– recipients. Weight loss is shown as a percentage of maximal weight, and LN counts refer to pooled axillary, inguinal, brachial, and mesenteric LN. Hearts were fixed in formaldehyde and paraffin sections were subjected to H&E staining. DO11+CD25– T cells for diabetes induction were purified from rag-sufficient DO11 x RIP-mOVA mice. Treg populations (CD4+CD25+DO11+) were purified (MoFlo) from CTLA-4-sufficient or -deficient DO11 x RIP-mOVA/rag–/– mice. Cells were injected i.v. into mOVA/rag–/– mice. Blood glucose was measured by Ascensia Elite XL meter (Bayer). Mice are typically considered diabetic following two consecutive readings >200 mg/dl.

In vitro suppression

Treg (CD4+CD25+DO11+) or T cells (CD4+CD25–DO11+) were purified from CTLA-4-sufficient or -deficient DO11 x RIP-mOVA/rag–/– mice as above and their ability to suppress the proliferation of Thy1.1+ wild-type CD4+CD25– cells (2.5 x 104) assessed. Cells were incubated at the indicated ratios in the presence of 1 µg/ml anti-CD3 and splenic CD19+ APC for 72 h, then the absolute number of Thy1.1 responder T cells was assessed by flow cytometry.

CD86 down-regulation

Four x 104 splenic CD11c+ cells were incubated with 8 x 104 CD4+CD25– T cells, an equivalent number of CD4+CD25+ Treg, or both. Anti-CTLA-4 Ab (50 µg/ml) or control hamster IgG was added as indicated. Forty-eight hours later, dendritic cell (DC) expression of CD86 and MHC class II were ascertained by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Treg are overrepresented in CTLA4–/– mice

CTLA-4-deficient mice succumb to a lymphoproliferative syndrome, resulting in their death around 3–4 wk of age (3, 4). Because these initial reports preceded availability of Foxp3+ Abs, Treg have not yet been rigorously analyzed in CTLA-4–/– mice. We used intracellular Foxp3 staining to quantify Treg in 15- to 17-day-old CTLA-4–/– mice and age-matched controls. Surprisingly, we found that Foxp3+ Treg were overrepresented in CTLA4–/– mice. The increased CD25 expression in these animals was previously thought to indicate activation associated with lymphoproliferation (4). However, our data demonstrate that this increase in CD25+ cells is almost entirely attributable to an increase in the Foxp3+ Treg population (Fig. 1A). CTLA-4–/– mice showed an increase in the percentage (Fig. 1B) and absolute number (Fig. 1C) of Foxp3+cells in both peripheral LN and spleen.


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 1. CTLA-4-deficient mice have increased numbers of Foxp3+ cells. LN, spleen (Sp), and thymus cells from 15- to 17-day-old CTLA4–/– mice (CT–/–) or littermate CTLA4+/– mice (CT+/–) were stained for CD4, Foxp3, and CD25. A, Representative FACS stain for Foxp3 and CD25 on LN cells. B, Percentage of CD4 cells that express Foxp3 in LN or spleen. Each symbol represents a single mouse. C, Absolute numbers of CD4+Foxp3+ or CD4+Foxp3– cells in LN or spleen. Bars show means from four to eight mice with SD. D, Representative thymus stains on 15-day-old CTLA4–/– mice or littermate controls. Lower panel is gated on CD4+CD8– cells: the percentage of Foxp3+ within this population was not significantly different between CTLA4–/– mice and littermate controls (4.62% ± 0.58 and 4.73% ± 0.75 respectively, n = 4). E, Fold increase in absolute number of CD4+Foxp3+ and CD4+Foxp3– cells between CTLA4–/– mice and littermate controls. Fold increase was calculated using mean values from four to eight mice.

 
CTLA4–/– Treg show unaltered thymic selection but increased peripheral proliferation

The augmented Treg population in CTLA4–/– mice could potentially reflect increased thymic selection or enhanced proliferation in the periphery. To resolve this issue, we first examined Treg numbers in the thymus. Although thymic development appears grossly normal in CTLA-4–/– animals (16, 17), Foxp3+ Treg have not previously been measured. To this end, we stained thymus cells from CTLA-4–/– mice for CD4, CD8, and Foxp3. We found that the proportion of single-positive CD4 cells expressing Foxp3 in the thymus of CTLA-4–/– mice was not significantly different from that seen in CTLA-4+/– littermate controls (Fig. 1D). Thus, the absolute number of Treg was increased in the periphery, but not the thymus of CTLA-4–/– animals (Fig. 1E). Interestingly, although the CD4+Foxp3– population was clearly expanded in CTLA-4–/– mice, consistent with the activation and expansion of effector T cells, the fold increase in the CD4+Foxp3+ compartment was numerically larger (Fig. 1E). This suggested that rather than being diluted out by the proliferating effector cells, the CD4+Foxp3+ population contributed to lymphadenopathy and splenomegaly in these animals.

Given the unaltered thymic selection of Treg in CTLA-4–/– mice, we examined whether the augmented Foxp3+ population could be due to increased Treg proliferation. Spleen sections from 15- to 17-day-old CTLA-4–/– animals or CTLA-4+/– littermates were stained for CD4, Foxp3, and Ki67, the nuclear protein expressed by proliferating cells. The Foxp3+ cells in CTLA-4–/– mice clearly showed increased proliferation compared with their control counterparts (Fig. 2A). Similar data were obtained when Ki67 was measured by intracellular staining and flow cytometry (Fig. 2B). In control CTLA-4+/– littermates, CD4+Foxp3+ Treg proliferated more than CD4+Foxp3– cells, consistent with the increased BrdU incorporation exhibited by the CD4+CD25+ subset in vivo (18). Indeed, in unmanipulated mice, the CD4+CD25+ Treg population exhibits a surprising capacity for proliferation, apparently reflecting their continuous encounter with self-Ags (19). The proportion of Treg in the cell cycle was clearly increased in CTLA-4–/– mice compared with CTLA-4+/– littermates. Thus, the absence of CTLA-4 (Fig. 2B) resulted in enhanced Treg proliferation and an enlarged peripheral Treg compartment.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 2. Foxp3+ cells in the periphery of CTLA-4-deficient mice show increased proliferation. A, Spleen sections from 17-day-old CTLA4–/– mice or littermate controls (CTLA4+/–) were stained for CD4 (blue), Foxp3 (red), and Ki67 (green). Proliferating Treg appear yellow. Lower panel shows scoring of stained sections for the percentage of Foxp3+ cells that are Ki67+. Each symbol represents a different field of view and each column a different mouse. In brief, ≥5 fields were scored per mouse. Mean values were significantly different (p < 0.05). B, Spleen cells from 17-day-old CTLA4–/– mice or littermate controls (CTLA4+/–) were stained for CD4 and intracellular Foxp3 and Ki67. Plots are gated on CD4+Foxp3+ cells. Treg proliferation in age-matched wild-type mice (CTLA-4+/+) was similar to that seen in CTLA-4+/– mice. Data are representative of two (A) or more than five (B) independent experiments.

 
CTLA-4 regulates proliferation of OVA-specific Treg

Interpretation of data obtained from CTLA4–/– animals is complicated by the lethal lymphoproliferative syndrome they develop. To overcome this, CTLA-4–/– mice can be bred onto a TCR-transgenic background (DO11.10), directing their specificity to a non-self Ag (OVA). To preclude rearrangements of second TCRs that might recognize self-Ags, these animals can be maintained on a rag-deficient background. DO11/CTLA4–/–/rag–/– mice do not develop lymphoproliferative syndrome and their peripheral T cells bear a naive phenotype (20). Although this model system has proved useful in dissecting the role of CTLA-4 in non-Treg (20, 21), it cannot be used to elucidate the role of CTLA-4 in Foxp3+ Treg since DO11/rag–/– animals do not develop Treg (22). We therefore crossed DO11/CTLA4–/–/rag–/– mice with mice that expressed OVA under the control of the RIP. Coexpression of the transgenic TCR and its Ag in this manner has previously been shown to permit Treg development (23). This strategy allowed us to study Treg in mice that lack CTLA-4 but have an intact CD28 pathway. Moreover, since in the TCR-transgenic system both CTLA-4–/– animals and their wild-type controls were maintained on a rag-deficient background, only a single TCR can be expressed. This permitted analysis of a single clone of Treg in the presence or absence of CTLA-4. In DO11+ mice that did not express the RIP-mOVA Ag, DO11 T cells did not develop into Treg, regardless of whether or not they expressed CTLA-4 (Fig. 3A, left half). In Ag-positive mice, a fraction of DO11 T cells developed into Foxp3+ Treg; however, neither the percentage nor absolute number of Foxp3+ cells were altered by CTLA-4 deficiency (Fig. 3A, right half, B, and C). The absence of an effect of CTLA-4 on Treg selection in the thymus was observed in both polyclonal (Fig. 1) and TCR- transgenic (Fig. 3) systems. Thus, CTLA-4 signaling is not an obligate step in Treg differentiation, nor does it limit the size of the Treg population selected against a defined self-Ag. We next examined whether CTLA-4 expression limited Treg proliferation in the periphery. A key feature of the TCR- transgenic system is that Ag availability is restricted anatomically by the RIP transgene, such that T cells at most peripheral sites do not encounter Ag. This differs from the situation in the nontransgenic CTLA-4–/– animals that exhibit global T cell activation in the periphery. Accordingly, Treg in peripheral lymphoid organs of DO11 x RIP-mOVA/rag–/– mice remained largely undivided even in the absence of CTLA-4 (Fig. 3D, upper panel). However, Treg isolated from the pancreas exhibited greater proliferation in mice that lacked CTLA-4. These data support the idea that CTLA-4 signaling controls Treg proliferation in response to encounter with self-Ags.


Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 3. Analysis of Ag-specific Treg deficient in CTLA-4. Treg were quantified by Foxp3 staining in DO11 x RIP-mOVA/rag–/– mice sufficient or deficient for CTLA-4. Ag-negative DO11 mice were used as controls. All mice were rag gene deficient. A, Representative Foxp3 staining in gated CD4+ (spleen) or CD4+CD8– (thymus (Thy)) cells from 3-wk-old animals. B, Percentage of CD4 cells (CD4+CD8– in thymus) that express Foxp3 at the indicated time after birth in DO11 x RIP-mOVA/rag–/– or DO11 x RIP-mOVA/ctla4–/–rag–/– mice. Each symbol represents an individual mouse. C, Absolute number of CD4+CD8–Foxp3+ (thymus) or CD4+Foxp3+ cells (spleen) in 6-wk-old mice of the indicated genotype. D, Pooled pancreata from DO11 x RIP-mOVA/rag–/– or DO11 x RIP-mOVA/ctla4–/–rag–/– mice were digested and stained for CD4, Foxp3, and Ki67. Plots are gated on CD4+ cells and are representative of two similar experiments using pooled pancreas tissue from two to three animals.

 
Ab-mediated CTLA-4 blockade augments Treg proliferation in BALB/c mice

Clinical relevance of the CTLA-4 pathway centers on the use of anti-CTLA-4-blocking Abs to augment antitumor immune responses (24, 25). We therefore tested whether Ab-mediated CTLA-4 blockade in normal animals enhanced Treg proliferation, as predicted by our findings in the CTLA-4 knockout mouse. Mice treated with anti-CTLA-4 Ab showed increased Ki67 within CD4+Foxp3+ cells 8 days later (Fig. 4A). In the steady state, Treg would be expected to be the initial cellular target of anti-CTLA-4 Ab since they constitutively express CTLA-4 (8, 9), whereas this protein is activation induced in Foxp3– T cells. To dissect the kinetics with which Treg and T cells responded to anti-CTLA-4 treatment, we undertook a time course analysis. Three days following initiation of anti-CTLA-4 blockade, Foxp3+ Treg showed significantly increased proliferation (21.5 ± 3.1% vs 13.7 ± 3.2% for control Ab), whereas the proliferation of Foxp3– T cells at this time point was not significantly different from that seen in control Ab-treated animals (2.6 ± 0.4% vs 2.4 ± 0.6%; Fig. 4B). Although the Foxp3– population did show an increase in proliferation following anti-CTLA-4 blockade, this manifested later then for the Foxp3+ population. By day 8, there was a trend toward slightly increased proliferation of Foxp3– cells in mice that had received anti-CTLA-4 Ab (3.9 ± 1.8% vs 2.3 ± 0.6%) but this trend was not significant in our time course until day 14. Consistent with an initial effect on Treg, Foxp3+ cells had increased CD69 expression as early as 24 h following anti-CTLA-4 administration (Fig. 4, C and D). In contrast, no induction of CD69 was detectable on Foxp3– T cells at this time point (Fig. 4, C and D). Thus, blocking CTLA-4 Abs can target Treg populations in vivo and trigger an increase in Treg proliferation.


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 4. Anti-CTLA-4 Ab increases Treg proliferation in vivo. A, BALB/c mice were injected with 500 µg of anti-CTLA-4 Ab (or control hamster IgG) on days 0, 2, and 6 and LN were harvested on day 8. Cells were stained for CD4 and intracellular Foxp3 and Ki67. Representative FACS stains showing Ki67 expression in BALB/c LN cells gated on CD4+Foxp3+ or CD4+Foxp3– cells. Proliferation of CD4+Foxp3+ cells was increased in mice treated with anti-CTLA-4 Ab compared with control Ab (30.68 ± 0.9% vs 13.85 ± 2.6%, respectively; n = 4). B, BALB/c mice were treated with twice weekly injections of anti-CTLA-4 Ab or control Ab and analyzed at the indicated time points. The percentage of Ki67+ cells within the CD4+Foxp3+ (Treg) population and the CD4+Foxp3– (T cell) population in LN is shown. Each bar shows the mean value for three to six individual mice with SD (**, p < 0.005). Values from control Ab-treated mice were similar at all time points and are presented as a single pooled control (individual controls were used for statistical analysis). C, CD69 staining in gated CD4+Foxp3+ Treg and CD4+Foxp3– T cells 24 h following injection of BALB/c mice with 500 µg of anti-CTLA-4 Ab or control Ab. D, Mean values with SD for multiple mice treated as in C (*, p < 0.01; n = 4).

 
Ag-specific Treg deficient in CTLA-4 fail to suppress autoimmune diabetes

Our findings raised the paradox that a fatal lymphoproliferative syndrome develops in the CTLA-4 knockout mouse despite an increase in Treg. Two potential resolutions for this paradox can be envisaged: first, that CTLA-4 deficiency renders Treg pathogenic (by releasing the brakes on a population that already bears high affinity for self-Ags) and, second, that CTLA-4 is required for Treg suppression. To test whether Treg lacking CTLA-4 were pathogenic, we took advantage of the fact that injection of CTLA-4–/– lymphocytes into rag-deficient recipients can transfer the lymphoproliferative syndrome (26). We assessed the ability of purified CD4+CD25– or CD4+CD25high cells from CTLA4–/– animals to transfer disease in this system. As expected, introduction of LN cells or purified CD25– cells from CTLA-4–/– mice into rag–/– recipients induced the lymphoproliferative syndrome with associated weight loss (Fig. 5A). Since myocarditis is a feature of CTLA-4 deficiency (3, 4), we also examined heart sections from recipient mice. Histological analysis showed clear evidence of lymphocytic infiltration in recipients of whole LN cells or purified CD25– cells from CTLA-4–/– mice (Fig. 5B). In contrast, CD25– cells from wild-type mice did not induce substantial weight loss at this time point (<4% weight loss) and did not cause detectable infiltration of the heart (data not shown). Importantly, recipients of CD4+CD25high cells from CTLA-4–/– mice remained healthy and did not exhibit weight loss (Fig. 5A) or heart infiltration (Fig. 5B). Thus, the augmented population of CD25+ cells in CTLA-4–/– mice was not directly responsible for immune-mediated destruction. We therefore tested the second possibility that CTLA-4 is necessary for Treg suppression in vivo. To assess the requirement for CTLA-4 in Treg function, we took advantage of the fact that Ag-specific Treg lacking CTLA-4 can be purified from our TCR-transgenic mice. Accordingly CD4+CD25+ cells were isolated from DO11 x RIP-mOVA/rag–/– mice that were either CTLA-4 sufficient or deficient. Foxp3 staining confirmed equivalent Treg purity within the two populations (Fig. 6A). We tested the ability of these Treg to control diabetes in an adoptive transfer model (27). Disease was induced by transfer of OVA-specific CD25– cells into mice expressing OVA in the pancreas and the ability of cotransferred regulatory populations to suppress diabetes was assessed. Mice that received CTLA-4-sufficient Treg were completely protected from diabetes (Fig. 6B). Strikingly, cotransfer of CTLA-4-deficient Treg was completely ineffective at preventing diabetes induction (Fig. 6B). The failure of CTLA-4-deficient Treg to control diabetes did not reflect decreased Treg survival or trafficking since transferred CTLA4-deficient Treg could be readily detected in the pancreas of recipient animals (Fig. 6C). Intriguingly, parallel analysis of CTLA-4–/– Treg in vitro showed that they retained their suppressive capacity in this setting (Fig. 6D). To further explore the basis for the failure of CTLA-4–/– Treg to control diabetes, we assessed their expression of IL-10 and surface TGFβ (Fig. 6E). No difference in IL-10 expression was observed between wild-type and CTLA-4–/– Treg. Furthermore, CTLA-4–/– Treg expressed higher levels of surface TGFβ than their wild-type counterparts, consistent with previous findings from the Bluestone laboratory using polyclonal Treg (11). Since defects in IL-10 and TGFβ did not appear to account for the compromised in vivo function of CTLA-4–/– Treg, we sought alternative explanations. We investigated the hypothesis that Treg-expressed CTLA-4 could deplete APC of costimulatory ligands. Strikingly, we found that the presence of Treg resulted in a marked down-regulation of CD86 expression on APC and that this could be blocked by anti-CTLA-4 Ab (Fig. 7). Expression levels of MHC class II on APC was unaltered by Treg. CD86 down-regulation was observed regardless of whether B cells or DC were used as APC, although the magnitude of down-regulation was greater with the latter (mean decrease in mean fluorescence intensity was 43.5% with DC vs 29.0% with B cells, n = 3). Because CD86 is a major costimulator of T cell responses, these data suggest that Treg may use CTLA-4 to alter the capacity of APC to elicit effective T cell activation. Collectively, these data support an obligate role for Treg-expressed CTLA-4 in the regulation of an anti-islet immune response and suggest that this reflects a capacity for Treg to influence APC in a CTLA-4-dependent manner.


Figure 5
View larger version (85K):
[in this window]
[in a new window]

  		FIGURE 5. Treg deficient in CTLA-4 are not inherently pathogenic. One to 2 x 106 LN cells or purified CD4+CD25– or CD4+CD25high LN cells from 15- to 17-day-old CTLA-4–/– mice were injected into rag–/– recipients. A, Percentage weight loss and LN cellularity are shown 3–4 wk following transfer. Columns show mean data with SD for three to five mice. B, H&E staining of heart from rag–/– mice that received CTLA-4–/– whole LN, CD4+CD25– cells, or CD4+CD25high cells.

 

Figure 6
View larger version (45K):
[in this window]
[in a new window]

  		FIGURE 6. Ag-specific Treg deficient in CTLA-4 lack regulatory function in vivo. A, Foxp3 staining in DO11+CD4+CD25+ LN cells from DO11+mOVA+ rag–/– or DO11+mOVA+ ctla4–/–rag–/– mice. B, Blood glucose levels in mOVA/rag mice that received 0.15 x 106 DO11+CD25– cells alone or in combination with 0.15 x 106 CD4+CD25+ cells purified from DO11+mOVA+ rag–/– or DO11+mOVA+ ctla4–/–rag–/– mice. Six mice are shown per experimental group, except for the CD25– alone group where only two are shown for clarity. Diabetes was observed for zero of six recipients of wild-type (wt) Treg, six of six recipients of CTLA-4–/– Treg, and six of six recipients of CD25– cells alone. Transfer of CTLA-4–/– Treg alone did not induce diabetes. C, Digested pancreas samples from recipients of wild-type or CTLA-4–/– DO11+ Treg 22 days after transfer were stained for CD4 and Foxp3. Absolute cell numbers were calculated as the mean value for three recipients: for CD4+Foxp3– cells, these were 1648 and 4719 (for recipients of wild-type and ctla4–/– Treg, respectively) and for CD4+Foxp3+ cells these were 335 and 440 (for recipients of wild-type and ctla4–/– Treg, respectively). D, 2.5 x 104 Thy1.1+CD4+CD25– cells were incubated with wild-type or ctla4–/– CD4+CD25+ cells (upper panel) or control CD25– cells (lower panel) in the presence of 1 µg/ml anti-CD3 and APC. Seventy-two hours later, the absolute number of Thy1.1+ responder cells was assessed by flow cytometry. Results are expressed as a percentage of the maximum cell number and graphs show the mean value and SD from three independent experiments. E, LN cells from wild-type or ctla4–/– DO11+mOVA+ rag–/– mice were stained for surface TGFβ and secreted IL-10 as described in Materials and Methods. Plots are gated on CD4+Foxp3+ (Treg) or CD4+Foxp3– (T cells).

 

Figure 7
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 7. CTLA-4-dependent CD86 down-regulation by Treg. Four x 104 splenic CD11c+ cells were incubated for 48 h with 8 x 104 CD4+CD25– T cells alone (T), 8 x 104 CD4+CD25+ cells, or both. In brief, 50 µg/ml anti-CTLA-4 Ab or control hamster IgG was added as indicated. Expression of CD86 and MHC class II levels on gated CD11c+ cells was assessed by flow cytometry and representative plots (A) and mean fluorescence intensity (MFI) values (B) are shown. Data show one representative experiment of three performed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The lymphoproliferative syndrome associated with CTLA-4 deficiency is considered a classic example of immune dysfunction involving uncontrolled proliferation of pathogenic T cells. The data presented here extend our interpretation of the CTLA-4–/– phenotype to encompass dysregulated Treg proliferation and function. In fact, the fold increase in the Treg population in these animals is greater than the fold increase of Foxp3– cells. In the setting of CTLA-4–/– deficiency, given the global lymphocyte activation, Foxp3– T cells are likely to produce cytokines that further promote Treg homeostasis. However, our short-term anti-CTLA-4 blockade experiments in normal mice suggest that the steady-state Treg turnover seen in vivo (19) is subject to continuous inhibition via CTLA-4.

One implication of our data is that individuals with polymorphisms that compromise CTLA-4 function could have more Treg but a reduced capacity to suppress. Thus, defects in CTLA-4 may effectively uncouple Treg number and function. Such a scenario could potentially contribute to the augmented Treg numbers sometimes observed in autoimmune diseases (28, 29).

Tuning the magnitude of immune responses by manipulating the CTLA-4 pathway has long been a major therapeutic goal. However, the initial rationale for this approach, namely, the blockade of inhibitory pathways in effector T cells (30) has been complicated by the emergence of the Treg field. Accordingly, the design of strategies to target the CTLA-4 pathway needs to take into account potential effects on Treg in addition to effector T cells. In particular, the preferential expression of CTLA-4 on Foxp3+ Treg has prompted great interest in the potential role of CTLA-4 in this population. In this respect, CTLA-4 blockade has recently been shown to increase Treg numbers in a mouse melanoma model (31) and in prostate cancer patients (32). Such findings could be construed as discouraging, since they suggest regulation is boosted hand-in-hand with immunity after CTLA-4 blockade. However, we provide evidence that Treg suppression in vivo is compromised in the absence of CTLA-4. This raises the possibility that Treg expanded by CTLA-4 blockade may show impaired regulation. Thus, CTLA-4 blockade may enhance antitumor responses both by increasing effector T cell activation and simultaneously decreasing Treg function. Our data are seemingly at odds with the finding that Treg isolated from anti-CTLA-4-treated mice (31) or humans (32) retain suppressive activity in vitro. However, intriguingly we found that CTLA-4–/– Treg from our transgenic animals were capable of suppressing T cell proliferation when tested in vitro (Fig. 6D), despite their inability to control pathology in vivo. Thus, the capacity to regulate immune responses in vivo is not always accurately predicted from in vitro suppression assays, as previously documented for IL-10 and TGFβ (33, 34). The discrepancy between in vitro and in vivo suppression assays may reflect the fact that Treg are clearly equipped with multiple modes of action (35), any of which may be sufficient for suppression in a controlled in vitro environment. Consistent with this, it has previously been shown that the ability of CTLA-4–/– Treg to suppress in vitro is partially dependent on TGFβ (11), suggesting that this pathway can compensate for the lack of CTLA-4. In contrast, the mechanistic requirements for in vivo suppression may be more stringent and may vary depending on the nature of the immune response and the tissue site.

The generation of TCR-transgenic Treg lacking CTLA-4 allowed us to test the role of this pathway for the first time in a setting uncomplicated by lymphoproliferation or interrupted CD28 signaling. Our data reveal a clear requirement for Treg-expressed CTLA-4 in the regulation of autoimmunity in an adoptive transfer model of diabetes. A role for CTLA-4 in Treg function is supported by the observation that CTLA-4 and Foxp3 need to be expressed on the same cell to provide optimal protection from the lymphoproliferative disease associated with deficiency of either pathway (36). One possibility is that the roles of CTLA-4 in controlling proliferation and suppression are inherently linked such that proliferation impairs suppression; further studies are warranted to investigate this idea. However, we favor the idea that proliferation per se is not sufficient to turn off Treg suppressive function. This view is based on the fact that congenically marked Ag-specific Treg tracked in vivo show efficient suppression despite extensive proliferation (37).

We consider it likely that the requirement for CTLA-4 in Treg function varies depending on the disease setting. Notably, CTLA-4-deficient Treg have been shown to be capable of preventing colitis induced by the transfer of CD4+CD45RBhigh cells into rag-deficient recipients (12). Interestingly, suppression by CTLA-4–/– Treg in this setting was highly dependent on IL-10, which was not the case for wild-type Treg. Thus, pathological responses that can be controlled by IL-10 may not require Treg expression of CTLA-4. In line with this idea, it has recently been shown that mice lacking IL-10 specifically in Treg develop colitis but do not exhibit lymphoproliferation or systemic autoimmunity (38). This implicates IL-10 as an important effector molecule for Treg function within the colon, but suggests that other effector mechanisms (e.g., CTLA-4) can substitute for IL-10 at other sites. The corollary of this argument is that CTLA-4 might be essential for Treg function in some tissues but not others. In this regard, knockdown of CTLA-4 by RNA interference triggers a focused autoimmunity of the pancreas (39), suggesting a nonredundant role for the CTLA-4 pathway in the control of autoimmunity in this tissue.


   Acknowledgments
 
We are grateful to J. Verhagen for critical reading of this manuscript and D. Withers for conjugation of CD4 mAb.


   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 a Medical Research Council Career Development Fellowship (to L.S.K.W.). L.E.C., holds a Biotechnology and Biological Sciences Research Council/Astrazeneca Case Studentship and E.M.S. and G.A.R. are supported by the University of Birmingham Scientific Projects Committee. Back

2 E.M.S. and C.J.W. contributed equally to the work. Back

3 Address correspondence and reprint requests to Dr. Lucy S. K. Walker, Medical Research Council Centre for Immune Regulation, University of Birmingham Medical School, Birmingham, B15 2TT, U.K. E-mail address: L.S.Walker{at}bham.ac.uk Back

4 Abbreviations used in this paper: Treg, regulatory T cell; RIP, rat insulin promoter; LN, lymph node; DC, dendritic cell; mOVA, membrane OVA. Back

Received for publication June 27, 2008. Accepted for publication October 23, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Gough, S. C., L. S. Walker, D. M. Sansom. 2005. CTLA4 gene polymorphism and autoimmunity. Immunol. Rev. 204: 102-115. [Medline]
  2. Kavvoura, F. K., J. P. Ioannidis. 2005. CTLA-4 gene polymorphisms and susceptibility to type 1 diabetes mellitus: a HuGE review and meta-analysis. Am. J. Epidemiol. 162: 3-16. [Abstract/Free Full Text]
  3. 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]
  4. Waterhouse, P., J. M. Penninger, E. Timms, A. Wakeham, A. Shahinian, K. P. Lee, C. B. Thompson, H. Griesser, T. W. Mak. 1995. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270: 985-988. [Abstract/Free Full Text]
  5. Krummel, M. F., J. P. Allison. 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182: 459-465. [Abstract/Free Full Text]
  6. Schneider, H., J. Downey, A. Smith, B. H. Zinselmeyer, C. Rush, J. M. Brewer, B. Wei, N. Hogg, P. Garside, C. E. Rudd. 2006. Reversal of the TCR stop signal by CTLA-4. Science 313: 1972-1975. [Abstract/Free Full Text]
  7. Buhlmann, J. E., S. K. Elkin, A. H. Sharpe. 2003. A role for the B7-1/B7-2:CD28/CTLA-4 pathway during negative selection. J. Immunol. 170: 5421-5428. [Abstract/Free Full Text]
  8. Read, S., V. Malmstrom, F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192: 295-302. [Abstract/Free Full Text]
  9. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192: 303-310. [Abstract/Free Full Text]
  10. Bachmann, M. F., G. Kohler, B. Ecabert, T. W. Mak, M. Kopf. 1999. Cutting edge: lymphoproliferative disease in the absence of CTLA-4 is not T cell autonomous. J. Immunol. 163: 1128-1131. [Abstract/Free Full Text]
  11. Tang, Q., E. K. Boden, K. J. Henriksen, H. Bour-Jordan, M. Bi, J. A. Bluestone. 2004. Distinct roles of CTLA-4 and TGF-β in CD4+CD25+ regulatory T cell function. Eur. J. Immunol. 34: 2996-3005. [Medline]
  12. Read, S., R. Greenwald, A. Izcue, N. Robinson, D. Mandelbrot, L. Francisco, A. H. Sharpe, F. Powrie. 2006. Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo. J. Immunol. 177: 4376-4383. [Abstract/Free Full Text]
  13. Thornton, A. M., E. M. Shevach. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188: 287-296. [Abstract/Free Full Text]
  14. Oida, T., L. Xu, H. L. Weiner, A. Kitani, W. Strober. 2006. TGF-β-mediated suppression by CD4+CD25+ T cells is facilitated by CTLA-4 signaling. J. Immunol. 177: 2331-2339. [Abstract/Free Full Text]
  15. Thornton, A. M., C. A. Piccirillo, E. M. Shevach. 2004. Activation requirements for the induction of CD4+CD25+ T cell suppressor function. Eur. J. Immunol. 34: 366-376. [Medline]
  16. Waterhouse, P., M. F. Bachmann, J. M. Penninger, P. S. Ohashi, T. W. Mak. 1997. Normal thymic selection, normal viability and decreased lymphoproliferation in T cell receptor-transgenic CTLA-4-deficient mice. Eur. J. Immunol. 27: 1887-1892. [Medline]
  17. Chambers, C. A., D. Cado, T. Truong, J. P. Allison. 1997. Thymocyte development is normal in CTLA-4-deficient mice. Proc. Natl. Acad. Sci. USA 94: 9296-9301. [Abstract/Free Full Text]
  18. Hori, S., M. Haury, J. J. Lafaille, J. Demengeot, A. Coutinho. 2002. Peripheral expansion of thymus-derived regulatory cells in anti-myelin basic protein T cell receptor transgenic mice. Eur. J. Immunol. 32: 3729-3735. [Medline]
  19. Fisson, S., G. Darrasse-Jeze, E. Litvinova, F. Septier, D. Klatzmann, R. Liblau, B. L. Salomon. 2003. Continuous activation of autoreactive CD4+CD25+ regulatory T cells in the steady state. J. Exp. Med. 198: 737-746. [Abstract/Free Full Text]
  20. Greenwald, R. J., V. A. Boussiotis, R. B. Lorsbach, A. K. Abbas, A. H. Sharpe. 2001. CTLA-4 regulates induction of anergy in vivo. Immunity. 14: 145-155. [Medline]
  21. Eggena, M. P., L. S. Walker, V. Nagabhushanam, L. Barron, A. Chodos, A. K. Abbas. 2004. Cooperative roles of CTLA-4 and regulatory T cells in tolerance to an islet cell antigen. J. Exp. Med. 199: 1725-1730. [Abstract/Free Full Text]
  22. Itoh, M., T. Takahashi, N. Sakaguchi, Y. Kuniyasu, J. Shimizu, F. Otsuka, S. Sakaguchi. 1999. Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J. Immunol. 162: 5317-5326. [Abstract/Free Full Text]
  23. Walker, L. S., A. Chodos, M. Eggena, H. Dooms, A. K. Abbas. 2003. Antigen-dependent proliferation of CD4+CD25+ regulatory T cells in vivo. J. Exp. Med. 198: 249-258. [Abstract/Free Full Text]
  24. Leach, D. R., M. F. Krummel, J. P. Allison. 1996. Enhancement of antitumor immunity by CTLA-4 blockade. Science 271: 1734-1736. [Abstract]
  25. Peggs, K. S., S. A. Quezada, A. J. Korman, J. P. Allison. 2006. Principles and use of anti-CTLA4 antibody in human cancer immunotherapy. Curr. Opin. Immunol. 18: 206-213. [Medline]
  26. Tivol, E. A., J. Gorski. 2002. Re-establishing peripheral tolerance in the absence of CTLA-4: complementation by wild-type T cells points to an indirect role for CTLA-4. J. Immunol. 169: 1852-1858. [Abstract/Free Full Text]
  27. Sarween, N., A. Chodos, C. Raykundalia, M. Khan, A. K. Abbas, L. S. Walker. 2004. CD4+CD25+ cells controlling a pathogenic CD4 response inhibit cytokine differentiation, CXCR-3 expression, and tissue invasion. J. Immunol. 173: 2942-2951. [Abstract/Free Full Text]
  28. van Amelsfort, J. M., K. M. Jacobs, J. W. Bijlsma, F. P. Lafeber, L. S. Taams. 2004. CD4+CD25+ regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid. Arthritis Rheum. 50: 2775-2785. [Medline]
  29. Liu, W., A. L. Putnam, Z. Xu-Yu, G. L. Szot, M. R. Lee, S. Zhu, P. A. Gottlieb, P. Kapranov, T. R. Gingeras, B. Fazekas de St Groth, et al 2006. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ Treg cells. J. Exp. Med. 203: 1701-1711. [Abstract/Free Full Text]
  30. Allison, J. P., A. A. Hurwitz, D. R. Leach. 1995. Manipulation of costimulatory signals to enhance antitumor T-cell responses. Curr. Opin. Immunol. 7: 682-686. [Medline]
  31. Quezada, S. A., K. S. Peggs, M. A. Curran, J. P. Allison. 2006. CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J. Clin. Invest. 116: 1935-1945. [Medline]
  32. Kavanagh, B., S. O'Brien, D. Lee, Y. Hou, V. Weinberg, B. Rini, J. P. Allison, E. J. Small, L. Fong. 2008. CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependant fashion. Blood 112: 1175-1183. [Abstract/Free Full Text]
  33. Powrie, F., J. Carlino, M. W. Leach, S. Mauze, R. L. Coffman. 1996. A critical role for transforming growth factor-β but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RBlowCD4+ T cells. J. Exp. Med. 183: 2669-2674. [Abstract/Free Full Text]
  34. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, F. Powrie. 1999. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190: 995-1004. [Abstract/Free Full Text]
  35. Tang, Q., J. A. Bluestone. 2008. The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol. 9: 239-244. [Medline]
  36. Chikuma, S., J. A. Bluestone. 2007. Expression of CTLA-4 and FOXP3 in cis protects from lethal lymphoproliferative disease. Eur. J. Immunol. 37: 1285-1289. [Medline]
  37. Billiard, F., E. Litvinova, D. Saadoun, F. Djelti, D. Klatzmann, J. L. Cohen, G. Marodon, B. L. Salomon. 2006. Regulatory and effector T cell activation levels are prime determinants of in vivo immune regulation. J. Immunol. 177: 2167-2174. [Abstract/Free Full Text]
  38. Rubtsov, Y. P., J. P. Rasmussen, E. Y. Chi, J. Fontenot, L. Castelli, X. Ye, P. Treuting, L. Siewe, A. Roers, W. R. Henderson, Jr, et al 2008. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity 28: 546-558. [Medline]
  39. Chen, Z., J. Stockton, D. Mathis, C. Benoist. 2006. Modeling CTLA4-linked autoimmunity with RNA interference in mice. Proc. Natl. Acad. Sci. USA 103: 16400-16405. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
L. E. Jeffery, F. Burke, M. Mura, Y. Zheng, O. S. Qureshi, M. Hewison, L. S. K. Walker, D. A. Lammas, K. Raza, and D. M. Sansom
1,25-Dihydroxyvitamin D3 and IL-2 Combine to Inhibit T Cell Production of Inflammatory Cytokines and Promote Development of Regulatory T Cells Expressing CTLA-4 and FoxP3
J. Immunol., November 1, 2009; 183(9): 5458 - 5467.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. Wu, U. Rauch, E. Korpos, J. Song, K. Loser, P. R. Crocker, and L. M. Sorokin
Sialoadhesin-Positive Macrophages Bind Regulatory T Cells, Negatively Controlling Their Expansion and Autoimmune Disease Progression
J. Immunol., May 15, 2009; 182(10): 6508 - 6516.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schmidt, E. M.
Right arrow Articles by Walker, L. S. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schmidt, E. M.
Right arrow Articles by Walker, L. S. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS