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Division of Molecular Immunology, Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH 45229
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Abstract |
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E
7 integrin is necessary for the homing of Treg at site of Leishmania major infection. The vast majority of Treg present in the dermis at steady-state conditions or during L. major infection express the E chain (CD103) of E7. Genetically susceptible BALB/c mice that lack CD103 become resistant to infection, a phenotype that is associated with a poor capacity of Treg to be retained in the infected site. Such susceptible phenotype can be restored when Treg from wild-type mice were transferred in CD103–/– mice. The central role of CD103 in Treg retention was further demonstrated by usage of blocking Abs against CD103 and the transfer of Treg purified from CD103–/– mice. Our results strongly suggest that this molecule is induced and maintained on Treg following or just prior to their arrival in tissues. Furthermore, the expression of CD103 and the subsequent retention of Treg in tissues is highly regulated by their exposure to Leishmania Ag and the level of activation of the APCs they encounter. Thus, CD103, by controlling Treg retention, can contribute to the outcome of chronic infection by Leishmania. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Using an intradermal low dose model of Leishmania infection, we have previously shown that Treg are essential for the development and maintenance of cutaneous infection with L. major in resistant C57BL/6 mice (13). Treg rapidly accumulate at sites of L. major infection favoring the early parasite expansion. During the acute phase of the infection, which coincides with the expression of effector immune responses, the capacity of Treg to accumulate at site of infection is sharply reduced. After control of the infection, Treg accumulate again in the infected tissue, suppress effector T cells, thus enabling the establishment of parasite persistence (13). In this model, the balance between Treg and effector cells is critical to the expression of immunity. In consequence, such equilibrium has to be tightly controlled. A role for Treg in the pathogenesis of Leishmania infection is not restricted to the resistant strains. In susceptible BALB/c mice, cells that suppress L. major protective immunity have been shown to belong to an IL-4 and IL-10 producing population of cells with regulatory T cell phenotype that also inhibited colitis (14). In this susceptible strain, the removal of Treg transiently exacerbated the Th2 response but eventually led to a better control of the infection (15, 16, 17, 18).
In this report, we addressed the mechanisms controlling Treg retention in Leishmania-infected sites. The integrin E7 has been previously shown to be expressed at the surface of 30% of the Treg in lymphoid tissues (19). Recent data indicated that this integrin also defines a subset of CD4+CD25+ and CD4+CD25– with strong suppressive properties and specific migratory patterns (20, 21). The expression of E (CD103) is positively regulated by TGF- (22), a cytokine that is highly expressed at the vicinity of mucosal tissues and at sites of inflammation. The primary ligand of E7 is E-cadherin (23, 24), an epithelial homophilic adhesion molecule, highly expressed in the skin, site of L. major infection, where it is expressed at the surface of keratinocytes and Langerhans cells (25). Those results support the idea that CD103 could contribute to Treg accumulation at the site of Leishmania infection.
In this report, we showed that CD103 is necessary for the retention of Treg at the site of L. major infection. We showed that virtually all Treg found in the dermis were expressing this molecule. BALB/c CD103–/– mice became resistant to the infection, a phenotype that correlates with a poor recruitment of Treg at site of infection. Our results strongly suggest that this molecule is induced and maintained on Treg following or just prior to their arrival in tissues. Furthermore, we show that the expression of this molecule and the subsequent retention of Treg in tissues are highly regulated by their exposure to Leishmania Ag and the level of activation of the APC they encounter. All together, our results suggest that CD103 plays an important role in the retention of Treg during the course of the infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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C57BL/6, BALB/c, and C.B-17 SCID mice (6–8 wk old) were purchased from Charles River Laboratories. Rag2–/– and B6.SJL (Ly5.1) mice were obtained through the Taconic National Institute of Allergy and Infectious Disease (National Institutes of Health) Exchange Program. Fc
R–/– BALB/c mice (26) were kindly provided by Dr. J. Kohl (Children’s Hospital, Research Foundation, Cincinnati, OH). CD103–/– BALB/c mice (27) were kindly provided by Dr. G. Hadley (University of Maryland Medical School, Baltimore, MD). All mice were maintained in Children’s Hospital Research Foundation Animal Facility under pathogen-free conditions.
Infection protocol
L. major clone V1 (MHOM/IL/80/Friedlin) promastigotes were grown at 28°C in medium 199 supplemented with 20% heat-inactivated FCS (HyClone), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 25 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 µg/ml hemin (in 50% triethanolamine), and 2 µg/ml d-biotin (M199/S). Infective-stage promastigotes (metacyclics) of L. major were isolated from stationary cultures using a Ficoll gradient as previously described (28). Mice were infected in the ear dermis with 103 or 105 L.major metacyclic promastigotes using a 27-gauge 1/2 needle in a volume of 10 µl.
Parasite quantitation
Parasite loads in the ears were determined as previously described (29). Briefly, the ventral and dorsal sheets of the infected ears were separated, deposited dermal side down in RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, and liberase Cl enzyme blend (0.5 mg/ml; Roche). Ears were incubated for 40 min at 37°C. The sheets were dissociated in RPMI 1640 with 10% serum and 0.05% DNase I (Sigma-Aldrich) using a medimachine (BD Bioscience) according to the manufacturer’s protocol. The tissue homogenates were filtered using a 50-µm syringe filter (Falcon Products) and serially diluted in a 96-well flat-bottom microtiter plate containing biphasic medium prepared using 50 µl of NNN medium containing 20% of defibrinated rabbit blood overlaid with 100 µl of M199/S. The number of viable parasites in each ear was determined from the highest dilution at which promastigotes could be grown out after 7 days of incubation at 28°C. The number of parasites was also determined in the local draining lymph nodes (LN) (retromaxillar). The LN were mechanically dissociated and parasite load was determined by limiting dilution as described above.
Analysis of lymphocytes
Single cell suspensions from the ear dermis and LN were obtained as described above. For the analysis of surface markers and intracytoplasmic cytokines cells were stimulated with L. major-infected bone marrow-derived dendritic cells (BMDC) as a source of Ag for 16 h. The cells were cultured for an additional 6 h with brefeldin A (10 µg/ml) (13) and then fixed in 4% paraformaldehyde. Before staining, cells were incubated with an anti-FcIII/II receptor and 10% normal mouse serum in PBS containing 0.1% BSA, 0.01% NaN3. Cells were permeabilized and stained for the surface markers CD3 (145-2 C11), TCR- (H57-597), CD4 (RM4-5), CD25 (PC6C1), CD8 (53-6.7), and CD103 (M290) and for the cytokines IFN- (XNG1.2) and IL-10 (JE56-5H4). Incubations were conducted for 30 min on ice. The isotype controls used were rat IgG2b (A95-1) and rat IgG2a (R35-95). All Abs were purchased from BD Pharmingen. The data were collected and analyzed using CellQuest software and a FACSCalibur flow cytometer (BD Biosciences). For each sample, at least 100,000 cells were analyzed. The frequency of CD4+ and CD8+ T cells was determined by gating on CD3+ or TCR-+ cells.
In some experiments, CD4+CD25+ and CD4+CD25– T cells from retromaxillar LN of naive or Leishmania-infected mice were purified by cell sorting as previously described (30). Purified cells were incubated at a ratio of 5 lymphocytes for 1 BMDC, bone marrow-derived macrophages (M
) (31) or inflammatory M (29) infected or not with L. major (ratio of 5 parasites for 1 cell) with or without 100 ng/ml LPS from Salmonella typhimurium (Sigma-Aldrich), 10 µg/ml anti-CD40 (1C10; eBioscience) or 10 ng/ml TGF-1 (R&D Systems). At 18 h, cells were collected and surface expression of CD103 on the lymphocyte population was evaluated by flow cytometry.
Cytokine assays
For cytokine measurements in culture supernatants, pooled cells from draining LN were resuspended in RPMI 1640 containing FBS/penicillin/streptomycin at 6 x 106 cells/ml, and 200 µl was plated in U-bottom 96-well plates. Cells were incubated at 37°C in 5% CO2 with uninfected or L. major-infected BMDC or alternatively 50 µg/ml soluble Leishmania Ag for 48 h. IFN-, IL-10, and IL-4 production were analyzed by ELISA (R&D Systems) according to the manufacturer’s protocol.
Adoptive cell transfers
CD4+CD25+ or CD4+CD25– T cells were purified from cell sorting as previously described (32). In some cases, CD4+ T cells were pre-enriched by negative selection using magnetic beads, CD4+ T cell isolation kit from Miltenyi Biotec. The T cell subsets were >98% pure as analyzed by flow cytometry. In some experiments, Treg were sorted as CD103– cells. Purified cell subsets were transferred i.v. into mice (RAG–/– or SCID) at the same time as the mice were infected in the ear with L. major. For intradermal injection of lymphocytes, CD4+ T cells from naive wild-type or CD103–/– BALB/c mice or 8 wk-infected BALB/c mice were negatively selected using magnetic beads isolation kit (Miltenyi Biotec, Auburn, CA). Isolated cells were labeled with CFSE (Molecular Probes) before being injected under a volume of 10 µl in the dermis in the presence or absence of 5 mg/ml blocking Ab (M290; BD Pharmingen) or isotype control (BD Pharmingen). Both Abs were purchased endotoxin free. After 18 h, dermal cells were collected and labeled for flow cytometry analysis.
Suppressive assay
CD4+CD25– T cells isolated from naive or chronically infected wild-type or CD103–/– BALB/c mice (4 mo infection) were incubated in PBS containing 1.25 µM CFSE for 5 min at room temperature. The suppression assay was set up in a 48-well plate in 500 µl of complete culture medium per well. CD25– T cells and BMDC were seeded at 5 x 104 and 7 x 104 cells, respectively. CD25+ T cells (5 x 104) were added or not into the wells where the CD25– T cells were activated with 0.5 µg/ml soluble anti-CD3 (clone 145-2C11). After a 5-day culture at 37°C, 5% CO2 and culture supernatant were collected for cytokine ELISA. Cells were collected and fixed with 4% (w/v) paraformaldehyde in PBS. Fc receptors were blocked with 24G2 cell line culture supernatant, and cells were stained with anti-CD4 (clone RM4-5) and anti-TCR- chain (clone H57-597) antibodies. Cell acquisition was performed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). Cell proliferation was quantitated for the CD4+TCR-+ population by analyzing the CFSE fluorescence pattern with ModFit LT software (Verity Software House).
Statistical analysis
Statistical analyses were performed using Prism software (GraphPad). Dual comparisons were made using the unpaired Student’s t test. All data from parasite numbers were log transformed before statistical tests were conducted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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We had previously shown that Treg accumulates at sites of Leishmania infection (13). Based on previous work showing that a subset of Treg expressed high levels of the integrin E (CD103) (19), we postulated that CD103 (the E chain of the E7 integrin) may contribute to the retention of Treg at the site of Leishmania infection. To address this question we followed the expression of CD103 in the dermis and regional LN during the course of the infection in resistant C57BL/6 and susceptible BALB/c mice. We found that the expression of CD103 was differentially expressed between CD4+CD25– T cells and Treg in the dermis of C57BL/6 and BALB/c mice (Fig. 1). In both mouse strains, all of the CD4+CD25+ T cells from the dermis at steady-state condition expressed CD103 (Fig. 1A and data not shown). In C57BL/6 mice, during the acute phase (5 wk), at a time in which Treg stop accumulating in the dermis (13), the proportion of Treg expressing CD103 decreases from 96% at steady-state condition to 76%. The mean fluorescence intensity (MFI) of CD103 expression by Treg decreased also significantly in intensity (from an MFI of 540 to 200, Fig. 1B) suggesting the presence of Treg with a wider range of CD103 expression at this stage of the infection. During the chronic phase of the infection, CD103 expression increased (up to 450 at 20-wk postinfection, Fig. 1B).
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The pattern of expression of this molecule in the regional LN followed the one observed in the infected site (Fig. 1A). Of note, while a small percentage of CD25– T cells expressed CD103 at steady-state condition, after infection the expression of this molecule became restricted to the CD25+ subset (Fig. 1A). The expression of CD103 at the surface of Treg in regional LN increased during the chronic phase (from 28% to 47%) and kept increasing (in proportion and intensity) with the age of the mice (up to 70% of the Treg and MFI of 580 at 1 year postinfection, data not shown). In contrast to resistant C57BL/6 mice, in susceptible BALB/c mice infected with a low dose of parasite (103), the number of cells expressing a regulatory T cell phenotype (CD4+CD25+CTLA-4+CD45RBlow) increased in a constant manner during the course of the infection (data not shown). The vast majority of Treg in the dermis of BALB/c mice expressed CD103 (over 80% at all time points), while <20% of the CD4+CD25– T cell expressed this marker (data not shown). The intensity of CD103 expression on the surface of intradermal Treg was significantly higher than on the surface of CD4+CD25– T cells at steady-state condition and was sustained during the course of the infection (Fig. 1C).
We next addressed whether similar patterns of expression were observed for other molecules that had been previously shown to contribute to T cell homing (reviewed in Ref. 33). At different time points postchallenge, both intradermal subsets of CD4+ lym-phocytes, Treg (CD4+CD25+), and effector populations (CD4+CD25–) expressed high levels of CD44, CD18 (2 chain of LFA-1) and CD11a (L chain of LFA-1) (data not shown). Both subsets expressed CD49d (4 chain of VLA-4 and LPAM-1) and CD61 (3 chain of 3v and 3iib) between 20 and 30%. Both subsets were negative for CD62L. Thus, only the expression of CD103 appeared to be restricted to Treg during the course of the infection.
CD103–/– mice are resistant to infection with L. major
The sustained expression of CD103 in BALB/c mice suggested that an impairment in the expression of this molecule may affect their susceptible phenotype. To determine the functional impact of CD103 ablation on Treg, we infected wild-type or CD103–/– BALB/c mice with two doses of Leishmania; a high dose of 105 parasites that induces a strong Th2 response with an exacerbated susceptibility phenotype (34) and a low dose of 103 parasites producing a non-healing phenotype but with a less polarized response and milder lesions (35). Strikingly, the difference between wild-type and CD103–/– mice depended on the inoculum size. At a high dose of parasites, both wild-type and CD103–/– mice developed a severe non-healing phenotype (Fig. 2A) with a comparable number of parasites at sites of infection and similar levels of IL-4 and IL-10 production in response to Leishmania Ag (data not shown). At this dose of parasites, no differences were observed in the number and phenotype of intradermal lymphocytes at different time points postinfection (data not shown). In contrast, at a lower dose of infection (103) CD103–/– mice were strongly resistant to infection compared with wild type. The lesion size was significantly reduced at all time points and CD103–/– mice resolved their infection after 9 wk of infection. (Fig. 2, A and B). Parasite number was also significantly reduced in the dermis and the draining at 8 wk postinfection (Fig. 2C). Such phenotype correlates with a shift in the nature of the cytokines produced by draining LN cells in response to Leishmania Ag from moderate IFN- and a high level of IL-4 for wild-type mice and a higher IFN- production at 4 and 8 wk and no detectable IL-4 for the CD103–/– mice at 8 and 12 wk postinfection (Table I). After 12 wk of infection, the level of IFN- was comparable between the two strains whereas IL-10 production was reduced in CD103–/– mice (Table I). The number of intradermal CD4+ lymphocytes releasing IFN- in response to Leishmania Ag was also enhanced in the dermis of CD103–/– mice (16% vs 9% in wild-type mice at 4 wk postinfection, data not shown). CD103–/– mice have previously been described to have a reduced number of intradermal lymphocytes at steady-state condition (36). At 4 wk postinfection the number of Treg was significantly reduced in the dermis of the CD103–/– mice whereas only a trend was observed for the CD25– cells (Fig. 3). At 8 wk postinfection, at a time when CD103–/– mice had efficiently controlled their parasite load, the number of both populations was dramatically reduced compared with wild-type mice (Fig. 3). A reduction in antigenic load at site of infection can explain that both populations of T cells were reduced at this time point. This reduction of Treg cells was restricted to the dermis since the peripheral LN of CD103–/– mice contained a similar number of Treg at steady-state conditions and a similar proportion of Treg were found in the LN during the infection (data not shown). To determine whether the phenotype observed in CD103–/– mice could be due to an impairment in the function of Treg, CD4+CD25+ T cells from naive or chronically infected wild-type or CD103–/– mice, cells were purified and assessed for their capacity to suppress effector T cells proliferation and cytokine release. The CD4+CD25+ cells from wild-type and CD103–/– naive mice suppressed equally the proliferation as well as both IL-4 and IFN- release of effector lymphocytes in response to anti-CD3 stimulation (Table II). When CD4+CD25+ T cells were purified from mice infected for 3 mo, Treg from CD103–/– mice were still able to suppress effector T cell proliferation and cytokines release whereas CD4+CD25+ T cells from infected wild-type mice suppressed efficiently the release of cytokines by effector cells but poorly suppressed their proliferation (Table II). Thus, Treg suppressive function is preserved in CD103–/– mice.
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A previous report showed that the number of intradermal lymphocytes was reduced in CD103–/– mice (36). However, the expression of CD103 is not restricted to CD4+ T lymphocytes since this molecule can also be expressed on subsets of CD8+ T cells, 
T cells, dendritic cells (DC), and mast cells (37, 38, 39, 40, 41, 42, 43, 44). Thus, impairment in intradermal lymphocytes homing could be the consequence of another cell defect. To directly address the role of CD103 in the retention of intradermal lymphocytes, we purified CD4+ T cells from wild-type or from CD103–/– naive BALB/c. Cells were labeled with CFSE and inoculated intradermally in BALB/c naive mice. The following day, the number of CFSE-labeled CD4+CD25– or CD4+CD25+ T cells retained in the dermis was quantified using flow cytometry analysis (Fig. 4A). The number of CD4+CD25+ T cells (but not CD4+CD25– T cells) retained in the dermis was significantly reduced when CD4+ T cells originated from CD103–/– compared with wild-type mice (Fig. 4B). To confirm that such impairment was not due to a developmental defect of T cells in CD103–/– mice, we used blocking anti-CD103 Ab in vivo. We used BALB/c mice deficient for the common Fc receptor chain to prevent nonspecific binding of the blocking anti-CD103 Abs (IgG2a) through their IgG Fc receptor (26). Negatively selected CD4+ T cells from retromaxillar LN were CFSE labeled and injected intradermally in the presence of 5 µg of anti-CD103 (M290) or isotype control. The following day, the number of lymphocytes that were retained in the injected ear was evaluated. As shown in Fig. 4C, the number of both CD4+CD25– and CD4+CD25+ T cells was dramatically reduced when anti-CD103 was injected with CD4+ T cells compared with isotype control. Similar results were obtained when Treg were obtained from the dermis of BALB/c mice infected for 8 wk (85% reduction of Treg number in the presence of blocking Ab compared with isotype control, data not shown). Interestingly, while CD4+CD25+ T cells constituted only 10% of the original inoculums, after 1 day, approximately 50% of the cells retained were CD25+ suggesting that, at condition of homeostasis, Treg are preferentially retained in the dermis compared with CD4+CD25– T cells. One day after intradermal injection, >70% of the CD4+ T cells retained in the skin were CD103 + (data not shown).
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Since in CD103–/– mice other cells that could contribute to Leishmania susceptibility may be affected, we directly addressed the capacity of CD103–/– lymphocytes to migrate into the skin upon infection. Purified CD4+CD25+ T cells from wild-type or CD103–/– mice were transferred i.v. into SCID mouse recipients and at the same time the mice were infected intradermally with L. major. At 3 wk postinfection and transfer, the infected tissues were collected. The number of T cells able to accumulate in the dermis was strongly reduced when CD4+CD25+ T cells were purified from CD103–/– compared with wild-type mice (Fig. 5). In contrast, the engraftment of Treg from CD103–/– was comparable to wild type in regional LN. These data formally demonstrate that in the absence of CD103, Treg are selectively impaired in their capacity to be retained at site of infection.
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We next determine whether CD103 was required for Treg entrance at site of infection. We purified CD4+CD25+ from congenic (Ly5.1+) mice as CD103 negative and co-injected them with CD4+CD25– cells from wild-type background (Ly5.2). Since CD25 can be down-regulated posttransfer, this approach allows us to track cells according to their origin (13). Lymphocytes were co-transferred i.v. into Rag–/– mice at the time of L. major infection. At 3 wk postinfection and transfer, the dermal cells were collected and the phenotype of the cells analyzed by flow cytometry. The cells were gated on TCR--positive events (Fig. 6, left panel) and CD4+CD25+ or CD4+CD25– events (Fig. 6, middle panel) to reproduce the gating strategy that was used in wild-type mice. The vast majority of the cells still expressing CD25 posttransfer originated from regulatory T cells background as indicated by their expression of the congenic marker Ly5.1 (Fig. 6, right panel). Of Treg still expressing CD25, 70% had acquired CD103 expression (Fig. 6). Following transfer, 35% of dermal Treg lost their expression of CD25. Interestingly, the loss of CD25 expression on those cells also correlated with a poor expression of CD103.
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The surface expression of CD103 on Treg is highly regulated
Since the expression of CD103 decreased during the course of the infection in resistant C57BL/6 mice (Fig. 1), we determined whether the exposure of Treg to a pro-inflammatory or a deactivating environment could modulate the expression of this molecule. We purified Treg from the LN of chronically infected mice and exposed them to BMDC or M infected or not with Leishmania. Interestingly, when Treg purified from chronically infected mice were exposed to Leishmania-infected DC, the expression of CD103 was increased in percentage (from 43 to 71%, Fig. 7A) and intensity (from an MFI of 55 to 86) (Fig. 7B). On the other hand, when Treg were exposed to BMDC in the presence of LPS, the level of expression of CD103 was significantly decreased from an MFI of 86 to 10 (Fig. 7B). Likewise, CD103 expression at the surface of Treg was down-regulated following exposure to DC that were activated with anti-CD40 agonist Ab (clone 1C10, data not shown). TGF- can enhance CD103 expression on T cells (22). Consistently, if Treg were exposed to infected BMDC in the presence of TGF-, the expression of CD103 was enhanced in proportion (from 71 to 92%, data not shown) and intensity (from 95 to 140, data not shown). Since previous report showed that Treg can express TLRs and in particular TLR-4 (45), we tested the direct role of LPS on CD103 expression by Treg. A direct exposure of Treg to LPS failed to induce down-regulation of CD103 (Fig. 7B) demonstrating that such effect was mediated by activated DC and not through direct interaction of LPS with Treg. Inflammatory M or M derived from bone marrow were unable to either enhance or down-regulate CD103 expression under those various conditions (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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E chain (CD103) of the E7 integrin plays a critical role in the retention of Treg at the site of Leishmania infection. We and others have shown previously that the outcome of chronic infection by L. major was tightly controlled by the equilibrium between Treg and effector T cells (13, 16, 17, 30). Here we report that CD103 can contribute to the local control of this equilibrium. Genetically susceptible mice that lack CD103 became resistant to the infection, a phenotype that was associated with an impairment of Treg to accumulate at sites of infection. Our results demonstrate that CD103 is not necessary for the entrance of Treg in tissues but induced upon or just prior to their arrival in the infected dermis and required for Treg retention. The expression of CD103 is highly regulated by the environment, with pro-inflammatory signals inducing its down-regulation. This report is, to our knowledge, the first direct demonstration that a molecule can selectively contribute to Treg homing at sites of infection.
The CD103 molecule is the E chain of the E7 integrin whose principal ligand is E-cadherin, an epithelial homophilic adhesion molecule. Expression of CD103 at very high levels is a hallmark of intraepithelial lymphocytes residing in the gut wall and other epithelial compartments such as the skin and lung (46). This molecule is also expressed at high levels by subsets of mucosal mast cells, mucosal DC, and a subset of DC from lymphoid organs (37, 38, 39, 40, 41, 42, 43, 44). The first suggestion that CD103 could be involved in T cell homing came from the observation that in CD103 deficient mice, the number of T cells was reduced in the skin and intestinal epithelia (36). Previous studies indicate that CD103 expression also characterizes a subset of peripheral CD8+ T cells with a unique capacity to access the epithelial compartment of organ allograft (27, 47). Recently, it has been shown that CD103 was also expressed at the surface of natural CD4+CD25+ T cells from lymphoid organs, 30% of which express this molecule (19). Treg expressing CD103 efficiently prevent the development of colitis in the SCID model, although those cells display rather poor suppressive capacity in vitro (20). This molecule also allows to define subsets of regulatory T cells (CD25+ or CD25–) present in lymphoid organs with a distinct profile of adhesion molecules (21). Those subsets, following activation in vitro, were able to migrate to sites of inflammation and control the intensity of the local response (21). Thus, those studies demonstrated that CD103 allows identification of subsets of regulatory T cells, but a direct role for the CD103 molecule in Treg retention remained to be addressed.
Our results demonstrate that CD103 is required for Treg retention at steady-state condition and during Leishmania infection. At steady-state conditions, we have shown that half of the lymphocytes homing in the dermis express a regulatory phenotype with a high level of CD25, glucocorticoid-induced TNF receptor, and CTLA-4 expression (13). The skin is a highly exposed organ that requires multiple levels of regulation in which regulatory T cell accumulation may be instrumental. In this study, we showed that the vast majority of dermal Treg also expressed high levels of CD103 and that this molecule was required for the homing of Treg in the dermis. Blockade with anti-CD103 Ab prevents Treg retention in the dermis, and Treg purified from CD103–/– are directly impaired in their capacity to accumulate or be retained in the dermis of naive mice.
Our results strongly suggest that CD103 is also required for the homing of Treg during the course of infection with Leishmania. We have previously shown that in genetically resistant mice, Treg are preferentially attracted to sites of L. major infection during parasite expansion and following clinical cure (30). Such retention correlates with a strong expression of CD103 at the surface of Treg. Genetically susceptible mice that lack CD103 are resistant to L. major infection, a phenotype that is associated with a poor capacity of Treg to accumulate in the infected skin. Although we cannot exclude that other parameters may contribute to the resistant phenotype observed in CD103–/– mice, a direct role for CD103 in Treg retention is supported by the fact that Treg from CD103–/– transferred into SCID mice are directly impaired in their capacity to accumulate at the site of Leishmania infection.
Previous reports supported a role for Treg in the establishment of the susceptible phenotype in BALB/c mice. For instance, the CD4+ T cells that suppress L. major immunity in BALB/c have been shown to belong to an IL-4 and IL-10 producing population of cells CD45RBlow that also inhibited colitis (14). In addition, the biweekly administration of anti-CD25 during the first 4 wk of infection renders BALB/c mice resistant to L. major infection (15). More recently, it was shown that in BALB/c mice, the removal of Treg initially enhanced Th2 phenotype but eventually led to a better control of the infection (16, 17, 18). In our low dose infection model, the reduction of Treg in the infected dermis (and not the regional LN) of CD103–/– mice was associated with a shift toward a self-controlled Th1 phenotype. A possible explanation for the role that Treg play in this polarization would be that, in susceptible mice, Treg at the site of infection by maintaining a deactivating environment, modulate the innate response in a way that favors a Th2 polarization. A direct confirmation that Treg favor Th2 responses in our model came from the fact that the transfer of Treg from wild-type mice was sufficient to restore a Th2-susceptible phenotype in CD103–/– mice. This result is in accordance with recently published observations showing that during Schistosoma mansoni infection, Treg suppress Th1 responses allowing for the development of a protective Th2 response (48). Interestingly, despite a reduction of Treg at sites of infection, no substantial difference in IL-10 production by LN cells was detectable between the wild-type and CD103–/– mice until 12 wk postinfection. Since Treg are a major source of IL-10 in various parasitic model (13, 48, 49), this finding is in apparent contradiction with a reduced role for Treg in CD103–/– mice during Leishmania infection. On the other hand, in BALB/c mice infected with Leishmania other populations of T cell have been shown to produce IL-10 (50). In addition, Treg from LN of CD103–/– mice display strong suppressive function in vitro as assessed by their capacity to inhibit proliferation and cytokine production by effector T cells. Thus, although the number of Treg is greatly reduced in the dermis of infected CD103–/– mice, the number and function of Treg in regional LN is not affected.
Several types of regulatory cells exist some of which are induced in response to infectious challenge (e.g., Tr1 or TH3) and some of which are judged as natural regulators (51). In our present study, we have primarily addressed the role of natural Treg; however, during chronic infections in BALB/c mice, we cannot exclude that both natural and inducible Treg contribute to the susceptible phenotype. For instance, natural Treg are more likely to contribute to the early events occurring during infection whereas both subsets could control later stage of the infection. Indeed, Treg extracted from LN of chronically infected mice were less able to suppress effector cells than naive natural Treg, suggesting that other populations were present in the CD25+ fraction. Nevertheless, our results suggest that regardless of their origin, Treg are not retained in the absence of CD103.
The requirement for CD103 expression does not apply to effector T cells or to Treg when high inflammatory conditions are present. When LPS was injected intradermally, the population of T cells mobilized in the dermis was CD103 negative. Likewise, the effector T cell population mobilized in Leishmania-infected dermis did not express this molecule. In addition, both populations of lymphocytes can be efficiently recruited in the dermis of CD103–/– mice following inflammatory stimuli (LPS) or when the mice were infected with a high dose of parasites. Thus, a role for CD103 appears to be mostly restricted to Treg when homeostasis must be restored. We cannot exclude that a fraction of conventional CD4+ T cells also require CD103 for their retention. Indeed, half of the CD25– cells at steady-state condition (but not during infection) expressed this marker but with lesser intensity than Treg. In addition, use of anti-CD103-blocking Abs also reduced the number of CD25– cells that were retained in the dermis. To determine whether those cells are derived from regulatory T cells or conventional CD4+ T cells remains to be addressed.
A role for CD103 in the retention of Treg is supported by previous observations (21). In a model of inflammatory skin disorder, mice reconstituted with CD4+ T cells from CD103–/– mice develop more severe lesions compared with mice reconstituted with CD4+ T cells from wild-type mice (52). In addition, CD103–/– mice tend to develop spontaneous skin disorders (52). In humans, the expression of E7 is also associated with some clinical cutaneous disorders such as T cell lymphoma, lichen planus, or atopic dermatitis (40, 53, 54, 55). All together, these results suggest that a dysregulation of CD103 expression correlates with a disruption of the local immunoregulation of the skin leading to excessive pathological responses. Thus, by controlling Treg tropism, CD103 could contribute to the control of peripheral homeostasis and the pathological process due to microbial infection.
Our results suggest that CD103 does not define a subset of regulatory T cells with distinct properties but rather that this molecule is rapidly induced and maintained on Treg following or just prior to their arrival in tissues. Using transfer experiments, we showed that Treg purified as CD103 negative can enter efficiently at the site of infection and express this marker. In addition, our results demonstrate that the only cells able to express high levels of CD103 originated from the natural Treg population. The acquisition of this marker following or just prior to migration in tissues is also observed for CD8+ T cells that respond to donor alloantigens in the graft site (47). Interestingly, the loss of CD25 expression that is observed on Treg upon homeostatic proliferation (13) is also associated in our study with a reduction in CD103 expression in Leishmania-infected sites. Nevertheless, a small proportion of transferred Treg that became CD25– still expressed CD103. Previous results published by Huehn et al. (21) suggest that CD4+CD25–CD103+ cell subsets isolated from lymphoid organs have undergone repetitive cell divisions. Consistent with this hypothesis, aging mice have an increased number of cells expressing CD103 with functional suppressive function (56). Thus, this subset may have originated from CD25+CD103+ Treg cells that have undergone massive proliferation. All together these results suggest that CD103 may be a better predictor of the natural Treg origin than CD25 and could allow purification of Treg that have been previously exposed to Ag in the periphery. Such an approach may be particularly useful in human studies in which peripheral blood is the most accessible compartment.
Although Treg control the intensity of effector immune responses, their functions also have to be controlled. Such control occurs either by direct inhibition of their function or by overriding their suppressive effects on effector cells (2). Although the homing property of Treg remains poorly defined, their capacity to be selectively retained at sites where regulation is required may represent an important factor in the control of peripheral homeostasis. We had previously shown that both effector T cells and Treg could enter at primary and secondary sites of infection with similar efficiency (30) suggesting that the regulation of Treg accumulation is not at the level of recruitment but more likely at the level of differential retention. In this report, our results suggest that the encounter with the microenvironment and the subsequent regulation of CD103 will dictate the capacity of Treg to be retained. Previous reports demonstrated that the E subunit of the integrin E7 is transcriptionally regulated by TGF- (22). Supporting this observation, in susceptible BALB/c mice (in which high levels of TGF-, IL-4, and IL-10 are produced (57, 58)), Treg maintained a high level of expression of CD103 during the course of the infection. Thus, a deactivating environment that dominates in the context of an early infection, a chronic infection, or a highly susceptible phenotype will favor CD103 expression at the surface of Treg. In vitro, exposure of Treg purified from chronically infected mice to L. major-infected DC led to an increase of CD103 expression suggesting that the parasite itself manipulates its environment to favor Treg retention. It is therefore tempting to speculate that Treg expressing CD103 found in lymphoid organs is enriched in Treg that are specific for Ags that are presented in tissues enriched in deactivating cytokines such as the gut, the lung, or the skin. In contrast, when Treg were exposed to infected DC in the presence of proinflammatory stimuli (e.g., LPS, anti-CD40), CD103 expression was sharply down-regulated. Our results in vivo mirror those observations; CD103 expression at the surface of Treg is down-regulated in resistant C57BL/6 mice during the acute phase of the infection. During this phase, a proportion of Treg in the dermis are negative for CD103, and the overall intensity of fluorescence decreased compared with steady-state conditions, which suggests the presence of Treg with a wider expression of CD103. During this phase, parasite invades DC, an event that induces DC activation (59). In contrast with lymphoid cells, when Treg were purified from the infected dermis, the expression of CD103 could not be further modulated in vitro. Thus, those results suggest that during the acute phase of the infection, Treg that are patrolling through the infected site would not encounter conditions that favor the expression of this marker. Similarly, Treg that were elicited by the intradermal injection of high levels of LPS had a reduced expression of CD103. Thus, our results suggest that the expression of CD103 and the subsequent retention of Treg in tissues are highly regulated by the level of activation of the microenvironment they encounter.
Integrin-mediated cell adhesion with the extracelluar matrix and neighboring cells can profoundly influence a variety of signaling events including those involved in mitogenesis, survival, and differentiation (reviewed in Ref. 60). For instance, the role of CD103 as a costimulatory molecule has been previously shown for thymocytes (61). E-cadherin, the ligand of E7, is highly expressed at the surface of keratinocytes and Langerhans cells in the skin, site of L. major infection. Understanding the functional consequences of the interaction between E7 and its natural ligand on Treg function and survival remains to be addressed.
In this report, we propose that the regulation of Treg accumulation at sites of infection is not dictated by their capacity to enter but more likely by the capacity of Treg to be selectively retained or alternatively to better survive/proliferate locally. Creating a microenvironment that favors Treg retention may represent a fundamental strategy to favor microbial survival. Thus, Leishmania, like other pathogens known to produce chronic infection, triggers the production of deactivated cytokine (TGF-, IL-10) by the cells they infect. Treg patrolling through tissues could encounter Ag in a context that will or will not favor their local retention providing for an additional level of control of Treg function. All together, our results suggest that the integrin E7 could be used as a therapeutic target to manipulate the expression of local immunity.
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1 This work was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health Research Grant R01AI057992-01 (to Y.B.), Cincinnati Children’s Hospital Medical Center and the Ellison Medical Foundation. G.S. is supported by the Coodenacao de Aperfeicoamento de Pessoal de Nivel Superior. 
2 I.S. and S.K.R. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Yasmine Belkaid, at his current address: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Drive, Bethesda, MD 20852. E-mail address: ybelkaid{at}niaid.nih.gov
4 Abbreviations used in this paper: Treg, regulatory T cell; LN, lymph node; DC, dendritic cell; BMDC, bone marrow-derived DC; M, macrophages; MFI, mean fluorescence intensity.
Received for publication November 16, 2004. Accepted for publication January 30, 2005.
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) from ear cells during the course of the infection of self-healing C57BL/6 mice. The values are expressed as the difference of MFI between anti-CD103 staining and isotype control ± SD, four mice per time point; **, statistically significant differences in MFI between Treg at steady-state condition and at 5 wk postinfection, p < 0.001). C, Intensity of CD103 expression at the surface of TCR-
MFI between anti-CD103 staining and isotype control ± SD, n = 3. This data is representative of three distinct experiments. Asterisks represent statistically significant differences between indicated groups (**, p < 0.001; ***, p < 0.0001). C, CD4+ T cells mobilized by LPS injection are CD103 low or negative. C57BL/6 mice were injected intradermally with PBS, or 100 ng of LPS. At 18 h postinjection, dermal cells were extracted, and the phenotype of the collected cells was analyzed by flow cytometry. Events were gated on TCR-