Functional Analysis of Effector and Regulatory T …Isolation of T-cell subsets. The separation of T-cell subsets for transfers and in vitro stimulation was performed as follows. Cells

INFECTION AND IMMUNITY, May 2008, p. 1908–1919 Vol. 76, No. 50019-9567/08/$08.00�0 doi:10.1128/IAI.01233-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Functional Analysis of Effector and Regulatory T Cells in a ParasiticNematode Infection�

Sebastian Rausch,1 Jochen Huehn,2 Dennis Kirchhoff,3 Justyna Rzepecka,1 Corinna Schnoeller,1Smitha Pillai,1 Christoph Loddenkemper,4 Alexander Scheffold,3 Alf Hamann,2

Richard Lucius,1 and Susanne Hartmann1*Department of Molecular Parasitology, Humboldt-University, Berlin 10115, Germany1; Experimental Rheumatology, Charite Berlin,

Berlin 10117, Germany2; Immunomodulation Group, Deutsches Rheuma-Forschungszentrum Berlin, Berlin 10117,Germany3; and Pathology/RCIS, Charite Berlin, Berlin 12200, Germany4

Received 7 September 2007/Returned for modification 10 October 2007/Accepted 15 February 2008

Parasitic nematodes typically modulate T-cell reactivity, primarily during the chronic phase of infection. Weanalyzed the role of CD4-positive (CD4�) T effector (Teff) cells and regulatory T (Treg) cells derived from micechronically infected with the intestinal nematode Heligmosomoides polygyrus. Different CD4� T-cell subsetswere transferred into naıve recipients that were subsequently infected with H. polygyrus. Adoptive transfer ofconventional Teff cells conferred protection and led to a significant decrease in the worm burdens of H.polygyrus-infected recipients. Roughly 0.2% of the CD4� T cells were H. polygyrus specific based on expressionof CD154, and cells producing interleukin 4 (IL-4) and IL-13 were highly enriched within the CD154�

population. In contrast, adoptive transfer of Treg cells, characterized by the markers CD25 and CD103 and thetranscription factor Foxp3, had no effect on the worm burdens of recipients. Further analysis showed that soonafter infection, the number of Foxp3� Treg cells temporarily increased in the inflamed tissue while effector/memory-like CD103� Foxp� Treg cells systemically increased in the draining lymph nodes and spleen. Inaddition, Treg cells represented a potential source of IL-10 and reduced the expression of IL-4. Finally, underin vitro conditions, Treg cells from infected mice were more potent suppressors than cells derived from naıvemice. In conclusion, our data indicate that small numbers of Teff cells have the ability to promote hostprotective immune responses, even in the presence of Treg cells.

Infections with parasitic nematodes have been shown tomarkedly modulate the host’s immune system as a means toescape immune effector mechanisms and to ensure parasitesurvival within an immunocompetent host (35, 55). Highlyeffective immune evasion mechanisms are key elements of thelong-lasting persistence of parasitic worms. Both parasite-spe-cific and nonspecific immune suppression are well documented(19, 47, 60). Usually, acute infection stimulates antigen-specificT-cell proliferation, but with increasing exposure to parasiteantigens, the immune system becomes hyporesponsive (34).

Immune responses against gastrointestinal nematodes in-clude strong immunoglobulin G1 (IgG1) and IgE responses,eosinophilia, intestinal mastocytosis, goblet cell hyperplasia,and smooth-muscle hypercontractility (1, 12, 70). Th2-driveneffector mechanisms were shown to be protective against gas-trointestinal helminths, whereby interleukin 13 (IL-13) andIL-4 play an important role in primary, as well as secondary,infections (7, 13, 15, 53). Recently, alternatively activated mac-rophages have been introduced as an IL-4-dependent effectorpopulation essential for protective immunity to challenge in-fections with Heligmosomoides polygyrus (4). However, there isa lack of information on the activity of CD4-positive (CD4�)T-cell subsets during the chronic phase of primary nematodeinfections (when T-cell reactivity is suppressed) and on the

T-cell subsets that contribute to host-protective or parasite-beneficial immune responses. In contrast, the interplay of T-cell subsets and regulation of effector responses in bacterial(28) and protozoan infections, like those with Leishmania (9,38, 66) and Plasmodium (21, 44) species, are well defined.

It has been suggested that nematode infections reduce bothTh1- and Th2-mediated responses by profoundly influencingregulatory pathways (5, 35). Regulatory T (Treg) cells representa subset of CD4� T cells that are critically involved in balanc-ing the reactivity of the immune system and preventing auto-immunity (40, 50). Alongside their role in preventing autoim-mune reactions, Treg cells have been shown to control excessiveinflammatory responses against pathogens (20, 48). On theother hand, a strict control of T effector (Teff) cell responses byTreg cells can promote pathogen persistence (9, 28, 44, 67).Some cell markers used to identify Treg cells are CD25, glu-cocorticoid-induced tumor necrosis factor receptor family-re-lated gene (GITR), and the transcription factor forkhead boxtranscription factor P3 (Foxp3). In addition, the integrin�E(CD103)�7 is a marker for a subset of highly potent effector/memory-like Treg cells, and CD4� CD103� cells were found tobe the most potent suppressors of inflammatory processes indisease models, such as colitis and arthritis (22, 23, 30, 54).Here, we used CD25, CD103, and Foxp3 to identify Treg cells.For the characterization of parasite-specific Teff cells, we usedCD154, a marker recently shown to exhibit exquisite specificityfor antigen-activated T cells (15, 26).

To investigate the roles of different CD4� T-cell subsetsduring the chronic phase of a primary nematode infection, we

* Corresponding author. Mailing address: Department of MolecularParasitology, Humboldt-University Berlin, Philippstr. 13, 10115 Berlin,Germany. Phone: (49) 30 2093 6450. Fax: (49) 30 2093 6051. E-mail:[email protected].

� Published ahead of print on 3 March 2008.

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used the gastrointestinal nematode H. polygyrus, which residesin the proximal third of the mouse duodenum for up to severalmonths during primary infection. Mice become infected byingestion of infective larvae (L3) that invade the duodenalwall, where development to the L4 stage takes place. L4 reen-ter the gut lumen and mature to adults, which are chronicallymaintained during primary infection (42). We used H. poly-gyrus infection to address the role of CD4� Teff cells and Treg

cells with regard to worm expulsion and the role of Treg cells inmodulating Teff cell function. Our data demonstrate that adop-tive transfer of Teff cells leads to protective immune responsesin which antigen-specific CD4� T cells produce predominantlyIL-4 and IL-13. In contrast, CD4� Treg cells from chronicinfection show no effect on the adult worm burden after adop-tive transfer, although they are highly potent suppressors invitro.

MATERIALS AND METHODS

Mice, parasites, and infection. H. polygyrus was maintained by serial passage inBALB/c mice. The infective larvae (L3) were obtained from feces culture, ex-tensively washed, and stored in distilled water at 4°C. BALB/c and C57BL/6 micewere purchased from the Bundesinstitut fur Risikobewertung, Berlin, Germany.C57BL/6-Tg(CAG-EGFP)C15-001-FJ001Osb mice were a gift of M. Okawa,Osaka University. Male mice at 7 to 10 weeks of age were infected with 200 L3using a gavage tube. Infection was surveyed by fecal egg count. The worm burdenwas determined by collecting adult worms from the small intestine on the day ofdissection. Animals were housed and handled following national guidelines andas approved by our animal ethics committee.

Preparation of adult worm antigen. Soluble worm antigen was prepared fromadult worms kept in culture in RPMI medium containing 100 U/ml penicillin and100 �g/ml streptomycin for 24 h. The worm material was homogenized andsonicated (1 min; 60 W) on ice in phosphate-buffered saline (PBS) (pH 7.4). Thehomogenate was centrifuged (20 min; 20,000 � g; 4°C), and the supernatant waspassed through a 0.4-�m filter (Schleicher & Schuell, Germany) for sterilization.The protein content was determined by the bicinchoninic acid test (Pierce).Antigen extracts were stored at �80°C until they were applied.

Antibodies, staining, and sorting reagents. The following antibodies and sec-ondary reagents were purchased from BD Biosciences (Heidelberg, Germany):�CD4 fluorescein isothiocyanate (FITC)/PerCP (RM4-5), �CD103 phyco-erythrin (PE) (M290), �CD25 allophycocyanin (APC)/PerCP-Cy5.5 (PC61),�CD3e (145-2C11), �CD28 (37.51), �CD40 (HM40-3), �IL-10 PE (JES5-16E3),�IL-4 FITC (11B11), �-gamma interferon (�IFN-�) FITC (XMG1.2), �CD8 PE(53-6.7), SA-PE-Cy7, and SA-APC. �CD103 biotin (M290), �CD19 FITC (ID3),�FcR II/III (2.4G2), and �-digoxigenin PE were kindly provided by the GermanArthritis Research Center (Berlin, Germany). �CD154 (CD40L) APC (MR1)was obtained from Miltenyi Biotec (Bergisch-Gladbach, Germany). �IL-13(38213.11) was obtained from R&D systems (Wiesbaden, Germany) and coupledwith digoxigenin at the German Arthritis Research Center. Foxp3 staining wasperformed using the PE �-mouse Foxp3 staining set (clone FJK-16S) purchasedfrom eBioscience (San Diego, CA). �CD4, �CD90, �PE, and �APC microbeads,as well as FITC MultiSort kits, were obtained from Miltenyi Biotec.

Phenotypic analysis of lymphocytes by flow cytometry. Mesenteric lymph nodecells (MLNC) and splenocytes from naıve and H. polygyrus-infected mice weredissociated by passing organs through a steel mesh in PBS, pH 7.4, containing0.2% bovine serum albumin. In some experiments, intraepithelial lymphocytes(IEL) and lamina propria lymphocytes (LPL) were isolated from the smallintestine. After visible Peyer’s patches were removed, the small intestine wasopened longitudinally; washed in PBS, pH 7.4; and incubated in RPMI at 37°C,150 rpm for 40 min. The small intestine was washed twice in PBS, and thesupernatants were collected for isolation of IEL. The organ was cut into piecesand incubated (40 to 50 min; 37°C; 150 rpm) with collagenases VIII and D (40�g/ml each; both from Sigma), and then tissue was removed with a mesh. Thesupernatants were spun, and the cells were layered on a column of Percoll (GEHealthcare, Uppsala, Sweden) with a 40%-70% gradient. The cells were spun atroom temperature and 2,200 � g for 20 min, and then collected from theinterphase, washed, and kept in PBS-0.2% bovine serum albumin. For detectionof changes in lymphocyte composition, cell suspensions (1 � 106 total cells) werestained with �CD4, �CD8, and �CD19 monoclonal antibodies (MAbs). Treg cells

were detected by staining them for CD4, CD25, and CD103. Nonspecific bindingof the MAbs was blocked by the addition of �FcgRII/III (20 �g/ml). Intracellulardetection of Foxp3 was performed according to the manufacturer’s instructions.For intracellular detection of CD154 and cytokines, cells were fixed in PBScontaining 2% formaldehyde for 15 min at room temperature. After permeabi-lization with 0.5% saponin (Sigma), the cells were blocked with whole rat IgG(0.1 mg/ml) for 15 min at 4°C to reduce nonspecific binding of MAbs and stainedwith �CD154 and two of the �-mouse cytokine MAbs for 30 min at 4°C. Forcombined detection of CD154 and Foxp3, CD154 was stained on the cell surfacedirectly during in vitro stimulation in complete culture medium as describedbelow (see “Culture conditions”). Cytometric analysis was performed usingFACSCalibur or LSRII (BD Biosciences) and FlowJo software (Tree Star, Inc.).

Isolation of T-cell subsets. The separation of T-cell subsets for transfers and invitro stimulation was performed as follows. Cells were stained for CD25 (APC)and CD103 (PE). CD25� and CD103� cells were enriched by the AutoMACSmagnetic separation system using �APC and �PE magnetic beads. For isolationof the different regulatory subsets, the bead-positive fraction was stained withFITC-labeled �CD4, and the CD4� CD25� CD103� cells and CD4� CD25�

CD103� cell subsets were separated using a FACS Diva cell sorter (BD, Hei-delberg, Germany). After complete removal of cells expressing CD25 and/orCD103, the negative fraction was used to isolate conventional CD4� T cells using�CD4 beads. Naıve splenocytes depleted of T cells using �CD90 beads wereirradiated (30 Gy) and used as antigen-presenting cells for in vitro assays. Forsome adoptive transfers, the whole CD4� CD103� subset (irrespective of CD25expression) was isolated. Therefore, cells were stained for CD4 (FITC), CD25(APC), and CD103 (PE). CD4� cells were isolated using the FITC-MultiSort kitby AutoMACS. After removal of the beads, CD103� cells were isolated fromCD4� cells using �PE beads.

Culture conditions. Cell cultures were performed in cRPMI (BioChrom, Ber-lin, Germany) containing 10% fetal calf serum, 20 mM L-glutamine, 100 U/mlpenicillin, and 100 �g/ml streptomycin as quadruplicates on 96-well plates. Cul-ture of complete and Treg cell-depleted MLNC was performed with 3.5 � 105

cells for 72 h and a concentration of 12 �g/ml of adult worm antigen, and thenthe supernatants were harvested for cytokine detection. Culture of regulatorysubsets and CD4� CD25� CD103� responder cells for detection of polyclonalresponse to �CD3 stimulation (1 �g/ml) was performed with 2.5 � 104 CD4�

cells and 5 � 104 antigen-presenting cells per well. The cells were incubated for48 h, followed by the addition of 1 �Ci of [methyl-3H]thymidine (AmershamPharmacia Biotech, Gent, Belgium) per well for 20 h to measure proliferation. Incoincubation assays, the indicated numbers of Treg cells were added to naıveresponder cells and antigen-presenting cells and treated as described above. Insome assays, bone marrow-derived dendritic cells (DC) were used as antigen-presenting cells, together with sorted CD4� T-cell populations. For the gener-ation of DC, bone marrow was isolated from tibias and femurs of 6- to 8-week-old naıve BALB/c mice, and the cells were kept in 24-well plates at aconcentration of 1.5 � 106/ml in cRPMI supplemented with 20 ng/ml of granu-locyte-macrophage colony-stimulating factor (PeproTech, Hamburg, Germany)for 6 days, followed by incubation with 10 �g/ml of adult worm antigen for 12 h.Control cells were left untreated. DC (1 � 104) were cultured for 72 h with 5 �104 T cells, and then the supernatants were harvested for cytokine enzyme-linkedimmunosorbent assay (ELISA) and the cells were snap-frozen in liquid nitrogenand stored at �80°C for real-time PCR. Recombinant mouse IL-2 (10 ng/ml;PeproTech, Hamburg, Germany) and 1 �g/ml �CD28 were added to somecultures for optimal stimulation. For measurement of CD154 (CD40L) expres-sion of T cells and detection of cytokines in antigen-specifically activated cells,splenocytes and MLNC from infected animals were incubated in 24-well plates ata concentration of 4 � 107 cells per ml with 20 �g/ml of adult worm antigen and1 �g/ml �CD28 for 12 h. To survey cytokine production, brefeldin A (5 �g/ml;Sigma) was added after the first 2 h of stimulation. After 12 h, the cells werewashed and prepared for flow-cytometric analysis as described above. For sur-face staining of CD154, cells were incubated as indicated above but without theaddition of brefeldin A. �CD154-APC, �FcR II/III MAbs (20 �g/ml), whole ratIgG (10 �g/ml), and �CD40 (to avoid rapid removal of CD154 from the cellsurface after binding of CD40 expressed on antigen-presenting cells) were addedto the culture. After 12 h, the cells were washed and stained for CD4, CD103,and Foxp3. We omitted staining of CD25 in these assays due to the unreliabilityof the marker with respect to Treg characterization after restimulation.

Histology. Tissue samples from the proximal third of the small intestines ofnaıve and H. polygyrus-infected mice were fixed in 4% phosphate-buffered for-malin, embedded in paraffin, and used for cross sections. Immunohistology forFoxp3-expressing cells was performed as described elsewhere (32, 33). Foxp3�

cells were counted in 10 high-power fields (40-fold magnification) randomlydistributed in sections from each animal (Peyer’s patch areas were excluded).

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Cytokine analysis and quantitative PCR. IL-4, IL-10, and IFN-� in cell culturesupernatants were quantified using OptEIA ELISA kits (BD Biosciences) ac-cording to the manufacturer’s instructions. IL-13 and active transforming growthfactor �1 (TGF-�1) were detected using DuoSets from R&D Systems. ActiveTGF-�1 was analyzed in culture supernatants without acidification. Transcriptquantification by real-time PCR of IL-4 and IL-10 in distinct CD4� Teff and Treg

populations was performed after coincubation of T cells with naıve or H. polygy-rus antigen-pretreated bone marrow-derived DC (see “Culture conditions”above). RNA extractions were performed using the RNeasy Mini Kit (Qiagen,Hilden, Germany), followed by digestion of DNA using the RNase-free DNaseset (Qiagen) according to the manufacturer’s instructions. RNA was reversetranscribed using the TaqMan reverse transcription reagent (Applied Biosys-tems, Warrington, United Kingdom) and oligo(dT)s. Quantitative real-time PCRwas performed with the 7300 Real-Time PCR System (Applied Biosystems)using TaqMan reagents (Applied Biosystems). PCR amplifications were done intriplicates containing 3 �l of cDNA, 2 �l of 20� TaqMan-labeled primer mix-ture, and 10 �l of 2� TaqMan PCR buffer. The 20� TaqMan primer mixtureconsisted of two unlabeled PCR primers (900 nM [each] final concentration) andone 6-carboxyfluorescein dye-labeled TaqManMGB probe (250 nM final con-centration). All primers were obtained from Applied Biosystems (IL-4 assayidentifier, Mm00445259_m1; IL-10 assay identifier, Mm00439616_m1; GAPDH[glyceraldehyde-3-phosphate dehydrogenase] assay identifier, Mm99999915_g1).Real-time PCR was performed using the following conditions: 10 min of dena-turation at 95°C, followed by 40 amplification cycles of 15 s at 95°C and 60 s at60°C. The relative amounts of IL-10 and IL-4 mRNA were normalized to theendogenous reference GAPDH. Quantification of transcripts in cells cultured inthe presence of DC pretreated with H. polygyrus antigen was done relative to cellscultured with naıve DC using the 2��CT method as described elsewhere (31).

Adoptive-transfer experiments. Sorted CD4� T-cell subpopulations (5 � 105

cells per animal) were injected intraperitoneally into naıve mice in 0.2 ml ofsterile PBS. Control animals received PBS only. One day after transfer, the micewere infected with approximately 200 L3 larvae. Four weeks after infection,animals were sacrificed, and the number of adult worms in each animal wasdetermined and calculated as a percentage of the exact dose of applied L3 (setas 100%). The success of the infection was determined by surveying the fecal eggoutput starting on day 10 postinfection (p.i.). To survey cell survival and to tracetransferred cells, the mice received 1 � 107 carboxyfluoroscein succinimidyl ester(CFSE)-labeled or enhanced green fluorescent protein (EGFP)-expressingCD4� cells. Reanalysis by flow cytometry was performed 6 days after transfer tothe spleen, MLN, and small intestine.

Statistical analysis. Statistical analysis was performed using GraphPad Prismsoftware (San Diego, CA). Statistical significance as indicated in the figurelegends was analyzed by either the Mann-Whitney test or analysis of variance(ANOVA), in combination with Bonferroni posttests.

RESULTS

Analysis of lymphocytes in MLNs of mice chronically in-fected with H. polygyrus. To gather information about changesoccurring in the MLN after infection with H. polygyrus, we

analyzed the lymphocyte composition of the MLN in thechronic phase of infection (28 days p.i.). The absolute cellnumbers obtained from the MLN showed a 5.2-fold increase(P 0.05) in infected compared to naıve animals (Table 1).CD4� and CD8� T cells, as well as B cells, were analyzed byflow cytometry staining. We found a 4.7-fold (P 0.05) in-crease in absolute CD4� and a 4.3-fold (P 0.05) increase inabsolute CD8� T-cell numbers. The strongest increase in totalnumbers, however, was found for B cells (8.3-fold; P 0.05)(Table 1). Interestingly, the number of CD25� CD103� T cellsincreased 7.8-fold (P 0.05) within the CD4� T-cell compart-ment, surpassing the outgrowth of CD25� CD103� T cells(5.1-fold; P 0.05) and CD25� CD103� Teff cells (4.5-fold;P 0.05) (Table 1).

Analysis of Treg cells during the course of infection. Tofurther characterize the regulatory CD4� T-cell compartment,we analyzed the frequency of Treg cells in the MLNs andspleens of H. polygyrus-infected mice at different time pointsafter infection. Flow cytometry analysis was performed afterstaining of CD4, CD25, CD103, and the transcription factorFoxp3. By detecting CD25 and CD103, we were able to distin-guish between CD4� CD25� CD103� naturally occurring andCD4� CD25� CD103� effector/memory-like Treg cells (Fig.1A). Foxp3 expression levels were 90% in CD4� CD25�

CD103� cells at most time points analyzed (naıve mice and 3,21, and 28 days p.i.), with the exception of 6 days p.i., showinga significant decrease in Foxp3� cells within this compartment(naıve, 95.75% � 0.05%; 6 days p.i., 86.72% � 1.11%; P 0.03), arguing for an increased proportion of recently activatedeffector cells present in MLNC at this time point (Fig. 1B). TheCD4� CD25� CD103� subset displayed Foxp3 expression lev-els of 96% at all time points analyzed (Fig. 1B and notshown), while few (1.8%) CD4� CD25� CD103� cells ex-pressed Foxp3 (not shown). Comparing percentages of CD4�

Foxp3� cells between naıve and infected animals (3, 6, 21, and28 days p.i.) revealed no significant differences (Fig. 1E and notshown). Thus, the increase in total Treg cells during infectionreflects the increase in CD4� cell numbers. However, we de-tected significant changes concerning the frequency of effector/memory-like Treg cells after infection with H. polygyrus. Withinthe MLN draining the site of infection, we determined a sig-

TABLE 1. Total MLNC numbers and lymphocyte composition of MLNs from naıve and H. polygyrus-infected animals

Cell typeNo.

Increase (fold)Naıve 28 days p.i.

CD4� 9.32 � 106 � 1.40 � 106b 43.54 � 106 � 5.17 � 106b,c 4.7CD25� CD103�a 8.28 � 106 � 1.21 � 106 36.93 � 106 � 4.35 � 106c 4.5CD25� CD103�a 0.79 � 106 � 0.12 � 106 4.06 � 106 � 0.29 � 106c 5.1CD25� CD103�a 0.21 � 106 � 0.06 � 106 1.64 � 106 � 0.28 � 106c 7.8

CD8� 2.89 � 106 � 1.01 � 106 12.71 � 106 � 3.19 � 106c 4.3

B cells (CD19�) 4.67 � 106 � 2.12 � 106 38.99 � 106 � 15.54 � 106c 8.3

Total 1.72 � 107 � 0.29 � 107 8.96 � 107 � 1.1 � 107c 5.2

a CD4� T-cell subset.b Mean � standard error of the mean of four (naıve) and five (28-day p.i.) animals. Similar data were obtained in four experiments.c Statistical significance for comparison of naıve to infected animals as determined by the Mann-Whitney test (P 0.05).

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FIG. 1. Comparison of Treg numbers in naive and H. polygyrus (H.p.)-infected mice. (A) Treg cells were detected by flow cytometry based onsurface expression of CD4, CD25, and CD103. Shown are plots derived from CD4� MLNC of naıve and chronically infected animals. The dataare representative of four animals per group and four independent experiments. (B) The predominantly regulatory phenotype of CD4� CD25�

CD103� (top) and CD4� CD25� CD103� (bottom) cells derived from MLNs could be confirmed by intracellular staining of Foxp3. Four animalsper group were analyzed in two independent experiments. (C and D) The frequencies of CD25� CD103� cells within CD4� lymphocytes asdetected in MLNs (C) and spleens (D) of animals at different time points after infection (black bars) compared to naıve controls (open bars). Thedata were derived from four animals per group, and analysis of Treg cell numbers was performed at least twice for each time point. Means plusstandard errors of the mean (SEM) are shown. (E) Frequencies of total Foxp3� cells within CD4� lymphocytes from MLN. Means plus SEM offour animals per group are shown. The data are representative of two independent experiments. (F) Proportion of CD25� CD103� and CD25�

CD103� cells in CD4� Foxp3� cells. Means plus SEM of four animals per group are shown. The data are representative of two independentexperiments. (G) Cross sections of the proximal third of the small intestine were stained for Foxp3-expressing cells. Foxp3� Treg cells were foundin the epithelium and in the lamina propria (depicted by black arrowheads). The white arrowheads indicate tissue-dwelling larvae of H. polygyrusat day 6 p.i. (H) Significant increases in Treg cells within the intestine were detected on days 3, 6, and 12 p.i. Stained cells in 10 high-power fields(HPF) (40-fold magnification) per animal were counted. Means of single animals (closed circles) and means of groups (horizontal lines) are shown.�, significant difference between naıve and infected animals as determined by the Mann-Whitney test (P 0.05).

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nificant increase (P 0.03) in CD103� Treg percentages asearly as day 6 days p.i. in comparison to naıve animals (Fig.1C). Within the spleen, a significantly increased percentagewas detected from day 12 p.i. onward (P 0.03) (Fig. 1D). Thehigher frequency of Treg cells expressing CD103 was stable inboth lymphoid compartments until the chronic phase of infec-tion (Fig. 1C and D) and returned to the level of naıve micewhen adult worms were expelled after 8 to 12 weeks (data notshown). The level of CD25� CD103� Treg cells showed nosuch increase (data not shown). When analyzing the ratio ofCD103� and CD103� Treg cells after gating on all Foxp� cells,we rather determined a decrease in CD103� cells in infectedanimals at the chronic stage, counterbalancing the increase inCD103� cells (Fig. 1F). This finding argues for a conversion ofnaturally occurring Treg cells into effector/memory-like Treg

cells, probably directly related to the infection.To test whether the changes in Treg numbers found in the

lymphatic organs were also reflected at the site of inflamma-tion, we detected Foxp3-expressing cells in sections of theproximal third of the small intestine (Fig. 1G). In clear contrastto the findings in lymphatic organs, we detected a transientmaximal accumulation of Foxp3� cells at day 6 p.i. (P 0.016),which gradually returned to the basal level seen in naıve ani-mals at later time points of infection (Fig. 1H). The kinetics ofFoxp3� cells was mirrored by a maximal production of IL-10and TGF-�1 in the small-intestinal tissue at day 6 p.i. (data notshown). Taken together, these data show that Treg cells with aneffector/memory-like phenotype increase strongly during infec-tion with H. polygyrus in the MLN and spleen, while Foxp3-

expressing cells accumulate only transiently at the site of in-fection.

Cytokine production of complete and CD25/CD103-depletedMLNC. MLNC of H. polygyrus-infected mice were isolated atdifferent time points after infection and cultured in the pres-ence of H. polygyrus antigen to analyze their cytokine profile.Our data show that the key Th2 cytokines IL-4 and IL-13 inparticular, as well as IL-10, were readily produced in highconcentrations during the early phase of infection (6 days p.i.),accompanied by a weak IFN-� response (Fig. 2A to D). Similaramounts of cytokines were found during the acute phase (12days p.i.), except for IFN-�, which was only marginally pro-duced at this time point (Fig. 2A). At the chronic phase ofinfection (28 days p.i.), all cytokines analyzed were produced insmaller amounts, especially IL-13 and IL-10. Although it didnot reach statistical significance, the trend toward lower cyto-kine production by MLNC from the chronic phase argues fora generally down-regulated parasite-specific response. ActiveTGF-�1 was detected only in very small amounts (15 pg/ml)irrespective of the time point (data not shown). Interestingly,depletion of cells with a mainly regulatory phenotype (carryingthe surface markers CD25 and CD103) (Fig. 2E) resulted indrastic changes in cytokine production of the remainingMLNC (Fig. 2A to D). First, in the early phase of infection (6days p.i.), all cytokines analyzed were produced in smalleramounts after depletion of CD25� and CD103� cells. Thefinding of lower IL-4, IL-13, IL-10, TGF-�1 (not shown), andIFN-� levels in cultures depleted of cells carrying CD25 and/orCD103 argues for depletion of not only Treg cells, but also

FIG. 2. Cytokine production of MLNC during H. polygyrus infection. (A to D) The cytokine response of MLNC after restimulation with H.polygyrus adult worm antigen was analyzed with cells from animals at days 6, 12, and 28 p.i. The cytokine response of complete MLNC (open bars)was compared to that of MLNC depleted of cells expressing CD25 and CD103 (hatched bars). IFN-� (A), IL-4 (B), IL-13 (C), and IL-10(D) production was analyzed. The means plus standard deviations (SD) of three cell pools of two animals each are shown. �, statistical significancecomparing cytokine responses before and after depletion as determined by two-way ANOVA, followed by Bonferroni posttests (P 0.05). Thedata are representative of two independent experiments. (E) The efficiency of Treg depletion was determined by flow cytometry. The expressionof CD25 and CD103 (upper row) and Foxp3 (lower row) in CD4� cells before (left) and after (right) depletion is shown. (F) The relative expressionof IL-4 and IL-10 mRNAs by CD4� T-cell subsets after coincubation with H. polygyrus-primed DC was assessed at day 6 p.i. Means plus SD ofthree measurements are shown.


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recently activated Teff cells at this early time point. We couldconfirm the presence of effector cells within the CD4� CD25�

CD103� subset at 6 days p.i. by detection of Foxp3 expression(showing a decline at 6 days p.i.) (Fig. 1B) and by analyzingIL-4 and IL-10 mRNA levels in different CD4� T-cell sub-sets kept in cocultures with DC presenting H. polygyrus antigens(Fig. 2F). We determined that CD4� CD25� CD103� T cellsexpressed large amounts of IL-4 and IL-10 transcripts at thisearly time point, indicating the presence of Teff and Treg cellswithin this population. In contrast, the CD4� CD25� CD103�

Treg population from the early infection exclusively expressedhigh levels of IL-10 (Fig. 2F). As we did not analyze thesegregation of the IL-10 production to Foxp� and Foxp3� cellswithin the CD4� CD25� CD103� compartment, we cannotexclude a contribution of recently activated effector cells to theIL-10 production at the early time point. Hence, the depletionof cells expressing CD25 and/or CD103 at the early time pointremoved a significant proportion of recently activated Teff cells,as well as Treg cells, as seen by diminished production of theTh2 cytokines IL-4, IL-13, and IL-10 by the remaining cells.The depletion had no effect with respect to IL-10 production atlater time points (Fig. 2D), arguing that cells other than Treg

cells may also represent important IL-10 sources during theacute and chronic phases. Interestingly, the IL-4 response inthe chronic phase of infection was more vigorous after deple-tion, indicating a suppressive effect of Treg cells on the Th2response in the chronic phase of infection (Fig. 2B).

Adoptive transfer of CD4� T cells from H. polygyrus-infectedmice. To analyze the functions of different CD4� T-cell sub-

sets, we performed adoptive transfers of CD4� Teff cells incomparison to CD4� Treg subsets derived from chronically H.polygyrus-infected mice (28 days p.i.). Teff cells (CD4� CD25�

CD103�) and CD4� T cells with a regulatory phenotype,namely, CD4� CD25� CD103� and CD4� CD25� CD103� Tcells, were transferred to naıve recipients. Control animalsreceived PBS only. One day after transfer, the recipients wereinfected with a defined dose of larvae. The purity of Teff cellswas 98% for expression of CD4�, with 3% remainingCD25� and/or CD103� cells, whereas the Treg compartmentswere 90% CD4� CD25� CD103� cells and 89% CD4�

CD25� CD103� cells, respectively (Fig. 3A). Four weeks afterinfection, the adult worm burdens of recipients and controlanimals were assessed. We detected a significant reduction of43.7% (P 0.008) in the worm burden in animals receiving Teff

cells (CD4� CD25� CD103�) compared to the PBS controlgroup (Fig. 3B). In contrast, transfer of CD4� CD25� CD103�

T cells from naive mice had no influence (data not shown),indicating that the reduction was due to parasite-specific Teff

cells. The protective role could be solely ascribed to CD4� Tcells, as the transfer of CD4� cells had no effect (data notshown). Animals receiving CD4� CD25� CD103� Treg cells(comprising 90% Foxp3� cells) had worm burdens compa-rable to that of the PBS control. Similarly, the transfer ofCD4� CD25� CD103� Treg cells (comprising 95% Foxp3�

cells) did not result in significant changes. In contrast, transferof a heterogeneous T-cell population containing Treg and Teff

cells, namely, CD4� CD103� T cells (with a purity of 95%CD4� cells and 85% CD103� cells but comprising only

FIG. 3. Influence of adoptive CD4� T-cell transfer on adult worm burden. T cells of the indicated subtypes (5 � 105) obtained from MLNs andspleens of chronically H. polygyrus-infected mice were transferred to recipients that were subsequently infected with H. polygyrus larvae. Controlanimals received PBS only. (A) Purity of transferred cells as determined by flow cytometry. Representative data from one of three independentexperiments are shown. (B) Adult worm burden in recipients 28 days p.i. Worm counts are shown as percentages of the number of applied larvae.Group sizes varied between 5 and 20 animals. The data originated from three individual experiments. Individual worm counts and medians areshown. The asterisks show statistical significance as determined by a Kruskal-Wallis test followed by a Mann-Whitney test: �, P 0.05; ��, P 0.01. (C) Tracing of transferred cells in C57BL/6 mice receiving 1 � 107 CD4� cells from chronically infected EGFP-expressing donors. Therecipients were infected with H. polygyrus the following day. Examples of flow cytometry plots derived from splenocytes, MLNC, and small-intestinal IEL and LPL 6 days after transfer are shown. (D) Percentages of EGFP� cells within lymphocytes of recipients. Means plus standarddeviations for four animals are shown. The data are representative of two experiments.


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about 70% Foxp3� cells) led to a significant decrease in wormnumbers (P 0.03) comparable to pure CD4� Teff cells (Fig.3B). We verified cell survival in separate experiments usingCFSE-labeled CD4� cells (data not shown). In addition, weperformed transfers with EGFP-expressing CD4� cells fromchronically infected donors. The recipients were infected thefollowing day. Six days after transfer, spleen and MLN, as wellas IEL and LPL from the small intestine, were analyzed fortransferred cells (Fig. 3C and D). We were able to traceEGFP� cells in all analyzed organs with no significant accu-mulation in any of the compartments. The slightly elevatednumbers of EGFP� cells within MLNC compared to spleno-cytes merely reflects the higher CD4� T-cell numbers found inlymph nodes (Fig. 3D). We also analyzed percentages of Treg

cells according to expression of CD25, CD103, and Foxp3within the EGFP� population and could show that Treg cellssurvived the transfer (data not shown).

Hence, CD4� Teff cells from the chronic phase of an H.polygyrus infection transferred protection to naıve recipients incontrast to naturally occurring or effector/memory-like Treg

cells, which had no intrinsic effect on worm development. Fur-thermore, transfer of a mixed T-cell population comprisingeffector and regulatory T cells showed that Treg cells were notefficient in suppressing the protective effect mediated by Teff

cells.Cytokine production of CD4� T-cell subsets in the chronic

phase of infection. We then analyzed the cytokine productionof the CD4� T-cell subsets used for the transfer experiments togain information about mediators involved in worm expulsionor establishment. H. polygyrus-treated DC were used as anti-gen-presenting cells to stimulate the T-cell subsets. We deter-mined that Teff cells (CD4� CD25� CD103�) released IL-4and IL-13 in the presence of H. polygyrus-primed DC in con-trast to Treg cells (CD4� CD25� CD103� or CD4� CD25�

CD103�) (Fig. 4A and B). However, CD4� CD25� CD103� Tcells, in particular, released significant amounts of IL-10, ar-guing for the effector/memory-like Treg cell subset as an im-portant source of IL-10 (Fig. 4C). Incubation of the CD4�

subsets with naıve DC resulted in marginal release of cytokinesby T cells, clearly showing the antigen specificity of the cyto-kine response. Analysis of active TGF-�1 in culture superna-tants revealed only marginal increases in cultures with antigen-loaded DC compared to those with naıve DC (data not shown).The generally lower cytokine levels in cultures of separated Teff

cells compared to complete MLNC cultures (Fig. 2) is probablydue to fewer CD4� T cells present in DC cocultures than inpreparations of whole MLNC and to other cytokine-producingcells present in MLNC cultures, like basophils, mast cells, andeosinophils (16).

While only CD4� CD25� CD103� T cells produced IL-10 inan antigen-specific manner, we found that both CD25-express-ing T-cell subsets produced large amounts of IL-10 when op-timized conditions were provided by addition of exogenousIL-2 and enhanced costimulation through �CD28 antibodies(Fig. 4F). Similarly, IL-4 and IL-13 were produced in muchlarger amounts almost exclusively by CD4� CD25� CD103�

Teff cells after enhanced costimulation was provided (Fig. 4Dand E). Levels of active TGF-�1 were only marginally affected(data not shown). None of the CD4� T-cell subsets from H.polygyrus-infected mice was found to produce relevant amounts

of the Th1 cytokine IFN-� (data not shown). Hence, our dataindicate a dominant and parasite-specific Th2 response byCD4� effector cells from the chronic phase of infection, whileeffector/memory-like CD4� CD25� CD103� Treg cells proba-bly produce IL-10 in response to H. polygyrus antigen.

Distribution of antigen-specific CD4� T cells and their cy-tokine production. To assess the distribution of H. polygyrus-specific CD4� T cells within the Teff and Treg cell populations,we examined the expression of CD154 as a marker of antigen-specific CD4� T-cell activation (15, 26). As the detection ofCD154 directly ex vivo is not possible, we used an optimized invitro protocol (26). Comparison of CD4� T cells from spleenand MLN showed that in both sites, about 0.2% (MLN, 0.233 �0.036 naıve versus infected [P 0.001]; spleen, 0.246 � 0.040naıve versus infected [P 0.0003]) of CD4� CD103� Teff cellsexpressed CD154 when restimulated with adult worm antigenin vitro (Fig. 5A and B). By additional detection of CD103 andFoxp3, we were able to distinguish between CD154 expressionon Teff and Treg cells.

Interestingly, when analyzing the level of CD154 on CD103�

cells not coexpressing Foxp3 and therefore not of regulatoryphenotype, we found clearly higher percentages of antigen-specific cells within this Teff subpopulation in MLNs (1.701% �0.306% CD4� CD103� versus CD4� CD103�; P 0.0006)and spleens (0.586% � 0.144% CD4� CD103� versus CD4�

CD103�; P 0.01) (Fig. 5A and C) of infected animals in

FIG. 4. Cytokine production by CD4� T-cell subsets. CD4� T cellswere isolated from pooled MLNC and splenocytes of eight mice in thechronic phase of infection (28 days p.i.) according to the indicatedsurface marker expression. T-cell subsets were incubated for 72 h withnaıve bone marrow-derived DC (nDC) or DC pretreated with H.polygyrus adult worm antigen (HpDC). (A to C) Release of IL-4 (A),IL-13 (B), and IL-10 (C) was detected by ELISA. Means plus standarddeviations (SD) of triplicate determinations are shown, and the dataare representative of three independent experiments. (D to F) Releaseof IL-4 (D), IL-13 (E), and IL-10 (F) after addition of recombinantmouse IL-2 and �CD28 to cultures. Mean values plus SD of triplicatedeterminations of one of two independent experiments with similarresults are shown.


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comparison to noninfected mice. In contrast, only low levels ofCD154 expression were detected on Foxp3� Treg cells fromMLN (0.03%) and spleen (0.05%) (data not shown). Thesedata indicate that mice that received T cells sorted for coex-pression of CD4 and CD103 (irrespective of CD25 expression)in transfer studies (Fig. 3B) received a highly enriched Treg

population that still contained a small number of antigen-specific effector cells not expressing Foxp3. The percentage ofantigen-specific effectors within the transferred CD4�

CD103� cell compartment was as low as 0.2%, according toCD154 expression, comparable to the values for antigen-spe-cific cells within the CD4� CD103� effector compartment.These CD103� effectors may have led to protection, eventhough a large number of Treg cells was cotransferred, arguingfor an insufficient capacity of the Treg cells to control theantigen-specific effectors.

We next determined the cytokine production of the antigen-specific CD154� CD4� T cells. Investigation of IL-4, IL-13,IL-10, and IFN-� production after in vitro restimulation (Fig.5D) revealed a dominant Th2 response in CD154� CD4� Tcells of chronically infected mice, characterized by high expres-sion levels of IL-4 (24.55% � 1.32%), IL-13 (10.48% �1.99%), or both (10.26% � 1.02%) (Fig. 5D and E). Only lowlevels of IFN-�-producing cells were detected in the antigen-specific T-cell compartment (4.47% � 0.82%), and the fre-quencies of IL-10-producing cells were hardly distinguishablefrom nonspecific background (1.60% � 0.37%) (Fig. 5D andF). These data again indicate that the lower worm burden inrecipients receiving CD103-expressing CD4� T cells might bedue to cotransfer of antigen-specific effector cells that wereable to produce IL-4 and IL-13, thereby mediating worm ex-pulsion.

Suppressive effect of Treg cells from H. polygyrus-infectedmice in vitro. To further investigate Treg cells from worm-infected animals, we analyzed the suppressive activities ofsorted Treg subsets in vitro. CD4� CD25� CD103� and CD4�

CD25� CD103� Treg cells were isolated from naıve and worm-infected mice in the acute (12 days p.i.) and chronic (28 daysp.i.) phases of infection. The cells were added to Treg-depletedCD4� T cells from naıve mice, which were stimulated poly-clonally by �CD3 antibodies. CD4� CD25� CD103� Treg cellsfrom infected mice suppressed the proliferation of naıve re-sponder CD4� T cells more vigorously than their counterparts

FIG. 5. Distribution of antigen-specific CD4� T cells (CD154�)and their cytokine responses. (A) Shown are examples of fluorescence-activated cell sorter plots of MLNC from naıve mice (upper row) andH. polygyrus-infected mice at day 28 p.i. (lower row). The cells wererestimulated with H. polygyrus antigen in vitro and stained for CD4,CD154, CD103, and Foxp3. CD4� cells were plotted for expression ofCD103 and Foxp3 (left). The middle plots show CD154 expressionwithin the CD4� CD103� Foxp3� effector population. The right-handplots show CD154 expression by CD4� CD103� Foxp3� effectors. (Band C) Frequencies of CD154-expressing cells within the CD4�

CD103� Foxp3� (B) and CD4� CD103� Foxp3� (C) effector popu-lations of MLNs (black bars) and spleens (open bars) after restimula-tion in vitro. The data were obtained from eight infected and sevennaıve mice. Means plus standard errors of the mean (SEM) are shown.The data are representative of three independent experiments. (D)Intracellular staining of cytokines within restimulated CD4� T cellsfrom spleens (28 days p.i.). The cells were gated for expression of CD4and CD154 (left), and IL-4 and IL-13 (upper row) or IFN-� and IL-10(lower row) were determined in CD4� CD154� cells (center) andCD4� CD154� cells (right). The cytometry plots shown are represen-tative of a group of eight infected mice. (E) IL-4/IL-13 and IFN-�/IL-10 (F) responses in CD4� CD154� cells after restimulation with H.polygyrus antigen. Means plus SEM of eight animals are shown. Theasterisks show statistical significance comparing cells from naıve andinfected animals as determined by the Mann-Whitney test: ��, P 0.01; ��, P 0.001.


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from naıve controls (Fig. 6A and Table 2). For the lowest ratioof CD4� CD25� CD103� Treg cells to responder cells (1:20),reflecting the in vivo situation, we found that Treg cells fromthe chronic phase of infection exhibited the highest suppressiveefficiency (P 0.001 compared to naıve cells and day 12 p.i.).The CD4� CD25� CD103� Treg cells derived from infectedanimals were more efficient in mediating suppression thanCD4� CD25� CD103� Treg cells (P 0.03 for all testedratios) (Fig. 6B). As expected, both Treg subsets showed ananergic phenotype after �CD3 stimulation (data not shown).These data clearly indicate the high in vitro suppressive capac-ity of CD4� CD25� CD103� Treg cells derived from the

chronic phase of infection with regard to activation and pro-liferation of CD4� T cells.

DISCUSSION

In this study, we investigated the roles of different CD4�

T-cell subsets in a chronic intestinal worm infection. Adoptivetransfer of CD4� cells from the chronic phase of an H. polygy-rus infection containing small numbers of antigen-specific Teff

cells led to significant protection in recipients. In contrast,adoptive transfer of Treg cells did not alter the worm burden,although Treg cells significantly suppressed CD4� T-cell pro-liferation in vitro. Hence, our data clearly point to the impor-tance of CD4� Teff cells in mediating host protection andworm expulsion. Our data provide evidence for the followingtraits of Teff/Treg interaction. First, Teff cells that arise during aprimary H. polygyrus infection exhibit the ability to mediateprotection. Second, Teff cells persist in chronically infectedmice, despite strong cellular suppression (10, 49, 52). Third,about 0.24% of CD4� Teff cells are antigen-specific cells, basedon expression of CD154, containing highly enriched frequen-cies of IL-4- and IL-13-producing cells responding to parasiteantigens. Fourth, Treg cells are probably an important source ofIL-10 during chronic infection. Fifth, elevated numbers of ef-fector/memory-like Treg cells persist in lymphatic organs untilthe chronic phase, and Treg cells derived from infected animalsshow an augmented suppressive capacity in vitro.

Our study revealed a significant and permanent increase ineffector/memory-like (CD103�) Treg cell numbers in lymphaticorgans as early as day 6 p.i. However, only a transient increasein Treg cells was detected at the site of inflammation, whichmight represent a host reaction to control inflammatory re-sponses induced by innate immune and Teff cells. The peak ofTreg cells in the small intestine at day 6 p.i. is in accordancewith the time point of intense inflammation around tissue-invading larvae, characterized by accumulation of mainly gran-ulocytes and, to a lesser extent, macrophages and CD4� T cells(4, 43). Interestingly, it has been shown that Treg cells not onlycontrol inflammatory responses directly or indirectly driven byTeff cells, but also suppress innate immune responses in theabsence of Teff cells (36). Therefore, it is conceivable that Treg

cells induced by the nematodes are recruited to modulateinnate and adaptive immune responses. The resulting immu-nosuppression could control excessive pathology and favorprolonged parasite survival. Helminth-induced Treg cells havebeen shown to be involved in immunomodulation in various

FIG. 6. Suppressive capacities of Treg cells in vitro. CD4� CD25�

CD103� and CD4� CD25� CD103� Treg cells were purified fromsplenocytes and MLNC of H. polygyrus-infected (12 and 28 days p.i.)and naıve mice. CD4� CD25� CD103� responder cells were isolatedfrom naıve animals. (A and B) The suppressive capacities of CD4�

CD25� CD103� Treg cells (A) and of CD4� CD25� CD103� Treg cells(B) from naıve mice (open bars) and infected animals at the acutephase (12 days p.i.) (gray bars) or chronic phase (28 days p.i.) (blackbars) of infection were analyzed. The ratios of Treg cells and responderCD4� T cells are indicated. Proliferation of CD4� T cells after stim-ulation with �CD3 antibodies was detected by [3H]thymidine uptake.Means plus standard errors of the mean of quintuple determinationsare shown. The data shown are representative of two independentexperiments.

TABLE 2. Statistical analysis of suppressive efficiencies of Treg cell subsets from naive and H. polygyrus-infected mice

Treg/CD4Respa

ratio

CD4� CD25� CD103�b CD4� CD25� CD103�b

Naıve vs. 12days p.i.

Naıve vs.28 days p.i.

12 vs. 28days p.i.



12 vs. 28days p.i.

1:20 NS P 0.001 P 0.01 P 0.001 P 0.001 P 0.0011:10 P 0.001 P 0.001 NS P 0.001 P 0.001 NS1:5 NS P 0.01 NS P 0.001 P 0.001 NS1:2 P 0.001 NS P 0.05 NS NS NS1:1 NS NS NS NS NS NS

a Ratio of Treg cells to naıve CD4� responders.b Treg cell subset analyzed for suppressive efficiency. Statistical significance as determined by two-way ANOVA, followed by a Bonferroni posttest. NS, not significant.


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helminth infections, such as human infections with Onchocercavolvulus (51) or murine infections with Litomosoides sigmodon-tis (59), H. polygyrus (14), and Schistosoma mansoni (6, 29, 37).The general notion is that during helminth infections, Treg cellsmight have the function of facilitating parasite survival anddiminishing immunopathology (34, 35), but their concrete roleis not yet fully elucidated.

We provide evidence that during the early phase of infec-tion, activated Teff and Treg cells are both important sources ofIL-10. However, during the chronic phase of infection, Treg

cells probably represent the main T-cell source of IL-10.Hence, our data indicate that not only Treg cells, but alsoeffector cells provide IL-10 that could have a role in dampen-ing exaggerated inflammation, as shown previously for Th1 andTh2 effector cells (3, 8, 25, 61). Whether Treg cells in nematodeinfections mediate suppression via IL-10, TGF-�, or both isstill under investigation (8, 27, 39, 59). Increases in TGF-�1expression by CD4� and CD4� cells and surface-boundTGF-�1 on CD4� cells, as well as increased plasma levels ofactive TGF-�1 during infection with H. polygyrus, have beenshown by others (14, 56, 68). In our system, Treg cells from thechronic stage of infection seem to exert their effects via IL-10,supported by the fact that we detected only low levels of activeTreg-derived TGF-�1. Of note, our analysis of the TGF-�1production by CD4� T-cell subsets is restricted to the secretedactive form, and currently we cannot exclude differences withrespect to the secreted inactive form of the molecule. By an-alyzing tissue samples from the small intestine, we found ele-vated TGF-�1 levels at the site of infection in the early phase(6 days p.i.) (data not shown), perhaps indicating tissue repairin this phase of active inflammation.

In addition to T cells, B cells can also serve as major sourcesof IL-10 (45, 46), and B cells in protozoan and helminth in-fections have been shown to produce IL-10 in response topathogen-derived antigens (2, 17, 46). To date, few data on Bcells as a source of IL-10 in intestinal nematode infections areavailable. We detected large increases in B-cell numbers inMLNs of infected animals, and further analysis of these cells aspossible source of IL-10 during H. polygyrus infection isneeded.

This study showed that especially CD4� CD25� CD103� Tcells from mice chronically infected with H. polygyrus produceIL-10 in a parasite-specific manner. The high frequency ofFoxp3-expressing cells within this T-cell population stronglysuggests effector/memory-like Treg cells as a parasite-specificsource for IL-10, although we cannot completely rule out thepossibility that Foxp3� effector cells or adaptive Treg cells maycontribute to the detected IL-10 production. In mice infectedwith S. mansoni, CD4� CD25� T cells, whether Foxp3� orFoxp3�, have been identified as sources of IL-10 (6, 58).

We revealed a disproportionate increase in CD25� CD103�

Treg cells within the CD4� T-cell compartments of infectedanimals. Similar to what has been described for infection withS. mansoni (6), we detected only minor changes in the totalTreg cell numbers as determined from Foxp3 expression inlymphatic organs. However, we found a significant increase incells expressing CD103 within the regulatory T-cell compart-ment, arguing for a specific role of these effector/memory-likeTreg cells in modulating the immune response to H. polygyrus.To test this hypothesis, we performed suppression assays with

responder CD4� T cells from naıve mice. These in vitro assaysdemonstrated that especially CD4� CD25� CD103� Treg cellsisolated from the chronic phase of infection strongly sup-pressed the proliferation of CD4� T cells when added in aphysiological ratio of Treg to responder cells (1:20). A partic-ularly potent suppressive capability of CD4� CD25� CD103�

Treg cells in comparison to CD4� CD25� CD103� Treg cells invitro and in vivo has also been shown by others (30, 57). Weexpected that adoptive transfer of Treg cells might interferewith the primary Th2 response to H. polygyrus, entailing in-creased worm burdens, as studies on protective immunityagainst H. polygyrus have shown that severe combined immu-nodeficient mice lacking T and B cells and mice depleted ofCD4� T cells harbor higher worm burdens than wild-type oruntreated controls (63, 64). However, in spite of the highsuppressive capacity of CD103� Treg cells in vitro, our adop-tive-transfer model did not reveal an influence of Treg cells onthe worm burden in vivo. Transfer of CD4� CD25� CD103�

Treg cells, as well as CD4� CD25� CD103� Treg cells, did notalter worm numbers, whereas transfer of a mixture of Treg andTeff cells (CD4� CD103� T cells) significantly lowered wormburdens, arguing against a major control of effector responsesby Treg cells in vivo. The discrepancy between the suppressiveactivity of CD103� Treg cells in vitro and the failure to inter-fere with worm expulsion in vivo might be due to multiplefactors. First, Treg cells might be involved in suppressing pa-thology, but not in facilitating worm survival. Second, thetransferred CD4� CD103� T-cell population represented aheterogeneous pool of Treg and Teff cells containing the lowestpercentage of Foxp3� cells (70%) in comparison to theCD4� CD25� CD103� and CD4� CD25� CD103� subsets(both �90%). Third, reduction of the adult worm burden inrecipients receiving CD4� CD103� T cells coincided with asignificant proportion of parasite-specific effector cells withinthe CD103� Foxp3� cell compartment, as shown by expressionof CD154 after antigen stimulation. Finally, the conditions invitro versus in vivo were clearly different. In vitro, Treg cellsdrastically inhibited the proliferation of naıve CD4� responderT cells after polyclonal stimulation, whereas in vivo, Treg cellshad to combat antigen-specifically activated Teff cells.

We have provided data on the distribution and cytokineprofile of parasite-specific CD4� cells using the markerCD154, recently shown to exhibit exquisite specificity for an-tigen-activated CD4� cells in the human and mouse systems(15, 26). Our approach revealed low percentages (1%) ofantigen-specific cells in the chronic phase, producing predom-inantly IL-4 and IL-13. Although cytokine-producing CD4� Tcells were also detected at low percentages among CD154�

cells, the responders to worm antigens were highly enrichedwithin the CD154� population. In contrast, much higher fre-quencies of IL-4-producing cells are described for infectionswith H. polygyrus in GFP–IL-4 reporter mice (41). Severalfactors may have led to the marked discrepancies. First, Mohrset al. reported that not all GFP� cells secrete IL-4 in responseto stimulation with H. polygyrus antigen extracts and providedevidence that CD4� T cells recently producing IL-4 in vivo areimpaired in their cytokine response in vitro (41). This findingmight, in part, explain the lower frequencies of antigen-specificIL-4 producers detected in our in vitro assays. Second, differ-ent conditions, such as the infection time point analyzed, the


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source of antigen-presenting cells, in vitro cell numbers duringrestimulation, or antigen dose, might have led to an underes-timation of specific CD4 T-cell numbers based on CD154 ex-pression. However, despite the marked difference between thefrequencies of antigen-specific CD4� T cells presented hereand for the IL-4 reporter system, our data show that theCD154 technique facilitates the analysis of a broader spectrumof pathogen-specific cytokine responses in a nonmanipulatedimmune system.

Adoptive transfer of CD4� cells did not show an effect onthe worm burden (data not shown). This observation furthersupports the concept that resistance to gastrointestinal hel-minths is dependent on CD4� Th2-type immune responses, asshown in animal models with H. polygyrus or Nippostrongylusbrasiliensis (11, 64, 65), as well as in humans infected withAscaris lumbricoides and Trichuris trichiura (24, 62).

Hence, our study is in line with the recent finding thatmemory CD4� T cells develop after infection with the gastro-intestinal nematode Trichuris muris and mediate protectionagainst rechallenge (69). In a recent publication introducingalternatively activated macrophages as an effector populationessential for protective immunity to challenge infections withH. polygyrus, it was shown that memory Th2 cells derived fromH. polygyrus-cured mice transferred protection against a pri-mary infection (4). The protective effect of Th2 memory cellswas more pronounced than what we found in our transfermodel using Teff cells from an ongoing infection. It would beinteresting to investigate whether differences in, e.g., the hom-ing receptor repertoires or susceptibility to induced cell deathbetween memory and effector T cells might be responsible forthis phenomenon.

In conclusion, our data indicate that in our model system,tipping the balance of effector T cells during infection stronglyinfluences the survival of parasitic nematodes, while Treg cellsmay have functions that are not directly related to worm per-sistence.

ACKNOWLEDGMENTS

The study was supported by Deutsche Forschungsgemeinschaft SFB650 to S.H., R.L., J.H., and A.H.

We thank B. Sonnenburg and M. Muller for excellent technicalassistance and K. Raba and T. Kaiser for FACS sorting.

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Functional Analysis of Effector and Regulatory T …Isolation of T-cell subsets. The separation of T-cell subsets for transfers and in vitro stimulation was performed as follows. Cells