Pronounced Phenotype in Activated Regulatory T Cellsduring a Chronic Helminth Infection
Laura E. Layland,*,† Jorg Mages,* Christoph Loddenkemper,‡ Achim Hoerauf,†
Hermann Wagner,* Roland Lang,*,x and Clarissa U. Prazeres da Costa*
Although several markers have been associated with the characterization of regulatory T cells (Tregs) and their function, no studies
have investigated the dynamics of their phenotype during infection. Since the necessity of Tregs to control immunopathology has
been demonstrated, we used the chronic helminth infection model Schistosoma mansoni to address the impact on the Treg gene
repertoire. Before gene expression profiling, we first studied the localization and Ag-specific suppressive nature of classically
defined Tregs during infection. The presence of Foxp3+ cells was predominantly found in the periphery of granulomas and isolated
CD4+CD25hiFoxp3+ Tregs from infected mice and blocked IFN-g and IL-10 cytokine secretion from infected CD4+CD252 effector
T cells. Furthermore, the gene expression patterns of Tregs and effector T cells showed that 474 genes were significantly regulated
during schistosomiasis. After k-means clustering, we identified genes exclusively regulated in all four populations, including Foxp3,
CD103, GITR, OX40, and CTLA-4—classic Treg markers. During infection, however, several nonclassical genes were upregulated
solely within the Treg population, such as Slpi, Gzmb, Mt1, Fabp5, Nfil3, Socs2, Gpr177, and Klrg1. Using RT-PCR, we confirmed
aspects of the microarray data and also showed that the expression profile of Tregs from S. mansoni-infected mice is simulta-
neously unique and comparable with Tregs derived from other infections. The Journal of Immunology, 2010, 184: 713–724.
It is an uncontested fact that Foxp3+ regulatory T cells (Tregs)are an essential immune population that prevent autoimmu-nity, control immune homoeostasis, and limit chronic in-
flammatory disorders, such as inflammatory bowel disease andschistosomiasis (1, 2). There are now several classes of Treg pop-ulations that express an abTCR, which in itself designates a form ofAg-specificity. Natural thymic-derived regulatory T cells (nTregs)are generated by strong agonist ligands and require costimulation.That theAg specificity of those cells is tightly regulated reflects theirrole in the periphery: controlling unwarranted responses to “self”(3–5). In contrast, generation of peripherally induced Treg pop-ulations appears more effective in the absence of costimulation, asrecently described using resting B cells (6).Peripherally inducedor inducibleTregpopulationscanbegrouped
into IL-10–secreting Tr1 cells (3, 7) or TGF-b2producing Th3 cells(1, 4). Tr1 cells have recently been shown to play a role in con-trolling autoimmune diseases (8–10), although they are morereadily associated with allergy and helminth diseases because oftheir ability to induce B cells to secrete the noninflammatory IgG4subclass (11–13). Further studies have shown that thismechanism is
dependent on GITR–GITRL interaction and TGF-b, although thesource of TGF-b remains unclear (13). The role of the latter cyto-kine in all Treg populations remains elusive despite strong evidenceregarding the necessity of TGF-bRII on suppressed cells (6, 14).Furthermore, TGF-b constitutes a regulatory switch that, in com-bination with other cytokines, can reprogram effector T cell (Teff)differentiation along different pathways (15). There is a plethora ofresearch on themechanisms throughwhich inducible Tregs developand function (16), and it is becoming increasingly clear that thegeneration of Treg subsets depends not only on the status of theimmune system per se, but also the effected organs and the type ofunderlying disease or stimulus: autoimmune, infectious, or immu-nization. Many models have described the need for surface ex-pression markers of Tregs such as CTLA-4, GITR, and cAMP, andtheir capacity to suppress either activated T cell or B cells. Never-theless, little is known about the fundamental changes that occurwithin a specific Treg population during a chronic infection orwhether such alterations occur at all.Our previous work has focused on the tropical helminth Schis-
tosoma mansoni, and the most important species S. mansoni, S. ja-ponicum, and S. hematobium infect 250–300 million peopleworldwide. This blood fluke has a complicated lifecycle.Within thedefinite mammalian host, the parasite undergoes no fewer thanthree different life-stages. The ensuing immunopathology doesnot arise from the worms themselves, but from the abundanteggs that are released from fecund females. In order for themiracidium-residing life form within the egg to find its snailhost, the eggs must penetrate into the intestinal lumen to beexcreted with the stool. As a result, there is an inflammatoryresponse, and CD4+ T cell-mediated granulomas develop andeventually cause detrimental host damage in both the liver andintestine. Using our well-established Schistosoma-mouse model,we have expanded previous studies that demonstrated a majorrole for Tregs (CD4+CD25hiFoxp3+) during schistosomiasis (17–19). We have also shown that schistosome-specific Tregs expandduring the infection and are essential for controlling the host’simmune response to the parasite: absence of Treg-enhanced
*Institute for Medical Microbiology, Immunology and Hygiene, Technische Univer-sitat Munchen, Munich; †Institute for Medical Microbiology, Immunology and Par-asitology, University Clinic Bonn, Bonn; ‡Institut fur Pathologie, ChariteUniversitatsmedizin Berlin, Campus Benjamin Franklin, Berlin; and xInstitute ofClinical Microbiology, Immunology and Hygiene, University Hospital Erlangen,Erlangen, Germany
Received for publication May 7, 2009. Accepted for publication November 2, 2009.
This work was supported by Grants DFG CO-457/1-3 and SFB Tr22 from the Ger-man Research Council.
Address correspondence and reprint requests to Dr. Clarissa U. Prazeres da Costa,Institute for Medical Microbiology, Immunology and Hygiene, Technische Uni-versitat Munchen, Trogerstrasse 30, 81675 Munich, Germany. Email address:[email protected]
Abbreviations used in this paper: Ct, threshold cycle; Klrg1, killer cell lectin-likereceptor G1; MLN, mesenteric lymph node; Mt1, metallothionein-1; Nrp-1, neuro-pilin-1; Penk1, preproenkephalin 1; SEA, soluble egg Ag; Slpi, secretory leukopro-tease inhibitor 1; Teff, effector T cell; Treg, regulatory T cell.
Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
granuloma pathology and disruption of the finely tuned Th1/Th2balance (2). These endogenous immunoregulatory mechanismsare currently believed to be stimulated by components of thehelminth (20), but the underlying mechanisms remain unclear.Although the identification and isolation of Treg populations are
well established using Foxp3, little is known about the way inwhich these cells orchestrate both molecular and cellular eventsduring chronic infections. Thus, the major focus of this study was toascertain whether the gene expression profiles of Tregs were alteredduring an ongoing infection and whether the expression of thesemolecules could be a basis to understand Treg function in vivo.Using DNA microarray technology, we have compared the geneexpression profile of Teff and Treg populations from naive andschistosome-infected mice. We demonstrated that a total of 474probe sets were significantly regulated. Furthermore, within theTreg population isolated from infected mice, cluster analysisrevealed that exclusively 64 genes were upregulated and 50 geneswere downregulated when compared with Tregs isolated fromnaive mice. Interestingly, within this cluster we did not find geneexpression of classical Treg markers such as IL-10 or TGF-b, butrather granzyme B, secretory leukoprotease inhibitor 1 (Slpi),metallothionein-1 (Mt1) and killer cell lectin-like receptor G1(Klrg1), effector molecules implied in the literature to be involvedin regulating inflammatory processes (21–24).
Materials and MethodsExperimental infection
Female specific pathogen-free C57BL/6 mice were purchased from HarlanWinkelmann (Borchen, Germany). Mice were infected s.c. with 100 cer-cariae from a Brazilian strain of S. mansoni from Biomphalaria glabratasnails (Brazilian origin), kept in the institute’s laboratory, and killed afternine weeks of infection. Litomosoides sigmodontis infection was per-formed as described previously; mice were killed 30 d postinfection (25).Candida albicans was injected i.v. with a sublethal dose and analyzed after10 d of infection. All animal studies were approved by the local ethicalreview committee.
Soluble egg Ag preparation and parasite assessment
Sterile soluble egg Ag (SEA) were prepared according to standard pro-cedures and as described previously (2, 26). Livers from infected mice werehomogenized and digested overnight with 1 mg/m collagenase VIII(Sigma-Aldrich Chemie, Taufkirchen, Germany) and 312 U/ml DNAse(Sigma-Aldrich). The homogenate was then passed through a filter, re-peatedly washed with endotoxin-free PBS (Sigma-Aldrich), and centri-fuged over a Percoll 0.25 M sucrose gradient (Amersham BiosciencesEurope, Braunschweig, Germany) to yield a pellet of purified eggs. Eggswere then extensively washed in 1 mM EDTA/PBS (Sigma-Aldrich),counted, and homogenized with a pestle and mortar in 1 ml PBS for 20min. The homogenate was then ultracentrifuged at 100,000 3 g for 2 h at4˚C, and the resulting supernatant was immediately frozen at 280˚C.Preparations were determined to be free of contaminating endotoxin usingthe FDA-standard LAL-assay (ACILA, Weiterstadt, Germany). Theamount of worms per individual mouse was determined after a lethal doseof pentobarbital and portal vein perfusion. Following perfusion, male andfemale worms were collected and identified by macroscopic examination.In order not to miss any worms, livers and intestines were thoroughlyexamined under a plate microscope. Estimates of parasite burden, in-cluding eggs per organ, were performed as described previously (2, 25).Individual samples of livers and intestines were removed and digested with5% KOH at 37˚C to release the eggs out of the tissues. The number of eggsin each organ were counted and divided through the total amount of femaleworms per individual mouse
Cytokine milieu, Th responses, and Ag-specific suppression
Cytokine milieu. To compare the immunological responses in infected mice,weighed liver samples from individual mice were homogenized in RPMI1640 medium and centrifuged for 10 min at 13,000 rpm. The resultingsupernatant was then tested for the presence of Th cytokines using com-mercial ELISA kits (R&D Systems, Minneapolis, MN).
Bulk mesenteric lymph node cell responses. In 96-well, round-bottomedplates, erythrocyte-depleted mesenteric lymph node (MLN) cells (53 105)from individually infected or naive mice were plated with or without SEA(25 mg/ml) in triplicate. After 72 h, the supernatants were removed andtested for IL-10, IL-4, IL-13, or IFN-g cytokine by ELISA.
Ag-specific suppression assays. Spleen-derived CD4+CD252 and CD4+
CD25hi T cell populations were isolated using the MoFlo cell sorter(Beckmann-Coulter, Krefeld, Germany) from 9-wk infected C57BL/6mice.Thereafter, responderCD4+CD252T cells (13 105) were incubated for 72 hwith 25 mg/ml SEA and 23 105 naive irradiated APC (isolated using MHCclass II microbeads from Miltenyi Biotech [Bergish Gladbach, Germany]).In some cocultures, 1 3 105 Tregs were also added. Ag-specific cytokinelevels were measured via ELISA. Experiments were performed in replicatesof six and repeated at least twice to ensure reproducibility.
Immunohistology
For double immunoenzymatic labeling of Foxp3/CD3, 4-mm–thick serialsections were cut, deparaffinized, and subjected to a heat-induced epitoperetrieval step before incubationwithAbs. Sectionswere immersed in sodiumcitrate buffer solutions at pH 6.0 and heated (100˚C) in a high-pressurecooker for 2 min. The slides were rinsed in cool running water, washed inTBS (pH 7.4), and blocked using a commercial peroxidase-blocking reagent(DakoCytomation, Carpinteria, CA). After a 30-min incubation with a goatpolyclonal Ab against the C terminus of the Foxp3 protein (ab2481, dilution1:50; Abcam Limited, Cambridge, U.K.), slides were incubated further withbiotin-conjugated rabbit anti-goat and the EnVision peroxidase kit (Dako-Cytomation). Sections were then incubated for 30 min with the second Abagainst CD3 (clone UCHT1, 1:25; DakoCytomation) and visualized by thealkaline phosphatase–anti-alkaline phosphatase method (27). Alkalinephosphatase was revealed using Fast Red (Roche, Mannheim, Germany) aschromogen. Microscopic analysis was performed using a Leitz microscope(Leica Microsystems, Wetzlar, Germany).
Isolation of CD4+CD25hi T cells and Foxp3 staining
Bulk lymphocyte cells from the spleens of infected or naive mice wereisolated following standard erythrocyte-depletion protocols. T cell pop-ulations were then enriched by depleting MHC class II+ cells using specificmicrobeads (Miltenyi). Thereafter, cells were subjected to an Fc blockingstep using anti-CD16/CD32 and after thorough washing stained witha combination of anti-CD4 allophycocyanin and anti-CD25 PE Abs (Na-tutec, Frankfurt, Germany). After an additional washing step, populationsof CD4+CD252 and CD4+CD25hi T cells were isolated using the MoFlocell sorter. A fraction of isolated cells were tested for levels of intracellularFoxp3 using FITC-labeled anti-Foxp3 Ab (Natutec). Fluorescence wasanalyzed using a FACSCalibur flow cytometer and CELLQuest software(BD Biosciences, San Jose, CA).
Microarray studies
Total RNA was prepared from MoFlo-separated Treg and Teff spleen cellpopulations using the RNA isolation kit from Qiagen (Hilden, Germany).Biological replicates of independent sorts from two naive mice and threeinfected mice were used for microarray analysis using the AffymetrixGeneChip platform (HighWycombe, Bucks, U.K.). Per sample, 200 ng totalRNA was processed using Ambion’s MessageAmp II Biotin Enhanced la-beling kit according to the manufacturer’s instructions. Biotinylated cRNAwas hybridized onMOE430A 2.0 GeneChips that were stained, washed, andscanned by following Affymetrix’s standard procedures. The probe leveldata (CEL files) were processed for global normalization and generation ofexpression values using the robust multiarray average algorithm of the “affypackage” in the R software environment (http://www.R-project.org). Toidentify differentially regulated genes, a combination offiltering for absolute(max-min. 80) and relative (max/min. 2) changes in average expressionsignals across all conditions, and statistical testing (LimmaF-test, p, 0.005)(1) was applied (28). Correction for multiple testing was done using themethod described by Benjamini and Hochberg (29). The resulting 474 probesets passing these filtering criteria were then subjected to k-means clustering(usingR), or to hierarchical clustering and visualization procedures using theSpotfireDecisionSite FunctionalGenomics software (Tibco, PaloAlto, CA).Normalized expression values were deposited in the Gene Expression Om-nibus repository as series numberGSE 17580 (www.ncbi.nlm.nih.gov/geo/).
Real-time RT-PCR detection of mRNA
In brief, RNA was prepared from spleen-derived T cell populations (CD4+
CD25hi and CD4+CD252) or organ samples using the RNA isolation kit fromQiagen (Hilden, Germany) and transcribed into cDNA using Superscript IIreverse transcriptase (Invitrogen,Karlsruhe,Germany).All real-timeRT-PCRs
were performed on the Light Cycler 480 System (Roche, Mannheim, Ger-many). Fold changes of selected genes of individual samples were relativelyquantified using threshold cycle (Ct) values and calculated using the ddCTmethod normalizing b-actin values. For organ samples, relative expressionvalues were calculated as 2^(-(Ct(Slpi)-Ct(actin)). Primers were designed(Metabion, Martinsried, Germany) and prevalidated probe sequences se-lected using the Universal Probe Library System and Software (Roche,Basel, Switzerland; cat. no. 04683641001) and are shown in Table II.
Statistical analysis
Statistical differences were analyzed by either ANOVA or Student’s t testusing GraphPad Prism software (San Diego, CA).
ResultsFoxp3+T cells are presentwithinS.mansoni-induced granulomasand control pathology
Histological preparations from the livers and intestines of naive andS. mansoni-infected C57BL/6 mice were subjected to immunohis-tochemical analysis to identify the location of Foxp3+ T cells in thetarget organs (Fig. 1a–d). As shown, Foxp3+ T cells are presentwithin granulomas found in the livers of infected mice (Fig. 1a).Interestingly, no detectable Foxp3+ T cells could be detected in liversections from naive mice (Fig. 1b), strongly indicating that there isa definite influx into the liver of these regulatory cells during in-fection. This finding confirms previous studies that have identifiedelevated mRNA levels of Foxp3 in the liver throughout infection (2,19, 30). The virtual absence of Foxp3+ T cells in naive livers was instark contrast to the situation in the intestines (Fig. 1d), whereFoxp3+ T cells could be readily detected in the layers of the mucosal
wall and within the villi. Fig. 1c further shows that during infectionthere are Foxp3+ T cells within the intestinal granulomas. On closerexamination, these Tregs were more prominent in the peripheralcircumference of the granulomas (Fig. 1e). Interestingly, areas oforgan that contained clusters of granulomas contained Foxp3+
T cells only around the edges of the cluster, indicating that the Tregsencircle and thus control granulomagrowth—the samephenomenoncould also be observed on histological analysis of human granulo-mas (data not shown). In a previous study, we demonstrated thatdepletion of CD4+CD25+ Tregs during infection elicits uncontrolledimmunopathology and Th responses (2). Thus, we also stainedpreparations from these infected groups of mice and observed thatthere is a distinct lack of Foxp3+ T cells in the granulomas (Fig. 1f).Thesefindings confirmedour notion thatCD25 is still a relevantTregmarker during infection. We therefore continued to use this markerfor isolation of Tregs for the following array analysis.
Immunological analysis of S. mansoni-infected mice andcharacterization of isolated Tregs
CD4+CD252 Teffs or CD4+CD25hi Tregs from the spleens of 9-wkC57BL/6 S. mansoni-infected or age-related control mice were iso-lated using theMoFlo cell sorter.We selectedmice that showed similarlevels of infection; this was determined by increased liver or spleenweights (Fig. 2A) and a similar number of eggs within the livers andintestines (Fig. 2B). The in situ cytokine milieu within infected liverswas also tested via ELISA and compared with uninfected controls(Fig. 2C). All tested parameters showed equal results in the infectedmice. In addition, restimulation of bulk MLNs with SEA clearly
FIGURE 1. Foxp3+ T cells reside within the pe-
riphery of S. mansoni-induced granulomas. Histolog-
ical sections (4 mm) from infected and naive livers (a,
b) or intestines (c, d) were immunohistochemically
stained for Foxp3 as indicated by arrows in (c). e,
Quantification of Foxp3+ T cells. Twenty granulomas
per section per mouse were analyzed for the quantity
and location of Foxp3+ T cells within the liver and
intestine. Foxp3+ T cells found residing within the
circumference of the granulomas were designated
peripheral, and those inside the granulomas were
classified as central. Analysis was determined on ten
infected mice and statistics were determined using the
Student’s t test. f, Evidence of necessary Treg control
during schistosomiasis: absence of Foxp3+ T cells in
showed that cells prepared from individually infected mice producedsimilar levels of Th1 (IFN-g) and Th2 (IL-10, IL-4, and IL-13) re-sponses (Fig. 2D). Fig. 2A–D shows data collected from infectedmiceused for microarray analysis and those used to corroborate microarraydata via PCR, which are described in the following sections. Fig. 2Edemonstrates our strategy for sorting regulatory cells. In brief, MHC-class II depleted spleen cell populations from individual mice weresubjected to cell sorting after labeling with anti-CD4 allophycocyaninandanti-CD25PEAbs. Populations ofCD4+CD252andCD4+CD25hi
T cell populations were obtained using the gates described in Fig. 2E.Thereafter, cells were immediately subjected to RNA preparation
protocols. To ensure that the Treg population was Foxp3+, some cellswere stained for this marker via intracellular staining. Fig. 2E alsoshows that, whereas only 0.18% of the CD4+CD252 T cells wereFoxp3+, 98% of the CD4+CD25hi T cells expressed Foxp3. To clarifywhether isolated Tregs possessed a suppressive capacity, we cocul-tured CD4+CD25hi cells with CD4+CD252 Teffs in a 1:1 setting andrestimulated them with SEA. Both Ag-specific Th1 (IFN-g) and Th2(IL-10) responses from effector cells were dampened in the presenceof Tregs isolated from the spleen (Fig. 2F). Similar results were ob-tained using Tregs isolated from the MLNs (data not shown). Finally,reflecting the flow cytometric analysis of Tregs and Teffs from initial
FIGURE 2. Immunological status of 9-wk-infected mice and characterization of isolated Foxp3+ T cells. To ensure that cells used for microarray analysis
were isolated from mice that were similarly infected, individual mice were analyzed for (A) haptomegalgy and splenomegaly: increased organ weight;
(B) parasite burden: total number of eggs in liver and intestine following KOH digestion of organ; (C) in situ cytokine milieu of liver and (D) Th responses
of bulk MLNs cells from individual mice following restimulation with SEAs. Cytokine levels were measured after 72 h of stimulation. Data in A–D
are from individually assessed mice and show the mean 6 SD of the specified parameters. E, Isolation strategy Teff and Treg populations using the MoFlo
cell sorter and the degree of intracellular Foxp3+ in the subsets after separation. F, Ag-specific suppression of effector CD4+ T cells by Treg following
restimulation with SEAs. Isolated CD4+CD25hi Treg (1 3 105) from the spleens of infected mice were cocultured with spleen-derived CD4+CD252 Teffs
(1 3 105) with spleen-derived irradiated APC (2 3 105) and SEAs for 72 h. Thereafter, levels of IL-10 (right) and IFN-g (left) were measured by ELISA.
Bars represent mean 6 SD of two suppression assays and show values of six individually tested Treg populations. G, Initial gene expression pattern
following microarray analysis of spleen cell populations. To observe whether the isolated CD4+CD25hi and CD4+CD25– T cell populations were correctly
separated, we studied the initial gene expression profile following subjection to microarray analysis. Bars show the gene levels of classical Treg markers
such as Foxp3 and IL-10. One can observe that the Treg populations were high for Foxp3 and IL-10, but low in IL-4 whereas Teffs were high for IL-4.
microarray data (Fig. 2E) further showed that sorted Tregs from thespleen gave.40-fold and 20-fold higher signals for Il2ra and Foxp3transcripts than Teffs; expression of IL-10 was much higher in Tregs,whereas Teffs strongly expressed IL-4 (Fig. 2G).
Comparison of differential gene expression within CD4+ T cellpopulations of schistosome-infected and naive mice
After determining that the isolated cell populationswere indeedTregsor Teffs, we performed gene expression profiling using AffymetrixGeneChips on spleen-derived T cells isolated from control andinfected mice to determine whether the Treg transcriptome is alteredduring infection. To ensure the overall reproducibility of the system,correlation coefficientson theunfilterednormalizedexpressionvaluesof all samples were determined. We observed a high degree of con-cordanceamongbiologicalreplicateswithlowaberrationtoeachother.For example, in the replicatesofTregorTeff populations (naiveversusinfected), the correlationcoefficientwas0.99–0.993 (data not shown).For identification of transcripts regulated during infection or
between Tregs and Teffs, we applied a combination of statisticaland change filters that identified 474 probes sets as differentiallyexpressed. Fig. 3 portrays a hierarchical cluster analysis for the fourdifferent spleen cell populations. The dendrogram clearly demon-strates that despite infection, gene expression profiles within thecellular subsets remained closely related. However, application ofa k-means clustering algorithm, to group these regulated transcriptsaccording to similarity in expression patterns, identified eight mainclusters. Using these clusters, we were then able to distinguishcharacteristic differences between naive and infected Treg and Teffpopulations (Fig. 4A). Table I describes selected genes found withinthese groups, which contained between 30 and 100 transcripts.Of specific interest were the genes regulated within the Teff
population isolated frominfectedmice (cluster 7); these included IL-4, IFN-g, Granzyme K, and CCL5. Clusters 5 and 6 show genesupregulated in Tregs isolated from naive and infected mice. Asexpected, genes that have been linked toTregphenotype or function,such as Foxp3, CTLA-4, CD83, neuropilin-1 (Nrp-1), IL-2ra, andGITR (Tnfrsf18), could be identified. Cluster 4 shows genes thatwere exclusively upregulated in Tregs after 9 wk of infection andincluded genes such asCD103, IL-10,Gzmb, andKlrg1. In addition,we also identified genes that have not been previously associatedwith Treg function or activity; these included Slpi, Mt1, CCL1,Socs2, preproenkephalin 1 (Penk1), and an asparagine synthetase. Inaddition, we identified a cluster of genes that were specificallydownregulated in the infected Treg population (cluster 2); theseincluded transcription factors such as zinc finger protein 207, Foxp1,cytoprotective heat shock protein 1b, and actin-binding Utrn.The main focus of this work was to identify genes specifically
upregulated in Tregs during infection. To confirm the geneshighlighted in the cluster analysis (cluster 4), mRNA quantificationby Light Cycler RT-PCR was performed to compare Slpi, Gzmb,Mt1, Fabp5, Klrg1, Nfil3, Socs2, and Gpr177 in infected and naiveTregs (Fig. 4B) (Table II). The data shows the expression of thesegenes in regulatory T cells isolated from the spleens of additionalinfected or age-related mice. The infectious status of these extrainfected mice compared with those used for the microarrayanalysis can be observed in Fig. 2A–D. In confirmation with themicroarray data, all tested genes were upregulated.
Tregs isolated from 9-wk-infected mice possess a specific genesignature
Previous expression profiling on naive Tregs has associated a certainpanel of genes with no known regulatory function (31). Many ofthose were also observed in the analysis of naive and infected Tregsin this study and are shown in clusters 5 and 6. Expanding on this
panel, we further included genes that have been associated withTreg function or activity (Fig. 5). In addition, we also mined themicroarray data for cytokines, especially those essential in S. man-soni responses. The data shown are the average normalized signalintensity values found within the three tested replicates. On closercomparison of Tregs from infected and naive mice, it is clear that nolarge differences could be observed in the regulation of markersincluding Foxp3, IL-2 receptor a, CTLA-4, Nrp1, and CD83. Incontrast, markers such as Klrg1, GITR (Tnfrsf18), OX40 (Tnfrsf4),and Gzmb were more strongly upregulated in Treg populationsisolated from infected mice. These cells also expressed more IL-10,but interestingly no differential regulation of TGF-b could be found.In contrast, Teff populations did not express any of these markers,but those from infected mice showed higher expression of IFN-gand IL-4 (confirmed also in cluster 7). Interestingly, in regard to theexpression of TLRs only TLR1 was found to be expressed on Treg.In contrast to previously publishedwork,which demonstrated TLR2surface expression on Tregs, we observed no differences in TLR2gene expression in Tregs or Teffs (32).Finally, we were interested in whether the genes that were upregu-
lated inTregs from infectedmicewere specific toS.mansoni infection,upregulated during other helminth infections, or found in Tregs duringany infection.Wechose the infectionmodelsCandidaalbicans and thefilarial helminthmodelLitomosoides sigmodontis andafter isolationof
FIGURE 3. Differential gene expression in Teff or Treg populations.
C57BL/6 mice were infected with S. mansoni for 9 wk. From both infected
and naive mice, total RNAwas prepared from isolated populations of CD4+
CD252 (Teff) and CD4+CD25hi (Treg) from the spleen and processed for
gene expression analysis using Affymetrix GeneChips. Following stringent
statistical and fold-change filters, 474 genes were differentially expressed to
a significant degree. Expression data from T cell populations isolated from
three individualmice per groupwere normalized using z-scores and analyzed
by hierarchical clustering. Each row in the heatmap represents one probe set,
and the columns stand for the analyzed cell population. A blue square in-
dicates lower expression, and higher expression is depicted in yellow, all
relative to a row-wise mean (black). The column dendrogram shows the
degree of similarity between the sorted cell populations.
Tregs from the spleen studied the expression of six genes using RT-PCR. Fig. 6 shows expression levels of positive control genes such asFoxp3 and IL-10 and negative control gene GATA3. No large differ-ences could be observed in the fold change of CD4/CD25 in the for-mer, whereas downregulation was observed in the latter. In therandomly-selected genes for comparative studies (Capg, Penk1, andTnfrsf9), there was surprisingly more overlap in the expression ofgenes between Candida albicans and schistosome-infected mice thanbetween the two helminth groups. Moreover, Tregs from C. albicans-infected mice showed even higher levels of expression. Interestingly,only Capg, described as a possible cancer suppressor gene (33), wasspecifically upregulated in Tregs from infected animals. Naturally,these hand-selected genes do not reflect the overall expression patternof infected Tregs. Nevertheless, they provide an impression about howthe gene expression patterns in Tregs change during different in-fections—that is, genes that are general for infection and genes that arespecific for controlling a particular ongoing infection. More in-depthanalysiswill be required to isolate genes that overlap and those that arespecific for a certain infection.
DiscussionTo survive within the host, parasites have developed complexstrategies to evade effective immune responses. Helminths excel atmanipulating the fine balance between effector responses and areknown to maneuver the hosts’s own regulatory mechanisms, re-sulting in immune suppression and long-term chronic infection in
the majority of individuals (30, 34). It is currently believed thatsuch evasive mechanisms include immune regulation and can havean effect on both the innate and adaptive branches of the immunesystem. In our previous study, we focused on the question whetherCD42CD25+ Tregs might be an active player in dampening ef-fective immune responses by the host; we found that depletion ofthose cells lead to enhanced and more vigorous immune responses(2). In this study, using Foxp3 as a marker of Tregs, we show thepresence of such cells within both liver and intestinal granulomasand confirm our earlier work, because no Foxp3+ cells could belocated in the granulomas of anti-CD25 Ab-treated mice (Fig. 1f).Interestingly, during normal infection we further observed thatthese cells operate on the periphery of the granulomas. On closerstudy, it was apparent that the immune cell content of the gran-uloma changed during development (35, 36). Based on size andfibrotic appearance we can identify three stages of granulomadevelopment. For example, “young” granulomas (i.e., those thatare just beginning to form around the egg) contain mainly T cells,macrophages, and some eosinophils. The latter population thenproceeds to saturate the “middle-aged” granulomas and can rep-resent up to 90% of the actual cellular content. “Old” granulomas(i.e., those in which the eggs have been destroyed and displaya strong fibrotic appearance) contain hardly any eosinophils andare instead dominated by an influx of Foxp3+ T cells. Althoughnot fully confirmed, we believe that there is an inverse relationshipbetween the number of eosinophils and Foxp3+ T cells.
FIGURE 4. Identification of biologically rele-
vant genes through clustering and confirmation of
gene upregulation in organs and Tregs isolated
from S. mansoni-infected mice. To assess the dy-
namics of specifically regulated genes in all four
fractionated populations, the 474 probe sets were z-
score normalized and used for a k-means cluster
analysis. A, Eight different clusters were obtained.
Numbers 124depict the different sorted spleen cell
populations: CD252 naive, CD25+ naive, CD252
infected, and CD25+ infected. The number of
transcripts in each cluster can be identified. Lines
show the distribution of the transcripts around the
mean in each cluster. B, Confirmation of upregu-
lated genes in cluster 4. RNAwas extracted fromall
four mouse spleen-derived CD4+ T cell pop-
ulations, transcribed into cDNA, and analyzed for
their expression levels of the depicted eight genes
using real-time PCR. Fold change was calculated
according to the ddCT method and values .1 in-
dicate induction of depicted genes in Tregs over
Teffs from individual naive or infected mice. Fold
change values #1 indicate no gene regulation or
downregulation of indicated genes. C, Upregula-
tion of Slpi in livers and intestines of S. mansoni-
infected mice. RNA was isolated from liver and
intestine samples fromnaive and infectedC57BL/6
mice and transcribed into cDNA. Samples were
then analyzed for their expression levels of Slpi and
b-actin using real-time PCR. Relative expression
values were calculated as 2^(-(Ct(Slpi)-Ct(actin)).
Our previous results (2) and the successful use of the anti-CD25Ab in other in vivo infection systems (37–39) encouraged us to usethis surface-marker for the isolation of Tregs. Currently, littleinformation is available on the function of Tregs and their geneticvariability during a chronic infection. Thus, we have focused ourstudy on the question of whether the gene-expression profile ofdistinct T cell populations changes during infection and whethernew genes characterizing activated Treg could be defined. Weused DNA microarray technology (Affymetrix GeneChip MouseGenome 430 2.0 Array) to address this question and createda strategy to isolate Foxp3+ T cells that were as pure as possible.The basis for the Treg-defining criteria was results obtained fromthe analysis of Tregs sorted from Foxp3-GFP mice that demon-strated that CD25hi cells are indeed 97% Foxp3-positive, whereasthe Foxp3-negative fraction is within the CD25lo section of the setgates (31). Because of this analysis, we decided to omit the CD4+
CD25lo population in our Treg analysis. In addition, as shown inour previous studies, the majority of Teffs that reacted stronglytoward the specific SEA were contained within the CD4+CD252
fraction. In this study, we clearly show that whereas Teffs fromschistosome-infected mice respond to SEA, Tregs do not. Instead,these regulatory cells actually suppress responding effector cellsin an Ag-specific manner.Upon evaluation of the microarray data, the 474 regulated genes
were grouped into eight clusters using a k-means cluster algorithm.This approach allowed us to assess the dynamics of specificallyregulated genes in all four fractionated populations. Detailed listsof the genes upregulated in the specific populations are listed inTable I. The transcriptional profile of CD4+CD25hi T cells clearlydiffers from that of the CD4+CD25- T cells. As shown in cluster 7the gene profile in Teffs is strongly cytokine-related (e.g., IL-4,
IFN-g, IL-21), although other factors such as chemokines, cell-cycle components and facets of apoptosis are also present (Cxcr6,Ccl5, PD-1, Casp1, T-bet, and Cxcr6). These genes have beenfound before in other microarray studies of this population, al-though cells were derived from naive mice (31, 40). Of interest,PD-1 has been shown to play a role in schistosome infection,because PD-1 expression levels on CD4+ T cells correlate with theseverity of morbidity as assessed through hypersplenomegalysyndrome (41). CCl5 (RANTES)-expressing cells are actuallyfound during Th1-priming conditions and reflect the fact thatsorted cells were isolated from mice that were still in the mixed Thphase (Th1 and Th2) of infection (42)—a classical hallmark ofschistosomiasis. Interestingly, many upregulated genes within theactivated Teff population belong to the family of serine proteases.These cells exhibit a highly proliferative potential in vitro andhave been shown to be significantly increased in infected spleens,livers, and MLNs (2, 30). In addition, the ratio of CD252/CD25+
cells does not change during infection; only the absolute numbersincrease, implying that the CD25 molecule is only transientlyupregulated in the Teff populations as described previously (43).Strikingly, not a single Treg-associated gene was upregulated inthis population. For example, IL-2 was upregulated only in Teff,albeit on a lower scale. The genes that were induced during in-fection in both Treg and Teff populations formed an interestingcluster. Of course, any overlapping upregulated genes cannot beused to differentiate between the two populations during the in-fection. Among them were the inhibitory receptor gp49A (44),proteoglycan 2, Caspase-4, Ccr5 and 2, and Ctla2a. Interestingly,there was a complete absence of any genes previously associatedwith the Treg signature. These data may indicate the assumedplasticity of T cell development (16) during immune responses.
Table I. (Continued )
Probe Set Gene Title Gene Symbol Fold Change 1 Fold Change 2
1419043_a_at Interferon inducible GTPase 1 Iigp1 3.6 1.71419083_at Tumor necrosis factor (ligand) superfamily, member 11 Tnfsf11 3.4 1.01448471_a_at Cytotoxic T lymphocyte-associated protein 2 a Ctla2a 3.4 4.91453851_a_at Growth arrest and DNA-damage-inducible 45 g Gadd45g 3.2 1.71450852_s_at Coagulation factor II (thrombin) receptor F2r 3.1 2.31421186_at Chemokine (C-C motif) receptor 2 Ccr2 3.0 3.1
Description of the cluster-analysis: cluster 1 FC1, naiveCD4+CD25- versus naiveCD4+CD25hi; cluster 1 FC2, infCD4+CD25- versus nfCD4+CD25hi; cluster 2 FC1,naiveCD4+CD25hi versus infCD4+CD25hi; cluster 3 FC1, naiveCD4+CD25hi versus infCD4+CD25hi; cluster 3 FC2, naiveCD4+CD25- versus infCD4+CD252; cluster 4 FC1,naiveCD4+CD25hi versus infCD4+CD25hi; cluster 4 FC2, infCD4+CD25- versus CD4+CD25hi; cluster 5 FC1, naiveCD4+CD25- versus naiveCD4+CD25hi; cluster 5 FC2,infCD4+CD25- versus infCD4+CD25hi; cluster 6 FC1, naiveCD4+CD252 versus naiveCD4+CD25hi; cluster 6 FC2, infCD4+CD25- versus infCD4+CD25hi; cluster 7 FC1,naiveCD4+CD25- versus infCD4+CD252; cluster 8, FC1; naiveCD4+CD25- versus infCD4+CD252; and cluster 8 FC2, naiveCD4+CD25hi versus infCD4+CD25hi.
Asns, asparagine synthetase.
Table II. Sequences of primers and probe numbers used for real-time PCR
The most interesting cluster comprises those genes that are sig-nificantly upregulated in the Treg population during infection. Themechanisms of Treg-mediated suppression are still poorly un-derstood, but three distinct categories are currently described: cell–cell contact and chemokines such as CD103 and ADAM8 (45); localsecretion of inhibitory cytokines/molecules Socs2, Gzmb, Mt1, andIL-10 (7, 45–47); and local competition for growth factors (Il-2r) andinduction of apoptosis (48, 49). However, the underlying pathwaysmight not bemutually exclusive. The genes found in cluster 4 displaythe genes that were selectively upregulated in CD4+CD25hi cellsduring infection. We were able to identify many Treg-associatedmarkers that have been previously described within naive Foxp3+ Tcells and in Foxp3-transduced CD4+CD252 T cells (24, 48, 50, 51).In addition, wewere also able to detect upregulation of newgenes notpreviously described with Treg function or activity. A completelynovel candidate was Slpi, which was actually our most strongly-regulated gene in thesecells.Slpi is a serine protease inhibitor that hasantiinflammatory properties and can modulate dendritic cell activityand thereby suppress T cell proliferation, although Slpi in this anal-ysis was dendritic cell-derived (22). Ongoing studies have recently
demonstrated that this marker is also elevated in the livers and in-testines of infected mice (Fig. 4C). Gzmb was also one of the mostprominently upregulated genes and has previously been shown to bereleased by in vitro-activated Tregs and functionally drives apoptosisin naive B cells (48). In accordance with other publications, thecomplete absence of TGF-b regulation in infected Tregs or any otherpopulation indicates that the function of thismoleculemight be betterevaluated on the protein level, because the studies performed in thisstudy cannot rule out the possibility of posttranslational regulation(52). This was not the case with IL-10, because it was significantlyupregulated during infection in the Tregs. These results overlap inpart with a study by Fontenot et al. that analyzed Tregs from naivemice that carried a GFP reporter in the Foxp3 locus (31).The identities of the genes that were upregulated in CD4+CD25hi
cells in naive and infected mice (clusters 5 and 6) were consideredproof that the sorted cells were indeed Tregs. This also demon-strates that this method of cell sorting is valid and can be appliedto investigate Tregs in the absence of other genetically modifiedmarkers like the Foxp3-GFP in mice. Naturally, IL-2ra, Foxp3,Ctla-4, and Tnfrsf18 were among the most strongly upregulated
FIGURE 5. Treg signature during in-
fection. We selected characteristic genes that
have been previously associated with Tregs
isolated from naive mice (31). These genes
are collectively depicted under the heading
Treg signature. Data show gene expression
levels of all four CD4+ T cell populations
isolated from the spleen cells of naive or in-
fected mice. The levels of cytokines, which
are classically associated with schistosome
infection, are also shown from the microarray
analysis. Symbols represent mean 6 SD from
three biological replicates.
FIGURE 6. Specific gene upregulation in Treg during infection. CD4+CD25hi and CD4+CD252 T cells were isolated from the spleens of naive mice or
C57BL/6 mice infected with S. mansoni (Schisto), Litomosodies sigmodontis (Lito), or Candida albicans (Caal). RNAwas extracted from both CD4+ T cell
populations from infected and naive mice, transcribed into cDNA, and analyzed for expression patterns of six genes: Foxp3, IL-10, Gata3 (upper panel),
Capg, Tnfrsf9, and Penk1 (lower panel). Graphs show the fold change according to the ddCT method. Values .1 indicate induction of depicted genes
within the Treg population as analyzed to the Teff population. Bars represent mean 6 and SD from T cell subsets isolated from eight naive mice and six
mice per infection group. PCR assays were performed three times to ensure reproducibility. Statistics were performed using the Student’s t test.
genes. Others in the current study, such as Ebi3, Socs2, Nrp1,Gpr83, CD10, and Folr4, have been described in gene-expressionanalysis before or were only recently identified as contributing toTreg function (42, 51, 53–55). We are currently analyzing thefunctional relevance of new genes like Penk-1, Mt1, and Slpi,especially within infectious systems. Our preliminary data usingtwo other infectious systems clearly identified the overlapping, butdistinctive Treg phenotype induced during infection. As a sideremark, C57BL/6 mice are not fully permissive to infection withL. sigmodontis; thus, when compared with S. mansoni-infectedmice, they are probably not in a comparable chronic-infectionstate, which might explain the weaker correlation of these genes.Nevertheless, it is becoming increasingly clear that Tregs usea vast array of mechanisms to exert their function.
AcknowledgmentsWe thank M. Schiemann for their support during isolation of T cell popu-
lations and A. Servatius for technical assistance.
DisclosuresThe authors have no financial conflicts of interest.
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