Transient depletion of regulatory T cells in transgenicmice reactivates virus-specific CD8+ T cells andreduces chronic retroviral set pointsKirsten K. Dietzea, Gennadiy Zelinskyya, Kathrin Gibberta, Simone Schimmera, Sandra Francoisa, Lara Myersb,Tim Sparwasserc, Kim J. Hasenkrugb, and Ulf Dittmera,1

aInstitute for Virology, University Clinics Essen, University of Duisburg-Essen, 45122 Essen, Germany; bLaboratory of Persistent Viral Diseases, RockyMountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; and cInstitute for InfectionImmunology, TWINCORE, 30625 Hannover, Germany

Edited* by Irving L. Weissman, Stanford University, Palo Alto, CA, and approved November 18, 2010 (received for review October 13, 2010)

Although chronic infectionswith viruses such as HIV and hepatitis Cvirus have been associated with regulatory T cell (Treg)-mediatedsuppression of virus-specific CD8+ T-cell activity, no causal relation-ship between Tregs and chronic viral set points has been estab-lished. Using transgenic mice in which Tregs can be selectivelyablated, we now show that transient depletion of Tregs duringa chronic retroviral infection allows exhaustedCD8+ T cells to regainantiviral functions, including secretion of cytokines, production ofcytotoxic molecules, and virus-specific cytolytic activity. Further-more, short-term Treg ablation resulted in long-term reduc-tions in chronic virus loads. These results demonstrate that Treg-mediated immunosuppression can be a significant factor in themaintenance of chronic viral infections and that Treg-targetedimmunotherapy could be a valuable component in therapeutic strat-egies to treat chronic infectious diseases.

cytotoxic T cells | retrovirus | friend virus

The ability of viruses to establish chronic infections requiresone or more mechanisms to escape the destruction of in-

fected cells by CD8+ cytotoxic T lymphocytes (CTLs) (1, 2). Onesuch mechanism appears to be the induction of regulatory T cells(Tregs), a specialized subset of CD4-expressing T cells that cansuppress the proliferation and/or function of effector T cells (3).Viral induction of Tregs was implicated as a mechanism of virus

escape in studies using the model of Friend retrovirus (FV) in-fection of mice (4). In a recent paper, we demonstrated that virus-induced Tregs dampened the peak CD8+ T-cell response duringacute FV infection and that depletion of Tregs resulted in reducedacute viral loads (5). Thus, prevention of Treg-mediated sup-pression of CD8+ T cells was a viable immunotherapy during theearly phase of retroviral infection. A critical question remaining,however, was whether Treg depletion would promote the reac-tivation of previously suppressed and functionally exhaustedCD8+ T cells during an established chronic infection. Such animmunotherapeutic approach for treating chronic infectionswould be a major advance but would depend on whether CD8+

T-cell exhaustion was reversible. Answers to these outstandingissues are important because several studies have indicatedprobable roles for Treg-mediated suppression of effector T-cellresponses in human chronic infections, including HIV and hep-atitis C virus (6), which account for well over 200 million infec-tions worldwide according to the World Health Organization.Although Tregs have been associated with chronic viral infectionsand T-cell exhaustion, no causal relationship between Treg-mediated immunosuppression and the maintenance of chronicviral loads has yet been established, primarily because of the dif-ficulty in specifically eliminating Tregs.To address this issue experimentally, we took advantage of

depletion of regulatory T cell (DEREG) mice that express thediphtheria toxin (DT) receptor and GFP under the control of theTreg-specific Foxp3 promoter (7). Injection of DT into these

animals specifically eliminates Foxp3-expressing Tregs, althoughleaving the overall composition of other lymphocyte subsets in-tact (8, 9). Foxp3-negative Tregs, which have been found in mice(10, 11), cannot be depleted in this model.

Results and DiscussionWe used these DEREG mice to establish chronic FV infections,which are associated with an expanded population of activatedbut not virus-specific Tregs in lymphoid tissues (5, 12–14). Thesevirus-induced Tregs can actively suppress the antiviral effectorfunctions of FV-specific CD8+ T cells, as has been shown byadoptive transfer experiments using T-cell receptor (TCR) trans-genic (Tg) CD8+ T cells that recognize an immunodominant FVepitope (15). At 6–8 wk post-FV infection, the mice were injectedwith DT, which resulted in 98% depletion of GFP+ Tregs at 1 wkpostdepletion, a similar depletion efficacy as in naive mice or miceacutely infected with FV (5, 8). After Treg depletion, a more thantwofold increase in the mean frequency of activated (CD43+)CD8+ T cells was detected in the lymph nodes and spleen (Fig.1A), where most infected cells were found during chronic FV in-fection (Fig. S1). The mean absolute number of CD43+CD8+ Tcells per spleen increased from 9.1 × 106 to 26.7 × 106 after Tregablation. The CD43+CD8+ T cells also had increased expressionof CD69 and CD44 and were negative for CD62L (5). To dem-onstrate virus specificity of the augmented T-cell response, CD8+

T cells were stained with class I tetramers (16) specific for theimmunodominant FV epitope DbGagL (17). Treg ablationresulted in an ∼2.5-fold increase in the mean frequency of tetra-mer-positive CD8+ T cells in lymph nodes and spleen comparedwith nondepleted controls (Fig. 1B). The mean absolute numberof these cells per spleen also increased from 2.5 × 106 to 6.0 × 106.It was not clear why there was a wide range of CD8+ T-cellreactivation levels in individual mice, because all the mice hadsimilar levels of Treg depletion. Analysis of individual miceshowed that relative increases in tetramer-positive cells werereflected by corresponding increases in the whole population ofactivated CD8+ T cells (CD43+). The rapid increase in virus-specific CD8+ T cells strongly suggested an increase in pro-liferative capacity following relief of Treg-mediated suppression.Indeed, activated CD8+ T cells, including tetramer-positive CD8+

T cells from both lymph nodes and spleens of Treg-depleted mice,incorporated significantly more BrdU than cells from untreated

Author contributions: K.K.D., G.Z., K.J.H., and U.D. designed research; K.K.D., G.Z., K.G.,S.S., S.F., and L.M. performed research; T.S. contributed new reagents/analytic tools;K.K.D., K.G., and L.M. analyzed data; and K.K.D., G.Z., K.J.H., and U.D. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: ulf.dittmer@uni-due.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1015148108/-/DCSupplemental.

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mice (Fig. 1 C and D), indicating that significant proliferationhad occurred.It has been shown that virus-specific CD8+ T cells with effector

phenotype (tetramer-positive CD43+) are functionally exhaustedduring chronic FV infection (18). To investigate whether Tregdepletion during chronic infection could influence functionalproperties as well as numbers of effectors, activated CD8+ T cellswere first analyzed for production of the cytokines IFN-γ, TNF-α,and IL-2. Depletion resulted in significantly more activated CD8+

T cells producing IFN-γ, TNF-α, and IL-2 (Fig. 2A). CD8+ T cellsproducing multiple cytokines have been associated with non-progression in HIV infection (19), and we observed a meanfourfold increase in the percentage of CD8+ T cells producingthree different cytokines in the lymph nodes of Treg-depletedmice compared with nondepleted controls (Fig. 2B).Cytolytic capacity is severely suppressed in exhausted CD8+ T

cells from chronically infected mice (18), so we analyzed severalmeasurements of this critical function in Treg-depleted mice. By1 wk following Treg depletion, significantly more activated CD8+

T cells from lymph nodes and spleen expressed granzyme B (Fig.2C), a cytotoxic molecule known to be important in immunecontrol of both FV (20) and HIV (21). This was also found forthe subset of tetramer-positive CD8+ T cells recognizing theimmunodominant FV epitope (Fig. 2D). Increased granzyme Blevels correlated with increased frequencies of CD8+ T cellsexpressing the degranulation marker CD107a (22, 23) (Fig. 2E),suggesting increased cytotoxic activity after Treg ablation. Totest directly for improved cytotoxic activity on release of Treg-

mediated suppression, an in vivo cytotoxicity assay was per-formed using lymphocytes loaded with the FV immunodominantepitope (5, 17). As expected, less than 10% killing of peptide-loaded targets was observed in chronically infected control mice.In contrast, Treg-depleted mice averaged ∼37% killing of peptide-loaded targets in the spleen at only 2 h postinjection. Repre-sentative flow cytometric histograms as well as the mean data areshown (Fig. 2F). These data are consistent with the granzyme Band CD107a staining and demonstrate the reactivation of potentcytolytic CD8+ T cells during chronic FV infection followingTreg depletion.The most likely explanation for the rapid increase in the

numbers of functionally active virus-specific CD8+ T cells fol-lowing Treg depletion was that loss of suppression allowed re-versal of CD8+ T-cell dysfunction.Based on previous results demonstrating that Treg numbers

are very low in the livers of chronically infected mice and thatCD8+ T cells in that tissue are not suppressed (24), an adoptivetransfer experiment was devised to test the reversibility of Treg-mediated suppression of CD8+ T cells. Genetically labeled(Thy1.1), virus-specific, CD8+ T cells from FV-specific TCR Tgmice were adoptively transferred into chronically infected miceto become suppressed for 1 wk (15). The Thy1.1 donor cells werethen sorted from the spleens and adoptively transferred intodifferent chronically infected hosts to determine if cells thattrafficked to the liver would regain function. At 4–5 d post-transfer, CD8+ T cells that homed back to the spleen retaineda suppressed phenotype as measured by lack of granzyme B

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Fig. 1. Treg depletion results in proliferation of activated virus-specific CD8+ T cells. Frequencies of activated (positive for the activation-induced isoform ofCD43) CD8+ T cells from spleens and lymph nodes of naive mice, chronically infected mice (chron), and chronically infected mice treated with DT (A) andtetramer-positive CD43+CD8+ T cells (B) and tetramer-positive CD8+ BrdU+ T cells (C) in chronically infected mice and chronically infected mice treated with DTare shown as calculated by cell counting and flow cytometry as previously described (5). Each dot represents an individual mouse. Data were pooled fromthree to five independent experiments with similar results. Differences between the groups were statistically significant by an unpaired t test (*P < 0.05; **P <0.005; ***P < 0.0005). (D) Representative dot plots for BrdU incorporation by activated CD8+ T cells and tetramer-positive CD8+ T cells. The percentages ofCD8+ T cells that were BrdU− but positive for CD43 or MHC class I tetramers are given in the upper left quadrants. The percentages of CD8+ T cells that wereBrdU+ and positive for CD43 or MHC class I tetramers are given in the upper right quadrants.

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production (Fig. 3A) and CD107a expression (Fig. 3B). In con-trast, almost half of the cells that homed to the liver produced

granzyme B and expressed cell surface CD107a (Fig. 3 A and B).Thus, Treg-mediated suppression of CD8+ T cells was reversible,

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Fig. 2. Treg depletion results in functional reactivation of CD8+ T cells of chronically infected mice. (A) Frequencies of IFN-γ–, TNF-α–, and IL-2–producingCD43+CD8+ T cells from spleens and lymph nodes of chronically infected mice (chron) and chronically infected mice treated with DT are shown. Each dotrepresents an individual mouse. Numbers were calculated by cell counting and flow cytometry using fluorescent staining as described previously (5). Datawere pooled from three to five independent experiments with similar results, and differences between the groups were analyzed by an unpaired t test (**P <0.005; ***P < 0.0005). (B) Mean percentages of activated CD8+ T cells producing IFN-γ, TNF-α, IL-2, and combinations of these cytokines using data pooledfrom two independent experiments (n = 8). (C) Frequencies of CD43+CD8+ T cells producing granzyme B (GzmB+). (D) Frequencies of tetramer-positive CD8+ Tcells producing granzyme B. (E) Frequencies of CD43+CD8+ T cells expressing degranulation marker CD107a. (C–E) Data were pooled from three to five in-dependent experiments with similar results, and differences between the groups were analyzed by an unpaired t test (*P < 0.05; **P < 0.005; ***P < 0.0005).(F) Representative histograms and mean percentages showing differential killing of unloaded cells (CFSElow) vs. cells loaded with the FV DbGagL peptide(CFSEhi) in an in vivo cytotoxicity assay. Data were pooled from spleen cells of four mice per group, and the difference between the groups was statisticallysignificant by an unpaired t test (*P < 0.05).

2422 | www.pnas.org/cgi/doi/10.1073/pnas.1015148108 Dietze et al.

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dependent on microenvironmental signals that correlated withlow Treg concentrations.The initiation of cytotoxic CD8+ T-cell function and the

production of antiviral cytokines suggested that Treg depletioncould result in diminished chronic viral loads. Levels of chronicinfection were compared between Treg-depleted and controlanimals using infectious center assays. At 7 d following the firstTreg depletion, there was a significant reduction of virus loads inthe lymph nodes averaging 4.5-fold (Fig. 4A). There was alsoa statistically significant reduction in the spleen averaging 1.8-fold. Importantly, reduced chronic viral loads were maintainedfor 5–8 wk following cessation of depletion (Fig. 4A). Thus,transient relief of Treg-mediated suppression resulted in long-term reduction of viral set points during this chronic retroviralinfection. To prove formally that the reactivation of exhaustedCD8+ T cells mediated the reduction in chronic viral loads, wedepleted CD8+ T cells with the monoclonal antibody 169.4 inchronically infected mice that were ablated for Tregs. CD8+ T-cell depletion prevented the reduction in viral set points in thespleen (Fig. 4B) indicating that CD8+ T cells indeed mediatedthe reduction of chronic FV loads in these mice.Although the primary purpose of our studies was to prove

formally whether Tregs influenced chronic viral loads, the posi-tive results also have obvious implications for therapeuticstrategies to treat chronic infections. A sustained reduction ofchronic viral loads following a short period of therapy would bedesirable for many chronic viral infections in humans, especiallyfor HIV, where the chronic viral set points strongly influence

progression to AIDS (25). As in chronic FV infection, the ac-cumulation of Tregs in HIV-infected lymphoid tissues correlatesboth with the dysfunction of virus-specific CD8+ T cells (19, 26)and with local viral loads (27), suggesting that their ablationmight result in long-term nonprogression of patients with HIV. Itmust be remembered, however, that unlike FV, HIV paradoxi-cally causes a disease characterized not only by immunosup-pression but by immune hyperactivation, which is detrimental tosurvival (28, 29). Because Tregs likely limit HIV-induced im-mune hyperactivation (30) as well as suppress HIV-specific im-mune responses (31, 32), it is hard to predict if Treg ablationwould potentially delay or exacerbate disease progression. Thus,careful studies in simian immunodeficiency virus-infected mac-aques are requisite before Treg immunotherapy is considered forHIV infections. Because Tregs are important in controlling au-toimmune diseases (33), another obvious concern with Tregablation therapy is the possibility of inducing immunopathology.Importantly, transient depletion of Tregs in FV-infected mice fora period of up to 10 d did not lead to any clinical signs of im-munopathology (5). Genetic factors play a major role in both

Fig. 3. Virus-specific CD8+ T cells regain function after release of Tregsuppression. Reactivation of adoptively transferred virus-specific CD8+ T cellstrafficking to the liver but not to the spleen. (A) Mean percentages of Thy1.1CD8+ T cells producing granzyme B (Left) and a representative dot plot ofgranzyme B expression of cells from the spleen and liver (Right). Resultswere obtained from five experiments with a total of 10 recipient mice. Themean percentage of granzyme B+ cells from the spleens was 9.2 ± 1.2%, andthat from the livers was 46.8 ± 6.2%, (*P < 0.0001 by a paired t test). (B)Mean percentages of Thy1.1 CD8+ T cells expressing cell surface CD107a(Left) and a representative dot plot of CD107a expression of cells from thespleen and liver (Right). Results were obtained from two experiments witha total of 7 recipient mice. The mean percentage of CD107a+ cells from thespleens was 8.9 ± 1.7%, and that from the livers was 43.6 ± 5.7% (*P =0.0007 by a paired t test).

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Fig. 4. Sustained reduction of viral loads during chronic infection afterablation of Tregs. (A) Lymph nodes and spleens from chronically infected(chron) control mice, chronically infected Treg-depleted mice euthanized 1d after the third DT injection, and chronically infected Treg-depleted miceeuthanized 5–8 wk after the third DT injection were analyzed for viral loadsby infectious center assays. Each dot represents an individual mouse. Datawere pooled from three to five independent experiments with similarresults. Differences between the groups were statistically significant by anunpaired t test (*P < 0.05; **P < 0.005). (B) Chronically infected mice weredepleted for Tregs, CD8+ T cells, or Tregs and CD8+ T cells, or they werenondepleted. Spleen cells of these animals were analyzed for viral loads byinfectious center assays at 1 d after the last injection of DT and/or the CD8a-depleting antibody 169.4. Each dot represents an individual mouse. Differ-ences between the groups were statistically significant by an unpaired t test(*P < 0.05; ns, not significant).

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mouse and human autoimmune diseases, however, and theC57BL/6 adult mice used in these studies are relatively resistantto the development of autoimmune diseases (34, 35). Never-theless, evidence of the safety of transient Treg ablation has beenprovided by clinical trials in patients with cancer, where Tregswere selectively depleted by application of a recombinant IL-2–DT fusion protein (ONTAK) (36). The temporary reduction inTregs by ONTAK augmented tumor-specific T-cell responsesafter vaccination of patients with tumor antigens, and thetreatment did not induce serious clinical side effects.Our current study shows that transient depletion ofTregs is a very

potent method to restore virus-specific CD8+ T-cell responses ina chronic retroviral infection. In recent experiments, anotherapproach was used to reactivate CD8+ T cells in chronic lympho-cytic choriomeningitis virus infections by blocking inhibitory cellsurface receptors on CD8+ T cells (37, 38). This approach wasnot successful in mice chronically infected with FV (39), however,indicating that different therapeutic approaches may have to betailored to the specific mechanisms leading to CD8+ T-cell dys-function in various infections. Even in chronic FV infections, morethan onemechanism of suppressionmay be in place as indicated bythe failure ofTreg ablation to eliminate chronicFVcompletely. It isalso possible that a rapid replenishment of Tregs prevented com-plete viral clearance, however. As evidence of the latter, at 5 wkpost-Treg depletion when viral loads were still reduced, the Tregcompartment was restored and only very little activity of CD8+

Tcells was detectable (Fig. S2).Therefore,multiple rounds ofTreg-targeted immunotherapy, or its combination with other therapies,such as antiviral drugs or therapeutic vaccines, could be necessaryto achieve complete virus clearance, the ultimate goal in thetreatment of chronic viral infections.

MethodsMice. Inbred C57BL/6 (B6), Thy1.1, andDEREG (7)miceweremaintained underpathogen-free conditions. The DbGagL TCR Tg mice (15) carrying the GFPgene (40) were on a C57BL/6 background. Experiments were done using mice(H-2b/b, Fv1b/b, and Fv2r/r) or Tgmice back-crossed on C57BL/6 background thatare resistant to FV-induced leukemia. All mice were female and aged 8–16 wkat the beginning of the experiments. All animal procedures were performedin compliance with animal care and use committee protocols.

Virus and Viral Infection. The FV stock used in these experiments was FVcomplex containing B-tropic Friend murine leukemia helper virusand polycythemia-inducing spleen focus-forming virus (41). The stock wasprepared as a 10% (wt/vol) spleen cell homogenate from BALB/c mice infected14 d previously with 3,000 spleen focus-forming units of noncloned virusstock. Experimental mice were injected i.v. with 0.5 mL of PBS containing15,000 spleen focus-forming units of FV. The virus stock contained lactatedehydrogenase-elevating virus to enhance chronic FV infection.

Infectious Center Assays. Infectious center assays were performed as describedpreviously (42).

Cell Surface and Intracellular Staining by Flow Cytometry. Cell surface stainingwas performed using Becton Dickinson reagents. T-cell antibodies were asfollows: anti-CD43 (1B11), anti-CD8 (53-6.7), Thy1.1 (OX-7), and anti-CD107a(1D4B). Dead cells (7AAD+) were excluded from all analyses. Intracellulargranzyme B (monoclonal anti-human granzyme B antibody allophycocyanin(APC)-conjugated, clone GB12; Caltag Laboratories) staining was performedas described (14). Intracellular staining for IFN-γ, TNF-α, and IL-2 was per-formed as described (8). Foxp3 expression was detected by intracellularstaining using the anti-mouse/rat Foxp3 antibody (clone FJK-16s) and theFoxp3 staining set (eBioscience). Data were acquired on an LSR II flowcytometer (Becton Dickinson) from 100,000–500,000 lymphocyte-gatedevents per sample. Analyses were done using FACSDiva software (BectonDickinson) and FlowJo software (Treestar).

Lymphocyte Depletion. To deplete Tregs, DEREG mice were injected i.p. withDT (Merck) diluted in endotoxin-free PBS. A total of 0.5 μg of DT was in-oculated every third day for three times. The treatment depleted over 97%of the CD4+ eGFP+ T cells in all investigated organs of DEREG mice. For de-pletion of CD8+ T cells, chronically infected mice were treated i.p. everyother day for four times with 500 μL of supernatant fluid obtained fromhybridoma cell line 169.4 producing a CD8a-specific monoclonal antibody(43). The treatment depleted over 96% of the CD8+ T cells in the spleen.

Tetramers and Tetramer Staining. For detection of Db-GagL-specific CD8+ Tcells, nucleated lymph nodes and spleen cells were stained with APC-labeledanti-CD8 (Ly-2) (eBioscience), FITC-labeled CD43 (1B11) (Becton Dickinson), andphycoerythrin-labeled MHC class I H2-Db tetramers specific for FV GagLpeptide (Beckman Coulter) (16, 17).

In Vivo Cytotoxicity Assay. The in vivo CTL assay described by Barber et al. (44)was modified to measure cytotoxicity in FV-infected mice (5).

BrdU Staining. BrdU incorporation was detected using the BrdU flow kitprotocol (Becton Dickinson) as described (5).

CD8+ T-Cell Reactivation. (B10 × A.BY) F1 mice (Th1.2) were chronicallyinfected with 1,500 spleen focus-forming units of FV/lactate dehydrogenase-elevating virus as described (45) and then adoptively transferred with 106

Thy1.1 CD8+ T cells from virus-specific TCR Tg mice (15). Seven days afteradoptive transfer, Thy1.1 donor cells were sorted from the spleen usinga FACSAria cell sorter (Becton Dickinson). The suppressed status of the CD8+

T cells was confirmed by lack of granzyme B production and CD107a ex-pression, and 2.5–3.5 × 106 purified CD8+ donor cells (depending on yield)were injected i.v. into chronically infected Thy1.2 mice. Four to five daysfollowing adoptive transfer into the secondary recipients, spleen and liverlymphocytes were isolated from tissues of perfused mice (to remove bloodlymphocytes) and analyzed by flow cytometry as described above.

Statistical Analysis. Statistical data were derived by using the nonparametric ttest (Prism software; GraphPad Software Inc.).

ACKNOWLEDGMENTS. This work was supported by the German ResearchAssociation (Deutsche Forschungsgemeinschaft), Transregio 60 project B4,and, in part, by the Division of Intramural Research, National Institute ofAllergy and Infectious Diseases, National Institutes of Health.

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