Human CD4+ CD25+ Regulatory T Cells Control T-Cell Responses to Human Immunodeficiency Virus and Cytomegalovirus Antigens

Regulatory T (T_R) cells maintain tolerance to self-antigensand control immune responses to alloantigens after organ transplantation.Here, we show that CD4⁺ CD25⁺ human T_R cells suppress virus-specificT-cell responses. Depletion of T_R cells from peripheral bloodmononuclear cells enhances T-cell responses to cytomegalovirusand human immunodeficiency virus antigens. We propose that chronicviral infections lead to induction of suppressive T_R cells thatinhibit the antiviral immune response.

INTRODUCTION

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Introduction

CD4⁺ CD25⁺ regulatory T cells (T_R cells) are a subset of circulatingCD4⁺ T cells with suppressive properties (21, 24). They werefirst identified in mice as cells capable of maintaining self-toleranceby suppressing autoreactive T cells (1, 22), or suppressingalloreactive immune responses after organ transplantation (8,15, 23). Human T_R cells have been identified and characterizedin peripheral blood and the thymus (2, 13, 18, 25). Althoughthe phenotypic characterization of T_R cells is still incomplete,CD25 has been used to distinguish a functionally relevant suppressiveT-cell subpopulation.

T_R cells develop in the thymus as cells that recognize antigenswith high affinity yet escape negative selection (14). T_R cellscan also be induced in the periphery after antigen activationand are termed adaptive T_R (4). These adaptive T_R cells canbe induced in vitro by cytokine priming and coculture with immaturedendritic cells (6, 12, 29) and in vivo after repeated exposureto superantigen (7). In addition to suppressing auto- and alloreactiveT cells, T_R cells can also suppress immune responses to humantumors and to bacterial and acute viral infections in animalmodels (3, 26, 28). However, it is not known whether T_R cellssuppress immune responses in human chronic viral infections.Here we show that depletion of CD25⁺ T cells from peripheralblood mononuclear cells (PBMC) augments T-cell immune responsesto cytomegalovirus (CMV) and human immunodeficiency virus (HIV)antigens. T_R cells may play an important role in controllingand suppressing the immune responses in chronic viral diseases.

MATERIALS AND METHODS

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Materials and Methods

Study samples. Blood samples from healthy blood donors were obtained underapproved University of California—San Francisco Committeeon Human Research Institutional Review Board protocols. Bloodsamples from HIV-infected patients were obtained from 16 adultpatients recruited after informed consent from the UCSF Optionscohort (median CD4 T-cell count = 544 cells/µl; range,483 to 799 cells/µl; and median viral load, 26,700 copies/ml;range, 1945 to >500,000 copies/ml). All but one of the patientswere infected within 1 year prior to blood sampling, and noneof the patients were currently under antiretroviral treatment.PBMC from healthy seronegative donors and HIV-infected individualswere isolated from heparinized whole blood by Ficoll-Paque PLUSdensity gradient centrifugation (Amersham Pharmacia, Uppsala,Sweden). The cells were washed twice in RPMI 1640 (MediaTech,Herndon, Va.) supplemented with 15% fetal calf serum (FCS) (GeminiBioProducts, Woodland, Calif.). PBMC not assayed immediatelywere frozen in FCS containing 10% dimethyl sulfoxide (Sigma-Aldrich,St. Louis, Mo.). Frozen PBMC were thawed, washed twice in RPMI1640 supplemented with 15% FCS, and incubated overnight at 37°Cprior to use.

Phenotyping of CD25⁺ regulatory T cells (T_R). Briefly, fresh or frozen PBMC were washed once in phosphate-bufferedsaline (PBS) containing 1% bovine serum albumin (BSA) (Sigma-Aldrich,St. Louis, Mo.) and stained with fluorescently labeled antibodiesfor CD3-peridinin chlorophyll protein (PerCP), CD4-fluoresceinisothiocyanate (FITC), CD8-allophycocyanin (APC), CD25-FITCor -phycoerythrin (PE), CD38-APC or -PE, and/or HLA-DR—APC(BD BioSciences, San Jose, Calif., and BD BioSciences PharMingen,San Diego, Calif.) for 20 min at 4°C. The cells were thenwashed twice with PBS containing 1% BSA, fixed in 1% paraformaldehyde,acquired on a flow cytometer (FACSCalibur; BD BioSciences),and analyzed using FlowJo software (Tree Star, San Carlos, Calif.).

Depletion of CD25⁺ cells. CD25⁺ cells were purified with MACS CD25 MicroBeads (MiltenyiBiotec, Auburn, Calif.). Briefly, fresh or frozen PBMC werewashed twice in PBS containing 0.5% BSA and 2 mM EDTA, resuspendedin 80 µl of PBS containing 0.5% BSA-2 mM EDTA and 20 µlof MACS CD25 MicroBeads per 10⁷ total PBMC, and incubated for15 min at 6 to 12°C. PBMC were washed twice in PBS containing0.5% BSA and 2 mM EDTA and applied to a magnetic column on aMidiMACS separation unit (Miltenyi Biotec). CD25⁺ and CD25^-T-cell fractions were collected. The CD25⁺ cell fraction contained>90% CD4⁺ T cells. In some experiments the CD25⁺ cell fractionwas purified to >99% CD4⁺ T cells by cell sorting after stainingwith monoclonal antibodies to CD3 and CD4 (FACSvantage; BD Biosciences).

Tetramer staining. PBMC were washed once in PBS containing 1% BSA and stained withCD8-FITC, CD3-PE, and APC-conjugated HLA A*02 CMV pp65 Tetramer(NLVPMVATV) (Beckman Coulter Immunomics, San Diego, Calif.).After staining, the cells were washed twice in PBS containing1% BSA and fixed in 1% paraformaldehyde.

Cytokine flow cytometry. PBMC, PBMC depleted of CD25⁺ T cells, or PBMC cocultured withCD25⁺ T cells were stimulated with staphylococcal enterotoxinB (SEB) (Sigma-Aldrich), CMV pp65 peptide (NLVPMVATV) (ResgenInvitrogen, Carlsbad, Calif.), HIV type 1 (HIV-1) Gag p24 (ProteinSciences, Meriden, Conn.), HIV-1 SF2 p55 Gag (National Institutesof Health AIDS Research and Reference Reagent Program), HIVGag p55 peptide mix (BD BioSciences PharMingen), Human CMV virallysate (Advanced Biotechnologies, Inc., Columbia, Md.), or withRPMI 1640 supplemented with 15% FCS and incubated for 18 h.All antigens were used at a final concentration of 2.5 to 5µg/ml. Brefeldin A (Sigma-Aldrich) was added at a finalconcentration of 5 µg/ml for the last 6 h of incubation.Cells were washed in PBS containing 2 mM EDTA, fixed in 1% paraformaldehyde,and permeabilized in FACS permeabilizing solution (BD BioSciences)for 10 min prior to being stained with CD8-APC, CD3-PerCP, gammainterferon (IFN- ${gamma}$ )-PE, and tumor necrosis factor alpha (TNF- ${alpha}$ )-FITC(BD BioSciences). The cells were washed twice in PBS containing1% BSA and fixed in 1% paraformaldehyde before being acquiredon a flow cytometer (FACSCalibur; BD Biosciences) and analyzedusing FlowJo software (Tree Star).

RESULTS AND DISCUSSION

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Results and Discussion

CD4⁺ CD25⁺ T_R cells inhibit superantigen-induced IFN- ${gamma}$ expression in both CD4 and CD8 T cells. To assess the potential inhibitory capacity of T_R cells, wecompared IFN- ${gamma}$ expression in PBMC after superantigenic stimulationwith or without CD25⁺ T cells. Depletion of CD25⁺ cells dramaticallyaugmented the IFN- ${gamma}$ expression in both the CD4 and CD8 T-cellcompartments (Fig.1a). When autologous CD25⁺ cells were addedback to a culture of PBMC depleted of CD25⁺ T cells, the IFN- ${gamma}$ expression was suppressed in a dose-dependent manner (Fig. 1b).The suppressive activity was found in the CD4⁺ T-cell compartmentof the CD25⁺ cell fraction (Fig. 1c), and the suppression ofcytokine expression was not due to dilution of responding cells(data not shown). Similar results were obtained by analyzingTNF- ${alpha}$ expression (data not shown).

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FIG. 1. T_R cells suppress superantigen-induced cytokine production. IFN- ${gamma}$ expression in T cells in PBMC was compared with that of T cells in PBMC depleted of CD25⁺ cells (a). The cultures were stimulated for 18 h with SEB. Brefeldin A was added for the last 5 h to promote cytokine accumulation. Means ± standard errors of the mean are shown (n = 3). (b) CD25⁺ T cells were added back into PBMC depleted of CD25⁺ cells at increasing ratios. The CD25⁺ T cells were stained with CFSE before being added back and were gated out of the analysis. (c) CD25⁺ T cells and sorted CD25⁺ CD4⁺ T cells (>99% pure) were added back into PBMC depleted of CD25⁺ cells. Means ± standard errors of the mean are shown (n = 2).

Suppressive CD25⁺ T cells can be induced from CD25^- T cells by activation with superantigen alone. The CD4⁺ T-cell population in peripheral blood of healthy blooddonors contains about 10 to 15% CD4⁺ CD25⁺ T cells (5). Therole of these cells in controlling and suppressing autoreactiveT cells is now well established, and the induction of the T_Rcells has been shown to take place in the thymus (10, 14, 19).However, induction of suppressive T cells specific for exogenousantigens that are not expressed in the thymus is likely to takeplace in the periphery. Therefore, we hypothesized that T_R cellscan be generated in vitro from peripheral CD25^- cells. We depletedCD25⁺ T cells from PBMC (Fig. 2a), labeled the remaining cellswith 6-carboxyfluorescein succinimidyl ester (CFSE), and culturedthem for 7 days in medium containing SEB. At day 7, 40 to 60%of the T cells expressed CD25, as did a large majority of theproliferating cells (Fig. 2b). Next, we wanted to test whetherthe induced CD25⁺ cells had suppressive properties. When theCD25⁺ cells were added to PBMC stimulated with SEB, they suppressedIFN- ${gamma}$ expression in the responding T cells in a dose-dependentmanner. No suppression was observed when the CD25^- cells wereadded to the culture (Fig. 2c). From these results, we hypothesizedthat there may be an expansion of T_R cells in conditions withpersistent immune activation. HIV infection leads to chronicimmune activation and is associated with increased frequencyof CD38⁺ CD8⁺ T cells (Fig. 3a). However, in a cross-sectionalstudy, we did not observe a higher percentage of CD4⁺ CD25⁺T cells in peripheral blood in HIV-infected individuals (n =10) than in healthy blood donors (n = 10) (Fig. 3b). This maybe due to redistribution of CD4⁺ CD25⁺ T cells to lymphoid tissues.Alternatively, the suppressive T cells occurring in peripheralblood may represent a heterogenic population of CD4 T cells.In mice, CD4⁺ CD69⁺ T cells have been shown to possess suppressiveactivity (11). This population only partly overlapped with theCD4⁺ CD25⁺ T-cell subset. It is, therefore, possible that usingCD25 expression to identify suppressive regulatory CD4⁺ T cellsunderestimates the real frequency of suppressive T cells inhealthy as well as HIV-infected individuals.

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FIG. 2. Suppressive CD25⁺ T cells can be induced from PBMC depleted of CD25⁺ T cells after activation with SEB. PBMC were depleted of CD25⁺ cells (a), labeled with CFSE, and cultured in the presence of SEB for 7 days (b). At day 7 (c), the CD25⁺ and CD25^- cell fractions were added into fresh PBMC cultures from the same donor. The cocultures were stimulated for 18 h with SEB. Brefeldin A was added for the last 5 h to promote cytokine accumulation. The CFSE-stained cells added into the fresh PBMC at day 7 were gated out of the analysis. Representative data are shown.

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FIG. 3. The frequency of T_R cells in HIV patients is unaltered. PBMC from HIV patients (n = 10) and healthy subjects (n = 10) were stained with fluorochrome-labeled monoclonal antibodies and analyzed for the frequencies of CD4⁺ CD25⁺ T cells and CD8⁺ CD38⁺ T cells. Means ± standard errors of the mean are shown.

CD4⁺ CD25⁺ T_R cells suppress antiviral immune responses. To investigate whether circulating CD25⁺ T cells suppress immuneresponses to viral antigens, we compared the IFN- ${gamma}$ expressionof CMV-pp65-specific CD8⁺ T cells with or without depletionof CD25⁺ T cells. The frequency of HLA-A2/CMV pp65 tetramer⁺CD8⁺ T cells was 0.57%, whereas the percentage of respondingIFN- ${gamma}$ ⁺ CD8⁺ T cells in the PBMC culture was 0.26% (Fig. 4a).Thus, less than 50% of the CD8⁺ T cells specific for this antigenresponded with IFN- ${gamma}$ expression in this individual. However,when the PBMC culture was depleted of CD25⁺ T cells, the frequencyof IFN- ${gamma}$ ⁺ CD8⁺ T cells increased to 0.59%, similar to the frequencyidentified with the CMV pp65 tetramer. When CD25⁺ T cells wereadded back, the IFN- ${gamma}$ expression was suppressed to the levelobserved before depletion.

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FIG. 4. T_R cells suppress antiviral immune responses. PBMC from a healthy CMV-infected individual were stained with a CMV pp65 tetramer and with cell surface markers (a). The frequency of tetramer-positive CD8⁺ T cells was compared with the frequency of IFN- ${gamma}$ -expressing T cells in PBMC, PBMC cultures depleted of CD25⁺ cells, and PBMC cultures depleted of CD25⁺ cells to which the depleted cells were added back in a 1:3 ratio. PBMC from an HIV-infected subject were stimulated with HIV antigens (b). The frequencies of IFN- ${gamma}$ - and TNF- ${alpha}$ -expressing T cells in PMBC cultures and PBMC cultures depleted of CD25⁺ cells were compared. Representative data are shown.

To further assess the degree of immunosuppression of antiviralimmune responses, we measured intracellular IFN- ${gamma}$ and TNF- ${alpha}$ expressionin T cells in response to several HIV antigens in PBMC culturesand in PBMC cultures depleted of CD25⁺ T cells. In all HIV-infectedsubjects, the anti-HIV immune response was considerably increasedin both the CD4 and CD8 T-cell populations after depletion ofCD25⁺ T cells (Fig. 4b and 5). These results indicate that suppressiveCD25⁺ T cells suppress the immune response to chronic viralantigens.

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FIG. 5. Depletion of T_R cells enhances the antiviral immune response to HIV. PBMC from HIV-infected subjects (n = 6) were stimulated with HIV and CMV antigens. The frequencies of IFN- ${gamma}$ - and TNF- ${alpha}$ -expressing T cells in PMBC cultures and PBMC cultures depleted of CD25⁺ cells were compared. The cells were gated on the CD3⁺ CD8^- and CD3⁺ CD8⁺ T cells. The shaded area in each graph represents the level of detection. Note: the scale on the y axis differs in the panels.

Based on these results, we propose that chronic viral infectionleads to induction in the periphery of a T_R cell populationwhich is involved in the suppression of antiviral immune responses.T_R cells may thereby impair an otherwise successful immune response.Our data support a mechanism of antigen-specific induction ofT_R cells, which then exert suppression in a nonspecific manner(9, 27). In HIV infection, HIV-induced T_R cells could potentiallycontribute to the generalized immunosuppression (16, 17, 20),and it is possible that manipulation of T_R cells could helprestore antigen-specific immune responsiveness in chronic viralinfections, such as HIV infection.

ACKNOWLEDGMENTS

We gratefully acknowledge assistance from Gerald Spotts andPaul Bradley in obtaining samples.

This work was supported by NIH grant U01 AI41531 (to D.F.N.and F.M.H.). D.F.N. is an Elizabeth Glaser Scientist of theElizabeth Glaser Pediatric AIDS Foundation. J.M. is supportedby a scholarship from the Swedish Research Council and the SwedishFoundation for International Cooperation in Research and HigherEducation (STINT).

FOOTNOTES

* Corresponding author. Present address: The Biotechnology Centre of Oslo, University of Oslo, P.O. Box 1125, Blindern, N-0317 Oslo, Norway. Phone: 47-22840521 or 47-92066046. Fax: 47-22840506. E-mail: aandahl{at}basalmed.uio.no.

REFERENCES

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