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The Journal of Immunology, 2008, 180, 1729 -1736
Copyright © 2008 by The American Association of Immunologists, Inc.
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NK Cells Lyse T Regulatory Cells That Expand in Response to an Intracellular Pathogen1

Sugata Roy*,{dagger}, Peter F. Barnes*,{dagger},{ddagger}, Ankita Garg*,{dagger}, Shiping Wu*,{dagger}, David Cosman§ and Ramakrishna Vankayalapati2,*,{dagger}

* Center for Pulmonary and Infectious Disease Control, {dagger} Departments of Microbiology and Immunology and {ddagger} Department of Medicine, University of Texas Health Center, Tyler, TX 75708l; and § Amgen, Seattle, WA 98101


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We evaluated the capacity of NK cells to influence expansion of CD4+CD25+FoxP3+ regulatory T cells (Tregs) in response to microbial Ags, using Mycobacterium tuberculosis as a model. We previously found that Tregs expand when CD4+ cells and monocytes are exposed to M. tuberculosis. Addition of NK cells that were activated by monokines (IL-12, IL-15, and IL-18) or by exposure to M. tuberculosis-stimulated monocytes reduced Treg expansion in response to M. tuberculosis. NK cell inhibition of Treg expansion was not mediated through IFN-{gamma}. Activated NK cells lysed expanded, but not freshly isolated Tregs. Although monokines increased NK cell expression of the activating receptors NKp46, NKG2D, 2B4, CD16, and DNAM-1, only anti-NKG2D and anti-NKp46 inhibited NK cell lysis of expanded Tregs. Of five NKG2D ligands, only UL16-binding protein 1 (ULBP1) was up-regulated on M. tuberculosis-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs. M. tuberculosis-stimulated monocytes activated NK cells to lyse expanded Tregs, and this was also inhibited by anti-NKG2D and anti-ULBP1, confirming the physiological relevance of this effect. Our study identifies a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer cells are large granular lymphocytes that can kill infected cells without prior sensitization and play an important role in innate immunity to microbial pathogens. NK cells mediate protection against viruses, bacteria, and parasites through destruction of infected cells and by secretion of cytokines that shape the adaptive immune response (1, 2, 3, 4, 5). NK cells participate in dendritic cell maturation (6, 7) and are an important early source of IFN-{gamma}, which is critical for activation of macrophages (8, 9). Therefore, NK cells may favor clonal expansion of Th1 cells, processes that are important for elimination of many intracellular pathogens. Our published data indicate that NK cells lyse Mycobacterium tuberculosis-infected monocytes and alveolar macrophages through the NKp46 receptor and NKG2D (10), and NK cells contribute to the capacity of CD8+ T cells to produce IFN-{gamma} and to lyse M. tuberculosis-infected monocytes (11).

Recently, attention has been focused on regulatory T cells (Tregs),3 a subset of CD4+ T cells that express CD25 and FoxP3 (12), which can inhibit IFN-{gamma} production by T cells. Many studies have shown that Tregs play a central role in down-regulating the immune response to organ transplants and tumors, preventing the development of autoimmunity (13) and dampening the immune response to intracellular pathogens (14, 15). Recent studies also showed that Tregs inhibit NK cell cytolytic activity via TGF-β, but do not inhibit production of IFN-{gamma} by NK cells stimulated by IL-2R{gamma} chain-dependent cytokines (16). Increased numbers and activity of Tregs are also associated with depressed NK cell activity in several diseases (17, 18, 19). However, the effects of NK cells on Tregs in healthy persons during a response to microbial Ags are unknown. In the current study, we found that NK cells inhibit Treg expansion through direct lysis of expanded Tregs, and investigated the mechanisms involved in this lysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patient population

Blood was obtained from 20 healthy tuberculin reactors. All studies were approved by the Institutional Review Board of the University of Texas Health Center (Tyler, TX) and informed consent was obtained from all participants.

Abs and other reagents

For flow cytometry, we used FITC anti-CD4, allophycocyanin anti-CD25, PE anti-Foxp3, PE-Cy5 anti-Foxp3 (all from eBioscience), FITC anti-CD14, FITC anti-DNAM-1, FITC anti-CD16, PE anti-NKp46, PE anti-2B4, allophycocyanin anti-NKG2D, and PE anti-CD127 (all from BD Biosciences).

In some cases, indirect immunolabeling was performed using anti-human mAb to MHC class I-related chain (MIC)A/B (BD Biosciences), UL16-binding protein (ULBP)1, ULBP2, and ULBP3 (M295, M312, and M551, respectively; Amgen), and anti-human MHC class I primary Ab (W6/32; DakoCytomation) and a FITC goat anti-mouse secondary Ab (Southern Biotechnology Associates). After gating on live cells, the mean fluorescence intensity (MFI) of stained cells was measured.

For neutralization, we used mAbs to IFN-{gamma}, MICA/B, NKG2D, NKp46, DNAM-1 CD16, and CD244 (BD Biosciences) and mAb to MICA/B, ULBP1, ULBP2, and ULBP3 (Amgen). Isotype control Abs were also used (BD Biosciences). In some experiments, rIL-12, rIL-15 (both from R&D Systems), and rIL-18 (MBL International) were added to the cells. M. tuberculosis whole cell lysates (WCL) were obtained from Dr. J. Belisle (Colorado State University, Fort Collins, CO), and heat-killed M. tuberculosis Erdman was provided by Dr. P. Brennan (Colorado State University, Fort Collins, CO).

Immunolabeling of intracellular Foxp3

Surface staining to detect CD4+, CD25+, and CD127+ cells and intracellular staining to detect Foxp3+ cells was performed, using the Cytofix/Cytoperm Plus kit (eBioscience). Controls for each experiment included cells that were unstained, cells to which FITC-, allophycocyanin-, or PE-conjugated mouse IgG had been added, and cells that were single stained, either for the surface marker or for Foxp3. We gated on CD4+ lymphocytes, and determined the percentages of CD25+ and Foxp3+ cells. For some experiments, we gated on Foxp3+ cells to detect CD127low cells, using a FACSCalibur (BD Biosciences).

Isolation of NK cells, monocytes, CD4+ cells, and CD8+ cells

PBMC were isolated by centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech). For NK cell isolation, CD3+ cells were depleted from PBMC with magnetic beads conjugated to anti-CD3 (Miltenyi Biotec), and from the negative cell fraction, CD56+ cells were isolated by positive selection with magnetic beads conjugated to anti-CD56 (Miltenyi Biotec). The positive cells were 95–100% CD56+ and 95–97% CD3–, as measured by flow cytometry, and were used as effector cells. Monocytes and CD4+ cells were isolated with magnetic beads conjugated to anti-CD14 or -CD4 (Miltenyi Biotec), respectively. Positively selected cells were >95%+, as measured by flow cytometry.

Culture of NK cells

In some experiments, freshly isolated NK cells were cultured in the presence of complete RPMI 1640 and 10% heat-inactivated human serum (Atlanta Biologicals) and IL-12, IL-15, and IL-18 (each at 3 ng/ml) for 72 h to obtain activated NK cells. In other experiments, freshly isolated NK cells from healthy tuberculin reactors were cultured in 12-well plates at 2 x 106 cells/well in RPMI 1640 containing penicillin and 10% heat-inactivated human serum, in the presence of 0.5 x 106 autologous monocytes/well and M. tuberculosis WCL (tuberculosis (TB) lysate) (5 µg/ml). After 72 h, CD56+ cells were isolated and designated as TB lysate-activated NK cells. These activated NK cells were cultured with CD4+ cells or used as effector cells against Treg targets.

Culture of CD4+ cells and monocytes

Freshly isolated CD4+ cells were cultured in 12-well plates at 2 x 106 cells/well in RPMI 1640 containing penicillin (Invitrogen Life Technologies) and 10% heat-inactivated human serum, with or without 2 x 105 autologous monocytes/well. CD4+ cells and monocytes were cultured in the presence or absence of M. tuberculosis WCL (5 µg/ml) at 37°C in a humidified 5% CO2 atmosphere. After 3 days, CD4+CD25+ cells were isolated.

Isolation of Tregs

Using the Treg isolation kit (Miltenyi Biotec), CD4+CD25+ cells were isolated from PBMC and designated as "freshly isolated Tregs." Similarly, CD4+CD25+ cells were isolated from culture of CD4+ cells and TB lysate-stimulated monocytes, and designated as "expanded Tregs." In both cases, CD4+ cells were negatively selected by incubation with a mixture of mAbs against CD19, CD14, CD8, and {gamma}{delta} TCR, followed by positive selection with anti-CD25 microbeads to select CD25+ cells. Seventy-five percent of the freshly isolated and expanded CD4+CD25+ cells and <5% of the CD4+CD25– cells were Foxp3+. In some experiments, Tregs were isolated by depleting CD127+ cells. To deplete CD127+ cells, CD4+ cells were negatively selected, as outlined above, and from the negative fraction, CD127+ cells were removed with anti-CD127 biotin-labeled Ab and anti-biotin beads. The negative fraction was highly enriched for Tregs, and ~85% of the CD127– cells were Foxp3+, as measured by flow cytometry.

Cytotoxicity assay

NK cell-mediated cytotoxicity against freshly isolated Tregs, expanded Tregs, and CD4+CD25– cells was assayed in a 51Cr-release assay, using standard methods (20). Briefly, CD4+ cells and monocytes were cultured in the presence or absence of M. tuberculosis WCL for 3 days and cells were labeled overnight with 100 µCi sodium chromate. Expanded CD4+CD25+ cells and CD4+CD25– cells or CD127+ and CD127– cells were isolated, as outlined above. Fresh CD4+CD25+ cells and CD4+CD25– cells from PBMC were isolated, using the Treg isolation kit, and were labeled with 100 µCi sodium chromate. Target cells were washed three times, and triplicate wells of 104 cells/well were mixed with effector cells at an E:T ratio of 20:1 in 200 µl of RPMI 1640 with 10% heat-inactivated human serum. Ten hours after incubation, 100 µl of supernatant was removed from each well, and radioactivity was measured in a gamma counter. The percent-specific lysis was calculated using: 100 x (experimental release – spontaneous release/maximum release – spontaneous release).

In some experiments, cytotoxicity of activated NK cells against expanded CD4+CD25+ and CD4+CD25– cells was performed, using a flow cytometry-based cytotoxicity kit (Cell Technology). Briefly, expanded CD4+CD25+ and CD4+CD25– cells were labeled with CFSE, before using them as target cells for activated NK cells. Ten hours after incubation, a DNA-binding, fluorescent dye, 7-aminoactinomycin D (7-AAD), which stains dead cells, was added to the cultures to measure cell death. The percent lysis was calculated as: 100 x (CFSE and 7-AAD double-positive cells/total number of CFSE-positive cells). Spontaneous cell death of target cells was calculated and subtracted as a background control.

Statistical analysis

Results are shown as the mean ± SE. Comparisons between groups were performed by a paired or unpaired t test, as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NK cell-mediated inhibition of Treg expansion

We recently found that culture of CD4+ cells with M. tuberculosis-stimulated monocytes results in expansion of CD4+CD25+FoxP3+ cells, and that these cells inhibit IFN-{gamma} production by CD4+ and CD8+ cells, indicating that they are Tregs (21). Human Th1 responses are inhibited by Tregs (13) but stimulated by NK cells (8, 9, 11). Because NK cells and Tregs have opposing effects, we asked whether NK cells reduced expansion of Tregs. At day 0, CD56+CD3– cells from eight healthy tuberculin reactors were isolated by immunomagnetic selection, cultured with IL-12, IL-15, and IL-18 for 48 h, and washed twice to remove the monokines. These were designated as monokine-activated NK cells. At day 3, more blood was obtained from the same donor, CD3–CD56+ NK cells, CD14+ and CD4+ cells were isolated from PBMC by immunomagnetic selection, and cultured under four conditions: 1) CD14+ and CD4+ cells alone; 2) TB lysate-activated CD14+ and CD4+ cells; 3) TB lysate-activated CD14+, CD4+ and freshly isolated CD56+CD3– cells; 4) TB lysate-activated CD14+, CD4+ and monokine-activated CD56+CD3– cells. After 3 days, the percentages of CD4+CD25+Foxp3+ cells were measured by flow cytometry. The total number of viable cells under these four conditions, as measured by trypan blue exclusion, varied by 10% or less, and the percentage of CD4+ cells varied by 5% or less (data not shown), so changes in CD4+CD25+FoxP3+ cells, expressed as a percentage of CD4+ cells, reflect changes in absolute numbers of CD4+CD25+FoxP3+ cells. Confirming our previous findings (21), TB lysate significantly expanded CD4+CD25+Foxp3+ cells. Addition of freshly isolated NK cells did not affect Treg expansion, but monokine-activated NK cells markedly inhibited Treg expansion (540 ± 124 vs 1420 ± 109 cells per 104 CD4+ cells, p < 0.001, Fig. 1a).


Figure 1
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  		FIGURE 1. NK cell-mediated inhibition of Treg expansion. a, Monokine-activated NK cells inhibit Treg expansion. Freshly isolated CD4+ cells and autologous monocytes from healthy tuberculin reactors were cultured at a ratio of 9:1, in the presence or absence of TB lysate (WCL, 5 µg/ml). To some M. tuberculosis-activated CD14+ and CD4+ cells, monokine-activated or freshly isolated CD3-CD56+ cells were added at a ratio of one CD3–CD56+ cell:one CD14+ cell:nine CD4+ cells. After 3 days, the percentages of CD4+CD25+Foxp3+ cells were determined by flow cytometry, and expressed as the number of CD4+CD25+Foxp3+ cells per 104 CD4+cells. Mean values and SEs are shown for results obtained with eight donors. b, TB lysate-stimulated monocytes activate NK cells to inhibit expansion of Tregs. Freshly isolated CD4+ cells and autologous monocytes from healthy tuberculin reactors were cultured at a ratio of 9:1, in the presence or absence of TB lysate (WCL, 5 µg/ml). Some TB lysate-stimulated cells were cultured with NK cells from the same donors that had been preactivated by culture with TB lysate-stimulated autologous monocytes for the preceding 72 h at a ratio of one CD3–CD56+ cell:one CD14+ cell:nine CD4+ cells. After 3 days, the percentages of CD4+CD25+Foxp3+ cells were measured by flow cytometry and expressed as the number of CD4+CD25+Foxp3+ cells/104 CD4+ cells. Mean values and SEs are shown for results obtained with eight donors.

 
Because monokine-activated NK cells reduced the expansion of Tregs, we next wished to determine whether NK cells activated through a more physiologic stimulus could also inhibit Treg expansion. We therefore activated NK cells from eight healthy tuberculin reactors by culturing them with TB lysate-stimulated monocytes for 72 h. These NK cells were then added to TB lysate-activated CD14+ and CD4+ cells, and after 3 days, the percentages of CD4+ CD25+ Foxp3+ cells were measured by flow cytometry. Addition of TB lysate-activated NK cells significantly inhibited Treg expansion in response to TB lysate-activated monocytes (1420 ± 100 to 500 ± 130 cells per 104 CD4+ cells, p < 0.001, Fig. 1b). In four healthy tuberculin reactors, addition of NK cells cultured with TB lysate without monocytes, or addition of NK cells cultured with monocytes without TB lysate, had no effect on Treg expansion (1370 ± 61 vs 1380 ± 40 and 1250 ± 34 cells per 104 CD4+ cells, respectively, p > 0.1).

NK cell-mediated inhibition of Treg expansion is not mediated through IFN-{gamma}

The above findings suggest that monokine-activated NK cells inhibit expansion of Tregs. NK cells are an important early source of IFN-{gamma}, and M. tuberculosis-activated NK cells produce IFN-{gamma} (3). To determine whether NK cells inhibit Treg expansion through IFN-{gamma}, we cultured CD14+, CD4+, and CD3–CD56+ NK cells from four healthy tuberculin reactors under experimental conditions described in Fig. 1, in the presence or absence of anti-IFN-{gamma}. As in Fig. 1, monokine-activated NK cells inhibited M. tuberculosis-expanded CD4+CD25+Foxp3+ cells (1550 ± 130 to 810 ± 102 cells per 104 CD4+ cells, p < 0.001, Fig. 2) and inhibition was not affected by anti-IFN-{gamma} (810 ± 102 cells per 104 CD4+ cells vs 790 ± 104 cells per 104 CD4+ cells, p > 0.1, Fig. 2).


Figure 2
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  		FIGURE 2. Effect of anti-IFN-{gamma} on NK cell-mediated inhibition of Treg expansion. Freshly isolated CD4+ cells from healthy tuberculin reactors were cultured with monocytes at a ratio of 9:1, with or without TB lysate (WCL). To some TB lysate-activated CD14+ and CD4+ cells, monokine-activated CD3–CD56+ cells were added at a ratio of one CD3–CD56+ cell:one CD14+ cell:nine CD4+ cells. Some cells were cultured with anti-IFN-{gamma} (10 µg/ml). After 3 days, the percentages of CD4+CD25+Foxp3+ cells were determined by flow cytometry, and expressed as the number of CD4+CD25+Foxp3+ cells per 104 CD4+ cells. Mean values and SEs are shown for results obtained with four donors.

 
Previously, we found that PGE2 is one of the soluble factors produced by M. tuberculosis-stimulated monocytes that contributes to expansion of Tregs (21). To determine whether monokine-activated NK cells affect PGE2 production by monocytes, we cultured monocytes, CD4+ cells and activated NK cells at a ratio of 9:1:1, with or without heat-killed M. tuberculosis, for 3 days and measured PGE2 levels by ELISA. In three healthy tuberculin reactors, NK cells did not inhibit PGE2 production by autologous monocytes (1407 ± 271 vs 1678 ± 452 pg/ml, p > 0.1).

NK cells lyse M. tuberculosis-expanded Tregs

NK cells lyse infected cells without prior sensitization and play an important role in innate immunity to microbial pathogens. To determine whether NK cells lyse M. tuberculosis-expanded Tregs, we used three different target cells: 1) PBMC were immunomagnetically depleted of CD14+, CD19+, CD8+, and {gamma}{delta}+ cells, followed by positive selection with anti-CD25, as described in Materials and Methods. This yielded CD4+CD25+ cells which we designated as "freshly isolated Tregs"; 2) CD4+ cells from the same donor were cultured with TB lysate-activated autologous CD14+ monocytes to expand Tregs, as in Figs. 1 and 2, followed by immunomagnetic depletion of non-CD4+ cells and positive selection with anti-CD25 to yield CD4+CD25+ cells, which we designated as "expanded Tregs"; 3) during isolation of expanded Tregs, the CD4+ cells remaining after positive selection with anti-CD25 were designated as CD4+CD25– cells. These three cell populations were labeled with 51Cr, as described in Materials and Methods, and used as target cells. Effector cells were monokine-activated CD56+CD3– NK cells or freshly isolated NK cells. In five healthy tuberculin reactors, monokine-activated NK cells lysed TB lysate-expanded Tregs, but not freshly isolated Tregs (specific lysis of 19 ± 2.3% vs 2.6 ± 1.6%, respectively; p < 0.001, Fig. 3a), nor Treg-depleted CD4+CD25– T cells (specific lysis of 0.5 ± 0.5%). Freshly isolated NK cells did not lyse any target cells, indicating that only monokine-activated NK cells lyse TB lysate-expanded Tregs. To confirm that the CD4+CD25+ cells were being lysed, we used a flow cytometry-based cytotoxicity method in which target cells are labeled with CFSE and stained with 7-AAD, which stains dead cells, as outlined in Materials and Methods. In two healthy tuberculin reactors, monokine-activated NK cells showed specific lysis of 14 ± 2% of expanded CD4+CD25+ cells, compared with 6 ± 2% of expanded CD4+CD25– cells. To ensure that this effect was specific for NK cells, we cultured CD8+ cells, isolated from PBMC of six donors, with IL-12, IL-15, and IL-18 for 3 days, then used them as effector cells, with TB lysate-expanded Tregs as targets. No lytic activity was observed (data not shown).


Figure 3
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  		FIGURE 3. Cytotoxicity of NK cells against autologous Tregs. a, Activated NK cells lyse expanded Tregs. Freshly isolated and monokine-activated NK cells from healthy tuberculin reactors were cultured with freshly isolated Tregs, TB lysate-expanded T regs, or CD4+CD25– cells, at an E:T ratio of 20:1. Mean values and SEs for results obtained with five donors are shown. b, Activated NK cells lyse CD127– cells. Monokine-activated NK cells from healthy tuberculin reactors were cultured with TB lysate-expanded CD127+ and CD127– cells, at an E:T ratio of 20:1. Mean values and SEs for results obtained with three donors are shown.

 
To confirm that NK cells specifically lyse Tregs, we isolated Tregs using a method that did not depend on selection of CD25+ cells, taking advantage of recent findings that Tregs express low levels of the IL-7R, CD127 (22, 23, 24). We first cultured TB lysate-stimulated CD14+ cells and CD4+ cells to expand Tregs. Cells were then immunomagnetically depleted of CD14+, CD19+, CD8+, and {gamma}{delta}+ cells to yield CD4+ cells, followed by positive selection with CD127 to yield CD4+CD127+ and CD4+CD127– cells. For six healthy tuberculin reactors, 85% of the CD4+CD127– cells were Foxp3+ and <5% of the CD4+CD127+ cells were Foxp3+. For three healthy tuberculin reactors, we measured activated NK cell-mediated cytotoxicity against CD4+CD127+ and CD4+CD127– target cells. Activated NK cells lysed CD127– but not CD127+ cells (specific lysis of 42 ± 5% vs 1 ± 0.4%, respectively; p < 0.001, Fig. 3b).

MHC class I expression on expanded Tregs

NK cells lyse autologous cells with reduced MHC class I expression because class I molecules engage inhibitory receptors on NK cells (25). To determine whether MHC class I expression was reduced on TB lysate-expanded Tregs, we performed immunostaining with an anti-MHC class I Ab. In cells obtained from six healthy tuberculin reactors, there was no significant difference in the MFIs of expanded Tregs (1340 ± 195), expanded CD4+CD25– cells (1227 ± 224), freshly isolated Tregs (1006 ± 175) and freshly isolated CD4+CD25– cells (958 ± 84, Fig. 4). Because the secondary anti-mouse Abs used to measure MHC class I expression may have bound to positively selected CD25+ cells, this may have artifactually increased the MFIs of MHC class I staining for Tregs, compared with CD4+CD25– cells. Therefore, we obtained freshly isolated and expanded Tregs from two healthy tuberculin reactors by negatively selecting CD4+ cells with the Treg isolation kit, followed by negative selection with anti-CD127. After staining with anti-MHC class I Abs, MFIs of freshly isolated CD4+CD127+ and CD4+CD127– cells were 443 ± 25 and 379 ± 50, respectively. M. tuberculosis-expanded CD4+CD127+ and CD4+CD127– cells also expressed similar levels of MHC class I (670 ± 52 and 762 ± 38).


Figure 4
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  		FIGURE 4. MHC class I expression on CD4+CD25+ and CD4+CD25– cells. CD4+ cells and autologous CD14+ monocytes from six healthy tuberculin reactors were cultured with M. tuberculosis WCL (5 µg/ml). After 3 days, CD4+ cells were negatively selected, followed by positive selection with anti-CD25 to yield CD4+CD25+-expanded Tregs and expanded CD4+CD25– cells. A parallel procedure was used to select CD4+CD25+ freshly isolated Tregs and CD4+CD25– cells from PBMC. Immunolabeling of freshly isolated Tregs, TB lysate-expanded T regs, and their corresponding CD4+CD25– fractions was performed, using a primary anti-MHC class I mAb and a FITC goat anti-mouse secondary Ab. The MFI of MHC class I-positive cells was determined by flow cytometry. Mean values and SEs for results obtained with six donors are shown.

 
Expression of activating receptors by NK cells

The capacity of human NK cells to lyse targets is controlled by a balance of activating and inhibitory receptors, the latter binding to MHC class I molecules on target cells and inhibiting target cell lysis. Our findings in Fig. 3a indicate that monokine-activated NK cells, but not freshly isolated NK cells, lyse Tregs. Therefore, we compared the expression of different NK cell-activating receptors on these two NK cell populations. NK cells from five healthy tuberculin reactors were cultured with IL-12, IL-15, and IL-18 for 72 h and receptor expression was measured by flow cytometry. Freshly isolated autologous NK cells were used as controls. We found that culture of NK cells with IL-12, IL-15, and IL-18 up-regulated expression (MFI) of the activating receptors NKp46 (80 ± 4.7 vs 45 ± 4.7), NKG2D (101 ± 7.7 vs 50 ± 4.2), DNAM-1(77 ± 7.4 vs 48 ± 6.1), CD16 (141 ± 7.7 vs 60 ± 7.4), and 2B4 (73 ± 5.7 vs 42 ± 6.1), compared with freshly isolated NK cells (Fig. 5).


Figure 5
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  		FIGURE 5. Expression of activating receptors by freshly isolated and monokine-activated NK cells. NK cells from five healthy tuberculin reactors were cultured with IL-12, IL-15, and IL-18 for 48 h and receptor expression was measured by flow cytometry. Freshly isolated autologous NK cells were used as controls. A representative result is shown for each activating receptor. The red lines show staining of freshly isolated NK cells; the green lines show staining of monokine-activated NK cells.

 
NK cell lysis of TB lysate-expanded Tregs is mediated through NKp46 and NKG2D

To determine the relative contribution of the different NK cell receptors to lysis of expanded Tregs, we incubated monokine-activated NK cells from six healthy donors with neutralizing mAb to five NK cell receptors, before addition of expanded Tregs. Anti-NKG2D and -NKp46 reduced the percent-specific lysis of expanded Tregs from 22 ± 2.6% to 6.7 ± 2.7% and 8.3 ± 2.6%, respectively (p < 0.001, Fig. 6). In contrast, neutralization of CD16, DNAM-1, and 2B4 did not reduce lysis of expanded Tregs. Culture of NK cells with Abs to the NK cell-activating receptors overnight did not have differential effects on NK cell survival, as measured as by counting the total number of cells and by trypan blue exclusion (data not shown). Therefore, the effects of anti-NKp46 and -NKG2D were not due to toxicity of the Abs.


Figure 6
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  		FIGURE 6. Effect of neutralizing Abs to NK-activating receptors on the capacity of NK cells to lyse M. tuberculosis-expanded Tregs. Monokine-activated NK cells from six healthy donors were preincubated with either Ab or isotype control Ab (all at 10 µg/ml) for 1 h on ice, and cultured with TB lysate-expanded T regs in the presence of the same Ab. Mean values with SEs for results obtained with six donors are shown.

 
ULBP1 is the major NKG2D ligand expressed by expanded Tregs

The data above suggest that NKp46 and NKG2D are the dominant receptors that mediate lysis of TB lysate-expanded Tregs. NKG2D recognizes a variety of ligands, such as MICA, MICB, and the proteins ULBP1, ULBP2, and ULBP3, which are normally expressed at low levels but are up-regulated during stress (26). To identify the ligands for NKG2D on TB lysate-expanded Tregs, we measured cell surface ligand expression on expanded Tregs, freshly isolated Tregs and their corresponding CD4+CD25– populations from six healthy donors by flow cytometry. ULBP1 expression was up-regulated on expanded Tregs, compared with expanded CD4+CD25– cells (MFI of 43 ± 7.7% vs 15 ± 1.5%; p < 0.05, Fig. 7 and Table I) and freshly isolated Tregs (MFI of 15.3 ± 2.2%). There was a marginal increase in ULBP3 expression on expanded Tregs, compared with expanded CD4+CD25– cells (MFI of 21.7 ± 4.5% vs 13.5 ± 1.3%; p > 0.1, Fig. 7 and Table I) and freshly isolated Tregs (MFI of 12 ± 1.8%, p = 0.03). ULBP2 and MICA/B expression increased slightly on expanded Tregs compared with expanded CD4+CD25– cells and freshly isolated Tregs, but these differences are not statically significant (Fig. 7 and Table I).


Figure 7
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  		FIGURE 7. Expression of NKG2D ligands by freshly isolated Tregs, TB lysate-expanded T regs, and their corresponding CD4+CD25– cell fractions. CD4+ cells and autologous monocytes from six healthy tuberculin reactors were cultured with TB WCL (5 µg/ml). After 3 days, CD4+CD25+-expanded Tregs and CD4+CD25– cells were isolated by negative selection of CD4+ cells, followed by positive selection of CD25+ cells, as outlined in Materials and Methods. Using the same method, autologous Tregs and CD4+CD25– cells were also isolated from PBMC. Freshly isolated CD4+CD25+ (red line), TB lysate-expanded CD4+CD25+ (green line), fresh CD4+CD25– (orange), and TB lysate-expanded CD4+CD25– (blue) cell fractions were stained with Abs to MICA/B, ULBP1, ULBP2, and ULBP3 as primary Abs and a FITC goat anti-mouse secondary Ab. TB lysate-expanded CD4+CD25+ cells were also stained with the secondary Ab alone (black). Cells from six donors showed similar results, and one representative experiment is shown.

 

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  		Table I. Expression of NKG2D ligands by freshly isolated Tregs, TB lysate-expanded Tregs, and their corresponding CD4+CD25– cell fractionsa

 
Effect of anti-ULBP1 on NK cell-mediated lysis of expanded Tregs

To confirm that ULBP1 was the major ligand responsible for NK cell-mediated lysis of expanded Tregs, we cultured monokine-activated NK cells from five healthy donors with TB lysate-expanded Tregs, in the presence of Abs to ULBP1, ULBP2, ULBP3, or MICA/B or isotype control Abs (all at 10 µg/ml). Only anti-ULBP1 significantly inhibited NK cell-mediated lysis of expanded Tregs (specific lysis of 27 ± 4% vs 10 ± 2%, p < 0.001, Fig. 8). Culture of NK cells with Abs to the NK cell-activating receptors overnight did not have differential effects on NK cell survival, as measured by counting the total number of cells and by trypan blue exclusion (data not shown). Therefore, the effect of anti-ULBP1 was not due to toxicity of the Ab.


Figure 8
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  		FIGURE 8. Effects of Abs to MICA/B and ULBPs on NK cell-mediated lysis of TB lysate-expanded T regs. Monokine-activated NK cells from healthy tuberculin reactors were preincubated with Abs to MICA/B, ULBP1, ULBP2, ULBP3, or isotype control Ab (all at 10 µg/ml) for 1 h on ice, and cultured with TB lysate-expanded T regs in the presence of the same Ab. Mean values with SEs for results obtained with five donors are shown.

 
TB lysate-stimulated monocytes activate NK cells to lyse expanded Tregs through NKG2D and ULBP1

The experiments above demonstrate that monokine-activated NK cells lyse expanded Tregs through NKG2D and ULBP1. To determine whether these molecules also mediated lysis of Tregs by NK cells activated by the more physiologic stimulus of TB lysate-exposed monocytes, we cultured NK cells from five healthy tuberculin reactors with autologous monocytes for 72 h in the presence of TB lysates, and used them as effectors against TB lysate-expanded Tregs. Similar to findings in Fig. 3, monocyte-activated NK cells lysed expanded Tregs (29 ± 3% lysis, Fig. 9) and this was inhibited by anti-NKG2D and -ULBP1 (7 ± 3%, 8 ± 2%, p < 0.001 for both, compared with values treated with isotype control Abs, Fig. 9).


Figure 9
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  		FIGURE 9. Effect of neutralization of NKG2D and ULBP1 on the capacity of monocyte/TB lysate-activated NK cells to lyse M. tuberculosis-expanded T regs. NK cells from healthy tuberculin reactors were activated by culture with TB lysate-stimulated monocytes. NK cells were then preincubated with either Ab or isotype control Ab (all at 10 µg/ml) for 1 h on ice, and cultured with M. tuberculosis-expanded T regs in the presence of the same Ab. Mean values with SEs for results obtained with five donors are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Induction of the Th1-mediated immune response is vital for host defense against various intracellular pathogens, including M. tuberculosis (27, 28). Exposure to M. tuberculosis can elicit protective immunity, but can also induce expansion of CD4+CD25+FoxP3+ Tregs, which inhibit Th1 responses (21, 29, 30). In this report, we identified NK cells as potential modulators of Tregs during the immune response to M. tuberculosis infection. We found that activated NK cells reduce Treg expansion by direct lysis of TB lysate-expanded Tregs through the NK cell-activating receptors, NKp46 and NKG2D. Of five NKG2D ligands, only ULBP1 was up-regulated in TB lysate-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs, suggesting that lysis was mediated through interactions between NKG2D and ULBP1. As far as we are aware, these represent the first studies demonstrating that activated NK cells can lyse Ag-expanded Tregs. Our results suggest that NK cells may play an important role during the early phases of the immune response to M. tuberculosis and other intracellular pathogens by inhibiting Treg expansion.

Tregs influence the immune response to parasitic, bacterial, viral, and fungal pathogens. The effects may be favorable or harmful to the host, depending on the pathogen and stage of infection. In Leishmania infection, Tregs persist at the site of infection, are essential for parasite persistence but favor host immunity to exogenous reinfection (15, 31). Tregs favor the host in Helicobacter infection (32, 33), but have harmful effects in malaria, HIV, CMV, and hepatitis C infections (34, 35, 36, 37, 38). Tregs prevent efficient clearance of M. tuberculosis in infected mice (29). Tuberculosis patients had increased Treg numbers, and depletion of Tregs enhanced M. tuberculosis-induced IFN-{gamma} production by PBMC, suggesting that Tregs inhibit an effective immune response (30, 39). Therefore, to maintain immunity to M. tuberculosis and other pathogens, immunoregulatory mechanisms must eliminate or inhibit the function of Tregs. However, information regarding the nature of these immunoregulatory mechanisms is limited.

Murine gamma-herpesvirus 68 infection reduces Treg numbers and inhibits their function, although the underlying mechanisms are undefined (40). The human T cell lymphotropic virus type I-associated virus-encoded tax protein inhibits Foxp3 expression and suppressor function (41). TNF-{alpha} inhibits Foxp3 expression and activity of human Tregs (42), and IL-6 can down-modulate Treg development and activity in a murine model of asthma (43). Our current findings identify an additional mechanism by which Tregs can be eliminated through direct lysis by NK cells.

Increased numbers and activity of Tregs are associated with depressed NK cell activity in cancer and bone marrow transplantation (17, 18, 19). One recent study demonstrated that Tregs inhibit NK cell function in a manner that depends on the presence of TGF-β and the absence of stimulation with IL-2, IL-15, and TLR4 (16), suggesting that Tregs inhibit NK cell function only during specific phases of the immune response. Similarly, we found that NK cells only inhibited Treg expansion under specific conditions. NK cells activated by monokines or by TB lysate-stimulated monocytes inhibited Treg expansion in response to M. tuberculosis. In contrast, freshly isolated NK cells did not inhibit Treg expansion. It is intriguing to speculate that, under normal physiologic conditions, NK cells are quiescent, but following infection, they undergo cytokine-dependent activation and inhibit Treg expansion. During the early phases of the immune response to intracellular pathogens, NK cells may inhibit Treg expansion, and favor development of a Th1 response. In tuberculosis patients, decreased NK cell activity and increased Treg numbers have been noted (3, 30, 39), and our current findings suggest that reduced NK cell function may contribute to expansion of Tregs.

Tregs include natural Tregs, which respond to self Ags and maintain self-tolerance, and adaptive Tregs, which respond to foreign Ags and maintain homeostatic control over adaptive immune responses (44) We found that activated NK cells lyse M. tuberculosis-expanded (adaptive) Tregs, but not freshly isolated (natural) Tregs. Our results suggest that activated NK cells regulate the frequency of adaptive Tregs but do not affect the Tregs which maintain self tolerance. The key effector functions of NK cells are cytokine production and cytotoxicity (3, 8, 9). NK cells produce IFN-{gamma} in response to M. tuberculosis (3, 45), but IFN-{gamma} did not mediate NK cell inhibition of Treg expansion (Fig. 2). PGE2 produced by M. tuberculosis stimulated monocytes is critical for expansion of Tregs in healthy tuberculin reactors (21), but our results indicate that NK cell-mediated inhibition of Treg expansion was not through inhibition of PGE2 production. Instead, NK cells directly lyse expanded Tregs. NK cells destroy target cells with reduced surface expression of MHC class I (25), but this was not the mechanism for NK cell lysis of expanded Tregs (Fig. 4). Expression of several NK cell-activating receptors were up-regulated on monokine-activated NK cells, but only neutralization of NKp46 and NKG2D abrogated the capacity of NK cells to inhibit expansion of Tregs (Fig. 6), indicating that these receptors played the dominant roles in lysis. Our study defines new mechanisms by which NKp46 and NKG2D can contribute to control of intracellular infections by inhibiting expansion of Tregs.

ULBPs and MICA/B are ligands for NKG2D that are expressed at low levels by many tissues (26), and expression is up-regulated on cells stressed by infection or malignant transformation (10, 46). Although NKG2D ligand expression has been described primarily in epithelial cells and mononuclear phagocytes (10, 47), T cells can also express these ligands. Treatment of mouse T cell blasts with a DNA-damaging agent increases NKG2D ligand expression, and increased expression correlates with NKG2D-dependent lysis by IL-2-activated NK cells (48). In addition, activated NK cells can recognize and lyse syngeneic activated T cells in a NKG2D-dependent manner (49). We found that TB lysate-expanded Tregs, but not freshly isolated Tregs, have increased expression of ULBP1, enhancing their susceptibility to NKG2D-mediated lysis. These findings provide the first evidence that expression of ULBP1 varies during the course of expansion of CD4+CD25+FoxP3+ T cells, and that ULBP1 up-regulation contributes to NK cell elimination of these T cells. Microarray analyses of murine primary FoxP3+ cells and a hybridoma transfected with FoxP3 did not identify any murine NKG2D ligands as one of the genes up-regulated by FoxP3 (50, 51). Nevertheless, studies in mice may not reflect events that occur in human cells during expansion in response to microbial Ag. It will be important to determine whether FoxP3 directly or indirectly increases transcription of ULBP1, or if it leads to mobilization of ULBP1 protein from the cytoplasmic compartment to the cell surface, as has been described during treatment of cells with hydrogen peroxide (47).

Although freshly isolated NK cells express NKp46 and NKG2D on their surface (10), we found that they did not lyse TB lysate-expanded Tregs. One possibility is that the magnitude of NKp46 and NKG2D expression on freshly isolated NK cells is insufficient to trigger significant lysis. Another possibility is that a second costimulatory signal is required to induce NK cell lysis of Tregs. For example, OX40 ligand is selectively induced on IL-15-activated NK cells after stimulation through NKG2D (52).

In summary, we identified a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response during microbial infections. NK cells can eliminate Ag-expanded Tregs through the NKp46 and NKG2D receptors, and this is mediated at least in part by expression of ULBP1 on the surface of expanded Tregs. These interactions should be taken into account in developing vaccines against M. tuberculosis and other intracellular pathogens, and in designing immunotherapeutic modalities that use Tregs to control autoimmune diseases.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Institutes of Health (AI054629 and A1063514), the Cain Foundation for Infectious Disease Research, and the Center for Pulmonary and Infectious Disease Control. P.F.B. holds the Margaret E. Byers Cain Chair for Tuberculosis Research. Back

2 Address correspondence and reprint requests to Dr. Ramakrishna Vankayalapati, Center for Pulmonary and Infectious Disease Control, University of Texas Health Center, 11937 U.S. Highway 271, Tyler, TX 75708-3154. E-mail address: Krishna.vankayalapati{at}uthct.edu Back

3 Abbreviations used in this paper: Treg, regulatory T cell; MIC, MHC class I-related chain; ULBP, UL16-binding protein; TB, tuberculosis; MFI, mean fluorescence intensity; 7-AAD, 7-aminoactinomycin D; WCL, whole cell lysate. Back

Received for publication April 26, 2007. Accepted for publication November 25, 2007.


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 Introduction
 Materials and Methods
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 Disclosures
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