Pathogen Expand in Response to an Intracellular NK Cells Lyse T
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The Journal of Immunology NK Cells Lyse T Regulatory Cells That Expand in Response to an Intracellular Pathogen1 Sugata Roy,*† Peter F. Barnes,*†‡ Ankita Garg,*† Shiping Wu,*† David Cosman,§ and Ramakrishna Vankayalapati2*† 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-␥. 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. The Journal of Immunology, 2008, 180: 1729–1736. atural killer cells are large granular lymphocytes that can Many studies have shown that Tregs play a central role in kill infected cells without prior sensitization and play an down-regulating the immune response to organ transplants and N important role in innate immunity to microbial patho- tumors, preventing the development of autoimmunity (13) and gens. NK cells mediate protection against viruses, bacteria, and dampening the immune response to intracellular pathogens (14, parasites through destruction of infected cells and by secretion of 15). Recent studies also showed that Tregs inhibit NK cell cy- cytokines that shape the adaptive immune response (1–5). NK cells tolytic activity via TGF-, but do not inhibit production of participate in dendritic cell maturation (6, 7) and are an important IFN-␥ by NK cells stimulated by IL-2R␥ chain-dependent cy- early source of IFN-␥, which is critical for activation of macro- tokines (16). Increased numbers and activity of Tregs are also phages (8, 9). Therefore, NK cells may favor clonal expansion of associated with depressed NK cell activity in several diseases Th1 cells, processes that are important for elimination of many (17–19). However, the effects of NK cells on Tregs in healthy intracellular pathogens. Our published data indicate that NK cells persons during a response to microbial Ags are unknown. In the lyse Mycobacterium tuberculosis-infected monocytes and alveolar current study, we found that NK cells inhibit Treg expansion macrophages through the NKp46 receptor and NKG2D (10), and through direct lysis of expanded Tregs, and investigated the NK cells contribute to the capacity of CD8ϩ T cells to produce mechanisms involved in this lysis. IFN-␥ and to lyse M. tuberculosis-infected monocytes (11). Recently, attention has been focused on regulatory T cells Materials and Methods (Tregs),3 a subset of CD4ϩ T cells that express CD25 and Patient population FoxP3 (12), which can inhibit IFN-␥ production by T cells. 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 *Center for Pulmonary and Infectious Disease Control, †Departments of Microbiol- ogy and Immunology and ‡Department of Medicine, University of Texas Health participants. Center, Tyler, TX 75708l; and §Amgen, Seattle, WA 98101 Abs and other reagents Received for publication April 26, 2007. Accepted for publication November 25, 2007. For flow cytometry, we used FITC anti-CD4, allophycocyanin anti-CD25, The costs of publication of this article were defrayed in part by the payment of page PE anti-Foxp3, PE-Cy5 anti-Foxp3 (all from eBioscience), FITC anti- charges. This article must therefore be hereby marked advertisement in accordance CD14, FITC anti-DNAM-1, FITC anti-CD16, PE anti-NKp46, PE anti- with 18 U.S.C. Section 1734 solely to indicate this fact. 2B4, allophycocyanin anti-NKG2D, and PE anti-CD127 (all from BD 1 This work was supported by grants from the National Institutes of Health (AI054629 Biosciences). and A1063514), the Cain Foundation for Infectious Disease Research, and the Center In some cases, indirect immunolabeling was performed using anti- for Pulmonary and Infectious Disease Control. P.F.B. holds the Margaret E. Byers human mAb to MHC class I-related chain (MIC)A/B (BD Biosciences), Cain Chair for Tuberculosis Research. UL16-binding protein (ULBP)1, ULBP2, and ULBP3 (M295, M312, and 2 Address correspondence and reprint requests to Dr. Ramakrishna Vankayalapati, M551, respectively; Amgen), and anti-human MHC class I primary Ab Center for Pulmonary and Infectious Disease Control, University of Texas Health (W6/32; DakoCytomation) and a FITC goat anti-mouse secondary Ab Center, 11937 U.S. Highway 271, Tyler, TX 75708-3154. E-mail address: Krishna. (Southern Biotechnology Associates). After gating on live cells, the mean [email protected] fluorescence intensity (MFI) of stained cells was measured. ␥ 3 Abbreviations used in this paper: Treg, regulatory T cell; MIC, MHC class I-related For neutralization, we used mAbs to IFN- , MICA/B, NKG2D, NKp46, chain; ULBP, UL16-binding protein; TB, tuberculosis; MFI, mean fluorescence in- DNAM-1 CD16, and CD244 (BD Biosciences) and mAb to MICA/B, tensity; 7-AAD, 7-aminoactinomycin D; WCL, whole cell lysate. ULBP1, ULBP2, and ULBP3 (Amgen). Isotype control Abs were also used (BD Biosciences). In some experiments, rIL-12, rIL-15 (both from R&D Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 Systems), and rIL-18 (MBL International) were added to the cells. M. www.jimmunol.org 1730 NK CELLS LYSE Tregs tuberculosis whole cell lysates (WCL) were obtained from Dr. J. Belisle were labeled with 100 Ci sodium chromate. Target cells were washed (Colorado State University, Fort Collins, CO), and heat-killed M. tuber- three times, and triplicate wells of 104 cells/well were mixed with effector culosis Erdman was provided by Dr. P. Brennan (Colorado State Univer- cells at an E:T ratio of 20:1 in 200 l of RPMI 1640 with 10% heat- sity, Fort Collins, CO). inactivated human serum. Ten hours after incubation, 100 l of supernatant was removed from each well, and radioactivity was measured in a gamma Immunolabeling of intracellular Foxp3 counter. The percent-specific lysis was calculated using: 100 ϫ (experi- ϩ ϩ ϩ mental release Ϫ spontaneous release/maximum release Ϫ spontaneous Surface staining to detect CD4 , CD25 , and CD127 cells and intracel- ϩ release). lular staining to detect Foxp3 cells was performed, using the Cytofix/ In some experiments, cytotoxicity of activated NK cells against ex- Cytoperm Plus kit (eBioscience). Controls for each experiment included ϩ ϩ ϩ Ϫ panded CD4 CD25 and CD4 CD25 cells was performed, using a flow cells that were unstained, cells to which FITC-, allophycocyanin-, or PE- cytometry-based cytotoxicity kit (Cell Technology). Briefly, expanded conjugated mouse IgG had been added, and cells that were single stained, ϩ ϩ ϩ Ϫ ϩ CD4 CD25 and CD4 CD25 cells were labeled with CFSE, before us- either for the surface marker or for Foxp3. We gated on CD4 lympho- ϩ ϩ ing them as target cells for activated NK cells. Ten hours after incubation, cytes, and determined the percentages of CD25 and Foxp3 cells. For ϩ a DNA-binding, fluorescent dye, 7-aminoactinomycin D (7-AAD), which some experiments, we gated on Foxp3 cells to detect CD127low cells, stains dead cells, was added to the cultures to measure cell death. The using a FACSCalibur (BD Biosciences). percent lysis was calculated as: 100 ϫ (CFSE and 7-AAD double-positive Isolation of NK cells, monocytes, CD4ϩ cells, and CD8ϩ cells cells/total number of CFSE-positive cells). Spontaneous cell death of target cells was calculated and subtracted as a background control. PBMC were isolated by centrifugation over Ficoll-Paque (Amersham Phar- macia Biotech). For NK cell isolation, CD3ϩ cells were depleted from Statistical analysis PBMC with magnetic beads conjugated to anti-CD3 (Miltenyi Biotec), and Ϯ from the negative cell fraction, CD56ϩ cells were isolated by positive Results are shown as the mean SE. Comparisons between groups were selection with magnetic beads conjugated to anti-CD56 (Miltenyi Biotec). performed by a paired or unpaired t test, as appropriate. The positive cells were 95–100% CD56ϩ and 95–97% CD3Ϫ, as measured by flow cytometry, and were used as effector cells. Monocytes and CD4ϩ Results cells were isolated with magnetic beads conjugated to anti-CD14 or -CD4 NK cell-mediated inhibition of Treg expansion Ͼ ϩ (Miltenyi Biotec), respectively. Positively selected cells were 95% ,as ϩ measured by flow cytometry. We recently found that culture of CD4 cells with M. tuberculo- sis-stimulated monocytes results in expansion of CD4ϩCD25ϩ Culture of NK cells ϩ FoxP3 cells, and that these cells inhibit IFN-␥ production by In some experiments, freshly isolated NK cells were cultured in the pres- CD4ϩ and CD8ϩ cells, indicating that they are Tregs (21). Human ence of complete RPMI 1640 and 10% heat-inactivated human serum (At- Th1 responses are inhibited by Tregs (13) but stimulated by NK lanta Biologicals) and IL-12, IL-15, and IL-18 (each at 3 ng/ml) for 72 h cells (8, 9, 11).