Expansion of human NK-22 cells with IL-7, IL-2, and IL-1β reveals intrinsic functional plasticity

Marina Cella, Karel Otero, and Marco Colonna1

Department of Pathology and , Washington University School of Medicine, St. Louis, MO 63110

Communicated by Emil R. Unanue, Washington University, St. Louis, MO, May 1, 2010 (received for review February 24, 2010) Natural killer-22 (NK-22) cells are a human NK cell subset situated In this study we investigated the that are required for in mucosal-associated lymphoid tissues that specialize in IL-22 the expansion of NK-22 cells isolated from human tonsils. We secretion in response to IL-23. Here we investigated the found that IL-7, IL-2, and IL-1β promoted survival and pro- requirements for NK-22 cell expansion. IL-7 maintained the sur- liferation of NK-22 cells but induced different functional pro- vival of NK-22 cells and IL-22 production in response to IL-23 but files, indicating that NK-22 cells are functionally flexible and may was insufficient to induce robust expansion. Proliferation of NK-22 contribute to immune responses in the gut by different mecha- cells was increased markedly by adding either IL-1β or IL-2 to nisms. Additionally, we observed that NK-22 cells release the IL-7 and was even stronger in the presence of IL-1β plus IL-2. In B-cell survival factor, B-cell activating factor belonging to the contrast to IL-7, continuous culture in IL-1β and IL-2 modified TNF family (BAFF), suggesting a potential role for NK-22 cells NK-22 cytokine profiles. IL-1β promoted constitutive IL-22 secre- in promoting B-cell–mediated immunity in the mucosae. tion rather than acute IL-22 production in response to IL-23 and induced IL-17 in some cells. IL-2 reduced secretion of IL-22 and Results IL-17, increasing production of IFN-γ and leukemia inhibitory fac- Human CD56+NKp44+ NK Cells Are Functionally Heterogeneous. We tor. Functional deviation toward IFN-γ production also was in- previously defined NK-22 cells in human tonsils and Peyer’s duced by continuous culture in IL-23. These results demonstrate patches based on the expression of CD56 and NKp44 and the the functional plasticity of NK-22 cells, which may allow flexible capacity to produce IL-22 in response to IL-23 (1). To refine the responses to different pathogens. Finally, we found that NK-22 phenotype of NK-22 cells, we isolated CD56+ cells from tonsils, cells released the B-cell survival factor, B-cell activating factor be- stimulated them with IL-23, and analyzed them by multicolor longing to the TNF family (BAFF), suggesting a potential role of flow cytometry for intracellular content of IL-22 and expression – NK-22 cells in promoting B-cell mediated mucosal immunity. of NKp44 as well as various cell-surface markers, such as CCR6, the αE CD103, and the ectonucleotidase CD39. All B-cell activating factor belonging to the TNF family | IL-22 | mucosal IL-22–producing cells expressed NKp44 and the re- immunity | ceptor CCR6; in contrast, none expressed CD103 (Fig. S1). Some but not all IL-22–producing cells expressed CD39. Thus, e recently identified a subset of natural killer (NK)-like cells CD56+NKp44+ cells include at least two functionally distinct − Win human tonsils, Peyer’s patches, and appendix, which ex- subsets: CCR6+CD103 cells, which secrete IL-22 in response to press CD56 and the NKp44. These cells specialize in the IL-23, and CD103+ cells, which do not produce IL-22 and have secretion of IL-22 in response to IL-23; therefore we designated an undefined function. The presence of these subsets, together − them as “NK-22” cells (1, 2). NK-22 cells also produce IL-26 and with the classical CD56+NKp44 NK cells, reveals a consider- the leukemia inhibitory factor (LIF). All these cytokines play an able heterogeneity of NK cells in mucosal-associated lymphoid important role in the defense of mucosal barriers against patho- tissues. Importantly, the refined phenotype of NK-22 cells as − gens (3, 4). NK-22 cells have remarkable similarities with T-helper CD56+NKp44+CCR6+CD103 cells allows the isolation of these IMMUNOLOGY (TH) 17 T cells: Both cell types secrete IL-22, express the che- cells with little or no contamination by other NK cell subsets, mokine receptor CCR6, migrate in response to CCL20, and ex- facilitating their functional characterization. press the transcription factors retinoic-acid–related orphan recep- tor γt(RORγt) and aryl hydrocarbon receptor. The latter is Human NK-22 Cells Proliferate in Response to IL-2 Plus IL-7. We next investigated the cytokine requirements for the survival and ex- required for production of IL-22 (5). However, in contrast to TH17 cells, NK-22 cells isolated from the tonsils do not produce IL-17. pansion of NK-22 cells in vitro. In mouse, the development of + + γ + + γ The developmental pathway that generates human NK-22 cells NKp46 CD127 ROR t IL-22 cells requires the common γ β is unclear. In lymphoid tissues, classical NK cells develop through chain of cytokine receptors ( c) and IL-7 but not the chain of five distinct stages (6); stage 3 NK cells produce IL-22 (7), whereas IL-2/IL-15 receptors (CD122) or IL-15 (10, 13). Consistent with stage 4 and 5 NK cells produce IFN-γ. Thus, it is possible that NK- mouse data, we found that primary NK-22 cells express the IL-7 receptor α chain (CD127) (Fig. S2), and therefore we hypothe- 22 cells correspond to stage 3 NK cells. However, NK-22 cells also fi are similar to human lymphoid tissue-inducer cells (LTi), which sized that IL-7 might be suf cient to maintain and/or expand NK-22 cells in vitro. To test this hypothesis, we sorted CD56+ are present in the lymphoid aggregates of fetal mesentery, and to + + − LTi-like cells, which are found in tonsils. Thus, NK-22 cells also NKp44 CCR6 CD103 cells from tonsils (Fig. S3) and cultured them in IL-7. After 1 week, we observed only a modest (2-fold) may develop from LTi and LTi-like cells (8, 9). The murine equivalent of NK-22 cells has been identified in gut-associated lymphoid tissues as NKp46+CD127+RORγt+ cells – Author contributions: M. Cella and M. Colonna designed research; M. Cella and K.O. (1, 10 12). Their development depends on the common gamma performed research; M. Cella, K.O., and M. Colonna analyzed data; and M. Colonna wrote chain (γc) of cytokine receptors, IL-7, RORγt, and the intestinal the paper. microbiota (10, 11, 13) but does not require IL-15 or the β chain of The authors declare no conflict of interest. IL-2 and IL-15 receptors (CD122), which are essential for the Freely available online through the PNAS open access option. – development of classical NK cells. Thus, in the mouse IL-22 1To whom correspondence should be addressed. E-mail: [email protected]. + + γ + producing NKp46 CD127 ROR t cells and classical NK cells This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. appear to develop through different pathways (13). 1073/pnas.1005641107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1005641107 PNAS | June 15, 2010 | vol. 107 | no. 24 | 10961–10966 Downloaded by guest on September 25, 2021 increase in number, suggesting that IL-7 is sufficient for cell sur- factor κ B ligand (RANKL) (Fig. 1G), which may facilitate the vival but is a weak inducer of proliferation (Fig. 1A). To facilitate interaction of NK-22 cells with antigen-presenting cells and/or a robust expansion of NK-22 cells, we supplemented IL-7 with IL- epithelial cells that express RANK (16). Overall, we conclude 2, which is commonly used to expand classical NK cells in vitro. In that the phenotype of NK-22 cells cultured in IL-7 or IL-2 plus these conditions we observed a 7- to 10-fold increase in cell IL-7 closely resembles that of primary NK-22 cells, although IL-2 number within a week (Fig. 1A), which was caused by augmented appears to increase the frequency of cells expressing NKG2D proliferation, as assessed by BrdU incorporation assay (Fig. S4). and CD94. Thus, IL-2 plus IL-7 promotes robust NK-22 cell expansion. Phenotypic analysis of NK-22 cells cultured in IL-7 or IL-2 IL-2 Deviates the Polarization of NK-22 Cells Toward the Production of γ plus IL-7 confirmed the expression of CD56, NKp44, and CCR6 IFN- . To examine the cytokine produced by NK-22 cells cultured (Fig. 1B). Cells also expressed high amounts of CD117 (c-Kit), in IL-7 or in IL-2 plus IL-7, we stimulated them with IL-23 and NKp46, CD161 (NKRP1-A), and CD96 (Fig. 1C). Additionally, measured the intracellular content of IL-22 as well as IL-22 se- creted in the culture supernatant. We also measured the in- we observed the unprecedented expression of CD33, CD146, tracellular content of IFN-γ, which is produced by conventional CD164, and CD147 (Fig. 1D). Many of these molecules have NK cells, and CCL20, a chemokine that is preferentially released been shown to mediate adhesion (14, 15). Cells cultured in IL-2 by NK-22 cells (1). Given the expression of CD121a on NK-22 plus IL-7 expressed more NKG2D and CD94 than cells cultured β E cells, we included IL-1 with or without IL-23 as stimuli. More- in IL-7 alone (Fig. 1 ), whereas other NK cell receptors, such over, in some experiments, we stimulated cells with phorbol 12- as CD16, 2B4, and KIRs, were undetectable in both culture myristate 13-acetate (PMA)/ionomycin. NK-22 cells cultured in conditions. IL-7 alone produced IL-22 in response to IL-23 (Fig. 2 A and B); We previously reported the expression of the receptor for IL-1 additionally, IL-1β strongly synergized with IL-23 in inducing (CD121a) in the transcriptome of primary NK-22 cells (1). We IL-22 secretion, whereas IL-1β alone had no effect. NK-22 cells fi con rmed CD121a expression at the level on primary cultured in IL-7 alone produced no IFN-γ or CCL20 (Fig. 2A). In NK-22 cells (Fig. S2) and showed that it is maintained after control experiments, peripheral blood NK cells produced no culture in IL-7 with or without IL-2 (Fig. 1F). Cultured NK-22 IL-22 in response to various stimuli (Fig. 2C). cells also expressed the receptor for IFN-γ (CD119) (Fig. 1F). NK-22 cells cultured in IL-2 plus IL-7 showed a different Thus, NK-22 cells can respond to IL-1β and IFN-γ. Moreover, polarization profile. The frequencies of IL-22–producing cells in cultured NK-22 cells expressed receptor activator for nuclear response to IL-23, IL-1β plus IL-23, and PMA/I were reduced in comparison with those of cells grown in IL-7 alone (Fig. 3A). Moreover, a discrete subset of cells produced IFN-γ after stim- ulation with IL-23 plus IL-1β and PMA/ionomycin. Stimulation with IL-1β plus IL-23 also induced cells producing both IL-22 and IFN-γ (Fig. 3A). Despite the production of IFN-γ, NK-22 cells cultured in IL-2 plus IL-7 continued to express RORγt mRNA, which was undetectable in classical NK cells or CD103+ tonsil NK cells cultured in IL-2 (Fig. 3B). Finally, only cells stimulated with PMA/ionomycin made CCL20 at low frequency

Fig. 1. Impact of IL-7 and IL-2 on proliferation and phenotype of NK-22 − cells. CD56+NKp44+CCR6+CD103 cells were sorted from tonsils and cultured Fig. 2. NK-22 cells cultured in IL-7 maintain their capacity to produce IL-22 in IL-7 alone or in IL-2 plus IL-7. After 7 days of culture cells were analyzed for in response to IL-23. CD56+NKp44+CCR6+CD103− cells were sorted from cell numbers (A). After 10–15 days of culture, we measured cell surface ex- tonsils and cultured in IL-7 for 10–15 days. Cells were left untreated or pression of CD56, NKp44, and CCR6 (B); NKp46, CD161, CD117, and CD96 (C); stimulated with IL-1β, IL-23, or both and were analyzed for (A) frequencies of CD33, CD164, CD146, and CD147 (D); CD94, NKG2D, CD16, and 2B4 (E); the IL-22–, CCL20-, and IFN-γ–producing cells (by intracellular staining) and (B) receptors for IL-1 (CD121a) and IFN-γ (CD119) (F); and RANKL (presented in release of IL-22 in the supernatants 18 h after stimulation (by ELISA). Data two-color analysis versus NKp44) (G). Empty and filled profiles represent presented here were obtained from one tonsil specimen representative of staining with control and specific antibodies, respectively. Data shown here three. (C) For comparison, we measured intracellular IL-22 and CCL20 con- were obtained from one tonsil specimen representative of three. tents of peripheral blood NK cells in response to IL-1β, IL-23, or both.

10962 | www.pnas.org/cgi/doi/10.1073/pnas.1005641107 Cella et al. Downloaded by guest on September 25, 2021 Fig. 4. IL-1β plus IL-7 induces proliferation of NK-22 cells and constitutive production of IL-22. Identical numbers of CD56+NKp44+CCR6+CD103− cells sorted from tonsils were cultured in IL-7, IL-1β plus IL-7, or IL-2+IL-7. After 10 days of culture we analyzed (A) cell numbers; (B) expression of CD121a and Fig. 3. NK-22 cells cultured in IL-2 plus IL-7 exhibit limited IL-22 production in CD56; (C) release of IL-22 in cell-culture supernatants by ELISA; (D)in- − response to IL-23 but produce IFN-γ.CD56+NKp44+CCR6+CD103 cells were tracellular IL-22, IFN-γ, and CCL20 after stimulation with IL-1β, IL-23, IL-1β plus IMMUNOLOGY sorted from tonsils and cultured in IL-2 plus IL-7 for 10–15 days. Cells were IL-23, and PMA/I by flow cytometry; (E) intracellular IFN-γ after stimulation stimulated with IL-1β,IL-23,IL-1β plus IL-23, or PMA/ionomycin (PMA/I) and with IL-12; (F) RORC mRNA by RT-PCR. Empty and filled profiles in B represent were analyzed for (A) production of IL-22, CCL20, and IFN-γ by intracellular staining with control and specific antibodies, respectively. Data presented in D staining and (B) expression of RORC mRNA by RT-PCR analysis. Data presented and E were obtained from the same tonsil specimen used for Figs. 2A and 3A in A were obtained from the same tonsil specimen used for Figs. 2A and 4 D to facilitate comparisons. PB NK, peripheral blood NK cells; TS, tonsil. and E to facilitate comparisons. PB NK, peripheral blood NK cells; TS, tonsil. IL-2 plus IL-7 (Fig. 4A and Fig. S4). The cell-surface phenotype of (Fig. 3A). Overall, these results suggest that NK-22 cells have cells expanded in IL-1β plus IL-7 was remarkably similar to that of functional plasticity. Although NK-22 cells in the presence of primary NK-22 cells or NK-22 cells cultured in IL-2 plus IL-7. One IL-7 mainly produce IL-22 in response to IL-23 with or without exception was the reduced expression of CD121a, probably IL-1β, the addition of IL-2 reduces the frequency of IL-22– reflecting the engagement and down-regulation of CD121a by IL- producing cells and increases that of IFN-γ–producing cells. 1β present in the culture medium (Fig. 4B). Another noticeable However, NK-22 cells grown in IL-2 plus IL-7 remain distinct exception was the reduced expression of CD56. from classical NK cells, because they continue to express RORγt. To characterize functionally NK-22 cells cultured in IL-1β plus The IL-2–induced deviation of NK-22 cells toward IFN-γ pro- IL-7, we investigated cytokine production by ELISA and in- duction mirrors the previously observed capacity of IL-2 to in- tracellular staining. Supernatants of cells grown in IL-1β and IL- terfere in the development of TH17 T cells, facilitating the 7 constitutively contained higher amounts of IL-22 than super- generation of IFN-γ–producing TH1 T cells (17). natants of cells cultured in IL-7 plus IL-2 or in IL-7 alone (Fig. 4C). However, only a few cells produced IL-22 when stimulated IL-1β Plus IL-7 Expands Human NK-22 Cells. Given the expression of with IL-23 with or without IL-1β, as evidenced by intracellular the receptor for IL-1 on both primary and cultured NK-22 cells staining (Fig. 4D). It is possible that culture in IL-1β promotes (Fig. S2 and Fig. 1F), we attempted to culture primary NK-22 cells a continuous release of IL-22, decreasing the responsiveness of using IL-1β. Although IL-1β alone did not support NK-22 pro- NK-22 cells to stimulation with IL-23 with or without IL-1β. Only liferation, IL-1β plus IL-7 expanded NK-22 cells as vigorously as PMA/ionomycin induced IL-22 in a significant subset of NK-22

Cella et al. PNAS | June 15, 2010 | vol. 107 | no. 24 | 10963 Downloaded by guest on September 25, 2021 cells (10–20% in different experiments) and CCL20 in some cells. The physiological stimuli that induce CCL20 in NK-22 cells remain unknown. In contrast to cells cultured in IL-2 and IL-7, cells grown in IL- 1β plus IL-7 did not produce IFN-γ in response to any of the stimuli used to elicit IL-22 (Fig. 4D). Additionally, no IFN-γ was produced in response to IL-12, whereas NK-22 cells cultured in IL-2 plus IL-7 were responsive to IL-12 and produced IFN-γ (Fig. 4E). Finally, cells grown in IL-1β and IL-7 exhibited high levels of expression of RORγt (Fig. 4F). We conclude that IL-1β together with IL-7 induces the proliferation of NK-22 cells and maintains their expression of RORγt and their capacity of pro- ducing IL-22.

Some NK-22 Cells Grown in IL-1β Plus IL-7 Acquire the Capacity to Produce IL-17. Because it recently was shown that IL-1β drives proliferation of TH17 T cells (18), we investigated whether NK- 22 cells cultured in IL-1β and IL-7 also produce IL-17. We found that cells expressed IL-17 mRNA (Fig. 5A). Furthermore, stim- ulation of cells with PMA/ionomycin promoted the release of IL- 17 in culture supernatant (Fig. 5B) and induced a small but measurable percentage of cells containing intracellular IL-17 (Fig. 5C). In contrast, stimulation with IL-23 or IL-23 plus IL-1β did not elicit IL-17 production. The physiological stimuli that trigger IL-17 production remain unknown. Cells maintained in IL-7 alone did not produce IL-17, indicating that IL-1β is nec- essary to induce IL-17 production.

Functional Polarization Induced by IL-1β Plus IL-7 Is Modified by IL-2 and IL-23. We next determined the stability of the functional polarization induced by IL-1β plus IL-7. Highly purified tonsil NK-22 cells were cultured initially in IL-1β plus IL-7 or in IL-7 alone. After 12 days, we added IL-2 and continued the cultures for 8 additional days. As a result of this change in culture con- ditions, we observed a clear switch in cytokine production from IL-22 and IL-17 to IFN-γ both by ELISA and intracellular staining (Fig. 5 D and E). These results corroborate the plasticity of NK-22 cells, which can produce IL-22 and IL-17 or IFN-γ in response to IL-7 plus IL-1β or to IL-2, respectively. Because IL-23 has been shown to expand TH17 T cells and pro- mote their terminal differentiation (19), we investigated whether we could expand IL-17–producing NK cells by adding IL-23 to IL-1β and IL-7 throughout the culture. IL-23 did not affect cell recovery (Fig. 6A). However, it induced up-regulation of CD94 and CD56 (Fig. 6B) and marked expansion of IL-22–producing cells (Fig. 6C). IL-17–producing cells were undetectable. In con- trast, IL-23 increased the frequency of IFN-γ–producing cells as well as cells producing both IFN-γ and IL-22. These IFN-γ– producing cells were not responsive to IL-12. The ability of IL-23 to enhance the production of IL-22 and IFN-γ and to inhibit IL-17 further supports the functional flexibility of NK-22 cells.

IL-1β and IL-2 Synergize in Expanding NK-22 Cells. Because both IL-2 and IL-1β induced NK-22 proliferation, we investigated whether these cytokines could cooperate in expanding NK-22 cells. Cul- Fig. 5. NK-22 cells produce IL-17, IL-22, or IFN-γ depending on the cytokine mi- ture of NK-22 cells with IL-1β plus IL-2 induced a striking and + + + − β lieu. CD56 NKp44 CCR6 CD103 cells were sorted from tonsils and cultured in IL- sustained cell growth in comparison with culture with IL-1 plus 1β plus IL-7 or in IL-7 alone for 12 days. Cells then were divided into two aliquots: IL-7, IL-2 plus IL-7, or IL-2 alone (Fig. S4). Cells produced IL- One aliquot was cultured in the original medium; IL-2 was added to the culture 22 and/or IFN-γ (Fig. S5) but not IL-17 and expressed NKG2D medium of the second aliquot. Both cultures were continued for 8 additional days. (Fig. S6), similar to NK-22 cells cultured in IL-2 plus IL-7. Thus, Cells were analyzed for (A) expression of IL-17 mRNA (by RT-PCR); (B and D) release IL-2 and IL-1β synergize in expanding NK-22 cells, although the of IL-17 in culture supernatants 8 h after stimulation with PMA/I or IL-1β plus IL-23 presence of IL-2 deviates the functional profile of NK-22 cells (by ELISA); (C and E) intracellular content of IL-17, IL-22, and IFN-γ after stimulation fl toward that of classical NK cells. with PMA/I (by ow cytometry). Data presented here were obtained from one tonsil specimen representative of three. PB NK, peripheral blood NK cells. IL-2 Promotes NK-22 Cell Secretion of Leukemia Inhibitory Factor. We previously reported that NK-22 cells produce LIF (1), a cytokine sured LIF released in supernatants by ELISA. The amount of that has been reported to protect the mucosal barriers (20). To LIF secreted by NK-22 cells was modest when cells were cultured determine whether cultured NK-22 cells produce LIF, we mea- with IL-7 or IL-7 plus IL-1β but increased considerably when IL-

10964 | www.pnas.org/cgi/doi/10.1073/pnas.1005641107 Cella et al. Downloaded by guest on September 25, 2021 Fig. 7. Cultured NK-22 cells release LIF and BAFF. (A)CD56+NKp44+ − Fig. 6. Addition of IL-23 to IL-1β and IL-7 in the culture medium enhances CCR6+CD103 cells were initially cultured in IL-7 or in IL-1β plus IL-7 for 12 days. + + + − NK-22 production of IL-22 and IFN-γ. CD56 NKp44 CCR6 CD103 cells were Cells were divided into two aliquots: One was cultured in the original medium; IL- sorted from tonsils and cultured in IL-1β plus IL-7 or in IL-1β plus IL-7 plus 2 was added to the second aliquot. After 6 additional days of culture, LIF was IL-23 for 10 days. Cultures were analyzed for (A) cell numbers; (B) expression quantified in the supernatants by ELISA. (B) Release of soluble BAFF by NK-22 cells of CD56 and CD94; and (C) intracellular IL-2 and IFN-γ after stimulation with in different culture conditions. Supernatants were tested 3 days after the last PMA/I. Empty and filled profiles in B represent staining with control and change of medium by ELISA. Note that CD103+ tonsil NK cells do not produce specific antibodies, respectively. Data shown here were obtained from one detectable amounts of BAFF. (C) Cells were cultured as described in A,andrelease tonsil specimen representative of three. of soluble BAFF in supernatants was quantified by ELISA. Data presented here were obtained from one tonsil specimen representative of three. 2 was added to the medium (Fig. 7A). We conclude that secre- tion of LIF is mainly a feature of NK-22 cells exposed to IL-2. Interestingly, we found that ≈50% of cultured NK-22 cells ex-

pressed CXCR5 (Fig. S7). This chemokine receptor drives cells IMMUNOLOGY NK-22 Cells Release Soluble BAFF. Transcriptional profiling of pri- to migrate toward the B-cell area of lymphoid aggregates (22). mary tonsil NK-22 cells versus conventional NK cells had shown Altogether, the release of soluble BAFF and the expression previously that NK-22 cells express the B-cell survival and pro- of CXCR5 suggest that NK-22 cells may contribute to B-cell– liferation factor BAFF (1). However, we could not detect BAFF mediated mucosal immunity. protein on the cell surface of primary NK-22 cells. Because BAFF is cleaved from the cell surface and released in the extracellular Discussion medium as a soluble protein (21), it was possible that BAFF might This study demonstrates that IL-7, IL-2, and IL-1β have different be cleaved rapidly from primary NK-22 cells and released in the impacts on the proliferation of human NK-22 cells. IL-7 is suf- lymphoid tissue. We tested this hypothesis by measuring BAFF in ficient to mediate the survival of NK-22 cells but not their the supernatants of NK-22 cells cultured in vitro with different expansion. IL-2 promotes strong proliferation of NK-22 cells. cytokine combinations. As controls, we tested the supernatants of β + + IL-1 alone has little impact on NK-22 cell proliferation but NKp44 CD103 tonsil NK cells cultured in IL-2. NK-22 cells promotes strong expansion of NK-22 cells together with IL-7 and β cultured in IL-7 or in IL-1 plus IL-7 constitutively released very an even stronger expansion with IL-2. Interestingly, it was shown large amounts of BAFF in the absence of any stimulation (Fig. 7B). recently that IL-1β drives a T-cell receptor-independent pro- Cells cultured in IL-2 plus IL-7 showed a reduced release of BAFF, liferation of TH17 T cells (18). Thus, our demonstration that although the amount of BAFF was still remarkable compared NK-22 cells require IL-1β for expansion further underscores the + + B with that released by NKp44 CD103 cells (Fig. 7 )orperipheral remarkable similarities between TH17 and NK-22 cells. It should blood NK cells. These results suggest that NK-22 cells constitu- be noted that IL-1β also synergizes with IL-23 in stimulating tively release BAFF and that IL-2 partially decreases this capacity. NK-22 cells to produce IL-22 or IFN-γ, depending on whether To corroborate that IL-2 inhibits BAFF release, we measured NK-22 cells are cultured in IL-7 or IL-2 plus IL-7, respectively. BAFF in the culture supernatant of NK-22 cells that were cul- Thus, IL-1β signaling appears to have a crucial role in triggering tured first with IL-7 or IL-7 plus IL-1β and then were switched to cytokine secretion in NK-22 cells. medium containing IL-2. Release of BAFF was markedly re- Another important conclusion of our study is that NK-22 cells duced after IL-2 was added to the culture medium (Fig. 7C). maintain a certain degree of plasticity that allows different func-

Cella et al. PNAS | June 15, 2010 | vol. 107 | no. 24 | 10965 Downloaded by guest on September 25, 2021 tional polarizations. Cells exposed to IL-7 alone most faithfully cells and conventional NK cells suggest that they may be de- recapitulate the polarization of primary NK-22 cells, particularly velopmentally distinct cell subsets. However, in the presence of the selective production of IL-22 in response to IL-23. Cells cul- IL-2, NK-22 cells and conventional NK cells exhibit similar func- tured with IL-1β plus IL-7 also produce IL-22; however, they se- tions, such as IFN-γ production. It is likely that in certain patho- crete IL-22 constitutively rather than in response to stimuli. logical conditions both NK-22 and classical NK cells converge in β Moreover, some of the cells cultured with IL-1 plus IL-7 promoting IFN-γ–mediated immune responses and, probably, cy- β fi produce IL-17. Thus, IL-1 modi es the polarization of primary totoxicity, despite deriving from distinct developmental pathways. NK-22 cells, inducing the capacity to produce IL-17. Although we NK-22 cells have been proposed to contribute to gut innate did not detect any IL-17 production in primary NK-22 cells, this defense through the secretion of IL-22, IL-26, and LIF (1). This feature was observed in fetal and adult LTi-like cells cultured in study also indicates that NK-22 cells may contribute to mucosal vitro, which closely resemble our NK-22 cells in phenotype (8). adaptive immunity. NK-22 cells release large amounts of soluble We envision that there may be yet undefined pathological con- BAFF, particularly when grown in IL-7 and in IL-7 plus IL-1β, ditions in which dendritic cells or other cells release large amounts of IL-1β, inducing IL-17 secretion by NK-22 cells. and express CXCR5, which can direct NK-22 cells toward the Remarkably, culture of NK-22 cells with IL-2–containing me- B-cell areas of lymphoid aggregates. Thus, NK-22 cells may con- dium induced the appearance of cells producing IFN-γ and ex- tribute to adaptive responses, promoting B-cell survival in lym- pressing the NK cell cytotoxicity receptors NKG2D and CD94. phoid aggregates and enhancing the generation of protective Moreover, NK-22 cells cultured with IL-2 plus IL-7 became re- antibodies, such as mucosal IgA. sponsive to IL-12. Because NK-22 cells cultured in vitro with IL-2 plus IL-7 retained the expression of RORγt transcript, we envi- Materials and Methods γ– Cell Preparation and Sorting. Single-cell suspensions of tonsil cells were pre- sion that the IFN- producing cells derive, at least in part, from + the functional deviation of NK-22 cells. Although the initial pared as previously described (1). CD56 cells were enriched by microbeads − CD56+NKp44+CCR6+CD103 cells were fairly homogenous, we magnetic isolation (Miltenyi Biotec). Cells were stained with anti-CD56, cannot exclude the possibility that some of the IFN-γ–producing -NKp44, -CCR6, and -CD103, and sorted on a FACSAria II (BD Biosciances). cells may derive from the expansion of another NK cell subset Cell Culture, Cytokines, Flow Cytometry, and Quantification of mRNA. Sorted included within the original population. Consistent with the func- cells (2–4 × 104/well) were plated in 96 U-bottom plates in RPMI medium tional plasticity of NK-22 cells, a recent study showed that LTi- supplemented with 10% FCS (HyClone), GlutaMAX I, kanamycin, sodium − like cells isolated from human tonsils and cultured in IL-2 secrete pyruvate, nonessential amino acids (all from Invitrogen), and 10 5 M γ IFN- (9). Interestingly, our results show that IL-2 also may be an β-mercaptoethanol. IL-7 (50 ng/mL; Peprotech), IL-1β (50 ng/mL; Peprotech), important factor in promoting the secretion of LIF, a cytokine IL-23 (50 ng/mL; R&D), and IL-2 (1,000 U/mL; Roche) were added for long- that has been shown to protect epithelial cells against bacterial term culture or acute stimulation. BrdU incorporation was measured by the infections (20). The functional deviation of NK-22 cells toward APC BrdU Flow Kit (BD). Human ELISA IL-22 construction was purchased IFN-γ production is reminiscent of that of TH17 T cells, which also from Antigenix America. ELISAs for BAFF and LIF were purchased from R&D. fl can convert into IFN-γ–producing TH1 T cells (23–26). Further A detailed description of the antibodies used for ow cytometry is given in supporting the functional flexibility of NK-22 cells, we found that the SI Text. RORC, IL-17A, and GAPDH mRNA levels were quantified by real- continuous presence of IL-23 in addition to IL-1β and IL-7 in the time PCR, using the procedure and primers described in the SI Text. culture medium enhanced NK-22 cell production of IFN-γ and IL-22 and suppressed IL-17. These effects of IL-23 on NK-22 ACKNOWLEDGMENTS. We thank Todd Fehniger, Tom Hannan, Melissa fi cell polarization are remarkably similar to those observed when Swiecki, and Susan Gil llan for critically reviewing the manuscript and β the Cell Sorting Facility of the Department of Pathology and Immunology, IL-23 is used to culture TH17 cells in the absence of TGF- (24). Washington University School of Medicine, for excellent cell sorting. This Whether NK-22 cells can be classified as NK cells has been work was supported by Grant A1067854 from the National Institute of Al- matter of debate (27). Our data on RORγt expression in NK-22 lergy and Infectious Diseases Center for HIV/AIDS Vaccine Immunology.

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