articles 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function

Julia Parrish-Novak*, Stacey R. Dillon†, Andrew Nelson†, Angie Hammond*, Cindy Sprecher*, Jane A. Gross†, Janet Johnston†, Karen Madden*, Wenfeng Xu*, Jim West‡, Sara Schrader*, Steve Burkhead*, Mark Heipel*, Cameron Brandt*, Joseph L. Kuijper*, Janet Kramer*, Darrell Conklin§, Scott R. Presnell§, Jon Berry‡, Faith Shiota†, Susan Bort†, Kevin Hambly†, Sherri Mudri†, Chris Clegg†, Margaret Moorek, Francis J. Grant§, Catherine Lofton-Dayk, Teresa Gilbertk, Fenella Raymondk, Andrew Ching§, Lena Yaok, Deb Smithk, Philippa Websterk, Theodore Whitmorek, Mark Maurerk, Kenneth Kaushansky¶, Rick D. Holly* & Don Foster*

Departments of * Functional Cloning, † Immunology, ‡ Biochemistry, § Biomolecular Informatics, and k Genetics, ZymoGenetics, Inc., 1201 Eastlake Avenue E., Seattle, Washington 98102, USA ¶ Division of Hematology, Box 35 7710, University of Washington, School of Medicine, 1959 NE Pacific Street, Seattle, Washington 98195, USA ......

Cytokines are important in the regulation of and immune responses, and can influence lymphocyte development. Here we have identified a class I receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

Although primary among the (ILs) this receptor is probably a signalling subunit. IL21R has highest and haematopoietic growth factors is low, these are amino-acid sequence similarity to IL2R␤ and IL4R␣. We subse- structurally related1 and fold into a classic ‘four-helix-bundle’ quently cloned murine IL21R from a mouse splenocyte library, and structure that is representative of the family. Most cytokines bind found that it shares overall structure and functional motifs with to cells and transduce signals through either the class I or the class II human IL21R (Fig. 1a). cytokine receptors. The class II cytokine receptors include the IL21R was localized to human 16p11 using the GeneBridge 4 receptors for IL10 and the , whereas the class I cytokine radiation hybrid mapping panel (Research Genetics, Inc.). In receptor family includes the receptors for IL2, IL3, IL4, IL5, IL6, IL7, addition, the IL21R sequence was found in genomic sequence IL9, IL11, IL12, IL13 and IL15, as well as the haematopoietic growth from two overlapping bacterial artificial clones factors, leptin and growth hormone2. Class I cytokine receptors that mapped to 16p11 (GenBank accession nos AC002303 and are capable of signal transduction through homodimerization have AC004525). Comparison of the IL21R cDNA sequence with this been used in the past to design biological activity assays to screen for genomic sequence revealed that the IL21R spans about 20 kilo- sources of the cognate ligand, and to facilitate either purification or bases (kb) of genomic DNA, and lies about 65 kb from the IL4R␣ expression cloning of the ligand3. gene. Here we describe a new class I cytokine receptor, IL21R. We Mpl, the receptor for thrombopoietin (TPO), delivers a prolif- describe the use of a proliferation assay, based on BaF3 cells4 erative signal on homodimerization in the presence of TPO7.To expressing IL21R, to screen for potential interactions with known determine whether the intracellular domain of IL21R is capable of cytokines, to identify a source of the cognate ligand, and to clone the signalling as a homodimer, we constructed a chimaeric receptor ligand, IL21. Preliminary biological characterization indicates that using the extracellular and transmembrane domains of Mpl fused to IL21 may be involved in haematopoietic development and differ- the intracellular domain of IL21R. This chimaeric receptor was entiation of natural killer cells in conjunction with IL15, in the transfected into BaF3 cells4, which are IL3-dependent and not activation and proliferation of B cells co-stimulated through CD40 normally responsive to TPO. Positive control cells were transfected and also in the co-stimulation of T cells. with full-length Mpl. Both the full-length Mpl and the chimaeric receptor transduced proliferative signals in the BaF3 cells in Receptor cloning and assay development response to TPO (data not shown). The proliferative signal deliv- We identified an expressed sequence tag (EST) containing a pre- ered by the homodimeric Mpl/IL21R chimaera in BaF3 cells dicted signal peptide and a predicted amphipathic helix. Sequencing suggests that a ligand for IL21R may exert a proliferative effect on of a full-length clone revealed a cDNA coding for a member of the cells expressing the full-length receptor, thus providing an activity class I cytokine receptor family. This cDNA, designated IL21R, assay for ligand cloning. contains an open reading frame encoding a 538-amino-acid protein that has structural motifs common to class I cytokine receptors2,5. Ligand functional cloning Extracellular motifs include a single cytokine recognition module, We expressed full-length IL21R in BaF3 cells, creating the cell line two pairs of conserved cysteine residues, and a ‘WSXWS’ motif. The BaF3/IL21R. Known human and murine cytokines were tested for intracellular domain contains strong intracellular signalling motifs, activity, and only those that also stimulated parental BaF3 cells including classical box 1 and box 2 motifs6–10, which indicate that supported growth of BaF3/IL21R cells. Conditioned media from

NATURE | VOL 408 | 2 NOVEMBER 2000 | www.nature.com © 2000 Macmillan Magazines Ltd 57 articles more than 100 different primary and immortalized cell types were assay cell line. Proliferation was determined using an Alamar blue tested for activity, and conditioned medium from PMA (phorbol assay (Accumed). Each positive conditioned medium was re- 12-myristate 13-acetate)/ionomycin-activated human peripheral assayed with and without sIL21R. Results showed that each positive CD3+ T cells induced proliferation of BaF3/IL21R cells. The condi- pool contained a proliferative factor that was neutralized by soluble tioned medium was inactive on wild-type BaF3 cells, indicating receptor. Sequencing of individual positive clones from each pool specificity for cells expressing IL21R. This proliferative response was revealed that all contained inserts encoding the same polypeptide, suppressed by the addition of soluble IL21R (sIL21R), indicating which we have designated interleukin-21 (IL21) to reflect both its that sIL21R is capable of binding to the proliferative factor (ligand) structural relationship to other interleukins and its biological in the conditioned medium. activities. A cDNA library was constructed from sorted CD3+ human T cells that had been activated for 13 h with PMA and ionomycin. Pools of Ligand structure 250 clones each were transfected into baby hamster kidney (BHK) The sequence of human IL21 cDNA contains an open reading frame cells, and conditioned media were transferred to the BaF3/IL21R that encodes a polypeptide precursor of 162 amino acids. The signal

a mIL21R MPRGPVAALLLLILHGAWSCLDLTCYTDYLWTITCVLETRSPNPSILSLTWQDEYEELQD 60 X::: .:.::::.:.:.:.: ::.:::::: :..:.:: .. .::.:.:::::.::::.: hIL21R MPRGWAAPLLLLLLQGGWGCPDLVCYTDYLQTVICILEMWNLHPSTLTLTWQDQYEELKD

mIL21R QETFCSLHRSGHNTTHIWYTCHMRLSQFLSDEVFIVNVTDQSGNNSQECGSFVLAESIKP 120 ..: ::::::.::.:: ::::: . .:..:..: ::.:::::: :::::::.::::::: hIL21R EATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVNITDQSGNYSQECGSFLLAESIKP

mIL21R APPLNVTVAFSGRYDISWDSAYDEPSNYVLRGKLQYELQYRNLRDPYAVRPVTKLISVDS 180 :::.::::.:::.:.::: :.:..:. :.:.::::::::::: ::.::.: ::::::: hIL21R APPFNVTVTFSGQYNISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDS

mIL21R RNVSLLPEEFHKDSSYQLQVRAAPQPGTSFRGTWSEWSDPVIFQTQAGEPEAGWDPHMLL 240 :.::::: ::.:::::.:::::.: ::.:..:::::::::::::::..: ..::.::.:: hIL21R RSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLL

mIL21R LLAVLII-VLVFMGLKIHLPWRLWKKIIWAPVPTPWAPVPTPESFFQPLYREHSGNFKKWVNTPFTAS 299 :: ..:. . .: .::.: ::::::::::::: ::.:::.::.:: :::.. ::.:::::..:::.: hIL21R LLLLVIVFIPAFWSLKTHPLWRLWKKIIWAWA-VPSPVPSPERFFMPLYKGCSGDFKKWVGAPFTGS

mIL21R SIELVPQSSTTTSAL-----HLSLYPAKEKKFPGLPGLEEEQLECDGMSEQLECDGMSEPGHWCIIPLAA 354 :.:: : :....:.: v :^. ::: ....:.. .: ..:.::...:.::...:. : : :. hIL21R SLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAEELVESDGVPKLVESDGVPKPSFW---PTAQ

mIL21R GQAVSAYSEERDRPYGLVSIDTVTVGDAEGLCVWPCSCEDDGYPAMNLDAGRESGPNSED 414 . . ::::::::::::::::::::: :::: :.::::::::::::..:::: :..:. :: hIL21R NSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLED

mIL21R LLLVTDPAFLSCGCVSGSGLRLGGSPGSLLDRLRLSFAKEGDWTADPTWRTGSPGGGSES 474 :: .... :::::::... :::. :::::::. ..:...::... .: . :::: ::: hIL21R PLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSES

mIL21R EAGSP-PGLDMDTFDSGFAGSDCGSPVE------TDEGPPRSYYLRQLRQWVVRTPPPVDSGAQSS 529 ::::: .:::::::::::.::::.:::: .:::::::::::::::::: .:: ..:.:.: hIL21R EAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYYLRQLRQWVVIPPPLSSPGPQAS

b hIL-21 ------MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLV mIL-21 ------MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLD **** *:: * *:: hIL-15 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI-EDLI hIL-4 ------MGLTSQLLPPLFFLLACAGNFVHGHKCD-ITLQEIIKTLNSLTEQKT hIL-2 ------MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGIN

hIL-21 PEF------LPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKR mIL-21 PEL------LSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRR : :: : *: :: : * *: * hIL-15 QSMHIDAT------LYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIL hIL-4 LCTELTVTDI------FAASKNTTE----KETFCRAATVLRQFYSHHEKDTRCLGATAQQF hIL-2 NYKN------PKLTRMLTFKFYMPKKATE---LKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDL

hIL-21 KPPSTNAGRRQKHRL------TCPSCDSYEKK--PPKEFLERFKSLLQKMIHQHLSSRTHGSEDS mIL-21 RLPARRGGKKQKHIA------KCPSCDSYEKR--TPKEFLERLKWLLQKMIHQHLS : * * : :* *: : ::::* : ***: : hIL-15 ANNSLSSNGNVTESG------CKECEELEEK--NIKEFLQSFVHIVQMFINTS hIL-4 HRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS hIL-2 ISNINVIVLGLKGSE------TTFMCE-YADETATIVEFLNRWITFCQSIISTL

Figure 1 Amino-acid sequences of IL21R and IL21. a, Alignment of the amino-acid underlined and in boldface, is present near the C terminus. b, Alignment of human and sequences of murine and human IL21R. Overall identity is 62%. Signal sequence is murine IL21 with related cytokines. Identities (colon) or similarities (asterisk) between underlined. Both pairs of cysteines (shown in bold) and the WSEWS motif (underlined) are either human or murine IL21 and human IL15 are indicated. Mature N termini are fully conserved in the mouse. Box 1 and Box 2 (shown in bold) are less conserved, but indicated by open boxes. Potential N-linked glycosylation sites are underlined. conform to the overall consensus sequences. A consensus Stat3-binding site28,

58 © 2000 Macmillan Magazines Ltd NATURE | VOL 408 | 2 NOVEMBER 2000 | www.nature.com articles peptidase cleavage rules11 predict a cleavage site after Gly 31, leaving and NK cells using biotinylated IL21. Resting CD23+ B cells from a mature N terminus that corresponds to our observed N terminus three different donors show positive staining, which is blocked in for recombinant human IL21 protein (data not shown). The mature each case by 15-fold excess unlabelled IL21 (Fig. 3a). Although polypeptide has a predicted relative molecular mass of 15,000 receptor expression was not consistently detectable by flow cyto- (Mr 15K), and consists of a 131-residue four-helix-bundle cytokine metry on peripheral NK cells, we were able to detect it on the NK cell domain with significant homology to IL2, IL4 and IL15 (Fig. 1b). line NK-92 (ref. 14) (Fig. 3b), and on fresh NK cells by polymerase IL21 and IL15 share two pairs of cysteine residues in identical chain reaction with reverse transcription (RT–PCR) (data not positions, one pair that is conserved in IL2, IL4 and granulocyte- shown). We carried out flow cytometry on a series of mouse and macrophage colony-stimulating factor (GMCSF), and one that is human cell lines. In addition to NK-92, cell lines found to express unique to IL21 and IL15. This structural consistency, together with IL21R included Raji (human Burkitt lymphoma), Jurkat (human T- the relatively high degree of amino-acid homology, indicates that cell leukaemia), IM-9 (human B-cell line), HS Sultan (human B-cell IL15 is the closest structural relative of IL21. line) and A20 (mouse B-cell lymphoma). IL21R expression was Using the Stanford G3 radiation hybrid panel (Research Genetics, not detected on EL4 (mouse thymoma) or K562 (human T-cell Inc.), we mapped the human IL21 gene to 4q26-q27, roughly 180 kb leukaemia) (Fig. 3b; and data not shown). from the IL2 gene. The IL15 gene lies more distal in the cluster. The Expression of IL21 ligand in normal tissues was not detectable by presence of these three highly related in the same chromo- northern analysis (data not shown); however, the sources from somal region suggests that they may have arisen by gene duplication. which human and mouse IL21 cDNAs were cloned suggested that it is probably produced by activated peripheral T cells. Quantitative Murine IL21 RT–PCR analysis was used to measure activation-induced changes A search of nucleic-acid databases using the human IL21 sequence in IL21 message levels in purified human peripheral T cells. As CD3+ revealed a partial, unspliced expressed sequence tag (EST1483966) T cells express IL21 transcripts on stimulation with PMA and representing the murine orthologue of IL21. We obtained a cDNA ionomycin, CD4+ and CD8+ T-cell subsets were isolated and clone by screening a library constructed from activated CD90+ stimulated either with PMA plus ionomycin or with an immobi- splenocytes. Murine IL21 cDNA encodes a precursor with a signal lized anti-CD3 antibody. CD4+ T cells are stimulated to express IL21 peptide and a 122-amino-acid mature protein with 57% identity under both sets of conditions; however, expression is significantly to human IL21 (Fig. 1b). The sequences of human and murine IL21 are especially well conserved in the regions predicted to encode ␣-helices A and D, regions that in other cytokines are important in a 50 1,12 CD23+ receptor interactions . Both human and murine IL21 have an 40 CD3+ acidic amino acid in position Asp 13 of the mature peptide, 30 corresponding to Glu 9 in IL4, which is involved in the primary 20 Counts high-affinity interaction between IL4 and IL4R␣12. Human and 10 murine IL21 also contain a conserved Gln 114, equivalent to 0 100 101 102 103 104 0 1 2 3 4 Gln 141 of IL2, which has been implicated in the interaction 10 10 10 10 10 between IL2 and IL2R␥c (ref. 13). 50 CD56+ 40 CD14+ Tissue distribution 30 Analysis of northern blots from a variety of human and murine 20 Counts tissues (Clontech) revealed IL21R transcripts only in lymphoid 10 tissues, including spleen, thymus, peripheral blood lymphocytes 0 0 1 2 3 4 (PBLs) and lymph node (Fig. 2). In situ hybridization using 10 10 10 10 10 100 101 102 103 104 antisense probes specific to two different regions of IL21R resulted in positive signals in a subset of cells within normal tonsil, lymph b 120 120 node and thymus, and in the lamina propria of duodenal villi, as IM-9 NK-92 well as in fibrotic (but not normal) lung (data not shown). To characterize the specific cell types expressing surface IL21R, we Counts carried out flow cytometry on resting and activated peripheral B, T Counts

0 0 100 101 102 103 104 100 101 102 103 104 AVID-PE AVID-PE 120 120 HL-60 JURKAT PBLs Trachea Kidney Prostate Testis Ovary Colon Liver Thyroid Spinal cord node Lymph gland Adrenal Small intestine Pancreas Thymus Brain Skeletal muscle Bone marrow Bone marrow Heart Lung Spleen Stomach ... Placenta ...... Counts Size Counts (kb) 9.5 0 0 7.5 100 101 102 103 104 100 101 102 103 104 4.4 AVID-PE AVID-PE

2.4 Figure 3 Flow cytometric analysis of IL21R expression. Human peripheral blood lymphocytes (a) or cell lines (b) were stained with antibodies to the markers indicated, and/or with 2 ␮gml−1 biotinylated IL21 (bold lines). Binding was competed using Figure 2 Expression analysis of IL21R in normal tissues. Northern analysis was performed 30 ␮gml−1 unlabelled IL21 (dashed lines). Thin lines represent secondary reagent only on multiple-tissue northerns (human, human III, human IV; Clontech). Filters were (Streptavidin APC). Other reagents used are anti-CD23-PE, anti-CD3-CyChrome, anti- exposed for 48 h at −80 ЊC. Bottom panel shows hybridization with a human transferrin CD14-PE and anti-CD56-FITC. Results shown in a are representative of three different receptor probe. donors.

NATURE | VOL 408 | 2 NOVEMBER 2000 | www.nature.com © 2000 Macmillan Magazines Ltd 59 articles higher in the samples stimulated with anti-CD3 (Fig. 4a). To further To assess the activity of IL21 on lymphoid progenitors, we co- define the conditions that induce IL21 expression, we purified cultured human non-adherent bone marrow progenitor cells with peripheral T cells from a different donor and stimulated with and human IL21 in combination with various other anti-CD3 either alone or in combination with anti-CD28. The cytokines. Although IL21 alone does not induce significant prolif- combination of anti-CD3 and anti-CD28—conditions that best eration of marrow progenitors, it does synergize with IL15, con- mimic an antigen encounter in vivo—provided higher induction of sistently causing at least a twofold increase in cell proliferation as IL21 expression than that seen with anti-CD3 alone in this assay compared with cultures containing stem cell factor and IL15 alone (Fig. 4b). The small increase in message seen in stimulated CD8+ (data not shown). Flow cytometric analysis of these expanded cells cells in both experiments is probably due to contaminating CD4+ for lineage-specific markers revealed expression of the NK cell cells (1–3%). CD19+ B cells and CD14+ monocytes did not express marker CD56 (data not shown). − IL21 under any stimulation condition (data not shown). Other groups have shown that CD3 CD56+ NK cells can be generated from 21-day cultures of human CD34+ haematopoietic Biological activities progenitor cells (HPCs) supplemented with IL15 in the absence of Because resting B lymphocytes express IL21R (see Fig. 3a), we stromal cells, an effect that is enhanced by the addition of Flt3L15,16. designed assays to determine the effects of IL21 on B-cell prolifera- Given our observation that CD56+ NK cells grow out of bone tion. Although IL21 alone has no mitogenic effect on isolated marrow cultures stimulated with IL15 and IL21, we undertook an human B cells (data not shown), it significantly co-stimulates analysis of the effect of IL21 on the development of NK cells from proliferation induced by anti-CD40 monoclonal antibodies HPCs. We isolated CD34+ human HPCs (95% pure) and cultured (Fig. 5a). In contrast, IL21 inhibits proliferation induced by the them either with Flt3L with and without IL21, or with Flt3L plus combination of plate-bound anti-IgM antibodies and IL4 (Fig. 5b). IL15 with and without IL21. Cells were collected on day 15 to assess Both effects are reversed by the addition of sIL21R to the culture. their NK markers by flow cytometry (Fig. 7a) and also their capacity − We also tested IL21 in T-cell proliferation assays and found that it to lyse MHC K562 cells in a standard 51Cr-release assay (Fig. 7b). As co-stimulates anti-CD3-activated murine thymocytes (Fig. 6a), previously reported16, the combination of IL15 and Flt3L alone − leading to an accelerated outgrowth of CD8+CD4 cells (data not induced the outgrowth of CD56+ cells. In contrast, IL21 and Flt3L shown). We did not observe significant levels of proliferation of alone did not significantly expand the CD56+ fraction. In cultures thymocytes in response to IL21 in the absence of anti-CD3. IL21 exposed to the combination of Flt3L, IL15 and IL21, however, there also co-stimulates anti-CD3-activated mature murine (Fig. 6b) and was a significant increase in total cell numbers and the proportion of human (Fig. 6c) T cells. In each case, addition of sIL21R blocked this cells that were CD56+CD16bright, when compared to cultures sup- IL21-dependent proliferation (data not shown). Notably, our early plemented with Flt3L and IL15 alone. The cells growing out of experiments suggested that the proliferative effect of IL21 is subtler these Flt3L, IL15, IL21 cultures were differentiated NK cells, as they on human T cells than on murine T cells. We reasoned that this were large and granular and expressed both CD56 and CD16, but might be a function of the prior activation state of the human did not express CD3 (Fig. 7a; and data not shown). Furthermore, T cells, as murine T cells are less likely to be pre-activated in vivo. these cells exhibited significantly higher effector function at day 15 Indeed, when we separated CD45RA+ (naive) and CD45RO+ than those cells grown with only IL15 and Flt3L (Fig. 7b). After (memory) human T cells and assayed them for proliferative 21 days in culture, however, the cells cultured in the presence of responses to IL21, we found that naive, but not memory, T cells Flt3L and IL15 (without IL21) had gained lytic activity, but − were co-stimulated by IL21 (Fig. 6c; and data not shown). To remained CD16low/ , consistent with previous reports15,16 (data not compare the activity of IL21 to that of related cytokines on shown). murine T cells, we performed a cross-titration of IL21 and IL2, We also tested whether IL21 can act on mature NK cells. We IL7 or IL15, in the presence or absence of a suboptimal concentra- isolated CD56+ cells from fresh human peripheral blood mono- tion of immobilized anti-CD3 monoclonal antibody (Fig. 6d). nuclear cell (PBMCs) (Fig. 7c), and cultured them for 1–2 days with − Unlike its related counterparts, IL21 has no significant proliferative and without 30 ng ml 1 IL2, IL15 or IL21. The cells cultured with effect on T cells in the absence of anti-CD3 or other stimuli (Fig. 6d; IL21 exhibited enhanced lytic activity on K562 target cells, although and data not shown). However, it does act in concert with IL2, IL15, the effect of IL21 was never quite as pronounced as that of IL2 or and, to a lesser extent, with IL7 to enhance T-cell proliferation, IL15 (Fig. 7d). Treatment of NK cells with combinations of IL21 and either with or without anti-CD3 stimulation (Fig. 6d).

50- 7- ) a

–3 b a b *** 6- 10 40-

1.8 ** 1.8 × 1.6 1.6 ** 5- 1.4 1.4 30- 1.2 1.2 4- * 1.0 *** 1.0 0.8 0.8 20- 3- * IL21/rRNA 0.6 IL21/rRNA 0.6 0.4 2- 0.4 10- 0.2 0.2 * 1- H]TdR uptake (c.p.m.

0 0 3 *** 12345 15 [ *** 12345 5 123 4 0- 0- Anti-CD40 Anti-CD40 Anti-IgM Anti-IgM Anti-IgM + CD4+ CD8+ CD4+ CD8+ + IL21R alone + IL4 IL4 + IL21R Figure 4 Quantitative RT–PCR analysis of IL21 expression in lymphocyte subsets. Figure 5 Effect of IL21 on the proliferative responses of human B cells. B cells were a, Stimulation conditions are resting (lane 1); 3.5 h anti-CD3 (lane 2) or PMA plus purified from peripheral blood of normal human donors and cultured either in the ionomycin (lane 3);16 h anti-CD3 (lane 4) or PMA plus ionomycin (lane 5). b, Stimulation absence (open bars) or in the presence (filled bars) of 10 ng ml−1 IL21. a, Anti-CD40 and conditions are resting (lane 1); 2 h anti-CD3 (lane 2); and anti-CD3 plus anti-CD28 for 2 h 25 ␮gml−1 sIL21R were used as additional stimuli as indicated. b, Combinations of (lane 3), 4 h (lane 4) or 16 h (lane 5). Vertical bars represent the ratio of IL21 message in immobilized anti-IgM, IL4 and 25 ␮gml−1 sIL21R were used as additional stimuli as each sample to that of 18S ribosomal RNA in the same sample. Values plotted represent indicated. Values plotted represent the mean value (Ϯ s.d.) obtained from triplicate the mean (Ϯ s.d.) obtained from triplicate assays. Asterisk, P Ͻ 0.05; two asterisks, P Ͻ cultures. Similar results were obtained in four independent experiments. Asterisk, 0.005; three asterisks, P Ͻ 0.001. P Ͻ 0.05; two asterisks, P Ͻ 0.005; three asterisks, P Ͻ 0.001.

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IL2 or IL15 led to an additive effect on lytic function (data not Given the structural similarity between IL21 and the IL15/IL4/IL2 shown). family of cytokines, together with the structural relationship between IL21R and both IL2R␤ and IL4R␣, it is reasonable to Discussion speculate that, although the IL21R receptor appears to be capable of We have cloned a cytokine–receptor pair, interleukin-21 and IL21R, signal transduction through homodimerization (as is IL4R␣17), it that both regulates the proliferation of mature B and T cells in may physiologically associate in a heteromeric receptor with response to activating stimuli, and mediates expansion of NK cell IL2R␥c. The IL2R␥c receptor subunit participates in forming the populations from bone marrow. IL21R is expressed in peripheral receptors for IL15, IL4, IL7, IL9 and IL2 (for review, see ref. 18). Our B cells as well as in cell lines of B, T and NK lineages. IL21 is preliminary data suggest that IL2R␥c may indeed be a part of the expressed in a highly controlled manner, by CD4+ T cells that IL21 receptor complex; further characterization is in progress. have been activated using stimuli that closely mimic an antigen IL21 promotes expansion of mature B cells in response to encounter. stimulation through CD40. Interactions between CD40 and its

a 300 mIL-21(ng ml–1) 0

) 250 * –3 1.2 d + anti-CD3 no anti-CD3 10 µ –1 × 200 6 (0.25 g ml ) 500 100 30 150 * 400 80

* 100 * 300 60

* 200 40

H]TdR uptake (c.p.m. 50 3 * 0 100 20 1 0.3 0.1 0 0 0 b 0 2 10 50 0 2 10 50 –1 Concentration mIL-2 (ng ml–1) * * mIL-21(ng ml ) 400 * )[ 0 500 100 –3 )

0.4 –3 10 ×

10 400 80 300 2 ×

10 300 60 * 200 50 200 40 *

100 * 100 20 H]TdR uptake (c.p.m. 3 [ H]TdR uptake (c.p.m. 3 [ 0 0 * 0502 10 50 0 2 10 0 * 1 0.2 0.04 0 Concentration mIL-7 (ng ml–1) c 500 500 200 hIL-21(ng ml–1) 400 ) * 0

–3 * 150 400 * *

10 2 300 × 10 100 300 50 200 * 50 100 200 * * * 0 0 * 0 2 10 50 0 2 10 50

H]TdR uptake (c.p.m. 100 3 [ Concentration hIL-15 (ng ml–1) *** 0 0.6 0.2 0.07 0 Concentration anti-CD3 (µg ml–1)

Figure 6 IL21 co-stimulates anti-CD3 activated thymocytes and mature T cells. a, Whole described in Methods. These enriched naive T cells (75% CD3+CD45RA+,1% C57Bl/6 thymocytes were assayed for proliferation in response to plate-bound anti-CD3⑀ CD3+CD45RO+, 4% CD11b+, 5% CD20+) were tested in triplicate cultures for monoclonal antibody in the absence or presence of increasing concentrations of mIL21, proliferation in a 3-day assay to immobilized anti-human CD3 monoclonal antibody, in the as indicated. Values plotted represent the mean value (Ϯ s.d.) obtained from triplicate presence or absence of human IL21, as indicated. Control T cells stimulated with PMA cultures. Asterisks indicate significant values (P Ͻ 0.05 by Student’s t-test) above those and ionomycin incorporated an average of 615 × 103 c.p.m. Results shown are from wells lacking IL21. Data shown are representative of results from six experiments. representative of three experiments. d, Mature murine T cells were purified as in b and Thymocytes stimulated with 2 ng ml−1 PMA and 500 ng ml−1 ionomycin as a positive incubated in the absence (right) or presence (left) of 0.25 ␮gml−1 anti-CD3⑀ monoclonal control incorporated 445 × 103 c.p.m. in the same time period. b, Mature murine T cells antibodies, along with increasing amounts of mIL2 (top), mIL7 (middle) or hIL15 (bottom) were enriched from pooled C57Bl/6 spleen and lymph nodes by depletion of CD19+ B in a 3-day proliferation assay. A cross-titration of 0 (filled circles), 2 (open circles), 10 cells. The resulting cell population (78% CD3+ and 3% B220+) was assayed as in a.T (filled squares) or 50 ng ml−1 (open squares) mIL21 was also included, to test for synergy cells stimulated with PMA and ionomycin incorporated 625 × 103 c.p.m. Results shown and/or competition among the related cytokines. Results shown are representative of are representative of five experiments. c, Human CD45RA+ T cells were isolated as three experiments.

NATURE | VOL 408 | 2 NOVEMBER 2000 | www.nature.com © 2000 Macmillan Magazines Ltd 61 articles ligand, CD40L, are an integral part of the communication between role in regulation of the immune response by promoting the B cells and helper T cells in mounting thymus-dependent (TD) expansion of helper T cells and by eliciting the appropriate immune responses, and are vital in generation of memory B cells responses from neighbouring activated B cells. and germinal centres (for reviews, see refs 19, 20). Defects in CD40L IL21 promotes expansion and differentiation of NK cells from lead to X-linked hyper-IgM syndrome, a primary immunodefi- bone marrow progenitors in vitro, in synergy with Flt3L and IL15. ciency with characteristics including absence of germinal centres The most striking result is the development of CD16bright, highly and increased incidence of autoimmunity (for review, see ref. 21). In lytic NK cells by 15 days in culture in the presence of IL21. In contrast to its synergistic effect with CD40 stimulation, IL21 agreement with previous reports15,16, we also observed the develop- − completely inhibits proliferation of B cells stimulated with anti- ment of lytic CD16low/ cells from CD34+ marrow cells after 21 days IgM and IL4. This suggests that signalling through IL21R can differ in culture in the presence of only Flt3L and IL15 (data not shown). markedly, depending on the co-stimuli encountered by the CD16 is thought to mediate antibody-dependent cellular cytotoxi- during an immune response. city (for review, see ref. 22), and its expression on IL21-treated NK In addition to its effects on B cells, IL21 promotes enhanced cells reflects an enhanced maturational state. expansion of T cells co-stimulated with anti-CD3. In humans, the NK cells are lymphoid cells that participate in the innate immune response to IL21 appears to be limited to naive CD45RA+ T cells, system’s surveillance of malignant cells and thus may help to control suggesting either that IL21R is downregulated on activated/memory metastatic cancer. These cells are also essential for natural immunity T cells, or that the signalling cascade downstream of IL21R in naive against microbial pathogens, in particular viruses; they influence compared with memory T cells is distinct. As activated CD4+ T cells the graft-versus-leukaemia effect after bone marrow transplanta- also produce IL21, this cytokine may have an autocrine/paracrine tion; and they are involved in the regulation of haematopoiesis (for

a b

60 3 65 3 flt-3L 40 flt-3L+IL-15 flt-3L+IL-21 flt-3L flt-3L+ IL-21 30 flt-3L+IL-15+IL-21

36 0.1 32 0.1 20

57 4 561 10

flt-3L+ % Specific lysis flt-3L+ IL-15+ IL-15 IL-21 0

CD16 28 11 9 25 –10 1 0.1 0.01 Effector : Target CD56 (corrected for % CD56+)

c d 100 Media Total PBMC CD56+ purified 80 IL-2 2 10 1 58 IL-15 60 IL-21 82 13 40

20

CD16 6 28 % Specific lysis 0 CD56 –20 100 10 1 0.1 Effector : Target (corrected for % CD56+)

Figure 7 IL21 synergizes with IL15 and Flt3L to enhance the proliferation and cells in a standard 4-h assay. The effector-to-target ratio was corrected for the differentiation of NK cells from CD34+ bone marrow progenitors, and alone enhances percentage of CD56+ cells in each culture. c, Mature CD56+ NK cells were purified from the effector function of mature NK cells. a, Human CD34+ bone marrow cells (95% pure) fresh human PBMCs by positive selection, stained with anti-CD56-PE and anti-CD16- were cultured for 15 days in the presence of 2 ng ml−1 Flt3L (open circles), Flt3L plus CyChrome monoclonal antibodies, and analysed for purity by flow cytometry. 20 ng ml−1 hIL15 (filled circles), Flt3L plus 15 ng ml−1 hIL21 (open squares) or Flt3L plus d, Purified CD56+ NK cells shown in c were cultured in media alone (open squares), or hIL15 plus hIL21 (filled squares). An aliquot of cells from each culture condition was with 30 ng ml−1 of hIL2 (circles), hIL15 (triangles) or hIL21 (filled squares) for 24 h, and stained and analysed by flow cytometry for surface expression of CD56 and CD16 (all cells then tested for lytic function in the assay described in b. No significant differences were were CD3−). Data were gated on viable cells based on their light scatter characteristics; noted in the number of cells recovered from each culture condition. Similar results were the percentages of cells in each quadrant are indicated. Results shown are representative achieved when the cells were cultured for 48 h before assay. Results shown are of four experiments using four different bone marrow donors. b, Cells shown in a were representative of five experiments using four different blood donors. collected, washed and assayed for their ability to lyse 51Cr-labelled K562 (MHC−) target

62 © 2000 Macmillan Magazines Ltd NATURE | VOL 408 | 2 NOVEMBER 2000 | www.nature.com articles reviews, see refs 22–25). The contrasting effects of IL21 on the Each well was pulsed with 1 ␮Ci [3H]thymidine on day 2–4, and plates were collected proliferation of B cells, depending on their mode of activation, as and counted 16–18 h later. well as its effects on NK cell maturation and on T cells, suggest that it B-cell proliferation assays may be an important regulator of the . We purified human peripheral B cells with anti-CD19 magnetic beads (Miltenyi Biotec). Note added in proof: Following acceptance of this article, a paper For anti-IgM stimulation, plates were pre-coated with anti-human IgM (Southern Biotech − 29 Ⅺ Associates) and cultures were supplemented with 10 ng ml 1 IL4 (R&D Systems). Anti- was published describing the receptor . − human CD40 monoclonal antibody (Genzyme) was added to cultures at 1 ␮gml 1. Proliferation was assessed by the incorporation of [3H]thymidine (1 ␮Ci per well) during Methods the last 16 h of a 120-h culture. We carried out treatments in triplicate. Library construction − Received 19 July; accepted 13 September 2000. Human CD3+ PBMNC and Balb/C CD90+ splenocytes were activated using 10 ng ml 1 − PMA and 0.5 ␮gml 1 ionomycin (Calbiochem). We isolated RNA using Rneasy Midi Kit 1. Wlodawer, A., Pavlovsky, A. & Gustchina, A. Hematopoietic cytokines: similarities and differences in (Qiagen) and mRNA using MPG mRNA purification kit (CPG Inc.). cDNA was the structures, with implications for receptor binding. Protein Sci. 2, 1373–1382 (1993). synthesized using a modified Gubler–Hoffman method26 and cloned into expression 2. Cosman, D. The hematopoietin receptor superfamily. Cytokine 5, 95–106 (1993). vector pZP7NX. 3. Lok, S. et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of production in vivo. Nature 369, 565–568 (1994). Protein expression and purification 4. Palacios, R. & Steinmetz, M. IL3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo. Cell 41, 727–734 (1985). The extracellular portion of IL21R was cloned into an expression vector with a C-terminal 5. Bazan, J. F. Structural design and molecular evolution of a cytokine receptor superfamily. Proc. Natl Glu–Glu (EE) tag and expressed in BHK cells. Protein was purified from conditioned Acad. Sci. USA 87, 6934–6938 (1990). media using anti-EE G-sepharose and POROS HQ-50 columns (PerSeptive BioSystems), 6. Vigon, I. et al. Characterization of the murine Mpl proto-oncogene, a member of the hematopoietic and then Sephadex S200 (Pharmacia) and ActiClean Etox (Sterogene) columns. We cloned cytokine receptor family: molecular cloning, chromosomal location and evidence for a function in cell human IL21 into pDC312 (ref. 27) and expressed it in suspension-adapted CHO DG44 growth. Oncogene 8, 2607–2615 (1993). cells. Mouse IL21 was cloned into pZP9 and expressed in BHK cells. Conditioned media 7. Skoda, R. C. et al. Murine c-mpl: a member of the hematopoietic receptor superfamily were separated using Poros 50 HS columns (PerSeptive BioSystems). Human IL21 was that transduces a proliferative signal. EMBO J. 12, 2645–2653 (1993). then purified on a Sephacryl S-200 column (Pharmacia). Mouse IL21 was purified using 8. Murakami, M. et al. Critical cytoplasmic region of the signal transducer gp130 is Poros AL/sIL21R columns, and then Sephacryl S-200. conserved in the cytokine receptor family. Proc. Natl Acad. Sci. USA 88, 11349–11353 (1991). 9. Drachman, J. G. & Kaushansky, K. Dissecting the thrombopoietin receptor: Functional elements of Real-time PCR the Mpl cytoplasmic domain. Proc. Natl Acad. Sci. USA 94, 2350–2355 (1997). + + 10. Gurney, A. L., Wong, S. C., Henzel, W. J. & De Sauvage, F. J. Distinct regions of c-Mpl cytoplasmic Human PBMCs were isolated from fresh whole blood. We purified CD4 and CD8 cells domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation. Proc. by positive selection using a MACS magnetic separation column (Miltenyi Biotec). Purity Natl Acad. Sci. USA 92, 5292–5296 (1995). + + was assessed by flow cytometry; CD4 cells were 85% and 97% pure, and CD8 cells were 11. von Heijne, G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 84% and 81% pure in the assays shown in Fig. 4a and b, respectively. We carried out real- 4683–4690 (1986). time quantitative RT–PCR using the ABI PRISM 7700 sequence detection system (PE 12. Hage, T., Sebald, W. & Reinemer, P. Crystal structure of the interleukin-4/receptor ␣ chain complex Ј Ј Ј Applied Biosystems). Primers were (5 -tgtgaatgac ttggtccctg aa-3 ) and (5 -aacaggaaaa reveals a mosaic binding interface. Cell 97, 271–281 (1999). agctgaccac tca-3Ј). The TaqMan probe (5Ј-tctgccagct ccagaagatg tagagacaaa c-3Ј) was 13. Zurawski, S. M. et al. Definition and spatial location of mouse interleukin-2 residues that interact with synthesized by PE Applied Biosystems. Levels of IL21 mRNA were determined by analysis its heterotrimeric receptor. EMBO J. 12, 5113–5119 (1993). of total RNA samples using the one-step RT–PCR method (PE Applied Biosystems). RNA 14. Gong, J. H., Maki, G. & Klingemann, H. G. Characterization of a human cell line (NK-92) with from BHK cells expressing human IL21 was used to generate a standard curve for each phenotypical and functional characteristics of activated natural killer cells. Leukemia 8, 652–658 assay. Relative IL21 mRNA levels were determined by the standard curve method (user (1994). bulletin no. 2, PE Applied Biosystems) using 18S ribosomal RNA (TaqMan ribosomal 15. Mrozek, E., Anderson, P. & Caligiuri, M. A. Role of interleukin-15 in the development of human RNA control reagents protocol, PE Applied Biosystems) measurements to normalize. CD56+ natural killer cells from CD34+ hematopoietic progenitor cells. Blood 87, 2632–2640 (1996). 16. Yu, H. et al. Flt3 ligand promotes the generation of a distinct CD34+ human Purification of bone marrow HPCs progenitor that responds to interleukin-15. Blood 92, 3647–3657 (1998). 17. Kammer, W. et al. Homodimerization of interleukin-4 receptor ␣ chain can induce intracellular We prepared CD34+ cells from fresh human bone marrow mononuclear cell preparations signaling. J. Biol. Chem. 271, 23634–23637 (1996). (Poietics Technologies) using a CD34 magnetic selection system (Dynal). Mononuclear 18. Sugamura, K. et al. The interleukin-2 receptor ␥ chain: Its role in the multiple cytokine receptor cells were washed, resuspended in PBS/2% BSA, and incubated with anti-CD34 magnetic Њ + complexes and development in XSCID. Annu. Rev. Immunol. 14, 179–205 (1996). beads for 30 min at 4 C. After washing to remove non-rosetted cells, the CD34 cells were 19. Grewal, I. S. & Flavell, R. A. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16, released by incubation with polyclonal antibody to the Fab portion of the CD34 111–135 (1998). Њ monoclonal, 15 min at 37 C. Cells purified by this technique have an average purity of 20. Foy, T. M., Aruffo, A., Bajorath, J., Buhlmann, J. E. & Noelle, R. J. Immune regulation by CD40 and its 95%, as assessed by flow cytometry. ligand gp39. Annu. Rev. Immunol. 14, 591–617 (1996). 21. Callard, R. E., Armitage, R. J., Fanslow, W. C. & Spriggs, M. K. CD40 ligand and its role in X-linked NK cytotoxicity assays hyper-IgM syndrome. Immunol. Today 14, 559–564 (1993). Aliquots of bone marrow cultures were stained for flow cytometric analysis with 22. Robertson, M. J. & Ritz, J. Biology and clinical relevance of human natural killer cells. Blood 76, 2421– anti-CD3-FITC, anti-CD56-PE and anti-CD16-CyChrome monoclonal antibodies 2438 (1990). (PharMingen), and analysed on a FACSCalibur using CellQuest software (Becton 23. Whiteside, T. L., Vujanovic, N. L. & Herberman, R. B. Natural killer cells and tumor therapy. Curr. Top. Dickinson). We examined NK-mediated target cytolysis by standard 51Cr-release assay. Microbiol. Immunol. 230, 221–244 (1998). Effector cells were prepared from human CD34+ bone marrow cells cultured for 15– 24. Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. & Salazar-Mather, T. P. Natural killer cells in 21 days, or human CD56+ PBMCs cultured for 1–2 days, with various combinations of antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17, 189–220 cytokines, and then collected, washed, counted, mixed at various concentrations with (1999). 5 × 10351Cr-labelled K562 cells in 96-well round-bottomed plates, and incubated for 4 h 25. Williams, N. S. et al. Natural killer cell differentiation: insights from knockout and transgenic mouse at 37 ЊC. After incubation, half the supernatant from each well was collected for models and in vitro systems. Immunol. Rev. 165, 47–61 (1998). 26. Gubler, U. & Hoffman, B. J. A simple and very efficient method for generating cDNA libraries. Gene determination of radioactivity. The percentage of specific 51Cr release was calculated from 25, 263–269 (1983). the formula 100 × (X −Y)/(Z − Y), where X is 51Cr release in the presence of effector cells, 27. Morris, A. et al. in Animal Cell Technology (ed. Carrondo, M. J. T.) 529–534 (Kluwer Academic, The Y is the spontaneous release in the absence of effectors, and Z is the total 51Cr release from Netherlands, 1997). target cells incubated with 0.5% Triton X-100. 28. Stahl, N. et al. Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors. Science 267, 1349–1353 (1995). T-cell proliferation assays 29. Dzaki, K., Kikly, K., Michalovich, D., Young, P. R. & Leonard, W. J. Cloning of a type I cytokine For mouse assays, T cells from C57Bl/6 mice were isolated from pooled splenocytes and receptor most related to the IL-2 receptor ␤ chain. Proc. Natl Acad. Sci. USA 97, 11439–11444 (2000). lymph node lymphocytes and depleted of CD19+ B cells using a MACS column (Miltenyi Biotech). Whole thymocytes were also collected. Cells were cultured in triplicate with Acknowledgements −1 murine IL21 (0–30 ng ml ) in 96-well flat-bottomed plates (pre-coated with anti-CD3 We thank J. Lehner, J. Rosser, S. McMillen, S. Matthews, J. Coleman, S. Sexson, A. Adan, monoclonal antibody 2C11; PharMingen) for 3 d at 37 ЊC. Each well was pulsed with 1 ␮Ci 3 W. Lint, K. Hodges, L. Smith, J. Rodriguez, C. Noriega, B. Dedinsky, K. Kontor, J. Gray, [ H]thymidine on day 2, and plates were collected and counted 16 h later. For human M. Rixon and R. Hoffman for technical assistance; and P. McKernan, J. Kelly, S. Jaspers, assays, whole blood was collected from a healthy donor and depleted of red blood cells + G. McKnight, G. Rosenberg, K. Swiderek, D. Prunkard, B. Persson, W. Kindsvogel and using Ficoll Paque gradients (Amersham Pharmacia). To enrich for naive T cells, CD11b T. Bukowski for project advice. Nomenclature has been approved by HUGO. monocytes and CD19+ B cells were first depleted using a MACS column, and then positively selected for CD45RA+ cells. These T cells were cultured in triplicate with human Correspondence and requests for materials should be addressed to D.F. − IL21 (0–50 ng ml 1) in 96-well flat-bottomed plates (pre-coated where indicated with (e-mail: [email protected]). Sequences described have been submitted to GenBank under anti-human CD3 monoclonal antibody UCHT1; PharMingen) for 3–5 days at 37 ЊC. accession nos AF254067, AF254068, AF254069 and AF254070.

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