Natural Killer Cell Development and Function Precede αβ T Cell Differentiation in Mouse Fetal Thymic Ontogeny This information is current as James R. Carlyle, Alison M. Michie, Sarah K. Cho and Juan of September 24, 2021. Carlos Zúñiga-Pflücker J Immunol 1998; 160:744-753; ; http://www.jimmunol.org/content/160/2/744 Downloaded from References This article cites 64 articles, 37 of which you can access for free at: http://www.jimmunol.org/content/160/2/744.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 24, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Natural Killer Cell Development and Function Precede ab T Cell Differentiation in Mouse Fetal Thymic Ontogeny1 James R. Carlyle, Alison M. Michie, Sarah K. Cho, and Juan Carlos Zu´n˜iga-Pflu¨cker2 Natural killer (NK) cells mediate MHC-unrestricted cytolysis of virus-infected cells and tumor cells. In the adult mouse, NK cells are bone marrow-derived lymphocytes that mature predominantly in extrathymic locations but have also been suggested to share a common intrathymic progenitor with T lymphocytes. However, mature NK cells are thought to be absent in mouse fetal ontogeny. We report the existence of thymocytes with a mature NK cell phenotype (NK1.11/CD1172) as early as day 13 of gestation, approximately 3 days before the appearance of CD41/CD81 cells in T lymphocyte development. These mature fetal thymic NK cells express genes associated with NK cell effector function and, when freshly isolated, display MHC-unrestricted cytolytic activity in vitro. Moreover, the capacity of fetal thymic NK cells for sustained growth both in vitro and in vivo, in addition to their close phenotypic resemblance to early precursor thymocytes, confounds previous assessments of NK lineage Downloaded from precursor function. Thus, mature NK cells may have been inadvertently included in previous attempts to identify multipotent and bipotent precursor thymocytes. These results provide the first evidence of functional NK lymphocytes in mouse fetal ontogeny and demonstrate that NK cell maturation precedes ab T cell development in the fetal thymus. The Journal of Immunology, 1998, 160: 744–753. igration of fetal liver-derived hemopoietic precursors pression of the winged-helix nude (whn) gene (10, 11). However, http://www.jimmunol.org/ to the early fetal thymic rudiment occurs by day 12 of the development of NK cells is thymus independent, and these mouse gestation. The earliest hemopoietic cells to col- cells are present at normal to elevated levels in athymic nude mice M 3 onize the thymus, thymic lymphoid progenitors (TLPs), are mul- as well as in mice defective in the ability to rearrange genes en- tipotent lymphoid-committed precursors capable of giving rise to coding the Ag receptors (severe combined immune deficiency the B, T, thymic dendritic, and NK cell lineages but lack signifi- (SCID) and RAG-deficient mice) (12–15). Nonetheless, in addi- cant potential for myeloid and other hemopoietic cells (1, 2). Soon tion to peripheral sites for NK lymphopoiesis, NK cell develop- after exposure to the thymic microenvironment, these precursors ment can occur within the thymus, and these cells have been sug- rapidly lose B lymphoid potential and become committed to the gested to share a common thymic progenitor with T lymphocytes by guest on September 24, 2021 T/NK lineages (3). Subsequently, a wave of thymocyte differen- within the TLP population (1, 2, 16–19). Previous studies pro- tiation is established in the fetal thymus, marked by the ordered vided evidence for, but failed to define, a proposed bipotent thymic appearance of various developmental stages along the pathway to progenitor for T and NK cells (16, 20–22); additionally, these mature T cells (1, 2, 4). In fetal thymic ontogeny, the development reports did not outline the earliest stages of NK cell development of a defined subset of gd T cells precedes that of conventional ab in fetal ontogeny. Instead, these investigations demonstrated that T cells (5). However, the ordered appearance of NK cells remains various purified populations of thymocytes can give rise to either unknown within the context of thymocyte development, and func- T or NK cells under different in vitro or in vivo conditions (16, tional NK cells are thought to be absent in mouse fetal ontogeny. 20–22). Importantly, none of these studies addressed the possibil- NK cells are responsible for mounting MHC-unrestricted cytol- ity that NK cells derived from populations of precursor thymo- ysis of virus-infected and transformed cells (6–9). The develop- cytes, upon i.v. injection or in vitro culture, represented an out- ment of mature peripheral ab and gd T cells is thymus-dependent growth of an already existent subset of mature NK cells. and does not occur efficiently in mice that fail to develop a proper To investigate these questions, we analyzed day 13 to 15 mouse thymic epithelium (nu/nu, or nude mice) due to a defect in ex- fetal thymocytes, which contain precursors for all lymphoid lin- eages, but no mature ab T or B lymphocytes, and have an overall CD32/CD42/CD82 triple-negative (TN) phenotype (1, 2, 4). Re- Department of Immunology, University of Toronto, Toronto, Ontario, Canada cently, we reported the identification of a novel population of thy- Received for publication August 27, 1997. Accepted for publication October mocytes that serve as common committed progenitors for T and 3, 1997. NK lymphocytes (3). These precursors display both the NK1.1 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 molecule (NKR-P1C) of NK cells (23, 24) as well as the CD117 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (c-kit) molecule characteristic of hemopoietic precursors (17, 25– 1 1 This work was supported by grants from the Medical Research Council of Can- 27). We now report the identification of NK1.1 thymocytes with ada (MRC) and the National Cancer Institute of Canada (to J.C.Z.-P.), and by an a mature NK cell phenotype, lacking expression of CD117. These MRC studentship (to J.R.C) and an MRC scholarship (to J.C.Z.-P.). fetal thymic NK cells are evident as early as day 13 to 14 of 2 Address correspondence and reprint requests to Dr. J.C. Zu´n˜iga-Pflu¨cker, Depart- ment of Immunology, University of Toronto, Medical Sciences Building, Toronto, gestation, express genes associated with NK cell effector function, Ontario, M5S 1A8 Canada. E-mail address: jc.zuniga.pfl[email protected] and display MHC-unrestricted cytolytic activity directly ex vivo. 3 Abbreviations used in this paper: TLP, thymic lymphoid progenitor; FTLP, fetal Strikingly, despite the above functional characteristics and lack of TLP; FTNK: fetal thymic NK1.11 progenitor; FTOC: fetal thymic organ culture; CD117 surface expression, these mature NK cells possess a com- HSA: heat stable antigen; RAG: recombination-activating gene; SCF: stem cell posite phenotype similar to early precursor thymocytes, including factor; TN, triple negative; FT, fetal thymocytes; Sw, Swiss.NIH; dGuo, 1 2 1 deoxyguanosine. a CD44 (Pgp-1), CD25 (IL-2Ra), CD16/32 (FcgRIII/II), Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 745 1 2 1 1 2 CD24low (HSA), CD90 / (Thy-1), CD122 (IL-2Rb), CD2 / GCA TAC AGG; NKR-P1 (genes 2, 34, 40) 59, AAG GTA CAC ATT (LFA-2), CD52 (Ly-1), and TN phenotype. Many of these char- GCC AGA CAT; NKR-P1A (gene 2) 39, GTA GAC ATG GCT CAG TGA 9 acteristics have been previously used in an attempt to define bi- TTG; NKR-P1B (gene 34) 3 , GGA CAG GGG AGA GAT GGA GAT; NKR-P1C (gene 40, NK1.1) 39, GAG TCA ACG AAT GGA AAG GAA; potent T/NK precursor cells as well as early T lineage precursors Ly-49A 59, TTC TGC TTC CTT CTT CTG GTA; Ly-49A 39, TGT GTT (16, 22). We now demonstrate directly that fetal thymic NK cells CAA GGC AAG TTT AGA; Ly-49C 59, AGA CCA GAA AAA CGC are capable of substantial growth, both in vitro and in vivo, con- CAA CTT; Ly-49C 39, TTC ACT GTT CCA TCT GTC CTG; perforin 59, tributing significantly to the NK cell reconstitution potential of ATG TTC CCC AGT CGT GAG AGG; perforin 39, AAG GTG GAG TGG AGG TTT TTG; CD95L (Fas-ligand) 59, AAG AGA ACA GGA GAA precursor-phenotype thymocytes upon adoptive transfer. Thus, NK ATG GTG; CD95L 39, AGA TTT GTG TTG TGG TCC TTC. cell progenitor activity reported in previous studies that failed to exclude fetal thymic NK cells may have stemmed from an out- 51Cr-release cell-mediated cytotoxicity assay growth of pre-existing mature NK cells, in addition to bona fide Single-cell suspensions from freshly isolated day 15 fetal thymocytes from NK cell precursor activity. These results indicate that the early timed-pregnant C57BL/6 mice and adult RAG-22/2 mice were sorted for 2 1 fetal thymus is completely capable of supporting NK lineage de- a CD3 /CD90 (Thy-1) phenotype with or without NK1.1 expression.