90 | NOVEMBER 2001 | VOLUME 1 www.nature.com/reviews/immunol

H I G H L I G H T S

Haematopoiesis is the processwhereby mature blood cells of dis-tinct lineages are produced frommulitpotent haematopoietic stemcells (HSCs). Despite decades ofstudy, the origin of these HSCs dur-ing mammalian embryogenesis hasremained unknown. Reporting inImmunity, Cumano and colleaguesshow that, in mice, HSCs are gener-ated in an intraembryonic region,termed the splanchnopleura (Sp),and not from the extraembryonicyolk sac (YS) blood islands.

During vertebrate embryogene-sis, haematopoietic cells first appearwithin the extraembryonic YS. Asthe bone marrow and fetal liver (themain haematopoietic organs inmammals) require an input ofexogenous haematopoietic precur-sors to generate differentiated prog-eny, it was suggested that HSCsoriginate in the YS and later migrateto the fetal liver. However, theintraembryonic Sp region has alsobeen shown to have haematopoieticactivity.

The differentiation potential ofhaematopoietic precursors from theYS and Sp, separated from mouse

embryos before circulation, hasbeen investigated previously by theauthors. These experiments estab-lished that lymphoid potential isrestricted to precursors of intra-embryonic origin. In transplanta-tion experiments using normal,irradiated mice as recipients, nei-ther precursors from the YS or fromSp were able to reconstitute anadult haematopoietic compartment.As this might have been due totechnical limitations of the systemused, the authors developed a neworgan culture and cell-transfer sys-tem to investigate this further. Theprotocol involved culturing the Spand YS, again separated before theonset of blood circulation, for 4days (to ensure a sufficient numberof HSCs were available), beforetransplanting the precursors intorecombinase activating gene (Rag)–/–

or Rag –/– crossed with common γ-chain (Rag γc –/–) mice. Rag –/– micelack T and B cells (so reducing pos-sible competition between donorand recipient precursors) andRagγc–/– mice additionally lack nat-ural killer cells (which might killhaematopoietic precursors).

Natural killer (NK) cells are often called‘innate lymphocytes’ as they combine innaterecognition with the effector mechanisms ofT lymphocytes — cytokine secretion(interferon-γ; IFN-γ) and cytotoxic killing.However, in terms of responses to infection,NK cells might be more similar tolymphocytes than previously thought.A report from Dokun et al. in NatureImmunology indicates that, similar to T andB cells, antigen-specific NK cells selectivelyexpand in response to infection.

NK cells identify their targets byintegrating signals from their inhibitory andactivation receptors. But not all NK cells arecreated equal. Although their antigenreceptors are invariant, NK cell subsets carrydistinct complements of receptors.Previously, this group showed that, in mice,an activation receptor of the Ly49 family,Ly49H, confers specific protection against

murine cytomegalovirus (MCMV). Thisimplies that NK cells are capable of virus-specific recognition.

So, are Ly49H+ NK cells preferentiallyactivated by MCMV? The present studyassessed this in two ways — by scoring forIFN-γ production through intracellularcytokine staining and by measuring NK cellproliferation using a bromodeoxyuridine(BrdU)-incorporation assay. In the early phaseof the response to MCMV there is a burst ofIFN-γ production by NK cells, which peaks at36 hours. However, at 2 days post-infection,there are comparable percentages of Ly49H+

and Ly49H– NK cells producing IFN-γ, andthere was no difference in the proliferation ofthe two subsets. By contrast, the authors foundthat by day 6 post-infection, there has been anoutgrowth of Ly49H+ NK cells, and these cellsproliferate to a much greater degree than theirLy49H– counterparts.

But is this preferential proliferationactually driven by Ly49H recognition ofMCMV? Vaccinia virus infection, whichinduces NK cell activation, did not cause theselective proliferation of Ly49H+ cells,indicating this might indeed be an MCMV-specific response. In addition,administration of Ly49H antibodies wasshown to inhibit bulk NK cell expansion inresponse to MCMV, which shows thatLy49H has a direct role in MCMV-triggeredNK cell activation.

This study provides a new model of NK cellactivity in viral infection. Yet it remains to beseen whether this delayed, preferentialproliferation of NK cells is itself importantfor antiviral immunity.

Jen BellReferences and links

ORIGINAL RESEARCH PAPER Dokun, A. O. et al. Specificand nonspecific NK cell activation during virus infection. Nature Immunol. 2, 951–956 (2001)FURTHER READING Cerwenka, A. & Lanier, L. L. Naturalkiller cells, viruses and cancer. Nature Rev. Immunol. 1, 41–49(2001)WEB SITEWayne Yokoyama’s lab: http://www.hhmi.org/research/investigators/yokoyama.htm

Natural killer selection

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Stem cell origins

H A E M ATO P O I E S I S

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NATURE REVIEWS | IMMUNOLOGY VOLUME 1 | NOVEMBER 2001 | 91

The ‘missing-self ’ hypothesis, formulated byKlaus Kärre and colleagues in 1986, proposed that natural killer (NK) cells seek out and destroy cells that have lost expression of majorhistocompatibility complex (MHC) class Iantigens. Two recent papers, published in Natureand in Proceedings of the National Academy ofSciences, now show that NK cells can rejecttumour cells that express ligands for the activatingNK receptor NKG2D, despite the expression ofMHC class I molecules by the tumour cells.

The formulation of the missing-self hypothesispredicted the existence of receptors on NK cellsthat inhibit their activity and which recognizeMHC class I molecules. Many of these receptorshave now been identified. Recent work has alsoidentified several activating NK receptors,including the lectin-like molecule NKG2D, whoseengagement provides dominant activating signalsto the NK cell. Previous work by Tom Spies’ groupshowed that NK cells can kill NKG2D ligand-expressing cells in vitro. The mouse ligands forNKG2D are retinoic acid early inducible-1 (Rae-1)and H60, which are expressed by some tumourcells, but not by normal adult cells.

Both groups looked for direct evidence tosupport the idea that tumour cells ectopicallyexpressing ligands for NKG2D could stimulateantitumour responses by NK cells. The Rauletgroup used a retroviral expression system toexpress Rae1β and H60 in three mouse tumourcell lines that express MHC class I molecules —EL4 thymoma cells, RMA T-cell lymphoma cells(which were used in the original Kärre study) andB16-BL6 melanoma cells. Transduced EL4 andB16-BL6 cells that were injected subcutaneouslyinto recipient mice were rapidly and completely

rejected. Tumour cells were rejected in wild-typemice depleted of CD8+ T cells and in Rag –/– mice(which lack T and B cells), but grew in Rag –/– micedepleted of NK cells, indicating that conventionalNK cells are responsible for the rejection. Rae1β-or H60-transduced RMA cells were also rejectedin wild-type mice, but rejection required bothCD8+ T cells and NK cells.

The Lanier group also used the RMA cells toinvestigate NK cell responses. RMA cells stablytransfected with Rae1γ or Rae1δ were injectedintraperitoneally into recipient mice. Miceinjected with mock-transfected cells developedtumours and died, whereas mice challenged withtransfected cells rejected tumour cells in an NK-cell dependent manner.

So, NK cells can reject tumour cells expressingNKG2D ligands, despite MHC class I expression.But do mice primed with NKG2D ligand-expressing tumour cells develop an adaptive T-cell response against subsequent challenge bynon-transduced parental tumour cells? This iswhere the results from the two groups differ.Raulet and colleagues found that NKG2D ligand-negative tumour cells were rejected by micepreviously challenged with transduced tumourcells, and that this was a CD8+ T-cell-dependentprocess. By contrast, Lanier and colleagues foundthat mice that had rejected Rae1γ-transfectedRMA cells were unable to reject parental tumourson re-challenge.

These results show that NK cells can, and do,participate in rejection of MHC class-I bearingtumour cells and, although there are somediscrepancies in the results, this approach mightbe effective for tumour vaccine development.

Elaine BellReferences and links

ORIGINAL RESEARCH PAPERS Diefenbach, A., Jensen, E. R.,Jamieson, A. M. & Raulet, D. H. Rae1 and H60 ligands of the NKG2Dreceptor stimulate tumour immunity. Nature 413, 165–171 (2001) |Cerwenka, A., Baron, J. L. & Lanier L. L. Ectopic expression of retinoicacid early inducible-1 gene (RAE-1) permits natural killer cell-mediatedrejection of a MHC class I-bearing tumor in vivo. Proc. Natl Acad. Sci.USA 98, 11521–11526 (2001)WEB SITESLewis Lanier’s lab: http://cc.ucsf.edu/people/lanier_lewis.htmlDavid Raulet’s lab: http://mcb.berkeley.edu/labs/raulet/

Targetingtumour cells

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Ragγc–/– mice injected with YS cellsgenerated donor-derived myeloidcells, but this reconstitution was tran-sient, and after 3 months donor-derived progeny were no longerobserved. No lymphocytes were everdetected in these mice. Therefore,YScells cannot generate lymphocytes,but can provide short-term myeloidreconstitution. By contrast, after injec-tion of Sp-derived cells into Ragγc–/–

recipients, donor-derived myeloidcells, as well as B and T cells were gen-erated. These myeloid and lymphoidcells were still present 8 months afterinjection, indicating that Sp-derivedpercursors can provide long-termreconstitution of recipient mice.

The authors conclude that theonly cells capable of adult long-termhaematopoiesis are from the Spregion. So, during mouse embryo-genesis, HSCs are generated intra-embryonically and do not derivefrom the YS.

Jenny Buckland

References and linksORIGINAL RESEARCH PAPER Cumano, A. et al.Intraembryonic, but not yolk sac hematopoieticprecursors, isolated before circulation, providelong-term multilineage reconstitution. Immunity15, 477–485 (2001)

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