Development of innate CD4؉ ␣-chain variable segment 24 (V␣24) natural killer T cells in the early fetal thymus is regulated by IL-7

Johan K. Sandberg*†‡, Cheryl A. Stoddart*, Fabienne Brilot*, Kimberly A. Jordan*, and Douglas F. Nixon*

*Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94103; and †Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, 14186 Stockholm, Sweden

Edited by Phillipa Marrack, National Jewish Medical and Research Center, Denver, CO, and approved March 17, 2004 (received for review September 17, 2003) Natural killer (NK) T cells are innate CD1d-restricted immune cells Classical MHC-restricted human T cells go through a complex involved in regulation of immune tolerance, tumor immunity, and process of maturation and selection in the thymus before they immunity to infectious pathogens. Human ␣-chain variable gene can join the peripheral repertoire (reviewed in ref. 29). The segment 24 (V␣24) NK T cells exist in the periphery as two earliest thymic precursors express CD34 and lack CD1a, and the functionally distinct subsets: one CD4؉ and one CD4؊ subset. transition to a CD34ϩ CD1aϩ stage is strongly associated with T However, the developmental pathway of human V␣24 NK T cells cell commitment (30). Through several discrete stages, these is not well understood. Here, we show that V␣24 NK T cells develop precursors develop into CD4ϩ/ϩ or CD8ϩ/ϩ cells, with rear- in the fetal thymus. The relative number of intrathymic NK T cell ranged TCR␣ and TCR␤ , which express the specific TCR precursors decline in a linear manner with gestational age, and and are subject to induction of self-tolerance through positive they are very rare in the neonatal thymus, indicating that these and negative selection before being allowed to mature into CD4ϩ cells preferentially develop in the early fetal thymus. Their restric- or CD8ϩ cells and leave the thymus (reviewed in refs. 29 and 31). tion element, CD1d, is expressed by a vast majority of thymocytes. Survival, proliferation, and expansion of thymocytes are sup- A majority of intrathymic V␣24 NK T cell progenitors are CD4؉, ported by IL-7 (32), which is produced by stromal cells in the whereas a minority are CD4͞8؉/؉. CD4؉ V␣24 NK T cell precursors thymus and appears to play a role at several stages of T cell show features of mature NK T cells, such as high levels of their development (33, 34). semiinvariant T cell receptor and CD3 and some expression of In mice, CD1d-restricted V␣14 NK T cells depend on the CD161, whereas the CD4͞8؉/؉ precursors seem less mature. The thymus for their development (35–38). However, the site and IL-7 shows a biphasic effect on V␣24 NK T cell progenitors pathway of development and maturation of human V␣24 NK T in fetal thymic organ culture, with high doses driving proliferation cells are unknown. Investigation of these pathways and sites is of immature CD161- progenitors and low doses supporting survival important both for the basic understanding of the biology of and maturation. Thus, the data demonstrate that human V␣24 NK these cells and for potential therapeutic interventions to restore T cells of the CD4؉, but not the CD4؊, subset develop in the early or improve V␣24 NK T cell function in diseases in which these fetal thymus. Furthermore, data suggest an intrathymic pathway cells are lost or impaired. Here, we have found that precursors of CD4؉ V␣24 NK T cell development that is regulated by IL-7. of the CD4ϩ V␣24 NK T cell subset can be identified in the early human fetal thymus. Their relative numbers decrease in an uman natural killer (NK) T cells are innate immune cells inverse linear correlation with gestational age, and they appear that express the NK cell marker CD161 and possess a to be very rare in the neonatal thymus. Furthermore, we have H ␣ semiinvariant T cell receptor (TCR) with uniform use of ␣-chain studied the developmental stages of V 24 NK T cell in the variable gene segment 24 (V␣24), paired with ␤-chain variable thymus, the response to IL-7 in fetal thymus organ culture gene segment 11 (V␤11) (1–3). They recognize glycolipids (FTOC), and their maturational stage in relation to NK T cells presented by CD1d expressed by presenting cells such as in cord and peripheral blood. The data indicate that human ␣ ϩ Ϫ dendritic cells, and they respond by producing , includ- V 24 NK T cells of the CD4 , but not the CD4 , subset develop ing IFN-␥, tumor necrosis factor ␣, and IL-4 (1, 4, 5). The early in the fetal thymus, a finding compatible with early seeding of these cells to peripheral sites during fetal development. primary role of CD1d-restricted NK T cells is thought to be the ϩ regulation of immune responses, and several reports (6–11) Furthermore, data suggest an intrathymic pathway of CD4 ␣ support a role for NK T cell dysfunction in the pathogenesis of V 24 NK T cell development that is regulated by IL-7. autoimmune diseases and cancers, as well as in murine models Methods of autoimmunity (12–15). They are lost in HIV-1 infection in (16–18) and lymphocytic choriomeningitis virus infec- Human Subjects and Tissues. Heparinized samples of whole blood tion in mice (19), and they can be activated to participate in from healthy donors and cord blood were obtained after in- defense against hepatitis B virus and cytomegalovirus in mice formed consent. Peripheral blood mononuclear cells were iso- ͞ (20, 21). lated by Ficoll Paque Plus density-gradient centrifugation (Am- Human V␣24 NK T cells can be subdivided into CD4ϩ and ersham Biosciences). Fetal thymus tissue was obtained from CD4Ϫ subsets that appear to be both functionally and pheno- Advanced Bioscience Resources (Alameda, CA). The study was typically distinct, with differences in homing receptors and cytokine profiles (16, 22–26). The balance between these two subsets is most probably important for the immunoregulatory This paper was submitted directly (Track II) to the PNAS office. ␣ Abbreviations: ␣-GalCer, ␣-galactosylceramide; APC, allophycocyanin; FTOC, fetal thy- role of the V 24 NK T cell compartment. Another interesting mus organ culture; NK, natural killer; TCR, T cell receptor; V␣24, ␣-chain variable gene feature of NK T cells is their uniform memory T cell-like segment 24. phenotype with expression of CD45RO and CD28 and frequent ‡To whom correspondence should be addressed at: Center for Infectious Medicine, De- expression of CCR5 (27, 28). This phenotype may be linked to partment of Medicine, Karolinska Institutet, Huddinge University Hospital F59, 14186 their role as innate immune cells, lending them the ability to Stockholm, Sweden. E-mail: [email protected]. respond rapidly to CD1d-restricted stimuli. © 2004 by The National Academy of Sciences of the USA

7058–7063 ͉ PNAS ͉ May 4, 2004 ͉ vol. 101 ͉ no. 18 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0305986101 Downloaded by guest on October 2, 2021 based on protocols approved by the local institutional review board.

Flow Cytometry. The frequency of V␣24 NK T cells in peripheral blood, cord blood, and thymocyte suspensions, as well as the expression of surface markers, were assessed by four-color flow cytometry. The following reagents were used: anti-V␣24 phy- coerythrin and anti-V␤11 FITC and biotin conjugates, and anti-IL-7R␣ FITC were obtained from Immunotech (Marseilles, France); streptavidin–allophycocyanin (APC), anti-CD3 peri- dinin–chlorophyll , anti-CD4 APC, anti-CD8 peridinin– chlorophyll protein, anti-CD45RO APC, anti-CD62L APC, anti-CD1d phycoerythrin, anti-CCR5 APC, anti-CD161 FITC, anti-CD161 APC, anti-HLA class 1 APC, and anti-HLA-DR APC were obtained from PharMingen. Single-cell suspensions of thymocytes were stained for 30 min on ice in PBS buffer supplemented with 5% FCS. Samples were analyzed on a FACSCalibur (Becton Dickinson) instrument by using CELLQUEST software. We collected 1 ϫ 106 lymphocyte events per sample.

FTOC. Fetal thymus tissue was dissected into pieces of Ϸ2mm3 so that every piece was likely to contain cortex and have similar cellularity. Three pieces of tissue were placed on sterile poly- carbonate membrane filters (Millipore) placed on gel foam sponge (Upjohn) rafts in 700 ␮l of Yssel’s medium containing 1% human serum (Gemini Biological Products, Calabasas, CA) in 24-well plates. Graded doses of IL-7 (0–250 ng͞ml) (R & D Systems) were added to the culture medium. Medium and cytokines were changed on days 1 and 3. On day 7, thymus fragments were harvested individually with a sterile micropipette tip. A single-cell suspension was made by placing the fragment into a sterile nylon-mesh bag, submerging the bag in PBS with 2% FCS in a 60-mm tissue culture dish, and dispersing the tissue between the nylon layers with forceps. The medium from cultures was collected separately and analyzed for cellular content. Cells that were recovered from tissue and medium were Fig. 1. Detection of V␣24 NK T cell progenitors responsive to ␣-GalCer in stained for flow cytometry in a 96-well V-bottom plate. human fetal thymus depends on gestational age. (a) Flow cytometric assess- ment of V␣24ϩ V␤11ϩ NK T cells in adult peripheral blood and fetal and ␣ postnatal thymus. (b) CD1d expression in fetal thymocytes, with data gated on V 24 NK T Cell Expansion Culture. FTOC was performed as de- Ϫ Ϫ CD1d-high and CD3 (I), CD1d-high and CD3-low (II), and CD1d and CD3-high scribed above, and cells leaving the tissue were collected in the (III). (c)V␣24ϩ V␤11ϩ cells collected in FTOC supernatant after culture expand ϫ 6 surrounding medium at day 7. Next, we cultured 1 10 cells for in response to stimulation with ␣-GalCer (␣GC) over an 8-day culture. (d) The 8 days in Yssel’s medium supplemented with 1% human serum frequency of V␣24ϩ V␤11Ϫ non-NK T cells is shown as an internal-reference in the presence of 1 ϫ 105 irradiated and ␣-galactosylceramide population. V␣24ϩ V␤11ϩ thymocytes were enumerated in thymi by flow (␣-GalCer)-pulsed (1 ␮g͞mlfor1hat37°C) CD1d-transfected cytometry at gestational ages of 13–20 weeks (n ϭ 18). Solid line shows the DT407 cells. The culture was analyzed for V␣24 NK T cells by linear regression of all data (R ϭ 0.74; P Ͻ 0.001). flow cytometry on days 4 and 8.

Statistical Analysis. Data were analyzed by descriptive statistics, or negative subset, in particular by CD4͞8ϩ/ϩ cells but also in paired t test, and linear regression by using SIGMASTAT software small numbers of CD4ϩ CD3Ϫ/Ϫ cells and CD4͞8Ϫ/Ϫ CD3Ϫ cells

(SPSS, Chicago). (Fig. 1b). IMMUNOLOGY The dual expression of V␣24 and V␤11 TCR segments Results accurately identifies CD1d-restricted NK T cells that recognize V␣24 NK T Cell Progenitors in the Human Fetal Thymus. Data pre- ␣ ␣ and respond to the prototypical CD1d-presented antigen -Gal- sented in a recent study (39) indicated that V 24 NK T cells were Cer in human peripheral blood (24, 40, 41). To demonstrate that not present in the postnatal human thymus. This finding was ϩ ϩ V␣24 V␤11 thymocytes in the fetal thymus respond to interpreted to suggest that these cells develop in a thymus- ␣-GalCer, we used a FTOC system, in which pieces of tissue were independent manner (39). An alternative explanation is that V␣24 NK T cells are produced at a specific time during cultured for 7 days, and cells that had exited the tissue were harvested from the surrounding medium. These FTOC tissue development or even late in postnatal life. To investigate these ␣ possibilities, we obtained fetal, postnatal, and adolescent thymic ‘‘emigrants’’ were enriched in V 24 NK T cells as compared with tissue and analyzed these tissues for the presence of V␣24ϩ the thymic tissue, and the cells expanded 50-fold in response to ϩ ␣ V␤11 NK T cells by flow cytometry. By this assay, we could -GalCer over an 8-day culture (Fig. 1c). In contrast, the ␣ ϩ ␤ Ϫ confirm that V␣24 NK T cells were very rare or absent from the frequency V 24 V 11 non-NK T cells in the culture did not postnatal and adolescent thymi (Fig. 1a). In contrast, V␣24ϩ change in response to ␣-GalCer. V␤11ϩ cells were clearly detectable in the fetal thymus at a Together, these data demonstrate that the V␣24 NK T cell gestational age of 13 weeks (Fig. 1a). CD1d, the ligand of V␣24 ligand CD1d is expressed in the thymus and that V␣24 NK T cells NK T cells, was highly expressed by thymocytes in the CD3 low are generated in the human fetal thymus.

Sandberg et al. PNAS ͉ May 4, 2004 ͉ vol. 101 ͉ no. 18 ͉ 7059 Downloaded by guest on October 2, 2021 Fig. 2. Identification of CD4ϩ and CD4͞8ϩ/ϩ V␣24 NK T cell progenitors in human fetal thymus. (a) Detection of V␣24 NK T cell progenitors in V␣24͞V␤11 TCR low and high stages by flow cytometry. (b) Assessment of CD4, CD8, and CD161 expression in TCR low and TCR high subsets. One representative of 16 experiments is shown.

V␣24 NK T Cell Progenitors Appear Early During Development. The striking difference in V␣24 NK T cell progenitor frequency between fetal and postnatal thymus prompted a more careful analysis of these cells in the fetal stage of development. We obtained 18 fetal thymi from gestational ages of 13–20 weeks. Strikingly, V␣24 NK T progenitors were most frequent in the early tissue specimens, and numbers showed an inverse linear Fig. 3. Expression of maturation markers in the developing V␣24 NK T cell correlation with gestational age over the 13- to 20-week span progenitors. Flow cytometric assessment of maturation markers and func- (R ϭ 0.74; P Ͻ 0.001) (Fig. 1d). The data indicate that tional receptors CD3 and CD161 (a), as well as CD62L, CD45RO, CCR5, and ͞ ϩ/ϩ ϩ development of V␣24 NK T cells preferentially occurs in an early HLA-A,B,C (b)inCD48 and CD4 stages of NK T cell development, as phase of thymic development. However, these results do not compared with the overall thymocyte population, are shown. One represen- tative of 10 experiments is shown. exclude the possibility of V␣24 NK T cell development in the postnatal thymus. CD3-low, and CD161Ϫ immature V␣24 NK T cell progenitors to Stages of V␣24 NK T Development and Maturation. These findings ϩ ͞ ␣ mature CD4 , TCR CD3-high cells that start to express CD161 prompted us to identify stages of intrathymic V 24 NK T and CD62L before they exit into the periphery. progenitor maturation. Careful examination of V␣24 and V␤11 flow cytometry dot plots revealed that cells were either high or Biphasic Regulation of V␣24 NK T Cell Progenitors by IL-7. IL-7 low in their expression of the invariant TCR (Fig. 2a). Cells with ϩ supports survival, proliferation, and expansion of thymocytes in high expression of the invariant TCR were predominantly CD4 , ϩ ϩ both humans and mice (32–34). Staining for IL-7R␣ clearly and low TCR expression was associated with a CD4͞8 / showed that intrathymic V␣24 NK T cell progenitors, as well as phenotype (Fig. 2b). These two populations would fit into one ϩ ϩ ϩ the vast majority of total thymocytes, expressed IL-7R␣ (Fig. immature CD4͞8 / stage and one more mature CD4 stage 4a). We, therefore, investigated the effects of this cytokine on similar to those identified for regular MHC-restricted T cells ␣ (29). Of the invariant TCR-high NK T cell progenitors, 20–30% V 24 NK T cell development in an FTOC system. Pieces of fetal ␣ thymic tissue were cultured for 7 days in the presence of 0–250 expressed CD161, a marker of mature V 24 NK T cells in the ͞ periphery (Fig. 2b). Expression of CD161 did not show any ng ml IL-7, and thymocyte and NK T cell numbers were correlation with gestational age (data not shown). CD161 was determined by total cell counts and flow cytometry. Low doses ϩ ϩ ͞ not expressed in the CD4͞8 / stage, again suggesting that these in the 0–5ngml range had moderate effects on the total ϩ ϩ ␣ cells are less mature (Fig. 2b). CD4͞8 / NK T cell progenitors absolute number of V 24 NK T cell progenitors retrieved from expressed intermediate levels of CD3, whereas the CD4ϩ cells the tissue cultures, whereas high-dose IL-7 treatment increased ␣ expressed high levels (Fig. 3a). Expression of CD161 was found the number of V 24 NK T cell progenitors in the tissue at day only in CD4ϩ NK T cell progenitors (Fig. 3a). Similarly, HLA 7(P ϭ 0.03; Fig. 4b) and resulted also in increased V␣24 NK T class I and to some extent CD62L were up-regulated in the CD4ϩ cell progenitor numbers in the surrounding medium (P ϭ 0.03; stage (Fig. 3b). The RO isoform of CD45 remained high Fig. 4c). However, the percentage of V␣24 NK T cell progenitors throughout both double-and single-positive stages, and CCR5 retrieved from the media fraction of cultures was the highest at expression was undetectable (Fig. 3b). These data support a 5 and 25 ng͞ml IL-7 (P ϭ 0.004 and P ϭ 0.001, respectively), model of intrathymic maturation from the CD4͞8ϩ/ϩ, TCR͞ suggesting that these doses preferentially supported survival of

7060 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0305986101 Sandberg et al. Downloaded by guest on October 2, 2021 Fig. 4. Biphasic regulation of V␣24 NK T cell development by IL-7. (a) Expression of IL-7 receptor ␣ in the thymus. Analysis of NK T cell progenitor numbers and phenotype after 7 days FTOC with 0–250 ng͞ml IL-7. The number of V␣24 NK T cell progenitors recovered in tissue is shown in b, and the medium surrounding the tissue (c) was quantified by cell counting and flow cytometric analysis. (d) Percentage of V␣24 NK T cell progenitors in the medium. Percentages of NK T cell precursors expressing a CD161ϩ (e) and a CD4͞8ϩ/ϩ phenotype (f) after a 7-day FTOC with graded doses of IL-7 are given. Figures represent data from six independent experiments. *, P Ͻ 0.05; **, P Ͻ 0.005, compared with culture without IL-7, as determined by paired t test.

these cells over the general thymocyte population (Fig. 4d). We CCR5 at high levels. These data suggest that V␣24 NK T cells next analyzed expression of the maturation marker CD161 on differentiate further in the periphery after leaving the thymus, and the developing TCR-high V␣24 NK T cell progenitors in the that the CD4Ϫ V␣24 NK T cells frequently found in peripheral FTOC. Low doses of IL-7 promoted up-regulation of CD161 blood develop at an alternative site, at another developmental (P ϭ 0.03 and P ϭ 0.006), whereas the 250 ng͞ml dose resulted stage, or under particular circumstances. in less CD161 expression as compared with untreated control FTOC (P ϭ 0.02) (Fig. 4e). In line with this shift in V␣24 NK T Discussion cell progenitor phenotype, high-dose IL-7 treatment shifted the CD1d-restricted V␣24 NK T cells are innate immune cells with phenotype of these progenitors toward an immature CD4͞8ϩ/ϩ the capacity to influence various diseases. Loss or dysfunction of phenotype (P ϭ 0.0; Fig. 4f). Thus, intrathymic V␣24 NK T cell these cells occurs in autoimmune conditions (6–8, 10), cancer progenitors respond to IL-7 in a biphasic manner: high doses of (9, 11), and viral infections (16–18, 42). Investigation of the IL-7 promote proliferation of these cells and maintain them in pathways and sites for development and differentiation of human an immature state, whereas low doses of IL-7 support survival V␣24 NK T cells is, therefore, important both for the basic and promote expression of markers associated with mature V␣24 understanding of the biology of these cells and for potential NK T cells. therapeutic interventions to restore or improve V␣24 NK T cell IMMUNOLOGY function in certain diseases. We have shown here that human Continued Maturation of V␣24 NK T Cells After Leaving the Thymus. CD4ϩ V␣24 NK T cells develop in the thymus. NK T cell Peripheral blood V␣24 NK T cells in adults contain both CD4ϩ and progenitors are most frequent in the thymus at an early gesta- CD4Ϫ subsets that are functionally distinct (16, 22–26). However, tional age, but their relative number declines with age, and they the intrathymic V␣24 NK T progenitors are almost exclusively are very rare or absent in postnatal thymus. The size of the CD4ϩ in their CD3͞TCR-high stage, and virtually no CD4͞8Ϫ/Ϫ thymus, and thus the total cellularity, increase sharply during progenitors can be observed (Fig. 2b). This observation prompted development of the fetus. The higher percentage of NK T cell a more careful comparison of the intrathymic NK T progenitors, progenitors during early fetal age and the subsequent drop in and NK T cells in cord blood and in adult peripheral blood frequency are, therefore, probably a reflection of the timing mononuclear cells (Fig. 5). Cord blood V␣24 NK T cells were rather than an absolute measure of NK T cell development, predominantly CD161ϩ albeit not to the same extent as in adult indicating that there is preferential development of V␣24 NK T peripheral blood. However, the cord blood V␣24 NK T cells were cells in the early fetal thymus. An early seeding of immunoregu- similar to intrathymic progenitors in that they were all CCR5Ϫ and latory V␣24 NK T cells from the thymus to the developing CD4ϩ, whereas many peripheral blood NK T cells were CD4Ϫ and peripheral makes sense from a functional per- CCR5ϩ.V␣24 NK T cells of all compartments were CD45ROϩ, spective in that their early presence could be involved in estab- indicative of the memory T cell-like phenotype, but most of the lishing immunological tolerance at peripheral sites. In line with peripheral blood V␣24 NK T cells lacked CD62L and expressed this notion, murine V␣14 NK T cells are involved in regulating

Sandberg et al. PNAS ͉ May 4, 2004 ͉ vol. 101 ͉ no. 18 ͉ 7061 Downloaded by guest on October 2, 2021 proliferation, as well as thymocyte differentiation, respectively (44). In addition, IL-7R␣ associates with the common ␥-chain that is required to initiate expression of the CD161 homologue NK.1.1 in the mouse thymus (45). Thus, the effects of IL-7 on thymic development are complex and involve several signaling pathways that together may account for the dose-dependent effects of IL-7 on V␣24 NK T cell development. Mature intrathymic progenitors and cord blood V␣24 NK T cells are almost exclusively CD4ϩ. In adult peripheral blood, however, close to 50% of NK T cells are CD4Ϫ with no or low expression of CD8. This observation leaves several possibilities: the CD4Ϫ subset may be a separate lineage that is extrathymi- cally derived, they may develop in the thymus at a later devel- opmental stage, or the CD4Ϫ V␣24 NK T cells may be derived from the CD4ϩ subset. If the last possibility is correct then the CD4Ϫ NK T cell could be a late-stage-activated NK T cell. This model is supported by the effector-like phenotype of CD4Ϫ NK T cells, with high levels of CD11a and CCR5 and loss of CD62L, and by the recent observation that human subjects with an expanded V␣24 NK T cell compartment have a preferential expansion of CD4Ϫ NK T cells (28). If this model is correct, then CD4 could be viewed as a V␣24 NK T cell differentiation marker that is lost upon maturation into a late-stage effector-type cell. However, other possibilities clearly exist, and further investiga- tions are required to determine the developmental origin of CD4Ϫ V␣24 NK T cells. Development of human V␣24 NK T cells in the thymus displays both similarities and differences to V␣14 NK T cell Fig. 5. Shifts in V␣24 NK T cell phenotype after leaving the thymus. Expres- development in the mouse. One major difference is that intra- sion of CD161, CD62L, CD45RO, and CCR5 in NK T cell progenitors defined by thymic NK T cell progenitors are frequent in the adult mouse V␣24 and V␤11 in the TCR high CD4ϩ intrathymic stage in cord-blood V␣24 NK (35–37), whereas in humans, the V␣24 NK T cell progenitors are T cells and adult peripheral blood V␣24 NK T cells, as determined by four-color found in the early fetal thymus and are very hard to find in the flow cytometry, are shown. postnatal thymus. The two species, thus, appear to be different in the timing of NK T cell development. Another striking difference is that both CD4ϩ and CD4Ϫ NK T cell progenitors tolerance to self expressed in the eye (43) and the are found in the mouse thymus (35, 36), whereas we detect only pancreas (12–15). The NK T cell ligand CD1d is ubiquitously ϩ ϩ ϩ the CD4 subset in the human fetal thymus. The site or timing expressed by CD4͞8 / thymocytes and most likely functions as Ϫ of CD4 NK T cell maturation or development, thus, seems to the selecting ligand for the developing NK T cell progenitors, differ between the two species. reminiscent of what is believed to occur in the mouse (37). ϩ Here, we have shown that human CD4 V␣24 NK T cells Because V␣24 NK T cells are defined by their TCR, the develop in the thymus. We have observed that V␣24 NK T cell earliest stage of development at which thymocytes destined to progenitors are most frequent in the thymus at an early gestational become NK T cells can be identified is in the CD3͞TCR low stage, but their frequency declines with age, and they are rare in the stage. Our data suggest that NK T cell progenitors develop Ϫ ϩ ϩ ϩ postnatal thymus. Surprisingly, the CD4 V␣24 NK T cells com- through CD4͞8 / and CD4 stages similar to the overall monly found in adult peripheral blood are not found in the fetal pathway of classical T cells (29). However, single-positive NK T ␣ progenitors are clearly different in that they are uniformly thymus. IL-7 regulates V 24 NK T cell development in a biphasic ϩ manner such that high doses promote proliferation of these cells in CD4 . In addition, they appear to initiate CD161 expression in ͞ ϩ/ϩ what may be the final maturation step before leaving the thymus. an immature CD4 8 state, whereas low doses can support ␣ survival and drive expression of the CD161 marker characteristic of Unlike classical T cells, V 24 NK T cells do not switch CD45 ␣ isoform expression from RO to RA to obtain the classical naı¨ve mature V 24 NK T cells. These results shed on the develop- CD45RAϩ CD62Lϩ phenotype, but maintain the CD45ROϩ mental pathway of CD1d-restricted NK T cells and are important ϩ both for the understanding of the basic biology of these cells and for CD62L central memory T cell-like phenotype in cord blood. ␣ IL-7 plays a key role in the development of T cells in both potential therapeutic interventions to restore or improve V 24 NK humans and mice (32–34). We find that IL-7 regulates V␣24 NK T cell function in certain diseases. T cell progenitors in FTOC in a biphasic manner such that high We thank Kirin Brewery Company, Japan, for the kind gift of the doses, likely to occur only in the local micro environment, ␣ promote proliferation of these cells in an immature CD4͞8ϩ/ϩ -GalCer; Karen Beckerman for access to cord blood samples; Jose Rivera for technical assistance; and Alf Grandien and Maire Quigley for state. In contrast, low doses of IL-7 rather support survival and careful reading of the manuscript. This work was supported by National promote expression of the maturation marker CD161. IL-7R␣ ͞ Institutes of Health Grants AI52731 and AI54018, the Elizabeth Glaser signals via both the phosphatidylinositol 3-kinase protein kinase Pediatric AIDS Foundation, the Swedish Research Council, and the B pathway and a signal transducer and activator of transcription Swedish Foundation for Strategic Research. D.F.N. is an Elizabeth 5-dependent pathway, which regulate thymocyte survival and Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.

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