CD16 Cross-Linking Blocks Rearrangement of the TCRb Locus and Development of ab T Cells and Induces Development of NK Cells from Thymic Progenitors Scott K. Durum,1* Chong-Kil Lee,* Theresa M. Geiman,† William J. Murphy,† and Kathrin Muegge† Mouse thymocytes normally develop into T lymphocytes, but the embryonic thymus also contains precursor cells capable of developing into NK cells. Here, we describe conditions that induce pro-T cells to develop into NK cells. CD16 is expressed on thymic pro-T cells. We observed that CD16 cross-linking during culture of embryonic thymic organs suppressed rearrangement of the TCRb locus (but did not inhibit TCRg locus rearrangement). Rearrangement of the TCRb locus is normally required for development to the CD41CD81, and this development was also suppressed by CD16 cross-linking. The ability of CD16 crosslinking to block abT cell development was not attributable to toxic effects, but rather was accompanied by promotion of development into NK cells, identified based on molecular and functional criteria. These results suggest that common lymphoid precursors can respond to environmental signals to commit to the abT vs NK developmental pathways. The Journal of Immunology, 1998, 161: 3325–3329.
T
cell precursors are initially produced outside the thymus in hemopoietic tissues, including the yolk sac and fetal liver of the embryo and later in the bone marrow of the adult. Very small numbers of these precursors (,100; see Ref. 1) arrive in the thymus and begin a complex process involving gene rearrangements, proliferation, differentiation, and negative and positive selection (for reviews see Ref. 2). The early intrathymic precursors have been extensively characterized in terms of surface phenotype; however, a number of questions remain regarding their degree of commitment to the T cell lineage (reviewed in Ref. 3). T lymphocytes and NK cells share a number of similarities in surface markers, and it has been suggested that they might develop from a common precursor (reviewed in Ref. 4). Evidence consistent with a common precursor comes from knockout of the Ikaros transcription factor (5), the common cytokine receptor gc chain (6), Jak3 (7), or overexpression of CD3e (8), any of which results in a deficiency of both T lymphocytes and NK cells (as well as B lymphocytes in the first two examples). Most NK cells are produced in the bone marrow, whereas only a small proportion of mature NK cells have been reported in the murine thymus (9). However, the murine embryonic thymus at day 14 (d14)2 of gestation has previously been shown to contain cells that can develop into NK cells if injected i.v. (10). These intrathymic NK precursors share many phenotypic markers with T cell precursors, but whether they are identical has not been established. As indicated (3, 11), it remains to be determined whether individual murine thymocytes have a dual capacity, the ability to become either T or *Laboratory of Immunoregulation, Division of Basic Sciences, National Cancer Institute, and †Science Applications International Corporation, National Cancer Institute, Frederick, MD 21702 Received for publication March 4, 1998. Accepted for publication June 1, 1998. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1
Address correspondence and reprint requests to Dr. Scott K. Durum, National Cancer Institute, Building 560 Room 31-71, Frederick, MD 21702-1201. E-mail address:
[email protected] 2
Abbreviation used in this paper: d, day (e.g., d14).
Copyright © 1998 by The American Association of Immunologists
NK, or whether the population of early thymocytes contains two committed cell types. Single progenitor cells from the human thymus have been used to generate colonies, some of which have dual potential, although the frequency of dual-potential cells could not be determined (12). In the course of examining various stimuli that influence the association of thymocytes with extracellular matrix (13) and promote gene rearrangements (14), we noted functional effects of antiCD16 Abs. CD16, the low affinity FcR, is expressed on embryonic thymocytes at d14 of gestation (15) and has not previously been ascribed a functional activity in these cells. Under certain culture conditions (discussed below), we observed that treatment with antiCD16 blocked rearrangement of the TCRb locus and also blocked T cell development, whereas NK cell development was promoted by anti-CD16.
Materials and Methods Mice C57BL/6 mice were bred at the Animal Production Area (APA), National Cancer Institute-Frederick Cancer Research and Development Facility, Frederick, MD. To produce timed pregnancies, mice were mated overnight, the following day being considered gestational day 1. After 14 days of gestation, mothers were killed by CO2 asphyxiation, embryos by chilling on ice, and thymuses were removed using a dissecting microscope.
Thymic organ culture For generation of large cell numbers, the thymic lobes were placed in submersion culture in 50 ml of medium in 96-well plates, one lobe per well, in either the presence of anti-CD16 (2.4G2) for NK conditions, or the absence of anti-CD16 (abT cell conditions). Cells were harvested after 48 h (for PCR analysis of TCR gene rearrangement) or after 5 days (for flow microfluorometry analysis or NK assay).
Flow analysis Following organ culture, cell surface staining for CD4, CD8, TCRab, TCRgd, and NK1.1 (PharMingen, San Diego, CA) and scatter profile were performed on an Epics Profile (Coulter, Hileah, FL). 0022-1767/98/$02.00
3326
T/NK PRECURSOR
FIGURE 2. Anti-CD16 inhibition of TCRb rearrangement in different mouse strains. Thymic organs obtained from C57BL/6 or C3H embryos were cultured as described in the legend to Figure 1. Analysis for Vb3 rearrangement was performed by PCR.
FIGURE 1. CD16 cross-linking inhibits rearrangement of TCRb. Thymic lobes were removed from C57BL/6 mouse embryos on d14 following fertilization. A, CD16 expression was analyzed by flow microfluorometry. The hatched area contains cells stained by anti-CD16, and the clear area shows staining by an isotype-matched control. B, Thymic lobes were placed in submersion cultures with IgG2b (which inhibits TCRb rearrangement). Anti-CD16 Fab fragments were added as competitors. After culture for 5 days, PCR was used to assess Vb rearrangement. Controls for DNA loading consisted of internal primers for Vb8. C, Lobes were placed in submersion culture with the indicated concentrations of anti-CD16 and analyzed for rearrangement of Vb8 and Vb6.
PCR for TCR gene rearrangement Following organ culture, DNA was extracted and assayed by PCR for rearranged Vb3, -6, and -8 and Vg3 genes and compared with controls for DNA loading using internal V region primers as described (14, 16).
NK assay YAC-1 target cells were labeled by incubation for 1 h at 37° with Na51CrO4 (150 mCi; DuPont, Boston, MA). Target cells were washed and plated into round-bottom wells (Corning, New York, NY) at a concentration of 5 3 103 cells/well. Effector cells were added at the indicated concentrations in medium with human IL-2 (500 U/ml). After 5 h,. specific 51 Cr release was calculated as follows: {[cpm (exp) 2 cpm (spontaneous)/ cpm (maximum) 2 cpm (spontaneous)]} 3 100.
Results We initially observed that Abs of different specificities could block rearrangement of the TCRb locus during thymic organ culture under certain culture conditions (see Discussion). One example of this “nonspecific” effect of Abs is shown in Figure 1. This inhibitory effect is based on interaction of the added Igs with CD16, a
low affinity FcR, which is expressed on most thymocytes at d14 of gestation (Fig. 1A). Inhibition of TCRb rearrangement by an IgG2b Ab was reversed when Fab fragments of the 24G.2 Ab (which recognizes the FcR CD16 and CD32) were added as competitors (Fig. 1B). This confirms that an FcR mediates inhibition by Igs, and it has been shown that CD16 (but not CD32) is the FcR expressed at this stage in early T cell development (10). Crosslinking CD16 with bivalent 24G.2 Ab effectively inhibited TCRb rearrangement under these conditions (Fig. 1C), confirming that CD16 can deliver this signal. Two different mouse strains were examined for the inhibitory effect of anti-CD16 on TCRb rearrangement, as shown in Figure 2. Although the C3H strain appears to be less inhibited by anti-CD16, this is probably more a matter of timing, because some rearrangement had already occurred in the starting population before organ culture. Nevertheless, this shows that CD16 cross-linking is effective, in two different mouse strains, in abolishing or reducing TCRb rearrangement. A number of different Vb genes have been tested, including Vb3, -6, and -8, as well as several J regions from both clusters, and all were equally inhibited by CD16 cross-linking. The TCRg locus undergoes rearrangement at about the same pro-T cell stage as does the TCRb locus. Nevertheless, anti-CD16 treatment had much less of an inhibitory effect on the g locus than on the b locus, as shown in Figure 3. Thus, anti-CD16 does not inhibit all VDJ recombination in pro-T cells, suggesting that it does not antagonize the recombinase machinery per se but, rather, affects the target genes. Rearrangement of the TCRb gene is normally required for pro-T cells to develop to the CD41CD81 stage. This differentiation was greatly inhibited by anti-CD16, as shown in Figure 4A. The inhibition of development was not associated with killing thymocytes by the anti-CD16, based on several criteria. Rag-1 gene expression was maintained after CD16 treatment, as shown in Figure 4B, which not only shows that pro-T cells remained viable, but also explains how the TCRg gene could undergo rearrangement (Fig. 3). Other evidence that anti-CD16 exerted its effect by nonlethal mechanisms are shown by the absence of apoptotic DNA (Fig. 4C) and the recovery of reasonable numbers of cells from cultured thymic organs (Fig. 4D). Note that DNA fragmentation was examined at early time points after addition of CD16, because it has
The Journal of Immunology
3327
FIGURE 3. Anti-CD16 does not block rearrangement of the TCRg locus. C57BL/6 thymic organ culture was performed as described in the legend to Figure 1. Rearrangement of Vg3 to Jg1 and control internal primers are shown. A positive control for rearranged TCRg is d16 fetal thymus, and negative controls are the 3T3 cell line and d14 fetal liver. Two concentrations of DNA (1 mg and 0.3 mg) were added to the PCR; the lower dose is indicated by the asterisk.
been shown that apoptotic thymocytes are rapidly phagocytosed by macrophages, leading to complete DNA degradation at later times (17). Pro-T cells have the capacity to develop into NK cells if placed under suitable conditions. Since CD16 is present on NK cells and can activate numerous functions in these cells, we considered the possibility that cross-linking CD16 on the pro-T cell might induce development toward the NK lineage. This proved to be the case, as shown in Figure 5. Three phenotypic and functional criteria were used to show that CD16 promoted differentiation to the NK lineage, scatter profile of large granular cells (Fig. 5A), expression of NK1.1 (Fig. 5B), and the ability to lyse the NK-sensitive target cell YAC-1 (Fig. 5C). To boost lytic function, IL-2 was added to mixture of effector and target cells. Expression of CD16 also persisted on these cells (not shown). Another criterion of NK cells is the absence of TCRb rearrangements (Figs. 1–3), which is also a very sensitive measure of the presence of T cells and reflects the potency of inhibition of T cell development. Thus, CD16 cross-linking, while inhibiting abT cell development, promoted NK development as further supported by comparing the numbers of each cell type detected per thymic lobe (Table I). Before culture, one d14 lobe contained ;30,000 cells (none of which were ab, gd, or NK cells). As shown, inclusion of anti-CD16 for 5 days of thymic organ culture reduced the number of abT cells 10-fold and increased the number of NK cells 20-fold, whereas generation of gdT cells was relatively unaffected.
Discussion We observed that CD16, which is expressed on pro-T cells, can deliver signals that inhibit rearrangement of TCRb, completely repress abT cell development, and promote NK development. We have performed additional studies3 demonstrating that individual pro-T cells have the potential to develop into either T or NK cells, and that CD16 cross-linking can direct this development. Taken together, our results support the theory that development from a common lymphoid stem cell can be directed by environmental signals along one or the other pathway. The physiologic significance of the CD16 effects shown here remain a matter of speculation but could represent a mechanism for confining the TCRb gene rearrangement event to certain com3 C.-K. Lee, K. Kim, T. M. Geiman, W. J. Murphy, K. Muegge, and S. K. Durum. Cloning thymic precursors for ab, gd, and NK lineages. Submitted for publication.
FIGURE 4. T cell development from d14 thymus is suppressed under conditions favoring NK cell development. C57BL/6 thymic lobes were placed in submersion cultures with anti-CD16 at the indicated concentrations. A, After 5 days, T cell development was assessed by staining with anti-CD4 and anti-CD8 and flow microfluorometry. B, After 2 days, Rag-1 expression was assayed by RT-PCR. C, At the indicated times, DNA fragmentation was assessed by agarose gel electrophoresis and ethidium bromide stain. Apoptotic control DNA is from the thymus of mice injected i.v. with anti-CD3 Ab. D, Recovery of cells from thymic lobes was determined following culture with anti-CD16.
partments in the thymus. Thus, pro-T cells that have escaped the correct microenvironment may engage a CD16 ligand that switches off the TCRb gene rearrangement. The only ligand that we currently know for CD16 is aggregated Ig, which elicited our original finding; others, however, may exist. The shift from ab T to NK development induced by anti-CD16 could occur in two different ways. One model is that CD16 crosslinking acts on the lymphoid precursor, suppressing ab T cell development and stimulating NK development. A second model is that anti-CD16 does not act directly on the lymphoid precursor, but upon its daughter cells. Thus, the lymphoid precursor divides unequally, one daughter being committed to the T lineage, the other to the NK lineage, and CD16 cross-linking inhibits ab development from the former and stimulates NK development from the latter. Our data do not clearly favor one or the other of these models. The shift from ab T cell to NK development was observed using two different protocols: culturing a thymic lobe in the presence of anti-CD16 in a microtiter well (submersion culture) as shown; or reconstituting an irradiated thymic lobe (by hanging drop) with precursors and placing it in a floating culture.3 There was little effect of anti-CD16 on T cell development of an intact thymic lobe in floating culture (not shown); this is a more optimal culture system for abT cell development than the submersion culture, and perhaps the anti-CD16 cannot penetrate the organ to reach sufficient levels, whereas thymocytes in suspension (during hanging drop reconstitution) receive a saturating dose. The timing of anti-CD16 addition was critical, its presence at the initiation of hanging drop reconstitution producing complete abT to NK shift,
3328
T/NK PRECURSOR
FIGURE 5. NK cell development from d14 thymus. C57BL/6 thymic lobes were cultured as described in the legend to Figure 1 under conditions favoring T cell vs NK cell development. A, Scatter profile was determined. B, Cells were stained with biotinylated NK1.1 and avidin-phycoerythrin. C, After 5 days of culture, cells were assayed in the presence of IL-2 (500 U/ml) for NK activity on YAC-1 target cells labeled with 51 Cr in a 4-h release assay.
whereas delaying its addition by 36 h greatly reduced its efficacy (not shown), probably because precursors became committed to the abT cell lineage during this period.3 CD16 is expressed on most thymocytes at d14 and as yet has no known function in T cell development. Cross-linking CD16, which
Table I. Effect of anti-CD16 on recovery of different cell types following thymic organ culture a Cells Recovered Culture Addition
Nil Anti-CD16
ab T cells
40,717 (3,573) 415 (81)
b
gd T cells
NK cells
6,679 (2,184) 6,072 (1,323)
1,246 (1,164) 26,868 (2,506)
a C57BL/6 embryonic thymus (d14) was cultured 5 days without (nil) or with anti-CD16 (75 mg/ml). Approximately 30,000 cells were present in each thymic lobe prior to culture. b Recovery per lobe, the mean of eight experiments in which 10 lobes were pooled for each determination. SD is shown in parentheses. Cell type was determined by staining with Abs against TCRab, TCRgd, and NK1.1.
induced a shift from ab T cell development into NK development, did not trigger detectable Ca1 influx (not shown) or apoptosis nor did it repress Rag-1 gene expression. Anti-CD16 blocked ab T cell development from CD252 cells,3 perhaps due to blocking rearrangement of the TCRb locus. There was little blocking effect on TCRg locus rearrangement (or on gd T cell development). The effects on TCRb rearrangement and abT cell development have been observed in .50 experiments. Thus, CD16 cross-linking could have a specific effect on the accessibility of the b locus to the recombinase complex (discussed in Ref. 18). It has long been thought that mechanisms exist that govern accessibility of rearranging loci, and the enhancers of each locus have been implicated in regulating this accessibility. An example of an extrinsic signal that differentially regulates the accessibility of rearranging loci is the IL-7R, which induces accessibility of the TCRg 4 S. Durum, S. Candeias, H. Nakajima, W. Leonard, A. Baird, L. Berg, and K. Muegge. Defective rearrangement of the TCRg locus in murine thymocytes deficient in IL-2Ra, gc, or Jak3 is associated with methylation of the locus, a reduced production of sterile transcripts and can be overcome by treatment with the specific dealetylase inhibitor Trichostatin A. Submitted for publication.
The Journal of Immunology locus (but not the TCRb locus) to cleavage by Rag proteins (Refs. 18 and 19; and Footnote 4). Thus, CD16 signals may have the opposite effect, masking the TCRb locus but leaving the TCRg locus accessible to Rag cleavage. Mature NK cells express CD16 and can be activated by CD16 cross-linking to secrete cytokines and lyse target cells (reviewed in Ref. 20). Thus, CD16 can trigger intracellular events in NK cells and perhaps delivers similar intracellular signals leading to NK differentiation from a common lymphoid progenitor. Is the NK pathway normally a default pathway for cells that fail to rearrange the TCRb locus? This seems unlikely, since Rag knock-out mice, which cannot produce rearrangements, do not preferentially generate NK cells. Thus, it seems likely that CD16 actively induces NK development in our system.
3329
7.
8.
9.
10.
11.
12.
Acknowledgments We thank Rodney Wiles for technical assistance, Louise Finch for FACS analysis, Dr. W. Fogler for Abs, Drs. L. Mason and R. Hornung for help with NK assays, and Drs. B. J. Fowlkes, J. Ortaldo, J. O’Shea, J. Oppenheim, and D. Longo for comments on the manuscript.
References 1. Scollay, R., J. Smith, and V. Stauffer. 1986. Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus. Immunol. Rev. 91:129. 2. Storb, U., and A. M. Kruisbeek. 1996. Lymphocyte development. Curr. Opin. Immunol. 8:155. 3. Shortman, K., and L. Wu. 1996. Early T lymphocyte progenitors. Annu. Rev. Immunol. 14:29. 4. Lanier, L. L., H. Spits, and J. H. Phillips. 1992. The developmental relationship between NK cells and T cells. Immunol. Today 13:392. 5. Georgopoulos, K., M. Bigby, J. H. Wang, A. Molnar, P. Wu, S. Winandy, and A. Sharpe. 1994. The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143. 6. Cao, X., E. W. Shores, J. Hu-Li, M. R. Anver, B. L. Kelsall, S. M. Russell, J. Drago, M. Noguchi, A. Grinberg, E. T. Bloom, et al. 1995. Defective lymphoid
13.
14. 15.
16.
17. 18. 19.
20.
development in mice lacking expression of the common cytokine receptor g chain. Immunity 2:223. Park, S. Y., K. Saimo, T. Takahashi, M. Osawa, H. Arase, N. Hirayama, K. Miyake, H. Nakauchi, T. Shirasawa, and T. Saito. 1995. Developmental defects of lymphoid cells in Jak3 kinase-deficient mice. Immunity 3:771. Wang, B, C. Biron, J. She, K. Higgins, M.-J. Sunshine, E. Lacy, N. Lonberg, and C. Terhorst. 1994. A block in both early T lymphocyte and natural killer cell development in transgenic mice with high-copy numbers of the human CD3E gene. Proc. Natl. Acad. Sci. USA 91:9402. Garni-Wagner, B. A., P. L. Witte, M. M. Tutt, W. A. Kuziel, P. W. Tucker, M. Bennett, and V. Kumar. 1990. Natural killer cells in the thymus: studies in mice with severe combined immune deficiency. J. Immunol. 144:796. Rodewald, H.-R., P. Moingeon, J. L. Lucich, C. Dosiou, P. Lopez, and E. L. Reinherz. 1992. A population of early fetal thymocytes expressing FcgRII/ III contains precursors of T lymphocytes and natural killer cells. Cell 69:139. Leclercq, G., B. Debacker, M. DeSmedt, and J. Plum. 1996. Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells. J. Exp. Med. 184:325. Sanchez, M., M. E. Muench, M. G. Roncarolo, L. L. Lanier, and J. H. Phillips. 1994. Identification of a common T/natural killer cell progenitor in human fetal thymus. J. Exp. Med. 180:569. Kitazawa, H., K. Muegge, R. Badolato, J.-M. Wang, W. E. Fogler, D. K. Ferris, C.-K. Lee, S. Candeias, M. R. Smith, J. J. Oppenheim, and S. K. Durum. 1997. Interleukin-7 activates a4b1 integrin in murine thymocytes. J. Immunol. 159: 2259. Muegge, K., M. Vila, and S. K. Durum. 1993. Interleukin-7: a cofactor for V(D)J recombination of the T cell receptor b gene. Science 261:93. Rodewald, H.-R., K. Awad, P. Moingeon, L. D’Adamio, D. Rabinowitz, Y. Shinkai, F. W. Alt, and E. L. Reinherz. 1993. FcgRII/III and CD2 expression mark distinct subpopulations of immature CD42CD82 murine thymocytes: in vivo developmental kinetics and T cell receptor b chain rearrangement status. J. Exp. Med. 177:1079. Cande´ias, S., K. Muegge, and S. K. Durum. 1996. Junctional diversity in signal joints from T cell receptor b and d loci via terminal deoxynucleotidyl transferase and exonucleolytic activity. J. Exp. Med. 184:1919. Surh, C. D., and J. Sprent. 1994. T-cell apoptosis detected in situ during positive and negative selection in the thymus. Nature 372:100. Cande´ias, S., K. Muegge, and S. K. Durum. 1997. IL-7 receptor and VDJ recombination: trophic versus mechanistic actions. Immunity 6:501. Cande´ias, S., J. J. Peschon, K. Muegge, and S. K. Durum. 1997. Defective T-cell receptor g gene rearrangement in interleukin 7 receptor knockout mice. Immunol. Lett. 57:9. Ravetch, J. V., and J.-P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.
