Temporal Predisposition to βα and δγ Fates in the Pablo Pereira, Laurent Boucontet and Ana Cumano This information is current as J Immunol 2012; 188:1600-1608; Prepublished online 11 of September 30, 2021. January 2012; doi: 10.4049/jimmunol.1102531 http://www.jimmunol.org/content/188/4/1600 Downloaded from

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Temporal Predisposition to ab and gd T Cell Fates in the Thymus

Pablo Pereira, Laurent Boucontet, and Ana Cumano

How T cell progenitors engage into the gd or ab T cell lineages is a matter of intense debate. In this study, we analyzed the differentiation potential of single from wild-type and TCRgd-transgenic mice at two sequential early developmental stages. Double-negative (DN) 3 progenitors from both wild-type and transgenic mice retain the capacity to engage into both pathways, indicating that full commitment is only completed after this stage. More importantly, DN2 and DN3 progenitors from TCRgd transgenic mice have strong biases for opposite fates, indicating that developmentally regulated changes, other than the production of a functional TCR, altered their likelihood to become a gd or an ab T cell. Thus, unlike the differentiation in other hematopoietic lineages, T cell progenitors did not restrict, but rather switch their differentiation potential as they developed. The

Journal of Immunology, 2012, 188: 1600–1608. Downloaded from

cell development in the thymus is determined by cis-acting tiation is the silencing of TCRg genes and the rearrangement of genetically defined developmental programs and trans- TCRa genes, which results in the excision of the TCRd locus, thus T acting interactions with stromal cells, other thymocytes, precluding TCRgd expression in ab-lineage cells (17, 18). In and soluble factors (1, 2). ab T cells and gd T cells, defined by the normal animals, most DP cells carry productive TCRb-chains,

chains they use to form their clonotypic receptors, develop in the suggesting that they originated from cells that expressed a pre- http://www.jimmunol.org/ thymus from a common progenitor. These progenitors are found TCR at the DN3 developmental checkpoint [these cells are usually among double-negative (DN) CD42CD82 thymocytes. Based on referred to as “b-selected” cells and the checkpoint is referred the expression of CD25, CD44, and CD117, progenitor thymo- to as “b-selection” (7)]. Notably, TCRgd can also mediate b- cytes can be organized according to the following maturation selection–like differentiation to the DP stage. This is most evident sequence: CD252CD44+CD117+ (DN1) → CD25+CD44+CD117+ in animals unable to express a functional pre-TCR, in TCRgd Tg (DN2 or pro-T cells) → CD25+CD442CD1172 (DN3 or pre- mice in vivo (8, 10, 19–23), and in cultures in which TCRgd- T cells) → CD252CD442CD1172 (DN4) (3). Completed TCRg expressing progenitor cells are allowed to develop in vitro (24– and TCRd rearrangements are already detectable at the DN2 26). Like b-selected DP cells, gd-selected DP cells silence TCRg stage and terminate at the DN3 stage (4, 5). At this point, Vb to gene expression and undergo TCRa rearrangements, indicating by guest on September 30, 2021 DJb rearrangements begin (6). Progression beyond the DN3 stage that they are bona fide ab-lineage cells (21). The absence of requires surface expression of either TCRgd or a pre-TCR (7, 8), productive TCRb rearrangements in gd-selected DP cells, to- composed of a TCRb-chain paired with an invariant pre–TCRa- gether with their silencing of TCRg genes, preclude them from chain (9). Formation of a productive TCRgd allows the develop- expressing TCR/CD3 complexes at that stage, resulting in an ment of gd T cells that remain DN (8, 10, 11). Development of abortive differentiation. Lack of intracytoplasmic TCRb expres- gd-lineage cells is accompanied by increased transcription of sion (TCRbic) and surface CD3 expression can be used to dis- TCRd and, presumably, TCRg genes (12). However, TCRgd is not tinguish them from b-selected DP cells. Thus, high levels of strictly required for the differentiation of gd-lineage cells, as TCRgd and simultaneous expression of CD4 and CD8 define the evidenced by the fact that early expression of a TCRab (mostly earliest populations that unequivocally represent commitment to seen in TCRab-transgenic [Tg] mice) produces TCRab+ DN cells the gd and ab lineages, respectively (27, 28). with characteristics of gd T cells (13–16). By contrast, ab-lineage Models of ab and gd lineage commitment vary in the extent to cells subsequently differentiate to the DN4 and CD4+CD8+ which TCRgd and the pre-TCR are proposed to instruct com- double-positive (DP) stages. Concomitant with DP cell differen- mitment (instructive models) or reinforce a previous commitment event (selective models) (1). Selective models propose that lineage commitment is stochastic and results in a binary choice prior to Institut Pasteur, Unite´ Lymphopoı¨ese and INSERM U668, 75015 Paris, France TCR expression, whereby daughter cells commit to one or the Received for publication September 2, 2011. Accepted for publication December 5, other lineage. Instructive models, by contrast, propose that lineage 2011. commitment follows TCR expression. The strict instructive model This work was supported by institutional funding and by the grant “” cannot be easily reconciled with observations that TCRgd can from Agence National de le Recherche and a grant from the Ligue de Recherche contre le (both to A.C.). drive the generation of ab-lineage–committed DP cells or that early expression of a Tg TCRab produces TCRab+ DN cells with Address correspondence and reprint requests to Dr. Pablo Pereira, Unite´ Lympho- poı¨ese, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail characteristics of gd T cells, described above. It was recently address: [email protected]. proposed that the strength of the signal, rather than the TCR The online version of this article contains supplemental material. isoform, determines lineage selection, with strong TCR signals Abbreviations used in this article: B6, C57BL/6; DN, double negative; DP, double promoting a gd-like fate and weaker signals promoting ab T cell positive; Eb, TCRb enhancer; FTOC, fetal thymus organ culture; KO, knockout; differentiation through DP intermediates (29). Although they are TCRbic, intracytoplasmic TCRb expression; Tg, transgenic; WT, wild-type. usually considered to favor the instructive model, experiments Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 supporting the signal strength model (30, 31) are compatible with www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102531 The Journal of Immunology 1601 selective models, as well (32–34). More importantly, variations in Cell surface labeling was performed, as described (39). Cells were ana- signal capacity of TCRs may interfere with commitment, as well lyzed in a FACSCalibur, FACSCanto I, or FACSCanto II (Becton Dick- as with selective events that may be different in developing ab inson, Franklin Lakes, NJ) and analyzed with either CELLQuest pro software (BD Bioscience) or FlowJo software. For sorting, thymocytes and gd lineage cells (33, 34). More recent experiments showed were stained with FITC-labeled anti-CD44, PE-labeled anti-CD25, allo- that a fraction of TCRgd+ DN3-like cells, which can give rise to phycocyanin-labeled anti-CD117, and a mixture of biotin-labeled Abs ab-lineage cells in vitro, could be diverted to the gd T cell lineage specific for CD3, CD4, CD8, CD19, CD11b, CD11c, NK1.1, TCRb, when a strong TCR signal was mimicked by TCR cross-linking and TCRd, followed by PE-Cy7–labeled streptavidin and sorted with a MoFlow cell sorter (Cytometrix, Fort Collins, CO). Unless otherwise (25), thus providing further support for this model. However, specified, these biotin-labeled Abs were used to define the lin2 cells in our concerns with this interpretation have been put forward (34). It experiments. seems difficult to reconcile these two models (33–37), suggesting Cell cultures that they are incomplete. A key question in the development of ab and gd T cells and OP9-DL1 cells were produced in the laboratory and maintained in Opti- germane to lineage commitment is the definition of the exact point MEM media (Life Technologies) supplemented with 2-ME, antibiotics at which the two lineages diverge [i.e., the point(s) of commit- (Life Technologies), and 10% FCS. Cocultures of progenitor cells were performed in the same culture media with a monolayer of 2000 OP9-DL1 ment]. Selective and instructive models predict that commitment cells/well and the indicated numbers of progenitor cells. Media were occurs before or after TCR expression, respectively. Using the supplemented with mouse rIL-7. In the kinetic experiments, 500–1000 OP9-DL1 coculture system (38), it was proposed that commitment OP9-DL1 cells were added at some point after removing half of the cells initiates at the DN2 stage and is completed at the DN3 stage. This for analysis. was based on the finding that single DN3 cells give rise to either Downloaded from TCRab+ or TCRgd+ cells, but rarely both (24). However, the Results absence of CD4 and CD8 expression analysis precludes definitive Normal development of ab-lineage cells in adult Tg-gd mice conclusions with regard to ab/gd-lineage commitment, particu- We described several aspects of the development of T cells in larly because TCRgd expression in progenitor cells is known to Vg1Vd6 Tg-gd mice (39, 40). The rearranged chains were iso- result in the production of both gd and DP cells (24). Furthermore, lated as large cosmids, thus ensuring physiological expression of these experiments do not allow the discrimination of true lineage the transgenes (39). Compared with WT mice, Tg-gd mice dis- http://www.jimmunol.org/ commitment from ab T cell fate consequent to the inability of played comparable numbers of ab-lineage DP and single-positive a progenitor cell to form a TCRgd as the result of unsuccessful thymocytes and an ∼5-fold increase in the number of gd thymo- rearrangement at their TCRg/TCRd loci. Such discrimination can cytes (Fig. 1A,1B). Numbers of early T cell progenitors also only be made by the analysis of progenitor cells capable of compared well between WT and Tg-gd mice (Fig. 1C–E), indi- expressing TCRgd. In this study, we analyzed, at the clonal level, cating that the transgenes resulted in no loss of progenitors, which the potential of progenitors cells isolated from wild-type (WT) was then compensated for during development. Cells bearing the and Tg-gd mice to differentiate into ab- and gd-lineage cells Tg TCR appear to be bona fide gd-lineage cells, as evidenced by in vitro. Our results identified a successive change in their ability to respond to TCR ligation in a manner similar to WT content that alters the likelihood of a cell becoming a gd or ab gd T cells (Supplemental Fig. 1), as well as by a number of phe- by guest on September 30, 2021 T cell, emphasized by DN2 and DN3 progenitors with an identical notypic and molecular criteria that have been used in the past to TCRgd displaying divergent fate outcomes when assessed at the differentiate ab- and gd-lineage cells (39) clonal level. They also indicated the existence of mechanisms operating in vivo that preclude expression of a TCRgd in many Clonal analysis of T cell progenitors from WT and Tg-gd mice progenitor cells, thus favoring and allowing ab-lineage develop- To analyze the developmental potential of progenitor cells, we used ment in WT and Tg-gd animals, respectively. the OP9-DL1 culture system, which allows the differentiation of single progenitor cells to both ab and gd cell lineages. Unlike an Materials and Methods earlier report (24), we determined ab and gd T cell lineage po- Mice tential by the presence, within the progeny, of DP and TCRgd+ cells, respectively. C57BL/6 (B6) mice were obtained from Iffa-Credo (L’Abresle, France). B6 CD45.1, B6 mice Tg for rearranged Vg1Jg4Cg4 and Vd6Dd2Jd1 chains Limiting-dilution analyses of DN2 and DN3 cells isolated from (Tg-gd) (39), pTa-deficient mice, and TCRb enhancer (Eb)-deficient mice WT and Tg-gd mice are shown in Fig. 2A. About one in two DN2 (23) were maintained in our animal facilities. All animal procedures were cells and one in nine DN3 cells could develop to a sizable clone, in compliance with the guidelines of the committee on animal experi- regardless of whether they originated from normal or Tg-gd mice. mentation of the Institut Pasteur and were approved by the French Ministry of Agriculture. These frequencies compared well with previously published fre- quencies in WT mice (24, 41). The different response of DN2 and Radiation chimeras DN3 cells likely reflects the fact that DN2 cells proliferate in re- Irradiated (1200 rad) B6 mice were injected with a total of 2 3 106 T cell- sponse to IL-7 before differentiating, whereas DN3 cells require depleted marrow cells from CD45.1 Tg-gd mice and CD45.2 WT a functional pre-TCR/TCR to differentiate further (38, 41). How- mice at different ratios. T cell depletion was performed by one-step killing ever, the presence of functionally rearranged TCRg and d genes at 37˚C for 45 min with anti-Thy1 (J1j), anti-CD4 (RL.174), and anti-CD8 results neither in an advantage nor in a disadvantage of precursor (H0-2.2) mAbs and rabbit complement (Cedarlane Laboratories, Hornby, ON, Canada), followed by centrifugation over a density gradient using frequencies at these developmental stages, suggesting that many Lympholyte M (Cedarlane Laboratories). DN3 cells do not express the Tg TCR (see later discussion). When the progenies of single DN2 and DN3 cells from WT mice Abs, staining, and cell sorting were analyzed (see Fig. 1B for strategy and definition of sorting Fluorochrome-labeled anti-CD25 (PC61), anti-CD44 (IM7), anti-CD117 gates), we unexpectedly observed that no progenitor generated (2B8), anti-CD3ε (145-2C11), anti-Cd (GL3), anti-TCRb (H57-597), progeny containing exclusively gd T cells (Fig. 2B). About half of anti-NK1.1 (PK136), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-CD11c (N418), anti–Gr-1 (RB6-8C5), anti-CD19 (1D3), anti the DN2 cells gave rise to both DP and gd T cells, whereas the CD45.1 (A20), and anti-CD45.2 (1D4) mAbs and streptavidin were pur- remainder generated exclusively DP cells. Single DN3 cells gave chased from BD Bioscience, eBioscience, or BioLegend (San Diego, CA). rise to progeny containing only DP cells (75%) or DP cells and gd 1602 ab AND gd T LINEAGE DIVERGENCE Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 1. Development of ab- and gd-lineage cells in Tg-gd mice. A, Flow cytometric analyses of WT and Tg-gd thymocytes for CD4 and CD8 (top panels) or CD3 and TCRgd (bottom panels). Numbers indicate frequencies of cells in the quadrants (top panels) or the frequency of gd thymocytes (bottom panels). B, Total number of ab-lineage DP and single-positive cells and gd-lineage cells in the thymus of WT and Tg-gd mice. Each symbol represents an individual mouse. DN thymocytes from WT (C) and Tg-gd mice (D) were stained with FITC-labeled anti-CD44, PE-labeled anti-CD25, allophycocyanin- labeled anti CD117, and a mixture of lineage-specific biotin-labeled Abs specific for CD3, CD4, CD8, CD19, CD11b, CD11c, TCRb, TCRgd, and NK1.1, followed by streptavidin–PE–Cy7. Left panels, CD44 and CD117 profiles of lin2 thymocytes showing the gate used to define CD117 bright cells. Right panels, CD25 and CD44 profiles of lin2 CD117 bright cells (top panels) or lin2 cells (bottom panels) and the gates used to define the DN1 to DN4 populations. Numbers indicate frequencies of cells in the indicated gate. E, Total number of the indicated progenitor cells in the thymus of WT and Tg-gd mice. Each symbol represents an individual mouse.

T cells (25%) (Fig. 2B,2C). Thus, DN2 and DN3 cells produce clonal analyses performed in fetal thymus organ cultures (FTOC; similar progeny, with the only difference being an increase in the Fig. 2D). Furthermore, many DP cells originating from Tg-gd frequency of cells giving rise exclusively to ab-lineage cells as DN2 cells were not b selected, as suggested by their lack of development progresses. This could be due, at least in part, to the TCRbic chains (Fig. 2E). Perhaps more surprising was the finding accumulation of nonfunctional TCRg and/or TCRd rearrange- that none of the DN3 cells from Tg-gd mice produced progenies ments in DN3 cells that renders them and their progeny incapable containing exclusively gd-lineage cells, in clear contrast with their of expressing a TCRgd. DN2 counterparts (Fig. 2B). DN3 cells from Tg-gd mice gave rise Similar analyses performed with progenitors from Tg-gd mice to progeny resembling those produced from WT DN3 cells (Fig. also produced unexpected results. First, ∼70% of DN2 cells from 2C), albeit with a higher frequency of clones giving rise to cells of Tg-gd mice gave rise to exclusively gd cells, whereas none gen- both lineages (Fig. 2B), indicating that the apparent ab-lineage erated exclusively ab-lineage cells (Fig. 2B,2C). The lack of commitment of WT DN3 cells is due, at least in part, to the in- ab-only progeny from Tg-gd DN2 cells, as well as that of gd-only ability of these cells to express a TCRgd. progeny from WT DN2 cells, was not a result of using the OP9- Collectively, these results are not readily compatible with pre- DL1 culture system, because their absence was also observed in commitment models that consider the ab/gd lineage decision as The Journal of Immunology 1603 Downloaded from http://www.jimmunol.org/

FIGURE 2. Clonal analysis of DN2 and DN3 T cell progenitors from WT and Tg-gd mice in OP9-DL1 cultures or FTOC. Sorted DN2 cells (lin2CD17+ CD44+CD25+) or DN3 cells (lin2CD172CD442CD25+) from adult WT and Tg-gd mice were cultured at the indicated cell concentrations with OP9-DL1 in the presence of IL-7 (A–C) or in FTOC (D, E) and analyzed for the presence of TCRgd (gd-lineage cells) and CD4 and CD8 (ab-lineage cells) between 7 and 14 d after the initiation of the cultures. A, Limiting-dilution analysis in which the indicated number of cells was seeded in 48–96 replicates, analyzed as above, and plotted as the fraction of negative wells. B, Total number of wells displaying gd-lineage cells (gd), ab-lineage cells (DP), or cells of both lineages (Both) in progenies of the indicated progenitor cells, expressed as a percentage of T cell-reconstituted wells in OP9-DL1 cocultures. Data represent a minimum of 63 clones per population of progenitor cells analyzed in different experiments. C, Representative stainings of the types of clones obtained in the analyses shown in B. D, Same as B, but single DN2 cells were seeded in FTOC, and their progeny were analyzed at day 12. Data from 55 reconstituted lobes (22 with WT and 33 with Tg-gd DN2). E, Intracellular TCRb expression in DP cells from D. Data are shown as the fraction of cells expressing by guest on September 30, 2021 intracellular TCRb among DP cells. Each symbol represents a single reconstituted lobe. a binary choice prior to TCR expression, because neither a pro- cells from Tg-gd mice produced ∼10-fold less DP cells and ∼5- genitor cell that gave rise exclusively to gd-lineage cells in WT fold more gd T cells than did WT DN2 or DN3 cells (Fig. 3, top mice nor a progenitor cell that gave rise exclusively to ab-lineage right panels). Furthermore, most DP cells originating from Tg-gd cells in Tg-gd mice could be identified in these analyses. Rather, DN2 cells never exhibited high levels of CD3 at the cell surface they indicated that the potential to develop into gd-orab-lineage (compare the fraction of cells expressing high levels of CD3 cells varies with the developmental stage of the progenitor cell. among TCRgd-negative cells in DN2 cultures from WT and Tg-gd This was most evident in progenitors from Tg-gd mice, which mice; Fig. 3), suggesting that they were gd selected rather than b showed marked bias for developing into gd-lineage cells at the selected. Because of the silencing of TCRg genes and their ab- DN2 stage and into ab-lineage cells at the DN3 stage. sence of functional TCRb-chains, gd-selected DP cells do not express TCR/CD3 complexes. Of note, DN2 cells from Tg-gd Developmental progression is accompanied by decreased mice differentiated into DP cells with faster kinetics than did DN2 gd-lineage developmental potential cells from WT mice, a likely consequence of the gd selection To better understand the differences between WT and Tg-gd mediated by TCRgd in some of their progeny. progenitors and to provide quantification of their developmental To identify a more physiological setting in which only signals potential, we cultured small numbers (100 cells) of adult DN2 and mediated through polyclonal gdTCRs that rely on endogenous DN3 cells from WT and Tg-gd mice on OP9-DL1 monolayers and rearrangements for their expression could drive T cell differenti- analyzed the development of gd and DP cells at different time ation, we analyzed the developmental potential of DN2 and DN3 points. As previously reported (24), WT DN2 and DN3 cells cells from Eb-knockout (KO) mice in similar assays (Supple- differentiated efficiently into DP cells and gd T cells. WT DN2 mental Fig. 2A). Like DN2 cells from Tg-gd mice, Eb-KO DN2 cells displayed delayed kinetics and a slightly greater efficiency of cells produced progeny consisting of roughly equal numbers of gd differentiation into gd-lineage cells than did WT DN3 cells (Fig. T cells and TCRbic-negative DP cells. Interestingly, gd T cells 3), probably due to their more immature state and lower extent of developed with similar kinetics in cultures from Eb-KO and WT TCRg and TCRd rearrangements. DN3 cells from Tg-gd mice DN2 cells, indicating that gdTCRs are expressed with similar differentiated into DP cells with similar efficiency and kinetics as kinetics and efficiency in both strains. Also similar to Tg-gd DN3 did WT DN3 cells, as well as into gd T cells with a 5-fold higher cells and different from their DN2 counterparts, DN3 cells from efficiency and with accelerated kinetics compared with DN3 cells Eb-KO mice produced ∼20-fold more DP cells than gd T cells, from WT mice (Fig. 3, bottom right panels). By contrast, DN2 although their progression beyond the DN3 stage was mediated 1604 ab AND gd T LINEAGE DIVERGENCE

FIGURE 3. Development of gd- and ab-lineage cells from WT and Tg-gd T cell progenitors in OP9- DL1 cultures. One hundred DN2 cells (lin2CD117+ CD44+CD25+) or DN3 cells (lin2CD1172CD442 CD25+) isolated from adult WT and Tg-gd mice were cultured with OP9-DL1 in the presence of IL-7. On the indicated days, half of the cultures were harvested and analyzed by flow cytometry for the presence of TCRgd (gd-lineage cells) and CD4 and CD8 (ab- lineage cells), as indicated. Right panels, Absolute cell numbers of gd cells and DP cells originating in these cultures from DN2 (top panels) or DN3 (bottom panels) progenitor cells. One representative experi- ment of four is shown. Downloaded from

through gdTCRs. Of note, differentiation of DN3 cells was more DN2 cells (Fig. 5A) and 4 DN3 cells (Fig. 5B) in which cells from inefficient in Eb-KO mice than in WT or Tg-gd mice, and 10-fold both lineages were detected. CD4 versus CD8 and CD3 versus more DN3 cells were required to obtain progenies of similar size, TCRd profiles of progeny from four representative DN2 cells and http://www.jimmunol.org/ indicating that expression of a gdTCR in DN3 cells is rather in- four DN3 cells at different time points are shown in Supplemental efficient. Fig. 3. The appearance of gd T cells in cultures originating from When we performed similar kinetic studies including DN1 and single DN2 cells preceded that of DP cells by ∼2 d in every clone two transitional populations (DN2–3 [lin2CD117intCD44intCD25+] analyzed. Conversely, in cultures originating from DN3 cells, DP and DN3–4 [lin2CD1172CD442CD25int]) and plotted the ratio cells preceded gd cells by $2 d. The late development of gd cells of the maximum number of gd-toab-lineage DP cells generated from DN3 cells was independent of whether differentiation be- from those cultures, two important points become apparent (Fig. yond the DN3 stage occurred through b or gd selection, as evi- 4). First, this ratio was more or less constant until the DN2–3 denced by the high or low levels, respectively, of CD3 expressed transitional stage, and it decreased linearly thereafter, identifying at the cell surface of the developing DP cells (Fig. 5C). However, by guest on September 30, 2021 a window from the late DN2 stage to the DN3 stage during which the nature of the selecting receptor influenced the total number of the differential production of gd-orab-lineage cells takes place. gd-lineage cells that developed in these cultures (Fig. 5B,5C). The decreased ratio in DN3 cells compared with that of earlier These results demonstrated intrinsic differences between DN2 and progenitors was due to decreased production of gd T cells rather DN3 progenitors with regard to their ability to differentiate into than to increased production of DP cells (Fig. 3), indicating that gd-orab-lineage cells in WT mice. They also revealed the developmental progression is accompanied by decreased gd- lineage cell production. Second, a similar pattern with parallel slopes was observed in progenitor cells from WT, Tg-gd, and Eb- KO mice (Fig. 4, Supplemental Fig. 2B), indicating that the de- crease in gd-lineage developmental potential, along with T cell differentiation, is independent of the likelihood of a progenitor cell to productively rearrange TCRg-andTCRd-chains that coexpress at the cell surface, as well as of the expression of a functional pre-TCR; therefore, this decrease is likely to be de- velopmentally regulated. These results are most compatible with the notion of a developmental switch occurring before the DN3 stage, which alters the likelihood of progenitor cells developing as gd-orab-lineage cells. However, differential proliferation and/or survival of gd T cells originating from DN2 or DN3 cells in cul- ture could also explain, at least in part, these results. Differentiation of DP and gd cells from DN2 or DN3 cells occurs with opposite kinetics The gd bias of DN2 cells, compared with the ab bias of DN3 FIGURE 4. Loss of gd-lineage potential as T cell progenitors advance in development. One hundred DN1 (lin2CD117+CD44+CD252), DN2 (lin2 cells, in Tg-gd mice predicts a difference in the kinetics at which 2 2 CD117+CD44+CD25+), DN2–3 (lin CD117intCD44intCD25+), DN3 (lin cells of the two lineages will be generated from DN2 and DN3 CD1172CD442CD25+), and DN3–4 transitional cells (lin2CD1172 cells in WT mice in vitro. To test this prediction, we cloned DN2 CD442CD25int) isolated from WT and Tg-gd mice were cultured and and DN3 cells in OP9-DL1 coculture system and analyzed the analyzed on different days, as in Fig. 3. Data represent the ratio of the generation of gd T cells and DP cells at different time points in maximum number of gd-lineage cells (gdTCR+ cells) and ab-lineage cells independent clones. Fig. 5 shows the results for progeny of 10 (DP cells) obtained in these cultures. The Journal of Immunology 1605

FIGURE 6. Surface expression of TCRgd in progenitors from WT and Tg-gd mice. DN thymocytes from WT and Tg-gd mice were stained with FITC-labeled anti-CD3, PE-labeled anti-Cd, allophycocyanin-labeled anti Downloaded from CD117, PE-Cy7–labeled anti-CD25, allophycocyanin-Cy7–labeled anti- CD44, and a mixture of lineage-specific biotin-labeled Abs specific for CD4, CD8, CD19, CD11b, CD11c, TCRb, and NK1.1, followed by streptavidin-Pacific blue. A, CD25 and CD117 profiles of lineage-negative thymocytes from WT (left panel) and Tg-gd (middle panel). The definition of DN2-like to DN4-like cells is depicted in the right panel. B, Surface

TCRgd expression in the indicated DN-like populations, as defined above, http://www.jimmunol.org/ from WT (filled graphs) and Tg-gd mice (dashed lines).

nisms operating in vivo and precluding expression of TCRgd in the majority of progenitor cells. Of note, TCRd+ cells that fell into the DN4-like gate represented the majority of TCRgd thymocytes, which express no or low levels of c-kit, CD25, and CD44. The same TCRgd induces the differentiation of ab- and

gd-lineage cells in vivo by guest on September 30, 2021 FIGURE 5. Different predisposition of DN2 and DN3 cells to generate The above results help to explain the apparently normal devel- gd-andab-lineage cells in vitro. DN2 cells (one cell/well) and DN3 cells opment of ab-lineage cells in Tg-gd mice (Fig. 1). In these ani- (five cells/well) from WT mice were cultured in OP9-DL1 monolayers in the mals, lack of expression of the transgenes in many progenitor cells presence of IL-7. On the indicated days, half of the cultures were harvested allows normal TCRb rearrangements and, consequently, normal and analyzed by flow cytometry for the presence of TCRgd (gd-lineage differentiation of ab-lineage cells (Fig. 7A). To investigate cells) and CD4 and CD8 (ab-lineage cells). Developmental kinetics of DP whether lineage-potential switch during development observed ab-lineage cells and gd-lineage cells from cloned DN2 (A) or DN3 (B)cells in vitro operates in vivo, we crossed Tg-gd mice with mice defi- from WT mice. C, CD4/CD8 and CD3/TCRd profiles of progenies of two representative DN3 cells after 10 d in culture. D, CD3 expression of the cient in pre-TCR assembling (pTa-KO and Eb-KO mice, which gd-negative population of the two clones depicted in Fig. 4C. lack pTa-chains or TCRb-chains, respectively). We reasoned that, if this were the case, stochastic expression of the transgenes at different developmental points should result in the development of plasticity of DN3 cells, which, although initially prone to develop both gd T cells and ab-lineage DP cells mediated by a unique as ab-lineage cells, produce progeny that can revert this pheno- TCR. As shown in Fig. 7B, compared with their non-Tg litter- type and develop as gd cells. A similar plasticity of progenitors mates, both pTa-KO Tg-gd mice and Eb-KO Tg-gd mice con- after TCRgd expression was shown recently (25). tained 6- and 40-fold more gd and DP thymocytes, respectively. Thus, as previously shown for other TCRgd Tg lines (21), our Tg- Most progenitor cells never express TCRgd, even in Tg-gd gd TCR induced the differentiation of both ab- and gd-lineage mice cells in vivo. In vitro DN2 cells from adult Tg-gd mice differentiate primarily into gd-lineage cells. The same cells in vivo produce normal Environmental independence of the size of the gd thymocyte numbers of ab-lineage cells, presumably through DN3 interme- population diates. A partial solution to this paradox came from the analysis of The lack of TCRgd expression in many progenitor cells observed surface expression of TCRgd in different subsets of progenitor above could be part of the normal developmental program in the cells. Timing and levels of expression of TCRgd were similar in adult thymus that favors the generation of ab cells over gd cells. WT and Tg-gd mice, being detectable for the first time at the DN3 Alternatively, it may result from dysregulation imposed by the stage (Fig. 6). Furthermore, only a small fraction of DN3 and TCR transgenes, which notably increases the number of progen- DN3–DN4 transitional cells expressed detectable levels of surface itors capable of expressing a functionally identical TCRgd that TCRgd, even in Tg-gd mice (1% and 1.9% of DN3 cells and 5.5% may compete for factors or niches that, although not limiting in and 19.3% of DN3–DN4 cells in WT and Tg-gd mice, respec- WT animals, become limiting in Tg-gd mice. To distinguish these tively; Fig. 6B). These results indicated the existence of mecha- possibilities, we generated radiation bone marrow chimeras that 1606 ab AND gd T LINEAGE DIVERGENCE

FIGURE 7. The same TCRgd induces the differentiation of ab- and gd-lineage cells in vivo. A, Intracellular TCRb staining of DN3 and DP cells from WT and Tg-gd mice. Numbers represent mean 6 SD of positive cells in six individual mice/group analyzed at 6–8 wk of age. B, Numbers of DP cells (left panel) and gd thymocytes (right panel)inWT,Eb-KO, and pTa-KO mice expressing TCRgd transgenes (Tg-gd) or not (LM). Data are mean 6 SD of three to six mice/group analyzed individually at 3–4 wk of age. were reconstituted with mixtures of WT and Tg-gd bone marrow the fact that not all combinations of TCRg and TCRd are cells at different ratios. We reasoned that if niches or factors limit expressed at the cell surface (11, 42), results in many daughter the development of gd thymocytes in Tg animals, we should find cells being unable to express TCRgd. However, a major impli- Downloaded from a similar number of Tg-gd T cells in all of the chimeras, irre- cation of this finding is that progenitor cells that fail to express spective of the proportion of WT and Tg progenitors. As shown in a selectable TCRgd still retain the potential to differentiate into Fig. 8, there was a linear correlation between the number of WT ab-lineage cells. and Tg-gd thymocytes developing in the chimeras compared with A second important observation is that, during the DN2 to DN3 the number of WT and Tg progenitors injected. This result ex- transition, there are intrinsic changes in the progenitors that alter

cluded any possible effect of environmental factors in limiting the their likelihood to differentiate into gd-orab-lineage cells. This is http://www.jimmunol.org/ number of gd thymocytes in Tg-gd mice. Rather, it indicated that best illustrated by our experiments showing that DN2 and DN3 the number of gd thymocytes present in Tg-gd mice reflects the cells with identical TCRs have different T cell fate outcomes when maximum number of gd-lineage cells that could develop when assessed at the clonal level (Fig. 2); however, it is also evident in all progenitors carry functionally rearranged TCRg- and TCRd- progenitors from WT animals (Fig. 4) and in those from Eb-KO chains. mice that are unable to form a pre-TCR and that, therefore, can only signal through TCRgd (Supplemental Fig. 2). It could be Discussion argued that this loss of gd-lineage potential during development The data presented in this article revealed three important obser- does not reflect intrinsic changes in developing cells but rather vations that are germane to ab/gd lineage commitment. First, a successful depletion of gd-committed cells from a heteroge- by guest on September 30, 2021 although we were unable to identify a progenitor cell from WT neous population of gd- and ab-committed cells that commit mice that would develop exclusively as gd cells in either the OP9- stochastically during the DN2 to DN3 transition. However, such DL1 system or in FTOC, such a progenitor was evident in the a mechanism cannot explain the high frequency of DN2 cells from DN2 population of Tg-gd mice. This suggested that commitment Tg-gd mice that differentiate exclusively into gd-lineage cells to the gd-lineage occurs only after expression of TCRgd, con- (and the absence of DN2 cells that differentiate exclusively into tradicting selective models of ab/gd lineage choice, which state ab-lineage cells in the same mouse), without postulating that that a binary choice occurs before rearrangement of the TCR ab-lineage commitment is accompanied by the repression of genes. The finding that there are no gd committed DN2 cells in TCRgd expression. But this postulate is incompatible with the WT mice, in contrast to their Tg counterparts, is not unexpected. development of gd-selected DP cells in vivo and in vitro. Im- The stochastic nature of the rearrangement process, together with portantly, the predisposition to differentiate into ab-lineage cells is maintained for some time after expression of TCRgd, as evi- denced by the fact that about one fifth of the TCRgd+ DN3 cells retain the potential for both ab and gd T cell differentiation (25). The third important observation is that there is an absence of surface expression of TCRgd in the majority of cells transitioning from DN2 to DN4 stages, even when they carry functionally rearranged TCRg and TCRd chains known to pair at the cell surface. This is unlikely to be a Tg artifact, because the number of gd T cells that develop in the thymus is a direct function of the likelihood that a progenitor will assemble and express a selectable TCRgd at the cell surface, both in the chimeras shown in Fig. 8, as well as in non-Tg animals carrying only one or two functional alleles of Cd (43), thus excluding a possible role for environmental factors in limiting the number of gd thymocytes in Tg-gd mice. FIGURE 8. The size of the gd thymocyte population in WT and Tg-gd Furthermore, mRNA expression of the Tg Vg1 chain in progenitor mice is independent of extrinsic factors. Irradiated B6 mice were recon- stituted with 2 3 106 T cell-depleted bone marrow cells from CD45.2 WT cells coincides with the time when the TCRg locus opens for mice and CD45.1 Tg-gd mice, at the indicated ratios. Depicted are num- rearrangement in WT mice (39), and the ontogenic time at which bers of gd thymocytes of WT or Tg-gd origin analyzed 10 wk after re- protein expression of the Tg TCR is detectable (Fig. 6) parallels constitution. Each symbol represents the mean of three mice analyzed that of endogenous TCRgd, indicating a seemingly normal ex- individually. pression of the Tg chains in Tg-gd mice. Similar mechanisms are The Journal of Immunology 1607 likely to operate in WT animals. Thus, only 0.5–1% of WT DN3 DN3 progenitors in vitro and the fact that most DN3 from Tg-gd cells express detectable levels of surface TCRgd (12, 25), whereas mice are not yet lineage fixed are best explained within this 15–20% of the same DN3 population expresses intracellular scenario. Thus, although consistent with stochastic models of TCRb-chains and, consequently, the pre-TCR (44, 45), frequen- ab/gd-lineage commitment, these results place the commitment cies also reported in preselected DN3 cells (12, 44). Although point after expression of the TCR. Alternatively, one may consider TCRgd requires two successful rearrangement events, as opposed that the strength of signal resulting from expression of TCRgd in to one for the pre-TCR, the expected frequency of pre-TCR– gd-selected DN3 cells moves cells away from their initial expressing cells should not exceed that of TCRgd-expressing cells ab-lineage pathway and commits them to the gd lineage. As by .2-fold (11). Thus, mechanisms precluding surface expression discussed elsewhere (34), the signal-commitment model cannot of TCRgd are required to explain the 10–40-fold higher frequency accommodate the fact that the same TCRgd can drive the gener- of pre-TCR–expressing cells over TCRgd-expressing cells in the ation of DP and TCRgd+ cells without invoking heterogeneity in WT DN3 population. The molecular nature of these mechanisms the signal received by progenitor cells expressing identical TCRs is unknown, but they may be related to the relatively low TCRg at the same developmental stage and that can only be stochastic in transcription previously found in DN2 and DN3 cells from Tg-gd nature. mice (39). These mechanisms are likely unrelated to the change in In conclusion, our results indicated two points of commitment lineage potential discussed above. Their major consequence is that during T cell differentiation. The first one is developmentally ab-lineage differentiation is favored over gd-lineage differentia- regulated and sequential, rather than binary, allowing a fraction of tion in adult mice. progenitor cells to develop as gd-lineage cells during a develop-

We propose a revised model for T cell-lineage determination that mental window. After that, the ab lineage of development is fa- Downloaded from integrates these results and observations documented in the liter- vored but not fixed, and commitment to the gd lineage is still ature. Our results indicated that DN2 cells are predisposed to possible. The model of ab/gd-lineage commitment presented in differentiate into gd cells and, as they advance through develop- this article provides a rationale for the fact that, in normal animals, ment to the DN3 stage, progressively lose gd-lineage potential DN2 cells are endowed with a set of genes that is important for while acquiring ab-lineage potential. During the DN2 to DN3 proper gd T cell function but that is no longer expressed at the

transition, expression of TCRgd results in gd cell differentiation, DN3 and DN4 stages (16, 53). It can also accommodate hetero- http://www.jimmunol.org/ as originally proposed by Pardoll et al. (46) and Allison and geneity in the DN2 population that has been interpreted as support Lanier (47). Likely, the differentiation of gd cells at this stage is for stochastic models of lineage development (54), because de- fast, such that putative intermediate populations are difficult to creased levels of expression of IL-7R mostly correlate with the detect in steady-state analyses ex vivo. The gd cells developing DN2 to DN3 transition. It also accommodates the existence of gd through this pathway initially express CD117 and represent ∼5% cells expressing functional TCRb-chains (45) due to late devel- of the total gd thymocytes in WT animals and ∼50% in Tg-gd opment of gd cells after b selection (52). Finally, it proposes mice at any given time point (39). Therefore, c-kit expression may heterogeneity in the development of gd T cells that might be be a marker of newly generated gd cells that develop from DN2– linked to the development of different functional subsets (55). DN3 transitional cells. Because of the potential loss of c-kit ex- by guest on September 30, 2021 pression, the contribution of this pathway to the total gd com- Acknowledgments partment is difficult to determine. However, the finding that 50– We thank Bruno Silva-Santos, Dan Pennington, and Adrian Hayday for dis- 70% of the gd thymocytes contain incomplete DJb (44, 48) re- cussion and critical reading of previous versions of the manuscript. arrangements strongly suggests that more than half of the gd thymocytes differentiated before the onset of V-DJb rearrange- Disclosures ments at the DN3 stage. The authors have no financial conflicts of interest. By contrast, DN3 cells are predisposed toward ab-lineage differentiation. 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