Antibodies and Flow Cytometry.

Anti–δ TCR (GL-3), anti–β TCR (H57), anti-CD3ε (500A2), anti–IL-7Rα (A7R34), anti-CD45.2 (104), and anti-CD45.1 (A20) mAbs were purified and conjugated with FITC or biotin using standard protocols. Anti-CD44–FITC, anti-CD25–PE, anti–c-kit (CD117)-allophycocyanin (APC), anti-CD8–Tricolor mAbs, and streptavidin-Tricolor were purchased from Caltag. Anti-CD44–Cy5, anti-CD11c–biotin, anti-CD25–FITC, anti-CD45.2 (Ly5.2)–FITC, anti–c-kit–FITC, anti–IL-7Rα (CD127, B12-1)–biotin, anti–mouse I-A^b–PE mAbs, and strepavidin-PE were supplied by BD PharMingen. Anti-CD4–613, anti-CD8–613, anti-CD25–613, and strepavidin-613 were acquired from GIBCO BRL. Rabbit polyclonal antibody against IL-7Rα (D20) was supplied by Santa Cruz Biotechnology, Inc. A7R34 rat anti–mouse IL-7Rα mAb was a gift from Dr. S.-I. Nishikawa and was obtained from Dr. M. Kondo (Stanford University, Palo Alto, CA). Anti–rabbit IgG–biotin and anti–bromodeoxyuridine (BrdU)–FITC were purchased from Vector Laboratories and Becton Dickinson, respectively. Incorporation of BrdU into dividing thymocytes in vivo was carried out by injecting mice intraperitoneally twice at a 2-h interval with 0.9 mg of BrdU 3 4. Thymocytes from injected mice were analyzed 1 h later by flow cytometry. For the analysis of IL-7Rα expression on thymic precursor populations, CD4⁻CD8⁻ thymocytes were isolated by rabbit and guinea pig complement–mediated lysis of CD4⁺ and/or CD8⁺ thymocytes using anti-CD4 (RL172) and CD8 (AD4-15) ascites fluids. After one or two rounds of complement lysis >99% pure double negative (DN) cells were obtained. Cells were incubated with 2.4G2 to block Fc receptors, then stained with mAbs specific for CD25-613, CD44-Cy5, IL-7Rα–biotin/strepavidin-PE, and CD3ε-FITC (or c-kit–FITC), and analyzed by four-color flow cytometry on an XL-MCL flow cytometer (Beckman Coulter). Since virtually all CD25⁺ DN cells were CD3⁻, the combination of anti-CD25–FITC/CD44-Cy5/IL-7Rα (B12-1)–biotin-PE and CD8-613 or c-kit–APC mAbs was used for the analysis of pro-T and pre-T subsets. IL-7R⁺ and IL-7R^neg-lo pro-T cells were sorted with an ELITE cell sorter (Beckman Coulter) using >99% pure DN thymocytes stained with the mAbs as above and then gating on CD44⁺CD25⁺ (3% of total DN thymocytes) population and sorting for the cells expressing high levels of IL-7R (25–30% of total pro-T cells) and non- or low expressing cells (20–25% of total pro-T cells). Of the anti–IL-7Rα mAbs, B12-1 gave the best separation and was used for all sorting experiments. Cell yield for each pro-T cell subset was 3–5 × 10³ cells/mouse.

Cell Culture, Fetal Thymic Organ Culture, and Intrathymic Injection.

Sorted pro-T cells were cultured for 4 d at 10⁵ cells/ml in medium (RPMI 1640 supplemented with 10% FCS, 50 μM 2-ME, 2 mM l-glutamine, 20 mM Hepes, and antibiotics) containing 10 ng/ml of rIL-7 (Genzyme) or a 1:100 dilution of cell culture supernatants from J558 plasmacytoma cells transfected either with IL-7 or stem cell factor (SCF) cDNAs 19. For fetal thymic organ culture (FTOC), 1.5 × 10⁴ viable, sorted cells were used in a 2-d hanging drop culture to reconstitute fetal day 14 thymic lobe that had been previously depleted of resident thymocytes by treatment with 1.35 mM 2′-deoxyguanosine for 5 d. Repopulated thymi were transferred to a Transwell plate and cultured for 9–24 d. For some experiments, thymi from B6-Ly5.1 mice were used to check for host thymocyte survival in FTOC. Invariably, recipient thymocytes were not detected during FTOC in these experiments. At the early phase of culture (days 9–11) three to five thymi were pooled to obtain sufficient cells for flow cytometric analysis using appropriate mAbs. At a later phase of culture (day 21–22) individual thymi were analyzed. For intrathymic injection, sorted cells from B6 mice were washed and resuspended in PBS/0.1% BSA and injected into thymi of 4–6-wk-old sublethally irradiated (750 rads) B6-Ly5.1 congenic mice. For pro-T cells and pro-T subsets ~2 × 10⁴ sorted viable cells were injected. For pre-T cells ~2 × 10⁵ cells were injected. Reconstituted thymi were analyzed by flow cytometry 8–11 d after injection. DCs were isolated as described 20. In brief, thymi were digested for 1 h at 37°C in serum-free medium containing 1.6 mg/ml collagenase and 0.1% DNase. DCs were incubated with biotinylated anti-CD11c mAb and subsequently with streptavidin-coupled magnetic beads. They were then positively selected by magnetic cell sorting (MACS; Miltenyi Biotec) and analyzed by flow cytometry after staining with Ly5.2 (donor)-FITC and streptavidin-Tricolor that binds to the remaining free biotin of CD11c mAb on DCs.

IL-7Rα⁺ and IL-7Rα^neg-lo Pro-T Cells Exhibit Differences in Developmental Potential.

The developmental potential of sorted IL-7Rα⁺ and IL-7Rα^neg-lo pro-T cells (from B6-Ly5.2 mice) was examined after intrathymic injection into adult B6-Ly5.1 mice. 7–11 d after engraftment, the reconstituted thymi were analyzed for the content of differentiated γδ⁺ cells and CD4⁺CD8⁺ (αβ lineage) cells that had arisen from donor cells (Fig. 3 A, and Table ). Strikingly, the two pro-T cell populations differed substantially in their lineage potential. In the experiment shown in Fig. 3 A, IL-7Rα⁺ pro-T cells gave rise to 12-fold more γδ thymocytes than did IL-7R^neg-lo pro-T cells; the average difference was fivefold (Table ). Unseparated pro-T cells exhibited an intermediate capacity to generate γδ cells (Table ). Conversely, IL-7R^neg-lo pro-T cells gave rise to more αβ lineage cells than did IL-7Rα⁺ cells, by an average of more than fivefold numerically, although the difference was less dramatic when expressed as a percentage of donor-derived cells (Table ). Overall, the average ratio of the percentage of CD4⁺CD8⁺ cells and the percentage of γδ⁺ cells in individual reconstituted mice was >13-fold lower with IL-7Rα⁺ pro-T cells than with IL-7R^neg-lo pro-T cells (Fig. 3 B). When the host cell types were analyzed in the same animals, the difference in the ratio was limited to twofold (Table ). It is important to note that the sorted donor pro-T cell subpopulations differentiated in the presence of a large excess (20–100-fold; Table ) of developing host thymocytes. Thus, thymic “niches” and other undefined factors that might influence development of the cells should be equivalently available to both populations of injected cells. Therefore, the different outcomes observed indicate that the IL-7Rα⁺ and IL-7R^neg-lo pro-T cell subpopulations differ intrinsically in their developmental potential.

Open in a separate window

Figure 3

Intrathymically injected pro-T cell subsets displayed distinct γδ versus αβ lineage developmental potential. (A) Representative profiles of thymocytes generated from donor (Ly5.2⁺) precursor cells. CD4/CD8 profiles of donor-type thymocytes, γδ TCR expression on gated donor-type CD4⁻CD8⁻ thymocytes, and αβ TCR expression on donor thymocytes are illustrated. Host thymocyte profiles are presented for a mouse injected with saline alone. Percentages in brackets represent the percentage of γδ TCR⁺ cells of all donor thymocytes. In the profiles compared, IL-7Rα¹ and IL-7Rα^neg-lo pro-T cells generated similar numbers of donor-derived thymocytes. (B) The ratio of αβ to γδ thymocytes generated from the respective sorted populations 8–11 d after injection. The bars represent averages of αβ/γδ thymocyte ratio of individual mice (see Table ). Total donor thymocyte numbers were higher in the thymi injected with IL-7R^neg-lo pro-T cells, but the broad range (0.2–10.6%) of donor cell reconstitution level in each experimental group makes the interpretation of this difference ambiguous. (C) Representative profiles of five independent experiments show similar proportions of DCs generated from the pro-T subsets (average proportions of donor DCs in CD11c⁺ population ± SEM: IL-7Rα^neg-lo, 10.4 ± 2.6%; IL-7Rα⁺, 12.2 ± 2.4%). Thymic DCs were purified from mice 9 d after intrathymic injection of pro-T cells or no cells (mock), as indicated. The cells were analyzed for CD11c expression, which identifies all DCs, and for Ly-5.2 expression, which distinguishes DCs of donor (Ly-5.2⁺) versus host (Ly-5.2⁻) origin. Purified DCs (all MHC class II⁺) from mice with 4% and 1.5–1.9% donor cell reconstitution from injected IL-7Rα^neg-lo and IL-7Rα⁺ pro-T cells, respectively, are shown. For comparison, cells from a mouse that underwent the same surgical procedure but was not injected are presented (mock).

Intrathymically injected pre-T cells efficiently differentiated into αβ lineage cells, but yielded extremely few γδ cells (Table ; Fig. 3A and Fig. B). In some experiments pre-T cells yielded no detectable γδ T cells. Hence the αβ/γδ ratio for pre-T cells was 10-fold higher than that of IL-7R^neg-lo pro-T cells, and 100-fold higher than IL-7Rα⁺ pro-T cells. The results suggest that most pre-T cells are restricted to the αβ lineage. It should also be noted that ~10 times as many pre-T cells as IL-7R^neg-lopro-T cells had to be injected to generate similar numbers of αβ lineage cells (Table ). The much lower cell generative capacity of pre-T cells compared with pro-T cells excludes the possibility that contaminating pre-T cells can account for the αβ lineage preference of the IL-7R^neg-lo pro-T cell subset.

In contrast to the apparent γδ/αβ lineage bias in pro-T subsets, injected IL-7Rα⁺ and IL-7Rα^neg-lo pro-T cell subsets, like unseparated pro-T cells 17, generated similar proportions of lymphoid DCs (Ly5.2 donor type, MHC class II⁺ and CD11c⁺; Fig. 3 C). Thus, the two pro-T subsets do share some developmental properties associated with an early T progenitor population, but specifically differ in their ability to generate γδ versus αβ lineage cells.

The lineage bias observed in the pro-T subsets developing in the adult thymic environment in vivo was recapitulated in FTOC. Host thymocyte-depleted E14 thymi were repopulated with sorted pro-T cells. At a relatively early stage of the cultures (days 9–11), IL-7Rα⁺ pro-T cells yielded approximately three times the percentage of γδ thymocytes as IL-7Rα^neg-lo pro-T cells (Fig. 4 A and Table ). Conversely, IL-7Rα⁺ pro-T cells generated fewer αβ lineage cells than IL-7Rα^neg-lo cells, although the difference was not striking. The total numbers of thymocytes generated from the two pro-T subsets in FTOC were not significantly different (Table ). Thus, as was observed in the intrathymic injection studies, the average ratio of αβ/γδ lineage cells generated by IL-7Rα^neg-lo pro-T cells was higher than that of IL-7Rα⁺ pro-T cells, in this case by a factor of five on average (Table ). At a later stage of FTOC (21–22 d) both input populations yielded somewhat lower percentages of γδ cells and higher percentages of αβ lineage cells, probably because of greater expansion of αβ lineage cells. Nevertheless, the IL-7Rα^neg-lo pro-T cells still yielded fewer γδ cells and more αβ lineage cells than IL-7Rα⁺ pro-T cells, resulting in an average threefold difference in the αβ/γδ ratio (Table ). Thus, the lineage bias of the two pro-T cell subsets is apparent at both early and late stages of thymic reconstitution.

Open in a separate window

Figure 4

The sorted pro-T subsets display distinct γδ/αβ lineage developmental potential in FTOC. (A) Thymocytes from FTOCs reconstituted with sorted pro-T cell subsets were analyzed for γδ TCR and CD4/CD8 expression after 11 d of culture. Representative profiles are from three thymic lobes that were pooled. Many of the cells are accumulating on the axes of the profiles as a result of four-color compensation settings. (B) Similar transgene expression in sorted pro-T cell subsets from low 1 2 transgene copy line as determined by semiquantitative RT-PCR using transgene-specific primers. Numbers indicate approximate cell equivalents. Two additional experiments yielded comparable results. No PCR products were detected when the RT step was omitted. (C) Thymocytes from FTOCs reconstituted with sorted pro-T cell subsets from high copy G8 transgenic mice were analyzed for γδ TCR and CD4/CD8 expression after 10 d of culture. The levels of IL-7Rα expression on the pro-T progenitor populations in the transgenic mice were not notably different from those in nontransgenic littermates. Three thymic lobes of each type were pooled for analysis.

Are the Pro-T Cell Subsets Sequentially Related Populations That Exhibit Distinct TCR Rearrangement Potential, but No Other Intrinsic Lineage Bias?

One possibility is that the IL-7Rα⁺ pro-T cell population precedes the IL-7Rα^neg-lo population in a developmental sequence. If cells at the IL-7Rα⁺ stage rearrange TCR-γ and -δ genes in preference to TCR-β genes, the population would exhibit a relative bias in favor of γδ cell development. Failure to successfully initiate γδ lineage development at this stage would result in subsequent differentiation of IL-7Rα^neg-lo pro-T cells. At this stage TCR-γ, -δ, and -β genes may rearrange more or less equivalently. Equivalent rearrangement of the three genes would marginally favor αβ lineage development, which requires only one in-frame rearrangement (of TCR-β) as opposed to the two in-frame rearrangements (of γ and δ) required for γδ lineage development. This scheme differs substantially from models in which the two populations represent divergent paths, though it does imply different lineage potential in the two populations due to an early preference for TCR-γ and -δ rearrangements.

Several lines of evidence are inconsistent with this sequential model. First, if all developing T cells traverse the IL-7Rα⁺ pro-T cell stage, the latter cells should ultimately yield αβ and γδ lineage progeny in physiological proportions, i.e., a ratio of 100 or more, and should not exhibit a significant bias for γδ lineage development. In fact, the IL-7Rα⁺ pro-T cell subset yielded an αβ/γδ ratio of ~15 (Fig. 3 B). The discrepancy is unlikely to reflect differences in developmental kinetics in the populations, because the lineage bias was evident at both early and late times after initiation of FTOCs (Table ). In contrast, unseparated pro-T cells yielded αβ and γδ progeny in physiological proportions. A second persuasive argument against the sequential scheme arises from its prediction that many cells in the IL-7Rα^neg-lo population should have rearrangements at the TCR-δ and/or -γ loci. This is because the majority (67%) of the TCR-γ and -δ rearrangements that would occur at the “early” IL-7Rα⁺ stage are predicted to be nonproductive. Many cells with productive rearrangements of one of the genes (γ or δ) would have nonproductive rearrangements of the other, and some cells would have only nonproductive rearrangements at both loci. All of these cells would be unable to differentiate into γδ cells. If they have no intrinsic lineage bias they should then convert to the “subsequent” IL-7Rα^neg-lo population, contributing numerous chromosomes with rearranged TCR-γ and -δ alleles. In strong contrast to this expectation, the latter population is essentially devoid of cells with TCR-δ rearrangements (Fig. 2 E), and contains a very low frequency of rearranged TCR-γ alleles similar to that in the IL-7Rα⁺ population. Yet, αβ lineage cells are known to contain high levels of TCR-γ and -δ rearrangements, most of which are nonproductive 30 31 32. It is likely that these rearrangements occur subsequent to the IL-7Rα^neg-lo stage rather than before. These data argue against the proposal that IL-7Rα⁺ pro-T cells preferentially undergo TCR-γ and -δ rearrangements before differentiating into IL-7Rα^neg-lo pro-T cells. Although the results do not rule out all conceivable models in which the two subsets are sequentially related, the simplest interpretation is that the two subsets represent alternative, as opposed to sequential, developmental stages.

Is Differential IL-7R Signaling in Pro-T Cells Responsible for the Observed Lineage Bias?

Previous reports demonstrate that IL-7R signaling plays a unique role in γδ cell development, as it promotes rearrangement and expression of TCR-γ genes but is not necessary for rearrangement or expression of the other TCR genes 18 27 28 33. Thus, an obvious possibility was that the bias of the IL-7Rα⁺ subset for γδ lineage development resulted from enhanced rearrangement and expression of TCR-γ genes in this subset. Conversely, the αβ lineage bias of the IL-7Rα^neg-lo pro-T cell subset might be because of impaired rearrangement and expression of TCR-γ genes. However, several lines of evidence indicate that IL-7Rα^neg-lo pro-T cells and pre-T cells have the capacity to support high levels of TCR-γ gene expression, despite the low or absent levels of IL-7Rs on these cells. First, the low copy prerearranged TCR-γ transgene, whose transcription was previously shown to be IL-7Rα dependent, was expressed at a similar level in the pro-T subsets (Fig. 4 B). Second, in pre-T cells, endogenous TCR-γ rearrangements are abundant, and these are expressed at a high level despite negligible IL-7R on these cells (data not shown). Third, Vγ2-Jγ1 rearrangements, although very rare at the pro-T cell stage, are equally represented in the two pro-T cell subpopulations as determined by semiquantitative PCR (data not shown). Finally, it is notable that αβ lineage cells contain very high levels of mostly nonproductive TCR-γ rearrangements 34 35; it is therefore implausible to argue that rearrangement of TCR-γ genes is impaired in the progenitors of these cells. Collectively these data suggest either that IL-7Rα signaling at an earlier stage of development allows TCR-γ gene activation at a later stage, or that low levels of IL-7R are sufficient to activate the locus at the pro- and pre-T cell stages.

Although these arguments suggest that the two pro-T cell subsets are equally capable of activating the TCR-γ locus, we sought a more direct test of whether the lineage bias was attributable to impaired TCR-γ gene expression. The results indicated that the lineage bias was still evident even in the presence of high levels of functional TCR γ chain in both pro-T subsets, directed by a high copy transgene (Fig. 4 C). These findings represent a strong argument against the possibility that the lineage bias of the pro-T cell subsets results from differential TCR-γ gene activation as a consequence of distinct IL-7Rα levels. Nevertheless, IL-7Rα levels correlate with lineage-biased subsets in young adult mice. It should be noted that pro-T cells in newborn mice uniformly express high levels of IL-7Rα (data not shown). Thus, the correlation between IL-7R levels on pro-T cells and lineage potential may hold true only in a homeostatic steady-state thymic environment.

We thank Drs. C. Chambers, E. Robey, L. Berg, and R. Vance for helpful discussions and review of the manuscript; P. Schow, H. Nolla, R. Vance, and C. McMahon for technical assistance with flow cytometric cell sorting; and A. Lazar for providing B6 mice.

This work was supported by grants to D.H. Raulet from the National Institutes of Health. J. Kang was a research fellow of the National Cancer Institute of Canada supported by the Canadian Cancer Society. A. Volkmann was supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation.