The Proportion of NK Cells Expressing Each Ly49 Receptor Increases during Ontogeny.
Cells with the NK phenotype (NK1.1+CD3−) were enriched from splenocytes of B6 mice of various ages, and the percentage of NK cells that stained with each of three Ly49 receptor-specific antibodies was assessed. The percentage of NK cells expressing any particular Ly49 receptor increased over ontogeny, reaching appreciable levels by 2–3 wk of age, and reaching adult levels at 6–8 wk of age (Fig. ). A similar pattern was observed for NK1.1+CD3− cells from the bone marrow, except that smaller percentages stained with the antibodies at each age point studied (data not shown).
Figure 1 Ontogeny of expression of Ly49 receptors by splenic NK cells. Early splenic NK1.1+CD3− cells do not express Ly49 receptors and gradually acquire them during postnatal life. The proportion of NK1.1+CD3− cells (± (more ...)
The timing of the appearance of Ly49+
NK cells in the spleen closely matches the appearance of NK activity in the mouse as assayed by their competence to reject bone marrow grafts (17–22 d; reference 29
) or as assayed by the appearance of in vitro NK activity in the spleen (18–26 d; references 14
). These findings raised the possibility that the NK cells found in younger (<3 wk) mice, few of which express Ly49 receptors, are largely inactive. However, it is not known whether the Ly49−
NK cells in young mice are deficient in functional activity, as suggested by Hackett et al. (14
), or are simply too infrequent to mediate substantial NK cell activity, as suggested by their lower absolute cell numbers in the spleen during early life (data not shown).
Acquisition of Ly49 Receptor Expression by NK1.1+ Ly49A−C−G2−I− Cells.
NK cells that express the tested Ly49 receptors could arise directly from NK1.1− progenitor cells. Alternatively, they could arise from NK1.1+CD3− cells that do not express these Ly49 receptors. We employed a cell transfer system to assess whether NK1.1+CD3− cells that do not express the tested Ly49 receptors can subsequently acquire them.
Splenocytes from 40–150 B6 mice of 18–45 d of age were pooled, enriched for NK cells, and then stained and sorted for NK1+CD3− Ly49A−C−G2−I− cells. Sorted populations were reanalyzed at the completion of the sort; the reanalysis of a representative experiment is shown in Fig. a. Sorted cells were injected i.v. into B6 Ly5.1 mice 2–3 h after the mice were sublethally irradiated. After 10 d, the spleens of the host mice were excised and splenocytes were stained without enrichment. Donor cells were recognized by an anti-Ly5.2 antibody, and generally constituted ~0.5% of the splenocytes (Fig. , b and c). The donor cells were generally >90% NK1.1+. The number of donor-type cells recovered ranged from 2–20% of the number injected, with an average of ~10%.
Figure 2 Ly49A−C−G2−I− NK cells express some Ly49 receptors after in vivo transfer. Ly49A−C−G2−I− NK cells were purified by cell sorting from B6-derived, NK-enriched splenocytes and then transferred (more ...)
10 d after transfer, ~90% of the donor-derived cells were NK1.1+
cells, suggesting that the donor NK phenotype is quite stable. No CD3+
donor cells were detected in the spleen and no donor cells of any type were detected in the thymuses of the recipient mice. Significantly, although essentially none of the donor-derived cells expressed Ly49A, Ly49C/I, or Ly49G2 at the time of transfer, an appreciable fraction expressed Ly49C/I (Fig. e
) or Ly49G2 (Fig. f
) after transfer. Surprisingly, none of the donor cells expressed Ly49A after transfer (Fig. d,
and see below).
The results of 11 replicates of this experiment are summarized in the first line of Table , including two experiments in which donor cells of B6 origin were injected into BALB.B mice, and one experiment in which donor cells of B6 Ly5.1 origin were injected into B6 mice. Ly49C/I and Ly49G2 receptors were consistently expressed by a significant fraction of cells after transfer, whereas no significant expression of Ly49A was observed in any of the experiments. Control mice were routinely injected with HBSS in parallel and then analyzed at the same time that host mice were analyzed. Few or no cells that stained with the donor-specific antibodies were detected in these mice. Furthermore, control antibodies specific for the host cell marker did not stain the donor-derived cells, indicating that the staining procedure was specific for donor-type cells. These results suggest that Ly49C/I and Ly49G2 expression was induced on a fraction of transferred splenic NK1.1+
cells, whereas Ly49A was not. It is notable that Ly49C and Ly49I receptors are H-2b
–reactive receptors (31, 32; Hanke, T., and D.H. Raulet, unpublished data), and hence self-reactive in these experiments, whereas Ly49G2 is not H-2b
). Therefore, the capacity of a receptor to be expressed after transfer is not dependent on it being self-specific.
Summary of Transfer Experiments with Ly49A−C−G2−I− NK Cells or Bone Marrow Cells
Expression of a Ly49 Receptor Is Stable upon In Vivo Transfer.
As shown above, Ly49A−C−G2−I− NK cells can give rise to Ly49C/G2/I+ NK cells. One possible interpretation of this observation is that cells are constantly turning Ly49C, Ly49G2, and/or Ly49I on and off. To address this possibility, we purified populations of NK cells expressing Ly49A, Ly49C/I, or Ly49G2. These populations were transferred individually into Ly5-congenic mice. In representative experiments, NK1.1+ cells expressing Ly49A (Fig. a), Ly49C/I (Fig. b), or Ly49G2 (Fig. c) were purified. Cells from each sort were injected into sublethally irradiated B6 Ly5.1 mice, and splenocytes from the recipient mice were analyzed after 10 d. As can be readily seen, the large majority of cells of donor origin continued to express the selected receptor even 10 d after in vivo transfer (Fig. ). Whether or not this stability extends beyond 10 d remains untested.
Figure 3 Cells expressing particular Ly49 receptors before transfer continue to express them after in vivo transfer. B6 NK cells sorted for expression of the Ly49A (a), Ly49C/I (b), or Ly49G2 (c) receptors were transferred into B6-Ly5 congenic mice. Analyses (more ...) Failure of Ly49A−C−G2−I− NK Cells to Acquire Ly49A after Transfer.
It was surprising that donor NK1.1+ Ly49A− C−G2−I− cells specifically failed to express Ly49A after transfer. The Ly49A stain was effective, because host-derived Ly49A+ NK cells were readily detectable in the same samples (not shown). Furthermore, Ly49A+ NK cells were easily detected among donor-type cells after injection of unsorted, enriched NK cells regardless of whether or not these cells had been stained in the same manner as was used for the sorting experiments (not shown). Also, Ly49A+ NK cells were readily detectable when sorted Ly49A+ NK cells were injected (Fig. a). Thus, the failure to detect Ly49A+ NK cells after transfer of Ly49A−C−G2−I− NK cells does not reflect an inability to detect Ly49A expression.
The possibility was considered that the failure to detect Ly49A+ NK cells after transfer of NK1.1+Ly49A− cells reflected the relatively advanced age (18–45 d old in different experiments) of the donor mice. However, transferred Ly49A−C−G2−I− NK cells from 4–10-d-old donors also failed to produce significant numbers of Ly49A+ NK cells (Table ). Since most endogenous Ly49A+ cells appear in normal mice after 10 d of age (Fig. ), it appears unlikely that precursors of Ly49A+ NK cells have already disappeared by this age.
Another possibility was that acquisition of Ly49A is delayed compared with the other receptors and had not yet occurred by 10 d after transfer. However, even 20 d after transfer of Ly49A−C−G2−I− NK cells no Ly49A+ NK cells could be detected (Table ). In normal mice, Ly49A expression reaches nearly adult levels within 20 d of birth (Fig. ). There was also the possibility that Ly49A expression occurs immediately after transfer and is subsequently rapidly extinguished. However, as shown above, transferred Ly49A+ NK cells persist and faithfully continue to express Ly49A 10 d after transfer (Fig. a). Therefore, it is unlikely that the expression of Ly49A after transfer is transient. Finally, it seemed possible that irradiated mice represent a nonpermissive environment for differentiation of Ly49A+ NK cells from Ly49A− progenitor cells. However, irradiated mice reconstituted with NK cell–depleted bone marrow cells generated normal numbers of donor-type Ly49A+ NK cells 12 wk after reconstitution (Table ). Similar results were obtained when fetal liver cells were transferred (data not shown).
The above experiments and considerations suggest that irradiated mice are permissive for acquisition of Ly49A by differentiating NK cells, and that the failure to detect these cells after transfer of Ly49−NK1.1+ cells is unlikely to reflect inadequate detection procedures. It thus appears likely that splenic NK1.1+CD3− cells are competent to initiate expression of some Ly49 receptors (Ly49C/I and Ly49G2), whereas initiation of Ly49A expression by these cells either does not occur or is grossly delayed (see Discussion).
Successive Expression of Ly49 Receptors.
Although the donor cells in the above transfer experiments were Ly49A−C− G2−I− cells, it is highly likely that at least some of them expressed one of the at least five other members of the Ly49 receptor family, for which specific antibodies were not available. With this in mind, two nonmutually exclusive explanations can be considered for the initiation of Ly49C/I and Ly49G2 receptor expression after transfer of Ly49A− C−G2−I− NK cells. The first is that the Ly49-expressing cells after transfer were all derived from transferred NK1.1+ progenitor cells that did not express any Ly49 receptors. The competing possibility is that some or all of the Ly49C/ I+ and Ly49G2+ cells after transfer were derived from NK cells that already expressed other Ly49 family members at the time of transfer. If this were the case, it would imply that expression of new Ly49 receptors on NK lineage cells occurs rather gradually and successively.
To investigate whether Ly49 receptors can be expressed successively, we purified Ly49A+C−G2−I− cells (referred to below as Ly49A single positive cells) and transferred them to Ly5 congenic mice. After 10 d, an appreciable fraction of the cells expressed Ly49C/I or Ly49G2 (Fig. , Table ). In other experiments, Ly49A−C−G2+I− cells (referred to below as Ly49G2 single positive cells) were purified and transferred. 10 d after transfer, an appreciable fraction of these cells expressed Ly49C and/or Ly49 (Table ). Thus, splenic NK cells that already express at least one Ly49 receptor were capable of expressing new Ly49 receptors after in vivo transfer. These data suggest that Ly49 receptors may be expressed successively during NK cell development. The newly expressed receptors that we observed included receptor(s) that are self-specific (Ly49C and/or Ly49I) and a receptor that is not self-specific (Ly49G2). Thus, there was no correlation between the presence of a MHC class I ligand for the newly expressed receptor and the ability of the NK cell to initiate its expression. However, when Ly49G2+ NK cells were transferred, few or none of them expressed Ly49A 10 d after transfer (Table ). These data extend the earlier results indicating that transferred NK1.1+Ly49A− cells do not give rise to Ly49A+ cells.
Figure 4 Ly49A single positive (Ly49A+C−G2−I−) NK cells express Ly49C/I and Ly49G2 after in vivo transfer. Ly49A+C−G2−I− NK cells were purified by cell sorting from B6-derived, NK-enriched splenocytes (more ...)
NK Cells Already Expressing an Ly49 Receptor Remain Capable of Expressing New Receptors After In Vivo Transfer
Ly49-expressing Cells after Transfer Are Not Derived from Contaminating Ly49C/I or G2+ Cells or NK1.1− Cells in the Transferred Population.
The previous data suggest that NK1.1+ CD3− cells that do not express a Ly49 receptor can acquire it after in vivo transfer. However, it remained possible that the Ly49C/I or G2+ cells after transfer were derived not from the majority Ly49A−C−G2−I− NK1.1+ CD3− population, but from contaminating cells in the inoculum, which might selectively survive or expand after transfer. One possibility was that the Ly49C/I or G2+ cells after transfer were derived from small numbers of contaminating Ly49C/I or G2+ cells in the inoculum. To address this possibility, the inoculum population (Ly49A−C−G2−I− NK cells from B6 mice) was purposefully contaminated with a defined percentage of sorted Ly49C/I or G2+ NK cells from B6-Ly5.2, as diagrammed in Fig. . The Ly5 difference allowed us to evaluate the contribution of the contaminants to the resulting Ly49C/I or G2+ NK cells after transfer. To distinguish both types of donor cells from host-type NK cells after transfer, the cells were inoculated into irradiated BALB.B recipients that fail to express NK1.1 on their NK cells. Thus, cells that expressed NK1.1 were sure to be of donor origin, and whether they were derived from the added Ly49C/I or G2+ contaminant population could be ascertained based on Ly5 expression.
Figure 5 Experimental design to assess contribution of cellular contaminants to Ly49C/I or G2+ populations present after cell transfer. Ly49A−C−G2−I− NK1.1+ cells were purposefully contaminated with tracer Ly5 (more ...)
Post-sort analysis indicated that the Ly49A−
NK cells purified from B6 (Ly5.2) splenocytes contained
0.1% Ly49C/I or G2+
NK contaminants (not shown). This population was spiked with sorted Ly49C/I or G2+
NK cells from B6-Ly5.1 mice, so as to achieve 2% contamination, a >20-fold excess (2%/
0.1%) compared with B6-derived contaminants (Table ). After flow cytometric analysis to confirm the contamination level, the cell mixture was inoculated into BALB.B mice. Although the contaminant population accounted for
95% of the Ly49C/I or G2+
NK cells at the time of injection, 10 d after transfer they accounted for only ~15% of the resulting Ly49C/I+
NK cells and 5–20% of the resulting Ly49G2+
cells (Table ). These data indicate that contaminating Ly49C/I or G2+
NK cells in the inoculum cannot account for the large majority of Ly49C/I or G2+
NK cells in the population after transfer, especially when it is recalled that the Ly5.1+
contaminating cells were added at a >20-fold excess compared with contaminating B6 origin Ly49C/I or G2+
cells. Therefore, the large majority of Ly49C/I or G2+
NK cells after transfer were derived from Ly49A−
cells in the inoculum.
Contribution of Added Contaminants to NK Cell Populations after In Vivo Transfer
A similar strategy (Fig. ) was used to address the possibility that the Ly49C/I or G2+ NK cells observed after transfer arose from contaminating NK1.1− cells in the inoculum. The spleen contains hematopoietic stem cells and possibly other more restricted lymphoid progenitors, so it was possible that these cells differentiated into Ly49C/I or G2+ NK cells after transfer. Post-sort analysis indicated that a purified population of B6-derived Ly49A−C−G2−I− NK cells contained ~2.1% NK1.1− cells, a value similar to that seen in other experiments (e.g., 1.4% in Fig. ). NK1.1− splenocytes from B6-Ly5.1 mice were added to this population so as to achieve 5% contamination, a 2.5-fold excess compared with the NK1.1− contaminants already present. The added NK1.1− splenocytes had been sorted from a population that was initially enriched for NK cells using the same procedure as was used for the NK1.1+ population. After flow cytometric analysis to confirm the cell composition (not shown) the mixture was injected into irradiated BALB.B mice. 10 d after transfer, NK1.1+ (donor) cells were tested for expression of Ly5 markers (Fig. ). Although 70% of the contaminating NK1.1− cells in the inoculum were from the Ly5.1+ donor, essentially none (<2%) of the donor-derived NK1.1+ cells after transfer were Ly5.1+. Significant numbers of Ly5.1+ cells were observed in only one of the two recipients, and these were overwhelmingly NK1.1− (Fig. ). None of the Ly5.1+ cells after transfer detectably expressed Ly49C/I or Ly49G2. We conclude that neither contaminating NK1.1− precursors, nor contaminating Ly49C/I or G2+ cells, can account for the vast majority of Ly49C/I or G2+, NK1.1+ cells observed after transfer of Ly49A−C−G2−I− NK1.1+ cells. It follows that the Ly49C/I or G2+ cells after transfer were derived from NK1.1+ Ly49A−C−G2−I− cells in the inoculum.
Figure 6 NK cells do not arise from splenic NK1.1− contaminants in the starting Ly49A−C−G2−I− NK population. Cells were purified, mixed and inoculated into BALB.B mice as described in Fig. . NK cells of (more ...)