Figure 1. NK cells developing in vitro from early thymic progenitors progressively acquire Ly49 molecules recognized by the mAb 4D12

Fresh E14 thymocytes contain very few [<1%] Ly49E-expressing cells

as judged by staining with the 4D12 mAb that recognizes Ly49E and C

[note histogram plot shown at high magnification]

Following 2d of primary culture in IL4+PMA there are essentially

no detectable cells that stain with the 4D12 mAb

However, within a few days of transfer to IL2 [this plot is after 3 days]

4D12⁺ cells appear. Note, though, that at this stage the intensity of

staining of 4D12⁺ cells is low

By 6 days after transfer to IL2 the proportion of 4D12⁺ cells has increased

but the level of expression is still low

By 16 days after transfer to IL2 there is both a further increase in the

proportion of 4D12⁺ cells and a marked heterogeneity in the level

of expression, some cells now being 4D12^hi

After 40 days of culture in IL2 most cells strongly express the

Ly49 molecule(s) recognized by 4D12

Control staining of cultured adult splenic NK cells with 4D12.

Most of the positive cells express Ly49C

Conclusions

1. Fresh E14 thymocytes contain <1% of cells that stain with the anti-Ly49 mAb 4D12

2. During intial culture in IL4+PMA there is no development of 4D12⁺ cells

3. 4D12⁺ cells appear slowly and progressively during subsequent culture in IL2

4. The 4D12⁺ cells that appear initially express only low levels of the Ly49 molecule(s)

recognized by 4D12

5. Most long term lines and clones of NK cells derived from thymic progenitors uniformly

express high levels of these Ly49 molecules [data not shown]

6. Because the only known Ly49 molecules recognized by 4D12 are Ly49C and Ly49E

and NK cells developing in vitro do not express Ly49C, it is highly likely that the

Ly49 molecules detected on NK cells developing in vitro are Ly49E