216 4e FORUM D'IMMUNOLOGIE

D I S C U S S I O N

;I. ~l. T. 0 w e n :

There are two issues arising from the papers tha t I whould like to comment on.

1) Di]ficullies in oblaining primary generation of B cells in vitro.

I fully support the view of Nossal tha t the development of systems for reproduc- ing pr imary B cell genesis in vitro is of great importance if we are to understand the cellular and molecular mechanisms involved. I think tha t we must distin- guish between two processes here: (a) the differentiation of pre-B cells (and perhaps their immediate precursors) into B cells and (b) the generation of pre-B cells from self-renewing stem cells. The former, i. e. the maturation of pre-B cells, can be readily obtained in vitro (e. g. Melchers, 1977); however, the latter, i. e. the continuous production of pre-B cells from stem cells, is difficult to obtain. Like Nossal, I suspect tha t IL-3 will not be adequate to support the lat ter --- after all, IL-3 has been shown to support proliferation of pre-B cell clones and already-generated foetal liver pre-B cells (Palacios el al., 1984), but I am not aware of evidence tha t it will support the generation of new pre-B cells from self-renewing stem cells. The serum factors of NZB mice and tha t derived from a patient with cyclic neutropenia, both described by Kincade, are of considerable interest. Again, they would seem to promote proliferation/ maturat ion of pre-B cells ra ther than the continuous production of pre B-cells from stem cells.

The main claim to having achieved the challenge of Nossal seems to belong to Whitlock el al. (1984). They have modified the Dexter culture system (where many others have failed to produce pre-B cells) and claim tha t the), can obtain long term bone marrow cultures which produce pre-B cells. However, some caution may be justified because the methodology requires selection at various stages and, in particular, cultures go through a ~ crisis ~ phase which might imply tha t the pre-B cells which are produced eventually are a minor component of normal B lymphopoiesis.

2) Phenolypic and [unclional tlif[erences between pre-B cells of [celal liver and pre-B cells o[ adull marrow. Kincade points to a number of interesting differences between B-cell pre-

cursors in embryos and adults. For example, Lyb-2 is expressed on adult but not embryonic cells. I am not sure to what extent these differences might reflect a predominence of very immature cells at the foetal stage, while in adult marrow, many more ~ mature ~ pre-B cell types might be present. Thus, the lat ter are likely to be analysed in marrow, but the former in foetal l i v e r - however, both types would be present in both locations.

However, the issue raised by Zharhary el al. is unlikely to be explained so readily. They point to the striking difference in terms of receptor repertoire development between B-cell precursors in perinatal liver and spleen as opposed to adult marrow. The specificity repertoire of pre-B cells of the neonate is restricted, whereas tha t of adult bone marrow sIg- B-cell precursors is not. They go as far as to suggest tha t the differences are such as to indicate tha t the two cell populations may belong to distinct B-cell lineages. However, an alternative. admit tedly highly speculative suggestion is made in my paper, namely, tha t some pre-B cells generated in foetal liver might clonally expand independently of antigen, in other sites prior to entering the peripheral recirculating pool of

B-CELL ONTOGENY 217

B cells. I discussed the possibility in the context of Reynold 's observations on Peyer ' s patches of the foetal lamb, but it is possible tha t such expansion might occur within adult marrow. Clonal expansion of this typc would make sense if point mutat ions within V genes were to contribute substantially to the pr imary repertoire. I cntircly agree with Zharhary et al. t ha t further analyses of their observation is very impor tan t to our understanding of pr imary B-cell differen- tiation.

Re/erences.

MELCIIERS, F., Europ. J. Immunol . , 1977, 7, 467-481. PALACIOS, 1~., HENSON, G., STEINMETZ, M. & McKEARN, J. P., Nature (Lond.),

1984, 309, 126-131. WIIITLOCK, C. A., I:~OBEFITSON, D. & WITTI~], O. N., J. Immunol . Methods, 1984,

67, 353-369.

P. W. Kineade:

- - Truf fa-Bachi el al.

One point made in this article is t ha t pre-B cells ,( are characterized by a considerable proliferative capacity )). This may be true in the sense tha t even memorv B cells can divide many times but the available information indicates tha t c~+,slg - pre-B cells do not Extensively self renew within adult bone marrow (Landreth et at., J . immunol . , 1982, 127, 2027). On the other hand, if the factor- dependent pre-B cells maintained by Whitlock and Witte or Palacios el al. are normal, the potential for replication without significant differentiation is there. I t will be impor tan t to learn if, during ontogeny, during regeneration from irradiation, etc. the cycling of pre-B cells can be modified.

- - Nossal .

The report by Palacios el al. describing IL-3 dependent pre-B cell lines caught the attention of m a n y of the contributors to this forum, i agree with Professor Nossal that this is a very interesting molecule with multiple effects in culture, but it seems unlikely tha t iL is the sole regulator of B lymphocyte precursor formation. Our experience has been tha t purified IL-3, unlike Landre th ' s cyclic neutropcnia factor(s), has not induced 14.8-' or cW'- cells in culture from earlier precursors. You might expect a multipoietin to have tlfis function. None of our factors support replication of an IL-3-dependent cell line and it is my understan- ding tha t IL-3 has not been demonstrable in either the Dexter or Whitlock- Wit te culture systems.

We obtained evidence several years ago tha t two different and presumably non-lymphoid (14.8-) cell types augment the formation of B cells in culture (J . l m m u n o l . , 1981, 127, 255). Characterization of such potentially impor tan t microenvironmental components has not progressed because prel iminary surveys did not reveal them to be positive for Ia or Mac-1 antigens. Depletion of regu- la tory cells should be a t t empted with additional monoclonal antibodies before culture and correlations should be sought between their number and/or activity in various dysregulated conditions. We can speculate without evidence tha t one of these is hyperact ive or overabundant in young NZB mice. Similary: the func- tion of one of them could abnormally increase in the cyclic neutropenm patient. Investigations of this type comprise our approach to detinition of the c~ black box ,.

- - Zharharg.

Zharhary and colleagues review a convincing body of evidence which indi- cates tha t s lg- precursors within adult bone marrow are eapable of generating