Cloning Cells of the Immune System
N. A. Mitchison    Hämatol. Bluttransf. Vol 26

The purpose of this introductory note is to explain why thc immunological papers in this collection conccntrate on cloning. Thcrc are threc good reasons for choosing cloning as an appropriate subject at the present time. One is that cloning provides a valuablc means of acquiring information about the working of the immune system. Another is that scveral of the technical problems which prcvented satisfactory cloning have just been solved, so that rapid progress can and is being made. The third is that application of the new procedurcs is providing insight into leukaemia. The value of cloning follows from the way in which the immune system is arranged as a loose population of cells which traffic from place to placc interacting through transient contacts and soluble factors. In consequencc cells are differentiated from one another not by their anatomical position and connections but by their genetic and epigenctic makeup. There is a strong contrast here between the nervous and immune systems, the two most complcx and highly integrated systems of thc body which otherwise share many fcatures in common. No doubt clones from thc nervous system can provide intcrcsting information about such topics as receptor function and metabolic control, but they cannot bc expected to tell us directly how neurones work. This is not true of Iymphocytcs and to a lesscr cxtent, of antigen-prcscnting cells. hcrc we can cxpect clones to exprcss all major functions. The major technical problem has been to find ways of keeping cells alive and multiplying outsidc the body. The first step forward was to maintain clones of B cells under antigenic stimulation in irradiated mice (Askonas and Williamson 1972). For B cells thc in vitro problem has now largcly bccn solved by thc 294 Haematology and Blood Transfusion Vol26 Modern Tre!lds i!l Huma!l Leukcmia IV Edited by Neth. Gallo, Graf, Ma!l!lweilcr, Wi!lkler Spri!lgcr- Verlag Berlin Heidelbcrg I 98 1 hybridoma technique (Köhler and Milstein 1976 ). Monoclonal antibodies produced by this technique turn out to bc immensely powerful tools in biochemistry, cell biology, and medicine. Their application is well exemplified by Bcverley's study of stem cell surface markers described in this volume. Thc hybridoma revolution is sweeping all beforc it, leaving only little room for alternatives such as thc immortalization of human immunoglobulin-sccreting cells by Epstein-Barr virus infection (Steinitz et al. 1977). IFor T cells, hybriomas have thus for proved less successful. Our own expericnce has bcen that immunoregulatory activity can bc maintained in this way for a while, but tends to decline in an unpredictablc and uncontrollable way (Kontiainen ct al. 1978). Other laboratories find the same dccline. On the other hand, T cells are proving highly amenable to less drastic cloning procedurcs. One such procedure is to maintain them on T ccll growth factor (TCGF). Another is to restimulate cultures with antigen at intervals. Both of these proccdures arc discussed and evaluated in detail at the International Congress of Immunology this year, and the latter is wcll exemplified by Hengartner's study dcscribed hcrc. Our approach (Czitrom ct al. 1980) has been to gcnerate allospccific helper T cells by stimulation in vitro. Our prcvious work had shown that the adoptive sccondary response in micc could be successfully adapted for the study of helper T cells dirccted at cell surface antigens. We generate helper T cells by alloantigen-induced proliferation in vitro directed at Ik (A.TH anti-A.TL) and test for their ability to help in vivo primed B cells directed at Db (A.TH anti-B 10) in an adoptive secondary response with 2000- R irradiated boosting antigen -a cell carrying both the Ik and the Ob antigens [B10.A(2R)]. Helper T cells did increase the anti-Ob response, as judged by CrSl cytotoxicity titrations 9 days after cell transfer. The in vitro generated specific helper T cells in primary and repeatedly stimulated mixed lymphocyte cultures were more effective in helping these B cell responses than equivalent helper T cells induced by in vivo priming. Similar results have been obtained with helper T cells boosted in vitro and directed at H-minor antigens (CBA anti-B10.Br) in helping in vivo primed B cells directed at Thy .1 (CBA anti-AKR). Thus, we are still at an early stage in our attempt to generate clones. The point of our approach is that it utilizes a powerful and important group of antigens, the murine alloantigens, at the expense of having to use a rather cumbersome assay forfunction , How far will these approaches take us with leukaemia? The use of TCGF for growing leukaemic and normal lymphocytes in vitro are just beginning to be explored and will be made easier by the purification of the agent as here described by Gallo. TCGF is itself both a candidate agent and a target for therapy in immunological diseases, including leukaemia. Lymphocytes can be generated in vitro with the capacity to kill MHC-identical human leukaemic cells (Sondel et al. 1976). There are still many questions about these cells, such as their relationship with natural killer (NK) cells. These can surely best be answered by cloning. On the B cell side, the main application of monoclonal antibodies to leukaemia thus far has been in ( 1) the identification of markers on lymphocyte subsets and their use in defining leukaemic phenotypes, topics discussed here by Greaves and (2) the characterization of transformation proteins such as ppSRC6o (for references see Mitchison and Kinlen 1980). Some fascinating questions are beginning to arise in ontogeny as one attempts to relate the stages of lymphocyte development to events affecting immunoglobulin genes. At what stage, for instance, do VH and VL genes move to their "differentiated" position close to J and C genes ? If, as seems likely in the mouse at least, V H genes are expressed ( as idiotypes ) earlier than V L genes, why does the intervening interval (the pre-B cell) last so long? Could it be that movement of V II is a difficult and dangerous process for the cell, as the evidence of mistaken movements on the unexpressed chromosome suggests; if so, may not the rapid proliferation of pre-B cells represent a mechanism for expanding a premium cell before it has to undergo the equally costly business of moving a VI gene? Such speculations may at least begin to explain why so many ALLs are of pre-B types (this discussion of pre-B cells draws on M. Cooper's unpublished data and is derived from discussion with him),


Askonas EA, Williamson AR (1972) Factors affecting the propagation of a B cell clone forming antibody to the 2,4-dinitrophenyl group, Eur J Immunol 2'487-493- Czitrom AA, Yeh Ming. Mitchison NA ( 1980) Allospecific helper T cells generated by alloantigenic stimulation in vitro In Preud'homme JL, Hawken VAL (eds) Abstracts tbe 4th international congress of immunology, Paris, 1980, Acadcmic Press, New York -Kbhler C. Milstein C ( 1976) Derivation of specific antibody-producing tissuc culture and tumour lines by cell fusion, Eur J Immunol 6'511-519- Kontiainen S, Simpson E, Bohrer E, Beverley PCL, Herzenberg LA, Fitzpatrick WC, Vogt P, Torano A, McKenzie IFC, Feldmann M (1978) T cell lines producing antigenspecific suppressor factor, Nature 274'477-480 -Mitchison NA, Kinlen L ( 1980) Present concepts in immune surveillance, In' Fougereau M, Dausset J (eds) Immunology 1980 (Proceedings of the 4th international congress of immunology, Paris, 1980) Academic Press, New York pp 641-650- Sondel PM. OErien C, Porter L, Schlossman SF, Chess L ( 1976) Cell-mediated destruction of human leukaemic cells by MHC identical lymphocytes' requirement for a proliferative trigger in vitro, J Immunol 117.2197-2203 -Steinitz M, Klein G, Koskimies F, Mäkelä O ( 1977 ) EE virus induced E lymphocyte cell lines producing specific antibody Nature 269420-422 295