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Lymphopoiesis and lymphokines
Immunology Today September 1983 Lymphopoiesis and lymphokines from Roland Scollay Steamboat Springs recently played host to a meeting on normal and neoplastic hematopoiesis*, which included some interesting results from studies on lymphopoiesis. H. Cantor (Harvard) discussed the induction of m R N A for an antigenbinding protein on T inducer ceils. Inducer clones were activated by their specific antigen, 'induced' m R N A was prepared by subtractive hybridization with non-activated cells of the same clone, and a cDNA was made from this induced mRNA. In Northern analysis of a n u m b e r of other clones, some of the cDNA bound only to R N A from other inducer clones, not to RNAs from other T cells. Translation products from the cDNA could be separated into three bands, one of which bound specifically to the inducing antigen, and was recognized by a clone-specific antibody directed against that clone (putative anti-T-ceil idiotype). This raised the interesting question of the induction of the T-cell receptor after antigen stimulation, but little detail was presented on the antigen binding by the translated protein, so the validity of this aspect could not be assessed. I. Weissman (Stanford) discussed the hypothesis that cells which form turnouts from non-acutely transforming viruses are stimulated through their antigen receptor, the tumours arising from precursors with receptors specific for virus antigen. By this hypothesis, virus binding should provide an access to the antigen receptor. However, as Weissman pointed out, a number of antibodies which block virus binding specifically on one tumour (behaving like an antiidiotype) cross-react with molecules such as Thy 1 and gp 70 on other tumours, even though conventional anti-Thy 1 or anti-gp 70 do not block virus binding on *The U C LA symposium ' N o r m a l and Neoplastic Hematopoiesis' was held in Colorado between 22 M a r c h and 2 April 1983.
the original tumour. He suggested, therefore, that this approach may not provide a means of isolating the T-cell receptor. Weissman also discussed another receptor system, that of the lymphocyte receptors for vascular endothelium which play a role in lymphocyte migration. He described a monoclonal antibody (MEL14), whidh binds to lymphocytes and which inhibits the in-vitro adherence of lymphocytes to the high endothelial venules of peripheral lymph nodes, but not of Peyer's patches. It also inhibits
migration of lymphocytes or tumour cells to peripheral lymph nodes in vivo but not to Peyer's patches, so the antibody probably recognizes one of a n u m b e r of lymphocyte surface molecules involved in the control of lymphocyte traffic. Interestingly, although MEL-14 did not bind to most thymocytes (thymocytes generally lack the ability to migrate to lymphoid tissues), a small n u m b e r of scattered ceils in the thymic cortex were strongly labelled with the antibody and Weissman suggested that these were the cortical precursors of peripheral T ceils. Thymic lymphopoiesis was also discussed. F. Lepault (Villejuif) and I. continued on p. 245
On getting a T-cell clone and being assured you have one* from Fritz Bach Cellular i m m u n o l o g y has for years b e e n p l a g u e d b y the heterogeneity i n function a n d specificity of a n t i g e n r e c o g n i t i o n p r e s e n t w i t h i n the p o p u l a t i o n s of l y m p h o c y t e s available for study. T h e ability to clone single T cells from those p o p u l a t i o n s is therefore a most useful technology because the progeny in a n y one clone will be of a single functional subtype and, a t t h e very least, will have the great specificity associated w i t h monoclonality. C l o n i n g is b e i n g widely used to p u s h f o r w a r d our understanding of both the antigens that elicit response and the cells t h a t respond. M y purpose here, however, is not to review those very exciting applications but rather to address the sometimes thorny issues of the d e f i n i t i o n of clonality, h o w it is achieved, a n d the circumstances in w h i c h ' p r o o f ' of clonality is or is not essential to the i n t e r p r e t a t i o n of data. A clone is defined as a population of cells arising from a single precursor cell. In practice, however, it is extremely difficult to be certain that in a culture one truly has a clone defined in this manner. Several different methods have been used for cloning. Growth of 'colonies' from cells plated in soft agar is one. However, it can be very difficult to determine the likelihood that the cells in any one colony arose from a single precursor. Cells can migrate in soft agar and can come together even across some dis* While one can never be completely certain that a clone is present, one can estimate the probability of this being so.
tance; thus two cells (of the same or different functional subtype) can initiate the growth of a colony. It could be argued that fffew enough cells are plated initially into the dish, the probability of two cells clustering in this way is very, small indeed. But it is just this vagueness of conclusion that one wishes to avoid in cloning. Instead, it is highly desirable to provide an accurate statement of the probability of clonality for those instances in which clonality is essential to the interpretation of the data, or, at the very" least, to use a method in which uncertainties can be rather easily evaluated. continued on p. 245
© 1983.El~ier SciencePublishe~ BV., Amsterdam 0167- 4919/83/$01.00
the original tumour. He suggested, therefore, that this approach may not provide a means of isolating the T-cell receptor. Weissman also discussed another receptor system, that of the lymphocyte receptors for vascular endothelium which play a role in lymphocyte migration. He described a monoclonal antibody (MEL14), whidh binds to lymphocytes and which inhibits the in-vitro adherence of lymphocytes to the high endothelial venules of peripheral lymph nodes, but not of Peyer's patches. It also inhibits
migration of lymphocytes or tumour cells to peripheral lymph nodes in vivo but not to Peyer's patches, so the antibody probably recognizes one of a n u m b e r of lymphocyte surface molecules involved in the control of lymphocyte traffic. Interestingly, although MEL-14 did not bind to most thymocytes (thymocytes generally lack the ability to migrate to lymphoid tissues), a small n u m b e r of scattered ceils in the thymic cortex were strongly labelled with the antibody and Weissman suggested that these were the cortical precursors of peripheral T ceils. Thymic lymphopoiesis was also discussed. F. Lepault (Villejuif) and I. continued on p. 245
On getting a T-cell clone and being assured you have one* from Fritz Bach Cellular i m m u n o l o g y has for years b e e n p l a g u e d b y the heterogeneity i n function a n d specificity of a n t i g e n r e c o g n i t i o n p r e s e n t w i t h i n the p o p u l a t i o n s of l y m p h o c y t e s available for study. T h e ability to clone single T cells from those p o p u l a t i o n s is therefore a most useful technology because the progeny in a n y one clone will be of a single functional subtype and, a t t h e very least, will have the great specificity associated w i t h monoclonality. C l o n i n g is b e i n g widely used to p u s h f o r w a r d our understanding of both the antigens that elicit response and the cells t h a t respond. M y purpose here, however, is not to review those very exciting applications but rather to address the sometimes thorny issues of the d e f i n i t i o n of clonality, h o w it is achieved, a n d the circumstances in w h i c h ' p r o o f ' of clonality is or is not essential to the i n t e r p r e t a t i o n of data. A clone is defined as a population of cells arising from a single precursor cell. In practice, however, it is extremely difficult to be certain that in a culture one truly has a clone defined in this manner. Several different methods have been used for cloning. Growth of 'colonies' from cells plated in soft agar is one. However, it can be very difficult to determine the likelihood that the cells in any one colony arose from a single precursor. Cells can migrate in soft agar and can come together even across some dis* While one can never be completely certain that a clone is present, one can estimate the probability of this being so.
tance; thus two cells (of the same or different functional subtype) can initiate the growth of a colony. It could be argued that fffew enough cells are plated initially into the dish, the probability of two cells clustering in this way is very, small indeed. But it is just this vagueness of conclusion that one wishes to avoid in cloning. Instead, it is highly desirable to provide an accurate statement of the probability of clonality for those instances in which clonality is essential to the interpretation of the data, or, at the very" least, to use a method in which uncertainties can be rather easily evaluated. continued on p. 245
© 1983.El~ier SciencePublishe~ BV., Amsterdam 0167- 4919/83/$01.00